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.

Injectable Micelle-Incorporated Hydrogels for the Localized Chemo-Immunotherapy of Breast Tumors.

Although immune checkpoint blockade (ICB) holds potential for the treatment of various tumors, a considerable proportion of patients show a limited response to ICB therapy due to the low immunogenicity of a variety of tumors. It has been shown that some chemotherapeutics can turn low-immunogenic tumors into immunogenic phenotypes by inducing a cascade of immune responses. In this paper, we synthesized an injectable micelle-incorporated hydrogel, which was able to sequentially release the chemotherapeutic gemcitabine (GEM) and the hydrophobic indoleamine 2, 3-dioxygenase inhibitor, d-1-methyltryptophan (d-1MT) at tumor sites. The hydrogel was formed via the thiol-ene click reaction between the thiolated chondroitin sulfate and the micelle formed by amphiphilic methacrylated Pluronic F127, in which hydrophobic d-1MT was encapsulated in the core of the F127 micelles and the hydrophilic GEM was dispersed in the hydrogel network. The successive release of chemotherapeutics and immune checkpoint inhibitors at tumor tissues will first promote the infiltration of cytotoxic T lymphocytes and subsequently induce a robust antitumor immune response, ultimately exerting a synergetic therapeutic efficacy. In a 4T1 tumor-bearing mice model, our results showed that the combination of chemotherapy and immunotherapy through the micelle-incorporated hydrogel triggered an effective antitumor immune response and inhibited tumor metastasis to the lung. Our results highlight the potential of the injectable micelle-incorporated hydrogel for the localized chemo-immunotherapy in the treatment of breast tumors.

Synthesis, structure and midkine binding of chondroitin sulfate oligosaccharide analogues.

The preparation of chondroitin sulfate (CS) oligosaccharide mimetics, more easily synthesized than natural sequences, is a highly interesting task because these compounds pave the way for modulation of the biological processes in which CS is involved. Herein, we report the synthesis of CS type E analogues which present easily accessible glucose units instead of glucuronic acid (GlcA) moieties. NMR experiments and molecular dynamics simulations showed that the 3D structure of these compounds is similar to the structure of the natural CS-E oligosaccharides. In addition, fluorescence polarization (FP) and saturation transfer difference NMR (STD-NMR) experiments revealed that the synthesized CS-like derivatives were able to interact with midkine, a model heparin-binding growth factor, suggesting that the presence of the GlcA carboxylate groups is not essential for the binding. Overall, our results indicate that the synthesized glucose-containing oligosaccharides can be considered as functional and structural CS mimetics.

Synthesis and characterization of poly(vinyl alcohol)/chondroitin sulfate composite hydrogels containing strontium-doped hydroxyapatite as promising biomaterials.

Novel poly(vinyl alcohol)/chondroitin sulfate (PVA/CS) composite hydrogels containing hydroxyapatite (HA) or Sr-doped HA (HASr) particles were synthesized by a freeze/thaw method and characterized aiming towards biomedical applications. HA and HASr were synthesized by a wet-precipitation method and added to the composite hydrogels in fractions up to 15 wt%. Physical-chemical characterizations of particles and hydrogels included scanning electron microscopy, energy-dispersive spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, thermogravimetry, porosity, compressive strength/elastic modulus, swelling degree, and cell viability. Particles were irregular in shape and appeared to have narrow size variation. The thermal behavior of composite hydrogels was altered compared to the control (bare) hydrogel. All hydrogels exhibited high porosity. HA/HASr particles reduced total porosity without reducing pore size. The mechanical strength was improved as the fraction of HA or HASr was increased. HASr particles led to a faster water uptake but did not interfere with the total hydrogel swelling capacity. In cell viability essay, increased cell growth (above 120%) was observed in all groups including the control hydrogel, suggesting a bioactive effect. In conclusion, PVA/CS hydrogels containing HA or HASr particles were successfully synthesized and showed promising morphological, mechanical, and swelling properties, which are particularly required for scaffolding.

Fabrication of chondroitin sulfate calcium complex and its chondrocyte proliferation in vitro.

Chondroitin sulfate (CS)-calcium complex (CSCa) was fabricated, and the structural characteristics of CSCa and its proliferative bioactivity to the chondrocyte were investigated in vitro. Results suggested calcium ions could bind CS chains forming polysaccharide-metal complex, and the maximum calcium holding capacity of CSCa reached 4.23 %. Characterization of CSCa was performed by EDS, AFM, FTIR, UV, XRD and 1H-NMR. It was found that calcium ions were integrated with CS by binding the sulfate or carboxyl groups. The thermal properties analysis indicated CSCa had a good thermal stability by TGA and DSC. CSCa could interact the calcium-sensing receptor increasing the intracellular calcium ions and influence the cell cycle. The TGF-ß1 secretion induced by CSCa could activate the TGF-ß/Smads pathway and change the genes associated proliferation expression ultimately leading to the chondrocyte proliferation. This research probably has an important implication for understanding the effect of CSCa on bone care as food supplements.

Synthesis of structurally defined chondroitin sulfate: Paving the way to the structure-activity relationship studies.

Chondroitin sulfate (CS) is one of the major and widespread glycosaminoglycans, a family of structurally complex, linear, anionic hetero-co-polysaccharides. CS plays a vital role in various normal physiological and pathological processes, thus, showing varieties of biological activities, such as anti-oxidation, anti-atherosclerosis, anti-thrombosis, and insignificant immunogenicity. However, the heterogeneity of the naturally occurring CS potentially leads to function unspecific and limits further structure-activity relationship studies. Therefore, the synthesis of CS with well-defined and uniform chain lengths is of major interest for the development of reliable drugs. In this review, we examine the remarkable progress that has been made in the chemical, enzymatic and chemoenzymatic synthesis of CS and its derivatives, providing a broad spectrum of options to access CS of well controlled chain lengths.

(Semi)-Synthetic Fucosylated Chondroitin Sulfate Oligo- and Polysaccharides.

Fucosylated chondroitin sulfate (fCS) is a glycosaminoglycan (GAG) polysaccharide with a unique structure, displaying a backbone composed of alternating N-acetyl-d-galactosamine (GalNAc) and d-glucuronic acid (GlcA) units on which l-fucose (Fuc) branches are installed. fCS shows several potential biomedical applications, with the anticoagulant activity standing as the most promising and widely investigated one. Natural fCS polysaccharides extracted from marine organisms (Echinoidea, Holothuroidea) present some advantages over a largely employed antithrombotic drug such as heparin, but some adverse effects as well as a frequently found structural heterogeneity hamper its development as a new drug. To circumvent these drawbacks, several efforts have been made in the last decade to obtain synthetic and semi-synthetic fCS oligosaccharides and low molecular weight polysaccharides. In this Review we have for the first time collected these reports together, dividing them in two topics: (i) total syntheses of fCS oligosaccharides and (ii) semi-synthetic approaches to fCS oligosaccharides and low molecular weight polysaccharides as well as glycoclusters displaying multiple copies of fCS species.

Recent advance in delivery system and tissue engineering applications of chondroitin sulfate.

Chondroitin sulfate (CS) is a naturally derived bioactive macromolecule and the major component of extracellular matrix (ECM), which widely distributed in various organisms and has attracted much attention due to their significant bioactivities. It is regarded as a favorable biomaterial that has been applied extensively in field of drug delivery and tissue engineering due to its property of non-poisonous, biodegradation, biocompatible and as a major component of ECM. The present article reviews the structure and bioactivities of CS, from the preparation to structure analysis, and emphatically focuses on the biomaterial exertion in delivery system and tissue engineering. At the same time, the present application status and prospect of CS are analyzed and the biomaterial exertion of CS in delivery system and various tissue engineering are also comparatively discussed in view of biomaterial development.

A Study for the Access to a Semi-synthetic Regioisomer of Natural Fucosylated Chondroitin Sulfate with Fucosyl Branches on N-acetyl-Galactosamine Units.

Fucosylated chondroitin sulfate (fCS) is a glycosaminoglycan found up to now exclusively in the body wall of sea cucumbers. It shows several interesting activities, with the anticoagulant and antithrombotic as the most attractive ones. Its different mechanism of action on the blood coagulation cascade with respect to heparin and the retention of its activity by oral administration make fCS a very promising anticoagulant drug candidate for heparin replacement. Nonetheless, its typically heterogeneous structure, the detection of some adverse effects and the preference for new drugs not sourced from animal tissues, explain how mandatory is to open an access to safer and less heterogeneous non-natural fCS species. Here we contribute to this aim by investigating a suitable chemical strategy to obtain a regioisomer of the natural fCS polysaccharide, with sulfated l-fucosyl branches placed at position O-6 of N-acetyl-d-galactosamine (GalNAc) units instead of O-3 of d-glucuronic acid (GlcA) ones, as in natural fCSs. This strategy is based on the structural modification of a microbial sourced chondroitin polysaccharide by regioselective insertion of fucosyl branches and sulfate groups on its polymeric structure. A preliminary in vitro evaluation of the anticoagulant activity of three of such semi-synthetic fCS analogues is also reported.

The synthesis and biological evaluation of chondroitin sulfate E glycodendrimers.

Aim: Chondroitin sulfate (CS) is a class of highly sulfated polysaccharides that possess many important biological functions. The heterogeneity of CS limits pharmacological research and leads to ambiguous mechanisms. Thus, glycomimetics are demanded as replacement of natural polysaccharides to explore important biological processes. Results & methodology: Here the preparation of CS glycodendrimers is reported as well as their use as CS mimetics to regulate the NF-κB pathway. Multivalent presentation of sugar epitopes on appropriate dendrimer scaffolds increased the suppression of the NF-κB pathway. The interaction between CS-E molecules and TNF-α was examined by nuclear magnetic resonance technology. Conclusion: Overall, the glycodendrimer reported here may be potentially employed as molecular tool to investigate the biological functions of CS.

Efficient weapon for protracted warfare to malaria: A chondroitin sulfate derivates-containing injectable, ultra-long-lasting meshy-gel system.

Progress at elimination of malaria is limited by the challenges of reaching large rural population and ensuring patient adherence to adequate pharmacologic treatment. In the present study, a novel material (octadecylamine modified chondroitin sulfate) was synthesized, to fabricate a long acting release meshy gel system as an efficient weapon for protracted warfare to malaria. Ivermectin loaded meshy gels (IVM-MG) composed of different amount of phospholipids, triglyceride and modified chondroitin sulfate were formulated. They were in aqueous state with low viscosity before injection, but rapidly turned into gel state with significantly increased viscosity upon exposure to an aqueous environment after injection. In vitro study proved a sustained released effect in different releasing media. In vivo study showed no irritation at injection site and slowly drug release over a 30-day release period in rat model. Among the three IVM-MG formulations, IVM-MG-3 with the highest amount of octadecylamine modified chondroitin sulfate presented the highest viscosity increase after solution-gel transition, the least initial burst release, and the longest sustained release effect over 30 days in rat model. Furthermore, by using mathematical models, IVM-MG system could boost the efficacy of mass drug administration toward malaria elimination goals. Meshy gel systems for long-acting drug delivery have the potential to revolutionize treatment options for malaria and other diseases of which treatment adherence is essential for their efficacy.

Synthesis of chondroitin sulfate magnesium for osteoarthritis treatment.

Magnesium chondroitin sulfate (MgCS) has been fabricated and characterized in this study. We investigated its morphology, composition as well as structure. The results verify that the sodium of sodium chondroitin sulfate (CS) is successfully replaced by magnesium and formed a polysaccharide-metal ion complex. To evaluate the clinical potential of MgCS, cell proliferation and apoptosis test were conducted. The results reveal that MgCS could effectively increase the proliferation and decrease the apoptosis of osteoarthritis (OA) chondrocytes. Moreover, real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to evaluate the gene expression level. RT-qPCR analysis suggests that MgCS could significantly increase the expression of COLII and decrease the expression of IL-1ß and iNOS in OA chondrocytes. Furthermore, significant upregulation of Bcl-2 mRNA expression and downregulation of the expression of apoptosis related gene p53 were observed. Thus, it is indicated that MgCS should have great potentials in OA treatment.

Controlling methacryloyl substitution of chondroitin sulfate: injectable hydrogels with tunable long-term drug release profiles.

Drug delivery systems capable of local sustained release of small molecule therapeutics remain a critical need in many fields, including oncology. Here, a system to create tunable hydrogels capable of modulating the loading and release of cationic small molecule therapeutics was developed. Chondroitin sulfate (CS) is a sulfated glycosaminoglycan that has many promising properties, including biocompatibility, biodegradation and chemically modifiable groups for both covalent and non-covalent bonding. CS was covalently modified with photocrosslinkable methacryloyl groups (CSMA) to develop an injectable hydrogel fabrication. Utilizing anionic groups, cationic drugs can be adsorbed and released from the hydrogels. This study demonstrates the synthesis of CSMA with a varying degree of substitution (DS) to generate hydrogels with varying swelling properties, maximum injection force, and drug release kinetics. The DS of the synthesized CSMA ranged from 0.05 ± 0.02 (2 h reaction) to 0.28 ± 0.02 (24 h reaction) with a DS of 1 representing 100% modification. The altered DS resulted in changes in hydrogel properties with the swelling of 20% CSMA hydrogels ranging from 42 (2 h reaction) to 13 (24 h reaction) and injection forces ranging from 18 N (2 h reaction) to 94 N (24 h reaction). The release of sunitinib, an oncology therapeutic that inhibits intracellular signaling by targeting multiple receptor tyrosine kinases, ranged from 18 µg per day (2 h reaction) to 9 µg per day (24 h reaction). While decreasing the DS increased the hydrogel swelling and rate of therapeutic release, it also limited the hydrogel fabrication range to only those containing 10% or higher CSMA. Blended polymer systems with poly(vinyl alcohol)-methacrylate (PVAMA) were fabricated to stabilize the resulting hydrogels via attenuating the swelling properties. Release profiles previously unattainable with the pure CSMA hydrogels were achieved with the blended hydrogel formulations. Overall, these studies identify a method to formulate tunable CSMA and blended CSMA/PVAMA hydrogels capable of sustained release of cationic therapeutics over six weeks with applications in oncology therapeutics.

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

A holistic approach to unravelling chondroitin sulfation: Correlations between surface charge, structure and binding to growth factors.

Chondroitin sulfate (CS) is a relevant family of polysaccharides that participates in a large variety of biological events that are related to neural processes by regulating various growth factors through the pattern and degree of sulfation of the polysaccharide. However, their own complexity makes their optimization for biomedical applications a difficult undertaking. Thus, a different perspective has to be taken. Herein, we show that the particular sulfate distribution within the disaccharide repeating-unit plays a key role in the binding of growth factors (GFs). In particular, this disposition modulates the surface charge of the helical structure that, interestingly, has a significant influence on the binding capacity of CSs with several GFs. This fact should be carefully considered in the design of new ligands with improved activity as GFs ligands.

Preparation and in vitro evaluation of novel cross-linked chondroitin sulphate nanoparticles by aluminium ions for encapsulation of green tea flavonoids.

Chondroitin sulphate is a sulphated glycosaminoglycan biopolymer composed over 100 individual sugars. Chondroitin sulphate nanoparticles (NPs) loaded with catechin were prepared by an ionic gelation method using AlCl3 and optimised for polymer and cross-linking agent concentration, curing time and stirring speed. Zeta potential, particle size, loading efficiency, and release efficiency over 24 h (RE24%) were evaluated. The surface morphology of NPs was investigated by scanning electron microscopy and their thermal behaviour by differential scanning calorimetric. Antioxidant effect of NPs was determined by chelating activity of iron ions. The cell viability of mesenchymal stem cells was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide assay and the calcification of osteoblasts was studied by Alizarin red staining. The optimised NPs showed particle size of 176 nm, zeta potential of -20.8 mV, loading efficiency of 93.3% and RE24% of 80.6%. The chatechin loaded chondroitin sulphate NPs showed 70-fold more antioxidant activity, 3-fold proliferation effect and higher calcium precipitation in osteoblasts than free catechin.

Synthesis and anticoagulation studies of "short-armed" fucosylated chondroitin sulfate glycoclusters.

Fucosylated chondroitin sulfate (FuCS) is a structurally complex glycosaminoglycan found in sea cucumbers with a wide spectrum of biological activities, among which anticoagulant activity is particularly attractive for the development of alternative anticoagulant drugs with decreased adverse effects and risks of bleeding. Previous studies show that FuCS glycomimetics bearing several trisaccharide epitopes displayed promising anticoagulant activity and did not change the mode of action of FuCS. To simplify synthetic difficulty of high valent glycoclusters and obtain candidate compounds with relatively low molecular weights, here we report the synthesis of two FuCS glycoclusters with low valence and more compact structures. Anticoagulation studies showed that these simplified "short-armed" glycoclusters demonstrated comparable potency with "long-armed" high valent glycoclusters, offering a concise approach for the development of novel anticoagulant agents.

Synthesis of Fucosylated Chondroitin Sulfate Nonasaccharide as a Novel Anticoagulant Targeting Intrinsic Factor Xase Complex.

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan, and its oligosaccharides exhibit excellent anticoagulant activity with lower risks of adverse effects and bleeding. Herein we report a facile approach to the synthesis of FuCS hexa- and nonasaccharides on the basis of the enzymatic degradation of chondroitin over 12 linear steps. As compared with a clinical low-molecular-weight heparin drug (enoxaparin), the nonasaccharide synthesized in this study displayed similar APTT activity and selective intrinsic factor Xase complex inhibitory activity ((12.9±0.83) nm) by binding to factor IXa with high affinity, thus offering promise for the development of new anticoagulant agents targeting the intrinsic coagulation pathway.

Injectable and self-crosslinkable hydrogels based on collagen type II and activated chondroitin sulfate for cell delivery.

Injectable hydrogels are attractive and alternative scaffolds for cell delivery because they could form in situ, simulate natural tissue and fill any shape of defect. This study aimed at fabricating injectable, self-crosslinkable and biomimetic hydrogels based on collagen type II (Col II) and activated chondroitin sulfate (CS-sNHS) under physiological conditions without the addition of any catalysts or crosslinking agents. The inner morphology of hydrogels was detected by scanning electron microscopy, and it showed that fibrous structure formed in the hydrogels. The gelation time, water absorption capacity and the mechanical property of hydrogels were closely related to the weight ratio of Col II and CS-sNHS in hydrogels. Chondrocytes were encapsulated into these hydrogels, and the effect of hydrogels on survival, proliferation, morphology of cells and remolding of extracellular matrix was investigated. The results demonstrated that chondrocytes survived well and showed round or oval morphology in these hydrogels, in addition, the matrix in hydrogels had been remolded and the collagen fibers displayed periodic alternation of light and shade. These results implied that the injectable and self-crosslinkable hydrogels were alternative carriers for chondrocyte delivery.

Synthesis of Azide-Modified Chondroitin Sulfate Precursors: Substrates for "Click"- Conjugation with Fluorescent Labels and Oligonucleotides.

Azidopropyl-modified precursors of chondroitin sulfate (CS) tetrasaccharides have been synthesized, which, after facile conversion to final CS structures, may be conjugated with alkyne-modified target compounds by a one-pot "click"-ligation. RP HPLC was used for the monitoring of the key reaction steps (protecting group manipulation and sulfation) and purification of the CS precursors (as partially protected form, bearing the O-Lev, O-benzoyl, and N-trichloroacetyl groups and methyl esters). Subsequent treatments with aqueous NaOH, concentrated ammonia, and acetic anhydride (i.e., global deprotection and acetylation of the galactosamine units) converted the precursors to final CS structures. The azidopropyl group was exposed to a strain-promoted azide-alkyne cycloaddition (SPAAC) with a dibenzylcyclooctyne-modified carboxyrhodamine dye to give labeled CSs. Conjugation with a 5'-cyclooctyne-modified oligonucleotide was additionally carried out to show the applicability of the precursors for the synthesis of biomolecular hybrids.