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）衍生的幼稚外泌体向内皮祖细胞（EPC）的位点的增强递送。 使用脉冲聚焦超声（pFUS）治疗中风，而不会对周围脑组织造成损伤。 该研究的初步结果还表明，EPC外泌体的增强递送（通过pFUS）引起了 与EPC相比，在28天内显著降低每搏输出量并改善脑血流量 外泌体仅处理动物。因此，没有纳米气泡的pFUS可以用作增强纳米气泡的方法。 在中风部位递送IV施用的外泌体。 我们已经开发了一个平台，使用非肿瘤HEK 293细胞制造工程化外泌体， 并表达特异性细胞靶向肽以及治疗探针。这项建议的目的是 利用我们的平台开发工程化外泌体，携带神经元特异性狂犬病病毒糖蛋白（RVG） 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），以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肽和靶向RVG肽的工程化外泌体可能是靶向RVG肽和靶向RVG肽。 治疗性Ngb蛋白可以被递送到缺血性中风的部位 使用pFUS增强，以改善卒中后外泌体治疗的益处。假设是 测试的具体目标如下： 具体目标1：研究工程化外泌体的特征和体内生物分布 在有或没有pFUS的中风区域中静脉内（IV）施用的外来体。 将研究工程化外泌体的特征，以确定大小、zeta电位和分子量。 使用根据我们公开的技术， 方法.然后，携带Ngb的外泌体在表面上或在管腔土pFUS中的生物分布将是 通过SPECT-CT在中风部位使用In-111标记的相应外泌体测定。我们还将 确定具有或不具有RVG肽的外来体的分布。 具体目标2：研究递送工程化外泌体后中风的恢复 带有或不带有pFUS的Ngb。 外泌体将在中风24小时后通过pFUS递送;中风恢复将使用MRI确定，并且外泌体将在中风后24小时内被释放。 将进行一系列短期和长期行为结果测试，然后进行免疫组化 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.