Anti-catabolic drug anchored cationic exosomes for cartilage targeting and repair

项目摘要 骨关节炎（OA）与严重的关节疼痛、炎症和慢性软骨退化相关。 间充质干细胞（MSC）衍生的外泌体正在成为OA的有希望的治疗剂，因为它们携带 蛋白质和遗传物质，诱导再生过程，如细胞迁移，增殖，分化 矩阵合成它们在跨多种关节组织和细胞类型的生物和运输串扰中的作用， 但仍不清楚。此外，外泌体脂质双层的负电荷阻碍了它们的渗透 带负电荷的软骨软骨的高负固定电荷密度提供了独特的 利用静电相互作用来增强组织内转运、摄取和保留的机会 使它们带正电。我们设计了一种两亲性的软骨穿透阳离子 肽（CP），由于其最佳电荷，可以迅速扩散通过整个组织厚度，被摄取 细胞，并在健康和关节炎软骨中长时间结合。该项目将工程 软骨靶向MSC-外泌体，锚定CP和抗分解代谢OA生物制剂IL-1 Ra（IL-1 抑制剂）。目前，广泛的基因工程方法用于生产 定制的外泌体封装生物制剂，这可能会损害其内在组成，使其 临床翻译综合症该项目将使用简单的一步合成法将CP和IL-1 Ra接枝到 外泌体脂质双层。因此，CP-外泌体可以使用软骨作为药物仓库和靶细胞，从而增强软骨细胞的增殖。 最佳负载的IL-1 Ra对其受体的可用性，同时保留其内在的治疗潜力。 目的1构建CP移植MSC-外泌体（CP-Exo）并研究其在软骨内的转运特性 在健康和关节炎的状态下。使用细胞因子跨多种细胞类型的转运串扰和摄取 将研究挑战的软骨细胞和滑膜细胞共培养物，以了解它们的治疗效果。 优点来自软骨或滑膜靶向或两者。目的2合成融合IL-1 Ra的重组脂质体 其将以不同密度锚定在外泌体双层上以形成混合载体IL-1 Ra-CP-Exo。其 将使用细胞因子激发的软骨-滑膜外植体共培养物评价生物活性，并与 游离IL-1 Ra和未修饰的外来体。目标3将描述关节动力学、软骨内摄取和 CP-Exo在健康和损伤的大鼠膝关节中的生物分布，以及IL 1-Ra-CP-Exo在抑制 将使用创伤后OA的大鼠模型评价损伤诱导的分解代谢信号传导。该项目铺路 利用外泌体的内在治疗潜力进行软骨修复以及其可定制的方法 开发作为药物载体，允许可调节的软骨内运输特性，易于药物锚定 和抗体的可控装载。这个项目的成功 可以使外泌体作为无细胞、非免疫原性的药物递送平台快速临床翻译， 软骨和其他带负电荷的组织，如半月板、椎间盘、眼睛等。

Project Summary Osteoarthritis (OA) is associated with severe joint pain, inflammation, and chronic cartilage degeneration. Mesenchymal stem cells (MSCs) derived exosomes are emerging as promising therapeutics for OA as they carry proteins and genetic materials that induce regenerative processes like cell migration, proliferation, differentiation and matrix synthesis. Their role in biological and transport crosstalk across multiple joint tissues and cell types, however, remains unclear. Additionally, the negative charge of exosome lipid bilayer hinders their penetration into the negatively charged cartilage. The high negative fixed charge density of cartilage offers a unique opportunity to utilize electrostatic interactions to enhance intra-tissue transport, uptake, and retention of exosomes by making them positively charged. We have designed an amphipathic cartilage penetrating cationic peptide (CP) that can rapidly diffuse through full tissue thickness due to their optimal charge, be up-taken by cells, and bind within for extended periods in both healthy and arthritic cartilage. This project will engineer cartilage targeting MSC-exosomes anchored with CPs and with an anti-catabolic OA biologic, IL-1Ra (IL-1 inhibitor) in optimal concentrations. Currently, extensive genetic engineering approaches are used to produce customized exosomes encapsulating biologics, which may compromise their intrinsic composition making their clinical translation complex. The project will use a simple one-step synthesis of grafting CP and IL-1Ra on exosome lipid bilayer. CP-exosomes can thus use cartilage as a drug depot and target cells thereby enhancing the availability of optimally loaded IL-1Ra to its receptors while preserving their intrinsic therapeutic potential. Aim 1 will engineer CP grafted MSC-exosome (CP-Exo) and characterize its intra-cartilage transport properties in healthy and arthritic states. Their transport crosstalk and uptake across multiple cell types using cytokine challenged chondrocyte and synovial cell co-cultures will be studied to understand whether their therapeutic benefits arise from cartilage or synovium targeting or both. Aim 2 will synthesize recombinant lipid fused IL-1Ra that will be anchored in different densities on exosome bilayer to form a hybrid vehicle, IL-1Ra-CP-Exo. Its bioactivity will be evaluated using cytokine challenged cartilage-synovium explant co-cultures and compared with free IL-1Ra and unmodified exosomes. Aim 3 will characterize joint kinetics, intra-cartilage uptake and biodistribution of CP-Exo in healthy and injured rat knees, and bio efficacy of IL1-Ra-CP-Exo in suppressing injury induced catabolic signaling will be evaluated using rat models of post traumatic OA. The project paves way for utilizing the intrinsic therapeutic potential of exosomes for cartilage repair as well as for its customizable development as a drug carrier allowing for adjustable intra-cartilage transport properties, easy drug anchoring and controllable loading of a variety of pro-chondrogenic protein drugs and antibodies. The success of this project can enable rapid clinical translation of exosomes as a cell-free, non-immunogenic platform for drug delivery to cartilage and other negatively charged tissues like meniscus, intervertebral discs, eye etc.