nanoscale polymer carriers to deliver chemotherapeutic agents to tumours

Review

10.1517/14712598.5.12.1557 © 2005 Ashley Publications ISSN 1471-2598 1557

Ashley Publicationswww.ashley-pub.com

Delivery

Nanoscale polymer carriers to deliver chemotherapeutic agents to tumoursMateja Cegnar, Julijana Kristl & Janko Kos†

†University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, 1000 Ljubljana, Slovenia

Nanoscale polymer carriers have the potential to enhance the therapeuticefficacy of antitumour drugs as they can regulate their release, improve theirstability and prolong circulation time by protecting the drug from elimina-tion by phagocytic cells or premature degradation. Moreover, nanoscalepolymeric carriers are capable of accumulating in tumour cells and tissuesdue to enhanced permeability and retention effect or by active targetingbearing ligands designed to recognise overexpressed tumour-associatedantigens. The diversity in the polymer structures being studied as drugcarriers in cancer therapy allows an optimal solution for a particular drug tobe provided regarding its delivery and efficacy, and thus the patient’s qualityof life. This review is focused on the different types of nanoscale polymercarriers used for the delivery of chemotherapeutic agents and on the factorsthat affect their cellular uptake and trafficking.

Keywords: cancer, drug delivery, nanoparticles, polymer, targeting

Expert Opin. Biol. Ther. (2005) 5(12):1557-1569

1. Introduction

Cancer is a complex set of proliferative diseases arising in most cases via multisteppathways and involving an accumulation of genetic and epigenetic changes.Abnormal cells that divide uncontrollably have the ability to infiltrate and destroynormal body tissue. They can spread throughout the body, forming metastases,which constitute a major cause of death in cancer patients.

Although the mechanisms of cancer development and progression are still notwell understood, alterations in some fundamental biological mechanisms, such ascell growth and signalling, immune response, apoptosis, tissue remodelling,angiogenesis and so on, can be definitively related to malignant diseases. A largenumber of molecules involved in these processes have recently been identified astargets for diagnostic or therapeutic intervention. Although new diagnosticapproaches and new selective antitumour drugs have significantly improved thesuccess of therapy and the quality of life of the patients, cancer is still the secondmost frequent cause of mortality. New strategies to cure malignant diseases, thus,constitute one of the most challenging fields in modern science. In addition to thediscovery of new therapeutic targets and the application of new synthetic andbiological molecules, improvement of their pharmacokinetic and pharmaco-dynamic characteristics would enhance the efficacy of the therapy. Nanoscalepolymeric carriers are capable of stabilising and protecting these activecompounds, and of delivering them to the site of action, thus providing an idealtool to achieve progress in combating cancer.

1. Introduction

2. Cancer therapy

3. Why nano?

4. Polymers as drug carriers in

cancer chemotherapy

5. Factors affecting polymeric

carriers uptake by cancer cells

6. Nanoscale polymeric carriers

in radiotherapy

7. Expert opinion and conclusions

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Nanoscale polymer carriers to deliver chemotherapeutic agents to tumours

1558 Expert Opin. Biol. Ther. (2005) 5(12)

Figure 1. Interstitial, vascular and cellular levels for targeting cancer tissue.DDS: Drug delivery system; Enz: Enzymes; ∆mtb: Changes in metabolism; Rp: Cell surface tumour receptor.

Rp

Interstitial level

∆ mtb

mediator

regulationfailure

acidic products

Normal tissue

lymph node

low pH, high pressure, and defective lymphatic drainage

leaky tumourendothelium, and extravasation of DDS

normal vessels have a tight endothelium

Vascular level Cellular level

Tumour tissue

polymer carrying a drug molecules

EnzEnzEnzEnzEnz

2. Cancer therapy

Malignant tissue, a target for therapeutic intervention, isrecognised at three different levels: vascular, interstitial andcellular (Figure 1) [1-3].

The vascularisation of tumours is heterogeneous, showingregions of necrosis or haemorrhage, as well as regions thatare densely vascularised to sustain adequate supplies ofnutrient and oxygen for rapid tumour growth (angiogen-esis). Tumour blood vessels are abnormal in several ways,including a high proportion of proliferating endothelialcells, an increased tortuosity, a deficiency in pericytes and anaberrant basement membrane formation, all resulting inenhanced vascular permeability [3-5].

The tumour interstitional compartment is composedpredominantly of collagen and an elastic fibre network.Interstitial fluid and macromolecular constituents(hyaluronate and proteoglycans) are interspersed within thecrosslinked structure, forming a hydrophilic gel. Theinterstitium, unlike most normal tissues, is also character-ised by lower pH, a high interstitional pressure leading to anoutward convective interstitial fluid flow, and by theabsence of an anatomically well-defined functioninglymphatic network.

The cellular compartment is categorised by alterations inbasic cellular mechanisms such as cell growth, apoptosis, orcellular transport mechanisms such as the P-glycoproteinefflux system or multi-drug resistance-associated protein, bothresponsible for multi-drug resistance (MDR).

Surgery is often the first course of action to remove atumour, but when the disease has already evolved and meta-stases are spread through the body, systemic chemotherapy,radiotherapy or a combination of both are essential. However,this classical treatment of malignant diseases often faces resist-ant tumours, and directions towards new treatment strategiesare being considered [6,7]:

• development of more effective low molecular weightanticancer drugs; combinatorial chemistry andhigh-throughput in vitro screening are used to select thebest candidates from natural and synthetic compounds

• identification, by genomic and proteomic studies, of newtargets for chemotherapy arising from altered molecularmechanisms in tumour cells, such as apoptosis, signal trans-duction pathways, tumour vascularisation, modulation ofextracellular matrix and so on; frequently, therapeuticagents proposed in this field are peptides and proteins

• development of gene therapy and application of

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Cegnar, Kristl & Kos

Expert Opin. Biol. Ther. (2005) 5(12) 1559

oligonucleotides such as small interfering RNA or aptamers• development of antitumour vaccines and stimulation of

antitumour immune responses• development of improved drug delivery systems (DDSs) to

selectively localise chemotherapeutic agents in tumourtissue, and to ensure their sustained and controlled release

Cytotoxic drugs are usually more effective for malignant cellswith a higher proliferation rate. However, some normal cellswith rapid turnover, such as bone marrow cells and intestinalepithelium cells, may also be seriously affected. Therefore, forsuccessful chemotherapy, tumour cytokinetics have to beconsidered, as cancer drugs possess either cell type-, cell cycle-or cell growth phase-specific activity. In addition, most anti-cancer drugs are highly hydrophobic and, hence, are notsoluble in water. Adjuvants have to be used for their adminis-tration, and this may cause serious side effects, some of whichare life-threatening, as in case the solvent Camphor EL(polyoxyethylated castor oil) for the drug paclitaxel, whichcauses hypersensitivity reaction, nephrotoxicity, neurotoxicityand cardiotoxicity [8-10].

The dosage of chemotherapeutic agents is also an impor-tant issue in chemotherapy. Rapid renal clearance of lowmolecular weight drugs often demands high-frequencydosing, which usually causes fluctuations in blood concentra-tion. In spite of achieving an initial response in chemotherapy,a drug often fails in long-term treatment because of the devel-opment of drug resistance by cancer cells. Resistance candevelop against multiple anticancer drugs with certain specialtypes of molecular structure, that is, MDR [11].

The use of peptides and proteins, or gene fragments andpolynucleotides as anticancer agents is also hampered bytheir rapid elimination from the circulation, mainly due toenzymatic degradation by proteases or DNA/RNAases. Inaddition, they are eliminated from the circulation byreticuloendothelial cells and can often stimulate an immuneresponse due to their multi-potent activity. The specificityof some of these drugs is also only partial, and theirwidespread distribution into non-target organs may causesevere side effects. The delivery of these macromolecules totarget cells in tumours can constitute a significant problem,compared with low molecular weight chemotherapeuticagents. Moreover, the targets for gene fragments and manyproteins are intracellular, and the low permeability of cellmembranes to macromolecules is an additional obstacle forthe development of gene- or protein-based drugs.Furthermore, endosomal escape, cytosolic trafficking, ineffi-cient nuclear uptake and transgene expression are additionalbarriers that gene-based drugs must overcome for successfulgene therapy [8,12].

Effective solution of these problems requires close collabo-ration between clinicians, scientists and engineers. The availa-bility of several potent anticancer agents for clinical useunderlines the fact that inadequate delivery is the single mostimportant factor delaying their optimal application.

3. Why nano?

Size is one of the main physicochemical properties that plays akey role in determining the therapeutic behaviour of thedelivery system [8]. Colloidal carriers can be formulated in afluidised form with a liquid and administered intravenously.The smallest capillaries in the body are 5 – 6 µm in diameter.Thus, the size of particles has to be < 5 µm to preventembolism [13]. Nanosized delivery systems, compared withthose of micrometer size, can penetrate much deeper intotissue through fine capillaries, crossing the fenestrationpresent in the epithelial lining (e.g., liver), and are generallytaken up more efficiently by cells [14,15]. The latter could be anadvantage for transport through the gastrointestinal mucosaand blood–brain barrier.

Much effort has been done to evaluate the uptake ofnanoparticles (NPs) through intestinal tissue, in particularto enhance the bioavailability of large drug molecules [16,17].The uptake of nanocarriers is size-dependent [18]. Particles100 – 300 nm in size have been shown to be taken up signif-icantly better than larger particles [19]. Interestingly, particles< 50 nm diameter exhibited lower cellular uptake [20],suggesting a lower limit beyond which size no longer plays akey role in the endocytic process.

When administered intravenously, transport via the inter-endothelial cell junction may contribute to the accumulationof drug in adjacent endothelial tissue in the case of aberranttumour vasculature [5,15,19]. Altered vasculature enables theenhanced permeation and retention (EPR) effect, firstdocumented by Maeda and co-workers [21]. The effectivenessof EPR can be attributed to two factors: the extent of extrava-sation of macromolecules or colloidal carriers due to leakinessof tumour vasculature and poor blood flow, and the extent ofintratumoural retention depending on impaired lymphaticdrainage, interstitional tumour pressure and the structure ofthe drug carrier [4]. The particle size is determined by the poresize of tumour leaky capillary, reported to be between 200 nmand 1.2 µm, depending on the tumour model [4,22].

Using different mouse and human tumour models, theextent of EPR-mediated targeting has been reported to bedependent on tumour size. Smaller tumours exhibitedhigher vascular permeability and, consequently, accumula-tion of higher drug cargo [23]. From this point of view, nano-scale DDSs are appropriate for treating micrometastases thatare difficult to be eradicated by using conventional therapy.EPR-mediated targeting also depends on the type oftumour, being successful in highly vascularised tumourssuch as Kaposi’s sarcoma [4]. Thus, translating the EPRphenomenon into the clinical setting could provide auniversal gateway for the selective delivery of macromolecu-lar drugs by using polymeric carriers. These systems have theadvantage that they can be tailored in terms of molecularweight, structure and size to allow optimal extravasation andintratumoural retention. However, EPR-mediated targetingis less effective in poorly vascularised tumours, particularly

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in the outer, actively growing tumour mass. In these casesother methods of delivery, including intratumoural injectionsof NPs are superior [14,24].

4. Polymers as drug carriers in cancer chemotherapy

Anticancer drug delivery can be improved by specificpolymeric formulations that can:

• bring hydrophobic drugs into the liquid phase (especiallyfor low molecular weight drugs)

• reduce renal filtration and prolong circulation lifetime byincreasing the size

• protect the drug (especially proteins and gene fragments)from premature degradation

• shield and mask the surface of the drugs to avoid their uptakeby macrophages and the reticuloendothelial system (RES)

• reduce the respiration rate of drug efflux by MDR cells• enhance drug delivery by passive targeting exploring the

tumour’s own structure and EPR• enhance drug delivery by active targeting to overexpressed

tumour-associated antigens

Several strategies have been explored to deliver a drug to aspecific site, and delivery utilising polymers appears to be thesimplest approach. When using polymers, the followingconcerns should be considered:

• any toxicity of polymers (biocompatibility) and of theirdegradation products in the body (degradability)

• the feasibility of formulation and overall cost of thepolymeric DDS

• predicted mechanism of the release and problems associatedwith release, that is, release failure or dose dumping

• administration and acceptance by patients [25]

The polymers used in drug delivery can be broadly dividedinto two types: non-biodegradable and biodegradable.

Non-biodegradable polymers pose problems of toxicity,mostly due to their accumulation in the body. The basicmechanism of drug release from non-degradable matrices isdiffusion, dependent on polymer permeability and drugcharacteristics. Thus, the high molecular weight or poorlysoluble drugs are not amenable for such non-degradable poly-mer DDSs [26]. To overcome these problems, development ofbiodegradable polymers began in the early 1970s and, in thelast decade, has made notable progress in clinical application,especially in the area of cancer [27]. They can be degraded tobiocompatible or non-toxic products that are removed fromthe body by normal physiological pathways. The drug releasefrom biodegradable polymers depends on the structure of thepolymeric system, its pore size and degradation of the materialitself. The biodegradation can be enzymatic, chemical orsimply hydrolytic (Table 1) [26,27].

Polymers that have been used clinically include syntheticpolymers: polyethylene glycol (PEG), polystyrene-maleicanhydride copolymer, N-(2-hydroxypropyl)-methacrylamidecopolymer (HPMA), polyvinyl pyrrolidone, polyethyleneimine(PEI), polyamidoamines; natural polymers: dextran (α-1,6 poly-glucose), dextrin (α-1,4 polyglucose); and pseudosyntheticpolymers, such as the man-made polyamino acids: polylysine,polyglutamic acid, polymalic acid and polyaspartamides [23,28].

Advanced polymer chemistry and engineering are produc-ing increasingly intricate polymer structures, including multi-valent polymers, branched polymers, graft polymers,dendrimers, dendronised polymers, block copolymers,star-like polymers and hybrid glyco- and peptide polymericderivatives [23]. Their advantage is a better defined chemicalcomposition, tailored surface multivalency and definedthree-dimensional architecture (Figure 2).

4.1 Particulate drug carriersParticulate drug carriers are nanosized molecular assembliesthat entrap the drug physically in a loading space (generallythe core of the particle), thus isolating the drug from theenvironment and providing a higher degree of protectionfrom enzymatic inactivation than the polymeric conjugates(discussed later). Several polymeric particulate nanocarrierscan be identified: polymeric NPs, block copolymeric micelles,dendrimers and polyplexes (Figure 3) [29].

Apart from the efficacy of the drug, the therapeutic effectof a polymeric DDS depends on the structural and physico-chemical properties of the polymeric system. The key featuresof the drug-loaded system are size and size distribution,surface and bulk morphology, surface chemistry and charge,drug encapsulation efficiency, and the physical and chemicalstatus of the drug within the DDS [8].

Particulate drug carriers can be characterised due to theirphysicochemical properties, such as particle size, sizedistribution, composition, charge, hydrophobicity, orbiopharmaceutical properties, including the rate of drug

Table 1. Classification of biodegradable polymers.

Polymers Degradation mechanism

Naturalproteins: albumin, globulin, gelatine, collagen, casein

enzymes: proteases, collagenases, etc.

polysaccharides: starch, cellulose, chitosan, dextran, alginate

enzymes: amylases, alginases, etc., pH

Synthetic polyorthoesters ester hydrolysis, esterases

polyanhydrides hydrolysis

polyamides hydrolysis

polyalkylcyanoacrilates hydrolysis

polyesters (lactides/glycolides, polycaprolactones)

ester hydrolysis, esterases

polyphosphazenes hydrolysis, dissolution

pseudo-polyamino acids proteases

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encapsulation or drug release, biodistribution, bioavailabilityand so on.

Laser light scattering or photon correlation spectroscopyare used for the determination of particle size and sizedistribution; scanning electron microscopy, transmissionelectron microscopy and atomic force microscopy formorphological properties; X-ray photoelectron spectroscopy,Fourier transform infrared spectroscopy and nuclearmagnetic resonance spectroscopy for surface chemistry; anddifferential scanning calorimetry for thermodinamic proper-ties. For characterisation of biopharmaceutical properties ofnanoparticles, fluorescence-based techniques are becomingthe most convenient [30].

4.1.1 Polymeric nanoparticlesNanoparticles are solid, colloidal particles consisting ofmacromolecules, and vary in size from 10 to 1000 nm. Thedrug is either dissolved, entrapped, adsorbed or encapsulatedin the polymer matrix. Depending on the method of prepara-tion it is possible to distinguish between nanospheres andnanocapsules. Nanocapsules are vesicular systems in which thedrug is confined to a cavity surrounded by a polymermembrane, whereas nanospheres are matrix systems in whichthe drug is physically and uniformly dispersed in a matrix [13].

The early nanoparticles were formulated mainly from variousderivatives of poly(alkylcyanoacrylate) [31-34]. Experiments withanticancer drugs, such as doxorubicin, actinomycin and vinblas-tine, either incorporated in or associated with different poly-cyanoacrylate NPs, showed that the polymer type,hydrophobicity, biodegradation profile and drug properties(molecular weight, charge, localisation in NPs, adsorbed orincorporated) have a great influence on the drug distributionpattern, as they are delivered mainly to the mononuclear phago-cytes system (MPS). Such conventional NPs, once in the blood-stream, are rapidly opsonised and massively cleared bymacrophages, a fact that could be exploited in the chemotherapyof macrophage-infiltrated tumours, such as hepatocarcinoma,

bronchopulmonary tumours and myelomas [3,35,36]. Otherside effects, such as myelosuppression, could also be expectedbecause NPs target bone marrow, also a part of the MPS.Addressing anticancer drug-loaded NPs to other tumours isusually not possible due to their very short circulation time(the mean half-life of conventional NPs is 3 – 5 min followingintravenous administration).

A great deal of work has been devoted to developingso-called ‘stealth’ NPs, which are ‘invisible’ to macrophages,preventing unwanted immune responses, having a prolongedcirculation half-life and, thus, allowing selective extravasationat pathological sites (tumour or regions of inflammation).Key factors in reducing the opsonisation of NPs by macro-phages are high curvature (by reducing NP size below100 nm) and hydrophilic surface. For example, drugsincorporated in polyvinylpyrrolidone NPs and chistosan NPs(50 – 60 nm and 100 nm, respectively, and both hydrophilic)exerted an improved chemotherapeutic effect [37,38]. Signifi-cant progress has been made by coating conventional NPsurfaces with hydrophilic polymers (polyethylene glycol,poloxamines, poloxamers and polysaccharides). Such a coat-ing provides a dynamic ‘cloud’ of hydrophilic and neutralchains at the NP surface, which repels plasma proteins.Hydrophilic polymers can be introduced at the surface eitherby adsorption of surfactants or by chemical coupling to blockor branched copolymers [3,39-41].

4.1.2 Block copolymer micellesAttention has been drawn to various amphiphilic blockcopolymers that can self-associate to form micelles in aqueoussolution. Polymer micelles have advantages over conventionalsurfactant micelles in that they are more stable in physiologi-cal solution. Micelles have a fairly narrow size distribution inthe nanometre range and are characterised by their uniquecore–shell architecture, in which hydrophobic segments ofcopolymers are segregated by a hydrophilic exterior, which, inaddition, offers stealth characteristics [42].

Block copolymer micelles are useful for the systemic deliv-ery of water-insoluble drugs at concentrations greater thantheir water solubility [29]. In most block micelles composed ofamphiphilic copolymers, hydrophobic drugs are retained inthe hydrophobic core of the micelles by hydrophobic inter-action. The interaction between the core-forming segment ofthe block copolymer and drugs can also be a metal–ligandcoordination bond and electrostatic interaction. For example,polyethylene glycol-block-polyaspartic acid and cisplatin(cis-chlorodiamineplatinum(II)) form micelles with a core ofthe polymer–metal complex through ligand exchange fromchloride to the carboxyl group of the polyaspartic acid. Inaddition, it was also shown that block copolymers with theoppositely charged polyelectrolyte segment, poly(ethyleneglycol)-block-poly(lysine) and poly(ethylene glycol)-block-poly(aspartic acid), spontaneously associate to form micelleswith a core composed of polyion complex of polylysine andpolyaspartic acid segments [36].

Figure 2. Architecture of novel polymers.

Graft

Dendrimer

MultivalentStar

Dendronised polymer

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A final feature that makes block polymers attractive fordrug delivery applications is the fact that their chemicalcomposition, total molecular weight, block length ratio andcharge can easily be changed, allowing the size and morphol-ogy of the micelles to be controlled. The introduction offunctional groups, either on the hydrophobic or hydrophilicblock, allows crosslinking of the micelles. This generallyincreases the stability even at concentrations much below thecritical micellar concentration, and affects the permeabilityand release of the drug from the micelles [43-45].

4.1.3 DendrimersFirst discovered in the early 1980s, dendrimers are anotherfamily of particulate carriers of great interest. They are highlybranched, globular macromolecules with many arms emanat-ing from a central core. They can be synthesised to createnanoscale structures with precise control of size and shape, low

polydispersity index, and a highly functionalised outer surfacethat can be derivatised with various ligands. They aremonodisperse, symmetrical macromolecules built around asmall molecule or arranged in a linear polymer core usingconnectors and branching units (dendronised polymer). Inter-action of a dendrimer macromolecule with its environment iscontrolled predominately by the terminal groups. By modify-ing these, the interior of a dendrimer may be made hydrophilicand the exterior surface hydrophobic, or vice versa [46].

Dendrimers can be synthesised starting from the centralcore and working outward towards the periphery (divergentsynthesis) or in a top-down approach starting from the outer-most residues (convergent synthesis). As dendrimers are builtfrom multifunctional monomers, each layer or generation ofbranching units doubles or triples the number of peripheralfunctional groups [29]. Dendrimers have some unique proper-ties related to their globular shape and the presence of the

Figure 3. Schematic presentation of polymer carriers with drug molecules.

Polymer particulate carriers

Hydrophilic block

Cationic block

DNA

Polyplex: polymer–DNA complexDendrimersystem

Block copolymermicelles

Nanoparticles

Figure 4. PLGA nanoparticles loaded with cystatin, a protein inhibitor of cysteine proteases [67]. A: Scanning electronmicroscopy of PLGA cystatin-loaded nanoparticles. B: Fluorescence microscopy of highly proliferating and invasive MCF 10 cells, treatedwith PLGA nanoparticles, loaded with Alexa 488-labelled cystatin. After 30 min, nanoparticles were concentrated in lysosomes (whitespots). C: Fluorescence microscopy of Caco-2 cells treated in the same way as in B. Negligible amounts of nanoparticles were internalisedin 30 min.PLGA: Poly(DL-lactide-co-glycolide).

1 µm 20 µm 20 µm

A B C

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internal cavities. The most important is their good loadingcapacity, as drug molecules can be loaded within the macro-molecule interior, in the core and cavities between branches,or, in addition, attached to the surface groups in the case of‘polymer therapeutics’ (see section 4.2). Initial studies ofdendrimers as potential delivery systems focused on their useas unimolecular ‘dendritic boxes’ for non-covalent encapsula-tion of drug molecules. An advantage of using dendrimersrather than polymeric micelles is that their structure remainsglobular at all concentrations because the hydrophobicsegments are covalently connected. However, the drawback isthat it is difficult to control the release of molecules from thedendrimer core. In some cases, harsh conditions are required,whereas in others the encapsulated drug is not well retainedand the molecules are released relatively rapidly [47].

Anticancer drugs, such as 5-fluorouracil (5-FU), methotrexateor doxorubicin, that have been successfully entrapped indendrimers showed a burst release remaining failure of systemictoxicity. Introduction of stabilising poly(ethylene oxide) chainson the dendrimer slowed down the release of low molecularweight drugs. A promising approach to control the drug releaseand additionally localise it to tumour tissue involvespH-sensitive dendrimers. Quaternised poly(propylyene immine)dendrimer involves the introduction of quaternary ammoniumgroups at the external surface, which retains the drug within thecavities, but, in a slightly acidic environment such as in tumourtissue, the internal nitrogens are protonated, releasing theentrapped drug from the dendrimer [48].

4.1.4 PolyplexesPolyplexes are considered as non-viral vectors consisting of acationic polymer that interacts with negatively charged DNA byelectrostatic interaction, leading to hydrophobic collapse withparticle formation in the nanometre range. This compactionprotects the DNA from nucleases and reduces its dimensions.Polycationic carriers are either naturally occurring proteins suchas histones or protamines, or chemically synthesised compoundssuch as polylysine, polyarginine, polyhistidine, PEI, polyami-doamine or polymethacrylates. Most of the synthetic com-pounds allow modification of the molecular weight, polymerarchitecture and ligand attachment [49-52].

The net polyplex charge remains positive net value, whichenables the carrier to interact efficiently with negativelycharged cell membranes and enter the cell by endocytosis. Toreduce nonspecific interactions with blood cells and toenable the polyplexes to circulate in the bloodstream, thesurface of the polyplexes can be masked by hydrophilicpolymers to give the stealth characteristics. Targetingcell-surface receptors of tumours by introducing cell-bindingligands into polyplexes is an attractive approach to achievingspecific binding and internalisation [53,54].

4.2 Polymer therapeuticsThe drug can be entrapped in the polymer matrix, adhered tothe particle surface or covalently bound to the polymer. The

latter has been termed ‘polymer therapeutics’. The phrase wascoined by regulatory authorities, and the complexesconsidered as ‘new chemical entities’ rather than as drugdelivery formulations [4,23].

Linear polymer therapeutics are simplified polymer–drugconjugates consisting of a linear polymer covalently linked toa drug molecule. The ideal polymer should be hydrophilic toensure water solubility and must contain functional groupsfor covalent linkage of the drug. The polymer should be inertor biodegradable and protect the drug from rapid eliminationin the body [4,48]. Typically, the drug is attached to thepolymer by a biodegradable linker that defines the release ofthe drug and the localisation of the conjugate.

Spacers that are susceptible either to very specific proteoly-sis, or to a less specific pH-controlled hydrolysis or reduction-sensitive hydrolysis can be used to link the drug to the polymerbackbone [4,23,48,54]. A very successful formulation, made byKopecek and co-workers [55], was doxorubicin conjugated toHPMA through a Gly-Phe-Leu-Gly peptidyl spacer. Thistetrapeptide is stable in the circulation, but is cleaved by thelysosomal cysteine proteases, which are elevated in manyhuman tumours [56], thus helping to improve targeting. Lessspecific targeting of polymer–drug conjugates to tumour tissuehave been constructed using an acid-sensitive spacer such ascis-aconityl, hydrazone, acid sensitive ester or Schiff-base. Sucha spacer enables release of the drug in the tumour due to theacid environment in the tumour interstitium or in endosomes(pH 5 – 6) and lysosomes (pH 4 – 5).

Reduction-sensitive linkers can be achieved by using adisulfide-bonded spacer. Glutathione (GSH) is abundant incells, especially those stimulated by xenobiotics (such ascancer cells during chemotherapy). Its intracellular concentra-tion is 300 times higher than in the blood, so that the conju-gate is stable in the circulation, but dissociates intracellularlyafter endocytosis [48].

In these various ways antitumour conjugates have beenmade for daunomycin, doxorubicin, paclitaxel melphalan,mesochlorin, emitine, platinates, chlorambucil, streptomycin,mitomycin, mitoxantrone and others [4,48,57].

Globular-shaped polymer therapeutics are additionallyorganised supramolecular structures such as micelles. Theirmain contribution arises from the various amphiphilic blockcopolymers used, displaying a better protection of bounddrug than linear conjugates, and much more sustained drugrelease [58]. Examples are conjugates of doxorubicin withdi-block copolymer of poly(lactic acid) and methoxy-PEG [48],or with a copolymer of polyaspartic acid and PEG [23,29,59].Dendrimers are also globular-shaped polymer therapeutics. Inthis case the drug loading can be tuned by varying the numberof dendrimers, and release of the drug can be controlled byincorporating cleavable linkages between the drug anddendrimer. Examples include polyamidoamine dendrimer,polyarylether dendrimer and polyester dendrimer (based on2,2-bis(hydroxymethyl)propionic acid or on glycerol andsuccinic acid), which carry cisplatin, 5-FU or doxorubicin [47].

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5. Factors affecting polymeric carriers uptake by cancer cells

5.1 Passive and active targetingPassive targeting of nanoscale polymeric carriers and theaccumulation of their drug cargo in tumour tissue can beascribed mainly to their prolonged circulation time and theEPR effect. Drug carriers can also be targeted actively tocancer tissue by introducing ligands that are recogniseduniquely by receptors or certain other proteins expressed oncancer cells, also known as tumour-associated antigens(TAAs) [60]. TAAs can be various molecules, such asreceptors, enzymes, glycoproteins, structural proteins andso on, localised predominantly on the cell surface. Usefulligands for their targeting can be antibodies, engineeredantibodies, nanobodies, tumour-associated peptides,enzyme inhibitors, receptor agonists and antagonists oraptamers. Humanised monoclonal antibodies, alreadyapproved for the therapy of particular cancer types [61], aredesigned to bind and inactivate TAAs such as HER-2 orepidermal growth factor receptor, and they are alsopromising molecules for the active targeting of nanoscalepolymeric carriers [62].

5.2 Cellular uptakeUptake of polymeric carriers into cells occurs by clathrin-mediated endocytosis, a process that involves the invaginationof the membrane to form small, membrane-bound vesiclesknown as endosomes, coated by the protein clathrin [63-65].Endocytosis may be classified as pinocytosis or phagocytosis.Pinocytosis results in internalisation of material contained inthe extracellular fluid into the endosomes. The endosomalcontent is either recycled and circulated back to the plasmamembrane, or it enters an alternative pathway in whichendosomes fuse with the primary lysosomes containinghydrolytic enzymes and an acid environment, leading to theformation of secondary lysosomes. Phagocytosis is morecommonly defined as the triggering of uptake of particularmaterial by specialised cells of the reticuloendothelial systemknown as macrophages.

In general, tumour cells exhibit enhanced endocyticactivity [66], probably due to their higher metabolic activityand proliferation rate – see Figure 4. By using polymericcarriers, endocytosis can be potentiated by the introductionof positive charges or hydrophobic residues, which promoteinteractions with negatively charged membranes or lipidcomponents of the cell surface. However, these groups haveto be introduced with caution, as excess can favournonspecific uptake.

Once internalised, the polymeric carriers remain confinedwithin the vesicles unless released by a triggered mechanism(see intracellular trafficking). Lysosomotropic internalisationof DDSs is thus inevitable and is desirable for lysosomaltargeting, but drugs whose desired destination is other thanthe lysosomes require a vesicular escape.

5.3 Modulation of molecular signalling pathways by nanocarriersFollowing internalisation by endocytosis, the polymericcarrier–drug complex can enter pathways that differ fromthose of the free drug. Recognition of the polymer-bound/encapsulated drug depends on the polymer rather than thedrug, as the latter is shielded by the polymer matrix. Thepolymer may interact with various effector molecules andtrigger differiential expression of genes, crucial for MDR, celldefence and cell death mechanisms [68,69].

MDR is the refractory response of cancer cells to theaction of a variety of structurally unrelated drugs, and is oneof the primary causes for failure of cancer chemotherapy.MDR1 and MRP genes encode for the ATP-dependentefflux pumps P-glycoprotein (P-gp) and MDR protein,which actively pump out a drug that has been internalisedby diffusion. MDR is related to cell detoxification systems,such as the glutathione–glutathione-S-transferase pathway,which counteracts the toxicity of the drugs by conjugating themto reduced glutathione (GSH). Other mechanisms contributingto MDR act by entrapping the drug in the intracellular vesicles,preventing it from reaching the target site, by repairing damagein DNA or by modulating apoptotic genes, for example,suppressor p53 and apoptotic protein Bcl-2 [63,68,70].

A drug conjugated to or incorporated in the polymernetwork has been demonstrated to be less sensitive to MDRthan the free drug. Pluronic polymer [69-71] can inhibit anefflux pump such as P-gp, decreasing the level of GSH. Asthese pumps are energy-dependent, their inhibition isrelated to energy depletion by abolition of pump-associatedATPase activity. As the polymer mediates direct and indirectenergy depletion, efflux pump operation fails, and the resist-ant cell consequently becomes more sensitive to chemother-apeutic agents. In addition, the polymeric carrier mayphysically sequester the drug from the efflux pump at theplasma membrane [72].

Molecular signalling, mediated by polymeric carriers, canalso modulate cellular processes such as the mode of celldeath. Comparing free and bound drug, it was discovered thatthe polymer can induce apoptosis by a cumulative stimulationof caspases, the enzymes that are activated by various intrinsicand extrinsic factors and are capable of triggering an expand-ing cascade of proteolytic activity within the cell [69]. Thiseventually results in the digestion of structural proteins in thecytoplasm, chromosomal DNA degradation and/or phago-cytosis of the cell [73]. In addition, treatment of cancer withpolymeric drug carriers has been correlated to a decrease invascular permeability, regulated by vascular endothelialgrowth factor (VEGF). In contrast, for a free drug, such asdoxorubicin, an upregulation of VEGF was observed, whichresulted in increased permeability. The lower permeability ofcancer vessels has the benefit of directing a polymeric-bounddrug to other locations in a tumour, thus achieving morehomogenous and more effective distribution. Another differ-ence observed for free and bound/encapsulated drug is the

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time-dependent response of the latter. Polymeric carriers exerta delayed response that can shut down the stress responsemechanism and dictate a different subcellular pathway. Atime-dependent response may define an important role forDDSs, either in ensuring release of the drug or triggeringother molecular signalling systems for specific actions [74].

5.4 Vesicular versus cytoplasmic traffickingAs discussed above, drugs that are not active towards subcellu-lar structures generated in the endocytic process have toescape the vesicular capture. As the endosome matures it fuseswith a lysosome, and the polymeric DDS is thus exposed todigestive enzymes and an acidic environment. These lyso-somal enzymes, particularly proteases, can also be a target forantitumour therapy [75]. They degrade the components of theextracellular matrix, a proteolytic event associated with earlytumour development, as it affects tumour cell proliferationand angiogenesis, and with dissemination of tumour cellsfrom primary tumours. Several classes of proteases areinvolved in this process, and matrix remodelling may occurextracellularly, intracellularly or be a combination of both [76].The authors have shown that poly(D,L-lactic-co-glycolic acid)polymeric carriers can be an effective tool for rapid delivery ofprotease inhibitors into tumour cells, impairing their lyso-somal proteolytic activity and, consequently, their invasivepotential [77]. The addition of protease inhibitors to a poly-meric DDS may also enhance the stability of other drugsprior to their vesicular escape.

The endolysosomal escape of the drug carriers can berealised by tailoring the complex to respond to the vesicularenvironment. Nori and Kopecek [63] suggested a specificspacer in polymer drug conjugates that would release thedrug from the carrier in the acidic environment, enabling itto diffuse to cytoplasmic targets (see section 4.2, Polymertherapeutics). Such a strategy is useful for low molecularweight drugs that are stable over a range of physiologicalconditions and that can also penetrate the endosomemembrane. However, proteins or oligonucleotides pose agreater challenge due to their susceptibility to enzymaticcleavage and degradation in acid environments. For thesesubstances, various approaches have been suggested to facili-tate their cytoplasmic delivery. Escape from the endosomemay be triggered by introducing fusogenic peptides into theDDS [78]. In this case the proteins are able to formmembrane channels in response to the low pH. They usuallycontain charged and hydrophobic domains that are respon-sible for conformational changes following protonation andmembrane insertion, leading to disruption of the endosome.Pore formation and membrane destabilisation was proposedas most probable mechanism.

Endosomal escape may also be accomplished by endosomo-lytic polymers, such as PEI, which are believed to causemembrane rupture by the proton sponge mechanism [79]. Thepresence of large numbers of amine groups confers a highbuffering capacity on the polymer. As the low pH of the

lysosome is maintained by a proton pump, buffering by PEIcalls for an increased entry of protons. The influx is accompa-nied by the co-transport of chloride ions, further increasing theosmolarity of the lysosome. Water is then taken up and inducesthe swelling and eventual rupture of the endosome. Anotheradvantage of DDSs with high buffer capacities is that thepro-enzymes are not activated at the higher pH.

Other strategies, independent of endocytic processes, canalso be used to enhance the delivery of protein or oligonucleo-tide drugs to the cytoplasm. They include physical or chemicaltechniques, such as electroporation, ionophoresis or the use ofdetergents, which alter or destroy the integrity of the cellmembrane. Although these methods can enable the transportof drugs through the membranes, they usually affect cellviability and possess limited clinical applications. A newsolution that meets this challenge has been the discovery ofcell-penetrating peptides [80]. These peptides are not only ableto translocate into the cell, but also aid the transport of diversesubstances, ranging from small molecules to particulatematerials, into the cytoplasm and nucleus.

These peptides originate from transcription-activatingproteins, antennapaedia (from Drosophila) and the HIV-1 Tatprotein. A shorter sequence was confirmed to be responsiblefor the penetration, comprising between 7 and 30 amino acids,also referred to as the membrane translocating sequence orprotein transduction domain. Binding a cell-penetrating Tatpeptide to a polymeric drug conjugate resulted in a threefoldhigher cytotoxic activity of chemotherapeutic agent [63].

5.5 Nuclear traffickingMany chemotherapeutic agents elicit their action on geneticelements in the cancer cell by crosslinking or chelatingDNA, or by interfering with DNA replication, transcriptionor translation. Nuclear targeting is therefore a key factor inthe effectiveness of such drugs. However, the nuclearenvelope is a double-layered membrane, allowing the passivetransport of molecules < 9 nm only, and large molecules canenter the nucleus only by active transport with the aid of thenuclear pore complex (NPC). This consists of proteinsknown as nucleoporins that are arranged in the membranein a ring-like structure and regulated by special moleculescalled nuclear localisation signals (NLSs). NLSs generallycontain cationic amino acids with residues that can be phos-phorylated. When these molecules are activated they associ-ate with the NPC and, enabled by GTPase activity, thecomplex is translocated into the nucleus. Bonding NLSs tothe delivery systems has been reported to result in a 400- to2000-fold amplification of the cytotoxic activity of controlsystems lacking the NLS [81].

Enhanced delivery of genetic material to the cell nucleusmay also facilitate the application of gene therapy in cancerpatients. Utilisation of viruses in gene delivery is a commonapproach, as their intrinsic machinery enables theirpermeation through the nuclear envelope. However, theimmunogenicity and mutagenic nature of viral vectors

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constitutes a major limitation for their regular clinical use.Therefore, non-viral delivery systems, including nanoscalepolymeric carriers could be used to overcome these problems.For example, polyplexes, conjugated to NLS, have alreadybeen shown to increase the transfection efficiency oftherapeutic genes [81].

6. Nanoscale polymeric carriers in radiotherapy

6.1 Radionuclide-containing polymeric carriersThe use of radionuclides for the treating tumours has been aneffective alternative to other therapies such as chemotherapyand external radiation therapy [101]. Nanoscale or microscalepolymeric carriers have an advantage of delivering high dosesof radioactivity to the tumour area without causing anydamage to the surrounding tissues and organs. The mostroutinely used radiopharmaceuticals are 99mTc (Technetium),90Y (Yttrium), 188Re (Rhenium) and 166Ho (Holmium) (forreview see [82]), and their selection depends mostly on the typeof tumour and the treatment range.

Besides glass microspheres, polymer-based carriers, such aspolylactic acid and polyglycolic acid, their copolymers,poly(lactic-co-glycolic acid) and albumin, are preferred in thedelivery of radionuclides to tumours. Their advantage ininternal radiotherapy has been demonstrated in hepatic,spleen, bone, and head and neck carcinomas [82].

6.2 Boron neutron capture therapyBoron neutron capture therapy (BNCT) is the treatment oftumours based on a nuclear capture reaction [47]. When 10Bis irradiated with thermal neutrons, highly energetic α-parti-cles and 7Li ions are produced that are toxic to cells in thevicinity of boron atoms. To achieve a cytotoxic effect intumour cells, it is necessary to deliver 10B to tumour tissue ata concentration of ≥ 109 atoms per cell. The use ofpolyhedral borane cages, containing large numbers of boronatoms conjugated to antibodies against TAAs, has been aleading approach to this direction [83,84]. However, the directbinding of large amounts of boron can affect the reactivityof the antibody. In this case the use of polyvalentdendrimeric particulate carriers can very effectively increasethe delivery of boron to tumour cells while preserving thereactivity of the antibody. For example, polyamidoaminedendrimers, carrying 1100 boron atoms and conjugated tocetuximab, a humanised monoclonal antibody to epidermalgrowth factor (EGF) receptor, showed a tenfold higherconcentration in brain tumours than in normal braintissue [85]. The binding of cetuximab itself to EGF receptorreduces cell growth and proliferation, and provides togetherwith BNCT a synergistic antitumour effect. The successfulapplication of several other humanised monoclonal antibod-ies in cancer treatment [61] extends the number of possibletargets for specific delivery of boron-enriched particulatecarriers and opens new possibilities for BNCT.

7. Expert opinion and conclusions

The fascinating advance towards understanding the molecularmechanisms of the living world has opened new perspectives inbiology, biotechnology and medicinal chemistry. By exploringscientific breakthroughs, pharmaceutical and biotech companiesare introducing an increasing number of new candidate drugs foreffective and specific control of some of the most dangeroushuman diseases, such as cancer. However, the research is focusedpredominantly on the identification of proper targets and devel-opment of the most potent molecules for their inactivation,whereas the localisation of their action and specific delivery to thesite of pathological event have received less attention. In cancertreatment the chemotherapeutics are typical examples of suchpotent agents, being cytotoxic to tumour cells and causing regres-sion of tumour burden, but their usefulness is limited by alsoaffecting non-tumour tissues. Nanoscale polymer carriers enabletumour-targeted delivery of chemotherapeutics, they can signifi-cantly enhance therapeutic efficacy and improve the quality oflife of cancer patients by reducing undesirable side effects. Somerecent successes in the area, such as the FDA approval forAbraxane™, a NP form of albumin and paclitaxel, enabling50% more drug to be administrated to patients, support theapplication of nanoscale systems in cancer therapy.

The advantage of polymeric carriers is the diversity of theirstructure, which enables their adaptation to the properties ofthe drug and the target. By small changes in the chemicalstructure of the polymer, or different combinations of thepolymers, the physical properties of the delivery system can bemodified to optimise its function. The nanoscale size of thecarriers enables passive targeting of DDSs to tumours byexploiting the EPR effect and rapid internalisation by targetedcells. The efficacy of nanoscale polymer carriers can beimproved in different ways, for example, by using biodegrada-ble polymers, increasing the drug loading capacity, masking thecarriers to prolong their circulation half-time, by tailoring theirstructure to escape vesicular capture during endocytosis, or torelease the drug inside the tumour, making use of the changesin its microenvironment. However, in our opinion, the greatestneed in the application of nanoscale polymer carriers in cancertherapy is the development of active targeting. In addition to acytotoxic drug, the carrier should contain a ligand recognisingtumour-associated antigens that are overexpressed during theprogression of malignant disease. There are several ways oftargeting them; however, the most appropriate molecules forthis purpose are monoclonal antibodies, approved in recentyears for therapeutic application in cancer.

Acknowledgements

The authors thank Professor R Pain for critical reading ofthe manuscript. Research work was supported by theMinistry of High Education, Science and Technology of theRepublic of Slovenia, and partially by the 6th EU FrameworkIP project CancerDegradome.

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74. NISHIYAMA N, NORI A, MALUGIN A, KASUYA Y, KOPECKOVA P, KOPECEK J: Free and N-(2-hydroxypropyl)methacrylamide copolymer-bound geldanamycin derivate induce different stress responses in A2780 human ovarian carcinoma cells. Cancer Res. (2003) 63:7876-7882.

75. TURK V, KOS J, TURK B: Cysteine cathepsins (proteases) – On the main stage of cancer? Cancer Cell (2004) 5:409-410.

76. PREMZL A, ZAVAŠNIK-BERGANT V, TURK V, KOS J: Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro. Exp. Cell Res. (2003) 283(2):206-214.

77. CEGNAR M, PREMZL A, ZAVAŠNIK-BERGANT V, KRISTL J, KOS J: Poly(lactide-co-glycolide) nanoparticles as a carrier system for delivering cysteine protease inhibitor cystatin into tumour cells. Exp. Cell Res. (2004) 301:223-231.

• This paper showed an effective inhibition of intracellular proteases using delivery of inhibitor by polymer NPs.

78. DECOUT A, LABEUR C, GOETHALS M, BRASSEUR R, VANDEKERCKHOVE J, ROSSENEU M: Enhanced efficiency of a targeted fusogenic peptide. Biochimica Biophysica Acta – Biomembranes (1998) 1372:102-116.

79. LUNGWITZ U, BREUNIG M, BLUNK T, GÖPFERICH A: Polyethylenimine-based non-viral gene delivery. Eur. J. Pharm. Biopharm. (2005) 60(2):247-266.

80. DESHAYES S, MORRIS MC, DIVITA G, HEITZ F: Cell-penetrating peptides: tools for intracellular delivery of therapeutics. Cell Mol. Life Sci. (2005) 62:1839-1849.

81. MERDAN T, KOPECEK J, KISSEL T: Prospects for cationic polymers in gene and oliglonucleotiede therapy against cancer. Adv. Drug Deliv. Rev. (2002) 54:715-758.

•• This article summarises efforts on the application of polyplexes in cancer therapy.

82. SINHA VR, GOYEL V, TREHAN A: Radioactive microspheres in therapeutics. Pharmazie (2003) 59:419-426.

83. BARTH RF, CODERRE JA, VICENTE MG, BLUE TE: Boron neutron capture therapy of cancer: current status and future prospects. Clin. Cancer Res. (2005) 11:3987-4002.

• This article presents the current status and future prospects of boron neutron capture therapy of cancer.

84. NOVAK-DESPOT D, KOS J, SERŠA G, CEMAŽAR M, ŠKRK J, GUBENŠEK F: Boronated CDI 315B monoclonal antibody and its potential use in boron neutron capture therapy. In: Advances in Neutron Capture Therapy (Volume II), Chemistry and Biology. Larsen BB, Crawford J, Weinreich R (Eds.), Elsevier Science, Amsterdam, The Netherlands (1997):516-518.

85. HUANG SM, BOCK JM, HARARI PM: Epidermal growth factor receptor blockade with C225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of head and neck. Cancer Res. (1999) 59:1935-1940.

Patent101. GLAJCH JL, SINGH P: US6455024

(2002).

AffiliationMateja Cegnar, Julijana Kristl & Janko Kos†1

†Author for correspondence1University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, 1000 Ljubljana, SloveniaTel: +386 1 4769 604; Fax: +386 1 4258 031;E-mail: [email protected]

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nanoscale polymer carriers to deliver chemotherapeutic agents to tumours

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