Absorption Mechanism Of Baicalin Solid Lipid Nanoparticleson Caco-2 Cell Model

Baicalin(BA)is a flavonoid extracted from the dried rhizome of Scutellaria baicalensis Georgi.It has remarkable biological activity and is antibacterial,anti-inflammatory,anti-oxidant and anti-viral.Pharmacological effects such as immune regulation.The intramolecular hydrogen bond between flavonoids and glucuronide in BA molecular structure leads to poor water solubility and fat solubility,and the oral bioavailability is only 2.2%,which limits the clinical application.Solid lipid nanoparticles(SLN)are microparticle delivery systems with particle sizes ranging from10 to 1000 nm.They are characterized by low toxicity,good biocompatibility,biodegradability,and good Targeting,SLN is used to wrap poorly soluble drugs to increase their absorption in the body.In order to improve the oral bioavailability of BA,the previous research group prepared baicalin solid lipid nanoparticles(BA SLN)by loading it into SLN.SLN can improve the bioavailability of BA and improve the absorption of BA.However,the mechanism of SLN to improve BA absorption is still unclear.There are few studies on the oral absorption mechanism of BA SLN at home and abroad.Intestinal absorption is a key step in the process of drug absorption.The most commonly used model is the human colon adenocarcinoma Caco-2 cell model.After a period of culture,Caco-2 cells have similar morphology,marker enzyme expression,uptake and transport and permeability characteristics to intestinal epithelial cells,so they are often used for the study of drug absorption mechanism in small intestinal epithelial cells.Based on the previous work,this study established a Caco-2 cell uptake and transport model,investigated the uptake and transport characteristics of BA and BA SLN in intestinal epithelial cells,and preliminarily explained the mechanism of SLN improving BA intestinal absorption,in order to provide theoretical basis for BA SLN prescription design.Objective:To explore the uptake characteristics and transport mechanism of BA and BA SLN in Caco-2 cell model,and to elucidate the absorption mechanism of BA and BA SLN in intestinal epithelial cells.Method:An in vitro Caco-2 cell uptake and transport model was established to observe cell morphology,transmembrane resistance(TEER)and ratio of activity of alkaline phosphatase(ALP)on the apical side(AP)to the basal side(BL).Densification and polarization characteristics;the toxicity of BA and BA SLN to Caco-2 cells was detected by CCK-8 method and LDH method,and the safe concentration was determined.The determination method of BA in cells was established and the method was verified.The effects of time,concentration,temperature and endocytosis inhibitors on the Caco-2 cell uptake of BA and BA SLN were investigated.The inoculation mode and uptake characteristics of BA and BA SLN were investigated.The transport experiments showed that BA and BA SLN were in Caco-2 Apparent permeability coefficient(Papp)and efflux rate(ER)on cells;laser confocal microscopy to detect the distribution of Zona occluding-1(ZO-1);using P-glycoprotein(P-gp),multidrug resistant drug-associated protein 2(MRP2)and breast cancer resistance protein(BCRP)protein inhibitors were used to study the effects of efflux transporters on the transport of BA and BA SLN;Western blotting was used to detect ZO-1,P-gp,MRP2 and BCRP proteins.expression.Results:1.Establishment of Caco-2 cell uptake and transport model and toxicity testThe Caco-2 cells were cultured for 5-7 days,and observed under an inverted microscope.The fusion was basically achieved,and the boundary was clear and paved.After culturing for 14 days,the morphological observation showed that the cells were uniform and dense.The TEER of Caco-2 cells increased with the prolongation of culture time.At 17 and 21 days,the TEER values were 545.84?·cm ~2 and 556.98?·cm~2 respectively.The ratio of ALP activity between AP side and BL side of this cell model varied with the culture time.Prolonged and increased,the ratio was 5.05±0.09,9.10±0.25 when cultured for 14 and 21 days,respectively,which can be used for cell transport experiments.The CCK-8 method was used to detect the toxicity of BA and BA SLN on Caco-2 cells.The cell survival rate decreased with the increase of drug concentration.When BA and BA SLN concentrations were 0-150?g/mL,the cells were incubated for 24 h.Cell viability was not significantly different from the blank group;LDH method was used to detect the integrity of the cell membrane.The release rate of lactate dehydrogenase did not change significantly in the concentration range of 0-150?g/mL;the drug concentration reached 200?g/mL and above.The release rate of lactate dehydrogenase increased significantly.2.Uptake characteristics of baicalin solid lipid nanoparticles in Caco-2cell modelWhen the drug concentration was 150?g/mL,the uptake of BA and BA SLN in Caco-2 cells increased with time in 0-1 h.BA and BA SLN at 0-150?g/mL concentration in Caco-2 cells for 1 h,the uptake increased with the increase of concentration,and the cell uptake of BA SLN solution was significantly higher at each concentration.BA solution.When the drug concentration was 150?g/mL,the cells were incubated at 4°C and 37°C for 1 h,respectively.The uptake of BA and BA SLN in Caco-2 cells increased with the increase of temperature.When the endocytosis inhibitors?-cyclodextrin and chlorpromazine were present,there was no significant change in the uptake of BA on Caco-2 cells,while the uptake of BA SLN on Caco-2cells was significantly reduced.3.Transport mechanism of baicalin solid lipid nanoparticles in Caco-2cell modelCompared with the BA group,the Papp of the BA SLN group increased significantly from AP to BL in the Caco-2 cell monolayer,and the ER decreased significantly.The intercellular tight junction protein ZO-1 in the BA group and the BA SLN group showed a continuous shape,and Compared with the blank group,there was no significant difference in the expression of ZO-1 protein between BA group and BA SLN group;MRP2 inhibitor MK-571 and BCRP inhibitor KO-143 could reduce the efflux rate of BA,while BA SLN was not affected.The effect of protein inhibitors was not significantly affected by BA expression of MRP2 and BCRP compared with the blank group.BA SLN significantly decreased the expression of efflux protein MRP2and BCRP protein.Conclusion:1.The Caco-2 cell uptake and transport model was successfully established;the safe concentration of BA and BA SLN on cells was 0-150?g/mL.2.The uptake pattern of BA on Caco-2 cells may be passive diffusion and active transport;BASLN has passive diffusion and active transport on Caco-2 cells,which is affected by endocytosis inhibitors,BA SLN It can be inoculated by endocytosis,thereby increasing BA intake.3.BA SLN significantly increased the transmembrane transport capacity of BA,and BA SLN was unable to open tight junctions between cells,and its ability to increase drug transmembrane transport was independent of the cell bypass pathway.BA is susceptible to efflux protein MRP2 and BCRP,which is poorly absorbed by extracellular efflux.After BA is loaded into SLN,BA SLN can avoid the efflux of such efflux proteins,thereby reducing drug efflux.