Effect Of A New Nebulizer On Plasmid DNA Integrity And Gene Transfection

Background:Patient are suffered from some pulmonary geneticdisease which lowerd the quality of people’s life, such as asthma and alpha-1antitrypsin deficiency and which threaten people’s survive, shuch as lung cancer and cystic fibrosis. Gene therapy, the replacement of defective or absent genes within the cell, or gene therapeutics, the use of transient administration of genes to affect function or modulate responses, are approaches to the treatment of pulmonary diseases that are gaining credibility. The delivery of genes by inhalation holds promise for the treatment of a wide range of pulmonary and non-pulmonary disorders and offers numerous advantages over more invasive modes of delivery. Unfortunately, expensive cost of gene therapy, inefficient penetration of mucous barriers and inhibitory effects of surfactant,nebulisation shear forces,and other lung-specific features have generally resulted in a lack of therapeutic effect, and much of this work has diminished in recent years as a consequence. In this study, we introduce a modified nebulizer which not only provide an excellent protection to the plasmic DNA, but also enhance the effectiveness of gene transfection that provide a applicable device for aerosol gene therapy. Objective:Explore the effect of a new airway miniaturized nebulizer on the plasmid DNA (pDNA) integrity and animals gene transfection efficiency.Methods:In the first experiment, the effect of the new airway miniaturized nebulizer on pDNA integrity was tested.2ml pDNA(20μg/ml) was put into the sample pool of the new airway miniaturized nebulizer and a clinical commonly used jet nubulizer respectively. Aerosol samples were collected at the1st min,3rd min,5th min from the nozzle of the two nebulizers respectively and investigated with agarose gel electrophoresis. In the second experiment, the effect of gene transfection efficiency of the new airway miniaturized nebulizer on animals was studied.3.3μg PEI/pEGFP (polyethylenimine and plasmid DNA of green fluorescent protein complex formation which contain3.3μg pEGFP) was aerosolized to rats via the new airway miniaturized nebulizer and a clinical commonly used jet nubulizer respectively. Rats were sacrificed24h after aerosol treatment. The mRNA was abstracted from the lung of those rats, reverse transcribed into cDNA and determined by real-time PCR. PCR products were also investigated with agarose gel electrophoresis.Result:At the1st min,3rd min,5th min, the proportions of integrity pDNA of the new airway miniaturized nebulizer were (99.6±0.7)%,(100±1.2)%,(99.6±0.7)%respectively, and compared with the control group. The result was no significant difference between the two group (P>0.05, n=3). The effect of the ne w airway miniaturized nebulizer on plasmid DNA can be neglected. At the1st min,3rd min,5th min, the proportions of integrity pDNA of the clinical commonly used jet nubulizer were (70.3±1.5)%,(49.3±1.5)%,(32.7±0.6)%respectively, and compared with the control group. The results was significantly different between the two group (P<0.05, n=3). The proportion of integrity pDNA of the jet nubulizer was decline graduately accompany with time. The new nebulizer yielded382.1±101.1fold higher expression levels than the ordinary jet nebulizer (P<0.01, n=3)Conclution:The new airway miniaturized nebulizer provided an excellent protection on the plasmid DNA, increased the efficiency of gene transfection significantly, and provided a suitable device for aerosol gene therapy.