Fabrication and characterization of a folic acid-bound 5-fluorouracil loaded quantum dot system for hepatocellular carcinoma targeted therapy

Abstract

In the present study, we covalently coupled folic acid (FA) and 5-fluorouracil acetic acid (FUA) on the surface of quantum dots (QDs) to produce a tumor targeting drug delivery system, FA-QDs-FUA. The QDs not only act as hepatocellular carcinoma (HCC)-targeted delivery vehicles, but also play a key role in imaging. The structural and optical properties of as-prepared FA-QDs-FUA were characterized using UV-visible spectra, fluorescence spectra, infrared spectra, particle size and zeta potential. In vitro hemolysis activity, cytotoxicity and targeting specificity of the FA-QDs-FUA system were also evaluated. The in vivo anti-tumor efficacy of FA-QDs-FUA in tumor-bearing mice was investigated. The average particle size and zeta potential of FA-QDs-FUA was 220.28 nm and -13.3 mV, respectively. The drug-loading content of FA-QDs-FUA was 36.85% ± 1.61% (n = 3). The in vitro release profile of 5-FU from FA-QDs-FUA demonstrated a slow and sustained release behaviour as compared to free 5-FU drug. The results of the in vitro cellular experiment demonstrated that FA-QDs-FUA reduced cytotoxicity as compared to free 5-FU and targeted more easily hepatocellular carcinoma cells (SMMC-7721 and HepG2) than normal cells. Mice treated with FA-QDs-FUA showed superior tumor suppression compared to those treated with free 5-FU at 4.72 mg kg^-1 of 5-FU. Therefore, the FA-QDs-FUA system can be used as a promising candidate for improving 5-FU efficacy and tumor targeting specificity with limited toxicity.

Scheme 1. Schematic diagram of designing FA-conjugated QDs-FUA nanoparticles, targeting toward tumor.

Fig. 1. The characterization of FA-QDs-FUA. (A) The ultraviolet absorption spectrum of FA-QDs-FUA; (B) the fluorescence spectrum of FA-QDs-FUA; (C) the FTIR of FA-QDs-FUA, FA and FUA; (D) the SEM image of FA-QDs-FUA; (E) the particle size of FA-QDs-FUA.

Fig. 2. In vitro drug releases behaviour of FA-QDs-FUA at different pH (7.4, 6.8 and 5.0) values (n = 3).

Fig. 3. In vitro percent hemolysis of FA-QDs-FUA and free 5-FU at different concentration. (n = 3).

Fig. 4. In vitro cell viability studies (MTT assay) following the treatment of PEG-QDs and FA-PEG-QDs on L02 cells. All the results presented are the mean ± SD (n = 3) of three replicate analysis. *** corresponds to highly significant (p < 0.001), ** for significant (p < 0.01) values.

Fig. 5. In vitro cytotoxicity studies (MTT assay) following the treatment of FA-QDs-FUA and free 5-FU to L02 (A), HepG2 (B), and SMMC-7721 (C) cells for 24 h, 48 h and 72 h. All the results presented are the mean ± SD of three replicate analyses. *** corresponds to highly significant (p < 0.001), ** for significant (p < 0.01) values.

Fig. 6. (A) In vitro fluorescence microscopy images and (B) quantification of L02, HepG2, and SMMC-7721 cells after incubation with FA-PEG-QDs for 2 h. Original magnifications are 200×. The scale bar represents 20 μm. *** corresponds to highly significant (p < 0.001).

Fig. 7. Evaluation of ALT, AST, BUN, Cr, LDH, CK and CK-MB plasma levels after all the treatments of saline, free 5-FU, and FA-QDs-FUA. (n = 8) ** for significant (p < 0.01) values.

Fig. 8. Tumor growth inhibition after intravenous injection of FA-QDs-FUA and free 5-FU in tumor-bearing mice. Data are shown as the mean ± standard deviation (n = 8).*p < 0.05, **p < 0.01, statistically significant compared with normal saline control.

Fig. 9. H&E analyses of tumor, liver and kidney of nude mice bearing SMMC-7721 cell xenograft after treatments with saline, free 5-FU and FA-QDs-FUA for 24 days. Original magnifications are 400×. The scale bar represents 10 μm.

Fig. 10. The content of Cd derived from the drug carrier(ZnCdSe/ZnS QDs) which accumulated in the liver, tumor, lung, heart, kidney and spleen after tail vein i.v. injection with saline or FA-QDs-FUA (ND-not detected).