Nitric Oxide (NO) Loaded Gelatin Nanoparticles as Antimicrobial Therapeutics for the Treatment of Respiratory Infections

Respiratory infections (RI) represent a significant global challenge which place substantial burdens on healthcare systems. The widespread overuse and misuse of antimicrobials has led to the rise in antimicrobial resistance (AMR), which often leads to poor patient outcomes and increased mortality rates. This highlights the urgent need for novel antimicrobial therapies that can effectively treat RI, without contributing to AMR. Nitric oxide (NO) is a broad-spectrum antimicrobial that is endogenously produced by macrophages in response to pathogens. Due to its multiple antimicrobial mechanisms, NO has not been shown to contribute the emergence of AMR. However, its high reactivity represents a challenge for effective targeted delivery to infection sites. The focus of this thesis was to develop NO-releasing gelatin nanoparticles (GNP/NO), as an inhaled therapeutic for the treatment of RI. The initial stage of this research focused on the synthesis and optimisation of GNP using the 2-step desolvation method. Synthesis parameters such as pH, gelatin temperature and acetone temperature were evaluated for their impact on GNP characteristics. It was found that pH had the most significant effect on GNP characteristics. The nanoparticles were functionalised with the NO-donor, N-diazeniumdiolate to form GNP/NO and the NO release kinetics, antimicrobial and antibiofilm efficacy were investigated periodically over a 6 month period. Though total NO release did decrease over time, the particles maintained some antimicrobial efficacy against Staphylococcus aureus, Escherichia coli and Candida albicans under nutrient-poor conditions after 6 months. The feasibility of GNP/NO as an inhaled NO-delivery system was further evaluated. The aerodynamic diameter of nebulised GNP was 4.0 ± 0.3 μm, indicating the inhaled nanoparticles would be deposited within the lower respiratory lung tissue. Antimicrobial assessment of nebulised GNP/NO against two bacterial isolates commonly associated with RI, S. aureus and Pseudomonas aeruginosa demonstrated complete eradication of the bacteria in bronchoalveolar lavage supernatant. Cytocompatibility analysis also revealed GNP/NO to not be cytotoxic to human pharyngeal cells. These findings highlight the potential of GNP/NO as an alternative to traditional antibiotics for the treatment of RI, which would also help to mitigate the growing threat of AMR.