Preparation Of Polycation Nanostructured Lipid Carrier Based On Olein For Efficient Gene Delivery And Its Intracellular Transfer Mechanism

Efficient and safe gene transfer to mammalian cells remains the most challenging hurdle to successful gene therapy.Compared with viral vectors, nonviral gene transfer vectors are most attractive due to their fewer safety concerns and ease to preparation.In results,the development of novel efficient nonviral gene transfer vectors has attracted much attention although the efficiency is not quite satisfactory.In the present study,we try to merge the advantages of DOPE,cetylated PEI and olein together to make a novel nonviral vector--polycation nanostructured lipid carriers（PNLC） for efficient gene delivery.The myristylated PEI（600Da,1200Da,1800Da）,cetylated PEI（600Da, 1200Da,1800Da） and octadecylated PEI（600Da,1200Da,1800Da） was synthesized.PNLC composed of alkylated PEI,oil phase and phospholipids, was prepared by emulsion-solvent evaporation method.Transfection of plasmid pEGFP-N2 mediated by the PNLC/DNA complexes was performed in SPC-A1 cells,and the expressed green fluorescence protein（GFP） was determined to optimize the formulation of PNLC.Transfection efficiency of the optimized PNLC in SPC-A1 cells and CHO cells was evaluated by FACScan flow cytometer.With the help of specific inhibitors,the possible mechanism of PNLC was investigated.Finally,the PNLC/DNA complexes were fluorescently labeled and trafficked by laser confocal scanning microscopy in live SPC-A1 cells.At first,the myristylated PEI（600Da,1200Da,1800Da）,cetylated PEI（600Da, 1200Da,1800Da） and octadecylated PEI（600Da,1200Da,1800Da） was synthesized.PNLC was prepared by emulsion-solvent evaporation method. The particle size and zeta potential values were characterized by NICOMp^TMZLS380.PNLC was complexed with plasmid DNA at various N/P（PNLC/DNA） ratios.Transfection of the formed PNLC/DNA complexes in SPC-A1 cells was performed,and 48h after transfection,the fluorescence intensity of the expressed GFP in SPC-A1 cells was determined fluorometrically to optimize the formulation of PNLC.Firstly,the molar ratio of triolein/EPC was fixed at 1 and the molar ratio of alkylated PEI/EPC was adjusted to investigate the effect of alkylated PEI on the gene transfection efficiency.Results showed that when the alkylated PEI was cetylated PEI1200（1:6） and the molar ratio of the cetylated PEI1200（1:6）/EPC was 0.1 in the PNLC formulation,the expressed GFP fluorescence intensity in SPC-A1 cells was the highest.The alkylated PEI in the PNLC formulation was determined as P₁₂C₁₆（1:6）.Secondly,when the molar ratio of P₁₂C₁₆（1:6）/EPC was fixed at 0.1,triolein,tripalmitin,trimyristin, Miglycol M812N and Miglycol 818 were separately included into the PNLC formulation to investigate the effect of oil phase on the gene transfection. Results indicated that when the oil phase was triolein and the molar ratio of triolein/EPC was 1,the expressed GFP fluorescence intensity in SPC-A1cells was the highest.Triolein and its analogues such as diolein and monoolein was determined as the oil phase in the PNLC formulation.Thirdly,to investigate the effect of EPC and DOPE on the gene transfection,PNLC was prepared with P₁₂C₁₆（1:6）,olein（triolein,diolein or monoolein）,EPC or DOPE.The expressed GFP fluorescence intensity in SPC-A1 cells indicated that enhanced transfection efficiency was obtained after the addition of olein to the PNLC formulation,and the optimized PNLC formulation was shown in the table.PNLC Ingredients of the optimized PNLC formulationPTE Molar ratio of P₁₂C₁₆（1:6）/EPC was 0.1,molar ratio of triolein/EPC was 1PTD Molar ratio of P₁₂C₁₆（1:6）/DOPE was 0.1,molar ratio of triolein/DOPE was 0.8PDE Molar ratio of P₁₂C₁₆（1:6）/EPC was 0.1,weight ratio of diolein/EPC was 2PDD Molar ratio of P₁₂C₁₆（1:6）/DOPE was 0.1,weight ratio of diolein/DOPE was 0.75PME Molar ratio of P₁₂C₁₆（1:6）/EPC was 0.1,molar ratio of monoolein/EPC was 6PMD Molar ratio of P₁₂C₁₆（1:6）/DOPE was 0.1,molar ratio of monoolein/DOPE was 1.5The size distribution of the optimized PNLC/DNA complexes（N/P=10） was measured by NICOMP^TMZLS380,and the morphological character of the PNLC/DNA complexes was observed under atomic force microscopy（AFM）. Furthermore,the transfection of plasmid pEGFP-N2 mediated by the optimized PNLC in SPC-A1 cells and CHO cells was quantified by FACScan flow cytometer,effect of serum on the transfection efficiency was taken into account as well.Results indicated that the transfection efficiency of the PTE and PDE formulated PNLC was comparable with that of Lipofectamine ^TM2000, moreover,the transfection efficiency of the PME,PTD,PDD and PMD formulated PNLC was much higher.Transfection mediated by the optimized PNLC/DNA complexes in both cell lines was still effective in the presence of 10%serum.In the presence of 10%serum,the transfection efficiency of PTD and PDD formulated PNLC was 192%and 125%of that in the absence of serum in SPC-A1 cells,and not changed significantly in CHO cells.With the help of specific inhibitors,the endocyted pathway of the PNLC/DNA complexes in SPC-A1 cells was investigated.After the addition of specific inhibitors to the SPC-A1 cells,the cell uptake of the FITC labeled PNLC/DNA complexes was measured by FACScan flow cytometer and the expressed luciferase activity in SPC-A1 cells was determined and normalized with protein content.After the addition of chlopromazine to the SPC-A1 cells, the cell uptake of the PNLC/DNA complexes decreased and the expressed luciferase activity was only about 10%of that without the inhibitor;after the addition of filipinⅢto the SPC-A1 cells,the cell uptake and the expressed luciferase activity in SPC-A1 cells were not changed significantly.As the PNLC/DNA complexes were fluorescently labeled with TM-Rhodamine and the late endosome/lysosome was labeled with LysoTracker Green DND 26,the colocalization of the PNLC/DNA complexes and the late endosome/lysosome was observed under laser confocal scanning microscopy.We could deduce that the PNLC/DNA complexes were endocyted into the SPC-A1 cells via the clathrin-dependent pathway.Microtubules and the dynein and kinesin motor proteins were primarily responsible for trafficking of cargo and vesicles.To determine the effect of microtubules on the transfer of the PNLC/DNA complexes,the microtubules-depolymerizing agent,nocodazole,and the microtubules-stabilizing agent,paclitaxel,were added to the SPC-A1 cells. Resulted demonstrated that the gene expression in SPC-A1 cells was almost abolished after the addition of the inhibitors.With the help of the specific inhibitors to the motor proteins,effect of dynein and kinesin on the transfection efficiency of PNLC was investigated.It was showed that the transfection efficiency of the PNLC/DNA complexes was enhanced 50%after the addition of dynein inhibitor,sodium orthovanadate（SOV）,the decreased after the addition of the inhibitor of kinesin Eg5,monastrol.All the results demonstrated that mocrotubules and the kinesin and dynein motor proteins were responsible for the cytosolic transfer of the PNLC/DNA complexes in SPC-A1 cells.To determine the intracellular transport of the PNLC/DNA complexes in SPC-A1 cells,PNLC was fluorescently labeled with FITC and the plasmid DNA was fluorescently labeled with RM-Rhodamine.The formation of the PNLC/DNA complexes and the intracellular transport of the fluorescently labeled PNLC/DNA complexes were observed under laser confocal scanning microscopy.After endocyted into the SPC-A1 cells,the PNLC/DNA complexes was colocalized with the late endosome/lysosome and then transferred to the perinuclear region.Furthermore,the FITC labeled cetylated PEI1200（1:6） and the TM-Rhodamine labeled plasmid DNA appeared in the cell nucleus.We could deduce that after the endocytosis via the clathrin-dependent pathway, the PNLC/DNA complexes were transferred to the late endosome/lysosome after escape from the late endosome/lysosome,the PNLC/DAN complexes was presented to the perinuclear region,and then,the cetylated PEI and the plasmid was transferred in to the cell nucleus.