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1.
Bioelectrochemistry ; 131: 107369, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31706114

RESUMO

High-frequency irreversible electroporation (H-FIRE) is an emerging electroporation-based therapy used to ablate cancerous tissue. Treatment consists of delivering short, bipolar pulses (1-10µs) in a series of 80-100 bursts (1 burst/s, 100µs on-time). Reducing pulse duration leads to reduced treatment volumes compared to traditional IRE, therefore larger voltages must be applied to generate ablations comparable in size. We show that adjuvant calcium enhances ablation area in vitro for H-FIRE treatments of several pulse durations (1, 2, 5, 10µs). Furthermore, H-FIRE treatment using 10µs pulses delivered with 1mM CaCl2 results in cell death thresholds (771±129V/cm) comparable to IRE thresholds without calcium (698±103V/cm). Quantifying the reversible electroporation threshold revealed that CaCl2 enhances the permeabilization of cells compared to a NaCl control. Gene expression analysis determined that CaCl2 upregulates expression of eIFB5 and 60S ribosomal subunit genes while downregulating NOX1/4, leading to increased signaling in pathways that may cause necroptosis. The opposite was found for control treatment without CaCl2 suggesting cells experience an increase in pro survival signaling. Our study is the first to identify key genes and signaling pathways responsible for differences in cell response to H-FIRE treatment with and without calcium.


Assuntos
Cloreto de Cálcio/farmacologia , Morte Celular/efeitos dos fármacos , Eletroporação/métodos , Animais , Linhagem Celular Tumoral , Humanos , Hidrogéis , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
2.
Anticancer Res ; 39(11): 6193-6196, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704847

RESUMO

BACKGROUND/AIM: Carbohydrate antigen 19-9 (CA19-9) is a tumor marker for pancreatic cancer. Irreversible electroporation (IRE) is an experimental treatment modality for pancreatic cancer. The aim of this study was to evaluate whether percutaneous IRE lowers the CA19-9 level in pancreatic cancer and whether this correlates with improved overall survival. PATIENTS AND METHODS: Seventy-one patients with locally advanced pancreatic cancer or local recurrence after resection were treated. Patients with missing data, metastatic disease and normal serum CA19-9 before IRE were excluded. This left 35 cases for analysis. RESULTS: The median CA19-9 did not decrease in the cohort after IRE treatment (282 U/ml before versus 315 U/ml after; p=0.80). The 25th percentile of patients with the best CA19-9 response had improved overall survival compared to the 25th percentile with the worst response (mean 13.1 versus 8.1 months, respectively; p=0.01). CONCLUSION: IRE did not lower the level of CA19-9 in pancreatic cancer cases. However, a response in CA19-9 was correlated with improved survival.


Assuntos
Adenocarcinoma/sangue , Biomarcadores Tumorais/sangue , Antígeno CA-19-9/sangue , Eletroporação/métodos , Recidiva Local de Neoplasia/sangue , Neoplasias Pancreáticas/sangue , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Idoso , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Prognóstico , Taxa de Sobrevida
3.
Folia Histochem Cytobiol ; 57(4): 159-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31746453

RESUMO

INTRODUCTION: The extremely-low frequency electromagnetic field (ELFEMF) has been proposed for use in cancer therapy since it was found that magnetic waves interfere with many biological processes. Gold nanoparticles (Au-NPs) have been widely used for drug delivery during cancer in vitro studies due to their low cytotoxity and high biocompatibility. The electroporation of cancer cells in a presence of Au-NPs (EP Au-NPs) can induce cell apoptosis, alterations of cell cycle profile and morphological changes. The impact of ELFEMF and EP Au-NPs on morphology, cell cycle and activation of apoptosis-associated genes on Hep-2 laryngeal cancer cell line has not been studied yet. MATERIALS AND METHODS: ELFEMF on Hep-2 cells were carried out using four different conditions: 25/50 mT at 15/30 min, while Au-NPs were used as direct contact (DC) or with electroporation (EP, 10 pulses at 200V, equal time intervals of 4 sec). MTT assay was used to check the toxicity of DC Au-NPs. Expression of CASP3, P53, BAX and BCL2 genes was quantified using qPCR. Cell cycle was analyzed by flow cytometry. Hematoxylin and eosin (HE) staining was used to observe cell morphology. RESULTS: Calculated IC50 of DC Au-NPs 24.36 µM (4.79 µg/ml) and such concentration was used for further DC and EP AuNPs experiments. The up-regulation of pro-apoptotic genes (CASP3, P53, BAX) and decreased expression of BCL2, respectively, was observed for all analyzed conditions with the highest differences for EP AuNPs and ELFEMF 50 mT/30 min in comparison to control cells. The highest content of cells arrested in G2/M phase was observed in ELFEMF-treated cells for 30 min both at 25 or 50 mT, while the cells treated with EP AuNPs or ELFEMF 50 mT/15 min showed highest ratios of apoptotic cells. HE staining of electroporated cells and cells exposed to ELFEMF's low and higher frequencies for different times showed nuclear pleomorphic cells. Numerous apoptotic bodies were observed in the irregular cell membrane of neoplastic and necrotic cells with mixed euchromatin and heterochromatin. CONCLUSIONS: Our observations indicate that treatment of Hep-2 laryngeal cancer cells with ELFEMF for 30 min at 25-50 mT and EP Au-NPs can cause cell damage inducing apoptosis and cell cycle arrest.


Assuntos
Antineoplásicos/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Antineoplásicos/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Campos Eletromagnéticos , Eletroporação/métodos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Ouro/toxicidade , Humanos , Nanopartículas Metálicas/toxicidade , Regulação para Cima
4.
Nat Commun ; 10(1): 4730, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628330

RESUMO

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.


Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular/genética , Proliferação de Células/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Animais , Eletroporação/métodos , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante Heterólogo
5.
Curr Urol Rep ; 20(10): 63, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31478109

RESUMO

PURPOSE OF REVIEW: Although still considered experimental, focal irreversible electroporation (IRE) as a primary treatment for prostate cancer (PCa) is considered one of the most promising ablative technologies for focal therapy. This review provides a description of the principle of IRE for the treatment of PCa, combined with an overview of the recent research. RECENT FINDINGS: It has been almost a decade since the first human studies of focal IRE for PCa were trying to demonstrate its feasibility and safety, and recently new data are emerging regarding the functional and oncological outcomes. It was shown that the expected ablation efficacy of IRE is dependent on increased safety margins of > 9 mm and an uninterrupted IRE procedure, but these findings need further investigation in larger cohorts and randomized control trials (RCT). Recent data from larger cohorts with a longer follow-up of up to 12 months prove that focal IRE as primary treatment for localized PCa is indeed safe, has effective short-term oncological control in selected patients, and it has good functional outcomes by retaining urinary function and causing only mild erectile dysfunction.


Assuntos
Técnicas de Ablação/métodos , Eletroporação/métodos , Neoplasias da Próstata/terapia , Disfunção Erétil/etiologia , Humanos , Masculino , Margens de Excisão , Neoplasias da Próstata/patologia , Recuperação de Função Fisiológica
6.
Bioelectrochemistry ; 130: 107343, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31401517

RESUMO

Skin is a very suitable target for gene therapy and DNA vaccination due to its accessibility, its surface and its ability to produce transgenes. Gene electrotransfer (GET) to the skin is under development for clinical applications for DNA vaccine or local treatment such as wound healing. Local treatments are effective if the expression of the plasmid affects only the local environment (skin) by inducing an efficient concentration over a prolonged period. In this study, we evaluate the control of expression in the skin of a plasmid coding a fluorescent protein by its CpG (cytosine-phosphate-guanine motif) content. Two fluorescent reporter genes are evaluated: tdTomato and GFP. The expression is followed on the long term by in vivo fluorescence imaging. Our results show that GET mediated expression in the skin can be controlled by the CpG content of the plasmid. Long term expression (>120 days) can be obtained at high level with CpG-free constructs associated with a proper design of the electrodes where the field distribution mediating the gene electrotransfer is present deep in the skin.


Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Plasmídeos/administração & dosagem , Pele/metabolismo , Animais , Ilhas de CpG , DNA/genética , Eletrodos , Eletroporação/métodos , Feminino , Genes Reporter , Camundongos Endogâmicos C57BL , Plasmídeos/genética
7.
Molecules ; 24(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408997

RESUMO

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.


Assuntos
Eletroporação/métodos , Morfolinos/genética , Oligonucleotídeos Antissenso/genética , Interferência de RNA , Transfecção/métodos , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Cadeias alfa de Integrinas/antagonistas & inibidores , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Lipídeos/química , Camundongos , Camundongos Endogâmicos mdx , Morfolinos/síntese química , Morfolinos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Cultura Primária de Células , Proteínas do Complexo SMN/antagonistas & inibidores , Proteínas do Complexo SMN/genética , Proteínas do Complexo SMN/metabolismo
8.
Surgery ; 166(4): 503-508, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31416604

RESUMO

BACKGROUND: We have previously demonstrated in vitro cytotoxicity of mesothelin-chimeric antigen receptor autologous T cells against pancreatic cancer cells using lentiviral vectors, but these vectors pose safety concerns. Here, we incorporated Sleeping Beauty and minicircle design enhancements into interleukin-2-secreting natural NK-92MI cells to eliminate both bacterial and viral components and address inhibition by the tumor microenvironment. METHODS: Parental (conventional deoxyribonucleic acid)-mesothelin-chimeric antigen receptor and minicircle-mesothelin-chimeric antigen receptor vectors were electroporated into NK-92MI cells and engraftment was visualized by immunofluorescence analysis with protein-L staining. Interferon-γ and granzyme B secretion were measured by enzyme-linked immunosorbent assay from cocultures of parental-mesothelin-chimeric antigen receptors and minicircle-mesothelin-chimeric antigen receptors with human pancreatic cancer cells, and cytotoxicity of chimeric antigen receptor NK-92MI cells was tested against three pancreatic cancer cell lines. RESULTS: Cloning of mesothelin-chimeric antigen receptor Sleeping Beauty into a minicircle vector removed its bacterial backbone and reduced its size with improved electroporation efficiency. Chimeric antigen receptor engraftment, Interferon-γ and granzyme B secretion, and specific lysis against all three pancreatic cancer lines were significantly increased with minicircle-mesothelin-chimeric antigen receptor versus parental-mesothelin-chimeric antigen receptor NK-92MI cells. CONCLUSION: We provide proof of concept that allogeneic mesothelin-chimeric antigen receptor NK-92MI cells with hybrid Sleeping Beauty and minicircle technologies provide increased engraftment and cytotoxicity in vitro with potential safety benefits when translated to the clinical arena.


Assuntos
Morte Celular/imunologia , Proteínas Ligadas por GPI/farmacologia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Neoplasias Pancreáticas/patologia , Receptores de Antígenos Quiméricos/imunologia , Linhagem Celular Tumoral , Eletroporação/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias Pancreáticas/terapia , Sensibilidade e Especificidade , Microambiente Tumoral
9.
Int J Mol Sci ; 20(16)2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430949

RESUMO

Electrochemotherapy is an efficient method for the local treatment of cutaneous and subcutaneous metastases, but its efficacy as a systemic treatment remains low. The application of gene electrotransfer (GET) to transfer DNA coding for immune system modulating molecules could allow for a systemic effect, but its applications are limited because of possible side effects, e.g., immune system overactivation and autoimmune response. In this paper, we present the simultaneous electrotransfer of bleomycin and plasmid DNA as a method to increase the systemic effect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve efficient cell transfection while killing cells via the cytotoxic effect of bleomycin at later time points. We also show the dynamics of both cell electrotransfection and cell death after the simultaneous electrotransfer of bleomycin and plasmid DNA. Therefore, this method could have applications in achieving the transient, cell death-controlled expression of immune system activating genes while retaining efficient bleomycin mediated cell killing.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , DNA/genética , Plasmídeos/genética , Transfecção/métodos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Bleomicina/administração & dosagem , Células CHO , Morte Celular/efeitos dos fármacos , Cricetulus , DNA/administração & dosagem , Eletroporação/métodos , Expressão Gênica/efeitos dos fármacos , Plasmídeos/administração & dosagem
10.
IET Nanobiotechnol ; 13(6): 609-616, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31432794

RESUMO

Here, a rapid and easy transformation by electroporation technique for gene transfer in plants using cell penetrating amino nanocomplex (nanoplex) has been demonstrated in Nicotiana. Nanoplex was prepared using cell penetrating amino acids (CPAs) such as poly-L-lysine (PLL) and Argenine (Arg), in combination with the gold nanoparticles (AuNPs). PLLs-modified nanoplex with zeta potential of 34.2 ± 1.22 mV charge showed 63.3% efficiency for gene transformation in plant cells as compared to 60% when modified with Arg and the zeta potential was found to be 30.0 ± 0.83 mV; whereas, the transformation efficiency without nanoplex was found to be 6.6%. The findings indicate that the zeta potential of positively charged nanocomplex (AuNPs/CPAs/DNA/CPAs) increases the transformation efficiency because of their ability to protect the DNA from electroporation wave and endogenous enzyme damage. Transformation was confirmed by GUS assay and amplification of npt gene. This technique may open up new possibilities of gene transfer in plants, which will enable to produce large number of transgenic plants.


Assuntos
Eletroporação/métodos , Técnicas de Transferência de Genes , Ouro/química , Nanopartículas Metálicas/química , Plantas/genética , Transformação Genética/fisiologia , Agrobacterium tumefaciens , Células Cultivadas , DNA de Plantas/genética , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Tabaco/citologia , Tabaco/genética
11.
Bioelectrochemistry ; 130: 107342, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31404809

RESUMO

Electroporation is a method which uses an adequate number of electric pulses of enough amplitude, duration and number applied to cells, thus inducing transient permeabilization of the cell membrane. Due to possibility that microenvironment in applications of in vivo electroporation is slightly acidic, we studied the effects of slightly acidic electroporation and recovery medium. We observed no difference in the permeabilization threshold, detected by propidium iodide, of cells which were electroporated and allowed to recover in growth (pH 7.8) or acidic (pH 6.5) medium. In contrast, statistically significant difference was observed in survival of cells that were exposed to pulse amplitudes greater than permeabilization threshold. Survival of cells was greater if acidic electroporation and recovery medium were used, but acidic extracellular pH decreased gene electrotransfer efficiency. We also observed differences in morphology between cells that were electroporated and left to recover in growth medium and cells that were electroporated and left to recover in acidic medium. Our results imply that slightly acidic extracellular pH allows more efficient repair of damage that is induced on cell membrane during electroporation with high pulse amplitudes.


Assuntos
Permeabilidade da Membrana Celular , Eletroporação , Técnicas de Transferência de Genes , Animais , Células CHO , Sobrevivência Celular , Cricetulus , Eletroporação/métodos , Concentração de Íons de Hidrogênio
12.
Appl Microbiol Biotechnol ; 103(19): 7917-7929, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392376

RESUMO

The growth of antibiotic resistant microorganisms and the increasing demand for nonthermal antimicrobial treatment in the food and beverage industry motivates research into alternative inactivation methods. Pulsed electric fields (PEFs) provide an athermal method for inactivating microorganisms by creating nanometer-sized membrane pores in microorganisms, inducing cell death when the PEF duration and intensity are sufficient such that the pores cannot reseal after the PEFs through a process referred to as irreversible electroporation. While PEF inactivation has been studied for several decades, recent studies have focused on extending the technique to various liquids in the food industry and optimizing microorganism inactivation while minimizing adverse effects to the treated sample. This minireview will assess the biophysical mechanisms and theory of PEF-induced cellular interactions and summarize recent advances in applying this technology for microorganism inactivation alone and synergistically in combination with other technologies, including temperature, pressure, natural ingredients, and pharmaceuticals.


Assuntos
Desinfecção/métodos , Eletroporação/métodos , Viabilidade Microbiana , Bactérias/crescimento & desenvolvimento , Fenômenos Biofísicos , Fungos/crescimento & desenvolvimento
13.
In Vitro Cell Dev Biol Anim ; 55(8): 598-603, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31297696

RESUMO

The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P < 0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P < 0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8-16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Eletroporação/métodos , Edição de Genes , Genoma , Mutação/genética , Zigoto/metabolismo , Animais , Blastocisto/metabolismo , Bovinos , Embrião de Mamíferos/metabolismo , Taxa de Mutação
14.
Nat Protoc ; 14(8): 2452-2482, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31341289

RESUMO

Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.


Assuntos
Sistemas CRISPR-Cas/genética , Eletroporação/métodos , Edição de Genes/métodos , Animais , Feminino , Masculino , Camundongos , Microinjeções , Oviductos/fisiologia , Gravidez , RNA Guia/administração & dosagem , RNA Guia/genética , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética
15.
Bioelectrochemistry ; 130: 107328, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31306879

RESUMO

Blood vessels, the extracellular space, and the cell membrane represent physiologic barriers to nanoparticle-based drug delivery for cancer therapy. We demonstrate that electroporation (EP) can assist in the delivery of dye stabilized sorafenib nanoparticles (SFB-IR783) by increasing the permeability of endothelial monolayers, improving diffusion through the extracellular space in tumorspheres, and by disrupting plasma membrane function in cancer cells. These changes occur in a dose-dependent fashion, increasing proportionally with electric field strength. Cell death from irreversible electroporation (IRE) was observed to contribute to the persistent transport of SFB-IR783 through these physiologic barriers. In a model of mice bearing bilateral xenograft HCT116 colorectal tumors, treatment with EP resulted in the immediate and increased uptake of SFB-IR783 when compared with the untreated contralateral tumor. The uptake of SFB-IR783 was independent of direct transfection of cells through EP and was mediated by changes in vascular permeability and extracellular diffusion. The combination of EP and SFB-IR783 was observed to result in 40% reduction in mean tumor diameter when compared with sham treatment (p < .05) at the time of sacrifice, which was not observed in cohorts treated with EP alone or SFB-IR783 alone. Treatment of tumor with EP can augment the uptake and increase the efficacy of nanoparticle therapy.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/tratamento farmacológico , Sorafenibe/administração & dosagem , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Eletroporação/métodos , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Sorafenibe/farmacocinética , Sorafenibe/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos
16.
Int J Mol Sci ; 20(11)2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31174257

RESUMO

The gene delivery to skeletal muscles is a promising strategy for the treatment of both muscular disorders (by silencing or overexpression of specific gene) and systemic secretion of therapeutic proteins. The use of a physical method like electroporation with plate or needle electrodes facilitates long-lasting gene silencing in situ. It has been reported that electroporation enhances the expression of the naked DNA gene in the skeletal muscle up to 100 times and decreases the changeability of the intramuscular expression. Coelectransfer of reporter genes such as green fluorescent protein (GFP), luciferase or beta-galactosidase allows the observation of correctly performed silencing in the muscles. Appropriate selection of plasmid injection volume and concentration, as well as electrotransfer parameters, such as the voltage, the length and the number of electrical pulses do not cause long-term damage to myocytes. In this review, we summarized the electroporation methodology as well as the procedure of electrotransfer to the gastrocnemius, tibialis, soleus and foot muscles and compare their advantages and disadvantages.


Assuntos
Eletroporação/métodos , Técnicas de Transferência de Genes/normas , Músculo Esquelético/metabolismo , Animais , Eletroporação/normas , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Plasmídeos/genética , Plasmídeos/metabolismo
17.
RNA ; 25(9): 1118-1129, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31151992

RESUMO

Oligonucleotide drugs are experiencing greater success in the clinic, encouraging the initiation of new projects. Resources are insufficient to develop every potentially important project, and persuasive experimental data using cell lines close to disease target tissue is needed to prioritize candidates. Friedreich's ataxia (FRDA) is a devastating and currently incurable disease caused by insufficient expression of the enzyme frataxin (FXN). We have previously shown that synthetic nucleic acids can activate FXN expression in human patient-derived fibroblast cells. We chose to further test these compounds in induced pluripotent stem cell-derived neuronal progenitor cells (iPSC-NPCs). Here we describe methods to deliver oligonucleotides and duplex RNAs into iPSC-NPCs using electroporation. Activation of FXN expression is potent, easily reproducible, and potencies parallel those determined using patient-derived fibroblast cells. A duplex RNA and several antisense oligonucleotides (ASOs) with different combinations of 2'-methoxyethyl (2'-MOE), 2'-fluoro (2'-F), and constrained ethyl (cEt) were active, providing multiple starting points for further development and highlighting improved potency as an important goal for preclinical development. Our data support the conclusion that ASO-mediated activation of FXN is a feasible approach for treating FRDA and that electroporation is a robust method for introducing ASOs to modulate gene expressions in neuronal cells.


Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Neurônios/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos/metabolismo , RNA/metabolismo , Linhagem Celular , Eletroporação/métodos , Fibroblastos/metabolismo , Ataxia de Friedreich/metabolismo , Expressão Gênica/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
18.
Mater Sci Eng C Mater Biol Appl ; 102: 437-446, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31147014

RESUMO

Owing to poor skin permeability, the transdermal (TRD) drug delivery at the required therapeutic rate still remains an arduous task. In the present investigation, a novel TRD enhancement strategy was introduced using the synergistic effect of gold nanoparticle (GNP) and skin electroporation. Diclofenac sodium (DS) was selected as a model drug. An electro-sensitive patch was constructed using skin adhesive matrix, polyvinyl alcohol/poly(dimethyl siloxane)-g-polyacrylate. GNP/carbon nanotube nanocomposite (GNP-CNT) was incorporated into the matrix with GNP and CNT to enhance skin permeability and electrical conductivity, respectively. Varying the concentration of GNP-CNT, alters the thermomechanical properties, water vapor permeability (WVP), drug encapsulation efficiency (DEE) and drug release profile, building a possibility to fine-tune the properties of the device. The membrane constructed with 1.5% GNP-CNT displayed the highest DEE and thermomechanical properties. The TRD DS release study was performed in rat skin at different GNP-CNT contents and variable conditions of applied voltage. Incorporating GNP-CNT enhanced the DS permeation profile with the best performance exhibited by device containing 1.5% nanofillers at an applied bias of 10.0 V. Electroporation in conjugation with GNP remarkably destroys the stratum corneum (SC) barrier by disparate mechanisms involving the breakdown of multilamellar lipid system, generation of new aqueous pathway and thermal effect. Furthermore, the dramatic disruption of lipid barriers generated by applied voltage was efficiently stabilized by GNP in addition to the transient and reversible openings created by them. Finally the safety of the device was confirmed by cell viability assay and environmental stability test. The developed skin permeation approach may open new avenues in TRD drug delivery.


Assuntos
Sistemas de Liberação de Medicamentos , Eletricidade , Eletroporação/métodos , Ouro/química , Nanopartículas Metálicas/química , Absorção Cutânea , Administração Cutânea , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Diclofenaco/farmacologia , Composição de Medicamentos , Liberação Controlada de Fármacos , Humanos , Nanopartículas Metálicas/ultraestrutura , Nanocompostos/química , Nanocompostos/ultraestrutura , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestrutura , Permeabilidade , Ratos , Vapor , Propriedades de Superfície , Resistência à Tração , Termogravimetria
19.
Bioelectromagnetics ; 40(5): 331-342, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31179573

RESUMO

In this work, the electroporation phenomenon induced by pulsed electric field on different nucleated biological cells is studied. A nonlinear, non-local, dispersive, and space-time multiphysics model based on Maxwell's and asymptotic Smoluchowski's equations has been developed to calculate the transmembrane voltage and pore density on both plasma and nuclear membrane perimeters. The irregular cell shape has been modeled by incorporating in the numerical algorithm the analytical functions pertaining to Gielis curves. The dielectric dispersion of the cell media has been modeled considering the multi-relaxation Debye-based relationship. Two different irregular nucleated cells have been investigated and their response has been studied applying both the dispersive and non-dispersive models. By a comparison of the obtained results, differences can be highlighted confirming the need to make use of the dispersive model to effectively investigate the cell response in terms of transmembrane voltages, pore densities, and electroporation opening angle, especially when irregular cell shapes and short electric pulses are considered. Bioelectromagnetics. 2019;40:331-342. © 2019 Wiley Periodicals, Inc.


Assuntos
Eletroporação , Modelos Biológicos , Dinâmica não Linear , Algoritmos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Simulação por Computador , Campos Eletromagnéticos , Eletroporação/métodos
20.
Diagn Interv Radiol ; 25(4): 304-309, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31199286

RESUMO

PURPOSE: Irreversible electroporation (IRE) is a nonthermal tumor ablation technique that induces cell apoptosis while preserving extracellular architecture. Surgical clips and embolic agents may lie adjacent to, or within, the target lesion. It is unknown to date if IRE causes degradation to the embolic agents or surgical clips that may have adverse effects to patients. We aimed to examine the effects of the IRE on the morphology of various embolic agents and the effects of these agents to the ablation field using a previously validated vegetal model. METHODS: Metallic surgical clips and various metallic and nonmetallic embolic agents were inserted within the center of the tuber ablation field. Additionally, clips were inserted on the edge and outside the ablation field. One tuber was ablated as a control. Ablation settings were based on previous published experiments. Tubers were imaged with magnetic resonance imaging (MRI) 18-24 hours after ablation and the ablated field dimensions were measured. Nonmetallic embolic agents were examined microscopically by the pathologist. RESULTS: Nonmetallic agents did not affect the ablation pattern. Metallic implants, however, caused arcing of the ablation margins. There was no macroscopic or microscopic degradation to the agents after IRE. CONCLUSION: The ablation zone arced in the presence of surgical clips at the edge or outside the ablation margins; therefore, nearby critical structures may be susceptible to the effects of IRE. Furthermore, there was no physical degradation of the embolic agents or surgical clips, and this may have importance when considering IRE ablation of previously embolized lesions in vivo.


Assuntos
Técnicas de Ablação/instrumentação , Eletroporação/métodos , Embolização Terapêutica/instrumentação , Verduras/citologia , Técnicas de Ablação/efeitos adversos , Apoptose/fisiologia , Carcinoma Hepatocelular/cirurgia , Embolização Terapêutica/efeitos adversos , Humanos , Fígado/patologia , Fígado/cirurgia , Imagem por Ressonância Magnética/métodos , Tubérculos , Solanum tuberosum/citologia , Instrumentos Cirúrgicos/efeitos adversos
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