RESUMO
Nanosecond Pulsed Electric Fields (nsPEF) have the potential to treat a variety of cancer types including melanoma, pancreatic and lung squamous cancers. Recent studies show that nsPEF-based cancer therapy may be improved further with the assistance of moderate heating of the target. A feedback-looped heating system, utilizing a 980-nm fiber optic laser, was integrated into nsPEF electrodes for tumor ablation. The laser beam profile was determined to be Gaussian using a knife-edge technique. Thermal properties of the biological target were evaluated based on the treatment area, penetration depth and thermal distribution due to laser irradiation with or without nsPEF. Synergistic effects between nsPEF and the moderately elevated temperature at the target was observed, resulting in enhanced overall survival tumor regression up to 50% in the treatment of lung squamous cell cancer in mice.
Assuntos
Carcinoma de Células Escamosas/radioterapia , Raios Infravermelhos/uso terapêutico , Terapia a Laser/métodos , Neoplasias Pulmonares/radioterapia , Tratamento por Radiofrequência Pulsada/métodos , Pele/efeitos da radiação , Animais , Engenharia Biomédica , Linhagem Celular Tumoral , Feminino , Tecnologia de Fibra Óptica , Temperatura Alta/uso terapêutico , Camundongos , Camundongos Endogâmicos DBA , SuínosRESUMO
Nanosecond pulse stimulation as a tumor ablation therapy has been studied for the treatment of various carcinomas in animal models and has shown a significant survival benefit. In the current study, we found that moderate heating at 43°C for 2 minutes significantly enhanced in vitro nanosecond pulse stimulation-induced cell death of KLN205 murine squamous cell carcinoma cells by 2.43-fold at 600 V and by 2.32-fold at 900 V, as evidenced by propidium iodide uptake. Furthermore, the ablation zone in KLN205 cells placed in a 3-dimensional cell-culture model and pulsed at a voltage of 900 V at 43°C was 3 times larger than in cells exposed to nanosecond pulse stimulation at room temperature. Application of moderate heating alone did not cause cell death. A nanosecond pulse stimulation electrode with integrated controllable laser heating was developed to treat murine ectopic squamous cell carcinoma. With this innovative system, we were able to quickly heat and maintain the temperature of the target tumor at 43°C during nanosecond pulse stimulation. Nanosecond pulse stimulation with moderate heating was shown to significantly extend overall survival, delay tumor growth, and achieve a high rate of complete tumor regression. Moderate heating extended survival nearly 3-fold where median overall survival was 22 days for 9.8 kV without moderate heating and over 63 days for tumors pulsed with 600, 100 ns pulses at 5 Hz, at voltage of 9.8 kV with moderate heating. Median overall survival in the control groups was 24 and 31 days for mice with untreated tumors and tumors receiving moderate heat alone, respectively. Nearly 69% (11 of 16) of tumor-bearing mice treated with nanosecond pulse stimulation with moderate heating were tumor free at the completion of the study, whereas complete tumor regression was not observed in the control groups and in 9.8 kV without moderate heating. These results suggest moderate heating can reduce the necessary applied voltage for tumor ablation with nanosecond pulse stimulation.
Assuntos
Carcinoma de Células Escamosas/terapia , Eletroporação/métodos , Temperatura Alta/uso terapêutico , Terapia a Laser , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
BACKGROUND: Health care-associated opportunistic pathogens Staphylococcus aureus and Enterococcus faecium persist on dry environments and can contribute to organism transmission through contact. These organisms can be monitored on surfaces by culture, molecular methods, or metabolic assays. This study was designed to determine the kinetics of bacterial persistence on acrylonitrile butadiene styrene, a plastic commonly used in the manufacture of bedrails. MATERIALS AND METHODS: Polymerase chain reaction for genomic DNA was used to confirm the presence of bacteria cells on this plastic irrespective of viability. Bacterial viability was measured by culture, ATP quantification, and a metabolic assay at time points up to and longer than 1 year. RESULTS: Polymerase chain reaction confirmed the presence of bacteria on the plastic for the entire time period studied. However, S aureus culturability was reduced after 3 and 7 weeks; neither organism was culturable after 1 year. At 7 weeks, ATP levels were reduced for both organisms, paralleling S aureus culturability but indicating that ATP quantification did not predict E faecium culturability. S aureus-reducing potential was reduced after 7 weeks, whereas E faecium-reducing potential reached the level of fresh inoculum after 12 hours in broth culture. Low but significant levels of metabolic activity were detected for each organism after 1 year. CONCLUSIONS: S aureus and E faecium cells may retain viability on plastic for longer than 1 year.
Assuntos
Contaminação de Equipamentos , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/fisiologia , Plásticos , DNA Bacteriano , Genoma Bacteriano , Propriedades de Superfície , Fatores de TempoRESUMO
Irreversible electroporation (IRE) as a non-thermal tumor ablation technology has been studied for the treatment of pancreatic carcinoma and has shown a significant survival benefit. We discovered that moderate heating (MH) at 43 °C for 1-2 minutes significantly enhanced ex vivo IRE tumor ablation of Pan02 cells by 5.67-fold at 750 V/cm and by 1.67-fold at 1500 V/cm. This amount of heating alone did not cause cell death. An integrated IRE system with controllable laser heating and tumor impedance monitoring was developed to treat mouse ectopic pancreatic cancer. With this novel IRE system, we were able to heat and maintain the temperature of a targeted tumor area at 42 °C during IRE treatment. Pre-heating the tumor greatly reduced the impedance of tumor and its fluctuation. Most importantly, MHIRE has been demonstrated to significantly extend median survival and achieve a high rate of complete tumor regression. Median survival was 43, 46 and 84 days, for control, IRE with 100 µs, 1 Hz, 90 pulses and electric fields 2000-2500 V/cm and MHIRE treatment respectively. 55.6% of tumor-bearing mice treated with MHIRE were tumor-free, whereas complete tumor regression was not observed in the control and IRE treatment groups.
Assuntos
Hipertermia Induzida/instrumentação , Hipertermia Induzida/métodos , Neoplasias Experimentais/terapia , Neoplasias Pancreáticas/terapia , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets.
RESUMO
Gene electrotransfer (GET) is a proven and valuable tool for in vivo gene delivery to a variety of tissues such as skin, cardiac muscle, skeletal muscle, and tumors, with controllable gene delivery and expression levels. Optimizing gene expression is a challenging hurdle in preclinical studies, particularly for skin indications, due to differences in electrical conductivity of animal compared to human dermis. Therefore, the goal of this study was to develop an ex vivo model for GET using recellularized human dermis to more closely mimic human skin. Decellularized human dermis (DermACELL(®)) was cultured with human dermal fibroblasts and keratinocytes for 4 weeks. After one week of fibroblast culture, fibroblasts infiltrated and dispersed throughout the dermis. Air-liquid interface culture led to epithelial cell proliferation, stratification and terminal differentiation with distinct basal, spinous, granular and cornified strata. Firefly luciferase expression kinetics were evaluated after GET of recellularized constructs for testing gene delivery parameters to skin in vitro. Elevated luciferase expression persisted up to a week following GET compared to controls without electrotransfer. In summary, recellularized dermis structurally and functionally resembled native human skin in tissue histological organization and homeostasis, proving an effective 3D human skin model for preclinical gene delivery studies.
Assuntos
Derme/citologia , Células Epidérmicas , Técnicas de Transferência de Genes , Queratinócitos/citologia , Pele Artificial , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , Humanos , Masculino , Engenharia TecidualRESUMO
Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic pathogen responsible for infections at many body sites. In the last 10 years, clinical strains resistant to many or all commonly used antibiotics have emerged globally. With few antimicrobial agents in the pharmaceutical pipeline, new and alternative agents are essential. Platelets secrete a large number of proteins, including proteins with antimicrobial activity. In a previous study, we demonstrated that donor platelet supernatants and plasma significantly inhibited the growth of a reference strain of A. baumannii in broth and on skin. This inhibition appeared to be unrelated to the platelet activation state. In this study, we demonstrate that this growth inhibition extends to clinical multidrug resistant isolates. We also demonstrate that there is no relationship between this activity and selected platelet-derived antimicrobial proteins. Instead, the donor plasma components complement and alpha-2 macroglobulin are implicated.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/metabolismo , Atividade Bactericida do Sangue , Proteínas do Sistema Complemento/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Plasma/química , alfa-Macroglobulinas/metabolismo , Acinetobacter baumannii/fisiologia , HumanosRESUMO
Cold plasma is emerging as a potential method for medical applications. The current study assessed the efficacy of a novel cold plasma reactor based on shielded sliding discharge producing cathode-directed streamers generated in ambient air for the delivery of plasmid DNA. Experiments were performed with mouse melanoma cells (B16F10) and human keratinocyte cells (HaCaT) inoculated with plasmid DNA encoding luciferase. Quantitative results measured over a 72-h period displayed luciferase expression levels as high as 5-fold greater in cells exposed to plasma-activated air (PAA) than levels obtained from the inoculation of plasmid DNA alone (P < 0.05, P < 0.01). No effect on cell viability was observed. Delivery of plasmid encoding GFP to HaCaT cells seeded on polycaprolactone (PCL) scaffolds was confirmed by immunostaining. The use of cold plasma for DNA delivery is attractive as it provides a non-viral, non-invasive method where the electrode or the plasma itself never directly contacts the exposed site. The current device design provides localized DNA transfer using a novel technology. Our report suggests PAA warrants further exploration as an alternative or supplemental approach for DNA transfer.
Assuntos
Ar , DNA/metabolismo , Gases em Plasma , Plasmídeos/metabolismo , Transfecção/instrumentação , Transfecção/métodos , Animais , Linhagem Celular , Sobrevivência Celular , Genes Reporter , Humanos , Queratinócitos/fisiologia , Luciferases/análise , Melanócitos/fisiologia , CamundongosRESUMO
Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.
Assuntos
Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Plaquetas/metabolismo , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/microbiologia , Plasma Rico em Plaquetas/metabolismo , Ferimentos e Lesões/tratamento farmacológico , Ferimentos e Lesões/microbiologia , Acinetobacter baumannii , Animais , Modelos Animais de Doenças , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Ratos , Pele/lesões , Staphylococcus aureus , CicatrizaçãoRESUMO
Cold atmospheric plasma inactivates bacteria through reactive species produced from the applied gas. The use of cold plasma clinically has gained recent interest, as the need for alternative or supplementary strategies are necessary for preventing multi-drug resistant infections. The purpose of this study was to evaluate the antibacterial efficacy of a novel shielded sliding discharge based cold plasma reactor operated by nanosecond voltage pulses in atmospheric air on both biotic and inanimate surfaces. Bacterial inactivation was determined by direct quantification of colony forming units. The plasma activated air (afterglow) was bactericidal against Escherichia coli and Staphylococcus epidermidis seeded on culture media, laminate, and linoleum vinyl. In general, E. coli was more susceptible to plasma exposure. A bacterial reduction was observed with the application of air alone on a laminate surface. Whole-cell real-time PCR revealed a decrease in the presence of E. coli genomic DNA on exposed samples. These findings suggest that plasma-induced bacterial inactivation is surface-dependent.