Preliminary Study On The Tissue Distribution And Lymphatic Tanspoet Of Baicalin Nanoemulsion

Chronic Hepatitis B?CHB?,is a contagious disease that seriously endangers human health.At present,the main way to treat CHB is to take antiviral drugs.Hepatitis B Virus?HBV?not only hide in hepatocytes after being infected with liver,but also can be resided in the lymphatic system via lymphatic transport to form an extrahepatic virus reservoir,which makes CHB difficult to cure.Baicalin?BA?,is one main active flavonoids extracted from the dried root of Scutellaria baicalensis Georgi.Extensive studies have shown that BA can block the entry of HBV into host cells and release from host cells,and thereby inhibiting HBV replication.At present,BA tablets and capsules have been widely used for the treatment of acute and chronic hepatitis,and the curative effect is exact.Nanoemulsion is a new type of lymphatic targeted drug delivery system.Nanoemulsion is a ransparent and stable system,which is composed of surfactant,co-surfactant,oil phase and aqueous phase in a suitable ratio with droplets ranging from1-200 nm.After the nanoemulsion enters the intestinal epithelial cells,the biotriglycerides formed by the lipoprotein and the smooth endoplasmic reticulum synthesized by the rough endoplasmic reticulum of the intestinal wall cells are assembled into chylomicrons,followed by Golgi and drug-encapsulated drugs in the Golgi apparatus.The chylomicrons assemble to form vesicles,and finally the chylomicrons in the vesicles flow through the capillary lymphatics to the lymphatic circulation system.In our previous study,a stable W/O type BA nanoemulsion was prepared.This study to evaluate the targeting of BA nanoemulsion in lymphoid tissue through tissue distribution experiments in mice.The effect of chylomicrons on the distribution of BA in lymphoid tissues using a chylomicron flow blocking approach.Therefore,the transport pathway of BA nanoemulsion targeted to lymphoid tissues was initially clarified,which provides a theoretical basis for the research and application of lymphatic targeting agents.Objective:The tissue distribution of of BA nanoemulsion in mice after oral administration was investigated.The transport pathway of BA nanoemulsion to lymphoid tissue was preliminarily elucidated,which provided reference and basis for the study of lymphatic targeted agents.Method:The HPLC method was established to determine the content of BA in biological samples.For the tissue distribution study,the mices used in this study were randomly divided into two groups,intragastric administered with BA nanoemulsion and BA suspension.Afterward,the mices were scrificed and liver,brain,spleen,kidney,thymus,lung and lymph nodes were immediately removed at each time point.The tissues were extracted and analyzed by using the HPLC method.The pharmacokinetic parameters were analyzed by drug and statistics?DAS?version 2.0 software.Ovrall drug targeting index?TI?,targeting efficiency?TE?,and relative targeting efficiency?RTE?of BA nanoemulsion were calculated and compared with that of suspension to evaluate the tissue targeting property of BA nanoemulsion.The chylomicron flow blocking model was used and were orally administrated of BA nanoemulsion.Afterward,the rats were scrificed and liver,thymus,spleen and lymph nodes were immediately removed at each time point.The tissues were extracted and analyzed by using the HPLC method as described above.The pharmacokinetic parameters were analyzed by drug and statistics?DAS?version 2.0 software.The pharmacokinetic parameters were compared between the chylomicron blocked group and the chylomicron unblocked group.The effects of chylomicron-mediated transmembrane transport on the distribution of BA nanoemulsion in lymphoid tissue were investigated.The transport pathway of BA nanoemulsion to lymphoid tissue was preliminarily elucidated,Results:1.Establishment of analytical method for baicalin in biological samplesThe results of the methodological study showed that the separation between BA and rutin was greater than 1.5.The endogenous components in the biological samples did not interfere with the determination of the analyte and the internal standard.The specificity of the method was good.The BA in each tissue of mice was in the range of0.0388⁵.17?g/mL,the correlation coefficient was R>0.9996.The BA in the tissues of rats was in the range of 0.08¹0.16?g/mL,the correlation coefficient R>0.9997.For inter-assay and intra-assay precisions of analysis ranged from 1.38%to 6.52%.The extraction recovery and accuary ranged from 86.48 to 100.44%.For room temperature stability and freeze-thaw stability,the RSD was less than 5.69%.2.Tissue distribution of W/O type baicalin nanoemulsion miceThe results of targeted evaluation showed that the relative targeting efficiencies?RTE?of BA nanoemulsions in thymus,lymph nodes,spleen,lung,kidney,brain and liver tissues were 78.549%,56.626%,46.086%,37.542%,5.480%,2.117%,-43.938%respectively.The target index?TI?of BA nanoemulsion in thymus,lymph node,spleen,lung,kidney,brain and liver were 2.517,2.235,2.082,1.939,1.506,1.458,0.798,respectively.The RTE of BA nanoemulsion in thymus and lymph nodes was significantly increased compared with liver,kidney,brain and lung?p<0.01?.The RTE of BA nanoemulsion in spleen was significantly increased compared with liver,kidney and brain?p<0.01?.The TI of BA nanoemulsion was significantly increased in liver,kidney,brain and lung in thymus,lymph node and spleen?p<0.01?.3.Preliminary study on lymphatic transport pathway of W/O type baicalin nanoemulsion in ratsThe results of chylomicron blocking experiments showed that the AUC_?0-t?in the spleen tissue of the chylomicronunblocked group and the chylomicron-blocking group were 10.554±1.648?g/g*h and 5.754±0.912?g/g*h?p<0.05?,respectively.In thymus tissue,the AUC_?0-t?were 20.349±3.507?g/g*h and 13.805±3.061?g/g*h?p<0.05?,respectively.In lymph node tissue,the AUC_?0-t?were 8.938±1.336?g/g*h and6.217±0.828?g/g*h?p<0.05?,respectively.Conclusion:1.The HPLC analytical method in this study is accurate,stable and meets the requirements for biological sample;2.BA nanoemulsion can be targeted to lymphoid tissue after oral administration;3.BA nanoemulsion can enter the lymphatic system through chylomicron-mediated transmembrane transport,and accumulated in lymphoid tissue,which may be one of the main reasons for BA nanoemulsion target to lymphoid tissue.