Used To Enhance The In Vivo Gene Transfection Efficacy Of Linoleic Acid And Poly Apple Acid Pairs Grafted Chitosan Novel Nano Carrier

Proper gene vector was essential to realize the therapeutic potential of gene therapy. Chitosan, due to its biocompatibility and biodegradability, showed great promise in gene delivery. However further applications of chitosan were limited by low transfection efficiency, and hence chitosan should be chemically modified to overcome the bottlenecks of chitosan-based gene delivery systems. Linoleic acid and poly (malic acid) double grafted chitosan (LMC) polymers, which were synthesized according to our previous work and could be self-assembled into nanoparticles in aqueous phase, were promising in combining the advantages of hydrophilic and hydrophobic modifications to mediate satisfactory gene expression.Herein, LMC nanoparticles with different linoleic acid and poly (malic acid) grafting ratios were synthesized and model plasmids that expressed green fluorescent protein (pGFP) were encapsulated to investigate in vitro and in vivo gene transfection efficiency along with the effects on LMC/pEGFP nanocomplexes exerted by hydrophilic and hydrophobic modifications of LMC nanoparticles.1 The synthesis and characterization of LMC nanoparticlesLMC polymers with different linoleic acid and poly (malic acid) grafting ratios, LMC1, LMC2, LMC3, and LMC4, were synthesized through varying the feed ratios, and the grafting ratios, cytoxicity, the contact angle, size, and Zeta potential were characterized. The results indicated that the grafting ratios of linoleic acid were 23.3%, 46.6%,70.0%, and 73.3% while those of poly (malic acid) were 3%,3%,3%, and 7%, respectively. LMC nanoparticles were 150-300 nm in diameters, and Zeta potential measurements indicated a positive surface charge at pH 5.5 which enabled the encapsulation of anionic pEGFP. MTT assay suggested LMC nanoparticles exerted inappreciable cytotoxicity on HEK293 cells at the concentration below 1 mg/mL2 The preparation and characterization of LMC/pEGFP nanocomplexesLMC nanoparticles could condense pEGFP at pH 5.5, forming LMC/pEGFP nanocomplexes of which the loading efficiency exceeded 85%. LMC nanoparticles retained the pEGFP binding ability as compared to chitosan. LMC/pEGFP nanocomplexes were spherical and their sizes were slightly lower than LMC nanoparticles while increasing with higher weight ratios. The ability to resist non-specific protein adsorption as well as pEGFP release rates of LMC/pEGFP nanocomplexes were superior as compared to chitosan/pEGFP nanocomplexes, and increased with higher linoleic acid and poly (malic acid) grafting ratios. Higher linoleic acid grafting ratios and lower poly (malic acid) grafting ratios were favorable for DNase I degradation resistance, and hence except for LMC4/pEGFP nanocomplexes, LMC/pEGFP nanocomplexes demonstrated higher pEGFP protection capability as compared to chitosan/pEGFP nanocomplexes.3 Cell adsorption, cellular uptake, uptake mechanism, and cellular distribution of LMC/pEGFP nanocomplexesCell adsorption, cellular uptake, uptake mechanism, and cellular distribution of LMC/pEGFP nanocomplexes were investigated. Cell adsorption and cellular uptake percentages were promoted 0.8-3.8 and 1.7-3.2 folds by LMC/pEGFP nanocomplexes compared to chitosan/pEGFP nanocomplexes, respectively, and stronger cell adsorption and higher cellular uptake percentages correlated to higher linoleic acid grafting ratios and lower poly (malic acid) grafting ratios. Uptake mechanism assays indicated LMC/pEGFP nanocomplexes were internalized through energy-dependent, clathrin-mediated endocytosis pathway while were irrelevant of caveolin-mediated endocytosis and cytoskeleton reorganization. Higher linoleic acid grafting ratios were favorable for the relative higher distribution percentages in nuclei, and no apparent differences in the distribution of pEGFP among nuclei were observed between LMC3/pEGFP and LMC4/pEGFP nanocomplexes.4 In vitro and in vivo gene transfection efficiency of LMC/pEGFP nanocomplexesThe transfection efficiency of LMC/pEGFP nanocomplexes was investigated on HEK293 cells and the effects of weight ratios, serum, and post-transfection intervals on transfection effiency were also evaluated. LMC3/pEGFP nanocomplexes mediated the highest gene expression, and the transfection percentages reached 34.5% at the weight ratio of 12, which were 8.0-folds higher than those of chitosan/pEGFP nanocomplexes. LMC3/pEGFP nanocomplexes showed superior serum compatibility and a notable enhancement effects in gene expression were observed. Elevated transfection percentages with prolonged post-incubation times were observed with LMC3/pEGFP nanocomplexes and reached 40.8% at 72 h, which were 2.1 folds and 1.4 folds compared to those of 24 h and 48 h. In vivo transfection effiency of LMC3/pEGFP nanocomplexes was evaluated through intramuscular administration in mice. The results showed that the highest GFP expression was mediated by LMC3/pEGFP nanocomplexes (36.62±10.66 I/mg), which were 4.2-folds and 2.2-folds higher than those of PEI/pEGFP and chitosan/pEGFP nanocomplexes.