Restriction lift date: 9999-12-31
Processing, formulation and delivery strategies to ensure stable and efficacious siRNA medicines
Loading...
Files
Date
2026-05-21
Authors
Neary, Michael T.
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
Small interfering ribonucleic acid (siRNA) has significant potential for treating and preventing diseases with currently unmet clinical needs. Accordingly, considerable research and development has been conducted in the field of therapeutic siRNA culminating with the approval of the first siRNA product, Onpattro® in 2018. Nevertheless, therapeutic siRNA and its delivery vectors such as lipid nanoparticles (LNPs) present numerous challenges, not least their inherent instability. Hence, difficulties are encountered during both production of siRNA medicines and their administration to patients. The overarching aim of this thesis was to evaluate a selection of techniques for processing and delivering siRNA and to elucidate their impact on siRNA formulation stability and efficacy.
This research was initiated in Chapter 2 by focusing on microfluidics which was utilised throughout the thesis for combining siRNA with lipids to fabricate siRNA nanocarrier delivery systems. An in-depth literature review on nebulised RNA for pulmonary delivery helped to establish the objectives and experimental design in subsequent Chapters 3 and 4. The interplay between nebulised aerosol characteristics and siRNA nanocarrier properties was investigated by aerosolising a range of siRNA formulations with two Aerogen® Pro nebulisers with different volume median diameters (VMD). Nebulisation studies were further progressed with the novel application of a lipid-polymer hybrid formulation based on an ionisable amino-polyester (APE-LNPs) for nebulised siRNA delivery. Drop-on-demand piezoelectric inkjet printing (PIJP) was the key technique explored in Chapter 5 where it was used as a controlled method for dispensing droplets of siRNA. This dispensing technology was further utilised in Chapter 6 through integration with freeze-drying to develop a novel jet freeze-drying (jet FD) process for generating lyophilised siRNA microparticles. In parallel, conventional vial-based freeze-drying of siRNA-LNPs was also performed.
The results in Chapter 2 showed that the microfluidics process could be optimised via adjustment of influential mixing parameters for generating siRNA lipoplexes and LNPs used in subsequent nebulisation studies. Increasing lipid concentration in the organic phase was found to be a preferable means of scaling up output of stable siRNA nanocarriers rather than increase the batch volume.
In Chapter 3 it was noted that the aerosol performance i.e., emitted droplet size distribution was consistent across siRNA nanocarrier formulations and this was observed with both the High and Low VMD devices. Droplet sizing and cascade impaction results exhibited potential for achieving siRNA deposition in the lungs. Both nebuliser devices had a similar impact on siRNA nanocarrier physicochemical stability manifesting as aggregation in lipoplexes and reduced siRNA encapsulation efficiency (EE%) in LNPs. Additionally, formulation selection was generally more influential towards in vitro gene knockdown rather than the impact of nebulisation.
The instability of the siRNA lipoplexes and LNPs during nebulisation partly motivated Chapter 4’s work which applied hybrid APE-LNP nanocarriers for nebulised delivery of siRNA. The APE-LNPs showed notable physical resistance against loss of encapsulated siRNA induced by the nebuliser’s vibrating mesh. Further, droplet sizes suitable for inhalation and low cytotoxicity and high gene knockdown in A549 alveolar cells were achieved which overall indicates encouraging potential of APE-LNPs as a delivery vector for nebulised siRNA.
Chapter 5’s studies involving PIJP were performed to ascertain the impact of dispensing parameters on naked siRNA and siRNA-LNPs. The results showed that siRNA-LNP stability during dispensing was highly dependent on operating voltage as much greater instability was observed with lower actuation voltage. Regarding naked siRNA, PIJP-induced degradation was extensive at an siRNA concentration of 1 µg/mL however, this effect was not apparent at higher concentrations.
In Chapter 6, the novel jet FD method was successfully developed to fabricate lyophilised microparticles loaded with siRNA-LNPs. Spherical microparticles with a highly porous morphology were generated and were comparable in size to jetted droplets. Post reconstitution, siRNA-LNP properties were similar across samples prepared by the novel jet FD method and conventional lyophilised technique showing preservation of high siRNA EE% and siRNA integrity while critically, gene silencing activity was also maintained. Solid-state characterisation confirmed that the trehalose cryoprotectant used in the lyohilised samples was in an amorphous form, however, further freeze-drying cycle optimisation may be required to reduce residual moisture content.
In conclusion, this thesis makes a significant contribution to the knowledge surrounding therapeutic siRNA specifically, its processing, formulation strategies and delivery via nebulisation. Although, the potential for lung deposition was demonstrated across different siRNA formulations, nebulisation had a clear destabilising effect on siRNA nanocarriers, including both lipoplexes and LNPs. In this regard, the APE-LNPs are highly promising as a more robust siRNA delivery system, maintaining high transfection capability. Separately, the novel jet FD demonstrates considerable potential for processing siRNA formulations into a more stable lyophilised form, potentially enhancing their long-term storage and handling properties. Collectively, these findings offer numerous opportunities for further research involving siRNA therapeutics.
Description
Keywords
siRNA , Nanocarriers , Microfluidics , Nebulisation , Piezoelectric inkjet printing , Freeze-drying , Jet freeze-drying
Citation
Neary, M. T. 2026. Processing, formulation and delivery strategies to ensure stable and efficacious siRNA medicines. PhD Thesis, University College Cork.