Influence of the backbone chemistry and ionic functional groups of five pairs of oppositely charged polyelectrolytes on complex coacervation.

Complex coacervation plays an important role in various fields. Here, the influences of the backbone chemistry and ionic functional groups of five pairs of oppositely charged polyelectrolytes on complex coacervation were investigated. These pairs include synthetic polymers with aliphatic hydrocarbon backbones, peptides with amide bonds, and carbohydrates with glycosidic linkages. Despite sharing identical charged groups, specific pairs displayed distinct liquid/liquid and liquid/solid phase separations depending on the polyelectrolyte mixing ratio, buffer, and ionic strength. The coacervate phase boundary broadened in the orders: glycosidic linkages > amide backbone > aliphatic hydrocarbon backbone, and Tris-phosphate > Tris-acetate > Tris-chloride buffers. Coacervates prepared from polyelectrolytes with lower solubilities in water resisted disassembly at high salt concentrations, and their merge rate was slow. These observations suggest that the hydrophobic segments in polyelectrolytes interfere with the formation of complex coacervates; however, following coacervate formation, the hydrophobic segments render the coacervates stable and elastic.

Monodisperse polyhydroxyalkanoate nanoparticles as self-sticky and bio-resorbable tissue adhesives.

Monodisperse nanoparticles of biodegradable polyhydroxyalkanoates (PHAs) polymers, copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB), are synthesized using a membrane-assisted emulsion encapsulation and evaporation process for biomedical resorbable adhesives. The precise control over the diameter of these PHA particles, ranging from 100 nm to 8 µm, is achieved by adjusting the diameter of emulsion or the PHA concentration. Mechanical properties of the particles can be tailored based on the 3HB to 4HB ratio and molecular weight, primarily influenced by the level of crystallinity. These monodisperse PHA particles in solution serve as adhesives for hydrogel systems, specifically those based on poly(N, N-dimethylacrylamide) (PDMA). Semi-crystalline PHA nanoparticles exhibit stronger adhesion energy than their amorphous counterparts. Due to their self-adhesiveness, adhesion energy increases even when those PHA nanoparticles form multilayers between hydrogels. Furthermore, as they degrade and are resorbed into the body, the PHA nanoparticles demonstrate efficacy in in vivo wound closure, underscoring their considerable impact on biomedical applications.

Adsorptive chito-beads for control of membrane fouling.

Chitosan has excellent antimicrobial, adsorption, heavy metal removal, and adhesion properties, making it a good substitute for microplastic-based cleaners. Here, chitosan microbeads (chito-beads) of various sizes ranging from 32 µm to 283 µm were prepared via emulsion using a liquid on oil method and the feasibility of using them as an essential constituent in a chemical cleaning solution for a reverse-osmosis (RO) membrane-fouling-control process was assessed. Prior to the assessment the cleaning efficiency of a solution containing chito-beads, the interaction energy between chitosan and a representative organic foulant (humic acid (HA)) in a RO membrane fouling was analyzed using colloidal atomic force microscopy, and the strongest attraction between chitosan and HA was observed in an aqueous solution. When comparing the membrane cleaning efficiency of cleaning solutions with and without chito-beads, smaller chito-beads (32 µm and 70 µm) were found to have higher cleaning efficiency. Applications of chito-beads to the membrane cleaning process can enhance the cleaning efficiency through the physicochemical interaction with organic foulants. This study can widen the use of chito-beads as an additive to membrane chemical cleaning solutions to control membrane fouling in other membrane processes as well.

Spontaneous Transition of Spherical Coacervate to Vesicle-Like Compartment.

Numerous biological systems contain vesicle-like biomolecular compartments without membranes, which contribute to diverse functions including gene regulation, stress response, signaling, and skin barrier formation. Coacervation, as a form of liquid-liquid phase separation (LLPS), is recognized as a representative precursor to the formation and assembly of membrane-less vesicle-like structures, although their formation mechanism remains unclear. In this study, a coacervation-driven membrane-less vesicle-like structure is constructed using two proteins, GG1234 (an anionic intrinsically disordered protein) and bhBMP-2 (a bioengineered human bone morphogenetic protein 2). GG1234 formed both simple coacervates by itself and complex coacervates with the relatively cationic bhBMP-2 under acidic conditions. Upon addition of dissolved bhBMP-2 to the simple coacervates of GG1234, a phase transition from spherical simple coacervates to vesicular condensates occurred via the interactions between GG1234 and bhBMP-2 on the surface of the highly viscoelastic GG1234 simple coacervates. Furthermore, the shell structure in the outer region of the GG1234/bhBMP-2 vesicular condensates exhibited gel-like properties, leading to the formation of multiphasic vesicle-like compartments. A potential mechanism is proposed for the formation of the membrane-less GG1234/bhBMP-2 vesicle-like compartments. This study provides a dynamic process underlying the formation of biomolecular multiphasic condensates, thereby enhancing the understanding of these biomolecular structures.

A peptide of PilZ domain-containing protein controls wastewater-treatment-membrane biofouling by inducing bacterial attachment.

Membrane-based wastewater reclamation is used to mitigate water scarcity; however, irreversible biofouling is an elusive problem that hinders the efficiency of a forward-osmosis (FO) membrane-based process, and the protein responsible for fouling is unknown. Herein, we identified fouling proteins by analyzing the microbiome and proteome of wastewater extracellular polymeric substances responsible for strong irreversible FO-membrane fouling. The IGLSSLPR peptide of a PilZ domain-containing protein was found to recruit bacterial attachment when immobilized on the membrane surface while suppressing it when dissolved, in a similar manner to the Arg-Gly-Asp (RGD) peptide in mammalian cell cultures. Bacteria adhere to IGLSSLPR and poly-l-lysine-coated membranes with similar energies and exhibit water fluxes that decline similarly, which is ascribable to interaction as strong as electrostatic interactions in the peptide-coated membranes. We conclude that IGLSSLPR is the key domain responsible for membrane fouling and can be used to develop antifouling technology against bacteria, which is similar to the current usage of RGD peptide in mammalian cell cultures.

RNA-mediated demixing transition of low-density condensates.

Biomolecular condensates play a key role in organizing cellular reactions by concentrating a specific set of biomolecules. However, whether condensate formation is accompanied by an increase in the total mass concentration within condensates or by the demixing of already highly crowded intracellular components remains elusive. Here, using refractive index imaging, we quantify the mass density of several condensates, including nucleoli, heterochromatin, nuclear speckles, and stress granules. Surprisingly, the latter two condensates exhibit low densities with a total mass concentration similar to the surrounding cyto- or nucleoplasm. Low-density condensates display higher permeability to cellular protein probes. We find that RNA tunes the biomolecular density of condensates. Moreover, intracellular structures such as mitochondria heavily influence the way phase separation proceeds, impacting the localization, morphology, and growth of condensates. These findings favor a model where segregative phase separation driven by non-associative or repulsive molecular interactions together with RNA-mediated selective association of specific components can give rise to low-density condensates in the crowded cellular environment.

A sticky carbohydrate meets a mussel adhesive: Catechol-conjugated levan for hemostatic and wound healing applications.

The stickiest natural polysaccharide, levan, plays a role in metalloproteinase activation, which is an important step involved in the healing of injured tissue. However, levan is easily diluted, washed away, and loses adhesion in wet environments, which limits its biomedical applications. Herein, we demonstrate a strategy for fabricating a levan-based adhesive hydrogel for hemostatic and wound healing applications by conjugating catechol to levan. Prepared hydrogels exhibit significantly improved water solubilities, and adhesion strengths to hydrated porcine skin of up to 42.17 ± 0.24 kPa which is more than three-times that of fibrin glue adhesive. The hydrogels also promote rapid blood clotting and significantly faster healing of rat-skin incisions compared to nontreated samples. In addition, levan-catechol exhibited an immune response close to that of the negative control, which is ascribable to its significantly lower endotoxin level compared to native levan. Overall, levan-catechol hydrogels are promising materials for hemostatic and wound healing applications.

Cation-π Interactions Contribute to Hydrophobic Humic Acid Removal for the Control of Hydraulically Irreversible Membrane Fouling.

Hydraulically irreversible membrane fouling is a major problem encountered during membrane-based water purification. Membrane foulants present large hydrophobic fractions, with humic acid (HA) being a prevalent example of hydrophobic natural organic matter. Furthermore, HA contains numerous aromatic rings (π electrons), and its hydrophobic interactions are a major cause of irreversible membrane fouling. To address this issue, in this study, we used the cation-π interaction, which is a strong noncovalent, competitive interaction present in water. Because the strength of cation-π interactions depends on the combination of cations and π molecules, utilizing the appropriate cations will effectively remove irreversible fouling caused by hydrophobic HA. We performed macroscale experiments to determine the cleaning potential of the test cations, nanomechanically analyzed the changes in HA cohesion caused by the test cations using a surface force apparatus and an atomic force microscope, and used molecular dynamics simulations to elucidate the HA removal mechanism of test studied cations. We found that the addition of 1-ethyl-3-methylimidazolium, an imidazolium cation with an aromatic moiety, effectively removed the HA layer by weakening its cohesion, and the size, hydrophobicity, and polarity of the HA layer synergistically affected the HA removal mechanism based on the cation-π interactions.

eXtreme gradient boosting-based classification of bacterial mixtures in water and milk using wireless microscopic imaging of quorum sensing peptide-conjugated particles.

Numerous bacteria can cause water- and foodborne diseases and are often found in bacterial mixtures, making their detection challenging. Specific bioreceptors or selective growth media are necessary for most bacterial detection methods. In this work, we collectively used five quorum sensing-based peptides identified from bacterial biofilms to identify 10 different bacterial species (Bacillus subtilis, Campylobacter jejuni, Enterococcus faecium, Escherichia coli, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, Staphylococcus aureus, Vibrio parahaemolyticus) and their mixtures in water and milk. Four different machine learning classification methods were used: k-nearest neighbors (k-NN), decision tree (DT), support vector machine (SVM), and eXtreme Gradient Boosting (XGBoost). Peptides were crosslinked to submicron particles, and peptide-bacteria interactions on paper microfluidic chips caused the particle aggregation. A wireless, pocket fluorescence microscope (interfaced with a smartphone) counted such particle aggregations. XGBoost showed the best accuracy of 83.75% in identifying bacterial species from water samples using 320 different datasets and 91.67% from milk samples using 140 different datasets (5 peptide features per dataset). Each peptide's contribution to correct classification was evaluated. The results were concentration-dependent, allowing the identification of a dominant species from bacterial mixtures. Using XGBoost and the previous milk database, we tested 14 blind samples of various bacterial mixtures in milk samples, with an accuracy of 81.55% to predict the dominant species. The entire process could be completed within a half hour. The demonstrated system can provide a handheld, low-cost, easy-to-operate tool for potential hygiene spot-checks, public health, or personal healthcare.

Nanochitin and Nanochitosan: Chitin Nanostructure Engineering with Multiscale Properties for Biomedical and Environmental Applications.

Nanochitin and nanochitosan (with random-copolymer-based multiscale architectures of glucosamine and N-acetylglucosamine units) have recently attracted immense attention for the development of green, sustainable, and advanced functional materials. Nanochitin and nanochitosan are multiscale materials from small oligomers, rod-shaped nanocrystals, longer nanofibers, to hierarchical assemblies of nanofibers. Various physical properties of chitin and chitosan depend on their molecular- and nanostructures; translational research has utilized them for a wide range of applications (biomedical, industrial, environmental, and so on). Instead of reviewing the entire extensive literature on chitin and chitosan, here, recent developments in multiscale-dependent material properties and their applications are highlighted; immune, medical, reinforcing, adhesive, green electrochemical materials, biological scaffolds, and sustainable food packaging are discussed considering the size, shape, and assembly of chitin nanostructures. In summary, new perspectives for the development of sustainable advanced functional materials based on nanochitin and nanochitosan by understanding and engineering their multiscale properties are described.

Adhesion and Cohesion Differences between Catechol- and Pyrogallol-Functionalized Chitosan.

Marine-inspired phenolic compounds that exhibit underwater adhesion are used as biomedical adhesives under wet conditions. While these applications mainly use catechol and pyrogallol moieties that contain different numbers of hydroxyl groups on their benzene rings, how this difference affects adhesion and cohesion is not well understood. Herein, the chitosan backbone is functionalized with catechol and pyrogallol at similar modification rates (to give chitosan-catechol (CS-CA) and chitosan-pyrogallol (CS-GA), respectively) and their interaction energies are compared by using a surface forces apparatus (SFA). The phenolic moieties decrease the rigidity of the chitosan chain and increase solubility; consequently, CS-CA and CS-GA are more cohesive and adhesive than chitosan at pH 7.4. Moreover, the additional hydroxyl group of GA provides a further interacting chance; hence, CS-GA is more cohesive and adhesive than CS-CA. This study provides in-depth insight into interactions involving chitosan derivatives bearing introduced phenolic moieties that will help to develop biomedical adhesives.

Electroconductive, Adhesive, Non-Swelling, and Viscoelastic Hydrogels for Bioelectronics.

As a new class of materials, implantable flexible electrical conductors have recently been developed and applied to bioelectronics. An ideal electrical conductor requires high conductivity, tissue-like mechanical properties, low toxicity, reliable adhesion to biological tissues, and the ability to maintain its shape in wet physiological environments. Despite significant advances, electrical conductors that satisfy all these requirements are insufficient. Herein, a facile method for manufacturing a new conductive hydrogels through the simultaneous exfoliation of graphite and polymerization of zwitterionic monomers triggered by microwave irradiation is introduced. The mechanical properties of the obtained conductive hydrogel are similar to those of living tissue, which is ideal as a bionic adhesive for minimizing contact damage due to mechanical mismatches between hard electronics and soft tissues. Furthermore, it exhibits excellent adhesion performance, electrical conductivity, non-swelling, and high conformability in water. Excellent biocompatibility of the hydrogel is confirmed through a cytotoxicity test using C2C12 cells, a biocompatibility test on rat tissues, and their histological analysis. The hydrogel is then implanted into the sciatic nerve of a rat and neuromodulation is demonstrated through low-current electrical stimulation. This hydrogel demonstrates a tissue-like extraneuronal electrode, which possesses high conformability to improve the tissue-electronics interfaces, promising next-generation bioelectronics applications.

Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins.

Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine's ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine's ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems.

Sum-Frequency Vibrational Spectroscopic Study of the Cation-π Interaction: Amine and Guanidine.

The cation-π interaction is an interaction between a positively charged cation and π electrons in an aromatic group of a molecule. It is considered to play key roles in signal transduction, stabilization of the protein structure, enzyme catalysis in biology, and wet adhesion and biomolecular condensation. In this study, octadecylguanidine hydrochloride (ODG) and octadecylamine (ODA) having guanidine and amine headgroups, respectively, are found to interact with π molecules (phenol or indole) as investigated by sum-frequency vibrational spectroscopy. ODG is unstable and does not form a neat monolayer on the water surface. However, after adding π molecules into subphase water, it becomes more stable against dissolution as evidenced by the appearance of its CHx peaks and a CH peak of the aromatic ring in the sum-frequency spectrum. Unlike ODG, ODA forms a stable monolayer on the water surface at a neutral pH. After adding π molecules into the solution, the amine-π interaction promotes the protonation of the amine headgroup and the penetration of the π molecules makes the ODA monolayer more disordered. Indole is found to be more effective in binding with the ODG as compared to phenol.

Ionic fluid as a novel cleaning agent for the control of irreversible fouling in reverse osmosis membrane processes.

While a variety of chemical cleaning strategies has been studied to control fouling in membrane-based water treatment processes, the removal of irreversible foulants strongly bound on membrane surfaces has not been successful. In this study, we firstly investigated the diluted aqueous solutions of ionic fluid (IF, 1-ethyl-3-methylimidazolium acetate) as a cleaning agent for three model organic foulants (humic acid, HA; bovine serum albumin, BSA; sodium alginate, SA). The real-time monitoring of cleaning progress by optical coherence tomography (OCT) showed that fouling layer was dramatically swelled by introducing IF solution and removed by shear force exerted during cleaning. This phenomenon was induced due to the pre-existing interactions between organic foulants were weakened by the intrusion of IF into the fouling layer, which was analyzed by the measurement of adhesion forces using atomic force microscopy (AFM). In the experiments with model foulants and wastewater effluent, IF was added to alkaline cleaning agents (NaOH) to verify the applicability to be supplemented in commercial cleaning agents, and resulted in the significantly enhanced control of irreversible membrane fouling. Implication of utilizing recyclable IF with negligible volatility is that environmental effects of membrane cleaning solutions could be minimized by decreasing usage of cleaning chemicals, while increasing the cleaning efficiency.

Resolving the Mutually Exclusive Immune Responses of Chitosan with Nanomechanics and Immunological Assays.

Multifaceted functions displayed by both pro- and anti-inflammatory properties of chitosan hinder its effective development as an immunomodulatory agent. Herein, the contributions of the bending stiffness of chitosan with regard to its immune regulatory properties toward inflammation are investigated. The anti-inflammatory properties of chitosan molecular weight (MW) with a shorter (≈1 kDa) or longer (≈15 kDa) than the persistent length (LP ) are compared using immunological assays and nanomechanics-based experiments on the surface forces apparatus (SFA). Interestingly, 1 kDa chitosan significantly enhances the generation of anti-inflammatory regulatory T cells (Tregs) through the Dectin-1-dependent pattern recognition receptor (PRR) on antigen-presenting cells. SFA analyses also show a similar trend of interaction forces between chitosan and diverse PRRs depending on their MW. The results obtained in the immunological and nanomechanical experiments are consistent and imply that the binding features of PRRs vary depending on the MW of chitosan, which may alter immune activity. In accordance, in vivo administration of only 1 kDa represses inflammatory responses and suppresses the progression of experimental colitis. This study elucidates a previously unexplored bending stiffness-dependent immune regulatory property of chitosan and suggests the applicability of low MW (rod-like) chitosan as a pharmaceutical ingredient to treat diverse inflammatory disorders.

Molecular mechanisms mediating stiffening in the mechanically adaptable connective tissues of sea cucumbers.

Mutable collagenous tissues (MCTs) from echinoderms (e.g., sea stars, sea urchins) possess the remarkable ability to change their mechanical properties rapidly and reversibly thanks to the release of effector molecules regulating the number of cross-links between collagen fibrils. Among these effector molecules, tensilin has been identified as a stiffening factor in sea cucumber MCTs. Since its discovery and description twenty years ago, tensilin orthologs have been identified in a few sea cucumber species but no novel information about its molecular mode of action has been reported. In this study, using a combination of in silico analyses, we identified the tensilin present in the dermis of Holothuria forskali, Hf-(D)Tensilin. Anti-peptide antibodies showed that this protein is localised in the secretory granules of type 2 juxtaligamental-like cells, a MCT specific cell type. We then used the bacterium E. coli to produce recombinantly Hf-(D)Tensilin and confirmed its stiffening effect on pieces of the dermis and its aggregation effect on collagen fibrils extracted from the sea cucumber dermis. To investigate how tensilin can cross-bridge collagen fibrils, truncated recombinant tensilins were also produced and used in combination with various compounds. Results suggest that two types of interactions contribute to the aggregation effect of tensilin on the fibrils: (1) the N-terminal NTR TIMP like domain of the protein interacts strongly with sulfated GAGs attached to the surface of the collagen fibrils, and (2) the C-terminal part of the protein is involved in its dimerisation/oligomerisation through ionic but possibly also cation-π and hydrophobic interactions.

Counterplotting the Mechanosensing-Based Fouling Mechanism of Mussels against Fouling.

Marine organisms react to various factors when building colonies for survival; however, severe accumulation of diverse organisms on artificial structures located close to water causes large industrial losses. Herein, we identify a concept in the development of antifouling surfaces based on understanding the surface stiffness recognition procedure of mussel adhesion at the genetic level. It was found that on a soft surface the combination of decreased adhesive plaque size, adhesion force, and plaque protein downregulation synergistically weakens mussel wet adhesion and sometimes prevents mussels from anchoring, mainly due to transcriptional changes within the mechanosensing pathway and the adhesive proteins in secretory glands. In addition, the use of soft substrates or antagonists of surface mechanosensing behavior suppresses mussel fouling significantly.

Strong, Multifaceted Guanidinium-Based Adhesion of Bioorganic Nanoparticles to Wet Biological Tissue.

Gluing dynamic, wet biological tissue is important in injury treatment yet difficult to achieve. Polymeric adhesives are inconvenient to handle due to rapid cross-linking and can raise biocompatibility concerns. Inorganic nanoparticles adhere weakly to wet surfaces. Herein, an aqueous suspension of guanidinium-functionalized chitin nanoparticles as a biomedical adhesive with biocompatible, hemostatic, and antibacterial properties is developed. It glues porcine skin up to 3000-fold more strongly (30 kPa) than inorganic nanoparticles at the same concentration and adheres at neutral pH, which is unachievable with mussel-inspired adhesives alone. The glue exhibits an instant adhesion (2 min) to fully wet surfaces, and the glued assembly endures one-week underwater immersion. The suspension is lowly viscous and stable, hence sprayable and convenient to store. A nanomechanic study reveals that guanidinium moieties are chaotropic, creating strong, multifaceted noncovalent bonds with proteins: salt bridges comprising ionic attraction and bidentate hydrogen bonding with acidic moieties, cation-π interactions with aromatic moieties, and hydrophobic interactions. The adhesion mechanism provides a blueprint for advanced tissue adhesives.

Exfoliated bentonite/alginate nanocomposite hydrogel enhances intestinal delivery of probiotics by resistance to gastric pH and on-demand disintegration.

In this study, we developed Lactobacillus rhamnosus GG (LGG)-encapsulating exfoliated bentonite/alginate nanocomposite hydrogels for protecting probiotics by delaying gastric fluid penetration into the nanocomposite and their on-demand release in the intestine. The pore size of the bentonite/alginate nanocomposite hydrogels (BA15) was two-fold smaller than that of alginate hydrogel (BA00). Following gastric pH challenge, the survival of LGG in BA15 decreased by only 1.43 log CFU/g as compared to the 6.25 log CFU/g decrease in alginate (BA00). Further, the internal pH of BA15 decreased more gradually than that of BA00. After oral administration in mice, BA15 maintained shape integrity during gastric passage, followed by appropriate disintegration within the target intestinal area. Additionally, a fecal recovery experiment in mice showed that the viable counts of LGG in BA15 were six-fold higher than those in BA00. The findings suggest the exfoliated bentonite/alginate nanocomposite hydrogel as a promising platform for intestinal delivery of probiotics.