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

应用脉冲聚焦超声 (pFUS) 递送工程治疗性外泌体来治疗缺血性中风

• 批准号: 10741954
• 负责人: ALI SYED ARBAB
• 金额: $ 42.35万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741954
• 关键词: Animals; Antibody Formation; Area; Biodistribution; Biogenesis; Biological; Blood - brain barrier anatomy; Brain; Brain Hypoxia-Ischemia; Carrier Proteins; Cells; Central Nervous System; Cerebrovascular Circulation; Characteristics; Chimeric Proteins; DNA; Engineering; Focused Ultrasound; Goals; Heat shock proteins; Human Anti-Mouse Antibody; Immunoelectron Microscopy; Immunohistochemistry; Indium-111; Injections; Investigation; Ischemia; Ischemic Stroke; Laboratories; Legal patent; Lesion; Ligands; Lipids; Liquid substance; Magnetic Resonance Imaging; Mediating; Membrane Proteins; Mesenchymal Stem Cells; Methods; Microbubbles; Mus; Neuronal Hypoxia; Neurons; Nucleic Acids; Outcome; Oxidative Stress; Peptides; Permeability; Pharmaceutical Preparations; Physiologic pulse; Proteins; Publishing; Recovery; Reperfusion Therapy; Reporting; Research Personnel; Resolution; Site; Small Interfering RNA; Specificity; Stroke; Stroke Volume; Surface; Techniques; Testing; Therapeutic; Tissue Sample; Toxic effect; behavioral outcome; biomaterial compatibility; brain tissue; endothelial stem cell; engineered exosomes; exosome; immunogenicity; immunoreaction; improved; in vivo; intravenous administration; nanobubble; nerve stem cell; neurobehavioral; neuroglobin; post stroke; rabies virus glycoprotein G; single photon emission computed tomography; stem cells; stroke recovery; targeted delivery; targeted treatment; technology platform; therapy outcome; white matter; zeta potential

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.

我们已经显示出增强的内皮祖细胞（EPC）衍生为幼稚外泌体到达的位置的增强 使用以脉冲为中心的超声（PFU）中风，而不会损害周围的脑组织。 该研究的初步结果还表明，EPC外泌体的增强（由PFU）引起 与EPC相比 外泌体仅处理过动物。因此，没有纳米泡的PFU可以用作增强的方法 在现场中风的静脉注册外泌体的递送。 我们已经开发了一个平台，以使用携带的无肿瘤的HEK293单元格制造工程外泌体 并表达特定的细胞靶向肽以及治疗问题。该提议的目标是 使用我们的平台开发工程外泌体来携带神经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， 神经蛋白（NGB），以保护神经元免受缺氧/缺血性和氧化应激相关的伤害 缺血性中风。我们假设携带靶向RVG肽和的工程外泌体 治疗性NGB蛋白可以传递到缺血性中风部位，这些外泌体的递送将 使用PFU可以增强中风后外泌体疗法的益处。假设将是 以以下特定目的进行测试： 特定目的1：研究工程外泌体和体内生物分布的特征 静脉注射（IV）在具有或没有PFU的卒中区域的外泌体。 将研究工程外泌体的特征，以确定尺寸，Zeta电位和 根据我们出版 方法。然后，在表面或腔内或腔内中携带NGB的外泌体的生物分布将是 使用Spect-CT在中风部位使用IN-111标记的相对外泌体确定。我们也会 确定有或没有RVG肽的外泌体的分布。 特定目的2：研究工程外泌体后，调查中风的恢复 带有或不带有PFU的NGB携带。 中风24小时后，PFU将送出外泌体；中风恢复将使用MRI确定，A 将进行短期和长期行为结果测试的电池，然后进行免疫组织化学 在脑组织样品上。