Design of nanofibre based adsorbent for extracellular vesicle isolation

研究背景细胞外囊泡（Extracellular vesicles，EVs）是自然界中在多细胞环境中传递信息的载体。相对最近的注意力集中在这些实体的治疗潜力。这是由于对它们的生物学重要性的理解增加以及其他载体策略如病毒和非病毒载体（最值得注意的是脂质纳米颗粒（LNP））的成功所驱动的EV（也称为外来体）正在以天然和工程化形式进行积极开发。在这两种情况下，对产品特性的理解都具有挑战性。EV是复杂的细胞产物，由具有一系列嵌入蛋白质的脂质包膜组成，并在其中携带货物。单个细胞可以通过多种途径制造囊泡，最感兴趣的途径之一是内体途径（其产生外泌体），但即使在一个细胞中也会产生囊泡群体。了解这种异质性是一个未满足的需求。因此，表征方法必须处理一个巨大的范围内的多样性，以及复杂性和不稳定的性质EVs.AimIn这个项目中，我们将研究使用纳米纤维为基础的吸附作为一种手段，以分离EV人口用于这些新生产品的表征和制造。目前用于分离和富集外泌体的方法不能产生高功能产量的EV。它们也没有区分电动汽车的不同尺寸和不同类型，例如，外泌体、微泡、微粒、凋亡小体。该研究计划涉及开发一种用于分离外泌体的新方法，该方法可以通过在离子交换色谱系统中以高，中，低密度应用用亲和配体修饰的专用，高孔隙率材料来解决这些问题。该材料引起低剪切，因此与来自其它分离方法的高剪切有关，目的1）建立EV上游过程模型和相关分析，使得可以使用相关生物测定可靠地确定产物质量和质量。2）通过理解以下内容来确定结合ECOIEX层析期间EV产率损失的机制：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组分。3）通过以下方式确定结合层析期间产物质量损失的机制：a.将不同的纯化方法与产品损失联系起来。将不同的饲料材料/ EV种群与产品损失相关联。基于上述内容合理操作纯化条件。

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.