Biocompatible Reduction and pH Dual-Responsive Core Cross

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Biocompatible Reduction and pH Dual-Responsive Core CrossLinked Micelles Based on Multi-Functional Amphiphilic LinearHyperbranched Copolymer for Controlled Anticancer Drug Delivery Kun Tian, Xu Jia, Xubo Zhao, and Peng Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01051 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 12, 2017

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Molecular Pharmaceutics

Biocompatible Reduction and pH Dual-Responsive Core Cross-Linked Micelles Based on MultiFunctional Amphiphilic Linear-Hyperbranched Copolymer for Controlled Anticancer Drug Delivery Kun Tian, Xu Jia, Xubo Zhao, and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ABSTRACT: Novel strategy has been developed for fabricating the biocompatible reduction and pH dual-responsive core cross-linked (CCL) micelles as drug delivery system (DDS) for the controlled anticancer drug delivery, via the atom transfer radical polymerization (ATRP) of tertbutyl acrylate (tBA) with N,N'-bis (acryloyl)cystamine (BACy) as crosslinker and a multifunctional amphiphilic linear-hyperbranched copolymer as macroinitiator, which was synthesized via the self-condensing vinyl co-polymerization (SCVCP) of tert-butyl acrylate (tBA) and pchloromethylstyrene (CMS) with a poly(ethylene glycol) (PEG) based initiator (mPEG-Br). The hydrolyzed core cross-linked (HCCL) micelles were obtained as DDS for doxorubicin (DOX) by hydrolysis the tBA units into acrylic acid (AA) ones. The in vitro release performance showed that higher GSH concentration and/or lower pH value would lead to a faster and more efficient

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DOX release, meaning their reduction and pH dual-responsiveness. Therefore, the proposed HCCL micelles are expected to be potential anticancer drug-carriers for tumor microenvironment responsive controlled delivery.

Keywords: drug delivery system; core cross-linked micelles; amphiphilic linear-hyperbranched copolymer; reduction and pH dual-responsive; controlled release

INTRODUCTION Since the core cross-linked (CCL) micelles were synthesized by Wooley’ group for the first time in 1996,1 they have attracted more and more interest in diagnosis and treatment of cancer in the last decades,2,3 owing to their simple fabrication, and unique core-shell structure with stable morphology and narrower diameter distribution, especially as a successful solution for the dilemma of extracellular stability versus intracellular drug release responding to the tumor pathological micro-environmental factors, such as acidic pH values4-6, higher reductant level7-11, and the both.12 At the same time, their nanoscaled particle size is beneficial to accumulating in tumor tissues via the enhanced permeability and retention (EPR) effect.13 These features especially make the CCL micelles grow into potential smart polymeric cargo as drug delivery system (DDS) for overcoming multidrug resistance in cancer therapy.12,14 As drug-carriers, the actual applicability and feasibility of CCL micelles are mostly based on the monomers, crosslinking agents and/or initiators used in their synthesis procedure. To resolve the dilemma of extracellular stability versus intracellular drug release, the acid-labile4-6 or reduction cleavable linkages7-11 have been developed as the crosslinking units for the CCL micelles, especially the disulfide bond-containing crosslinking agents such as N,N'bis(acryloyl)cystamine (BACy), which could introduce the additional reduction responsiveness to


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the DDSs for accelerating drug release triggered by high level of the reducing agent at the tumor tissues.15,16 On the other hand, initiators are another important factor on the structural stability and multi-response capability of the CCL micelles. In order to introduce biocompatibility, the PEG-based macroinitiators have been widely used, as well as the PEG-based macromonomers such as poly (ethylene glycol) monomethyl ether methacrylate) (PEGMA).17 Frechet et al. firstly described the principle of self-condensing vinyl co-polymerization (SCVCP) by synthesizing the hyperbranched polymers using 3-[(1-chloroethyl)ethenyl]benzene as an inimer.18 Muller synthesized the acrylate-based hyperbranched polymers via SCVCP of 2(2-bromoisobutyryloxy)ethyl methacrylate (BIEM) as an inimer with (diethylamino)ethyl methacrylate (DEAEMA).19 After quaternization with MeI, novel cationic hyperbranched polyelectrolytes were obtained for viable tailored materials with unique properties for various applications, such as chemical sensing and gene delivery. The hyperbranched polymers own porous structure and plentiful active end groups in surface, which has been used for the intermolecular interaction-induced self-assembly.20 Also, their highly branched structure endows the polymer chains hardly twining, which could improve solubility of polymer.21 In addition, the plentiful active end groups in the hyperbranched polymers could be used as the initiating groups for the graft polymerization. However, there is no report on the CCL micelles prepared with the hyperbranched polymer-based macroinitiator, although the hyperbranched polymer-based macroinitiator has been successfully used for the surface-initiated grafting polymerization to achieve a high percentage of grafting.22 Recently, linear-dendritic prodrug was designed by conjugating Doxorubicin (DOX) and lipoic acid onto mPEG-b-PAMAM copolymer owing to the plentiful end functional groups of PAMAM.23 Then cross-linked micellar prodrug nanoparticles were obtained by crosslinked the prodrug with dithiothreitol (DTT) after self-assembly, for the pH and reduction dual-responsive


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drug delivery. Here, a multi-functional amphiphilic linear-hyperbranched copolymer was synthesized via the SCVCP of tert-butyl acrylate (tBA) and p-chloromethylstyrene (CMS) from a poly(ethylene glycol) (PEG) based initiator (mPEG-Br) (Scheme 1), and used as a macroinitiator for the atom transfer radical polymerization (ATRP) of tert-butyl acrylate (tBA) and N,N'-bis (acryloyl)cystamine (BACy) to fabricate a reduction responsive CCL micelles. After hydrolyzing the tBA units into acrylic acid (AA) ones, novel cytocompatible reduction and pH dualresponsive core cross-linked (HCCL) micelles were designed as DDS for the tumor intracellular triggered release of DOX (Scheme 2).

Scheme 1. Preparation of the multi-functional amphiphilic linear-hyperbranched copolymer via SCVCP.


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Scheme 2. Preparation of the HCCL micelles via ATRP and their drug loading and tumor intracellular triggered release behavior.

EXPERIMENTAL SECTION Materials and reagents. Methoxy poly(ethylene glycol) (mPEG-OH, Mn=2000) was purchased from Beijing Chemical Works. 2-Bromoisobutyryl bromide (97.0%) was purchased from TCI. Triethylamine (TEA) was stirred with CaH2 for 48 h in order to remove any trace of water. Cuprous bromide (Cu(I)Br) (Tianjin Guangfu Fine Chem. Res. Inst.) was treated by glacial acetic acid for 12 h in the dark, then washed with anhydrous ether, anhydrous ethanol and acetone. Tert-


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butyl acrylate (tBA) was stirred with CaH2 overnight and distilled under reduced pressure. pChloromethylstyrene (CMS) was extracted with 5% NaOH solution and dried by anhydrous Na2SO4. 1,1,4,7,7-Pentamethyldiethylenetriamine (PMDETA, >98%) was purchased from Tokyo KASET Kogyo. Co., Ltd. Doxorubicin hydrochloride (DOX⋅HCl, 99.4%) was provided from Beijing Huafeng United Technology Co. Ltd. All other reagents were analytical reagent grade and the deionized water was used for all experiments and sample preparations.

Synthesis procedure. mPEG-Br. mPEG-Br was synthesized as macroinitiator according to the procedure reported previously:24 Firstly, in order to remove any trace water, mPEG-OH (20 g, 0.01 mol) was dissolved in 250 mL of dried toluene, and 50 mL toluene was distilled out at 75 °C under reduced pressure. Then, TEA (2.79 mL, 0.02 mol) was added into the reacting flask. The flask was placed at ice bath until the temperature inside went to 0 °C. 2-Bromoisobutyl bromide (2.54 mL, 0.02 mol) was slowly added using constant separating funnel and the mixture was stirred for 48 h at room temperature. After that, most of toluene was distilled out by rotary evaporation before the crude polymer was achieved by precipitating into a 10-times volume of ether. The precipitate was dried in vacuum at 40 °C overnight and then dissolved in saturated solution of NaHCO3. Finally, the mPEG-Br macroinitiator was obtained by extracting with dichloromethane for three times, and distilling by rotary evaporation.

Amphiphilic linear-hyperbranched copolymer. The typically SCVCP technique was used to synthesize the linear-hyperbranched copolymer.18 Typically, mPEG-Br (1.0 g, 0.5 mmol), tBA (1.09 mL, 7.5 mmol), CMS (1.06 mL, 7.5 mmol), PMDETA (0.105 mL, 0.5 mmol) and cyclohexanone (2 mL) were added into a Schlenk flask. Then, Cu(I)Br (72 mg, 0.5 mmol) was


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added after several freeze-pump-thaw cycles. The reaction was conducted with stirring for 24 h at 90 °C. Finally, the mixture was exposed to air and 5 mL tetrahydrofuran (THF) was added in order to dissolve the product. Neutral alumina column was used to remove residual catalyst. All solvent was removed by rotary evaporation, and the glutinous crude product was dissolved in 2 mL acetone and precipitated into deionized water at 0°C. The product was obtained by centrifugation and then dried in vacuum at 40 °C.

CCL micelles. N,N'-bis(acryloyl)cystamine (BACy) was synthesized according to the method reported previously.25 The amphiphilic linear-hyperbranched copolymer synthesized above was used as the multi-functional macroinitiator for the fabrication of the CCL micelles through the ATRP technique, with the molar ratio of [macroinitiator]:[tBA] of 1:100 and [tBA]/[BACy] molar ratio of 100:5 and 100:2, respectively. Take the first situation as example, macroinitiator (0.4 g, 0.1 mmol), tBA (1.45 mL, 10 mmol), BACy (0.13 g, 0.5 mmol) and PMDETA (21 µL, 0.1 mmol). Cu(I)Br (14.4 mg, 0.1 mmol) was added into a mixed solution of 4 mL methanol and 2 mL cyclohexanone. After two freeze-pump-thaw cycles, the flask was heated in an oil bath at 90 °C for 9 h. Then the product was purified by passing a neutral alumina column, dissolved in 2 mL acetone, precipitated into deionized water, and dried in vacuum at 40 °C.

HCCL micelles. The CCL micelles (1.0 g) were dispersed into CH2Cl2 (4 mL), then trifluoroacetic acid (TFA, 1 mL) was added in order to hydrolyze the ester groups in the tBA units. Stirring in the dark for 4 h, the solvent was removed by rotary evaporation. The hydrolyzed CCL (HCCL) micelles were obtained after dried in vacuum at 40 °C.


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Drug loading and controlled release. 10 mg HCCL micelles were dispersed into 4 mL DOX⋅HCl aqueous solution (pH 5.0, 1.0 mg/mL) with ultrasonic treatment in the dark for 12 h, and then the dispersion was stirred for 48 h in the dark at room temperature, after adjusting to pH 7.4 with dilute NaOH aqueous solution. The DOX-loaded micelles were obtained by centrifugation while the supernatant solution was measured by a Lambda 35 UV-vis spectrophotometer at a wavelength of maximum absorbance (480 nm, Figure S1), to calculate the DOX-loading capacity (DLC) and DOX-loading efficiency (DLE) according to the formula below: DLC (%) =

initial mass of DOX - mass of DOX in supernatan t × 100% mass of HCCL micelles

DLE (%) =

initial mass of DOX - mass of DOX in supernatant × 100% initial mass of DOX

For the in vitro controlled release, the dialysis tubes with molecular weight cut off of 14000, containing 5 mL phosphate buffer saline (PBS) dispersion of the DOX-loaded micelles (10 mg) with different pH values (5.0 or 7.4) and different levels of reductant (10 µM or 10 mM GSH or DTT) respectively, were placed into 120 mL of the same buffer solution in an IS-RSD3 incubation shaker at 37°C. After mildly shaking at 37 °C for certain time, 5 mL dialysate was taken out and measured by UV-vis spectrophotometer at 480 nm for the purpose of getting the DOX concentration gradient and the drug release profile, and 5 mL fresh PBS buffer solution was added to keep the dispersion volume constant.

Cellular toxicity and uptake. In a Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), SKOV3 cells (1×104 cells/well) were seeded in 96-well cell plates. Then medium was removed and 100 µL of the HCCL dispersion (with or without DOX)


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was added. After cultured for 24 h at 37 °C and 5% CO2, 10 µL of 5 mg/mL MTT [3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] was added to the cell plates in order to get the cell viability. 4 h later, each well was added with 100 µL lysis solution (95 µL 10% SDS, 5 µL isobutyl alcohol and 1.0 µL concentrated hydrochloric acid). After the plates were shaken for 20 min, their absorbance was measured by the Enzyme-linked Immunosorbent Assay Appliance at the wavelength of 570 nm. Confocal laser-scanning microscopy (CLSM) was used to investigate the cellular uptake behavior. Firstly, the cell nuclei of SKOV3 cells were treated by Hoechst 33258 for coloration. The cells seeded in a 6-well plate were bred in medium contains 100 µL of PBS. Then, 0.50 mL 20.0 µg/mL of the dispersion of the DOX-loaded HCCL micelles was added into the well plate. After washing the cells with PBS, the cellular uptake behavior was observed by CLSM at 480 nm (DOX) and 405 nm (Hoechst).

Characterization. Chemical structure of the hyperbranched polymers was characterized by 1HNMR spectrometer (JEOL, ECS 400M), using dimethyl sulfoxide-d6 as solvent. FT-IR spectra of the CCL and HCCL micelles were characterized in 4000-400 cm-1 wavenumber range with a Nicolet 360 FTIR spectrometer, with the KBr pellet method. Morphology of the CCL and HCCL micelles was observed using transmission electron microscopy analysis (TEM, JEM-1200EX). The hydrodynamic diameter and distribution of the CCL and HCCL micelles were analyzed using the dynamical mode (dynamic light scattering (DLS)) on a Light Scattering System BI200SM device (Brookhaven Instruments) equipped with a BI-200SM goniometer, a BI-9000AT correlator, a temperature controller, and a coherent INOVA 70C argon ion laser at 20°C. DLS


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measurements are performed using a 135-mW intense laser excitation at 514.5 nm at a detection angle of 90° at 25 °C. Due to the CCL micelles were difficult to be dispersed well into water directly, they were dispersed into 1 mL ethanol, and then the ethanol dispersions were mixed into 100 mL water for the TEM and DLS analysis. As for the HCCL micelles, they were dispersed into water directly for the TEM and DLS analysis. DOX release profiles were measured by a UV-vis spectrophotometer (TU-1901) at wavelength of 480 nm at 37 °C.

Figure 1. 1 H NMR spectrum of mPEG-Br (A) and the amphiphilic linear-hyperbranched copolymer (B).

RESULTS AND DISCUSSION Amphiphilic linear-hyperbranched copolymer. Firstly, mPEG-Br was synthesized as the macroinitiator for the SCVCP. As showed in the 1H NMR spectrum of the mPEG-Br (Figure 1A), the 2-bromoisobutyryl group was revealed by the appearance of the chemical shift at δ=1.9


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ppm. Additionally, the chemical shift at δ=3.6 ppm was due to the protons in O-CH2- of PEG, while the one at δ=3.4 ppm was due to the protons in the O-CH3 end groups of mPEG. The relative peak area ratio of the two signals at δ=1.9 ppm and δ=3.4 ppm was about 2.1:1, near to the theoretical number ratio (2:1) of the two kinds of protons in the different chemical environments as the end groups of the desired product, mPEG-Br. It indicated that mPEG-OH has been converted completely into mPEG-Br. Then the amphiphilic linear-hyperbranched copolymer was synthesized via the SCVCP technique, in which mPEG-Br, tBA and CMS were mixed with molar ratio of 1:15:15. From the 1

H NMR spectrum of the product (Figure 1B), the chemical shifts around δ=7.5 ppm were

attributed to the aromatic protons in benzene while that at δ=3.6 ppm of O-CH2- in PEG was remained. Besides, the strong signal at δ=1.5 ppm was due to the tert-butyl group of PtBA. The chemical shift at δ=4.7 ppm revealed the existence of the chloromethyl protons (-CH2Cl) of the CMS units, and those at δ=5.3 and δ=5.8 ppm meant the vinyl protons (CH2=CH-) of the CMS units. These evidences showed that the multi-functional amphiphilic hyperbranched-linear copolymer had been synthesized successfully. Moreover, the relative peak area showed that average about 4 tBA and 10 CMS units had been copolymerized with one macroinitiator mPEGBr. It can be calculated that the amphiphilic linear-hyperbranched copolymer owns an average relative molecular weight of 4040,26 which was proved by the GPC analysis result (Mn = 4150, PDI = 1.07).

Hydrolyzed core cross-linked (HCCL) micelles. In order to investigate the effect of the structure on the size and morphology of the final micelles, three kinds of CCL micelles were prepared under different cases: (i) [tBA]/[BACy] molar ratio of 100:5 in the mixture solvent of 4 mL methanol and 2 mL cyclohexanone, (ii) [tBA]/[BACy] molar ratio of 100:5 in the mixture


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solvent of 2 mL methanol and 1 mL cyclohexanone, and (iii) [tBA]/[BACy] molar ratio of 100:2 in the mixture solvent of 2 mL methanol and 1 mL cyclohexanone, with the same amount of the amphiphilic linear-hyperbranched copolymer as multi-functional macroinitiator. The products were distinguished with DLS technique (Figure 2). Comparing the latter two cases, the concentration of monomers decreased as the solvent amount increased, which would reduce the number of molecules involved in the crosslinking reaction. It’s easy to see that the product from the first case owned the two hydrodynamic diameter and distinguishable size distribution (40 and 70 nm, Figure 2a), compared to the others. As for the latter two cases, the swelling ability of the micelles was strengthened with the decrease in the crosslinking ratio, resulting into an increase in their hydrodynamic diameter. So the product from the third case owned the maximum hydrodynamic diameter (around 270 nm, Figure 2c). Such big particle is not beneficial to the application as DDS, because that a diameter between 30 nm and 200 nm is desired to produce long-circulating nanoparticles that can accumulate inside tumor tissues.27 Therefore, the CCL micelles obtained from the second case, which possessed the narrowest diameter distribution and appropriate average hydrodynamic diameter (Dh=80 nm, Figure 2b), were chosen for the further application.

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To gain the carboxyl groups to provide the pH-responsive characteristic, the CCL micelles were then hydrolyzed with trifluoroacetic acid (TFA) to introduce the AA units into the hydrolyzed CCL (HCCL) micelles. As shown in the FT-IR spectra (Figure S2), the presence of the -COOH groups was proved by the two absorbance peaks at 3300 cm-1 and 1680 cm-1. Also, the absorbance peak of the ester group disappeared and the absorbance of –CH3 and –CH2groups at around 2900 cm-1 became weak, meaning that the PtBA blocks had been transformed into PAA completely. After the hydrolyzation of the ester groups in the CCL micelles in organic solvent, the products showed bigger particle size of about 110 nm with relatively narrow particle size distribution than those of the CCL micelles (Figure 3), also revealing the successful hydrolysis. There was no small particle matter in the TEM analysis, also indicating that the CCL


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micelles had been successfully crosslinked to form stable structure with BACy as crosslinker during the ATRP, although the characteristic absorbance of amide groups in BACy units was overlapped by the stronger absorbance peak of the carboxyl groups in the FT-IR spectrum. The HCCL micelles contained plentiful carboxyl acid groups, so they can swell significantly in water, with hydrodynamic diameter of about 380 nm (Figure 2d).

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Figure 4. Variation of the average hydrodynamic diameter (Dh) and scattered light intensity of the HCCL micelles as a function of the pH values of their aqueous dispersions.

The pH-responsive characteristic of the HCCL micelles was also investigated with DLS technique. The average hydrodynamic diameter (Dh) of the HCCL micelles kept at a low value (in the range of 160-180 nm) in the lower pH region (3-4) (Figure 4). When pH was higher than 4, along with the increase in pH value, there was a significant increase of Dh (to 320 nm) as well as the decrease of the scattered light intensity (23 kcps to 7 kcps), due to the pKa value of carboxyl acid groups in PAA about 4.7.28 Within high pH region, the carboxyl acid groups in PAA segments would dissociate into carboxylate anions, and then their chains would mutually repulsive because of the electrostatic exclusion. Thus, the biggest diameter (about 500 nm) and lowest scattered light intensity (about 4 kcps) were achieved above pH 9.0.

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(c) Figure 5. Diameter dynamic distribution of the HCCL micelles after being treated with 10 mM GSH for 3 h (a), 5 h (b), and 12 h (c).

DLS technique was also used for the characterization of the reduction-triggered disintegration of the HCCL micelles, after they were treated with 10 mM GSH in pH 7.4 PBS solution for different times. The results are presented in Figure 5. The part with Dh of about 70 nm should be the shrunk HCCL micelles due to the effect of the ionic strength. With the extension of the treating time, the matter with bigger Dh appeared as the aggregates of the polymer blocks, and their Dh increased from 160 nm to 250 nm, and finally 370 nm from 3 h to 5 h and further to 12 h, respectively. Furthermore, the matter with Dh of about 50 nm was formed within 12 h. These phenomena demonstrated that the HCCL micelles have been destroyed by treating with GSH, revealing the reduction-triggered disintegration characteristic of the HCCL micelles.

DOX-loading and in vitro release. To evaluate the drug-loading possibility of the HCCL micelles, doxorubicin (DOX), which possesses a wide antitumor spectra against hematopoietic


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malignancies and solid tumors,29 was chosen as a model drug. The HCCL micelles possessed plentiful carboxyl acid groups while there is an amino group in one DOX molecule. Thus, DOX is expected to be loaded onto the HCCL micelles as an alkaline molecules (with pKa of 8.25) via the strong electrostatic interaction.30 The DLC and DLE were analyzed by UV spectrophotometer to be 18.4% and 46.0%, respectively.

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It is well-known that the pH value of cancer cells’ living environment is tend to be 4.5-5.5 while that of normal cells’ living environment is tend to be around 7.4. To simulate the drugs’ behavior in human cells’ living environment, pH 7.4 and 5.0 PBS solutions were chosen as release media for the in vitro release performance. From Figure 6, the cumulative releases at pH 5.0 and 7.4 were 28.8% and 16.8% within 24 h, respectively. At acidic media, free carboxylic acid groups were generated by protonation effect and the electrostatic interaction between DOX


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and micelles was weakened, leading to the faster drug release.31,32 In addition, the higher solubility of DOX at acidic media is also beneficial to its release. From the practical situation of cancer cells , not only pH values but also the concentrations of reducing agent such as glutathione (GSH) and DL-dithiothreitol (DTT) in both cell nucleu and cytosol should be considered. GSH has already been proved to play an important role in the control of the reductive environment in cells.33 The control was achieved by concentration difference between intra- and extracellular and it has been found that the former concentration is about 1000 times higher than the latter.15 Based on this reason, disulfide bonds, which could be cleaved off by reducing agent, have been introduced by BACy in the synthesis process for the unique reduction-responsive drug controlled release performance.34 Thus, the crosslinked structure of the HCCL micelles could be cleaved off, favoring the diffusion of DOX out the HCCL micelles. So PBS solutions containing GSH or DTT were chosen to investigate the influence of reducing agent on the in vitro release. In pH 5.0 media, it was apparent that the DOX release rate in PBS with 10 mM GSH was far higher than that in PBS without GSH. After 3 h of drug-releasing, the DOX cumulative release in the former situation (55.1%) was about five times higher than that in the latter one (11.3%). After 24 h, the final cumulative release came to 77.8% and 28.8%, respectively. The HCCL micelles were mostly destroyed due to disulfide bonds had been broken down by GSH, and DOX loaded in micelles was transformed into free molecule. As for the other reducing agent DTT, the DOX cumulative release came to 21.6% in initial 3 h and then increased to 61.2% in the end. It can be seen that GSH possessed more powerful reductive ability than DTT, due to the synergistic effect of GSH containing an amino group. It can replace DOX in its electrostatic interaction with PAA segment.35 Furthermore, the cationic GSH in acidic media could also generate repulsive force toward DOX. The two factors would accelerate


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the release of DOX. In fact, GSH is the major redox factor which decides the reductive capacity of cells in cells’ living environment.36 For mimicking the microenvironment of the normal cells, pH 7.4 PBS solutions with 10 µM GSH or DTT were used as in vitro releasing media to predict the drug leakage during the blood circulation. pH 7.4 PBS solution with 10 µM GSH led to a faster DOX release rate and higher final cumulative release (19.3%) than that with 10 µM DDT or without GSH/DDT. Replacing GSH by DTT, the cumulative release was down to 17.7%. Compared with pH 7.4 PBS solution without any reducing agent (16.8%), there was only a slight increase. It is due to the fact that only little disulfide bonds in the crosslinking structure had been cleaved off in presence of the low reductant level. The fast DOX release in pH 5.0 PBS with 10 mM GSH (mimicking the tumour microenvironment) and low cumulative release in pH 7.4 PBS solution with 10 µM GSH (mimicking the normal tissue microenvironment) demonetrated that the HCCL micelles could release DOX upon the tumour microenvironment, while less leakage in normal tissue microenvironment. These features are expected to not only improve the anticancer efficiency of the anticancer drugs, but also reduce their toxic side effect to the normal tissues. Higuchi and Korsmeyer-Peppas equation models were used to simulate and analyze the DOX release behavior in different conditions. Parameters which obtained by equation models were shown in Figure S3. It was easy to conclude that the linear coefficient which fitted by equation models were relative ideal, with almost all R2 values were about 0.9. The k values at pH 5.0 were more than 1, so they were diffusional release while the others followed non-diffusional release mechanism, according to Higuchi theory.37 The reason would be the fast release under acidic conditions. From the Korsmeyer-Peppas models, all n values were above 0.5, meaning all conditions were non-Fickian diffusion.38 Because the amount of diffusion was proportional to time, the release rate was proportional to the cumulative release.


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In vitro cytotoxicity and cellular uptake. Ovarian cancer SKOV3 cells were used to evaluate the in vitro cytotoxicity of the HCCL micelles. As shown in Figure 7, the viability of the cells cultured with the HCCL micelles decreased from 96.4% to 86.4%, with the concentration of the HCCL micelles increased from 5 µg/mL to 40 µg/mL. It demonstrated that the free HCCL micelles had shown superior cytocompatibility. As for the cells treated with free DOX or the DOX-loaded HCCL micelles, the viability decreased from 44.9% to 7.1% or 54.2% to 40.6% respectively, with increasing the DOX dosage from 5 µg/mL to 40 µg/mL. Considering that the cumulative DOX release from the DOX-loaded HCCL micelles was near 80% in the case, the DOX-loaded HCCL micelles showed similar cancer cell killing capacity as the free DOX.

DOX-loaded HCCL micelles HCLL micelles Free DOX

100

Cell viability (%)

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80 60 40 20 0 0

10

20

30

40

50

Dosage (µg/mL)

Figure 7. In vitro cytotoxicity of the DOX-loaded HCCL micelles, HCCL micelles and free DOX. Values are expressed as mean ± SD (n = 3).

The confocal laser-scanning microscopy (CLSM) technique was used to investigate the cellular uptake of the DOX-loaded HCCL micelles. It is worth noting that strong DOX red


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fluorescence was located in cell nuclei after 12 h incubation with the DOX-loaded HCCL micelles (Figure 6), meaning the efficient cellular uptake of the DOX-loaded HCCL micelles and remarkable intracellular release of DOX. The result proved that the DOX-loaded HCCL micelles were successfully transported into SKOV3 cells and the loaded DOX was released from the micelles with the synergetic effect of GSH and pH inside the tumor cells, and mainly accumulated in nuclei.39

(a)

(b)

(c) Figure 8. CLSM images of the cell nucleus: dyed by Hoechst (a), DOX in cells (b) and merged image (c).


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CONCLUSIONS In summary, novel cytocompatible reduction and pH dual-responsive core cross-linked micelles were designed on the basis of an amphiphilic linear-hyperbranched copolymer, which was synthesized via SCVCP and used as the multi-functional macroinitiator for the ATRP of tBA with BACy as cross-linking agent, followed by the hydrolysis of their tBA units into AA units. Due to the AA units in the hydrolyzed core cross-linked (HCCL) micelles, DOX-loading capacity of 18.4% was achieved via the electrostatic interaction. The DOX-loaded HCCL micelles exhibited a faster DOX release in the acidic media with higher GSH concentration (mimicking the tumor intracellular microenvironment), while less drug leakage in the neutral media with low GSH concentration (mimicking the normal physiological medium), demonstrating that the HCCL micelles could not only improve the anticancer efficiency of the anticancer drugs, but also reduce their toxic side effect to the normal tissues. The cytotoxicity showed that the free HCCL micelles possessed excellent cytocompatibility, while the DOX-loaded HCCL micelles exhibited the similar cancer cell killing capability as the free DOX. The CLSM result proved that the DOXloaded HCCL micelles could be successfully transported into SKOV3 cells and the loaded DOX was released and mainly accumulated in nuclei. All these features make the HCCL micelles potential DDS for targeted and controlled anticancer drug delivery.

AUTHOR INFORMATION Corresponding Author. * Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes. The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This project was granted financial support from the Program for New Century Excellent Talents in University of the Ministry of Education (Grant no. NCET-09-0441).

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For Table of Contents Use Only

Biocompatible Reduction and pH Dual-Responsive Core Cross-Linked Micelles Based on Multi-Functional Amphiphilic Linear-Hyperbranched Copolymer for Controlled Anticancer Drug Delivery Kun Tian, Xu Jia, Xubo Zhao, and Peng Liu*


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Biocompatible Reduction and pH Dual-Responsive Core Cross

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