Transport Mechanisms Of Polymannuronic Acid And Polyguluronic Acid Across Caco-2 Cell Monolayers

As sodium alginate oligosaccharides,polymannuronic acid(PM)and polyguluronic acid(PG)have been reported to have excellent biological activities.The activity of PM and PG is closely related to the mode of administration,and the oral drug delivery system is the most widely used and most accessible manner among the various routes of administration.Therefore,a detailed knowledge of the oral transport of PM and PG is important for its biological activities.It has been widely recognized that the primary means of intestinal transport of drugs are the paracellular pathway and the transcellular pathway.On the one hand,it is difficult for polysaccharide biomacromolecules to penetrate the tight junction(TJ)between cells due to their relatively high molecular weight(Mw),which severely limits their transport through the paracellular pathway.On the other hand,transport of polysaccharide biomacromolecules through the transcellular pathway is complicated and involves endocytosis of polysaccharide molecules through the apical membrane,internalization in the cells,and ultimately exocytosis from the basolateral membrane.To investigate the transport in the gastrointestinal tract,FITC-PM and FITC-PG were synthesized by fluorescein isothiocyanate(FITC)labeling,the transport mechanisms of FITC-PM and FITC-PG across the intestinal epithelial cell monolayers(Caco-2 cell monolayers)were then investigated.The experimental process and results are as follows1.Fluorescence labeling of PM and PGPM and PG were chemically modified with tyramine and conjugated with FITC to synthesize FITC-PM and FITC-PG.The results of agarose gel electrophoresis and fluorescence spectrophotometry showed that PM and PG had been successfully labeled by FITC.The result of high-performance gel permeation chromatography showed that the Mw of PM and PG increased slightly after fluorescent labeling,which was the result of conjugation of tyramine and FITC groups on PM and PG molecules.The measurement experiments of fluorescence intensity showed that the fluorescent labeling rates of FITC-PM and FITC-PG were 12.66%and 9.52%.2.Transport mechanisms of FITC-PM and FITC-PGA rapid 7-day Caco-2 cell monolayer model was successfully established using 0.4 μg/mL puromycin,and then the effects of transport time,transport concentration,transport temperature,and inhibitor/siRNA technology on the transport PM ans PG were investigated in this model.The results of the investigation of the time showed that the transport of FITC-PM and FITC-PG increased with the prolong of time,but stopped the transfer at 240 min.The reason for this phenomenon was that the transport and efflux rates were relatively balanced at 240 min.The results of the investigation on the transport concentration showed that their transport amounts were constant regardless of the concentration,while their apparent permeability coefficient(Papp)values were negatively correlated with both transportation and concentration,indicating that their transport was canier-saturated.The results of transport temperature experiments showed that low temperature could significantly inhibit the transport of FITC-PM and FITC-PG(the relative transport percentage has been reduced by 90%and 81%),indicating that their transport was energy-dependent.The results of investigation of transport inhibitors showed that with the addition of chlorpromazine,dynasore,methyl-β-cyclodextrin(MβCD)or 5-(N-ethyl-N-isopropyl)amiloride(EIPA),the relative transport percentage of FITC-PM was reduced by 19%,65%,34%and 61%,and the relative transport percentage of FITC-PG was reduced by 58%,47%,22%and 20%,which indicated that both of them entered Caco-2 cells via the macropinocytosis pathway and the clathrin and caveolae(or lipid raft)-mediated endocytosis pathway.After silencing the clathrin heavy chain,dynamin-II or caveolin-1 using siRNA technology,the results of drug transport experiments showed that the relative transport percentage of FITC-PM was reduced by 19%,80%and 27%,the relative transport percentage of FITC-PG was reduced by 66%,30%,and 24%,which further indicated that clathrin,dynamin-II,and caveolin-1 were essential for FITC-PM and FITC-PG to enter Caco-2 cells.The results of the intracellular inhibitors experiment showed that with the addition of monensin and bafilomycin AI,the relative transport percentage of FITC-PM was decreased by 66%and 54%,and the relative transport percentage of FITC-PG was decreased by 40%and 64%,which indicated that the endosome maturation process was essential for the transport of PM and PG.The addition of nocodazole reduced the relative transport percentage of FITC-PM by 39%,but had no effect on the relative transport percentage of FITC-PG,indicating that tubulin was also involved in the transport of PM in Caco-2 cells,but may not be involved in the transport of PG.3.Effect of absorption enhancer on the transport of FITC-PM and FITC-PGThe result of the effect of chitosan(CS)on the TJ of Caco-2 cell monolayer showed that CS could reversibly open TJ between cells.Subsequently,FITC-PM or FITC-PG and CS were combined and it was found that the Papp values of FITC-PM and FITC-PG in CS-treated cells were 1.9-fold and 2.6-fold larger than those of the control group,indicating that the addition of CS significantly increased the transport of FITC-PM and FITC-PG.The results of western blotting experiments showed that CS turned on TJ may be related to the decreased expression of TJ related proteins(mainly CLDN4 and occludin proteins)In summary,PM and PG was fluorescently labeled with FITC,and then the transport mechanisms of FITC-PM and FITC-PG across the monolayer of intestinal epithelium were studied.The results demonstrated that the transport of FITC-PM and FITC-PG into epithelial cells was time-and energy-dependent,which was mediated by the macropinocytosis pathway and the clathrin-and caveolae(or lipid raft)-mediated endocytic pathway.The transport process of FITC-PM and FITC-PG in Caco-2 cells depended on the acidification of endosomes and involved lysosomes.Tubulin mediated the transport of FITC-PM,but not of FITC-PG.Moreover,CS promoted the transport of FITC-PM and FITC-PG by reversibly opening the TJ.This study provides a comprehensive understanding of the mechanisms of PM and PG absorption and transport in small intestinal epithelial cells.