In situ preparation of water-soluble ginsenoside Rh2-entrapped bovine serum albumin nanoparticles: in vitro cytocompatibility studies

Abstract

The present study investigates a simple and convenient one-step procedure for the preparation of bovine serum albumin (BSA)-Rh2 nanoparticles (NPs) at room temperature. In this work, ginsenoside Rh2 was entrapped within the BSA protein to form BSA-Rh2 NPs to enhance the aqueous solubility, stability, and therapeutic efficacy of Rh2. The physiochemical characterization by high-performance liquid chromatography, nuclear magnetic resonance, Fourier transform infrared spectroscopy, field emission transmission electron microscopy, dynamic light scattering, and thermogravimetric analysis confirmed that the prepared BSA-Rh2 NPs were spherical, highly monodispersed, and stable in aqueous systems. In addition, the stability of NPs in terms of different time intervals, pHs, and temperatures (20°C-700°C) was analyzed. The results obtained with different pHs showed that the synthesized BSA-Rh2 NPs were stable in the physiological buffer (pH 7.4) for up to 8 days, but degraded under acidic conditions (pH 5.0) representing the pH inside tumor cells. Furthermore, comparative analysis of the water solubility of BSA-Rh2 NPs and standard Rh2 showed that the BSA nanocarrier enhanced the water solubility of Rh2. Moreover, in vitro cytotoxicity assays including cell viability assays and morphological analyses revealed that Rh2-entrapped BSA NPs, unlike the free Rh2, demonstrated better in vitro cell viability in HaCaT skin cell lines and that BSA enhanced the anticancer effect of Rh2 in A549 lung cell and HT29 colon cancer cell lines. Additionally, anti-inflammatory assay of BSA-Rh2 NPs and standard Rh2 performed using RAW264.7 cells revealed decreased lipopolysaccharide-induced nitric oxide production by BSA-Rh2 NPs. Collectively, the present study suggests that BSA can significantly enhance the therapeutic behavior of Rh2 by improving its solubility and stability in aqueous systems, and hence, BSA-Rh2 NPs may potentially be used as a ginsenoside delivery vehicle in cancer and inflammatory cell lines.

Keywords: bovine serum albumin; cancer; ginsenoside Rh2; inflammation; solubility; stability.

Figure 1

Schematic illustration of the BSA-Rh2 NPs synthesis and application mechanism for anticancer and anti-inflammatory efficacy. Abbreviations: BSA, bovine serum albumin; NPs, nanoparticles.

Figure 2

HPLC analysis of BSA-Rh2 NPs as compared with standard Rh2. Abbreviations: BSA, bovine serum albumin; NPs, nanoparticles; HPLC, high-performance liquid chromatography.

Figure 3

¹H NMR analysis of BSA-Rh2 NPs (A), standard Rh2 (B) and standard BSA (C). Abbreviations: BSA, bovine serum albumin; NPs, nanoparticles.

Figure 4

FT-IR pattern of BSA-Rh2 NPs corresponding to standard BSA. Abbreviations: BSA, bovine serum albumin; NPs, nanoparticles.

Figure 5

FE-TEM shape, morphology, SEAD and FFT characterization of BSA-Rh2 NPs. Notes: Particles size at 200 nm (A), at 10 nm (B), FFT of NPs (C and D), SEAD pattern of NPs (E). Abbreviations: FE-TEM, field-emission transmission electron microscope; SEAD, selected area (electron) diffraction; FFT, fast fourier transform; BSA, bovine serum albumin; NPs, nanoparticles.

Figure 6

Characterizations of BSA-Rh2 NPs by particle size analysis (A), zeta potential analyzer (B), and TGA analysis (C), respectively. Abbreviations: BSA, bovine serum albumin; NPs, nanoparticles.

Figure 7

Time dependent stability of BSA-Rh2 NPs using particles size analysis, with respect to different time interval (A) and pH conditions (B). Abbreviations: BSA, bovine serum albumin; NPs, nanoparticles.

Figure 8

Solubility of free ginsenoside Rh2 and BSA-Rh2 NPs in water, their corresponding microscopic image and HPLC graph of supernatant, respectively. Abbreviations: BSA, bovine serum albumin; HPLC, high-performance liquid chromatography; NPs, nanoparticles.

Figure 9

In vitro efficacy of BSA-Rh2 NPs, cell cytotoxicity in HaCaT skin cells (A), A549 lung cancer cell lines (B), HT29 colon cancer cells (C), Hoechst 33258 staining in A549 cells (D), and HT29 colon cancer cells (E). Notes: Apoptotic cells are indicated with yellow arrows, Scale bar, 10 μm. Data shown represent the mean values of three experiments ± SD. *P<0.05, **P<0.01, ***P<0.001 vs control. Abbreviations: BSA, bovine serum albumin; NPs, nanoparticles.

Figure 10

In vitro efficacy of BSA-Rh2 NPs, in RAW 264.7 (murine macrophage) cell lines (A) and inhibition of LPS induced NO production assay (B). Note: Data shown represent the mean values of three experiments ± SD. **P<0.01, ***P<0.001 vs control. Abbreviations: BSA, bovine serum albumin; LPS, lipopolysaccharide; NO, nitric oxide; NPs, nanoparticles.