RESUMO
Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.
Assuntos
Archaeoglobus fulgidus , Proteínas de Membrana , Desnaturação Proteica , Dodecilsulfato de Sódio , Termodinâmica , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/farmacologia , Archaeoglobus fulgidus/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Espectrometria de Fluorescência , Estabilidade Proteica , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Naftalenossulfonato de Anilina/química , Naftalenossulfonato de Anilina/metabolismo , Triptofano/química , Triptofano/metabolismo , Cobre/química , Cobre/metabolismo , Dobramento de Proteína , Conformação ProteicaRESUMO
Mucormycosis is an extremely aggressive fungal disease with a high mortality rate, especially in people with compromised immune systems. Most cases of mucormycosis are caused by the fungus Rhizopus oryzae. The treatments used are based on high doses of antifungals, associated with surgical resections, when it is possible. However, even with this aggressive treatment, the estimated attributable mortality rate is high. There is therefore a need to develop adjuvant treatments. Photodynamic Inactivation (PDI) may be an auxiliary therapeutic option for mucormycosis. Due to the lack of reports in the literature on the morphology and photodynamic inactivation of R. oryzae, characterization of the fungus using Confocal Microscopy and Transmission Electron Microscopy, and different protocols using Photodithazine® (PDZ), a chlorin e6 compound, as a photosensitizer, were performed. The fungus growth rate under different concentrations and incubation times of the photosensitizer and its association with the surfactant Sodium Dodecyl Sulphate (SDS) was evaluated. For the hyphae, both in the light and dark phases, in the protocols using only PDZ, no effective photodynamic response was observed. Meanwhile with the combination of SDS 0.05% and PDZ, inhibition growth rates of 98% and 72% were achieved for the white and black phase, respectively. In the conidia phase, only a 1.7 log10 reduction of the infective spores was observed. High concentration of melanin and the complex and resistant structures, especially at the black phase, results in a high limitation of the PDI inactivation response. The combined use of the SDS resulted in an improved response, when compared to the one obtained with the amphotericin B treatment.
Assuntos
Fármacos Fotossensibilizantes , Rhizopus oryzae , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Rhizopus oryzae/efeitos dos fármacos , Porfirinas/farmacologia , Porfirinas/química , Fotoquimioterapia , Antifúngicos/farmacologia , Antifúngicos/química , Dodecilsulfato de Sódio/farmacologia , Dodecilsulfato de Sódio/química , Luz , Testes de Sensibilidade MicrobianaRESUMO
Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.
Assuntos
Adenosina Trifosfatases , Biocatálise , Cobre , Detergentes , Micelas , Dodecilsulfato de Sódio , Adenosina Trifosfatases/metabolismo , Archaeoglobus fulgidus/enzimologia , Biocatálise/efeitos dos fármacos , Calorimetria , Cobre/metabolismo , Detergentes/farmacologia , Hidrólise/efeitos dos fármacos , Legionella pneumophila/enzimologia , Dodecilsulfato de Sódio/farmacologia , Temperatura , TermodinâmicaRESUMO
Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-ß -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER > 400 Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p > 0.05) on the permeability of furosemide (1.80 × 10-5 ± 4.55 × 10-7 cm/s); however, metformin permeability (1.36 × 10-5 ± 1.25 × 10-6 cm/s) increased significantly (p < 0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-ß -cyclodextrin (w/v) (3.5-fold), 10 and 20 µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p < 0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p < 0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.
Assuntos
Adenocarcinoma , Quitosana , Neoplasias Colorretais , Metformina , Humanos , Células CACO-2 , Quitosana/farmacologia , Furosemida/farmacologia , Dodecilsulfato de Sódio/farmacologia , Metformina/farmacologia , Permeabilidade , Absorção IntestinalRESUMO
In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non-ionic (0.1% v/v triton X-100, t-octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation-driven set-up. After 48 h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold-like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72 h on normal insulin-producing cells (MIN-6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non-toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source.
Assuntos
Detergentes , Insulinas , Animais , Antibacterianos/farmacologia , Colágeno/metabolismo , DNA/metabolismo , Matriz Extracelular Descelularizada , Detergentes/química , Detergentes/metabolismo , Detergentes/farmacologia , Etanol/farmacologia , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Cabras , Insulinas/análise , Insulinas/metabolismo , Insulinas/farmacologia , Camundongos , Octoxinol/análise , Octoxinol/metabolismo , Octoxinol/farmacologia , Pâncreas , Estudos Prospectivos , Dodecilsulfato de Sódio/análise , Dodecilsulfato de Sódio/metabolismo , Dodecilsulfato de Sódio/farmacologia , Suínos , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Engineered replacement materials have tremendous potential for vascular applications where over 400,000 damaged and diseased blood vessels are replaced annually in the United States alone. Unlike large diameter blood vessels, which are effectively replaced by synthetic materials, prosthetic small-diameter vessels are prone to early failure, restenosis, and reintervention surgery. We investigated the differential response of varying 0%-6% sodium dodecyl sulfate and sodium deoxycholate anionic detergent concentrations after 24 and 72 h in the presence of DNase using biochemical, histological, and biaxial mechanical analyses to optimize the decellularization process for xenogeneic vascular tissue sources, specifically the porcine internal thoracic artery (ITA). Detergent concentrations greater than 1% were successful at removing cytoplasmic and cell surface proteins but not DNA content after 24 h. A progressive increase in porosity and decrease in glycosaminoglycan (GAG) content was observed with detergent concentration. Augmented porosity was likely due to the removal of both cells and GAGs and could influence recellularization strategies. The treatment duration on the other hand, significantly improved decellularization by reducing DNA content to trace amounts after 72 h. Prolonged treatment times reduced laminin content and influenced the vessel's mechanical behavior in terms of altered circumferential stress and stretch while further increasing porosity. Collectively, DNase with 1% detergent for 72 h provided an effective and efficient decellularization strategy to be employed in the preparation of porcine ITAs as bypass graft scaffolding materials with minor biomechanical and histological penalties.
Assuntos
Artéria Torácica Interna , Alicerces Teciduais , Animais , Detergentes/farmacologia , Duração da Terapia , Matriz Extracelular/química , Humanos , Dodecilsulfato de Sódio/farmacologia , Suínos , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
The iontophoresis delivery of insulin (INS) remains a serious challenge due to the low permeability of the drug through the skin. This work aims to investigate the potential of water-soluble polypyrrole nanoparticles (WS-PPyNPs) as a drug donor matrix for controlled transdermal iontophoresis of INS. WS-PPyNPs have been prepared via a simple chemical polymerization in the presence of sodium dodecyl sulfate (SDS) as both dopant and the stabilizing agent. The synthesis of the soluble polymer was characterized using field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), fluorescence spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. The loading mechanism of INS onto the WS-PPyNPs is based on the fact that the drug molecules can be replaced with doped dodecyl sulfate. A two-compartment Franz-type diffusion cell was employed to study the effect of current density, formulation pH, INS concentration, and sodium chloride concentration on anodal iontophoresis (AIP) and cathodal iontophoresis (CIP) of INS across the rat skin. Both AIP and CIP delivery of INS using WS-PPyNPs were significantly increased compared to passive delivery. Furthermore, while the AIP experiment (60 min at 0.13 mA cm-2) show low cumulative drug permeation for INS (about 20.48 µg cm-2); the CIP stimulation exhibited a cumulative drug permeation of 68.29 µg cm-2. This improvement is due to the separation of positively charged WS-PPyNPs and negatively charged INS that has occurred in the presence of cathodal stimulation. The obtained results confirm the potential applicability of WS-PPyNPs as an effective approach in the development of controlled transdermal iontophoresis of INS.
Assuntos
Insulina/farmacologia , Iontoforese/métodos , Nanopartículas/química , Pele/efeitos dos fármacos , Administração Cutânea , Animais , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Humanos , Microscopia Eletrônica de Varredura , Polímeros/química , Polímeros/farmacologia , Pirróis/química , Pirróis/farmacologia , Coelhos , Pele/ultraestrutura , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/farmacologia , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Água/químicaRESUMO
Quercus infectoria (nutgall) has been reported to possess antimicrobial activities against a wide range of pathogens. Nevertheless, the biofilm removal effect of nutgall extract has not been widely investigated. In this study, we therefore evaluated the effect of nutgall extract in combination with cetrimonium bromide (CTAB) against preformed biofilm of Salmonella Typhimurium on polypropylene (PP) and stainless steel (SS) coupons in comparison with other sanitizers. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of nutgall extract and surfactants (CTAB and sodium dodecyl sulfate; SDS) were assessed. CTAB showed a more efficient antimicrobial activity than SDS and was selected to use in combination with nutgall extract for removing biofilm. To determine the biofilm removal efficacy, the PP and SS coupons were individually submerged in 2x MBC of nutgall extract (256 mg/ml) + 2x MBC of CTAB (2.5 mg/ml), nutgall extract alone (256 mg/ml), CTAB alone (2.5 mg/ml), distilled water, and 100 ppm sodium hypochlorite for 5, 15, and 30 min. The remaining sessile cells in biofilm were determined. Overall, the greatest biofilm removal efficacy was observed with nutgall extract + CTAB; the biofilm removal efficacy of sanitizers tended to increase with the exposure time. The SEM analysis demonstrated that S. Typhimurium biofilm on PP and SS coupons after exposure to nutgall extract + CTAB for 30 min displayed morphological alterations with wrinkles. This study suggests nutgall extract + CTAB may be an alternative to commonly used sanitizers to remove biofilm from food contact surfaces in the food industry and household.
Assuntos
Biofilmes/efeitos dos fármacos , Cetrimônio/farmacologia , Extratos Vegetais/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Contagem de Colônia Microbiana , Descontaminação/métodos , Microbiologia de Alimentos , Testes de Sensibilidade Microbiana , Tumores de Planta , Polipropilenos , Quercus/química , Aço Inoxidável , Tensoativos/farmacologiaRESUMO
The present study was assessed to determine the in vivo toxic effects of a cationic surfactant, cetylpyridinium chloride (CPC), and an anionic surfactant, sodium dodecyl sulfate (SDS) in terms of oxidative stress biomarkers in benthic oligochaete worm Tubifex tubifex for 14 days. The investigation demonstrated that sublethal concentrations of CPC (0.0213, and 0.0639 mg L-1) and SDS (1.094 and 3.092 mg L-1)induced paramount alterations in the oxidative stress enzymes in Tubifex tubifex. Superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH), and glutathione peroxidase (GPx) exhibited an initial notable increase in their activities in the surfactants exposed worms at 1d and 7d of exposure period followed by consequential reduction at 14d exposure period with respect to control, while catalase (CAT) and malondialdehyde (MDA) activities markedly incremented gradually throughout the exposure periods. Through the construction of the correlation matrix and integrated biomarker response (IBR), the effects of CPC and SDS on Tubifex tubifex were distinguished. These results indicate that exposure to these cationic and anionic surfactants modulates the levels of oxidative stress enzymes in Tubifex tubifex.
Assuntos
Cetilpiridínio/farmacologia , Oligoquetos/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Animais , Ânions/química , Biomarcadores/metabolismo , Catalase/metabolismo , Cátions/química , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Metalotioneína/metabolismo , Oligoquetos/metabolismo , Oligoquetos/fisiologia , Dodecilsulfato de Sódio/química , Superóxido Dismutase/metabolismo , Tensoativos/químicaRESUMO
Tissue engineering vascular grafts (TEVGs) constructed by decellularized arteries have the potential to replace autologous blood vessels in bypass surgery for patients with cardiovascular disease. There are various methods of decellularization without a standard protocol. Detergents approaches are simple, and easy control of experimental conditions. Non-ionic detergent Triton X-100 and ionic detergent sodium dodecyl sulfate (SDS) are the most commonly used detergents. In this study, we used Triton X-100 and SDS with different concentrations to decellularize porcine carotid arteries. After that, we investigated the acellular effect and mechanical properties of decellularized arteries to find a promising concentration combination for decellularization. Results showed that any detergents' combination would damage the inherent structure of extracellular matrix, and the destruction increased with the increase of detergents' concentration. We concluded that the decellularization approach of 0.5% Triton X-100 for 24 h combined with 0.25% SDS for 72 h could help to obtain decellularized arteries with minimum destruction. This protocol may be able to prepare a clinically suitable vascular scaffold for TEVGs.
Assuntos
Prótese Vascular , Engenharia Tecidual , Animais , Artérias Carótidas , Detergentes/farmacologia , Matriz Extracelular , Octoxinol/farmacologia , Dodecilsulfato de Sódio/farmacologia , Suínos , Alicerces TeciduaisAssuntos
Corantes/farmacologia , Expressão Gênica/efeitos dos fármacos , Tinturas para Cabelo/química , Heme Oxigenase-1/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Aminopiridinas/farmacologia , Antraquinonas/farmacologia , Compostos Azo/farmacologia , Cresóis/farmacologia , Dimetil Sulfóxido/farmacologia , Dinitroclorobenzeno/farmacologia , Eugenol/farmacologia , Células HaCaT , Heme Oxigenase-1/genética , Humanos , Hidroquinonas/farmacologia , Técnicas In Vitro , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Naftoquinonas/farmacologia , Fenilenodiaminas/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Pirogalol/farmacologia , RNA Mensageiro/metabolismo , Resorcinóis/farmacologia , Dodecilsulfato de Sódio/farmacologiaRESUMO
Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.
Assuntos
Derme Acelular , Engenharia Tecidual/métodos , Útero/citologia , Animais , Ácido Desoxicólico/farmacologia , Matriz Extracelular/ultraestrutura , Feminino , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Modelos Anatômicos , Octoxinol/farmacologia , Preservação de Órgãos , Perfusão , Ovinos , Dodecilsulfato de Sódio/farmacologia , Soluções/toxicidade , Artéria Uterina , Útero/irrigação sanguíneaRESUMO
Bacteria in biofilms are more resistant to antibacterial agents than bacteria in planktonic form. Hence, antibacterial agents should be able to eradicate biofilms to ensure the best outcomes. Little is known about how well many antibacterial agents can disrupt biofilms. In this study, we compared sodium lauryl sulfate (SDS), rhamnolipids (RHL), and N-acetylcysteine (NAC) for their ability to eradicate mature biofilms and inhibit new biofilm formation against Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans. SDS and RHL effectively inhibited formation of five bacterial biofilms in a dose-dependent manner, even at concentrations below the minimal inhibitory concentrations (MICs), suggesting that their antibiofilm activities are unrelated to their antibacterial activities. In contrast, NAC at certain concentrations promoted biofilm formation by all bacteria except P. aeruginosa, whereas at supra-MIC concentrations, it inhibited biofilm formation against the four bacteria, suggesting that its antibiofilm activity depends on its antibacterial activity. NAC was ineffective at eradicating mature H. pylori biofilms, and it actually promoted their formation at concentrations >10 mg/mL. Our results suggest that RHL is superior at eradicating biofilms of H. pylori, E. coli, and S. mutans; SDS is more effective against S. aureus biofilms; and NAC is more effective against P. aeruginosa biofilms. Our results may help determine which antibiofilm agents are effective against certain bacterial strains and develop agents effective against specific bacterial threats.
Assuntos
Acetilcisteína/farmacologia , Biofilmes/efeitos dos fármacos , Glicolipídeos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Meios de Cultura/química , Meios de Cultura/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Helicobacter pylori/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Plâncton/efeitos dos fármacos , Plâncton/crescimento & desenvolvimento , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/crescimento & desenvolvimentoRESUMO
INTRODUCTION: Tissue loss as a consequence of congenital anomalies, trauma, malignancy, or gangrene represents a major health care problem in the United States. Because younger individuals are disproportionately affected, the costs are magnified over time and the resultant individual and societal effects are tremendous. The currently available options to restore soft tissue defects are associated with donor site morbidities. Vascularized composite allotransplantation may provide form, function, and esthetics without a donor site; however, it comes with the significant risk associated with toxic immunosuppression (Biomaterials. 2015;61:246-256, Ann Plast Surg. 2015;75(1):112-116, Transplantation. 2009;88(2):203-210). Engineered tissues offer promise in finding viable alternatives to allograft and autologous tissues. In this study, we present our simple and quick method to decellularize a muscle without disrupting the vascular network integrity or the extracellular matrix. Optimizing the decellularization process is a crucial step toward creating an "off-the-shelf" flap that can be used for soft tissue reconstruction. METHODS: The superficial gracilis muscle of 20 rats were harvested on their circulation and decellularized using perfusion with Krebs-Henseleit buffer and sodium dodecyl sulfate for 6 hours. These flaps were evaluated by gross morphology, histology, DNA quantification, integrity of the vascular network, scanning electron microscopy, and transmission electron microscopy. RESULTS: All samples were decellularized successfully as determined by DNA content and histological analysis for cellular content. The vascular network was preserved in all samples. CONCLUSIONS: We present a quick, simple, and affordable method to decellularize a muscle flap through the vascular network. Our proposed method is efficient and can be completed in a significantly shorter time when compared with other methods. It is also safe and does not affect integrity of tissue, and this is essential for a reliable recellularization.
Assuntos
Músculo Grácil/transplante , Dodecilsulfato de Sódio/farmacologia , Lesões dos Tecidos Moles/cirurgia , Retalhos Cirúrgicos/transplante , Engenharia Tecidual/métodos , Derme Acelular , Animais , Materiais Biocompatíveis , Sistema Livre de Células , Modelos Animais de Doenças , Músculo Grácil/citologia , Humanos , Perfusão/métodos , Ratos , Sensibilidade e Especificidade , Coleta de Tecidos e ÓrgãosRESUMO
We describe herein a chitosan nanocarrier for drug delivery applications obtained through the self-assembly of carboxymethyl-hexanoyl chitosan and dodecyl sulfate (CHC-SDS). Nanocapsules with spherical morphology were obtained in phosphate buffer at pHâ¯7.4. These CHC-SDS nanocapsules showed no toxicity toward Jurkat cells (acute lymphoblastic leukemia) and were used to encapsulate a new pyrazoline (H3TM04) with antileukemia activity. The samples were characterized by dynamic light scattering (DLS) and Laser Doppler Micro-Electrophoresis. The encapsulation efficiency was higher than 96% (293.6⯵gâ¯mL-1) and the H3TM04-loaded nanocapsules (CHC-SDS-H) had a negative surface charge (-29.8⯱â¯0.7â¯mV) and hydrodynamic radius of around 84â¯nm. For the first time, CHC-SDS-H were formed and the antitumoral cancer activity was proved. The in vitro assays showed the controlled release of H3TM04 from the CHC-SDS-H nanocapsules in phosphate buffer pHâ¯7.4. The H3TM04 release data were described by the power law model, indicating that H3TM04 delivery occurred via an erosion mechanism. The cytotoxicity assays with Jurkat and K-562 cells (acute myeloid leukemia) demonstrated that the CHC-SDS-H nanocapsule decreases the half maximal inhibitory concentration (IC50). The study showed that CHC-SDS nanocapsules represent a promising nanocarrier for pyrazoline derivates that could be applied in leukemia therapy.
Assuntos
Antineoplásicos , Portadores de Fármacos , Leucemia/tratamento farmacológico , Nanopartículas , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Quitosana/análogos & derivados , Quitosana/química , Quitosana/farmacocinética , Quitosana/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Humanos , Células Jurkat , Células K562 , Leucemia/metabolismo , Leucemia/patologia , Nanopartículas/química , Nanopartículas/uso terapêutico , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/farmacocinética , Dodecilsulfato de Sódio/farmacologiaRESUMO
Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.
Assuntos
Antiprotozoários/farmacologia , Cetrimônio/farmacologia , Citotoxinas/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Antiprotozoários/química , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cetrimônio/química , Citotoxinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Concentração Inibidora 50 , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Simulação de Dinâmica Molecular , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Compostos de Amônio Quaternário/química , Dodecilsulfato de Sódio/química , Marcadores de Spin , Ácidos Esteáricos/química , Tensoativos/químicaRESUMO
Abstract Background: Pericardium tissue allograft can be used for surgical repair in several procedures. One of the tissue engineering strategies is the process of decellularization. This process decreases immunogenic response, but it may modify the natural extracellular matrix composition and behavior. Objective: The aim of this study was to evaluate the effectiveness of cell removal, maintenance of extracellular matrix properties and mechanical integrity of decellularized human pericardium using a low concentration solution of sodium dodecyl sulfate. Methods: Decellularization was performed with sodium dodecyl sulfate and ethylenediaminetetraacetic acid. Histological analysis, DNA quantification, evaluation of glycosaminoglycans and collagen were performed. Biomechanical assay was performed using tensile test to compare the decellularization effects on tissue properties of tensile strength, elongation and elastic modulus. P < 0.05 was considered significant. Results: There was reduction in visible nuclei present in pericardium tissue after decellularization, but it retained collagen and elastin bundles similar to fresh pericardium. The DNA contents of the decellularized pericardium were significantly reduced to less than 511.23 ± 120.4 ng per mg of dry weight (p < 0.001). The biomechanical assay showed no significant difference for fresh or decellularized tissue. Conclusion: The decellularization process reduces cell content as well as extracellular matrix components without changing its biomechanical properties.
Resumo Fundameto: O enxerto de pericárdio pode ser usado em muitos procedimentos de correção cirúrgica. Uma das estratégias da engenharia tecidual é o processo de descelularização. No entanto, embora esse processo diminua a resposta imunogênica, a descelularização pode modificar tanto o comportamento como a composição da matriz extracelular natural. Objetivos: Avaliar a eficácia da descelularização usando baixa concentração de dodecil sulfato de sódio na remoção celular, na manutenção das propriedades da matriz extracelular e na integridade mecânica do pericárdio humano descelularizado. Métodos: A descelularização foi realizada com dodecil sulfato de sódio e ácido etilenodiamino tetra-acético. Foi realizada análise histológica, quantificação de DNA, e avaliação de glicosaminoglicanos e colágeno. O estudo biomecânico foi conduzido pelo teste de tração para comparar os efeitos da descelularização sobre as propriedades teciduais de resistência à tração, alongamento e módulo de elasticidade. Foi considerado um valor de p < 0,05 como estatisticamente significativo. Resultados: Observou-se uma redução na quantidade de núcleos presentes no pericárdio após a descelularização, apesar de manter quantidades similares de feixes de elastina e de colágeno. As concentrações de DNA do pericárdio descelularizado foram significativamente reduzidas para menos que 511,23 ± 120,4 ng por mg de peso seco (p < 0,001). O teste biomecânico não apontou diferenças entre os tecidos fresco e descelularizado. Conclusão: A descelularização reduziu a concentração de células bem como os componentes da matriz extracelular sem afetar suas propriedades biomecânicas.
Assuntos
Humanos , Adolescente , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Pericárdio/citologia , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Separação Celular/métodos , Engenharia Tecidual/métodos , Pericárdio/efeitos dos fármacos , Fenômenos Biomecânicos , Medicina Regenerativa , Alicerces TeciduaisRESUMO
The pH of the skin surface increases with age and thus reduces epidermal barrier function. Aged skin needs appropriate skin care to counterbalance age-related pH increase and improve barrier function. This confirmatory randomized study investigated the efficacy of water-in-oil (w/o) emulsions with either pH 4 or pH 5.8 in 20 elderly subjects after 4 weeks of treatment. After the treatment, the skin was challenged with a sodium dodecyl sulphate (SDS) solution in order to analyze barrier protection properties of both formulations. The pH 4 w/o emulsion resulted in a significantly lower skin pH compared with the pH 5.8 w/o emulsion and an improved skin hydration after 4-week treatment. Further, the pH 4 emulsion led to more pronounced improvements in length of intercellular lipid lamellae, lamellar organization as well as lipid levels than the pH 5.8 emulsion. Following SDS-induced barrier damage to the skin, the pH of all test areas increased, but the area treated with the pH 4 emulsion showed the lowest increase compared with baseline. In addition, even after the SDS challenge the skin area treated with the pH 4 emulsion still maintained a significantly increased length of intercellular lipid lamellae compared with the beginning of the study. This study provides evidence that topical application of a w/o emulsion with pH 4 reacidifies the skin in elderly and has beneficial effects on skin moisturization, regeneration of lipid lamellae and lipid content. Application of a pH 4 emulsion can improve the epidermal barrier as well as the stratum corneum organization in aged skin.
Assuntos
Cosméticos/administração & dosagem , Epiderme/metabolismo , Envelhecimento da Pele/efeitos dos fármacos , Perda Insensível de Água/efeitos dos fármacos , Administração Cutânea , Idoso , Método Duplo-Cego , Emulsões , Epiderme/efeitos dos fármacos , Epiderme/ultraestrutura , Espaço Extracelular/diagnóstico por imagem , Espaço Extracelular/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Óleos/química , Permeabilidade/efeitos dos fármacos , Envelhecimento da Pele/fisiologia , Dodecilsulfato de Sódio/farmacologia , Resultado do Tratamento , Água/químicaRESUMO
This study investigated the effect of surfactants associated with sodium fluoride (NaF) on enamel erosion prevention, using an erosion-remineralization in vitro model. Sodium lauryl sulfate (SLS), polysorbate 20 (P20), and cocoamidopropyl betaine (CAPB) were tested, at concentrations of 1.0 and 1.5%, and associated or not with NaF (275 ppm). The control groups were distilled water and the NaF solution. Bovine enamel samples (n = 12) were prepared and submitted to a 5-day cycling model: acid challenge (0.3% citric acid, pH 2.6, 4×/day), human saliva (2 h, 4×/day), and the treatment solutions (2 min, 2×/day). The protective potential of the agents against initial erosion was assessed by microhardness and the surface loss by profilometry. Enamel surface wettability was determined by goniometry, protein adsorption was measured by spectroscopy (FTIR), and the KOH-soluble fluoride was quantified. Goniometry showed that SLS and CAPB increased enamel wettability. No differences were found among the surfactants regarding protein adsorption. Microhardness showed that SLS reduced NaF protection. P20 (1 and 1.5%) and CAPB 1.5% presented a protective effect, but lower than the NaF solution. Profilometry showed that CAPB protected enamel, but no agent associated with NaF promoted a higher protection than the NaF solution alone. KOH-soluble fluoride analysis showed that all surfactants reduced the fluoride adsorption on the enamel surface. Therefore, the surfactants tested (except for P20) changed the enamel surface energy. The SLS decreased the protective potential of NaF on initial erosion, but no tested agent interfered with the protective effect of NaF on enamel erosive wear.
Assuntos
Betaína/análogos & derivados , Cariostáticos/farmacologia , Esmalte Dentário/patologia , Polissorbatos/farmacologia , Dodecilsulfato de Sódio/farmacologia , Fluoreto de Sódio/farmacologia , Tensoativos/farmacologia , Erosão Dentária/prevenção & controle , Adsorção/efeitos dos fármacos , Análise de Variância , Animais , Betaína/química , Betaína/farmacologia , Bovinos , Ácido Cítrico/efeitos adversos , Esmalte Dentário/efeitos dos fármacos , Dureza , Polissorbatos/química , Saliva/fisiologia , Dodecilsulfato de Sódio/química , Tensoativos/química , Erosão Dentária/induzido quimicamente , Molhabilidade/efeitos dos fármacos , Espectroscopia por Absorção de Raios XRESUMO
Infections by small ruminant lentiviruses (SRLVs) affect goats and sheep causing chronic multisystemic diseases that generate great economic losses. The caprine lentivirus (CLV) and the ovine lentivirus (OLV) present tropism for cells of the monocyte/macrophage lineage, which are directly associated with the main route of transmission through the ingestion of milk and colostrum from infected animals. In this manner, controlling this route is of paramount importance. Currently, researches have investigated the use of chemical additives in milk that can preserve colostrum or milk and inactivate microbiological agents. Among the compounds, sodium dodecyl sulfate (SDS) has been shown to be satisfactory in the chemical inactivation of HIV and CLV in milk, and also as a biocide in goat colostrum.(AU)
As lentiviroses de pequenos ruminantes (LVPRs) são infecções que afetam caprinos e ovinos, causando doenças multissistêmicas crônicas, ocasionando grandes perdas econômicas. Os agentes causadores, lentivírus caprino (LVC) e o lentivírus ovino (LVO), apresentam tropismo por células da linhagem monocítico--fagocitária, as quais estão diretamente associadas à principal via de transmissão, por meio da ingestão de leite e colostro provindos de animais infectados. Desse modo, o controle por esta via é de suma importância. Atualmente, pesquisas vêm sendo desenvolvidas para o uso de aditivos químicos no leite, que possam conservar o colostro ou leite, e inativar agentes microbiológicos presentes. Dentre estes, o dodecil sulfato de sódio (SDS) vem apresentando resultados satisfatórios na inativação química do HIV e LVC em leite, e ainda como biocida em colostro caprino.(AU)