In vivo targeted gene transfer by non-viral vectors is subjected to anatomical constraints depending on the route of administration. Transfection efficiency and gene expression in vivo using non-viral vectors is also relatively low. We report that in vivo electropermeabilization of the liver tissue of rats in the presence of genes encoding luciferase or ~-galactosidase resulted in the strong expression of these genetic markers in rat liver cells. About 31)-40% of the rat liver cells electroporated expressed the ~galactosidase genetic marker 48 h after electroporation. The marker expression was also detected at least 21 days after transfection at about 5% of the level 48 h after electroporation. The results indicate that gene transfer by electroporatinn in vivo may avoid anatomical constraints and low transfection efficiency.
Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.
Electroporation is a physical event that temporarily reduces cell membrane barrier properties. Diminished membrane barrier properties are achieved by exposing cells to pulsed electric fields. When a cell has been treated with electric fields it is possible for extracellular agents to gain access to the cell interior. This process has been used in vivo to increase the uptake of chemotherapeutic agents by tumor cells which results in dramatically higher response rates than when drug is used alone. This type of treatment is called electrochemotherapy (ECT); bleomycin is most often used as the drug for this type of treatment. It was hypothesized that electroporation could be used to augment the cytotoxicity of other anticancer agents. Therefore, this study was performed in order to screen 44 different combinations of drug and cell type in vitro to identify drugs that may have higher cytotoxicity when combined with electroporation. Results from seven cell types indicate that the IC50 of bleomycin can be reduced by a factor of 100-5000 when electroporation is used to facilitate internalization. The IC50 values of cisplatin and carboplatin could be reduced by factors ranging from 3 to 13 in six different cell lines as a result of electroporation. These IC50 reductions in multiple cell lines suggest that cisplatin and carboplatin may be effective in vivo as part of ECT treatment.
Crystalline silicon carbide (SiC) and silicon (Si) biocompatibility was evaluated by directly culturing three mammalian cell lines on these semiconducting substrates. Cell proliferation and adhesion quality were studied using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and fluorescent microscopy. The reported results show that SiC is indeed a more biocompatible substrate than Si. The surface wettability of SiC and Si samples was evaluated through static contact angle measurements, which provided interesting information regarding the influence of different cleaning procedures on the SiC surfaces. The cell proliferation data are discussed in light of the contact angle measurements results. This joint analysis leads to interesting conclusions that may help to uncover the main factors that define a semiconductor's biocompatibility.
Crystalline silicon carbide (SiC) and silicon (Si) biocompatibility was evaluated in vitro by directly culturing three skin and connective tissue cell lines, two immortalized neural cell lines, and platelet-rich plasma (PRP) on these semiconducting substrates. The experiments were performed specifically for the three adopted SiC polytypes, namely 3C-, 4H- and 6H-SiC, and the results were compared to those obtained for Si crystals. Cell proliferation and adhesion quality were studied using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and fluorescent microscopy. For the neural cells studied AFM was also used to quantify the filopodia and lamellipodia extensions on the surface of the tested materials. Fluorescent microscopy was also used to assess platelet adhesion to the semiconductor surfaces where significantly lower values of platelet adhesion to 3C-SiC was observed compared to Si. The reported results show that SiC is indeed a more biocompatible substrate than Si. While there were some differences among the degree of biocompatibility of the various SiC polytypes tested, SiC appears to be a highly biocompatible material in vitro that is also somewhat hemocompatible. This extremely intriguing result appears to put SiC into a unique class of materials that is both bio- and hemo-compatible and is, to the best of our knowledge, the only semiconductor with this property.
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