Simultaneous and independent electrotransfer of DNA and small molecules and its dependence on external medium conductivity

Elektroporacijos metodas gali būti pritaikytas mažos molekulinės masės junginių arba DNR pernašai per ląstelės membraną. Metodo efektyvumą lemia elektrinio lauko ir išorinės terpės parametrai, vienas iš kurių yra išorinės terpės savitasis laidumas. Šiame darbe buvo tirta elektroporacijos terpės savitojo laidumo įtaka molekulių elektropernašai į ląsteles ir sąveika tarp vienalaikiškai vykstančios DNR ir mažų molekulių pernašos per membraną. Buvo nustatyta, kad išorinėje terpėje esanti DNR pagerina tiek mažos molekulinės masės junginių pernašą į ląsteles, tiek ir tokių junginių išnašą iš ląstelių. Taip pat buvo parodyta, kad išorinės terpės savitojo laidumo padidėjimas pablogino tiek DNR, tiek ir mažų molekulių pernašos į ląsteles efektyvumą. Šį skirtumą bandyta paaiškinti elektrinių laukų poveikyje vykstančios elektrodeformacijos priklausomybė nuo išorinės terpės ir citoplazmos savitųjų laidumų santykio: išorinės terpės savitajam laidumui esant mažesniam nei citoplazmos, elektrinio lauko poveikyje susidaro ištemptasis sferoidas, išorinės terpės savitajam laidumui esant didesniam nei citoplazmos, susidaro suplotasis sferoidas. Pritaikus matematinį modeliavimą buvo parodyta, kad ant suplotųjų sferoidų paviršiaus sukuriamas mažesnis transmembraninis potencialas, nei ant ištemptųjų sferoidų paviršiaus.The method of electroporation can be utilized for the transfer of small molecular mass molecules or DNA across the cell membrane. The effectiveness of the method is dependent on the parameters of the electric field and the external medium, including the conductivity of the external medium. This work assessed the effect of external medium conductivity on the electrotransfer of small molecules and DNA to the cells and the interaction between simultaneous electrotransfer of DNA and small molecules across the plasma membrane. It was shown that the DNA in the external medium increases the efficiency of small molecule electrotransfer both to and from the cells. It was also shown that the increase in the external medium conductivity decreased the efficiency of both DNA and small molecule electrotransfer to the cells. This difference is explained by the dependence of cell electrodeformation dependence on the ratio of conductivities of external medium and the cytoplasm: when the external medium has lower conductivity than the cytoplasm, prolate spheroid is formed, and when the external medium has higher conductivity than the cytoplasm, oblate spheroid is formed. Mathematical modelling was employed to show that lower transmembrane potential is formed on the surface of oblate spheroids in comparison to the prolate spheroids.Gamtos mokslų fakultetasBiologijos katedr

Research on Molecular Electrotransfer to Chinese Hamster Ovary (CHO) Cells Dependent on Medium Conductivity

Šiame darbe buvo tirta bleomicino, etidžio bromido, propidžio jodido ir liuciferazę koduojančios plazmidės elektropernašos į CHO ląsteles priklausomybė nuo terpės laidumo. Naudotose parametrų ribose ląstelių gyvybingumas nebuvo paveikiamas. Mažų molekulių elektropernašos efektyvumas netiesiškai mažėjo, didinant elektroporacijos terpės laidumą. DNR elektrotransfekcija taip pat buvo didžiausia mažo laidumo terpėje, bet nesilpnėjo, didinant elektroporacijos terpės laidumą virš 0,3 S/m.This work assesses efficiency of bleomycin, ethidium bromide, propidium iodide and luciferase-encoding plasmid electrotransfer to CHO cells dependent on electroporation medium conductivity. Cell viability was unaffected by medium conductivity in the settings used. Molecule electrotransfer efficiency is non-linearly decreased when medium conductivity is higher. DNA electrotransfection is highest in low conductivity medium, but does not decrease when medium conductivity increases above 0,3 S/m.Gamtos mokslų fakultetasVytauto Didžiojo universiteta

The dependence of the cell electrotransfection efficiency on the electrosensitization induced membrane changes and the time of DNA addition to the electroporation medium

Electroporation – a physical method that temporarily disrupts the membranes and increases the cell membrane permeability – has been known for a few decades. The application of short, high voltage (kV/cm) electric pulses leads to the formation of transient pores that allow the passage for different types of molecules into the cell. It has been applied for numerous uses in the technology and medicine. One of these applications is the gene electrotransfection, when the electroporation is used for the delivery of DNA to the cells. It is well known that the delivery of the DNA to the cells is much more complex in comparison to the delivery of small molecules. The aim of this work was to compare the dependence of cell electrotransfection efficiency on the electrosensitization induced membrane changes and the time of DNA addition to the electroporation medium. All the experiments were done with Chinese Hamster Ovary (CHO) cells. The cells were electroporated using combinations of 1400 V/cm, 100 μs electric pulses. 100 μg/ml of the plasmid coding for green fluorescent protein (pGFP) was used to evaluate the cell electrotransfection efficiency. Cell fluorescence was measured 24 hours after the transfection using flow cytometer (BD Accuri C6). The cells were electroporated once and then again after 10, 15 or 20 minutes, and pGFP was added to the solution either before the electric pulses or during the waiting time. The results show that the addition of the time-delayed electric pulses increases the transfection efficiency in comparison to the settings used for the initial electroporation only if the plasmid is present in the medium before the first electroporation. If the plasmid is added to the solution during waiting time, the electrotransfection efficiency is reduced, signaling electro de-sensitizationBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

The dependence of molecular transmembrane electrotransfer efficiency on medium conductivity and osmotic pressure

The electrotransfer efficiency was evaluated for different external medium conductivities, osmotic pressures and electric pulse voltages. It was found that increase in conductivity or decrease in electric pulse strength decreases electrotransfer efficiency. Decrease in osmotic pressure tends to decrease electrotransfer efficiencyBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Dependence of CHO and DC-F3 cell electrotransfection on conductivity of electoporation medium

Electroporation is a process when cell membrane permeability is increased after transmembrane voltage is lifted with external electrical fields. Thereafter exogenous molecules can penetrate through cell membrane. When plasmid DNA is electrotansfered and migrates to nucleus cell become electrotransfected. The aim of this study was to evaluate CHO and DC3F cell electrotransfection efficiency dependence on electroporation media conductivity. CHO and DC-3F cell suspension with luciferase coding plasmid (LUC) was electroporated with 1200 V/cm 100 μs various number pulses at 1 Hz in SMEM (1.3 S/m) and EP (0.1 S/m) electroporation media. Results showed better CHO electrotransfection with low conductivity electroporation medium. Opposite results were obtained with DC-3F cell line electrotransfection. Cell size and the concentration of charged carriers are proposed as an explanation to electrotransfection differences on CHO and DC- 3F cell linesBiologijos katedraVytauto Didžiojo universiteta

The effect of external DNA on the efficiency of bi-directional small molecule electrotransport

ONLINE ISSN: 2335-8718Electroporation is a widely used physical method for increasing the cell membrane permeability. After the application of electric pulses, transient electropores are formed, allowing both the leakage of compounds from the cell (electroextraction) and the delivery of small molecules (electrotransfer) and DNA (electrotransfection) to the cell. For certain clinical applications, simultaneous delivery of both small molecules (e.g. anticancer drugs) and DNA could be advantageous. However, the information about the effects of DNA on small molecule transport is very scarce. We investigated the effect externally provided DNA has on the small molecule transport across electroporated cell membrane. For the determination of electroextraction, we used Chinese Hamster Ovary (CHO) cells loaded with green fluorescent calcein dye, and measured the loss of intracellular calcein fluorescence using flow cytometry. For the determination of small molecule electrotransport, we delivered anticancer drug bleomycin to CHO cells, and evaluated the efficiency of bleomycin electrodelivery using clonogenic assay. Our results show that both the outward flux of calcein and the inward flux of bleomycin were positively affected by the presence of extracellular DNA during the application of the electric field. These findings suggest that a combined treatment of chemotherapy and gene therapy might be a feasible approach towards the increase in the treatment efficiency of cancerBiologijos katedraVytauto Didžiojo universiteta

Enhancement of drug electrotransfer by extracellular plasmid DNA

This work was supported by the grant (DOTSUT–09.3.3-LMT-K-712- 01-0188) from the Research Council of LithuaniaElectroporation is a widely established method for molecular delivery across electric field perturbed plasma membrane. It can be used as a non-viral DNA transfection method, or as a way to achieve small molecule delivery to or extraction from cells. We examined the possibility of combining the DNA delivery to the cells with small molecule transport across electroporated plasma membrane. The results show that the presence of DNA in electroporation medium increases the extraction of fluorescent dye calcein from calcein-AM loaded cells as well as the delivery of small-molecule drug bleomycin to the cells. We propose that these results may have implications in enhanced drug delivery using electroporation both in vivo and in clinicsAplinkos tyrimų centrasBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

The interdependence of Calcein electroextraction and DNA electrotransfer when processes are performed simultaneously

Electroporation, as a cell membrane permeabilization phenomenon to various molecules, occur when cells are affected with electric field. Induced molecule diffusion through electroporated membrane is termed electrotransfer. These induced processes are employed for molecule extraction from cells as well as DNA electrotranfer to the cells. However it is not much known how molecule electroextraction is affected with DNA electrotransfer when performed simultaneously. To test this we have used chinese hamster ovary (CHO) cell line as an object. For electroextraction we used calcein-AM. Once freely diffused inside the cells, calcein-AM is metabolized into calcein. Due to its hydrophilic properties calcein cannot diffuse back to the medium through cell membrane. However calcein extraction is observed after temporary compromising cell membrane with electroporation. DNA electrotransfer was performed using PGL-4 Luciferase coding plasmid. Calcein extraction was measured with flow cytometer (BD Acura-C6). Luciferase protein relative concentration was measured with microplate reader (GENios Tecan). Electroporation was performed with one HV (1400 V/cm 100 μs) pulse. Obtained results indicated an inverse dependency between DNA transfection and calcein electroextraction. When calcein extraction was measured a highly statisticaly significant increase (P<0.001) of extracted calcein was observed when there was 100 μg/ml plasmid in medium compared with electroextraction when there is no plasmid inside the medium. When DNA transfection efficiency was measured using same experiment setup an inverse results were obtained. With plasmid concentration of 100 μg/ml in cell suspension a statistical significant decrease of transfection efficiency was observed compared with electrotransfection when there is no calcein inside the cell.[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Different cell viability assays following electroporation in Vitro

ISBN 978-3-319-32885-0, 9783319267791Exposure of cells to high intensity electric fields can lead to death of some cells due to irreversible electroporation or due to electroporation-induced secondary effects. If cells are exposed to these fields in the presence of various exogenous molecules, cell death can also be induced due to their activity in the cells. In all these cases, mechanisms of cell death as well as the time periods over which death of these cells occur are highly variable. Therefore, application of different cell viability assays can lead to inconsistent results, either depending on the assay itself or the application time. The aim of this chapter is to provide a brief presentation of viability assays that have been employed in electroporation-based applications and provide a short discussion on choosing the specific cell viability assay to properly interpret the obtained results. Since most of cell viability assays (all tetrazolium salt-based assays, resazurin assay, calcein acetoxymethyl ester (calcein AM) assay, crystal violet (CV) assay, evaluation of necrosis and/or apoptosis, etc.) are applied at time points when death of some cells in the treated population is still in progress, they cannot provide precise viability results. Therefore, those who seek to evaluate cell viability as precisely as possible should rely on end-point clonogenic assay. Other cell viability assays can be applied in parallel with clonogenic assay or at least should be tested in pilot experiments before they are employed routinely in a specific set of experimentsBiochemijos katedraBiologijos katedraVytauto Didžiojo universiteta

The dependence of induced transmembrane potential on the cell electrodeformation and its correlation with cell electrotransfection efficiency

Electroporation is a widely established method that uses electric fields to enhance molecular transport across plasma membrane. Nowadays, it is used for drug and gene delivery into the cells and tissues for therapeutic purposes and for the extraction of bioactive compounds from tissues during food processing. In our study we have investigated efficiency of molecular electrotransfer in dependence of medium conductivity and presence of plasmid DNA in an electroporation medium. We have observed the inverse dependence between the conductivity of electroporation medium and the efficiency of the molecular transport to the Chinese Hamster Ovary cells. We have seen that efficiency of small molecule electrotransfer decays when the electroporation medium conductivity increases. We observed the same results when transfecting the cells with plasmid DNA. We propose that this dependency may at least in part be caused by the differential electrodeformation of the cells in the external media that have higher or lower conductivity than the cell cytoplasm. [...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta