A Novel Oral Astaxanthin Nanoemulsion from Haematococcus pluvialis Induces Apoptosis in Lung Metastatic Melanoma

Abstract

Astaxanthin (AST) is a naturally occurring xanthophyll carotenoid having the potential to be used as an anticancer agent; however, the human body has a low bioavailability of AST due to its poor solubility in the water phase. Therefore, we applied D-α-tocopheryl polyethylene glycol succinate (TPGS) as an emulsifier and natural edible peanut oil to form a steady oil-in-water (O/W) nanoemulsion loaded with AST (denoted as TAP-nanoemulsion). TAP-nanoemulsions were stable without the droplet coalescence against thermal treatments (30-90°C), pH value changes (over a range of 2.0-8.0), and ionic strength adjustments (at NaCl concentrations of 100-500 mM) measured by dynamic light scattering (DLS). AST within TAP-nanoemulsion was released up to 80% in a simulated intestinal enzymatic fluid in vitro, and the overall recovery rate was fairly consistent in the Caco-2 cellular model. In order to further evaluate in vivo melanoma inhibitory experiments, we injected the fluorescent-stained B16F10 cells into female C57BL/6 mouse tail veins and treated TAP-nanoemulsion in an oral gavage. qRT-PCR and Western blot demonstrated that TAP-nanoemulsion triggered effectively the apoptosis pathway, including enhancements of cleaved caspase-3 and caspase-9, ataxia-telangiectasia mutated kinase (ATM), and p21WAF1/CIP1 (p21) and decreases of B-cell lymphoma 2 (Bcl-2); cyclins D, D1, and E; mitogen-activated protein kinase (MEK); extracellular signal-regulated kinases (ERK); nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB); and matrix metallopeptidase-1 and metallopeptidase-9 (MMP-1 and MMP-9) in both gene and protein expressions. In conclusion, this study suggests that TAP-nanoemulsion with the oral treatment has a positive chemotherapy effect in melanoma with lung metastases in vivo. As far as we know, this is the first time to demonstrate that an antioxidant in nanoparticle administration cures lung metastatic melanoma.

Figure 1

The influences on the physical stabilities of TAP-nanoemulsion. (a) Different TPGS concentrations, 2.5–15.0 mg/mL; (b) various pH values, pH 2.0–8.0; (c) diverse NaCl concentrations, 0–500 mM; and (d) various temperatures, 30–90°C. (e) Cryo-FESEM images of TAP-nanoemulsion.

Figure 2

The cellular viability of foreskin fibroblasts was measured by MTT assay after (a) TPGS, (b) peanut oil, and (c) TAP-nanoemulsion treatments for 24 h. The data was represented with mean values ± SD of three independent experiments performed. ^∗p < 0.05 as compared with the vehicle control group.

Figure 3

The ROS production percentage was enhanced by PMA (20 μg/mL) and reduced via TAP-nanoemulsion administration measured by flow cytometry.

Figure 4

(a) Bioaccessibility of TAP-nanoemulsion after each step of an in vitro gastrointestinal mimic digestion. (b) Permeability of TAP-nanoemulsion across the Caco-2 monolayer with various concentrations (0–26.6 μg/mL). Each value was represented as the mean values ± SD (n = 3).

Figure 5

In vivo behavior, TAP-nanoemulsion administrated against metastatic melanoma in the lung of B16F10-bearing C57BL/6 mice. (a) Changes in mouse body weight after different treatments. (b) Mouse morbidity-free survival efficacy with different treatments following the vehicle control, tumor-only, and TAP-nanoemulsion groups. (c) The evaluations of organ weights observed from the three groups. (d) The mRNA expressions associated with the three groups in qRT-PCR assay. (e) TAP-nanoemulsion inhibited lung metastatic melanoma through induction of apoptosis-related proteins by Western blotting. We took α-tubulin as our experimental loading control. Data are representative of 3 experiments. Blue: blank control; yellow: tumor only; red: TAP-nanoemulsion treatment groups. ^∗p < 0.05 as compared with the tumor-only group.

Figure 6

Proposed schematic diagram of TAP-nanoemulsion biofunction.