Development of NAD+ loaded nanoparticles as a safe and efficient strategy to combat sepsis.

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Project Summary Sepsis is a complex disorder caused by a dysregulated host response to infection. Current sepsis therapeutic strategies do not adequately address immune dysregulation and endothelial dysfunction. NAD+ can potentially be an efficient therapeutic molecule for sepsis, but its therapeutic efficacy is hindered by its inability to pass through the cell membrane. Extracellular NAD+ has to be degraded into NAD+ precursors (e.g., nicotinamide and nicotinamide riboside), which can be taken up by cells and subsequently enhance intracellular NAD+ biosynthesis. However, this conversion process is inefficient. Such a limitation in NAD+ intracellular transportation drastically decreases the bioactivity of NAD+ and necessitates an extremely high dose for effective therapy. We aim to develop an innovative, safe, and effective sepsis therapy utilizing NAD+ delivery nanoparticles (NPs), which can directly (i.e., without being converted to NAD+ precursors) and efficiently replenish the cellular NAD+ pool, in combination with a broad-spectrum antibiotic. To achieve this goal, we formulated a family of NAD+ or NAD+/antibiotic (e.g., rifampicin (Rif)) loaded lipid (LP)-coated calcium phosphate nanoparticles (NPs) (coined as NAD+ loaded NPs including NAD+-LP-CaP and NAD+-Rif-LP-CaP). The NAD+ loaded NPs possess a number of desirable properties including high loading content, high stability, pH-responsive drug release profiles, and endosomal escape capability. Despite the therapeutic potential of NAD+, there is no prior report on in vivo studies using NAD+ NPs for therapeutic studies including sepsis. Our preliminary data has demonstrated that NAD+-LP- CaP can suppress the release of pro-inflammatory cytokines and prevent inflammation-induced cell death and endothelium disruption. Therefore, NAD+ loaded NPs can potentially help maintain homeostasis of both the immune system and the vascular system. Our NAD+ loaded NPs successfully treated LPS- and bacteria-induced sepsis in vivo. They were able to accumulate in the sepsis injured organs and mitigate multiple organ injury. Compared with free NAD+, NAD+ loaded NPs showed significantly improved therapeutic efficacies. For this proposed work, we plan to further optimize the NAD+ loaded NPs to achieve even higher drug loading content and efficiency (Aim 1). The therapeutic mechanism of NAD+-LP-CaP will be studied in vitro in order to gain a better understanding of how the NAD+ loaded NPs suppress inflammation and also protect cells including immune cells and endothelial cells from inflammation-induced cell damage (Aim 2). Finally, we will systematically determine the safety, pharmacokinetics, and therapeutic efficacy of the NAD+ loaded NPs in two representative mouse sepsis models, and also study the NP’s impact on immune and vascular homeostasis (Aim 3). If successful, this proposed study will create an innovative, safe, and effective therapeutic approach for treating sepsis. A translational success of this paradigm-shift therapy for sepsis could significantly improve the prognosis of this serious disease that causes one in five deaths around the world and decrease the tremendous financial burden it brings to the healthcare systems (e.g., around $41.5 billion cost in Medicare in the US).