Studies On Amphiphilic Cationic Peptide Stearate For Gene Delivery

Cationic peptide stearate indicates considerable potential in gene delivery for efficient cell-membrane penetrating and DNA condensing ability. In this study, cationic peptide stearate/pDNA(STR-Pep/pDNA) and PEG modified cationic peptide stearate/pDNA(PEG-STR-Pep/pDNA) were engineered as gene delivery systems. The physicochemical properties, in vitro transfection efficiency, in vivo anti-tumor activity and endosomal escape ability were evaluated.The chemical conjugation was realized through the amidation reaction between the amino of cationic peptide and the carboxyl of stearic acid. Aldehyde PEG was modified through the rection between the aldehyde group and the side chain amino of cationic peptide. The chemical structures were comfirmed by NMR and the critical micelle concentration in aqueous medium was determined by by pyrene fluorescence method. The particle size and morphology were observed by TEM. The cytotoxicity of cationic peptide stearate and PEG modified peptide stearate were investigated by MTT assay.Prepared STR-Pep/pDNA and PEG-STR-Pep/pDNA complex nanoparticles were observed by TEM to evaluation the particle size and morphology characteristics. Gel retardation assay was performed for examination of the condensation ability of polymer with DNA by electrophoresis. The expressions of green fluorescent proteins mediated by STR-Pep/pEGFP and PEG-STR-Pep/pEGFP complexes were observed and measured by inverted fluorescence microscopy and flow cytometry in HEK293, SKOV-3, MCF-7cells. pGL-3was also used to evaluate the transfection efficiencies of STR-Pep/pDNA and PEG-STR-Pep/pDNA complexes. Meanwhile, to confirm the endosomal escape ability of cationic peptide, pDNA was labeled with5-FAM fluorescence (green fluorescence), lysosomes and endosomes were labeled with Lyso-Tracker. The intracellular process and endosomal escape of STR-Pep/pDNA complex were observed by confocal laser scanning microscopy. In vivo anti-tumor activity was evaluated using pPEDF as therapy gene in SKOV-3bearing nude mice.Since STR-Pep and PEG-STR-Pep have prominent micelle characteristic and could mediate effective gene transfection, the anti-tumor co-therapy of drug loaded micelles and therapy gene was taken into consideration. In this study, doxorubicin was used as model drug and was loaded into STR-Pep, PEG-STR-Pep micelles by dialysis method. The DOX content in micelles were measured through ultrafiltration centrifugal-organic solvent extraction. The tumor cells inhibition of STR-Pep/DOX and STR-Pep-PEG/DOX micelles were measured against SKOV-3, A549, HepG2cells. STR-Pep/DOX/pDNA and PEG-STR-Pep/DOX/pDNA complex were also prepared to evaluate the transfection efficiency in HepG2cells.The result shows STR-Pep and PEG-STR-Pep have stable physicochemical properties. The CMC value of STR-Pep and PEG-STR-Pep micelles was182μg/mL and212μg/mL. The particle sizes were approximately10-20nm and50-60nm. STR-Pep and PEG-STR-Pep micelles could condense pDNA tightly at low weight ratio to form stable nanoparticles. STR-Pep/pDNA and PEG-STR-Pep/pDNA complex nanoparticles successfully mediated effective gene transfection in different cell lines. In certain cells the transfection efficiencies were close to or even higher than that of LipofectamineTM2000. In vivo anti-tumor activity was obtained by STR-Pep/pDNA and PEG-STR-Pep/pDNA complex nanoparticles. Meanwhile, the DOX encapsulation efficiencies of STR-Pep and PEG-STR-Pep micelles were54.1%and60.8% respectively. STR-Pep/DOX/pEGFP and PEG-STR-Pep/DOX/pEGFP complex could mediate in vitro gene transfection despite the efficiency need to be improved.