Delivery of DNA–Cationic Liposome Complexes by Small-Particle Aerosol

Abstract

Aerosol delivery of gene therapy for treatment of lung diseases allows topical treatment of the airways with DNA concentrations not obtainable by systemic administration. We have investigated delivery of cationic liposomes complexed to plasmid DNA in a small particle aerosol. Plasmid cDNA–DMRIE/DOPE complexes were nebulized using either an Aerotech II or Puritan-Bennett 1600 (PB1600) nebulizer. Reservoir sampling showed that DNA–DMRIE/DOPE complexes were damaged to a significant degree during nebulization, such that activity of transfected gene was diminished. Of the nebulizers analyzed, DNA–DMRIE/DOPE complexes were more stable in the PB1600. The loss of effective transfection by DNA–DMRIE/DOPE, as detected by decreased reporter gene activity in A549 lung cells, was consistent with denaturation of the DMRIE/DOPE. In contrast, nebulized DNA–DOSPA/DOPE complexes retained complete ability to transfect. Adjustments to flow rate and reservoir volume of the PB1600 allowed a longer period of delivery of active DNA–DMRIE/DOPE particles. DNA–DMRIE/DOPE was radiolabeled with Technetium-99m (^99mTc), nebulized, and the output captured in either an Andersen Sampler (AS) (Andersen, 1958) cascade impactor particle size analyzer or an all glass impinger. cDNA–cationic lipid complexes were detected in size ranges of 0.4–10 μm, with most particles found between 1–2 μm. Aerosol output was consistent from 0 to 5 min. These results show the feasibility of aerosol delivery of DNA–cationic lipids for the purposes of gene therapy to the lung. DNA–cationic lipid complexes were nebulized using either of two nebulizers with differently designed jets, the Aerotech II or Puritan Bennett 1600. Damage to the DNA–DMRIE/DOPE complexes during nebulization was shown by a decrease in activity of the transfected gene. In contrast, DNA–DOSPA/DOPE was not significantly damaged by nebulization in the PB1600 nebulizer. Damage to DNA–DMRIE/DOPE complexes was consistent with the separation of the DOPE from the DNA–DMRIE occurring with the reflux of the DNA–lipid through the nebulizer jets. These results indicate that a more stable DNA–cationic lipid complex is desirable for aerosol delivery of gene therapy. Effective DNA–DMRIE/DOPE particles could be delivered for a longer period of time by decreasing the flow rate and increasing the reservoir volume. Using Technetium-99m-labeled complexes, the particle size of the nebulized DNA–DMRIE/DOPE complexes was shown to be primarily in the 1 to 2-μm range, which would target the lower airways.