Facile Fabrication of Nanoparticles with Dual-Targeting Ligands for Precise Hepatocellular Carcinoma Therapy In Vitro and In Vivo.

Efficient hepatocellular carcinoma (HCC) therapy remains a significant challenge due to the unsatisfactory targeting efficiency of nanoparticles (NPs) with either a passive targeting or a single active targeting property. Although a dual-targeting mechanism-based strategy can promote the partial targeting efficiency, most of the reported NPs with dual-targeting properties generally suffer from sophisticated chemical design, multistep synthesis, and purification procedures, leading to batch-to-batch variation and difficulties in scalable production. To develop a facile yet efficient strategy toward dual-targeting ligand-functionalized NPs for precise HCC therapy and potential clinical translation, folic acid (FA) was readily introduced as a hydrophobic and targeting component to a hydrophilic macromolecular prodrug, galactosylated chitosan-5-fluorouracil acetic acid (GC-FU), to afford FA-GC-FU formulation that can self-assemble into NPs driven by the solubility variation of FA and GC-FU without the necessity of previously used physical cross-linking. The resulting nanoparticles of FA-GC-FU can target the overexpressed asialoglycoprotein receptors (ASGPRs) and folate receptors (FRs) on the surface of HCC cells, respectively, via the FA and lactobionic acid (LA) residues exposed on the surface of the NPs, leading to the maximized targeting efficiency of HCC and minimized nonspecific uptake by normal hepatocytes in vitro and in vivo. Therefore, this study not only developed a simple yet efficient strategy toward a facile fabrication of NPs with dual-targeting ligands but also presented a precise therapeutic platform for HCC with great potential for clinical translation.

In vitro and in vivo evaluation of macromolecular prodrug GC-FUA based nanoparticle for hepatocellular carcinoma chemotherapy.

A novel type of macromolecular prodrug delivery system is reported in this research. The N-galactosylated-chitosan-5-fluorouracil acetic acid conjugate (GC-FUA) based nanoparticle delivery system was evaluated in vitro and in vivo. Biocompatibility of GC-FUA-NPs was screened by BSA adsorption test and hemolysis activity examination in vitro. Cytotoxicity and cellular uptake study in HepG2 and A549 cells demonstrated that compared to free 5-Fu, the GC-FUA-NPs play great function in killing cancer cells for the cell endocytosis mediated by asialoglycoprotein receptor (ASGPR), which overexpresses on the cell surface. Pharmacokinetics study further illustrated that the drug-loaded nanoparticles has a much longer half-time than free 5-Fu in blood circulation in Sprague-Dawley (SD) rats. Tissue distribution was investigated in Kunming mice, and the result showed that the GC-FUA-NPs have a long circulation effect. The obtained data suggested that GC-FUA-NP is a very promising drug delivery system for efficient treatment of hepatocellular carcinoma.

In vitro and in vivo evaluation of pectin-based nanoparticles for hepatocellular carcinoma drug chemotherapy.

The fabrication and evaluation of a natural pectin-based drug delivery system are reported in this study. The drug delivery system displays specific active targeting ability to hepatocellular carcinoma due to the presence of excess galactose residues in the polymer structure as the natural targeting ligands. The system was prepared under very mild conditions in an aqueous medium containing Ca(2+) and CO3(2-) ions, generating uniform pectin-based nanoparticles with an average diameter of 300 nm, and the drug-loading content of anticancer drug 5-fluorouracil (5-FU) is around 24.8%. Cytotoxicity study of the 5-FU-loaded nanoparticles (5-FU-NPs) in HepG2 and A549 cell lines demonstrated their greater potency in killing cancer cells with overexpressed asialoglycoprotein receptor (ASGPR) on the cell surface, compared to that of the free drug. Pharmacokinetics study using Sprague-Dawley (SD) rats further confirmed that the drug-loaded nanoparticles showed a much longer half-life in the circulation fluids than the free drug. Tissue distribution was investigated on Kunming mice, and the results also demonstrated that the 5-FU-NPs has a long circulation effect. Taken together, the pectin-based drug delivery systems exhibit size-induced prolonged circulation as well as ASGP receptor-mediated targeting ability to cancer cell lines; therefore, it is a promising platform for the treatment of hepatocellular carcinoma.

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin.

AIM: To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. METHODS: HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor gamma (PPARgamma), NF-kappaB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. RESULTS: MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose-dependent manner, with little effect on growth of L-02 cells, and when IC(50) was measured as 8.45 micromol/L and 191.55 micromol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC(50) was 9.27 micromol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 micromol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 micromol/L ADFMChR than when treated with 30.0 micromol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 micromol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 micromol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 micromol/L GW9662, a blocker of PPARgamma. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 micromol/L ADFMChR, PPARgamma and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-kappaB expression decreased; however, pre-incubation with 10.0 micromol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 micromol/L ADFMChR on PPARgamma and NF-kappaB protein expression in HepG2 cells. CONCLUSION: ADFMChR induces apoptosis of HepG2 cell lines by activating PPARgamma, inhibiting protein expression of Bcl-2 and NF-kappaB, and increasing Bax expression.