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

项目摘要 败血症是一种复杂的疾病，由宿主对感染的反应失调引起。目前脓毒症的治疗 策略没有充分解决免疫失调和内皮功能障碍。NAD+可能会 是一种有效的脓毒症治疗分子，但其治疗效果因其无法通过而受到阻碍 通过细胞膜。胞外NAD+必须被降解成NAD+前体(例如，烟酰胺和 烟酰胺核苷)，它可以被细胞摄取，从而促进细胞内NAD+的生物合成。 然而，这种转换过程效率很低。这种对NAD+细胞内转运的限制 降低NAD+的生物活性，需要极高的剂量才能有效治疗。 我们的目标是利用NAD+递送纳米粒(NPs)开发一种创新、安全和有效的脓毒症治疗方法。 它可以直接(即，不转化为NAD+前体)并有效地补充细胞内的NAD+ 泳池，与广谱抗生素相结合。为了实现这一目标，我们制定了NAD+或 NAD+/抗生素(例如，利福平(Rif))负载脂质(LP)包裹的磷酸钙纳米粒(NPs)(称为 作为NAD+携带的NP，包括NAD+-LP-CAP和NAD+-Rif-LP-CAP)。NAD+加载的NP具有多个 具有理想的性能，包括高载药量、高稳定性、对pH敏感的药物释放曲线，以及 内体逃逸能力。尽管NAD+具有治疗潜力，但之前还没有关于体内研究的报告 使用NAD+NPs进行包括脓毒症在内的治疗研究。我们的初步数据表明，NAD+-LP- CAP可以抑制促炎细胞因子的释放，防止炎症诱导的细胞死亡和 内皮细胞破坏。因此，NAD+负载的NPs可以潜在地帮助维持两者的动态平衡 免疫系统和血管系统。我们的NAD+负载纳米粒成功治疗了内毒素和细菌诱导的 体内败血症。它们能够在脓毒症损伤的器官中蓄积，减轻多器官损伤。 与游离NAD+相比，负载NAD+纳米粒的治疗效果显著提高。 在这项拟议的工作中，我们计划进一步优化负载NAD+的NPs，以实现更高的载药量 内容和效率(目标1)。NAD+-LP-CAP的治疗机制将在体外进行研究，以期 更好地了解NAD+负载的NPs如何抑制炎症并保护细胞，包括 免疫细胞和内皮细胞免受炎症诱导的细胞损伤(目标2)。最后，我们将系统地 确定NAD+负载纳米粒在两个有代表性的患者中的安全性、药代动力学和治疗效果 建立小鼠脓毒症模型，并研究NP对免疫和血管内稳态的影响(目标3)。 如果成功，这项拟议的研究将创造一种创新、安全和有效的治疗方法。 败血症。这种范式转换疗法治疗脓毒症的翻译成功可以显著改善预后。 这种严重的疾病导致全世界五分之一的人死亡，并减少了巨大的经济损失 它给医疗系统带来了负担(例如，美国的联邦医疗保险成本约为415亿美元)。

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).