NanoBio&Med 2014 Abstracts Book by Phantoms Foundation

nov. 18-21, barcelona (spain) www.nanobiomedconf.com

abstracts book organisers

Foreword Organising

Committee

Sponsors

Exhibitors

Speakers

Posters

101

On behalf of the Organizing Committee, we take great pleasure in welcoming you to Barcelona (Spain) for the NanoBio&Med 2014 International Conference. The NanoBio&Med 2014, after successful editions organized within ImagineNano in Bilbao 2011 & 2013, is going to present the most recent international developments in the field of Nanobiotechnology and Nanomedicine and will provide a platform for multidisciplinary communication, new cooperations and projects to participants from both science and industry. Emerging and future trends of the converging fields of Nanotechnology, Biotechnology and Medicine will be discussed among industry, academia, governmental and non-governmental institutions. NanoBio&Med 2014 will be the perfect place to get a complete overview into the state of the art in those fields and also to learn about the research carried out and the latest results. The discussion in recent advances, difficulties and breakthroughs will be at his higher level. This year, an industrial forum will also be organized to promote constructive dialogue between business and public leaders and put specific emphasis on the technologies and applications in the nanoBioMed sector. We are indebted to the following Scientific Institutions, Companies and Government Agencies for their financial support: Universitat de Barcelona, Institute for Bioengineering of Catalonia (IBEC), The Centre for BioNano Interactions (CBNI), ICEX Espa帽a Exportaci贸n e Inversiones, NanoSciences Grand Sud-Ouest, NanoMed Spain and the health campus of the University of Barcelona (HUBc). In addition, thanks must be given to the staff of all the organising institutions whose hard work has helped planning this conference.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Antonio CORREIA President of the Phantoms Foundation (Spain) Dietmar PUM Deputy Head of the Biophysics Institute – BOKU (Austria) Josep SAMITIER Director of the Institute for Bioengineering of Catalonia – IBEC (Spain)

NanoBio&Med2014 november 18-21, 2014 - Barcelona (Spain)

The Centre for BioNano Interactions (CBNI) is a multi-disciplinary platform for Nanotoxicology and NanoMedicine. The Centre for Bio NanoInteractions is Irelandâ&#x20AC;&#x2122;s National Platform for BioNanoInteraction science, and draws together specialists from its Universities, Institutes and companies. We are one of the worldâ&#x20AC;&#x2122;s leading Centres of knowledge for bionanointeractions applied to the fields of nanosafety, nanobiology and nanomedicine, and we are pioneering many of the new techniques and approaches in the arena. We have strong links and co-operations with academia, institutions, industry, and governments world-wide. We seek to set standards through commitment to excellence in research and innovation, blended with caution and attention to detail in the science, and public dissemination. We appreciate the multilateral responsibilities to promote knowledge, economic development, and above all the advancement of these in a safe and sustainable manner. As such, the Centre is founded on principles of integrity and transparency in all of its activities. More info: www.ucd.ie/cbni

IBEC is a non-profit foundation established at the end of 2005 by the Ministries of Innovation, Universities and Enterprises and Health of the Generalitat de Catalunya (Autonomous Government of Catalonia), by the University of Barcelona (UB) and by the Technical University of Catalonia (UPC). The governing body of IBEC is its Board of Trustees, composed of members of the four founding institutions. IBEC's Board of Trustees receives advice from the director of the institute and from the International Scientific Committee. IBEC's International Scientific Committee plays a key role in the activities of the institute, focusing especially on the selection and evaluation processes of the research group leaders. The International Scientific Committee is composed of international renowned scientists in different bioengineering fields, as well as prestigious professionals in key areas within the activities of IBEC, such as research results valorization or medical technologies validation. IBEC is funded by its founding institutions, by competitive research projects, whether national or international, and by R&D contracts with companies. More info: www.ibecbarcelona.eu

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Speakers list: Alphabetical order (by surname) Authors Alcaraz, Jordi (University of Barcelona, Spain) Aberrant mechanical microenvironment in lung cancer probed at the micro- and nano-scales

Barrán Berdón, Ana Lilia (Universidad Complutense de Madrid, Spain) Calix[4]arene TMAC4 as efficient non-viral vector in gene therapy

Benita, Simon (The Hebrew University of Jerusalem, Israel) Improved Oral Absorption of Exenatide using a Novel Nanoencapsulation and Microencapsulation Approach

Benny, Ofra (The Hebrew University of Jerusalem, Israel) Nanomicelles for Targeting the Tumor Microenvironment

Blázquez, María (INKOA SISTEMAS, SL, Spain) Selection of Nanotechnology enabled products for nano-release assessment throughout their life cyle in the NANOSOLUTIONS project

Brossel, Rémy (Cell Constraint & Cancer SA, France) Action of mechanical Cues in vivo on the Growth of a subcutaneously grafted Tumor: Proof of Concept

Cassinelli, Nicolás (nanoScale Biomagnetics, Spain) Setting a standard on magnetic heating of nanoparticles for bioapplications

Cognet, Laurent (CNRS & Université de Bordeaux, France) Single-molecule and super-resolution microscopies in biology: taking the best of fluorescent dyes, gold nanoparticles and carbon nanotubes

Contant, Sheila ((IQAC) /CSIC /CIBER-BBN, Spain) Preparation of Colloidal Dispersions of Magnetic Nanoparticles Coated with Biocompatible Polymers

Daban, Joan-Ramon (Universitat Autònoma de Barcelona, Spain) Use of Nanomechanical Data to Validate a Supramolecular Multilayer Model That Explains the Dimensions, Topology, and Physical Properties of Condensed Metaphase Chromosomes

Della Pia, Eduardo Antonio (University of Copenhagen/Nano Science Center, Denmark) Conducting polymers as a versatile platform for protein nanoarrays technologies

Domènech, Oscar (University of Barcelona, Spain) Lipid composition modulates nanomechanics of transmembrane proteins

Dostalek, Jakub (Austrian Institute of Technology, Austria) Plasmonic biosensors advanced by functional hydrogels

Elezgaray, Juan (Université Bordeaux, France) Localized, DNA based logical circuits as components for biodetection

Eritja, Ramon (IBMB-CSIC, Spain) Development of modified siRNA for gene silencing

Espejo Rodriguez, Consuelo (OEPM, Spain) How can I protect my invention? BioMed Patents

Fornaguera, Cristina (Institut de Química Avançada de Catalunya (IQAC-CSIC) & CIBER-BBN, Spain) Polymeric nanoparticles, prepared from nano-emulsion templating, as novel advanced drug delivery systems crossing the Blood-Brain Barrier

Franzese, Giancarlo (Universitat de Barcelona, Spain) Kinetics of the protein corona assembly on nanoparticlesinetics of the protein corona assembly on nanoparticles

Gierlinger, Notburga (BOKU, Austria & ETH Zurich, Switzerland) Imaging molecular structure of plant cells by Confocal Raman microscopy

Gorostiza, Pau (ICREA & IBEC, Spain) Optopharmacology to regulate endogenous proteins with light

Guari, Yannick (Université Montpellier II, France) Cyano-bridged coordination polymers nanoparticles as contrast agents for Biomedical Imaging

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Page Oral

Oral

Keynote

Oral

Keynote

Oral

Keynote

Oral

Keynote

Authors

Page

Kanioura, Anastasia (Institute of Nuclear and Radiological Sciences & Technology, Energy & Safety, NCSR “Demokritos", Greece) Effect of nanoscale surface roughness on the adhesion and proliferation of normal skin fibroblasts and HT1080 fibrosarcoma cells

Katakis, Ioanis (Universitat Rovira i Virgili, Spain) Screen printed superhydrophobic surfaces as enablers for Capillarity-Driven Biodetection Devices for Food Safety and Clinical Analysis: towards ASSURED sensors

Lagunas, Anna (Institut de Bioenginyeria de Catalunya (IBEC), Spain) Large-scale dendrimer-based uneven nanopatterns of RGD towards improved architectural networks in chondrogenesis

Llop Roig, Jordi (CIC biomaGUNE, Spain) Following the degradation and biological fate of polymeric poly (lactic-co-glycolic acid) nanoparticles

Marco, M.-Pilar (IQAC-CSIC/CIBER-BBN, Spain) Biofunctional surfaces for Multiplexed Diagnostic Platforms using Site-Encoded DNA Strategies

Mendoza, Ernest (Universitat Politècnica de Catalunya / Goldemar, Spain) From Basic Research to an Industrial Product: The case of Goldemar

Misawa, Masaki (National Institute of Advanced Industrial Science and Technology (AIST), Japan) Radiosensitizing Effect of Gold Nanoparticles under kV- and MV- X-ray Irradiations

Navajas, Daniel (Universitat de Barcelona / IBEC and CIBER of Respiratory Diseases, Spain) Nanomechanics of the extracellular matrix of lung and heart tissues

Paez Aviles, Cristina (University of Barcelona, Spain) Cross-cutting KETs: Innovation and Industrialization challenges for Nanobiotechnology and Nanomedicine towards Horizon 2020

Paoli, Roberto (Institute for Bioengineering of Catalonia (IBEC), Spain) DC studies of Layer-by-layer nanopores electrical properties tuning on Polycarbonate Membranes

Pardo Jimeno, Julian (University of Zaragoza, Aragon Centre for Biomedical Research (CIBA), Spain) Bona fide induction of apoptosis in transformed cells during photothermal therapy using gold nanoprisms

Pla-Roca, Mateu (Institute for Bioengineering of Catalonia (IBEC), Spain) Nanotechnology Platform at the Institute for Bioengineering of Catalonia: description of capabilities and examples

Polo, Ester (CBNI / University College Dublin, Ireland) A microscopic molecular basis for Nanoparticle Interactions with Organisms

Porath, Danny (Hebrew University of Jerusalem, Israel) The Quest for Charge Transport in single Adsorbed Long DNA-Based Molecules

Puntes, Victor (Institut Català de Nanociència i Nanotecnologia (ICN2) & ICREA, Spain) Nanomedicine 2.0

Rigat-Brugarolas, Luis G. (Institite for Bioengineering of Catalonia (IBEC) & CIBER-BBN, Spain) Developing new tools for drug testing: introducing a microfluidic platform mimicking the spleen for future pharmacological trials

Rodea Palomares, Ismael (Universidad Autónoma de Madrid, Spain) PAMAM dendrimers internalizes quickly in microalgae and cyanobacteria causing toxicity and oxidative stress

Sáenz, Juan José (Universidad Autónoma de Madrid, Spain) Speckle fluctuations resolve the interdistance between incoherent point sources in complex media

Sánchez, Samuel (Max Planck Institute for Intelligent Systems, Germany) Self-powered microbots towards a “Fantastic Voyage”

Scheffold, Frank (University of Fribourg, Switzerland) Scattering based bead-microrheology applied to biomaterials

Schroeder, Avi (Technion - Israel Institute of Technology, Israel) Targeted drug delivery and personalized medicine

Schwartz Navarro, Simo (Vall d'Hebron Hospital - CIBBIM, Spain) Personalized Cancer Nanomedicine. CLINAM 2014

Serrano Núñez, Juan Manuel (Sesderma Laboratories, Spain) Liposomes: Topical and Oral Bioavailability

Shoseyov, Oded (The Hebrew University of Jerusalem, Israel) Nano crystalline cellulose-protein composites: Super performing biomaterials for tissue engineering and regenerative medicine

Soares, Paula (Universidade Nova de Lisboa, FCT-UNL, Portugal) Studies on thermal and magnetic properties of iron oxide nanoparticles for magnetic hyperthermia application

Stremersch, Stephan (Ghent University, Belgium) Hijacking nature's own communication system: evaluation of extracellular vesicles as a siRNA delivery vehicle

Swersky Sofer, Nava (ICA, Israel) From Science to Product, in Israel and Beyond

Oral

Keynote

Invited

Keynote

Invited

Oral

Keynote

Oral

Keynote

Oral

Invited

Keynote

Oral

Keynote

Oral

Keynote

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Authors Tatkiewicz, Witold I. (Institut de Ciència de Materials de Barcelona (ICMAB-CSIC) & CIBER-BBN, Spain) 2D Microscale Surface Engineering with Novel Protein based Nanoparticles for Cell Guidance

Toca Herrera, Jose Luis (BOKU / Institute for Biophysics, Austria) Atomic force microscopy, life sciences and soft matter

Unciti Broceta, Juan Diego (Centre for Genomics and Oncological Research (GENYO), Spain) Multiplicity of Nanofection: a New Index to Assess Nanoparticle Cellular Uptake

Uriarte, Juan José (Universitat de Barcelona, Institut d’Investigacions Biomèdiques August Pi Sunyer & CIBER de Enfermedades Respiratorias, Spain)

Page Oral

Keynote

Oral

Keynote

Oral

Invited

Oral

Nanomechanics of Decellularized Lung and in Vivo Lung Elastance in a Murine Model of Marfan Syndrome

Veciana, Jaume (Institut de Ciència de Materials de Barcelona (CSIC)-CIBER-BBN, Spain) Supramolecular organizations as novel nanomedicines for drug delivery

Vicendo, Patricia (Laboratoire des IMRCP, Université de Toulouse, France) Polymeric micelles nanovectors for photodynamic therapy applications: From the structure to the activity

Vila, Mercedes (University of Aveiro, Portugal) Nanographene-oxide mediated hyperthermia for cancer treatment

Wajs, Ewelina (Universitat Rovira i Virgili, Spain) Host-guest engineered stimuli-responsive nanocapsules

Yudina, Tetyana (The Catalan Institute of Nanoscience and Nanotechnology (ICN2), Spain) Nanoceria

Zambelli, Tomaso (ETH Zurich, Switzerland) FluidFM: combining AFM and microfluidics for single-cell perturbation in vitro

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Keynote

100

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Aberrant mechanical microenvironment in lung cancer probed at the micro- and nanoscales Jordi Alcaraz, Marta Puig, Marta Gabasa, Roberto Lugo, Roland Galgoczy Unit of Biophysics, School of Medicine, University of Barcelona, Casanova 143, Barcelona, Spain jalcaraz@ub.edu The lung is a moderately soft organ with unique mechanical properties that are necessary for breathing. However, the appearance of a desmoplastic stroma rich in activated fibroblasts/myofibroblasts and pro-fibrotic extracellular matrix (ECM) components in lung tumors strongly suggests that the cellular microenvironment becomes abnormally stiff in lung cancer. To test this hypothesis, we have analyzed the expression of fibrillar collagens (a major pro-fibrotic ECM component) in vivo by picrosirius red staining, and have assessed the mechanical consequences of enhanced collagen density ex-vivo by atomic force microscopy (AFM). Likewise, we have determined the fraction of activated fibroblasts in vivo, and the mechanical consequences of such activation in culture at the nano- and micro-scale by AFM. Finally, we have examined cell-ECM mechanical interactions in fibroblasts at the nano-scale with microfabricated flat-ended-AFM tips designed to mimic few cell-ECM adhesion sites. The biological consequences of aberrant cell-ECM mechanical interactions in tumors were further analyzed in terms of fibroblast accumulation, which is a major hallmark of solid tumors in the lung and other organs. Normal lung parenchyma exhibited weak collagen staining and short collagen fibers, whereas tumor samples exhibited abundant collagen staining that was frequently organized into long and straight fibril bundles indicative of mechanical tension. Likewise, fibril bundles were often organized in a parallel fashion (see dashed squares) [1], which has been previously shown to

NanoBio&Med2014

render stiffer tissues. To examine the mechanical effects of increased collagen deposition, we probed the Young´s elastic modulus (E) of collagen gels with increasing concentration by AFM. E scaled with collagen density according c to E ~ c2. This dependence is well captured by current models of semiflexible semidiluted polymers in which the fibril length is larger than the pore or mesh size [2]. In normal lung parenchyma, fibroblasts were sparsely located, and α-SMA+ cells corresponding to smooth muscle cells were largely restricted to the perivasculature (see black arrows). In contrast, lung tumors exhibited a stroma rich in peritumoral α-SMA+ fibroblasts. These features were used to assess the relative amount of tumor stroma as the percentage of αSMA+ area, which elicited ~25% [1]. To mimic fibroblast activation in culture, most fibroblasts required exogenous TGF-b1, which is a potent fibrotic cytokine commonly upregulated in the tumor microenvironment. Treatment of fibroblasts with TGF-b1 induced de novo expression of α-SMA incorporated into stress fibers in culture. Concomitantly to these cytoskeletal alterations, TGF-b1 treatment increased the Young´s modulus more than 2-fold with respect to untreated cells. To investigate the critical molecular machinery involved in cell-ECM force transmission, we microfabricated flat-ended AFM tips with a constant cross-section area of ~1 μm2, and coated them with an RGD peptide, which is an adhesive domain found in many ECM components that is specifically recognized by

november 18-21, 2014 - Barcelona (Spain)

ECM integrin receptors. The RGD-coated flatended tip was brought to contact with the surface of a single fibroblasts up to a moderate force, hold for 30 s to enable the formation of focal adhesion precursors, and retracted until the contact was lost. We found a marked increase in both cell stiffnes and cell-ECM adhesion when using RGD-coated tips, but not RGE or other nonintegrin specific coatings. Such cell stiffening and adhesion strengthening were abrogated upon inhibiton of actin but not microtubule polymerization, revealing that local fibroblast mechanoresponses requires integrin-mediated rearrangements of the actin cytoskeleton [3]. To examine the pathological consequences of abnormal integrin mechanosensing in fibroblasts, we analyzed how extracellular stiffening comparable to that expected within the tumor microenvironment altered fibroblast behavior in an integrin-specific fashion. We found that fibroblast density markedly increased in gels with a tumor-like rigidity compared to soft gels with normal-like rigidity values. Remarkably, such cell density increase was abrogated upon inhibition of beta1 integrin mechanosensing through FAKpY397, which is the most abundant component of fibroblast integrin receptors [1].

References [1] Puig M, Lugo R, Gabasa M, GimĂŠnez A, VelĂĄsquez A, Galgoczy R, et al. Matrix stiffening and Î˛1 integrin drive fibroblast accumulation in lung cancer in a subtypedependent fashion. Mol Cancer Res. 2014;(in press). [2] Alcaraz J, Mori H, Ghajar CM, Brownfield D, Galgoczy R, Bissell MJ. Collective epithelial cell invasion overcomes mechanical barriers of collagenous extracellular matrix by a narrow tube-like geometry and MMP14dependent local softening. Integr Biol (Camb). 2011;3:1153-66. [3] Acerbi I, Luque T, Gimenez A, Puig M, Reguart N, Farre R, et al. Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells. PLoS One. 2012;7:e32261.

In summary, we have collected evidence supporting that the tumor microenvironment is much stiffer than the normal lung parenchyma and that activated fibroblasts are largely responsible for such tissue hardening. Of note, we have also obtained evidence of a positive feedback loop in which activated fibroblasts increase tissue hardening, which in turns stimulates fibroblast accumulation in a beta1integrin dependent fashion, which is a hallmark of lung cancer.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Calix[4]arene TMAC4 as efficient non-viral vector in gene therapy A.L. Barrán-Berdón1*, Belén Yélamos2, Luis García-Rio3, E. Aicart1 and E. Junquera1 1 Grupo de Química Coloidal y Supramolecular, Universidad Complutense de Madrid, Madrid, Spain 2 Dpto. Bioquímica y Biología Molecular I, Universidad Complutense deMadrid, Madrid, Spain 3 Centro Singular de Investigación en Química Biológica y Materiales Moleculares. Universidad de Santiago de Compostela, Praza do Obradoiro, s/n 15782-Santiago de Compostela, Spain anbarran@pdi.ucm.es One of the major challenges in the gene therapy process is to find efficiently non-viral gene carriers. Several studies have been done in order to increase the number of compounds able to compact, protect and transport nucleics acids into the cell. The development of several kinds of macrocycles such calixarenes open a new way in the non-viral vector tools in gene therapy. Calixarenes are very promising in gene delivery applications for several reasons: their synthesis is relatively easy, they present low toxicity levels and, possessing two clearly distinct chemical regions, allow an efficient region-selective chemistry.[1-3] Complexes prepared by mixing the gene vector (formed by calix[4]arene TMAC4 and the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), at several molar fractions, α) with plasmid pEGFP-C3 (pDNA) or linear double-stranded calf thymus DNA (ctDNA). A wide biophysical and biochemical characterization was performed including zeta potential, gel electrophoresis, SAXS, cryo- TEM, fluorescence microscopy and cell viability/cytotoxicity to establish a structurebiological activity relationship. The study was performed at several compositions, , between calixarene and DOPE, and at several effective charge ratios, ρeff, (between the gene vector and the DNA) of the complex.

diffractograms correspond to nanoaggregates formed by a lamellar structure at any α. CryoTEM studies reveal the presence of cluster-type and finger print multilamellar structures. Finally, the biochemical studies in vitro show that complexes TMAC4/DOPE-pDNA present moderate transfection efficiency and good cell viability in HEK293T cells lines. Therefore, the reported complexes can be considered as potential DNA vectors for gene therapy in vivo.

References [1] V. Bagnacani, F. Sansone, G. Donofrio, L. Baldini, A. Casnati, R. Ungaro, Organic Letters, 10 (2008) 3953-3956. [2] R. Rodik, A. Klymchenko, Y. Mely, V. Kalchenko, Journal of Inclusion Phenomena and Macrocyclic, (2014) 1-12. [3] R.V. Rodik, A.S. Klymchenko, N. Jain, S.I. Miroshnichenko, L. Richert, V.I. Kalchenko, Y. Mely, Chemistry, 17 (2011) 5526-5538.

Electrochemical studies (zeta potential and gel electrophoresis) confirm that pDNA is efficiently compacted by the TMAC4/DOPE system. Structural characterization by SAXS shows that

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Figures Figure 1. a) Molecular structure of the calix[4]arene TMAC4. b) Plot of zeta potential (ď ş) against the complex composition (L/D) of TMAC4/DOPE-pDNA at several molar fractions, ď Ą. c) Fluorescence micrograph of transfected HEK293T cells.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Improved Oral Absorption of Exenatide using a Novel Nanoencapsulation and Microencapsulation Approach Liat Kochavi-Soudry, Taher Nassar and Simon Benita The Institute for Drug Research of the School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel

Oral delivery of peptides remains challenging and continues to be one of the most attractive alternatives to their parenteral delivery. Despite intensive efforts invested over the last two decades, no commercial solution has yet emerged due to several drawbacks and hurdles associated with the poor intestinal membrane permeability of these hydrophilic macromolecules, instability in the gut and rapid metabolism. Therefore, the development of sophisticated delivery systems for oral administration of peptidic drugs still remains an attractive scientific challenge. Exenatide is a 39amino-acid peptide approved as an adjunctive therapy for patients with type-2 diabetes failing to achieve glycemic control with oral antidiabetic agents. Exenatide is injected subcutaneously (SC) twice a day and can induce pain and possible infections at the sites of injection that could adversely affect patient compliance. A once a week injection of exenatide has been developed but still suffers from the aforementioned drawbacks. In the present research, we propose a nano/microencapsulation process of the hydrophilic bio macromolecule to protect and control exenatide release. This unique strategy should facilitate the controlled release of the exenatide-loaded nanoparticles (NPs), as opposed to the release of the dissolved drug, in the vicinity of the mucosa in an attempt to avoid GI acidic and proteolytic enzymes degradation of the peptide. The first line of protection was achieved by loading the peptide into primary NPs. Different types of NPs were prepared;

NanoBio&Med2014

bovine serum albumin (BSA) NPs cross-linked with glutaraldehyde, BSA mixed with dextran NPs cross linked with sodium trimetaphosphate and conjugation of exenatide to poly lactic-coglycolic acid (PLGA) NPs. The second line of protection was achieved following encapsulation of the primary NPs within microcapsules consisting of a blend of Eudragit L 55-100 and hydroxypropyl methyl cellulose (HPMC) using a spray drying technique. The primary NPs and microcapsules containing exenatide NPs were imaged by Cryo-TEM and SEM respectively. The mean diameter of the cross-linked NPs ranged between 50-100nm or 300-500 nm depending on the cross-linker and matrix. The PLGA NPs mean diameter was 90150nm. The zeta potential value was around 45mV and the encapsulation yield was above 30-40% irrespective of the formulation. The in vitro release kinetic profiles showed that it was possible to reduce the burst release depending on the type of formulation up to 20% followed by a gradual slow release over 6-8 h. The pharmacokinetic results allowed to identify an optimal formulation based on dextran/BSA cross-linked NPs embedded in microparticles which elicited significant plasma levels following oral administration in rats. The marked increase in the oral bioavailability of such a formulation is promising, confirming that the peptide was not markedly degraded during the manufacturing process and the transit via the gastro-intestinal tract.

november 18-21, 2014 - Barcelona (Spain)

Nanomicelles for Targeting the Tumor Microenvironment Ofra Benny Institute for Drug Research, Faculty of Medicine The Hebrew University of Jerusalem, Jerusalem, Israel ofrab@ekmd.huji.ac.il Tumor metastases are the principal cause of mortality in the majority of cancer patients. A hospitable tumor microenvironment, of which the vascular system is a significant component, is crucial in the implantation of disseminated tumor cells. Angiogenesis, the formation of new blood vessels, is a multifactorial process that is critical for tumor progression and metastasis. Anti-angiogenic compounds, has been widely investigated as a strategy to treat cancer. However, several of these drugs are limited by poor pharmacological properties, such as low bioavailability, undesired biodistribution and short half-life necessitating their use in high intravenous doses which expose the patients to adverse size-effects due to off-target activity. To overcome these drug limitations, we developed a formulation of self-assembled nanomicelles composed of short di-block polymers, polyethylene glycol-polylactic acid (PEG-PLA), for conjugating small molecule drugs. We present a case of re-formulating a broad spectrum anti-angiogenic drug from the fumagillin family which originally had several clinical limitations. In the new formulation, unlike the free compound, the drug showed high oral availability, improved tumor targeting and reduced toxicity. Dramatic anti-cancer activity was obtained in eight different tumor types (60-90% growth inhibition) in mice, and, importantly, the treatment was able to prevent liver metastases due to the shift from intravenous to oral administration. The activity was associated with reduction of microvessel density and increased tumor apoptosis. Nanomicelle drug delivery system has been shown to be an efficient approach for improving

pharmacological properties of drugs and for better targeting the tumor-microenvironment.

References [1] Benny O, Fainaru O, Adini A, Cassiola F, Bazinet L, Adini I, Pravda E, Nahmias Y, Koirala S, Corfas G, D'Amato RJ, Folkman J. Nat Biotechnol. 2008 Jul;26(7):799-807. [2] Benny O, Pakneshan P. Cell Adh Migr. 2009 Apr-Jun;3(2):224-9.

Figures

Figure 1. Nanomicelles for treating tumors by targeting tumor microenvironment (A) diagram showing the selfassembled di-block copolymer nanomicelles. (B) AFM image of PEG-PLA Nanomicelles. (C) tumor growth inhibition of subcutaneous Lewis Lung Carcinoma.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Selection of nanotechnology enabled products for nano-release assessment throughout their life cyle in the NANOSOLUTIONS project 1

María Blázquez1, I. Unzueta1, E. Fernández-Rosas2, A. Vílchez2 and S. Vázquez-Campos2 RTD Department, INKOA SISTEMAS, SL, Ribera de Axpe 11, 48950 Erandio Bizkaia, Spain 2 LEITAT Technological Center, C/Innovació, 2, 08225 Terrassa, Spain maria@inkoa.com

The hazard evaluation of engineered nanomaterials (ENMs) needs to take into consideration that the initially synthesized ENMs will not remain unaltered during their life cycle. The intentional introduction of surface modifications to ENMs is a common practice prior to the incorporation of these ENMs in other products. Later, during the use or end of life phases, other transformation processes may take place, so that if ENMs are released they may share few characteristics with the initially synthesized ENMs. Possible changes include surface coating, irreversible embedding in matrices, dissolution, agglomeration and aggregation, surface charge modification, whereas factors underlying the occurrence of these changes include aging, mechanical stress, chemical stress and/or interactions with biota in the environment in most cases in a combined manner. The main goal of the present work has consisted on the selection of nano-enabled products and the evaluation of their life cycle to study the release of ENMs in different phases, within the framework of the NANOSOLUTIONS FP7 European research project. This project ultimately aims at identifying and elaborating those characteristics of ENMs that determine their biological hazard potential by providing a means to develop a safety classification of ENMs.

NanoBio&Med2014

According to the specific processes undergone by the selected applications, the life cycle of the ENMs -beyond manufacturing stage- has been evaluated. Thereafter, the life cycle stages that are most likely to result in the transformation of the ENMs and/or to result in the release of ENMs have been identified prioritizing normal use conditions (releases generated in accidental scenarios have not been considered). The outcome of present work has enabled the definition of realistic laboratory scaled simulation processes to be undertaken in the execution of the project. Preliminary findings on nanoadditivated commercial fabrics are also introduced. Acknowledgements: The authors would like to thank NANOSOLUTIONS consortium. The project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement #309329

november 18-21, 2014 - Barcelona (Spain)

Action of mechanical Cues in vivo on the Growth of a subcutaneously grafted Tumor: Proof of Concept Rémy Brossel Cell Constraint & Cancer SA, Le mas l’Hermite, 331, chemin de la Poterie, Raphèle-les-Arles, France brossel.remy@gmail.com The cancerous tumor tissue and its extracellular matrix are subject to mechanical signals. The role of pressure in tumor transformation and growth as well as in the appearance of metastasis is more and more understood. Hence the effect of constraint/stress on tumor growth has been widely explored in vitro in 3dimension cell culture. The proof of concept delivered by the present work shows the effect of a constraint field in vivo on tumor growth. Nude mice were grafted subcutaneously with a mix of ferric nanoparticles and MDA MB 231 cells. The nanoparticles with a diameter of 100 nm rapidly spread around the growing tumor. The field of constraint was applied through the magnetized nanoparticles located around the tumor. It was generated by the action of a magnetic field gradient on the nanoparticles using permanent magnets located outside the animal. A very statistically significant difference (p=0.015) was observed between the volume of tumors with nanoparticles around and subjected to a field of constraint for 2 hours/day for 21 days and observed to day 59 or more, and the volume of tumor of the three control groups. This experiment provides the first evidence of an action of mechanical signals on the growth of tumor in vivo, in animal. These results confirm in vivo the results previously obtained in vitro on 3-dimension tissue culture models.

[2] Murphy MF, et al. (2013) Evaluation of a nonlinear

References

[13]

[1] Cross SE, et al. (2008) AFM-based analysis of human metastatic cancer cells. Nanotechnology 19(38):384003.

[3] [4]

[5]

[6]

[7] [8] [9] [10] [11] [12]

[14]

Hertzian-based model reveals prostate cancer cells respond differently to force than normal prostate cells. Microsc Res Tech 76(1):36-41. Remmerbach TW, et al. (2009) Oral cancer diagnosis by mechanical phenotyping. Cancer Res 69(5):1728-1732. Fuhrmann A, et al. (2011) AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells. Phys Biol 8(1):015007. Canetta E, et al. (2014) Discrimination of bladder cancer cells from normal urothelial cells with high specificity and sensitivity: combined application of atomic force microscopy and modulated Raman spectroscopy. Acta Biomater 10(5):2043-2055. Indra I (2012) Mechanical forces and tumor cells: insight into the biophysical aspects of cancer progression. Wayne State University Dissertations. http://digitalcommons.wayne.edu/oa_dissertations/4 13:Paper 413. Xu W, et al. (2012) Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells. PLoS One 7(10):e46609. Plodinec M, et al. (2012) The nanomechanical signature of breast cancer. Nat Nanotechnol 7(11):757-765. Lekka M, et al. (2012) Cancer cell recognition-mechanical phenotype. Micron 43(12):1259-1266. Ingber DE, Madri JA, & Jamieson JD (1981) Role of basal lamina in neoplastic disorganization of tissue architecture. Proc Natl Acad Sci U S A 78(6):3901-3905. Tse JM, et al. (2012) Mechanical compression drives cancer cells toward invasive phenotype. Proc Natl Acad Sci U S A 109(3):911-916. Lee GY, Kenny PA, Lee EH, & Bissell MJ (2007) Threedimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4(4):359-365. Jonietz E (2012) Mechanics: The forces of cancer. Nature 491(7425):S56-57. Baish JW & Jain RK (2000) Fractals and cancer. Cancer Res 60(14):3683-3688.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

[15] Bizzarri M, et al. (2011) Fractal analysis in a systems [16] [17]

[18]

[19] [20] [21]

biology approach to cancer. Semin Cancer Biol 21(3):175-182. D'Anselmi F, et al. (2011) Metabolism and cell shape in cancer: a fractal analysis. Int J Biochem Cell Biol 43(7):1052-1058. Stein GS, et al. (1999) Implications for interrelationships between nuclear architecture and control of gene expression under microgravity conditions. FASEB J 13 Suppl:S157-166. Xu R, Boudreau A, & Bissell MJ (2009) Tissue architecture and function: dynamic reciprocity via extra- and intra-cellular matrices. Cancer Metastasis Rev 28(1-2):167-176. Ingber DE (2006) Cellular mechanotransduction: putting all the pieces together again. FASEB J 20(7):811827. Paszek MJ, et al. (2005) Tensional homeostasis and the malignant phenotype. Cancer Cell 8(3):241-254. Montel F, et al. (2011) Stress clamp experiments on multicellular tumor spheroids. Phys Rev Lett 107(18):188102.

Figure 1B. Close up of the West and East sides with force vector Iron

Tumor

Tumor MDA MB 231, Perls special stain, x100-Important labeling of peritumoral areas

Figure 2. Spreading of the nanoparticles around a subcutaneous grafted tumor

Figures

East

West

Figure 3. Growth curve of the tumors in the 4 groups Figure 1A. Schematic representation of the experimental setup with the animal (Magnets tumor not at scale)

Volume (mm3)

Median(Q1; Q3)

(Min; Max)

Mean (±std)

Treated (N=7)

529 (502; 840)

346; 966

646±235

Controls (N=33)

1,334 (758; 1784)

256; 2106

1,250±282

Significance (p value) Significant (p=0.015) IC 95% 579 (124; 1,099)

Table 1 - Tumor volume measured on D59+tumors

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Setting a standard in magnetic heating of nanoparticles for bioapplications Nicolรกs Cassinelli nanoScale biomagnetics. Calle Panamรก 2, Local , 50012 Zaragoza, Spain contact@nbnanoscale.com Magnetic nanoparticles (MNPs) with functionalized surfaces are bringing novel and promising ways to treat deadly diseases such as cancer. They have multiple applications that range from magnetic hyperthermia, localized drug delivery and release, to tissue engineering and new materials. MNPs are designed to attack, with high specificity, a given tissue, challenging researchers in solving biochemical and physiological issues. Depending on the success in such a challenge, cancer specific hyperthermia and drug delivery protocols could be developed. Clinical success of these techniques has been delayed for several years in an important part because of the lack of reliable and compliant specific instrumentation. The Spanish company nanoScale Biomagnetics, formed in 2008 as a Spin Off coming from the University of Zaragoza, entered the market in 2010 with a set of instruments and accessories that can be described as the first high end resource for magnetic heating experiments with MNPs, from material characterization to in vivo experiments. With the common goal of developing the use of the technique, nB works with customers and institutions from all around the globe in the search of new standards and tools.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Single-molecule and super-resolution microscopies in biology: taking the best of fluorescent dyes, gold nanoparticles and carbon nanotubes Laurent Cognet Laboratoire Photonique Numérique et Nanosciences (LP2N). IOGS, CNRS et Université de Bordeaux Institut d’Optique d’Aquitaine, rue François Mitterrand. 33405 Talence cedex, France laurent.cognet@u-bordeaux.fr The optical microscopy of single molecules has recently been beneficial for many applications, in particular in biology. It allows a subwavelength localization of isolated molecules and subtle probing of their spatio-temporal nano-environments on living cells. It also allows designing innovative strategies to obtain superresolved optical images i.e. with resolution below the diffraction limit. For many single-molecule microscopy applications, more photostable nanoprobes than fluorescent ones are desirable. For this aim, we developed several years ago far-field photothermal methods based on absorption instead of luminescence. Such approaches do not suffer from the inherent photophysical limitations of luminescent objects and allows the ultra-sensitive detection and spectroscopy of tiny absorbing individual nano-objects such as gold nanoparticles down to 5 nm in cells or carbon nanotubes. In order to access confined cellular environment (adhesion sites, synapses etc...), I will present our current efforts to reduce the functional nano-objects sizes as well as to use new near infrared nanoprobes. The second part of my presentation will be dedicated to the presentation of superresolution microscopy methods. It is indeed crucial to study a large ensemble of molecules on a single cell while keeping the subwavelength localization provided by single

NanoBio&Med2014

molecule microscopy. In order to study the dynamical properties of endogenous membrane proteins found at high densities on living cells we developed a new single molecule super-resolution technique, named uPAINT. Interestingly, uPAINT does not require the use of photo-activable dyes allowing easy multi-color super-resolution imaging and single molecule tracking. Different applications of uPAINT will be presented, in particularly the first demonstration of super-resolution imaging of functional receptors in interaction. This last result was obtained combining super-resolution microscopy and single molecule FRET.

References [1] Super-resolution microscopy approaches for live cell imaging. A. Godin, B. Lounis, L. Cognet Biophys. J., 107 (2014) 1777. [2] Hyper-bright Near-Infrared Emitting Fluorescent Organic Nanoparticles for Single Particle Tracking. E. Genin, Z. Gao, J. Varela, J. Daniel, T. Bsaibess, I. Gosse, L. Groc, L. Cognet, M. Blanchard-Desce Adv. Mat 26 (2014) 22582261. [3] Identification and super-resolution imaging of ligand-activated receptor dimers in live cells. P. Winckler, L. Lartigues, G.Gianonne, F. De Giorgi, F. Ichas, J-B. Sibarita, B. Lounis and L. Cognet Sci. Rep., 3 (2013) 2387.

november 18-21, 2014 - Barcelona (Spain)

[4] A highly specific gold nanoprobe for live-cell single-molecule imaging. C. Leduc, S. Si, , J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis Nano Lett. 13, 4, (2013) 1489-1494. [5] Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. O. Rossier, V. Octeau, J.B. Sibarita, C. Leduc, B. Tessier, D. Nair, V. Gatterdam, O. Destaing, C. Albigès-Rizo, R. Tampé, L. Cognet, D. Choquet, B. Lounis & G. Giannone Nat. Cell Biol. 14 (2012) 1057-1067.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Preparation of Colloidal Dispersions of Magnetic Nanoparticles Coated with Biocompatible Polymers S. Contant, C. Solans* Institute for Advanced Chemistry of Catalonia (IQAC)/CSIC/CIBER BBN, Barcelona, Spain conxita.solans@iqac.csic.es In recent years much interest has been dedicated to the use of magnetic nanoparticle dispersions for the diagnostic and treatment of diseases [1]. The colloidal dispersions need to have several characteristics such as biocompatibility, colloidal stability, suitable size and narrow size distribution to be used in biomedical applications. The size of the nanoparticles has an influence not only in the magnetic properties and colloidal stability but also in the possibility of crossing biological barriers such as cell junctures and membranes [2]. To assure biocompatibility, magnetic nanoparticles require an appropriate coating. Besides making the dispersions biocompatible, the coating is essential because it protects magnetic nanoparticle surface from oxidation, increases blood circulation time, provides specificity for biological target sites and gives steric repulsion acting as a barrier against the interaction between the particles thereby providing colloidal stability [3, 4]. The objective of this work was to prepare dispersions of magnetic nanoparticles coated with biocompatible polymers showing colloidal and physicochemical characteristics suitable for biomedical applications. Three different polymers were used for coating: polyacrylic acid (PAA), polyethylene glycol (PEG) and polyethylene glycol bisamine (PEG bisamine). Iron oxide nanoparticles were used as core. The coated nanoparticle dispersions were characterized in terms of stability, iron content, size and morphology of the nanoparticles and amount of coating. The dispersions prepared

NanoBio&Med2014

showed stability over a month, concentrations of iron over 5mg/ml, and resulted in coated nanoparticles with small diameters (around 100nm by DLS and 10nm by TEM) and concentrations of organics between 5 and 13%. The results showed that the coated magnetic nanoparticles could be suitable for further biomedical applications.

References [1] Hilger, I., Kaiser, W. A. Nanomedicine, 7 (2012) 1443-1459. [2] Salas, G., Veintemillas-Verdaguer, S., Morales, M. P. International Journal of Hyperthermia, (2013) 1-9. [3] Wahajuddin, Arora, S. International Journal of Nanomedicine, 7 (2012) 3445-3471. [4] Reddy, L. H., Arias, J. L., Nicolas, J., Couvreur, P. Chemical Reviews, 112 (2012) 5818â&#x2C6;&#x2019;5878.

november 18-21, 2014 - Barcelona (Spain)

Use of Nanomechanical Data to Validate a Supramolecular Multilayer Model That Explains the Dimensions, Topology, and Physical Properties of Condensed Metaphase Chromosomes Joan-Ramon Daban Dep. de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain joanramon.daban@uab.es In the cell nucleus, genomic DNA molecules are associated with histone proteins and form long chromatin filaments containing many nucleosomes. The three-dimensional organization of these giant DNA molecules is undoubtedly the most challenging topological problem of structural biology. Previous TEM and AFM studies from our laboratory showed that, during cell division, chromatin filaments are folded into multilayer planar structures [1,2], in which DNA forms a two-dimensional network with a good flexibility and mechanical strength [3]. This discovery led to the thin-plate model in which we proposed that condensed chromosomes are formed by many stacked layers of chromatin oriented perpendicular to the chromosome axis [4]. More recently we found that multilayered plates can be selfassembled from chromatin fragments obtained by micrococcal nuclease digestion of metaphase chromosomes [5]. This finding, together with nanotechnology results showing that self-assembly of different structures of biological origin can produce complex micrometer-scale materials [6-8], suggested that chromosomes could be self-organizing structures. This communication shows that if chromosomes are considered as typical supramolecular assemblies, using the nanomechanical data obtained in other laboratories [9,10] and basic energetic considerations, it is possible to explain the

geometry and physical properties of condensed chromosomes. Metaphase chromosomes of different animal and plant species show great differences in size, ranging from 2 to 27 µm in length, and from 0.3 to 1.3 µm in diameter. The observed chromosome sizes are dependent on the amount of DNA that they contain (from 35 to 7450 Mb), but in all cases chromosomes are elongated cylinders that have relatively similar shape proportions: the average value of the length to diameter ratio (L/D) is 13. This study demonstrates that it is possible to explain this morphology by considering that chromosomes are self-organizing supramolecular structures formed by stacked layers of planar chromatin having different nucleosome-nucleosome interaction energies in different regions [11] (see figure). The nucleosomes in the periphery of the chromosome are less stabilized by the attractive interactions with other nucleosomes and this generates a surface potential that destabilizes the structure. Chromosomes are smooth cylinders (scheme a) because this morphology has a lower surface energy than structures having irregular surfaces (scheme b). The symmetry breaking produced by the different values of the surface energies in the telomeres and in the lateral surface (εT > εL) explains the elongated structure of the chromosomes.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

The results obtained by other authors in nanomechanical studies of chromatin [9] and chromosome [10] stretching have been used to test the proposed supramolecular structure [11]. It is demonstrated quantitatively that internucleosome interactions (εnn) between chromatin layers (scheme c) can justify the work required for elastic chromosome stretching. Chromosomes can be considered as hydrogels with a lamellar liquid crystal organization. These hydrogels have outstanding elastic properties because, in addition to the covalent bonds of the DNA backbone, they have attractive ionic interactions between nucleosomes that can be regenerated when the chromosome suffers a deformation. This self-healing capacity has been observed in nanotechnology studies of other hydrogels stabilized by ionic interactions [12]. In the cell, this may be useful for the maintenance of chromosome integrity during cell division. Finally, since early studies indicated that chromosomes are helically coiled [13], it is possible that each chromosome is formed by a single helicoidal plate [2,11]; the successive turns of a helicoid (scheme d) are equivalent to the stacked layers considered in the original thin-plate model. The flat plates seen in our micrographs and a helicoidal plate are topologically equivalent. They can be converted into each other without changing their mean curvature; the plane and the helicoid are both minimal surfaces (their mean curvature is zero). A continuous helicoidal plate has good mechanical properties and allows a homogenous organization of chromatin that precludes the random entanglement of the genomic DNA molecules. Furthermore, this chromatin organization can explain the morphology of the chromosome bands used in cytogenetic analyses for the diagnosis of cancer and hereditary diseases

NanoBio&Med2014

References [1] I. Gállego, P. Castro-Hartmann, JM. Caravaca, S. Caño and JR. Daban, Eur. Biophys. J., 38 (2009) 503. [2] P. Castro-Hartmann, M. Milla and JR. Daban, Biochemistry, 49 (2010) 4043. [3] I. Gállego, G. Oncins, X. Sisquella, X. FernándezBusquets and JR. Daban, Biophys. J., 99 (2010) 3951. [4] JR. Daban, Micron, 42 (2011) 733. [5] M. Milla and JR. Daban, Biophys, J., 103 (2012) 567. [6] GM. Whitesides and B. Grzybowski, Science, 295 (2002) 2418. [7] WJ. Chung, JW. Oh, K. Kwak, BY. Lee,… and SW. Lee, Nature, 478 (2011) 364. [8] T. Guibaud, E. Barry, MJ. Zakhary, M. Henglin,… and Z. Dogic, Nature, 481 (2012) 348. [9] Y. Cui and C. Bustamante, Proc. Natl. Acad. Sci. USA., 97 (2000) 127. [10] MG. Poirier and JF.Marko, J. Muscle Res. Cell Motil., 23 (2002) 409. [11] JR. Daban, J. Royal Soc. Interface, 11 (2014) 20131043. [12] JY. Sun, X. Zhao, WRK. Illeperuma, O. Chaudhuri,… and Z. Suo, Nature, 489 (2012) 133. [13] E. Boy de la Tour and UK. Laemmli, Cell, 55 (1998) 937. Acknowledgements: Research supported in part by grant BFU2010-18939 from the Ministerio de Economía y Competitividad

november 18-21, 2014 - Barcelona (Spain)

Figures

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Conducting polymers as a versatile platform for protein nanoarrays technologies Eduardo Antonio Della Pia1*, N. Lloret1, J. Holm2, M. Zoonens3, J.-L. Popot3, J. Nygård2, K. L. Martinez1 1 Bio-Nanotechnology Laboratory, University of Copenhagen, Copenhagen, Denmark 2 Niels Bohr Institute & Nano-Science Center, Copenhagen, Denmark 3 C.N.R.S./Université Paris-7 UMR 7099, Institut de Biologie Physico-Chimique Paris, France martinez@nano.ku.dk and dellapiaea@nano.ku.dk Micro- and nano-arrays of biomolecules such as DNA, peptides and proteins offer exciting opportunities in both basic and applied research (i.e. diagnostics, drug screening and drug discovery) [1]. High-density miniaturized biochips can increase assays sensitivity and throughput while reducing sample consumption and processing time [1, 2]. While DNA micro-arrays are currently being realized and are showing all their potential in genomic applications, protein arrays are still in their infancy due to the delicate nature of proteins and their challenging interaction with solid substrates [1, 2]. Even though substantial advances in nano-patterning techniques have been achieved and protein nano-patterns have been realized using dip-pen lithography, electron beam lithography or nanografting, examples of proteins nano-arrays are still rare and without evidence of proteins activity and stability [1, 2]. Here we report a versatile platform for spatially and selective functionalization of electrically contacted gold micro- and nano-structures with biological molecules such as proteins. The method is based on the electrochemical functionalization of the gold surfaces with conducting polymers bearing biotin or metal ion units [3]. We first demonstrate that biotinbinding molecules such as streptavidin or histidine-tagged proteins can be selectively immobilized on the polymeric film [4, 5]. We then show that protein multiplexed nano-arrays can be successfully prepared by sequential polymerizations and biomolecular

NanoBio&Med2014

immobilizations. The platform can be further used to immobilize complex membrane proteins stabilized in amphipathic polymers (amphipols) [6]. In fact, by taking advantage of the high affinity between biotin and streptavidin, we immobilize distinct membrane proteins onto different electrodes via amphipols modified with a biotin tag (biotinylated amphipols, Figure 1) [7]. Antibody-recognition events indicate that the membrane proteins are stably anchored to the substrate and that the electropolymerization is compatible with their protein-binding activity. Finally we take advantage of the good conductivity properties of the conducting polymers and measure the direct electron transfer properties of a redoxactive membrane protein bound to the substrate [8]. The platform described here is a first step for fabricating functional arrays of membrane proteins and we believe it will be a candidate of choice to produce electronically transduced nano-biosensors.

References [1] Wu, Chien‐Ching, David N. Reinhoudt, Cees Otto, Vinod Subramaniam, and Aldrik H. Velders., "Strategies for Patterning Biomolecules with Dip‐Pen Nanolithography." Small 7.8 (2011): 989-1002 [2] Christman, Karen L., Vanessa D. Enriquez-Rios, and Heather D. Maynard. "Nanopatterning proteins and peptides." Soft Matter 2.11 (2006): 928-939.

november 18-21, 2014 - Barcelona (Spain)

[3] Cosnier, Serge, and Michael Holzinge."Electrosynthesized polymers for biosensing." Chemical Society Reviews 40.5 (2011): 2146-2156. [4] Della Pia, Eduardo Antonio, Jeppe V. Holm, Noemie Lloret, Christel Le Bon, Jeam-Luc Popot, Manuela Zoonens, Jesper Nygård and Karen Laurence Martinez. “A step closer to membrane protein multiplexed nanoarrays using biotin-doped polypyrrole” ACS nano 8.2 (2014): 1844-1853. [5] Della Pia, Eduardo Antonio, Caroline Lindberg, Maeva Vignes, Martinez. "A novel surface for strong and reversible immobilization of histagged proteins” Manuscript in preparation. [6] Zoonens, Manuela, and Jean-Luc Popot. "Amphipols for each season." The Journal of membrane biology (2014): 1-38. [7] Della Pia, Eduardo Antonio, Randi Westh Hansen, Manuela Zoonens, and Karen L. Martinez. "Functionalized amphipols: a versatile toolbox suitable for applications of membrane proteins in synthetic biology." The Journal of membrane biology (2014): 1-12. [8] Laursen, Tomas, Peter Naur, and Birger Lindberg Møller. "Amphipol trapping of a functional CYP system." Biotechnology and applied biochemistry 60.1 (2013): 119-127.

Figures

Figure 1. Fluorescent microscopy image of three gold electrodes functionalized with biotin-doped polypyrrole film and (top and central electrode) streptavidin and neutravidin Oregon Green (bottom electrode). By successive polymerization and protein incubation, the membrane protein tOmpA trapped in biotinylated amphipols and NBD fluorescent-labelled amphipols was immobilized on the top electrode and the membrane protein bacteriorhodopsin trapped in biotinylated amphipols and Alexa 647 fluorescent-labelled amphipols was immobilized on the central electrode. The image is obtained by overlaying fluorescence images obtained in three different channels. Scale bar is 5 μm.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Lipid composition modulates nanomechanics of transmembrane proteins Domènech, Ò., Vázquez-González, M.L., de Robles, B., Montero, M.T. and Hernández-Borrell, J. University of Barcelona, Avda. Joan XXIII s/n, Barcelona, Spain odomenech@ub.edu There is a need to investigate the increasingly emergent problem of antibiotic resistance because of the social impact and economic consequences [1]. The use of new nanoscale techniques opens new perspectives in the study of the mechanisms involved in the generation of resistances [2]. Particularly, multidrug efflux pumps are under the spotlight to understand the molecular and physicochemical basis of the efflux mechanism to decrease the antibiotic concentration inside the bacterium. We used lactose permease (Lac Y) from Escherichia coli as a paradigm for the secondary transport proteins that couple the energy stored in an electrochemical ion gradient to a concentration gradient (ß-galactoside/H+ symport) to study the effect of the lipid matrix in its nanostructure. Firstly we characterized with the Atomic Force Microscope (AFM) the nanomechanics of the lipids in Supported Lipid Bilayers (SLBs) mimicking the lipid composition of bacteria (Figure 1). Secondly we incorporate the protein

to the lipid bilayers and investigate the changes produced when modifying the lipid environment (Figure 2). We found that proteins were segregated into liquid-crystalline phases (L) whilst the forces needed to extend a single protein were higher when the unsaturation in the hydrocarbon chains of the lipids decreased. This fact could be related to the lateral pressure on the protein in the lipid bilayer evidenced during the unfolding of a single protein when pulling it with the AFM tip.

References [1] Alanis, A.J., Archives of Medical Research, 36 (2005) 697. [2] Longo, G., Alonso-Sarduy, L., Marques Rio, L., Bizzini, A., Trampuz, A., Notz, J., Dietler, G., Kasas, S. Nature Nanotechnology, 8 (2013) 522.

Figures Figure 1. Nanomechanics of lipids forming SLBs mimicking E. coli inner membrane. Comparative of the phase diagram obtained with DSC and AFM.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Figure 2. AFM topographic image and height profile analysis of a SLB composed of POPE:POPG (3:1,mol/mol) with LacY at a LPR (w/w) of 0.5 (Z scale = 15 nm) (A). Insert in A presents a magnified image (470 Ă&#x2014; 280 nm, Z = 3 nm) where domains with LacY can be distinguished from domains without LacY. Histograms present the distribution of forces of domains with LacY (red) and domains without LacY (green) for Fy (B) and Fadh (C). Fittings to a Gaussian distribution are represented in solid lines.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Plasmonic biosensors advanced by functional hydrogels Jakub Dostalek1, Ulrich Jonas2,3, Christian Petri1,2, Nityanand Sharma1,4 1 AIT-Austrian Institute of Technology, Muthgasse 11, Vienna 1190, Austria 2 University of Siegen, Adolf-Reichwein-Strasse 2, Siegen 57076, Germany 3 Foundation for Research and Technology Hellas, P.O. Box 1527, 71110 Heraklion, Crete, Greece 4 Nanyang Technological University, Centre for Biomimetic Sensor Science, Singapore 637553 Rapid and sensitive detection of biomarkers is of a key interest in the field of medical diagnostics. The paper will present current advances in optical biosensors for the analysis of trace amounts of biomolecules that combine plasmonic metallic nanostructures and hydrogel materials. When post-modified with ligands for the specific capture of target analyte, these materials can serve as a matrix for specific capture of target analyte on the surface with good resistance to unspecific sorption of other molecules present in complex samples. The capture of target analyte in the hydrogel matrix can be probed by evanescent field of guided waves. Depending on the thickness of the hydrogel matrix (from around hundred nanometers to several micrometers in swollen state), it can be probed by surface plasmons with probing depth adjusted from around hundred nanometers (regular surface plasmons) to about micrometer (long range surface plasmons) or even above (by waveguide modes supported by hydrogel layer itself) [1, 2]. In addition, matrices prepared from hydrogels that are responsive to external stimulus can be advantageous for plasmonic sensors relying on surface plasmons with highly confined field distribution as they can be collapsed after the capture of the analyte into the plasmonic hotspot where the maximum field strength occurs. Examples of the implementation of hydrogel materials for the direct refractometric detection of small molecules by using antibody and molecularly imprinted polymer nanoparticles will be discussed based on spectroscopy of guided waves [3, 4]. In addition,

NanoBio&Med2014

surface plasmon-enhanced fluorescence spectroscopy biosensors that take advantage of responsive hydrogel binding matrices will be presented with the limit of detection at low femtomolar concentrations [5]. Acknowledgements: Authors gratefully acknowledge partial support from the Austrian Science Fund (FWF) through the project ACTIPLAS (P 244920-N20) and by the International Graduate School Bio-Nano-Tech, a joint Ph.D. program of the University of Natural Resources and Life Sciences Vienna (BOKU), the Austrian Institute of Technology (AIT), and the Nanyang Technological University (NTU).

References [1] A. Aulasevichet al, Macromolecular Rapid Communications, vol. 30, pp. 872-877, 2009. [2] J. Dostalek et al., Plasmonics, vol. 2, pp. 97106, 2007. [3] N. Sharma et al., Macromolecular Chemistry and Physics, in press, 2014. [4] Q. Zhang et al., Talanta, vol. 104, pp. 149-154, 2013. [5] Y. Wanget al., Analytical Chemistry, vol. 81, pp. 9625-9632, 2009.

november 18-21, 2014 - Barcelona (Spain)

Localized, DNA based logical circuits as components for biodetection Juan Elezgaray CBMN, UMR 5248, Allée Saint Hilaire, Bat. B14. 33600 Pessac, France juan.elezgaray@u-bordeaux.fr Recent advances in the field of molecular programming [1,2] have shown that enzymefree, DNA based circuits can be designed to perform the basic steps of molecular detection. Those include amplification and transduction of non nucleic-acid inputs. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this presentation, I will consider simple examples of these circuits when they are attached to platforms (DNA origamis [3]). After discussing their thermodynamic properties [4], I will show that these platforms can be used to precisely control the interaction between different gates. As expected, constraining distances between gates leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. In particular, strand displacement without toehold needs to be taken into account. Finally, I will present recent results showing how aptamers [5] can be interfaced with DNA origamis to generalize the triggering of DNA circuits by non nucleic-acid inputss

[3] P. W. K. Rothemund (2006) Folding DNA to create nanoscale shapes and patterns, Nature, 440, 297. [4] J.M. Arbona, J.P. Aimé and J. Elezgaray, Cooperativity in the annealing of DNA origamis, J. Chem. Phys. 138, 015105. [5] Durand, G., Lisi, S., Ravelet, C., dausse, E., Peyrin, E. and Toulmé, J-J (2014) Riboswitches Based on Kissing Complexes for the Detection of Small Ligands, Ang. Chem. Int. Ed. 53, 6942-6945.

References [1] Zhang,D.Y. and Seelig,G. (2011) Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem., 3, 103–113. [2] Li, B., Ellington, A.D. and Che, X. (2011) Rational, modular adaptation of enzyme-free DNA circuits to multiple detection methods, Nucl. Ac. Res., 39, e110.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Development of modified siRNA for gene silencing Santiago Grijalvo, Anna Aviñó, Montserrat Terrazas, Adele Alagia, Ramon Eritja Instituto de Química Avanzada de Cataluña (IQAC), CSIC, CIBER-BBN, Barcelona, Spain ramon.eritja@iqac.csic.es With the advent of RNA interference as a means to silence gene expression, small interfering RNA (siRNA) oligonucleotides have been recognized as powerful tools for targeting mRNAs and eliciting their gene inhibitory properties [1]. Small RNA duplexes are recognized by a protein complex called RISC provoking the specific degradation of messenger [2]. As a consequence of this discovery, siRNA oligonucleotides are now being intensively investigated as potential therapeutic agents for various biomedical indications [3]. siRNA are not readily taken up into tissues and are also susceptible to degradation by nucleases in the blood. For these reasons the interest in the design and preparation of modified RNA derivatives that are more stable, easier to produce at large scale and with a higher cellular uptake it is of vital importance to improve RNAi limitations [3].

[3] J. K. Watts, G. F. Deleavey, M. J. Damha. Drug Discovery Today 2008, 13, 842-855 [4] M. Terrazas, S.M. Ocampo, J. C. Perales, V. Márquez, R. Eritja. ChemBioChem 2011, 15, 1056-1065 [5] S. Grijalvo, S. M. Ocampo, J. C. Perales, R. Eritja. J. Org. Chem. 2010, 75, 6806-6813

Specifically we will show the development of siRNAs carrying conformationally restricted pseudonucleosides [4] as well as the synthesis and properties of siRNA conjugates with molecules that may enhance cellular uptake such as peptides, lipids and intercalating agents [5].

References [1] T. M. Rana. Nature Reviews Mol. Cell Biol. 2007, 8, 23-36 [2] S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl. Nature 2001, 411, 494-498

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

How can I protect my invention? BioMed Patents Consuelo Espejo Rodríguez Spanish Patent Office Examiner consuelo.espejo@oepm.es Inventions are one of the most powerful intangible values within a company, and Industrial Property can be the most effective way of protection. The objective of this lecture is to give an overview of what and how the inventions (particularly the nanoinventions applied in Phama and Bio fields) can be protected. We will also answer some questions such as:  

   

 



Do the industrial property titles expire and are territorially limited? What I should and I shouldn’t do before making available any important technical information? What are the patent requirements in nanotechnology? How Pharma and Bio inventions are evaluated by the Patent Offices? What are the exceptions to patentability in Biotechnology? Did you know that all nanopatents are classified in a specific technical “drawer” and that you have free access to all of them? Have you heard about the Nanopharma Technological alerts? How to search in Espacenet, ChemBL (Chemical structure drawing) and NCBI (Mesh)? Is there any tips for drafting a successful patent?

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Polymeric nanoparticles, prepared from nano-emulsion templating, as novel advanced drug delivery systems crossing the Blood-Brain Barrier 1

Cristina Fornaguera1, Aurora Dols-Pérez1, Gabriela Calderó1, M.José García-Celma2, Conxita Solans1 Institut de Química Avançada de Catalunya (IQAC-CSIC) and CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/ Jordi Girona 18 – 26, 08034, Barcelona, Spain 2 Department of Pharmacy and Pharmaceutic Technology, University of Barcelona, Barcelona, Spain cristina.fornaguera@iqac.csic.es

Introduction The administration of drugs to the Central Nervous System (CNS) is a key issue for the treatment of neural diseases. The intravenous administration, compared to the highly invasive intracranial administration, represents a promising alternative. However, due to the presence of the Blood-Brain Barrier (BBB), most drugs do no reach the CNS, thus producing low therapeutic efficiencies [1]. In this context, the need for effective drug delivery systems to the CNS is still a challenge. Polymeric nanoparticles constitute a promising strategy to target drugs through the BBB using the intravenous route. Diverse types of molecules have been previously used for the nanoparticle functionalization to target the BBB, such as permeabilization agents to achieve a passive targeting or monoclonal antibodies against receptors in the BBB to achieve an active targeting [2]. However, current approaches are not sufficiently efficient on the BBB crossing [3]. Therefore, to develop polymeric nanoparticles that efficiently cross the BBB, non-toxic, biocompatible and biodegradable materials are required, together with a safety method of preparation [3]. The use of a preformed polymer instead of the in situ polymerization and the nano-emulsion templating technology

NanoBio&Med2014

constitutes an interesting and versatile strategy [4]. Nano-emulsions are fine emulsions with droplet sizes typically between 20 – 200 nm, showing high kinetic stability against sedimentation / creaming and a transparent to translucent appearance [5]. Their preparation by low-energy emulsification methods represents an alternative to high-energy methods not only for obtaining nano-emulsions with smaller and less polydisperse droplets, with an energy and cost efficient procedure, but also due to the high versatility of achieving nano-emulsions with the desired characteristics. Among low-energy methods, the phase inversion composition (PIC) method is advantageous for the pharmaceutical industry [5] because it can be performed under mild conditions (e.g. mild temperatures). Once nanoemulsions are prepared, the formation of nanoparticles is achieved by solvent evaporation (schematic representation of nanoparticle production methodology on Figure 1). Objectives The aim of this work was to design polymeric nanoparticles from nano-emulsions templating that efficiently cross the BBB, once administered by the intravenous route.

november 18-21, 2014 - Barcelona (Spain)

Results Poly-(lactic-co-glycolic acid) (PLGA) nanoparticles were designed by the PIC nanoemulsification method followed by solvent evaporation. Nano-emulsions were stabilized with the polysorbate 80 surfactant, since previous studies reported its ability to enhance BBB permeability. Loperamide was incorporated into nanoparticles, prior to nanoemulsion formation, with the aim to study the BBB nanoparticles crossing via in vivo analgesia measurements, since the drug loperamide hydrochloride (LOP) produces a central analgesia, but it does not cross the BBB by itself. Nanoparticle surface was further functionalized with the anti-transferrin receptor monoclonal antibody (anti-TfR mAb), overexpressed in the BBB. Polymeric nano-emulsions were obtained in the electrolyte solution (W) / polysorbate 80 (O) / [4wt% PLGA + 0.1wt% LOP in 20/80 ethanol/ethyl acetate] system, at 25ºC. Nanoemulsions with 90wt % of water content, with an O/S ratio of 70/30 were chosen due to the compromise between the low surfactant content and sizes appropriate for the intravenous administration (around 120 nm). Polymeric nanoparticles, formed by solvent evaporation from template nano-emulsions, showed hydrodynamic radii of around 100 nm and negative surface charges. Loperamide encapsulation efficiency was found to be very high (>99wt%) and the in vitro release profile from nanoparticles was sustained, as compared with the drug in aqueous solution. The covalent binding of the anti-TfR mAb to the nanoparticle surface was successfully achieved by means of the carbodiimide reaction. A concentration step was required to achieve therapeutic loperamide concentrations. In vitro toxicity determinations demonstrated that nanoparticles were nonhemolytic neither non-toxic at the in vivo required concentrations. Central analgesia was measured in vivo by means of the hot plate test. The passive targeting of the BBB by non-

functionalized nanoparticles produced slight analgesic effects, while the active BBB targeting by the anti-TfR mAb produced a marked analgesia (50% more than the basal level). Therefore, it could be concluded that the formulated nanoparticles, functionalized with the anti-TfR mAb constitute a promising alternative to deliver drugs to the CNS by the intravenous route of administration.

References [1] Kabanov A.V., Gendelman H.E., Progress in Polymer Science, 32 (2007) 1054 – 1082. [2] Wohlfart S., Gelperina S., Kreuter J., J Control Release, 161(2) (2012) 264. [3] Tosi G., Fano R.A:, Bondioli L., Badiali L., Benassi R., Rivasi F., Ruozi B., Forni F., Vandelli A., Nanomedicine, 6(3) (2011) 423 – 436. [4] Anton N., Benoit J.P., Saulnier P., J Control Release, 128(3) (2008) 185. [5] Solans C., Solè I., Curr Opin Colloid Interf Sci, 17 (2012) 246.

Figures

Figure 1. Schematic representation of the whole process for the nanoparticles production.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Kinetics of the protein corona assembly on nanoparticlesinetics of the protein corona assembly on nanoparticles Giancarlo Franzese Departament de Fisica Fonamental, Universitat de Barcelona. Marti i Franques 1.Barcelona - Spain gfranzese@ub.edu Nanoparticles (NPs) in the extracellular matrix are immediately coated by layers of biomolecules forming a "protein corona". The protein corona gives to the NPs a "biological identity" that regulates the NP-cell interaction. Therefore, the cell uptake of the NPs is strongly affected by the protein corona. For this reason learning to predict the biological identities of NPs based on a partial experimental knowledge is essential to foresee a priori the safety implications of a NP for human health and, more in general, the environment.

in plasma, or because they are the most abundant in the corona of silica NPs. Our results are compared with experiments made under the same conditions showing that the approach has a predictive power [4].

To this goal we propose a multiscale approach that, adopting numerical techniques from allatoms simulations [1] to coarse-grained models for protein-protein [2] and protein-NP interactions [3], accounts for the effect of interfaces on the hydration layer [4,5] in the description of proteins [6] and NPs in water [7]. The approach allows us to predict the protein corona assembly based on a partial experimental knowledge of the protein affinities for NPs with a specific physico-chemical composition and the size [8].

[2]

Specifically, we study, by numerical simulations, the competitive adsorption of proteins on a NP suspended in blood plasma as a function of contact time and plasma concentration. We consider the case of silica NPs in a "simplified" blood plasma made of three competing proteins: Human Serum Albumin, Transferin and Fibrinogen. These proteins are of particular interest because they have a high concentration

[6]

NanoBio&Med2014

References [1] M. Bernabei, G. Franzese et al. in preparation; see also T.

[3] [4]

[5]

[7]

[8]

Kesselring, G. Franzese, S. V. Buldyrev, H. J. Herrmann, H. E. Stanley, Nanoscale Dynamics of Phase Flipping in Water near its Hypothesized Liquid-Liquid Critical Point, Scientific Reports (Nature Publishing Group) 2, 474 (2012). L. Xu, S. V. Buldyrev, H. E. Stanley, G. Franzese, Homogeneous Crystal Nucleation Near a Metastable Fluid-Fluid Phase Transition, Physical Review Letters 109, 095702 (2012). P. Vilaseca, K.A. Dawson, G. Franzese, Understanding and modulating the competitive surface-adsorption of proteins, Soft Matter, 9, 6978 (2013). M. G. Mazza, K. Stokely, S. E. Pagnotta, F. Bruni, H. E. Stanley, G. Franzese, More than one dynamic crossover in protein hydration water, Proceedings of the National Academy of Sciences of the USA 108, 19873 (2011). V. Bianco and G. Franzese, Critical behavior of a water monolayer under hydrophobic confinement, Scientific Reports (Nature Publishing Group) 4, 4440 (2014). G. Franzese, and V. Bianco, Water at Biological and Inorganic Interfaces, Food Biophysics, 8, 153 (2013). E. G. Strekalova, M. G. Mazza, H. E. Stanley, and G. Franzese, Large decrease of fluctuations for supercooled water in hydrophobic nanoconfinement, Physical Review Letters 106, 145701 (2011). O. Vilanova, J.J. Mittag, P. M. Kelly, S. Milani, K.A. Dawson, J. R채dler, G. Franzese, Predicting the kinetics of Protein-Nanoparticle corona formation in model plasma, in preparation.

november 18-21, 2014 - Barcelona (Spain)

Imaging molecular structure of plant cells by Confocal Raman microscopy Notburga Gierlinger1,2, Batirtze Prats Mateu1, Barbara Stefke1, Ursula L端tz-Meindl3 1 Dep. of Materials Science and Process Engineering, BOKU-University of Natural Resources and Life Sciences, Peter Jordan Str. 82, 1190 Vienna, Austria 2 Institute for Building Materials, ETH Zurich, Zurich, Switzerland 3 Cell Biology Dep., Plant Physiology Division, University of Salzburg, Salzburg, Austria burgi.gierlinger@boku.ac.at During the last years Confocal Raman microscopy evolved as a powerful method to get insights into chemistry and structure of plant cells and cell walls with a spatial resolution of around 300 nm. Two-dimensional spectral maps can be acquired of selected areas and Raman images calculated by integrating the intensity of characteristic spectral bands or by using multivariate data analysis methods. This enables direct visualization of changes in the molecular structure and analyzing the spectra laying behind the chemical images reveals detailed insights into cell wall chemistry and structure [1-4]. Insights have been gained into the design of plant cell walls to achieve movement in wooden parts of trees or in roots by means of gelatinous fibers. Plant cell walls are based on cellulose microfibrils embedded in a matrix of hemicelluloses and lignin. The orientation of the cellulose microfibrils (alignment with respect to the fiber axis) on the nanolevel, the arrangement of different layers on the microlevel, as well as the amount of lignin determine mainly properties and functionalities. These parameters are elucidated in-situ in context with the microstructure and reveal thus the design of e.g. so called gelatinous fibers. Almost pure cellulose has been identified as the main swelling core of this fibers, functionalized by a small outer lignified layer with high microfibril angle [4-7].

Recently the potential of method has also been shown on the algal model system Micrasterias denticulata. The changes in the molecular structure within the different cell organelles and structures can be followed as well as the changes in the outer cell wall during growth (Figure 1). Funding: Austrian Science Fund (FWF): START Project Nr. Y-728-B1

References [1] Gierlinger, N et al. Nature Protocols (2012) [2] Gierlinger, N et al. Journal of Experimental Botany 62 (2) (2010) 587-595. [3] Gierlinger, N. Frontiers in Plant Science (2014) doi: 10.3389/fpls.2014.00306 [4] Gierlinger, N. & Schwanninger, M. Plant Physiology 140, (2006) 1246-1254. [5] Goswami, L et al., Plant Journal. 56-4 (2008) 531-538.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Figures

Figure 1. Raman spectroscopic image of Micrasterias denticulata (160 x 160 Âľm, 0.5 Âľm step size, 532 nm WITec300RA). Based on the 102 400 Raman spectra images were calculated with the help of non-negative matrix factorization (NMF), a method to evaluate distribution maps of different components and demixed basis spectra. The different colours represent the different basis spectra (components). The blue colour represents the outer cellulosic cell wall, which is more highlighted in the old half of the cell (lower part of the image) due to higher cellulose amount and crystallinity than in the newly formed young part (upper smaller side). In the inner part the red colour corresponds to starch and highlights the small round pyrenoids in the older cell half, which are embedded in the chloroplast. Proteins, pectins and fats are coloured in green and yellow.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Optopharmacology to regulate endogenous proteins with light Pau Gorostiza IBEC (Institut de Bioenginyeria de Catalunya) & ICREA. Parc CientĂfic de Barcelona, Barcelona pau@icrea.cat The development of light-regulated drugs (optopharmacology) has important applications to neuronal receptors and enables the remote stimulation of neurons without genetic manipulation. Controlling drug activity with light offers the possibility of enhancing pharmacological selectivity with spatial and temporal regulation, thus enabling highly localized therapeutic effects and precise dosing patterns. Recent advances of the laboratory will be presented, including the development and characterization of the first photoswitchable allosteric modulator of a G protein-coupled receptor.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Cyano-Bridged coordination polymers nanoparticles as contrast agents for Biomedical Imaging Y. Guari, J. Larionova and J. Long Institut Charles Gerhard, UMR 5253 CNRS-UM2-ENSCM-UM1, Université Montpellier II Place Eugène Bataillon, 34095, Montpellier Cx 5, France From 1704, year of the discovery of the oldest coordination polymer, Prussian blue, to now, many cyano-bridged coordination polymers were synthesised and extensively studied. This research field remains very active with the development of materials featuring magnetic, photomagnetic, sorption or catalytic properties. Signiﬁcant parts of the current research activity on these materials is devoted to the synthesis and study of size and shape controlled cyanobridged coordination polymer materials at the nanoscale [1]. These nanomaterials have the same advantages as the corresponding bulk materials. Among them may be mentioned the versatility of precursors that can be assembled, the adjustable porosity and the possibility to combine several properties within a single nano-object [2]. In addition, the ease of synthesis of these nanoparticles under mild conditions allows control of their size, shape and sometimes their organization and thus control over their properties. We will illustrate the latest developments made in our research group on synthetic methodologies that we developed for the preparation of nano-objects or nanocomposites of these materials and magnetic, magneto-optic or sorption properties associated therewith. We will also address the potential application of cyano-bridged coordination polymers nanoparticles in the field of medical imaging. Keywords: Prussian blue, nanoparticles, medical imaging.

NanoBio&Med2014

References [1] (a) S. P. Moulik, G. C. De, A. K. Panda, B. B. Bhowmik, A. R. Das, Langmuir 1999, 15, 8361; (b) S. Vaucher, M. Li, S. Mann, Angew. Chem., Int. Ed. 2000, 39, 1793; (c) J. Larionova, Y. Guari, C. Sangregorio, Ch. Guérin, New J. Chem. 2009, 33, 1177; (d) F. Volatron, L. Catala, E. Riviere, A. Gloter, O. Stephan and T. Mallah, Inorg. Chem., 2008, 47, 6584. [2] (a) G. Maurin-Pasturel, J. Long, Y. Guari, F. Godiard, M.-G. Willinger, Ch. Guérin, J. Larionova Angew. Chem. Int. Ed. 2014, 53, 3872; (b) M. Perrier, S. Kenouche, J. Long, T. Kalaivani, J. Larionova, C. Goze-Bac, A. Lascialfari, M. Mariani, N. Baril, C. Guérin, B. Donnadieu, A. Trifonov, Y. Guari Inorg. Chem., 2013, 52, 13402.

Figures

Figure 1. Biomedical imaging using cyano-bridged coordination polymer nanoparticle.

november 18-21, 2014 - Barcelona (Spain)

Effect of nanoscale surface roughness on the adhesion and proliferation of normal skin fibroblasts and HT1080 fibrosarcoma cells Kanioura A.1, Bourkoula A.1, Tsougeni K.2, Petrou P.1, Kletsas D.3, Tserepi A2, Gogolides E.2, Kakabakos S.1 1 Institute of Nuclear and Radiological Sciences & Technology, Athens, Greece 2 Institute of Nanoscience & Nanotechnology, NCSR “Demokritos”, Athens, Greece 3 Institute of Biosciences and Applications, NCSR “Demokritos”, Aghia Paraskevi, Athens, Greece nkanioura@ipta.demokritos.gr Separation and enrichment of cancer cells from a mixture with normal cells are important steps for cancer diagnosis. The methods used for the separation of cancer from normal cells are based on observation of morphological features, labeling of the cells with specific markers or differences in physical properties between cancer and normal cells (e.g. cell size, density, adhesion, dielectric properties) [1]. Surface nanotopography, as it has been reported in the literature, affects cell adhesion, proliferation and viability [2]. However, there are few reports about the potential of such nanostructured surfaces for separation/enrichment of cancer cells from mixtures with normal ones [3]. Here, we investigated the effect of surface nanotexturing on the adhesion, viability, and proliferation of normal fibroblasts and fibrosarcoma HT1080 cancer cells on thin PMMA films nanotextured through oxygen plasma etching in comparison to flat untreated surfaces. Randomly nanotextured PMMA film surfaces were prepared following a published procedure [4]. Briefly, a 25% (w/w) PMMA solution was spin coated on Si wafers at 1500 rpm followed by baking for 1.5 h at 150 oC. The films were treated with O2 plasma. The etching conditions were: bias voltage: -100 Volts; electrode temperature: 15 oC; etching time: 3 min; source power: 1900 W; pressure: 0.75 Pa and oxygen flow: 100 sccm in a Helicon Plasma reactor (MET system, Adixen). The plasma treated surfaces (Fig. 1)

along with untreated ones were then used as substrates to culture 10000 cells/ml normal fibroblasts or HT1080 cells for periods of 1 and 3 days. The adhered cells were fixed and stained with phalloidin-Atto 488 (F-actin) and DAPI (nucleus) for cell counting and observation using an epifluorescence microscope, as described previously [5]. It was found that on the untreated surfaces after 1-day culture the number of adhered cells per surface area was approx. 1200 and 1000 cells/cm2 for the HT1080 and the normal fibroblasts, respectively. After 3 day culture the HT1080 cell population increased 4 times on these surfaces and the normal fibroblasts 1.5 times. On the other hand, concerning O2 plasma treated surfaces after 1-day culture approx. 2500 HT1080 cells and 800 normal fibroblasts had been adhered per sq. cm. A significant finding was that after 3-day culture the HT1080 cells population per surface area was increased almost 4 times (9800 cells/cm2) whereas, the number of normal fibroblasts was decreased by 20% (640 cells/cm2) compared to 1-day culture. In addition, as it is depicted in Fig. 2A and B the morphology of normal fibroblasts on the nanotextured surfaces was considerably affected after 3-day culture, as witnessed by the excessive distortion of cytoskeleton, compared to the untreated surfaces. In contrast, surface nanotexturing did not influence the morphology of HT1080 cells (Fig. 3A and B). The reduced cell population of normal fibroblasts on the rough

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

surfaces after 3-day culture was not due to apoptosis as it was proved through staurosporine assay. Therefore to explain this finding we performed focal points staining using fluorescently labeled anti-vinculin antibody. It was found that the number of focal points per normal fibroblast cell was reduced by 40% whereas, that of HT1080 was increased by 30% on the nanotextured surfaces compared to the untreated ones. Therefore the decreased number and the distortion of the cytoskeleton of normal fibroblasts on the nanotextured surfaces could be ascribed to the considerable decrease of the focal points formation, which affects cell adhesion and viability. In conclusion, taking into account that adhesion and proliferation of normal skin fibroblasts is inhibited on O2 plasma nanotextured PMMA surfaces in contrast to HT1080 cells, these surfaces could be useful for the enrichment and isolation of fibrosarcoma cells derived from tissues suspected for neoplasias and help to improve cancer diagnosis.

References [1] J.H. Kim, J.S. Kim, H. Choi, S.M. Lee, B.H. Jun, K.N. Yu, E. Kuk, Y.K. Kim, D.H. Jeong, M.H. Cho, Y.S. Lee, Anal. Chem. 78 (2006) 6967-6973. [2] K. Anselme, P. Davidson, A.M. Popa, M. Giazzon, M. Liley, L. Ploux, Acta Biomater. 6 (2010) 3824-3846. [3] K.W. Kwon, S.S. Choi, S.H. Lee, B. Kim, S.N. Lee, M.C. Park, P. Kim, S.Y. Hwang, K.Y. Suh, Lab on a Chip 7 (2007) 1461-1468 [4] E. Gogolides, V. Constantoudis, D. Kontziampasis, K. Tsougeni, G. Boulousis, M. Vlachopoulou, A. Tserepi, J. Phys. D: Appl. Phys. 44 (2011) 174021. [5] D. Kontziampasis, A. Bourkoula, P.Petrou, A. Tserepi, S. Kakabakos, E. Gogolides, Proceedings of SPIE 8765 (2013) 87650B.

NanoBio&Med2014

Figures

Figure 1. SEM images of O2 plasma treated PMMA surfaces at bias voltage 100V for 3 min.

Figure 2. Fluorescence microscope images of normal skin fibroblasts cultured for 3 days on untreated flat PMMA surfaces (A) or plasma treated surfaces (100 V, 3 min) (B). Cytoskeleton (F-actin) was stained with phalloidin-Atto488 and cells nuclei with DAPI.

Figure 3. Fluorescence microscope images of HT1080 fibrosarcoma cells cultured for 3 days on untreated flat PMMA surfaces (A) or plasma treated surfaces (100 V, 3 min) (B). Cytoskeleton and cells nuclei were stained as described in Fig. 2.

november 18-21, 2014 - Barcelona (Spain)

Screen printed superhydrophobic surfaces as enablers for Capillarity-Driven Biodetection Devices for Food Safety and Clinical Analysis: towards ASSURED sensors Ioanis Katakis Bioengineering and Bioelectrochemistry Group, Universitat Rovira i Virgili,. Tarragona, Spain ioanis.katakis@urv.cat Lab-on-Chip (LOC) concepts are usually realized as microsystems fabricated by microfabrication technologies of varying degrees of complexity and operated by control equipment that commonly require external power sources, fluid movement devices, and detection systems. Investment in purpose-built manufacturing lines, for micron or sub-micron featured microsystems and sophisticated control apparatus is justified for high throughput analytical tasks based on limitedvolume samples. Most analytical needs in food safety and decentralized diagnostics/theranostics are not limited by the available sample volume and are not high throughput in nature; rather it is cost and ease of use that eventually decide their large scale adoption. A convenient alternative for the realization of such application-oriented LOC concepts is to manufacture simple, basic microsystems by 3-D screen printing. Such elemental microsystems can be operated almost autonomously: fluid movement can be achieved through capillary action, and both fluidic control and detection by electrochemistry. Screen printing manufacturing requires a simple and relatively low cost production line and provides the flexibility to incorporate different materials in the 3D design accommodating both structural and actuation or detection elements. We demonstrate that basic unit operations such as dissolution, separation, mixing, reaction, flow manipulation and detection can be satisfactorily realized and controlled for most detection applications with low power requirements.

activated sludge. In a particular product developed, Salmonella could be detected in poultry meat extracts with limit of detection of 10-20 CFUs within 15 hours of sampling. When proteins need to be detected by immunochemical methods in lateral flow-type devices rendered by the 3-D screen printing method, we demonstrate that flow control is crucial for signal development and successful immunorecognition. We provide such flow control with electrochemically activated stop/go printed microvalves that modulate the hydrophilicity of the device walls. We thus achieve successful detection of ď ˘-lactoglobulin (a potential food allergen) or HCG (a pregnancy indicator). We therefore present a simple to manufacture, generic, low cost, and easy to use technology platform that can tackle a variety of analytical problems. We discuss how these technologies in combination with nanochemical solutions can provide a possible platform towards ASSURED diagnostics /theranostics. Acknowledgements: This work was made possible through support by the Spanish Ministry of Science and Innovation (BIO2010-20359 MICROCAP) and by the Catalan agency of support of University research (AGAUR grant 2010VALOR00063 SALMONELLA TRUST).

We applied such simple architectures in integrated devices that can detect pathogens in food and

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Large-scale dendrimer-based uneven nanopatterns of RGD towards improved architectural networks in chondrogenesis Anna Lagunas IBEC (Institut de Bioenginyeria de Catalunya) Parc Científic de Barcelona, Barcelona alagunas@ibecbarcelona.eu Cartilage damage is the main cause of joint disorders, having a huge impact on an increasingly ageing population. Cartilage inability to spontaneous repair and regenerate has stimulated clinical and experimental work towards optimal cartilage regeneration. Transplantation of mesenchymal stem cells (MSCs), which have a vast proliferative capacity and differentiation potential, has emerged as a promising strategy to treat joint defects. However direct implantation of undifferentiated MSCs without any preconditioning lead to calcification of the implanted cells, fibrogenesis and heterotopic tissue formation in the cartilage [1]. As in most biological systems showing multilevel organization with cross-level interdependence, extensive cell-cell communication networks are formed during cartilage development. In the initial stages of chondrogenesis MSCs condensation takes place, leading to a marked decrease of the intercellular space, and the occurrence of a large number of cell-to-cell contacts of the gapjunction (GJ) type. Signaling in multi-cellular networks is strongly influenced by the system architecture: in conventional culture systems of chondrogenic differentiation of MSCs, a hyalinelike, zonal-distributed cartilage structure, in which nearly cylindrical cells are aligned and connected side-by-side and end-to-end along the proximal-distal axis of the limb, is not sustained; instead, irregularly shaped cells spread randomly, resulting in randomly distributed cell junctions.

NanoBio&Med2014

In this sense it is of crucial importance to provide an appropriate cell environment that allows the establishment and maintenance of cell-to-cell interactions during the different stages of MSCs differentiation, and that while still favoring strong cell anchorage, allows the subsequent transplantation and release in the injured area. Taking advantage of a recently developed dendrimer-based large-scale nanopatterning approach [2], surfaces of poly-Llactic acid (PLA) nanopatterned with cell adhesive dendrimers, at different initial bulk concentrations, were used as substrates for chondrogenesis. Surface nanopatterning is applied to modulate cell-biomaterial interaction in order to better mimic cartilage architecture.

References [1] Cui, J. H., Park, S. R., Park, K., Choi, B. H., Min, B. H. Preconditioning of mesenchymal stem cells with low-intensity ultrasound for cartilage formation in vivo. Tissue Eng. 2007; 13: 351-60. [2] Lagunas, A., Castaño, A. G., Artés, J. M., Vida, Y., Collado, D., Pérez-Inestrosa, G., Gorostiza, P., Claros, S., Andrades, J. A., Samitier, J. Largescale dendrimer-based uneven nanopatterns for the study of local RGD density effects on cell adhesion. Nano Research 2014; 7: 399-409.

november 18-21, 2014 - Barcelona (Spain)

Figures

Figure 1. (a) AFM image (scale bar = 250 nm) of nanopatterned dendrimers. Inset: magnified phase image of one of the nanodomains (scale bar = 50 nm). (b) dmin probability contour plot showing regions of high local ligand density. Color scale: dmin values in nm. (c) Fluorescent micrograph of a fibroblast after 4.5 h in culture on the nanopatterns. Inset: magnified portion of FAs formed at the cell periphery. Scale bar = 20 Îźm. (d) Optical microscope image showing early hMSCs cell condensation on nanopatterns (chondrogenesis, day 3).

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Following the degradation and biological fate of polymeric poly (lactic-co-glycolic acid) nanoparticles Jordi Llop, Marco Marradi, Pengfei Jiang, María Echeverría, Shan Yu, Boguslaw Szczupak, Maria Puigivila, Vanessa Gómez-Vallejo and Sergio E. Moya. CIC biomaGUNE, Paseo Miramón 182, 20009 San Sebastián, Spain jllop@cicbiomagune.es Due to their small size and unique physicchemical properties, nanoparticles (NPs) have been proposed as diagnostic, therapeutic or even theragnostic tools. By appropriate multifunctionalization, NPs can be administered systemically and directed towards specific organs or tissues, providing thus enhanced therapeutic/diagnostic efficacy while reducing significantly undesired side- or toxicological effects [1].

of core-shell NPs after intravenous administration in rodents. Fe3O4/poly(lactic-coglycolic acid) (PLGA)/Bovine serum albumin (BSA) NPs (Figure 1) were simultaneously labelled with 111In, which was entrapped into the Fe3O4 crystal lattice, and 125I, which was covalently attached to the tyrosine residues of BSA. Both isotopes emit gamma rays with different energies (171 and 245 keV for 111In, 35.5 keV for 125I).

When moving to in vivo applications, the determination of the pharmacokinetic properties and biological fate of NPs is of paramount importance, both to assess potential toxicological effects and to anticipate therapeutic efficacy. However, NPs are extremely difficult to detect and quantify once distributed in a biological system. One alternative to overcome this problem consists of labeling the NPs with radionuclides that can lead to their detection with ultra-high sensitivity using in vivo imaging techniques such as Positron Emission Tomography (PET) or Single Photon Emission Computerized Tomography (SPECT) [2,3]. Of note, radiolabelling and subsequent imaging studies provide information about the loci of the radionuclide, but no information about the radiochemical integrity or the chemical stability of the NPs is obtained.

In a first step, biodistribution studies were performed in mice using dissection/gamma counting. With that aim, dual-labelled NPs (containing c.a. 111 kBq of 125I and 370 kBq of 111 In) were administered to animals, which were sacrificed at different time points (5 min-48h), the organs were harvested and the gamma emission spectra for each organ and blood were analyzed using a multichannel analyzer. Progressive accumulation of 125I in the thyroid glands, the intestine and the bladder, together with preferential accumulation of 111In in other major organs such as the lungs and the liver, suggest a fast degradation of the NPs after administration.

Here, we present an unprecedented duallabeling strategy to assess simultaneously the pharmacokinetic properties and biological fate

NanoBio&Med2014

The results were confirmed by in vivo SPECT studies, using a microSPECZT-Visio SPECT-CT system. Labelled NPs (containing c.a. 7.4 MBq of each radionuclide) were administered via the tail vein and static images were acquired at 1, 24 and 48 hours after administration. Reconstruction of the images in different energy windows was performed, and energy-resolved

november 18-21, 2014 - Barcelona (Spain)

images to determine the loci of both isotopes over time were obtained. As for dissection/counting studies, images showed progressive accumulation of 125I in the thyroid gland, and elimination of this isotope mainly via urine and intestine. This pattern is compatible with a progressive dissociation of the protein (BSA) from the NPs and subsequent detachment of 125I.

Figures

The strategy reported here, based on incorporation of two different gamma emitters (with different emission energies) followed by imaging studies with energy discrimination, might be applied to the determination of the biodistribution pattern and biological fate of a wide range of core-shell NPs.

References [1] Janib SM, Moses AS, MacKay JA, Adv Drug Deliv Rev 62 (2010) 1052-1063. [2] Pérez-Campaña C, Gómez-Vallejo V, Puigivila M, Martin M, Calvo-Fernández T, Moya SE, Ziolo RF, Reese T, Llop J. ACS Nano 7(4) (2013) 3498-3505. [3] Frigell J, Garcia I, Gómez-Vallejo V, Llop J, Penades S. J Am Chem Soc 136(1) (2014) 449457.

Figure 1. TEM image of Oleic acid-coated iron oxide nanoparticles encapsulated with PLGA and stabilized with BSA.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Biofunctional Surfaces for Multiplexed Diagnostic Platforms using Site‐Encoded DNA Strategies M.‐Pilar Marco Nanobiotechnology for Diagnostic (Nb4D) group, IQAC‐CSIC, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER‐BBN), Barcelona, Spain pilar.marco@cid.csic.es Advances in genomics and proteomics, point to a future in which clinical diagnostic will be based on molecular signatures characteristic of the health/disease status of the individuals, for which simultaneous detection of multiple biomarkers will be required. Moreover, future trends in medicine demand for rapid, reliable diagnostic technologies able to assist doctors on a more personalized and efficient medicine. In this respect, research in micro/nanobiotechnologies may allow the development of a new generation of improved diagnostic devices based on novel biosensing systems. Biosensors are devices responding to biomolecular recognition events occurring at the surfaces of particular micro/nanostructured materials, known as transductors, which defined physical properties are influenced by those specific events. To achieve this goal there is the need to construct homogeneous, organized, biocompatible and stable functional biohybrid surfaces in which the bioreceptor and the material behave as a single unit. However, preparation of reliable bioreceptor protein multiplexed platforms is still a challenge due to the molecular variability and complex nature of proteins (different hydrophobicities, acidic or basic characters, functionality, etc.). Thus, development of protein microarray technology has not been as straightforward as the DNA microarray technology. Unlike nucleic acids, which are relatively homogeneous in terms of structural and electrostatic properties, proteins can be extremely diverse regarding chemical structure and biological properties. Preventing

NanoBio&Med2014

protein denaturation and maintaining structural conformations and biofunctionality, while constructing these biohybrid surfaces that will act as transductors, are key issues. An alternative to circumvent these limitations is the use of oligonucleotide probes with well‐known sequences and their subsequent hybridization with their complementary oligonucleotides previously immobilized on the surface. Examples on the use of this strategy, known as DNA‐Directed Immobilization (DDI), to develop fluorescence site‐encoded DNA addressable microarrays and biosensors platforms based on distinct principles will be presented.

Figures

Figure 1. DDI schematic immunosensor chip.

november 18-21, 2014 - Barcelona (Spain)

approach of

a LSPR

From Basic Research to an Industrial Product: The case of Goldemar Ernest Mendoza Goldemar Solutions, c /Baldiri Reixac 15, 08028 Barcelona, Spain ernest.mendoza@goldemar.com Over the last years there has been a massive research effort in the field of nanotechnology. These developments are already in the market and many more to come in the near future. However, the process to bring an outcome from the basic research to an industrial product is long and complicated. With this talk I will first introduce our company and the nanotechnology product that we have developed in the field of cleantech. Also, I would like to explain the process that we have followed to bring our product to the market. Not only from a technological point of view but also from a market, investment and financial prospective.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Radiosensitizing Effect of Gold Nanoparticles under kV- and MV- X-ray Irradiations Masaki Misawa1, Shota Kuribayashi2, Masashi Hayano2, Yukiko Iwata2, Masanori Sato2, Katsuhide Fujita1 1 National Institute of Advanced Industrial Science & Technology (AIST) Ibaraki 305-8564, Japan 2 Komazawa University 1-23-1 Komazawa, Setagaya-ku, Tokyo 154-8525, Japan m.misawa@aist.go.jp Introduction: As a possible radiosensitizer in radiotherapy, we investigated the generation of reactive oxygen species (ROS) from dispersed gold nanoparticles (AuNPs) with average diameters of 5-60nm under clinical Xray irradiation[1][2]. The same AuNPs were added to the cultures of HeLa cells and their survivability was measured. Contribution of ROS generation to the cell survivability was discussed. Materials and Methods: Concentrations of AuNPs (BBI solutions) were changed at 0, 36, 72, and 144μM in 96 multi-well plates. ROS generation was measured by a fluorescent reagent Aminophenyl fluorescein ( APF, Sekisui Medical), which is sensitive to hydroxyl radicals (OH•). The integrated X-ray doses were varied from 1 to 10.0 Gy. A Mitsubishi linac (Model:EXL15SP) was operated at 10MV with a dose rate of 1Gy/min. Survivability of HeLa cells were measured by absorbance of WST-1 (Roche) at 440nm.

cultures for 24hours, X-ray doses up to 10Gy were given. The cell survivability was decreased as the X-ray doses. Sensitizing effect was observed over the entire dose range for 5nm AuNPs, and over the low doses up to 5Gy for 20nm and 40nm AuNPs. Sensitizing effect was not observed for 60nm AuNPs over the entire dose range. Conclusion: AuNPs function as a possible x-ray sensitizer by causing damage with a augmented effect of OH•. Particles size can be a key factor in ROS generation and cell damage.

References [1] Misawa M, Takahashi J., Nanomedicine 7(5) (2011), 604-14. [2] Takahashi J., Misawa M., Radiation Physics and Chemistry 78(11) (2009) 889-98.

Results and discussion: APF fluorescent intensity indicated that ROS generation for 20 – 80nm Au colloids was greater than that of distilled water by a factor of 5-7 in a concentration-dependent way (Fig.1). Because of APF’s specific sensitivity, we consider that OH• was a major species generated under x-ray irradiation. Regardless of the same mass density, ROS generation in 5nm and 10nm colloids was suppressed. With the addition of 75μM 5-60nm AuNPs in HeLa cell

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Figures

Figure 1. APF fluorescent intensity indicates ROS generation. Over 20nm AuNPs showed enhanced ROS generation relative to distilled water by a factor of 5-7.

Figure 2. Decrease in HeLa cell survivability as the X-ray dose. 5-40nm AuNPs showed sensitization effect.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Nanomechanics of the extracellular matrix of lung and heart tissues Daniel Navajas Institute for Bioengineering of Catalonia, School of Medicine - University of Barcelona, and CIBER of Respiratory Diseases. Barcelona, Spain dnavajas@ub.edu Cells sense and actively respond to mechanical features of their microenvironment. Moreover, mechanical cues have been shown to mediate critical cell functions including proliferation, differentiation, gene expression, contraction, and migration. Therefore, a precise definition of the mechanical properties of the extracellular matrix (ECM) is needed to further our understanding of the cell-microenvironment interplay. We use atomic force microscopy (AFM) to study nanomechanical properties of lung and heart ECM. Thin slices (10-20 m thick) of decellularized rat lung parenchyma and mouse heart left ventricle are probed with a custom-built AFM attached to an inverted optical microscope. The Young’s modulus (E) of the ECM is computed by fitting the tip-ECM contact model to force-indentations curves recorded on the ECM. The complex shear modulus (G*) is measured by placing the tip at an operating indentation of 500 nm and superimposing small amplitude (75 nm) multifrequency oscillations composed of sine waves (0.1-11.45 Hz). G* is computed in the frequency domain from the complex ratio between oscillatory force and indentation. We found that lung ECM exhibits scale-free rheology with a storage modulus (G’, real part of G*) increasing with frequency as a weak power law [1]. G’ values in the lung parenchyma ECM ranged from 6 kPa in the alveolar septum to 15 kPa in the pleural membrane. The loss modulus (G’’, imaginary part of G*) displayed a parallel frequency dependence at low frequencies, but increased more markedly at higher frequencies. We assessed the effect of different decellularization procedures on the local

NanoBio&Med2014

stiffness of the acellular lung by measuring E at different sites of rat lungs subjected to four decelularization protocols with/without perfusion through the lung circulatory system and using two different detergents [2]. Lung matrix stiffness revealed considerable inhomogeneity, but conventional decellularization procedures did not result in substantially different local stiffness. We measured E of ECM in healthy and bleomycininduced fibrotic mouse lungs [3]. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous and increased according to the degree of the structural fibrotic lesion. We also studied ECM nanomechanics of different histological regions of the left ventricle wall of healthy and infarcted mouse hearts [4]. The ECM of the ventricular wall was 2-fold stiffer than the lung parenchyma with G’ ranging from 10 kPa in the epicardium and collagen-rich regions of the myocardium to 30 kPa in elastinrich regions of the myocardium. Importantly, infarcted ECM showed a predominant collagen composition and was 3-fold stiffer than collagen rich regions of the healthy myocardium. ECM rheology of both lung and heart tissues was very well characterized by a two power law model composed of a weak power law with an exponent 0.05, accounting for a viscoelastic solid regime dominant at physiological frequencies, and a second power law with an exponent of 3/4, accounting for a viscoelastic liquid regime at high frequencies. Our AFM measurements define intrinsic mechanical properties of the ECM at the length scale in which cells sense and probe their microenvironment. Regional changes in

november 18-21, 2014 - Barcelona (Spain)

mechanical properties of the ECM could provide differential mechanical cues to regulate the spatial distribution, differentiation and function of lung and heart cells. Acknowledgements: Funded in part by the Spanish Ministry of Economy and Competitiveness grant FISPI11/00089.

References [1] ] Luque T, Melo E, Garreta E, Cortiella J, Nichols J, Farré R, Navajas D. Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy. Acta Biomater 2013, 9:6852-9. [2] Melo E, Garreta E, Luque T, Cortiella J, Nichols J, Navajas D, Farré R. Effects of the Decellularization Method on the Local Stiffness of Acellular Lungs. Tissue Eng Part C 2013; 20:412-22. [3] Melo E, Cardenes N, Garreta E, Luque T, Rojas M, Navajas D, Farré R. Inhomogeneity of local stiffness in theextracellular matrix scaffold of fibrotic mouse lungs. J Mech Behav Biomed Mater 2014; 37:186–195. [4] Andreu I, Luque T, Sancho A, Pelacho B, Iglesias-García O, Melo E, Farré R, Prósper F, Elizalde MR, Navajas D. Heterogeneous micromechanical properties of the extracellular matrix in healthy and infarcted hearts. Acta Biomater 2014, 10:3235-42.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Cross-cutting KETs: Innovation and Industrialization challenges for Nanobiotechnology and Nanomedicine towards Horizon 2020 Cristina Paez-Aviles1, Esteve Juanola-Feliu1, Josep Samitier1,2,3 Dep. of Electronics, Bioelectronics and Nanobioengineering Research Group (SIC-BIO) University of Barcelona. Barcelona, Spain. 2 IBEC-Institute for Bioengineering of Catalonia, Nanosystems Engineering for Biomedical Applications Research Group, Baldiri Reixac 10-12, 08028 Barcelona, Spain 3 CIBER-BBN-Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine,María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain cpaezaviles@el.ub.edu 1

Integration between Key Enabling Technologies (KETs) will be essential for competitiveness and innovation in Europe in the coming years. In this context, the new European Commission’s initiative Horizon 2020, the biggest financial program for Research and Innovation aims to finance different Risk Management Projects going “from fundamental research to market innovation”. This involves the entire innovation chain focusing on the research and development of crosscutting KETs, which are among the priorities of the Horizon 2020 Framework strategy. This strategy identifies the need for the EU to facilitate the industrial deployment of KETs in order to make its industries more innovative and globally competitive [1]. Horizon 2020 aims to redefine the cooperation in funding and scientific research by turning scientific breakthroughs into innovative products and services with over 74 billion € budget [2]. Is emphasized on three main pillars: Scientific Excellence, Society Challenges and Industrial Leadership. This last one aims to support SMEs in the industrial development and application of KETs [3], which have been selected according to the economic criteria, economic potential, capital intensity,

NanoBio&Med2014

technology intensity, and their value adding enabling role: Nanotechnology, Micro and Nano Electronics, Photonics, Advanced Materials, Biotechnology Industry, and Advanced Manufacturing Systems. Nanotechnology, is expected to make a rapid impact on society [4],[5]. After a long R+D incubation period, several industrial segments are already emerging as early adopters of nanotech-enabled products and findings suggest that the Bio&Health market is among the most challenging field for the coming years. Nanotechnology is also considered multidisciplinary since it is not restricted to the realm of advanced materials, extending also to manufacturing processes, biotechnology, pharmacy, electronics and IT, as well as other technologies [6]. These characteristics allow the connection to a diversified set of industries [7], implying that nanotechnologies can be involved directly or indirectly in the other five remaining KETs. This strong interdisciplinary character, combined with the possibility of manipulating a material atom by atom, opens up unknown fields and provides an endless source of innovation and creativity.

november 18-21, 2014 - Barcelona (Spain)

While each KET already has huge potential for innovation individually, their cross-fertilization is particularly important to offer even greater possibilities to foster innovation and create new markets. The concept of cross-cutting KETs refers to the integration of different key enabling technologies in a way that creates value beyond the sum of the individual technologies for developing innovative and competitive products, goods and services that can contribute to solving societal challenges. The global market volume in KETS is 646 billion euros and substantial growth is expected of approximately 8% of EU GDP by 2015. At present, the emerging sector of applied nanotechnology is addressed to the biomedicine (nanobiotechnology and nanomedicine) [8], starting to show a promising impact in the health sciences principally in three main areas: Diagnostics, Therapeutics and Regenerative Medicine (Figure 1) [9], [10]. Nanomedicine is considered a long-term play in the market [11]. In fact, the global nanotechnology market is anticipated to grow around 19% by year during 2013-2017 [12]. The expected market size related to radical innovation-based nanomedicines will be 1.000 M€ in 2020 and 3.000 M€ in 2025 [13]. In this context H2020 will spend 9.7% of the total budget in Health, demographic change and wellbeing.

the successful use of KETs potential. This is meant to be the “European industrial Renaissance” by covering the whole value of chain lab-to-market as the principal aim of H2020 where market is the main starting point.

References [1] European Commission, Brussels, 2009. [2] D. Kalisz and M. Aluchna, Eur. Integr. Stud., vol. 6, (2012) 140–149. [3] E. Commision, 2012. [4] ECSIP consortium, Copenhagen, 2013. [5] M. C. Roco and W. S. Bainbridge, J. Nanoparticle Res., vol. 7, (2005) 1–13. [6] N. Islam and K. Miyazaki, Technovation, vol. 27, no. 11, (2007) 661–675. [7] T. Nikulainen and C. Palmberg,Technovation, vol. 30, no. 1, (2010) 3–11. [8] K. Miyazaki and N. Islam, vol. 30, no. 4, (2010) 229–237. [9] European Technology Platform on Nanomedicine, 2013. [10] European Commission, RO-cKETs - multiKETs Pilot Lines Conference, 2014. [11] T. Flynn and C. Wei, Nanomedicine, vol. 1, no. 1, (2005) 47–51. [12] RNCOS, 2013. [13] European Commission, 2009. [14] K. Debackere, R D Manag., vol. 30, no. 4, (2000) pp. 323–328..

Translation of innovation and time-to-market reduction are important challenges on this framework. Nanomedicine firms have focused primarily on the science and less on the commercial applications resulting difficult to bring products into the market [11]. This remarks the existence of a gap between the current high levels of scientific performance and the industrial competitiveness [14]. The Commission states that bridging the so called “Valley of Death” to upscale new KET technology based prototypes to commercial manufacturing, often constitutes a weak link in

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Figures

Figure 1. Fields for cross-cutting KETs innovations in the Health and Healthcare Domain.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

DC studies of Layer-by-layer nanopores electrical properties tuning on Polycarbonate Membranes Roberto Paoli, Maria Bulwan, Antoni Homs-Corbera, Josep Samitier Institute for Bioengineering of Catalonia (IBEC), c/ Baldiri Reixac, 10-12, 08028 Barcelona, Spain rpaoli@ibecbarcelona.eu Nanoporous membranes have numerous potential biological and medical applications that involve sorting, sensing, isolating, and releasing biological molecules [1]. Recent advances in nanoscience are making possible to precisely control morphology as well as physical and chemical properties of the pores in nanoporous materials. Different researches showed that transport selectivity through solidstate nanopores can be effectively modulated by changing the size [2], the charge [3–5] and the polarity of the pores [6–8] or by using tethered receptors that are capable of selective molecular recognition [9]. Surface modification techniques are often used in order to achieve those results, as they can alter both physical and chemical properties. We investigated how polyelectrolyte layer-bylayer (LBL) surface modification can be used to change the characteristics of nanoporous membranes. Studies were performed with a custom made three-dimensional multilayer microfluidic device able to fit membrane samples. The device allowed us to efficiently control LBL films deposition over blank low-cost commercially available polycarbonate tracketched (PCTE) membranes. We have demonstrated pore diameter reduction and deposition of the layers inside the pores through confocal and SEM images. Posterior impedance studies served to study the effect of the LBL charges to the net inner nanopore surface charges. Measurements were performed using Phosphate Buffer Saline as

conductive medium. DC results generally show dependence between the electrical resistance and the increasing number of layers. Adding layers on pore surface decreases the pore mean aperture, resulting in a diminution of ions flux and thus of electric current across the membrane. Measurements have also demonstrated contrasted behaviors depending on the number and parity of deposited opposite charge layers. PCTE membranes are originally negatively charged and results evidenced higher impedance increases for paired charges LBL depositions. Impedance decreased when an unpaired positive layer was added. Following Electrical Double Layer theory we hypothesize that charges in the buffer tend to redistribute in the solution, reorganizing near the pores surfaces and creating an opposing charges layer which alters local conductivity.

References [1] S. P. Adiga, C. Jin, L. A. Curtiss, N. A. MonteiroRiviere, R. J. Narayan, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol., 1, (2013), 568–81. [2] K. B. Jirage, J. C. Hulteen, C. R. Martin, Science (80-. )., 278, (1997), 655–658. [3] S. B. Lee, C. R. Martin, Anal. Chem., 73, (2001), 768–775. [4] K.-Y. Chun, P. Stroeve, Langmuir, 17, (2001), 5271–5275. [5] G. Wang, B. Zhang, J. R. Wayment, J. M. Harris, H. S. White, J. Am. Chem. Soc., 128, (2006), 7679–7686.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

[6] J. C. Hulteen, K. B. Jirage, C. R. Martin, J. Am. Chem. Soc., 120, (1998), 6603–6604. [7] K. B. Jirage, J. C. Hulteen, C. R. Martin, Anal. Chem., 71, (1999), 4913–4918.

[8] E. D. Steinle, D. T. Mitchell, M. Wirtz, S. B. Lee, V. Y. Young, C. R. Martin, Anal. Chem., 74, (2002), 2416–2422. [9] S. B. Lee, D. T. Mitchell, L. Trofin, T. K. Nevanen, H. Söderlund, C. R. Martin, Science (80-. )., 296, (2002), 2198–2200.

Figures

Figure 1. SEM images of polycarbonate membrane: on the left, not covered by polymers; on the right, covered by polymers.

Figure 2. Measured resistance values comparison between different functionalizations of 200nm pore size membranes. Resistance tends to increase with the number of deposited layer, but measurements related to an odd number of deposited layers reveal a negative offset.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Bona fide induction of apoptosis in transformed cells during photothermal therapy using gold nanoprisms Julián Pardo, M. Pérez-Hernández, P. del Pino, S. G. Mitchell, B. Pelaz, E. M Gálvez and J. M. de la Fuente Instituto Universitario de Nanociencia de Aragon (INA), Universidad de Zaragoza, Zaragoza, Spain Fundación ARAID, Spain Aragón Health Research Institute (IIS Aragón), Biomedical Research Centre of Aragón (CIBA), Zaragoza, Spain Instituto de Carboquímica, CSIC, 50018 Zaragoza, Spain pardojim@unizar.es Gold nanoparticles (NPs) are promising vehicles to specifically deliver drugs to cancer cells and in addition to their use in drug targeting, they can be used as “heaters” during photothermal therapy of solid carcinomas using near-infrared (NIR) laser light [1,2]. We have previously shown that functionalization of gold nanoprisms (NPRs) with glucose selectively enhances their cellular uptake in transformed cells [3]. During the last years several types of NPs have been used to kill tumoural cells, although in most cases the type of cell death (necrosis, apoptosis , autophagy, etc.) induced has not been clearly identified so far. Here we will present data that unequivocally show that apoptosis is really induced in transformed cells during photothermal therapy using gold NPRs. In addition, we will show for the first time the molecular mechanism of apoptosis during photothermal therapy in transformed cells following irradiation with NIR laser light [4]. To this aim we have established conditions to readily induce apoptosis on mouse embryonic fibroblast (MEF) cells transformed with the SV40 virus and analyzed the mechanism of apoptosis using MEFs from different knock out mice, which are deficient in proteins involved in the different routes of apoptosis (Bak and Bax, Bid, caspase3 or caspase-9). Our results show that “hot” NPRs activate the intrinsic mitochondrial pathway of apoptosis mediated by Bak and Bax through the activation of the BH3-only protein Bid and that apoptosis and cell death is

dependent on the presence of both caspase-9 and caspase-3. Our findings demonstrate how the functionalization and dose of NPRs, as well as laser power density and irradiation time exposure, must be regulated to specifically induce apoptotic cell death. Moreover the molecular mechanism presented here may help to predict the efficacy of NP-based photothermal therapy to treat cancer patients.

References [1] B. Pelaz, V. Grazu, A. Ibarra, C. Magen, P. del Pino, J.M. de la Fuente, Langmuir, 2012, 28, 8965. [2] C. Bao, N. Beziere, P. del Pino, B. Pelaz, G. Estrada, F. Tian, V. Ntziachristos, J. M. de la Fuente and D. Cui, Small, 2012, 9, 68. [3] M. Pérez-Hernández, P. del Pino, B. Pelaz, E. M. Galvez, J. M. de la Fuente, J. Pardo (under review). [4] M. Pérez-Hernández, P. del Pino, S. G. Mitchell, B. Pelaz, E. M Gálvez, J. M. de la Fuente, Julián Pardo (under review).

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Nanotechnology Platform at the Institute for Bioengineering of Catalonia: description of capabilities and examples Mateu Pla-Roca Institute for Bioengineering of Catalonia (IBEC) Baldiri Reixac 10-12 (Ed. ClĂşster), 08028, Barcelona, Spain nanotechnology@ibecbarcelona.eu The Nanotechnology Platform, a core facility of the Institute for Bioengineering of Catalonia (IBEC) is an accessible and versatile research facility featuring 100 m2 of class 10,000 cleanroom space and laboratories offering state-ofthe-art equipment for the fabrication and characterization of microdevices and micro/nanostructures. Our mission is to facilitate advanced research support by providing services in the fields of micro/nanofabrication and nanotechnology for all academic and industrial researchers. Some of the many areas of application include lab-ona-chip (LOC), materials science, tissue engineering, optics and biomaterials. IBECâ&#x20AC;&#x2122;s Nanotechnology Platform offers scientific and technological support that includes the design, characterization and development of microdevices and micro/nanostructures so academic researchers and companies alike may use the platform to develop their innovative ideas.

Figures

Figure 1. (A) Chemical imaging/analysis of surfaces using TOF-SIMS (B) Fabrication of microdluidic chips and structuration of materials at the (C) micro and (D) nanoscale. (E) Biocompatible polymeric surface micropatterned with a fluorescent protein.

Our experience in giving support to research groups and practical examples will be introduced during the presentation.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

The Quest for Charge Transport in single Adsorbed Long DNA-Based Molecules Danny Porath Institute of Chemistry and Center for Nanoscience and Nanotechnology The Hebrew University of Jerusalem, 91904 Israel danny.porath@mail.huji.ac.il DNA and DNA-based polymers have been at the focus of molecular electronics owing to their programmable structural versatility. The variability in the measured molecules and experimental setups, caused largely by the contact problem, has produced a wide range of partial or seemingly contradictory results, highlighting the challenge to transport significant current through individual DNAbased molecules. A well-controlled experiment that would provide clear insight into the charge transport mechanism through a single long molecule deposited on a hard substrate has never been accomplished. In this lecture I will report on detailed and reproducible charge transport in G4-DNA, adsorbed on a mica substrate. Using a novel benchmark process for testing molecular conductance in single polymer wires, we observed currents of tens to over 100 pA in many G4-DNA molecules over distances ranging from tens to over 100 nm, compatible with a long-range thermal hopping between multi-tetrad segments. With this report, we answer a long-standing question about the ability of individual polymers to transport significant current over long distances when adsorbed a hard substrate, and its mechanism. These results may re-ignite the interest in DNA-based wires and devices towards a practical implementation of these wires in programmable circuits

References [1] "Direct measurement of electrical transport through DNA molecules", Danny Porath,

Alexey Bezryadin,Simon de Vries and Cees Dekker, Nature 403, 635 (2000). [2] "Charge Transport in DNA-based Devices", Danny Porath, Rosa Di Felice and Gianaurelio Cuniberti, Topics in Current Chemistry Vol. 237, pp. 183-228 Ed. Gary Shuster. Springer Verlag, 2004. [3] “Direct Measurement of Electrical Transport Through Single DNA Molecules of Complex Sequence”, Hezy Cohen, Claude Nogues, Ron Naaman and Danny Porath, PNAS 102, 11589 (2005). [4] “Long Monomolecular G4-DNA Nanowires”, Alexander Kotlyar, Nataly Borovok, Tatiana Molotsky, Hezy Cohen, Errez Shapir and Danny Porath, Advanced Materials 17, 1901 (2005). [5] “Electrical characterization of self-assembled single- and double-stranded DNA monolayers using conductive AFM”, Hezy Cohen et al., Faraday Discussions 131, 367 (2006). [6] “High-Resolution STM Imaging of Novel Poly(G)-Poly(C)DNA Molecules”, Errez Shapir, Hezy Cohen, Natalia Borovok, Alexander B. Kotlyar and Danny Porath, J. Phys. Chem. B 110, 4430 (2006). [7] "Polarizability of G4-DNA Observed by Electrostatic Force Microscopy Measurements", Hezy Cohen et al., Nano Letters 7(4), 981 (2007). [8] “Electronic structure of single DNA molecules resolved by transverse scanning tunneling spectroscopy”, Errez Shapir et al., Nature Materials 7, 68 (2008). [9] “A DNA sequence scanned”, Danny Porath, Nature Nanotechnology 4, 476 (2009). [10] “The Electronic Structure of G4-DNA by Scanning Tunneling Spectroscopy”, Errez

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Shapir, et.al., J. Phys. Chem. C 114, 22079 (2010). [11] â&#x20AC;&#x153;Energy gap reduction in DNA by complexation with metal ionsâ&#x20AC;?, Errez Shapir, G. Brancolini, Tatiana Molotsky, Alexander B. Kotlyar, Rosa Di Felice, and Danny Porath, Advanced Maerials 23, 4290 (2011). [12] "Quasi 3D imaging of DNA-gold nanoparticle tetrahedral structures", Avigail Stern, Dvir Rotem, Inna Popov and Danny Porath, J. Phys. Cond. Mat. 24, 164203 (2012). [13] "Comparative electrostatic force microscopy of tetra- and intra-molecular G4-DNA", Gideon I. Livshits, Jamal Ghabboun, Natalia Borovok, Alexander B. Kotlyar, Danny Porath, Advanced materials 26, 4981 (2014). [14] "Long-range charge transport in single G4DNA molecules", Gideon I. Livshits et. al., Nature Nanotechnology, Advanced Online Publication (2014).

Figures

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

NANOMEDICINE 2.0 Victor Puntes Institut CatalĂ de Nanotecnologia (ICN), Campus de la UAB, 08193 Bellaterra, Spain victor.puntes@icn.cat Last decade has seen a flourishment in the study of the properties of inorganic nanoparticles for medical applications. Nanoparticles display properties that are strongly determined by both morphology and environment and in the physico-chemical context where they are immersed, therefore allowing to monitor and manipulate biological states. In fact, inorganic nanoparticles behave as "artificial atoms", since their high density of electronic states -which controls many physical properties- can be extensively and easily tuned by adjusting composition, size and shape and used in biological environments. In fact, nanotechnologyâ&#x20AC;&#x2122;s ability to shape matter on the scale of molecules is opening the door to a new generation of diagnostics, imaging agents, and drugs for detecting and treating disease at its earliest stages. But perhaps more important, as I will show, it is enabling researchers to combine a series of advances, creating thus nanosized particles that may contain drugs designed to kill tumours together with targeting compounds designed to home-in on malignancies, and imaging agents designed to light up even the earliest stage of cancers. In fact, a description of cancer in molecular terms seems increasingly likely to improve the ways in which human cancers are detected, classified, monitored, and (especially) treated, and for that, nanoparticles, which are small and therefore allows addressing molecular structures in an unique manner, may be especially useful for those tasks.

be changing thanks to the intense research efforts and exemplary bold initiatives to develop cancer nanotechnology [2]. As a consequence, more than a dozen nanoparticle-based imaging agents and therapeutics are either on the market, in clinical trials, or awaiting clinical trials [3,4]. Similarly, the use of superparamagnetic nanoparticles for photoablation (hyperthermia) of brain tumours is already applied in the clinic [5].

References [1] ] P.A Kiberstis et al., Celebrating a Glass HalfFull. Science 312 (2006) 1157 [2] Gallego, O., & Puntes, V. What can nanotechnology do to fight cancer? Clin. Transl. Oncol., 8, (2006) 788â&#x20AC;&#x201C;795. [3] Avnesh S., Thakor, Sanjiv S, Gambhir. Nanooncology: The future of cancer diagnosis and therapy CA: A Cancer Journal for Clinicians 63 (2013) 395 ; [4] R. Juliano Nanomedicine: is the wave cresting? Nature Reviews Drug Discovery 12 (2013), 171 [5] http://www.magforce.de/en/home.html.

When almost 10 years ago, Science magazine dedicated the special issue on nanotechnology on cancer treatment [1], clinicians and pharmaceutics did not consider it as a real alternative yet. Currently, this perception may

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Figures

Figure 1. Normally, not all that is conceived becomes reality, however, what becomes reality, has been previously in the imagination. The magic bullet, a way to drive drugs towards the target avoiding effects to the rest of the body. The fantastic voyage, scientist and a submarine are miniaturized to go and do medical work inside the body. Their fight against the immune system is epic. Both precluded developments on nanomedicine.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Developing new tools for drug testing: introducing a microfluidic platform mimicking the spleen for future pharmacological trials L.G. Rigat-Brugarolas1,2, A. Elizalde3, H.A. del Portillo3,4, A. Homs-Corbera1,2 and J. Samitier1,2,5 1 Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Spain 2 Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain 3 Barcelona Centre for International Health Research (CRESIB, Hospital Clínic - UB), Spain 4 Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain 5 Department of Electronics, Barcelona University (UB), Spain lrigat@ibecbarcelona.eu Constant evolution and improvements on areas such as tissue engineering, microfluidics and nanotechnology have made it possible to partially close the gap between conventional in vitro cell cultures and animal model-based studies. A step forward in this field concerns organ-on-chip technologies, capable of reproducing the most relevant physiological features of an organ in a microfluidic device.

Similar in structure to a large lymph node, the spleen is a complex three-dimensional branched vasculature exquisitely adapted to perform different functions containing closed/rapid and open/slow microcirculations, compartmentalized parenchyma and sinusoidal structure forcing erythrocytes to squeeze through interstitial slits (IES) before reaching venous circulation. [4]

Research in microfluidic devices that represents organ models is still in its infancy, but offers a tantalizing glimpse into future of drug testing and biological hypotheses evaluation. [1]

Taking into account these features, we designed and developed a multilayered microfluidic device of the first ever functional human splenon-on-a-chip, mimicking the hydrodynamic behavior of the spleen's red pulp, to evaluate and simulate its activities, mechanics and physiological responses. Different physiological features have been translated into engineering elements that can be combined to integrate a biomimetic splenon model [5] (the minimal functional unit of the spleen).

Drug testing in animal models is timeconsuming, costly, and often does not accurately predict the adverse effects in humans. Toward a more reliable output, several platforms, in the interface between nanobio and tissue engineering, have been developed in the past years [2,3] with the aim to supplement or supplant animal studies or at least try to prioritize them. Nevertheless, no one developed before a spleen-like platform for studying the importance of this organ in differente haematological diseases.

This biochip-based platform should allow a deeper understanding of the underlying mechanisms of Plasmodium parasite infection and contribute to vaccine development and drug testing of malaria and other hematological disorders. Preliminary results showed significant statistical differences in terms of cell

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

deformability between old vs. fresh RBCs (p=0.001) and non-parasitized vs. P. Yoelii parasitized reticulocytes (p=0.006) when passing through the 2 µm constrictions simulating the IES. Still, additional challenges remain before these in vitro models can be used in applications such as diagnostics, but they could be the future of drug testing and biological platforms. Acknowledgements: Part of this work was financially supported by the technology transfer program of the Fundación Botín and by the Explora Program of the Ministry of Economy and Competitiveness of the Government of Spain. We thank David Izquierdo and Miriam Funes for their help in this project.

References [1] [2] [3] [4]

D. Huh et al. Science (2010) 328, 1662-1668. D. Huh et al. Sci Transl Me (2012) 4, 159ra147. A. Neswith et al. Lab Chip (2014) 14, 3925-3936. A.J. Bowdler, The complete spleen (2010) 2nd edition. [5] L.G. Rigat-Brugarolas et al. Lab Chip (2014) 14, 1715-1724.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

PAMAM dendrimers internalizes quickly in microalgae and cyanobacteria causing toxicity and oxidative stress Rodea-Palomares I.1,2, Gonzalo S.2, Rosal R.2 Leganés F.1 & Fernandez-Piñas F.1 1 Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid. Madrid, Spain 2 Departamento de Ingeniería Química, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain ismael.rodea@uam.es 1. Introduction Poly(amidoamine) (PAMAM) dendrimers are hyper-branched polymeric, nanoscale molecules with exceptional properties that make them attractive for a variety of biomedical and technological applications [1]. Dendrimers are considered “perfect” polymers due to their symmetry, and are classified according to their “generation” “G”, which accounts for the number of “layers” of polymer forming the dendrimer. Each generation doubles molecular weight and surface functional groups. Furthermore they are susceptible of a variety of surface functionalizations. Despite their promising applications, they have been found to be toxic to mammalian cells depending on generation and surface functionalization and their possible adverse effects for aquatic life, and especially for microalgae are largely unknown. In the present work we chose generation G2, G3 and G4 native –NH2 (cationic) and NH-C-(CH2OH)3 (-OH) (anionic) surface functionalized PAMAM dendrimers in order to study the dependency of polyamidoamine (PAMAM) dendrimer toxicity on generation and surface functionalization. As model organisms we chose a green microalga (Chlamydomonas reindhartii) and a cyanobacterium (Anabaena PCC7120). We have applied a multi-method approach to get insight into the toxic mechanisms of action of PAMAM dendrimers on both C. reindhartii and Anabaena sp. PCC 7120 including physicochemical characterization of PAMAM dendrimers in culture media, and

different physiological techniques.

and

cell

biology

2. Materials and methods Materials and physicochemical characterization. Amineand hydroxyl terminated G2, G3 and G4 PAMAM ethylenediamine core dendrimers were used (Sigma-Aldrich). The size distribution of nanoparticles was obtained using dynamic light scattering (DLS). Zeta potential was measured via electrophoretic light scattering. Growth inhibition experiments were performed with C. reindhartii and Anabaena sp. PCC 7120 following the standard OECD TG 201. Detection of reactive oxygen species (ROS): DCF was used as indicator of intracellular ROS formation. C4BODIPY was used for evaluating lipid peroxidation. Internalization studies: PAMAMAlexa Fluor 488 conjugates were prepared following the standard protocol (A30006, Molecular probes). The Alexa Flour 488 reactive dye has a tetrafluorophenyl (TFP) ester which reacts efficiently with primary amines. AntiAlexa fluor 488 Rabbit IgG Fraction (A-11094, Molecular probes), was used In order to discriminate surface bound and truly internalized dendrimers. Fluorescence studies were performed by flow cytometry and confocal microscopy. Ultrastructure alterations were studied by transmission electron microscopy (TEM).

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

3. Results and discussion 3.1. Toxicity of PAMAM dendrimers in cyanobacteria and microalgae All the cationic dendrimers (native â&#x20AC;&#x201C;NH2) proved toxic to both the green alga and the cyanobacterium. G2 and G3 Anionic dendrimers (-OH surface functionalized) were nontoxic, however, G4-OH proved toxic for both organisms. When toxicity is referred to mass concentration (mg/L), cationic dendrimers showed similar toxicity, apparently irrespective of generation (size). However, considering the large differences in molecular weight of the tested dendrimers, concentrations expressed on a molar basis revealed a clear relationship between dendrimer generation and toxicity for both organisms. 3.2. Toxicity of PAMAM dendrimers correlated with oxidative stress Increasing evidences indicate that nanoparticles in general can generate reactive oxygen species (ROS) and subsequently oxidative stress which might eventually lead to cell damage and cell death [2]. When the ability of the tested anionic and cationic dendrimers to elicit oxidative stress was evaluated by fluorometry, flow citometry and confocal microscopy we found that the strong differences in toxicity between anionic and cationic PAMAM dendrimers correlated with alterations in the ROS metabolism in both organisms. Figure 1 showed, as an example, ROS induction kinetics along the experimental lapse time (0 h-72 h) of Anabaena exposed to G2-OH and G2-NH2. Interestingly, neither DCF fluorescence (general ROS indicator), nor Bodipy fluorescence (lipid peroxidation) colocalized with photosynthetic structures of both organisms even when lipid peroxidation was observed in C. reinhardtii based on flow cytometry analysis, suggesting that the photosynthetic machinery is neither affected nor the origin of the observed oxidative stress

NanoBio&Med2014

which is in disagreement with previous studies [3, 4]. 3.3. PAMAM dendrimers were internalized very fast and presented low retention times in cell envelopes. We made a time course of dendrimers internalization. In the cyanobacterium, the three dendrimers were quickly taken up (80% of G2 and G3 and 100% of G4 after 10 min). In the green alga, dendrimer uptake was slower with 100% uptake after 2 h, G4 dendrimer uptake was slightly quicker than the other two dendrimers. The experiments with the antiAlexa antibody showed that in both organisms the Alexa Fluor-dendrimer conjugates were largely internalized even at the shorter time assayed (10 min). Interestingly, similar to the ROS results, no co-localization of Alexa fluor488 and photosynthetic membranes was found supporting the hypothesis that oxidative stress is neither affecting nor coming from the photosynthetic machinery. Furthermore, derdrimers were found to target mitochondria producing mitochondrial peroxidation. It has been found that dendrimers are internalized in different animal and human cell systems[5]; however, to our knowledge, this is the first time that PAMAM dendrimer internalization is confirmed in algae and cyanobacteria. Conclusions Cationic (-NH2) PAMAM dendrimers presented a generation-dependent increasing toxicity in both organisms. Anionic (-OH) PAMAM of generation G2 and G3 were non toxic, however, G4-OH presented a similar level of toxicity to G4NH2 in both organisms. Internalization of PAMAM dendrimers was observed by the first time in microalgae and cyanobacteria. Internalization was very fast (after 10 min of exposure) and with low retention time in cell envelopes of both organisms. Toxicity correlated with oxidative stress and dendrimer internalization. However, the photosintetic

november 18-21, 2014 - Barcelona (Spain)

machinery seemed to be unafected, and most probably was not involved in oxidative stress. Acknowledgements: This study was supported by the Community of Madrid grants S-0505/AMB/0321and S-2009/AMB/1511and by the Spanish Ministry of Science grant CGL201015675/BOS and CTM2008-04239/TECNO and CTM2008-00311/TECNO.

References [1] Svenson, S. and D.A. Tomalia, Dendrimers in biomedical applicationsâ&#x20AC;&#x201D;reflections on the field. Advanced Drug Delivery Reviews, 2005. 57(15): p. 2106-2129. [2] Nel, A., et al., Toxic Potential of Materials at the Nanolevel. Science, 2006. 311(5761): p. 622627. [3] Petit, A.-N., et al., Effects of a cationic PAMAM dendrimer on photosynthesis and ROS production of Chlamydomonas reinhardtii. Nanotoxicology, 2012. 6(3): p. 315-326. [4] Petit, A.-N., et al., Toxicity of PAMAM dendrimers to Chlamydomonas reinhardtii. Aquatic Toxicology, 2010. 100(2): p. 187-193. [5] Albertazzi, L., et al., Dendrimer Internalization and Intracellular Trafficking in Living Cells. Molecular Pharmaceutics, 2010. 7(3): p. 680688.

Figures

Figure 1. ROS induction kinetics (DCF fluorescence 488/528nm) in Anabaena PCC7120 exposed to increasing concentrations of anionic (-OH) and cationic (-NH2) G2 PAMAM dendrimers along the experimental lapse time (72h).

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Speckle fluctuations resolve the interdistance between incoherent point sources in complex media R. Carminati1, G. Cwilich2, L.S. Froufe-Pérez3 and J.J. Sáenz4,5 1 ESPCI ParisTech, PSL Research University, CNRS,Institut Langevin, 1 rue Jussieu, Paris, France 2 Department of Physics, Yeshiva University, 500 W 185th Street, New York, New York 10033, USA 3 Department of Physics, University of Fribourg, Chemin du Mus ee 3, CH-1700, Fribourg, Switzerland 4 Condensed Matter Physics Center (IFIMAC), Depto. de Física de la Materia Condensada and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, 28049 Madrid, Spain 5 Donostia International Physics Center (DIPC),Paseo Manuel Lardizabal 4, San Sebastian, Spain juanjo.saenz@uam.es We propose a method to capture the interaction between two identical sources in a scattering environment, based only on the measurement of intensity fluctuations [1]. The principle of the method is schematically illustrated in Fig. 1, and is based on the analysis of the intensityintensity correlation function and the intensity fluctuations in the speckle pattern formed by two identical and mutually incoherent point sources. This approach permits in principle to monitor the relative distance between the sources in the range 10-500 nm, with a precision that is not limited by diffraction, but by the microstructure of the scattering medium. A key issue affecting subwavelength imaging methods is the optical transparency of the media surrounding the light emitters. Taking advantage of the transparency of cells, fluorescence microscopy uniquely provides noninvasive imaging of the interior of cells and allows the detection of specific cellular constituents through fluorescence tagging. However, certain biological tissues or softmatter systems (such as foams or colloidal suspensions) look turbid due to intense scattering of photons traveling through them [2]. The image formed at a given point in the observation plane consists in a superposition of multiple fields, each arising from a different scattering sequence in the medium. This gives

NanoBio&Med2014

rise to a chaotic intensity distribution with numerous bright and dark spots known as a speckle pattern, producing a blurred image carrying no apparent information about the source position [3]. Techniques to measure the distance between individual nano-objects without actually imaging their position exist [4], Fluorescence Resonance Energy Transfer (FRET) being the most widespread example [5]. It relies on the near-field energy transfer between two fluorophores (donor and acceptor) emitting at different wavelengths. The FRET signal (e.g. the ratio between the intensities emitted by the donor and the acceptor at different wavelengths) depends on the donor-acceptor distance in the range 2 ∼ 10 nm. As such, it is not very sensitive to scattering problems. However, determining distances between two emitters in the range of 10 to 500 nm in a scattering medium still remains a challenging problem, not accessible either by fluorescence microscopy or FRET techniques. Our main goal here is to introduce a new approach to obtain information about the relative distance between two identical incoherent point sources in a disordered environment, based on the analysis of the fluctuations of the emitted light. This is an issue

november 18-21, 2014 - Barcelona (Spain)

of much interest, for example, in the study of conformational changes in biomolecules in living tissues.

Figures

References [1] R. Carminati, G. Cwilich, L.S. Froufe-Pérez, and J.J. Sáenz, arXiv preprint, arXiv:1407.5222v2 [2] A. Yodh and B. Chance, Physics Today 48, 34 (1995). [3] J.C. Dainty (ed.) Laser Speckle and Related Phenomena (Springer-Verlag, Berlin, 1975); J.W. Goodman, Speckle Phenomena in Optics: Theory and Applications (Roberts  & Company, Englewood, 2007). [4] X. Michalet and S. Weiss, Proc. Nat. Acad. Sci. USA  103, 4797 (2006). [5] For a recent review see the Special Issue Förster Resonance Energy Transfer in ChemPhysChem 12, Issue 3, (2011) and references therein.

Figure 1. The intensity radiated by two incoherent point sources in a complex medium form a speckle pattern that fluctuates in both space and time. The speckle fluctuations encode the relative distance between the sources [After Ref. 1]

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Self-powered microbots towards a “Fantastic Voyage” Samuel Sánchez Max Planck for Intelligent Systems, Stuttgart, Germany

Self-powered micro-motors are currently subject of a growing interest due to their visionary but also potential applications in robotics, biosensing, nanomedicine, microfluidics, and environmental field [1]. These micromotors are autonomous since they do not need external sources of energy in order to move. Instead, self-powered microrobots propel by decomposition of the fuel where they swim.

[4] Soler, Ll. et al., ACS Nano, 7, 9611 (2013), b) J. Orozco, et al Angew.Chem., Int. Ed., 52, 13276; (2013) [5] (a) Gao, W. and Wang, J., ACS Nano, 8, 3170 (2014). (b) Soler, L. and Sanchez, S. Nanoscale, 6, 7175 (2014).

These tiny motors swim through the water and can clean up contaminants or can swim through blood to one day transport medicines to a targeted part of the body -like taken from a science fiction movie Fantastic Voyage-. Those artificial nanomotors act collectively [2] reacting to external stimuli like chemotactic behaviour [3] and are capable to clean polluted water [4,5]. Future operations of autonomous intelligent multi-functional nanomachines will combine the sensing of hazardous chemicals using bio-inspired search strategies. With continuous innovations we expect that manmade nano/microscale motors will have profound impact upon in several fields such as drug delivery, biosensing and environmental remediation, among other visions.

References [1] Sanchez, Soler and Katuri, Angew. Chem. Int. Ed., 2014. DOI: 10.1002/anie.201406096 [2] Solovev, A. A. et al., Nanoscale, 5, 1284 (2013) [3] Baraban, L. et al., Angew. Chem. Int. Ed., 52, 5552. (2013), b) S. Saha, et.al., Phys. Rev. E, 89, 062316, (2014); c) Y. Hong, N. M.K. et al, Phys. Rev. Lett., 99, 178103 (2007)

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Scattering based bead-microrheology applied to biomaterials Frank Scheffold Department of Physics and Fribourg Center for Nanomaterials University of Fribourg, 1700 Fribourg, Switzerland

The quantitative stochastic description of Brownian motion of spherical micro- and nanobeads in complex fluids has laid the foundations for the invention of tracer microrheology, a powerful, noninvasive method that allows the measurement of mechanical properties over an extended range of frequencies using all optical instrumentation [13]. Over the last 20 years the method has been applied to a large range of materials ranging from foodstuff, dispersions, slurries to polymer solutions and surfactant based systems [1-4]. Since only small volumes are required the method is particularly well suited for applications in the field of biomaterials and bioactive substances [5-7].

preparation protocols can be used and the samples are held in common cylindrical or rectangular glass cuvettes filled with fluid volumes of typically 0.2-1 ml. Moreover these methods can resolve extremely fast displacements on the order of microseconds with a sub-nanometer resolution.

The basic idea of optical microrheology is to study the response of small (colloidal) particles embedded in the system under study. The particle can be added as tracer particles or can be naturally present in the system (such as oil droplets in an emulsion or fat droplets and protein micelles in yoghurt). The motion of the embedded probe particles can either be controlled actively, e.g. using optical tweezers or one can analyze the thermal motion of the particles. Both approaches can provide quantitative information about the viscous and viscoelastic properties of the surrounding fluid. Scattering techniques such as diffusing wave spectroscopy (DWS) or dynamic light scattering (DLS) are some of the most popular techniques to probe passive (thermal) particle motion remotely. These laser-based techniques offer the advantage to provide an ensemble average of the probe particle motion within about one minute measurement time. Standard sample

[1] T. G. Mason and D. A. Weitz, Optical Measurements of Frequency-Dependent Linear Viscoelastic Moduli of Complex Fluids, Phys. Rev. Lett. 74, 1250 (1995). [2] N. Willenbacher, C. Oelschlaeger, M. Schöpferer, P. Fischer, F. Cardinaux and F. Scheffold, Broad Bandwidth Optical and Mechanical Rheometry of Wormlike Micelle Solutions, Physical Review Letters 99, 68302 (2007) [3] P. Domínguez-García, Frédéric Cardinaux, Elena Bertseva, László Forró, Frank Scheffold, Sylvia Jeney, Accounting for inertia effects to access the high-frequency microrheology of viscoelastic fluids, submitted, arXiv:1408.4181 [cond-mat.soft] [4] F. Cardinaux, H. Bissig, P. Schurtenberger and F. Scheffold, Optical microrheology of gelling biopolymer solutions based on diffusive wave spectroscopy, Food Hydrocolloids 20, 325-331 (2007)

Here I will briefly review the methodology and instrumentation and then discuss applications to biomaterials and foodstuff [5], for probing protein interactions/aggregation and for the study three-dimensional assemblies of cell clusters [6,7]

References

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

[5] C. Oelschlaeger, M. Cota Pinto Coelho, and N. Willenbacher, Chain Flexibility and Dynamics of Polysaccharide Hyaluronan in Entangled Solutions: A High Frequency Rheology and Diffusing Wave Spectroscopy Study, Biomacromolecules 14, 3689 (2013). [6] B. Fabry, G. N. Maksym, J. P. Butler, M. Glogauer, D. Navajas, and J. J. Fredberg, Scaling the microrheology of living cells, Phys. Rev. Lett. 87, :148102 (2001) [7] F. Scheffold, J. Frith and J. Cooper White, Diffusing Wave Spectroscopy of Concentrated Mesenchymal Cell Suspensions, under preparation.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Targeted drug delivery and personalized medicine Avi Schroeder Technion â&#x20AC;&#x201C; Israel Institute of Technology, Israel avids@technion.ac.il The field of medicine is taking its first steps towards patient-specific care. Our research is aimed at tailoring treatments to address each personâ&#x20AC;&#x2122;s individualized needs and unique disease presentation. Specifically, we are developing nanoparticles that target disease sites, where they perform a programmed therapeutic task. These systems utilize molecular-machines and cellular recognition to improve efficacy and reduce side effects. Two examples will be described: the first involves a nanoscale theranostic system for predicting the therapeutic potency of drugs against metastatic cancer. The system provides patient-specific drug activity data with singlecell resolution. The system makes use of barcoded nanoparticles to predict the therapeutic effect different drugs will have on the tumor microenvironment. The second system makes use of enzymes, loaded into a biodegradable chip, to perform a programed therapeutic task â&#x20AC;&#x201C; surgery with molecular precision. Collagenase is an enzyme that cleaves collagen, but not other tissues. This enzyme was loaded into the biodegradable chip and placed in the periodontal pocket. Once the collagenase releases from the chip, collagen fibers that connect between the teeth and the underlying bone are relaxed, thereby enabling enhanced orthodontic corrective motion and reducing pain. This new field is termed BioSurgery. The clinical implications of these approaches will be discussed.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Personalized Cancer Nanomedicine. CLINAM 2014 Simo Schwartz Vall d'Hebron Hospital - CIBBIM. P. de la Vall d'Hebron, 119-129. 08035 Barcelona, Spain It has been hypothesized that drug delivery by nanoparticles may well circumvent the resistance machinery of cancer stem cells (CSC). To be able to study efficacy of nanomedicines in population of CSC, we first developed an in vitro model in which CSC are tagged by a fluorescent reporter gene under the control of a CSC specific promoter. Using this system, we demonstrated that while bulk cancer cells die, CSC population augments after paclitaxel (PTX) treatment. We then investigated the prospects of different targeted and non-targeted delivery systems loaded with PTX and functionalized with specific antibodies against cancer stem cell populations in regular breast cancer cell lines, as well as in our CSC models. Our data shows that reducing tumor resistance of cancer stem cells might be related to specific active targeting of DDS and not attributed to a general mechanism of action of nanomedicines.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Liposomes: Topical and Oral Bioavailability Julio Cortijo, Patricia Almudéver Folch, Juan Manuel Serrano Núñez Sesderma Laboratories, Polígono Industrial Rafelbuñol C/Massamagrell 3, Rafelbuñol 46138 Valencia, Spain j.serrano@sesderma.com Liposomes are small vesicles composed of one or more lipid bilayers. The size can go from 30nm up to several microns. Liposomes can encapsulate hydrophilic solutes in the aqueous core and lipophilic solutes in the membrane. These vesicles can be classified according to their size and number of bilayers: Multilamellar (100-10.000nm), Small Unilamellar (less than 100nm), Large Unilamellar (100-500nm). Sesderma manufactures very uniform, unilamellar liposome populations of between 50150nm. The advantages of liposomes are that, the structure is very similar to biological membranes and thus, are biodegradable and non toxic, they can reach the deepest layers of the skin, they provide a sustained release of the active ingredients, they prevent the oxidation and degradation of the ingredients and they show higher efficiencies at lower concentrations. We have carried out three different experiments on topical bioavailability: liposome penetration through skin, hair follicles and nails. All the ingredients used to prepare the liposomes are classified as GRAS (generally recognized as safe). In the first one, we compared the permeation capacity through human skin, using a Franz Diffusion Cell, of two different substances encapsulated and not encapsulated in liposomes: fluorescein and sodium ascorbate. Aliquots were taken from the receptor chamber at different times. The concentration of sodium ascorbate was determined by high performance liquid chromatography with ultraviolet detection (HPLCUV) and that of fluorescein by spectrofluorimetry. The results were as follows:

These results might be due to the nature and size of the active ingredients, and the characteristics of the

layers of the skin. The epidermis is a stratified layer with plenty of cells, this is why liposomes can get through it easier than the ingredients in solution. Fluorescein can diffuse faster through the epidermis than sodium ascorbate because fluorescein is more lipophilic than sodium ascorbate. The dermis has less cells and more fibres, and has a greater aqueous volume, so the preparation that permeates faster is that of sodium ascorbate solution due to its hydrophilic nature and small size. Finally, we can confirm that liposomes help substances pass through the skin. In the second case, liposome ability to go across the follicular canal was assayed with liposomal fluorescein. The skin samples were extracted from human scalp and the equipment used was the same as in the prior experiment: Franz Diffusion Cell. Pictures were taken at different times with a fluorescence microscope. We concluded that the follicular canal is an excellent penetration enhancer; a liposome reservoir is formed, facilitating its pass through the hair follicle and into the dermis. In the third experiment, we assessed the penetration capacity of liposomal fluorescein on one hand and a solution of fluorescein on the other hand, through human nails. The equipment utilized was a Franz Diffusion Cell with a coupling device for nails. Aliquots were taken from the receptor chamber at different times and the concentrations of fluorescein were determined by spectrofluorimetry. Pictures were also taken with a fluorescence microscope.The results showed that the maximum quantity of absorption for both formulations was obtained after 2 days in contact with the products. The concentration of fluorescein (2.96 ±1. 0.2 μg/cm²) for the liposomal formulation was 2.5 times higher than the solution (1.22 ± 0.2 μg/cm²). However, the permeability constant is very similar for both preparations: fluorescein solution (0.006 ± 0.002 cm²/s) and liposomal fluorescein (0.008 ± 0.001 cm²/s). We could also observe that there was an increase in the thickness of the nail treated with

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

liposomal fluorescein whilst there were no changes observed in the nail treated with the solution of fluorescein. Ascorbic Acid Oral Pharmacokinetics in Rats Aim: Compare the pharmacokinetics of two sodium ascorbate formulations: - Sodium ascorbate solution (extemporaneously prepared). - Sodium ascorbate encapsulated in liposomes. Method: The day prior to the administration of the formulations, 12 Wistar rats (280-310 g) were cannulated in the jugular vein to allow blood sampling at preset times. A volume of 2.5 mL of each fresh formulation was administered orally as single dose with intraoesophageal cannula. Six replicates were performed for each formulation. 200 μl blood samples were taken at the following times: 0 , 15 , 30, 45 , 60, 90 , 120 minutes and 3, 4 , 5, 6 , 7, 8 , 10, 12 , 23, 26 hours . The samples were centrifuged at 2000g for 5 min to obtain plasma which was immediately deproteinized with icecold MPA 10% (metaphosphoric acid). The samples were filtered through a pore diameter of 0.45μm. The analytical method used to measure vitamin C (sodium ascorbate) was HPLC-UV with UV detection at 254nm. The mobile phase used consisted of a KH2PO4 (0.1M) solution: ACN (95:5) at a pH of 2. As the stationary phase, the column Sherisorb ODS1 5uM 25x0.4mm was used and the selected flow rate was 1 ml / min. The injection volume used was 60 μL. Results:

Conclusions: Liposomes enable a better control of the release of the drug in plasma and maintains it for a longer period of time. A liposomal formulation of sodium ascorbate requires a smaller dose to reach the desired plasma concentration and, therefore, the desired therapeutic effect.

References [1] ELKEEB, R., ALIKHAN, A., ELKEEB, L., HUI, X. & [2]

[3]

[4] [5] [6] [7]

MAIBACH, H. I. 2010. Transungual drug delivery: current status. Int J Pharm, 384, 1-8. ISHIDA, A., OTSUKA, C., TANI, H. & KAMIDATE, T. 2005. Fluorescein chemiluminescence method for estimation of membrane permeability of liposomes. Anal Biochem, 342, 338-40. KARLSEN, A., BLOMHOFF, R. & GUNDERSEN, T. E. 2005. High-throughput analysis of vitamin C in human plasma with the use of HPLC with monolithic column and UV-detection. J Chromatogr B Analyt Technol Biomed Life Sci, 824, 132-8. KLIGMAN, A. M. & CHRISTOPHERS, E. 1963. Preparation of Isolated Sheets of Human Stratum Corneum. Arch Dermatol, 88, 702-5. O'GOSHI, K. & SERUP, J. 2006. Safety of sodium fluorescein for in vivo study of skin. Skin Res Technol, 12, 155-61. SZNITOWSKA, M. & BERNER, B. 1995. Polar pathway for percutaneous absorption. Curr Probl Dermatol, 22, 164-70. TORRES-MOLINA, F., ARISTORENA, J. C., GARCIACARBONELL, C., GRANERO, L., CHESA-JIMENEZ, J., PLA-DELFINA, J. & PERIS-RIBERA, J. E. 1992. Influence of permanent cannulation of the jugular vein on pharmacokinetics of amoxycillin and antipyrine in the rat. Pharm Res, 9, 1587-91.

Figure 1. Plasma concentration versus time after oral administration of 250 mg of sodium ascorbate formulated in an extemporaneous solution (black line) or in liposomes (green line). Mean ± SEM, n = 6.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Nano crystalline cellulose-protein composites: Super performing biomaterials for tissue engineering and regenerative medicine Oded Shoseyov The Robert H Smith Institute of Plant Science and genetics. The Faculty of Agriculture The Hebrew University of Jerusalem, Israel A platform technology that brings together the toughness of cellulose nano-fibers from the plant kingdom, the remarkable elasticity and resilience of resilin that enables flees to jump as high as 400 times their height from the insect kingdom, and the adhesion power of DOPA, the functional molecule of mussels that enable it to bind tightly under water to organic and inorganic matter from the marine kingdom and all that combined with Human Recombinant Type I collagen produced in tobacco plants; SUPERPERFORMING BIOMATERIALS. Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance proteincarbohydrate composite material. Plant cell walls also present durable composite structures made of cellulose, other polysaccharides, and structural proteins. Plant cell wall composite exhibit extraordinary strength exemplified by their ability to carry the huge mass of some forest trees. Inspired by the remarkable mechanical properties of insect cuticle and plant cell walls we have developed novel composite materials of resilin and NanoCrystalline Cellulose (resiline-NCC) that display remarkable mechanical properties combining

strength and elasticity. We produced a novel resilin protein with affinity to cellulose by genetically engineering a cellulose binding domain into the resilin. This CBD-Resilin enable, interfacing at the nano-level between the resilin; the elastic component of the composite, to the cellulose, the stiff component. Furthermore, chemical and enzymatic modifications of the composite are developed to produce DOPAResiline-NCC which confers adhesive and sealant properties to the composite. As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, and bone reconstruction, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and posttranslational modifications. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen,

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Combining collagen at the nano-scale with resilin to produce fibers resulted in super-performing fibers with mechanical properties which exceed that of natural fibers.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Studies on thermal and magnetic properties of iron oxide nanoparticles for magnetic hyperthermia application Paula Soares, Isabel Ferreira and Jo達o Paulo Borges Department of Materials Science, FCT-UNL, Campus de Caparica, 2829-516 Caparica, Portugal pi.soares@campus.fct.unl.pt Hyperthermia is an old technique which is recognized as a possible treatment option for cancer [1]. Cancer is a severe disease and currently is one of the leading causes of morbidity and mortality in the world, while chemo- and radiotherapy present several side effects due to their lack of specificity to the cancer type and the development of drug resistance. Iron oxide nanoparticles are having been extensively investigated for several biomedical applications such as hyperthermia and magnetic resonance imaging for cancer treatment. In this context, a work was performed comparing the effect of surfactants on the stability and the heating ability of iron oxide colloids. Iron oxide nanoparticles were synthetized through chemical precipitation and stabilized using two surfactants: sodium citrate and oleic acid. The as-prepared nanoparticles were characterized by several techniques and their heating ability was evaluated using different sample concentrations and field intensities. Hysteresis loops measured at temperatures 10 and 315 K for coated iron oxide nanoparticles are shown in Fig. 1. Comparing the effect of sodium citrate and oleic acid it is possible to observe that oleic acid is reducing the magnetic moments at the surface of the nanoparticles probably due to the diamagnetic contribution of the surfactant volume. For higher concentrations of oleic acid it seems to be an increase in the SPA values.

Hyperthermia results (Fig. 2) show a strong reduction on the ILP value when oleic acid is added to the colloids, while for sodium citrate this reduction is not so pronounced. However, ILP values are within the literature values for commercial iron oxide nanoparticles. These results show oleic acid has a more severe effect on the magnetic properties and heating ability of the nanoparticles. This effect is probably due to the surfactant viscosity and the size of the molecule that is higher than sodium citrate

References [1] Soares, Paula IP; Alves, Ana MR; Pereira, Laura CJ; Coutinho, Joana T; Ferreira, Isabel MM; Novo, Carlos MM; Borges, Jo達o PMR. Journal of colloid and interface science, 419 (2014) 46-51. [2] IP Soares, Paula; MM Ferreira, Isabel; AGBN Igreja, Rui; MM Novo, Carlos; PMR Borges, Jo達o; Recent patents on anti-cancer drug discovery,7(1) (2012) 64-73. [3] Soares, PIP; Dias, SJR; Novo, CMM; Ferreira, IMM; Borges, JP, Mini reviews in medicinal chemistry, 12(12) (2012) 1239-1249. [4] Baptista, Ana; Soares, Paula; Ferreira, Isabel; Borges, Joao Paulo; ,Nanofibers and nanoparticles in biomedical applications, Bioengineered Nanomaterials, 93, 2013,CRC Press [5] Soares, Paula I. P., Ferreira, Isabel M.M., Borges, Jo達o P.M.R., Topics in Anti-Cancer Research, Vol. 3; Bentham Science Publishers, 2014, Chapter 9, 342383.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Figures

Figure 1. Magnetization vs. applied magnetic field for Iron oxide nanoparticles coated with sodium citrate (a) and oleic acid (b)

Figure 2. – Intrinsic loss power (ILP) vs. Surfactant concentration (left image – sodium citrate, Right image – Oleic acid) for three field intensities with a frequency of 418.5 kHz.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Hijacking nature's own communication system: evaluation of extracellular vesicles as a sirna delivery vehicle Stephan Stremersch1, K. Braeckmans1,2, R. E. Vandenbroucke3, S. De Smedt1, K. Raemdonck1 1 Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, Ghent, Belgium 2 Center for Nano-and Biophotonics, Ghent University, Ghent, Belgium 3 Inflammation Research Center, VIB – Ghent University, Ghent, Belgium Stephan.Stremersch@UGent.be In order to exploit the therapeutic potential of small interfering RNA (siRNA), it is key to overcome the various intra- and extracellular barriers imposed by the human body. To this end, siRNA therapeutics are commonly packaged in an appropriate nanosized drug carrier.[1] Recently it was discovered that extracellular vesicles (EVs), i.e. lipid membranesealed nanosized particles, act as nature’s own nucleic acid delivery system.[2] EVs are secreted by every cell type and have been shown to contain a variety of biological molecules, including miRNA, which can be transferred to other cells leading to phenotypic changes. For this reason, interest has surged towards evaluating these vesicles as a new personalized drug delivery platform for therapeutic nucleic acids, such as siRNA. Yet, to date a major impediment in using EVs as a carrier for siRNA in the clinic is the lack of a suitable procedure for efficient and reproducible siRNA loading.[3] In this work we aimed to develop and thoroughly characterize methods for loading isolated EVs with siRNA. EVs were purified from conditioned cell culture medium derived from a B16F10 melanoma cell line by (density gradient) ultracentrifugation. The presence of EVs was confirmed by means of cryo-TEM imaging, immunoblot detection of EV-specific markers and via their typical size and buoyant density. In a first effort towards intrave¬sicular loading of exogenous siRNA, we critically evaluated a

previously reported method based on electroporation of an EV/siRNA mixture with the aim to induce transient pores in the EV membrane, hence allowing the siRNA to migrate through the lipid bilayer. Using this approach, siRNA encapsulation efficiencies up to 25% were reported.[4] Importantly, duplication of these experiments in our hands under identical experimental conditions revealed that the afore-mentioned siRNA encapsulation was largely due to unspecific aggregate formation, independent of the presence of extracellular vesicles.[5] The latter aggregates resulted from the interaction of multivalent cations, released from the metal electrodes in the electroporation cuvettes, with hydroxyl anions present in the electroporation buffer and were shown to coprecipitate siRNA. After blocking aggregate formation no significant encapsulation of siRNA could be measured. Taken together, these results dem¬onstrate the necessity for alternative methods to load EVs with siRNA and the importance of including the correct controls to properly assess loading efficiencies in biological vesicles. Next, we developed a new loading approach in which siRNA modified with a cholesterol moiety, was used to ally siRNA to the EV lipid membrane. The association of siRNA on the surface of EVs was shown using different methods based on gel electrophoresis, an antigen-specific bead based assay and

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

iodixanol density gradient ultracentrifugation. Moreover, we clearly demonstrated that this approach required the use of chemically stabilized siRNA, due to the presence of significant nuclease activity in the isolated EV sample. Furthermore, we could confirm that EV cell uptake was not affected by the siRNA incorporation and compared the functional siRNA delivery capacity with an anionic, fusogenic liposome in a monocyte/DC cell line (JAWSII). Interestingly, we observed that the fusogenic liposomes clearly outperformed the EVs in terms of siRNA delivery. Finally, we also compared the gene silencing capacity of the cholesterol-siRNA inserted in the EV membrane with that of the endogenously present, intravesiclular miRNA’s. Therefore, a total miRNA profiling of the purified EVs was done by a nCounter® miRNA expression assay. Target mRNA’s in JAWSII cells of the most abundant miRNA’s identified in the melanoma EVs were evaluated for specific post-transcripitional gene suppression and compared to specific gene silencing of the pan-leucocytic marker CD45 via the loaded cholesterol siRNA. To conclude we can state that electroporation, in contrast to previous reports, is not a feasible technique for loading siRNA in isolated EVs. Instead we developed a new approach based on a cholesterol modified siRNA to efficiently and reproducibly load EVs with exogenous small nucleic acids (graphical abstract). Finally, we compared the functional siRNA delivery potential between EVs and a classic, fusogenic liposome and between exogenous siRNA and endogenous miRNA.

[3] P. Vader, S.A. Kooijmans, S. Stremersch, K. Raemdonck, Therapeutic delivery, 5 (2014) 105-107. [4] L. Alvarez-Erviti, Y.Q. Seow, H.F. Yin, C. Betts, S. Lakhal, M.J.A. Wood, Nat. Biotech., 29 (2011) 341-U179. [5] S.A. Kooijmans, S. Stremersch, K. Braeckmans, S.C. De Smedt, A. Hendrix, M.J. Wood, R.M. Schiffelers, K. Raemdonck, P. Vader, J.Control.Release, 172 (2013) 229-238.

Figures

Figure 1. EVs released by B16F10 melanoma cells were purified via density gradient ultracentrifugation. Next, two methods for exogenous siRNA loading were evaluated. Electroporation appeared not a feasible loading technique, cholesterol mediated siRNA loading on the other hand, provided efficient and reproducible loading. Finally, these cholesterol-siRNA loaded EVs were evaluated for cell uptake and functional siRNA delivery.

References [1] K. Raemdonck, R.E. Vandenbroucke, J. Demeester, N.N. Sanders, S.C. De Smedt, Drug discov. today, 13 (2008) 917-931. [2] H. Valadi, K. Ekstrom, A. Bossios, M. Sjostrand, J.J. Lee, J.O. Lotvall, Nat. Cell Biol., 9 (2007) 654-U672.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

2D Microscale Surface Engineering with Novel Protein based Nanoparticles for Cell Guidance Witold I. Tatkiewicz1,2, Joaquin Seras-Franzoso2,3,4, Elena García-Fruitós2,3, Esther Vazquez2,3,4, Nora Ventosa1,2, Imma Ratera1,2, Antonio Villaverde2,3,4 and Jaume Veciana1,2 1 Dep. of Molecular Nanoscience and Organic Materials, Institut de Ciencia de Materials de Barcelona (CSIC), Bellaterra, 08193 Barcelona, Spain 2 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, Barcelona, Spain 3 Ins. de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Barcelona, Spain 4 Dep. of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain wtatkiewicz@icmab.es / iratera@icmab.es / jveciana@icmab.es The term “inclusion bodies” (IBs) was coined to describe optically opaque moieties present in cell lumen. They have aspect of refractile particles of up to a few hundred nanometers and about 2 μm3 of size when observed by optical microscopy and as electron-dense aggregates without defined organisation by transmission electron microscopy [1]. The history of IBs turned when they were recognized as a prospective biomaterial with desirable properties. Being a product derived from biological synthesis, it is fully biocompatible and preserves the functionality of the embedded protein [2]. In a course of investigation it was revealed that IBs size, geometry, stiffness, wettability, z-potential, bioadhesiveness, density/porosity etc. can be easily fine tuned by control over basic parameters of their production: harvesting time, host genetic background and production conditions (e.g. temperature, pH) In addition, their production and downstream processes are fully scalable, cost effective and methodologically simple [3]. It is widely accepted, that cell´s responses, such as positioning, morphological changes, proliferation, motility and apoptosis are the result of complex chemical, topographical and biological stimuli. Here we will show the

application of IBs as a functional biomaterial for engineering two dimensional substrates for cell guidance. We have cultivated fibroblast cells on supports patterned with IBs derived from green fluorescent protein (GFP) or human basic fibroblast growth factor (FGF). Two methodologies of pattern deposition were applied: microcontact printing (μCP) optimized for use with aqueous colloidal suspensions and a novel, template-free technique based on the coffee-drop effect due to a convective selfassembly (Figure 1) [4]. The first technique was applied in order to deposit IBs with high resolution geometrical patterns of various shapes and sizes. Then we have investigated how cells react to IBs geometrical distribution. Parameters such as orientation morphology and positioning were thoroughly investigated based on rich statistical data delivered by microscopy image treatment (Figure 2). The second technique has been recently developed in order to deposit complex and well-controlled two dimensional IB´s patterns with concentration gradients for the study of cell motility (Figure 3). Cell movement cultivated on such substrates was characterized and quantified based on confocal microscopy time-lapse acquisitions [5,6].

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

In both cases a deep statistical data treatment was preformed to characterize macroscopic responses of cells when grown over nanoscale profiles made with IBs concluding that cell proliferation is not only dramatically stimulated but cell also preferentially adhere to IBs-rich areas, align, elongate and move according to such IBs geometrical cues. These findings prove the potential of surface patterning with functional IBs as protein-based nanomaterials for tissue engineering and regenerative medicine among other promising biomedical applications

Ratera I., Veciana J., Villaverde A., Nanomedicine, 7 (2012) 79. [6] Tatkiewicz W.I., Seras-Franzoso J., García-Fruitós E., Vazquez E., Ventosa N., Peebo K., Ratera I., Villaverde A., Veciana J., ACSNano, 7 (2013) 4774.

Figures

References [1] (a) Villaverde A., Carrio M.M., Biotechnol. Lett., 25 (2003) 1385. (b) García-Fruitós E., RodríguezCarmona E., Diez-Gil C., Ferraz R.Mª, Vázquez E., Corchero J.L., Cano-Sarabia M., Ratera I., Ventosa N., Veciana J., Villaverde A. Adv. Mater., 21 (2009) 4249. (c) Cano-Garrido, O., Rodríguez-Carmona E., Díez-Gil C., Vázquez E., Elizondo E., Cubarsi R., Seras-Franzoso J., Corchero J.L., Rinas U., Ratera I.; et al. Acta Biomater. 9 (2013) 6134. [2] García-Fruitós E., Vazquez E., Díez-Gil C., Corchero J.L.; Seras-Franzoso J., Ratera I., Veciana J., Villaverde A., Trends Biotechnol., 30 (2012), 65. [3] (a) García-Fruitos E., Seras-Franzoso J., Vazquez E., Villaverde A., Nanotechnology, 21 (2010) 205101. (b) Vazquez E., Corchero J. L., Burgueno J.F., SerasFranzoso J., Kosoy A., Bosser R., Mendoza R., Martínez-Láinez J.M., Rinas U., Fernandez E., RuizAvila L., García-Fruitós E., Villaverde A., Adv. Mater., 24 (2012) 1742. (c) Vazquez E., Roldán M., Diez-Gil C., Unzueta U., Domingo-Espín J., Cedano J., Conchillo O., Ratera I., Veciana J., Daura X., FerrerMiralles N., Villaverde A., Nanomedicine., 5 (2010) 259. [4] (a) Han W., Lin Z., Angew. Chem. Int. Ed., 51 (2012) 1534 (b) Hanafusa T., Mino Y., Watanabe S., Miyahara M.T., Advanced Powder Technology, 25 (2014) 811 [5] (a) Díez-Gil C., Krabbenborg S., García-Fruitós E., Vazquez E., Rodríguez-Carmona E., Ratera I., Ventosa N., Seras-Franzoso J., Cano-Garrido O., Ferrer-Miralles N., Villaverde A., Veciana J., Biomaterials, 31 (2010) 5805. (b) Seras-Franzoso J., Díez-Gil C., Vazquez E., García-Fruitós E., Cubarsi R.,

NanoBio&Med2014

Figure 1. Schematic illustration of particle deposition. Particles are pinning to the substrate on the edge of meniscus, where the evaporation is more intense. Image adapted from reference [4b].

Figure 2. IBs striped (top) and random (bottom) pattern are compared. On the left; representative confocal microscopy images of cells cultivated on such patterns are presented. On the right; the overall orientation distribution of cells is presented. It is clearly seen, that cells are guided by the stripped pattern and they orient themselves along its geometry, whereas no predominant orientation of cells can be observed in the case of random pattern.

Figure 3. Example of GFP-derived IBs gradient pattern deposited by a controlled convective self-assembly technique. Left: fluorescence microscopy image, right: IBs concentration calculated based on fluorescence intensity.

november 18-21, 2014 - Barcelona (Spain)

Atomic force microscopy, life sciences and soft matter Jos矇 L. Toca-Herrera Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 11, A-1190 Vienna, Austria In this contribution, I would like to present the different use and development in atomic force microscopy (AFM) focusing primarily in the fields of life sciences and soft matter. In particular, we will see in which way the AFM is used as an imaging machine to characterize macromolecules at different interfaces or to follow crystallization processes. Furthermore, the use of the AFM as a mechanical machine it will be presented. In this part, I will talk about molecular forces, elasticity of proteins and cell mechanics [1,2]. Finally, different possibilities to combine the scanning probe microscopy with other microscopy techniques such as fluorescence microscopy, RICM and STED will be mentioned [3].

References

Figures

Figure 1. Left: force relaxation experiment after treating the cells with cytochalasin. Note that the grey line (after actin disruption with cytochalasin) decays faster than the black one (control). Middle: creep compliance experiment on the same cell before and after cytochalasin treatment. Note that the deformation of the cell is larger (grey line) after cytochalasin treatment. Right (above): optical image of the control MCF-7 cells. Right (below): optical image of the cells treated with cytochalasin. Figure adapted from [2].

[1] ]. S. Moreno-Flores, R. Benitez, M. dM Vivanco

and J. L. Toca-Herrera. Stress relaxation and creep on living cells with the atomic force microscope: a means to calculate elastic moduli and viscosities of cell components. Nanotechnology 21 (2010) 445101. [2] K. A. Melzak, G. R. Lazaro, A. HernandezMachado, I. Pagonabarraga, J. M. Cardenas D覺az de Espada and J. L. Toca-Herrera. AFM measurements and lipid rearrangements: evidence from red blood cell shape changes. Soft Matter, 8 (2012) 3716. [3] S. Moreno-Flores and J. L. Toca-Herrera. Hybridizing Surface Probe Microscopies: Toward a Full Description of the Meso- And Nanoworlds. CRC Press. 2013. Boca Raton. FL.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Multiplicity of Nanofection: a New Index to Assess Nanoparticle Cellular Uptake Juan D. Unciti-Broceta, Victoria Cano-Cortés, Patricia Altea Manzano, Salvatore Pernagallo, Juan J. Díaz-Mochón, Rosario M. Sánchez-Martín. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government (GENTO). PTS Granada,Avenida de la Ilustración, 114.18016 Granada, Spain Fac. de Farmacia, Dep. de Química Farmacéutica y Orgánica, Uni. de Granada, 18071 Granada, Spain Nanogetic S. L. Calle Marqués de los Vélez 2, 6ºA. 18005 Granada, Spain DestiNA Genomica S.L. Avenida de la Innovación, 1. 18100 Armilla (Granada), Spain juandiego.unciti@genyo.es Engineered nanoparticles (ENPs) for biological applications are produced from functionalized nanoparticles (NPs) after undergoing multiple coupling and cleaning steps, giving rise to an inevitable loss of NPs in final compositions. Herein, we present a simple method to quantify the number of ENPs per microliter using standard spectrophotometers and volumes of up to one microliter. Light going through NP suspensions is scattered via reflection, refraction and diffraction phenomenon and the amount of the scattered light depend on the number of NPs found in suspensions. By measuring optical densities (OD) at 600 nm of different polystyrene NP suspensions of three different sizes (100 nm, 200 nm and 460 nm), linear correlations between OD600 and number of NPs were found for each NP size. These calibration curves can then be applied to estimate the number of ENP compositions of a particular NP size and material (Figure 1). To exemplify the method, we introduced the number of ENPs versus number of cells as a new parameter to report cellular uptake assays where capacities of cells to uptake beads or NPs (“nanofection”) need to be assessed. This parameter allows us to introduce “multiplicity of nanofection 50” (MNF50) index, which is defined as the number of NPs per cell needed to “nanofect” 50% of a given cell type, as a measure of the capacity of a cell type to uptake certain ENPs. Three mammalian cell lines were tested with 200 nm Cy5-PEG-NPs and, following

NanoBio&Med2014

flow cytometry analysis, each of them presented different MNF50, being MDA MB 231 mammalian breast cancer cell line the one with a lower MNF50 and therefore with a higher uptaking capacity of these ENPs (Figure 2). Median of fluorescence intensity (MFI) of Cy5 positive cells analysis showed a linear behavior with different slopes for each cell line which is also a parameter to assess cell capacities for NPs uptaking (Figure 3). Furthermore, if we compare MFI increments (ΔMFI=MFI sample/MFI untreated) same results were obtained (Figure 4). A deeper study of ΔMFI showed a surprising data, from the closest ratio to their MNF50, the increase of the ΔMFI is doubled when the NPs number are doubled, something which is not observed when ratios lowers than their MNF50 are used. Importantly, this effect is the same for the three studied cell lines. Therefore, when MNF50 is reached, the nanofection rate is constant and proportional to the number of nanoparticles used with cell lines presenting similar behavior. Nowadays the efficiency of many NPs-based delivery systems of bioactive cargoes are related to solid content (w/V) of NPs per cell. This method allows introducing a new parameter to analyze cellular uptake by reporting nanoparticle number versus cells number (multiplicity of nanofection). Based on these data we believe that the number of NPs per cell could be reported rather than weight of NPs per cell in any cell-based assays using NPs. The implementation of the Multiplicity of

november 18-21, 2014 - Barcelona (Spain)

nanofection (MNF) will improve dramatically the efficiency of any nanoparticle-based devices.

References [1] V. Wagner, A. Dullaart, A. K. Bock and A. Zweck,

Nature biotechnology, 2006, 24, 1211-1217. [2] E. Elinav and D. Peer, ACS nano, 2013, 7, 28832890. [3] M. Goldsmith, L. Abramovitz and D. Peer, ACS nano, 2014, 8, 1958-1965. [4] T. Lammers, S. Aime, W. E. Hennink, G. Storm and F. Kiessling, Accounts of chemical research, 2011, 44, 1029-1038. [5] Y. Namiki, T. Fuchigami, N. Tada, R. Kawamura, S. Matsunuma, Y. Kitamoto and M. Nakagawa, Accounts of chemical research, 2011, 44, 1080-1093. [6] J. G. Borger, J. M. Cardenas-Maestre, R. Zamoyska and R. M. Sanchez-Martin, Bioconjugate chemistry, 2011, 22, 1904-1908. [7] M. Bradley, L. Alexander, K. Duncan, M. Chennaoui, A. C. Jones and R. M. SanchezMartin, Bioorganic & medicinal chemistry letters, 2008, 18, 313-317. [8] J. M. Cardenas-Maestre, A. M. Perez-Lopez, M. Bradley and R. M. Sanchez-Martin, Macromolecular bioscience, 2014, DOI: 10.1002/mabi.201300525. [9] N. Gennet, L. M. Alexander, R. M. SanchezMartin, J. M. Behrendt, A. J. Sutherland, J. M. Brickman, M. Bradley and M. Li, New biotechnology, 2009, 25, 442-449. [10] S. Kunjachan, F. Gremse, B. Theek, P. Koczera, R. Pola, M. Pechar, T. Etrych, K. Ulbrich, G. Storm, F. Kiessling and T. Lammers, ACS nano, 2012, 7, 252-262. [11] R. Sanchez-Martin, V. Cano-Cortes, J. A. Marchal and M. Peran, Methods in molecular biology (Clifton, N.J.), 2013, 1058, 41-47. [12] R. M. Sanchez-Martin, L. Alexander and M. Bradley, Annals of the New York Academy of Sciences, 2008, 1130, 207-217. [13] R. M. Sanchez-Martin, M. Cuttle, S. Mittoo and M. Bradley, Angewandte Chemie

(International ed. in English), 2006, 45, 54725474. [14] S. Sengupta and A. Kulkarni, ACS nano, 2013, 7, 2878-2882. [15] A. Tsakiridis, L. M. Alexander, N. Gennet, R. M. Sanchez-Martin, A. Livigni, M. Li, M. Bradley and J. M. Brickman, Biomaterials, 2009, 30, 5853-5861. [16] R. M. Sanchez-Martin, M. Muzerelle, N. Chitkul, S. E. How, S. Mittoo and M. Bradley, Chembiochem : a European journal of chemical biology, 2005, 6, 1341-1345. [17] J. Shi, A. R. Votruba, O. C. Farokhzad and R. Langer, Nano letters, 2010, 10, 3223-3230. [18] R. M. Yusop, A. Unciti-Broceta, E. M. Johansson, R. M. Sanchez-Martin and M. Bradley, Nature chemistry, 2011, 3, 239-243. [19] A. Unciti-Broceta, J. J. Díaz-Mochón, R. M. Sánchez-Martín and M. Bradley, Accounts of chemical research, 2012, 45, 1140-1152. [20] D. Schneider and K. Lüdtke-Buzug, in Magnetic Particle Imaging, eds. T. M. Buzug and J. Borgert, Springer Berlin Heidelberg, 2012, vol. 140, ch. 19, pp. 117-122. [21] K. Y. Win and S. S. Feng, Biomaterials, 2005, 26, 2713-2722. [22] B. D. Chithrani, A. A. Ghazani and W. C. Chan, Nano letters, 2006, 6, 662-668. [23] B. D. Chithrani and W. C. Chan, Nano letters, 2007, 7, 1542-1550. [24] V. V. Savchenko, A. G. Basnakian, A. A. Pasko, S. V. Ten and R. Huang, in Computer graphics, eds. E. Rae and V. John, Academic Press Ltd., 1995, pp. 437-447. [25] W. Lee, J. Sodek and C. A. McCulloch, Journal of cellular physiology, 1996, 168, 695-704. [26] C. H. Park and M. A. Latina, Investigative ophthalmology & visual science, 1993, 34, 2228-2236. [27] in Light Scattering by Particles in Water, eds. M. Jonasz and G. R. Fournier, Academic Press, Amsterdam, 2007, DOI: http://dx.doi.org/10.1016/B978-0123887511/50000-4, pp. vii-x.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

[28] F. Alexis, E. Pridgen, L. K. Molnar and O. C.

Farokhzad, Molecular pharmaceutics, 2008, 5, 505-515.

Figures

Figure 1

Figure 2

Figure 3

Figure 4

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Nanomechanics of Decellularized Lung and in Vivo Lung Elastance in a Murine Model of Marfan Syndrome J.J. Uriarte1,3,4, P.N. Nonaka1, N. Campillo1,2,4, Y. Mendizabal5, E. Sarri5, G. Egea5, R.Farre1,3,4; D. Navajas1,2,4 1 Unitat de Biofísica i Bioingeniería, Facultat de Medicina, Univ. de Barcelona, Barcelona,Spain 2 Institut de Bioenginyeria de Catalunya, Barcelona, Spain 3 Institut d’Investigacions Biomèdiques August Pi Sunyer, Barcelona, Spain 4 CIBER de Enfermedades Respiratorias, Madrid, Spain 5 Dept. Biologia Cel·lular, Immunologia i Neurociències. Fac. de Medicina, UB, Barcelona, Spain dnavajas@ub.edu Marfan syndrome (MFS) is an autosomal dominant disorder caused by mutations in the gene (FBN1) encoding fibrillin-1, the major component of extracellular matrix (ECM) microfibrills. In the pathogenesis of MFS, matrix metalloproteases and over activity of TGF-β are directly involved. The syndrome carries an increased risk of aneurysm and dissection of the ascending aorta, and alterations in eyes, skeleton and lungs. Although lung mechanics in MFS could be affected by changes in elastic and tensile strength of connective tissue, there are no data available on the effects of this monogenetic disease in lung mechanics. The aim of this work is to assess whether lung scaffold stiffness and in vivo lung elastance is affected in a Marfan mouse model. Twelve 9 month-old C57BL/6 mice (6 healthy controls and 6 Marfan mice heterozygous for an Fbn1 allele encoding a cysteine substitution, Fbn1(C1039G/+) were used. Control and Marfan mice were intraperitoneally anesthetized (urethane, 1.5 g/kg), paralyzed (pancuronium bromide, 0.1 mg/kg) and subjected to volume-control mechanical ventilation (100 breaths/min, 0.30 ml tidal volume). Subsequently, the chest wall was opened and a positive end-expiratory pressure of 2 cmH2O was applied. The signals of tracheal pressure and flow during mechanical ventilation were recorded at the entrance of the tracheal cannula and lung elastance was determined by conventional linear regression.

The animals were euthanized by exsanguination, the left lung lobule was excised and decellularized with a conventional protocol based on freezing/thawing cycles and sodium dodecyl sulfate detergent. Acellular lung slices (12 micron thick) were obtained in order to measure nanomechanics (Young’s modulus) of different regions of the lung scaffold (alveolar septum, tunica adventitia and tunica intima) with atomic force microscopy using pyramidal cantilevers (nominal spring constant 0.03 N/m) at an operating indentation of 500 nm. Marfan mice exhibited an in vivo lung elastance that was 42% lower than controls (21.7±2.7 and 37.1±2.5 cmH2O/ml, respectively; mean±SEM; p<0.05). Remarkably, no significant differences were found in the local stiffness of the acellular lung between Marfan mice and controls: 36.4±3.7 vs 38.4±10.0 kPa, 63.2±17.5 vs 48.2±6.8 kPa and 125.2±10.2 vs 119.8±23.7 kPa in the alveolar septum and the lung vessels tunicae adventitia and intima, respectively. In conclusion, these data suggest that the higher in vivo compliance observed in Marfan lungs are not caused by a softening of the acellular lung scaffold, as demonstrated by AFM measurements of the local nanomechanical properties of the extracellular matrix of the lung. These changes could be attributed to alterations in the 3-D structure of the lung.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

References [1] Luque T, Melo E, Garreta E, Cortiella J, Nichols

J, Farré R, Navajas D. Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy. Acta Biomaterialia 9 (2013) 6852–6859. [2] Melo E, Cardenes N, Garreta E, Luque T, Rojas M, Navajas D, Farré R. Inhomogeneity of local stiffness in the extracellular matrix scaffold of fibrotic mouse lungs. J Mech Behav Biomed Mater 2014; 37: 186–195. [3] Neptune ER, Frischmeyer PA, Arking DE, Myers L, Bunton TE, Gayraud B, Ramirez F, Sakai LY, Dietz HC. Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nat Genet. 2003 Mar; 33(3):407-11. [4] Cañadas V, Vilacosta I, Bruna I, Fuster V. Marfan Syndrome. Part 1: Pathophysiology and diagnosis. Nat Rev Cardiol. 2010 May; 7 (5): 256-65.

Figure 2. Local stiffness at different acellular lung parenchyma of control (white) and Marfan mice (gray). Mean ± SE. There are no significant differences.

Figures

Figure 1. Effective elastance (E) computed from control (white) and Marfan mice (gray) during in vivo conventional mechanical ventilation. Mean ± SE. Asterisk indicates p<0.05.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Supramolecular organizations as novel nanomedicines for drug delivery Jaume Veciana Institut de Ciència de Materials de Barcelona (CSIC) and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Campus Universitari de Bellaterra, Cerdanyola, Spain vecianaj@icmab.es The objective of this lecture is to give a broad overview of how nanotechnology is impacting in some areas of medicine and pharmacology. This lecture will report the advantages of nanoparticulate molecule-based organizations for drug delivery. It has been reported that polymeric nanoparticles and nanovesicles are efficient drug carriers that can significantly help to develop new drug delivery routes, and more selective and efficient drugs with a higher permeability to biological membranes and with controlled released profiles, as well as to enhance their targeting towards particular tissues or cells [1-2]. The potential of nanotechnology «bottom-up» strategies, based on molecular self-assembling, is much larger than that of «top-down» approaches for the preparation of such nanosized supramolecular organizations. For instance, by precipitation procedures it should be possible to control particle size and size distribution, morphology and particle supramolecular structure. However, conventional methods from liquid solutions have serious limitations and are not adequate for producing such nanoparticulate materials at large scale with the narrow structural variability, high reproducibility, purity and cost needed to satisfy the high-performance requirements and regulatory demands dictated by the USA and European medicine agencies. On the contrary, using compressed solvent media it is possible to obtain supramolecular materials with unique physicochemical characteristics (size, porosity, polymorphic nature morphology, molecular self-assembling, etc.) unachievable with

classical liquid media. The most widely used CF is CO2, which has gained considerable attention, during the past few years as a «green substitute» to organic solvents. Due to such properties, during the past few years CFs based technologies are attracting increasing interest for the preparation of nanoparticles and nanovesicles with application in nanomedicine. In this lecture a simple one-step and scale-up methodology for preparing multifunctional nanovesicle-drug conjugates will be presented. This method is readily amenable to the integration/encapsulation of multiple bioactive components, like peptides, proteins, enzymes, into the vesicles in a single-step yielding sufficient quantities for clinical research becoming, thereby, nanocarriers to be used in nanomedicine. A couple of examples of novel nanomedicines for solving serious diseases, prepared by this methodology, will be presented and their advantages discussed [3-4]

References [1] M. E. Davis, Z. Chen, D. M. Shin, Nature Reviews-Drug Discovery 2008, 7, 771-782. [2] J. A. Hubbell, R. Langer, Nature Materials, 2013, 12, 963-966. [3] N. Ventosa, L. Ferrer-Tasies, E. Moreno-Calvo, M. Cano, M. Aguilella, A. Angelova, S. Lesieur, S. Ricart, J. Faraudo, J. Veciana. Langmuir, 2013, 29, 6519-6528. [4] I. Cabrera, E. Elizondo, E. Olga; J. Corchero, M. Mergarejo, D. Pulido, A. Cordoba, E. Moreno-Calvo, U. Unzueta, E. Vazquez, I. Abasolo, S. Schwartz, A. Villaverde, F. Albericio, M. Royo, M. Garcia, N. Ventosa, J. Veciana. Nano Letters, 2013, 13, 3766-3774.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Polymeric micelles nanovectors for photodynamic therapy applications: From the structure to the activity P. Vicendo1, A.F. Mingotaud1, L. Gibot2 , A. Lemelle1, U. Till1,2,3, B. Moukarzel1, M.P. Rols2, C. Chassenieux4, M. Gaucher5 and F. Violleau5 1 Université de Toulouse; UPS/CNRS; IMRCP, Toulouse Cedex 9, France 2 Université de Toulouse, IPBS-CNRS UMR 5089, 205 Route de Narbonne, Toulouse Cedex, France 3 Technopolym, Institut de Chimie de Toulouse, 118 route de Narbonne, Toulouse Cedex 9, France. 4 Université de Toulouse, Institut National Polytechnique de Toulouse – Ecole d’Ingénieurs de Purpan, Département Sciences Agronomiques et Agroalimentaires, UPSP/DGER 115, 75 voie du TOEC, BP 57611, F-31076 Toulouse Cedex 03, France 5 LUNAM Université, Université du Maine, IMMM UMR CNRS 6283 Département PCI, Avenue Olivier Messiaen, 72085 Le Mans Cedex 09, France vicendo@chimie.ups-tlse.fr The recent development of light irradiation systems has facilitated the emergence of new therapies based on light-sensitive drugs. However, photosensitizers have a tendency to self-associate in physiologic environment, leading to a loss of their physical properties. Hence, nanometric formulations have been assessed, because this limits self-association and enables accumulation in solid tumors owing to enhanced permeability and retention effect (EPR). In this study, we present first a thorough characterization of polymeric micelles based on light scattering and Asymmetrical Flow Field Flow Fractionation. In a second step, we examine their efficiency as photosensitizer vectors using 2D or 3D tumor model namely spheroids. Polymeric micelles were formed from 4 different amphiphilic block copolymers: poly(ethylene oxide-bε-caprolactone) 2000-2800, poly(ethylene oxide-b-ε-caprolactone) 50004000, poly(ethylene oxide-b-polystyrene) 31002200 and poly(ethylene oxide-b-(D,L)-lactide) 2400-2000. The micelles have been characterized by static and dynamic light

NanoBio&Med2014

scattering, electron microscopy and asymmetrical flow field-flow fractionation. This showed that all systems led to polymeric selfassemblies having a size close to 20nm and a neutral surface. They were shown to be stable upon ageing and dilution, even in the presence of various blood components such as globulins or albumin, which is essential for a possible application as vectors. Cytotoxicity and phototoxicity in the presence of Pheophorbide a as photosensitizer were then characterized both on 2D and 3D cell culture. PDT on spheroids enabled to corroborate results from 2D, showing that encapsulation of Pheophorbide yielded a strong increase of photocytotoxicity. However, small differences for the nanovectors were highlighted: PEO-PCL 2000-2800 being the most efficient in 2D, whereas PEO-PDLLA 24002000 was the best for 3D tests. The obtained results will be discussed in relation with the ones obtained in physical chemistry characterizations. Only a thorough physico-chemical characterization coupled to in vitro experiments

november 18-21, 2014 - Barcelona (Spain)

may enable a critical analysis of possible vectors. The polymeric micelles chosen in this study were observed to yield a strong efficiency in PDT, but the differences observed between 2D and 3D systems show that a great care should be taken when testing such vectors.

References [1] L. Gibot, A. Lemelle, U. Till, B. Moukarzel, A.-F.

Mingotaud, V. Pimienta, P. Saint-Aguet, M.-P. Rols, M. Gaucher, F. Violleau, C. Chassenieux, P. Vicendo, Biomacromolecules 15(4) (2014) 1443-1455.

Figures

Figure 1. Polymers and PS used

Figure 2. Example of tumor spheroid macroscopic aspect after PDT procedure with PEO-PS micelles loaded with pheophorbide a.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Nanographene-oxide mediated hyperthermia for cancer treatment Mercedes Vila TEMA-NRD, Mechanical Engineering Department and Aveiro Institute of Nanotechnology (AIN), University of Aveiro, 3810-193 Aveiro, Portugal mvila@ua.pt One of the new trends of nanomedicine is the application of nanoparticles for targeting tumors to achieve localized tumor cell destruction while producing minimal side effects on healthy cells, but their application will not be feasible without a previous understanding of vector-cell/tissue interactions, possible toxicity and accumulation risks. [1] The enhanced permeability and retention effect (EPR), provoked by the angiogenesis process of tumors, allows preferential concentration of nanosystems on the tumor periphery, making hyperthermia mediated by these systems a potential efficient therapy for producing confined tumoral cell death. [2] It can induce lethal damage to cellular components at temperatures above 40 ยบC and cancerous cells are subsequently removed by macrophages, causing the tumor to diminish. Although hyperthermia is a well-known concept, little is known about the type of damage, cell death and secondary effects that these nanosystems mediated therapies can provoke locally. Amongst hyperthermia potential agents, nano graphene oxide (nGO) has been proposed due to its strong Near-Infrared (NIR 700-1100 nm range) optical absorption ability and its unique 2-dimensional aspect ratio. [3] Restricting all dimensions at nanoscale could allow unique performing when compared to any other nanoparticle, but it is mandatory to deeply study the hyperthermia route and the kind of nGO-cell interactions induced in the process.

NanoBio&Med2014

By optimizing the nGO synthesis, it is possible to diminish the initial cell-particle interactions to reduce possible future toxicity in healthy cells.[3,4] Cell internalization kinetics (specifically for targeting tumoral osteoblasts on a bone cancer model) were established for producing a safe and efficient tumor cell destruction avoiding damage on untreated cells as well as an evaluation of the nature of tumor destruction that could be produced by this hyperthermia treatment.[5,6] The type of cell damage and toxicity produced by NIR laser irradiation was evaluated as a function of exposure time and laser power in order to control the temperature rise and consequent damage in the nGO containing cell culture medium. The results showed that cell culture temperature (after irradiating cells with internalized nGO) increases preferentially with laser power rather than with exposure time. Moreover, when laser power is increased, necrosis is the preferential cell death (Fig. 1). The results suggested that controlling the type of cell death, the threshold for producing soft or harmful damage could be precisely controlled and so, the increase of cytokine release to the medium, having this a direct impact on immune system reactions. Moreover, nGO cell exposure did not stimulate proinflammatory cytokine secretion [7] and nanoparticles incorporation by different cell types either in the absence or in the presence of eight endocytosis inhibitors, showed that macropynocitosis is the general mechanism of nGO internalization, but it can also entry through clathrin-dependent mechanisms in hepatocytes and macrophages. [8].

november 18-21, 2014 - Barcelona (Spain)

References [1] Day ES, Morton JG, West JL. Nanoparticles for

[2]

[3]

[4]

[5]

[6]

[7]

[8]

Thermal Cancer Therapy J Biomed Eng 2009;131:074001. Gonçalves G, Vila M, Portolés MT, Vallet-Regi M, Gracio J, Marques PAAP. Nano-Graphene Oxide: a potential platform for cancer therapy and diagnosis. Advanced Healthcare Materials. (2013) 2(8):1072-90 Vila M, Portolés MT, Marques PAAP, Feito MJ, Matesanz MC, Ramírez-Santillán et al. Cell uptake survey of pegylated nano graphene oxide. Nanotechnology 2012; 23:465103. Gonçalves G, Vila M, Bdikin I, de Andrés A, Emami N, Ferreira RAS, Carlos LD, Grácio J, Marques PAAP. Breakdown into nanoscale of graphene oxide: Confined hot spot atomic reduction and fragmentation Nature Scientific Reports, 2014, In press Vila M.,.Matesanz M.C, Gonçalves G., Feito M.J., Linares J., Marques P.A.A.P., Portolés M.T., Vallet-Regi M. Triggering cell death by nanographene oxide mediated hyperthermia. Nanotechnology 2014 (25) 035101 Matesanz MC, Vila M, Feito MJ, Linares J, Gonçalves G, Vallet-Regi M et al. The effects of graphene oxide nanosheets localized on factin filaments on cell cycle alterations. Biomaterials 2013; 34: 1562-9. Feito MJ, Vila M, Matesanz MC, Linares J, Gonçalves G, Marques PAAP, Vallet-Regí M, Rojo JM, Portolés MT. In vitro evaluation of graphene oxide nanosheets on immune function J Colloid Interface Sci. 432 (2014) 221228; Linares J, Matesanz MC, Vila M, Feito MJ, Gonçalves G, Vallet-Regí M, Marques PAAP, Portolés MT. Endocytic Mechanisms of Graphene Oxide Nanosheets in Osteoblasts, Hepatocytes and Macrophages ACS Appl. Mater. Interfaces, 6 (2014) 13697−13706

Figures

Figure 1. Morphology evaluation by confocal microscopy of cultured human SAOS-2 osteoblasts in the presence of GOs, before (left) and after 7 min of 1.5 W/cm2 laser irradiation showing necrotic cells (right).

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Host-guest engineered stimuli-responsive nanocapsules Ewelina Wajs1, Thorbjørn Terndrup Nielsen2, Alex Fragoso1 1 Nanobiotechnology & Bioanalysis Group, Universitat Rovira I Virgili, Tarragona, Spain 2 Department of Biotechnology, Aalborg University, 9000 Aalborg, Denmark ewelinamaria.wajs@urv.cat The supramolecular self-assembly of materials through host-guest interactions is a powerful tool to create non-conventional materials. Thus, biodegradable nanocapsules with redox-/or light-responsibility were fabricated with noncovalent interactions between βCD and ferrocene (Fc)/or αCD and azobenzene (Azo) units. Different biocompatible polymers, dextran-βCD (βCD-Dex) and dextranferrocene (Fc-Dex), dextran-αCD (αCD-Dex) and dextranazobenzene (Azo-Dex) were assembled in alternating way on gold nanoparticles of two different sizes (100 and 400 nm). The gold nanoparticles were removed by chemical degradation and rhodamine B (RhB) was encapsulated inside the carriers as a model drug. The encapsulation process of the dye molecules was accelerated by oxidation step or by UV-light of the nanocapsules wall, thus enabling easier and faster diffusion through the polymer layers. Confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), atomic force microscopy (AFM), RAMAN spectroscopy, UV-spectroscopy and dynamic light scattering (DLS) measurements were employed for the characterization of the nanocapsules.

Figures

Figure 1. Schematic representation of the formation of LbL self-assembled nanocapsules via host-guest interactions between complementary βCD and Fc appended dextran polymers (the same methodology was applied for the αCD/Azo appended dextran polymers).

*Financial support from Ministerio de Economía y Competitividad, Spain (grant BIO2012-30936 to A.F.) is gratefully acknowledged.

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Nanoceria Tetyana Yudina, Eudald Casals, V铆ctor Puntes The Catalan Institute of Nanoscience and Nanotechnology (ICN2), Edifici CIN2, 08193 Bellaterra (Barcelona), Spain tetyana.yudina@gmail.com Cerium oxide nanoparticles (CeO2NPs or nanoceria) is an inorganic material with many of applications and more to come. Besides being rather a chemically inert ceramic, its flurite-like electronic structure (Fig. 1) confers it a variety of interesting properties, making nanoceria one of the most interesting NPs in industry and biomedical research. What makes nanoceria very appealing is its high capacity to buffer electrons from an oxidant/reducing environment, which is due to its easy ability of being oxidized and reduced, from Ce3+ to Ce4+ and vice versa [1], followed by the capture or release of oxygen, or oxygen species (as OH路). Since redox reactions are an important class of chemical reactions encountered in everyday processes, CeO2 NPs are widely used in a range of industrial applications as combustion of fuels, environmental remediation [2], water purification [3], catalysis [4] and many others. A special interesting case is metabolism where partial reduction of oxygen produces byproducts, known as reactive oxygen species (ROS) including superoxide anion (O2-), hydrogen peroxide (H2O2) and the hydroxyl radical (OH路). On one hand, ROS are an antibacterial tool in case of infection; on the other hand, high amounts of ROS are toxic for humans and the environment. Unfortunately, the heightened levels of ROS can damage significantly cellular integrity, by inducing chronic inflammation, lipid peroxidation, DNA damage, damage of oxidation sensitive proteins, or even trigger cell death (apoptosis) by a metabolic flux disruption. Therefore, the oxygen storage capacity of CeO2NPs becomes highly useful to remove them as soon as they

are generated, in situation of ROS disbalance. That property makes nanoceria a potential candidate as a therapeutic tool in prevention and treatment of a wide range of human diseases with ROS disbalance, such as: cancer, diabetes mellitus, cardiovascular disease (CDV), age-related macular degeneration (AMD) and ophthalmology. Moreover, the overproduction of ROS is critical in neurodegeneration, including Alzheimer, Parkinson, Huntington, schizophrenia among others. What is clear is that there is a strong correlation between the cellular effects of the NPs and their engineering, including the preparation method, morphology (size, shape, surface composition, contaminants) and aggregation state of the nanoparticles [5]. Regarding its uses in medicine, morphology determines biodistribution and reactivity, therefore, for the hard task of performing precise work within the biological machinery, a fine morphological control of CeO2 nanoparticles and their aggregation state is needed, since it drives the reactivity, colloidal stability, interaction with proteins and pharmacokinetics of the nanoparticles within the organism. Up to now many of labored protocols of nanoceria synthesis have been described, such as high-temperature thermolysis of cerium salts, mechanochemical reactions, gas-phase methods, non-isotermal precipitation, supercritical synthesis methods, hydrothermal synthesis, sol-gel, flame-spray pyrolysis and solvothermal method, between others. Many of them require multiple steps, as use of high

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

temperatures, refluxing, sonication or product drying. Nowadays, the wet-chemical preparations have become one of the most widely used methods of synthesis of CeO2 nanoparticles. Even so, an obtaining of pure and monodisperced CeO2 nanoparticles, with a reproducible size-control is still a challenge, since its is a highly reactive nanomaterial and tends to suffer changes in size, morphology and aggregation state (Fig.2).

Figures

Therefore, we focused our study in an understanding of the kinetic behavior of the formation of nanoceria, in order to be able to control the purity, size and aggregation state of the obtained material. In this study we describe a preparation of CeO2 nanoparticles in aqueous phase by a kinetic control of Ce3+ oxidation at room temperature. We also try to give explanations of the nucleation, selective attachment and aggregation phenomena of the nanoceria and propose storage conditions suitable for their bio-medical or industrial purposes. The sizedependent reactivity, scalability and biocompatibility are also analyzed.

Figure 1. Electronic structure of CeO2NPs fluorite structure, containing 8 coordinate Ce4+ and 4 coordinate O2â&#x20AC;&#x201C;.

References [1] Cafun, J.D. et al., ACS Nano, 7(2013) 10726-32. [2] Sajith, V., Sobhan C.B., Peterson G.P.,

Advances in Mechanical Engineering, Vol 2010 (2010) 1. [3] Mellaerts, R. et al., Rsc Advances, 3(3) (2013) 900-909. [4] Popa-Wagner, A. et al., Oxid Med Cell Longev, 2013 (2013) 963520. [5] Dowding, J.M. et al., Acs Nano, 7(6) (2013) 4855-4868.

NanoBio&Med2014

Figure 2. HR-TEM imaging of CeO2NPs, showing the instability of nanoceria, reflected in phenomenas as selective attachment (red brackets in (a) and (b)), morphological changes of the NPs (blue brackets in (a) and (c)) and aggregation (d).

november 18-21, 2014 - Barcelona (Spain)

FluidFM: combining AFM and microfluidics for single-cell perturbation in vitro Tomaso Zambelli Laboratory of Biosensors and Bioelectronics, ETH Zurich, Switzerland

Glass micropipettes are the typical instrument for intracellular injection, patch clamping or extracellular deposition of liquids into viable cells. The micro pipette is thereby slowly approached to the cell by using micro manipulators and visual control through an optical microscope. During this process, however, the cell is often mechanically injured which leads to cell death and failure of the experiment. To overcome these challenges and limitations of this conventional method we developed the FluidFM technology, an evolution of standard AFM microscopy combining nanofluidics via cantilevers with integrated microfluidic channel [1]. The channel ends at a well-defined aperture at the apex of the AFM tip while the other extremity is connected to a reservoir. The instrument can therefore be regarded as a multifunctional micropipette with force feedback working in liquid environment.

Figures

Figure 1. a) Scheme of the FluidFM. b) Two fluorescent viruses ejected from a microchanneled cantilever. c) A yeast attached by underpressure at the aperture of a microchanneled cantilever.

We are focussing on three applications for single-cell biology [2]: i) cytosolic and intranuclear injection, ii) cell adhesion, and iii) single virus deposition on cell surfaces. At the same time we are using the FluidFM as lithography tool in liquid [3].

References [1] A. Meister et al, Nano Lett (2009) 9:2501 [2] O. Guillaume-Gentil et al, Trends Biotech

(2014) 32:381 [3] R.R. Gr端ter et al, Nanoscale (2013) 5:1097.

100

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Poster list: Alphabetical order (by surname) Authors Almendral-Parra, María Jesús Sara Sánchez Paradinas and Víctor Barba Vicente

Ariza-Sáenz, Martha Vega E., Espina M., Gómara M.J, Egea M.A, Haro I., Garcia M.L.

Country Poster title Spain

Biomolecule-Quantum Dot systems for biological applications: Size-controlled aqueous synthesis of CdS Quantum Dots in homogeneous phase with BSA as capping ligand.

Spain

Optimization and characterization of poly (lactic-co-glycolic acid) nanoparticles loaded with an HIV-1 inhibitor synthetic peptide.

Spain

Nucleic Acids for biosensing applications.

Spain

Silver nanoparticles downregulates p53 activation and induce desacetilation of histone 3 in epithelial human lung cancer epithelial cells.

Spain

Magnetoliposomes for magnetic resonance imaging.

Spain

In vitro and in vivo evaluation of TiO2 oral absorption.

Spain

Synthesis of modified dendrimers and conjugation with selected apoptosis inhibitors.

Belgium

Monitoring the intracellular dynamics of polystyrene nanoparticles in lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking.

Spain

Interaction of targeted of magnetoliposomes with Hela epithelial carcinoma and 3T3 fibroblasts cell lines.

Spain

Thermal stability of a cationic solid lipid nanoparticle (cSLN) formulation as a possible biocompatibility indicator.

Spain

PLGA nanoparticles as advanced imaging nanosystems.

Spain

Continuous synthesis of silver nanoparticles using green chemicals and microreactors and its evaluation as bactericidal agents.

Aviñó, Anna César Sánchez, Mar Oroval, Laura Carrascosa, Ramón Martínez-Máñez, Laura Lechuga and Ramon Eritja

Blanco Perez, Jordi Daisy Lafuente, Domènec Sánchez, José Luis Domingo, Mercedes Gómez

Busquets, M. Antònia Joan Estelrich, Josep Queralt, Montserrat Gallardo

Cabellos, Joan Gemma Janer, Ezequiel Mas del Molino, Elisabet Fernández-Rosas, Socorro VásquezCampos

Corredor, Miriam Ignacio Alfonso, Dietmar Appelhans, Angel Messeguer

Deville, Sarah Rozhin Penjweini, Nick Smisdom, Kristof Notelaers, Inge Nelissen, Jef Hooyberghs, Marcel Ameloot

Estelrich, Joan M. Carmen Moran, M. Antònia Busquets

Fàbregas, Anna Montserrat Miñarro, Josep Ramon Ticó, Encarna García-Montoya, Pilar Pérez-Lozano, Josep Mª Suñé-Negre

Feiner-Gracia, Natàlia C. Fornaguera, A. Dols-Perez, M.J. GarcíaCelma, C. Solans

Giorello, Antonella Santiago Ibarlín, Esteban Gioria, José Luis Hueso, Victor Sebastián, Manuel Arruebo, Laura Gutierrez and Jesús Santamaría

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

101

Authors Gómara, María José Aimee Vasconcelos, Estefanía Vega, Yolanda Pérez, María Luisa García, Isabel Haro

Grijalvo, Santiago Adele Alagia, Gustavo Puras, Jon Zárate, Jose Luis Pedraz and Ramon Eritja

Country Poster title Spain

Biodegradable polymeric nanoparticles modified with cell penetrating peptides as an effective ocular drug delivery system.

Spain

Cationic vesicles based on non-ionic surfactant and synthetic aminolipids mediate delivery of antisense oligonucleotides into mammalian cells.

Spain

Gold Nanoparticles Supported on Nanoparticulate Ceria as a Powerful Agent against Intracellular Oxidative Stress.

Iran

Application of magnetic chitosan nano particles for antiAlzheimer drug delivery systems.

Spain

The interferences of nanomaterials with hemoglobin a handicap to study hemocompatibility.

Spain

Nanoscale conductance imaging of electronic materials and redox proteins in aqueous solution.

Spain

Gemini Amphiphilic Pseudopeptides for Encapsulation and Release of Hydrophobic Molecules.

Spain

ph-responsive “onion nanospheres” coming from ionic liquid crystal pamam dendrimers.

Belgium

Modulation of dendritic cell sensitization by combined exposure to allergens and nanoparticles.

Spain

Toxicity assays of nebulized gold nanoparticles with potential applications in the development of nanopesticides.

Spain

Cerium oxide nanoparticles reduce portal hypertension and show antiinflammatory properties in CCl4-treated rats.

Spain

Bridging Research and Industrial Production towards H2020: Future challenges for Nanomedicine with a multi-KET approach.

Germany

Accessing the Nanoparticle Corona in Pulmonary Surfactant.

Spain

2D Microscale Engineering of Novel Protein based Nanoparticles for Cell Guidance.

Spain

Long-term exposures to low doses of cobalt nanoparticles induce cell-transformation enhanced by oxidative damage.

Herance, José Raúl María Gamón, Cristina Menchón, Roberto Martín, Nadezda Apostolova, Milagros Rocha, Victor Manuel Victor, Mercedes Alvaro, Hermengildo García

Khanmohammadi Mohammadreza Hamideh Elmizadeh

Llanas, Hector Sordé A. , Mitjans M. , Vinardell M.P.

López Martínez, Montse J. M. Artés, I. Díez-Perez, F. Sanz and P. Gorostiza

Lotfallah, Ahmed H. Ignacio Alfonso,M. Isabel Burguete and Santiago V. Luis

Marcos, Mercedes Silvia Hernández-Ainsa, Joaquín Barberá, José Luis Serrano, Teresa Sierra

Nelissen, Inge Birgit Baré, Sarah Deville, An Jacobs, Nathalie Lambrechts, Peter Hoet

Ochoa-Zapater, María Amparo J. Querol-Donat, F.M. Romero, A. Ribera, G. Gallello, A. Torreblanca, M.D. Garcerá

Oró Bozzini, Denise G. Fernández-Varo, V. Reichenbach, T. Yudina, E. Casals, G. Casals,B. González de la Presa, V. Puntes, W. Jiménez

Paez-Aviles, Cristina Esteve Juanola-Feliu, Josep Samitier

Raesch, Simon S. Stefan Tenzer, Wiebke Storck, Christian Ruge, Ulrich F. Schaefer, Claus-Michael Lehr

Ratera, Imma Witold I. Tatkiewicz, Joaquin Seras-Franzoso, Elena García-Fruitós, Esther Vazquez, Nora Ventosa, Antonio Villaverde and Jaume Veciana

Rubio Lorente, Laura Balasubramanyam Annangi, Jordi Bach, Gerard Vales, Laura Rubio, Ricardo Marcos, Alba Hernández

102

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

Authors Saenz del Burgo, Laura Jesús Ciriza, Gorka Orive, Rosa María Hernández, Jose Luis Pedraz

Country Poster title Spain

Graphene oxide application in cell microencapsulation for bioartificial organ development.

Spain

Tramadol Hydrochloride Released from Lipid Nanoparticles: Studies on Modelling Kinetics.

Spain

Polycationic Silicon Phthalocyanines as Photosensitizers for Photodynamic Therapy and Photodynamic Inactivation of Microorganisms.

Sánchez, Elena Helen Alvarado, Prapaporn Boonme, Guadalupe Abrego, Tatiana Andreani, Monica Vazzana, Joana F. Fangueiro, Catarina Faggio, Carla Silva, Sajan José, Antonello Santini, María Luisa Garcia, Ana C. Calpena, Amélia M. Silva, Eliana B. Souto

van de Winckel, Eveline Andrés de la Escosura, Tomás Torres

NanoBio&Med2014

november 18-21, 2014 - Barcelona (Spain)

103

Biomolecule-Quantum Dot systems for biological applications: Size-controlled aqueous synthesis of CdS Quantum Dots in homogeneous phase with BSA as capping ligand. María Jesús Almendral Parra1, Sara Sánchez Paradinas2, Víctor Barba Vicente1. 1

Departamento de Química Analítica, Nutrición y Bromatología. Facultad de Ciencias Químicas. Universidad de Salamanca. Plaza de la Merced, s/n. 37008, Salamanca. Telefon: +34 923294483. e-mail: almendral@usal.es. 2 Institut für Physikalische Chemie und Elektrochemie .Leibniz Universität Hannover. Schneiderberg 39.30167 Hannover. Raum: 2 07. Telefon: +49 511 762 16076. e-mail: sara.sanchez@pci.unihannover.de.

The literature contains few works reporting on the in situ generation of a reagent for the obtaining of nanocrystals with quantum characteristics. Yang and Xiang1 have described the aqueous synthesis of nanocrystals of CdS using CdSO4 and Na2S2O3 as precursors in the presence of thioglycerol as a dispersant. However, in that experimental work the authors failed to study the changes in size of the nanocrystals as a function of different conditions and, additionally, the dispersive behaviour of thioglycerol is small, since colloidal solutions of CdS are obtained and these have low stability. The same precursors and the same dispersant were used by Unni et al. for the synthesis of nanocrystals of CdS spiked with Zn2+ or Cu2+, with the above drawback of the low capacity of the dispersant to stabilize the solutions in which the nanocrystals are formed. Serum albumins have been used as a model protein for many and diverse biophysical, biochemical and physicochemical studies. Due to the high homology between bovine serum albumin (BSA) and Human Serum Albumin (HSA) it is possible to investigate systems aiming at future applications in medicine and biology. In this work, we reported the bioconjugation of CdS Quantum dots directly with bovine serum albumin (BSA) as capping ligand via an aqueous route. In an earlier work2 by our team, we performed the synthesis of CdS nanocrystals in aqueous medium starting out from the precursors CdCl2 and Na2S and using mercaptoacetic acid as the dispersant. We performed an in-depth study of several variables that affect size, surface state, fluorescence and stability of the aqueous solutions containing the CdS nanocrystals. In the present work we describe a similar set of experiments, but with a fundamental difference in that the S2- ion was generated in situ from the precursor thioacetamide CH3C(S)NH2, which was slowly hydrolyzed in basic aqueous solution. Cd(ClO4)2.6H2O was used as the precursor of Cd2+ and bovine serum albumin(BSA) was employed as capping ligand. We study the variables affecting the hydrolysis rate of CH3C(S)NH2 (pH, temperature). For these variables we studied the evolution of the size of the nanoparticles (NPs) with the time, the surface characteristics governing their fluorescence properties and their stability. We compared the above characteristics of the NPs of CdS obtained with both methods, deducing the advantages conferred by synthesis in homogeneous phase.

Y. J. Yang, J. W. Xiang. Template-free synthesis of CuS nanorods with a simple aqueous reaction at ambient conditions. Appl. Phys. A: Mater Sci. Proc. 81 (2005) 1351-1353 2 M. J. Almendral Parra, A. Alonso Mateos, S. Sánchez Paradinas, J. F. Boyero Benito, E. Rodríguez Fernández and J. J. Criado Talavera. Procedures for controlling the size, structure and optical properties of CdS Quantum Dots during synthesis in aqueous solution. Journal of Fliorescence 22 (2012) 59-69.

OPTIMIZATION AND CHARACTERIZATION OF POLY (LACTIC-CO-GLYCOLIC ACID) NANOPARTICLES LOADED WITH AN HIV-1 INHIBITOR SYNTHETIC PEPTIDE 1,2

Ariza-Sáenz M , Vega E , Espina M , GómaraM.J , Egea M.A 2

Haro I , Garcia, ML

1. Department of Physical Chemistry, Institute of Nanoscience and Nanotechnology, Faculty of Pharmacy, University of Barcelona, Avda Joan XXIII s/n 08028 Barcelona, Spain. 2. Unit of Synthesis and Biomedical Applications of Peptides, IQAC-CSIC, Jordi Girona 18-26 08034 Barcelona, Spain marisagarcia@ub.edu Introduction: It has been reported previously that patients coinfected with HIV and GB virus C (GBV-C) have a prolonged survival. Recently, it has been shown that the GBV-C envelope glycoproteins are enabling to interfere with HIV-1 fusion and entry (1). We report herein the synthesis of a peptide inhibitor of HIV-1 derived form the envelope E1 protein of GBV-C virus and the preparation, optimization and in vitro characterization of poly (lactic-co-glycolic acid) PLGANanoparticles (NPs) encapsulating this peptide sequence. Methods: The peptide was synthesized by the Fmoc solid phase peptide synthesis method (2), purified by High Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry (MALDI-Tof). Peptide-loaded PLGA NPs were prepared by a double emulsion/solvent evaporation technique (3). After selecting the factors that influenced physicochemical properties of the peptide-NPs, a three factors, five-level 3 central composite rotatable design 2 + star was applied to optimize the formulation. The factors selected were the volume of the inner aqueous phase, the concentration of PLGA and the concentration of peptide. The main interactions and effect of these independent variables were studied on particle size, polydispersity index (PDI), and entrapment efficiency (EE). The selected standard conditions were set as 0.8% (w/v) of PLGA; 0.025% (w/v) of peptide; 2.5% (w/v) of Poly (vinyl alcohol) PVA; 0.5 mL of inner aqueous phase; 30 seconds of primary emulsion and 90 seconds of secondary emulsion. The mean size (Z) and PDI were determined by photon correlation spectroscopy and the EE was assessed determining the non-entrapped peptide by HPLC. The destabilization process of the formulation selected was evaluated using a Turbiscan Lab Expert ®. Results: The experimental responses of a total of 16 formulations resulted in a mean size nanoparticle diameter ranging from 209 to 471 nm, with polydispersity index from 0.06 to 0.4 and EE of peptide values ranging from 13% to 84%. The formulation with 0.86 % (w/v) of PLGA; 0.025% (w/v) of peptide; 2.5% (w/v) of PVA; 0.5 mL of the inner aqueous phase, was found suitable for obtaining a high entrapment efficiency (84%) with an adequate average size of 262 nm and unimodal size distribution. The figure 1 shows the three-dimensional response surface of diagram corresponding to effects of peptide and polymer on the entrapment efficiency of peptide-NPs. As illustrated, the highest E.E was obtained with peptide concentration around of 0.025 %(w/v) and polymer concentration around 0.86 %(w/v), thus being these parameters considered relevant in the encapsulation process. Turbiscan data showed that the Peptide-NPs formulation selected has a good stability during more than 72 hours (Figure 2). Conclusion: The results obtained suggested WKDW WKH GRXEOH HPXOVLRQ VROYHQW HYDSRUDWLRQ LV D VXLWDEOH PHWKRG IRU SHSWLGHV¶ HQWUDSPHQW Besides, the factorial design is a valuable tool to provide screening trials useful to select an optimized formulation with a minimum number of experiments. References 1. Mohr, EL, Stapleton, JT. GB virus type C interactions with HIV: the role of envelope glycoproteins. J. Virol. Hepatol. 2009; 16: 757-768 2. Fmoc solid phase peptide synthesis. A practical approach. W.C. Chan and P.D. Whire Eds. Oxford University Press Inc. New York, 2000. 3. Zhang JX, Zhu KJ. An improvement of double emulsion technique for preparing bovine serum albumin-loaded PLGA microspheres. J Microencapsul. 2004; 21 (7):775-85.

Figure 1. Surface response diagram of entrapment efficiency.

(a)

(b)

Figure 2. Turbiscan profiles for a Peptide-NPs sample. (a). Transmission level (%) versus high cell (mm); (b) Backscattering (%) versus high cell (mm).

Nucleic acids for biosensing applications Anna Aviñó, César Sánchez*, Mar Oroval#, Laura Carrascosa*, Ramón Martínez-Máñez#, Laura Lechuga*, and Ramon Eritja Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26 08034 Barcelona, Spain # Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Unidad Mixta Universidad Politécnica de Valencia - Universidad de Valencia, Spain. *Nanobiosensors and Bioanalytical Applications Group ± CIBER-BBN and Research Center on Nanoscience and Nanotechnology (CIN2) CSIC, Spain aaagma@cid.csic.es Abstract DNA biosensors are small devices which intimately couple biological recognition element interacting with the target analyte with a physycal transducer that translates the biorecognition event into a useful electrical signal. Common transducing elements are optical, electrochemical or masssensitive. DNA biosensors, based on nucleic acid recognition processes, are being developed towards the goal of rapid, simple and inexpensive testing of genetic or infectious deseases. In one hand, we present a biosensing approach for the label-free detection of nucleic acid sequences with special emphasis on targeting RNA sequences with secondary structures or microRNAs that are involved in several deseases. The approach is based on selecting 8-aminopurine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. On the other hand, the design of stimuli-responsive nanoscopic gated systems involving biomolecules has recently attracted great attention. Capped materials have been mainly used in drug delivery applications. However in sensing are less common. Among different biomolecules that could act as caps, nucleic acids aptamers are especially attractive for the design of gated nanosensors for sensing applications. Specifically, we have prepared an aptamergated delivery system (S1-TBA) for the fluorogenic detection of thrombin. The sensing mechanism arises from the high affinity between an aptamer (TBA) and itV WDUJHW SURWHLQ Į-thrombin). References [1] Aviñó A.Frieden, M Morales J.C García de la Torre B. Ramón Güimil García1, Azorín F. Gelpí,J.L. Orozco,M González C. and Ramon Eritja, Nucleic Acids Res. 30 ( 2002), , 2609-2619 [2] Laura G. Carrascosa S. Gómez-Montes, A. Aviñó, A. Nadal, M. Pla, R. Eritja and L. M. Lechuga, Nucl. Acids Res. 2 (2012), 11. [3] Oroval, M. Climent, E Coll, C. Eritja R. Aviñó A. Marcos M.D. Sancenón F. Matínez-Máñez R. and Amorós P, Chem. Commun. 49 (2013), 5480 -582A

Figures

DETECTION OF RNA SEQUENCES USING PARALLEL TAIL CLAMPS AS BIORECEPTORS TO FORM STABLE TRIPLEX STRUCTURES IN A SPR BIOSENSOR

DETECTION OF THROMBIN USING AN APTAMER ±GATED DELIVERY SYSTEM

Silver nanoparticles downregulate p53 activation and induce desacetylation of histone 3 in human lung cancer epithelial cells Jordi Blanco, Daisy Lafuente, Domènec Sánchez, José Luis Domingo, Mercedes Gómez Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Universitat Rovira i Virgili, 43201 Reus, Spain. jordi.blanco@urv.cat

Abstract Nanomaterials have been widely used in recent years in aerospace engineering, nanoelectronics, environmental remediation, medical health care, and consumer products. Silver nanoparticles (AgNPs) are one of the most commonly used nanomaterials, because possess potent antibacterial and antifungal characteristics. AgNPs have been used extensively as an antimicrobial agent in cosmetics, textiles and the food industry, as well as a disinfectant for medical devices and for coating home appliance [1]. The emerging number of consumer products containing AgNPs and increasing enviromental concentration, have led to concerns, because nanoparticles may pose a risk for humans and the environment. The main ways by which people may be exposed to AgNP are by inhalation, dermal contact, and oral ingestion. The absorbed AgNPs can pass through the respiratory or gastrointestinal tracts and stored in many organs such as lung, liver, spleen, kidney and the central nervous system. There is growing evidence that AgNPs are highly toxic in terms of cytotoxicity, genotoxicity, and oxidative stress [3]. The present study evaluated the cytotoxic effects of AgNPs (20 nm of diameter coated with 0.3% of PVP) in A549 cells. A549 cells were exposed to 0, 25, 50, 100 and 200 µg/mL of AgNPs along 72 hours. AgNPs caused cell death in a dose- and time- dependent manner (Figure 1). Cell death induced at high doses was positively correlated with a down regulation of the expression and phosphorylation of p53 protein and acetylation of histone 3 (H3, Figure 2). Contrarily, the expression of total H3 protein was overexpressed at high doses. The desacetylation of H3 at high doses of AgNPs suggests that epigenetic changes could be happening into the chromatin. These sugestion are reinforced by the morphologic changes observed in A549 at high doses. In the same way, downregulation of the expression of p53 could be also due to a desacetylation of lysine residues which lead to its proteosomal degradation. The down regulation of p53 could lead to a deregulation of cell cycle and could induce arrest in S phase and thereby increase of the expression of histones proteins. The knowledge of the mechanisms by which AgNPs induce these changes could help to better understanding how nanoparticles could induce cancer cells death.

References [1] Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao AJ, Quigg A, Santschi PH, Sigg L: Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 2008, 17:372-386. [2] Chen X, Schluesener HJ: Nanosilver: a nanoproduct in medical application. ToxicolLett 2008, 176:112. [3] Kim S, Choi JE, Choi J, Chung KH, Park K, Yi J, Ryu DY: Oxidative stressdependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol In Vitro 2009, 23:1076-1084.

Figures

Figure 1.

Figure2.

Magnetoliposomes for magnetic resonance imaging 1,3

1,3

Joan Estelrich , Josep Queralt , Montserrat Gallardo , M. Antònia Busquets 2 1 Departament de Fisicoquímica. Departament de Fisiologia. Facultat de Farmàcia. UB. Avda Joan XXIII, s/n 08028 Barcelona 3 Institut de Nanociència i Nanotecnologia (IN2UB) joanestelrich@ub.edu Abstract The development of superparamagnetic iron oxide nanoparticles (SPIONS) has opened a new and attractive approach towards the molecular imaging of living subjects because they overcome not only many of the current limitations in diagnosis but also in the treatment and management of human diseases [1]. The combination of imaging modalities based on the use of SPIONS such as magnetic resonance imaging (MRI), optical imaging (OI) or positron emission tomography (CT) have been developed to visualize pathological situations. Among these techniques, MRI is one of the most powerful, and non-invasive modality of diagnosis due to its high soft tissue contrast, spatial resolution, and penetration depth. MRI images result from the spatial identification of hydrogen nuclei. The contrast in the images comes from local differences in spin relaxation kinetics along the longitudinal T1 (spinlattice) and transverse T2 (spin-spin) relaxation times [2]. Contrast agents alter the signal intensity by selectively shortening the hydrogen relaxation times of the tissues and are used to improve the sensitivity and specificity of MRI. In particular, SPIONS have emerged as T2 contrast agents because their enhancement of the negative contrast, thus showing darker images of the regions of interest. This contrast is strongly related to the SPIONS coating being the most widely used dextran and its derivatives. However, these coatings have raised several controversies mainly because of their weak physisorption on the nanoparticle surface and toxicology associated to the coating [3]. To overcome these drawbacks, the SPIONS can be incorporated into lipid vesicles thus obtaining magnetoliposomes (MLs). MLs have special interest because of their low cytotoxicity, enhanced versatility and target biodistribution. To gain understanding on the magnetic relaxation processes involved in contrast generation by MLs, we seek an analysis of the impact that coating has on the relaxivity of MLs. With this purpose, we have prepared magnetite coated with polyethylene glycol (PEG) [4] and then, the resulting ferrofluid has been encapsulated into liposomes of different lipid composition in order to analyze the influence of size, physical state of the phospholipid bilayer and medium of dispersion of MLs in the MRI parameters [5]. Thus, dimyristoyl phosphatidylcholine (DMPC), dipalmytoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC) and dioleoyl phosphatidylcholine (DOPC) were chosen to study the effect of chain length and membrane physical state on T1 and T2 relaxations. In addition, ferrofluid was incorporated into DMPC:Cholesterol (CHOL) at different molar rations. Finally, the influence of size on MRI relaxivities was analyzed with non-extruded (multilamellar liposomes, MLVs) and extruded MLs (LUVs). Samples were prepared at iron concentrations ranging from 0.2 to 1.2 mM in agar (5%) fantoms as to mimic human tissues, in 8 x 6 cm wells plate (Figure 1). MRI experiments were conducted on a 7.0 T BioSpec 70/30 horizontal animal scanner (Bruker BioSpin, Ettlingen, Germany), equipped with a 12 cm inner diameter actively shielded gradient system (400 mT/m) and a receiver/transceiver coil covering the whole mouse volume. The sample was placed in a Plexiglas holder. Tripilot scans were used for accurate positioning of the sample in the isocenter of the magnet. T1 relaxometry maps were acquired by using RARE (rapid acquisition with rapid enhancement) sequence applying 9 repetition times = 354.172, 354.172, 500, 700, 1000, 1400, 2000, 3000 and 6000 ms, echo time = 10 ms, RARE factor = 1 average, number of slices = 6 for vertical view, field of view = 69.9 x 60 mm, matrix size = 128 x 128 pixels, resulting in a spatial resolution of 0.546 × 0.469 mm in 1 mm slice thickness. For the T2 relaxometry maps, MSME (multi-slice multi-echo) sequence was used with a repetition time = 4764.346 ms with 16 echo times corresponding to 14.11, 28.21, 42.32, 56.43, 70.54, 84.64, 98.75, 112.86, 126.97, 141.07, 155.18, 169.29, 183.4, 197.5, 211.61 and 225.72 ms, 1 average, number of slices = 6 for vertical view, field of view = 69.9 x 60 mm, matrix size = 448 x 384 pixels, resulting in a spatial resolution of 0.156 x 0.156 mm in 1 mm slice thickness. Data were processed using the Paravision 5.1 software (Bruker, BioSpin, Ettlingen, Germany). In contrast to the T2 relaxation, T1 relaxation depends on a fast proton exchange between the bulk water phase with slow T1 relaxation, and protons at the surface of magnetic particle aggregates where T1 relaxation is fast. Since residence time of protons is roughly proportional to the square of the particle size, this can explain the lower values of the MLs compared with the values described in the literature [6].

DMPC DMPC DMPC DMPC DMPC DMPC [Fe] [Fe] [Fe] [Fe] [Fe] [Fe] 0,20 mM 0,20mM 0,14 mM 0,14 mM 0,08 mM 0,08 mM DPPC DPPC DPPC DPPC DPPC DPPC [Fe] [Fe] [Fe] [Fe] [Fe] [Fe] 0,20mM 0,20mM 0,14 mM 0,14 mM 0,08 mM 0,08 mM DSPC DSPC DSPC DSPC DSPC DSPC [Fe] [Fe] [Fe] [Fe] [Fe] [Fe] 0,20mM 0,20mM 0,14 mM 0,14 mM 0,08 mM 0,08 mM DOPC DOPC DOPC DOPC DOPC DOPC [Fe] [Fe] [Fe] [Fe] [Fe] [Fe] 0,08 mM 0,08 mM 0,14 mM 0,14 mM 0,20mM 0,20mM DMPC/chol DMPC/chol DMPC/chol DMPC/chol DMPC/chol DMPC/chol 95:5 95:5 90:10 90:10 80:20 80:20 [Fe] [Fe] [Fe] [Fe] [Fe] [Fe] 0,08 mM 0,08 mM 0,08 mM 0,08 mM 0,08 mM 0,08 mM

Figure 1. Top: MRI Maps of T1 (left) and T2 (right). Below: samples of the above maps. MLVs. multilamellar magnetoliposomes with the corresponding mM iron concentration ([Fe]). DMPC: dimyristoyl phosphatidylcholine; DPPC: dipalmytoyl phosphatidylcholine; DSPC: distearoyl phosphatidylcholine, DOPC: dioleoyl phosphatidylcholine and CHOL: cholesterol. Control samples of ferrofluid (FF) were also analyzed as control (Figure not shown). 3.0

0.200 0.175 0.150

2.6

T2/ s

T1/ s

2.8

2.4

0.125 0.100 0.075 0.050

2.2

0.025 2.0 0.00

0.02

0.04

0.06

[Fe]/mM

0.08

0.10

0.000 0.00

0.02

0.04

0.06

0.08

0.10

[Fe]/mM

Figure 2. T1 and T2 relaxation times of ferrofluid (blue), large unilamellar (red) and multilamellar (black) magnetoliposomes of DMPC Acknowledgements. The authors are grateful for the financial support given by the Spanish Ministerio de Economía y Competitividad (MINECO) to the project MAT2012-36270-C04-03. References [1] C. Corrot, P. Robert, JM Idee, M. Port, Adv. Drug Deliv. Rev. 58 (2006) 1471-1504. [2] H. Fattahi, S. Laurent, F. Liu, N. Arsalani, L. Vander Elst, R.N. Muller, Nanomedicine 6 (2011) 529544. [3] S. Mornet, J. Portier, E. Doguet. J. Magn. Magn. Matter 293 (205) 127-134. [4] S.García-Jimeno, J. Estelrich, Colloids Surf. A, 420 (2013) 74-81. [5] R. Sabaté, R. Barnadas-Rodríguez, J. Callejas-Fernández, R. Hidalgo-Alvarez, J. Estelrich, Int. J. Pharm. 347 (2008) 156-162. [6] M. Hoedenius, C. Würth, J. Jayapaul, J.E. Wong, T. Lammers, J. Gätjens, S. Arns, N. Mertens, I. Slabu, G. Ivanova, J. Bornemann, M. De Cuyper, U. Resch-Genger, F. Kiessling. Contrast Media Mol. Imaging 7 (2011) 59-67.

In vitro and in vivo evaluation of TiO2 oral absorption Joan Cabellos, Gemma Janer, Ezequiel Mas del Molino, Elisabet Fernández-Rosas, Socorro VásquezCampos LEITAT, Innovació 2, Terrassa, SPAIN jcabellos@leitat.org Abstract The Caco-2 monolayer permeation test is a widely used in vitro test to predict oral absorption of organic compounds, particularly by the pharmaceutical industry (Artursson et al. 2001). A few reports also exist on the use of this test system for NMs (e.g., Al-Jubory and Handy 2012; Antunes et al. 2013; Jin et al. 2013). But the predictive value of the data obtained in such test is still unclear. The main processes that determine uptake for chemicals are diffusion and active transport through membrane transporters (Grassi 2007), and the Caco-2 monolayer permeation test is a good model for both processes. However, these are not the main processes governing the oral uptake of particles. Three main pathways for absorption of particles across intestinal barriers have been described. First, paracellular transport can take place for small molecules that can pass the tight junctions and the pore-diameter which is reported to be around 0.6–5 nm (Ruenraroengsak et al. 2010). Second, transcytosis may appear across enterocytes, but mostly across M-cells located in the Peyer’s patches (Powell et al. 2010). An third, particles (nano and micro sized) can be transported across degrading enterocytes (Hillyer and Albrecht 2001; Volkheimer 1993). The three mechanisms that have been described for NMs oral absorption could theoretically occur in the Caco-2 model, particularly if this could be modified to incorporate M-cells. In the present report, we have used a single type of TiO2 NPs (spherical, 18 ± 8 nm diameter, surface area was 89.8 m2/g) in different in vitro and in vivo systems in order to better understand how results from these studies can be extrapolated to other cell types or levels of organization. In particular, we describe the results of five studies: an in vitro cell uptake using A549 cells, an in vitro permeation test using differentiated Caco-2 cells, an in vitro permeation test using a coculture of differentiated Caco-2 cells and M-cells, and two in vivo oral absorption tests in rats (with and without fasting conditions prior to administration). To overcome the analytical challenges associated to the tracking of unlabelled TiO2 NPs, both inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) were used. TiO2 NPs were present in form of agglomerates of different sizes within cytoplasmic vesicles of A549 cells after 72 hours exposure. In none of the cells evaluated, particles were found freely in the cytoplasm or in the nucleus. This efficient cell internalization and the fact that NPs were found as aggregates in cytoplasmic vesicles is consistent with previous reports in a variety of human cell lines. A very small proportion of the NPs was able to cross the differentiated Caco-2 cell membrane (below or close to the detection limit, i.e., 0.1 ppm or 0.4% of the applied concentration). In an attempt to increase the biological relevance of this permeation model, we introduced Raji cells to induce the differentiation of Caco-2 cells into M-cells. The Caco-2/M-cell coculture model was established and characterized through measuring TEER values during the differentiation process, and performing morphological (histological sections and scanning electron microscopy; Figure 1) and immunostaining (ZO1 and ocludin tight junction proteins). All data confirmed that a proportion of Caco-2 cells consistently differentiated into M-cells. In such system, a very low permeation rate for TiO2 NP was recorded, although qualitatively higher (higher frequency of values above the detection limit) than that for Caco-2 cell monocultures. The readily internalization in A549 cells and in most cell lines contrasts with an extremely low absorption by Caco-2 cells in our study and the low uptake by these cells in the study by Fisichella et al. (2012). The fact that we used the same TiO2 NPs for in vitro studies with both A549 and Caco-2 cells indicate that these differences are related to the cell line properties and not to the TiO2 NPs used. The apparent contradiction is probably related to the cell membrane morphology of the latter. Differentiated Caco-2 cells are polarised cells with microvilli in their apical side (where exposure takes place). To evaluate in vivo absorption Sprague-Dawley male rats were administered the vehicle or the TiO2 NPs by oral gavage. Two independent experiments were performed, one in which administration of the NPs was performed in fasting conditions and one without food restrictions. Administered rats were weighted and observed to assess clinical signs of toxicity on the day of administration and on the following day, before sacrifice. At termination, spleen, liver, small and large intestines, and mesenteric lymph nodes were removed. The intestines were carefully washed with phosphate buffer to remove their

content. Peyer’s patches were excised and separated from the rest of the small intestine. The caecum was separated from the rest of the large intestine. One of the Peyer’s patches and a piece of smooth small intestine were immediately preserved in buffered glutaraldehyde-paraformaldehyde for later TEM analyses. The remaining samples were kept at -20°C until acid digestion and analysis by ICP-MS. There was no detectable increase in titanium levels in any of the tissues evaluated 24 hours after the administration of 100 mg/kg TiO2 NPs, regardless of the food restriction conditions (only some tissues under fasting conditions: Peyer’s patches, smooth small intestine, and mesenteric lymph nodes). Smooth sections and Peyer’s patch sections of the small intestine of the animals that received TiO2 NPs without food restrictions were examined by TEM. No TiO2 NPs were observed in the smooth sections. In contrast, we did observe at least one cell containing considerable amounts of TiO2 NPs aggregates in a Peyer’s patch section (Figure 2). In this cell, the TiO2 NPs were not surrounded by membranes, but they were freely distributed in the cytoplasm. We did not observe NPs inside mitochondria or the nucleus. The low bioavailability of TiO2 NPs in this report contrasts with the relatively high oral bioavailability study reported by Jani et al. (1994). We had hypothesized that these differences could be due to the fact that Jani et al. (1994) administered the particles after several hours of fasting (Janer et al., 2014), but the experiment that we conducted in fasting conditions does not support such hypothesis. In summary, we showed that A549 cells readily uptake the TiO2 NPs used in this study. The results were consistent with most literature reports for TiO2 NPs and other type of NMs, suggesting a limited modulating effect of the physicochemical properties of NMs on cell uptake. However, such rapid uptake contrasted with a very low oral absorption in the in vitro and in vivo studies that we performed. The results of this study support that M-cells play an important role in the absorption of nanoparticles, and suggest that the Caco-2/M-cell coculture model is a more relevant model for the prediction of oral absorption of nanoparticles than the Caco-2 monoculture model. References Al-Jubory AR, Handy RD, Nanotoxicology, 7 (2013) 1282-301. Antunes F, Andrade F, Araujo F, Ferreira D, Sarmento B, Eur J Pharm Biopharm 83 (2013) 427-35. Artursson P, Palm K, Luthman K, Adv Drug Deliv Rev 46 (2001) 27-43. Grassi M GG, Lapasin R, Colombo I, A Physical And Mathematical Approach. CRC Press. (2007). Powell JJ, Faria N, Thomas-McKay E, Pele LC, J Autoimmun 34 (2010) J226-33. Jin X, Zhang ZH, Li SL, Sun E, Tan XB, Song J, Jia XB, Fitoterapia 84 (2013) 64-71. Ruenraroengsak P, Cook JM, Florence AT, J Control Release 141 (2010) 265-76. Hillyer JF, Albrecht RM, J Pharm Sci 90 (2001) 1927-36. Volkheimer G. Pathologe 14 (1993) 247-52. Fisichella M, Berenguer F, Steinmetz G, Auffan M, Rose J, Prat O, Part Fibre Toxicol 9 (2012) 18. Jani PU, McCarthy DE, Florence AT, Int. J. Pharmaceutics 105 (1994) 157-168. Janer G, Mas del Molino E, Fernández-Rosas E, Fernández A, Vázquez-Campos S. Toxicol Lett. 228 (2014) 103-10. Figures

2 Pm Figure 1. SEM images of Caco-2/Raji cocultures. Caco-2 cells show dense microvilli and contrast with larger M-cells with only rudimentary microvilli (M).

Figure 2. TEM micrograph showing the presence of TiO2 nanoparticles in a cell from a Peyer’s patch section. The arrows point to some of the NP aggregates.

Synthesis of modified dendrimers and conjugation with selected apoptosis inhibitors Miriam Corredor1, Ignacio Alfonso1, Dietmar Appelhans2, Angel Messeguer1 1

Dep. Chemical Biology and Molecular Modelling, IQAC-CSIC C/ Jordi-Girona, 18-26 08034 Barcelona (Spain) 2 Dep. Bioactive and Responsive Polymers, Leibniz-Institut für Polymerforschung Dresden Hohe Straȕe, 6 D-01069 Dresden (Germany) miriam.corredor@iqac.csic.es Apoptosis is a biological process relevant to different human diseases stated that is regulated through protein-protein interactions and complex formation.[1] One point of regulation is the formation of a multiprotein complex known as apoptosome.[2] In our group, it has been previously reported a peptidomimetic compound bearing a 3-substituted-piperazine-2,5-dione moiety and a seven-membered ring moiety as potent apoptotic inhibitors.[3] We reduced the conformational freedom of the exocyclic tertiary amide bond of the diketopiperazine by an isosteric substitution of a triazole. For one of the proposed structures a ȕ-lactam compound was isolated, that resulted to be the most potent inhibitor.[4] At this point, we wanted to conjugate our potential drugs with a polymer that could offer a more specific intracellular transport and release to reach the molecular target. Dendritic polymers are widely used as multifunctional materials with specific properties for potential biomedical and pharmaceutical applications. These multifunctional macromolecules have been used as carrier systems of drugs in the study of bio-interaction against different bio-active molecules and systems.[5] The most important drawback of these types of dendrimers is their toxicity due to the positive charge on their surface. Thus, a high generation of poly(propylene imine) dendrimers with densely organized oligosaccharide shells in which each peripheral amino group is modified by two chemically coupled oligosaccharide units has been reported.[6] This attachment resulted in much lower cytotoxicity towards different cell lines.[7] In this work, 5th generation PPI dendrimers modified with maltose units were synthesized and coupled with previously mentioned modified small molecules which have shown activity as potential apoptosis inhibitors. NH2 NH 2 NH2

H2N 2 H2N H2N NH2 NHNH 2 NH 2 H2 N H2 N NH2 H2 N NH2 N H2 N NH2 N N N N NH2 H2 N HN N H2N 2 NH2 N N N N H2N N NH2 N H2N N N N NH2 N N H2N N NH2 N N N NH2 HH22NN N N N N N N NH2 N H2N N NH2 H2N N N N N NH2 H2 N N NH2 H 2N N N N N N N N NH2 H2N N NH 2 N N H2N N N NH2 N H2N N N N NH 2 N N H2 N NH2 N N N N H2 N NH 2 N NHNH 2 2 H2N N N N N H2N NH N 2 H2NH N NH 2 NH2 NH2 2 H 2N NHNH 2 2 H2N NH2 NH2 NH

G5-PPI

NH2

NH2 NH2

NHS/ EDC

TEA / DMSO

NH2

Figure 1: Coupling of a small molecule with a dense-shell glycodendrimer bearing an amino-terminal group.

References [1] Mondragón, L.; Orzáez, M.; Sanclimens, G.; Moure A.; Armiñán, A.; Sepúlveda, P.; Messeguer, A.; Vicent, M. J.; Pérez-Payá. J. Med. Chem. 51 (2008) 521-529. [2] Martin, A. G.; Nguyen, J.; Wells, J. A.; Fearnhead, H. O. Biochem. Biophys. Res. Comm. 319 (2004) 944-950. [3] Moure, A.; Sanclimens, G.; Bujons, J.; Masip, I.; Alvarez-Larena, A.; Pérez-Payá, E.; Alfonso, I.; Messeguer, A. Chem. Eur. J. 17 (2011) 7927-7939. [4] Corredor, M.; Bujons, J.; Orzáez, M.; Sancho, M.; Pérez-Payá, E.; Alfonso, I.; Messeguer, A. Eur. J. Med. Chem. 63 (2013) 892-896. [5] Boas, U.; Heegaard, P.M; Chem. Soc. Rev. 33 (2004) 43-63. [6] Klajnert, B.; Appelhans, D.; Komber, H.; Morgner, N.; Schwarz, S.; Richter, S.; Brutschy, B.; Ionov, M.; Tonkikh, A. D.; Bryszewska, M.; Voit, B. Chem. Eur. J. 14 (2008) 7030-7041. [7] Arima, H.; Chihara, Y.; Arizono, M.; Yamashita, S.; Wada, K.; Hirayama, F.; Uekama, K. J. Control Release 116 (2006) 64-74.

Monitoring the intracellular dynamics of polystyrene nanoparticles in lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking i, ii

i, ii

Sarah Deville , Rozhin Penjweini , Nick Smisdom , Kristof Notelaers , Inge Nelissen , Jef ii, iii i Hooyberghs , Marcel Ameloot i Biomedical Research Unit, Hasselt University, Diepenbeek, Belgium. ii Flemish Institute For Technological Research (VITO), Mol, Belgium iii Theoretical Physics, Hasselt University, Diepenbeek, Belgium sarah.deville@vito.be Abstract Interactions of nanoparticles (NPs) with living cells and resulting biological responses are largely dependent on NP uptake processes, intracellular transport and their complex behaviours. In order to demonstrate the applicability of image (cross-) correlation spectroscopy based techniques for monitoring the intracellular dynamics of NPs, 100 nm fluorescently stained carboxylated polystyrene (PS) NPs were used to expose in vitro cultured human lung epithelial A549 cells, thus mimicking NP exposure in the lungs. Transport direction, transport velocity and diffusion of PS NPs were determined to acquire more insights in the intracellular transport following NP uptake. To complement these ensemble average techniques, PS NP motions were also analysed by single particle tracking. Hereby, distinct clusters are registered and tracked frame by frame allowing access to individual PS NP dynamics. Potential dynamic interactions of NPs with the nucleus, mitochondria, early endosomes, late endosomes and lysosomes were also explored. PS NPs directed motions were shown to be highly dependent on the microtubule-assisted transport and were strongly associated with the endolysosomal compartment. Image (cross-) correlation analyses were shown to be a powerful tool for determining the kinetic behaviour of NPs inside the cell. References [1] Penjweini, R.; Smisdom, N.; Deville, S.; Ameloot, M., Biochimica et Biophysica Acta (BBA) Molecular Cell Research, 1843 (5) (2014), 855-865.

Interaction of targeted of magnetoliposomes with Hela epithelial carcinoma and 3T3 fibroblasts cell lines. 1,3

2,3

1,3

Joan Estelrich , M. Carmen Moran , M. Antònia Busquets 2 Departament de Fisicoquímica. Departament de Fisiologia. Facultat de Farmàcia. UB. Avda Joan XXIII, s/n 08028 Barcelona 3 Institut de Nanociència i Nanotecnologia (IN2UB) joanestelrich@ub.edu

Abstract

309

108

231

Counts

In the last years, the development of iron oxide magnetic nanoparticles (IONPs) has significantly increased with respect to other nanosized particles because of their attractive properties as theranostic agents [1]. These systems combine both, therapeutic and diagnostic properties. In order to improve their versatility and biodisponibility, IONPs can be incorporated into liposomes, resulting in a new kind of nanoscale system, known as magnetoliposomes (MLs) [2]. MLs, which are biodegradable and highly versatile especially in composition, have opened great expectations for the development of personalized medicine [3]. Any biomedical use of MLs entails thorough understanding of their toxicology, establishment of principles and test procedures to ensure safe manufacture and usage, and comprehensive information about their safety and potential hazard [4]. In this way, we have designed MLs appropriate for theranostic applications. However, previously to any biomedical application, the lack of inherent toxicity must be checked. To this end, the following study has been performed according to the following steps: i) synthesis of IONPs; ii) incorporation of IONPs into liposomes of different lipid composition and, iii) analysis of the cytotoxicity and internalization of MLs in cell models. IONPs coated with polyethylene-glycol (PEG) were synthesized by the coprecipitation method according to the procedure described elsewhere [5]. As far as the lipid composition is concerned, three different lipid mixtures have been prepared, namely, a) bare liposomes: dimyristoylphosphatidylcholine (DMPC)/cholesterol (CHOL): 8:2; b) bare liposomes with PEG (DMPC/CHOL/PEG: 8:2:0.3) and; c) functionalized MLs or bare liposomes with the cyclic RGD peptide (DMPC/CHOL/PEG/RGDc: 8:2:0.3:0.03). For internalization studies, MLs were decorated with the fluorescent label 0.05% Rhodamine-B. The model cells chosen for the study were 3T3 fibroblasts and Hela epithelial carcinoma cell lines. Both cells are rich in integrin membrane proteins but they are different concerning which kind of ligand is recognized. In this way, 3T3 is rich in collagen-receptor integrins, whereas HeLa in RGD-receptor integrins. Therefore, the rationale of MLs compositions was the selective targeting of the functionalized MLs towards HeLa cells. Thus, bare and PEG-MLs are considered control MLs with no affinity for the above mentioned cells. Results obtained by confocal microscopy and flow cytometry were concordant with the possibility of the formation of the so called protein corona around the MLs [6]. Potser referenciar les figures 1 I 2 al text.

154

0 100

101

102 FL 4 Log

103

104

0 100

101

102 FL 4 Log

103

Figure 1. Flow cytometry of control (black); bare MLs of DMPC/CHOL/Rho-PE (80:20:0.05) (red) and, functionalized MLs DMPC/CHOL/PEG/RGDc/Rho-PE (80:20:3: 0.3:0.05) incubated 4h with 3T3 cells (left) or Hela cells (right).

104

Figure 2. Laser confocal microscopy images of bare (left) and functionalized (right) MLs upon incubation for 4h with 3T3 cells (top) and Hela cells (bottom). Membrane cell was labeled with Alexa; the nucleus with DAPI and magnetoliposomes with Rhodamine B. References [1] D. Ho, X.L. Sun, S.H. Sun, Accounts of Chemical Research 44 (2011) 875-882. [2] N. Crawley, M. Thompson, Analytical Chemistry 86 (1) (2014) 130-160 [3] H. Fattahi, S. Laurent, F. Liu, N. Arsalani, L. Vander Elst, R.N. Muller, Nanomedicine 6 (2011) 529544. [4] Arora, S.; Rajwade, J.M., Paknikar, K.M. Toxicol. Applied Pharmacol. 258 (2012) 151-165 [5] S.GarcĂa-Jimeno, J. Estelrich, Colloids Surf. A, 420 (2013) 74-81. [6] M.P. Monopoli, Aberg, C. A. Salvati, K. Dawson, Nature Nanotech. 7 (2012) 779-786 Acknowledgements. MAB and JE are grateful for the financial support to the project MAT2012-36270C04-03 and, MCM to the project MAT2012-38047-C02-01 given by the Spanish Ministerio de EconomĂa y Competitividad (MINECO). MCM acknowledges the support of the MICINN (Ramon y Cajal contract RyC 2009-04683).

Thermal stability of a cationic solid lipid nanoparticle (cSLN) formulation as a possible biocompatibility indicator Anna Fàbregas, Montserrat Miñarro, Josep Ramon Ticó, Encarna García-Montoya, Pilar PérezLozano, Josep Mª Suñé-Negre Drug Development Service (SDM). Dept. Pharmacy and Pharmaceutical Technology. Faculty of Pharmacy (Universitat de Barcelona), Avda. Joan XXIII, s/n 08028, Barcelona, Spain afabregas@ub.edu

Abstract Cationic solid lipid nanoparticles have become a non-viral delivery system for nucleic acid transfection and further genomic regulation and delivery of encapsulated drugs [1]. A formulation of cationic solid lipid nanoparticles intended for gene delivery [2] has been analyzed in terms of thermal stability at different temperatures. The aim is to determine in a short-term study the influence of temperature on particle size and surface potential, in order to assess what is the best temperature that contributes to maintain cSLN without or low aggregation and proper surface potential [3]. Short-term thermal storage study can serve as well for an approach to behavior at physiological temperature when the study is carried out at 37 ºC. The cSLN formulation consists of stearic acid, octadecylamine and surfactant Poloxamer 188 [2]. Thermal behaviour is studied at 4 ºC, 25 ºC and 37 ºC. The cSLN are synthesized using the hot microemulsification method [4]. Then, the nanoparticles are distributed in vials and stored at the temperatures mentioned above. The particle size determinations are carried out in a Mastersizer 2000 laser diffractometre (Malvern Instruments, UK) and Z potential values are determined on a Zetasizer Nano-Z (Malvern Instruments, UK). Both measures are performed daily during a week. The results are represented graphically (figure 1), and show the evolution of this formulation at the different temperatures in terms of particle size (given as surface weighted mean in nm) and surface charge (given as Z potential in mV). Mean value and standard deviation (table 1) show that at 37 ºC, these nanoparticles suffer the lowest variation both in particle size and Z potential. Thus, cSLN formulation presents a thermal behavior which results in a stable state at 37 °C in comparison to 25 ºC and 4 ºC, with particle size and Z potential showing slightly changes, then indicating that at this temperature the formulation is still able during a week for acid nucleic binding. Additionally, while 37 ºC corresponds to physiological temperature at which cSLN would be administered, it may be taken into consideration as a possible indicator of biocompatibility, although the influence of other variables such as thermal behavior after nucleic acid binding should be taken into account in further studies. It can be concluded that regarding low tendency to aggregation or modification of surface potential in the first days after its synthesis when stored at 37 ºC, these cSLN may represent a proper non-viral delivery system following nucleic acid binding intended for immediate and short-term administration.

References [1] Ekambaran P et al., Scientific & Chemical Communications, 2 2012 80-102. [2] Fàbregas et al., International Journal of Pharmaceutics, 1-2 2014 270-279. [3] Vauthier C et al., European Journal of Pharmaceutics and Biopharmaceutics, 2 2008 466-475. [4] Mehnert W et al. Advanced Drug Delivery Reviews, 64 2012 83-101.

Figure 1. Graphical representation of changes on particle size and surface potential as a function of time. Z Potential (mV)

Surface weighted mean (nm) Days

4 ºC

25 ºC

37 ºC

Days

4 ºC

25 ºC

37 ºC

269

217

236

27.7

35.8

32.5

124

339

119

29.9

35.6

30.1

27.1

24.0

31.6

126

55194

113

237

40709

132

34.9

39.2

34.2

206

126742

121

38.6

37.0

35.2

65070

58070

122

34.2

43.7

40.2

86870

75887

282

29.9

31.6

27.3

Mean

21843

51023

161

Mean

31.7

35.3

33.0

37507

44112

4.2

6.2

4.1

Table 1. Values of particle size and surface potential at different temperatures during 7 days.

PLGA nanoparticles as advanced imaging nanosystems N. Feiner-Gracia, C. Fornaguera, A. Dols-Perez, M.J. García-Celma, C.Solans 1

QCI group, Institut de Química Avançada de Catalunya (IQAC-CSIC), C/ Jordi Girona 18-26, 08034, Barcelona, Spain 2 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) 3 Departament de Farmàcia i Tecnologia Farmacèutica, Universitat de Barcelona (UB), Av/ Joan XXIII S/N, 08028, Barcelona, Spain

natalia.feiner@iqac.csic.es Abstract Introduction Polymeric nanoparticles (NP) are of increasing interest in the biomedical field. They represent a promising strategy for in vivo diagnosis as medical imaging nanosystems [1]. Due to the possibility of functionalizing nanoparticle surface, these systems can be vectorized to the tissue of interest. In addition, if they include a fluorescent dye nanoparticle tracking can be monitored. Biocompatible, biodegradable and safety materials are required for the preparation of nanoparticles intended for biomedical applications. Therefore, poly(lactic-co-glycolic acid) (PLGA) polymer is appropriate to prepare polymeric nanoparticles using nano-emulsion templating, which is a simple, well-known and versatile method. Nano-emulsions are colloidal systems with droplet size in the range of 20-200 nm. The phase inversion composition method (PIC), a low energy emulsification method, is a suitable methodology to prepare nanoemulsions for pharmaceutical applications as the process can be performed at mild temperature [2]. Following, polymeric nanoparticles can be easily obtained from polymeric O/W nanoemulsions by solvent evaporation, if the oil component (internal phase) of nano-emulsions consist in a preformed polymer dissolved in a volatile organic solvent. The fluorescent dye can be solubilized in the oil phase prior to nano-emulsion formation to enhance high loading efficiency. Objectives The aim of this work was to obtain biomedical imaging systems appropriate for intravenous administration. Results O/W polymeric nano-emulsions were prepared in a system PBS/ polysorbate80 surfactant/ [4% PLGA and 0.1% fluorescent dye in an organic solvent]. The organic solvent consisted in ethyl acetate or 80/20 ethyl acetate/ethanol. The fluorescent dyes selected were Coumarin 6 (C6) and Rhodamine 6G due to their non-toxic character, appropriate to be used in the biomedical field and also due to their solubility characteristics in the oil phase of the selected system. Nanoemulsions were prepared by the PIC method, at 25ºC. Nanoparticles (Figure 1) were obtained from nano-emulsion templating. Both (nano-emulsions and nanoparticles) were characterized using Zeta Potential (surface charge), dynamic light scattering (DLS, hydrodynamic size) and visual aspect (stability). Nanoparticles and their template nano-emulsions showed hydrodynamic radii below 100 nm and negative surface charges. Nanoparticles sizes were lower than those of their template nano-emulsions. The stability of the nanoparticles allows their use as medical imaging systems. Moreover, the encapsulation efficiency achieved was nearly complete, for both fluorescent dyes, attributed to the nanoparticle preparation method. The fluorescent release was studied for nanoparticle dispersions and for an aqueous and a micellar solutions, for comparative purposes. The Rhodamine 6G release from nanoparticles was slower

than that from the aqueous solution (Figure 2), which is of great interest due to the fact that nanoparticles will reach the target tissue before the fluorescent dye begins to be released.

Conclusion The formulated polymeric nanoparticles are promising as fluorescent delivery systems for biomedical applications.

References [1] S. Mura, P. Couvrer, Advanced Drug Delivery Reviews, 64 (2012) 1394-1416 [2] G. Calder贸, MJ Garc铆a-Celma, C. Solans, J of Colloid and Interface Science, 535 (2) (2010) 406-411

Figures

Figure 1. Visual appearance of nanoparticle dispersions. From left to right: free-NP, C6-NP, Rho 6G-NP

1.2

% released

1 0.8 0.6 0.4

Nanoparticle

0.2

Micellar solution Aqueous solution

0 0

100

150 Time (h)

200

250

300

Figure 2. Release of Rhodamine 6G as a function of time for nanoparticles, micellar solution and aqueous solution

Continuous synthesis of silver nanoparticles using green chemicals and microreactors and its evaluation as bactericidal agents 3

1,3

1,2

Santiago Ibarlín , Esteban Gioria , Antonella Giorello , José Luis Hueso , Victor Sebastián , 1,2 3 1,2 Manuel Arruebo , Laura Gutierrez and Jesús Santamaría 1

Instituto de Nanociencia de Aragón (INA), University of Zaragoza, Spain 2Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain 3 Instituto de Investigaciones en Catálisis y Petroquímica (INCAPE), UNL – CONICET, Santiago del Estero 2829, Santa Fe, 3000, Argentina antonellagiorello@gmail.com Silver nanoparticles have been extensively studied in medicine and microbiology mainly because of their bactericidal properties [1]. There are many methods for the synthesis of these nanoparticles but only a few of them are really reproducible and use protocols and reactants that are environmentally friendly. In this work, the synthesis of silver nanoparticles using green reactants is described. Moreover, a continuous production method is proposed based on the use of microreactors [2]. Green chemicals such as glucose and starch are used as reducer and stabilizing agents, respectively. Different synthesis parameters such as reactant ratios and temperatures are thoughtfully evaluated and optimized to maximize the production of silver nanoparticles. The synthetized materials are fully characterized by TEM, UV.Vis and XPS. Likewise, the bactericidal activity of selected nanoparticles has been evaluated against Escherichia coli. References [1] Knetsch, M.L.W. and L.H. Koole. Polymers. New Strategies in the Development of Antimicrobial Coatings: The Example of Increasing Usage of Silver and Silver Nanoparticles, 2011) p.340366. [2] Sebastian, V., M. Arruebo, and J. Santamaria. Small. Reaction Engineering Strategies for the Production of Inorganic Nanomaterials, (2014) p. 835-853.

AgNO3 Aging hose

Microreactor Syringe pump Glucose + Starch Collection flask

Thermostatic bath

Figure 1. Scheme of continuous synthesis device.

Figure 2. TEM micrographs of AgNp synthesized with Ag / Gluc = 1: 5 at 40°C.a: Flow, b: Batch.

Figure 3. Results of the exposure of different concentrations of the selected particles (1.25, 5 y 15 ppm) 6 against E.coli (10 UFC/ml). Positive control (+: culture medium with bacterias) and negative control (B19 and –F19: culture medium with nanoparticles) were also made.

Biodegradable polymeric nanoparticles modified with cell penetrating peptides as an effective ocular drug delivery system María José Gómara1, Aimee Vasconcelos1, Estefanía Vega2, Yolanda Pérez3, María Luisa García2, Isabel Haro1 1. Unit of Synthesis and Biomedical Applications of Peptides, IQAC-CSIC, Jordi Girona, 18-26 08034 Barcelona, Spain. 2. Department of Physical Chemistry, Institute of Nanoscience and Nanotechnology, Faculty of Pharmacy, University of Barcelona, Avda Joan XXIII s/n 08028 Barcelona, Spain. 3. NMR Unit, IQAC-CSIC, Barcelona, Spain. isabel.haro@iqac.csic.es

Abstract The bioavailability of ophthalmic drugs in aqueous solutions is usually low due to their rapid elimination after mucosal instillation; a consequence of reflex blinking and tear drainage, as well as of the presence of the corneal barrier. In fact, only 5% of the applied dose reaches intraocular tissues after corneal penetration [1]. Research into biomaterials has therefore included the use of biodegradable polymeric nanoparticles (NPs) in ocular drug delivery; one of the most promising applications of NPs, as they offer a controlled release profile of a drug which is entrapped in the polymeric matrix [2,3]. These are advantages that suggest that the required therapeutic effects could easily be achieved [4]. Over the years, the potential of a variety of synthetic biodegradable polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their copolymer, poly(lactic-co-glycolic acid) (PLGA), for the production of NPs has been extensively explored due to their biocompatibility, biodegradability and mechanical strength [5]. On the other hand, flurbiprofen (FB) is a non-steroidal anti-inflammatory drug which has been introduced into ocular therapy recently not only for the management of inflammatory diseases that affect ocular structures, but also for use during eye surgery. FB has previously been formulated in PLGA NPs by Vega et al. [6], who achieved good stability and appropriate physicochemical properties for ocular administration, without causing ocular irritancy at any level. Recently, a novel cell penetrating peptide (CPP) for ocular delivery was reported (peptide for ocular delivery; POD) that is capable of transporting both small and large molecules across the plasma membrane [7]. The main aim of this work is to improve the corneal epithelium penetration of NPs composed of PLGAPEG by means of conjugating POD, the final objective being to achieve a longer sustained release of FB which has been used as an example of NSAID drug. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The physicochemical and biopharmaceutical properties of the resulting NPs (morphology, in vitro release, cell viability and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eye after topical instillation of sodium arachidonate. PLGA-PEG-POD-NPs exhibited suitable entrapment efficiency and sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (HET-CAM) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD-NPs are promising vehicles for ocular drug delivery. References [1] Zhang W, Prausnitz MR, Edwards A. J Control Release, 99 (2004) 241-258. [2] Pignatello R, Bucolo C, Spedalieri G, Maltese A, Puglisi G. Biomaterials, 23 (2002) 3247-3255. [3] Pignatello R, Bucolo C, Ferrara P, Maltese A, Puleo A, Puglisi G. Eur J Pharm Sci, 16 (2002) 43-46. [4] Dillen K, Weyenberg W, Vandervoort J, Ludwig A. Eur J Pharm Biopharm, 58 (2004) 539-549. [5] Deshpande AA, Heller J, Gurny R. Crit Rev Ther Drug Carrier Syst, 15 (1998) 381-420. [6] Vega E, Egea MA, Valls O, Espina M, García ML. J Pharm Sci. 95 (2006) 2393-2405. [7] Johnson LN, Cashman SM, Kumar-Singh R. Mol Ther, 16 (2007) 107-114.

Figure

15 * ** ***

NPs

*** *** ***

*** ***

******

***

******

***

18 0

15 0

12 0

21 0

Ocular inflamation score

Time (min) SA

Ocufen 速

PLGA-PEG-NPs

PLGA-PEG-POD-NPs

Comparison of anti-inflammatory efficacy of PLGA-PEG-NPs, PLGA-PEG-POD-NPs and Ocufen速 in the prevention of ocular inflammation induced by SA in the rabbit eye. Values expressed as mean 賊SD. * P<0.05, **P<0.01 and ***P<0.001 significantly lower than the inflammatory effect induce by AS. ($P<0.05, $$ P<0.01 and $$$P<0.001 significantly lower than anti-inflammatory efficacy of Ocufen速.

Cationic vesicles based on non-ionic surfactant and synthetic aminolipids mediate delivery of antisense oligonucleotides into mammalian cells 1

Santiago Grijalvo, Adele Alagia, Gustavo Puras, Jon Zรกrate, Jose Luis Pedraz, and Ramon Eritja

Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Department of Chemical and Biomolecular Nanotechnology and Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain. 2 NanoBioCel group, University of the Basque Country (EHU-UPV), Vitoria and Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) recgma@cid.csic.es Abstract A formulation based on a synthetic aminolipid containing a double-tailed with two saturated alkyl chains along with a non-ionic surfactant polysorbate-80 has been used to form lipoplexes with an antisense oligonucleotide capable of inhibiting the expression of Renilla luciferase mRNA [1]. The resultant lipolexes were characterized in terms of morphology, zeta potential, average size, stability and electrophoretic shift assay. The lipoplexes did not show any cytotoxicity in cell culture up to 150 mM concentration. The gene inhibition studies demonstrated that synthetic cationic vesicles based on nonionic surfactant and the appropriate aminolipid play an important role in enhancing cellular uptake of antisense oligonucleotides obtaining promising results and efficiencies comparable to commercially available cationic lipids in cultured mammalian cells (Figure 1). Based on these results, this amino lipid moiety could be considered as starting point for the synthesis of novel cationic lipids to obtain potential non-viral carriers for antisense and RNA interference therapies. References [1] Santiago Grijalvo, Adele Alagia, Gustavo Puras, Jon Zรกrate, Jose Luis Pedraz and Ramon Eritja, Colloids and Surfaces B: Biointerfaces 119, (2014) 30-37.

Figure 1

Gold Nanoparticles Supported on Nanoparticulate Ceria as a Powerful Agent against Intracellular Oxidative Stress 1,2

José Raúl Herance, María Gamón, Cristina Menchón, Roberto Martín, Nadezda Apostolova, 1 1 3 3 Milagros Rocha, Victor Manuel Victor, Mercedes Alvaro, Hermengildo García. 1

Foundation for the Promotion of Healthcare and Biomedical Research in the Valencian Community (FISABIO)/ University Hospital Doctor Peset (Service of Endocrinology), Juan de Garay 19-21, 46017, 2 Valencia, Spain. Institut d'Alta Tecnologia-PRBB/ CIBER-BBN/CRC-Centre d'Imatge Molecular, Dr. 3 Aiguader 88, 08003, Barcelona, Spain. Instituto de Tecnología Química CSIC-UPV/Departamento de Química, Universidad Politécnica de Valencia, Av. De los Naranjos s/n, 46022 Valencia. jrherance@yahoo.es Abstract Ceria-supported gold nanoparticles are prepared exhibiting peroxidase activity and acting as radical traps. Au/CeO2 shows a remarkable biocompatibility as demonstrated by measuring cellular viability, proliferation, and lack of apoptosis for two human cell lines (Hep3B and HeLa). The antioxidant activity of Au/CeO2 against reactive oxygen species (ROS) is demonstrated by studying the cellular behavior of Hep3B and HeLa in a model of cellular oxidative stress. It is determined that Au/CeO2 exhibits higher antioxidant activity than glutathione, the main cytosolic antioxidant compound, and its CeO2 carrier. Overall the result presented here shows the potential of implementing well-established nanoparticulated gold catalysts with remarkable biocompatibility in cellular biology. References [1] Menchón C, Martín R, Apostolova N, Victor VM, Alvaro M, Herance JR, García H., Small, 8 (2012) 1895. Figures

Fig 1. Effect of Au/CeO2 (20 µg/ml), CeO2 (20 µg/ml) and glutathione (100 µM) on Rotenone-induced ROS production. Bar charts showing DCFH fluorescence in Hep3B cells.

Fig 2. Effect of Au/CeO2 and the carrier CeO2 on cellular proliferation and viability in Hep3B and HeLa cells (a). Cell count over 3 days by static cytometry (data represented as mean ± S.E.M, n= 3). (b) MTT assay of exponentially growing cells after 24 h of culture (data represented as mean ± S.E.M, n= 5-6) were analyzed by Student´s t-test, significance vs control * p<0.05.

Fig 3. Assessment of apoptosis in He3B cells after 24 h incubation with the nanoparticles or the positive apoptotic control, staurosporine (STS). (a) Representative histograms (Bivariate Annexin V/PI analysis) of untreated control, the carrier CeO2 and 1µM STS-treated cells. The table shows the % of each subpopulation for all the conditions studied. (b) Summary histogram of AnnexinV and PI fluorescence data and (c) nuclear morphology changes (mean Hoechst fluorescence and nuclear area). Data (mean ± S.E.M, n=4) was analyzed by Student´s t-test significance vs control * p<0.05 and *** p<0.001.

Application of magnetic chitosan nano particles for anti-Alzheimer drug delivery systems Mohammadreza Khanmohammadi, Hamideh Elmizadeh Chemistry Department, Faculty of Science, IKIU, Qazvin, Iran m.khanmohammadi@sci.ikiu.ac.ir Abstract Nanoparticles have become an important area of research in the field of drug delivery because they have the ability to deliver a wide range of drugs to different areas of the body at appropriate times [1]. Polymers used to form nanoparticles can be two types, hydrophobic and hydrophilic. Nanoparticles based on hydrophilic polymers such as chitosan are appropriate candidates for drug delivery systems [2±4]. Chitosan nanoparticles and magnetic chitosan nanoparticles can be applied as delivery systems for the anti-Alzheimer drug tacrine. Investigation was carried out to elucidate the influence of process parameters on the mean particle size of chitosan nanoparticles produced by spontaneous emulsification. The method was optimized using design of experiments (DOE) by employing a 3-factor, 3-level Box±Behnken statistical design. This statistical design is used in order to achieve the minimum size and suitable morphology of nanoparticles. Also, magnetic chitosan nanoparticles were synthesized according to optimal method. The designed nanoparticles have average particle size from 33.64 to 74.87nm, which were determined by field emission scanning electron microscopy (FE-SEM). Drug loading in the nanoparticles as drug delivery systems has been done according to the presented optimal method and appropriate capacity of drug loading was shown by ultraviolet spectrophotometry. Chitosan and magnetic chitosan nanoparticles as drug delivery systems were characterized by Diffuse Reflectance Fourier Transform Mid Infrared spectroscopy (DR-FTMIR). References [1] M.L. Hans, A.M. Lowman, Biodegradable nanoparticles for drug delivery and targeting, Curr. Opin. Solid State Mater. Sci. 6 (2002) 319±327.. [2] E. Lee, J. Lee, I.H. Lee, M. Yu, H. Kim, S.U. Chae, S. Jon, Conjugated chitosan as a novel platform for oral delivery of paclitaxel, J. Med. Chem. 51 (2008) 6442±6449. [3] A. Trapani, J. Sitterberg, U. Bakowsky, T. Kissel, The potential of glycol chitosan nanoparticles as carrier for low water soluble drugs, Int. J. Pharm. 375 (2009) 97±106. [4] Y. Zhang, M. Huo, J. Zhou, D. Yu, Y. Wu, Potential of amphiphilically modified low molecular weight chitosan as a novel carrier for hydrophobic anticancer drug: synthesis, characterization, micellization and cytotoxicity evaluation, Carbohydr. Polym. 77 (2009) 231±238.

FT-IR Spectra chitosan nanoparticles (1) and magnetic chitosan nanoparticles (2)

The interferences of nanomaterials with hemoglobin a handicap to study hemocompatibility Llanas H, SordĂŠ A, Mitjans M, Vinardell MP Departament de Fisiologia, Facultat de FarmĂ cia, Av. Joan XXIII s/n, 08028 Barcelona, Spain hectorllanasmarco@gmail.com Abstract The interactions of nanomaterials with membrane cells are an important research area because such interactions are critical in many applications such as biomedical imaging, drug delivery, disease diagnostics and DNA/protein stricter probing [1]. More and more nanomaterials are designed for biological applications, and this raises new concerns about the safety of nanotechnology [2,3]. Nanotechnology-derived devices and drug carriers are emerging as alternatives to conventional smallmolecule drugs, and in vitro evaluation of their biocompatibility with blood components is a necessary part of early preclinical development. Special attention should be paid to the interaction of nanomaterials (NMs) with erythrocytes and for this reason the haemolysis assay is recommended as a reliable test for material biocompatibility [4]. The method used was the hemolysis assay as described in previous papers [5] and adapted to the study of NMs. Briefly, red blood cells obtained by centrifugation from fresh blood were incubated at room temperature for 1, 3 and 24 hours with different concentrations of the different nanomaterials studied. At the end of the incubation period, tubes were centrifuged and the amount of hemoglobin on the supernatant has been determined by spectroscopy at 540 nm to determine the percentage of hemolysis induced by the chemicals, compared to red blood cells totally hemolysed. We have used red blood cells from human, rat and rabbit. One of the possible limitations of the hemolysis assay is the absorption of the NMs at 540 nm and this should be discarded [6,7]. Another the possible interference of the nanomaterials with the endpoint of the hemoglobin determination is the adsorption of the hemoglobin by the nanomaterial and/or the protein denaturation. In order to study these possible interferences we have exposed the hemoglobin obtained from erythrocytes by hypotonic haemolysis to the nanomaterials under study. The haemoglobin spectrum was recorded with an UV/visible spectrophotometer. We have studied different nanomaterials such as nano aluminum oxide as nanopowder (13 and 50 nm) and nanowires, zinc oxide nanopowder (50 and 100nm) (Sigma-Aldrich) and a commercial preparation of hydroxyapatite (nanoXIM.CarePasteÂŽ, supplied by Fluidinova). This is a highly dispersed hydroxyapatite aqueous paste specially designed to be incorporated in high performance Oral Care products, with special highlight in toothpastes and mouthwashes aiming enamel remineralization and reduction of teeth sensitivity. The hemolysis phenomena is usually concentration-dependent (higher test concentration induces higher hemolysis). In some cases this is not observed, a decrease in the hemolysis is observed when the concentrations of the test substances increase. This can be observed in the hemolysis induced by Al2O3 nanowire after 3 hours incubation (Figure 1). This effect could be done by the adsorption of the hemoglobin by the nanowire and this could be demonstrated by the spectrum of hemoglobin treated with the nanowire (Figure 2). Clearly, we can observe that the spectrum is not modified, then there is no denaturation and the decrease in absorbance could be attributed to the adsorption phenomena. Similarly, we have observed this phenomenon with a commercial hydroxyapatite preparation (nanoXIM) (Figures 3). In figure 4 we can observe the decrease in the supernatant color and increase in pellet color with increasing concentrations of the nanomaterial. In the case of nano zinc oxide we can observe a significant color change and the hemoglobin spectrum shows an alteration due to the protein denaturation induced by the nanoparticle at higher concentration. This effect is not observed with lower concentrations.

References [1] Verma A, Stellaci F, Small 6 (2010) 12. [2] Nel E, Madler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, Klaessig F, Castranova V, Thompson M, Nat. Mater. 8 ( 2009) 543. [3] Ai J, Biazar E, Jafarpour M, Montazeri M, Majdi A, Aminifard S, Zafari M, Akbari HR, Rad HG. Int J Nanomedicine. 6 (2011) 1117. [4] Lu S, Duffin R, Poland C, Daly P, Murphy F, Drost E, Macnee W, Stone V, Donaldson K. Environmental Health Perspectives 117 (2009) 241. [5] Nogueira DR, Mitjans M, Infante MR, Vinardell MP. Acta Biomaterialia 7 (2011) 2846. [6] Dobrovolskaia M.A., Clogston J.D, Neun B.W., Hall J.B., Patri A.K., MacNeil S. Nanoletters 8 (2008) 2180. [7] Neun BW, Dobrovolskaia MA.Methods Mol Biol. 697 (2011) 215. Figures

Figure 1: Hemolysis induced by nanowire of Al2O3 after 3 hours incubation

Figure 2: Rabbit hemoglobin spectrum and effect of Al2O3 nanowire at 60 and 80 mg/ml after 3 hours incubation time

Figure 3: Human hemoglobin spectrum and effect of nanoXIM after 24 hours incubation time (31 to 1.5 mg/mL)

Figure 4: Human erythrocytes treated with increasing concentration of nanoXIM. The supernatant shows decrease in color with concentration and the pellet shows the adsorption of hemoglobin

Figure 5: Human hemoglobin spectrum and effect of nano ZnO 100 nm after 24 hours incubation time at 37ยบC. Spectrum alteration after treatment with 2 mg/mL

Nanoscale conductance imaging of electronic materials and redox proteins in aqueous solution M. López-Martínez

1,2,3

, J. M. Artés , I. Díez-Perez

1,3

1,2,3

, F. Sanz

and P. Gorostiza

1,2,4,*

Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 15-21, 08028 Barcelona, Spain Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) 3 Physical Chemistry Department, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain 4 Institució Catalana de Recerca i Estudis Avançats (ICREA) 2

(*) pau@icrea.cat Electron Transfer (ET) plays essential roles in chemistry and biology, as it is involved in electrochemical reactions and in crucial biological processes such as cell respiration and photosynthesis. ET takes place between redox proteins and in protein complexes, and it displays an outstanding efficiency and environmental adaptability. Although the fundamental aspects of ET processes are well understood, more experimental methods are needed to determine electronic pathways, especially in complex systems like organic and inorganic nanostructures as well as in biomolecules like nucleic acids and proteins. Understanding how ET works is important not only for fundamental reasons, but also for the potential technological applications of these redox-active nanoscale systems. Electrochemical Scanning Tunneling Microscopy (ECSTM) is an excellent tool to study electronic 1 materials and redox molecules including proteins . It offers atomic or single molecule resolution and allows working in aqueous solution, in nearly physiological conditions in the case of proteins, and under full electrochemical control. Beyond imaging, ECSTM allows performing current-voltage and currentdistance tunneling spectroscopy. We have adapted this spectroscopy mode of ECSTM to include a sinusoidal voltage modulation to the STM tip, and current measurement by means of a lock-in amplifier, 2 which renders a signal that is proportional to the differential conductance dI/dV of the studied surface . We have used this setup to record for the first time spatially resolved, differential conductance images under potentiostatic control (differential electrochemical conductance (DECC) imaging). We have validated and optimized the technique using an iron electrode, whose reversible oxidation in borate 3 buffer is well characterized (Figure 1). We have applied DECC imaging to gold Au <111> surfaces coated with P. Aeruginosa Azurin, a redox metalloprotein with a copper center involved in the respiratory chain of denitrifying bacteria. Azurin can be immobilized on single crystal Au <111> surfaces via a dithiol covalent bond, and it has become a 4 model system to study biological ET processes . DECC imaging provides simultaneously the surface topography and local conductance with a resolution of a few nanometers, and reveals regions with different conductance within the protein. The characterization of conduction pathways in redox proteins at the nanoscale would enable important advances in biochemistry and would cause a high impact in 5 the field of nanotechnology . This method can be used to study more complex biosystems, like multicenter redox proteins and protein redox complexes, and lead to a deeper understanding of their electronic properties and ET pathways. References 1

2 3 4

Friis, E. P. et al. An approach to long-range electron transfer mechanisms in metalloproteins: In situ scanning tunneling microscopy with submolecular resolution. Proceedings of the National Academy of Sciences of the United States of America 96, 1379-1384, doi:10.1073/pnas.96.4.1379 (1999). Robinson, R. S. & Widrig, C. A. Differential conductance tunneling spectroscopy in electrolytic solution. Langmuir 8, 2311-2316, doi:10.1021/la00045a039 (1992). Diez-Perez, I., Guell, A. G., Sanz, F. & Gorostiza, P. Conductance maps by electrochemical tunneling spectroscopy to fingerprint the electrode electronic structure. Analytical Chemistry 78, 7325-7329, doi:10.1021/ac0603330 (2006). Artes, J. M., Diez-Perez, I., Sanz, F. & Gorostiza, P. Direct Measurement of Electron Transfer Distance Decay Constants of Single Redox Proteins by Electrochemical Tunneling Spectroscopy. Acs Nano 5, 2060-2066, doi:10.1021/nn103236e (2011). Artes, J. M., Diez-Perez, I. & Gorostiza, P. Transistor-like Behavior of Single Metalloprotein Junctions. Nano letters 12, 2679-2684 (2012).

Figures

ph-UHVSRQVLYH ³RQLRQ QDQRVSKHUHV´ FRPLQJ IURP LRQLF OLTXLG FU\VWDO SDPDP GHQGULPHUV 1,3

Silvia Hernández-Ainsa , Joaquín Barberá , Mercedes Marcos , José Luis Serrano , Teresa 1 Sierra 1 . Instituto de Ciencia de Materiales de Aragón (ICMA), Universidad de Zaragoza±CSIC, Pedro 2 Cerbuna 12, 50009, Zaragoza, Spain. . Instituto Universitario de Nanociencia (INA), 3 Universidad de Zaragoza, Pedro Cerbuna 12, 50009, Zaragoza, Spain. . Current address: Biological and Soft Systems, Cavendish Laboratory, JJ Thomson Avenue, Cambridge, CB3 0HE. Liquid crystal order allows the control of the supramolecular arrangement, thus providing a powerful tool to obtain ordered structures capable of executing a function. For instance, many lyotropic liquid crystals have been studied as bioinspired synthetic materials mimicking cellular membranes. In this way, supramolecular self-assembly in water constitutes an active topic of research because the possibility to produce a variety of nanoobjets with different shapes. These structures have opened a wide range of potential applications in fields as different as Material Science or Biomedicine. Among the great variety of molecules investigated in this context, dendrimers posses some specific characteristics that make them of great relevance to address the desired self-assembly process. Nearly all amphiphilic dendrimers for guest encapsulation have been obtained by means of covalent bonds, which is laborious and require several purification steps. The preparation of dendrimers by non-covalent self-assembly processes, such as the ionic interaction, is a very convenient synthetic method due to its simplicity, (it is made in one-step) and its versatility because variation of the functional groups can easily be carried out. Some of these ionic dendrimers have shown liquid crystalline properties. With the aim to obtain amphiphilic molecules a series of ionic amphiphilic dendrimers constituted by the grafting of poly(amidoamine) (PAMAM) of different generations (G=0-4) with linear carboxylic acids bearing hydrophobic chains has been prepared. Almost all of the compounds present liquid crystalline behaviour as shown by differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and X-ray diffractometry (XRD) studies. Smectic A mesomorphism has been found for most of the compounds and a rectangular columnar mesophase is displayed for the highest generation compound at low temperature. Interestingly these amphiphilic dendrimers are also capable to self-assemble in water depending on their hydrophobic/hydrophilic balance forming some nanoobjects. In most of the cases these nanoobjects resemble nanospheres whose morphology has been studied by means of transmission electronic microscopy (TEM). The stability of these nanospheres is disrupted in acid or basic media and their amphiphilic nature makes them suitable for trapping both hydrophobic ȕ-carotene) and hydrophilic (Rhodamine B) molecules. These features make these easy to synthesize systems promising and versatile candidates as molecular nanocarriers for a number of biomedical and technological applications.

50 n m

a b c d a), b), c) Representative TEM images of nanospheres formed by self-assembly in water of the ionic dendrimer derived from PAMAM and myristic acid., d) POM textures of G4(C14) at 59ºC in the first cooling process.

Gemini Amphiphilic Pseudopeptides for Encapsulation and Release of Hydrophobic Molecules a

Ahmed H.Lotfallah, Ignacio Alfonso, M. Isabel Burguete and Santiago V. Luis

Departamento de Química Inorgánica y Orgánica, Universiat Jaume I, Avenida Sos Baynat b s/n, Castellón, Spain. Departamento de Química Biológica y Modelización Molecular, Instituto de Química Avanzada de Cataluña (IQAC-CSIC), Jordi Girona 18-26, Barcelona, Spain. E-mail: hajjaj@qio.uji.es Abstract: Gemini amphiphilic pseudopeptides (GAPs) are non-biogenic peptide like molecules [1], able to self-assemble into well-ordered nanostructures through the cooperative action of polar (Hbonding and dipole-dipole) and non-polar (van der Waals) interactions [2-4]. In acidic medium, GAPs are able to form vesicles which have been studied in the solid state (SEM, TEM and AFM; Fig. 1) and in the liquid state (optical fluorescent microscope, Fig. 3). This vesicular morphology is attributed to the hydrophobic interactions which play a major role in the stability of the folding state [3]. In addition, GAPs provide o/w emulsion that remains stable for months and also shows good stability toward the acidic pH and centrifugation effect Fig. 3&2. The capability of this system to encapsulate hydrophobic molecules such as dimethylanthracene (DMA) and dansyldiethyl amine (DEA) was evaluated by fluorescence spectroscopy and microscopy respectively. The results showed that the DMA fluorescence was highly enhanced after 24 hours and I1/I3 fluorescence intensity ratio increased by almost 0.6 Fig. 4. Additionally, DEA was efficiently incorporated into the inner hydrophobic core of GAPs vesicles rendering green colored balls Fig 2. Ultimately, such system can be enzymatically disassembled resulting in the destruction of vesicles and release of its contents Fig. 5 [5]. Therefore, the GAPs here considered are promising system for drug delivery. References: [1] Fundamentals of Protein Structure and Function" Engelbert Buxbaum, Springer, 1 edition, 2007, ISBN:0387263527. [2] (a) S. Cavalli, F. Albericio, A. Kros, Chem. Soc. Rev., 2010, 39, 241. (b) R. J.Brea, C.Reiriz, J. R. Granja, Chem. Soc. Rev., 2010, 39, 1448. (c) I. W. Hamley, Soft Matter, 2011, 7, 4122. [3] J. Rubio, I. Alfonso, M. I. Burguete, S V. Luis, Soft Matter, 2011, 7, 10737.

[4] J. Rubio, I. Alfonso, M. I. Burguete, S V. Luis, Chem. Commun., 2012, 48, 2210. [5] S. Bai, C. Pappas, S. Debnath, P. J. M. Frederix, J. Leckie, S. Fleming, R. V. Ulijin 2014, 8, 7005. Figures:

Fig. 1 Micrographs of GAPs model grown from 1:1 MeOH : H2O + HCl captured by (a) SEM, (b) AFM and (c) TEM techniques.

Fig. 2 Fluorescent microscope images of o/w GAPs encapsulated DEA emulsion; Long term stability (a) after 1 week, (b) after 1 month, (c) after 3 months.

Fig. 3 Optical microscope images of o/w GAPs emulsion after centrifugation at 3000 rpm for 30 minutes; Mechanical stability test (a) 5 min, (b) 15 min, (c) 30 min.

Fig. 4 Fluorescence spectroscopy of Dimethyl anthracene; (a) free DMA, (b) encapsulated DMA.

Fig. 5 Optical microscope images of o/w GAPs emulsion; (a) without thermolysin, (b,c) after adding 1.5 mg/mL thermolysin.

Modulation of dendritic cell sensitization by combined exposure to allergens and nanoparticles a

a,b

Inge Nelissen , Birgit Baré , Sarah Deville , An Jacobs , Nathalie Lambrechts , Peter Hoet a

Flemish Institute for Technological Research (VITO), Applied Bio&molecular Systems, Mol, Belgium b Hasselt University, Biomedical Research Institute (BIOMED), Diepenbeek, Belgium c Catholic University Leuven (KULeuven), Lung Toxicology Unit, Leuven, Belgium inge.nelissen@vito.be

Abstract The adjuvant activity of air pollution particles in allergic airway sensitization is well known, but a similar role of manufactured nanoparticles in allergic sensitization has not been clarified. The goal of our study was to assess the possible alteration of an allergen-induced sensitization response by gold nanoparticles (NPs) through in vitro studies. +

Immature myeloid dendritic cells (CD34-DC), differentiated from human cord blood-derived CD34 progenitor cells, were incubated in the presence of subtoxic concentrations of two sensitizing compounds and citrate-stabilized 50-nm gold NPs (4.4 µg/ml) for 24 hours, either as separate inducers or as a mixture. The chemical sensitizer nickel sulphate (NiSO 4, 160 and 430 µg/ml) and a whole Der p protein allergen mixture (20, 100 and 200 µg/ml) were used as model allergens. Activation and maturation of CD34-DC were studied as indicators of a sensitization response by measuring cell surface expression of the antigen-presenting HLA-DR receptor, the co-stimulatory molecules CD80, CD86 and CD83, and the integrin CD11c using flow cytometry.

Exposure of CD34-DC to NPs induced significant upregulation of the three co-stimulatory molecules as compared to dispersant treated cells. Der p alone did not stimulate any of the studied cell surface markers, but when co-incubated with the NPs it was observed to significantly inhibit NP-induced CD34DC activation in a dose-dependent way. Sole exposure to NiSO4 significantly upregulated CD86 and CD83, while downregulating CD80 expression in CD34-DC. When NiSO4 and gold NPs were combined during co-exposure, we observed a cell activation pattern and levels similar to those induced by NiSO 4 alone, and thus significantly lower than an additive effect of both inducers (Figure). These results indicate that gold NPs interfere with the allergens in the CD34-DC culture, resulting in decreased sensitizing effects. This may either be mediated via a physico-chemical or immune regulatory mechanism. Further investigation will enhance our insight in the possible impact that nanoparticles may pose to our health. Figure

Mixture effect of Au-NPs and NiSO4 on a sensitization response in CD34-DCs. Cells were co-exposed to Au-NPs (4.4 µg/ml) and NiSO4 (430 µg/ml) for 24 hours, harvested, and analysed for surface marker expression. Mean log2 SI ± standard deviation (N=5) are shown (log2 SI of solvent control (SC) = 0). Significantly altered expression compared to respective SC is indicated with * (p<0.05) and ** (p<0.005) only for conditions with |SI|>1.5.

Toxicity assays of nebulized gold nanoparticles with potential applications in the development of nanopesticides a

M.A. Ochoa-Zapater *, J. Querol-Donat , F.M. Romero , A. Ribera , G. Gallello , A. Torreblanca , M.D. a Garcerá . a

Departamento de Biología Funcional y Antropología Física, Universitat de València, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain. b Instituto de Ciencia Molecular, Universitat de València, Catedrático José Beltrán, 2. 46980, Paterna, Valencia, Spain. c Departamento de Química Analítica, Universitat de València, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain. * ozama@alumni.uv.es

Abstract In recent years, nanotechnology applications in agriculture had led to the development of a wide range of plant protection products described as nanopesticides: these products include polimer based formulations [1], formulations containing inorganic nanoparticles [2] and nanoemulsions [3]. The main reasons for the development of these products are the growing need for alternative pesticides to prevent damage on non-target organisms and delay the development of resistances [4]. Moreover, some of these alternative pesticides can benefit from these nanoformulations, which can provide delivery systems for active ingredients with reduced solubility, as well as increase stability and protect them from premature degradation [5]. The toxicity of nebulized gold nanoparticles (AuNPs), which could be functionalized for the formulation of nanopesticides, was tested in two laboratory reared insect species, the german cockroach Blattella germanica, considered an important urban pest with serious implications in public health [6], and the milkweed bug Oncopeltus fasciatus. AuNPs were synthetized following the methodology described by Bastús et al. [7] and characterized by UV-Vis and Transmission Electron Microscopy (TEM). Adult insects (15 females and 15 males, aged 1-6 days) were exposed to 1mL and 2mL of AuNPs in sodium citrate with the aid of a nebulizer based system (figure 1), with times of total exposure ranged between 15 to 90 minutes (table 1). Mortality rates were monitored 24, 48, 72 and 96 hours post-treatment, and enzymatic activities related to oxidative stress and insecticide resistance [8], such as glutathione Stransferases (GSTs) and esterases (p-NPA), were measured in exposed insects frozen immediately after nebulization and insects frozen at 96h post-treatment. Also, a comparison between the obtained activity rates and results from our previous studies in tarsal contact toxicity bioassays were made for the two insect species. Finally, in order to study the persistence of nanoparticles in treated insects, inductively coupled spectroscopy (ICP-OES) was performed in insects frozen at times 0 and 96 hours after AuNPs exposure. References [1] Adak T, Kumar J, Shakil NA, Walia S, J. Environ. Sci. Health B, 47 (3) (2012) 217±25. [2] Song M-R, Cui S-M, Gao F, Liu Y-R, Fan C-L, Lei T-Q, Liu D-C, J. Pestic. Sci., 37 (3) (2012) 258±60. [3] Kumar RSS, Shiny PJ, Anjali CH, Jerobin J, Goshen KM, Magdassi S, Mukherjee A, Chandrasekaran N, Environ. Sci. Pollut. Res., 20 (4) (2013) 2593±602. [4] Bhattacharyya A, Bhaumik A, Rani PU, Mandal S, Epidi T, Afr. J. Biotechnol., 9 (2010) 3489±3493. [5] Nguyen HM, Hwang IC, Park JW, Park HJ, J. Microencapsul., 29 (6) (2012) 596±604. [6] Roberts J, Br. Med. J., 312 (1996) 1630. [7] Bastús NG, Comenge J, Puntes V, Langmuir, 27 (2001) 11098-11105. [8] Hemingway J, Ranson H, Annu. Rev. Entomol., 45 (2000) 371-391.

Figures Table 1. Total exposure time to nebulized AuNPs Treatment

Volume (mL)

Duty (%)a

Exposure time (h:m:s) Blattella germanica Mean SD AuNP 1 100 0:15:15 0:00:05 50 0:16:59 0:00:13 5 0:46:01 0:01:51 AuNP 2 100 0:16:50 0:00:06 50 0:20:26 0:00:03 5 1:14:40 0:02:15 a % of nebulized solution per cycle (1 cycle equals 6 seconds)

Oncopeltus fasciatus Mean SD 0:14:53 0:00:17 0:16:19 0:00:08 0:46:15 0:00:31 0:16:55 0:00:34 0:20:02 0:00:03 1:18:27 0:02:15

Figure 1. Adult cockroaches being exposed to nebulized AuNPs in the nebulization chamber

CERIUM OXIDE NANOPARTICLES REDUCE PORTAL HYPERTENSION AND SHOW ANTIINFLAMMATORY PROPERTIES IN CCl4-TREATED RATS D. OrĂł1, G. FernĂĄndez-Varo1,3, V. Reichenbach1, T. Yudina2 , E. Casals2, G. Casals1,B. GonzĂĄlez de la Presa1, V. Puntes2, W. JimĂŠnez1,3. 1

Biochemistry and Molecular Genetics Service, Hospital ClĂnic de Barcelona, IDIBAPS, Centro de InvestigaciĂłn BiomĂŠdica en Red de Enfermedades HepĂĄticas y Digestivas (CIBERehd), 2Institut CatalĂ de Nanotecnologia (ICN), Bellaterra, Spain, 3Department of Physiological Sciences I, University of Barcelona, Barcelona, Spain. dnise_2@hotmail.com Background and Aims. During the last few years nanoparticles (NPs) have emerged as a new technology allowing enhanced levels of precision in treating disease. Cerium oxide (CeO2) NPs have proven to behave as free radical scavenger and/or antiinflammatory agents. However, whether CeO2NPs are of therapeutic value in liver disease is not known. We assessed the organ distribution, subcellular localization, metabolic fate and systemic and hepatic effects of the iv administration of CeO2NPs to CCl4-treated rats. The aim of the study was to determine whether CeO2NPs display hepatoprotective properties in experimental liver disease. Methods. Organ and subcellular distribution of NPs was assessed using magnetic resonance imaging (MRI) and transmission electron microscopy (TEM), respectively. The metabolic fate of CeO2NPs was investigated by measuring daily urinary and fecal excretion of Ce (ICP-MS). The systemic and hepatic effects of NPs were assessed in CCl4-treated rats receiving CeO2NPs (0.1 mg/kg, n=10) or vehicle (n=15) twice weekly for two weeks and CCl 4 treatment was continued for 8 additional weeks. Thereafter, mean arterial pressure (MAP) and portal pressure (PP) were assessed and serum samples obtained to measure standard hepatic and renal function tests. Liver samples were also obtained to evaluate mRNA expression of genes related to LQIODPPDWRU\ RU YDVRDFWLYH DFWLYLW\ PDFURSKDJH LQILOWUDWLRQ ÄŽ-smooth muscle actin ÄŽ-SMA) expression and hepatic apoptosis. Results. More than 90% of the NPs were located in the liver and spleen 30 min after administration. The remaining targeted lungs and kidneys. No NPs were located in the brain. CeO2NPs were internalized by parenchymal cells and found in either, peroxisomes or free in the cytoplasm. Most NPs were excreted by the urine. CeO 2NPs ameliorated systemic inflammatory biomarkers (LDH: 879Âą229 vs 392Âą67, ALT: 1287Âą419 vs 304Âą40 U/L; p<0.05) and improved PP (9.9Âą0.4 vs 8.2Âą0.4 mm Hg, p<0.05) without affecting MAP. A marked reduction in mRNA abundance of iQIODPPDWRU\ F\WRNLQHV 71)ÄŽ 53Âą11.1 vs 18.3Âą4.7, ,/ Č&#x2022; 61.6Âą10 vs 31.2Âą5.4; p<0.05), iNOS: (537Âą158 vs 94Âą35, p<0.05) and ET-1 (14.2Âą2.7 vs 6.3Âą1.9, p<0.05), infiltration of macrophages (29.5Âą0.8 vs 25.7Âą0.7 cells/field) and protein expression of caspase-3 (21Âą2.9 vs 7.1Âą2.7 DAU, p<0.05) DQG ÄŽ-SMA (7.1Âą0.3 vs 5.8Âą0.3 %, p<0.01) was observed in the liver of rats receiving CeO2NPs. Conclusions. CeO2NPs administration to CCl 4-treated rats protects against chronic liver injury by markedly attenuating the intensity of the inflammatory response and reducing portal hypertension, thereby suggesting that CeO2NPs may be of therapeutic value in chronic liver disease.

Bridging Research and Industrial Production towards H2020: Future challenges for Nanomedicine with a multi-KET approach 1

Cristina Paez-Aviles , Esteve Juanola-Feliu , Josep Samitier

1,2,3

Department of Electronics, Bioelectronics and Nanobioengineering Research Group (SIC-BIO), University of Barcelona, Martí i Franquès 1, Planta 2, 08028 Barcelona, Spain. cpaezeviles@el.ub.edu 2 IBEC-Institute for Bioengineering of Catalonia, Nanosystems Engineering for Biomedical Applications Research Group, Baldiri Reixac 10-12, 08028 Barcelona, Spain 3 CIBER-BBN-Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine, María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain

Abstract It is stated that pilot production builds the bridge between research and industrial production since this activity is among technology and commercialization. However, pilot scalability is considered a bottleneck in the way to commercialization, even more in the Health domain where scalability is more complex. In this context, the new European Commission’s initiative Horizon 2020, the biggest financial program for Research and Innovation, plans to finance different Risk Management Projects going “from fundamental research to market innovation” involving the entire innovation chain. H2020 is particularly focused on the research and development of Key Enabling Technologies (KETs), which are among the priorities of the framework strategy, that identifies the need for the EU to facilitate the industrial deployment of KETs in order to make its industries more innovative and globally competitive [1]. Six KETs have been selected according to an economic criteria, economic potential, capital intensity, technology intensity, and their value adding enabling role: Nanotechnology, Micro and Nano Electronics, Photonics, Advanced Materials, Biotechnology Industry, and Advanced Manufacturing Systems[2]. While each KET already has huge potential for innovation individually, their cross-fertilization is particularly important to offer even greater possibilities to foster innovation and create new markets. Integration between KETs will be essential for create jobs in industry, improve competitiveness and innovation, and at the same time address today’s burning societal challenges in Europe in the coming years. The concept of cross-cutting KETs refers to the integration of different key enabling technologies in a way that creates value beyond the sum of the individual technologies for developing innovative and competitive products, goods and services oriented to solve societal needs. The global market volume in KETS is €646 billion and substantial growth is expected of approximately 8% of EU GDP by 2015. In this context, Horizon 2020 will invest €5.96 billion in the industry sector for the development of the KETs and about 1/3 of this budget will be assigned to projects integrating different KETs [3]. Most high tech pilot production problems are inherently multi-KETs. The scale up of nanomedicines for clinical testing is severely hindered by a lack of knowledge about how and where to manufacture such entities according to Good Manufacturing Practice (GMP) and taking into account the medical regulatory requirements. The Commission states that bridging the so called “Valley of Death” to upscale new KET technology based prototypes to commercial manufacturing, often constitutes a weak link in the successful use of KETs potential[4]. Translation of innovation and time-to-market reduction are important challenges on H2020. After a long R+D incubation period, several industrial segments are already emerging as early adopters of nanotech-enabled products and findings suggest that the Bio&Health market is among the most challenging field for the coming years. As a major application of Nanotechnology, the field of Nanomedicine fits naturally amongst the Key Enabling Technologies defined by the European Commission. It is considered multidisciplinary since it is not restricted to the realm of advanced materials, extending also to manufacturing processes, biotechnology, pharmacy, electronics and IT, as well as other technologies [5]. These characteristics allow the connection to a diversified set of industries [6]. Inherent interactions exist between these sectors and could be mutually beneficial in terms of research innovation (Fig. 1). For example, the use of quantum dots and shape-shifting nanomaterials for medical applications could greatly benefit from the latest progress in photonics, and nanomedicine

sensors from biotechnology and biological pores. Additionally, new medical therapies enabled by Nanotechnology and Advanced Materials, can contribute to personalised health care. This strong interdisciplinary character, combined with the possibility of manipulating a material atom by atom, opens up unknown fields and provides an endless source of innovation and creativity in the healthcare domain.

Fig. 1: A Multi-KET approach of Nanomedicine: common R&D topics with the KETs [7]

References [1] [2] [3] [4] [5] [6] [7]

European Commission, Brussels, 2009. B. Aschhoff, D. Crass, K. Cremers, C. Grimpe, F. Brandes, F. Diaz-lopez, R. K. Woolthuis, M. Mayer, and C. Montalvo, 2010 E. Commision, 2013. M. De Heide, M. Butter, D. Kappen, A. Thielmann, A. Braun, M. Meister, D. Holden, F. Livese, E. O. Sullivan, C. Hartmann, M. Zaldua, N. Olivieri, L. Turno, M. Deschryvere, J. Lehenkari, P. Ypma, P. Mcnally, and M. De Vries, 1–58, 2013 N. Islam and K. Miyazaki, Technovation, vol. 27, no. 11, (2007) 661–675. T. Nikulainen and C. Palmberg,Technovation, vol. 30, no. 1, (2010) 3–11. European Technology Platform on Nanomedicine, 2013.

Accessing the Nanoparticle Corona in Pulmonary Surfactant 1,2

Simon S. Raesch , Stefan Tenzer , Wiebke Storck , Christian Ruge , Ulrich F. Schaefer , Claus1,2 Michael Lehr 1

Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken, Germany 2 Department of Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland, 66123 Saarbruecken, Germany 3 Institute for Immunology, University Medical Center of Mainz, 55101 Mainz, Germany Simon.Raesch@mx.uni-saarland.de Abstract Nanoparticles (NP) that come in contact with a biological fluid are opsonized by biomolecules such as proteins, which build a “corona”. This time-dependent layer of adherent biomolecules typifies the actual biological identity of the NP. Considering the many different NP with varying surface modifications which are produced worldwide, differences in resulting corona seem plausible and were identified in the coronas on NP in plasma [2, 3]. An attractive pharmaceutical target for various nanoparticles is the lung, as the air-blood barrier is a less 2 than 2 µm thin layer with an enormous alveolar surface area larger than 100m . The vast amount of potentially polluted air, which passes through the lung, makes an effective maintenance system essential. In the alveolar region cells are only covered by a thin pulmonary surfactant (PS) layer and clearance is mainly carried out by alveolar macrophages. PS, secreted by type II alveolar cells, allows gas diffusion and its surface tension lowering effect is thus essential for stability of the alveoli during a breathing cycle. The surfactant layer consists of approximately 90% lipids (mainly phospholipids, especially DPPC) and 10% proteins with about half of them being surfactant specific proteins (SP). Before NP are either taken up by the alveolar cells or ingested by macrophages, they are coated by the PS building a lipid-protein-“corona”. So far, it remains to be elucidated whether the fate of inhaled NP depends on the coating obtained from the surfactant layer, though there is evidence for the influence of the SP on macrophage uptake [1]. Although understanding the surfactant-NP interaction is fundamental for the fate of NP in the lung, there is, so far, no reproducible method for the analysis of the NP-corona. The unique composition, structure, and properties of the lipid-rich PS require different and more advanced analytical methods for the assessment of the NP-corona in the deep lung. Hence, we used a native pulmonary surfactant preparation, isolated from porcine lungs, for the development of a method to access the lipid-protein-corona. Centrifugation, magnetic separation and density centrifugation were compared with three magnetic model particles (Phosphatidylcholine-, PEGcoated, and plain PLGA). Magnetic separation of NP was found to be superior to the other common techniques. SDS-PAGE showed the impact of hydrophobicity on the PS corona and was verified by advanced labelfree proteomics.

References [1] Ruge, C. A. et al., PLoS ONE, 7(7) (2012) e40775 [2] Monopoli, M. P. et al., J. Am. Chem. Soc., 133(8) (2011) 2525–34 [3] Tenzer, S. et al., Nat. Nanotechnol., 8(Oct) (2013) 772–781 Graphical Abstract

2D Microscale Engineering of Novel Protein based Nanoparticles for Cell Guidance 1,2

2,3,4

2,3

Witold I. Tatkiewicz, Joaquin Seras-Franzoso, , Elena García-Fruitós, Esther Vazquez, 1,2 1,2 2,3,4 1,2 Ventosa, Imma Ratera, Antonio Villaverde, and Jaume Veciana

2,3,4

Nora

Department of Molecular Nanoscience and Organic Materials, Institut de Ciencia de Materials de Barcelona (CSIC), Bellaterra, 08193 Barcelona, Spain, 2 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193 Barcelona, Spain, 3 Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain, 4 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain, wtatkiewicz@icmab.es, iratera@icmab.es, jveciana@icmab.es Abstract 7KH WHUP ³LQFOXVLRQ ERGies´ (IBs) was coined to describe optically opaque moieties present in cell 3 lumen. They have aspect of refractile particles of up to a few hundred nanometers and about 2 ȝP of size when observed by optical microscopy and as electron-dense aggregates without defined organisation by transmission electron microscopy.[1] The history of IBs turned when they were recognized as a prospective biomaterial with desirable properties. Being a product derived from biological synthesis, it is fully biocompatible and preserves the functionality of the embedded protein [2]. In a course of investigation it was revealed that IBs size, geometry, stiffness, wettability, z-potential, bio-adhesiveness, density/porosity etc. can be easily fine tuned by control over basic parameters of their production: harvesting time, host genetic background and production conditions (e.g. temperature, pH) In addition, their production and downstream processes are fully scalable, cost effective and methodologically simple.[3] It is widely accepted, that cell´s responses, such as positioning, morphological changes, proliferation, mottility and apoptosis are the result of complex chemical, topographical and biological stimuli. Here we will show the application of IBs, as a functional biomaterial for engineering two dimensional substrates for cell guidance. We have cultivated fibroblast cells on supports patterned with IBs derived from green fluorescent protein (GFP) or human basic fibroblast growth factor (FGF). Two methodologies of pattern deposition were applied: microcontact printing (ǋCP) optimized for use with aqueous colloidal suspensions and a novel, template-free technique based on the coffee-drop effect due to a convective self-assembly (Figure 1).[4] The first technique was applied in order to deposit IBs with high resolution geometrical patterns of various shapes and sizes. Then we have investigated how cells react to IBs geometrical distribution. Parameters such as orientation morphology and positioning were thoroughly investigated based on rich statistical data delivered by microscopy image treatment (Figure 2)The second technique has been recently developed in order to deposit complex and well-controlled two dimensional IB´s patterns with concentration gradients for the study of cell motility (Figure 3). Cell movement cultivated on such substrates was characterized and quantified based on confocal microscopy time-lapse acquisitions.[5,6] In both cases a deep statistical data treatment was preformed to characterize macroscopic responses of cells when grown over nanoscale profiles made with IBs concluding that cell proliferation is not only dramatically stimulated but cell also preferentially adhere to IBs-rich areas, align, elongate and move according to such IBs geometrical cues.. These findings prove the potential of surface patterning with functional IBs as protein-based nanomaterials for tissue engineering and regenerative medicine among other promising biomedical applications. References [1]

[2]

(a) Villaverde A., Carrio M.M., Biotechnol. Lett., 2003, 25, 1385 (b) E. García-Fruitós, E. Rodríguez-Carmona, C. Diez-Gil, R. Mª Ferraz, E. Vázquez, J. L. Corchero, M. Cano-Sarabia, I. Ratera, N. Ventosa, and J. Veciana, A. Villaverde Adv. Mater., 2009, 21, 4249 (c) Cano-Garrido, O.; Rodríguez-Carmona, E.; Díez-Gil, C.; Vázquez, E.; Elizondo, E.; Cubarsi, R.; Seras-Franzoso, J.; Corchero, J. L.; Rinas, U.; Ratera, I.; et al.. Acta Biomater. 2013, 9, 6134. García-Fruitós E., Vazquez E., Díez-Gil C., Corchero J.L.; Seras-Franzoso J., Ratera I., Veciana J., Villaverde A., Trends Biotechnol., 2012, 30, 65

[3]

[4] [5]

[6]

(a) García-Fruitos E., Seras-Franzoso J., Vazquez E., Villaverde A., Nanotechnology, 2010, 21, 205101 (b) Vazquez E., Corchero J. L., Burgueno J.F., Seras-Franzoso J., Kosoy A., Bosser R., Mendoza R., Martínez-Láinez J.M., Rinas U., Fernandez E., Ruiz-Avila L., García-Fruitós E., Villaverde A., Adv. Mater., 2012, 24, 1742 (c) E. Vazquez , M. Roldán , C. Diez-Gil , U. Unzueta, J. Domingo-Espín, J. Cedano, O. Conchillo, I. Ratera, J. Veciana , X. Daura, N. Ferrer-Miralles, A. Villaverde, Nanomedicine., 2010, 5, 2, 259 (a) Han W., Lin Z., Angew. Chem. Int. Ed., 2012, 51, 1534 (b) Hanafusa T.,Mino Y., Watanabe S., Miyahara M.T., Advanced Powder Technology 2014, 25, 811 (a) C. Díez-Gil, S. Krabbenborg, E. García-Fruitós, E. Vazquez, E. Rodríguez-Carmona, I. Ratera, N. Ventosa, J. Seras-Franzoso, O. Cano-Garrido, N. Ferrer-Miralles, A. Villaverde, J. Veciana, Biomaterials, 2010, 31, 5805 (b) J. Seras-Franzoso, C. Díez-Gil, E. Vazquez , E. García-Fruitós , R. Cubarsi, I. Ratera, J. Veciana, A. Villaverde, Nanomedicine, 2012, 7(1):79-93 W. I. Tatkiewicz, J. Seras-Franzoso, E García-Fruitós, E. Vazquez, N. Ventosa, K. Peebo, I. Ratera, A. Villaverde, J.Veciana, ACSNano, 2013, 7(6), 4774

Figures

Figure 1. Schematic illustration of particle deposition. Particles are pinning to the substrate on the edge of meniscus, where the evaporation is more intense. Image adapted from reference [4b].

Figure 3. Example of GFP-derived IBs gradient pattern deposited by a controlled convective selfassembly technique. Left: fluorescence microscopy image, right: IBs concentration calculated based on fluorescence intensity.

Long-term exposures to low doses of cobalt nanoparticles induce cell-transformation enhanced by oxidative damage. Laura Rubio: Balasubramanyam Annangi, Jordi Bach, Gerard Vales, Laura Rubio, Ricardo Marcos, Alba Hernández. Universitat Autònoma de Barcelona, Campus Bellaterra Facultat de Biociències, Departament de Genètica i Microbiologia, Barcelona, Spain laura.rubio@uab.cat Abstract A great effort is being done by the scientists to increase our knowledge on the role of nanoparticlesassociated genotoxic and carcinogenic effects [1, 2]. Although important research activity took place in this area for >10 years, most of the findings concerning to the genotoxic and cell transforming potential of NPs are limited to short-term in vitro studies [3, 4]. Comprehensively, acute or short-term studies use high environmentally irrelevant single doses to study the adverse effects of engineered NPs, which could not be enough to draw plausible conclusions about the potential human health risk of NPs exposure [5]. Until now, only a few in vitro long-term exposure studies have been carried out with NPs [6,7,8,9]. Keeping this in mind, it seems necessary to increase the amount of in vitro investigations focusing on long-term or chronic exposures at sub-toxic doses. Despite the usefulness of cobalt nanoparticles (CoNPs) in various fields [10], they are also potentially harmful to humans. Some studies have found that in vitro acute exposure of CoNPs induce oxidative stress, DNA damage, morphological transformation and inflammatory responses in different cell types, among other kind of effects [3, 11, 12, 13, 14,15]. However, there is no conclusive information available on the in vitro carcinogenic potential of CoNPs under chronic settings so far. In vitro cell transformation assays have been proposed as alternatives to long term animal studies. In fact, OECD has specific JXLGHOLQHV RQ µµ&HOO WUDQVIRUPDWLRQ DVVD\V IRU GHWHFWLRQ RI FKHPLFDO FDUFLQRJHQV¶¶ [16] with accumulated evidence that the cellular and molecular processes involved in vitro cell transformation are similar to those of in vivo carcinogenesis [17]. In this study, we have evaluated the cell transforming ability of cobalt nanoparticles (CoNPs) after longterm exposures (12 weeks) to sub-toxic doses (0.05 and 0.1Ԝ µg/mL). To get further information on whether CoNPs-induced oxidative DNA damage is relevant for CoNPs carcinogenesis, the cell lines +/+ selected for the study were the wild-type mouse embryonic fibroblast (MEF Ogg1 ) and its isogenic í í Ogg1 knockout partner (MEF Ogg1 ), unable to properly eliminate the 8-OH-dG lesions from DNA. Our initial short-term exposure experiments demonstrate that low doses of CoNPs are able to induce í í reactive oxygen species (ROS) and that MEF Ogg1 cells are more sensitive to CoNPs-induced acute toxicity and oxidative DNA damage. On the other hand, long-term exposures of MEF cells to sub-toxic doses of CoNPs were able to induce cell transformation, as indicated by the observed morphological cell changes, significant increases in the secretion of metalloproteinases (MMPs) and anchorageindependent cell growth ability, all cancer-like phenotypic hallmarks. Interestingly, such changes were í í significantly dependent on the cell line used, the Ogg1 cells being particularly sensitive. Altogether, the data presented here confirms the potential carcinogenic risk of CoNPs and points out the relevance of ROS and Ogg1 genetic background on CoNPs-associated effects. References [1] Arora S, Rajwade JM, Paknikar KM. Toxicol Appl Pharmacol. 258(2012):151±65. [2] Becker H, Herzberg F, Schulte A, Kolossa-Gehring M. Int J Hyg Environ Health 214(2011):231±8. [3] Colognato R, Bonelli A, Ponti J, Farina M, Bergamaschi E, Sabbioni E, et al. Mutagenesis 23(2008):377±82. [4] Ponti J, Broggi F, Mariani V, De Marzi L, Colognato R, Marmorato P, et al. Nanotoxicology 7(2013):221±33.

[5] Hristozov DR, Gottardo S, Critto A, Marcomini A. Nanotoxicology 6(2012):880±98. [6] Hackenberg S, Scherzed A, Technau A, Froelich K, Hagen R, Kleinsasser N. J Biomed Nanotechnol 9(2013):86±95. [7] Huang S, Chueh PJ, Lin YW, Shih TS, Chuang SM. Toxicol Appl Pharmacol 241(2009):182±94. [8] Jacobsen NR, Saber AT, White P, Møller P, Pojana G, Vogel U, et al. Environ Mol Mutagen 48(2007):451±61. [9] Kocbek P, Teskac K, Kreft ME, Kristl J. Small 6(2010):1908±17. [10] Horev-Azaria L, Kirkpatrick CJ, Korenstein R, Marche PN, Maimon O, Ponti J, et al. Toxicol Sci 122(2011):489±501. [11] Magaye R, Zhao J, Bowman L, Ding M. Exp Ther Med 4(2012):551±61. [12] Papageorgiou I, Brown C, Schins R, Singh S, Newson R, Davis S, et al. Biomaterials 28(2007): 2946±58. [13] Papis E, Gornati R, Prati M, Ponti J, Sabbioni E, Bernardini G. 2007. Toxicol Lett 170(2007):185± 92. [14] Petrarca C, Perrone A, Verna N, Verginelli F, Ponti J, Sabbioni E, et al. Int J Immunopathol Pharmacol 19(2006):11±14. [15] Ponti J, Broggi F, Mariani V, De Marzi L, Colognato R, Marmorato P, et al. Nanotoxicology 7(2013):221±33. [16] Vasseur P, Lasne C. Mutat Res 744(2012):8±11. [17] Combes R, Balls M, Curren R, Fischbach M, Fusenig N, Kirkland D, et al. ATLA 27(1989):745±67.

Graphene oxide application in cell microencapsulation for bioartificial organ development Laura Saenz del Burgo*, Jesús Ciriza*, Gorka Orive, Rosa María Hernández, Jose Luis Pedraz NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain The Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) * Both authors contributed equally to the development of the present work joseluis.pedraz@ehu.es Abstract Cell microencapsulation represents a great promise for the development of new long-term drug delivery systems. However, several challenges need to be overcome before it can be translated extensively into the clinic. For instance, the long term cell survival inside the microcapsules. On this regard, graphene oxide has shown to promote the proliferation of different cell types both in two and three dimension cultures. Therefore, we planned to combine the use of graphene oxide together with the cell microencapsulation technology and analyze the biocompatibility of this chemical compound with cells within alginate-poly-L-lysine (APA) microcapsules. We have been able to produce 200 µm-diameter APA microcapsules with increasing concentrations of graphene oxide in their inside and prove that the physical chemical parameters of the traditional microcapsules were no modified. Moreover, microcapsules containing graphene oxide enhanced the viability of the encapsulated cells, providing another step for the future pre-clinical application of graphene oxide in combination with cell microencapsulation. References [1] Orive, G.; Santos, E.; Pedraz, J. L.; Hernandez, R. M., Adv Drug Deliv Rev, 67-68 (2013) 3-14. [2] Basta, G.; Montanucci, P.; Luca, G.; Boselli, C.; Noya, G.; Barbaro, B.; Qi, M.; Kinzer, K. P.; Oberholzer, J.; Calafiore, R., Diabetes Care, 34(11) (2011) 2406-9. [3] Goenka, S.; Sant, V.; Sant, S., J Control Release, 173 (2013) 75-88. [4] Lee, W. C.; Lim, C. H.; Shi, H.; Tang, L. A.; Wang, Y.; Lim, C. T.; Loh, K. P., ACS Nano, 5(9) (2011) 7334-41. [5] Li, N.; Zhang, Q.; Gao, S.; Song, Q.; Huang, R.; Wang, L.; Liu, L.; Dai, J.; Tang, M.; Cheng, G., Sci Rep, 3 (2013) 1604. [6] Ruiz, O. N.; Fernando, K. A.; Wang, B.; Brown, N. A.; Luo, P. G.; McNamara, N. D.; Vangsness, M.; Sun, Y. P.; Bunker, C. E., ACS Nano 5(11) (2011) 8100-7. Figures

Figure 1.- Microscopy images of bright field (A) and fluorescence after calcein ethidium staining (B) from microcapsules containing graphene oxide [1) without oxide graphene, 2) 10 µg/ml, 3) 25 µg/ml, 4) 50 µg/ml and 5) 100 µg/ml] and C2C12 myoblasts 4 days after encapsulation. Scale bar 100 µm.

Figure 2.- Viability of encapsulated C2C12 myoblasts in alginate microcapsules containing different concentrations of graphene oxide [0-100 Âľg/ml]. A) Metabolic activity measured in the cell counting kit 8 (CCK8) assay and B) Membrane integrity measured by the lactate dehydrogenase activity (LDH) assay, both expressed as the ratio between day 8 and 1 after microencapsulation.

Tramadol Hydrochloride Released from Lipid Nanoparticles: Studies on Modelling Kinetics Elena SĂĄnchezÂŞ, Helen Alvaradoa,b*, Prapaporn Boonmec, Guadalupe Abregoa,b, Tatiana Andreanid,e, Monica Vazzanaf, Joana F. Fangueirob, Catarina Faggiof, Carla Silvag, Sajan JosĂŠh, Antonello Santinii, MarĂa Luisa Garciaa, Ana C. Calpenab AmĂŠlia M. Silvad,e, Eliana B. Soutoj* a Department

of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain b Department of Biopharmacy and Pharmacology, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain cFaculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand dDepartment of Biology and Environment, University of TrĂĄs-os Montes e Alto Douro, Portugal; eCentre for Research and Technology of Agro-Environmental and Biological Sciences, Portugal; fDepartment of Biological and Environmental Sciences, University of Messina, Italy; gCenter for Nanotechnology and Smart Materials, Portugal; hDepartment of Pharmaceutical Sciences, Mahatma Gandhi University, India; iDepartment of Pharmacy, University of Napoli, Italy; jDepartment of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra

*E-mail address corresponding author: hl_alvarado@yahoo.com; ebsouto@ff.uc.pt Keywords: tramadol hydrochloride, HPLC, lipid nanoparticles, kinetic model A reverse-phase (RP) high performance liquid chromatography (HPLC) method was developed according to the International Conference on Harmonisation (ICH) guidelines, for the determination of tramadol hydrochloride (THC) from lipid nanoparticles (LNs). THC is an opioid analgesic drug, mainly acting on the central nervous system (CNS) and structurally related to codeine and morphine, but clinically 10-fold less potent than codeine and 6000-fold less than morphine. It was developed in 1970s and is currently in use for the management, treatment and relief of moderate to severe pain conditions. A method for the preparation of THC standard and N1,N1-dimethylsulfanilamide (used as the internal standard) has been described. HPLC analysis was performed on a 250x4 mm chromatographic column with LiChrospher 60 RP-selectB 5-Č?P 0HUFN XVLQJ acetonitrile:0.01 M phosphate buffer, pH 2.8 (3:7, v/v) as mobile phase. Fluorescence detection was done at 296 nm (THC) and at 344 nm (N1,N1-dimethylsulfanilamide). THCloaded LNs dispersions were produced by hot high pressure homogenization technique, using CompritolÂŽ 888ATO as solid lipid, stabilized with 3% (w/w) PhospholiponÂŽ 80H and 1% (w/w) TyloxapolÂŽ as surfactants. Particles ranging between 79.4Âą0.3 and 144.6Âą14 nm in size were obtained, with a mean zeta potential of -10.2Âą1.2 mV. Four kinetic models (i.e., zero order, Higuchi, Baker-Lonsdale and KorsmeyerÂąPeppas) were selected to fit the data to describe the THC release profile from LNs. The in vitro release profile of THC from LNs was compared with that from the commercial oral suspension (TramalÂŽ), in pH 6.8 phosphate buffer. Commercial THC suspension depicted a 100% release in the first hour; whereas for LNs, a biphasic sustained release profile was observed. According to the obtained R2 values, Korsmeyer-Peppas model was reported as the best fit modelling kinetic

profile for THC release from LNs. The recorded n = 0.63 value typical for anomalous nonFickian transport is in agreement with the biphasic mechanism of drug release from LNs.

Polycationic Silicon Phthalocyanines as Photosensitizers for Photodynamic Therapy and Photodynamic Inactivation of Microorganisms Eveline van de Winckel, Andrés de la Escosura, Tomás Torres Departamento de Química Orgánica, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, España eveline.vandenwinckel@uam.es Abstract Photodynamic therapy (PDT) is a form of therapy that uses light-sensitive compounds, which upon [1] selective exposure to light become toxic to targeted malignant and other diseased cells . Since its incidental discovery in 1900, photodynamic therapy (PDT) and all related aspects, ranging from its mechanism of action, the different photosensitizers that can be employed and its clinical applications have been studied in great detail. In general, it is well-known that three components are required for PDT to occur; a photosensitizer, oxygen and a light source. In the presence of oxygen, irradiation of the photosensitizer of choice can lead to the generation of singlet oxygen, which is a powerful, indiscriminate oxidant that reacts with a variety of biological molecules. Singlet oxygen is indeed the main reactive oxygen species (ROS) in [2] PDT, responsible for the destruction of tumor cells, bacteria, viruses, etc. . Following the absorption of light, the photosensitizer is transformed from its ground singlet state (S0) into an electronically excited triplet state (T1) via a short-lived excited singlet state (S1). The excited triplet can undergo two kinds of reactions as shown by the Jablonski diagram depicted in Fig 1. Firstly, it can participate in an electron-transfer process with a biological substrate to form radicals and radical ions that, after interaction with oxygen, can produce oxygenated products (type I reaction). Alternatively, it can undergo a photochemical process known as a type II reaction, which results in the conversion of 3 1 stable triplet oxygen ( O2) into the short-lived but highly reactive singlet oxygen ( O2) species, the putative cytotoxic agent. Phthalocyanines are an important class of non-natural organic pigments that have received [3] considerable attention in the field of PDT .Our focus will be centered on the design of novel silicon phthalocyanines with different substitution patterns in their axial positions (Fig 2) as new photosensitizers for their use in photodynamic therapy. For different and multipurpose reasons, it has been chosen to incorporate a series of polyamine ligands on one face of the phthalocyanine core, while on the other face a series of hydrophobic ligands are incorporated. In this way, the obtained photosensitizer molecules will be amphiphilic, which is a desirable characteristic to facilitate their possibility to cross cell membranes and improve their cell uptake. Another reason to incorporate various polyamine chains in one of the ligands is the fact that under physiological pH these polyamine chains will be protonated. This is useful to target Gram negative bacteria, which, in contrast to Gram positive bacteria that only possess an inner cell membrane and peptidoglycan layer, also possess an outer negatively charged cell membrane. For this reason, for the photosensitizer to be able to cross this membrane it is required to be positively charged. Furthermore, polyamines are naturally occurring compounds that play multifunctional roles in a number of cell processes including cell proliferation and differentiation. Rapidly dividing cells such as tumor cells require large amounts of polyamines to sustain the rapid cell division. Part of these materials can be biosynthesized internally, while the majority is imported from exogenous sources through active and specific polyamine transporters (PAT). These features have led to the use of polyamines as potent vectors for the selective delivery of [4] chemotherapeutic and DNA-targeted drugs into cancer cells . In summary, we have designed and are currently preparing a library of ligands to incorporate in the silicon (IV) phthalocyanines, making use of different substitution patterns in their axial positions. An overview of the series of ligands to incorporate can be seen in Fig 2. The resulting amphiphilic phthalocyanines are expected to be non-aggregated in aqueous media, because of their axial substituents, and are expected to have an enhanced photoinduced singlet oxygen generation in the pH range from 5 to 7.

References [1] T. Hasan et al. Photochem. Photobiol. Sci., 3 (2004) 436卤450 [2] A. Almeida et al., Bioorg. & Med. Chem., 21 (2013) 4311-4318 [3] a) J.R. Cast贸n et al., Chem. Sci., 5 (2014) 575-581 b) J.L.M. Cornelissen et al., J. Am. Chem. Soc., 133(18) 2011 6878-6881 [4] a) D.K.P. Ng et al., J. Med. Chem., 54 (2011) 320-330 , b) D.K.P. Ng et al., Chem. Comm., 49 (2013) 4274-4276 Figures

Fig 1. Jablonsky diagram that illustrates the photophysical and photochemical processes occurring when a photosensitizer (PS) is irradiated for PDT purposes.

Fig 2. General structure of novel silicon phthalocyanine photosensitizers with different substitution patterns in their axial positions, and a representation of several of the ligands to be incorporated (top: series of polycationic ligands, bottom: series of hydrophobic ligands)

Calle Alfonso Gomez 17 Planta 2 - Loft 16 28037 Madrid (Spain) info@phantomsnet.net www.phantomsnet.net