Studies On Nanotechnologies To Improve The Oral Absorption Of Curcumin

ObjectiveCaco-2 cells model and in situ intestinal perfusion model in rat were used to explore the influences on improving the oral absorption of Curcumin（CUR）by different nanomedicines like curcumin polymer micelle（CUR-PMs）,curcumin liposomes（CUR-Lipo）and curcumin nanoemulsion（CUR-NE）.In order to provide the theoretical basis for the preparation of oral nanomedicines of CUR.Methods1.Establishment of the analysis method of CUR HPLC and HPLC-MS/MS2.Preparation and characterization of curcumin nanomedicinesThe preparation of CUR polymer micelle,CUR liposomes and CUR nanoemulsion with easy preparation,good repeatability and high stability were optimized by encapsulation efficiency,drug-loading and drug release in vitro.3.Experiments of cellular uptake and transport in Caco-2 cell.To establish Caco-2 cell uptake and transfer test mode.IncuCyte ZOOM kinetic energy dynamic and HPLC method were applied to investigate the uptake and transport characteristics of CUR and nanoparticles in Caco-2 cells and also research the effects of different protein inhibitors and temperature on endocytosis.4.Intestinal absorption in rat with in situ sinkle-pass perfusion modelThe absorption rate constant（Ka）and the apparent permeability coefficient(P_app)were selected to evaluate the absorption of CUR and its nanao-preparations in duodenum,jejunum and ileum as well as the influence of concentration on the absorption in duodenum.Results1.The HPLC and HPLC-MS/MS method was established to determine the content of CUR in following tests,Caco-2 cell uptake test and the analysis of in situ intestinal perfusion in rats.2.The average particle sizes of CUR-PMs,CUR-Lipo and CUR-NE were 14.38±0.59nm（PDI 0.192±0.010）,68.88±5.34 nm（PDI 0.231±0.020）and 78.41±1.08 nm（PDI 0.157±0.010）,respectively.The encapsulation efficiency of CUR-PMs,CUR-Lipo and CUR-NE were 89.0±2.18%,95.5±1.33%and 67.8±7.21%.All of the nanocarriers can fit preparations requirement and meanwhile meet the following experiments.In the 48 h release experiment,the cumulative release of the three nanomedicines was curcumin liposomes（CUR-Lipo,47.6%）>curcumin nanoemulsion（CUR-NE,25.9%）>curcumin polymer micelles（CUR-PMs,17%）.3.Absorption and transport studies in Caco-2 cells.（1）The fluorescence intensity in Caco-2 cells was time and concentration-dependent.The fluorescence intensity of CUR noparticles was obviously higher than that of curcumin,and the values of fluorescence intensity increased with time,furthermore the trend of CUR-nanoparticles were obvious downward than CUR slowly.The rate of fluorescence intensity descent within 24 h was CUR>CUR-PMs>CUR-NE>CUR-Lipo.（2）The uptake rate of the CUR solution reached the maximum at 45min,and it increased with the increase of concentration.In addition,the uptake rate of CUR-nanoparticles up to the maximum at 60 min,and at 40μmol/L,the intake of CUR-PMs,CUR-Lipo and CUR-NE was greater than that of the CUR solution.（both with significant difference）.When the concentration reached 100μmol/L,the CUR-NE could still significantly improve the uptake rate of the CUR（P<0.01）,while the intake of CUR-Lipo and CUR-PMs appeared to be saturated with the increase of concentration.（3）β-cyclodextrins,amiloride and chlorpromazine had significant effect on the uptake of the three nano-preparations,and quercetin had no significant influence on the uptake of three nano-preparations.The effect of low temperature on the uptake of CUR-PMs and CUR-Lipo was significant（P<0.01）while had no effect on the uptake rate of CUR-NE.（4）In the transport experiment in Caco-2 cells,the P_apppp value of the CUR was significantly increased by the CUR-PMs and the CUR-NE（P<0.05）,while the concentration saturation was observed.4.Intestinal absorption in rat with in situ intestinal perfusion model showed that CUR and its nanomedicines had the best absorption in duodenum.Moreover,CUR-PMs and CUR-Lipo significantly improved the Ka value and P_apppp value of the CUR,and with statistical significance.The uptake saturation of CUR-PMs and CUR-Lipo occurred at high concentration（160μg/ml）.It indicated that the absorption of CUR-PMs and CUR-Lipo was primary active transport.ConclusionThree CUR-nanoparticles can improve the intake of CUR,among them,the endocytpsispathwayofCUR-PMsandCUR-Lipoincludeclathrin-mediated,macropinocytosis and caveolae-mediated endocytosis.Combined with the absorption saturation phenomenon in the transport assay and intestinal perfusion experiment,the absorption of CUR-PMs and CUR-Lipo were given priority to with active transport way.The intake,Ka and P_apppp values of CUR-NE increased with the concentration,but the clathrin-mediated endocytpsis pathway inhibitors（β-cyclodextrin）decreased the uptake ralue,so the CUR-NE may involved clathrin-mediated endocytpsis and macropinocytosis pathway,but had passive transport predominantly.The formulations of CUR-PMs,CUR-Lipo and CUR-NE are simple and easy to prepare,which can improve the stability of the CUR and the oral absorption and showed the sustained release..It is more efficient to improve the CUR absorption by the CUR-PMs（endocytosis pathway）and the CUR-NE（passive transport）.Therefore,micelles and nanoemulsion are suitable as the nano-carriers of CUR,and the micelles have certain advantages because of their high drug loading.