In vitro analysis of the possibility to simultaneously transfer bleomycin and plasmid DNA in cells via electroporation

Electroporation is a process when as a result of electric fields effect induced transmembrane potential create transient pores in the plasma membrane, leading to a large increase in drug transport, delivery of macromolecules, a quick drug effect onset. Hence, anticancer drugs can be easily transported into targeted tumour cells by using this technique. Nowadays, electrochemotherapy is used in clinics for the cancer treatment. Electrochemotherapy is a process, when electroporation is combined with the injection of cytotoxic drugs (e.g. bleomycin (BLM). A single molecule of bleomycin can cause around 8–10 DNA breaks causing the cell death, which explains its high cytotoxicity when present inside the cell. However, it is only a local response. In order to initiate a systemic response rate, one has to induce immune response. One way to do it is to transfect a gene that express a compound that enhance immune response. In this study, we show the possibility to simultaneously transfer bleomycin and plasmid DNA in cells via electroporation. Chinese hamster ovary (CHO) cell line was used for experiments. The final concentration of BLM in experiments ranged from 0.1 ng/ml to 20 ng/ml. For that different concentrations of anticancer drugs were used together with pDNA transfection. pMAX FGP coding plasmid in concentraton of 200 μg/ml was used. Electroporation was performed by using combination of 1 electric pulse that induced electric fields at the amplitude of 1400 V/cm for the duration of 100 μs. Cells were electroporated in laboratory made EP medium (pH 7.1, conductivity 0.1 S/m, osmolarity 270 mOsm). Afterwards, comet assay was performed to evaluate DNA damage. In addition, clonogenic assay was done to evaluate cell viability. Transfection efficiency was measured using flow cytometry (BD accuri C6).[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta