title: Simulated lung transfection by nebulization of liposome cDNA complexes using a cascade impactor seeded with 2-CFSME0-cells.
abstract: The aim of this study was to devise a simulation aerosol system for quasirealistic gene transfection that could eventually be used to study the characteristics of aerosol delivery, stability, delivery efficiency, and expression efficacy of gene products. It consisted of (1) a PARI aerosol generator and PARI LL jet; (2) an Andersen cascade impactor with a calibrated vacuum pump, fitted with a glass "throat," nebulizer in which stages were seeded with pulmonary cells of interest (e.g., 2-CFSME0-); and (3) a hot room set to 37 degrees C and approximately 70% relative humidity. Cell viability remained at 95% to 99%. A prostaglandin G/H synthase (PGH)- and a human alpha 1-antitrypsin (AAT)-expressing plasmid, respectively, driven by a cytomegalovirus promoter (pCMV4-PGH, pCMV4-AAT) and a heat-insensitive placental alkaline phosphatase (PAP)-expressing plasmid driven by a Rous sarcoma virus promoter (pRSV-PAP) were employed; cationic liposomes consisted of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoylphosphatidylethanolamine (DOTMA/DOPE) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol/DOPE (DC-Chol/ DOPE). The fluorescent dye Toto-1 was used to visualize aerosol distribution and to monitor cellular uptake. Alternatively, pCMV4-PGH deposited onto impactor stages covered with nitrocellulose membranes was hybridized with random primer-32P-radiolabeled pCMV4-PGH and autoradiographed. The mass median aerodynamic diameter (MMAD) of the plasmid, liposomes, and liposome-plasmid complexes and their effect on the mass output were monitored. A majority of gene product was delivered to stages 1 through 5, corresponding to an area ranging from the pharynx to the terminal bronchi, excluding the alveolar space. A corresponding, although very low, transfection of cells with pRSV-PAP was found, with the majority of transfected cells on stages 4 and 5. The MMAD was significantly affected by the presence of the DNA constructs alone or by DNA constructs complexed with cationic liposomes; the control phosphate buffered saline (PBS) MMAD of 2.3 microns increased to 3.5 microns for DC-Chol liposomes and 4.5 microns for the DC-Chol/PGH complex; DOTMA-based liposomes and liposome DNA complexes precipitated during aerosolization. Mass output was reduced for cationic liposomes from 0.61 g/min (PBS control) to 0.35 g/min. Large plasmid (pRSV-PAP, 10.1 kb) was more rapidly degraded by aerosolization than smaller plasmid (pCMV4-AAT, 6.2 kb) although complexation with cationic liposomes provided some protection in both cases.
authors.affiliation: Advanced Therapies, Inc., Novato, California, USA.
authors.forename: H
authors.initials: H
authors.lastname: Schreier
authors.forename: L
authors.initials: L
authors.lastname: Gagné
authors.forename: J T
authors.initials: JT
authors.lastname: Conary
authors.forename: G
authors.initials: G
authors.lastname: Laurian
mesh_terms: D000336:Aerosols / Q000008:administration & dosage*; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D002138:Calibration; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000235:genetics / Q000628:therapy; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004337:Drug Carriers; D015870:Gene Expression; D015316:Genetic Therapy; D006801:Humans; D008081:Liposomes; D008168:Lung / Q000166:cytology / Q000187:drug effects; D008954:Models, Biological; D009330:Nebulizers and Vaporizers; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods*