RESUMO
Cellular response upon nsPEF exposure depends on different parameters, such as pulse number and duration, the intensity of the electric field, pulse repetition rate (PRR), pulsing buffer composition, absorbed energy, and local temperature increase. Therefore, a deep insight into the impact of such parameters on cellular response is paramount to adaptively optimize nsPEF treatment. Herein, we examined the effects of nsPEF ≤ 10 ns on long-term cellular viability and growth as a function of pulse duration (2-10 ns), PRR (20 and 200 Hz), cumulative time duration (1-5 µs), and absorbed electrical energy density (up to 81 mJ/mm3 in sucrose-containing low-conductivity buffer and up to 700 mJ/mm3 in high-conductivity HBSS buffer). Our results show that the effectiveness of nsPEFs in ablating 3D-grown cancer cells depends on the medium to which the cells are exposed and the PRR. When a medium with low-conductivity is used, the pulses do not result in cell ablation. Conversely, when the same pulse parameters are applied in a high-conductivity HBSS buffer and high PRRs are applied, the local temperature rises and yields either cell sensitization to nsPEFs or thermal damage.
Assuntos
Neoplasias , Esferoides Celulares , Humanos , Temperatura , EletricidadeRESUMO
Cold Atmospheric Plasma (CAP) is an emerging technology with great potential for biomedical applications such as sterilizing equipment and antitumor strategies. CAP has also been shown to improve skin wound healing in vivo, but the biological mechanisms involved are not well known. Our study assessed a possible effect of a direct helium jet CAP treatment on keratinocytes, in both the immortalized N/TERT-1 human cell line and primary keratinocytes obtained from human skin samples. The cells were covered with 200 µL of phosphate buffered saline and exposed to the helium plasma jet for 10−120 s. In our experimental conditions, micromolar concentrations of hydrogen peroxide, nitrite and nitrate were produced. We showed that long-time CAP treatments (≥60 s) were cytotoxic, reduced keratinocyte migration, upregulated the expression of heat shock protein 27 (HSP27) and induced oxidative cell stress. In contrast, short-term CAP treatments (<60 s) were not cytotoxic, did not affect keratinocyte proliferation and differentiation, and did not induce any changes in mitochondria, but they did accelerate wound closure in vitro by improving keratinocyte migration. In conclusion, these results suggest that helium-based CAP treatments improve wound healing by stimulating keratinocyte migration. The study confirms that CAP could be a novel therapeutic method to treat recalcitrant wounds.
Assuntos
Gases em Plasma , Proteínas de Choque Térmico HSP27/metabolismo , Hélio/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Queratinócitos/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Estresse Oxidativo , Fosfatos/metabolismo , Gases em Plasma/uso terapêuticoRESUMO
High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects upon exposure of healthy and tumor-bearing mice. In field experiments, the exposure to 1.5 GHz narrow-band electromagnetic fields with the incident amplitude peak value level in the range of 40 kV/m and 150 MHz wide-band electric fields with the amplitude peak value in the range of 200 kV/m, did not alter healthy and tumor-bearing animals' growth, nor it had any impact on cutaneous murine tumors' growth. While we did not observe any noticeable behavioral changes in mice during the exposure to narrow-band signals when wide-band HPEM signals were applied, mice could behave in a similar way as they respond to loud noise signals: namely, if a mouse was exploring the cage prior to signal application, it returned to companion mates when wide-band HPEM signals were applied. Moreover, the effect of wide-band signals was assessed on normal blood vessels permeability in real-time in dorsal-chamber-bearing mice exposed in a pilot study using wide-band signal applicators. Our pilot study conducted within the applicator and performed at the laboratory scale suggests that the exposure to wide-band signals with the amplitude of 47.5 kV/m does not result in increased vessel permeability.
Assuntos
Comportamento Animal , Permeabilidade Capilar , Neoplasias Experimentais/metabolismo , Ondas de Rádio , Animais , Feminino , Camundongos , Neoplasias Experimentais/patologiaRESUMO
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.
Assuntos
Eletroporação , Animais , Transporte Biológico , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Terapia Genética , Humanos , Camundongos , Microscopia , Neoplasias/terapia , Análise de Célula ÚnicaRESUMO
Cellular life strongly depends on the membrane ability to precisely control exchange of solutes between the internal and external (environmental) compartments. This barrier regulates which types of solutes can enter and leave the cell. Transmembrane transport involves complex mechanisms responsible for passive and active carriage of ions and small- and medium-size molecules. Transport mechanisms existing in the biological membranes highly determine proper cellular functions and contribute to drug transport. The present chapter deals with features and electrical properties of the cell membrane and addresses the questions how the cell membrane accomplishes transport functions and how transmembrane transport can be affected. Since dysfunctions of plasma membrane transporters very often are the cause of human diseases, we also report how specific transport mechanisms can be modulated or inhibited in order to enhance the therapeutic effect.
Assuntos
Transporte Biológico , Membrana Celular/fisiologia , Canais Iônicos/metabolismo , Condutividade Elétrica , Canais Iônicos/químicaRESUMO
Six conjugates of benzoxazole and green fluorescent protein chromophore that differ by the length of their alkyl chain (from C1 to C16) are investigated. They exhibit rigidofluorochromism and clear aggregation-induced emission enhancement (AIEE) behavior with emission in the orange-red that is specific to the solid state. A preparation method based on solvent exchange is used to prepare particles. The self-association properties of these molecules depend on the length of the alkyl chain. Microfibers, platelets, and rounded microparticles are successively obtained by increasing the chain length. The same method is used to prepare nanoparticles (NPs) that are fully characterized. In particular, homogeneous populations of stable NPs measuring around 70 nm are obtained with the analogs whose chains contain four to eight carbon atoms. The behavior with respect to living cells is also influenced by the nature of the compounds. Only the dyes with intermediate hydrophobicity are efficiently uptaken by both normal and tumor cells, and fluorescence only originates from dispersed dye molecules. There is no evidence for incorporation of NPs into cells. This work shows that small variations of the chemical structure must be taken into account for making the best use of AIEE compounds in view of precise applications.
Assuntos
Benzoxazóis/química , Proteínas de Fluorescência Verde/química , Nanopartículas/química , Corantes Fluorescentes/química , Microscopia Eletroquímica de Varredura , Nanopartículas/ultraestruturaRESUMO
BACKGROUND: In the context of spore contamination involved in bio-terrorism and food preservation, the development of new techniques for spore inactivation is an important challenge. RESULTS: Here, a successful application of electric arc discharges resulting in spore death was reported. Two types of electric arcs were compared, different with respect to their durations. The discharges with 0.5 µs duration induced a small inactivation area of 0.6 % of surface treated around their point of entry into the sample, while those with 20 µs duration induced a much larger inactivation area from 7 to 55 % of surface treated roughly proportional to the number of discharges delivered. In particular, 50 discharges of 20 µs duration induced inactivation in more than 55% of surface treated at an inactivation rate above 3.6 log10. CONCLUSIONS: These results are promising and warrant developing electric arcing as a novel method for spore inactivation.
Assuntos
Bacillus pumilus/fisiologia , Esporos Bacterianos/fisiologia , Contagem de Colônia Microbiana , Eletricidade , Viabilidade Microbiana , ÁguaRESUMO
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.
Assuntos
Polímeros/química , Micelas , Fotoquimioterapia , Fármacos FotossensibilizantesRESUMO
Drug delivery by nanovectors involves numerous processes, one of the most important being its release from the carrier. This point still remains unclear. The current work focuses on this point using poly(ethyleneglycol-b-ε-caprolactone) micelles containing either pheophorbide-a (Pheo-a) as a fluorescent probe and a phototoxic agent or fluorescent copolymers. This study showed that the cellular uptake and the phototoxicity of loaded Pheo-a are ten times higher than those of the free drug and revealed a very low cellular penetration of the fluorescence-labeled micelles. Neither loaded nor free Pheo-a displayed the same cellular localization as the labeled micelles. These results imply that the drug entered the cells without its carrier and probably without a disruption, as suggested by their stability in cell culture medium. These data allowed us to propose that Pheo-a directly migrates from the micelle to the cell without disruption of the vector. This mechanism will be discussed.
Assuntos
Portadores de Fármacos/química , Lactonas/química , Polietilenoglicóis/química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Clorofila/análogos & derivados , Clorofila/química , Clorofila/metabolismo , Clorofila/farmacologia , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Liberação Controlada de Fármacos , Células HCT116 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Lactonas/metabolismo , Lactonas/farmacologia , Micelas , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologiaRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células CHO , Linhagem Celular , Permeabilidade da Membrana Celular , Sobrevivência Celular/fisiologia , Cricetulus , Eletroporação/métodos , Fosfatidilcolinas/metabolismoRESUMO
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.
Assuntos
Derme/citologia , Eletroporação/métodos , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Engenharia Tecidual , Colágeno/metabolismo , Derme/metabolismo , Fibroblastos/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Microscopia Eletrônica de TransmissãoRESUMO
The interdependencies of the two main processing parameters affecting "electroporation" (electric field strength and pulse duration) while using pulse duration in the range of milliseconds and microseconds on the permeabilization, inactivation, and extraction of pigments from Chlorella vulgaris was compared. While irreversible "electroporation" was observed above 4 kV/cm in the millisecond range, electric field strengths of ≥10 kV/cm were required in the microseconds range. However, to cause the electroporation of most of the 90 % of the population of C. vulgaris in the millisecond (5 kV/cm, 20 pulses) or microsecond (15 kV/cm, 25 pulses) range, the specific energy that was delivered was lower for microsecond treatments (16.87 kJ/L) than in millisecond treatments (150 kJ/L). In terms of the specific energy required to cause microalgae inactivation, treatments in the microsecond range also resulted in greater energy efficiency. The comparison of extraction yields in the range of milliseconds (5 kV, 20 ms) and microseconds (20, 25 pulses) under the conditions in which the maximum extraction was observed revealed that the improvement in the carotenoid extraction was similar and chlorophyll a and b extraction was slightly higher for treatments in the microsecond range. The specific energy that was required for the treatment in the millisecond range (150 kJ/L) was much higher than those required in the microsecond range (30 kJ/L). The comparison of the efficacy of both types of pulses on the extraction enhancement just after the treatment and after a post-pulse incubation period seemed to indicate that PEF in the millisecond range created irreversible alterations while, in the microsecond range, the defects were a dynamic structure along the post-pulse time that caused a subsequent increment in the extraction yield.
Assuntos
Carotenoides/isolamento & purificação , Carotenoides/metabolismo , Permeabilidade da Membrana Celular/efeitos da radiação , Chlorella vulgaris/fisiologia , Eletricidade , Eletroporação/métodos , Carotenoides/efeitos da radiação , Clorofila/isolamento & purificação , Clorofila/metabolismo , Clorofila/efeitos da radiaçãoRESUMO
Lactose-derived catanionic vesicles offer unique opportunities to overcome cellular barriers. These potential nanovectors, very easy to formulate as drug delivery systems, are able to encapsulate drugs of various hydrophilicity. This article highlights versatile interaction mechanisms between these catanionic vesicles, labeled with hydrophilic and amphiphilic fluorescent probes, and a mammalian cell line, Chinese Hamster Ovary. Confocal microscopy and flow cytometry techniques show that these vesicles are internalized by cells through cellular energy dependent processes, as endocytosis, but are simultaneously able to spontaneously fuse with cell plasma membranes and release their hydrophilic content directly inside the cytosol. Such innovative and polyvalent nanovectors, able to deliver their content via different internalization pathways, would positively be a great progress for the coadministration of drugs of complementary efficiency.
Assuntos
Endocitose , Fusão de Membrana , Membranas Artificiais , Animais , Células CHO , Cátions , Linhagem Celular , Membrana Celular/metabolismo , Separação Celular , Cricetulus , Citosol , Sistemas de Liberação de Medicamentos , Citometria de Fluxo , Corantes Fluorescentes/química , Glicolipídeos/química , Cinética , Lactose/química , Microscopia Confocal , TensoativosRESUMO
Biological membranes are weakly permeable to hydrophilic molecules and ions and electric pulses can induce their transient permeabilization, but this process is not well characterized. We directly assay the electropermeabilization process, on the minimum model of lipid vesicles, by using a highly sensitive fluorescence method based on manganese ion transport. The approach gives access, at the single-lipid self-assembly level, to the transmembrane potential needed to detect divalent ion permeabilization on supramolecular giant unilamellar lipid vesicles. The critical values are strongly dependent on the lipid composition and are observed to vary from 10 to 150 mV. These values appear to be much lower than those previously reported in the literature for cells and vesicles. The detection method appears to be a decisive parameter as it is controlled by the transport of the reporter dye. We also provide evidence that the electropermeabilization process is a transient transition of the lipid self-organization due to the loss of assembly cohesion induced by bioelectrochemical perturbations of the zwitterionic interface with the solution.
Assuntos
Lipídeos de Membrana/química , Membrana Celular/metabolismo , Transporte de Íons , Manganês/metabolismoRESUMO
Electropermeabilization is a physical method that uses electric field pulses to deliver molecules into cells and tissues. Despite its increasing interest in clinics, little is known about plasma membrane destabilization process occurring during electropermeabilization. In this work, we took advantage of atomic force microscopy to directly visualize the consequences of electropermeabilization in terms of membrane reorganization and to locally measure the membrane elasticity. We visualized transient rippling of membrane surface and measured a decrease in membrane elasticity by 40%. Our results obtained both on fixed and living CHO cells give evidence of an inner effect affecting the entire cell surface that may be related to cytoskeleton destabilization. Thus, AFM appears as a useful tool to investigate basic process of electroporation on living cells in absence of any staining or cell preparation.
Assuntos
Membrana Celular/química , Citoesqueleto/química , Animais , Células CHO , Permeabilidade da Membrana Celular , Cricetinae , Elasticidade , Eletroporação , Microscopia de Força AtômicaRESUMO
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.
Assuntos
Portadores de Fármacos/química , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Polímeros/química , Técnicas de Cultura de Células/métodos , Clorofila/análogos & derivados , Clorofila/química , Clorofila/farmacologia , Portadores de Fármacos/toxicidade , Estabilidade de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Células HCT116/efeitos dos fármacos , Humanos , Lactonas/química , Luz , Micelas , Fármacos Fotossensibilizantes/farmacologia , Poliésteres/química , Polietilenoglicóis/química , Espalhamento de Radiação , Relação Estrutura-AtividadeRESUMO
Electrotransfer is a method by which molecules can be introduced into living cells via plasma membrane electropermeabilization. Here, we show that electropermeabilization affects the lateral mobility of Rae-1, a GPi anchored protein. Our results suggest that 10-20 % of the membrane surface is occupied by defects or pores and that these structures propagate rapidly (<1 min) over the cell surface. Electrotransfer of plasmid DNA (pDNA) also affects the lateral mobility of Rae-1. Furthermore, we clearly show that, once inserted into the plasma membrane, pDNA is completely immobile and excludes Rae-1; this indicates that the pDNA molecules are tightly packed together to form aggregates occupying at least the outer leaflet of the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Eletroporação , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , DNA/metabolismo , Plasmídeos/genética , PorosidadeRESUMO
Electroporation is a physical method of transferring molecules into cells and tissues. It takes advantage of the transient permeabilization of the cell membrane induced by electric field pulses, which gives hydrophilic molecules access to the cytoplasm. This method offers high transfer efficiency for small molecules that freely diffuse through electrically permeabilized membranes. Larger molecules, such as plasmid DNA, face several barriers (plasma membrane, cytoplasmic crowding, and nuclear envelope), which reduce transfection efficiency and engender a complex mechanism of transfer. Our work provides insight into the way electrotransferred DNA crosses the cytoplasm to reach the nucleus. For this purpose, single-particle tracking experiments of fluorescently labeled DNA were performed. Investigations were focused on the involvement of the cytoskeleton using drugs disrupting or stabilizing actin and tubulin filaments as the two relevant cellular networks for particle transport. The analysis of 315 movies (~4,000 trajectories) reveals that DNA is actively transported through the cytoskeleton. The large number of events allows a statistical quantification of the DNA motion kinetics inside the cell. Disruption of both filament types reduces occurrence and velocities of active transport and displacements of DNA particles. Interestingly, stabilization of both networks does not enhance DNA transport.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Rastreamento de Células , Citoplasma/metabolismo , Eletroporação , Plasmídeos/metabolismo , Transfecção , Tubulina (Proteína)/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células CHO , Permeabilidade da Membrana Celular , Cricetulus , Citoesqueleto , Depsipeptídeos/farmacologia , Humanos , Microscopia de Fluorescência , Paclitaxel/farmacologia , Plasmídeos/genética , Tiazolidinas/farmacologiaRESUMO
The understanding of the mechanisms involved in DNA electrotransfer in human skin remains modest and limits the clinical development of various biomedical applications, such as DNA vaccination. To elucidate some mechanisms of DNA transfer in the skin following electroporation, we created a model of the dermis using a tissue engineering approach. This model allowed us to study the electrotransfection of fibroblasts in a three-dimensional environment that included multiple layers of fibroblasts as well as the self-secreted collagen matrix. With the aim of improving transfection yield, we applied electrical pulses with electric field lines perpendicular to the reconstructed model tissue. Our results indicate that the fibroblasts of the reconstructed skin tissue can be efficiently permeabilized by applied millisecond electrical pulses. However, despite efficient permeabilization, the transfected cells remain localized only on the surface of the microtissue, to which the plasmid was deposited. Second harmonic generation microscopy revealed the extensive extracellular collagen matrix around the fibroblasts, which might have affected the mobility of the plasmid into deeper layers of the skin tissue model. Our results show that the used skin tissue model reproduces the structural barriers that might be responsible for the limited gene electrotransfer in the skin.
Assuntos
DNA , Eletroporação , Humanos , Transfecção , Eletroporação/métodos , DNA/genética , Plasmídeos/genética , Colágeno/genética , FibroblastosRESUMO
Electroporation, a technique that uses electrical pulses to temporarily or permanently destabilize cell membranes, is increasingly used in cancer treatment, gene therapy, and cardiac tissue ablation. Although the technique is efficient, patients report discomfort and pain. Current strategies that aim to minimize pain and muscle contraction rely on the use of pharmacological agents. Nevertheless, technical improvements might be a valuable tool to minimize adverse events, which occur during the application of standard electroporation protocols. One recent technological strategy involves the use of high pulse repetition rate. The emerging technique, also referred as "high frequency" electroporation, employs short (micro to nanosecond) mono or bipolar pulses at repetition rate ranging from a few kHz to a few MHz. This review provides an overview of the historical background of electric field use and its development in therapies over time. With the aim to understand the rationale for novel electroporation protocols development, we briefly describe the physiological background of neuromuscular stimulation and pain caused by exposure to pulsed electric fields. Then, we summarize the current knowledge on electroporation protocols based on high pulse repetition rates. The advantages and limitations of these protocols are described from the perspective of their therapeutic application.