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Microalgal metabolites as anti-cancer/anti-oxidant agents reduce cytotoxicity of elevated silver nanoparticle levels against non-cancerous vero cells

Heavy metal pollution has become a major concern globally as it contaminates eco-system, water networks and as finely suspended particles in air. In this study, the effects of elevated silver nanoparticle (AgNPs) levels as a model system of heavy metals, in the presence of microalgal crude extracts (MCEs) at different ratios, were evaluated against the non-cancerous Vero cells, and the cancerous MCF-7 and 4T1 cells. The MCEs were developed from water (W) and ethanol (ETH) as green solvents. The AgNPs-MCEs-W at the 4:1 and 5:1 ratios (v/v) after 48 and 72 h treatment, respectively, showed the IC50 values of 83.17–95.49 and 70.79–91.20 μg/ml on Vero cells, 13.18–28.18 and 12.58–25.7 μg/ml on MCF-7; and 16.21–33.88 and 14.79–26.91 μg/ml on 4T1 cells. In comparison, the AgNPs-MCEs-ETH formulation achieved the IC50 values of 56.23–89.12 and 63.09–91.2 μg/ml on Vero cells, 10.47–19.95 and 13.48–26.61 μg/ml on MCF-7; 14.12–50.11 and 15.13–58.88 μg/ml on 4T1 cells, respectively. After 48 and 72 h treatment, the AgNPs-MCE-CHL at the 4:1 and 5:1 ratios exhibited the IC50 of 51.28–75.85 and 48.97–69.18 μg/ml on Vero cells, and higher cytotoxicity at 10.47–16.98 and 6.19–14.45 μg/ml against MCF-7 cells, and 15.84–31.62 and 12.58–24.54 μg/ml on 4T1 cells, respectively. The AgNPs-MCEs-W and ETH resulted in low apoptotic events in the Vero cells after 24 h, but very high early and late apoptotic events in the cancerous cells. The Liquid Chromatography-Mass Spectrometry-Electrospray Ionization (LC-MS-ESI) metabolite profiling of the MCEs exhibited 64 metabolites in negative ion and 56 metabolites in positive ion mode, belonging to different classes. The microalgal metabolites, principally the anti-oxidative components, could have reduced the toxicity of the AgNPs against Vero cells, whilst retaining the cytotoxicity against the cancerous cells.


Introduction
Heavy metals are elements occurring naturally, and have high atomic weight and a density of at least 5 times the density of water. The presence and wide distribution of heavy metals in the environment are attributed to the anthropogenic activities such as mining and electro-plating industries, and also from agricultural and electronic wastes. There have been major concern on the potential effects of heavy metal pollution on the environment and human health that constant monitoring has become an essential part of the integrated remediation strategies [1,2]. The toxicity of heavy metals depends on dosage, exposure route and duration, and chemical types. Exposure and impacts on individuals vary based on gender, age, genetics, and nutritional uptake. Heavy metals such as cadmium, arsenic, chromium, mercury, and lead, have high degree of toxicity, and are known to cause damage to many organs, even at low levels. These are among the priority metals of public health concern and classified as human carcinogens according to the International Agency for Research on Cancer and the U.S. Environmental Protection Agency [3].
Toxic level of heavy metals in drinking water and marine organisms such as fish, and in the food chains from unsustainable agricultural practices, and also released from vehicles and fumes could be among the factors that contribute towards the spike in cancer incidence globally. Breast cancer is the most prevalent cancer in women and early stage diagnosis to identify cancerous cells could decrease the mortality rates in the long term. The screening methods include mammography, raselmis suecica is rich in carotenoids, chlorophyll, α-Tocopherol, and other vitamins, and has been mostly used as a portion of live food for shrimp larvae, bivalves, artemia, and rotifers [22]. Fresh water Chlorella vulgaris is used as food additive and in pharmaceutical applications, and is rich in nucleic acid, protein, chlorophylls, carotenoids, minerals, vitamins (B12), and carbohydrate content [23]. The high cytotoxicity of the AgNPs on the Vero, MCF-7, and 4T1 cells has been reported. However, the AgNPs, in co-application with the T. suecica-CHL at the 2:1 ratio, have exhibited no toxicity on the Vero cells, though the cytotoxicity on the MCF-7 and 4T1 cells are retained [7]. The effects of elevated levels of AgNPs with the MCEs from W and ETH extracts (AgNPs-MCEs-W and ETH) at the 3:1, 4:1 and 5:1 ratios, on the non-cancerous and cancerous cells, have not been reported before. This study was based on the hypothesis that the elevated level of the AgNPs should be more cytotoxic to both the cancerous and non-cancerous cells. The presence of microalgal natural compounds from the green solvent extracts; or the MCEs rich in antioxidant activities, could reduce the toxicity of the elevated AgNPs level on the non-cancerous cells, whilst retaining the cytotoxicity on the cancerous cells.
The objectives of this study were to determine the cytotoxicity of the elevated AgNPs as the model heavy metal contaminants, in the presence of N. oculata, T. suecica and Chlorella sp.-W, ETH and CHL, at the 3:1, 4:1 and 5:1 ratios (AgNPs:MCEs, v/v), against the non-cancerous Vero cells, and the cancerous MCF-7 and 4T1 cells. The cytotoxic activities were confirmed with the flow cytometric and apoptotic biomarker analyses. The bioactive compounds of the MCEs-W and ETH were analysed by the LC-MS-ESI technique, and compared with the different solvent extracts from CHL, HEX, and MET.

Cultivation and extraction of microalgae
The cultivation and extraction of microalgae have been described before [7]. The N. oculata, T. suecica and Chlorella sp., used in the present study, were morphologically characterized, and taxonomically identified by the Fisheries Research Institute of Malaysia, Kuala Muda, Kedah, Malaysia, under the guidance of Dr. Mohd Fariduddin Othman. The N. oculata species was further molecularly identified by using the 18S rRNA, rbcL gene, and the internal transcribed spacer (ITS) region of the ribosomal RNA transcription units. The partial 18S rRNA sequence, partial rbcl gene, and ITS region were determined, showing 97-99% similarity to Nannochloropsis oculata, as confirmed by the sequence alignment and phylogenetic tree analysis, and deposited into the GenBank with accession numbers HQ201714, HQ201713, and HQ201712, respectively [24]. The extraction of microalgae was as being reported before [7].

Preparation of AgNPs-MCEs ratio
The detail of the biosynthesis and characterization of AgNPs, and the isolation, identification and culture of Lactobacillus plantarum for AgNPs biosynthesis have been described elsewhere [8].
For the preparation of the AgNPs:MCEs ratio, 10 mg of AgNPs were dissolved in 1 ml of dimethylsulfoxide (DMSO) (10 mg/ml stock), and 10 mg of MCEs-CHL, ETH and W were dissolved in 1 ml DMSO (10 mg/ml stock). Various concentrations of AgNPs and MCEs were prepared (3.125-100 μg/ml) for single applications. For co-applications, each stock solution of AgNPs and MCEs was mixed to give the final total concentration of 10 mg/ml at 3:1, 4:1 and 5:1 ratios (AgNPs:MCEs (w/ w)) ( Table 1). The Eco-AlgaeAgNano™-W and ETH were compared with the AgNPs-MCEs-CHL. Preliminary studies on the MCEs-MET and HEX (data not shown) showed no significant cytotoxicity on the MCF-7 and 4T1 cells, while the MCEs-CHL showed moderate cytotoxicity. So, subsequent studies were based on the comparison between the MCEs-CHL and MCEs-W and ETH. The ratios of 3:1, 4:1 and 5:1 were selected based on the preliminary studies carried out with the 1:1, 1.5:1, 2:1, 1.5:3 ratios (data not shown). The highest ratio at 2:1 was cytotoxic against the MCF-7 cells, but exhibited very low or no cytotoxicity against the 4T1 cells.

Cell lines
The MCE-7 (ATCC ® HTB-22T M ) and Vero cells (ATCC ® CCL-81™) were obtained from the Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Terengganu, Malaysia, while the 4T1 cells were from ATCC (ATCC ® CRL-2539™). The Vero cells are the most common mammalian continuous cell line used in research and suitable for the screening assays of natural products, in vitro studies [25,26,27,28,29,30], and are usually used in cancer studies as normal cell lines [27]. Vero cells have been tested extensively in the production of the vaccine and found to be free from the symptomatic agents which could have reduced the sensitivity of the tests. The cell-line does not produce tumors at the passage level used to form the vaccine but could have had the possibility of spreading tumor at much higher passage level [31,32]. The progeny of Vero cells is aneuploid and continuous, which contains an abnormal number of chromosomes. A persistent cell lineage can be replicated through numerous cycles of mitosis and does not become aging [33]. Vero cells are interferon-deficient, and unlike the normal mammalian cells, they do not secrete αor β-interferon (IFN) when infected with viruses. However, they still have an α/β IFN receptor, and exhibit responses when the recombinant IFN is added to the culture media [34]. The Vero cells are therefore used in this study as the normal cell line control due to this susceptibility to different types of chemical compounds, microbes, and toxins.

Cell culture
The Minimum Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% sodium pyruvate, 1% penicillin streptomycin and 1% Non-essential amino acid was used for the culture of MCF-7 and Vero cells. The Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS 100 unit/ml penicillin and 100 mg/ml streptomycin without phenol red, and 2 mM L-glutamine was used to culture 4T1 cells. The cells were maintained in a humidified atmosphere, containing 5% CO 2 and at 37 C. The media was changed every 2-3 days, by replacing the old media with the fresh one. Trypsin-EDTA was used to trypsinize the cells for cell counting to assess the viability, proliferation, and confluency.

MTT assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is based on the ability of a mitochondrial dehydrogenase enzyme in the viable cells to cleave the tetrazolium rings of the pale yellow MTT and form formazan crystals in purple color [35]. The number of surviving cells therefore is directly proportional to the level of the formed formazan [36]. The cytotoxicity of the AgNPs and MCEs single and co-application on the MCF-7 (5 Â 10 4 ) and 4T1 (2 Â 10 4 ) and Vero (4 Â10 4 ) cells were assayed after 24, 48 and 72 h. One hundred μL of the cell suspension were pipetted into each of the 96-well plate, to allow cell attachment and proliferation. After overnight incubation, the cells were treated with various concentrations (3.125-100 μg/ml) of MCEs and AgNPs for single and co-applications. Each well was subjected to an MTT assay following the previously reported methods [7,37].

Morphological characterization
The MCF-7, 4T1 and Vero cells were seeded for 24 h and the media was later changed with the fresh media containing AgNPs and MCEs at the IC 50 levels established for 72 h treatment. The negative Control was without treatment. The cell morphology was observed under inverted microscope.

Determination of apoptosis
Flowcytometric analyses were carried out to determine whether the cell death in breast cancer and Vero cells was due to apoptosis or necrosis. The MCF-7, 4T1 and Vero cell were cultured in 75 cm 3 flasks for 24 h, and then treated with the AgNPs-MCEs single and co-application. After 24 h, the cells were harvested by trypsinization and then resuspended in 1 ml Binding buffer and centrifuged at 300Âg for 10 min. The cell pellet was re-suspended in 100 μL of 1Â Binding Buffer (per 10 6 cells). Ten μL of fluorescence labeled Annexin V-FITC was loaded per 10 6 cells, mixed well and incubated for 15 min in the dark at room temperature. The cells were washed by adding 1 ml of 1Â Binding Buffer per 10 6 cells and centrifuged at 300Âg for 10 min. The cell pellet was resuspended in 500 μL of 1Â Binding Buffer per 10 6 cells. Finally, 5 μL of Propidium iodide (PI) solution was added immediately prior to flow cytometric analysis (Beckman Coulter CytoFLEX, USA). The negative Controls were the untreated cells [7]. The Phosphatidylserine (PS) levels, expressed in Relative Fluorescence Unit (RFU) in the cells from the untreated Controls, served as a baseline indicator for normal PS levels [38].

Cell-cycle analysis
The method used was as described before [7]. The breast cancer and Vero cells were cultured in 75 cm 3 flasks and incubated for 24 h. After incubation, the cells were harvested by trypsinization and then resuspended with 1 ml of PBS addition and centrifugation at 1200 rpm and 4 C. The pelleted cells were re-suspended in 0.3 ml of PBS buffer. The cells (1 Â 10 6 ) were fixed by gently adding 700 μL of cold ethanol (70%) drop wise to the tube containing 300 μL of cell suspension in PBS, and vortexed gently. The fixed cells in the tube were left on ice for 1 h (or a few days at 4 C), centrifuged, washed 1 time with cold PBS, re-centrifuged, and the cell pellet re-suspended in 250 μL of PBS. Five μL of 10 mg/ml RNAse A was added (the final concentration being 0.2-0.5 mg/ml). After incubation at 37 C for 30 min, ten μL of 1 mg/ml PI solution was added (the final concentration being 10 μg/ml). Finally, the sample was kept in the dark at 4 C until analysis by flow cytometry.

ADP/ATP ratio
The procedure was carried out as previously described [7]. The breast cancer and Vero cells were cultured on white 96-well plates at 10 4 cells/well for 24 h and then treated with desired apoptosis inducer (including the untreated cells). After 24 and 48 h, the culture medium was removed from the plate, 50 μL of Nucleotide Releasing Buffer (10 3 -10 4 cells) was added to help gently loosen the membrane, so that the ATP will leak out of the cell without complete cell lysis, and incubated for 5 min at room temperature with gentle shaking. One hundred μL of ATP Monitoring Enzyme was added to the Control and the sample in the white 96-well plate. and the background luminescence (Data A) was read. Fifty μL of cells were transferred into the luminometer plate and treated with the Nucleotide Releasing Buffer. After 5 min, the sample in the luminometer or luminescence capable plate reader (Data B) was read, and then the sample was read after 10 min incubation at room temperature (Data C). Later, the 10X ADP-Converting enzyme was diluted 10-fold with the Nucleotide Releasing Buffer, and 10 μL of 1X ADP Converting Enzyme was added. The samples were again read after 5 min (Data D). The data was analysed as follows:

Caspase 3/7
Caspase 3/7 (effector caspases) is related to the onset of the "death cascade" and are responsible for initiating the degradation stage of apoptosis such as DNA fragmentation, cell contraction and membrane blebbing [39]. It thus signifies the important cell entry point into the The MCEs were analyzed using a LC-MS (LCMS-IT-TOF, SHIMADZU, Japan), equipped with an electrospray ionization interface in the positive and negative mode. The separation was performed in a SunFire C18 column (150 mm Â 2.2 mm Â 3.5 μm, Waters, Milford, MA) using the gradient solvent system ( Table 2) as follows: -Solvent A (acetonitrile, 0.1% formic acid) and Solvent B (water, 0.1% formic acid). The flow rate was set at 0.2 ml/min, and the injection volume was 20 μL, 13 L/min dry gas flow (N 2 ), 30 psi nebulizer pressure, 350 C drying gas temperature, and 4500 V capillary voltage. The full-scan MS was recorded for 400-700 m/z. The raw data was first transferred into centroid mode data of mzXML format using MSConvert tool from Proteowizard software (http://proteowizard.sourceforge.net/). The metabolites were identified and further confirmed by comparing the retention time and the MS spectra with the commercially available standard or matching the accurate mass information with the online Metabolomics Workbench database (www.metabolomicsworkbench.org), METLIN (metlin.sc ripps.edu) and HMDB (www.hmdb.ca).

Statistical analysis
All experiments were carried out in triplicate and the results were expressed as the means AE standard deviation (SD). Two-way ANOVA model was used for the analysis of statistical significance by using GraphPad Prism software (version 6, CA, USA), and a p < 0.05 threshold was considered to indicate the significant differences (Tukey's test).  [41,42]: IC 50 20 μg/mL ¼ high cytotoxicity, IC 50 21-45 μg/ml ¼ moderate cytotoxicity, IC 50 46-100 μg/ml ¼ low cytotoxicity, and IC 50 > 100 μg/ml or no IC 50 estimated ¼ non-cytotoxic (as shown in Table 3). All the co-application formulations had exhibited much lower or reduced cytotoxic effects on the Vero cells, as compared to the single AgNPs (p < 0.05).

Apoptotic and cell-cycle analyses
The untreated (control) cells showed intact and regular shape. The cells appeared large, rounded, healthy looking and attached to each other, with no apoptotic bodies or necrotic cells ( Figure 3). The AgNPs-MCEs-W and ETH treatments did not exhibit any significant changes in the morphology and survival of the non-cancerous Vero cell line ( Figure  4). The AgNPs-MCEs treatment however had resulted in cell morphological changes in MCF-7 and 4T1 cells where the cells became rounded up, shrinking in size, and detached from the monolayer surface of the wells (as shown by the arrows) ( Figures 5 and 6). The number of cells was also lower than the Control, with some cells showing membrane blebbing and the formation of apoptotic bodies observed as round or oval masses of cytoplasm, much smaller than the original cells. These are the initial characteristic features of apoptotic cell death.
However, the ADP/ATP ratio can be used as a complementary method and as an indicator of apoptosis, but not to give the actual level of induction. The higher ratio in the MCEs than the AgNPs after 48 h may suggest that it gives better indication of the late apoptosis. For 4T1 cells (Figure 13b benzonic acid esters were tentatively identified. In the absence of analytical standards, the compound identification and confirmation may depend on the chromatographic and spectral information like the spectra, retention time, the presence of fragment ion as reported in the previous studies, or available on-line databases such as Metabolomics Workbench (www.metabolomicsworkbench.org) or Metlin (metlin.sc ripps.edu). The microalgal metabolites identified and analysed in our study, including fatty acids, lipids, sterol, and natural pigments, are important as pharmaceutical agent and in nutrient-dense food industry [43].

Fatty acids
For negative ion mode, five metabolites were tentatively identified as fatty acids (Table 5) assigned as arachidoyl dodecanoate (t R ¼ 29  Table 4 shows that Stearidonoyl-CoA (t R ¼ 1.39) (m/z 544.94) was identified in MCEs-W, while Table 5 shows its presence in T. suecica and Chlorella sp.-MET, and Chlorella sp.-ETH. Stearoyl-CoA desaturase (SCD) which stimulates the de-saturation of D9-cis for a group of fatty acyl-CoA substrates, is the main and highly regulated enzyme required for the biosynthesis of mono-unsaturated fatty acids (MUFA) [44]. The metabolite octadecanoic acid trichloroethyl ester (t R ¼ 28.58) at m/z 414.18 was observed in MCEs-CHL and HEX but not detected in MCEs-MET, ETH and W, while octadecanoic acid trichloroethyl ester isomer (t R ¼ 32.08) was observed in MCEs-W and in all MCEs except N. oculata-CHL. For positive ion, three metabolites were identified as fatty acids (Table 6)

Other compounds
The metabolites identified in negative ion mode included 3 flavones, 2 flavonoids and 1 prenylated chalcone. Qancaonin Q (t R ¼ 15.11) and gancaonin Q isomer (t R ¼ 15.     19) were identified at m/z 533.14, in N. oculata-W, T. suecica-ETH, Chlorella sp.-ETH; and N. oculata-W, respectively. Other ions were observed but the obtained MS data did not allow their complete identification (unknown compounds).

Discussion
Antitumor activity of microalgal compounds has been attributed to their lipophilicity that can cross the membranes and interact with proteins involved in apoptosis. The compounds could stimulate DNAdependent DNA polymerase inhibition, alter cyclins expression or most of the transduction pathway, trigger immune response [54], and also induce cytotoxicity against different cancer cell-lines [55,56]. The mechanisms for AgNPs-induced cytotoxicity on the cancer cells are attributable to the oxidative stress, mitochondrial damage, induction of apoptosis and DNA damage [57]. The biogenic AgNPs have been proposed as the adjuvant for the antibiotic activities in the treatment of infectious diseases caused by the bacteria [58]. The mechanisms involve the bonding reaction between the NPs and the bioactive molecules in which the hydroxy and amino groups can interact with the large surface     area of the AgNPs via chelation [59]. The AgNPs may damage the cell metabolism, disulfide bond formation, and iron homeostasis, resulting in cell death [60]. This may also increase the production of reactive oxygen species (ROS) and membrane permeability to induce the activity of a wide range of bioactive molecules. In the case of gram-negative bacteria, the antibiotic activity in several metabolic states may be promoted, or the antibiotic ability towards a resistant bacterial strain may be restored [59]. The type of solvent and extraction procedures could affect the composition and biological effects of the extracts [61]. The major advantage of AgNPs-MCEs-W, ETH is the use of water and ethanol as green solvents to produce the MCEs. CHL is not environmentally-friendly and also toxic. Both ETH and W, on the other hand have low toxicity as compared to the other organic solvents [62]. ETH is known as a good solvent for polyphenol extraction and safe for human consumption [63]. The W extracts may have more non-phenolic compounds or contain phenolic compounds that have a smaller amount of active groups than the other solvents [64]. In successive extraction, this can be complementary. The Padina gymnospora water extracts show high inhibition of lipid oxidation in the liposomes, while the ethanol extracts show a pro-oxidative activity. Although the water extracts have less phenolic content and low radical scavenging activity as compared to the ethanolic extracts, the iron chelating activity is reportedly high [65]. The highest cytotoxicity of AgNPs-MCEs-W, ETH on the breast cancer can be correlated to the increased early and late apoptotic events, increased sub G1 (apoptotic events) with enhanced biomarker activities. The incorporation of the MCEs in the AgNPs-MCEs-W, ETH formulation, using water or ethanol as extracting solvent, was proven effective to reduce the cytotoxicity of the AgNPs on the non-cancerous healthy cells. This could potentially reduce the side-effects from the AgNPs application during theranostics cancer treatment.

Carotenoids
Apo-4 0 -lycopenoate 23.86 461 As the most comprehensive and largest libraries currently available contain only a small part of the endogenous metabolites found in the biological samples, the number of unidentified metabolites may be three times greater than the number of specific metabolites, even after comprehensive data processing [67]. For the negative ion mode in our study, the MCEs-W exhibited the presence of fatty acids stearidonoyl-CoA and octadecanoic acid trichloroethyl ester isomer. However, while the MCEs-ETH exhibited octadecanoic acid trichloroethyl ester isomer, only Fucoxanthin, violaxanthin and their derivatives were the two interesting compounds detected that could be explored further as anticancer agents or for malignant tumor inhibition [47,48].
Other species such as N. gaditana has been characterized by the high lipids, around 65 and 70% (dry basis) [68], and the carotenoids such as      β-carotene, astaxanthin, neoxanthin, canthaxanthin, zeaxanthin and violaxanthin [69]. Lutein, foloxanthin, zanthophils, neoxanthin, estrogen luroxanthin and anthraxanthin in T. suecica extracts have exhibited antioxidant activity and strong repair mechanism in the human lung cancer cell line (A549). The tissue repair effects are demonstrated in the reengineered human skin tissue cells "EpiDerm™" [70]. The most abundant antioxidants in the Chlorella extracts are the carotenoids, chlorophyll, chlorophyll a and b, phenophytes a and lutein [71]. The most abundant carotenoids include α and β-carotene, zeaxanthin, lutein, neoxanthin and phylaxanthin, while the ketocarotenoids like canthaxanthin and astaxanthin are found in some Chlorella species [72]. Carotenoids in general exhibit anti-cancer, anti-obesity, anti-inflammatory, and anti-diabetic effects and have potential to cure heart disease [73].
Violaxanthin isolated from C. ellipsoidea has been proposed as a suitable candidate to tackle inflammatory disorders and identified as exhibiting anti-proliferative and pro-apoptotic effects against HCT116 human colon cancer cells [74]. Fucoxanthin and its metabolites, fucoxanthinol and halocynthiaxanthin, have shown anti-inflammatory, anti-mutagenic, anti-obesity, anti-neoplastic, anti-diabetic activity [48], and antioxidant activity with the decomposition of ROS such as OH and O 2À [75]. Fucoxanthin isomers including 13Z and 13 0 Z isomers also have high anti-proliferative activity in different cancer cell lines [49]. Several fatty amides, amino acids and vitamins were detected in the MCEs-W and ETH, to further promote the antioxidant activities of the MCEs in our study. The negative ion mode detected N-oleoyl glutamine and geranylgeranylcysteine in all MCEs; and geranylgeranylcysteine isomer in MCEs-W. The phylloquinone or vitamin K1 was identified in both MCEs-W and ETH; and all trans-retinyl palmitate was observed in N. oculata-ETH. For positive ion mode, phylloquinone was detected only in MCEs-W; ascorbyl stearate and ascorbyl stearate isomer only in N. oculata-W and T. suecica-W; riboflavin cyclic-4 0 ,5 0 -phosphate in N. oculata-W; and β-tocopherol in Chlorella sp.-W. The amides including termitomycamide B isomer was found in MCEs-ETH; termitomycamide B isomer in T. suecica-ETH and Chlorella sp.-ETH; and termitomycamide B and Narachidonoyl glutamic acid in T. suecica-ETH. The vitamin β-tocopherol isomer was detected in MCEs-ETH and riboflavin cyclic-4 0 ,5 0 -phosphate in Chlorella sp.-ETH.
In our study, presqualene diphosphate, an intermediate in the biosynthesis of terpenoid [53], is found in Chlorella sp.-ETH. Squalene was detected in N. oculata-CHL and HEX and T. suecica-CHL, but none in the MCEs-W and ETH. Squalene, a triterpene and a precursor for the synthesis of cholesterol and steroid hormones in human body, does not induce scavenging activity or significantly affect the cell proliferation rates, cell cycle profile or apoptosis in human epithelial cells MCF10A and breast cancer cells (MDA-MB-231 and MCF-7) [76]. However, squalene in a dose-dependent manner, affects intracellular ROS level reduction, prevents oxidative injury from H 2 O 2 and protects against DNA damage in epithelial MCF10A cells. Interestingly no similar effects of squalene are observed in the MCF7 and MDA-MB-231 cancer cells. Squalene, found in large quantities in virgin olive oils, has been suggested to be partially responsible for the low incidence of breast cancer in mediterranean diet groups. Other compound such as prenylated chalcone (Paratocarpin E) which has significant anti-cancer effects [51], is identified in N. oculata-W and T. suecica-W. Two phenols, 2-methoxy-6-(all-trans-heptaprenyl) phenol and 2-methoxy-6-(all-trans-heptaprenyl) phenol isomer, were observed in MCEs-ETH. Geranylgeranyl diphosphate (GGPP), one of the key isoprenoids for conversion into plant growth compounds, was found in N. oculata-W, N. oculata-ETH and T. suecica-W, and GGPP isomer was detected in Chlorella sp.-ETH. Didecyl phthalate, the benzoic acid esters derived from the oxidation of fatty acids in marine organisms, was found in the MCEs-W and T. suecica-ETH. Canthiumine, a cyclic peptide, was detected in the MCEs-ETH. Pyrimidine 2 0 -deoxyribonucleoside diphosphates, identified as dUDP, was detected in N. oculata-ETH; and phytanoyl CoA, a coenzyme A for the side chain of the chlorophyll [53], in N. oculata-ETH and Chlorella sp.-ETH.
The flavonoid quercetin 7-methyl ether 3,3 0 ,4 0 -trisulfate was detected in T. suecica-ETH; erycristin in MCEs-ETH and kolaflavanone in N. oculata and T. suecica-ETH. Several flavonoid-like compounds such as gancaonin Q, 6-prenylapigenin, 6,8-diprenyleriodictyol, and 4hydroxylon-chocarpin isolated from Dorstenia genus, exhibit antiproliferative activities against leukemia and solid cancer cells, as well as AML12 normal hepatocytes, by arresting the cell cycle, inducing apoptosis through Caspase 3/7, and anti-angiogenic activities [77]. Amino acid profile of Chlorella pyrenoidosa includes phenylalanine, lysine, valine, threonine, methionine, histidine, leucine, isoleucine, and tryptophan. The contents of glutamine, asparagus and lysine have been reduced in Chlorella species and the total content of amino acids also decrease, a result of changes in the protein content [78]. The amino acids derived from oxaloacetate such as aspartate, lysine, asparagine, isoleucine, and threonine have also shown changes under temperature treatments [79]. The high anticancer activities of AgNPs-MCEs co-application against breast cancer in our study, suggests the ability of the AgNPs to interact with the metabolites in the MCEs to confer selective cytotoxicity on the breast cancer cells, but not on the non-cancerous Vero cells. The AgNPs may act as an adjuvant to the MCEs, and the antioxidative compounds, principally the carotenoids, may lessen the toxicity on the Vero cells [7]. The lower number of metabolites in the MCEs-W did not affect the efficacy with the AgNPs, suggesting that key metabolites are important and enough to retain the cytotoxicity on MCF-7 and 4T1 cells, without affecting the Vero cells.
Other studies have reported the use of metal and non-metal based codelivery of drugs. The AgNPs-based co-delivery of oseltamivir (Ag@OTV) has reportedly inhibited the H1N1 influenza virus activity through the ROS-mediated signaling pathways and, AKT and p53 phosphorylation activation [80]. The selenium-based nanoparticle for drug delivery has also been successful in inhibiting the virus activity. The surface functionalization of selenium NPs by amantadine (Se@AM) is designed to reflect the drug resistance induced by the influenza virus infection. The Se@AM which exhibits reduced toxicity, significantly prevents the ability of H1N1 influenza to infect the host cells by the inhibition of neuraminidase activity [81]. The surface of SeNPs loaded with arididol (Se@ARB) with lower toxicity has obviously prevented the H1N1 virus infection. The Se@ARB interferes with the reaction between the H1N1 influenza virus and the host cells by inhibiting hemagglutinin (HA) and neuraminidase (NA) activity. Se@ARB can block H1N1 from infecting MDCK cells and prevent DNA fragmentation, chromatin condensation, and impede the ROS generation. The in vivo study further suggests that the Se@ARB could inhibit the lung infection in H1N1-infected mice [82]. Our study may have great implications in the theranostic application of the AgNPs for effective diagnosis and therapeutics during cancer treatment. The potential applications and the side-effects with the AgNPs treatments remain the major challenges to be resolved before it can be considered safe.

Conclusions
The AgNPs-MCEs-W, ETH at the 4:1 and 5:1 ratios showed much lower or no cytotoxic effects on the Vero cells whilst retaining the cytotoxicity on the MCF-7 and 4T1 cells, comparable to or better than the AgNPs-MCEs-CHL. There were low apoptotic events observed in Vero cells with the AgNPs-MCEs co-application, but dose dependent effects were exhibited in the MCF-7 and 4T1 cell lines with increased early and late apoptosis, cell cycle arrest in all phases and apoptotic events in sub G1 phase. The induction of apoptosis in the breast cancer cells was confirmed with enhanced the level of ADP/ATP ratios and Caspase 3/7 activities after 24 and 48 h treatments. Not all metabolites had the same LC-MS ionization mode, as some were detected only with either the negative or positive mode. Others such as apo-4 0 -lycopenoate, all-transβ-carotene, 15,15 0 -dihydroxy-β-carotene, protoporphyrin IX, ascorbyl palmitate and heptasiloxane and hexadecamethyl, can be detected in both modes based on the polarity and chemical properties. Some of the metabolites were detected in all species and all extracts, while others were detected only in one species or extract. Less number of metabolites was detected in the W extracts such as stachyose, methyl pyrophaeophoribide, ascorbyl stearate and ascorbyl stearate isomer. Fucosterol, protoporphyrin IX, didecyl phthalate, presqualene diphosphate and heptasiloxane, hexadecamethyl were detected only in ETH extracts. However, the presence of MCEs-W and ETH with elevated AgNPs level showed reduced toxicity against the non-cancerous cells whilst retaining the potent anticancer activities against breast cancer cells. This finding may be of great interest for the theranostic applications during cancer treatment.

Declarations
Author contribution statement H. Hussein: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
M. Maulidiani: Analyzed and interpreted the data.
M. Abdullah: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.