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

When we collected blood to measure levels of serum cytokines, we observed, qualitatively, differences in blood viscosity in mice treated with the unmodified bPEI nanoparticles. This inspired us to evaluate if cationic nanoparticles influence blood properties after IV injection. 30 minutes after mice were treated with unmodified bPEI nanoparticles, hematocrit rose from 40% to 80%. At the same time point, hemoglobin concentration rose from 12 to 25 g/dL. In contrast, no changes in hematocrit or hemoglobin concentration were observed in IAA-modified bPEI nanoparticle-treated mice after 30 minutes (Fig. 3F-G) . Both unmodified and IAA-modified bPEI nanoparticles increased the neutrophil fraction in white blood cells from 0 to 15% after 30 minutes. This neutrophil fraction remained high with mice treated with unmodified bPEI nanoparticles after 150 minutes, but returned to baseline in mice treated with IAA-modified bPEI nanoparticles (Fig. 3H) . The increase in hematocrit prompted us to evaluate if the unmodified bPEI nanoparticles were initiating a coagulation cascade by hemolyzing red blood cells, which can induce adenosine diphosphate release and subsequently activate platelets. [30] Neither bPEI nanoparticle formulation induced a significant amount of hemolysis when incubated with whole blood at a concentration of 125 µg/mL, which is twice as high as the blood pool concentration of bPEI nanoparticles immediately after IV injection in our experiments (60 µg/mL; Supplementary Fig. 4B ). We then hypothesized that high hematocrit All rights reserved. No reuse allowed without permission.