Interactions of amphiphilic polyoxazolines formulated or not in lipid nanocapsules with biological systems: Evaluation from membrane models up to in vivo mice epidermis.

In this study, we evaluated the potential of amphiphilic polyoxazolines (POx) to interact with biological membranes thanks to models of increasing complexity, from a simple lipid bilayer using giant unilamellar vesicles (GUV), to plasma membranes of three different cell types, fibroblasts, keratinocytes and melanocytes, which are found in human skin. Upon assessing an excellent penetration into GUV membranes and cultured cells, we addressed POx's potential to penetrate the murine skin within an in vivo model. Exposure studies were made with native POx and with POx encapsulated within lipid nanocapsules (LNC). Our findings indicate that POx's interactions with membranes tightly depend on the nature of the alkyl chain constituting the POx. Saturated C16POx insert rapidly and efficiently into GUV and plasma membranes, while unsaturated C18:2POx insert to a smaller extent. The high amount of membrane-inserted saturated C16POx impacts cell viability to a greater extent than the unsaturated C18:2POx. The in vivo study, performed on mice, showed an efficient accumulation of both POx types in the stratum corneum barrier, reaching the upper epidermis, independently of POx's degree of saturation. Furthermore, the formulation of POx into lipid nanocapsules allowed delivering an encapsulated molecule, the quercetin, in the upper epidermis layers of murine skin, proving POx's efficacy for topical delivery of active molecules. Overall, POx proved to be an excellent choice for topical delivery, which might in turn offer new possibilities for skin treatments in diseases such as psoriasis or melanomas.

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.

Post-pulse addition of trans-cyclohexane-1,2-diol improves electrotransfer mediated gene expression in mammalian cells.

Electric field mediated gene transfer is facing a problem in expression yield due to the poor transfer across the nuclear envelope. Trans-cyclohexane-1,2-diol (TCHD) was shown to significantly increase chemically mediated transfection by collapsing the permeability barrier of the nuclear pore complex. We indeed observed a significant increase in expression by electrotransfer when cells are treated post pulse by a low non toxic concentration of TCHD. This was obtained for different pulsing conditions, cell strains and plasmid constructs. An interesting improvement in cell viability can be obtained. This can significantly enhance the non-viral gene electrical delivery.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of ß2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process.

A double-pulse approach for electrotransfection.

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.

Imaging living cells surface and quantifying its properties at high resolution using AFM in QI™ mode.

Since the last 10 years, AFM has become a powerful tool to study biological samples. However, the classical modes offered (imaging or tapping mode) often damage sample that are too soft or loosely immobilized. If imaging and mechanical properties are required, it requests long recording time as two different experiments must be conducted independently. In this study we compare the new QI™ mode against contact imaging mode and force volume mode, and we point out its benefit in the new challenges in biology on six different models: Escherichia coli, Candida albicans, Aspergillus fumigatus, Chinese hamster ovary cells and their isolated nuclei, and human colorectal tumor cells.

First explanations for differences in electrotransfection efficiency in vitro and in vivo using spheroid model.

Electro-gene-therapy is a promising technique for cancer treatment. However, knowledge about mechanism of gene transfer with electric field in tumor is limited. Whereas in vitro electrotransfection is efficient, gene expression in tumoral cells in vivo is weak. To determine reasons for this difference and unravel gene transfer mechanisms, we propose to use multicellular tumor spheroid as a tridimensional model ex vivo. Comparison of efficiency between cell in suspension and cells in spheroid allow highlighting fundamental differences. For classical electrical conditions (consisting in 10 pulses of 500V/cm, 5ms, 1Hz), suspension cells present a transfection rate of 23.75%±2.450 SEM. In the same conditions on spheroid, although plasmid DNA coding GFP interact with half of electrically permeabilized cells, less than 1% of cells are expressing the transgene. First answers to in vivo electrotransfection failure are given: cell mortality due to electric field is responsible of this low transfection rate, as tridimensional and multicellular structure that prevents DNA passage. These results show that spheroid is reproducing in vivo situation. Validation of spheroid as a relevant model for electrotransfection study opens ex vivo optimization possibility before in vivo assay.

Successful treatment of equine sarcoids with cisplatin electrochemotherapy: a retrospective study of 48 cases.

REASONS FOR PERFORMING STUDY: Sarcoids are the commonest form of equine skin tumour. Several therapeutic measures have been described but none is considered to be universally effective. Electrochemotherapy (ECT) is a new anticancer therapy that utilises electrical field pulses to induce increased cell membrane permeability to antitumour hydrophilic drugs, such as cisplatin. The increased intracellular concentration of the drugs has a significant therapeutic benefit. The procedure has not been previously reported in a large number of horses. OBJECTIVE: To validate ECT as a novel alternative treatment for equine sarcoids. METHODS: A retrospective study evaluating the efficacy of cisplatin ECT in the treatment of equine sarcoids was performed. Electrochemotherapy treatments were applied under general anaesthesia at 2 week intervals with or without prior excision or debulking. Electric pulses were directly applied to the lesions following intra-tumoural injections of an aqueous solution of cisplatin. RESULTS: One-hundred-and-ninety-four sarcoids on 34 horses, 2 ponies, 11 donkeys and one mule were treated with ECT. The 4 year nonrecurrence rate was 97.9% for animals (47/48) and 99.5% (193/194) for tumours. When ECT was used as a single treatment, a significant influence of tumour size (ρ= 0.55) on the number of treatments required for cure was shown. When prior surgery was performed, there was a significant influence (P<0.001) of the excision quality (complete or incomplete) and the healing mode (closed or open wound) on the number of treatments. The most common adverse effect was a slight oedematous reaction for lesions located on thin skin regions. CONCLUSION AND CLINICAL RELEVANCE: Results demonstrate that ECT, with or without concurrent tumour debulking, is an effective alternative for treatment of equine sarcoids.

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.

Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells.

The RNA interference-mediated gene silencing approach is promising for therapies based on the targeted inhibition of disease-relevant genes. Electropermeabilization is one of the nonviral methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is effective in the field of gene silencing by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. When added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cells. Therefore, we report that an electric field acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The transfer kinetics of siRNA are compatible with the creation of nanopores, which are described with the technique of synthetic nanopores. The mechanism involved was clearly specific for the physico-chemical properties of the electrotransferred molecule and was different from that observed with small molecules or plasmid DNA.

Pre-treatment of cells with pluronic L64 increases DNA transfection mediated by electrotransfer.

Gene transfer into muscle cells is a key issue in biomedical research. Indeed, it is important for the development of new therapy for many genetic disorders affecting this tissue and for the use of muscle tissue as a secretion platform of therapeutic proteins. Electrotransfer is a promising method to achieve gene expression in muscles. However, this method can lead to some tissue damage especially on pathologic muscles. Therefore there is a need for the development of new and less deleterious methods. Triblock copolymers as pluronic L64 are starting to be used to improve gene transfer mediated by several agents into muscle tissue. Their mechanism of action is still under investigation. The combination of electrotransfer and triblock copolymers, in allowing softening electric field conditions leading to efficient DNA transfection, could potentially represent a milder and more secure transfection method. In the present study, we addressed the possible synergy that could be obtained by combining the copolymer triblock L64 and electroporation. We have found that a pre-treatment of cells with L64 could improve the transfection efficiency. This pre-treatment was shown to increase cell viability and this is partly responsible for the improvement of transfection efficiency. We have then labelled the plasmid DNA and the pluronic L64 in order to gain some insights into the mechanism of transfection of the combined physical and chemical methods. These experiences allowed us to exclude an action of L64 either on membrane permeabilization or on DNA/membrane interaction. Using plasmids containing or not binding sequences for NF-κB and an inhibitor of NF-κB pathway activation we have shown that this beneficial effect was rather related to the NF-κB signalling pathway, as it is described for other pluronics. Finally we address here some mechanistic issues on electrically mediated transfection, L64 mediated membrane permeabilization and the combination of both for gene transfer.

In vitro gene transfer by electrosonoporation.

Among the nonviral methods for gene delivery in vitro, electroporation is simple, inexpensive and safe. To upregulate the expression level of transfected gene, we investigated the applicability of electrosonoporation. This approach consists of a combination of electric pulses and ultrasound assisted with gas microbubbles. Cells were first electroporated with plasmid DNA encoding-enhanced green fluorescent protein and then sonoporated in presence of contrast microbubbles. Twenty-four hours later, cells that received electrosonoporation demonstrated a four-fold increase in transfection level and a six-fold increase in transfection efficiency compared with cells having undergone electroporation alone. Although electroporation induced the formation of DNA aggregates into the cell membrane, sonoporation induced its direct propulsion into the cytoplasm. Sonoporation can improve the transfer of electro-induced DNA aggregates by allowing its free and rapid entrance into the cells. These results demonstrated that in vitro gene transfer by electrosonoporation could provide a new potent method for gene transfer.

A 3D in vitro spheroid model as a way to study the mechanisms of electroporation.

Electropermeabilization is a physical method to deliver molecules into cells and tissues. Clinical applications have been successfully developed for antitumoral drug delivery and clinical trials for gene electrotransfer are currently underway. However, little is known about the mechanisms involved in this transfer. The main difficulties stem from the lack of single cell models which reliably replicate the complex in vivo environment. In order to increase our understanding of the DNA electrotransfer process, we exploited multicellular tumor spheroids as an ex vivo model of tumor. We used confocal microscopy to visualize the repartition of permeabilized cells in spheroids subjected to electric pulses. Our results reveal that even if cells can be efficiently permeabilized with electric fields, including those cells present inside the spheroids, gene expression is by contrast limited to the external layers of cells. Taken together, these results, in agreement with the ones obtained in tumors, indicate that the spheroid model is more relevant to an in vivo situation than cells cultured as monolayers. They validate the spheroid model as a way to study electro-mediated gene delivery processes.

Control by pulse parameters of DNA electrotransfer into solid tumors in mice.

Electrotransfer (electroporation) is recognized as one of the most promising alternatives to viral vectors for transfection of different tissues in vivo for therapeutic purposes. We evaluated the transfection efficiency of reporter genes (green fluorescent protein and luciferase) in murine subcutaneous tumors using different combinations of high-field (HV) (600-1400 V cm(-1), 100 mus, 8 pulses) and low-field (LV) (80-160 V cm(-1), 50-400 ms, 1-8 pulses) pulses and compared it to protocol using eight identical pulses of 600 V cm(-1) and 5 ms duration (electro-gene therapy, EGT). Expression of GFP was determined using a fluorescent microscope and flow cytometry and expression of luciferase by measuring its activity using a luminometer. The EGT protocol yielded the highest expression of both reporter genes. However, a careful optimization of combinations of HV and LV pulses may result in similar transfection as EGT pulses. With the combination protocol, relatively high fields of LV pulses were necessary to obtain comparable transfection to the EGT protocol. Expression of reporter genes was higher in B16 melanoma than in SA-1 fibrosarcoma. Our data support the hypothesis that both electropermeabilization and electrophoresis are involved in electrotransfer of plasmid DNA, but demonstrate that these components have to happen at the same time to obtain significant expression of the target gene in tumors.

Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin.

Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommendations for gene transfer to various types of tissues.

Membrane perturbation by an external electric field: a mechanism to permit molecular uptake.

Electropermeabilisation is a well established physical method, based on the application of electric pulses, which induces the transient permeabilisation of the cell membrane. External molecules, otherwise nonpermeant, can enter the cell. Electropermeabilisation is now in use for the delivery of a large variety of molecules, as drugs and nucleic acids. Therefore, the method has great potential in the fields of cancer treatment and gene therapy. However many open questions about the underlying physical mechanisms involved remain to be answered or fully elucidated. In particular, the induced changes by the effects of the applied field on the membrane structure are still far from being fully understood. The present review focuses on questions related to the current theories, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses recent findings using molecular dynamics simulations as well as experimental studies of the effect of the field on membrane components.

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.

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.

Induction of apoptosis by electrotransfer of positively charged proteins as Cytochrome C and Histone H1 into cells.

Cytochrome C (Cyt. C) is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown mechanism, but regulated by the bcl-2 family of proteins. The linker Histone H1 is another basic protein with highly conservative structure, composition, and equal molecular weight, not changed during the evolution. An attempt was made to understand better the apoptotic processes by electroloading of leukemic cells, such as K562, HL-60, and SKW3, and human lymphocytes with positively charged proteins, such as Cyt. C, Histone H1, and methylated BSA albumin (mBSA). The triggering apoptotic processes followed by MTT test, FACS analysis, and DNA fragmentation after the electrotransfer of these proteins into the cells were observed. Histone H1 and mBSA induce the release of Cyt. C from rat liver mitochondria. Cytochrome C release was higher when mitochondria were in "high-energy" state. It is supposed that release of Cyt. C from mitochondria is due to the mechanical rupture of the outer mitochondrial membrane, rich in negatively charged groups, predominately due to cardiolipin. The reason for the morphological rupture of the outer mitochondial membrane could be the rigidification and segregation of the membrane and the destroyed membrane asymmetries of both monolayers in the presence of positively charged proteins at higher linear charges such as Histone H1. We suggested that Histone H1, at a given moment of activated signaling for apoptosis, could be not transported to the nucleus and could lead to the release of Cyt. C from the mitochondria in the cytoplasm. It is temping to speculate that Histone H1 has other physiological extranuclear functions involved in apoptosis.

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.