Comb-like Amphiphilic Copolymers Bearing Acetal-Functionalized

Article pubs.acs.org/Biomac

Comb-like Amphiphilic Copolymers Bearing Acetal-Functionalized Backbones with the Ability of Acid-Triggered Hydrophobic-toHydrophilic Transition as Effective Nanocarriers for Intracellular Release of Curcumin Junqiang Zhao,†,‡,⊥ Haiyang Wang,§,⊥ Jinjian Liu,∥ Liandong Deng,§ Jianfeng Liu,∥ Anjie Dong,†,‡,§ and Jianhua Zhang*,‡,§ †

School of Materials Science and Engineering, Tianjin University, Tianjin, 300072, People’s Republic of China Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin University, Tianjin, 300072, People’s Republic of China § Synergetic Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People’s Republic of China ∥ Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, 300192, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: The pH-responsive micelles have enormous potential as nanosized drug carriers for cancer therapy due to their physicochemical changes in response to the tumor intracellular acidic microenvironment. Herein, a series of comb-like amphiphilic copolymers bearing acetal-functionalized backbone were developed based on poly[(2,4,6trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate-co-poly(ethylene glycol) methyl ether methacrylate] [P(TTMA-co-mPEGMA)] as effective nanocarriers for intracellular curcumin (CUR) release. P(TTMA-co-mPEGMA) copolymers with different hydrophobic−hydrophilic ratios were prepared by one-step reversible addition fragmentation chain transfer (RAFT) copolymerization of TTMA and mPEGMA. Their molecular structures and chemical compositions were confirmed by 1H NMR, Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). P(TTMA-co-mPEGMA) copolymers could self-assemble into nanosized micelles in aqueous solution and displayed low critical micelle concentration (CMC). All P(TTMA-comPEGMA) micelles displayed excellent drug loading capacity, due to the strong π−π conjugate action and hydrophobic interaction between the PTTMA and CUR. Moreover, the hydrophobic PTTMA chain could be selectively hydrolyzed into a hydrophilic backbone in the mildly acidic environment, leading to significant swelling and final disassembly of the micelles. These morphological changes of P(TTMA-co-mPEGMA) micelles with time at pH 5.0 were determined by DLS and TEM. The in vitro CUR release from the micelles exhibited a pH-dependent behavior. The release rate of CUR was significantly accelerated at mildly acidic pH of 4.0 and 5.0 compared to that at pH 7.4. Toxicity test revealed that the P(TTMA-co-mPEGMA) copolymers exhibited low cytotoxicity, whereas the CUR-loaded micelles maintained high cytotoxicity for HepG-2 and EC-109 cells. The results indicated that the novel P(TTMA-co-mPEGMA) micelles with low CMC, small and tunable sizes, high drug loading, pHresponsive drug release behavior, and good biocompatibility may have potential as hydrophobic drug delivery nanocarriers for cancer therapy with intelligent delivery.

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INTRODUCTION Due to poor solubility, low stability, rapid elimination, and undesirable toxicity, a great many anticancer drugs are often limited in their therapeutic capabilities. Therefore, there has always been an urgent demand to develop drug delivery systems to improve the pharmacokinetics and biodistribution of drugs.1−3 Owing to their unique structures and properties, polymeric nanoparticles self-assembled from amphiphilic © 2013 American Chemical Society

copolymers represent a promising carrier for delivery of various drugs. They are capable of solubilizing hydrophobic drugs, avoiding rapid renal exclusion, prolonging circulation, and facilitating passive accumulation at pathological sites.4 HowReceived: July 24, 2013 Revised: October 5, 2013 Published: October 10, 2013 3973

dx.doi.org/10.1021/bm401087n | Biomacromolecules 2013, 14, 3973−3984

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Scheme 1. Illustration of pH-Sensitive Micelles Self-Assembled from P(TTMA-co-mPEGMA) Comb-like Copolymers with AcidCleavable Cyclic Benzylidene Acetals for Active Loading and pH-Triggered Release of CUR

ity.27,28 In some previous studies, cyclic benzylidene acetals have been introduced into linear amphiphilic block copolymers, and their self-assemblies have achieved significant success in improving drug efficacy.25 However, the micelles self-assembled from linear block copolymers displayed low resistance to dilution-induced disassembly in vivo and relatively small loading capacity.30−33 In contrast to the normal linear block copolymers, some nonlinear copolymers,34,35 such as toothbrush-like, comb-like, and star-like or dendrimers, have been proven to exhibit significantly different properties from their linear analogues. Among these amphiphilic polymers with more complex macromolecular architectures, the comb-shaped amphiphiles have obtained a greater amount of attention in biomedical applications, because of their unique compositional flexibility and the simplicity of preparation.36 Besides the melt rheology, mechanical behavior, and thermal property, combshaped amphiphilic copolymers show different solubility and aggregation behavior in water.37 Comb-shaped amphiphiles have great steric hindrance for both the soluble and insoluble components, which can assemble forming looser and larger aggregates by the intramolecular aggregation mechanism.38 These well-defined aggregates perhaps can provide greater drug encapsulation abilities and unique drug release kinetics.30,34 Nevertheless, very little research has been focused on pHsensitive polymeric nanoparticles assembled from comb-shaped amphiphiles containing acid-cleavable links, especially cyclic benzylidene acetals. Therefore, it is imperative to construct acetal-functionalized comb-shaped copolymers and explore their potential as functional nanocarriers for controlled drug delivery. Undoubtedly, such studies will also improve our comprehension of the structure−property relationship in drug delivery systems. Curcumin [CUR, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione; Scheme 1] is a hydrophobic polyphenolic compound derived from dietary spice turmeric. CUR has been proven to possess antioxidant, anti-inflammatory, antimicrobial, and antitumor effect, but its application in cancer therapy is limited because of its low aqueous solubility, rapid systemic elimination, and thus poor bioavailability.39−41

ever, despite the fact that tremendous progress has been achieved, few of the nanocarriers produce optimal outcomes, due to lack of controllable drug release inside targeted cells. To address the challenges, considerable efforts have been devoted to the development of new nanocarriers that are responsive to internal and external stimuli, such as pH, temperature, redox, enzyme, light, and so on.5−9 Considering the various pH gradient in the living body, pHresponsiveness has attracted the most considerable research interest. The physiological pH of blood and normal tissues is about 7.4, but the extracellular pH in tumor tissues is about 6.8 and the intracellular endo/lysosomal pH ranges from 4.0 to 6.5. The differences in pH between cancer tissues and normal tissues are advantageous for the specific targeting tumor and controlled release drugs at the tumor site.10−12 The polymers containing amines or carboxylic groups with different chemical structures could undergo a solubility change, structure degradation, or de-cross-linking in response to pH decrease.13,14 Another popular approach is to incorporate acidlabile linkages, such as acetal,15,16 orthoester,17,18 hydrazine,19 oxime bonds,20 and cis-acotinyl,21 which can be rapidly cleaved under mildly acidic conditions, resulting in the formation of a new hydrophilic polymer chain. The hydrophobic−hydrophilic transition would destroy the nanostructure of polymeric nanocarriers, leading to the release of their payloads. In recent years, much attention has been paid to the acid-cleavable cyclic benzylidene acetals.16,22−29 Compared with other acid sensitive linkages, they possess several favorable characteristics. First, cyclic benzylidene acetals contain an aromatic ring, which will contribute to micelle formation due to the hydrophobicity of aromatic rings. Moreover, most hydrophobic drugs contain aromatic or cyclic rings. The aromatic groups as the hydrophobic moiety cause a strong π−π interaction with the drug molecules, which would lead to higher drug encapsulation.25 Second, the cyclic benzylidene acetals can be rapidly changed into polar diol moieties within the physiologically accessible pH range (4.0−6.5), causing the desired hydrophobic-to-hydrophilic transformation.22 And third, polymers containing cyclic benzylidene acetals exhibit low cytotoxic3974


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Scheme 2. Preparation of P(TTMA-co-mPEGMA) via One-Step RAFT Copolymerization

a solvent and tetramethylsilane (TMS) as the internal standard. The molecular weight and molecular weight polydispersity index (Mw/Mn) of the polymers were measured with a Waters 1515 gel permeation chromatographer (GPC, Waters company, Milford, USA) equipped with refractive index detector, using PLgel MIXED C (MW 200−3M), PLgel C (MW500−20K) and PLgel C (MW4−400K) column with molecular weight range 100−500 000 calibrated with polystyrene standards. THF was used as an eluent at a flow rate of 1.0 mL/min. Dynamic laser scattering (DLS) measurements were performed on a Brookhaven BI-200SM (Brookhaven Instruments Co., Holtsville, USA) at λ = 532 nm with a fixed detector angle of 90°. TEM measurements were performed on a JEOL JEM-1011 transmission electron microscope with an accelerating voltage of 100 kV. A drop of the micelle solution (1.0 mg/mL) was deposited onto a 230 mesh copper grid coated with carbon and allowed to dry in air at 25 °C before measurements. Synthesis of P(TTMA-co-mPEGMA). A series of comb-like P(TTMA-co-mPEGMA) copolymers with different chemical compositions were synthesized by one-step RAFT copolymerization of mPEGMA and TTMA, as shown in Scheme 2. Typically, mPEGMA (0.40 g, 0.40 mmol), TTMA (0.40 g, 1.10 mmol), AIBN (1.64 mg, 10.0 μmol), CPDTC (9.90 mg, 40.0 μmol), and THF (6.50 mL) were added into a 25 mL Schlenk tube. The mixture was degassed through three freeze−pump−thaw cycles. The polymerization tube was placed in a thermostatic water bath at 60 °C for about 24 h under nitrogen atmosphere, which was finally quenched by immersing the tube into liquid nitrogen. And then the reaction mixture was precipitated into an excess of cold n-hexane. The obtained product was dried overnight in a vacuum oven at 25 °C, yield: 92.5%. CMC Measurement. CMC was measured by steady-state fluorescent-probe methodology using pyrene as a probe on a Varian fluorescence spectrophotometer at 25 °C.43,44 Sample solutions for fluorescence investigation were prepared as described previously. The copolymer concentrations in this experiment varied from 1.0 × 10−6 to 1.0 mg/mL. The final pyrene concentration in copolymer solution was kept at 6.0 × 10−7 M. These solutions were shaken vigorously and then allowed to equilibrate at 25 °C for at least 24 h. The excitation spectra of pyrene with various copolymer concentrations were measured at the detection emission wavelength (λem = 373 nm). The CMC value was obtained from the intersection of the tangent to the horizontal line of I337/I330 with relative constant value and the diagonal line with rapidly increased I337/I330 ratio. Formation and Characterization of Micelles. P(TTMA-comPEGMA) copolymer micelles were prepared by a nanoprecipitation method.45 Typically, the copolymer (10.0 mg) was dissolved in 1.0 mL of THF. Then, the polymer solution was dropwise added to 10.0 mL phosphate buffer solution (PBS, 10 mM, pH 7.4) under magnetic stirring. The micelles were formed immediately, and the resulting solution was stirred overnight to allow complete evaporation of THF at room temperature. The core−shell nanostructure of P(TTMA-comPEGMA) micelles was confirmed by 1H NMR measurements. The freeze-dried powder of P(TTMA-co-mPEGMA) micelles was dis-

In this study, we purposed a new strategy for controlled intracellular release of CUR by the use of comb-like amphiphilic copolymers with acid-cleavable acetal-functionalized backbone (Scheme 1). The pH-sensitive amphiphilic copolymers, poly[(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate-co-poly(ethylene glycol) methyl ether methacrylate] [P(TTMA-co-mPEGMA)], were synthesized by one-step reversible addition fragmentation chain transfer (RAFT) copolymerization, as shown in Scheme 2. The obtained comb-like copolymers were comprised of hydrophilic poly(ethylene glycol) (PEG) pendent chains and a hydrophobic backbone based on cyclic benzylidene acetals. Their chain structures and chemical compositions were characterized by 1H NMR, Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC). The self-assembly behaviors and the CUR release profiles from copolymer aggregates under different pH solutions were investigated. The cell uptake and cytotoxicity of CUR-loaded P(TTMA-comPEGMA) micelles were also determined. The results confirmed that the comb-shaped copolymers P(TTMA-comPEGMA) bearing acetal-functionalized backbones possessed good properties for application as smart nanomedicine platforms for anticancer drug delivery.

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EXPERIMENTAL SECTION

Materials. 2,4,6-Trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate (TTMA) was synthesized according to a previous report by Grinstaff (Scheme S1, Supporting Information).24 The structure of TTMA was confirmed by 1H NMR (Figure S1). 2,4,6Trimethoxybenzaldehyde (98%), methacryloyl chloride (97%), 1,1,1tris(hydroxymethyl) ethane (99%), p-toluenesulfonic acid monohydrate (97%), n-dodecylthiol (99%), carbon disulfide (99%), pyrene (99%), azobisisobutyronitrile (AIBN, 98%), fetal bovine serum (FBS), and curcumin (CUR, 98%) were used as received from Aldrich. Tetrahydrofuran (THF), diethyl ether, ethyl acetate, dichloromethane (DCM), hexane, triethylamine, sodium hydride, solid iodine, and anhydrous magnesium sulfate were provided by Jiangtian company (Tianjin, China). Poly(ethylene glycol) methyl ether methacrylate macromonomer (mPEGMA, Mn = 1000 g/mol, MW/Mn = 1.2, Alfa Aesar) were purified by passing through an alkaline aluminum oxide column to remove inhibitor. AIBN was recrystallized twice from ethanol. 2-Cyanopropan-2-yl dodecyl trithiocarbonate (CPDTC) was synthesized according to the described procedure (Scheme S2),42 which was confirmed by 1H NMR (Figure S2). Characterizations. Fourier transform infrared spectroscopy (FTIR) was carried out using KBr disks in the region of 4000−500 cm−1 by using BIO-RAD FT-IR 3000 (BIO-RAD Company, Hercules, USA). 1H NMR spectra of the products were recorded on a Varian Inova-500 M instrument (Varian Inc., Palo Alto, USA) with CDCl3 as 3975


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persed into D2O with the concentration of 1.0 mg/mL for 1H NMR measurement. The solutions of freeze-dried P(TTMA-co-mPEGMA) micelles in CDCl3 was also examined as control. The size, size distribution, and morphology of the micelles were determined by DLS and TEM, respectively. The micelles suspension was filtered through a 450 nm syringe filter before measurements. Meanwhile, the colloidal stability of the P(TTMA-co-mPEGMA) micelles in PBS 7.4 with or without 10% FBS were investigated by DLS.46 pH-Triggered Hydrolysis of Acetal Groups in the Micelles. The acetal hydrolysis was followed by UV/vis spectroscopy by measuring the absorbance at 292 nm, according to the previous reports by Frechet and co-workers.24 P(TTMA-co-mPEGMA) micelle solutions (1.0 mg/mL) were prepared and equally divided into three groups. Each test tube contained 2.0 mL micelles solution. Their pH were adjusted to 4.0 and 5.0 by addition of 4.0 M pH 4.0 and 5.0 acetate buffer or maintained at pH 7.4 using phosphate buffer, respectively. The solutions were shaken at 37 °C. At desired time intervals, 80 μL aliquot was taken out and diluted with 3.5 mL phosphate buffer (10 mM, pH 7.4). The absorbance at 292 nm was monitored. At the end, all the samples were completely hydrolyzed by the addition of two drops of concentrated HCl and were measured again to determine the absorbance at 100% hydrolysis, which was used to calculate the degree of acetal hydrolysis. Acetal hydrolysis of P(TTMA-co-mPEGMA) micelles was further tracked by steady-state fluorescent-probe methodology using pyrene as a probe on a Varian fluorescence spectrophotometer at 25 °C. First, water-insoluble pyrene was encapsulated into P(TTMA-co-mPEGMA) micelles (10 mL, 1.0 mg/mL), and the final pyrene concentration in micellar solution was 11.2 μg/mL. The fluorescence intensity was measured in time by a Varian fluorescence spectrophotometer under pH 7.4 5.0, and 4.0 conditions.47 Size Changes of Micelles in Response to Acetal Hydrolysis. The changes in size of P(TTMA-co-mPEGMA) micelles in response to acetal hydrolysis were followed by DLS. The micelles solutions under pH 7.4 and 5.0 were prepared as above. The samples were gently stirred at 37 °C. The changes in micelle size were monitored in time by DLS. Drug Loading and in Vitro pH-Triggered Release. CUR was used as a model drug for drug loading. CUR-loaded P(TTMA-comPEGMA) micelles were prepared by a nanoprecipitation technique.46 For example, P(TTMA-co-mPEGMA) (10.0 mg) and CUR (1.0 mg) were mixed in 2.0 mL of THF, followed by dropwise addition of 10 mL phosphate buffer (10 mM, pH 7.4). To completely evaporate THF, the solution was stirred overnight and then vacuumed for 1 h. Then, the solution was filtered and lyophilized for determination of drug loading content (DLC) and drug loading efficiency (DLE). The drug loaded micelles was dissolved in THF and analyzed with a UV/ vis spectrophotometer at 425 nm using a standard curve method.48 The DLC and DLE of the drug loaded micelles were calculated according to eqs 1 and 2, respectively:

out for UV/vis measurement and replenished with an equal volume of fresh media. The amount of released drug in the incubation medium was quantified by UV/vis spectrophotometer. Cumulative release of CUR from P(TTMA-co-mPEGMA) micelles in PBS (pH 7.4) containing 2% (w/w) Tween 80 for high loading percentages of the CUR (19.3%) was also studied. The release experiments were conducted in triplicate. The results presented are the average data with standard deviations. Cell Uptake Studies. The cellular uptake of CUR was studied toward human hepatoma (HepG-2) cells using fluorescence microscopy. Cells were seeded onto glass coverslips in a six-well culture plate at a density of 1 × 104 cells/well. After 24 h culture, the cells were treated with free CUR (10 μg/mL) or CUR-loaded micelles (10 μg/mL, equivalent CUR concentration) for 2 h. After incubation, the cell monolayers were rinsed three times with 1 mL PBS (10 mM, pH 7.4) to remove excess micelles or free CUR. Fresh PBS (10 mM, pH 7.4) was added to the plates and the cells were viewed and imaged under a confocal laser scanning microscope (CLSM, Leica AF 6500, Leica Microsystems GmbH, Germany). Similarly, for time-dependent cellular uptake studies of free CUR and CUR-loaded micelles, the glass coverslips were removed from the incubator at predetermined time intervals, and the cells were processed using the above confocal studies protocol. Cell Viability Assays. The cytotoxicity of P(TTMA-co-mPEGMA) micelles was studied by MTT assays using HepG-2 cells and esophageal carcinoma (EC-109) cells. In brief, the cells were seeded in 96-well plates at 8000 cells per well in 100.0 μL of complete Dulbecco’s modified Eagle’s medium (DMEM) and incubated at 37 °C in a 5% CO2 atmosphere for 24 h, followed by removing the culture medium and adding micelle solutions at different concentrations (0−1.0 mg/mL). The cells were subjected to MTT assay after being incubated for another 48 h. The absorbance of the solutions was measured on a Bio-Rad 680 microplate reader at 570 nm. The Cellular proliferation inhibition of the CUR-loaded micelles against HepG-2 and EC-109 cells was also evaluated in vitro by MTT assay. All aspects were operated under a condition similar to that for P(TTMA-comPEGMA) micelles. After washing the cells with PBS, the free CUR or CUR-loaded micelles were added at different CUR concentrations (0−100.0 μg/mL). Then all the treatment processes were identical to the previous procedure as mentioned above.

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RESULTS AND DISCUSSION Synthesis and Characterization of P(TTMA-co-mPEGMA). The smart stimuli-responsive drug nanocarriers based on amphiphilic polymers with acid cleavable bonds can offer some unique advantages for efficient intracellular release of anticancer drugs, as they can allow for active delivery of the encapsulated payloads to specific cancer cells. The aim of this study was to design and prepare a series of novel comb-like amphiphilic polymer P(TTMA-co-mPEGMA) bearing hydrophilic PEG as the side chains and containing acid-labile acetal groups in the hydrophobic backbone. The mPEG as corona-forming blocks can provide biocompatibility and a stealth effect on the obtained micelles. Meanwhile, the hydrophobic PTTMA backbone can be selectively hydrolyzed into a hydrophilic backbone in the acidic environment, leading to the destruction of the nanostructure and the rapid intracellular drug release. A series of P(TTMA-co-mPEGMA) polymers were obtained by one-step RAFT copolymerization of mPEGMA and TTMA monomers using CPDTC as RAFT agent because of its wellcontrolled ability for the RAFT polymerizations of methacrylate derivatives.42 The hydrophobic−hydrophilic ratios in P(TTMA-co-mPEGMA) polymers were regulated by the molar mass ratio of TTMA to mPEGMA in the initial feed. The chain structures and chemical compositions of the copolymers were characterized by FT-IR, 1H NMR, and GPC and were shown in Figure 1, Figure 2, and Figure S3, respectively. It can be seen

DLC (%) = (amount of loaded drug) /(amount of drug − loaded micelle) × 100%

(1)

DLE (%) = (amount of loaded drug) /(total amount of feeding drug) × 100%

(2)

The release profiles of CUR from P(TTMA-co-mPEGMA) micelles were investigated at 37 °C in three different media, i.e., (a) acetate buffer, pH 4.0; (b) acetate buffer, pH 5.0; (c) phosphate buffer, pH 7.4. The concentrations of the release media were 10 mM. The above prepared CUR-loaded micelles were divided into into three groups (each 5.0 mL). Their pH were adjusted to 4.0 or 5.0 using acetate buffer or maintained at pH 7.4 using phosphate buffer. Then, 5.0 mL of CUR-loaded micelle dispersions (at low theoretical DLC of 5.0%) and immediately transferred to a dialysis tube with a MWCO of 3.5 kDa. The dialysis tube was immersed into 25 mL of corresponding buffer containing Tween-80 (0.5% w/w) at 37 °C under gentle shaking. At desired time intervals, 5.0 mL of release media was taken 3976


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Figure 1. FT-IR spectra of TTMA, mPEGMA, and P(TTMA-co-mPEGMA).

mPEGMA. In addition, the signals of methane, methylene and methyl protons in backbone (1.15−2.35 ppm, a, b, c, e, f) were present in the spectra of P(TTMA-co-mPEGMA). And the characteristic peaks of PTTMA were observed at about δ 6.09 (n), δ 5.92 (p), and 3.78−3.90 ppm (l), respectively. These results confirmed the formation of P(TTMA-co-mPEGMA). The peak intensities of the hydrogen protons on the phenyl ring of TTMA (at about at δ 6.09, n), ethylene protons of the PEG (at about δ = 3.65 ppm, h) and methylene protons of the CPDTC (at about δ = 1.25, e) can be used to calculate the chemical composition and Mn value of P(TTMA-co-mPEGMA). The GPC curves of P(TTMA-co-mPEGMA) were shown in Figure S3. It can be found that all of the polymers showed symmetrical and unimodal GPC curve with narrow molecular weight distributions (MW/Mn < 1.30). The molecular weights that were calculated from the ratio of 1H NMR peak area and obtained by GPC were listed in Table 1. It was found that the molecular weight calculated from 1H NMR and the molecular weight determined by GPC were both well-agreement with the theoretical values, indicating the well-controlled nature of this RAFT copolymerization. Consequently, all the results demonstrated that comb-like copolymers P(TTMA-co-mPEGMA) with precisely controlled architecture and predetermined chain composition were synthesized via one-step RAFT copolymerization. Preparation and Characterization of P(TTMA-comPEGMA) micelles. CMC as the fundamental parameter of the micellization behavior of amphiphilic copolymers in dilute aqueous solutions was determined by a fluorescence technique with pyrene as a probe. It has been reported that fluorescence spectra of pyrene solutions contain a vibrational band exhibiting

Figure 2. 1H NMR spectrum of P(TTMA-co-mPEGMA) in CDCl3.

from Figure 1 that the P(TTMA-co-mPEGMA) exhibited characteristic peaks of both PTTMA and mPEGMA, such as the aromatic −C−H and −C−C− stretching vibrations at 2945, 1609, and 738 cm−1, the asymmetric deformation vibrations of −C−H of CH3 at 1367 cm−1and the stretching vibrations of −O−CH3 at 1030 cm−1, respectively. Moreover, the typical signals of the methylene (CH2), carbonyl (−CO) and ether bond (C−O−C) at 2875, 1735, and 1110 cm−1 can also be found in FT-IR spectrum of P(TTMA-co-mPEGMA). The 1H NMR spectra of P(TTMA-co-mPEGMA) copolymers with different compositions were shown in Figure 2. The sharp peaks at 3.35 ppm (terminal OCH 3 , i), 3.65 ppm (OCH2CH2O, h) and 4.15 ppm (C(O)-O−CH2, g) can be attributed to the methoxyl and methylene protons of the Table 1. Characterization of P(TTMA-co-mPEGMA) copolymers

TTMA/mPEGMA (number ratio)a

a

P(TTMA-co-mPEGMA)

Mn,PTTMA/ Mn,P(mPEGMA)a

feeding

productb

Mnb (g/mol)

Mnc (g/mol)

Mw/Mnc

I II III

10000/6000 10000/10000 10000/15000

27.0/6.0 27.0/10.0 27.0/15.0

26.2/5.8 26.0/9.5 26.0/14.3

15640 19270 24070

15800 20500 25300

1.21 1.24 1.29

The theoretical values. bCalculated from 1H NMR. cDetermined by GPC. 3977


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high sensitivity to the polarity of the pyrene environment.49,50 The CMC was obtained when the ratio of pyrene fluorescence intensities at 337 and 330 nm (I337/I330) was plotted against the logarithm of copolymers concentrations, as shown in Figure S4. According to the technique, the CMC values of P(TTMA-comPEGMA)-I, P(TTMA-co-mPEGMA)-II and P(TTMA-comPEGMA)-III were determined to be 0.18, 0.29, and 0.45 μg/mL, respectively. An increase in the P(mPEGMA) content from 37.5% to 60% resulted in an increase in the CMC, which was consistent with the general trend for amphiphilic copolymers where the CMC increases as the hydrophilic content increases. It was worth pointing out that the content of the hydrophilic P(mPEGMA) segments only slightly affected the CMC, which may be because the main driving force behind the formation of micelles in water is the hydrophobic interactions of hydrophobic segments. In addition, P(TTMAco-mPEGMA) polymers exhibited low values of CMC, resulting mainly from high hydrophobicity of aromatic rings.30,51 Due to the relatively low CMC values, the micelles self-assembled from P(TTMA-co-mPEGMA) comb-like copolymers would provide good stability in solution, even after extreme dilution by the larger volume of systemic circulation in the body.49 As amphiphilic copolymers, P(TTMA-co-mPEGMA) had the ability to self-assemble into micelle-like aggregates with a hydrophobic core (PTTMA) and a hydrophilic corona (mPEG) in water. The microstructure of freeze-dried P(TTMA-co-mPEGMA)-II micelles dispersed in D2O was confirmed by high-resolution 1H NMR spectroscopy, as shown in Figure 3. The chemical shifts of mPEG side chains

Table 2. Characterization of P(TTMA-co-mPEGMA) Micelles P(TTMA-co-mPEGMA)

size (nm)a

PDIa

CMC (μg/mL)b

I II III

112.5 ± 3.0 138.2 ± 4.5 192.7 ± 9.6

0.12 ± 0.03 0.16 ± 0.04 0.19 ± 0.07

0.18 0.29 0.45

a

Obtained by DLS measurements. microscopy using pyrene as a probe.

b

Determined by fluorescence

particle size. However, P(TTMA-co-mPEGMA) polymers with higher mPEG content generally have relatively high PDI values. This can be attributed to the difference in mPEG chain organization during nanoparticles formation. The higher mPEG content in graft polymers could reduce the interactions of hydrophobic segments and create a steric barrier around PTTMA core, thus leading to forming looser and larger aggregates.30,52 TEM was also used to examine the morphology of P(TTMA-co-mPEGMA) copolymers micelles, as shown in Figure 4. TEM images revealed that P(TTMA-co-mPEGMA) can form stable nanoparticles with a well-defined spherical shape and a homogeneous size distribution around 90−120 nm depending on composition. An increase in the particle size can be obtained by increasing mPEG content, which was consistent with the DLS results (Table 2). The smaller sizes observed by TEM observations as compared to that determined by DLS were most likely due to shrinkage of hydrophilic shells upon drying samples.53,54 These results demonstrated that the size of the P(TTMA-co-mPEGMA) copolymers micelles can be easily tailored by changing the hydrophobic/hydrophilic chain ratio. P(TTMA-co-mPEGMA) micelles with an inner hydrophobic core and a hydrophilic corona (mPEG) were expected to have a good physical stability. The diameter changes of the P(TTMAco-mPEGMA) micelles in PBS 7.4 with or without 10% FBS were investigated by DLS and shown in Figure S5. All P(TTMA-co-mPEGMA) micelles were stable in a PBS solution with or without 10% FBS, as there was no significant shift in their size with time as determined by DLS. It has been confirmed that the mPEG chains around the hydrophobic core of the micelle can serve to minimize interactions with proteins, enzymes, and cells and thus provide the stability to micelles, reduce systemic clearance rates, and prolong circulation half-life in vivo.46,52 pH-Triggered Dissociation of the Copolymer Micelles. It is well-known that the hydrolysis of acetal moieties is rapid at low pH values and relatively stable in neutral or alkaline media. The hydrolysis of cyclic benzylidene acetals in P(TTMA-comPEGMA) copolymers micelles was investigated at different pH values of 4.0, 5.0, and 7.4 at 37 °C. According to a previous study,27 the extent of acetal hydrolysis was conveniently determined by using UV/vis spectroscopy via monitoring the absorbance at 292 nm, which is the characteristic absorbance of the hydrolysis product (2,4,6-trimethoxybenzaldehyde), as shown in Figure 5A−C. The results in Figure 5D showed that the hydrolysis rate of acetals in all P(TTMA-co-mPEGMA) micelles was highly pH-dependent, which were in agreement with previous reports.25,26 Typically, P(TTMA-co-mPEGMA)II micelles under physiological conditions (pH 7.4) was relatively stable and the acetal hydrolysis rate was extremely low (11.3% in 72 h). However, P(TTMA-co-mPEGMA)-II micelles at mild acid (pH 4.0 and 5.0) showed rapid acetal hydrolysis with acetal half-lives of approximately 2.84 and 9.75 h at pH 4.0 and 5.0, respectively. It also can be seen from

Figure 3. 1H NMR spectra of P(TTMA-co-mPEGMA)-II micelles in the solvent of D2O (A) and CDCl3 (B).

and PTTMA moieties both appeared in the 1H NMR spectrum of P(TTMA-co-mPEGMA)-II in CDCl3. However, in the medium of D2O, the only chemical shifts of methoxyl and methylene protons in the PEG moieties were observed around 3.17 ppm (terminal OCH3) and 3.48 ppm (OCH2CH2O), demonstrating that the PTTMA backbone had a strong tendency to aggregate and form an inner hydrophobic core that was covered by an external corona of mPEG. P(TTMA-co-mPEGMA) micelles were further determined by DLS measurements and TEM observations. DLS measurements showed that nanosized polymeric aggregates ranging from 112.5 to 192.7 nm were formed in aqueous solution (Table 2 and Figure 4). Moreover, the increase of the hydrophilic mPEG segments resulted in an increase of the 3978


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Figure 4. Size distributions of P(TTMA-co-mPEGMA)-II (A, B) and -III (C, D) copolymer micelles determined by DLS and TEM micrograph.

Figure 5. pH-Triggered hydrolysis of acetals in P(TTMA-co-mPEGMA)-II micelles measured by UV/vis spectrophotometer at pH 7.4 (A), 5.0 (B), and 4.0 (C). Hydrolysis kinetics of P(TTMA-co-mPEGMA) copolymers at different pH (D).

the pyrene can reflect the disassembly rate of the polymeric micelles. As shown in Figure 6, the fluorescence intensity of pyrene in micelles under pH 7.4 conditions remained relatively stable but was gradually decreased with incubation time under pH 5.0 and 4.0. These results further suggested P(TTMA-comPEGMA) can form into pH-sensitive micelles with the ability of rapid disassembly under mildly acid conditions, which should be very beneficial to facilitate the release their payloads. Hydrophobic PTTMA backbone of P(TTMA-co-mPEGMA) bearing acetal groups can be selectively hydrolyzed into a hydrophilic backbone in the acidic environment. The acidtriggered hydrophobic to hydrophilic transition can lead to the morphological changes of the nanostructure. The nanostructure

Figure 5D that the rate of hydrolysis of P(TTMA-comPEGMA) was increased with the increase of mPEG content. As mentioned above, the higher mPEG content can result in the formation of looser and larger aggregates, which should be beneficial to the diffusion of water molecules and thus enhance the polymer degradation.30 The acetal hydrolysis of P(TTMA-co-mPEGMA) micelles at varied pH (7.4, 5.0, and 4.0) was further tracked by the use of steady-state fluorescent-probe methodology using pyrene as a probe. Pyrene is a highly polarity-dependent fluorescence probe, which displays very weak fluorescence in aqueous solution but shows highly fluorescent in hydrophobic environment.43 As a result, the change in the fluorescence intensity of 3979


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Figure 6. pH-Triggered hydrolysis of acetals in P(TTMA-co-mPEGMA)-II micelles measured by steady-state fluorescent-probe methodology using pyrene as a probe at pH 7.4, 5.0, and 4.0.

P(TTMA-co-mPEGMA)-II micelles were degraded into watersoluble unimers as a result of complete acetal hydrolysis.27 Chen and Zhong et al. reported that the acetal-functionalized PEG-b-poly(mono-2,4,6-trimethoxybenzylidene-pentaerythritol carbonate) diblock micelles can maintain micellar structures even after complete acetal hydrolysis,25 which was different with acid-induced disassembly of comb-like P(TTMA-comPEGMA)-II copolymer micelles. The latter may be better for enhanced intracellular drug delivery. Encapsulation and in Vitro pH-Triggered Release. CUR-loaded P(TTMA-co-mPEGMA) micelles were prepared through a single-step nanoprecipitation method. Adding P(TTMA-co-mPEGMA) and CUR codissolved THF into water resulted in the coprecipitation of P(TTMA-co-mPEGMA) and CUR. In this process, P(TTMA-co-mPEGMA) selfassembled into core−shell structured micelles with coreencapsulated CUR. In our experimental condition, the results showed that all P(TTMA-co-mPEGMA) micelles showed remarkably high loading efficiencies toward CUR (DLE > 94%), even in the case of high DLC (DLC ≈ 20%). One of possible reasons is the strong π−π conjugate actions of benzene and hydrophobic interaction between the PTTMA and CUR.55,56 In addition, due to the intramolecular aggregation mechanism, the comb-shaped polymers can form looser and larger aggregates, perhaps giving rise to greater drug encapsulation abilities.30,52 To optimize the process parameters, we studied the effect of different theoretical drug loading on the properties of the resultant micelles, and the results were presented in Table 3. The micelle size became smaller at a low DLC level (about 5%) likely due to the existence of effective

and size changes of P(TTMA-co-mPEGMA)-II micelles in response to cyclic benzylidene acetal hydrolysis were followed by DLS measurements, as shown in Figure 7. Little size change

Figure 7. Time dependence of the size changes of P(TTMA-comPEGMA)-II micelles during acetal hydrolysis at pH 7.4 and 5.0.

of P(TTMA-co-mPEGMA)-II copolymer micelles was observed over 72 h at pH 7.4, which can be ascribed to the relatively stability of acetal groups in neutral media. However, under otherwise the same conditions, the micelles expanded in pH 5.0 acetate buffer from 138 nm to about 350 nm in 6 h and reaching about 455 and 600 nm in 12 and 24 h, respectively. Furthermore, the large number of 9.0 nm particles were detected following 72 h incubation at pH 5.0, indicating that

Table 3. Characteristics of CUR-Loaded P(TTMA-co-mPEGMA) Micelles in PBS 7.4 P(TTMA-co-mPEGMA)

theoretical DLC (%)

I

5 10 15 20 5 10 15 20 5 10 15 20

II

III

a

DLCa (%) 4.90 9.76 14.2 18.8 4.92 9.88 14.6 19.3 4.94 9.86 14.8 19.6

± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.08 0.15 0.25 0.05 0.10 0.18 0.23 0.04 0.10 0.17 0.20

DLE (%) 98.0 97.6 94.7 94.0 98.4 98.8 97.3 96.5 98.8 98.6 98.7 98.0

± ± ± ± ± ± ± ± ± ± ± ±

0.75 0.10 1.30 1.16 0.92 1.02 1.25 1.13 0.84 1.03 1.23 1.08

size (nm)b 110.4 125.6 143.1 159.1 130.6 159.2 165.5 194.3 180.6 218.4 225.3 247.3

± ± ± ± ± ± ± ± ± ± ± ±

2.3 2.8 3.1 3.9 3.2 5.0 7.2 1.5 2.5 3.7 5.7 8.7

PDIb 0.13 0.14 0.15 0.17 0.15 0.18 0.18 0.21 0.15 0.17 0.19 0.20

± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.03 0.07 0.06 0.05 0.06 0.05 0.07 0.03 0.05 0.06 0.08

Evaluated by UV/vis measurements. bMeasured by DLS at a concentration of 1.0 mg/mL. 3980


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Figure 8. (A) pH-Triggered release of CUR from P(TTMA-co-mPEGMA) micelles in response to varied pH at theoretical DLC of 5%. (B) Cumulative release of CUR from P(TTMA-co-mPEGMA)-II micelles for different loading percentages of CUR at different pH.

Figure 9. TEM micrographs of the CUR-loaded P(TTMA-co-mPEGMA)-II micelles in acetate buffer under pH 5.0 at 0 h (A) and after 6 h (B).

in the release medium under pH 5.0 at 0 h and after 6 h at 37 °C were investigated by TEM, as shown in Figure 9. At initial stage (0 h), P(TTMA-co-mPEGMA)-II micelles exhibited spherical nanostructure with an average diameter of about 105 nm and a narrow size distribution (Figure 9A). However, the remarkable swelling of micelles was observed when the CUR-loaded P(TTMA-co-mPEGMA)-II micelles was incubated in acetate buffer under pH 5.0 after 6 h (Figure 9B). As mentioned above, the hydrophobic PTTMA segments can be converted into hydrophilic segments in the mildly acidic environment, leading to swelling of the micelles and thus enhancing the release of CUR. It should further be noted that drug release rate slightly increased with increasing mPEGMA content in P(TTMA-co-mPEGMA) copolymers. 49.3%, 51.0%, and 56.0% of CUR was released in 10 h at pH 5.0 from P(TTMA-co-mPEGMA)-I, -II, and -III micelles, respectively. This can be attributed to the fact that the higher mPEGMA content resulted in forming looser micelles and thus a slightly faster hydrolysis. The effect of different DLC on the CUR release was investigated and shown in Figure 8B. As the DLC of CUR in P(TTMA-co-mPEGMA)-II micelles increased from 4.92% to 19.3%, higher initial burst and more sensitive CUR release were observed. The higher DLC can lead to a higher concentration gradient and thus facilitate the diffusion and release of drug. In sum, CUR-loaded P(TTMA-co-mPEGMA) micelles presented a pH-dependent CUR release profile, which should be beneficial for tumor treatment. Most of loaded CUR will remain in micelles for a considerable length of time when CUR-loaded P(TTMA-co-mPEGMA) micelles stay in the

interactions between micellar core and CUR. When the theoretical DLC was further increased, the particle size and PDI increased with rising of CUR theoretical DLC in the feed. For instance, CUR-loaded P(TTMA-co-mPEGMA)-II micelles had low PDI of 0.15−0.21 and particle sizes ranging from 130.6 to 194.3 nm depending on CUR loading levels. It was worth pointing out that P(TTMA-co-mPEGMA) with different mPEGMA content displayed somewhat similar drug-loading capacity (Table 3). This is may be due to the fact that the drugloading capacity for hydrophobic drugs mainly depended on the inner hydrophobic core of micelles.52 The release profiles of CUR from different P(TTMA-comPEGMA) micelles were studied at 37 °C for a low DLC (about 5%) at pH 7.4, 5.0, and 4.0 using a dialysis method in vitro, as shown in Figure 8A. The results showed that release of CUR from all three comb-like copolymer micelles was obviously faster at acidic pH (5.0 and 4.0) than at physiological pH (7.4). For instance, only approximately 20.9% of CUR was released from CUR-loaded P(TTMA-co-mPEGMA)-II micelles within 24 h, indicating that CUR-loaded P(TTMA-comPEGMA)-II micelles was relatively stable under physiological conditions (pH 7.4). However, the release of CUR was significantly accelerated at pH 5.0 and pH 4.0 with 37.4% and 78.5% of CUR released from P(TTMA-co-mPEGMA)-II micelles in 6 h. The significant increase in CUR release in the acidic medium can be ascribed to that the pH-induced cyclic benzylidene acetals hydrolysis resulted in the swelling and disassembly of P(TTMA-co-mPEGMA) micelles. The nanostructure changes of P(TTMA-co-mPEGMA)-II micelles 3981


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Figure 10. Fluorescence microscopy images of HepG-2 cells incubated with free CUR and CUR-loaded P(TTMA-co-mPEGMA)-II micelles for 2, 4, and 8 h at equivalent CUR concentration of 10 μg/mL. The scale bars correspond to 75 μm for big views, and 25 μm for small views.

fluorescence intensity was found in the cells treated with CURloaded micelles. This may be due to that the low endo/ lysosomal pH would cause cleavage of acetals in P(TTMA-comPEGMA)-II micelles and sustain drug release for a longer period of time. Consequently, the results demonstrated that P(TTMA-co-mPEGMA) copolymer micelles with pH-responsive cyclic benzylidene acetals had a high potential as the carrier for intracellular CUR delivery. In order to investigate the biocompatibility of P(TTMA-comPEGMA) copolymer micelles, the in vitro cytotoxicities of micelles toward HepG-2 and EC-109 cells were evaluated using a MTT assay. As shown in Figure 11, the viabilities of cells treated with blank P(TTMA-co-mPEGMA)-II micelles for 48 h

plasma at normal physiological conditions (pH 7.4). However, the loaded CUR will be rapidly released due to the acidic environment in endocytic compartments (pH 4.0−6.5), when CUR-loaded P(TTMA-co-mPEGMA) micelles are taken up by the tumor cells via a endocytosis process.12 Intracellular CUR Release and Cellular Proliferation Inhibition. Because CUR exhibits green fluorescent, it allows us to easily monitor the cellular uptake and intracellular distribution of free CUR and CUR-loaded P(TTMA-comPEGMA)-II micelles by using CLSM. Fluorescence microscopy images of HepG-2 cells incubated with free CUR and CUR-loaded micelles at equivalent CUR concentration of 10 μg/mL were shown in Figure 10. Weak fluorescence was observed mainly in the cytoplasm of the cells after 2 h of incubation with free CUR, indicating that few CUR molecules entered the cells and mainly accumulated in the cytoplasm. By contrast, when the cells were incubated with CUR-loaded micelles, much stronger fluorescence intensity appeared in the cytoplasm with a stronger fluorescence in the nucleus. The enhanced cellular uptake of CUR by CUR-loaded micelles could be attributed to the fact that nanosized micelles were more readily internalized by an endocytosis mechanism, whereas free CUR was transported into cells by a passive diffusion mechanism.57 In addition, the microscopic studies also demonstrated the cells treated with free CUR showed maximum fluorescence at initial treatment (as observed after 2 h of incubation) but the fluorescence intensity was gradually decreased with time (as observed after 4 or 8 h of incubation). Compared with the cells treated with free CUR, the profound

Figure 11. Cytotoxicity of P(TTMA-co-mPEGMA)-II micelles to HepG-2 and EC-109 cells. The cells were incubated with micelles for 48 h. Data are presented as the average ± standard deviation (n = 5). 3982


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Figure 12. Cellular proliferation inhibition of CUR-loaded P(TTMA-co-mPEGMA)-II micelles and free CUR as a function of CUR dosages toward HepG-2 cells (A) and EC-109 cells (B). The cells were incubated with CUR-loaded micelles or free CUR for 48 h. Data are presented as the average ± standard deviation (n = 5).

were evaluated at all test concentrations up to 1.0 mg/mL. The results revealed that the comb-like P(TTMA-co-mPEGMA) copolymer micelles were practically nontoxic (cell viability >93%) and could be safely used as biocompatible carriers for efficient intracellular drug delivery. The ability to inhibit the cell proliferation of CUR-loaded P(TTMA-co-mPEGMA)-II micelles was also evaluated in HepG-2 and EC-109 cells. As shown in Figure 12, all the studied cell lines showed a typical dose−response sigmoidal curve. The in vitro half maximal inhibitory concentration (IC50) is the quantitative measurement for the cell toxicity induced by CUR. These IC50 values were calculated from the obtained sigmoidal curves of all the studied cell lines and the results demonstrated that CUR-loaded P(TTMA-co-mPEGMA)-II micelles had low IC50 values of 3.13 and 3.98 μg/mL for HepG-2 and EC-109 cells, respectively, which were lower than those obtained with free CUR under otherwise the same conditions (IC50 = 5.50 and 4.98 μg/mL for HepG-2 and EC-109 cells, respectively). On the one hand, the IC50 values for HepG-2 were lower than those reported for nanopartical CUR formulations (22.0 μg/mL58 and 6.63 μg/ mL59). On the other hand, the research on nanoparticle-based CUR formulations for cellular proliferation inhibition of EC109 cells was still rarely reported. The results here indicated that CUR-loaded P(TTMA-co-mPEGMA) micelles had obvious inhibitory effect on EC-109 cells. The high cellular proliferation inhibition of CUR-loaded micelles indicated that CUR has been efficiently delivered and intracellular release for HepG-2 and EC-109 cells. Above results confirmed that P(TTMA-co-mPEGMA) micelles with acid-cleavable cyclic benzylidene acetal cores can efficiently load and deliver CUR into tumor cells, achieving high drug efficacy.

microscopy observation also indicated that more CUR was delivered and released into the cytoplasm of HepG-2 cells treated with CUR-loaded P(TTMA-co-mPEGMA) micelles. And MTT assays revealed that CUR-loaded P(TTMA-comPEGMA) micelles showed higher antitumor activity for HepG-2 and EC-109 cells than free CUR. The results demonstrated that pH-sensitive P(TTMA-co-mPEGMA) micelles were able to actively load hydrophobic anticancer drug CUR and efficiently deliver CUR into tumor cells, achieving high cellular proliferation inhibition. Meanwhile, P(TTMA-comPEGMA) micelles displayed low cytotoxicity up to a concentration of 1.0 mg/mL. Therefore, the pH-sensitive comb-like copolymer micelles with acid-cleavable cyclic benzylidene acetal cores are highly promising as smart carriers for triggered intracellular delivery of hydrophobic anticancer drugs.

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ASSOCIATED CONTENT

S Supporting Information *

Additional synthesis processes of TTMA and CPDTC and their characterizations by 1H NMR spectra. GPC trace and CMC measurement data of P(TTMA-co-mPEGMA). The stability of P(TTMA-co-mPEGMA) micelles in PBS pH 7.4 with or without 10% FBS. 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 address: [email protected]; Tel: +86 22 27890706; Fax: +86-22-27890706.

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Present Address *

Key Laboratory of Systems Bioengineering, Ministry of Education of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People’s Republic of China.

CONCLUSIONS A series of comb-like amphiphilic copolymers P(TTMA-comPEGMA) bearing acetal-functionalized backbone with the ability of acid-triggered hydrophobic to hydrophilic transition were designed and synthesized by one-step controlled RAFT copolymerization of TTMA and mPEGMA. P(TTMA-comPEGMA) with low CMC values can readily self-assemble into nanosized micelles in aqueous solution. Due to acidtriggered hydrolysis of acetals, the obtained micelles can undergo swelling and disassembly. The morphological changes of P(TTMA-co-mPEGMA) micelles at mildly acidic pH of 4.0 and 5.0 can result in rapid drug release. The in vitro CUR release from the micelles was significantly accelerated when solution pH decreased from 7.4 to 5.0 or 4.0. Confocal

Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants from the National Natural Science Foundation of China (No. 51103097, 81371667) and Specialized Research Fund for the Doctoral Program of Higher Education of China (20120032110013). 3983


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Comb-like Amphiphilic Copolymers Bearing Acetal-Functionalized

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