Temperature-Sensitive Polymersomes for Controlled Delivery of

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Temperature-Sensitive Polymersomes for Controlled Delivery of Anticancer Drugs Fei Liu,†,# Veronika Kozlovskaya,†,# Srikanth Medipelli,† Bing Xue,† Fahim Ahmad,‡ Mohammad Saeed,‡ Donald Cropek,§ and Eugenia Kharlampieva*,†,∥ †

Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States Department of Biochemistry and Molecular Biology, Drug Discovery Division, Southern Research, Birmingham, Alabama 35205, United States § U.S. Army Engineer Research and Development Center, Construction Engineering Research Laboratory, Champaign, Illinois 61826, United States ∥ Center for Nanoscale Materials and Biointegration, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States ‡

S Supporting Information *

ABSTRACT: We report on a novel type of triblock copolymer polymersomes with temperature-controlled permeability within the physiologically relevant temperature range of 37−42 °C for sustained delivery of anticancer drugs. These polymersomes combine characteristics of liposomes, such as biocompatibility, biodegradability, monodispersity, and stability at room temperature, with tunable size and thermal responsiveness provided by amphiphilic triblock copolymers. The temperaturesensitive poly(N-vinylcaprolactam)n-poly(dimethylsiloxane)65-poly(N-vinylcaprolactam)n (PVCLn-PDMS65-PVCLn) copolymers with n = 10, 15, 19, 29, and 50 and polydispersity indexes less than 1.17 are synthesized by controlled RAFT polymerization. The copolymers are assembled into stable vesicles at room temperature when the ratio of PVCL to the total polymer mass is 0.36 < f < 0.52 with the polymersome diameter decreasing from 530 to 40 nm as the length of PVCL is increased from 10 to 19 monomer units. Importantly, the permeability of polymersomes loaded with the anticancer drug doxorubicin can be precisely controlled by PVCL length in the temperature range of 37−42 °C. Increasing the temperature above the lower critical solution temperature of PVCL results in either gradual vesicle shrinkage (n = 10 and n = 15) or reversible formation of beadlike aggregates with no size change (n = 19), both leading to sustained drug release. All temperature-triggered morphological changes are reversible and do not compromise the structural stability of the vesicles. Finally, concentration- and time-dependent cytotoxicity of drug-loaded polymersomes to human alveolar adenocarcinoma cells is demonstrated. Considering the high loading capacity (∼40%) and temperature responsiveness in the physiological range, these polymer vesicles have considerable potential as novel types of stimuli-responsive drug nanocarriers.

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INTRODUCTION Polymer nanostructures assembled from amphiphilic block copolymers have been intensively studied for anticancer drug delivery because their small size (≤200 nm) allows for accumulation in tumors because of the enhanced permeability and retention arising from leaky cancerous vasculature.1−5 In this respect, systems that are capable of changing their morphologies and releasing encapsulated therapeutics in response to external stimuli are of special interest6−9 as they can exploit the lower pH (pH 5−6.8) and elevated temperatures (40−42 °C) that exist in tumors due to the high glycolysis rate and fast proliferation, respectively.10−16 Moreover, for thermosensitive polymer nanovectors, an enhanced therapeutic effect can be achieved due to nanovehicle aggregation within the tumor vascularity. Temperaturetriggered polymer nanocarrier aggregation can be attained in solid tumors because of an elevated endogenous temperature or © XXXX American Chemical Society

by external heat produced with focused ultrasound and hyperthermia.1 ABA triblock copolymers possessing regions of hydrophilicity (A block) bordering a hydrophobic copolymer center (B block) can self-assemble into polymer vesicles (polymersomes) and mimic natural lipid bilayers within a cell membrane.17,18 However, unlike liposome-based carriers, which may exhibit short half-lives, low stabilities toward oxidation, and may suffer from leakage and fusion,19 polymeric vesicles demonstrate a variety of morphologies, long-term stabilities, and physicochemical properties that may be controlled by the high diversity of block copolymer structures.20−24 Received: August 6, 2015 Revised: November 6, 2015

A

DOI: 10.1021/acs.chemmater.5b03048 Chem. Mater. XXXX, XXX, XXX−XXX

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Chemistry of Materials

motivated by the need for the development of nanocarriers with reversible change in vesicle membrane permeability and without risks for structural integrity, factors that are crucial for controlled release in effective drug therapy. We have chosen poly(N-vinylcaprolactam) (PVCL) as hydrophilic temperature-sensitive blocks attached to a long hydrophobic PDMS core block to generate a temperatureresponsive bolaamphiphile (an amphiphilic molecule that has hydrophilic blocks at both ends of a sufficiently long hydrophobic chain). PVCL is well-known for its excellent biocompatibility, stability against hydrolysis, and complexation ability.39 In contrast to PNIPAM, PVCL possesses a classical Flory−Huggins thermoresponsive phase diagram with a continuous coil-to-globule phase transition from 36 to 50 °C, depending on molar mass and concentration.40,41 This feature allows for convenient control of PVCL temperature sensitivity by varying its molecular weight. The temperature response of PVCL can also be controlled through functionalization of the vinylcaprolactam (VCL) monomer before copolymerization.32 As is known, the polydispersity of a thermosensitive copolymer block can broaden its volume phase transition temperature range; however, the recent development of controlled polymerization reactions has enabled the synthesis of PVCL homopolymers and copolymers with precisely controlled molecular weights and chemical structures.42−45 Herein, we demonstrate the first example of thermally responsive PVCLn-PDMS65-PVCLn triblock copolymer polymersomes with finely controlled permeability within the physiologically relevant temperature range of 37−42 °C. We integrate characteristics of liposomes, such as biocompatibility, biodegradability, monodispersity, and assembly at room temperature, with the reversible temperature-responsiveness of PVCL to design a new type of nanovesicle for sustained drug release in tumor lesions. In contrast to conventional thermosensitive polymersomes, the reported vesicles are assembled at room temperature and experience reversible morphological changes, such as shrinking or coagulation, at T > LCST that lead to enhanced permeability and drug release while maintaining their structural stability. The PVCLn-PDMSm-PVCLn triblock copolymers with n = 10, 15, 19, 29, and 50 and polydispersity indexes less than 1.17 are attained by controlled RAFT polymerization of PVCL using bifunctional bis(hydroxyalkyl) poly(dimethylsiloxane) with m = 65 as a macro chain transfer agent (CTA). The initial size of the polymersomes obtained at room temperature and the temperature-induced size and permeability changes of the polymer vesicles are controlled by varying the PVCL chain length and are analyzed with dynamic light scattering (DLS) and transmission electron microscopy (TEM). The PVCL10PDMS65-PVCL10 and PVCL15-PDMS65-PVCL15 polymersome membrane permeabilities are studied via temperature-induced release of the anticancer drug doxorubicin (DOX, Mw = 543.5 g mol−1) from the vesicles within the physiologically relevant temperature range of 25−42 °C using UV−visible spectroscopy. The concentration- and time-dependent effect on cell viability is studied by coculturing DOX-loaded polymersomes with human alveolar adenocarcinoma A549 cells. Considering their high loading capacity (∼40%) and temperature response in the physiological range, these polymer vesicles have considerable potential as novel types of stimuli-responsive drug nanocarriers as well as offer new percepts for fundamental studies on thermo-triggered assemblies in solution.

Temperature-responsive lower critical solution temperature (LCST) polymers have been appended to the hydrophobic blocks of amphiphilic block copolymers with poly(Nisopropylacrylamide) (PNIPAM) being the most ubiquitous LCST polymer.25 When a hydrophilic LCST polymer segment is attached to another hydrophilic block, these block copolymers can dissolve in water at temperatures under the LCST and self-associate to stable morphologies, such as micelles and vesicles, upon heating.26−29 Thus, poly(ethylene oxide)-block-poly(N-isopropylacrylamide) (PEO-b-PNIPAM) diblock copolymers with low polydispersity (PDI ≤ 1.2) synthesized with reversible addition-fragmentation chain-transfer (RAFT) polymerization produced stable polymer vesicles at T ≥ 37 °C only when the weight fraction of the hydrophilic block was less than 21%.26 Another PNIPAM-based diblock copolymer, poly(N-(3-aminopropyl)methacrylamide hydrochloride)-block-PNIPAM, resulted in vesicular aggregates upon heating its aqueous solutions to 45 °C.28 These vesicles dissolved upon cooling unless the N-(3-aminopropyl)methacrylamide hydrochloride units were cross-linked via the formation of an ionically paired polyelectrolyte complex with an anionic homopolymer.28 Double hydrophilic block copolymers based on thermosensitive poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) have been shown to self-assemble into polymersomes at ∼39 °C; however, PDMAEMA blocks were able to exhibit LCST behavior only at an extreme pH of 11 when the polymer segments became neutral.30 Thus, current approaches utilize elevated temperature as a critical point to form vesicles. However, the polymersomes obtained by temperature-induced self-assembly at T > 36 °C may have a limited use with thermal therapies as they require a temperature decrease, sometimes lower than the physiological temperature, to disassemble the polymer vesicle membrane and release the therapeutic cargo. For structural integrity and a reversible response to be provided, the vesicles assembled above the polymer LCST are chemically cross-linked, which limits their biodegradability. Thermosensitive micelles made from amphiphilic block copolymers could be a good alternative as they release drugs from the hydrophobic core at T > LCST upon collapse of the thermosensitive hydrated micelle corona.31−34 However, poor stability, relatively low loading capacity, and challenging functionalization hinders micelle applications for controlled drug delivery. Conversely, if the temperature-sensitive block is conjugated to hydrophobic polymer blocks, the resulting copolymers can form assemblies at room temperature, which can further aggregate into complex morphologies at high temperatures.32−34 The biggest challenge with synthetic liposomes (polymersomes) is insufficient control over permeability through the hydrophobic vesicle membrane. For addressing the problem, passive channel proteins have been inserted into the hydrophobic domain of the polymeric vesicle wall composed of poly(2-methyl-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-oxazoline) (PMOXAnPDMSm-PMOXAn) linear copolymers to render the polymersome membrane permeable.35−38 However, the process of channel protein production, purification, and integration into polymersome membranes can be rather difficult and expensive. In our work, we hypothesize that LCST polymers designed as hydrophilic blocks on amphiphilic block copolymers can be used to achieve and control permeability through the hydrophobic polymersome membrane upon collapse of the hydrated membrane segments at their LCSTs. We are B


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Assembly of PVCLn-PDMS65-PVCLn Vesicles. The vesicles were prepared using nanoprecipitation or thin film hydration methods.46 Briefly, for nanoprecipitation, 400 μL of an ethanol solution of PVCLnPDMS65-PVCLn at 0.5 mg mL−1 was added dropwise to 2 mL of deionized (DI) water at room temperature and stirred for 6 h, followed by dialysis of the mixture in water for 24 h to remove ethanol. To prepare polymersomes using a thin film hydration method, we formed thin films of copolymers by slow evaporation of CH2Cl2 from 0.5 mg mL−1 block copolymer solutions in CH2Cl2 using a rotary evaporator. The thin copolymer films were hydrated in DI water for 24 h under stirring followed by a double extrusion through a 0.1 μm pore size polycarbonate membrane using an Avanti mini-extruder (Avanti Polar Lipids, Incorporated). Loading of Doxorubicin (DOX) into PVCLn-PDMS65-PVCLn Vesicles. The DOX-loaded vesicles were fabricated using the nanoprecipitation method similarly to that described above. Briefly, 400 μL of ethanol solution of a triblock copolymer (0.5 mg mL−1) was added to 2 mL of doxorubicin hydrochloride aqueous solution (0.5 mg mL−1) followed by stirring for 6 h. Then, mixtures were transferred into a Float-A-Lyzer tube (Spectrum Laboratories) with a MWCO of 3000 Da and dialyzed in water for 48 h to remove free DOX and ethanol. The DOX loading capacity (40%, w/w) was quantified based on the weight percentage of DOX in vesicles and analyzed by UV−visible spectroscopy (Varian Cary 50; Agilent Technology). Briefly, dialyzed DOX-loaded vesicles were lyophilized and weighed followed by exposure to methanol to extract DOX. The polymer matter was separated from solution by centrifugation at 8000 rpm for 10 min. The loaded DOX amount was determined by measuring the absorbance of the solution at 490 nm based on the DOX calibration curve.46 Release of DOX from DOX-Loaded PVCLn-PDMS65-PVCLn Vesicles. The DOX release experiments were carried out using the membrane dialysis method.26 Briefly, 1 mL of DOX-loaded vesicle solution was placed in Float-A-Lyzer dialysis tubes (MWCO 3000 Da; Spectrum Laboratories), which were immersed in 50 mL of PBS buffer solution at pH 7.4 (HyClone). The dialysis was carried out in an incubator (Precision Scientific Company) under shaking (80 rpm) at temperatures of 25, 30, 35, 37, 40, and 45 °C. The dialysis media was withdrawn at a certain time interval and was replaced with the same volume of fresh PBS buffer solution. The concentration of DOX released into the dialysis media was determined by UV−visible spectroscopy (Varian Cary 50) and a DOX calibration curve. Dynamic Light Scattering (DLS). The vesicle size was measured using a Nano-ZS Zetasizer (Malvern) equipped with a He−Ne laser (663 nm). The average diameter of the assembled particles was obtained from three independent runs (15 measurements each). The particle size distribution was evaluated by standard deviation values (polydispersity index, PDI). Transmission Electron Microscopy (TEM). TEM imaging of the vesicles was performed using an FEI Tecnai T12 Spirit TWIN TEM microscope operated at 80 kV. For TEM imaging at room temperature, 7 μL of a vesicle solution at room temperature was dropped onto an argon plasma-treated, Formvar/carbon-coated copper grid (200 mesh, TED Pella). After 30 s, the excess solution was blotted off with a Kimwipe paper. The copper grid with the sample was stained with 1% uranyl acetate for 1 min followed by blotting off excess liquid and air-drying. For TEM imaging after the exposure of vesicle solutions to high temperatures, 7 μL of samples were preheated to 55 °C and dropped on a preheated argon plasmatreated, Formvar/carbon-coated copper grid; the excess solution was blotted off, and the TEM grid was dried at 55 °C for 2 h. Then, the TEM grid was stained with 1% uranyl acetate for 1 min. The excess liquid was blotted off and allowed to air-dry before imaging. Cell Studies. Human alveolar adenocarcinoma A549 cells were used for vesicle cytotoxicity and cell viability studies. Cells were seeded in 96-well plates at a density of 2 × 104 cells per well in Dulbecco’s modified Eagle’s medium (DMEM; high glucose) supplemented with 10% heat-inactivated fetal bovine serum and allowed to grow to near confluency. Subsequently, the cells were incubated with empty or DOX-loaded PVCL10-PDMS65-PVCL10 vesicles. For cytotoxicity

EXPERIMENTAL SECTION

Materials. 2,2′-Azobis(2-methylpropionitrile) (AIBN) (Aldrich) was recrystallized from methanol prior to use. N-Vinylcaprolactam (VCL) (Sigma) was distilled under low pressure before polymerization. Tetrahydrofuran (Fisher) was distilled before use. Potassium ethyl xanthate, 2-bromopropionyl bromide, diethyl ether, hexane, dichloromethane, calcium sulfate anhydrous, and dialysis tubing (MWCO of ∼5 K) were purchased from Fisher Scientific and used as received. Bis(hydroxyalkyl) poly(dimethylsiloxane) (PDMS65, Mn = 5600 Da) was obtained from Sigma-Aldrich. Doxorubicin hydrochloride (>99%) was purchased from LC Laboratories. Preparation of Bis(hydroxyalkyl) Poly(dimethylsiloxane) Macro-Chain Transfer Agent (X-PDMS65-X). The PDMS macroCTA was synthesized from bis(hydroxyalkyl) poly(dimethylsiloxane) (PDMS65) by a two-step reaction.44 First, PDMS65 terminated with dihydroxyl groups (10 g, 2 mmol) and triethylamine (TEA, 1.67 mL, 12 mmol) were mixed in a 250 mL round-bottom flask with 100 mL of anhydrous CH2Cl2 in an ice bath. A solution of 2-bromopropionyl bromide (2 mL, 18 mmol) in anhydrous CH2Cl2 (20 mL) was added dropwise to the mixture over 2 h. The solution was then allowed to warm to room temperature and was stirred for 24 h. The precipitate was separated by filtration, 300 mL of CH2Cl2 was added, and the solution was washed successively with 1 M HCl solution (3 times, 50 mL), 1 M NaOH solution (3 times, 50 mL), and deionized (DI) water (2 times, 50 mL) and then dried with anhydrous calcium sulfate. The polymer solution was concentrated in a rotary evaporator and precipitated in acetone. The final product Br-PDMS-Br was dried overnight under vacuum at room temperature (93% yield, 8.9 g). 1H NMR (400 MHz, CDCl3, δ): 4.41 (q, 1H, CHBr), 4.3 (t, 2H, -OCH2), 3.7 (t, 2H, -OCH2CH2-), 3.4−3.5 (m, 2H, -CH2CH2), 1.85 (d, 3H, -CHCH3), 1.6 (m, 2H, -CH2CH2CH2-), 0.5 (m, 2H, -CH2-Si), 0 (m, 195H, -Si-CH3). The number-average molecular weight Mn of BrPDMS-Br was 5290 Da (1H NMR). Second, Br-PDMS-Br (3 g, 0.6 mmol) was dissolved in CH2Cl2 (50 mL) in a 100 mL round-bottom flask and mixed with pyridine (0.8 mL, 4 mmol). Potassium ethyl xanthogenate (0.32 g, 2 mmol) in 20 mL of CH2Cl2 was added dropwise. The mixture was then stirred at room temperature for 24 h. After the precipitate was collected by filtration, the crude product was dissolved in 200 mL of CH2Cl2. The organic solution was washed successively with 1.0 M HCl solution (3 times, 50 mL), 1.0 M NaOH solution (3 times, 50 mL), and deionized water (2 times, 50 mL) and then dried with anhydrous CaSO4. The polymer solution was concentrated to 20 mL with a rotary evaporator and dried overnight under vacuum at room temperature to obtain XPDMS-X, where X stands for a xanthate group, -S-CS-OC2H5 (2.6 g, 86.7% yield). 1H NMR (400 MHz, CDCl3, δ) on one side: 4.7 (q, 2H, CH2CH3), 4.41 (q, 1H, CHBr), 4.3 (t, 2H, -OCH2-), 3.7 (t, 2H, -OCH2CH2-), 3.4−3.5 (m, 2H, -CH2CH2), 1.65 (m, 3H, -CHCH3), 1.45 (t, 3H, -CH2CH3), 0.5 (m, 2H, -CH2-Si), 0 (m, 195H, -Si-CH3). Mn of X-PDMS-X was estimated to be 5370 Da based on the 1H NMR calculation of repeating units. Preparation of Poly(N-vinylcaprolactam) n -block-poly(dimethylsiloxane)65-block-poly(N-vinylcaprolactam)n (PVCLnPDMS65-PVCLn). In a typical RAFT polymerization,39 X-PDMS-X macro-CTA (1 g, 0.2 mmol), AIBN (33 mg, 0.2 mmol), VCL (2.3 g, 16 mmol), and freshly distilled tetrahydrofuran (4 mL) were added to a 25 mL Schlenk flask equipped with a magnetic stirring bar. The mixed solution was degassed by five freeze−pump−thaw cycles under argon followed by heating at 60 °C to initiate the polymerization. Samples were periodically withdrawn by a syringe to monitor the extent of polymerization. After a certain amount of time, the reaction was immediately quenched in a dry ice bath. Samples were dissolved in acetone, transferred to dialysis tubes (MWCO = 5000 Da), and dialyzed for 2 days in methanol. Monomer conversion and purified molecular weight were determined by 1H NMR. 1H NMR (400 MHz, CDCl3, δ) of a purified polymer: 4.2−4.6 (broad peak, -CH- in poly(N-vinylcaprolactam)), 3.0−3.5 (broad peak, -CH2(N)CH2), 2.1− 2.6 (broad peak, -C(O)-CH2-), 1.3−2.1 (broad peak, -CH2CH2CH2-), 0 (broad peak, CH3(Si) in poly(dimethylsiloxane)). C


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Chemistry of Materials studies of empty polymersomes, DOX-free vesicles at 0.25 mg mL−1 were prepared first and then diluted to result in 5, 1, 0.5, and 0.1 μg mL−1 final concentrations, which were confirmed by determining the remaining polymer mass after freeze-drying of the DOX-free polymersome solutions. DOX-loaded vesicle suspensions with an overall DOX concentration of 90 μg mL−1 was diluted so that the DOX concentration was 5, 1, 0.5, or 0.1 μg mL−1 prior to being dispensed into the respective wells. Free DOX solution with the same concentration was used as a positive control, and nontreated cells were used as a negative control. Plates were then incubated at 37 °C with humidity of ≥85% and an air/CO2 ratio of 95/5%. After 24, 48, and 72 h, liquid medium was aspirated, the cells were rinsed three times with 1× PBS for 5 min each, and cell viability was quantified in each well by adding 100 μL of Cell-Titer Glo reagent (Promega) and measuring the luminescence in an Envision plate reader (PerkinElmer). Prior to cell viability measurements, images of the cells in each well were captured for visual control. Each experiment was carried out in triplicate. For confocal microscopy analysis, transmission and fluorescent optical sections were collected with a Nikon A1R+ confocal laser microscope equipped with a 100× oil immersion objective using a TRITC filter set. Multiple optical sections were imaged for each microscopic field.

and 16 h of reaction, respectively. The composition of the triblock copolymers was analyzed using 1H NMR analysis. The number-average molecular weights of bis(hydroxyethyl)oxypropyl poly(dimethylsiloxane) (PDMS65) were calculated based on the ratio between the integrals at δ = 0.3 ppm (-SiCH3 protons in the PDMS block repeat units) and at δ = 0.6 ppm (the methylene protons in the end groups) (Figure S1). The PVCL block length was determined by comparing integrals at δ = 4.5 ppm (>CH-; the methanetriyl protons of the PVCL blocks on both ends) and at δ = 0.3 ppm (the methyl protons for -Si-CH3 in the PDMS block). The length of a single PVCL block was calculated as the half of all PVCL repeat units. Gel permeation chromatography (GPC) in THF revealed a unimodal distribution of triblock copolymer elution peaks with a very low polydispersity ranging from 1.13 to 1.17 (Figure 2b; Table 1). The number-average molecular weights obtained from GPC analysis were much lower but in a similar range when compared to those calculated using 1H NMR data (Table 1). These results indicate that the molecular weight of PVCL blocks can be precisely controlled by the feeding ratio of VCL monomer to the RAFT agent, which is in good agreement with previous reports on RAFT polymerization of PVCL.42 Self-Assembly of Temperature-Responsive PVCLnPDMS65-PVCLn Polymer Vesicles: Control of Vesicle Size and Morphology. To explore the self-assembly of temperature-responsive PVCLn-PDMS65-PVCLn triblock copolymers, we used the nanoprecipitation method: the copolymers were dissolved in ethanol at 0.5 mg mL−1, added to 2 mL of deionized (DI) water at room temperature, and stirred for 6 h. The ethanol was removed from the aqueous solution by dialyzing the mixture in DI water for 24 h. Figure 3 shows TEM images of vesicles made from PVCL10PDMS65-PVCL10 (Figure 3a), PVCL15-PDMS65-PVCL15 (Figure 3b), and PVCL19-PDMS65-PVCL19 (Figure 3c). One can see that all of the tested block copolymers produced vesicular morphologies with a darker periphery and a hollow nonstained interior, implying that the membrane remained intact and nonpermeable toward a TEM staining dye. The block copolymers with larger PVCL chains (n = 29 and n = 50) did not produce vesicles but rather assembled into micelles with an average diameter below 30 nm as evidenced from DLS measurements (not shown). The PVCL10-PDMS65-PVCL10, PVCL15-PDMS65-PVCL15, and PVCL19-PDMS65-PVCL19 copolymers could also form polymer vesicles when assembled via the thin film hydration method where a thin layer of the copolymers was hydrated in DI water under stirring followed by extrusion through a 0.1 μm pore size polycarbonate membrane (Figure S3).47 The average polymersome diameter was 210, 142, and 59 nm for polymersomes with PVCL n = 10, 15, and 19, respectively. We found that polymersomes with PVCL10 and PVCL15 blocks had an average hydrodynamic size of 530 nm (PDI = 0.054) and 220 nm (PDI = 0.033) after 24 h of preparation using the nanoprecipitation method and were stable for 72 h as measured by DLS (Figure 3d, e). In contrast, the initial average hydrodynamic size for PVCL19-PDMS65PVCL19 vesicles was 164 nm (PDI = 0.037), but after preparation, the size decreased to 40 nm (PDI = 0.025) within 72 h (Figure 3f), resulting in highly monodisperse vesicles as evidenced by TEM analysis (Figure 3c). The polymersomes obtained via thin film hydration were smaller than those produced by nanoprecipitation, which is explained by the fact that thin film hydration vesicles were pushed through small pores of a membrane during extrusion. As is known, the

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RESULTS AND DISCUSSION Synthesis of Thermoresponsive Triblock Copolymers of Poly(N-vinylcaprolactam)n-b-poly(dimethylsiloxane) 65 -b-poly(N-vinylcaprolactam) n (PVCLn-PDMS65-PVCLn ). Temperature-sensitive PVCL nPDMS65-PVCLn triblock copolymers were synthesized by RAFT polymerization of N-vinylcaprolactam (VCL) using bis(hydroxyethyl)oxypropyl poly(dimethylsiloxane) macrochain transfer agent (X-PDMS65-X) in tetrahydrofuran at 60 °C with AIBN as a free radical source (Figure 1). The macro-

Figure 1. Temperature-sensitive PVCLn-PDMS65-PVCLn triblock copolymers were synthesized by RAFT copolymerization of Nvinylcaprolac tam (VC L) with bis(hy drox yalky l) poly(dimethylsiloxane) macro-chain transfer agent (X-PDMS65-X) prepared via modification of PDMS65 with 2-bromopropionyl bromide (1) and potassium ethyl xanthogenate (2).

chain transfer agent X-PDMS65-X was prepared via modification of bis(hydroxyethyl)oxypropyl poly(dimethylsiloxane) bearing 65-repeat units (PDMS65) with 2-bromopropionyl bromide (Figure 1(1); Figure S1a), followed by a subsequent conversion of Br-PDMS65-Br into X-PDMS65-X macro-chain transfer agent using potassium ethyl xanthogenate (Figure 1(2); Figure S1b). The chain length of PVCL was regulated by varying the reaction time, and PVCLn-PDMS65-PVCLn triblock copolymers with n = 10, 15, 19, 29, and 50 were obtained after 2, 3, 4, 8, D


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Figure 2. (a) 1H NMR spectra of the temperature-responsive triblock copolymers of PVCLn-PDMS65-PVCLn with n = 10, 15, 19, 29, and 50. (b) GPC traces of PVCLn-PDMS65-PVCLn triblock copolymers (A: PVCLn; B: PDMS65) with n = 10, 15, 19, 29, and 50.

Table 1. Number-Average Molecular Weights (Mn), Polydispersity (Đ), and Chemical Composition of the Copolymers

a

ABA copolymer

Mn (1H NMR), Da

Mn (GPC)a, Da

Đ (GPC)

PVCL, wt %

PVCL:PDMS ratio

PVCL10-PDMS65-PVCL10 PVCL15-PDMS65-PVCL15 PVCL19-PDMS65-PVCL19 PVCL29-PDMS65-PVCL29 PVCL50-PDMS65-PVCL50

7780 9170 10280 13060 18900

5034 5209 5540 7093 11947

1.13 1.15 1.15 1.17 1.15

36 45 52 62 79

1:3.5 1:2.1 1:1.7 1:1.1 1:0.7

The GPC number-average molecular weights were obtained using polystyrene linear standards.

increasing f values.39,50 Because of this, these copolymers were excluded from further analysis. Temperature-Induced Size and Permeability Changes in PVCLn-PDMS65-PVCLn Polymersomes. PVCL chains can collapse upon heating due to the destruction of hydrogen bonds of VCL with water and enhanced hydrophobic interactions between PVCL segments.51 We explored the temperature-dependent behavior of the PVCLn-PDMS65PVCLn triblock copolymer polymersomes in water at temperatures ranging from 25 to 55 °C using DLS. Figure 4 demonstrates that the average hydrodynamic sizes of PVCL10PDMS65-PVCL10 and PVCL15-PDMS65-PVCL15 triblock copolymer vesicles significantly decrease from ∼500 nm (PDI = 0.055) to ∼380 nm (PDI = 0.039) and from ∼210 nm (PDI = 0.072) to ∼175 nm (PDI = 0.045), respectively, when their

formation of various self-assembled morphologies from block copolymers in water can be predicted by the ratio of the hydrophilic fraction to the total copolymer mass (f).13 A block copolymer is expected to self-assemble into vesicles when this ratio is in the range from approximately 0.25 to 0.4 and to form spherical micelles when this ratio is larger than 0.5.26,48,49 We found that this ratio equaled 0.36 in the case of PVCL10PDMS65-PVCL10 and increased to 0.45 and to 0.52 for PVCL15PDMS65-PVCL15 and PVCL19-PDMS65-PVCL19, respectively, corresponding to the formation of vesicular structures, which agrees with the TEM results (Figure 3a−c). The block copolymers with larger hydrophilic block lengths of PVCL29 (f = 0.62) and PVCL50 ( f = 0.79) did not produce polymer vesicles, which is consistent with the reported trends on the progression from vesicular to micellar morphologies with E


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evidenced from the temperature trend measurements using DLS (Figure 4h). The possible explanation for this “secondary assembly” can be that, although the PVCL19 blocks can still collapse above the PVCL phase transition temperature, they remain long enough to have substantial interactions with the PVCL chains from other polymersomes. The much smaller size of PVCL19-PDMS65-PVCL19 vesicles can probably favor the stability of such aggregates in solution. The results of the reversible size changes of the polymersomes imply that although the hydration and dehydration of PVCL chains linked to the hydrophobic PDMS blocks are the main driving force for the observed volume changes of the polymer vesicles, the structural integrity of the polymersomes remains intact because the water-insoluble PDMS also contributes to the reversible dimensional transitions. Large and reversible size changes upon heating were also reported for PDMAEMA-b-PNIPAM53 and poly(2-cinnamoylethyl methacrylate)-b-PNIPAM54 polymersomes upon covalent crosslinking of the PNIPAM corona and are attributed to the LCST behavior of the resultant stable PNIPAM network. The degree of the PVCL-PDMS65-PVCL vesicle volume change in this work decreases from 1.3- to 1.2-fold with an increasing PVCL chain length from 10 to 15 and an undetectable size decrease in the case of PVCL19. This result can be explained by the increased interchain entanglements for longer PVCL blocks leading to a smaller free volume available for their temperatureinduced collapse and, consequently, to a less pronounced temperature response,55 which translates to the PVCLPDMS65-PVCL vesicle volume changes. As we have recently demonstrated, the temperature-induced collapse of PVCL blocks at the PVCL’s LCST can lead to distortions in the hydrophobic micelle core, forcing the release of encapsulated drug from micelles.32 A similar phenomenon can be expected in the case of a drug encapsulated within a PVCL-PDMS-PVCL polymer vesicle. We investigated the temperature-triggered release of the anticancer drug doxorubicin (DOX) from PVCL10-PDMS65-PVCL10, PVCL15PDMS65-PVCL15, and PVCL19-PDMS65-PVCL19 polymersomes at 25 °C and under conditions mimicking a tumor microenvironment of 42 °C (pH 7.4, 0.01 M phosphate). All three polymersome systems kept the drug encapsulated with no release for a 10 h experimental time (Figure 5a). In contrast, when the temperature was elevated to 42 °C, DOX was gradually released from the polymersomes with the accumulative DOX release of 86 ± 4%, 29 ± 3%, and 11 ± 1% from PVCL 10 -PDMS 65 -PVCL 10, PVCL 15-PDMS 65 -PVCL 15, and PVCL19-PDMS65-PVCL19, respectively, in 10 h. Apparently, the observed drug release at 42 °C is due to the phase transition of hydrophilic PVCL polymersome inner and outer coronas at its LCST, resulting in a localized and reversible disturbance of the polymersome membrane. In Figure 5b, the temperatureinduced increase in PVCLn-PDMS65-PVCLn polymersome permeability toward DOX is summarized schematically. The increasing temperature to or above that of the PVCL phase transition can lead to a gradual collapse of PVCL block chains, which can result in a local PVCL chain aggregation and leads to an overall decrease in the polymersome size and an increase in membrane “perforation”, causing release of DOX from the polymersome cavity. Temperature-Dependent Release of DOX from PVCL10-PDMS65-PVCL10 and PVCL15-PDMS65-PVCL15 Polymersomes. Because PVCL-PDMS65-PVCL polymersomes demonstrated the release of DOX in a physiologically relevant

Figure 3. TEM images of (a) PVCL10-PDMS65-PVCL10, (b) PVCL15PDMS65-PVCL15, and (c) PVCL19-PDMS65-PVCL19 prepared at T = 26 °C and imaged after 72 h. (d−f) Sizes of the produced vesicles in (a−c), respectively, 24 and 72 h after preparation as measured with dynamic light scattering.

solutions were gradually heated to 55 °C (Figure 4a, d). Remarkably, the size changes were completely reversible when the solutions were cooled back to 25 °C as demonstrated by their temperature trend profiles (Figure 4b, e). A similar size decrease of ∼1.7-fold was recently reported for PDAEMA-bPNIPAM polymersomes of 75 nm in size when the temperature was increased from 25 to 50 °C.52 The TEM analysis of the samples prepared from the vesicle solutions preheated at 55 °C confirmed the presence of the polymersomes, which kept their spherical morphology and did not aggregate (Figure 4c, f). In contrast, the PVCL19-PDMS65-PVCL19 polymersomes obtained from the triblock copolymer with longer PVCL19 blocks showed a reverse temperature trend, demonstrating a slight increase in the average hydrodynamic sizes of the structures upon gradual heating from 25 to 55 °C (Figure 4h). As seen from the light scattering intensity profile measured at T = 25 and 55 °C, the average hydrodynamic size (∼120 nm) of the structures present in solution did not change significantly, but the light scattering intensity increased in the size range from 300 to 800 nm, and the PDI increased from 0.037 to 0.083 when the solution was heated to 55 °C (Figure 4g). TEM analysis revealed the presence of chain-like structures at T = 55 °C unlike the well-separated spherical polymersomes observed for the sample at T = 25 °C (Figure 3c). These results suggest that smaller PVCL19-PDMS65-PVCL19 polymersomes tend to produce “chains of beads” at elevated temperatures. However, this process does not result in vesicle fusion and this “secondary assembly” is completely reversible and dissociates back to primary polymersomes after cooling back to T = 25 °C as F


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Figure 4. Temperature-dependent size trends (a, d, g) and sizes (b, e, h) of PVCL10-PDMS65-PVCL10 (top), PVCL15-PDMS65-PVCL15 (middle), and PVCL19-PDMS65-PVCL19 (bottom) vesicle solutions as measured with DLS. TEM images of the ABA copolymer solutions prepared at 55 °C with PVCL10 (c), PVCL15 (f), and PVCL19 (i) temperature-responsive hydrophilic blocks.

temperature range (T ≤ 42 °C), we hypothesized that the release from polymersomes should be limited below the LCST of a PVCL block and only become prominent and relatively fast above it when PVCL collapses and causes defects in the PDMS shell. To test this hypothesis, we investigated the release of drug from DOX-loaded polymersomes at 25, 30, 35, 37, 40, and 45 °C and at pH 7.4 during 10 h. The PVCL19-PDMS65PVCL19 polymer vesicles were excluded from the study because of the low DOX amount released at higher temperatures (Figure 5a). Figure 6a shows that at T < 30 °C the cumulative DOX release from PVCL10-PDMS65-PVCL10 vesicles was negligible in the time frame of the measurement (below 10%) and slightly increased to 26 ± 3% at 35 °C with no further increase at 37 °C. However, the 3-fold increase in the cumulative DOX release compared to that at 37 °C was achieved at 40 °C resulting in 76 ± 5% of DOX in the released media. A nearly identical release of DOX (87 ± 10%) was achieved at 45 °C, implying that the temperature-induced PVCL chain collapse occurred in the range between 37 and 40 °C. With longer PVCL15 block chains, however, the temperature threshold at which the increased DOX release was observed from the polymersomes was found to be in the range between 35 and 37 °C (Figure 6b). Furthermore, it is important to note that the largest amount of DOX released from PVCL15 vesicles (31 ± 2% at 45 °C) is only a third of that of PVCL10 (Figure 6a, b). This result also reflects the effect of

increasing PVCL block length on the decreased degree of the PVCL-PDMS65-PVCL vesicle volume change due to the increased interchain entanglements for longer PVCL blocks as discussed above. The resulting smaller free volume available for the temperature-induced collapse of PVCL chains may lead to fewer disturbances in the hydrophobic PDMS layer and, therefore, a lesser amount of DOX release. Overall, the fact that water-soluble, hydrophilic DOX can escape from the polymersome by passing through the hydrophobic PDMS layer at elevated temperatures along with the fact that the vesicle remains intact after DOX release implies that transient pores are indeed created in the PVCL-PDMS-PVCL membrane at elevated temperatures. The release data was analyzed according to the experimentally established Peppas−Ritger equation, mt/mf = ktn, where mt and mf are DOX release at time t and infinite time, respectively, k is a kinetic constant characteristic of the macromolecular system and the drug, and n is the release exponent indicative of the transport mechanism.56 The kinetic constants were extracted from linear logarithmic fits (Figure S3) and plotted as a function of temperature (Figure 6c) from which the LCST values of the PVCL blocks in PVCL10PDMS65-PVCL10 and PVCL15-PDMS65-PVCL15 polymersomes were calculated as the inflection points of the obtained temperature-dependent release rates. The calculations resulted in 40 and 37 °C for the PVCL phase transition in PVCL10G


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nontoxic to the cancer cells. This result agrees with previous reports on biocompatibility and noncytotoxicity of PVCL-based homopolymers and block copolymers,57,58 nanogels,59 and hydrogel capsules.60 In contrast, when DOX-loaded PVCL10PDMS65-PVCL10 polymersomes were incubated with the cells, the cell viability decreased gradually within 72 h of incubation (Figure 7b, c). After 24 and 48 h of incubation, the cell viability decreased from 85 ± 1% to 59 ± 5% and from 71 ± 1% to 50 ± 2% for the vesicles containing 0.1 and 0.5 μg mL−1 of DOX, respectively, demonstrating the effect of a higher DOX loading concentration on the decreased cell viability (Figure 7b, c). However, after 48 h of exposure, the cell viability did not decrease further, implying that DOX release was saturated for 0.1 and 0.5 μg mL−1 DOX-loaded vesicles after 48 h. In contrast to low DOX-loaded polymersomes, the samples containing 1 and 5 μg mL−1 of DOX continued killing the cells during the whole 72 h incubation period with the cell cytotoxicity reaching more than 75 and 97%, respectively. The cell viability for these samples decreased from 62 ± 1% to 34 ± 1% to 24.7 ± 0.4% for 1 μg mL−1 DOX-loaded polymersomes and from 52 ± 1% to 6.2 ± 0.3% to 2 ± 1% for 5 μg mL−1 DOX-loaded vesicles after 24, 48, and 72 h of treatment, respectively (Figure 7b, c). Higher anticancer activity observed for carrier-mediated delivery of DOX has been welldocumented previously.61−63 Carrier-mediated drug uptake can enhance drug concentration in a tumor cell unlike the case when a free drug with the same dosing gradually permeates to the cell.64−66 Similarly, a sustained intracellular release of DOX from loaded vesicles has been reported to be more efficient at killing cancer cells, leading to a lower cell viability compared to that of DOX alone.64 Figure 8 demonstrates the representative confocal microscopy images of the cells incubated for 24 and 48 h with DOXloaded PVCL10-PDMS65-PVCL10 polymersomes (Figure 8a−c and d−f, respectively) and free DOX solution (Figure 8g−i and j−l, respectively). The red fluorescence from DOX within A549 cytosolic space after 24 h indicates effective DOX-vesicle transport into the cancer cells. After a 48 h incubation, bright red DOX fluorescence is observed within the cell nuclei for both DOX-vesicles and DOX-treated cells, confirming the successful release of DOX from the vesicles and its delivery to the cytoplasm and to cell nuclei. Despite the fact that the PVCL10-PDMS65-PVCL10 polymersomes are capable of releasing only up to 27 ± 2% of DOX at 37 °C (Figure 6a), there is a possible degradation of the ester bonds between the PVCL and PDMS blocks in the triblock copolymer chains in the acidic environment (pH 90 °C.26 In Vitro Cell Cytotoxicity and Release of Doxorubicin into Cells. To evaluate the therapeutic properties of DOXloaded PVCL10-PDMS65-PVCL10 vesicles, we incubated human alveolar adenocarcinoma A549 cells with DOX-free or DOXloaded vesicles with various concentrations of DOX at pH 7.4 and 37 °C for various incubation times (24, 48, and 72 h), and their cytotoxicity to the cells was quantified using Cell-Titer Glo assay (see Experimental Section). Free DOX solution (PBS, pH 7.4, 37 °C) with the concentration matching that in the vesicle aliquots was used as a positive control for cell cytotoxicity, whereas untreated cells were used as a negative control. The viability of the cells incubated with the DOX-free PVCL10-PDMS65-PVCL10 vesicles did not decrease after 24, 48, or 72 h and stayed above 90% even after 72 h of incubation (Figure 7a) indicating that without DOX the vesicles were H


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Figure 6. Time-dependent release of DOX from DOX-loaded (a) PVCL10-PDMS65-PVCL10 and (b) PVCL15-PDMS65-PVCL15 vesicles at 25 °C (square), 30 °C (pentagon), 35 °C (down-triangle), 37 °C (up-triangle), 40 °C (circle), and 45 °C (rhomb). (c) Temperature-dependent DOX release rate for PVCL10-PDMS65-PVCL10 and PVCL15-PDMS65-PVCL15 DOX-loaded polymersomes.

Figure 7. Viability of human alveolar adenocarcinoma A549 cells (%) after incubation with various concentrations of (a) DOX-free PVCL10PDMS65-PVCL10 vesicles for 24, 48, and 72 h, and (b, c) DOX-loaded PVCL10-PDMS65-PVCL10 vesicles (DOX-vesicles) and DOX solution for (b) 24, (c) 48, and (d) 72 h at 37 °C. Each data point represents an average of three replicates ± SD (*p < 0.008, **p < 0.02, ***p < 0.002, ****p < 0.0001). I


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Figure 8. Optical and confocal microscopy images of human alveolar adenocarcinoma A549 cells after incubation with 5 μg mL−1 of DOX-loaded PVCL10-PDMS65-PVCL10 polymersomes (DOX-vesicles; a−f) and free DOX (DOX; g−l) for 24 (a−c and g−i) and 48 h (d−f, and j−l) at 37 °C and pH 7.4. DOX emits a red fluorescence signal. The scale bar is 10 μm in all images.

polymersomes at T > 37 °C. Thus, in vivo evaluation of drug release from the temperature-sensitive DOX-loaded PVCLPDMS-PVCL polymersomes will have to be performed.

free volume available for PVCL temperature-induced collapse. We also demonstrate for the first time that the permeability of the PVCLn-PDMS65-PVCLn triblock copolymer polymersomes toward the small-molecular weight anticancer drug doxorubicin (DOX) can be precisely controlled in the physiologically relevant temperature range of 37−42 °C. The fact that watersoluble, hydrophilic DOX can escape from the polymersome by passing through the hydrophobic PDMS layer at elevated temperatures, along with the fact that the vesicle remains intact after DOX release, implies that transient pores are created in the PVCL-PDMS-PVCL membrane at elevated temperatures. In addition, we show concentration- and time-dependent cytotoxicity of DOX-loaded polymersomes in human alveolar adenocarcinoma cells. Considering their high loading capacity (∼40%) and temperature response in the physiological range, these polymer vesicles have considerable potential as novel types of stimuli-responsive drug nanocarriers.

3. CONCLUSIONS We report on the synthesis of novel temperature-responsive poly(N-vinylcaprolactam)n-poly(dimethylsiloxane)65-poly(N-vinylcaprolactam)n (PVCLn-PDMS65-PVCLn) triblock copolymers with n = 10, 15, 19, 29, and 50 and low polydispersity indexes synthesized by controlled RAFT polymerization of PVCL using bifunctional bis(hydroxyalkyl) poly(dimethylsiloxane) as a macro-chain transfer agent. We show for the first time the assembly of PVCLn-PDMS65-PVCLn triblock copolymers into stable polymersomes of a submicrometer size at room temperature when the PVCL hydrophilic ratio is 0.36 < f < 0.52 and show that the polymersome size can be controlled by the PVCL chain length. The obtained polymeric vesicles show a temperature-induced size decrease that is dependent on the PVCL block length. The degree of the PVCL-PDMS65-PVCL vesicle volume changes decreases from 1.3- to 1.2-fold with increasing PVCL chain length from PVCL10 to PVCL15, to an undetectable size decrease in the case of PVCL19, which is due to the increased interchain entanglements for longer PVCL blocks leading to a smaller

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.5b03048. J


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Chemistry of Materials 1

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H NMR spectra for Br-PDMS65-Br and its bixanthate derivative X-PDMS65-X; hydrodynamic sizes of PVCLnPDMS65-PVCLn (n = 10, 15, 19) prepared using a thin film hydration method; Peppas−Ritger linear fits of doxorubicin release from doxorubicin-loaded PVCLPDMS-PVCL polymersomes at various temperatures; and hydrodynamic sizes of PVCL-PDMS-PVCL polymersomes at pH 7.5 and pH 3 at 37 °C (0.01 phosphate buffer) after 24 h (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

F.L. and V.K. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF-DMR 1306110 (E.K.), by the U.S. Army Corps of Engineers (CERL, W9132T-12-2-0005), and by funds from Southern Research (M.S.). Aaron Alford (UAB) is acknowledged for technical assistance. The UAB Transmission Electron microscopy facility and High Resolution Imaging facility (Confocal laser scanning microscopy) are also acknowledged.

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