Simulation Of Nanoparticles In Chinese Medicine Decoction And Mechanism Investigation On The Promotive Berberine Absorption In Intestine

Objective:The chitosan（CS）nanoparticles were perpared to simulate the polysaccharide microparticle system in Chinese Medicine decoction.The Caco-2 cell model was established to evaluate the effects of particle with various size distributions on berberine（Ber）uptake/transport in intestine,and the related mechanisms.Methods:（1）The water-soluble chitosan were used as raw material,and chitosan particles with different sizes were prepared by ionic cross-linking.The particle sizes and surface potentials were measured by the Laser Scattering Particle Size Analyzer,and the morphology of the particles were observed by the Scanning Electron Microscope.（2）The Caco-2 cell model was established,then using electorn microscopy,transepithelial electrical resistance and the apparent permeability coefficients(P_app)of fluorescein and propranolol hydrochloride to measuer the integrity and compactness of the monolayer cell.（3）The MTT method was used to determine the toxic concentration of CS nanoparticles and Ber.The effects of particle sizes on cell uptake of berberine were investigated by the high performance liquid chromatography（HPLC）and the BCA protein assay kit.The Caco-2 model was used to study the bidirectional transport of Ber in AP→BL and BL→AP.（4）The transport of Ber at 4℃and 37℃by Caco-2 were investigated.sodium azide（Na N₃）,chlorpromazine（CPZ）,methyl-β-cyclodextrin（MβCD）,amiloride hydrochloride（EIPA）and cytochalasin D（CD）had been respectively used as the inhibitors of energy,Clathrin-mediated endocytosis,Caveolin/lipid structure-mediated endocytosis,macrolithiasis pathway and actin microfilament,and been used to investigate the mechanisms of the promotive effects of CS particles on Ber absorption in intestine.Results:（1）The CS particles,named as P1,P2 and P3,are uniform,with sizes of（450±34）,（318±14）and（670±57）nm and surface potentials of（20.2±1.6）,（17.1±0.4）and（12.8±1.0）m V,respectively.（2）The Caco-2 cell model has been established successfully and exhibits clearly observe microvilli and tight junction under electron microscope.The transmembrane resistance value is（387±13）Ω·cm².The P_appof fluorescein and propranolol hydrochloride are（8.09±2.23）×10^-7cm/s and（7.06±0.80）×10^-6cm/,respectively.（3）With analyses of MTT,the appropriate concentrations of CS nanoparticles and Ber are 15μg/m L and 10μg/m L,respectively,with survival cell rates of 88%and 78%,after 4 h treatment.The Ber uptakes in particle groups of P1,P2,P3 are（0.9259±0.0648）,（0.6441±0.0029）and（0.7764±0.0980）mg Ber/g protein,respectively,with（0.4654±0.0782）mg Ber/g protein in the control group.In the transport experiment,the particle group exhibits Papp_{（AP→BL）}and Papp_{（BL→AP）}of 4.16×10^-6and 1.48×10^-6cm/s,respectively;while in control group the Papp_{（AP→BL）}and Papp_{（BL→AP）}are 0.55×10^-6and 1.48×10^-6cm/s,respectively.（4）At 4 and 37°C,the Ber uptakes in particle group are（0.2863±0.0274）and（0.9259±0.1199）mg/g,respectively.In endocytosis inhibitors of Na N₃,CPZ,MβCD,EIPA and CD,the Ber uptakes are（0.5539±0.0009）,（0.5167±0.0062）,（0.6033±0.0537）,（0.8848±0.0500）and（0.8385±0.0500）mg/g,respectively,（0.9259±0.1199）mg/g of Ber uptake in the control group.Conclusion:CS particles with size of（450±34）nm have the strongest promotion on Ber uptake in intestine and can facilitate the transport of Ber from AP to BL.These promotive effects are energy dependent and associated with the combination of caveolin-mediated endocytosis and giant pinocytosis.