Collagen/Curdlan composite sponge for rapid hemostasis and skin wound healing.

Collagen's unique properties promise hemostatic potential, but its sponge form's stability and mechanics need improvement. In this study, we developed a series of homeostatic sponges by co-assembling collagen and curdlan at different ratios into hydrogels, followed by freeze-drying treatment. The incorporation of curdlan into collagen sponges has been found to significantly enhance the sponge's properties, including increased porosity, elevated water uptake, improved elasticity, and enhanced resistance to degradation. In vitro cytotoxicity and hemolysis assays have demonstrated the biocompatibility and nontoxicity of composite sponges. In mouse liver perforation and incision models, the composite sponges achieved rapid coagulation within 67 s and 75 s, respectively, outperforming gauze and gelatin sponge in reducing blood loss. Furthermore, composite sponges demonstrated superior wound healing potential in mice full-thickness skin defects model, with accelerated healing rates observed at days 3, 7, and 14 compared to the control group. Overall, collagen/curdlan composite sponge show promise for hemostasis and wound healing applications.

Self-Assembling Gelatin-Curdlan Fibril Hydrogels for Oriented Neural Cell Growth.

The ex vivo replication of the highly helical and fibril structures of load-bearing soft tissue is a challenging goal for the study of hydrogels. Inspired by nature, we prepared tissue-like physical gels based on curdlan and gelatin by self-assembly. The hybrid gels have a flexible fibril-matrix architecture, and the fibril orientation is highly tunable. The tensile strength of the gels can be tuned from ∼1.1 to ∼16.5 MPa. The coil-helix transition and nanofibril formation process in the self-assembly system was thoroughly investigated. These helical gels exhibit excellent cell compatibility, which supports adhesion and oriented growth of neural cells. Furthermore, the oriented nanofibrils in the gel are found to be associated with an upregulated expression of regeneration-related genes like N-cadherin (Cdh2) and neural growth factor (NGF). Owing to the strength and biomimetic structure, these gels have great potential in tissue engineering applications.

Preparation and Characterization of Gluten/SDS/Chitosan Composite Hydrogel Based on Hydrophobic and Electrostatic Interactions.

Gluten is a natural byproduct derived from wheat starch, possessing ideal biocompatibility. However, its poor mechanical properties and heterogeneous structure are not suitable for cell adhesion in biomedical applications. To resolve the issues, we prepare novel gluten (G)/sodium lauryl sulfate (SDS)/chitosan (CS) composite hydrogels by electrostatic and hydrophobic interactions. Specifically, gluten is modified by SDS to give it a negatively charged surface, and then it conjugates with positively charged chitosan to form the hydrogel. In addition, the composite formative process, surface morphology, secondary network structure, rheological property, thermal stability, and cytotoxicity are investigated. Moreover, this work demonstrates that the change can occur in surface hydrophobicity caused by the pH-eading influence of hydrogen bonds and polypeptide chains. Meanwhile, the reversible non-covalent bonding in the networks is beneficial to improving the stability of the hydrogels, which shows a prominent prospect in biomedical engineering.

Extraction and Characterization of Pepsin- and Acid-Soluble Collagen from the Swim Bladders of Megalonibea fusca.

There is a growing demand for the identification of alternative sources of collagen not derived from land-dwelling animals. The present study explored the use of pepsin- and acid-based extraction protocols to isolate collagen from the swim bladders of Megalonibea fusca. After extraction, these acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) samples respectively were subjected to spectral analyses and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization, revealing both to be comprised of type I collagen with a triple-helical structure. The imino acid content of these ASC and PSC samples was 195 and 199 residues per 1000 residues, respectively. Scanning electron microscopy demonstrated that samples of freeze-dried collagen exhibited a compact lamellar structure, while transmission electron microscopy and atomic force microscopy confirmed the ability of these collagens to undergo self-assembly into fibers. ASC samples exhibited a larger fiber diameter than the PSC samples. The solubility of both ASC and PSC was highest under acidic pH conditions. Neither ASC nor PSC caused any cytotoxicity when tested in vitro, which met one of the requirements for the biological evaluation of medical devices. Thus, collagen isolated from the swim bladders of Megalonibea fusca holds great promise as a potential alternative to mammalian collagen.

Cell membrane-camouflaged DOX-loaded ß-glucan nanoparticles for highly efficient cancer immunochemotherapy.

Biomimetics plays an important role in cancer treatment since it can prolong the circulation of nanoparticles, enhance their delivery and retention in target tissues, and reduce the systemic toxicity of drugs and their carriers. In this study, we developed a biomimetic nanosystem consisting of chemotherapeutic and immunotherapeutic agents wrapped in cell membranes. Specifically, the anti-tumor drug doxorubicin (DOX) was loaded into a bacterial-derived immunomodulatory agent (low molecular weight curdlan, lCUR), and the lCUR-DOX was further wrapped in the red blood cell membrane for camouflage and prolonged circulation. The successful preparation of the lCUR-DOX@RBC nanosystem was supported by various optical and morphological characterizations. In vitro studies indicated that the nanosystem can escape uptake by macrophages, inhibit the invasion of tumor cells, and reprogram M2 macrophages with an immunosuppressive phenotype into M1 macrophages with an immunopromoting phenotype via the MAPK signaling pathway while promoting the phagocytosis of macrophages. In vivo studies showed that the nanosystem effectively inhibits tumor growth in the A-375 tumor-bearing mouse model. Taken together, the above results support further development of the lCUR-DOX@RBC platform for cancer immunochemotherapy in clinical applications.

Single-helical formyl ß-glucan effectively deliver CpG DNA with poly(dA) to macrophages for enhanced vaccine effects.

Single helical ß-glucan is a one-dimensional host that can form a hybrid helix with DNAs/RNAs as delivery systems. However, unmodified ß-glucan has a gelling tendency and a single helical conformation is challenging to obtain. Therefore, in this study, we developed a ß-glucan formyl derivative with stable single helical conformation and no gelling tendency. Circular dichroism studies found that the formyl-ß-glucan could form a hybrid helix with DNA CpG-poly(dA). The hybrid helix delivery system showed improved activation on antigen-presenting cells, thereby upregulating the mRNA and protein levels of inflammatory factors, and had an immune-enhancing effect on ovalbumin (OVA) immunized mice. These results indicate that formyl-ß-glucan can be developed as a non-cationic supramolecular DNA delivery platform with low toxicity and high efficiency.

Macrophage-Targeted Berberine-Loaded ß-Glucan Nanoparticles Enhance the Treatment of Ulcerative Colitis.

Aim: This study focuses on constructing of an anti-inflammatory drug delivery system by encapsulation of berberine in the ß-glucan nanoparticles and evaluates its effect on treating ulcerative colitis. Methods: ß-Glucan and the anti-inflammatory drug berberine (BER) are self-assembled into nanoparticles to construct a drug delivery system (GLC/BER). The interaction between the drug and the carrier was characterized by circular dichroism, ultraviolet-visible spectroscopy, and dynamic light scattering. The anti-inflammatory effect of the GLC/BER was evaluated through a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model and a sodium sulfate (DSS)-induced C57BL/6 mouse ulcerative colitis model. Results: The GLC/BER nanoparticles have a particle size of 80-120 nm and a high encapsulation efficiency of 37.8±4.21%. In the LPS-induced RAW264.7 macrophage inflammation model, GLC/BER significantly promoted the uptake of BER by RAW264.7 cells. RT-PCR and ELISA assay showed that it could significantly inhibit the inflammatory factors including IL-1ß, IL-6 and COX-2. Furthermore, GLC/BER shows inhibiting effect on the secretion of pro-inflammatory factors such as IL-1ß and IL-6, down-regulating the production of nitrite oxide; in animal studies, GLC/BER was found to exert a relieving effect on mice colitis. Conclusion: The study found that GLC/BER has an anti-inflammatory effect in vitro and in vivo, and the GLC carrier improves the potency and bioavailability of BER, providing a new type of nanomedicine for the treatment of colitis.

Laminarin acetyl esters: Synthesis, conformational analysis and anti-viral effects.

Chemical modification of polysaccharides is important for expanding their applications and gaining new insights into their structure-property relationships. Here we reported the synthesis, characterization, and anti-viral activities of laminarin acetyl derivatives. The chemical structure and chain conformation of acetylated laminarin were characterized by FT-IR, H1 NMR, AFM, UV-vis spectrum, and induced circular dichroism based on a modified Congo Red assay (ICD-CR assay). The inhibition effect of laminarin and its acetyl derivatives on HSV-1 was evaluated by viral plaque assay and virus-associated DNA/protein change. Acetylation modification was found to trigger the conformation transition of laminarin from triple helix to single helix, and the extent of transition can be tuned by the degree of substitution. The single helical acetylated laminarins were found to be stable in neutral aqueous solution and exhibited no cytotoxicity. However, the acetylated laminarin exhibited declined antiviral activity after modification.

Antifracture, Antibacterial, and Anti-inflammatory Hydrogels Consisting of Silver-Embedded Curdlan Nanofibrils.

The bacterial exopolysaccharide Curdlan has a unique collagen-like triple helical structure and immune-modulation activities. Although there have been several types of Curdlan gels reported for antibacterial or wound healing purposes, none of them exhibit favorable mechanical properties for clinically applicable wound healing materials. Herein, we present a two-step approach for preparing Ag-embedded Curdlan hydrogels that are highly soft but are very stretchable compared with common polysaccharide-based hydrogels. Ag ions were first reduced in a diluted Curdlan solution to form AgNP-decorated triple helices. Then, the aqueous solution consisting of Curdlan/Ag nanoparticles was mixed with a dimethyl sulfoxide solution consisting of a high concentration of Curdlan. This mixing triggered the conformation transformation of Curdlan random coils into triple helices, and then the helices were further packed into semicrystalline nanofibrils of ∼20 nm in diameter. Due to the presence of semicrystalline fibrils, this novel Curdlan hydrogel exhibits a fracture strain of ∼350% and fracture stress of ∼0.2 MPa at a water content of ∼97%. This nanofibril hydrogel supported the attachment, spreading, and growth of fibroblasts and effectively inhibited the growth of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Moreover, the hydrogels downregulated NO production and proinflammatory gene expression levels in lipopolysaccharide (LPS)-stimulated macrophages but did not change the anti-inflammatory gene expression levels in IL-4-stimulated macrophages. In an animal study, these hydrogels accelerated wound healing in a bacteria-infected mice skin wound model. These results validate the further development of Curdlan/AgNPs nanofibril hydrogels in clinical wound management.

Proangiogenic Peptide Nanofiber Hydrogels for Wound Healing.

Rapid vascularization is vital for dermal regeneration, nutrient and nutrition transfer, metabolic waste removal, and prevention of infection. This study reports on a series of proangiogenic peptides designed to undergo self-assembly and promote angiogenesis and hence skin regeneration. The proangiogenic peptides comprised an angiogenic peptide segment, GEETEVTVEGLEPG, and a ß-sheet structural peptide sequence. These peptides dissolved easily in ultrapure water and rapidly self-assembled into hydrogels in a pH-dependent manner, creating three-dimensional fibril network structures and nanofibers as revealed by a scanning microscope and a transmission electron microscope. In vitro experiments showed that the peptide hydrogels favored adhesion and proliferation of mouse fibroblasts (L929) and human umbilical vein endothelial cells (HUVECs). In particular, many connected tubes were formed in the HUVECs after 8 h of culture on the peptide hydrogels. In vivo experiments demonstrated that new blood vessels grew into the proangiogenic peptide hydrogels within 2 weeks after subcutaneous implantation in mice. Moreover, the proangiogenic-combined hydrogels exhibited faster repair cycles and better healing of skin defects. Collectively, the results indicate that the proangiogenic peptide hydrogels are a promising therapeutic option for skin regeneration.

Chiral Active ß-Glucan Nanoparticles for Synergistic Delivery of Doxorubicin and Immune Potentiation.

BACKGROUND: ß-glucans are chiral polysaccharides with well-defined immunological properties and supramolecular wrapping ability of its chiral feature. However, the exploitation of chiral properties of these nanoparticles in drug delivery systems was seldom conducted. METHODS: ß-glucan molecules with different chain lengths were extracted from yeast Saccharomyces cerevisiae and thereafter modified. In a conformation transition process, these ß-glucan molecules were then self-assembled with anti-cancer drug doxorubicin into nanoparticles to construct drug delivery systems. The chiral interactions between the drug and carriers were revealed by circular dichroism spectra, ultraviolet and visible spectrum, fourier transform infrared spectroscopy, dynamic light scattering and transmission electron microscope. The immune-potentiation properties of modified ß-glucan nanoparticles were evaluated by analysis of the mRNA expression in RAW264.7 cell model. Further, the antitumor efficacy of the nanoparticles against the human breast cancer were studied in MCF-7 cell model by cellular uptake and cytotoxicity experiments. RESULTS: ß-glucan nanoparticles can activate macrophages to produce immune enhancing cytokines (IL-1ß, IL-6, TNF-α, IFN-γ). A special chirality of the carriers in diameter of 50~160 nm can also associate with higher drug loading ability of 13.9% ~38.2% and pH-sensitive release with a change of pH from 7.4 to 5.0. Cellular uptake and cytotoxicity experiments also prove that the chiral-active ß-glucan nanoparticles can be used in anti-cancer nanomedicine. CONCLUSION: This work demonstrates that ß-glucans nanoparticles with special chiral feature which leading to strong immunopotentiation ability and high drug loading efficiency can be developed as a novel type of nanomedicine for anti-cancer treatment.

Berberine-Loaded Nanostructured Lipid Carriers Enhance the Treatment of Ulcerative Colitis.

PURPOSE: Berberine (BBR), a major ingredient extracted from Coptis chinensis, is a natural drug with limited oral bioavailability. We developed nanostructured lipid carriers (NLCs) as a delivery system for enhanced anti-inflammatory activity of BBR against ulcerative colitis (UC). METHODS: BBR-loaded nanostructured lipid carriers (BBR-NLCs) prepared via high-pressure homogenization were evaluated for particle size, zeta potential, drug entrapment efficiency, drug loading, drug release, toxicity, and cellular uptake. The anti-UC activities of free and encapsulated BBR were evaluated in a DSS-induced acute model of UC in mice. RESULTS: Spherical BBR-NLCs were prepared with a particle size of 63.96± 0.31 nm, a zeta potential of +3.16 ± 0.05 mV, an entrapment efficiency of 101.97±6.34%, and a drug loading of 6.00±0.09%. BBR-NLCs showed excellent biocompatibility in vivo. Cellular uptake experiments showed that BBR-NLCs improved uptake of BBR by RAW 264.7 cells and Caco-2 cells. Oral administration of BBR-NLCs significantly alleviated colitis symptoms (DAI, colon length, spleen swelling, MPO activity) through inhibition of NF-κB nuclear translocation, decreased expression of pro-inflammatory cytokines (IL-1ß, IL-6, MMP-9, CX3CR1, COX-2, TERT), and increased expression of the tight junction protein ZO-1. CONCLUSION: BBR-loaded NLCs improved colitis symptoms, which suggested that this may be a novel formulation for treatment of UC.

Preparation and biological evaluation of a novel agarose-grafting-hyaluronan scaffold for accelerated wound regeneration.

At present, seeking an effective dressing for wound regeneration has drawn considerable interest. In this paper, a novel agarose-grafting-hyaluronan (Ag-g-HA) scaffold was synthesized for rapid wound healing. Elemental analysis results showed that the HA grafting rate of Ag-g-HA was ∼69%. Ag-g-HA remained bioactive to accelerate cell proliferation and stimulate secretion of TNF-α for macrophagocyte RAW 264.7, and collagen I and collagen III for fibroblast 3T3. An i n vivo study demonstrated that Ag-g-HA showed a faster repair cycle and a better skin histological structure for a full-thickness skin defect. The collagen I, collagen III and TNF-α secreted by mice for Ag-g-HA were similiar to HA. Ag-g-HA showed a similiar biological activity to HA but had a longer degradation time through its improved insolubility. These findings demonstrate that the Ag-g-HA scaffold accelerated wound healing, and could be a promising novel scaffold for tissue engineering and regenerative medicine.

Dynamic and Hierarchically Structured Networks with Tissue-like Mechanical Behavior.

Collagen is the most abundant structural protein in soft tissues, and the duplication of its structure and mechanics represents a key challenge to nanotechnology. Here we report a fibrous supramolecular network that can mimic nearly all of the aspects of collagen from dynamic hierarchical architecture to nonlinear mechanical behavior. This complex self-assembly system is solely based on a glucose polymer: curdlan, which is synthesized by bacteria and can form a similar triple helix as collagen. Triggered by solvent and temperature cues, free curdlan chains wind into superhelical trimers, and the trimers then bundle hexagonally into nanofibers of 20-40 nm in diameter. The fibers are interconnected in a water-rich 3D network structure. The network is highly dynamic and stress-responsive, which can shift from isotropic to anisotropic organization by the winding/unwinding of stress-induced interfiber triple helical net-points. Mechanical tests show that these nanofiber networks exhibit similar nonlinear elasticity as collagenous tissues including skin and tendon. The supramolecular networks also display a very wide range of tensile strength from ∼60 KPa to ∼50 MPa depending on the specific network organization. These biomimetic and dynamic supernetworks may have applications in tissue engineering, drug delivery systems, artificial skin, and soft robotics.

A comparative study on agarose acetate and PDLLA scaffold for rabbit femur defect regeneration.

The development of degradable polymer scaffolds is a key issue in bone regeneration. Poly(D, L-lactide) (PDLLA) and its derivatives have usually been applied to the construction of degradable scaffolds, but these scaffolds had problems with acidic degradation products and quick loss of mechanic strength during the later degradation, which usually led to scaffold collapse and cavity formation because of the slower rate of bone regeneration. In the present paper, a polysaccharide derivative, agarose acetate (AGA), was synthesized and a novel porous AGA scaffold was successfully developed through a salt-leaching process. The AGA scaffold had over 90% porosity without swelling in water, and compared to collapse and acidic products of PDLLA scaffold during degradation, the AGA scaffold maintained a stable morphology and a nearly neutral pH value over 18 months' degradation in PBS. A bone mesenchymal stem cells (BMSCs) adhesion and proliferation experiment showed that more cells adhered to the AGA scaffold than to the PDLLA scaffold. A subcutaneous implant test showed that the AGA scaffold slowly degraded and did not cause an inflammatory response surrounding the implantation lesion site. AGA scaffold was implanted into femur defects in New Zealand white rabbits to test its in vivo performance. Results indicated that the AGA scaffold accelerated the process of bone regeneration compared to the PDLLA group and, with time, new bone was formed from the margin toward the center of the scaffolds, and the scaffold left in place retained its porous structure without collapsing. Meanwhile, the AGA scaffold showed a low degradation rate and kept its shape during the in vivo degradation compared to the PDLLA scaffold. This performance could have the benefit of integrated regenerative bone being formed instead of cavities due to the quickly degraded scaffold disappearing. These results demonstrate that the AGA scaffold has significant potential in bone regeneration applications.

Self-Assembly of Core-Corona ß-Glucan into Stiff and Metalizable Nanostructures from 1D to 3D.

The development of self-assembly strategies for well-studied biopolymers is an important route to complex and functional nanostructures. Here, we report the self-assembly of a stiff polysaccharide, formylated yeast ß-glucan, into multiple highly ordered nanostructures from 1D to 3D. This polysaccharide could fold into a two-component helix that consisted of a rod-like helical core and flexible coronas. Annealing in formic acid can trigger the cross-linking of the coronas, resulting in the packing of helices into rod-like, sheet-like, or tube-like supramolecular nanostructures. The specific morphology of the resultant assemblies can be controlled by different annealing conditions such as annealing speed or polymer concentrations. Owing to the presence of reductant formyl groups, these ß-glucan nanostructures can reduce silver ions in situ, leading to the guided assembly of ultrathin silver nanowires, silver-polymer nanorods, and silver-polymer necklaces.

Mechanically stable surface-hydrophobilized chitosan nanofibrous barrier membranes for guided bone regeneration.

The use of chitosan based nanofiber membranes in guided bone regeneration (GBR) is limited by its uncontrolled swelling and mechanical instability in aqueous environments. This paper describes the significantly improved stability and properties of surface butyrylated chitosan nanofiber (BCSNF) membranes that greatly enhance their potential in GBR. The BCSNF membranes exhibited an overall degree of substitution of 1.61, an average diameter of 99.3 ± 33.7 nm, and a 75% decrease in swelling with an approximate doubling in suture pull out strengths as compared to unmodified fibers in aqueous environment. In a five week phosphate-buffered saline-lysozyme degradation study, it was found that the remaining mass fraction of BCSNF membranes was 11.5% more than that of unmodified fibers. In vitro, the BCSNF membranes were found to support the adhesion and proliferation of fibroblasts and were cell occulusive. In vivo, the BCSNF membranes were found to significantly improve the regeneration of a rat calvarial critical size defect in a 12 week healing period and showed better barrier function than commercially available collagen membranes with little soft tissue penetration through the membranes. Taken together, these data provide strong scientific evidence for use of BCSNF membranes in GBR applications.

O-acylation of chitosan nanofibers by short-chain and long-chain fatty acids.

Chitosan nanofibers (CSNFs) have potential applications in biomaterials, oil recovery and food packaging, but their instability in moist environment has limited their full utilization. Here we report that CSNFs can be O-acylated in a post-electrospinning treatment by using pyridine as catalyst and short-chain (C2, C3, C4, C5 and C6) and long-chain (C8 and C12) fatty acid anhydrates as acylation agents. The effects of O-acylation to CSNFs were analyzed in detail. FT-IR, 1H NMR and elemental analysis indicated that the hydroxyl groups of chitosan in CSNFs were acylated in 2h. XRD spectra indicated that the O-acylation modification altered the crystal structure of the native fibers and the acyl substituents packed in a laterally aligned and layered structure. SEM examinations showed that the acylation modification could effectively control the fibrous structure of CSNFs and improve their stability in moist environment. The O-acylated CSNFs generally have an average diameter about 100nm except for laurelated CSNFs (∼200nm). Water contact angle measurement indicated that the wetting properties of O-acylated CSNFs were affected by the length of acyl side chains. This fiber acylation strategy can tune the material properties of CSNFs and expand their potential applications.

Tile-based self-assembly of a triple-helical polysaccharide into cell wall-like mesoporous nanocapsules.

Tile-based self-assembly is a robust system in the construction of three-dimensional DNA nanostructures but it has been rarely applied to other helical biopolymers. ß-Glucan is an immunoactive natural polymer which exists in a triple helical conformation. Herein, we report that ß-glucan, after modification using two types of short chain acyl groups, can self-assemble into tiles with inactivated sticky ends at the interface of two solvents. These tiles consist of a single layer of helices laterally aligned, and the sticky ends can be activated when a few acyl groups at the ends are removed; these tiles can further pack into mesoporous nanocapsules, in a similar process as the sticky DNA tiles pack into complex polyhedral nano-objects. These nanocapsules were found to have targeted effects to antigen presenting cells in a RAW264.7 cell model. Our study suggests that tile-based self-assembly can be a general strategy for helical biopolymers, and on fully exploiting this strategy, various new functional nanostructures will become accessible in the future.