Targeting Nanoparticles for Drug Delivery to Renal Graft Endothelium during Ex Vivo Normothermic Perfusion

项目总结 排斥反应仍然是同种异体移植物丢失的主要原因，其频率和严重程度因围术期的原因而加剧。 移植物手术缺血再灌注损伤(IRI)。对这种联系的机械解释是，IRI 移植物内皮细胞(EC)增强其刺激宿主免疫系统的能力，加剧 拒绝。体外常温灌流(EVNP)是一种实验性的方法，最先用于肾脏 由我们在剑桥大学的合作者进行的移植，通过改善 组织的代谢状态。我们假设这一过程可以同时用于改善IRI 并在器官植入前通过向移植物EC输送siRNA来减少排斥反应。我们已经选择了肿瘤 作为siRNA分子靶点的肿瘤坏死因子受体1(TNFR1)及其关键接头蛋白Tradd 这些实验中的敲除是因为这些分子介导了EC的肿瘤坏死因子反应 促进炎症和细胞死亡，这是肾脏IRI的重要过程，但这一R21的目标仅限于 SiRNA敲除效果的优化。目前，siRNA在临床上的传递受到不完全的限制 击倒的程度和持续时间很短，因为无法有效地定位感兴趣的细胞，以及 不适当的细胞目标。在这里，我们建议进行原则证明研究，以证明抗体- 共轭、可降解的聚合物纳米颗粒(NP)可以被设计来克服这些限制。我们的 具体目标是：(1)优化多功能、可降解NPs的配方，使用已建立的和 用于siRNA负载、siRNA缓释和mAb偶联的新型可降解聚合物；(2)鉴定 培养人结合和摄取结合NP的最佳EC特异性单抗或单抗组合 耶鲁大学的EC在体外流动，接受EVNP的已故供者肾脏，后者将由 我们在剑桥大学的合作者拥有独立于此R21应用程序的资源； 成功完成这些目标将为创新方法提供概念验证，以减少 通过体外靶向移植物的新型药物输送载体进行同种异体排斥反应，使宿主免受 需要过度的免疫抑制。

PROJECT SUMMARY Rejection remains the major cause of allograft loss and its frequency and severity are exacerbated by peri- operative ischemia/reperfusion injury (IRI) of the graft. A mechanistic explanation of this connection is that IRI of graft endothelial cells (EC) enhances their ability to stimulate the host immune system, exacerbating rejection. Ex vivo normothermic perfusion (EVNP) is an experimental approach, pioneered for renal transplantation by our collaborators at the University of Cambridge, to reduce graft injury by improving the metabolic state of the tissue. We hypothesize that this procedure can simultaneously be used to ameliorate IRI and reduce rejection by delivery of siRNA to graft EC prior to organ implantation. We have selected tumor necrosis factor receptor 1 (TNFR1) and its key adaptor protein, TRADD, as molecular targets for siRNA knockdown in these experiments because these molecules mediate tumor necrosis factor- responses of EC that promote inflammation and cell death, important processes in renal IRI, but the goal of this R21 is limited to optimization of the effect of siRNA knockdown. siRNA delivery in the clinic is currently limited by incomplete extent and short duration of knockdown, by inability to efficiently target the cells of interest and by effects of inappropriate cellular targets. Here, we propose proof-of-principle studies to demonstrate that antibody- conjugated, degradable polymer nanoparticles (NP) can be designed to overcome these limitations. Our specific aims are: (1) to optimize formulation of multifunctional, degradable NPs using both established and novel degradable polymers for siRNA loading, siRNA sustained release and mAb conjugation; (2) to identify the best EC specific mAb or combination of mAb for binding and uptake of conjugated NP by cultured human EC under flow in vitro at Yale and in deceased donor kidneys undergoing EVNP, the latter to be performed by our collaborators at the University of Cambridge with resources independent of this R21 application; Successful completion of these aims will provide proof-of-concept for an innovative approach to reduce allograft rejection by ex vivo targeting the graft with novel drug delivery vehicles, sparing the host from the need to excessive immunosuppression.