Fibrin-Based Nanoparticles as a Novel Sealant for Vascular Anastomosis

项目摘要 血管吻合术是一种重要的外科技术，其中紧密间隔的缝线用于连接血管。 这个过程经常用于器官移植，创伤修复和心血管手术。然而，血管 吻合术是耗时的并且与严重的并发症和长的恢复时间相关。纤维蛋白胶的应用 吻合术改善了手术效果，缩短了手术时间，但这些胶水通常不实用 因为它们的浓缩配方可以产生高密度凝胶，工作时间短，细胞渗透率低， 存储限制。我们已经开发了纤维蛋白为基础的纳米粒子（FBN），我们已经用来提供生长因子 促进体内愈合。与传统的纤维蛋白胶不同，FBN是预聚合的， 纤维蛋白/凝血酶浓度。由于它们的胶体结构和上述性质，FBN表现出可调的 凝胶化，增加的细胞浸润，室温储存和增强的药物递送能力-包括 成纤维细胞生长因子2（FGF 2），一种已知的血管修复刺激物。本提案的目的是优化， 具有可调生长的FBN外科密封剂的可涂敷和贴片制剂的表征和体内分析 因子释放预期这些FBN密封剂将表现出比当前纤维蛋白胶更好的功能性， 更长的工作时间、延长的室温稳定性、靶向生长因子递送和改善的 治愈的结果。目的1优化FBN贴片和流动性胶的配方。这将通过 表征FBN浓度、凝血酶浓度和所用辅因子的变化对 胶水的力学和功能。测试将探测聚合和降解动力学、结构， 机械性能、密封能力和安全特性。使用的方法包括流变仪、显微镜、机械 测试和新的离体和微流体测定。目标2将优化FBN对FGF 2的负载/释放， 在体外表征FGF 2-FBN密封剂对血管伤口愈合的作用。FGF 2负载效率和释放 将测定FBN密封剂的特性，并与高密度散装纤维蛋白胶进行比较。内皮细胞和成纤维细胞 划痕试验和伤口闭合试验将用于评估体外愈合结果。目标3将比较FBN 制剂（凝胶和贴剂;未加载和加载有FGF 2）与当前纤维蛋白胶的比较，使用体内兔模型， 颈动脉吻合术血管造影将用于表征取样组织的血管形态和组织学 将用于评价愈合、再狭窄和增生的体征。这个建议的使用FBNs将导致一个新的 具有改善的工作时间和可调的药物递送特性的外科密封剂，其具有上级的伤口愈合性能， 更快的手术时间、更少的并发症和更好的恢复。这项技术还将增加可访问性 通过消除冷链要求，使其在各种全球卫生应用中的使用更加开放。培训 本项目所需的相关研究技术和临床技能将有助于申请人的成功 在她职业生涯的下一个阶段，她将成为一名独立的医生科学家。

PROJECT SUMMARY Vascular anastomosis is an important surgical technique whereby closely spaced stitches are used to connect blood vessels. This process is used frequently in organ transplantation, trauma repairs, and cardiovascular surgery. However, vascular anastomosis is time consuming and associated with serious complications and long recovery times. Use of fibrin glue in anastomosis has led to improved surgical outcomes and shorter operating times, but these glues are frequently impractical as their concentrated formulations create high-density gels with short working times, low cellular infiltration, and cold storage limitations. We have developed fibrin-based nanoparticles (FBNs) which we have used to deliver growth factors and promote healing in vivo. Unlike traditional fibrin glues, FBNs are pre-polymerized and use physiologically relevant fibrin/thrombin concentrations. Owing to their colloidal structure and the aforementioned properties, FBNs exhibit tunable gelation, increased cellular infiltration, room temperature storage, and enhanced drug delivery capabilities – including of fibroblast growth factor 2 (FGF2), a known stimulant of vascular repair. The objective of this proposal is the optimization, characterization and in vivo analysis of paintable and patch formulations of an FBN surgical sealant with tunable growth factor release. It is expected that these FBN sealants will demonstrate better functionality than current fibrin glues, with the benefit of longer work time, extended stability at room temperature, targeted growth factor delivery, and improved healing outcomes. Aim 1 will optimize the formulation of patch and flowable FBN glues. This will occur through characterization of the effects of changes in FBN concentration, thrombin concentration, and cofactor used, on the mechanics and functionality of the glues. Testing will probe polymerization and degradation dynamics, structure, mechanical properties, sealant ability, and safety profile. Modalities used will include rheometry, microscopy, mechanical testing, and novel ex vivo and microfluidic assays. Aim 2 will optimize the loading/release of FGF2 by FBNs and characterize the effects of FGF2-FBN sealants on vascular wound healing in vitro. FGF2 loading efficiency and release characteristics of FBN sealants will be determined and compared to high-density bulk fibrin glues. Endothelial and fibroblast scratch tests and wound closure assays will be used to assess healing outcomes in vitro. Aim 3 will compare FBN formulations (gel and patch; unloaded and loaded with FGF2) to current fibrin glues using an in vivo leporine model of carotid artery anastomosis. Angiography will be use to characterize vascular morphology and histology of sampled tissues will be used to evaluate signs of healing, restenosis, and hyperplasia. This proposal’s use of FBNs will lead to a novel surgical sealant with improved work time and tunable drug delivery profiles that boasts superior wound healing – allowing for faster operating times, fewer complications, and improved recovery. This technology will also increase the accessibility of surgical glues by removing cold-chain requirements, opening up their use in varied global health applications. Training in the associated research techniques and clinical skills required for this project will contribute to the success of the applicant during the next phases of her career on the path to becoming an independent physician scientist.