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

With the end goal of designing cationic nanoparticles for nucleic acid transfection, we tested if IAA-modified bPEI and chitosan nanoparticles could transfect siRNA. The PD-L1 gene was targeted in B16-F10 melanoma cells, which is an intriguing therapeutic target that could potentially reduce tumor-related immune suppression. [32] PD-L1 was first induced in melanoma cells through treatment with interferon-g at a concentration of 50 ng/mL. Treatment of PD-L1-induced cells with unmodified bPEI or chitosan nanoparticles yielded two observations. First, nanoparticles with PD-L1 target siRNA could reduce PD-L1 gene expression relative to nanoparticles with negative control siRNA. Second, unmodified bPEI and chitosan nanoparticles with negative control siRNA upregulated PD-L1 expression over PD-L1induced cells without nanoparticles. IAA-modified nanoparticles with negative control siRNA, on the other hand, did not upregulate PD-L1 expression. Since PD-L1 can be upregulated in response to TLR4 activation, these data further support that IAA modification reduces the inflammatory response to cationic nanoparticles ( Supplementary Fig. 9A-D) . [33] We also evaluated if bPEI nanoparticles could deliver functional luciferase mRNA (mLuc). First, we delivered mLuc with bPEI nanoparticles to HELA cells and measured luminescence from cell lysates after 5 hours. Normalized luminescence signal from IAAmodified bPEI nanoparticle-treated cells with mLuc was four times higher than from cells treated with unmodified bPEI nanoparticles with mLuc ( Supplementary Fig. 10A ). Following this study, we evaluated if IAA-modified bPEI nanoparticles could deliver functional mLuc in vivo. Three hours after IV delivery of luciferase mRNA, we observed a 20-fold increase in BLI signal in the lungs relative to untreated mice. This level of BLI signal remained high after 6 hours ( Supplementary Fig. 10B ). The equivalent dose of unmodified bPEI nanoparticles caused severe acute toxicity within 30 minutes, so luciferase expression was not measured.