UTILIZATION OF MODIFIED SURFACTANT-ASSOCIATED PROTEIN-B FOR DELIVERY OF DNA TO AIRWAY CELLS IN CULTURE

Pulmonary surfactant lines the airway epithelium and creates a potenti al 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 th e airway, we hypothesized that SP-B could be modified to deliver DNA t o airway cells. We have modified native bovine SP-B by the covalent li nkage 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 th e modified SP-B. Transfection efficiency was determined by transfectio n of pulmonary adenocarcinoma cells (H441) in culture with the test pl asmid pCPA-RSV followed by measurement of activity of the reporter gen e encoding chloramphenicol acetyltransferase (CAT). Transfections were performed with DNA-protein complexes using poly(lysine)10kDa-SP-B ([L ys]10kDa-SP-B) or poly(lysine)3.3kD.SPB ([Lys]3.3kDa-SP-B), and result s were compared with transfections using unmodified poly(lysine).DNA, unmodified SP-B.DNA, or DNA only. For [Lys]10kDa-SP-B.pCPA-RSV prepara tions, CAT activity was readily detectable above the background of [LY S]3.3kDa-SP-B or unmodified SP-B. The SP-B-poly(lysine) conjugates wer e effective over a broad range of protein-to-DNA molar ratios, althoug h they were optimal at approximately 500:1-1000:1. Transfection effici ency varied with the tested cell line but was not specific to airway c ells. Addition of replication-defective adenovirus to the [Lys]10kDa-S P-B.pCPA-RSV complex enhanced CAT activity about 30-fold with respect to that produced by the [Lys]10kDa-SPB.pCPA-RSV complex alone. This in crease suggests routing of the adenoviral.[Lys]10kDa-SP-B.pCPA-RSV com plex through an endosomal pathway. Effects of covalent modification on the secondary structure of SP-B were examined by Fourier transform in frared spectrometry (FTIR). Results of FTIR indicated that the conform ation of [Lys]10kDa-SP-B was comprised primarily of alpha-helical stru cture compared with a predominately aggregated structure of unmodified poly(lysine). We conclude that poly(lysine) conjugates of SP-B effect ively deliver DNA in vitro and may have utility as DNA delivery vehicl es to the airway in vivo.