Iana Tsoneva

DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles.

Abstract We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 μm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. “Short” DNAs (oligonucleotide 21b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (Cendo) of d-erythro-sphingosine (Sph+) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below Cendo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. Cendo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph+ GUVs and about 20 mol% for SOPC/Sph+ or eggPC/Sph+ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes.

Sphingosine-mediated electroporative DNA transfer through lipid bilayers

When the cationic sphingosine is present in planar lipid bilayers, the adsorption of pDNA is enhanced and the electroporatability of the bilayer is facilitated. Furthermore, pDNA, adsorbed to lipid bilayers composed of diphytanoyl lecithin and d‐sphingosine causes electroporative channel‐like events of conductance 1 to 7 pS, provided the voltage polarity is correct and the voltage is high enough, V m≥+30 mV. The data suggest electrotransport of pDNA through the bilayer, mediated by transient complexes between DNA and the lipids in the pore edges of elongated, electropercolated hydrophilic pore zones. The results are the basis for the optimisation of electroporative transfer of DNA or oligonucleotides to cells and tissue.

Dip patch clamp currents suggest electrodiffusive transport of the polyelectrolyte DNA through lipid bilayers

Planar lipid bilayers formed from monolayers of diphytanoyl lecithin (DPhL) were found to interact with plasmid DNA (5.6 kbp; M(r) = 3.7 x 10(6)) leading to an increase in the conductance of the membrane. The association of DNA with a lipid bilayer greatly facilitates the transport of the small ions of the main salt KCl. The appearance of long-lived current levels, for instance, of 27.6 pA at Vm = +60 mV membrane voltage, where the actual contact (adsorption) is electrophoretically enhanced, suggests a locally conductive DNA/lipid interaction zone where parts of the DNA strand may be transiently inserted in the bilayer, leaving other parts of the DNA probably protruding out from the outer surface of the bilayer. At Vm = -60 mV, where DNA can be electrophoretically moved away from the membrane, the membrane current is practically zero. This current asymmetry is initially also observed at higher voltages, for instance at 200 mV. However, if the voltage sign (Vm = +200 mV) is changed after a transient positive current (approximately 15 pA) was observed, there is also now (at Vm = -200 mV) a finite negative current at the negative membrane voltage. Thus, it appears that at Vm = +200 mV the adsorbed parts of the polyelectrolyte DNA are not only transiently inserted in, but actually also electrophoretically pulled through, the porous zones onto the other membrane side leaving the bilayer structure basically intact. These data provide direct electric evidence for the electrophoretic transport of a highly charged and hydrated macromolecule, probably together with the associated gegen-ions, through the thin hydrophobic film of the lipid bilayer.

Electrodelivery of Drugs into Cancer Cells in the Presence of Poloxamer 188

In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.

New Insights in the Gene Electrotransfer Process: Evidence for the Involvement of the Plasmid DNA Topology

Electropermeabilization is a non-viral method that can be used to transfer plasmid DNA (pDNA) into cells and tissues. According the applications and considered tissues, this safe method can be less efficient than the viral approaches. Biophysical mechanisms of gene electrotransfer are not entirely known. Contrary to small molecules that have direct and fast access to the cytoplasm, pDNA is electrophoretically pushed towards the permeabilized membrane where it forms a complex before being transferred into the cytoplasm. In order to understand the biophysical mechanisms of gene electrotransfer and in this way to improve it, we investigated the dependence of the topology of pDNA i.e. linear versus supercoiled on both pDNA/membrane interaction and gene expression. Our results revealed that: i) even if pDNA/membrane interactions are only slightly affected by the topology of pDNA, ii) gene transfer and expression are strongly influenced by it. Indeed, the linearization of pDNA leads to a decrease in the transfection level.

New Modality for Electrochemotherapy of Surface Tumors

ABSTRACT Electrochemotherapy is based on electroporation, which creates aqueous pathways in the cell membrane as a result of applied short intensive electric field usually generated by high voltage electrical pulses. The phenomenon enhances the simultaneous administration of cytotoxic drugs. Recently, several researchers forewarn of some adverse effects that could occur with electrochemotherapy of tumors located close to the heart. We developed equipment Chemipulse III, consisting of portable low-cost electroporator, QRS synchronizer and module for long-term recording of pulse parameters. It is designed for data collection of the pulse parameters and the impedances of treated patients with surface tumors without risk of heart complications but with enhanced means for electrical safety of both patient and physician. The new instrument is with battery supply, enhanced protection against electrical hazards both for the patient and the physician, autonomy providing for more than 200 shocks with one battery charging. The data could be used to support the optimization of the voltage shock setting and the evaluation of the procedure effectiveness. The results obtained show high efficiency of the improved approach (new modality) for electrochemotherapy. Thirty-seven patients with a total of 47 lesions of Ca basocellulare, Ca spinocellulare and Kaposi Sarcoma were treated with electrochemotherapy using intralesional application of bleomycin and new developed equipment Chemipulse III. The electrotreatment was done by 16 biphasic rectangular pulses, 50+50 μs duration each, with a 20 μs interval between both phases and a pause of 880 μs between the bipolar pulses. The equipment with QRS synchronizer is mandatory in cases of electrochemotherapy of patients with pacemakers and patients with heart problems. Erythema and slight edema at the site of the treated lesions occurred in most of the patients and disappeared in a few days, one week at most.

In Vitro Evaluation of Elastic Multiblock Co-polymers as a Scaffold Material for Reconstruction of Blood Vessels

There is a need to create cell- and histocompatible implant materials, which might temporarily replace the mechanical function of a native tissue for regenerative therapies. To match the elastic behavior of the native tissue two different multiblock co-polymers were investigated: PDC, consisting of poly(p-dioxanone) (PPDO)/poly(ε-caprolactone) (PCL), and PDD, based on PPDO/poly((adipinate-alt-1,4-butanediol)-co-(adipinate-alt-ethylene glycol)-co-adipinate-alt-diethylene glycol) (Diorez). PDC is capable of a shapememory effect. Both multiblock co-polymers show an improved elasticity compared to materials applied in established vascular prosthesis. PDD is softer than PDC at 20°C, while PDC maintains its elasticity at 37°C. Thermodynamic characteristics indicate a more polar surface of PDD. Low cell adhesion was found on surfaces with low molar free energy of hysteresis (ΔG) derived from contact angle measurements in wetting and dewetting mode and high cell adhesion on high-ΔG surfaces. An increasing content of PCL in PDC improved cell adhesion and spreading of human umbilical vein endothelial cells. The prothrombotic potential of PDD is higher than PDC. Finally, it is concluded that PDC is a promising material for vascular tissue engineering because of its improved elastic properties, as well as balanced prothrombotic and anti-thrombotic properties with endothelial cells.

Effective production by electrofusion of hybridomas secreting monoclonal antibodies against Hc-antigen of Salmonella

Abstract Hybridomas secreting monoclonal antibodies (Mab) were generated by electrofusion of splenic lymphocytes from Balb/c mice immunized with Hc-antigen of Salmonella with mouse myeloma cells (x63-Ag8.653). The fusion efficiency was expressed by the number of antibody secreting hybridomas/106 lymphocytes. The results obtained show that: (1) the number of antibody secreting hybridomas produced by electrofusion is one order of magnitude higher than with the polyethylene glycol (PEG) technique; (2) pretreatment of lymphocytes with electrical pulses leads to an additional increase in the fusion yield; (3) treatment with protease does not change the yield; (4) electrofusion resulted in both IgG- and IgM-secreting hybridomas. It is supposed that the increased fusion efficiency when using lymphocytes pretreated with electrical pulses is due to their increased “fusogenicity”.

Treatment of Melanoma by Electroporation of Bacillus Calmette-Guerin

ABSTRACT Electrochemotherapy could be considering as a good method for treatment of cutaneous and subcutaneous tumor lesions. Here we report the case of 59 year-old woman with melanoma malignum cutis, who developed after axillary dissection, cutaneous metastasis not appropriate for surgical operation. Electrochemotherapy with BCG vaccine resulted in good local control of the disease, with a complete response of treated melanoma lesion.

Electrical breakdown of protoplast membranes under different osmotic pressures

Abstract The electrical breakdown of membranes depends on their tension. The cell membrane tension increases with decreasing osmolarity of the solution. We measured the breakdown voltage of pea protoplasts in hypo-, iso- and hypertonic mannitol solutions. In hypotonic solutions, near the isotonic point, the breakdown voltage decreased strongly with increasing osmotic pressure difference. For lower osmolarities, the breakdown voltage did not change so significantly. In hypertonic solutions, the protoplasts broke at the same voltage as in isotonic ones. We developed a theory, based on the fluctuation waves mechanism of membrane rupture, which describes the experimental results approximately. It does not, however, fit the data for solutions of low osmolarity. Possible mechanisms, not taken into account by the theory, and applications to biotechnology are discussed.