Construction And Evaluation Of A Novel Mannose Receptor-based Non-viral Gene Delivery Vehicle

Object:This study aims to synthesize spermine-mannan (SM) specific for Mannose Receptor, and will further characterize SM and SM/DNA complexes. The SM-based delivery system will be applied for systemic gene delivery to acheive liver targeting, and will also to be applied on mannose receptor expressed cells for invitro transfection to acheive a promoted efficiency. Moreover the mechanism of how SM-based delivery system functions will also be investigated.Method:SM was synthesized with varied grafted ratio and was characterized using FTIR, DSC, H’-NMR and Elementary analysis. MTT assay was used to optimize the cytotoxicity of SM and the transfection efficiency was optimized using Luciferase report plasmid. The particle size&zeta potential of optimized grafted SM/DNA complex was measured using zetasizer, and the morphology was observed under TEM. Invivo distribution of SM/FITC-DNA was evaluated using Invivo-imaging system. And the particle size and transfection efficiency of SM/DNA was measured to evaluated the interference of serum existence. The slice of HE staining was done for the evaluation of invivo toxicity in liver. As for invivo transfection efficiency, it was evaluated using EGFP reporter plasmid. Furthermore, The cytotoxicity of SM-based delivery system was evaluated on four cell lines using MTT assay, and the transfection efficiency was also evaluated using Luciferase reporter plasmid. The specificity of SM to MMR was confirmed by competition assay, inhibition assay and calcium chelation assay. For the investigation of intracellular transport and endosomal escape, SM and DNA were both labeled and observed at certain time point using Confocal Laser Microscopy. The SM based system was also utilized in bone marrow derived dendritic cells, and the uptake of SM/FITC on BMDC was measured by Flow Analysis Cytometry, and the transfection was also done using EGFP plasmid.Conclusion:We successfully synthesized SM, characterized it and optimized the vehicle in cytotoxicity and transfection efficiency with varied grafted ratio. And SM/DNA complex was characterized as a stable system for DNA delivery. SM-based delivery system was proved to be liver targeting and could prolong the gene accumulation in liver. The system was also confirmed to be less interfered by serum. The optimized SM-based delivery system was further confirmed to be sufficient in hepatic transfection with almost no side-effect. Moreover, The SM-based delivery system is a biocompatible vehicle with superior transfection efficiency on MMR+cells compared with other transfection agents. It was confirmed that the SM-based system is MMR specific. The intracellular transport and endosomal escape of the vehicle was also clearly described.