Double-Network Hydrogel with Strengthened Mechanical Property for Controllable Release of Antibacterial Peptide.

Developing double-network (DN) hydrogels with high mechanical properties and antibacterial efficacy to combat multidrug-resistant bacterial infections and serve as scaffolds for cell culture still remains an ongoing challenge. In this study, an ion-responsive antibacterial peptide (AMP) (C16-WIIIKKK, termed as IK7) was synergistically combined with a photoresponsive gelatin methacryloyl (GelMA) polymer to fabricate a biocompatible DN hydrogel. The GelMA-IK7 DN hydrogel showed enhanced mechanical properties in contrast to the individual IK7 and GelMA hydrogels and demonstrated substantial antibacterial efficacy. Further investigations revealed that the DN hydrogel effectively inhibited bacterial growth by the controlled and sustained release of the IK7 peptide. In addition, the formation of the DN hydrogel was also found to protect AMP IK7 from rapid degradation by proteinase K. Our findings suggested that the developed GelMA-IK7 DN hydrogel holds great potential for next-generation antibacterial hydrogels for three-dimensional cell culture and tissue regeneration.

Controllable formation of bulk perfluorohexane nanodroplets by solvent exchange.

Perfluorocarbon (PFC) nanodroplets have rapidly developed into useful ultrasound imaging agents in modern medicine due to their non-toxic and stable chemical properties that facilitate disease diagnosis and targeted therapy. In addition, with the good capacity for carrying breathing gases and the anti-infection ability, they are employed as blood substitutes and are the most ideal liquid respirators. However, it is still a challenge to prepare stable PFC nanodroplets of uniform size and high concentration for their efficient use. Herein, we developed a simple and highly reproducible method, i.e., propanol-water exchange, to prepare highly homogeneous and stable perfluorohexane (PFH) bulk nanodroplets. Interestingly, the size distribution and concentration of formed nanodroplets could be regulated by controlling the volume fraction of PFH and percentage of propanol in the propanol-water mixture. We demonstrated good reproducibility in the formation of bulk nanodroplets with PFH volume fractions of 1/2000-1/200 and propanol percentage of 5-40%, with uniform particle size distribution and high droplet concentration. Also, the prepared nanodroplets were very stable and could survive for several hours. We constructed a ternary phase diagram to describe the relationship between the PFH volume ratio, propanol concentration, and the size distribution and concentration of the formed PFH nanodroplets. This study provides a very useful method to prepare uniform size, high concentration and stable PFC nanodroplets for their medical applications.

Gelation of a Pentapeptide in Alcohols.

Properties of solvents such as polarity and H-bond-forming ability are critical for the formation of an organogel and have a significant impact on the gel behavior, as solvents are the majority of organogel systems. However, so far, there is still a lack of systematic studies regarding the effects of molecular structures of solvents on the characteristics of organogels. Motivated by revealing such a relationship, in this paper, we studied the morphologies of assemblies, gelation behaviors, and secondary structures of a pentapeptide termed EAF-5 in a wide variety of alcohols. The side chains and lengths of carbon chains of the solvent molecules were found to play a critical role in the self-assembly and gelation of EAF-5. EAF-5 was capable of self-assembling into fibers and entangling into a network in alcohols including ethanol, propanol, butanol, n-pentanol, and n-hexanol, which further immobilized the corresponding alcohols to form gels. In these organogels, increasing ß-sheet secondary structures of the peptides were formed by introducing side chains and extending the length of primary alcohol molecules. We hypothesized that alcohol molecules with extended lengths and side chains reduced the gelator-solvent interactions and promoted the gelator-gelator interactions, resulting in the self-assembly of EAF-5 into fibril structures and development of gels. These findings provide a new sight into the interactions between gelators and solvents and are helpful for designing peptide-based organogelators.

The effects of nanobubbles on the assembly of glucagon amyloid fibrils.

Some recent studies have shown that the surface and interface play an important role in the assembly and aggregation of amyloid proteins. However, it is unclear how the gas-liquid interface affects the protein assembly at the nanometer scale although the presence of gas-liquid interfaces is very common in in vitro experiments. Nanobubbles have a large specific surface area, which provides a stage for interactions with various proteins and peptides on the nanometer scale. In this work, nanobubbles produced in solution were employed for studying the effects of the gas-liquid interface on the assembly of glucagon proteins. Atomic force microscopy (AFM) studies showed that nanobubble-treated glucagon solution formed fibrils with an apparent height of 4.02 ± 0.71 nm, in contrast to the fibrils formed with a height of 2.14 ± 0.53 nm in the control. Transmission electron microscopy (TEM) results also showed that nanobubbles promoted the assembly of glucagon to form more fibrils. Thioflavin T (ThT) fluorescence and Fourier transform infrared (FTIR) analyses indicated that the nanobubbles induced the change of the glucagon conformation to a ß-sheet structure. A mechanism that explains how nanobubbles affect the assembly of glucagon amyloid fibrils was proposed based on the above-mentioned experimental results. Given the fact that there are a considerable amount of nanobubbles existing in protein solutions, our results indicate that nanobubbles should be considered for fully understanding the protein aggregation events in vitro.

Self-assembly of pentapeptides in ethanol to develop organogels.

Organogels have a wide variety of applications in the fields of chemistry, electricity, biomedicine and environmental engineering, which call for robust strategies for designing and developing novel organogelators. Here, we reported a pentapeptide, ECAYF, which was capable of forming a self-healing ethanol gel exhibiting viscoelastic and solid-like properties. The ethanol gel of ECAYF was stable for at least several months, suggesting strong non-covalent interactions between ethanol and the peptide in the gel. In the ethanol gel, self-assembled peptide fibrils were found to immobilize the ethanol molecules for gelation. Results also suggested that the EAF-5 peptide adopted H-bonding ß-sheet secondary structures, which further assembled into fibrils. Meanwhile, the self-assembly of the ECAYF peptide in mixtures of differently fractioned ethanol and H2O was observed, which clearly indicated that ethanol promoted the assembly of ECAYF in the solutions. These findings are helpful in understanding the roles of organic solvents as well as the complicated interactions between the solvent and gelator molecules in gelation.

Novel tyrosinase inhibitory peptide with free radical scavenging ability.

Tyrosinase is a key enzyme involved in melanin synthesis. Therefore, various tyrosinase inhibitors have been screened by researchers in recent years. In the present study, we discovered a novel tyrosinase inhibitor, a peptide ECGYF (named EF-5), with free radical scavenging ability. The effect of tyrosinase inhibition by EF-5 was stronger than that of arbutin and glutathione, when analyzed both in vitro (IC50: 0.46 mM) and in vivo. The UV-Vis absorption and circular dichroism spectroscopies indicated that EF-5 interacted with tyrosinase in a different way as that of glutathione. The results of molecular docking showed that the binding between EF-5 and tyrosinase was determined majorly by hydrogen bonds and hydrophobic interactions. EF-5 had also retained its ability to scavenge both hydroxyl and superoxide radicals in vitro and was found to be nontoxic to cells, as revealed by the MTT assay. These features suggested that the EF-5 peptide may serve as a safe and effective alternative as a tyrosinase inhibitor.

Biostable hydrogels consisting of hybrid ß-sheet fibrils assembled by a pair of enantiomeric peptides.

The assembly of chiral peptides facilitates the formation of diverse supramolecular structures with unique physicochemical and biological properties. However, the effects of chirality on peptide assembly and resulting hydrogel properties remain underexplored. In this study, we systematically investigated the assembly propensity, morphology, and biostability of mixture of a pair of enantiomeric peptides LELCLALFLF (ECF-5) and DEDCDADFDF (ecf-5) at various ratios. Results indicate the development of ß-sheet fibrils, ultimately leading to the formation of self-supporting hybrid hydrogels. The hydrogel formed at a ratio of 1:1 exhibits a significantly lower storage modulus (G') than of the ratios of 0:1, 1:3, 3:1 and 1:0 (nD/nL; same below). Kink-separated fragments of approximately 100 nm in length predominate at ratios of 1:3 and 3:1, compared with the smooth fibrils at other ratios, probably attributed to an alternating arrangement of the co-assembled and self-assembled peptide fragments. The introduction of ecf-5 to the hybrid hydrogels improves resistance to proteolytic digestion and maintains commendable biocompatibility in both MIN6 and HUVECs cells. These findings provide valuable insights into the development of hydrogels with tailored properties, positing them potential scaffolds for 3D cell culture and tissue engineering.

Gram-selective antibacterial peptide hydrogels.

The human microbiome plays fundamental roles in human health and disease. However, widely used broad-spectrum antibiotics severely disrupt human-related microbial communities, eventually leading to resistant bacteria, posing a growing threat to global medical health. Antimicrobial peptides (AMPs) are promising antimicrobial agents that barely cause bacterial resistance. Excellent broad-spectrum antimicrobial activities have been achieved using hydrogels self-assembled from AMPs, but there is still a lack of AMP hydrogels that can target Gram-positive and Gram-negative bacteria. Herein, several hydrogels self-assembled from AMPs, termed IK1, IK3, and IK4, were designed and synthesized. In vitro antibacterial results indicated that the IK1 and IK4 hydrogels specifically targeted Gram-positive and Gram-negative bacteria, respectively, while the IK3 hydrogel targeted both Gram-positive and Gram-negative bacteria. The desired broad-spectrum or Gram-selective AMP hydrogels are believed to be obtained through the rational design of the hydrophilicity, hydrophobicity, and charge properties of the peptide molecules. Good in vivo Gram-selective antibacterial properties and the ability to promote wound healing have been demonstrated via treating mouse wound models with these AMP hydrogels. We believe that these Gram-selective AMP hydrogels could potentially have important applications in treating common recurring infections.

Correction: Gram-selective antibacterial peptide hydrogels.

Correction for 'Gram-selective antibacterial peptide hydrogels' by Yangqian Hou et al., Biomater. Sci., 2022, 10, 3831-3844, https://doi.org/10.1039/D2BM00558A.

Antimicrobial d-Peptide Hydrogels.

Biofilms are widely involved in human lives, such as in medical infection, environmental remediation, and industrial processes. However, the control of the biofilm has still been a challenge because of its strong drug resistance. Here, we designed and synthesized an amphipathic antimicrobial peptide (Ac-DKDHDHDQDKDLDVDFDFDADK-NH2 (KKd-11)) that was composed of d-amino acids (DAAs). KKd-11 was found to self-assemble into a hydrogel with an improved long-term antimicrobial ability and a better antiprotease activity as compared to the hydrogel formed by Ac-LKLHLHLQLKLLLVLFLFLALK-NH2 (KK-11). Our results indicated that KKd-11 was not only able to inhibit the formation of biofilms but also could effectively damage preformed mature biofilms and kill the bacteria within the biofilms. Besides, cell viability assays indicated that the KKd-11 peptide had very good biocompatibility. We think d-peptide hydrogels may have great potential in the treatment of biofilm-induced infections.

Generating Bulk Nanobubbles in Alcohol Systems.

Bulk nanobubbles (NBs) have attracted wide attention due to their peculiar physicochemical properties and great potential in applications in various fields. However, so far there are no reports on bulk NBs generated in pure organic systems, which we think is very important as NBs would largely improve the efficiency of gas-liquid mass transfer and facilitate chemical reactions to take place. In this paper, we verified that air and N2 NBs could be generated in a series of alcohol solutions by using various methods including acoustical cavitation, pressurization-depressurization, and vibration. The experiments proved that NBs existed in alcohol solutions, with a highest density of 5.8 × 107 bubble/mL in propanol. Our results also indicated that bulk NBs could stably exist for at least hours in alcohol systems. The parameters in generating NBs in alcohols were optimized. Our findings open up an opportunity for improving gas-liquid mass transfer efficiency in the field of the chemical industry.

Macrochirality of Self-Assembled and Co-assembled Supramolecular Structures of a Pair of Enantiomeric Peptides.

Although macrochirality of peptides' supramolecular structures has been found to play important roles in biological activities, how macrochirality is determined by the molecular chirality of the constituted amino acids is still unclear. Here, two chiral peptides, Ac-LKLHLHLQLKLLLVLFLFLALK-NH2 (KK-11) and Ac-DKDHDHDQDKDL DVDFDFDADK-NH2 (KKd-11), which were composed entirely of either L- or D-amino acids, were designed for studying the chiral characteristics of the supramolecular microstructures. It was found that monocomponent KK-11 or KKd-11 self-assembled into right- or left-handed helical nanofibrils, respectively. However, when they co-assembled with concentration ratios varied from 1:9 to 9:1, achiral nanowire-like structures were formed. Both circular dichroism and Fourier transform infrared spectra indicated that the secondary structures changed when the peptides co-assembled. MD simulations indicated that KK-11 or KKd-11 exhibited a strong propensity to self-assemble into right-handed or left-handed nanofibrils, respectively. However, when KK-11 and KKd-11 were both presented in a solution, they had a higher probability to co-assemble instead of self-sort. MD simulations indicated that, in their mixtures, they formed nanowires without handedness feature, a good agreement with experimental observation. Our results shed light on the molecular mechanisms of the macrochirality of peptide supramolecular microstructures.

Reactive Oxygen Species Scavenging and Biodegradable Peptide Hydrogel as 3D Culture Scaffold for Cardiomyocytes.

Myocardial ischemia-reperfusion produces a large amount of reactive oxygen species (ROS), which damage the myocardial tissue. Therefore, localized scavenging of ROS from the myocardial tissue would reduce its damage and avoid metabolic abnormalities caused by systemic ROS. In this study, a free radical scavenging and biodegradable supramolecular peptide (ECAFF, named as ECF-5) hydrogel was designed as a culture scaffold for cardiomyocytes. The peptide hydrogel significantly preserved the migration and proliferation of cardiomyocytes and reduced their damage from oxidative stress. In addition, the hydrogel degraded during cell growth, which implies that it may avoid thrombosis of the capillaries in practical use and provide the opportunity for the cells to attach to each other and form a functional tissue. The hydrogel can be used as a 3D culture scaffold for cardiomyocyte culture and allow cardiomyocytes to grow into tissue-like cell spheres. The excellent nature of the ECF-5 hydrogel enables it to have broad applications in the biomedical field in the future.

Peptides Co-Assembling into Hydrangea-Like Microstructures.

Supramolecular assembly in vitro is a simple and effective way to produce multi-level biostructures to mimic the self-assembly of biomolecules in organisms. The study on peptide assembly behaviors would benefit a lot to understand what goes on in life, as well as in the construction of plenty of functional biomaterials that have potential applications in various fields. Since cellular microenvironments are crowded and contain various biomolecules, studying protein and peptide co-assembly is of great interest. Here, we introduced the co-assembly of 5-FAM-ELVFFAE-NH2 (EE-7) and (CY5)-KLVFFAK-NH2 (KK-7), which are sequences derived from the core of the amyloid ß (Aß) peptide, a key protein in Alzheimer's diseases. Morphologic studies employing atomic force microscopy and scanning electron microscopy indicated that the co-assembled entities had a novel hydrangea-like microstructure, in contrast to micro-sheet structures formed from monocomponent EE-7 or KK-7, respectively. Fluorescence co-localization experiments confirmed that the hydrangealike microstructures were indeed made of both EE-7 and KK-7. We suggest that the formation of the hydrangea-like microstructures is driven by both the electrostatic and hydrophobic interactions between EE-7 and KK-7. A molecular mechanism has been provided to explain the formation of the hydrangea-like microstructures.