Endocytosis And Exocytosis Differences Between Active And Negative Targeting Gold Nanoparticles And Related Mechanisms

Malignant tumor is considered to be one of the most serious diseases and the main cause of death worldwide.Cancer therapy has now become a global conundrum.Even though some active targeting therapeutic nanomedicines are under clinical trials,there are still no products available in commerce.Among the diverse factors that may influence the therapeutic outcomes,the exocytosis of targeted drug delivery systems(TDDS)and its relationship with the corresponding receptor receive little attentions.In this study,the endocytosis and exocytosis differences between active targeting and negative targeting preparations were compared,especially to illustrate the relationship between the exocytosis and the corresponding receptors.we have no idea if and tried to find out whether there exists the exocytosis of ligand-modified nanomedicine and what the relationship with the efflux of receptor.In this study,αvβ3 integrin was used as receptor model.Cyclic pentapeptides cRGDfK was selected to prepare active targeted particles(cRGDfK-PEG-AuNPs)and AuNPs modified without cRGDfK was prepared as the negative targeted particles.The pharmaceutical properties,endocytosis,exocytosis of the particles and the recycling of the receptor were studied,in order to find out the associations between them.It is expected these findings could provide guidance for studies on active TDDS.The dipolymers CM-PEG-S-S-PEG-CM was synthesized through polymerization by commercially available polymers HS-PEG-CM.cRGDfK modified linkers were obtained through amidation reaction.TLC analysis was used to monitor the reaction process.Both the dipolymers and the cRGDfK conjugated polymers were successfully synthesized determined by MALDI-TOF-MS and the conjugation rate of cRGDfK was about 95%determined by HPLC analysis.Gold nanoparticles(AuNPs)were prepared through the reduction of tetrachloroauric acid trihydrate by using NaBH4 as the reductants.cRGDfK-PEG-AuNPs and PEG-AuNPs were obtained through the formation of Au-S bond confirmed by XPS method.The average particles size of unmodified AuNPs determined by dynamic light scattering(DLS)was about 40nm and increased to 50nm-60nm when conjugated to linkers.The transmission electron microscope(TEM)images of cRGDfK-PEG-AuNPs and PEG-AuNPs showed spheroidal morphology.Visible spectrophotometry was established to quantify the Au content of the two kinds of AuNPs solution.The cRGDfK density on the surface of the cRGDfK-PEG-AuNPs was about 74.8%estimated through elemental analysis.The impact of serum on the intracellular trafficking of cRGDfK-Alexa Fluor(?) 555 conjugates was investigated by using transferrin-Alexa Fluor(?) 488 conjugates as the intracellular trafficking pathway markers.For the intracellular trafficking of Tfn-AF488,both the serum-free DMEM group and the complete-DMEM group all indicated that Tfn accumulated in the perinuclear area after the treatment of Tfn-AF488 for about 10min.There were no evident differences between the two groups.As to cRGDfK-AF555,for the serum-free group,cRGDfK seemed to travel a similar routine as the Tfn had done.Interestingly,for the complete-DMEM group,the peptide seemed to recycle back to the leading lamella within the 15 minutes’ incubation.The pentapeptide cRGDfK performed quite different trafficking pathways under the two culture conditions.The serum-containing DMEM was selected in the following experiments.Determined by sulforhodamine B(SRB)assay,both PEG-AuNPs and cRGDfK-PEG-AuNPs,as well as inhibitors did not decrease the viability of U87 cells and MCF-7 cells.The endocytosis of both PEG-AuNPs and cRGDfK-PEG-AuNPs all exhibited a time-dependent manner.cRGDfK-PEG-AuNPs were proved to be endocytosed by U87 cells more quickly.The competition experiment as well as the endocytosis experiment in MCF-7 cells all proved that the endocytosis of cRGDfK-PEG-AuNPs was αvβ3-dependent.The endocytosis pathway study showed that,both clathrin-dependent endocytosis and micropinocytosis were utilized by cRGDfK-PEG-AuNPs to enter U87 cells,while PEG-AuNPs took advantage of lipid raft-dependent especially caveolin-dependent route.Furthermore,cdc 42 GTPase might play an important role during the uptake of PEG-AuNPs in U87 cells.Determined by ICP-MS analysis,exocytosis could be obviously observed in cRGDfK-PEG-AuNPs group and the maximum efflux occurred at 7.5min.As expected,no such trends were observed in PEG-AuNPs group.When the exocytosis experiment was carried out in the presence of primaquine or using MCF-7 cell as the cell model,the evident efflux of cRGDfK-PEG-AuNPs at 7.5min was disappeared.The exocytosis of PEG-AuNPs in U87 cells was not affected.The intracellular trafficking of avβ3 integrin was detected by ELISA analysis.The results showed that in the complete DMEM,some of the αvβ3 integrins had recycled back to the cell membrane after the 5 minutes’incubation.At the end of 7.5min,much more labeled αvβ3 integrins had returned back.Then a relative clear conclusion could be ascertained that,along with the increased internalization via αvβ3 integrin-mediated endocytosis,the receptor-dependent exocytosis was also non-negligible.