title: Therapeutic aerosol bioengineering of siRNA for the treatment of inflammatory lung disease by TNFα gene silencing in macrophages.
abstract: The development of small interfering RNA (siRNA) to silence specific genes offers a new means of understanding and treating a range of respiratory diseases, including inflammatory lung disease. The alveolar macrophage (AM) is a key component of the inflammatory process in the lungs, associated with high levels of gene expression in inflammatory lung disease and therefore an attractive target for therapeutic siRNA. Delivery of siRNA to macrophages presents a significant delivery challenge, as fully differentiated alveolar macrophages are difficult to access and transfect. In this study we engineered particles suitable for inhalation that would efficiently transfect macrophages postinhalation. The process for encapsulation of siRNA in poly(lactic-co-glycolic acid) microparticles (MPs) was optimized using a double emulsion technique, and the resulting particles were characterized for size, shape, aerosol characteristics, encapsulation efficiency, and integrity of encapsulated siRNA. The cell uptake of the siRNA-loaded microparticles was determined by flow cytometry, confocal laser scanning microscopy (CLSM), and high-content analysis (HCA) with MPs capable of transfecting up to 55% of cells. Anti-TNFα siRNA-MPs were then prepared to study the functional activity of encapsulated siRNA in LPS-stimulated macrophages as a model of inflammation. The anti-TNFα siRNA-MPs were able to decrease TNFα expression by 45% over 48 h in the differentiated human monocytic cell line THP-1 compared to negligible knockdown using commercial transfection reagents and offered significant, sustained siRNA knockdown of TNFα in primary monocytes for up to 72 h.
authors.affiliation: School of Pharmacy and §Department of Physiology, Royal College of Surgeons in Ireland , Dublin 2, Ireland.
authors.forename: Ciara
authors.initials: C
authors.lastname: Kelly
authors.forename: Awadh B
authors.initials: AB
authors.lastname: Yadav
authors.forename: Ciaran
authors.initials: C
authors.lastname: Lawlor
authors.forename: Katie
authors.initials: K
authors.lastname: Nolan
authors.forename: Joanne
authors.initials: J
authors.lastname: O'Dwyer
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Greene
authors.forename: Noel G
authors.initials: NG
authors.lastname: McElvaney
authors.forename: Neeraj
authors.initials: N
authors.lastname: Sivadas
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Ramsey
authors.forename: Sally-Ann
authors.initials: SA
authors.lastname: Cryan
mesh_terms: D000336:Aerosols; D057005:Bioengineering; D002454:Cell Differentiation; D002470:Cell Survival; D004337:Drug Carriers; D016503:Drug Delivery Systems; D004655:Emulsions; D020868:Gene Silencing; D006801:Humans; D007223:Infant; D007249:Inflammation / Q000235:genetics / Q000473:pathology / Q000628:therapy; D019344:Lactic Acid / Q000737:chemistry*; D007948:Leukemia, Monocytic, Acute / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D008171:Lung Diseases / Q000235:genetics / Q000473:pathology / Q000628:therapy; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008863:Microspheres; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D000336:Aerosols; D004337:Drug Carriers; D004655:Emulsions; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid
keywords: TNFα; aerosol; high-content cell analysis; macrophages; microparticles; siRNA