The Cellular Uptake Route Of Epirubicin-loaded Folate-Conjugated Pullulan Aacetate Nanoparticles

BackgroundWith the development of nanotechnology,a few of nanomaterial-based products have shown promise in cancer treatments and some of them including nanoparticles,liposomes,and polymer-drug conjugates have been approved for the clinical research.Pullulan is a flexible water soluble,neutral linear polysaccharide consisting of ?-1,6 linked maltotriose residues and has been widely applied in the pharmaceutical fields.Due to its excellent bioproperties,e.g,non-toxicity,biocompatibility,biodegradability,non-immunogenicity and non-antigenicity,pullulan has been recently used as the carrier material to prepare the targeted drug and/or gene delivery systems.In our previous reports,folate conjugated pullulan acetate nanoparticles(FPA NPs)that we previously prepared had excellent drug loading and sustained drug release properties,and showed the potential to improve the bioavailability of the loaded drug in vivo.But,the tumor targeted nano-drug carriers based on pullulan and the cellular internalization needed to be further studied and developed.Objective1.The cellular internalization and intracellular delivery of nanoparticle carriers are very important to exert the anticancer effects of the loaded drugs.In this study,we further evaluated the cytotoxicity and the cellular uptake of FPA NPs in in three human cancer cell lines(Hela,MCF-7 and HepG2 cells),with different folate-receptor(FR)expression levels.2.To clarify the the cellular uptake route and immune escape mechanism of nanoparticles in Kupffer cells(KC)and to preliminarily evaluate the potential of FPA NPs as a targeted nano-drug carrier.Methods1.Folate conjugated pullulan acetate nanoparticles(FPA NPs)and Epirubicin-loaded FPA NPs(FPA/EPI NPs)were prepared by dialysis method.The particle size was determined by dynamic light scattering(DLS)and zeta-potential was also performed by the same instrument.The morphology of nanoparticles was observed using transmission electron microscopy(TEM).The drug loading,encapsulation efficiency and in vitro drug release profiles of FPA/EPI NPs have been evaluated.2.We assessed the cytotoxicities of blank FPA NPs in Hela,MCF-7 and HepG2 cells with different FR expression levels using MTS assay to preliminarily evaluate the biosafety of this nanoparticle carrier.Meanwhile,several endocytic inhibitors including chlorpromazine(CPZ),chloroquineto(CQ),amiloride(AMR),nystatin(NY)and folate(FOL),as a FR inhibitor,were selected to study the potential endocytosis mechanisms of FPA/EPI NPs in Hela,MCF-7 and HepG2 cells.3.To investigate the cellular internalization mechanisms of FPA/EPI NPs,their uptakes in KC were further detected in the presence of different uptake inhibitors,including CPZ,CQ,AMR,NY,and free FOR using the fluorescence spectrophotometry methods.Meanwhile,pyrrolidine dithiocarbamate(PDTC)as the inhibitor of NF-?B channel,were selected to quantitative study the secretion of inflammatory cytokines(TNFa,IL-1?,IL-6)using ELISA method.Results1.FPA/EPI NPs were prepared by the dialysis method.The TEM image showed that blank FPA NPs and FPA/EPI NPs both had a regular spherical shape with compact structure,and their size,determined by the dynamic light scattering method,were(204.2±10.9)nm and(273.4±11.0)nm,respectively,with the relatively narrow size distributions.The EPI loading content and encapsulation efficiency in FPA/EPI NPs were(6.45±1.04)% and(72.45±11.50)%,respectively.EPI exhibited a rapid initial drug release during the first 12 h,and then followed by a significant sustained release from FPA/EPI NPs.2.The results of the cytotoxicities of blank FPA NPs suggested that FPA NPs showed no significant toxicities in three different cancer cells at the concentrations used in this study.Furthermore,the results from the confocal microscopic images that EPI carried by FPA NPs could successfully enter into cancer cells,but could not be completely released during 2-hour incubation period.Among these non-specific mechanisms,the clathrinmediated endocytosis and the cell macropinocytosis obviously played more important roles in the cellular internalization of FPA/EPI NPs.After pretreatments with free folate,the uptakes of FPA/EPI NPs in Hela and MCF-7 cells decreased respectively to 57.8% and 70.0% compared to the controls without adding any endocytic inhibitors,but the uptake in HepG2 cells did not significantly change.3.KC cells were extracted from SD rat liver by perfusion,enzymatic digestion and gradient centrifugation methods successfully.CQ significantly inhibited the KC uptake of FPA/EPI NPs.After pretreatments with combined application of CQ and PDTC,the inflammatory cytokines(TNF-a,IL-1?,IL-6)decreased significantly(***P <0.001).Conclusion1.In this study,we successfully prepared FPA/EPI NPs with a high drug encapsulation efficiency,sustained release profiles.2.The in vitro cytotoxicities results demonstrated that FPA NPs,used as the drug carrier,were relatively biosafe.The cytotoxicities results of FPA/EPI NPs showed that drug loaded had nanoparticles significantly higher cytotoxicity and more potent internalization capability in the FR-overexpressed cancer cells.Multi mechanisms appeared to be involved in the cellular internalization of FPA/EPI NPs,but the FRmediated endocytosis was only observed in cancer cells with FR-expression levels.3.CQ significantly inhibited the KC uptake of FPA/EPI NPs.After pretreatmenting with combined application of CQ and PDTC,the three inflammatory cytokines decreased significantly.4.In summary,FPA NPs,being used as a novel carrier for EPI,displayed significantly enhanced in vitro anticancer activities in cancer cells with FR-expression levels,indicating their potential applications in cancer chemotherapy.