Efficient Fabrication of Photosensitive Polymeric Nano-objects via an

Mar 13, 2017 - An ingenious formulation of RAFT dispersion polymerization based on photosensitive monomers of 2-nitrobenzyl methacrylate (NBMA) and ...
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Efficient Fabrication of Photosensitive Polymeric Nano-objects via an Ingenious Formulation of RAFT Dispersion Polymerization and Their Application 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: An ingenious formulation of RAFT dispersion polymerization based on photosensitive monomers of 2-nitrobenzyl methacrylate (NBMA) and 7-(2methacryloyloxy-ethoxy)-4-methyl-coumarin (CMA) is reported herein. Various morphologies, such as spherical micelle, nanoworm, lamella, and vesicle, are fabricated at up to 20% solids content. Photoinduced cleavage of the NBMA moieties and dimerization of the coumarin moieties are simultaneously triggered upon UV (365 nm) irradiation. The former endows the cores of the nano-objects with abundant carboxyl groups, resulting in the transformation of the hydrophobic cores to hydrophilic ones. The latter induces the core-cross-linking of the nanoobjects, which endows the nano-objects with enhanced structural stability and prevents the nanoparticle-to-unimer disassembly. The resultant nano-objects exhibit superior structural stability and excellent performances for drug delivery, including high drug loadings, pH-stimuli release, and high-efficient endosomal escape.

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INTRODUCTION In recent years, polymerization-induced self-assembly and reorganization (PISR) has become a powerful strategy for fabrication of polymeric nano-objects with predictable morphologies. PISR strategy, which combines polymerization and self-assembly in one-pot, efficiently simplifies the fabrication of polymeric nano-objects and is conducted at high concentrations, making their realistic production in largescale possible.1−7 Although several living polymerization techniques have been reported for PISR,8−10 reversible addition−fragmentation chain transfer (RAFT) polymerization is chosen in the majority of literature examples,11−18 in which a solvophilic macromolecular chain transfer agent is used as a polymerization control agent and stabilizer. The chemical nature of the stabilizer dictates the surface chemistry, and even influences the stability of the nano-objects.2−4 A wide range of polymeric nano-objects with various functional stabilizers at surfaces have been fabricated in the past years,19−30 which are receiving increasing attention in a broad range of materials applications. Potential applications of the polymeric nano-objects in the field of drug delivery have come into particular focus recently. The site-specific drug release at the pathological site is highly desirable for cancer chemotherapy. If triggered drug release can be achieved in response to the specific microenvironmental conditions in tumors, improving drug bioavailability and decreasing side effects will be realized. It is well-known that the microenvironments in endosomes−lysosomes compartments and extracellular fluids of tumor tissue are mildly acidic.31 Thus, many drug release systems triggered by mildly © XXXX American Chemical Society

acidic media have been designed for pathological site-specific drug release.32−35 In addition, the structural integrity of the nanovehicles is also an important concern for their practical applications as drug nanocarriers. The nanocarriers are subjected to high dilution and larger shear force upon intravenous injection, which may result in dissociation of the polymeric nanocarriers simultaneously with the undesirable release of the drugs before reaching the pathological site. Shell cross-linking and core cross-linking strategies have been reported to enhance the structural integrity of the nanocarriers.36−40 However, fabrication of the intelligent polymeric nanovehicles is usually low efficient via the traditional self-assembly strategy. Such self-assembly is usually conducted at relatively high dilution (the copolymer concentration is typically less than 1%), which severely limits the scaled-up production of the polymeric nano-objects.41−44 The recently emerging polymerization-induced self-assembly and reorganization strategy may potentially solve this problem due to the one-step polymerization to generate nano-objects with controllable morphologies at high concentrations.1−7 Stimuli-responsive morphology transitions of the PISR-generated polymeric nano-objects, due to the adjustable volume fractions of the core and stabilizer blocks, are reported by several groups.45−55 Recently, guest cargos are encapsulated into the vesicles during the process of PISR, and controlled release is realized during the thermoresReceived: December 20, 2016 Revised: February 24, 2017

A

DOI: 10.1021/acs.biomac.6b01887 Biomacromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Fabrication of Photolabile Polymeric Nano-objects by Polymerization-Induced Self-Assembly and Reorganization (PISR)

Scheme 2. (A) (a) UV-triggered concurrent core cross-linking and decomposing of the nano-objects; (b) electrostatic interaction-induced encapsulation of DOX with high drug loadings; (c) pH-triggered drug release due to protonation of the nanocarriers, which breaks the electrostatic interaction. (B) Proposed mechanism of high-efficient endosomal escape of the DOX-loaded nano-objects via protonation of the nanocarriers in the acidic endosome.

ponsive vesicles−spheres transitions.56,57 While both postpolymerization and in situ cross-linking strategies have been reported to improve the structural stability of PISR-generated nano-objects,58−64 cross-linking always hinders the morphology transition and thus prevents the release of guest cargos. Although the field of PISR is rapidly growing, the PISRgenerated drug delivery systems are less reported,65 presumably due to the special requirement for a soluble monomer that produces an insoluble polymer. In practice, relatively few monomers are amenable to the above criterion. Therefore, developing new dispersion polymerization formulations for PISR is essential to expand the scope of this unique approach. Efficient preparation of intelligent polymeric nanocarriers is highly desirable for nanomedicine. Herein, we reported a new dispersion polymerization formulation based on the photosensitive monomers 2-nitrobenzyl methacrylate (NBMA) and 7-(2-methacryloyloxyethoxy)-4-methyl-coumarin (CMA) to fabricate polymeric nano-objects (Scheme 1), which exhibit excellent performances in drug delivery after postpolymerization photoirriadiation, such as pH-responsive release, robust structures, remarkably high drug loadings, and rapid endosomal escape rate (Scheme 2). Four types of nano-objects, spherical micelle, nanoworm, lamella, and vesicle, were fabricated simultaneously with the polymerization at up to 20% solids content. Upon UV-

irradiation, dimerization of the coumarin moieties resulted in core-cross-linking of the nano-objects, which endows the nanoobjects with robust structures. Unlike the previous reports36−40 that cross-linking usually leads to compromised membrane permeability, the membrane permeabilities of the nano-objects herein are enhanced due to the simultaneously cleavage of the NBMA moieties upon UV-irradiation, which induces the formation of abundant carboxyl groups and thus gives rise to the hydrophobic-to-hydrophilic transitions of the core-forming blocks. Due to the core-cross-linking structures, the double hydrophilic block copolymer nano-objects did not conduct the nanoparticle-to-unimer disassembly as identified by TEM. The anticancer drug DOX was efficiently encapsulated into the core of the nano-objects by the electrostatic interaction between −COO− and −NH3+ groups with remarkably high drug loading content (up to 24.9 wt %) and drug loading efficiency (up to 82.9%). Intracellular drug delivery experiments show that the DOX-loaded nanocarriers exhibit rapid release of DOX into the cell nucleus and effective anticancer activity.

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EXPERIMENTAL SECTION

Materials. Doxorubicin hydrochloride (DOX, 98%) was obtained from adamas-beta. Methacryloyl chloride (95%), triethylamine (99%), 2-nitrobenzyl alcohol (≥98%), Dulbecco’s modified Eagle’s medium (DMEM, Hyclone), and fetal bovine serum (FBS, Hyclone) were purchased from Aladdin and used as received. N-(2-Hydroxypropyl) B

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Encapsulation of DOX into the Nano-objects. The encapsulation of drugs into the four types of nano-objects was carried out as follows. The powders of the nano-objects (10 mg) were mixed with DOX·HCl (3 mg) in distilled water (3 mL) and then stirred at room temperature overnight. The unloaded drugs were removed by dialysis against distilled water. The water was renewed every 0.5 h until the fluorescence of DOX outside the dialysis tube was negligible. The whole procedure was conducted in the dark. The drug loading efficiency (DLE) and drug loading content (DLC) in the nano-objects were determined by the fluorescence spectroscopy of the nano-objects dispersions before and after dialysis and calculated using the following equations.

methacrylamide (HPMA), 7-(2-hydroxyethoxy)-4-methylcoumarin, and 4-(4-cyanopentanoic acid) dithiobenzoate (CPDB) were synthesized according to the previous reports.66−68 All other reagents of analytical grade (Shanghai Chemical Reagents Co.) were used as received. Synthesis of the Macro RAFT Agent Poly(N-(2hydroxypropyl)methacrylamide) (PHPMA). HPMA (10.0 g, 70 mmol), CPDB (279 mg, 1 mmol), AIBN (33 mg, 0.2 mmol), and ethanol (20 mL) were mixed in a 50 mL polymerization tube. The tube was sealed under high vacuum after three freeze−evacuate−thaw cycles, and then the tube was placed in a preheated oil bath at 70 °C while stirring. After polymerization for 20 h, the reaction mixture was cooled to room temperature, and then diluted with ethanol. The mixture was precipitated into excess diethyl ether while stirring. The above precipitation procedure was repeated for three times. The precipitate was collected by filtration, and then dried under a vacuum oven at room temperature overnight. PHPMA was obtained as a red solid of 2.4 g. Synthesis of 7-(2-Methacryloyloxyethoxy)-4-methylcoumarin (CMA). 7-(2-Hydroxyethoxy)-4-methylcoumarin (4.4 g, 20.0 mmol) and triethylamine (4.0 g, 40.0 mmol) were mixed with CH2Cl2 (150 mL). The mixture was cooled in an ice−water bath while stirring for 15 min. Methacryloyl chloride (4.2 g, 40.0 mmol) diluted with CH2Cl2 (30 mL) was slowly added into the mixture in 1 h. The reaction was allowed to proceed for further 24 h at room temperature. The solution was diluted with 200 mL of dichloromethane and then washed with distilled water three times (3 × 100 mL). The organic phase was dried over anhydrous MgSO4 overnight. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography using CHCl3 as the running solvent to afford white powder in 85% yield. Synthesis of 2-Nitrobenzyl Methacrylate (NBMA). 2-Nitrobenzenemethanol (15.3 g, 0.1 mol), triethylamine (12.1 g, 0.12 mol), and CH2Cl2 (80 mL) were mixed and then stirred in an ice−water bath for 15 min. Methacryloyl chloride (12.5 g, 0.12 mol) diluted with CH2Cl2 (30 mL) was added into the mixture over 1 h, and then the mixture was reacted at room temperature for 24 h. After filtering the triethylammonium salts, the solvent (CH2Cl2) was removed by rotary evaporation. The crude product was purified by column chromatography using ethyl acetate/hexane (1/6, v/v) as the running solvent. The final product was obtained as a greenish-yellow oil in 87% yield after removing the solvent. RAFT Dispersion Copolymerization of NBMA and CMA Using PHPMA as Macro RAFT Agent. A typical procedure of the RAFT dispersion copolymerization at 15% solids content is as follows: PHPMA26 (40 mg, 10−2 mmol), NBMA (177 mg, 0.8 mmol), CMA (58 mg, 0.2 mmol), AIBN (0.33 mg, 2 × 10−3 mmol), and ethanol (1.83 g) were added into a glass tube with a magnetic bar. The glass tube was sealed under vacuum after being degassed by three freeze− pump−thaw cycles, and then the sealed tube was placed in an oil bath at 70 °C while stirring. The polymerization was quenched after 24 h. For the sake of description convenience, the above sample is simply denoted as H26N100-15%, wherein H26 denotes the PHPMA26, N100 represents the targeting degree of polymerization (DP) of P(NBMAco-CMA) is 100, and the number after the short line denotes the total solids concentration (15% in this case). For other samples listed in Table S1, the same procedure of the RAFT dispersion polymerization was conducted besides varying the total solids concentration and the target DP of P(NBMA-co-CMA). The molar ratio of AIBN/PHPMA26 remained 1/5 for all the RAFT dispersion polymerizations. Photoinduced Dimerization of CMA and Decomposition of NBMA in the Nano-objects. Parts of the obtained nano-objects (spherical micelles, nanoworms, lamellae, and vesicles) were diluted to 50 mg/mL with distilled water and then irradiated with UV (365 nm, 0.1 mW/cm2 and 1 cm from the lamp to sample) for 2 h with the exception of spherical micelles for 1 h. The resultant mixtures were dialyzed against ethanol for 2 days to remove the decomposed products of NBMA, and then dialyzed against distilled water for removal of ethanol. The final products were obtained by lyophilization.

DLE (%) =

weight of drug encapsuled in the nano‐objects weight of the drug in feed × 100%

DLC (%) =

weight of drug encapsuled in the nano‐objects weight of nano‐objects × 100%

In Vitro pH-Regulated Drug Release. The pH-regulated drug release was conducted according to the following protocol. The dispersion of DOX-loaded nano-objects was divided into six parts, and each part was transferred into a new dialysis tube (molecular weight cutoff: 3500 Da) immediately. The drug release process was carried out by dialyzing the dispersions of DOX-loaded nano-objects in the dialysis tube against 80 mL of buffer solution at different pH values (20 mM; pH 7.4 or pH 5.4) under gentle stirring. An aliquot of the external buffer solution (2 mL) was removed and replaced with an equal volume of fresh media periodically. The amount of the DOX released from the nano-objects was quantified based on the fluorescence emission at 595 nm against a standard calibration curve. The drug release experiment at each pH value was done in triplicate, and the data are shown as the mean value plus a standard deviation (±SD). In Vitro Cytotoxicity Assay. HeLa cells were chosen to evaluate the in vitro cytotoxicity via the MTT assay. The cells were first cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 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 (8000 cells/well) in 200 μL of complete DMEM and incubated at 37 °C in CO2/air (5:95) for 24 h. The media was replaced by fresh DMEM containing the drug-loaded or drug-free nano-objects with different concentrations. After 48 h of incubation, the MTT reagent (in 20 μL PBS, 5 mg/mL) was added to each well. The cell was further incubated for 4 h, and then the medium in each well was removed and replaced by 100 μL of dimethyl sulfoxide (DMSO). The plate was gently agitated for 15 min, and then the cell viability was measured by reading the absorbance at 570 nm using a microplate reader. Each experiment was conducted in quadruplicate, and the data are expressed as the mean value plus standard deviation (±SD). Cellular Uptake and Intracellular Drug Delivery. The HeLa cells were seeded in a 4-well cell culture plate (3 × 104 cells/well) in 300 μL of DMEM and cultured in DMEM supplemented 10% FBS in a CO2/air (5:95) incubator at 37 °C for 24 h. The four types of DOXloaded nano-objects were added at a final DOX concentration of 3 μg/ mL. After incubation for 1 and 3 h, the cells were washed with phosphate-buffered saline (PBS) for three times. Then the cells were visualized with an LSM510 confocal laser scanning microscope. Characterization. The 1H NMR spectra were carried out on a Bruker DMX300 spectrometer (resonance frequency of 300 MHz), and DMSO-d6, CDCl3, and CD3OD were used as the solvent. The molecular weight (Mn) and Mw/Mn were determined with a Waters 150C gel permeation chromatograph (GPC) equipped with two Ultrastyragel columns in series and an RI 2414 detector (set at 30 °C); DMF was used as eluent with a flow rate of 1.0 mL/min, and a series of monodispersed polystyrene standards with molecular weights ranging from 800 to 400000 g/mol were used in the calibration. C

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Table 1. Molecular and Morphological Characterization of PHPMA26-P(NBMA-co-CMA)x Prepared by RAFT Dispersion Polymerization, and the Summary of Final Monomer Conversions of the RAFT Dispersion Polymerization with Various of the Targeting DP of P(NBMA-co-CMA) and the Total Solids Content Sample codea

Conversion (%)b

DPb

Mn,theory (g/mol)b

Mn,GPC (g/mol)c

Mw/Mnc

D (nm)d

PDId

Morphologye

H26N20-10 H26N30-10 H26N40-10 H26N60-10 H26N80-10 H26N100-10 H26N20-15 H26N30-15 H26N40-15 H26N60-15 H26N80-15 H26N100-15 H26N20-20 H26N30-20 H26N40-20 H26N60-20 H26N80-20 H26N100-20

96.1 95.4 90.4 85.4 80.8 76.0 92.5 91.2 89.3 78.1 76.2 73.1 94.9 92.8 90.5 82.2 78.6 76.0

19 29 36 51 65 76 19 27 36 47 61 73 19 28 36 49 63 76

8500 10800 12500 16000 19300 21900 8500 10400 12500 15100 18300 21200 8500 10400 12500 15500 18800 21900

6700 8700 10500 14200 17300 26700 6600 8600 10400 13900 17800 28300 6500 8700 10200 14800 19700 26900

1.31 1.41 1.44 1.49 1.56 1.77 1.29 1.34 1.39 1.45 1.62 1.85 1.32 1.40 1.48 1.54 1.65 1.83

25.6 91.1 128.5 202.8 480.34 177.8 32.1 119.3 237.9 146.5 505.9 178.8 93.9 130.1 150.4 168.2 379.4 186.4

0.08 0.12 0.17 0.20 0.28 0.13 0.09 0.16 0.20 0.11 0.34 0.20 0.18 0.21 0.15 0.19 0.32 0.14

S NW NW NW, L La, V V S NW NW, La La La, V V NW NW La La La, V V

a

A brief notation of H26Nx-y was used for each sample, wherein H26 denotes PHPMA26, N represents P(NBMA-co-CMA), x is the targeting DP of P(NBMA-co-CMA), and y represents the total solids content. bThe monomer conversion, the DP of P(NBMA-co-CMA), and the Mn,theory of PHPMA-P(NBMA-co-CMA) were calculated according to the 1H NMR. cThe Mn and Mw/Mn of PHPMA26-P(NBMA-co-CMA)x were obtained by GPC. dThe sphere-equivalent intensity-average diameter and the polydispersity of the nano-objects were determined by LLS. eThe morphology formed in the RAFT dispersion polymerization was identified by TEM, wherein S = spherical micelles, NW = nanoworms, La = lamellae, and V = vesicles.

synthesized at first, and their 1H NMR spectra and UV−vis spectra are shown in Figure S3, Figure S4, and Figure S5, respectively. Then, NBMA and CMA with a molar ratio of 8/2 were used as the comonomers in the RAFT dispersion polymerization. Both NBMA and CMA have good solubility in ethanol at the polymerization temperature (70 °C), but the in situ formed P(NBMA-co-CMA) are ethanol-immiscible, so polymerization induces self-assembly of the copolymer PHPMA-P(NBMA-co-CMA). As in the previous reports,11 the total solid contents and the chain length ratios of the coreforming block to the shell-forming block significantly influenced the final morphology. In this study, DP of the shell-forming block (the macro RAFT agent) remained constant, the total solid contents (10 to 20%, w/w) and DPs of the core-forming P(NBMA-co-CMA) block were systematically varied, and the results are listed in Table 1. As the DP of the P(NBMA-co-CMA) block increases at 15% w/w solids content, spherical micelles (Figure 1A), nanoworms (Figure 1B), the mixture of nanoworms and lamellae (Figure 1C), pure lamellae (Figure 1D), the mixture of lamellae and vesicles (Figure 1E), and pure vesicles (Figure 1F) were successively obtained as shown in the TEM images of Figure 1. A “phase diagram”, which may serve as a “roadmap” for the reproducible fabrication of morphologically pure nano-objects, was constructed as shown in Figure 1G by systematically varying the copolymer solids content and DP of the P(NBMAco-CMA) block (Table 1). The similar morphological transitions from spheres to worms to lamellae to vesicles, along with the mixed phases observed between the four pure phases, were captured at each of the solids contents. At lower solids content, the higher DPs of the core-forming blocks are often required for the morphology evolution (Figure 1G). These phenomena are well in accordance with the previous

The transmission electron microscope (TEM) observations were conducted on a Hitachi H-800 TEM at an accelerating voltage of 100 kV. The samples for TEM were prepared by depositing the dispersion of the nano-objects (10 μL) on copper grids. A dynamic laser light scattering (LLS) spectrometer (Zetasizer Nano ZS90, Malvern Instruments Ltd., Malvern, UK) equipped with a He−Ne laser (4.0 mW, 633 nm) was used to measure the diameter of the nano-objects at 25 °C and a fixed angle of 90°, and all the data were averaged over three measurements. The ζ potentials of the aqueous dispersions of polymeric nano-objects were determined using a Zetasizer Nano-ZS 90 (Malvern Instruments Ltd., Malvern, UK). All the data were averaged over three measurements. UV−vis absorption spectra were obtained with a TU-1901 UV−vis spectrophotometer. Fourier transform infrared (FT-IR) spectra were acquired using a Bruker VECTOR-22 IR spectrometer over 64 scans. A Shimadzu RF-5301PC luminescence spectrometer was used to measure the fluorescence spectra at room temperature, and the slit width for excitation and emission was set at 10 nm. Confocal laser scanning microscopy (CLSM) images were recorded on a Leica TCS SP5 microscope.

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RESULTS AND DISCUSSION Fabrication of Photosensitive Polymeric Nano-objects via RAFT Dispersion Polymerization. Poly(N-(2hydroxypropyl) methacrylamide) was synthesized by RAFT polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) using 4-(4-cyanopentanoic acid) dithiobenzoate (CPDB) as the RAFT agent. Well-defined PHPMA with Mn = 5100 and Mw/Mn = 1.28 was obtained as shown in Figure S1. The DP of PHPMA was calculated to be 26 based on the integration ratio of the methine proton signal (g) of HPMA units and the aromatic proton signals (a, b, and c) in the 1H NMR spectrum of Figure S2. The obtained PHPMA26 was used as macro-RAFT agent in the following RAFT dispersion polymerization. For fabricating photosensitive polymeric nanoobjects, photosensitive monomers CMA and NBMA were D

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further demonstrates successful cleavage of the 2-nitrobenzyl group from the NBMA units. After UV irradiation, the ζ potentials for all four types of nano-objects were decreased by approximately 12 mV (Figure S8), owing to the formation of carboxyl groups via photoinduced cleavage of 2-nitrobenzyl. Therefore, the nano-objects after UV irradiation are mainly composed of the PHPMA-P(MAA-co-CMA). It is reasonable to speculate that the nano-objects without enough core-crosslinking degree will dissociate or partially dissociate in distilled water due to good solubility of the core-forming P(MAA-coCMA) block in aqueous solution. However, the TEM images in Figure 2 show that, after UV irradiation, all four types of nano-

Figure 1. TEM images of the PHPMA 26-P(NBMA-co-CMA)x copolymer nano-objects obtained by RAFT dispersion polymerization: (A) H26N20-15% (S), (B) H26N30-15% (NW), (C) H26N40- 15% (NW and La), (D) H26N60-15% (La), (E) H26N80-15% (La and V), (F) H26N100-15% (V), wherein H26 represents PHPMA26 and N denotes P(NBMA-co-CMA), the number subscript of N denotes the targeting DP of P(NBMA-co-CMA), 15% is the solids content. (G) “Phase diagram” constructed by systematically varying the DP of the P(NBMA-co-CMA) block and the copolymer solids content. S = spherical micelles, NW = nanoworms, La = lamellae, and V = vesicles.

reports, which may be due to the much more frequent inelastic collisions resulting in rapid internanoparticle fusion at higher solids content.11 Photoinduced Concurrent Core Cross-linking and Decomposing of the Photosensitive Polymeric Nanoobjects. Photoinduced (UV 365 nm) cleavage of NBMA or 2nitrobenzyl acrylate (NBA) moieties to form carboxyl groups has been well-documented.69−73 If NBMA or NBA is used as the hydrophobic units to fabricate the nano-objects via selfassembly of an amphiphilic copolymer, photoirradiation will result in dissociation of the nano-objects due to the hydrophobic-to-hydrophilic transition of the core-forming block. 72 However, for the nano-objects composed of PHPMA-P(NBMA-co-CMA) herein, under UV-irradiation, the photoinduced cleavage of NBMA moieties simultaneously occurred with the dimerization of coumarin moieties. The former resulted in the hydrophobic-to-hydrophilic transition of the core-forming block, and the latter led to the core-crosslinking which prevented the nanoparticle-to-unimer disassembly (Scheme 2A). Figure S6 shows that the absorption band of coumarin moieties at around 320 nm dramatically decreased after UV irradiation, and exceeding 60% of the coumarin moieties took part in dimerization within 2 h for all four kinds of nano-objects. At the same time, the absorption band at around 270 nm, which is attributed to the NBMA, also significantly reduced after UV irradiation, illustrating that the photoinduced decomposition of NBMA occurred. Upon UVirradiation, both of the absorption bands at around 320 and 270 nm decreased with time (Figure S6B); around 43%, 63%, and 83% of the coumarin moieties took part in dimerization and about 57.1%, 67.4%, and 82% of the NBMA decomposed after 0.5 h, 1 h, and 2 h of UV-irradiation. The Fourier transform infrared (FT-IR) spectra show that the asymmetric N−O stretch at 1530 cm−1 for all four types of nano-objects dramatically decreased after UV irradiation (Figure S7), which

Figure 2. TEM images of the four types of nano-objects (spherical micelles, nanoworms, lamellae, and vesicles) after UV irradiation: (A) H26N20-15% (spherical micelles), (B) H26N30-15% (nanoworms), (C) H26N60-15% (lamellae), and (D) H26N100-15% (vesicles).

objects keep their corresponding morphologies well, because UV irradiation induces dimerization of the coumarin moieties, leading to robust structures of the nano-objects. All four types of cross-linked nano-objects, which are mainly composed of PHPMA-P(MAA-co-CMA), are applied in the following studies, including drug loading, pH-regulated drug release, in vitro cytotoxicity evaluation, and intracellular drug delivery. The “nano-objects” without additional instruction in the following text represent the ones after UV irradiation. Encapsulation and in Vitro Release of DOX. The anticancer drug DOX was loaded into the nano-objects by mixing DOX and the nano-object in aqueous solution and then stirring overnight (Scheme 2A). For the commonly used approach to encapsulate the drugs, the drugs and block copolymers were mixed in organic solvents (such as tetrahydrofuran, 1,4-dioxane and dimethyl sulfoxide, etc.), and then water was added into the mixture and the drugs were encapsulated into the nanoparticles during micellization.41,43 Herein, the nano-objects were prefabricated and the DOX was spontaneously encapsulated into the nano-objects via the electrostatic interaction between PMAA and DOX, and none of the organic solvents (most of them are toxic) was needed in the process of DOX encapsulation. Figure 3A and Figure S9 show that remarkably high drug loading content (DLC, up to 24.9 wt %) and drug loading efficiency (DLE, up to 82.9%) were obtained herein. Both DLC and DLE increased gradually E

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cytotoxicity at their concentrations up to 500 μg/mL (Figure 3C), which is consistent with the well-documented biocompatibility of PHPMA.66,74 However, by treating HeLa cells with the DOX-loaded nano-objects, dramatic decrease of the cell viability was observed (Figure 3D). The cytotoxicity of four types of DOX-loaded nano-objects has the following order (Figure 3D): DOX-loaded spherical micelles < DOX-loaded nanoworms < DOX-loaded lamellae < DOX-loaded vesicles, which have the same order with the increase of DLC (Figure 3A), and it is reasonable to speculate that this cytotoxicity difference is induced by their DLCs. Intracellular Drug Release of the DOX-Loaded Nanoobjects. Confocal laser scanning microscopy (CLSM) was used to study the intracellular drug release behaviors of the DOX-loaded nano-objects. The nuclei of HeLa cells were stained with DAPI (blue). Remarkably significant DOX fluorescence was observed in the nuclei of HeLa cells following 1 h of incubation with DOX-loaded nano-objects (Figure 4,

Figure 3. (A) Drug loading content (DLC) and (B) pH-regulated drug release of the four types of nano-objects. In vitro cytotoxicity of (C) the drug-free (the data on the x-axis are the concentrations of nano-objects) and (D) the DOX-loaded nano-objects (the data on the x-axis are the concentrations of DOX-loaded nano-objects) determined by MTT assay against HeLa cells. S = spherical micelles, NW = nanoworms, La = lamellae, V = vesicles.

with morphological change of the nano-objects from spherical micelles to nanoworms to lamellae to vesicles. This phenomenon is due to increasing of the DPP(MAA‑co‑CMA) (in other words, increased content of carboxyl groups) with the changes of spherical micelles to nanoworms to lamellae to vesicles, which is consistent with the encapsulation mechanism; that is, electrostatic interaction between PMAA and DOX induces drug loading. To further confirm this mechanism, we measured the ζ potentials of the four types of nano-objects, and all of their ζ potentials increased after encapsulation of DOX due to the consumption of −COO− groups (Figure S8). The spontaneous encapsulation of DOX into the nano-objects also illustrates that the nano-objects after UV irradiation are permeable. In contrast, mixing of DOX and the nano-objects without UV irradiation in the aqueous solution leads to negligible DLC (0.16%) due to the absence of electrostatic interaction and the hydrophobic cores. The above result illustrated that the nano-objects with hydrophobic cores (without UV irradiation) have poor permeability. In consideration of the intracellular endosomes/lysosomes mildly acidic microenvironment, we investigated the in vitro drug release at pH 5.4. The DOX released from the DOXloaded nano-objects exceeded 50% within 3 h and reached about 80% within 24 h for all four types of nanocarriers (Figure 3B). However, the release of encapsulated DOX was greatly retarded at pH 7.4, and the cumulative release within 24 h was less than 12.8% for all four types of nanocarriers (Figure 3B). The pH regulated drug release is due to protonation of the PMAA segments at acidic conditions, which breaks the electrostatic interaction between −NH3+ of DOX and −COO− of P(MAA-co-CMA), leading to acceleration of the DOX release. The above results imply that these DOX-loaded nanocarriers are capable of effectively minimizing the undesired DOX leakage during blood circulation (pH 7.4) and significantly enhancing intracellular (acidic microenvironment) drug release once upon cellular uptake. Cytotoxicity Evaluation. In vitro cytotoxicity evaluation shows that the DOX-free nano-objects exhibit negligible

Figure 4. CLSM images of HeLa cells after incubation with DOXloaded lamellae (3 μg/mL equivalent DOX) for 1 and 3 h. The cell nuclei were stained by DAPI (blue).

Figures S10−S12). For all four types of DOX-loaded nanoobjects, the longer incubation time (3 h) resulted in stronger DOX fluorescence in the nuclei of HeLa cells (Figure 4, Figures S10−S12). It is crucial for DOX to accumulate in the cell nucleus to get anticancer activity via interaction with genomic DNA and induce pharmacological responses. Usually, during cell uptake, the DOX-loaded nano-objects prefer to integrate into the cell membrane to form many small endosomes with the entrapped nanocarriers, which hampers the intracellular drug delivery.75−77 So nanocarriers with high-efficient endosomal escape are very important. Herein, negligible DOX fluorescence remaining in the cytoplasm and significant DOX accumulation in the cell nucleus were observed following both 1 and 3 h incubation with the DOX-loaded nano-objects, which revealed the rapid endosomal escape and effective intracellular drug delivery performance of all four types of DOX-loaded nano-objects (Figure 4, Figures S10−S12). Once upon cell uptake, the protonation of the −COO− group in the acidic endosomal microenvironment induces a rapid flux of ions and water into the subcellular compartment, resulting in osmotic swelling and endosomal rupture. The protonation of the −COO− group also breaks the electrostatic interaction between the −COO− groups and DOX, resulting in the subsequently fast release of DOX. So the DOX escapes rapidly from the endosome into the intracellular environment, which results in F

DOI: 10.1021/acs.biomac.6b01887 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

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efficient intracellular drug release and significant DOX accumulation in the cell nucleus. The DOX-loaded nanoobjects exhibit excellent performances in anticancer activity.

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CONCLUSION In summary, we reported an ingenious formulation of PISR by introducing photosensitive monomers NBMA and CMA for the RAFT dispersion polymerization to fabricate polymeric nano-objects, which exhibit excellent performances in drug delivery after postpolymerization photoirriadiation. Various morphologies, such as spherical micelle, nanoworm, lamella, and vesicle, with photolabile cores are fabricated at up to 20% solids content. UV irradiation induces concurrent linear-tocross-linked and hydrophobic-to-hydrophilic core transitions, resulting in the formation of nano-objects with superior stability and permeability. The resultant nano-objects exhibit excellent performances in drug delivery, such as remarkably high drug loadings, pH-responsive release, and high-efficient endosomal escape. In consideration of the high efficiency and the potential scalability of PISR, this superior self-assembly method may well ultimately prove to be the preferred route for fabrication of intelligent drug delivery systems, which will be of great interest for nanomedicine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b01887. 1 H NMR spectra of PHPMA, CMA, and NBMA; GPC traces of PHPMA; UV−vis absorption spectra; Fourier transform infrared (FT-IR) spectra; ζ potential; and DLE (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

Cai-Yuan Pan: 0000-0003-2859-3621 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support from the National Natural Science Foundation of China under contract No. 21525420 and 21374107, China Postdoctoral Science Foundation (BH2060000011), and the Fundamental Research Funds for the Central Universities (WK 2060200012 and WK 2060200015) is gratefully acknowledged.

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DOI: 10.1021/acs.biomac.6b01887 Biomacromolecules XXXX, XXX, XXX−XXX