Green - synthesized silver nanoparticles with aqueous extract of green algae Chaetomorpha ligustica and its anticancer potential

: Marine green algae are rich in various bioactive components with known anticancer activity. Some anti cancer drugs present in green algae are in clinical trials nowadays. Algae - mediated silver nanoparticles ( AgNPs ) have been of a great interest in cancer treatment due to their unique physico - chemical properties. In this study, we evaluate the anticancer e ﬃ ciency of marine alga Chaetomorpha ligustica collected from the Arabian Gulf against colon cancer cell lines HT29 and HCT116. The anti cancer potential of biosynthesized AgNPs from C. ligustica extract is also reported. Fourier transform infrared ( FTIR ) spectroscopy and gas chromatography - mass spectrometry analyses were used to identify the phytoconstituents pre sent in algae extract. The synthesized AgNPs were con ﬁ rmed via UV - Vis spectroscopy, whereas their morphology and stability were recorded by transmission electron micro scopy ( TEM ) , zeta potential, and zetasizer. We recorded absorption peak at 420 nm; TEM images showed an average size of 8.8 nm, whereas zeta potential and zetasizer study showed aggregation of nanoparticles. FTIR spectroscopy peaks of C. ligustica AgNPs were a little di ﬀ erent from those of the C. ligustica extract. Both extracts showed cytotoxicity against cancer cell lines in a dose - dependent manner, but nanoparticles were found to be more toxic than algae extract. HT29 was found to be more sensitive than HCT116. For the ﬁ rst time, species of C. ligustica have been used and reported for the synthesis of nano particles. C. ligustica and its biogenic nanoparticles need to be scaled up for many biomedical applications especially in cancer research. - diphenyltetrazolium bromide ) assay. Absorbance was measured at 490 nm using a scanning microliter - well spectrophotometer. The color intensity is correlated with the number of healthy living cells.


Introduction
Herbal medicine in the form of extracts, powders, brews, or gels has been used to treat various ailments both in humans and animals [1]. Primary health care through herbal medicine is a very common practice in developed and in developing countries [2]. Cancer is a major cause of death in humans, and colorectal cancer is the third most diagnosed form of this disease all over the world [3]. A large number of preventive therapeutic studies are going on all over the world to find a way to ameliorate cancer incidence and/or reduce cancer mortality in human populations [4]. An enhanced search for natural compounds that can help in preventing cancer is being explored keenly. Most of the herbal medicines are derived from plants, but some algae are being focused on due to their novel bioactive compounds [5]. Algae are heterogeneous organisms, existing as brown (Phaeophyta), red (Rhodophyta), and green (Chlorophyta) and are mainly divided as microalgae and macroalgae. Various bioactive metabolites with potent cytotoxicity against different kinds of cancers are being explored from marine macroalgae or seaweeds [6]. Some recent studies reported many novel anticancer drugs present in seaweeds [7][8][9][10] and Kahalalide F, an antitumor marine drug, present in green algae are in clinical trials nowadays [11].
Chaetomorpha, one of the genera of green algae, are macroscopic unbranched filaments of cylindrical-shaped cells and are mostly found in the intertidal zone of the seas. The Chaetomorpha sp. are a rich source of antioxidants and possess drug properties against various human diseases [12]. Currently, many biomedical compounds present in Chaetomorpha sp. are being explored for various cancer therapeutic treatments such as chemo-protective agents, anticancer and drug-delivery systems [13,14]. Chaetomorpha sp. are a rich source of proteins, carbohydrates, fatty acids, pigments, and secondary metabolites, such as phenols, sulfated polysaccharides, and halogenated compounds [15]. Besides being strong antitumor, antiviral, antibacterial, and cytotoxic agents, these compounds have the ability to convert metal ions into metal nanoparticles due to their reducing and capping properties [16].
Biological methods of preparing nanoparticles are less toxic with low cost as compared to physical or chemical methods [17]. Being rich in bioactive compounds, plants are preferred for the green synthesis of metallic nanoparticles [18]. Plant-mediated metal nanoparticles are either synthesized intracellular, that is, inside plant tissue or extracellular by using plant extract [19,20]. Biomolecules present in plants significantly influence the quality and quantity of metal nanoparticles and thus determine their application approach. Due to unique surface chemistry, charge, energy, and spatial dimensions, metal nanoparticles offer outstanding applications in many fields of biomedicine [21]. They are widely used in drug-delivery systems, in vitro diagnostic tests, as biosensors and antimicrobial and anticancer agents [22,23]. Metal nanostructures are used as cargo molecules for imaging and gene delivery in many anticancer therapies. Metal nanoparticles can hinder some signal cascades that are responsible for the development and pathogenesis of tumors. Biogenic metal nanoparticles are found to be more cytotoxic against human cancer cells as compared to normal cells [22].
Algae-mediated silver nanoparticles (AgNPs) have been of a great interest in biomedicine especially cancer treatment and for targeted delivery of various anticancerous drugs [16]. This study examined the in vitro anticancer potential of marine alga Chaetomorpha ligustica collected from the Arabian Gulf against two colon cancer cell lines HCT116 and HT 29. Biologically synthesized AgNPs mediated by water extract of C. ligustica were also reported in this study.

Collection of algae
The green algae, C. ligustica, was collected from the shore of the Arabian Gulf. It was washed properly with distilled water and then shade dried at room temperature. Once dry, it was powdered in an electric blender. One gram of powder was dissolved in 100 mL of sterile autoclaved water and left on a magnetic stirrer for 48 h at 4°C. Then, the mixture was filtered by using Whitman No. 1 filter paper. The obtained extract was stored at −80°C for further experiments.

Fourier transformed infrared (FTIR)
Perkin Elmer FTIR-Spectrometer Spectrum (Spectrum BX, USA) was used to identify the functional groups present in algae extract. FTIR was measured in the range from 4,400 to 400 cm −1 . The spectra (16 scans per spectrum) were collected with a spectral resolution of 16.0 cm −1 with an interval of 2.0 cm −1 .

Gas chromatography-mass spectrometry (GC-MS)
The phytochemical analysis of hexane extract of C. ligustica was accomplished by GC-MS analysis. GC Column TRACE™ TR-35MS (Thermo Fisher Scientific, USA) with the film thickness of 0.25 µm, length of 30 m, and diameter of 0.25 mm was used. The carrier gas was helium (99.9999% purity) at a flow rate of 1.2 mL·min −1 . The injector temperature was set to 250°C. The injection mode was split less, and the injection volume was 1.0 μL. The GC column temperature was programed as an initial temperature of 60°C, held for 3 min, increased at a rate of 10°C·min −1 to 300°C, and held for 5 min.

Biogenically synthesized AgNPs from C. ligustica extract
AgNPs were synthesized by dissolving silver nitrate (AgNO 3 ) solution in C. ligustica extract with the concentration of 5 mM AgNO 3 in the mixture. The reduction of silver ions to AgNPs was observed by a color change from light green to dark brown.

Characterization of biogenically synthesized AgNPs
Prepared nanoparticles were characterized as described below before treating the cells.

UV-Vis spectrophotometer
Prepared nanoparticles were initially confirmed by observing a UV-Visible spectrophotometer from 300 to 600 nm with a peak at 420 nm by using the UV-Vis spectrophotometer (Thermo Scientific 1500, USA).

Transmission electron microscopy (TEM)
The shape and size of the nanoparticles were determined by using TEM. A grid coated with copper was used to place the silver nanoparticle sample to be imaged under TEM (JEOL JEM-1400 Plus, Japan).

Zetasizer and zeta potential
The average size of nanoparticles was recorded by Zetasizer Nano Series ZS (ZEN3600), UK by using the technique of dynamic light scattering (DLS) and zeta potential by electrophoretic light scattering (ELS).

Cell lines
In this study, two types of colon cancer cell lines, HT 29 and HCT116, were included. According to ATCC, the Global Bio-resource Center, HCT116 is derived from carcinoma-primary and HT29 adenocarcinoma-primary.

Expression level of genes
Cells were divided into three groups as control or untreated, C. ligustica extract (50% cytotoxic) treated, and C. ligustica AgNPs (50% cytotoxic) treated for 24 h.

Determination of cell viability
Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Absorbance was measured at 490 nm using a scanning microliter-well spectrophotometer. The color intensity is correlated with the number of healthy living cells.

Nucleic acid isolation
After treatment, RNA samples were extracted from two different colon cancer cell lines by using an RNeasy mini kit (Qiagen, DE). All extractions were performed following the manufacturer's procedure.

cDNA preparation
cDNA synthesis was performed by reverse transcription of total purified by using the high-capacity cDNA reverse transcription kit (Applied Biosystems, USA). The cDNA synthesis was performed according to the manufacturer's procedure. The obtained cDNA samples were stored at −20°C.

Expressional level of genes
Power SYBR ® Green PCR Master Mix 2X from Applied Biosystems ® (Life Technologies, USA) was used to perform quantitative real-time PCR. All colon cancer cell lines were analyzed in triplicate, and gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH); a nontemplate negative control was included in each run to check background signal. Primer sequences and annealing temperature are listed in Table 1.

Statistical analysis
Statistical analyses were carried out using GraphPad Prism ® 9.0 statistical software (GraphPad Inc., USA) and Microsoft Excel ® . An independent t-test was used to evaluate the difference in gene expression between the different stages of colon cancer cell lines and treated and untreated colon cancer cell lines. The level of significance used throughout was p < 0.05.

Cell viability
Results of cytotoxic effects of C. ligustica extract and C. ligustica AgNPs against human colon cancer cell lines HT29 and HCT116 after treatment are presented in Figure 4. C. ligustica extract was found more toxic at 10 μg·mL −1 as compared to C. ligustica AgNPs, but with the increase in concentration C. ligustica AgNPs showed higher cytotoxicity against both the cell lines.

Expressional level of ATM, ATR, CHK1,
and CHK2 in cell lines treated with C. ligustica extract and C. ligustica

AgNPs extract
As shown in Figure 5, the expression level of all the four genes (ATM, ATR, CHK1, and CHK2) was increased with treatment in HT29. In HCT116, the expression level of ATM was decreased with C. ligustica extract and AgNP treatment, but we found a slight increase in gene expression levels of other genes on treatment as shown in Figure 5.

Discussion
Green algae are being explored for drug properties for the fight against many diseases, including colorectal cancers, as they are found rich in many therapeutic compounds that can induce apoptosis through different molecular mechanisms [24,25]. The cytotoxic potential of various marine macroalgae against the growth of many kinds of cancer cell lines has been reported in the literature [25][26][27][28][29]. We explored both C. ligustica extract and C. ligustica AgNPs for cytotoxicity. FTIR and GC-MS analyses were used to identify the phytoconstituents present in algae extract.

FTIR of C. ligustica extracts
FTIR analysis is one of the most reliable and sensitive methods used by researchers to identify the biomolecules involved in the green synthesis of nanoparticles from metal ions [30,31]. C. ligustica extract was found rich in active metabolites (Figure 1) as shown by IR spectrum peaks, which may have supported the green synthesis process of AgNPs [15]. Our results suggested that compounds with hydroxyl, alkene, carboxyl, and amide groups of the protein, polysaccharides, and phenols present in C. ligustica extract were likely involved in the synthesis process as some peak shifts were seen in C. ligustica Fucoxanthin is reported to induce G1 cell-cycle arrest and apoptosis in various cancer cell lines and in animal models of cancer [33]. Eucalyptol is an active anticancer compound that induces cell death through reactive oxygen species-mediated apoptosis [34,35]. Propanoic acid, milbemycin B, and rhodopin detected in C. ligustica extract are achieving a lot of interest due to their cytotoxic properties against various cancer cell lines [36][37][38]. Several biochemical and microscopic experiments revealed the anticancer potential of methanone [39].

Characterization of C. ligustica AgNPs
AgNPs were successfully synthesized from C. ligustica extract. It is well known that the formation of AgNPs is initially confirmed by recording a sharp surface plasmon resonance (SPR) in the range from 350 to 500 nm; however, the position of the peak is decided by the shape and size of the nanoparticle formed [40,41]. The absorption peak at 420 nm (Figure 3a) confirms SPR in AgNPs [41]. These results confirm the particle size below 100 nm with a small degree of polydispersity, which is in agreement with our zetasizer and TEM results. Overall, the size of the nanoparticle was satisfactory as the polydispersity index was 0.013, indicating little aggregation. The TEM is a perfect tool to study the morphology of particles at the nanoscale [42]. Our TEM images were clearly showing spherical shapes with particle sizes in the range from 2 to 12 nm.

Cell viability
Both C. ligustica extract and its biogenic AgNPs showed cytotoxicity against cancer cell lines in a dose-dependent manner, but nanoparticles were found to be more toxic than the algae extract. Our results generally agree with many studies that highlight the efficacy of AgNPs against different types of cancer cell lines [26,27]. HT29 was found to be more sensitive than HCT116. Our results are well supported by Gurunathan et al. [28] reporting plantbased nanoparticles are more effective against HCT116 as compared to HT29.
4.5 Expressional level of ATM, ATR, CHK1, and CHK2 in cell lines treated with C. ligustica extract and C. ligustica AgNPs extract We analyzed the gene expression of ATM, ATR, CHK1, and CHK2 in both the cell lines after treating the cells with C. ligustica extract and its biogenic AgNPs. All these genes are involved in DNA damage response and cell cycle checkpoints. The gene expression of ATM was significantly increased in HT29 but slightly decreased in HCT116 after treatment. The ATM gene expression is considered a predictive biomarker in colorectal cancer. Decreased expression levels of ATM are associated with poor survival in colon cancer patients [43]. The loss of ATM expression is directly related to chromosomal instability and worse survival in colorectal cancer associated [44]. The expression level of ATR, CHK1, and CHK2 was significantly increased in both cell lines, but it was more prominent in HT29. All cell lines have their unique characteristics depending on the origin from tumor tissue samples of a patient [45,46]. HT29 is derived from a female adenocarcinoma-primary, whereas HCT116 is derived from male carcinoma-primary [47]. HT29 has a deficient p53 gene expression, but HCT116 has mutations in PI3KCA and K-RAS genes [48]. Recent report by Khorrami et al. states that biogenic nanoparticles can interrupt the metabolic pathway of cancer cells even at the very lowest concentration [49].

Conclusion
To the best of our knowledge, bio-reduction of silver ions by C. ligustica aqueous extract has been reported for the first time. The produced AgNPs show peak at 420 nm in the UV-Vis spectra; TEM images showed particle size in the range from 2 to 12 nm. Quick formation of AgNPs in presence of C. ligustica extract showed the presence of active metabolites, which was confirmed by FTIR and GC-MS analyses. GC-MS profile showed some anticancer compounds in C. ligustica extracts. C. ligustica and its biogenic nanoparticles have good potential to be explored for anticancer compounds. C. ligustica and its biosynthesized AgNPs hold huge potential for pharmaceuticals mainly for cancer therapies.