| As nanomaterials have been widely used in biology, medicine and drug delivery systems, the biosafety of nanomaterials have attracted increasing attention due to its unique physicochemical properties and biological activities. In this study, taking poly(ethylene glycol)-poly(?-caprolactone)(PEG-PCL) and poly(ethylene glycol)-poly(D,L-lactide)(PEG-PDLLA) as the research objects, the effects of PEG-PCL and PEG-PDLLA block polymers or in the form of micelles on the activities of CYP450 enzymes in liver were investigated, which could provide a basis knowledgement for the design and safety evaluation of this polymeric nanocarriers.Firstly, different of various molecular weight of hydrophilic/hydrophobic block segment polymers(mPEG2k-PCL2 k, mPEG2k-PCL3.5k, mPEG2k-PCL5 k, mPEG2k-PCL10 k, mPEG5k-PCL5 k and mPEG2k-PDLLA2k) were selected as the materials, and the blank polymeric micelles were prepared by thin-film dispersion and solvent evaporation.method. The results of Fluorescence Spectrophotometry, Particle Size Analyzer and transmission scanning electron microscope(TEM) showed that,the critical micelle concentration(CMC) of polymers ranged from 0.89 to 4.00 ?g·mL-1. The particle sizes were less than 100 nm, which were uniform and electrically neutral.Secondly, the above block polymers were incubated with specific probe substrates of CYP450 isozymes in both rat and human liver microsomes, and the inhibitory effects of polymeric micelles on seven isozymes(CYP1A1/B2?CYP1A2?CYP2B1?CYP2C6?CYP2C11?CYP2D2?CYP3A1/2) in rats and isozymes(CYP1A2?CYP2B6?CYP2C8?CYP2C9?CYP2C19?CYP2D6?CYP3A4) in human were evaluated by determining the metabolites using a robust liquid mass spectrometry(LC-MS/MS) assay. No change was observed below CMC(<10 ?g·m L-1). However, when the concentrations of block polymers were higher than CMC, significant inhibitory effects were produced on CYP450 enzymes in a concentration-dependent manner and had difference between rat and human:(1) With the same molecular weight of PCL, when the PEG molecular weight increased, the inhibition intensity of the polymeric micelles of mPEG2k-PCL2 k, mEG2k-PCL3.5k, mPEG2k-PCL5 k and mPEG2k-PCL10 k on the rat and human CYP450 isozymes decreased gradually. The mPEG2k-PCL2 k polymeric micelles showed the strongest inhibition on CYP1A2 in rat and CYP2B6 in human with an IC50 values of 0.06 mg·mL-1 and 0.37 mg·mL-1, respectively. However, the mPEG2k-PCL10 k polymeric micelles showed a strongest inhibition on CYP2C11 in rat and CYP3A4(substrate for midazolam) in human with an IC50 values of 0.48 mg·m L-1 and 1.50 mg·mL-1, respectively.(2) Hydrophilic segment with a higher molecular weight of mPEG2k-PCL5 k polymeric micelles showed a stronger inhibitory effect than mPEG5k-PCL5 k polymeric micelles on CYP2C6 and CYP3A1/2(substrate for testosterone) in rat liver microsomes with the same hydrophobic segment, whereas was weaker on seven CYP450 isoforms in human liver microsomes.(3) The inhibition of mPEG2k-PCL2 k polymer on CYP450 isoforms were stronger than mPEG2k-PDLLA2 k polymeric micelles with a similar molecular weight in both rat and human liver microsomes.MTT experiment and the activity of LDH, AST, ALT and ALP in cell culture medium was tested to study the toxicity of above block polymers in rat primary hepatocytes. The results showed that compared to the control group, there was no significant difference in MTT test and ALT level in 72 h; during 24 h to 72 h, the levels of AST and ALP increased in different degrees(1.0~1.7 times), especially significant during 48 h to 72 h.Finally, a cocktail probes assay and LC-MS/MS assay were emplyed to investigate the effects of polymeric micelles on CYP450 isozymes activities in primary rat hepatocytes. With the concentration ranged from 0.1 to 1000 ?g·m L-1 or 0.01 to 100 ?g·mL-1, all the polymer showed an obvious induction on CYP1A2, especially at the highest concentration(1000 mg·mL-1)(P<0.001). The polymers of mPEG2k-PCL2 k, mPEG2k-PCL3.5k, mPEG2k-PCL5 k, mPEG2k-PCL10 k, mPEG2k-PDLLA2k) had significant induction on the activity of CYP1A1/B2 and CYP3A1/2(P< 0.05, 0.01 or 0.001), but no obvious concentration dependence was observed. Besides, mPEG5k-PCL5 k polymer significantly induced CYP3A1/2 activity, while the activities of CYP2C6 increased when the concentration was 0.1 ?g·m L-1(P<0.05). The induction intensity of CYP2B1 decreased with an increase of the concentrations of polymers, and the effects on the activity of CYP2C6 inducted in low concentration, while expressed as inhibition or no change at higher concentration. Among those, the strongest inhibition was observed on CYP3A1/2.In summary, mPEGx-PCLy and mPEG2k-PDLLA2 k block polymeric micelles play a certain role in both inhibition and induction of CYP450 isozymes activity, based on the various factors including: concentrations of micelles, molecular weight of hydrophilic/hydrophobic block segment, hydrophobic block composition, the type of isozymes and species. In addition, with an increase of incubation time, all the micelles above may have toxicity on primary rat hepatocytes. This study had a certain guiding significance for safety evaluation on poly(ethylene glycol)-polyesters block polymers in vivo, and it also suggests that the potential interaction between CYP450 substrate drugs and the combination of those polymeric nanocarriers. |
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