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Emerging evidence suggests that bioengineered nanoparticles can be used as immunomodulatory agents. Using murine and non-human primate vaccination models, we have identified novel cationic lipid nanoparticles (LNPs) that significantly enhance antigen-specific B-cell, CD4+T cell, and most notably, CD8+ T-cell responses. Importantly, we found that LNP-containing vaccine formulations resulted in protective efficacy in a virus challenge model, as well as, in syngeneic murine tumor models. In-vivo imaging and immunophenotyping studies showed that LNPs prolong antigen retention at the injection site and enhance antigen migration to the lymph nodes. Unbiased screening of LNPs ability to activate innate immune sensors showed that LNPs selectively activate Toll-Like Receptor-2 (TLR2). Using genetic ablation, and antibody mediated blockade, we uncovered that TLR2-MyD88 pathway is required for LNPs ability to boost immune responses in-vivo. Ex-vivo and in-vitro studies using dendritic cells (DC) showed that neutralization of TLR2 does not impact the LNP mediated increase in antigen uptake. However, LNP dependent enhancement of DC maturation, antigen processing & presentation, and chemokine production, were significantly reduced when TLR2 was neutralized. Pharmacological inhibition TIRAP, and CD14-MD2, further confirmed the need for the activation of TLR2-MyD88 pathway in LNP’s functionality. Future studies are aimed at utilizing chosen Merck chemical inhibitors of innate immune kinases to further interrogate the TLR2-MyD88 pathway’s involvement in LNP functionality. Such detailed knowledge of LNP’s mode of action will significantly influence the future design of LNPs for vaccines and immune oncology applications.

In order to rapidly screen and select lead candidates for in vivo evaluation of lipid nanoparticles (LNPs) for systemic small interfering RNA (siRNA) delivery, an in vitro assay amenable to high-throughput screening (HTS) is developed. The strategy is to mimic the in vivo experience of LNPs after systemic administration, such as interactions with serum components, exposure to endosomal pH environments, and interactions with endosomal membrane lipids. It is postulated that the amount of siRNA released from LNPs after going through these treatments can be used as a screening tool to rank order the effectiveness of siRNA delivery by lipid nanoparticles in vivo. LNPs were incubated with 50% serum from different species (i.e. mouse, rat, or rhesus) at 37°C. The resulting samples were then reacted with anionic, endosomal-mimicking lipids at different pHs. The amount of siRNA released from LNPs was determined using spectrophotometry employing the fluorescent indicator SYBR Gold. Our results indicated that the amount of siRNA liberated was highly dependent upon the species of serum used and the pH to which it was exposed. LNPs treated with mouse serum showed higher levels of siRNA release, as did those subjected to endosomal pH (6.0), compared to physiological pH. Most interestingly, a good correlation between the amount of siRNA released and the in vivo efficacy was observed. In conclusion, an in vitro siRNA release assay was developed to screen and rank order LNPs for in vivo evaluation.