Laure Gibot

Polymeric Micelles Encapsulating Photosensitizer: Structure/Photodynamic Therapy Efficiency Relation

Various polymeric micelles were formed from amphiphilic block copolymers, namely, poly(ethyleneoxide-b-ε-caprolactone), poly(ethyleneoxide-b-d,l-lactide), and poly(ethyleneoxide-b-styrene). The micelles were characterized by static and dynamic light scattering, electron microscopy, and asymmetrical flow field-flow fractionation. They all displayed a similar size close to 20 nm. The influence of the chemical structure of the block copolymers on the stability upon dilution of the polymeric micelles was investigated to assess their relevance as carriers for nanomedicine. In the same manner, the stability upon aging was assessed by FRET experiments under various experimental conditions (alone or in the presence of blood proteins). In all cases, a good stability over 48 h for all systems was encountered, with PDLLA copolymer-based systems being the first to release their load slowly. The cytotoxicity and photocytotoxicity of the carriers were examined with or without their load. Lastly, the photodynamic activity was assessed in the presence of pheophorbide a as photosensitizer on 2D and 3D tumor cell culture models, which revealed activity differences between the 2D and 3D systems.

Antitumor drug delivery in multicellular spheroids by electropermeabilization.

Electrochemotherapy (ECT) is a physical technique that allows cytotoxic molecules to be efficiently released in tumor cells by inducing transient cell plasma membrane permeabilization. The main antitumoral drugs used in ECT are nonpermeant bleomycin and low permeant cisplatin. The method is nowadays applied in clinics as a palliative treatment. In order to improve it, we took advantage of a human 3D multicellular tumor spheroid as a model of tumor to visually and molecularly assess the effect of ECT. We used bleomycin and cisplatin to confirm its relevance and doxorubicin to show its potential to screen new antitumor drug candidates for ECT. Confocal microscopy was used to visualize the topological distribution of permeabilized cells in 3D spheroids subjected to electric pulses. Our results revealed that all cells were efficiently permeabilized, whatever their localization in the spheroid, even those in the core. The combination of antitumor drugs and electric pulses (ECT) led to changes in spheroid macroscopic morphology and cell cohesion, to tumor spheroid growth arrest and finally to its complete apoptosis-mediated dislocation, mimicking previously observed in vivo situations. Taken together, these results indicate that the spheroid model is relevant for the study and optimization of electromediated drug delivery protocols.

Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model

Background Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy–and normal cell sensitivity. Methods Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). Results The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p<0.0001) or electrochemotherapy using bleomycin (p<0.0001). Strikingly, the size of normal fibroblast spheroids was neither affected after calcium electroporation nor electrochemotherapy using bleomycin, indicating that calcium electroporation, like electrochemotherapy, will have limited adverse effects on the surrounding normal tissue when treating with calcium electroporation. The intracellular ATP level, which has previously been shown to be depleted after calcium electroporation, was measured in the spheroids after treatment. The results showed a dramatic decrease in the intracellular ATP level (p<0.01) in all four spheroid types—malignant as well as normal. Conclusion In conclusion, calcium electroporation seems to be more effective in inducing cell death in cancer cell spheroids than in a normal fibroblast spheroid, even though intracellular ATP level is depleted in all spheroid types after treatment. These results may indicate an important therapeutic window for this therapy; although further studies are needed in vivo and in patients to investigate the effect of calcium electroporation on surrounding normal tissue when treating tumors.

Progress and prospects: the use of 3D spheroid model as a relevant way to study and optimize DNA electrotransfer.

The use of electrotransfer to deliver therapeutic agents such as cytotoxic drugs and nucleic acids to cells and tissues has been successfully developed over the last decade. This strategy is promising for the systemic secretion of therapeutic proteins, vaccination and gene therapy. The safe and efficient use of this physical method for clinical purposes requires knowledge of the mechanisms underlying the DNA electrotransfer and expression phenomena. Despite the fact that the pioneering work on plasmid DNA electrotransfer to cells was initiated 30 years ago, many of the underlying mechanisms remain elucidated. While efficient in vitro, the method faces a lack of efficiency in packed tissues. Until now, the great majority of studies have been performed on cells in 2D cultures in Petri dishes or in suspension. However, these studies cannot get access to the tissue-specific architecture and organization present in 3D living tissues. In this context, 3D cell culture models are more relevant concerning in vivo cell organization since cell-cell contacts are present as well as extracellular matrix. The aim of this review is to describe the relevance of using spheroid as a model to address and improve the electrotransfer processes.

Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue

DNA electrotransfer is a successful technic for gene delivery. However, its use in clinical applications is limited since little is known about the mechanisms governing DNA electrotransfer in the complex environment occurring in a tissue. The objectives of this work were to investigate the role of the extracellular matrix (ECM) in that process. Tumor ECM composition was shown to modulate in vivo gene electrotransfer efficiency. In order to assess the effects of ECM composition and organization, as well as intercellular junctions and communication, in normal tissue response to electric pulses, we developed an innovative three-dimensional (3D) reconstructed human connective tissue model. 3D human dermal tissue was reconstructed in vitro by a tissue engineering approach and was representative of in vivo cell organization since cell–cell contacts were present as well as complex ECM. This human cell model presented multiple layers of primary dermal fibroblasts embedded in a native, collagen-rich ECM. This dermal tissue could become a useful tool to study skin DNA electrotransfer mechanisms. As proof of the concept, we show here that the cells within this standardized 3D tissue can be efficiently electropermeabilized by milliseconds electric pulses. We believe that a better comprehension of gene electrotransfer in such a model tissue would help improve electrogene therapy approaches such as the systemic delivery of therapeutic proteins and DNA vaccination.

Crosslinked polymeric self-assemblies as an efficient strategy for photodynamic therapy on a 3D cell culture

In order to compare the efficiency of crosslinked nano-vectors in the field of photodynamic therapy (PDT) both on 2D and 3D cell cultures, various polymeric crosslinked self-assemblies based on poly(ethyleneoxide-b-e-caprolactone) have been synthesized by radical polymerization of acrylate end-functionalized polymers. Crosslinked self-assemblies obtained from the reaction of the functionalized polymers with ethyleneglycoldimethacrylate (EGDMA) were compared to chain-end polymerized and to unreacted ones. Polymeric micelles with a size between 10 and 20 nm were obtained, as well as an elongated system with a length close to 100 nm. They all have been characterized by Transmission Electron Microscopy and Dynamic Light Scattering but also by Asymmetrical Flow Field-Flow Fractionation in order to prove that they consisted of pure self-assemblies. Chain-end polymerization or crosslinking did not induce any change in morphology nor strong size modification. After post-encapsulation of a photosensitizer, namely Pheophorbide a, the systems have been examined for their potential use in PDT on HCT-116 and FaDu human tumor cell lines both in 2D and 3D cultures. The crosslinked vectors were observed to be the most efficient on both cell lines cultivated in 3D spheroids, whereas unreacted or chain-end polymerized ones presented a lower activity. This was different from the trend observed in 2D cell cultures where an uncrosslinked micelle was observed to be efficient at a lower concentration compared to its chain-end polymerized or crosslinked analogue. The different synthesized self-assemblies also allowed assessing the influence of polymer chain length and shape on PDT efficiency. The molecular weight of the polymer did not lead in our case to efficiency change, for similar size and surface characteristics. As for the shape effect, the elongated self-assembly was not observed in our case to be more efficient than spherical micelles. Crosslinked polymeric vectors are therefore promising vectors for 3D tumor treatment.

Gene transfer by pulsed electric field is highly promising in cutaneous wound healing

Introduction: Tremendous progress has been achieved during the last decades in electroporation-based technologies for medicine. Understanding the basic underlying mechanisms of gene delivery opens the way for clinical gene therapy and DNA vaccination. Areas covered: This review focuses on the use of gene electrotherapy in cutaneous tissue repair and how it affects healing. Expert opinion: Gene electrotherapy is safe, efficient and promising as shown by the increasing number of publications reporting evidence for its potential in wound healing. Going deeper into the mechanisms of DNA delivery and expression as well as into skin regeneration at the molecular, cellular and tissues levels will help make it an attractive approach for the treatment of skin pathologies in general.

Self-assembled polymeric vectors mixtures: characterization of the polymorphism and existence of synergistic effects in photodynamic therapy.

The objective of this work was to assess the relation between the purity of polymeric self-assemblies vectors solution and their photodynamic therapeutic efficiency. For this, several amphiphilic block copolymers of poly(ethyleneoxide-b-ε-caprolactone) have been used to form self-assemblies with different morphologies (micelles, worm-like micelles or vesicles). In a first step, controlled mixtures of preformed micelles and vesicles have been characterized both by dynamic light scattering and asymmetrical flow field flow fractionation (AsFlFFF). For this, a custom-made program, STORMS, was developed to analyze DLS data in a thorough manner by providing a large set of fitting parameters. This showed that DLS only sensed the larger vesicles when the micelles/vesicles ratio was 80/20 w/w. On the other hand, AsFlFFF allowed clear detection of the presence of micelles when this same ratio was as low as 10/90. Subsequently, the photodynamic therapy efficiency of various controlled mixtures was assessed using multicellular spheroids when a photosensitizer, pheophorbide a, was encapsulated in the polymer self-assemblies. Some mixtures were shown to be as efficient as monomorphous systems. In some cases, mixtures were found to exhibit a higher PDT efficiency compared to the individual nano-objects, revealing a synergistic effect for the efficient delivery of the photosensitizer. Polymorphous vectors can therefore be superior in therapeutic applications.

Importance of endogenous extracellular matrix in biomechanical properties of human skin model

The physical and mechanical properties of cells modulate their behavior such proliferation rate, migration and extracellular matrix remodeling. In order to study cell behavior in a tissue-like environment in vitro, it is of utmost importance to develop biologically and physically relevant 3D cell models. Here, we characterized the physical properties of a single cell type growing in configurations of increasing complexity. From one human skin biopsy, primary dermal fibroblasts were isolated and seeded to give monolayer (2D model), spheroid (3D model poor in extracellular matrix) and tissue-engineered cell sheet (3D model rich in endogenous extracellular matrix). Living native human dermis tissue was used as a gold standard. Nanomechanical and viscoelastic properties at the cell scale were measured by atomic force microscopy (AFM) while biphoton microscopy allowed collagen detection by second harmonic generation and scanning electron microscopy helped in model morphological characterization. In all models, fibroblasts presented a similar typical elongated cell shape, with a cytoskeleton well-arranged along the long axis of the cell. However, elastic moduli of the tissue-engineered cell sheet and native dermis tissue were similar and statistically lower than monolayer and spheroid models. We successfully carried out AFM force measurements on 3D models such as spheroids and tissue-engineered cell sheets, as well as on living native human tissue. We demonstrated that a tissue-engineered dermal model recapitulates the mechanical properties of human native dermal tissue unlike the classically used monolayer and spheroid models. Furthermore, we give statistical evidence to indicate a correlation between cell mechanical properties and the presence of collagens in the models studied.

How Imaging Membrane and Cell Processes Involved in Electropermeabilization Can Improve Its Development in Cell Biology and in Clinics

Cell membranes can be transiently permeabilized under the application of electric pulses. This process, called electropermeabilization or electroporation, allows hydrophilic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. The method is nowadays used in clinics to treat cancers. Vaccination and gene therapy are other fields of application of DNA electrotransfer. A description of the mechanisms can be assayed by using different complementary systems with increasing complexities (models of membranes, cells cultivated in 2D and 3D culture named spheroids, and tissues in living mice) and different microscopy tools to visualize the processes from single molecules to entire animals. Single-cell imaging experiments revealed that the uptake of molecules (nucleic acids, antitumor drugs) takes place in well-defined membrane regions and depends on their chemical and physical properties (size, charge). If small molecules freely cross the electropermeabilized membrane and have a free access to the cytoplasm, larger molecules, such as plasmid DNA, face physical barriers (plasma membrane, cytoplasm crowding, nuclear envelope) which reduce transfection efficiency and engender a complex mechanism of transfer. Gene electrotransfer indeed involves different steps that include the initial interaction with the membrane, its crossing, transport within the cytoplasm, and finally gene expression. In vivo, additional very important effects of electric pulses are present such as blood flow modifications. The full knowledge on the way molecules are transported across the electropermeabilized membranes and within tissues is mandatory to improve the efficacy and the safety of the electropermeabilization process both in cell biology and in clinics.