Internalization of mRNA lipoplexes by dendritic cells

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Lipoplexes are crucial for delivering mRNA into cells, particularly dendritic cells (DCs).
• Understanding mRNA lipoplex internalization pathways in DCs is vital for optimizing T cell-based immunotherapies, including those for HIV.
• Previous studies suggest various endocytic pathways mediate lipoplex uptake, but the specific mechanisms for transfection-effective uptake remain unclear.

Purpose of the Study:

• To elucidate the endocytic pathways responsible for mRNA lipoplex internalization by dendritic cells (DCs).
• To determine which internalization routes lead to effective protein expression and subsequent T cell stimulation.
• To investigate the role of macropinocytosis, phagocytosis, and clathrin/caveolae-mediated endocytosis in DC transfection.

Main Methods:

• Confocal microscopy was used to visualize the colocalization of fluorescently labeled mRNA lipoplexes with late endosomes in DCs.
• Inhibitors targeting specific endocytic pathways (phagocytosis, macropinocytosis, clathrin-mediated, and caveolae-mediated endocytosis) were employed.
• Cellular uptake and protein expression levels were quantified following treatment with various endocytic inhibitors.

Main Results:

• One-third of mRNA lipoplexes colocalized with late endosomes.
• Inhibitors of macropinocytosis and phagocytosis (Cytochalasin D) significantly reduced lipoplex internalization (50%) and protein expression (70%).
• Inhibitors targeting other pathways showed varied effects, with Genistein and Dynasore strongly reducing protein expression but not overall uptake, suggesting macropinocytosis and/or phagocytosis are key for transfection-effective uptake.

Conclusions:

• Transfection-effective internalization of mRNA lipoplexes by DCs predominantly occurs via macropinocytosis and/or phagocytosis.
• These pathways are critical for achieving therapeutic protein expression from delivered mRNA.
• The findings provide insights into optimizing lipid-based mRNA delivery systems for enhancing immune responses.