Cholesterol-coated gold nanorods as an efficient nano-carrier for chemotherapeutic delivery and potential treatment of breast cancer: in vitro studies using the MCF-7 cell line

Abstract

Gold nanorods (GNRs) have a recognized role in treatment of cancers as efficient nanocarriers for chemotherapeutic drug delivery. In this study, GNRs modified with cholesterol-PEG were employed as a nanocarrier for a hydrophobic compound having a promising phosphatidylinositol 3-kinase (PI3Kα) inhibitory activity. The acquired nanocomplex was characterized by optical and infra-red (IR) absorption spectroscopies, in addition to hydrodynamic size and zeta potential. Glide docking and superposing of docked poses of the hydrophobic ligand and cholesterol moiety demonstrated that hydrophobic interactions drive the conjugation and attachment of the ligand to the cholesterol moiety of the nanocarrier. In vitro release study under a cellular environment indicates that the presence of cells has enhanced the release and the cellular uptake of the conjugated ligand. Furthermore, the anti-proliferative assay of the nanocomplex revealed potent cytotoxicity over a low concentration range of the nanocomplex against MCF-7 breast cancer cells compared to the free compound or the nanocarrier alone. Analysis of cellular death modality by flow cytometry showed that the nanocomplex has a rapid effect on cell death, as cells went toward the late apoptotic/necrotic stage rapidly and proportionally to the increase of the nanocomplex concentration. The overall results propose that cholesterol-decorated GNRs could be considered as a promising nanocarrier for hydrophobic drugs to achieve efficient delivery and potential therapy against breast cancer cells.

Fig. 1. Illustration demonstrates the nanocomplex (A) of Chol-GNRs conjugated with Compound A (B) by non-covalent hydrophobic interactions Compound A and the cholesterol moiety that linked to a thiolated PEG (C).

Fig. 2. Characterization of GNRs, Chol-GNRs, PEG-GNRs, Chol-A-GNRs and PEG-A-GNRs. (A) Optical absorption spectra of GNRs, Chol-GNRs, PEG-GNRs and Chol-PEG-SH dispersed in water. An extra peak was observed in the optical spectrum of Chol-GNRs at ∼250 nm which confirms the successful surface modification. (B) Optical absorption spectra of GNRs, Compound A in PBS, Chol-A-GNRs and PEG-A-GNRs. An extra absorption peak was observed at ∼297 for Chol-A-GNRs which confirms the successful conjugation of Compound A to Chol-GNRs. (C) Hydrodynamic sizes and effective surface charges of GNRs, Chol-GNRs, Chol-A-GNRs, PEG-GNRs and PEG-A-GNRs. The size of Chol-GNRs was increased due to functionalization with Chol-PEG-SH, however, no significant change in the size and charge of Chol-A-GNRs upon conjugation with Compound A. (D) TEM image of GNRs that confirms a rod-shape of the nanoparticles having average length and width of ∼52.1 nm and ∼12.5 nm, respectively, and an AR of ∼4. (E) TEM image of Chol-GNRs that confirms the rod-shape of the nanoparticles having average length and width of ∼54 nm and ∼12.5 nm, respectively, and an AR of ∼4.

Fig. 3. Infra-red spectra of Compound A, Chol-GNRs and Chol-A-GNRs revealed similarities in the core structures' peaks with slight shifts suggesting successful conjugation of Compound A to the cholesterol moiety of GNRs by hydrophobic interactions occurred between their structural backbones.

Fig. 4. Glide docking and complex formation of Compound A/PI3Kα (A) and cholesterol/PI3Kα (B).

Fig. 5. Superposing of docked poses of cholesterol and Compound A in 3D models. The best docked poses of Compound A and cholesterol are extracted and overlaid to each other. The hydrophobic interaction drives the attachment of Compound A to cholesterol moiety where hydrophobic carbon backbone motifs match their peers in each compound due to their similar hydrophobic core structures. Cholesterol is depicted in pink (A), yellow (B) and white color (C). Compound A carbon atoms are represented in green color, blue color for nitrogen atoms (N), and red color for oxygen (O).

Fig. 6. In vitro release profile of Compound A from the nanocomplex (Chol-A-GNRs) in PBS and in tissue culture medium (A). Release profile of Compound A from the nanocomplex (Chol-A-GNRs) under cellular environment (B). Percentages of cellular uptake of Chol-GNRs and PEG-GNRs into MCF-7 breast cancer cells and human dermal fibroblasts (C). Data are presented as mean ± SD, n = 3. Unpaired t-test was used to assess the differences; * represent p < 0.05.

Fig. 7. Estimation of IC ₅₀ of free Compound A over the concentration range of 0.97–500 μg mL⁻¹ against MCF-7 breast cancer cells (results present the average and standard deviation of three determinations).

Fig. 8. Anti-proliferative activity of the nanocomplex (Chol-A-GNRs), free Compound A, and the nanocarrier (Chol-GNRs) against MCF-7 breast cancer cells (A) and human dermal fibroblasts (B). The nanocomplex demonstrates potent anti-proliferative activity against MCF-7 cells over low range of concentrations compared to human dermal fibroblasts. Data are given as mean ± SD, n = 3. Unpaired t-test was used to assess the differences; * represent p < 0.05, ** represents p < 0.01.

Fig. 9. Flow cytometry assay of apoptosis of the control untreated MCF-7 cells (A); MCF-7 cells incubated for 24 h in the presence of 4.0 μg mL⁻¹ (B) and 8.0 μg mL⁻¹ (C) of the nanocomplex and stained with FITC-conjugated annexin V and PI-stained. The dot plot for each sample was divided into four quadrants to indicate viable cells (lower left quadrant), early apoptotic cells (lower right quadrant), necrotic cells (upper left quadrant), and late apoptotic cells (upper right quadrant). A significant difference was observed in the percentage of necrotic and late apoptotic cells upon treating the MCF-7 cells with 8.0 μg mL⁻¹ and 4.0 μg mL⁻¹ of the nanocomplex, respectively compared to the untreated cells (P < 0.05). Data are presented as mean ± SD, n = 3 (D). Unpaired t-test was used to assess the differences; * represent p < 0.05. (A)–(C) Plots are examples of one run of the analysis.

Fig. 10. Flow cytometric analysis of cell cycle parameters. MCF-7 cells were incubated for 24 h; in the presence of 8.0 μg mL⁻¹ of the nanocomplex (A) and without additive of treatment (untreated) (B). Cells were stained with propidium iodide for analysis by flow cytometry. The cells treated with the nanocomplex showed 100% arrest at G0/G1 phases. A statistical difference was observed between MCF-7 cells treated with the nanocomplex and their control untreated cells (P < 0.05). Data are given as means, n = 3, and unpaired t-test was used to assess the difference.