Nanoparticle drug delivery (NDDS) is a novel system in which the drugs are delivered to the site of action by small particles in the nanometer range. Natural or synthetic polymers are used as vectors in NDDS, as they provide targeted, sustained release and biodegradability. Here, we used the chitosan and hepatoma cell-specific binding molecule, glycyrrhetinic acid (GA), to synthesize glycyrrhetinic acid-modified chitosan (GA-CTS). The synthetic product was confirmed by Fourier transformed infrared spectroscopy (FT-IR) and 1H-nuclear magnetic resonance (1H-NMR). By combining GA-CTS and 5-FU (5-fluorouracil), we obtained a GA-CTS/5-FU nanoparticle, with a particle size of 217.2 nm, a drug loading of 1.56% and a polydispersity index of 0.003. The GA-CTS/5-FU nanoparticle provided a sustained release system comprising three distinct phases of quick, steady and slow release. We demonstrated that the nanoparticle accumulated in the liver. In vitro data indicated that it had a dose- and time-dependent anti-cancer effect. The effective drug exposure time against hepatic cancer cells was increased in comparison with that observed with 5-FU. Additionally, GA-CTS/5-FU significantly inhibited the growth of drug-resistant hepatoma, which may compensate for the drug-resistance of 5-FU. In vivo studies on an orthotropic liver cancer mouse model demonstrated that GA-CTS/5-FU significantly inhibited tumor growth, resulting in increased survival time.

Biodegradable polymer nanoparticle drug delivery systems provide targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability and fewer side effects. These drug delivery systems are widely used for delivering cytotoxic agents. In the present study, we synthesized GC/5-FU nanoparticles by combining galactosylated chitosan (GC) material with 5-FU, and tested its effect on liver cancer in vitro and in vivo. The in vitro anti-cancer effects of this sustained release system were both dose- and time-dependent, and demonstrated higher cytotoxicity against hepatic cancer cells than against other cell types. The distribution of GC/5-FU in vivo revealed the greatest accumulation in hepatic cancer tissues. GC/5-FU significantly inhibited tumor growth in an orthotropic liver cancer mouse model, resulting in a significant reduction in tumor weight and increased survival time in comparison to 5-FU alone. Flow cytometry and TUNEL assays in hepatic cancer cells showed that GC/5-FU was associated with higher rates of G0–G1 arrest and apoptosis than 5-FU. Analysis of apoptosis pathways indicated that GC/5-FU upregulates p53 expression at both protein and mRNA levels. This in turn lowers Bcl-2/Bax expression resulting in mitochondrial release of cytochrome C into the cytosol with subsequent caspase-3 activation. Upregulation of caspase-3 expression decreased poly ADP-ribose polymerase 1 (PARP-1) at mRNA and protein levels, further promoting apoptosis. These findings indicate that sustained release of GC/5-FU nanoparticles are more effective at targeting hepatic cancer cells than 5-FU monotherapy in the mouse orthotropic liver cancer mouse model.

GC/5-FU nanoparticles can significantly inhibit the growth of liver cancer in mice via the p53 apoptosis pathway, and relieve the side effects and immunosuppression of 5-FU.

While chitosan (CS) has been researched widely as a non-viral vector, its usefulness has been limited by its low cell specificity and transfection efficiency. Therefore, we successfully synthesized galactosylated chitosan (GC) and complexed it with an enhanced green fluorescent protein plasmid (pIRES-EGFP) for transfection into cultured H22 cells (murine hepatic cancer cell line) using various GC/EGFP (N/P) charge ratios. Maximal gene transfection rates detected by flow cytometry occurred at an N/P ratio 5:1. Compared with those of lipofectin/EGFP and naked pIRES-EGFP, GC/EGFP complexes show a very efficient cell-selective transfection to hepatocytes. The MTT assay detected relatively low cytotoxicity in cells transfected with GC. A recombinant plasmid granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin (IL) 21 (pIRES/GM-CSF-IL21) was successfully constructed and GC/GM-CSF-IL21 nanoparticles (average diameter, 82.1 nm) were administered via the tail vein of mice with liver metastasis of colon cancer model, for 5 consecutive days. The GC/GM-CSF-IL21 nanoparticles exhibited hepatocyte and passive tumor specificity, increased therapeutic efficacy compared to control groups, promoted leukocytes to aggregate in tumor tissues, and activated the cytotoxicity of natural killer (NK) cells and cytolytic T lymphocyte (CTL). Our results indicate that GC can be used in gene therapy to improve transfection efficiency and can be used as an immunological stimulant in vivo.

Vector plays an important role in hepatic targeted drug delivery system. In this study, a novel material as a liver targeting vector, dual-ligand modified chitosan (GCGA) composed of chitosan (CTS), glycyrrhetinic acid (GA) and lactobionic acid (LA), was designed and synthesized by an orthogonal experiment with two-step synthesis under mild conditions. The synthesized final product was characterized and confirmed by FTIR and (1)H-NMR spectroscopy, and DS of GA and LA in CTS were measured to be 13.77 and 16.74 mol% using (1)H-NMR, respectively. The cytotoxicity of CTS and GCGA was concentration dependent which was inverse proportion to the cell viability by MTT assay using L929 cell line, and inhibitory concentration 50% (IC50) was 0.2 mg/ml for GCGA. The in vitro targeting efficiency and the in vitro cellular uptake were investigated. Compared with CTS NPs and GA-CTS NPs, GCGA NPs showed good cell specificity to BEL-7402 cells via the dual-ligand-receptor-mediated recognition, leading to a higher affinity to BEL-7402 cells. The results suggested that GCGA described here has the potential to be used as an effective vector for hepatic targeted drug therapy.