Synthesis of Photo-and pH Dual-Sensitive Amphiphilic Copolymer

Aug 11, 2016 - Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China. ABSTRACT: Novel photo- and ...
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Synthesis of Photo- and pH Dual-Sensitive Amphiphilic Copolymer PEG43-b‑P(AA76-co-NBA35-co-tBA9) and Its Micellization as LeakageFree Drug Delivery System for UV-Triggered Intracellular Delivery of Doxorubicin Xubo Zhao, Mingzhu Qi, Shuo Liang, Kun Tian, Tingting Zhou, Xu Jia, Jiagen Li, 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 photo- and pH dual-sensitive amphiphilic copolymers containing photolabile o-nitrobenzyl (NB) groups were designed via combination of ATRP, hydrolyzation, and simple esterification reaction and self-assembled into stimuli-regulated amphiphilic micelles in aqueous solution. On the basis of the optimization of the morphology and particle size of the micelles via modulating the number of the photocleavable o-nitrobenzyl acrylate (NBA) units, the unique ones assembled from PEG43-bP(AA76-co-NBA35-co-tBA9) with an average hydrodynamic diameter (Dh) of 163 nm was selected as a potential drug delivery system (DDS) for UV-triggered delivery of doxorubicin (DOX). The micelles possessed a favorable drug-loading capacity (DLC) of 27.5%, with the hydrodynamic diameter of 213 nm after DOXloading. Most importantly, the DOX-loaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles exhibited a cumulative DOX release ratio of only 3.69% in the simulated physiological medium within 6 days without UV-irradiation, indicating their potential as leakage-free DDS. As in the acidic media mimicking the tumor microenvironment, a high cumulative DOX release ratio of 74.70% was achieved within 6 days after UV-irradiation for 20 min, showing a sustained release behavior. Under UV-irradiation, the photolabile o-nitrobenzyl moieties were cleaved off, the amphiphilic copolymer transformed into a water-soluble polymer, favoring the metabolism of drug carriers, and the micelles were demicellized to accelerate the drug release in a triggered or ondemand manner. KEYWORDS: drug delivery system, UV-triggered delivery, leakage-free, micelle, photosensitive amphiphilic copolymer

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INTRODUCTION

Among the photolabile molecules, o-nitrobenzyl (NB) derivatives with an intramolecular rearrangement or photochemical cleavage reactions upon irradiation with UV light, have been widely used in designing photoresponsive systems,12 such as photodegradable hydrogels, thin-film patterning, selfassembled monolayers, photocleavable block copolymers, and bioconjugates. They have attracted more and more interest as a potential photoresponsive DDS for UV-triggered release. Besides the o-nitrobenzyl derivatives have been utilized as cross-linkers for polymeric nanoparticles13,14 and layer-by-layer engineered microcapsules,15 recently the UV-responsive micelles have been intensely investigated as a DDS due to their advantages, especially their excellent flexibility and control of their structure and subsequent properties. Anilkumar et al. reported photosensitive micelles to trigger cell death, in which a photolabile nitrobenzyl linkage could be cleaved upon photoirradiation to release a cytotoxic nitrosobenzaldehyde deriva-

Fabrication of stimuli-sensitive nanoplatforms with external and physiological triggers, either exogenous (temperature, ultrasound, light, electric, or magnetic effect) or endogenous (pH, redox potential, enzymes, or glucose), has attracted extensive attention, especially as the drug delivery system (DDS) for tumor treatment.1−3 Among them, light has been considered to be potentially useful in biomedical applications by controlling chemistry in space and time. The light-responsive nanoplatforms can be used to facilely and precisely control the pharmacokinetic fate and activity of the loaded bioactive molecules (e.g., proteins or nucleic acids), without introducing any intrusive chemical stimulation.4−8 Furthermore, the light-responsiveness is very useful in vivo due to its efficient response to irradiation, which penetrates innocuously into tissue. Many photochemical systems have been developed to fulfill the physiological demands, mainly through UV-activation.9 Presently, degradable polymeric nanoplatforms may be integrated with clinic-related and multiple physiological stimuli to achieve on-demand drug release kinetics, which remains challenging for biomedical applications.6,10,11 © XXXX American Chemical Society

Received: July 21, 2016 Accepted: August 11, 2016

A

DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of Amphiphilic Copolymer PEG43-b-P(AA-co-NBA-co-tBA) and Detachment of Nitrobenzene after UVIrradiation

Scheme 2. Self-Assembly, DOX-Loading, Disassembly and Release DOX after UV-Irradiation

tive.16 Wu et al. designed and synthesized a multiresponsive nitrobenzene-based random copolymer by the copolymerization of the temperature/acid dual-sensitive monomer (DMAEMA) and the light-sensitive monomer (NBM). It self-assembled into micelles with tunable morphology responding to the stimuli, endowing a controlled release of the loaded molecule (Nile red).17 Yuan and Guo synthesized novel UV and thermo dualresponsive amphiphilic nitrobenzene-based copolymers (PNBM-b-P(MEO2MA-co-OEGMA)) via a two-step atom transfer radical polymerization (ATRP). After self-assembly into micelles in water, the controlled release of the encapsulated Nile red could be manipulated by altering the temperature and/ or exposing the UV-irradiation.18 Du et al. reported photo and thermo dual-responsive biodegradable polymers, amphiphilic polyaspartamide derivatives (NB-g-PHPA-g-mPEG), for anticancer drug (paclitaxel) delivery.19 Meanwhile, Sullivan and coworkers fabricated the cationic diblock copolymers containing onitrobenzyl groups for tailorable complexation with DNA and light-responsive release.20 Liu and Dong synthesized novel PNBC-b-PEO block copolymers which could assemble as micelles in a water solution. As DDS, the release of DOX could be controlled by altering the light-irradiation time, due to gradually photocleaving of the

PNBC cores.21 Liu’s group fabricated dynamic covalent shellcross-linked (SCL) micelles with a photocaged chemotherapeutic drug (camptothecin (CPT)) in the core through comicellization of two amphiphilic diblock copolymers, P(CL-g-CPT)-bP(OEGMA-co-MAEBA)-CPT and PCL-b-P-(OEGMA-coMAEBA-co-FA), followed by cross-linking with pH-labile acylhydrazone and reductant-cleavable disulfide linkages.22 The conjugated CPT could be released effectively upon photoirradiation, and its diffusion could be adjusted with the acid and reductant levels. Importantly, negligible CPT release (∼1.2%) could be released at pH 7.4 without UV-irradiation, due to the covalent conjugation. The result demonstrated that the premature drug leakage, in the cases in which drugs were loaded via noncovalent interactions, could be completely prevented by the proposed “photo-caging” strategy. In the present work, novel photo- and pH dual-sensitive amphiphilic copolymer PEG43-b-P(AA76-co-NBA35-co-tBA9) was designed and synthesized via ATRP of tert-butyl acrylate (tBA) with a poly(ethylene glycol) (PEG)-based macroinitiator, followed by hydrolyzation of the partial tBA units into acrylic acid (AA), and esterification with o-nitrobenzyl alcohol to introduce the photolabile o-nitrobenzyl acrylate (NBA) units (Scheme 1). The copolymer could easily form micelles in water, B

DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

PEG43-b-P(AA-co-NBA-co-tBA). A serious of PEG43-b-P(AA-coNBA-co-tBA) copolymers were prepared using an esterification reaction between the carboxyl group of PEG43-b-P(AA111-co-tBA9) and the hydroxyl group of o-nitrobenzyl alcohol. Typically, 1.067 g (0.095 mmol) of PEG43-b-P(AA111-co-tBA9) was suspended in 10 mL of DMSO. EDCI (2 equiv to carboxyl groups), 0.1 equiv of DMAP, and 0.5 equiv of o-nitrobenzyl alcohol were added to the above solution and stirred at room temperature for 24. The suspension was dialyzed (MWCO: 1000) against excess DMSO for 48 h to remove any residual EDC, NBA, and DMAP. Subsequently, the dialysis tube was transferred into excess deionized water to completely remove DMSO. Finally, the obtained product was separated by lyophilization. For comparison, the PEG43-b-P(AA-co-NBA-co-tBA) copolymers with different NBA contents were also synthesized with 0.7 equiv. or 0.9 equiv. of o-nitrobenzyl alcohol using the similar steps. Micellization and DOX-Loading. Ten milligrams of PEG43-bP(AA-co-NBA-co-tBA) copolymer was dissolved into 10 mL of DMSO, and the solutions were dialyzed (MWCO: 1000) against excess water for 48 h to achieve the micelles. The DOX-loaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles were prepared by dialyzing (MWCO: 1000) a 10 mL DMSO solution containing 10 mg of PEG43-b-P(AA76-co-NBA35-co-tBA9) copolymer and 5 mg of DOX against excess water for 48 h. Then the DOX-loaded micelles were separated by centrifugation. In Vitro Controlled Release. Ten milliliters of DOX-loaded micelles dispersion in phosphate-buffered saline (PBS, pH 7.4 or pH 5.0) was dialyzed (MWCO of 10 000) in 120 mL relevant PBS at 37 °C with or without UV-irradiation (365 nm, 1000 W, at a distance of 50 cm for 20 min). 5.0 mL of solution was taken at certain intervals for measuring the drug concentrations in the dialysates using a UV spectrophotometer. And 5.0 mL fresh relevant PBS was replenished after each sampling, in order to keep the solution volume constant. The cumulative release was denoted as the percentage of the released DOX over time. Analysis and Characterizations. The 1H NMR spectra were recorded with a Bruker 400 MHz NMR spectrometer at room temperature. A Bruker IFS 66 v/s infrared spectrometer was used for the Fourier transform infrared (FT-IR) spectroscopy analysis in the range of 400− 4000 cm−1 with a resolution of 4 cm−1, by KBr pellet. The number-average molecular weight (Mn) and polydispersity index (PDI) of the copolymers were measured by gel permeation chromatography (GPC) technique, with THF as eluent (1.0 mL min−1) at 35 °C, after calibration with polystyrene standards. The morphologies of the micelles and those after UV-irradiation were characterized with a JEM-1200 EX/S transmission electron microscope (TEM). The samples were stained with 2% phosphotungstic acid. A dynamic light scattering (DLS) technique was used to analyze the micelle size, with a Light Scattering System BI-200SM device (Brookhaven Instruments), using a 135 mW intense laser excitation at 532 nm at a detection angle of 90° using the aqueous dispersions of the samples directly at 25 °C. The zeta potentials of the micelles before and after DOX-loading were measured with a Zetasizer Nano ZS (Malvern Instruments Ltd., U.K.). The drug-loading and in vitro release performance was evaluated with a PerkinElmer Lambda 35 UV−vis spectrometer at 480 nm at room temperature. The drug loading capacity (DLC) and drug encapsulation efficiency (DEE) were assessed as the mass ratio of the loaded DOX in the micelles and the micelles, and the mass ratio of DOX-loaded in the micelles and the total feeding DOX, respectively. The cumulative release (%) at a particular time (t) can be calculated with the following equation:

in which the photocleavable o-nitrobenzyl (NB) groups play a role as the phototriggering switch for the disassembly of the micelles, and subsequently the remaining AA units are beneficial for drug-loading via electrostatic interaction and pH responsive controlled release performance. As a drug carrier for doxorubicin (DOX), the drug release could be efficiently triggered by UV light. Upon UV-irradiation, photolabile o-nitrobenzyl (NB) groups in the copolymer could be cleaved off and the copolymer transformed into the water-soluble ones. As a result, the DOXloaded micelles disassembled, and the loaded DOX molecules via electrostatic interaction were released in the simulated tumor microenvironment (Scheme 2). Notably, the cumulative release was only 3.69 wt % within 6 days at physiological media without UV-irradiation. The near leakage-free characteristic of the proposed photo- and pH dual-sensitive DDS could efficiently avoid premature drug release during blood circulation.

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EXPERIMENTAL SECTION

Materials and Reagents. Poly(ethylene glycol) monomethyl ether (PEG43-OH) was purchased from Beijing Kaizheng Bioeng. Development Co. Ltd. Doxorubicin hydrochloride (DOX) was obtained from Beijing Huafeng United Technology Co. Ltd. 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCl) was purchased by Fluorochem. N-Hydroxylsuccinimide (NHS) was provided from Aladdin Chem. Co. Ltd. Tetrahydrofuran (THF) was refluxed over sodium (Na) and distilled in N2 atmosphere. Triethylamine (TEA) and toluene were dried over CaH2 for 48 h at room temperature and distilled under reduced pressure. Tert-butyl acrylate (tBA), o-nitrobenzyl alcohol, and 4-dimethylaminopyridine (DMAP) were purchased from J & K Chem. Ltd. N,N,N″,N″-Pentamethyl diethylenetriamine (PMDETA, 99%), trifluoroacetic acid (TFA, 97%), and 2-bromoisobutyryl bromide (BIBB, 98%, Aladdin) were used directly without any pretreatment. All other reagents (analytic reagent, Tianjin Chem. Co. Ltd.) were used directly. Double-distilled water was used throughout. PEG43-Br. Ten grams of PEG43-OH was dissolved in 150 mL of toluene, and then approximately 40 mL of toluene was removed with traces of water by azeotropic distillation under reduced pressure. 2.5 mL of TEA was added to the solution, and 2.0 mL of BIBB was added dropby-drop with magnetic stirring at 0 °C within 40 min. After mild stirring overnight at room temperature, most toluene was distilled out under reduced pressure, and the product was separated by precipitation in excess cold ether. After being dried under vacuum, the crude product was dissolved in 20 mL of pH 8−9 NaHCO3 aqueous solution, and extracted with CH2Cl2. The organic phase was gathered and dried over MgSO4, the resultant macroinitiator (PEG43-Br) was obtained by complete distillation of the solvent under reduced pressure.23 PEG43-b-PtBA120. PEG43-b-PtBA120 was prepared via the ATRP of tBA with the macroinitiator PEG43-Br. 2.000 g (0.965 mmol) of PEG43Br was dissolved into 6 mL of anhydrous THF. After gassing and degassing in N2 atmosphere, 0.167 g (0.965 mmol) of PMDETA, 17.316 g (135.100 mmol) of tBA, and 0.138 g (0.965 mmol) of CuBr were added in turn under degassing by freeze−pump−thaw in N 2 atmosphere. Then the polymerization was performed at 45 °C for 8 h. The mixture was diluted with THF and passed an alumina column to remove the copper catalyst. Finally, the PEG43-b-PtBA120 copolymer was separated by precipitation in cold ether and dried under vacuum overnight at room temperature.23 Here the tBA conversion was determined to be 89% by 1 H NMR analysis. PEG43-b-P(AA111-co-tBA9). Three grams of PEG43-b-PtBA120 was dissolved into 30 mL of dichloromethane, and TFA (5-fold molar excess to the tBA units) was added and stirred at room temperature for 24 h. Then, most of the TFA and dichloromethane were removed by distillation under reduced pressure with a rotary evaporator. Subsequently, the hydrolyzed copolymer, PEG43-b-P(AA111-co-tBA9), was dialyzed (MWCO: 1000) against excess water for 48 h to completely remove any residual TEA and dichloromethane. Finally, the obtained product was lyophilized and stored at 4 °C.

t−1

cumulative release (%) =

CtV0 + V ′ ∑i = 1 Ci M total

× 100%

where Mtotal represents the total DOX content in dialysis tubes before release, V0 and V′ are the initial release media volume and the collected release media volume at certain time intervals (here, V0 = 120 mL and V′ C

DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. 1H NMR spectra of PEG43-OH, PEG43-Br, PEG43-b-PtBA120, hydrolysate PEG43-b-P(AA111-co-tBA9), and the three amphiphilic copolymers with different components of NBA (PEG43-b-P(AA76-co-NBA35-co-tBA9), PEG43-b-P(AA70-co-NBA41-co-tBA9), and PEG43-b-P(AA56-co-NBA55-cotBA9)). = 5 mL), respectively; Ct represents the DOX concentration in release media at certain time t.

combination of ATRP, hydrolyzation, and esterification, as shown in Scheme 2. In the 1H NMR spectrum of PEG43-OH (Figure 1), the characteristic signals at δ = 3.65 ppm and δ = 3.38 ppm were assigned to the inner methylene protons which are adjacent to oxygen (b, OCH2) and the terminal methyl protons (a, OCH3), respectively. As for the macroinitiator PEG43-Br, the 2-bromoisobutyryl groups was revealed by the

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RESULTS AND DISCUSSION Synthesis and Characterization of Block Copolymers. Novel amphiphilic copolymers, PEG43-b-P(AA-co-NBA-co-tBA), with different components of NBA were prepared by a D

DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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which the characteristic absorbance at 1729 cm−1 was ascribed to the carbonyl group of acrylic acid units of the hydrolysate PEG43b-P(AA111-co-tBA9).23 Furthermore, the strength reduction of the characteristic absorbance at 1404 cm−1 also revealed the hydrolysis of the PtBA block. Finally, amphiphilic copolymers, PEG43-b-P(AA-co-NBA-cotBA), were prepared using simple esterification in DMSO solution. In order to optimize the morphology and diameter of the PEG43-b-P(AA-co-NBA-co-tBA) micelles, three different amounts of o-nitrobenzyl alcohol were introduced. Calculated from the integral area ratios of the chemical shifts at δ = 3.65 ppm (b) and δ = 7.41−8.22 ppm (f) of 1.23, 1.05 and 0.78, the photoresponsive amphiphilic copolymers with different components of NBA (PEG43-b-P(AA76-co-NBA35-co-tBA9), PEG43-bP(AA77-co-NBA41-co-tBA9), and PEG43-b-P(AA70-co-NBA41-cotBA9)) were successfully prepared. The new strong characteristic absorbance at 1534 cm−1 (asymmetric NO stretch), 1348 cm−1 (symmetric NO stretch), and 864 cm−1 (CNO2 stretch) of the photosensitive NB groups revealed the successful conjugation of NB groups to form NBA units.21 Micellization. The photoresponsive amphiphilic copolymer were composed of the hydrophobic (tBA and NBA units) and hydrophilic (PEG block and AA units) segments, so they could self-assemble into unique micelles. After micellization via solvent dialysis, the size and morphology of the micelles were tracked by TEM and DLS techniques in pH 7.4 aqueous solution. The effect of hydrophobic segments on the size and morphology of the micelles was discussed with different contents of the photosensitive NB groups. The PEG43-b-P(AA76-co-NBA35-co-tBA9) copolymer formed spherical micelles with particle size of 92 ± 5 nm, by TEM analysis (Figure 4). Increasing the hydrophobic photosensitive NBA units from 35 of the PEG43-b-P(AA76-co-

characteristic chemical shift of the methyl protons (c, C(Br) CH3) at δ= 1.94 ppm. The integral area ratio of the two characteristic signals at δ = 1.94 ppm and δ = 3.38 ppm was calculated to be 2.02, demonstrating the complete transformation of PEG43-OH into PEG43-Br. In the FT-IR analysis, a new characteristic absorbance of ester carbonyl stretching vibration emerged at 1738 cm−1 (Figure 2).

Figure 2. FTIR spectra of (a) PEG43-OH, (b) PEG43-Br, (c) PEG43-bPtBA120, (d) PEG43-b-P(AA111-co-tBA9), and (e) PEG43-b-P(AA76-coNBA35-co-tBA9).

After the ATRP of tBA, the chemical shift at δ = 1.44 ppm (d) represented the methyl protons in the t-butyl groups, revealing the successful polymerization of tBA. Comparing the integral areas of the characteristic signals d and b, the polymerization degree of tBA in the diblock copolymer PEG43-b-PtBA was calculated to be 120. FT-IR analysis also confirmed the successful preparation of the PEG43-b-PtBA120 diblock copolymer. The absorbance at 1304 cm−1 was ascribed to t-butyl groups in the PEG43-b-PtBA120 diblock copolymer (Figure 2). The numberaverage molecular weight (Mn) and the polydispersity index of the PEG43-b-PtBA120 were 13 469 and 1.17, respectively, according to GPC results (Figure 3).

Figure 3. GPC traces of PEG43-OH and PEG43-b-PtBA120.

Subsequently, the PtBA block was hydrolyzed in a controlled manner in dichloromethane solution with TFA. The hydrolysis degree could be accessed as the signal area ratio of the two chemical shifts at δ = 3.65 ppm (b) and δ = 1.44 ppm (d). Compared with the PEG43-b-PtBA120, the hydrolysis degrees of 92.5% of the PtBA block (PEG43-b-P(AA111-co-tBA9)) was obtained in the presence of TFA with a 5-fold molar excess to the tBA units. In the FT-IR analysis, the absorbance at 1738 cm−1 of carbonyl group of PEG43-b-PtBA120 divided into two peaks, in

Figure 4. TEM images of the PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles (a), PEG43-b-P(AA70-co-NBA41-co-tBA9) micelles (b), PEG43-bP(AA56-co-NBA55-co-tBA9) micelles (c), disassembled PEG43-b-P(AA76co-NBA35-co-tBA9) micelles after UV-irradiation for 20 min (d), DOXloaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles (e), and disassembled DOX-loaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles after UV-irradiation for 20 min (f). E

DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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6). Meanwhile, after UV-irradiation, the clear dispersion of the PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles changed into a

NBA35-co-tBA9) to 41 and 55 of the copolymers PEG43-bP(AA70-co-NBA41-co-tBA9) and PEG43-b-P(AA56-co-NBA55-cotBA9), the hydrophobic AA units decreased, and the particle size decreased from 92 ± 5 nm to 79 ± 8 nm to 64 ± 4 nm, respectively. Meanwhile, increasing the contents of the hydrophobic photosensitive NBA units from 35 to 41 and 55, the micellar morphology changed from smooth spheres to spheres with protuberance, to flower-like micelles. This phenomenon might be attributed to shrinking of the hydrophobic monomer, further causing surface collapse, with the increasing contents of the hydrophobic units. Furthermore, the hydrodynamic diameter of the PEG43-bP(AA76-co-NBA35-co-tBA9) micelles, PEG43-b-P(AA70-co-NBA41co-tBA9) micelles, and PEG43-b-P(AA56-co-NBA55-co-tBA9) micelles also decreased from approximately 163 nm, to 129 and 100 nm with increasing of contents of photosensitive NBA units, as shown in Figure 5. More importantly, the morphology of

Figure 6. Typical hydrodynamic diameter distributions of the PEG43-bP(AA76-co-NBA35-co-tBA9) micelles before (a) and after (b) 365 nm UV-irradiation for 20 min. The inserts are the digital photographs of the dispersions before (c) and after (d) 365 nm UV-irradiation for 20 min.

yellow, cloudy suspension due to the poor water-solubility of the cleaved o-nitrobenzyl alcohol molecules, as shown in the inserted digital photographs (Figure 6). Notably, the UV−vis spectrometer was utilized to track the exfoliation of the o-nitrobenzyl alcohol, as shown in Figure 7.

Figure 5. Typical hydrodynamic diameter distributions of the PEG43-bP(AA56-co-NBA55-co-tBA9) micelles (a), PEG43-b-P(AA70-co-NBA41-cotBA9) micelles (b), PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles (c), DOX-loaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles (d).

all three PEG43-b-P(AA-co-NBA-co-tBA) micelles exhibited high stability with uniform monodispersity in PBS. Both the TEM and DLS demonstrated that the size of the PEG43-b-P(AA-co-NBAco-tBA) micelles could be regulated with contents of the photosensitive NBA units. The PEG43-b-P(AA76-co-NBA35-cotBA9) micelles were chosen as the drug delivery system for further investigation, due to their unique morphology. Demicellization under UV-Irradiation. Due to the introduction of photosensitive o-nitrobenzyl groups, the PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles were endowed with excellent photosensitive properties under typical UVirradiation. Owing to the large absorption coefficient of the onitrobenzyl group, the PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles presented a fast photocleavage reaction upon 365 nm UV-irradiation, typically within 20 min.24 Upon UV-irradiation, the o-nitrobenzyl alcohol groups could be cleaved from the PEG43-b-P(AA76-co-NBA35-co-tBA9) copolymer, and the amphiphilic copolymer changed into the water-soluble copolymer PEG43-b-P(AA111-co-tBA9). As a result, the PEG43-b-P(AA76-coNBA35-co-tBA9) micelles dissociated, as shown in Scheme 2. In the TEM analysis, the spherical morphology of the PEG43-bP(AA76-co-NBA35-co-tBA9) micelles (Figure 4a) disappeared after UV-irradiation for 20 min, and a small fragment appeared in Figure 4d. Furthermore, there are some small matters and large aggregation in the DLS analysis of the PEG43-b-P(AA76-coNBA35-co-tBA9) micelles after UV-irradiation for 20 min (Figure

Figure 7. UV/vis spectra of the PEG43-b-P(AA111-co-tBA9), PEG43-bP(AA76-co-NBA35-co-tBA9), detachment of nitrobenzyl groups from the PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles after UV-irradiation for 20 min at the same concentration, and the DOX-loaded PEG43-b-P(AA76co-NBA35-co-tBA9) micelles.

Owing to cloudy suspension, the absorption of the dispersion of the PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles dramatically increased under UV-irradiation, demonstrating that the pendant o-nitrobenzyl groups had been gradually photocleaved from the hydrophobic cores, which is consistent with the DLS analysis. All these results demonstrated that the PEG43-b-P(AA76-co-NBA35co-tBA9) micelles exhibited excellent photosensitive property. More importantly, this remarkable photosensitive property make the PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles occurring hydrophilic transformation and further dissociation under typical stimuli, such as UV-irradiation. The excellent stimuli-sensitive property ensures that the smart PEG43-b-P(AA76-co-NBA35-coF

DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces tBA9) micelles possessing a remarkable stimuli-triggered drug release characteristic, which reduces the undesirable leakage of anticancer drugs in normal tissues, and increases therapeutic efficacy in tumor tissues. Drug-Loading and in Vitro Triggered Release. As a potential DDS, the drug-loading and in vitro triggered release performance of the proposed PEG43-b-P(AA76-co-NBA35-cotBA9) micelles were investigated. Owing to the random sequential structure of the hydrophilic AA units and hydrophobic NBA and tBA units, the amphiphilic PEG43-b-P(AA76-co-NBA35co-tBA9) copolymer should form the core−shell micelles with the PEG blocks as shells and the random copolymer bloks as cores, which could be swollen in water due to the existence of the AA units. So the model drug (DOX) could be efficiently encapsulated in the micelles via electrostatic interaction with the AA units and the hydrophobic interaction with the NBA and tBA units. After micellization in the presence of DOX, the DOXloaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles were obtained with DLC and DEE of 27.5% and 55.0%, respectively. The DOX-loading via electrostatic interaction with the AA units could be revealed by the zeta potentials, which increased from −34.69 to −20.75 mV after DOX loading. Meanwhile, the new absorption appeared at 515 nm in the UV−vis spectrum of the micelles after drug-loading. After DOX-loading, the particle size and hydrodynamic diameter of the PEG43-b-P(AA76-coNBA35-co-tBA9) micelles increased from 92 ± 5 nm to 116 ± 14 nm (Figure 4), and from 163 to 213 nm (Figure 5), respectively. More importantly, in comparison with the PEG43-b-P(AA76-coNBA35-co-tBA9) micelles after UV-irradiation, the DOX-loaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles were more easily dissociated into small fragments under the same conditions, as shown in Figure 4f. Such a feature would benefit quick UVtriggered DOX release in the tumor tissues. The cumulative DOX release from the DOX-loaded micelles was only 3.69 wt % within 6 days at physiological media (Figure 8). As for an acidic medium, the cumulative release ratio increased to 10.09 wt % at pH 5.0. It is higher than the physiological media due to the enhanced solubility of DOX as well as the faster diffusion of DOX. To further explore the UVtriggered drug release performance, the drug release experiments were conducted under UV-irradiation at 365 nm for 20 min at

pH 5.0. A high cumulative release of 74.70 wt % was achieved within 6 days, and the drug release showed a sustained release behavior in the last 5 days. However, the drug release without UV-irradiation occurred at the early stage of about 2 days. So we speculate that the DOX release mainly comes from the DOX molecules loaded in the PEG shells via hydrogen bond and those loaded in the surface layer of the hydrophobic cores. The results demonstrated that the proposed PEG43-b-P(AA76-co-NBA35-cotBA9) micelles possessed an excellent UV-triggered release property. As a DDS for cytotoxic anticancer drugs, they could efficiently inhibit the premature DOX leakage during blood circulation, owing to their improved stability at pH 7.4 without a UV-trigger. As in the tumor microenvironment after UVirradiation for 20 min, most DOX could be released quickly and then in a sustained release model. The results indicated that the photo- and pH dual-sensitive micelles possessed an ideal structure for a DDS with reduced side effects.

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CONCLUSIONS In the present work, a facile strategy has been established for the photo- and pH dual-sensitive micelles as a leakage-free drug delivery system (DDS) for UV-triggered controlled release of the cytotoxic anticancer drug (DOX), based on an amphiphilic copolymer containing photolabile o-nitrobenzyl (NB) groups. The amphiphilic copolymer could easily assemble into micelles, and their content of the hydrophobic photocleavable onitrobenzyl acrylate (NBA) units could modulate the morphology and diameter of the resultant micelles. Most importantly, the photocleavable o-nitrobenzyl acrylate (NBA) units acted as the phototriggering switch for the drug release, due to the disassembly of the micelles upon UV-irradiation. As a DDS, the DOX-loaded PEG43-b-P(AA76-co-NBA35-co-tBA9) micelles with a DLC of 27.5% showed negligible DOX release in the simulated physiological medium within 6 days without UVirradiation, while a high cumulative DOX release ratio of 74.70% at the simulated tumor microenvironment after UV-irradiation for 20 min with a sustained release model. The unique phototriggering characteristic makes the micelles a potentially smart stimuli-responsive nano-DDS for controlled delivery of hydrophobic anticancer drugs.

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AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: 86 0931 8912582. E-mail: [email protected] (P.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was granted financial support from the National Nature Science Foundation of China (Grant No. 20904017), the Program for New Century Excellent Talents in University (Grant No. NCET-09-0441), and the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant No. J1103307).

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Figure 8. Cumulative DOX release from the DOX-loaded PEG43-bP(AA76-co-NBA35-co-tBA9) micelles in simulated body fluids at pH 7.4 and 5.0, and after 365 nm UV-irradiation for 20 min. Results show the UV−vis measurements at room temperature at 485.00 nm for DOX. G

DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.6b08935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX