CN110585426A - 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗 - Google Patents

一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗 Download PDF

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CN110585426A
CN110585426A CN201910766153.XA CN201910766153A CN110585426A CN 110585426 A CN110585426 A CN 110585426A CN 201910766153 A CN201910766153 A CN 201910766153A CN 110585426 A CN110585426 A CN 110585426A
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张闯年
王晓莉
孔德领
孙洪范
裴萌月
徐蓉
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Abstract

本发明涉及一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127‑136肽（GPC3127‑136，AMFKNNYPSL）为免疫抗原，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，载体材料、免疫抗原和佐剂之间的质量比是1:1～10:0.3～0.8。本发明的纳米疫苗能够增加树突状细胞（dendritic cell，DC）对抗原和佐剂的胞吞量，上调DC表面共刺激分子及促进TNF‑a、IL‑6等细胞因子分泌，利于激发机体产生有效的免疫应答，且其原料廉价易得、制备方法简单易重复、易于大规模加工生产，能够增强肝癌的免疫治疗效果，具有良好的应用前景。

Description

一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗
技术领域
本发明涉及生物医领域，尤其是涉及一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法。
背景技术
肝癌的发病率及死亡率在逐年增加，严重危害人类健康和安全。肝癌免疫治疗是通过重新激活机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种治疗方法。
肿瘤疫苗在肿瘤免疫治疗中占有重要地位，肿瘤疫苗经体内抗原提呈细胞处理、加工，并将抗原信息提呈给T细胞，从而激发机体免疫反应。树突状细胞（dendritic cell，DC）是体内已知功能最强的专职抗原提呈细胞，是启动、调控和维持免疫应答的中心环节。
寻找有效的抗原对于肿瘤疫苗的研发至关重要。磷脂酰肌醇蛋白聚糖3（Glypican3，GPC3）是一种肝癌胚胎蛋白，其在大多数原发性肝癌细胞中高表达，但在正常肝脏和良性肝脏病变组织中检测不到，因此，GPC3可以作为肿瘤抗原或治疗靶点。GPC-3127-136肽同样能够诱导产生细胞毒T细胞（CTL）反应，亦可作为肝癌特异性抗原，且多肽更易于合成，便于生产，纯度更高，价格也更低廉。
疫苗中的抗原以游离状态进入机体，会被体液稀释、酶降解，使得DC对抗原的摄取量极低，难以诱导机体产生有效的免疫应答。纳米疫苗可以保护抗原不被快速降解和清除，通过对纳米粒子结构进行设计还可以实现对抗原的靶向输送和控制释放，进而提高DC对抗原的摄取量、延长抗原的作用时间，利于激发机体产生有效的免疫应答。
聚乙烯亚胺（PEI）是一种水溶性聚合物,分子链上存在的伯、仲、叔胺基，使其呈碱性和阳离子活性,对阴离子物质有较好的亲和性和吸附性。天然多糖海藻酸钠（ALG）是一种线性阴离子多糖，具有良好的生物相容性和生物可降解性，对人体无毒，廉价易得，已在食品、医疗、生物技术和药学等领域得到了广泛的应用。疫苗佐剂(Adjuvant)是能够非特异性地改变或增强机体对抗原的特异性免疫应答。佐剂还能够诱发机体产生长期、高效的免疫反应，同时又能减少抗原的用量，降低肿瘤疫苗的生产成本。
现有的肿瘤疫苗对肿瘤的免疫治疗效果较低，且肿瘤疫苗的原料成本高，制备方法复杂，不利于大规模加工生产。
发明内容
本发明设计了一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法，其解决的技术问题是现有的肿瘤疫苗对肿瘤的免疫治疗效果较低，且肿瘤疫苗的原料成本高，制备方法复杂，不利于大规模加工生产。
为了解决上述存在的技术问题，本发明采用了以下方案：
一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127-136肽（GPC3127-136，AMFKNNYPSL）为免疫抗原，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，所述载体材料、所述免疫抗原和所述佐剂之间的质量比是1:1～10:0.3～0.8。
进一步，所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的粒径为100nm～1000nm。
进一步，所述海藻酸钠和所述聚乙烯亚胺之间的质量比是1:0.4～3。
进一步，所述海藻酸钠的分子量为8000-50000。
进一步，所述聚乙烯亚胺的分子量为3000-25000。
一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的制备方法，包括以下步骤：
步骤一，将GPC3127-136水溶液和聚乙烯亚胺水溶液混合得到PC3127-136和聚乙烯亚胺混合溶液，再将海藻酸钠水溶液和CpG水溶液混合得到海藻酸钠和CpG混合溶液；
步骤二，在搅拌条件下，将所述海藻酸钠和CpG混合溶液滴加到所述GPC3127-136和聚乙烯亚胺混合溶液中，继续搅拌得到所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗。
静电相互作用在步骤二中，海藻酸钠和CPG带负电，PEI和多肽带正电，滴加行程纳米粒原理就是静电相互作用。
进一步，步骤一中，所述GPC3127-136水溶液的浓度为0.5 mg/ml～4 mg/ml；所述聚乙烯亚胺水溶液的浓度为0.5 mg/ml～3 mg/ml；所述CpG水溶液的浓度为0.5 mg/ml～4mg/ml；所述海藻酸钠水溶液的浓度为0.5 mg/ml～2 mg/ml。
进一步，步骤一中，所述GPC3127-136水溶液和所述聚乙烯亚胺水溶液之间的质量比为1:0.05～0.5;所述海藻酸钠水溶液和所述CpG水溶液之间的质量比为1:0.8～1.5。
进一步，步骤二中，将所述海藻酸钠和CpG混合溶液滴加到所述GPC3127-136和聚乙烯亚胺混合溶液中后继续搅拌的时间为1min～15min。
该应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法具有以下有益效果：
（1）本发明的纳米疫苗能够增加树突状细胞（dendritic cell，DC）对抗原和佐剂的胞吞量，上调DC表面共刺激分子及促进TNF-a、IL-6等细胞因子分泌，利于激发机体产生有效的免疫应答，且其原料廉价易得、制备方法简单易重复、易于大规模加工生产，能够增强肝癌的免疫治疗效果，具有良好的应用前景。
（2）本发明应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法中，纳米疫苗能够同时递送抗原和佐剂，同时能够增加DC对抗原和佐剂的胞吞量，从而提高肿瘤的免疫治疗效果。
（3）本发明应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法中，制备的纳米粒子原料廉价易得、制备方法简单易重复、易于大规模加工生产，具有良好的应用前景。
附图说明
图1：本发明实施例1中得到的抗原和佐剂共递送纳米疫苗的粒径分布图；
图2：本发明实施例1中DC表面共刺激分子CD40、CD80以及CD86的荧光强度柱状图；
图3：本发明实施例2中得到的抗原和佐剂共递送纳米疫苗的粒径分布图；
图4：本发明实施例2中DC分泌的TNF-a、IL-6浓度柱状图；
图5：本发明实施例3中得到的荧光标记纳米疫苗的粒径分布图；
图6：本发明实施例3中DC对荧光标记纳米疫苗的胞吞共聚焦结果图。
具体实施方式
下面结合图1至图6，对本发明做进一步说明：
本发明应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127-136肽（GPC3127-136，AMFKNNYPSL）为免疫抗原，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的粒径为100nm～1000nm，所述载体材料、所述免疫抗原和所述佐剂之间的质量比是1:1～10:0.3～0.8，海藻酸钠和聚乙烯亚胺之间的质量比是1:0.4～3，海藻酸钠的分子量为8000-50000，聚乙烯亚胺的分子量为3000-25000。
应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的制备方法，包括以下步骤：
步骤一，将GPC3127-136水溶液和聚乙烯亚胺水溶液混合得到PC3127-136和聚乙烯亚胺混合溶液，再将海藻酸钠水溶液和CpG水溶液混合得到海藻酸钠和CpG混合溶液，其中GPC3127-136水溶液的浓度为0.5 mg/ml～4 mg/ml；聚乙烯亚胺水溶液的浓度为0.5 mg/ml～3 mg/ml；CpG水溶液的浓度为0.5 mg/ml～4 mg/ml；海藻酸钠水溶液的浓度为0.5 mg/ml～2 mg/ml；GPC3127-136水溶液和聚乙烯亚胺水溶液之间的质量比为1:0.05～0.5;海藻酸钠水溶液和CpG水溶液之间的质量比为1:0.8～1.5。
步骤二，在搅拌条件下，将海藻酸钠和CpG混合溶液滴加到GPC3127-136和聚乙烯亚胺混合溶液中，继续搅拌1min～15min得到应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗。
实施例一：
纳米疫苗的制备：
将GPC3127-136水溶液（1mg/ml，0.5ml）和PEI水溶液（0.5mg/ml，0.2ml）混合，再将ALG水溶液（0.5mg/ml，0.5ml）和CpG水溶液（0.5mg/ml，0.5ml）混合，搅拌条件下，将ALG和CpG混合溶液滴加到GPC3127-136和PEI的混合溶液中，搅拌5min，得到抗原和佐剂共递送纳米疫苗。
如图1所示,本实施例获得的纳米疫苗的粒径分布比较均一，平均粒径为111.9nm。
纳米疫苗促DC成熟评价：
收集第六天未成熟的的DC，按106个细胞每孔，将DC铺于24孔板，贴壁2小时后，加入纳米疫苗，GPC3127-136浓度为10微克每毫升，继续培养48小时。轻吹细胞收集于离心管中，离心，细胞进行流式抗体标记，利用流式细胞仪检测DC表面共刺激分子CD40、CD80以及CD86的表达。
如图2所示，DC与纳米疫苗共培养后，DC表面共刺激分子CD40、CD80以及CD86产生的荧光强度更强，说明纳米疫苗对DC促成熟效果更好。
实施例二：
纳米疫苗的制备：
将GPC3127-136水溶液（2mg/ml，1ml）和PEI水溶液（2mg/ml，0.3ml）混合，再将ALG水溶液（2mg/ml，0.1ml）和CpG水溶液（2mg/ml，0.1ml）混合，搅拌条件下，将ALG和CpG混合溶液滴加到GPC3127-136和PEI的混合溶液中，搅拌2min，得到抗原和佐剂共递送纳米疫苗。
如图3所示，本实施例获得的纳米疫苗的粒径分布比较均一，平均粒径为173.7nm。
纳米疫苗促DC细胞因子分泌评价：
收集第六天未成熟的的DC，按106个细胞每孔，将DC铺于24孔板，贴壁2小时后，加入纳米疫苗，GPC3127-136浓度为10微克每毫升，继续培养48小时。轻吹细胞收集于离心管中，离心，收集上清液，酶联免疫吸附测定（ELISA）检测上清液中TNF-a、IL-6的含量，图4是DC 分泌的TNF-a、IL-6浓度柱状图。
如图4所示，DC与纳米疫苗共培养后，DC分泌的TNF-a、IL-6含量较与游离抗原和佐剂组明显增多，说明纳米疫苗刺激DC细胞因子分泌效果更好。
实施例三：
荧光标记纳米疫苗的制备：
将异硫氰酸荧光素标记的GPC3127-136水溶液（4mg/ml，2ml）和PEI水溶液（0.5mg/ml，1ml）混合，再将ALG水溶液（2mg/ml，0.2ml）和罗丹明荧光素标记的CpG水溶液（1mg/ml，0.5ml）混合，搅拌条件下，将ALG和罗丹明荧光素标记的CpG混合溶液滴加到异硫氰酸荧光素标记的GPC3127-136和PEI的混合溶液中，搅拌3min，得到抗原和佐剂共递送荧光标记纳米疫苗。
如图5所示，本实施例获得的纳米疫苗的粒径分布比较均一，平均粒径为224nm。
DC对荧光标记纳米疫苗的胞吞评价：
按106个细胞每皿，将DC铺于共聚焦皿中，贴壁24小时后，加入含有荧光标记的纳米疫苗的培养基，GPC3127-136浓度为10微克每毫升，再培养24小时。用PBS洗3次细胞，多聚甲醛固定，Lamp-1染溶酶体，DAPI染细胞核，用共聚焦显微镜观察DC对荧光标记纳米疫苗的胞吞共聚焦结果。
如图6所示，纳米疫苗能够同时增加DC对多肽抗原和佐剂的胞吞量，有利于提高免疫响应。
上面结合附图对本发明进行了示例性的描述，显然本发明的实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (5)

1.一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127-136肽（GPC3127-136，AMFKNNYPSL）为免疫抗原，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，所述载体材料、所述免疫抗原和所述佐剂之间的质量比是1:1～10:0.3～0.8。
2.根据权利要求1所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的粒径为100nm～1000nm。
3.根据权利要求1或2所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：所述海藻酸钠和所述聚乙烯亚胺之间的质量比是1:0.4～3。
4.根据权利要求3所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：所述海藻酸钠的分子量为8000-50000。
5.根据权利要求1-4中任何一项所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：所述聚乙烯亚胺的分子量为3000-25000。
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