Effects of alkali pretreatment of silk fibroin on microstructure and properties of hydroxyapatite–silk fibroin nanocomposite

Nanocomposites comprising hydroxyapatite (HAp) and silk fibroin (SF) were synthesized from Ca(OH)2 suspension co-dispersed with SF fine particles and H3PO4 solution via a wet-mechanochemical route. The SF particles were modified with an alkali solution to increase contact points between HAp phase and SF matrix. HAp crystallites grow more preferentially along c-axis on alkali pretreated SF substrates. The composites exhibit porous microstructure with 70% of open porosity and about 70% of the interpores ranging from 40 to 115 microm in diameter. The peak shifts in amide II band of SF indicate that the chemical interactions between HAp crystals and SF matrix are intensified by the alkali pretreatment of SF. The stronger inorganic-organic interactions promote the formation of three-dimensional network extending throughout the composites, bringing about an increase of 63% in the Vickers hardness to the composite.     

Formation of spherical protein nanoparticles without impacting protein integrity

Protein formulation at the nanoscale is challenging because of protein susceptibility to chemical and physical degradation during processing. Herein, we present a straightforward method to prepare spherical protein nanoparticles by co-lyophilizing five structurally different enzymes (horseradish peroxidase, carbonic anhydrase, lysozyme, subtilisin Carlsberg and α-chymotrypsin) with methyl-β-cyclodextrin followed by suspension of the powders in ethyl acetate. The size distribution was narrow and varied from 88 ± 14 to 148 ± 16?nm as determined by dynamic light scattering. Scanning and transmission electron micrographs confirmed the size and spherical morphology of the protein nanoparticles. Residual activities for all enzymes tested were 100% upon dissolving the nanoparticles in buffer and no insoluble aggregates were formed.     

An ordered electrospun polycaprolactone–collagen–silk fibroin scaffold for hepatocyte culture

Nanofiber scaffolds are widely used as the platform for three-dimensional culture of hepatocytes in vitro. The pore size of scaffolds plays an important role in promoting the infiltration and proliferation of hepatocyte. We show that the average pore size of electrospun scaffold increases from ~ 7.6 to 13.2 μm, while the average fiber diameter decreases from ~ 2.0 to 1.5 μm when collected by probe array collectors. Though increase in pore size decreases the tensile stress of scaffolds, it leads to enhance the proliferation and attachment of hepatocytes. Specifically, a 6 × 6 array scaffold which was prepared by probe collector was orderly arrayed. Compared with the conventional scaffold, the pore size of the arrayed scaffold doubled and the hydrophilicity was improved. When HepG₂ cells were seeded on the arrayed scaffold, cells showed superior adhesion ability, better cell morphology and three-dimensional growth. These results indicated that the ordered 6 × 6 array scaffold has the potential as a suitable substratum for in vitro culture of hepatocytes.     

Synthesis of protein–gold nanoparticle hybrid and gold nanoplates in protein aggregates

A straightforward and economically viable approach was developed to biomimetic synthesis of gold nanocrystals by using casein micelles (CMs) without additional reductant. The UV–vis, TEM, SAED, FTIR, DLS and XRD techniques were employed to systematically characterize Au nanocrystals synthesized. Isotropic gold nanoparticle (GNP) and gold nanoplates in good yields (up to 90%) with different sizes can be obtained easily by adjusting the experimental condition. Spherical nanoparticles were obtained with tunable mean sizes at higher pH and casein concentrations. The high colloidal stability of the spherical GNP is attributed to the formation of CM/GNP hybrid under some experimental condition. At lower pH, reaction temperature and casein concentrations, single-crystalline gold nanoplates in good yields (up to 90%) are obtained. The growth of these nanostructures is attributed to an interplay between the faceting tendency of the protein molecules/micelles and the growth kinetics. More importantly, the morphological evolution of large gold nanoplates at different reaction times has been followed, and compared with some earlier protein systems, different formation mechanisms in casein micelles are obtained. The results demonstrate that both the property of individual protein molecules and protein aggregates play important roles in controlling the formation of gold nanocrystals by using amphiphilic protein.     

Enzymatic degradation behavior and cytocompatibility of silk fibroin–starch–chitosan conjugate membranes

The objective of this study was to investigate the influence of silk fibroin and oxidized starch conjugation on the enzymatic degradation behavior and the cytocompatability of chitosan based biomaterials. The tensile stress of conjugate membranes, which was at 50 Megapascal (MPa) for the lowest fibroin and starch composition (10 weight percent (wt.%)), was decreased significantly with the increased content of fibroin and starch. The weight loss of conjugates in α-amylase was more notable when the starch concentration was the highest at 30 wt.%. The conjugates were resistant to the degradation by protease and lysozyme except for the conjugates with the lowest starch concentration. After 10 days of cell culture, the proliferation of osteoblast-like cells (SaOS-2) was stimulated significantly by higher fibroin compositions and the DNA synthesis on the conjugate with the highest fibroin (30 wt.%) was about two times more compared to the native chitosan. The light microscopy and the image analysis results showed that the cell area and the lengths were decreased significantly with higher fibroin/chitosan ratio. The study proved that the conjugation of fibroin and starch with the chitosan based biomaterials by the use of non-toxic reductive alkylation crosslinking significantly improved the cytocompatibility and modulated the biodegradation, respectively.     

Fabrication and characterization of curcumin-loaded silk fibroin/P（LLA-CL） nanofibrous scaffold

Curcumin exhibited excellent properties including antioxidant, anti- inflammatory, antiviral, antibacterial, antifungal, anticancer, and anticoagulant activities. In this study, curcumin was incorporated into silk fibroin （SF）/poly（L-lactic acid-co-e- caprolactone） （P（LLA-CL）） nanofibrous scaffolds via electrospinning, and changes brought about by raising the curcumin content were observed： SEM images showed that the average nanofibrous diameter decreased at the beginning and then increased, and the nanofibers became uniform; FTIR showed that the conformation of SF transforming from random coil form to β-sheet structure had not been induced, while SF conformation converted to β-sheet after being treated with 75% ethanol vapor; XRD results confirmed that the crystal structure of （P（LLA-CL）） had been destroyed; The mechanical test illustrated that nanofibrous scaffolds still maintained good mechanical properties. Further, curcumin-loaded nanofibrous scaffolds were evaluated for drug release, antioxidant and antimicrobial activities in vitro. The results showed that curcumin presented a sustained release behavior from nanofibrous scaffolds and maintained its free radical scavenging ability, and such scaffolds could effectively inhibit S. aureus growth （〉 95%）. Thus, curcumin-loaded SF/P（LLA-CL） nanofibrous scaffolds might be potential candidates for wound dressing and tissue engineering scaffolds.     

Effect of eggshell and silk fibroin on styrene–ethylene/butylene–styrene as bio-filler

SEBS (poly(styrene-b-ethylene/butylene-b-styrene)) biodegradable composites reinforced with various eggshell contents and silk fibroin are prepared by melt processing technique followed by different characterization techniques. Compare with SEBS/eggshell composites, the SEBS/eggshell/silk composites exhibit the reduction in particle size and increase interface interaction within the matrix. Thermal stability also increases in quantity.     

Minimum protein oscillator based on multisite phosphorylation∕dephosphorylation

The authors propose a novel minimum oscillator whereby a protein with multiple phosphorylation sites directly embedded in a negative feedback loop can exhibit oscillation. They demonstrate that if the fully phosphorylated substrate inhibits the first phosphorylation step in a cooperative manner, multisite substrates can exhibit oscillatory behaviour at the presence of a kinase and phosphatase. With a fixed number of sites, the non-linearity of the negative feedback and the substrate∕enzyme ratio must be above certain threshold values to generate undamped oscillation. There is an inverse relationship between the number of phosphorylation sites and the minimum non-linearity of the negative feedback required for oscillation; that is, the ultrasensitivity and time delay rooted in multisite phosphorylation compensate for the explicit non-linearity in the negative feedback. The period and amplitude of oscillation are mainly determined by the number of phosphorylation sites and the substrate∕enzyme ratio. The authors' results suggest that a multisite protein can be exploited for the construction of a synthetic protein oscillator featuring simplicity, robustness and tunability.     

Viruses and virus-like protein assemblies—Chemically programmable nanoscale building blocks

Supramolecular proteins are generated using a limited set of twenty amino acids, but have distinctive functionalities which arise from the sequential arrangement of amino acids configured to exquisite three-dimensional structures. Viruses, virus-like particles, ferritins, enzyme complexes, cellular micro-compartments, and other supramolecular protein assemblies exemplify these systems, with their precise arrangements of tens to hundreds of molecules into highly organized scaffolds for nucleic acid packaging, metal storage, catalysis or sequestering reactions at the nanometer scale. These versatile protein systems, dubbed as bionanoparticles (BNPs), have attracted materials scientists to seek new opportunities with these pre-fabricated templates in a wide range of nanotechnology-related applications. Here, we focus on some of the key modification strategies that have been utilized, ranging from basic protein conjugation techniques to more novel strategies, to expand the functionalities of these multimeric protein assemblies. Ultimately, in combination with molecular cloning and sophisticated chemistries, these BNPs are being incorporated into many applications ranging from functional materials to novel biomedical drug designs.      

Nano-scaled hydroxyapatite/polymer composite I. Coating of sintered hydroxyapatite particles on poly(γ-methacryloxypropyl trimethoxysilane)-grafted silk fibroin fibers through chemical bonding

The inorganic-organic composite consisting of nano-scaled hydroxyapatite (HAp) and silk fibroin (SF) fibers was prepared through covalent linkage to develop a novel biomaterial for a soft-tissue-compatible material. The preparation of the composite was conducted through the three-step procedure consisting of chemical modification using 2-methacryloxyethyl isocyanate (MOI) monomer to introduce vinyl groups on SF, poly(gamma-methacryloxypropyl trimethoxysilane) (MPTS) graft-polymerization on SF, and coupling process between the surface of polyMPTS-grafted SF and HAp nano-particles. The amount of the graft-polymerization of polyMPTS through vinyl groups was well controlled by the reaction time. The nano-crystals were subsequently coated on the grafted fibers by heating at 120 degrees C for 2 h in a vacuum. The crystalline structure of the SF substrate did not change in the procedure. In the SEM observation of the composite surface, it was found that the bonded nano-crystals were separated and partially aggregated with several crystals attached on the SF fiber surface. The HAp particles adhered more strongly on the SF surface with separation or aggregation of several crystals than on the surface of the original SF after ultrasonic treatment.     

Characterization and evaluation of β-glucan formulations as injectable implants for protein and peptide delivery

Context: Injectable implants are biodegradable, syringeable formulations that are injected as liquids, but form a gel inside the body due to a change in pH, ions or temperature. Objective: To investigate the effect of polymer concentration, pH, ions and temperature on the gel formation of β-glucan, a natural cell-wall polysaccharide derived from barley, with particular emphasis on two-phase system formation after addition of dextran or PEG. Materials and methods: Oscillation viscometry was used to evaluate the gel character by measuring flow index (N), storage (G') and loss (G″) moduli. Two-phase systems were further characterized for hardness and syringeability using a texture analyzer. Finally, in vitro release characteristics were determined using Franz diffusion cells. Results: Oscillation viscometry revealed that only addition of dextran or PEG resulted in distinct gel formation. This was seen by a decrease in N after polymer addition. Moreover, hardness (in g) of the gels increased significantly (p?相似文献   

Nanoparticulate strategies for the treatment of polyglutamine diseases by halting the protein aggregation process†

Polyglutamine (polyQ) diseases are a class of neurodegenerative disorders that cause cellular dysfunction and, eventually, neuronal death in specific regions of the brain. Neurodegeneration is linked to the misfolding and aggregation of expanded polyQ-containing proteins, and their inhibition is one of major therapeutic strategies used commonly. However, successful treatment has been limited to date because of the intrinsic properties of therapeutic agents (poor water solubility, low bioavailability, poor pharmacokinetic properties), and difficulty in crossing physiological barriers, including the blood–brain barrier (BBB). In order to solve these problems, nanoparticulate systems with dimensions of 1–1000?nm able to incorporate small and macromolecules with therapeutic value, to protect and deliver them directly to the brain, have recently been developed, but their use for targeting polyQ disease-mediated protein misfolding and aggregation remains scarce. This review provides an update of the polyQ protein aggregation process and the development of therapeutic strategies for halting it. The main features that a nanoparticulate system should possess in order to enhance brain delivery are discussed, as well as the different types of materials utilized to produce them. The final part of this review focuses on the potential application of nanoparticulate system strategies to improve the specific and efficient delivery of therapeutic agents to the brain for the treatment of polyQ diseases.     

Characterization and evaluation of β-glucan formulations as injectable implants for protein and peptide delivery

Context: Injectable implants are biodegradable, syringeable formulations that are injected as liquids, but form a gel inside the body due to a change in pH, ions or temperature.

Objective: To investigate the effect of polymer concentration, pH, ions and temperature on the gel formation of β-glucan, a natural cell-wall polysaccharide derived from barley, with particular emphasis on two-phase system formation after addition of dextran or PEG.

Materials and methods: Oscillation viscometry was used to evaluate the gel character by measuring flow index (N), storage (G′) and loss (G″) moduli. Two-phase systems were further characterized for hardness and syringeability using a texture analyzer. Finally, in vitro release characteristics were determined using Franz diffusion cells.

Results: Oscillation viscometry revealed that only addition of dextran or PEG resulted in distinct gel formation. This was seen by a decrease in N after polymer addition. Moreover, hardness (in g) of the gels increased significantly (p?<?0.001) from 3.65?±?0.43 to 34.30?±?8.90 (dextran) and 805.80?±?5.30 (PEG) 24?h after polymer addition. In vitro release profiles showed significantly (p?<?0.05) reduced AUC_{0–8 h}, k and percentage of drug released from two-phase systems compared to β-glucan dispersions, with the PEG system resulting in the lowest amount released over 8?h (15.1?±?1.6%).

Discussion: The unfavorable mixing enthalpy and higher water affinity of PEG resulted in the formation of a dense β-glucan gel.

Conclusion: 1.5% (w/w) β-glucan combined with PEG at a ratio of 1:3 seemed to be the most promising injectable formulation with respect to fastest gel formation, increased hardness and sustained release.     

Effects of thermal treatments on protein adsorption of Co–Cr–Mo ASTM-F75 alloys

Post-manufacturing thermal treatments are commonly employed in the production of hip replacements to reduce shrinkage voids which can occur in cast components. Several studies have investigated the consequences of these treatments upon the alloy microstructure and tribological properties but none have determined if there are any biological ramifications. In this study the adsorption of proteins from foetal bovine serum (FBS) on three Co–Cr–Mo ASTM-F75 alloy samples with different metallurgical histories, has been studied as a function of protein concentration. Adsorption isotherms have been plotted using the surface concentration of nitrogen as a diagnostic of protein uptake as measured by X-ray photoelectron spectroscopy. The data was a good fit to the Langmuir adsorption isotherm up to the concentration at which critical protein saturation occurred. Differences in protein adsorption on each alloy have been observed. This suggests that development of the tissue/implant interface, although similar, may differ between as-cast (AC) and heat treated samples.     

Engineering of poly(ε-caprolactone) microcarriers to modulate protein encapsulation capability and release kinetic

Drug delivery applications using biodegradable polymeric microspheres are becoming an important means of delivering therapeutic agents. The aim of this work was to modulate the microporosity of poly(ε-caprolactone) (PCL) microcarriers to control protein loading capability and release profile. PCL microparticles loaded with BSA (bovine serum albumin) have been de novo synthesized with double emulsion solvent evaporation technique transferred and adapted for different polymer concentrations (1.7 and 3% w/v) and stabilizer present in the inner aqueous phase (0.05, 0.5 and 1% w/v). SEM (scanning electron microscope) and CLSM (confocal laser scanning microscope) analysis map the drug distribution in homogeneously distributed cavities inside the microspheres with dimensions that can be modulated by varying double emulsion process parameters. The inner structure of BSA-loaded microspheres is greatly affected by the surfactant concentration in the internal aqueous phase, while a slight influence of polymer concentration in the oil phase was observed. The surfactant concentration mainly determines microspheres morphology, as well as drug release kinetics, as confirmed by our in-vitro BSA release study. Moreover, the entrapped protein remained unaltered during the protein encapsulation process, retaining its bio-activity and structure, as shown through a dedicated gel chromatographic analytical method.     

Graphene quantum dots rescue protein dysregulation of pancreatic β-cells exposed to human islet amyloid polypeptide

Nano Research - The amyloid aggregation of peptides and proteins is a hallmark of neurological disorders and type 2 diabetes. Human islet amyloid polypeptide (IAPP), co-secreted with insulin by...     

Surface energy of hydroxyapatite and β-tricalcium phosphate ceramics driving serum protein adsorption and osteoblast adhesion

The main objective of this work was to evaluate the specific role of calcium phosphates surface energy on serum protein adsorption and human osteoblast adhesion, by isolating chemical effects from those caused by topography. Highly dense phosphate ceramics (single-phase hydroxyapatite HA and β-tricalcium phosphates β-TCP) presenting two distinct nano roughnesses were produced. Some samples were gold-sputter coated in order to conveniently mask the surface chemical effects (without modification of the original roughness) and to study the isolated effect of surface topography on cellular behavior. The results indicated that the nano topography of calcium phosphates strongly affected the protein adsorption process, being more important than surface chemistry. The seeding efficacy of osteoblasts was not affected nor by the topography neither by the calcium phosphate chemistries but the β-TCP chemistry negatively influenced cell spreading. We observed that surface hydrophobicity is another way to change protein adsorption on surfaces. The decrease of the polar component of surface energy on gold-coated samples leaded to a decreased albumin and fibronectin adsorption but to an increased cell adhesion. Overall, this work contributes to better understand the role of topography and surface chemistry of calcium phosphates in serum protein adsorption and osteoblast adhesion.