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Colloids and Surfaces B: Biointerfaces 136 (2015) 160–167

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb

reduction-degradable polymer prodrug for cisplatin delivery:reparation, in vitro and in vivo evaluation

houfeng Wang, Huili Liu, Xiaoming Shu, Li Zheng, Lijuan Chen ∗

tate Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy,hengdu 610041, PR China

r t i c l e i n f o

rticle history:eceived 12 May 2015eceived in revised form 14 August 2015ccepted 5 September 2015vailable online 9 September 2015

a b s t r a c t

We describe the development of novel reduction-sensitive therapeutic micelles consisting of polyethy-lene glycol-poly-(l-glutamic acid) (PEG-PLG), and conjugate with dithiodipropionic-Pt (IV) (DTDP-Pt).The drug delivery polymeric micelles lie in the covalent conjugation of each cisplatin drug to the PLGchains through a disulfide bond. The resulting micelles show well-controlled cisplatin loading yield,excellent reduction-responsive drug release kinetics, and enhanced in vitro cytotoxicity against cancer

eywords:olymer prodrugisplatinrug deliverytimuli-responsive

cells. The in vivo studies on the subcutaneous human ovarian carcinoma SKOV-3 xenograft model con-firmed that PEG-P(LG-DTDP-Pt) micelles showed significant antitumor activity and reduced side effects,compared with free cisplatin. This characteristic drug release profile holds the promise to suppress cancercell by rapidly releasing a high dose of chemotherapy drugs inside the tumor cells, thereby improvingthe therapeutic efficacy of the drug payload.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Cisplatin has been widely used in the clinic to treat a varietyf cancers such as head and neck, ovarian, breast, bladder andmall cell lung cancer because of its potent activity to cross-linkNA upon entering the cells [1–4]. However, cisplatin is vulnera-le to attack by plasma proteins, particularly those containing thiolroups, such as human serum albumin [5]. It has been reported thatne day after cisplatin administration, 65–98% of the platinum inhe blood was protein-bound [6,7]. This undesirable protein bind-ng deactivated the drugs, leading to less therapeutic efficacy, andccounted for some severe side effects of cisplatin therapy [8,9].cquired resistance and unpleasant side effects, much attentionas been paid to platinum(IV)-based compounds [10–13]. A rela-ionship between the structure and the anti-cancer activity wasroposed [14–16].

Binding a drug to macromolecular scaffolds (polymer–drugonjugates) considerably increase its stability, thereby signifi-antly altering the biodistribution of the drug-conjugate [17–19].

ntravascular half-life for instance is greatly extended, particularlyor polymer conjugates larger than the glomerular filtration cut-ff [20]. Drug concentrations in tumor tissue are increased due to

∗ Corresponding author.E-mail address: chenlijuan125@163.com (L. Chen).

ttp://dx.doi.org/10.1016/j.colsurfb.2015.09.010927-7765/© 2015 Elsevier B.V. All rights reserved.

the EPR (enhanced permeability and retention) effect as a result ofhigh rates of leakage from the neovasculature of tumor tissue [21].Among all the drug carriers, self-assembled polymeric micelles ofpoly(ethylene glycol)-poly(amino acids) (PEG-PAA) have emergedas one of the most promising platforms for improved antitumordrug delivery and have been widely studied in preclinical andclinical trials, owing to their excellent biodegradability and bio-compatibility [22]. Typically, cell uptake of polymeric micelles havebeen shown to occur via endocytosis [23], whereby higher molecu-lar weight conjugates are trafficked to the cytoplasm, consequentlythe chemotherapeutic agents are released from the polymer back-bone and diffuse into cytoplasm or nucleus to inhibit cell growthor kill the cell directly. Drug activity in this case typically requiresrelease of the drug from the carrier via labile bonds.

Herein, we chose biodegradable polyethylene glycol-poly-(l-glutamic acid) (PEG-PLG) as the copolymer backbone reacted withdithiodipropionic-Pt (IV) conjugate (DTDP-Pt). Since the disulfidebond is specifically cleavable by GSH, which is abundant in thetumor cell cytoplasm [24]. The final micelles were triggered bytumor extracellular reduction for enhanced cellular uptake andanticancer efficiency of anticancer drug (Scheme 1). The PEG-P(LG-DTDP-Pt) micelles were stable and drug leakage free withoutreducing agent. The antitumor efficacy in SKOV-3 xenograft model

showed Pt (IV)-loaded micelles reduced systemic toxicity andenhanced antitumor efficacy compared with free cisplatin, indi-cating its great potential for efficient cancer chemotherapy.

Z. Wang et al. / Colloids and Surfaces B: Biointerfaces 136 (2015) 160–167 161

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Scheme 1. Preparation of PEG-P(LG-DTDP-Pt)

. Experimental

.1. Materials

�-Methoxy-�-hydroxy-poly(ethylene glycol) (PEG, Mn = 5000),isplatin (II), Hoechst33342, Coumarin-6 (C6) and MTT (3-(4,5-imethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) wereurchased from Sigma. Ethanolamine (EA), 3, 3′-dithiodipropioniccid (DTDP), ethanolamine (EA) and glutathione (GSH) wereurchased from alfa aesar. �-Benzyl-l-glutamate-N-carbox- yan-ydride (BLG-NCA) was synthesized as described in previous work25]. N,N-Dimethylforma-mide (DMF), dichloromethane (DCM)nd dimethyl sulfoxide (DMSO) were stored over 4A molecularieve and purified by vacuum distillation with CaH2. All chemicalsnd reagents were used without further purification.

NMR spectra were recorded on Bruker Avance 400 spectrometerBruker Company, Germany) using DMSO-d6 as solvent. Mass spec-ra (MS) were measured by ESI-Q-TOF Priemier mass spectrometerMicromass, Manchester, UK). X-ray photoelectron spectroscopyXPS Kratos XSAM800, Britain) was recorded using AlKa (1486.6 eV)s the radiation source. Gel permeation chromatography (GPC) wasarried out on PL gel column (HLC-8320GPC, Japan), polystyrenetandards were used as calibration standards.

.2. Synthesis of DTDP-Pt

Dithiodipropionic anhydride (DTDPA) was synthesized accord-ng to previously published protocol [26]. Cisplatin(II) was oxidizedy oxygen peroxide into complex 1, and the latter was reacted withTDPA to form DTDP-Pt. The Pt (IV) conjugated to the DTDPA iss

hown in Scheme 2. Briefly, 0.2 g complex 1 (0.6 mM) in 16 mL dryMSO was added 0.06 g DTDPA (0.6 mM) and the reaction mix-

ure were stirred at room temperature for 48 h. The solution wasyophilized and 10 mL acetone was added to precipitate a light yel-ow solid, it was washed several times with acetone and diethylther, and dried under reduced pressure. DTDP-Pt was isolated in0% yield. MS(ESI-Q-TOF): 713.0728[M]+, 338.0611[M + 2Na]2+.

.3. Synthesis of the polymers PEG-P(LG-DTDP-Pt)

Firstly, PEG-NH2 was prepared according to literature [27]..89 g PEG-NH2 and 0.7 g BGlu-NCA (the molar ratio of mPEG-H2 to BGlu-NCA was 1:15) were dissolved in 10 mL dry DMF and

tirred at 45 ◦C for 48 h under N2 atmosphere. The product wasrecipitated in cold diethyl ether, filtered, and dried under reducedressure. Subsequently, 1.5 g PEG-PBLG copolymer was dissolved

n dichloroacetic acid (10 mL), then HBr/acetic acid (33 wt.%, 3 mL)as added for the removal of the �-benzyl group. After stirring at

0 ◦C for 12 h, the mixture was precipitated into ice diethyl ether.he precipitates were redissolved in DMF and then dialyzed against

es and the possible mechanism of their action.

distilled water (MWCO = 3500 Da), then freeze-dried to give theproduct PEG-PLG. Overall yield was 80.1%.

1 g PEG-PLG (1.7 mM caboxyl groups) was dissolved in 20 mLdry DMF in a flask, to which 0.3 g EA (5 mM), 0.7 g EDCI (3.5 mM),0.39 g HOBT (3.5 mM) and 0.122 g DMAP (1 mM) were added. Thereaction mixture was stirred at room temperature for 48 h andthen precipitated by ice ethyl ether to obtain PEG-P(LG-OH). Then,0.132 g DTDP-Pt (0.249 mM) was first dissolved in 15 mL dried DMFunder stirring in a flask. Thereafter, 0.2 g PEG-P(LG-OH) (0.165 mMhydroxyl group), 0.7 g EDCI (3.5 mM), and 0.122 g DMAP (1 mM)were added into the mixed solution. The reaction was carried out atroom temperature for 48 h. And then the reaction solution was pre-cipitated by ice ethyl ether, and the solid product was collected byfiltration and vacuum-dried to get white powders. Then the prod-uct was dissolved in DMF and the solution was dialyzed againstwater to remove un-reacted cisplatin(IV), and finally lyophilized toobtain PEG-P(LG-DTDP-Pt).

2.4. Preparation and characteristics of Pt(IV)-loadedPEG-P(LG-DTDP-Pt) micelles

Pt(IV)-loaded micelles were prepared by a dialysis method.Briefly, 200 mg PEG-P(LG-DTDP-Pt) were dissolved in 10 mL DMSO,and then 10 mL PBS (pH 7.4) was added under stirring. The mix-ture was stirred for another 6 h and then dialyzed against distilledwater using a dialysis bag (MWCO = 3500 Da). After 5000 rpm cen-trifuged for 3 min, the supernatant solution was filtered througha 0.45 mm filter to gain a clarified solution, and then the resul-tant Pt(IV)-loaded micelles were stored at 4 ◦C for use. LyophilizedPt(IV)-loaded micelles were dissolved in aqua regia, then dilutedwith PBS, the amount of Pt in the solution was determined byICP-OES. The particle size distribution of the micelles was evalu-ated by dynamic light scattering (DLS) with a Zetasizer Nano ZS-90instrument (Malvern Instruments, Malvern, UK). The morphologyof Pt(IV)-loaded micelles were examined using a transmission elec-tron microscope (TEM, H-6009IV, Hitachi, Japan). To investigate thestability of Pt(IV)-loaded micelles, the micelles in PBS (pH 7.4) con-taining 10% fetal bovine serum were incubated at 37 ◦C for 1 day, 3days and 7 days, then the particle size were measured.

2.5. In vitro drug release

To determine the behavior of Pt from the Pt(IV)-loaded micelles,10 mL of the micellar solution was added into the dialysis mem-brane tubing (3.5 kDa, Spectrum/Por), and immersed in 25 mL ofPBS (pH 7.4) which contained 10 mM GSH and 150 mM NaCl. The

dialysis was conducted at 37 ◦C in a shaking culture incubator. Andat predetermined intervals, the release mediums were all pipedout and replaced by fresh buffer. Then the release mediums werelyophilized, redissolved and measured concentration by ICP-OES.

162 Z. Wang et al. / Colloids and Surfaces B: Biointerfaces 136 (2015) 160–167

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Scheme 2. Synthetic scheme for the pr

.6. Cell culture and animals

Human hepatoma HepG2 cells and human ovarian carcinomaKOV-3 cells were obtained from the American Type Culture Col-ection (ATCC; Manassas, VA, USA). Cells were maintained in DMEMInvitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovineerum (FBS; Invitrogen), 100 U/mL penicillin (Invitrogen) and00 �g/mL streptomycin (Invitrogen).

BALB/c nude mice (4–6 weeks old) were purchased fromuaFukang Biological Technology Company (Beijing, China) andaintained at the Animal Center of State Key Laboratory of Biother-

py of Sichuan University for 2 weeks of adaptive feeding prior tohe start of experiment. The experimental protocol was approvedy the Ethics Review Committee for Animal Experimentation ofichuan University.

.7. Cell uptake study

In order to visualize the Pt(IV)-loaded micelles taken by theepG2 cell lines, the cells incubated with C6 labeled micelles werexamined. 1 × 105 cells were placed in 6-well plates and incubatedvernight in 2 mL DMEM medium supplemented with 10% FBS at7 ◦C in 5% CO2. Cells were treatment with free C6 and C6 labeledicelles (5 �g mL−1) for 4 h. Then cells were washed with PBS and

xed with 4% (w/v) formaldehyde in PBS. The cell nuclei weretained with Hoechst33342, following the manufacturer’s instruc-ions. The coverslips were placed onto the glass microscope slides,nd C6 uptake was visualized using a laser scanning confocal micro-cope (LSCM) (Leica TCS SP5, Germany).

.8. Cytotoxicity assay

Cell cytotoxicity of Pt(IV)-loaded micelles was evaluated by MTTssay using HepG2 and SKOV-3 cell lines. 3000 cells in 100 �L

tion of PEG-P(LG-DTDP-Pt) copolymer.

DMEM containing 10% FBS, 100 units/mL penicillin and 100 �g/mLstreptomycin were seeded in 96-well plates and incubated for12 h at 37 ◦C in 5% CO2. These cells were treatment with DMSO,PEG-PLG copolymer, free cisplatin and PEG-P(LG-DTDP-Pt) micelleswas evaluated for 24 h and (or) 48 h, respectively. The biggestDMSO concentration in cisplatin solution was 0.76% (v/v). Afterincubation, 20 �L MTT (5 mg/mL, dissolved in PBS, pH 7.4) wasadded to each well and incubated for another 4 h. Then removedthe incubated medium, added 150 �L DMSO to each well andgently shook for 10 min at room temperature. Absorbance wasmeasured at 570 nm using a Spectramax M5 Microtiter Plate Lumi-nometer (Molecular Devices, US). Set the absorbance value ofuntreated cells to be 100%. Each experiment was repeated in trip-licate.

2.9. In vivo antitumor efficiency

The antitumor efficacy was evaluated in the SKOV-3 xenograftBALB/c nude mice model. The mice were randomly divided into fourgroups (n = 5), and the freshly harvested SKOV-3 cells (1 × 107cellsmouse−1, 100 �l injection−1) were injected into the right flank ofeach mouse. When the tumors grew to 50 mm3, the mice wererandomly divided into three groups (n = 5), and this day was des-ignated as day 0. Three groups of mice were treated with thefollowing treatments: saline (control group) or a dose of 3.0 mg/kg(with respect to Pt) of free cisplatin and Pt(IV)-loaded micelles. Themice were injected intravenously three times via the tail vein atday 0, 3, 6. The antitumor activity was evaluated in terms of thetumor volumes, which was calculated using the following equa-

tion: V = a × b2/2, where a and b are major and minor axes of thetumors measured by a caliper, respectively. The body weight wasmeasured simultaneously as an indicator of the systemic toxic-ity.

Z. Wang et al. / Colloids and Surfaces B: B

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ig. 1. 1H NMR spectra of (A) DTDP-Pt, (B) PEG-PBLG, (C) PEG-P(LG-DTDP-Pt) inMSO-d6.

.10. Statistical analysis

All experiments were performed at least three times andxpressed as means ± SD. Data were analyzed for statistical sig-ificance using Student’s test. p < 0.05 was considered statisticallyignificant.

. Results and discussion

.1. Synthesis and characterization of PEG-P(LG-DTDP-Pt)opolymer

The synthetic route to PEG-P(LG-DTDP-Pt) is shown in Scheme 2.TDP was selected as a disulfide donor due to its capability to beonveniently converted to DTDPA which has high reactivity towardhe reagents carrying hydroxyl. Cisplatin was oxidized with H2O2 toorm c,c,t-[PtCl2(NH3)2(OH)2] (complex 1), and then was allowedo react with DTDPA, to yield DTDP-Pt. Fig. 1A depicts the 1H NMRpectrum of DTDP-Pt, the peaks of CH2− of DTDP at 2.1 and.3 ppm, and −NH3 of cisplatin at 6.5 ppm. XPS and ESI-Q-TOF weresed to confirm the synthesis of DTDP-Pt (Figs. S1 and S2 in Sup-orting information). Wide scan and the of Pt 4f is shown in Fig. S1.

less binding energy shift was observed means the transformingt (II) to Pt (IV). The major multi-peaks centered at 713.0728[M]+

ass–charge ratios verified that the mean MWs of DTDP-Pt were13.0728 Da.

PEG-PBLG block copolymer was prepared by the ring open-ng polymerization of BLG-NCA using mPEG-NH2 as the initiator,ollowed by deprotection of �-benzyl in HBr/acetic acid to fromEG-PLG. They were used to react with DTDP-Pt to gain the final

iointerfaces 136 (2015) 160–167 163

PEG-P(LG-DTDP-Pt) conjugates. The length of PBLG block was usingfeed molar ratio of PEG-NH2 to BLG-NCA at 1:15. Fig. 1B showsthe 1H NMR spectrum of PEG-PBLG as the representative copoly-mer, the resonances at ı = 3.6 and 3.3 ppm were attributed to themethylene protons and end methoxy protons of PEG CH2CH2–and CH3–, respectively. The peaks at ı = 4.2 were assigned to theprotons of the PBLG backbone C(O)CH(CH2–)NH . The methyleneprotons of PBLG side groups CH2–CH2–CO gave characteristicsignals at ı = 1.97 and 2.01 ppm. The resonances at ı = 5.0 and 7.1were the �-benzyl groups C6H5– and C6H5CH2–. As indexed inFig. 1C, the 1H NMR spectrum of the final cisplatin prodrug PEG-P(LG-DTDP-Pt) included most characteristic resonance peaks ofthe PEG-PBLG polymer and DTDP-Pt, for example, the character-istic peaks of NH3 cisplatin at 6.5 ppm and 3.6 and 3.3 ppm forthe methylene protons and end methoxy protons, however, thepeaks at 5.0 ppm were ascribed to the �-benzyl groups disappeared.The formation of PEG-P(LG-DTDP-Pt) conjugates was further con-firmed by the GPC measurements. Typical GPC chromatogramsare shown in Fig. S3, the Mn of PEG-PLG and PEG-P(LG-DTDP-Pt)were 7802 gmoL−1 and 11,802 gmoL−1 respectively. Both copoly-mers exhibited a relatively narrow PDI of around 1.03–1.26, but thePEG-P(LG-DTDP-Pt) copolymers showed reduced retention time,indicating higher molecular weights of the PEG-P(LG-DTDP-Pt)after clicking with DTDP-Pt.

3.2. Preparation and characteristics of PEG-P(LG-DTDP-Pt)micelles

The DTDP-Pt is insoluble in water, so the copolymer PEG-P(LG-DTDP-Pt) can self-assemble into micelles with the P(LG-DTDP-Pt)as the inner core, and the PEG block as the hydrophilic shell. DLSand TEM were used to experimentally assess self-assembly prop-erties of the Pt(IV)-loaded micelles (Fig. 2). DLS clearly revealedformation of micellar structures with a mean diameter of 106 nm,the polydispersion index (PDI) of micelles was about 0.12 (Fig. 2A).TEM confirmed the distinct outline of polymeric aggregates but ata significantly smaller size (70 nm on average). In contrast to DLSdeterminations, which are performed using aqueous suspension,TEM analysis requires dried samples. The large difference betweenthe experimentally determined average diameter of these micellesby DLS and TEM might be attributed to both the dehydration effectand existing aggregation.

The zeta potential distribution of the micelles was measuredas shown in Fig. 2B, the mean zeta potential was −10.6 mV, andit has been reported that negatively charged carriers have shownpotential for protein resistance and exhibited a prolonged bloodcirculation time for in vivo applications [28]. The data of stabilityinvestigation of Pt(IV)-loaded micelles in 10% fetal bovine serumwere shown in Fig. 2D, and the DLS results revealed that micelleskept stable at least 7 days at 37 ◦C with stable particle size, thismight attribute to amphiphilic PEG-PLG enhanced stability of themicelles.

The Pt loaded into the PEG-P(LG-DTDP-Pt) micelles mainly bychemical conjugation, which means the loading capacity connectsto the content of PLG in the micelles. To a specific micelles, the Ptloading capacity of micelles could reach to 9.6% measured by ICP-OES. The accumulative drug release behaviors of PEG-P(LG-DTDP-Pt) micelles in Fig. 3A. The absence of GSH was showed slow gradualprofiles, only18.6% of Pt was released in 40 h. As we expect, Pt wasrapidly released from the micelle in the presence of 10 mM GSH,for example, ca. 80% Pt was released in 40 h. It was thought thatthe structure of PEG-P(LG-DTDP-Pt) micelle was destroyed due to

the degradation of disulfide bonds in polymer backbone under areduction environment.

To assess the functionality of this redox-induced release mech-anism, time-dependent changes in nanomicelles size were studied

164 Z. Wang et al. / Colloids and Surfaces B: Biointerfaces 136 (2015) 160–167

Fig. 2. (A) Size, (B) zeta potential distributions, and (C) transmission electron microscopy of Pt-loaded micelles in PBS. (D) The stability of micelles size upon exposure to 10%fetal bovine serum as determined by DLS.

Fig. 3. (A) Cisplatin release from the Pt-loaded micelles in the presence of GSH and no GSH in PBS at 37 ◦C. (The data are mean ± SD, n = 3). (B) The size changes of micellesin response to GSH in PBS (pH 7.4) determined by DLS.

Z. Wang et al. / Colloids and Surfaces B: Biointerfaces 136 (2015) 160–167 165

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Fig. 4. Fluorescence images of HepG2 cells after incubation with free C6 an

y DLS in the presence and absence of 10 mM GSH (Fig. 3B). Aontrol experiment without GSH revealed insignificant change inverage nanomicelles size over 24 h underlining chemical stabil-ty. In contrast, alterations in PEG-P(LG-DTDP-Pt) micelles behavior

ere observed following inclusion of 10 mM GSH. Within 2 h, theverage micelles size increased from 106 nm to about 160 nm.he formation of larger aggregates with mean diameters 250 nmas observed in 4 h. PEG-P(LG-DTDP-Pt) was turned into a highly

swollen” micelle, the partially solvated DTDP-Pt core of whichas much more expanded upon unpacking the tight enclosure of

he hydrophobic interlayer. We hypothesize that this size increases the consequence of an increasingly hydrophobic nature of the

icelles following reductive detachment of the DTDP-Pt inner coreFig. 4).

.3. Cellular uptake of PEG-P(LG-DTDP-Pt) micelles

It is well known that free cisplatin quickly enters nuclei afterell uptake [29], whereas cisplatin transported in cytoplasm verylowly [30]. In order to visualize the micelles taken by the cells, theuorescent C6 can serve as a probe for the composite micelles. Theells incubated with free C6 and C6 labeled micelles were exam-ned by LSCM. Fig. 5 shows the fluorescence microscopy imagesf HepG2 cells at 1 h and 4 h post administration of free C6 and C6abeled micelles. Because the cells were thoroughly washed, the flu-rescence of C6 is believed to come from the micelles taken insidehe cells. The cell nucleus was stained by Hoechst33342. The cellsptake of C6 was enhanced as time increased, however, lower greenuorescence was observed in the cells both in 1 h and 4 h incuba-ion for free C6 compared to C6 labeled micelles, indicating that

he more green fluorescence was distributed in the cytoplasm by6 labeled micelles. The results suggesting that PEG-P(LG-DTDP-t) micelles could be successfully internalized by tumor cells viandocytosis.

oaded micelles for 1 h and 4 h. C6 is shown in green and cell nuclei is blue.

3.4. Cytotoxicity of PEG-P(LG-DTDP-Pt) micelles

The in vitro cytotoxicity of DMSO, PEG-PLG copolymer, free cis-platin and PEG-P(LG-DTDP-Pt) micelles was evaluated in HepG2and SKOV-3 cells using MTT assay. As shown in Fig. 5A and B, theviability of two cells treated with DMSO (0.76% v/v), no markedcytotoxicities were detected in two cells. The cell viabilities ofPt(IV)-loaded micelles were evaluated after 24 h or 48 h incuba-tion, PEG-PLG copolymer and free cisplatin were used as control. Asshown in Fig. 5C and D, PEG-PLG copolymer was 90–100% at all testwith the Pt concentrations(1:6), revealing the low toxicity and goodcompatibility of copolymer to cells. Pt(IV)-loaded micelles exhib-ited dose- and time-dependent cell proliferation inhibition for bothtwo cells. The results also show that Pt(IV)-loaded micelles appearto induce higher antitumor effect compared with free cisplatin.For HepG2 cells, ic50 (i.e., inhibitory concentration to produce50% cell death) of Pt(IV)-loaded micelles was determined as 0.91and 0.25 �g ml−1 after 24 or 48 h incubation time, respectively,which were lower than those observed for free cisplatin (1.38 and0.73 �g ml−1 after 24 or 48 h incubation time, respectively). A sim-ilar result was observed in SKOV-3 cells (6.05 and 1.34 �g ml−1 forPt(IV)-loaded micelles after 24 h or 48 h incubation time, respec-tively, lower than those of 16.92 and 5.71 �g ml−1 for free cisplatinafter 24 or 48 h incubation time, respectively. The results implythat Pt(IV)-loaded micelles are more effective than free cisplatinfor cancer therapy.

3.5. In vivo antitumor efficacy of PEG-P(LG-DTDP-Pt) micelles

To evaluate the antitumor activity of Pt(IV)-loaded micelles, effi-

cacy studies were performed in mice bearing SKOV-3 xenografts.Seven days after inoculation with SKOV-3 cells, mice were treatedwith free cisplatin and Pt(IV)-loaded micelles at 3 mg kg−1 Pt equiv-alents for a total of two doses via the tail vein. The tumor volumes

166 Z. Wang et al. / Colloids and Surfaces B: Biointerfaces 136 (2015) 160–167

Fig. 5. In vitro cytotoxicity of DMSO, PEG-PLG copolymer, free cisplatin and Pt-loaded micelles was evaluated in HepG2(A and C) and SKOV-3 (B and D) cells using MTT assayfor 24 h and 48 h. The molar ratio of PEG-PLG: Pt = 1:6. (The data are mean ± SD, n = 3).

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ig. 6. Effect of free cisplatin and Pt-loaded micelles on tumor volume (A), body we dose of 3.0 mg/kg (with respect to Pt) or saline were administered via tail vein on

nd the body weights were measured. As shown in Fig. 6A, theumor volumes in the control group (PBS) increased rapidly to1000 mm3 in 18 days, and all cisplatin treatment groups decreasedumor growth rates (p < 0.05) compared with the control group. Inddition, the mice treated with Pt(IV)-loaded micelles had a sig-ificantly lower mean tumor volume (p < 0.01) than the mice inhe free cisplatin groups at the same dose. For example, 18 daysfter injection, the average tumor volume in the free cisplatin andt(IV)-loaded micelles treated mice at 3 mg kg−1 Pt equivalents hadeached 750 and 300 mm3, respectively. The body weight loss is anmportant indicator for evaluating drug-related toxicity. As shownn Fig. 6B, treatment with free cisplatin at 3 mg kg−1 Pt resulted inhe greatest body weight loss (24%), which revealed that free drugad significant treatment-related toxicities. In contrast, the treat-ent with Pt(IV)-loaded micelles appeared to be well tolerated and

aused almost no decrease in body weight.

The above results demonstrated that Pt(IV)-loaded micelles

xhibited enhanced antitumor activity over free cisplatin in termsf the tumor volume regression and greatly reduced the toxic-ty of cisplatin. One reason for the enhanced in vivo antitumor

B) of mice bearing SKOV-3 ovarian carcinoma xenograft. PBS was used as a control., 3, 6, (The data are mean ± SD, n = 5)

efficacy might be the enhanced accumulation at the tumor site ofthe Pt (IV)-loaded micelles, due to the EPR effect. In addition, thefast release of cisplatin from Pt(IV)-loaded micelles at the tumorsite may contribute to the enhanced antitumor efficacy.

4. Conclusions

In conclusion, a reduction-responsive PEG-P(LG-DTDP-Pt) pro-drug was synthesized as a new cisplatin delivery vehicle. Thedrug delivery system is that the cisplatin analogue prodrug wascovalently linked to the PLG chains through disulfide bond. Wedemonstrated that the Pt(IV)-loaded micelles had well-controlledcisplatin loading yield and showed excellent reduction respon-sive drug release kinetics, leading to enhanced in vitro cytotoxicityagainst tumor cells. The in vivo studies demonstrated that Pt(IV)-loaded micelles exhibited increased tumor accumulation, reduced

toxicity and higher antitumor efficacy compared with free cisplatinat the same dose. Thus, as an environmentally sensitive drug deliv-ery vehicle, these micelles can potentially minimize the drug lossduring their circulation in the blood, where the reduction value is

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ow, and trigger rapid intracellular drug release when the micellesre endocytosed by the cancer cells. This characteristic drug releaseinetics may improve the therapeutic efficacy of the drug pay-oad.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.09.10.

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