The functional role of Cys3-Cys4 loop in hydrophobin HGFI.

Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic-hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3-Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3-Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water-air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3-Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3-Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic-hydrophilic interfaces.

Development of smart packaging film incorporated with sodium alginate-chitosan quaternary ammonium salt nanocomplexes encapsulating anthocyanins for monitoring milk freshness.

This study focused on the preparation, functionality, and application of smart food packaging films based on polyvinyl alcohol (PVA) and anthocyanins (ACNs) -loaded sodium alginate-chitosan quaternary ammonium salt (HACC-SA) nanocomplexes. The average encapsulation rate of anthocyanins-loaded nanocomplexes reached 62.51 %, which improved the hydrophobicity and water vapor barrier of the PVA film. FTIR confirmed that the nanocomplexes were immobilized in the PVA film matrix by hydrogen bonding, which improved the mechanical properties of the film. The SEM and XRD results demonstrated that the HACC-SA-ACNs nanocomplexes were uniformly distributed in the film matrix and the crystallinity of PVA was decreased. The P/HACC-SA-ACNs film showed a significant response to buffers of pH 2-13 and high color stability after 21 days of storage compared to the P/ACNs film. Furthermore, the color of the composite film changed from purple to red as the milk freshness decreased during 72 h of milk freshness monitoring, indicating that the P/HACC-SA-ACNs films were suitable and promising for application as smart packaging materials.

Preparation and characterization of PVA/arginine chitosan/ZnO NPs composite films.

In this study, arginineated chitosan (ACS) was used as a soft brain membrane and chelating agent to synthesize ACS-ZnO NPs, and then ACS and ACS-ZnO NPs were added to a polyvinyl alcohol (PVA) matrix as an antimicrobial agent to form films by casting. The formation and structural morphology of ACS and ACS-ZnO NPs were investigated by applying FTIR, 1H NMR, XRD, EDS, SEM, and TEM techniques, and ACS has shown better water solubility. The cytotoxicity experiments of ACS and ACS-ZnO NPs on A549 cells showed that both had good cytocompatibility. The incorporation of ACS and ACS-ZnO NPs improves the composite film's mechanical properties, water barrier, and oxygen barrier and exhibits excellent antibacterial activities against S. aureus and E. coli. More importantly, in addition to extending the shelf life of cherry tomatoes, the composite film is also biodegradable to some degree. Therefore, polyvinyl alcohol films based on ACS and ACS-ZnO NPs added as antimicrobial agents have great potential for food packaging applications.

The study of self-regulating α-TCP based composite by micro/nano scaled silk fibroin and α-CSH on physicochemical and biological properties of bone cement.

A novel self-hardening α-tricalcium phosphate (α-TCP) bone cement complexed with different content of α-calcium sulfate hemihydrate (α-CSH) and micrometer hydroxyapatite mineralized silk fibroin (HA-SF) using micro/SF as curing liquid has been investigated in this work, which was capable of tunable setting time, degradation, mechanical property and ability to anti-washout. After addition 0 ∼ 25% α-CSH to the α-TCP cement with SFFs as curing liquid, it shortened the setting time of the modified composite to 10 ∼ 30 min. Furthermore, the addition of SFFs improved the compressive strength of the composite from 5.41 MPa to 9.44 MPa. The composites with both Na2HPO4 and SFFs as curing liquid showed good anti-collapse performance. The weight loss ratio of bone cement was -0.18 ∼ 12.08% in 4 weeks when the content of α-CSH in α-TCP/α-CSH was between 0 ∼ 25 wt%. During the degradation of α-CSH, the amorphous α-TCP were deposited as hydroxyapatite to formed a plate-like products on the surface of composite. Compared to the composite with Na2HPO4 solution as the curing liquid, alkaline phosphatase (ALP) activity of the composites using SFFs as curing liquid were maintained at high levels on the 14th day especially when the Ca/P ratio was 1.7. This study provides a theoretical basis for the regeneration of bone defects guided by bone cement materials.

The self-regulating on cohesion properties of calcium phosphate/ calcium sulfate bone cement improved by citric acid/sodium alginate.

Calcium phosphate cement (CPC) has attracted extensive interest from surgeons and materials scientists. However, the collapsibility of calcium phosphate cement limits its clinical application. In this work, a gel network of SA-CA formed by the reaction of citric acid (CA) and sodium alginate (SA) was introduced into the α-TCP/α-CSH composite. Furthermore, a high proportion of α-CSH provided more calcium sources for the system to combine with SA forming a gel network to improve the cohesion property of the composite, which also played a regulating role in the conversion of materials to HA. The morphology, physicochemical properties, and cell compatibility of the composites were studied with SA-CA as curing solution. The results show that SA-CA plays an important role in the compressive strength and collapse resistance of bone cement, and its properties can be regulated by changing the content of CA. When CA is 10 wt%, the mechanical strength is the highest, reaching 12.49 ± 2.03 MPa, which is 265.80% higher than water as the solidifying liquid. In addition, the cell experiments showed that the samples were not toxic to MC3T3 cells. The results of ALP showed that when SA-CA were used as curing solution, the activity of ALP was higher than that of blank sample, indicating that the composite bone cement could be conducive to the differentiation of osteoblasts. In this work, the α-CSH/α-TCP based composite regulated by gel network of SA-CA can provide a promising strategy to improve the cohesion of bone cement.

Preparation, characterization and food packaging application of nano ZnO@Xylan/quaternized xylan/polyvinyl alcohol composite films.

Xylan could be considered as a good potential candidate for food packaging film because of the vast source and biodegradability, however, its application was restricted by the drawbacks of poor film-forming property, humidity sensitivity, weak mechanical strength and poor antibacterial property. In this paper, xylan was firstly modified by quaternization to improve the film-forming property, then ZnO nanoparticles encapsulated by xylan (nano ZnO@Xylan) was prepared by nanoprecipitation method, finally a series of biodegradable composite films were prepared using quaternized xylan and polyvinyl alcohol with incorporation of nano ZnO@Xylan. The surface morphology, molecular structure and crystallography structure of the films were characterized. The addition of nano ZnO@Xylan decreased water vapor permeability and solubility, meanwhile obviously increased the ultraviolet shielding performance as well as the antibacterial properties of the films. The bacteriostasis rate of the films against E. coli and S. aureus reached up to 99 %. Furthermore, the preservation time of cherry tomatoes covered with ZnO@Xylan/QX/PVA films was extended to at least 21 days. In conclusion, all the results ensure that the fabricated composite films have considerable promising application in the food packaging industry.

Gallic acid functionalized chitosan immobilized nanosilver for modified chitosan/Poly (vinyl alcohol) composite film.

Food spoilage caused by bacterial growth is a serious threat to human health. Silver nanoparticles (AgNPs) have antibacterial activity against various pathogenic microorganisms, but their rapid release often leads to cumulative toxicity. In this paper, silver nanoparticles (AgNPs@CG) were reduced and immobilized in situ using gallic acid-functionalized chitosan (CG) as a reducing and stabilizing agent to achieve a long-lasting and stable controlled release of Ag+. The AgNPs@CG was incorporated into the CG/PVA composite film. The results showed that the release of Ag+ was only 0.686 ± 0.022 mg·L-1 after seven days, which had a long-lasting antibacterial effect on E. coli and S. aureus. In addition, CG/PVA/AgNPs-2 composite film can significantly improve the freshness preservation effect in fresh-cut apple preservation applications. In conclusion, CG/PVA/AgNPs composite film has potential applications as effective and safe packaging material to extend the shelf life of food products.

The preparation and study on properties of calcium sulfate bone cement combined tuning silk fibroin nanofibers and vancomycin-loaded silk fibroin microspheres.

In this study, a bioactive composite material based on calcium sulfate hemihydrate (CSH) bone cement was studied, which use calcium sulfate dihydrate (CSD) as coagulant and silk fibroin nanofibers (SFF) solution as the curing liquid, further loaded vancomycin silk fibroin microspheres (SFM/VCM). The drug release effect of bone cements caused by tuning weight content of SFM/VCM (0.5, 1, 2%) and the concentration of silk fibroin solution (SFS) (20, 60, 100 mg/mL) used for preparation of SFM was studied in this article. Scanning electron microscope (SEM) demonstrated that the average diameter of microspheres gradually increased and the setting time was prolonged with the concentration of SFS increasing. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) were used to analyze the composition of composite materials. The result of compressive strength revealed that the composites contained 0.5% SFM/VCM showed better mechanical performance independent on the concentration of microspheres and the cumulative drug release percentage of all composites were less than 55% after 4 weeks. The drug-loading bone cement possesses not only injectability but also sustained release capability which has a promising prospect in the field of bone substitute material.

Polylactic acid/polyvinyl alcohol-quaternary ammonium chitosan double-layer films doped with novel antimicrobial agent CuO@ZIF-8 NPs for fruit preservation.

ZIF-8, a subclass of metal organic frameworks (MOFs), was employed as the CuO carriers because of its high surface areas and good dispersibility. A novel antibacterial agent CuO@ZIF-8 was synthesized by environmentally-friendly direct calcination strategy, and introduced into the composite double-layer films for packing materials. The double-layer films were prepared via solution casting method with polylactic acid (PLA) and polyvinyl alcohol (PVA)-quaternary ammonium chitosan as the matrix of outer layer and inner layer, respectively; and CuO@ZIF-8 nanoparticles were introduced into the PVA-quaternary ammonium chitosan layer. The double-layer films exhibited superior antibacterial activity resulted from the uniform dispersion of CuO by ZIF-8 carriers. The elongation at break was enhanced and up to 17.13%, about 2.4-fold that of PLA films. Meanwhile, the films provided low water vapor permeability and strong UV-barrier ability which were attributed to the lay-by-layer casting, CuO@ZIF-8 doping and TiO2 addition. Cherry tomato preservation experiment revealed that the composite films retarded the growth of harmful microorganisms on the fruit surface. MTT assay confirmed the cytocompatibility of the films. The easily fabricated double-layer films presented potential possibility in the field of biodegradable food packaging.

The study of mechanical and drug release properties of the mineralized collagen/polylactic acid scaffold by tuning the crystalline structure of polylactic acid.

Open bone fractures in clinical are not only difficult to heal but also at a high risk of infections. Annual cases of fractures which result from osteoporosis amount to approximately 9 million. The objective of this study is to load the antibiotic drug of vancomycin and tune its controlled delivery on a bone repair scaffold material of Mineralized Collagen/poly(lactic acid) (MCP) via changing the crystallinity of poly(lactic acid) to achieve inhibiting infection while repairing defects. We explored the crystallization process of the material during molding and prepared non-crystalline MCP1, MCP2, MCP3 and MCP4 by rapid freeze forming and crystalline MCP5 by tuning temperature decreasing rate. This method can control the micropore structure of the material; and the material changes from brittleness to toughness, which greatly enhances the control of mechanical properties. The drug release behavior of the material was studied for 28 days. Furthermore, the antibacterial property of the material was tested by the zone of inhibition, which shows the material good bacteriostasis. The controllable MCPs are expected to be substitutes for the treatment of infectious bone defects applying to clinical practical treatment.

Preparation, characterization and antibacterial activity of a novel soluble polymer derived from xanthone and O-carboxymethyl-N, N, N-trimethyl chitosan.

In this contribution, a novel soluble and antibacterial polymer, O-xanthonyl-chitosan (CTMC-Xan), was synthesized successfully by grafting 1,3-dihydroxy-xanthone (Xan) to the side chains of O-carboxymethyl-N, N, N-trimethyl chitosan (CTMC). The chemical structure and physical properties of the polymer were analyzed by 1H NMR, FT-IR spectra, UV spectra and XRD. The results showed that Xan could covalently bond with the carboxyl groups of CTMC by esterification at a grafting ratio of 9.1%. XRD patterns indicated that CTMC-Xan does not exhibit crystallization. The solubility tests showed that CTMC-Xan was completely dissolved and stable in neutral solution but unstable in acid or basic conditions. Moreover, it was found that the antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) of CTMC-Xan was much stronger than that of Xan and CTMC, and the minimal bactericidal concentration (MBC) was 125 µg·mL-1. Due to the enhanced solubility and antibacterial activity, CTMC-Xan could potentially serve as a desirable biomaterial for food and pharmaceutical applications.

The effect of calcium phosphate and silk fibroin nanofiber tuning on properties of calcium sulfate bone cements.

Calcium sulfate (CS) bone cements have been used as bone substitutes for a long time, but their clinical use is currently limited due to their rapid degradation rate and brittleness. This work aimed to study the effect of α-tricalcium phosphate (α-TCP) and silk fibroin nanofibers (SFF) on CS bone cements. The bone cements were prepared from α-CS hemihydrate (α-CSH), calcium sulfate dihydrate (CSD; as a setting accelerator) and varying α-TCP contents (0%, 5%, 10%, 15%, 20% and 25%), with SFF solution or deionized water as the solidification solution at the same liquid/solid ratio. Scanning electron microscopy, particle size distribution, x-ray diffraction and Fourier transform infrared spectroscopy were used to measure the composition and characterize the properties of the materials. The compressive strength, setting time and weight loss rate of samples were also tested. Cytotoxicity was evaluated by a Cell Counting Kit-8 assay. The results suggest that the tuning of α-TCP and SFF has an important role in determining the compressive strength and degradation rate of CS bone cements, and the properties could be changed by varying the content of α-TCP. Moreover, cell experiments showed no toxicity of the samples towards MC3T3 cells. Thus, the materials prepared from α-CSH, CSD, α-TCP and SFF in this work could provide the basis for research into CS-based bone repair materials.

Calcium sulfate bone cements with nanoscaled silk fibroin as inducer.

Both nanostructures and conformations of different protein/polysaccharide additives have critical influence on the performance of calcium sulfate (CS) bone cements. Silk fibroin (SF) as matrix and additives has been introduced to develop bone scaffolds and cements. Here, ß-sheet-rich SF nanofibers (SFF) was used to tune the solidification of CS, achieving better mechanical and biological properties. The ratio of SFF was adjusted to further optimize CS functions. Compared to that regulated with natural silk fibers (NSF) and SF solutions (SFS), the SFF-induced CS showed smaller size and more filament structures. Better mechanical properties were achieved, suggesting the superiority of the SFF as the solidifying solution to combine with α-calcium sulfate hemihydrate (α-CSH) at the same liquid/solid (L/S) ratio. Scanning electron microscope, X-ray diffraction, Fourier transform infrared spectroscopy, setting time, porosity, mechanical performance test, degradation performance test, and water resistance test were used to demonstrate the properties of this bone repair cement. Cell culture experiments in vitro was used to evaluate the biocompatibility of this composited material. In conclusion, the results demonstrated that nanofibers was a better form of SF in the modification of CSH cement. And the research conducted in this article on improving the mechanical and biological properties of CSH would supported the reference for later clinical experiments. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2611-2619, 2019.

Investigation of the relationship between the rodlet formation and Cys3-Cys4 loop of the HGFI hydrophobin.

We used protein fusion technology to transplant the Cys3-Cys4 loop of HGFI (a class I hydrophobin from Grifola frondosa) into a nonamyloidogenic hydrophobin HFBI (a class II hydrophobin from Trichoderma reesei) and replace the corresponding amino acids between Cys3 and Cys4 in this protein to identify whether this loop renders it amyloidogenic. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the mutant protein HFBI-AR could form amphipathic membranes by self-assembling at the hydrophilic mica and hydrophobic polystyrene surfaces. This property enabled the mutant protein to alter the surface wettabilities of polystyrene and mica as well as to change the elemental composition of siliconized glass. Atomic force microscopy (AFM) measurements indicated that, unlike class I hydrophobins, no amyloid-like rodlets were observed on the mutant protein HFBI-AR coated mica surface. Moreover, the Cys3-Cys4 region could not catalyze the mutant protein HFBI-AR to drive intermolecular association and formation of a cross-ß rodlet structure to resist depolymerization in organic solvents when it self-assembled at water-air interfaces. These results demonstrate that the Cys3-Cys4 loop is not the major determinant that initiates HGFI to form rodlets or account for the unique properties of the proteins.

Poly(ε-caprolactone) modified with fusion protein containing self-assembled hydrophobin and functional peptide for selective capture of human blood outgrowth endothelial cells.

Human blood outgrowth endothelial cells (HBOECs)-specific binding peptide, TPSLEQRTVYAK (TPS), was proposed to be applied on autologous cell therapy for treating cardiovascular diseases. Hydrophobins, as a family of self-assembly proteins originated from fungi, have demonstrated unique characteristics to modulate surface properties of other materials coated with these amphiphilic proteins in previous studies. In this report, a fusion protein which was composed of class I hydrophobin HGFI originated from Grifola frondosa and functional peptide TPS was expressed by Pichia pastoris expression system. Then, we purified this fusion protein by ultrafiltration and reverse-phase high performance liquid chromatography. Water contact angle, X-ray photoelectron spectroscopy measurements indicated that the surface properties of hydrophobin were greatly preserved by this fusion protein while comparing with wild HGFI. Cell binding assay showed that this fusion protein demonstrated specific binding property to HBOECs while coating on biodegradable poly(ε-caprolactone) (PCL) grafts in the presence of fetal bovine serum, whereas HGFI-coated PCL non-selectively enhanced all types of cells attachments. Methylthiazol tetrazolium assay was employed to verify the cytocompatibility of this fusion protein-based material. This work presented a new perspective to apply hydrophobin in tissue engineering and regenerative medicine and provided an alternative approach to study endothelial progenitor cells.