Experimental Study On Brain Targeting And Anti-tumor Effects And Mechanism Of Quercetin Nanoliposomes On C6Glioma

Objective:To prepare quercetin nanoliposomes (QUE-NL) and investigate its properties and the oral absorption character in rat. To investigate the absorption character of QUE-NL in brain tissue and plasma in mice and rat, and measure the pharmacokinetic parameters in rats and the existence form of QUE-NL in plasma and absorption character in rat brain tissue.Methods:Preparation of QUE-NL by the emulsification-evaporation and low temperature curing method and the optimum formulation with the optimal conditions which oil phase were mainly made of Glyceryl behenate (ATO), soy lecithin and cholesterol, and aqueous phase was made of poloxamer, PEG2000-DPSE and Tween80. The morphology of QUE-NL was examined by transmission electron microscope (TEM) and the particle diameter distribution was determined by laser particle size analyzer, and the drug loading and encapsulation efficiency of QUE-NL was determined by HPLC. The determination methods of quercetin in the nanoliposomes and in the mixture of gastrointestinal contents and feces were established by HPLC. Determination of oral absorption rate of QUE-NL in gastrointestinal in rat by HPLC. The serial blood samples were collected at designed time points and determination of quercetin in plasma and drug targeting index (DTI) of brain tissue in mice by HPLC. The pharmacokinetic parameters were calculated with the software3P97a. The determination of quercetin existence form in plasm and drug targeting index (DTI) of brain tissue in rat by HPLC. Result:The mean diametre of QUE-NL was172.63nm, and the nanoparticles morphology of QUE-NL was sphere or spherical.The entrapment efficiency was85.90%and the drug loading rate was7.25%. The oral absorption of QUE-NL in rat was better than that of quercetin suspension and common liposomes. The absorption of quercetin and common liposomes all increased with concentration of quercetin and showed linear correlation. Meanwhile, the absorption of QUE-NL with the increase concentration of quercetin and showed no linear correlation. The distribution of QUE-NL in mice brain tissue was better than that of quercetin suspension and common liposomes, and drug targeting index (DTI) of QUE-NL values were more than1in brain tumor tissue. The plasma concentration and area under the curve (AUC) of QUE-NL in rat was obviously higher than that of quercetin suspension. Volume of distribution (Vd) and plasma clearances of QUE-NL were lower than that of free quercetin. Quercetin mainly was in faglycone and isorhamnetin state, and the distribution of QUE-NL in rat brain tissue was better than that of quercetin suspension and common liposomes and drug targeting index (DTI) of QUE-NL values were more than1in brain tumor tissue.Conclusion:QUE-NL can improve the oral absorption of quercetin in mice and rat with optimal preparation of QUE-NL in this experiment. QUE-NL can prolong the circle time of quercetin in plasma for longer half-life and larger AUC and the results show that QUE-NL has long circulating effects. Quercetin was obsorbde in plasma in a combinative form but not in free state, QUE-NL can improve the absorption of quercetin in mice and rat brain tissue, which showed good brain targeting in mice and rat, so as to provide experimental foundation for further application for treatment of glioma. Objective:To study on the effects of quercetin nanoliposomes (QUE-NL) on proliferation and apoptosis on C6glioma cells in vitro. To study the role of mitochondrial pathway in the apoptosis or necrosis in C6glioma cell lines induced by QUE-NL. To investigate the effects and mechanism of QUE-NL induced apoptosis on C6glioma cells through of JAK2/STAT3signaling pathway. Furthermore, provided the evidence for further development and supplement of quercetin using for anti-glioma.Methods:Using emulsification-evaporation and low temperature curing method to prepare quercetin nanoliposomes.The cultured cells were divided into QUE and QUE-NL treatment groups according to the concentrations of QUE, blank control and DMSO as control groups. MTT assay was used to observe the proliferation on the cells treated for12,24,48and72h.Given the concentration of QUE-NL50、100、200、400umol/L,taking quercetin and temozolomide as control. The apoptosis and necrosis of C6glioma cells was observed by flow cytometry (FCM) after the cells were respectively treated with QUE and QUE-NL for24h. Detected the activity of LDH to study the cytotoxicity effect of quercetin on rat C6glioma cell. Detected the level of ROS,SOD and MDA and the activity of Caspase-3、8、9on glioma C6cell to explore the possible mechanism of rat C6glioma cell apoptosis and necrosis.To detect the effect of quercetin nanoparticles promoting on rat C6glioma cell apoptosis and necrosis by flow cytometry analysis technical. Detected the activity of LDH to study the cytotoxicity effect of quercetin on rat C6glioma cell. Detected the change of mitochondrial membrane potential by flow cytometry, and the proteins express of Caspase-3,8,9by western-blot methods to explore the possible mechanism of apoptosis and necrosis on rat glioma C6cell induced by QUE-NL through mitochondrial pathway.Given the concentration of quercetin nanoliposomes (the final concentrations were50,100,200and400μmol.L-1respectively), taking quercetin and temozolomide as control. The supernatant was collected to measure the secretion of cytokine TNF-a, IL-6and IL-8by ELISA, and measure expression of JAK2. The changes of the protein p53and Bcl-2were detected respectively after C6glioma cells were treated with QUE and QUE-NL for24h200μmol.L-1were detected by Western-blot methods, and to explore the possible mechanism of rat glioma C6cell apoptosis and necrosis induced by QUE-NL through JAK2/STAT3pathway.Results:With the augmentation of quercetin nanoliposomes and the extension of the treated time, the C6cell growth was inhibited, the OD values decreased (P<0.05) and the cell numbers were cut down. Quercetin nanoliposomes test groups have higher proportion of apoptotic cells and dead cells than blank control group and the proportion of dead cell increased significantly. QUE-NL groups have higher proportion of apoptotic and necrosIs cells than blank test groups and the proportion of necrosis cell increased significantly. The quantities of LDH released by rat C6glioma cells are significantly increased. The level of ROS, SOD and MDA all changed significantly. The proportion of apoptotic cells with Caspase-3、9activity increased, indicating that QUE-NL induced the apoptotic cells via mitochondrial pathway. QUE-NL induced cell death(necrosis) in C6glioma cells in a dose-and time-dependent manner, high concentrations of quercetin nanoliposome (QUE-NL) can induce necrotic cell death of which was distinct from apoptosis and autophagy. QUE-NL-induced mitochondrial membrane potential loss, cytochrome C released and had no effects on caspase activations, however, reductions in ATP levels and increases in LDH activity indicated that QUE-NL stimulated necrotic cell death.The proapoptotic protein of Bax and p53protein increased evidently, at the same time Bcl-2protein expression decreased after cultivated with QUE-NL by western blotting. The decreased expression of Bcl-2protein and the increased expression of p53protein were also observed after treatment with QUE. We also detected the activation of (JAK2/STAT3) and the inhabitation of JAK2regulator STAT3inhibited QUE-NL-induced C6glioma necrotic cell death upstream of the mitochondria. In addition, application of STAT3-specific inhibitors for defined periods showed that inhabited p53protein and activation of JAK2between24and36h. Furthermore, STAT3inhibitors failed to significantly inhibit QUE-NL-induced down-regulating of STAT3and p-STAT3protein on C6glioma cell death. Conclusion:Quercetin nanoliposomes have obviously cytotoxic on glioma C6cell and higher proportion of apoptotic cells and the proportion of necrosis cells increased significantly with different concertration of QUE-NL than blank control groups. Inhibitory effect of QUE-NL on C6cells was proved to be dependent on the treated time and the dose of QUE-NL. The possible mechanism of QUE-NL induced apoptosis and necrosis of C6glioma cells may regulate the level of ROS,SOD and MDA, and the induced apoptosis effect of C6glioma cells was implemented by activating mitochondrial pathway. C6glioma cells which treated with QUE-NL exhibited a cellular pattern related with necrosis and were not apoptosis and was characterized by caspase independence.Quercetin nanoliposomes can induce rat glioma C6cell apoptosis and promote cell necrosis, mitochondrial and JAK2/STAT3pathway are essential for QUE-NL-induced C6glioma necrosis and the JAK2/STAT3cascade has a crucial role in QUE-NL-induced C6glioma cell death. Quercetin nanoliposomes regulated JAK2/STAT3pathway resulted in down regulation of JAK2, STAT3, p-STAT3and Bcl-2protein expression, up-regulation of p53protein expression and ROS throug mitochondrial pathway may be the mained mechanisms for anti-glioma. Objective:To investigate anti-tumor effects of quercetin nanoliposomes on the growth of C6glioma in vivo. To compare the anti-glioma and toxic effect between QUE-NL and quercetin suspension, and evaluate the character of brain targeting.Methods:Utility of stereodirected instrument to establish the in situ in vivo C6glioma model by inoculate C6glioma cells into the cerebral cortex of SD rat, and investigate the effect of quercetin nanoliposomes on the rat C6glioma model. The C6glioma rats were divided into different groups by random selection,which were treated with different doses of QUE-NL or quercetin suspension (at dose of25mg/kg, and50mg/kg, every day for3weeks), temozolomide, blank nanoliposomes respectivyly after established the rat C6glioma model for1week. The size of tumor was measured and the living state and weight of C6glioma rats were observed. The concentration of quercetin in plasma and DTI of brain tumor tissues was determined by HPLC. The protein expression of JAK2、STAT3、p53and survivin was detected by immunohistochemistry methods. The parameters concerning heart, liver and kidney function were measured and the morphology of organ tissues was observed pathologically.Results:Compared with the control groups, the tumor volume was reduced obviously (P<0.05) in groups of QUE-NL and quercetin suspension at dose of25and50mg/kg can inhibit the growth of glioma in rat, and temozolomide also can inhibit the growth of glioma. There are significant different inhibit growth of tumor between quercetin nanoliposomes and quercetin suspension at dose of25,50mg/kg (P<0.05), and QUE-NL can inhibit the growth of glioma in rat more effective than quercetin suspension.There are stronger growth inhibition against C6glioma and no significant difference (P>0.05) in the groups of QUE-NL and temozolomide at dose of25and50mg/kg.The result of immunohistochemistry showed that the protein expression of JAK2STAT3and survivin was strongly positive in rat C6glioma model group, and the protein expression of p-STAT3increased significantly. The protein expression of STAT3and p-STAT3decreased significantly and protein expression of p53increased significantly in QUE-NL group.QUE-NL can increase significantly quercetin content and drug targeting index (DTI) values were more than1in brain tumor tissue, there are significant difference compared with quercetin suspension (P<0.05), which indicated that QUE-NL modified by polysorbate80can enhance quercetin pass the blood-brain barrier.QUE-NL and quercetin suspension have no obviouly effect on the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (y-GT) in C6glioma rats, there were no changes in liver and kidneys function. While the activity of serum ALT increased significantly and liver damage were observed in temozolomide adminwastrated group. QUE-NL also showed made no morphologic changes to heart, liver, kidneys and spleen in C6glioma rats.Conclusion:It was demonstrated that QUE-NL can enhance effect of anti-glioma with lower dosage. The side-effect of QUE-NL was less than the same dosage of quercetin. QUE-NL had good targeting efficiency and DTI in C6glioma rats.