Article pubs.acs.org/Biomac

Asymmetrical Polymer Vesicles with a “Stealthy” Outer Corona and an Endosomal-Escape-Accelerating Inner Corona for Efficient Intracellular Anticancer Drug Delivery Qiuming Liu,† Jing Chen,† and Jianzhong Du* School of Materials Science and Engineering, Key Laboratory of Advanced Civil Engineering Materials of Ministry of Education, Tongji University, 4800 Caoan Road, Shanghai, 201804, China S Supporting Information *

ABSTRACT: The efficient intracellular drug delivery is an important challenge due to the slow endocytosis and inefficient drug release of traditional delivery vehicles such as symmetrical polymer vesicles, which have the same coronas on both sides of the membrane. Presented in this paper is a noncytotoxic poly(ethylene oxide)-block-poly(caprolactone)block-poly(acrylic acid) (PEO113-b-PCL132-b-PAA15) triblock copolymer vesicle with an asymmetrical structure. The biocompatible exterior PEO coronas are designed for stealthy drug delivery; The pH-responsive interior PAA chains are designed for rapid endosomal escape and enhanced drug loading efficiency. The hydrophobic PCL vesicle membrane is for biodegradation. Such asymmetrical polymer vesicle showed high doxorubicin (DOX) loading efficiency and good biodegradability under extracellular enzymatic conditions. Compared with three traditional symmetrical vesicles prepared from PEO113-b-PCL110, PEO43-b-PCL98-b-PAA25, and PAA21-b-PCL75 copolymers, the DOXloaded asymmetrical PEO113-b-PCL132-b-PAA15 polymer vesicles exhibited rapid endocytosis rate and much faster endosomal escape ability, demonstrating promising potential applications in nanomedicine.

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INTRODUCTION During the past decades, a range of tumor-targeting drug delivery vehicles such as micelles,1 liposomes,2 and polymer vesicles3−16 have been developed for cancer chemotherapeutics. Usually, block copolymer vesicles are hollow spheres, which have a hydrophobic membrane with hydrophilic coronas randomly stretched on its interior and exterior without any preferred direction, leading to a symmetrical corona structure if the curvature effect is neglected.17−19 They have been widely investigated for drug release because of their large hydrophilic cavity, suitable membrane barrier, feasibility for chemical functionalization, and physiological applications.7,20 Asymmetrical vesicles that process different coronas on both sides of the membrane have also been theoretically predicted21 and experimentally trapped.22−24 For example, Eisenberg and coworkers had experimentally observed that PEO-b-PCL-bPAA triblock copolymer could self-assemble into asymmetrical vesicles with a long PAA outer corona and a short PEO inner corona to encapsulate protein.22 However, the high charge density of PAA exposed to the exterior of the vesicles may result in substantial cell death. Recently, Armes et al. reported that poly(ethylene oxide)-block-poly(2-(diisopropylamino)ethyl methacrylate)-block-poly(2-(dimethyldimethylamino)ethyl methacrylate) (PEO-b-PDPA-b-PDMA) self-assembled into asymmetrical vesicles with a PEO outer corona, showing better biocompatibility in DNA intracellular delivery.24 However, those nonbiodegradable asymmetrical PEO113-bPDPA84-b-PDMA13 vesicles were taken up by cells at a slow rate, requiring up to 48 h for complete internalization. Recently, © 2014 American Chemical Society

Zhong and coworkers reported biodegradable chimaeric polymer vesicles based on asymmetrical PEG-b-PCL-b-PDEA triblock copolymers for protein delivery.25 They also reported pH-sensitive degradable chimaeric polymer vesicles based on asymmetrical PEG-b-PTTMA-b-PAA triblock copolymers for DOX delivery.26 Those vesicles showed high drug loading efficiency (DLE) and efficient delivery and release into the cells. However, there are some challenges in cancer therapy using some traditional polymer vesicles as a drug delivery carrier: (1) Their slow endocytosis rate may result in toxicity to normal cells due to the diffusion of anticancer drugs outside the cancer cells.27 (2) They usually have poor endosomal escape ability after getting inside the cells as a drug delivery vehicle.9,28 (3) They usually exhibit low loading efficacies for hydrophilic anticancer drugs such as DOX·HCl.5,29 Therefore, to develop a new polymer vesicle with a rapid endocytosis rate, accelerated endosomal escape ability and high DLE will be of great interest for nanomedicine. To solve the above problems, herein we report a noncytotoxic biodegradable pH-sensitive asymmetrical polymer vesicle based on PEO113-b-PCL132-b-PAA15 triblock copolymer (Polymer 4 in Table 1) for rapid cellular internalization and efficient intracellular anticancer drug delivery. The membrane of the asymmetrical vesicle is composed of biocompatible and biodegradable PCL. Biocompatible PEO chain is designed as Received: May 10, 2014 Revised: June 30, 2014 Published: July 7, 2014 3072

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Table 1. Properties of Block Copolymers and Corresponding Vesicle Structure polymera

compositionb

Mn,NMRb

Mn,GPCc

Mw/Mnc

f PEOd

f PAAd

vesicle structure

1 2 3 4 5

PEO43-b-PCL78 PEO43-b-PCL98-b-PAA25 PEO113-b-PCL110 PEO113-b-PCL132-b-PAA15 PAA21-b-PCL75

10 800 15 000 17 500 18 200 10 300

25 800 14 800 23 100 17 900 12 600

1.19 1.26 1.23 1.20 1.26

0.18 0.13 0.29 0.24 0.00

0.00 0.13 0.00 0.06 0.16

symmetrical symmetrical symmetrical asymmetrical symmetrical

a

Polymers 2 and 4 are synthesized from the precursor polymers 1 and 3, respectively. bCompositions of copolymers are determined by 1H NMR in CDCl3. cDetermined by DMF GPC calibrated with near-monodisperse PEO standards. dCalculation of relative volume fractions of each block is shown in the Supporting Information. See Figures S1−S9 for details.

the outer corona because it is stealthy to immune system. The pH-responsive PAA is designed as the inner corona of the polymer vesicle for rapid endosomal escape.30,31 Furthermore, those PAA chains can enhance the loading and stabilization of the anticancer drug DOX·HCl via electrostatic interactions between them. The main difference between this polymer and Eisenberg’s copolymer lies in the relative block length of PEO and PAA. In our paper, the biocompatible PEO is designed as the outer corona of the asymmetrical vesicles rather than the inner corona in their work. It is noteworthy that this design in the asymmetrical vesicles may lead to great improvements in their biocompatibility, drug loading capacity, endocytosis rate, and endosomal escape ability. To reveal the advantage of this asymmetrical polymer vesicle, we also prepared DOX-loaded symmetrical vesicles based on PEO113-b-PCL110, PEO43-b-PCL98-b-PAA25, and PAA21-b-PCL75 block copolymers as the controls. (See Figures S1−S9 in the Supporting Information for syntheses and characterization.) Scheme 1 compares the underlying mechanism of DOX-loaded asymmetrical vesicles with symmetrical vesicles for intracellular drug delivery. During the blood circulation, the asymmetrical vesicle (red arrows indicated) can be internalized via fast endocytosis, thereby initiating the protonation of inner PAA coronas. This process also induces a rapid flux of ions and water into the subcellular compartment, eventually resulting in the rupture of the endosomal membrane, thus triggering the efficient and rapid release of the encapsulated DOX·HCl to the cell nucleus.32 DOX·HCl-loaded vesicles with asymmetrical or symmetrical corona structures exhibit different performance in cellular internalization, endosomal escape, drug release, and biodegradation. (1) The symmetrical PEO113-b-PCL110 vesicles are trapped in endosome and difficult to escape after cellular uptake. (2) The asymmetrical PEO113-b-PCL132-b-PAA 15 vesicles (red arrows indicated) can be internalized via fast endocytosis. They can also rapidly escape from endosome due to the protonation of the inner PAA coronas at low pH, thus triggering the efficient and rapid release of the encapsulated DOX·HCl to the cell nucleus. They can also be degraded and escape from cell by exocytosis. (3) The symmetrical PEO43-bPCL98-b-PAA25 vesicles with some outside PAA coronas undergo slow endocytosis because of the repulsive electrostatic interaction. (4) The symmetrical PAA21-b-PCL75 vesicles become unstable and precipitated after DOX loading due to the lack of PEO.

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Scheme 1. Illustration of DOX-Loaded Asymmetrical Polymer Vesicles (Red Arrows Indicated) for Efficient Intracellular Drug Delivery with Three Traditional Symmetrical Vesicles (Blue Arrows Indicated) as Controlsa

a

Blue: stealthy, biocompatible, and hydrophilic PEO. Purple: biodegradable and hydrophobic PCL. Green: pH-sensitive PAA (hydrophilic when deprotonated at higher pH). Claret: hydrophilic anticancer drug DOX in its HCl salt form. Orange: negative charges on PAA chains after deprotonation of −COOH. Chemical Industry) monomer was purified through a silica column to remove the inhibitor before use. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo in Japan. N,N,N′,N″,N″-Pentamethyldiethylenetriamine (PMDETA; 98%) and 2-bromoisobutyryl bromide were obtained from Aladdin Chemistry (Shanghai, China). Stannous 2ethylhexanoate (Sn(Oct)2, ∼95%), THF, CH2Cl2, anisole, and nhexane were purchased from Aladdin and used as received. Syntheses of Block Copolymers. The syntheses of block copolymers with different chains and degrees of polymerizations were provided in the Supporting Information (Figures S1−S9). Self-Assembly of Block Copolymer into Vesicles. PEO-b-PCLb-PAA copolymer vesicles were prepared by a solvent switching method. In brief, to continuously stirred deionized water (18.0 mL) was dropwise added 9 mL of THF solution of PEO-b-PCL-b-PAA (30 mg) by a gastight syringe. Then, the solution was transferred to a dialysis tube to dialyze against deionized water for 2 days by changing water three times each day to remove THF. Determination of the Critical Vesiculation Concentration. The critical vesiculation concentration (CVC) of the copolymer was estimated by fluorescence spectroscopy using pyrene as a hydrophobic

EXPERIMENTAL SECTION

Materials. Poly(ethylene oxide) methyl ether (MeO−PEO−OH; Mn = 1900 and 5000) was purchased from Alfa Aesar and dried azeotropically with toluene to remove traces of water. ε-Caprolactone (CL, Aldrich) was dried azeotropically using anhydrous toluene prior to use. Triethylamine (TEA) was dried by refluxing over CaH2 and distilled prior to use. tert-Butyl acrylate (tBA; purchased from Tokyo 3073

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fluorescence probe. The probe can be partitioned preferably in the vesicle membrane, causing changes in the photophysical properties of the vesicles under investigation. A stock solution of pyrene was made by dissolving pyrene (2 mg, 0.010 mmol) in acetone (12.5 mL) to form a 7.911 × 10−4 M solution. The pyrene solution (10 μL) was dropped into the empty centrifuge tubes and the acetone was evaporated after several hours in airing chamber. The block copolymer vesicle solution was serially diluted with deionized water from the concentration of 0.94 to 0.022 μg/mL. Each polymer solution (2 mL) was transferred to a vial containing pyrene and stirred overnight. The final pyrene concentration in the polymer solutions reached 1.236 × 10−7 M (which is less than the pyrene saturation concentration in water). Fluorescence determinations were made by exciting samples at 334 nm, using a 3 nm slit width for excitation and a 1.5 nm slit width for emission. Emission wavelengths were scanned from 350 to 500 nm. The intensities of the I3 (383 nm) to I1 (371 nm) vibronic bands were evaluated for each sample, and the ratios of I3/I1 were plotted against the logarithm of the concentration of each polymeric sample. The CVC was taken as the intersection of two regression lines calculated from the linear portions of the graphs. In Vitro Stability of Vesicles in Phosphate-Buffered Saline. The hydrodynamic diameter and the size distribution of the asymmetrical vesicles prepared from PEO 113-b-PCL 132 -b-PAA15 incubated in phosphate-buffered saline (PBS) (0.01 M at pH 7.4) for 7 days were evaluated by dynamic light scattering (DLS). Buffering Capacity Study. Relative buffering capacities of the asymmetrical PEO113-b-PCL132-b-PAA15 vesicles and the symmetrical PEO113-b-PCL110 vesicles were compared using acid−base titration.33 Initially, the pH value of the 0.33 mg/mL vesicle solutions was adjusted to pH 10 using 0.1 M NaOH. Sequentially, pH 1.5 HCl aliquots were added, and the pH value was measured by a pH meter after each addition. Drug Loading and in Vitro Release. The controlled release of drug was achieved according to the following protocol.34 9.0 mL of THF solution of 30.0 mg copolymer was dropwise added to 18.0 mL of fresh water solution of 3 mg DOX·HCl with continuous stirring in 2 h. When THF was removed by rotary evaporator at room temperature, this DOX-loaded polymer vesicles solution was further stirred for 12 h. Then, the unloaded free drug was removed by dialysis using a dialysis tube (cut off Mn = 8000−14 000) according to a reported procedure.3 The dialysis tube was immersed in 1000 mL of deionized water and dialyzed at 20 °C with a stirring rate of 300 r/min (in a 1000 mL beaker). Fresh dialysis medium was renewed five times in 2.5 h (0.5 h each), and the whole procedure was performed in the dark. Fluorescence spectroscopy before and after dialysis was to calculate DLE. The vesicle/DOX mixture after removing free drug was divided into six parts and immediately transferred to a new dialysis tube (cutoff Mn = 8000−14000). The final drug release process was carried out by dialyzing 5.0 mL of DOX-loaded vesicles in the dialysis tube against 100 mL tris buffer (0.01 M; pH 7.4 or pH 5.0) in a beaker (250 mL) at 37 °C and 190 r/min of stirring rate. The volume of liquid in the beaker (outside of the dialysis tube) was ensured around 100 mL during the measurement. At different time intervals, the liquid in the beaker was measured with fluorescence spectroscopy (excitation at 461 nm and emission at 591 nm), and the cumulative release curve of DOX was obtained. The calibration curve was reported in the recent paper of our group.34 The DLC (drug loading content) and DLE were calculated according to the following equation.

fetal bovine serum (FBS) for 24 h at 37 °C in a humidified 5% CO2containing atmosphere. Then 20 μL of polymer vesicles, DOX-loaded vesicles and free DOX·HCl solutions with different concentrations were added and incubated with cells for another 72 h, respectively. Untreated cells served as a control group. At the end of the treatment, CCK-8 dye was added to each well, and the plates were incubated for another 1 h at 37 °C. Subsequently, the absorbance was measured by dual wavelength spectrophotometry at 450 and 630 nm using a microplate reader. Each treatment was repeated five times. The relative cell viability (%) was determined by comparing the absorbance at 450 nm with control wells containing only cell culture medium. Flow Cytometrical Analysis. HeLa cells were inoculated in a 24well plate (105 cells/well), which were preincubated for 24 h in a cell culture incubator at 37 °C in a 5% CO2-containing atmosphere. Then, various vesicles with 1.0 μg/mL DOX loading were added to the wells and then coincubated with the HeLa cells for 5 min, 20 min, and 1 h in the incubator, respectively. Following incubation, cells were washed with PBS and incubated for 10 min with 0.1% trypsin to detach them and to remove surface-bound material. After the incubation, one volume of serum was added to stop the trypsin treatment and 10 volumes of PBS were added to detach the cells completely. The cell suspension was centrifuged at 800 g, washed with PBS, centrifuged again, and resuspended in 500 μL of PBS. The positive rate and mean intensity were analyzed by flow cytometry (Beckman MofloXDP, 484 nm/591 nm). A minimum of 10 000 events/sample was analyzed. To quantify effects of various treatments on cellular uptake, the median of cell fluorescence distribution in experiment was normalized to the cell fluorescence distribution median in untreated control. Free DOX was performed as positive control. Intracellular Release of DOX·HCl. The cellular uptake and intracellular release behaviors of DOX-loaded asymmetrical vesicles were followed with confocal laser scanning microscopy (CLSM) using HeLa cells. 1 × 105 HeLa cells were seeded on the four-well glass bottom culture dish (627870, Cellview, Greiner Bio-one) using DMEM supplemented with 10% FBS for 24 h. The media were aspirated and replaced by 500 μL of fresh DMEM supplemented with 10% FBS. 50 μL of DOX-loaded asymmetrical vesicles (5.0 μg/mL) was added. The cells were incubated with DOX-loaded vesicles for 1, 2, or 24 h at 37 °C in a humidified 5% CO2-containing atmosphere. The culture media were removed, and the cells were rinsed three times with PBS. The cells were fixed with 4% paraformaldehyde and the cell nucleuses were stained with DAPI. CLSM images of cells were obtained using CLSM. Enzymatic Biodegradation of Asymmetrical Block Copolymer Vesicles. Biodegradation of the asymmetrical vesicles was in situ conducted in the DLS sample cells at 37 °C. In a typical experiment, 150 μL of Pseudomonas cepacia lipase aqueous solution (2.0 mg/mL) was directly added to 2.85 mL of vesicles dispersion (0.53 mg/mL). The asymmetrical vesicle solution with and without lipase was degraded on a shaking platform at 37 °C; then, the derived count rates of the vesicle solutions by DLS at different time intervals were evaluated. Characterization. DMF Gel Permeation Chromatography. The molecular weight of the PEO-b-PCL-b-PtBA triblock copolymer, PCLb-PtBA diblock copolymer, the PEO-Br, and PEO-b-PCL-Br macroinitiators were assessed by gel permeation chromatography (GPC), which was carried out with a Waters Breeze 1525 GPC analysis system with two PL mix-D columns using poly(ethylene oxide) (PEO, purchased from TOSOH) as standard. The mobile phase was DMF with 0.5 M LiBr at a flow rate of 1.0 mL min−1 and 40 °C. 1 H NMR Spectra. Proton nuclear magnetic resonance (1H NMR) spectra were recorded using Bruker AV 400 MHz spectrometers, with CDCl3 as solvent and TMS as standard at room temperature. DLS Studies. DLS studies of aqueous polymer vesicle and DOXloaded vesicle solutions were determined using Zetasizer Nano-ZS 90 (Malvern Instruments, Worcestershire, U.K.) at a fixed scattering angle of 90°. Each reported measurement was conducted for three runs. All aqueous aggregates solutions were analyzed using disposable cuvettes. The data were processed by cumulative analysis of the experimental

DLC (%) = (weight of drug encapsulated in vesicles/weight of polymer) × 100% DLE (%) = (weight of drug encapsulated in vesicles /weight of drug in feed) × 100%

Cytotoxicity Test. The cytotoxicity of the polymer vesicles and DOX-loaded vesicles against normal liver cells (L02) and HeLa cells were evaluated by measuring the inhibition of cell growth using the CCK-8 assay. First, L02 or HeLa cells were seeded with equal density in each well of 96-well plates (4000 cells/well) in 100 μL of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% 3074

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correlation function, and particle diameters were calculated from the computed diffusion coefficients using the Stokes−Einstein equation. Zeta Potential Studies. Zeta potential studies of aqueous polymer vesicle solution were determined using a Zetasizer Nano-ZS 90 (Malvern Instruments) equipped with a multipurpose autotitrator (MPT-2) at various pH. No background electrolyte was added. Each reported measurement was conducted for three runs. Transmission Electron Microscopy. TEM images were obtained using a JEOL JEM-2100F instrument at 200 kV equipped with a Gatan 894 Ultrascan 1 k CCD camera. 5 μL of diluted vesicles suspension was dropped onto a carbon-coated copper grid and was negatively stained with 1% phosphotungstic acid (pH 7.0) to prepare TEM samples. The water droplet was allowed to evaporate slowly under ambient conditions before measurement. Atomic Force Microscopy. AFM was employed to verify the hollow structure of the asymmetrical vesicles. The sample solutions in water at concentrations of 0.03 and 0.4 mg/mL, respectively, were dropped (10 μL) onto the substrate and dried at room temperature for 12 h. The fresh silicon wafer was used as the sample substrate, which was washed with acetone four times before sample preparing. The observation was conducted on a Seiko (SPA-300HV) instrument operating in tapping mode at 200−400 kHz drive frequency. Fluorescence Measurements. Fluorescence experiments were carried out to monitor the self-assembly procedure of PEO113-bPCL132-b-PAA15 with pyrene as internal probe (λex = 334 nm) via a Lumina Fluorescence Spectrometer (Thermo Fisher).

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much shorter PAA chains are located in its interior to minimize the interfacial tension and enhance the vesicle curvature.23,38,39 DLS (see Figure S12 in the Supporting Information) revealed the asymmetrical and symmetrical vesicles had an intensity-averaged hydrodynamic diameter (Dh) of around 100 to 150 nm with narrow distributions. The size of such asymmetrical and symmetrical vesicle is well suitable for biomedical applications.40 Transmission electron microscopy (TEM) studies in Figure 1 clearly confirmed the hollow

RESULTS AND DISCUSSION

Preparation of Block Copolymers. Five AB diblock and ABC triblock copolymers (Table 1) were synthesized by sequential ring-opening polymerization (ROP) of CL and atom transfer radical polymerization (ATRP) of tBA monomers, according to the modified procedures.35−37 PEO-b-PCL-b-PAA triblock copolymers with various chain lengths were synthesized in four steps as illustrated in Figure S1 in the Supporting Information: (1) A monohydroxy-functional diblock copolymer precursor (PEO-b-PCL−OH) was synthesized by ROP of CL in anhydrous toluene using a monohydroxy-capped PEO−OH macroinitiator. (2) Esterification of the terminal hydroxyl group was achieved by using 2bromoisobutyryl bromide to afford PEO-b-PCL-Br, the desired macroinitiator for ATRP. (3) The PEO-b-PCL-b-PtBA triblock copolymer was synthesized by ATRP at 60 °C, using CuBr/ PMDETA as the catalyst system and PEO-b-PCL-Br as the macroinitiator and tBA as the monomer. (4) The target triblock copolymer PEO-b-PCL-b-PAA was synthesized by hydrolysis of the tert-butyl ester groups of PtBA in trifluoroacetic acid (TFA)/DCM at room temperature. Notably, the GPC traces of PEO-b-PCL, PEO-b-PCL-b-PAA, and PAA-b-PCL copolymers showed a unimodal distribution with low polydispersities ranging from 1.19 to 1.33. Also, the 1H NMR analyses of the key polymers were discussed in the Supporting Information. Preparation of Asymmetrical and Symmetrical Block Copolymer Vesicles. During the self-assembly process at pH 6.8, for the copolymers without obvious hydrophilic asymmetry, the hydrophilic PEO and PAA chains were statistically expressed at both the interior and exterior of the PCL vesicle membrane to form traditional symmetrical vesicles. However, the PEO113-b-PCL132-b-PAA15 triblock copolymer with an obvious hydrophilic asymmetry (long PEO and short PAA chains) self-assembles into asymmetrical vesicles above a low CVC of 0.91 μg/mL (Figure S11 in the Supporting Information). The long PEO chains should be preferentially segregated to the exterior of the PCL membrane, whereas the

Figure 1. TEM images of vesicles prepared from (A) PEO113-bPCL110, (B) PEO113-b-PCL132-b-PAA15, (C) PEO43-b-PCL98-b-PAA25, and (D) PCL75-b-PAA21 block copolymer at pH 7.0. The polymer vesicles were stained by phosphotungstic acid.

structure of vesicles prepared from PEO113-b-PCL110, PEO113-bPCL132-b-PAA15, PEO43-b-PCL98-b-PAA25, and PCL75-b-PAA21 at pH 7.0. (See Section 3.1 in the SI for discussions.) The corresponding number-averaged diameters of these four kinds of vesicles were consistent with the DLS results. Moreover, AFM study further confirmed the vesicle formation (Figure 2). The marked area had a width of 140 nm, which was much higher than the height of 5.6 nm. This is due to the collapse of those soft and deformable vesicles on the silicon substrate. In addition, as shown in Figure S13 in the Supporting Information, the discrepancy between the diameters appearing on the AFM images (∼140 nm) and the hydrodynamic sizes (∼100 nm) was ascribed to the flattening effect on the silicon wafer during the AFM measurement.35 Zeta potential studies on the vesicles in Figure 3 provided strong evidence of the asymmetry of PEO113-b-PCL132-b-PAA15 triblock copolymer vesicles. As the electrostatic potential is at, or very close to, the surface of the shear plane, it is particularly sensitive to the chemical structure of the chains expressed at the outer surface of the vesicles. For example, for the PAA21-bPCL75 diblock copolymer, there is no doubt that the hydrophilic PAA chains are located on both the internal and external surfaces of the symmetrical vesicles. The addition of base strongly affects the zeta potentials of PAA21-b-PCL75 vesicles due to the deprotonation of PAA. In contrast, the zeta potentials of symmetrical PEO113-b-PCL110 diblock copolymer vesicles are approximately zero due to the nonionic PEO chains in the outer membrane. Because the PEO113-bPCL132-b-PAA15 triblock copolymer has a significantly larger relative volume fraction of PEO than PAA ( f PEO = 0.24, f PAA = 0.06, as shown in Table 1), it forms asymmetrical vesicles and 3075

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Figure 2. AFM images of asymmetrical vesicles prepared from PEO113-b-PCL132-b-PAA15 (Polymer 4) at pH 7.0. (A) Height contrast, (B) phase contrast, and (C) height profile along the scan line marked in panel A. The diameter/height aspect ratio is in agreement with a hollow structure.

volume fractions ( f PEO = f PAA = 0.13, Table 1), leading to nearly even distribution on both the interior and exterior of the vesicle. To evaluate the physiological stability of the asymmetrical vesicles prepared from PEO113-b-PCL132-b-PAA15 triblock copolymer, the vesicle in PBS was monitored through DLS in vitro for more than 80 h. Figure S13 in the Supporting Information shows the effect of the time on the hydrodynamic diameters of the asymmetrical vesicles in 0.01 M PBS at pH 7.4, in which there is no obvious change even after being incubated for 80 h. The phenomenon demonstrated the high stability of the asymmetrical vesicles in PBS and suggested potential stability during the in vivo circulation. Buffering Capacity. The endosomal escape ability of drug delivery vehicles after cellular internalization is still a great challenge.20 The proton sponge effect of a synthetic drug delivery vehicle has been reported to be a key factor in the swelling of endosomes, the escaping into the cytosol and the overall drug release.41 A significant buffering capacity of polymers that could be protonated at low pH increases the endosomal escape rate as a consequence of the remarkable acidification within this subcellular compartment.42 The asymmetrical vesicles prepared from PEO113-b-PCL132-bPAA15 in this study acted as a proton sponge because of the protonation of PAA chains under endosome low-pH condition (pH 5.0 to 6.0). Therefore, the buffering capacities of the asymmetrical and symmetrical vesicle solutions were tested by serial addition of HCl aliquots. The results from these titration experiments are summarized in Figure 4A. Compared with the symmetrical vesicles, the asymmetrical vesicles displayed much better buffering capacity attributed to its PAA chains that contain a large amount of −COOH groups. Enhanced Drug Loading and on Demand Release. Our asymmetrical vesicles with PAA chains preferentially located on the inside of vesicle membrane may improve the DLE of DOX·

Figure 3. Zeta potentials versus pH curves obtained for the aforementioned vesicles illustrating the membrane chain spatial segregation. (a) Triangles: symmetrical vesicles prepared from PEO113-b-PCL110 (Polymer 3). (b) Diamonds: asymmetrical vesicles prepared from PEO113-b-PCL132-b-PAA15 (Polymer 4). (c) Circles: symmetrical vesicles prepared from PEO43-b-PCL98-b-PAA25 (Polymer 2). (d) Stars: symmetrical vesicles prepared from PAA21-b-PCL75 (Polymer 5).

consequently exhibits a virtually identical zeta potential independent of the pH variation to that obtained for PEO113b-PCL110 vesicles (without PAA). Thus, the anionic PAA chains in PEO113-b-PCL132-b-PAA15 triblock copolymer are preferentially segregated within the interior of the vesicles, while the PEO chains are expressed at the membrane exterior. In addition, symmetrical vesicles prepared from PEO43-b-PCL98-bPAA25 triblock copolymer have a characteristic electrophoretic signature that lies between symmetrical vesicles prepared from PEO113-b-PCL110 or PAA21-b-PCL75 diblock copolymers because both the PEO and the PAA chains have identical 3076

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Figure 4. (A) Buffering capacity of 0.33 mg/mL asymmetrical vesicles (prepared from Polymer 4) and symmetrical vesicles (prepared from Polymer 3) solutions were adjusted to pH 10 by 0.1 M NaOH. Subsequently, aliquots of pH 1.5 HCl were added into the vesicle solutions. (B) pHdependent DOX release profiles of DOX-loaded asymmetrical vesicles (Polymer 4) and symmetrical vesicles (Polymers 2 and 3) in 0.01 M tris buffer at pH 7.4 and 5.0 and 37 °C.

Table 2. Characterization of DOX-Loaded Asymmetrical and Symmetrical Vesicles vesicle structure

composition

DLC (wt %)a

DLE (%)

size (nm)b

PDIb

ξDV (mV)c

ξV (mV)d

symmetrical asymmetrical symmetrical

PEO113-b-PCL110 PEO113-b-PCL132-b-PAA15 PEO43-b-PCL98-b-PAA25

3.27 3.79 4.12

32.7 38.0 41.2

94.6 107.3 114.1

0.126 0.125 0.172

−4.51 −3.92 −10.9

−5.34 −6.86 −19.8

Theoretical DLC was 10 wt %. bSize and PDI of DOX-loaded vesicles. cξ of DOX-loaded vesicles. dξ of vesicles. The size, PDI, and ξ are determined by DLS at 25 °C and 0.5 mg/mL.

a

Figure 5. Cytotoxicity of asymmetrical and symmetrical vesicles against (A) L02 cells and (B) HeLa cells at various concentrations. (C) Antitumor activity and (D) IC50 values of free DOX·HCl and DOX-loaded vesicles. L02 and Hela cells were incubated with polymer vesicles, DOX·HCl, or DOX-loaded vesicles for 48 h. The relative cell viabilities were determined by CCK-8 assays (n = 5).

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Figure 6. Intracellular efficiency of asymmetrical and symmetrical vesicles at various treatment times by flow cytometry. (A) DOX positive rate in HeLa cells. (B) DOX average intensity in HeLa cells. (C) Fluorescence-activated cell sorter (FACS) results after 5 min of coincubation with different vesicles and free DOX.

slower release rate than asymmetrical vesicles. After 16 h, the DOX release contents and release rates of both kinds of vesicles were almost the same. The DOX release content was much higher at pH 5.0 than that at pH 7.4 for the same DOX-loaded vesicles. The pHsensitive drug release relies on the protonation effect of PAA chains at low pH, which could break up the interaction between −COO− of PAA and −NH3+ of DOX·HCl and also could make the DOX more water-soluble, thus leading to the increased release of DOX in aqueous solution. Therefore, the pHsensitive asymmetrical vesicles prepared from PEO113-bPCL132-b-PAA15 are able to not only efficiently load DOX· HCl via the electrostatic interaction but also rapidly release it in response to endo/lysosomal pH, which makes them unique and particularly appealing for intracellular release. Cytotoxicity Study. The cytotoxicities of the asymmetrical Polymer 4 and symmetrical Polymers 2 and 3 vesicles against normal liver cells (L02) and HeLa cells were evaluated by using a sensitive colorimetric CCK-8 (Cell Counting Kit-8) assay over 48 h, whereby dehydrogenase activities were determined via the reduction of WST-8 (2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt) to a yellow-colored product (formazan). The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. L02 and HeLa cells were treated with the three kinds of polymer vesicles at various concentrations from 62.5 to 1000 μg/mL, respectively. The cell viabilities were calculated using the ratio of the numbers of L02 and HeLa cells of the treated group over the untreated control (Figure 5). As expected, the asymmetrical PEO113-b-PCL132-b-PAA15 and the symmetrical PEO113-bPCL110 vesicles treated with L02 and HeLa cells remained higher metabolic activities among the three groups at various concentrations after 48 h. Especially at 1.0 mg/mL, the cytotoxicity of the asymmetrical PEO113-b-PCL132-b-PAA15 and

HCl due to the electrostatic interactions between PAA and DOX. (See Figure S10 in the Supporting Information.) The in vitro drug encapsulation and release of doxorubicin (DOX)loaded asymmetrical PEO113-b-PCL132-b-PAA15 triblock copolymer vesicles were studied with three traditional symmetrical vesicles as controls: (1) PEO43-b-PCL98-b-PAA25 with longer PAA chains; (2) PEO113-b-PCL110 without PAA chains; and (3) PAA21-b-PCL75 without PEO chains. The theoretical DLC was set to be 10%. Table 2 showed that the actual DLCs for the PEO113-b-PCL110, PEO113-b-PCL132-b-PAA15, and PEO43-bPCL98-b-PAA25 vesicles were 3.27, 3.79, and 4.12%, respectively. With the volume fractions of PAA chains ( f PAA) increased from 0, 0.06 to 0.13, the DLEs of three kinds of vesicles were increased from 32.7, 38.0, to 41.2%. However, the DOX-loaded vesicles prepared from PAA21-b-PCL75 with the highest PAA volume fraction (f PAA = 0.16) precipitated from aqueous solution, as confirmed by the DLS study (Figure S14 in the Supporting Information). This is consistent with the hypothesis of electrostatic interaction-enhanced drug loading mechanism. (See Figure S10 in the Supporting Information.) To further confirm this hypothesis, we studied the zeta potentials of vesicles before and after loading DOX·HCl at pH 7.4 (Table 2). After the DOX·HCl was loaded onto the PAA chains of the vesicle corona, the ξ of vesicles prepared from PEO113-b-PCL110, PEO113-b-PCL132-b-PAA15, and PEO43-bPCL98-b-PAA25 increased from −5.34, −6.86, and −19.8 mV to −4.51, −3.92, and −10.9 mV, respectively, due to the consumption of −COO− groups. The in vitro kinetic release profiles of DOX-loaded vesicle solutions were carried out in 0.01 M tris buffer at 37 °C and pH 7.4 and 5.0, respectively. As shown in Figure 4B, in the first 12 h at pH 7.4, DOX was released from the asymmetrical PEO113b-PCL132-b-PAA15 vesicles faster than that from the symmetrical PEO43-b-PCL98-b-PAA25 vesicles, possibly because more DOX· HCl was loaded on the exterior of the PEO43-b-PCL98-b-PAA25 vesicle membrane via electrostatic interaction, leading to a 3078

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Figure 7. CLSM images of HeLa cells incubated with DOX-loaded asymmetrical vesicles prepared from PEO113-b-PCL132-b-PAA15 (Polymer 4) for 1, 2, and 24 h. For each panel, images from left to right show DOX·HCl fluorescence in cells (red), cell nucleus stained by DAPI (blue), and overlays of two images. It indicated rapid internalization of asymmetrical vesicles, fast release of DOX·HCl, and efficient kill of HeLa cells.

were indicated as DOX fluorescence positive ratio and average intensity at different time. The DOX-loaded asymmetrical vesicles consistently showed the highest positive ratio (Figure 6A) and average intensity (Figure 6B) among the three vesicles, which is comparable to the free DOX·HCl. What’s more, the rapid intracellular rate of DOX-loaded asymmetrical vesicles is also demonstrated by the 96% positive ratio of HeLa cells only after 5 min (Figure 6C). These results confirmed that the asymmetrical Polymer 4 vesicles possess the higher efficacy for drug delivery than the other two symmetrical vesicles. To further investigate the intracellular drug release behaviors of DOX-loaded vesicles, we used CLSM to study the DOXloaded asymmetrical Polymer 4 vesicles against HeLa cells. The cell nucleus was stained with DAPI (blue). As shown in Figure 7, remarkably, significant DOX·HCl fluorescence was observed in the nucleus of HeLa cells following 1 h incubation with DOX-loaded asymmetrical vesicles, indicating the rapid internalization of vesicles and efficient release of DOX·HCl inside cells. The longer incubation time (2 h) resulted in stronger DOX·HCl fluorescence in the nucleus of HeLa cells. It is critical for DOX·HCl to be released from vesicles and accumulate in the nucleus for anticancer activity via interaction with genomic DNA. Thus, after rapid uptake, the relatively high DOX·HCl concentrations resulted in cell death within 24 h

the symmetrical PEO113-b-PCL110 vesicles is significantly lower than the PEO43-b-PCL98-b-PAA25 vesicles (p < 0.01, n = 5), and the cell viabilities are more than 95%. This is because only the highly biocompatible PEO chains (without PAA) are expressed outside the vesicles membrane for the asymmetrical PEO113-bPCL132-b-PAA15 and the symmetrical PEO113-b-PCL110 vesicles. In contrast, for the symmetrical PEO43-b-PCL98-b-PAA25 vesicles, both PEO and PAA are located outside the vesicle membrane, leading to higher cytotoxicity than the asymmetrical vesicles. Overall, the asymmetrical vesicles from PEO113-bPCL132-b-PAA15 block copolymers showed an excellent noncytotoxicity to normal L02 cells and HeLa cancer cells at various concentrations (≤1000 μg/mL). Intracellular Efficiency and Drug Release. The intracellular efficiencies of the DOX-loaded vesicles against HeLa cells were investigated by flow cytometry. Polymers 2, 3, and 4 with the same DOX·HCl loading concentration (1.0 μg/mL) were added to HeLa cells. Meanwhile, 1.0 μg/mL of free DOX·HCl was tested as the positive control. After 5 min, 20 min, and 1 h of coincubation, HeLa cells were trypsinized and washed with PBS to remove most of the materials adherent to the cell surface. Then the samples were submitted for fluorescenceactivated cell sorter (FACS, Beckman MofloXDP) analysis at 484 nm/591 nm. The DOX-loaded vesicles uptake efficacies 3079

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incubation, as evidenced by the live-cell fluorescence in Figure 7 and the decrease in cell viability in Figure 5C,D. Usually, after long blood circulation, the DOX-loaded vesicles prefer to integrate into asymmetrical cell membrane.43 Then, the host cell membrane invaginates, forming many small endosomes with the encapsulated DOX-loaded vesicles. Once the DOX-loaded vesicles are successfully internalized into the targeted cells via endocytosis, their PAA coronas are protonated due to the more acidic cytosomal environment (pH ≈ 5 to 6).44 Subsequently, the anticancer drug, DOX·HCl, which is absorbed by the electrostatic interaction, will be quickly released within the endosomal organelle. At the same time, the increased osmotic pressure in the endosome leads to momentary swelling and subsequent disruption. In both cases, the DOX and vesicles will escape from the endosome into the intracellular environment, resulting in an efficient intracellular drug release. Several different endocytic pathways such as phagocytosis, pinocytosis and receptor-mediated endocytosis45 share the same feature that the cell uptake rate during the endocytosis is strongly dependent on the binding efficacies of vesicles to the cell membrane.11,46,47 The flow cytometric results revealed that the neutral asymmetrical vesicles prefer to integrate into the host cell membrane with asymmetrical phospholipid and protein membranes. Such integration is responsible for the antitumor activity of the DOX-loaded asymmetrical PEO113-bPCL132-b-PAA15 vesicles as efficient as the free DOX·HCl. In contrast, for the symmetrical PEO43-b-PCL98-b-PAA25 vesicles, their lowest cancer cell killing efficiency suggests that the DOXloaded vesicle with a negative charge cannot be efficiently internalized into the cell due to the electrostatic repulsion.48,49 Antitumor Activity Study. The antitumor activities of DOXloaded vesicles were investigated in HeLa cells by CCK-8 assay. Interestingly, the DOX-loaded asymmetrical vesicles from PEO113-b-PCL132-b-PAA15 copolymer retained the highest drug efficacy comparable to the free DOX·HCl and were consistently higher than the DOX-loaded PEO113-b-PCL110 and PEO43-b-PCL98-b-PAA25 symmetrical vesicles at various concentrations (Figure 5C). Furthermore, as shown in Figure 5D, the IC50 (inhibitory concentration to produce 50% cell death; calculated by SPSS) of DOX·HCl from the DOX-loaded asymmetrical PEO113-b-PCL132-b-PAA15 vesicles was estimated to be 0.487 μg/mL (equiv. 12.85 μg/mL of DOX-loaded asymmetrical vesicles), which was close to that of the free DOX·HCl (0.431 μg/mL) and much lower than those from the DOX-loaded PEO113-b-PCL110 (0.618 μg/mL) and PEO43-bPCL98-b-PAA25 symmetrical vesicles (1.076 μg/mL). The high antitumor activity of DOX-loaded asymmetrical vesicles indicates that the DOX·HCl has been delivered and released into the nucleus of HeLa cells with a high efficiency. In Vitro Enzymatic Biodegradation. The asymmetrical vesicles were subjected to enzymatic degradation of the central PCL block by Pseudomonas lipases. PCL is well known for its biodegradability, but its nonenzymatic degradation is very slow through a surface erosion mechanism.50−52 When immersed into the lipase solutions or implanted in vivo, much greater rates of chain scission and mass loss are observed.53 As shown in Figure 8, the biodegradation process can be monitored by measuring the decrease in the derived count rate of DLS with time, which is proportional to both the molecular weight and the concentration of vesicles.54 In this study, the asymmetrical PEO113-b-PCL132-b-PAA15 vesicles were subjected to enzymatic degradation of the central PCL block by Pseudomonas lipases, a

Figure 8. Derived count rate variation during enzymatic degradation and a macroscopic view after degradation of the asymmetrical PEO113b-PCL132-b-PAA15 (Polymer 4) vesicles without (circle, a) and with (square, b) Pseudomonas cepacia lipase at pH 7.4 and 37 °C. The concentrations of vesicles and lipase were 0.5 and 0.1 mg/mL, respectively. The derived count rate was measured by DLS.

family of enzymes showing a high activity for ester chain scission.53 At a shaking rate of 100 rpm and 37 °C, the polymer chains seemed to be broken, as the derived count rate decreased quickly. Finally, the polymer vesicle was degraded into PEO, PAA, and hydroxycarboxylic acid after several days. Overall, many factors should be considered to evaluate the performance of a drug delivery system, such as DLE, waterdispersibility, biocompatibility, biodegradability, cell-uptake rate, endosomal escape ability, and so on. Table 3 is an assessment of different kinds of DOX-loaded vesicles and free DOX reported in this paper. The asymmetrical vesicle shows much better performance than other drug delivery systems in this paper. The DLC and DLE of the asymmetrical vesicles are high but not the highest compared with the symmetrical vesicles with more PAA chains. However, the asymmetrical vesicles offer many other advantages such as faster endocytosis rate and better compatibility and endosomal escape ability than the symmetrical ones.

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CONCLUSIONS In summary, we have successfully developed a biocompatible and pH-sensitive asymmetrical vesicle based on PEO113-bPCL132-b-PAA15 triblock copolymer. Compared with symmetrical vesicles, this DOX-loaded asymmetrical vesicle demonstrates promising potential applications in nanomedicine and the following advantages: (1) The vesicles possess much better biocompatibility and are intrinsically stealthy due to the much longer PEO chains expressed on their outer surface. (2) They have high loading capacity of DOX·HCl because of the electrostatic interactions between DOX·HCl and the shorter PAA chains located in the interior of the vesicle without blemishing their biocompatibility. (3) They can rapidly get into the cell membrane by endocytosis, followed by the disappearance of the electrostatic interactions between DOX·HCl and PAA chains due to the protonation of −COO− groups within the endosomal acidic environment. Subsequently, DOX can be rapidly and efficiently released into the cell nucleus. Moreover, the method for preparing such asymmetrical polymer vesicle can be extended to prepare a range of biomedical materials. For example, some MRI contrast agents, such as superparamagnetic iron oxide nanoparticles, may be precipitated in the inner PAA coronas of the asymmetrical vesicles for cancer theranostics. 3080

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Table 3. Assessment of DOX-loaded Asymmetrical and Symmetrical Vesicles and Free DOXa properties

a

structure

composition

DLE

water-dispersibility

biocompatibility

biodegradability

cell-uptake rate

endosomal escape ability

asymmetrical symmetrical symmetrical symmetrical

PEO113-b-PCL132-b-PAA15 PEO43-b-PCL98-b-PAA25 PEO113-b-PCL110 PAA21-b-PCL75 DOX·HCl

+ ++ − +++

+ + + − +

++ + ++

+ + +

++ − ++

+ −

−

“+” means good. the more “+” there are, the better the property is. “−” means bad. No evaluation was performed in the blank area. (14) Zhu, Y. Q.; Fan, L.; Yang, B.; Du, J. Z. ACS Nano 2014, 8, 5022−5031. (15) Liu, Q. M.; Zhu, H. S.; Qin, J. Y.; Dong, H. Q.; Du, J. Z. Biomacromolecules 2014, 15, 1586−1592. (16) Du, J. Z.; Chen, Y. M. Angew. Chem., Int. Ed. 2004, 43, 5084− 5087. (17) Zhu, Y. Q.; Liu, L.; Du, J. Z. Macromolecules 2013, 46, 194−203. (18) Du, J. Z.; Willcock, H.; Patterson, J. P.; Portman, I.; O’Reilly, R. K. Small 2011, 7, 2070−2080. (19) Ghoroghchian, P. P.; Li, G. Z.; Levine, D. H.; Davis, K. P.; Bates, F. S.; Hammer, D. A.; Therien, M. J. Macromolecules 2006, 39, 1673− 1675. (20) Lomas, H.; Canton, I.; MacNeil, S.; Du, J.; Armes, S. P.; Ryan, A. J.; Lewis, A. L.; Battaglia, G. Adv. Mater. 2007, 19, 4238−4243. (21) Jiang, Y.; Chen, T.; Ye, F. W.; Liang, H. J.; Shi, A. C. Macromolecules 2005, 38, 6710−6717. (22) Wittemann, A.; Azzam, T.; Eisenberg, A. Langmuir 2007, 23, 2224−2230. (23) Stoenescu, R.; Meier, W. Chem. Commun. 2002, 3016−3017. (24) Blanazs, A.; Massignani, M.; Battaglia, G.; Armes, S. P.; Ryan, A. J. Adv. Funct. Mater. 2009, 19, 2906−2914. (25) Liu, G. J.; Ma, S. B.; Li, S. K.; Cheng, R.; Meng, F. H.; Liu, H. Y.; Zhong, Z. Y. Biomaterials 2010, 31, 7575−7585. (26) Du, Y. F.; Chen, W.; Zheng, M.; Meng, F. H.; Zhong, Z. Y. Biomaterials 2012, 33, 7291−7299. (27) Chiang, W. H.; Ho, V. T.; Huang, W. C.; Huang, Y. F.; Chern, C. S.; Chiu, H. C. Langmuir 2012, 28, 15056−15064. (28) Lee, R. J.; Wang, S.; Turk, M. J.; Low, P. S. Bioscience Rep. 1998, 18, 69−78. (29) Choucair, A.; Soo, P. L.; Eisenberg, A. Langmuir 2005, 21, 9308−9313. (30) Lee, N. S.; Lin, L. Y.; Neumann, W. L.; Freskos, J. N.; Karwa, A.; Shieh, J. J.; Dorshow, R. B.; Wooley, K. L. Small 2011, 7, 1998−2003. (31) Zhang, F. W.; Elsabahy, M.; Zhang, S. Y.; Lin, L. Y.; Zou, J.; Wooley, K. L. Nanoscale 2013, 5, 3220−3225. (32) Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J. J. Controlled Release 2011, 151, 220−228. (33) Cai, X. J.; Dong, C. Y.; Dong, H. Q.; Wang, G. M.; Pauletti, G. M.; Pan, X. J.; Wen, H. Y.; Mehl, I.; Li, Y. Y.; Shi, D. L. Biomacromolecules 2012, 13, 1024−1034. (34) Chen, W. Q.; Du, J. Z. Sci. Rep. 2013, 3, DOI: 10.1038/ Srep02162. (35) Du, J. Z.; Armes, S. P. Langmuir 2009, 25, 9564−9570. (36) Du, J. Z.; Armes, S. P. Soft Matter 2010, 6, 4851−4857. (37) Du, J. Z.; Armes, S. P. Langmuir 2008, 24, 13710−13716. (38) Luo, L. B.; Eisenberg, A. Angew. Chem., Int. Ed. 2002, 41, 1001− 1004. (39) Terreau, O.; Luo, L. B.; Eisenberg, A. Langmuir 2003, 19, 5601−5607. (40) Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Muller, M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Nat. Mater. 2010, 9, 101−113. (41) Moreira, C.; Oliveira, H.; Pires, L. R.; Simoes, S.; Barbosa, M. A.; Pego, A. P. Acta Biomater. 2009, 5, 2995−3006. (42) Sonawane, N. D.; Szoka, F. C.; Verkman, A. S. J. Biol. Chem. 2003, 278, 44826−44831.

Overall, our asymmetrical vesicle exhibits fast endocytosis rate, rapid endosomal escape ability, efficient DOX loading, and rapid intracellular drug release on demand as well as excellent biodegradability, suggesting their promising potential applications in nanomedicine.

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ASSOCIATED CONTENT

S Supporting Information *

Full synthetic details and more discussions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

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

Q.L. and J.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.D. is supported by Shanghai 1000 Plan, Eastern Scholar Professorship, NSFC (21174107 and 21374080), Ph.D. Program Foundation of Ministry of Education (20110072110048), Fok Ying Tong Education Foundation (132018), and the fundamental research funds for the central universities.

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REFERENCES

(1) Tockary, T. A.; Osada, K.; Chen, Q. X.; Machitani, K.; Dirisala, A.; Uchida, S.; Nomoto, T.; Toh, K.; Matsumoto, Y.; Itaka, K.; Nitta, K.; Nagayama, K.; Kataoka, K. Macromolecules 2013, 46, 6585−6592. (2) Lian, T.; Ho, R. J. Y. J. Pharm. Sci. 2001, 90, 667−680. (3) Ren, T. B.; Liu, Q. M.; Lu, H.; Liu, H. M.; Zhang, X.; Du, J. Z. J. Mater. Chem. 2012, 22, 12329−12338. (4) Du, J. Z.; Fan, L.; Liu, Q. M. Macromolecules 2012, 45, 8275− 8283. (5) Zhou, C. C.; Wang, M. Z.; Zou, K. D.; Chen, J.; Zhu, Y. Q.; Du, J. Z. ACS Macro Lett. 2013, 1021−1025. (6) Sun, L.; Du, J. Z. Polymer 2012, 53, 2068−2073. (7) Du, J. Z.; O’Reilly, R. K. Soft Matter 2009, 5, 3544−3561. (8) Du, J. Z.; Tang, Y. Q.; Lewis, A. L.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 17982−17983. (9) Hu, X.; Hu, J.; Tian, J.; Ge, Z.; Zhang, G.; Luo, K.; Liu, S. J. Am. Chem. Soc. 2013, 135, 17617−17629. (10) Salva, R.; Le Meins, J. F.; Sandre, O.; Brulet, A.; Schmutz, M.; Guenoun, P.; Lecommandoux, S. ACS Nano 2013, 7, 9298−9311. (11) Massignani, M.; LoPresti, C.; Blanazs, A.; Madsen, J.; Armes, S. P.; Lewis, A. L.; Battaglia, G. Small 2009, 5, 2424−2432. (12) Du, J. Z.; O’Reilly, R. K. Macromol. Chem. Phys. 2010, 211, 1530−1537. (13) Zhu, Y. Q.; Wang, F. Y. K.; Zhang, C.; Du, J. Z. ACS Nano 2014, DOI: 10.1021/nn502386j. 3081

dx.doi.org/10.1021/bm500676e | Biomacromolecules 2014, 15, 3072−3082

Biomacromolecules

Article

(43) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug. Discovery 2005, 4, 581−593. (44) Kim, T.; Rothmund, T.; Kissel, T.; Kim, S. W. J. Controlled Release 2011, 152, 110−119. (45) Conner, S. D.; Schmid, S. L. Nature 2003, 422, 37−44. (46) Zhang, X. P.; Jin, Y. J.; Pllummer, M. R.; Pooyan, S.; Gunaseelan, S.; Sinko, P. J. Mol. Pharmaceut 2009, 6, 836−848. (47) Amand, H. L.; Bostrom, C. L.; Lincoln, P.; Norden, B.; Esbjorner, E. K. BBA Biomembranes 2011, 1808, 1860−1867. (48) Ziegler, A. Adv. Drug Deliver Rev. 2008, 60, 580−597. (49) Fuchs, S. M.; Raines, R. T. Biochemistry 2004, 43, 2438−2444. (50) Kweon, H.; Yoo, M. K.; Park, I. K.; Kim, T. H.; Lee, H. C.; Lee, H. S.; Oh, J. S.; Akaike, T.; Cho, C. S. Biomaterials 2003, 24, 801−808. (51) Atzet, S.; Curtin, S.; Trinh, P.; Bryant, S.; Ratner, B. Biomacromolecules 2008, 9, 3370−3377. (52) Zhang, Q.; Remsen, E. E.; Wooley, K. L. J. Am. Chem. Soc. 2000, 122, 3642−3651. (53) Herzog, K.; Muller, R. J.; Deckwer, W. D. Polym. Degrad. Stab. 2006, 91, 2486−2498. (54) Sanson, C.; Schatz, C.; Le Meins, J. F.; Brulet, A.; Soum, A.; Lecommandoux, S. Langmuir 2010, 26, 2751−2760.

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