Oxidation-Responsive OEGylated Poly-l-cysteine and Solution

Materials and Instrumentals. All solvents were purchased from Beijing ...... Guyton , K. Z.; Kensler , T. W. Br. Med. Bull. 1993, 49, 523– 44. [PubM...
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Oxidation-Responsive OEGylated Poly‑L‑cysteine and Solution Properties Studies Xiaohui Fu, Yinan Ma, Yong Shen, Wenxin Fu, and Zhibo Li* Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: The oxidation-responsive behaviors of OEGylated poly-L-cysteine homopolypeptides, that is, poly(LEGxMA-C)n, were investigated. These poly-L-cysteine derivatives adopted mixed conformation in water, in which the βsheet accounted for a significant proportion. Upon oxidation, the thioethers in polypeptide side chains were converted to polar sulfone groups, which triggered the secondary structure transition from β-sheet preferred conformation to random coil. Accordingly, the increase of side-chain polarity together with conformation changes increased samples’ water solubility and cloud point temperature. Using mPEG45-NH2 as macroinitiator, we synthesized PEG45-b-poly(L-EG2MA-C)22 diblock copolymer via ring-opening polymerization (ROP) of L-EG2MA-C N-carboxyanhydride (NCA). The PEG45-b-poly(L-EG2MA-C)22 was able to self-assemble into spherical micelles in aqueous solution, and the micelles could undergo an oxidation-triggered disassembly due to the oxidation-responsive thioethers. Such a new class of oxidation-responsive polypeptides might provide a promising platform to construct inflammation targeting drug delivery systems.

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INTRODUCTION Stimuli-responsive polymers have attracted great research interests considering their promising applications in bio- and nanotechnology.1−5 For instance, they can be used to mimic adaptive biological systems to construct smart drug delivery systems or to amplify signals in biosensors.2,6−8 Compared to conventional polymers, biodegradable stimuli-responsive polymers are of critical importance for biomedical applications given their advantages in biocompatibility and biodegradability.5,9−12 Several biodegradable polymers have been investigated, such as polyesters,13−15 poly(amino ester)s,11,16,17 poly(organo phosphazene)s,18 and poly(amino acid)s.5,9,19 Among them, polypeptides are unique because they can not only be modified with different functional groups but also offer a conformation specific stimuli-responsive mechanism arising from their ordered secondary structures, for example, α-helix versus β-sheet.5,6,20 Great progresses were recently made to prepare conformation switchable polypeptide materials, including those that respond to pH variation,21 light,22−24 and bioactive molecules.25,26 For ionic polypeptides such as poly(L-lysine) or poly(L-glutamic acid), pH variation can switch their conformation from random coil to α-helix accompanied by a decrease of hydrophilicity and solubility in water. Using this naturally responsive feature, researchers prepared pH responsive aggregates such as micelles and vesicles.5,10,27−32 Recently, OEGylated poly-L-glutamates were demonstrated to have a thermal-responsive property in water33−35 and their thermalresponsive property depended not only on the subunit structures, but also on polypeptide conformation.35 Further© 2014 American Chemical Society

more, the stimuli-triggered conformation and phase transitions lead to unique self-assembly behaviors, making these materials promising candidates for construction of controlled drug and gene delivery systems.10,21,36 Compared to the massive amount of literature reported on pH-responsive polypeptide materials, only a few studies concentrated on oxidation-responsive polypeptides. Recently, Kramer and Deming reported the preparation of sugarfunctionalized poly-L-cysteine, which can be oxidized using hydrogen peroxide to induce a helix-to-coil transition without loss of water solubility.6 They also found that block copolypeptides containing galactosylated poly-L-cysteine segments adopting either α-helical or disordered conformation gave different assembly morphologies.37 Considering their promising applications as controlled drug carriers in oxidation stress environments, it is of significance to investigate the oxidation-responsive polypeptide materials, which could associate conformation and solubility with external stimuli. Herein, we explored the aqueous solution properties of the oxidation-sensitive OEGylated poly-L-cysteine homopolypeptides, that is, poly(L-EGxMA-C)n, with x being 2, 3, 4/5, or 8/9. We found that conversion of thioether into polar sulfoxide or sulfone group can not only transform polypeptide conformation, but also change sample solubility, for example, from hydrophobic to hydrophilic or to thermal-responsive. Taking advantage of such characteristics, we prepared PEG45-b-poly(LReceived: January 12, 2014 Revised: February 12, 2014 Published: February 14, 2014 1055

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spectra were recorded on a Hitachi F2500 luminescence spectrometer at the detection wavelength (λem) of 390 nm. Synthesis of PEG45-b-poly(L-EG2MA-C)22 Diblock Copolymer. The detailed preparations of S-(2-(2-methoxyethoxy)ethoxy) isobutyrate-L-cysteine (L-EG2MA-C) and corresponding L-EG2MA-C NCA were reported elsewhere.46 Their structures were verified by 1 H NMR. For L-EG2MA-C (Figure S6a): 1H NMR (400 MHz, D2O) δ 4.35 (t, 2H), 3.96 (dd, 1H), 3.83 (t, 2H), 3.74 (t, 2H), 3.66 (t, 2H), 3.42 (s, 3H), 3.20 (dd, 1H), 3.07 (dd, 1H), 2.99−2.79 (m, 3H), 1.29 (d, 3H). For L-EG2MA-C NCA: 1H NMR (400 MHz, CDCl3) δ 7.24 (d, 1H), 4.58−4.37 (m, 2H), 4.26−4.12 (m, 1H), 3.84−3.70 (m, 2H), 3.69−3.63 (m, 2H), 3.60−3.51 (m, 2H), 3.37 (s, 3H), 3.21−3.08 (m, 1H), 2.95−2.66 (m, 4H), 1.24 (d, 3H); FTIR (THF): 2933, 1852, 1784, 1730, 1651, 1096, 924 cm−1. The PEG45-b-poly(L-EG2MA-C)22 was synthesized by ROP of L-EG2MA-C NCA using mPEG45-NH2 as macroinitiator (Figure S1). Typically, 72 mg of mPEG45-NH2 was dried in a 50 mL Schlenk flask at 100 °C in high vacuum for 5 h. Then, 5 mL of anhydrous THF was injected into the flask using a syringe followed by addition of 6 mL L-EG2MA-C NCA solution in THF (100 mg/mL). The mixture was stirred at 40 °C for 3 days and the consumption of NCA was confirmed by FTIR. The sample mixtures were precipitated into ethyl ether, collected by centrifugation, and dried under reduced pressure to give the product as a white solid with 71% yield. Oxidation. The oxidization experiments followed reported procedure.6 For the preparation of poly(L-EGxMA-CO), poly(LEGxMA-C) samples at 20 mg/mL were dissolved in a mixed aqueous solution of 1% acetic acid and 1% H2O2. The mixture was heated to 38 °C for 6 h under stirring. Excess sodium thiosulfate (1 M) was then added to reduce unreacted H2O2. The solution mixture was transferred to 3500 MWCO dialysis tubing, and dialyzed against Millipore water for 3 days with water changing twice per day. The solution was lyophilized to yield partial oxidized products, poly(L-EGxMA-CO). For the preparation of poly(L-EGxMA-CO2), poly(L-EGxMA-C) samples at 20 mg/mL were dissolved in a mixture of 5% acetic acid and 10% H2O2 in DI water. The mixture was heated to 38 °C for 16 h under stirring. Excess sodium thiosulfate (1 M) was added to reduce unreacted H2O2. The solution mixture was transferred to 3500 MWCO dialysis tubing and dialyzed against Millipore water for 3 days with water changing twice per day. Dialyzed polypeptides were lyophilized to yield completely oxidative products, poly(L-EGxMACO2). Critical Micellization Concentration (CMC) Measurements. CMC was measured by fluorescence spectroscopy using pyrene as probe. Typically, 60 μL of acetone solution of pyrene (2.5 × 10−5 M) was added to the 5 mL plastic centrifuge tube, and then the acetone was evaporated in air. A total of 3 mL of polymer aqueous solution at prescribed concentrations was introduced into the tubes and pyrene concentration in all the samples was about 6 × 10−7 M, which was slightly lower than the saturation solubility of pyrene in water. These solutions were shaken vigorously and then allowed to equilibrate at room temperature for at least 24 h. The excitation spectra of pyrene with different PEG45-b-poly(L-EG2MA-C)22 concentrations were measured at the detection emission wavelength (λem = 390 nm). The CMC value was obtained from the intersection of the tangent to the linear concentration-dependent section and the concentrationindependent section of I333/I331. Drug Loading and Release. DOX was used as a model drug for drug loading and the preparation processes were as follows. DOX·HCl was dissolved in deionized water and the pH was adjusted to about 9.6 with 0.1 M NaOH. The DOX was collected by centrifugation, washed with distilled water three times, and followed by lyophilization in the dark. DOX-loaded PEG45-b-poly(L-EG2MA-C)22 micelles were prepared by the dialysis method and the DOX-free micelles were also prepared with the same procedures. Briefly, PEG45-b-poly(L-EG2MAC)22 (10.0 mg) and DOX (2.0 mg) were mixed in 1.0 mL of DMF and 4 mL of acetone. The solution was placed in a bath sonicator for 30 min to dissolve DOX. Then, 5.0 mL of deionized water was added dropwisely to the solution under stirring. The mixture was stirred at room temperature overnight, the organic solvent was removed by

EG2MA-C) diblock copolypeptide, which can form nanoscopic micelles showing an oxidation triggered drug-release profile. It was known that oxidation is an ubiquitous feature of inflamed tissues, where a number of phagocyte-generated oxidants, for example, reactive oxygen species (ROS) and reactive nitrogen species (RNS), can fulfill both roles of signaling and toxic molecules.38 These particular ROS, such as hydrogen peroxide, superoxide, OH radical, and so on, are generally acknowledged being the main contributors to the intra- and extracellular redox potentials. They are also associated stress conditions and signaling cascades in injury, cancer, atherosclerosis, diabetes, and other diseases.38−41 Considering that inflammatory cells often exhibit more intracellular oxidative environment than normal ones,38,42,43 we believe that such unique oxidationresponsive polypeptide materials are promising for preparing inflammation targeting drug delivery systems or being used as sacrificial materials to protect sensitive payloads from damage induced by oxidizing conditions.

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EXPERIMENTAL SECTION

Materials and Instrumentals. All solvents were purchased from Beijing Chemical Co. Hexane, tetrahydrofuran (THF), and dichloromethane (DCM) were purified by first purging with dry nitrogen, followed by passing through columns of activated alumina. N,NDimethylformamide (DMF; anhydrous, 99.8%, packaged under argon in resealable ChemSeal bottles) was obtained from Alfa Aesar. LCysteine was purchased from GL Biochem (Shanghai) Ltd. Methacryloyl chloride, tri(ethylene glycol) monomethyl ether, oligo(ethylene glycol) methyl ether methacrylates (with formulate weight being 188, 300, or 475, which were denoted as EG2MA, EG4/5MA, or EG8/9MA, respectively), and sodium thiolsulfate were purchased from Aladdin reagent. Pyrene and the ω-methoxy-poly(ethylene glycol) amine (mPEG-NH2, Mn = 2000 Da and PDI = 1.1) were purchased from Aldrich. The average degree of polymerization (DP) for mPEGNH2 is about 45, determined from 1H NMR. Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing Huafeng United Technology Corporation. Deionized water (18 MΩ·cm) was obtained from a Millipore Milli-Q purification unit. All commercial reagents were used as received without further purification, unless otherwise stated. 1H NMR spectra were recorded on Bruker AV400 FT-NMR spectrometer using CDCl3 or D2O as solvents. All infrared spectroscopy measurements were performed using a Nicolet Avatar 330 FT-IR spectrometer. The solution samples were cast on KBr plates. Tandem size exclusion chromatography/laser light scattering (SEC/LLS) was performed at 50 °C using an SSI pump connected to Wyatt Optilab DSP and Wyatt DAWN EOS light scattering detectors with 0.02 M LiBr in DMF as eluent at flow rate of 1.0 mL/min. All SEC/LLS samples were prepared at concentrations of about 5 mg/mL. Circular dichroism (CD) spectra were recorded on an Applied Photophysics Chirascan CD spectrometer. The solution at concentration of 0.5 mg/mL was placed into a quartz cell with a path length of 1.0 mm. The secondary structures were analyzed on DICHROWEB using CONTIN-LL.44,45 The cloud points (CPs) were measured by monitoring the transmittance of a 500 nm light beam through a quartz sample cell at a concentration of 2 mg/mL on a Shimadzu UV−vis spectrometer. The heating or cooling rate was 0.5 °C/min. The CP was defined as the temperature corresponding to 50% transmittance of aqueous solution during the heating process. Transmission electron microscopy (TEM) samples were examined with a JEM2200FS TEM (200 keV). TEM samples were prepared by casting dilute sample solution on carbon coated TEM grids and air-dried at room temperature before measurements. Dynamic light scattering (DLS) measurements were performed using LLS spectrometer (ALV/DLS/ SLS-5322F) equipped with a multi-τ digital time correlator (ALV5000) and a He−Ne Laser (λ = 632.8 nm) at an angle of 90°. The sample concentrations were 1 mg/mL. All solutions were filtered through a 0.45 μm PVDF filter prior to measurements. Fluorescence 1056

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dialysis using a dialysis bag (MWCO = 3500 Da) against deionized water for 48 h with the aqueous media refreshed six times, and the whole procedure was performed in the dark. Then, the solution was filtered and lyophilized. The entrapment capacity (EC) and entrapment efficiency (EE) were calculated by the following equations:

EC% =

amount of DOX in micelle × 100% amount of DOX‐loaded micelle

EE% =

amount of DOX in micelle × 100% total amount of feeding DOX

In vitro drug release profiles of DOX-loaded micelles were investigated in PBS (0.01 mol/L, pH 7.4) containing 0.16 M NaCl. The DOX-loaded micelle solid was suspended in 2.0 mL of release medium and transferred into a dialysis bag (MWCO = 3500 Da). The release experiment was initiated by placing the end-sealed dialysis bag into 15.0 mL of release medium at 37 °C with continuous shaking at 100 rpm. At predetermined intervals, 3.0 mL of external release medium was sampled and an equal volume of fresh release medium was replaced. The DOX amount was determined by UV−vis spectroscopy at 485 nm using the standard curve method. All of the release experiments were carried out in triplicate, and the averaged results were reported.

Figure 1. 1H NMR spectra of (a) poly(L-EG3MA-C)26, (b) poly(LEG3MA-CO)26, and (c) poly(L-EG3MA-CO2)26 in CDCl3.

(−CH2SCH2CH(CH3)−). After being partially oxidized to sulfoxide groups, a slightly higher-frequency shift of the peaks corresponding to the protons of methyl, methylene and methine groups around the sulfur atoms (−CH2SCH2CH(CH3)−) was observed (Figure 1b), indicating a polarity increase of these functional groups. Further oxidation to sulfone polymers, the methyl peaks further shifted to 1.38 ppm as well as the resonances corresponding to methylene and methine groups shift to 3.05−3.53 ppm (Figure 1c). Meanwhile, we found no polymer backbone degradation after oxidation (Figure S7). We have demonstrated that the OEGlated poly-L-cysteine derivatives, that is, poly(L-EGxMA-C)n, displayed thermalresponsive behaviors in water only when the repeat unit of OEG side chains was between 3 and 5.46 We first applied turbidimetry to determine the influence of oxidation on corresponding cloud points (CP). Figure 2 shows the plot of

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RESULTS AND DISCUSSION We previously reported the preparation of OEGylated poly-Lcysteine homopolypeptides via combination of Michael-type reaction and ROP of NCA.46 The Michael-type reaction formed thioether bonds in the side chains of polypeptides. Depending on the side chain length, these OEGylated poly-Lcysteines can display thermal-responsive properties in water. It was known that the thioethers can be selectively oxidized into either sulfoxide or sulfone groups (Scheme 1). Oxidation of Scheme 1. Selective Oxidation of Thioethers into Sulfoxide and Sulfone Groups

thioethers can increase molecular polarity, which can induce physical property change.47−49 We first investigated the change of solution properties after oxidation. Note that the OEGylated poly-L-cysteines used here were synthesized previously and their molecular parameters were reported elsewhere. Due to the difficulty of NCA purifications, most samples had relatively low molecular weights but the molecular weight distributions of the obtained samples were relatively narrow. Their corresponding degrees of polymerization (DP) were denoted as the subscript.46 The oxidized samples were denoted as poly(LEGxMA-CO) and poly(L-EGxMA-CO2) for partially oxidized sulfoxide polymers and fully oxidized sulfone polymers, respectively. The corresponding structures were confirmed by 1 H NMR (Figure 1). Figure 1a shows the 1H NMR spectrum of poly(L-EG3MA-C)26, in which the resonance at 1.24 ppm corresponds to the methyl in β position to −S− moieties and the broad peaks between 2.58 and 3.18 ppm are attributed to methylene and methine in α and β position to sulfur atoms

Figure 2. Plots of transmittance as a function of temperature for aqueous solutions (2 mg/mL) of poly(L-EG2MA-CO2)71, poly(LEG3MA-C)26, poly(L-EG3MA-CO)26, poly(L-EG3MA-CO2)26, poly(LEG4/5MA-C)54, and poly(L-EG4/5MA-CO)54.

transmittance versus temperature for poly(L-EGxMA-C)n aqueous solution with different oxidizing status. For poly(LEG2MA-CO2)71, the transmittance decreased from 100 to 40% when the temperature increased from 30 to 70 °C. If we defined 50% transmittance as CP, we can determine the CP being about 58 °C. Before oxidation, poly(L-EG2MA-C)71 was insoluble in water at room temperature. Similar results were observed for poly(L-EG3MA-C)26 sample. The corresponding 1057

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Figure 3. CD spectra of (a) poly(L-EG2MA-C)71 and (b) poly(L-EG3MA-C)26 as a function of their degree of oxidation.

Figure 4. TEM images of (a) the self-assembled PEG45-b-poly(L-EG2MA-C)22 micelle, (b) DOX-loaded PEG45-b-poly(L-EG2MA-C)22 micelle, and (c) the Rh and size distributions of the corresponding micelles in aqueous solution at 25 °C.

CP increased from about 50 to 55 and to 80 °C when poly(LEG3MA-C)26 was oxidized into poly(L-EG3MA-CO)26 and then into poly(L-EG3MA-CO2)26, respectively. Also, oxidizing poly(LEG4/5MA-C)54 into poly(L-EG4/5MA-CO)54 caused CP increase from 65 to 72 °C. Further oxidizing into poly(L-EG4/5MACO2)54 caused complete disappearance of thermal-responsive properties. From the above discussion, oxidation of sulfide groups to sulfoxide or sulfone groups increased the solubility of the resulting polypeptides, which accounted for the increase of CP. In particular, the CP of sulfoxide polymers was about 5 and 7 °C higher than that of poly(L-EG3MA-C)26 and poly(LEG4/5MA-C)54, respectively. The CP of sulfone polymers, however, was above 30 °C higher than that of corresponding poly(L-EGxMA-C)n. Besides solubility, a change of side chain polarity can also induce transition of secondary structures.6 Thus, we further investigated the effects of oxidation on the secondary structures of OEGylated poly-L-cysteine derivatives using CD spectroscopy. Previous studies showed that OEGylated poly-L-cysteine derivatives formed mixed conformation with high β-sheet content. Figures 3 and S2 compared the CD spectra of poly(L-EGxMA-C), poly(L-EGxMA-CO), and poly(L-EGxMA-CO2). Apparently, all four samples did not show obvious conformation changes upon oxidation to intermediate poly(L-EGxMA-CO). However, all four samples adopted random coil conformation after fully oxidized into poly(L-

EGxMA-CO2), as revealed by CD measurements. Such transition was similar to poly(glyco-CO2) homopolypeptides.6 The reason we assumed was that sulfoxide functionality with intermediate polarity was not sufficient to destabilize the ordered conformation (e.g., α-helix or β-sheet). On the contrast, oxidation of the thioethers to sulfones in poly(LEGxMA-C)s was not only able to destabilize the α-helices, but also the β-strands, and consequently caused a transition to disordered conformation. These results further demonstrated that oxidative switching of the chain conformation was indeed a critical factor for the enhanced solubility and increased CP of OEGylated poly-L-cysteine derivatives. For the above four poly(L-EGxMA-C)n samples, we found that oxidation of poly(L-EG2MA-C)n can induce a hydrophobic to hydrophilic transition. We then considered using such feature to construct oxidation-responsive micelles, which can be disassembled on demand. Moreover, such oxidation-responsive characteristics can be applied to realize controlled release of cargo molecules encapsulated in the aggregates in response to oxidizing reagents.43,50 We then synthesized a PEG45-b-poly(LEG2MA-C)22 diblock copolymer, and its structure was confirmed by NMR and SEC/LLS (Table S1). In this design, nonionic PEG was used as hydrophilic block, while oxidationresponsive poly(L-EG2MA-C)n block was initially used as hydrophobic block (Figure S1). When dispersed in water, 1058

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poly(L-EG3MA-CO2) (Figure 1), we can conclude that most of the diblock copolymer has been successfully oxidized to PEG45b-poly(L-EG2MA-CO2)22. We have previously described that the oxidation of thioethers to sulfones in poly(L-EGxMA-C)s was able to cause a conformation transition. Circular dichroism analysis (Figure 6) of the block copolymer confirmed that poly(L-EG2MA-C)22

PEG45-b-poly(L-EG2MA-C)22 formed spherical micellar assemblies revealed by TEM (Figure 4a). The CMC of PEG45-bpoly(L-EG2MA-C)22 was determined to be around 4.6 × 10−3 mg/mL by the fluorescent probe method using pyrene as the probe (Figure S4). The average diameter of the aggregates was about 97 nm, indicated from DLS measurements (Figure 4c). To explore the oxidation-responsive behavior of PEG45-bpoly(L-EG2MA-C)22 copolymer micelles, we used H2O2 as a model oxidizing agent. The macroscopic change of its aqueous solution before and after oxidation can be obviously observed from the optical photos in Figure 5. Pristine PEG45-b-poly(L-

Figure 6. Circular dichroism spectra of PEG45-b-poly(L-EG2MA-C)22 (solid line) and PEG45-b-poly(L-EG2MA-CO2)22 (dashed line). Figure 5. Turbidity measurement on PEG45-b-poly(L-EG2MA-C)22 micellar dispersions (1 mg/mL) during exposure to H2O2.

segment in diblock adopted mainly β-sheet (β-sheet 41%, random coil 37%, α-helix 3%, and turn 19%) in water. In contrast, PEG45-b-poly(L-EG2MA-CO2)22 was predominantly disordered random coil. The reason we believed was due to the sulfone groups, which had a strong interaction with water. Such strong interactions would disrupt hydrophobic packing of the polypeptide side chains to destabilize the β-strand conformation and, consequently, increased polypeptides’ solubility in water.6 The polarity of polypeptide side chains was important in terms of determing samples’ solubility. For example, protonated poly(L-lysine) was readily soluble in water while adopting random coil. Deprotonated poly(L-lysine) formed helical structure but became insoluble in water. Here, such process of oxidizing thioethers into sulfone mimics can not only switch the chain conformation but also realize oxidative disintegration of aggregates arising from polarity change of side chains. Given the oxidative disintegration of micellar aggregates, the PEG45-b-poly(L-EG2MA-C)22 can be used to prepare an oxidation-responsive drug delivery system since some inflammatory cells often exhibit oxidation-stressed environments compared with the normal cells. As a proof of this concept, we encapsulated Doxorubicin, which is a widely used clinical anticancer drug,51 as a model hydrophobic drug to investigate the release profile from the PEG-polypeptide hybrid micelles. The DOX-loaded nanoparticles were characterized by means of UV-vis, TEM, and DLS. Determined by UV-vis spectroscopy, the EC and EE of PEG45-b-poly(L-EG2MA-C)22 micelle was about 12.7 and 63.5 wt %, respectively. After being loaded with DOX, the PEG45-b-poly(L-EG2MA-C)22 still formed spherical micelles with similar sizes as controlled ones (Figure 4a,b). Figure 4c shows the hydrodynamic radius (Rh) distribution of DOX-loaded micelles in aqueous solution, and the average Rh was about 96 nm.

EG2MA-C)22 diblock copolymer formed cloudy micellar dispersion, but the dispersion became transparent upon addition of H2O2 solution. The reason we presumed was due to the oxidation of the hydrophobic thioethers to either sulfoxide or sulfone functionalities, both of which would increase hydrophilicity of poly(L-EG2MA-C)22 segment. Such changes resulted into the disruption of micellar aggregates and ultimately to complete dissolution of copolymer with the presence of excess H2O2 (Figure 5). The increase of oxidant concentration can speed up the reaction as well as phase transition. For example, the turbidity of micellar dispersion dropped faster with 10 vol % H2O2 than the dispersion with 5 vol % H2O2 (Figure 5). The former dispersion turned into completely clear after about 5 h compared to about 10 h for the latter dispersion. These results indicated that we can tune the dissociation rate of PEG45-b-poly(L-EG2MA-C)22 micelles by using different amounts of oxidant. Moreover, TEM characterization also suggested nearly complete disappearance of micelles after exposure to H2O2 (Figure S5). 1 H NMR spectrometry was applied to confirm the structure change after oxidation. Figure S6 compares the 1H NMR spectra of PEG45-b-poly(L-EG2MA-C)22 micellar dispersions before and after oxidation. Before oxidation, the peak of the methyl protons in β position to sulfur atoms was at ∼1.25 ppm and the peaks of the methylene and methine groups around the sulfur atoms were between 2.58 and 3.17 ppm. After oxidation, the appearance of a broad peak at ∼1.36 ppm, corresponding to the methyl in β position to −SOx− moieties, indicated the occurrence of oxidation. Meanwhile, the resonances of the previous methylene and methine groups upfielded to 2.80−3.30 ppm. Comparing the changes with the above-mentioned 1H NMR spectra of poly(L-EG3MA-C), poly(L-EG3MA-CO), and 1059

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effectively increase the release rate and the accumulated release, indicating the suitability of the micelles for practical application.

The release profiles of DOX from DOX-loaded micelles in PBS with various concentrations of H2O2 or Fe2+ were studied. As shown in Figure 7, all cases had an initial quick release of

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ASSOCIATED CONTENT

* Supporting Information S

Additional CD, SEC/LLS, NMR spectra, and TEM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors appreciate financial support from the NSFC Funding for Distinguished Young Scholar (51225306) and the CAS-CSIRO Cooperative Research Program (GJHZ1408).

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Figure 7. Oxidation-responsive release studies of DOX from PEG45-bpoly(L-EG2MA-C)22 nanoparticles in PBS, PBS solutions containing 0.1% (v/v) H2O2, 0.3% (v/v) H2O2, and 0.3% (v/v) H2O2 with 36 μmol/L FeSO4·7H2O, respectively.

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DOX and the initial release rate increased with increasing H2O2 concentration or adding Fe2+ in the release medium. We can see that all the release reached the equilibrium after 10 h. For the DOX release in PBS without H2O2 or Fe2+, 35.6% DOX was released at the end of the examination. Meanwhile, the more H2O2 was added into the medium, the more DOX was released from the micelles. Furthermore, in the presence of a catalytic amount of Fe2+, which is also existing in vivo and has a similar concentration, could further accelerate the release rate and increase the accumulative release. That is because Fe2+ can increase the reactivity of H2O2.52 The accumulated DOX releases after 55 h were about 37.9, 43.3, and 65.5% in PBS containing 0.1% (v/v) and 0.3% (v/v) H2O2 and 0.3% (v/v) H2O2 with 36 μmol/L FeSO4·7H2O, respectively. These results indicated that the as-prepared oxidation-responsive micelles were feasible to trigger release of cargos from these carriers in oxidative stressed environments, which commonly exist in injury, cancer, and other diseases.39−41

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CONCLUSIONS In summary, we investigated the oxidation-responsive properties of a series of OEGlated poly-L-cysteine derivatives. With the presence of oxidant, the side-chain linkages underwent oxidation-induced transformation from less-polar thioethers to more-polar sulfone groups. The change of the side-chain structure triggered the conformation transitions from β-sheet preferred conformation to random coil. The conformation change and the increased polarity together contributed to the rise of water solubility and corresponding CP of poly(LEGxMA-C)n. Given these properties, we successfully prepared oxidation-responsive PEG-polypeptide micelles, which can be easily disintegrated by oxidizing substances, such as H2O2. DOX, as a model drug, was loaded into the oxidationresponsive micellar cores. The in vitro release results revealed that the release rate of DOX from micelles can be modulated by addition of oxidant. Moreover, catalytic amount of Fe2+ can 1060

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