CN110638690B - 一种人工外泌体复合物的制备方法及应用 - Google Patents
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Abstract
本发明属于生物化学技术领域，具体涉及一种人工外泌体复合物的制备方法及应用。该复合物的制备原料为线性聚乙烯亚胺(PEI)和透明质酸(HA)。本发明的HA‑PEI制备过程，可以得到线性的聚乙烯亚胺，而目前大部分聚乙烯亚胺均为支链化的；HA‑PEI制备方案，可以定量制备特定分子量的HA‑PEI；操作步骤可控，可以规模化工业生产；HA‑PEI可通过透明质酸的配体活性及线性聚乙烯亚胺的透膜性，促进皮肤深层细胞对包裹的活性物质的吸收。
Description
技术领域
本发明属于生物化学技术领域，具体涉及一种人工外泌体复合物的制备方法及应用。
背景技术
护肤品的有效性主要在于活性物质的吸收效率，目前市面上有效的活性物质递送介质很少。线性聚乙酰亚胺因同时具有亲水性基团和亲脂性基团，可通过亲水性基团在内亲脂性基团在外的形式形成双分子层，将物质包裹形成微囊体，微囊体可利用自身的膜融合性与细胞膜融合并递送包含物到细胞内。但由于该微囊体是利用的膜融合性特征，故不具有细胞选择性。透明质酸又名玻尿酸，是天然的保湿润滑剂，由于透明质酸分子量大，不能有效被深层皮肤细胞吸收，护肤持久性有限。
发明内容
本公开的一方面解决的一个技术问题是提供一种新型人工外泌体复合物的制备方法及应用，目的在于融合线性聚乙烯亚胺和透明质酸，利用透明质酸的配体活性与聚乙烯亚胺的透膜活性使二者协同作用用于护肤产品，促进活性物质被深层皮肤有效吸收。
为实现上述目的，本发明采用如下技术方案，本发明一种人工外泌体复合物，该复合物的制备原料为线性聚乙烯亚胺(PEI)和透明质酸(HA)。
进一步的，所述明质酸为D-葡萄糖醛酸及N-乙酰葡糖胺组成的双糖单位，分子量在400—1800000之间，以透明质酸或者透明质酸钠形式。
进一步的，所述线性聚乙烯亚胺为聚(2-乙基-2-唑啉)经过硫酸水溶液加热水解而得到的，分子量在1000—100000之间。
进一步的，还包含酰胺键。
进一步的，人工外泌体复合物配置是以水溶液的形式，浓度为1mg/mL—1g/mL之间。
一种上述人工外泌体复合物的制备方法，包括以下步骤：
a.45克聚(2-乙基-2-唑啉)溶于200mL 30％的硫酸水溶液中，同时搭建加热回流装置。
b.加热反应液至回流状态，同时检测蒸馏出的溶液的pH，主要是判断反应副产物丙酸。丙酸可以与水共沸，直至蒸出溶剂不再有丙酸，反应可以停止，该反应大约需要7天。
c.反应中止后，反应体系降温至冰浴条件，同时机械搅拌。用氢氧化钠溶液调节pH至中性，再补加一部分氢氧化钠，过量至pH偏碱性。
d.调节pH过程中，会缓慢析出固体。
e.过滤，并用大量蒸馏水洗涤。直至滤液pH由碱性至中性，即可中止洗涤。
f.过滤出的固体，室温条件下进行真空干燥，直至没有任何水分残余，该过程大约需要7天。
g.取600mg的HA于烧杯1中加入300mL的dd水中配成溶液A，再取12g的PEI于烧杯2中加入300ml的dd水配成溶液B，将溶液A加入到溶液B中，加入1M Hcl调节溶液pH等于6.5得到溶液C。取6.3mmol的EDC和6.3mmol的HOBt溶于15mL的dd水和15mL的DMSO形成的混合溶剂形成溶液D，将溶液C和溶液D加入1.5L的圆底烧瓶中，常温下搅拌24h，用1M的NaOH溶液调节pH等于7得到反应液。
h.将反应液加入到活化过的透析袋(8000-14000KD)中，在100mM的Nacl溶液中透析2d，在体积分数为25％的乙醇中透析1d，最后在纯水中透析1d得到HA-PEI液，冷冻干燥两天得到白色海绵状物质HA-PEI。
i.将干燥好的HA-PEI，取出10克分散到50mL的蒸馏水中，添加稀盐酸溶液至pH7.1。随后，加入蒸馏水定容至100mL。
j.0.2微米滤膜对该溶液进行无菌化处理，即可得到浓度100mg/mL的HA-PEI母液。
一种人工外泌体复合物的应用，将人工外泌体复合物配置成可通过透明质酸受体显著促进深层皮肤对活性物质的吸收的制剂。
进一步的，所述剂型为临床上可接受的任一剂型。
进一步的，人工外泌体复合物为所述促进深层皮肤吸收活性物质的唯一活性成分的制剂，或该复合物与其他物质一起制备所述促进皮肤吸收活性物质的制剂。
本发明与现有技术相比，具有如下几个优势：
1)本发明的HA-PEI制备过程，可以得到线性的聚乙烯亚胺，而目前大部分聚乙烯亚胺均为支链化的。
2)本发明的HA-PEI制备方案，可以定量制备特定分子量的HA-PEI。
3)本发明操作步骤可控，可以规模化工业生产。
4)本发明的HA-PEI可通过透明质酸的配体活性及线性聚乙烯亚胺的透膜性，促进皮肤深层细胞对包裹的活性物质的吸收。
附图说明
图1是根据本公开的一个方面的人工外泌体对皮肤刺激性检测(Control：对照组；Exocrinetrater：人工外泌体组)；
图2是根据本公开的一个方面的人工外泌体对Hacat细胞形态的影响(Control：对照组；Exocrinetrater：人工外泌体组)；
图3是根据本公开的一个方面的人工外泌体对Hacat细胞的增殖影响(Control：对照组；Exocrinetrater：人工外泌体组)；
图4是根据本公开的一个方面的人工外泌体对pCDNA3.1-eGFP递送效率检测结果；
图5是根据本公开的一个方面的人工外泌体递送途径检测结果(DAPI：细胞核染料；Exocrinetrater：人工外泌体；Mitochondria：线粒体)；
图6是根据本公开的一个方面的人工外泌体对大鼠皮肤活性的影响(Control：对照组；Exocrinetrater：人工外泌体组)。
具体实施方式
下面将结合具体的实施方案对本发明进行进一步的解释，但并不局限本发明。
下述实施例中，如无特殊说明，所用方法为常规方法，所用试剂都来源于市售商品。
实施例1.人工外泌体复合物对皮肤刺激性检测
检测方法：
招募10名志愿者，使用新配置的浓度为1mg/mL的人工外泌体均匀涂抹在左手手腕内侧，右手手腕内侧涂抹ddH2O作为对照组，涂抹后静置20分钟，期间持续观察手腕内侧变化情况，并记录志愿者是否有痛、痒等刺激性感觉。
实验结果如图1所示。
表1.人工外泌体对皮肤测试统计结果
实验结论：
10名志愿者在涂抹人工外泌体20分钟内均未观察到皮肤受刺激后的红肿现象，且10名志愿者均无刺激性痛、痒等感觉。
实施例2.人工外泌体的细胞毒性检测
检测方法：
培养人永生化表皮细胞Hacat，消化细胞并铺到96孔板内，5000cells/孔。设置人工外泌体梯度浓度孵育Hacat细胞，继续培养48小时，期间观察细胞的形态并最终使用MTT方法检测对细胞增殖的影响。
实验结果如图2、3所示。
实验结论：
人工外泌体在细胞层面未表现出明显的细胞毒性，且对细胞的增殖无影响。
实施例3.人工外泌体对活性物质递送效率的检测
检测方法：
以人工外泌体为载体，在Hacat细胞上检测对绿色荧光质粒pCDNA3.1-eGFP递送到细胞内的活性。
实验结果如图4所示。
实验结论：
人工外泌体具有较好地包被活性物质效果，且可以有效的将活性物质递送到细胞内。
实施例4.人工外泌体对活性物质递送途径的检测
检测方法：
为进一步检测人工外泌体对活性物质的递送途径，染色细胞内的线粒体、溶酶体等细胞器，同时利用人工外泌体递送绿色荧光质粒pCDNA3.1-eGFP，通过荧光的定位信息来分析活性物质的递送区域。
实验结果如图5所示。
实验结论：
结果表明人工外泌体可将活性物质直接递送到线粒体部位，推测可通过该递送途径，调理线粒体功能，促进线粒体功能的稳定性，并最终达到抗炎修复的作用。
实施例5.人工外泌体对大鼠皮肤活性影响的检测
检测方法：
实验动物为Vista大鼠，使用羧甲基纤维素钠配包裹有活性物质的人工外泌体，使人工外泌体浓度为1mg/mL。持续一周在大鼠背部涂抹人工外泌体，对照组涂抹溶有活性物质的羧甲基纤维素钠凝胶。一周后，取材大鼠背部皮肤组织，做免疫荧光检测表皮细胞活性蛋白Cytokeratin的表达情况，通过荧光的强弱来反馈人工外泌体的功能性渗透作用。
实验结果如图6所示。
实验结论：
上皮细胞处于活性状态时会表达膜蛋白Cytokeratin，免疫荧光实验可通过绿色荧光的强弱来反映Cytokeratin的表达量进而反馈表皮细胞的活性。从结果看，不含人工外泌体的活性物质不能有效刺激Cytokeratin的表达，即不能有效激活细胞活性；而添加人工外泌体后皮肤组织细胞高表达Cytokeratin，人工外泌体激活了表皮细胞的活性，有利于细胞对活性物质的吸收。而且，实验结果表明，人工外泌体可有效激活皮肤深层细胞的活性。由于人工外泌体含有透明质酸成分，我们推测皮肤浅表层的角化层、透明细胞层等细胞因不能有效表达透明质酸受体，而不能有效吸附人工外泌体，使活性成分深层递送到含有透明质酸受体的棘细胞层和基底层，使护肤效果更有效。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (7)
1.人工外泌体复合物的制备方法，其特征在于：该复合物的制备原料为线性聚乙烯亚胺(PEI)和透明质酸(HA)，所述复合物的制备方法包括以下步骤：
a.45克聚(2-乙基-2-唑啉)溶于200 mL 30％的硫酸水溶液中，同时搭建加热回流装置；
b.加热反应液至回流状态，同时检测蒸馏出的溶液的pH，主要是判断反应副产物丙酸，丙酸可以与水共沸，直至蒸出溶剂不再有丙酸，反应可以停止，该反应需要7天；
c.反应中止后，反应体系降温至冰浴条件，同时机械搅拌，用氢氧化钠溶液调节pH至中性，再补加一部分氢氧化钠，过量至pH偏碱性；
d.调节pH过程中，会缓慢析出固体；
e.过滤，并用大量蒸馏水洗涤，直至滤液pH由碱性至中性，即可中止洗涤；
f.过滤出的固体，室温条件下进行真空干燥，直至没有任何水分残余，该过程需要7天；
g.取600 mg的HA于烧杯1中加入300 mL的dd水中配成溶液A，再取12 g的PEI于烧杯2中加入300 mL 的dd水配成溶液B，将溶液A加入到溶液B中，加入1M HC l调节溶液pH等于6.5得到溶液C；取6.3 mmol的EDC和6.3 mmol的HOBt溶于15 mL的dd水和15 mL的DMSO形成的混合溶剂形成溶液D，将溶液C和溶液D加入1.5 L的圆底烧瓶中，常温下搅拌24 h，用1M的NaOH溶液调节pH等于7得到反应液；
h.将反应液加入到活化过的透析袋(8000-14000kD) 中，在100 mM的NaC l溶液中透析2 d，在体积分数为25％的乙醇中透析1 d，最后在纯水中透析1 d得到HA-PEI液，冷冻干燥两天得到白色海绵状物质HA-PEI；
i.将干燥好的HA-PEI，取出10克分散到50 mL的蒸馏水中，添加稀盐酸溶液至pH7.1，随后，加入蒸馏水定容至100 mL；
j.0.2微米滤膜对该溶液进行无菌化处理，即可得到浓度100 mg/mL的HA-PEI母液。
2.根据权利要求1所述的人工外泌体复合物的制备方法，其特征在于：所述透明质酸为D-葡萄糖醛酸及N-乙酰葡糖胺组成的双糖单位，分子量在400- 1800000之间，以透明质酸或者透明质酸钠形式。
3.根据权利要求1所述的人工外泌体复合物的制备方法，其特征在于：所述线性聚乙烯亚胺为聚(2-乙基-2-唑啉)经过硫酸水溶液加热水解而得到的，分子量在1000- 100000之间。
4.根据权利要求1所述的人工外泌体复合物的制备方法，其特征在于：人工外泌体复合物配置是以水溶液的形式，浓度为1 mg/mL- 1 g/mL之间。
5.一种根据权利要求 1 所述的 人工外泌体复合物的应用，其特征在于：将人工外泌体复合物配制成可通过透明质酸受体促进皮肤对活性物质的吸收的制剂。
6.根据权利要求5所述的应用，其特征在于：所述剂型为临床上可接受的任一剂型。
7.根据权利要求5所述的应用，其特征在于：人工外泌体复合物为所述促进皮肤吸收活性物质的唯一活性成分的制剂，或该复合物与其他物质一起制备所述促进皮肤吸收活性物质的制剂。
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