Nanomedicine for targed drug delivery

FEATURE ARTICLE

www.rsc.org/materials | Journal of Materials Chemistry

Nanomedicine for targeted drug delivery Do Kyung Kim* and Jon Dobson* Received 9th February 2009, Accepted 24th April 2009 First published as an Advance Article on the web 4th June 2009 DOI: 10.1039/b902711b In recent years, nanoparticles have played an ever increasing role in biomedical research and clinical applications. The unique physical properties of nanomaterials are being exploited in the field of nanomedicine for applications as diverse as drug delivery and targeting, MRI contrast enhancement, gene therapy, biomarkers, targeted hyperthermia and many others. This review focuses on the design, synthesis and unique properties of nanoparticles used in nanomedicine as well as on clinical uses for both diagnosis and treatment of disease.

1. Introduction The application of nanotechnology to biology and medicine— bionanotechnology—is a growing field. Though research in this area has undergone rapid expansion in the last decade, many applications have not yet made it to routine clinical use.

However, the potential impact of nanotechnology is broad as nanoscale structures are sufficiently tiny to facilitate unique interactions with most biological components (e.g., viruses, organelles, proteins, DNA) at the molecular level. Particularly impressive achievements are being made in the fields of diagnosis and detection by MRI1 and fluorescent quantum dots,2 targeted and controlled drug/gene delivery,3 and cancer treatment by hyperthermia,4 and this work has given rise to a new, emerging discipline known as nanomedicine.

Within the field of cancer research, there is great scope for applying new techniques in nanomedicine to the diagnosis and

Institute for Science & Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom ST4 7QB. E-mail: d.k.kim@pmed. keele.ac.uk; j.p.dobson@bemp.keele.ac.uk

Do Kyung Kim received the Ph.D. degree in materials chemistry (nano-bio) from the Royal Institute of Technology (KTH) in Sweden, 2002. He was a Postdoctoral Fellow in the Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT), in 2003. He is currently a lecturer in the Institute of Science and Technology in Medicine (ISTM, rated 5A in the 2001 RAE exercise), Keele University, U.K. His research interests include target oriented drug delivery systems, nanocomposites, quantum dots, and bulk production of nanomaterials for energy applications.

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treatment of these diseases. Conventional anticancer modalities such as surgery, radiotherapy, chemotherapy, hormones and immunotherapy have provided improvements to the successful treatment of neoplastic disease. However, each of these treatments has advantages and disadvantages, consequently combined treatment modalities are often recommended to achieve the optimum effects. Chemotherapy, for example, is a whole body treatment which is administered either orally or intravenously. This results in the systemic distribution of cytotoxic, chemotherapeutic compounds which can be more effective for the treatment of micrometastases. Unfortunately, the systemic distribution of cytotoxic compounds usually results in more serious side-effects (anemia, vomiting, diarrhea, nausea, decreased infection resistance, and an increased likelihood of hemorrhaging, hair loss), some of which can be life-threatening, compared to surgery or radiotherapy, and these treatments are often used in combination. The major goal of targeted therapies is to reduce the side-effects which result from systemic distribution of cytotoxic drugs in order to more effectively control cancer cell proliferation or tumor angiogenesis.5 The past few decades have seen great improvements in chemotherapy, leading to increases in survival rates, but there is

Jon Dobson received a B.Sc. and M.Sc. from the University of Florida, and a Ph.D. from the Swiss Federal Institute of Technology (ETH-Zurich) in 1991.

He did his postdoctoral research in the Department of Physics, Institute of Geophysics, ETHZurich, before taking a lectureship in Biophysics in the Department of Physics at the University of Western Australia.

He is currently a Professor in Biophysics and Biomedical Engineering at Keele University and Eminent Scholar/Visiting Professor at the University of Florida.

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still a critical need to develop more refined therapies which increase survival and the quality of life for cancer patients. The enormous technical advances that have occurred in the area of basic cancer research have not yet been paralleled by similar improvements in treatment results. Thus, current research is more focused on a better understanding of the pathophysiology of neoplastic diseases at the cellular and molecular level in order to develop more specific cellular and molecular targeting therapies.

Despite advances in cancer therapies, side effects of systemically administered drugs repeatedly remain a dose-limiting factor in therapeutic protocols. Self-directed localization of therapeutic agents to the disease-affected target tissue has been proposed to overcome this problem. For tumor targeting, disease-specific, tumor-associated antigens have been exploited for this purpose,6 but the selective uptake of chemotherapeutic agents by tumor tissues remains a great challenge. For this reason, massive efforts have been devoted to the development of ‘‘smart drugs’’ that can be directed to tumor cell-specific enzymes and surface receptors.

This is an area where bionanotechnology is making great strides.

At present, there are numerous anticancer agents available in the clinic. These anticancer agents/drugs have an elimination half-life which results in a decrease of the therapeutic potential and side-effects such as bone marrow depression and gastrointestinal damage. Novel strategies have been suggested to decrease the toxicity of active molecules by targeting the specific tumor site, where the drug can selectively bind to the targeted tissue at a cellular and/or sub-cellular level to influence its therapeutic effects. Chemotherapeutic activity can be enhanced by using macromolecules as a vector to control the release rate of anticancer agents. For example, polyalkylcyanoacrylate nanoparticles as drug delivery systems (DDSs) play an important role in the incorporation of anticancer drugs as they can enhance the drug’s concentration in the tumor sites and decrease drug levels in the heart, thus avoiding some side-effects.7 Polymeric nanoparticles are being used to control the loaded anticancer agent/drug at the targeted site. In a general sense, polymeric nanoparticles as used in nanomedicine consist of core-shell nanocapsules that may incorporate therapeutic agents either within the particle or attached to the outside. Several kinds of more basic inorganic and metallic nanoparticles without the sophisticated structures may also be used since their intrinsic physical properties may be exploited for targeting. Superparamagnetic iron oxide nanoparticles (SPION) in particular can be used as both a targeting agent and a therapeutic by combining their responses to external AC and DC magnetic fields.8 In spite of major advances in the development of small-scale devices, however, the majority of DDSs continue to employ small molecules administrated orally, transdermally, parenterally, or through the nose or lung. Target-oriented DDSs, due to their specificity, should allow for the use of lower doses of anti-cancer agents, reducing the side-effects associated with systemic distribution.9 The development of novel, smart biomaterials is already beginning to have an enormous effect on nanomedicine.10 Many synthesis techniques used to produce nanoparticles for cancer therapy have focused on empirical analysis for the development of controlled-release anticancer agents and the increasing demand for multi-functional vectors in nanomedicine still represents a major fabrication challenge. However, design This journal is ª The Royal Society of Chemistry 2009

criteria can be proposed which integrate several aspects; (i) theoretical and practical consideration of the novel phenomena resulting from the particle’s composition and small size, (ii) design of complex or composite structures with specific morphologies and surface chemistry required for multi-functionality, (iii) generation and assembly of new molecular and macromolecular structures using suitable processing methods, (iv) methods of incorporation of pharmaceutical agents to be delivered to the target cells by active or passive targeting, and (v) modification of the particles’ surfaces and interfaces which render them suitable for interaction with the pathological sites.

Moreover, novel concepts are needed for the development of nanoparticles for cancer-oriented drug delivery systems (CoDDS) with core-shell or mesoporous structures. These carrier vectors then can be programmed to respond in a variety of ways to external stimulation, e.g. pH, ionic strength, temperature, ultrasound, radiation, magnetic fields, UV-light etc. The development of suitable nanostructures, methodologies for drug incorporation, methodologies for controlling release rates, toxicology and biological activity therefore should be considered during the design phase.

2. Design of nanoparticles for cancer therapy and their prerequisites 2.1 Nanomaterials in biological systems Nanomedicine is concerned with the development of advanced, multifunctional, and even smarter ‘‘smart’’ materials for specific applications in this highly integrated field. Recently, these interdisciplinary concepts have been converging at the intersection of nanotechnology and molecular biotechnology. They are closely associated with surface chemistry and the physical properties of nanomaterials, the topics of bio-organic and bioinorganic chemistry, and the various aspects of molecular biology, recombinant DNA technology and protein expression, and immunology.

The development of micro/nanospheres has become an important area of research as such systems enable the controlled-release of cytotoxic drugs directly into the pathological sites. They also make it possible to transport therapeutic agents into sites of inflammation or neoplastic diseases. The in vivo application of nanomaterials require an understanding of the fundamental mechanisms of their behavior in biological systems. The effectiveness of targeted nanomedicine can be evaluated by considering several phenomena depending on injection sites or extravascular routes. For intravenous injection the primary factors will be the distribution, elimination and metabolism of nanomaterials in vivo, whereas extravascular injection includes more factors such as cellular uptake (endocytosis; phagocytosis and pinocytosis, receptor-meditated endocytosis).

Endocytosis is a process whereby cells absorb materials (small particles, molecules and liquids) from the extracellular space by engulfing them with their cell membrane. This mechanism is used by all cells in the body as many substances important to cellular function are polar and consist of large molecules, and thus cannot pass through the hydrophobic plasma membrane. Internalization of the nanomedicine at J. Mater. Chem., 2009, 19, 6294–6307 | 6295

undesired sites due to endocytosis can be eliminated by applying stealth coatings on the surface of nanomaterials via manipulation of the surface chemistry. Generally, when these nanoparticles are injected intravenously, they will be captured by macrophages of the mononuclear phagocyte system (MPS; liver, spleen, lungs and bone marrow). Once in the bloodstream, surface non-modified nanoparticles (conventional nanoparticles) are rapidly taken up and massively cleared by the macrophages.11 The stealth coating can both enhance the antitumor efficacy, with a high concentration of the therapeutic agents localized in pathological sites, and prolong the agent’s half-life in the blood.

One of the most important characteristics for nanomedicines is their biocompatibility, especially with respect to their surface chemistry. Biocompatibility of nanomaterials can be improved by modifying the terminal groups on the surface of the nanomaterials as well as their constituents. Obviously, it is important to stabilize the nanomaterials sterically by chemical modification or by attaching an outer shell, which minimizes the recognition by the mononuclear phagocyte system (MPS) in the reticuloendothelial system (RES).12 Essential properties of any multifunctional nanomedicine carrier are its durability and long-circulating pharmaceutical effects. One of the main reasons for producing long-circulating drugs is to maintain the required therapeutic level of the pharmaceutical agent in the blood for extended time periods.

Subsequently, long-circulating, drug-containing microparticulates or large macromolecular aggregates will accumulate slowly (the enhanced permeability and retention—EPR effect— also termed ‘‘passive’’ targeting or accumulation via an impaired filtration mechanism) in pathological sites with affected and leaky vasculature, and facilitate controlled release in those areas.

In addition, the prolonged circulation half-life produces better targeting effects for specific ligand-modified drugs and drug carriers allowing more time for their interaction with the pathological sites due to the larger number of passages of pharmaceuticals through the target.13

2.2 Nanomaterials as potential carrier vectors of therapeutic agents The extracellular matrix biology, cell receptors and immunology should be considered during the development of artificial synthetic nanomedicines for CoDDSs, together with an understanding of how the human body reacts to the specific substances. It should be possible to incorporate hydrophobic/ hydrophilic drugs with active surface modifications into the nanomedicine by the attachment of active functional ligands for targeting specific organs, receptors, etc.

Nanoparticles (Fig. 1A). Colloids are representative of nanomaterials stabilized in solution to prevent uncontrolled size growth, aggregation, and flocculation. Utilization of colloidal processing leads to attractive novel concepts for the preparation of advanced nanostructured materials. Several parameters of the colloidal systems have been considered, such as temperature, osmolality and pH of the polymerization medium, which could influence the characteristics (morphology and morphometry, drug content, melting point transition or the enthalpy of transition) and stability of the nanoparticles. Based on their unique mesoscopic physical, chemical, thermal and mechanical properties, nanoparticles offer great potential for many biomedical applications, including bioanalysis and bioseparation, tissue-specific drug therapy applications, gene and radionuclide delivery.14 Many studies have been focused on colloidal processing of polymeric, inorganic and metallic materials through chemical methods. The candidate nanoparticles for nanomedicine may be either amorphous or crystalline and may have particular physical characteristics, such as optical, magnetic, fluorescence, and electric properties. With semiconductor Quantum Dots (QDs), i.e.

CdS/CdSe, as the particle sizes decrease, quantum effects cause the material to fluoresce under ultra-violet or infrared light. By modifying the functional groups on the surface, the bioactivity of QD nanocrystals can be directed. Consequently, the luminescent effects of QDs can contribute to fluorescence-based techniques in

Fig. 1 Generalized schematic representation of nanomedicine constituents. For example, A can be a nanomaterial core such as an iron oxide, B represents surface functional groups, C is a biocompatible layer such as polymer or novel metal, and D is a functional group to conjugate further active targeting agents such as an antibody. Any flexible combination such as A + B, A + B + C, A + D and A + B + C + D provides the potential to construct a nanomedicine.

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Table 1 Examples of nanoscale platforms for nanomedicine (Fig. 1A) Composition

Size (nm)

Applications

Ref.

Metals Au Ag Pt Co

2–150 1–80 1–20 1–50

Drug and gene delivery Antibody tagged marker Sensors and electrodes Magnetic separation, drug targeting

16 17 18 19

Fluorescent labeling Fluorescent labeling Infrared photodetectors Biomedical devices for nerve tissue monitoring Photoluminescence Nonlinear optics Photoluminescence

20 21 22 23

MR contrast agent, Drug Delivery, Hyperthermia MR contrast agent, Drug Delivery

27

1–10

Drug/Gene delivery

29

100–250 1–10

Drug/Gene delivery Drug/Gene delivery, MR contrast agent

30 31

Semiconductors (Quantum Dots) CdX(X ¼ S, Se, Te) 1–20 ZnX(X ¼ S, Se, Te) 1–20 PbS 2–18 3–50 TiO2 ZnO GaAs, InP Ge Magnetic Fe–O

1–30 1–15 6–30

Fe–Pt

2–10

Nanotube (NT) Carbon NTs Polymer Liposomes Dendrimer

6–40

24 25 26

28

biomedicine. A major drawback, however, is that compounds containing cadmium are cytotoxic, even at low concentrations, and will accumulate in organisms and ecosystems. One possible explanation for its cytotoxicity is that it interferes with the action of zinc-containing enzymes and the cytotoxic effects of the nanomaterials themselves is a rather controversial and topical issue at present.15 Thus the potential cytotoxicity of the core materials should be considered when developing nanomedicines.

Table 1 gives an overview of some of the examples of nanoparticles being developed for use as nanomedicines.

Magnetic nanoparticles or QDs are particularly attractive as core materials when there is a requirement to monitor the distribution of the sample after injecting intravenously or via an extravascular route. Furthermore, magnetic nanoparticles can be non-invasively traced by in vivo MRI techniques and QDs can be monitored by in vivo/vitro near-IR. Consequently the experimental/therapeutic aims and conditions, such as in-vivo/in-vitro and instrumentation techniques, should be considered during the design of proper nanomedicines.

After selecting suitable core materials, the colloidal behavior of the nanoparticles in the dispersion medium should be considered. They can be constructed to absorb, conjugate and encapsulate therapeutic agents inside or outside. The nanoparticles can be prepared by various chemical synthesis routes, such as microemulsion, chemical co-precipitation, thermal reduction and polymerization etc. Although the as-prepared nanoparticles may be dispersed within the colloid, the particles may agglomerate and precipitate when transferred into PBS buffer solution due to the presence of different ion species in the medium and changes in the zeta-potential of the particles. When the particles are designed for intravenous injection, the surface This journal is ª The Royal Society of Chemistry 2009

charge of the particles in the biological media is an important parameter to be considered.

Surface modifications (Fig. 1B and D). Uncoated nanoparticles as carrier vectors are not highly suitable due to concerns associated with the complicated preparation of stable colloidal suspensions with low cytotoxicity. As a consequence, surface modification with biocompatible substances is essential.

Integrating organic molecules with inorganic nanostructures has yielded exciting results in the field of nanomedicine. Organic molecules have been used as surface coatings to prevent unnecessary nanoparticle aggregation, as molecules to direct nanoparticle assembly, and as homing devices to target nanostructures to specific biological sites. Furthermore, organic molecules can enhance functional capabilities in nanostructured materials.

The ability to assemble nanostructures requires accurate control of the particle’s surface chemistry, where functional molecules (carboxyl, hydroxyl, thiol, or amine) can be directly assembled onto the surface of nanomaterials. The surface of metallic nanoparticles such as Au or Pt colloids can be easily modified with recognition biomolecules such as weak ligands that can be easily desorbed onto the surface. For instance, proteins and transferrin can be physically adsorbed onto the surface of citrate-stabilized Au nanoparticles through ionic and hydrophobic interactions as well as dative binding. However, modification of the surface structure of most inorganic nanoparticles is rather more complicated than metallic nanoparticles, thus additional coating steps with dual functional agents like a (3-aminopropyl) trimethoxysilane (APTMS) have been proposed to activate the reactive amine groups. An amphiphilic polymeric layer has been introduced onto the surface of QDs to activate the carboxyl groups. The primary amine group in biomolecules can be covalently bonded to the amphiphilic polymer with a crosslinking agent, EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), forming an amide bond linkage between the QDs and the biomolecules.

Three different types of magnetic colloids can be prepared through various stabilization methods (Fig. 2). The first

Fig. 2 Schematic representation of three different types of magnetic colloids: (a) small (typically 12 nm) core-shell structure magnetite nanoparticles with their magnetic dipole–dipole interactions screened by a layer of surfactant; (b) prepared in a polymeric matrix with a primary agglomeration size of 35 nm; (c) MPEG modified SPION (primary agglomeration size is 120 nm).32–34

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magnetic colloid is prepared by coating the magnetic core with a suitable surfactant such as sodium oleate (anionic surfactant, CH3(CH2)7CH]CH(CH2)7COONa). The second stabilization method is produced by the formation of nanocomposites consisting of magnetic nanoparticles distributed throughout a matrix and then the grafting of a non-magnetic coating, e.g. polymeric starch. The presence of polymeric starch hinders the cluster growth after nucleation during the formation of SPIONs. These polymeric networks cover a large number of continuously formed iron oxide monodomains and hold them apart against attracting forces by surface tension. In addition, the formation of a polymeric layer on the surface of the magnetite nanoparticles prevents further crucial oxidation, which affects phase transformation. The third magnetic colloid suspension is stabilized, in a similar manner to that of starch, by MPEG immobilization on the SPION surface.

The introduction of amine groups on the surface of SPION has been performed by the two procedures depicted in Fig. 3: (a) silanization and (b) chemisorption processes. The silanization process is based on the covalent binding of APTMS to SPION. The silane coupling agent, usually called an organosilane, has the following structure: RxSiY(4 x). Silicon is located at the centre of the molecule and contains organic functional groups R (vinyl, amine, chloro etc.) and other functional groups Y (methoxy, ethoxy etc.). The inorganic groups Y of the molecule hydrolyze to silanol and form a metal hydroxide or siloxane bond with the inorganic material. The organic group can be connected covalently with organic materials, such as proteins, PEG, biomolecules etc. The surface modification with LAA is based on the chemical adsorption process. LAA is an amine acid and is used as a chelating agent in SPIONs. LLA on the surface of SPIONs acts as a small intermediary molecule that can ensure the availability of a complementary attachment site for functional biomolecules.

After LAA is chemisorbed onto the surface of the SPION, the coupling between amine acid and the biomolecules can involve chemical bonds. With this procedure, a number of biomolecules can be grafted onto the SPION. Fig. 3 (c) and (d) also show schematic views of the BSA immobilization process. The BSAcoated SPION prepared by direct coprecipitation is based on the adsorption of protein followed by covalent binding via carbodiimide activation (Fig. 3c). An attempt to immobilize BSA on APTMS-functionalized SPION has also been performed (Fig. 3d).

The surface layer (few or several monolayers) is distinctly different from that of the core material in both composition and structure. Again, such particles are categorized as core-shell structures. The thickness of the surface layer may be thin or thick depending on the functionality required. In a broad perspective these particles can also be considered as composite nanoparticles.

However, the term nanocomposite generally refers to materials consisting of a dispersion of nanoparticles within a suitable matrix. The most common example of nanocomposites is the precipitation of core nanoparticles within a nanoporous polymer structure. It is interesting to note that the fundamental properties of the polymeric materials can be dramatically altered as a result of the dispersion of few percent of inorganic nanoparticles.

Polymeric nanomaterials. There has been considerable interest in developing biodegradable nanoparticles as effective drug delivery devices.35 Biodegradable polymers are polymers that can be degraded and/or catabolized, eventually to carbon dioxide and water, by microorganisms (bacteria, fungi, etc.) under natural environments.36 However, due to the development of a wide variety of synthetic biocompatible polymers, the definition has been altered to include many artificially synthesized polymeric materials. Needless to say, components of the

Fig. 3 Schematic view of surface modification of magnetic nanoparticles: (a) silanization process; (b) chemisorption process; (c) carbodiimide activation; (d) immobilization of BSA on APTMS-modified SPION.

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degraded polymers should not be toxic and should not promote the generation of harmful substances within the body.

Biodegradable polymers can be classified into three major categories: 1) polyesters produced by microorganisms; 2) natural polysaccharides (i.e. chitosan,37–41 dextran42); 3) artificially synthesized polymers, especially aliphatic polyesters (i.e. poly lactide (PLA),43,44 poly (lactide-r-glycolide) (PLGA),45 and poly 3-caprolactone (PCL)44), polyamide (i.e. poly L-lysine,46) and others such as poly(methylmethacrylate) (PMMA)47 and poly(ethyl-2-cyanoacrylate) (PECA)48 which are also being developed as nanoparticles for the same purpose.

Biodegradable polymers are not only limited to medical devices and wound dressings, but are also used for the fabrication of scaffolds in tissue engineering,49 and as DDSs for controlled-release of 5-fluorouracil,37 cisplatin,50 lidocaine,51–53 indomethacin,44 taxol,54 4-nitroanisole,55 dexamethasone,56 radioactive compounds,57 peptides,58 and proteins59–64 with characteristic release rates at the specific target sites. For the purpose of DDSs, interest has focused on the use of particle formulations prepared from aliphatic polyesters due to their biocompatibility and resorbability. Chemical modification of pharmaceutical nano-carriers with certain synthetic polymers, such as poly(ethylene glycol) or PEG, is the most frequently used way to prolong their circulation time as drug carriers. On a biological level, coating nanoparticles with PEG sterically hinders the interactions of blood components with their surface and reduces the binding of plasma proteins with PEGylated nanoparticles. This prevents drug carrier interaction with opsonins and slows down their fast capture by RES.65 Multifunctional smart nanoparticles. These have been designed in order to meet the need for the fabrication of nanoparticles with a higher degree of complexity. Further to the core-shell structure, nanoparticles with structures similar to nanocomposites have been fabricated (nanobeads). The single bead consists of a nanocomposite core, where one or fewer nanoparticles are dispersed into the matrix. Several possible combinations of organic and inorganic particles can be dispersed within the core matrix structure. Each of the dispersed components can be selected to achieve a specific function or property of the particle.

The surface layer can combine both physical (e.g. diffusion control) and chemical (e.g. allowing certain conjugation chemistries) functionality to the particles. In this way, it is possible to ‘‘program’’ the nano-carriers with multiple functionalities suitable for performing tasks that can be triggered under specific conditions. For example, it is possible to fabricate such nanocarriers that can be magnetically stimulated or localized for target oriented controlled drug release. The release rate of the therapeutic drug can be controlled by changing the matrix of the nano-carriers or through the control of the porosity of a suitable shell layer on the surface of the bead. The nanobeads can be programmed to be responsive to the environment, e.g. small variations in temperature, or pH. Though the fabrication of these advanced generation nanoparticles is complex they have great potential for DDSs. The design and fabrication of biochemically functionalized superparamagnetic iron oxide nanoparticles and near-infrared light absorbing nanoparticles is of particular interest for cancer targeting and therapy applications. The processing of This journal is ª The Royal Society of Chemistry 2009

nanoparticles with controlled properties, such as chemical properties (composition of the bulk, interaction between the particles, and surface charge) and structural properties (crystalline or amorphous structure, size, and morphology), is the main feature in designing the nanoprecursors (nanoparticles/nanotube/nanolayer). The development of supramolecular, biomolecular, and dendrimer chemistries for engineering substances of ˚ ngstr€ A om and nanoscale dimensions has been encouraged for requirements in nanotechnology. The emerging disciplines of nanoengineering, nanoelectronics, and nanobioelectronics require suitably sized and functional building blocks to construct their architectures and devices.66 Magnetic nanoparticles are also of particular interest as highgradient external magnetic fields exert a force on them, and thus they can be manipulated or transported to specific pathological sites by applying an external magnetic field.60 They also have tunable sizes, so their dimensions can match either that of a virus (20–500 nm), a protein (5–50 nm) or a gene (2 nm wide and 10– 100 nm long). In addition, superparamagnetic particles are of interest because they do not retain any magnetism after removal of the magnetic field, thus reducing the potential for aggregation and blockage within the vasculature.

The most promising candidates for smart drug carriers as nanomedicines are polymeric nanomaterials. In general, a polymer which tends to lose mass over time within a living organism is called an absorbable, resorbable, or bioabsorbable, as well as a biodegradable polymer. In comparison with the strict definition, biodegradable polymers require the enzymes of microorganisms for natural hydrolytic or oxidative degradation.

Regardless of the degradation behavior, this terminology applies to both enzymatic and non-enzymatic hydrolysis. The physiochemical properties of the materials should be considered to remove toxins from the drugs in the patient’s body as quickly as possible. Such engineered ‘smart’ nanomaterials are strong candidates for drug detoxification because the size and surface modification of the nanomaterials are the key factors in preventing further damage to the patient’s healthy organs.45 As one of the stimuli-sensitive polymers (SSPs), poly(N-isopropylacrylamide) (PNIPAAm) is well known as a thermosensitive polymer due to its distinct phase transition at a specific LCST at 32 C in water.67–71 PNIPAAm is hydrophilic below the lower critical solution temperature (LCST); however, it becomes hydrophobic when it is heated up above the LCST. PNIPAAm has been consistently investigated as it has ‘smart’ characteristics and it is being developed for nanomedicine in the form of micelles,67 tablets,70 and hydrogels.

A new class of temperature-programmed ‘shell-in-shell’ structures with two different copolymers synthesized by a modified-double-emulsion method (MDEM) was reported as an advanced generation nanocarrier.43 Thermosensitive inner shells composed of poly(N-isopropylacrylamide-co-D,L-lactide) (PNIPAAm-PDLA) with a lower critical solution temperature (LCST) can be fabricated. This novel technique allows for the construction of a delivery vector with programmable release rates for any kind of hydrophilic chemotherapeutic agents into the polymeric nanocapsules. The drug release rates are governed by several parameters which only involve the PLLA-PEG outer shell, such as the volumetric ratio between the organic phase and aqueous phase, the interaction parameters between the J. Mater. Chem., 2009, 19, 6294–6307 | 6299

therapeutic agents and the core domain, tacticity of the copolymer, the encapsulation efficiency, and so on.

Generally, a hydrophilic protein such as BSA can be encapsulated in the polymeric spheres using a double-emulsion method (DEM), a so-called ‘water-in-oil-in-water’ (w/o/w) emulsion method.61,71,72 However, the DEM has a disadvantage with respect to the stability of amphiphilic polymer spheres because both the inner shells and the outer shells are composed of the same species of copolymer. As for the MDEM, two different kinds of copolymers are sequentially incorporated in the organic phase to promote an enhanced stability of the spheres. In this way, the inner shells can be prepared with PNIPAAm-PDLA diblock copolymers and the outer shells can be prepared with PLLA-PEG diblock copolymers respectively.

For another class of thermosensitive nano-carriers, poly[(NIPAAm-r-AAm)-co-lactic acid] (PNAL) has been reported.73 Au nanoparticles can be directly self-assembled on the surface of PNAL nanospheres by virtue of primary amino groups coming from acrylamide (AAm) molecules of PNAL diblock terpolymer.

The primary amino groups can be strongly bound to noble metals such as gold or silver. Therefore, the ‘shell’ domain of Au@PNAL becomes an affinity site for biomolecules to be conjugated. Furthermore, the LCST of poly(N-isopropylacrylamide-r-acrylamide) (PNA) was modulated from 32

C up to approximately 36 C through the manipulation of the ratio between NIPAAm and AAm units. This nanostructure is expected to serve as a synchronous delivery system by virtue of its Au-modified surface and hydrophobic inner core site.

3. Active targeting of tumors/cancer cells In general, the surface molecules of tumors/cancer are overexpressed compared to normal, healthy cells. The exhibiting surface-specific molecules could be used to regulate cellular processes and are therefore referred to as tumor associated antigens. These overexpressed antigens differ from those on the normal cells and thus can be targeted and used for therapy by an antibody or a receptor ligand to which a toxic substance has been coupled. Antibodies, most frequently monoclonal antibodies (mAbs), are used to target tumor-specific structures in several ways (Fig. 4). Generally, antibodies conjugated to a DDS, which can load the chemotherapeutical agents such as ricin, genistein and pseudomonas exotoxin A (ETA) in nanocapsules, have been suggested to effectively eradiate tumor cells. Additionally, a superantigen-conjugated antigen can be used to enhance the immunoreactions toward the pathological cells. However, the use of antibodies (most mAbs are derived from mice) can provoke an immune response. Consequently, a humanized chimeric antibody is produced by modifying the non-binding part of the antibodies with human parts to enhance tolerance. In some cases, when a smaller targeting agent is preferred, only the binding part of the antibody, the Fab fragment75 can be used.

The smallest parts of the antibody, the variable regions, or socalled single-chain fragments (ScFv), are among the most frequently used ligands. Because mAb fragments lack the Fc domain that binds to Fc receptors on phagocytic cells (resulting in the RES scavenging particles bearing whole mAbs), particulates derivatized with mAb fragments have increased circulation times in the blood compared to particulates derivatized with 6300 | J. Mater. Chem., 2009, 19, 6294–6307

whole mAbs.76 The frequent use of mAbs or mAb fragments as ligands for particulate drug carriers is the result of a well established and relatively facile conjugation chemistry that produces derivatives that retain full binding activity. mAb fragments have the additional advantage that they can be expressed as recombinant proteins in prokaryotic cells, a procedure that facilitates a more cost-effective massive production than the production of whole mAbs in eukaryotic cells.77 Targeting toxic therapeutic agents to pathological sites or to sites of inflammation of the endothelium through binding to receptors over-expressed on the surface of cancer cells can diminish the systemic toxicity and enhance the effectiveness of the targeted compounds. Small molecule-targeted therapeutics have a number of advantages over toxic immunoconjugates; better tumor penetration, lack of neutralizing host immune response and superior flexibility by selecting the proper drug components with optimal specificity, potency and stability during circulation.78 Therefore, actively targeted particulates represent the second generation of particulate drug carriers; the first comprises particles not derivatized with a ligand. Non-targeted particle-drug formulations demonstrate relative tumor selectivity as a consequence of ‘passive’ targeting, and several have already been approved for clinical use. As a result of the increased permeability of endothelial barriers in tumor blood vessels and the lack of effective lymphatic drainage from the tumor, passive targeting results in the selective extravasation and accumulation of particulates or other macromolecules in tumor tissues. However, active targeting is expected to lead to higher intratumoral accumulation and, in the case of targeting with internalizing ligands, to higher intracellular concentrations of the drug.79 For the potential targeting of liver cancer cells, a novel carrier was prepared with paclitaxel-loaded nanoparticles (P/NPs) composed of poly(g-glutamic acid) and poly(lactide) and further conjugation of galactosamine on the prepared nanoparticles (Gal-P/NPs). In in vitro studies, both the P/NPs and the Gal-P/ NPs had a similar release profile to paclitaxel. The inhibition of HepG2 cell growth by the Gal-P/NPs was comparable to that of a clinically available paclitaxel formulation (Phyxol ), while the P/NPs displayed significantly less activity (p <0.05).80 Folic acid has been covalently conjugated to 66 nm liposomes via spacers of various lengths to target the liposomes to KB cells expressing folate receptors. The binding of folate-PEG liposomes to KB cells could be competitively inhibited by excess free folate or by antiserum against the folate receptor, demonstrating that the interaction is mediated by the cell surface folate-binding protein. These folate-PEG liposomes show potential for delivering large quantities of low molecular weight compounds nondestructively into folate receptor-bearing cells.81 The use of phage display peptide libraries to identify novel, site-specific ligands has significantly contributed to the increased use of peptides acting as ligands. When coupled to the anticancer drug Doxorubicin, two of these peptides, one containing an av integrin-binding Arg-Gly-Asp motif and the other an Asn-GlyArg motif, enhanced the efficacy of the drug against human breast cancer xenografts in nude mice and also reduced its toxicity.82 Moreover, a phage screening procedure was introduced for better tumor-homing to targets that are accessible to circulating phage.

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Fig. 4 Tumor cell with some of the targeted structures and examples of their targeting moieties.74

Homing and binding to tumor-derived cell suspensions indicated that LyP-1 also recognizes an osteosarcoma xenograft, and spontaneous prostate and breast cancers in transgenic mice, but not other tumor xenografts. These results suggest that tumor lymphatics carry specific markers and that it may be possible to specifically target therapies into tumor lymphatics.83 Certain tumors, including many that are found in the lung, overexpress the CD44 cell surface marker. CD44 is a receptor that binds to hyaluronan (HA), a carbohydrate consisting of b1,3 N-acetyl glucosaminyl-b1,4 glucuronide. Eliaz and Szoka have shown that the incorporation of phosphatidylethanolamine lipid derivatives-containing HA oligosaccharides (HA-PE) into liposomes could target drug-containing liposomes to tumor cells that express CD44. HA targeted liposome (HAL)-delivered Doxorubicin (DOX) was significantly more potent than the unencapsulated DOX in cells expressing high levels of CD44. This suggests that HALs can be a functional targeted drug carrier for treatment of CD44 expressing tumors.84 Liposomal nano-carriers loaded with Doxorubicin and bearing controlled numbers of both folic acid and a monoclonal antibody against the EGFR were designed as a dual-ligand system which can target tumor cells while sparing off-target cells.

Typically overexpressed multiple types of surface receptor were designed to enhance the selectivity of the targeted nano-carriers.

This approach was examined in the human KB cell line, which over-expresses both folate receptor (FR) and the epidermal growth factor receptor (EGFR).85 EGF-conjugated magnetoliposomes were used to target the overexpressed EGFR in tumor cells. These are liposomes that have magnetic nanoparticles embedded in their bilayer, allowing for selective heating and release of a drug when the magnetoliposome is exposed to an AC magnetic field. If a tumor cell overexpresses EGFR by 5-fold, then each of its endosomes will have five times more receptors than those of a normal healthy cell. Therefore, the tumor cell’s endosome has a five times greater chance of containing one EGFbound component and a 25 times greater chance of containing both components.74

4. Passive targeting of tumors/cancer in vivo The coupling of low molecular weight drugs with a high molecular weight polymer provides for so-called ‘‘passive tumor targeting’’. This strategy increases drug accumulation in solid This journal is ª The Royal Society of Chemistry 2009

tumors generally governed by the enhanced permeability and retention (EPR) effect. The passive targeting system does not require a special targeting moiety and is therefore substantially simpler to construct compared to DDSs. Instead, passive targeting can be applied based on specific conditions at the targeted site (tumor or tumor-bearing organ).86 Tumor vessels have a wide lumen, whereas tumor tissues have poor lymphatic drainage. This anatomical defect, along with functional abnormalities, results in extensive leakage of blood plasma components, such as macromolecules, nanoparticles and lipid particles, into the tumor tissue. Moreover, the slow venous return in tumor tissue and the poor lymphatic clearance mean that macromolecules are retained in the tumor, whereas extravasation into tumor interstitium continues, a process termed the enhanced permeability and retention effect.87 By harnessing this unique characteristic (the EPR effect) of solid tumors, the selective delivery of macromolecular anticancer agents to the pathological sites has become a reality. The disorganized pathology of angiogenic tumor vasculature with its discontinuous endothelium leads to hyperpermeability to circulating macromolecules, and the lack of effective tumor lymphatic drainage leads to subsequent macromolecular accumulation.88 Chemotherapy with HT-1080 bearing mice was used to investigate this drug targeting strategy and the cause of side effects of dextran-peptide-methotrexate conjugates. In these experiments, passive targeting was facilitated by the prolonged plasma circulation and higher tissue accumulations of both types of conjugates compared to free methotrexate. Independent of the peptide sequence of the linker, the ratio of drug accumulation at the tumor versus drug accumulation at the major site of side effects (small intestine) for either conjugate was increased by the EPR effect. The tumor targeting effect of the dextran-peptidemethotrexate conjugate was dominantly due to passive targeting and EPR.89 The antitumor effect of poly(ethylene glycol)–camptothecin conjugate (PEG–CPT) was studied in a nude mouse model of human colon cancer xenografts. The conjugation of the low molecular weight anticancer drug CPT with low solubility to high molecular weight, water-soluble PEG polymer provided several advantages over the native drug. It offered better uptake by the targeted tumor cells and substantially enhanced apoptosis and antitumor activity of the conjugated drug in the tumor along with decreased apoptosis in the liver and kidneys as compared with the native drug.86 In addition to the molecular manipulation of therapeutic agents, surface-modified nanoparticles with stealth coatings can be used as passive targeting agents. This allows them to selectively extravasate in pathological sites, like tumors or inflamed regions with a leaky vasculature. As a result, such long-circulating nanoparticles are able to directly target most of the tumors outside the MPS regions.90 The core nanomaterials can be prepared with mesoporous structures using MCM41 or CNT. This system could incorporate the low molecular weight chemotherapeutic agents such as DNA/RNA and can be applied to gene therapy. Antifouling polymer, poly(TMSMA-rPEGMA),-coated SPIONs were also synthesized.

Another passive EPR approach is to exploit the clear visualization of leaky areas at the early stages of cancer/tumor growth by injecting superparamagnetic iron oxides (SPION) as MR J. Mater. Chem., 2009, 19, 6294–6307 | 6301

contrast agents in vivo. When the anti-biofouling polymer-coated SPIONs were incubated with macrophage cells, uptake was significantly lower in comparison to that of the popular contrast agent, Feridex I.V., suggesting that the polymer-coated SPION can circulate for longer periods in the plasma, escaping uptake by the RES such as macrophages. Indeed, when the coated SPIONs were administered to tumor xenograft mice by intravenous injection, the tumor could be detected in T2-weighted MR images within 1 h as a result of the accumulation of the nanomagnets within the tumor site. Although the poly(TMSMA-rPEGMA)-coated SPIONs do not have any targeting ligands on their surface, they are potentially useful for cancer diagnosis in vivo.1 Dextran and its derivatives have been used as attractive coating materials for inorganic nanoparticles such as SPIONs due to their biocompatible and hydrophilic characteristics. For example, Palmacci and Josephson91 synthesized cross-linked superparamagnetic iron oxides (CLIOs) with epichlorohydrin, which makes a continuous coating of SPIONs. CLIOs show good stability and biocompatibility while the cross-linking process does not affect particle size and magnetic properties of the colloids. However, the conjugation of bioactive molecules to dextran-coated SPIONs such as CLIOs is still very difficult and strongly relies on the generation of additional functional groups such as amine and carboxyl groups. In order to generate amine groups on a dextran coating, cross-linked superparamagnetic iron oxides (CLIOs) have been aminated by adding concentrated ammonia at 37 C overnight for further attachment of Tat peptides, a membrane translocation signal, through N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). However amine groups generated by this method are unstable, which may result in release of the attached Tat peptide.

Versatile, ultra-small superparamagnetic iron oxides (VUSPIOs) based on maghemite and partially oxidized dextran, have been conjugated to poly(ethyl glycol) (PEG), a protein resistant molecule. But generation of aldehyde groups breaks the glucose unit of the dextran backbone. Carboxymethyl dextran (CMD) that contains carboxylic groups as functional groups can be easily modified with versatile bioactive molecules such as peptides, proteins and oligonucleotides. Therefore, CMD is of particular interest as a coating material for SPIONs. Though the use of magnetic micro- and nanoparticles as drug carriers for cancer therapy was first proposed decades ago,92 the technology has yet to prove effective in the clinic. Early animal studies were promising using magnetic nanoparticles as both drug carriers and for targeted magnetic fluid hyperthermia.93 Following on from the success of magnetic targeting and hyperthermia in animal trials, there has been a handful of clinical studies aimed at moving the technology into humans.94 One of the main reasons for the lack of clinical success of magnetic targeting is that the forces required to trap and target magnetic nanoparticle carriers circulating in the bloodstream are difficult to achieve for tumors deep in the tissue.95–97 Recent efforts to overcome this obstacle have focused on such novel variations as implantable magnets98 and, more recently, the use of magnetic nanoparticle-loaded macrophages to enhance targeting.

In this latter approach, the innate propensity of macrophages to respond to chemical signals from tumors was harnessed to increase the proportion of magnetic nanoparticle-loaded 6302 | J. Mater. Chem., 2009, 19, 6294–6307

macrophages which can carry associated chemotherapeutic compounds and/or apoptosis genes.99 By increasing the proportion of therapeutically armed macrophages, it should be possible to target the non-vascularized hypoxic core of solid tumors100 and subsequently employ magnetic fluid hyperthermia to destroy the carriers and remaining tumor cells.

5. Nanomedicine for controlled release of therapeutic agents at the target site Although biomaterials (biologically derived components) are useful for new medical treatments, critical problems with biocompatibility, mechanical properties, degradation and numerous other issues remain. Stealth properties and responsiveness such as pH, temperature, specificity and other critical problems should be resolved in order to satisfy the prerequisites.

Generally, the drug release mechanisms from micro/nanoparticles should consider the following factors: (1) surface desorption; (2) diffusion through particle pores; (3) diffusion through intact polymers; (4) diffusion through water swollen polymers; (5) surface or bulk erosion of the polymeric matrix.101 In addition, certain stimuli-sensitive functions such as pH sensitivity,73 redox potential, external magnetic field,97 temperature,67 ionic strength and ligand–receptor interactions could be introduced to enhance the long-circulating and targeted pharmaceutical nano-carriers. For example, the intratumoral pH value in solid tumors may drop to 6.5, i.e. one pH unit lower than in normal blood (7.4) because of hypoxia and massive cell death inside the tumor, and drops still further inside cells, especially inside endosomes (5.5 and even below).102 Enzymes can catalyze the cross-linking of the polymer chains to form a continuous, three-dimensional matrix for hydrogels for tissue engineering, wound healing, and DDSs. In addition, enzyme-responsive surfaces can direct the attachment of cells, and enzyme-responsive polymeric hydrogel beads have potential as a matrix for DDSs.103 The possibility of delivering cytotoxic agents directly into tumor cells gives several advantages: loss of the drug in the bloodstream and upon the liposome–cell interaction are minimized and the preparation of drug-loaded nanoparticles becomes simpler.

To be effective, there are a number of attributes that the material must possess, including the ability to condense therapeutic molecules to a size of less than 150 nm so that it can be taken up by receptor-mediated endocytosis—the ability to be taken up by endosomes in the cell and to allow therapeutic molecules to be released in active form, and to enable it to travel to the cell’s nucleus. Moreover, gene therapy is gaining popularity as a medical treatment for cancer, tumors, Alzheimer’s disease, diabetes etc, however, the clinical efficacy is lower than expected due to the detergent effect. When it is administered directly into the blood vessel or lesion, the therapeutic molecules are taken up by other healthy organs/cells and the residual time in biological systems is less than 24 h, thus the pharmacological action is diminished. Also, it has been reported that the therapeutic molecules taken up by healthy organs/cells undergo mutation and may cause other, more serious diseases, such as cancer. Most antitumor agents are hydrophilic compounds and, therefore, cannot be retained within the membrane. Thus, the use This journal is ª The Royal Society of Chemistry 2009

of prodrug forms of anticancer agents to alter the phase behavior of the chemicals is becoming more popular.

5.1.

Alkylating agents

Alkylating reagents are chemical reagents that have an alkyl group such as propyl in place of a nucleophilic group in a molecule. Alkylating reagents include a number of cytotoxic drugs, some of which react specifically with N7 of the purine ring resulting in depurination of DNA. These alkylating drugs interact with DNA and prevent the division of the cells.

The alkylation of DNA bases can cause disruption of the replication mechanism of the cell. The nitrogen bases in DNA molecules are nucleophilic and can be easily alkylated. If the N–H groups are replaced by N–R groups then the DNA base pairing is disrupted and can lead to cellular dysfunction. This should have an effect on the replication of cancerous cells, thus leading to a slow-down or stoppage of the growth of the cancer.

Chlorambucil is one of the well-know anticancer agents for blood cancers and acts to reduce the number of blood cells. It is also used to treat other cancers such as lymphomas. The chemical structure of Chlorambucil is an aromatic derivative of mechlorethamine and is closely related in structure to melphalan. Its therapeutic effects are the slowest acting and it is generally least toxic among the alkylating agents. Alkylation of DNA results in breaks in the DNA molecule as well as cross-linking of the twin strands, thus interfering with DNA replication and transcription of RNA. Like other alkylators, chlorambucil is cell cycle phasenonspecific.104,105 Leroux et al. demonstrate that polymeric nanoparticles can be loaded with chlorambucil (8.52% m/m) with an entrapment efficiency of 60%. Nanoparticles as small as 70 nm in diameter can be produced by increasing the percentage of poly(vinyl alcohol) to 27.5% in the external phase. The particle size can be controlled by using gelatin instead of poly(vinyl alcohol) and the smallest nanoparticles, with an average size of 700 nm, can be obtained.106 Chitin-based biodegradable microspheres also have been investigated for their ability to encapsulate chlorambucil as a model drug. The polymer sphere can be prepared by directly blending chitin with different contents of poly(D,L-lactide-coglycolide 50:50) (PLGA 50/50) in dimethylacetamide–lithium chloride solution, followed by coagulating in water via wet phase inversion. In the initial stage, the drug-release rate increases with increased chitin content due to the hydration and surface erosion of the hydrophilic chitin phase; however, the following stage of slow release is sustained for several days, mainly due to the bulk hydrolysis of hydrophobic PLGA phase.27 Cyclophosphamide is a cyclic phosphamide ester of mechlorethamine. It is transformed via hepatic and intracellular enzymes to active alkylating metabolites, acrolein and phosphoramide mustard. Cyclophosphamide causes prevention of cell division primarily by cross-linking DNA strands. This anticancer agent is applicable to breast cancer, lung cancer, multiple myeloma, mycosis fungoides, neuroblastoma and retinoblastoma etc. It must be handled carefully as it is considered to be highly carcinogenic in humans. Cyclophosphamide-loaded polybutylcyanoacrylate nanospheres were investigated to obtain a suitable and tolerated ocular delivery device for therapeutic This journal is ª The Royal Society of Chemistry 2009

applications involving treatment of severe ocular inflammatory processes that localize in the anterior chamber of the eye.107,108 Local delivery of 4-hydroperoxycyclophosphamide (4HC derived from cyclophosphamide), was achieved via a controlledrelease biodegradable polymer to determine whether the use of a polymer vector can enhance efficacy. Ninety Fischer 344 rats implanted with 9L or F98 gliomas were treated with an intracranial polymer implant containing 0% to 50% loaded 4HC in the polymer. Long-term survival for more than 80 days was 40% in the 4HC-treated rats versus 30% in the BCNU-treated rats. It can be concluded that 4HC-impregnated polymers provided an effective and safe local treatment for rat glioma.109 Carmustine (BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea) is a highly lipophilic nitrosourea compound which undergoes hydrolysis in vivo to form reactive metabolites. These metabolites cause alkylation and cross-linking of DNA. Nitrosoureas generally lack cross-resistance with other alkylating agents.104,105 The US Food and Drug Administration (FDA) approval of Gliadel in 1996 represents the first new treatment approved for brain tumors in over 20 years. It has also been approved by numerous regulatory agencies worldwide. Gliadel is a polymer– drug combination that delivers the chemotherapeutic agent carmustine directly to the site of a brain tumor via controlled release from a biodegradable matrix.110 In order to compare the effectiveness of lipid microspheres to Gliadel , Takenaga incorporated BCNU into lipid microspheres by homogenizing a soybean oil solution of BCNU with egg yolk lecithin. Compared to the corresponding conventional dose of BCNU, the lipid microsphere-encapsulated BCNU significantly enhanced antitumor activity with reduced toxicity in mice with L1210 leukemia. [14C]Triolein uptake by L1210 leukemia cells was increased by incorporation into microspheres. The nanospheres showed longer in vivo half-life due to the avoidance of cellular uptake by the reticuloendothelial system, resulting in higher accumulation at the tumor sites.111 5.2.

Antimetabolic agents

Cytarabine is metabolized intracellularly into its active triphosphate form (cytosine arabinoside triphosphate). This metabolite then damages DNA by multiple mechanisms, including the inhibition of alpha-DNA polymerase, inhibition of DNA repair through an effect on beta-DNA polymerase, and incorporation into the DNA. The latter mechanism is probably most important. Cytotoxicity is highly specific for the S phase of the cell cycle.104,105 Ellena et al. investigated the distribution of phospholipid and triglyceride molecules in the membranes forming a non-concentric vesicular network within a multivesicular lipid particle (MLP). MLP formulations exhibited controlled release of encapsulated pharmaceuticals on timescales of a few days to a few weeks. The MLP can be synthesized by a double emulsification process with a neutral lipid such as a triglyceride. MLP formulations with the antineoplastic agent cytarabine encapsulated in the aqueous compartments were prepared that further contained [C-13]carbonyl-enriched triolein. This rational approach can be used to develop MLP formulations with variable rates of sustained release, modulated by changes in the distribution of various phospholipids and triglycerides.112 J. Mater. Chem., 2009, 19, 6294–6307 | 6303

Fluorouracil (FU) was developed in 1957 based on the observation that tumour cells utilized the base pair uracil for DNA synthesis more efficiently than did normal cells of the intestinal mucosa. It is a fluorinated pyrimidine that is metabolized intracellulary to its active form, fluorodeoxyuridine monophophate (FdUMP). The active form inhibits DNA synthesis by inhibiting the normal production of thymidine. Fluorouracil is cell cycle phase-specific (S-phase).104 5-Fluorouracil (5-FU)-loaded poly (L-lactide) (PLLA) or its carbonate copolymer microspheres were prepared by a modified oil-in-oil (o/o) emulsion solvent evaporation technique. Using this modified process, microspheres with various particle sizes can be prepared with high 5FU entrapment efficiency (about 80%). In vitro drug release experiments showed a burst release of 5-FU from PLLA microspheres, followed by a sustained release over 50 days. In the case of other vectors, poly (L-lactide-co-1,3-trimethylene carbonate) (PLTMC) and poly (L-lactide-co-2,2-dimethyl-1,3trimethylene carbonate) (PLDTMC), the drug release rate can be prolonged to more than 60 days.113 Roullin et. al. developed 5-FU-loaded poly(L-lactide-co-glycolide) (PLGA) microspheres to deliver the therapeutic agents into the CNS for stereotactic intracerebral implantation.37 In-vivo experiments with C6 glioma-bearing rats showed promising results—the median survival time was doubled.114 A phase I–II pilot study was conducted on 8 patients with high-grade glioma who underwent surgical removal before 5-FU-loaded microspheres were implanted. After 18 months the patients’ survival rate and welfare was improved.115 Microsphere fate and the 5-FU diffusion area from these particles in the brain was also investigated depending on the locations of the inserted drugloaded microspheres. [3H] 5-FU microspheres were used to evaluate diffusion areas from the implantation site.37 Another approach for controlled DDSs into the brain has been developed using implantable, biodegradable microspheres. The strategy was evaluated initially to provide localized and sustained delivery of the radiosensitizer 5-FU after patients underwent surgical resection of malignant glioma.116 Methotrexate and its active metabolites compete for folate binding sites of the enzyme dihydrofolate reductase. Folic acid must be reduced to tetrahydrofolic acid by this enzyme in order for DNA synthesis and cellular replication to occur. Competitive inhibition of the enzyme leads to blockage of tetrahydrofolate synthesis, depletion of nucleotide precursors, and inhibition of DNA, RNA and protein synthesis. Methotrexate is cell cycle phasespecific (S phase).104,117 Methotrexate can be widely employed for breast cancer, bladder cancer and head and neck cancer etc.

ABA-type triblock copolymers of poly(trimethylene carbonate)-poly(ethylene glycol)-poly (trimethylene carbonate) were synthesized by ring-opening polymerization. The anticancer drug methotrexate was loaded into the core-shell structure of polymeric nanoparticles with a diameter of 50–160 nm. Generally, the release rate of methotrexate from the nanoparticles was shown to be comparatively faster than that of microsphere systems due the higher surface area and smaller particle size.118 Hydrophilic gelatin nanoparticles were also prepared which incorporated the methotrexate by solvent evaporation techniques based on single water-in-oil (W/O) emulsion with glutaraldehyde as a cross-linking agent. The methotrexate loaded gelatin particles were in the range of 100–200 nm mean diameter.119 6304 | J. Mater. Chem., 2009, 19, 6294–6307

5.3.

Anticancer antibiotics

At low concentrations actinomycin D inhibits DNA-directed RNA synthesis and at higher concentrations DNA synthesis is also inhibited. All types of RNA are affected, but ribosomal RNA is more sensitive. Actinomycin D binds to double-stranded DNA, permitting RNA chain initiation but blocking chain elongation. Binding to the DNA depends on the presence of guanine. It is applicable to the treatment of testicular, ovarian, and germ cell cancers. Isobutylcyanoacrylate nanoparticles loaded with actinomycin D were shown to concentrate preferentially in rat mesangial cells and to increase the drug’s uptake in these cells in vitro and in vivo, as compared to the free drug. Drug targeting by nanoparticles of renal cells and macrophages may be possible, resulting in a lowering of the critical level of drug dosage in tubular cells and a reduction of tubular toxicity.120 The effects of actinomycin D-loaded polymethylcyanoacrylate nanoparticles on the growth of a transplantable soft tissue sarcoma were also investigated in a rat model. Actinomycin Dloaded polymethylcyanoacrylate nanoparticles showed a greater inhibitory action than the free drug on the growth of the S250 sarcoma but the nanoparticles alone did not demonstrate any significant antitumor effect.121 This study demonstrated that 24 h after injection, adsorbed actinomycin D is 5.6-, 44- and 64-fold more concentrated than the free drug in muscle, spleen and liver respectively.122 Bleomycin is an antineoplastic antibiotic. It is used to treat several types of cancer, including cervical and uterine cancer, head and neck cancer, testicular and penile cancer, and certain types of lymphoma. Bleomycin causes DNA strand scission through formation of an intermediate metal complex requiring a metal ion cofactor such as copper or iron. This action results in inhibition of DNA synthesis, and to a lesser degree, in inhibition of RNA and protein synthesis. The drug is cell-cycle specific for G phase, M-phase and S phase.123 The manipulation of the physicochemistry of water-soluble polymers such as glycolchitosan can be used to create hybrid materials for drug delivery and gene delivery with biocompatibility. Glycol chitosan modified by the attachment of a strategic number of pendant fatty acid groups (11–16mol%) assembles into unilamellar, polymeric vesicles in the presence of cholesterol. An ammonium sulphate gradient bleomycin (MW 1400), for example, can be efficiently loaded onto these polymeric vesicles to yield a bleomycin-topolymer ratio of 0.5 units mg 1.124 Bleomycin has been conjugated to carbon nanoparticles as a new DDS for the treatment of digestive cancer. In this way, higher levels of anticancer drug can be localized to the regional lymph nodes and the injection site compared to distribution of the drug in aqueous solution. In 12 patients with histologically proven carcinoma, bleomycin-conjugated carbon nanoparticles were injected endoscopically into the primary lesions. Endoscopic injection of this dosage formulation shows that it can control these digestive cancers in patients in whom operation is contraindicated.125 Formulations of ultra-deformable liposomes containing bleomycin (Bleosome ) also have been reported and proposed for topical treatment of skin cancer.126 Bleosome exerted a lethal effect on human keratinocytes cell lines and a cell line derived from a primary carcinoma in vitro when it is loaded with This journal is ª The Royal Society of Chemistry 2009

sufficient bleomycin. The cell line, derived from squamous cell carcinoma, seemed to be more susceptible to Bleosome than HPV-immortalised keratinocytes (NEB-1).127 Daunorubicin is an anthracycline antibiotic which damages DNA by intercalating between base pairs resulting in uncoiling of the helix, ultimately inhibiting DNA synthesis and DNAdependent RNA synthesis.128 A tumor-targeting daunorubicin liposome, DaunoXome , is commercially available and its beneficial effects are well reported. The daunorubicin liposome product, (DaunoXome ) is a formulation of daunorubicin in small unilamellar vesicles (SUVs) composed of highly pure distearoylphosphatidylcholine (DSPC) and cholesterol in a 2:1 mole ratio. Several countries have approved DaunoXome for use in treating Kaposi’s sarcoma (KS) in HIV-positive patients.

Preclinical investigations indicate that DaunoXome increases in vivo daunorubicin tumor delivery by about 10-fold over conventional drugs, yielding a comparable increase in therapeutic efficacy.129

6. Conclusions Nanomaterials can contribute enormously in the area of nanomedicine and this contribution is likely to become more significant in future. Specific, target-oriented DDSs are showing promising results along with diminished side-effects and increased therapeutic effects. Both active and passive targeting techniques will enhance the localization of the therapeutic agents in pathological sites. Furthermore, degradation/release rates, and surface activation/functionalization is being actively investigated for optimization of these systems.

The selective uptake of chemotherapeutic agents by tumor tissues is a great challenge since anticancer agents themselves are not specific to target sites. Active targeting techniques using nanomaterials as carrier vectors can and will be improved via focused design, the incorporation of theoretical considerations, and logical approaches to the production of more specific cancer cell recognition systems. New design strategies for nanovectors as nanomedicines should be developed in order to move these systems into the clinic and to realize their main benefits— decreased side-effects and more effective treatment. Novel techniques for the effective loading of active molecules and surface activation (i.e. antibodies and functional groups) for active targeting are essential to improve the therapeutic effectiveness. As we write this, the next generation of intelligent smart biomaterials for use in nanomedicine are being intensively investigated and developed and this research is already having an enormous effect on nanomedicine as a new research field.

Acknowledgements This work was financially supported by Engineering and Physical Sciences Research Council (EPSRC) CoDDS projects (EP/ E016944/1).

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