CN111588911B - 一种缓释外泌体的复合材料及其制备方法与应用 - Google Patents

一种缓释外泌体的复合材料及其制备方法与应用 Download PDF

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CN111588911B
CN111588911B CN202010452334.8A CN202010452334A CN111588911B CN 111588911 B CN111588911 B CN 111588911B CN 202010452334 A CN202010452334 A CN 202010452334A CN 111588911 B CN111588911 B CN 111588911B
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王淑芳
齐春晓
刘语菲
李承乾
王彪
祁玉婷
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Nankai University
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Abstract

本发明公开了一种缓释外泌体的复合材料及其制备方法和应用，其特点在于利用天然存在的酶促反应(谷氨酰胺转氨酶TG2催化的蛋白连接)将外泌体化学连接在生物支架材料上起到长期稳定缓慢释放的目的。制备方法包括支架材料的制备，外泌体悬液的获取，以及外泌体的负载。该复合支架避免化学交联剂残留对机体的损伤，负载过程不会对外泌体生物活性产生影响，不破坏外泌体膜结构完整性，对于外泌体治疗效果的保证奠定基础，可缓释外泌体，延长体内的作用时间，对于不能反复手术移植病例提供了长期有效的治疗方法。

Description

一种缓释外泌体的复合材料及其制备方法与应用
技术领域
本发明涉及一种缓释外泌体的方法，具体说是利用谷氨酰胺转氨酶2(TG2)将细胞分泌的外泌体与材料中的蛋白质进行交联，从而达到缓释外泌体的目的，属于生物材料及生物医学工程领域。
背景技术
外泌体是一种可被多种细胞分泌的小囊泡，内含大量的生物活性因子，如核酸、蛋白质等，在细胞交流中扮演重要角色，并在生理和病理过程中发挥作用。将外泌体负载到生物支架材料中移植到损伤性、缺损性疾病部位表现出很好地促进组织再生、调控宿主免疫环境能力。但是外泌体产量较低，因此能够使外泌体在支架材料中缓释的技术至关主要。TG2是一种人体细胞普遍存在的具有催化作用的蛋白质分子，通过催化谷氨酰胺残基和赖氨酸残基形成共价键将含有两种氨基酸残基的蛋白、多肽进行连接。TG2介导的蛋白连接使一种天然存在的酶促反应，不对蛋白活性产生影响，形成的共价键能够被细胞分泌的金属蛋白酶(MMPs)降解从而使连接的蛋白分离。本发明利用TG2催化蛋白连接的活性，将外泌体连接到含有谷氨酰胺残基和/或赖氨酸残基的生物支架材料中以实现外泌体的化学负载，进而实现外泌体的缓释。
发明内容
本发明针对于外泌体易流失、无法长效发挥作用的问题，利用TG2将外泌体与含有谷氨酰胺残基或赖氨酸残基的生物材料交联制备复合支架材料，使外泌体能长期稳定负载到支架上并达到缓释效果，提高其在组织工程中应用效率，发挥更好的治疗效果；所述生物材料包括丝素蛋白、胶原蛋白、明胶等；所述外泌体包括特定条件下功能化的脂肪间充质干细胞分泌的外泌体、骨髓间充质干细胞分泌的外泌体、脐带间充质干细胞分泌的外泌体、M2型巨噬细胞分泌的外泌体中的一种或几种的混合物。其具体制备方法包括以下步骤：
1)支架材料制备：将待负载生物材料配制成合适浓度，在加入交联剂后置于-20℃反应24h，转移至冷冻干燥机冷冻干燥24h，获得具有多孔结构的生物支架；
2)外泌体悬液的获取：培养间充质干细胞至第三代，通过炎症因子、药物、小分子或缺氧刺激细胞48h获取功能性细胞，更换无血清培养基培养24h收取细胞培养上清；通过梯度离心去除细胞碎片以及较大的囊泡；采用超速离心的方法100 000g离心70min，弃上清，加入PBS再次100 000g离心30min，弃上清，加入200μL PBS将沉淀重悬即外泌体悬液；
3)外泌体的负载：将外泌体悬液与1-5％(w/v)TG2酶混合后注入复合支架至饱和，37℃条件下反应30min即可将外泌体负载到多孔支架内部。
经TG2催化的复合支架具有缓释外泌体的作用，可用于生物支架的体内移植后治疗效果的长期维持，优先解决外泌体负载后的突释问题。
本发明与现有技术相比突出优点在于：
1)材料选取上，生物可降解的天然蛋白催化剂拥有较好的生物相容性。该催化剂完成催化作用外游离出来，不参与构成产物，最终可通过洗涤的方式将其从支架材料彻底，不会因为交联剂残留对组织造成损伤。
2)制备工艺上，利用天然的酶促反应将外泌体与复合材料中的蛋白质进行共价连接，实现外泌体的稳定负载，不会对外泌体生物活性产生影响，不破坏外泌体膜结构完整性，对于外泌体治疗效果的保证奠定基础。
3)产物功能上，制备的复合支架材料可缓释外泌体，延长体内的作用时间，对于不能反复手术移植病例提供了长期有效的治疗方法。
具体实施例
实施例1：
1)支架材料制备：7％明胶和0.25％戊二醛混合液置于-20℃反应24h，转移至冷冻干燥机冷冻干燥24h，获得具有多孔结构的明胶多孔支架；
2)外泌体悬液的获取：培养脂肪间充质干细胞至第三代，在培养基中加入20ng/mLIFN-γ刺激细胞48h获取炎性间充质干细胞，更换无血清培养基培养24h收取细胞培养上清；通过梯度离心去除细胞碎片以及较大的囊泡；采用超速离心的方法100 000g离心70min，弃上清，加入PBS再次100 000g离心30min，弃上清，加入200μL PBS将沉淀重悬即外泌体悬液；
3)将外泌体悬液与2％(w/v)TG2酶混合后注入复合支架至饱和，37℃条件下反应30min即可将外泌体负载到多孔支架内部。
实施例2：
1)支架材料制备：称取蚕丝溶于9.3M的溴化锂溶液中配成配制20％(w/v)的蚕丝溶液，置于水浴锅中60℃加热4h后，采用3500D透析袋透析48h；随后，更换20％g/mL的PEG溶液继续透析6-10h得到浓缩丝素蛋白溶液；采用10000rpm，4℃，离心20分钟后除去杂质，并通过称重法确定浓缩丝素蛋白浓度，加入适量蒸馏水配制成浓度为6％；加0.25％戊二醛后，置于-20℃反应24h，转移至冷冻干燥机冷冻干燥24h，获得具有多孔结构的明胶多孔支架；
2)培养骨髓间充质干细胞至第三代，在2％氧气环境下培养细胞48h，更换无血清培养基培养24h收取细胞培养上清；通过梯度离心去除细胞碎片以及较大的囊泡；采用超速离心的方法100 000g离心70min，弃上清，加入PBS再次100 000g离心30min，弃上清，加入200μL PBS将沉淀重悬即外泌体悬液；
3)将外泌体悬液与2％(w/v)TG2酶混合后注入复合支架至饱和，37℃条件下反应30min即可将外泌体负载到多孔支架内部。
实施例3：
1)支架材料制备：2％胶原蛋白和0.135μM氢氧化纳混合液置于-20℃反应24h，转移至冷冻干燥机冷冻干燥24h，获得具有多孔结构的胶原多孔支架；
2)外泌体悬液的获取：培养脂肪间充质干细胞至第三代，在培养基中加入20ng/mLIFN-γ刺激细胞48h获取炎性间充质干细胞，更换无血清培养基培养24h收取细胞培养上清；通过梯度离心去除细胞碎片以及较大的囊泡；采用超速离心的方法100 000g离心70min，弃上清，加入PBS再次100 000g离心30min，弃上清，加入200μL PBS将沉淀重悬即外泌体悬液；
3)将外泌体悬液与2％(w/v)TG2酶混合后注入复合支架至饱和，37℃条件下反应30min即可将外泌体负载到多孔支架内部。
实施例4：
1)支架材料制备：7％明胶和0.25％戊二醛混合液置于-20℃反应24h，转移至冷冻干燥机冷冻干燥24h，获得具有多孔结构的明胶多孔支架；
2)外泌体悬液的获取：巨噬细胞接种于T75细胞培养瓶中，含有300nM PMA的培养基诱导24小时后，细胞贴壁后换成含有20ng/mL IL-6和IL-13培养基诱导细胞48h获取M2型巨噬细胞，更换无血清培养基培养24h收取细胞培养上清；通过梯度离心去除细胞碎片以及较大的囊泡；采用超速离心的方法100 000g离心70min，弃上清，加入PBS再次100 000g离心30min，弃上清，加入200μL PBS将沉淀重悬即外泌体悬液；
3)将外泌体悬液与2％(w/v)TG2酶混合后注入复合支架至饱和，37℃条件下反应30min即可将外泌体负载到多孔支架内部。
实施例5：
取外泌体负载支架或外泌体与支架混合物加入到4.0mL PBS中置于垂直混悬仪上，37℃条件下检测外泌体释放效率，分别于4h、12h、24h、2d、4d、7d取反应液200μL补充PBS200μL。反应结束后，用CD9 ELISA试剂盒检测反应液中CD9浓度。结果如表1显示，外泌体经TG2负载到支架材料后具有缓释作用，释放作用能够持续7d。
实施例6：
采用3％的戊巴比妥钠进行麻醉(静脉注射40mg/每kg体重)，随后采用无菌角膜环钻在双膝关节滑车沟中央制造直径3mm深，0.5mm宽的软骨缺损并移植外泌体负载支架或外泌体物理吸附支架。术后，连续肌肉注射青霉素(80000U)3天。分别在4周和12周处死动物收集膝关节，根据ICRS scoring system对样本的缺损填充情况、表明平滑度以及组织整合情况进行评测并评分。样本置于EDTA脱钙液(pH 7.2)中在37℃摇床80转/分钟条件下进行脱钙，每3天更换一次脱钙液，脱钙4-6周。以细针穿刺样本非缺损区以检查脱钙程度，直至穿刺有类似突破湿润报纸的感觉时，提示脱钙完成，样本换至PBS中漂洗。漂洗后的样本进行脱水、石蜡包埋后，石蜡组织切片机进行6μm石蜡切片。切片常规65℃烤片、脱蜡、复水后进行H/E染色观察细胞的分布，甲苯胺蓝和番红O的染色用于评价粘多糖的生成，免疫组化染色评价软骨层支架II型胶原生成。
结果：根绝软骨整体评分，TG2介导的外泌体负载组能更有效的修复软骨缺损，缺损修复率达95.2％，物理负载组为64.9％，软骨成分分析显示TG2介导的外泌体负载组能更有效的促进软骨粘多糖和2型胶原的产生。以上结果证明本发明制备的外泌体负载支架起到了缓释作用，延长了作用时间，具有更优良的治疗效果。
表1.TG2介导的外泌体负载支架与外泌体物理负载支架性质比较
Figure BSA0000209669570000041
Figure BSA0000209669570000051

Claims (2)

1.一种缓释外泌体的复合材料，是利用谷氨酰胺转氨酶2将外泌体与含有谷氨酰胺残基或赖氨酸残基的生物材料交联制备的复合支架材料；所述生物材料选自丝素蛋白、胶原蛋白、明胶；所述外泌体包括特定条件下功能化的脂肪间充质干细胞分泌的外泌体、骨髓间充质干细胞分泌的外泌体、脐带间充质干细胞分泌的外泌体、M2型巨噬细胞分泌的外泌体中的一种或几种的混合物。
2.一种如权利要求1所述的缓释外泌体的复合材料制备方法，包括如下步骤：
1)支架材料制备：将待负载生物材料配制成合适浓度，在加入交联剂后置于-20℃反应24h，转移至冷冻干燥机冷冻干燥24h，获得具有多孔结构的生物支架；
2)外泌体悬液的获取：培养间充质干细胞至第三代，通过炎症因子、药物、小分子或缺氧刺激细胞48h获取功能性细胞，更换无血清培养基培养24h收取细胞培养上清；通过梯度离心去除细胞碎片以及较大的囊泡；采用超速离心的方法100 000g离心70min，弃上清，加入PBS再次100 000g离心30min，弃上清，加入200μL PBS将沉淀重悬即外泌体悬液；
3)外泌体的负载：将外泌体悬液与1-5％(w/v)谷氨酰胺转氨酶2混合后注入复合支架至饱和，37℃条件下反应30min即可将外泌体负载到多孔支架内部。
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