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Smart Asymmetric Vesicles with Triggered Availability of Inner Cell-Penetrating Shells for Specific Intracellular Drug Delivery Junjie Li, Shiyan Xiao, Yixuan Xu, Shuai Zuo, Zengshi Zha, Wendong Ke, Chuanxin He, and Zhishen Ge ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017

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ACS Applied Materials & Interfaces

Smart

Asymmetric

Vesicles

with

Triggered

Availability of Inner Cell-Penetrating Shells for Specific Intracellular Drug Delivery Junjie Li,†,1 Shiyan Xiao, †,1 Yixuan Xu,1 Shuai Zuo,1 Zengshi Zha,1 Wendong Ke,1 Chuanxin He,*,2 and Zhishen Ge*,1 1

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and

Engineering, University of Science and Technology of China, Hefei 230026, Anhui, China 2

College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen,

Guangdong, 518060, People’s Republic of China.

KEYWORDS:

vesicles, matrix metalloproteinases, cell-penetrating, chemotherapy, and

intracellular delivery

ACS Paragon Plus Environment

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ABSTRACT

Smart nanocarriers attract considerable interest in the filed of precision nanomedicine. Dynamic control of the interaction between nanocarriers and cells offers the feasibility that in situ activates cellular internalization at the targeting sites. Herein, we demonstrate a novel class of enzymeresponsive asymmetric polymeric vesicles self-assembled from matrix metalloproteinase (MMP)-cleavable peptide-linked triblock copolymer, poly(ethylene glycol)-GPLGVRG-b-poly(εcaprolactone)-b-poly(3-guanidinopropyl methacrylamide) (PEG-GPLGVRG-PCL-PGPMA), in which the cell-penetrating PGPMA segments asymmetrically distribute in the outer and inner shells with the fractions of 9% and 91%, respectively. Upon treatment with MMP-2 to cleave the stealthy PEG shell, the vesicles undergo morphological transformation into fused multi-cavity vesicles and small nanoparticles, accompanied by redistribution of PGPMA segments with 76% exposed to the outside. The vesicles after dePEGylation show significantly increased cellular internalization efficiency (~ 10 times) as compared to the original ones due to the triggered availability of cell-penetrating shells. The vesicles loading hydrophobic anticancer drug paclitaxel (PTX) in the membrane exhibit significantly enhanced cytotoxicity against MMPoverexpressing HT1080 cells and multicellular spheroids. The proposed vesicular system can serve as a smart nanoplatform for in situ activating intracellular drug delivery in MMP-enriched tumors.


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INTRODUCTION Smart nanocarriers are of considerable interest in the field of precision nanomedicine for maximizing the therapeutic efficacy and minimizing adverse side effects.1-4 The complicated physiological conditions require an optimal nanocarrier to adjust its properties responding to special physiological environments at the varying delivery stages. It should be noted that various physicochemical properties of the nanoparticles affect the interaction with cells, including chemical composition, morphology, size, and surface properties.5-8 Dynamic regulation of the interaction between drug-loading nanoparticles and cells is desirable for efficient site-specific intracellular drug delivery. For anticancer drug delivery, a variety of property-changeable nanocarriers (e.g. size, shape, or surface charge) have been developed for tumor tissue or intracellular delivery through responding to the tumor microenvironments.3,4,9 For example, as one of ingenious strategies, burying or protecting ligands (e.g. cell penetrating peptide, folate) in the nanocarriers during delivery journey and activating them at the targeting sites achieved dynamic control of intracellular internalization and enabled the ligands to work more specifically and efficiently.10-14 Nevertheless, more effective dynamic control of the interaction between nanoparticles and cells still remains a great challenge. Polymeric vesicles (polymersomes) not only provide cytomimetic models but also can be used as potent drug nanocarriers with various distinct advantages over conventional polymeric drug delivery systems (e.g. micelle).15-19 The aqueous interior and hydrophobic membrane endow vesicles the ability to load both hydrophilic and hydrophobic drugs simultaneously.20 Diverse structural design and incorporation of stimuli-responsive polymers allow vesicles to undergo


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triggered structural adjustment or morphological transformation.21-30 Drug release from the stimuli-responsive vesicles can be triggered by the specific biological signals.24,31-33 Nevertheless, dynamic control of cellular uptake of vesicular nanoparticles through adjustment of the interaction with cells was rarely investigated. Matrix metalloproteinases (MMP) as a family of hydrolytic enzymes play an important role in tumor growth and metastasis, which are overexpressed in tumor tissue and have been exploited as the stimuli for tumor imaging and drug release.11,12,34-42 Herein, we prepared a well-defined MMP-cleavable peptide-bridged triblock copolymer, poly(ethylene glycol)-GPLGVRG-b-poly(εcarprolactone)-b-poly(3-guanidinopropyl

methacrylamide)

(PEG-GPLGVRG-PCL-PGPMA),

which was utilized to self-assemble into polymeric vesicles. The asymmetric sturcture of the vesicle with the majority of PGPMA block distributed in the inner shells (~ 90%) was verified (Scheme 1). After dePEGylation in the presence of MMP-2, morphological and architectural transformation occurred with rearrangement of PGPMA segments and 76% PGPMA exposed to the outside. Given that PGPMA segment is a cell-penetrating peptide (CPP)-mimicking polymer, improved cellular uptake was demonstrated due to the surface property transition from stealthy PEGylation to CPP functionalization. Hydrophobic anticancer drug paclitaxel (PTX) loaded in the membranes of the polymersomes shows significantly high cytotoxicity against the MMPoverexpressing HT1080 cells and multicellular spheroids. The proposed innovative polymeric vesicular system is able to be utilized as smart nanocarriers for in situ activable intracellular drug delivery in MMP-enriched tumors.


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Scheme 1. Schematic illustration of the smart asymmetric polymeric vesicle with MMPtriggered dePEGylation followed by structural transformation and availability of inner cellpenetrating shells to in situ activate intracellular delivery of hydrophobic anticancer drug paclitaxel (PTX). EXPERIMENTAL SECTION Materials. Tin(II) 2-ethylhexanoate (Sn(Oct)2), copper(I) bromide (CuBr, 99%), N,N,N',N'',N''pentamethyldiethylenetriamine (PMDETA, 98%), anhydrous N,N-dimethylformamide (DMF), N,N'-dicyclohexylcarbodiimide

(DCC,

98%),

4-dimethylaminopyridine

(DMAP,

98%),

fluorescein isothiocyanate (FITC), azobisisobutyronitrile (AIBN, 99%), and poly(ethylene glycol) methyl ether (average Mn 5,000) were purchased from Sigma-Aldrich and used as received. α-Methoxy-ω-azido-poly(ethylene glycol) (PEG113-N3, Mn = 5000, Mw/Mn = 1.05) was obtained from JenKem Technology Co., Ltd. (Beijing, China). ε-Carprolactone (CL) was dried over CaH2 and distilled before use. Benzene and tetrahydrofuran (THF) were dried by refluxing over sodium/benzophenone and distilled. N-(3-aminopropyl) methacrylamide (APMA) was


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purchased from Polysciences Inc. and TCI Development Co., Ltd., respectively. 4-Cyano-4-(2phenylethane sulfanylthiocarbonyl) sulfanyl pentanoic acid (CPSSPA)43 and 3-guanidinopropyl methacrylamide (GPMA)44 were synthesized according to the reported method. PTX (98%) was purchased from klamar®. Fetal bovine serum (FBS), trypsin, and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO and used as received. 3-(4,5-Dimethylthiazol-2yl)2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI, 94%), and fluorescein diacetate (FDA) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Recombinant human MMP-2 (purity > 95%, the specific activity > 1000 pmoles min-1 µg-1) was purchased from Sino Biological Inc. (Beijing, China). Peptide G(propargylglycine)PLGVRG (alkynyl-GPLGVRG) (purity 95.36% from HPLC, ESI-MS: calcd. for (C31H51N10O8 + H)+: 692.82; found: 692.7) was obtained from ChinaPeptides Co., Ltd. (Shanghai, China). All other commercially available solvents and reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Human fibrosarcoma cell line, HT1080, was purchased from Shanghai Institute of Cell Biology (Shanghai, China). Characterization. All 1H NMR spectra were recorded on a Bruker AV300 NMR 300 MHz spectrometer using CDCl3, dimethyl sulphoxide-d6 (DMSO-d6), or D2O as the solvent. The molecular weight (MW) and molecular weight distribution (Mw/Mn) were determined by gel permeation chromatography (GPC) equipped with a G1310B Iso. pump, a G1316A PLgel column, and a G1362A differential refractive index detector. The eluent was DMF with 1 g L-1 LiBr at a flow rate of 1.0 mL min-1. A series of low-polydispersity polyethylene glycol (PEG) standards were employed for calibration. Particles sizes, and particle size distributions were conducted on a zeta-potential analyzer with dynamic laser light scattering (DLS), equipped a Malvern Zetasizer Nano ZS90, a He-Ne laser (633 nm), and 173o collecting optics. All data were


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averaged over three measurements. Reversed-phase HPLC (RP-HPLC) analysis was performed on a Shimadzu HPLC system, equipped with a LC-20AP binary pump, a SPD-20A UV-Vis detector, and a Symmetry C18 column. Confocal laser scanning microscopy (CLSM) images were acquired using Leica TCS SP5 microscope (Germany). UV-vis spectroscopy and fluorescence spectroscopy were recorded by UV-2401PC UV-VIS Spectrophotometer (Sahimadzu Corporation, Japan) and F-4600 Fluorescence Spectrophotometer (Hitachi, Japan), respectively. For preparation of cryogenic transmission electron microscopy (cryo-TEM) samples, 3 µL of sample solutions was applied to the hydrophilized TEM grid, blotted and vitrified in an automated vitrification robot (FEI VitrobotTM marker IV) by plunging into liquid ethane. This process was performed with the environmental chamber of the Vitrobot conditioned to 100 % humidity and 4 °C to prevent temperature and drying artefacts. Images were obtained with a Tecnai G2 Spirit Biotwin electron microscope at 120 kV. Sample preparation (Scheme S1) Synthesis of PEG-GPLGVRG-NH2. The peptide alkynyl-GPLGVRG (0.1 g, 0.144 mmo1), PEG113-N3 (0.6 g, 0.12 mmol), PMDETA (62 mg, 0.36 mmol), and anhydrous DMF (3 mL) were charged into a 5-ml Schlenk flask. The mixture was degassed by a freeze-pump-thaw cycle and backfilled with N2. CuBr (52 mg, 0.36 mmol) was introduced as a solid under the protection of N2. The reaction system was degassed by three freeze-pump-thaw cycles again and sealed under vacuum. Then, the Schlenk flask was placed in a preheated oil bath at 40 °C. After 24 h, the resulting mixture was precipitated into diethyl ether. After filtration and drying in a vacuum oven overnight at room temperature, the powder was dissolved in water and dialyzed against distilled water for three days using a dialysis bag (MWCO: 3500 Da). The solution was lyophilized, affording PEG-GPLGVRG-NH2 as a white powder (0.52 g, yield: 76.1%; Mn,GPC = 6.5 kDa,


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Mw/Mn = 1.06). 1H NMR and Fourier Transform infrared spectroscopy (FT-IR) analysis of PEGGPLGVRG-NH2 are shown in Figure S1. Synthesis of PEG-GPLGVRG-PCL. Block copolymer, PEG-GPLGVRG-PCL, was prepared via the ring-opening polymerization (ROP) of CL which was initiated by the terminal amino groups of PEG-GPLGVRG-NH2. Briefly, PEG-GPLGVRG-NH2 initiator was lyophilized from benzene to obtain the anhydrous white powder. Then, PEG-GPLGVRG-NH2 (114 mg, 0.01 mmol), CL (0.7 g, 3 mmol), Sn(Oct)2 (0.66 mL, 1 mg mL-1 in benzene), anhydrous THF (4 mL ), and benzene (10 mL) were charged into a 20-ml Schlenk flask. After lyophilization, the mixture was backfilled with N2, followed by addition of 4 mL anhydrous THF. The reaction system was sealed under vacuum and subsequently stirred for reaction at 80 °C. After 18 h, the resulting mixture was precipitated into diethyl ether. After repeating the above dissociation-precipitation cycle twice, the final product was dried in a vacuum oven, yielding a white solid (0.44 g, yield: 54%; Mn,GPC = 30.3 kDa, Mw/Mn = 1.18). The actual the degree of polymerization (DP) of PCL segment was determined to be 180 by 1H NMR analysis in DMSO-d6 (Figure S2A). Thus, the polymer was denoted as PEG114-GPLGVRG-PCL180. Synthesis of PEG-GPLGVRG-PCL-based macro-RAFT agent. PEG-GPLGVRG-PCL-based macro-RAFT agent was synthesized through DCC coupling reaction. Briefly, CPSSPA (34 mg, 0.1 mmol), DCC (21 mg, 0.1 mmol), and DMAP (0.12 mL, 1 mg mL-1 in dichloromethane) were dissolved in 2 mL anhydrous DMF and stirred for 30 min. Then, the block copolymer, PEG114GPLGVRG-PCL180 (0.26 g, 0.01 mmol) in dichloromethane (8 mL) was added and stirred for 24 h at room temperature. The resulting mixture was evaporated to dryness, re-dissolved in ethyl acetate, filtered and concentrated by rotary evaporation. The concentrated mixture was precipitated into diethyl ether. The above dissolution-precipitation cycle was repeated four times.


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The final product was dried in a vacuum oven, yielding a pale yellow solid (0.25 g, yield: 86%; Mn,GPC = 30.4 kDa, Mw/Mn = 1.17). 1H NMR analysis of PEG-GPLGVRG-PCL-based macroRAFT agent is shown in Figure S2B. Synthesis of PEG-GPLGVRG-PCL-PGPMA. Triblock copolymer, PEG-GPLGVRG-PCLPGPMA,

was

prepared

via

reversible

addition-fragmentation

chain-transfer

(RAFT)

polymerization of GPMA monomer using PEG-GPLGVRG-PCL-based macro-RAFT agent as the chain transfer agent. Briefly, PEG-GPLGVRG-PCL-based macro-RAFT agent (265 mg, 0.01 mmol), AIBN (0.23 mg, 0.0014 mmol), GPMA (56 mg, 0.3 mmol), and 1,4-dioxane/DMSO (0.5 mL/2 mL) were charged into a 5-mL Schlenk flask. The reaction system was degassed by three freeze-pump-thaw cycles and sealed under vacuum. Then, the Schlenk flask was placed in a preheated oil bath at 80 °C. After 12 h, the reaction mixture was precipitated into cold diethyl ether. The precipitate was collected, dissolved in dichloromethane and repeated the dissolutionprecipitation process twice. The final product, PEG-GPLGVRG-PCL-PGPMA, was dried in a vacuum oven, yielding a pale solid powder (0.24 g, yield: 75%; Mn,GPC = 33.1 kDa, Mw/Mn = 1.19). Actual DP of PGPMA segment was determined to be 13 by 1H NMR analysis in DMSOd6 (Figure S2C). Thus, the polymer was denoted as PEG114-GPLGVRG-PCL180-PGPMA13. As a control, a triblock copolymer without MMP-responsive peptide (GPLGVRG), PEG114-PCL155PGPMA15 (Mn,GPC = 30.5 kDa, Mw/Mn = 1.15) was also synthesized according to similar procedures using PEG114-OH as the initiator for ROP of CL, followed by RAFT polymerization of GPMA. Synthesis of FITC-labelled PEG-GPLGVRG-PCL-PGPMA. For labelling by FITC, small amount of amino-group-containing monomers, APMA, was copolymerized with GPMA using PEG-GPLGVRG-PCL-based macro-RAFT agent as the chain transfer agent. The triblock


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copolymer, PEG114-GPLGVRG-PCL180-P(GPMA0.85-co-APMA0.15)16 (Mn,GPC = 34.2 kDa, Mw/Mn = 1.21), was obtained via similar procedures for PEG-GPLGVRG-PCL-PGPMA preparation using PEG-GPLGVRG-PCL-based macro-RAFT agent as the chain transfer agent. Then, fluorescent tagging of PEG114-GPLGVRG-PCL180-P(GPMA0.85-co-APMA0.15)16 was performed through the reaction between amino groups and FITC. Briefly, FITC (27 mg, 0.07 mmol), PEG114-GPLGVRG-PCL180-P(GPMA0.85-co-APMA0.15)16 (0.1 g, 0.0035 mmol), trimethylamine (5 mg, 0.05 mmol) were dissolved in THF (10 mL), followed by stirring in the dark overnight at room temperature. Then, the solution was concentrated by rotary evaporation, followed by precipitation into diethyl ether. The above dissolution–precipitation cycle was repeated twice. After vacuum drying, the final product, FITC-labelled PEG-GPLGVRG-PCL-PGPMA was obtained as a slightly yellow powder. The number of fluorescein per block copolymer was calculated to be 2 based on ultraviolet-visible absorption. FITC-labelled PEG-PCL-PGPMA were also synthesized according to similar procedures. Preparation

of

MMP-responsive

PEG-GPLGVRG-PCL-PGPMA

asymmetric

polymersomes. MMP-responsive asymmetric polymersomes (MR-Vesicle) based on the triblock copolymer, PEG-GPLGVRG-PCL-PGPMA, were constructed using the thin-film rehydration method as reported by Discher et al.45 Typically, 10 mg PEG-GPLGVRG-PCL-PGPMA triblock copolymer was dissolved in 1 mL chloroform and the solution was slowly evaporated to form a thin film, followed by drying under vacuum overnight. Then, the film was hydrated with 1 mL PBS (10 mM, pH 7.4) for 12 h under vigorous stirring at 60 °C. FITC-labelled PEG-GPLGVRGPCL-PGPMA polymersomes, PEG-PCL-PGPMA polymersomes (NR-Vesicle), and FITClabelled PEG-PCL-PGPMA polymersomes were also prepared according to the same protocol.


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Preparation of drug-loaded polymersomes. Hydrophilic fluorescent dye calcein was encapsulated into polymersomes during thin-film rehydration. Briefly, the prepared PEGGPLGVRG-PCL-PGPMA film was hydrated with 1 mL PBS (10 mM, pH 7.4) containing 0.5 mg calcein for 12 h under vigorous stirring at 60 °C. Free calcein was removed through Millipore ultrafiltration (molecular weight cutoff: 50 kDa). The concentration of calcein was determined by fluorescence spectroscopy. Hydrophobic anticancer drug, paclitaxel (PTX), is loaded into the polymersome post-vesicle formation by the method as reported by Discher et al.45,46 Typically, PTX dissolved in methanol (50 mg mL-1) was slowly added at 10% v/v into the polymersome solutions (10 mg mL-1). After constant stirring for 1 h, the unentrapped drug was removed through Millipore ultrafiltration (molecular weight cutoff: 50 kDa). Drug loading contents were measured using fluorescence intensity and HPLC (mobile phase: acetonitrile/H2O (1:1, v/v), 1 mL/min) for calcein and PTX, respectively, which were determined to be 9.7% and 4.5%, respectively. FITC fluorescence quenching experiment. The asymmetric architecture of MR-Vesicle and dePEGylation-induced redistribution of PGPMA were confirmed by FITC fluorescence quenching experiment using iodide ion (I-) as a quencher.47,48 Freshly prepared FITC-labelled MR-Vesicle solution (0.5 mg mL-1) was incubated with MMP-2 (1 µg mL-1) for 24 h at 37 oC, followed by addition of various amounts of KI (from 0 to 1 M). Fluorescence spectroscopy was recorded by F-4600 Fluorescence Spectrophotometer (Hitachi, Japan). Meanwhile the Freshly prepared FITC-labelled MR-Vesicle solution (0.5 mg mL-1) was also added various amounts of KI (from 0 to 1 M) and the fluorescence intensity was measured. Drug release of calcein and PTX co-loaded polymersomes. Freshly prepared calcein and PTX co-loaded MR-Vesicle and NR-Vesicle solutions (0.5 mg mL-1) were incubated at 37 °C in the


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absence or presence of MMP-2 (1 µg mL-1). At different time intervals, an aliquot of solution was taken from the incubation medium, followed by ultracentrifugation using Millipore ultrafiltration tube (MWCO: 10 kDa). RP-HPLC was used to estimate the PTX release amount (mobile phase: acetonitrile/H2O (1:1, v/v), 1 mL/min, UV-vis detection-wavelength at 217 nm for PTX). The release profiles of calcein were quantified by Fluorescence spectroscopy using standard curves. In vitro cellular uptake. HT-1080 cells were seeded at a density of 1 × 105 cells/well into a 35mm glass-bottom culture dish (NEST, China) and incubated overnight in 2 mL DMEM medium with 10% FBS at 37 °C with 5% CO2 humidified atmosphere. The medium was replaced with fresh DMEM with 10% FBS, followed by addition of the PBS solution of FITC-labelled MRVesicle and NR-Vesicle at FITC concentration of 1 mM and incubated for 24 h. As a control, cells were treated with MMP inhibitor (doxycycline, 25 µM) for 4 h prior to addition of MRVesicle.49 The cells were washed three times with ice-cold PBS followed by detachment by trypsin, then harvested and resuspended in PBS. The suspended cells were applied to flow cytometry measurements. Data were analyzed with Flowjo software. In vitro cytotoxicity. HT-1080 cells were seeded in 96-well plates at a density of 1 × 104 cells/well in 100 µL DMEM with 10% FBS at 37 °C with 5% CO2 humidified atmosphere. After 24 h incubation, the medium was replaced with fresh DMEM with 10% FBS, followed by addition of different drug formulations at various concentrations of PTX. After 24 h incubation, the culture medium was replaced with fresh medium and another 48 h incubation was continued. For MTT assay, 20 µL MTT solution (5 mg mL-1 in PBS buffer) was added and incubated for 4 h, followed by removal of the medium and addition of 200 µL DMSO to dissolve the formed


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purple formazan crystals. Finally, the plate was shaken for 15 min, followed by measurement of the absorbance at 490 nm by a microplate reader. As for live/dead assay, the medium was replaced with 100 µL PBS, followed by staining FDA and PI for live and dead cells, separately. Briefly, 1 µL FDA solution (1 mg mL-1 in DMSO) was added to each well and incubated for 20 min for staining live cells. Subsequently, each well was washed with PBS twice, followed by addition of 100 µL PBS. PI solution (2 µL, 1 mg mL-1 in DMSO) was added to each well followed by 10 min incubation for staining dead cells. Finally, each well was washed with PBS twice and then transferred for observation under fluorescent microscope. Cellular uptake and cytotoxicity in 3D multicellular spheroids (MCS). In order to produce 3D multicellular spheroids, 96-well spheroid microplates with unique round well-bottom geometry were used. Briefly, 100 µL of HT-1080 cell suspension (2 × 104 cells mL-1 DMEM medium) were added into 96-well spheroid microplates, followed by incubation for 48 hours at 37 °C with 5% CO2 humidified atmosphere. Formation of multicellular spheroid was monitored by fluorescent microscope (Olympus inverted microscope IX-71). The distribution of samples in 3D spheroids was conducted. The prepared spheroids were treated with the PBS solution of FITC-labelled MR-Vesicle and NR-Vesicle at FITC concentration of 1 mM for 24 h. Thereafter, the spheroids were carefully washed with cold PBS without agitation and observed with CLSM. In 3D MCS viability assay, the prepared 3D spheroids were treated with different drug formulations and the diameters of spheroids were measured every day after images were taken from optical microscopy. 3D live/dead assay was also conducted according to the similar procedures. For quantification of 3D spheroids viability, adenosine triphosphate (ATP) content


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was measured based on the protocol from Promega CellTiter-Glo® 3D Cell Viability Assay. The final results were reported in terms of percentage (%) compared to the cells without addition of drugs. Meanwhile, the spheroids were trypsinized into single cells and stained with Annexin VFITC and PI using the Annexin V-FITC apoptosis detection kit. Quantitative apoptotic measurements were performed by flow cytometric analysis. Statistical analysis. All data were presented as means ± standard deviation (s.d.). Statistical analysis was conducted by Student’s t-test for two groups, and one-way analysis of variance for multiple groups. A value of P less than 0.05 was considered statistically significant. RESULTS AND DISCUSSION Triblock copolymer synthesis and self-assembly. MMP-cleavable peptide-bridged triblock copolymers, PEG-GPLGVRG-PCL-PGPMA, were designed with a relatively higher molecular weight (MW) of PEG and a lower MW of PGPMA as hydrophilic blocks and PCL as the hydrophobic block (Scheme S1). Initially, the azido-terminated PEG (PEG-N3) was conjugated by alkynyl-GPLGVRG peptide via click reaction. Quantitative conjugation was confirmed by 1H NMR and FT-IR analysis (Figure S1). The terminal amino group was subsequently utilized to initiate ROP of CL, and DP of PCL was determined to be 180 (Figure S2A). The terminal hydroxyl groups were further transformed into a macro-RAFT agent with a high yield as evidenced by comparing the integrals of benzene proton signals (c) and PEG peak (a) (Figure S2B). Furthermore, the macro-RAFT agent was utilized to perform RAFT polymerization of GPMA with a DP of 13 (Figure S2C). GPC characterization of PEG-GPLGVRG-NH2, PEGGPLGVRG-PCL, and PEG-GPLGVRG-PCL-PGPMA confirmed narrow MW distributions and gradual shift to higher MWs without initiator shoulders for PEG-GPLGVRG-PCL and PEGGPLGVRG-PCL-PGPMA indicating the high-efficiency initiation of polymerization (Figure S3).


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As a control, the triblock copolymer PEG114-PCL155-PGPMA15 which lacks a peptide linkage was also synthesized. To label the PGPMA segments, amino group-containing APMA monomer was added for copolymerization with GPMA during RAFT polymerization followed by reaction with FITC. The average number of fluorescein molecules per polymer chain was calculated to be two based on ultraviolet-visible absorption. Self-assembly of the triblock copolymer into polymeric vesicles was performed via the thinfilm rehydration method.25,50 MMP-responsive PEG114-GPLGVRG-PCL180-PGPMA13 vesicle (MR-Vesicle) and non-responsive PEG114-PCL155-PGPMA15 vesicle (NR-Vesicle) were formed, respectively. Cryo-TEM characterization was used to observe the morphology of MR-Vesicle, which show spherical shape with a diameter of 159 ± 35 nm. The clear contrast between the dark edge and hollow interior indicates apparent vesicular structure and the membranes thickness is estimated to be 8 ± 1 nm (Figure 1A,B, inset). DLS revealed that the average diameter was 180 nm with relatively narrow unimodal size distribution (Figure S4A). The inconsistence between the cryo-TEM and DLS results is ascribed to the vesicular hydrated outer PEG layer of DLS samples. To further investigate the architecture of the polymersomes, we performed the fluorescence quenching experiments using iodide ion (I-) as a quencher based on MR-Vesicle that consists of FITC-labelled PGPMA segments.51,52 Various amounts of KI were added and the fluorescence of fluorescein was measured. The results of F0/F versus I- concentration are shown in Figure 2 (0 h), where F0 and F represent the fluorescence intensity without and with addition of I-, respectively. The data were fitted according to Stern-Volmer equation:

F0 ΦK[I− ] = 1+ F 1+ (1−Φ)K[I− ]

(1)


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where K and Φ are the Stern-Volmer quenching constant and the fraction of accessible chromophores, respectively. The best-fit Φ value is 0.09 ± 0.005, which indicates that over 90% FITC molecules are inaccessible to I-. The inaccessible FITC molecules from PGPMA of MRVesicle are presumably located in the inner shells of the polymersomes. Therefore, the polymersome MR-Vesicle possesses an asymmetric architecture with ~ 90% PGPMA distributed in the inner shells and ~ 10% in the outer shells.26,50,52-55

Figure 1. Cryo-TEM images of MR-Vesicle (A, B) before and (C, D) after treatment with MMP2 (1 µg mL-1) for 24 h. The macroscopic appearances of MR-Vesicle solutions are shown in the insets.


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Figure 2. Steady-state fluorescence quenching by iodide ion (I-) for FITC-labelled MR-Vesicle (0.5 mg mL-1). Fluorescence quenching experiments of MR-Vesicle were performed at two separated tubes with the same amount of MR-Vesicle solutions: one was performed without addition of MMP-2 (0 h), and the other one was incubated with MMP-2 (1 µg mL-1) for 24 h followed by addition of iodide ion (I-). The best-fit Φ values were obtained by Matlab. MMP-responsive behavior of the vesicles. To evaluate the MMP-responsive behavior of MRVesicle in the presence of MMP-2, an aliquot of solution was taken out at predetermined time intervals and lyophilized for GPC characterization.41 The results showed that the peak areas of PEG segments increased steadily (Figure 3A). Quantified PEG release showed that rapid dePEGylation occurred within first 8 h followed by slow dePEGylation rate (Figure 3B). After 24 h, ~ 90% PEG shells were cleaved. The dePEGylation-induced morphological transformation of MR-Vesicle was investigated by cryo-TEM. At 24 h, some fused multi-cavity vesicles and small nanoparticles can be clearly distinguished (Figure 1C,D). After treatment with MMP-2 for 24 h, turbidity of the solution remained stable without apparent precipitation (Figure 1, insets). Time-dependent DLS analysis in the presence of MMP-2 revealed that the sizes of MR-Vesicle first decreased slightly and then increased after 6 h (Figure S4B).


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The dePEGylation-induced structural transformation of MR-Vesicle was subsequently investigated by the fluorescence quenching experiments upon addition of iodide ion (I-). After incubation of FITC-labelled MR-Vesicle with MMP-2 (1 µg mL-1) for 24 h, various amounts of KI were added and the fluorescence of FITC was measured. After fitting by Stern-Volmer equation, the best-fit Φ value was 0.76 ± 0.03 for MR-Vesicle after treatment with MMP-2 for 24 h (Figure 2, 24 h), which indicates that 76% FITC are accessible to I- compared with only 9% before treatment. The FITC molecules on PGPMA of MR-Vesicle located in the inner shells of the vesicles are inaccessible to I-, while inversion of PGPMA to the external surfaces can expose the FITC to I- in the aqueous solution, accompanied by efficient fluorescence quenching. Thus, dePEGylation of MR-Vesicle results in morphological transformation accompanied by inversion of cell-penetrating PGPMA to the outside. Despite of strong hydrophobic interactions between long PCL blocks in the membranes of MR-Vesicle, the hydrophobic PCL portions after dePEGylation are then exposed to water and the strong repulsion between PCL and solvent likely induces energetically unstable vesicles. The further aggregation, morphological transformation, and structural rearrangement occur with 76% PGPMA exposed to the outside solution.

Figure 3. (A) GPC traces of the eluted PEG for varying incubation times of MR-Vesicle in the presence of MMP-2 (1 µg mL-1). (B) Quantification of released PEG as a function of incubation time.


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Dissipative particle dynamics (DPD) simulation. To understand the mechanism of the structural transformation of the vesicles, dissipative particle dynamics (DPD) simulation was employed to investigate the transformation behaviors of MR-Vesicle with MMP-triggered dePEGylation.56 A model molecule A3B6C2 was built to capture the essential feature of PEGGPLGVRG-PCL-PGPMA, with A3 and C2 representing the hydrophilic PEG and PGPMA blocks of the vesicle, respectively, and B6 as the hydrophobic PCL block (Figure 4 and Figure S5).57 Asymmetric vesicles with spherical shapes and A as outer shells and C as inner shells were firstly achieved after a relaxation then long equilibrium simulation of the originally built models. The resulting vesicles were then applied to study the structure transformation after representative fractions (75% or 100%) of dePEGylation after cleavage of the A-B bond to mimic the cleavage of peptides (Figure S5). A typical process of the structure transformation is depicted, for spherical vesicle with A being cleaved. Our simulations indicate that some of the B6C2 molecules could gradually undergo an inversion to reach a state with the hydrophilic C portion to the outside. This inversion results in the packing rearrangement of B6C2, and spontaneously forms a bilayer membrane to expose their hydrophilic C shells to solvent. During this process, the monocavity vesicle can be formed. Notably, some dissociated small polymeric micelles with B as the cores and C as the shells can be observed during the mophological transition for the system with 75% dePEGylation. However, considering that the vesicles are not kept individually from others, therefore the kinetics of the structure transformation of the vesicle after dePEGylation should be affected by its neighboring vesicles. The dynamic processes of structural reorganization were further studied via simulations for the systems with two and four vesicles (Figure 4 and Figures S6, S7). In the multiple vesicle system, hydrophobic effect induces the contact between the vesicles shortly after


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dePEGylation, and forms bilayers between the vesicle cavities. First occurrence of B6C2 molecules translocation from the inner membrane leaflet to the outside is observed at ~ 950 and ~ 250 τ (simulation time unit) for the two and four vesicle systems with 100% dePEGylation, respectively. This time scale at a simulation temperature of kBT = 0.64 is significantly shorter than the corresponding single vesicle system (~ 1800 τ). Moreover, because of the developed stress between the opposite layers coming from the attachment of vesicles, a transient pore is formed on the membrane layer of one vesicle in the systems with 75% or 100% dePEGylation. Diffusive translocation of B6C2 molecules then occurrs spontaneously on a very short time scale, accompanied by the leakage of interior solvent. This pore-mediated translocation of B6C2 and their lateral diffusion on the outer membrane layer of the “vesicle aggregate” stabilizes the system, and greatly facilitates the equilibrium of the vesicles between the inner and outer leaflets. Note that this pore-mediated inversion mechanism is only observed in the multiple vesicle systems, and do not occur in the single vesicle system. These results demonstrate that the interactions between the vesicles after dePEGylation presumably play a more important role for the multi-vesicle systems in the inversion of B6C2 molecules forming the final structure with the majority of C molecules located at the outer shells. During these processes, the multi-cavity vesicles are formed.


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A

A3B6C2

B

0τ

300 τ

950 τ

1250 τ

1500 τ

2560 τ

3550 τ

C

0τ

100 τ

250 τ

400 τ

600 τ

2750 τ

Figure 4. DBD simulation of the morphological transformation. (A) The schematic model A3B6C2 for one PEG-GPLGVRG-PCL-PGPMA molecule. (B) The dynamic processes of the structure transformation for two vesicle systems with 100% dePEGylation. (C) Sequential snapshots for four vesicles with 100% dePEGylation. The two systems are simulated at a temperature of kBT=0.64. In general, the generated nanoparticle morphologies judged from the simulation results can be found in the cryo-TEM images incuding fused vesicles with one or multiple inner cavities and small-zise nanoparticles (Figures 1, 4, and Figures S5-S7). Thus, although the real process of morphological transformation after dePEGylation for MR-Vesicle may be more complicated, the simulated processes can help us to understand the inversion of PGPMA shells from inner shells to the outside. MMP-triggered drug release and cytotoxicity. To validate the feasibility of the MMPresposnive vesicles as smart nanocarriers, a hydrophilic fluorescent dye (calcein) and a


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hydrophobic anticancer drug (PTX) were used as model molecules to be co-encapsulated into MR-Vesicle with the loading content level of 9.7% and 4.5%, respectively. Drug release profiles of PTX and calcein from MR-Vesicle during morphological transformation were investigated (Figure 5). Apparently, the hydrophilic payloads are released rapidly with ~ 80% after 12 h in the presence of MMP-2. After dePEGylation, the polymersomes lose their structural integrity so that the solutions inside inner cavities leak rapidly. On the other hand, the hydrophobic drugs loaded in the membranes exhibit much slower release rate with lower than 30% release after 24 h incubation with MMP-2. The turbulence during morphological transformation facilitates the release of hydrophilic payloads but does not induce the rapid release of the hydrophobic drugs. PGPMA polymers as a CPP mimic have been demonstrated to nonspecifically promote cellular uptake.44 With the dePEGylation of MR-Vesicle and more PGPMA blocks exposing to the outside, enhanced cellular uptake is anticipated and the majority of hydrophobic drugs left in the nanoparticles should be delivered into the cells efficiently. We first evaluated the cellular uptake by HT1080 cells which overexpress MMP-2 and MMP-7.58 Under our cell culture conditions, the concentrations of MMP-2 and MMP-7 were determined to be 67 ± 8 ng mL-1 and 81 ± 11 ng mL-1, respectively, in the culture medium of HT1080 using MMP enzyme linked immunosorbent (ELISA) assay. Using FITC-labelled polymersomes, CLSM images show stronger fluorescence intensity and primary cytosolic localization of MR-Vesicle as compared to NR-Vesicle with weak intracellular fluorescence intensity and distribution predominantly in lysosomes (Figure 6A). Flow cytometry analysis further revealed that the cellular uptake of MR-Vesicle was ~ 10 times higher than that of NR-Vesicle group and MR-Vesicle group with the inhibitor (Figure 6B).


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Figure 5. The release profiles of hydrophilic calcein and hydrophobic PTX from MR-Vesicle in the presence of MMP-2 (1 µg mL-1). Mean ± s.d., n = 3. To evaluate cytotoxicity, the block copolymers of MR-Vesicle were first demonstrated to display negligible cytotoxicity towards HT1080 cells at a high concentration (Figure S8). Subsequetly, MTT assay and live/dead cell staining method were used to examine the cell viability. Against HT1080 cells, PTX-loaded MR-Vesicle show significantly higher cytotoxicity than that of PTX-loaded NR-Vesicle, and even higher than that of free PTX molecules (Figure 6C and Figure S9). The half maximal inhibitory concentration (IC50) value of the PTX-loaded MR-Vesicle was determined to be 33.1 ng mL-1 compared with 80.2 ng mL-1 for free PTX. In contrast, for MR-Vesicle with the addition of the MMP inhibitor or NR-Vesicle, the IC50 values of PTX were both high as 160 ng mL-1. These results indicate that the cell-penetrating shellsmediated intracellular delivery could promote the cellular uptake and improve therapeutic efficacy of the loaded anticancer drugs.


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Figure 6. (A) CLSM observation of intracellular distribution of FITC-labeled MR-Vesicle and NR-Vesicle after incubation with HT1080 cells for 24 h. LysoTracker Red and 4′,6-diamidine-2′phenylindole dihydrochloride (DAPI) were used to stain lysosome and nucleus, respectively. (B) Flow cytometry analysis of the cellular internalization of FITC-labelled NR-Vesicle and MRVesicle after incubation with HT1080 cells for 24 h at FITC-equivalent concentration of 1 mM. (C) PTX concentration-dependent cytotoxicity of various PTX formulations (a: PTX; b: PTXloaded NR-Vesicle; c: PTX-loaded MR-Vesicle; d: PTX-loaded MR-Vesicle + MMP inhibitor (doxycycline, 25 µM)) against HT1080 cells. Mean ± s.d., n = 4. Penetration and cytotoxicity in multicellular spheroids. To investigate the tissue binding and diffusion of MR-Vesicle, we established HT1080 MCS as a three-dimension in vitro tumor model.59,60 After incubation for 24 h with FITC-labelled MR-Vesicle, markedly higher binding and penetration ability was observed as shown in the equatorial cross-section slices (Figure 7A). In sharp contrast, NR-Vesicle only distributed at the periphery of the spheroids. After treatment with PTX-loaded MR-Vesicle for 12 days, the sizes of the MCS were reduced significantly


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whereas NR-Vesicle-treated MCS showed little size decrease (Figure S10). We further measured the ATP content of the spheroids at day 12 (Figure 7B). MR-Vesicle group possessed an ATP content of approximately 10% compared to the control group, indicating low viability of the cells inside the spheroids. Flow cytometry analysis further confirmed that the fraction of apoptotic cells was more than 95% (Figure S11). In sharp contrast, the groups of PTX-loaded NR-Vesicle and free PTX showed over 80% ATP content and higher fractions of live cells (~ 70%). The live/dead cell staining assay further confirmed that the majority of cells were killed in the spheroids treated by PTX-loaded MR-Vesicle, whereas free PTX and PTX-loaded NR-Vesicle can only induce cell death preferentially at the periphery of the spheroids (Figure 7C). This remarkable potency of MR-Vesicle can be ascribed to the high-efficiency binding, penetration, and cellular internalization ability with dePEGylation and more available cell-penetrating PGPMA segments after structural transformation.

Figure 7. (A) Fluorescence distribution of FITC-labelled NR-Vesicle or MR-Vesicle in HT1080 MCS after 24-h incubation. The inset curves represent the relative fluorescence intensity along the corresponding guide lines. (B) Relative ATP contents of the spheroids after treatment with PBS (a, control), PTX (b), PTX-loaded NR-Vesicle (c), PTX-loaded MR-Vesicle (d) at the PTX-


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concentration of 35 ng mL-1 on day 12. Mean ± s.d., ***P < 0.005 (t-test). (C) Representative images of the spheroids by live/dead cell staining at day 12 with live cells stained by fluorescein diacetate (FDA) (green) and dead cells by propidium iodide (PI) (red) after treatment with PBS (a, control), PTX (b), PTX-loaded NR-Vesicle (c), PTX-loaded MR-Vesicle (d). CONCLUSIONS In summary, we demonstrated the enzyme-activable asymmetric polymeric vesicles with triggered availability of cell-penetrating shells. Peptide-bridged triblock copolymers, PEGGPLGVRG-PCL-PGPMA, were successfully prepared and utilized for self-assembly into asymmetric vesicles with the majority of PGPMA segments (~ 90%) distributed in the inner shells. Under MMP-2 treatment, dePEGylation and structural transformation with more PGPMA segments exposed to the outside (76%) were verified by both experimental investigation and computer simulation, which resulted in improved affinity to the cells. PTX-loaded polymersomes showed significantly higher cellular internalization efficiency and cytotoxicity towards MMPoverexpressing HT1080 cells and MCS. Therefore, this unique smart polymeric vesicle system shows high-efficiency protection of cell-penetrating molecules by the asymmetric architecture and in situ activating them by MMP trigger, which promises efficient in vivo applications as smart nanocarriers for site-specific intracellular drug delivery. ASSOCIATED CONTENT Supporting Information DPD simulation procedures, synthetic routes for the preparation of PEG-GPLGVRG-PCLPGPMA, 1H NMR and GPC characterization of the polymers, DLS characterization of MRVesicle, more DPD simulation results, cytotoxicity by live/dead cell staining, growth profiles


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and annexin V/PI assay for HT1080 spheroids. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Email: [email protected] (Z. Ge), [email protected] (C. He). Author Contributions † These authors contributed equally to the work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We gratefully acknowledge financial support from National Natural Scientific Foundation of China (NNSFC) Project (21674104) and the Fundamental Research Funds for the Central Universities (WK3450000002). REFERENCES (1) Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M. A Review of Stimuli-Responsive Nanocarriers for Drug and Gene Delivery. J. Controlled Release 2008, 126, 187-204. (2) Kamaly, N.; Yameen, B.; Wu, J.; Farokhzad, O. C. Degradable Controlled-Release Polymers and Polymeric Nanoparticles: Mechanisms of Controlling Drug Release. Chem. Rev. 2016, 116, 2602-2663.


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