ABSTRACT
Pancreatic bioengineering is a potential therapeutic alternative for type 1 diabetes (T1D) in which the pancreas is decellularized, generating an acellular extracellular matrix (ECM) scaffold, which may be reconstituted by recellularization with several cell types to generate a bioartificial pancreas. No consensus for an ideal pancreatic decellularization protocol exists. Therefore, we aimed to determine the best-suited detergent by comparing sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), and Triton X-100 at different concentrations. Murine (n=12) and human pancreatic tissue from adult brain-dead donors (n=06) was harvested in accordance with Institutional Ethical Committee of the University of São Paulo Medical School (CEP-FMUSP) and decellularized under different detergent conditions. DNA content, histological analysis, and transmission and scanning electron microscopy were assessed. The most adequate condition for pancreatic decellularization was found to be 4% SDC, displaying: a) effective cell removal; b) maintenance of extracellular matrix architecture; c) proteoglycans, glycosaminoglycans (GAGs), and collagen fibers preservation. This protocol was extrapolated and successfully applied to human pancreas decellularization. The acellular ECM scaffold generated was recelullarized using human pancreatic islets primary clusters. 3D clusters were generated using 0.5×104 cells and then placed on top of acellular pancreatic slices (25 and 50 µm thickness). These clusters tended to connect to the acellular matrix, with visible cells located in the periphery of the clusters interacting with the ECM network of the bioscaffold slices and continued to produce insulin. This study provided evidence on how to improve and accelerate the pancreas decellularization process, while maintaining its architecture and extracellular structure, aiming at pancreatic bioengineering.
Subject(s)
Deoxycholic Acid , Detergents , Pancreas , Sodium Dodecyl Sulfate , Tissue Engineering , Tissue Scaffolds , Animals , Detergents/chemistry , Detergents/pharmacology , Humans , Pancreas/cytology , Mice , Sodium Dodecyl Sulfate/pharmacology , Deoxycholic Acid/pharmacology , Deoxycholic Acid/chemistry , Tissue Scaffolds/chemistry , Tissue Engineering/methods , Octoxynol/chemistry , Extracellular Matrix , Diabetes Mellitus, Type 1 , Microscopy, Electron, Scanning , Decellularized Extracellular Matrix/chemistryABSTRACT
Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.
Subject(s)
Saponins , Trehalose , Saponins/chemistry , Trehalose/chemistry , Immunoprecipitation/methods , Animals , Detergents/chemistry , Protein Binding , Humans , Octoxynol/chemistryABSTRACT
Polycyclic aromatic hydrocarbons (PAHs), as persistent environmental pollutants, often reside in nonaqueous-phase liquids (NAPLs). Mycobacterium sp. WY10, boasting highly hydrophobic surfaces, can adsorb to the oil-water interface, stabilizing the Pickering emulsion and directly accessing PAHs for biodegradation. We investigated the impact of Triton X-100 (TX100) on this interfacial uptake of phenanthrene (PHE) by Mycobacteria, using n-tetradecane (TET) and bis-(2-ethylhexyl) phthalate (DEHP) as NAPLs. Interfacial tension, phase behavior, and emulsion stability studies, alongside confocal laser scanning microscopy and electron microscope observations, unveiled the intricate interplay. In surfactant-free systems, Mycobacteria formed stable W/O Pickering emulsions, directly degrading PHE within the NAPLs because of their intimate contact. Introducing low-dose TX100 disrupted this relationship. Preferentially binding to the cells, the surfactant drastically increased the cell hydrophobicity, triggering desorption from the interface and phase separation. Consequently, PAH degradation plummeted due to hindered NAPL access. Higher TX100 concentrations flipped the script, creating surfactant-stabilized O/W emulsions devoid of interfacial cells. Surprisingly, PAH degradation remained efficient. This paradox can be attributed to NAPL emulsification, driven by the surfactant, which enhanced mass transfer and brought the substrate closer to the cells, despite their absence at the interface. This study sheds light on the complex effect of surfactants on Mycobacteria and PAH uptake, revealing an antagonistic effect at low concentrations that ultimately leads to enhanced degradation through emulsification at higher doses. These findings offer valuable insights into optimizing bioremediation strategies in PAH-contaminated environments.
Subject(s)
Biodegradation, Environmental , Mycobacterium , Octoxynol , Phenanthrenes , Surface-Active Agents , Phenanthrenes/chemistry , Phenanthrenes/pharmacology , Phenanthrenes/metabolism , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacology , Mycobacterium/metabolism , Mycobacterium/drug effects , Mycobacterium/chemistry , Octoxynol/chemistry , Emulsions/chemistry , Alkanes/chemistry , Alkanes/metabolism , Hydrophobic and Hydrophilic InteractionsABSTRACT
Phase separation and aggregation behaviour of triton X-100 (TX-100) and bovine serum albumin (BSA) mixture were investigated using cloud point and UV-visible spectroscopic techniques. The effects of various hydrotropes (HYTs) - namely, sodium salicylate (SS), sodium benzoate (SB), glycerol (Glyc), and 4-aminobenzoic acid (4-ABA) - on the cloud point (CP) of TX-100 + BSA were determined. The obtained CP values for the mixed system in the presence of HYTs followed the order: The measured critical micellization concentration (CMC) values of the TX-100 + BSA mixture were found to be significantly altered with varying amounts of BSA. The calculated free energy of clouding and micellization indicated the non-spontaneous nature of the phase transition and the spontaneous association of the TX-100 + BSA mixture. The non-spontaneity of phase separation decreased with increasing concentrations of HYTs. The enumerated values of ∆Hco and ∆Sco were consistently recorded as negative and positive magnitudes, respectively, in all aqueous HYTs media. The clouding process occurred due to a combination of hydrophobic and electrostatic interactions. The binding constant of the mixed system was determined employing the UV-vis spectroscopic method using the Benesi-Hildebrand equation.
Subject(s)
Octoxynol , Serum Albumin, Bovine , Spectrophotometry, Ultraviolet , Serum Albumin, Bovine/chemistry , Octoxynol/chemistry , Animals , Cattle , Hydrophobic and Hydrophilic Interactions , Protein Aggregates , Micelles , Phase Transition , Surface-Active Agents/chemistry , Phase SeparationABSTRACT
Surfactant pollution is escalatitheng in eutrophic waters, but the effect of surfactant charge properties on the physiological and biochemical properties of toxin-producing microalgae remains inadequately explored. To address this gap, this study explores the effects and mechanisms of three common surfactants-cetyltrimethylammonium bromide (CTAB, cationic), sodium dodecyl sulfate (SDS, anionic), and Triton X-100 (nonionic)-found in surface waters, on the agglomeration behavior, physiological indicators, and Microcystin-LR (MC-LR) release of Microcystis aeruginosa (M. aeruginosa) by using UV-visible spectroscope, Malvern Zetasizer, fluorescence spectrometer, etc. Results suggest that charge properties significantly affect cyanobacterial aggregation and cellular metabolism. The CTAB-treated group demonstrates a â¼5.74 and â¼9.74 times higher aggregation effect compared to Triton X-100 and SDS (300 mg/L for 180 min) due to strong electrostatic attraction. Triton X-100 outperforms CTAB and SDS in polysaccharide extraction, attributed to its higher water solubility and lower critical micelle concentration. CTAB stimulates cyanobacteria to secrete proteins, xanthohumic acid, and humic acids to maintain normal physiological cells. Additionally, the results of SEM and ion content showed that CTAB damages the cell membrane, resulting in a â¼90% increase in the release of intracellular MC-LR without cell disintegration. Ionic analyses confirm that all three surfactants alter cell membrane permeability and disrupt ionic metabolic pathways in microalgae. This study highlights the relationship between the surface charge properties of typical surfactants and the dispersion/agglomeration behavior of cyanobacteria. It provides insights into the impact mechanism of exogenous surfactants on toxic algae production in eutrophic water bodies, offering theoretical references for managing surfactant pollution and treating algae blooms.
Subject(s)
Microcystins , Microcystis , Surface-Active Agents , Microcystins/chemistry , Microcystins/metabolism , Microcystis/drug effects , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacology , Octoxynol/chemistry , Octoxynol/pharmacology , Sodium Dodecyl Sulfate/chemistry , Sodium Dodecyl Sulfate/pharmacologyABSTRACT
Photodynamic therapy (PDT) is an effective method for bacterial infection control in root canals of teeth with a broad-spectrum antibacterial activity. However, its application in root canal treatment is limited due to its inefficiency under hypoxic conditions and dentin staining. Triton X-100 (TX) shows great potential in enhancing the efficiency of antimicrobial agents through improving bacterial membrane permeability. The present study employed a combination of toluidine blue O (TB)-mediated PDT with TX to target the Enterococcus faecalis (E. faecalis), a bacterium with strong resistance to various antibacterial agents and mostly detected in infected root canals. PDT combined with TX showed enhanced antibacterial efficiency against both planktonic cells and biofilms of E. faecalis. At the same time, TX enhanced the antibacterial effect in dentinal tubules and reduced the incubation time. Mechanism studies revealed that TX improved reactive oxygen species (ROS) production through increasing the proportion of TB monomers. Additionally, increased membrane permeability and wettability were also observed. The findings demonstrated the PDT combined with TX could be used as a highly effective method for the root canal disinfection of teeth.
Subject(s)
Anti-Bacterial Agents , Biofilms , Enterococcus faecalis , Octoxynol , Photochemotherapy , Reactive Oxygen Species , Enterococcus faecalis/drug effects , Photochemotherapy/methods , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Biofilms/drug effects , Octoxynol/chemistry , Octoxynol/pharmacology , Reactive Oxygen Species/metabolism , Tolonium Chloride/pharmacology , Tolonium Chloride/chemistry , Humans , Microbial Sensitivity Tests , Dental Pulp Cavity/microbiology , Dental Pulp Cavity/drug effects , Photosensitizing Agents/pharmacology , Photosensitizing Agents/chemistryABSTRACT
Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.
Subject(s)
Extracellular Matrix , Hydrogels , Muscle, Skeletal , Animals , Hydrogels/chemistry , Swine , Extracellular Matrix/metabolism , Tissue Engineering/methods , Decellularized Extracellular Matrix/chemistry , Mice , alpha-Galactosidase/immunology , alpha-Galactosidase/metabolism , Deoxycholic Acid/chemistry , Octoxynol/chemistryABSTRACT
Triton X-100 (TX-100) is a membrane-disrupting detergent that is widely used to inactivate membrane-enveloped viral pathogens, yet is being phased out due to environmental safety concerns. Intense efforts are underway to discover regulatory acceptable detergents to replace TX-100, but there is scarce mechanistic understanding about how these other detergents disrupt phospholipid membranes and hence which ones are suitable to replace TX-100 from a biophysical interaction perspective. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) techniques in combination with supported lipid membrane platforms, we characterized the membrane-disruptive properties of a panel of TX-100 replacement candidates with varying antiviral activities and identified two distinct classes of membrane-interacting detergents with different critical micelle concentration (CMC) dependencies and biophysical mechanisms. While all tested detergents formed micelles, only a subset of the detergents caused CMC-dependent membrane solubilization similarly to that of TX-100, whereas other detergents adsorbed irreversibly to lipid membrane interfaces in a CMC-independent manner. We compared these biophysical results to virus inactivation data, which led us to identify that certain membrane-interaction profiles contribute to greater antiviral activity and such insights can help with the discovery and validation of antiviral detergents to replace TX-100.
Subject(s)
Detergents , Phospholipids , Polyethylene Glycols , Octoxynol/pharmacology , Octoxynol/chemistry , Detergents/pharmacology , Detergents/chemistry , Phospholipids/chemistry , Micelles , Antiviral Agents/pharmacology , Lipid Bilayers/chemistryABSTRACT
Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.
Subject(s)
Detergents , Extracellular Matrix , Animals , Swine , Detergents/chemistry , Detergents/metabolism , Detergents/pharmacology , Extracellular Matrix/metabolism , Octoxynol/chemistry , Octoxynol/metabolism , Octoxynol/pharmacology , Muscle, Skeletal , Sodium Dodecyl Sulfate/chemistry , Sodium Dodecyl Sulfate/metabolism , Sodium Dodecyl Sulfate/pharmacology , Tissue Scaffolds , Tissue Engineering/methodsABSTRACT
PURPOSE: This study aims to decellularized caprine pericardium tissue with varied non-ionic surfactant and anionic detergent concentrations. METHODS: Protocol A consists of 1%, 0.5%, and 0.25% (w/v) sodium dodecyl sulphate (SDS). Protocol B uses 1%, 0.5%, and 0.25% (w/v) Triton X-100. Protocol C comprised 0.5% SDS + 0.5% Triton X-100, 0.5% + 0.25%, and 0.25% SDS + 0.5% Triton X-100. RESULTS: Protocol B left a few countable cells in the pericardium tissue, but treatments A and C removed all cells. DNA quantification also demonstrated that protocol B had the most leftover DNA after decellularization. The pericardium tissue treated with an equal combination of anionic detergent and non-ionic surfactant preserves the matrix. However, changing the anionic detergent-non-ionic surfactant ratio disrupted the microstructure. Protocol A decreased pericardium tissue secant modulus (p < 0.05). Protocol B-treated pericardium tissue matched native tissue secant modulus and ultimate tensile stress. Protocol C strengthened pericardium tissue. CONCLUSION: The intact extracellular matrix and biomechanical properties like native tissues require optimal chemical doses and combinations.
Subject(s)
Goats , Octoxynol , Pericardium , Sodium Dodecyl Sulfate , Pericardium/drug effects , Pericardium/cytology , Animals , Octoxynol/pharmacology , Octoxynol/chemistry , Sodium Dodecyl Sulfate/pharmacology , Decellularized Extracellular Matrix/chemistry , Decellularized Extracellular Matrix/pharmacology , Detergents , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacology , DNA , Biomechanical Phenomena , Elastic ModulusABSTRACT
Cloud-point extraction (CPE) is a pre-treatment technique for the extraction and preconcentration of different chemical compounds, such as metal ions, pesticides, drugs, phenols, vitamins etc., from various samples. CPE is based on the phenomenon of two phases (micellar and aqueous) forming after the heating of an aqueous isotropic solution of a non-ionic or zwitterionic surfactant above the cloud-point temperature. If analytes are added to the surfactant solution under suitable conditions, they should be extracted into the micellar phase, also called the surfactant-rich phase. Recently, the traditional CPE procedure is being increasingly replaced by improved CPE procedures. In this study, recent advances in CPE over the last three years (2020 - 2022), including the application of various innovative approaches, are reviewed. In addition to the basic principle of CPE, alternative extraction media in CPE, CPE supported by various auxiliary energies, a different modified CPE procedure and the use nanomaterials and solid-phase extraction in combination with CPE are presented and discussed. Finally, some future trends for improved CPE are presented.
Subject(s)
Surface-Active Agents , Water , Octoxynol/chemistry , Surface-Active Agents/chemistry , Solid Phase Extraction , PhenolsABSTRACT
The fungicide chlorothalonil (CTL) and its metabolite hydroxy chlorothalonil (OH-CTL) constitute a risk of soil and water contamination, highlighting the need to find suitable soil remediation methods for these compounds. Surfactants can promote the bioavailability of organic compounds for enhanced microbial degradation, but the performance depends on soil and surfactant properties, sorption-desorption equilibria of contaminants and surfactants, and possible adverse effects of surfactants on microorganisms. This study investigated the influence of five surfactants [e.g., Triton X-100 (TX-100), sodium dodecyl sulphate (SDS), hexadecyltrimethylammonium bromide (HDTMA), Aerosol 22 and Tween 80] on the sorption-desorption, degradation, and mobility of CTL and OH-CTL in two volcanic and one non-volcanic soil. Sorption and desorption of fungicides depended on the sorption of surfactants on soils, surfactants' capacity to neutralize the net negative charge of soils, surfactants' critical micellar concentration, and pH of soils. HDTMA was strongly adsorbed on soils, which shifted the fungicide sorption equilibria by increasing the distribution coefficient (Kd) values. Contrarily, SDS and TX-100 lowered CTL and OH-CTL sorption on soils by decreasing the Kd values, which resulted in an efficient extraction of the fungicide compounds from soil. SDS increased the degradation of CTL, especially in the non-volcanic soil (DT50 values were 14 and 7 days in natural and amended soils, with final residues <7% of the initial dose), whereas TX-100 enabled an early start and sustenance of OH-CTL degradation in all soils. CTL and OH-CTL stimulated soil microbial activities without noticeable deleterious effects of the surfactants. SDS and TX-100 also reduced the vertical transport of OH-CTL in soils. Results of this study could be extended to soils in other regions of the world because the tested soils represent widely different physical, chemical, and biological properties.
Subject(s)
Fungicides, Industrial , Soil Pollutants , Surface-Active Agents/chemistry , Soil/chemistry , Adsorption , Octoxynol/chemistry , Soil Pollutants/analysisABSTRACT
Decellularization by chemical approaches has harmful effects on extracellular matrix (ECM) proteins, and damages lots of functional peptides and biomolecules present in the ultrastructure. In this study, we employed a combination of chemical and physical decellularization methods to overcome these disadvantages. The induced osmotic pressure by hypertonic/hypotonic solutions dissociated and removed most of cellular membranes significantly without any detergent or chemical agent. In total, 0.025% trypsin solution was found adequate to remove the remaining debrides, and ultimately 1% Triton X-100 was utilized for final cleansing. In addition, conducting all the decellularization processes at 4 °C yielded an ECM with least damages in the ultrastructure which could be inferred by close mechanical strength and swelling ratio to the native vessel, and high quality and quantity of cell attachment, migration and proliferation which were examined by optical microscopy and scanning electron microscopy (SEM) of the histology samples. Moreover, the obtained biological scaffold (BS) had no cytotoxicity according to the MTT assay, and this scaffold is storable at -20 °C. Employing bioreactor for concurrent cyclic tensile and shear stresses improved the cell migration into pores of the BS and made the cells and the scaffold compact in analogous to native tissue. As opening angle test showed by decellularizing of the blood vessel, the residual stress dropped significantly which revealed the role of cells in the amount of induced stress in the structure. However, intact and healthy ECM explicitly recovered upon recellularization and beat the initial residual stress of the native tissue. The tensile test of the blood vessels in longitudinal and radial directions revealed orthotropic behavior which can be explained by collagen fibers direction in the ECM. Furthermore, by the three regions of the stress-strain curve can be elucidated the roles of cells, elastin and collagen fibers in mechanical behavior of the vascular tissues.
Subject(s)
Extracellular Matrix , Tissue Engineering , Tissue Engineering/methods , Extracellular Matrix/metabolism , Biomimetics , Octoxynol/chemistry , Collagen/chemistry , Tissue Scaffolds/chemistryABSTRACT
Understanding the pathways of solubilization of lipid membranes is of high importance for their use in biotechnology and industrial applications. Although lipid vesicle solubilization by classical detergents has been widely investigated, there are few systematic structural and kinetic studies where different detergents are compared under varying conditions. This study used small-angle X-ray scattering to determine the structures of lipid/detergent aggregates at different ratios and temperatures and studied the solubilization in time using the stopped-flow technique. Membranes composed of either of two zwitterionic lipids, DMPC or DPPC, and their interactions with three different detergents, sodium dodecyl sulfate (SDS), n-dodecyl-beta-maltoside (DDM), and Triton X-100 (TX-100), were tested. The detergent TX-100 can cause the formation of collapsed vesicles with a rippled bilayer structure that is highly resistant to TX-100 insertion at low temperatures, while at higher temperatures, it partitions and leads to the restructuring of vesicles. DDM also causes this restructuring into multilamellar structures at subsolubilizing concentrations. In contrast, partitioning of SDS does not alter the vesicle structure below the saturation limit. Solubilization is more efficient in the gel phase for TX-100 but only if the cohesive energy of the bilayer does not prevent sufficient partitioning of the detergent. DDM and SDS show less temperature dependence compared to TX-100. Kinetic measurements reveal that solubilization of DPPC largely occurs through a slow extraction of lipids, whereas DMPC solubilization is dominated by fast and burst-like solubilization of the vesicles. The final structures obtained seem to preferentially be discoidal micelles where the detergent can distribute in excess along the rim of the disc, although we do observe the formation of worm- and rodlike micelles in the case of solubilization of DDM. Our results are in line with the suggested theory that bilayer rigidity is the main factor influencing which aggregate is formed.
Subject(s)
Detergents , Micelles , Detergents/chemistry , Lipid Bilayers/chemistry , Dimyristoylphosphatidylcholine , Kinetics , Octoxynol/chemistry , SolubilityABSTRACT
HYPOTHESIS: Although solubilization of lipid membranes has been studied extensively, questions remain regarding the structural pathways and metastable structures involved. This study investigated whether the non-ionic detergent Triton X-100 follows the classical solubilization pathway or if intermediate nanostructures are formed. EXPERIMENTS: Small angle X-ray and neutron scattering (SAXS/SANS) was used in combination with transmission electron cryo-microscopy and cryo-tomography to deduce the structure of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles and Triton X-100. Time-resolved SAXS and dynamic light scattering were used to investigate the kinetics of the process. FINDINGS: Upon addition of moderate detergent amounts at low temperatures, the lipid vesicles implode into ordered rippled bilamellar disc structures. The bilayers arrange in a ripple phase to accommodate packing constraints caused by inserted TX-100 molecules. The collapse is suggested to occur through a combination of water structure destabilization by detergents flipping across the membrane and osmotic pressure causing interbilayer attraction internally. The subsequently induced ripples then stabilize the aggregates and prevent solubilization, supported by the observation that negatively charged vesicles undergo a different pathway upon TX-100 addition, forming large bicelles. The findings demonstrate the richness in assembly pathways of simple lipids and detergents and stimulate considerations for the use of certain detergents in membrane solubilization.
Subject(s)
Detergents , Surface-Active Agents , Octoxynol/chemistry , Detergents/chemistry , Scattering, Small Angle , X-Ray Diffraction , Lipids/chemistry , Lipid Bilayers/chemistryABSTRACT
The influence of sodium hexadecyl sulfate on the nature of the fluorescence spectra of rhodamine 6G in an aqueous solution and a solution of nonionic Triton X-100 was investigated. The change in the nature of the emission spectra is explained by the formation of hydrophobic stoichiometric and sub-stoichiometric reagent-surfactant associates. Stabilization of the colloid-chemical state and reduction of the total turbidity of rhodamine 6G-anionic surfactant associate solutions with the addition of nonionic surfactant as a modifier were registered. The method of modification of the rhodamine 6G-sodium hexadecyl sulfate system with a nonionic surfactant was used in the development of conditions for the fluorescence determination of protein substances in physiological solutions. The concentration conditions for the use of the modified reagent system rhodamine 6G-anionic surfactant-nonionic surfactant for the fluorescence determination of albumin in urine were optimized.
Subject(s)
Albumins , Surface-Active Agents , Octoxynol/chemistry , Fluorescence , Surface-Active Agents/chemistry , SodiumABSTRACT
Clouding behavior and thermodynamic properties for the TX 100 + BSA mixture were investigated in aqueous and aq. alcoholic media. In an aqueous environment, the values of cloud point (CP), at which a clear solution becomes cloudy, for TX 100 decreases with augmentation of the concentration of BSA. The reverse result was obtained in the aq. alcoholic media. In this study, we have used ethanol (EtOH), 1-propanol (1-PrOH), and 2-butanol (2-BuOH) as alcohols. The changes of CP values in alcoholic media have been obtained in the following order: CPH2O+EtOH > CPH2O+2-BuOH > CPH2O+1-PrOH. The standard free energy (∆Gco), standard enthalpy (∆Hco), and standard entropy (∆Sco) changes of clouding were derived at CP. The ΔGc0 values of TX 100 + BSA decreases in the aqueous and alcoholic media with increasing the concentration of BSA and alcohol. This process becomes endothermic in the aq. alcoholic media. Different thermodynamic properties of transfer and entropy-enthalpy compensation parameters for the phase partitioning of the TX 100 + BSA mixture have been calculated and assessed properly.
Subject(s)
Polyethylene Glycols , Water , Octoxynol/chemistry , Water/chemistry , Thermodynamics , Alcohols/chemistryABSTRACT
The separation of boron in nuclear fuels by cloud point extraction (CPE) has been a challenge due to high acidity of digested sample solutions. High acidity hampers the coacervation of micelles. As a result, the cloud point temperature increases and thus could cause the inevitable loss of boron as volatile species. Herein we have proposed a novel CPE-assisted colorimetric method for the quantification of traces of boron (B) in uranium-based fuels. A 1:1 mixture of 2-ethyl hexane-1,3-diol (EHD) and curcumin dispersed in Triton X-114 surfactant was used in the proposed CPE process. We had investigated several compounds to act as micelle surface modifiers. Among them, only bromine water (Br2) was found not only to lower the cloud point temperature (CPT, from 80 °C to 42 ± 2 °C) but also resulted in the quantitative recovery of boron (≥95%). The CPE of boron from uranium matrix in a 2.0 mol L-1 HCl medium was suitable for direct chemical quality assurance of routine uranium-based fuels. The molar extinction coefficient of the boron-EHD-curcumin complex was found to be 4.75 × 105 L mol-1 cm-1 (λmax at 458 nm) in N,N-dimethyl formamide medium. The linear dynamic range and detection limit of the proposed analytical procedure were calculated to be 10-150 ng mL-1 and 0.8 ng mL-1 respectively. The proposed analytical methodology was validated by analysis of three in-house working reference materials of uranium. Determination of traces of boron in two uranium dioxide and two metallic uranium samples were found to demonstrate the applicability of the method. The relative standard deviation of the proposed method was found to be of 3-5%.
Subject(s)
Curcumin , Uranium , Boron , Chemical Fractionation/methods , Micelles , Octoxynol/chemistry , Spectrophotometry/methodsABSTRACT
Decellularization has been applied to many tissues and organs to obtain biomaterials for applications in tissue engineering. In this study, decellularization and characterization of chicken skin was performed to provide comprehensive information and in-depth details on this material as a potential tissue scaffold. Application of Triton X-100 and sodium dodecyl sulfate (SDS) on tissues at different time intervals as two decellularization protocols were compared according to various aspects, such as removal of cellular components, DNA quantification, protection of extracellular matrix (ECM), mechanical properties, and cytocompatibility, to find the optimum technique during preparation of decellularized scaffolds. The results showed that treatment with SDS revealed better results when compared with Triton X-100 regarding the preservation of tissue structure and morphology, although there was no difference in the efficiency of decellularization. In general, the tissues decellularized with SDS demonstrated higher levels of cytocompatibility and better mechanical properties in comparison with samples treated with Triton X-100. In conclusion, this study revealed that decellularized chicken skin is a cheap, abundant, and biocompatible material that supports cell attachment, growth, and proliferation. Therefore, it could be used as a proper candidate to prepare scaffolds for further studies on tissue engineering, especially for skin tissue engineering.
Subject(s)
Tissue Engineering , Tissue Scaffolds , Animals , Tissue Engineering/methods , Octoxynol/chemistry , Chickens , Extracellular Matrix/chemistryABSTRACT
Detergent-mediated virus inactivation (VI) provides a valuable orthogonal strategy for viral clearance in mammalian processes, in particular for next-generation continuous manufacturing. Furthermore, there exists an industry-wide need to replace the conventionally employed detergent Triton X-100 with eco-friendly alternatives. However, given Triton X-100 has been the gold standard for VI due its minimal impact on protein stability and high inactivation efficacy, inactivation by other eco-friendly detergents and its impact on protein stability is not well understood. In this study, the sugar-based detergent commonly used in membrane protein purification, n-dodecyl-ß- d-maltoside was found to be a promising alternative for VI. We investigated a panel of detergents to compare the relative VI efficacy, impact on therapeutic quality attributes, and clearance of the VI agent and other impurities through subsequent chromatographic steps. Detergent-mediated inactivation and protein stability showed comparable trends to low pH inactivation. Using experimental and modeling data, we found detergent-mediated product aggregation and its kinetics to be driven by extrinsic factors such as detergent and protein concentration. Detergent-mediated aggregation was also impacted by an initial aggregation level as well as intrinsic factors such as the protein sequence and detergent hydrophobicity, and critical micelle concentration. Knowledge gained here on factors driving product stability and VI provides valuable insight to design, standardize, and optimize conditions (concentration and duration of inactivation) for screening of detergent-mediated VI.