Cholesterol Kip Lulan Self-assembled Nanoparticles Interact With Liver Cancer Cell Research

As one of the natural polymer materials, polysaccharides possess good biocompatibility and biological safety. Hydrophobically modified polysaccharides, which can self-assemble into nanoparticles with an inner hydrophobic core and an outer shell of hydrophilic groups in aqueous media, have been recognized as a promising drug carrier due to long-circulation, good stability and passive targeting characteristics in vivo. Understanding the interaction between nanoparticles and cells, has important scientific significance and application value in understanding life processes in cellular level, mechanisms of drug action and gene therapy, which provides the basis for developing more safe and effective nano-sized drug carrier.In this study, covalent conjugation with fluorescein isothiocyanate (FITC) yielded stably labeled CHSP (FITC-CHSP), which was subsequently employed to prepare nanoparticles by dialysis. The effect of nanoparticle concentration, incubation time and temperature on the cellular uptake of the nanoparticles was systematically evaluated, and the cellular uptake mechanism and subcellular distribution of CHSP nanoparticles were also studied. In addition, the cytotoxicity and cellular uptake of CHSP nanoparticles as drug carrier of doxorubin (DOX) was discussed. Furthermore, the subcellular distribution of drugs was visualized and quantified. The main contents and results of this work were as follows:1. Preparation and characterization of FITC-CHSP and FITC-CHSP self-assembled nanoparticlesCovalent conjugation with FITC yielded stably labeled CHSP (FITC-CHSP), which was characterized by FT-IR. FITC-CHSP was successfully formulated into nanoparticles (mean particle size63.0±1.9nm) by dialysis with an almost spherical shape.2. Cellular uptake mechanism and subcellular distribution of CHSP nanoparticlesCytotoxicity assay clearly indicated that CHSP nanoparticles did not show significant toxicity against HepG2cells. The effect of nanoparticle concentration, incubation time and temperature on the cellular uptake of the nanoparticles was systematically evaluated by fluorometry, and the results suggested that cellular uptake of nanoparticles was concentration-, time-, and temperature-dependent. In vitro experiments with endocytic inhibitors revealed that clathrin-mediated endocytosis and macropinocytosis were involved in the internalization of CHSP nanoparticles. The subcellular distribution study demonstrated that CHSP nanopartciles were entrapped in the lysosomes at1h after incubation, while colocalization of nanopartticles with either the Golgi apparatus or the endoplasmic reticula was not observed throughout the entire course of the study.3. Preparation of DOX-loaded CHSP nanoparticles and in vitro drug release studyDOX, as a model drug, was physically entrapped into CHSP nanoparticles by dialysis method. DOX-loaded CHSP self-assembled nanoparticles were almost spherical in shape and their size increased from185.6to226.4nm with feed weight ratio of DOX to CHSP increased. The DOX-loading capacity ranged from10.31%to30.79%, which can be regulated by feed weight ratio of DOX to CHSP, and encapsulation efficiency ranged from71.2to88.3%. The release profile in vitro demonstrated that DOX showed sustained release over72h, and DOX release rate decreased with increase in the pH value of media.4. Uptake of DOX-loaded CHSP nanoparticles by HepG2cells in vitroCytotoxicity assay revealed that both DOX-loaded CHSP nanoparticles and free DOX showed cytotoxicity against HepG2cells, and cytotoxicity was enhanced with increasing DOX equivalent. In addition, the half maximal inhibitory concentration (IC50) of DOX-loaded CHSP nanoparticles and free DOX in HepG2cells were0.99μg/mL and2.36μg/mL, respectively, which demonstrated that DOX-loaded CHSP nanoparticles exhibited more cytotoxic activity against HepG2cells than free DOX alone. Flow cytometry analysis showed that cellular uptake of DOX increased with increasing the incubation time, and DOX-loaded CHSP nanoparticles enhanced uptake by cells when incubated more than2h.Subcellular disribution of DOX was visualized and quantified by CLSM and In Cell Developer software, respectively. The results showed that due to the different uptake mechanisms of the free drug and drug-loaded nanoparticles, free DOX was mainly distributed in the nucleus at30min after incubation, and DOX in the whole cell and nucleus were both increased with incubation time increased. However, DOX-loaded CHSP nanoparticles was mainly distributed in the cytoplasm at30min after incubation, and entrapped in the lysosomes at1h after incubation. Meawhile, DOX in the nucleus was increased significantly, and DOX in the whole cell, nucleus and lysosomes were all increased. Furthermore, DOX in the whole cell and nucleus in the cells treated with DOX-loaded CHSP nanoparticles were more than that in the free DOX treated cells, respectively, at1h after incubation. However, content of DOX in the Golgi apparatus, or the endoplasmic reticula, or the mitochodrion was low, and there was no significant difference with incubation time increased.In conclusion, CHSP nanoparticles had good biocompatibility, and could be fast uptaken by clathrin-mediated endocytosis and macropinocytosis. In addition, uptake ability of DOX-loaded nanoparticles by HepG2cells was stronger than that of free DOX, and DOX-loaded CHSP nanoparticles exhibited more cytotoxic activity against HepG2cells than free DOX alone, which indicated that DOX-loaded CHSP nanoparticles could achieve high efficacy at lower doses compared with free DOX. Our CHSP nanoparticles may serve as a drug carrier for hepatoma therapy.