Date of Award

2013

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Sally-Ann Cryan

Second Supervisor

Dr. James Barlow

Keywords

Lung Diseases, RNA, Small Interfering

Abstract

Local delivery of short-interfering RNA (siRNA) to the lungs represents a promising means of treating a range of pulmonary conditions such as acute lung injury (ALI) and chronic obstructive pulmonary disorder (COPD). However, effective pulmonary delivery of siRNA remains hampered by a range of host defence mechanisms. Furthermore, pharmaceutical issues that are currently impeding pulmonary siRNA therapy include cytotoxicity, inefficient rates of delivery and low levels of post-delivery siRNA stability. One strategy to overcome these issues involves encapsulation of siRNA in nanoparticles. These can potentially facilitate intracellular delivery to target cells, overcome the mucus barrier and ensure stable delivery. Successful siRNA mediated therapy for respiratory disease is also dependent, however, on integration with an effective means of delivery to the appropriate area of the lungs. Herein, we have developed a range of gene delivery vectors (GDVs) suitable for inhalation for enhanced siRNA delivery to lung epithelial cells.

A range of novel Poly(ethyleneimine)-Poly(ethylene) glycol (PEI-PEG) co-polymers, aminemodified cyclodextrin (SC12CD) and PEGylated peptidomimetic constructs (AOC95/99) were combined with siRNA to form siRNA nanoparticles. These were then characterised by developing high throughput, multi-parameter screening methods. These performed a detailed examination of siRNA uptake efficiency and cytotoxicity in Calu-3 bronchial epithelial cells. In vitro siRNA knockdown following siRNA nanoparticle delivery was assessed in undifferentiated and fully differentiated, mucus-producing Calu-3 cell culture. The effect of nébulisation on siRNA nanoparticle transfection efficiency was determined using PEILPEG(IOkDa) in an integrated in vitro device-cell culture model. Following this, the ability of PEI-LPEG(IOkDa) IL-8- siRNA nanoparticles to modulate IL-8 in vitro and in vivo were assessed following the establishment of an LPS-stimulated rat model of pulmonary inflammation.

Results of these experiments found that PEGylation of PEI, particularly 10kDa linear PEG, resulted in significant (pin vivostudies, reductions in IL-8 gene expression were observed in lung epithelial cells but were lost at a protein level. Early indications were of PEGylation being associated with pro-inflammatory effects, however there was no significant increase compared to unmodified PEI and positive controls.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

12,475 KB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2013.

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