Study On The Cellular Transportation And Multidrug Resistant Mechanism Of Polyamidoamine Dendrimers In Tumor Cells

Recently,the cytological studies of drug carriers have been attracting more attention.The transportation of carriers can influence the pharmacodynamics and guide the rational design of drug delivery systems.Dendrimers have emerged as one of the most promising innovative polymeric nanocarriers for different therapeutic categories of bioactive agents due to their advantages of monodispersity,nano-size,branched-architecture and peripheral charge.Compared with other PAMAM dendrimers,because of the unique property of a positive charged on the surface,PAMAM-NH2 dendrimers have more widely application.In addition to encapsulating a chemotherapeutic drug in the interior,PAMAM-NH2 can bind negative DNA/RNA molecules via an electrostatic interaction and be used as gene vectors.In this study,the HepG2,MCF-7 and MCF-7/ADR cell were selected as the model cell lines.We performed systematic analysis of the cellular apoptosis,cellular uptake,intracellular transportation and efflux of PAMAM-NH2 dendrimers.Our aim was to support the rational design of PAMAM-NH2 based drug delivery systems,especially to reverse MDR.The cytotoxicty of PAMAM-NH2 in vitro was evaluated using MTT method.PAMAM-NH2 exhibits significant concentration-and time-dependent toxicity against HepG2,MCF-7 and MCF-7/ADR cells.At low concentrations(1-50 μg/mL),the viabilities of HepG2,MCF-7 and MCF-7/ADR cells at 48 h and 72 h were more than 80%,and the PAMAM-NH2 showed low cytotoxicity against HepG2,MCF-7 and MCF-7/ADR cells.At high concentrations(100-1000 lμg/mL)at 72 h,the viabilities of the three cells incubated with PAMAM-NH2 sharply decreased.10 μg/mL of PAMAM-NH2 was not toxic to HepG2,MCF-7 and MCF-7/ADR cells,and could be used in other studies in this study.We carried out the cell cycle and mitochondrial membrane potential analysis,measurement of intracellular reactive oxygen species(ROS)and caspase activities assay,to explore the mechanism of apoptosis caused by PAMAM-NH2 in HepG2,MCF-7 and MCF-7/ADR cells.The high concentrations of PAMAM-NH2 can induce the caspase-dependence mitochondria mediated apoptosis pathway,and stimulate the G0/G1 cell cycle into sub-Gl.To investigate the different cellular uptake,intracellular transportation and efflux of PAMAM-NH2by HepG2,MCF-7 cells and MCF-7/ADR cells,we ensured that about 13.67%of the amine groups on surface of the PAMAM dendrimers were modified with FITC.Using flow cytometer,we investigated the influence of time and concentration on cellular uptake rate of PAMAM-NH2.The results shown that the uptake of PAMAM-NH2 was time-and concentration-dependent.The cellular uptake efficiency of PAMAM-NH2 finally reaches plateaus in HepG2,MCF-7 cells and MCF-7/ADR cells.When the concentration of PAMAM-NH2 reached 50,the uptake of PAMAM-NH2 approached saturation in HepG2,MCF-7 cells and MCF-7/ADR cells.The final uptake rates of PAMAM-NH2 in three cells were different.The tested final uptake rate of PAMAM-NH2 in HepG2 cells was highest and that in MCF-7/ADR cells was lowest.The different tested PAMAM-NH2 uptake efficiency between HepG2 cells and MCF-7 cells was due to the different binding at HepG2 cells and MCF-7 cells surface.The different tested PAMAM-NH2 uptake efficiency between MCF-7 cells and MCF-7/ADR cells come from two factors.On the one hand,the binding at the MCF-7 and MCF-7/ADR cells was different.On the other hand,the excretion from the MCF-7/ADR cells might be higher than that from MCF-7 cells.Through the endocytosis inhibition studies,we found that uptake pathways of PAMAM-NH2 dendrimers were different in the three cells.In HepG2 cells,the PAMAM-NH2 is taken up by clathrin-mediated endocytosis.In MCF-7 and MCF-7/ADR cells,the PAMAM-NH2 is taken up by clathrin-mediated endocytosis and macropinosomes.In comparison,the clathrin-mediated endocytosis played more important role in the intermalization of PAMAM-NH2 in MCF-7 cells,while the macropinosomes was more important for the internalization of PAMAM-NH2 in MCF-7/ADR cells.Through investigating the intracellular PAMAM-NH2 with the addition of sucrose and pre-incubation with low temperature,we found that the PAMAM-NH2 could be taken up by the energy-dependent formation of nanoscale membrane holes in addition to the endocytosis.To investigate the contribution of the Golgi complex,endoplasmic reticulum(ER)and endosomal acidification to the endoeytosis process of PAMAM-NH2,brefeldin A,monensin and bafilomycin Al were added as inhibitory agents.The endocytosis of PAMAM-NH2 is a facile procedure and is independent of the intracellular process in HepG2,MCF-7 cells and MCF-7/ADR cells.The ER and Golgi complex exerted negligible effects on the endocytosis of PAMAM-NH2,and only the lysosomes participated in the intermalization process of PAMAM-NH2.After staining the cell organelles with different fluorescence probes,we found the PAMAM-NH2 could co-localize with lysosomes,mitochondria,nucleus and microtubules in HepG2,MCF-7 cells and MCF-7/ADR cells.The ratios of PAMAM-NH2 localized in the nucleus of three cells were different:HepG2 cells>MCF-7 cells>MCF-7/ADR cells.The results of pixel intensity analysis of the colocalization and endo-lysosomal membrane integrity assay revealed that compared with cancer cells,the PAMAM-NH2 more difficultly escaped from lysosomes in multidrug resistant cancer cells.The results of flow cytometer assay indicated that the exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in HepG2 and MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells.The remaining PAMAM-NH2 in the HepG2,MCF-7 and MCF-7/ADR cells increased with the addition of brefeldin A,monensin and bafilomycin Al,indicating that these three processes,namely,transportation from the ER to the Golgi complex,transportation from the Golgi complex to the plasma membrane and endosomal acidification,participated in the exocytosis of PAMAM-NH2 in HepG2,MCF-7 and MCF-7/ADR cells.The results about the exocytosis of dendrimers by LSCM revealed that in HepG2 and MCF-7/ADR cells,the ER and Golgi complex have the same contribution to the exocytosis of PAMAM-NH2,while in MCF-7 cells,the Golgi complex have more contribution to the exocytosis of PAMAM-NH2.The PAMAM-NH2 could be rapidly eliminated from the nucleus,but portion of PAMAM-NH2 was retained in the cytoplasm and could not be excreted in three cells.Using the PAMAM-OH as the control,we explored the reasons for the different results about cytotoxicity,time-dependent uptake,colocalization with lysosomes and the exocytosis of PAMAM-NH2 in MCF-7 cells and MCF-7/ADR cells.We found that the above results of PAMAM-OH were the same in MCF-7 cells and MCF-7/ADR cells.The PAMAM-NH2 may not bypass P-gp and MDR-associated protein,resulting in the exocytosis by P-gp and MDR-associated protein.The PAMAM-OH was only taken up by endocytosis in resistant cancer cells,so it can bypass P-gp and MDR-associated protein.Therefore,the multidrug resistance(MDR)in tumor cells could influence the PAMAM-NH2 because of the terminal amino.