Search Results (62)

Gene therapy is a groundbreaking strategy in regenerative medicine, enabling precise cellular behavior modulation for tissue repair. In situ nucleic acid delivery systems aim to directly deliver nucleic acids to target cells or tissues to realize localized genetic reprogramming and avoid issues like donor cell dependency and immune rejection. The key to success relies on biomaterial-engineered delivery platforms that ensure tissue-specific targeting and efficient intracellular transport. Viral vectors and non-viral carriers are strategically modified to enhance nucleic acid stability and cellular uptake, and integrate them into injectable or 3D-printed scaffolds. These scaffolds not only control nucleic acid release but also mimic native extracellular microenvironments to support stem cell recruitment and tissue regeneration. This review explores three key aspects: the mechanisms of gene editing in tissue repair; advancements in viral and non-viral vector engineering; and innovations in biomaterial scaffolds, including stimuli-responsive hydrogels and 3D-printed matrices. We evaluate scaffold fabrication methodologies, nucleic acid loading–release kinetics, and their biological impacts. Despite progress in spatiotemporal gene delivery control, challenges remain in balancing vector biocompatibility, manufacturing scalability, and long-term safety. Future research should focus on multifunctional “smart” scaffolds with CRISPR-based editing tools, multi-stimuli responsiveness, and patient-specific designs. This work systematically integrates the latest methodological advances, outlines actionable strategies for future investigations and advances clinical translation perspectives beyond the existing literature. Full article

This study examines the environmental challenges posed by azo-dye pollutants and aluminum industrial waste. Aluminum oxide nanoparticles (P/Al₂O₃-NPs) were produced using a green method that utilized pharmaceutical packaging waste as an aluminum source and marine algae extract (Padina pavonica) as reducing and stabilizing agents and that was characterized by XRD, EDX, SEM, TEM, and zeta potential. Batch biosorption studies were performed to assess the effectiveness of P/Al₂O₃-NPs in removing CR dye from aqueous solutions. The results demonstrate that the particle sizes range from 58.63 to 86.70 nm and morphologies vary from spherical to elliptical. FTIR analysis revealed Al–O lattice vibrations at 988 and 570 cm⁻¹. The nanoparticles displayed a negative surface charge (−13 mV) and a pH_zpc of 4.8. Adsorption experiments optimized parameters for CR dye removal, achieving 97.81% efficiency under native pH (6.95), with a dye concentration of 30 mg/L, an adsorbent dosage of 0.1 g/L, and a contact time of 30 min. Thermodynamic studies confirmed that the process is exothermic and spontaneous. Kinetic data fit well with the pseudo-second-order model, while equilibrium data aligned with the Langmuir isotherm. The adsorption mechanism involved van der Waals forces, hydrogen bonding, and π–π interactions, as supported by the influence of pH, isotherm data, and FTIR spectra. Overall, the study demonstrates the potential of eco-friendly P/Al₂O₃-NPs to efficiently remove CR dye from aqueous solutions. Full article

The fibrin matrix of thrombi is intertwined with neutrophil extracellular traps (NETs) containing histones that render resistance to fibrinolysis. During NET formation, histones are citrullinated. Our study addresses the question of whether citrullination modifies the fibrin-stabilizing effects of histones. We studied the structure and viscoelastic properties of fibrin formed in the presence of native or citrullinated H1 and core histones by scanning electron microscopy, clot permeation, and oscillation rheometry. The kinetics of fibrin formation and its dissolution were followed by turbidimetry and thromboelastometry. Co-polymerizing H1 with fibrin enhanced the mechanical strength of the clots, thickened the fibrin fibers, and enlarged the gel pores. In contrast, the addition of core histones resulted in a reduction in the fiber diameter, and the pores were only slightly larger, whereas the mechanical stability was not modified. Plasmin-mediated fibrinogen degradation was delayed by native and citrullinated core histones, but not by H1, and the action of des-kringle1-4-plasmin was not affected. Plasmin-mediated fibrinolysis was inhibited by native and citrullinated core histones, and this effect was moderated when the kringle domains of plasmin were blocked or deleted. These findings suggest that in NET-containing thrombi that are rich in core histones, alternative fibrinolytic enzymes lacking kringle domains are more efficient lytic agents than the classic plasmin-dependent fibrinolysis. Full article

The underlying structural features of the mismatch between catalytic and cytostatic properties in L-asparaginase from Rhodospirillum rubrum (RrA) and three of its mutants were investigated. The rationale for selecting the specific mutations (RrA_{A64V, E67K}; RrA_{R118H, G120R}; RrA_{E149R, V150P, F151T}) is to elucidate the role of inter-subunit interaction in RrA and its impact on catalytic efficiency and stability. Bioinformatic modeling revealed a predominantly negative surface charge on RrA with limited positive charge clusters in the vicinity of the interface region. Thus, some negatively charged groups were replaced with positively charged ones to enhance the electrostatic interactions and stabilize the enzyme quaternary structure. RrA_{A64V, E67K} and RrA_{R118H, G120R} additionally contained an N-terminal 17-amino acid capsid peptide derived from the bacteriophage T7 (MASMTGGQQMGRGSSRQ), which could potentially affect the conformational stability of theenzymes. Circular dichroism (CD) spectroscopy was applied to the kinetic parameters analysis of Asn hydrolysis and showed that native RrA displayed a V_max of 30 U/mg and a K_M of 4.5 ± 0.5 mM. RrA_{E149R, V150P, and F151T} exhibited a substantially increased V_max of 57 U/mg. The catalytic efficiency of V_max/K_M also improved compared to the native enzyme: the V_max/K_M increased from approximately 7 U/mg × mM⁻¹ (for the native enzyme) to 9 U/mg × mM⁻¹ for Mut3. Other mutants exhibited less pronounced changes. Thermo-denaturation studies allowed us to determine the phase transition parameters of the RrA variants in comparison with commercial reference sample EcA. RrA_{A64V, E67K} and RrA_{R118H, G120R} exhibited the most favorable phase transition parameters, with melting temperatures (T_m) of 60.3 °C and 59.4 °C, respectively, exceeding that of the wild-type RrA (54.6 °C) and RrA_{E149R, V150P, F151T} (52 °C). The EcA demonstrated a slightly superior thermal stability, with a T_m of 62 °C. The mutations showed a significant effect on protein stability during trypsinolysis. Therefore, RrA_{E149R, V150P, F151T} showed higher resistance (45% activity remaining after 30 min of trypsin exposure) compared to the native RrA retained 20% activity. EcA preparations exhibited lower stability to trypsinolysis (losing over 90% activity in 15 min). The cytostatic effects were evaluated using MTT assays against K562 (leukemic) and A549 (lung carcinoma) cell lines. The MTT assays with K562 cells revealed that RrA_{E149R, V150P, F151T} (IC₅₀ of 10 U/mL) and RrA_{R118H, G120R} (IC₅₀ of 11.5 U/mL) exhibited superior antiproliferative activity compared to native enzymes RrA (IC₅₀ of 15 U/mL) and EcA (24 U/mL). RrA_{E149R, V150P, F151T} showed the most significant improvement in cytostatic activity. The results obtained indicate that the substitutions in RrA_{E149R, V150P, F151T} resulted in the improvement of the enzyme biocatalytic properties and an increase in the resistance to aggregation and trypsinolysis. This highlights the role of electrostatic interactions in stabilizing the oligomeric structure of the enzyme, which eventually translates into an improvement in cytostatic efficiency and antiproliferative forces. Full article

ATP analogues are essential tools in enzymology and structural biology, but the structural and functional implications of their chemical modifications on nucleotide-binding proteins are often underappreciated. To address this, we evaluated a panel of ATP analogues, focusing on thiosubstituted and fluorinated molecules, using the AAA+ ATPase p97 as a benchmark system. Hydrolysis stability and impact on protein conformation, binding modes, and kinetics of enzymatic catalysis were assessed by protein-detected methyl NMR and ligand-detected ¹⁹F NMR in solution, as well as ³¹P solid-state NMR of nucleotides within protein sediments. ATPγS and AMP-PNP emerged as the most suitable analogues for preserving pre-hydrolysis states over extended periods, despite undergoing gradual hydrolysis. In contrast, both AMP-PCP and α/β-thiosubstituted analogues failed to induce native protein conformations in p97. Notably, we demonstrate a novel real-time NMR setup to explore the effect of nucleotide mixtures on cooperativity and the regulation of enzymes. Additionally, aromatic fluorine TROSY-based ¹⁹F NMR shows promise for direct ligand detection in solution, even in the context of large macromolecular complexes. These findings provide critical guidance for selecting ATP analogues in functional and structural studies of nucleotide-binding proteins. Full article

(1) Background: The aim of the work was to investigate the influence of selected physico-chemical factors on the solubility and release rate of CT (cryptotanshinone) in alcohologels. (2) Methods: The alcohologels of methylcellulose (MC), hydroksyethylcellulose (HEC), polyacrylic acid (PA) and polyacrylic acid crosspolymer (PACP) with CT were prepared and/or doped with native potato starch (SN) and modified citrate starches (SM2.5 and SM10). The analytical methods included evaluation of CT release profiles, Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and electrospray ionization mass spectrometry (ESI-MS), and scanning electron microscope (SEM) images were performed. (3) Results: The release and decomposition kinetics of CT in relation to the phosphate buffer solution (PBS) and methanol were observed. The amount of cryptotanshinone (CT) released into PBS was significantly lower (2.5%) compared to its release into methanol, where 22.5% of the CT was released into the model medium. The addition of SM2.5 to the alcohologel significantly increased the CT content to 70% in the alcohologel preparation containing NaOH (40%), and this enhanced stability was maintained for up to two months. The ATR-FTIR exhibited interactions between PA and 2-amino-2-methyl-1,3-propanediol (AMPD) as well as between PA and NaOH in case of the alcohologels. Moreover, it indicated the interaction between CT and NaOH. PXRD diffractograms confirmed the FTIR study. (4) Conclusions: The study observed the influence of a number of factors on the solubility and release rate of CT, as: alkalizers and their concentration, SM2.5 addition. The transition of CT in the presence of NaOH to the tanshinone V sodium (T-V sodium) form was suspected. Full article

The search for more sustainable reaction conditions has been necessary to obtain more selective processes. In this context, laccases have gained great notoriety in recent years. However, these enzymes are unstable in organic solvents and have low thermal stability. Alternatively, conjugation with PEG (PEGylation) can be essential to overcome these problems. In this work, the commercial laccase from Myceliophthora thermophila (LacMT) was subjected to PEGylation with PEG functionalized as succinimidyl carbonate (mPEG-SC), followed by assessing its thermal stability and catalytic activity. Mono-PEGylated LacMT derivatives were obtained, with less than 50% of the enzyme remaining in its native form. In addition, 10% of the bi-PEGylated species was successfully obtained according to gel electrophoresis analysis. The PEGylated derivatives showed a significantly reduced ABTS oxidation activity (98 ± 3 U/mg) compared to the native LacMT (407 ± 9 U/mg) but higher than the control enzyme without PEGylation (51 ± 2 U/mg), demonstrating that the addition of activated PEG to the protein resulted in better protection against the harmful action of the pH change required in the process. PEGylated LacMT retained more than twice the initial activity of the native protein at 40 °C during 24 h. In addition, PEGylated LacMT exhibited kinetic changes, whereas the catalytic turnover rate (k_cat) of the PEGylated enzyme was reduced by 27% compared to the control. These findings are being reported for the first time. This sets precedents for constructing efficient catalytic systems involving laccases since no immobilized biocatalyst or commercial conjugate contains these proteins. Full article

The high amount of starch in fruits is responsible for its post-processing cloudiness. In the current study, α-amylase from porcine pancreases was immobilized onto carboxymethyl cellulose/polyacrylamide (CMC/PAM) hydrogel. This in-house-built CMC/PAM-Amy hydrogel offers a more efficient and sustainable solution for apple juice clarification. To acquire the best immobilization efficiency, the concentration of glutaraldehyde crosslinker was optimized. Biocatalytic characterization studies were brought into consideration for free and immobilized α-amylase. The synthesized native and immobilized CMC/PAM-Amy hydrogels were also characterized using SEM, FTIR and XRD. Under ideal circumstances, the activity of CMC/PAM-Amy was up to 604 μmolmin⁻¹, and its immobilization efficiency was 96.29 ± 1.15%. A kinetic parameters study resulted in a conspicuously lowered Km value for immobilized amylase, signifying its higher affinity for its substrate. CMC/PAM-Amy showed a half-life (t_1/2) 3.5 times higher than free-Amy at 50, 55 and 60 °C. The higher values of the inactivation rate constant (k_d), free energy of inactivation (ΔG*), enthalpy of inactivation (ΔH*) and change in entropy (ΔS*) of CMC/PAM-Amy manifested the enhanced thermal stability of amylase after immobilization. A reusability study revealed that immobilized amylase retained roughly 70% of its initial catalytic activity after six successive repetitions of the process. CMC/PAM-Amy displayed improved recycling ability operational stability and biocatalytic activity, rendering it an auspicious tool in decreasing the starch content of crude apple juice to about 61% of its total starch content before treatment. Moreover, the values of Brix, viscosity, acidity and turbidity were also decreased in CMC/PAM-Amyclarified apple juice. Therefore, immobilized amylases with other industrial enzymes could be an efficient tool for potential industrial application. Full article

Using free microorganisms for industrial processes has some limitations, such as the extensive consumption of substrates for growth, significant sensitivity to the microenvironment, and the necessity of separation from the product and, therefore, the cyclic process. It is widely acknowledged that confining or immobilizing cells in a matrix or support structure enhances enzyme stability, facilitates recycling, enhances rheological resilience, lowers bioprocess costs, and serves as a fundamental prerequisite for large-scale applications. This report summarizes the various cell immobilization methods, including several synthetic (polyvinylalcohol, polyethylenimine, polyacrylates, and Eudragit) and natural (gelatin, chitosan, alginate, cellulose, agar–agar, carboxymethylcellulose, and other polysaccharides) polymeric materials in the form of thin films, hydrogels, and cryogels. Advancements in the production of well-known antibiotics like penicillin and cephalosporin by various strains were discussed. Additionally, we highlighted cutting-edge research related to strain producers of peptide-based antibiotics (polymyxin B, Subtilin, Tyrothricin, varigomycin, gramicidin S, friulimicin, and bacteriocin), glusoseamines, and polyene derivatives. Crosslinking agents, especially covalent linkers, significantly affect the activity and stability of biocatalysts (penicillin G acylase, penicillinase, deacetoxycephalosporinase, L-asparaginase, β-glucosidase, Xylanase, and urease). The molecular weight of polymers is an important parameter influencing oxygen and nutrient diffusion, the kinetics of hydrogel formation, rigidity, rheology, elastic moduli, and other mechanical properties crucial for long-term utilization. A comparison of stability and enzymatic activity between immobilized enzymes and their free native counterparts was explored. The discussion was not limited to recent advancements in the biopharmaceutical field, such as microorganism or enzyme immobilization, but also extended to methods used in sensor and biosensor applications. In this study, we present data on the advantages of cell and enzyme immobilization over microorganism (bacteria and fungi) suspension states to produce various bioproducts and metabolites—such as antibiotics, enzymes, and precursors—and determine the efficiency of immobilization processes and the optimal conditions and process parameters to maximize the yield of the target products. Full article

Curcumin is a hydrophobic bioactive compound, and its incorporation into lipid-based carriers can enhance its bioaccessibility and maintain its stability over time. Pickering emulsions are long-term stability systems, effective for encapsulation, protection, and delivery of bioactive compounds. This study aimed to produce Pickering oil-in-water (O/W) emulsions stabilized by cassava starch nanoparticles (native or modified by heat–moisture treatment (HMT)) with high kinetic stability to encapsulate curcumin. The effect of curcumin incorporation on emulsion features was also assessed, as well as curcumin stability over time. Native starch nanoparticles (NSNPs) were not effective stabilizers in the concentration range of 0.8 to 4 wt%. Otherwise, modified starch nanoparticles (HSNPs) at 4 wt% produced a long-term stability Pickering emulsion, which was used to encapsulate curcumin (0.07 wt%). Confocal laser scanning microscopy (CLSM) showed that HSNPs were located at the droplet’s interface. The interfacial tension for HSNPs exhibited initial values from 40 to 33 mN/m, quickly reaching equilibrium. These findings suggest that HSNPs exhibit low surface activity and the stabilization mechanism of emulsion is based on steric hindrance. The stabilization by steric hindrance is supported by the low zeta potential value (−5.39 mV). Stable emulsions showed shear thinning behavior, and the power-law model demonstrated excellent fit to experimental data (R² ≥ 0.998). The addition of curcumin reduced the interfacial tension, droplet size, apparent viscosity, and consistency index, indicating that this bioactive compound can also act at the interface. After 60 days, curcumin degradation was fully avoided. Our findings indicated that HSNP-stabilized Pickering emulsions can protect encapsulated curcumin from degradation. Full article

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (K_M = 78 µM, k_cat = 0.67 s⁻¹). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications. Full article

Amyloidosis is a group of protein misfolding diseases, which include spongiform encephalopathies, Alzheimer’s disease and transthyretin (TTR) amyloidosis; all of them are characterized by extracellular deposits of an insoluble fibrillar protein. TTR amyloidosis is a highly debilitating and life-threatening disease. Patients carry less stable TTR homotetramers that are prone to dissociation into non-native monomers, which in turn rapidly self-assemble into oligomers and, ultimately, amyloid fibrils. Liver transplantation to induce the production of wild-type TTR was the only therapeutic strategy until recently. A promising approach to ameliorate transthyretin (TTR) amyloidosis is based on the so-called TTR kinetic stabilizers. More than 1000 TTR stabilizers have already been tested by many research groups, but the diversity of experimental techniques and conditions used hampers an objective prioritization of the compounds. One of the most reliable and unambiguous techniques applied to determine the structures of the TTR/drug complexes is X-ray diffraction. Most of the potential inhibitors bind in the TTR channel and the crystal structures reveal the atomic details of the interaction between the protein and the compound. Here we suggest that the stabilization effect is associated with a compaction of the quaternary structure of the protein and propose a scoring function to rank drugs based on X-ray crystallography data. Full article

The use of nanoparticle systems for the controlled release of growth factors is a promising approach to mimicking of the biochemical environment of native tissues in tissue engineering. However, sustaining growth factor release inside an appropriate therapeutic window is a challenge, particularly in bioprinted scaffolds. In this study, a chitosan-coated alginate-based nanoparticle system loaded with hepatocyte growth factor was developed and then incorporated into bioprinted scaffolds. The release kinetics were investigated with a focus on identifying the impact of the chitosan coating and culture conditions. Our results demonstrated that the chitosan coating decreased the release rate and lessened the initial burst release, while culturing in dynamic conditions had no significant impact compared to static conditions. The nanoparticles were then incorporated into bioinks at various concentrations, and scaffolds with a three-dimensional (3D) structure were bioprinted from the bioinks containing human pulmonary fibroblasts and bronchial epithelial cells to investigate the potential use of a controlled release system in respiratory tissue engineering. It was found that the bioink loaded with a concentration of 4 µg/mL of nanoparticles had better printability compared to other concentrations, while the mechanical stability of the scaffolds was maintained over a 14-day culture period. The examination of the incorporated cells demonstrated a high degree of viability and proliferation with visualization of the beginning of an epithelial barrier layer. Taken together, this study demonstrates that a chitosan-coated alginate-based nanoparticle system allows the sustained release of growth factors in bioprinted respiratory tissue scaffolds. Full article

Composite collagen gels with hyaluronic acid are developed tissue-engineered structures for filling and regeneration of defects in various organs and tissues. For the first time, phytic acid was used to increase the stability and improve the mechanical properties of collagen gels with hyaluronic acid. Phytic acid is a promising cross-linker for collagen hydrogels and is a plant-derived antioxidant found in rich sources of beans, grains, and oilseeds. Phytic acid has several benefits due to its antioxidant, anticancer, and antitumor properties. In this work, studies were carried out on the kinetics of the self-assembly of collagen molecules in the presence of phytic and hyaluronic acids. It was shown that both of these acids do not lead to collagen self-assembly. Scanning electron microscopy showed that in the presence of phytic and hyaluronic acids, the collagen fibrils had a native structure, and the FTIR method confirmed the chemical cross-links between the collagen fibrils. DSC and rheological studies demonstrated that adding the phytic acid improved the stability and modulus of elasticity of the collagen gel. The presence of hyaluronic acid in the collagen gel slightly reduced the effect of phytic acid. The presence of phytic acid in the collagen gel improved the stability of the scaffold, but, after 1 week of cultivation, slightly reduced the viability of mesenchymal stromal cells cultured in the gel. The collagen type I gel with hyaluronic and phytic acids can be used to replace tissue defects, especially after the removal of cancerous tumors. Full article

To date, most research on amyloid aggregation has focused on describing the structure of amyloids and the kinetics of their formation, while the conformational stability of fibrils remains insufficiently explored. The aim of this work was to investigate the effect of amino acid substitutions on the stability of apomyoglobin (ApoMb) amyloids. A study of the amyloid unfolding of ApoMb and its six mutant variants by urea has been carried out. Changes in the structural features of aggregates during unfolding were recorded by far-UV CD and native electrophoresis. It was shown that during the initial stage of denaturation, amyloids’ secondary structure partially unfolds. Then, the fibrils undergo dissociation and form intermediate aggregates weighing approximately 1 MDa, which at the last stage of unfolding decompose into 18 kDa monomeric unfolded molecules. The results of unfolding transitions suggest that the stability of the studied amyloids relative to the intermediate aggregates and of the latter relative to unfolded monomers is higher for ApoMb variants with substitutions that increase the hydrophobicity of the residues. The results presented provide a new insight into the mechanism of stabilization of protein aggregates and can serve as a base for further investigations of the amyloids’ stability. Full article