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

Accurate prediction and subsequent minimization of in vivo systemic toxicity requires a critical and comprehensive understanding of how nanoparticles interact with a variety of biological components after in vivo delivery, e.g., with serum proteins, blood and immune cells, and cells in various tissues like the liver, lung, and spleen. It is also critical to understand how they distribute to organs and in specific cell-types to trigger the innate immune response. In this study, we identify the distinct contributors to the in vivo toxicity responses induced by two widely-reported cationic nanoparticles for nucleic acid delivery -branched polyethyleneimine (bPEI) and chitosan. We found that the mechanism of in vivo systemic toxicity is a combination of innate immune responses, complement reactions, and hepatic and hematological toxicities -and the relative contribution of these differs with the type of polymer. We then compared in vivo systemic toxicity to these nanoparticles after modification with the small molecule imidazole-acetic-acid (IAA), which modifies primary-amines on the polymers and introduces secondary and tertiary amines. We hypothesized that such modification would reduce innate immune activation by downregulating interactions with the Toll-like receptor 4 (TLR4). While we observed reduced TLR4-mediated systemic toxicity with the IAA-modified nanoparticles, we also observe a significant impact on other measures of the systemic toxicity response -which include in vivo hepatotoxicity, complement activation, and changes in hematological properties.