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信使 RNA(mRNA)是蛋白质合成中从基因到核糖体的遗传信息的瞬时载体[1],相对于 DNA 治疗, 有更多优势,例如,无整合到宿主基因组中而导致插入突变的风险、比DNA更能获得持久预测蛋白表达动力学蛋白表达,且体外合成 mRNA 相对容易等。然而,由于mRNA不稳定性,其需要一个递送载体保护,免受核酸酶降解的同时还要使其被细胞吞噬,核酸胞内释放并翻译成蛋白。目前已上市的mRNA疫苗,大多采用的是脂质纳米粒(LNPs)载体[2-4]。LNP有4个重要组成部分:结构性脂质、胆固醇、阳离子脂质(或可电离脂质体)和隐形脂质。其中,阳离子脂质或可电离脂质是将带负电的mRNA能够装载到LNP必不可少的组成部分。Lipo8000是新型的阳离子脂质体转染试剂,转染效率和Lipo3000基本一致,适用于将DNA和RNA转染入真核细胞,对多种细胞具有高转染效率,常作为脂质纳米粒载核酸药物研究的对照试剂 [5-6]。DLin-MC3-DMA被认为是最有效的阳离子脂质体之一,具有“低毒高效”的优势[7],2018年全球首个上市的siRNA产品Onpattro就是采用DLin-MC3-DMA作为载体[8]。DLin-MC3-DMA在酸性条件下呈正电性,而生理pH条件(pH值为7.4)下呈电中性,现已成为制备肝脏靶向siRNA/LNP系统的“标准”脂质材料。但其递送mRNA的能力尚未见到报道。本研究以DLin-MC3-DMA作为阳离子脂质构建脂质纳米粒,并以Lipo8000为对照对其体外递送mRNA的能力进行考察,为后续肿瘤基因治疗研究提供参考。
Construction and in vitro evaluation of an LNP system for mRNA delivery
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摘要:
目的 构建脂质纳米粒DLin-LNP,以EGFP-mRNA为模型药,考察DLin-LNP对于mRNA的体外递送能力。 方法 采用薄膜水化法制备DLin-LNP, 并进一步制备 DLin@mRNA,对纳米粒进行表征,使用激光扫描共聚焦显微镜观察脂质纳米粒胞内的分布情况,以RM-1细胞为模型考察胞内转染情况。 结果 成功制备了脂质纳米粒DLin-LNP,其粒径为(151.1±2.1) nm,空载电位为(23.7±0.5) mV。DLin-LNP在RM-1细胞中转染mRNA效率较高,其毒性远低于市售脂质体Lipo8000,且 DLin-LNP脂质纳米粒稳定性好。 结论 DLin-LNP具有高转染效率和安全性,且稳定性好,可作为mRNA递送载体,为后续脂质纳米粒肿瘤治疗中的应用提供依据。 -
关键词:
- 脂质纳米粒 /
- DLin-MC3-DMA /
- 信使核糖核酸 /
- 药物递送
Abstract:Objective To construct lipid nanoparticles DLin-LNP for mRNA delivery. Methods DLin-LNP was prepared by thin film hydration method, and DLin-LNP/mRNA was further constructed by using EGFP-mRNA as model drug. The particle size, zeta potential, and appearance morphology were measured. Furthermore, the intracellular distribution and transfection of DLin-LNP/mRNA in RM-1 cells was investigated by laser scanning confocal microscope. Results DLin-LNP was successfully prepared. The average particle size was about (151.1±2.1) nm, the no-load potential was (23.7±0.5) mV. The cytotoxicity of DLin-LNP was far lower than that of the commercially available liposomal Lipo8000. The results of transfection experiment indicated that DLin-LNP has high transfection efficiency for mRNA delivery with low cytotoxicity and good stability. Conclusion DLin-LNP could become a potential mRNA vector for gene therapy. -
Key words:
- LNP /
- DLin-MC3-DMA /
- mRNA /
- drug delivery
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