Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways

Randall Toy; Pallab Pradhan; Vijayeetha Ramesh; Nelson C. Di Paolo; Blake Lash; Jiaying Liu; Emmeline L. Blanchard; Philip J. Santangelo; Dmitry M. Shayakhmetov; Krishnendu Roy

Endosomal escape of unmodified and IAA-modified bPEI nanoparticles was evaluated by measurement of their co-localization with intracellular compartments (CD63, EEA1 -an early endosomal marker, LAMP1 -a lysosomal marker, clathrin, and caveolin) after 2 hours of treatment. Incomplete overlap with intracellular compartments was observed with both unmodified and IAA-modified nanoparticle formulations (Supplementary Fig. 8A ). This indicates that bPEI nanoparticles, regardless of IAA modification, are able to escape into the cytoplasm to facilitate delivery of mRNA or siRNA. We confirmed that the changes in innate immune activation were not due to differences in cellular uptake of the bPEI and IAA-modified bPEI nanoparticles (Supplementary Fig. 8B ).