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Utilization of Modified Surfactant-Associated Protein B for Delivery of DNA to Airway Cells in Culture
John E. Baatz, Michael D. Bruno, Peter J. Ciraolo, Stephan W. Glasser, Barry R. Stripp, Kathleen L. Smyth and Thomas R. Korfhagen
Proceedings of the National Academy of Sciences of the United States of America
Vol. 91, No. 7 (Mar. 29, 1994), pp. 2547-2551
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2364271
Page Count: 5
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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-[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 α-helical structure compared with a predominately 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.
Proceedings of the National Academy of Sciences of the United States of America © 1994 National Academy of Sciences