chondroitin-sulfates and 1-ethyl-3-(3-dimethylaminoethyl)carbodiimide

Folic acid (FA) was selected to link with macromolecules for the selective and specific delivery of doxorubicin (DOX) to folate receptor (FR)-positive tumor cells, because of the high binding affinity of FA to the tumor-associated FR. We synthesized folate-mediated chondroitin sulfate (FA-PEG-ChS) for tumor cell targeting and non-folate-mediated naproxen-linked chondroitin sulfate (Nap-PEG-ChS) for comparison. Both the aforementioned polymers contain a PEG1000 spacer. We encapsulated an anticancer agent, DOX, during the formation of complexes with chitosan. Polyelectrolyte complexes (PEC) grafted with a fluorescent dye (FITC) served as a platform for online imaging cellular internalization. FR-positive KB and FR-deficient A549 cancer cells were tested. The concentration to kill 50% of the cells (IC(50)) of DOX-loaded FA-complex was 1.53 μg/ml, in comparison to 0.91 μg/ml of free DOX. The overlaid fluorescent images of DOX and FITC on confocal laser scanning microscopy demonstrated the co-internalization of DOX and the complex nanoparticles into the cytoplasm of KB cells followed by a gradual release of DOX.

Topics: Antineoplastic Agents; Carbodiimides; Cell Survival; Chondroitin Sulfates; Doxorubicin; Drug Carriers; Endocytosis; Folic Acid; Folic Acid Transporters; Humans; KB Cells; Polyethylene Glycols

The use of primary hepatocyte cultures as in vitro models for studying xenobiotic metabolism and toxicity is limited by the loss of liver-specific differentiated functions with time in culture and the inability of the cells to proliferate. The aim of this study was to investigate the effect of incorporating 20% chondroitin-6-sulphate (Ch6SO4), a glycosaminoglycan (GAG), into collagen gels (0.3% w/v) and crosslinking the gels with either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression of glutathione-S-transferases (GSTs) and the activity of cytochrome P450 in hepatocytes cultured for 48 hours and 7 days. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion. Cell homogenates were immunoblotted against class alpha and pi GST subunits. To measure cytochrome P450 activity, testosterone hydroxylation was assessed. Viability of the cultured cells was assessed by confocal laser scanning microscopy using the vital stain carboxyfluorescein diacetate (CFDA). Cells cultured on gels crosslinked with EDAC were dead by 48 hours as judged by lack of CFDA-derived fluorescence and absence of GST bands on the immunoblots. The viability and morphology of the cells were unaffected by any of the other components of the substrata tested. Expression of GSTs indicated that the hepatocyte phenotype was stable for at least 48 hours. The addition of GAG did not improve the phenotype at either 48 hours or 7 days in culture, but the combination of GAG and DAH crosslinking improved GST expression in the 7-day cultures. However, the hepatocyte cytochrome P450 activity did not show any improvement on any of the gels. The combination of GAG and DAH crosslinking provided the most stable substratum environment in terms of GST expression in hepatocytes.

Topics: Animals; Carbodiimides; Cell Culture Techniques; Cell Survival; Cells, Cultured; Chondroitin Sulfates; Collagen; Cross-Linking Reagents; Culture Media; Cytochrome P-450 Enzyme System; Diamines; Drug Combinations; Gels; Glutathione Transferase; Hepatocytes; Male; Phenotype; Rats; Rats, Sprague-Dawley

The soluble form of the syndecan-1 heparan sulfate proteoglycan acts as a tumor suppressor molecule that inhibits growth and induces apoptosis of some cancer cell lines in vitro. Analogs of syndecan-1 were produced by carbodiimide (EDAC) conjugation of glycosaminoglycan (GAG) chains to a protein scaffold, thereby generating synthetic proteoglycans that were evaluated for anticancer properties. Surprisingly, when analyzing activities of the controls, we discovered that EDAC modified GAG chains inhibit myeloma cell viability even in the absence of protein. Here, we describe the production and the activities of these novel molecules called neoglycans. The GAG chains heparin and chondroitin sulfate (CS) were exposed to EDAC to generate the neoglycans neoheparin and neoCS, respectively. Heparin and CS in the absence of EDAC modification have no effect or a slight growth promoting effect on cancer and normal cell lines. However, neoheparin and neoCS substantially reduce cell viability by induction of apoptosis of myeloma and breast cancer cells in vitro. NeoCS when injected directly into breast tumors growing in nude mice reduces or abolishes their growth without causing apparent toxicity to the adjacent normal tissue. The neoglycans need not be continuously present in cell cultures because a short pulse exposure is sufficient to reduce cell viability. NeoCS fractions purified by size exclusion chromatography reduce myeloma cell viability, confirming the specificity of neoglycan activity. Collectively, the results of this study demonstrate the anticancer activities of this new class of GAG chain-based molecules and provide the foundation for future development of neoglycans as novel therapeutic agents.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carbodiimides; Cell Division; Cell Survival; CHO Cells; Chondroitin Sulfates; Cricetinae; Dogs; Drug Screening Assays, Antitumor; Female; Glycosaminoglycans; Heparin; Humans; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Multiple Myeloma; Proteoglycans; Syndecan-1; Syndecans; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Artificial skin substitutes based on autologous keratinocytes cultured on collagen-based substrata are being developed for grafting onto patients with severe burns. The properties of the substratum can be manipulated by crosslinking the collagen with the glysocaminoglycan, chondroitin-6-sulphate (Ch6SO4), carbodiimides and polyamines. Biological stability, assessed by resistance to collagenase, was increased by incorporation of Ch6SO4, but crosslinking with the carbodiimides, 1-ethyl-3-(dimethylaminopropyl)carbodiimide and 1,1-carbonyldiimidazole or the polyamines, putrescine or diaminohexane, had little further benefit. Contraction of the collagen gels occurred to a greater extent when seeded with fibroblasts than with keratinocytes. The extent of contraction by either cell type was not influenced by the presence of Ch6SO4 in the gel, but the carbodiimides, and to a lesser extent the polyamines, limited cell-mediated contraction, particularly that mediated by fibroblasts. Optimum substratum composition for artificial skin substitutes will involve a compromise between the desired attributes of biological stability, rate of contraction, mechanical strength, biocompatibility and promotion of cell growth.

Topics: Biocompatible Materials; Carbodiimides; Cells, Cultured; Chondroitin Sulfates; Collagen; Cross-Linking Reagents; Drug Stability; Fibroblasts; Gels; Humans; Imidazoles; Keratinocytes; Skin, Artificial

This study demonstrates the effect of the glycosaminoglycans, hyaluronic acid and chondroitin-6-sulphate (Ch6SO4), diamines and a carbodiimide cross-linking agent on the growth of human epidermal cells on collagen gels. Ch6SO4 incorporated into collagen gels stimulated cell growth rate, but the effect was found to be inconsistent. We found that approximately 50% of the incorporated Ch6SO4 in the gels leached out into the growth medium after the first 3 d in culture, and this is thought to lead to the inconsistent cell growth response. In order to minimize the elution of Ch6SO4 from the gels and thereby maximize its effect on the growth of the keratinocytes, 1-100 micrograms ml-1 Ch6SO4 was added in the medium. The results showed that Ch6SO4 at these concentrations in the medium did not stimulate the cell growth on either plain collagen gels or gels containing 20% Ch6SO4. As an alternative strategy, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and diamines (putrescine or diaminohexane) were used to immobilize Ch6SO4 onto the collagen gels and to cross-link the gels. The cross-linking process partially prevented the elution of Ch6SO4 from the gels. Interestingly, only putrescine, not diaminohexane, promoted the growth of keratinocytes on the cross-linked plain collagen gels. We proposed to develop an artificial skin substitute containing putrescine as a growth factor for the human epidermal cells.

Topics: Analysis of Variance; Carbodiimides; Cell Division; Cells, Cultured; Chondroitin Sulfates; Collagen; Cross-Linking Reagents; Drug Carriers; Gels; Glycosaminoglycans; Hexanes; Humans; Hyaluronic Acid; Keratinocytes; Putrescine; Skin, Artificial; Urea