Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael-type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of l-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young\'s moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel\'s mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver\'s natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications.

The thermo-responsive behavior of Poly(N-isopropylacrylamide) makes it an ideal candidate to easily embed cells and allows the polymer mixture to be injected. However, P(NiPAAm) hydrogels possess minor mechanical properties. To increase the mechanical properties, a covalent bond is introduced into the P(NIPAAm) network through a biocompatible thiol-ene click-reaction by mixing two polymer solutions. Co-polymers with variable thiol or acrylate groups to thermo-responsive co-monomer ratios, ranging from 1% to 10%, were synthesized. Precise control of the crosslink density allowed customization of the hydrogel\'s mechanical properties to match different tissue stiffness levels. Increasing the temperature of the hydrogel above its transition temperature of 31 °C induced the formation of additional physical interactions. These additional interactions both further increased the stiffness of the material and impacted its relaxation behavior. The developed optimized hydrogels reach stiffnesses more than ten times higher compared to the state of the art using similar polymers. Furthermore, when adding cells to the precursor polymer solutions, homogeneous thermo-responsive hydrogels with good cell viability were created upon mixing. In future work, the influence of the mechanical micro-environment on the cell\'s behavior can be studied in vitro in a continuous manner by changing the incubation temperature.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy), enzymes responsible for the hydrolysis of carbohydrates. In recent decades, many natural compounds and their bioinspired analogues have been studied as α-Glu and α-Amy inhibitors. However, no studies have been devoted to the evaluation of α-Glu and α-Amy inhibition by the neolignan obovatol (1). In this work, we report the synthesis of 1 and a library of new analogues. The synthesis of these compounds was achieved by implementing methodologies based on: phenol allylation, Claisen/Cope rearrangements, methylation, Ullmann coupling, demethylation, phenol oxidation and Michael-type addition. Obovatol (1) and ten analogues were evaluated for their in vitro inhibitory activity towards α-Glu and α-Amy. Our investigation highlighted that the naturally occurring 1 and four neolignan analogues (11, 22, 26 and 27) were more effective inhibitors than the hypoglycemic drug acarbose (α-Amy: 34.6 µM; α-Glu: 248.3 µM) with IC5O value of 6.2-23.6 µM toward α-Amy and 39.8-124.6 µM toward α-Glu. Docking investigations validated the inhibition outcomes, highlighting optimal compatibility between synthesized neolignans and both the enzymes. Concurrently circular dichroism spectroscopy detected the conformational changes in α-Glu induced by its interaction with the studied neolignans. Detailed studies through fluorescence measurements and kinetics of α-Glu and α-Amy inhibition also indicated that 1, 11, 22, 26 and 27 have the greatest affinity for α-Glu and 1, 11 and 27 for α-Amy. Surface plasmon resonance imaging (SPRI) measurements confirmed that among the compounds studied, the neolignan 27 has the greater affinity for both enzymes, thus corroborating the results obtained by kinetics and fluorescence quenching. Finally, in vitro cytotoxicity of the investigated compounds was tested on human colon cancer cell line (HCT-116). All these results demonstrate that these obovatol-based neolignan analogues constitute promising candidates in the pursuit of developing novel hypoglycemic drugs.

Injectable hydrogels have wide applications in clinical practice. However, the development of tough and bioadhesive ones based on biopolymers, along with biofriendly and robust crosslinking strategies, still represents a great challenge. Herein, we report an injectable hydrogel composed of maleimidyl alginate and pristine gelatin, for which the precursor solutions could self-crosslink via mild Michael-type addition without any catalyst or external energy upon mixing. This hydrogel is tough and bioadhesive, which can maintain intactness as well as adherence to the defect of porcine skin under fierce bending and twisting, warm water bath, and boiling water shower. Besides, it is biocompatible, bioactive and biodegradable, which could support the growth and remodeling of cells by affording an extracellular matrix-like environment. As a proof of application, we demonstrate that this hydrogel could significantly accelerate diabetic skin wound healing, thereby holding great potential in healthcare.

In this study, functional Pluronic F127 precursors were designed and synthesized for the preparation of thermosensitive hydrogels. Using linear Pluronic thioacetate and Pluronic multi-acrylate precursors, F127-based hydrogels were prepared through thioacetate deprotection-mediated Michael-type addition. The properties of these gels were compared to those obtained through free radical crosslinking of F127 diacrylate. Temperature was found to have a clear influence on gel swelling as a result of F127 thermoresponsiveness. The macromolecular architecture and functionality of the precursors were also optimized and characterized in terms of gelation kinetics and drug diffusion. In vitro tests were conducted on fibroblasts and endothelial cells to assess their response to cellular adhesion with Pluronic gels that were functionalized with an RGD peptide or pretreated with serum proteins to promote cell adhesion. This study provides a method for creating tailored hydrogels suitable for various biomedical applications, such as soft-tissue engineering, cell encapsulation, wound healing, and sustained delivery of therapeutic molecules.

The unavoidable presence of acrylamide in foods has fuelled the search for a suitable food additive, one that can successfully mitigate dietary acrylamide levels without changing food quality or compromising the health of consumers. The purpose of this study was to investigate the effect of a sulphur-based additive and amino acid, methionine, on acrylamide reduction. Differential scanning calorimetry, supported by chromatographic measurements, has shown that methionine interacts with acrylamide at a possible optimum temperature of 160°C, thereby disfavouring acrylamide polymerisation. Analysis of the methionine-acrylamide interaction via density functional theoretical modelling (DFT/6-31 + G(d)/RCAM-B3LYP) revealed that methionine\'s reducing effect may be driven by a Michael-type conjugation of the vinyl group of acrylamide at both the sulphur atom (∆Gf = -53 kJ mol-1) and the amino group (∆Gf = -11.84 kJ mol-1) of methionine. The former conjugation pathway results in a product that is more thermodynamically feasible.

As some proteins are known to interact with sulfated and phosphated biomolecules such as specific glycosaminoglycans, this study derives from the hypothesis that sulfonate and phosphonate groups on solid polymer surfaces might cause specific interfacial interactions. Such surfaces were prepared by plasma polymerization of heptylamine (HA) and subsequent grafting of sulfonate or phosphonate groups via Michael-type addition of vinylic compounds. Adsorption of the proteins fibrinogen, albumin (HSA) and lysozyme on these functionalised plasma polymer surfaces was studied by XPS and quartz crystal microbalance with dissipation (QCM-D). It was also studied whether pre-adsorption with HSA would lead to a passivated surface against further adsorption of other proteins. XPS confirmed grafting of vinyl sulfonate and vinyl phosphonate onto the amine surface and showed that the proteins adsorbed to saturation at between 1 and 2 h. QCM-D showed rapid and irreversible adsorption of albumin on all three surfaces, while lysozyme could be desorbed with PBS to substantial extents from the sulfonated and phosphonated surfaces but not from the amine surface. Fibrinogen showed rapid initial adsorption followed by slower additional mass gain over hours. Passivation with albumin led to small and largely reversible subsequent adsorption of lysozyme, whereas with fibrinogen partial displacement yielded a mixed layer, regardless of the surface chemistry. Thus, protein adsorption onto these sulfonated and phosphonated surfaces is complex, and not dominated by electrostatic charge effects.

Owe to unique advantages of heterogeneous catalytic reactions, there is increasing interest to use this type of chemical transformations in organic synthesis. Among various heterogeneous catalytic systems, magnetic supported ionic liquids are emerging ones in the chemical synthesis. As a result, this research focuses on developing an efficient magnetically recyclable catalytic system for Michael-type addition reaction based on quaternized γ-Fe2O3@cellulose ionomer. Core-shell structured magnetite cellulose nanosphere was synthesized by one step precipitation route and further modified with epichlorohydrin and hexamethylenetetramine. Anion exchange reaction was performed with polytungstophosphate. Synthesized nanocatalyst was characterized with FESEM, FTIR, VSM, EDX and TEM techniques. Vinyl pyridine and three types of functional groups i.e., hydroxyl, thiol, and amine were employed to evaluate the catalyst performance. Results showed that the addition reaction promoted up to 95% within 2h reaction time at moderate temperature (50°C) moreover the nanocatalyst showed good recyclability after three catalytic run as the reaction efficiency was >80% at the end of the third cycle which confirmed high efficiency of the presented system as a green heterogeneous catalyst to synthesis intermediate organic compounds.

The new fluorescent probe 4-hydroxy-3-((2E,4E)-5-phenylpenta-2,4-dienoyl)-2H-chromen-2-one (probe 1) was designed and synthesized for selective detection of sulfite. The fluorescence intensity of the probe was decreased only in the presence of HSO3-; all other anions assessed resulted in an increased fluorescence response. Hence, probe 1 acts as a highly selective sensor for HSO3-. This sulfite sensitivity can also be readily monitored visually, as once treated with sulfite the solution shows a marked color change from yellow to colorless. Moreover, probe 1 can be conveniently used as a signal tool to determine the HSO3- levels in various sugar samples.

The enzyme 4-oxalocrotonate tautomerase (4-OT) encoded by the xylH gene is a part of the degradation pathway of aromatic compounds in Pseudomonas putida mt-2. 4-OT was described to catalyze Michael-type addition of acetaldehyde to β-nitrostyrene, and the whole cell system based on recombinantly expressed 4-OT has been developed previously. In this study biocatalytic process based on Escherichia coli whole cells expressing 4-OT was significantly improved using immobilization and ex situ product recovery strategies. Whole cell immobilization in alginate beads was applied in biocatalytic production of 4-nitro-3-phenyl-butanal from β-nitrostyrene and acetaldehyde. Immobilized biocatalyst showed wider pH activity range and could tolerate twofold higher initial concentrations of substrate in comparison to the free whole cell biocatalyst. Beads retained their initial activity over 10 consecutive biotransformations of the model reaction and remained suitable for the repetitive use with 85% of the initial activity after two months of storage. Bioprocess was further improved by utilizing Amberlite XAD-2 hydrophobic resin for the product recovery. With this modification, the amount of organic solvent was reduced 40-fold in comparison to previously reported method making this biocatalytic process greener.