Acta Biomaterialia 10 (2014) 2159–2168

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Acta Biomaterialia

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Reduction and pH dual-bioresponsive crosslinked polymersomesfor efficient intracellular delivery of proteins and potent inductionof cancer cell apoptosis

http://dx.doi.org/10.1016/j.actbio.2014.01.0101742-7061/� 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel./fax: +86 512 65880098.E-mail addresses: fhmeng@suda.edu.cn (F. Meng), zyzhong@suda.edu.cn

(Z. Zhong).

Huanli Sun, Fenghua Meng ⇑, Ru Cheng, Chao Deng, Zhiyuan Zhong ⇑Biomedical Polymers Laboratory and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering andMaterials Science, Soochow University, Suzhou 215123, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 July 2013Received in revised form 21 December 2013Accepted 8 January 2014Available online 15 January 2014

Keywords:PolymersomesDual-sensitiveCrosslinkingProtein deliveryCancer therapy

The clinical applications of protein drugs are restricted because of the absence of viable protein deliveryvehicles. Here, we report on reduction- and pH–sensitive crosslinked polymersomes based on thepoly(ethylene glycol)–poly(acrylic acid)–poly(2-(diethyl amino)ethyl methacrylate) (PEG–PAA–PDEA)triblock copolymer for efficient intracellular delivery of proteins and the potent induction of cancer cellapoptosis. PEG–PAA–PDEA (1.9–0.8–8.2 kg mol�1) was synthesized by controlled reversible addition-fragmentation chain transfer polymerization and further modified with cysteamine to yield the thiol-containing PEG–PAA(SH)–PDEA copolymer. PEG–PAA(SH)–PDEA was water-soluble at acidic and physio-logical pH but formed robust and monodisperse polymersomes with an average size of �35 nm uponincreasing the pH to 7.8 or above followed by oxidative crosslinking. These disulfide-crosslinked poly-mersomes, while exhibiting excellent colloidal stability, were rapidly dissociated in response to 10 mMglutathione at neutral or mildly acidic conditions. Notably, these polymersomes could efficiently loadproteins like bovine serum albumin and cytochrome C (CC). The in vitro release studies revealed that pro-tein release was fast and nearly quantitative under the intracellular-mimicking reducing environment.Confocal microscopy observations showed that these dual-sensitive polymersomes efficiently releasedfluorescein isothiocyanate-CC into MCF-7 cells in 6 h. Most remarkably, MTT assays showed that CC-loaded dual-sensitive polymersomes induced potent cancer cell apoptosis, in which markedly decreasedcell viabilities of 11.3%, 8.1% and 52.7% were observed for MCF-7, HeLa and 293T cells, respectively, at aCC dosage of 160 lg ml�1. In contrast, free CC caused no cell death under otherwise the same conditions.These dual-bioresponsive polymersomes have appeared as a multifunctional platform for active intracel-lular protein release.

� 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

The clinical applications of protein drugs are restricted by theabsence of viable protein delivery vehicles [1–7]. Recently, poly-mersomes with large aqueous compartments that can accommo-date and protect proteins from degradation have emerged as oneof the most ideal protein nanocarriers [8–13]. For instance, poly-mersomes have been explored for use in the loading and controlleddelivery of various proteins, including insulin [12,14,15], hemoglo-bin [16,17], myoglobin [18], immunoglobulin G [19], ovalbumin[20], aquaporin Z [21] and cytochrome C (CC) [22]. These polymer-somes, however, in general exhibit low protein loading capacity

and involve the use of organic solvents that might lead to the dena-turation or deactivation of proteins. In recent years, chimaericpolymersomes [23,24] and pH-sensitive polymersomes [25–27]that possess polyion blocks have been developed for efficient load-ing of proteins under aqueous conditions.

It should further be noted that polymersomes, like other self-assembled systems, are challenged by the stability dilemma, i.e.they tend to dissociate or aggregate as a result of extensive dilutionand/or massive interaction with blood pool, thus leading to prema-ture drug release and low tumor targetability [28,29]. The stabilityof polymersomes could be improved by using a variety of cross-linking strategies [30–34]. However, overly stable polymersomesare not optimal due to drug release being restricted in the tumorcells, resulting in a compromised therapeutic effect. We recentlyreported that disulfide-crosslinked thermosensitive polymersomesbased on the poly(ethylene glycol)–poly(acrylic acid)–poly(N-iso-

2160 H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168

propylacrylamide) (PEG–PAA–PNIPAM) triblock copolymer,though displaying excellent colloidal stability with restrainedprotein release under physiological conditions, underwent a rapiddisassembly and protein release behavior in an intracellular-mim-icking reductive environment and in cancer cells [35,36]. In recentyears, disulfide-crosslinking that is prone to cleavage in anintracellular environment, due to the existence of a high reducingpotential in the cytoplasm and cell nucleus, has appeared as a mostelegant approach to address stability and the intracellular releasedilemma [37–40].

In this paper, we report on reduction and pH dual–biorespon-sive disulfide-crosslinked polymersomes based on the water-solu-ble poly(ethylene glycol)–poly(acrylic acid)–poly(2-(diethylamino)ethyl methacrylate) triblock copolymer thiol derivative(PEG–PAA(SH)–PDEA) for efficient intracellular delivery of proteinsand potent induction of cancer cell apoptosis (Scheme 1). In recentyears, dual-stimuli responsive nanocarriers have emerged as ad-vanced systems for better controlled drug delivery [41]. PEG andPAA are among the few polymers approved by the US Food andDrug Administration for use in biomedical devices [42], whilePDEA with pH-sensitivity and proton sponge effect has beenwidely investigated for biomedical applications [43–45]. The pen-dant thiol groups undergo facile crosslinking via oxidative reactionthrough exposure in air. Notably, both polymersome formation andthe crosslinking process do not involve any organic solvents, cata-lysts and byproducts. Here, the preparation of reduction- and pH–bioresponsive disulfide-crosslinked polymersomes, loading andreduction/pH triggered release of proteins and intracellular proteinrelease behaviors, as well as the anti-tumor activity of CC-loadedcrosslinked polymersomes, were investigated.

2. Experimental section

2.1. Materials

Methoxy PEG (Mn = 1.9 kg mol�1, Fluka) was dried by azeotro-pic distillation from toluene. 2,20-azobisisobutyronitrile (AIBN,98%, J&K) was recrystallized twice from methanol. Acrylic acid

Scheme 1. Illustration of reduction- and pH-bioresponsive disulfide-crosslinked polymtriggered intracellular release of proteins.

(AA, 99%, Alfa Aesar) was distilled before use. 2-(diethyl-amino)ethyl methacrylate) (DEA, 99%, Aldrich) was purified bypassing through a basic alumina column. 4-cyanopentanoic aciddithionaphthalenoate (CPADN) was synthesized according to thedescribed procedure for 4-cyanopentanoic acid dithiobenzoate[46]. PEG-CPADN was synthesized by an amidation reaction ofPEG-NH2 and CPADN, similar to the synthesis of PEG-SS-CPADNmacro-reversible addition-fragmentation chain transfer (RAFT)agent [26]. Cystamine dihydrochloride (cystamine�2HCl, >98%, AlfaAesar), triethylamine (Et3N, 99%, Alfa Aesar), dicyclohexyl carbodi-imide (DCC, 99%, Alfa Aesar), N-hydroxysuccinimide (NHS, 98%,Alfa Aesar), dithiothreitol (DTT, 99%, Merck), glutathione (GSH,99%, Roche), 5,50-dithiobis(2-nitrobenzonic acid) (DTNB, 99%, AlfaAesar), fluorescein isothiocyanate (95%, Fluka), CC from equineheart (Sigma), and bovine serum albumin (BSA) V fraction (>98%,Roche) were used as received.

2.2. Characterization

Proton nuclear magnetic resonance (1H NMR) spectra were re-corded on a Unity Inova 400 spectrometer operating at 400 MHzusing deuterated water (D2O) as a solvent. The chemical shiftswere calibrated against residual solvent signals of D2O. The molec-ular weight and polydispersity (PDI) of copolymers were deter-mined with a Waters 1515 gel permeation chromatography(GPC) instrument equipped with three ultra-hydrogel columns fol-lowing an INLINE precolumn and a differential refractive-indexdetector. The measurements were performed using 0.3 M NaAcaqueous solution (pH 4.4) as the eluent at a flow rate of0.5 ml min�1 at 30 �C and a series of narrow poly(ethylene oxide)standards for calibration of the columns. The size and size distribu-tion of polymersomes were determined using dynamic light scat-tering (DLS). Measurements were carried out at 25 �C using aZetasizer Nano-ZS from Malvern Instruments equipped with a633 nm He–Ne laser using back-scattering detection. Transmissionelectron microscopy (TEM) was performed using a Tecnai G220TEM operated at an accelerating voltage of 120 kV. The sampleswere prepared by dropping 10 ll of polymersomes dispersion

ersomes based on PEG–PAA(SH)–PDEA triblock copolymer for efficient loading and

H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168 2161

(0.2 mg ml�1) on the copper grid followed by staining with phos-photungstic acid. The images of polymersomes and cellular uptakewere taken on a confocal laser scanning microscope (CLSM; TCSSP2).

2.3. Synthesis of PEG–PAA–PDEA triblock copolymer via RAFTpolymerization

The PEG–PAA–PDEA triblock copolymer was synthesized viasequential RAFT polymerization of AA and DEA using PEG-CPADNas a macro-RAFT agent and AIBN as a radical source. Briefly, to aSchlenk reaction vessel equipped with a magnetic stirrer wascharged with PEG-CPADN (0.20 g, 0.087 mmol), AIBN (1.5 mg,0.009 mmol), AA (0.075 g, 1.045 mmol) and dioxane (2.3 ml). After30 min of degassing with a nitrogen flow, the reaction vessel wassealed and immersed in an oil bath thermostated at 70 �C. Thepolymerization was allowed to proceed for 48 h and then cooledto room temperature (RT). A sample was taken to determine theAA conversion and content by 1H NMR. Subsequently, AIBN(1.8 mg, 0.011 mmol), DEA (1.05 g, 5.642 mmol) and dioxane(1.9 ml) were charged into the reaction system at RT under a nitro-gen atmosphere. After degassing for 15 min, the reaction vesselwas sealed and immersed in an oil bath thermostated at 70 �Cagain and the polymerization was allowed to proceed for 72 h.The resulting PEG–PAA–PDEA copolymer was isolated by precipi-tation in cold n-hexane, filtered and dried in vacuo followed by fur-ther purification through dissolving in deionized water and dialysisagainst deionized water (molecular weight cutoff (MWCO) 3500,pH 3.5). The final product was dried by lyophilization for 48 h.Yield: 80%. 1H NMR (400 MHz, D2O): 3.66 (m, PEG methyleneprotons), 3.38 (s, CH3O–), 0.9–1.26 (methyl of PDEA); 1.91 (s,–CH2C(CH3)–); 2.80, (–N(–CH2CH3)2), 3.02, (–OCH2CH2N–); 4.18(–OCH2CH2N–)). Mn (1H NMR) = 1.9–0.8–8.2 kg mol�1, Mn (GPC) =12.9 kg mol�1, PDI = 1.43.

2.4. Synthesis of thiol-containing PEG–PAA(SH)–PDEA triblockcopolymer

The thiol-containing PEG–PAA(SH)–PDEA triblock copolymerwas prepared by treating PEG–PAA–PDEA with cystamine followedby reduction with DTT. Briefly, under a nitrogen atmosphere, PEG–PAA–PDEA (0.312 g, 0.029 mmol), DCC (0.177 g, 0.858 mmol) andNHS (0.050 g, 0.434 mmol) were dissolved in 15 ml dimethyl sulf-oxide (DMSO) and reacted for 24 h at RT. Then a DMSO solution ofcystamine dihydrochloride (0.322 g, 1.430 mmol) and Et3N(0.306 g, 3.024 mmol) was added under a nitrogen atmosphere.The reaction was allowed to proceed for 24 h. The cystamine-mod-ified copolymer was recovered by dialysis against DMSO anddeionized water, respectively. The product was dried by lyophiliza-tion for 48 h.

Under a nitrogen atmosphere, cystamine-modified PEG–PAA–PDEA (0.08 g) and DTT (0.15 g, 0.2 M) were dissolved in 5 ml waterand stirred for 24 h at RT. The thiol-containing PEG–PAA(SH)–PDEA copolymer was isolated via dialysis against deionized water(MWCO 3500) under a nitrogen atmosphere followed by freeze-drying for 48 h. The thiol content of the PEG–PAA(SH)–PDEAcopolymer was determined by the Ellman test.

2.5. Formation and crosslinking of polymersomes

The polymersomes were prepared via adjusting an acidic solu-tion of the PEG–PAA(SH)–PDEA copolymer (pH 3.0, 1 mg ml�1) topH 7.8 or above using 0.1 M NaOH. The average sizes of polymer-somes at different pHs were determined by DLS. To obtain disul-fide-crosslinked polymersomes, polymersome dispersion wasincubated at 37 �C under constant shaking for 5 h before dialysis

against phosphate buffer (PB, 10 mM, pH 8.0). The colloidal stabil-ity of disulfide-crosslinked polymersomes against large volumedilution, high salt concentration and adding organic solvent wasinvestigated via DLS.

2.6. CLSM observation of polymersomes co-loaded with Nile red andfluorescein isothiocyanate (FITC)

In order to verify the vesicular structure, polymersomes weremade as described above in the presence of FITC. After extensivedialysis (MWCO 3500 Da) to remove free FITC, 20 ll of Nile redin acetone was added to give a final Nile red concentration of10�6 M. The solution was stirred for 3 h at 37 �C to evaporate ace-tone. CLSM images were taken using a confocal microscope (TCSSP2).

2.7. Reduction- and pH-triggered destabilization of disulfide-crosslinked polymersomes

The size change of dual-bioresponsive crosslinked polymersomes inresponse to mildly acidic and/or reductive conditions was followed byDLS measurements. Briefly, disulfide-crosslinked polymersome disper-sion was divided into five aliquots of 0.5 ml, which were adjusted intofive different conditions: (i) PB (10 mM, pH 8.0), (ii) PB (10 mM, pH7.4) with 0.04 mM GSH, (iii) PB (10 mM, pH 7.4) with 10 mM GSH, (iv)acetate buffer (10 mM, pH 5.0) with 0.04 mM GSH and (v) acetate bufferwith 10 mM GSH. The disulfide-crosslinked polymersome dispersionwas placed in a shaking bed at 200 rpm and 37 �C. The sizes of polymer-somes were monitored in time by DLS.

2.8. Encapsulation of FITC-labeled proteins

Protein-loaded crosslinked polymersomes were readily prepared byadjusting the pH of PEG–PAA(SH)–PDEA and protein aqueous solution atpH 6.0–8.0 followed by incubation at 37 �C under constant shaking for5 h and extensive dialysis (MWCO 500 kDa) against PB (10 mM, pH8.0) to remove free proteins. In brief, FITC-labeled CC (FITC–CC) or BSA(FITC–BSA) solution in water was added to PEG–PAA(SH)–PDEA copoly-mer solutions in deionized water at pH 6.0 (polymer concentration1 mg ml�1) to obtain protein/polymer weight ratios of 5–50 wt.%. Thesolution pH was adjusted to 8.0 followed by shaking at 37 �C for 5 h topromote oxidative crosslinking. Free proteins were removed by dialysisagainst PB (10 mM, pH 8.0) for 20 h at RT in the dark with a minimum offive changes of media.

To determine protein loading content (PLC) and protein loadingefficiency (PLE), thus-prepared protein-loaded disulfide-crosslinked polymersomes were disrupted by treating with10 mM DTT for 0.5 h, adjusting the pH to �2.5 using 0.5 M HCl,dilution to 3 ml using acetate buffer (10 mM, pH 4.0) and shakingat 37 �C for 4 h to ensure the complete release of proteins. Theamount of FITC-labeled proteins was determined using fluores-cence measurements (FLS920, excitation at 492 nm, emission at518 nm) based on the calibration curve with known concentrationsof FITC-labeled proteins. PLC and PLE were calculated according tothe following formula:

PLCðwt:%Þ¼ ðweight of loaded protein=weight of copolymerÞ� 100%

PLEð%Þ¼ ðweight of loaded protein=weight of protein in feedÞ� 100%

2.9. In vitro release of FITC-labeled proteins

The in vitro release of FITC–BSA and FITC–CC from disulfide-crosslinked polymersomes was investigated using a dialysis meth-od (MWCO 500 kDa) at 37 �C under four different conditions, i.e.

2162 H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168

phosphate buffered saline (PBS) buffer (pH 7.4, 10 mM, 150 mMNaCl) with 0.04 mM DTT, PBS buffer with 10 mM DTT, acetate buf-fer (pH 5.0, 10 mM, 150 mM NaCl) with 0.04 mM DTT and acetatebuffer with 10 mM DTT. Briefly, 0.5 ml of protein-loaded disulfide-crosslinked polymersome dispersion in PB (10 mM, pH 8.0) wasdialyzed (MWCO 500 kDa) against 20 ml of the above release med-ia. At desired time intervals, 6 ml of release media was taken outand replenished with an equal volume of corresponding fresh med-ia. The amounts of released proteins as well as proteins remainingin the dialysis tube were determined by fluorescence measure-ments (FLS920, excitation at 492 nm, emission from 500 to600 nm). The release experiments were conducted in triplicate,and the results presented are the average data with standarddeviations.

2.10. Confocal microscopy observation of MCF-7 cells incubated withFITC–CC loaded disulfide-crosslinked polymersomes

MCF-7 cells were plated on microscope slides in a 24-well plate(5 � 104 cells per well) under 5% CO2 atmosphere at 37 �C usingDulbecco’s modified Eagle’s medium (DMEM) supplemented with10% fetal bovine serum (FBS), 1% L-glutamine, antibiotics penicillin(100 IU ml�1), and streptomycin (100 lg m–1) for 24 h. 50 ll ofFITC–CC loaded disulfide-crosslinked polymersomes or free FITC–CC (FITC–CC dosage: 160 lg ml�1) was added. After incubationfor 3 or 6 h, the culture medium was removed and the cells onmicroscope plates were washed three times with PBS. The cellswere fixed with 4% paraformaldehyde for 15 min and washed threetimes with PBS. The cell nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI, blue) for 15 min and washed three times withPBS. Images of cells were obtained using a Nikon Digital EclipseC1si CLSM (Nikon).

2.11. MTT assay

The anti-tumor activity of CC-loaded disulfide-crosslinked PEG–PAA(SH)–PDEA polymersomes was evaluated in HeLa, MCF-7 and293T cells via MTT assays. The cells were seeded in a 96-well plateat a density of 1 � 104 cells per well in 90 ll of DMEM supple-mented with 10% FBS, 1% L-glutamine, antibiotics penicillin(100 IU ml�1), and streptomycin (100 lg ml�1) for 24 h. The med-ium was aspirated and replaced by 90 ll of fresh medium. 10 llof CC-loaded disulfide-crosslinked polymersomes or free CC in PB(10 mM, pH 8.0) was added to yield final CC concentration of 80and 160 lg m–1. The cells were cultured for another 48 h, and10 ll of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbro-mide (MTT) solution in PBS (5 mg ml�1) was added. The cells wereincubated for 4 h. The medium was aspirated, the MTT-formazangenerated by live cells was dissolved in 150 ll of DMSO and theabsorbance at a wavelength of 570 nm of each well was measuredusing a BioTek microplate reader. The relative cell viability (%) wasdetermined by comparing the absorbance at 570 nm with controlwells containing only cell culture medium. Data are presented asaverage ± SD (n = 4). The cytotoxicity of blank disulfide-crosslinkedpolymersomes was determined in a similar way.

3. Results and discussion

3.1. Synthesis of water soluble PEG–PAA(SH)–PDEA triblock copolymer

The thiol-containing PEG–PAA(SH)–PDEA triblock copolymerwas obtained in two steps (Scheme 2). Firstly, the PEG–PAA–PDEAtriblock copolymer was synthesized in one pot via sequential RAFTpolymerization of AA and DEA in dioxane at 70 �C using PEG-CPADN (Mn = 1.9 kg mol�1) as a macro-RAFT agent and AIBN as a

radical source. CPADN is a versatile RAFT agent, through whichwe have prepared well-defined block copolymers including PEG–P(HEMA-co-AC) [47], PDMA–PCL–PDMA [48] and PEG–PHPMA[49]. The 1H NMR spectrum of crude polymerization productshowed that conversion of AA monomer was nearly complete(>98%) after 2 days of polymerization as revealed by negligible sig-nals at d 5.93–6.35 attributable to the residual vinyl protons of AAand that PAA had a degree of polymerization (DP) of 11. The PEG–PAA–PDEA triblock copolymer was obtained with a final yield of�80%. 1H NMR also displayed (besides peaks attributable to PEG(d 3.64) and PAA (d 1.64 and 2.38)) signals at d 0.91, 1.18, 1.91,2.91, 3.16 and 4.23 assignable to the PDEA block (Fig. 1). The Mn

of the PDEA block was calculated to be 8.2 kg mol�1 by comparingthe integrals of signals at d 4.23 (PDEA methylene protons next tothe ester group) and 3.64 (PEG methylene protons). GPC measure-ments revealed that the PEG–PAA–PDEA block copolymer had aunimodal distribution with an Mn of 12.9 kg mol�1 and a moderatepolydispersity index of 1.43, confirming successful synthesis of thePEG–PAA–PDEA copolymer. Then, the PEG–PAA–PDEA copolymerwas reacted with cystamine via carbodiimide chemistry followedby treating with DTT and extensive dialysis under a nitrogenatmosphere, to furnish the thiol-containing PEG–PAA(SH)–PDEAtriblock copolymer. The Ellman test showed that PEG–PAA(SH)–PDEA had on average about eight free thiol groups per polymerchain. Taking the 1H NMR results, a PEG content of 17.4 wt.% wasdetermined for the PEG–PAA(SH)–PDEA triblock copolymer.

3.2. Formation of reduction- and pH–sensitive crosslinkedpolymersomes

The PEG–PAA(SH)–PDEA copolymer could be readily dissolvedin water at physiological pH or below (pH 6 7.4) with a hydrody-namic diameter of 6.9–8.4 nm (Fig. 2A). However, upon increasingsolution pH, a sharp transition was observed at pH 7.4–7.8, where-in nanoparticles with an average size of �36 nm were formed dueto deprotonation of the PDEA block (Fig. 2A). The size of nanopar-ticles changed little up to pH 9.0. Interestingly, upon decreasing pHusing 0.1 M HCl, these nanoparticles maintained their sizes frompH 9.0 to 7.2 and swelled to �110 nm, rather than dissolution,upon further decreasing the pH to 2.8 (Fig. 2A), indicating thatthese nanoparticles have been crosslinked by air during the pHadjusting process. In comparison, under a nitrogen or reducingenvironment, thus-formed nanoparticles were disassembled intounimers upon decreasing the pH to 2.8. In the following studies,crosslinked nanoparticles were prepared via increasing the pH ofPEG–PAA(SH)–PDEA solution from 3.0 to 8.0 followed by shakingat 37 �C for 5 h and extensive dialysis for 20 h. DLS showed thatcrosslinked nanoparticles had an average size of 33 nm with alow PDI of 0.16, and swelled to �80 nm after adjusting the pH to2.5 (Fig. 2B). The PEG–PAA(SH)–PDEA block copolymer had a lowPEG content of 17.4 wt.%, which favors the formation of vesicularstructures [10,50]. TEM micrography confirmed that these nano-particles had a vesicular structure with a mean particle size closeto that determined by DLS (Fig. 2C). Moreover, CLSM observationof giant crosslinked polymersomes co-loaded with Nile red (hydro-phobic) and FITC (hydrophilic) clearly showed that Nile red wasdistributed uniformly in the membrane while FITC was distributedin the aqueous lumen, in accordance with a vesicular structure(Fig. 2D). It is evident that polymersomes are readily formed fromthe PEG–PAA(SH)–PDEA triblock copolymer.

The stability of the disulfide-crosslinked polymersomes againstlarge volume dilution, high salt concentration and organic solventswere investigated via DLS measurements. The results showed thatcrosslinked polymersomes were stable against 100-fold dilutionwith PB (Fig. 3A) as well as against a high salt concentration ofup to 2 M NaCl (Fig. 3B). Notably, these crosslinked polymersomes

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Scheme 2. Synthesis of PEG–PAA(SH)–PDEA triblock block copolymer. Conditions: (i) AIBN, dioxane, 70 �C, 48 h; (ii) AIBN, dioxane, 70 �C, 72 h; (iii) DCC, NHS, DMSO, 30 �C,48 h; and (iv) H2O, RT, 24 h.

H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168 2163

maintained their structure and swelled to �70 nm following theaddition of a 10-fold volume of dioxane (Fig. 3C). These resultsdemonstrated that disulfide-crosslinked PEG–PAA(SH)–PDEA poly-mersomes have excellent colloidal stability. Interestingly, in thepresence of 10 mM GSH at pH values of either 7.4 or 5.0, polymer-somes were dissociated into unimers (less than 10 nm) in 1.7 h(Fig. 3D), indicating that crosslinked PEG–PAA(SH)–PDEA polymer-somes are prone to fast de-crosslinking and dissociation under theintracellular reductive condition. The resulting water-soluble poly-mers might be excreted from the body. In comparison, crosslinkedpolymersomes only swelled to 41 and 65 nm at pH values of 7.4and 5.0, respectively, after 14 h in the presence of 0.04 mM GSH(mimicking the extracellular environment). It should be noted thatcrosslinking density could have an influence on the stability andsize of nanoparticles. This paper serves to demonstrate the proof-of-concept that dual-bioresponsive polymersomes could be readilyformed from the PEG–PAA(SH)–PDEA copolymer and applied forefficient delivery of apoptotic proteins. In the following, we willstudy in detail different factors, including crosslinking densitieson nanoparticle properties.

9 8 7 6 5 4 3 2 1 0

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Fig. 1. 1H NMR spectrum (400 MHz) of PEG–PAA–PDEA triblock copolymer in D2O.

3.3. Loading and triggered release of proteins

In the following, loading and release of FITC–BSA and FITC–CCwere studied. Protein-loaded crosslinked polymersomes were fac-ilely prepared by adjusting the pH of PEG–PAA(SH)–PDEA and pro-tein aqueous solution at pH 6.0–8.0 followed by shaking at 37 �Cfor 5 h and extensive dialysis to remove free proteins. The resultsshowed that both FITC–BSA and FITC–CC were efficiently loadedinto crosslinked polymersomes with high protein loading efficien-cies of 64–100% at theoretical protein loading contents of 5–50wt.% (Table 1). High protein loading contents of 34.7 and 32.1wt.% were obtained for FITC–BSA and FITC–CC, respectively. Theefficient protein loading was most likely due to the presence ofeffective electrostatic and/or hydrogen bonding interactions be-tween proteins and PDEA blocks of PEG–PAA(SH)–PDEA duringthe polymersome formation. Similar results have been reportedfor several different systems containing either carboxylic acid oramino groups [36,51,52]. It should be noted that low protein load-ing level is a bottleneck for many current protein carriers, includ-ing liposomes and polymersomes [53–55]. These protein-loaded

crosslinked polymersomes had small sizes of 32.6–38.8 nm andlow PDIs of 0.12–0.23 (Table 1). Moreover, zeta potential measure-ments showed that protein-loaded crosslinked polymersomes hadnegative surface charges of –1.8- –9.3 mV in PB, corroborating thatPDEA chains are located inside the polymersomes.

The in vitro protein release studies were performed at 37 �C infour different media, i.e. PBS buffer (pH 7.4, 10 mM, 150 mM NaCl)with 0.04 mM DTT, PBS buffer with 10 mM DTT, acetate buffer (pH5.0, 10 mM, 150 mM NaCl) with 0.04 mM DTT and acetate bufferwith 10 mM DTT. Remarkably, the results showed that FITC–BSAwas quantitatively released in 11 h at pH 7.4 in the presence of10 mM DTT (Fig. 4A). In contrast, only 50% of FITC–BSA was re-leased at pH 7.4 with 0.04 mM DTT. Moreover, protein releasewas also low (31 or 36%) at pH 5.0, with either 0.04 mM or10 mM DTT, supporting the fact that crosslinked polymersomesare stable under acidic conditions. Similar release profiles werealso observed for FITC–CC, in which 80% and 34–36% of proteinwas released in 11 h at pH 7.4 in the presence of 10 mM DTT andall three control conditions, respectively (Fig. 4B). Therefore, cross-linked PEG–PAA(SH)–PDEA polymersomes can not only efficientlyload proteins under mild conditions but also quickly release pro-teins under a reductive condition mimicking that of the cytoplasm

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Fig. 2. pH-Induced formation of nano-sized polymersomes from PEG–PAA(SH)–PDEA triblock copolymer. (A) PEG–PAA(SH)–PDEA polymersome sizes as a function of pH. (B)Size distribution profiles of PEG–PAA(SH)–PDEA copolymers at pH 3.0 (unimers), polymersomes at pH 8.0 before and after 5 h exposure to the air and crosslinkedpolymersomes at pH 2.5. (C) TEM micrograph of disulfide-crosslinked polymersomes. (D) CLSM image of giant polymersome co-loaded with Nile red and FITC.

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ume

(%)

Size (nm)

pH 8.0, 0 hpH 7.4, 10 mM GSH, 1.7 hpH 5.0, 10 mM GSH, 1.7 hpH 7.4, 0.04 mM GSH, 14 hpH 5.0, 0.04 mM GSH, 14 h

A B

DC

Fig. 3. The colloidal stability and stimuli-responsive property of crosslinked PEG–PAA(SH)–PDEA polymersomes studied by DLS measurements. (A) Stability against extensivedilution. (B) Stability against high salt concentration. (C) Stability against organic solvent. (D) Reduction and pH-triggered change of polymersome sizes.

2164 H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168

Table 1Loading of FITC–BSA and FITC–CC into PEG–PAA(SH)–PDEA polymersomes.a

Proteins Protein/polymer feed ratio (wt.%) PLC (wt.%) PLE (%) Size (nm) PDI f-potentialb (mV)

– 0 – – 33.0 0.16 �5.7 ± 0.8FITC–BSA 5 5.0 �100 36.9 0.21 �6.7 ± 0.5

10 10.0 �100 38.5 0.23 �7.5 ± 0.120 15.9 79.6 36.0 0.18 �7.6 ± 1.250 34.7 69.3 33.6 0.19 �9.3 ± 0.5

FITC–CC 5 5.0 �100 38.8 0.16 �3.3 ± 0.210 8.2 81.8 35.9 0.12 �1.9 ± 0.220 13.4 66.8 34.8 0.15 �2.9 ± 0.650 32.1 64.2 32.6 0.15 �1.8 ± 0.4

a Final polymersome concentration was about 0.8 mg ml�1.b Measured in PB at pH 8.0.

H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168 2165

and cell nucleus. These results further indicate that encapsulatedproteins maintain their structure and do not form large aggregates.

3.4. Intracellular protein release and anti-tumor activity of CC-loadeddual-sensitive polymersomes

The cellular uptake and intracellular protein release behaviorsof FITC–CC loaded crosslinked PEG–PAA(SH)–PDEA polymersomeswere studied using CLSM in MCF-7 cells. Interestingly, strong FITC–CC fluorescence was observed in MCF-7 cells following 3 h incuba-tion with FITC–CC loaded crosslinked polymersomes (Fig. 5A).FITC–CC was distributed throughout the whole cell, including cellnuclei at a prolonged incubation time of 6 h (Fig. 5B). The localiza-tion of protein in the cell nuclei was further confirmed by XZ andYZ images of MCF-7 cells (Fig. 6). Protein-loaded polymersomes

0 2 4 6 8 10 120

20

40

60

80

100

pH 7.4, 10 mM DTTpH 5.0, 10 mM DTTpH 7.4, 0.04 mM DTTpH 5.0, 0.04 mM DTT

Cum

ulat

ive

rele

ase

(%)

Time (h)

0 2 4 6 8 10 120

20

40

60

80

100

Cum

ulat

ive

rele

ase

(%)

Time (h)

pH 7.4, 10 mM DTTpH 5.0, 10 mM DTTpH 7.4, 0.04 mM DTTpH 5.0, 0.04 mM DTT

A

B

Fig. 4. Reduction and/or pH triggered release of proteins from crosslinked PEG–PAA(SH)–PDEA polymersomes: (A) FITC–BSA and (B) FITC–CC.

are most probably taken up by cells via the endocytosis mecha-nism. The proton sponge effect of PDEA would facilitate endosomalescape of polymersomes. In the cytosol, polymersomes are proneto decrosslinking and dissociation, resulting in fast cytoplasmicprotein release. In contrast, little FITC–CC fluorescence was de-tected in MCF-7 cells following 6 h incubation with free FITC–CCunder otherwise the same conditions (Fig. 5C), in line with low cel-lular uptake of proteins [56]. These results demonstrate that dual-bioresponsive crosslinked PEG–PAA(SH)–PDEA polymersomes areable to efficiently deliver and release proteins into cancer cells.

It’s of crucial importance for protein carriers that released pro-teins maintain their biological activity. Herein, horse heart CC,which is known to play a significant role in programmed cell death[57], was selected as a model therapeutic protein. It has been re-ported that intracellularly injected CC [58] and intracellular re-leased CC from nanoparticles [59,60] could cause effective cellapoptosis. We have demonstrated that membrane ionizable poly-mersomes based on PEG–PTMC derivatives could deliver CC intoMCF-7 cells, resulting in enhanced anti-tumor activity as compared

Fig. 5. CLSM images of MCF-7 cells incubated with FITC–CC loaded crosslinkedPEG–PAA(SH)–PDEA polymersomes or free FITC–CC (dosage: 160 lg ml�1). (A)FITC–CC loaded polymersomes, 3 h. (B) FITC–CC loaded polymersomes, 6 h. (C) FreeFITC–CC, 6 h. For each panel, images from left to right show FITC–CC fluorescence incells (green), cell nuclei stained by DAPI (blue) and overlays of two images. Thescale bars correspond to 20 lm in all the images.

XZ

YZ

XZ

YZ

XZ

YZ

Fig. 6. XZ and YZ images of MCF-7 cells following 6 h incubation with FITC–CC loaded crosslinked PEG–PAA(SH)–PDEA polymersomes (dosage: 160 lg ml�1). The scale barscorrespond to 20 lm in all the images.

0

20

40

60

80

100

120

160 µg/mL80 µg/mL

*********

293T

HeLa

MCF-7

293T

HeLa

MCF-7

*** *****

Cel

l Via

bilit

y (%

)

CC-loaded Xlinked polymersomesfree CC

0

20

40

60

80

100

120

0.80.40.30.20.1

Cel

l via

bilit

y (%

)

Concentration (mg/mL)

MCF-7 cellsHeLa cells293T cells

B

A

Fig. 7. MTT assays in MCF-7, HeLa and 293T cells. (A) Antitumor activity of CC-loaded crosslinked PEG–PAA(SH)–PDEA polymersomes or free CC (dosage: 80 and160 lg ml�1) after 48 h incubation. (B) Cytotoxicity of crosslinked PEG–PAA(SH)–PDEA polymersomes. Data are presented as the average ± standard deviation(n = 4). (Student’s t-test, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001).

2166 H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168

to free CC [51]. The anti-tumor activity of CC-loaded disulfide-crosslinked polymersomes was studied using MTT assays in threedifferent cells, i.e. MCF-7, HeLa and 293T cells. Remarkably, our re-sults showed that CC-loaded crosslinked polymersomes provokedsignificant cell apoptosis following 48 h incubation at a CC dosageof 80 or 160 lg ml�1, corresponding to a polymersome concentra-tion of 0.135 or 0.27 mg ml�1 (Fig. 7A). It is interesting to notethat cancerous cells, MCF-7 and HeLa cells, exhibited muchgreater sensitivity to CC-loaded crosslinked polymersomes than

non-cancerous 293T cells. The lower cytotoxicity of protein-loadedpolymersomes observed for non-cancerous cells is probably due totheir slower uptake of polymersomes as well as a lower GSH con-centration than for cancer cells. For instance, 34.4%, 15.3% and70.5% cell viabilities were observed in MCF-7, HeLa and 293T cells,respectively, at a CC dosage of 80 lg ml�1. At a higher CC dosage of160 lg ml�1, cell viabilities of MCF-7 and HeLa cells were furtherdecreased to 11.3% and 8.1%, respectively, which were much lowerthan that observed for 293T cells (52.7% cell viability). The highanti-tumor activity of CC-loaded polymersomes demonstrates thatencapsulated proteins well maintain their structure and function.In contrast, free CC did not induce any cell apoptosis in all threetypes of cells (98–110% cell viabilities) under otherwise the sameconditions, in accordance with poor cellular uptake of free CC.Importantly, MTT assays revealed that crosslinked PEG–PAA(SH)–PDEA polymersomes were practically non-toxic to MCF-7 and HeLacells (cell viabilities > 83%) up to a tested concentration of0.8 mg ml�1 while they were non-toxic to 293T cells up to0.3 mg ml�1 (cell viability > 90%) (Fig. 7B), indicating that PEG–PAA(SH)–PDEA polymersomes possess good biocompatibility.These results confirm that dual reduction- and pH-bioresponsivecrosslinked PEG–PAA(SH)–PDEA polymersomes are able to effi-ciently deliver and release CC into cancer cells, resulting in effec-tive cell apoptosis.

4. Conclusions

We have demonstrated for the first time that reduction- andpH-bioresponsive disulfide-crosslinked polymersomes based onthe PEG–PAA(SH)–PDEA triblock copolymer can efficiently loadand deliver apoptotic proteins to cancer cells, resulting in potentinhibition of cancer cells. These dual-sensitive crosslinked poly-mersomes offer several unique advantages for protein delivery:(i) they are formed under mild conditions via adjusting the acidicsolution of the PEG–PAA(SH)–PDEA copolymer to pH 8.0 followedby auto-crosslinking in the air, wherein no organic solvent or cat-alyst is required, leading to minimal protein denaturation; (ii) theypossess excellent protein loading capacity; (iii) they are highly sta-ble with inhibited protein release under physiological environ-ments due to the interfacial disulfide-crosslinking ofpolymersomes; (iv) they rapidly dissociate into unimers underintracellular-mimicking reductive conditions due to reduction-triggered de-crosslinking, resulting in fast and efficient protein re-lease; and importantly (v) released proteins exhibit potent apopto-tic activity, causing effective inhibition of cancer cells. These dualreduction- and pH-bioresponsive crosslinked polymersomes pro-vide an attractive strategy for intracellular protein release.

H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168 2167

Acknowledgements

This work is financially supported by research grants from theNational Natural Science Foundation of China (NSFC 51003070,51103093, 51173126, 51273137 and 51273139), the National Sci-ence Fund for Distinguished Young Scholars (NSFC 51225302),Innovative Graduate Research Program of Jiangsu Province (GrantNo. CXZZ11_0094) and a Project Funded by the Priority AcademicProgram Development of Jiangsu Higher Education Institutions.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1–7, andschemes 1 and 2 are difficult to interpret in black and white. Thefull colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2014.01.010.

References

[1] Berndt N, Hamilton AD, Sebti SM. Targeting protein prenylation for cancertherapy. Nat Rev Cancer 2011;11:775–91.

[2] Leader B, Baca QJ, Golan DE. Protein therapeutics: a summary andpharmacological classification. Nat Rev Drug Discov 2008;7:21–39.

[3] Torchilin V. Tumor delivery of macromolecular drugs based on the EPR effect.Adv Drug Deliv Rev 2011;63:131–5.

[4] Torchilin VP, Lukyanov AN. Peptide and protein drug delivery to and intotumors: challenges and solutions. Drug Discov Today 2003;8:259–66.

[5] Wadia JS, Dowdy SF. Transmembrane delivery of protein and peptide drugs byTAT-mediated transduction in the treatment of cancer. Adv Drug Deliv Rev2005;57:579–96.

[6] Morishita M, Peppas NA. Is the oral route possible for peptide and protein drugdelivery? Drug Discov Today 2006;11:905–10.

[7] Petersen LK, Sackett CK, Narasimhan B. High-throughput analysis of proteinstability in polyanhydride nanoparticles. Acta Biomater 2010;6:3873–81.

[8] Meng FH, Zhong ZY. Polymersomes spanning from nano- to microscales:advanced vehicles for controlled drug delivery and robust vesicles for virus andcell mimicking. J Phys Chem Lett 2011;2:1533–9.

[9] Discher DE, Ortiz V, Srinivas G, Klein ML, Kim Y, Christian D, et al. Emergingapplications of polymersomes in delivery: from molecular dynamics toshrinkage of tumors. Prog Polym Sci 2007;32:838–57.

[10] Lee JS, Feijen J. Polymersomes for drug delivery: design, formation andcharacterization. J Control Release 2012;161:473–83.

[11] Meng FH, Zhong ZY, Feijen J. Biomacromolecules 2009;10:197–209.[12] Christian DA, Cai S, Bowen DM, Kim Y, Pajerowski JD, Discher DE.

Polymersome carriers: from self-assembly to siRNA and proteintherapeutics. Eur J Pharm Biopharm 2009;71:463–74.

[13] Liu G-Y, Chen C-J, Ji J. Biocompatible and biodegradable polymersomes asdelivery vehicles in biomedical applications. Soft Matter 2012;8:8811–21.

[14] Kim H, Kang YJ, Kang S, Kim KT. Monosaccharide-responsive release of insulinfrom polymersomes of polyboroxole block copolymers at neutral pH. J AmChem Soc 2012;134:4030–3.

[15] Xiong XY, Li YP, Li ZL, Zhou CL, Tam KC, Liu ZY, et al. Vesicles from pluronic/poly(lactic acid) block copolymers as new carriers for oral insulin delivery. JControl Release 2007;120:11–7.

[16] Rameez S, Alosta H, Palmer AF. Biocompatible and biodegradablepolymersome encapsulated hemoglobin: a potential oxygen carrier.Bioconjugate Chem 2008;19:1025–32.

[17] Li B, Chen G, Meng F, Li T, Yue J, Jing X, et al. A novel amphiphilic copolymerpoly(ethylene oxide-co-allyl glycidyl ether)-graft-poly(e-caprolactone):synthesis, self-assembly, and protein encapsulation behavior. Polym Chem2012;3:2421–9.

[18] Kishimura A, Koide A, Osada K, Yamasaki Y, Kataoka K. Encapsulation ofmyoglobin in PEGylated polyion complex vesicles made from a pair ofoppositely charged block Ionomers: a physiologically available oxygen carrier.Angew Chem, Int Ed 2007;46:6085–8.

[19] Fu Z, Ochsner MA, de Hoog H-PM, Tomczak N, Nallani M.Multicompartmentalized polymersomes for selective encapsulation ofbiomacromolecules. Chem Commun 2011;47:2862–4.

[20] Stano A. Scott EA, Dane KY, Swartz MA, Hubbell JA. Tunable T cell immunitytowards a protein antigen using polymersomes vs. solid-core nanoparticles.Biomaterials 2013;34:4339–46.

[21] Kumar M, Grzelakowski M, Zilles J, Clark M, Meier W. Highly permeablepolymeric membranes based on the incorporation of the functional waterchannel protein Aquaporin Z. Proc Natl Acad Sci USA 2007;104:20719–24.

[22] Hvasanov D, Wiedenmann J, Braet F, Thordarson P. Induced polymersomeformation from a diblock PS-b-PAA polymer via encapsulation of positivelycharged proteins and peptides. Chem Commun 2011;47:6314–6.

[23] Liu G, Ma S, Li S, Cheng R, Meng F, Liu H, et al. The highly efficient delivery ofexogenous proteins into cells mediated by biodegradable chimaericpolymersomes. Biomaterials 2010;31:7575–85.

[24] Zhang Y, Wu F, Yuan W, Jin T. Polymersomes of asymmetric bilayer membraneformed by phase-guided assembly. J Control Release 2010;147:413–9.

[25] Canton I, Massignani M, Patikarnmonthon N, Chierico L, Robertson J, RenshawSA, et al. Fully synthetic polymer vesicles for intracellular delivery ofantibodies in live cells. FASEB J 2013;27:98–108.

[26] Zhang J, Wu L, Meng F, Wang Z, Deng C, Liu H, et al. PH and reduction dual-bioresponsive polymersomes for efficient intracellular protein delivery.Langmuir 2012;28:2056–65.

[27] Anraku Y, Kishimura A, Oba M, Yamasaki Y, Kataoka K. Spontaneous formationof nanosized unilamellar polyion complex vesicles with tunable size andproperties. J Am Chem Soc 2010;132:1631–6.

[28] Du J, O’Reilly RK. Advances and challenges in smart and functional polymervesicles. Soft Matter 2009;5:3544–61.

[29] Deng C, Jiang Y, Cheng R, Meng F, Zhong Z. Biodegradable polymeric micellesfor targeted and controlled anticancer drug delivery: promises, progress andprospects. Nano Today 2012;7:467–80.

[30] Chambon P, Blanazs A, Battaglia G, Armes SP. How does cross-linking affect thestability of block copolymer vesicles in the presence of surfactant? Langmuir2012;28:1196–205.

[31] Du J, Armes SP. PH-responsive vesicles based on a hydrolytically self-cross-linkable copolymer. J Am Chem Soc 2005;127:12800–1.

[32] Gaitzsch J, Appelhans D, Grafe D, Schwille P, Voit B. Photo-crosslinked and pHsensitive polymersomes for triggering the loading and release of cargo. ChemCommun 2011;47:3466–8.

[33] Yang X, Grailer JJ, Rowland IJ, Javadi A, Hurley SA, Matson VZ, et al.Multifunctional stable and pH-responsive polymer vesicles formed byheterofunctional triblock copolymer for targeted anticancer drug deliveryand ultrasensitive MR imaging. ACS Nano 2010;4:6805–17.

[34] Holowka EP, Deming TJ. Synthesis and crosslinking of L-DOPA containingpolypeptide vesicles. Macromol Biosci 2010;10:496–502.

[35] Xu H, Meng F, Zhong Z. Reversibly crosslinked temperature-responsive nano-sized polymersomes: synthesis and triggered drug release. J Mater Chem2009;19:4183–90.

[36] Cheng R, Meng F, Ma S, Xu H, Liu H, Jing X, et al. Reduction and temperaturedual-responsive crosslinked polymersomes for targeted intracellular proteindelivery. J Mater Chem 2011;21:19013–20.

[37] Meng F, Hennink WE, Zhong Z. Reduction-sensitive polymers andbioconjugates for biomedical applications. Biomaterials 2009;30:2180–98.

[38] Cheng R, Feng F, Meng F, Deng C, Feijen J, Zhong Z. Glutathione-responsivenano-vehicles as a promising platform for targeted intracellular drug and genedelivery. J Control Release 2011;152:2–12.

[39] Meng F, Cheng R, Deng C, Zhong Z. Intracellular drug release nanosystems.Mater Today 2012;15:436–42.

[40] Sun H, Meng F, Cheng R, Deng C, Zhong Z. Reduction-sensitive degradablemicellar nanoparticles as smart and intuitive delivery systems for cancerchemotherapy. Expert Opin Drug Deliv 2013;10:1109–22.

[41] Cheng R, Meng F, Deng C, Klok H-A, Zhong Z. Dual and multi-stimuliresponsive polymeric nanoparticles for programmed site-specific drugdelivery. Biomaterials 2013;34:3647–57.

[42] Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev DrugDiscov 2003;2:214–21.

[43] Hu Y, Litwin T, Nagaraja AR, Kwong B, Katz J, Watson N, et al. Cytosolic deliveryof membrane-impermeable molecules in dendritic cells using pH-responsivecore–shell nanoparticles. Nano Lett 2007;7:3056–64.

[44] Biggs S, Sakai K, Addison T, Schmid A, Armes SP, Vamvakaki M, et al. Layer-by-layer formation of smart particle coatings using oppositely charged blockcopolymer micelles. Adv Mater 2007;19:247–50.

[45] Yang YQ, Zhao B, Li ZD, Lin WJ, Zhang CY, Guo XD, et al. PH-sensitive micellesself-assembled from multi-arm star triblock co-polymers poly(e-caprolactone)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) for controlledanticancer drug delivery. Acta Biomater 2013;9:7679–90.

[46] Thang SH, Chong YK, Mayadunne RTA, Moad G, Rizzardo E. A novel synthesis offunctional dithioesters, dithiocarbamates, xanthates and trithiocarbonates.Tetrahedron Lett 1999;40:2435–8.

[47] Chen W, Zheng M, Meng F, Cheng R, Deng C, Feijen J, et al. In situ formingreduction-sensitive degradable nanogels for facile loading and triggeredintracellular release of proteins. Biomacromolecules 2013;14:1214–22.

[48] Zhu C, Jung S, Luo S, Meng F, Zhu X, Park TG, et al. Co-delivery of siRNA andpaclitaxel into cancer cells by biodegradable cationic micelles based onPDMAEMA-PCL-PDMAEMA triblock copolymers. Biomaterials2010;31:2408–16.

[49] Wei R, Cheng L, Zheng M, Cheng R, Meng F, Deng C, et al. Reduction-responsivedisassemblable core-cross-linked micelles based on poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-lipoic acid conjugates for triggeredintracellular anticancer drug release. Biomacromolecules 2012;13:2429–38.

[50] Ahmed F, Discher DE. Self-porating polymersomes of PEG-PLA and PEG-PCL:hydrolysis-triggered controlled release vesicles. J Control Release2004;96:37–53.

[51] Li S, Meng F, Wang Z, Zhong Y, Zheng M, Liu H, et al. Biodegradablepolymersomes with an ionizable membrane: facile preparation, superior

2168 H. Sun et al. / Acta Biomaterialia 10 (2014) 2159–2168

protein loading, and endosomal pH-responsive protein release. Eur J PharmBiopharm 2012;82:103–11.

[52] Wang X, Sun H, Meng F, Cheng R, Deng C, Zhong Z. Galactose-decoratedreduction-sensitive degradable chimaeric polymersomes as a multifunctionalnanocarrier to efficiently chaperone apoptotic proteins into hepatoma cells.Biomacromolecules 2013;14:2873–82.

[53] Kirby C, Gregoriadis G. Dehydration-rehydration vesicles: a simple method forhigh yield drug entrapment in liposomes. Nat Biotechnol 1984;2:979–84.

[54] Cerritelli S, Velluto D, Hubbell JA. PEG-SS-PPS: reduction-sensitive disulfideblock copolymer vesicles for intracellular drug delivery. Biomacromolecules2007;8:1966–72.

[55] O’Neil CP, Suzuki T, Demurtas D, Finka A, Hubbell JA. A novel method for theencapsulation of biomolecules into polymersomes via direct hydration.Langmuir 2009;25:9025–9.

[56] Torchilin V. Intracellular delivery of protein and peptide therapeutics. DrugDiscov Today Tech 2008;5:e95–e103.

[57] Green DR, Reed JC. Mitochondria and Apoptosis. Science 1998;281:1309–12.[58] Zhivotovsky B, Orrenius S, Brustugun OT, Doskeland SO. Injected cytochrome c

induces apoptosis. Nature 1998;391:449–50.[59] Santra S, Kaittanis C, Perez JM. Cytochrome c encapsulating theranostic

nanoparticles: a novel bifunctional system for targeted delivery of therapeuticmembrane-impermeable proteins to tumors and imaging of cancer therapy.Mol Pharmaceut 2010;7:1209–22.

[60] Kim SK, Foote MB, Huang L. The targeted intracellular delivery of cytochrome Cprotein to tumors using lipid-apolipoprotein nanoparticles. Biomaterials2012;33:3959–66.