Nano Today (2013) 8, 313—331

Available online at www.sciencedirect.com

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REVIEW

Anthracycline nano-delivery systems to overcomemultiple drug resistance: A comprehensive review

Ping Maa, Russell J. Mumpera,b,∗

a Center for Nanotechnology in Drug Delivery, Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy,University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAb UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Received 13 March 2013; received in revised form 22 April 2013; accepted 29 April 2013Available online 10 June 2013

KEYWORDSAnthracyclines;Nanoparticles;Multi-drug resistance

Summary Anthracyclines (doxorubicin, daunorubicin, and idarubicin) are very effectivechemotherapeutic drugs to treat many cancers; however, the development of multiple drugresistance (MDR) is one of the major limitations for their clinical applications. Nano-deliverysystems have emerged as the novel cancer therapeutics to overcome MDR. Up until now, manyanthracycline nano-delivery systems have been developed and reported to effectively circum-vent MDR both in vitro and in vivo, and some of these systems have even advanced to clinicaltrials, such as the HPMA-doxorubicin (HPMA-DOX) conjugate. Doxil, a DOX PEGylated liposomeformulation, was developed and approved by FDA in 1995. Unfortunately, this formulation doesnot address the MDR problem. In this comprehensive review, more than ten types of devel-

oped anthracycline nano-delivery systems to overcome MDR and their proposed mechanisms arecovered and discussed, including liposomes; polymeric micelles, conjugate and nanoparticles;peptide/protein conjugates; solid-lipid, magnetic, gold, silica, and cyclodextrin nanoparticles;and carbon nanotubes.

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© 2013 Elsevier Ltd. All righ

Anthracyclines in cancer treatment

Anthracyclines are among the most effective and commonlyused chemotherapeutic drugs [1]. The mechanisms of

∗ Corresponding author at: John A. McNeill Distinguished Pro-fessor, Center for Nanotechnology in Drug Delivery, Division ofMolecular Pharmaceutics, UNC Eshelman School of Pharmacy, CB#7355, 100G Beard Hall, University of North Carolina at Chapel Hill,Chapel Hill, NC 27599-7355, USA. Tel.: +1 919 966 1271;fax: +1 919 966 6919.

E-mail address: mumper@email.unc.edu (R.J. Mumper).

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1748-0132/$ — see front matter © 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.nantod.2013.04.006

served.

ntitumor activity of anthracyclines are well characterizednd documented, wherein anthracyclines are able to diffusecross the cell membrane, intercalate between DNA baseairs, target topoisomerase II (TOPO II), and induce cellpoptosis [2]. The first anthracyclines of doxorubicin (DOX,ig. 1) and daunorubicin (DNR, Fig. 1) were isolated from theacterium of Streptomyces peucetius, which could producered pigment and were found to have good activity againsturine tumors back to the 1950s [3,4]. DOX is widely used

o treat various cancers, including leukemia, Hodgkin’s lym-homa, bladder and breast cancers, etc., while DNR is usedo treat some types of leukemias, such as acute myeloideukemia (AML) and acute lymphocytic leukemia (ALL). In

314 P. Ma, R.J. Mumper

Abbreviations

Ab antibodyABC ATP-binding cassetteAFM atomic force microscopeALL acute lymphocytic leukemiaAML acute myeloid leukemiaAOT Aerosol OT

TM

AS ODN antisense oligodeoxynucleotideASO antisense oligonucleotidesATP adenosine triphosphateAu NPs gold nanoparticlesAUC area under the curveBAX BCL-2-associated X proteinBCL-2 B-cell lymphoma 2BCRP breast cancer resistance proteinBrij 78 polyoxyl 20-stearyl etherBrTet 5-bromotetrandrineBSA bovine serum albuminBUDP bilirubin uridine diphosphateCbm carbamateCHOL cholesterolCL cardiolipinCmax maximum concentrationc-Myc v-myc myelocytomatosis viral oncogene

homologCNT carbon nanotubesCPP cell penetrating peptideCSO chitosan oligosaccharideCyA cyclosporine ADAF 5-dodecanoylaminofluoresceinDiIC18(3) 1,1′-dioctadecyl-3,3,3′,3′-

tetramethylindocarbocyanine perchloratediGly glycylglycineDiOC18(3) 3,3′-dioctadecyloxacarbocyanine

perchlorateDNA deoxyribonucleic acidDNR daunorubicinDOPA 1,2-dioleoyl-sn-glycero-3-

phosphoethanolamineDOTAP N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-

trimethyl-ammonium methylsulfateDOX doxorubicinDPA N,N-diisopropylethylenediamineDPPC dipalmitoyl-phosphatidylcholineDPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerolDSAA N,N-distearyl-N-methyl-N-2-(N′-arginyl)

aminoethyl ammonium chlorideDSPC 1,2-distearoyl-sn-glycero-3-phosphocholineEGFR epidermal growth factor receptorELP elastin-like polypeptideEPC egg phosphatidylcholineEPR enhanced permeability and retentionE-wax emulsifying waxFA fatty acidFA-CS folate-chitosanFITC fluorescein isothiocyanateFRET förster resonance energy transfer

GST glutathione S-transferaseh hourHCC hepatocellular carcinomaHER2 human epidermal growth factor receptor 2HIF1A hypoxia-inducible factor 1�HPESO hydrolyzed polymer of epoxidized soybean oilHPLC high-performance liquid chromatographyHPMA N-(2-hydroxypropyl)methacrylamideHSP heat-shock proteinIC50 half maximal inhibitory concentrationICP-MS inductively coupled plasma mass spectrome-

tryIDA idarubicinIGF-1R type 1 insulin-like growth factor receptori.v. intravenouskg kilogramLA linoleic acidLHRH-R luteinizing hormone-releasing hormone

receptorLPD cationic liposome-polycation-DNALPD-II anionic liposome-polycation-DNAmAb monoclonal antibodyMAL maleimideMDR multiple drug resistancemg milligrammin minutemL milliliterMNSP mesoporous silica nanoparticlesMPA mercaptopropionic acidmPEG methoxy poly(ethylene glycol)Mr molecular massmRNA messenger RNAMRP multidrug resistant proteinMTD maximum tolerated doseNC nanocapsuleng nanogramNLC nanostructured lipid carriersNP nanoparticlePACA poly(alkyl cyanoacrylate)PC phosphatidylcholinePCL poly(�-caprolactone)PCL-b-PEO polycaprolactone-block-poly(ethylene

oxide)pDbB poly(butyl methacrylate)PDLLA poly(D,L-lactic acid)pDMAEMA poly(dimethylaminoethyl methacrylate)PDTC pyrrolidinedithiocarbamatePEG poly(ethylene glycol)PEG-DSPE polyethylene glycol-

distearoylphosphatidylethanolaminePEG-PDLLA monomethoxy poly(ethylene glycol)-

block-poly(D,L-lactic acid)PEI polyethyleniminePEO-b-PCL poly(ethylene oxide)-block-

poly(�-caprolactone)PEO-PPO-PEO poly(ethylene oxide)-poly(propylene

GalN N-acylated galactosamineGSH reduced glutathioneGSSG oxidized glutathione

oxide)-poly(ethylene oxide)P-gp P-glycoproteinPhe L-phenylalanine

Anthracycline nano-delivery systems to overcome multiple drug resistance 315

PHIM/f pH insensitive micelles with folatePHSM/f pH sensitive micelles with folatePIBCA polyisobutylcyanoacrylatePIHCA polyisohexylcyanoacrylatePLA-TPGS poly(L-lactide)-vitamin E TPGSPLGA poly(lactic-co-glycolic acid)PLK1 polo-like kinase 1pLLA/PEG poly(L-lactic acid)-b-PEGPluronic P85 poly(oxyethylene-b-oxypropylene-b-

oxyethylene)P(MDS-co-CES) poly{N-methyldietheneamine

sebacate)-co-[(cholesteryl oxocarbonylamidoethyl) methyl bis(ethylene) ammoniumbromide] sebacate}

p.o. per ospolyHis/PEG poly(L-histidine)-b-PEGPPLA 4-armed porphyrin-polylactidepSMA poly(styrene-alt-maleic anhydride)PVA poly(vinyl alcohol)PX paclitaxelRef referenceRES reticuloendothelial systemRGD4C integrin �v�3-specific ligandRNA ribonucleic acidROS reactive oxygen speciesSA stearic acids.c. subcutaneousSCCHN squamous cell carcinoma of head and neckshRNA short hairpin RNAsiRNA small interfering RNASLN solid lipid nanoparticleSolutol HS15 polyethylene glycol 660 hydroxystearateSTDC sodium taurodeoxycholateSTS sodium tetradecyl sulfatet1/2 half-lifeTet tetrandrineTHA tetraheptylammoniumTK1 thymidine kinase 1TLC thin layer chromatographyTNF tumor necrosis factorTopo topoisomeraseTPGS D-�-tocopheryl polyethylene glycol 1000 suc-

cinateTRAIL TNF-related apoptosis-inducing ligandtriGly glycylglycylglycine�g microgram�L microliterVEGF vascular endothelial growth factorVIP vasoactive intestinal peptide

F

ottameugpafa

lopwafcmobMctpadriTieagaA

�2m �2-microglobulin

order to find better anthracyclines, a great deal of researchhas been conducted to establish the structure—activityrelationship of anthracyclines, and this research has guidedidentification and synthesis of better anthracyclines. In last

two decades there have been hundreds of DOX and DNRanalogs reported in different laboratories wherein therehave been chemical modifications of their tetracyclic ring,side chain, and/or aminosugar [5,6]. However, only a few

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igure 1 Chemical structures of DOX, DNR, and IDA.

f the analogs have been approved for clinic use and amonghem, idarubicin (IDA, 4-demethoxydaunorubicin, Fig. 1) ishe most successful alternative to DNR [7]. Idarubicin waspproved by the US FDA in 1990 [4]. The absence of theethoxy group at position 4 of IDA results in significantly

nhanced lipophilicity, which results in more rapid cellularptake, superior DNA-binding capacity, and consequentlyreater cytotoxicity as compared to DOX and DNR [2]. Thehysicochemical properties of these three anthracyclinesre summarized in Table 1. For use in the clinic, they areormulated as hydrochloride salt forms dissolved in anqueous solution for intravenous injection.

Unfortunately, the clinical use of anthracyclines has beenimited by their severe cardiotoxicity and the developmentf multiple drug resistance (MDR) [8,9]. In general, manyatients achieve a complete remission when initially treatedith anthracyclines; however, ∼70% of the patients eventu-lly experience a relapse of the disease, and the treatmentailure is mainly due to MDR. The MDR mechanisms of anthra-yclines are complicated and not fully understood. Theost established mechanism of resistance is over-expression

f drug efflux proteins, particularly members of the ATP-inding cassette (ABC) superfamily: P-glycoprotein (P-gp,DR1), multidrug resistance protein 1 (MRP1), and breastancer resistance protein (BCRP). Anthracyclines are knowno be efficient substrates for ABC transporters. For exam-le, P-gp, a membrane transporter encoded by MDR1 gene,ctively pumps anthracyclines out of the cells resulting inrug resistance [10,11]. Baekelandt et al. reported the cor-elation of P-gp expression and response rate in a studynvolving 73 patients with advanced ovarian cancer [12].hey found that P-gp negative patients responded signif-

cantly better to chemotherapy (p < 0.001), and the P-gpxpression was clearly a predictor of both overall (p = 0.045)nd progression free (p = 0.006) survival, which indicated P-p expression was a marker for chemotherapy resistancend prognosis in advanced ovarian cancer. In addition toBC transporters, other cellular mechanisms of resistance

ave also been reported, such as alteration in TOPO II,ree-radical formation, up-regulation of B-cell lymphoma 2BCL-2) family members, down-regulation of tumor suppres-or protein p53, etc. [13—16].

316 P. Ma, R.J. Mumper

Table 1 Physicochemical properties of DOX, DNR, and IDA.

DOX DNR IDA

Formula C27H29NO11 C27H29NO10 C26H27NO9

Molecular weight (g/mol) 543.52 527.52 497.49Water solubility (mg/mL) 92.8 39.2 35.6Log P 1.27 1.68 2.10pKa ∼8.4 10.3 8.5Melting point (◦C) 204—205 208—209 173—174Half-life (h) 55 18.5 22

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of empty liposomes was probably due to the high bindingaffinity (1.6 × 106 M−1) of positively-charged DOX to thenegatively-charged CL. However, neither pretreatmentwith empty liposomes before drug treatment nor thecombination of vincristine and empty liposomes couldreverse MDR, which suggested DOX must be incorporated orcomplexed with liposomes to overcome MDR. The authorssuggested the DOX in liposomes would alter intracyto-plasmic vesicles to transport DOX in MDR cells, and themodulation of MDR could be due to the increase of drugaccumulation or the intracellular drug redistribution inMDR cells. Rahman et al. prepared DOX-loaded liposomescomposed of CL/PC/CHOL (molar ratio 2:10:6.8) and com-pared the cytotoxicity of the liposomes versus free DOX inP-gp resistant HL-60/VCR and its parental HL-60 cell lines

Figure 2 Summary of the proposed cellular mechanisms ofanthracycline NPs to overcome MDR. (1) NPs interact with ormodify plasma membrane and therefore change the membranestructure and induce membrane permeability; (2) NPs do notenter cells; instead free drugs are released to plasma mem-brane and then diffuse into cells; (3) NPs directly interact withand inhibit P-gp; (4) NPs bypass, but do not inhibit P-gp; (5)NPs enter into cells via receptor-mediated endocytosis; (6) NPsenter into cells via endocytosis, phagocytosis, or micropinocy-tosis; (7) NPs down-regulate P-gp, MRP, BCL-2, and HSP-70; (8)

Protein binding (%) 70

Non-cellular resistance mechanisms have also proposednd are attributed to the unique vasculatory of solid tumors17—19]. The vasculature of solid tumors is characterizeds heterogeneous, where the blood vessels are dilated andortuous. The interstitial fluid pressure of solid tumors isncreased than normal tissues, which is due to the higher vas-ular permeability and the absence of lymphatic system. Inddition, solid tumors have an acidic environment and lackutrients and oxygen, all of which help to induce resistanceo cytotoxic drugs.

Nanoparticle (NP) delivery systems have been shown toe promising carriers to improve the therapeutic effect ofnthracyclines mainly due to the enhanced permeability andetention (EPR) effect in solid tumors, while they minimizeystemic exposure, enhance drug efficacy and reduce non-pecific toxicity [20—22]. Nano-delivery systems have alsoeen shown to increase the circulation time of drugs inlood, thereby increasing the ability of drugs to reach theirites of action. Doxil, a DOX-encapsulated polyethylene gly-ol (PEG)-coated liposome formulation with the particle sizef ∼100 nm, was approved in 1995 for the treatment ofvarian cancer, AIDS-related Kaposi’s sarcoma, and multipleyeloma. The DOX PEGylated liposomes demonstrate slowerlasma clearance rate, prolonged circulation time in blood,nd decreased volume of distribution than either traditionalOX liposomes or free DOX. Importantly, Doxil was proved toave less cardiotoxicity as compared to free DOX [23—25].owever, the liposome formulation has not addressed MDRhich continues to be a major hurdle in cancer therapy.

n order to overcome MDR, various nano-delivery systemsave been developed and evaluated both in vitro and inivo. In this comprehensive review, different nano-deliveryystems for the delivery of anthracyclines as well as theirechanisms to overcome MDR are addressed (Table 2 and

ig. 2).

nthracycline nanoparticles to overcome MDR

iposomes

hierry and his co-workers developed DOX-encapsulatediposomes with cardiolipin/phosphatidylcholine/cholesterolCL/PC/CHOL) and demonstrated that both DOX-

ncapsulated liposomes and free DOX spiked into auspension of empty liposomes could reverse MDR andad comparable activity in MDR Chinese hamster LZ cells26]. The efficacy of free DOX spiked into a suspension

NPs up-regulate BAX, p53, and caspase-3; (9) NPs generate ROS;(10) NPs deplete ATP; (11) very small NPs enter into the nucleus;(12) NPs dysregulate mitochondrial function or activate mito-chondrial independent apoptotic pathways.

Anthracycline nano-delivery systems to overcome multiple drug resistance 317

Table 2 Summary of the proposed mechanisms of anthracycline NPs to overcome MDR.

Platform Composition Mechanism Status Ref.

Liposome CL, PC, CHOL Interact with P-gp, modify plasma membrane in vitro [27,28]CL, PC, CHOL Increase drug accumulation, intracellular drug

redistributionin vitro [26]

DSPE-PEG, CHOL, DPPC, DPPG Direct inhibit ATPase, alter raft lipidcomposition, reduce lipid raft-associated P-gp

in vitro [30]

DSPC, CHOL PSC 833 (P-gp inhibitor) in vivo [31]EPC, CHOL, PEG-DSPE Verapamil (P-gp inhibitor) in vitro [32]EPC, CHOL, mPEG-DSPE,MAL-PEG-DSPE

Verapamil (P-gp inhibitor), transferrin(targeting)

in vitro [33]

EPC, CHOL, DSPE-PEG, DPPC MDR1 ASO, BCL-2 ASO, endocytosis, membranefusion

in vivo [34]

DOTAP MRP1 siRNA, BCL-2 siRNA in vitro [36]DSAA, DOTAP, DOPA, CHOL,DSPE-PEG, DSPE-PEG-AA

DSAA (induce ROS, inhibit MDR transporters,enhance drug uptake), VEGF siRNA (increasedrug uptake and targeting), c-Myc siRNA(improve therapeutic effect anddown-regulate MDR)

in vivo [37]

Polymeric NP PIBCA PIBCA and its degradation products change ormodify cell membrane, massive drug diffusionfrom NPs saturates P-gp NPs do not enter thecells

in vitro [39]

PACA NP-cell interaction on cell surface, formdrug-polycyanoacrylic acid ion-pair complex,cyclosporine A (P-gp inhibitor)

in vitro [40,45,46]

PIHCA Bypass but not direct inhibit P-gp in vitro [42,43]AOT-alginate Methylene blue (inhibit P-gp and generate ROS) in vitro [48,49]PPLA Porphyrin (photosensitizer), TPGS (P-gp

inhibitor)in vitro [53]

Stearyl-modified dextran Bypass P-gp in vitro [47]PLGA Curcumin (increase drug retention in the

nucleus; down-regulate P-gp and BCL-2)in vitro [51]

PLGA Receptor-mediated endocytosis (HER2) in vitro [52]Polymeric micelles Pluronic P85 Interact with P-gp, change cell membrane

structure, induce cell membrane permeabilityin vitro [54]

PEO-PPO-PEO Endocytosis, sensitize cells in vitro [55]PLA-TPGS Inhibit P-gp, enhance drug cellular uptake,

promote drug to translocate into the nucleusin vitro [56]

PLGA-PEG-folate TPGS (P-gp inhibitor) in vitro [57]PEG-polyphosphazene Endocytosis, pH-sensitive polymer (disrupt

endosomes by proton-sponge effect and/orinteract between polymer and endosomemembrane)

in vitro [62]

CSO-FA Interact with cell membrane, alkyl side chainon chitosan introduces perturbation effect,fatty acids form hydrophobic microdomainsnear shell surface

in vitro [63—65]

PolyHis/PEG (orpolyHis/PEG-folate),pLLA/PEG-folate

Receptor-mediated endocytosis (folate),trigger drug release at low pH (pH-sensitive),interact between polyHis group of the micelleand endosome membrane

in vivo [66—69]

PEO-b-PCL RGD4C (targeting), TAT (cell-penetrationpeptide), MDR1 siRNA

in vivo [77]

PEG-PDLLA Drug released to plasma membrane and theninternalized into cells, PEG-induced fusion tocell membrane

in vitro [79]

PCL-PEO Endocytosis in vitro [80,81]Pluronic L61 Facilitate drug entry into the nucleus, increase

drug cellular uptake, inhibit drug effluxin vitro [82]

318 P. Ma, R.J. Mumper

Table 2 (Continued )

Platform Composition Mechanism Status Ref.

Polymer conjugate HPMA Inhibit P-gp and �2m, lysosomally degradablelinker (GFLG), endocytosis, down-regulateP-gp, MRP, BCL-2, HSP-70, etc.

phase I/II [83—89]

Dextran Endocytosis, bypass P-gp phase I [95—97]PEG-modified dendrimer Endocytosis, rupture endosomes

(proton-sponge effect)in vitro [94]

Magnetic NP Fe3O4, ZnO P-gp inhibitor or competitive P-gp substrate(Fe3O4), interact between NPs and cellmembrane, tetrandrine (P-gp inhibitor),up-regulate BAX, p53, caspase-3, inhibitBCL-2, down-regulate P-gp, shRNA (targeting)

in vivo [140—147]

Carbon nanotube — Controllable and sustained drug release, P-gpantibody (targeting)

in vitro [161]

CD NP — Interaction between polymer and P-gp, inhibitP-gp

in vitro [162]

Peptide/proteinconjugate

TAT Bypass but not inhibit P-gp in vitro [100,101]maurocalcine, penetratin, TAT Active mitochondrial independent apoptotic

pathwaysin vitro [102—104]

Vectocell Internalization in vitro [105,106]Penetratin, SynB1 Bypass P-gp, interact between conjugate and

cell membranein vitro [107]

Transferrin Bypass P-gp (conjugate slowly dissociates afterbinding to cell membrane), receptor-mediatedendocytosis, interact between conjugate andDNA

in vitro [108,109]

IGF-1R mAb Receptor-mediated endocytosis, escape P-gprecognition

in vivo [115]

[D-Lys6]LHRH Not by receptor-mediated endocytosis,down-regulate ErbB/HER receptors, disruptG-protein signaling

in vivo [116—118]

AS-ODN High drug accumulation, inhibit P-gp (AS-ODN) in vivo [119,120]BSA Endocytosis, conjugate degrades in lysosomes in vivo [121—123]diGly, triGly, GSH, GSSG Rapid drug uptake, high drug accumulation in vitro [124]Poly-D-Lysine, poly-L-lysine Endocytosis, poly-L-lysine digested by

lysosomesin vitro [125]

SLNs Emulsifying wax, Brij 78, TPGS Inhibit P-gp, deplete ATP, increase drugretention

in vivo [128,133]

Monostearin, oleic acid Inhibit P-gp, high affinity between lipids orNLC and cell membrane

in vitro [135]

Stearic acid, Pluronic F68,HPESO

Not inhibit or bypass P-gp, not alter cellmembrane permeability, drug released fromoutside of cell and then simple passivediffusion, phagocytosis, GG918 (P-gp inhibitor)

in vitro [136—138]

Gold NP — Change or modify cell membrane properties,dysregulate mitochondrial function

in vitro [152]

— Internalization, NPs even enter the nucleus in vitro [153]— Drug—NP complex formation, phagocytosis,

simple diffusionin vitro [154,155]

Silica NP — Endocytosis, bypass P-gp in vivo [157]— PEI (proton sponge effect), P-gp siRNA in vitro [158]— Inhibit P-gp, micropinocytosis in vivo [159]

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27,28]. The results showed that the IC50 values of freeOX (30 nM) and DOX liposomes (20 nM) were comparable

n HL-60 cells, while in HL-60/VCR cells the IC50 valuesf free DOX and DOX liposomes were 0.9 and 0.17 �M,

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localization, BCL-2 siRNA in vitro [160]

espectively, which indicated the liposome formulation was-fold more toxic than free DOX in HL-60/VCR cells. Theechanisms of DOX liposomes to overcome MDR were inves-

igated, and it was concluded that the empty liposomes

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Anthracycline nano-delivery systems to overcome multiple d

can directly interact with P-gp based on their competitiveinhibition of [3H]-vincristine binding to P-gp. In addition, themembrane fluidity of the resistant cells was different fromthat of the sensitive cells. Therefore, it was concluded thatliposomes likely interact with and modify the environmentof the plasma membrane, resulting in more drug uptakein resistant cells. In contrast, Hu et al. formulated threedifferent DOX liposomes; however, none of them showedMDR circumvention in vitro in rat glioblastoma cells, and theempty liposomes were unable to inhibit [3H]-azidopine bind-ing to P-gp [29]. It was suggested that the different resultswere due to the different compositions in the liposomeformulations where they included lower amounts of lipidsand higher DOX/lipid molar ratios. In addition, the origin ofthe lipids in liposomes was different. All of these may leadto avoidance of the interaction between lipids/liposomesand cell plasma membrane. More recently, Riganti et al.formulated DOX-containing anionic liposomes (Lipodox)and demonstrated that the Lipodox was significantly moreeffective than free DOX in resistant HT29-dx cells [30]. TheP-gp inhibition mechanisms of Lipodox were summarized intwo aspects: (1) indirect effect, which is due to the inter-action between liposomes and cell membrane (e.g. changein the composition of lipid rafts and P-gp localization); (2)direct effect, which is due to the direct interaction betweenliposome and P-gp (e.g. direct inhibition of ATPase activity).

Co-delivery of DOX and a P-gp inhibitor was also reportedto overcome MDR. Krishna et al. developed DOX liposomeswith 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) andCHOL at the lipid molar ratio of 55:45 [31]. The DOX lipo-somes or free DOX (i.v.) and P-gp inhibitor PSC 833 (p.o.)were co-administered in normal BDF1 mice. It was foundthat with p.o. administration of PSC 833, the maximum tol-erated dose (MTD) was reduced by 2.5—3-fold with freedrug while only 20% reduction for DOX liposomes comparedto i.v. alone. This suggested the DOX liposomes were lesstoxic than free DOX. Furthermore, in a murine P388/ADRsolid tumor model, the tumor inhibition of DOX liposomescombined with PSC 833 was comparable to the sensitiveP388/WT tumors, while a modest modulation was observedfor the co-administration of free DOX with PSC 833 at theMTD. It was also confirmed that the antitumor efficacy wasPSC 833 dependent because the DOX liposomes alone pro-vided significantly less activity. It should be noted that theDOX liposomes demonstrated a comparable pharmacokineticprofile and tissue biodistribution with or without PSC 833p.o. administration, while free DOX altered pharmacoki-netics in the presence of PSC 833. Similarly, Wang et al.co-encapsulated DOX and another P-gp inhibitor, verapamil,into stealth liposomes composed of egg phosphatidylcholine(EPC), CHOL, and PEG2000-DSPE (molar ratio 50:45:5) [32].The results showed the stealth liposomes with DOX and ver-apamil overcame MDR in vitro in both DOX-resistant ratprostate cancer cell line Mat-LyLu-B2 and human uterus sar-coma MES-SA/DX5 cell line, while the stealth liposomes withDOX alone were not effective enough to reverse MDR. To fur-ther target the tumor cells, the Robert group synthesizedtransferrin immunoliposomes encapsulating both DOX and

verapamil (Tf-L-DOX/VER), and this formulation increasedthe cytotoxicity by 5.2- and 2.8-fold over that of L-DOX/VERand Tf-L-DOX, respectively, in DOX-resistant K562 leukemiacells [33].

tbtp

resistance 319

Since the mechanisms of MDR are multifactorial, thedeal delivery system should address different MDR path-ays. In order to do so, the Minko group developed aomplex liposome system which included: (1) a chemother-peutic drug of DOX; (2) antisense oligonucleotides (ASOs)argeted to MDR1 mRNA; and (3) ASOs targeted to BCL-2RNA [34]. They showed this complex system was more

oxic in vitro in resistant A2870/AD human ovarian carci-oma cells when compared to free DOX, DOX liposomes,nd DOX liposomes with either one type of ASOs. In addi-ion, the complex liposomes were shown to be internalizednto the cancer cells both in vitro and in vivo and even pen-trated into the nucleus. However, the mechanisms wereot clear. It was also suggested that both membrane fusionnd endocytosis may be involved in liposome internaliza-ion into the tumor cells. Subsequently, the Minko groupuccessfully prepared a series of complex liposomes foro-delivery of DOX and ASO targeted to hypoxia-inducibleactor 1� (HIF1A) mRNA [35] or siRNA targeted to MRP1nd BCL-2 mRNA [36]. All of the liposome systems with theombination of DOX and ASO or siRNA showed enhancedhemotherapeutic efficacy in resistant cells both in vitrond in vivo. Chen et al. developed even more complex DOXiposome systems, namely cationic liposome-polycation-DNALPD) and anionic liposome-polycation-DNA (LPD-II), andhowed a significant antitumor inhibition in an NCI/ADR-ES xenograft mouse model [37]. With their DOX liposomeystems, they co-delivered the following cargos to over-ome MDR: (1) a guanidinium-containing cationic lipid,,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammo-ium chloride (DSAA), which could induce reactive oxygenpecies (ROS), inhibit MDR transporters, and enhance DOXptake in NCI/ADR-RES cells; (2) a vascular endothelialrowth factor (VEGF) siRNA, to increase DOX uptake andherapeutic efficacy via targeting tumor vasculature, dis-upting local blood supply and blocking angiogenesis; (3) aherapeutic c-Myc siRNA, where the c-Myc is a well-knownncogene and shown to positively control the expression ofDR. Thus, the silencing of c-Myc may result in both a direct

herapeutic effect and down-regulation of MDR.

olymeric nanoparticles

he Couvreur group entrapped DOX into biodegradableolyisobutylcyanoacrylate (PIBCA) polymers to form DOX-IBCA NPs and showed the complete reversion of drugesistance in vitro in several resistant cell lines [38]. Theaser microspectrofluorometry technique was utilized tonvestigate the mechanisms of the NPs to overcome MDR.t was proposed that the DOX-PIBCA NPs entered the cellsy endocytosis, and DOX was transported to the lysosomesnd released close to the nuclear membrane, followed bynteraction with DNA. It was also suggested that the DOX-IBCA NPs bypassed the P-gp pump which was probably dueo the molecular structure or the ionic charge of the NPs.nterestingly, this group suggested in another paper that theOX-PIBCA NPs did not enter the cells by endocytosis path-ay at all [39]. In contrast, the results demonstrated that

he NPs were first adsorbed on the cell membrane, followedy the degradation of polymer close to cell membrane, andhe drug was then released and entered the cells by simpleassive diffusion. Compared to free DOX, the massive DOX

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20

oncentration gradient from PIBCA NPs saturated P-gp andts pharmacological function. It was also suggested thatIBCA or its degradation products modified the cell mem-rane, which led to the permeation of more DOX into cells.n addition to NP-cell direct interaction, another mecha-ism was proposed wherein DOX formed ion pairs with theolyalkylcyanoacrylate (PACA) degradation product of poly-yanoacrylic acid. This DOX-polycyanoacrylic acid ion-pairomplex increased the apparent lipophilicity of DOX, andllowed the drug entering the cells bypass the recognitionf P-gp. It was concluded that the reversal of MDR with DOX-ACA NPs was the result of both the adsorption of NPs onhe cell surface and the formation of DOX-polycyanoacryliccid ion-pair complex at the plasma membrane [40].

Henry-Toulmé et al. demonstrated that DOX-olyisohexylcyanoacrylate (DOX-PIHCA) NPs were notndocytosed by the cells, which supported the results fromhe Couvreur group [41]. Barraud and co-workers also devel-ped DOX-PIHCA NPs and compared their antitumor efficacyersus free DOX both in vitro and in vivo in a resistantepatocellular carcinoma (HCC) model [42]. The IC50 of NPsas reduced by 1.5—4.5-fold in in vitro studies in several

esistant HCC cells. In vivo HCC transgenic mouse model,OX-PIHCA NPs had significantly improved tumor inhibitoryffect compared to free DOX (p = 0.01). The mechanisms byhich DOX-PIHCA NPs bypass P-gp efflux were the same asOX-PIBCA NPs discussed above. In similar, the Robert grouprepared DOX-PIHCA NPs and showed complete reversalf MDR in resistant C6 0.001 cells [43]. It was found thatnly drug tightly associated with DOX-PIHCA NPs overcame-gp resistance but empty NPs did not, while the emptyiposomes alone blocked P-gp function, which indicated theifferent mechanisms to overcome P-gp resistance betweenhe nano-delivery systems. It was also suggested that theechanism by which DOX-PIHCA NPs bypassed P-gp rather

han direct inhibit P-gp. In order to investigate whetherree DOX or DOX-PIHCA NPs used different mechanisms tocquire MDR, two human tumor cell lines, K562 and MCF-7,ere selected and DOX concentration in both formulationsas gradually increased. It was found that DOX-PIHCA NPsere more difficult to generate resistant cell lines and-gp expression was consistently lower than that in freeOX-selected cells, while breast cancer resistance proteinBCRP) expression was in a reverse order. These suggestedifferent mechanisms may be involved in the acquisitionf drug resistance [44]. Soma et al. prepared PACA NPso-encapsulated of DOX and cyclosporine A (CyA, a P-gpnhibitor) and showed that the NPs had the most effectiveell growth inhibition compared to other combinationsf both drugs in solution or NPs with single drug alone inesistant P388/ADR cells [45,46].

In addition to DOX-PACA NPs, Susa et al. successfully for-ulated DOX into a stearylamine-modified dextran NPs andemonstrated enhanced drug accumulation in the nucleusompared to free DOX in several resistant osteosarcomaells [47]. It was found that the fluorescence of free DOXas mainly in the nucleus in sensitive cells but mainly in theytoplasm in resistant cells, while the drug distribution of NP

ormulation was mainly in the nucleus even in resistant cells.his indicated that the NP formulations were able to deliverOX into the nucleus in resistant cells and the mechanismsay be due to bypassing of P-gp. Khdair et al. co-delivered

fmao

P. Ma, R.J. Mumper

OX and methylene blue into Aerosol OT (AOT)-alginatePs and this combination therapy significantly increasedhe in vitro cytotoxicity in resistant NCI/ADR-RES cells andmproved tumor growth inhibition in vivo [48,49]. Methy-ene blue is a photosensitizer and it is suggested to generateOS and inhibit P-gp, although the mechanism is not fullynderstood [50]. It was hypothesized the P-gp inhibition andnduced ROS of methylene blue increased the cytotoxicity ofOX in resistant cancer cells. Misra et al. also proposed dualrugs of DOX and curcumin co-encapsulated into poly(lactic-o-glycolic acid) (PLGA) NPs [51]. The application ofurcumin helped the retention of DOX in the nucleus, as wells down-regulated the expression of P-gp and BCL-2 in K562ells. The combination of both drugs in NP formulations hadnhanced in vitro cytotoxicity compared to single drug alonen either solution or NP formulations. Lei developed non-argeted and HER2 antibody conjugated DOX-loaded PLGAPs and compared the cellular uptake and cytotoxicity toree DOX in resistant ovarian SKOV-3 and uterine MES-SA/Dx5ells [52]. The results showed higher cellular uptake of tar-eted NPs than both of free drug or non-targeted NPs inKOV-3 cells. It was suggested that the major mechanismf targeted PLGA NPs was receptor-mediated endocytosis.hieh et al. developed more complex DOX NPs, where thehemotherapeutic agent DOX and a photosensitizer wereo-incorporated into 4-armed porphyrin-polylactide (PPLA)Ps with D-�-tocopheryl polyethylene glycol 1000 succinateTPGS, a P-gp inhibitor) coated on the NP surface [53]. Itas concluded the combined agents showed a synergisticffect and increased DOX delivery to the nucleus in resistantCF-7/ADR cells.

olymeric micelles

lakhov et al. demonstrated that DNR-loadedoly(oxyethylene-b-oxypropylene-b-oxyethylene) (Pluronic85) block copolymer micelles increased the cytotoxicity upo 3- and 700-fold greater than free DNR in sensitive SKOV3nd resistant SKVLB cells, respectively [54]. Based on theesults from the cytotoxicity, influx and efflux, and drug-opolymer binding studies, it was hypothesized that theopolymer affected P-gp function by direct interaction with-gp and/or the structure change of the plasma membrane.

n addition, the copolymer may induce the permeabilityf cell membrane. Lee et al. developed DOX-encapsulatedoly(ethylene oxide)-poly(propylene oxide)-poly(ethylenexide) (PEO-PPO-PEO) micelles and the micelle formulationxhibited 15-fold greater cytotoxicity compared to freeOX in MCF-7 cells [55]. The flow cytometry analysisnd confocal images suggested the micelles entered theells via the endocytosis pathway. Once inside cells, theicelles were initially localized in endosomes and DOX was

hen released in a sustained manner in the cytosol. Theopolymer itself may also contribute to sensitization of cellsnd to the enhancement of DOX-induced cell apoptosis. Lind co-workers synthesized poly(L-lactide)-vitamin E TPGSPLA-TPGS) block copolymer and used this as the carrier

or DOX [56]. The results indicated that the PLA-TPGSicelles inhibited P-gp, enhanced drug cellular uptake,

nd facilitated translocation of DOX into the nucleus, allf which were responsible for MDR circumvention. Zhao

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Anthracycline nano-delivery systems to overcome multiple d

and co-workers added TPGS into PLGA-PEG-folate poly-meric micelles and showed increased DOX cellular uptakecompared to the micelles without TPGS, which may be dueto the P-gp inhibition of TPGS [57]. Other possible mech-anisms of TPGS to overcome MDR may include inhibitionof efflux pump ATPase and substrate binding, generationof ROS, and alteration of membrane fluidity [58—61].Zheng et al. synthesized pH-sensitive polymers by linkingN,N-diisopropylethylenediamine (DPA) onto the backboneof PEGylated polyphosphazene [62]. DOX was entrapped inthe polymer to form self-assembled micelles and the IC50

value of this formulation was 60-fold lower than free DOXagainst resistant MCF-7/ADR cells. The cellular uptake andintracellular distribution of DOX micelles were evaluated byconfocal microscopy, and it was found that much more DOXwas in the nucleus compared to free DOX and the majorityof free DOX was entrapped in the intracellular compart-ments. Furthermore, DND-26 (an acidic organelle-selectivefluorescent probe) was incorporated into the micelles toinvestigate whether the pH-sensitive polymeric micellescould help DOX escape from endosomes and lysosomes. Theresults demonstrated the fluorescence of DND-26 micelleswas spread over the cells while free DND-26 was mainlylocated in the endosomes and lysosomes. Pre-incubation ofthe polymer solution followed by the addition of free DND-26showed that the polymer and free DND-26 entered the cellsvia endocytosis and passive diffusion pathways, respectively.All these suggested the pH-sensitive polymeric micellesdisrupted endosomes and released the drug after they wereendocytosed into the cells, and that the mechanisms may bedue to the proton-sponge effect and/or polymer-endosomalmembrane interaction. Yuan et al. synthesized linoleic acid-grafted chitosan oligosaccharide (CSO-LA) and incorporatedDOX into CSO-LA micelles [63,64]. The results demon-strated a significant enhancement in the internalization ofthe micelles in both sensitive MCF-7 and K562 and theirresistant cells compared to free DOX. In addition, paclitaxel(PX) and DOX were successfully co-loaded in the stearicacid-grafted CSO micelles and the results showed that thisformulation was able to completely reverse MDR in resistantcells [65]. The following mechanisms were proposed forthe fatty acid-grafted CSO micelles (CSO-FA) to overcomeMDR: (1) positively-charged CSO-FA micelles facilitate theinteraction between micelles and negatively-charged cellmembrane; (2) the alkyl side chain on chitosan backbonefavors its fusion and hydrophobic interactions with cellmembrane and this effect is alkyl chain dependent; (3) thefatty acid forms hydrophobic microdomains near the surfaceof the micelle due to the stereo resistance effect, whichenhances the hydrophobicity of the micelle and furtherfavor the internalization of micelles into cells because ofthe lipophilic property of the cell membrane.

The Bae group developed DOX-loaded pH-sensitive poly-meric micelles with folate (PHSM/f), which was a mixtureof two block copolymers of poly(L-histidine)(Mn: 5K)-b-PEG (Mn: 2K) (polyHis/PEG) or polyHis/PEG with folate(polyHis/PEG-folate) and poly(L-lactic acid)(Mn: 3K)-b-PEG(Mn: 2K)-folate (pLLA/PEG-folate) [66—68]. The mixed

copolymers in the micelle formulation improved the micellestability in pH 7.4 due to the hydrophobic properties ofpLLA [69]. This formulation overcame MDR both in vitro andin vivo in several resistant cell models. The results showed

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resistance 321

hat the micelles entered the cells via folate receptor-ediated endocytosis pathway, and then DOX escaped from

ndosomes and was released into the cytoplasma due tohe positively-charged polyHis which fused and destabi-ized the negatively-charged endosomal membrane at lowH of 6.5—7.2. To better understand the mechanism ofhe micelles, FITC-pLLA/PEG with folate (FITC-pLLA/PEG-olate) was synthesized as a pH-insensitive micelle controlPHIM/f). In contrast to PHSM/f, PHIM/f was found to beostly entrapped into sub-organelles, such as endosomes,

nd had much less antitumor efficacy compared to PHSM/f.aken together, the active folate receptor-mediated endo-ytosis, triggered drug release at low pH, and the interactionetween polyHis group and endosomal membrane may beesponsible for the MDR reversal effect. Later on, it wasoticed that a fraction of the loaded DOX was releasedn tumor extracellular space (pH 6.5—7.2) before activelynternalized into the cells. Therefore, the released drug hadhe potential to be pumped out by P-gp and attenuated itsfficacy. To prevent DOX releasing in tumor extracellularpace, L-phenylalanine (Phe) was introduced to the copoly-er of polyHis/PEG to form poly(His-co-Phe)/PEG [70,71].he Phe group in the copolymers significantly dropped theKa values and allowed the destabilization of micelles atven the lower pH of 6 to avoid the tumor extracellular pH.he mixture of poly(His-co-Phe)/PEG and pLLA/PEG-folateormed the second generation of pH-sensitive micelles whichrecisely target the early endosomes at pH of 6.

It has been shown that an NF-KB inhibitor enhancedumor cell sensitivity of apoptosis induced by chemother-peutic agents, such as DOX and PX [72,73]. Fan et al.o-loaded DOX and pyrrolidinedithiocarbamate (PDTC,n NF-KB inhibitor) in folate-chitosan (FA-CS) polymericicelles, and the micelles showed significantly lower IC50

alues and enhanced cellular uptake in resistant cells74]. Lee et al. co-delivered human tumor necrosis factorTNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) andOX with self-assembled micelles from cationic copolymerf poly{N-methyldietheneamine sebacate)-co-[(cholesterylxocarbonylamido ethyl) methyl bis(ethylene) ammo-ium bromide] sebacate} (P(MDS-co-CES)) [75]. It wasemonstrated that the co-delivery of DOX and TRAILn P(MDS-co-CES) micelles entered the cells via receptor-ediated endocytosis and enhanced the cytotoxicity against

esistant tumor cells. Benoit et al. developed cationicicelles from copolymers of poly(dimethylaminoethylethacrylate) (pDMAEMA) and poly(butyl methacrylate)

pDbB) [76]. DOX was loaded into the hydrophobic coref pDbB. An siRNA against polo-like kinase 1 (PLK1), aene up-regulated in many cancers and responsible for cellycle progression, was condensed with positively-chargedDMAEMA. A pH-sensitive copolymer of poly(styrene-alt-aleic anhydride) (pSMA) was further complexed withositively-charged PLK1 siRNA/DOX micelles to form aernary complex for escape of the drug from endosomes.he co-delivery of PLK1 siRNA and DOX using this ternaryomplex system exhibited synergistic effect in resistantCI/ADR-RES cells. Xiong et al. constructed poly(ethylene

xide)-block-poly(�-caprolactone) (PEO-b-PCL) micelles forhe co-delivery of MDR1 siRNA and DOX [77]. Two ligands,ntegrin �v�3-specific ligand (RGD4C) and TAT peptide, werettached on the shell of the micelles for active targeting

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nd cell-penetration purpose, respectively. This multifunc-ional polymeric micellar system was able to deliver both ofOX and MDR1 siRNA into intracellular compartments, andvercame P-gp-mediated resistance in vitro and targetedv�3-positive tumors in vivo. Nakanishi et al. prepared aolymeric micelle formulation of NK911 for DOX. NK911 con-isted of block copolymers of PEG (Mw: 5K) and poly(asparticcid) (∼30 units) [78]. DOX was partially conjugated tohe side chain of aspartic acid (∼45%) to enhance theydrophobicity of the inner core of the micelles. Therefore,wo types of DOX, i.e. incorporated DOX and conjugatedOX, were in NK911 formulation. However, the conjugatedOX did not show any antitumor activity. The preclinicaltudies demonstrated much stronger tumor inhibitory effectgainst several tumor models compared to free DOX andurrently NK911 is in a phase I trial.

In order to better understand the mechanism ofolymeric micelle mediated drug delivery, Chen et al.nvestigated the cellular uptake of monomethoxyoly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-DLLA) micelles [79]. Fluorescein isothiocyanate (FITC) wassed to label micelle itself and 1,1′-dioctadecyl-3,3,3′,3′-etramethylindocarbocyanine perchlorate (DiIC18(3)) wassed as a hydrophobic model molecule encapsulated in theicelle. It was found that the cellular uptake of DiIC18(3) wasuch faster than that of FITC-labeled PEG-PDLLA micelles,hich indicated their different cell entry pathways. More-ver, förster resonance energy transfer (FRET) imagingnd spectroscopy were utilized to monitor the cellularptake of PEG-PDLLA micelles in real time loaded with aRET pair of DiIC18(3) and 3,3′-dioctadecyloxacarbocyanineerchlorate (DiOC18(3)). The FRET results confirmed thatoth of the hydrophobic dyes were entrapped in the core ofhe micelles and were subsequently released to the plasmaembrane and then internalized by the cells. PEG on the

hell of the micelles facilitated the release of the dyesecause of the PEG-induced fusion to the cell membrane.n contrast, Allen et al. evaluated polycaprolactone-block-oly(ethylene oxide) (PCL20-b-PEO44) copolymer micelles inC12 cells and the results strongly suggested the micellesntered the cells via endocytosis pathway based on a seriesf cellular uptake studies [80]. Savic et al. triple-labeledCL-b-PEO micelles, nucleus, and plasma membrane (orytoplasmic organelles, such as mitochondria, Golgi,tc.) and showed that the micelles were endocytosednto the cells and distributed into several cytoplasmicompartments, including mitochondria, Golgi apparatus,ut not the nucleus [81]. 5-DodecanoylaminofluoresceinDAF), a model molecule, was incorporated into PCL-b-PEOicelles and the results suggested the micelle formulation

nhanced the delivery of the agent into the cells andncreased its efficacy. Venne et al. demonstrated that theOX-loaded poly(oxypropylene)-poly(oxyethylene) blockopolymer pluronic L61 micelles enhanced by 290- and00-fold the cytotoxicity in resistant CHRC5 and MCF-7/ADRells, respectively, but were comparable with free DOXn their matched sensitive cell lines [82]. The micelleormulation was found to shift the distribution of DOX

rom the cytoplasmic compartment to the nucleus, and theopolymer increased the drug uptake and inhibited the drugfflux. All of these contributed to the ability of pluronic61 micelles to overcome MDR.

[

co

P. Ma, R.J. Mumper

olymer conjugates

he Kopecek and Duncan groups collaboratively developedwo N-(2-hydroxypropyl)methacrylamide (HPMA)-DOX con-ugates, namely PK1 and PK2, and both of the conjugatesere tested in phase I/II clinical trials [83]. The HPMA-DOXonjugates had the following three components: (1) a water-oluble polymeric carrier of HPMA; (2) an anticancer drug ofOX; (3) biodegradable polymer-drug linker. In the PK1 con-ugate, the linker was a tetrapeptide of GFLG, which wastable in the blood circulation but susceptible to cleavagey enzymes in the lysosomes. In contrast, PK2 had additionalalactose residues that were recognized by the asialogly-oprotein receptor on hepatocytes for targeted therapy ofepatocellular carcinoma. The IC50 value of HPMA-DOX con-ugate in resistant A2780/AD cells was only about 20% higherhan in sensitive A2780 cells (the resistance index of freeOX in A2780/AD cells: 40), which indicated that the conju-ate formulation at least partially overcame P-gp-mediatedesistance. The analysis of P-gp gene expression showed thatree DOX at high doses induced P-gp expression in sensitive2780 cell, while the HPMA-DOX conjugate inhibited P-gpnd �2-microglobulin (�2m) genes in resistant A2780/ADells.

The mechanisms of action of HPMA-DOX conjugates haveeen extensively studied and well established [84—89]. ThePMA-DOX conjugates entered the cells via endocytosisathway and DOX was then released from the lysosomes inhe perinuclear regions due to the lysosomally-degradablepacer of GFLG between DOX and the polymer. Then, theeleased DOX entered the nucleus and exerted its pharma-ological function. The conjugates demonstrated prolongedlood circulation in vivo, and enhanced tumor-to-bloodatio as a function of time, and enhanced Cmax at 48 h inumors after i.v. administration. All three of these param-ters indicated that the conjugates passively accumulatedn tumor tissues via the EPR effect. It is important toote that the particle size of HPMA-DOX conjugates wasess than 10 nm. Interestingly, the concentration gradientf HPMA-DOX conjugates was found to be decreased fromhe perinuclear region to the plasma membrane. In con-rast, the concentration gradient of free DOX was in thepposite direction, where it decreased from the plasmaembrane to the perinuclear region. Consequently, DOX in

he conjugate formulation had increased ability to interactith nuclear DNA and/or topoisomerase II. In addition, freeOX up-regulated MDR genes such as MDR1 and MRP, whilehe conjugates overcame MDR1 and down-regulated MRP.ree DOX also activated various cell detoxification mech-nisms, while HPMA-DOX conjugate down-regulated BCL-2,eat-shock protein 70 (HSP-70), glutathione S-transferase �GST-�), bilirubin uridine diphosphate (BUDP) transferases,opoisomerase II� and II�, and thymidine kinase 1 (TK1)enes. With the exposure of HPMA-DOX conjugate, cellpoptosis, lipid peroxidation, and DNA damage were signif-cantly higher compared to free DOX. For more details onhe design, efficacy, safety, and mechanisms of action ofPMA-DOX conjugates, please refer to referenced articles

84—89].Omelyanenko et al. synthesized targetable HPMA-DOX

onjugates containing N-acylated galactosamine (GalN)r monoclonal OV-TL 16 antibodies (OV-TL 16 Ab) [90].

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Anthracycline nano-delivery systems to overcome multiple d

Fluorescence confocal microscopy studies demonstratedthat both of the targeted conjugates had a similar fatewhen incubated with the cells, where the conjugates wererecognized and internalized into the cells, localized in thelysosomes and DOX was then released from the polymer andeventually diffused from the cytoplasma into the nucleus.St’astny et al. designed HPMA-DOX conjugates with differ-ent targeting moieties, including anti-CD71, antithymocyteglobulin, anti-CD4, and transferrin, and compared their abil-ity to reverse MDR in CEM/VLB cells [91]. Anti-CD4 targetedHPMA-DOX conjugate demonstrated the weakest ability toovercome resistance and this was probably due to the poorinternalization of anti-CD4 molecule. It was hypothesizedthat receptor-mediated endocytosis was a very importantfactor for the MDR reversal effect of HPMA-DOX targetedconjugates and this effect was targeting moiety depen-dent. Nan et al. prepared targeted HPMA-DOX conjugatescontaining a peptide sequence of WHYPWFQNWAMA, tobind surface-specific receptor of Hsp47/CBP2 which wasover-expressed in human squamous cell carcinoma of headand neck (SCCHN) [92]. Interestingly, both targeted andnon-targeted conjugates demonstrated the less cellularuptake and lower cytotoxicity than free DOX in sensitiveSCCHN cells. This indicated the endocytosis process of theconjugates was slower than the rapid passive diffusion offree DOX in sensitive SCCHN cells. In contrast, both targetedand non-targeted conjugates exhibited significantly highercellular uptake and more potent than free DOX in resis-tant SCCHN cells. Moreover, targeted conjugates showedhigher cellular uptake than non-targeted conjugates. Takentogether, all the studies suggested the targeted HPMA-DOXconjugates had the potential to treat the resistant head andneck cancer. Bidwell et al. developed a thermally targetedelastin-like polypeptide (ELP) DOX conjugate to overcomeMDR in resistant MES-SA/Dx5 and NCI/ADR-RES cells [93].This DOX conjugate contained four functional domains: (1)an anticancer agent of DOX; (2) GFLG, a tetrapeptide linker,which could facilitate DOX release from lysosomes;(3) TAT,a cell penetrating peptide; (4) ELP, a thermal-responsivepolypeptide as the vehicle for DOX. It was found to bebeneficial to use ELP as a drug carrier compared to HPMAin PK1, based on the fact that ELP macromolecules accu-mulated in tumors and this accumulation may be furtherenhanced by thermal targeting.

In addition to HPMA-DOX conjugate, Kono et al. conju-gated DOX to PEG modified poly(amidoamine) dendrimerswith either an amide or hydrazone linkage [94]. The resultsdemonstrated the acid-labile hydrazone linkage was veryimportant to exhibit the antitumor efficacy in resistantcells. It was hypothesized the DOX-dendrimer conjugate wastaken up into cells via endocytosis and entrapped into sub-cellular acidic compartments of endosomes and lysosomes.The endosomes were ruptured by DOX-dendrimer via pro-ton sponge effect, and the hydrazone linkage was brokenin acidic environment and DOX was then released from thedendrimer to exert its pharmacological action.

The Fong group applied a Schiff base covalent bondformation strategy to synthesize dextran-DOX conjugates

[95—97]. Both free DOX and the conjugate were localizedmainly in cytoplasmic compartments in resistant KB-V1cells but for different reasons. Free DOX was difficult todiffuse into the nucleus due to the P-gp efflux, while the

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resistance 323

extran-DOX conjugate was excluded from the nucleus dueo its large size. The P-gp seemed to be only effective if theolecular weight of drug conjugates was less than 70 kDa.his was consistent with the findings that the central poreize of P-gp was 5 nm, while the effective size of 70 kDaextran was ∼5 nm [98]. Therefore, dextran-DOX conjugatearger than 70 kDa was not a good P-gp substrate and hadetter accumulation in the nucleus. Furthermore, the crit-cal size of dextran for DOX accumulation was calculated as03 kDa based on the relative cytotoxicity of dextran-DOXonjugate in sensitive and resistant KB cells. The DOX cou-led to 70 kDa dextran, i.e. dextran-DOX conjugate (AD-70,OX-OXD), was tested in a phase I clinical trial and the MTDf the conjugate was found to be 40 mg DOX/m2 [99].

eptide/protein conjugates

iang et al. synthesized a TAT-DOX conjugate and evalu-ted its cellular uptake and intracellular distribution inCF-7 cells [100]. Both the conjugate and free DOX were

ransported into the cells but with different intracellularistribution, where free DOX was mainly in the nucleushile the most of the conjugate was located in the per-

nuclear and cytoplasmic regions. On the other hand, theytotoxicity of the conjugate and free DOX was comparable.his suggested DOX also have activity in cytoplasma. Theesearch supported that the activity of DOX was not onlyue to its inhibition of DNA synthesis, but also due to itsnteraction with cytoplasmic components to cause cell apo-tosis [101]. Importantly, the cytotoxicity of the conjugateas about 8—10-fold higher than free DOX in both resistantCF-7/ADR and AT3B-1 cells. To understand the mechanismsf TAT-DOX conjugate to overcome MDR, the intracellularOX concentration was measured in sensitive and resistantCF-7 cells. About 90% of the free DOX accumulated in

ensitive cells but dropped to only 5% in resistant cells,ndicating a strong P-gp efflux in resistant cells. In contrast,he conjugate had 58.6% retention in resistant cells. Inddition, neither the mixture of DOX and TAT nor verapamila P-gp inhibitor) affected the cytotoxic properties of theAT-DOX conjugate, which suggested the MDR reversal ofhe conjugate was bypassed but not inhibited. Aroui ando-workers designed three DOX-cell penetrating peptideCPP) conjugates, namely maurocalcine, penetratin, andAT, and compared the cytotoxicity to free DOX in differentensitive and resistant cells [102—104]. All three conjugatesisplayed similar efficacy which was about 5-fold moreytotoxic than free DOX in resistant cells. In general,PPs are used as cell impermeable compounds, while theenefits of CPPs applied to the cell membrane permeableompound DOX may be due to improved stability, facilitatedell compartment targeting and DNA binding, alterationfflux pathways and detoxification reactions of DOX. Tonderstand the mechanisms, BCL-2 and BCL-XL proteinxpressions were determined in the cells treated with DOXonjugates or free DOX since anti-apoptotic BCL-2 familyas known to control mitochondria membrane permeability.

owever, the results showed no difference between theonjugate and free DOX. To determine if other apoptoticathways were responsible for mitochondrial permeabiliza-ion, the DOX-CPP conjugates were studied in MDA-MB-231

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ells over-expressing of BCL-2. It was found that the DOXonjugate was 5-fold more toxic than free DOX in resistantDA-MB-231/BCL-2 cells, which indicated that the DOX-CPPonjugate activated multiple apoptotic pathways otherhan mitochondrial events. It should be noted that free DOXas mainly localized in the nucleus and the DOX conjugateas localized in the cytosol. The alteration of intracellularistribution of DOX conjugate may contribute the mitochon-rial independent apoptotic pathways. Meyer-Losic et al.onjugated DOX to another CPP, Vectocell, via differentinkers [105]. This peptide originated from human proteinith 15—23 amino acid residues, and the studies showed

t was internalized by different mechanisms [106]. Whenhemically stable bonds were utilized to the conjugate withectocell, e.g. at the C14 position of the thioether or the3′ position of the amide group of DOX, the in vivo activityas minimal, which was probably due to inhibition of the

nteraction between DOX and DNA caused by Vectocell.he best linker was found to be at the C14 position ofOX with an ester bond, and this Vectocell-DOX conjugatead significantly greater antitumor efficacy compared toree DOX both in vitro and in vivo in colon and breastumor models. The mechanism of the improved therapeuticndex of Vectocell-DOX conjugate was not clear. However,ecause the conjugate had lower charge-to-mass ratio, iteemed to be different than other CPPs, such as TAT.

Mazel et al. coupled DOX to two different peptides,amely penetratin or SynB1, to obtain two different DOX-eptide conjugates [107]. The results demonstrated the IC50

alue of the conjugates was about 20-fold less than free DOXn resistant K652/ADR cells, and the conjugates had similarellular uptake in both sensitive and resistant cells. All ofbove results suggested that conjugate entered the resistantells in a way not recognized by P-gp, although the mech-nism was unknown. It was known that the amino group onOX was an important substrate group for P-gp recognitionnd since the coupling of DOX to peptide was taken placet this amino position. Therefore, the conjugate may enterhe resistant cells bypass of P-gp efflux. Interestingly, theytotoxicity of the conjugate was less than that of free DOXn sensitive cells, indicating some loss of DOX activity whichas probably due to the covalent binding between DOX andeptide. It should be noted that the conjugates in the stud-es were not susceptible to hydrolysis because succinate andhioether were used as the linkers for SynB1 and penetratin,espectively. When substituted succinate linker to disulfide,OX-SynB1 conjugate was more potent. The DNA bindingtudies showed that the conjugate intercalated with DNA.ther mechanisms, such as interaction between conjugatend cell membrane, may be involved in the induction of theell apoptosis by the conjugate.

Fritzer et al. synthesized transferrin-DOX conjugate andemonstrated that it was much more potent than free DOXn resistant K562/ADR, HL-60/ADR, KB-C1 and KB-V1 cells108,109]. Since a Schiff based coupling strategy was uti-ized to prepare transferrin-DOX conjugate, the conjugateid not undergo hydrolysis at acidic pH in the endocyticompartment. It was stable at least for 2 weeks at 37 ◦C

t pH 3.0 without detectable free DOX. The fluorescenticroscope studies showed that the cell membrane, but

ot DNA, was the target of the conjugate. The mechanismsf transferrin-DOX conjugate were proposed where the

rMco

P. Ma, R.J. Mumper

onjugate slowly dissociated and released DOX in a sustainedanner after binding to cell membrane, which prolonged

he effect of DOX on cell membrane and caused membraneamage. In this way, P-gp was unable to circumvent the func-ion of transferrin-DOX conjugate. In contrast, Lai et al.uggested a different mechanism of transferrin-DOX con-ugate where the conjugate entered the cells and mainlyocalized in the cytoplasma [110]. It was pointed out that theiscrepancy may be due to the different fluorescent label-ng, where DOX fluorescence was quantified in Lai’s studieshile fluorescence-labeled transferrin was used for Fritzer’s

tudy. The increased cytotoxicity of transferrin-DOX con-ugate over that of free DOX was partly explained by theonjugate bio-reductive processes and ROS generation inytoplasma. The ability of the transferrin-DOX conjugate tovercome MDR was further confirmed by Łubgan, where theonjugate was 4- and 200-fold more cytotoxic than free DOXn sensitive HL-60 and resistant HL-60/ADR leukemia cells,espectively [111]. Interestingly, when Munns et al. inves-igated transferrin-DOX conjugate in sensitive MGH-U1 andesistant MGH-U1R bladder cancer cell lines, it was foundhat the conjugate did not overcome resistance [112]. It isorthy to mention that the mass spectrometry data demon-

trated that the conjugate did not dissociate at all. To under-tand whether the integral conjugate form was active or not,ransferrin-negative TRVb and transferrin-positive TRVb-1ells were utilized as the controls. The results showed com-arable cytotoxicity for the transferrin-DOX conjugate andree DOX, indicating that both of the DOX forms were equallyctive. It is known that the lipid composition and fluidity ofell membrane are different between resistant and sensi-ive cells, and this membrane acquired drug resistance mayxplain the transferrin-DOX conjugate failing to overcomeesistance in MGH-U1R bladder cancer cells [113,114].

Guillemard et al. linked DOX to a mAb specifically recog-izing the type 1 insulin-like growth factor receptor (IGF-1R)115]. This IGF-1R-DOX conjugate had more than a 200-foldnhanced therapeutic index compared to free DOX in vitron resistant KB-V cells, and significantly reduced the tumorurden in vivo in a KB-V xenograft mouse model. Unlikehe transferrin-DOX conjugate, IGF-1R-DOX conjugate wasnternalized into cells via receptor-mediated endocytosisnd DOX was released into the perinuclear and cytoplasmicegions farther away from the P-gp pump and thereforeeducing the likelihood for efflux. Again, the amino groupn DOX was used to couple with mAb. Therefore, thisonjugate was able to escape P-gp recognition. Based onhe fact that the luteinizing hormone-releasing hormoneeceptor (LHRH-R) is found in >50% of human breast can-ers, Bajo et al. chemically coupled DOX to [D-Lys6]LHRHo generate a cytotoxic conjugate, called AN-152 [116].he AN-152 demonstrated significantly antitumor efficacy

n vivo compared to other controls in a DOX-resistantX-1 xenograft mouse model. The following mechanismsf AN-152 were proposed to overcome resistance in MX-1umors: (1) AN-152 could significantly reduce HER2 andER3 levels but not EGFR, while free DOX had no effect onhese receptors. Therefore, down-regulation of ErbB/HER

eceptor family members may contribute to circumventDR; (2) the receptor-mediated endocytosis pathway of theonjugate via targeting to LHRH-R may not be the reasonf its escape from the efflux pump because the resistance

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of DOX in MX-1 tumors is known not to be mediated by thetransport system; (3) AN-152 significantly decreased mRNAlevels of G�11 and G�12 but free DOX did not [117]. Bothof the above G-proteins are known to couple to LHRH-Rand regulate cell growth [118]. The disruption in G-proteinsignaling by AN-152 also contributed to circumvent MDR.

Ren et al. conjugated DOX to an antisense oligodeoxynu-cleotide (AS ODN, 5′-TCCTCCATTGCGGTCCCCTT-3′) on its3′-phosphate group [119,120]. The conjugate significantlyenhanced the stability of both DOX and AS ODN in bio-logical fluid in vitro, and increased binding affinity of ASODN to its complementary sequence. The intracellular accu-mulation of AS ODN was much higher in the conjugateform compared to free AS ODN, which was mainly due tothe improved lipophilicity of AS ODN in conjugate. ThisAS ODN-DOX conjugate demonstrated significantly improvedantitumor efficacy, and markedly inhibited P-gp expressionand mRNA levels compared to AS ODN or DOX alone bothin vitro and in vivo in a resistant KB-A-1 cell model.

The Ohkawa group developed a bovine serum albumin(BSA)-DOX conjugate and investigated the conjugate bothin vitro and in vivo in sensitive AH66P and resistant AH66DRcells [121—124]. The results demonstrated the BSA-DOXconjugate had similar cytotoxicity in vitro in both AH66Pand AH66DR cells, which indicated the complete rever-sal of MDR (free DOX resistant index: 200). It was alsofound that DOX concentration in the cell remained rel-atively high even after 36 h. The treatment of BSA-DOXconjugate in rats in a resistant AH66DR model led to sig-nificantly prolonged survival compared to free DOX. All theresults indicated this BSA-DOX conjugate had the poten-tial to overcome MDR and it was suggested the conjugateentered the cells via endocytosis pathway and the drug wasthen slowly released from lysosomes. Furthermore, bothof the drug concentration and molecular mass (Mr) of theinternalized BSA-[14C]DOX conjugate in different subcellularcompartments (lysosomes, cytosol, nucleus, and mitochon-dria) were measured by a liquid scintillation counter andHPLC gel filtration, respectively. Interestingly, the accumu-lation of the conjugate markedly increased in lysosomes inresistant AH66DR cells as a function of time up to 24 h,while significantly enhanced accumulation in mitochondriabut moderate increase in lysosomes and the nucleus wasobserved in sensitive AH66P cells. In both of the cell lines,a total of three peaks with Mr from 3—70 kDa were iden-tified in lysosomes, one peak with Mr < 2 kDa was in thenucleus and mitochondria, two peaks with one <2 kDa andthe other one >500 kDa were determined in the cytosol. Nofree DOX was found in any compartments. The two peaksin the cytosol suggested that the smaller one (<2 kDa) maybe the conjugate degradation product released from lyso-somes, and the larger one (>500 kDa) may be the complex ofthe BSA-DOX conjugate or its degradation product with tubu-lin or other unknown proteins. It should be noted that thesmaller peaks < 2 kDa were found in the nucleus, mitochon-dria and cytosol. In addition, based on the fact that theaccumulation of the DOX BSA-[14C]DOX conjugate increasedin lysosomes in AH66DR cells, all of which indicated the BSA-

DOX conjugate degraded in lysosomes and resultant activeadducts < 2 kDa were responsible for the antitumor efficacyin resistant AH66DR cells. To confirm the lysosomal degrada-tion products from the conjugate exhibited cytotoxic effect,

NtaN

resistance 325

oly-D-lysine-DOX and poly-L-lysine-DOX conjugates wereested. The cellular uptake of both conjugate was similar,ut only poly-L-lysine-DOX conjugate showed the cytotoxic-ty because poly-L-lysine was digested by lysosomal enzymesut poly-D-lysine did not [125]. Later on, four DOX-peptideonjugates with Mr < 2 kDa, namely glycylglycine (diGly), gly-ylglycylglycine (triGly), reduced glutathione (GSH), andxidized glutathione (GSSG), were synthesized and evalu-ted the cytotoxicity effect in both AH66P and AH66DR cells.iGly-DOX and triGly-DOX demonstrated the same cytotox-city as free DOX in both of the cell lines, GSSG-DOX hadhe same cytotoxicity as BSA-DOX conjugate in both cells,nd GSH-DOX showed 9- and 7.5-fold more cytotoxic activ-ty than BSA-DOX conjugate against AH66P and AH66DR cells,espectively. The highest cytotoxicity of GSH-DOX amongll DOX conjugates was due to the rapid uptake and highccumulation in resistant AH66DR cells.

olid lipid nanoparticles

ang et al. developed DOX-loaded solid lipid NPs (SLNs)ith glyceryl caprate (Capmul MCM C10) as the lipid core,olyethylene glycol 660 hydroxystearate (Solutol HS15) ashe surfactant, and curdlan as the shell forming material126,127]. The DOX SLNs enhanced the cellular uptake to7.1- and 21.6-fold at 1 and 2 h, respectively, and increasedpoptotic cell death determined by crystal violet stainingssay, when compared to free DOX in resistant MCF-7/ADRells. In addition, the SLNs did not induce hemolytic activityn human erythrocytes which indicated the safety of theormulation. It was concluded that the DOX SLNs had theotential to overcome MDR. The Mumper group successfullyrepared DOX and IDA SLNs from warm microemulsionrecursors using emulsifying wax as the oil phase, andolyoxyl 20-stearyl ether (Brij 78) and TPGS as the co-urfactants [128—132]. Anionic ion-paring agents of sodiumaurodeoxycholate (STDC) and sodium tetradecyl sulfateSTS) were applied to neutralize the cationic anthracyclinesnd enhance the drug entrapment in SLNs. The DOX SLNsad significantly improved antitumor efficacy than free DOXoth in vitro and in vivo in a resistant P388/ADR cell model,ut IDA SLNs did not demonstrate any benefit compared toree IDA, which may be due to the more lipophilic propertyf IDA. The mechanisms of the DOX SLNs overcoming MDRere investigated and it was concluded that the MDR

eversal of SLNs may due to the P-gp inhibition by Brij 78nd TPGS, and ATP depletion by Brij 78 [133]. It is knownhat SLNs have some potential limitations, such as low drugoading capacity, burst drug release behavior, and potentialrug expulsion upon storage. To avoid the above limitations,he incorporation of liquid lipid to solid lipid, a secondeneration of SLN — nanostructured lipid carriers (NLC),as developed and found to enhance imperfections of SLNsnd achieved more space for drug molecules, thus improvedrug loading [134]. Zhang et al. applied monostearin as theolid lipid and oleic acid as the liquid lipid to construct DOX

LC [135]. This DOX NLC exhibited greater in vitro cyto-oxicity compared to free DOX in both resistant MCF-7/ADRnd SKOV3-TR30 cells. The high affinity between lipids orLC and the cell membrane, competitive inhibition of P-gp,

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ll of above contributed the increased intracellular drugoncentration and overcame MDR.

Wong et al. developed a novel polymer-lipid hybrid DOXLN system which was composed of hydrolyzed polymer ofpoxidized soybean oil (HPESO), stearic acid, pluronic F68,nd DOX [136]. HPESO was applied not only to achieve moreniform and spherical particles, but also to enhance DOXartition in the SLN thereby increasing drug loading capac-ty. The cytotoxicity of DOX SLNs was evaluated in resistantDA435/LCC6/MDR1 cells, and the results showed SLNs were

ignificantly more potent than free DOX. The mechanismsf DOX SLNs were investigated and proposed as the fol-ows: (1) DOX is released from DOX SLNs outside the cellsut the cytotoxicity is increased; (2) DOX-SLNs enter intohe cells and DOX is then released from SLNs inside theells, resulting in higher cytotoxicity. In the meanwhile,he following mechanisms were ruled out: (1) blank SLNsnd/or excipients inhibit or bypass the MDR proteins basedn the fact that the combination of blank SLNs and DOXr DOX-HPESO complex did not show significant cytotoxicffect in MDR cells; (2) the lipid components in the SLNslter permeability of cell membrane. These two findings areuite different compared to nano-delivery systems discussedreviously, which suggested the reversal of MDR activitiesas diversified and carrier dependent. Later, endocytosis

nhibition and fluorescent image studies of SLNs were per-ormed to better understand the mechanisms of the cellularrug uptake [137]. The results suggested the phagocytosisathway was involved in SLN internalization and DOX asso-iated with SLN bypassed the P-gp efflux in resistant cells.t was proposed that the DOX in SLNs probably entered theells with the combination of simple passive diffusion ofeleased drug from the carrier outside of cells and phago-ytosis. The released drug outside of cells may in parte effluxed by P-gp, however, DOX that entered cells viahagocytosis would be entrapped in cells and difficult toe pumped out by P-gp. In another study, the co-deliveryf DOX and GG918 (Elacridar, a lipophilic and non-ionic P-p inhibitor) in the SLNs showed enhanced DOX cellularptake than any forms of DOX/GG918 combination [138].huhendler et al. developed polymer-lipid hybrid SLN withyristic acid, HPESO, pluronic F68, PEG100SA, PEG40SA,

nd both DOX and mitomycin C were simultaneously loadedn the SLNs [139]. The SLNs demonstrated to be 20—30-foldore toxic in resistant MB435/LCC6/MDR1 cells compared to

ree DOX.

agnetic nanoparticles

hen et al. investigated how Fe3O4 magnetic NPs facilitatedNR to overcome MDR in vitro in sensitive and resistant562 cells [140]. To increase the interaction between NPsnd lipid portion of cell membrane, tetraheptylammoniumTHA) was coated on the NPs. Confocal fluorescence, atomicorce microscope (AFM), and electrochemical studies wereerformed to evaluate the synergistic effects of NPs on theptake of DNR in K562 cells. The observations confirmed

he THA-coated Fe3O4 NPs interacted with cell membranend significantly enhanced the uptake of DNR in resistant562 cells. The similar size of THA capped Ni magneticPs were applied as a control, but the NPs showed much

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P. Ma, R.J. Mumper

ess efficacy in terms of DNR cellular uptake in both sen-itive and resistant cells, indicating the unique propertyf THA capped Fe3O4 NPs to facilitate the DNR uptake.he following mechanisms were proposed for THA cappede3O4 NPs to overcome MDR: (1) Fe3O4 NPs may functions the inhibitor or competitive substrate for MDR asso-iated proteins (e.g. P-gp); (2) the interaction betweenHA capped Fe3O4 NPs and cell membrane. Later on, bothOX and tetrandrine (Tet, a P-gp inhibitor) were loaded

n Fe3O4 magnetic NPs and the results suggested the syn-rgetic reversal effect in resistant K562/A02 cells [141].nterestingly, Tet-Fe3O4 NPs were able to decrease by 100-old the MDR1 mRNA level but could not reduce the totalmount of P-gp, indicating P-gp function was blocked. Thectivity of a modified Tet, 5-bromotetrandrine (BrTet), waslso evaluated and it was demonstrated to have better effi-acy than Tet in resistant K562/A02 cells both in vitro andn vivo [142—145]. The BrTet-Fe3O4 NPs demonstrated thebility to down-regulate MDR1 mRNA level and P-gp expres-ion. The combination of DNR and BrTet-Fe3O4 NPs also hadignificantly greater antitumor efficacy than any controlsn vitro in resistant K562/A02 cells and in vivo in xenograftude mice. It was confirmed this NP system inhibited BCL-2xpression, up-regulated BCL-2-associated X protein (BAX),53 and caspase-3 proteins in resistant K562/A02 xenograftumors, all of which contributed to synergetic effect ofhe NPs to overcome MDR. Furthermore, short hairpin RNAshRNA) targeted the sequences of 3491—3509, 1539—1557,nd 3103—3121 nucleotide of MDR1 mRNA was constructed146]. The in vitro data suggested the combination ofDR1 shRNA and Fe3O4 magnetic NPs was more efficient

o reverse MDR and less toxic in resistant K562/A02 cellsSimilarly, DNR-loaded ZnO NPs were shown to have greaterytotoxicity compared to free DNR in resistant K562/A02ells [147].

Kievit et al. developed a complex DOX NP formulation,here polyethylenimine (PEI)-DOX was first constructed viapH-sensitive hydrazone linkage and this PEI-DOX was then

onjugated to PEG-coated superparamagnetic iron oxidePs [148]. PEI was used to serve as a docking moleculeor DOX to achieve high drug loading and help it escaperom endosomes. The results showed the complex NPs wereapidly taken up in both sensitive and resistant rat glioma C6ells, and significantly enhanced drug retention and greaterytotoxicity compared to free DOX in resistant C6 cells.n addition, DOX had the fastest release profile at acidicH, which indicated the cleavage of hydrazone linkage.aken together, this DOX complex NPs overcame MDR initro.

old nanoparticles

old NPs (Au NPs) have been widely used as biomedicalmaging and biosensors [149]. Because they are biocompat-ble, small size, high stability and tissue permeability, AuPs are also served as effective drug delivery carriers andrugs could be associated on the NPs by physical adsorp-

ion, ionic bonding, and/or covalent bonding [150,151].u et al. conjugated DOX onto PEGylated Au NPs via aisulfide bond (Au-PEG-SS-DOX), and the NPs showed thereater intracellular drug uptake than free DOX in resistant

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HepG2-R cells, which was confirmed by both confocal imagesand inductively coupled plasma mass spectrometry (ICP-MS)[152]. Interestingly, the DOX was released from lysosomesand reached the cytoplasma but not the nucleus, whichimplied that the cytotoxic function of Au-PEG-SS-DOX wasnot through its interaction with nuclear DNA. It was sug-gested the ability of NPs to overcome MDR may be relatedto the cell membrane properties, and NPs may dysregulatemitochondrial function in cytoplasma thus inducing the cellapoptosis. Zhang et al. linked DOX onto Au NPs via an amidebond to form DOX-Au NPs with ultrasmall particle size of2.7 nm [153]. The DOX-Au NPs were observed to be non-toxic and expected to be cleared by kidney within hours.The DOX-Au NPs were internalized into the cells and evenentered into the nucleus as seen by confocal and electronmicroscopy, which was probably due to its small size. TheNPs were demonstrated to have about 20- and 6-fold greatercytotoxicity and faster action than free DOX, respectively,in resistant B16 melanoma cells. The DOX-Au NPs werealso sensitive to resistant HeLa cells over-expressing BCL-2,which was probably due to the entry of NPs into the nucleusand DNA damage caused by released DOX. The Fu groupdeveloped 3-mercaptopropionic acid capped Au NPs (MPA-capped Au NPs), and the NPs significantly facilitated DNRuptake compared to free DNR in resistant K562/ADM cellsand the enhanced intracellular DNR fluorescence was mainlylocated on cell membrane [154,155]. Interestingly, the AuNPs without MPA functional group did not show facilitatedeffect, suggesting this functional group played an importantrole in the enhanced DNR accumulation on cell membrane.It was suggested that MPA-capped Au NPs and free DNRformed a complex via electrostatic interaction thus facil-itating drug penetration into the cells by simple diffusionand phagocytosis. Au NPs may also interact with proteinsor other components on the cell membrane and circum-vent MDR. Wang et al. conjugated DOX onto Au NPs via anacid-labile hydrazone linker (DOX-Au Hyd NPs) and NPs wereconfirmed to enter the cells via energy-dependent endo-cytosis, specifically, both caveolae- and clathrin-mediatedendocytosis, and DOX was then released from the NPs tocytoplasma and the nucleus [156]. The DOX-Au Hyd NPshad significantly enhanced intracellular drug uptake andless efflux, and dramatically increased cytotoxicity com-pared to free DOX in resistant MCF-7/ADR cells. DOX-Au NPswith a carbamate linker was prepared as a control (DOX-Au Cbm NPs) and the linkage was stable so that DOX wasnot released. In contrast, DOX-Au Cbm NPs demonstratedsimilar cytotoxicity as free DOX in MCF-7/ADR cells, indicat-ing that the drug release from NPs was important to exertactivity.

Silica nanoparticles

Huang et al. covalently conjugated DOX onto mesoporoussilica nanoparticles (MNSP) via hydrazone linkage (DOX HydMNSP) [157]. The DOX Hyd MNSP demonstrated significantlyinduced apoptosis both in vitro and in vivo in resistant MES-

SA/Dx-5 cells compared to the controls. It was suggestedthe MNSP entered the cells via endocytosis and bypassedP-gp efflux pump. It was also claimed that this was thefirst report that MNSP overcame MDR in vivo. Meng et al.

Q�f

resistance 327

ngineered MNSP to simultaneously deliver DOX and MDR1iRNA [158]. The surface of MNSP was functionalized with

phosphonate group which allowed DOX binding insidehe MNSP via electrostatic action and this functional groupoated with the cationic polymer of PEI which was furtheromplexed with anionic MDR1 siRNA. The dual deliveryf DOX and MDR1 siRNA with MNSP significantly enhancedntracellular and intranuclear DOX concentration comparedo free DOX or DOX MNSP without siRNA in resistant KB-V1ells. It was suggested that the DOX was released fromysosomes via proton sponge mechanism which was sup-orted by the findings that the addition of NH4Cl inhibitedOX release and entry into the nucleus. Shen et al. loadedOX in MNSP and this DOX MNSP showed 8-fold more potentnd dramatically enhanced drug intracellular uptake anduclear accumulation than free DOX in resistant MCF-7/ADRells in vitro [159]. The DOX concentration of DOX MNSP was.12- and 5.11-fold greater than that of free DOX at 0.5 andh, respectively, in xenograft MCF-7/ADR tumor-bearingude mice. It was the first report that the MNSP itself inhib-ted P-gp expression based on its ability to down-regulate-gp levels. The mechanism of MNSP entry into the cellsas through micropinocytosis pathway and once the NPs

nternalized into the cell, MNSP may bypass P-gp becauset was too large to be effluxed. All of the above probablyontributed to the MNSP ability to overcome MDR. Chent al. co-delivered DOX and BCL-2 siRNA in MNSP, whereOX was entrapped inside the pore of the MNSP and BCL-2iRNA was complexed with MNSP modified polyamidoamineendrimers [160]. The results demonstrated that the MNSPsnhanced drug cytotoxicity by up to 132-fold greater thanhat of free DOX in resistant A2780/AD human ovarianancer cells, and that the significantly increased antitumorfficacy was probably due to the suppression of BCL-2RNA and perinuclear localization of DOX via MNSP delivery

arrier.

arbon nanotubes

i et al. coupled a P-gp antibody to functionalized carbonanotubes via a diimide-activated amidation reaction forargeting purpose, and then loaded DOX on the remainingurface of the carbon nanotubes via physical adsorption161]. The physical adsorption between DOX and nanotubesaximally preserved the molecule integrity because of the

hemical bond avoidance. In addition, the release of DOXrom nanotubes improved in a controlled manner upon expo-ure of DOX nanotubes under near-infrared radiation. It wasroposed that the controllable and sustained release of DOXy near-infrared radiation and specific P-gp targeting werehe main reasons that the nanotube overcame MDR in resis-ant human leukemia K562R cells. It was also suggested thathe P-gp antibody conjugated on the nanotubes provideduge stereohindrance for P-gp recognition of DOX thus sup-ressed the efflux of DOX by P-gp.

yclodextrin nanoparticles

iu and co-workers designed a delivery system of novel-CD-centered star-shaped amphiphilic polymers (sPEL/CD)or DOX [162]. To construct the sPEL/CD complex, mPEG

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nd PLA were reacted to form linear mPEG-PLA (mPEL)s the arms, and then �-CD served as the core to obtainPEL/CD by an arm-first method. The drug loading ofOX was as high as 18% with an entrapment efficiency of4%, which was probably due to the presence of PLA toncrease hydrophobic interaction between polymer andrug as well as enlarge �-CD interspaces to accommodateore DOX. The DOX-loaded sPEL/CD had 3-fold decreased

C50 compared to free DOX in resistant MCF-7/ADR cells.ecause it was reported that pluronic block copolymersere able to prevent MDR in cancer cells, it was hypoth-sized the mPEG-PLA block segment in sPEL/CD complexad similar effect due to its structural similarity to pluronic163]. The interaction between polymer and P-gp maye another explanation of sPEL/CD system to reverseDR.

onclusions and future perspective

nthracyclines are very effective chemotherapeutic drugso treat various cancers. However, severe cardiotoxicity andhe development of MDR are the major limitations for thepplication of anthracyclines in clinic.

Nano-delivery systems have emerged as the novel can-er therapeutics to overcome some of the limitations ofnthracyclines. With the optimal particle sizes and surfaceroperties, NPs may be able to passively target anthracy-lines into the tumor tissues via the EPR effect, escaperom RES recognition, prolong circulation time in blood,nd improve the drug distribution in the body. Doxil, aOX PEGylated liposome formulation, was approved in 1995.his formulation demonstrated slower plasma clearance,nhanced circulation and half-life, decreased cardiotoxic-ty compared to free DOX. However, it does not addressDR issue. To date, many nano-delivery systems haveeen developed and reported, such as liposome formula-ions, polymeric NPs, solid lipid NPs, mesoporous silica NPs,agnetic NPs, polymer—drug conjugates, to effectively cir-

umvent MDR both in vitro and in vivo. Some of these systemsave even been advanced to clinical trials, for examplehe HPMA-DOX conjugate. However, MDR is very compli-ated and multifactorial. In addition, the MDR mechanismsre not fully understood. Therefore, it is better to addressifferent MDR pathways in the nano-delivery systems. Forgiven particular NP system, ideally it not only inhibits

r bypasses efflux pump resistance, such as P-gp, BCRPnd MRP1, but also circumvents non-pump resistance, suchs BCL-2, p53, etc. Moreover, in addition to the passivelyargeting, the active targeting using ligands may furthermprove the anticancer efficacy in resistant tumors whileecrease the toxicity in normal tissues.

Although nano-delivery systems are promising in cancerherapy, there remain many challenges for these systems.otably, it is difficult to characterize nano-delivery systems

n vivo. As a consequence, there continues to be a lack ofnderstanding of in vivo NP stability and in vivo drug releaseechanisms. Also, the EPR effect is likely exaggerated in

umans. However, nano-delivery systems with particle sizess small as <40 nm may have a better chance to passivelyccumulate into tumors. Furthermore, the long-term toxic-ty of nano-materials is largely unknown. Nevertheless, the

P. Ma, R.J. Mumper

uture of nano-delivery systems remains exciting and willertainly advance to address MDR in cancer therapy.

cknowledgments

he authors are supported, in part, by Award Number U54A151652 from the National Cancer Institute. The content

s solely the responsibility of the authors and does notecessarily represent the official views of the Nationalancer Institute or the National Institutes of Health.

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rug

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and commercialization. Dr. Mumper received

Anthracycline nano-delivery systems to overcome multiple d

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Ping Ma received his B.S. degree in Pharma-ceutics in 2000 from China PharmaceuticalUniversity, Nanjing, China. Ma earned his M.S.degree in Industrial Pharmacy in 2006 fromthe University of Toledo, Toledo, OH. Sub-sequently, he began his Ph.D. studies in Dr.Russell J. Mumper’s laboratory and receiveda Ph.D. degree in Pharmaceutics in 2012 fromthe University of North Carolina at ChapelHill with a focus on the development of lipid-based nanoparticle formulations for improved

ancer treatment. Currently, Dr. Ma is working for Hospira Inc.

Russell J. Mumper is the Vice Dean and theJohn A. McNeill Distinguished Professor at theUNC Eshelman School of Pharmacy at the Uni-versity of North Carolina (UNC) at Chapel Hill.Dr. Mumper has 25 years of experience in thepharmaceutical/biotechnology industries andacademia with expertise in advanced drugdelivery systems and product development

his Ph.D. in Pharmaceutics/Drug Delivery anda B.A. in Chemistry from the University of

entucky, Lexington, KY.