Activation Of PXR-P-gp/Mrp2 Axis Involved In The Accelerated Blood Clearance Phenomenon Induced By Pegylated Liposomes

Liposome（Lip）modified by polyethylene glycol（Polyethylene glycol,PEG）process prolonged circulation time in the body and reduced clearance rate by mononuclear macrophages,it is widely used in the design and development of nanodrug delivery system^[1,2].However,when PEG-modified liposome（PEG-Lip）was repeated intravenous injected into the same body（rat,beagle,or rhesus,etc.）,the elimination of PEG-Lip in the blood is accelerated accompanied by the significantly shortened circulation time and increased drug accumulation in liver and spleen tissue,which is known as"accelerated blood clearance phenomenon"（ABC phenomenon）.ABC phenomenon has been receiving much attention,while the underlying mechanisms have not been fully elucidated,which significantly hinders the effectiveness of PEG-Lip.Previous research in our group found that the pregnane X receptor（PXR）is activated in the ABC phenomenon and mediates the upregulation of the downstream target gene CYP450（CYP3A1）metabolic enzyme expression and activity,but the detailed role of PXR activation is not fully clear.Given the rapid accumulation and clearance characteristics of PEG-Lip in the liver during the ABC phenomenon,this study was undertaken to investigate the roles of hepatic efflux transporters,the major downstream target genes of PXR,including P-glycoprotein（P-gp）and multidrug resistance protein 2（Mrp2）.Both played a significant role in the hepatic clearance process.This study found significantly augmented nuclear co-localization of the PXR-retinoid X receptor alpha（RXRα）transcriptionally active heterodimer in the ABC phenomenon and followed by upregulated levels of P-gp and Mrp2.Further findings revealed that the efflux of their shared substrate doxorubicin（DOX）was also increased in primary hepatocytes isolated from rats repeatedly injected with PEG-Lips,which were inhibited by P-gp and Mrp2inhibitors,respectively^[8].Meanwhile,these findings are in agreement with the results of pretreatment with the specific PXR inducer dexamethasone.This study choosed a commonly used anti-tumor drug,spontaneous fluorescent doxorubicin hydrochloride（DOX）as the model drug.PEGylated liposomal Doxorubicin hydrochloride（PEG-DOX-Lip）were prepared by surface modification with biocompatible PEG₂₀₀₀-DSPE,then the role of PXR-P-gp/Mrp2 signaling axis in PEG-Lip induced ABC phenomenon was investigated in vitro and in vivo based on the basis of the observation that this liposome inducing ABC phenomenon in rats.Objective:Based on the nuclear receptors regulated transporter,this study was conducted to investigate the role of transporter proteins in ABC phenomenon and to provide new ideas to elucidate the mechanism of ABC phenomenon.Methods:Firstly,PEG-DOX-Lip was prepared,and the optimal prescription was screened by unifactor investigation and orthogonal experiments,the the quality of PEG-DOX-Lip was evaluated by measuring particle size,zeta potential,Polydispersion index,（PDI）and encapsulation efficiency（EE%）,morphology and in vitro release.Secondly,DOX concentration in rat plasma samples was analyzed by using combined liquid chromatography-mass spectrometry（LC-MS/MS）technical,then pharmacokinetic parameters and distribution characteristics of PEG-DOX-Lip in rats was selected as indicators to investigate the plasma concentration and hepatosplenic fluorescence distribution of PEG-DOX-Lip after repeated administration and the effect of time interval between the two injections on the strength of the ABC phenomenon,a optimal time interval that inducing the most significant ABC phenomenon was selected for further study.Thirdly,Western blotting,RT-q PCR and immunofluorescence were used to study the effects of repeated injected PEG-Lip on PXR expression and nuclear translocation in rats,and the induction of PXR on ABC phenomenon was further verified by the addition of PXR-specific inducer.Finally,The hepatocytes of rats that subjected to single injection or repeated injection of PEG-Lip rats were isolated by a two-step collagenase in situ perfusion method,and the P-gp and Mrp2 expression levels in hepatocytes was analyzed by using flow cytometry immunofluorescence.To further investigate the efflux transporter activity of isolated primary hepatocytes towards P-gp/Mrp2 substrate drugs and to examine the involvement of P-gp and Mrp2,hepatocytes were incubated with P-gp substrate and Mrp2 substrate with or without the addition of P-gp or/and Mrp2 inhibitor,then the efflux capacity of hepatocytes toward fluorescent substrate was analyzed by using flow cytometry to verify the role of PXR-P-gp/Mrp2 signaling axis in the induction of ABC phenomenon by PEG-liposomes.Results:（1）PEG-DOX-Lip prepared by optimal prescription has a average particle size of（152.5±2.1）nm,PDI of 0.173±0.019,Zeta potential value of（-27.2±0.5）m V,and EE%of 80%,indicating that the of prepared PEG-DOX-Lip has a narrow particle size distribution,uniform dispersion and the stable system.Transmission electron microscopy（TEM）result showed the smooth spherical or spherical-like structure of PEG-DOX-Lip.（2）Both he administration intervals between two injections from 1 d to7 day could induce ABC phenomenon,but the most obvious ABC phenomenon was observed in the 3 d group,consistent with the strongest accumulation of DOX in liver and spleen tissues.Therefore,3 day of time interval between two injections was chosen for the subsequent experiments.（3）Compared with the single injection group,after preperitoneal injection of dexamethasone（DEX）,a specific inducer of PXR,the pharmacokinetic behavior and fluorescence distribution of PEG-DOX-Lip were similar to those induced by repeated injection,and PXR underwent significant nuclear translocation in rat liver tissues in the repeated injection group and the DEX group.Moreover,the expression of hepatic PXR m RNA and protein in the rats in the two groups was significantly increased compared with that in the single injection group,and the expression of Retinoid X receptorα（RXRα）protein in the nucleus of rats in the repeated injection group was significantly increased,which was similar to the results of immunofluorescence colocalization staining,that is,hepatic PXR-RXRαwas significantly colocalized in the nucleus.At the same time,compared with single injection,the expression levels of m RNA and Mrp2 in the DEX group and the repeated injection group increased significantly,which was consistent with the quantitative analysis results of immunofluorescence images.It is suggested that PXR may be involved in the occurrence of ABC phenomenon by regulating the transcription of P-gp and/or Mrp2,thereby accelerating the clearance of PEG-Lip in secondary injections and changing its distribution.（4）Firstly,the expression of CK-18 in isolated hepatocytes was verified,and the results showed that all primary hepatocytes in all groups were successfully extracted;Secondly,from the results of flow cytometry,the protein expression levels of hepatocytes P-gp and Mrp2 in the DEX group and the repeated injection group were higher than those in the single injection group,which was consistent with the immunofluorescence image results.Moreover,after co-incubation of hepatocytes with P-gp substrate and Mrp2 substrate in the DEX group and repeat injection group,the efflux capacity of fluorescent substrate was higher than that of single injection group.Finally,hepatocytes in the DEX group and the repeat injection group could increase the intracellular accumulation of the substrate drug DOX and reduce the efflux after adding P-gp and or Mrp2 inhibitors,respectively,so as to verify the role of the PXR-P-gp/Mrp2 signaling axis in the phenomenon of PEGylated liposomes inducing ABC phenomenon.Conclusion:PEG-Lip injection a few days in advance could act as an exogenous ligand to activate the nuclear receptor PXR,which upregulated PXR transcription and protein levels and promoted nuclear co-localization of PXR-RXRα,as well as enhanced transcription and protein expression of the downstream target genes P-gp and Mrp2 and their exocytotic activity on substrate molecules,revealing an important PXR-P-gp/Mrp2axis in the ABC phenomenon changes,which may be one of the metabolic mechanisms for the rapid clearance of secondary injected PEG-DOX-Lip from the organism during the ABC phenomenon.