Effects Of MPEG_2k-PCL_x Block Polymers On The Activity Of Hepatic Uptake Transporters OATP And NTCP

Amphiphilic block polymeric micelles have gained much attention in recent years for their extensive application in drug delivery due to their good solubilization ability,targeting ability,high stability and good biocompatibility.Hepatic drug transporters can play an important role in modulating drug distribution,metabolism and excretion,thus affecting the pharmacokinetics and/or pharmacodynamics of a drug.Clinically relevant drug-drug interactions mediated by transporters are of increasing interest in drug development.Polymeric micelles would accumulate in the liver after administration into blood veins.While considerable research has been conducted focusing on the efflux transporters less is known about the relationship between polymeric micelles and hepatic uptake transporters.It has been reported that polymer micelles would affect the activity of efflux transporters like P-glycoprotein（P-gp）which is likely related to the structures of hydrophobic segments.In this paper,the block polymers which have the same hydrophilic segment with the different hydrophobic segments ranging from a variety of molecular weight were chosen to investigate the effects of block polymers and polymeric micelles on the activity of hepatic transporters including OATP/Oatp and NTCP/Ntcp in vitro and in vivo.Furthermore,the influence of block polymeric micelles on the gene expression was examined by real-time PCR technique.Firstly,the micelles were prepared by film dispersion(mPEG_2k-PCL_2k,mPEG_2k-PCL_3.5k,mPEG_2k-PCL₅)and solvent-evaporation(mPEG_2k-PCL_7.5k,mPEG_2k-PCL_10k)method,respectively.The size distribution and morphology of the micelles were characterized by the dynamic light scattering（DLS）and transmission electron microscope（TEM）.The critical micelle concentrations（CMC）were determined by fluorescence probe method with the pyrene.Along with the increasing molecular weight of the micelles,the particle sizes enhanced ranging from 20 nm to 120 nm,while the CMC values were decreased from 2.1 to1.0μg/m L.The micelles were a near spherical uniform shape.The biodistribution of polymeric micelles in mice was monitored by in vivo imaging system showed that the DiR loaded micelles were mainly accumulated in the liver compared with other organs.Secondly,HEK293 cells stably expressing human OATP1B1 were constructed by gene transfection technology.Western blotting assay showed that the hOATP1B1-transfected HEK293 cells over-expressed OATP1B1 protein,and PIT accumulation in hOATP1B1-transfected HEK293 cells was 2.79 fold than vector-transfected HEK293 cells.These results demonstrated that hOATP1B1-transfected HEK293 cells were successfully established.The MTT assay was used to study the effects of block polymers on the viability of cells showing that only mPEG_2k-PCL_7.5kand mPEG_2k-PCL_10kpolymeric micelles at the highest concentration（10 mg/mL）with toxicity.The fluorescence resonance energy transfer（FRET）technique was used to investigate the stability in vitro and the intracellular integrity of the micelles.The results revealed that the（DiO+DiI）-micelles remained stable in 37℃for 24 h and the intracellular micelles kept integrity within 12 h.hOATP1B1-transfected HEK293 cells were used to estimate the impacts of block polymers and micelles on the uptake function of OATP1B1.When incubated with cells for 1 h,PIT showed an increase in accumulation with increasing molecular weight of the hydrophobic segment followed by a decline in accumulation.mPEG_2k-PCL_3.5kpolymeric micelle performed the smallest IC₅₀value（3.481mg/m L）with the strongest inhibitory effect on OATP1B1.To further investigate the influence of block polymeric micelles on the function of OATP/Oatp in vivo,the plasma concentration of PIT in rats was determined by LC-MS/MS after injected mPEG_2k-PCL_3.5kpolymeric micelle intravenously via tail for 1 d or 7 d,respectively.Compared with the control group,the area under concentration-time curve（AUC）enhanced while the clearance rate（Cl/F）reduced significantly.The polymeric micelles might inhibit the uptake activity of OATPs,which resulted in the reduction of PIT in liver cells along with the increase of PIT in plasma.Real-time PCR techniques were implemented to explore the effect of polymeric micelle on the expression of genes.No significant change was observed in the protein expression of Oatp1b2、Ntcp、Mrp2、Mrp3、Mrp4 and the gene levels of rOatp1a4,r Oatp1b2,rOatp2b1,rNtcp while only the Bsep protein and rOatp1a1 gene level was significantly reduced after 7d administration.Finally,hNTCP-transfected HEK293 cells were utilized to investigate the effect of block polymers and micelles on the activity of NTCP transporter.Interestingly,the results presented that the polymeric micelles except for mPEG_2k-PCL_10kwould facilitate the accumulation of intracellular T-CA in the presence of Na⁺.Induction was enhanced followed the increasing of concentrations and the prolong of incubation time.For most of the substrates of NTCP are endogenous bile acids,BA homeostasis in vivo was investigated to obtain the relationship between the polymeric micelles and hepatic transporter NTCP/Ntcp.Histopathological section photos of liver and biochemical indices changes in plasma or liver from mice after injected the micelle solution by tail vein for 7 d performed no significant influence on liver function.However,the total bile acids（TBA）in liver and the concentrations of several bile acids in plasma and liver were elevated,which might be explained that mPEG_2k-PCL_3.5k.5k polymeric micelles promoted the uptake function of NTCP/Ntcp related to the in vitro assay.In conclusion,the relationship between polymeric micelles and hepatic transporters like OATP/Oatp and NTCP/Ntcp was evaluated in this thesis.Polymeric micelles might have a potential to inhibit OATP/Oatp or induce NTCP/Ntcp,which was likely involved in the concentrations of micelles,the molecular weight of the hydrophobic segment of block polymer and the incubation time.This study provided a reliable guiding significance for safe and rational application of block polymers and suggested the potential drug-drug interation（DDI）between polymeric micelles and hepatic transporter substrate drugs.