Engineering Surface Coatings for Localized Delivery of Therapeutic Extracellular Vesicles

项目摘要 PI的长期研究目标是设计用于组织工程的细胞-材料界面， 再生医学和医疗植入物，特别关注骨再生。细胞外囊泡 (EV)在过去的十年中，治疗多种病理的基于β-受体的疗法越来越受到关注。 尽管已经显示来自各种载体系统的EV递送在临床前诱导组织再生， 然而，在治疗性EV的有效和受控递送方面仍然存在关键差距。这项建议旨在 设计表面涂层系统，促进组织工程和再生的局部EV递送 医药应用。这项研究的核心假设是，表面介导的EV递送 与推注或全身EV递送相比，调节细胞摄取和信号传导。 本课题的具体研究目标是：（1）研究静电和受体在细胞内的作用， 配体相互作用在吸附和释放电动汽车/从表面涂层;（2）调查的影响， 与推注EV输送相比，基于表面EV输送;以及（3）证明来自 表面涂覆的组织工程支架在大鼠脊柱融合模型中改善体内组织再生。EVs 将衍生自间充质干细胞/基质细胞（MSC），并表征其大小、表面电荷和 蛋白质标记物MSC-EV与由ECM蛋白组成的表面涂层之间的相互作用， 将在各种条件（pH、R-L抑制剂、盐）下分析多糖和带电聚合物 筛选）。EV摄取 效率和内吞途径将在几种细胞类型（MSC，HUVEC，HEK 293）中进行分析， 通过荧光显微镜和流式细胞术（+/-内吞途径）与推注EV递送的比较 抑制剂）。细胞粘附、增殖和血管生成分化和免疫调节也将被讨论。 体外分析。从表面涂覆的骨水泥递送的EV的体内骨形成能力和融合功效 将在大鼠后外侧腰椎融合模型中通过手动触诊、放射照相 评分、体积显微计算机断层扫描（µCT）和免疫组织化学，与未涂层相比 支架和推注输送。这项工作的结果将大大促进对材料如何 特性和基于表面的EV递送影响细胞EV摄取、粘附、增殖和分化。 此外，该提案将能够开发有效的治疗EV表面涂层， 对于用于许多不同治疗，包括软骨再生， 糖尿病伤口愈合、心肌梗塞以及肌腱和肌肉修复。

PROJECT SUMMARY The PI’s long-term research goal is to engineer the cell-material interface for applications in tissue engineering, regenerative medicine, and medical implants, with a particular focus on bone regeneration. Extracellular vesicle (EV) - based therapies have been of increasing interest in the past decade to treat a variety of pathologies. Although EV delivery from various carrier systems has been shown to induce tissue regeneration in preclinical models, there remains a critical gap in efficient and controlled delivery of therapeutic EVs. This proposal aims to engineer a surface coating system that promotes localized EV delivery for tissue engineering and regenerative medicine applications. The central hypothesis underlying this research is that surface-mediated EV delivery modulates cellular uptake and signaling in comparison to bolus or systemic EV delivery. The specific research objectives of this proposal are to: (1) Investigate the roles of electrostatic and receptor- ligand interactions in the adsorption and release of EVs to/from surface coatings; (2) Investigate the effects of surface-based EV delivery in comparison to bolus EV delivery; and, (3) Demonstrate that EV delivery from surface-coated tissue engineering scaffolds improves tissue regeneration in vivo in a rat spinal fusion model. EVs will be derived from mesenchymal stem/stromal cells (MSCs) and characterized for size, surface charge, and protein markers. Interactions between MSC-EVs and surface coatings composed of ECM proteins, polysaccharides and charged polymers will be analyzed under various conditions (pH, R-L inhibitors, salt screening) via fluorescence labeling studies and quartz crystal microbalance with dissipation analyses. EV uptake efficiency and endocytic pathways will be analyzed in several cell types (MSCs, HUVECs, HEK293s) in comparison to bolus EV delivery via fluorescence microscopy and flow cytometry (+/- endocytic pathway inhibitors). Cellular adhesion, proliferation, and angiogenic differentiation and immunomodulation will also be analyzed in vitro. In vivo bone forming capacity and fusion efficacy of EVs delivered from surface-coated scaffolds will be evaluated in the rat posterolateral lumbar fusion model via manual palpation, radiographic scoring, volumetric microcomputed tomography (µCT) and immunohistochemistry, in comparison to uncoated scaffolds and bolus delivery. Results from this work will significantly advance understanding of how material properties and surface-based EV delivery impact cellular EV uptake, adhesion, proliferation and differentiation. Additionally, this proposal will enable development of effective therapeutic EV surface-coatings that can tailored for a wide variety of scaffolds and/or implants for many different therapies, including cartilage regeneration, diabetic wound healing, cardiac infarction, and tendon and muscle repair.