Study On The In Vivo Behaviors Of Herpetrione Amorphous Nanoparticles Based On Self-indentification System

Bolengguazi（BL）,as a well-known Tibetan medicine,is one of commonly used drugs by Tibetan healers for treating hepatitis B.As one of its main active ingredients,Herpetrione（Her）,is an insoluble lignan component,belonging to BCSII class,with low bioavailability.Nanosuspension（NS）is a submicron colloidal dispersion system of pure drugs,which dispersed in a crystalline or amorphous form and stabilized by small amount of stabilizer.If the drug is dispersed in an amorphous form,it can also be called amorphous nanoparticles（ANPs）.NS has been successfully used to overcome the problems of poor solubility of active pharmaceutical ingredients.In previous study,our research group formulated Her into amorphous nanoparticles（Her-ANPs）,and found that its cumulative release and bioavailability were significantly improved as compared to the bulk drug.However,the key scientific issues whether Her-ANPs is absorbed as molecules or integral nanoparticles after oral administration and how to improve bioavailability as well as what is the in vivo behavior of Her-ANPs following intravenous delivery still remain unclear.In this study,an environment-responsive fluorophores with water-initiated aggregation-caused quenching（ACQ）properties was embedded in the Her-ANPs by fluorescence hybridization technology to construct a hybrid Her-ANPs and then explore the in vivo fate of Her-ANPs,with a view to explain its biological characteristics,and further expand the insight into the in vivo performance of ANPs.Her-ANPs with different particle sizes were successfully prepared by anti-solvent precipitation method and optimized by single-factor experiment.The particle size of Her-ANPs was about 200 nm（Her-ANPs-200）and 450 nm（Her-ANPs-450）,respectively,with PDI less than 0.2.Scanning electron microscopy results showed that Her-ANPs-200 and Her-ANPs-450 were both spherical in shape.DSC and XRD results showed that Her bulk drugs and Her-ANPs were all amorphous,indicating nanocrystallization would not affect the physical state of Her.The results of in vitro verification experiments showed that the fluorescence quenching sensitivity of Her-ANPs was strong,and the drug dissolution of Her-ANPs had a good correlation with the fluorescence quenching of ACQ fluorescent probes.The changes of ACQ fluorescence signal of fluorescent probes can indicate changes of Her-ANPs,identify the integrity structure of Her-ANPs accurately and sensitively.So it can be used to study the in vivo fate of Her-ANPs.The distribution,retention and degradation of Her-ANPs after oral administration were studied by animal living imaging and Ex vivo tissue imaging.The study found that oral administration of Her-ANPs with two different particle sizes in rats did not rapidly dissolve and release in vivo,but could be retained in the gastrointestinal tract for 8 h.After oral administration,the fluorescence signal began to accumulate in liver1h later and the fluorescence signal could be retained in issue for 24-36 h.However the biodistribution of the Her-ANPs to liver is of less intensity.which may be attributed to that only small amount of Her-ANPs was absorbed.No fluorescence was observed in other organs,which may be attributed to that only small amount of Her-ANPs was absorbed and the absorbed Her-ANPs released fast.Moreover,single pass intestinal perfusion and intestinal frozen sections observationfound that fluorescence signals can be observed on both the lumen side（AP）and the basal side（BL）of the intestine.The result indicated that Her-ANPs can be taken up by the intestinal epithelial cells as intact form,and Her-ANPs-450 with larger size was more likely taken up by the ileum.The surface of drug increased after nanocrystallization,which extended its residence time in the gastrointestinal tract.In summary,ANPs improve oral absorption of Her by small amount of ANPs entering the systemic circulation as intact form,and extending the contact time with the GIT.Three cell models,including Caco-2 cell model,Caco-2/HT29-MTX co-culture cell model and Caco-2/HT29-MTX/Raji co-culture cell model,were established for the study of cell uptake and transmembrane transport of Her-ANPs.The study found that both integral Her-ANPs-200 and Her-ANPs-450 can be taken up by enterocyte with small amounts.Her-ANPs-200 with smaller particle size were more easily taken up in epithelial cells,because the smaller ones may reduce its interaction with the lattice structure of cells mucus,making it easier to penetrate the mucus layer.However,in the transport experiments of the three kinds of cells,no fluorescent signal was observed in the receiving pool,which may be caused by the small amount of uptake and transport of Her-ANPs.The results further proved that Her-ANPs with different particle sizes were absorbed into the cells in the form of whole ANPs after oral administration and affected by the mucus layer.ANPs with small particle sizes can improve the absorption of oral drugs by increasing the penetration of the mucus barrier.Through in vitro release,pharmacokinetics and pharmacodynamics experiments,the effects of Her-ANPs on release,oral bioavailability and pharmacodynamics were compared,and the difference in particle size was discussed..The in vitro release test results showed that,compared with bulk drug,the in vitro release of Her from Her-ANPs was significantly increased（P<0.05）.The particle size of Her-ANPs was negatively correlated with the release,and the smaller ANPs had a larger specific surface area,leading to an increase in the in vitro release of Her.The pharmacokinetic results showed that the AUC_（0-t）and C_maxof Her-ANPs with two particle sizes were significantly higher than that of Her（P<0.01）,indicating Her-ANPs can significantly improve the bioavailability.This is mainly because Her-ANPs significantly reduced the particle size of the Her,increased the dissolution of Her and its bioadhesion to the gastrointestinal tract,promoted Her absorption,and thus improved Her oral bioavailability.In addition,the significantly prolonged MRT_（0-t）was also conducive to the increase of bioavailability.The AUC_（0-t）and C_maxof Her-ANPs-200 were higher than Her-ANPs-450,indicating that the small particle size was more conducive to the improvement of bioavailability.The pharmacodynamic results showed that Her and Her-ANPs had a protective effect on CCl₄-induced acute liver injury.The decrease of MDA、IL-1β,IL-6 and TNF-αand the increase of SOD and GSH-Px suggested that the protective effect of Her on liver injury in mice may be related to enhancing the activity of antioxidant enzymes and reducing lipid peroxidation products.The therapeutic effect of Her-ANPs with two particle sizes was more significant than that of the bulk drug.The main reason is that after Her being prepared into Her-ANPs,the absorption in the body increases,thereby enhancing its hepatoprotective effects.The differences in the distribution and degradation kinetics of particles in the body after the injection of Her-ANPs of different particle sizes were compared via small animal living imaging,pharmacokinetics and tissue distribution kinetics studies.The results showed that after injection of Her-ANPs-200 and Her-ANPs-450,fluorescence signals can be observed in whole body,indicating that the drug circulated through the blood and was distributed throughout the body and maintained for at least 36 hours.There were some differences between the two particle size groups:Her-ANPs-200 with smaller particle size can enter the blood circulation faster.However,Her-ANPs-450 had a longer residence time in the blood,the drug dissolved slowly,and the fluorescence signal intensity decreased slowly.The results showed that Her-ANPs can be taken up by various organs and had a longer residence time after injection.In addition,the fluorescence signal in the liver of the Her-ANPs-450group was significantly stronger than that of the Her-ANPs-200,the retention time is also longer.It showed that Her-ANPs-450 is more likely to reside in the organs.The concentration of Her in biological samples was determined by HPLC.The results showed that the method was specific and accurate,and could be used for the determination of Her in biological samples.The results of pharmacokinetic studies showed that Her-ANPs with two particle sizes distributed rapidly and widely in the heart,liver,spleen,lung,and kidney tissues,and Her-ANPs at different concentrations were detected.The AUC_（0-t）of each organ after injection of Her-ANPs-200 and Her-ANPs-450 was in the following order:liver>lung>spleen>kidney>heart,indicating that Her-ANPs are more likely to accumulate in the organs of RES（liver,spleen,lung）after injection.The uptake of Her-ANPs-450 in liver,spleen and lung tissues was higher than that of Her-ANPs-200.Slower dissolution rate and enhanced recognition by RES might explain the enhanced accumulation of Her-ANPs-450.The tissue distribution kinetics of Her-ANPs particles were analyzed.The absolute bioavailability of Her-ANPs-200/Her-ANPs-450 after oral administration calculated by TRE value was 2.41%and 1.75%,respectively.The absolute bioavailability of Her-ANPs-200/450 calculated by ARE value was 0.74%and 0.66%,respectively.The contributions of Her-ANPs-200/Her-ANPs-450 calculated by TRE value was 7.12%and 7.82%,respectively.The contribution of integral Her-ANPs calculated by ARE value was 2.19%（Her-ANPs-200）and 2.95%（Her-ANPs-450）.In summary,the study verified the feasibility of using ACQ fluorescent probes to study the fate of ANPs of lignans in vivo.After oral administration,integral HPE-ANPs could be taken up by intestinal epithelial cells.At the same time,the absorption of oral drugs can be improved by extending the contact time with the gastrointestinal tract and increasing the penetration ability of the mucus barrier.After injection of Her-ANPs,the particles were more likely to be accumulated in the RES organs（liver,spleen,lung）,suggesting that it had a certain RES organ-targeting,so it was beneficial for the treatment of ailments conflicting any RES organs.