DISTRIBUTION OF DNA AND ALGINATE IN PURULENT CYSTIC-FIBROSIS SPUTUM -IMPLICATIONS TO PULMONARY TARGETING STRATEGIES

Cystic fibrosis (CF) patients frequently experience recurring airway i nfections characterized by thick, viscous sputum. The consistency and nature of these purulent secretions may produce a significant barrier to the successful delivery of drugs and gene therapy vectors designed to treat CF. We have carried out a series of in vitro studies to deter mine the distribution of two macromolecular components typically prese nt in purulent sputum, bacterial alginate and neutrophil-derived DNA. Sputum samples were obtained from hospitalized CF patients. DNA and al ginate were disrupted, respectively, by the in vitro additions of huma n recombinant deoxyribonuclease I (rhDNase) or alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa. N-acetyl-L-cysteine ( acetylcysteine) was similarly used to collapse the mucin matrix of the se samples for comparison. Using a centrifugation-based rheological me thod known as the compaction assay, a greater maximal response was obs erved for rhDNase compared to alginate lyase treatment. A simultaneous addition of these enzymes to purulent sputum produced an additive com paction response. Electron microscopy was used to identify alginate an d DNA components within the mucin matrix of sputa and to evaluate chan ges following treatment with high concentrations of alginate lyase or rhDNase. DNA was more widely distributed throughout purulent samples t han alginate. Differences in the distribution of DNA and alginate may explain, at least in part, the larger compaction response to rhDNase v ersus alginate lyase treatment. An improved understanding of DNA and a lginate distribution within purulent CF sputum may lead to improvement s in drug and vector delivery to airway epithelial cells.