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

We investigated the in vivo immune response to bPEI and chitosan nanoparticles and determine how IAA modification of the nanoparticles would alter the immune response. Each nanoparticle formulation was synthesized through an ionic gelation process with sodium tripolyphosphate (unmodified bPEI nanoparticles = bPEI-TPP, IAA-modified bPEI nanoparticles = bPEI-IAA-TPP, unmodified chitosan nanoparticles = Ch-TPP, IAA-modified chitosan nanoparticles = Ch-IAA-TPP; Fig. 1B ). [24] Both PEI and chitosan have been reported to activate TLR4 in macrophages, leading to the secretion of IL-12, nitric oxide, and tumor necrosis factor alpha (TNF-a). [25] [26] [27] In addition, chitosan triggers DNA release from mitochondria in vitro, which results in stimulator-of-interferon-gene (STING) pathway-mediated upregulation of antiviral signaling through production of interferons and pro-inflammatory cytokines. [28] We hypothesized that these innate immune reactions could be reduced by modification of the primary amines in these polymers to secondary and tertiary amines -which could, at the same time preserve or enhance their buffering capacity and thus delivery efficacy. To achieve this, we conjugated IAA to bPEI or chitosan using a carbodiimide chemistry (Fig. 1C ). [11] Using nuclear magnetic resonance spectroscopy (NMR), we determined that 12% of available reaction sites on the bPEI polymer were modified with IAA ( Supplementary Fig. 1A ). Nanoparticles composed of unmodified or IAA-modified bPEI were slightly less than 100 nm in size ( Supplementary Fig. 1B ). Both unmodified and IAA-modified bPEI nanoparticles had a similar, slightly negative zeta potential (~-10 mV) at pH 7.4. To quantify IAA modification of chitosan, we measured absorbance by spectrophotometry at 230 nm (Supplementary Fig. 2A ).