H2S-releasing nanoemulsions: a new formulation to inhibit tumor cells proliferation and improve tissue repair

Abstract

The improvement of solubility and/or dissolution rate of poorly soluble natural compounds is an ideal strategy to make them optimal candidates as new potential drugs. Accordingly, the allyl sulfur compounds and omega-3 fatty acids are natural hydrophobic compounds that exhibit two important combined properties: cardiovascular protection and antitumor activity. Here, we have synthesized and characterized a novel formulation of diallyl disulfide (DADS) and α-linolenic acid (ALA) as protein-nanoemulsions (BAD-NEs), using ultrasounds. BAD-NEs are stable over time at room temperature and show antioxidant and radical scavenging property. These NEs are also optimal H2S slow-release donors and show a significant anti-proliferative effect on different human cancer cell lines: MCF-7 breast cancer and HuT 78 T-cell lymphoma cells. BAD-NEs are able to regulate the ERK1/2 pathway, inducing apoptosis and cell cycle arrest at the G0/G1 phase. We have also investigated their effect on cell proliferation of human adult stem/progenitor cells. Interestingly, BAD-NEs are able to improve the Lin- Sca1+ human cardiac progenitor cells (hCPC) proliferation. This stem cell growth stimulation is combined with the expression and activation of proteins involved in tissue-repair, such as P-AKT, α-sma and connexin 43. Altogether, our results suggest that these antioxidant nanoemulsions might have potential application in selective cancer therapy and for promoting the muscle tissue repair.

Keywords: antioxidants; cancer; garlic; hydrogen sulfide; omega-3 fatty acid.

Figure 1. Morphological characterization of NEs

Optical micrographs of (A) BAD- and (B) BD-NEs; (C) size distribution of BAD-NEs; fluorescence micrographs of BAD-NEs stained with (D) nile red, (E) prodan and (F) FITC. Obtained at 100× of magnification, scale bars = 10 μm.

Figure 2. Ultrastructural properties of NEs

Representative HR-SEM micrographs of BAD-NEs captured at 10 kV (A) and (B) and at 5 kV (C). Scale bars are: 10 μm in (A) and 1 μm in (B) and (C).

Figure 3. Chemical characterization of NEs

RP-HPLC chromatograms of (A) BD-NEs and BAD-NEs after solubilization in 40% CH₃CN and 0.05% TFA v/v, obtained using C₁₈ column at 0.8 ml/min flow rate and the following gradient: 0–5 min, 0%; 5–25 min, 60%; 25–55 min, 90% and 55–75 min 90% of solv. (B) (80% of CH₃CN and 0.1% of TFA v/v). The peaks are identified comparing the retention times of the single compounds obtained by RP-HPLC analysis performed at the same conditions.¹H NMR spectra of (B) BD-NEs and (C) BAD-NEs in CDCl₃/CD₃OD mixture performed at 25°C.

Figure 4. H₂S slow-releasing by NEs

H₂S-release detected by methylene blue assay at different concentrations of (A) BAD-NEs, (B) BD-NEs and (C) DADS in DMSO, in the presence of 1 mM DTT in 50 mM Tris-HCl, pH 7.4 buffer. The H₂S concentrations are calculated using a calibration curve obtained at different concentrations of Na₂S (see also Supplementary Figure S2). Each bar represents the ± SD of three experiments.

Figure 5. Antioxidant properties of BAD-NEs

(A) Inhibition of the pDNA cleavage by BAD-NEs, 0.5 μg of pDNA in buffer 50 mM Tris HCl buffer, pH 7.4, after 30 min of UV irradiation at 254 nm (lane 1), in the absence (lane 2) and in the presence of 1 μl of BAD-NE (30 mM DADS and 32 mM ALA) (lane 3); 0.5 μg pDNA after 30 min of incubation with 100 μM CuCl₂ and 10 mM ascorbic acid in 50 mM Tris HCl, pH 7.4, buffer at 37°C in the absence (lane 4) and in the presence (lane 5) of 1 μl of BAD-NE. (B) 0.5 μg of pDNA after addition of 100 μM CuCl₂ and 10 mM ascorbic acid in 50 mM Tris HCl, pH 7.4, buffer after 0 (lane 1) and 30 min (lane 2) of incubation at 37°C alone and in the presence of BAD-NE (lanes 3 and 4) or ALA-DADS (AD) mixture (lanes 5 and 6), ALA (A) (lanes 7 and 8) and DADS (lanes 9 and 10). The ALA and DADS concentrations are the same in all samples; (C) SDS-PAGE of 25 μl of BD-NE (lane 2) BAD-NE (lane 3) and MBs (lane 4) obtained using 5% w/v of BSA solution; (D) inhibition of the free-radical polymerization of PEG-fibrinogen hydrogel after addition of 0.1% w/v of Irgacure^® 2959 photo-initiator and 5 min of UV (365 nm, 5 mW/cm²) exposure in the presence (BAD) or in the absence (CTRL) of 8% v/v BAD-NE.

Figure 6. Effects of BAD-NE on cell viability of MCF-7 cancer cell line

(A) Cell viability of MCF-7 cell line after 24 h of treatment with different concentrations of BAD-NE and 50 μM DADS. The BAD concentrations are expressed as DADS concentration in BAD-NE. (B) Cell viability of MCF-7 cell line over the time (at 6 h, 24 h and 48 h) in the presence of BAD-NE with 50 μM of DADS. (C) Fluorescence microscope micrographs of MCF-7 cells after 24 h of treatment with BAD-NE (with 50 μM of DADS), the nucleus are stained with PI solution, obtained with mag. 100×, scale bar = 10 μm; (D) effects on cell viability of MCF-7 cells after 48 h of treatment with BAD-NE, BD-NE and DADS, at 50 μM of DADS concentration; (E) cell cycle distribution of the alive MCF-7 cells after 48 h of treatment with BAD-NE (with 50 μM of DADS) obtained by FACS analyses. The p values were < 0.05 with respect to the control using One-way-ANOVA (n = 3 or 5 experiments as biological replicas).

Figure 7. Anti-proliferative effects of the NEs on HuT 78 cancer cell line

(A) Cell viability and mortality of HuT 78 cell line after 24 h of treatment with BAD-NE, BD-NE and DADS, at 50 μM of DADS concentration. (B) Cell viability over the time after 24 and 48 hours of treatment with BAD-NE, BD-NE and DADS (50 μM of DADS). The p values were < 0.05 with respect to the control (n = 3 or 5 experiments as biological replicas).

Figure 8. Western blotting analysis of MCF 7 cell line after 24 h of treatment with BAD-NE

(A) Expression of cleaved form of caspase-3 and p21 protein after BAD-NE (BAD) and BD-NE (BD) treatment; (B) Expression of ERK1/2, AcH3, p21 and GAPDH proteins (gray box indicates to unrelated lanes on the same blot, see Figure 3S; (C) expression of phosphorylated form of ERK1/2, P-ERK1/2 dimer and β-actin proteins. The relative densitometries of the samples with respect to the control have been obtained after normalization of the concentrations with respect to GAPDH or β-actin concentrations. Each bar represents the ± SD of three experiments as biological replicas.

Figure 9. Stability of the BAD-NEs over time

(A) Optical micrograph of BAD-NE after one month at room temperature, obtained at mag. 100×, scale bar =10 μm; (B) representative HR-SEM micrograph of BAD-NEs kept for 1 month at room temperature, obtained at 10 kV, scale bar = 1 μm; (C) cell mortality of MCF-7 after 48 h of treatment with BAD-NEs kept for 1 month at room temperature (about 25°C) (the estimated concentration of DADS was 15 μM). (D) Cell viability of MCF-7 cells after 48 h of treatment with fresh preparation of BAD-NE and stored one year at –80°C (with 50 μM of DADS).

Figure 10. Effects of BAD-NE on cell viability of Lin^– Sca-1⁺ hCPC line

(A) Cell viability of hCPC after 3 days of growth in the presence of BAD-NE (BAD) and DADS (50 μM of DADS). The p value < 0.05; n = 7 biological replicas; (B) fluorescence micrographs of hCPCs cultured for 3 days in the presence of BAD-NE. The nuclei are stained with Hoeschst 33342 (in blue) and the expressions of α-sma (in red) and Cx43 (in green) proteins are detected. Scale bars = 20 μm; (E) western blotting analysis of Lin^- Sca-1⁺ hCPC line after 3 days in the presence of BAD-NE, the expression of Akt, P-Akt , α-sma and GAPDH and β-tubulin proteins are assessed. The relative densitometries of P-Akt, α-sma of the treated with respect to the control have been obtained after normalization of the concentrations with respect to β-tubulin concentration.

Figure 11. Schematic illustration of BAD-NE preparation and its effects on cancer and adult progenitor stem cells