Fabrication of Reductive-Responsive Prodrug Nanoparticles with

Aug 22, 2016 - A highly efficient strategy, polymerization-induced self-assembly (PISA) for fabrication of the polymeric drug delivery systems in canc...
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Fabrication of Reductive-Responsive Prodrug Nanoparticles with Superior Structural Stability by Polymerization-Induced SelfAssembly and Functional Nanoscopic Platform for Drug Delivery Wen-Jian Zhang, Chun-Yan Hong,* and Cai-Yuan Pan* CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *

ABSTRACT: A highly efficient strategy, polymerizationinduced self-assembly (PISA) for fabrication of the polymeric drug delivery systems in cancer chemotherapy is reported. Diblock prodrug copolymer, PEG-b-P(MEO2MA-co-CPTM) was used as the macro-RAFT agent to fabricate prodrug nanoparticles through PISA. The advantages of fabricating intelligent drug delivery system via this approach are as following: (1) Simultaneous fulfillment of polymerization, selfassembly, and drug encapsulation in one-pot at relatively high concentration (100 mg/mL); (2) Almost complete monomer conversion allows direct application of the resultant prodrug nanoparticles without further purification; (3) Robust structures of the resultant prodrug nanoparticles, because the cross-linker was used as the comonomer, resulted in core-cross-linking simultaneously with the formation of the prodrug nanoparticles; (4) The drug content in the resultant prodrug nanoparticles can be accurately modulated just via adjusting the feed molar ratio of MEO2MA/CPTM in the synthesis of PEG-b-P(MEO2MA-co-CPTM). The prodrug nanoparticles with similar diameters but various drug contents were obtained using different prodrug macro-CTA. In consideration of the long-term biological toxicity, the prodrug nanoparticles with higher drug content exhibit more excellent anticancer efficiency due to that lower dosage of them are enough for effectively killing HeLa cells.

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INTRODUCTION Polymeric drug delivery systems for cancer chemotherapy have attracted more and more attention during the past decades, partially due to the limitations of small molecule drugs, such as severe side effects, limited stability, poor water solubility, unsatisfactory biodistribution, and pharmacokinetics.1−3 Generally, amphiphilic block copolymers are used to fabricate nanocarriers with core−shell nanostructures loading small chemotherapeutic drugs in the cores. The hydrophilic shell, such as polyethylene glycol (PEG), can effectively prevent anticancer drugs in the core from prematurely interacting with the biological environment, protect the drug from premature degradation, minimize nonspecific protein adsorption during blood circulation and capturing by the reticuloendothelial system (RES). The majority of these nanocarriers demonstrate passive targeting capabilities due to the characteristic features of tumor biology that facilitate accumulation of nanocarriers in the tumor through the enhanced permeability and retention (EPR) effect.4−8 At designing the nanocarriers, to achieve site-specific drug release at the pathological site should be always considered. Since the reductive and mildly acidic intracellular microenvironment of the tumor cells, lots of pH or reductive responsive nanocarriers have been reported.9−14 © XXXX American Chemical Society

The structural integrity of the nanocarriers is another important issue for designing polymeric drug delivery system.15,16 The polymeric nanocarriers are usually subjected to high dilution and large shear force during blood circulation. When the polymer concentration is reduced to below the critical micelles concentration (CMC), the polymeric nanocarriers tend to dissociate, which induces undesirable drug leakage. To enhance stability of the polymeric nanocarriers, shell cross-linking (SCL) and core cross-linking (CCL) strategies have been proposed for efficient drug delivery, even subjecting to severe biological microenvironment.6,17−19 Conventionally, the polymeric drug nanocarriers are prepared through postpolymerization self-assembly of amphiphilic block copolymers, during which the anticancer drugs were encapsulated into the cores of nanocarriers. Such selfassembly is usually conducted at relatively high dilution (the polymer concentration is typically less than 1%). In addition, further purification for removal of the unencapsulated drugs is usually necessary.20−22 For fabrication of the nanocarriers with robust structures, additional procedure for core/shell crossReceived: June 4, 2016 Revised: August 2, 2016

A

DOI: 10.1021/acs.biomac.6b00819 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules linking is necessary.9,23 In recent years, polymerization-induced self-assembly (PISA) strategy has become a powerful tool for the fabrication of polymeric nanoparticles (NPs),24−33 which is due to the much high fabricating efficiency including very high fabricating concentration, combination of the polymerization and the self-assembly in one-pot, almost complete monomer conversion leading to no further purification for the following applications. What is more, NPs with a robust structure can be obtained just by adding cross-linker as comonomer in the polymerization system without any additional postpolymerization procedure.34,35 A few literatures have reported the encapsulation of guest cargos, such as proteins and nile red, into the vesicles during the PISA,36−38 but fabricating nanocarriers with performance of controlled release in response to biological milieu still remains a great challenge. Davis et al. fabricated NPs by PISA for drug delivery in 2014,39 but postPISA drug conjugation and core-cross-linking should be conducted. Moreover, styrene was used as the monomer for PISA, which is incompatible for bioapplication, and further purification after PISA should be performed to remove the unreacted styrene due to the low monomer conversion. To solve the above problems, we designed superiorly stable prodrug NPs with three layer structure as shown in Scheme 1.

Scheme 2. Proposed Mechanism of Reductive-Responsive CPT Parent Drug Release from the Prodrug NPs

content is enough for effectively killing HeLa cells, which is beneficial to reduce the long-term biological toxicity.

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Scheme 1. Fabrication of Reductive-Responsive Prodrug NPs with Robust Structure by Polymerization-Induced SelfAssembly (PISA) Using PEG-b-P(MEO2MA-co-CPTM) as the Macro-CTA

EXPERIMENTAL SECTION

Material. Methoxypolyethylene glycol amine (PEG113-NH2, aladdin), N-hydroxysuccinimide (98%, aladdin), camptothecin (98%, chengdu tianyuan natural product Co., Ltd.), benzyl methacrylate (BzMA, 98%, energy chemical), cystamine dihydrochloride (99%, energy chemical), methacryloyl chloride (95%, aladdin), Dulbecco’s modified Eagle’s medium (DMEM, Hyclone), fetal bovine serum (FBS, Hyclone) and glutathione reduced form (GSH, ≥98%, SigmaAldrich) were used as received. 2-(2-Methoxyethoxy)ethyl methacrylate (MEO2MA, Mn ∼ 188 g/mol, 95%) was purchased from SigmaAldrich and purified by passing over a neutral Al2O3 column to remove the inhibitor. N,N′-Azobis(isobutyronitrile) (AIBN, 98%, aladdin) was recrystallized from ethanol prior to use. 2-((2-Hydroxyethyl)disulfanyl)ethyl methacrylate (HSEMA) and 4-(4-cyanopentanoic acid) dithiobenzoate (CPDB) were synthesized according to the literature.40,41 All other reagents of analytical grade were used without further purification. Synthesis of 4-Cyano-4-((thiobenzoyl)sulfanyl) Pentanoic Succinimide Ester (SCPDB). N-Hydroxysuccinimide (2.52 g, 21.9 mmol) and 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPDB, 6.11 g, 21.9 mmol) were dissolved in anhydrous dichloromethane (20 mL). After stirring at ice−water cooling bath for 30 min, a solution of dicyclohexylcarbodiimide (DCC; 4.53 g, 21.9 mmol) in anhydrous dichloromethane (10 mL) was added dropwise into the above mixture over 30 min. The reaction mixture was stirred at 20 °C in dark for 24 h and then washed with distilled water twice. The organic layer was dried over anhydrous Na2SO4. After removing the Na2SO4 by filtration, the solvent was removed using a rotary evaporator. The crude product was purified by column chromatography using a mixed eluent of n-hexane/ethyl acetate = 4:1 (v/v). A red solid was obtained in 86% yield. Synthesis of Poly(ethylene glycol) Dithiobenzoate (PEG113CPDB). All glassware was dried at 100 °C overnight to remove a trace of water before carrying out the reaction. SCPDB (0.45 g, 1.2 mmol) was dissolved in anhydrous dichloromethane (10 mL). A solution of PEG113-NH2 (5 g, 1 mmol) in anhydrous dichloromethane (15 mL) was added dropwise into the SCPDB solution over 1 h. After stirring at room temperature for 12 h, the reaction solution was poured into excess diethyl ether to afford some pink precipitate. The precipitate was collected by filtration and then dissolved in dichloromethane, and the precipitation process was repeated for an additional two times. The degree of dithiobenzoate functionalization was approximately 96% as calculated according to the 1H NMR data (Figure 1).

The redox-sensitive core is cross-linked via copolymerization of benzyl methacrylate (BzMA) and N,N-cystaminebismethacrylamide (CBMA); the middle layer is the anticancer drug, camptothecin (CPT)-containing copolymer, in which CPT is linked to the polymer chains via reductive-sensitive linkage; and the outer layer is hydrophilic poly(ethylene glycol) which is used for stabilize the NPs. Thus, the drug NPs are superiorly stable even in good solvens, highly soluble in water and reductive-sensitive. Such prodrug NPs are fabricated through RAFT dispersion polymerization of (BzMA) and cross-linker, CBMA using diblock prodrug copolymer PEG-b-P(MEO2MAco-CPTM) as the macro-RAFT agent (macro-CTA), so the prodrug NPs are formed simultaneously with core-cross-linked. The CPT contents in the macro-CTA can be accurately modulated just via adjusting the feed molar ratio of CPTM/ MEO2MA, and further the drug content in the resultant prodrug NPs can be controlled by using different CPT contents of macro-CTA. Cleavage of the conjugated CPT drug within the prodrug NPs can be effectively triggered under reductive microenvironments, such as high level of GSH in the cytosol. Scheme 2 shows the mechanism of reductive-responsive CPT parent drug release from the prodrug NPs. Cell viability assays reveal that the lower dose of the prodrug NPs with higher drug B

DOI: 10.1021/acs.biomac.6b00819 Biomacromolecules XXXX, XXX, XXX−XXX

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In Vitro Redox-Regulated Drug Release. The redox-regulated drug release was performed as the follows. The four different prodrug NPs were respectively transferred into the dialysis bags (molecular weight cutoff: 3500 Da) and dialyzed against 200 mL of PBS buffer (10 mM, pH 7.4) with glutathione (GSH) at different concentrations (0.01, 5, and 10 mM) at 37 °C. An aliquot of the buffer solution (2 mL) outside of the dialysis bags was sampled and replaced with equal volume of fresh buffer solution at the predetermined intervals. The concentration of the CPT released from the prodrug NPs was evaluated using the fluorescence spectrometer (excitation wavelength: 340 nm, emission wavelength: 440 nm). In Vitro Cytotoxicity Assay. The in vitro cytotoxicity was determined via MTT assay. The cells were first cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with streptomycin (100 μg/mL), penicillin (100 units/mL), and 10% fetal bovine serum (FBS) at 37 °C in a CO2/air (5:95) incubator for 2 days. For MTT assay, HeLa cells were seeded in a 96-well plate with an initial density of 8000 cells/well in 100 μL of complete Dulbecco’s modified Eagle’s (DMEM) medium supplemented with 10% fetal bovine serum (FBS), and then cultured in a CO2/air (5:95) incubator at 37 °C for 24 h. The cells were then treated with the prodrug or drug-free NPs at various concentrations. Cells without nanomaterials treatment were used as control. After 48 h of incubation, the media were removed and replace by fresh DMEM media containing 0.5 mg/mL MTT reagent. After further 4 h of incubation, the medium in each well was replaced by 150 μL of dimethyl sulfoxide (DMSO) to dissolve the obtained blue formazan crystals. The absorbance at 570 nm was measured to evaluate the cell viability. Cellular Internalization of the Prodrug NPs Observed by CLSM. HeLa cells were seeded in a cell culture plate with an initial density of 3 × 104 cells/well in 300 μL of DMEM medium supplemented with 10% fetal bovine serum (FBS) and then cultured in a CO2/air (5:95) incubator at 37 °C for 24 h. The media were removed and replaced by the fresh DMEM medium (supplemented with 10% FBS) containing prodrug NPs at a final CPT concentration of 10 μg/mL. After incubation for 4, 8, and 16 h, respectively, the media were removed and the cells were washed with PBS three times. The intracellular distribution of drug was observed by a LSM510 confocal laser scanning microscope. Characterization. The 1H NMR spectra were measured on a Bruker DMX300 spectrometer (300 MHz) using CDCl3 as the solvent and tetra-methylsilane as the internal reference. Molecular weight (Mn) and Mw/Mn were measured using a Waters 150C gel permeation chromatography (GPC), equipped with RI 2414 detector (set as 30 °C) and two Ultrastyragel columns in series, monodispersed polystyrene standards were used in calibration, and tetrahydrofuran (THF) was used as eluent at a flow rate of 1.0 mL/min. Transmission electron microscope (TEM) characterizations were conducted on a Hitachi H-800 electron microscope at an accelerating voltage of 100 kV. Dynamic light scattering characterizations were performed on a Laser Light Scattering (LLS) spectrometer, Zetasizer Nano ZS90 (Malven Instruments Ltd., Malvern, U.K.), equipped with a He−Ne Laser (4.0 mW, 633 nm) at 25 °C and a fixed angle of 90°. UV−vis spectra were obtained by a TU-1901 UV−vis spectrophotometer. The fluorescence spectra were acquired on a Shimadzu RF-5301PC luminescence spectrometer, and the slit widths were both set at 10 nm for excitation and for emission. Confocal laser scanning microscopy (CLSM) observations were conducted using a Leica TCS SP5 microscope.

Figure 1. 1H NMR spectrum of PEG113-CPDB in CDCl3. Synthesis of Reduction-Responsive Monomer (CPTM). Camptothecin (CPT, 5.0 g, 14.4 mmol) and DMAP (5.26 g, 43.1 mmol) were mixed with anhydrous dichloromethane (300 mL) under an argon atmosphere. Triphosgene (1.49 g, 5.0 mmol) was added into the above mixture, and then the mixture was stirred at room temperature for 30 min. HSEMA (4.01 g, 15.8 mmol) was diluted with anhydrous dichloromethane (50 mL) and then added dropwise into the above mixture via a constant pressure funnel. After stirring at room temperature for 12 h, the mixture was concentrated by a rotary evaporator. The crude product was purified by silica column chromatography using ethyl acetate as the running solvent. A pale solid powder was obtained in 83% yield (6.97 g). Synthesis of N,N-Cystaminebismethacrylamide (CBMA). Cystamine dihydrochloride (9.0 g, 40 mmol) was dissolved in 80 mL of water. Aqueous solution of sodium hydroxide (16 mL, 10 M) was added into the above solution, and then the mixture was stirred in ice−water bath for 20 min. Methacryloyl chloride (8.36 g, 80 mmol, in 10 mL of dichloromethane) was added dropwise into the above mixture at 0 °C, while a white precipitate was formed. After continuously stirring for 3 h, the reaction mixture was filtered, and the solid was collected and then washed with deionized water for three times. The product was obtained by crystallization from ethyl acetate in 47% yield. Synthesis of Diblock Copolymer PEG-b-P(MEO2MA-coCPTM) with Various Contents of CPTM. PEG113-CPDB (0.53 g, 0.1 mmol), AIBN (1.64 mg, 10−2 mmol), and CPTM/MEO2MA, with a total molar mass of 4.5 mmol and various molar ratios of 0/1, 1/8, 1/ 4, and 1/2, were dissolved into 1,4-dioxane (5 mL), and then the resultant solutions were respectively added into four 10 mL polymerization tubes. The tubes were sealed under vacuum after three freeze−evacuate−thaw cycles. Then the polymerizations were conducted at 70 °C while stirring for 24 h. After rapid cooling to room temperature, the reaction mediums were added dropwise into the excess diethyl ether to obtain a precipitate. The precipitation processes were repeated for three times. The final products were obtained by drying the precipitates under vacuum for 24 h. Fabrication of Core-Cross-Linked Prodrug NPs with Controllable Drug Content. A typical procedure of the RAFT dispersion polymerization for fabrication of the prodrug NPs is as follows: PEGb-P(MEO2MA-co-CPTM) (145 mg, 0.01 mmol), AIBN (0.328 mg, 2 × 10−3 mmol), BzMA (70 mg, 0.40 mmol), CBMA (29 mg, 0.10 mmol), and ethanol/water (2.44 g, the mass ratio of 7/3) were added into a 5 mL glass tube with a magnetic bar. The tube was sealed under vacuum after three freeze−evacuate−thaw cycles. After the polymerization was conducted at 70 °C for 24 h, the reaction was quenched by rapidly cooling to room temperature. For the RAFT dispersion polymerizations using other three macro-CTAs, the same procedures, the same total solids content (10%), and the same molar ratio of AIBN/PEG-b-P(MEO2MA-co-CPTM) (1/5) were used.

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RESULTS AND DISCUSSION Preparation and Characterization of Prodrug Diblock Copolymer PEG-b-P(MEO2MA-co-CPTM). To avoid destruction of the dithiobenzoate during the reaction between PEG113-NH2 and 4-cyano-4-((phenylcarbono-thioyl)thio) pentanoic acid (CPDB), a feasible and highly efficient method is the reaction of succinimide activated ester with amino group of PEG113-NH2 at mild condition.42 Thus, the first step is the synthesis of the succinimide-modified cyanopentanoate dithioC

DOI: 10.1021/acs.biomac.6b00819 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules benzoate (SCPDB) by the esterification reaction of Nhydroxysuccinimide and CPDB. The 1H NMR spectrum of the SCPDB is shown in Figure S1. The aromatic proton signals appear at δ = 7.3−8.0 ppm (signals d−f). Signals b (δ = 2.5− 2.8 ppm, 3.0 ppm) and c (δ = 1.9 ppm) are, respectively, attributed to the methylene protons of pentanoic acid and methyl protons adjacent to the nitrile group. The signal at δ 2.9 ppm (a) is assigned to the methylene protons of succinimide, and quantification ratios of the signals’ integral values are in accordance with the structures of SCPDB. All these data support successful preparation of the SCPDB. Then the PEG113-CPDB macro-RAFT agent was prepared by the reaction of SCPDB with the methoxypolyethylene glycol amine (PEG113-NH2), which has been proved to be a superior method for synthesis of the PEG113-CPDB owing to high extent of end-group modification.42,43 Figure 1 shows the 1H NMR spectrum of the resultant PEG113-CPDB. The proton signals at δ = 7.3−8.0 ppm (f−h), 2.3−2.7 ppm (d), and 1.9 ppm (e) are, respectively, attributed to the aromatic protons, two methylene protons, and methyl protons of the CPDB unit; the ethylene and methyl proton signals of the PEG113 appear at δ = 3.67 (b) and 3.4 ppm (a), respectively. The signal at δ = 6.5 ppm (c) is ascribed to the amido proton, so, the amidation reaction between the N-hydroxysuccinimide-activated ester and the amino group of PEG113-NH2 is successful. Based on integral values of the aromatic proton signals (f−h) and the PEG proton signal (b), an average degree of the dithiobenzoate functionalization is 96%, and the highly capping efficiency is consistent with the previous reports.42,43 Camptothecin (CPT), which is isolated from Chinese Camptotheca acuminata (happy tree), was selected as the model drug due to its significant anticancer activity. For preparation of the reductive-sensitive prodrug through copolymerization, the first step is synthesis of the CPTcontained monomer with disulfide (CPTM), which was synthesized according to the procedure described in the literature.44 All proton signals of the CPTM are marked in the 1H NMR spectrum of Figure S2, which supports that synthesis of the CPTM is successful. The prodrug diblock copolymer, PEG-b-P(MEO2MA-coCPTM) was synthesized by copolymerization of CPTM and MEO2MA using PEG113-CPDB as macro-RAFT agent, as shown in Scheme S1. The drug content in the diblock copolymers can be modulated by the feed molar ratio of CPTM/MEO2MA. Herein, the feed molar ratios of CPTM/ MEO2MA = 1/2, 1/4 and 1/8 with a constant total molar content were used to prepare the prodrug diblock copolymers. Figure 2 shows the 1H NMR spectra of the resultant PEG-bP(MEO2MA-co-CPTM)s. The proton signal at δ = 4.1 ppm (n), which is assigned to the ester methylene of MEO2MA, increases clearly with the feed molar ratio of CPTM/MEO2MA decreasing from 1/2 to 1/4 and to 1/8. The actual molar ratio of CPTM/MEO2MA in the PEG-b-P(MEO2MA-co-CPTM) were calculated according to the 1H NMR spectra by comparing the integral values of the ester proton signal (l) of the CPTM units to that of the ester proton signal (n) of the MEO2MA units, and the results are listed in Table 1. It can be seen that the actual molar ratios of CPTM/MEO2MA in the resultant PEG-b-P(MEO2MA-co-CPTM) are approximately consistent with the feed molar ratio, so, the drug content in the prodrug can be controlled by the feed molar ratio of CPTM/MEO2MA. When the feed molar ratios of CPTM/ MEO2MA increase from 1/8 to 1/4 and to 1/2, the resultant

Figure 2. 1H NMR spectra of prodrug diblock copolymers, PEG-bP(MEO2MA-co-CPTM) prepared from the copolymerizations with various feed molar ratios of (A) CPTM/MEO2MA = 1/2, (B) CPTM/ MEO2MA = 1/4, (C) CPTM/MEO2MA = 1/8.

CPT contents in the PEG-b-P(MEO2MA-co-CPTM) increase from 13.4 to 22.7%, and then to 36.3% (Table 1). GPC traces of the PEG-b-P(MEO2MA-co-CPTM)s shown in Figure 3 reveal that the three prodrug macro-CTAs with different drug contents have similar molecular weights. For the sake of brevity, PEG-b-P(MEO2MA-co-CPTM)s synthesized with the feed molar ratios of CPTM/MEO2MA = 1/8, 1/4, and 1/2 are simply denoted as macro-CTA1, macro-CTA2, and macroCTA3, respectively. Fabrication of Prodrug NPs with Controllable Drug Content and Superior Structural Stability. The core-crosslinked prodrug NPs were prepared by RAFT dispersion polymerization of CBMA and BzMA in a mixture of ethanol and water. The first step is synthesis of cross-linker CBMA, which was synthesized according to our previous reported method,45 and its structure was characterized by 1H NMR. Successful synthesis of CBMA is supported by the 1H NMR spectrum (Figure S3): vinyl proton signals appear at δ = 5.45 and 5.72 ppm (b); ethylene proton signals next to imide nitrogen and adjacent to sulfur, respectively, appear at 3.65 (d) and 2.92 (e) ppm; methyl proton signal appears at 1.95 ppm (a). Then all three macro-CTAs (macro-CTA1, macro-CTA2, and macro-CTA3) were used to fabricate prodrug NPs via onepot RAFT dispersion polymerization of BzMA/CBMA with a feed molar ratio of 8/2 (Scheme 1). A mixture of ethanol/water with a mass ratio of 7/3 was used as the reaction media. Both of the monomers (BzMA and CBMA) and the macro-CTA (PEGb-P(MEO2MA-co-CPTM)) are well soluble in the reaction media, but the in situ formed P(BzMA-co-CBMA) by polymerization has poor solubility in the mixture. So the PISA occurs in this system. Because the drug was conjugated on the macro-CTA, it is envisioned that drug loading was achieved during the NPs formation. When the RAFT dispersion polymerizations were, respectively, conducted using macroD

DOI: 10.1021/acs.biomac.6b00819 Biomacromolecules XXXX, XXX, XXX−XXX

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Table 1. Summary of the Feed Molar Ratio of CPTM/MEO2MA for Preparation of Prodrug Macro-CTAs, the Actual Molar Ratio of CPTM/MEO2MA, the CPT Content in the Resultant Macro-CTAs, the CPT Content in the Resultant Prodrug NPs, and the Diameter/PDI of the Resultant Prodrug NPs samplea prodrug NPs-1 prodrug NPs-2 prodrug NPs-3 NPs-4

feed molar ratio of CPTM/MEO2MA

molar ratio of CPTM/MEO2MA in the macro-CTAs

CPT content in the prodrug macro-CTAs/%

CPT content in the prodrug NPs/%

diameter/nmb (PDI)

1/8

1/9.02

13.4

4.2

36.3 (0.119)

1/4

1/4.65

22.7

7.4

37.2 (0.111)

1/2

1/2.32

36.3

12.8

36.8 (0.095)

0/1

0

0

0

36.6 (0.087)

a

The prodrug NPs-1, -2, and -3 were, respectively, fabricated using macro-CTA1, macro-CTA2, and macro-CTA3. NPs-4 are drug-free spherical micelles that were fabricated using PEG-b-PMEO2MA (macro-CTA4). bThe intensity-average diameter and polydispersity of the NPs determined by DLS.

shown in Figure S5. The morphology of prodrug NPs in THF still remained very well (Figure S5), which indicates superior structural stability of the prodrug NPs due to the core-crosslinking during the PISA. DLS diameters of the prodrug NPs in THF are nearly twice as big as them in water (Figure S6), which indicates that the core of prodrug NPs are swollen instead of dissolving in THF due to the core-cross-linking structure. All the above results verify that the prodrug NPs with robust structure are efficiently fabricated via PISA strategy and the drug contents in the resultant prodrug NPs can be accurately modulated via adjusting the feed molar ratio of MEO2MA/CPTM during preparation of the prodrug macroCTA (PEG-b-(PMEO2MA-co-CPTM)). Redox-Regulated Drug Release. The proposed release mechanism of parent drug CPT from the NPs upon reductive milieu is shown in Scheme 2.44 In vitro drug release experiments for the obtained prodrug NPs with different drug contents were performed at various concentrations of the GSH (0.01, 5, and 10 mM). High performance liquid chromatography (HPLC) was used to characterize the released drug and the pure CPT. Figure S7 shows that the pure CPT and the released drug from NPs-3 have the same retention time, which illustrates that the released CPT has the same structure of its parent CPT. The cumulative CPT release was evaluated using the fluorescence spectrometer. In the presence of 0.01 mM of GSH, less than 5% cumulative CPT was released upon incubation for 48 h, as shown in Figures 5 and S8, which reveals a minimal premature drug release during blood circulation. After incubation for 48 h in the presence of 5 or 10 mM GSH, higher cumulative CPT release was achieved: exceeding 35% CPT release for 5 mM GSH and 42% for 10 mM GSH (Figures 5 and S8), which is reasonable because the cleavage rate of the disulfide bond increases with concentration

Figure 3. GPC traces of the PEG-CPDB (black line) and the PEG-bP(MEO2MA-co-CPTM) fabricated with various feed molar ratios of CPTM/MEO2MA: 1/8 (blue line), 1/4 (green line), and 1/2 (red line).

CTA1, macro-CTA2, and macro-CTA3, almost complete monomer conversions (>99%) were achieved within 24 h of polymerization, as judged by 1H NMR spectra (the vinyl proton signals of the monomers are almost invisible, the data are not shown), and NPs with the similar diameter were obtained as characterized by DLS and TEM (Table 1 and Figure 4). Due to the superior hydrophilic PEG blocks on the

Figure 4. TEM images of the prodrug NPs fabricated by RAFT dispersion polymerizations using different PEG-b-(PMEO2MA-coCPTM)s as the macro-CTA: (A) macro-CTA1; (B) macro-CTA2; (C) macro-CTA3.

shell, these nanoparticles also dispersed well in water. TEM images in Figure S4 show that shape and dispersity of the nanoparticles are not affected by their transfer from ethanol/ water (7/3) to pure water. The parent CPT content in the resultant prodrug NPs are modulated by using different macroCTAs, which can be calculated to be 4.2, 7.4, and 12.8% when the macro-CTA1, macro-CTA2, and macro-CTA3 are used, respectively. TEM images of the prodrug NPs in tetrahydrofuran (THF, which is a good solvent for all of the blocks) are

Figure 5. In vitro CPT release profiles of the prodrug NP-3 at various concentrations of the GSH. E

DOI: 10.1021/acs.biomac.6b00819 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 6. In vitro cytotoxicity of the NPs with different concentrations of the NPs: (A) the drug-free NPs (fabricated using macro-CTA4), (B) the NPs fabricated using prodrug macro-CTAs (black line: macro-CTA1, red line: macro-CTA2, green line: macro-CTA3).

to evaluate cytotoxicity of the NPs directly after the polymerization without any further purification procedures. Figure 6A shows that the NPs fabricated using macro-CTA4 are almost noncytotoxic up to a concentration of 1000 μg/mL, which is in accordance with the well-recognized biocompatibility of PEG.5,6 Meanwhile, the above results indicate that the ethanol used in the PISA has negligible cytotoxicity due to the very low content after high dilution (Note: the solid content of the obtained NPs is 100 mg/mL, and the highest NP concentration for cytotoxicity evaluation is 1 mg/mL, so, at least 100 times dilution of the obtained NP dispersions by the PBS buffer is necessary for the cytotoxicity evaluation). The prodrug NPs, which were directly obtained from the RAFT dispersion polymerization using the macro-CTA1, the macroCTA2, and the macro-CTA3 without any further purification procedure, were added to HeLa cells, and the results are shown in Figure 6B. Dramatically decreased cell viability was observed as the concentration increasing of all the three prodrug NPs (Figure 6B). Because of the three prodrug NPs having similar diameter (Table 1 and Figure 4), influence of the NPs size on their anticancer activities can be ignored. The IC50s (inhibitory concentration for 50% cell viability) of the three prodrug NPs are 145 μg/mL for prodrug NPs-1 (equiv 6.09 μg/mL of CPT), 85 μg/mL for prodrug NPs-2 (equiv 6.29 μg/mL of CPT), and 50 μg/mL for prodrug NPs-3 (equiv 6.40 μg/mL of CPT), respectively; thus, the cytotoxicity of three prodrug NPs has the following order: prodrug NPs-1 (CPT content: 4.2%) < prodrug NPs-2 (7.4%) < prodrug NPs-3 (12.8%), which is the same order with increase of the CPT contents. However, the CPT doses at the IC50 are very close to each other (6.09−6.40 μg/mL) for the three prodrug NPs, which further indicate that the cytotoxicity are majorly induced by CPT, and the NPs with high CPT content release more CPT molecules in the same duration resulting in high cytotoxicity, that is, they exhibit more excellent anticancer efficiency. So, the less prodrug NPs are needed to kill the HeLa cells, and less polymeric matrix are remained after drug delivery, which is more appreciate for cancer chemotherapy concerning the long-term toxicity of polymeric nanocarriers in the organism. The above results reveal that all the three prodrug NPs exhibit excellent performance of intracellular drug release. To further investigate the intracellular drug release behaviors of the prodrug NPs, confocal laser scanning microscopy (CLSM) was used to study the internalization of the prodrug NPs by HeLa cells. The CPT emission was used directly to visualize cellular uptake of the NPs without additional fluorescence labeling. So the amount of CPT-conjugated NPs internalized by HeLa cells should in principle be proportional to the fluorescence intensity. The three prodrug NPs with

increase of the GSH. All three CPT release profiles in Figures 5 and S8 display two stages of the CPT release: faster release rate within an initial 5−7 h and then the release rate decreasing, such as, for the drug release of prodrug NPs-3 in the presence of 10 mM GSH, the release rates before and after 7 h are 36.7 and 7.5 μg/h, respectively. Thus, upon cell internalization, the drug release can be efficiently enhanced subjecting to high concentration of GSH. As previous reports, 19,40 high concentration of GSH also induced cleavage of the reductivesensitive cross-linker and then dissociation of the NPs. However, due to the hydrophobic cores of the NPs herein, the GSH is difficult to diffuse into the cores of the NPs in aqueous solution leading to very slow degradation of the core cross-linker. When the degradation of NPs-3 was conducted in a mixed solvent of THF/water (9/1, v/v, a little of water is used to dissolve the GSH), which is a good solvent for both of the blocks in the NPs. The degradation of NPs was observed, and the diameter of the NPs-3 reduced from 67.1 to 4.3 nm (detected by LLS) after stirring for 24 h in the presence of 10 mM GSH. This is because the GSH is easy to diffuse into the swollen core of NPs resulted in fast dissociation of the NPs. It is reasonable to speculate that, in the high dilution environment, such as in body fluids, the GSH can also diffuse into the core of NPs and induced the degradation of them. Cytotoxicity Evaluation. In vitro cytotoxicity was evaluated against HeLa cells using MTT assay. Before studying cytotoxicity of the prodrug NPs, it is necessary to evaluate the cytotoxicity of the nanocarriers without drug conjugation because well-biocompatible nanocarriers are highly desired for reducing the side effect in drug delivery. The macro-CTA4 without CPT-conjugation was synthesized by RAFT polymerization of MEO2MA using PEG-CPDB as the macro RAFT agent. 1H NMR spectrum of the resultant polymer shown in Figure S9 demonstrates that successful chain extension of the PEG-CPDB was realized to get diblock copolymer PEG-bPMEO2MA (macro-CTA4). As previously reported,28 the molecular weight of macro-CTA has significant influence on the size of the NPs fabricated through PISA strategy. For fabricating nanocarriers (without drug conjugation) with similar size to the above-mentioned prodrug NPs, it is expected to get macro-CTA4 with the molecular weight similar to the other three macro-CTAs. For this purpose, the same polymerization conditions were conducted. Macro-CTA4 with Mn = 15600 was obtained (Figure S10), which is approximately equal to the other three macro-CTAs (Figure 3). As expected, the NPs fabricated using macro-CTA4 have similar size to the other three prodrug NPs (Table 1, Figures 4 and S11), which is reasonable owing to the similar molecular weights of the macroCTAs and the core-forming block. Then MTT assay was used F

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Biomacromolecules equivalent CPT content (10 μg/mL) were used to incubate HeLa cells. As expected, no obvious difference on cell internalization of the three prodrug NPs at all the incubation time (4, 8, and 16 h) were observed due to similar size of the NPs (Figure 7). After incubation with the prodrug NPs for 4 h,

efficient approach for fabrication of the polymeric drug delivery systems in cancer chemotherapy.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b00819. 1 H NMR of the succinimide-modified cyanopentanoate dithiobenzoate (SCPDB), reduction-cleavable camptothecin (CPT) prodrug monomer (CPTM), cross-linker (CBMA), and the drug-free macro-CTA PEG-bPMEO2MA, DLS of the prodrug NPs, a part of TEM images, GPC trace, drug release profiles (Figures S1− S11), and the scheme of synthesize prodrug macro-CTA PEG-b-P(MEO2MA-co-CPTM) (Scheme S1; PDF).

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Figure 7. CLSM images recorded for HeLa cells after incubation for 4, 8, and 16 h with the (A) prodrug NPs-1, (B) prodrug NPs-2, and (C) prodrug NPs-3.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

only weak blue fluorescence was observed, but the fluorescence intensity remarkably increased after 8 h of incubation, which reveals excellent cell internalization of the prodrug NPs. It is crucial for parent CPT to be released from the prodrug NPs and then the released CPT molecules are accumulated in the cell nucleus for anticancer activity via interaction with genomic DNA to induce pharmacological responses. Significant blue fluorescence is observed within the nucleus of HeLa cells after incubation of 16 h with all the three prodrug NPs, which indicates that the highly reductive intracellular microenvironment should be responsible for the efficient CPT cleavage from the prodrug NPs and nuclear accumulation of the drugs (Figure 7). Thus, we report a superior strategy of fabricating the prodrug NPs for effectively anticancer chemotherapy.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China under Contract Nos. 21525420 and 21374107, the Fundamental Research Funds for the Central Universities (WK 2060200012 and WK 2060200015), and China Postdoctoral Science Foundation (BH2060000011).

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REFERENCES

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CONCLUSION Reductive-responsive prodrug NPs with controllable drug content and superior structural stability have been fabricated through the PISA strategy. In one-pot RAFT dispersion polymerization at relatively high concentration (100 mg/mL), polymerization, self-assembly, and drug encapsulation are simultaneously realized, while almost complete monomer conversion can be achieved, which allows direct application of the resultant prodrug NPs without further purification, this is beneficial for commercialization becuase the purification procedures are always time-consuming and uneconomic. The core-cross-linked prodrug NPs are obtained just by use of the cross-linker as comonomer in the polymerization, no further postpolymerization procedures are needed. The drug content in the resultant prodrug NPs can be accurately modulated by adjusting the feed molar ratio of MEO2MA/CPTM in preparation of the PEG-b-P(MEO2MA-co-CPTM). Cell viability studies reveal that the NPs without CPT conjugation are almost nontoxic, whereas CPT-conjugated NPs exhibit remarkably high cytotoxicity due to the reductive-responsive release of CPT. Compare to the lower CPT content NPs, the NPs with higher drug content exhibit more excellent anticancer efficiency, this will be beneficial to reduce the long-term biological toxicity because less polymeric matrix remains after effectively killing HeLa cells. Therefore, this study provides an G

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DOI: 10.1021/acs.biomac.6b00819 Biomacromolecules XXXX, XXX, XXX−XXX