The Application Of PLA-PEG Nanoparticles In Carrying Antitumor Drug And The Effect Of PLA-PEG Nanoparticles On Mouse Hepatic Cells

Objective: In order to enlarge galenic pharmacy of epirubicin PLA—PEG nanoparticles, we prepared epirnbicin-loaded PLA-PEGnanoparticles. In order to investigate the biocompatibility of PLA-PEGnanoparticles with cultured cells and organs. We analyzed the changes ofgene profile of mouse liver after a large dose injection of blank PLA-PEGnanoparticles (42.04 mg/kg, i.v.). We analyzed the effect of ATP-bindingcassette (ABC) transporters on nanoparticles hepatic metabolism and cellmembrane transport. Also we produce the mechanism of cell effiux ofPLA-PEG nanoparticles by hepatic cells.Methods: In our experiments, we prepared doxorubicin-loadedPLA-PEG nanoparticles by the method of uniform design, also analyzedthe morphology, physico-chemical property and pharmaceutic index, . Weanalyzed the effect ofPLA-PEG nanoparticles on cell viability by themethod of MTT assay. In situ cell apoptosis assay was used to investigatethe potential damage of blank PLA-PEG nanoparticles to importantorgans. SOD, NOS, and MDA activity assay was used to investigate theantioxygen ability afeter large dose injection.Then Kunming mice weretreated with large dose of PLA-PEG nanoparticles (42.04mg/kg, i.v) andafter 4 days total RNA was isolated to elucidate patterns of gene expression using a mouse cDNA-microarray (Super Array).Results: Blank PLA-PEG nanoparticles have been successfullyprepared and MTT assay suggested that the nanoparticles with HepG2cell co-culture model do not cause significant changes in membraneintegrity in controlled concentration range (0.001 mg/ml to 0.1 mg/ml).Immunohistochemical analysis demonstrated that large dose of PLA-PEGnanoparticles injection (42.04mg/kg, i.v) didn't induce hepatic cellsapoptosis. From biochemical assay experiments, although the levels ofSOD decreased and that of MDA, NOS increased after treatment withlarge dose of PLA-PEG nanoparticles injection (42.04mg/kg, i.v), butthey were all not significant (p＞0.05). Results of in vitro accumulationand efflux experiments indicated that about 51%-52 %( 51.5% and 52.0%)intracellular PLA-PEG nanoparticles was expulsed aider mouse primaryhepatocytes reached a saturation uptake of nanoparticles during theconcentration range of 750μg/ml to 1000μg/ml. Treatment withnanoparticles resulted in over-expression of a lot of ATP-binding cassette(ABC) transporters, especially two ABC transporters (ABCA8 andABCC5/MRP5) and down-regulation of GSTP1, in comparison with thecontrol. ABCA8 could extrude low molecular weight polymers afterPLA-PEG nanoparticles hydrolysis outside the cells. We also discoveredthat ABCC5 expressed multidrug resistance protein 5 (MRP5) to pumpout conjugate (GS-X) of PLA-PEG nanoparticles with GSH. The results were confirmed by RT-PCR.Conclusion: The biocompatibility of PLA-PEG nanoparticles withcultured cells and organs is very well. The uptake of naoparticles byhepatic cells is limited. The nanoparticles in cells are pumped bytransporters after saturation uptake. The results suggested that ABCtransporters (especially ABCA8) pump out the polymers after hydrolysisfrom mouse hepatic cells and large dose of PLA-PEG nanoparticles makemouse hepatic cells gain drug resistance to PLA-PEG nanoparticles.