Production, characterization and cytotoxicity effects of silver nanoparticles from Cystseira myrica

: 27 Background: A green, eco-friendly approach to biosynthesizing silver nanoparticles has been reported for 28 marine macroalga Cystseira myrica extract as a reducing agent. 29 Methods: Algal extract was prepared from the marine brown seaweed Cystoseira myrica. Different pH and 30 temperature impact the green synthesis of silver nanoparticles suggesting that the synthesis depends greatly on 31 pH and temperature. Silver nanoparticles have been detected by modifying the color of the algal extract and 32 verified by the use of UV-vis and other characterizations. The Structure and characters of synthesized 33 nanoparticles were confirmed using TEM, DLS, XRD, and FTIR. Cytotoxicity of the biosynthesized 34 nanoparticles using provided cell lines of breast carcinoma cells (MCF-7) and human hepatocellular carcinoma 35 cells (HepG2). 36 Results: Shape of silver nanoparticles at pH 9, 75°C for 30 min and was found to be suitable for the 37 biosynthesis process and the AgNPs exhibited a characteristic absorption peak at 434nm. High resolution 38 Electron Microscope Transmission (HR-TEM) reported polydisperse and spherical shapes ranging from 8 to 15 39 nm. High attractive and repulsive forces between each nanoparticle were recorded with an average zeta potential 40 value of approximately −29.3 mV for C. myrica extract NPs. The X-ray diffraction study revealed the crystalline 41 structure of silver nanoparticles. FT-IR has shown the bioreduction of silver ions to silver nanoparticles through 42 biomolecules found in C. myrica extract. Silver nanoparticles have been found to have anticancer activity. The 43 cytotoxicity assay was studied against MCF-7 and HepG2 at various concentrations (100, 50, 25, 12.5, 6.25, 44 3.125, 1.56, 0.78, 0.39, 0.2 and 0.1 μg/mL ). By increasing the concentration of AgNPs from 0.1 to 100 μg/mL 45 the maximum percentage of viability against MCF-7 and HepG2 cell line decreased from 94.55 ± 7.55 to 19.879 46 ± 0.503 and from 78.56 ± 11.36 to 25.81 ± 2.66 after time exposure respectively. 47 Conclusions: The silver nanoparticles from Cystoseira myrica have cytotoxicity activity against MCF-7 breast carcinoma cell line and HepG2 human hepato cellular carcinoma cell line. 13 14 present proposed to assess the ability of marine macroalgae to synthesis silver nanoparticles and their cytotoxic effect against tumor cells. The cytotoxic effects of silver nanoparticles be attributed


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Minerals, polysaccharides, polyunsaturated fatty acids, and vitamins are abundant in Phaeophyceae algae [3] 57 Furthermore, these organisms exhibit significant quantities of secondary metabolites with pharmacological 58 interest, such as phenolic compounds, terpenoids, and alkaloids, which have been associated to interesting 59 biological activities, such as anticancer and neuroprotective properties [2,4].

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Preparation of algal extract

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The brown algae Cystoseira myrica used in this work is a member of Phaeophyta. Cystoseira myrica was 84 collected from Hurghada coastal along the Red sea, Egypt. The marine brown seaweed Cystoseira myrica was 85 cleaned with seawater to remove impurities. The seaweed was carried in sterile polythene bags to the laboratory.

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In the laboratory, the sample was washed with tap water triplicate to remove dirt, sand, epiphytes and then with 87 deionized water three times, to remove any metallic compounds until the pH of the wash solution was equal to 88 deionized water. It was dried in the shade at room temperature until constant weight. The powdered dried algal 89 materials were ground in an electric mortar to get the powder form and passed through 0.2 mm sieve. About 5 90 gm of powdered alga was added with 50 ml double distilled water in 250 ml conical flask, mixed well on a 91 rotary shaker for 1 hour and then boiled from 5-10 min about 60-80°C [11], the crude extract obtained was 92 filtered and used as reducing and stabilizer agent (stored at 4°C for experimental use).

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To study the effect of different conditions on the synthesis of silver nanoparticles, the reaction solution 95 was incubated at 30,60,90,120 and 150 min. To study pH effect, the pH of the reaction solution was adjusted 96 to (5, 7 and 9) in order to study the experiments [12] by using NaOH (0.1N) or HCL (0.1N) [13] after adjusting 97 pH value at 9. The reaction temperature was carried at 25°C and by heating in the water bath at 50 and 75°C

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Cu kal radiation at a 2 angle, the X-ray generator was operated at a voltage of 45kv and a current of 30mA.

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The spectra were measured using an FT-IR spectrometer (FT/IR-6100 type A) in the wavelength range of 130 4000 to 400 nm -1 . The biomolecules in the algal extract of Cystoseira myrica that are responsible for the 131 reduction of silver ions to create nanoparticles are identified using FT-IR.

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The present study was proposed to assess the ability of marine macroalgae to synthesis silver nanoparticles    3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65 8 solution. The color variations indicate a reduction in Ag + to Ag 0 [27]. UV spectroscopy was used to detect the 179 formation of AgNPs throughout time (fig1). At low pH (5) the broad band was recorded and the presence of 180 broad band indicated AgNPs aggregation ( fig.1.a.). The neutral pH 7 was also reported to support the 181 phycofabrication of the nanoparticles. The present study reported that an increase in pH accelerates the time of 182 reduction of silver ions and stabilizes the silver nanoparticles by adsorbing on them. In the alkaline medium, 183 the stabilizing and reducing ability of Cystoseira myrica extract is increased. The absorbance increased as the 184 pH values ranged from 5 to 9, and a narrow SPR band at a lower wavelength was seen (434nm). Haglan et al,

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(1:9), no change in color was showed and the UV-vis data observe no absorbance peak. A broad absorption 207 peak appears at 415nm after 120 and 150 min as shown in Fig. 2(a). After 120 and 150 minutes, a broad 208 absorption peak appears at 415nm, as illustrated in Fig. 2 (a). The absorbance peak of silver nanoparticles 209 becomes stronger at 50°C, as seen in Fig. 2 (b), and the position blue shifts from 429 nm to 426.5 nm. Results 210 recorded showed that with increasing temperature, after 30 minutes at 75°C, the typical absorbance peaks 211 became stronger and blue shifted, with a peak at 413.5nm. As the reaction time increases, a broad absorbance 212 band forms on the higher wavelength side, corresponding to the agglomeration of some silver nanoparticles.

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The synthesis rate increased with an increase in reaction temperature up to 75 °C at the optimal AgNO3   to depend on their physical shape representing their antitumor activity [35]. The most common instrument for 227 determining the distribution of size profile of small particles (nano-scale) in suspension is the dynamic light 228 scattering (DLS). Therefore, the DLS curves of the silver nanoparticles are shown in Fig. 3(b). The size 229 distribution curves of the prepared silver nanomaterial showed that the average size is less than 100 nm on an 230 average of 8-15nm as confirmed with the HRTEM. Moreover, zeta potential is a measure of the magnitude of 231 the charge repulsion/attraction, known to affect stability, between the particles in solution. Therefore, its 232   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65 determination brings detailed insight into the reasons for materials aggregation, coagulation, or flocculation in 233 suspensions, which is critically important during water treatment processes. In this work, zeta potential 234 measurement shows that silver nanoparticle has a zeta potential value around −29.3 mV. These zeta potential 235 values indicate that these Silver NPs have higher colloidal stability in the aqueous solution, which is probably 236 due to a stronger repulsion behavior between single particles in polar solution (i.e., water) [36].

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Crystal characterization: X-Ray analysis

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The extract of brown algae Cystoseira myrica is a source for eco-friendly, cost effective, nonhazardous 288 stable nanoparticles. Silver nanoparticles (AgNPs) from the algal extract were observed within 30 min at 75°C.

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 Availability of data and material: The datasets used and/or analyzed during the current study are 308 available from the corresponding author on reasonable request.