Article

PEG-b-PCL Copolymer Micelles with the Ability of pH-controlled Negativeto-positive Charge Reversal for Intracellular Delivery of Doxorubicin Hongzhang Deng, Jinjian Liu, Xuefei Zhao, Yuming Zhang, Jianfeng Liu, Shuxin Xu, Liandong Deng, Anjie Dong, and Jianhua Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501290t • Publication Date (Web): 17 Oct 2014 Downloaded from http://pubs.acs.org on October 22, 2014

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PEG-b-PCL Copolymer Micelles with the Ability of pH-controlled Negative-to-positive Charge Reversal for Intracellular Delivery of Doxorubicin

Hongzhang Deng †, #, Jinjian Liu §, Xuefei Zhao†, Yuming Zhang§, Jianfeng Liu§, Shuxin Xu†, Liandong Deng†, Anjie Dong†, #, Jianhua Zhang†,* †

Department of Polymer Science and Technology and Key Laboratory of Systems

Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China §

Tianjin Key Laboratory of Radiation Molecular and Molecular Nuclear Medicine, Institute of

Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, 300192, China #

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China

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Abstract The application of PEG-b-PCL micelles was dampened by their inherent low drug loading capability and relatively poor cell uptake efficiency. In this study, a series of novel PEG-b-PCL copolymers methoxy poly(ethylene glycol)-b-poly(ε-caprolactone-co-γ-dimethyl maleamidic acid -ε-caprolactone) (mPEG-b-P(CL-co-DCL)) bearing different amounts of acid-labile βcarboxylic amides on the polyester moiety were synthesized. The chain structure and chemical composition of copolymers were characterized by 1H-NMR, FT-IR and GPC. mPEG-b-P(CL-coDCL) with critical micellar concentrations (CMC) of 3.2 ~ 6.3 µg/mL could self-assemble into stable micelles in water with diameters 100 to 150 nm. Doxorubicin (DOX), a cationic hydrophobic drug, was successfully encapsulated into the polymer micelles, achieving a very high loading content due to electrostatic interaction. Then the stability, charge-conversional behavior, loading and release profiles, cellular uptake and in vitro cytotoxicity of free drug and drug-loaded micelles were evaluated. The β-carboxylic amides functionalized polymer micelles are negatively charged and stable in neutral solution but quickly become positively charged at pH 6.0, due to the hydrolysis of β-carboxylic amides in acidic conditions. The pH-triggered negative-to-positive charge reversal not only resulted in a very fast drug release in acidic conditions, but also effectively enhanced the cellular uptake by electrostatic absorptive endocytosis. The MTT assay demonstrated that mPEG-b-P(CL-co-DCL) micelles were biocompatible to HepG2 cells while DOX-loaded micelles showed significant cytotoxicity. In sum, the introduction of acid-labile β-carboxylic amides on the polyester block in mPEG-bP(CL-co-DCL) exhibited great potentials for the modifications in the stability in blood circulation, drug solubilization and release properties, as well as cell internalization and intracellular drug release. Keywords: amphiphilic copolymer micelles; charge-reversal; PEG-b-PCL; drug delivery; DOX

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INTRODUCTION Amphiphilic block copolymers can self-assemble into well-defined, nanoscale-sized core/shell structures, i.e., polymeric micelles. Due to the excellent solubilization capacity, relatively high stability and prolonged circulation in the blood, polymeric micelles represent very promising carriers for drug delivery.1-4 Particularly, nanocarriers assembled from the block copolymers composing of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) have received widespread attention over the past decades.5-8 PEG with excellent biocompatibility forming the hydrophilic corona in the micelles can reduce nonspecific adhesion to blood components and increase blood circulation times.9,10 PCL has several particular properties, such as biocompatibility and biodegradability, which were extensively studied as hydrophobic core in the micelles to load hydrophobic drug. However, PEG-b-PCL as drug delivery system still have some drawbacks, such as poor drug loading capability and untriggered release, as well as relatively poor uptake efficiency due to the stealth properties of the PEG.11-14 The introduction of functional groups to the PCL segment of PEG-b-PCL block copolymer has been proved to be an effect way to tune the properties of polymers and modulate the chemical, physical and biological properties of the resultant micelles. A variety of functional groups, such as aromatic,15,16 cyclic ketal group,17,18 carbonyl,19 and hydroxyl20 have been introduced onto the PCL segment to improve the performance of PEG-b-PCL micelles. However, most of these modifications focused just on improving the drug loading and release properties, without simultaneously overcoming the inherently poor cell uptake and endosome escape of PEG-b-PCL micelles. The physiological pH of blood and normal tissues is about 7.4, but the extracellular pH in tumor tissues is often acidic and the intracellular endo/lysosomal pH ranges from 4.0 to 6.5. The difference in pH between cancer tissues and normal tissues is advantageous for the specific

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targeting tumor and controlled release at the tumor site.21,22 The polymers containing amine or carboxylic groups with different chemical structures were the most representative pH-responsive polymers, which could undergo solubility change or structure degradation in response to pH decrease.23 However, it has been confirmed that the charge of the micelles exerted a great impact on their in blood circulation, membrane transduction and nuclear localization functions.24-26 Micelles with negatively charged carboxyl groups can avoid blood clearance and reduce toxicity, but they tend to be repelled by the negative cellular membrane. On the contrary, the positively charged micelles generated from polymers containing amines have higher affinity with negatively charged cell membranes, which is effective to enhance cell internalization. However, positive micelles always have serious aggregation and rapid clearance after injection duo to the nonspecific interactions with blood components. Therefore, an ideal nanocarrier should be negative or uncharged in the physiological environment to reduce toxicity and prolong blood circulation time, but would activate the cationic charges in tumor tissues and their intracellular compartments to promote the cellular uptake and assist the endosomal escape.25,27,28 β-Carboxylic amides have a characteristic pH-responsive hydrolysis. They are stable and negatively charged at neutral pH, but they will quickly hydrolyze at acidic environments, such as cancerous tissues and lysosomes, to regenerate the cationic primary amines.25,27 Therefore, the polyelectrolytes bearing acid-labile β-carboxylic amides can maintain a neutral to negatively charged nature in the physiological environment. However, in the tumor tissues or their intracellular compartments, the acidic microenvironment would trigger the charge conversion. In addition, the β-carboxylic amides modified polymers can exploit the charge interactions with cationic drugs, such as doxorubicin (DOX), to achieve high drug-loading capacity and loading efficiency.29,30 Moreover, an abrupt change in the interaction with counter-ions by generating a

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repulsive electrostatic force owing to the charge conversion can accelerate drug release. Apparently, this pH-induced charge-reversal property can realize some ideal functions, such as controlled drug loading and release, promoted cellular uptake and endosomal escape, which has recently been widely used to design drug delivery systems. For example, Kataoka et al synthesized the charge-converting block copolymer using the citraconic amide as a pH-sensitive charge group. The formed micelles selectively released the drug promptly in response to the acidic conditions in the intracellular endosomal compartments.27,31,32 Shen et al. have also applied the negative-to-positive charge-reversal technique to design conjugates and polymeric micelles, which could effectively prolong circulation time in blood and enhance the cellular uptake of the micelles25,33,34. These results have proved the functions of pH-sensitive chargeconversion concept in improving the drug delivery efficiency. Nevertheless, most of the chargeconversional nanomedicines studied previously were limited to use β-carboxylic acid groups to reversibly mask the primary and secondary amine groups of cationic polymers, such as poly(ethylene imine) and polylysine. Unfortunately, the in vivo applications of these polycations were greatly limited due to their high cytotoxicity to cells.35-37 As one of the most widely used biopolymers for drug delivery, PEG-b-PCL has excellent biocompatibility. However, to the best of our knowledge, very few studies have been carried out to introduce pH-sensitive chargeconversion function to PEG-b-PCL micelles. In this study, a series of novel PEG-b-PCL copolymers methoxy poly(ethylene glycol)-b-poly(εcaprolactone-co-γ-dimethyl maleamidic acid -ε-caprolactone) (mPEG-b-P(CL-co-DCL)) bearing different amounts of acid-labile β-carboxylic amides on the PCL block were prepared. The chain structure and chemical composition of copolymers were characterized by 1H-NMR, FT-IR and GPC. mPEG-b-P(CL-co-DCL) could self-assemble into stable micelles in water, which were

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observed by TEM and dynamic light scattering. As one of the most widely used chemotherapy, the cationic hydrophobic DOX was chosen as a drug model and successfully encapsulated into the micelles. Subsequently, the stability, charge-conversional behavior, loading and release profiles, cellular uptake and in vitro cytotoxicity were evaluated. As shown in Scheme 1, the anionic carboxylic groups in mPEG-b-P(CL-co-DCL) can specifically interact with cationic DOX via electrostatic interaction to achieve high loading content and loading efficiency. Moreover, the DOX-loaded ionic complex micelles can respond to the tumor extracellular and intracellular acid microenvironment by pH-triggered charge-reverse, which is expected to simultaneously prolong blood circulation time in blood, facilitate the cell internalization and control intracellular drug release, thus greatly improving the delivery efficiency of DOX.

Scheme 1. Illustration of charge-conversional behavior of mPEG-b-P(CL-co-DCL) micelles for active loading and triggered release of DOX.

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EXPERIMENTAL Materials γ-(carbamic acid tert-butyl ester)-ε-caprolactone (CATCL) was synthesized according to a previous method38 and the detailed information was given in the Supplementary Material. Methoxy poly (ethylene glycol) (mPEG, Mn = 5.0×103g/mol), stannous octoate (Sn(Oct)2), εcaprolactone

(ε-CL),

4-(tert-butyloxycarbonylamino)

cyclohexanone

and

m-

chloroperoxybenzoic acid were purchased from Sigma-Aldrich (Milwaukee, USA). Prior to use, PEG was dried by an azeotropic distillation in toluene and ε-CL was dried over calcium hydride (CaH2) for 48 h at room temperature and distilled under reduced pressure. Trifluoroacetic acid (TFA) and 2,3-dimethylmaleic anhydride (DMMA) purchased from GL Biochemical Co., Ltd (Shanghai, China) were used as received. Tetrahydrofuran (THF) was dried by refluxing over Na metal under an argon atmosphere before use. Dichloromethane (DCM) was purified by distillation with CaH2. Doxorubicin (DOX.) was purchased from Wuhan Hezhong Biochemical manufacturing Co., Ltd (Wuhan, China). Dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and other reagents were commercially available from Jiangtian Chemical Co., Ltd. (Tianjin, China) and used as received. Synthesis and characterization of mPEG-b-P(CL-co-DCL) Synthesis of mPEG-b-P(CL-co-DCL) was accomplished in three steps in Scheme 2: (I) ringopening polymerization of CL and CATCL with mPEG to prepare methoxy poly(ethylene glycol)-b-poly(ε-caprolactone-co-γ-carbamic acid tert-butyl ester-ε-caprolactone) (mPEG-P(CLco-CATCL)); (II)hydrolysis of mPEG-P(CL-co-CATCL) by using TFA to prepare methoxy poly(ethylene

glycol)-b-poly(ε-caprolactone-co-γ-amine-ε-caprolactone)

(mPEG-b-P(CL-co-

ACL)); (III) amidation reaction between mPEG-b-P(CL-co-ACL) and DMMA to prepare

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mPEG-b-P(CL-co-DCL). The detailed synthesis process was given in the Supplementary Material.

Scheme 2. Synthesis route of mPEG-b-P(CL-co-DCL)

Successful preparation of mPEG-b-P(CL-co-DCL) was confirmed by using nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC) analyses. 1H-NMR spectra were recorded on a Varian INOVA500MHz nuclear magnetic resonance instrument (Varian Inc., Palo Alto, USA) using CDCl3, DMSO-d6, and D2O as a solvent and tetramethylsilane (TMS) as the internal standard. FT-IR was carried out using Bio-Rad FT-IR 3000 (Bio-Rad, USA). The number-average molecular weight (Mn) and polydispersity index

(Mw/Mn) of

copolymers

were determined

by gel

permeation

chromatography (GPC) using a Malvern Viscotek GPC max system. THF was used as the eluting solvent with a flow rate of 1 mL/min and polystyrene was used as standard for calibration. The critical micelle concentration (CMC) was measured by a steady state fluorescent-probe methodology using pyrene as probe on a Varian fluorescence spectrophotometer at room temperature according to our previous studies.40,41

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Preparation and characterization of mPEG-b-P(CL-co-DCL) micelles mPEG-b-P(CL-co-DCL) micelles were prepared by dialysis method. Typically, mPEG-b-P(CLco-DCL) (100 mg) was dissolved in 5 mL of DMF. The solutions were dialyzed for 48 h in a dialysis bag with MWCO of 3500 Da using 1000 mL deionized water, which was replaced every 4 h. The size, size distribution (PDI) and zeta potential (ζ) of micelles were performed using a laser particle size analyzer (Zetasizer Nano, Malvern, UK). The average of three measurement results was taken. Morphologies of micelles were observed under a Hitachi H600 transmission electron microscopy (TEM) system at an operated voltage of 75 kV. For TEM measurement, the sample was prepared by adding a drop of micelles solution onto the copper grid, and then the sample was air-dried and measured at room temperature. Protein adsorption Bovine serum albumin (BSA) was used as a model protein to examine protein adsorption capacity of mPEG-b-P(CL-co-DCL) micelles according to a previous study.42 The micelles were incubated with a solution of BSA in PBS (pH 7.4 or pH 6.0) at 37 oC with the final concentration of the micelles and BSA at 1 mg/mL and 0.5 mg/mL for 12 h, respectively. Then each sample was centrifuged to precipitate the protein-adsorbed micelles. The concentration of residual BSA in PBS solution was measured using UV-Vis spectroscopy (Beckman DU 640, USA), and then the amount of BSA adsorbed on the micelles was obtained. Stability monitored by an optical analyzer The pH dependent stability assay was performed using an optical analyzer (Turbiscan Expert®, Formulaction, France) according to the previous studies.43,44 The micelles (20 mL, 5 mg/mL) were allocated in a cylindrical glass tube and measurements were carried out using a pulsed near infrared LED source at a wavelength of 880 nm. The light which was transmitted (T) and

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backscattered (BS) through the whole height of sample was recorded. A latex suspension and silicon oil were used as reference standards. The experimental data were obtained in percentage to the reference standards. The variation of BS (△BS) and T (△T) signals linked to the droplet volume fraction (migration) or size (coalescence) were observed. The samples were scanned every 1 h for 12 h. The Turbiscan Lab software was used for data elaborated. Experiments were carried at the room temperature. Drug loading and release experiments Typically, mPEG-b-P(CL-co-DCL) copolymers or mPEG-b-PCL copolymers and DOX were dissolved in DMF, and then the mixture solution was dropwise added to double distilled water under magnetic stirring. The mixture was placed in a dialysis bag (MWCO = 3500 Da) and free dialyzed against a phosphate buffer (pH 7.4) to form micelles. The amount of DOX was determined by UV-Vis spectrophotometer. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated from the following equations:

weight of loaded drug × 100% ------- [1] weight of drug - loaded micelles weight of loaded drug DLE(%) = × 100% ------- [ 2] weight of drug in feed DLC (%) =

In vitro release profiles of DOX from the polymer micelles were investigated at 37 oC in buffer solutions (pH 7.4 and 5.4) by use of a dialysis tube (MWCO = 3500 Da). At regular time intervals, 10 mL release medium was withdrawn and replaced with an equal volume of fresh medium. The DOX concentration in the samples was calculated based on the absorbance intensity at 488 nm with UV-vis spectroscopy. The cumulative drug release percentage (Er) was calculated by the following equation:

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n −1

Er（%） =

Vt ∑ Ci + V 0Cn 1

m

×100%------- [3]

DOX

Where mDOX represents the amount of DOX in the micelles, V0 is the whole volume of the release media (V0 = 40 mL), Vt is the volume of the replaced media (Vt= 10 mL), and Cn represents the concentration of DOX in the sample. Cell Uptake Studies The cellular uptake of DOX, DOX-loaded mPEG-b-P(CL-co-DCL) micelles and mPEG113-bPCL77 micelles was observed by fluorescence microscope using HepG2 cells. HepG-2 cells were seeded in a 6-well plate at a density of 1 × 104 cells per well. When the cells reached about 80% confluence, the cells were treated with free DOX, DOX-loaded mPEG-b-P(CL-co-DCL) micelles or mPEG113-b-PCL77 micelles at an equivalent DOX concentration of 10 µg/mL in fresh culture medium at pH 7.4 or 6.0, respectively. After incubation for 4 h, the above incubated cells were thoroughly washed three times with PBS to remove excess free DOX or DOX-loaded micelles solution. Thereafter, the cells were fixed with 4 % formaldehyde for 30 min at room temperature and then the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). After replacement with PBS, the fluorescence images were obtained by using fluorescence microscope (Baxter, United States). Cell Viability Assays The biocompatibility and cytotoxicity of mPEG-b-P(CL-co-DCL) micelles and mPEG113-bPCL77 micelles as control were evaluated by MTT assays using HepG-2 cells. The cells were plated in a 96-well plate (5×103 cells/well) using RPMI-1640 medium supplemented with 10 % fetal bovine serum, 1 % L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 µg/mL) in a 5% CO2 atmosphere for 24 h. After removing the culture medium and adding

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mPEG113-b-PCL77 or mPEG-b-P(CL-co-DCL) micelles solutions with PBS (pH 7.4) at different concentrations, the cells were cultured at 37 oC in an atmosphere containing 5 % CO2 for another 24 h. The medium was aspirated and replaced by 100 µL fresh medium. A total of 20 µL MTT solution (5 mg/mL) was added. And then the cells were incubated for another 4 h. Afterward, the medium was completed removed and 200 µL of DMSO was added to each well to extract the formazan products formed by viable cells. The absorbance of the solutions was measured on a Bio-Rad 680 microplate reader at 570 nm. The relative cell viability (%) was determined by comparing the absorbance at 570 nm with control, which contained only culture medium. Data are presented as average ± SD (n=4).

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RESULT AND DISCUSSION Synthesis and characterization of mPEG-b-P(CL-co-DCL) Due to its poor water solubility, short biological half-life, severe cardio-toxicity and nonspecific distribution, an urgent need at all times exists to explore intelligent drug delivery system for the delivery of DOX to improve the anticancer effect and safety. Polymer micelles assembled from PEG-b-PCL have been reported for DOX delivery and have achieved significant success in improving drug efficacy and reducing side effects. However, the poor drug loading content, untriggered release, and relatively poor uptake of PEG-b-PCL micelles limited their applications.11-14,45 The introduction of acid-induced labile β-carboxylic amides on the polyester block of PEG-b-PCL copolymers is expected to overcome these limitations and render them ideal for encapsulation and delivery of hydrophobic cationic drugs.

β-Carboxylic amides-

functionalized mPEG-b-P(CL-co-DCL) was readily synthesized in three steps, as shown in Scheme 2. Firstly, the ring-opening polymerization of CL and CATCL with mPEG was conducted to prepare mPEG-b-P(CL-co-CATCL). Then the pendant carbamic acid tert-butyl ester groups in CATCL units were hydrolyzed into amino-groups by TFA to produce mPEG-bP(CL-co-ACL). Subsequently, the amino-groups in mPEG-b-P(CL-co-ACL) was amidated with DMMA to produce mPEG-b-P(CL-co-DCL). The whole reaction was implemented in mild reaction conditions without tedious protection and deprotection. The number of β-carboxylic amides groups on hydrophobic segment could be well-controlled by adjusting the amount of CATCL in the initial feed. Considering the mild reaction conditions, simple manipulations and controllable process, the procedure described here provided a promising method to introduce βcarboxylic amides to the polyester of mPEG-b-PCL. The chemical structures and compositions of copolymers were characterized with 1H-NMR, FT-

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IR and GPC, as shown in Figure 1, Figure 2, and Figure S2, S3, S4 and S5 in Supplementary Material. It can be seen from Figure 1 and Figure S2 that the mPEG-b-P(CL-co-CATCL), mPEG-b-P(CL-co-ACL) and mPEG-b-P(CL-co-DCL) exhibited characteristic peaks of both PEG and PCL. For example, the sharp single peak at 3.63 ppm was assigned to the methylene protons of the PEG. Peaks at 1.42, 1.65, 2.32, and 4.07 ppm were attributed to methylene protons in PCL units, respectively. Compared with the 1H-NMR spectra of mPEG-b-P(CL-co-CATCL) and mPEG-b-P(CL-co-ACL),

1

H-NMR spectrum of mPEG-b-P(CL-co-DCL) showed the

characteristic methyl peak at 1.84 ppm (-C(CH3)=C(CH3)-). Moreover, the relative intensity of methyl peak at 1.84 ppm was increase with the increase of the repeating units of DCL. These results confirmed the formation of mPEG-b-P(CL-co-DCL).The number of repeating units of CL and DCL in mPEG-b-P(CL-co-DCL) was controlled by adjusting the feed ratio of CL to CATCL (Table 1 and Table S1, S2). The repeating units of DCL and molecular weights were calculated by comparing the integral areas of characteristic peaks of ethylene protons (-CH2-CH2-O-, at 3.63 ppm) of PEG, methylene protons (-CH2-CH2-CH2-CH2-CH2-O-C(=O)-, at 1.65 ppm ) of CL units, methyl (-C(CH3)=C(CH3)-, at 1.84 ppm). The Mn and composition of mPEG-b-P(CLco-DCL) obtained from 1H-NMR spectrum was close to the theoretical value (Table 1). Further evidence for the preparation of mPEG-b-P(CL-co-DCL) was offered by FT-IR spectra, as shown in Figure 2. It can be seen that all the block copolymers showed the characteristic peaks of both mPEG and PCL, such as -CH3 at 2976 cm-1, methylene (-CH2-) at 2860 cm-1, -C=O at 1750 ~ 1735 cm-1 and -C-O-C- at 1070 cm-1. In addition, the appearance of a very broad band of -COOH in the region 3300-2500 cm-1 and characteristic peaks corresponding to the -C=C- at 1620 cm-1 and 916 cm-1 were observed in the spectra of mPEG-b-P(CL-co-DCL), indicating that the dimethyl maleamidic acid groups were generated in mPEG-b-P(CL-co-DCL) copolymers. The

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GPC traces of the three different mPEG-b-P(CL-co-DCL) copolymers in Figure S4 all showed a unimodal peak, which confirmed that the mPEG-b-P(CL-co-DCL) copolymers possessed a monomodal molecular weight distribution. Their molecular weight (Mn) and polydispersity index (Mw/Mn) were determined by 1H-NMR and GPC, and summarized in Table 1. The Mn values of mPEG-b-P(CL-co-DCL) copolymers obtained by GPC were higher than those calculated by 1

HNMR. This may be ascribed to that the GPC characterizations of the samples were carried out

using polystyrene as standards.46 These results demonstrated that multifunctional mPEG-b-P(CLco-DCL) could be synthesized facilely with well-controlled numbers of β-carboxylic amides groups.

Figure 1. 1H-NMR spectra of mPEG113-b-P(CL50-co-CATCL15), mPEG113-b-P(CL50-co-ACL15), mPEG113-b-P(CL45-co-DCL9), and mPEG113-b-P(CL47-co-DCL29) in CDCl3.

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Figure 2. FT-IR spectra of mPEG113-b-P(CL50-co-CATCL15) (A), mPEG113-b-P(CL50-co-ACL15) (B), mPEG113-b-P(CL47-co-DCL29) (C) and mPEG113-b-P(CL45-co-DCL9) (D)

Table 1. Structure and composition of mPEG-b-P(CL-co-DCL) Polymers

Mn (g/mol)

a

mPEG113-b-P(CL45-co-DCL9)

Theory 12200

mPEG113-b-P(CL48-co-DCL21) mPEG113-b-P(CL47-co-DCL29)

1

b

MW/Mnb

H-NMR 12430

GPC 17900

15302

14958

19000

1.36

17028

17148

23000

1.25

1.27

a

The composition calculated from 1H-NMR of mPEG-b-P(CL-co-DCL).

b

MW/Mn measured by GPC

Preparation and characterization of mPEG-b-P(CL-co-DCL) micelles CMC is one of the most physicochemical parameter of the micellization behavior of amphiphilic copolymers in dilute aqueous solution. The CMC of mPEG-b-P(CL-co-DCL) and mPEG-b-PCL in water was determined by fluorescence technique with pyrene as a probe, as shown in Figure 3 and summarized in Table 2. The CMC values of mPEG113-b-P(CL45-co-DCL9), mPEG113-bP(CL48-co-DCL21) and mPEG113-b-P(CL47-co-DCL29) were determined to be 3.2 µg/mL, 4.6

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µg/mL and 6.3 µg/mL, respectively. The increase of the DCL content resulted in an increase in the CMC. In addition, it can be observed that the CMC of all mPEG-b-P(CL-co-DCL) was higher than that of mPEG-b-PCL with similar structure and composition. These results may be due to the fact that the introduction of the dimethyl maleamidic acid groups to the polyester of mPEG-b-PCL decreased the hydrophobicity of polyester chains. It was well-known that the main driving force of forming micelles in water is the hydrophobic interactions of hydrophobic segments. Therefore, the CMC value of mPEG-b-P(CL-co-DCL) was increased with the increase of the number of DCL units . The amphiphilic mPEG-b-P(CL-co-DCL) can self-assemble into micelles with a hydrophobic core (P(CL-co-DCL)) and a hydrophilic corona (mPEG) in water. The microstructure of freeze-dried mPEG-b-P(CL-co-DCL) micelles dispersed in D2O was proved by using high-resolution 1H-NMR spectroscopy, as shown in Figure 4. The 1H-NMR spectrum of mPEG113-b-P(CL48-co-DCL21) in CDCl3 exhibited the chemical shifts of mPEG side chains and P(CL-co-DCL) moieties. However, in the medium of D2O, the only chemical shifts of methoxyl and methylene protons of mPEG moieties were observed, demonstrating that the P(CL-co-DCL) chains have a great tendency to aggregate and form a hydrophobic core. mPEGb-P(CL-co-DCL) micelles were further determined by laser particle size analyzer measurements and TEM observations, as shown in Figure 5, Figure S6 and summarized in Table 2. Laser particle size analyzer measurements showed that mPEG-b-P(CL-co-DCL) yielded micelles with an average size ranging from 115 to 151 nm and a low polydispersity index (PDI< 0.2). The size of mPEG-b-P(CL-co-DCL) micelles were higher than that of mPEG-b-PCL. Moreover, the diameter of mPEG-b-P(CL-co-DCL) micelles increased with the increase of DCL units. The results can be ascribed to that the introduction of dimethyl maleamidic acid groups decreased the hydrophobicity of PCL segment and created a steric barrier around PCL core, thus leading to

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forming looser and larger aggregates. TEM was also used to observe the morphology of mPEGb-P(CL-co-DCL) micelles, which revealed that mPEG-b-P(CL-co-DCL) can form stable micelles with a well-defined spherical shape and a homogeneous size distribution around 100 ~ 130 nm depending on composition. Moreover, the increase of particle size of micelles with increasing DCL content was also observed, which was consistent with the laser particle size analyzer results. Compared with the size determined by dynamic light scattering, the smaller sizes observed by TEM observations were most likely due to shrinkage of hydrophilic shells upon drying samples.40,47 These results indicated that mPEG-b-P(CL-co-DCL) micelles can be obtained and their size can be easily tailored by adjusting the content of DCL in copolymer. In addition, the results in Figure 5 and Table 2 indicated that the particle size of DOX-loaded mPEG-b-P(CL-co-DCL) micelles was slightly smaller than that of DOX-free micelles. Generally, like the case of mPEG-b-PCL, the non-covalent incorporation of drugs into the micelle hydrophobic core would increase the particle size of micelles. This kind of abnormal phenomenon can be attributed to the fact that the electrostatic interactions between DOX and DCL units of mPEG-b-P(CL-co-DCL) make the structure of the micellar core more compact.

Figure 3. Fluorescence emission spectra of pyrene in the presence of decreasing concentrations of mPEG-b-P(CL-co-DCL) at pH 7.4 (A) and Plot of I337/I333 in the excitation spectrum versus the concentrations of copolymers in water (B).

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Figure 4. 1H-NMR spectra of mPEG113-b-P(CL48-co-DCL21) micelles in the solvent of D2O (A) and (B) CDCl3

Table 2. Characteristics of DOX-free and DOX-loaded mPEG-b-P(CL-co-DCL) micelles

mPEG113-b-P(CL45-co-DCL9)

DOX-free micelles CMC Size (nm)a PDI a (mg/mL)b 115 ± 11 3.2×10-3 0.17

mPEG113-b-P(CL48-co-DCL21)

136 ± 13

4.6×10-3

0.20

121 ± 14

0.17

10.6

71

mPEG113-b-P(CL47-co-DCL29)

151 ± 12

6.3×10-3

0.18

136 ± 12

0.15

12.6

84

mPEG113-b-PCL77

105 ± 9

0.9× 10-3

0.11

127 ± 15

0.13

3.9

26

Copolymers

a

DOX-loaded micelles DLC DLE Size (nm)a PDI (%) (%) 0.16 109 ± 8 8.7 58

Determined using Laser particle size analyzer at 25°C in PBS (10 mM, pH 7.4).

b

measured by a fluorescence technique with pyrene as a probe.

c

Feed ration of DOX to polymers was 15 mg/100 mg.

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Figure 5. Size distribution and morphology of DOX-free (A) and DOX-loaded (B) mPEG113-bP(CL48-co-DCL21) micelles determined by Laser particle size analyzer and TEM. The scale bar of the TEM was 0.2 µm.

Figure 6. Variation of backscattering data (△BS) of mPEG113-b-P(CL48-co-DCL21) micelles at pH 7.4 and pH 6.0 with or without BSA measured along the height axis of the sample tube. Data from bottom to top are given for different periods of time.

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Stability of mPEG-b-P(CL-co-DCL) micelles The physical and biological stability of charged polymeric micelles is of particular importance for their pharmaceutical applications, as the stability not only determines the shelf life, but also directly affects the absorption and physiological distribution of drug.48 The physical and biological stability of mPEG-b-P(CL-co-DCL) micelles at pH 7.4 and pH 6.0 with or without BSA was monitored by an optical analyzer Turbiscan. The variation of backscattering data (∆BS), depended on the mean particle size and their volume fraction, can be used to reflect the stability of micelles.43,44 The ∆BS as a function of the sample height for mPEG113-b-P(CL48-co-DCL21) micelles were shown in Figure 6. At pH 7.4 with or without BSA, all of the △BS profiles were close to the baseline value during the entire time of analysis. This result showed that no aggregation or sedimentation occurred along the entire tube. Although the introduction of the dimethyl maleamidic acid groups to the polyester of mPEG-b-PCL decreased the hydrophobicity of polyester chains and increased CMC values, the mPEG-b-P(CL-co-DCL) micelles at pH 7.4 can achieve to a very stable dispersion state. The good stability may arise from the combination of steric stabilization by PEG chains and the electrostatic stabilization due to the adequate surface charge of polymeric micelles. It has been proved that the PEG chains around the hydrophobic core can serve to minimize interactions with proteins, providing the stability to micelles.48,49 In addition, the repulsive electrostatic force between negatively charged micelles and BSA proteins can prevent the occurrence of flocculation or coagulation to maintain the stability. However, as shown in Figure 6 B and D, the △BS profiles of mPEG113-b-P(CL48-coDCL21) micelles at pH 6.0 were slightly increased, indicating a decrease in micelle stability. Especially at pH 6.0 with BSA, the △BS profiles increased gradually to about 5% with the increase of analysis time up to 12 h, indicating the appearance of abundant aggregation. The

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dimethyl maleamidic acid groups, under acidic environments, would be quickly hydrolyzed and changed into cationic primary amines

25,27

. The change, on the one hand, broke the hydrophilic

and hydrophobic balance that is critical for micellar stability.48 As a result, the stability of mPEG-b-P(CL-co-DCL) micelles at pH 6.0 was decreased. On the other hand, the chargereversal from negative to positive resulted in the strong adsorption of BSA to the micelle surface, thereby significantly decreasing the stability of polymeric micelles in BSA solution. The effect of pH on the BSA adsorption on the mPEG-b-PCL and mPEG-b-P(CL-co-DCL) micelles was investigated and shown in Figure S7. mPEG-b-PCL micelles at pH 7.4 and pH 6.0 both exhibited very low protein adsorption amount. The amount of BSA adsorption on all mPEG-bP(CL-co-DCL) micelles at pH 7.4 was low and close to the value of mPEG-b-PCL micelles. However, the pH value had a decisive effect on BSA adsorption on the mPEG-b-P(CL-co-DCL) micelles. The amount of adsorbed BSA on all mPEG-b-P(CL-co-DCL) micelles at pH 6.0 was significantly higher than that at pH 7.4, due to the charge-reversal demonstrated in Figure 7. The stability studies in the presence of BSA indicated that the mPEG-b-P(CL-co-DCL) micelles were stable and hemocompatible at physiological pH42, allowing the applications in drug delivery. ζ-Potential of mPEG-b-P(CL-co-DCL) micelles Acid-labile β-carboxylic amides in the polyester moiety rendered the surface charge of mPEG-bP(CL-co-DCL) micelles able to be reversed to positive from negative at acidic pH. In order to evaluate the ability of pH-triggered charge reversal, the effect of pH on ζ-potential of DOX-free and DOX-loaded mPEG113-b-P(CL48-co-DCL21) micelles at physiological environment (pH 7.4) and tumor microenvironment (pH 6.0) was measured and shown in Figure 7. At pH 7.4, DOXfree and DOX-loaded mPEG113-b-P(CL48-co-DCL21) micelles had an initial ζ-potential of about 13.9 and -6.6 mV, respectively. A marked decrease in ζ-potential of DOX-loaded micelles can be

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attributed to the consumption of the carboxyl groups by the positive DOX.50 It can also be observed that both DOX-free and DOX-loaded mPEG113-b-P(CL48-co-DCL21) micelles at pH 7.4 remained negatively charged even after 12 h. This can be ascribed to the inertness of negatively charged dimethyl maleamidic acid groups at pH 7.4, which has been proven by many previous studies.25,34,51 However, at pH 6.0, the ζ-potential of DOX-free and DOX-loaded mPEG113-bP(CL48-co-DCL21) micelles changed sharply from negative to high positive in a short time. It only took about 0.4 h for DOX-free micelles and about 0.9 h for DOX-loaded micelles to become positively charged. Compared with DOX-free micelles, DOX-loaded mPEG113-bP(CL48-co-DCL21) micelles had a slower charge reversal rate at pH 6.0, which may be due to the effect of the loaded DOX. Some studies have confirmed that cationic DOX and carboxyl derivatives can form very stable ionic complexes.29,30,52 In addition, these results indicated the charge reversal process was very fast due to the quick and effective detachment of the acid-labile β-carboxylic amides. These results were in agreement with the previously reports.34,45 Du et al found that 30% of the dimethyl maleamidic acid groups incubated at pH 6.8 would be hydrolyzed and transformed into amino groups in 10 min.34 Zhou et al also confirmed that, at pH 6.0, the time for 50% of the dimethyl maleamidic acid groups to hydrolyze was about 10 min.35 In sum, these results demonstrated the pH-tunable charge reversal process of mPEG-b-P(CL-coDCL) micelles. They are negatively charged at physiological pH but would activate the cationic charges in the acidic tumor extra- and intracellular environment. Obviously, this characteristic is very suitable for delivering anticancer drug, as it can be used to control drug release and effectively enhance tumor cellular internalization of drug-loaded micelles.

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Figure 7. ζ-Potential of DOX-free (A) and DOX-loaded (B) mPEG113-b-P(CL48-co-DCL21) micelles at pH 7.4 and pH 6.0.

Drug loading and release DOX, as a model chemotherapeutic agent was used to evaluate the loading and releasing properties of mPEG-b-P(CL-co-DCL) micelles. DOX-loaded mPEG-b-P(CL-co-DCL) micelles were prepared at a polymer concentration of 1 mg/mL. The characteristic of the DOX-loaded micelles, including size, size distribution, loading content and encapsulation efficiency were summarized in Table 2. The micelle sizes slightly decreased after loading different amount of DOX. As mentioned above, DOX can interact with carboxyl of DCL units by electrostatic attraction, leading to a more compact structure of micellar core and thus a smaller size of

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micelles. All the PDI remained in a narrow range of 0.15-0.19. Compared with mPEG-b-PCL, mPEG-b-P(CL-co-DCL) micelles showed a much higher drug loading capacity. The drug loading content and encapsulation efficiency of mPEG-b-P(CL-co-DCL) micelles were nearly 3 ~ 4 times higher than that of mPEG-b-PCL micelles. Moreover, the drug loading content and encapsulation efficiency of mPEG-b-P(CL-co-DCL) micelles increased with increasing the content of DCL units. These phenomena can be due to the electrostatic interaction between DOX and carboxyl. The carboxyl derivatives, such as oleic acid, polyglutamic acid and polymalic acid, have widely been used to enhance the DOX-loading capacity and efficiency.29,30,53 The in vitro drug release studies of DOX-loaded mPEG-b-P(CL-co-DCL) micelles were carried out under the physiological environment (pH 7.4) and endosomal compartments (pH 5.4) using a dialysis method at a concentration of 1.0 mg/mL. As shown in Figure 8, the release of DOX from all copolymer micelles was obviously faster at acidic pH (5.4) than at pH 7.4. At pH 7.4, only about 20% of DOX was released within 24 h from mPEG-b-PCL micelles, whereas about 10 ~ 13% of DOX was released from mPEG-b-P(CL-co-DCL) micelles. In addition, no significant difference in the drug release among mPEG-b-P(CL-co-DCL) micelles with different content of DCL units in polymers was observed. The very slow drug release from mPEG-bP(CL-co-DCL) micelles, as the consequence of electrostatic attraction between DOX and carboxyl in the micellar core, indicated that DOX-loaded mPEG-b-P(CL-co-DCL) micelles was relatively stable under physiological conditions. However, the release rate of DOX was significantly accelerated at pH 5.4. About 31.4% and nearly 100% of DOX was released from mPEG-b-PCL micelles and mPEG-b-P(CL-co-DCL) micelles in 24 h, respectively. Especially, it can be observed that the DOX release rate at pH 5.4 from mPEG-b-P(CL-co-DCL) micelles was extremely high, particularly in the initial phase. The cumulative release of DOX was

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approximately 90% in 6 h. The drug release behavior from polymeric micelles depended on drug diffusion rate from the micelles, micelle stability and the interaction between the drug and the core-forming block. The accelerated drug release from mPEG-b-PCL micelles can attribute to the higher solubility of DOX in acidic media, leading to faster drug diffusion from micelles to media.54 The significant increase in DOX release from mPEG-b-P(CL-co-DCL) micelles in the acidic medium can be ascribed to that the pH-induced hydrolysis of the dimethyl maleamidic acid groups resulted in a quick charge reversal, as demonstrated above. Owing to the charge conversion, a repulsive electrostatic force will generate in the DOX-loaded mPEG-b-P(CL-coDCL) micelles, which can greatly accelerate the release rate of drug. These results were in agreement with the previous reports performed by Kataoka et al.24,31 They observed 97% of drug was released in 5 h at pH 5.5 from charge reversal micelles prepared from citraconic amidefunctionalized copolymers.24 It can be found that drug release rate slightly decreased with increasing DCL content in mPEG-b-P(CL-co-DCL) copolymers. 89.3%, 85.1%, and 76.4% of DOX was released in 4 h at pH 5.4 from mPEG113-b-P(CL45-co-DCL9) micelles, mPEG113-bP(CL48-co-DCL21) micelles and mPEG113-b-P(CL47-co-DCL29) micelles, respectively. This may be due to the fact that the higher DCL content resulted in forming stronger interaction and a slightly slower hydrolysis. In addition, it was worth pointing out that the detached dimethyl maleic acids (DMA) are negative and thus they are likely to electrostatically interact with the positive DOX to form DOX-DMA ionic complexes. Moreover, DOX-DMA ionic complexes should be much more water-soluble than DOX, due to highly water solubility of DMA. In sum, DOX-loaded mPEG-b-P(CL-co-DCL) micelles showed a pH-dependent DOX release profile, which should be beneficial for tumor treatment. Most of loaded DOX will remain in micelles for a long time when DOX-loaded mPEG-b-P(CL-co-DCL) micelles stay in the plasma at normal

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physiological conditions (pH 7.4). However, the pH-sensitive micelles will promptly release DOX by sensing the change of pH corresponding to the acidic conditions in the intracellular endosomal compartments (pH 4.0 - 6.5), when DOX-loaded micelles are taken up by the tumor cells via an endocytosis process.

Figure 8. In vitro release of DOX from mPEG-b-P(CL-co-DCL) micelles at pH 7.4 and pH 5.4.

In vitro Cell Uptake To further demonstrate the effect of charge reverse ability of mPEG-b-P(CL-co-DCL) micelles on the cellular internalization process, the cell uptake of free DOX, DOX-loaded mPEG113-bPCL77 micelles and DOX-loaded mPEG113-b-P(CL48-co-DCL21) micelles at pH 7.4 and 6.0 were investigated on HepG2 cells by the use of fluorescence microscopy. DOX could show an intrinsic red fluorescence. Blue fluorescence represents cell nucleus stained with DAPI. Merged images displayed the overlay of red fluorescence of DOX and blue DAPI staining of nuclei. As shown in Figure 9 (A), the cells exposed to free DOX showed an obvious fluorescence signal in the nucleus (evidenced by purple dots in nucleus, a sign of co-localization of DOX with DAPI)

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and a relatively weak fluorescence signal in cytoplasm (evidenced by red dots of DOX signal). This can be explained that the cellular uptake mechanism of free DOX was basically through passive diffusion. Fluorescence microscopy images of HepG-2 cells incubated with DOX-loaded

mPEG113-b-PCL77 micelles and DOX-loaded mPEG113-b-P(CL48-co-DCL21) micelles were shown in Figure 9 (B) and (C). It can be observed that the DOX fluorescence intensity in the tumor cells treated with DOX-loaded mPEG113-b-PCL77 micelles and DOX-loaded mPEG113-b-P(CL48-co-

DCL21) micelles was much higher than those treated with free DOX. It was widely accepted that micelles are more effectively internalized into cells by an endocytosis procedure, rather than by passive diffusion of the free drug. Even negatively charged particles can be internalized by cells through caveolae-mediated endocytosis, in spite of the unfavorable interaction between the particles and the negatively charged plasma membrane55-57 Therefore it was not surprising to observe that the cellular uptake efficiency of DOX-loaded micelles at pH 7.4 and pH 6.0 was higher than that of free DOX. In addition, no significant difference was observed in the cellular uptake at different pH values of free DOX and DOX-loaded mPEG113-b-PCL77 micelles. However, the remarkably more efficient cellular uptake occurred in DOX-loaded mPEG113-b-P(CL48-co-DCL21) micelles at pH 6.0. This can be ascribed to the occurrence of electrostatic attraction between micelles and cell membrane due to charge reversal from negative to positive as a consequence of the hydrolysis of β-

carboxylic amides at acidic pH, thus enhancing the cellular internalization. Moreover, it can also be observed that the cells treated with DOX-loaded micelles showed a much broad red fluorescence distribution both in cytoplasm and nucleus. In particular, for the cells treated with DOX-loaded

mPEG113-b-P(CL48-co-DCL21) micelles at pH 6.0, the DOX fluorescence was detected primarily in the cytoplasm. This kind of intracellular drug distribution can be due to the rapid endo-lysosomal escape of micelles into cytosol following their uptake56. Many studies have demonstrated that charge reversal of micelles selectively in the acidic pH of endo-lysosomal compartment is proposed as the

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mechanism responsible for the escape of micelles from endo-lysosomes55,57. These results demonstrated that pH-triggered charge reversal micelles for intracellular drug delivery could significantly enhance cellular uptake and facilitate endo-lysosomal escape, leading to a high therapeutic efficacy.

Figure 9. Representative fluorescence microscopy images of HepG2 cells incubated with free DOX (A), DOX-loaded mPEG113-b-PCL77 micelles (B), and DOX-loaded mPEG113-b-P(CL48co-DCL21) micelles (C) at equivalent DOX concentrations of 10 mg/mL at pH 7.4 and 6.0 respectively. The images from left to right were DOX fluorescence in cells, Cell nuclei stained by DAPI, Merged images and Enlarged views of the boxed areas in the merged images. The scale bars correspond to 75 µm for big views, and 25 µm for small views. Cell Viability Assays The in vitro biocompatibility and cytotoxicity of mPEG113-b-PCL77 and mPEG113-b-(PCL48-coDCL21) toward HepG-2 cells were evaluated using a MTT assay. As shown in Figure 10 (A), the viability of cells treated with both polymer micelles for 24 h was evaluated at all test concentrations up to 1.0 mg/mL. PEG-b-PCL polymer has been demonstrated to be

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biocompatible and non-toxic. In the tumor cell cytotoxicity test, the mPEG113-b-(PCL48-coDCL21) micelles exhibited similar biocompatibility with mPEG113-b-PCL77, without any significant cytotoxicity against HepG-2 cells, which indicated that mPEG-b-P(CL-co-DCL) could be safely used as biocompatible carriers for efficient intracellular drug delivery. The viability of HepG2 cells after incubation with free DOX, DOX-loaded mPEG113-b-PCL77 micelles and DOX-loaded mPEG113-b-(PCL48-co-DCL21) micelles was evaluated and showed in Figure 10 (B). All the studied cell lines showed a typical dose-response sigmoidal curve. The in vitro half maximal inhibitory concentration (IC50) values were 0.49, 0.62 and 1.17 µg/mL for free DOX, DOX-loaded mPEG113-b-(PCL48-co-DCL21) micelles and DOX-loaded mPEG113-b-PCL77 micelles, respectively. The DOX-loaded mPEG113-b-PCL77 micelles showed a much lower cytotoxicity than that of free DOX, but DOX-loaded mPEG113-b-(PCL48-co-DCL21) micelles showed comparable antitumor activity to the free DOX. The lower cytotoxicity of DOX-loaded mPEG113-b-PCL77 micelles may be due to the incomplete drug release from micelles and delayed nuclear uptake.55 Compared with DOX-loaded mPEG113-b-PCL77 micelles, DOX-loaded mPEG113-b-(PCL48-co-DCL21) micelles showed a higher inhibition in cellular proliferation, which was mostly attributable to the accelerated drug release in acidic intracellular compartments, enhanced cellular uptake and improved endosomal escape, as evidenced by both in vitro DOX release and cellular uptake studies. Above results confirmed that mPEG-b-P(CLco-DCL) micelles with the function of acid-trigged charge reversal can efficiently load and deliver DOX into tumor cells, which are highly promising as efficient nanocarriers for intracellular delivery of anticancer drugs, especially for cationic hydrophobic drugs.

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Figure 10. Viability of HepG2 cells after incubation for 24 h with mPEG113-b-PCL77 and mPEG113-b-P(CL48-co-DCL21) micelles at various concentrations (A). Viability of HepG2 cells after being incubated with free DOX, DOX-loaded mPEG113-b-PCL77 micelles and DOX-loaded mPEG113-b-P(CL48-co-DCL21) micelles for 24 h (B)

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CONCLUSION In this work, a series of multifunctional PEG-b-PCL copolymers bearing acid-labile β-carboxylic amides-modified PCL chain with the ability of acid-triggered charge-reversal from negative to positive were designed and synthesized. The mPEG-b-P(CL-co-DCL) copolymers with relatively low CMC values can readily self-assemble into stable nano-sized micelles in aqueous solution. Moreover, due to the electrostatic interaction between DOX and carboxyl, the mPEG-b-P(CL-coDCL) micelles exhibited a very high drug-loading capacity and loading efficiency. The dimethyl maleamidic acid groups on the polyester segment of mPEG-b-P(CL-co-DCL) can be hydrolyzed under acidic environment and resulted in a quick charge reversal, which had great influence on the drug release profiles, cell internalization process and endo-lysosomal escape. The in vitro DOX release from mPEG-b-P(CL-co-DCL) micelles was significantly increased when solution pH decreased from 7.4 to 5.4. The fluorescence microscopy observation also indicated that more DOX was delivered and released into the cytoplasm of HepG-2 cells treated with DOX-loaded mPEG-b-P(CL-co-DCL) micelles in culture medium at pH 6.0. In addition, the mPEG-b-P(CLco-DCL) micelles showed a very low cytotoxicity up to a concentration of 1.0 mg/mL, and MTT assays revealed that DOX-loaded mPEG-b-P(CL-co-DCL) micelles showed similar cytotoxicity to HepG-2 cells compared with the free DOX. These results demonstrated that mPEG-b-P(CLco-DCL) micelles with pH-induced charge-reversal function were able to actively load cationic DOX, and efficiently deliver them into tumor cells, achieving high cellular proliferation inhibition. These results also indicated that the introduction of acid-labile β-carboxylic amides on the polyester block in mPEG-b-P(CL-co-DCL) should be a very promising approach to improve the properties of mPEG-b-PCL micelles.

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ASSOCIATED CONTENT Supporting Information Structure and composition of γ-(carbamic acid tert-butyl ester)-ε-caprolactone (CATCL) and 1HNMR spectra of CATCL in CDCl3 used in this paper. Structure and composition of mPEG-bP(CL-co-CATCL) used in this paper and 1H-NMR spectra of mPEG-b-P(CL-co-ACL) in DMSO-d6 used in this paper. Structure and composition of mPEG-b-P(CL-co-DCL) and mPEGb-P(CL-co-ACL) used in this paper. The FT-IR spectra of mPEG-b-P(CL-co-CATCL) and mPEG-b-P(CL-co-ACL). GPC elution chromatograms of mPEG-b-P(CL-co-CATCL), mPEG-bP(CL-co-ACL) and mPEG-b-P(CL-co-DCL) copolymers. GPC elution chromatograms of mPEG-b-P(CL-co-DCL) copolymers. Size distribution and morphology of mPEG-b-P(CL45-coDCL9) micelles and mPEG113-b-P(CL47-co-DCL29) micelles. BSA adsorption on the polymeric micelles at pH 7.4 and pH 6.0 for 12 h. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected]; Fax: +86 22 27890710; Tel: +86 22 27890707. Present Address a

Department of Polymer Science and Technology and Key Laboratory of Systems

Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Note The authors declare no competing financial interest. ACKNOWLEDGMENT

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This work was supported by a grant from the National Natural Science Foundation of China (Number 51103097, 81371667, 31271073, 81171371 and 31470963).

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