Abstract
Background
Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition that is characterised by the combination of chronic airflow obstruction and emphysema. It is the third leading cause of mortality worldwide and the fourth in Aotearoa New Zealand, with the incidence of mortality being two times higher in Māori population than that of other ethnic groups. Cigarette smoking is the most common and significant risk factor for COPD. Currently, there is no cure for COPD, with existing pharmacological and non-pharmacological therapies primarily focusing on providing symptomatic relief and reducing exacerbations. There remains an unmet need for the development of new therapeutic agents that addresses the complete pathophysiology of COPD, targeting its molecular mechanisms.
MicroRNA (miRNA) are small, non-coding ribonucleic acid (RNA) that regulates post-transcription gene-expression and have been widely linked to a range of disorders including COPD. Altered miRNAs have been identified in COPD, making them potential biomarkers and therapeutic targets. The systemic delivery of free miRNA however poses a significant challenge due to poor cellular uptake and nuclease-mediated degradation. To overcome this limitation, a nanoparticle-based formulation is a promising approach for miRNA delivery that can be formulated into inhaled dry powder formulation using the spray freeze drying method.
Purpose
The long-term aim of this research theme is to identify altered miRNA associated with the pathophysiology of COPD and to restore their expression levels by delivering miRNA mimics or inhibitors through an inhalable formulation directly to the lungs. This thesis aims to:
1. Identify altered miRNAs associated with COPD and evaluates the biomarker potential in the Aotearoa New Zealand population associated with cigarette smoking.
2. Fabricate a cationic liposome nanoparticulate delivery system for miRNA delivery.
3. Develop a spray freeze drying method and formulate a carrier-based inhaled dry powder formulation intended for pulmonary delivery of miRNA.
Methods
The thesis is divided into four experimental chapters (Chapters 2 – 5). Chapter 2, exosomes were isolated from the Aotearoa New Zealand human plasma from never smokers and smoking individuals using size exclusion chromatography. The miRNA expression of the selected miRNAs (miRNA-146a, miRNA-134 and miRNA-15b) was quantified using reverse transcription polymerase chain reaction (RT PCR) analysis to evaluate the altered expression between the two groups. Chapter 3 demonstrates the fabrication of cationic liposomes with miRNA using the microfluidics method. The miRNA-loaded cationic liposomes were characterised for their physicochemical properties and in vitro cytotoxicity and efficacy on lung epithelial cells. Chapter 4 details the development and optimisation of the spray freeze drying method for producing highly porous inhaled dry powder formulation. The influence of L-leucine and the height between the atomising nozzle and the surface of the liquid nitrogen was assessed on aerosolisation performance of ceftazidime spray freeze dried powder formulation. Chapter 5 optimises the ratio of N-acetyl-L-cysteine (NAC) and excipients, inulin and L-leucine, of a highly porous inhaled spray freeze dried formulation using response surface methodology. This includes assessing the impact on total production yield, water content and fine particle fraction.
Results
Chapter 2 identifies three miRNAs and were selected for possible biomarker potential based on the pathological changes in COPD, namely, miRNA-146a for inflammation, miRNA-134 for mucus hypersecretion and miRNA-15b for airway remodelling. The RT PCR analysis demonstrated a significant upregulation in the expression of plasma-derived exosomal miRNAs (miRNA-146a, miRNA-134 and miRNA-15b) associated with cigarette smoking. This association might be linked to the pathophysiology of COPD, and may serve as potential biomarkers in the Aotearoa New Zealand population.
Chapter 3 demonstrates the successful incorporation of miRNA into cationic liposomes using microfluidics with desired physicochemical properties having a cationic zeta potential of +25 mV, a particle size of <200 nm, uniform particle size distribution and encapsulation efficiency of 90%. The cationic liposome loaded with miRNA demonstrates enhanced transfection on lung epithelial cell lines. This work identifies that the high amount of cationic liposomes loaded with miRNA may be toxic to the lung epithelial cells and needs further investigations.
Chapter 4 demonstrates the successful method development of spray freeze drying laboratory-based setup and fabrication of an inhalable ceftazidime spray freeze dried powder formulation. The results showed that the addition of 20% w/w L-leucine in the formulation resulted in particles with highly porous, fragile and spherical morphology with improved fine particle fraction of 76% compared to without L-leucine-containing formulation (61%) while keeping the height variable constant. There was no correlation between fine particle fraction and height between the atomising nozzle and the surface of the liquid nitrogen.
Chapter 5 demonstrates that excipients played a major role in inhaled NAC spray freeze dried powder formulation, where inulin provides structural integrity and L-leucine acts as an aerosolisation enhancer. The results demonstrated the production of highly porous and good physicochemical properties (spherical morphology, water content <5%, no drug-excipient interactions) and fine particle fraction of 61%.
Conclusions
This thesis identifies altered miRNAs associated with COPD and demonstrates their potential as biomarker agents in the Aotearoa New Zealand population associated with cigarette smoking. The fabrication of miRNA-loaded cationic liposomes show potential for an effective delivery of miRNA to lung epithelial cells, however, cytotoxicity remains a concern at higher doses. The successful method development of spray freeze drying provides a workable method to produce inhalable dry powder formulations, paving the way for inhaled miRNA delivery.