title: Utilization of modified surfactant-associated protein B for delivery of DNA to airway cells in culture.
abstract: Pulmonary surfactant lines the airway epithelium and creates a potential barrier to successful transfection of the epithelium in vivo. Based on the functional properties of pulmonary surfactant protein B (SP-B) and the fact that this protein is neither toxic nor immunogenic in the airway, we hypothesized that SP-B could be modified to deliver DNA to airway cells. We have modified native bovine SP-B by the covalent linkage of poly(lysine) (average molecular mass of 3.3 or 10 kDa) to the N terminus of SP-B and formed complexes between a test plasmid and the modified SP-B. Transfection efficiency was determined by transfection of pulmonary adenocarcinoma cells (H441) in culture with the test plasmid pCPA-RSV followed by measurement of activity of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfections were performed with DNA.protein complexes using poly(lysine)10kDa-SP-B ([Lys]10kDa-SP-B) or poly(lysine)3.3kDa-SP-B ([Lys]3.3kDa-SP-B), and results were compared with transfections using unmodified poly(lysine).DNA, unmodified SP-B.DNA, or DNA only. For [Lys]10kDa-SP-B.pCPA-RSV preparations, CAT activity was readily detectable above the background of [Lys]3.3kDa-SP-B or unmodified SP-B. The SP-B-poly(lysine) conjugates were effective over a broad range of protein-to-DNA molar ratios, although they were optimal at approximately 500:1-1000:1. Transfection efficiency varied with the tested cell line but was not specific to airway cells. Addition of replication-defective adenovirus to the [Lys]10kDa-SP-B.pCPA-RSV complex enhanced CAT activity about 30-fold with respect to that produced by the [Lys]10kDa-SP-B.pCPA-RSV complex alone. This increase suggests routing of the adenoviral.[Lys]10kDa-SP-B.pCPA-RSV complex through an endosomal pathway. Effects of covalent modification on the secondary structure of SP-B were examined by Fourier transform infrared spectrometry (FTIR). Results of FTIR indicated that the conformation of [Lys]10kDa-SP-B was comprised primarily of alpha-helical structure compared with a predominantly aggregated structure of unmodified poly(lysine). We conclude that poly(lysine) conjugates of SP-B effectively deliver DNA in vitro and may have utility as DNA delivery vehicles to the airway in vivo.
authors.affiliation: Department of Pediatrics, Medical University of South Carolina, Charleston 29425-3313.
authors.forename: J E
authors.initials: JE
authors.lastname: Baatz
authors.forename: M D
authors.initials: MD
authors.lastname: Bruno
authors.forename: P J
authors.initials: PJ
authors.lastname: Ciraolo
authors.forename: S W
authors.initials: SW
authors.lastname: Glasser
authors.forename: B R
authors.initials: BR
authors.lastname: Stripp
authors.forename: K L
authors.initials: KL
authors.lastname: Smyth
authors.forename: T R
authors.initials: TR
authors.lastname: Korfhagen
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001980:Bronchi / Q000166:cytology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004274:DNA, Recombinant / Q000494:pharmacology*; D003673:Defective Viruses / Q000235:genetics; D004337:Drug Carriers / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006801:Humans; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D011107:Polylysine / Q000494:pharmacology*; D011487:Protein Conformation; D011510:Proteolipids / Q000494:pharmacology*; D011663:Pulmonary Surfactants / Q000494:pharmacology*; D012136:Respiratory Syncytial Viruses / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D004274:DNA, Recombinant; D004337:Drug Carriers; D010714:Phosphatidylethanolamines; D011510:Proteolipids; D011663:Pulmonary Surfactants; D011107:Polylysine; C020888:1,2-dielaidoylphosphatidylethanolamine; D015500:Chloramphenicol O-Acetyltransferase