Engineering Surface Coatings for Localized Delivery of Therapeutic Extracellular Vesicles

用于治疗性细胞外囊泡局部递送的工程表面涂层

• 批准号: 10719257
• 负责人: Christina Andrea Holmes
• 金额: $ 17.06万
• 依托单位: FLORIDA AGRICULTURAL AND MECHANICAL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-07 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10719257
• 关键词: 3-Dimensional; Adhesions; Adsorption; Animal Model; Architecture; Atomic Force Microscopy; Biocompatible Materials; Biological Assay; Bolus Infusion; Bone Regeneration; Cell Adhesion; Cell Communication; Cell secretion; Cells; Charge; Clinical; Confocal Microscopy; Development; Electrostatics; Engineering; Environment; Enzyme-Linked Immunosorbent Assay; Equilibrium; Extracellular Matrix; Extracellular Matrix Proteins; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Goals; Hydrogels; Immunohistochemistry; Implant; In Vitro; Label; Lateral; Ligands; Lipids; Liver Regeneration; Manuals; Mediating; Mesenchymal; Modeling; Morphology; Muscle; Myocardial Infarction; Natural regeneration; Nucleic Acids; Organ; Osteogenesis; Palpation; Pathology; Pathway interactions; Play; Polymers; Polysaccharides; Pre-Clinical Model; Proliferating; Property; Proteins; Quartz; Rattus; Regenerative Medicine; Research; Research Proposals; Reverse Transcriptase Polymerase Chain Reaction; Role; Scanning Electron Microscopy; Signal Transduction; Sodium Chloride; Spinal Fusion; Stains; Stromal Cells; Surface; System; Tendon structure; Testing; Therapeutic; Tissue Engineering; Tissues; Vesicle; Western Blotting; Work; absorption; bone; cartilage regeneration; cell type; clinical application; clinical translation; diabetic wound healing; extracellular vesicles; immunoregulation; improved; improved outcome; in vivo; in vivo regeneration; inhibitor; insight; interest; light scattering; mechanical properties; medical implant; nanoparticle; nanoscale; preclinical study; protein biomarkers; radiological imaging; receptor; regenerative therapy; repaired; scaffold; screening; stem; surface coating; therapeutically effective; tissue regeneration; tissue repair; tomography; uptake; zeta potential

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.

项目概要 PI 的长期研究目标是设计细胞-材料界面以应用于组织工程、 再生医学和医疗植入物，特别关注骨再生。 在过去的十年中，基于 EV 的疗法在治疗各种病理方面引起了越来越多的兴趣。 尽管在临床前研究中，各种载体系统的 EV 递送已被证明可以诱导组织再生 模型中，在有效和受控的治疗性 EV 输送方面仍然存在严重差距。该提案旨在 设计一种表面涂层系统，促进组织工程和再生的局部 EV 输送 这项研究的核心假设是表面介导的 EV 传递。 与推注或全身 EV 输送相比，可调节细胞摄取和信号传导。 本提案的具体研究目标是：（1）研究静电和受体的作用 (2) 研究配体相互作用对表面涂层吸附和释放EV的影响； 基于表面的 EV 输送与推注 EV 输送的比较；以及 (3) 证明 EV 输送来自 表面涂层的组织工程支架可改善大鼠脊柱融合模型的体内组织再生。 将源自间充质干细胞/基质细胞 (MSC)，并对其大小、表面电荷和特征进行表征 蛋白质标记物 MSC-EV 和由 ECM 蛋白质组成的表面涂层之间的相互作用， 多糖和带电聚合物将在各种条件下进行分析（pH、R-L 抑制剂、盐 筛选）通过荧光标记研究和石英晶体微天平与耗散分析。 将在多种细胞类型（MSC、HUVEC、HEK293）中分析效率和内吞途径 通过荧光显微镜和流式细胞术与推注 EV 递送进行比较（+/- 内吞途径 细胞粘附、增殖、血管生成分化和免疫调节也将受到影响。 体外分析了表面涂层递送的 EV 的体内骨形成能力和融合功效。 将通过手动触诊、放射照相在大鼠后外侧腰椎融合模型中评估支架 与未涂层相比，评分、体积微计算机断层扫描 (μCT) 和免疫组织化学 这项工作的结果将显着促进对材料如何进行的理解。 特性和基于表面的 EV 传递影响细胞 EV 的摄取、粘附、增殖和分化。 此外，该提案将有助于开发有效的治疗性电动汽车表面涂层，该涂层可以定制 用于多种不同疗法的支架和/或植入物，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。