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、炎症和慢性软骨变性有关。 间充质干细胞(MSCs)来源的外切体正在成为治疗骨性关节炎的有前途的药物 诱导细胞迁移、增殖、分化等再生过程的蛋白质和遗传物质 和矩阵合成。它们在跨越多种关节组织和细胞类型的生物和运输串扰中的作用， 然而，目前仍不清楚。此外，外切体脂双层的负电荷阻碍了它们的渗透。 进入带负电的软骨。软骨的高负电荷密度提供了独特的 利用静电相互作用增强组织内转运、摄取和滞留的机会 通过使外切体带正电荷。我们设计了一种能穿透阳离子的两亲性软骨 多肽(CP)，由于其最佳电荷，可以迅速扩散到整个组织厚度，被 细胞，并在健康和关节炎软骨中长时间结合在一起。这个项目将成为工程师 以CPS和抗分解代谢的OA生物IL-1ra(IL-1)为靶向MSC-exosome的软骨 抑制剂)的最佳浓度。目前，广泛的基因工程方法被用于生产 定制的外切体封装生物制剂，这可能会损害其固有组成，使其 临床翻译复合体。该项目将使用简单的一步法合成接枝CP和IL-1ra 外切体脂双层。因此，CP-exosome可以将软骨作为药库和靶细胞，从而增强 最佳负载的IL-1ra对其受体的可用性，同时保持其固有的治疗潜力。 Aim 1将设计CP嫁接的MSC-Exosome(CP-Exo)并表征其软骨内转运特性 处于健康和关节炎状态。它们利用细胞因子在多种细胞类型之间的传输串扰和摄取 将对软骨细胞和滑膜细胞共培养进行研究，以了解它们的治疗作用 好处来自软骨或滑膜靶向或两者兼而有之。AIM 2将合成重组脂融合IL-1ra 它将以不同的密度锚定在外切体双层上，形成一种混合动力载体IL-1Ra-CP-Exo。它的 将使用细胞因子刺激的软骨-滑膜外植体共培养来评估生物活性，并与 游离IL-1ra和未经修饰的外切体。目标3将描述关节动力学、软骨内摄取和 Cp-Exo在健康和损伤大鼠膝关节中的生物分布及IL1-Ra-Cp-Exo的抑制作用 损伤诱导的分解代谢信号将使用创伤后骨性关节炎的大鼠模型进行评估。该项目铺平了道路 利用外切体的内在治疗潜力进行软骨修复及其可定制化的方法 作为药物载体的开发允许可调节的软骨内传输特性，易于药物锚定 并可控制负载多种促软骨蛋白药物和抗体。这个项目的成功 可以实现外切体的快速临床翻译，作为无细胞、非免疫原性的药物输送平台 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.