Design and development of miRNA and stem cell based micro/nano systems as lung disease therapy
Creators: name: Alhasan, Layla Mehdi
Publisher: name: RMIT University
Year: 2024

description: Patients suffering from lung diseases are often treated with lung transplantation, which is considered a definitive therapy offered for patients with end-stage lung failure. Because of this, it improves survival rates besides boosting the quality of life for lung transplant recipients. However, lung transplantation suffers from a number of flaws, mainly a scarcity of organs and tissue donors. In this thesis, three approaches were attempted to address these issues. These were gene therapy, microfluidic -device -assisted stem cell delivery and tissue engineering. MicroRNA (miRNA) is emerging as a new class of gene therapeutics with the promise of healing many diseases. The applications of miRNA often require miRNA to be transfected efficiently inside cells. Chapter 3 of this work is particularly devoted to investigating the ability of a liquid marble (LM) miniature bioreactor to enhance miRNA transfection. To achieve this, suspended A549 lung cancer spheroids were first generated using liquid marble (LM) by taking advantage of the LM non-adhesive shell, thus promoting the cell-to-cell attachment that is essential for spheroid formation. Next, a tumour suppressor (miR-126) was transfected to spheroids inside LM. It was observed that the resulting miR-126 expression and the subsequent down-regulation in VEGF-A expression reached a level that cannot be reached using conventional methods, such as transfecting miR-126 to monolayer cells or transfecting miR-126 to spheroids generated using liquid suspension methods. There are multiple possible reasons for the greater miRNA expression observed in the LM bioreactor. First of all, miRNA is much smaller than many chemotherapeutic drugs and thus penetrated well into spheroids. Secondly, the PTFE shell possesses ultra-low surface free energy and so does not absorb and waste any miRNA through non-specific adsorption. Lastly, the hydrophobic interface present on the LM shell may play a role in promoting the movement of hydrophilic miRNA towards the centre of the LM where spheroids resided. Overall, this study demonstrates that LM can serve not only as a platform that produces tumour-like spheroids, but also as an efficient microbioreactor vessel that enhances miRNA transfection and outperforms conventional transfection vessels.<br><br>As part of this approach, a novel micro-centrifugation device was also developed to generate multicellular spheroids, as shown in Chapter 4, these spheroids are useful for lung cancer behaviour and drug screening studies. Intense acoustically driven micro-centrifugation flows were employed to enhance the assembly of multicellular spheroids in the microwells of a tissue culture plate. This ability to interface microfluidics with commonly used tissue culture plasticware is a significant advantage, mainly because it can be multiplied for high-throughput operation and allows the retention of existing analytical equipment designed to fit current laboratory formats. The micro-centrifugation flow induced in microwells coated with a low adhesive hydrogel rapidly enhanced the concentration of BT-474 cells, resulting in tight aggregates within a minute. This was considerably faster than the conventional hanging drop and liquid overlay methods, which typically require a day to maintain their viability. <br><br>Despite the promise of stem cell therapy for lung therapeutics and repair, there are very few viable means for directly delivering stem cells to locally target the respiratory airways via inhalation. This is not surprising given the significant challenges in aerosolising stem cells, particularly given their susceptibility to damage under the sizable stresses involved in the nebulisation process. The current study presents promising results using a microfluidic acoustic nebulisation platform in Chapter 5. This platform is not only low-cost and portable, but its high MHz order frequencies were effective in preserving the structural and functional integrity of mesenchymal stem cells (MSCs) during the nebulisation process. This was verified through an assessment of the viability, structure, metabolic activity, proliferation ability and genetic constitution of the nebulised MSCs using a variety of assays that included: cell viability staining, flow cytometry, reverse transcription, quantitative polymerase chain reaction and immuno-phenotyping. Given the novelty of inhaled stem cell therapy, comparisons with other delivery methods are difficult at present due to lack of comparable studies. More data for benchmarking are likely to become available in the near future due to the increasing interest in stem cell therapy, given the absence of viable alternative treatment régimes for respiratory ailments to date. Nonetheless, the results in this work provide compelling evidence that the SAW nebulisation platform, with its inherent benefits of low cost and portability, constitutes an attractive tool for the delivery of stem cells via inhalation for the treatment and repair of lung function.<br><br>In Chapter 6, a novel tissue scaffold composed of enzymatically cross-linked gelatin was developed. Essentially, mESCs were encapsulated in this scaffold simultaneously with the scaffold being cross-linked. A novel injectable hydrogel system composed of gelatin-3,4-dihydroxyhydrocinnamic acid (Gtn-DHHA) conjugates were cross-linked rapidly by laccase-mediated oxidation to form a hydrogel tissue scaffold which could simultaneously immobilise mESCs. This scaffold system supported the proliferation and differentiation of mESCs into lung epithelial cells in both the presence and absence of growth factors. The stiffness of the hydrogels was readily modulated by altering polymer precursor concentration. Gtn-DHHA hydrogel was maintained in air-liquid interface (ALI) conditions to mimic the lung micro- environment and mESC differentiation was evaluated by measuring the expression of E-cadherin and Foxa2, both of which are considered to be definitive endoderm markers. Furthermore, the expression of Pept2, Sema4F, Unc5b, and Ttf1 in hydrogel-encapsulated cells was assessed using the quantitative polymerase reaction (qPCR) and immunofluorescence assays. The expression of these markers was up-regulated in the presence and absence of growth factors, in particular under air-liquid interface by comparison with submerged and control cultures. This scaffold facilitated mESC differentiation into alveolar epithelial cells, even without growth factors, and is therefore a promising approach for lung repair.<br><br>In summary, this thesis first presents a novel, effective method—namely liquid marble— for better generation of multicellular spheroids to enhance miR-126 PEI nanoparticles transfection. It then introduces a novel method called a SAW device, which is also used to produce multicellular spheroids, and to generate MSC-laden aerosols to cure small lung damage. Finally, it presents a novel three-dimensional (3D) biodegradable scaffold as an alternative material for an extracellular matrix (ECM) for improved stem cell survival, proliferation, and differentiation. This is considered to be a critical step for in-vitro differentiation of ESCs into lung epithelial cells for lung repair and regeneration.