Sugar Dual Responsive Core-Cross-Linked PIC Micelles for

Aug 20, 2013 - Herein, a series of biocompatible, robust, pH/sugar-sensitive, core-cross-linked, polyion complex (PIC) micelles based on phenylboronic...
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pH/Sugar Dual Responsive Core-Cross-Linked PIC Micelles for Enhanced Intracellular Protein Delivery Jie Ren,† Yanxin Zhang,† Ju Zhang,‡ Hongjun Gao,† Gan Liu,† Rujiang Ma,† Yingli An,† Deling Kong,‡ and Linqi Shi*,† †

Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China ‡ Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300071, China S Supporting Information *

ABSTRACT: Herein, a series of biocompatible, robust, pH/sugar-sensitive, core-cross-linked, polyion complex (PIC) micelles based on phenylboronic acid−catechol interaction were developed for protein intracellular delivery. The rationally designed poly(ethylene glycol)-b-poly(glutamic acid-coglutamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and poly(ethylene glycol)-b-poly(L-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-L-lysine) (PEG-b-P(Lys-co-LysCA)) copolymers were successfully synthesized and self-assembled under neutral aqueous condition to form uniform micelles. These micelles possessed a distinct core-cross-linked core−shell structure comprised of the PEG outer shell and the PGlu/PLys polyion complex core bearing boronate ester cross-linking bonds. The cross-linked micelles displayed superior physiological stabilities compared with their non-crosslinked counterparts while swelling and disassembling in the presence of excess fructose or at endosomal pH. Notably, either negatively or positively charged proteins can be encapsulated into the micelles efficiently under mild conditions. The in vitro release studies showed that the release of protein cargoes under physiological conditions was minimized, while a burst release occurred in response to excess fructose or endosomal pH. The cytotoxicity of micelles was determined by cck-8 assay in HepG2 cells. The cytochrome C loaded micelles could efficiently delivery proteins into HepG2 cells and exhibited enhanced apoptosis ability. Hence, this type of core-cross-linked PIC micelles has opened a new avenue to intracellular protein delivery.

1. INTRODUCTION In the past decades, proteins and peptides that possess biological activities have emerged as potent medicines for various types of human diseases, including cancer and diabetes, due to their high specificity, high activity, and low side effect in comparison with small chemical drugs.1−3 However, the application of protein-based therapeutics is limited because of several drawbacks, such as their rapid elimination from the circulatory system, poor bioavailability, low cell permeability, and inefficient endosomal escape. Moreover, for many protein drugs (i.e., cytochrome C, BAX, and caspase-3), their impermeable nature hinders the transportation process into specific intracellular compartments, including nuclei and cytoplasm, where they can only exert therapeutic effects.4,5 The intracellular delivery of membrane impermeable proteins is, therefore, an essential research topic for protein therapeutic applications.6−8 In past years, various nanoscale delivery systems, including liposomes,9 nanocapsules,10,11 polymersomes,12,13 nanogels,14 inorganic nanoparticles,15 and micelles16,17 have been developed to enhance the uptake of proteins in target cells, and at the same time, to stabilize the encapsulated proteins in blood © XXXX American Chemical Society

circulation. Among these various types of nanocarriers, polyion complex (PIC) micelle, which has well-defined core−shell supramolecular structures formed through electrostatic interactions, has emerged as one of the most promising nanocarriers for efficient encapsulation and controlled delivery of watersoluble proteins.18−20 In comparison with other vectors, PIC micelles can reduce immune response, elongate circulation time, and protect the cargoes from enzymatic attack.21,22 Additionally, the self-assembly process of PIC micelle is usually conducted in neutral aqueous medium, which avoids the use of organic solvents, possibly leading to protein denaturation or deactivation.23 However, the low surface charge density of proteins and the electrostatic screening by the added salt could weaken the electrostatic interactions between charged segments and proteins,18,24 which are the main driving force for micelle formation and protein encapsulation. Thus, the major bottlenecks associated with the instability of PIC micelles and the Received: May 22, 2013 Revised: August 19, 2013

A

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poly(glutamic acid-co-glutamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and poly(ethylene glycol)-b-poly(L-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-L-lysine) (PEG-b-P(Lys-co-LysCA)) for intracellular protein delivery (Scheme 1).

premature burst release of protein cargoes in physiological environment have limited their applications as intracellular protein delivery carriers.25,26 Recently, significant attempts have been made to overcome these challenges. Several groups have reported on the stabilization of the PIC micelles by crosslinking the micellar core. Kataoka et al. reported a trypsin loaded core-cross-linked PIC micelle system using glutaraldehyde as the cross-linker.27 Shi et al. prepared a series of mixed shell PIC micelles with the core cross-linked by EDC·HCl, which would maintain stability in PBS buffer (pH 7.4 with 150 mM NaCl) in the presence of a physiologically relevant concentration of BSA.28 However, the use of these toxic crosslinking agents would lead to either protein denaturation or reduced cell viability, while the irreversibility of the crosslinking would significantly inhibit protein release at the site of action. Alternatively, endeavors were also made to strengthen the interactions between proteins and polyions by increasing the surface charge density of proteins. For example, Kataoka et al. developed a strategy on a physiologically stable, pHsensitive, polymeric nanocomplex by modifying cis-aconitic amide onto the lysine groups of cytochrome C.26 Our previous work has introduced anionic characteristics to the transcription factor (TF) by forming a specific anionic TF·DNA complex, resulting in an efficient encapsulation of proteins into nanoparticles.29 Whereas the utilization of recombinant technology or gene technology in these cases brought out either complicated preparation procedure or lack of universality, which lead to limitations in their practical applications. Consequently, novel and convenient strategies for improvement of the protein encapsulation as well as the carrier stability are still urgently needed. Recently, Li et al. has developed a novel class of dual pH value and diol-responsive cross-linked micelles using a facile cross-linking strategy based on boronic acid−catechol interactions.30 Benefiting from the cross-linking bonds, the micelles are able to maintain their micellar structures and retain the encapsulated hydrophobic drug under physiological conditions. Inspired by Li’s strategy, we introduced boronate ester crosslinking bonds based on phenylboronic acid−catechol interactions into the core of PIC micelles, assuming that the boronate ester bonds could tie up the PIC core and improve the structural stability of protein loaded PIC micelles under physiological salt condition. Meanwhile, as Li et al. and other previous works depicted, the boronate ester bonds formed by phenylboronic acid and catechol exhibit fast dual responsiveness to competing diols or acidic pH value.30−32 Thus, this type of rapid, efficiently cross-linking bonds would assist PIC micelle retaining its integrity under physiological conditions, while breaking down in response to the acidic endosomal environment or exogenous competing diols. It is worth noting that the binding process can be carried out under nearly neutral aqueous condition and needs no additional chemical agents, which will help maintaining the bioactivities of protein therapeutics during the micelle preparation. Additionally, as the cross-linking bonds form, the phenyl group contained boronate ester can introduce hydrophobic domains into the micellar core. Thus, the hydrophobic interactions between these domains and proteins will promote the protein encapsulation as well as enhance the stability of protein loaded micelles under physiological conditions, which has already been demonstrated by other previous works.14,24,33−35 Herein, we report on development of core-cross-linked pH/ sugar sensitive PIC micelles based on poly(ethylene glycol)-b-

Scheme 1. Illustration of Protein-Loaded Core-Cross-Linked PIC Micelle Formation and Intracellular Protein Delivery Triggered by Endosomal pH

We investigated the effect of boronate ester cross-linking bonds on the physiological stability of PIC micelles as well as their pH and sugar responsiveness. Model proteins were encapsulated into the micelles and their in vitro release behaviors were studied under diverse conditions. Moreover, the cytotoxicity, cellular uptake and apoptosis ability of the cytochrome C (CC)-loaded micelles were also determined using cell counting kit-8 (cck-8) assay, flow cytometry, and confocal laser scanning microscopy (CLSM), respectively.

2. EXPERIMENTAL SECTION 2.1. Materials. α-Methoxy-ω-aminopoly(ethylene glycol) (CH3OPEG113-NH2, Mw = 5000; Mw/Mn = 1.05) was purchased from Aladdin and dried under vacuum before used. β-Benzyl-L-glutamate Ncarboxyanhydride (BLG-NCA) and β-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride (Lys(Z)-NCA) were synthesized by the FuchsFarthing method using bis(trichloromethyl) carbonate (triphosgene).28,36 Insulin (Ins) and cytochrome C were purchased from Genview. Fluorescein-labeled insulin (FITC-Ins) and fluoresceinlabeled cytochrome C (FITC−CC) were synthesized according to literature procedures.15,36 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC, Sigma), trifluoroacetic acid (J&K Chemical), hydrogen bromide (HBr; 45% in acetic acid, J&K Chemical), 3-aminophenylboronic acid (APBA, Fluka), protocatechuic acid (PCA, J&K Chemical), alizarin red S (ARS, J&K Chemical), and other chemicals and solvents were used as received. The HepG2 cell line was kindly provided by Dr. Ju Zhang (Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China). Annexin V-FITC Apoptosis Detection Kit was purchased from KenGEN. 2.2. Synthesis of PEG-b-PGlu and PEG-b-P(Glu-co-GluPBA). PEG-b-PGlu was synthesized through deprotection of benzyl groups of PEG-PBLG, which was prepared by the ring-opening polymerization (ROP) of BLG-NCA monomer with PEG113-NH2 as the macroinitiator, as described previously.36 Briefly, BLG-NCA (6.312 g, 24 mmol) and mPEG-NH2 (2.0 g, 0.4 mmol) were dissolved into 50 mL of dry DMF in a round-bottom flask. The polymerization was performed at 30 °C under a dry argon atmosphere for 72 h. Then, the solution was precipitated into excessive diethyl ether. The crude product was further washed twice with diethyl ether and dried under B

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vacuum at room temperature for 24 h to obtain poly(ethylene glycol)b-poly(β-benzyl-L-glutamic) (PEG-b-PBLG; yield: 79%). Subsequently, PEG-b-PBLG (5.0 g) was dispersed in NaOH aqueous solution (1 M, 100 mL) and stirred for 48 h at room temperature for deprotection of benzyl groups, followed by neutralization with 1 M HCl aqueous solution. The mixed solution was concentrated by vacuum evaporation and dialyzed against distilled water using a dialysis bag (MWCO 7000 Da). PEG-b-PGlu was obtained after lyophilization (yield: 77%). PEG-b-P(Glu-co-GluPBA) copolymers were synthesized by coupling APBA to the pendent carboxyl group of PGlu block. Briefly, PEG-b-PGlu and EDAC (5.0 equivalent of carboxyl group) were dissolved in DMF and incubated at 4 °C for 30 min and then APBA was added. The feeding molar ratios of APBA to carboxyl group were 40, 60, and 80%, respectively. Then the reaction solution was stirred at room temperature for another 24 h and dialyzed against distilled water for 3 day using a dialysis bag (MWCO 7000 Da), followed by lyophilization to obtain PBA-contained polymers with different degrees of modification. 2.3. Synthesis of PEG-b-PLys and PEG-b-P(Lys-co-LysCA). PEG-b-PLys was synthesized through the ROP of Lys(Z)-NCA monomer with mPEG-NH2 as the macroinitiator and the deprotection of benzyl groups as described previously.28 In brief, a total of 7.36 g (24 mmol) of Lys(Z)-NCA was dispersed in 50 mL of DMF followed by addition of 2.0 g (0.4 mmol) of PEG113-NH2. The reaction mixture was stirred for 72 h at 35 °C under a dry argon atmosphere. The solvent was removed under reduced pressure. The resultant solid was dissolved in 20 mL of CHCl3 and precipitated into excessive diethyl ether. The precipitation was washed twice with diethyl ether and dried under vacuum at room temperature for 24 h to obtain 7.11 g (yield 76%) poly(ethylene glycol)-b-poly(ε-benzyloxycarbonyl-L-lysine) (PEG-b-PLys(Z)). Subsequently, 4.2 g PEG-b-PLys(Z) was dissolved in 40 mL of trifluoroacetic acid and stirred for 0.5 h. Then, 4 mL hydrogen bromide (HBr; 45% in acetic acid) was added into the solution and stirred for further 48 h at room temperature. The reaction mixture was diluted with 30 mL of distilled water and vigorously shaken with 200 mL of diethyl ether. The water phase was neutralized by sodium hydroxide and dialyzed against distilled water using a dialysis bag (MWCO 7000 Da). The aqueous solution of final product was lyophilized to obtain 1.92 g PEG-b-PLys (yield: 71%). Similar to the synthesis of PEG-b-P(Glu-co-GluPBA), the PEG-bP(Lys-co-LysCA) copolymers were synthesized by coupling PCA to the pendent primary amino group of PGlu block. In brief, PCA and EDAC (5.0 equivalent of carboxyl group) were dissolved in MES buffer (pH 5.5) and incubated at 4 °C for 30 min and then PEG-bPLys was added. The feeding molar ratios of PCA to amino group were 30, 50, and 90%, respectively. Then the final solution was stirred at room temperature for another 24 h and dialyzed against distilled water for 3 day using a dialysis bag (MWCO 7000 Da), followed by lyophilization to obtain CA contained polymers of different degrees of modification. 2.4. Preparation of Non-Cross-Linked Micelle (NCM) and Cross-Linked Micelles (CLMs). The formation of CLMs was simply carried out by mixing the aqueous solutions of the two kinds of copolymers. The PEG-b-P(Glu-co-GluPBA) copolymers were first dissolved into a NaOH solution (pH 10), followed by regulating the pH value to 7.4 using the HCl solution, while the PEG-b-P(Lys-coLysCA) with similar modified ratios were dissolved directly into water of pH 7.4. Then, a given volume of PEG-b-P(Lys-co-LysCA) solution was added dropwise into the PEG-b-P(Glu-co-GluPBA) solution in an electrically neutralized condition (i.e., [Glu]/[Lys] = 1:1) under vigorous stirring. The mixed solution became opalescent instantly due to the formation of micelles. The NCM was prepared via the same method as CLMs using PEG-b-PGlu and PEG-b-PLys. 2.5. Loading of Proteins. The protein-loaded micelles were readily obtained by first mixing the protein with the identically charged polyion aqueous solution, followed by addition of the oppositely charged polyion at neutral pH. Briefly, CC was added to the PEG-bP(Lys-LysCA) solution in deionized water at pH 7.4 (polymer concentration was fixed to 2 mg/mL) at varying protein/polymer

weight ratios (5−20 wt %). The mixed solution was stirred for 30 min at room temperature, followed by dropwise addition of PEG-b-P(Gluco-GluPBA) aqueous solution in an electrically neutralized condition and stirred overnight. Free proteins were removed by dialysis (MWCO 350 kDa) against PBS buffer (pH 7.4, 10 mM) for 24 h at room temperature with at least 6× change of media. Ins-loaded micelles were prepared via the same method, as depicted above. To determine protein loading content (PLC) and protein loading efficiency (PLE), protein-loaded micelles were lyophilized and disrupted by adding excess DMSO, which led to complete release of loaded proteins. The amount of FITC-labeled proteins was determined by fluorescence measurements based on the calibration curve with known concentrations of FITC-labeled proteins in DMSO. PLC and PLE were calculated according to the following formula: PLC(wt%) = (weight of loaded protein/weight of polymer) × 100%

PLE(%) = (weight of loaded protein/weight of protein in feed) × 100% 2.6. In Vitro Protein Release. The release of FITC-Ins and FITC−CC from core-cross-linked PIC micelles was investigated using a dialysis method at 37 °C in four different media, that is, phosphate buffered saline (PBS) buffer (pH 7.4, 10 mM, 150 mM NaCl), PBS buffer with 2 mg/mL glucose, PBS buffer with 50 mg/mL fructose, and acetate buffer (pH 5.0, 10 mM, 150 mM NaCl). Briefly, 0.4 mL of protein-loaded micelles in PBS buffer (pH 7.4, 10 mM) was dialyzed (MWCO 350 kDa) against 15 mL of the above release media in a water bath at 37 °C with a shaking rate of 120 rpm. At desired time intervals, 1 mL of release media was taken out and replenished with an equal volume of fresh media. The amounts of released proteins were determined by fluorescence measurements (excitation at 490 nm, and emission from 510 to 520 nm). The release experiments were conducted in triplicate, and the results presented are the average data with standard deviations. 2.7. cck-8 Assay for Cell Viability. HepG2 cells were seeded in a 96-well plate at an initial density of 5000 cells per well using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 mg/mL) for 24 h at 37 °C in humidified 5% CO2 atmosphere. After incubation of 24 h, the culture medium of each well was replaced by 200 μL of fresh medium containing various concentrations of micelles. A total of 24 h later, the culture media were replaced with 25 μL of cck-8 PBS solution (1 g/L final concentration) and the cells were further incubated for another 30 min. The optical absorbance was measured at 450 nm of each well using a microplate reader (Labsystem, Multiskan, Ascent, Finland). The cell viability (%) was determined by comparing the absorbance at 450 nm with control wells containing only cell culture medium. Data are presented as average ± SD (n = 6). 2.8. Cellular Uptake and Intracellular Protein Release Studies. The cellular uptake and intracellular release behaviors of FITC−CC loaded core-cross-linked PIC micelles were investigated by confocal laser scanning microscopy (CLSM) using HepG2 cells. The cells were plated on microscope slides in a 24-well plate (1 × 105 cells/ well) under 5% CO2 atmosphere at 37 °C using DMEM medium supplemented with 10% fetal bovine serum, antibiotics penicillin (100 IU/mL), and streptomycin (100 μg/mL) for 24 h. Then the cells were exposed to FITC−CC loaded micelles or free FITC−CC for 4 or 24 h at 37 °C in a humidified 5% CO2-containing atmosphere (FITC−CC dosage: 50 μg/mL). After the incubation, the culture medium was removed. The cells on microscope plates were fixed with 4% formaldehyde for 20 min and washed three times with PBS buffer. Then the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and washed six times with PBS buffer. The fluorescence images were obtained using confocal laser scanning microscope (TCS SP5). 2.9. Apoptotic Activity of CC-Loaded Cross-Linked Micelles. HepG2 cells were plated in a 24-well plate (5 × 104 cells/well) under C

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5% CO2 atmosphere at 37 °C using DMEM medium supplemented with 10% fetal bovine serum, antibiotics penicillin (100 IU/mL), and streptomycin (100 mg/mL) for 24 h. The cells were treated with CCloaded core-cross-linked PIC micelles or free CC for 12 or 24 h at 37 °C in a humidified 5% CO2-containing atmosphere (CC dosage: 50 μg/mL). To quantify the percentage of apoptotic cells, Annexin VFITC kit was used as described by the manufacturer (KenGEN, China). Briefly, HepG2 cells were digested with EDTA-free trypsin, washed twice with cold PBS, and resuspended in binding buffer at a concentration of 1 × 105 cells/mL. Then, the cells were stained with 5 μL of Annexin V-FITC solution and 10 μL of PI solution for 15 min at room temperature in the dark. At the end of the incubation, 400 μL of binding buffer was added, and the cells were analyzed immediately by flow cytometry (BD FACSCalibur, Mountain View, CA). 2.10. Characterizations. 1H NMR spectra were recorded on a Varian UNITY-plus 400 M NMR spectrometer at room temperature with tetramethylsilane (TMS) as an internal standard. The weightaverage molecular weight (Mw) and polydispersity (PDI) were measured by gel permeation chromatography (GPC) at 25 °C with a Waters 1525 chromatograph equipped with a Waters 2414 refractive index detector. GPC measurements were carried out using DMF as eluents with a flow rate of 1.0 mL/min, respectively. Poly(ethylene glycol) standards were used for calibration. Dynamic light scattering (DLS) experiments at a 90 °C scattering angle were performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 636 nm at required temperature. All samples were obtained by filtering through a 0.45 μm Millipore filter into a clean scintillation vial. Transmission electron microscopy (TEM) measurements were performed using a Philips T20ST electron microscope at an acceleration voltage of 100 kV. To prepare the TEM samples, the sample solution was dropped onto a carbon-coated copper grid and dried slowly at required temperature. The zeta potential values were measured on a Brookheaven Zeta PALS (Brookheaven Instrument, U.S.A.). The instrument utilizes phase analysis light scattering at 37 °C to provide an average over multiple particles. The ζ potential of the micelles was determined with a Zetasizer Nano-ZS from BrookHeaven Instruments. CLSM images were taken on a confocal microscope (TCS SP5). Figure 1. 1H NMR spectra (400 MHz, D2O) of PEG113-b-P(Glu-coGluPBA)45 and PEG113-b-P(Lys-co-PLysCA)43 with different modification degrees.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Copolymers. PEG-b-PGlu and PEG-bPLys were prepared via the ROP procedure of BLG-NCA and Lys-NCA with mPEG-NH2 as the macroinitiator in DMF and subsequent deprotection of benzyl group. The gel permeation chromatography (GPC) measurements revealed a unimodal peak with a Mn of 14.8 kDa and a PDI of about 1.34 for PEG-bPBLG. For PEG-b-PLys(Z), the Mn is 16.2 kDa and the PDI is about 1.38 (Figure S1). The two copolymers after deprotection were characterized by 1H NMR. As shown in Figure 1, the composition ratio of PEG-b-PGlu was calculated by the peak integration ratio of -OCH2CH2- protons of PEG at 3.59 ppm and -NHCHCO- protons of PGlu at 4.23 ppm, indicating the degree of polymerization (DP) of PGlu was 45. Likewise, by comparing of the peak integration of -OCH2CH2- protons of PEG at 3.59 ppm and -NHCHCO- protons of PLys at 4.18 ppm, the DP of PLys was calculated to be 43. The specific composition ratios of these two copolymers were designed on purpose to meet the demand of forming a nanocarrier for drug delivery. First, the choice of PEG with Mw of 5000 g/mol has been confirmed as an effective shield of nanocarriers for intravenous administration.22 Second, the PGlu and PLys blocks of several tens units were selected, of which lengths would be enough to offer charged segments for a PIC micelle. Third, it has been demonstrated by Kataoka et al. that only when a pair of PEG-b-polyions possess a matched chain length of oppositely charged segments, the mixing of their solutions

would lead to the formation of PIC micelles.37 Thus, these two ionic copolymers with the similar DPs would induce the micellization when mixed with each other. PEG-b-P(Glu-co-GluPBA) copolymers were synthesized by modification of PEG-b-PGlu with 3-aminophenylboronic acid (APBA) using EDAC as the coupling reagent at different carboxyl/APBA mole ratios. As shown in Figure 1A, the peaks between 7.0 and 7.5 were attributed to phenyl protons in pendent PBA groups. The modification degrees were calculated to be 16, 28, and 36%, respectively, by the peak intensity ratio between the phenyl protons of pendent phenylboronic group and the methylene protons of PEG. Similar to the synthesis process of PEG-b-P(Glu-co-GluPBA), the side chain amino group of PEG-b-PLys were modified by protocatechuic acid (PCA) at different amino/CA mole ratios to form PEG-bP(Lys-co-LysCA) with different degrees of modification of 17, 25, and 35%, respectively, determined by comparing the integrals of signals attributed to the phenyl protons of pendent catechol group and the methylene protons of PEG. 3.2. Formation of Core-Cross-Linked PIC Micelles. The phenylboronic acid−catechol interactions during the micelle formation were confirmed by a colorimetric assay based on the indicator of alizarin red S (ARS). As is well-known, ARS is a catechol dye exhibiting dramatic changes in fluorescence D

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intensity after forming esters with boronic acids.30 The fluorescence intensity of ARS at 580 nm increased significantly after incubation with PEG-b-P(Glu-co-GluPBA) (PBS buffer solution at pH 7.4), implying a binding interaction between ARS and the PBA group. When a series of PEG-b-P(Lys-coLysCA) aqueous solutions with varying concentrations were added, respectively, the fluorescence of ARS decreased remarkably with the increasing amounts of PEG-b-P(Lys-coLysCA) (0−0.20 mg/mL), as illustrated in Figure 2. This result

continuous decrease of Dh (118.8 to 85.3 nm), as depicted in Table 1. This trend of size discrepancy of CLMs with reduced cross-linking degrees can be explained as follows. During the cross-linking formation, the pendent PBA groups and CA groups bond with each other to pack the PIC core. Meanwhile, the hydrophobic phenyl groups were introduced into the core of PIC micelles. These two factors could synergetically render the PIC core more compact, leading to a size decrease of micelles. In addition, low polydispersities (PDI; 0.094−0.156) and nearly neutral zeta potentials (0.92−2.32 mV) suggested PEG shielding core−shell structures of CLMs. Transmission electron microscopy (TEM) micrographs demonstrated that the CLMs had mostly spherical morphology and size distributions close to those determined by DLS (inset in Figure 3A). 3.3. Stability Studies of Core-Cross-Linked PIC Micelles. It is well-known that the instability of PIC micelles under physiological salt condition is the main hindrance for their therapeutic application in drug delivery. Thus, the stability of NCM and CLMs was studied by monitoring the size (Dh) and relative light scattering intensity (I/I0) changes as functions of time measured by DLS measurement under simulative physiological salt condition (NaCl, 150 mM). It was clearly shown in Figure 4 that the relative light scattering intensity (I/ I0) of NCM decreased to about 30%, while the average size exceeded 2500 nm within 4 h, suggesting a swelling and dissociating fate of NCM under physiological salt condition, which was consistent with the depiction in previous works.25,35 As for CLM1 and CLM2 with low cross-linking degrees (16 and 25%), the increase of sizes as well as the relative light scattering intensities (I/I0) implied a swelling process without dissociation. These facts can be attributed to the maintenance of micelle structure packed by cross-linking bonds and hydrophobic interactions in the core of CLMs when the main driving force of PIC core formation was weakened by electrostatic screening. Thus, the CLMs exhibited better colloidal stability under physiological salt condition than their non-cross-linked counterparts. It is notable that when the crosslinking degree was increased to 35%, the core-cross-linked micelle (CLM3) could keep intact within at least 24 h, supported by the little change of the relative light scattering intensity (I/I0) and average size of CLM3 within 24 h. Because proteins existed in the serum may disrupt the micellar structure during the circulation in human blood vessel,38,39 it is necessary to investigate the stability of crosslinked micelles in the serum or plasma. Herein, we use 50% v/v bovine serum to simulate the blood circulation conditions. The DLS results showed that CLM3 could keep intact within at least 24 h, indicating that the stability of CLM3 in simulative blood circulation conditions is similar to that in PBS (Figure S2A). 3.4. pH and Sugar Responsiveness of the Core-CrossLinked PIC Micelles. Due to its superb stability under physiological salt condition, CLM3 was chosen as a model to investigate the pH/sugar sensitivity of CLMs by monitoring the variation of relative light scattering intensities (Figure 5A) and micelle sizes (Figure 5B) in response to diverse stimuli conditions. Because the interactions between PBA and CA groups may be influenced by blood glucose, the stability of CLM3 under simulative physiological glucose condition (∼2 mg/mL) was investigated. It is showed in Figure 5 that the relative light scattering intensity and size variation profiles of CLM3 as functions of time in the presence of glucose with a

Figure 2. Fluorescent intensity of ARS (0.1 mM) upon mixing with micelles formed by PEG113-b-P(Glu0.64-co-GluPBA0.36)45 (0.20 mg/ mL) with different ratios of PEG113-b-P(Lys0.65-co-LysCA0.35)43 (0.05− 0.20 mg/mL) in PBS buffer at pH 7.4. Excitation: 468 nm.

suggested that the pendent CA groups of PEG-b-P(Lys-coLysCA) would competitively replace ARS and combine with PBA groups, leading to the fabrication of cross-linking bonds in the PIC core. A series of core-cross-linked micelles were prepared by mixing equal molar ratios of PEG-b-P(Glu-co-GluPBA) and PEG-b-P(Lys-co-LysCA) with similar modification degrees at neutral pH and their characterizations were shown in Table 1. Table 1. Characterization of Core-Cross-Linked PIC Micelles and Non-Cross-Linked Micelle entry

cross-linkinga ratio (%)

sizeb (nm)

PDIb

ζ-potentialc (mV)

NCM CLM1 CLM2 CLM3

0 16 25 35

172.5 118.8 91.0 85.3

0.005 0.156 0.129 0.094

−0.21 ± 1.56 −0.92 ± 2.45 1.28 ± 0.43 2.32 ± 1.68

a

Determined by the amount of possible cross-linker in the micellar core. bThe average size and size distribution of micelles formed at 25 °C and pH 7.4 measured by DLS. cDetermined by Zeta PALS at 25 °C and pH 7.4.

Non-cross-linked micelle was formed via the same method as a control. These micelles differed in the ratios of possible crosslinkers per polymer (0, 16, 25, and 35%), which were denoted as NCM, CLM1, CLM2, and CLM3, respectively. Dynamic light scattering (DLS) measurements showed that the CLMs have average hydrodynamic diameters (Dh) of 118.8 to 85.3 nm (Figure 3A), which were much lower than those of their parent PIC micelle (NCM) formed by PEG-b-PGlu and PEG-b-PLys (172.5 nm). Interestingly, the increase of the amounts of crosslinkers (16−35%) in the core-cross-linked micelles led to a E

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Figure 3. Size distribution and TEM images (inset) of CLM3 (A), and CC-loaded (20%) CLM3 (B) measured by DLS and TEM.

Figure 4. Relative scattering light intensity (A) and size (B) variation of CLMs and NCM over 24 h in PBS buffer (10 mM, NaCl 150 mM) at 37 °C.

Figure 5. Relative scattering light intensity (A) and size (B) variation of CLM3 over 24 h in response to different environmental stimuli at 37 °C.

in several hours. The swelling and dissociating of CLM3 were also observed under acidic condition (pH 5.0) with rapid increase of micelle size (∼80 to ∼120 nm) and decrease in light scattering intensity (100% to ∼30%). That destabilization of CLMs under acidic conditions can be attributed to two factors. On one hand, the cross-linking boronate bonds are critically weakened under acidic conditions because the binding constant between CA and PBA groups at pH 5.0 was much lower than that at pH 7.4. On the other hand, the carboxyl groups of PGlu block are deionized at pH 5.0 because of their low pKa (∼5), resulting in a significantly reduction in the interaction between

concentration of 2 mg/mL are similar to those of CLM3 in the nonstimuli media without glucose. The stability of CLM3 under physiological glucose condition is most likely due to the much lower binding constant between glucose and PBA groups (4.6) compared with that between CA and PBA groups (830) at physiological pH.32 In contrast, in the presence of excess fructose (50 mg/mL), which has a much higher binding constant with PBA groups (210), CLM3 would experience a rapid swelling and dissociating process due to the efficient cleavage of cross-linking bonds. This is evidenced by the significant growth in micelle size (∼80 to ∼160 nm) and reduction in relative light scattering intensity (100% to ∼55%) F

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(Table 2). Zeta potential measurements displayed that they had nearly neutral surface charges (data not shown). TEM micrographs revealed that the CC loaded CLM3 had an almost spherical morphology with an average size about 90 nm (inset in Figure 3B). The little change of micelle sizes, morphology, PDIs, and surface charges implied that the encapsulation of protein cargoes had essentially no influences on the selfassembly of block copolymers. Next, FITC−CC and FITC-Ins loaded CLM3 were chosen for further investigations. To investigate stimuli-responsive protein release behaviors, protein release studies were carried out at 37 °C in four different media for FITC−CC (Figure 6A) and FITC-Ins (Figure 6B), respectively. These media are PBS buffer (pH 7.4, 10 mM, 150 mM NaCl), PBS buffer with 2 mg/ mL glucose, PBS buffer with 50 mg/mL fructose, and acetate buffer (pH 5.0, 10 mM, 150 mM NaCl). As expected, minimal protein (80%), suggesting its acceptable biocompatibility. 3.7. Cellular Uptake of Micelles and Intracellular Delivery of Cytochrome C. The cellular uptake and intracellular protein delivery behaviors of FITC−CC loaded CLM3 in human HepG2 cells were investigated using CLSM (Figure 8). Notably, distinct FITC fluorescence was observed in HepG2 cells following 4 h of incubation with FITC−CC loaded CLM3, while its intensity increased significantly after 24 h of incubation. In contrast, no FITC fluorescence was detected in HepG2 cells incubated with free FITC−CC for even 24 h, which was probably due to its low permeation across the cellular membrane. Higher power photomicrographs of the cellular uptake and intracellular distribution of the FITC−CC in both MCF-7 and HepG2 cells were shown in Figure S4, suggesting the identical results as those of Figure 8. These results demonstrate that the pH-sensitive micelles can be endocytosed by cancer cells and deliver protein cargoes into cytoplasm efficiently. Two factors may facilitate this process. On one hand, the cross-linked micelles are disassembled at endosomal pH value and release the protein cargoes. On the other hand, the protonation of primary amino groups on the

4. CONCLUSIONS In summary, we reported the design and synthesis of a novel class of pH/sugar responsive core-cross-linked polyion complex micelles self-assembled by PEG-b-P(Glu-co-GluPBA) and PEGb-P(Lys-co-LysCA). Benefiting from the cross-linking bonds and hydrophobic interactions, we have readily optimized the physiological stability of the resulting core-cross-linked PIC micelles as well as their disassembly in response to excess fructose or endosomal acidic pH value. Both negatively and positively charged model proteins can be efficiently capsulated into the core-cross-linked micelles under mild conditions and rapidly released in the presence of diols or acids. Cell culture H

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Figure 9. Contour diagram of Annexin V-FITC/PI flow cytometry of HepG2 cells incubated with free CC for 12 h (A) and 24 h (B), and with CCloaded CLM3 for 12 (C) and 24 h (D). CC dosage was fixed to 50 μg/mL.

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studies revealed that these biocompatible cross-linked micelles can load and deliver intact cytochrome C into the cytoplasm of cancer cells, exerting a superior cell apoptosis activity. Thus, this novel type of core-cross-linked PIC micelle can serve as a promising platform for intracellular protein delivery.

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ASSOCIATED CONTENT

S Supporting Information *

GPC trace of copolymers before deprotection, the stability and release behaviors of protein-loaded cross-linked micelles in the presence of bovine serum (50% v/v), and the fluorescent calibration curves of FITC-labeled proteins are available. In addition, the high power CLSM photomicrographs to show the cellular uptake and intracellular distribution of the FITCproteins are disclosed. 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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REFERENCES

ACKNOWLEDGMENTS

We thank the National Natural Science Foundation of China (Nos. 21274001, 91127045), the National Basic Research Program of China (973 Program, No. 2011CB932503), and PCSIRT (IRT 1257) for financial support. I

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