富含支化精氨酸的两亲性阳离子脂质肽体内外mRNA基因递送能力及安全性评价

mRNA delivery and safety evaluation of arginine-rich amphipathic cationic lipopeptides in vivo and in vitro

mRNA基因治疗因其可扩展、修饰,不需要进入细胞核及不整合宿主遗传基因等优势而备受关注。在基因治疗中,将mRNA安全有效地递送到细胞内对于基因治疗的成功至关重要。本研究基于前期设计合成的富含支化精氨酸的两亲性阳离子脂质肽(dendritic arginine&disulfide bond-containing cationic lipopeptide,RLS)基因载体在斑马鱼体内成功实现比商品化试剂Lipofectamine 2000高1.5倍的mRNA转染效果,并通过体外细胞毒性和斑马鱼体内生物安全性研究证实其具有良好的生物安全性。首先,通过核磁共振氢谱(1H NMR)及飞行时间质谱(MALDI-TOF)对阳离子脂质肽的化学成分进行表征。通过动态光散射粒度分析仪(DLS)测试的粒径和电位结果显示,在氮磷(N/P)比为20时,RLS与mRNA复合组装体形成平均粒径约220 nm、表面ζ电位约+21 mV的均匀纳米粒子。体外基因转染中,RLS在人胚肾293细胞(HEK293)和大鼠间充质干细胞(MSC)中的转染效率较Lipofectamine 2000分别提高1.2和3倍。此外,将RLS显微注射至斑马鱼胚胎后,通过评价生存率、孵化率和致畸率等指标,进一步证实了其在体内良好的安全性。综上所述,该富含支化精氨酸的两亲性阳离子脂质肽RLS具有优良的mRNA递送性能和安全性。

mRNA gene therapy has attracted much attention due to its advantages such as scalability,modification,no need to enter the nucleus and no integration of host genes.In gene therapy,safe and effective delivery of mRNA into cells is critical for the success of gene therapy.In this study,we designed and synthesized an amphiphilic cationic lipopeptide gene vector(dendritic arginine&disulfide bond-containing cationic lipopeptide,RLS)enriched with branched arginine.We achieved a 1.5-fold higher mRNA transfection efficiency in zebrafish compared to the commercial reagent Lipofectamine 2000,and confirmed its good biosafety by in vitro cytotoxicity and in vivo biosafety.First,we characterized the chemical composition of the cationic lipid peptides by nuclear magnetic resonance hydrogen spectroscopy(1H NMR)and time-of-flight mass spectrometry(MS).The results of particle size and potential tested by dynamic light scattering particle size analysis showed that at a nitrogen/phosphorus(N/P)ratio of 20,the RLS/mRNA composite assemblies formed homogeneous nanoparticles with an average particle size of about 220 nm and a surfaceζpotential of about+21 mV.In vitro gene transfection,the transfection experiments demonstrated that RLS exhibited 1.2-fold higher transfection efficiency in human embryonic kidney 293 cells(HEK293)and 3-fold higher transfection efficiency in rat mesenchymal stem cells(MSC)compared to Lipofectamine 2000.In addition,after microinjection of RLS into zebrafish embryos,we evaluated the survival,hatching,and teratogenicity rates,all of which confirmed its favorable in vivo safety profile.Thus,this amphiphilic cationic lipid peptide RLS,enriched with branched arginine,exhibits excellent mRNA delivery properties and safety.These findings highlight its potential as a promising gene therapy tool.