DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 microl of CHO cell suspension containing 100, 10 or 1 microg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 microg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 microg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 micros) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 microg/ml and fivefold at 1 microg/ml. At 10 microg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.

In this study we evaluated the influence of medium conductivity to propidium iodide (PI) and bleomycin (BLM) electroporation mediated transfer to cells. Inverse dependency between the extracellular conductivity and the efficiency of the transfer had been found. Using 1 high voltage (HV) pulse, the total molecule transfer efficiency decreased 4.67 times when external medium conductivity increased from 0.1 to 0.9 S/m. Similar results had been found using 2 HV and 3 HV pulses. The percentage of cells killed by BLM electroporation mediated transfer had also decreased with the conductivity increase, from 79% killed cells in 0.1 S/m conductivity medium to 28% killed cells in 0.9 S/m conductivity medium. We hypothesize that the effect of external medium conductivity on electroporation mediated transfer is triggered by cell deformation during electric field application. In high conductivity external medium cell assumes oblate shape, which causes a change of voltage distribution on the cell membrane, leading to lower electric field induced transmembrane potential. On the contrary, low conductivity external medium leads to prolate cell shape and increased transmembrane potential at the electrode facing cell poles.

These findings demonstrate that transfection is very susceptible to medium viscosity and highlights the importance of the electrophoretic component in experiments when a considerable transfection level is needed.

The aim of this study was to estimate the changes of autofluorescence and sensitized fluorescence under the effect of cosmetics. We used a method of fluorescence spectroscopy in vivo and examined the mouse skin covering the tumour. Analysis of fluorescence spectral changes was made after differentiation of the cosmetics according to its effects: i) inducing temporary changes of skin autofluorescence after absorbtion into skin (lipsticks, face powders, body lotions, mascaras); ii) permanently changing the fluorescence of the skin (collagen containing products). Cosmetics have been shown to be optically active and capable to alter the fluorescence of exogenously accumulated photosensitizers and endogenous tissue fluorophores.

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