CN113797151B - 一种含间充质干细胞外泌体的修护凝胶及其制备方法 - Google Patents
一种含间充质干细胞外泌体的修护凝胶及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种含间充质干细胞外泌体的修护凝胶及其制备方法,属于化妆品技术领域,该修护凝胶由外泌体冻干粉和凝胶辅助组分组成。本发明中对外泌体进行先冻干处理再添加使用,避免了外泌体直接使用时其他组分对外泌体的破坏,从而增强了修护凝胶对皮肤的修复性能;同时其具有低免疫原性低,在皮肤表面使用时不会造成过敏反应;添加甘油、透明质酸钠、燕麦β‑葡聚糖、海藻糖、神经酰胺、烟酰胺作为本发明的保湿体系,加强凝胶的保湿性和成膜性,促进有效成分的经皮吸收,最大程度发挥本发明的皮肤修护功能;马齿苋提取物的添加使本发明具有消炎抗菌功效;甘油、甘氨酸、海藻糖复配的冻干保护液可以保护外泌体在冷冻干燥中的生物活性。
Description
技术领域
本发明涉及化妆品技术领域,具体涉及到一种含间充质干细胞外泌体的修护凝胶及其制备方法。
背景技术
外泌体(exosome)是哺乳动物细胞通过“内吞-外排”过程分泌到细胞外的一类囊泡状结构,直径约为30-150nm。外泌体不仅存在于细胞培养液中,也存在于血液、尿液、羊水、母乳、精液、唾液、胸腔积液等体液中,其组成包括脂质双分子层以及细胞来源相关的蛋白质、RNA、DNA等物质。
现有的凝胶类化妆品虽然种类非常繁多,但是对过敏感性肌肤引起的过敏不能够起到很好的修复,且功效单一。同时现有技术中并未见添加外泌体的修护凝胶的研究报道。因此,为了满足消费者对外泌体修护凝胶的需求,提供一种含间充质干细胞外泌体的修护凝胶及其制备方法也就显得十分的有意义。
发明内容
针对上述的不足,本发明的目的是提供一种含间充质干细胞外泌体的修护凝胶及其制备方法,可有效解决现有凝胶类化妆品对皮肤修复效果差和功效单一的问题;该修护凝胶兼具有修复皮肤、抗炎杀菌和保湿效果,可更好满足消费者的需求。
为达上述目的,本发明采取如下的技术方案:
本发明提供一种含间充质干细胞外泌体的修护凝胶,由外泌体冻干粉和凝胶辅助组分组成;
其中,外泌体冻干粉由质量比为1~2:1~2的外泌体提取液和冻干保护剂组成。
进一步地,外泌体冻干粉和凝胶辅助组分的质量比为2~10:1~5。
进一步地,外泌体提取液和冻干保护剂的质量比为1:1。
进一步地,外泌体提取液通过以下方法制得:
收集P3-P7代的脐带干细胞培养上清,转移上清于离心管中进行第一次离心去活细胞,第二次离心去除死细胞,取离心后的上清过滤,即可得到外泌体提取液。
进一步地,外泌体提取液的制备方法中第一次离心的参数为:温度为2~5℃;转速为300~400xg;离心时长为10~15min。
进一步地,外泌体提取液的制备方法中第二次离心的参数为:温度为2~5℃;转速为2000~3000xg;离心时长为10~15min。
进一步地,冻干保护剂由以下质量份的原料组成:甘油1~5份、甘氨酸1~5份、海藻糖1~3份和无菌水1~3份。
进一步地,冻干保护剂由以下质量份的原料组成:甘油3份、甘氨酸3份、海藻糖2份和无菌水2份。
进一步地,外泌体冻干粉通过以下方法制得:
步骤(1):将外泌体提取液和冻干保护剂混合均匀得到外泌体冻干液;
步骤(2):将步骤(1)所得的外泌体冻干液于-80℃~-70℃预冻48-56h,然后将预冻好的外泌体冻干液真空冷冻干燥,即可得到外泌体冻干粉。
进一步地,凝胶辅助组分包括以下质量份的原料:甘油2~6份、燕麦β-葡聚糖2~6份、透明质酸钠2~5份、马齿苋提取物1~4份、神经酰胺1~2份、烟酰胺1~2份、海藻糖1~2份、Carbopol Ultrez 20 0.5~2份和无菌水5~10份。
进一步地,凝胶辅助组分包括以下质量份的原料:甘油4份、燕麦β-葡聚糖4份、透明质酸钠3份、马齿苋提取物2份、神经酰胺1份、烟酰胺1份、海藻糖1份、Carbopol Ultrez20 1份和无菌水5~10份。
本发明中马齿苋提取物可直接购买市售产品。
本发明还提供上述含间充质干细胞外泌体的修护凝胶的制备方法,包括以下步骤:
步骤(1):将外泌体冻干粉、透明质酸钠、甘油、马齿苋提取液、燕麦β-葡聚糖、烟酰胺和一半的无菌水加入容器中,搅拌均匀,得到溶液一;
步骤(2):将剩下的一半无菌水加入容器中,加热至70℃~80℃,然后加入Carbopol Ultrez 20搅拌均匀,冷却,得到均匀透明的凝胶状溶液二;
步骤(3):将步骤(2)所得的溶液二分批次加入步骤(1)所得的溶液一中,搅拌均匀,即可得到含间充质干细胞外泌体的修护凝胶。
综上所述,本发明具有以下优点:
1、本发明提供了一种含间充质干细胞外泌体的修护凝胶,该修护凝胶可以在皮肤表面形成一层保护薄膜,隔绝外界环境对皮肤的损害,同时添加外泌体可以对皮肤进行修护,维护皮肤状态。同时配合添加的透明质酸钠、甘油等作为本发明的保湿体系,添加马齿苋提取物做消炎抗菌体系。通过各组分间的协同作用,使得修护凝胶兼具有修复皮肤、抗炎杀菌和保湿效果,可更好满足消费者的需求。
2、本发明中选用的干细胞外泌体是一种高效的皮肤修复成分,同时来源于间充质干细胞,具有低免疫原性低,在皮肤表面使用时不会造成过敏反应;同时添加甘油、透明质酸钠、燕麦β-葡聚糖、海藻糖、神经酰胺、烟酰胺作为本发明的保湿体系,加强凝胶的保湿性和成膜性,促进有效成分的经皮吸收,最大程度发挥本发明的皮肤修护功能;马齿苋提取物的添加使本发明具有消炎抗菌功效;甘油、甘氨酸、海藻糖复配的冻干保护液可以保护外泌体在冷冻干燥中的生物活性。
3、本发明中对外泌体进行先冻干处理再添加使用,避免了外泌体直接使用时其他组分对外泌体的破坏,从而增强了修护凝胶对皮肤的修复性能。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明,即所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
因此,以下对提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。基于本发明的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
本例提供一种含间充质干细胞外泌体的修护凝胶,由外泌体冻干粉和凝胶辅助组分组成;
其中,外泌体冻干粉和凝胶辅助组分的质量比为3:5;
凝胶辅助组分包括以下质量份的原料:甘油4份、燕麦β-葡聚糖4份、透明质酸钠3份、马齿苋提取物2份、神经酰胺1份、烟酰胺1份、海藻糖1份、Carbopol Ultrez 20 1份和无菌水8份;
外泌体冻干粉由质量比为1:1的外泌体提取液和冻干保护剂组成;
外泌体提取液通过以下方法制得:
收集生长状况良好的P3-P7代的脐带干细胞培养上清,转移上清于离心管中离心(4℃,300xg,10min)去活细胞,(4℃,2000xg,10min)去除死细胞;取离心后的上清用0.22μm过滤器过滤即可得到外泌体提取液;
冻干保护剂由以下质量份的原料组成:甘油3份、甘氨酸3份、海藻糖2份和无菌水2份;
外泌体冻干粉通过以下方法制得:
步骤(1):将外泌体提取液和冻干保护剂混合均匀得到外泌体冻干液;
步骤(2):将步骤(1)所得的外泌体冻干液于-80℃预冻48h,然后将预冻好的外泌体冻干液真空冷冻干燥,即可得到外泌体冻干粉。
本例中含间充质干细胞外泌体的修护凝胶的制备方法,包括以下步骤:
步骤(1):将外泌体冻干粉、透明质酸钠、甘油、马齿苋提取液、燕麦β-葡聚糖、烟酰胺和一半的无菌水加入容器中,搅拌均匀,得到溶液一;
步骤(2):将剩下的一半无菌水加入容器中,加热至70℃,然后加入CarbopolUltrez 20搅拌均匀,冷却,得到均匀透明的凝胶状溶液二;
步骤(3):将步骤(2)所得的溶液二分批次加入步骤(1)所得的溶液一中,搅拌均匀,即可得到含间充质干细胞外泌体的修护凝胶。
实施例2
本例提供一种含间充质干细胞外泌体的修护凝胶,与实施例1的区别仅在于:凝胶辅助组分中Carbopol Ultrez 20为0.7份,其余参数及制备方法均相同。
实施例3
本例提供一种含间充质干细胞外泌体的修护凝胶,与实施例1的区别仅在于:冻干保护剂由以下质量份的原料组成:甘油5份、甘氨酸5份、海藻糖3份和无菌水3份;其余参数及制备方法均相同。
对比例1
本例提供一种含间充质干细胞外泌体的修护凝胶,与实施例1的区别仅在于:将外泌体提取液直接使用,即外泌体提取液未进行先冻干处理便与凝胶辅助组分配合使用;其余参数及制备方法均相同。
实验例
为验证本发明中含间充质干细胞外泌体的修护凝胶的皮肤修护功效,本例选择5组,每组5人分别编号为A、B、C、D、E,共25名年龄在20-40岁的每组受试者分别使用本发明实施例1-3和对比例1所得的修护凝胶30天。分别在使用开始前,使用10天、使用20天、使用30天分别使用VISIA-专业皮肤分析系统进行皮肤检测,检测指标包含:皱纹、毛孔、红色区、纹理。为方便统计,记录的是每次检测比初始值高/低的数值,同时收集受试者的使用感,测试结果如下表1-4所示。
表1实施例1所得含间充质干细胞外泌体的修护凝胶的测试结果
表2实施例2所得含间充质干细胞外泌体的修护凝胶的测试结果
表3实施例3所得含间充质干细胞外泌体的修护凝胶的测试结果
表4对比例1所得含间充质干细胞外泌体的修护凝胶的测试结果
通过表1-4的结果可以看出受试者皮肤的皱纹、毛孔、红色区、纹理在使用本发明产品后都有所好转,且随着使用时间的增长,情况越来越好。通过对受试者的面部观察可发现个受试者各自脸部情况都有所好转甚至消失,证明本发明的修护凝胶对皮肤有修护效果,修护效果随时间增加而增加,且明显优于对照组,说明本发明中对外泌体进行先冻干处理再添加使用,避免了外泌体直接使用时其他组分对外泌体的破坏,可有效增强修护凝胶对皮肤的修复性能。
以上内容仅仅是对本发明所作的举例和说明,所属本领域的技术人员不经创造性劳动即对所描述的具体实施例做的修改或补充或采用类似的方式替代仍属本专利的保护范围。
Claims (3)
1.一种含间充质干细胞外泌体的修护凝胶,其特征在于,所述修护凝胶由外泌体冻干粉和凝胶辅助组分组成;
其中,外泌体冻干粉由质量比为1~2:1~2的外泌体提取液和冻干保护剂制成;所述外泌体冻干粉通过以下方法制得:
步骤(1):将外泌体提取液和冻干保护剂混合均匀得到外泌体冻干液;
步骤(2):将步骤(1)所得的外泌体冻干液于-80℃~-70℃预冻48-56h,然后将预冻好的外泌体冻干液真空冷冻干燥,即得到外泌体冻干粉;
所述冻干保护剂由以下质量份的原料组成:甘油1~5份、甘氨酸1~5份、海藻糖1~3份和无菌水1~3份;
所述凝胶辅助组分包括以下质量份的原料:甘油2~6份、燕麦β-葡聚糖2~6份、透明质酸钠2~5份、马齿苋提取物1~4份、神经酰胺1~2份、烟酰胺1~2份、海藻糖1~2份、CarbopolUltrez 20 0.5~2份和无菌水5~10份;
所述含间充质干细胞外泌体的修护凝胶通过以下方法制得:
步骤(1):将外泌体冻干粉、透明质酸钠、甘油、马齿苋提取液、燕麦β-葡聚糖、烟酰胺和一半的无菌水加入容器中,搅拌均匀,得到溶液一;
步骤(2):将剩下的一半无菌水加入容器中,加热至70 ℃~80 ℃,然后加入CarbopolUltrez 20搅拌均匀,冷却,得到均匀透明的凝胶状溶液二;
步骤(3):将步骤(2)所得的凝胶状溶液二分批次加入步骤(1)所得的溶液一中,搅拌均匀,即得到含间充质干细胞外泌体的修护凝胶。
2.如权利要求1所述的含间充质干细胞外泌体的修护凝胶,其特征在于,所述外泌体提取液通过以下方法制得:
收集P3-P7代的脐带干细胞培养上清,转移上清于离心管中进行第一次离心去活细胞,进行第二次离心去除死细胞,取离心后的上清过滤,即得到外泌体提取液。
3. 如权利要求1所述的含间充质干细胞外泌体的修护凝胶,其特征在于,所述凝胶辅助组分包括以下质量份的原料:甘油4份、燕麦β-葡聚糖4份、透明质酸钠3份、马齿苋提取物2份、神经酰胺1份、烟酰胺1份、海藻糖1份、Carbopol Ultrez 20 1份和无菌水5~10份。
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