Efficient decellularization of human fetal kidneys through optimized SDS exposure.

Chronic kidney disease poses a significant threat to public health. Renal replacement therapy is the primary treatment option for end-stage kidney disease. However, there is a promising and relatively new method in regenerative medicine for creating a functional organ known as whole kidney decellularization. This method uses the intrinsic vasculature to perfuse the decellularizing agent into the tissue, effectively penetrating and removing cellular material. The regenerated bioscaffolds could serve as a source of organ donation. This study is focused on evaluating the effectiveness of various SDS exposures in decellularizing human fetal kidneys. The study included human fetal kidneys harvested from fetuses terminated before 14 weeks of gestational age. Kidneys were divided into six treatment groups based on SDS concentration and duration of perfusion. Decellularization, scanning electron microscopy, histopathological staining, immunofluorescent staining, and immunohistochemistry staining were performed to evaluate the adequacy of the process. The statistical analysis revealed that the SDS 0.1% treatment group had the highest collagen deposition after 24 h, significantly greater than the SDS 0.5% treatment group at 24 and 48 h. No significant differences were observed among the other treatment groups. The study concludes that the SDS 0.1% treatment group for 24 h was the most effective in terms of ECM content preservation and effective cell removal. This treatment showed better results than the other treatment groups and can be considered for future whole kidney decellularization studies.

Effect of silver nanoparticles foliar application on the nutritional properties of potato tubers.

The aim of presented study was to test nutritional properties of potato tubers and silver ions accumulation pattern after foliar application of silver nanoparticles (AgNPs) during potato vegetation. Potato plants were sprayed with different concentration of Ag nanoparticles (0.1, 1.0 and 10 mg·dm-3) synthesized with incorporation with sodium dodecyl sulphate (SDS) and sodium citrate as stabilizing agent. The lowest amounts of silver ions were transported to the tubers after spraying with AgNPs synthesized with SDS, rather than with citrate. Nevertheless silver ions accumulation in tubers was negligible. SDS method of synthesis was more favourable in terms of nutritional properties of potato tubers. The highest tested concentration of AgNPs_SDS had a favourable effect on a variety of macro- and micronutrients, ascorbic acid and soluble sugars. In turn, lower concentrations of AgNPs_SDS increased the content of phenolic compounds and free radical scavenging efficiency of tubers. These correlations were also confirmed by Principal Component Analysis.

Human ferritin nanocarriers for drug-delivery: A molecular view of the disassembly process.

Ferritins are natural proteins which spontaneously self-assemble forming hollow nanocages physiologically deputed to iron storage and homeostasis. Thanks to their high stability and easy production in vitro, ferritins represent an intriguing system for nanobiotechnology. Here we investigated the mechanism of disassembly and reassembly of a human recombinant ferritin constituted by the heavy chain (hHFt) exploiting a new procedure which involves the use of minimal amounts of sodium dodecyl sulfate (SDS) and assessed its effectiveness in comparison with two commonly used protocols based on pH shift at highly acidic and alkaline values. The interest in this ferritin as drug nanocarrier is related to the strong affinity of the human H-chain for the transferrin receptor TfR-1, overexpressed in several tumoral cell lines. Using different techniques, like NMR, TEM and DLS, we demonstrated that the small concentrations of SDS can eliminate the nanocage architecture without detaching the monomers from each other, which instead remain strongly associated. Following this procedure, we encapsulated into the nanocage a small ruthenium complex with a remarkable improvement with respect to previous protocols in terms of yield, structural integrity of the recovered protein and encapsulation efficiency. In our opinion, the extensive network of interchain interactions preserved during the SDS-based disassembly procedure represents the key for a complete and correct hHFt reassembly.

Which detergent is most suitable for the generation of an acellular pancreas bioscaffold?

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.

Impedance Spectroscopy Unveiled the Surfactant-Induced Unfolding and Subsequent Refolding of Human Serum Albumin.

Protein-surfactant interaction is a dynamic interplay of electrostatic and hydrophobic forces that ensues from the folding of a protein. We employ impedance spectroscopy (IS), a label-free method, to investigate the unfolding and refolding of human serum albumin (HSA), a globular plasma protein, in the presence of two surfactants: polysorbate-20 (Tween-20), a nonionic surfactant, and sodium dodecyl sulfate (SDS), an anionic surfactant. The equivalent electrical analog circuit was predicted from impedance spectra of HSA in an aqueous solution at physiological pH and room temperature, focusing on varying the concentration of codissolved surfactants. A change in the dielectric constant (ε') and ionic conductivity (κ) is observed by comparing the surfactant-treated protein samples to the bare surfactant solutions to assess the conformational changes induced by surfactants in HSA. Far-UV circular dichroism analysis revealed a decrease in α-helices and an increase in ß-sheets and random coils upon SDS addition, which were reversed by Tween-20. Dynamic light scattering supported the findings by measuring changes in the hydrodynamic diameter (dh) of HSA. Unfolding and refolding of HSA with surfactants were also observed through photoluminescence spectroscopy by examining the microenvironment surrounding the single tryptophan (W) within the protein, and the thermodynamic parameters were obtained using the modified Stern-Volmer equation. Our research explores the intriguing domain of protein-surfactant interactions, offering insights with promising applications across diverse biological processes and IS as a suitable alternative technique for investigating protein conformational changes by studying the electrical response of the samples.

Effects of sodium dodecyl sulfate, Sarkosyl and sodium lauroyl glutamate on the structure of proteins monitored by agarose native gel electrophoresis and circular dichroism.

We have studied binding properties of three detergents, i.e., sodium dodecyl sulfate (SDS), Sarkosyl and sodium lauroyl glutamate (SLG), to model proteins based on their effects on electrophoretic mobilities of the proteins using agarose native gel electrophoresis and circular dichroism (CD). This simple technology can evaluate the dissociative properties of bound detergents from the proteins and their effects on protein structure. SDS influenced the electrophoretic mobilities of all model proteins more strongly than the other two detergents, implying a stronger inclination for protein binding and subsequent alterations in protein structure or reductions in activity, which are supported by CD analysis. On the contrary, Sarkosyl and SLG showed weaker binding and interfered less with the structure and biological activities, indicating that these detergents may be useful for protein purification and analysis. It appeared that SLG was weaker in protein binding than Sarkosyl, although the effects of these two detergents appeared to depend on the proteins.

Interaction mechanism and compatibility studies of silk protein peptide (SPP) with the common surfactants SDS and DTAB.

The molecular interaction of low-molecular-weight SPP with common surfactants (SDS and DTAB) is a more complicated process than has been long believed. In this work, the interaction mechanism between SDS/DTAB and SPP was proposed using multiple methods including conductivity measurements, ST, UV-vis, FT-IR, DLS, fluorescence spectroscopy, and molecular docking simulations. Moreover, the foaming properties of the mixed systems were studied, and they were evaluated as cosmetics preservatives. The effects of various surfactant and protein concentrations and ratios on compatibility and functionality were studied. Based on the results, the mechanism of complex formation was identified as a cooperative van der Waals interaction followed by hydrophobic interaction and hydrogen bonding. A simpler head group leads to easier aggregation and interaction with the SPP, the formation of smaller-sized complexes, and a weaker impact on the fluorescence intensity. Thus, SDS monomers easily aggregate on SPP chains, leading to a stronger influence on the final secondary structure of SPP. This was confirmed by multiple spectroscopy methods. Comparing its single surfactant system, the SDS-SPP solution demonstrates better foaming power and the DTAB-SPP solution shows higher bacteriostatic activity. The good compatibility between SDS/DTAB and SPP can improve the functional properties of SDS or DTAB as well as lower the optimal concentration of each component. These results provide data and theoretical support for the design of cosmetic product formulas.

Moment analysis method for determination of rate constants of solute permeation across interface of spherical molecular aggregates by means of high-performance liquid chromatography.

A moment analysis method was developed for the study of solute permeation at the interface of spherical molecular aggregates. At first, new moment equations were developed for determining the partition equilibrium constant (Kp) and permeation rate constants (kin and kout) of solutes from the first absolute (µ1A) and second central (µ2C) moments of elution peaks measured by using high-performance liquid chromatography (HPLC). Then, the method was applied to the analysis of mass transfer phenomena of three solutes, i.e., hydroquinone, resorcinol, and catechol, at the interface of sodium dodecylsulfate (SDS) micelles. HPLC data were measured by using an ODS column and an aqueous phosphate buffer solution (pH = 7.0) as the mobile phase solvent. Pulse response experiments were conducted while changing SDS concentration (5 - 20 mmol dm-3) in the mobile phase under the conditions that the surface of ODS stationary phase was dynamically coated by SDS monomers. In order to demonstrate the effectiveness of the moment analysis method using HPLC, the values of Kp, kin, and kout were determined for the three solutes as 35 - 69, 2.4 × 10-8 - 1.4 × 10-6 m s-1, and 7.0 × 10-10 - 2.1 × 10-8 m s-1, respectively. Their values increase with an increase in the hydrophobicity of the solutes. The method has some advantages for the study of interfacial solute permeation of molecular aggregates. For example, neither immobilization nor chemical modification of both solute molecules and molecular aggregates is required when elution peaks are measured by using HPLC. Interfacial solute permeation takes place in the mobile phase without any chemical reaction or physical action on molecular aggregates. The values of Kp, kin, and kout were analytically determined from those of µ1A and µ2C by using the moment equations. The results of this study must contribute to the dissemination of an opportunity for studying the interfacial solute permeation of molecular aggregates to many researchers because of extremely high versatility of HPLC.

Impact of Surfactants on Cumulative Trypsin Activity in Bottom-Up Proteome Analysis.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

PBI derivatives/surfactant-based fluorescent ensembles: Sensing of multiple aminoglycoside antibiotics and interaction mechanism studies.

Fluorescent aggregates and ensembles have been widely applied in fabrication of fluorescent sensors due to their capacity of encapsulating fluorophores and modulating their photophysical properties. In the present work, fluorescent ensembles based on anionic surfactant SDS assemblies and perylene derivatives (PBIs) were particularly constructed. Three newly synthesized neutral PBI derivatives with different structures, PO, PC1 and PC2, were used for the purpose to evaluate probe structure influence on constructing fluorescent ensembles. The one with hydrophilic side chains, PO, experienced distinct photophysical modulation effect by SDS assemblies. The ensemble based on PO@SDS assemblies displayed effective fluorescence variation to antibiotic aminoglycosides (AGs). To improve cross-reactivity and discrimination capability of ensembles, a second probe, coumarin, was introduced into PO@SDS assemblies. The resultant ternary sensor, CM-PO@SDS, exhibited good qualitative and quantitative detection capabilities, and achieved differentiation of eight AGs and mixed AG samples both in aqueous solution and actual biological fluid, like human serum. Sensing mechanism studies revealed that hydrogen bonding, electrostatic and hydrophobic interactions are involved in the sensing process. This surfactant-based fluorescent ensemble provides a simple and feasible method for assessing AGs levels. Meanwhile, this work may provide some insights to design reasonable probes for constructing effective single-system based discriminative fluorescent amphiphilic sensors.

Evaluating the Expression of Efflux Pumps in Pseudomonas aeruginosa in Exposure to Sodium Dodecyl Sulfate, Didecyldimethylammonium Chloride, and Octenidine Dihydrochloride.

Emerging resistance of Gram-negative bacteria, including Pseudomonas aeruginosa, to commonly used detergents and disinfectant is encountering us with hazard. Inappropriate use of disinfectants has forced bacteria to gain resistance. The ability of bacteria to extrude substrates from the cellular interior to the external environment has enabled them to persist in exposure to toxic compounds, which is due to existence of transport proteins. Efflux pumps, in Gram-negative bacteria, are proteins responsible for exporting molecules outside of the cell, by crossing the two membranes. In this study, 40 P. aeruginosa strains from hospitals, clinics, and burn center laundries and 40 P. aeruginosa strains from urban laundries were collected. This study evaluated the minimum inhibitory concentration (MIC) level of sodium dodecyl sulfate (SDS), didecyldimethylammonium chloride (DDAC), and octenidine dihydrochloride (Od) in P. aeruginosa strains. The real-time PCR was carried out to evaluate the expression of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux system. The obtained results indicated a higher MIC level for SDS, DDAC, and Od in medical laundries. The sub-MIC level of DDAC and Od increased the expression level of MexAB-OprM, MexCD-OprJ, and MexXY-OprM in P. aeruginosa strains, suggesting that efflux pumps contribute to disinfectant resistance in P. aeruginosa.

Itch and Pain Behaviors in Irritant Contact Dermatitis Produced by Sodium Lauryl Sulfate in Mice.

Irritant contact dermatitis (ICD) is a nonspecific skin inflammation caused by irritants, leading to itch and pain. We tested whether differential responses to histamine-dependent and -independent pruritogens can be evoked in ICD induced by sodium lauryl sulfate (SLS). An ICD mouse model was established with 5% SLS in acetone versus a vehicle topically applied for 24 h to the cheek. Site-directed itch- and pain-like behaviors, occurring spontaneously and in response to mechanical, thermal, and chemical stimuli (histamine, ß-alanine, BAM8-22, and bradykinin) applied to the cheek, were recorded before (day 0) and after irritant removal (days 1, 2, 3, and 4). Skin inflammation was assessed through visual scoring, ultrasound, and measurements of skin thickness. SLS-treated mice exhibited hyperalgesia-like behavior in response to mechanical and heat stimuli on day 1 compared to the controls. SLS mice exhibited more spontaneous wipes (pain) but not scratching bouts (itch) on day 1. Pruritogen injections caused more scratching but not wiping in SLS-treated mice compared to the controls. Only bradykinin increased wiping behavior compared to saline. SLS-treated mice developed noticeable erythema, scaling, and increased skin thickness on days 1 and 2. SLS induced cutaneous inflammation and behavioral signs of spontaneous pain and itching, hyperalgesia to mechanical and heat stimuli and a chemical algogen, and enhanced itch response to pruritogens. These sensory reactions preceded the inflammation peak and lasted up to two days.

High-throughput preparation, scale up and solidification of andrographolide nanosuspension using hummer acoustic resonance technology.

The aim of this study was to rapidly develop a sufficiently robust andrographolide nanosuspension (AG-NS) system using hummer acoustic resonance (HAR) technology. The system can effectively improve the dissolution properties of AG, while having high stability and scale-up adaptability. The formulation of AG-NS was optimized in a high-throughput manner using HAR technology and the preparation process was optimized stepwise. Optimal AG-NS with Z-Ave = 223.99 ± 3.16 nm, PDI=0.095 ± 0.007 and zeta potential = -33.20 ± 0.58 mV was successfully prepared with Polyvinylpyrrolidone K30 and Sodium dodecyl sulfate. The optimal prescription was successfully scaled up 100 and 150 times using HAR technology, which was the initial exploration of its commercial scale production. AG-NS was solidified using freeze drying and fluid bed technology, respectively. The optimal AG-NS and its solidified products were exhaustively characterized using various analytical techniques. The high energy input of HAR technology and drying process converted part of the drug into the amorphous state. The in-vitro drug dissolution studies demonstrated relatively higher drug dissolution for AG-NS and its solidified products compared to controls at both the dissolution media (pH 1.2 buffer and pH 6.8 buffer). AG-NS and its solidified products successfully maintained their physical stability in short-term stability and accelerated stability experiments, respectively.

Application of Analytical Quality by Design to the development and validation of reduced and non-reduced capillary electrophoresis analytical procedures for mAb purity determination.

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a common analytical procedure used to quantitate critical quality attributes relating to the purity and glycosylation of monoclonal antibodies (mAbs). In this study, the application of an Analytical Quality by Design framework incorporating Design of Experiments was used to develop and validate both non-reduced (CE-NR) and reduced (CE-R) versions of this analytical procedure. Formal risk assessments were used to identify critical method attributes for optimization based on their potential impacts to performance criteria outlined in an analytical target profile. The resulting response surfaces connecting these critical factors to method performance were then utilized to generate a harmonized procedure to reduce execution risk across CE-R and CE-NR applications. Validation of these procedures according to regulatory guidelines support that they meet their required performance criteria, and a multivariate assessment of procedure robustness indicates that method parameters are in a sufficient state of control to ensure appropriate quantitation of mAb quality. Overall, this study demonstrates the utility of adopting an Analytical Quality by Design framework to leverage multidimensional knowledge from multiple critical method parameters to ensure an analytical procedure is fit-for-purpose.

Green micellar factorial design optimized first derivative synchronous spectrofluorimetric method for tripelennamine and diphenhydramine determination in pharmaceutical gel.

A green micellar synchronous spectrofluorimetric method was developed and validated for simultaneous determination of tripelennamine hydrochloride and diphenhydramine in bulk and combined pharmaceutical formulation. Synchronous fluorescence of tripelennamine hydrochloride and diphenhydramine was determined using Δλ = 60 nm. The first derivative of synchronous fluorescence was computed to resolve overlap in the synchronous fluorescence spectra. Tripelennamine hydrochloride was quantified at 375 nm, whereas diphenhydramine was quantified at 293 nm; each is the zero-crossing point of the other. As diphenhydramine exhibited weak native fluorescence, micelle enhancement upon incorporation of sodium dodecyl sulfate was considered. Two-level full factorial design was carried out to optimize experimental parameters. Optimum conditions involved using SDS (2% w/v) along with Teorell and Stenhagen buffer (pH 9). The method was found to be linear over the range 0.2-4.5 and 0.2-5 µg/mL for tripelennamine and diphenhydramine, respectively, with limits of detection 0.211 and 0.159 µg/mL. The method was successfully applied for simultaneous determination of tripelennamine hydrochloride and diphenhydramine in laboratory-prepared gel containing all possible excipients with mean percent recoveries ±SD 100.59 ± 0.79 and 98.99 ± 0.98 for tripelennamine hydrochloride and diphenhydramine, respectively. The proposed method was proved to be eco-friendly using different greenness assessment tools.

Evolution of the structure and interaction in the surfactant-dependent heat-induced gelation of protein.

The addition of a surfactant and/or an increase in temperature disrupt the native structure of proteins, where high temperature further results in protein gelation. However, in a mixed protein-surfactant system, surfactant concentration and temperature have been observed to exhibit both mutually associative and counter-balancing effects towards heat-induced gelation of protein-surfactant dispersion. This study is conducted on globular bovine serum albumin (BSA) protein and cationic surfactant dodecyl trimethyl ammonium bromide (DTAB), which interact strongly owing to their oppositely charged nature. The findings reveal that the BSA-DTAB suspension undergoes gelation with increasing temperature but only at lower concentrations of DTAB, where the presence of the surfactant facilitates gelation (associative effect). Conversely, as the surfactant concentration increases beyond a critical value, temperature-driven gelation of the BSA-DTAB system is completely inhibited, despite surfactant-induced protein denaturation (counter-balancing effect). To conceptualize these results, we compared them with observations made in a system comprising protein and a similarly charged surfactant, sodium dodecyl sulfate (SDS). It has been further demonstrated that the anionic surfactant (SDS) can restrict protein gelation at much lower concentration compared to the cationic surfactant (DTAB). The evolution of the structure and interaction during gel formation/inhibition has been examined to understand the underlying mechanism guiding these sol-gel transitions. We present a comprehensive phase diagram, encompassing the solution/gel states of the protein-surfactant dispersion, with respect to the dispersion temperature, surfactant concentration, and ionic behavior (anionic or cationic) of the surfactants.

In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 µg/mL propidium iodide in the separation medium (LOD 2 µg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 µg/mL, but using the local maximum at 100 µg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

Analytical quality by design-based development of a capillary electrophoresis method for Omeprazole impurity profiling.

Omeprazole (OME) is a proton pump inhibitor used to treat gastroesophageal reflux disease associated conditions. The current study presents an Analytical Quality by Design-based approach for the development of a CE method for OME impurity profiling. The scouting experiments suggested the selection of solvent modified Micellar ElectroKinetic Chromatography operative mode using a pseudostationary phase composed of sodium dodecyl sulfate (SDS) micelles and n-butanol as organic modifier in borate buffer. A symmetric three-level screening matrix 37//16 was used to evaluate the effect of Critical Method Parameters, including Background Electrolyte composition and instrumental settings, on Critical Method Attributes (critical resolution values, OME peak width and analysis time). The analytical procedure was optimized using Response Surface Methodology through a Central Composite Orthogonal Design. Risk of failure maps made it possible to define the Method Operable Design Region, within which the following optimized conditions were selected: 72 mM borate buffer pH 10.0, 96 mM SDS, 1.45 %v/v n-butanol, capillary temperature 21 °C, applied voltage 25 kV. The method was validated according to ICH guidelines and robustness was evaluated using a Plackett-Burman design. The developed procedure enables the simultaneous determination of OME and seven related impurities, and has been successfully applied to the analysis of pharmaceutical formulations.

Chronic skin damage induces small intestinal damage via IL-13-induced apoptosis.

The gut-skin axis has recently been widely recognized, and both the gut and skin have been found to affect each other through a bidirectional connection; however, the precise mechanisms remain to be elucidated. Therefore, we aimed to investigate the effects of chronic skin damage (CSD) on mouse intestines. Following the CSD model, 4% sodium dodecyl sulfate was applied to the back-shaved murine skin six times for 2 weeks after tape stripping. The small and large intestines were analyzed histologically and immunologically, respectively. Intestinal permeability was measured using fluorescein isothiocyanate-conjugated-dextran. The role of interleukin-13 (IL-13) in the ileum was investigated using an anti-IL-13 antibody. Apoptotic intestinal cells were analyzed using TUNEL staining. Villus atrophy was observed in the small intestine in the CSD model, along with increased permeability. Mast cells, but not T cells, eosinophils, or innate lymph cell-2, were increased in the intestinal mucosa. However, no significant changes were observed in the large intestine. mRNA expression of IL-13 was increased only in the ileum of the CSD model. Apoptotic intestinal epithelial cells were significantly increased in the ileum of the CSD model. Administration of an anti-IL-13 antibody ameliorated the intestinal damage caused by CSD, along with decreased apoptotic cells and mast cell infiltration. Skin damage causes morphological changes in the small intestine, accompanied by increased intestinal permeability, possibly through the IL-13-induced apoptosis of mast cells in the epithelium. Surfactant-mediated mechanical skin damage can cause a leaky gut.

Quantitatively measuring the cytotoxicity of viscous hydrogels with direct cell sampling in a micro scale format "MicroDrop" and its comparison to CCK8.

Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels.