Topical Application of Peptide-Chondroitin Sulfate Nanoparticles Allows Efficient Photoprotection in Skin.

In this article, we describe a method of delivery of chondroitin sulfate to skin as nanoparticles and demonstrate its anti-inflammatory and antioxidant role using UV irradiation as a model condition. These nanoparticles, formed through electrostatic interactions of chondroitin sulfate with a skin-penetrating peptide, were found to be homogenous with positive surface charges and stable at physiological and acidic pH under certain conditions. They were able to enter into the human keratinocyte cell line (HaCaT), artificial skin membrane (mimicking the human skin), and mouse skin tissue unlike free chondroitin sulfate. The preapplication of nanoparticles also exhibited reduced levels of oxidative stress, cyclobutane pyrimidine dimer formation, TNF-α, and so on in UV-B-irradiated HaCaT cells. In an acute UV-B irradiation mouse model, their topical application resulted in reduced epidermal thickness and sunburn cells, unlike in the case of free chondroitin sulfate. Thus, a completely noninvasive method was used to deliver a bio-macromolecule into the skin without using injections or abrasive procedures.

Oral Administration of Fucosylated Chondroitin Sulfate Oligomers in Gastro-Resistant Microcapsules Exhibits a Safe Antithrombotic Activity.

Fucosylated chondroitin sulfate (FCS) polysaccharide isolated from sea cucumber has potent anticoagulant activity. Based on its resistance to the enzymes present in vertebrates, it may serve as an anticoagulant and shows antithrombotic effects when delivered through gastro-resistant (GR) tablets. However, due to the multiple plasma targets of FCS polysaccharide in the coagulation pathway, bleeding can occur after its oral administration. In the current study, we used FCS oligomers, in particular a mixture of oligosaccharides having 6 to 18 saccharide units, as the active ingredient in GR microcapsules for oral anticoagulation. In a Caco-2 model, the FCS oligomers showed higher absorption than native FCS polysaccharides. Oral administration of FCS oligomer-GR microcapsules provided a dose-dependent, prolonged anticoagulant effect with a selective inhibition of the intrinsic coagulation pathway when compared with subcutaneous administration of FCS oligomers or oral administration of unformulated FCS oligomers or native FCS-GR microspheres. Continued oral administration of FCS oligomer-GR microcapsules did not result in the accumulation of oligosaccharides in the plasma. Venous thrombosis animal models demonstrated that FCS oligomers delivered via GR microcapsules produced a potent antithrombotic effect dependent on their anticoagulant properties in the plasma, while oral administration of unformulated FCS oligomers at the same dose exhibited a weaker antithrombotic effect than the formulated version. Oral administration of FCS oligomer-GR microcapsules resulted in no bleeding, while oral administration of native FCS-GR microcapsules resulted in bleeding (p < 0.05). Our present results suggest that a FCS oligomer-GR microcapsule formulation represents an effective and safe oral anticoagulant for potential clinical applications.

Polyvinylpyrrolidone microneedles for localized delivery of sinomenine hydrochloride: preparation, release behavior of in vitro & in vivo, and penetration mechanism.

Sinomenine (SIN) is an anti-inflammatory alkaloid derived from Sinomenium acutum, and the products sinomenine hydrochloride (SH) tablets and injections have been marketed in China to treat rheumatoid arthritis (RA). Oral administration of SH has shortcomings of gastrointestinal irritation and low bioavailability. The injection may require professional training and higher cost. It is of interest to develop an alternative form that is easier to administer and avoids the first-pass metabolism. In this study, SH-loaded dissolving microneedles (SH-MN) were fabricated using polyvinyl pyrrolidone and chondroitin sulfate with a casting method. In percutaneous permeation studies of In vitro, the cumulative permeation and permeation rate of SH-MN were 5.31 and 5.06 times higher than that of SH-gel (SH-G). In percutaneous pharmacokinetic studies, the values of the area under the curve after administration of SH-MN in the skin and blood were 1.43- and 1.63-fold higher than that of SH-G, respectively. In percutaneous absorption studies, SH-MN could absorb into tissue fluid; and dissolve after skin penetration. The drug was released along the channel and spread to surrounding skin tissue. After 4 h, the needle tip was almost completely dissolved, and the drug could penetrate to a depth of 200 µm under the skin. These results demonstrate that the SH-MN is an effective, safe, and simple strategy for transdermal SH delivery.

Comparative Analyses of Pharmaceuticals or Food Supplements Containing Chondroitin Sulfate: Are Their Bioactivities Equivalent?

INTRODUCTION: Oral supplementation of chondroitin sulfate (CS) and glucosamine (GlcN), symptomatic slow-acting molecules, is recommended by European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis and Musculoskeletal Diseases (ESCEO) and other European Union (EU) guidelines for the restoration of the articular cartilage surface in patients affected by osteoarthritis (OA). They are commercialized as pharmaceutical grade products and as food supplements in combination with plant extracts hyaluronic acid, methylsulfonylmethane, and other components. Food supplements do not need to undergo the strict regulatory controls of pharmaceutical grade products; thus, composition and contaminants that could be present may not be evidenced before commercialization and these uncertainties may give rise to concerns about the bioactivity of these formulations. METHODS: In this paper 10 different food supplements (FS) from diverse European countries were analyzed in comparison with two pharmaceutical grade products (Ph) using updated analytical approaches and biochemical cell-based assays. The purity, the titer, and the origin of CS in Ph and FS samples were initially assessed in order to successively compare the biological function. Both food supplements and pharmaceutical formulations were tested in vitro, using the same final CS concentration, on primary chondrocytes and synoviocytes in terms of (i) cell viability, (ii) activation of the NF-κB-mediated inflammation pathway, (iii) cartilage oligomeric matrix protein (COMP-2), IL-6, and IL-8 production. RESULTS: All the FS presented a certain insoluble fraction; the CS and the GlcN contents were lower than the declared ones in 9/10 and 8/10 samples, respectively. All FS contained keratan sulfate (KS) at up to 50% of the total glycosaminoglycan amount declared on the label. Primary cells treated with the samples diluted to present the same CS concentration in the medium showed cytotoxicity in 7/10 FS while Ph preserved viability and reduced NF-κB, COMP-2, and secreted inflammatory cytokines. CONCLUSION: Among all samples tested, the pharmaceutical grade products demonstrated effective modulation of biomarkers counteracting the inflammation status and improving viability and the physiological condition of OA human primary chondrocyte and synoviocyte cells. In contrast to that, most FS were cytotoxic at the tested concentrations, and only 3/10 of them showed similarities to Ph sample behavior in vitro. FUNDING: This work was partially supported by PON01_1226 NUTRAFAST, MIUR Ministero dell'Università e della Ricerca Scientifica. Bioteknet financed two short-term grants for graduate technicians. The journal's Rapid Service and Open Access fees were funded by IBSA CH.

Preparation of Chondroitin Sulfate-Glycyl-Prednisolone Conjugate Nanogel and Its Efficacy in Rats with Ulcerative Colitis.

A conjugate between chondroitin sulfate (CS) and glycyl-prednisolone (GP), named CS-GP, was produced by carbodiimide coupling at a high GP/CS ratio. CS-GP was not water-soluble and gave a nanogel (NG) in aqueous solution. Two types of nanogels, NG(I) and NG(II), with prednisolone (PD) contents of 5.5 and 21.1% (w/w), respectively, were obtained. They had particle sizes of approximately 280 and 570 nm, respectively, and showed negative ζ-potentials of approximately -40 mV. The PD release rate was slower in the nanogels than in a solution of CS-GP with a PD content of 1.4% (w/w). The PD release rate was slower in NG(II) than in NG(I), and was elevated at pH 7.4 than at pH 6.8. NG(II) was applied in vivo to rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis, and its therapeutic efficacy and pharmacokinetic features were investigated. The therapeutic efficacy of NG(II) was slightly better than that of PD alone. Drug delivery to the lower intestines was enhanced with NG(II). The CS-GP nanogel has potential as a potent DDS for the treatment of ulcerative colitis.

Coated nanostructured lipid carriers targeting the joints - An effective and safe approach for the oral management of rheumatoid arthritis.

Oral treatment of rheumatoid arthritis (RA) with the immunomodulator, leflunomide (LEF), is associated with systemic side effects namely immunosuppression and hepatotoxicity. Herein, attempts to improve LEF therapeutic outcomes via nanostructured lipid carriers (NLCs) targeting inflamed rheumatic joints were executed. LEF-NLCs coated with either chondroitin sulphate (CHS) or chitosan (CS) were around 250 nm in size with negative or positive charge, respectively. Particle coating was evidenced by TEM and FTIR analysis. NLCs generally ensured sustained release profile up to 21 days, particularly extended in coated formulations. In vivo pharmacokinetic study of LEF suspension, uncoated NLCs, CHS- and CS-coated NLCs was carried out. Following oral administration in RA-induced rat model, joint diameter, paw inflammation, liver functions were measured, in addition to histological examination of liver, kidney and joints. Results revealed improved joint healing and reduced hepatotoxicity following treatment with nanoencapsulated LEF compared to LEF suspension, whereby CHS-NLCs ensued the highest Cmax, AUC and lowest TNF-α level. The dual potential of CHS to achieve active targeting to CD44-receptor and hence maximize LEF concentration at the target site in addition to its synergistic effect in joint healing endow promises for a competent oral nanosystem for targeted drug delivery to the joints.

Characterization and bioactivity of self-assembled anti-angiogenic chondroitin sulfate-ES2-AF nanoparticle conjugate.

BACKGROUND: In the past few years, significant progress has been made in inhibiting neovascularization at the tumor site, cutting off the nutrient supply of the tumor, and inhibiting tumor growth and metastasis. However, many proteins/peptides have the disadvantage of poor stability, short half-life, and uncertain targeting ability. Chemical modification can be used to overcome these disadvantages; many polyethylene glycol-modified proteins/peptides have been approved by US FDA. The purpose of this study was to obtain a novel anti-angiogenic chondroitin sulfate (CS)-peptide nanoparticle conjugate with efficient anti-neovascularization and tumor targeting ability and an acceptable half-life. MATERIALS AND METHODS: The CS-ES2-AF nanoparticle conjugate was synthesized and characterized using 1H-nuclear magnetic resonance spectroscopy, transmission electron microscopy, and particle size and zeta potential analyzer. The anti-angiogenic ability was studied using MTT, migration, tube formation, and chick chorioallantoic membrane assays. The targeting ability of CS-ES2-AF was studied by ELISA, surface plasmon resonance, and bioimaging. The pharmacokinetics was also studied. RESULTS: The CS-ES2-AF could self-assemble into stable nanoparticles in aqueous solution, which significantly enhances its anti-neovascularization activity, tumor targeting more explicit, and prolongs its half-life. CONCLUSION: CS is an effective protein/peptide modifier, and CS-ES2-AF displayed good potential in tumor targeting therapy.

Novel Surface Coatings Using Oxidized Glycosaminoglycans as Delivery Systems of Bone Morphogenetic Protein 2 (BMP-2) for Bone Regeneration.

Tissue engineering of bone requires the delivery of growth factors in a localized, sustained manner. Here, chitosan is used as polycation, while heparin and chondroitin sulfate are employed either as native or oxidized polyanions for formation of multilayers by layer-by-layer technique. The use of oxidized heparin and oxidized chondroitin sulfate permits additional stabilization by cross-linking through imine bond formation between amino groups of polycations and aldehydes of oxidized glycosaminoglycans (oGAGs). Since these multilayers are highly hydrophilic, adhesion of C2C12 myoblasts is improved either by the use of a specific 4 + 9 pH regime with native glycosaminoglycans or a terminal collagen I layer in case of oGAGs. Adhesion and proliferation studies with C2C12 myoblasts, seeded either on bone morphogenetic protein (BMP-2) loaded or non-loaded multilayers, show that intrinsic cross-linking in oGAG-based multilayers supports cell adhesion, spreading, proliferation, and subsequent cell differentiation into osteoblasts. This is related to higher thickness and roughness of multilayers made of oGAGs compared to their native counterparts studied by ellipsometry and atomic force microscopy. Taken together, oGAG multilayer systems provide stable surface coatings and are useful as biocompatible reservoirs for sustained release of BMP-2, paving the way for coating implants and scaffolds for repair and regeneration of bone.

Enhancing the Intestinal Permeation of the Chondroprotective Nutraceuticals Glucosamine Sulphate and Chondroitin Sulphate Using Conventional and Modified Liposomes.

BACKGROUND: Liposomes are promising systems for the delivery of macromolecules and poorly absorbed drugs, owing to their ability to compartmentalize drugs, their biodegradability and biocompatibility. OBJECTIVE: The aim of the present study was to formulate and evaluate conventional and modified glucosamine sulphate (GluS) and chondroitin sulphate (CS) liposomal formulations, to enhance their oral permeation for the treatment of osteoarthritis (OA). METHOD: Liposomal formulations were prepared by the thin-film hydration method using two types of phospholipids; Epikuron 200© and Epikuron 200© SH, and three permeation enhancers; poloxamer 407, cetylpyridinium chloride, and sodium deoxycholate. In-vitro characterization of liposomal formulations was conducted in terms of entrapment efficiency, particle size, zeta potential, viscosity, physical stability and mucoadhesive strength. Surface morphology and vesicle shape, ex-vivo intestinal permeation, and histopathological studies were further carried out on the selected formulation. RESULTS: Results showed that the liposomal formulation containing sodium deoxycholate was the most optimum formula, showing high entrapment efficiency (60.11% for GluS and 64.10% for CS) with a particle size of 4.40 µm, zeta potential of -17.2 mV and viscosity of 2.50 cP. CONCLUSION: The aforementioned formula displayed the highest cumulative % permeated of GluS and CS through rabbit intestinal mucosa compared to the solution of drugs and other liposomal formulations (64.20% for GluS and 78.21% for CS) after 2 hours. There were no histopathological alterations in the intestinal tissue, suggesting the safety of the utilized liposomal formulation. In light of the above, liposomes can be considered promising oral permeation-enhancer system for GluS and CS, which is worthy of future bioavailability experimentation.

Efecto del condroitín sulfato en la sinovitis de pacientes con artrosis de rodilla / Effect of chondroitin sulphate on synovitis of knee osteoarthritic patients

Objetivo: Evaluar mediante ecografía el efecto del condroitín sulfato (CS) en la sinovitis de pacientes con artrosis (OA) de rodilla, y colaborar en el conocimiento de los mecanismos bioquímicos involucrados en la inflamación sinovial. Métodos: Estudio controlado, aleatorizado, ciego simple de 70 pacientes con OA de rodilla tratados durante 6 meses con CS o paracetamol (PCT). Los pacientes fueron visitados a tiempo basal, a las 6 semanas, y a los 3 y 6 meses para valorar el estado de su OA según los siguientes parámetros: sinovitis evaluada mediante ecografía (según definición de expertos OMERACT); dolor y función, mediante la escala visual analógica y el índice de Lequesne; y concentración de mediadores inflamatorios en suero y líquido sinovial, mediante ELISA. Resultados: El tratamiento con CS redujo en un 50% el número de individuos que presentaban sinovitis; sin embargo, se observó un incremento de un 123% en el grupo tratado con PCT. En los pacientes sin sinovitis inicial, se observó el establecimiento de esta en un 85,71 y 25% de los casos tratados con PCT y CS, respectivamente. Ambas terapias mejoraron la función articular, pero únicamente el tratamiento con CS produjo una mejora significativa del dolor al final del tratamiento. Se observó una asociación entre el tratamiento con CS y los cambios en la concentración de RANTES y UCN en el líquido sinovial. Conclusiones: El tratamiento con CS tiene un efecto mantenido beneficioso, previniendo la aparición de sinovitis o disminuyendo su presencia, así como reduciendo los síntomas de la artrosis. El PCT también mejora los síntomas clínicos, pero no tiene ningún efecto sobre la inflamación. Las variaciones observadas en la concentración de RANTES y UCN podrían estar relacionadas con el efecto antiinflamatorio asociado al tratamiento con CS (AU)

Objective: To evaluate by ultrasonography the effect of chondroitin sulfate (CS) on synovitis in patients with knee osteoarthritis (KOA). To collaborate in the understanding of the biochemical mechanisms involved in the synovial inflammation process. Methods: Randomized, single-blind, controlled trial involving 70 patients with primary KOA treated for 6 months with CS or acetaminophen (ACT). Evaluation of KOA status at baseline, 6 weeks, 3 and 6 months included: ultrasonography to assess synovitis (following the OMERACT expertise group definition), visual analogue scale and Lequesne index to measure pain and function, and ELISA to quantify inflammatory mediators in serum and synovial fluid. Results: Synovitis presence was reduced by 50% in the CS group while a 123% increase was observed in ACT group. Conversely, patients without initial synovitis and treated with ACT reached 85.71% synovitis onset, but only 25% in CS group. Both therapies improved articular function, but only CS resulted in significant pain improvement at the end of the treatment. Changes in RANTES and UCN synovial fluid concentration were associated with CS treatment. Conclusions: Treatment with CS had a sustained beneficial effect, preventing synovitis onset or reducing its presence as well as reducing KOA symptoms. ACT ameliorated clinical symptoms but had no effect on inflammation. The CS anti-inflammatory effect could be related to the observed changes in RANTES and UCN concentration (AU)

Efectos del condroitín sulfato sobre la respuesta cerebral a la estimulación dolorosa en pacientes con artrosis de rodilla. Estudio de resonancia magnética funcional aleatorizado, doble ciego y controlado con placebo / Effects of chondroitin sulfate on brain response to painful stimulation in knee osteoarthritis patients. A randomized, double-blind, placebo-controlled functional magnetic resonance imaging study

Introducción: La artrosis de rodilla es causa de dolor e incapacidad funcional. Uno de los problemas para evaluar la eficacia de los analgésicos ha sido la falta de medidas objetivas de dolor, aunque la resonancia magnética funcional (RMf) ha surgido como un medio útil para objetivar la respuesta del cerebro a la estimulación dolorosa. Hemos investigado el efecto del condroitín sulfato (CS) sobre la respuesta del cerebro a la estimulación dolorosa de la rodilla en pacientes con artrosis mediante RMf. Métodos: Veintidós pacientes recibieron CS (800mg/día) y 27 placebo y fueron evaluados inicialmente y después de 4 meses de tratamiento. En cada sesión de RMf se aplicó presión dolorosa sobre la interlínea de la rodilla y en la superficie de la rótula. El resultado se cuantificó como la atenuación de la respuesta cerebral a la estimulación dolorosa de la rodilla. Resultados: La RMf de la maniobra rotuliana mostró una reducción de la activación en la región de la sustancia gris periacueductal del mesencéfalo significativamente mayor durante el tratamiento con CS que en la condición de placebo. El grupo de CS, pero no el de placebo, mostró además una reducción de la activación en la representación cortical de la pierna tras el tratamiento. No se observaron efectos del CS con presión dolorosa sobre la interlínea de la rodilla. Conclusiones: La RMf fue sensible para objetivar los efectos del CS sobre la respuesta del cerebro a la presión dolorosa sobre el cartílago rotuliano-femoral, que es un resultado coherente con la acción conocida del CS sobre la regeneración de los condrocitos. El presente trabajo muestra nuevamente la utilidad de la RMf para objetivar los efectos del tratamiento en el dolor de origen artrósico (AU)

Introduction: Knee osteoarthritis is causing pain and functional disability. One of the inherent problems with efficacy assessment of pain medication was the lack of objective pain measurements, but functional magnetic resonance imaging (fMRI) has emerged as a useful means to objectify brain response to painful stimulation. We have investigated the effect of chondroitin sulfate (CS) on brain response to knee painful stimulation in patients with knee osteoarthritis using fMRI. Methods: Twenty-two patients received CS (800mg/day) and 27 patients placebo, and were assessed at baseline and after 4 months of treatment. Two fMRI tests were conducted in each session by applying painful pressure on the knee interline and on the patella surface. The outcome measurement was attenuation of the response evoked by knee painful stimulation in the brain. Results: fMRI of patella pain showed significantly greater activation reduction under CS compared with placebo in the region of the mesencephalic periaquecductal gray. The CS group, additionally showed pre/post-treatment activation reduction in the cortical representation of the leg. No effects of CS were detected using the interline pressure test. Conclusions: fMRI was sensitive to objectify CS effects on brain response to painful pressure on patellofemoral cartilage, which is consistent with the known CS action on chondrocyte regeneration. The current work yields further support to the utility of fMRI to objectify treatment effects on osteoarthritis pain (AU)

Chondroitin/doxorubicin nanoparticulate polyelectrolyte complex for targeted delivery to HepG2 cells.

Chondroitin (Chn) sulphate composed of N-acetyl galactoseamine units was chosen to target doxorubicin (DOX) to asialoglycoprotein receptors (ASGPRs) overexpressed in HepG2 cells of hepatocellular carcinoma (HCC). Two different ways of targeting the drug to the receptors were compared with each other; (i) by polyelectrolyte complex formation of DOX and Chn (DC), (ii) by loading the drug in gelatin nanoparticles (NPs) and then coating them by Chn. The characteristics of DC complexes were determined by Fourier transform infrared spectroscopy, differential scanning calorimetry and CHN analysis. The complexes and Chn coated NPs were characterised for their particles size, zeta potential, drug loading and release efficiency. The morphology of NPs was studied by transmission electron microscopy. The cytotoxicity of DC complex and Chn coated NPs were compared on HepG2 cells by MTT assay. The results showed that the cytotoxicity of both Chn coated gelatin NPs and DC complexes were significantly increased in comparison with free DOX. However, the presence of Chn did not have significant effect on the cytotoxicity of DOX loaded NPs. It was concluded that polyelectrolyte complex of DC could successfully target the drug to the hepatic ASGPRs and may be a simple promising way for targeted drug delivery in HCC.

Reduction-sensitive micelles self-assembled from amphiphilic chondroitin sulfate A-deoxycholic acid conjugate for triggered release of doxorubicin.

Reduction-sensitive chondroitin sulfate A (CSA)-based micelles were developed. CSA was conjugated with deoxycholic acid (DOCA) via a disulfide linkage. The bioreducible conjugate (CSA-ss-DOCA) can form self-assembled micelles in aqueous medium. The critical micelle concentration (CMC) of CSA-ss-DOCA conjugate is 0.047mg/mL, and its mean diameter is 387nm. The anticancer drug doxorubicin (DOX) was chosen as a model drug, and was effectively encapsulated into the micelles with high loading efficiency. Reduction-sensitive micelles and reduction-insensitive control micelles displayed similar DOX release behavior in phosphate buffered saline (PBS, pH7.4). Notably, DOX release from the reduction-sensitive micelles in vitro was accelerated in the presence of 20mM glutathione-containing PBS environment. Moreover, DOX-loaded CSA-ss-DOCA (CSA-ss-DOCA/DOX) micelles exhibited intracellular reduction-responsive characteristics in human gastric cancer HGC-27 cells determined by confocal laser scanning microscopy (CLSM). Furthermore, CSA-ss-DOCA/DOX micelles demonstrated higher antitumor efficacy than reduction-insensitive control micelles in HGC-27 cells. These results suggested that reduction-sensitive CSA-ss-DOCA micelles had the potential as intracellular targeted carriers of anticancer drugs.

Covalent and injectable chitosan-chondroitin sulfate hydrogels embedded with chitosan microspheres for drug delivery and tissue engineering.

Injectable hydrogels and microspheres derived from natural polysaccharides have been extensively investigated as drug delivery systems and cell scaffolds. In this study, we report a preparation of covalent hydrogels basing polysaccharides via the Schiff' base reaction. Water soluble carboxymethyl chitosan (CMC) and oxidized chondroitin sulfate (OCS) were prepared for cross-linking of hydrogels. The mechanism of cross-linking is attributed to the Schiff' base reaction between amino and aldehyde groups of polysaccharides. Furthermore, bovine serum albumin (BSA) loaded chitosan-based microspheres (CMs) with a diameter of 3.8-61.6µm were fabricated by an emulsion cross-linking method, followed by embedding into CMC-OCS hydrogels to produce a composite CMs/gel scaffold. In the current work, gelation rate, morphology, mechanical properties, swelling ratio, in vitro degradation and BSA release of the CMs/gel scaffolds were examined. The results show that mechanical and bioactive properties of gel scaffolds can be significantly improved by embedding CMs. The solid CMs can serve as a filler to toughen the soft CMC-OCS hydrogels. Compressive modulus of composite gel scaffolds containing 20mg/ml of microspheres was 13KPa, which was higher than the control hydrogel without CMs. Cumulative release of BSA during 2weeks from CMs embedded hydrogel was 30%, which was significantly lower than those of CMs and hydrogels. Moreover, the composite CMs/gel scaffolds exhibited lower swelling ratio and slower degradation rate than the control hydrogel without CMs. The potential of the composite hydrogel as an injectable scaffold was demonstrated by encapsulation of bovine articular chondrocytes in vitro. These results demonstrate the potential of CMs embedded CMC-OCS hydrogels as an injectable drug and cell delivery system in cartilage tissue engineering.

Polyethylene glycol-conjugated chondroitin sulfate A derivative nanoparticles for tumor-targeted delivery of anticancer drugs.

Polyethylene glycol (PEG)-decorated chondroitin sulfate A-deoxycholic acid (CSD) nanoparticles (NPs) were fabricated for the selective delivery of doxorubicin (DOX) to ovarian cancer. CSD-PEG was synthesized via amide bond formation between the NH2 group of methoxypolyethylene glycol amine and the COOH group of CSD. CSD-PEG/DOX NPs with a 247nm mean diameter, negative zeta potential, and >90% drug encapsulation efficiency were prepared. Sustained and pH-dependent DOX release profiles from CSD-PEG NPs were observed in dissolution tests. Endocytosis of NPs by SKOV-3 cells (CD44 receptor-positive human ovarian cancer cells), based on the CSA-CD44 receptor interaction, was determined by flow cytometry and confocal laser scanning microscopy (CLSM) studies. PEGylation of NPs also resulted in reduced drug clearance (CL) in vivo and improved relative bioavailability, compared to non-PEGylated NPs, as determined by the pharmacokinetic study performed after intravenous administration in rats. Developed CSD-PEG NPs can be a promising delivery vehicle for the therapy of CD44 receptor-expressing ovarian cancers.

Poly-ion Complex of Chondroitin Sulfate and Spermine and Its Effect on Oral Chondroitin Sulfate Bioavailability.

Chondroitin sulfate (CS) has been accepted as an ingredient in health foods for the treatment of symptoms related to arthritis and cartilage repair. However, CS is poorly absorbed through the gastrointestinal tract because of its high negative electric charges and molecular weight (MW). In this study, poly-ion complex (PIC) formation was found in aqueous solutions through electrostatic interaction between CS and polyamines-organic molecules having two or more primary amino groups ubiquitously distributed in natural products at high concentrations. Characteristic properties of various PICs generated by mixing CS and natural polyamines, including unusual polyamines, were studied based on the turbidity for PIC formation, the dynamic light scattering for the size of PIC particles, and ζ-potential measurements for the surface charges of PIC particles. The efficiency of PIC formation between CS and spermine increased in a CS MW-dependent manner, with 15 kDa CS being critical for the formation of PIC (particle size: 3.41 µm) having nearly neutral surface charge (ζ-potential: -0.80 mV). Comparatively, mixing tetrakis(3-aminopropyl)ammonium and 15 kDa of CS afforded significant levels of PIC (particle size: 0.42±0.16 µm) despite a strongly negative surface charge (-34.67±1.15 mV). Interestingly, the oral absorption efficiency of CS was greatly improved only when PIC possessing neutral surface charges was administered to mice. High formation efficiency and electrically neutral surface charge of PIC particles are important factors for oral CS bioavailability.

Preclinical evaluation of (99m)Tc labeled chondroitin sulfate for monitoring of cartilage degeneration in osteoarthritis.

PURPOSE: In previous in-vitro and ex-vivo studies we proved the specific uptake of (99m)Tc radiolabeled chondroitin sulfate (CS) in human articular cartilage. As a logical next step for the clinical use for imaging osteoarthritis we investigated in-vivo uptake of (99m)TcCS in dogs. PROCEDURES: The radiolabeling of CS Condrosulf (IBSA, Lugano, Switzerland) was performed using 25mg of CS and 20-40MBq/kg body weight of (99m)Tc by means of the tin method. In-vivo uptake of (99m)TcCS was evaluated in dogs (n=12, castrated males, 4-9years, with 15-51kg body weight). 6 healthy dogs served as controls and 6 with clinical and radiological signs of osteoarthritis in the carpal, elbow, and tarsal joint were examined. The tracer was i.v. injected into the external cephalic vein. The uptake was monitored after 2, 4, 6 and 24h in healthy and osteoarthritic dogs using a planar gamma camera by regional planar or whole body ventral and dorsal acquisition. For whole body scintigraphy animals were under general anesthesia, for planar under sedation only. RESULTS: In healthy control dogs we did not detect any specific uptake of (99m)TcCS in the cartilage. In contrast, in the diseased dogs suffering from osteoarthritis a significant, specific, persistent uptake between 4 and 6h in tarsal, carpal and cubital joints was documented. Median target (joint) to background (mid antebrachium) ratio (T/B) in the OA joints after 4, 6, and 24h was significantly higher than in healthy controls. Target to background ratio using soft tissue as a background (T/S) a similar significantly higher than in healthy controls. In all osteoarthritic joints we found a significant positive correlation (r=0.8, n=20) between grade of disease (I-III) and T/B. When matching radiographic (X ray) changes in osteoarthritic joints (grade II and III) we found also a maximal uptake of (99m)TcCS at the specific anatomical site of highest cartilage degeneration. None of the dogs experienced any side effects. CONCLUSION: These results suggest that (99m)TcCS might become a promising diagnostic tool for imaging osteoarthritis. More extensive and detailed examinations are required, however, before extending this methodology for application in humans.

Degradation of chondroitin sulfate by the gut microbiota of Chinese individuals.

Oral preparations of chondroitin sulfate (CS) have long been used as anti-osteoarthritis (anti-OA) drugs. However, little is known about the degradation of CS by human gut microbiota. In the present study, degradation profiles of CSA (the main constituent of CS drugs) by the human gut microbiota from six healthy subjects were investigated. Each individual's microbiota had differing degradation activities, but ΔUA-GalNAc4S was the end product in all cases. To elucidate the mechanisms underlying this phenomenon, different CSA-degrading bacteria were isolated from each individual's microbiota and tested for CSA degradation. In addition to Bacteroides thetaiotaomicron J1, Bacteroides thetaiotaomicron 82 and Bacteroides ovatus E3, a new CSA-degrading bacterium, Clostridium hathewayi R4, was isolated and characterized. Interestingly, at least two different CSA-degrading species were identified from each individual's gut microbiota. Predictably, these functional bacteria also had differing degradation rates, but still generated the same end product, ΔUA-GalNAc4S. In addition, the human fecal isolates produced different degradation profiles for CSC, CSD, and CSE, suggesting that CS could be readily metabolized to varying extents by diverse microbial consortiums, which may help to explain the poor bioavailability and unequal efficacy of CS among individuals in OA treatment.

Distinct CPT-induced deaths in lung cancer cells caused by clathrin-mediated internalization of CP micelles.

We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of poly(ε-caprolactone) (18.7 mol%), which self-assembled in water into a rod-like micelle to encapsulate hydrophobic camptothecin (CPT) in the core (micelle/CPT) for tumor-targeted drug delivery. As a result of the recognition of the micelle by CD44, the micelle/CPT entered CRL-5802 cells efficiently and released CPT efficaciously, resulting in higher tumor suppression than commercial CPT-11. In this study, H1299 cells were found to have a higher CD44 expression than CRL-5802 cells. However, the lower CD44-expressing CRL-5802 cells had a higher percentage of cell death and higher cellular uptake of the micelle/CPT than the higher CD44-expressing H1299 cells. Examination of the internalization pathway of the micelle/CPT in the presence of different endocytic chemical inhibitors showed that the CRL-5802 cells involved clathrin-mediated endocytosis, which was not found in the H1299 cells. Analysis of the cell cycle of the two cell lines exposed to the micelle/CPT revealed that the CRL-5802 cells arrested mainly in the S phase and the H1299 cells arrested mainly in the G2-M phase. A consistent result was also found in the evaluation of γ-H2AX expression, which was about three-fold higher in the CRL-5802 cells than in the H1299 cells. A near-infrared dye, IR780, was encapsulated into the micelle to observe the in vivo biodistribution of the micelle/IR780 in tumor-bearing mice. The CRL-5802 tumor showed a higher fluorescence intensity than the H1299 tumor at any tracing time after 1 h. Thus we tentatively concluded that CRL-5802 cells utilized the clathrin-mediated internalization pathway and arrested in the S phase on exposure to the micelle/CPT; all are possible reasons for the better therapeutic outcome in CRL-5802 cells than in H1299 cells.

A self-assembled system for tumor-targeted co-delivery of drug and gene.

A new cationic polymer eprosartan-graft-PEI (ESP) containing eprosartan (ES) and polyethylenimine 1.8K was successfully developed and employed as a safe gene vector to assemble a drug (ES) and gene co-delivery complex (ESP/pDNA). Chondroitin sulfate (CS) was then used as a coating polymer to shield the surface charge of ESP/pDNA complexes, as well as a tumor targeting entity to ensure specific delivery of CS/ESP/pDNA complexes. The CS/ESP/pDNA complexes with desirable particle size and zeta potential, exhibited amidase-responsive drug release and CS-mediated endocytosis in vitro. As compared with ESP/pDNA complexes, in vivo imaging results demonstrated decreased reticuloendothelial system uptake and remarkably increased tumor accumulation of CS/ESP/pDNA complexes. All these findings indicated the potential of CS/ESP/pDNA as a promising tumor-targeted drug and gene co-delivery system.