Nucleolin recognizing silica nanoparticles inhibit cell proliferation by activating the Bax/Bcl-2/caspase-3 signalling pathway to induce apoptosis in liver cancer

Abstract

Multifunctional nanocarrier platforms have shown great potential for the diagnosis and treatment of liver cancer. Here, a novel nucleolin-responsive nanoparticle platform was constructed for the concurrent detection of nucleolin and treatment of liver cancer. The incorporation of AS1411 aptamer, icaritin (ICT) and FITC into mesoporous silica nanoparticles, labelled as Atp-MSN (ICT@FITC) NPs, was the key to offer functionalities. The specific combination of the target nucleolin and AS1411 aptamer caused AS1411 to separate from mesoporous silica nanoparticles surface, allowing FITC and ICT to be released. Subsequently, nucleolin could be detected by monitoring the fluorescence intensity. In addition, Atp-MSN (ICT@FITC) NPs can not only inhibit cell proliferation but also improve the level of ROS while activating the Bax/Bcl-2/caspase-3 signalling pathway to induce apoptosis in vitro and in vivo. Moreover, our results demonstrated that Atp-MSN (ICT@FITC) NPs had low toxicity and could induce CD3⁺ T-cell infiltration. As a result, Atp-MSN (ICT@FITC) NPs may provide a reliable and secure platform for the simultaneous identification and treatment of liver cancer.

Keywords: AS1411; MSNs; apoptosis; hepatocellular carcinoma; proliferation.

SCHEME 1

Schematic of the synthesis and antitumor mechanism of Atp-MSN (ICT@FITC) NPs against liver cancer.

FIGURE 1

Synthesis and characterization of Atp-MSN (ICT@FITC) NPs (A) TEM images of MSNs (scale = 100 nm) (B) Atp-MSN (ICT@FITC) NPs (scale = 100 nm) (C) Hydrated particle size of the Atp-MSN (ICT@FITC) NPs (D) BET isotherm and BJH pore size distribution (inset) of Atp-MSN (ICT@FITC) NPs (E) Ultraviolet-visible (UV) absorption spectra of ICT, FITC, MSNs, and Atp-MSN (ICT@FITC) NPs, respectively.

FIGURE 2

FITC release from Atp-MSN (ICT@FITC) NPs and nucleolin detection (A) Detection of nucleolin (B) FITC was release from Atp-MSN (ICT@FITC) NPs at different temperatures (0°C and 37°C). Cellular uptake of Atp-MSN (ICT@FITC) NPs by HCCLM3 (C) and L02 (D) cells.

FIGURE 3

Atp-MSN (ICT@FITC) NPs inhibit HCC cell proliferation. Cytotoxicity studies in HCCLM3 (A) and HepG2 (B) HCC cells treated with different concentrations of MSNs and Atp-MSN (ICT@FITC) NPs for different lengths of time (n = 3). Colony formation (C) and soft agar colony formation assays (D) with HCCLM3, HepG2 and L02 cells treated with MSNs and Atp-MSN (ICT@FITC) NPs.

FIGURE 4

Atp-MSN (ICT@FITC) NPs effect on the apoptosis in tumor cells (A) and (E), Reactive oxygen species (ROS) produced after treatment with MSNs and Atp-MSN (ICT@FITC) NPs. DAPI (blue); DCF/ROS (green) (B) and (F), Annexin V-FITC and PI triple fluorescent staining showed cellular apoptosis after treatment with MSNs and Atp-MSN (ICT@FITC) NPs. DAPI: blue; Annexin V: green; PI: red (scale = 100 nm) (C) and (G), Immunoblotting for Bax, Bcl-2, caspase-3 and cleaved caspase-3 (normalized to GAPDH) (D) and (H), Quantification of Bax, Bcl-2, caspase-3 and cleaved caspase-3.

FIGURE 5

Atp-MSN (ICT@FITC) NPs inhibited HCC cell proliferation and induced apoptosis in vivo (A) Physical photographs of tumours from the Con, MSN and Atp-MSN (ICT@FITC) NP groups (B) Tumor growth curve of HCCLM3-bearing mice in three groups (n = 4), p < 0.01 (C) Evaluation of Bax, Bcl-2, caspase-3, cleaved caspase-3 and Ki67 protein expression by IHC (D) The average number of apoptotic cells detected by TUNEL immunofluorescence staining (E) Fluorescence image monitoring of immune cell infiltration (scale = 100 nm).

FIGURE 6

(A) H&E staining of the major organs from mice treated with different therapies (scale = 100 nm) (B) Mouse body weight changes (n = 4) (C) Mice hepatorenal function test (n = 4).