CN112915212A - 抑制淀粉样多肽聚集的功能化脂质体纳米材料及其制备方法和应用 - Google Patents

抑制淀粉样多肽聚集的功能化脂质体纳米材料及其制备方法和应用 Download PDF

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CN112915212A
CN112915212A CN202110128203.9A CN202110128203A CN112915212A CN 112915212 A CN112915212 A CN 112915212A CN 202110128203 A CN202110128203 A CN 202110128203A CN 112915212 A CN112915212 A CN 112915212A
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张苑
刘磊
张利伟
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Abstract

本发明公开了一种抑制淀粉样多肽聚集的功能化纳米脂质体制备方法，所述纳米材料包括脂质体以及连接在脂质体上识别淀粉样蛋白的多肽序列以及融合在脂质体上的多肽序列。本发明通过超声法制备得到的纳米材料具有良好的生物兼容性能够与Aβ共组装从而有效阻止其聚集，降低Aβ的神经毒性。从而实现协同治疗阿尔茨海默症，大大提高多肽纳米材料的治疗阿尔茨海默症的效率，具有广泛的应用前景。其有益效果在于：提供了一种新的治疗治疗蛋白错误折叠或加工类疾病的途径，成本低，具有靶向性，制备工艺简单，生物相容性好，细胞毒性低，不会对受体产生不良影响。

Description

抑制淀粉样多肽聚集的功能化脂质体纳米材料及其制备方法 和应用
技术领域
本发明属于高分子领域，涉及抑制淀粉样多肽聚集的功能性脂质体纳米材料制备及其应用。
背景技术
阿尔茨海默是最常见的与年龄相关的神经退行性疾病，主要临床表现为渐进性记忆障碍、认知功能障碍、人格改变及语言障碍等神经精神症状,严重影响社交、职业与生活功能,甚至最终失去生活能力,对人类健康和生活有着巨大威胁,造成家庭和社会的巨大经济负担。根据《年世界阿尔茨海默症报告》报道,到年为止,患该病的人数将激增至1.5亿。
在患者的神经细胞外发现有主要成分为淀粉样多肽Aβ斑块老年斑。Aβ肽在阿尔茨海默病患者脑内聚集形成具有神经毒性的寡聚体、原纤维和成熟纤维进而造成神经细胞凋亡，诱发了疾病。Aβ淀粉样多肽的错误折叠和自组装是阿尔茨海默病症(AD)的发病机理核心。研究表明，Aβ很容易聚集，形成具有β-sheet结构的Aβ聚集体，产生神经毒性，所以以Aβ为治疗靶点的研究已成为关注的热点。国内外抑制淀粉样多肽主要通过生物蛋白酶，无机纳米颗粒，金属离子，多金属氧酸盐，但这些物质生物相容性不好，因此开辟一种新的方案为阿尔茨海默症提供潜在治疗方法十分必要。
调研与研究发现KLVFF这段来源于淀粉样多肽本身的一段多肽序列能够特异性识别抑制淀粉样多肽的聚集，目前科研工作者已经做出了一些成果，KLVFF与有机吡啶分子结合·可以实现抑制Aβ，通过含有交联的KLVFF聚合层包裹牛血清蛋白抑制，或者将壳聚糖，聚乙二醇与功能性多肽通过自组装形成复合材料可以实现抑制Aβ聚集，研究表明这些对Aβ聚集有很好的抑制效果，但是有机吡啶分子没有表明其生物相容性，细胞毒性，而壳聚糖构建体系相对复杂，聚合物层在构建过程中用到丙烯酰胺等高分子材料，生物相容性都是未知的，基于此所以我们打算找到了一种制备过程简单，细胞毒性低，生物相容性好的纳米材料，来解决KLVFF多肽的稳定性问题。
脂质体是一种制备工艺简单，生物相容性好，细胞毒性低的纳米材料，美国FDA已经批准DSPE-2000、M-E5磷脂为医用辅料，是非常好的药物载体材料。将其进行表面修饰后包裹功能性纳米材料，形成生物复合材料，常用于疾病的治疗和研究。
如何制备将这两者功能性结合在一起的纳米材料成为本发明重点。
发明内容
本发明的目的在于提供一种具有抑制淀粉样多肽聚集的功能化纳米脂质体制备方法和和在阿尔茨海默症中的应用。在本发明中，所述功能性脂质体纳米材料制备简单，能够与淀粉样多肽Aβ共同组装从而阻止其聚集，阻止其生成寡聚体，原纤维和成熟纤维，从而实现治疗阿尔茨海默症。
为达到此发明目的，本发明采用以下技术方案:
本发明提供一种抑制淀粉样多肽聚集的功能性脂质体纳米材料，所述纳米材料包括脂质体以及连接在脂质体上的功能性多肽，所述功能性多肽由特异性识别淀粉样多肽的多肽序列，连接肽GSG，磷脂融合肽组成。
所述功能性多肽具有如下结构:RI-R2-R3，其中R1为特异性识别淀粉样多肽的多肽序列，R3为磷脂融合肽，R1和R3通过酰胺键与连接肽R2连接。
优选的，R1表示为KLVFF，结构式为:
Figure BDA0002924190850000021
优选的，R2表示为GSG，结构式为：
Figure BDA0002924190850000022
优选的，R3表示为FAEKFKEAVKDYFAKFWD，结构式为：
Figure BDA0002924190850000023
另一方面，本发明提供了一种抑制淀粉样多肽聚集的功能化脂质体纳米材料的制备方法，所述步骤如下：
(1)以二油酰基卵磷脂DOPC为原料，利用超声法得到脂质体；脂质体分子为：
Figure BDA0002924190850000024
(2)将功能性多肽加入缓冲溶液中，得到功能性多肽纳米材料溶液，与脂质体溶液混合自组装得到功能性脂质体纳米材料溶液。本发明中脂质体与R3发生膜融合，使得功能性多肽与脂质体结合在一起，得到功能性脂质体纳米材料。
其中功能性多肽与脂质体的摩尔比为1：4。
上述技术方案中抑制淀粉样多肽聚集的功能化脂质体纳米材料溶液的浓度为80μM。
上述技术方案中缓冲溶液为PBS缓冲溶液。
上述技术方案所述自组装在4℃静置下进行，静置时间为12h。另一方面，本发明提供了功能性脂质体纳米材料应用在制备阿尔茨海默症治疗药物中的方法。
本发明的有益效果为：
(1)本次发明制备的纳米材料具有良好的生物相容性，步骤少，制备简单，能够与淀粉样多肽Aβ共同组装从而阻止其聚集，降低生成寡聚体、原纤维和成熟纤维Aβ的神经毒性，从而为阿尔茨海默症提供潜在治疗方案。
(2)本发明制备的功能性脂质体纳米材料，可以靶向识别抑制Aβ聚集，对Aβ的识别能力良好，并可以有效阻止其聚集，大大提高多肽纳米材料的治疗阿尔茨海默症的效率，具有广泛的应用前景。
(3)本发明制备得到的功能性脂质体纳米材料细胞毒性低，能够与Aβ共组装从而有效阻止其聚集，降低Aβ的神经毒性，安全性高，具有广泛的应用前景。
附图说明
图1为功能化脂质体纳米材料的表征：a)脂质体和功能性脂质体的粒径分布结果b)脂质体和功能性脂质体的zeta电位结果c)功能性脂质体材料的CD实验结果d)脂质体的透射图像e)功能性脂质体的透射图像。
图2a为实施例1的Aβ孵育72h的透射电镜图，图2b为实施例1功能化脂质体纳米材料与Aβ共孵育72h的透射电镜图，图2c为Aβ孵育72h的AFM图像，图2d为实施例1功能化脂质体纳米材料与Aβ共孵育72h的AFM图像。
图3为实施例1功能性脂质体纳米材料与Aβ共孵育以及单独Aβ在激发波长440nm、发射波长480nm下测定的荧光强度曲线图。
图4为不同浓度功能化脂质体细胞毒性测定。
具体实施方式
下面结合具体实施例和说明书附图对本发明做进一步说明。
实施例1
(1)脂质体的制备：
通过超声法制备脂质体，以二油酰基卵磷脂DOPC为原料,先将10mg固体粉末溶于三氯甲烷中,振荡，待充分溶解后,将溶液按每瓶2mg分装在玻璃瓶中,将此含有的玻璃瓶置于真空干燥机中抽真空12h。在2mg的DOPC溶于40℃的2ml的PBS缓冲溶液，将其混合在离心管中，反复倒置震荡离心管使其混合均匀，将其超声1h，在4℃静置2h，完成脂质体的制备。
(2)功能化脂质体纳米材料的制备：
将5mg功能性多肽用1ml六氟异丙醇溶解，在25度恒温振荡器上振荡12h，保证多肽充分溶解，然后取50μl在真空干燥箱中烘干，接下来用500μl的PBS缓冲溶液溶解，形成160μM多肽溶液。取250μl的步骤一所制备的DOPC脂质体溶液稀释一倍形成640μM的脂质体溶液，将上述多肽溶液和脂质体溶液等体积振荡混合均匀，放入4℃冰箱中静置12h后，功能化纳米脂质体材料就制备完成。
实施例2
功能性脂质体纳米材料表征：
分别取1ml脂质体溶液和功能性脂质体纳米材料放入比色皿中，将比色皿放入仪器样品池中，进行粒径和电位测定。
分别取300μl功能性多肽溶液和功能性脂质体溶液放入比色皿，将比色皿放入仪器样品池中，进行圆二色谱测定
分别取10μl脂质体和功能性脂质体溶液滴在铜网上，静置10min，移液枪吸走多余液体，再取10μl染色剂滴在铜网上，静置4min后，吸走多余液体，再取10μl超纯水滴在铜网上，静置2min，吸走多余液体，样品在空气自然干燥2h，最后将铜网放入透射电镜样品室内进行形貌表征。
实施例3
功能化脂质体纳米材料抑制Aβ聚集体研究：
用实施例1制备的功能性脂质体纳米材料和PBS缓冲溶液分别将淀粉样多肽Aβ溶解，Aβ最终浓度为20μM，放置于37℃恒温摇床进行孵育，在孵育时间0h,2h,4h,6h,12h,24h,36h,48h,72h时取样品进行荧光测试，每个时间段具体操作如下，取25μl的上述样品溶液和25μl的THT(1mM)溶液,加入到100ul的PBS缓冲溶液中混合均匀，进行THT荧光强度测试。
分别取10μL孵育好的Aβ溶液和Aβ与纳米材料的共混液滴在云母上，静置10min用移液枪吸走多余液体，自然干燥后利用原子力显微镜进行表征。
功能性脂质体纳米材料细胞毒性测定：准备好PC12细胞悬浮液，向96孔板每孔加入5000个细胞，每孔100μL。培养12h，取出培养基，实验组每孔加入不同浓度的(10，20、40μg/mL)功能性脂质体纳米材料，不含细胞的那组进行同样处理，培养12h。然后加入CCK-8溶液然后通过450nm激发，690nm发射测试。
由图1a脂质体功能化修饰后粒径变小。图1b脂质体功能化修饰后电位增加，功能性多肽上只有KLVFF带正电，其余都为中性。图1c脂质体功能化修饰后二级结构发生改变，证明KLVFF暴露在脂质体外。图1d说明了脂质体的稳定性一般，图1e说明了加入了功能性多肽后形成的功能化脂质体纳米材料更加稳定。
图2的透射电镜图和AFM图像，显示了功能化脂质体纳米材料对Aβ聚集体有一定的抑制效果。
图3为实施例1功能性脂质体纳米材料与Aβ共孵育以及单独Aβ在激发波长440nm、发射波长480nm下测定的荧光强度曲线图，显示了功能化脂质体纳米材料对Aβ聚集体有一定的抑制效果。
图4为不同浓度功能化脂质体细胞毒性测定，证明了功能化脂质体纳米材料无细胞毒性。
申请人声明，本发明通过上述实施例来说明本发明的功能性脂质体纳米材料制备方法及应用，但本发明并不局限于上述实施例。对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (9)

1.抑制淀粉样多肽聚集的功能化脂质体纳米材料，其特征在于，所述纳米材料包括脂质体以及连接在脂质体上的功能性多肽，所述功能性多肽由特异性识别淀粉样多肽的多肽序列，连接肽GSG，磷脂融合肽组成。
2.如权利要求1所述的抑制淀粉样多肽聚集的功能化脂质体纳米材料，其特征在于，所述功能性多肽具有如下结构:R1-R2-R3，其中R1为特异性识别淀粉样多肽的多肽序列，R3为磷脂融合肽，R1和R3通过酰胺键与连接肽R2连接；
R1结构式为:
Figure FDA0002924190840000011
R2结构式为：
Figure FDA0002924190840000012
R3结构式为：
Figure FDA0002924190840000013
3.如权利要求1或2所述的抑制淀粉样多肽聚集的功能化脂质体纳米材料的制备方法，其特征在于，包括如下步骤：
(1)以二油酰基卵磷脂DOPC为原料，利用超声法得到脂质体；
(2)将功能性多肽加入缓冲溶液中，得到功能性多肽纳米材料溶液，与脂质体溶液混合自组装，得到抑制淀粉样多肽聚集的功能性脂质体纳米材料溶液。
4.如权利要求3所述的制备方法，其特征在于，步骤(1)中，脂质体的分子式为：
Figure FDA0002924190840000014
5.如权利要求3所述的制备方法，其特征在于，步骤(2)中，功能性多肽与脂质体的摩尔比为1：4。
6.如权利要求3所述的制备方法，其特征在于，步骤(2)中，抑制淀粉样多肽聚集的功能化脂质体纳米材料溶液的浓度为80μM。
7.如权利要求3所述的制备方法，其特征在于，步骤(2)中，缓冲溶液为PBS缓冲溶液。
8.如权利要求3所述的制备方法，其特征在于，步骤(2)中，自组装在4℃静置下进行，静置时间为12h。
9.将权利要求1或2所述的抑制淀粉样多肽聚集的功能化脂质体纳米材料在制备阿尔茨海默症治疗药物中的应用。
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