Local targeting in inflammatory bowel disease: Investigating the suitability of drug-loaded exosomes for oral delivery

炎症性肠病(IBD)可导致患者虚弱的身体和心理社会症状，并通过失学、旷课和医疗费用影响社会。据估计，全球有超过680万人患有IBD。IBD由溃疡性结肠炎(UC)和克罗恩病(CD)组成，这两种疾病是胃肠道的两种慢性复发性疾病。在UC中，炎症是持续的和广泛的，并干扰大肠或结肠的浅层粘膜。另一方面，CD的特点是更深更不稳定的炎症，可以发生在整个消化道。一线治疗策略包括使用小分子疗法，包括5-氨基水杨酸酯、皮质类固醇、全身免疫调节剂和JAK抑制剂。然而，由于这些方法的疗效有限，人们已经投入了大量的努力来开发治疗IBD的新型生物制剂，如抗肿瘤坏死因子-α和抗整合素抗体。这些全身治疗策略的缺点是严重的、有时是致命的不良反应。这些因素，加上复杂的给药方案，导致患者依从性差，这反过来又与不良的临床结果、增加的医疗成本以及增加的住院率和复发率相关。各种纳米药物制剂已被用于提高化学和生物分子药物的疗效和安全性。然而，这些系统的临床翻译受到它们从体内的快速清除以及所用材料的细胞毒性的限制。因此，一种受到越来越多关注的内源性纳米颗粒是外体；这些外体是纳米大小的脂双层封闭的细胞外小泡，由细胞释放，将脂质、蛋白质和核酸运送到邻近细胞。与人工合成的纳米粒相比，外切体具有明显的优势，如穿透深层组织的尺寸小，免疫原性有限，长循环的Zeta电位略为负，递送效率高，具有天然的靶向能力。到目前为止，遗传、抗癌和抗炎药物已经成功地通过外切体输送。在这些情况下，外切体会提高细胞毒药物的转染率，减少其副作用，并保护这些脆弱的分子免受药物清除的影响。它们还具有负Zeta电位的特点，使它们能够与带正电荷的炎症性肠病组织相互作用，使它们成为靶向口服药物输送的理想载体。这一创新项目旨在研制载药外体，用于将药物靶向口服给药到炎症肠道组织。我们将用体积排阻层析法和差速离心法分离外切体。然后，通过动态光散射(DLS)、纳米颗粒跟踪分析(NTA)、透射电子显微镜(TEM)和Western blotting对Exosome进行表征。将完成体外和体内的转基因研究，并将评估胃肠道粘液对外切体的影响。Exosome将通过电穿孔装载药物货物，并重复进行表征分析。它们最终将在体内进行治疗效果测试。

Inflammatory bowel disease (IBD) can result in debilitating physical and psychosocial symptoms for patients and affect society through loss of schooling, absenteeism, and health-care costs. More than 6.8 million people are estimated to be living with IBD worldwide. IBD comprises of ulcerative colitis (UC) and Crohn's disease (CD), two chronic, relapsing disorders of the gastrointestinal tract. In UC, inflammation is continuous and widespread and disturbs the superficial mucosal layer of the large intestine or the colon. CD on the other hand is characterized by deeper and more erratic inflammation that can occur throughout the entire digestive tract. First-line treatment strategies involve the use of small molecule therapeutics, including 5-aminosalicylates, corticosteroids, systemic immunomodulators and JAK inhibitors. However, due to the limited efficacy of these modalities, extensive effort has been put into developing novel biologics for the treatment of IBD, such anti-TNF-alpha and anti-integrin antibodies. The drawback of these systemic treatment strategies are the serious and sometimes fatal, adverse effects. These factors, coupled with complex dosing regimens, contribute to poor patient adherence that is in turn associated with poor clinical outcomes, increased healthcare costs, and increased rates of hospitalization and relapse. Various nano-based drug formulations have been used to improve the therapeutic efficacy and safety of chemical and biomolecular drugs. However, clinical translation of these systems is limited by their rapid clearance from the body, and the cytotoxicity of the materials used. Consequently, an endogenous nanoparticle that has been receiving increasing attention is exosomes; these are nanometre-sized lipid-bilayer-enclosed extracellular vesicles, which are released by cells to shuttle lipids, proteins and nucleic acids to neighbouring cells. Exosomes have distinct advantages over synthetic nanoparticles, such as their small size for penetration into deep tissues, limited immunogenicity, slightly negative zeta potential for long circulation, high delivery efficiency and natural targeting abilities. So far, genetic, anticancer and anti-inflammatory drugs have been successfully delivered by exosomes. In these cases, exosomes enhance the transfection efficiency of cytotoxic drugs and reduces their side effects, as well as protecting these fragile molecules from drug clearance. They are also characterised by a negative zeta-potential charge which enables them to interact with the positively charged inflamed inflammatory bowel disease tissue, making them ideal carriers for targeted oral drug delivery.This innovative project aims to formulate drug-loaded exosomes for targeted oral drug delivery to inflamed bowel tissue. We will isolate exosomes by size exclusion chromatography and differential centrifugation methods. The exosomes will then be characterised by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blotting. In-vitro and in-vivo transfection studies will be completed and an assessment of the effect of gastrointestinal mucus on the exosomes will be performed. The exosomes will be loaded with a drug cargo by electroporation and the characterisation assays repeated. They will finally be tested in-vivo for therapeutic efficacy.