CN113648288A - 一种红细胞膜包被功能性分子的纳米复合物、制法及应用 - Google Patents
一种红细胞膜包被功能性分子的纳米复合物、制法及应用 Download PDFInfo
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Abstract
本发明公开了一种红细胞膜包被功能性分子的纳米复合物制备及其应用;所述纳米复合物包括红细胞膜和功能性分子,所述功能性分子为mRNA、蛋白质以及小分子药物;本发明通过制备修饰后的红细胞,并提取红细胞膜包裹核酸类药物,其中红细胞膜磷脂等脂质成分可以使功能药物载体高效靶向脾脏,将药物递送至脾脏或肝脏细胞内并释放而发挥治疗作用,细胞膜增加了功能性药物在体内循环稳定性和细胞内递送效率;该纳米复合物的制备提取方法简单,适合大规模合成制备,且制备条件温和,效率高,粒径均匀,分散性好,具有良好靶向能力和高的生物相容性,具有高效、稳定的胞内递送效率。
Description
技术领域
本发明涉及一种红细胞膜包被功能性分子的纳米复合物,还包括所述纳米复 合物的制法及应用。
背景技术
目前,细胞膜仿生技术已经在药物递送、肿瘤治疗、免疫调节等多方面有了 广泛的应用。利用细胞膜包裹纳米复合物,大大提高了生物相容性,同时在体内 实现长效循环和靶向递送。而红细胞膜在体内的半衰期更长,在血液循环中的滞 留时间长达72小时,利用红细胞膜包裹的功能药物分子既具有纳米载体本身的理 化性质,又具有天然细胞的生物学性质,而且还可以捕获自身表面的病原体,中 和后将其呈递给脾脏免疫细胞,从而帮助人体抵抗感染。哈佛大学的Mitragotri 等人(Proc.Natl.Acad.Sci.,2020,117(30):17727-17736)利用红细胞的这种先天 能力构建了一种平台技术,称为Erythrocyte-Driven Immune Targeting(EDIT),这 些红细胞膜载体继承了原代细胞良好的生物相容性,其表面保留了的大量膜蛋白 是其体内长循环的重要因素。这一技术能将抗原递送到脾脏中的抗原呈递细胞, 从而产生免疫应答。该方法不仅能成功减缓小鼠癌性肿瘤的生长,还可以被用作 多种疫苗的生物相容性佐剂。
脾脏是人体最大的淋巴器官,激活脾脏区域的抗原呈递细胞能够激发全身的 抗肿瘤免疫反应,产生良好的抗肿瘤效果。然而,通过静脉给药且具有高效靶向 脾脏区域功能药物分子目前还面临多种困难。这是因为各类功能性分子作为外来 物质,一旦注射到体内,将会被快速清除,从而缩短了循环时间并限制了药物的 治疗效果,尽管可以通过聚合物对其进行表面修饰来改善,但会引起不同程度的 免疫副反应。此外,功能性分子虽然可以通过脂质体等材料包被被动靶向到特定 器官或肿瘤位点,但递送效率较低。
发明内容
发明目的:本发明针对现有技术中存在的脾脏或肝脏靶向药物循环时间短、 疗效差的问题,提供一种红细胞膜包被功能性分子的纳米复合物,还提供了上述 纳米复合物的制法及应用。
技术方案:本发明的所述的一种红细胞膜包被功能性分子的纳米复合物,所 述纳米复合物包括红细胞膜和功能性分子;所述功能性分子通过静电吸附和疏水 间作用力与细胞膜之间作用,从而被细胞膜包覆。
优选的,所述功能性分子为mRNA、蛋白质或小分子药物。
优选的,所述红细胞膜包被功能性分子的纳米复合物的粒径为98±0.3nm。
优选的,所述红细胞膜为叶酸或维生素A修饰的红细胞膜,所述功能性分子 修饰的红细胞膜,其制法为,使用叶酸或维生素A与DSPE进行反应得到的两种 产物可以直接插入细胞膜表面。
本发明公开了所述红细胞膜包被功能性分子的纳米复合物的制法,包括以下 步骤:
(1)红细胞膜的提取:离心血液,取下层血细胞,用磷酸盐缓冲盐溶液吹散 混匀,再离心洗涤,取压积红细胞,加入生理盐水,混匀至溶液变透明暗红,冷 藏放置后离心,出现乳白色沉淀并收集,即得到红细胞膜;
(2)红细胞膜的修饰:取步骤(1)制得的红细胞膜,加入叶酸或维生素A 进行红细胞的修饰,制得修饰后的红细胞膜。
(3)细胞膜包被功能性分子:取功能性分子溶于磷酸盐缓冲盐溶液,制得A 溶液,取步骤(2)制得的红细胞膜溶于乙醇-水的混合溶液,制得B溶液,超声 条件下,取A溶液加入B溶液中得混合溶液,在混合溶液中滴加磷酸盐缓冲盐溶 液,继续超声得所述红细胞膜包被功能性分子的纳米复合物。
优选的,步骤(1)中,所述离心,转速为11000~15000rpm,时间为10~20min。
优选的,步骤(3)中,所述A溶液的浓度为1~5mg/mL,所述B溶液的浓 度为1~5mg/mL的乙醇-水的混合溶液中,乙醇与水的体积比为1:1~3。
优选的,步骤(3)中,所述磷酸盐缓冲盐溶液的浓度为10mmol,所述混合 溶液与磷酸盐缓冲盐溶液的比例为1~10:1,所述继续超声时间为5~10min。
本发明还公开了所述红细胞膜包被功能性分子的纳米复合物的应用,所述红 细胞膜包被功能性分子的纳米复合物可应用于制备脾脏靶向的药物和肿瘤靶向的 药物。
优选的,所述红细胞膜包被功能性分子的纳米复合物还可应用于mRNA疫苗 的制备。
发明原理:本发明通过制备修饰后的红细胞,并提取红细胞膜包裹核酸类药 物,其中红细胞膜磷脂等脂质成分可以使功能药物载体高效靶向脾脏,将药物递 送至脾脏或肝脏细胞内并释放而发挥治疗作用,细胞膜增加了功能性药物在体内 循环稳定性和细胞内递送效率;通过利用红细胞膜表面存在的功能性蛋白,且红 细胞膜包被可以延长功能性分子的血液循环半衰期并避免被免疫系统识别的特 性,由于脾脏的抗原呈递细胞对红细胞碎片和衰老的红细胞具有极强的靶向吸附 能力,增强功能性分子的脾脏或肝脏主动靶向性增强。以红细胞膜包裹核酸类药 物mRNA为例,该mRNA功能药物载体采用反向纳米沉淀法合成,反应条件温 和,合成效率高,且粒径均匀,单一分散性好。
红细胞膜的包被一方面该纳米复合物稳定性好,具有高的生物相容性及良好 稳定性,另一方面红细胞膜的使用使得mRNA高效率靶向生物体脾脏,降低了 mRNA在血液循环中的过早释放的问题从而增加了mRNA在脾脏积累及释放, 一定程度上解决了目前mRNA疫苗的效率低下问题,增加了mRNA疫苗的保护 效力,同时,结合对于红细胞膜的修饰,通过实验验证,其中叶酸修饰的红细胞 膜用于靶向肿瘤,维生素A修饰的红细胞膜用于靶向肝脏,因此,将红细胞膜结 合功能性分子能够有效地达到靶向作用并发挥治疗作用。
有益效果:与现有技术相比,本发明具有如下显著优点:
(1)本发明所述红细胞膜包被功能性分子的纳米复合物,具有良好稳定性, 在常温条件下能较长时间保持结构稳定,降解不会产生有毒或有害物质,半衰期 长具有较大的比表面积;
(2)本发明所述的纳米复合物的制备方法,其红细胞膜包被的功能药物载体 采用反向纳米沉淀法合成,制备方法简单,反应条件温和,合成效率高,成本低, 有利于大规模生成,且粒径均匀,包裹细胞膜后粒径为98±0.3nm,单一分散性 好;
(3)所述的纳米复合物表面包被有红细胞膜,提高了生物相容性,避免了副 作用的发生,磷脂等脂质成分增加了功能性分子/红细胞膜的晚期内含体逃逸能 力,并增加了功能药物分子/细胞膜纳米颗粒稳定性和脾脏细胞内药物递送效率; 本发明通过开发和制备具有高效递送效率的功能药物分子胞内递送系统,为该类 药物的大规模临床应用创造了条件。
附图说明
图1为红细胞膜的TEM图;
图2为外部包裹有红细胞膜的mRNA纳米药物的TEM图;
图3为红细胞膜的粒径分布图;
图4为红细胞膜包被mRNA后的粒径分布图;
图5为红细胞膜包被的mRNA纳米药物在小鼠体内表达荧光素酶的荧光图;
图6为包裹红细胞膜的mRNA纳米药物在小鼠离体器官荧光素酶的荧光分布 图。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1
红细胞膜包裹mRNA功能药物分子靶向脾脏。
提取Balb/c小鼠红细胞膜:本实施例以提取Balb/c小鼠血液红细胞膜为例进 行说明,眼眶取血液5mL,再加入5ml PBS(0.01mol/L,pH 7.4),在4℃离心 (3000rpm,20min),小心吸取上层清液、中间白细胞和血小板层,将下层血细胞 层用磷酸盐缓冲盐溶液吹散混匀,再重复离心洗涤,取压积红细胞直接溶于40 倍体积预冷的破膜液中(低渗Tris-HCl0.01M pH=8.0,PMSF 0.02mM),混匀至 溶液变透明暗红,于4℃冰箱中放置2h后4℃下离心,弃去离心管内的暗红色上 清液,将红色絮状沉淀重新悬于破膜液中4℃,离心,重复洗涤三次,直至出现 乳白色沉淀并收集;
红细胞膜包裹mRNA:首先将mRNA溶解于PBS中(0.1mL),再将红细胞 膜溶解于乙醇:水(2:3)的溶液(0.2mL)中,超声条件下将mRNA溶液加入 该混合溶液,随后再在超声条件下,将(0.7mL)PBS缓慢滴入该混合液组装至 总体积为1mL,继续超声5min至组装完成。
实施例2
功能化红细胞膜包裹mRNA药物分子靶向肝脏
提取Balb/c小鼠血液红细胞膜为例进行说明,眼眶取血液5mL,再加入5ml PBS(0.01mol/L,pH 7.4),在4℃离心(3000rpm,20min),小心吸取上层清液、 中间白细胞和血小板层,将下层血细胞层用磷酸盐缓冲盐溶液吹散混匀,再重复 离心洗涤,取压积红细胞直接溶于40倍体积预冷的破膜液中(低渗Tris-HCl 0.01 M pH=8.0,PMSF 0.02mM),混匀至溶液变透明暗红,于4℃冰箱中放置2h后4℃ 下离心,弃去离心管内的暗红色上清液,将红色絮状沉淀重新悬于破膜液中4℃, 离心,重复洗涤三次,直至出现乳白色沉淀并收集。
功能化红细胞膜制备:将维生素A与DSPE置于DMF中,加入10%三乙胺 反应过夜后,经柱层析分离后得到产物,该产物与提取的细胞膜混合置于乙醇: 水=2:3的溶液中搅拌6h后得到维生素A修饰的红细胞膜
功能化红细胞膜包裹mRNA:首先将mRNA溶解于PBS中(0.1mL),再将 红细胞膜溶解于乙醇:水(2:3)的溶液(0.2mL)中,超声条件下将mRNA溶 液加入该混合溶液,随后再在超声条件下,将(0.7mL)PBS缓慢滴入该混合液 组装至总体积为1mL,继续超声5min至组装完成。
电镜观察:
将红细胞膜包裹前后的mRNA功能药物用电镜进行观察,即将步骤(1)及 步骤(3)的产物进行观察,图1为60倍下观察的mRNA功能药物的TEM图, 图2是60倍下观察的外部包裹有红细胞膜的mRNA纳米药物载体的TEM图。
通过布鲁克海文NanoDLS高灵敏粒度分析仪检测红细胞膜包裹mRNA前后 的水合粒径分布变化,如图3和图4所示为红细胞膜包裹mRNA前后的的水合粒 径分布变化图,从图4可以看出,红细胞膜包裹后,粒径增加,说明红细胞膜成 功包裹mRNA。
mRNA纳米药物靶向富集于脾脏细胞:
将红细胞膜包裹的mRNA功能药物以尾静脉注射方式打入Balb/c小鼠体内, 通过IVIS生物发光成像系统检测mRNA体内分布,分别于0h,2h,3h,4h,6h 检测小鼠体内生物发光强度,然后将小鼠进行离体,取心肝脾肺肾进行生物发光 检测,由图5和图6的结果表明,红细胞膜包裹的mRNA纳米药物主要靶向于脾 脏,且在3h后能够明显的看到mRNA在脾脏部位的富集,在6h以后通过解剖的 离体组织可以发现,该纳米系统在其他器官没有富集,只在脾脏部位有富集。
mRNA纳米药物靶向富集于肝脏细胞:
将功能化红细胞膜包裹的mRNA功能药物以尾静脉注射方式打入Balb/c小鼠 体内,通过IVIS生物发光成像系统检测mRNA体内分布,分别于0h,2h,3h, 4h,6h检测小鼠体内生物发光强度,功能化红细胞膜包裹的mRNA纳米药物主 要靶向于肝脏,且在2h后能够看到mRNA在脾脏部位的富集,在3h以后就能明 显的看到在肝脏部位有富集。
Claims (10)
1.一种红细胞膜包被功能性分子的纳米复合物,其特征在于,所述纳米复合物包括红细胞膜和功能性分子;所述功能性分子通过静电吸附和疏水间作用力与细胞膜之间作用,从而被细胞膜包覆。
2.根据权利要求1所述的红细胞膜包被功能性分子的纳米复合物,其特征在于,所述功能性分子为mRNA、蛋白质或小分子药物。
3.根据权利要求1所述的红细胞膜包被功能性分子的纳米复合物,其特征在于,所述红细胞膜包被功能性分子的纳米复合物的粒径为98±0.3nm。
4.根据权利要求1所述的红细胞膜包被功能性分子的纳米复合物,其特征在于,所述红细胞膜为叶酸或维生素A修饰的红细胞膜。
5.一种权利要求1所述的红细胞膜包被功能性分子的纳米复合物,其特征在于,包括以下步骤:
(1)红细胞膜的提取:离心血液,取下层血细胞,用磷酸盐缓冲盐溶液吹散混匀,再离心洗涤,取压积红细胞,加入生理盐水,混匀至溶液变透明暗红,冷藏放置后离心,出现乳白色沉淀并收集,即得到红细胞膜;
(2)红细胞膜的修饰:取步骤(1)制得的红细胞膜,加入叶酸或维生素A进行红细胞的修饰,制得修饰后的红细胞膜;
(3)细胞膜包被功能性分子:取功能性分子溶于磷酸盐缓冲盐溶液,制得A溶液,取步骤(2)制得的红细胞膜溶于乙醇-水的混合溶液,制得B溶液,超声条件下,取A溶液加入B溶液中得混合溶液,在混合溶液中滴加磷酸盐缓冲盐溶液,继续超声得所述红细胞膜包被功能性分子的纳米复合物。
6.根据权利要求5所述的制法,其特征在于,步骤(1)中,所述离心,转速为11000~15000rpm,时间为10~20min。
7.根据权利要求5所述的制法,其特征在于,步骤(3)中,所述A溶液的浓度为1~5mg/mL,所述B溶液的浓度为1~5mg/mL的乙醇-水的混合溶液中,乙醇与水的体积比为1:1~3。
8.根据权利要求5所述的制法,其特征在于,步骤(3)中,所述混合溶液与磷酸盐缓冲盐溶液的比例为1~10:1,所述继续超声时间为5~10min。
9.一种权利要求1所述的红细胞膜包被功能性分子的纳米复合物的应用,其特征在于,所述红细胞膜包被功能性分子的纳米复合物可应用于制备脾脏靶向的药物和肿瘤靶向的药物。
10.根据权利要求10所述的红细胞膜包被功能性分子的纳米复合物的应用,其特征在于,所述红细胞膜包被功能性分子的纳米复合物可应用于mRNA疫苗的制备。
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CN115006368A (zh) * | 2022-07-01 | 2022-09-06 | 重庆大学 | 细胞膜包被纳米药物及其应用 |
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