In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles

Abstract

Aim: To synthesize antisense oligonucleotides (ASODNs) of midkine (MK), package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma (HCC) growth using these nanoparticles.

Methods: HepG2 cell proliferation was analyzed in vitro using the 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction (PCR).

Results: The in vitro proliferation of HepG2 cells was significantly inhibited by the nanoparticles packaged with MK-ASODNs (NANO-ASODNs). Furthermore, the NANO-ASODNs significantly inhibited the growth of HCC in the mouse model.

Conclusion: NANO-ASODNs can significantly suppress the growth of HCC in vitro and in vivo.

Figure 1

Nano-assembly of MK-ASODNs and nanoliposomes and characterization of NANO-ASODNs. A: Schematic illustration of the self-assembly of MK-ASODN and nanoliposomes; B: TEM image of the empty nanoliposomes stained with 1% uranyl acetate; C, D: TEM image of the NANO-ASODNs.

Figure 2

Transduction and function of NANO-ASODNs in vitro. A: 0.2 μmol/L FAM-conjugated NANO-ASODNs transduced into HepG2 cells. The results were observed under a confocal microscope at indicted times of 6, 12 and 18 h; B: NANO-ASODNs down-regulated expression of MK mRNA; C: The proliferation of HepG2 cells was significantly inhibited by NANO-ASODNs.

Figure 3

NANO-ASODNs target the liver. The kinetic results of the NANO-ASODNs were observed through in vivo imaging systems at indicated times after NANO-ASODNs were injected through the tail vein. A: Free ASODNs did not concentrate within the liver and these ASODNs disappeared quickly; B: NANO-ASODNs were found to mainly target the liver (the arrow represents the NANO-ASODNs).

Figure 4

Morphological changes of HCC following treatment with NANO-ASODNs. The volume of HCC decreased significantly following treatment with 100, 50 and 25 mg/kg per day of NANO-ASODNs for 20 d. MK-ASODNs were the positive control. The PBS or free nanoparticles represent the negative controls.

Figure 5

Histopathological analysis. A: Tissue sections of the tumors from in situ xenograft HCC; B: Tissue sections of the tumors from nanoparticles; C: Tissue sections of the tumors from 5-FU (10 mg/kg per day); D: Tissue sections of the tumors treated with ASODNs 50 mg/kg per day; E-G: Tissue sections of tumors treated with NANO-ASODNs 100, 50 and 25 mg/kg per day of NANO-ASODN treated tumors, respectively; H: Tissue sections of the tumors treated with 5-FU (10 mg/kg per day) and 50 mg/kg per day of NANO-ASODNs. Regions showing an increase of necrosis and fibrosis were observed in the 5-FU or ASODN treatment groups (C-H, × 200) compared with the free nanoparticles and untreated groups (A and B, × 200).