Preliminary Study On The Mechanism Of Carboxymethyl Chitosan Enhancing Oral Drugs Trans-epithelium Absorption

Most eutherapeutic biomacromolecule drugs are difficult to enter the bloodstream by absorption of small intestine after oral administration, which greatly limit their clinical application. Chitosan (CS) attracts increasing concern in oral drug carriers because of its ability to open tight junction (TJ) between intestinal epithelial cells and facilitate paracellular transport of drugs. Chitosan/carboxymethyl chitosan (CS/CMCS) nanoparticles exhibit high efficiency in enhancing absorption of oral drugs because of its ability to promote paracellular transport of drug across intestinal epithelium and improve the drug oral bioavailability. The molecular mechanism of the interaction between carboxymethyl chitosan molecule and enterocytes is still not clear. In the present study, CMCSs with different carboxymethyl substitution degree were prepared, Caco-2 cell monolayers were used as trans-epithelium absorption model, the relationship between the molecular structure of carboxymethyl chitosan and their ability to enhance the absorption of oral drugs were analyzed.Three kinds of CMCS were synthesized with CS and chloroacetic acid by controlling reaction time, labled as CMCSI, CMCS2 and CMCS3. CMCS2 and CMCS3 showed better water solubility in neutral pH than CMCS1. The fourier transform infrared spectrum showed that carboxymethyl groups were covalently connected onto chitosan molecular skeleton in CMCS1, CMCS2 and CMCS3. Differences in the peak area of carboxyl of CMCS1, CMCS2 and CMCS3 reflected different carboxyl content. Molecular weight measurement results showed that the molecular weight of CS was 397.5 kDa, CMCS 1, CMCS2 and CMCS3 were 449.1 kDa, 540.7 kDa and 605.1 kDa respectively. The degree of carboxymethyl substitution of CMCS1, CMCS2 and CMCS3 were 36%,100% and 145% respectively. The molecular weight and carboxymethyl substitution degree increased with prolonging the reaction time. Substituent of carboxymethyl of CMCS1 and CMCS2 mainly took place on -OH group of C6 and the products were primary O-CMCS. The substitution reaction of CMCS3 happened on amino groups on the C2 after carboxyl groups on the C6 was saturated and the products were primary N,O-CMCS. The Ca2+ chelating ability of CMCS1, CMCS2 and CMCS3 were significantly higher than that of CS materials, and with the increasing degree of substitution of carboxymethyl, the chelating ability was gradually strengthened.Cytotoxicity of CS and CMCS with three degree of substitution were evaluated by MTT. The result of 72h showed that relative cell proliferation ratio of each group were all over 100%,which the concentration of extract were 0.0625mg/mL-1mg/mL, and also showed good compatibility. Moreover, CS, CMCS1, CMCS2 and CMC3 have a significant effect on promoting the growth of HUVEC, MEF and Caco-2 cell. The promotion was increased with high extract concentration within test concentration.Caco-2 cell dense monolayers intestinal absorption model was constructed successfully. The aspect of chitosan and different degree of substitution of CMCS to TEER was studied. The results of TEER showed that cells’ TEER values show the gradual rise in 1 h for the rapid decline after the treatments with extracts of CS in concentration of 0.25 mg/mL,0.5 mg/mL,1 mg/mL and three kinds of CMCS samples. TEER value decreases with increasing concentration of the material velocity increases, meanwhile TEER value increases with the biggest drop of carboxymethyl substitution degree of CMCS. The effects of all groups to Caco-2 cell dense monolayers have shown reversible. Especially, CMCS3 with 0.5 mg/mL and 1 mg/mL have obvious strong ability of open cell TJ.Tight junction protein Claudin-4 immune fluorescence studies showed that before the effects of CS (0.5 mg/mL) and CMCS3, a large number of claudin-4 protein exists on the cell membrane and TJs between cells were in a complete state. After 15 minutes reaction, significantly decreased the fluorescence signal, drastically reduced claudin-4 protein and intercellular TJ was opened. After 30 and 60 minutes reaction, significantly enhanced fluorescence signal, continued to increase claudin-4 protein and intercellular TJ gradually restored.Immunofluorescence results were consistent with the results of TEER. The tests confirmed that CMCS3 could open TJ structures between Caco-2 cells effectively, thereby increasing the permeability of the paracellular pathway. A large number of claudin-4 protein appearing could be inferred the CS and CMCS did not cause TJ irreparable damage.CMCS with higher carboxymethyl substitution degree showed higher opening ability of TJ, owing to inducing effect on the transfer and redistribution of claudin-4 protein in cytomembrane, as well as chelating Ca2+ within the cadherin, leading to the inactivation of cadherin, destroying the adherens junction (AJ), then opening the TJ. The present results are promising for the development of efficiency and safe oral drugs carries by accurate designing of carboxymethyl chitosan molecules.