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Self-Assembled Polymeric Nanomicelles as DeliveryCarriers for Antitumor Drug CamptothecinJunping Zeng a , Jiahui Yu b , Jin Huang a b & Peter R. Chang ca College of Chemical Engineering, Wuhan University of Technology , Wuhan , P. R. Chinab Institute of New Drug Innovation Research and Development, East China NormalUniversity , Shanghai , P. R. Chinac BioProducts and Bioprocesses National Science Program, Agriculture and Agri-FoodCanada , Saskatoon , Saskatchewan , CanadaAccepted author version posted online: 01 Aug 2011.Published online: 20 Jan 2012.

To cite this article: Junping Zeng , Jiahui Yu , Jin Huang & Peter R. Chang (2012) Self-Assembled Polymeric Nanomicellesas Delivery Carriers for Antitumor Drug Camptothecin, Journal of Dispersion Science and Technology, 33:2, 293-306, DOI:10.1080/01932691.2011.562407

To link to this article: http://dx.doi.org/10.1080/01932691.2011.562407

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Self-Assembled Polymeric Nanomicelles as DeliveryCarriers for Antitumor Drug Camptothecin

Junping Zeng,1 Jiahui Yu,2 Jin Huang,1,2 and Peter R. Chang31College of Chemical Engineering, Wuhan University of Technology, Wuhan, P. R. China2Institute of New Drug Innovation Research and Development, East China Normal University,Shanghai, P. R. China3BioProducts and Bioprocesses National Science Program, Agriculture and Agri-Food Canada,Saskatoon, Saskatchewan, Canada

Despite being highly recognized as an antitumor candidate due to its high potency in binding toDNA topoisomerase I and inhibiting of DNA relegation, full clinical application of camptothecinis unfortunately hampered by its poor solubility in aqueous medium and by the adverse effectscaused by its hydrolyzed product under physiological conditions. In an attempt to increase itseffective solubility, nanomicelles formed through self-assembly of copolymers by polymer-drugconjugate or by physical envelopment have recently been established to improve the efficacy ofmany drugs. This review provides the most up-to-date information available relating novelnanomicelles technology to the improvement and realization of the full potential of camptothecin.In particular, physicochemical and biological properties of camptothecin and its derivatives, thecontrolled factors of micelle formation, the techniques of drug encapsulation, and the structure-properties of nanomicelles are elucidated and discussed. Undoubtedly, polymer nanomicelle car-riers can be selectively delivered to tumors via the enhanced permeability and retention effect.Moreover, micelles with smart functions such as stimulus-responsive and specific drug targetingmay enhance the activity of potent bioactive compounds, facilitating their clinical applications.

Keywords Camptothecin, nanomicelle, physical encapsulation, polymer-camptothecinconjugate

1. INTRODUCTION

Camptothecin (CPT), an alkaloid, was first identifiedand purified from Camptotheca acuminata, a tree native toChina.[1] Interestingly, 20(S)-CPT exhibits anticanceractivity against a wide spectrum of human tumor cellsincluding lung, prostate, breast, colon, stomach, bladder,ovarian, and melanoma in animal models;[2] however, thetherapeutic application of unmodified CPT is hindered byits instability at neutral pH, poor solubility in aqueousmedium,[3] and toxicity.[4] In addition, the water-solublecarboxylate form of CPT can be excreted by the kidneysand it causes hemorrhagic cystitis, a severe adverse reactionto CPT administration. To overcome these drawbacks, CPT

or its derivative has been conjugated to, or enveloped by,various polymeric carriers to improve solubility, enhancethe stability of its lactone form, and reduce renal clearance.

To improve the specific delivery of drugs with low thera-peutic indices, several drug carriers including liposomes,[5]

microparticles,[6] nanoassociates,[7] nanoparticles,[8] drugpolymer-conjugates,[9] and polymeric micelles[10] have beenexplored. Polymeric micelles are currently recognized asone of the most promising modal drug carriers.[11]

Polymeric micelle-based anticancer drugs were originallydeveloped by Professor Kataoka et al. in late the 1980sand early 1990s.[12–14] Polymeric micelles have a uniquecore-shell structure in which an inner core, serving as a

Received 15 December 2010; accepted 13 January 2011.This research work was supported by the National Natural Science Foundation of China (20404014 and 50843031), 973 Projects of

Chinese Ministry of Science and Technology (2009CB930300), Program of Energy Research and Development (PERD) of Canada,Agricultural Bioproducts Innovation Program (ABIP) of Canada via the Pulse Research Network (PURENET), Shanghai Munici-pality Commission for Special Project of Nanometer Science and Technology (0952nm05300), Shanghai Municipality Commissionfor Non-governmental International Corporation Project (09540709000) and International Corporation Project (10410710000), andFundamental Research Funds for the Central Universities (Self-Determined and Innovative Research Funds of WUT 2010-II-022).

Address correspondence to Jin Huang, College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, P. R.China. E-mail: huangjin@iccas.ac.cn; and Peter R. Chang, BioProducts and Bioprocesses National Science Program, Agriculture andAgri-Food Canada, Saskatoon, SK S7N 0X2, Canada. E-mail: peter.chang@agr.gc.ca

Journal of Dispersion Science and Technology, 33:293–306, 2012

Copyright # [2012] Crown copyright

ISSN: 0193-2691 print=1532-2351 online

DOI: 10.1080/01932691.2011.562407

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nanocontainer for hydrophobic drugs with relatively lowstability, is surrounded by an outer shell of hydrophilicpolymers. Polymeric micelles have demonstrated longevityin the bloodstream and were effective in tumor accumu-lation after administration.[15–18] They are considerablymore stable than surfactant micelles and exhibit severaladvantages, such as a simple preparation, efficient drugloading without chemical modification of the parent drug,and controlled release of the drug.[19–22] The diameter ofthe micelles can be controlled within the range of 20 to100 nm to ensure that they do not pass through normal ves-sel walls; a reduced incidence of side effects from the drugsmay be expected due to the decreased distribution vol-ume.[23] Currently, although some questions may ariseregarding the stability in plasma, polymeric micelles seemto be one of the most advantageous carriers for the deliveryof water-insoluble anticancer drugs.[24]

Literature indicates that CPT can be incorporated intomicelles by polymer-drug conjugates or by physical encap-sulation through dialysis and emulsion techniques to over-come the solubility, stability, and unexpected toxicity issuesassociated with CPT. Polymer-CPT conjugates preparedusing conventional coupling chemistry have various hetero-geneities that may impact their pharmacological andpharmacokinetic properties in vivo. As a result, several dif-ferent types of conjugation between CPT and water-solublepolymers, such as polyethylene glycol (PEG),[25] poly(1-hydroxymethylethylene hydroxyl-methyl formal),[26]

poly[N-(2-hydroxypropyl) methacrylamide] (HPMA),[27]

and poly(L-glutamic acid) (PGA),[28] have been designedand evaluated as antitumor polymer prodrugs. Incorpor-ation of CPT into these polymer prodrugs was pursuedwith the intent of surmounting the limitations that precludebroad clinical application including poor solubility, rapidclearance, high systemic toxicity, and=or poor selectivitytoward cancer cells. The hydrophobic inner core of micellesformed by physical encapsulation serves as a nanocontai-ner for hydrophobic drugs, while the hydrophilic partserves as an interface between the bulk aqueous phaseand the hydrophobic domain. Consequently, the polymericmicelles maintain a satisfactory aqueous stability irrespec-tive of the high content of hydrophobic drug buried inthe inner core. The development of smart polymericmicelles, the properties of which change dynamicallybecause of their sensitivity to chemical or physical stimuli,is the most promising trend for targeted therapy with highefficacy and ensured safety.[11] These micelles respond topathological or physiological endogenous stimuli alreadypresent in the body, or to externally applied stimuli suchas temperature, light, or pH. A pH-triggered system isone where the drug release is expected to occur with thedestabilization of the micelle structure in response to theacidic pH of tumor tissue, as well as with the disruptionof intracellular compartments such as the endosome and

lysosome.[29] Disulfide cross-linking in the micelles is suffi-ciently stable in the oxidized atmosphere of the blood toenable prolonged circulation in the body, while thereductive intracellular environment leads to cleavage ofthe disulfide cross-linking.[30]

Drawing from a vast knowledge base, this review aimsto present a substantive overview of the self-assembly ofpolymer-CPT conjugates and of CPT nanoencapsulationby self-assembly of copolymers for antitumor drug deliveryapplications, including examples that show how the struc-ture and properties of nanomicelles affect cytotoxicity. Inthis review, physicochemical and biological properties ofcamptothecin and its derivatives (CPTs), the micelle forma-tion, the drug encapsulation process, and the functionsassociated with the structure or properties of nanomicellesin antitumor drug delivery systems will be discussed.

2. CAMPTOTHECIN

2.1. Camptothecin and Its Derivatives

CPT is a cytotoxic, quinoline alkaloid characterized by aplanar pentacyclic ring system (Figure 1).[2,31] While theA-D rings of CPT are necessary to maintain activity, mod-ifications are permissible[32]; however, the E-ring lactone isnecessary for activity by binding to topoisomerase I (TOPI),[33] while the carboxylate form is inactive on tumors.[34,35]

Unfortunately, at physiological pH most CPT moleculesexist in the inactive carboxylate form. It is generallybelieved that the 20-OH group participates in the increasedrate of lactone hydrolysis of CPT at physiological pH, andthat serum albumin preferentially binds the CPT carboxy-late form, which shifts the lactone=carboxylate equilibriumin favor of the carboxylate form.[36,37] Much effort hasfocused on stabilizing the lactone without compromisingcytotoxicity, and more recent structure-efficacy studieshave identified many compounds with improved solubilityand better antitumor activity. The A and B rings are themost tolerant to modification, and substitutions at posi-tions 7, 9, 10, and 11 often show good biological activityand improved physical or pharmacological features. Sub-stitution on rings C, D, and E is not well tolerated.[38]

Altering the C and D rings or substituting positions 12and 14 inactivates the molecules, and the inability of theanalogues to stabilize the covalent binary complex wasattributed to the disruption of planarity.[39] Substitution

FIG. 1. CPT in the lactone form and open carboxylate form. (Figure

available in color online.)

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on the E ring, where binding to TOP I occurs, underscoresthe importance of certain structural features to the mainte-nance of the activity of CPT.[39] For example enlargementof the ring to form the betahydroxy lactone improves stab-ility and drug activity. Additionally, modification of theC20 hydroxyl group through alkylation or acylation, thefavored method for linking CPT covalently to macromole-cules, has been shown to stabilize the lactone.[40]

Camptothecin derivatives have attracted a great deal ofresearch in an effort to deal with the solubility and stabilityissues associated with CPT. Herein, we focus only on deri-vatives with reported in vivo evaluation. Three styles canbe defined for modification at different points: QuinolineModification of CPT is the region most commonly modi-fied, and these derivatives (including the FDA approveddrugs irinotecan and topotecan among many others) showincreased solubility, lactone stability and antitumoractivity; E-Ring Modification has poor efficacy on themanipulation of lactone and very few have been reported,however, the homocamptothecins have offered promisewith E-ring stabilization and antitumor activity;20-Hydroxy-Linked Modification is the esterification oralkylation of the 20-hydroxyl group, which has been shownto increase lactone stability. While various ester derivativeshave been prepared, only a small number of 20-O-hydroxylmodifications have been tested in vivo.[40]

10-Hydroxycamptothecin (HCPT) Like CPT, HCPT asCPT derivative is naturally occurring.[41] The molecularstructure of HCPT (Figures 2A) includes an asymmetriccarbon with S-configuration in the lactone ring and a phe-nol group. It has a pKa value of 8.56, which is somewhatabove the physiological pH.[37]

7-ethyl-10-hydroxy-camptothecin (SN-38) SN-38(Figure 2B), a derivative of CPT, is an active metaboliteof Irinotecan (CPT-11), and has an advantage over itscamptothecin precursor in that it does not require acti-vation by the liver. SN-38 is approximately 200–2000-foldmore cytotoxic than CPT-11, and is up to 1000-fold morepotent than CPT-11 as a TOP I inhibitor against severaltumor cell lines.[42] However, therapeutic application ofunmodified SN-38 is hindered due to its poor solubilityin any pharmaceutically acceptable solvents.[43]

2.2. Physicochemical Properties and Biological Activity

CPT is a potent antitumor drug which has been usedextensively in traditional Chinese medicine. Due to the neg-ligible solubility of CPT in water however, the open-ringsalt form, which has a lower efficacy, was used and resultedin hemorrhagic cystitis and myelotoxicity. Typical chemicalfeatures of CPT are the planar aromatic five-ring systemwith a lactone moiety and an S-configuration at C-20.[44]

Because an intact lactone group is essential for interactionwith the DNA-enzyme complex, all CPTs presently in clini-cal trials possess an E-ring lactone.[44] This lactone moietyis chemically unstable and once placed in an aqueous sol-ution at physiological pH undergoes pH-dependent, revers-ible hydrolysis to a hydroxyl carboxylate form, which isdevoid of TOP I inhibitory activity (Figure 1),[45,46] highlytoxic and therapeutically inactive.[34,35,47] The hydrolysisrate is dependent on several factors including pH,[48] ionicstrength,[37] and protein concentration.[49,50] Preferentiallybinding to the carboxylate form of CPT, serum albuminserves as the driving force for shifting the lactone-carboxylate equilibrium toward the formation of carboxy-late.[34,35,47] In addition, increased temperature increasesthe rate of interconversion without affecting the equilib-rium itself. The in vivo equilibrium also depends onbinding to albumin.[44]

The potent biological activity of CPT has traditionallybeen exploited by humans for hunting, execution, warfare,and the treatment of diseases. Although the antitumoractivity of CPTs has been the focus of research groups inboth industrial and academic arenas,[51] CPTs have alsobeen studied as potent inhibitors of replication, transcrip-tion, and packing of double stranded DNA-containingadenoviruses, papovaviruses, and herpesviruses, and thesingle-stranded DNA-containing autonomous parvo-viruses.[52] Early reports detailing the prolongation of lifeand inhibition of solid tumor growth in animal tumor mod-els by CPT incited a flurry of research designed to ascertainthe action mechanisms. Topoisomerases are ubiquitousenzymes that solve topological problems generated bykey nuclear processes such as DNA replication, transcrip-tion, recombination, repair, chromatin assembly, and chro-mosome segregation.[51] TOP I is an essential enzyme thatrelaxes supercoiled DNA prior to transcription throughthe formation of a single strand break and relegation.[40]

It has been shown that CPT is capable of inhibitingDNA synthesis via strand scission and then cause cell deathduring the S-phase of the cell cycle.[39] CPT represents aparadigm for targeting macromolecular interactions,because it selectively targets TOP I by trapping the cata-lytic intermediate of the TOP I-DNA reaction, the cleavagecomplex. In fact, CPT slows down the dissociation of thesemacromolecules instead of preventing the binding of thetwo macromolecules they target (TOP I and DNA). The

FIG. 2. a) Molecular structure of the S-configurational isomer of

HCPT. b) Molecular structure of SN-38.

SELF-ASSEMBLED POLYMERIC NANOMICELLES AS CARRIERS FOR CPT 295

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activity of CPT underlines the usefulness of screening fordrugs that inhibit the dissociation of macromolecules.[53]

If properly developed, CPTs could prove to be powerfulantiviral drugs for several DNA viruses, which are causa-tive agents for a large number of diseases.[51]

3. NANOMICELLES

3.1. Mechanism of Micelle Formation andControlled Factors

Micelle formation occurs as a result of an attractiveforce that leads to the association of molecules and a repul-sive force that prevents unlimited growth of the micelles toa distinct macroscopic phase.[54,55] Amphiphilic copoly-mers are self-assembled when placed in a solvent that isselective for either the hydrophilic or the hydrophobicpolymer. The polymer exists as single chains only at verylow concentrations; as the concentration increases thepolymer chains start to associate to form micelles untilreaching a critical value called the critical micelle concen-tration (CMC). At or above the CMC, equilibrium willfavor micelle formation. Amphiphilic copolymers withhigh CMC may not be suitable as drug targeting devices,since they are unstable in an aqueous environment andeasily dissociate upon dilution.[24]

Whether the hydrophobic chain is randomly bound tothe hydrophilic polymer or grafted to one end of the hydro-philic chain determines the type of micelle formed. Themicellar size is determined mainly by the hydrophobicforces which sequester the hydrophobic chains in the core,and by the excluded volume repulsion between the chainswhich limits their size.[56] A difference in the balance ofthese two forces in random and end-modified copolymersmay account for the reason that micelles from randomlymodified polymers are smaller than end-modified poly-mers. Randomly modified polymers associate in a mannersuch that the hydrophobic and hydrophilic parts of thepolymer are entangled together allowing possible contactbetween the core and the aqueous media, and the hydro-philic chains forming the shell are less mobile.[57] Thehydrophobic terminals of the end-modified copolymersassociate to form micelles with water clusters around thehydrophobic segments excluded from the core. There isno direct interaction between the core and the hydrophilicshell, which remain as mobile linear chains in the micellarstructure.[56,57]

The length of the hydrophobic polymer chains affectsthe onset of micellization, while the effect of the hydrophi-lic chain length on the CMC is less pronounced.[54,58,59] TheCMC varies depending upon the chemical structure of thepolymer, as well as the molecular weights of each block ofthe copolymer. Higher molecular weight in general andhigher molecular weight of the hydrophobic block in parti-cular, corresponds to a lower CMC value. The effect of the

medium composition or the loaded drug on the CMC maybe difficult to predict, but the viscosity of the micellar coremay influence the physical stability of the micelles and drugrelease. Micellar size depends on several factors includingcopolymer molecular weight, relative proportion of hydro-philic and hydrophobic chains, and aggregation num-ber.[58,60,61] The size of micelles prepared by dialysis canbe affected by the organic solvent used to dissolve thepolymer.[62,63]

Polymers used to prepare thermo-responsive micellesexhibit a lower critical solution temperature (LCST), whichcan be defined as the temperature at which the polymerphase separates.[64] The polymer is soluble below theLCST, but it precipitates at temperatures above the LCST.Due to hydrophobic interactions that result in aggregationof the micelles, the diameter of these micelles rises rapidlyat temperatures above the LCST.[65] The micellar architec-ture is maintained after lowering the temperature below theLCST which shows that the effect of temperature on sizewas reversible.

3.2. Drug Encapsulation Techniques

Entrapment of drugs is generally done using direct dis-solution, dialysis, oil-in-water emulsion, solvent evapor-ation, salting-out or solution casting procedures.[66]

Direct dissolution The technique of direct dissolution isused mainly for moderately hydrophobic polymers (e.g.,PEG-PPO) and drugs, and involves dissolving the polymerand drug in an aqueous solvent. This method relies onheating the solution to promote micellization and it ishypothesized that the mechanism of micellization is a resultof dehydration of the hydrophobic core by heating.[67]

Direct dissolution is not generally effective for the prep-aration of most drug-loaded micelles because the solubilityof highly hydrophobic drugs in water is very low.

Dialysis The dialysis method involves dissolving thepolymer and drug in a water-miscible organic solvent, suchas ethanol,[68] acetone,[69] DMF,[70–72] THF,[71,72]

DMSO,[72] or DMAc,[72] and then removing the solventby dialysis against distilled water. The organic solvents uti-lized in the dialysis method are selective only for the hydro-philic portion of the polymer, while the hydrophobicportion of the polymer associates to form the micellar coreincorporating the insoluble drug during the process. Themain advantage of the dialysis procedure over the lattermethod (e.g., oil-in-water emulsion, solvent evaporation,salting-out and solution casting) is that the use of poten-tially toxic chlorinated solvents can be avoided.

Oil-in-water emulsion The oil-in-water (O=W) emulsionmethod employs a water-immiscible organic solvent (e.g.,ethyl acetate,[73] dichloromethane,[68,73,74] or chloroform[75] and water to form an emulsion of small organicsolvent droplets that serve as a template for micelleself-assembly. After dissolving the polymer and drug in a

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water-immiscible organic solvent, water is quickly added tothe solution. The mixture is then emulsified by sonicationor stirring to create nanosized droplets throughout theaqueous phase. The drug is enveloped at the droplet coreand hydrophilic blocks extend into the aqueous phase tostabilize the emulsion while the polymer assembles. Theorganic solvent is subsequently removed by evaporationto form drug-loaded micelles.

Solvent evaporation Similar to the O=W emulsionmethod, the solvent evaporation method uses sonication,stirring or both to overcome mass-transfer limitationsand remove the organic solvent by evaporation. In thismethod, however, the organic solvent such as acetone,[73,76]

has high water miscibility, low vapor pressure, and is easilyremoved.

Salting-out The salting-out method lies at the intersec-tion of the emulsion and solvent evaporation methods;similar to solvent evaporation, the polymer and drug aredissolved in a water-miscible organic solvent (e.g., acetone)in the salting-out process. A highly concentrated aqueouselectrolyte solution (up to 60wt% salt) which preventsmixing of water and organic solvent is then added to thepolymer solution to create bulk phase separation. Finally,distilled water is quickly added to the emulsion that wasprepared by stirring or sonication of the mixture, and theorganic solvent rapidly diffuses into the water phase toinduce the formation of nanomicelles.[77]

Solution casting Like the solvent evaporation method,in the solution casting method the polymer and drugare dissolved in an organic solvent, such as acetonitrile[78]

or DMF,[79] to create a thin film after removing thesolvent under nitrogen. Hot water is then added whilestirring to get a micelle with the drug encapsulated in thecore.

3.3. Functions of the Antitumor-Drug Delivery SystemAssociated with Structure and Properties ofNanomicelles

3.3.1. Passive Drug Targeting

Passive accumulation of macromolecules and othernanoparticles in solid tumors is a phenomenon which, asa potential biological target for tumor-selective drugdelivery, was probably overlooked for several years.[80]

Differences in the biochemical and physiological character-istics of healthy and malignant tissue are responsible forthe passive accumulation of nanomicelles in tumors, andthis feature has been termed the enhanced permeabilityand retention (EPR) effect and is depicted schematicallyin Figure 3. In passive targeting, nanomicelles can diffuseinto the tumor blood vessels that have a high proportionof proliferating endothelial cells; moreover, capillaries oftumor blood vessels are fenestrated and leaky, while capil-lary walls of normal tissue are much less permeable.[80]

Because of decreased lymphatic drainage, the nanomicellesare retained and can subsequently accumulate in tumors.With passive targeting, nanomicelles can preferentiallyaccumulate in the vicinity of the tumor mass on intra-venous administration. At the present time, several passivetargeted nanomicelles containing anticancer drugs (e.g.,doxorubicin (DOX), paclitaxel and cisplatin) are underpreclinical and clinical investigation,[81] The research insome studies have showed that some nanomicelles havereduced the adverse effects of anticancer drugs, mainlyreductions in nephrotoxicity[82] and pulmonary toxicity.[83]

Generally, nanomicelles increase drug efficiency by target-ing specific cells or organs, lowering the accumulation ofthe drug in healthy tissues and minimizing its toxicity,sometimes allowing higher doses to be administered.[24]

After intravenous administration, nanomicelles shouldhave a prolonged systemic circulation time, theoretically,due to their small size and hydrophilic shell which minimizeuptake by the mononuclear phagocyte system (MPS), andto their high molecular weight which prevents renalexcretion; however, the fact is that intact nanomicelles havebeen recovered from plasma several hours following intra-venous injection.[15,84] Possibly because extravasation ofliposomes from the vasculature is more difficult due totheir larger size, liposomes with similar surface featuresseem to have a longer circulation time than micelles.[85]

The size of the nanomicelles can be controlled within thediameter range of 20 to 100 nm, which resembles that ofnatural transporting systems (e.g., virus and lipoprotein),and allows efficient cellular uptake via endocytosis.[86]

The effect of size on nanomicelle biodistribution is alsoorgan specific[87]; thus, controlling the size of nanometersized delivery vehicles based on the aggregation ofhydrophobic polymers[88] or the self-assembly of the

FIG. 3. Schematic representation of the nanomicelles accumulating in

tumors by the EPR effect. (Figure available in color online.)

SELF-ASSEMBLED POLYMERIC NANOMICELLES AS CARRIERS FOR CPT 297

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hydrophobic polymer domain of the amphiphilic block-copolymers[20,89–92] is a key point for favored biodistribu-tion[73] and EPR. Passive targeting may reduce harmfuleffects to healthy tissues, but cannot eliminate them.

3.3.2. Active Drug Targeting

The EPR effect is considered to be a passive targetingmethod, but drug targeting could be further increased bybinding pilot molecules such as antibodies[93] or sugars,[94]

or by introducing a polymer sensitive to variations in tem-perature or pH.[24] Advances in molecular pharmacology,synthetic polymer chemistry, and nanotechnology haveled to the development from passive to active targetednanomicelles.[95] A number of specific interactions (suchas ligand–receptor and antibody–antigen binding) are uti-lized in the development of cancer-targeted nanomicelles,and result in preferential accumulation of nanomicelles incancer cells.[96] Active targeting of an anticancer drug isachieved by conjugating nanomicelles to ligand capableof recognizing antigens (or receptors) on the target tissue,so that drugs can be brought directly to the targeted abnor-mal organ, tissue, or cells, and activated by active drugtargeting.[97,98] Numerous ligands, such as antibodies(Herceptin, Mabthera, and Erbitux antibodies that recog-nize HER2=neu, CD20 and EGF receptors, respectively),small molecules (folic acid receptor is expressed on thesurface of cancer cells) or peptides (amino acid sequence[Arg–Gly–Asp] binds to tumor avb3 integrin), have beenused for active targeting.[99–103] Folate or transferrin target-ing is an interesting approach for cancer therapy becauseits receptors are often overexpressed on tumor cells: theexpression of folic acid receptors is observed in approxi-mately 89% of human ovarian cancers and in approxima-tely 20–50% of solid cancers originating from the kidney,lung, breast, bladder and pancreas.[104] For example, Yooand Park have formulated anticancer drug-loaded targetednanomicelles (PEG–poly[lactide-co-glycolide]) with a folicacid attachment to achieve folate receptor targeted deliveryof DOX.[105] Concurrently, a number of folates conjugatesincluding protein toxins, immune stimulants, chemothera-peutic agents, liposomes, nanoparticles, and imagingagents have been successfully prepared and delivered tofolate receptor-expressing pathologic cells.[106] Folic acidhas been utilized mostly as an active targeting ligand fortargeted nanomicelles incorporated with stimulus-responsive functions.[95]

Recently, further improvements to anticancer drugswere made in long-circulating and targeted nanomicellesby the addition of various stimulus-responsive functionssuch as pH, temperature, redox potential, magnetism andultrasound.[95] The addition of these stimulus-responsivefunctions to the nanomicelles could beneficially modifythe properties of the anticancer drug in the nanomicelles,for example by providing enhanced or controlled drug

release, improving cellular uptake, controlling the intra-cellular drug rate, or allowing for some physical activityin the target site (i.e., cancer cells) surroundings.[107,108]

Take thermo-response nanomicelles for example, theymay be utilized to enhance drug release and=or vasculartransport by local temperature changes. In an in vitrostudy, poly(N-isopropylacrylamide) (PNIPA)-poly(butyl-methacrylate) (PBMA) micelles, incorporating water-insoluble drug DOX, showed that the micelle formulationexpressed lower cytotoxicity toward bovine aorta endo-thelial cells than free DOX below the LCST (33�C). Attemperatures above the LCST, however, the activity ofthe micelle-drug conjugate was greater than that of freeDOX.[109]

4. SELF-ASSEMBLY OF POLYMER-CPT CONJUGATES

Because the physicochemical properties of anticancerdrugs are not usually optimal for their delivery into orthrough membranes, the topical application of anticancerdrugs is not always effective.[110] The main aims ofpolymer-drug conjugates are to overcome various barrierswhich hinder drug delivery, including improving watersolubility, regulating the drug release rate, reducing drugtoxicity, improving drug targeting, and synergistic drugaction. Polymer-drug conjugate implies the formation ofa covalent bond between specific groups on the drug andthe hydrophobic polymer of the core. Such bonds, suchas amide bonds, are resistant to enzymatic cleavage mainlydue to steric hindrance and cannot be readily hydrolyzedunless spacer groups are introduced.[111] In the prodrugapproach, once the prodrug has delivered the parent drug,one is left with a well-characterized and well-understoodmolecule with which to work.[110] The interactions of theprodrugs with body fluids can take place by a variety ofreactions because of the necessary conversion or activationof the prodrug to the parent drug molecules. In addition,the bioavailability of the polymer-drug conjugates can bemodified with solubilizing groups such as PEG to prolongblood circulation times. In order to eventually release thedrug, a stimulus-responsive cleavable linker, such asreduction-sensitive disulfide linkage, can be used. Thus,both of these approaches offer complementary advantagesto anticancer drug delivery to tumors.

A nanomicelle, a, b-poly[(N-carboxybutyl)-l-asparta-mide] (PBAsp)–CPT, was prepared by esterificationbetween PBAsp and 20-OH of CPT to enhance the solu-bility and stability of CPT in aqueous media.[112] The nano-micelle was spherical in shape and had good dispersity. Itssize, as measured by TEM, was about 50 nm and showed anarrow size distribution, with sizes larger than 100 nm, byDLS (Figure 4). The particle size of the resultingnanomicelles is between the pore size of a blood capillaryin normal tissue (8 nm) and that in tumor tissue

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(100–800 nm),[113] suggesting their potential as an efficientpassive target for tumor tissue. However, with an increasein CPT loading in PBAsp–CPT, the solubility sharplydecreased and the size of PBAsp–CPT nanomicellesshowed a tendency to increase. The in vitro cytotoxicityof PBAsp–CPT was evaluated with a mouse muscular cellline (L929) by MTT assay. The results showed that drugrelease of PBAsp–CPT nanomicelles displayed a linear sus-taining profile (Figure 5) as the ester bonds between thepolymer and CPT were hydrolyzed resulting in an essentialdecrease in cytotoxicity to the L929 cell line.

4.1. The Design of Nanomicelles for Long Circulating

In the body’s defense system, pharmaceutical nanocar-riers are easily opsonized and removed from circulationlong before completion of their function;[114] thus, the basicproperties of any multifunctional nanocarrier are its lon-gevity and its ability to circulate pharmaceuticals for a longtime. Because they maintain the required level of a pharma-ceutical agent in the blood for extended time intervals,long-circulating nanomicelles can slowly accumulate inpathological sites with affected and leaky vasculature andfacilitate drug delivery in those areas.[115–117] The most

frequent way to impart the longevity to drug carriersin vivo is to chemically modify pharmaceutical nanocar-riers with synthetic polymers, such as PEG. On the biologi-cal level, mechanisms for preventing opsonization by PEGinclude shielding of the charged surface, enhancing therepulsive interaction between nanomicelles and the bloodcomponent,[118] and increasing surface hydrophilicity.[119]

As a protecting polymer, PEG commonly exhibits attract-ive properties: prolonged half-life, high stability, watersolubility, low immunogenicity and antigenicity, high flexi-bility of its polymer chain, potential for specific cell target-ing, as well as minimum influence on specific biologicalproperties of modified pharmaceuticals.[120–123] Currently,many chemical approaches exist to synthesize activatedderivatives of PEG and to couple these derivatives with avariety of drugs and drug carriers.[121,124,125] mPEGylateda,b-poly (L-aspartic acid)-CPT (mPEG-graft-PAA-CPT)conjugates for the fabrication of mPEG-graft-PAA-CPTnanomicelles have been synthesized to enhance the stabilityand long-term circulation of CPT (Figure 6).[126] Becauseof the PEGylated copolymers’ biocompatibility and biode-gradability as biomaterials and drug delivery vehicles,[127]

the PEGylated polymer-CPT conjugates can provide pro-longed circulation times for drugs in the bloodstream ascompared to the nonconjugated drugs. The nanomicellesshowed good storage stability, even when incubated at37�C for 60 days, and they improved the stability of theCPT lactone form in aqueous media (Figure 7). A steadyrelease rate of CPT was maintained for 72 hours, suggest-ing great potential for mPEG-graft-PAA-CPT nano-micelles as a polymer CPT prodrug.

4.2. Design of Nanomicelles for Reduction Stimuli

Besides the traditional intelligent drug carriers based ontemperature and pH sensitive micelles, reduction-responsive biodegradable polymers have emerged as afascinating class of drug-loading materials (e.g., in the formof micelles[128,129] for intracellular triggered delivery andrelease of proteins and low molecular weight drugs.[130]

The disulfide linkage is a characteristic typical of thesereduction-sensitive polymers, and is usually located onthe main chain, side chain or cross-linker.[131] Cleavage ofthe disulfide linkage due to the thiol-disulfide exchangereaction is sensitive to the reduction conditions in thehuman body. In the body circulation and the extracellularenvironment, or on the cell surface, the disulfide bondsshowed stability in a low concentration of glutathionetripepetide (GSH), ca. 2�20 mM; in contrast, a high intra-cellular concentration of GSH (0.5�10mM) can lead toquick degradation of the disulfide linked polymers.[132]

Interestingly, these reduction-responsive polymers areespecially valuable for the triggered delivery of tumor-specific drugs owing to the concentration of GSH in tumortissue which is at least 4-fold over normal tissue.[131] As a

FIG. 4. a) TEM photographs and b) size distribution of

PBAsp-CPT20 micelles.[112] (Figure available in color online.)

FIG. 5. Cell viability of PBAsp-CPT20 nanomicelles at various CPT

concentrations against L929 cell line for 24 hours (mean �SD,

n¼ 6;�p< 0.05).[112]

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result, drugs conjugated or encapsulated with thesereducible polymers have enormous potential in formulatingdrug and gene intracellular delivery and release.

A reduction-sensitive polymer-CPT conjugate has alsobeen synthesized by successively reacting azide-functionalized CPT and poly(ethylene glycol) methyl ether(mPEG) with alkyne focal groups in disulfide-linkedpoly(amido amine) (SS-PAA). The mPEG-g-SS-PAA-CPTnanomicelles were then fabricated to improve the solubilityand stability of CPT in aqueous media (Figure 8).[130] Inthis research, because of the reduction degradability ofthe disulfide linker in the main chain of mPEG-g-SS-PAA-CPT, the CPT was sustainably released from nanomicelleswith linear gradual profiles (Figure 9). At the same time,

there was a gradual cleavage of polymer in the presenceof dithiothreitol (DTT) at concentrations that simulatedthe intracellular conditions; almost no change occurred atDTT levels corresponding to the extracellular conditions.Representative concentration-growth inhibition curves

FIG. 6. Synthesis scheme and self-assembly of mPEG-graft-PAA-CPT; TEM photographs of mPEG-graft-PAA-CPT.[126] (Figure available in color

online.)

FIG. 7. a) A355=A368 (the maximum UV absorption of CPT lactone

(kmax: 355nm) and carboxylate form (kmax: 368nm)) ratios of mPEG-

graft-PAA-CPT nanomicelles against incubation time in pH 7.4 PBS at

37�C; b) the UV adsorption spectra of CPT and mPEG-graft-PAA-

CPT.[126]. (Figure available in color online.)

FIG. 8. Formula and scheme for the disulfide-contained graft

copolymer-camptothecin conjugate and the assembled nanomicelles

observed in the TEM image.[130] (Figure available in color online.)

FIG. 9. In vitro release profiles of CPT from free drug and mPEG-g-

SS-PAA-CPT nanomicelle in pH 7.4 PBS at 37�C (means �SD, n¼ 3).[130]

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showed an essential decrease in cytotoxicity to mousemuscular cell line (L929) by MTT assay. These resultspresent a strategy for designing an antitumor polymerprodrug for tumor therapy.

5. CPT NANOENCAPSULATION BY COPOLYMERSELF-ASSEMBLY

Amphiphilic copolymers can self-assemble in aqueoussolution to form core-shell micellar nanostructures where acondensed and compact inner core (that serves as a nano-container for hydrophobic compounds) is shielded fromwater by the hydrophilic shell. The hydrophobic interiorof the structure enables efficient encapsulation of hydro-phobic molecules, such as CPTs, for drug delivery. Physicalencapsulation is more suitable for drug-loaded nanomicellesto controll release since drug release is governed by diffusionand does not depend on cleavage of a linker that may be bur-ied in the core of the micelle. When possible, incorporationof a drug by a physical procedure is the preferred methodbecause the release of a physically encapsulated drug fromthe micelle is easier, and encapsulation can be achieved bysimple mixing, avoiding the synthetic procedures necessaryfor covalent attachment of the linker and drug.[133] More-over, stimuli-responsive segments, such as pH stimuli ortemperature stimuli, have been used to form nanomicellesfor the effective and safer delivery of anticancer drugs.

In order to enhance the cellular uptake, solubility, andstability of SN-38 in aqueous media, amphiphilic brush-like polycations were synthesized to fabricate cationicnanomicelles, chitosan-graft-polycaprolactone (CS-g-PCL).[43] Some reports indicate that micelles with cationicshells have a better cell uptake ability due to electrostaticinteractions with the negatively-charged cell membranes.[11]

So far, several amphiphilic copolymers composed of catio-nic segments and hydrophobic moieties have been synthe-sized and assembled as nanomicelles. Cationic polymers,such as poly(ethyleneimine) (PEI),[134] poly(4-vinyl pyri-dine),[135,136] polylysine,[137,138] poly(N-methyldiethenea-mine sebacate),[139] and chitosan (CS), are adopted toconstruct cationic micelles. Due to the biocompatible, bio-degradable and nontoxic properties of CS, it has beeninvestigated extensively for drug and gene delivery, andfor other biomedical areas.[140–143] Graft copolymerizationof CS has become an attractive way to regulate the physicalproperties of CS. These nanomicelles have spherical shapeswith sizes ranging from 47 to 113 nm depending on thegrafting content of PCL in CS-g-PCL, suggesting theirpotential for passive targeting to tumor tissue and endocy-tosis. Compared with bare CS-g-PCL nanomicelles, thecorresponding SN-38-loaded nanomicelles, which encapsu-lated SN-38 using a lyophilization method, showedincreased particle sizes and slightly reduced zeta potentials(Figure 10). The drug encapsulation efficiency (EE), drug

loading (DL) and accumulative drug release could also becontrolled by adjusting the grafting PCL content in theCS-g-PCL. The stability of SN-38 in CS-g-PCL nanomi-celles was evaluated using reverse-phase HPLC underphysiological conditions (0.01mol=L PBS, pH 7.4, 37�C).More than 88% of the lactone ring was preserved whenSN-38 was incorporated into CS-g-PCL (1:24) nanomi-celles, while only 22.4% of SN-38 remained in the lactone

FIG. 10. Particle size determined by DLS and TEM, respectively: a)

bare CS-g-PCL (1:24) nanomicelles; b) SN-38-loaded CS-g-PCL (1:24)

nanomicelles.[43] (Figure available in color online.)

FIG. 11. Kinetic evaluation of the rate of lactone ring opening for (1)

free SN-38, and (2) SN-38-loaded CS-g-PCL (1:24) nanomicelle evaluated

by reversed-phase HPLC under physiological conditions (pH 7.4,

37�C).[43] (Figure available in color online.)

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ring form after 12-hours incubation in PBS (Figure 11).The in vitro cytotoxicity of free SN-38, bare nanomicelles,and SN-38-loaded nanomicelles was evaluated by MTTassay using mouse muscular cell line (L929). In comparisonwith free SN-38, the SN-38-loaded nanomicelles showeddecreased cytotoxicity against the L929 cell line, and thebare CS-g-PCL nanomicelles had very low toxicity. Theseresults indicated that nanomicelles based on cationicCS-g-PCL may be a candidate as a drug carrier forSN-38 and other hydrophobic drugs with improved deliv-ery and release properties.

5.1. Design of Nanomicelles for pH Stimuli

The extracellular pH of tumor tissue (as low as 6.8) issignificantly lower than the extracellular pH of normaltissue (pH 7.4)[144,145] owing to the glycolysis metabolismof cancer cells which causes the production of lactate andprotons in extracellular microenvironments.[146–148] Impor-tantly, the lower extracellular pH of tumor tissue facilitatesmetastasis, growth and invasion of tumors,[149–152] and hasoften been targeted for therapeutic strategies in the area ofdrug delivery. The difference in pH between normal tissueand tumor tissue can be used as a strategy for pH-sensitivedrug delivery. Nanomicelles delivery carriers, which hadbeen designed to present a micellization=demicellizationtransition at the acidic pH, have been studied in cancertreatment. The pH-responsive nanomicelles presented theendosomal pH-dependant demicellization after cellularuptake, and achieved a rapid stimulus-responsive drugrelease in the inner part of the tumor cells.

Min et al. designed pH-responsive polymer nanomi-celles, amphiphilic methyl ether poly(ethylene glycol)-poly(b-amino ester) (MEPG-PAE) block copolymers,using a Michael-type step polymerization.[153] Specifically,the pH-responsive MPEG-PAE micelles (pH-PMs) under-went a sudden demicellization in the acidic tumoral pHconditions, and exhibited a rapid release profile in the cellculture system and a very slow release rate under physio-logical conditions. The CPT encapsulated into the pH-PMsby a simple solvent casting method showed a rapid releaseof CPT in weakly acidic (pH 6.4) aqueous conditionsbecause of the sharp tumoral acid pH-responsivemicellization=demicellization transition. Importantly, com-pared with free CPT and CPT encapsulated PEG-PLLAmicelles, CPT-pH-PMs exhibited significantly increasedtherapeutic efficacy with minimal side effects on othertissues in breast tumor-bearing mice.

5.2. Design of Nanomicelles for Temperature Stimuli

The use of temperature-sensitive nanocarriers came nat-urally from the fact that many pathological areas demon-strate distinct hyperthermia,[108] and the technologies thatpermit site-specific elevation of temperature have led to thedevelopment of temperature-sensitive nanomicelles.[95]

There exist various means to heat the required area in thebody, and temperature can generally serve as a local stimulusboth within the tissue (inflammation is always accompaniedwith a local hyperthermia) and from the outside. Becausesome polymers are soluble below LCST and precipitatewhen the temperature increases above the LCST, they candamage the liposomal membrane during precipitation andallow for site-specific drug release from the nanomicelles.[154]

The usual representative of this class of polymers isPNIPA,[155] which has an LCST of 32�C. Polymeric micellescan be made temperature-sensitive by assembling amphiphi-lic copolymers,[156] and exact properties of such micelles canbe adjusted by chemical modification in such a way that themicelle destabilizes at temperatures above LCST andreleases the drug buried in its hydrophobic core.[157]

Yang et al. have synthesized new reverse thermo-responsive polymers, poly(ethylene oxide)–poly(propyleneoxide)(PEO-PPO) multiblock copolymers (poly(ether-carbonate)s), by covalent binding of PEG and poly(propylene glycol) chains using phosgene as the couplingunit.[158] Experimental results showed that the process ofanticancer drug HCPT release from well dispersed sphericalmicelles in vitro was composed of two steps: abrupt releaseand sustained release. In comparison with HCPT injection,the HCPT-loaded poly(ether-carbonate) micelles had awell-sustained release efficacy, which could be explainedby the viscosity increase of the copolymer solution withthe increase in temperature. After i.v. administration (2hours), the poly(ether-carbonate) micelles delivered HCPTmainly to the liver, and the concentration of HCPT in theliver (3.46 mg=g) was significantly higher than in othertissues and blood. In addition, the elimination half-life ofthe poly(ether-carbonate) micelles group was prolongedremarkably from 1.3 to 12.5 hours. Thus, the poly(ether-carbonate) micelles can be used as a drug deliverysystem for liver targeting and sustained drug release.

6. CONCLUSIONS AND PERSPECTIVES

We have summarized and discussed micelle incorpor-ation of CPTs by polymer-drug conjugates or by physicalenvelopment, which can accumulate in tumors via theEPR effect. Until recently, the EPR effect has not beenrecognized in the field of oncology; however, the micellarapproaches applied to CPT have shown a promising futurefor the development of anticancer therapy for several rea-sons including water solubility, stability, targeting, drugefficacy, and physicochemical properties. The incorpor-ation of water-insoluble CPT into nanomicelles wouldallow utilization of higher equivalent doses of CPT in can-cer treatment, and the research on nanomicelles for cancertherapy could be effective in CPT-resistant cancer cellsand lead to breakthroughs that enable their effectivetherapeutic application.

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Even though, as mentioned in this review, some prob-lems in the design and development of nanomicelles weresuccessfully overcome, more study is needed to develophighly effective and clinically acceptable nanomicelles. Asdiscussed in this review, the manner in which the micellesthemselves are prepared can be used to optimize drug load-ing, and to an extent, drug release. The challenge remainsto consistently prepare nanomicelles with uniform, predict-able size-distribution and high drug loading capacity whichremain stable upon dilution, but dissociate upon reachingthe target. Specifically, nanomicelles are needed that cansimultaneously overcome membrane-associated multi-drug-drug resistance and intracellular drug resistance,which are the ultimate causes of death in cancer patients.Interestingly, nanomicelles decorated with smart functionssuch as stimulus-responsive and specific cell-target abilitieswill lead to positive outcomes from in vitro and in vivostudies that can overcome the therapeutic challenges ofcancer. Unequivocally, comprehensive structure=propertystudies of nanomicelles would accelerate the practicaladministration and application of many pharmaceuticals.

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