Evaluation of Polycladia myrica mediated selenium nanoparticles (PoSeNPS) cytotoxicity against PC-3 cells and antiviral activity against HAV HM175 (Hepatitis A), HSV-2 (Herpes simplex II), and Adenovirus strain 2

1. Introduction

The use of material nanoparticles (NPs) with dimensions ranging from 1 to 100 nm and for a variety of applications that are distinct from those of the same materials’ bigger particles has attracted significant attention to nanotechnology in recent decades in a variety of scientific fields (Gunalan et al., 2012; Bhuyar et al., 2020). The trace element selenium is an essential micronutrient for the health of humans, animals, and microbes. It is also a food supplement and is thought to have cancer-prevention properties. Through selenoproteins, antioxidant defense, cell signal transmission, immunological modulation, and other metabolic activities, selenium is an essential trace mineral for preserving human health (Labunskyy et al., 2014). According to earlier research, a selenium deficit is directly linked to high cancer, infectious disease, and cardiovascular disease mortality rates (Rayman, 2012; Hatfield et al., 2014; Liu et al., 2017).

Many researchers have recently become interested in selenium nanoparticles (SeNPs) because of their biocompatibility, bioavailability, and low toxicity. Consequently, selenium nanoparticles are widely used in various biomedical applications and wastewater bioremediation due to their greater bioactivity (El-Shanshoury et al., 2020; Bisht et al., 2022). In general, physical, chemical, and biological techniques can be used to create selenium nanoparticles. However, biologically produced SeNPs are preferred for medicinal purposes because this technique is more compatible with human organs and tissues. Numerous researchers have investigated how their uses in biological systems are affected by their size, shape, and technique of manufacture (Kulkarni and Muddapur, 2014; Bisht et al., 2022). Kong (Kong et al., 2011) found that Selenium nanoparticles have been discovered to be safer than other Selenium compounds, including monomethylated Selenium, sodium selenite, and selenomethionine, its anticancer activity and intrinsic mechanisms remain unknown.

Due to their distinctive biological and physicochemical features, green nanomaterials have greatly developed in various relevant study fields over the past few decades (Khalifa et al., 2019). Depending on their intended uses and applications, nanoparticles could be created by various precursors. However, inorganic functional platforms are extremely important for concurrent diagnostics, antidote administration, and therapy (Alaqad and Saleh, 2016; Alshalalfeh et al., 2016). The manufacture of nanoparticles by chemical methods was seen to have some drawbacks, including a high energy requirement, toxic chemicals, high prices, and the generation of hazardous byproducts. This necessitates the creation of new biological eco-friendly effective approaches that rely on reducing agents from natural resources (Sharma et al., 2015). Using living organisms’ methods has some benefits, including being economical, easily accessible, non-toxic, and environmentally benign. Biomolecules were used in place of the conventional stabilizing and reducing agents (Ovais et al., 2018). Utilizing plants, animals, foods, agricultural waste, and microbes, as well as their various natural components to act as reducing, stabilizing, and capping agents, allow for the formation of physicochemical adsorption coating processes without the use of chemical reactions (Anwar et al., 2016; Arshad et al., 2017; Vincy et al., 2017; Anwar et al., 2021). Whether alive or dried, algae are an essential resource for producing nanoparticles. They come from various organic compounds that operate like a powerful agent for reducing target metals. “Bionanofactories” could be referred to algae (Arya et al., 2018). Due to the many valuable biological compounds that can create different nanoparticle patterns, algal species have been identified as an effective tool for nanoparticle creation. The biogenic production of nanoparticles using microalgae is a natural, safe, economical, ecologically responsible, non-toxic, low-temperature, and energy-efficient method used for various applications (Sharma et al., 2014). El-Sheekh et al. (2022) (used two blue-green algal strains; Oscillatoria sp. and Spirulina platensis for green synthesizing of Ag₂O|AgO-NPs and Au-NPs, respectively. Compared to other animals, algae are crucial for manufacturing nanoparticles (yeast, bacteria, and fungi) (Sharma et al., 2014; Rauwel et al., 2015; El-Sheekh and El-Kassas, 2016).

One of the most common cancers in men is prostate cancer (Jemal et al., 2007). Selenium (Se) is being studied more closely as a potential anticancer drug since it has been hypothesized to have cancer-prevention properties (Kong et al., 2011). Epidemiology, ecology, and medicine have revealed that se reduces the incidence of some malignancies, including breast, colon, lung, and prostate cancer (Helzlsouer et al., 2000; Klein, 2004; El-kassas and El-Sheekh, 2014; El-Sheekh et al., 2021). Ag, Au, TiO₂, CuCl₂, CeO₂, and SiO₂ NPs have been evaluated for their antiviral properties against a variety of viruses, including influenza virus H3N2 type-2, hepatitis B virus (HBV), foot-and-mouth disease virus, HIV-1, and H1N1, vesicular stomatitis virus dengue virus and HSV-1 (Rai et al., 2015; Orłowski et al., 2018; El-Sheekh et al., 2022). SeNPs have recently been investigated as antiviral agents for different types of fabrics. To achieve this, the authors of a recent polymer study used a flat screen-printing technique to graft SeNPs onto polyester fabric. They then measured the antimicrobial activity of the resulting material against SARS-CoV-2 Pseudomonas aeruginosa, Bacillus cereus, Salmonella typhi, and Escherichia coli. Following an MTT experiment, the researchers discovered that the printed polyester textiles with SeNPs exhibit high antiviral activity and are not harmful to human skin (Abou Elmaaty et al., 2022). It is worth saying that SeNPS were produced by different types of microalgae, like many strains of Cyanobacteria such as Anabaena variabilis and Spirulina platensis (Afzal et al., 2021; ElSaied et al., 2021), and only from Ulva lactuca (Chlorophyta) and Sargassum latifolium (Ochrophyta, Phaeophyceae) (El-Khateeb et al., 2019; Vikneshan et al., 2020; Makhlof et al., 2022) and for the first time in this research SeNPS are produced from the brown algae Polycladia myrica (formerlyCystoseira myrica) (formerly Polycladia myrica). So, this research aims to examine the antiviral and anticancer properties of Se-NPs mediated by P. myrica using the PC3 cell line, Herpes simplex II, Hepatitis A, and Adenovirus by MTT test, and the experiment was carried out in vitro.

2. Material and methods

2.1. Collection of algal samples

The brown seaweed Polycladia myrica was collected from the Gulf of Suez, Egypt coast. Dr. Fekry Ashour Mourad, a researcher at NIOF Egypt, identified the alga. All samples were identified as described previously and according to the methods of Aleem [Aleem, 1978; Aleem, 1993; Lipkin and Silva, 2002) and were confirmed using the Algae Base website (Guiry and Guiry, 2022). Sand and other foreign objects Sand and other foreign objects were removed from the algal samples by washing them under running fresh water. Before being brought to the lab, the seaweed was gathered and put in a polyethylene bag. The seaweed was washed three times with deionized water until the pH of the wash solution was comparable to deionized water. The seaweed was rinsed twice with distilled water to remove any metallic compound. The seaweed was collected, fully dried in the shade at room temperature, powdered in a mechanical grinder, and then put through a 0.2 mm screen, according to Soliman (Soliman et al., 2018).

2.2. Preparation and (FTIR) characterization of algal extract

A round flask containing 5 g of powdered alga and 50 mL of deionized water was thoroughly agitated on a rotary shaker for an hour before being allowed to boil at 70°C and then cool to room temperature (Hashemi et al., 2015). The extracted solution was filtered using filter paper Whatman No. 1. The filtrate was maintained at 4°C for additional research (Thamer et al., 2018). The spectra between 4000 and 400 nm^-1 were measured using an FTIR spectrometer (FT/IR-6100 type A). FTIR is used to find the bioactive substances in Polycladia myrica extract that cause SeNPs to be bio-reduced and become nanoparticles.

2.3. Preparation of Se nanoparticles

To find the ideal conditions, according to Kashyap (Kashyap et al., 2019), the extract of Polycladia myrica was mixed with 1mM Na₂SeO₄ (Sigma Aldrich Company, USA) solution at a mixing ratio (1:9). When the hue of the sodium selenite solution changed to orange-red after being stirred for 72 h in the dark at 25 2°C, phycosynthesized PoSeNPs formed (ElSaied et al., 2021). After centrifuging the mixture at 4400 g for up to 30 min, the NPS was cleaned with double distilled water before being treated with pure ethanol (Murugaboopathy et al., 2021). The finished product was baked at 50°C and kept in a sealed container, to be characterized for research or other purposes (Azizi et al., 2013)..

2.4. PoSeNPs characterization

2.4.1. UV spectroscopy characterization

A spectrophotometer was used to record the CsSeNPs solution’s UV-visible spectrum (Thermo Scientific Evolution TM 300) between 200 and 800 nm was used to measure the absorbance.

2.4.2. PoSeNPS' FTIR

FTIR was used to identify the potential active groups of biomolecules that regulate the capping and reduction of nanoparticles. At Al-Azhar University, the Regional Center for Mycology and Biotechnology in Egypt performed FTIR spectrum analysis using potassium bromide (KBr) as a test substance in the 450–4,000 cm^-1 wave numbers. The resulting peaks were plotted with wave number (cm¹) on the Y-axis and transmittance (percent) on the X-axis.

2.4.3. X-ray diffraction (XRD) spectrum

Utilizing the power of an X-ray diffractometer (XRD-6000, Shimadzu, Japan) with Cu-k radiation (= 1.5412) at 40 KV and 30 mA in the 2 range of 10-80° for purity investigation, the crystallinity and elemental composition of the generated Se-NPs were assessed and characterized.

2.4.4. Transmission electron microscope (TEM)

Using a transmission electron microscope (JEOL, JEM-2100, Japan) running at 200 kV accelerating voltage, the shape, and size of the phycosynthesized SeNPs were studied. Deionized water was used to dilute the reaction solution, and it was then sonicated (Branson-Sonifier 250, USA) for ten min (Tahmasebi et al., 2015). The sonicated sample was dropped onto copper grids coated with carbon before being dried in a vacuum for half an hour; then, we could take electron micrographs (Yue et al., 2004).

2.4.5. SEM scan

SEM stands for the scanning electron microscope. That scans with a high-energy electron beam in a raster scan pattern to bring the picture to the material. The signals produced by the electrons’ interactions with the atoms that potentially make up the sample provide information on its surface topography, composition, and other characteristics like electrical conductivity. Laminar airflow and UV light were used to disinfect SeNPs. Using sticky tape, the sterilized nanoparticles were carefully mounted on SEM stubs and evenly covered with gold. The sample was then put into the SEM’s sample chamber (JEOL JSM 6490 LV, Japan), and scanning was done at various magnifications between 15,000 and 35,000 and voltages between 20 and 30 kV (Šileikaitė et al., 2009; Razi et al., 2011).

2.4.6. EDX analysis

The biosynthesized selenium nanoparticles were dried and used for XRD analysis to confirm the crystalline nature and phase composition of nanoparticles synthesized using (JSM-IT100, JOEL, Japan) equipped with a LINXEYE detector, operated at 30 kV and 10 mA current with 2.2 KW Cu anode radiation (k = 1.54184 Å). To calculate the average size of the nanoparticles, Scherrer’s equation (D = kλ/β cos θ) was used, where k is a constant whose value is approximately 0.9, λ is the wavelength of the X-ray (1.54060), β is the width in radians of the peak due to the size effect which is calculated by the following equation: β =θ₁- θ₂×π/180 and θ is Bragg’s diffraction angle. Specifically, the particle size of the sample was estimated from the line width of the (300) XRD peak.

2.4.7. Zeta potential (ζ)

PoSeNPS surface charges were detected using zeta potential (Zeta plus, Brookhaven, USA). 25 µg of SeNPs samples were diluted in water ten times to prepare the sample, which was then sonicated for 15 min. at 20 Hz. Before measuring the zeta potential, the mixture was passed through a 0.22 m filter. NP aggregation was avoided by diluting SeNPs. Measurements ranged from -200 to +200 mV (ElSaied et al., 2021).

2.5. Antiviral activity test of SeNPs

2.5.1. Mammalian cell line

The American Type Culture Collection provided Vero cells taken from an African green monkey kidney (ATCC, Manassas, VA, USA). DMEM was used to grow Vero cells (Dulbecco’s Modified Eagle’s Medium), which contained 10% heat-inactivated foetal bovine serum (FBS), 1% L-glutamine, HEPES buffer, and 50 gram per ml gentamycin. All cells are subcultured 2 times weekly and reservation at thirty-seven °C in a moisturizer environment with 5% carbon dioxide (Vijayan et al., 2004).

2.5.2. Virus propagation

In confluent Vero cells, the cytopathogenic HAV HM175 strain of hepatitis A virus, herpes simplex type 2 virus (HSV-2), or adenovirus strain 2 was propagated and tested (Randazzo et al., 2018). The Spearman-Karber method was used to count the number of infectious viruses by calculating the dose of infectious tissue culture (TCID50) by using 8 wells of each dilution and 20 µL of inoculum in each well (Pinto et al., 1994).

2.5.3. Cytotoxicity evaluation

Vero cell lines were planted in 96-well plates for the cytotoxicity experiment at a cell density of 2×10⁵ cells in each ml per 100 L medium for growth. Following a day of injection, a new growth medium with different quantities of the tested material was introduced. Monolayers of the confluent cell were distributed into microtiter plates with 96-well and a flat-bottomed using a multichannel pipette (Falcon, Jersey, NJ, USA) in serial duplicated dilutions of the PoSeNPS starting with 3000 g/mL and decreasing down to two g/mL. 48 h were spent incubating the microtiter plates at 37°C in a moisture incubator with 5% carbon dioxide. For each concentration of the tested substance, 3 wells were employed. Cells as control were cultured with or without DMSO and without tested samples. It was discovered that the experiment was unaffected by the minimal aliquot of DMSO (maximum 0.1%) placed in each well. A MTT colorimetric technique was used to determine the viable cell yield following the conclusion of the incubation time (Mosmann, 1983; Riyadh et al., 2015). In a nutshell, the medium for culturing was withdrawn to be replaced with 100 µ of fresh medium devoid of phenol red. The stock solution with 12 mM MTT was added to each well which was prepared by adding 5 mg of MTT in 1 mL of PBS in the presence of the controls, which was untreated.

For 4 h, the 96-well plates were incubated at 37 °C with 5% carbon dioxide. After 85µl aliquot media were removed from each well, 50 µl DMSO was added and mixed with the pipette carefully then incubation was done at 37 °C for 10 min. The viable cells’ number was then determined by measuring the optical density at 590 nm using an ELISA reader (SunRise, TECAN, Inc., USA) to calculate the percentage of viability, where the mean optical density of wells treated with PoSeNPS is ODt, and the mean optical density of untreated cells is ODc. The viability curve of the Vero cell line followed by treatment with the specified drug is obtained by plotting a curve between tested compound concentration and surviving cells. The dose-response curve of each concentration was plotted using the Graphpad Prism software to obtain the 50% cytotoxic concentration (CC50), which is the dosage necessary to produce lethal effects in intact cells (San Diego, CA. USA). For further biological research, each compound’s maximum non-toxic concentration (MNT) was also established.

2.5.4. Evaluation of the antiviral activity

At the Regional Center for Mycology and Biotechnology, a cytopathic effect inhibition assay was used for the antiviral screening (RCMB, Al-Azhar University, Cairo, Egypt). The test was chosen to demonstrate the precise suppression of a biological process, specifically the cytopathic effect in sensitive mammalian cells, as evaluated by the MTT technique (Hu and Hsiung, 1989; Al-Salahi et al., 2015). In a nutshell, 96-well microtiter plates containing monolayers of (2×10⁵ cells/mL) the adhered Vero cells at the bottom of the wells were incubated for a day at 37°C in a moistened incubator with 5% carbon dioxide.

The SeNPS concentrate simultaneously treated cultures in a fresh maintenance medium after the plates had been rinsed with fresh Dulbecco’s Modified Eagle’s Medium and challenged with 104 doses of the virus; after this, they were incubated at 37°C for 2 days.

By SeNPS absence, infection controls and an untreated Vero cell control were created. For each concentration of the substance under examination, six wells were employed. The activity of the Antiviral was assessed by comparing the cytopathic inhibition effect to the control, and the level of protection provided to the cells by the test substance was calculated. Four replicates of each treatment were included in each evaluated independent experiment. In this assay technique, the positive controls were amantadine or acyclovir. Following the incubation period, the cells’ viability was estimated using the MTT assay, which was previously covered in the cytotoxicity section (Mosmann, 1983).

The rate of viral inhibition could be estimated as follows:

where A, B, and C indicate the absorbance of the SeNPS with virus-infected cells, the virus control absorbance, and the cell control absorbance, respectively.

2.5.5. Data analysis

Using these data and STATA modeling software, the dose that prevented 50% of viral infection (EC50) was calculated concerning virus control from the visual plots. The three tests’ median and standard error data show the percentages of viral inhibition concerning each virus under the test. The obtained curve by plotting the inhibition of the viral yield against the concentration of SeNPS was used to calculate EC₅₀ values directly. The ratio of CC₅₀ to EC₅₀ was used to calculate the selectivity index (SI), which was used to examine whether each chemical had enough antiviral activity to outperform its degree of toxicity (Alshalalfeh et al., 2016). This measure, known as a therapeutic index, was also employed to decide if a drug was worthy of further investigation. Active compounds had an SI-value of 2 or higher (Al-Salahi et al., 2015).

2.6. Cytotoxicity of SeNPS against PC-3

2.6.1. Mammalian cell lines

The American Type Culture Collection provided a human prostate cancer cell line (PC-3 cells) (ATCC, Rockville, MD).

2.6.2. Chemicals used

The foetal bovine serum, MTT, trypan blue dye, dimethyl sulfoxide (DMSO), and DMSO were purchased from Sigma (St. Louis, Mo., USA). Lonza was used to purchase RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin, and 0.25% Trypsin-EDTA (Belgium).

2.6.3. Propagation of cell line

Cell culturing was done on growth medium (RPMI-1640) supplemented with ten % inactivated foetal calf serum and 50% gentamycin. The cells were subcultured two to three times every 7 days and kept at 37°C in a moist environment with five % CO₂.

2.6.4. Cytotoxicity evaluation using viability assay

In Corning 96-well tissue culture plates, we suspend tumor cell lines in media with a concentration of 5×10⁴ cells per well for antitumor tests and then incubated for 24 h. To produce 10 concentrations of each chemical, the SeNPS were then put into 96-well plates (three duplicates). For every plate with 96 well, six-vehicle controls with media or 0.5% DMSO were run as a control. The MTT test assessed the number of viable cells after 24 h of incubation. In a nutshell, the RPMI 1640 medium without phenol red was replaced with 100 l of fresh culture medium for 96-well plates, and 10 µl of the stock solution of 12 mM MTT (5 mg of MTT in 1 mL of PBS) was set to each well that includes the untreated controls.

The plates with 96-well were then incubated for 4 h at 37°C with five % carbon dioxide. After 85µl aliquot of the media was removed from each well, 50µl of DMSO was added to each well with careful pipette mix, then incubation at 37°C for 10 min was done. The number of viable cells was then determined by measuring the optical density at 590 nm with an ELISA (SunRise, TECAN, Inc., USA). According to the following formula, the viability percentage was calculated [(ODt/ODc)]x100%, where ODt is the mean optical density of wells treated with SeNPS and ODc is the mean optical density of control untreated cells. After adding the given substance, the curve between viable cells and the concentration drug is displayed to determine the viability curve of each cancer cell line. Utilizing Graphpad Prism software (San Diego, CA, USA), from graphic plots of the dose-response curve for each conc, we could calculate the concentration of fifty % inhibition (IC₅₀), or the needed concentration to have harmful effects in fifty % of intact cells (Mosmann, 1983).

2.6.5. Microscopic observation of the prostate carcinoma (PC3) cell line treated with the PoSeNPS

The protocol for cytotoxic activity was followed during the conduct of this experiment. After the treatment at the tested concentration was complete, after removing the medium from the plates, the wells were washed three times with 300µL of phosphate-buffered saline (pH 7.2). We can fix the cells to the plate for fifteen min with 10% formalin at room temperature. After that, 100 µl of crystal violet with a concentration of 0.25% was used to make staining for the fixed cells for 20 min. After the stain was eliminated, we should rinse the plates with dH₂O to remove any remaining color, and then they were left to dry. Images showing morphological alterations compared to control cells were taken by using an inverted microscope (CKX41; Olympus, Japan), which was supplied with a digital camera for microscopy. At a magnification of 200x, the cytopathic effects (morphological changes) were visible.

3. Result and discussion

3.1. FTIR characterization of algal extract

According to the FTIR analysis of P. myrica extract (Figure 1), the presence of phenol or alcohol molecules is indicated by an absorption band at 3405 cm^-1 of strong stretching –OH (Silva et al., 2014; Hu et al., 2016; Mohamed et al., 2021). The peak at 2926 cm-1 represents the N=C=S stretching bond (isothiocyanate); also, the CH2 anti-symmetric stretch of methyl groups in lipids caused the absorption bands at 2977–2852 cm^-1 (Lu and Rasco, 2012). Due to the N-H bending vibration of secondary amines and the carbonyl unsaturated ketone amide (lipid, protein), the absorption bands at 1628 cm^-1 indicate the stretching vibration of -C=O- of Amide I. This agrees with Demir (Demir et al., 2015), who discovered that bands at 1739-1638 cm ¹ of the spectra show the stretching of C=C of phenyl compounds and the stretching of C=O of aromatic.

Figure 1 FTIR spectra of Polycladia myrica aqueous extract.

The lignin or asymmetric stretching vibration of nitro compounds (N-O) is indicated by the absorbance band at 1509 cm¹, representing the stretching vibration of C = C. Kubi and Kadla (Kubo and Kadla, 2005). Lopez (López et al., 2014) notice a small amount of lignin in seaweed cell walls. Furthermore, lignin’s cytotoxic properties against colon cancer were demonstrated by Lue (Lu et al., 1998). The absorption band indicated the existence of carboxylic acid (O-H bending) at 1423 cm^-1. Younger (Younger, 2014) It was well known that seaweed had many carboxylic acids, especially fatty acids. Deyab (Deyab et al., 2012). described the anticancer effects (in vitro) of several fatty acids, such as (oleic and palmitic acids) from seaweed. The primary and secondary amines’ N-H stretching is indicated by the 1260 absorbance band (ElSaied et al., 2021). According to Singh and ElSaied (Singh et al., 2016; ElSaied et al., 2021), the absorbance band at 1081 cm ¹ demonstrate the stretching of carbohydrates’ C-O, like pectin and starch, and the stretching vibration of C-N of aliphatic amines. 1128 cm¹ band is attributable to ACH₂OH groups of carbohydrates (Mordechai et al., 2001). Chowdhury discovered Fenugreek seed was high in the polysaccharide galactomannan in 1987 (Chowdhury et al., 1987). Plant extracts, including phenol and flavonol derivatives, function as reducing agents and nanoparticle stabilizers and have been implicated in the reduction of selenious acids to SeNPs (Huang et al., 2004).

The absorption bands at 600-716 cm^-1 (Phaeophyceae and Chlorophyta) show the stretching of C = S, which suggests sulfides in the tested algae. C-Br stretching peaks at 600 cm343 at 3405 cm^-1 belonging to the aqueous extract of C. myrica was shifted to the phycosynthesized represented alkyl halides compound (lipids) (Younger, 2014). Based on these findings, we discovered that P. myrica included a variety of phytochemicals, including phenol, alcohol, lipid, proteins, fatty acids, and other substances with anticancer and antiviral properties. Additionally, these phytochemicals might have helped sodium selenite convert to SeNAPS (Huang et al., 2007; Vikneshan et al., 2020).

3.2. Nanoparticles characterization

The results of SeNPS have been validated using seven characterization techniques: UV-Vis spectra, FTIR, XRD, TEM scanning, SEM scanning, EDX, and zeta potential.

3.2.1. SPR characteristic

Studies using UV-Vis spectra confirmed that sodium selenite’s color changed from colorless to ruby red (SeNPs), and that this color change was caused by the biosynthesis of SeNPs (Figure 2). As PoSeNPS decreased, the absorbance steadily climbed from 1.9 to 3. The largest beak, though, was discovered to be 350 nm.

Figure 2 UV-Visible spectrum of PoSeNPs.

3.2.2. PoSeNPS' FTIR analysis

To look into the functional groups responsible for the synthesis and stability of nanoparticles, the biosynthesized PoSeNPS were examined by FTIR analysis (Figure 3). Due to the aromatic rings’ -OH stretching and the presence of alcohol and phenol groups, the marker bands of SeNPs (powder) exhibit an absorption peak at 3300 cm^-1 and a peak at 2927 cm^-1 because of the stretching vibration of C-H of alkenes. These peaks demonstrate the presence of a biopolymer that is likely derived from the algal cell walls and is connected with the SeNPs (Fritea et al., 2017). Additionally, the amide I and II bands may be seen at 1652 cm^-1 for the C=O stretch of the ester group and 1543 cm^-1 for the N-H bending of the amines primary and secondary, respectively. Bands of 1321 cm^-1 and CH2 and CH3 groups exhibit asymmetric C-H bending (Fritea et al., 2017; ElSaied et al., 2021). According to the literature, another absorption band at 1036 cm^-1 shows the superposition of in-plane C-H bending and C-N stretching vibration of aliphatic amines and the distinctive Se-O stretching vibration (Kannan et al., 2014). At 698 and 468 cm^-1, further bending vibrations of the Se-O bond are highlighted. The peak at 3405 cm^-1 belonging to the aqueous extract of P. myrica was shifted to the phycosynthesized PoSeNPS peak at 3300 cm^-1, as shown in Figure 3 (Gunti et al., 2019). This indicated the interaction between Se and hydroxyl group from the P. myrica aqueous extract through hydrogen bonding which facilitated phycosynthesized CsSeNPs. Similar to this, the sharp peaks at 1628 and 1509 cm^-1, that responsible for the stretching of C=O and nitro asymmetric stretching vibration (N-O) compounds in the C. myrica extract, were both shifted to higher frequencies at 1652 and 1543 cm^-1, respectively, in phycosynthesized PoSeNPS, showing the interaction between carbonyl C=O stretch and N-O compounds of P. myrica aqueous extract with Se. The protein character of phycosynthesized PoSeNPS, which may be in charge of reduction and stabilization, is indicated by 2 absorption peaks of C-N stretching vibration of aliphatic amines and the N-H stretching of the primary and secondary amines at 1081 and 1260 cm^-1, respectively (Kokila et al., 2017). From the FTIR, it can be deduced that the bio-organic components of P. myrica extract, such as carbonyl-unsaturated ketone amides, amino acids, esters, and proteins, acted as potent capping and reducing agents on PoSeNPs (Liu et al., 2013). Similar findings have previously been published concerning these functional biochemical bonds (Suganya et al., 2015).

Figure 3 FTIR spectra of PoSeNPs.

3.2.3. XRD analysis

Utilizing the XRD techniques depicted in Figure 4, it was possible to identify SeNPS’s crystal structure and phase composition. The sample’s XRD pattern implies that it is nanocrystalline and closely resembles the typical selenium powder, indicating that selenium particles were formed. There is a specific diffraction peak in the range of 14–52.4° at the angle of 2θ, which is consistent with the diffraction peak of (JCPDS card number 65-1290). It can be inferred that the obtained particles are selenium. The diffraction peak in the pattern is very narrow, indicating that the synthesized nano-selenium particles are very small in size, crystalline, and with poor amorphous character. The XRD patterns obtained show the main peaks characteristic of crystalline SeNPS at 2θ values of 14°, 17.4°, 20.14°, 28.5°, 30°, 39.42°, 42.4°, and 52.4°(Figure 4) corresponding to the crystal planes (200), (220), (140), (220), (300), (226), (332) and (248), respectively. The average crystalline size of biogenic Se nanostructures measured by Scherrer’s equation was about 15.9 nm. The biological approach of SeNPs produced some background noise, which may have been caused by the presence of additional bioactive compounds in the P. myrica extract (Figure 4). The powder’s XRD pattern demonstrates that post-annealing is unnecessary to achieve the desired crystalline phase (ElSaied et al., 2021).

Figure 4 XRD spectrum of PoSeNPs.

3.2.4. Transmission Electron Microscopy (TEM) imaging

It has been established that the physical and chemical properties of the synthesized SeNPS, such as crystalline structure, shape, size dispersion, and surface condition, determine their applicability (Salih et al., 2016). SeNAPS were measured using TEM to establish their size and form, and the results showed that they ranged in size from 9.31 to 68.65 nm and had smooth spherical and semispherical shapes (Figure 5). These findings concur with those of (Vikneshan et al., 2020) and (ElSaied et al., 2021), who synthesized SeNPS using Ulva lactuca and Arthrospira platensis extracts. They discovered that the morphology of SeNPS using TEM was smooth, spherical/ball-shaped, and distributed uniformly.

Figure 5 Transmission Electron Microscope (TEM) photographs of PoSeNPs (A, B) with magnifying field 100nm and diameter.

3.2.5. SEM imaging

Figure 6 depicts the size of the SeNPS that P. myrica produced. The SeNPS’s SEM pictures revealed that it had a smooth surface and an oval-to-spherical form. 18.7 to 22.79 nm were found to be the particle size. The dynamic light scattering approach correlates with SEM analysis and is used for the size-distribution analysis.

Figure 6 Scanning Electron Microscope (SEM) photographs (A, B) of PoSeNPs with measuring the diameter of PoSeNPs.

3.2.6. EDX analysis

Se atoms were detected in the elemental composition analysis using EDX (atom % 0.11 ± 0.02; mass % 0.56 ± 0.10). (Figure 7). This study revealed the presence of selenium-containing nanostructures together with other EDX peaks like Si, Mg, Na, Ca, and O peaks, indicating that they were mixed precipitates of the selenium salt. XRD provided additional evidence that SeNPS were produced.

Figure 7 EDX analysis of PoSeNPs3.3.

3.2.7. Zeta potential (ζ)

ζ indicates an estimation of the created electric double layer in the solution by the surrounding ions, not the actual charge on each molecule. Because of the inter-particle electrostatic repulsion, NPs with ζ values between +30 mV to -30 mV typically display stability with high degrees (Jummes et al., 2020). The negative charge of the produced PoSeNPs was validated, indicating better stability of the NPs without aggregation. Zeta potential (Figure 8) was used in the current study to find the potential of the nanoparticles. A change of - 24.0 ± 5.62 mV was made to the zeta potential. With a particle size of 11.17–12.74 nm, SeNPs recorded a λ maximum at 545 nm. Further tests were conducted on the best nanocomposite (PoSeNPs), made from Na₂SeO₃ with extremely small particle sizes.

Figure 8 Zeta potential of the PoSeNPs.

3.3. Antiviral activity of the SeNPs

To detect the antiviral activity of PoSeNPs against HAV-10, HSV-2, and Adenovirus, MTT assay was used (Table 1) and VERO cell line (Table 2). Table 1 shows the Cytotoxic activity of PoSeNPs with Mammalian cells from African Green Monkey Kidney (Vero) cells detected under these experimental conditions with fifty % cell cytotoxic concentration (CC₅₀) = 220.53 ± 6.89 µg/mL. Table 1 shows that PoSeNPs have antiviral activity with MNCC (50 µg/mL) for all tested viruses. This cytotoxic activity was moderate (++) against the HAV-10 virus with (40.25 ± 2.61%) inhibition and weak (+) against HSV-2 and Adeno virus with (17.39 ± 1.45% and 8.64 ± 0.82%) inhibition, respectively. The best antiviral activity of PoSeNPs was against (HAV-10) with EC₅₀ and SI (63.81 ± 2.93 and 3.5), respectively. PoSeNPs show weak EC₅₀ in Inactive SI against both HSV-2 and Adenovirus. Xia (Xia et al., 2022) found that Se nanoparticles inhibit infection of Madin–Darby canine kidney (MDCK) cells with H1N1 by preventing chromatin condensation and DNA fragmentation. The antiviral activity of Se NPs against various other types of viruses is probably owing to the Se NPs’ direct binding to viral envelope glycoproteins, which prevents viral entry into the host cell, even though the mechanism of action has not been well studied (Lu et al., 2008; Fayaz et al., 2012).

Table 1 The antiviral effects of PoSeNPS when tested at (MNCC).

Table 2 The Cytotoxic activity against Mammalian cells from African Green Monkey Kidney (Vero) with 50% cell cytotoxic concentration (CC₅₀) = 220.53 ± 6.89 µg/ml.

3.4. PoSeNPs cytotoxic activity

Using vero cell lines, PC-3 cell lines were treated with varying doses of CsSeNPs (3.9–50 0 g/mL), and the cytotoxicity (Table 3 and Figures 9A, B) occurred in a concentration-dependent manner. Clinical trial preliminary findings indicate that dose and Se forms are essential for its anticancer effectiveness (Kong et al., 2011). All concentrations examined exhibit cytotoxic activity, except for 93.9-7.8-15.6 g/mL, which exhibits 100% cell viability and 0% inhibition.

Table 3 Inhibitory activity of PoSeNPS against prostate carcinoma cells (PC-3) with IC₅₀ = 123.51 ± 4.07 µg/mL.

Figure 9 (A) PC-3 cells non-treated (control) (B) PC-3 cells treated with PoSeNPS at 500 µg/mL concentration.

Kong (Kong et al., 2011) discovered that SeNPS inhibit prostate cancer cells’ growth partially via caspases-mediated apoptosis. For suppressing cancer growth apoptosis is an important mechanism, and caspases, in turn, induced apoptosis, as shown by the difference between the (9A) image (control cells without treatment and with 100% cell viability) and (9B) image (the cells with the highest PoSeNPS concentration500 µg/mL and 13.85% cell viability) and suppress androgen receptor transcriptional activity by its mRNA down-regulating and protein expression. Furthermore, selenium nanoparticles increase Akt kinase phosphorylation to be activated; therefore, phosphorylation promotes Akt-dependent androgen receptor, and regulation of Mdm2 degradation via the proteasome and prostate cancer pathway cell growth suppression by the disruption of androgen receptor, acting as a promising application in cancer therapy.

4. Conclusion

This study concluded that selenium nanoparticles synthesized by the brown alga Polycladia myrica have good antiviral activity against HAV-10 virus with an antiviral percent of 40.25%, despite weak antiviral activity against Adenovirus and HSV-2 with antiviral percent (8.64% and 17.39%), respectively. The cytotoxicity effect of these nanoparticles was determined against PC-3 with a maximum inhibitory percent of 80.53%. These nanoparticles have no hazardous effect against normal Vero cells as the viability percent was (78.39% and 49.23%) for Vero cells and PC-3 cells, respectively, at 125 µg/mL. As a suggestion, because each tool used for synthesizing green nanoparticles has its own characteristics, optimizing many factors like pH, materials concentration, and incubation time could increase obtaining more small-sized NPs.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Author contributions

MM carried out the experiments, constructed the Figurers and tables, and wrote the first draft of the manuscript. ME-S and HT shared the research idea, wrote, edited the manuscript, and revised the data and the whole work. All authors have read and agreed to the published version of the manuscript.

Funding

Under grant No IFPIP: 178-662-1443, this project was funded by Institutional Fund Projects, Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah.

Acknowledgments

This research work was funded by Institutional Fund Projects under grant no: (IFPIP: 178-662-1443). The authors gratefully acknowledge technical and financial support provided by the Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

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References