改性壳聚糖的制备及抑菌效果评价
摘要
壳聚糖(chitosan)是甲壳素脱乙酰基的降解产物,由α-氨基-D-葡胺糖通过β-1,4-糖苷键连结成的直链状多糖,不溶于水和碱溶液,可溶于大多数的有机酸中,具有许多独特的性质如无毒性、降解性、细胞亲和性和生物相容性等,是天然多糖中唯一的碱性多糖。由于大分子链上分布着许多氨基和羟基,能够形成分子内和分子间氢键,因而壳聚糖的结晶性较高、溶解性差,极大地限制了其应用。本研究通过对壳聚糖与环糊精进行复合或者制备壳聚糖纳米自聚体,以伊维菌素作为模型药物,目的是研制疏水性药物的载体。
本文采用戊二醛交联法制备环糊精-壳聚糖复合物,利用官能团偶联的方法将甲基聚乙二醇单甲醚接枝壳聚糖制备水溶性的壳聚糖衍生物。通过结构表征、载药性能分析评价了这些改性材料的性能。
利用金黄色葡萄球菌和大肠杆菌进行抑菌实验,结果表明分子量为十万左右,浓度为20mg/L的壳聚糖对两种菌抑菌效果较明显,改性后的壳聚糖对金黄色葡萄球菌,大肠杆菌抑菌效果均降低。
分别以p-环糊精(CD)及其与壳聚糖(CS)的复合物为载体对伊维菌素(IVM)进行包合。采用饱和溶液法制备IVM-β-CD包合物,用戊二醛交联法制备环糊精-壳聚糖复合物,采用包埋法包载伊维菌素并测定其包封率及载药量,并用差示扫描量热法(DSC), X-射线衍射(X-Ray),傅里叶红外光谱(FTIR),扫描电镜(SEM)对其性质进行了表征。结果表明β-CD-IVM主、客分子比为6：1,平均载药量为30.08%±0.11%,平均包封率为29.54%±1.21%。β-CD-CS-IVM平均载药量为10.2%±5.75%,平均包封率为15.13±3.43%。FTIR、X-射线衍射和DSC分析证实形成了β-CD-IVM及β-CD-CS-IVM包合物,表明β-CD-CS-IVM的载药量及包封率低于β-CD-IVM包合物。用电镜扫描观察了β-环糊精-壳聚糖微粒的表面结构,表明β-环糊精-壳聚糖微粒呈多孔性表面。β-环糊精-壳聚糖的载药量和包封率受β-环糊精/壳聚糖重量投料比,交联剂用量和温度影响。结果表明采用戊二醛做交联剂时,温度为60℃,重量投料比(β-CD/chitosan)为9：1,P-CD-CS的载药量最大。
本研究的另一部分工作是通过官能团偶联反应,合成甲基聚乙二醇单甲醚接枝壳聚糖(mPEG-g-CS),并使用DSC, X-Ray, FTIR, SEM对其进行了结构表征和性质研究,根据其性质制备了纳米自聚体,与β-环糊精-壳聚糖微粒相比在载药量和包封率均有所提高,分别是19.65%±2.05%,14.51%±1.36%,体外释放结果表明mPEG-g-CS纳米自聚体比β-CD-CS-IVM具有较好的缓释效果,在8个小时内累计释放98.26%。
Chitosan, which is a linear polysaccharide composed ofα-amino-D-glucan linked byβ-1,4-glycosidic bonds, is prepared by alkaline deacetylation of chitin. It is insoluble in water or alkaline solution, but can dissolve in majority of organic acids. Chitosan has many unique features sucn as nontoxicity, degradability, cell affinity and biocompatibility. Chitosan is the only alkaline polysaccharide with many amino and hydroxyl groups distributing along the macro-molecule chain, which can form intra-and inter-molecular hydrogen bonds. These features cause high crystallinity and low solubility of Chitosan, limiting its application enormously. This study compounded chitosan with cyclodextrin or prepared chitosan self-aggregates at nano-scale,using Ivermectin (IVM) as model drug, in order to design the carriers loading hydrophobic drugs.
Chitosan and cyclodextrin compounds were preparaed by cross-linking with glutaraldehyde. mPEG-graft-Chitosan was prepared through the functional group coupling's method. These materials were characterized structurally and assessd for drug-loading capacity.
Antibacterial experiment are carried against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), the result indicated that 20mg/L Chitosan with molecular weight about 100,000 had obviously bacteriostasis effect, the bacteriostasis effect of modified Chitosan against E.coli and S.aureus was all lower than that of non-modified chitosan.
β-cyclodextrin and Chitosan (CS) compound were used as carriers for IVM for inclusion. Theβ-cyclodextrin-IVM was prepared by saturated solution andβ-cyclodextrin-Chitosan was prepared by crossing linking with glutaraldehyde; Ivermectin was loaded by incubation method and the loading efficiency and association efficiency was determined.
The properties of them were characterized with the differential scanning calorimetry (DSC), X-beam diffraction (X-Ray), the Fourier infrared spectrum (FTIR), scanning electron microscope (SEM). The result indicates the proportion ofβ-CD and IVM was 6:1, the loading efficiency and association efficiency was 30.08%±0.11% and 29.54%±1.21%, Respectively. The average loading efficiency and association efficiency ofβ-CD-CS-IVM was 10.2%±5.75% and 15.13±3.43%, Respectively.β-CD-IVM andβ-CD-CS-IVM compound have been formed by FTIR, the X-beam diffraction and the DSC analysis confirmation,at the same time, the loading efficiency and association efficiency ofβ-CD-CS-IVM is lower thanβ-CD-IVM's. The surface texture ofβ-CD-CS was observed with the electron microscope scanning, indicated thatβ-CD-CS particle was porous. The average loading efficiency and association efficiency ofβ-CD-CS-IVM was affected by the proportion ofβ-CD and CS, time and temperature. The result indicated that when the glutaric dialdehyde was used as the crosslinking agent, the temperature is 60℃, the the weight proportion of (β-CD/Chitosan) is 9:1, The average loading efficiency ofβ-CD-CS was largest.
Another part of this study is synthesis of methyl polyethylene glycol monomethyl ether grafted Chitosan (mPEG-g-CS) through the coupling reaction of functional groups, and study its characterization of the structure and properties, according to which, nanoautopolymer of mPEG-g-CS is prepared. Compared withβ-cyclodextrin-Chitosan particles, the rate of drug loading and encapsulation efficiency increased to 19.65%±2.05% and 14.51%±1.36%, respectively, in vitro release results show that the sustained release effect of nano-autopolymer of mPEG-g-CS was better thanβ-CD-CS-IVM,which has cumulatively released 98.26% in 8 hours.
本文采用戊二醛交联法制备环糊精-壳聚糖复合物,利用官能团偶联的方法将甲基聚乙二醇单甲醚接枝壳聚糖制备水溶性的壳聚糖衍生物。通过结构表征、载药性能分析评价了这些改性材料的性能。
利用金黄色葡萄球菌和大肠杆菌进行抑菌实验,结果表明分子量为十万左右,浓度为20mg/L的壳聚糖对两种菌抑菌效果较明显,改性后的壳聚糖对金黄色葡萄球菌,大肠杆菌抑菌效果均降低。
分别以p-环糊精(CD)及其与壳聚糖(CS)的复合物为载体对伊维菌素(IVM)进行包合。采用饱和溶液法制备IVM-β-CD包合物,用戊二醛交联法制备环糊精-壳聚糖复合物,采用包埋法包载伊维菌素并测定其包封率及载药量,并用差示扫描量热法(DSC), X-射线衍射(X-Ray),傅里叶红外光谱(FTIR),扫描电镜(SEM)对其性质进行了表征。结果表明β-CD-IVM主、客分子比为6：1,平均载药量为30.08%±0.11%,平均包封率为29.54%±1.21%。β-CD-CS-IVM平均载药量为10.2%±5.75%,平均包封率为15.13±3.43%。FTIR、X-射线衍射和DSC分析证实形成了β-CD-IVM及β-CD-CS-IVM包合物,表明β-CD-CS-IVM的载药量及包封率低于β-CD-IVM包合物。用电镜扫描观察了β-环糊精-壳聚糖微粒的表面结构,表明β-环糊精-壳聚糖微粒呈多孔性表面。β-环糊精-壳聚糖的载药量和包封率受β-环糊精/壳聚糖重量投料比,交联剂用量和温度影响。结果表明采用戊二醛做交联剂时,温度为60℃,重量投料比(β-CD/chitosan)为9：1,P-CD-CS的载药量最大。
本研究的另一部分工作是通过官能团偶联反应,合成甲基聚乙二醇单甲醚接枝壳聚糖(mPEG-g-CS),并使用DSC, X-Ray, FTIR, SEM对其进行了结构表征和性质研究,根据其性质制备了纳米自聚体,与β-环糊精-壳聚糖微粒相比在载药量和包封率均有所提高,分别是19.65%±2.05%,14.51%±1.36%,体外释放结果表明mPEG-g-CS纳米自聚体比β-CD-CS-IVM具有较好的缓释效果,在8个小时内累计释放98.26%。
Chitosan, which is a linear polysaccharide composed ofα-amino-D-glucan linked byβ-1,4-glycosidic bonds, is prepared by alkaline deacetylation of chitin. It is insoluble in water or alkaline solution, but can dissolve in majority of organic acids. Chitosan has many unique features sucn as nontoxicity, degradability, cell affinity and biocompatibility. Chitosan is the only alkaline polysaccharide with many amino and hydroxyl groups distributing along the macro-molecule chain, which can form intra-and inter-molecular hydrogen bonds. These features cause high crystallinity and low solubility of Chitosan, limiting its application enormously. This study compounded chitosan with cyclodextrin or prepared chitosan self-aggregates at nano-scale,using Ivermectin (IVM) as model drug, in order to design the carriers loading hydrophobic drugs.
Chitosan and cyclodextrin compounds were preparaed by cross-linking with glutaraldehyde. mPEG-graft-Chitosan was prepared through the functional group coupling's method. These materials were characterized structurally and assessd for drug-loading capacity.
Antibacterial experiment are carried against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), the result indicated that 20mg/L Chitosan with molecular weight about 100,000 had obviously bacteriostasis effect, the bacteriostasis effect of modified Chitosan against E.coli and S.aureus was all lower than that of non-modified chitosan.
β-cyclodextrin and Chitosan (CS) compound were used as carriers for IVM for inclusion. Theβ-cyclodextrin-IVM was prepared by saturated solution andβ-cyclodextrin-Chitosan was prepared by crossing linking with glutaraldehyde; Ivermectin was loaded by incubation method and the loading efficiency and association efficiency was determined.
The properties of them were characterized with the differential scanning calorimetry (DSC), X-beam diffraction (X-Ray), the Fourier infrared spectrum (FTIR), scanning electron microscope (SEM). The result indicates the proportion ofβ-CD and IVM was 6:1, the loading efficiency and association efficiency was 30.08%±0.11% and 29.54%±1.21%, Respectively. The average loading efficiency and association efficiency ofβ-CD-CS-IVM was 10.2%±5.75% and 15.13±3.43%, Respectively.β-CD-IVM andβ-CD-CS-IVM compound have been formed by FTIR, the X-beam diffraction and the DSC analysis confirmation,at the same time, the loading efficiency and association efficiency ofβ-CD-CS-IVM is lower thanβ-CD-IVM's. The surface texture ofβ-CD-CS was observed with the electron microscope scanning, indicated thatβ-CD-CS particle was porous. The average loading efficiency and association efficiency ofβ-CD-CS-IVM was affected by the proportion ofβ-CD and CS, time and temperature. The result indicated that when the glutaric dialdehyde was used as the crosslinking agent, the temperature is 60℃, the the weight proportion of (β-CD/Chitosan) is 9:1, The average loading efficiency ofβ-CD-CS was largest.
Another part of this study is synthesis of methyl polyethylene glycol monomethyl ether grafted Chitosan (mPEG-g-CS) through the coupling reaction of functional groups, and study its characterization of the structure and properties, according to which, nanoautopolymer of mPEG-g-CS is prepared. Compared withβ-cyclodextrin-Chitosan particles, the rate of drug loading and encapsulation efficiency increased to 19.65%±2.05% and 14.51%±1.36%, respectively, in vitro release results show that the sustained release effect of nano-autopolymer of mPEG-g-CS was better thanβ-CD-CS-IVM,which has cumulatively released 98.26% in 8 hours.
引文
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