From Basic Principles of Protein-Polysaccharide Association to the Rational Design of Thermally Sensitive Materials.

Biology resolves design requirements toward functional materials by creating nanostructured composites, where individual components are combined to maximize the macroscale material performance. A major challenge in utilizing such design principles is the trade-off between the preservation of individual component properties and emerging composite functionalities. Here, polysaccharide pectin and silk fibroin were investigated in their composite form with pectin as a thermal-responsive ion conductor and fibroin with exceptional mechanical strength. We show that segregative phase separation occurs upon mixing, and within a limited compositional range, domains ∼50 nm in size are formed and distributed homogeneously so that decent matrix collective properties are established. The composite is characterized by slight conformational changes in the silk domains, sequestering the hydrogen-bonded ß-sheets as well as the emergence of randomized pectin orientations. However, most dominant in the composite's properties is the introduction of dense domain interfaces, leading to increased hydration, surface hydrophilicity, and increased strain of the composite material. Using controlled surface charging in X-ray photoelectron spectroscopy, we further demonstrate Ca ions (Ca2+) diffusion in the pectin domains, with which the fingerprints of interactions at domain interfaces are revealed. Both the thermal response and the electrical conductance were found to be strongly dependent on the degree of composite hydration. Our results provide a fundamental understanding of the role of interfacial interactions and their potential applications in the design of material properties, polysaccharide-protein composites in particular.

Proline-Tyrosine Ring Interactions in Black Widow Dragline Silk Revealed by Solid-State Nuclear Magnetic Resonance and Molecular Dynamics Simulations.

Selective one-dimensional 13C-13C spin-diffusion solid-state nuclear magnetic resonance (SSNMR) provides evidence for CH/π ring packing interactions between Pro and Tyr residues in 13C-enriched Latrodectus hesperus dragline silk. The secondary structure of Pro-containing motifs in dragline spider silks consistently points to an elastin-like type II ß-turn conformation based on 13C chemical shift analysis. 13C-13C spin diffusion measurements as a function of mixing times allow for the measurement of spatial proximity between the Pro and Tyr rings to be ∼0.5-1 nm, supporting strong Pro-Tyr ring interactions that likely occur through a CH/π mechanism. These results are supported by molecular dynamics (MD) simulations and analysis and reveals new insights into the secondary structure and Pro-Tyr ring stacking interactions for one of nature's toughest biomaterials.

Design and fabrication of a magnetic nanobiocomposite based on flaxseed mucilage hydrogel and silk fibroin for biomedical and in-vitro hyperthermia applications.

In this research work, a magnetic nanobiocomposite is designed and presented based on the extraction of flaxseed mucilage hydrogel, silk fibroin (SF), and Fe3O4 magnetic nanoparticles (Fe3O4 MNPs). The physiochemical features of magnetic flaxseed mucilage hydrogel/SF nanobiocomposite are evaluated by FT-IR, EDX, FE-SEM, TEM, XRD, VSM, and TG technical analyses. In addition to chemical characterization, given its natural-based composition, the in-vitro cytotoxicity and hemolysis assays are studied and the results are considerable. Following the use of highest concentration of magnetic flaxseed mucilage hydrogel/SF nanobiocomposite (1.75 mg/mL) and the cell viability percentage of two different cell lines including normal HEK293T cells (95.73%, 96.19%) and breast cancer BT549 cells (87.32%, 86.9%) in 2 and 3 days, it can be inferred that this magnetic nanobiocomposite is biocompatible with HEK293T cells and can inhibit the growth of BT549 cell lines. Besides, observing less than 5% of hemolytic effect can confirm its hemocompatibility. Furthermore, the high specific absorption rate value (107.8 W/g) at 200 kHz is generated by a determined concentration of this nanobiocomposite (1 mg/mL). According to these biological assays, this magnetic responsive cytocompatible composite can be contemplated as a high-potent substrate for further biomedical applications like magnetic hyperthermia treatment and tissue engineering.

A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation.

The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na2 CO3 -degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.

Lignin, an active component in the corn silk water extract, inhibits glycation.

The formation of advanced glycation end products is associated with aging and diabetic complications such as neuropathy, retinopathy, and nephropathy. Thus, the suppression of AGEs formation could prevent and/or treat their related disorders. Corn silk is used as a traditional medicine for the prevention of diabetic complications and treatment of edema in Japan and China. Previous studies revealed the anti-glycation activity of flavonoids in the methanolic extract of corn silk. The anti-glycation activity of the corn silk water extract was higher than that of the methanolic extract; however, the active components of the water extract remained unidentified. The purpose of this study is to make clear the components showing anti-glycation activity in the corn silk water extract and elucidated their structural characteristics. The evaluation of anti-glycation activity was carried out by enzyme-linked immunosorbent assay to detect glycated bovine serum albumin. Remarkable anti-glycation activity was observed in the > 3 kDa fraction. Reversed-phase HPLC analysis of this fraction showed broad peaks characteristic of high-molecular-weight polyphenols. Decomposition reactions did not provide evidence of condensed or acid-hydrolyzable tannins. Therefore, polyphenols contained in the corn silk water extract were considered to be lignin-carbohydrate complex. The 1H- and 13C-nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectroscopy spectra of the > 3 kDa fraction were in agreement with the values reported for lignin. Consequently, we concluded that lignin-carbohydrate complex is one of the active components against glycation in the corn silk water extract.

Dual-functional bioactive silk sericin for osteoblast responses and osteomyelitis treatment.

Sericin, a natural protein from silk cocoon, has been reported for various biological properties in the biomaterials field. Modified forms of sericin have been studied for bone tissue engineering, while its unmodified form has been scarcely reported. Therefore, the purpose of this study was to evaluate physical and biological properties of unmodified sericin for potential use in bone surgery. Sericin was extracted from silk cocoons using a chemical-free boiling method. Sericin extract showed distinct bands with molecular weight ranging from 25 to 42 kDa including smear bands. Fourier transform infrared spectra presented characteristic peaks of amide I, II, and III, confirming the chemical composition of sericin. Based on biological activity, sericin extract at a concentration of 40 µg/mL increased the proliferation of osteoblast cells up to 135%, compared with the untreated control. Moreover, increase in antibacterial activity against Staphylococcus aureus, both clinical isolates and the reference strain ATCC 29213, was demonstrated for sericin extract with normal saline, while no antibacterial activity was observed for sericin with broth. It was found that sericin with normal saline showed higher zeta potential than sericin without normal saline, indicating higher system stability. This was confirmed by the average particle size of sericin extract with NaCl (3,249.3±226.1 nm) showing approximately 10 times smaller than sericin solution (29,015.9 ± 8,085.6 nm). Furthermore, sericin extract at the minimal inhibitory concentration significantly reduced the biofilm formation of S. aureus up to 95%. The study indicates biological activities of sericin, which could be applied as a dual-functional bioactive material to support bone regeneration and treat bone infections.

Sustainable Antibacterial and Anti-Inflammatory Silk Suture with Surface Modification of Combined-Therapy Drugs for Surgical Site Infection.

Silk sutures with antibacterial and anti-inflammatory functions were developed for sustained dual-drug delivery to prevent surgical site infections (SSIs). The silk sutures were prepared with core-shell structures braided from degummed silk filaments and then coated with a silk fibroin (SF) layer loaded with berberine (BB) and artemisinin (ART). Both the rapid release of drugs to prevent initial biofilm formation and the following sustained release to maintain effective concentrations for more than 42 days were demonstrated. In vitro assays using human fibroblasts (Hs 865.Sk) demonstrated cell proliferation on the materials, and hemolysis was 2.4 ± 0.8%, lower than that required by ISO 10993-4 standard. The sutures inhibited platelet adhesion and promoted collagen deposition and blood vessel formation. In vivo assessments using Sprague-Dawley (SD) rats indicated that the coating reduced the expression of pro-inflammatory cytokines interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α), shortening the inflammatory period and promoting angiogenesis. The results demonstrated that these new sutures exhibited stable structures, favorable biocompatibility, and sustainable antibacterial and anti-inflammatory functions with potential for surgical applications.

Enhancing the In Vitro Biological Activity of Degraded Silk Sericin and Its Analog Metabolites.

Two sericins of high and low molecular weight (HS and LS) were prepared from commercial silkworm cocoon silk with a boiling water and Ca(OH)2 solution with ultrasonic treatments, respectively. This study first investigated the release concentration of the two abovementioned sericins in simulated saliva, gastric juice, and intestinal juice (pH 6.8, 2.0, and 7.4, respectively) within 10 h. The results showed that the order of sericin release rate and its amount in the simulated environment was gastric juice > saliva > intestinal juice. Second, the molecular weights of both sericin metabolites formed by in vitro enzymatic degradation were lower than 15 kDa. The α-glucosidase inhibitory activities of both sericins and their analog metabolites were positively correlated with their concentrations. The IC50 values of the HS- and LS-derived metabolites were 1.02 ± 0.12 mg/mL and 0.91 ± 0.15 mg/mL, respectively, which were five to seven times lower than those of both original sericins. The total antioxidant capacities and hydroxyl radical scavenging capacities of both metabolites were enhanced by one- to three-fold compared with HS and LS. These results indicate that both sericins, regardless of molecular size, have significantly enhanced antioxidant, superoxide free radical scavenging, and glycosidase inhibitory activities after simulated metabolism, and that LS is better than HS regardless of simulated digestion. These results confirm that sericin is important in the sustainable development and utilization of silk resources, especially the reduction in environmental pollution, and provides new ideas for the development of adjuvant treatments for diabetes and the development of foods with anti-hyperglycemic functions.

Subambient daytime radiative cooling textile based on nanoprocessed silk.

Decreasing energy consumption is critical to sustainable development. Because temperature regulation for human comfort consumes vast amounts of energy, substantial research efforts are currently directed towards developing passive personal thermal management techniques that cool the human body without any energy consumption1-9. Although various cooling textile designs have been proposed previously, textile-based daytime radiative cooling to a temperature below ambient has not been realized6-13. Silk, a natural protein fabric produced by moth caterpillars, is famous for its shimmering appearance and its cooling and comforting sensation on skin14-17. It has been recently recognized that silk, with its optical properties derived from its hierarchical microstructure, may represent a promising starting point for exploring daytime radiative cooling18-21. However, the intrinsic absorption of protein in the ultraviolet region prevents natural silk from achieving net cooling under sunlight. Here we explore the nanoprocessing of silk through a molecular bonding design and scalable coupling reagent-assisted dip-coating method, and demonstrate that nanoprocessed silk can achieve subambient daytime radiative cooling. Under direct sunlight (peak solar irradiance >900 W m-2) we observed a temperature of ~3.5 °C below ambient (for an ambient temperature of ~35 °C) for stand-alone nanoprocessed silks. We also observed a temperature reduction of 8 °C for a simulated skin when coated with nanoprocessed silk, compared with natural silk. This subambient daytime radiative cooling of nanoprocessed silk was achieved without compromising its wearability and comfort. This strategy of tailoring natural fabrics through scalable nanoprocessing techniques opens up new pathways to realizing thermoregulatory materials and provides an innovative way to sustainable energy.

Supplementation with a Natural Source of Amino Acids, Sil-Q1 (Silk Peptide), Enhances Natural Killer Cell Activity: A Redesigned Clinical Trial with a Reduced Supplementation Dose and Minimized Seasonal Effects in a Larger Population.

The aim of this study was to re-validate the changes in natural killer (NK) cell cytotoxicity and cytokines related to T cells after Sil-Q1 (SQ; silk peptide) supplementation in a larger pool of Korean adults with minimized daily dose of SQ and controlling seasonal influence compared to the previous study. A total of 130 subjects were randomly assigned (1:1) to consume either 7.5 g of SQ or placebo for 8 weeks. NK cell cytotoxicity and cytokines were measured at T0 (baseline) and T8 (follow-up). Comparing the NK cell cytotoxicity values at T0 and T8 within each group, the cytotoxicity at all effector cell (E) to target cell (T) ratios of 10:1, 5:1, 2.5:1, and 1.25:1 was significantly increased in the SQ group at T8. Additionally, significant differences in the changed value (Δ, subtract baseline values from follow-up values) comparison between the groups at E:T = 10:1, 5:1, and 2.5:1 were found. As a secondary endpoint, the interleukin (IL)-12 level in the SQ group was significantly increased for 8 weeks, and Δ IL-12 in the SQ group was greater than in the placebo group. In conclusion, the present study showed considerable practical implications of SQ supplementation. Thus, SQ is an effective and safe functional food supplement for enhancing immune function.

Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties.

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR® VSL2 and PONDR® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of ß-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher ß-sheet content had better fracture strength and Young's modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.

Chondrogenic differentiation of Wharton's Jelly mesenchymal stem cells on silk spidroin-fibroin mix scaffold supplemented with L-ascorbic acid and platelet rich plasma.

In this research, hWJ-MSCs were grown on silk scaffolds and induced towards chondrogenesis by supplementation with L-ascorbic acid (LAA) or platelet rich plasma (PRP). Silk scaffolds were fabricated with salt leaching method by mixing silk fibroin (SF) with silk spidroin (SS). The silk fibroin was obtained from Bombyx mori cocoon that had been degummed, and the silk spidroin was obtained from wild-type spider Argiope appensa. The effect of scaffold composition and inducer on cell proliferation was observed through MTT assay. The most optimal treatment then continued to be used to induce hWJ-MSC towards chondrogenic differentiation for 7 and 21 days. Scaffolds characterization showed that the scaffolds produced had 3D structure with interconnected pores, and all were biocompatible with hWJ-MSCs. Scaffold with the addition of 10% SS + 90% SF showed higher compressive strength and better pore interconnectivity in comparison to 100% silk fibroin scaffold. After 48 h, cells seeded on scaffold with spidroin and fibroin mix had flattened morphology in comparison to silk fibroin scaffold which appeared to be more rounded on the scaffold surface. Scaffold with 10% (w/w) of silk spidroin (SS) + 90% (w/w) of silk fibroin (SF) was the most optimal composition for cell proliferation. Immunocytochemistry of integrin ß1 and RGD sequence, showed that scaffold with SS 10% provide better cell attachment with the presence of RGD sequence from the spidroin silk which could explain the higher cell proliferation than SF100% scaffold. Based on Alcian Blue staining and Collagen Type II immunocytochemistry (ICC), cells grown on 10% SS + 90% SF scaffold with 10% PRP supplementation were the most optimal to support chondrogenesis of hWJ-MSCs. These results showed that the addition of spidroin silk from A. appensa. had impact on scaffold compressive strength and chondrogenic differentiation of hWJ-MSC and had the potential for further development of bio-based material scaffold in cartilage tissue engineering.

Characterization, antioxidant activities, and inhibition on α-glucosidase activity of corn silk polysaccharides obtained by different extraction methods.

The polysaccharides (CSPw, CSPc, CSPa, and CSPu) were prepared by hot water extraction, acid-assisted extraction, alkaline-assisted extraction, and ultrasound-assisted extraction from corn silk, respectively. High performance gel permeation chromatography (HPGPC), fourier-transform infrared (FT-IR) spectroscopy, and scanning electron microscopy (SEM) results indicated that the extraction methods had an obvious impact on the molecular weight, structure, and morphology of the CSPs. Among the four polysaccharides, CSPu showed the highest inhibitory α-glucosidase activity, which might be related to its smaller molecular weight. Furthermore, kinetics analyses revealed that CSPu had significant inhibition of α-glucosidase in a non-reversible and competitive manner. Fluorescence quenching analysis illustrated that the interaction mechanism of CSPu and α-glucosidase was claimed as a static quenching mechanism. Isothermal titration calorimetry (ITC) analysis showed that the main driving forces for the interaction of CSPu with α-glucosidase was hydrogen bonding and the binding interactions of them occurred spontaneously.

Structural Characterization of Black Widow Spider Dragline Silk Proteins CRP1 and CRP4.

Spider dragline silk represents a biomaterial with outstanding mechanical properties, possessing high-tensile strength and toughness. In black widows at least eight different proteins have been identified as constituents of dragline silk. These represent major ampullate spidroins MaSp1, MaSp2, MaSp', and several low-molecular weight cysteine-rich protein (CRP) family members, including CRP1, CRP2, and CRP4. Molecular modeling predicts that CRPs contain a cystine slipknot motif, but experimental evidence to support this assertion remains to be reported. To advance scientific knowledge regarding CRP function, we recombinantly expressed and purified CRP1 and CRP4 from bacteria and investigated their secondary structure using circular dichroism (CD) under different chemical and physical conditions. We demonstrate by far-UV CD spectroscopy that these proteins contain similar secondary structure, having substantial amounts of random coil conformation, followed by lower levels of beta sheet, alpha helical and beta turn structures. CRPs are thermally and pH stable; however, treatment with reagents that disrupt disulfide bonds impact their structural conformations. Cross-linking mass spectrometry (XL-MS) data also support computational models of CRP1. Taken together, the chemical and thermal stability of CRPs, the cross-linking data, coupled with the structural sensitivity to reducing agents, are experimentally consistent with the supposition CRPs are cystine slipknot proteins.

Potential of antimicrobial topical gel with synthesized biogenic silver nanoparticle using Rhodomyrtus tomentosa leaf extract and silk sericin.

OBJECTIVE: Silver nanoparticles synthesized using Rhodomyrtus tomentosa leaf extract and silk sericin were used to functionalize carbopol 940 gel for topical applications. RESULTS: UV-vis spectra presented surface plasmon resonance at 426 nm, transmission electron microscopy revealed that nanoparticles were spherical with an average size of 25-50 nm. X-ray diffraction presented crystalline silver nanoparticles with zeta potential of ≈ - 30 mV. FTIR spectra showed a reduction of silver nitrate indicated by the shift in -OH at 2958 cm-1. The silver nanoparticle demonstrated broad-spectrum antimicrobial activity against Gram-positive, Gram-negative and fungi with MIC ranging between 0.26 and 2.10 µg mL-1, respectively. MIC of hydrogel ranged between 1.05-2.10 µg mL-1 with cell viability of 89%. Spreadability and extrudability of gel were 9.3 ± 0.85 s and 19.85 ± 0.03%, respectively with first order of fickian diffusion. CONCLUSIONS: The silver nanoparticle gel exhibited an effective antimicrobial property, hence can be exploited for topical applications.

Conductive and enzyme-like silk fibers for soft sensing application.

A combination of supercritical carbon dioxide (scCO2) impregnation of pyrrole and sonochemical transformation of permanganate (KMnO4) was used to impart conductive and catalytic properties to silk fibers. The results indicated that the conductivity (from polypyrrole -PPy) and catalytic activities (from manganese dioxide -MnO2) were independent and complementary within the processing parameters used. The enhanced conductivity was attributed to scCO2 preferentially distributing the pyrrole monomers along with the silk internal fibrillar structure and hence, yielding a more linear PPy. The oxidative properties of the PPy-MnO2-silk hybrid showed an enzyme-like behavior for the degradation of hydrogen peroxide (H2O2) with a Km of about 13 mM and specific activity of 1470 ±â€¯75 µmol/min/g. Finally, we demonstrated that the PPy-MnO2-silk hybrid could be used as soft working electrodes for the simultaneous degradation and detection of H2O2.

Corn silk extract inhibit the formation of Nε-carboxymethyllysine by scavenging glyoxal/methyl glyoxal in a casein glucose-fatty acid model system.

Herein, corn silk extract and its flavonoids were used to inhibit the formation of Nε-carboxymethyllysine (CML) in a casein glucose-fatty acid model system. Under these optimum extraction conditions, nine major flavonoids were identified and quantified by HPLC-MS/MS. The percent inhibition of CML formation by corn silk extract was 76.57%. The inhibitory mechanism of corn silk extract toward CML formation was further investigated by examining the trapping of glyoxal/methyl glyoxal by the major flavonoids (5 mM) using HPLC-ESI-MS, and mono-, di-, and tri-adducts were found for some flavonoid compounds. The antioxidant activity of the corn silk extract was evaluated by the DPPH and ABTS assays. The scavenging activity of the corn silk extract for DPPH and ABTS was 84.38% and 89.11%, respectively. The results suggested that corn silk extract inhibited CML formation through glyoxal/methyl glyoxal scavenging or by its antioxidant activity attributed to its flavonoid content.

Temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome.

Interstitial cystitis (IC), also known as painful bladder syndrome, is a debilitating chronic condition with many patients failing to respond to current treatment options. Rapid clearance, mucosal coating, and tight epithelium create strong natural barriers that reduce the effectiveness of many pharmacological interventions in the bladder. Intravesical drug delivery (IDD) is the administration of therapeutic compounds or devices to the urinary bladder via a urethral catheter. Previous work in improving IDD for IC has focused on the sustained delivery of analgesics within the bladder and other small molecule drugs which do not address underlying inflammation and bladder damage. Therapeutic glycosaminoglycans (GAG) function by restoring the mucosal barrier within the bladder, promoting healing responses, and preventing irritating solutes from reaching the bladder wall. There is an unmet medical need for a therapy that provides both acute relief of symptoms while alleviating underlying physiological sources of inflammation and promoting healing within the urothelium. Semi-synthetic glycosaminoglycan ethers (SAGE) are an emerging class of therapeutic GAG with intrinsic anti-inflammatory and analgesic properties. To reduce SAGE clearance and enhance its accumulation in the bladder, we developed a silk-elastinlike protein polymer (SELP) based system to enhance SAGE IDD. We evaluated in vitro release kinetics, rheological properties, impact on bladder function, pain response, and bladder inflammation and compared their effectiveness to other temperature-responsive polymers including Poloxamer 407 and poly(lactic-co-glycolic acid)-poly(ethylene glycol). SAGE delivered via SELP-enhanced intravesical delivery substantially improved SAGE accumulation in the urothelium, provided a sustained analgesic effect 24 h after administration, and reduced inflammation.

Silk peptide treatment potentiates natural killer cell activity in vitro and induces natural killer cell maturation and activation in mouse splenocytes.

Context: Silk peptide from cocoons of silkworm (Bombyx mori L., Bombycidae) has been employed as a biomedical material and exhibits various bioactivities, including immune-modulating activity. Objective: We analyzed whether silk peptide exerts direct modulating effects on NK cells using an NK cell line in vitro and ex vivo splenocytes. We also attempted to delineate the mechanism underlying the modulation. Material and methods: In vitro activity of silk peptide on NK cells was determined by measurement of cytolytic activity against K562 cells at an effector-to-target ratio of 5:1 after incubation of NK-92MI cells with silk peptide (0-2000 µg/mL) for 48 and 72 h. Ex vivo activity of silk peptide on mouse splenic NK cells was determined similarly by using YAC-1 cells. Results: Treatment of NK-92MI NK cells with silk peptide (500-2000 µg/mL) significantly increased cytolytic activity on target cells by 2- to 4-fold. The same concentrations (500-2000 µg/mL) of silk peptide treatment also significantly enhanced the cytolytic activity of splenic NK cells against YAC-1 cells. Silk peptide treatment of IL-2-stimulated splenocytes induced enhanced expression of Th1, 2 and 17 cytokines including TNF-α, IFN-γ, IL-6, IL-4 and IL-17. Finally, ex vivo treatment with silk peptide on mouse splenocytes significantly enhanced the degree of NK cell maturation in a dose-dependent manner from 3.49 to 23.79%. Discussion and conclusions: These findings suggest that silk peptide stimulates NK cells, thereby influencing systemic immune functions and improving natural immunity. Thus, silk peptide could be useful as a complementary therapy in cancer patients.