Abstract
Diabetes mellitus is a chronic disease and is responsible for the development of several complications, such as diabetic wounds. Diabetic wound healing is characterized by an increase in hypoxia levels, poor angiogenesis, high inflammation levels, frequent bacterial infection and sometimes neuropathy. In order to explore a topical, targeted therapy to address this particular complication, this thesis attempted to implement the use of microRNAs (miRNAs) therapy in order to accelerate the wound healing process. miRNAs are a class of 19-25 nucleotides long, non-coding RNA molecules, involved in the regulation of post-transcriptional gene expression. Following literature review, miR-126 and miR-181b were chosen for this thesis project due to their pro-angiogenic and anti-inflammatory actions respectively. The miRNAs are often known to be delivered to their recipient cells by means of extracellular vesicles like exosomes. These exosomes, secreted by all kinds of cells, are very small in size (30-150 nm) and they also assist in cell to cell communication. Another type of delivery agent such as polymeric nanofibers have also been proven to be quite successful in the controlled delivery of therapeutic agents. However, the combined effect of miR-126 and miR-181b, administered in a controlled form on the healing of diabetic wounds have not been investigated before. Therefore, the goal of this thesis was to determine the effects of miR-126/miR-181b cocktail therapy in diabetic wound healing, facilitated by the exosome-nanofiber delivery system.
To achieve this, the first aim of this thesis was to isolate exosomes in vitro from primary HUVECs, followed by the transfection of the isolated exosomes with miR-126 and miR-181b mimics (Chapter 03). The process of exosome isolation has been varied, therefore making it imperative to optimise a suitable protocol for this particular project. Hence, three different methods were tried out for exosome isolation out of which, the ultrafiltration was then deemed to be the best suited method for exosome extraction for this thesis and was used for the rest of the project. Samples were further isolated using this process and subjected to various characterizations such as immunogold labelling and western blot analysis against some well-known markers (CD63, Alix, CD9 and Calnexin) of the exosomes. Once they were successfully characterized, the exosome samples were then transfected with miR-126 and miR-181b mimics to test for dose optimisations for the study. Transfection of the samples, followed by RT-PCR analysis revealed a significantly high level of the miR-126 and miR-181b mimics at 40 pmol dose. Moreover, a visual confirmation of the cellular uptake of these transfected exosomes was also obtained via confocal imaging. Thus, an optimized procedure for exosome isolation was successfully established and the transfection dose was also optimised in order to facilitate miRNA delivery within the endothelial cells.
The next aim of this thesis constituted the development of a stable nanofibrous scaffold in order to facilitate exosome mediated miRNA delivery within the cells (Chapter 04). Here, the electrospinning technique was applied in order to produce a nanofibrous patch that could retain and allow controlled delivery of the transfected exosomes into the target cells. Therefore, to produce such a scaffold, biomaterials like silk fibroin (SF), polyvinyl alcohol (PVA) and polylactic-co-glycolic acid (PLGA) were chosen following the literature review. The electrospinning parameters were first optimized by running two different layers (SF/PVA and PLGA) of electrospun fibers separately. The SF/PVA/PLGA fibers were then produced using the optimized electrospinning parameters, in a layer-by-layer fashion. These bilayer fibers were found to be bead-free, consistent with a uniform, average diameter of 308.3 ±14 nm when observed via SEM imaging. It was also noted that the two layers formed an integrated structure, thereby functioning as a unit. This was further supported by the Fourier Transform Infra-Red (FTIR) spectroscopy data, where slight shifts of characteristic absorption peaks for SF, PVA and PLGA fibers demonstrated a possible blend among the fibers. Additionally, this patch demonstrated a low swelling rate (around 28.6±0.8%), along with visible shrinkage in size on account of its hydrophobic nature. This shrinkage issue was overcome by implementing 3D printed, affordable polylactic acid (PLA) rings holding the edges of the patch. This resulted in significantly low degradation of the fibers, lasting up to 7 days with minor wear and tear, although gradual thinning was observed, as reflected in its loss of weight. This study therefore helped in producing a biocompatible scaffold that can be used to facilitate controlled release of miRNA-carrying exosomes into the target cells.
The final aim of this thesis was to determine the cellular uptake of the miRNA transfected exosomes and their effects on the hyperglycemic HUVECs in in vitro conditions (Chapter 05). The nanofibrous patch was incubated with the transfected exosomes and then used for further in vitro experiments to determine the effects of the miR-126 and miR-181b cocktail therapy on angiogenesis and inflammation in HUVECs. Unfortunately, the endothelial tube formation and the scratch assay were carried out with only the transfected exosomes as the presence of the patch posed a difficulty in the imaging of the cells.
From the data, it was clear that while individual treatments with miR-126 and miR-181b did not show significant effects on angiogenesis and cell migration under hyperglycemic conditions, the miRNA cocktail therapy exhibited promising results in inducing tube formation in HUVECs. This suggests a potential synergistic effect of the miRNA cocktail treatment, which warrants further investigation into the underlying mechanisms driving this enhanced response. The findings also highlighted the complex interplay between glucose levels, exosomes, and miRNA treatments in modulating endothelial cell functions. While high glucose levels were shown to impair tube formation, they paradoxically enhanced cell migration, possibly through the induction of MMPs and disruption of the endothelial cell signalling pathways. This unexpected result opens avenues for future research to better understand the intricate mechanisms involved and to optimize the miRNA cocktail therapy for enhanced wound healing and tissue repair under hyperglycemic conditions. Moving forward, expanding the sample size and fine-tuning the dosage of the miRNA cocktail treatment could provide deeper insights into its efficacy in promoting angiogenesis and cell migration. Additionally, investigating the potential role of exosomes in mediating the observed effects and elucidating the synergistic interactions between miR-126 and miR-181b will be crucial for advancing the development of innovative therapeutic strategies for vascular-related disorders. Additionally, the sample size of ELISA performed to determine the NF-κB levels in the treated HUVECs needs to be increased significantly in order to understand the effects of the treatments on this complex inflammatory mechanism.
In conclusion, it could be said that this project was the first of its kind to attempt a miR-126/miR-181b cocktail therapy on diabetic wound healing in an in vitro cell culture model. Although the effects of the cocktail therapy showed potential results only in terms of angiogenesis restoration it still needs to undergo further experimentations and optimisations in the future to be able to be implemented for diabetic wound healing treatment. This thesis, therefore, laid a foundation in exploring the possibility of the miR-126/miR-181b cocktail therapy in diabetic wound healing studies in the future.