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

Unmodified bPEI nanoparticles (BPEI-TPP) and IAA-modified bPEI nanoparticles (bPEI-IAA-TPP) were produced by mixing solutions of bPEI or bPEI-IAA with sodium tripolyphosphate in deionized water. The mass ratio of bPEI or bPEI-IAA to TPP was 3:2. Unmodified chitosan nanoparticles (chitosan-TPP) and IAA-modified chitosan nanoparticles were produced by mixing solutions of chitosan or chitosan-IAA with sodium tripolyphosphate in sodium acetate buffer (pH= 4.5). The mass ratio of chitosan or chitosan-IAA to TPP was 5:1. Following vortexing, the nanoparticle formulations were then put onto a rotator at room temperature for 30 minutes. After rotating, nanoparticles were centrifuged at 4000 g for 20 minutes in Amicon centrifuge filters (100 kD MWCO) and resuspended in 1 mL PBS. The size and zeta potential of the particles were measured using a Malvern Zetasizer Nano ZS. For fluorescent labeling, bPEI and chitosan nanoparticles were reacted with Vivotag-645 for 1 hour on a rotator in the All rights reserved. No reuse allowed without permission.