载药蛋白质/聚苯硼酸复合纳米微球制备及其释药性能研究

doi:10.6023/A22110464

摘要/Abstract

蛋白质纳米颗粒具有良好的生物相容性和生物降解性, 易于进行额外的表面修饰, 用作药物输送系统提高了生物利用度, 减少了药物分子的毒副作用. 本工作在利用苯硼酸基团与再生桑蚕丝蛋白(RSF)上相关侧基之间具有路易斯酸-碱配对反应的基础上, 通过3-丙烯酰胺苯硼酸(APBA)在RSF水溶液中原位聚合, 使RSF分子链重排形成微球并在表面负载抗炎中药, 制备了载药丝蛋白/聚苯硼酸纳米微球. 此尺寸分布均匀的微球直径约为550~600 nm, 表面光滑且在水中的分散性能良好; 对乔松素、杜鹃素和地奥司明三种药物的负载率分别为7.8%, 11.9%和13.4%, 包封率分别为75.0%, 89.1%和93.7%. 载药微球控制释放约7 d, 且缓释行为具有pH响应性. 丝蛋白/聚苯硼酸纳米微球与主体药物协同作用提高了自由基清除速度和清除效率, 优于直接给药组. 与此同时, 将RSF改换为牛血清白蛋白或明胶蛋白, 采用此方法也能制成尺寸分别为260和100 nm的白蛋白/聚苯硼酸微球或明胶蛋白/聚苯硼酸微球. 由此, 三种不同尺寸、电性和药物释放速率的蛋白质/聚苯硼酸纳米微球有望适应多种静脉注射和皮下或腹腔注射药物传输的需求.

关键词: 蛋白质, 苯硼酸, 抗炎药, 纳米载体, 控制释放

Protein nanoparticles (NPs), biocompatible and biodegradable, can be easily surface modified. In particular, amphiphilic proteins act as “surfactants” that help form microparticles while undergoing molecular chain rearrangement. These NPs have been successfully used as drug delivery systems, improving bioavailability and reducing the toxic effects of drug molecules. The use of regenerated silk protein (RSF) with m-acrylamidophenylboronic acid (APBA) composites as drug carriers for loading anti-inflammatory herbal extracts was reported. Firstly, a simple and rapid method was used to prepare silk protein/polyphenylboronic acid nanospheres, in brief, RSF solution and a certain amount of initiator were added to APBA solution, and the pH was adjusted by NaOH, and the polymerization was initiated by heating at 90 ℃ under nitrogen protection with stirring at 500 r/min. After 2 h of reaction, a milky solution was obtained, which formed silk protein/benzeneboronic acid nanospheres with hydrophobic interior and hydrophilic surface. The drug-loaded silk protein/polyphenylboronic acid nanospheres with an average size of 550 to 600 nm were prepared by mixing with the drug solution after dialysis and stirring at room temperature for 12 h to load the drug by adsorption. By the same method, drug-loaded albumin/polyphenylboronic acid microspheres and collagen/polyphenylboronic acid microspheres with sizes around 260 nm and 100 nm, respectively, could be prepared. The results observed by scanning and projection electron microscopy and dynamic light scattering showed that the drug-loaded silk protein/polyphenylboronic acid nanomicrospheres displayed regular spherical shape indicating smoothness and good dispersion with no obvious aggregation. The highest drug loading rate was about 13.4%, and the encapsulation rate was over 90%. Also, such drug-loaded nanospheres could achieve controlled release for about seven days and their slow release behavior was pH-responsive, with faster drug release in buffer solution at pH=5.5 than in buffer solution at pH=7.4. In addition, the synergistic interaction of the silk protein/polyphenylboronic acid nanomicrospheres with the subject drug improved its free radical scavenging rate and scavenging efficiency, which was superior to that of the direct drug delivery group. Thus, three protein/polyphenylboronic acid nanomicrospheres with different sizes, electrical properties and drug release rates may be adaptable to a wide range of intravenous and subcutaneous or intraperitoneal drug delivery needs and have great potential for clinical applications.

Key words: proteins, phenylboronic acid, anti-inflammatory drugs, nanocarriers, controlled release

引用此文

殷雪旸, 顾恺, 邵正中. 载药蛋白质/聚苯硼酸复合纳米微球制备及其释药性能研究[J]. 化学学报, 2023, 81(2): 116-123.

Xueyang Yin, Kai Gu, Zhengzhong Shao. Preparation of the Protein/Polyphenylboronic Acid Nanospheres for Drug Loading and Unloading[J]. Acta Chimica Sinica, 2023, 81(2): 116-123.

导出引用 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

分享此文

图/表 6

图1. RSF/聚苯硼酸纳米微球的扫描电镜(a)和透射电镜(b)图像以及分别负载乔松素(c, d)、杜鹃素(e, f)和地奥司明(g, h)的RSF/聚苯硼酸纳米微球的扫描电镜和透射电镜图像

Figure 1. SEM and TEM images of RSF-PAPBA (a, b), Pin@RSF-PAPBA (c, d), Far@RSF-PAPBA (e, f), and Dio@RSF-PAPBA (g, h) nanospheres

Sample	Hydrodynamic diameter/nm	PDI	Zeta potential/mV
RSF-PAPBA	540	0.04	–23.0
Pin@RSF-PAPBA	590	0.06	–31.0
Far@RSF-PAPBA	560	0.09	–32.5
Dio@RSF-PAPBA	570	0.07	–30.2
RSF	540	0.12	–23.3
Far@RSF	290	0.18	–34.4

表1. 载药前后RSF纳米微球的流体力学直径、多分散性指数和Zeta电位

Table 1. Hydrodynamic diameter (nm), polydispersity index (PDI) and Zeta potential (mV) of RSF nanospheres before or after drug loading

图2. (a) BSA-PAPBA、(b) Far@BSA-PAPBA、(c) GEL-PAPBA和(d) Far@GEL-PAPBA纳米微球的扫描电镜图像

Figure 2. SEM images of BSA-PAPBA (a), Far@BSA-PAPBA (b), GEL-PAPBA (c) and Far@GEL-PAPBA (d) nanospheres

Sample	Hydrodynamic diameter/nm	PDI	Zeta potential/mV
BSA-PAPBA	260	0.05	–24.2
Far@BSA-PAPBA	290	0.09	–46.2
GEL-PAPBA	170	0.04	+21.5
Far@GEL-PAPBA	180	0.06	+14.3

表2. 载药前后BSA-PAPBA和GEL-PAPBA纳米微球的流体力学直径、多分散性指数和Zeta电位

Table 2. Hydrodynamic diameter (nm), polydispersity index (PDI) and Zeta potential (mV) of BSA-PAPBA or GEL-PAPBA nanospheres before and after drug loading

图3. 负载药物的蛋白质/聚苯硼酸纳米微球在磷酸缓冲溶液中的释放曲线. (a) 负载杜鹃素的不同微球在pH=5.5时的释放曲线; (b) 负载杜鹃素的RSF-PAPBA 微球在不同pH下的释放曲线; (c, d) pH=7.4 和pH=5.5时, 载药RSF-PAPBA对乔松素、杜鹃素和地奥司明三种不同药物的释放曲线

Figure 3. Release profiles of drug loaded protein/polyphenylboronic acid nanospheres in phosphate buffer solution. (a) Release profiles of different microspheres loaded with farrerol at pH=5.5; (b) release profiles of RSF-PAPBA microspheres loaded with azathioprine at different pH; (c, d) release profiles of drug loaded RSF-PAPBA for three different drugs, namely, pinocembrin, farrerol and diosmin, at pH=7.4 and pH=5.5

图4. 药物及其对应的RSF-PAPBA载药纳米微球在30 min内的DPPH自由基清除效果的对比照片(a, c, e)和定量曲线(b, d, f)

Figure 4. The photographs (a, c, e) and quantitative curves (b, d, f) of the scavenging effects regarding DPPH radical of the corresponded drug and its RSF-PAPBA loaded nanospheres within 30 min

参考文献 47

[1]	Pahwa, R.; Goyal, A.; Jialal, I. Chronic Inflammation. Vol. 1, Ed.: Bansal, P., StatPearls, Petersburg, 2020, p. 7.
[2]	Luo, W. X.; Wen, G.; Yang, L.; Tang, J.; Wang, J. G.; Wang, J. H.; Zhang, S. Y.; Zhang, L.; Ma, F.; Xiao, L. L.; Wang, Y.; Li, Y. Theranostics. 2017, 7, 452. doi: 10.7150/thno.16677
[3]	Kamimura, M.; Furukawa, T.; Akiyama, S.; Nagasaki, Y. Biomater. Sci. 2013, 1, 361. doi: 10.1039/c2bm00156j
[4]	Davis, M. E.; Brewster, M. E. Nat. Rev. Drug Discov. 2004, 3, 1023. doi: 10.1038/nrd1576 pmid: 15573101
[5]	Lee, C. C.; MacKay, J. A.; Frechet, J. M. J.; Szoka, F. C. Nat. Biotechnol. 2005, 23, 1517. doi: 10.1038/nbt1171
[6]	Serajuddin, A. T. M. Adv. Drug Deliver. Rev. 2007, 59, 603. doi: 10.1016/j.addr.2007.05.010 pmid: 17619064
[7]	Akers, M. J. J. Pharm. Sci. 2002, 91, 2283. doi: 10.1002/jps.10154
[8]	Gregoriadis, G.; Wills, E. J.; Swain, C. P.; Tavill, A. S. Lancet. 1974, 303, 1313. doi: 10.1016/S0140-6736(74)90682-5
[9]	Juliano, R. L. Prog. Clin. Biol. Res. 1976, 9, 21. pmid: 1030801
[10]	Kobayashi, H.; Turkbey, B.; Watanabe, R.; Choyke, P. L. Bioconjugate Chem. 2014, 25, 2093. doi: 10.1021/bc500481x pmid: 25385142
[11]	Mitchell, M. J.; Jain, R. K.; Langer, R. Nat. Rev. Cancer. 2017, 17, 659. doi: 10.1038/nrc.2017.83 pmid: 29026204
[12]	Zheng, P.; Yang, L.; Chen, J. J.; Xu, W. G.; Li, G.; Ding, J. X. Chinese Chem. Lett. 2020, 31, 1178. doi: 10.1016/j.cclet.2019.12.001
[13]	Kosheli, T. M.; George, F. B.; Li, X. T.; Chen, Z. J.; He, W. Chinese Chem. Lett. 2022, 33, 587. doi: 10.1016/j.cclet.2021.08.020
[14]	Saha, A.; Pradhan, N.; Chatterjee, S.; Singh, R. K.; Trivedi, V.; Bhattacharyya, A.; Manna, D. ACS Appl. Nano Mater. 2019, 2, 3671. doi: 10.1021/acsanm.9b00607
[15]	Xie, M.; Fan, D.; Chen, Y.; Zhao, Z.; He, X.; Li, G.; Lan, P. Biomaterials. 2016, 103, 33. doi: 10.1016/j.biomaterials.2016.06.049
[16]	Wang, S.; Xu, T.; Yang, Y.; Shao, Z. ACS Appl. Mater. Interfaces. 2015, 7, 21254. doi: 10.1021/acsami.5b05335
[17]	Shao, Z. Z.; Vollrath, F. Nature. 2002, 418, 741. doi: 10.1038/418741a
[18]	Wu, M.; Yang, W.; Chen, S.; Yao, J.; Shao, Z.; Chen, X. J. Mater. Chem. B. 2018, 6, 1179. doi: 10.1039/C7TB03113K
[19]	Sun, N.; Lei, R.; Xu, J. H.; Kundu, S. C.; Cai, Y. R.; Yao, J. M.; Ni, Q. Q. J. Mater. Sci. 2019, 54, 3319. doi: 10.1007/s10853-018-3022-9
[20]	Yang, W. H.; Yu, S. Y.; Chen, S.; Liu, Y. Z.; Shao, Z. Z.; Chen, X. Acta Chim. Sinica. 2014, 72, 1164. (in Chinese) doi: 10.6023/A14080596
	(杨文华, 俞淑英, 陈胜, 刘也卓, 邵正中, 陈新, 化学学报, 2014, 72, 1164.) doi: 10.6023/A14080596
[21]	Wang, S. H.; Sun, L. N.; Cao, H.; Zhong, Y. M.; Shao, Z. Z. Acta Chim. Sinica. 2021, 79, 1023. (in Chinese) doi: 10.6023/A21050203
	(王苏杭, 孙灵娜, 曹涵, 钟一鸣, 邵正中, 化学学报, 2021, 79, 1023.) doi: 10.6023/A21050203
[22]	Huang, H.; Yang, D. P.; Liu, M. H.; Wang, X. S.; Zhang, Z. Y.; Zhou, G. D.; Liu, W.; Cao, Y. L.; Zhang, W. J.; Wang, X. S. Int. J. Nanomed. 2017, 12, 2829. doi: 10.2147/IJN.S128270 pmid: 28435261
[23]	Lu, H. X.; Noorani, L.; Jiang, Y. Y.; Du, A. W.; Stenzel, M. H. J. Mater. Chem. B. 2017, 5, 9591. doi: 10.1039/C7TB02902K
[24]	Yu, Z.; Yu, M.; Zhang, Z. B. Nanoscale Res. Lett. 2014, 9, 343. doi: 10.1186/1556-276X-9-343 pmid: 25114637
[25]	Azimi, B.; Nourpanah, P.; Rabiee, M.; Arbab, S. Iran Biomed. J. 2014, 18, 34.
[26]	Sahoo, N.; Sahoo, R. K.; Biswas, N.; Guha, A.; Kuotsu, K. Int. J. Biol. Macromol. 2015, 81, 317. doi: 10.1016/j.ijbiomac.2015.08.006
[27]	Kulkarni, S. A.; Feng, S. S. Pharm. Res. 2013, 30, 2512. doi: 10.1007/s11095-012-0958-3
[28]	Lee, B. R.; Ko, H. K.; Ryu, J. H.; Ahn, K. Y.; Lee, Y. H.; Oh, S. J.; Na, J. H.; Kim, T. W.; Byun, Y.; Kwon, I. C.; Kim, K.; Lee, J. Sci. Rep. 2016, 6, 35182. doi: 10.1038/srep35182
[29]	Ci, X.; Chu, X.; Wei, M.; Yang, X.; Cai, Q.; Deng, X. PLoS One. 2012, 7, e34634. doi: 10.1371/journal.pone.0034634
[30]	Fattori, V.; Rasquel-Oliveira, F. S.; Artero, N. A.; Ferraz, C. R.; Borghi, S. M.; Casagrande, R.; Verri, W. A. J. Nat. Prod. 2020, 83, 1018. doi: 10.1021/acs.jnatprod.9b00887 pmid: 32083866
[31]	Stephan, D. W. Acc. Chem. Res. 2015, 48, 306. doi: 10.1021/ar500375j
[32]	Chen, H.; Shao, Z. Z. Acta Polym. Sinica. 2018, 62, 987. (in Chinese)
	(陈红, 邵正中, 高分子学报, 2018, 62, 987.)
[33]	Chen, W.; Zhen, X.; Wu, W. Sci. China Chem. 2020, 63, 648. doi: 10.1007/s11426-019-9699-3
[34]	Wang, L. J.; Sheng, X. L.; Wang, J.; Zhang, Y. H. Chinese J. Org. Chem. 2021, 41, 567. (in Chinese) doi: 10.6023/cjoc202006060
	(王李娟, 盛显良, 王杰, 张玉辉, 有机化学, 2021, 41, 567.) doi: 10.6023/cjoc202006060
[35]	Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Kataoka, K. Nat. Nanotechnol. 2011, 6, 815. doi: 10.1038/nnano.2011.166 pmid: 22020122
[36]	Jiang, W.; Kim, B. Y.; Rutka, J. T.; Chan, W. C. Nat. Nanotechnol. 2008, 3, 145. doi: 10.1038/nnano.2008.30 pmid: 18654486
[37]	Reddy, S. T.; Van Der Vlies, A. J.; Simeoni, E.; Angeli, V.; Randolph, G. J.; O'Neil, C. P.; Hubbell, J. A. Nat. Biotechnol. 2007, 25, 1159. doi: 10.1038/nbt1332 pmid: 17873867
[38]	Wang, H.; Wang, S.; Su, H.; Chen, K. J.; Armijo, A. L.; Lin, W. Y.; Tseng, H. R. Angew. Chem. Int. Ed. 2009, 48, 4344. doi: 10.1002/anie.200900063 pmid: 19425037
[39]	Goodman, T. T.; Olive, P. L.; Pun, S. H. Int. J. Nanomed. 2007, 2, 265.
[40]	Tang, L.; Yang, X.; Dobrucki, L. W.; Chaudhury, I.; Yin, Q.; Yao, C.; Cheng, J. Angew. Chem. Int. Ed. 2012, 51, 12721. doi: 10.1002/anie.201205271 pmid: 23136130
[41]	Tang, L.; Yang, X.; Yin, Q.; Cai, K.; Wang, H.; Chaudhury, I.; Yao, C.; Zhou, Q.; Kwon, M.; Hartman, J. A.; Dobrucki, I. T.; Dobrucki, L. W.; Borst, L. B.; Lezmi, S.; Helferich, W. G.; Ferguson, A. L.; Fan, T. M.; Cheng, J. Proc. Natl. Acad. Sci. 2014, 111, 15344. doi: 10.1073/pnas.1411499111
[42]	Tan, H. Y.; Wang, N.; Li, S.; Hong, M.; Wang, X.; Feng, Y. Oxid. Med. Cell. Longev. 2016, 279, 5090.
[43]	Chen, L.; Deng, H.; Cui, H.; Fang, J.; Zuo, Z.; Deng, J.; Li, Y.; Wang, X.; Zhao, L. Oncotarget. 2018, 9, 7204. doi: 10.18632/oncotarget.23208
[44]	Matsumoto, A.; Sato, N.; Kataoka, K.; Miyahara, Y. J. Am. Chem. Soc. 2009, 131, 12022. doi: 10.1021/ja902964m pmid: 19663494
[45]	Wang, J.; Wu, W.; Zhang, Y.; Wang, X.; Qian, H.; Liu, B.; Jiang, X. Biomaterials. 2014, 35, 866. doi: 10.1016/j.biomaterials.2013.10.028
[46]	Wang, H.; Wang, K.; Sun, J.; Fang, G. Q.; Yao, Q. Q.; Wu, Z. Y. Chinese J. Org. Chem. 2018, 38, 1035. (in Chinese) doi: 10.6023/cjoc201709037
	(王浩, 王凯, 孙捷, 方桂迁, 姚庆强, 吴忠玉, 有机化学, 2018, 38, 1035.) doi: 10.6023/cjoc201709037
[47]	Cao, Z. B.; Chen, X.; Yao, J. R.; Huang, L.; Shao, Z. Z. Soft Matter. 2007, 3, 910. doi: 10.1039/b703139d