Hollow Polymeric Capsules from POSS-Based Block Copolymer for

Nov 11, 2016 - Bagher Eftekhari-Sis , Ali Akbari , Parisa Yekan Motlagh , Zahra Bahrami , Nasser Arsalani. Journal of Inorganic and Organometallic Pol...
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Hollow Polymeric Capsules from POSS-Based Block Copolymer for Photodynamic Therapy Zhenghe Zhang,† Yudong Xue,† Pengcheng Zhang,† Axel H. E. Müller,‡ and Weian Zhang*,† †

Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China ‡ Institut für Organische Chemie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany S Supporting Information *

ABSTRACT: A novel amphiphilic diblock copolymer, PHEMAPOSS-b-P(DMAEMA-co-CMA), was prepared via reversible addition−fragmentation chain transfer (RAFT) polymerization, where PHEMAPOSS block was first synthesized using a methacrylate monomer based on polyhedral oligomeric silsesquioxane (HEMAPOSS), and PHEMAPOSS was further utilized to prepare the block copolymer via RAFT copolymerization of 2(dimethylamino)ethyl methacrylate (DMAEMA) and reductioncleavable coumarin methacrylate (CMA) monomer. PHEMAPOSSb-P(DMAEMA-co-CMA) could self-assemble in water to form spherical micelles with POSS core and stimuli-responsive shell. The micelles were cross-linked by photodimerization of coumarin, and then hollow polymeric capsules could be finally obtained via etching the POSS core in the solution of hydrofluoric acid (HF). The morphologies of the micelles and hollow polymeric capsules were well characterized by TEM, SEM, and DLS. The hollow polymeric capsules are responsive to typical physiological stimuli such as pH, and redox potential, and could be further utilized in the encapsulation and release of tetraphenylporphyrin tetrasulfonic acid hydrate (TPPS) for photodynamic therapy (PDT). The in vitro release of TPPS-loaded polymeric capsules allowed a relatively low TPPS release at pH = 7.4. However, a burst release of TPPS was observed in the presence of 10 mM glutathione (GSH) at pH = 5.5. Confocal laser scanning microscopy (CLSM) confirmed that TPPS-loaded polymeric capsules could well improve the internalization rate in MCF-7 cells. According to the result of MTT assay, TPPS-loaded polymeric capsules demonstrated efficient PDT efficacy and low dark toxicity toward MCF-7 cells. Thus, TPPS-loaded polymeric capsules have presented potential application in PDT.

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INTRODUCTION Polyhedral oligomeric silsesquioxane (POSS), which is often regarded as one of inorganic silica nanoparticles with a threedimensional precise structure, has attracted great attention in the past decades. The typical POSS molecule of octasilsesquioxane (R8Si8O12, T8) consists of a rigid, cage-shaped inorganic silica core and eight organic corner groups,1 and these corner substituents can be conveniently modified by conventional organic chemistry, which provides promising opportunities to construct novel POSS-containing hybrid polymers with various enhanced advantageous properties including mechanical strength, thermal stability, oxidation resistance, etc.2−8 Recently, the living/controlled polymerization techniques have been developed in polymer science, and they also have been utilized to construct POSS-containing hybrid polymers with well-defined structures, including telechelic,10−22 star-shaped,23−27 dendrimers,28−30 block copolymers,31−36 and alternating copolymers.37−39 On account of the regular inorganic structure and excellent properties of POSS molecules, the self-assembly behavior of POSS-containing hybrid polymers has aroused great interest, © XXXX American Chemical Society

providing some interesting self-assembled morphologies. For example, Cheng et al. prepared polystyrene−(carboxylic acidfunctionalized POSS) (PS-APOSS), where the self-assembled morphologies of PS-APOSS could be varied from vesicles to wormlike cylinders and further to spheres with the increase of the ionization degree of the carboxylic acid.12 Li et al. synthesized a POSS end-capped PDMAEMA via ATRP and found a single self-assembled spherical micelle based on PDMAEMA-POSS, which could form a reversible complex micelle-on-micelle structure under external stimuli including pH and temperature.14 Wu et al. constructed a tadpole-shaped PEG-POSS-(azobenzene)7, which self-assembled into a large vesicle in water and underwent reversible smooth-curling transformation with varying UV and dark conditions.18 In recent years, we also prepared a series of POSS-containing hybrid polymers with a variety of well-defined structures,7 studied their self-assembly behavior in selective solvents, and achieved some novel interesting self-assembled morphologies Received: November 7, 2016

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Macromolecules Scheme 1. Illustration of Polymeric Capsules Preparation and the Process of Loading and Release of TPPS

Scheme 2. Synthesis of PHEMAPOSS-b-P(DMAEMA-co-CMA) Block Copolymer

such as ellipsoidal aggregates,19 giant capsules,40 complex spheres (pearl-necklace-like structure),36 dendritic cylinders,35 etc. Additionally, the self-assemblies based on well-defined POSScontaining polymers also presented some potential applications such as biomaterials,26,27,36,41−43 nanoprobes,16,18 lithography substrates,32 etc. Bai et al. fabricated fluorescent spherical micelles by self-assembly of a novel POSS end-capped PNIPAM in water. These micelles with high red fluorescence at 645 nm were thermoresponsive and could be used as biosensors, nanoprobes, etc.16 He et al. synthesized a series of thermoresponsive poly(PEGMA-co-PPGMA-co-POSSMA) via ATRP,44 which could form spherical micelles and further were used as artificial molecular chaperones for protecting protein protection from the thermal induced denaturation process. Gu et al. synthesized the star-shaped copolymers POSS-g-(PBLA-bPEG) with different groups including benzyl, carboxyl, and imidazole on PBLA blocks.25 Doxorubicin (DOX) was

encapsulated into the self-assembled spherical micelles formed by the copolymers, and the drug release behavior was evaluated. Although much effort about the assemblies based on POSScontaining polymers has been contributed in above-mentioned application fields, to the best of our knowledge, stimuliresponsive polymeric capsules constructed from POSScontaining polymers have not been constructed for drug delivery, especially photodynamic therapy (PDT). In this contribution we constructed one novel stimuliresponsive hollow polymeric capsules via the self-assembly of a well-defined POSS-containing block copolymer and further investigated its controlled release of the tetraphenylporphyrin tetrasulfonic acid hydrate (TPPS) photosensitizer for PDT. The preparation procedure of stimuli-responsive polymeric capsules is briefly presented in Scheme 1. The PHEMAPOSS-bP(DMAEMA-co-CMA) block copolymer was first prepared by RAFT polymerization, where PHEMAPOSS block was synthesized using a POSS-containing methacrylate monomer B

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Macromolecules

nitrogen by at least three freeze−pump−thaw cycles to eliminate the oxygen. After the flask was flame-sealed under vacuum, the polymerization was performed at 65 °C for 5 h. The polymerization was quenched by plunging the flask into liquid nitrogen. The reaction solution was precipitated into the freezing petroleum ether, and the final product was obtained after drying under vacuum at 45 °C for 24 h. Mn,GPC = 53 000 g mol−1, Mw/Mn = 1.28. Self-Assembly of PHEMAPOSS-b-P(DMAEMA-co-CMA) Block Copolymer in Aqueous Solution. In a typical process, PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer (10 mg) was first dissolved in 10 mL of THF (common solvent) with a concentration of 1 mg mL−1. Then distilled water was injected slowly into 1 mL of block copolymer THF solution at room temperature. The mixture solution was stirred for 4 h, and the micellar solution was obtained after dialyzing against distilled water for 3 days using cellulose ester dialysis membrane (MWCO = 3500). Finally the micellar concentration was 0.25 mg mL−1. Preparation of Hollow Polymeric Capsules. After selfassembly, the micellar solution was exposed to a 6 W high-pressure mercury UV bench lamp (λ = 365 nm) with different exposure times. The distance between the micellar solution and the UV lamp was about 1 cm. UV−vis spectroscopy was used to monitor the conversion of cross-linking. After about 24 h, the solution was freeze-dried into powder. 5 mg of dried micelles was dispersed in 5 mL of THF, and then 100 μL of 10% HF aqueous solution (caution! extremely corrosive) was added into the solution. The etching reaction was performed at room temperature. After 3 h, the treated micelles were dialyzed against water for 24 h (cellulose ester dialysis membrane: MWCO = 3500). Finally, a hollow polymeric capsule solution with a concentration about 1 mg mL−1 was obtained by adding a certain amount of water. TPPS Storage and Release. TPPS-loaded polymeric capsules were prepared as follows: 5 mg of hollow polymeric capsules were dispersed in 5 mL of phosphate buffer solution (PBS, pH = 7.4), followed by adding 2.5 mg of TPPS hydrate. After being stirred at room temperature for 24 h, the solution was centrifuged until the supernatant was color-free. The supernatant was collected, and TPPS in the supernatant was measured via fluorescence measurement at 420 nm relative to an established calibration curve. The encapsulation efficiency was calculated by subtracting the mass of TPPS in the supernatant from the total mass of TPPS. An in vitro drug release test was performed in three different phosphate buffer solutions (pH = 7.4, pH = 5.5, and pH = 5.5 with 10 mM GSH). Typically, 1 mg of TPPS-loaded polymeric capsules was dispersed into 1 mL of the above corresponding buffer and transferred into a dialysis bag (MWCO = 3500). Then the dialysis bag was immersed in 100 mL of release medium under stirring at 37 °C. At different intervals, 2 mL of dialysate was taken out and analyzed by fluorescence measurement, and then 2 mL of fresh buffer was added into the apparatus to keep the volume of the solution invariable. Cell Culture. The MCF-7 cells (human breast adenocarcinoma line) were fostered in Dulbecco’s modified Eagle’s medium (DMEM) with antibiotics (50 units mL−1 penicillin and 50 units mL−1 streptomycin) and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2. Intracellular Uptake. The cellular uptake of free TPPS and TPPSloaded polymeric capsules was checked by using confocal laser scanning microscopy (CLSM). MCF-7 cells were seeded on sterile cover glasses in a six-well plate with a density of 1 × 105 cells/well and incubated for 24 h. Then the culture medium was replaced by free TPPS or TPPS-loaded polymeric capsules containing fresh medium and incubated for 4 and 24 h, respectively. After a certain period of time, cells were washed with appropriate amount of PBS, fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, and then stained with 4′,6-diamidino-2-phenylindole (DAPI). Then fluorescence microscope (Nikon AIR) was employed to observe the fluorescence of these samples which incubated for different times. In Vitro Dark Cytotoxicity and Phototoxicity of TPPS-Loaded Polymeric Capsules. The MCF-7 cell suspension (200 μL) with a density of 10 000 cells/well was seeded into a 96-well plate and incubated for 24 h. For phototoxicity, the cells were treated with

(HEMAPOSS), and PHEMAPOSS was further utilized to prepare the block copolymer via RAFT copolymerization of 2(dimethylamino)ethyl methacrylate (DMAEMA) and reduction-cleavable coumarin methacrylate (CMA) monomer (Scheme 2). The block copolymer was self-assembled in water to form spherical micelles with a hybrid core and a responsive shell. In the second step, the cross-linking of the micellar shell was realized via photodimerization of coumarin units, and then the POSS core was etched using HF.45,46 Finally, pH-responsive polymeric capsules with disulfide bonds were obtained, and their controlled TPPS delivery and release for PDT was investigated in detail.

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EXPERIMENTAL SECTION

Materials. Cumyl dithiobenzoate (CDB, RAFT agent) was prepared according to the previous literature.47 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized twice from ethanol. Tetrahydrofuran (THF) was distilled over sodium prior to use. Triethylamine (TEA) and dichloromethane (DCM) were dried over calcium hydride (CaH2) and distilled before use. 2-(Dimethylamino)ethyl methacrylate (DMAEMA, 99%, Aladdin) was purified by passing through a column of basic aluminum oxide to remove inhibitors shortly before polymerization. Aminopropyl isobutyl polyhedral oligomeric silsesquioxane (AIPOSS, Hybrid Plastics), 2-hydroxyethyl methacrylate (HEMA, 96%, Aladdin), succinic anhydride (98%, Aladdin), 4-methylcoumarin (4-MC, 98%, Aladdin), 6-chloro-1hexanol (95%, Aladdin), methacryloyl chloride (95%, Aladdin), glutathione (GSH, 98%, Aladdin), 2,2′-dithiodiethanol (98%, Energy Chemical), tetraphenylporphyrin tetrasulfonic acid hydrate (TPPS Hydrate, 85%, TCI), and other reagents were used directly as received. The synthesis and characterization of POSS-containing monomer, HEMAPOSS, and reduction-cleavable coumarin methacrylate (CMA) monomer were separately described in the Supporting Information (Schemes S1, S2 and Figures S1−S5). Characterization. The 1H NMR measurement was performed on a BRUKER AV400 spectrophotometer using CDCl3 as the solvent and tetramethylsilane (TMS) as an internal reference. The number-average weight (Mn) and polydispersity index (Mw/Mn) were measured on a Waters 1515 gel permeation chromatograph (GPC) with polystyrene as the standards and THF as the eluent at a flow rate of 1 mL min−1. Transmission electron microscopy (TEM) analysis was performed on a JEOL JEM1400 electron microscope operated at 100 kV. The samples (0.25 mg mL−1) were prepared by directly dropping the selfassembled solution (0.25 mg mL−1) onto a carbon-coated copper grid and then dried at room temperature. Scanning electron microscopy (SEM) analysis was carried out using a JSM-7401F field emission scanning electron microscope at an acceleration voltage of 1 kV. The self-assembly aggregate solution was directly dropped onto a freshly cleaved mica and then dried in ambient atmosphere. Before the measurement, the sample was sputtered by gold. The dynamic light scattering (DLS) was performed on a BECKMAN COULTER Delasa Nano C particle analyzer with the wavelength of 532 nm at 25 °C and the scattering angle at 165°. Absorption spectra were recorded with a Shimadzu UV-2550 UV/vis spectrophotometer using a quartz cuvette with 1 cm beam path length. The fluorescence spectra (FS) was recorded on F-4500 fluorescence spectrophotometer at room temperature. Synthesis of PHEMAPOSS Homopolymer. PHEMAPOSS homopolymer was prepared via RAFT polymerization of HEMAPOSS monomer using CDB as the RAFT agent according to our previous work.36 Mn,GPC = 21 000 g mol−1, Mw/Mn = 1.15. Synthesis of PHEMAPOSS-b-P(DMAEMA-co-CMA) Block Copolymer. A typical synthesis for the PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer was as follows: PHEMAPOSS (0.2 g, 0.004 mmol), DMAEMA (0.5 g, 3.18 mmol), CMA monomer (0.2 g, 0.34 mmol), AIBN (22 μL, 10 mg mL−1 AIBN THF solution, 0.0013 mmol), and 1.5 mL of THF were placed into a dry flask with a magnetic stirring bar. The mixture was degassed and then filled with C

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Macromolecules various doses of TPPS and TPPS-loaded polymeric capsules in FBSfree DMEM for another 24 h. After the cells were illuminated by using a light-emitting diode (LED) lamp (100 mW cm−2) for 10 min, they were incubated for 24 h. Finally, 20 μL of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) solution (5 mg mL−1) was added into each well for incubation of another 4 h and followed by replace of 150 μL of DMSO to dissolve the formazan. Spectrophotometric microplate reader (Spectra Max) was used to detect 560 nm absorbance of formazan/DMSO solution. Cell viability (%) = (ODtest − ODbackground)/(ODcontrol − ODbackground) × 100, where ODtest and ODcontrol are the absorbance of formazan/DMSO solution which treated and untreated with samples, respectively. The in vitro dark cytotoxicity of free TPPS and TPPS-loaded polymeric capsules were monitored following the same procedure of phototoxicity described above but without irradiation.

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RESULTS AND DISCUSSION Synthesis of PHEMAPOSS-b-P(DMAEMA-co-CMA) Block Copolymer. The general strategy for synthesis of the Figure 3. Intensity-weighted hydrodynamic diameter distributions of PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer self-assembled micelles and z-average hydrodynamic diameters with a concentration of 0.25 mg mL−1 before and after cross-linking in water and after adding THF, as determined by DLS.

Figure 1. 1H NMR spectrum of PHEMAPOSS-b-P(DMAEMA-coCMA) block copolymer in CDCl3.

Figure 4. TEM (A, C) and SEM (B, D) images of (A, B) cross-linked micelles and (C, D) hollow polymeric capsules in aqueous solution.

novel HEMAPOSS monomer with long spacer between the methacrylate group and the POSS unit via a three-step procedure before. 36 The novel monomer HEMAPOSS efficiently decreases the steric hindrance in free-radical polymerization of POSS-based methacrylate monomers, and POSS-containing homopolymers (PHEMAPOSS) with a higher molecular weight could be achieved via RAFT polymerization. The polymerization was performed in toluene at 65 °C for 24 h using CDB as a RAFT agent and AIBN as an initiator. The molar ratio between HEMAPOSS, CDB, and AIBN was fixed to 50/1/0.3. The symmetrical GPC trace (Figure S6a) without shoulder or tail indicates that the RAFT polymerization of HEMAPOSS is well-controlled. The PHEMAPOSS homopolymer was also characterized by 1H

Figure 2. UV spectra of micellar solutions of PHEMAPOSS-bP(DMAEMA-co-CMA) block copolymer at different exposure times, which was under UV irradiation (365 nm).

PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer via RAFT polymerization is presented in Scheme 2. The first step was the synthesis of HEMAPOSS monomer and PHEMAPOSS homopolymer. Here, our group designed and synthesized a D

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Figure 5. In vitro release behavior of TPPS-loaded polymeric capsules at 37 °C in different release media: (a) pH = 7.4, 0 mM GSH, (b) pH = 5.5, 0 mM GSH, and (c) pH = 5.5, 10 mM GSH.

Figure 7. (A) Viability of MCF-7 cells attained by MTT assay with various concentrations of TPPS-loaded polymeric capsules and free TPPS in the dark. (B) Phototoxicity of MCF-7 cells against TPPSloaded polymeric capsules and free TPPS. (C) Viability of MCF-7 cells treated with and without 10 mM GSH-OEt and then incubated with different amounts of TPPS-loaded polymeric capsules. *P < 0.05 indicates significant differences.

Figure 6. CLSM images of MCF-7 cells incubated with free TPPS for 4 h (a) and 24 h (b) and with TPPS-loaded polymeric capsules for 4 h (c) and 24 h (d). The images from left to right are the cells with nuclear staining with DAPI, with TPPS fluorescence and merge of images.

The result shows that Mn,th,PHEMAPOSS and degree of polymerization (DPPHEMAPOSS) of the well-defined homopolymer PHEMAPOSS were 43 400 g mol−1 and 40, respectively. The PHEMAPOSS homopolymer was then used as a macroCTA to synthesize multistimuli-responsive PHEMAPOSS-bP(DMAEMA-co-CMA) block copolymer. The light-responsive coumarin monomer, CMA, which contains a disulfide unit was synthesized first. The 1H NMR spectrum of CMA confirms its chemical structure (Figure S5). The molar ratio between

NMR spectrum (Figure S7), and the number-average molecular weight (Mn,th,PHEMAPOSS) could be calculated by the monomer conversion from the formula Mn,th,PHEMAPOSS = [M]0/[CTA]0 × MHEMAPOSS × x + MCDB, where the MCDB, MHEMAPOSS, and x are the molecular weights of CDB and HEMAPOSS monomer and monomer conversion determined by 1H NMR, respectively. E

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Macromolecules DMAEMA, CMA, PHEMAPOSS, and AIBN was fixed to 795/ 85/1/0.3. After degassing, the polymerization was performed in THF at 65 °C for 5 h. Figure S6b shows the GPC trace of PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer. Compared to that of the macro-CTA (PHEMAPOSS), the monomodal peak shifted to a lower elution volume, and no peak of the residual macro-CTA was observed, which indicates the successful synthesis of the block copolymer. The 1H NMR spectrum of PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer is shown in Figure 1. By comparing the integration area of the signal at 7.42 ppm (a, aromatic proton from coumarin unit), 2.22 ppm (i, −OCH2CH2N(CH3)2) to that of 0.53 ppm (f, f′, −Si−CH2−), DPPDMAEMA and DPPCMA in P(DMAEMA-co-CMA) block were determined as DPPDMAEMA = (16I2.22)/(6I0.53) × DPPHEMAPOSS = 575 and DPPCMA = (16I7.42 )/I 0.53 × DP PHEMAPOSS = 95, respectively. The Mn,th,PHEMAPOSS‑b‑P(DMAEMA‑co‑CMA) of PHEMAPOSS40 -b-P(DMAEMA575-co-CMA95) block copolymer is 189 000 g mol−1. Preparation of Stimuli-Responsive Hollow Polymeric Capsules. In order to obtain the hollow polymeric capsules, the micellization of the block copolymer was preceded first. PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer with hydrophobic POSS, coumarin moieties, and hydrophilic PDMAEMA segment provided an opportunity to study its self-assembly behavior in water. The block copolymer was first dissolved in THF. Then, water was added slowly to above solution to induce the micellization of the block copolymer. In the wake of the addition of water, the solvent became gradually much more insoluble for the POSS moieties. Finally, the micelles could form with the water content reaching a critical value. The spherical micelles were cross-linked via photodimerization of the coumarin moieties under UV light (365 nm).46 The photodimerization process of coumarin was traced by the UV− vis spectrum at different exposure time (Figure 2). With the reaction time increasing, the characteristic absorption of coumarin moieties at about 320 nm clearly decreased, which indicates that the dimerization of coumarin double bonds occurred to form cyclobutane rings, and then the cross-linked shell of the micelles was obtained. After about 24 h, the main coumarin absorption peak at around 320 nm decreased only feebly upon UV irradiation, and the final dimerization degree was about 52%. This probable reason was that the photodimerization is bimolecular reaction, and there were only few coumarin moieties disorderly dispersed in the shell of the micelles; moreover, the micelles became more and more rigid with increasing cross-linking degree, which also hindered further photodimerization process.48 The morphology of these micelles was characterized by DLS, TEM, and SEM. Results from DLS suggested that the noncross-linked spherical micelles had a diameter of about 233 nm with the dispersity index (DI) of 0.26 (Figure 3), which is agreeable with that of the TEM image (Figure S8). After exposure to UV-light (365 nm) for 24 h, the size of the micelles decreased to 200 nm (DI = 0.29), which might be caused by a more compact structure of the micelles after cross-linking.48 In order to prove that 52% photodimerization degree of the coumarin moieties would be sufficient to maintain the integrity of these micelles formed after irradiation, THF, a nonselective solvent for PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer, was added into the micellar solution. DLS results revealed that the cross-linked micelles did not disassemble in THF, indicating the micelles had a good stability. It should be

noted that the diameter of these cross-linked micelles in THF measured by DLS was larger than that in water (387 nm, DI = 0.28), which is explained by swelling of these micelles in THF.43,48,49 TEM and SEM were also used to characterize the morphology of these cross-linked micelles at a concentration of 0.25 mg mL−1 in water, respectively. As shown in Figure 4A, the size in diameter of cross-linked micelles revealed by TEM was about 180 nm. Taking the size of these micelles into consideration, they should be composed of the aggregates of PHEMAPOSS-b-P(DMAEMA-co-CMA) block copolymer. Here, the size of the micelles determined by TEM was somewhat lower than that by DLS due to the fact that TEM gives a number-average and DLS gives a z-average, and the shrinkage of PDMAEMA shell upon drying. SEM images (Figure 4B) also verified the morphology of these cross-linked micelles, which had a diameter of about 220 nm. After the micelles were cross-linked via photodimerization of coumarin moieties by UV-light (365 nm), the hollow polymeric capsules could be obtained by etching the POSS core of crosslinked micelles in a HF/THF mixture solution. The formation of stimuli-responsive hollow polymeric capsules was separately revealed by TEM and SEM analysis. As shown in Figure 4C, the hollow structure of the as-obtained polymeric capsules is obviously observed from the TEM image, the inner diameter of polymeric capsules was about 70 nm, and the shell diameter was also approximately 70 nm. Moreover, the collapsed structure of polymeric capsules was also confirmed from the SEM images (Figure 4D), further suggesting that the POSS core had been removed and hollow polymeric capsules formed. Additionally, the diameter of hollow polymeric capsules (252 nm) detected by DLS was slightly larger than that of crosslinked micelles (200 nm) (Figure 3), which might be due to the fact that the PDMAEMA chains were no longer tethered by the POSS core in the spherical micelles and could swell further in water. It is well-known that PDMAEMA is a pH-responsive polymer; thus, the hollow polymeric capsules based on PDMAEMA also exhibited a unique responsive behavior to pH in aqueous solution. To study the stimuli-responsive behavior, the size of polymeric capsules was measured at different pH by DLS. Figure S9 shows the effect of pH on the size of these polymeric capsules, from which we could find that the size of these polymeric capsules increased with the decrease of pH value from 9.0 to 3.0. This could be explained that with the pH value decreasing, the PDMAEMA chains in polymeric capsules become gradually protonated, and the electrostatic repulsion among positively charged DMAEMA units increased. Reduction Release of TPPS from Hollow Polymeric Capsules. As a focal point of theranostics for tumor treatment, PDT administers and activates a photosensitizer such as porphyrin and its derivatives to its triplet state by light irradiation.50−53 Then the photosensitizer interacts with tissue oxygen to produce reactive oxygen species such as free radicals and singlet oxygen (1O2), which can exert oxidative damage to cause tumor cells death.54,55 Recently, we developed many kinds of stimuli-responsive photosensitizer release systems based on porphyrin-containing polymers, and their performance about PDT was studied.56−59 Here, to investigate the potential application of hollow polymeric capsules as a drug carrier, TPPS hydrate as a model porphyrin photosensitizer was chosen to evaluate the loading and releasing capacity of hollow polymeric capsules. TPPS-loaded polymeric capsules were obtained by incubating hollow polymeric capsules with TPPS hydrate (1:0.5 w/w) in the TPPS hydrate aqueous solution (1 F

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Macromolecules mg mL−1, pH = 7.4) at room temperature 24 h. In the TPPS hydrate loading process, the TPPS hydrate molecules were entrapped into hollow polymeric capsules by hydrogen bonds and electrostatic interactions between TPPS hydrate and polymeric capsules, and finally 15.4 wt % of TPPS hydrate was loaded by polymeric capsules. Considering the natural pH (pH = 7.4) of blood for sustaining human life, while extracellular matrix, endosomes, and lysosomes of tumor tissue have an acidic condition (ca. pH = 6.5−7.2, 5.0−5.5), three different conditions were applied to investigate the in vitro release behavior of TPPS-loaded polymeric capsules at 37 °C.60 In Figures 5a and 5b, TPPSloaded polymeric capsules were exposed to PBS solution with the pH of 7.4 and 5.5, which represented the normal blood and tumor cells environment, respectively. TPPS (10%) was released from polymeric capsules at pH = 7.4 within the first 24 h, while 27% of TPPS was released at pH = 5.5. The low leakage of TPPS from polymeric capsules at pH = 7.4 confirmed that the TPPS-loaded polymeric capsules could be of stabilization in blood circulation. The TPPS release was higher at pH = 5.5 due to the fact that polymeric capsules swell at the lower pH, leading to the faster release rate of TPPS molecules.43 As GSH extensively exists in tumor cells at approximately 2−10 mM, we simulated the redox environment by addition of 10 mM GSH in pH = 5.5 PBS to verify whether the degradation of TPPS-loaded polymeric capsules could trigger the TPPS release in the reductive environment.61 As shown in Figure 5c, obvious facilitation of TPPS release from polymeric capsules was observed at pH = 5.5 PBS when 10 mM GSH was added into the release media. The polymeric capsules released 40% of TPPS within 12 h and about 70% of TPPS after 48 h. These results indicate that TPPS release could be accelerated by the cleavage of disulfide bonds of the polymeric capsules in the intracellular reducing environment. Intracellular Drug Uptake and Localization. The cellular uptake and intracellular distribution of TPPS-loaded polymeric capsules in MCF-7 cells were further studied by CLSM. MCF-7 cells were cultured with free TPPS and TPPSloaded polymeric capsules with a TPPS concentration of 50 μg mL−1 for 4 and 24 h, respectively. DAPI with blue fluorescence was utilized to stain the nucleus to locate the predetermined cells. Moreover, under the treatment of free TPPS and TPPSloaded polymeric capsules, the red fluorescence of TPPS was mainly distributed in the cytoplasm of cells. As shown in Figure 6, the fluorescence intensity of cells treated with TPPS-loaded polymeric capsules was clearly more intense than that treated with free TPPS after 4 or 24 h. Meanwhile, no matter if MCF-7 cells were treated with free TPPS or TPPS-loaded polymeric capsules, the fluorescence intensity of cells treated for 24 h was higher than that for 4 h. These results suggested that TPPSloaded polymeric capsules were taken up by the cells through the cellular uptake process, which was not only different from the simple passive mechanism of free TPPS but also timedependent. In Vitro Dark Toxicity and Phototoxicity of TPPSLoaded Polymeric Capsules. To investigate the in vitro dark toxicity and phototoxicity of TPPS-loaded polymeric capsules, the cytotoxicity against cells was assessed by the MTT assay with or without light illumination. The cells in the dark without treatment were set to the viability of 100% as a reference. Without light treatment, both TPPS-loaded polymeric capsules and free TPPS exhibited no cytotoxicity against cells even with the TPPS concentration up to 50 μg mL−1 (Figure 7A).

The phototoxicity was investigated with light illumination. First of all, the phototoxicity of TPPS-loaded polymeric capsules occurred and became stronger with the increasing concentration of TPPS in polymeric capsules with the IC50 (calculated for TPPS concentration) of 18 μg mL−1, which was much higher than that of free TPPS. On the contrary, the free TPPS had low phototoxicity, and the result was in accordance with that of CLSM (Figure 7B). Second, some cells were selected as a contrast to be treated with 10 mM GSH-OEt for 2 h. Here, GSH-OEt can lead to increase cellular GSH levels via ethyl ester hydrolyzation of GSH-OEt in cytoplasm. As shown in Figure 7C, the cell viability of treated cells was lower than that without GSH-OEt. Obviously, with a high intracellular GSH concentration, disulfide bonds of TPPS-loaded polymeric capsules were easy to be cleaved, which led to release TPPS and decrease the cell viability. In summary, TPPS-loaded polymeric capsules were indeed a promising tool for the application of PDT.

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CONCLUSIONS Hollow polymeric capsules were successfully obtained by selfassembly of an amphiphilic PHEMAPOSS-b-P(DMAEMA-coCMA) block copolymer via RAFT polymerization, followed via photodimerization of coumarin and removal of the POSS core by HF. The polymeric capsules had the diameter of approximately 250 nm, according to SEM and TEM micrographs. Because of PDMAEMA as the shell and disulfide bonds in the coumarin-containing monomer, the polymeric capsules exhibited a unique responsive behavior to pH and could be degraded efficiently in the presence of reducing agents. The TPPS-loaded polymeric capsules were stable under physiological conditions and allowed the triggered release of the encapsulated TPPS at low pH and reducing agents (10 mM GSH) via cleavage of the disulfide bonds between the crosslinked polymer chains. The results of CLSM exhibited that TPPS-loaded polymeric capsules were more efficiently internalized by MCF-7 cells than free TPPS. According to the MTT assay, TPPS-loaded polymeric capsules with light illumination could effectively produce cytotoxic singlet oxygen to kill tumor cells. Thus, these hollow polymeric capsules present a potential application for PDT.

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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.macromol.6b02414.

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Experimental details (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel +86 21 64253033; Fax +86 21 64253033; e-mail [email protected] (W.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21074035 and 51173044) and Research Innovation Program of SMEC (No. 14ZZ065). G

DOI: 10.1021/acs.macromol.6b02414 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

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(19) Zhang, W. A.; Yuan, J. Y.; Weiss, S.; Ye, X. D.; Li, C. L.; Muller, A. H. E. Telechelic Hybrid Poly(acrylic acid)s Containing Polyhedral Oligomeric Silsesquioxane (POSS) and Their Self-Assembly in Water. Macromolecules 2011, 44 (17), 6891−6898. (20) Wu, K.; Huang, M.; Yue, K.; Liu, C.; Lin, Z.; Liu, H.; Zhang, W.; Hsu, C.-H.; Shi, A.-C.; Zhang, W.-B.; Cheng, S. Z. D. Asymmetric Giant “Bolaform-like” Surfactants: Precise Synthesis, Phase Diagram, and Crystallization-Induced Phase Separation. Macromolecules 2014, 47 (14), 4622−4633. (21) Zhang, W.; Mueller, A. H. E. A “Click Chemistry” Approach to Linear and Star-Shaped Telechelic POSS-Containing Hybrid Polymers. Macromolecules 2010, 43 (7), 3148−3152. (22) Huang, M.; Hsu, C.-H.; Wang, J.; Mei, S.; Dong, X.; Li, Y.; Li, M.; Liu, H.; Zhang, W.; Aida, T.; Zhang, W.-B.; Yue, K.; Cheng, S. Z. D. Selective assemblies of giant tetrahedra via precisely controlled positional interactions. Science 2015, 348 (6233), 424−428. (23) Ye, Y.-S.; Shen, W.-C.; Tseng, C.-Y.; Rick, J.; Huang, Y.-J.; Chang, F.-C.; Hwang, B.-J. Versatile grafting approaches to star-shaped POSS-containing hybrid polymers using RAFT polymerization and click chemistry. Chem. Commun. 2011, 47 (38), 10656−10658. (24) Zhang, W.-B.; He, J.; Yue, K.; Liu, C.; Ni, P.; Quirk, R. P.; Cheng, S. Z. D. Rapid and Efficient Anionic Synthesis of Well-Defined Eight-Arm Star Polymers Using OctavinylPOSS and Poly(styryl)lithium. Macromolecules 2012, 45 (21), 8571−8579. (25) Pu, Y.; Zhang, L.; Zheng, H.; He, B.; Gu, Z. Drug release of pHsensitive poly(L-aspartate)-b-poly(ethylene glycol) micelles with POSS cores. Polym. Chem. 2014, 5 (2), 463−470. (26) Yang, Y.-Y.; Wang, X.; Hu, Y.; Hu, H.; Wu, D.-C.; Xu, F.-J. Bioreducible POSS-Cored Star-Shaped Polycation for Efficient Gene Delivery. ACS Appl. Mater. Interfaces 2014, 6 (2), 1044−1052. (27) Fan, X.; Wang, Z.; He, C. ″Breathing’’ unimolecular micelles based on a novel star-like amphiphilic hybrid copolymer. J. Mater. Chem. B 2015, 3 (23), 4715−4722. (28) Pu, Y.; Chang, S.; Yuan, H.; Wang, G.; He, B.; Gu, Z. The antitumor efficiency of poly(L-glutamic acid) dendrimers with polyhedral oligomeric silsesquioxane cores. Biomaterials 2013, 34 (14), 3658− 3666. (29) Wang, X.; Yang, Y.; Gao, P.; Li, D.; Yang, F.; Shen, H.; Guo, H.; Xu, F.; Wu, D. POSS dendrimers constructed from a 1 -> 7 branching monomer. Chem. Commun. 2014, 50 (46), 6126−6129. (30) Li, D.; Niu, Y.; Yang, Y.; Wang, X.; Yang, F.; Shen, H.; Wu, D. Synthesis and self-assembly behavior of POSS-embedded hyperbranched polymers. Chem. Commun. 2015, 51 (39), 8296−8299. (31) Hirai, T.; Leolukman, M.; Jin, S.; Goseki, R.; Ishida, Y.; Kakimoto, M. A.; Hayakawa, T.; Ree, M.; Gopalan, P. Hierarchical Self-Assembled Structures from POSS-Containing Block Copolymers Synthesized by Living Anionic Polymerization. Macromolecules 2009, 42 (22), 8835−8843. (32) Hirai, T.; Leolukman, M.; Liu, C. C.; Han, E.; Kim, Y. J.; Ishida, Y.; Hayakawa, T.; Kakimoto, M.; Nealey, P. F.; Gopalan, P. One-Step Direct-Patterning Template Utilizing Self-Assembly of POSS-Containing Block Copolymers. Adv. Mater. 2009, 21 (43), 4334−4338. (33) Tan, B. H.; Hussain, H.; He, C. B. Tailoring Micelle Formation and Gelation in (PEG-P(MA-POSS)) Amphiphilic Hybrid Block Copolymers. Macromolecules 2011, 44 (3), 622−631. (34) Yang, C.; Deng, Y.; Zeng, B.; Yuan, C.; Chen, M.; Luo, W.; Liu, J.; Xu, Y.; Dai, L. Hybrid amphiphilic block copolymers containing polyhedral oligomeric silsesquioxane: Synthesis, characterization, and self-assembly in solutions. J. Polym. Sci., Part A: Polym. Chem. 2012, 50 (20), 4300−4310. (35) Hong, L.; Zhang, Z.; Zhang, W. Synthesis of Organic/Inorganic Polyhedral Oligomeric Silsesquioxane-Containing Block Copolymers via Reversible Addition-Fragmentation Chain Transfer Polymerization and Their Self-Assembly in Aqueous Solution. Ind. Eng. Chem. Res. 2014, 53 (26), 10673−10680. (36) Hong, L.; Zhang, Z.; Zhang, Y.; Zhang, W. Synthesis and SelfAssembly of Stimuli-Responsive Amphiphilic Block Copolymers Based on Polyhedral Oligomeric Silsesquioxane. J. Polym. Sci., Part A: Polym. Chem. 2014, 52 (18), 2669−2683.

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DOI: 10.1021/acs.macromol.6b02414 Macromolecules XXXX, XXX, XXX−XXX