CN114788896A - 一种全细胞肿瘤疫苗支架及其制备方法 - Google Patents
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Abstract
本发明提供一种全细胞肿瘤疫苗支架的制备方法，包括以下步骤：S1、配置支架基质材料、交联剂/光引发剂、肿瘤细胞、免疫佐剂的混合溶液；S2、固化S1得到的混合溶液，冷冻干燥后得到全细胞肿瘤疫苗支架。该支架制备过程简单，一步冻干法完成全细胞肿瘤疫苗抗原制备、支架制备和抗原装载。植入肿瘤切除部位，其中冻干的肿瘤细胞作为全细胞肿瘤抗原，辅以免疫佐剂，获得的具有大孔3D结构的疫苗支架可作为免疫发生中心招募树突状细胞并促进其成熟，从而激发抗肿瘤免疫反应，用于防止肿瘤术后复发和转移。
Description
技术领域
本发明涉及生物材料领域，具体涉及一种基于冷冻干燥技术的全细胞肿瘤疫苗支架及其制备方法。
背景技术
癌症严重威胁人类健康，基于肿瘤疫苗的免疫治疗具有极大的发展前途及应用潜力。尽管肿瘤疫苗发展迅速，但仍存在治疗效果有限、生产工艺复杂等问题。例如，树突状细胞疫苗需要在体外进行细胞分离、修饰和筛选，之后进行体内移植，导致生产周期长、价格昂贵，且超过90％的移植树突状细胞在归巢到淋巴结前死亡，使得效应T细胞的启动不足。肿瘤细胞疫苗、抗原或多肽疫苗等将树突状细胞在体内直接活化，但存在引发T细胞免疫应答能力有限、需反复注射等问题，极大地限制了其广泛应用。因此，开发新兴且便捷的肿瘤疫苗递送系统具有很强的临床应用前景和价值。利用生物材料递送疫苗、提高疗效是近几年研发的热点。相比于纳米材料，生物材料支架不仅可作为抗原和免疫调节生物制剂的递送系统，同时可原位募集免疫细胞。生物材料支架能够对抗原和免疫调节生物制剂的释放进行时间和空间的控制，从而实现对招募的树突状细胞进行原位诱导和活化，进而产生强有力的细胞毒性T淋巴细胞的免疫反应，提高抗肿瘤效果。
然而，目前基于生物材料支架的肿瘤疫苗制备过程繁琐，主要包括：肿瘤抗原的制备、生物材料支架的制备和抗原的装载。常用的肿瘤抗原包括特定抗原(如蛋白或多肽)，和全细胞抗原(如肿瘤细胞裂解物或放疗致死的肿瘤细胞)。蛋白/多肽疫苗通常只能靶向一种或几种肿瘤相关抗原，若要引发机体更为强烈的肿瘤特异性T细胞响应，往往需要在疫苗接种时混合多种抗原。由于肿瘤细胞上存在一系列突变的肿瘤抗原，全肿瘤细胞抗原比通用的肿瘤抗原免疫原性更强，可以协同增强机体产生特异性免疫应答。但全细胞肿瘤抗原的制备需特殊的处理，如反复冻融、放疗、化疗等。另外，在生物支架材料制作完成后，往往需要将肿瘤抗原进行额外担载，进一步使过程复杂化。因此，寻找更简单的方法制备基于生物材料支架的肿瘤疫苗意义重大。
发明内容
本发明针对现有技术中的不足，提供一种全细胞肿瘤疫苗支架的制备方法。
为实现上述目的，本发明采用以下技术方案：
一种全细胞肿瘤疫苗支架的制备方法，包括以下步骤：
S1、配置支架基质材料、交联剂/光引发剂、肿瘤细胞、免疫佐剂的混合溶液；
S2、固化S1得到的混合溶液，冷冻干燥后得到全细胞肿瘤疫苗支架。
为优化上述技术方案，采取的具体措施还包括：
进一步地，步骤S1中，所述支架基质材料为胶原、明胶、海藻酸钠、壳聚糖、透明质酸、明胶-甲基丙烯酸甲酯、海藻酸钠-甲基丙烯酸甲酯、壳聚糖-甲基丙烯酸甲酯、透明质酸-甲基丙烯酸甲酯中的一种或多种的混合。
进一步地，步骤S1中，所述交联剂/光引发剂为戊二醛、甲醛、钙盐、京尼平、丹宁酸、苯基-2,4,6-三甲基苯甲酰基膦酸锂(LAP)、2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮(2959)、2-羟基-2-甲基-1-苯基-1-丙酮(HMPP)中的一种或多种的混合。
进一步地，步骤S1中，所述免疫佐剂为咪喹莫特、CpG寡聚脱氧核苷酸、聚肌苷酸胞苷酸、单磷酰脂质A中的一种。
进一步地，步骤S2中，固化方法为光聚合固化或交联反应固化。
一种通过上述方法制备的全细胞肿瘤疫苗支架，该支架制备过程简单，一步冻干法完成全细胞肿瘤疫苗抗原制备、支架制备和抗原装载。植入肿瘤切除部位，其中冻干的肿瘤细胞作为全细胞肿瘤抗原，辅以免疫佐剂，获得的具有大孔3D结构的疫苗支架可作为免疫发生中心招募树突状细胞并促进其成熟，从而激发抗肿瘤免疫反应，用于防止肿瘤术后复发和转移。
本发明的有益效果是：常规的制备基于生物材料支架的肿瘤疫苗的方法主要包括肿瘤抗原的制备、生物材料支架的制备和抗原的装载，其制备过程十分繁琐；本发明首次通过一步冻干法，将全细胞肿瘤疫苗抗原制备、支架制备和抗原装载等过程一体化，直接将肿瘤细胞混入支架材料，得到了全细胞肿瘤疫苗支架，省去了繁琐的制备过程，提高了制备的效率；本发明选用全细胞肿瘤抗原，相比通用肿瘤抗原具有更强的抗原免疫性并协同增强机体产生特异性免疫应答；冻干的肿瘤细胞作为全细胞肿瘤抗原，辅以免疫佐剂，获得的具有大孔3D结构的疫苗支架可招募树突状细胞并促进其成熟，从而引发抗肿瘤免疫反应。此外，本发明制备的全细胞肿瘤疫苗支架涉及到的基质材料和交联剂/光引发剂均为常用试剂，具有易于获取的特点。
附图说明
图1是全细胞肿瘤疫苗支架的制备过程示意图；
图2为实施例1制备的4T1全细胞肿瘤疫苗支架的SEM照片；
图3为实施例1制备的4T1全细胞肿瘤疫苗支架的树突状细胞募集与激活；
图4为实施例1制备的4T1全细胞肿瘤疫苗支架的T细胞反应。
具体实施方式
实施例1 4T1全细胞肿瘤疫苗支架
制备：如图1所示，将明胶-甲基丙烯酸甲酯在60℃的去离子水中溶解，完全溶解后冷却至室温。将200μL明胶-甲基丙烯酸甲酯溶液(30％w/v，含有LAP 0.1％w/w)、50μL咪喹莫特溶液(240μg/mL)和50μL的小鼠乳腺癌4T1细胞溶液(4×107cells/mL)混合均匀，倒入模具，405nm光照后成胶，置于-80℃冰箱2小时后冷冻干燥得到4T1全细胞肿瘤疫苗支架。
表征：在场发射扫描电镜下观察4T1全细胞肿瘤疫苗支架的形貌，如图2所示，支架可见大孔3D结构和肿瘤细胞。
树突状细胞的募集和成熟：将4T1全细胞肿瘤疫苗支架和空白支架分别植入小鼠皮下，7天后取出4T1全细胞肿瘤疫苗支架和空白支架。将两种支架用胶原酶I(500μg/mL)和胶原酶IV(100μg/mL)于37℃下消化1小时，经过70μm的Falcon细胞过滤筛过滤得到单细胞悬液。得到的细胞用CD16/32抗体封闭，之后用有荧光标记的CD11c、CD80和CD86抗体染色，通过流式细胞术考察树突状细胞的招募和DC的成熟。流式结果分析(图3)可知相较于空白支架，4T1全细胞肿瘤疫苗支架中CD11c+细胞(树突状细胞)数量明显增多，且CD80+CD86+细胞(成熟的树突状细胞)所占比例明显增加。
T细胞反应：将4T1全细胞肿瘤疫苗支架和空白支架分别植入小鼠皮下，7天后取出淋巴结。用载玻片的磨砂面将淋巴结碾碎，经过70μm的Falcon细胞过滤筛过滤得到单细胞悬液。将得到的单细胞用CD16/32抗体封闭，后用有荧光标记的CD3、CD4和CD8a抗体染色，通过流式细胞术考察T细胞反应。流式结果分析(图4)可知相较于空白支架组，4T1全细胞肿瘤疫苗支架组淋巴结中CD3+CD4+细胞和CD3+CD8+细胞比例明显增加。
实施例2CT26全细胞肿瘤疫苗支架
制备：将明胶-甲基丙烯酸甲酯在60℃的去离子水中溶解，完全溶解后冷却至室温。配制明胶-甲基丙烯酸甲酯(10％w/v)、透明质酸-甲基丙烯酸甲酯(10％w/v)、HMPP(1％v/v)、单磷酰脂质A(400μg/mL)、小鼠结肠癌CT26细胞(1×108cells/mL)混合液，将300μL混合溶液倒入模具，紫外光光照后成胶，置于-20℃冰箱2小时，后冷冻干燥得到CT26全细胞肿瘤疫苗支架。
实施例3HepG2全细胞肿瘤疫苗支架
将壳聚糖溶解在0.5％的醋酸溶液中，得到浓度为2wt％的壳聚糖溶液，加入甘油磷酸钠调节pH值。将明胶溶解在蒸馏水中，得到溶度为4wt％的明胶溶液。将明胶与壳聚糖按照1:1(v/v)充分混合。将京尼平(0.5w/w)、人肝癌细胞HepG2(1×108cells/mL)、CpG寡聚脱氧核苷酸(250μg/mL)加入到明胶与壳聚糖的混合溶液中，将400μL混合溶液倒入模具，37℃放置后成胶，置于-80℃冰箱2小时，后冷冻干燥得到HepG2全细胞肿瘤疫苗支架。
实施例4Hela全细胞肿瘤疫苗支架
配制海藻酸钠(2％w/w)、聚肌苷酸胞苷酸(200μg/mL)、人宫颈癌Hela细胞(1×108cells/mL)混合液，加入氯化钙(0.5％w/w)混合均匀，将500μL混合溶液倒入模具，成胶后置于-20℃冰箱2小时，后冷冻干燥得到Hela全细胞肿瘤疫苗支架。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。
Claims (6)
1.一种全细胞肿瘤疫苗支架的制备方法，其特征在于，包括以下步骤：
S1、配置支架基质材料、交联剂/光引发剂、肿瘤细胞、免疫佐剂的混合溶液；
S2、固化S1得到的混合溶液，冷冻干燥后得到全细胞肿瘤疫苗支架。
2.根据权利要求1所述的一种全细胞肿瘤疫苗支架的制备方法，其特征在于，
步骤S1中，所述支架基质材料为胶原、明胶、海藻酸钠、壳聚糖、透明质酸、明胶-甲基丙烯酸甲酯、海藻酸钠-甲基丙烯酸甲酯、壳聚糖-甲基丙烯酸甲酯、透明质酸-甲基丙烯酸甲酯中的一种或多种的混合。
3.根据权利要求1所述的一种全细胞肿瘤疫苗支架的制备方法，其特征在于，
步骤S1中，所述交联剂/光引发剂为戊二醛、甲醛、钙盐、京尼平、丹宁酸、苯基-2,4,6-三甲基苯甲酰基膦酸锂、2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮、2-羟基-2-甲基-1-苯基-1-丙酮中的一种或多种的混合。
4.根据权利要求1所述的一种全细胞肿瘤疫苗支架的制备方法，其特征在于，
步骤S1中，所述免疫佐剂为咪喹莫特、CpG寡聚脱氧核苷酸、聚肌苷酸胞苷酸、单磷酰脂质A中的一种。
5.根据权利要求1所述的一种全细胞肿瘤疫苗支架的制备方法，其特征在于，
步骤S2中，固化方法为光聚合固化或交联反应固化。
6.一种如权利要求1-5中任一项方法制备的全细胞肿瘤疫苗支架。
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