Figure 1.
Establishment of the hepatic cancer mouse model.
(A) Liver cancer; (B) Normal mouse liver.
Figure 2.
Particle size and scanning electron microscope (SEM) image of GC/5-FU.
(A) Particle size graph showing the diameter of GC/5-FU (35.19±9.50 nm). (B) SEM image of GC/5-FU. The particles show spherical structure with a smooth surface and no adhesion between nanoparticles. (C) The in vitro release curve of nanoparticles in simulated body fluid (37°C, pH 7.4). A rapid release was observed from 0 h to 12 h, with a cumulative release percentage of 32.4%. Smooth slow-release occurred between Day 1 and 8, with a cumulative release percentage of 93.50%. During Day 8 to 10, the release reached a plateau, with a cumulative release percentage of 95.70% at Day 10.
Figure 3.
5-FU nanoparticle enhanced 5-FU to inhibit the growth of hepatic caner through ASGPR -mediated endocytosis in vitro.
(A) ASGPR protein expression was detected by western blot in SMMC-7721, SW480 and LO2 cell lines (n = 3). The ASGPR protein expression in SMMC-7721 and LO2 was stronger than with SW480 (P<0.01), the expression in SMMC-7721 increased significantly than those with SW480 (P<0.01). **P<0.01 compared to SW480, ##P<0.01; compared to LO2. (B) Concurrent focal images of SMMC-7721 cells after 4 h incubation with CS and GC nanoparticles. The strong green fluorescent bright spots in SMMC-7721 cells were observed in the GC nanoparticles using a laser scanning confocal microscope and only small amount of green fluorescent spots were found in the CS nanoparticles without galactosyl ligand modified chitosan. So it was confirmed that the GC nanoparticles targeted liver cancer cells and entered into cells via ASGPR-mediated endocytosis. (C) Comparison of the inhibition rates of 5-FU, CS/5-FU and GC/5-FU on SW480 and SMMC-7721 cells. Results are shown as a average ± means and standard deviation (n = 3). At the 24, 48 and 72 h time points, the rate of tumor inhibition rate decreased from in the order GC/5-FU-SMMC-7721 to 5-FU-SW480 to 5-FU-SMMC-7721 to CS/5-FU-SMMC-7721 to CS/5-FU-SW480 to GC/5-FU-SW480.
Table 1.
Comparison of IC50 of GC/5-FU, CS/5-FU and 5-FU for SW480 and SMMC-7721 cells (n = 3).
Figure 4.
5-FU nanoparticle had liver-targeting and enhanced 5-FU to inhibit the growth of hepatic caner through ASGPR in vivo.
(A) Expression of ASGPR mRNA in different tissues of hepatic cancer, liver, spleen, kidney, lung, muscle, heart and bowel in the orthotropic liver cancer mouse model evaluated by RT-PCR. Results are shown as means±SD (n = 3). (**P<0.01, compared with spleen, kidney, lung, muscle, heart and bowel; ## P<0.01, compared with liver). (B) 5-FU concentration in different tissues in mice were treated with i.v. 5-FU, CS/5-FU or GC/5-FU or, 5-FU determined 30 min post-injection. Results are shown as an means±SD (n = 3). Hepatic cancer tissue showed the highest 5-FU concentration, followed by liver tissue. (C) The ratio of 5-FU concentration in hepatic cancer cells relative to that in other tissues. The 5-FU concentration in hepatic cancer was 8.69-, 23.35-, 79.96- and 85.15-fold higher than that in normal liver tissue, kidney, heart and blood, respectively. (D) The weight of liver tumors measured on Day 10. Results are shown as means±SD deviation (n = 10). Mice received GC/5-FU,CS/5-FU, 5-FU, GC or PBS on Day 5 after establishing the transplant model. **P<0.01 compared to control or GC group; ##P<0.01 compared to 5-FU group, ▴P<0.05 compared to CS/5-FU group. (E)Body weight was monitored from Day 1 to 10 of treatment (n = 10). Body weigh decreased significantly more in the 5-FU group compared with the control, GC, CS/5-FU and GC/5-FU group. There was no difference in body weight between the GC/5-FU, CS/5-FU, GC and control groups. (F) Mouse survival. The median survival in the control, GC, 5-FU, CS/5-FU and GC/5-FU groups was 16, 17, 22, 29 and 35 days, respectively. The longest median survival time was seen in the GC/5-FU group.
Figure 5.
The effects of different treatments on cell cycle, proliferation index and apoptosis index by flow cytometry analysis of H22 cells.
(A) Quantification of cell cycle distribution and proliferation index of H22 cells. Percentage of cells in G0–G1 in the GC/5-FU,CS/5-FU and 5-FU groups was higher than in the control and GC groups, and the proliferation index decreased significantly in the other three groups (P<0.01). (B) Quantification of apoptosis of H22 in different treatment groups. The percentage of apoptotic cells in the GC/5-FU,CS/5-FU and 5-FU groups was significantly higher than in the control and GC groups (P<0.01). The percentage of apoptotic cells in the GC/5-FU group was also higher than in the 5-FU (P<0.01) and CS/5-FU groups (P<0.05). **P<0.01 compared with control or GC group; ##P<0.01 compared with the 5-FU group; ▴P<0.05 compared with the CS/5-FU group.
Figure 6.
Apoptosis, p53 and caspase-3 expression were detected in tumor tissues in mouse model.
(A) The quantification of the apoptotic index (AI) in hepatic cancer tissues. Apoptosis was detected using the TUNEL assay on the tumor sections. The tumor samples from the control and GC groups showed little apoptosis; the addition of 5-FU and CS/5-FU induced sporadic apoptosis, while GC/5-FU induced a high degree of apoptosis which showed a clustered distribution (C). The AI in the GC/5-FU, CS/5-FU and 5-FU groups was higher than that in the GC and control groups (P<0.01). The expression of AI in the GC/5-FU group increased more than in the CS/5-FU or 5-FU groups (P<0.01). (B) Quantification of p53 and caspase-3 expression detected by IHC. P53 (D) and Caspase-3 (E) staining in the control and GC groups showed a scattered cytoplasmic distribution pattern, appearing as dark yellow or dark brown. In the 5-FU, CS/5-FU and GC/5-FU groups, P53 and caspase-3 showed a sheet staining pattern, which was most marked in the GC/5-FU group. The expression of P53 and caspase-3 was higher in the 5-FU, CS/5-FU and GC/5-FU groups than in the control or GC groups. The highest increase was seen in the GC/5-FU group (P<0.01). **P<0.01 compared with control or GC group; ##P<0.01 compared with the 5-FU group; ▴▴P<0.01 compared with the CS/5-FU group.
Figure 7.
Expression of P53, caspase-3, Bax, Bcl-2 and PARP-1 in tumor tissues from mice.
(A) Tissue levels of P53 were measured by RT-PCR and western blot, and normalized to GAPDH. The expression of P53 was higher in the 5-FU, CS/5-FU and GC/5-FU groups than in the control and GC groups. The highest expression was seen in the GC/5-FU group. (B) Caspase-3 expression in individual tumor samples was determined by both RT-PCR and western blot analysis and results were normalized to GAPDH. The expression of PARP-1 displayed a increasing tendency from control to GC to 5-FU to CS/5-FU to GC/5-FU groups (P<0.01), with the most significant increase being seen in the GC/5-FU group. (C) Expression of Bcl-2 and Bax was quantified by both RT-PCR and western blot. The ratio of Bcl-2/Bax showed a decreasing tendency from control to GC to 5-FU to CS/5-FU to GC/5-FU groups (P<0.01). Specifically, the ratio in 5-FU, CS/5-FU and GC/5-FU was significantly lower than in the control and GC groups. The lowest ratio was observed in the GC/5-FU group (P<0.01). (D) PARP-1 expression in individual tumor samples was determined by RT-PCR and western blot analysis; results were normalized to GAPDH. The expression of PARP-1 displayed a decreasing tendency from control to GC to 5-FU to CS/5-FU to GC/5-FU groups (P<0.01). The lowest expression was seen in the GC/5-FU group. **P<0.01 compared to control or GC group; ##P<0.01 compared to 5-FU group; ▴P<0.05 compared to CS/5-FU group.