Preparation of Cas9 - RNP cell membrane vesicle －loaded biomimetic nanoparticles and its efficacy on the NLRP3 gene
10.19405/j.cnki.issn1000-1492.2023.03.001
- Author:
Dongqing Wu
1
;
Zhangyong Dan
1
;
Xiaoyan He
2
;
Huaqing Zhu
1
Author Information
1. Dept of Biochemistry and Laboratory of Molecular Biology of Anhui Medical University，Hefei 230032
2. Dept of College of Life Sciences，Anhui Medical University，Hefei 230032
- Publication Type:Journal Article
- Keywords:
cell membrane;
nanovesicles;
Cas9-RNP;
endocytosis;
gene knockdown
- From:
Acta Universitatis Medicinalis Anhui
2023;58(3):347-351
- CountryChina
- Language:Chinese
-
Abstract:
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 oflow-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@ MMswas 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.
- Full text:2024071610431709746载Cas9-RNP细胞膜囊...胞NLRP3基因的敲低作用_武冬青.pdf
