Synthesis, characterization, and in vitro evaluation of curcumin-loaded albumin nanoparticles surface-functionalized with glycyrrhetinic acid

Abstract

We have designed and developed curcumin (Ccn)-loaded albumin nanoparticles (BNPs) surface-functionalized with glycyrrhetinic acid (Ccn-BNP-GA) for GA receptor-mediated targeting. Ccn-BNP-GA was prepared by conjugating GA as a hepatoma cell-specific binding molecule onto the surface of BNPs. Ccn-BNP-GA showed a narrow distribution with an average size of 258.8±6.4 nm, a regularly spherical shape, an entrapment efficiency of 88.55%±5.54%, and drug loading of 25.30%±1.58%. The density of GA as the ligand conjugated to BNPs was 140.48±2.784 μg/g bovine serum albumin. Cytotoxicity assay results indicated that Ccn-BNP-GA was significantly more cytotoxic to HepG2 cells and in a concentration-dependent manner. Ccn-BNP-GA also appeared to be taken up to a greater extent by HepG2 cells than undecorated groups, which might be due to the high affinity of GA for GA receptors on the HepG2 cell surface. These cytotoxicity assay results were corroborated by analysis of cell apoptosis and the cell cycle. Further, Ccn-BNP-GA showed an approximately twofold higher rate of cell apoptosis than the other groups. Moreover, proliferation of HepG2 cells was arrested in G2/M phase based on cell cycle analysis. These results, which were supported by the GA receptor-mediated endocytosis mechanism, indicate that BNPs surface-functionalized with GA could be used in targeted cancer treatment with high efficacy, sufficient targeting, and reduced toxicity.

Keywords: albumin; curcumin; glycyrrhetinic acid; nanoparticles; surface-functionalized.

Figure 1

Differential scanning calorimetry for reagents. Notes: (A) Bovine serum albumin, (B) physical mixture, (C) curcumin, and (D) curcumin-loaded albumin nanoparticles. Abbreviation: DSC, differential scanning calorimetry.

Figure 2

Fourier transform infrared spectra for reagents. Notes: (A) GA, (B) bovine serum albumin, (C) N-hydroxysuccinimide ester of GA, and (D) albumin nanoparticles surface-functionalized with GA. Abbreviation: GA, glycyrrhetinic acid.

Figure 3

Transmission electron micrographs and size distribution of curcumin-loaded albumin nanoparticles (A, C) and curcumin-loaded albumin nanoparticles surface-functionalized with GA (B, D). Abbreviation: GA, glycyrrhetinic acid.

Figure 4

Flow curve on a Sephadex-50 column. Abbreviations: BNP, albumin nanoparticle; GA, glycyrrhetinic acid.

Figure 5

In vitro release profiles of Ccn-sus, Ccn-BNPs, and Ccn-BNP-GA. Abbreviations: Ccn-sus, curcumin suspension; Ccn-BNPs, curcumin-loaded albumin nanoparticles; Ccn-BNP-GA, curcumin-loaded albumin nanoparticles surface-functionalized with GA; GA, glycyrrhetinic acid.

Figure 6

In vitro cytotoxicity of Ccn-sus, Ccn-BNPs, Ccn-BNP-GA, and GA + Ccn-BNP-GA against HepG2 cells for 24 hours. Notes: Each point represents the mean ± standard deviation (n=6). *P<0.05 compared with Ccn-BNPs, ^#P<0.05 compared with Ccn-BNP-GA. Abbreviations: Ccn-sus, curcumin suspension; Ccn-BNPs, curcumin-loaded albumin nanoparticles; Ccn-BNP-GA, curcumin-loaded albumin nanoparticles surface-functionalized with GA; GA, glycyrrhetinic acid.

Figure 7

Photographs of HepG2 cells treated with curcumin suspension, curcumin-loaded albumin nanoparticles, curcumin-loaded albumin nanoparticles surface-functionalized with GA, and GA + curcumin-loaded albumin nanoparticles surface-functionalized with GA for 24 hours. Notes: (A) Control; (B) Ccn-sus; (C) Ccn-BNPs; (D) Ccn-BNP-GA; (E) GA+Ccn-BNP-GA. Abbreviations: Ccn-sus, curcumin suspension; Ccn-BNPs, curcumin-loaded albumin nanoparticles; Ccn-BNP-GA, curcumin-loaded albumin nanoparticles surface-functionalized with GA; GA, glycyrrhetinic acid.

Figure 8

Effect of free GA on the ability of Ccn-BNP-GA to bind to HepG2 cells over 24 hours. Notes: *P<0.05 compared with Ccn-sus, ^#P<0.05 compared with free GA + Ccn-BNP-GA. The results are expressed as the mean ± standard deviation (n=3). Abbreviations: Ccn-sus, curcumin suspension; Ccn-BNPs, curcumin-loaded albumin nanoparticles; Ccn-BNP-GA, curcumin-loaded albumin nanoparticles surface-functionalized with GA; GA, glycyrrhetinic acid.

Figure 9

Effect of curcumin-induced apoptosis in HepG2 cells exposed to various formulations for 24 hours. Notes: (A) Control, (B) curcumin suspension, (C) curcumin-loaded albumin nanoparticles, (D) curcumin-loaded albumin nanoparticles surface-functionalized with GA, and (E) GA + curcumin-loaded albumin nanoparticles surface-functionalized with GA. Abbreviations: GA, glycyrrhetinic acid; PI, propidium iodide.

Figure 10

Effect of 24 hours of treatment with curcumin on the cell cycle in HepG2 cells. Notes: (A) Control, (B) curcumin suspension, (C) curcumin-loaded albumin nanoparticles, (D) curcumin-loaded albumin nanoparticles surface-functionalized with GA, and (E) GA + curcumin-loaded albumin nanoparticles surface-functionalized with GA. Abbreviation: GA, glycyrrhetinic acid.