载Cas9-RNP细胞膜囊泡仿生纳米粒的制备及其对小鼠巨噬细胞NLRP3基因的敲低作用

Preparation of Cas9-RNP cell membrane vesicle-loaded biomimetic nanoparticles and its efficacy on the NLRP3 gene knockdown in mouse macrophage

目的通过使用小鼠巨噬细胞膜包裹Cas9核糖核蛋白复合物(RNP),制备Cas9-RNP仿生纳米粒Cas9-RNP@MMs,旨在利用此仿生纳米粒递送Cas9-RNP复合物用于基因编辑,并进一步在体外研究小鼠巨噬细胞RAW264.7对Cas9-RNP@MMs的内吞情况及其基因编辑效果,为开发低毒性的抑制NLRP3治疗靶点的仿生纳米粒载体提供证据。方法将提取的小鼠巨噬细胞细胞膜与制备的Cas9-RNP混合,超声后使用脂质体挤压仪挤压得到Cas9-RNP@MMs。使用纳米颗粒跟踪仪检测Cas9-RNP@MMs的颗粒直径,透射电子显微镜下观察Cas9-RNP@MMs的颗粒形态。激光共聚焦荧光显微镜成像分析细胞对Cas9-RNP@MMs的内吞情况。采用MTT法检测Cas9-RNP@MMs生物相容性。通过qPCR和Western blot检测NLRP3表达,来验证Cas9-RNP@MMs敲低NLRP3基因的效果。结果采用巨噬细胞细胞膜制备的Cas9-RNP@MMs平均粒径约为216 nm;激光共聚焦荧光显微镜下,Cas9-RNP@MMs能成功被RAW246.7细胞摄取;MTT检测结果显示Cas9-RNP@MMs处理的小鼠巨噬细胞RAW246.7具有良好的生物相容性;qPCR和Western blot检测显示,有两条NLRP3特异的向导RNA(sgRNA)通过Cas9-RNP@MMs介导,有良好的敲低NLRP3基因表达的效果。结论利用仿生纳米粒成功制备了内腔载有Cas9-RNP复合物的纳米级囊泡Cas9-RNP@MMs,Cas9-RNP@MMs具有良好的生物相容性并且可以被RAW246.7细胞高效内吞;含有NLRP3特异的sgRNA的Cas9-RNP@MMs能够特异性敲低NLRP3基因表达。

Objective Cas9-RNP biomimetic nanoparticles cas9-RNP@MMs were prepared by encapsulating the Cas9 Ribonucleoprotein complex(RNP)using mouse macrophage membranes,with the aim of utilizing this biomimetic nanoparticle to deliver the Cas9-RNP complex for gene editing,and further study the endocytosis of Cas9-RNP@MMs and its gene editing effect in mouse macrophage RAW264.7 in vitro,providing evidence for the development of low-toxicity biomimetic nanoparticle carriers that inhibit NLRP3 therapeutic targets.Methods The purified mouse macrophage membrane was mixed with the prepared cas9-RNP mixture,and after ultrasound,the CAS9-RNP@MMS was obtained by liposome extrusion instrument;The particle size of Cas9-RNP@MMs was measured by nanoparticle tracking analysis,and the particle morphology of Cas9-RNP@MMs was observed under transmission electron microscope.Laser confocal Fluorescence microscope imaging was used to analyze the endocytosis Cas9-RNP@MMs.The Biocompatibility of Cas9-RNP@MMs was measured by MTT assay.The expression of NLRP3 was detected by qPCR and Western blot to verify the knockdown effect of Cas9-RNP@MMs on NLRP3 gene.Results The average particle diameter of Cas9-RNP@MMs prepared from macrophages was about 216 nm.Under laser confocal fluorescence microscope,the Cas9-RNP@MMs could be successfully endocysed by Raw246.7 cell.MTT assay indicated that the Cas9-RNP@MMs-treated mouse macrophage RAW246.7 had good biocompatibility.qPCR and Western blot showed that two NLRP3-specific guide RNA were mediated by Cas9-RNP@MMs,with good effect of knockdown NLRP3 gene expression.Conclusion Nano-scale vesicles Cas9-RNP@MMs loaded with Cas9-RNP complexes were successfully prepared by biomimetic nanoparticles.Cas9-RNP@MMs have good biocompatibility and can be efficiently endocytosed by RAW246.7 cells.Cas9-RNP@MMs containing NLRP3-specific sgRNA can specifically knock down NLRP3 gene expression.