1. Introduction
Astaxanthin (AST) is a pigment synthesized from lycopene, part of the lutein family, and an oxidized carotenoid derivative [
1]. It is found in many of our favorite shellfish, salmon, trout, red snapper, shrimp, lobster, and eggs [
2]. AST has a number of important biological functions, including the prevention of oxidation in unsaturated fatty acids, ultraviolet radiation resistance, immune response, pigmentation, information transmission, and reproductive improvement [
3]. Among these functions, one of the most important properties of AST is its antioxidant effect. Studies have shown that its antioxidant properties exceed those of β-carotene and α-tocopherol. Given its outstanding antioxidant activity, many researchers believe that AST can protect the body against many oxidative-related common diseases, including cardiovascular diseases, different types of cancer, and some diseases of the immune system. Thus far, most of the applications of AST are for human nutrition and health [
4,
5]. Chen et al. reported that AST can inhibit the growth, migration, and invasion of melanoma A375/A2058 cells and downregulate the expression of MMP-1, -2, and -9 in a dose-dependent manner [
6]. AST has potential physiologic functions, such as relieving oxidative stress, anti-tumor, anti-inflammatory, anti-ultraviolet light oxidation, and anti-Helicobacter pylori infection [
7]. However, the 11 double-conjugated links in the AST structure restrict its stability, leading the volatile AST to be affected by unfavorable environmental factors, including heat, radiation, oxygen, and alkaline or acidic solutions [
8]. Moreover, its high lipophilicity and heat resistance limit its antioxidant effect in biomedical applications.
In recent years, there has been a great growth in the research of nanopharmaceutics. Nanopharmaceutics have advantages in morphology and size and can improve the performance and stability of drugs. Because it can use different types of raw materials to ensure specific properties, nanotechnology has received widespread attention in the pharmaceutical and food fields. In the food industries, nanotechnology can be used for preparing nanonutraceuticals, which can improve the shortcomings and improve the performance of nutritional components. However, relevant systems and good manufacturing practices are still needed to ensure the quality and safety of the final products [
9,
10]. Nowadays, nanotechnologies have been widely used to solve the AST instability issue. For instance, Rodriguez–Ruiz, et al. used sunflower oil as a green liquid lipid processed to synthesize a nanostructured lipid carrier (NLCs) loaded with natural AST. The antioxidant activity was then measured by the method of α-tocopherol-equivalent antioxidant capacity and compared with natural AST and α-tocopherol. The AST load studies showed that the recovery rate of AST from AST-loaded NLCs exceeded to 90%. The result indicated that the preparation could stabilize the AST molecule and maintain or even improve its antioxidant capacity [
11].
Protein secretion is important for eukaryotic cells. Unconventional protein secretion can secrete proteins without signal peptides or other cytoplasmic substances into the extracellular space, which may be responsible for extracellular vesicle (EV) formation [
12]. In various diameters, EVs can be divided into exosomes, microvesicles, and apoptotic bodies. Exosomes are 40–100 nm in diameter, whereas microbubbles and apoptotic bubbles are 100–350 and 500–1000 nm in diameter, respectively [
13]. Many studies on EVs focus on mammals as the main direction. Recently, plants were discovered to secrete EVs which play a major role in plant cell communication and immune regulation. They can protect plants against the invasion of pathogenic bacteria [
14]. Plant-derived exosome-like nanoparticles (PDENs) can be taken up by gut macrophages and interspecifically communicate by inducing various cytokines [
15]. Moreover, the extraction rate for PDENs is much higher than in mammalian cells [
16], illustrating their economic potential as a nanoplant [
17]. EVs transmit payloads to other species for cross-border communication. Their characteristics and transport capabilities improve the applicability of pharmaceutical transportation [
18]. PDENs and synthetic nanoparticles (such as liposomes and micelles) have common basic characteristics, including a similar lipid bilayer structure and the ability to carry hydrophilic or hydrophobic cargos [
19]. However, EVs are superior to synthetic nanoparticles in terms of nontoxicity, low immunogenicity, increased cellular absorption, greater stability in the gastrointestinal tract, and specific targeting capability [
20]. For example, PDENs have 10 times more specificity for tumor targeting than liposomes [
21], less potential for unnecessary gene or protein transfer prior to clinical application than nanoparticles derived from mammalian or bacterial cells, and almost no harmful immunogenic reactions [
22]. Furthermore, different from other delivery vehicles, PDENs have a controlled liquid phase film, which promotes their ability to resist surfactant dissolution. PDENs have also proven resistant to digestive enzymes (such as intestinal trypsin, pepsin, and bile extract). This important feature helps them protect the drug from resolving in the stomach or intestinal environment, leading to safe drug administration in the desired colon [
23].
Currently, the two major obstacles to cancer treatment are low specificity and high toxicity. To tackle these great challenges, researchers are striving to develop a range of new drug delivery systems. For example, scientists have successfully used ginger-derived PDENs to prepare nanocarriers to deliver doxorubicin to colon cancer cells. These nanocarriers internalize the colon-26 tumor model cells and inhibit their growth. Interestingly, the significant antitumor effect is not only derived from the anticancer activity of Adriamycin but also from the inhibitory effect of PDENs on oxidative stress [
24]. Reports showed that PDENs have no toxicity and good biocompatibility and can be produced economically on a large scale. Compared with synthetic liposomes, they are superior to liposomes in biocompatibility, safety, apoptosis, cell substrate impedance, and drug release [
25].
In our study, poly (lactic-co-glycolic acid) (PLGA)-encapsulated AST (AST@PLGA) nanoparticles were prepared by emulsion solvent volatilization method, and broccoli-derived extracellular vesicle (BEV)-coated AST@PLGA (AST@PLGA@BEVs) nanoparticles were prepared by ultrasound. The response surface methodology was used to optimize the drug loading. After characterizing the optimized nanoparticles, the in vitro anticancer activities of AST, AST@PLGA, and AST@PLGA@BEVs were contrasted and analyzed by HT-29 cells.
2. Materials and Methods
2.1. Materials
PLGA (lactide:glycolide 50:50; Mw 38,000–54,000) and Poly vinyl alcohol (PVA, P875084) were purchased from MACKLIN (Shanghai, China). Astaxanthin (AST, SML0982) was purchased from Sigma-Aldrich (St Louis, MO, USA). MTT Cell Proliferation and Cytotoxicity Assay Kits were purchased from Beyotime (Shanghai, China). Dichloromethane and Ethanol were purchased from SINOPHARM (Beijing, China).
2.2. Isolation and Purification of BEVs
Broccoli (1000 g) was washed with water, smashed, and filtered to prepare broccoli sap. The broccoli sap was isolated using differential centrifugation at 500×
g for 10 min, 2000×
g for 20 min, 5000×
g for 30 min, and 10,000×
g for 1 h at 4 °C. The supernatant used ultrahigh speed centrifugation at 150,000×
g for 2.5 h at 4 °C [
26]. The pellet was resuspended in phosphate-buffered saline (PBS).
2.3. Preparation of the Nanoparticles Loaded with AST
The AST@PLGA nanoparticles were prepared by the emulsion solvent volatilization method [
27]. The weighed AST and PLGA were dissolved in 1 mL of dichloromethane and then completely dissolved by ultrasound as an organic phase. The organic phase was slowly added to the PVA solution drop by drop, and then the emulsion was obtained by ultrasound. The emulsion was stirred on a magnetic stirrer for 3–4 h at room temperature and after centrifugation at 14,000 rpm for 40 min at 4 °C. The pellet was resuspended in PBS. Finally, BEVs and AST@PLGA were sonicated for 5 min in a water bath at 40% power.
2.4. Drug Loading (DL)
AST was added to the mixed solution of dichloromethane:ethanol (1:10,
v/
v, 1 mL) [
28]. The maximum absorption wavelength of the solution was measured using a UV-Vis spectrophotometer, BioTek, US. The absorbance of different contents of AST solution was detected at the maximum absorption wavelength, and a standard curve was constructed. The nanoparticle solution was lyophilized and dissolved in 1 mL of dichloromethane:ethanol (1:10) mixed solution, the absorbance was measured, and the content of AST was calculated according to the standard curve. DL was obtained using the following equation:
2.5. Response Surface Methodology (RSM)
The single-factor screening experiments that may affect the DL in the preparation process, including the ratios of drug to carrier and oil phase to water phase, the concentration of PVA, ultrasonic power, and ultrasonic time were conducted. Depending on the single-factor screening experiments, we selected the three factors that have the greatest influence on DL. RSM was adopted in the experimental design. The experiment was performed with three factors and three levels, including 17 runs, and DESIGN-EXPERT 10 software was used for the optimization study.
2.6. Particle Size and Zeta Potential
The AST@PLGA@BEV solution was diluted 50 times with ultra-pure water. Then, 1 mL of the diluted solution was added to the potential dish and particle-size dish. The particle size, polydispersity index (PDI), and zeta potential of the AST@PLGA@BEVs were determined using ES90 Nano, Malvern, UK. The samples were diluted with ultrapure-water to appropriate concentration. Each measurement was repeated three times at room temperature.
2.7. Transmission Electron Microscopy (TEM)
Transmission electron microscopy experiments used Tecnai 12, Philips, NL. Briefly, 10 μL of AST@PLGA@BEVs were added to dry copper net and stained with 2% phosphotungstic acid. After natural drying, Tecnai 12 was used to observe and then photograph the samples.
2.8. Cell Culture
Human colon cancer cells (HT-29) were cultured in 5% CO2 cell incubator at 37 °C in RPMI1640 medium supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum. After reaching 90% density, the cells were isolated by trypsin (including 0.25% EDTA) after the experiment.
2.9. Anticancer Activity In Vitro
HT-29 survival viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HT-29 cells were seeded in 96-well plates with 200 μL RPMI1640 for 24 h. Cells were pretreated with the same concentration of AST, AST@PLGA, and AST@PLGA@BEVs for 24 h. Each well was added with 10 μL MTT (5 mg/mL) and incubated in the cell incubator for 4 h. Then, 100 μL DMSO was mixed until the purple crystal dissolved completely under an ordinary optical microscope. The absorbance was detected at 570 nm.
2.10. Statistics
Each experiment was performed for at least three repeats. The results were presented as mean ± standard deviation. Statistical mean differences were evaluated using SPSS 25.0. The results were considered statistically significant if p < 0.05.