Aerosolization of Cationic Lipid:pDNA Complexes—In Vitro Optimization of Nebulizer Parameters for Human Clinical Studies

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Abstract
Previously, we have described the optimization of the aerosol delivery of a nonviral gene therapy vector to the lungs of rodents (Eastman et al., 1997b). Although aerosolizing cationic lipid:pDNA complexes into a whole-body exposure chamber resulted in high levels of reporter gene expression in the lungs of BALB/c mice, the conditions employed were not optimal for the delivery of lipid:pDNA complexes to the lungs of human patients. That is, the consumption rate of the material in the nebulizer, and thus the delivery time, were very slow and the aerosol was delivered in a continuous flow. Here we describe in vitro experiments used to develop a cationic lipid:pDNA aerosol with characteristics more suitable for delivery to the lungs of humans, as a necessary prerequisite for conducting a clinical study with human cystic fibrosis patients. Using cascade impactors and all-glass impingers, we have screened several commercially available nebulizers for their ability to deliver intact, respirable, active lipid:pDNA complexes in the shortest time possible, and have identified the Pari LC Jet Plus nebulizer as the optimal nebulizer that meets these criteria. Using this nebulizer in an intermittent mode to mimic breath actuation, consumption rates of approximately 0.6 ml/min of the cationic lipid:pDNA complexes (6 mM cationic lipid:8 mM pDNA) were obtained. The plasmid DNA remained intact and the complexes were shown to maintain activity throughout the nebulization run. Based on measurements of the nebulized dose and the mass median aerodynamic diameter, we calculate a delivered dose of ~ 22 μmol (7.2 mg) of pDNA for each 8 ml of cationic lipid:pDNA complex aerosolized to the lungs of a human patient. This dose should be sufficient to test the clinical efficacy of cationic lipid-mediated gene delivery for the treatment of cystic fibrosis. A series of jet nebulizers were screened for their ability to efficiently deliver cationic lipid:pDNA complexes as rapidly as possible while producing aerosol droplets of a respirable size. This screen identified the Pari LC Jet Plus nebulizer as being capable of delivering cationic lipid:pDNA complexes quickly. The characteristics of cationic lipid:pDNA aerosols produced by this nebulizer were studied in detail employing an intermittent delivery system to insure that the plasmid DNA remained intact and that the cationic lipid:pDNA complexes remained active throughout the entire aerosolization procedure. These studies revealed that the Pari LC Jet Plus nebulizer produced an aerosol of cationic lipid:pDNA complexes with characteristics that should allow for efficient delivery of the cationic lipid:pDNA complexes to the lungs of human patients for the purpose of conducting clinical studies for the treatment of cystic fibrosis.