Design of nanofibre based adsorbent for extracellular vesicle isolation

用于细胞外囊泡分离的纳米纤维吸附剂的设计

• 批准号: 2881719
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2881719
• 关键词: Design; nanofibre; based; adsorbent; extracellular

BackgroundExtracellular vesicles (EVs) are nature's vectors to shuttle information in multicellular environments. Relatively recently attention has focused on the therapeutic potential of these entities. This has been driven by increased understanding of their biological importance and the success of other vector strategies such as viral and non-viral vectors (most notably lipid nanoparticles (LNPs)).EVs (also referred to as exosomes) are under active development in native and engineered forms. In both cases understanding of the product characteristics is challenging. EVs are complex cellular products consisting of a lipid envelope with a range of embedded proteins and carrying a cargo within. A single cell can make vesicles by multiple pathways, the one of greatest interest is the endosomal pathway (which gives rise to exosomes), but even from in one cell a population of vesicles will be produced. Understanding this heterogeneity is an unmet need.Therefore characterisation methods have to deal with a huge range of diversity alongside the complexity and labile nature of EVs.AimIn this project we will investigate the use of nanofibre based adsorption as a means to separate EV populations for use in the characterisation and manufacture of these nascent products. Current approaches used to isolate and enrich for exosomes do not produce high functional yields of EVs. They also do not differentiate the different sizes of EVs and the different types as well, e.g., exosomes, exomeres, ectosomes, microvesicles, microparticles, apoptotic bodies. This programme of research involves development of a novel method for isolation of exosomes that can address those issues by applying a specialised, high porosity material modified with affinity ligands at a high, medium, and low density in an ion exchange chromatography system. The material causes low shear so issues with high shear from other separation methods, e.g., ultracentrifugation, that damage exosomes and compromise their function will be avoided.Objectives1) Establish a model EV upstream process and associated analytics such that product mass and quality using a relevant bioassay can be reliably determined.2) Determine mechanisms of EV yield loss during bind elute IEX chromatography by understanding:a. The kinetics of loss during processing.b. The manipulation of EV structure / population to determine the EV components associated with irreversible binding.3) Determine mechanisms of product quality loss during bind elute chromatography by:a. Correlating differing purification methodologies with product loss.b. Correlating differing feed materials / EV population with product loss.c. Rational manipulation of purification conditions based on the above.

背景细胞外囊泡（EV）是在多细胞环境中传递信息的自然载体。最近，人们的注意力集中在这些实体的治疗潜力上。这是由于对其生物学重要性的更多了解以及其他载体策略（例如病毒和非病毒载体（最著名的是脂质纳米颗粒（LNP））的成功而推动的。EV（也称为外泌体）正在天然药物中积极开发和工程形式。在这两种情况下，理解产品特性都是具有挑战性的。 EV 是复杂的细胞产品，由脂质包膜和一系列嵌入的蛋白质组成，并在其中携带货物。单个细胞可以通过多种途径产生囊泡，最令人感兴趣的途径之一是内体途径（产生外泌体），但即使在一个细胞中也会产生一群囊泡。了解这种异质性是一个未满足的需求。因此，表征方法必须处理大量的多样性以及电动汽车的复杂性和不稳定性质。目标在这个项目中，我们将研究使用基于纳米纤维的吸附作为分离电动汽车群体的一种手段。这些新兴产品的表征和制造。目前用于分离和富集外泌体的方法并不能产生高功能性的 EV 产量。它们也不区分 EV 的不同大小和不同类型，例如外泌体、外泌体、胞外体、微泡、微粒、凋亡小体。该研究计划涉及开发一种分离外泌体的新方法，该方法可以通过在离子交换层析系统中应用用亲和配体以高、中、低密度修饰的专用高孔隙率材料来解决这些问题。该材料导致低剪切，因此可以避免其他分离方法（例如超速离心）的高剪切问题，这些问题会损坏外泌体并损害其功能。 目标 1) 建立模型 EV 上游工艺和相关分析，以便使用相关的产品质量和质量可以可靠地确定生物测定。2) 通过了解以下内容确定结合洗脱 IEX 层析期间 EV 产量损失的机制：a．加工过程中损失的动力学。操纵 EV 结构/群体以确定与不可逆结合相关的 EV 成分。3) 通过以下方式确定结合洗脱色谱期间产品质量损失的机制：将不同的纯化方法与产物损失相关联。将不同的饲料材料/EV 群体与产品损失相关联。在此基础上合理操控纯化条件。