Enhancing the intestinal absorption of low molecular weight chondroitin sulfate by conjugation with alpha linolenic acid and the transport mechanism of the conjugates

The purpose of this report was to demonstrate the effect of amphiphilic polysaccharides based self assembling micelles on enhancing the oral absorption of low molecular weight chondroitin sulfate (LMCS) in vitro and in vivo, and identify the transepithelial transport mechanism of LMCS micelles across the intestinal barrier. Alpha lnolenic acid low molecular weight chondroitin sulfate polymers(a ALA LMCS) were successfully synthesized, and characterized by FTIR, 1HNMR, TGA per DSC, TEM, laser light scattering and zeta potential. The significant oral absorption enhancement and elimination half life (t1/2) extension of ALA LMCS2 in rats were evidenced by intra gastric administration in comparison with CS and LMCS. Caco 2 transport studies demonstrated that the apparent permeability coefficient (Papp) of ALA LMCS2 was significantly higher than that of CS and LMCS, and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, ALA LMCS micelles accumulated around the cell membrane and intercellular space observed by confocal laser scanning microscope (CLSM). Furthermore, evident alterations in the F actin cytoskeleton were detected by CLSM observation following the treatment of the cell mono layers with a ALA LMCS micelles, which further certified the capacity of a ALA LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Therefore, ALA LMCS2 with low cyto toxicity and high
Bio availability might be a promising substitute for CS in clinical use, such as treating osteoarthritis, atherosclerosis, etc. (Authors abstract)

Chondroitin sulfate (CS) ubiquitously distributes on cell surfaces and in the extra cellular matrix (ECM) of mammalian animals. CS is particularly abundant in bones, tendons, blood vessels, nerve tissues, and cartilage. CS, a sulfated glycosaminoglycan (GAG), is composed of a repeating disaccharide unit of glucuronic acid (GlcA) and N acetylgalactosamine (GaALAc), which is commonly sulfated at the C 4 and or C 6 of GaALAc in mammals. CS has various biological functions including anti oxidation, anti inflammation, anti atherosclerosis, immuno regulation, regulating cell adhesion and morphogenesis. The main problem affecting the efficacy CS is its poor intestinal absorption, resulting from its high molecular weight, charge density, as well as hydro philicity. Nano sized polymeric micelles have been successfully applied in oral drug delivery system. The objective here is to determine whether low molecular weight amphiphilic polysaccharides based self assembling micelles would enhance the oral absorption of CS. Esterification is considered as a method to improve the hydrophobicity for many polysaccharides. In this work, LMCS micelles were prepared using ALA as the hydrophobic chain of the micelle forming materials. The physicochemical characteristics of ALA LMCSs were investigated by FTIR, 1HNMR, TGA per DSC, TEM, laser light scattering and zeta potential. The oral bioavailability of ALA LMCS micelles in vivo was evaluated by determining the ALA LMCS concentrations in plasma levels following oral administration to rats in comparison with CS, and Caco 2 cell mono layers representing in vitro model of the intestinal epithelial barrier were used to determine the intestinal transport ability of ALA LMCS micelles. All these results indicated that the ALA LMCS micelles could enhance the intestinal absorption of CS. Furthermore, confocal laser scanning microscope (CLSM) was used to study the possible mechanisms, and found that the enhancement of absorption was depending on the paracellular pathway and endocytosis. The mean diameters of self aggregates of a ALA LMCSs were in the range of 78 to 117 nm. The CACs of a ALA LMCSs were in the range of 0.027 to 0.065mg per mL. The characteristics of narrow size distribution and relatively low CACs may contribute to the stability of the micelles upon dilution in vivo and in vitro and the improved oral bioavailability of CS. It was demonstrated that ALA LMCS2 has better absorption via oral administration than normal CS and LMCS. The oral absorption of CS derivatives were contributed to the conjugated ALA molecule constituent that could promote intestinal absorption by enhancing the hydrophobic properties of LMCS and increasing the interaction between LMCS and the intestinal membrane. Moreover, the transport of ALA LMCS2 micelles across intestinal epithelial barrier was proved via
paracellular pathway and endocytosis for the first time. In summary, this study suggested that ALA LMCS2 with low cytotoxicity and high oral bioavailability would be a promising substitute for CS. Furthermore, this study provided a simple set of strategies suitable for the oral delivery of macromolecules and polysaccharide drugs. (Editors comments)