Application of pulsed focused ultrasound (pFUS) to deliver engineered therapeutic exosomes for the treatment of ischemic stroke

我们已经显示了内皮祖细胞(EPC)来源的朴素外体的增强递送到 使用脉冲聚焦超声(PFUS)治疗中风，不会对周围脑组织造成损害。 研究的初步结果还表明，EPC外切体的增强递送(由pFUS)导致 与EPC相比，EPC在28天内显著减少了每搏量并改善了脑血流量 Exosome只治疗动物。因此，无纳米气泡的pFUS可以作为一种增强 在卒中部位注射静脉注射的外切体。 我们已经开发出一种平台，可以使用非肿瘤的HEK293细胞制造工程化的外切体 并表达特定的细胞靶向多肽和治疗性探针。这项提议的目标是 利用我们的平台开发携带神经元特异性狂犬病病毒糖蛋白(RVG)的工程化外切体 多肽作为中枢神经系统的靶向配体并携带靶向治疗探针， 脑红蛋白(NGB)，保护神经元免受缺氧/缺血和氧化应激相关的损伤 缺血性中风。我们假设携带靶向RVG多肽和RVG多肽的工程外体 治疗性NGB蛋白可以被输送到缺血性中风的部位，而这些外切体的输送将 使用pFUS加强治疗，以改善卒中后外显体治疗的益处。假设将是 测试的具体目标如下： 具体目的1：研究工程化外切体的特性及体内分布 在有或没有pFUS的卒中区域静脉(IV)注射外切体。 将对工程外切体的特性进行研究，以确定其大小、Zeta电位和 根据我们发表的技术，在管腔表面或管腔内存在多肽和蛋白质 方法：研究方法。那么携带NGB的外体在表面或在管腔±pFUS中的生物分布将是 用SPECT-CT在卒中部位用In-111标记的各自外显体测定。我们还将 确定含有或不含有RVG多肽的外切体的分布。 具体目标2：研究工程外切体交付后卒中的康复情况 携带NGB，有或没有pFUS。 Exosome将在中风后24小时由pFUS提供；中风的恢复将使用MRI来确定，并且 随后将进行一系列短期和长期行为结果测试，然后是免疫组织化学 在脑组织样本上。

We have shown enhanced delivery of endothelial progenitor cell (EPC) derived naïve exosomes to the sites of stroke using pulsed-focused ultrasound (pFUS) without causing damage to the surrounding brain tissues. Preliminary results from the study also showed that enhanced delivery of EPC exosomes (by pFUS) caused significantly decreased stroke volume and improved cerebral blood flow over 28 days compared to that of EPC exosomes only treated animals. Therefore, pFUS without nanobubbles can be used as a method to enhance the delivery of IV-administered exosomes at the site stroke. We have developed a platform to make engineered exosomes using non-tumorous HEK293 cells that carry and express specific cell-targeting peptides as well as therapeutic probes. The objectives of this proposal are to develop engineered exosomes using our platform to carry neuron-specific rabies virus glycoprotein (RVG) peptides as targeting ligands for the central nervous system and to carry a targeted therapeutic probe, neuroglobin (Ngb), to protect neurons from hypoxic/ischemic and oxidative stress-related insults following ischemic stroke. We hypothesize that engineered exosomes carrying both targeting RVG peptides and therapeutic Ngb protein can be delivered to the site of ischemic stroke and the delivery of these exosomes will be enhanced using pFUS to improve the benefits of post-stroke exosome therapy. The hypothesis will be tested with the following specific aims: Specific Aim 1: Investigate the characteristics of the engineered exosomes and in vivo biodistribution of intravenously (IV) administered exosomes in stroke areas with or without pFUS. Characteristics of the engineered exosomes will be investigated to determine the size, zeta potential, and the presence of peptide and protein on the surface or inside the lumen using techniques as per our published methods. Then biodistribution of exosomes carrying Ngb on the surface or in the lumen ±pFUS will be determined using In-111-tagged respective exosomes at the sites of stroke by SPECT-CT. We will also determine the distribution of exosomes with or without RVG peptides. Specific Aim 2: Investigate the recovery from a stroke following delivery of engineered exosomes carrying Ngb with or without pFUS. Exosomes will be delivered by pFUS after 24hrs of stroke; stroke recovery will be determined using MRI, and a battery of short- and long-term behavioral outcome tests will be performed followed by immunohistochemistry on brain tissue samples.