Strand Break Of Plasimd DNA Encapuslated In Giant Unilamellar Vesicle By Ultraviolet A Irradiation

Optical properties study of cell membranes have found highlighting at tiny concaves on cell membranes. This indicates that light can be converged by concave cell membrane. Spheral cell membrane has a bigger concave inside. If the light is converged into the cell and effect on the materials inside, it would be significant for new understanding the biological function of cell membrane.In this research, plasmid DNA was encapsulated in Giant Unilamellar Vesicle (GUV) and irradiated by Ultraviolet A (UVA)in to explore whether UVA can be converged by spherical GUV membrane which enhance the strand break of plasmid DNA inside.Purpose1. To study the effects of plasmid DNA concentration and molecular weight on GUV shape and encapsulation, and find out the best conditions for establishment of shperical membrane model GUV with DNA encapsulatedd.2. To Study the effect of UVA energy DNA damage and observe whether GUV can enhance the damage of plasmid DNA by UVA.Methods1. According to the different concentrations of the pGV102-NC plasmid DNA, the experiment was divided into groups:0.01 μmol/L group, 0.02 μmol/L group,0.04 μmol/L group,0.08 μmol/L group,0.12 μmol/L group,0.16 μmol/L group and the control group (deionized water). Then, according to the molecular weight of the plasmid DNA, the experiment was divided into groups:2868 bp group,6380 bp group,8889bp group,37428 bp group and the control group (deionized water). The formation, diameter distribution and encapsulation rate of the produced GUV were observed.2. According to the irradiation energy of UVA, the experiment was divided into groups:1.83 MJ/m2 group,3.69 MJ/m2 group,7.37 MJ/m2 group,14.74 MJ/m2 group,29.49MJ/m2 group, no-power irradiation group and enzyme group. The strand break of plasmid DNA was observed by Gel electrophoresis.. Then, we divided the samples of GUV into groups: irradiation plasmid DNA inside GUV group, irradiation plasmid DNA outside GUV group, irradiation plasmid DNA group, no-power irradiation plasmid DNA inside group, no-power irradiation plasmid DNA outside GUV group, no-power irradiation plasmid DNA group and enzyme group. The samples were irradiated with 1.83 MJ/m2 UVA, then the strand break of plasmid DNA was observed by Gel electrophoresis.Results1. There was little effect on the formation of GUV at the low DNA concentration (0.01～0.12μmol/L). But, with the increase of DNA concentration, the diameter distribution of GUV trended to 5-25 microns diameter. There was no GUV formed at high DNA concentration (0.16μmol/L). The encapsulation rate of GUV increased with increasing of DNA concentration from 0.01μmol/L to 0.08μmol/L. But the encapsulation rate of GUV decreased with increasing of DNA concentration from 0.08μmol/L to 0.12μmol/L. The GUV prepared had a higher encapsulation rate, good shape and high concentration DNA encapsulated at 0.04μmol/L. The molecular weight of DNA had little effect on the formation of GUV, but had great impacting on the encapsulation rate of GUV. The encapsulation rate of GUV increased with the increase of DNA molecular weight from 2868bp to 6380bp. But the encapsulation rate decreased with the increase of the molecular weight of DNA from 6380bp to 37428bp. When 37428bp DNA was used, the encapsulation rate was 0.2. The double-strand break of the plasmid DNA was observed and the amount of the double strand break increased with the UVA irradiation at dose form 1.83 MJ/m2 to 29.49 MJ/m2. The double-strand break of the plasmid DNA in GUV after irradiation with 1.83 MJ/m2 UVA was more than the plasmid DNA outside GUV.Conclusion:1. GUV can be prepared in plasmid DNA solutions at a certain concentration and molecular weight by electroformation. A certain amount of GUV with 15～35 microns diameter and 0.12±0.08μmol/L plasmid DNA encapsulated were obtained, which will offer spherical membrane model for study of the membrane biological function.2. Double strand break of plasmid of DNA can be induced by UVA irradiation with high doses, the spherical GUV can enhance the damage of plasmid DNA inside produced by UVA irradiation.