Investigating the Fate of MP1000-LPX In Vivo by Adding Serum to Transfection Medium

Background: Cationic liposomes (CLs) based messenger RNA (mRNA) vaccine has been a promising approach for cancer treatment. However, rapid lung accumulation after intraveous injection and significantly decreased transfection efficacy (TE) in serum substantially hamper its application. Objective: In this study, we attempt to investigate the fate of Mannose-PEG1000-lipoplex (MP1000-LPX) in vivo, a previous reported mRNA vaccine, and potential mechanism in it. Methods: MP1000-CLs and different type of MP1000-LPX were produced by previous method and characterized by dynamic light scattering (DLS). Organ distribution and Luc-mRNA expression of DiD loaded luciferase (Luc-mRNA)-MP1000-LPX were evaluated by IVIS Spectrum imaging system. Cellular transfection and uptake under serum-free and serum-containing conditions were analysed by flow cytometry and counted by FlowJo software. Results: MP1000-CLs had an average size of 45.3 ± 0.9 nm, a positive charge of 39.9 ± 0.9 mV. When MP1000-LPX formed, the particle size increased to about 130 nm, and zeta potential decreased to about 30 mV. All formulations were in narrow size distribution with PDI < 0.3. 6 h after intraveous injection, Luc-MP1000-LPX mostly distributed to liver, lung and spleen, while only successfully expressed Luc in lung. DC2.4 cellular transfection assay indicated serum substantially lowered TE of MP1000-LPX. However, the cellular uptake on DC2.4 cells was enhanced in the presence of serum. Conclusion: MP1000-LPX distributed to spleen but failed to transfect. Because serum dramatically decreased TE of MP1000-LPX on DC2.4 cells, but not by impeding its interaction to cell membrane. Serum resistance and avoidance of lung accumulation might be prerequisites for CLs based intravenous mRNA vaccines. Lay Summary: mRNA vaccine has been promising immunotherapy to treat cancer by delivering mRNA encoding tumor antigens to APCs and activating immune system against tumor cells. We are investigating the in vivo fate of MP1000-LPX, a CLs based mRNA vaccine. To see if serum causes the fate, we’ll be looking at the influence of serum on transfection and uptake efficacy of MP1000-LPX by DC2.4 cells experiments in vitro. Our findings will imply that serum inhibits transfection but not by decreasing uptake. Thus, we can ultilize serum to enhance transfection if we make intracellular process of MP1000-LPX successful.