Article pubs.acs.org/Biomac
Shape Transformation of Light-Responsive Pyrene-Containing Micelles and Their Influence on Cytoviability Haisheng Wang, Wenbo Zhang, and Changyou Gao* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China S Supporting Information *
ABSTRACT: The amphiphilic pyrene-containing random copolymers with light-responsive pyrene ester bonds were synthesized by copolymerizing 1-pyrenemethyl acrylate (PA) and N,N-dimethylacrylamide (DMA). The P(DMA-co-PA) copolymers formed spherical micelles in water, which were transformed into nanorods as a result of cleavage of the pyrene ester bonds under UV irradiation. In vitro culture with A549 cells and Raw cells showed that compared to the nonphotodegradable ones, the photodegradable P(DMA-co-PA) micelles caused significantly higher cytotoxicity under the same UV irradiation. The intracellular reactive oxygen species (ROS) level had a positive correlation with the cytotoxicity regardless of the cell types. The nonphotodegradable pyrene-containing micelles produced a lower level of ROS under UV irradiation. However, the photodecomposable P(DMA-co-PA) micelles produced a significant higher level of ROS under the same trigger of UV irradiation, which caused the shape transformation of micelles to nanorods and higher cytotoxicity simultaneously.
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INTRODUCTION The self-assembly has been widely applied for preparation of functional nanomaterials.1−6 Various self-assembly methods such as vapor deposition,7,8 template9,10 and classical selfassembly11−13 have been developed to construct supramolecular nanomaterials. During the last decades stimuliresponsive self-assembly has attracted much attention, which is widely used in nanotechnology, electronics, smart materials, and biomedical fields. It can be triggered by diverse environmental stimuli such as light,14 pH,15 and enzymes.16 The use of light as a trigger is particularly attractive as it is nondestructive, quickly switched, can be remotely controlled and be located at a precise spatial place.17 Therefore, photoresponsive self-assembly is considered as a reliable strategy in the controlled construction of smart nanomaterials. For instance, Zheng et al.18 made use of trans−cis photoisomerization functionality of sodium (4-phenylazo-phenoxy)acetate and achieved UV-induced self-assembly based on Nmethyl-N-cetylpyrrolidinium bromide. The wormlike micelles could be transformed into spherical micelles upon UV irradiation. Yagai et al.14 used diarylethenes (DAEs) to design photoresponsive supramolecular assemblies. Upon irradiation with UV and visible light alternatingly, a reversible morphology change between nanofibers and nanoparticles is realized due to the photoisomerization of DAEs. Recently, decomposition-induced assembly is developed to achieve controlled shape transformation of nanomaterials, leading to different nanostructures.19,20 For example, one© XXXX American Chemical Society
dimensional nanotubes or nanorods can be protruded from poly(allylamine hydrochloride) (PAH)-g-pyrene (Py) microcapsules through gradual hydrolysis of Schiff base bonds between PAH and 1-pyrenecarboxaldehyde (Py-CHO) in pH 0 or 2 solutions, respectively.19 The nanotubes or nanorods are consisted of Py-CHO by π−π stacking. Moreover, by substituting the Schiff base bonds with hydrazone bonds, the pyrene-containing micelles could be transformed into Py-CHO nanorods at relatively high pH such as pH 6.20 However, the Schiff base bonds and hydrazone bonds are all pH-responsive bonds, which may limit the accurate control and application of decomposition-induced self-assembly, in particular for biological and medical applications. Therefore, to improve the controllability and to generalize this method, it is desirable to make use of the photoresponsive bonds. One of the best choices is the pyrene ester bond, which is readily cleavable under 365 nm UV irradiation.21−23 Meanwhile, as a photosensitizing dye, pyrene derivatives can absorb photons and produce reactive oxygen species (ROS), exhibiting photogenotoxicity.24−26 This property can be used for photodynamic therapy, which involves adoption of a tumor localizing photosensitizing agent and the subsequent activation of the agent by light, leading to irreversible photodamage to tumor tissues.27 When pyrene derivatives in solution are taken Received: April 14, 2015 Revised: June 8, 2015
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DOI: 10.1021/acs.biomac.5b00497 Biomacromolecules XXXX, XXX, XXX−XXX
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water in a dialysis bag to remove ethanol, yielding the photodegradable micelles. Shape Transformation of Photodegradable Micelles in Water. The as-prepared P(DMA-co-PA) photodegradable micelles (0.25 mg/mL) were subjected to 365 nm UV light irradiation at the conditions of 20 mW·cm−2 for 1 h and 50 mW·cm−2 for 30 min, respectively. The resulting products were observed by transmission electron microscopy (TEM, JEM-1230). Shape Transformation of Photodegradable Micelles in Artificial Cytoplasm. The as-prepared P(DMA-co-PA) photodegradable micelles (0.25 mg/mL) were maintained in artificial cytoplasm at 37 °C under 365 nm UV light irradiation of 20 mW·cm−2 for 20 min. For all the experiments, the irradiation distance was fixed as 6.3 cm. The resulting products were observed by TEM. The artificial cytoplasm was prepared by sonicating suspension of A549 cells (106 per mL) for 30 min and then collecting the supernatant fluid through centrifugation. Cell Viability. A549 cells were cultured for 24 h at a density of 5 × 103 cells per well on a 96-well plate in a 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/ mL streptomycin at 37 °C in a 5% CO2 humidified incubator. The Group 1 was replaced with a fresh medium. The Groups 2 and 3 were replaced with a fresh medium containing the P(DMA-co-PA) photodegradable micelles (0.25 mg/mL), which were filtered to remove bacteria before use. The Groups 4 and 5 were replaced with a fresh medium containing 1 mg/mL nonphotodegradable micelles (the grafting ratio of pyrene was half of that of the P(DMA-co-PA), which were synthesized according to a reported approach20). After 12 h, the Groups 1, 3, and 5 were subjected to 365 nm UV light irradiation of 20 mW·cm−2 for 20 min, and the cells were cultured for another 24 h. To determine the cell viability, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, 5 mg/mL) was added to each well, and the cells were continuously cultured at 37 °C for 4 h. Dark blue formazan crystals generated by mitochondria dehydrogenase in viable cells were dissolved in dimethyl sulfoxide (DMSO) to measure the absorbance at 570 nm using a microplate reader (Bio-Rad Model 680). Three parallel experiments were conducted, and the data were normalized to that of the untreated control. Raw cells were treated in the same way by using DMEM instead of 1640 medium. Measurement of Intracellular Reactive Oxygen Species (ROS). The oxidation-sensitive probe DCFH-DA was employed to determine the intracellular ROS level. DCFH-DA is an amphiphilic nonfluorescent molecule that readily crosses cell membrane and is deacetylated by esterases and then oxidized to highly fluorescent 2′,7′dichlorfluorescein (DCF) in the presence of intracellular ROS.37 In this study, A549 cells were seeded on 48-well plates at a density of 2 × 104 cells per well and allowed to adhere for 24 h. The Group 1 was replaced with a fresh medium. The Groups 2 and 3 were replaced with a fresh medium containing the photodegradable micelles (0.25 mg/ mL). The Groups 4 and 5 were replaced with a fresh medium containing the nonphotodegradable micelles (1 mg/mL). After 12 h, the Groups 1, 3, and 5 were subjected to 365 nm UV light irradiation of 20 mW·cm−2 for 20 min. All the groups were then incubated with 10 μM DCFH-DA at 37 °C for 20 min in dark. At the end of the incubation, the cells were washed with PBS. The fluorescence of DCF was measured with flow cytometry (FACS Calibur, Becton Dickinson BD). Three parallel experiments were conducted, and the data were normalized to that of the untreated control. Raw cells were treated in the same way by using DMEM instead of 1640 medium. Characterizations. 1H Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DMX500 equipment by using N,N-dimethylacrylamide (DMSO-d6) as the solvent. The molecular weight and molecular weight distribution was measured on a Waters 1515 gel permeation chromatography (GPC) setup at 60 °C by using poly(methyl methacrylate) standards for calibration and DMF containing 0.05 M LiBr as eluent. Dynamic light scattering (DLS) was measured on a Zetasizer Nano-ZS from Malvern Instruments. The 365 nm UV light was generated by a UVEC-4 II system.
up by cells, subsequent exposure of the cells to light of the appropriate wavelength brings cell death. Thus, the micelles with pyrene ester bonds may also have the ability of killing cells upon photoexcitation. In this regard, the starting material, polymeric micelles, are playing an increasingly important role in diverse biological applications such as drug delivery, bioimaging, and diagnostics.28−31 Because of the advantages of reserving drugs in the hydrophobic core and prolonged circulation time, they have become promising nanocarriers for the controlled release of hydrophobic anticancer drugs.32,33 As a kind of nanomaterials, polymeric micelles can accumulate in the tumor tissues through an enhanced permeability and retention (EPR) effect and are able to concentrate into cells. The physiochemical properties (i.e., shape, size, and electric charge) of the micelles and the nature of the target cells such as either the phagocytosis or the other endocytic pathways (i.e., clathrin- and calveolae-mediated endocytosis) would have important impacts.34−36 After being internalized into cells, the stimuli-responsive micelles are destructed to release drugs32,33 or transformed into other structures20 upon corresponding triggers, killing the cells or influencing cell functions and behaviors. Herein the amphiphilic pyrene-containing random copolymers with pyrene ester bonds are synthesized by copolymerizing hydrophobic 1-pyrenemethyl acrylate (PA) with hydrophilic N,N-dimethylacrylamide (DMA), which form spherical micelles through self-assembly in aqueous solution. Compared with the traditional photoresponsive micelles, they possess the ability to transform into one-dimensional nanomaterials in a controllable manner. Moreover, the shape transformation process and the formed nanorods under irradiation of 365 nm UV causes extremely high cytotoxicity compared with those nonphotodegradable micelles.
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EXPERIMENTAL SECTION
Materials. 1-Pyrenemethanol (Py-CH2OH) and N,N-dimethylacrylamide were purchased from Sigma−Aldrich. Acryloyl chloride (AC) was purchased from TCI. Azodiisobutyronitrile (AIBN) was purchased from Aladdin. Other chemicals were of analytical grade and used as received. The water used in all experiments was prepared via a Millipore Milli-Q purification system and had a resistivity higher than 18.2 MΩ·cm. Synthesis of 1-Pyrenemethyl acrylate. A 0.4646 g (2 mmol) sample of Py-CH2OH was added to a solution of 0.840 mL (6 mmol) triethylamine in 20 mL of tetrahydrofuran (THF). A solution of 0.490 mL (6 mmol) of AC in 4 mL of THF was added dropwise under agitation at 0−10 °C. The reaction was then maintained at room temperature overnight. The resulting mixture was filtered to obtain the filtrate, which was dried under reduced pressure. The solid residue was washed with water and then dried in vacuum at 40 °C. Finally, PA was obtained with a yield of 79%. Synthesis of Pyrene-Containing Random Copolymers. A 0.0004 g (0.0024 mmol) sample of AIBN, 0.45 mL (4.38 mmol) of DMA, 0.0301 g (0.11 mmol) of 1-pyrenemethyl acrylate, and 3 mL of dimethylformamide (DMF) were mixed, degassed, and purged with nitrogen. After the copolymerization was carried out at 70 °C for 12 h, the reaction solution was cooled to −20 °C and added dropwise to excess icy diethyl ether to precipitate the P(DMA-co-PA) copolymers. The resulting copolymers were then sealed into a dialysis bag with a cutoff Mw of 14 kDa and were dialyzed against ethanol to remove the unreacted monomers, oligomers, and residual DMF. The obtained solution of the copolymers was then evaporated under reduced pressure to remove ethanol and further dried in vacuum at 60 °C. Finally, P(DMA-co-PA) copolymers were obtained with a yield of 27%. Synthesis of Photodegradable Micelles. The ethanol solution of the P(DMA-co-PA) copolymers was dialyzed against Millipore B
DOI: 10.1021/acs.biomac.5b00497 Biomacromolecules XXXX, XXX, XXX−XXX
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RESULTS AND DISCUSSION The amphiphilic P(DMA-co-PA) pyrene-containing random copolymers with photosensitive pyrene ester bonds were synthesized by a two-step procedure. First, PA was obtained by reacting Py-CH2OH with acryloyl chloride in THF at 0−10 °C. Its structure was verified with 1H NMR spectrum (Figure 1a). The chemical shifts at 5.96, 6.23, and 6.35 ppm are
The P(DMA-co-PA) random copolymers with pyrene ester bonds were synthesized by free radical polymerization. The hydrophobic 1-pyrenemethyl acrylate copolymerized with the hydrophilic DMA in DMF at 70 °C, with AIBN as the initiator. The 1H NMR spectrum of the copolymers (Figure 1b) shows the characteristic proton resonance peaks of both DMA units (δ 2.78, 2.86, 3.01 ppm) and pyrene groups (δ 8.11−8.33 ppm). The disappearance of the protons at δ 5.96, 6.23, and 6.35 ppm, which are assigned to the protons in carbon−carbon double bonds of 1-pyrenemethyl acrylate, further confirms the copolymerization of 1-pyrenemethyl acrylate and DMA. The ratio of pyrene-containing units in the copolymers was estimated as 4%, based on the integration peak areas of pyrene groups and DMA units (Supporting Information Figure S1). In addition, the molecular weight (Mn) of the copolymers was 2.93 × 104 Da with a PDI of 2.12, determined by GPC. Spherical photodegradable micelles were formed by transferring the P(DMA-co-PA) copolymers from ethanol to water slowly. DLS results show that the size of the photodegradable micelles was 39.2 nm. In addition, the particles were well dispersed in a dry state (Figure 2a). The pyrene ester bonds are already proved readily cleavable under irradiation of 365 nm UV.38−40 After the solution of the photodegradable micelles was subjected to 365 nm UV irradiation at 20 mW·cm−2 for 1 h, nanorods with a length of 200−300 nm were observed, accompanying with the disappearance of the spherical micelles (Figure 2b). In order to investigate the influence of light intensity on nanorods formation, the concentration of the photodegradable micelles was maintained unchanged and the light intensity was raised from 20 to 50 mW·cm−2. After irradiation for 30 min, nanorods with a length of 200−300 nm appeared with a more compact structure (Figure 2c), suggesting that stronger light intensity enhances the formation of nanorods. To mimic the milieu of cell compartments the shape transformation ability of the photodegradable micelles was further investigated in artificial cytoplasm at 37 °C.
Figure 1. 1H NMR spectra of (a) 1-pyrenemethyl acrylate and (b) its random P(DMA-co-PA) copolymers with DMSO-d6.
assigned to the resonance peaks of acrylate protons. The methylene protons at δ 5.92 ppm and the aromatic protons at δ 8.09, 8.18, 8.29, and 8.33 ppm are consistent with the structure of pyrene groups. Moreover, the integration ratios of all peak areas are in good accordance with the chemical structure of 1pyrenemethyl acrylate, confirming the successful synthesis of 1pyrenemethyl acrylate.
Figure 2. TEM images of P(DMA-co-PA) photodegradable micelles (0.25 mg/mL). (a) Before and after irradiation under 365 nm UV with a power output of (b) 20 mW·cm−2 for 1 h, and (c) 50 mW·cm−2 for 30 min in water, (d) 20 mW·cm−2 for 20 min in artificial cytoplasm at 37 °C, respectively. (e) Schematic illustration of photolysis of the P(DMA-co-PA) copolymers. (f) Schematic illustration of the shape transformation of the P(DMA-co-PA) photodegradable micelles in response to photo irradiation. C
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Figure 3. (a) Relative cytoviability of A549 and Raw cells after being incubated with (Groups 2−5) and without (Group 1) micelles for 12 h, treated at different conditions, and further incubated for 24 h. Group 1, photoirradiation only; Group 2, the P(DMA-co-PA) photodegradable micelles only; Group 3, the P(DMA-co-PA) photodegradable micelles and photoirradiation; Group 4, the nonphotodegradable micelles only; Group 5 the nonphotodegradable micelles and photoirradiation. The irradiation was conducted under 365 nm UV light with a power output of 20 mW·cm−2 for 20 min. The concentrations of the photodegradable and nonphotodegradable micelles were 0.25 and 1 mg/mL, respectively. (b) ROS level of A549 and Raw cells after being incubated and treated at the same conditions as illustrated in (a) for the 5 Groups.
efficient concentration of pyrene in the nonphotodegradable micelles was twice as that of the photodegradable ones. For A549 cells, the cytoviability of the Groups 1, 2, and 4 was all above 90%, revealing that the experimental UV irradiation, the photodegradable micelles or the nonphotodegradable micelles alone cause neglectable toxicity to A549 cells. However, the cytoviability of Group 3 was sharply reduced to 13%, indicating that the photodegradable micelles show significant cytotoxicity upon UV irradiation. By contrast, the cytoviability of Group 5 was maintained at 85%, showing that the nonphotodegradable micelles cause very trivial cellular toxicity. A similar alteration pattern was observed for the Raw cells, except that the nondegradable micelles in Group 5 caused rather high cytotoxicity upon UV irradiation. As a photosensitizing dye, pyrene derivative can absorb photons and produce reactive oxygen species (ROS).42−44 To explore the reason for the difference in cytotoxicity, intracellular ROS level was measured. Figure 3b shows that the intracellular ROS level had a positive correlation with the cytotoxicity regardless of the cell types, namely a higher ROS level was found in Group 3, and its value was highest for the Raw cells at the same conditions. Therefore, the pyrene-containing photodegradable micelles can produce rather high level of intracellular ROS to induce significant cytotoxicity under irradiation of UV light, and the efficacy of photosensitization depends on the decomposition ability of the pyrene-containing micelles as well. The nonphotodegradable pyrene-containing micelles produce a lower level of ROS under UV irradiation. However, the photodecomposable pyrene-containing micelles produce a significant higher level of ROS under the same trigger of UV irradiation, which causes the shape transformation of micelles to nanorods simultaneously. It is likely that the process of shape transformation inside cells, and the resulted nanorods are synergistically responsible for the higher production of ROS and thereby the higher cytotoxicity. Of course, the contribution of some unknown intermediate products during the shape transformation can not be ruled out. The present results are very appealing for cancer treatment, because as a carrier the P(DMA-co-PA) micelles can enhance the therapeutic effect in phototherapy. However, as pyrene is known to be cancer-genesis, the present protocol could be considered as a model study for future design of other types of light-triggered nanomaterials such as porphyrin nanostructures.
Incubation of the photodegradable micelles under 365 nm UV light irradiation of 20 mW·cm−2 for 20 min likewise resulted in formation of nanorods with a length of 200−300 nm (Figure 2d). As revealed by the 1H NMR spectrum (Supporting Information Figure S2), the nanorods had the same resonance peaks as those of the Py-CH2OH raw materials. The amount of remained polymers should be very small judging from the very weak peak assigned to PDMA. All the results suggest that the nanorods are formed via the transformation of the micelles triggered by UV irradiation. The transformation of the micelles into nanorods was further evidenced by UV−vis spectra (Supporting Information Figure S3) and fluorescence spectra (Supporting Information Figure S4), revealing the gradual photolysis of the pyrene ester bonds. In this process, the PyCH2OH molecules are gradually liberated from the photodegradable micelles (Figure 2e) to form Py-CH2OH seeds (driven by hydrophobic interaction and π−π stacking10,41) surrounded by the amphiphilic partially photolyzed P(DMA-coPA) copolymers, which prevent from formation of large irregular aggregates. Along with the further photolysis, more liberated Py-CH2OH molecules stack on the seeds until the PyCH2OH nanorods are formed (Figure 2f). Therefore, the applicability of the method of decomposition-induced assembly19,20 is verified through the formation of the nanorods from the photodegradable micelles under irradiation of 365 nm UV light. The cytoviability of the P(DMA-co-PA) photodegradable micelles was then assessed by MTT assay by using nonphotodegradable micelles as a control, which were synthesized according to a reported approach20 (Supporting Information Figure S5). In this regard, five groups (Figure 3a) were investigated. The Group 1 was treated with 365 nm UV irradiation only (20 mW·cm−2 for 20 min). The Group 2 was treated with the P(DMA-co-PA) photodegradable micelles only (0.25 mg/mL). The Group 3 was treated with 365 nm UV irradiation (20 mW·cm−2 for 20 min) after adding the P(DMAco-PA) photodegradable micelles (0.25 mg/mL). The Group 4 was treated with the nonphotodegradable micelles only (1 mg/ mL). The Group 5 was treated with 365 nm UV irradiation (20 mW·cm−2 for 20 min) after adding the nonphotodegradable micelles (1 mg/mL). Because the grafting ratio of pyrene in the P(DMA-co-PA) and P(DMA-co-PA) concentration were 2 and 1/4 times of the nondegradable micelles, respectively, the D
DOI: 10.1021/acs.biomac.5b00497 Biomacromolecules XXXX, XXX, XXX−XXX
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(6) Reincke, F.; Kegel, W. K.; Zhang, H.; Nolte, M.; Wang, D. Y.; Vanmaekelbergh, D.; Mohwald, H. Phys. Chem. Chem. Phys. 2006, 8, 3828−3835. (7) Liu, H. B.; Li, Y. L.; Xiao, S. Q.; Gan, H. Y.; Jiu, T. G.; Li, H. M.; Jiang, L.; Zhu, D. B.; Yu, D. P.; Xiang, B.; Chen, Y. F. J. Am. Chem. Soc. 2003, 125, 10794−10795. (8) Yoon, S. M.; Hwang, I.-C.; Kim, K. S.; Choi, H. C. Angew. Chem., Int. Ed. 2009, 48, 2506−2509. (9) Qiu, Y.; Chen, P.; Liu, M. J. Am. Chem. Soc. 2010, 132, 9644− 9652. (10) Zhang, X. J.; Zhang, X. H.; Shi, W. S.; Meng, X. M.; Lee, C.; Lee, S. J. Phys. Chem. B 2005, 109, 18777−18780. (11) Sun, X.; Qiu, L.; Cai, Z.; Meng, Z.; Chen, T.; Lu, Y.; Peng, H. Adv. Mater. 2012, 24, 2906−2910. (12) Kim, J.-K.; Lee, E.; Lee, M. Angew. Chem., Int. Ed. 2006, 45, 7195−7198. (13) Kar, S.; Wu, K.-W.; Hsu, I. J.; Lee, C.-R.; Tai, Y. Chem. Commun. 2014, 50, 2638−2641. (14) Yagai, S.; Iwai, K.; Yamauchi, M.; Karatsu, T.; Kitamura, A.; Uemura, S.; Morimoto, M.; Wang, H.; Wurthner, F. Angew. Chem., Int. Ed. 2014, 53, 2602−2606. (15) Wang, T. X.; Cai, Y. B.; Wang, Z. P.; Guan, E. J.; Yu, D. H.; Qin, A. J.; Sun, J. Z.; Tang, B. Z.; Gao, C. Y. Macromol. Rapid Commun. 2012, 33, 1584−1589. (16) Jiang, L. X.; Yan, Y.; Drechsler, M.; Huang, J. B. Chem. Commun. 2012, 48, 7347−7349. (17) Ercole, F.; Davis, T. P.; Evans, R. A. Polym. Chem. 2010, 1, 37− 54. (18) Yan, H.; Long, Y.; Song, K.; Tung, C. H.; Zheng, L. Q. Soft Matter 2014, 10, 115−121. (19) Wang, Z.; Mohwald, H.; Gao, C. ACS Nano 2011, 5, 3930− 3936. (20) Wang, H. S.; Yu, W.; Zhang, W. B.; Gao, C. Y. Macromol. Biosci. 2014, 14, 1748−1754. (21) Zhao, Y. Macromolecules 2012, 45, 3647−3657. (22) Schumers, J.-M.; Fustin, C.-A.; Gohy, J.-F. Macromol. Rapid Commun. 2010, 31, 1588−1607. (23) Jiang, J. Q.; Tong, X.; Zhao, Y. J. Am. Chem. Soc. 2005, 127, 8290−8291. (24) Mack, E. T.; Birzniece, D.; Veach, D. R.; Coyle, W.; Wilson, R. M. Bioorg. Med. Chem. Lett. 2005, 15, 2173−2176. (25) Dong, S.; Wang, S.; Stewart, G.; Hwang, H.-M.; Fu, P. P.; Yu, H. Int. J. Mol. Sci. 2002, 3, 937−947. (26) Hariharan, M.; Karunakaran, S. C.; Ramaiah, D.; Schulz, I.; Epe, B. Chem. Commun. 2010, 46, 2064−2066. (27) Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. J. Natl. Cancer Inst. 1998, 90, 889−905. (28) Lorenzo, C. A.; Bromberg, L.; Concheiro, A. Photochem. Photobiol. 2009, 85, 848−860. (29) McQuade, C.; Zaki, A. A.; Desai, Y.; Vido, M.; Sakhuja, T.; Cheng, Z. L.; Hickey, R. J.; Joh, D.; Park, S. J.; Kao, G.; Dorsey, J. F.; Tsourkas, A. Small 2015, 11, 834−843. (30) Rocha, L.; Paius, C. M.; Raicu, A. L.; Resmerita, E.; Rusu, A.; Moleavin, I. A.; Hamel, M.; Nichita, N. B.; Hurduc, N. J. Photochem. Photobiol., A 2014, 291, 16−25. (31) Lee, S. Y.; Lee, H.; In, I.; Park, S. Y. Eur. Polym. J. 2014, 57, 1− 10. (32) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113−131. (33) Xu, H. L.; Yao, Q.; Cai, C. F.; Gou, J. X.; Zhang, Y.; Zhong, H. J.; Tang, X. J. Controlled Release 2015, 199, 84−97. (34) Zhang, W. P.; Sun, J.; Fang, W. H.; Ai, X. Y.; Cai, C. F.; Tang, Y. L.; Su, X. N.; Feng, Z.; Liu, Y.; Tao, M. Y.; Yan, X. D.; Chen, G. L.; He, Z. G. Eur. J. Pharm. Sci. 2015, 66, 96−106. (35) Wang, J. L.; Li, L.; Du, Y. Q.; Sun, J.; Han, X. P.; Luo, C.; Ai, X. Y.; Zhang, Q.; Wang, Y. J.; Fu, Q.; Yang, Z. F.; He, Z. G. Mol. Pharmaceutics 2015, 12, 463−473.
On the other hand, it is particularly important for unveiling the function of smart micelles in drug delivery, because the structure variation of the smart carriers may bring some unpredictable effects that are often neglected and are indiscriminately attributed to the carried cargos. Attention thereby should be paid when the smart carriers are applied for delivery of functional substances, in particular for those with bioactive functions to improve the cytoviability and to endow the cells with biological functions, for example, gene transfection.
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CONCLUSIONS The amphiphilic P(DMA-co-PA) random copolymers with photoresponsive pyrene ester bonds were synthesized through copolymerization of hydrophobic 1-pyrenemethyl acrylate and hydrophilic DMA. The amphiphilic copolymers formed spherical photodegradable micelles in water, which were then transformed into nanorods accompanying with the cleavage of the pyrene ester bonds. The shape transformation of the photodegradable micelles took place similarly under biomimetic conditions of artificial cytoplasm. Under 365 nm UV irradiation, the photodegradable micelles cocultured with A549 cells resulted in significantly higher cytotoxicity than the nonphotodegradable ones. The intracellular ROS level had a positive correlation with the cytotoxicity regardless of the cell types. It is likely that the process of shape transformation inside cells, and the resulted nanorods, are synergistically responsible for the higher production of ROS and thereby the higher cytotoxicity. The results demonstrate that the P(DMA-co-PA) micelles possess higher cytotoxicity upon photoexcitation, especially to cancer cells. Special attention should be paid to the smart carriers when they are applied for delivery of functional substances into cells.
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ASSOCIATED CONTENT
S Supporting Information *
Integral of peak intensity in 1H NMR spectrum of P(DMA-coPA) random copolymers, FTIR spectra of formed nanorods and Py-CH2OH, UV−vis and fluorescence spectra monitoring the shape transformation process, and molecular structure of nonphotodegradable pyrene-containing micelles. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00497.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by Ph.D. Programs Foundation of Ministry of E ducation of China (20110101130005) and the Natural Science Foundation of China (51120135001).
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