Oral Absorption Mechanism Of Puerarin Nanocrystals Self-Stabilized Pickering Emulsion

Nanocrystals self-stabilized Pickering emulsions?NSSPE?was a new emulsion firstly prepared by our research team to improve oral bioavailability of poorly soluble ingredients from Chinese traditional medicine.In our preious study,puerarin nanocrystals self-stabilized Pickering emulsion?Pu-NSSPE?was successfully prepared.The pharmacokinetics after intragastric administration in rats confirmed that relativebioavailability of Pu-NSSPE compared with raw material,nanocrystals and surfactant emulsion was 262.43%,155.92% and 223.65%,respectively.However,the reason for the improving of oral bioavailability was not clear.Based on the previous studies,this study aimed to investigate absorption mechanisms of Pu-NSSPE after oral administration from the following three aspects,the changes of gastrointestinal status and drug release in vitro,intestinal absorption kinetics and cell absorption and transport.By Comparing with raw material,nanocrystals and surfactant emulsion,the mechanism for NSSPE to promote oral absorption of the poorly soluble drug could be understood.The specific research methods and results were as follows:?1?Preparation and properties of raw material suspension,nanocrystals suspension,surfactant emulsion and NSSPE for PuPreparation of four test samples:?1?Pu raw material suspension was obtained by adding Pu to water containing 0.3%CMC-Na whose pH was pre-adjusted to11.5,and sonicating it in water bath;?2?nanocrystals suspension was prepared by adding Pu to pure water with initial pH of 11.5 and homogenizing at 80 MPa after a high shearing;?3?surfactant emulsion was prepared as follows.Pu,Chuanxiong oil,Labrafil M 1944CS and Tween 80 was mixed by stirring.Then pure water with initial pH of 11.5 was added and pre-homogenized using the high-speed shearing machine followed by further homogenizing at 80 MPa.?4?NSSPE was obtained by mixing nanocrystals suspension prepared as above and oil phase consisted of Chuanxiong oil and Labrafil M 1944 CS and then homogenizing at 80 MPa.The properties of all samples were evaluated by particle size,droplet morphology,drug content pH and stability.The results showed that the raw material suspension subsided obviously 5 min after preparation.The nanocrystals had no significant difference in particle size and drug content within 1 d.Surfactant emulsion and NSSPE remained stable at room temperature for 14 d.The finial pH of all samples were range of 6-7.?2?State change and drug release in simulated gastrointestinal fluidThe gastrointestinal environment was simulated in vitro,and the state of NSSPE in the gastrointestinal tract was studied with surfactant emulsion as a control.The release rate of Pu from NSSPE in stimulated gastric liquld and stimulated intestinal liquld were compared with raw materials,nanocrystals and surfactant emulsion.Changes the state in the gastrointestinal tract:the emulsion and the stimulated gastric liquid were mixed at a ratio of 1:5 and 1:50,respectively.The pH was adjusted to 7.5 after shaking at 37? for 1 h,and the bile extract and trypsin were added.The oscillation continued at 37?for another 2 h.After mixing NSSPE with stimulated gastric liquid,the sample became clear.The longer was the time and the more stimulated gastric liquid,the more obvious was the clearing phenomenon.The particle size,fluorescence microscope and SEM showed that the green fluorescent of Pu on the surface of the emulsion droplets weakened,indicating that the stimulated gastric liquid may damage the structure of NSFPE droplet.By contrast,the effect of stimulated gastric liquid on surfactant emulsion was weak:it only became slightly clear after incubating with stimulated gastric liquid at a ratio of 1:50 for 1 h,and the particle size and droplets morphology had no obvious change.After entering the intestinal liquid,the surfactant emulsion and NSSPE showed similar changes:after entering the stimulated gastric liquid for 2 h,a large amount of drug crystals were observed besides the spherical emulsion droplets,and the particle size was reduced to about 0.5-1?m.The effect of gastrointestinal fluid on the zeta potential of surfactant emulsion and NSSPE showed the same trend:after entering the gastric liquid,the zeta potential of NSSPE and surfactant emulsion both increased significantly from-46.0±2.36 mV to 1.68±0.479mV and from-39.9±0.5 mV to-3.48±0.12 mV after 1 h,respectively.After entering the intestinal fluid,the zeta potential both significantly decreased,and after 2 hours,it was-33.10±0.52 mV and-44.57±2.47 mV for NSSPE and surfactant emulsion,respectively.Drug release:Among the four preparations,the cumulative release rates of raw material,nanocrystals,surfactant emulsion and NSSPE in stimulated gastric liquid were75.62%,99.30%,83.61%,80.97%,respectively,and the cumulative release rate of intestinal fluid was 74.60%,97.19%,79.00% and 76.54%,respectively.All drug release were consistent with the first-order kinetic equation.The release of NSSPE is slower than that of nanocrystals,confirming that the adsorption of nanocrystals at the interface of the emulsion is stronger.?3?Intestinal absorption kinetics of four preparations of puerarinThe rat intestinal absorption characteristic of Pu raw material suspension,nanocrystals,surfactant emulsion and NSSPE was studied by in situ single-pass intestinal perfusion.The Pu content of four samples in K-R solution were stable at 37?within 3 h and intestinal segment and the peristaltic pump tube didn't absrob Pu in four samples,which verified that the rat in situ single-pass intestinal perfusion could be used for the intestinal absorption kinetics study for these preperations of Pu.The K_a and P_apppp values of Pu-NSSPE decreased in the order of duodenum,jejunum,ileum and colon.The duodenum was significantly higher than the jejunum and ileum ?P<0.05?,and the colon?P<0.01?.There was no significant difference between the jejunum and the ileum.The absorption of NSSPE in each intestine was significantly higher than that of material drug and surfactant emulsion:the P_app of the duodenum,jejunum,ileum and colon of NSSPE increased by 2.22,2.05,0.70,1.73 times compared with material drug,and by 2.77,0.38,0.57,0.70 times compared with surfactant emulsion,respectively.Compared with nanocrystals,NSPPE increased P_app in the duodenum,jejunum and colon segments by 2.14,0.74,0.34,0.43 times.The order of absorption of the four preparations in the whole small intestine was as follows:NSSPE>nanocrystals>surfactant emulsion>material drug.?4?In vitro cell absorption and transportionThe cell uptake and transmembrane transportion mechanisms of Pu-NSSPE were studied on MDCK cell and Caco-2 cells.Pu aqueous solution and surfactant emulsion were used as controls.MDCK cell assay:the cell uptake rates of three formulations of Pu at different concentrations were compared.The results showed that the uptake rate of NSSPE in MDCK cells was significantly higher than that in water solution and surfactant emulsion at concentration of 100-200?g·mL^-1?P<0.05?.The transport experiment at 100?g·mL^-1 showed that the P_app of NSSPE across MDCK cells was also significantly higher than that of the solution group and the surfactant emulsion group?P<0.05?.This indicated that NSSPE had better absorption of Pu across MDCK cells than solution and surfactant emulsuion.The uptake and transport experiments of different concentrations of NSSPE showed that the cell uptake rate of NSSPE at 100,150 and 200?g·mL^-1 was 68.89%,101.04%and 113.16% higher than that of 50?g·mL^-1,respectively.There was no significant difference for P_app among three groups,and R_e was between 1.04-1.24.This indicated that the transport of NSSPE across MDCK cells was dominated by passive transport.Caco-2 cells assay:the uptake rate of Caco-2 cells to different concentrations of Pu-NSSPE,Pu aqueous solution and tween 80 stabilized emulsion was determined,and cell absorption at concentration of 100?g·mL^-1 was observed by laser confocal microscopy?CLSM?after Pu was labeled with Nile Blue.The results showed that the uptake rate of NSSPE by Caco-2 cells was significantly higher than that of aqueous solution and surfactant emulsion from 50 to 200?g·mL^-1?P<0.05?.In the CLSM test,more intake of Pu in Caco-2 cells for NSSPE was observed compared with Pu aqueous solution and tween 80 emulsion.Cellular uptake experiments with different inhibitors showed that genistein and indomethacin?cavein inhibitor?significantly inhibited the uptake of NSSPE by Caco-2 cells:the cell uptake rate was reduced by 25.46% and 20.17% compared with the control group without inhibitor,and the cell uptake rate was further reduced by 33.46% after combined application of genistein and indomethacin.However,uptake rate of chlorpromazine?meshin inhibitor?and amlilol?inhibitor of giant cellulite function?group were not significantly different from the control group.The transport experiment at 100?g·mL^-1 showed that the P_app of NSSPE was only significantly higher than that of the solution?P<0.05?,and there was no significant difference from surfactant emulsion.The directional transport experiments of NSSPE at different concentrations showed that the permeation rate of NSSPE across Caco-2 cells monolayer increased with increasing of concentration from 50?g·mL^-1 to 200?g·mL^-1.at 2 h.The AP?BL cumulative permeability of 200?g·mL^-1 was 1.99 times and 3.90times higher than that of 100 and 50?g·mL^-1,respectively.The P_app of AP?BL and BL?AP for NSSPE at the concentration of 100?g·mL^-1was 1.26 and 1.57 times of that of 50?g·mL^-1,respectively,but when the concentration was increased to 200?g·mL^-1,the P_app did not increase furthur?P>0.05?.R_e at all three concentrations was greater than 1.5.The above results indicated that both passive transport and active existed in the transport of NSSPE across Caco-2 cells monolayer.There was caveolin-mediated endocytosis in the process of cell uptake,but clathrin-mediated endocytosis and giant cell-drinking have little effect.The above results again showed that NSSPE could promote the oral absorption of Pu,especially the absorption in the duodenum,compared with the raw material suspension,NCS and surfactant emulsion,which may be related to the strong acidic environment of duodenum.The mechanism for NSSPE to promote oral absorptionof Pu may have nothing to do with the dissolution of Pu.There were both passive and active transmembrane transport,as well as caveolin-mediated endocytosis.