Folic acid-modified ginsenoside Rg5-loaded bovine serum albumin nanoparticles for targeted cancer therapy in vitro and in vivo

Abstract

Background and purpose: Ginsenoside Rg5 (Rg5), a triterpene saponin, extracted from the natural herbal plant ginseng, is one of the most potent anticancer drugs against various carcinoma cells. However, the therapeutic potential of Rg5 is limited by its low solubility in water, poor bioavailability, and nontargeted delivery. Therefore, we prepared folic acid (FA)-modified bovine serum albumin (BSA) nanoparticles (FA-Rg5-BSA NPs) to improve the therapeutic efficacy and tumor targetability of Rg5.

Methods: Various aspects of the FA-Rg5-BSA NPs were characterized, including size, polydispersity, zeta potential, morphology, entrapment efficiency (EE), drug loading (DL), in vitro drug release, thermal stability, in vitro cytotoxicity, cell apoptosis, cellular uptake, in vivo antitumor effects and in vivo biodistribution imaging.

Results: The FA-Rg5-BSA NPs showed a particle size of 201.4 nm with a polydispersity index of 0.081, uniform spherical shape, and drug loading of 12.64±4.02%. The aqueous solution of FA-Rg5-BSA NPs had favorable stability for 8 weeks at 4°C. The FA-Rg5-BSA NPs dissolved under acidic conditions. Moreover, the Rg5-BSA NPs and FA-Rg5-BSA NPs had advanced anticancer activity compared with Rg5 in MCF-7 cells, while poor cytotoxicity was observed in L929 cells. The FA-Rg5-BSA NPs facilitated cellular uptake and induced apoptosis in MCF-7 cells. In addition, in an MCF-7 xenograft mouse model, the in vivo antitumor evaluation revealed that FA-Rg5-BSA NPs were more effective in inhibiting tumor growth than Rg5 and Rg5-BSA NPs. The in vivo real-time bioimaging study showed that the FA-Rg5-BSA NPs exhibited superior tumor accumulation ability.

Conclusion: The results suggested that FA-Rg5-BSA NPs could serve as a promising system to improve the antitumor effect of Rg5.

Keywords: antitumor activity; drug delivery; ginsenoside Rg5; human MCF-7 breast cancer cells; nanoparticles.

Figure 1

Schematic illustration of preparation of FA-Rg5-BSA NPs. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles.

Figure 2

(A) Hydrodynamic size distributions, (B) Zeta potentials, (C) SEM and (D) TEM images of Rg5-BSA NPs (top) and FA-Rg5-BSA NPs (bottom), and (E) UV-vis absorption spectra determining the amount of FA in Rg5-BSA NPs. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Figure 3

Characterization of folic acid modification. Notes: (A) The FT-IR and (B) ¹H NMR spectra of BSA, Rg5, FA, Rg5-BSA NPs and FA-Rg5-BSA NPs. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles; FT-IR, fourier transform infrared spectroscopy; ¹H NMR, nuclear magnetic resonance.

Figure 4

The stability of Rg5-BSA NPs and FA-Rg5-BSA NPs over time. Notes: The stability of Rg5-BSA NPs (A) and FA-Rg5-BSA NPs (B) in water at 4 °C for 8 weeks. Data are represented as mean ± SD, n=3. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles; PDI, polydispersity index.

Figure 5

The release curve of Rg5 from Rg5-BSA NPs and FA-Rg5-BSA NPs. Notes: The in vitro cumulative release percentage of Rg5 from Rg5-BSA NPs and FA-Rg5-BSA NPs over 140 hrs at different pH values (pH 5 and 7.4).Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles.

Figure 6

In vitro cell viability assay. Notes: Cell viability of blank BSA NPs, FA-BSA NPs against MCF-7 cells (A) and L929 cells (B) after incubating for 72 hrs; cell toxicity of Rg5, Rg5-BSA NPs and FA-Rg5-BSA NPs against MCF-7 cells (C, E) and L929 cells (D, F) for 24 hrs and 48 hrs. Values are represented as mean ± SD. (n=3, *P<0.05, **P<0.01). Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles.

Figure 7

Cell apoptosis assay of MCF-7 cells treated with Rg5, Rg5-BSA NPs and FA-Rg5-BSA NPs by (A) Hoechst 33,342, AO/EB, and (B) Annexin V-FITC/PI staining. Note: Magnification 20× and scale bar is 100 µm. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles; AO/EB, Acridine orange/ethidium bromide.

Figure 8

The cellular uptake of FITC-labeled NPs treated with MCF-7 cells for 3 hrs was observed by CLSM (A) and flow cytometry (B). Note: Magnification 40× and scale bar is 50 µm. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles; FITC, fluorescein isothiocyanate; CLSM, confocal laser scanning microscopy.

Figure 9

Rg5, Rg5-BSA NPs and FA-Rg5-BSA NPs significantly inhibited the growth of MCF-7 xenograft in vivo. Notes: (A) The image of MCF-7 xenograft tumors with different treatments at 21 days. (B) Tumor volume and (C) body weight were measured every 3 days. (D) Tumor weight after 21 days treatment. p<0.05 was considered to have a significant difference. Different letters indicate significant differences between each group. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles.

Figure 10

The in vivo real-time biodistribution imaging. Notes: MCF-7 tumor-bearing mice treated with free DiR, Rg5-BSA NPs/DiR, and FA-Rg5-BSA NPs/DiR at 2 h, 8 h and 24 h. Abbreviations: BSA, bovine serum albumin; Rg5, ginsenoside Rg5; FA, folic acid; NPs, nanoparticles; DiR, dioctadecyl-tetramethyl indotricarbocyanine iodide.