Preparation and in vitro evaluation of biodegradable polypeptide gene vector / 第二军医大学学报

ABSTRACT

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 by1H 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. Results1H 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≥40. 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 h after transfected with LHR/pGL3 and LHRss/pGL3, and its toxicity was significantly lower than that of bPEI-25K, with the cell survival rates being about 25% at 24 h after transfected with 20 μg/mL bPEI-25K (P<0. 05). Conclusion The prepared lipoic acid modified polyarginine polypeptide nanocomplex is expected to become an efficient gene vector.