Abstract
Inflammatory bowel disease (IBD) is a chronic and progressive disorder with destructive inflammation in the gastrointestinal tract (GIT). RNA interference, which is mediated by small interfering RNA (siRNA), has been recognized as an efficient approach for downregulating the expression of tumour necrosis factor α (TNFα) at inflamed intestinal mucosa, thereby reduces inflammation and restores the damaged mucosa. Considering the site of drug action in IBD resides within the GIT itself, oral administration of medication is naturally the preferred and most efficacious choice. However, oral administration of siRNA is currently not possible since the physiological barriers in GIT pose significant challenges. Milk extracellular vesicles (mEVs), which could potentially resist in vitro digestion and possess the ability to transport across the intestinal epithelium, may serve as vehicles for oral delivery of anti-TNFα siRNA in IBD.This work focuses on the development of mEVs-based systems for oral siRNA delivery and the investigation of their potential for IBD therapy. Initially, mEVs were isolated by ultracentrifugation from bovine milk and purified by size exclusion chromatography (SEC). Expected size, remarkable yield, high purity, characteristic protein markers and typical morphology were exhibited among isolated mEVs. Thereafter, various strategies were assessed for loading siRNA into mEVs, and the most effective method (using a commercial transfection kit) achieved a loading efficiency above 20%.
The potential of mEVs as oral delivery systems for siRNA in IBD therapy was investigated in this study. The results showed that mEVs efficiently translocate across the Caco-2 intestinal epithelial model, which is not compromised by treatment with simulated intestinal fluids.
Significantly, two relevant in vitro human intestinal epithelial organoids (IEOs) models were initially created: a 3D apical-out IEO model and an IEO monolayer model. Unlike the conventional culture of IEOs whereby the apical surface is shielded in the interior of organoids, these models enabled the investigation of the apical-to-basolateral permeability of mEVs. mEVs demonstrated similar permeability through these highly human relevant models, demonstrating their potential for oral delivery. Furthermore, mEVs loaded with siRNA successfully induced (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene silencing in J774A.1 macrophages, confirming the therapeutic potential of mEVs delivery systems.
To improve the siRNA loading efficiency of mEVs, hybrid nanovesicles (‘hybridosomes’) based on mEVs and liposomes were developed in this work. Hybridosomes were fabricated using two related methods based on freeze-thawing fusion of mEVs and cationic liposomes. The systems were 180-230 nm and demonstrated efficient loading of siRNA cargo.
Hybridosomes exhibited significantly lower cytotoxicity in intestinal Caco-2 cells and superior stability in a fed-state simulated intestinal fluid compared to cationic liposomes. Furthermore, these systems significantly increased the transport of siRNA across the in vitro intestinal model, and hybridosomes loaded with GAPDH siRNA successfully induced transfection in J774A.1 macrophages. Importantly, anti-TNFα siRNA loaded-hybridosomes and -mEVs were both able to downregulate TNFα levels and relieve inflammation in an in vitro co-culture model of intestinal inflammation. In conclusion, this work demonstrates that mEVs and mEVs-mediated hybridosomes can either act as safe and effective systems for potential oral delivery of siRNA therapies in IBD.
| Date of Award | 1 May 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Driton Vllasaliu (Supervisor) & Maya Thanou (Supervisor) |