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M P Rols

Publications and source records attributed to M P Rols.

At least 19 recordsLinked to original sources
PubMed2006

Electrically-assisted nucleic acids delivery to tissues in vivo: where do we stand?

Electropulsation (electroporation) is a physical method for delivery of various molecules into the cells in vitro and in vivo. It is an expanding field due to its applicability in cancer therapy, where combined application of electric pulses and chemotherapeutic drugs is used for treatment of cutaneous and subcutaneous nodules of different malignancies. Another application of electropulsation in vivo is electrogene therapy, where after injection of naked plasmid DNA and delivery of electric pulses directly to the tissue the expression of gene of interest can be obtained. However, the transfection efficiency of this methodology in vivo is still lower than with viral vectors. Nevertheless, due to the lack of immunogenicity of the method, easiness of the preparation of large quantities of endotoxin free plasmid DNA, control and reproducibility of the method and the development of electropulsators approved for the clinical use, electrically-assisted nucleic-acid delivery holds a great potential for the clinical application. This aim of this minireview is to critically discuss the main limitations and obstacles associated with electrogene therapy and the failures and problems as well as the successes. Topics on electric field distribution in the tissue, electrode geometries, construction of plasmid, modulation of extracellular space, tissue damage, pro-inflammatory and immune response as well as blood flow modification associated with application of electric pulses and injection of naked DNA are presented with possible directions how to overcome these limitations. Furthermore, for successful electrogene therapy in clinical setting it is of utmost importance to elucidate the mechanisms of DNA transfer into the cells of tissues in vivo. This will enable appropriate selection of electric pulse parameters and plasmid DNA constructs for each particular intended use. In the long run, this review should encourage other scientists to consider electrically assisted gene delivery for gene therapy as it matures.

Age Factors↗
PubMed2005

Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of ?) knowledge.

Cell electropulsation is routinely used in cell Biology for protein, RNA or DNA transfer. Its clinical applications are under development for targeted drug delivery and gene therapy. Nevertheless, the molecular mechanisms supporting the induction of permeabilizing defects in the membrane assemblies remain poorly understood. This minireview describes the present state of the investigations concerning the different steps in the reversible electropermeabilization process. The different hypotheses, which were proposed to give a molecular description of the membrane events, are critically discussed. Other possibilities are then given. The need for more basic research on the associated loss of cohesion of the membrane appears as a conclusion.

Animals↗
PubMed2005

Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery.

Owing to their capacity to induce strong, sequence-specific, gene silencing in cells, short interfering RNAs (siRNAs) represent new potential therapeutic tools. This development requires, however, new safe and efficient in vivo siRNA delivery methods. In the present technical report, we show that electrically mediated siRNA transfer can suppress transgene expression in adult mice muscles. Using electropulsation for siRNA delivery opens the way for a targeted gene silencing on a broad range of tissues. Clinical applications of electropulsation for delivery of other classes of molecules are under trials. We reported that gene silencing was efficiently obtained in vivo in an adult mammal (mouse) with chemically synthesized siRNA after its electrical delivery. The associated gene silencing was followed on the same animal and lasted at least 11 days. Gene silencing was obtained in muscles not only on young adult mice but also on much older animals. No tissue damages were detected under our electrical conditions. Therefore, this method should provide an efficient approach for a localized delivery of siRNAs in various tissues and organs.

Animals↗
PubMed2004

In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression.

Electropulsation is one of the non-viral methods successfully used to transfer genes into living cells in vitro as in vivo. This approach shows promise in the field of gene and cellular therapies. The present paper first describes the factors controlling electropermeabilization to small molecules (< 4 kDa) and then the processes supporting DNA transfer in vitro. The description of in vitro events brings the attention of the reader to the processes occurring before, during, and after electropulsation of DNA and cells. Their developments for the in vivo processes are reported in the final part where the present and potential clinical applications are described.

Animals↗
PubMed2003

[Calcium and electropermeabilized cells].

Trains of short and intense electric pulses may induce a reversible local permeabilization on the membrane of the treated cells. Hydrophilic species can then almost freely cross the envelope and either enter or escape from the cytoplasm. The purpose of the present study was to investigate the possibility of introducing well defined amounts of Ca2+ ions within the cell. Chinese hamster ovary cells were used as a model system. When the pulsing buffer contained high levels of free Ca2+, the survival of cells was strongly affected. A 1 mM level was well tolerated. When cells were pulsed under moderated field conditions, it was observed that Ca2+ entered cells very rapidly (second time range). But the basic cytoplasmic level was set back spontaneously within a few minutes. The perspectives of this electrical injection are discussed for basic cell biology and high-throughput biotechnology.

Animals↗
PubMed2002

Electrochemotherapy of horses. A preliminary clinical report.

Sarcoids are skin spontaneous tumours detected in horses. It can be cured by chemotherapy by using cisplatin. A multisequence treatment must be performed. Problems are present due to the poor diffusion of the hydrophilic product in the tumours. Electropulsation is known to drastically enhance the effect of antitumoral drugs in vivo. Taking into account the very successful results of the group in Ljubljana (Slovenia), we started a research clinical program where electropulsation was applied after local cisplatin injection. The size of sarcoids is large (several centimeters). A specially designed set of wire contact electrodes was built. The distance between the electrodes was 0.9 cm and their length was 0.9 cm. The contact with the skin was obtained by a conductive paste. A PS15 Jouan Electropulsator was used to deliver eight pulses of 0.1 ms at a 1-Hz frequency with a 1.3-kV voltage. The animal was anesthesized. Intratumoral cisplatin injections were operated every 0.6 cm (0.2 ml at a 1-mg/ml concentration). Five minutes after the first drug injection, multiple electrotreatments were applied by moving the electrodes between the pulse applications. This allows the treatment of all the tumour surface. Several successive treatments were performed with a delay of 2 weeks between each. All lesions completely responded. The sarcoids disappear after only 2 or 3 electrochemotherapies. Objective responses were obtained in 100% of the treated lesions. All horses tolerated the treatment well. No adverse effect from the electric pulses was observed even in the case of a high number of pulses, or when several consecutive treatments were applied. No regrowth was observed in the 18 months follow-up period.

Animals↗
PubMed2002

Factors controlling electropermeabilisation of cell membranes.

Electric field pulses are a new approach for drug and gene delivery for cancer therapy. They induce a localized structural alteration of cell membranes. The associated physical mechanisms are well explained and can be safely controlled. A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. Electric field pulses with an overcritical intensity evoke a local membrane alteration. A free exchange of hydrophilic low molecular weight molecules takes place across the membrane. A leakage of cytosolic metabolites and a loading of polar drugs into the cytoplasm are obtained. The fraction of the cell surface which is competent for exchange is a function of the field intensity. The level of local exchange is strongly controlled by the pulse duration and the number of successive pulses. The permeabilised state is long lived. Its lifetime is under the control of the cumulated pulse duration. Cell viability can be preserved. Gene transfer is obtained but its mechanism is not a free diffusion. Plasmids are electrophoretically accumulated against the permeabilised cell surface and form aggregates due to the field effect. After the pulses, several steps follow: translocation to the cytoplasm, traffic to the nucleus and expression. Molecular structural and metabolic changes in cells remain mostly poorly understood. Nevertheless, while most studies were established on cells in culture (in vitro), recent experiments show that similar effects are obtained on tissue (in vivo). Transfer remains controlled by the physical parameters of the electrical treatment.

Animals↗
PubMed2001

Control by membrane order of voltage-induced permeabilization, loading and gene transfer in mammalian cells.

Cells can be transiently permeabilized by application of electric pulses. A direct consequence of this treatment is to create a new state in the membrane leading to DNA and protein transfers. A key step, in the interaction between macromolecules and the electropermeabilized membrane, is involved. We previously reported that membrane and DNA associated hydration and undulation forces appeared to be involved in this process by studying the effects of osmotic pressure. Effects of ethanol (EtOH) and L-alpha-lysophosphatidylcholine (lyso-PC), molecules known to affect membrane order and therefore undulation forces, were investigated on Chinese hamster ovary (CHO) cells. We used millisecond square wave pulses, conditions giving high efficiency for gene transfer. No effect was observed on cell permeabilization for small sized molecules. Only little change on electroloading of proteins such as R-phycoerythrin was obtained in presence of EtOH. But, a decrease (increase) in electrotransfection was observed for cells treated with EtOH (lyso-PC). Under our conditions, no additional effects of the chemical treatment were observed on cell viability and on membrane resealing. These results tentatively explained in terms of the effect of membrane order on membrane organization and interaction between molecules and membrane supports the existence of the plasmid-membrane interaction in the mechanism of electrically mediated gene transfer.

Animals↗
PubMed2001

Antitumor activity of 2',3'-dideoxycytidine nucleotide analog against tumors up-regulating DNA polymerase beta.

DNA polymerase beta (Pol beta), an error-prone DNA-synthesizing enzyme tightly down-regulated in healthy somatic cells, has been shown to be overexpressed in many human tumors. In this study, we show that treatment with the 2',3'-dideoxycytidine (ddC) nucleoside analog inhibited in vitro and in vivo the proliferation of Pol beta-transfected B16 melanoma cells, which up-regulate Pol beta compared with control isogenic cells. The administration of ddC also increased specifically the survival of mice bearing Pol beta-overexpressing B16 melanoma. When the phosphorylated form of ddC was electrotransfered into Pol beta-transfected melanoma, the cell growth inhibition was strengthened, strongly suggesting that the cytotoxic effect results from incorporation of the chain terminator into DNA. Using in vitro single- and double-stranded DNA synthesis assays, we demonstrated that excess Pol beta perturbs the replicative machinery, favors ddC-TP incorporation into DNA, and consequently promotes chain termination. Therefore, the use of chain terminator anticancer agents could be suitable for the treatment of tumors with a high level of Pol beta.

Animals↗
PubMed2000

Effect of serum on in vitro electrically mediated gene delivery and expression in mammalian cells.

In many cell systems, electric pulses can efficiently mediate gene transfer with a high level of expression in vitro. In vivo results have been reported where decrease in efficiency was obtained. The mechanisms involved in the process are unknown. Since, in vivo, the efficiency of non-viral methods of gene transfer is generally limited by the presence of serum, we report here the effect of serum on in vitro electrically mediated chinese hamster ovary cell membrane permeabilization, viability, gene transfer and expression. The results indicate that permeabilization and gene transfer are not inhibited by serum. By acting as a protector of cell viability, serum indeed increases gene transfer and expression.

Animals↗
PubMed2000

Electrochemotherapy of cutaneous metastases in malignant melanoma.

Electrochemotherapy is a new anticancer therapy in which transient permeabilization of cells by an electric field induces a significant increase in the bleomycin concentration and toxicity in tumour cells. We report a clinical study of electrochemotherapy in malignant melanoma. The main issues addressed were the effect of the size of the nodules, the optimization of the electrical parameters, and posttreatment clinical observations. Four patients were enrolled in the study. They received a 10 mg/m2 dose of bleomycin administered intravenously, followed by short, intense electric pulses applied directly to the skin at the tumour sites. Antitumour effects were obtained, especially in the smallest nodules. Objective responses were obtained in more than 90% of the 55 nodules treated, with a complete response rate of 9%. All patients tolerated the treatment well. No residual effects from the electric pulses were observed, even when a high number of pulses were required or when two consecutive treatments were applied. These results are encouraging and the study should be continued.

Adult↗
PubMed1998

Control by ATP and ADP of voltage-induced mammalian-cell-membrane permeabilization, gene transfer and resulting expression.

We have permeabilized and transfected mammalian cells by transient alteration of their native transmembrane electrical potential difference. Chinese hamster ovary cells were chosen as a model in order to study the electropermeabilization and electrotransfection processes. Propidium iodide was used to monitor permeabilization. A plasmid carrying the beta-galactosidase gene was used to follow direct gene transfer and expression by determining transient expression of the electrotransfered activity at the single-cell level. The effect of nucleotides on cell permeabilization and transfection was studied by altering the cytosolic ATP and ADP contents of cells either during the pulsation or during the period following it. Permeabilization and transfection are not regulated in the same way by the ATP and ADP levels. The permeabilization efficiency remains unaffected. Cell viability and the transfection yield are dramatically affected. While ADP is involved in the step of DNA transfer across the electropermeabilized plasma membrane, ATP controls other steps (cytoplasmic DNA migration towards the nucleus, expression). Our results prove, firstly, that membrane resealing is required but is not sufficient to preserve cell viability and, secondly, that transfection is a cell-mediated process and not only an electrophoretic step driven by the external field.

Adenosine Diphosphate↗
PubMed1998

Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.

Membrane electropermeabilization to small molecules depends on several physical parameters (pulse intensity, number, and duration). In agreement with a previous study quantifying this phenomenon in terms of flow (Rols and Teissié, Biophys. J. 58:1089-1098, 1990), we report here that electric field intensity is the deciding parameter inducing membrane permeabilization and controls the extent of the cell surface where the transfer can take place. An increase in the number of pulses enhances the rate of permeabilization. The pulse duration parameter is shown to be crucial for the penetration of macromolecules into Chinese hamster ovary cells under conditions where cell viability is preserved. Cumulative effects are observed when repeated pulses are applied. At a constant number of pulses/pulse duration product, transfer of molecules is strongly affected by the time between pulses. The resealing process appears to be first-order with a decay time linearly related to the pulse duration. Transfer of macromolecules to the cytoplasm can take place only if they are present during the pulse. No direct transfer is observed with a postpulse addition. The mechanism of transfer of macromolecules into cells by electric field treatment is much more complex than the simple diffusion of small molecules through the electropermeabilized plasma membrane.

Animals↗
PubMed1998

Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells.

Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer.

Animals↗
PubMed1998

Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy.

Electrochemotherapy (ECT) enhances the effectiveness of chemotherapeutic agents by administering the drug in combination with short intense electric pulses. ECT is effective because electric pulses permeabilize tumour cell membranes and allow non-permeant drugs, such as bleomycin, to enter the cells. The aim of this study was to demonstrate the anti-tumour effectiveness of ECT with bleomycin on cutaneous and subcutaneous tumours. This article summarizes results obtained in independent clinical trials performed by five cancer centres. A total of 291 cutaneous or subcutaneous tumours of basal cell carcinoma (32), malignant melanoma (142), adenocarcinoma (30) and head and neck squamous cell carcinoma (87) were treated in 50 patients. Short and intense electric pulses were applied to tumours percutaneously after intravenous or intratumour administration of bleomycin. The tumours were measured and the response to the treatment evaluated 30 days after the treatment. Objective responses were obtained in 233 (85.3%) of the 273 evaluable tumours that were treated with ECT. Clinical complete responses were achieved in 154 (56.4%) tumours, and partial responses were observed in 79 (28.9%) tumours. The application of electric pulses to the patients was safe and well tolerated. An instantaneous contraction of the underlying muscles was noticed. Minimal adverse side-effects were observed. ECT was shown to be an effective local treatment. ECT was effective regardless of the histological type of the tumour. Therefore, ECT offers an approach to the treatment of cutaneous and subcutaneous tumours in patients with minimal adverse side-effects and with a high response rate.

Adenocarcinoma↗
PubMed1998

In vivo electrically mediated protein and gene transfer in murine melanoma.

We show that efficient permeabilization of murine melanoma can be obtained in vivo by applying electric pulses. More than 80% of the cell population is affected as shown by the penetration of propidium iodide. A protein, beta-galactosidase, can be transferred and expressed into the cells by incorporating either the protein or a plasmid carrying the reporter gene with respective efficiencies of 20% and 4%. This is obtained by a direct injection of either the protein or the plasmid in the tumor, followed by the application of electric pulses with surface electrodes in contact with the skin. This approach is simple and safe to use, reproducible, and specific; moreover, it is potentially applicable to a wide variety of tissues, cell types, and animals.

Animals↗