Photosynthetic variation and detoxification strategies based on cadmium uptake, non-protein thiols, and secondary metabolites in Miscanthus sacchariflorus under cadmium exposure.

Miscanthus sacchariflorus is previously demonstrated to be a potential candidate for remediation of cadmium (Cd) pollution. To explore its resistance strategy to Cd, a hydroponic experiment was conducted to determine the variations of photosynthetic activity in leaves and physiological response in roots of this plant. Results showed that the root of M. sacchariflorus was the primary location for Cd accumulation. The bioconcentration factor in the roots and rhizomes was >1, and the translocation factor from underground to aboveground was <1. Throughout the experimental period, treatment with 0.06 mM Cd2+ did not significantly alter the contents of chlorophyll a, chlorophyll b, or carotenoid. By contrast, treatment with 0.15 and 0.30 mM Cd2+ decreased the contents of chlorophyll a, chlorophyll b, and carotenoid; caused the deformation of the chlorophyll fluorescence transient curve; reduced the photochemical efficiency of photosystem II; and increased the contents of non-protein thiols, total flavone, and total phenol. These results indicate that M. sacchariflorus has good adaptability to 0.06 mM Cd2+. Moreover, the accumulation of the non-protein thiols, total flavone, and total phenol in roots may promote the chelation of Cd2+, thus alleviating Cd toxicity. This study provides theoretical support for using M. sacchariflorus to remediate Cd-polluted wetlands.

Comparison of Techniques for the Quantitation of Reductive Aroma Compounds in White Wine: Links to Sensory Analysis and Cu Fractions.

Multiple analytical methodologies allow quantitation of H2S and methanethiol (MeSH) in wine, but confirmation that the determined concentrations are related to perceived off-aromas, or "reductive" faults, is yet to be provided. Fifty white wines underwent sensory evaluation and measurement of free and salt-treated H2S and MeSH concentrations by gas chromatography with sulfur chemiluminescence detection and/or gas detection tubes. The determined concentrations were compared across techniques and different analysis laboratories. Sulfhydryl off-odors in the wines were best described by boiled and rotten egg and natural gas/sewerage/durian aroma attributes. The wines with the highest ratings for both aromas had high concentrations of free H2S, free MeSH, and/or salt-treated MeSH but were unrelated to salt-treated H2S. The free sulfhydryl concentrations and their associated aromas appeared to be suppressed by specific Cu fractions in the wines. This study provides evidence of the relevant measures of reductive aroma compounds and their relation to off-odors and Cu fractions.

Moldable Tissue-Sealant Hydrogels Composed of In Situ Cross-Linkable Polyethylene Glycol via Thiol-Michael Addition and Carbomers.

Moldable tissue-sealant hydrogels were developed herein by combining the yield stress fluidity of a Carbomer and in situ cross-linking of 3-arm PEG-thiol (PEG-SH) and 4-arm PEG-acrylate (PEG-AC). The Carbomer was mixed with each PEG oligomer to form two aqueous precursors: Carbomer/PEG-SH and Carbomer/PEG-AC. The two hydrogel precursors exhibited sufficient yield stress (>100 Pa) to prevent dripping from their placement on the tissue surface. Moreover, these hydrogel precursors exhibited rapid restructuring when the shear strain was repeatedly changed. These rheological properties contribute to the moldability of these hydrogel precursors. After mixing these two precursors, they were converted from yield-stress fluids to chemically cross-linked hydrogels, Carbomer/PEG hydrogel, via thiol-Michael addition. The gelation time was 5.0 and 11.2 min at 37 and 25 °C, respectively. In addition, the Carbomer/PEG hydrogels exhibited higher cellular viability than the pure Carbomer. They also showed stable adhesiveness and burst pressure resistance to various tissues, such as the skin, stomach, colon, and cecum of pigs. The hydrogels showed excellent tissue sealing in a cecum ligation and puncture model in mice and improved the survival rate due to their tissue adhesiveness and biocompatibility. The Carbomer/PEG hydrogel is a potential biocompatible tissue sealant that surgeons can mold. It was revealed that the combination of in situ cross-linkable PEG oligomers and yield stress fluid such as Carbomer is effective for developing the moldable tissue sealant without dripping of its hydrogel precursors.

In situ crosslinkable multi-functional and cell-responsive alginate 3D matrix via thiol-maleimide click chemistry.

In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.

Did diet compliance and remission reduce oxidative stress in celiac patients?

OBJECTIVE: We aimed to examine the effect of remission status on thiol-disulfide homeostasis in celiac patients and thus to indirectly determine the effect of oxidative stress and inflammation caused by non-compliance with the diet. METHODS: Between February 2019 and December 2021, 117 patients diagnosed with celiac disease were included in this prospective randomized and controlled study. In addition to routine tests of celiac patients, thiol and disulfide measurements were made from the blood both at the beginning of the study and at the end of the first year. RESULTS: While 52 of the patients (44.4%) were in remission, 65 patients (55.6%) were not. There was an evident increase in native thiol levels of the patients who were initially not in remission but went into at the end of the first year (347.4±46.7 µmol/L vs. 365.3±44.0 µmol/L; p=0.001). Mean plasma disulfide levels of patients with celiac going into remission became reduced in the first year from the level of 14.5±5.1 µmol/L down to 8.9±4.2 µmol/L (p<0.001). In celiac patients who entered remission, disulfide and anti-tissue transglutaminase immunoglobulin A levels decreased in a correlation (r=0.526; p<0.001). CONCLUSION: Not being in remission in celiac disease leads to increased oxidative stress, and thiol-disulfide homeostasis is an indirect indicator of this. Additionally, providing remission in celiac patients reduces oxidative stress.

Investigation of oxidant-antioxidant status in patients treated with hirudotherapy: an experimental study.

Objectives: To examine the influence of hirudotherapy on parameters of oxidative stress. METHODS: The cross-sectional study was conducted from March 29 to September 29, 2021, at the Alanya Research and Training Hospital's Traditional and Complementary Medicine Application Centre, Turkey, and comprised adult volunteers of either gender. The participants were subjected to two sessions of hirudotherapy 4 weeks apart. Total antioxidant status, total oxidant status, oxidative stress index values, ischaemia-modified albumin level, paraoxonase 1, disulfide, native thiol, total thiol, and arylesterase levels were assessed at baseline and after the second hirudotherapy session. Data was analysed using SPSS 15. RESULTS: Of the 50 subjects, 30(60%) were females and 20(40%) were males. The overall mean age was 47.10±15.16 years. Oxidative stress, ischaemia-modified albumin and disulfide levels decreased, but not significantly (p>0.05). The reduction in disulfide levels was significant (p=0.021). CONCLUSIONS: Hirudotherapy, within its limitations, could reduce oxidative stress.

Cellular Distribution and Intracellular Localization of Different Sizes of Fluorescent Thiol-Organosilica Particles in Mouse Lungs.

We investigated the distribution of intratracheally administered thiol-organosilica (thiol-OS) particles in mouse lungs. Toward this end, single doses of thiol-OS particles containing fluorescein (140 nm in diameter) (F140) and rhodamine B (Rh) (Rh160, Rh280, Rh420, Rh640, and Rh1630 with diameters of 160, 280, 420, 640, and 1630 nm, respectively) were administered. After 24 h, fluorescence imaging revealed homogeneous fluorescence with a patchier pattern on the lung surface and no difference among the six particle sizes. Simultaneous dual administration of Rh and F140 particles did not reveal any size-dependent differences in the lung surface fluorescence. Fluorescence microscopy of the lung sections revealed a similar tissue distribution in the fluorescent areas of Rhs and F140. Some fluorescent areas showed one type of particle fluorescence or only one fluorescence. Cellular distribution of particles was observed in bronchoalveolar lavage cells and lung sections under a high magnification, and correlative light and electron microscopy revealed large cells with fluorescence corresponding to both particle types and small cells with fluorescence of individual particle types, indicating a cell-subset-dependent particle size effect. Rh280, Rh420, and Rh640 exhibited significant size effects and were taken up by alveolar macrophages. Extracellular particles were observed, indicating that saturation exceeded the particle dose threshold in the alveoli. F140 taken up by small and large macrophages colocalized with CD68, CD11c, and CD11b and correlated with CD11c. The size effect, intracellular localization, and extracellular distribution of particles provide insights into lung and systemic drug delivery.

Thiol-functionalized cellulose for mercury polluted water remediation: Synthesis and study of the adsorption properties.

Mercury pollution poses a global health threat due to its high toxicity, especially in seafood where it accumulates through various pathways. Developing effective and affordable technologies for mercury removal from water is crucial. Adsorption stands out as a promising method, but creating low-cost materials with high selectivity and capacity for mercury adsorption is challenging. Here we show a sustainable method to synthesize low-cost sulfhydrylated cellulose with ethylene sulfide functionalities bonded glucose units. Thiol-functionalized cellulose exhibits exceptional adsorption capacity (1325 mg g-1) and selectivity for Hg(II) over other heavy metals (Co, Cu, Zn, Pb) and common cations (Ca++, Mg++) found in natural waters. It performs efficiently across a wide pH range and different aqueous matrices, including wastewater, and can be regenerated and reused multiple times without significant loss of performance. This approach offers a promising solution for addressing mercury contamination in water sources.

Early proactive monitoring of DNA-thioguanine in patients with Crohn's disease predicts thiopurine-induced late leucopenia in NUDT15/TPMT normal metabolizers.

BACKGROUND: Thiopurine-induced leucopenia significantly hinders the wide application of thiopurines. Dose optimization guided by nudix hydrolase 15 (NUDT15) has significantly reduced the early leucopenia rate, but there are no definitive biomarkers for late risk leucopenia prediction. AIM: To determine the predictive value of early monitoring of DNA-thioguanine (DNATG) or 6-thioguanine nucleotides (6TGN) for late leucopenia under a NUDT15-guided thiopurine dosing strategy in patients with Crohn's disease (CD). METHODS: Blood samples were collected within two months after thiopurine initiation for detection of metabolite concentrations. Late leucopenia was defined as a leukocyte count < 3.5 × 109/L over two months. RESULTS: Of 148 patients studied, late leucopenia was observed in 15.6% (17/109) of NUDT15/thiopurine methyltransferase (TPMT) normal and 64.1% (25/39) of intermediate metabolizers. In patients suffering late leucopenia, early DNATG levels were significantly higher than in those who did not develop late leucopenia (P = 4.9 × 10-13). The DNATG threshold of 319.43 fmol/µg DNA could predict late leucopenia in the entire sample with an area under the curve (AUC) of 0.855 (sensitivity 83%, specificity 81%), and in NUDT15/TPMT normal metabolizers, the predictive performance of a threshold of 315.72 fmol/µg DNA was much more remarkable with an AUC of 0.902 (sensitivity 88%, specificity 85%). 6TGN had a relatively poor correlation with late leucopenia whether in the entire sample (P = 0.021) or NUDT15/TPMT normal or intermediate metabolizers (P = 0.018, P = 0.55, respectively). CONCLUSION: Proactive therapeutic drug monitoring of DNATG could be an effective strategy to prevent late leucopenia in both NUDT15/TPMT normal and intermediate metabolizers with CD, especially the former.

Energy Transfer-Based Recognition of Membrane Cholesterol by Controlling Intradistance of Linker.

Gold nanoparticles (AuNPs) are good candidates for donor material in energy transfer systems and can easily be functionalized with various ligands on the surface with Au-S bonding. Cyclodextrin (CD) forms inclusion complexes with fluorophores due to its unique structure for host-guest interaction. In this study, we fabricated ßCD-functionalized AuNPs using different lengths of thiol ligands and recognized cholesterol to confirm the energy-transfer-based turn-on fluorescence mechanism. AuNP-ßCD conjugated with various thiol ligands and quenched the fluorescein (Fl) dye, forming ßCD-Fl inclusion complexes. As the distance between AuNPs and ßCD decreased, the quenching efficiency became higher. The quenched fluorescence was recovered when the cholesterol replaced the Fl because of the stronger binding affinity of the cholesterol with ßCD. The efficiency of cholesterol recognition was also affected by the energy transfer effect because the shorter ßCD ligand had a higher fluorescence recovery. Furthermore, we fabricated a liposome with cholesterol embedded in the lipid bilayer membrane to mimic the cholesterol coexisting with lipids in human serum. These cellular cholesterols accelerated the replacement of the Fl molecules, resulting in a fluorescence recovery higher than that of pure lipid. These discoveries are expected to give guidance towards cholesterol sensors or energy-transfer-based biosensors using AuNPs.

Chemoselective Synthesis and Anti-Kinetoplastidal Properties of 2,6-Diaryl-4H-tetrahydro-thiopyran-4-one S-Oxides: Their Interplay in a Cascade of Redox Reactions from Diarylideneacetones.

2,6-Diaryl-4H-tetrahydro-thiopyran-4-ones and corresponding sulfoxide and sulfone derivatives were designed to lower the major toxicity of their parent anti-kinetoplatidal diarylideneacetones through a prodrug effect. Novel diastereoselective methodologies were developed and generalized from diarylideneacetones and 2,6-diaryl-4H-tetrahydro-thiopyran-4-ones to allow the introduction of a wide substitution profile and to prepare the related S-oxides. The in vitro biological activity and selectivity of diarylideneacetones, 2,6-diaryl-4H-tetrahydro-thiopyran-4-ones, and their S-sulfoxide and sulfone metabolites were evaluated against Trypanosoma brucei brucei, Trypanosoma cruzi, and various Leishmania species in comparison with their cytotoxicity against human fibroblasts hMRC-5. The data revealed that the sulfides, sulfoxides, and sulfones, in which the Michael acceptor sites are temporarily masked, are less toxic against mammal cells while the anti-trypanosomal potency was maintained against T. b. brucei, T. cruzi, L. infantum, and L. donovani, thus confirming the validity of the prodrug strategy. The mechanism of action is proposed to be due to the involvement of diarylideneacetones in cascades of redox reactions involving the trypanothione system. After Michael addition of the dithiol to the double bonds, resulting in an elongated polymer, the latter-upon S-oxidation, followed by syn-eliminations-fragments, under continuous release of reactive oxygen species and sulfenic/sulfonic species, causing the death of the trypanosomal parasites in the micromolar or submicromolar range with high selectivity indexes.

Acidic Derivatization of Thiols Using Diethyl 2-Methylenemalonate: Thiol-Michael Addition Click Reaction for Simultaneous Analysis of Cysteine and Cystine in Culture Media Using LC-MS/MS.

Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.

Redox proteomics in melanoma cells: An optimized protocol.

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.

Simple, ultrasensitive detection of superoxide anion radical mutations in melanoma mice with SERS microneedles.

Elevated levels of superoxide anion radicals (O2·-) have been implicated in the pathogenesis of a variety of diseases, such as cancer, inflammatory diseases and autoimmune diseases. To determine the O2·- concentration for assisting disease detection, a method based on surface-enhanced Raman scattering (SERS) combined with transparent polymer microneedles has been developed. Photocrosslinked NOA61 is used to prepare microneedles with sulfhydryl group, which can contribute to anchor gold nanoparticles (Au NPs) functionalized by p-mercaptobenzoic acid (PATP). This work successfully constructed SERS microneedles for in situ detection. A REDOX reaction occurred between PATP and O2·-, resulting in the formation of dimethylaminoborane (DMAB) and a subsequent change in Raman signal. Based on the quantitative relationship between the change of peak area ratio at 1042 cm-1 and 1077 cm-1 and the concentration change of O2·-, a standard curve with a linear range of 0-480 ng/mL was constructed. The SERS microneedles were effectively employed to track melanoma progression in mice, establishing a fundamental correlation between O2·- concentration and melanoma stage, as confirmed by ELISA. The benefits of this approach, including convenience, in situ applicability, and low cost, are anticipated to offer novel insights for non-invasive in situ detection, potentially enhancing disease monitoring and diagnosis.

An electrochemical aptasensor based on silver-thiolated graphene for highly sensitive detection of Pb2.

The presence of lead ions (Pb2+) in the environment not only leads to environmental contamination but also poses a significant risk to public health through their migration into food and drinking water. Therefore, the development of rapid and effective techniques for detection of trace amounts of Pb2+ is crucial for safeguarding both the environment and biosafety. In this study, an aptamer-based electrochemical sensor was developed for specific detection of Pb2+ by modifying a polylysine (PLL) coated silver-thiolated graphene (Ag-SH-G) nanocomposite (PLL/Ag-SH-G) on the surface of a glassy carbon electrode, which was further modified with gold nanoparticles (AuNPs) for attachment of aptamers (Apt) that specifically recognized Pb2+. The Ag-SH-G particles were synthesized using a one-step in situ method, resulting in significantly enhanced electrochemical properties upon incorporating Ag nanoparticles into the PLL/Ag-SH-G composite. Coating of the covalently or no-covalently bonded Ag-SH-G particles with PLL provides an excellent supporting matrix, facilitating the assembly of AuNPs and a thiol-modified aptamer for Pb2+. Under optimized conditions, Apt/AuNPs/PLL/Ag-SH-G/GCE exhibited excellent sensing performance for Pb2+ with a wide linear response range (10-1000 nM), a low detection limit (0.047 nM) and extraordinary selectivity. The sensor was employed and satisfactory results were obtained in river water, soil and vegetable samples for the detection of Pb2+.

Redox-Responsive "Catch and Release" Cryogels: A Versatile Platform for Capture and Release of Proteins and Cells.

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.

Light-triggered AND logic tetrapeptide dynamic covalent assembly.

We demonstrate light-triggered dynamic covalent assembly of a linear short tetrapeptide containing two terminal cysteine residues in an AND logic manner. A photobase generator is introduced to accomplish light-mediated pH regulation to increase the reduction potential of thiols in the tetrapeptide, which activates its oxidative polymerization through disulfide bonds. Interestingly, it is elucidated that under light irradiation, mere co-existence of photobase generator and the oxidizing agent permits the polymerization performance of this tetrapeptide. Hence, a light-triggered AND logic dynamic covalent assembly of a tetrapeptide is achieved. Further, upon redox response, the reversible aggregation and disaggregation can be transformed for numerous times due to the dynamic covalent feature of disulfide bond. As a comparison, no assembly occurs for a short peptide containing one terminal cysteine residue under the same stimuli condition. This work offers a new approach to remotely control programmable molecular assembly of short linear peptides based on dynamic covalent bond, holding great potential in wide bioapplications.

Characterization of low molecular weight sulfur species in seaweed from the Antarctic continent.

Antarctic seaweeds are vital components of polar marine ecosystems, playing a crucial role in nutrient cycling and supporting diverse life forms. The sulfur content in these organisms is particularly interesting due to its implication in biogeochemical processes and potential impacts on local and global environmental systems. In this study, we present a comprehensive characterization of seaweed collected in the Antarctic in terms of their total sulfur content and its distribution among different classes of species, including thiols, using various methods and high-sensitivity techniques. The data presented in this paper are unprecedented in the scientific literature. These methods allowed for the determination of total sulfur content and the distribution of sulfur compounds in different fractions, such as water-soluble and proteins, as well as the speciation of sulfur compounds in these fractions, providing valuable insights into the chemical composition of these unique marine organisms. Our results revealed that the total sulfur concentration in Antarctic seaweeds varied widely across different species, ranging from 5.5 to 56 g kg-1 dry weight. Furthermore, our investigation into the sulfur speciation revealed the presence of various sulfur compounds, including sulfate, and some thiols, which were quantified in all ten seaweed species evaluated. The concentration of these individual sulfur species also displayed considerable variability among the studied seaweeds. This study provides the first in-depth examination of total sulfur content and sulfur speciation in brown and red Antarctic seaweeds.

Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

Mucoadhesive polymers: Design of S-protected thiolated cyclodextrin-based hydrogels.

AIM: This study aims to design chemically crosslinked thiolated cyclodextrin-based hydrogels and to evaluate their mucoadhesive properties via mucosal residence time studies on porcine small intestinal mucosa and on porcine buccal mucosa. METHODS: Free thiol groups of heptakis(6-deoxy-6-thio)-ß-cyclodextrin (ß-CD-SH) were S-protected with 2-mercaptoethanesulfonic acid (MESNA) followed by crosslinking with citric acid. Cytotoxicity was assessed by hemolysis as well as resazurin assay. Hydrogels were characterized by their rheological and mucoadhesive properties. Ritonavir was employed as model drug for in vitro release studies from these hydrogels. RESULTS: The structure of S-protected ß-CD-SH was confirmed by IR and 1H NMR spectroscopy. Degree of thiolation was 390 ± 7 µmol/g. Hydrogels based on native ß-CD showed hemolysis of 12.5 ± 2.5 % and 13.6 ± 2.7 % within 1 and 3 h, whereas hemolysis of just 3.5 ± 2.8 % and 3.9 ± 3.0 % was observed for the S-protected thiolated CD hydrogels, respectively. Both native and S-protected thiolated hydrogels showed minor cytotoxicity on Caco-2 cells. Rheological investigations of S-protected thiolated ß-CD-based hydrogel (16.2 % m/v) showed an up to 13-fold increase in viscosity in contrast to the corresponding native ß-CD-based hydrogel. Mucosal residence time studies showed that thiolated ß-CD-based hydrogel is removed to a 16.6- and 2.4-fold lower extent from porcine small intestinal mucosa and porcine buccal mucosa in comparision to the native ß-CD-based hydrogel, respectively. Furthermore, a sustained release of ritonavir from S-protected thiolated ß-CD-based hydrogels was observed. CONCLUSION: Because of their comparatively high mucoadhesive and release-controlling properties, S-protected thiolated ß-CD-based hydrogels might be promising systems for mucosal drug delivery.