Aptamer Combined with Fluorescent Silica Nanoparticles for Detection of Hepatoma Cells

Abstract

Purpose: The purpose of this study is to develop a simple, effective method to label hepatoma cells with aptamers and then detect them using fluorescent silica nanoparticles (FSNPs).

Method: Streptavidin was conjugated to carboxyl-modified fluorescein isothiocyanate (FITC)-doped silica nanoparticles which were prepared by the reverse microemulsion method. The resulting streptavidin-conjugated fluorescent silica nanoparticles (SA-FSNPs) were mixed with hepatoma cells that had been labeled with biotin-conjugated aptamer TLS11a (Bio-TLS11a). The specificity and sensitivity of the nanoprobes were assessed using flow cytometry and fluorescence microscopy. Their toxicity was assessed in normal human liver cell cultures using the MTT assay, as well as in nude mice using immunohistochemistry.

Results: SA-FSNPs showed uniform size and shape, and fluorescence properties of them was similar to the free FITC dye. SA-FSNPs were able to detect aptamer-labeled hepatoma cells with excellent specificity and good sensitivity, and they emitted strong, photobleach-resistant fluorescent signal. SA-FSNPs showed no significant toxic effects in vitro or in vivo.

Conclusion: The combination of biotin-conjugated aptamers and SA-FSNPs shows promise for sensitive detection of hepatoma cells, and potentially of other tumor cell types as well.

Keywords: Aptamer; Cancer; Fluorescent nanoparticles; Hepatoma.

Fig. 1

Schematic illustration of highly sensitive detection of HepG2 hepatoma cells using a biotin-conjugated aptamer (Bio-TLS11a) and streptadivin-conjugated fluorescent silica nanoparticles (FSNPs)

Fig. 2

Characterization of SA-FSNPs. a Transmission electron micrograph of SA-FSNPs. b Fluorescence emission spectra of FITC dye and SA-FSNPs

Fig. 3

a Flow cytometric detection of HepG2 cells (a) or L02 cells (b) after incubation with SA-FSNPs or FITC-TLS11a or the combination of Bio-TLS11a and SA-FSNPs. b Quantitative analysis of HepG2 cells (a) or L02 cells (b). NS not significant. ^** P < 0.01, ^*** P < 0.001

Fig. 4

Fluorescence micrographs of HepG2 and L02 cells after incubation with SA-FSNPs or FITC-TLS11a or the combination of Bio-TLS11a and SA-FSNPs. SA-FSNPs and FITC were examined in the green channel, while DAPI-stained nuclei were examined in the blue channel

Fig. 5

Photostability of FITC-TLS11a and of the combination of Bio-TLS11a with SA-FSNPs. a Fluorescence micrographs of HepG2 cells labeled with the combination of Bio-TLS11a and SA-FSNPs (upper row) or with FITC-TLS11a alone (lower row) and then continuously irradiated for the indicated periods. b Quantitative analysis of fluorescence intensity after different irradiation periods. ^*** P < 0.001

Fig. 6

Toxicity of SA-FSNPs. a 293T and L02 cells in culture were incubated with various concentrations of SA-FSNPs, and cell viability was measured at 12, 24, and 48 h. b Nude mice were treated with PBS or SA-FSNPs, and sections from major organs were stained with hematoxylin-eosin and examined by light microscopy. Magnification, ×400