Effect Of Jet Nebulizer On Plasmid DNA Integrity And The Study Of Protective Methods

Background: Gene therapy shows great potential among the most innovative therapeutics in the coming future. There are numbers of factors lies in the way of successfully delivery of genetic drugs into the surface of airway and alveoli, such as: the weight of plasmid DNA, the type of vectors, and the damage to the plasmid DNA during aerosolization. Among those factors, the damage to the plasmid DNA due to shear forces which was generated during nebulization is crucial to the drug's therapeutic effects. The devices that has been applicated to the gene aerosolization experiment include vibrating mesh nebulizer, ultrasonic nebulizer, jet nebulizer, electrohydrodynamic delivery. Electrohydrodynamic delivery is still in laboratory phase. Vibrating mesh nebulizer is a newly developed generation device, which has not been widely used in clinics, and yet costs a lot. The jet nebulizer has by now the most popular device for drug delivery. This article aimed at exploring the relationship between damage to the plasmid DNA during nebulization and the aerosolization duration and the aerosolization amount. We also observed the protection effect of two kinds of polyplex commonly used. We hope our work may provide certain foundation for the lung gene therapy.Objective: To explore the effect of jet nebulizer on plasmid DNA (pDNA) integrity and the protective methods. Methods: In the experiment to determine the effect of nebulization time on pDNA integrity, 5ml pDNA solution was aerosolized. Aerosol samples were collected in the 1st min, 2nd min, 3rd min, 4th min, 5th min and the 10th min. In the experiment to determine the effect of aerosolized solution amount on pDNA integrity, 2 ml,4 ml,6 ml,8ml pDNA solution was aerosolized for 4 min respectively. Aerosol samples were collected in the last minute. In the experiment to determine the protective effectiveness of the two polyplex, 4 ml naked pDNA solution, 4ml PEI-DNA solution and 4 ml liposome-DNA solution was aerosolized for 10 min respectively. Aerosol samples were collected in the last minute. Degradation associated with the aerosolisation process was investigated with agarose gel electrophoresis.Results: As aerosolization time increased from 1min to 10min, the intact pDNA ratio decreased from (83.5±2.2)% to (37.1±2.8)%. As the pDNA solution amount added from 2 ml up to 8ml, the intact DNA ratio ascended from (32.1±3.5)% to (93.6±0.6)%. There was almost no damage to the PEI or cationic lipid protected pDNA solution.Conclusion: The damage to which jet nebulizer exerted on pDNA was related with time and volume. PEI and cationic lipids provided plasmid DNA with excellent protection, which laid a good foundation for the application of jet nebulizer in future gene aerosol treatment.