Aerosol delivery of plasmid DNA to the lungs offers the possibility of direct application of gene preparations to pulmonary surfaces as a means of treating a variety of genetic pulmonary disorders. However, the process of jet nebulization rapidly degrades naked DNA, viral vectors, and many lipid-based formulations. While complexing DNA with cationic lipids has been shown to significantly stabilize plasmid DNA, losses of biological activity often occur during nebulization, severely limiting the efficiency of aerosol delivery of many such complexes. In conjunction with the design of aerosol delivery systems appropriate for DNA delivery, we have developed formulations using polyethyleneimine (PEI, a polycationic polymer) and DNA that result in a high level of pulmonary transfection (10- to 100-fold greater than many cationic lipids) and are stable during nebulization. In addition, these PEI-based formulations exhibit a high degree of specificity for the lungs. The properties of PEI-based formulations that make them resistant to nebulization and efficient as DNA delivery vectors for pulmonary sites have been investigated. Potential applications of this technology, including the use of aerosolized PEI-DNA for genetic immunization, are discussed.

Aerosol gene delivery to the pulmonary system has vast potential for many diseases, including cystic fibrosis and lung cancer. We recently reported that polyethyleneimine (PEI), a cationic polymer, holds promise as a gene delivery vector for transfection in lung by aerosol. To further optimize the gene expression in the lung by aerosol, we utilized 5% CO(2) in air for the nebulization of PEI-DNA complexes. Five percent CO(2)-in-air gave a threefold higher gene expression compared to normal air using the chloramphenicol acetyl transferase (CAT) reporter gene delivered by Aerotech II nebulizer. The delivery of DNA by PEI was dose dependent with the highest expression obtained when 2 mg of DNA in 10 ml was nebulized at a PEI nitrogen:DNA phosphate (N:P) ratio of 10:1. The optimal N:P ratio for lung transfection was found to be between 10:1 and 20:1 using the CAT and luciferase reporter genes. The time-course studies showed the highest expression at 24 h after aerosol delivery and 40-50% of peak level was detectable even after a week. Tissue distribution indicates the expression to be specific to the lung with no detectable expression in any other tissue examined. Histological and biochemical analysis of lungs revealed no evidence of acute inflammation.

Gene therapy targeted at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and alpha-1 anti-trypsin deficiency. A variety of approaches such as intranasal or intratracheal instillation and aerosol delivery have been utilized to target genes to the airways. Polyethylenimine (PEI), a linear or branched polycationic polymer, has been used for delivery of genes to various organs. In this study, using fluorescein isothiocyanate (FITC)-labeled branched PEI, we initially examined the localization of PEI in the lungs after aerosol delivery to Balb/C mice. Further, after aerosol delivery of PEI-CAT DNA, in situ immunostaining for chloramphenicol acetyl transferase (CAT) protein was used to localize the transgene expression within the lungs. Immunohistochemistry for CAT, as well as localization of FITC-labeled PEI, revealed that after aerosol delivery, the PEI-DNA complexes deposit and subsequently transfect most of the epithelial cells in the conducting airways (including the peripheral airways). High levels of CAT were detected at 24 h after aerosol exposure and significant CAT expression was detected in the lungs up to 28 days after a single aerosol exposure. The data suggest that aerosol delivery of PEI-DNA complexes could be effective for the treatment of pulmonary diseases such as cystic fibrosis and alpha-1 anti-trypsin deficiency.

Recent technological advances and improved nebulizer designs have overcome many limitations of jet nebulizers. Newer devices employ a vibrating mesh or aperture plate (VM/AP) for the generation of therapeutic aerosols with consistent, increased efficiency, predominant aerosol fine particle fractions, low residuals, and the ability to nebulize even microliter volumes. These enhancements are achieved through several different design features and include improvements that promote patient compliance, such as compact design, portability, shorter treatment durations, and quiet operation. Current VM/AP devices in clinical use are the Omron MicroAir, the Nektar Aeroneb, and the Pari eFlow. However, some devices are only approved for use with specific medications. Development of "smart nebulizers" such as the Respironics I-neb couple VM technologies with coordinated delivery and optimized inhalation patterns to enhance inhaled drug delivery of specialized, expensive formulations. Ongoing development of advanced aerosol technologies should improve clinical outcomes and continue to expand therapeutic options as newer inhaled drugs become available.

9-NC liposome aerosol was strikingly effective in the treatment of three human cancer xenografts growing subcutaneously over the thorax in nude mice at doses much smaller than those traditionally used in mice administered by other routes.