Research in Focus: Nanomedicine and Drug Delivery

ISSUE THREE: JANUARY 2014

RESEARCH IN

FOCUS Nanomedicine and Drug Delivery Dr. Helen Burt, Dr. Kishor Wasan and Dr. Urs Hafeli

INTRODUCTION Nanomedicine is the medical discipline that investigates the use of nanotechnological devices, typically 10-200 nm in size.

Nanomedicine seeks to deliver a valuable set of research tools and clinically useful devices for use in advanced drug delivery systems, new therapies, and in in vivo imaging applications.

Nanomedical approaches to drug delivery centre on developing nanoscale particles or molecules to improve drug bioavailability and targeting. The future of nanomedicine will likely include self assembling biocompatible nanodevices that will detect, evaluate, treat and automatically report to the clinical doctor.

Nanoparticles, as drug delivery systems, can be designed to improve the pharmacological and therapeutic properties of drugs. The strength of nanoparticulate drug delivery systems is their ability to alter the pharmacokinetics and biodistribution of drugs.

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THE DR. HELEN BURT LAB Lab Members John Jackson, Lab Manager Dr. David Plackett, Research Associate Dr. Kevin Letchford, Part-time Assistant Professor Leon Wan, Graduate Student Rakhi Pandey, Graduate Student Aurora Chen, Graduate Student Donna Leung, Undergraduate Student In Whang, Undergraduate Student

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RESEARCH SUMMARY Research in Dr. Burt’s lab focuses on the development of polymeric drug delivery systems with a particular emphasis on nanomedicines. These systems provide benefits such as increased drug solubilization, controlled release and targeting for the treatment of conditions such as cancer, wound healing and infection.

SELECTED PROJECTS Superficial bladder cancer Objective: To develop mucoadhesive nanoformulations of taxane drugs for the intravesical treatment of bladder cancer.

Superficial bladder cancer is one of the most commonly diagnosed malignancies in North America.

This disease is commonly treated by surgical removal of the tumors followed by instillation of chemotherapy into the bladder via a urethral catheter (intravesical chemotherapy). Due to the short residence time of the instilled drug in the bladder and the extremely impermeable nature of the bladder wall, exposure of the drug to the tissue is severely limited. In a project led by Dr. Burt in collaboration with researchers in Chemistry, Pathology and Lab Medicine (Drs. Don Brooks and Jayachandran Kizhakkedathu), the Centre for Drug Research and Development, and clinicians in Urology (Drs. Martin Gleave and Alan So), we have developed a novel nanomedicine for the treatment of superficial bladder cancer.

This system is composed of highly branched biocompatible polymers called hyperbranched polyglycerols. These nanoparticles have been alkylderivatized to enable the solubilization of the poorly water soluble, anticancer drug docetaxel and further modified with amine groups that bind to the bladder wall and locally deliver the drug. When instilled intravesically into the bladder, these systems have been demonstrated to increase the uptake of the drug into the bladder tissue leading to marked improvements in the efficacy of docetaxel against bladder cancer.

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Nanoparticles for treatment of drug resistant tumors Objective: To use ultrasound to enhance the endocytosis of taxane-loaded nanoparticles (also containing a low molecular weight diblock copolymer inhibitor of p-glycoprotein) in order to reduce drug efflux and enhance cytotoxicity in drug resistant cells

Multidrug resistance (MDR) represents a major problem in cancer chemotherapy where cancer cells are resistant to a range of commonly used anticancer drugs. The major mechanism involved in MDR is the active transport of drugs out of the cells by efflux transport proteins, particularly p-glycoprotein (Pgp), located in the plasma membrane of the cells. In 2002.

Dr. Burt’s group was the first to develop a low molecular weight diblock polymeric inhibitor of Pgp. Since then, methods have been developed to locally and systemically deliver this inhibitor and drugs to resistant cells. In particular, we have been investigating the use of biocompatible and biodegradable amphiphilic block copolymers for the formation of nanoparticulate drug delivery systems for many years. These materials are particularly good at solubilizing poorly water soluble anticancer drugs such as paclitaxel and docetaxel, however; we have also shown that the polymeric Pgp inhibitor may be coencapsulated in these nanoparticles. Using the dual functionality of these block copolymers, we have created a nanoparticulate system that carries the cytotoxic agent as well as the inhibitor of Pgp. Additionally, we have demonstrated that ultrasound increases the RESEARCH IN FOCUS · 5

intracellular drug uptake of nanoparticles in drug resistant cancer cells.

In collaborative work with the Department of Mechanical Engineering at UBC, the effect of ultrasound produced by a micro-ultrasonic transducer on cellular uptake of drug loaded nanoparticles in MDR and drug sensitive cancer cell lines is being evaluated. Our strategy will involve using ultrasound to increase the uptake of nanoparticles into Pgp overexpressing cells thus creating a nanoparticle depot in the cells that will release the cytotoxic drug as well as the Pgp inhibitor.

Burn wound dressings for use in remote areas of Africa Objective: To develop an inexpensive anti-infective wound healing film for inclusion in a first response burn kit for use in remote African villages.

In the developing world and especially in areas of sub-Saharan Africa, the occurrence of burn wounds through accidents with open fires is a major health issue, particularly where children are concerned. In many such cases, it can take days to reach proper medical attention at a suitable hospital, during which time serious infection can take hold. In 2012, Dr. Robin Evans, a plastic surgeon from Campbell River, B.C., successfully obtained funding from Grand Challenges Canada to develop a simple and inexpensive burn wound dressing kit that could be made available in remote African villages.

In addition to the dressing, the kit includes pictorial instructions for use, rehydration salts and a cell phone for communication to the nearest health centre. Dr Evans requested input from the Burt group on suitable materials for the wound dressing and, over the past year, the group has worked on the potential use of biocompatible films composed of poly(vinyl alcohol) (PVA) and a silver salt as the core dressing material. The key idea has been the heat curing of PVA/silver films, which results in silver-based cross-linking of PVA and the reduction of silver to antibiotic, silver metal nanoparticles within the films. These outcomes meet two of the key project requirements: 1) a dressing material which can stay in place for 1-2 weeks before changing, and 2) sustained release of antibiotic silver over the same period. Silver release from such films prepared in the Burt laboratory has been characterized as a function of various processing conditions and, in cooperation with Dirk Lange in the UBC Department of Urological Sciences, the antibiotic activity of the films has been confirmed. Research on formulation optimization is continuing and connections with other project partners are now focused on scaled-up wound dressing manufacturing and information that may be required for 6 路 RESEARCH IN FOCUS

Health Canada approval.

Remotely actuated drug delivery implants for the treatment of diabetic retinopathy or prostate cancer Objective: To develop a small implantable polymeric device containing antiproliferative drugs that may be magnetically actuated to deliver exact doses of drugs on demand for the treatment of diabetic retinopathy or prostate cancer.

Diabetic retinopathy is the major cause of visual loss in middle-aged adults.

In proliferative diabetic retinopathy, unwanted proliferation of capillary cells in the retina compromises retina function, causing vision loss. Currently, delivery of therapeutic agents to the retina is via intravitreal injection of controlled release implants, which do not have the ability to stop or modulate release after injection. In collaboration with Dr. Mu Chaio (Department of Mechanical Engineering, UBC) and Dr. Nazly Pirmoradi (University of California, Berkeley), we have recently reported the development of a microelectromechanical system (MEMS) that delivers uniform doses of the antiproliferative drug docetaxel (DTX) on demand to the retina following placement of the device behind the eye. The flexible polymeric disc-shaped device measures approximately 1 cm (diameter) by 3 mm (thickness) and contains a reservoir of dry drug covered by a magnetic membrane containing a laser cut hole. A small external permanent magnet deflects the membrane so that fluid enters the reservoir, dissolves the drug up to the solubility limit (7懦g/ml) and when the magnet is released an exact amount of drug is delivered locally for diffusion through the sclera and into the retina. The device has been demonstrated to deliver effective doses of DTX to the target retina tissue in vivo and in human eyes ex vivo.

Using similar technology, this research team is also developing a device for the treatment of prostate cancer. Improved prostate cancer detection has led to more men being diagnosed at earlier ages. For men with prostate cancer undergoing active surveillance (AS) there are no treatment options, thus leading to poor psychological consequences. In this proposal a simple magnetically actuated drug delivery device may be non-invasively implanted in the prostate so that the patient may trigger a daily dose of docetaxel (DTX) using a magnetic rectal probe. It is proposed that this patientempowering treatment may release exact doses of drug to suppress tumor growth and positively prolong the duration of AS so that many older patients may never need radiation or surgical intervention. The device would be RESEARCH IN FOCUS 路 7

implanted using a surgical needle and oriented in alignment with the rectal cavity with the magnetic membrane closest to the cavity. DTX is an effective cytotoxic agent at concentrations as low as 10 ng/ml and it is anticipated that a single insertion of the magnetic rod in the rectum for 1 minute will release 200 ng of DTX from the device in the prostate. The device may be actuated at a distance of up to 4 cm with a preferred distance of 1-2 cm.

Nanocrystalline cellulose (cnc) as a new vehicle for controlled drug delivery Objective: To demonstrate the potential value of nanocellulose as a new material for controlled binding and release of therapeutic agents.

With initial support from FP Innovations and, subsequently, as a participant in the Arboranano network, the Burt group at UBC was the first to carry out and publish a thorough study of drug binding and release from nanocrystalline cellulose (CNC). This nanomaterial is attractive for this purpose because of its very high surface area-to-volume ratio, a surface charge allowing ionic bonding of drugs, and its often-reported biocompatibility. Furthermore, the hydroxyl groups on the surface of cellulose provide numerous opportunities for chemical modification and this in turn offers great versatility in terms of attaching a wide variety of pharmaceutical actives. Since the initial work, which showed that hydrophilic antibiotics such as tetracycline and doxorubicin could be bound to CNC and hydrophobic anti-cancer drugs such as paclitaxel and docetaxel could be bound to a quaternary ammonium salt-modified CNC, similar findings have been obtained with gentamicin, an aminoglycoside antibiotic, and with silver, which has attracted increased interest as an anti-infective because of concerns about the prevalence of multi-drug resistant bacteria. Further, we have demonstrated that drug/CNC combinations can be incorporated in electrospun polymer mats for potential application in areas such as wound healing and transdermal drug delivery. In more recent work, we have expanded our interests into the use of nanofibrillated cellulose (NFC), an alternative form of nanocellulose, and are also now exploring cationic forms of nanocellulose, which appear to have intrinsic antimicrobial activity. A review of nanocellulose and drug delivery has recently been submitted to an international journal and we are now looking for opportunities to fund further research in this exciting new field.

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THE DR. KISHOR WASAN LAB Lab Members Dr. Kristina Sachs-Barrable, Research Associate Dr. Fady Ibrahim, Postdoctoral Fellow Olena Sivak, Research Scientist/Technician Jinying Zhao, Research Scientist/Technician Jo-Ann Osei-Twum, Graduate Student Jenny Kim, Graduate Student Ankur Midha, Graduate Student Jacob Gordon, Graduate Student Carly Wong, Summer Student Riley Walsh, Summer Student Elaine Xu, Summer Student Gabriela Missassi, Summer Student Jocelyn Conway, Coordinator, NGDI UBC Raquel Baldwinson, Database Coordinator and Graduate Student, NGDI UBC

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RESEARCH SUMMARY

Over the past 11 years, the Wasan Lab has published a number of studies and has established the experimental methodologies necessary to justify the importance of investigating the role of lipids and lipoproteins in modifying the biological activity of water-insoluble drugs. With these research tools in place, the Wasan Lab has now demonstrated and provided the potential mechanisms by which water-insoluble drugs interact with lipids and lipoproteins and how these interactions impact on the absorption, distribution, efficacy, toxicity and metabolism of such compounds.

In the larger perspective, these studies have increased the understanding of the mechanisms involved in serum distribution of hydrophobic drugs.

In contrast to albumin protein binding, lipoprotein binding of drugs is often overlooked and so the role of lipoproteins as possible intravascular carriers for hydrophobic compounds and their involvement in modifying the biological effects of drugs is a novel and pharmaceutically important discovery. Recently, the U.S. Food and Drug Administration (Spring 2002) has suggested that lipoprotein-drug distribution studies should be considered as part of any new Investigational New Drug application that contains a hydrophobic compound. In addition, many pharmaceutical companies screen hydrophobic compounds for plasma lipoprotein distribution.

The Wasan Lab is actively engaged in the UBC-Neglected Global Diseases 10 路 RESEARCH IN FOCUS

Initiative and recently has published work in this area.

SELECTED PROJECTS Investigation into whether the lipoprotein binding of drugs (i.e.

Amphotericin B) modify their pharmacological toxicity and plasma disposition(i.e. pharmacokinetics) Collectively our research in this area has been cited over 350 times in the past decade. A number of key papers with several compounds (i.e.

Amphotericin B, Cyclosporine, Halofantrine, Nystatin, E5564, Clozapine) have been published demonstrating the importance of drug association with plasma lipoproteins.

Investigating the cardiovascular effects of a novel class of breast cancer agents called aromatase inhibitors in post-menopausal women A study that reported that the aromatase inhibitor, Letrozole, does not significantly alter serum cholesterol, HDL cholesterol, LDL cholesterol, triglycerides or Lp(a) in non-hyperlidiemic postmenopausal women with primary breast cancer treated up to 36 months following at least 5 years of adjuvant tamoxifen therapy. These findings further support the tolerability of extended adjuvant letrozole in postmenopausal women following standard tamoxifen therapy (Ann Oncol. 2005; 16(5):707-15).

Published research investigating the role of lipid profiles in schizophrenia therapy The first study reported that Risperidone augmentation of clozapine for treatment resistant schizophrenia offered no benefit for severity of symptoms, and may increase the risk for cognitive impairment and glucose dysregulation (accepted in The New England Journal of Medicine, November 8 2005; In Press February 2006).

The second study reported that modified in vitro lipid profiles can modify the lipoprotein distribution of clozapine (Am. J. Psychiatry 158:949-951, June 2001).

Amphotericin B for treatment of Visceral Leishmaniasis Physiological Role of P-Glycoprotein in Regulating the Gastrointestinal (GI) Absorption and Cellular Transport of Cholesterol Despite an increase in research, treatment options and public awareness RESEARCH IN FOCUS 路 11

of risk factors, cardiovascular disease remains the leading cause of death in Canada. Elevated plasma cholesterol levels, smoking and hypertension are some of the major contributing risk factors in this fatal disease. Although cessations of smoking and pharmaceutical treatment of hypertension are both effective means of reducing the risk of cardiovascular disease, a universally effective cholesterol-lowering treatment regime remains elusive.

The development of HMG-CoA reductase inhibitors (statins) in the early ‘80s was heralded as a solution to hypercholesterolemia, yet the rate of decrease in average total cholesterol levels among North Americans over the last decade has failed to meet expectations. Despite the widespread acceptance of dietary therapy and use of statins as means of reducing plasma cholesterol, the clinical promise of statins remains largely unfulfilled, indicating a need for alternate target mechanisms.

Cholesterol homeostasis in the cell is regulated by a complex set of mechanisms that include cholesterol biosynthesis, hydrolysis from lipoproteins internalized into lysosomes and bidirectional transport of cholesterol to the endoplasmic reticulum (ER) where cholesterol undergoes esterification, and from the ER to the plasma membrane (PM). Unlike any other cell type within the body, the intestinal absorptive cell is bathed at its apical surface with both dietary and biliary cholesterol. Transport of dietary cholesterol into the enterocyte contributes significantly to the plasma cholesterol pool and to maintaining whole body cholesterol balance.

Since absorbed cholesterol is likely the major source of cholesterol for the intestinal cell, understanding its transport and utilization is of importance and will provide valuable insights into the regulatory pathways of cholesterol.

With the continuous influx of cholesterol at the apical membrane, cholesterol synthesis and low-density lipoprotein receptor expression are suppressed.

Understanding the regulation of enterocyte cholesterol levels and the associated mechanisms of suppression may provide alternate targets for pharmaceutical lipid-lowering therapies. Class I P-glycoproteins [Pgp; encoded by the MDR-1 (humans) & MDR-1a/1b (mice) genes] are integral cell membrane proteins that were originally identified in multidrug-resistant tumor cells.

The original functions elucidated were to reduce intracellular concentrations of structurally diverse chemotherapeutic agents and to act as efflux transporters of xenobiotics from the small intestine enterocytes back into 12 ¡ RESEARCH IN FOCUS

the intestinal lumen resulting in decreased drug bioavailability. Preliminary studies have reported that nonspecific inhibitors of Pgp inhibit synthesis and esterification of cholesterol putatively by blocking trafficking of cholesterol from the PM to ER and that relative increases in Pgp within a given cell type are associated with increased accumulation of cholesterol.

These findings provide indirect evidence supporting a physiologic function for Pgp in regulating cholesterol homeostasis. However, direct evidence is required. The Wasan Lab is testing the hypothesis “that class I Pgp has a direct physiological function in regulating the gastrointestinal absorption and intracellular transport of cholesterol� by completing two specific aims: a) determining the cholesterol GI absorption and tissue distribution in MDR1a-/-/1b-/- knock-out mice fed low, standard and high cholesterol and fat enriched diets and b) determining whether modulation of MDR-1 Pgp expression and activity results in altered cholesterol uptake and intracellular transport from the PM to the ER within human intestinal cells.

Image: Infection cycle for the development of leishmaniasis RESEARCH IN FOCUS ¡ 13

THE DR. URS HAFELI LAB

Image: The Hafeli Lab

Lab Members Dr. Kathy Saatchi, Research Associate Dr. Thomas Schneider, Postdoctoral Fellow Chris (Tianxing) Gong, PhD Student Christian Buchwalder, PhD Student José de la Vega, PhD Student Mehrdad Bokharaei, PhD Student Sahan Ranamukhaarachchi, PhD Student Veronika Schmitt, PhD Student Dana Lambert, Graduate Student Nadja Mallock, Graduate Student René Pedroza, Graduate Student Tullio Esposito, Graduate Student Anna Löwa, Undergraduate Student Vitor Cid, Undergraduate Student

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RESEARCH SUMMARY

Research in the Hafeli Laboratory is primarily directed at fighting cancer with radioactive pharmaceuticals and the development of diagnostic radiopharmaceuticals to be used in different Nuclear Medicine procedures.

Dr. Hafeli also enjoys adapting nanotechnology and microtechnology for different drug delivery applications, including the development of painless microneedles and the use of microfluidics for the preparation of monosized microspheres. Under the guidance of Research Associate Dr. Kathy Saatchi, the Hafeli Laboratory has a strong chemistry base that encompasses organic, coordination and polymer chemistry. Using these tools, Dr. Hafeli’s research includes the entire spectrum of drug research from the synthesis and radiolabelling of new molecules; the preparation of drug delivery carriers such as nanoparticles, microspheres, antibodies, and polymers; the careful evaluation of drug delivery systems in vitro and in toxicology experiments; and finally, efficacy testing in different in vivo models using many different imaging modalities (SPECT, PET, CT, MRI, optical imaging).

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SELECTED PROJECTS Radiopharmaceuticals for cancer therapy and disease detection Radioactive drugs can be used for many therapeutic purposes such as cancer treatment or scar prevention. Radiopharmaceuticals, the general name for radiolabeled diagnostic and therapeutic agents, can take many different shapes including particles sized from tens of nanometers (= nanospheres) up to about 100 micrometers (= microspheres), viscous solutions and micellar/liposomal suspensions, sheets, and even metal implants such as stents (metal or plastic coils) or foils. Our lab is interested in preparing radioactively labelled drug delivery vehicles and using them to kill tumours and prevent their reoccurrence. The image to the left shows a radiopharmaceutical that we recently prepared and tested as a lung perfusion imaging agent. In a healthy animal, it perfectly outlines the lungs, without uptake in any other organ.

The main radioactive isotopes we are currently investigating are the betaemitters rhenium-188 (Re-188), yttrium-90 (Y-90), and their diagnostic counterparts technetium-99m (Tc-99m), indium-111 (In-111) and gallium-67 (Ga-67). Some PET isotope are also included, such as fluorine-18 (F-18), gallium-68 (Ga-68), and zirconium-89 (Zr-89). We also use other isotopes such as iodine-125 (I-125), iodine-123 (I-123), palladium-103 (Pd-103) and iridium-192 (Ir-192).

Diagnostic imaging with our new preclinical SPECT/PET/CT imager Together with Vesna Sossi (Physics) and Paul Schaffer (TRIUMF), we were successful in obtaining a Leaders Opportunity Fund grant titled “Hybrid microPET/SPECT and CT preclinical imaging.” This allowed us to purchase a MILABS VECTor/CT instrument to UBC that allows for simultaneous PET and SPECT imaging at resolutions not previously achievable. The instrument was installed in July 2012 at the new Centre for Comparative Medicine (CCM) and is connected to TRIUMF with a direct underground hydraulic tube line from their radioisotope production facility. This pressurized tube allows the delivery of vials with freshly prepared radioactive isotopes 16 · RESEARCH IN FOCUS

directly from TRIUMF to the imaging room in the CCM facility. Many of the radioisotopes used in imaging are short lived (e.g., C-11 has a halflife of 20 min, N-13 of 10 min) and are, thus, only useful with ultrarapid transport and chemistry. The new scanner will be used to develop novel radiopharmaceuticals, to investigate diseases, to carry out brain research, and to enhance imaging physics and processing.

Magnetic microspheres for targeted drug delivery One of the main problems with cancer therapy is not the lack of efficient drugs, but that these drugs are very difficult to concentrate in the tumour tissue without leading to toxic effects on neighbouring organs and tissues.

One solution is magnetic drug delivery with particulate carriers, a very efficient method of delivering a drug to a localized disease site.

The figure to the left highlights the concept of magnetic targeting by comparing it with systemic drug delivery. In magnetic targeting, a drug or therapeutic radioisotope is bound to a magnetic compound, injected into a patient’s blood stream, and then stopped with a powerful magnetic field in the target area. Depending on the type of drug, it is then slowly released from the magnetic carriers (e.g., release of chemotherapeutic drugs from magnetic microspheres) or confers a local effect (e.g., irradiation from radioactive microspheres; hyperthermia with magnetic nanoparticles).

Small amounts of drugs targeted magnetically to localized disease sites can thus possibly replace large amounts of freely circulating drug and reach effective and up to several-fold increased localized drug levels.

In our lab, we are currently investigating new ways of preparing uniform magnetic microspheres with a microchip based flow focusing method. In this method, the polymer is dissolved and pumped through an orifice where the liquid stream breaks up into monosized droplets. The solvent is then extracted into the surrounding water phase, so that each droplet turns into a microsphere made from biodegradable materials and appropriate RESEARCH IN FOCUS · 17

for human use. We are also interested in evaluating the toxicity of the whole microspheres and the small magnetic nanoparticles which give them their magnetic properties. These investigations are based on cell survival experiments and confocal microscopy investigations over time in cell cultures.

Development of microneedles for pain-free drug therapy and diagnosis In collaboration with Professor Boris Stoeber, Departments of Mechanical Engineering & Electrical and Computer Engineering at UBC, the Hafeli Lab is working on the use of silicon and polymer-based microneedles. Typically up to 500 懦m high, they allow for the penetration of the skin without touching the (deeper) nerve endings, and thus permit pain-free drug delivery to areas in the skin. From there, the drug can either diffuse to the blood capillaries which distributes it to other organs and tissues, or it can exert a local effect.

The best example for the latter is the use of vaccines bound to nanoparticles, which stay in the epidermis after painless injection and involve the immune system to generate protection against many different bacterial and viral diseases. Another novel use of microneedles is the pain-free extraction of interstitial fluid to determine the levels of drugs in the body of a patient.

Currently, the most effective hollow microneedles that the Hafeli lab prepares in the clean room at UBC are made from biocompatible metal and can be easily made into large arrays. To obtain the optimal microneedle shape that easily penetrates skin, an artificial skin model was made from different transparent layers, which together with a purpose-built test station, allows to optically observe the needle passing through the different skin layers while at the same time measuring the applied forces.

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