Study on cellular uptake mechanism and subcellular distribution of cholesterol-modified pullulan nanoparticles

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Objective To investigate the cellular uptake mechanism and subcellular distribution of cholesterol-modified pullulan (CHSP) nanoparticles by human hepatocellular carcinoma (HepG2) cells.Methods Fluorescein isothiocyanate (FITC) was conjugated to CHSP,and FITC-labeled CHSP (FITC-CHSP) nanoparticles were prepared by dialysis.The cytotoxicity of CHSP nanoparticles against HepG2 cells was evaluated by MTT assay.Meanwhile,several selective endocytosis inhibitors (chlorpromazine,filipin and amiloride) were selected to study the potential endocytosis mechanism.To study the subcellular distribution of CHSP nanoparticles at different incubation times,immunofluorescence staining was used to identify the Golgi apparatus,endoplasmic reticula (ER) and lysosomes,followed by confocal laser scanning microscopy(CLSM) observation.Results Covalent conjugation with FITC yielded stably labeled CHSP,which was successfully formulated into nanoparticles (mean particle size (63.0±1.9) nm) by dialysis with an almost spherical shape.MTT assay clearly indicated that the CHSP nanoparticles did not show significant toxicity in HepG2 cells.In vitro experiments with endocytic inhibitors revealed that clathrin-mediated endocytosis and macropinocytosis were both involved in the internalization of CHSP nanoparticles.The subcellular distribution study demonstrated that CHSP nanoparticles were entrapped in the lysosomes at 1 h after incubation,while colocalization of nanoparticles with either the Golgiapparatus or the ER was not observed during the entire course of the study.Conclusions CHSP nanoparticles may serve as a versatile carrier for intracellular delivery of therapeutic agents. Key words: Cholesterol-modified pullulan ; Nanoparticles ; Fluorescein isothiocyanate ; Uptake mechanism ; Subcellular distribution ;