荆豆凝集素介导的生物粘附脂质体作为口服疫苗载体的研究
发布时间：2018-09-10 20:42
【摘要】：本文制备了荆豆凝集素介导的生物粘附脂质体作为口服疫苗的载体。由于荆豆凝集素可以专属性粘附并靶向于小鼠肠道派伊尔氏结上的免疫细胞—M细胞,因此,可以诱发机体同时产生系统及粘膜免疫应答,并验证了该微粒体作为口服疫苗载体的可行性。 建立了考马斯亮蓝法测定模型抗原牛血清白蛋白(BSA)的含量；稳定性考察结果表明：温度、超声过程和光照对BSA溶液稳定性基本没有影响。 采用逆向蒸发法制备了牛血清白蛋白脂质体(LIP),以药物包封率和脂质体形态为指标,对影响脂质体制备的处方及工艺因素进行了考察,并用L9(34)正交设计实验对处方进行优化。按最优处方制备脂质体平均粒径为4.796±0.416Zeta电位为-15.4±2.6 mv,包封率为69.74%±3.41%。LIP体外释放曲线表明其具有一定缓释作用。 建立考马斯亮蓝法和红细胞凝集实验法测定荆豆凝集素(UEAI)的含量和活性。UEAI稳定性考察结果表明：4℃-80℃范围内,UEAI稳定；100℃时,随着时间延长,UEAI活性显著降低；pH2.0-10.0的PBS缓冲液中,UEAI溶液稳定。安全性评价结果表明：在浓度104.6-1046.5 mg·L-1范围内,UEAI对肠道是安全无刺激的,可以作为口服疫苗载体的修饰材料。同时,细胞毒性研究结果表明UEAI对Caco-2细胞具有一定的促进生长作用。 合成了胆固醇单琥珀酸酯(CHS),对其进行结构表征后,采用化学偶联技术制备了荆豆凝集素修饰的牛血清白蛋白脂质体(UEAI-LIP),平均粒径为5.129±1.671μm, Zeta电位为-21.4±1.5 mv；用Sepharose4B凝胶柱测定了UEAI-LIP连接率,最佳连接率为70.80%±2.28%。与LIP释药曲线比较可见：凝集素的修饰减少了药物突释,降低了药物释放速度。酶解稳定性考察结果表明：UEAI-LIP在人工胃液(含胃蛋白酶)和人工肠液(含胰蛋白酶)中脂质体表面有UEAI修饰会明显增加脂质体抗酶解能力,从而保证有更多抗原传递到免疫部位。 脂质体与小鼠胃肠道体内、外粘附性考察结果表明：与LIP相比,UEAI-LIP在含有PPs的小肠区域粘附力显著提高,且随表面修饰UEAI浓度的增加而增加。同时,单糖抑制实验表明UEAI与M细胞的特异性结合可被单糖a-L-岩藻糖竞争性抑制。 利用ELISA法来测定小鼠血清样本中的IgG含量和小肠分泌液样本中的IgA含量,从而确定诱发最佳免疫应答的脂质体处方。结果表明：①当抗原以不同载体口服免疫时：high-UEAI-LIP组诱发小鼠的IgG和IgA水平最高,其次是low-UEAI-LIP组和LIP组,最低的为BSA溶液组；②当抗原以不同粒径脂质体口服免疫时：平均粒径为137±18 nm的小粒径脂质体可以刺激机体的免疫系统产生更强的系统免疫应答,平均粒径为5.657±1.924μm的大粒径脂质体诱发高度的粘膜免疫应答；③当脂质体中包裹抗原剂量增加时,可以产生更多的IgA,起到更好的粘膜保护作用。④当BSA溶液以肌注方式免疫可产生高水平IgG；而脂质体以口服方式给药,则可产生较高IgA,即诱发充分的粘膜免疫应答。 以市售双价肾综合征出血热疫苗(HFRS)为模型抗原,对UEAI修饰脂质体作为口服疫苗载体的可行性进行验证。结果显示：将HFRS溶液以肌肉注射方式免疫小鼠,可激发机体免疫系统产生全身免疫应答；其所诱发的IgA水平在21天时,抗体水平基本达到高峰,但随后逐渐降低。而将抗原以UEAI修饰脂质体形式口服免疫后,可产生与肌注HFRS溶液相近IgG水平和更强IgA水平,并随免疫次数的增加,抗体IgA浓度明显提高,即可诱发高水平的粘膜免疫应答。
[Abstract]:The bioadhesive liposomes mediated by Jingdou agglutinin were prepared as the carriers of oral vaccine. Because Jingdou agglutinin can specifically adhere to and target the immune cells M cells in the intestinal Pair's node of mice, it can induce the simultaneous systemic and mucosal immune responses of the organism, and it has been proved that Jingdou agglutinin can be used as an oral vaccine carrier. Feasibility of vaccine vector.
The content of BSA was determined by Coomassie brilliant blue method. The results showed that temperature, ultrasonic process and illumination had little effect on the stability of BSA solution.
Bovine serum albumin liposomes (LIP) were prepared by reverse evaporation method. The formulation and technological factors affecting the preparation of liposomes were investigated by using drug encapsulation efficiency and liposome morphology as indexes. The prescription was optimized by L9(34) orthogonal design experiment. The average particle size of the liposomes prepared by the optimal prescription was 4.796+0.416 Zeta potential of - 15. 4 + 2.6 MV, the entrapment efficiency was 69.74% + 3.41%.LIP, the in vitro release curve showed that it had a certain sustained release effect.
The content and activity of UEAI were determined by Coomassie brilliant blue method and erythrocyte agglutination assay. The results of UEAI stability test showed that UEAI was stable in the range of 4 80 C, UEAI activity decreased significantly with time at 100 C, and UEAI solution was stable in PBS buffer with pH 2.0 10.0. In the range of concentration 104.6-1046.5 mg/L-1, UEAI is safe and non-irritating to the intestine, and can be used as a modified material for oral vaccine carrier.
Cholesterol MONOSUCCINATE (CHS) was synthesized and characterized. UAI-LIP modified by Jingdou agglutinin was prepared by chemical coupling technique. The average diameter of UEAI-LIP was 5.129+1.671 micron and the Zeta potential was -21.4+1.5 mv. 2.28%. Compared with the LIP release curve, the lectin modification reduced the drug burst release and the drug release rate. The results of enzymatic hydrolysis stability test showed that UEAI-LIP in artificial gastric juice (including pepsin) and artificial intestinal juice (including trypsin) could significantly increase the liposome anti-enzymatic hydrolysis ability, thus ensuring the stability of UEAI-LIP. More antigens are passed to the immune site.
Compared with LIP, the adhesion of UEAI-LIP to the small intestine containing PPs was significantly enhanced and increased with the concentration of surface modified UEAI. Simultaneously, the specific binding of UEAI to M cells was inhibited by monosaccharide a-L-fucose.
ELISA was used to determine the content of IgG in serum and the content of IgA in intestinal secretion of mice to determine the optimal liposome formulation for inducing immune response. When the antigen was orally immunized with liposomes of different sizes, the small size liposomes with an average diameter of 137 18 nm could stimulate the immune system to produce stronger systemic immune response, and the large size liposomes with an average diameter of 5.657 65 Increasing the dosage of encapsulated antigen can produce more IgA and play a better role in mucosal protection.
The feasibility of UEAI modified liposomes as oral vaccine carriers was validated by using commercially available bivalent hemorrhagic fever with renal syndrome vaccine (HFRS) as model antigen. The results showed that the immune system of mice immunized with HFRS solution by intramuscular injection could induce systemic immune response, and the level of IgA induced by HFRS could be detected at 21 days. After oral immunization in the form of UEAI modified liposomes, the levels of IgG and stronger IgA were similar to those in HFRS solution. With the increase of immunization times, the concentration of IgA increased significantly, which could induce a high level of mucosal immune response.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1
[Abstract]:The bioadhesive liposomes mediated by Jingdou agglutinin were prepared as the carriers of oral vaccine. Because Jingdou agglutinin can specifically adhere to and target the immune cells M cells in the intestinal Pair's node of mice, it can induce the simultaneous systemic and mucosal immune responses of the organism, and it has been proved that Jingdou agglutinin can be used as an oral vaccine carrier. Feasibility of vaccine vector.
The content of BSA was determined by Coomassie brilliant blue method. The results showed that temperature, ultrasonic process and illumination had little effect on the stability of BSA solution.
Bovine serum albumin liposomes (LIP) were prepared by reverse evaporation method. The formulation and technological factors affecting the preparation of liposomes were investigated by using drug encapsulation efficiency and liposome morphology as indexes. The prescription was optimized by L9(34) orthogonal design experiment. The average particle size of the liposomes prepared by the optimal prescription was 4.796+0.416 Zeta potential of - 15. 4 + 2.6 MV, the entrapment efficiency was 69.74% + 3.41%.LIP, the in vitro release curve showed that it had a certain sustained release effect.
The content and activity of UEAI were determined by Coomassie brilliant blue method and erythrocyte agglutination assay. The results of UEAI stability test showed that UEAI was stable in the range of 4 80 C, UEAI activity decreased significantly with time at 100 C, and UEAI solution was stable in PBS buffer with pH 2.0 10.0. In the range of concentration 104.6-1046.5 mg/L-1, UEAI is safe and non-irritating to the intestine, and can be used as a modified material for oral vaccine carrier.
Cholesterol MONOSUCCINATE (CHS) was synthesized and characterized. UAI-LIP modified by Jingdou agglutinin was prepared by chemical coupling technique. The average diameter of UEAI-LIP was 5.129+1.671 micron and the Zeta potential was -21.4+1.5 mv. 2.28%. Compared with the LIP release curve, the lectin modification reduced the drug burst release and the drug release rate. The results of enzymatic hydrolysis stability test showed that UEAI-LIP in artificial gastric juice (including pepsin) and artificial intestinal juice (including trypsin) could significantly increase the liposome anti-enzymatic hydrolysis ability, thus ensuring the stability of UEAI-LIP. More antigens are passed to the immune site.
Compared with LIP, the adhesion of UEAI-LIP to the small intestine containing PPs was significantly enhanced and increased with the concentration of surface modified UEAI. Simultaneously, the specific binding of UEAI to M cells was inhibited by monosaccharide a-L-fucose.
ELISA was used to determine the content of IgG in serum and the content of IgA in intestinal secretion of mice to determine the optimal liposome formulation for inducing immune response. When the antigen was orally immunized with liposomes of different sizes, the small size liposomes with an average diameter of 137 18 nm could stimulate the immune system to produce stronger systemic immune response, and the large size liposomes with an average diameter of 5.657 65 Increasing the dosage of encapsulated antigen can produce more IgA and play a better role in mucosal protection.
The feasibility of UEAI modified liposomes as oral vaccine carriers was validated by using commercially available bivalent hemorrhagic fever with renal syndrome vaccine (HFRS) as model antigen. The results showed that the immune system of mice immunized with HFRS solution by intramuscular injection could induce systemic immune response, and the level of IgA induced by HFRS could be detected at 21 days. After oral immunization in the form of UEAI modified liposomes, the levels of IgG and stronger IgA were similar to those in HFRS solution. With the increase of immunization times, the concentration of IgA increased significantly, which could induce a high level of mucosal immune response.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1
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