ph-responsive chimaeric pepsomes based on asymmetric...

pH-Responsive Chimaeric Pepsomes Based on AsymmetricPoly(ethylene glycol)‑b‑Poly(L‑leucine)‑b‑Poly(L‑glutamic acid)Triblock Copolymer for Efficient Loading and Active IntracellularDelivery of Doxorubicin HydrochloridePeipei Chen, Min Qiu, Chao Deng,* Fenghua Meng, Jian Zhang, Ru Cheng, and Zhiyuan Zhong*

Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College ofChemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, People’s Republic of China

*S Supporting Information

ABSTRACT: pH-Responsive chimaeric polypeptide-basedpolymersomes (refer to as pepsomes) were designed anddeveloped from asymmetric poly(ethylene glycol)-b-poly(L-leucine)-b-poly(L-glutamic acid) (PEG-PLeu-PGA, PEG islonger than PGA) triblock copolymers for efficient encapsu-lation and triggered intracellular delivery of doxorubicinhydrochloride (DOX·HCl). PEG-PLeu-PGA was convenientlyprepared by sequential ring-opening polymerization of L-leucine N-carboxyanhydride and γ-benzyl-L-glutamate N-carboxyanhydride using PEG-NH2 as an initiator followed bydeprotection. Pepsomes formed from PEG-PLeu-PGA hadunimodal distribution and small sizes of 64−71 nm depending on PLeu block lengths. Interestingly, these chimaeric pepsomeswhile stable at pH 7.4 were quickly disrupted at pH 5.0, likely due to alternation of ionization state of the carboxylic groups inPGA that shifts PGA blocks from hydrophilic and random coil structure into hydrophobic and α-helical structure. DOX·HClcould be actively loaded into the watery core of pepsomes with a high loading efficiency. Remarkably, the in vitro release studiesrevealed that release of DOX·HCl was highly dependent on pH, in which about 24.0% and 75.7% of drug was released at pH 7.4and 5.0, respectively, at 37 °C in 24 h. MTT assays demonstrated that DOX·HCl-loaded pepsomes exhibited high antitumoractivity, similar to free DOX·HCl in RAW 264.7 cells. Moreover, they were also potent toward drug-resistant MCF-7 cancer cells(MCF-7/ADR). Confocal microscopy studies showed that DOX·HCl-loaded pepsomes delivered and released drug into the cellnuclei of MCF-7/ADR cells in 4 h, while little DOX·HCl fluorescence was observed in MCF-7/ADR cells treated with free drugunder otherwise the same conditions. These chimaeric pepsomes with facile synthesis, efficient drug loading, and pH-triggereddrug release behavior are an attractive alternative to liposomes for targeted cancer chemotherapy.

■ INTRODUCTIONLiposomal doxorubicin with lower cardiotoxicity than freedoxorubicin hydrochloride (DOX·HCl) is currently applied foreffective treatment of various cancers in the clinics.1−3

However, the possible hand−foot syndrome of PEGylatedliposomal formulations limits their dose and substitution forDOX·HCl formulations. As for liposomes, polymersomescontain also a watery interior that can be used for loadingand delivery of hydrophilic drugs like DOX·HCl. Notably,polymersomes with typically a thicker membrane exhibit ingeneral better stability and lower premature drug release thanliposomes.4,5 Moreover, polymersomes have an intrinsic stealtheffect as conferred by their hydrophilic blocks like PEG.6−8 Incontrast to liposomes, polymersomes can be prepared fromblock copolymers with very different structures and molecularweights, allowing design and development of stimuli-sensitivebiodegradable polymersomes9−11 as well as tumor-homingpolymersomes.12,13 Notably, polymersomes has been used todeliver a variety of therapeutic drugs and proteins.14−16

Nevertheless, though polymersomes have a big waterycompartment, in practice they show usually poor drug loadingcapacity as well as low drug loading efficiency for water-solubleagents such as DOX·HCl and proteins.17 In the past years,different drug loading technologies including the trans-membrane gradient17−20 and nanoprecipitation methods21

have been developed to augment their drug encapsulationlevels. Notably, a novel strategy based on chimaeric polymer-somes has recently been developed to greatly improve loadingcapacity of hydrophilic drugs.22,23 The chimaeric polymersomesusually have polyion blocks in watery core, providingelectrostatic and hydrogen bonding interactions with hydro-philic drugs and proteins. However, the preparation ofasymmetric triblock copolymer often involves complex multi-step synthesis with low yields.

Received: January 27, 2015Revised: March 10, 2015Published: March 11, 2015

Article

pubs.acs.org/Biomac

© 2015 American Chemical Society 1322 DOI: 10.1021/acs.biomac.5b00113Biomacromolecules 2015, 16, 1322−1330

pubs.acs.org/Biomachttp://dx.doi.org/10.1021/acs.biomac.5b00113

Synthetic polypeptides with various components andarchitectures can be easily obtained through controlledpolymerization of α-amino acid N-carboxyanhydrides(NCAs).24,25 Moreover, these polypeptides might possess theappealing features of natural proteins including goodbiocompatibility, in vivo degradability, and unique secondaryand tertiary conformations and thus have been extensivelyemployed to design nanocarriers, including micelles, polymer-somes, and nanogels.26−29 For example, poly(N-2-(2-(2-methoxyethoxy)ethoxyl)acetyl-L-lysine)-poly(L-leucine) (P-(EG2-LL)-PLeu), PLL-poly(L-phenylalanine) (PLL-PPhe),and poly(L-methionine)-P(Leu/PPhe) were reported to formlarge peptide vesicles (referred to as pepsomes) with diametersof 1−50 μm.30−32 Lecommandoux et al. reported that PLL-poly(L-glutamic acid) (PLL15-PGA15) diblock copolypeptideswere self-assembled into vesicles with an average diameter ofaround 150 nm at pH < 4 or pH > 10.33 Gu et al. reported thatpepsomes with an average size of 300 to 800 nm formedthrough cooperative hierarchical self-assembly of globular PLLdendrimers and carboxyl-functionalized linear PLeu exhibited acomparable gene-transfection activity to 25 kDa PEI in theabsence of serum.34 It is known that there exist natural pHgradients in the tumor microenvironment (pH 6.5−7.2) and inthe endosomal/lysosomal compartments of tumor cells (pH4.0−6.5).35−37 Therefore, pH-responsive pepsomes have beendeveloped to achieve enhanced drug release in tumor tissue andcells.38−40 For example, pH-responsive, size-tunable, and stablevesicles were developed from amphiphilic poly(trimethylenecarbonate)-b-PGA (PTMC-PGA) copolymers.39 These vesiclescoloaded with DOX and superparamagnetic iron oxidenanoparticles (USPIO) revealed improved cytostatic effectstoward HeLa cells when a high frequency AC magnetic fieldwas applied.41 Hadjichristidis et al. reported that vesicles with adiameter of about 130 nm were developed from amphiphilicPLL-PBLG-PLL copolypeptides containing 19−75% PBLG atneutral pH for DNA encapsulation.38 Hammond et al.developed pH-responsive nanometer-scale vesicles based ondiisopropylamine/diethylamine functionalized PEG-poly(γ-propargyl-L-glutamate).42 Recently, Kataoka and Chen devel-oped pepsomes through the complexation of PEG-PGA with(1,2-diaminocyclohexane)platinum(II) (DACHPt) and DOX·HCl, respectively.43,44 These pepsomes achieved relatively highloading efficiency and loading content (over 10 wt %).Here, novel pH-responsive degradable chimaeric pepsomes

based on structurally well-defined poly(ethylene glycol)-b-poly(L-leucine)-b-poly(L-glutamic acid) (PEG-PLeu-PGA) co-

polymers were designed and developed for efficient encapsu-lation of DOX·HCl as well as activated drug release inside thetumor cells (Scheme 1). The molecular weight of PEG segmentwas devised higher than that of PGA segment so that PEGwould be primarily oriented at the outer periphery of pepsomeswarranting superb biocompatibility and shielding effect, whilePGA segment would be mostly located in the aqueous interiorof pepsomes affording high loading of DOX·HCl. Under theendosomal and lysosomal pH conditions, PGA would changefrom hydrophilic random coil structure into hydrophobic α-helical structure, resulting in destabilization and collapse ofpepsomes and enhanced release of loaded drug. The synthesisof pH-responsive degradable chimaeric pepsomes, loading andin vitro release of DOX·HCl as well as antitumor activity ofdrug-loaded pepsomes were investigated.

■ EXPERIMENTAL SECTIONMaterials. α-Methoxy-ω-amine-poly(ethylene glycol) (PEG-NH2,

Mn = 2.0 kg/mol, Jenkem Technology), L-leucine (GL Biochem(Shanghai) Ltd.), and Nε-benzyloxycarbonyl-L-glutamic acid (BLG,GL Biochem (Shanghai) Ltd.) were used as received. Tetrahydrofuran(THF) was dried by refluxing over sodium wire and distilled prior touse. N,N-Dimethylformamide (DMF) was dried with MgSO4 anddistilled under reduced pressure before use. Ethyl acetate andpetroleum ether (bp 60−90 °C) were refluxed with CaH2 anddistilled prior to use. Doxorubicin hydrochloride (DOX·HCl, 90%,Beijing Zhongshuo Pharmaceutical Technology Development Co.,Ltd.) was used as received. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4′6-diamidino-2-phenylindole dihydro-chloride (DAPI), and FITC-phalloidin were purchased from Sigmaand used as received. All the other reagents and solvents werepurchased from Sinopharm Chemical Reagent Co. Ltd. and used asreceived. L-Leucine N-carboxyanhydride (Leu-NCA) and γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) were synthesized accord-ing to the Fuchs-Farthing method using triphosgene.45,46

Characterization. 1H NMR spectra were recorded on a UnityInova 400 spectrometer operating at 400 M Hz using CF3COOD(TFA-d) as a solvent. The chemical shifts were calibrated against thesolvent signal. The molecular weight and polydispersity index of thecopolymers were determined by a Waters 1515 gel permeationchromatograph (GPC) instrument equipped with two linear PLgelcolumns (500 Å and Mixed-C) following a guard column and adifferential refractive-index detector. The measurements wereperformed using DMF as the eluant at a flow rate of 0.8 mL/min at40 °C and a series of narrow poly(methyl methacrylate)s standards forthe calibration of the columns. The size of pepsomes was determinedusing dynamic light scattering (DLS). Measurements were carried outat 25 °C by a Zetasizer Nano-ZS from Malvern Instruments equippedwith a 633 nm He−Ne laser using backscattering detection. The zetapotential of pepsomes was determined with a Zetasizer Nano-ZS from

Scheme 1. Illustration of Chimaeric Pepsomes Assembled from PEG-PLeu-PGA Asymmetric Triblock Copolymer for EfficientLoading and Activated Intracellular Release of DOX·HCl

Biomacromolecules Article

DOI: 10.1021/acs.biomac.5b00113Biomacromolecules 2015, 16, 1322−1330

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http://dx.doi.org/10.1021/acs.biomac.5b00113

Malvern Instruments. Transmission electron microscopy (TEM) wasperformed using a Tecnai G220 TEM operated at an acceleratingvoltage of 200 kV. The samples were prepared by dropping 10 μL of 1mg/mL pepsomes suspension on the copper grid followed by stainingwith 1 wt % phosphotungstic acid. Circular dichroism (CD) wasperformed in a quartz cuvette at 37 °C at a pepsome concentration of0.01 mg/mL using an AVIV 410 spectrophotometer (AVIVBiomedical Inc., Lakewood, NJ). CD spectra were recorded over awavelength range of 190−260 nm.Synthesis of PEG-PLeu-PBLG Triblock Copolymers. PEG-

PLeu-PBLG triblock polymers were synthesized by sequential ring-opening polymerization (ROP) of Leu-NCA and BLG-NCAmonomers in DMF using PEG-NH2 as an initiator. The following isa typical example on synthesis of PEG-PLeu52-PBLG copolymer.Under a nitrogen atmosphere, to a solution of Leu-NCA (392.5 mg,2.5 mmol) in DMF (8 mL) under stirring was quickly added a stocksolution of PEG-NH2 (100 mg, 0.05 mmol, monomer to initiator ratioof 50) in DMF (2 mL). After stirring at 35 °C for 72 h, a solution ofBLG-NCA (105.2 mg, 0.40 mmol) in DMF (2 mL) was added. Thepolymerization was allowed to proceed for another 72 h at 35 °C. Thecopolymer was isolated by precipitating in excess diethyl ether,purification through dissolving in dichloromethane and reprecipitatingin diethyl ether for twice, and drying at room temperature in vacuo for24 h. Yield: 90.0%. 1H NMR (400 MHz, TFA-d): δ 7.24 (s, 5H,-C6H5), 5.09 (s, 2H, C6H5CH2-), 4.67 (-COCHNH-), 3.88(-OCH2CH2O-), 3.55 (-OCH3), 2.46 (-CHCH2CH2CO-), 2.17,1.97 (-CHCH2CH2CO-), 1.72 (-CHCH2CH(CH3)2-), 1.62(-CHCH2CH(CH3)2-), 0.90 (-CHCH2CH(CH3)2-).Synthesis of PEG-PLeu-PGA Triblock Copolymers. In order to

remove the protecting group, PEG-PLeu52-PBLG (940 mg, 0.1 mmol)was dissolved in CF3COOH (9.4 mL), to which HBr/acetic acid (33wt %, 0.87 mL) was added. The reaction proceeded with stirring for 3h at 0 °C. The final product was isolated by precipitating in excess colddiethyl ether, redissolving in DMF and dialysis against distilled water,and freeze-drying. Yield: 88.0%. 1H NMR (400 M Hz, TFA-d): δ 4.67(-COCHNH-), 3.88 (-OCH2CH2O-), 3.55 (-OCH3), 2.63(-CHCH2CH2-), 2.28, 2.14(-CHCH2CH2-), 1.71 (-NHCHCH2CH-), 1.62 (-NHCHCH2CH-), 0.90 (-CH3).Preparation of Chimaeric Pepsomes. Chimaeric pepsomes

were prepared by dropwise addition of PB buffer to a DMF solution ofPEG-PLeu-PGA triblock copolymers under stirring at room temper-ature, followed by extensive dialysis against PB for 48 h using amembrane (MWCO 7000 Da). The medium was refreshed every 8 h.In order to verify their vesicular structure by confocal microscopy,pepsomes encapsulating Nile red were obtained through adding 20 μLof Nile red in acetone to the above chimaeric pepsomes (final Nile redconcentration = 1 × 10−6 M) followed by 3 h stirring at 37 °C toevaporate acetone. The confocal experiments were performed using aconfocal microscope (TCS SP2).The CAC was determined using pyrene as a fluorescence probe.

The concentration of polymer varied from 6.0 × 10−4 to 0.15 mg/mLand the concentration of pyrene was fixed at 0.6 μM. The fluorescencespectra were recorded using a FLS920 fluorescence spectrometer withthe excitation wavelength of 330 nm. The emission fluorescence at 372and 383 nm was monitored. The CAC was estimated as the cross-point when extrapolating the intensity ratio I372/I383 at low and highconcentration regions.Encapsulation of DOX·HCl. DOX·HCl-loaded pepsomes were

simply prepared by solvent exchange method. In a typical example, 0.1mL of PEG-PLeu-PGA copolymer solution in DMF was added to anaqueous solution of 0.9 mL of PB buffer and 0.063 mL of DOX·HClsolution (2 mg/mL) in PB at room temperature. After stirring for halfan hour, free DOX·HCl and DMF were removed by dialysis (MWCO7000 Da) against PB buffer for 48 h with at least 5 times change ofmedia. The whole procedure was performed in the dark.For determination of drug loading content, DOX·HCl-loaded

pepsomes were dissolved in DMF to release drug and analyzed withfluorescence spectroscopy (FLS 920, excitation at 480 nm). Thecalibration curve was obtained with DOX·HCl/DMF solutions withdifferent DOX concentrations. Drug loading content (DLC) and drug

loading efficiency (DLE) were calculated according to the followingformula:

= ×DLC(wt%)wt of loaded drug

total wt of polymer and loaded drug100%

= ×DLE(%)wt of loaded drugwt of drug in feed

100%

In Vitro Release of DOX·HCl. The release profiles of DOX·HClfrom chimaeric pepsomes were studied using a dialysis tube (MWCO12000) under shaking (100 rpm) at 37 °C in two different media, thatis, NaAc/HOAc (10 mM, pH 5.0) and PB (10 mM, pH 7.4). Therelease studies were performed at a pepsome concentration of 25 mg/L. Typically, 0.5 mL DOX·HCl-loaded pepsomes solution was dialyzedagainst 25 mL of release media. At certain time intervals, 5 mL ofrelease media was taken out and replenished with an equal volume offresh media. The amount of DOX·HCl released was determined byusing fluorescence (FLS920) measurement (excitation at 480 nm).The release experiments were conducted in triplicate and the resultspresented were the average data with standard deviations.

MTT Assays of Chimaeric Pepsomes. The antitumor activity ofDOX·HCl-loaded pepsomes was evaluated in RAW 264.7 cells andMCF-7/ADR cells by MTT assays. RAW 264.7 cells were cultured in a96-well plate (5 × 103 cells/well) using Dulbecco’s modified Eaglemedium. MCF-7/ADR cells were cultured in a 96-well plate (5 × 103

cells/well) using RPMI-1640 medium supplemented with 10% fetalbovine serum (FBS), 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 μg/mL) for 1 day. The medium wasaspirated and replaced by 80 μL of fresh medium supplemented with10% FBS. A total of 20 μL of DOX·HCl-loaded pepsomes in PB buffer(10 mM) was added to yield final pepsomes concentrations varyingfrom 1 × 10−3 to 20 μg/mL. The cells were cultured in an atmospherecontaining 5% CO2 for 2 day at 37 °C. The medium was aspirated andreplaced by 100 μL of fresh medium. A total of 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solu-tion (5 mg/mL) was added. The cells were incubated for another 4 h.The medium was aspirated, the MTT-formazan generated by live cellswas dissolved in 150 μL of DMSO, and the absorbance at a wavelengthof 490 nm of each well was measured using a microplate reader. Therelative cell viability (%) was determined by comparing the absorbanceat 490 nm with control wells containing only cell culture medium.Data are presented as average ± SD (n = 3). The cytotoxicity of blankpepsomes was determined in a similar way.

Intracellular Release of DOX·HCl. The cellular uptake andintracellular release behaviors of DOX·HCl-loaded pepsomes werefollowed with confocal laser scanning microscopy (CLSM) using RAW264.7 and MCF-7/ADR cells. RAW 264.7 cells were cultured in a 96-well plate (5 × 103 cells/well) using Dulbecco’s modified Eaglemedium. MCF-7/ADR cells were cultured on microscope slides in asix-well plate (5 × 105 cells/well) using RPMI-1640 mediumsupplemented with 10% FBS, 1% L-glutamine, antibiotics penicillin(100 IU/mL), and streptomycin (100 μg/mL). The cells wereincubated with DOX·HCl-loaded pepsomes in a humidified 5% CO2-containing atmosphere at 37 °C for 0.5−4 h. The culture medium wasremoved and the cells were rinsed three times with PBS. The cellnuclei were stained in blue with 4′,6-diamidino-2-phenylindole(DAPI), and the actin cytoskeleton was stained in green with FITC-phalloidin. The fluorescence images were obtained using confocalmicroscope (TCS SP2).

■ RESULTS AND DISCUSSIONSynthesis of PEG-PLeu-PGA Triblock Copolymers.

PEG-PLeu-PGA triblock copolymers were synthesized bysequential ROP of Leu-NCA and BLG-NCA monomersusing PEG-NH2 as an initiator followed by deprotection ofbenzyl groups. 1H NMR of PEG-PLeu-PBLG showedcharacteristic signals of PEG (δ 3.55 and 3.88), PLeu (δ 0.90,1.62, 1.72), and PBLG (δ 1.97, 2.17, 2.46, 5.09, 7.24; Figure



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1A). The degree of polymerization (DP) of PLeu and PBLGwas calculated by comparing the intensities of signals at δ 0.90

(methyl protons of PLeu) and δ 5.09 (benzyl methyleneprotons of PBLG) to δ 3.88 (methylene protons of PEG),respectively. The molecular weights determined by 1H NMRwere close to the design (Table 1). GPC measurements showedthat PEG-PLeu-PBLG copolymers had a low polydispersities of1.08−1.18. Moreover, the molecular weights determined fromGPC were close to those measured by 1H NMR, and increasedin parallel with the design. The benzyl groups in PEG-PLeu-PBLG copolymers were removed in CF3COOH with HBr/

HOAC at 0 °C for 3 h, generating PEG-PLeu-PGA copolymers.The disappearance of signals at δ 5.09 and 7.24 attributable tothe benzyl groups confirmed complete deprotection (Figure1B). Moreover, 1H NMR spectrum of PEG-PLeu-PGA revealedthat PLeu and PGA blocks had similar degrees of polymer-ization to their counterparts in the parent PEG-PLeu-PBLGcopolymer, indicating that polypeptide main chain was intactduring deprotection.

Formation and pH-Responsivity of Chimaeric Pep-somes. Chimaeric pepsomes were prepared from PEG-PLeu-PGA by solvent exchange method. Dynamic light scattering(DLS) measurements showed that PEG-PLeu-PGA copolypep-tides formed monodisperse pepsomes with low PDI of 0.15−0.17 and average hydrodynamic diameters from 64 to 71 nm forPEG-PLeu52-PGA (pepsome 1) and PEG-PLeu64-PGA (pep-some 2), respectively (Table 2). The small sizes of pepsomescould be attributed to the asymmetric structure of PEG-PLeu-PGA triblock copolypeptides that favors the formation of acurvature structure.23,47,48 TEM micrograph demonstrated thatPEG-PLeu-PGA chimaeric pepsomes had a vesicular structure,spherical morphology, and a size distribution close to thatdetermined by DLS (Figure 2A). The morphology ofpolypeptide-based block copolymer self-assembled structureswas recently reported to be not only dependent on themolecular composition, but also, to a larger extent, on thekinetic trapping of the system induced by the rigidity of the α-helical hydrophobic peptide segment during the solventdiffusion process.49 In addition, the intensity profiles of redfluorescence in a captured giant pepsome showed clearly thelocation of hydrophobic Nile Red in the membrane (Figure2B), further confirming their vesicular structure. PEG-PLeu-PGA pepsomes had negative surface charges (−24.5 ∼ −26.3mV), likely due to part of PGA chains located in the outerperiphery of pepsomes. Notably, these pepsomes revealed a lowcritical aggregation concentration (CAC) of 4.2−4.5 mg/L andexhibited excellent stability with little size change for over onemonth at physiological pH (pH 7.4) and 37 °C, which could beattribute to the α-helix assembly of PLeu in the membrane ofPEG-PLeu-PGA pepsomes.50

Interestingly, chimaeric pepsomes based on PEG-PLeu-PGAcollapsed under an acidic pH of 5.0, as revealed by TEM(Figure 2A), which is possibly due to a change of PGA blocksin pepsomes from hydrophilic random coil structure intohydrophobic α-helical structure upon decreasing pH.51 Thecircular dichroism (CD) analyses (Figure S2) revealed thatPEG-PLeu-PGA pepsomes in aqueous solution had obvious α-helical conformation belonging to PLeu blocks at pH 7.4. Thecontent of α-helix decreased rapidly when pH was lower than6.0 due to aggregation and precipitation of pepsomes. The DLSresults showed that these pepsomes swelled to over 1000 nm atpH 5.0 (Figure S3). Confocal laser scanning microscopy(CLSM) observations showed that the membrane of pepsomescontaining hydrophobic Nile Red was broken at pH 5.0 (Figure

Figure 1. 1H NMR spectra (400 MHz, CF3COOD) of PEG-PLeu52-PBLG (A) and PEG-PLeu52-PGA (B).

Table 1. Synthesis of PEG-PLeu-PBLG Triblock Copolymers

Mn (kg/mol)

entry copolymers design 1H NMRa GPCb Mw/Mnb yield (%)

1 PEG-PLeu52-PBLG 2−5.7−1.7 2−5.9−1.7 9600 1.18 902 PEG-PLeu64-PBLG 2−6.8−1.7 2−7.3−1.7 12400 1.08 91

aCalculated from 1H NMR (CF3COOD).bDetermined by GPC (eluent: DMF, standards: poly(methyl methacrylate)s, flow rate: 0.8 mL/min, 40

°C).



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2B), corroborating the pH-responsibility of PEG-PLeu-PGAchimaeric pepsomes.Loading and In Vitro Release of DOX·HCl. The loading

of DOX·HCl into PEG-PLeu-PGA pepsomes was performed bysolvent-exchange method. The results showed that thesechimaeric pepsomes had a high loading efficiency (DLE)ranging from 73.3 to 88.2% (Table 3), likely due to thepresence of ionic interactions between the carboxylic groups ofthe PGA blocks in the vesicular core and the amino groups ofDOX·HCl at neutral pH. Similar results were also observed inother polymersome systems that contained either carboxylic

acid or amino groups in the core.52,53 The DLE of pepsomesincreased slightly with increasing PLeu block lengths. Forexample, at a theoretical drug loading content (DLC) of 15 wt%, DLE of 80.7 to 84.4% were observed for pepsomes 1 and 2,respectively. DLS measurements exhibited that DOX·HCl-loaded pepsomes had small sizes of 57−87 nm and negativezeta potentials of −22.5 to −25.4 mV. The average sizes ofdrug-loaded pepsomes increased with increasing drug loadingcontents. For example, the average size of drug-loaded pepsome1 increased from 57 to 87 nm with increasing drug loadingcontents from 8.7 to 15.5 wt %.The in vitro release studies performed at 37 °C

demonstrated that DOX·HCl was released from chimaericpepsomes much faster at endosomal pH than at physiologicalpH (Figure 3). About 24.0 and 75.7% of drug was releasedfrom pepsome 1 at 37 °C in 24 h at pH 7.4 and 5.0,respectively. The prominent acceleration of drug release at pH5.0 could be attributed to a change of PGA protonation statethat results in disruption of pepsome structure as well asdecreased electrostatic interaction with DOX·HCl. In all cases,DOX·HCl was released in a controlled manner and initial burstdrug release was not observed. Under the same pH condition,DOX·HCl-loaded pepsome 1 displayed somewhat faster drugrelease than pepsome 2 counterparts, which is most likely dueto pepsome 2 having a thicker membrane than pepsome 1.These pH-responsive nanosystems are highly promising fortriggered intracellular drug delivery.54,55

Anti-Tumor Activity and Intracellular Drug ReleaseBehavior of DOX·HCl-Loaded Pepsomes. Due to theirexcellent biocompatibility, biodegradability, and unique hier-archical assembly properties, polypeptide-based nanocarriershave recently been developed and explored for the controlleddelivery of various anticancer drugs and proteins.56−58 Here,the antitumor activity of DOX·HCl-loaded pepsomes wasinvestigated in RAW 264.7 and MCF-7/ADR cells by MTTassays. The results showed that DOX·HCl-loaded pepsomeswere highly potent against RAW 264.7 cells, in which low half-maximal inhibitory concentrations (IC50) of 0.14 and 0.19 μg/mL, close to that of free DOX·HCl (0.11 μg/mL), wereobserved for DOX·HCl-loaded pepsome 1 and pepsome 2,respectively (Figure 4). Importantly, MTT assays indicated thatPEG-PLeu-PGA pepsomes were practically nontoxic in RAW

Scheme 2. Synthesis of PEG-PLeu-PGA CopolymerA

AReagents and conditions: (i) Leu-NCA, DMF, 35 °C, 72 h; (ii) BLG-NCA, DMF, 35 °C, 72 h; (iii) HBr/HOAc, CF3COOH, 0 °C.

Table 2. Preparation of Chimaeric PEG-PLeu-PGAPepsomes

entry pepsomes sizea (nm) PDIa ζ (mV) CACb (mg/L)

1 PEG-PLeu52-PGA 64 0.17 −24.5 4.52 PEG-PLeu64-PGA 71 0.15 −26.3 4.2

aSize and PDI of pepsomes were determined by DLS. bDetermined byfluorescence measurement.

Figure 2. Characterization of chimaeric pepsome 1 at pH 7.4 and 5.0:(A) TEM images; (B) CLSM micrograph of a giant pepsomeencapsulating Nile Red.



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264.7, MCF-7/ADR and L929 cells up to a testedconcentration of 0.5 mg/mL (Figure 5), supporting thatthese chimaeric pepsomes possess good biocompatibility.Nanoparticles normally enter cells through endocytosis, and

while contained in the endosomal compartment, they undergoan environmental pH decrease from early to late endo-somes.59,60 To examine the cellular uptake and intracellulardrug release behaviors, we treated RAW 264.7 cells with DOX·HCl-loaded pepsomes and investigated the intracellulartrafficking of DOX·HCl using confocal laser scanningmicroscopy (CLSM). The cell nuclei were stained with DAPI(blue). Remarkably, significant DOX·HCl fluorescence wasobserved in the nuclei of RAW 264.7 cells following 0.5 hincubation with DOX·HCl-loaded pepsome 1 (Figure 6A),indicating fast internalization of pepsomes and efficient releaseof DOX·HCl inside cells. Longer incubation time (e.g., 1 h, 2h) resulted in even stronger DOX·HCl fluorescence in thenuclei of RAW 264.7 cells (Figure 6B,C), which wascomparable to the cells incubated with free drug underotherwise the same conditions (Figure 6D). In addition, the

bright-field images indicated that RAW 264.7 cells underwentapoptosis following 2 h incubation with DOX·HCl-loadedpepsomes (Figure 6C). These results confirm that pH-responsive degradable chimaeric pepsomes can efficiently loadand deliver DOX·HCl into tumor cells, achieving highantitumor efficacy.Drug resistance, which accounts for a majority of cancer

treatment failure, poses a major hurdle in cancer chemo-therapy.61 Here, the antitumor activity of DOX·HCl-loadedpepsomes against drug resistant MCF-7/ADR cells wasinvestigated. In line with their high drug resistance, MCF-7/ADR cells following 48 h treatment with free DOX·HCl at aconcentration of 20 μg/mL had a cell viability of over 70%(Figure 7). In contrast, DOX·HCl-loaded pepsomes wereshown to effectively inhibit growth of MCF-7/ADR cells, inwhich IC50 of DOX·HCl-loaded pepsome 1 and pepsome 2 wasdetermined to be 6.67 and 10.9 μg/mL, respectively (Figure 7).The cellular uptake and intracellular drug release behaviors ofDOX·HCl-loaded pepsomes in MCF-7/ADR cells were studiedusing CLSM. DAPI (blue) and FITC-phalloidin were employedto stain the cell nuclei and cytoskeletons, respectively. Notably,obvious drug fluorescence was observed in the cytosol and cell

Table 3. Characterization of DOX·HCl-Loaded PEG-PLeu-PGA Pepsomes (0.5 mg/mL) in Phosphate Buffer

DLC (wt %)

pepsomes theory determineda DLE (%) sizeb (nm) PDIb ζ (mV)

pepsome 1 (PEG-PLeu52-PGA) 10 8.7 85.6 57 0.24 −22.515 12.5 80.7 72 0.23 −24.720 15.5 73.3 87 0.24 −23.1

pepsome 2 (PEG-PLeu64-PGA) 10 8.9 88.2 65 0.26 −23.115 12.9 84.4 79 0.25 −25.320 15.8 74.8 87 0.23 −25.4

aDetermined by fluorescence measurement. bSize and PDI of DOX·HCl-loaded pepsomes were determined by DLS.

Figure 3. pH-dependent release of DOX·HCl from PEG-PLeu-PGApepsomes at 37 °C.

Figure 4. Antitumor activity of DOX·HCl-loaded PEG-PLeu-PGApepsomes. RAW 264.7 cells were incubated with DOX·HCl-loadedpepsomes or free DOX·HCl for 48 h. The cell viability was determinedby MTT assays (n = 3).

Figure 5. Cytotoxicity of bare pepsomes determined by MTT assays inRAW 264.7, MCF-7/ADR and L929 cells. (A) Pepsome 1; (B)Pepsome 2. The cells were incubated with pepsomes for 48 h. Data arepresented as the average ± standard deviation (n = 3).



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nuclei of MCF-7/ADR cells following 1 h incubation withDOX·HCl-loaded PEG-PLeu-PGA pepsomes, indicating fastinternalization of DOX·HCl-loaded pepsomes and efficientrelease of DOX·HCl in MCF-7/ADR cells (Figure 8A). DOX·HCl fluorescence in the nuclei of MCF-7/ADR cells becamestronger at prolonged incubation times of 2 and 4 h (Figure8B,C). In contrast, little DOX·HCl fluorescence was observedfor MCF-7/ADR cells treated with free drug under otherwisethe same conditions (Figure 8D), likely due to P-glycoproteinmediated resistance in MCF-7/ADR cells.62,63

■ CONCLUSIONSWe have demonstrated that pH-responsive chimaeric pepsomesbased on PEG-PLeu-PGA triblock copolymer are able toactively load and efficiently deliver hydrophilic anticancer drugslike DOX·HCl into RAW 264.7 cells and drug-resistant MCF-7/ADR cells, achieving potent antitumor effects. Thesemultifunctional pepsomes have appeared as an attractivealternative to liposomes for targeted delivery of water-solubleanticancer drugs. They exhibit following several advantages: (i)they are readily formed with small sizes and definedmorphology from asymmetric PEG-PLeu-PGA triblock copo-lypeptides. PEG-PLeu-PGA can be conveniently prepared withcontrolled molecular weights and low polydispersities bysequential NCA polymerization followed by acid deprotection;(ii) they display remarkably high loading contents and loadingefficiencies toward hydrophilic anticancer drugs. Notably, todate, there are few nanosystems that can efficiently encapsulatewater-soluble drugs; (iii) they can be rapidly destabilized atendosomal pH, achieving efficient intracellular release of DOX·HCl and high antitumor activity in both RAW 264.7 and MCF-7/ADR cells; and (iv) they are intrinsically stealthed by PEG,which not only endows excellent biocompatibility but alsofacilitates design of active targeting systems. These chimaericpepsomes with facile synthesis, efficient loading of water-soluble anticancer drugs, and triggered intracellular drug releasebehavior have appeared as an attractive platform for targetedcancer chemotherapy.

Figure 6. CLSM images of RAW 264.7 cells incubated with DOX·HCl-loaded chimaeric pepsomes and free DOX·HCl (dosage 10 μg/mL). For each panel, images from left to right show cell nuclei stainedby DAPI (blue), DOX·HCl fluorescence in cells (red), bright-fieldimages of cells, and overlays of three images. The scale barscorrespond to 10 μm in all the images. (A) DOX·HCl-loadedpepsome 1, 0.5 h incubation; (B) DOX·HCl-loaded pepsome 1, 1 hincubation; (C) DOX·HCl-loaded pepsome 1, 2 h incubation; and(D) free DOX·HCl, 2 h incubation.

Figure 7. Antitumor activity of DOX·HCl-loaded PEG-PLeu-PGApepsomes. MCF-7/ADR cells were incubated for 48 h with DOX·HCl-loaded pepsomes or free DOX·HCl. The cell viability was determinedby MTT assays (n = 3).

Figure 8. CLSM images of MCF-7/ADR cells incubated with DOX·HCl-loaded chimaeric pepsomes and free DOX·HCl (dosage 20 μg/mL). For each panel, images from left to right show cell nuclei stainedby DAPI (blue), DOX·HCl fluorescence in cells (red), cytoskeletonstained by FITC-phalloidin (green) and overlays of three images. Thescale bars correspond to 12 μm in all the images. (A) DOX·HCl-loaded pepsome 1, 1 h incubation; (B) DOX·HCl-loaded pepsome 1,2 h incubation; (C) DOX·HCl-loaded pepsome 1, 4 h incubation; and(D) free DOX·HCl, 4 h incubation.



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■ ASSOCIATED CONTENT*S Supporting InformationGPC curves of PEG-PLeu-PBLG triblock copolymers, and CDand DLS results of pepsomes in response to change of pH. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*Tel./Fax: +86-512-65880098. E-mail: [email protected].*E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (NSFC 51273137, 51273139, and51473110), the National Science Fund for DistinguishedYoung Scholars (NSFC 51225302), Jiangsu Specially-Ap-pointed Professor Program, and a Project Funded by thePriority Academic Program Development of Jiangsu HigherEducation Institutions.

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