Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A.

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Chondroitin sulfate-modified tragacanth gum-gelatin composite nanocapsules loaded with curcumin nanocrystals for the treatment of arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease of yet undetermined etiology that is accompanied by significant oxidative stress, inflammatory responses,  and damage to joint tissues. In this study, we designed chondroitin sulfate (CS)-modified tragacanth gum-gelatin composite nanocapsules (CS-Cur-TGNCs) loaded with curcumin nanocrystals (Cur-NCs), which rely on the ability of CS to target CD44 to accumulate drugs in inflamed joints. Cur was encapsulated in the form of nanocrystals into tragacanth gum-gelatin composite nanocapsules (TGNCs) by using an inborn microcrystallization method, which produced CS-Cur-TGNCs with a particle size of approximately 80 ± 11.54 nm and a drug loading capacity of 54.18 ± 5.17%. In an in vitro drug release assay, CS-Cur-TGNCs showed MMP-2-responsive properties. During the treatment of RA, CS-Cur-TGNCs significantly inhibited oxidative stress, promoted the polarization of M2-type macrophages to M1-type macrophages, and decreased the expression of inflammatory factors (TNF-α, IL-1ß, and IL-6). In addition, it also exerted excellent anti-inflammatory effects, and significantly alleviated the swelling of joints during the treatment of gouty arthritis (GA). Therefore, CS-Cur-TGNCs, as a novel drug delivery system, could lead to new ideas for clinical therapeutic regimens for RA and GA.

Chemical Synthesis of Fucosylated Chondroitin Sulfate Tetrasaccharide with Fucosyl Branch at the 6-OH of GalNAc Residue.

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D-glucuronic acid but, recently, a novel structure with α-L-fucose linked to the 6-OH of N-acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O-sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure-activity relationship in the future.

Chondroitin sulphate modified MoS2 nanoenzyme with multifunctional activities for treatment of Alzheimer's disease.

Nano-MoS2 exhibit oxidoreductase-like activities, and has been shown to effectively eliminate excessive intracellular ROS and inhibit Aß aggregation, thus demonstrating promising potential for anti-Alzheimer's disease (anti-AD) intervention. However, the low water dispersibility and high toxicity of nano-MoS2 limits its further application. In this study, we developed a chondroitin sulphate (CS)-modified MoS2 nanoenzyme (CS@MoS2) by harnessing the excellent biocompatibility of CS and the exceptional activities of nano-MoS2 to explore its potential in anti-AD research. Promisingly, CS@MoS2 significantly inhibited Aß1-40 aggregation and prevented toxic injury in SH-SY5Y cells caused by Aß1-40. In addition, CS@MoS2 protected these cells from oxidative stress damage by regulating ROS production, as well as promoting the activities of SOD and GSH-Px. CS@MoS2 also modulated the intracellular Ca2+ imbalance and downregulated Tau hyperphosphorylation by activating GSK-3ß. CS@MoS2 suppressed p-NF-κB (p65) translocation to the nucleus by inhibiting MAPK phosphorylation, and modulated the expression of downstream anti- and proinflammatory cytokines. Owing to its multifunctional activities, CS@MoS2 effectively improved spatial learning, memory, and anxiety in D-gal/AlCl3-induced AD mice. Taken together, these results indicate that CS@MoS2 has significant potential for improving the therapeutic efficacy of the prevention and treatment of AD, while also presenting a novel framework for the application of nanoenzymes.

Future proofing of chondroitin sulphate production: Importance of sustainability and quality for the end-applications.

Chondroitin sulphates (CSs) are the most well-known glycosaminoglycans (GAGs) found in any living organism, from microorganisms to invertebrates and vertebrates (including humans), and provide several health benefits. The applications of CSs are numerous including tissue engineering, osteoarthritis treatment, antiviral, cosmetics, and skincare applications. The current commercial production of CSs mostly uses animal, bovine, porcine, and avian tissues as well as marine organisms, marine mammals, sharks, and other fish. The production process consists of tissue hydrolysis, protein removal, and purification using various methods. Mostly, these are chemical-dependent and are complex, multi-step processes. There is a developing trend for abandonment of harsh extraction chemicals and their substitution with different green-extraction technologies, however, these are still in their infancy. The quality of CSs is the first and foremost requirement for end-applications and is dependent on the extraction and purification methodologies used. The final products will show different bio-functional properties, depending on their origin and production methodology. This is a comprehensive review of the characteristics, properties, uses, sources, and extraction methods of CSs. This review emphasises the need for extraction and purification processes to be environmentally friendly and gentle, followed by product analysis and quality control to ensure the expected bioactivity of CSs.

Chondroitin Sulfate/Hyaluronic Acid-Blended Hydrogels Suppress Chondrocyte Inflammation under Pro-Inflammatory Conditions.

Osteoarthritis is characterized by enzymatic breakdown of the articular cartilage via the disruption of chondrocyte homeostasis, ultimately resulting in the destruction of the articular surface. Decades of research have highlighted the importance of inflammation in osteoarthritis progression, with inflammatory cytokines shifting resident chondrocytes into a pro-catabolic state. Inflammation can result in poor outcomes for cells implanted for cartilage regeneration. Therefore, a method to promote the growth of new cartilage and protect the implanted cells from the pro-inflammatory cytokines found in the joint space is required. In this study, we fabricate two gel types: polymer network hydrogels composed of chondroitin sulfate and hyaluronic acid, glycosaminoglycans (GAGs) known for their anti-inflammatory and prochondrogenic activity, and interpenetrating networks of GAGs and collagen I. Compared to a collagen-only hydrogel, which does not provide an anti-inflammatory stimulus, chondrocytes in GAG hydrogels result in reduced production of pro-inflammatory cytokines and enzymes as well as preservation of collagen II and aggrecan expression. Overall, GAG-based hydrogels have the potential to promote cartilage regeneration under pro-inflammatory conditions. Further, the data have implications for the use of GAGs to generally support tissue engineering in pro-inflammatory environments.

Hydrogel formats to model potential drug interactions occurring at the subcutaneous injection site.

We have previously developed an in vitro instrument, termed subcutaneous injection site simulator (SCISSOR), that can be used to monitor release properties of an active pharmaceutical ingredient (API) and formulation components of a medicine designed for SC injection. Initial studies to validate the SCISSOR instrument applications used a simple hyaluronic acid (HA) hydrogel to monitor early release events. We now report a type of cross-linked HA that can, when combined with HA, provide a hydrogel (HA-XR) with optical clarity and rheological properties that remain stable for at least 6 days. Incorporation of 0.05-0.1 mg/mL of collagens isolated from human fibroblasts (Col F), bovine type I collagen (Col I), chicken collagen type II (Col II), or chondroitin sulphate (CS) produced HA or HA-XR hydrogel formats with optical clarity and rheological properties comparable to HA or HA-XR alone. HA + Col F hydrogel had a much greater effect on release rates of 70 kDa compared to 4 kDa dextran, while Col F incorporated into the HA-XR hydrogel accentuated differences in release rates of prandial and basal forms of insulin as well as decreased the release rate of denosumab. A hydrogel format of HA + Col I was used to examine the complex events for bevacizumab release under conditions where a target ligand (vascular endothelial growth factor) can interact with extracellular matrix (ECM). Together, these data have demonstrated the feasibility of using a cross-linked HA format to examine API release over multiple days and incorporation of specific ECM elements to prepare more biomimetic hydrogels that allow for tractable examination of their potential impact of API release.

Simultaneous Rosiglitazone Release and Low-Density Lipoprotein Removal by Chondroitin Sodium Sulfate/Cyclodextrin/Poly(acrylic acid) Composite Adsorbents for Atherosclerosis Therapy.

Atherosclerosis (AS) is characterized by the accumulation of substantial low-density lipoprotein (LDL) and inflammatory response. Hemoperfusion is commonly employed for the selective removal of LDL from the body. However, conventional hemoperfusion merely focuses on LDL removal and does not address the symptom of plaque associated with AS. Based on the LDL binding properties of acrylated chondroitin sodium sulfate (CSA), acrylated beta-cyclodextrin (CD) and acrylic acid (AA), along with the anti-inflammatory property of rosiglitazone (R), the fabricated AA-CSA-CD-R microspheres could simultaneously release R and facilitate LDL removal for hemoperfusion. The AA and CSA offer electrostatic adsorption sites for LDL, while the CD provides hydrophobic adsorption sites for LDL and weak binding sites for R. According to the Sips model, the maximum static LDL adsorption capacity of AA-CSA-CD-R is determined to be 614.73 mg/g. In dynamic simulated perfusion experiments, AA-CSA-CD-R exhibits an initial cycle LDL adsorption capacity of 150.97 mg/g. The study suggests that the weakened inflammatory response favors plaque stabilization. The anti-inflammatory property of the microspheres is verified through an inflammation model, wherein the microsphere extracts are cocultured with mouse macrophages. Both qualitative analysis of iNOS\TNF-α and quantitative analysis of IL-6\TNF-α collectively demonstrate the remarkable anti-inflammatory effect of the microspheres. Therefore, the current study presents a novel blood purification treatment of eliminating pathogenic factors and introducing therapeutic factors to stabilize AS plaque.

Combination Nano-Delivery Systems Remodel the Immunosuppressive Tumor Microenvironment for Metastatic Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer for which effective therapies are lacking. Targeted remodeling of the immunosuppressive tumor microenvironment (TME) and activation of the body's immune system to fight tumors with well-designed nanoparticles have emerged as pivotal breakthroughs in tumor treatment. To simultaneously remodel the immunosuppressive TME and trigger immune responses, we designed two potential therapeutic nanodelivery systems to inhibit TNBC. First, the bromodomain-containing protein 4 (BRD4) inhibitor JQ1 and the cyclooxygenase-2 (COX-2) inhibitor celecoxib (CXB) were coloaded into chondroitin sulfate (CS) to obtain CS@JQ1/CXB nanoparticles (NPs). Then, the biomimetic nanosystem MM@P3 was prepared by coating branched polymer poly(ß-amino ester) self-assembled NPs with melittin embedded macrophage membranes (MM). Both in vitro and in vivo, the CS@JQ1/CXB and MM@P3 NPs showed excellent immune activation efficiencies. Combination treatment exhibited synergistic cytotoxicity, antimigration ability, and apoptosis-inducing and immune activation effects on TNBC cells and effectively suppressed tumor growth and metastasis in TNBC tumor-bearing mice by activating the tumor immune response and inhibiting angiogenesis. In summary, this study offers a novel combinatorial immunotherapeutic strategy for the clinical TNBC treatment.

Collagen short nanofiber-embedded chondroitin sulfate-hyaluronic acid nanocomposite: A cartilage-mimicking in situ-forming hydrogel with fine-tuned properties.

In situ-forming hydrogels that possess the ability to be injected in a less invasive manner and mimic the biochemical composition and microarchitecture of the native cartilage extracellular matrix are desired for cartilage tissue engineering. Besides, gelation time and stiffness of the hydrogel are two interdependent factors that affect cells' distribution and fate and hence need to be optimized. This study presented a bioinspired in situ-forming hydrogel composite of hyaluronic acid (HA), chondroitin sulfate (CS), and collagen short nanofiber (CSNF). HA and CS were functionalized with aldehyde and amine groups to form a gel through a Schiff-base reaction. CSNF was fabricated via electrospinning, followed by fragmentation by ultrasonics. Gelation time (11-360 s) and compressive modulus (1.4-16.2 kPa) were obtained by varying the concentrations of CS, HA, CSNFs, and CSNFs length. The biodegradability and biocompatibility of the hydrogels with varying gelation and stiffness were also assessed in vitro and in vivo. At three weeks, the assessment of hydrogels' chondrogenic differentiation also yields varying levels of chondrogenic differentiation. The subcutaneous implantation of the hydrogels in a mouse model indicated no severe inflammation. Results demonstrated that the injectable CS/HA@CSNF hydrogel was a promising hydrogel for tissue engineering and cartilage regeneration.

Recent progress in marine chondroitin sulfate, dermatan sulfate, and chondroitin sulfate/dermatan sulfate hybrid chains as potential functional foods and therapeutic agents.

Chondroitin sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid chains are natural complex glycosaminoglycans with high structural diversity and widely distributed in marine organisms, such as fish, shrimp, starfish, and sea cucumber. Numerous CS, DS, and CS/DS hybrid chains with various structures and activities have been obtained from marine animals and have received extensive attention. However, only a few of these hybrid chains have been well-characterized and commercially developed. This review presents information on the extraction, purification, structural characterization, biological activities, potential action mechanisms, and structure-activity relationships of marine CS, DS, and CS/DS hybrid chains. We also discuss the challenges and perspectives in the research of CS, DS, and CS/DS hybrid chains. This review may provide a useful reference for the further investigation, development, and application of CS, DS, and CS/DS hybrid chains in the fields of functional foods and therapeutic agents.

SARS-CoV-2 spike protein-ACE2 interaction increases carbohydrate sulfotransferases and reduces N-acetylgalactosamine-4-sulfatase by p38 MAPK.

Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.

Fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus alleviate the intestinal barrier injury and oxidative stress damage in vitro and in vivo.

This study aimed to investigate the alleviative effects of fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus (fCSSc) on the intestinal barrier injury and oxidative stress damage in vitro and in vivo. The results showed that fCS-Sc protected the intestinal barrier and improved the antioxidant function in H2O2 damaged Caco-2 cells via up-regulating the tight junction proteins and activating Keap1-Nrf2-ARE antioxidant pathway. Furthermore, administration fCS-Sc could ameliorate the weight loss and spleen index decrease in Cyclophosphamide (Cy) treated mice, improve the expressions of ZO-1, Claudin-1, Nrf2, SOD, and NQO-1 in Cy damaged colon tissue, showing significant protective effects against intestinal barrier damage and oxidative stress in vivo. fCS-Sc intervention also alleviated the gut microbiota disorder though increasing the richness and diversity of intestinal bacteria, regulating the structural composition of gut microbiota. fCS-Sc promoted the relative abundance of beneficial microbiota and inhibited the growth of harmful bacteria. This study provided a theoretical basis for the application of fCS-Sc as a prebiotic in chemotherapy.

CD44 targeted-chondroitin sulfate nanoparticles: Fine-tuning hydrophobic groups to enhance in vitro pH-responsiveness and in vivo efficacy for advanced breast cancer treatment.

The design of tumor-targeting nanoparticles with precisely controlled physical-biological properties may improve the delivery of chemotherapeutic agents. This study introduces pH-sensitive chondroitin sulfate-cholesterol (ChS-Chol) nano-assemblies for targeted intracellular doxorubicin (Dox) delivery in breast cancer treatment. Various ChS-Chol copolymers were synthesized, yielding self-assembling nanostructures with adjustable lipophilic content. In an aqueous environment, the ChS-Chol conjugates could form self-assembled nanostructures with a narrower size variation and a high negative potential. Moreover, the carriers would rapidly disassemble and release Dox in response to acidic pH. The in vitro cytotoxicity assay exhibited concentration-related anti-proliferation activity with Dox-loaded nanoparticles against 4T1, MCF-7, and MDA-MB-231 breast cancer cells. The nanoparticles demonstrated enhanced early apoptosis induction, efficient cellular uptake, and improved prevention of tumor cell proliferation compared to free Dox. In vivo results showcased significant tumor growth inhibition, underscoring the potential of these nanoparticle-based drug delivery systems for breast cancer therapy. The study emphasizes tailored nanocarrier design, leveraging pH-responsiveness and precise hydrophobic tuning to achieve targeted and potent therapeutic effects in the fight against breast cancer.

Chondroitin sulfate and caseinophosphopeptides doped polyurethane-based highly porous 3D scaffolds for tendon-to-bone regeneration.

Tendon disorders are common injuries, which can be greatly debilitating as they are often accompanied by great pain and inflammation. Moreover, several problems are also related to the laceration of the tendon-to-bone interface (TBI), a specific region subjected to great mechanical stresses. The techniques used nowadays for the treatment of tendon and TBI injuries often involve surgery. However, one critical aspect of this procedure involves the elevated risk of fail due to the tissues weakening and the postoperative alterations of the normal joint mechanics. Synthetic polymers, such as thermoplastic polyurethane, are of special interest in the tissue engineering field as they allow the production of scaffolds with tunable elastic and mechanical properties, that could guarantee an effective support during the new tissue formation. Based on these premises, the aim of this work was the design and the development of highly porous 3D scaffolds based on thermoplastic polyurethane, and doped with chondroitin sulfate and caseinophosphopeptides, able to mimic the structural, biomechanical, and biochemical functions of the TBI. The obtained scaffolds were characterized by a homogeneous microporous structure, and by a porosity optimal for cell nutrition and migration. They were also characterized by remarkable mechanical properties, reaching values comparable to the ones of the native tendons. The scaffolds promoted the tenocyte adhesion and proliferation when caseinophosphopetides and chondroitin sulfate are present in the 3D structure. In particular, caseinophosphopeptides' optimal concentration for cell proliferation resulted 2.4 mg/mL. Finally, the systems evaluation in vivo demonstrated the scaffolds' safety, since they did not cause any inflammatory effect nor foreign body response, representing interesting platforms for the regeneration of injured TBI.

Distinct mechanisms underlying the therapeutic effects of low-molecular-weight heparin and chondroitin sulfate on Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder influenced by various factors, including age, genetics, and the environment. Current treatments provide symptomatic relief without impeding disease progression. Previous studies have demonstrated the therapeutic potential of exogenous heparin and chondroitin sulfate in PD. However, their therapeutic mechanisms and structure-activity relationships remain poorly understood. In this study, low-molecular-weight heparin (L-HP) and chondroitin sulfate (L-CS) exhibited favorable therapeutic effects in a mouse model of PD. Proteomics revealed that L-HP attenuated mitochondrial dysfunction through its antioxidant properties, whereas L-CS suppressed neuroinflammation by inhibiting platelet activation. Two glycosaminoglycan (GAG)-binding proteins, manganese superoxide dismutase (MnSOD2) and fibrinogen beta chain (FGB), were identified as potential targets of L-HP and L-CS, and we investigated their structure-activity relationships. The IdoA2S-GlcNS6S/GlcNAc6S unit in HP bound to SOD2, whereas the GlcA-GalNAc4S and GlcA-GalNAc4S6S units in CS preferred FGB. Furthermore, N-S and 2-O-S in L-HP, and 4-O-S, 6-O-S, and -COOH in L-CS contributed significantly to the binding process. These findings provide new insights and evidence for the development and use of glycosaminoglycan-based therapeutics for PD.

High gastrointestinal digestive stability endows chondroitin sulfate-soluble undenatured type II collagen complex with high activity: Improvement of osteoarthritis in rats.

Previously, we prepared a chondroitin sulfate-soluble undenatured type II collagen complex (CS-SC II) with low salt content. This paper further explored the differences between CS-SC II and SC II in terms of gastrointestinal digestive characteristics and osteoarthritis (OA) improvement. In vitro and in vivo experiments showed that the gastric digestive stability of CS-SC II was high under both pH 2.0 and pH 3.0, the α1 chain and triple helix structure of type II collagen retained >60 %. However, SC II had high gastric digestive stability only under pH 3.0. Furthermore, intestinal digestion had little effect on α1 chains of CS-SC II and SC II, and distribution experiments showed that they might exert their biological activities in the intestine. CS-SC II had obvious improvement in OA rats at 1.0 mg/kg/d, that is, the joint swelling was significantly reduced and the weight-bearing ratio of the right hind limb was increased to 49 %, which was close to that of 4.0 mg/kg/d SC II. The wear of articular cartilage, Mankin and OARSI scores of rats in CS-SC II group were significantly reduced. The effects of low-dose CS-SC II on the proportion of regulatory T cells (Treg), mRNA expression of OA key biomarkers (Il6, Ccl7, MMP-3 and MMP13) and signaling pathway genes (NF-κB, AKT or AMPKα) were comparable to those of high-dose SC II. These results showed that CS-SC II might have greater potential to improve OA at a lower dose than SC II due to its high gastrointestinal digestive stability at a wide range of pH conditions.

Construction of a dual-component hydrogel matrix for 3D biomimetic skin based on photo-crosslinked chondroitin sulfate/collagen.

The main challenge in the field of 3D biomimetic skin is to search for a suitable hydrogel matrix with good biocompatibility, appropriate mechanical property and inner porosity that can support the adhesion and proliferation of skin cells. In this study, photocurable chondroitin sulfate methacrylate (CSMA) and collagen methacrylate (CoLMA) synthesized from chondroitin sulfate (CS) and type I collagen I (CoL) in the dermal matrix were used to construct a photo-crosslinked dual-component CSMA-CoLMA hydrogel matrix. Due to the toughening effect of the dual-component, the CSMA-CoLMA hydrogel improved the intrinsic brittleness of the single-component CSMA hydrogel, presented good mechanical tunability. The average storage and elasticity modulus could reach 3.3 KPa and 30.3 KPa, respectively, which were close to those of natural skin. The CSMA-CoLMA hydrogel with a ratio of 8/6 showed suitable porous structure and good biocompatibility, supporting the adhesion and proliferation of skin cells. Furthermore, the expression of characteristic marker proteins was detected in the epidermal and dermal bi-layered models constructed with the hydrogel containing keratinocytes and fibroblasts. These results suggest that the dual-component CSMA-CoLMA hydrogel has promising potential as a matrix to construct 3D biomimetic skin.

Identification and structural characterization of a novel chondroitin sulfate-specific carbohydrate-binding module: The first member of a new family, CBM100.

Chondroitin sulfate is a biologically and commercially important polysaccharide with a variety of applications. Carbohydrate-binding module (CBM) is an important class of carbohydrate-binding protein, which could be utilized as a promising tool for the applications of polysaccharides. In the present study, an unknown function domain was explored from a putative chondroitin sulfate lyase in PL29 family. Recombinant PhCBM100 demonstrated binding capacity to chondroitin sulfates with Ka values of 2.1 ± 0.2 × 106 M-1 and 6.0 ± 0.1 × 106 M-1 to chondroitin sulfate A and chondroitin sulfate C, respectively. The 1.55 Å resolution X-ray crystal structure of PhCBM100 exhibited a ß-sandwich fold formed by two antiparallel ß-sheets. A binding groove in PhCBM100 interacting with chondroitin sulfate was subsequently identified, and the potential of PhCBM100 for visualization of chondroitin sulfate was evaluated. PhCBM100 is the first characterized chondroitin sulfate-specific CBM. The novelty of PhCBM100 proposed a new CBM family of CBM100.

Thermally stabilized chondroitin sulfate-hemoglobin nanoparticles and their interaction with bioactive compounds.

The preparation of nanoparticles (NPs) based on hemoglobin (Hb) with a fully biocompatible methodology is presented. The spontaneous formation of electrostatic complexes of Hb with chondroitin sulfate (CS) at pH 4 in the polysaccharide/protein mass ratio regime where charge neutrality is met leads to spherical nanostructures with monomodal hydrodynamic radii distribution in the range of 50-100 nm. The integrity of the electrostatic complexes is disturbed at pH 7 as the net electric charge of Hb is very low. Treating the NPs at mildly elevated temperature stabilizes them against the pH increase taking advantage of Hb's ability of unfolding and self-associating upon thermal treatment. The NPs surface charge is pH-tunable and changes from positive to strongly negative upon pH increase to 7 proving the presence of negative surface patches of Hb and CS segments in their exterior. The α-helix content of Hb does not change significantly by thermal treatment. The NPs are found to bind the bioactive compounds curcumin and ß-carotene and are stable in solutions with high salt content. This investigation introduces a straightforward method to formulate Hb in NPs with possibilities in the nanodelivery of nutrients and drugs.