Synthesis and efficient hepatocyte targeting of galactosylated chitosan as a gene carrier in vitro and in vivo

Abstract

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.

5-Fluorouracil Nanoparticles Inhibit Hepatocellular Carcinoma via Activation of the p53 Pathway in the Orthotopic Transplant Mouse Model

“…We also confirmed that this nanoparticle material was a high selectivity for the liver and was associated with low cytotoxicity [27]. In the present study, we synthesized GC/5-FU nanoparticles by combining the GC material with 5-FU, and tested its effect on liver cancer in vitro and in vivo.…”

Preliminary pharmacology of galactosylated chitosan/5-fluorouracil nanoparticles and its inhibition of hepatocellular carcinoma in mice

“…Figure 4A shows that in normal mice the 5-FU concentration in the same tissue showed a significant difference between 5-FU and GC/5-FU into the liver in vitro and in vivo. In the present study, we also confirmed that this nanoparticle material has a high selectivity for the liver, more stability and sustain release and a low cytotoxicity, 19 and we synthesized GC/5-FU nanoparticles by combining the GC material with 5-FU, GC/5-FU nanoparticles is a sustained release, its peak time, half-life time, mean residence time (MRT) and area of under curve (AUC) were longer or more than those of 5-FU. Meanwhile, distribution of the GC/5-FU difficult to take effect on the mice.…”

Activation of cellular immunity and marked inhibition of liver cancer in a mouse model following gene therapy and tumor expression of GM-SCF, IL-21, and Rae-1

“…Previously, we studied recombinant plasmids that expressed both GM-CSF and IL-21 in a mouse model of orthotopic liver cancer by intravenous tail vein injection [13]. This construct markedly blocked the growth of tumors and enhanced both NK cell and CTL activity.…”