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Collagen nanofibrous materials have become integral to tissue engineering due to their exceptional properties and biocompatibility. Dehydrothermal crosslinking (DHT) enhances stability and maintains structural integrity without the formation of toxic residues. The study involved the crosslinking of electrospun collagen, applying DHT with access to air and under vacuum conditions. Various DHT exposure times of up to 72 h were applied to examine the time dependance of the DHT process. The DHT crosslinked collagen was subsequently chemically crosslinked using carbodiimides. The material crosslinked in this way evinced elevated Young’s modulus values and ultimate tensile strength values, a lower swelling rate and lower shrinkage ratio during crosslinking, and a higher degree of resistance to degradation than the material crosslinked solely with DHT or carbodiimides. It was shown that the crosslinking mechanism using DHT occupies different binding sites than those using chemical crosslinking. Access to air for 12 h or less did not exert a significant impact on the material properties compared to DHT under vacuum conditions. However, concerning longer exposure times, it was determined that access to air results in the deterioration of the properties of the material and that reactions take place that occupy the free bonding sites, which subsequently reduces the effectiveness of chemical crosslinking using carbodiimides.

The photosystem I (PSI) of the green alga Chlamydomonas reinhardtii associates with 10 light-harvesting proteins (LHCIs) to form the PSI-LHCI complex. In the context of state transitions, two LHCII trimers bind to the PSAL, PSAH and PSAO side of PSI to produce the PSI-LHCI-LHCII complex. In this work, we took advantage of chemical crosslinking of proteins in conjunction with mass spectrometry to identify protein–protein interactions between the light-harvesting proteins of PSI and PSII. We detected crosslinks suggesting the binding of LHCBM proteins to the LHCA1-PSAG side of PSI as well as protein–protein interactions of LHCSR3 with LHCA5 and LHCA3. Our data indicate that the binding of LHCII to PSI is more versatile than anticipated and imply that LHCSR3 might be involved in the regulation of excitation energy transfer to the PSI core via LHCA5/LHCA3.

In this study, we developed a method to fabricate chitosan-based network polysaccharides via the condensation between amino groups in water-soluble chitosan (WSCS) and a carboxylate-terminated maltooligosaccharide crosslinker. We previously reported on the fabrication of network-polysaccharide-based macroscopic hydrogels via the chemical crosslinking of water-soluble chitin (WSCh) with the crosslinker. Because the molecular weight of the WSCS was much smaller than that of the WSCh, in the present investigation, the chemical crosslinking of the WSCS with the crosslinker was observed at the nanoscale upon the condensation between amino and carboxylate groups in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide, affording nano-sized chitosan-based network polysaccharides. The occurrence of the crosslinking via the formation of amido linkages was supported by the IR analysis and 1H NMR measurements after the dissolution via acid hydrolysis in DCl/D2O. The products formed nanogels, whose sizes depended on the amino/carboxylate feed ratio. The nanoscale morphology and size of the products were evaluated via scanning electron microscopy, dynamic light scattering analyses, and transition electron microscopy. In the present study, we successfully developed the method to fabricate nanogel materials based on network polysaccharide structures, which can practically be applied as new polysaccharide-based 3D bionanomaterials.

The photosystem I (PSI) of the green alga Chlamydomonas reinhardtii associates with 10 light-harvesting proteins (LHCIs) to form the PSI-LHCI complex. In the context of state transitions, two LHCII trimers bind to the PSAL, PSAH and PSAO side of PSI to produce the PSI-LHCI-LHCII complex. In this work, we took advantage of chemical crosslinking of proteins in conjunction with mass spectrometry to identify protein-protein interactions between the light-harvesting proteins of PSI and PSII. We detected crosslinks suggesting the binding of LHCBM proteins to the LHCA1-PSAG side of PSI as well as protein-protein interactions of LHCSR3 with LHCA5 and LHCA3. Our data indicate that the binding of LHCII to PSI is more versatile than anticipated and imply that LHCSR3 might be involved in the regulation of excitation energy transfer to the PSI core via LHCA5/LHCA3.

For the effective clinical antibacterial application of biomaterials, such as for wound management and tissue repair, the biomaterials need to show proper antibacterial capability as well as non-cytotoxicity. Furthermore, the material needs to have suitable mechanical characteristics for further medical use. Chitosan hydrogel is a potential candidate for various antibacterial biomedical applications due to its amine functionalities that lead to antimicrobial characteristics. Nevertheless, its antimicrobial capability is dependent upon the degree of protonation of amine groups caused by the pH value. Moreover, its mechanical compressive strength may not be high enough for clinical use if not chemically or physically crosslinked. This study utilized a novel chemical crosslinker, mercaptosuccinic acid, to improve its mechanical characteristics. The natural antibacterial agent, cinnamaldehyde, was grafted onto the crosslinked chitosan to improve its antimicrobial capability. Meanwhile, to take advantage of the thiol functionality in the mercaptosuccinic acid, the bactericidal silver nanoparticles were incorporated through silver-thiol covalent bounding. NMR analyses indicated the chitosan was successfully mercaptosuccinic acid-crosslinked and grafted with cinnamaldehyde at different ratios. Combined the results from the mechanical assessment, swelling experiments, antimicrobial assessment, and cytotoxicity assay, the chitosan hydrogel with the highest crosslinked degree and grafted with cinnamaldehyde and silver nanoparticles is of great promise for further clinical uses.

Skeletal muscles exhibit excellent properties due to their well-developed microstructures. Taking inspiration from nature that thick filaments and thin filaments are linked by “cross-bridges”, leading to good stability and ion transport performance of muscles. In this work, extracted poplar lignin and microcrystalline cellulose (MCC) were connected by biomimetic covalent bonds, akin to biological muscle tissue, in which isophorone diisocyanate was used as the chemical crosslinking agent. Then, poplar lignin–MCC was mixed with polyacrylonitrile to serve as the precursor for electrospinning. The results show that due to the effective covalent-bond connection, the precursor fibers possess excellent morphology, smooth surface, good thermal stability, and high flexibility and toughness (average elongation-at-break is 51.84%). Therefore, after thermal stabilization and carbonization, derived lignocellulose-based carbon fibers (CFs) with a reduced cost, complete fiber morphology with a uniform diameter (0.48 ± 0.22 μm), and high graphitization degree were obtained. Finally, the electrodes fabrication and electrochemical testing were carried out. The results of electrochemical impedance spectroscopy (EIS) indicate that the Rs and Rct values of CFs supercapacitors are 1.18 Ω and 0.14 Ω, respectively. Results of cyclic voltammetry (CV) and galvanostatic charge–discharge (GCD) suggest that these CFs demonstrate great application potential in electrochemical materials.

E. coli histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU–DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity (σ = −0.06–0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA (σ = −0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5–6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.

The interfacial compatibility of the natural filler and synthetic polymer is the key performance characteristic of biocomposites. The fillers are chemically modified, or coupling agents and compatibilisers are used to ensure optimal filler-polymer compatibility. Hence, we have investigated the effect of compatibilisation strategies of olive pits (OP) flour content (10, 20, 30, and 40%wt.) filled with recycled low-density polyethylene (rLDPE) on the chemical, physical, mechanical, and thermal behaviour of the developed biocomposites. In this study, we aim to investigate the filler-polymer compatibility in biocomposites by employing novel strategies for the functionalisation of OP filler and/or rLDPE matrix. Specifically, four cases are considered: untreated OP filled rLDPE (Case 1), treated OP filled rLDPE (Case 2), treated OP filled functionalised rLDPE (Case 3), and treated and functionalised OP filled functionalised rLDPE (Case 4). In general, the evaluation of the performance of biocomposites facilitated the application of OP industrial waste as an eco-friendly reinforcing agent for rLDPE-based biocomposites. Furthermore, surface treatment and compatibilisation improved the properties of the developed biocomposites over untreated filler or uncoupled biocomposites. Besides that, the compatibilisers used aided in reducing water uptake and improving thermal behaviour, which contributed to the stability of the manufactured biocomposites.

The rheological changes that occur during the chemical gelation of semidilute solutions of chitosan in the presence of the low-toxicity agent glyceraldehyde (GCA) are presented and discussed in detail. The entanglement concentration for chitosan solutions was found to be approximately 0.2 wt.% and the rheological experiments were carried out on 1 wt.% chitosan solutions with various amounts of GCA at different temperatures (25 °C and 40 °C) and pH values (4.8 and 5.8). High crosslinker concentration, as well as elevated temperature and pH close to the pKa value (pH ≈ 6.3–7) of chitosan are three parameters that all accelerate the gelation process. These conditions also promote a faster solid-like response of the gel-network in the post-gel region after long curing times. The mesh size of the gel-network after a very long (18 h) curing time was found to contract with increasing level of crosslinker addition and elevated temperature. The gelation of chitosan in the presence of other chemical crosslinker agents (glutaraldehyde and genipin) is discussed and a comparison with GCA is made. Small angle neutron scattering (SANS) results reveal structural changes between chitosan solutions, incipient gels, and mature gels.

Physically crosslinked hydrogels with thixotropic properties attract considerable attention in the biomedical research field because their self-healing nature is useful in cell encapsulation, as injectable gels, and as bioinks for three-dimensional (3D) bioprinting. Here, we report the formation of thixotropic hydrogels containing nanofibers of double-hydrophobic elastin-like polypeptides (ELPs). The hydrogels are obtained with the double-hydrophobic ELPs at 0.5 wt%, the concentration of which is an order of magnitude lower than those for previously reported ELP hydrogels. Although the kinetics of hydrogel formation is slower for the double-hydrophobic ELP with a cell-binding sequence, the storage moduli G′ of mature hydrogels are similar regardless of the presence of a cell-binding sequence. Reversible gel–sol transitions are demonstrated in step-strain rheological measurements. The degree of recovery of the storage modulus G′ after the removal of high shear stress is improved by chemical crosslinking of nanofibers when intermolecular crosslinking is successful. This work would provide deeper insight into the structure–property relationships of the self-assembling polypeptides and a better design strategy for hydrogels with desired viscoelastic properties.

Nanogels represent an innovative platform for tunable drug release and targeted therapy in several biomedical applications, ranging from cancer to neurological disorders. The design of these nanocarriers is a pivotal topic investigated by the researchers over the years, with the aim to optimize the procedures and provide advanced nanomaterials. Chemical reactions, physical interactions and the developments of engineered devices are the three main areas explored to overcome the shortcomings of the traditional nanofabrication approaches. This review proposes a focus on the current techniques used in nanogel design, highlighting the upgrades in physico-chemical methodologies, microfluidics and 3D printing. Polymers and biomolecules can be combined to produce ad hoc nanonetworks according to the final curative aims, preserving the criteria of biocompatibility and biodegradability. Controlled polymerization, interfacial reactions, sol-gel transition, manipulation of the fluids at the nanoscale, lab-on-a-chip technology and 3D printing are the leading strategies to lean on in the next future and offer new solutions to the critical healthcare scenarios.

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

Stretchable and tough hydrogels have drawn a lot of attention recently. Due to their unique properties, they have great potential in the application in areas such as mechanical sensing, wound healing, and drug delivery. In this review, we will summarize recent developments of stretchable and tough hydrogels in these areas.

Hydrogels are three-dimensional networks composed of hydrated polymer chains and have been a material of choice for many biomedical applications such as drug delivery, biosensing, and tissue engineering due to their unique biocompatibility, tunable physical characteristics, flexible methods of synthesis, and range of constituents. In many cases, methods for crosslinking polymer precursors to form hydrogels would benefit from being highly selective in order to avoid cross-reactivity with components of biological systems leading to adverse effects. Crosslinking reactions involving the thiol group (SH) offer unique opportunities to construct hydrogel materials of diverse properties under mild conditions. This article reviews and comments on thiol-mediated chemoselective and biocompatible strategies for crosslinking natural and synthetic macromolecules to form injectable hydrogels for applications in drug delivery and cell encapsulation.

Chemical grafting or crosslinking of polyimide chains are known to be feasible approaches to increase polymer gas-pair selectivity and specific gas permeance. Different co-polyimides; 6FDA-ODA and 6FDA-ODA:DABA were synthesized using a two-step condensation method. Six different cross-linkers were used: (i) m-xylylene diamine; (ii) n-ethylamine; and (iii) n-butylamine, by reacting with 6FDA-ODA’s imide groups in a solid state crosslinking; while (iv) ethylene glycol monosalicylate (EGmSal); (v) ethylene glycol anhydrous (EGAn); and (vi) thermally labile iron (III) acetylacetonate (FeAc), by reacting with DABA carboxyl groups in 6FDA-ODA:DABA. The gas separation performances were evaluated by feeding an equimolar CO2 and CH4 binary mixture, at a constant feed pressure of 5 bar, at 25 °C. Fractional free volume (FFV) was calculated using Bondi’s contribution method by considering the membrane solid density property, measured by pycnometer. Other characterization techniques: thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) were performed accordingly. Depending on the type of amine, the CO2/CH4 selectivity of 6FDA-ODA increased between 25 to 100% at the expense of CO2 permeance. We observed the similar trend for 6FDA-ODA:DABA EGmSal-crosslinked with 143% selectivity enhancement. FeAc-crosslinked membranes showed an increment in both selectivity and CO2 permeability by 126% and 29% respectively. Interestingly, FeAc acted as both cross-linker which reduces chain mobility; consequently improving the selectivity and as micro-pore former; thus increases the gas permeability. The separation stability was further evaluated using 25–75% CO2 in the feed with CH4 as the remaining, between 2 and 8 bar at 25 °C. We also observed no CO2-induced plasticization to the measured pressure with high CO2 content (max. 75%).

The rheological changes that occur during the chemical gelation of semidilute solutions of chitosan in the presence of the low-toxicity agent glyceraldehyde (GCA) are presented and discussed in detail. The entanglement concentration for chitosan solutions was found to be approximately 0.2 wt.% and the rheological experiments were carried out on 1 wt.% chitosan solutions with various amounts of GCA at different temperatures (25 °C and 40 °C) and pH values (4.8 and 5.8). High crosslinker concentration, as well as elevated temperature and pH close to the pK a value (pH ≈ 6.3-7) of chitosan are three parameters that all accelerate the gelation process. These conditions also promote a faster solid-like response of the gel-network in the post-gel region after long curing times. The mesh size of the gel-network after a very long (18 h) curing time was found to contract with increasing level of crosslinker addition and elevated temperature. The gelation of chitosan in the presence of other chemical crosslinker agents (glutaraldehyde and genipin) is discussed and a comparison with GCA is made. Small angle neutron scattering (SANS) results reveal structural changes between chitosan solutions, incipient gels, and mature gels.

Nanogels represent an innovative platform for tunable drug release and targeted therapy in several biomedical applications, ranging from cancer to neurological disorders. The design of these nanocarriers is a pivotal topic investigated by the researchers over the years, with the aim to optimize the procedures and provide advanced nanomaterials. Chemical reactions, physical interactions and the developments of engineered devices are the three main areas explored to overcome the shortcomings of the traditional nanofabrication approaches. This review proposes a focus on the current techniques used in nanogel design, highlighting the upgrades in physico-chemical methodologies, microfluidics and 3D printing. Polymers and biomolecules can be combined to produce ad hoc nanonetworks according to the final curative aims, preserving the criteria of biocompatibility and biodegradability. Controlled polymerization, interfacial reactions, sol-gel transition, manipulation of the fluids at the nanoscale, lab-on-a-chip technology and 3D printing are the leading strategies to lean on in the next future and offer new solutions to the critical healthcare scenarios.

Hydrogels are three-dimensional networks composed of hydrated polymer chains and have been a material of choice for many biomedical applications such as drug delivery, biosensing, and tissue engineering due to their unique biocompatibility, tunable physical characteristics, flexible methods of synthesis, and range of constituents. In many cases, methods for crosslinking polymer precursors to form hydrogels would benefit from being highly selective in order to avoid cross-reactivity with components of biological systems leading to adverse effects. Crosslinking reactions involving the thiol group (SH) offer unique opportunities to construct hydrogel materials of diverse properties under mild conditions. This article reviews and comments on thiol-mediated chemoselective and biocompatible strategies for crosslinking natural and synthetic macromolecules to form injectable hydrogels for applications in drug delivery and cell encapsulation.