CN112618515A - 一种负载外泌体的口服结肠靶向给药的聚合物的制备方法 - Google Patents
一种负载外泌体的口服结肠靶向给药的聚合物的制备方法 Download PDFInfo
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Abstract
本发明公开了一种负载外泌体的口服结肠靶向给药的聚合物的制备方法，属于生物医药领域。本发明提供的这种负载外泌体用于口服结肠靶向给药的层层自组装聚合物是由壳聚糖季铵盐(HTCC)、氧化魔芋葡甘聚糖(OKGM)以及间充质干细胞来源的外泌体(MSCs‑EXO)通过层层包裹和逐层封装(LbL)复合而成的(MSCs‑EXO)‑(HTCC/OKGM)n自组装聚合物，具有较好的生物相容性、稳定性、安全性、抗炎性及缓释性能；可作为载药系统搭载外泌体对溃疡部分进行修复。
Description
技术领域
本发明涉及一种负载外泌体的口服结肠靶向给药的聚合物的制备方法，属于生物医药领域。
背景技术
疡性结肠炎(ulcerative colitis，UC)是一种慢性非特异性肠道炎症性疾病，病变主要累及乙状结肠、直肠的黏膜和黏膜下层，也可延伸至降结肠，甚至整个结肠。临床表现主要为腹泻﹑黏液脓血便腹痛等症状。疡性结肠炎可发病常在青春晚期和成年早期，平均年龄为17～40岁。由于疡性结肠炎的病因尚不不清，病情也易反复发作，病程漫长，迁延不愈，且具有癌变倾向并伴随多种肠外症状，因此，WHO将其确定为现代难治愈的疾病之一。
目前疡性结肠炎的手术治疗目前尚无规范化的标准术式。中医上的药物治疗常常采取保留灌肠给药的方式，西药主要选择氨基水杨酸类、肾上腺皮质激素类、免疫抑制剂类等药物进行治疗。现有的治疗疡性结肠炎的传统药物往往面临药效持续时间短、抗炎作用不持久的问题，还容易存在病情易复发、靶向性较差且容易导致一些不良反应等一系列问题。
因此，开发出一种具有较高安全性、稳定性和持久抗炎性较强靶向性的负载外泌体的口服结肠靶向给药的聚合物，对于治疗溃疡性结肠炎而言具有良好的市场前景。
发明内容
为了实现上述目的，本发明提供了一种负载外泌体的口服结肠靶向给药的聚合物的制备方法，由壳聚糖季铵盐和氧化魔芋葡甘聚糖作为外壳，通过层层自组装的方式将负载外泌体的口服结肠靶向给药进行包覆，获得了一种安全性高、稳定性好、具有持久抗炎性、靶向性较强的可用于治疗溃疡性结肠炎的负载外泌体的口服结肠靶向给药层层自组装聚合物系统。
本发明首先提供了一种负载外泌体的口服结肠靶向给药的聚合物的制备方法，所述方法步骤包括：
(1)收集间充质干细胞培养上清液，通过差速离心法提取外泌体；
(2)在魔芋葡甘聚糖水溶液中加入氧化剂高碘酸盐后搅拌反应，之后加入乙二醇、透析，固液分离取上清液，冷冻干燥制备得到氧化魔芋葡甘聚糖；
(3)将步骤(2)制备得到的氧化魔芋葡甘聚糖和壳聚糖季铵盐分别溶解于PBS缓冲液中得到氧化魔芋葡甘聚糖溶液和壳聚糖季铵盐溶液；
(4)将壳聚糖季铵盐溶液与外泌体恒定旋转20-30分钟，用PBS洗涤，离心，收集沉淀，得到(MSCs-EXO)-HTCC聚合物；
(5)将步骤(4)得到的(MSCs-EXO)-HTCC聚合物与氧化魔芋葡甘聚糖溶液恒定旋转20-30分钟，用PBS洗涤，离心，得到(MSCs-EXO)-(HTCC/OKGM)聚合物；
重复步骤(4)和(5)，即可制备得到负载干细胞用于口服结肠靶向给药的层层自组装聚合物，(MSCs-EXO)-(HTCC/OKGM)n聚合物，n为包裹层数。
进一步的，所述步骤(1)中收集的间充质干细胞上清液为第三代间充质干细胞融合至70-80％时，更换无血清培养基，培养48h收集的细胞上清液。
进一步的，所述步骤(1)中所述差速离心法的具体操作为：将收集的间充质干细胞上清液300g-500g离心10min-15min，收集上清；1800-2000g离心10min-15min，收集上清；10000g-11000g离心60min-70min，收集上清；100000g-110000min离心60min-70min，收集沉淀；最后将外泌体沉淀重悬于适量PBS中，100000g-110000min离心60min-70min，收集沉淀，得到纯化的外泌体。进一步的，所述步骤(1)中差速离心均在4℃下进行。
进一步的，所述氧化魔芋葡甘聚糖的制备具体包括：在500mL去离子水中加入5gKGM粉末，搅拌溶解，滴加10mL0.5mol/L高碘酸钠水溶液，40℃避光搅拌4h；之后向反应混合物中加入10mL乙二醇搅拌2h，以中和未反应的高碘酸盐；将溶液用透析膜(MWCO:12,000-14,000)透析3天，直到渗析液中不含碘酸盐；将反应产物以2500r/min离心20min，取上清液，真空冷冻干燥获得OKGM，将干燥的样品储存在干燥器中以供下一步使用。
进一步的，所述步骤(3)中所用的壳聚糖季铵盐(HTCC)和氧化魔芋葡甘聚糖(OKGM)在PBS中溶解的终浓度均为0.01-1.0mg/mL。
进一步的，所述步骤(4)和(5)中，PBS为磷酸缓冲盐溶液，pH为7.2-7.4，洗涤2～3次。
进一步的，所述外泌体与壳聚糖季铵盐溶液的质量体积比为200-500μg：1-3mL；其中，所述外泌体溶解于PBS中。
进一步的，所述氧化魔芋葡甘聚糖溶液与壳聚糖季铵盐溶液的体积比为l-3：1-3。
进一步的，优选重复步骤(4)和(5)1～3次，最优选重复2次，制备得到(MSCs-EXO)-(HTCC/OKGM)2聚合物。
本发明提供了上述制备方法制备得到的负载干细胞用于口服结肠靶向给药的层层自组装聚合物。
本发明提供了包含所述负载干细胞用于口服结肠靶向给药的层层自组装聚合物的药物或食品。
本发明提供了所述负载干细胞用于口服结肠靶向给药的层层自组装聚合物在制备用于治疗溃疡性结肠炎的药物中的应用。
本发明取得的有益效果：
(1)本发明采用LbL技术制备的(MSCs-EXO)-(HTCC/OKGM)n自组装聚合物的，具有较好的生物相容性、稳定性、安全性、抗炎性及缓释性能。
(2)本发明中，HTCC和OKGM可通过缓控式释放MSCs-EXO，利用MSCSs-EXO归巢性能，能够有效的促进溃疡部位组织的再生和修复。
具体实施方式
以下通过具体实施例和对比例对本发明作进一步的具体说明，但应该理解本发明并不受这些内容所限制。
稳定性检测：将未包被的外泌体和LbL包被的外泌体置于模拟胆汁溶液以及模拟胃液中，于37℃水浴2h。2h后，通过离心收集外泌体，洗涤2次后进行结构完整性检测，主要是膜结构是否完整。
粘膜粘附能力检测：新鲜分离的猪小肠被清洗并切片。LbL包被的或未包被的外泌体用特异性荧光标记，预先固定于小肠内壁，然后将在37℃孵育1h，使用IVIS成像观察分析。
实施例1
(1)间充质干细胞来源外泌体的提取：当第三代间充质干细胞融合至70-80％时，更换无血清培养基，培养48h，收集细胞上清液。使用差速离心法提取外泌体，具体操作为：在4℃下，将收集的间充质干细胞上清液300g离心10min，收集上清；20000g离心10min，收集上清；10000g离心70min，收集上清；100000g离心70min，收集沉淀；最后将外泌体沉淀重悬于适量PBS中，100000g离心70min，收集沉淀，得到纯化的外泌体。
(2)OKGM的合成：在500mL去离子水中加入5gKGM粉末，搅拌溶解，滴加10mL0.5mol/L高碘酸钠水溶液，40℃避光搅拌4h。之后向反应混合物中加入10mL乙二醇搅拌2h，以中和未反应的高碘酸盐。将溶液用透析膜(MWCO:12,000-14,000)透析3天，直到渗析液中不含碘酸盐。将反应产物以2500r/min离心20min，取上清液，真空冷冻干燥获得OKGM，将干燥的样品储存在干燥器中以供下一步使用。
(3)(MSCs-EXO)-(HTCC/OKGM)n自组装聚合物的合成：将壳聚糖季铵盐(HTCC)和氧化魔芋葡甘聚糖(OKGM)在PBS中溶解，终浓度均为0.1mg/mL。将阳离子聚合物壳聚糖季铵盐和外泌体在室温下恒定慢速旋转混合30min，洗涤2-3次，得到(MSCs-EXO)-HTCC聚合物。之后将阴离子聚合物氧化魔芋葡甘聚糖与MSCS-HTCC在室温下恒定慢速旋转混合30min，得到(MSCs-EXO)-(HTCC/OKGM)自组装聚合物。
再重复上述步骤(3)1～2次，分别得到(MSCs-EXO)-(HTCC/OKGM)2自组装聚合物、(MSCs-EXO)-(HTCC/OKGM)3自组装聚合物。
对上述制备得到的(MSCs-EXO)-(HTCC/OKGM)、(MSCs-EXO)-(HTCC/OKGM)2和(MSCs-EXO)-(HTCC/OKGM)3分别进行稳定性、粘膜粘附性能检测。
稳定性检测：(MSCs-EXO)-(HTCC/OKGM)在模拟胃液中两小时膜结构遭到破坏，不足以保护外泌体完全免受胆盐或胃酸的侵害。而(MSCs-EXO)-(HTCC/OKGM)2暴露在37℃的模拟胃液中或模拟胆盐溶液的聚合物可防止酸性和胆盐的侵蚀达2h，可见，具有良好的稳定性。(MSCs-EXO)-(HTCC/OKGM)3的稳定性更好。
粘膜粘附检测的实验结果表明，对于(MSCs-EXO)-(HTCC/OKGM)2而言，1h后可检测到LbL包裹的外泌体水平较裸露的外泌体高出了近三倍，2h后仍可高出两倍以上，6h内LbL包裹的外泌体水平明显高于裸露的外泌体，12h后由于外泌体达到饱和，此种差异逐渐减小。由此可见，LbL包裹的(MSCs-EXO)-(HTCC/OKGM)2的粘膜粘附能力更强，在肠道中停留的时间更久。而(MSCs-EXO)-(HTCC/OKGM)3包裹的外泌体释放所需的时间延长，其释放延迟了超过4h。
对比例1
对比例1为实施例1步骤(1直接提取得到的外泌体，未进行后续的封装过程。
检测结果如下：当暴露在37℃的模拟胃液中时，普通的、未包裹HTCC/OKGM的外泌体结构被破坏。
对比例2
其余步骤和实施例1相同，不对魔芋葡甘聚糖进行氧化，按照实施例1的方式直接利用魔芋葡甘聚糖和壳聚糖季铵盐进行自组装。
研究发现，二者结合无法实现自组装过程，原因是魔芋葡甘聚糖的水溶液粘稠，流动性差，无法进行后续实验。
对比例3
当壳聚糖季铵盐浓度小于0.01mg/mL时，壳聚糖季铵盐所带正电荷不足以支持与外泌体的结合；当壳聚糖季铵盐浓度大于1mg/mL时，壳聚糖季铵盐对外泌体显示出了一定的细胞毒性。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种负载外泌体的口服结肠靶向给药的聚合物的制备方法，其特征在于，所述方法步骤包括：
(1)收集间充质干细胞培养上清液，通过差速离心法提取外泌体；
(2)在魔芋葡甘聚糖水溶液中加入氧化剂高碘酸盐后搅拌反应，之后加入乙二醇、透析，固液分离取上清液，冷冻干燥制备得到氧化魔芋葡甘聚糖；
(3)将步骤(2)制备得到的氧化魔芋葡甘聚糖和壳聚糖季铵盐分别溶解于PBS缓冲液中得到氧化魔芋葡甘聚糖溶液和壳聚糖季铵盐溶液；
(4)将壳聚糖季铵盐溶液与外泌体恒定旋转20-30分钟，用PBS洗涤，离心，收集沉淀，得到(MSCs-EXO)-HTCC聚合物；
(5)将步骤(4)得到的(MSCs-EXO)-HTCC聚合物与氧化魔芋葡甘聚糖溶液恒定旋转20-30分钟，用PBS洗涤，离心，得到(MSCs-EXO)-(HTCC/OKGM)聚合物；
重复步骤(4)和(5)，即可制备得到负载干细胞用于口服结肠靶向给药的层层自组装聚合物，(MSCs-EXO)-(HTCC/OKGM)n聚合物，n为包裹层数。
2.根据权利要求1所述的制备方法，其特征在于，所述氧化魔芋葡甘聚糖的制备具体包括：在500mL去离子水中加入5gKGM粉末，搅拌溶解，滴加10mL0.5mol/L高碘酸钠水溶液，40℃避光搅拌4h；之后向反应混合物中加入10mL乙二醇搅拌2h，以中和未反应的高碘酸盐；将溶液用透析膜(MWCO:12,000-14,000)透析3天，直到渗析液中不含碘酸盐；将反应产物以2500r/min离心20min，取上清液，真空冷冻干燥获得OKGM，将干燥的样品储存在干燥器中以供下一步使用。
3.根据权利要求1或2所述的制备方法，其特征在于，所述步骤(3)中壳聚糖季铵盐和氧化魔芋葡甘聚糖在PBS中溶解的终浓度均为0.01-1.0mg/mL。
4.根据权利要求1～3任一项所述的制备方法，其特征在于，所述步骤(4)和(5)中，PBS为磷酸缓冲盐溶液，pH为7.2-7.4，洗涤2～3次。
5.根据权利要求1～4任一项所述的制备方法，其特征在于，所述外泌体与壳聚糖季铵盐溶液的质量体积比为200-500μg：1-3mL。
6.根据权利要求1～5任一项所述的制备方法，其特征在于，所述氧化魔芋葡甘聚糖溶液与壳聚糖季铵盐溶液的体积比为l-3：1-3。
7.根据权利要求1～6任一项所述的制备方法，其特征在于，重复步骤(4)和(5)1～3次。
8.权利要求1～7任一项所述的制备方法制备得到的负载外泌体的口服结肠靶向给药的聚合物。
9.包含权利要求8所述的负载外泌体的口服结肠靶向给药的聚合物的药物或食品。
10.权利要求8所述的负载外泌体的口服结肠靶向给药的聚合物在制备用于治疗溃疡性结肠炎的药物中的应用。
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| CN114376987A (zh) * | 2021-12-07 | 2022-04-22 | 安徽中医药大学 | 用于治疗溃疡性结肠炎的结肠靶向纳米粒及其制备方法 |
| CN114376987B (zh) * | 2021-12-07 | 2023-09-22 | 安徽中医药大学 | 用于治疗溃疡性结肠炎的结肠靶向纳米粒及其制备方法 |
| CN115089724A (zh) * | 2022-06-13 | 2022-09-23 | 江南大学 | 一种用于口服结肠靶向给药的外泌体-聚合物杂化纳米颗粒的制备方法及应用 |
| CN115089724B (zh) * | 2022-06-13 | 2024-03-01 | 江南大学 | 一种用于口服结肠靶向给药的外泌体-聚合物杂化纳米颗粒的制备方法及应用 |
| CN115737699A (zh) * | 2022-11-22 | 2023-03-07 | 百欧派(天津)生物技术有限公司 | 干细胞衍生制剂及其制备方法和应用 |
| CN115737699B (zh) * | 2022-11-22 | 2024-03-08 | 百欧派(天津)生物技术有限公司 | 干细胞衍生制剂及其制备方法和应用 |
| JP7497094B1 (ja) | 2023-09-07 | 2024-06-10 | 株式会社 バイオミメティクスシンパシーズ | 腸内細菌叢改善のための組成物及びその応用 |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2616153A (en) | 2023-08-30 |
| CN112618515B (zh) | 2021-09-24 |
| GB202307931D0 (en) | 2023-07-12 |
| WO2022142448A1 (zh) | 2022-07-07 |
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