Electrochemotherapy is an e cient method for the local treatment of cutaneous and subcutaneous metastases, but its e cacy as a systemic treatment remains low. The application of gene electrotransfer (GET) to transfer DNA coding for immune system modulating molecules could allow for a systemic e ect, but its applications are limited because of possible side e ects, e.g., immune system overactivation and autoimmune response. In this paper, we present the simultaneous electrotransfer of bleomycin and plasmid DNA as a method to increase the systemic e ect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve e cient cell transfection while killing cells via the cytotoxic e ect of bleomycin at later time points. We also show the dynamics of both cell electrotransfection and cell death after the simultaneous electrotransfer of bleomycin and plasmid DNA. Therefore, this method could have applications in achieving the transient, cell death-controlled expression of immune system activating genes while retaining e cient bleomycin mediated cell killing.

Intramuscular plasmid DNA injection results in long-term but low and variable expression of the injected genes. Optimization is difficult because the mechanism of naked DNA uptake by the cells in vivo is not yet determined. Here we used injections of plasmid DNA encoding luciferase to further characterize this mechanism. We analyzed the kinetics of naked DNA uptake by means of DNase I or heparin injections, using the level of luciferase expression as the indicator of DNA uptake. We demonstrated that in vivo heparin inhibits DNA uptake without affecting the expression of DNA internalized by means of electric pulses. Inhibition by heparin is dose dependent and compatible with the competition for the binding to a receptor. As shown also with DNase 1, DNA uptake by muscle cells is slow: a progressive accumulation of the DNA in the myofibers can be found for at least 4 hours after naked DNA injection. Physical presence of DNA molecules during the uptake period, but not later, was confirmed by the facilitation of DNA uptake with appropriate electric pulses. Therefore, uptake proceeds for the entire time during which intact DNA is present in the extracellular compartment. Our results support evidence for a DNA uptake mechanism based on receptor- mediated endocytosis.

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 s) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of 51Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.