Date of Award

11-2011

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Sally-Ann Cryan

Second Supervisor

Dr Caroline Jefferies

Keywords

Lung Diseases, RNA Interference, Gene Therapy

Abstract

RNA Interference (RNAi) allows specific and potent knockdown of target genes and interest now lies beyond its use as a molecular biology tool and in its potential as a therapeutic to mediate gene silencing in diseased cells. Targeted local delivery of small interfering RNA (siRNA) to the lungs via inhalation offers a unique opportunity to treat a range of previously unbeatable or poorly controlled respiratory conditions. Alveolar macrophages are the first line of defence against inhaled toxins and pathogens and are essential for the initiation of the inflammatory response. Targeting these cells provides a means of manipulating the immune response of the lungs for the treatment of diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and asthma. However, macrophages are a difficult cell type to transfect. Consequently a delivery system that will enhance uptake as well as specifically target alveolar macrophages would be beneficial for the treatment of respiratory inflammatory conditions and has received notable attention in recent years. Herein we have developed targeted liposomes and microparticles (MP) suitable for inhalation for optimal siRNA delivery to alveolar macrophages.

Anionic and mannosylated liposomes and uncoated and gelatin coated poly(lactic-co-glycolic acid) (PLGA) microparticles targeted macrophages via scavenger receptors (SRs), mannose receptors (MRs) and size and charge related phagocytosis, respectively. Mannosylated cholesterol analogues Mann-C2-Chol, Mann-C4-Chol and Mann-C6-Chol, differing in linker lengths, were synthesised and incorporated into neutral liposomes. Formulations of liposomes and microparticles were optimised for efficient siRNA encapsulation and screened for uptake, toxicity and immunogenicity in vitro using high content cell analysis (HCA) methods that were specifically developed. HCA determined uptake of targeted anionic 1,2-dioleoyl-sn-glycero-3- phospho-L-serine (DOPS) and mannosylated (Mann-C6-Chol) composed liposomes between 200 and 400nm and uncoated PLGA microparticles to be optimal in macrophage cells.

Significant knockdown of tumour necrosis factor-alpha (TNFa) in lipopolysaccharide (LPS) stimulated cells was mediated via DOPS liposomes and uncoated PLGA microparticles. Additionally, mannosylated liposomes appeared to activate macrophage mannose receptors in a concentration and linker dependent manner and reduce inflammation. In general liposomes and microparticles were non-toxic and non-immunogenic compared to positive controls. However exceptions included high doses of DOPS liposomes which significantly reduced cell viability in RAW 264.7 cells, significantly increased nuclear factor kappa B (NFkB) activity and induced pro-inflammatory cytokines in differentiated THP-1 cells after 24 hours. However, mannosylated liposomes induced a potent inflammatory response in vivo but effects were localised to the lungs, in vivo reductions of TNFa in bronchoalveolar lavage fluid (BALF) following LPS challenge were observed in mice treated with TNFa targeted naked siRNA and encapsulated in mannosylated liposomes.

Creative Commons License

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

File Size

6.78 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2011.

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