壳聚糖及其衍生物的修饰对pH敏感型PLGA微粒性质及其在巨噬细胞内分布的影响
|
摘要
研究了壳聚糖及其衍生物的修饰对pH敏感型聚乳酸-羟基乙酸(PLGA)微粒性质及其在巨噬细胞中分布的影响.通过微孔膜乳化技术与复乳溶剂挥发法相结合制备了壳聚糖及其衍生物修饰的pH敏感型PLGA微粒,用单因素法对pH敏感型PLGA微粒的制备工艺进行了优化,以Bradford protein assay法测定微粒的蛋白含量,用扫描电子显微镜观察了微粒的形态结构,用马尔文粒径仪测定了微粒的粒径和zeta电位,用台盼蓝染色法测定了微粒的细胞毒性,并利用荧光标记法确定了微粒在巨噬细胞内的分布.结果发现,4种微粒粒径为1 000～1 200 nm;壳聚糖及其衍生物修饰后的微粒包封率明显上升,zeta电位显著升高,壳聚糖修饰的微粒主要分布在细胞质,而未被修饰的微粒主要分布在溶酶体内.
This study investigated the effect of surface modification with chitosan and its derivatives on the characteristics of pH-sensitive poly(lactic-co-glycolic acid)(PLGA) microparticles and their distribution in macrophages.By combination of microporous shirasu porous glass and double emulsion-solvent evaporation method,the pH-sensitive PLGA microparticles modified by chitosan and its derivatives were prepared.The single factor method was used to optimize the preparation of pH-sensitive PLGA microparticles.The protein content of the microparticles was determined by Bradford assay.The morphological structure of the microparticles was observed with a scanning electron microscope.The particle size and zeta potential of the microspheres were measured with a Malvern particle size analyzer and the cytotoxicity of the microparticles was determined by trypan blue staining.The distribution of the microparticles within macrophages was determined by a fluorescent labeling method.The results showed that the size of four kinds of particles was 1 000～1 200 nm.Compared with unmodified microparticles,entrapment efficiency and zeta potential of the microparticles modified with chitosan and its derivatives significantly increased.The microparticles modified with chitosan distributed primarily in cytoplasm,whereas the unmodified microparticles mainly distributed in lysosomes.
This study investigated the effect of surface modification with chitosan and its derivatives on the characteristics of pH-sensitive poly(lactic-co-glycolic acid)(PLGA) microparticles and their distribution in macrophages.By combination of microporous shirasu porous glass and double emulsion-solvent evaporation method,the pH-sensitive PLGA microparticles modified by chitosan and its derivatives were prepared.The single factor method was used to optimize the preparation of pH-sensitive PLGA microparticles.The protein content of the microparticles was determined by Bradford assay.The morphological structure of the microparticles was observed with a scanning electron microscope.The particle size and zeta potential of the microspheres were measured with a Malvern particle size analyzer and the cytotoxicity of the microparticles was determined by trypan blue staining.The distribution of the microparticles within macrophages was determined by a fluorescent labeling method.The results showed that the size of four kinds of particles was 1 000～1 200 nm.Compared with unmodified microparticles,entrapment efficiency and zeta potential of the microparticles modified with chitosan and its derivatives significantly increased.The microparticles modified with chitosan distributed primarily in cytoplasm,whereas the unmodified microparticles mainly distributed in lysosomes.
引文
[1] LI Z,XIONG F F,HE J T.Surface-functionalized pH-responsive Poly(lactic-co-glycolic Acid)-based Microparticles for Intranasal Vaccine Delivery:Effect of Surface Modification with Chitosan and Mannan[J].Elsevier,2006,109:24-34.doi:10.1016/j.ejpb.2006.08.012
[2] KE C J,SU T Y,CHEN H L,et al.Smart Multifunctional Hollow Microspheres for the Quick Release of Drugs in Intracellular Lysosomal Compartments[J].Angew Chem Int Ed,2011,123:8236-8239.doi:10.1002/ange.201102852
[3] HEFFERNAN M J,KASTURI S P,YANG S C,et al.The Stimulation of CD8+ T Cells by Dendritic Cells Pulsed with Polyketal Nanoparticles Containing Ion-paired Protein Antigen and Poly(inosinic Acid)-poly(cytidylic Acid)[J].Biomaterials,2009,30:910-918.doi:10.1016/j.biomaterials.2008.10.034
[4] WILSON J T,KELLER S,MANGANIELLO M J,et al.pH-responsive Nanoparticles Vaccines for Dualdelivery of Antigens and Immunostimulatory Oligonucleotides[J].ACS Nano,2013(5):3912-3925.doi:10.1021/nn3054662
[5] HAMDY S,HADDADI A,HUNG R W,et al.Targeting Dendritic Cells with Nano-particulate PLGA Cancer Vaccine Formulations[J].Adv Drug Deliv Rev,2011,63(5):943-955.doi:10.1016/j.addr.2011.05.021
[6] LELEUX J,ROY K.Micro and Nanoparticle-based Delivery Systems for Vaccine Immunotherapy:An Immunological and Materials Perspective[J].Adv Healthcare Mater,2013,2(1):72-94.doi:10.1002/adhm.201200268
[7] 郑金琪,王思玲,苏德森.脂质体膜修饰物的应用进展[J].沈阳药科大学学报,2003,20(6):460-464.doi:10.3969/j.issn.1006-2858.2003.06.021ZHENG Jinqi,WANG Siling,SU Desen.Application Advance in the Modifying Compounds of the Membranes of Liposomes[J].Journal of Shenyang Pharmaceutical University,2003,20(6):460-464.
[8] 邹家龙,罗顺德,韩瑞玲.壳聚糖表面修饰PLGA纳米粒对小鼠骨髓系树突细胞交叉递呈的影响[J].中国医院药学杂志,2013,33(3):192-195.doi:10.13286/j.cnki.chinhosppharmacyj.2013.03.021ZOU Jialong,LUO Shunde,HAN Ruiling.Effects of Chitosan Coating PLGA Nanoparticles on Cross-presentation by Murine Bone Marrow-derivde Dendritic Cells[J].Chin Hosp Pharm,2013,33(3):192-195.
[9] TRIF M,FLORIAN P E,ROSEANU A,et al.Cytotoxicity and Intracellular Fate of PLGA and Chitosan-coated PLGA Nanoparticles in Madin-darby Bovine Kidney (MDBK) and Human Colorectal Adenocarcinoma (Colo 205) Cells[J].SFB,2015,103A:3599-3611.doi:10.1002/jbm.a.35498
[10] NAKASHIMA T,SHIMIZU M,MASATO K.Membrane Emulsification by Microprous Glass[J].Key Eng Mater,1991,61:513-516.doi:10.4028/www.scientific.net/KEM.61-62.513
[11] WEI Q,WEI W,LAI B.Uniform-sized PLA Nanoparticles:Preparation by Premix Membrane Emulsification[J] Int J Pharm,2008,359(3):294-297.doi:10.1016/j.ijpharm.2008.03.027
[12] GHADERI R,ARTURSSON P,CARLFORS J.Preparation of Biodegradable Microparticles Using Solution-enhanced Dispersion by Supercritical Fliuds (SEDS)[J].Pharm Res,1999(6):676-681.doi:10.1023/a.1018868423309
[13] CHOUINID F,BUCZKOWSKI S,LENAERTS V.Poly(alkyl-cyanoacrylate) Nanocapsules:Physicochemical Characterization and Mechanism of Formation[J].Pharm Res,1994(11):869-874.doi:10.1023/a.1018938026615
[14] CASTELLANOS I J,ALAZZAM W,GRIEBENOW K.Effect of the Covalent Modification with Poly(ethylene Glycol) on Alphachymotrypsin Stability Upon Encapsulation in Poly(lactic-coglycolic) Microspheres[J].J Pharm Sci,2005,94:327-340.doi:10.1002/jps.20243
[15] 周香莲,贺进田,周志涛,等.外水相中NaCl对S/O/W法制备的牛血清白蛋白PLGA缓释微粒性质的影响[J].药学学报,2010,45(8):1057-1063.doi:10.16438/j.0513-4870.2010.08.019ZHOU Xianglian,HE Jintian,ZHOU Zhitao,et al.Effect of NaCl in Outer Water Phase on the Characteristics of BSA Loaded PLGA Sustained-release Microspheres Fabricated by A Solid-in-oil-in-water Emulsion Technique[J].Acta Pharm Sin,2010,45(8):1057-1063.
[16] EMAMI J,HAMISHEHKAR H,NAJAFABADI A R,et al.A Novel Approach to Prepare Insulin-loaded Poly(lactic-co-glycolic Acid) Microcapsules and the Protein Stability Study[J].J Pharm Sci,2009,98(4):1712-1731.doi:10.1002/jps.21544
[17] 田瑞,王连艳,吴颉,等.快速膜乳化法制备粒径均一的PLGA微粒和微囊[J].过程工程学报,2009,9(4):754-762.doi:10.3321/j.issn.1009-606x.2009.04.021TAN Rui,WANG Lianyan,WU Jie,et al.Preparation of Uniform Size PLGA Microparticles and Microcapsules by Premix Membrane Emulsification[J].JCIE,2009,9(4):754-762.
[18] 马方奎.基于壳聚糖纳米载体的构建及其水解释药研究[D].青岛:中国海洋大学,2013.doi:10.7666/d.D328948MA Fangkui.Chitosan-PLGA Nanocarrier Systmes for Hydrolytic Erosion and Drug Release[D].Qingdao:Ocean University of China,2013.
[2] KE C J,SU T Y,CHEN H L,et al.Smart Multifunctional Hollow Microspheres for the Quick Release of Drugs in Intracellular Lysosomal Compartments[J].Angew Chem Int Ed,2011,123:8236-8239.doi:10.1002/ange.201102852
[3] HEFFERNAN M J,KASTURI S P,YANG S C,et al.The Stimulation of CD8+ T Cells by Dendritic Cells Pulsed with Polyketal Nanoparticles Containing Ion-paired Protein Antigen and Poly(inosinic Acid)-poly(cytidylic Acid)[J].Biomaterials,2009,30:910-918.doi:10.1016/j.biomaterials.2008.10.034
[4] WILSON J T,KELLER S,MANGANIELLO M J,et al.pH-responsive Nanoparticles Vaccines for Dualdelivery of Antigens and Immunostimulatory Oligonucleotides[J].ACS Nano,2013(5):3912-3925.doi:10.1021/nn3054662
[5] HAMDY S,HADDADI A,HUNG R W,et al.Targeting Dendritic Cells with Nano-particulate PLGA Cancer Vaccine Formulations[J].Adv Drug Deliv Rev,2011,63(5):943-955.doi:10.1016/j.addr.2011.05.021
[6] LELEUX J,ROY K.Micro and Nanoparticle-based Delivery Systems for Vaccine Immunotherapy:An Immunological and Materials Perspective[J].Adv Healthcare Mater,2013,2(1):72-94.doi:10.1002/adhm.201200268
[7] 郑金琪,王思玲,苏德森.脂质体膜修饰物的应用进展[J].沈阳药科大学学报,2003,20(6):460-464.doi:10.3969/j.issn.1006-2858.2003.06.021ZHENG Jinqi,WANG Siling,SU Desen.Application Advance in the Modifying Compounds of the Membranes of Liposomes[J].Journal of Shenyang Pharmaceutical University,2003,20(6):460-464.
[8] 邹家龙,罗顺德,韩瑞玲.壳聚糖表面修饰PLGA纳米粒对小鼠骨髓系树突细胞交叉递呈的影响[J].中国医院药学杂志,2013,33(3):192-195.doi:10.13286/j.cnki.chinhosppharmacyj.2013.03.021ZOU Jialong,LUO Shunde,HAN Ruiling.Effects of Chitosan Coating PLGA Nanoparticles on Cross-presentation by Murine Bone Marrow-derivde Dendritic Cells[J].Chin Hosp Pharm,2013,33(3):192-195.
[9] TRIF M,FLORIAN P E,ROSEANU A,et al.Cytotoxicity and Intracellular Fate of PLGA and Chitosan-coated PLGA Nanoparticles in Madin-darby Bovine Kidney (MDBK) and Human Colorectal Adenocarcinoma (Colo 205) Cells[J].SFB,2015,103A:3599-3611.doi:10.1002/jbm.a.35498
[10] NAKASHIMA T,SHIMIZU M,MASATO K.Membrane Emulsification by Microprous Glass[J].Key Eng Mater,1991,61:513-516.doi:10.4028/www.scientific.net/KEM.61-62.513
[11] WEI Q,WEI W,LAI B.Uniform-sized PLA Nanoparticles:Preparation by Premix Membrane Emulsification[J] Int J Pharm,2008,359(3):294-297.doi:10.1016/j.ijpharm.2008.03.027
[12] GHADERI R,ARTURSSON P,CARLFORS J.Preparation of Biodegradable Microparticles Using Solution-enhanced Dispersion by Supercritical Fliuds (SEDS)[J].Pharm Res,1999(6):676-681.doi:10.1023/a.1018868423309
[13] CHOUINID F,BUCZKOWSKI S,LENAERTS V.Poly(alkyl-cyanoacrylate) Nanocapsules:Physicochemical Characterization and Mechanism of Formation[J].Pharm Res,1994(11):869-874.doi:10.1023/a.1018938026615
[14] CASTELLANOS I J,ALAZZAM W,GRIEBENOW K.Effect of the Covalent Modification with Poly(ethylene Glycol) on Alphachymotrypsin Stability Upon Encapsulation in Poly(lactic-coglycolic) Microspheres[J].J Pharm Sci,2005,94:327-340.doi:10.1002/jps.20243
[15] 周香莲,贺进田,周志涛,等.外水相中NaCl对S/O/W法制备的牛血清白蛋白PLGA缓释微粒性质的影响[J].药学学报,2010,45(8):1057-1063.doi:10.16438/j.0513-4870.2010.08.019ZHOU Xianglian,HE Jintian,ZHOU Zhitao,et al.Effect of NaCl in Outer Water Phase on the Characteristics of BSA Loaded PLGA Sustained-release Microspheres Fabricated by A Solid-in-oil-in-water Emulsion Technique[J].Acta Pharm Sin,2010,45(8):1057-1063.
[16] EMAMI J,HAMISHEHKAR H,NAJAFABADI A R,et al.A Novel Approach to Prepare Insulin-loaded Poly(lactic-co-glycolic Acid) Microcapsules and the Protein Stability Study[J].J Pharm Sci,2009,98(4):1712-1731.doi:10.1002/jps.21544
[17] 田瑞,王连艳,吴颉,等.快速膜乳化法制备粒径均一的PLGA微粒和微囊[J].过程工程学报,2009,9(4):754-762.doi:10.3321/j.issn.1009-606x.2009.04.021TAN Rui,WANG Lianyan,WU Jie,et al.Preparation of Uniform Size PLGA Microparticles and Microcapsules by Premix Membrane Emulsification[J].JCIE,2009,9(4):754-762.
[18] 马方奎.基于壳聚糖纳米载体的构建及其水解释药研究[D].青岛:中国海洋大学,2013.doi:10.7666/d.D328948MA Fangkui.Chitosan-PLGA Nanocarrier Systmes for Hydrolytic Erosion and Drug Release[D].Qingdao:Ocean University of China,2013.