RESUMEN

The combined application of electric fields and ultrasonic waves has shown promise in controlling cell membrane permeability, potentially resulting in synergistic effects that can be explored in the biotechnology industry. However, further clarification on how these processes interact is still needed. The objective of the present study was to investigate the atomic-scale effects of these processes on a DPPC lipid bilayer using molecular dynamics simulations. For higher electric fields, capable of independently forming pores, the application of an ultrasonic wave in the absence of cavitation yielded no additional effects on pore formation. However, for lower electric fields, the reduction in bilayer thickness induced by the shock wave catalyzed the electroporation process, effectively shortening the mean path that water molecules must traverse to form pores. When cavitation was considered, synergistic effects were evident only if the wave alone was able to generate pores through the formation of a water nanojet. In these cases, sonoporation acted as a mean to focus the electroporation effects on the initial pore formed by the nanojet. This study contributes to a better understanding of the synergy between electric fields and ultrasonic waves and to an optimal selection of processing parameters in practical applications of these processes.

Asunto(s)

Electroporación , Membrana Dobles de Lípidos , Simulación de Dinámica Molecular , Ondas Ultrasónicas , Membrana Dobles de Lípidos/química , Electroporación/métodos , Electricidad , Permeabilidad de la Membrana Celular , 1,2-Dipalmitoilfosfatidilcolina/química

RESUMEN

Electroporation is a vital process that facilitates the use of modern recombineering and other high-throughput techniques in a wide array of microorganisms, including non-model bacteria like plant growth-promoting bacteria (PGPB). These microorganisms play a significant role in plant health by colonizing plants and promoting growth through nutrient exchange and hormonal regulation. In this study, we introduce a sequential Design of Experiments (DOE) approach to obtain highly competent cells swiftly and reliably for electroporation. Our method focuses on optimizing the three stages of the electroporation procedure-preparing competent cells, applying the electric pulse field, and recovering transformed cells-separately. We utilized a split-plot fractional design with five factors and a covariate to optimize the first step, response surface methodology (RSM) for the second step, and Plackett-Burman design for two categorical factors and one continuous factor for the final step. Following the experimental sequence with three bacterial models, we achieved efficiencies 10 to 100 times higher, reaching orders of 105 to 106 CFU/µg of circular plasmid DNA. These results highlight the significant potential for enhancing electroporation protocols for non-model bacteria.

Asunto(s)

ADN , Transformación Bacteriana , Plásmidos , Electroporación/métodos , Plantas , Bacterias/genética

RESUMEN

Escherichia coli bacterium is a rod-shaped organism composed of a complex double membrane structure. Knowledge of electric field driven ion transport through both membranes and the evolution of their induced permeabilization has important applications in biomedical engineering, delivery of genes and antibacterial agents. However, few studies have been conducted on Gram-negative bacteria in this regard considering the contribution of all ion types. To address this gap in knowledge, we have developed a deterministic and stochastic Brownian dynamics model to simulate in 3D space the motion of ions through pores formed in the plasma membranes of E. coli cells during electroporation. The diffusion coefficient, mobility, and translation time of Ca2+, Mg2+, Na+, K+, and Cl- ions within the pore region are estimated from the numerical model. Calculations of pore's conductance have been validated with experiments conducted at Gustave Roussy. From the simulations, it was found that the main driving force of ionic uptake during the pulse is the one due to the externally applied electric field. The results from this work provide a better understanding of ion transport during electroporation, aiding in the design of electrical pulses for maximizing ion throughput, primarily for application in cancer treatment.

Asunto(s)

Electroporación , Escherichia coli , Transporte Iónico , Transporte Biológico , Electroporación/métodos , Iones

RESUMEN

Reversible electroporation is a suitable technique to aid the internalization of medicaments in cancer tissues without inducing permanent cellular damage, allowing the enhancement of cytotoxic effects without incurring in electric-driven necrotic or apoptotic processes by the presence of non-reversible aqueous pores. An adequate selection of electroporation parameters acquires relevance to reach these goals and avoid opposite effects. This work applies the Method of Fundamental Solutions (MFS) for drug transport simulations in electroporated cancer tissues, using a continuum tumor cord approach and considering both electro-permeabilization and vasoconstriction effects. The MFS algorithm is validated with published results, obtaining satisfactory accuracy and convergence. Then, MFS simulations are executed to study the influence of electric field magnitude [Formula: see text], number of electroporation treatments [Formula: see text], and electroporation time [Formula: see text] on three assessment parameters of electrochemotherapy: the internationalization efficacy accounting for the ability of the therapy to introduce moles into viable cells, cell-kill capacity indicating the faculty to reduce the survival fraction of cancer cells, and distribution uniformity specifying the competence to supply drug homogeneously through the whole tissue domain. According to numerical results, when [Formula: see text] is the reversibility threshold, a positive influence on the first two parameters is only possible once specific values of [Formula: see text] and [Formula: see text] have been exceeded; when [Formula: see text] is just the irreversibility threshold, any combination of [Formula: see text] and [Formula: see text] is beneficial. On the other hand, the drug distribution uniformity is always adversely affected by the application of electric pulses, being this more noticeable as [Formula: see text], [Formula: see text], and [Formula: see text] increases.

Asunto(s)

Electroporación , Neoplasias , Humanos , Electroporación/métodos , Neoplasias/patología , Electricidad , Algoritmos , Apoptosis

RESUMEN

Electroporation has emerged as a suitable technique to induce the pore formation in the cell membrane of cancer tissues, facilitating the cellular internalization of chemotherapeutic drugs. An adequate selection of the electric pulse characteristics is crucial to guarantee the efficiency of this technique, minimizing the adverse effects. In the present work, the dual reciprocity boundary element method (DR-BEM) is applied for the simulation of drug transport in the extracellular and intracellular space of cancer tissues subjected to the application of controlled electric pulses, using a continuum tumour cord approach, and considering both the electro-permeabilization and vasoconstriction phenomena. The developed DR-BEM algorithm is validated with numerical and experimental results previously published, obtaining a satisfactory accuracy and convergence. Using the DR-BEM code, a study about the influence of the magnitude of electric field (E) and pulse spacing (dpulses) on the time behavior and spatial distribution of the internalized drug, as well as on the cell survival fraction, is carried out. In general, the change of drug concentration, drug exposure and cell survival fraction with the parameters E and dpulses is ruled by two important factors: the balance between the electro-permeabilization and vasoconstriction phenomena, and the relative importance of the sources of cell death (electric pulses and drug cytotoxicity); these two factors, in turn, significantly depend on the reversible and irreversible thresholds considered for the electric field.

Asunto(s)

Neoplasias , Humanos , Supervivencia Celular , Neoplasias/tratamiento farmacológico , Electroporación/métodos , Simulación por Computador , Membrana Celular

RESUMEN

The convective delivery of chemotherapeutic drugs in cancerous tissues is directly proportional to the blood perfusion rate, which in turns can be transiently reduced by the application of high-voltage and short-duration electric pulses due to vessel vasoconstriction. However, electric pulses can also increase vessel wall and cell membrane permeabilities, boosting the extravasation and cell internalization of drug. These opposite effects, as well as possible adverse impacts on the viability of tissues and endothelial cells, suggest the importance of conducting in silico studies about the influence of physical parameters involved in electric-mediated drug transport. In the present work, the global method of approximate particular solutions for axisymmetric domains, together with two solution schemes (Gauss-Seidel iterative and linearization+successive over-relaxation), is applied for the simulation of drug transport in electroporated cancer tissues, using a continuum tumor cord approach and considering both the electropermeabilization and vasoconstriction phenomena. The developed global method of approximate particular solutions algorithm is validated with numerical and experimental results previously published, obtaining a satisfactory accuracy and convergence. Then, a parametric study about the influence of electric field magnitude and inlet blood velocity on the internalization efficacy, drug distribution uniformity, and cell-kill capacity of the treatment, as expressed by the number of internalized moles into viable cells, homogeneity of exposure to bound intracellular drug, and cell survival fraction, respectively, is analyzed for three pharmacokinetic profiles, namely one-short tri-exponential, mono-exponential, and uniform. According to numerical results, the trade-off between vasoconstriction and electropermeabilization effects and, consequently, the influence of electric field magnitude and inlet blood velocity on the assessment parameters considered here (efficacy, uniformity, and cell-kill capacity) is different for each pharmacokinetic profile deemed.

Asunto(s)

Electroquimioterapia , Neoplasias , Humanos , Electroquimioterapia/métodos , Células Endoteliales , Neoplasias/tratamiento farmacológico , Electroporación/métodos , Electricidad

RESUMEN

This study takes a step in understanding the physiological implications of the nanosecond pulsed electric field (nsPEF) by integrating molecular dynamics simulations and machine learning techniques. nsPEF, a state-of-the-art technology, uses high-voltage electric field pulses with a nanosecond duration to modulate cellular activity. This investigation reveals a relatively new and underexplored phenomenon: protein-mediated electroporation. Our research focused on the voltage-sensing domain (VSD) of the NaV1.5 sodium cardiac channel in response to nsPEF stimulation. We scrutinized the VSD structures that form pores and thereby contribute to the physical chemistry that governs the defibrillation effect of nsPEF. To do so, we conducted a comprehensive analysis involving the clustering of 142 replicas simulated for 50 ns under nsPEF stimuli. We subsequently pinpointed the representative structures of each cluster and computed the free energy between them. We find that the selected VSD of NaV1.5 forms pores under nsPEF stimulation, but in a way that significant differs from the traditional VSD opening. This study not only extends our understanding of nsPEF and its interaction with protein channels but also adds a new effect to further study.

Asunto(s)

Electricidad , Electroporación , Electroporación/métodos , Terapia de Electroporación , Corazón

RESUMEN

A specific pulsed electric field protocol can be used to induce electroporation. This is used in the food industry for yeast pasteurization, in laboratories for generic transfer and the medical field for cancer treatment. The sensing of electroporation can be done with simple 'instantaneous' voltage-current analysis. However, there are some intrinsic low-frequency phenomena superposing the electroporation current, such as electrode polarization. The biological media are non-homogeneous, giving them specific characterization in the broad frequency spectrum. For example, the cell barrier, i.e., cell membrane, causes so called ß-dispersion in the frequency range of tens to thousands of kHz. Electroporation is a dynamic phenomenon characterized by altering the cell membrane permeability. In this work, we show that the impedance measurement at certain frequencies could be used to detect the occurrence of electroporation, i.e., dielectric dispersion modulated sensing. This approach may be used for the design and implementation of electroporation systems. Yeast suspension electroporation is simulated to show changes in the frequency spectrum. Moreover, the alteration depends on characteristics of the system. Three types of external buffers and their characteristics are evaluated.

Asunto(s)

Electroporación , Saccharomyces cerevisiae , Membrana Celular/metabolismo , Impedancia Eléctrica , Electroporación/métodos , Suspensiones

RESUMEN

Electroporation is a method for the introduction of molecules (usually nucleic acids) into a cell, consisting of submitting the cells to high-voltage and short electric pulses in the presence of the exogenous DNA/molecule. It is a versatile method, adaptable to different types of cells, from bacteria to cultured cells to higher eukaryotes, and thus has applications in many diverse fields, such as environmental biology, biotechnology, genetic engineering, and medicine. Electroporation has some advantages over other genetic transformation strategies, including the simplicity of the method, a wide range of adjustable parameters (possibility of optimization), high reproducibility and avoidance of the use of chemicals toxic to cells. Here we describe an optimized electroporation procedure for the industrially important fungus Acremonium chrysogenum, using germinated conidia and fragmented young mycelium. In both cases, the transformation efficiency was higher compared to the conventional polyethylene glycol (PEG)-mediated transformation of protoplasts.

Asunto(s)

Electroporación/métodos , Hongos/genética , Acremonium/genética , Biotecnología/métodos , Ingeniería Genética/métodos , Micelio/genética , Polietilenglicoles/química , Protoplastos , Reproducibilidad de los Resultados , Transformación Genética/genética

RESUMEN

Quantum dots (QDs) have attracted considerable attention as fluorescent probes for life sciences. The advantages of using QDs in fluorescence-based studies include high brilliance, a narrow emission band allowing multicolor labeling, a chemically active surface for conjugation, and especially, high photostability. Despite these advantageous features, the size of the QDs prevents their free transport across the plasma membrane, limiting their use for specific labeling of intracellular structures. Over the years, various methods have been evaluated to overcome this issue to explore the full potential of the QDs. Thus, in this review, we focused our attention on physical and biochemical QD delivery methods-electroporation, microinjection, cell-penetrating peptides, molecular coatings, and liposomes-discussing the benefits and drawbacks of each strategy, as well as presenting recent studies in the field. We hope that this review can be a useful reference source for researches that already work or intend to work in this area. Strategies for the intracellular delivery of quantum dots discussed in this review (electroporation, microinjection, cell-penetrating peptides, molecular coatings, and liposomes).

Asunto(s)

Colorantes Fluorescentes/administración & dosificación , Puntos Cuánticos/administración & dosificación , Animales , Péptidos de Penetración Celular/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Electroporación/métodos , Colorantes Fluorescentes/análisis , Humanos , Liposomas/química , Microinyecciones/métodos , Puntos Cuánticos/análisis

RESUMEN

Functional studies on echinoderms have been reduced to the use of pharmacological treatments. The ability to modulate the genetic expression of regenerating tissues can elucidate potential effectors during this process. Here we describe an effective transfection protocol that allows the introduction of Dicer-substrate interference RNAs (DsiRNAs) for the modulation of gene expression and its characterization during regeneration.

Asunto(s)

Interferencia de ARN , Pepinos de Mar/genética , Pepinos de Mar/fisiología , Animales , Electroporación/métodos , Regulación de la Expresión Génica , ARN Interferente Pequeño/genética , Regeneración , Pepinos de Mar/anatomía & histología , Transfección/métodos

RESUMEN

Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength (P < 0.05). Increase in the sperm damaged plasma membrane was observed with increasing field strength (P < 0.05). ROS and sperm mitochondrial functionality did not present a negative response after the electroporation (P > 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells.

Asunto(s)

ADN/administración & dosificación , Electroporación/métodos , Mitocondrias/fisiología , Especies Reactivas de Oxígeno/metabolismo , Espermatozoides/fisiología , Pez Cebra/fisiología , Animales , Supervivencia Celular , Fertilización In Vitro/métodos , Técnicas de Transferencia de Gen , Masculino , Motilidad Espermática , Transfección/métodos

RESUMEN

Electroporation is a widely used method consisting of application of high-voltage, short-duration electric pulses to increase cell membrane permeability, allowing cellular internalization of medications. In this work, the influence of two primary parameters, voltage level (V) and pulse spacing (N), on electroporation efficiency, uniformity and aggressiveness, as quantified by the total mass transport to viable cells, intracellular concentration gradients and an aggressiveness factor introduced here, is studied by means of numerical simulations of drug transport in electroporated tissues. The global method of approximate particular solutions (Global MAPS) is used to solve the governing equations, together with domain scaling, singular value decomposition and smoothing algorithms, to address the ill-conditioning of the final system and suppress small scale oscillations. The accuracy of Global MAPS is evaluated by comparing the initial extracellular concentration, Ce , and final intracellular concentration, Ci , with previous finite volume method results, obtaining similar behavior of Ce and Ci along the tissue domain, with some differences for Ci in high-gradient zones. According to the Global MAPS results, the influence of V and N on Ci is only significant over a certain range, within which the largest drug transport to viable cells occurs. In general, both electroporation efficiency and aggressiveness change in nonuniform manner with V and decrease with N, whereas the electroporation uniformity decreases as V increases and N decreases. The contour plots obtained here can be considered useful tools to compare electroporation-based treatments in terms of their efficiency, aggressiveness and uniformity, assisting in the selection of a suitable treatment plan for cancer.

Asunto(s)

Algoritmos , Electroporación/métodos , Simulación por Computador

RESUMEN

Skeletal muscle has a remarkable capacity to regenerate after injuries mainly due to a reservoir of precursor cells named satellite cells (SCs), which are responsible for after-birth growth and response to lesions, either by exercise or disease. Upon injury, the regenerative response includes SCs exit of quiescence, activation, proliferation, and fusion to repair or form new myofibers. This process is accompanied by inflammation, with infiltration of immune cells, primarily macrophages. Every phase of regeneration is highly regulated and orchestrated by many molecules and signaling pathways. The elucidation of players and mechanisms involved in muscle degeneration and regeneration is of extreme importance, especially for therapeutic strategies for muscle diseases.Here we are proposing a model of muscle injury induced by electroporation, which is an efficient method to induce muscle damage in order to follow the steps involved in degeneration and regeneration. Three days after electroporation, the muscle shows prominent signals of degeneration, like areas of necrosis and infiltration of macrophages, followed by regeneration, observed by the presence of centrally nucleated myofibers. After 5 days the regeneration is very active, with small dMyHC positive fibers. Fifteen days later, we observe a general regeneration of the muscle, with fibers with increased diameter after 60 days. This methodology is an easy and simple alternative to induce muscle lesion. It can be employed to study alterations in gene expression and the process of satellite cell recruitment, both in healthy and dystrophic/myopathic animal models for muscular dystrophy.

Asunto(s)

Electroporación/métodos , Regeneración Tisular Dirigida/métodos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/lesiones , Regeneración/fisiología , Animales , Modelos Animales de Enfermedad , Macrófagos/inmunología , Ratones , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patología , Distrofias Musculares/patología , Células Satélite del Músculo Esquelético/metabolismo

RESUMEN

Adoptive immunotherapy of cancer using T cells expressing chimeric antigen receptors (CARs) is now an approved treatment for non-Hodgkin lymphoma (NHL) and B cell acute lymphoblastic leukemia (B-ALL), inducing high response rates in patients. The infusion products are generated by using retro- or lentiviral transduction to induce CAR expression in T cells followed by an in vitro expansion protocol. However, use of viral vectors is cumbersome and is associated with increased costs due to the required high titers, replication-competent retrovirus (RCR) detection and production/use in a biosafety level 2 culture rooms, and additional quality control tests. Nonviral methods, like the Sleeping Beauty transposon system, can stably integrate in the genome of target cells and can be delivered using straightforward methods like electroporation. This chapter describes a protocol for T cell genetic modification using Sleeping Beauty transposon system and electroporation with the Lonza Nucleofector II device for the stable expression of CAR molecules in T lymphocytes.

Asunto(s)

Elementos Transponibles de ADN , Vectores Genéticos/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Linfocitos T/metabolismo , Antígenos CD19/inmunología , Antígenos de Neoplasias/inmunología , Técnicas de Cultivo de Célula , Electroporación/métodos , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Humanos , Inmunoterapia Adoptiva/métodos , Linfocitos T/inmunología , Transposasas

RESUMEN

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.

Asunto(s)

Adenina Fosforribosiltransferasa/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Chlamydomonas reinhardtii/genética , Genes Reporteros/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Electroporación/métodos , Edición Génica/métodos , Plásmidos/genética , ARN Guía de Kinetoplastida/genética , Ribonucleoproteínas/genética

RESUMEN

This article presents experimental and computational results of electroporation in rat liver. The experiments were performed using different forms of electrodes and waveforms of applied electric pulses. For the numerical simulation, the electroporation model proposed by Ramos and Weinert in a previous publication was used. Dynamic adjustments were used for obtaining a good modeling of the electric current. A single set of model parameters was obtained to fit the simulated current response for different waveforms and electrodes. These parameters were obtained with the use of a genetic algorithm that minimized the error between the simulated and experimental currents. The electroporation model with dynamic adjustment proved to be an appropriate simulation tool to predict the tissue conductivity during stimulation by intense electrical fields.

Asunto(s)

Simulación por Computador , Electroporación/métodos , Hígado/fisiología , Modelos Biológicos , Algoritmos , Animales , Conductividad Eléctrica , Electricidad , Electrodos , Electroporación/instrumentación , Diseño de Equipo , Ratas Wistar

RESUMEN

Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant ß-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated. The electroporation of germinated conidia and young mycelium was found to be appropriate for transforming A. chrysogenum with higher transformation efficiencies than those obtained with the conventional protoplast-based transformation procedures. The developed electroporation strategy is fast, simple to perform, and highly reproducible and avoids the use of chemicals toxic to cells. Electroporation of young mycelium represents an alternative method for transformation of fungal strains with reduced or no sporulation, as often occurs in laboratory-developed strains in the search for high-yielding mutants for industrial bioprocesses.

Asunto(s)

Acremonium/genética , Electroporación/métodos , Transformación Genética , Acremonium/efectos de los fármacos , Acremonium/metabolismo , Cefalosporinas/biosíntesis , Farmacorresistencia Bacteriana , Viabilidad Microbiana , Micelio/efectos de los fármacos , Micelio/genética , Micelio/metabolismo , Fleomicinas/farmacología , Protoplastos/fisiología , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo

RESUMEN

The efforts for the development and testing of vaccines against Trypanosoma cruzi infection have increased during the past years. We have designed a TcVac series of vaccines composed of T. cruzi derived, GPI-anchored membrane antigens. The TcVac vaccines have been shown to elicit humoral and cellular mediated immune responses and provide significant (but not complete) control of experimental infection in mice and dogs. Herein, we aimed to test two immunization protocols for the delivery of DNA-prime/DNA-boost vaccine (TcVac1) composed of TcG2 and TcG4 antigens in a BALB/c mouse model. Mice were immunized with TcVac1 through intradermal/electroporation (IDE) or intramuscular (IM) routes, challenged with T. cruzi, and evaluated during acute phase of infection. The humoral immune response was evaluated through the assessment of anti-TcG2 and anti-TcG4 IgG subtypes by using an ELISA. Cellular immune response was assessed through a lymphocyte proliferation assay. Finally, clinical and morphopathological aspects were evaluated for all experimental animals. Our results demonstrated that when comparing TcVac1 IDE delivery vs IM delivery, the former induced significantly higher level of antigen-specific antibody response (IgG2a + IgG2b > IgG1) and lymphocyte proliferation, which expanded in response to challenge infection. Histological evaluation after challenge infection showed infiltration of inflammatory cells (macrophages and lymphocytes) in the heart and skeletal tissue of all infected mice. However, the largest increase in inflammatory infiltrate was observed in TcVac1_IDE/Tc mice when compared with TcVac1_IM/Tc or non-vaccinated/infected mice. The extent of tissue inflammatory infiltrate was directly associated with the control of tissue amastigote nests in vaccinated/infected (vs. non-vaccinated/infected) mice. Our results suggest that IDE delivery improves the protective efficacy of TcVac1 vaccine against T. cruzi infection in mice when compared with IM delivery of the vaccine.

Asunto(s)

Enfermedad de Chagas/prevención & control , Electroporación/métodos , Vacunas Antiprotozoos/administración & dosificación , Vacunación/métodos , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Linfocitos T CD8-positivos/inmunología , Enfermedad de Chagas/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Celular , Inmunización Secundaria , Inmunoglobulina G/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Vacunas Antiprotozoos/inmunología , Absorción Cutánea , Trypanosoma cruzi/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología

RESUMEN

Physical methods such as electroporation have been used to improve the DNA uptake efficiency of sperm cells. This study aims to develop an efficient capillary-type electroporation method for incorporation of exogenous DNA into bovine cryopreserved sperm cells with minimal detrimental effects for later use in SMGT. Electroporation of the samples was performed in 2 different groups (with 1 µg of DNA and without DNA transfection) and under five different voltages: 500 V, 600 V, 700 V, 800 V and 900 V. Non-electroporated sperm cells (with and without DNA) were used as control. Kinetics parameters were determined using computer assisted semen analyses, whereas membrane integrity, fluidity, mitochondrial function and DNA uptake were evaluated by flow cytometry. Results revealed that all tested voltages reduced electroporated sperm motility (P < 0.05) when compared to the control (non-electroporated cells). Mitochondrial function results showed no statistical difference among groups. Similarly, groups electroporated with lower (500 V, 600 V and 700 V) voltages showed no difference in cell membrane integrity and fluidity. Groups electroporated at higher voltages (800 V and 900 V) demonstrated negative effects in cells membrane integrity when compared to other groups and control. Also, all electroporated groups demonstrated significant higher percentages of transfected sperm cells when compared to the control group (P < 0.05). Under the recommendation of using voltages up to 600 V, this method represents a safe and efficient alternative for electroporation of bovine spermatozoa.

Asunto(s)

Electroporación/métodos , Preservación de Semen , Espermatozoides/fisiología , Animales , Bovinos , Criopreservación , Masculino , Análisis de Semen , Motilidad Espermática/fisiología