Elisabeth Bellard

Drug delivery by electropulsation: Recent developments in oncology.

Electro-permeabilisation allows the free access of polar compounds to the cytoplasm by a reversible alteration of the cell membrane. It is now used in clinics for the eradication of cutaneous solid tumors. New developments predict its future applications for other anti-cancer treatments.

A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor

A monoclonal anti-CD160 antibody inhibits the growth of new vessels in pathological ocular and tumor neoangiogenesis but not in healthy tissues.

Membrane disorder and phospholipid scrambling in electropermeabilized and viable cells.

Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field.

Giant lipid vesicles under electric field pulses assessed by non invasive imaging

We present experimental results regarding the effects of electric pulses on giant unilamellar vesicles (GUVs). We have used phase contrast and coherent anti-Stokes Raman scattering (CARS) microscopy as relevant optical approaches to gain insight into membrane changes under electropermeabilization. No addition of exogenous molecules (lipid analogue, fluorescent dye) was needed. Therefore, experiments were performed on pure lipid systems avoiding possible artefacts linked to their use. Structural membrane changes were assessed by loss of contrast inside the GUVs due to sucrose and glucose mixing. Our observations, performed at the single vesicle level, indicate these changes are under the control of the number of pulses and field intensity. Larger number of pulses enhances membrane alterations. A threshold value of the field intensity must be applied to allow exchange of molecules between GUVs and the external medium. This threshold depends on the size of the vesicles, the larger GUVs being affected at lower electric field strengths than the smaller ones. Our experimental data are well described by a simple model in which molecule entry is driven by direct exchange. The CARS microscopic study of the effect of pulse duration confirms that pulses, in the ms time range, induce loss of lipids and membrane deformations facing the electrodes.

LNA-based Oligonucleotide Electrotransfer for miRNA Inhibition

Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease development and progression and therefore represent a potential new class of therapeutic targets. However, an effective and safe clinical approach for miRNA inhibition remains elusive, primarily due to the lack of effective delivery methods. We proposed to inhibit miRNA by electrotransferring an antisense DNA oligomer containing locked nucleic acids (LNAs) (LNA/DNA oligomer). We observed that electropulsation (EP) led to a strong cellular uptake of LNA/DNA oligomer. The LNA/DNA oligomer electrotransfer mechanism and intracellular localization were visually investigated in real time at the single-cell level. Cyanine 5-labeled oligonucleotide entered exclusively during pulse application on the side of the permeabilized cell membrane facing the cathode, driven by electrophoretic forces. Minutes after the electrotransfer, the LNA/DNA oligomer diffused into the nucleus. EP provided the anti-miRNA oligomer with immediate and direct access to its cytoplasmic mature miRNA target and/or its nuclear precursor miRNA target. We then demonstrated using a LNA/DNA oligomer anti-miR34a that LNA/DNA oligomer electrotransfer decreased the level of the miR34a target and induced its functional inhibition. Our findings show that using the electrotransfer technique for LNA-based oligonucleotide delivery is a promising therapeutic strategy to silence deleterious miRNAs overexpressed in diseases.

Fluorescence imaging agents in cancerology.

Fluorescence imaging agents in cancerology Background. One of the major challenges in cancer therapy is to improve early detection and prevention using novel targeted cancer diagnostics. Detection requests specific recognition. Tumor markers have to be ideally present on the surface of cancer cells. Their targeting with ligands coupled to imaging agents make them visible/detectable. Conclusions. Fluorescence imaging is a newly emerging technology which is becoming a complementary medical method for cancer diagnosis. It allows detection with a high spatio-temporal resolution of tumor markers in small animals and in clinical studies. In this review, we focus on the recent outcome of basic studies in the design of new approaches (probes and devices) used to detect tumor cells by fluorescence imaging.

Direct Validation of Aptamers as Powerful Tools to Image Solid Tumor

Visualization of cancer cells requires distinguishing malignant from normal cells by objective criteria with high specificity. For several years, tumor markers expressed on the surface of cancer cells have been characterized as cancer signatures, and their labeling with specific imaging probes has revolutionized cancer diagnosis. This specific labeling is also an important tool in surgery tumor ablation. The present study considers the tumor labeling potential of an aptamer that specifically recognizes the epithelial cancer biomarker mucin1 (MUC1). This anti-MUC1 aptamer was investigated in vitro in a three-dimensional (3D) environment and compared to an anti-MUC1 antibody for its capacity to visualize cancer cells. Multicellular spheroids of breast cancer MCF-7 cells were used as tumor models and anti-MUC1 fluorescent aptamer and antibody were visualized by fluorescence imaging. Results showed that the antibodies interacted only with cells located on the surface of the spheroid, whereas the anti-MUC1 aptamers were able to penetrate inside these 3D tumor models and thereafter internalized into the cancer cells. Due to their lack of immunogenicity and their facility to be chemically modified, aptamers may replace advantageously the use of antibodies in diagnosis based on imaging setup thanks to their specific detection of cancer cells without invasive surgical procedures or during clinical intraoperative intervention.

Transgene expression of transfected supercoiled plasmid DNA concatemers in mammalian cells

*Correspondence to: Jean-Michel Escoffre, Justin Teissie, CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077, Toulouse, France. E-mail: jean-michel.escoffre@ipbs.fr; justin.teissie@ipbs.fr We read with interest the article by Maucksch et al. [1], who analysed transgene expression of transfected supercoiled 4.7 kb monomeric and 9.4 kb dimeric plasmid concatemer in Jurkat T cells using electroporation. The authors reported that the relative number of electrotransferred gene copies per nucleus and plasmid expression efficiency, respectively, were 1.6and 3.5-fold higher for enhanced green fluorescent protein (EGFP)-dimer than for EGFP-monomer. However, the transfection rates considering the number of transfected cells were significantly lower for EGFP-dimer than for EGFPmonomer. Their conclusions were that more dimers were introduced in fewer cells. The relative number of gene copies introduced in the different cellular compartments (cytoplasm and nucleus) was evaluated using a fluorescence approach, which we described some years ago [2]. This took advantage of the spontaneous binding of TOTO dyes to DNA. Therefore, Maucksch et al. [1] have used noncovalent markers (SYTO-13 and TOTO-1) to determine the biodistribution of plasmids into the cells after electroporation. TOTO is virtually nonfluorescent in solution, but forms highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1 : 4, with a 1000-fold fluorescence enhancement. The dsDNA–TOTO complexes are completely stable to electrophoresis on agarose and acrylamide gels [3]. However, TOTO-1 is a dye capable of bisintercalation, although it interacts with dsDNA and ssDNA with a similar high affinity. Binding of the dye partially unwinds the DNA by distorting and elongating the helix. An external binding mode, where the dipole of the dye molecule is aligned with the DNA grooves, may be more important. TOTO-1 dye will bind to almost any sequence in dsDNA. TOTO binding with DNA is an equilibrium, where the dye can unbind if free DNA is added (i.e. equilibrium displacement) [4]. This is indeed the case when plasmids bearing TOTO enter in the cytoplasm where free DNA is present. Furthermore, TOTO can freely leak from the cytoplasm to the nucleus (where, again, free DNA is present) in a way similar to that reported for propidium iodide [4]. TOTO labelling DNA comprises a good tool for assaying the early steps of DNA entry into cells but cannot be used to investigate the process over a long period time. If this method is used to detect plasmid/membrane interaction (Figure 1), this approach is not adapted to study the intracellular traffic of plasmid. Indeed, in living or fixed cells, the fluorescent marker can dissociate from plasmid and label intracellular nucleic acids. This comprises the traffic of TOTO. Consequently, the visualization and quantification of plasmid biodistribution into cells can be biased. In our studies, to eliminate these artefacts, we used a dye covalently bound to the plasmid after checking that the level of protein expression was not affected. Indeed, we used the covalently rhodamine-labelled plasmid (pGeneGrip; Gene Therapy Systems, San Diego, CA, USA) to observe the cell traffic of fluorescent-labelled plasmid after electroporation [2]. Golzio et al. [2] demonstrated that this plasmid/membrane interaction is one of the key steps for obtaining gene expression. During the application

Electrochemotherapy guided by intraoperative fluorescence imaging for the treatment of inoperable peritoneal micro-metastases.

Surgery is often the first therapeutic indication in cancer. Patient survival essentially depends on the completeness of tumor resection. This is a major challenge, particularly in patients with peritoneal carcinomatosis (PC), where tumors are widely disseminated in the large peritoneal cavity. These small tumors can be difficult to visualize and are often positioned in delicate locations, further increasing the risk of producing serious tissue/organ damage during their ablation. We propose an innovative therapeutic approach based on intraoperative fluorescence (IF) guided electrochemotherapy (ECT) for the treatment of peritoneal micro-metastases. ECT combines the effects of tissue electro-permeabilization (EP) with the administration of an antimitotic agent (bleomycin) that has poor permeability across intact membranes. IF significantly improves the detection of small tumor lesions. ECT is clinically validated for the treatment of cutaneous tumors in animals and humans, but this is the first time that it has been used along with IF imaging for the targeted treatment of peritoneal metastases in a preclinical model. We set up a murine model of PC that develops secondarily to the resection of a distant primary tumor. Tumor growth and metastasis were finely monitored by non-invasive multimodal imaging (bioluminescence and 3D fluorescence/microCT). Once metastases were detected, mice were randomized into three groups: the ECT group (bleomycin injected intravenously followed by EP) and 2 control groups (bleomycin alone and EP alone). Twenty four hours after the intravenous injection of the tumor targeting agent Angiostamp™700, mice in all groups underwent an abdominal surgery for metastases exploration assisted by fluorescence imaging with the Fluobeam®700 portative device. EP was applied to every nodule detected by IF, except in the bleomycin control group. After surgery, the metastatic invasion was tracked by bioluminescence imaging. In mice treated with bleomycin or EP alone, the metastatic load progressed very rapidly and mice showed no significant difference in lifespan compared to non-operated mice (median lifespan: 27days vs. 25days, respectively). In contrast, the mice treated with ECT displayed a decreased metastatic load and an increased survival rate (median lifespan: 34days). These results provide evidence that IF guided ECT is an effective approach for the treatment of inoperable intraperitoneal micro-metastases.

A Double-Pulse Approach For Electrotransfection

Abstract Gene transfer and expression can be obtained by delivering calibrated electric pulses on cells in the presence of plasmids coding for the activity of interest. The electric treatment affects the plasma membrane and induces the formation of a transient complex between nucleic acids and the plasma membrane. It results in a delivery of the plasmid in the cytoplasm. Expression is only obtained if the plasmid is translocated inside the nucleus. This is a key limit in the process. We previously showed that delivery of a high-field short-duration electric pulse was inducing a structural alteration of the nuclear envelope. This study investigates if the double-pulse approach (first pulse to transfer the plasmid to the cytoplasm, and second pulse to induce the structural alteration of the envelope) was a way to enhance the protein expression using the green fluorescent protein as a reporter. We observed that not only the double-pulse approach induced the transfection of a lower number of cells but moreover, these transfected cells were less fluorescent than the cells treated only with the first pulse.