Keywords

Polyarginine Polypeptide Plasmid Dna Transfection Gene Transfer Techniquespolyarginine Polypeptide Plasmid Dna Transfection Gene Transfer Techniques

Abstract

Objective To prepare a lipoic acid-modified polyarginine peptide nanocomplex for gene delivery system, and to examine its transfection efficiency and cytotoxicity on human embryonic kidney cell line HEK293 cells. Methods Using different amounts of cysteine as cross-linking agents, four types of reduced lipoic acid-modified cross-linked polyarginine histidine (LHRss) with different cross-linking degrees were synthesized, and the synthesis was identified using 1H NMR and gel chromatography. LHRss. Plasmid DNA (pDNA) and LHRss were self-assembled with different nitrogen to phosphorus ratios (N/P) to form nanocomplexes. The particle size and zeta potential of the complex were measured with a particle size analyzer. The effect of the vector LHRss on pDNA was measured by gel retardation electrophoresis. Wrapping ability. LHRss/pDNA nanocomplexes were co-cultured with HEK293 cells to examine the cellular uptake of complexes with different cross-linking degrees and related gene transfection, and determine the cytotoxicity of different nanocomplexes to HEK293 cells. As a result, the LHRss polypeptide was successfully synthesized through structural identification. The particle size distribution of the assembled nanocomplexes is uniform. When N/P ¡Ý 40, the zeta potential of LHRss3/pDNA and LHRss4/pDNA complexes is greater than 30 mV. Gel retardation electrophoresis results showed that when the N/P value was 5, LHRss3 could completely wrap pDNA. When the N/P value is 40, the uptake and transfection efficiency of LHRss3/pDNA by HEK293 cells is higher than that of the other three complexes and monomeric lipoic acid-modified polyarginine histidine (LHR); among them, LHRss3/pGL3 The average fluorescence intensity of the complex was approximately 3.98 times that of the LHR/pGL3 complex, and the difference was statistically significant (P<0.05). Cytotoxicity experiments showed that after transfection of HEK293 cells with LHR/pGL3 and LHRss/pGL3 with different cross-linking degrees for 24 hours, the cell survival rates were above 80%, and their toxic effects were lower than those of bPEI-25K (transfected with 20 ¦Ìg/mL bPEI-25K The cell survival rate after staining the cells for 24 hours was about 25%, P<0.05). Conclusion The prepared lipoic acid-modified polyarginine peptide nanocomplex is expected to become an efficient gene carrier. Objective To prepare a lipoic acid modified polyarginine polypeptide nanocomplex for gene delivery system, and to observe its transfection efficiency and cytotoxicity on HEK293 cells. Methods We synthesized four disulfide cross-linked lipoic acid modified polyarginine peptide and histidine (LHRss) at different cross-linked degrees using different mol fraction of L-cysteine as cross-linking agent. The construction of LHRss was characterized by 1H nuclear magnetic resonance (1H NMR) and gel permeation chromatography. The LHRss/plasmid DNA (pDNA) nanocomplexes were self-assembled with LHRss and pDNA at different nitrogen/phosphorus (N/P) ratios. The size and zeta potential of LHRss/pDNA nanocomplexes were characterized by particle size analyzer, and the pDNA enrichment capability was determined by electrophoretic mobility shift assay (EMAS). Then, the intracellular uptake and gene transfection efficiency of LHRss/pDNA nanocomplexes in HEK293 cells were investigated. CCK-8 method was used to determine the cytotoxicity of LHRss/pDNA nanocomplexes on HEK 293 cells. Results 1H NMR results showed that LHRss was successfully synthesized. The nanocomplexes had a uniform distribution of particle size, and the zeta potential of LHRss3/pDNA and LHRss4/pDNA nanocomplexes were more than 30 mV when N/P¡Ý40. EMAS results showed that pDNA could be completely wrapped by LHRss3 when N/P=5 . When N/P=40, the intracellular uptake and transfection efficiency of LHRss3/pDNA nanocomplex by HEK293 cells was significantly higher than that of other three nanocomplexes and lipoic acid modified polyarginine peptide and histidine (LHR)/pDNA; the mean fluorescence intensity of LHRss3/pGL3 nanocomplexes was approximately 3.98 times that of the LHR/pGL3 nanocomplex (P<0.05). Cytotoxicity results showed that the cell survival rates were more than 80% at 24   For further details regarding this article and its research, please do not hesitate to contact our team for assistance. Original research done by Hu Chuling, Gu Fenfen, Tai Zongguang, Fang Jiwei, Gao Yuan, Gao Shen  

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