Effect Of Anionic PEGylated Poly- Peptide On Gene Transfection Mediated By Glycolipid Conjugate Micelles

Chitosan-g-stearic acid (CS) micelle has been demonstrated as an effective gene caeeier in vitro and in vivo. As a chitosan-based cationic polymer, CS is capable of condensing negatively charged DNA by ionic interactions and being advantages for DNA protection and excellent cellular internalization. However, some major deficiencies of CS, e.g., endosomal escape inefficiency and vector unpacking difficulty, result in low transfection efficiencies. In this research, poly(ethylene glycol)-b-poly(γ-glutamic acid) (PG) was incorporated into CS-based gene delivery system and CS/PG/pDNA ternary gene delivery systems were then prepared to improve the gene transfection efficiency.The CS copolymers were synthesised via the reaction of stearic acid (SA) carboxyl groups with chitosan amine groups in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). PEGylated chitosan-g-stearic acid (PCS) conjugates were obtained by coupling reactions of primary amine groups of CS conjugates and mPEG-ALD aldehyde side groups. The chemical composition and substitute degrees of amino groups (SD) were comfirmed by NMR and 2,4, 6-trinitrobenzene sulfonic acid (TNBS) method. The critical micelle concentrations (CMC) of CS and PCS were determined by fluorescence spectroscopy using pyrene as a probe. The zeta potential of the micelles and their corresponding gene delivery systems were measured by dynamic light scattering, and the size distribution and morphologies were examined by transmission electronic microscopy (TEM). PG was incorporated into a CS-based gene delivery system and CS/PG/pDNA gene delivery systems were prepared by ionic interaction. CS and PEGylated CS (PCS) micelles were introduced to prepare binary gene delivery systems for use as controls. Gel retardation assays were performed for the examination of the condensation and DNase I protection ability of polymers to pDNA by electrophoresis. Using enhanced green fluorescence protein (EGFP)-encoded pDNA (pEGFP-C1) as a reporter gene, the effect of PG on the gene transfection efficiency mediated by CS-based delivery system was investigated using HEK293 and EC-1 cells as model cells. The transfection capability was evaluated by an inverted fluorescence microscope or a confocal laser scanning microscope (CLSM) and quantitated by flow cytometry. The cellular uptake and intracellular trafficking of RITC-labelled gene delivery systems were observed by a CLSM. For the quantification of cellular uptake, HEK293 cells treated with RITC-labelled gene delivery systems were analysed by flow cytometry. The abilities of endosomal escape and pDNA release were quantitated by CLSM. Using macrophages RAW264.7 as model cells, cellular uptake was measured to verify the merit of PEG protection of CS/PG/pDNA from reticuloendothelial system (RES) clearance.The results showed that SA and/or PEG were successfully grafted onto the chitosan chain. The SD values of CS and PCS were 7.1% and 11.0%, respectively. The CMC values of CS and PCS micelles were 72.0 and 89.0μg/mL, respectively. Their zeta potentials were 18.7±0.6 and 16.3±1.6 mV, respectively. Both CS and PCS micelles showed irregular spherical in shape.CS micelles have powerful pDNA compact ability, and stable CS/pDNA gene delivery systems were formed when the N/P ratio was or above 1. The incorporation of PG into CS/pDNA gene delivery systems did not affect the pDNA compact ability of CS and showed protection effect to pDNA against DNase I. Importantly, PG could increase the gene transfection efficiency, which was affected by mixing methods of CS/PG/pDNA ternary gene delivery systems. The transfection efficiencies mediated by CS/PG/pDNA gene delivery systems against HEK293 and EC-1 cells reached up to 40.8% and 11.6%, respectively, which were much higher than that of CS/pDNA (1.3% and 4.0%) and PCS/pDNA gene delivery systems (0.8% and 2.4%). Besides a non-specific charged-mediated binding to cell membranes, the incorporation of PG into CS/pDNA gene delivery systems significantly enhanced their cellular uptake of HEK293 and EC-1 cells by γ-PGA-specific receptor-mediated pathway.In addition, the incorporation of PG in CS/pDNA gene delivery systems significantly enhanced their cellular uptake of HEK293 and EC-1 cells, as well as improved the endosomal escape and vector unpacking intracellular. However, the incorporation of PG reduced cellular uptake of CS/PG/pDNA gene delivery systems in macrophage (RAW264.7 cells). It was further demonstrated that besides a non-specific charged-mediated binding to cell membranes, there was a specific y-PGA specific receptor-mediated pathway involved in the internalization of CS/PG/pDNA gene delivery systems. PG had the capability of combining protons under the low pH condition of the endolysosomes and promoted their endosomal escape. After escape, the other non-protonated carboxyl groups could weaken the affinity between the pDNA and CS to maintain loosened the structure of CS/PG/pDNA gene delivery systems, resulted in the promotion of pDNA release from the gene delivery systems. However, the incorporation of PG reduced the cellular uptake of CS/PG/pDNA gene delivery systems in macrophages (RAW264.7 cells), which demonstrated that PEGylation could protect CS/PG/pDNA gene delivery systems from RES clearance.