Finding the sweet spot between complexation and cytosolic release with different molecular weight branched polyethyleneimine polymers for mRNA delivery.

Messenger RNA (mRNA) is a promising therapeutic for genetic defects, gene editing and vaccines. The success of mRNA therapies largely depends on the optimal delivery vehicles. Although lipid nanoparticles are the leading option, some polymers exhibited attractive properties in mRNA delivery. In this study, branched polyethylene-imine (PEI) with molecular weight (Mw) of 1.8 kDa, 10 kDa and 25 kDa were explored for mRNA delivery. The complexation, dissociation and stability of bPEI/mRNA polyplexes were examined by gel electrophoresis. In vitro cellular uptake and transfection efficiency were evaluated in human cancer cell lines by using fluorescently labeled mRNA and reporter mRNA encoding enhanced green fluorescence protein. An oligonucleotide dequenching assay was used to visualize the endosomal escape of polyplexes. The mRNA full complexation started from N/P ratio 5, independent of the Mw of PEI polymers. In SKOV-3 cells, 10 kDa PEI exhibited the most promising transfection efficiency. While 1.8 kDa bPEI/mRNA polyplexes failed to escape from endosomal compartments, 25 kDa bPEI/mRNA polyplexes did endosomal escape, but less mRNA dissociation in the cytosol. In HeLa cells, cellular uptake and transfection efficiency increased through increasing either the N/P ratio or Mw of bPEI. However, the delivery efficiency of bPEI as an mRNA carrier remains relatively low and depends on balancing the appropriate Mw and N/P ratio for each specific cell type. Since cellular uptake was consistently high, the limiting factors for transfection efficiency are most likely endosomal escape and the intracellular release of functional mRNA, both of which appear to be strongly cell‑type dependent.