CN105726485A - 一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法 - Google Patents
一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法 Download PDFInfo
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Abstract
本发明涉及一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，将卵磷脂、胆固醇、十八胺混合后溶于乙醇中，旋转蒸发，得到脂质体膜，然后加入抗原蛋白溶液，超声分散，得到负载抗原蛋白的免疫醇质体；利用层层自助装技术，将负载抗原蛋白的免疫醇质体滴加到透明质酸(HA)溶液中，搅拌，得到混合溶液，然后滴加到半乳糖化的壳聚糖(GC)溶液中，搅拌，即得。本发明制备的(GC)修饰的免疫醇质体具有高变形性、高包封率、能够软化角质并完整地渗透皮肤(从而免疫机体使之获得相应抗性)、对皮肤无刺激，性质稳定以及制备方法简单等优点，是构建经皮免疫贴剂的理想载体。
Description
技术领域
本发明属于免疫醇质体的制备领域，特别涉及一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法。
背景技术
经皮免疫(Transcutaneousimmunization，TCI)是近年发展起来的一种新型免疫接种策略，它是将抗原和佐剂局部应用于皮肤而诱导产生全身免疫反应的新方法，可以克服目前注射接种方式存在顺应性差、免疫效率低、易产生耐受等的缺陷，有着广阔的应用前景。目前限制其应用的主要障碍是皮肤表层角质层的屏障作用，使疫苗的大分子抗原物质难以通过皮肤表层而进入富含抗原提呈细胞(Antigen-presentingcells，APCs)的活性表皮层和真皮层，因此在不牺牲皮肤完整性的条件下研究一种新型的经皮免疫系统是开发经皮免疫的关键所在。
醇质体(ethosomes)不仅具有低毒性、低免疫性、良好的细胞相容性、可同时装载水溶性、脂溶性和大分子药物等优点，因而在药物递送尤其是透皮给药领域受到广泛关注。溶解在醇质体磷脂层中的高浓度的醇可增强膜的柔性性和流动性,使其在传递过程中可以变形，因此可以通过比其本身更小的间隙，透过紊乱的角质层。另外，醇质体可与细胞膜发生融合,透过细胞膜将药物直接释放到细胞内发挥作用。以醇质体作为经皮免疫载体，可有效的将抗原递送至皮肤的抗原呈递细胞引发免疫应答反应。
在皮肤的活性表皮层存在着树突状细胞-朗格汉斯细胞(未成熟的树突状细胞)，树突状细胞表面有甘露糖受体，该受体可以识别半乳糖基上的环结构，因此，在载有抗原的醇质体表面修饰半乳糖化的壳聚糖((GC))可以增加该系统的靶向性，从而增强免疫效应。透明质酸((HA))和所制得的(GC)都具有良好的生物相容性且带有相反电荷，利用层层自组装技术可以将其静电吸附于醇质体表面，所得的(GC)修饰的免疫醇质体不仅有良好的透皮效率还可以主动靶向树突状细胞从而引发免疫应答，使机体获得特定抗性。
综上所诉，基于醇质体制备技术和层层自组装技术可制得一种半乳糖化壳聚糖修饰的免疫醇质体，能够有效经皮递送抗原，使机体获得相应免疫能力。
发明内容
本发明所要解决的技术问题是提供一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，本发明操作简便，制备时间短且条件温和，利于大批量生产；所得到的免疫醇质体不仅有良好的透皮效率还可主动靶向树突状细胞有效引发免疫反应，方便应用于免疫接种领域。
本发明的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，包括：
(1)将卵磷脂、胆固醇、十八胺混合后溶于乙醇中，旋转蒸发，得到脂质体膜，然后加入抗原蛋白溶液，超声分散，得到负载抗原蛋白的免疫醇质体；
(2)利用层层自组装技术，将负载抗原蛋白的免疫醇质体滴加到透明质酸(HA)溶液中，搅拌，得到混合溶液，然后滴加到半乳糖化的壳聚糖(GC)溶液中，搅拌，即得半乳糖化壳聚糖修饰的免疫醇质体。
步骤(1)中加入十八胺的目的是使醇质体带正电。
醇质体的制备方法为薄膜分散法。
所述步骤(1)中卵磷脂、胆固醇、十八胺的质量比为25:2.5:1。
所述步骤(1)中抗原蛋白溶液的溶剂为乙醇水溶液，其中乙醇:水的体积比为1:9-3:7。
所述步骤(2)中透明质酸(HA)、半乳糖化的壳聚糖(GC)溶液的浓度均为0.5mg/mL，溶剂均为水。
所述步骤(2)中抗原蛋白的免疫醇质体、透明质酸(HA)、半乳糖化的壳聚糖(GC)的体积比为4：2：1。
步骤(2)中层层自组装技术具体为：醇质体带正电，透明质酸(HA)带负电，(GC)带正电，所以先在醇质体表面吸附一层(HA)后再吸附一层(GC)。
醇质体表面每吸附一层(GC)或(HA)都要用磁力搅拌器剧烈搅拌。
所述步骤(2)中半乳糖化的壳聚糖制备具体为：乳糖酸和壳聚糖在TEMED/HClbuffer溶液里进行反应，然后透析、冷冻干燥，得到半乳糖化的壳聚糖(GC)，其中乳糖酸和壳聚糖的摩尔比为1:1。
半乳糖化的壳聚糖制备过程中加入EDC/NHS引发反应。
所述反应时间为14-72h；透析时间为2-3天。
所述TEMED/HClbuffer溶液的pH为4.7。
所述步骤(2)中搅拌时间均为2-4h。
本发明主要采用基于醇质体制备技术和层层自组装技术，于负载抗原蛋白的免疫醇质体表面吸附先后(HA)和(GC)制得一种(GC)修饰的免疫醇质体，该醇质体能够有效经皮递送抗原蛋白，可应用于免疫接种领域。
有益效果
(1)本发明操作方便，实验装置简易，制备时间短且条件温和，利于大批量生产；
(2)本发明制备的半乳糖化壳聚糖修饰的免疫醇质体稳定性好，可长时间保存；
(3)本发明制备的半乳糖化壳聚糖修饰的免疫醇质体无皮肤刺激性，有良好的生物相容性，可高效透皮并主动靶向树突状细胞从而引发免疫反应，方便应用于免疫接种领域。
附图说明
图1是实施例所得不同物质修饰负载鸡卵清白蛋白的醇质体的电势分布图；其中a图为负载鸡卵清白蛋白的醇质体的电势分布图；b图为HA修饰后的醇质体的电势分布图；c图为GC修饰后的醇质体的电势分布图；d图为三种醇质体的电势分布对比图；
图2是实施例所得不同物质修饰负载鸡卵清白蛋白的醇质体的粒径分布图；其中a图为负载鸡卵清白蛋白的醇质体的粒径分布图；b图为HA修饰后的醇质体的粒径分布图；c图为GC修饰后的醇质体的粒径分布图；d图为三种醇质体的粒径分布对比图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
将卵磷脂、胆固醇和十八胺以质量比为25:2.5:1的比例混合溶于乙醇中，旋转蒸发得到一层脂质体膜，将鸡卵清白蛋白加入30％(v/v)乙醇水溶液中得到0.5mg/mL的抗原蛋白溶液，将该溶液加入脂质体膜的圆底烧瓶中搅拌30min，超声分散后即得到负载鸡卵清白蛋白的醇质体；
将2.0g乳糖酸(LA)溶解于50mlTEMED/HClbuffer(PH4.7)，再由NHS(0.14g)和EDC(0.6g)的混合物激活。最后将1.0g壳聚糖(13.7mmol)以LA的当量摩尔比加入到溶解液中。室温反应72h，将产物置于1.4kD的透析袋中透析2-3天，冷冻干燥后得到半乳糖化的壳聚糖(GC)。
将透明质酸(HA)和半乳糖化壳聚糖(GC)分别充分溶解于去离子水中，浓度为0.5mg/mL，利用层层自组装技术将所得的负载鸡卵清白蛋白的醇质体2mL先缓慢滴加到透明质酸(HA)溶液1mL中，剧烈搅拌2h得到透明质酸修饰的负载鸡卵清白蛋白的醇质体，再将该混合溶液缓慢滴加到半乳糖化的壳聚糖(GC)溶液1.5mL中剧烈搅拌2h，即得半乳糖化壳聚糖(GC)修饰的负载鸡卵清白蛋白的免疫醇质体。
Claims (10)
1.一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，包括：
(1)将卵磷脂、胆固醇、十八胺混合后溶于乙醇中，旋转蒸发，得到脂质体膜，然后加入抗原蛋白溶液，超声分散，得到负载抗原蛋白的免疫醇质体；
(2)利用层层自助装技术，将负载抗原蛋白的免疫醇质体滴加到透明质酸HA溶液中，搅拌，得到混合溶液，然后滴加到半乳糖化的壳聚糖GC溶液中，搅拌，即得半乳糖化壳聚糖修饰的免疫醇质体。
2.根据权利要求1所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述步骤(1)中卵磷脂、胆固醇、十八胺的质量比为25:2.5:1。
3.根据权利要求1所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述步骤(1)中抗原蛋白溶液的溶剂为乙醇水溶液，其中乙醇:水的体积比为1:9-3:7。
4.根据权利要求1所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述步骤(2)中透明质酸HA、半乳糖化的壳聚糖GC溶液的浓度均为0.5mg/mL，溶剂均为水。
5.根据权利要求1所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述步骤(2)中抗原蛋白的免疫醇质体、透明质酸HA、半乳糖化的壳聚糖GC的体积比为4：2：1。
6.根据权利要求1所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述步骤(2)中半乳糖化的壳聚糖制备具体为：乳糖酸和壳聚糖在TEMED/HClbuffer溶液里进行反应，然后透析、冷冻干燥，得到半乳糖化的壳聚糖GC，其中乳糖酸和壳聚糖的摩尔比为1:1。
7.根据权利要求6所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：半乳糖化的壳聚糖制备过程中加入EDC/NHS引发反应。
8.根据权利要求6所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述反应时间为14-72h；透析时间为2-3天。
9.根据权利要求6所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述TEMED/HClbuffer溶液的pH为4.7。
10.根据权利要求1所述的一种半乳糖化壳聚糖修饰的免疫醇质体的制备方法，其特征在于：所述步骤(2)中搅拌时间均为2-4h。
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| CN111296426B (zh) * | 2020-03-31 | 2021-09-24 | 大连理工大学 | 以非磷脂类脂质体作为载体的纳米农药剂型的制备方法及其应用 |
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