Effects Of MPEG_2k-PCL_x Polymeric Micelles On The Function Of Organic Cationic Transporter 1-3 And Its Mechanism

Objective:In this study,the amphiphilic polymer mPEG_2k-PCL_x was used as the research object to investigate the effects of five different polymeric micelles with the same hydrophilic segment and hydrophobic segment of different molecular weight on the function of organic cationic transporter 1-3（OCT1-3）and the preliminary mechanism.Methods:（1）mPEG_2k-PCL_x polymeric micelles were prepared and characterized for its physical,chemical and morphological properties.The in vitro stability and cytotoxicity were further investigated.MDCK cells stably transfected with hOCT1-3were used as the model to explore the inhibitory effects of polymeric micelles on the uptake of TEA and metformin mediated by hOCT1-3.（2）SD rats were injected by single and multiple dose of mPEG_2k-PCL_x polymeric micelles through tail vein to investigate the influence of micelles on the pharmacokinetics,tissue distribution and hypoglycemic effects of metformin,a substrate drug of OCTs.（3）mPEG_2k-PCL_xpolymeric micelles loaded with fluorescent probe Nile red were prepared.Three kinds of non-transfected cells（L02,HEK293,Caco-2）were used as models to investigate the endocytosis mechanism of micelles and the effects of micelles on cell membrane potential.Finally,the molecular mechanism of micelles affecting OCTs at the gene and protein levels were investigated by real-time fluorescent quantitative polymerase chain reaction（RT-qPCR）and Western blot,respectively,to explore the potential mechanism of micelle inhibition of transporter function.Results:（1）In vitro results showed that mPEG_2k-PCL_x micelles inhibited the transport function of hOCT1-3 above the critical micelle concentration（CMC）,and the inhibition decreased gradually with the increase of hydrophobic molecular weight.Among the five polymeric micelles,mPEG_2k-PCL_2k had the strongest inhibitory effect on hOCT1-3,while among the three hOCTs,the micelles had the most significant inhibitory effect on hOCT1 with IC₅₀ values of 0.11-0.28 mg/mL.（2）In vivo results showed that mPEG_2k-PCL_2k could affect the intestinal absorption,liver distribution and renal excretion of metformin by inhibiting the transport function of OCTs,significantly increasing the plasma concentration of metformin,and decreasing the CL and V_ss.In the oral glucose tolerance test（OGTT）,micelles attenuated the hypoglycemic effects of metformin and reduced fasting insulin levels.（3）The uptake of mPEG_2k-PCL_xmicelles by cells is a time-and energy-dependent process,with little involvement of macropinocytosis mechanism,which mainly enters cells through clathrin-and caveolin-mediated endocytosis.The micelles with closer hydrophilic/hydrophobic ratio and smaller particle size are more likely to be absorbed by cells.Mechanism studies showed that micelles could induce membrane depolarization in a short time,and down regulate transporter gene and protein levels in a long time.Conclusions:mPEG_2k-PCL_x micelles could inhibit the substrate uptake mediated by hOCT1-3 in vitro,which is related to OCTs subtype and hydrophobic segment chain length;it has an effect on the pharmacokinetics and pharmacodynamics of the substrate in vivo,suggesting that there is a potential risk of nano carrier-drug interactions（NDIs）when nano drugs are combined with organic cationic drugs.Therefore,in order to reduce the occurrence of adverse reactions,it is necessary to select appropriate carrier materials for the development of nano drugs.