Preparation and Properties of Egg White Dual Cross-Linked Hydrogel with Potential Application for Bone Tissue Engineering.

In this study, an egg white dual cross-linked hydrogel was developed based on the principle that the external stimulus can denature proteins and cause them to aggregate, forming hydrogel. The sodium hydroxide was used to induce gelation of the egg white protein, subsequently introducing calcium ions to cross-link with protein chains, thereby producing a dual cross-linked hydrogel. The characteristics of the dual cross-linked hydrogels-including the secondary structure, stability, microstructure, swelling performance, texture properties, and biosafety-were investigated to determine the effects of calcium ion on the egg white hydrogel (EWG) and evaluate the potential application in the field of tissue engineering. Results showed that calcium ions could change the ß-sheet content of the protein in EWG after soaking it in different concentrations of CaCl2 solution, leading to changes in the hydrogen bonds and the secondary structure of polypeptide chains. It was confirmed that calcium ions promoted the secondary cross-linking of the protein chain, which facilitated polypeptide folding and aggregation, resulting in enhanced stability of the egg white dual cross-linked hydrogel. Furthermore, the swelling capacity of the EWG decreased with increasing concentration of calcium ions, and the texture properties including hardness, cohesiveness and springiness of the hydrogels were improved. In addition, the calcium cross-linked EWG hydrogels exhibited biocompatibility and cell-surface adhesion in vitro. Hence, this work develops a versatile strategy to fabricate dual cross-linked protein hydrogel with biosafety and cell-surface adhesion, and both the strategy and calcium-egg white cross-linked hydrogels have potential for use in bone tissue engineering.

Fabrication of tumor-targeting composites based on the triple helical ß-glucan through conjugation of aptamer.

Herein the nucleic acid aptamers were attached to the polydeoxyadenylic acid (poly(dA)) tail for improving the tumor-targetability and cellular internalization of s-LNT/poly(dA) composite composed of two single chains of triple helical ß-glucan lentinan (s-LNT) and one poly(dA) chain. The in vitro results demonstrate that the cellular uptake of s-LNT/poly(dA) composites in MCF-7 cancer cells was enhanced effectively after attaching the aptamer. The as-prepared fluorescin isothiocyanate (FITC)-labelled LNT (LNT-FITC) through grafting was used for tracing the enhanced tumor-targetability of the composites. As a result, the cellular internalization of the LNT-FITC into MCF-7 and 4T1 cancer cells was further increased by the aptamer conjugated to poly(dA). Meanwhile, the in vivo experiments further demonstrate more s-LNT/poly(dA)-aptamer composites were effectively accumulated at the tumor site compared with s-LNT alone. This work provides a novel strategy for fabricating triplex ß-glucan as delivery vectors with active tumor-targetability.

A Novel Strategy for Treating Inflammatory Bowel Disease by Targeting Delivery of Methotrexate through Glucan Particles.

Therapy of inflammatory bowel disease (IBD) has been a difficult task in the medical field. There is a great clinical need for more effective treatments for IBD. Herein, a targeted oral delivery system of yeast glucan particles (YGPs) carrying a clinically used anti-inflammatory drug methotrexate (MTX) to the inflamed sites in IBD mice for therapy is reported. In the findings, MTX is effectively loaded into YGPs through re-precipitation followed by gelation reaction of alginate to obtain the composite YGPs/MTX, which are internalized into RAW264.7 macrophage cells through dectin-1 and CR3 receptors. Furthermore, YGPs/MTX can suppress the proliferation of macrophage cells efficiently, leading to down-regulation of pro-inflammatory cytokines induced by lipopolysaccharides. Additionally, YGPs accumulate in the inflammation site of colitis mice, enabling YGPs/MTX to target the inflammatory site, significantly improve the efficacy of MTX, and reduce the cytotoxicity of MTX. Therefore, the YGPs-based drug delivery system provides a new strategy for MTX application in the clinical treatment of IBD.

Inhibition of tumor growth by ß-glucans through promoting CD4+ T cell immunomodulation and neutrophil-killing in mice.

ß-glucans are polysaccharides comprising ß-D-glucoses with various bioactivities. Herein, we extracted three ß-glucans from Lentinus edodes with different sources and assessed their antitumor activities on a mice model with intragastric, intraperitoneal and intratumoral injection. Three polysaccharides were shown to have the same chemical structure of ß-(1,3)-glucan with ß-(1,6) branches, and exhibited S-180 tumor-suppressing ability with good safety. It was found that ß-glucans up-regulated CD4+ T cell level in lymphoid organs decreased by tumor-burden, indicating promotion of immunomodulation. ß-glucans targeted tumors in vivo even after oral or intraperitoneal injection. Furthermore, ß-glucans not only targeted to lymphoid organs and increased CD4+ T cells number, but also enhanced CD4+ T cells and neutrophils populations in tumors. It was proposed that ß-glucans promoted CD4+ T cell immunomodulation and neutrophils infiltration into tumors, leading to tumor growth inhibition. These findings reveal that ß-glucans can be used as an effective agent for cancer immunotherapy.

Nanoplatform Constructed from a ß-Glucan and Polydeoxyadenylic Acid for Cancer Chemotherapy and Imaging.

A nanoplatform carrying doxorubicin (Dox) for cancer therapy and a dye for imaging was developed based on a natural triple helix ß-glucan (t-LNT) and polydeoxyadenylic acid (poly(dA)). The t-LNT-Dox conjugates were prepared through Schiff-base reaction between the aldehyde group in the oxidized t-LNT and the amino group of Dox, the single chains (s-LNT-Dox) of which interacted with the poly(dA)-dye to form a composite s-LNT-Dox/poly(dA)-dye through hydrogen bonding between s-LNT and poly(dA). t-LNT-Dox was confirmed to acid-responsively release Dox in vitro, showing enhanced cytotoxicity against HeLa cancer cells with time. It was confirmed that Dox and the dye could be simultaneously delivered into HeLa cells or the tumors with a prolonged duration time. Furthermore, LNT-Dox conjugates effectively inhibited tumor growth and decreased adverse effects of the free Dox in vivo. Hence, this work develops a new strategy to fabricate the nanoplatform for therapy and imaging using a natural polysaccharide.

Orally Delivered Antisense Oligodeoxyribonucleotides of TNF-α via Polysaccharide-Based Nanocomposites Targeting Intestinal Inflammation.

Tumor necrosis factor alpha (TNF-α) is usually regarded as a potential target for inflammatory bowel disease therapy. Herein, a promising strategy for effective delivery of phosphorothioated antisense oligodeoxyribonucleotide of TNF-α (PS-ATNF-α), targeting the intestinal inflammation based on the interaction of the single chain of triple helical ß-glucan (s-LNT) with poly-deoxyadenylic acid [poly(dA)], and the colon-specific degradation of chitosan-alginate (CA) hydrogel, is reported. The target gene of PS-ATNF-α, with a poly(dA) tail through a disulfide bond (-SS-), interacts with s-LNT to form a rod-like nanocomposite of s-LNT/poly(dA)-SS-PS-ATNF-α, which significantly inhibits lipopolysaccharide (LPS)-induced TNF-α at the protein level by 38.2% and mRNA level by 48.9% in RAW264.7 macrophages. The nanocomposites carried by the CA hydrogel with the loading amount of 83.5% are then orally administered and specifically released to the inflamed intestine, followed by internalization into intestinal cells such as macrophages, to reduce TNF-α production by 36.4% and dextran sulfate sodium-induced inflammation by decreasing myeloperoxidase and malondialdehyde. This study defines a new strategy for the oral delivery of antisense oligonucleotides to attenuate inflammatory response, demonstrating a notable potential for clinical applications in intestine-inflammation-targeted therapy.

Yeast ß-Glucan Suppresses the Chronic Inflammation and Improves the Microenvironment in Adipose Tissues of ob/ob Mice.

Inflammation in visceral adipose tissues (VATs) contributes to the pathology of diabetes. This study focused on the inflammatory regulation in VATs by a yeast ß-1,3-glucan (BYG) orally administered to ob/ob mice. BYG decreased pro-inflammatory modulators of TNF-α, IL-6, IL-1ß, CCL2, and SAA3, and increased anti-inflammatory factors of Azgp1 (2.53 ± 0.02-fold change) at protein and/or mRNA levels (p < 0.05). Remarkably, BYG decreased the degree of adipose tissue macrophages (ATMs) infiltration to 82.5 ± 8.3%, especially the newly recruited ATMs. Interestingly, BYG increased the protective Th2 cell regulator GATA3 (7.72 ± 0.04-fold change) and decreased immunosuppressors IL-10 and IL-1ra, suggesting that BYG elicited inflammation inhibition via stimulating immune responses. Additionally, BYG increased the gut microbiota proportion of Akkermansia from 0.07% to 4.85% and improved the microenvironment of VATs through decreasing fibrosis and angiogenesis. These findings suggest that BYG has anti-inflammatory effect in diabetic mice, which can be used as a food component and/or therapeutic agent for diabetes.

Non-covalent interaction between CA-TAT and calf thymus DNA: Deciphering the binding mode by in vitro studies.

CA-TAT, a novel peptide analog, was modified at the N-terminus of TAT (47-57), the cell-penetrating peptide transacting activator of transcription, by attaching cecropin A (1-7). CA-TAT, TAT (47-57), and cecropin A (1-7) were synthesized using standard Fmoc solid-phase peptide synthesis procedures, purified using reversed-phase high performance liquid chromatography (RP-HPLC), and characterized using ESI-MS. CA-TAT demonstrated antibacterial activities against bacteria with low hemolysis (MHC > 128 µM). The minimum inhibitory concentration (MIC) values of CA-TAT were in the range of 1-16 µM, which completely inhibited both gram-positive and gram-negative bacteria. The interactions between CA-TAT or TAT (47-57) and calf thymus DNA (ct-DNA) were investigated using multi-spectroscopic techniques and viscometry. The results showed that both CA-TAT and TAT (47-57) can interact with DNA via the minor groove-binding mode, and binding constant was calculated to be 2.83 × 105 L mol-1 at 310 K, which is lower than that with the classical intercalation binder ethidium bromide (EB). Compared with TAT (47-57) or cecropin A (1-7), CA-TAT combined with DNA much closer. The study results suggest that CA-TAT can be used as a novel antibacterial peptide in the development of new antibiotics because of its antibacterial activity that targets intracellular DNA.

Progress in rigid polysaccharide-based nanocomposites with therapeutic functions.

Polysaccharides are naturally occurring biological macromolecules that are envisaged as promising substitutes of non-degradable polymers due to their outstanding inherent properties of biodegradability, biocompatibility, low-cost, and availability. Their utilization in the development of nanostructured hybrid materials has numerous advantages in theranostics, the integrated approach of therapeutics and diagnostics. In particular, some rigid polysaccharides occur in nature, which can self-assemble into ordered hierarchical structures, facilitating the controllable fabrication of various nanocomposites. These rigid polysaccharides, including Lentinan, Curdlan, Schizophyllan, Scleroglucan, Auricularian, and yeast glucan, possess the linear ß-(1→3)-d-glucan as the backbone with or without ß-(1→6)-linked glucose as the branch, having diverse biological activities. This review focuses on the incorporation of nanoparticles, such as gold (AuNPs), silver (AgNPs), selenium (SeNPs), silica, carbon nanotubes, homo-polynucleotides, bio-imaging probes, and drugs, into different rigid polysaccharide matrices through self-assembly and summarizes their biological functions as well as the correlation to their conformations. Additionally, we addressed the use of the as-engineered polysaccharide nanocomposites as effective therapeutic agents, and the challenges or ambiguity issues concerning further practical clinic therapeutic applications.