Application of Biosynthesized Silver Nanoparticles Against a Cancer Promoter Cyanobacterium , Microcystis aeruginosa

Cancer is already a major health problem. It is the second main cause of mortalities in the modern world after heart diseases, with more than 10 million new cases every year. This outline is expected to rise in the next few decades. In fact, around one in three people will be diagnosed with cancer throughout their lifetime (Siegel et al., 2012). The lifestyle changes suggest that the burden of neoplasia will become heavier over time, especially with increasing obesity and aging of what are now still youthful populations (Salim et al., 2009). The importance of environmental exposure to contaminants has also been


Introduction
Cancer is already a major health problem.It is the second main cause of mortalities in the modern world after heart diseases, with more than 10 million new cases every year.This outline is expected to rise in the next few decades.In fact, around one in three people will be diagnosed with cancer throughout their lifetime (Siegel et al., 2012).The lifestyle changes suggest that the burden of neoplasia will become heavier over time, especially with increasing obesity and aging of what are now still youthful populations (Salim et al., 2009).The importance of environmental exposure to contaminants has also been management have led to the enhancement of eutrophication in water bodies all over the world (Carmichael, 2007).conditions can lead to cyanobacterial blooms, which are characterized by excessive proliferation of cyanobacterial

Application of Biosynthesized Silver Nanoparticles Against a Cancer Promoter Cyanobacterium, Microcystis aeruginosa
Mostafa Mohamed El-Sheekh*, Hala Yassin El-Kassas cells that produce their toxins (deFigueiredo et al., 2004).Within the large family of cyanobacteria, the cancer promoting, Microcystis aeruginosa is the most common bloom-forming species that is able to produce cyanotoxin known as microcystin.Microcystin is one of the best studied classes of cyanobacterial toxins (Ouellette and Wilhelm 2003;Chen et al., 2009).
The risk of exposure to dissolved toxin immediately after the peak of a bloom must be addressed because cyanotoxins can persist even though the bloom has dissipated (Lawton et al., 1994).Prolonged exposure to sublethal doses of microcystin has been epidemiologically linked to primary liver cancer in humans (Yu, 1995).selective uptake by hepatocytes through the membrane transport family, Organic Anion Polypeptide Transporters (OAPT) that mediates the uptake and elimination of numerous xenobiotics (Hagenbuch and Meier, 2003;Fischer et al., 2005).Microcystin-LR, inhibits protein phosphatases 1 and 2A (PP1 and PP2A), which are serine/ threonine phosphatases, sustains the phosphorylation of proteins and induces tumor promotion mediated through signal transduction, resulting in development of tumors.Humans are often exposed to these tumor promoters in the environment, the relationship between human cancer development and the presence of PP1 and PP2A inhibitors Suganuma, 2009).
Besides the liver, the effects of microcystins on other human organs including the kidneys and intestine (Hagenbuch and Meier, 2003), brain (Maidana et al., 2006), lungs (Soares et al., 2007) and reproductive system (Ding et al., 2006) have been investigated.
However, natural products including plant extracts and herbs have been used as medicine sources (Abu-Rabia, 2005;Liu et al., 2012).Nanotechnology opens new science, manufacturing and various technologies.Among all metallic nanoparticles, silver NPs have proved to be the most effective against large variety of organisms including cyanobacteria (Gong et al., 2007).The biological system for biosynthesis of silver NPs must be consisting of environmentally acceptable solvent system, eco-friendly reducing and capping agents (Vigneshwaran et al., 2007;Xie et al., 2007), high-yield, low cost, non-toxic and environmentally benign procedures (Thakkar et al., 2010).
At present, only few studies have investigated the interactions of silver NPs with cyanobacteria.Therefore, the rationale of this work was to investigate algae mediated synthesis of in vivo silver NPs, using three selected microalgae species belonging to different groups namely, Spirulina platensis, Chlorella vulgaris and Scenedesmus obliquus.The bio-reduction of AgNO 3 ions in solution of biosynthesized silver NPs was determined against the cancer promoter, Microcystis aeruginosa which can be contaminated by this cyanobacteruim in swimming pools.

Source of algal strains and production of biomass
The cyanobacterium Spirulina platensis and two green algae (Chlorella vulgaris and Scendesmus obliquus) were kindly provided from Phycology Laboratory, Faculty of Science, Alexandria University.Microcystis aeruginosa was obtained from Phycology laboratory, Faculty of Science, Tanta University, Egypt.S. platensis was cultured in BG-11 medium for 15 days.Green algae were cultured the production of biomass, exponentially growing algae culture was transferred into fresh sterile medium [10% (v/v) of inoculums], incubated at 28±2 o C and day/night rhythm (16\8), respectively.M. aeruginosa was cultured in and Stanier, 1968), and kept in controlled conditions of

In vivo Synthesis of silver NPs and their characterization
There were two methods used for the in vivo synthesis cells from logarithmic phase were centrifuged at 4000 rpm for 20 min.The supernatant was removed and the biomass was washed with sterile deionized water to remove traces by taking 5 gm of algae biomass from an exponential of 1 mM aqueous AgNO 3 solution (pH 7) (Sudha et al., et al. (2010), the different algae species were individually cultured along with 1 mM AgNO 3 solution and kept in the previously mentioned conditions for two weeks.

Characterization of silver NPs
The properties and structure of silver NPs have been characterized by means of vis-UV spectra of the solution, Transmission Electron Microscope (TEM) and Energy-Dispersive analysis X-ray (EDX) spectrum as well as Fourier Transform Infrared Red spectroscopy (FTIR) spectral Analysis.
The bio-reduction of pure Ag + ions in aqueous solution was detected by sampling of aliquots (0.2 mL) of the colloidal suspension, and then diluting the samples with 2 mL deionized water and subsequently measuring Vis-UV spectra of the resulting diluents using UV-6800 UV\Vis maxima were scanned at the wavelength of 200-800 nm.
TEM analysis was employed to visualize the size and shape of silver NPs.TEM micrographs were taken by at the Electron Microscope Unit-Faculty of Science-Alexandria University.
The presence of elemental silver as well as the other elementary compositions of the silver NPs was detected.Energy-dispersive analysis X-ray (EDX) analysis was carried out at 20 KV by X-ray micro-analyzer (Module Scanning electron microscopy. Samples of the aqueous solution of the silver NPs were prepared by centrifugation at 10,000 rpm for 30 min.The pellet was lyophilized and subjected to FTIR analysis by KBR pellets (FTIR grade) method (Kasthuri et al., 2009).The spectrum was recorded in the range of 500 to 4000 cm -1 .

Cytotoxic potentials of the biosynthesized silver NPs against M. aeruginosa
Aliquots of 30 mL of the M. aeruginosa culture in to 0.1, 1 and 10 mgL -1 of the biosynthesized silver NPs and kept in controlled conditions of continuous light (45 and total chlorophyll were determined.The experiments were conducted in triplicate and results are shown as the mean with standard deviations.

Optical microphotographs of M. aeruginosa
Morphological changes in M. aeruginosa culture exposed to 0.1, 1.0 and10 mg/L of silver NPs for 24h were determined using a Reichert microscope (Austria-Nr.365475) and recorded with a Pixelink camera.

Determination of total chlorophyll
Total chlorophyll was measured according to Tandeau de Marsac (1977); 2 ml of the culture were centrifuged at 15.000 g for 10 min in a 2 ml reaction tube.Then 1900 resuspended using an ultrasonic bath.Afterwards 900 at 15.000g for 10 minutes and absorption was measured ml)=(Abs 650×13.9)/2ml.

Evaluation of algal culture viability
The M. aeruginosa cells viability was determined Pictures were recorded using the USB camera with image capture software that is available for recording images.

Biosynthesis of silver NPs
The biosynthesized silver NPs using the different analysis at various nm.The colour change was due to excitation of Surface Plasmon Vibration which indicated the formation of silver NPs (Figure 1).

Characterization of silver NPs
UV-Visible (UV-Vis) spectral analysis Plasmon band indicated the production of silver NPs at ~400 nm for S. platensis and C. vulgaris as well as Sc.obliquus, respectively.These results revealed that the silver NPs were dispersed in the aqueous solution with no evidence for aggregation (Figure 1).
Transmission electron microscope (TEM) the structural view and determination of the size of nanoparticles under the TEM as shown (Figure 2).Most of the biosynthesized silver NPs seem to be spherical in morphology and well distributed with the mean average size of 20.8±4, 8.2±3 and 8.8±2 nm for S. platensis, C. vulgaris and Sc.obliquus, respectively.Energy-dispersive analysis X-ray (EDX) spectrum Energy-dispersive analysis X-ray (EDX) spectra with the absorption peak in the range of 3 to 4 keV as outlined in (Figure 3).However, some of the additional peaks for C, Cu, N, O, P, Mg, S, and Ca were observed.Fourier Transform Infra-red (FTIR) analysis analyses were carried out to identify the possible biomolecules responsible for the reduction of silver ions and the capping of the bioreduced silver NPs synthesized by different micro algal species.Figure 4(a) shows FTIR spectrum of silver NPs synthesized using S. platensis.The peak at 3396 cm -1 is mainly due to N-H stretching vibration presence of secondary amines (protein, lipid), the peak at 1639 cm -1 can be assigned to the protein amide I band, mainly v(C=O) stretching and may be due to the N-H

The cytotoxic activity of the biosynthesized silver NPs M. aeruginosa cells
Cell viability assay cyanobacteruim, M. aeruginosa exposed to different concentrations of the biosynthesized silver NPs for control (Figure 5).Large aggregates were observed in the algal culture under the stress of 10 mgL -1 for all the biosynthesized silver NPs.reveal a reduction in viable cells after 24 h treatment.Growth of M. aeruginosa under the stress of 1 to 10mgL - 1 biosynthesized silver NPs resulted in a significant reduction of viable cells (Figures 6A & 6B).The exposure to 1mgL -1 0.5 and a % 51.1±0.4 decrease of viable cells, for those silver NPs produced by S. platensis, C. vulgaris, and Sc.obliquus, respectively.As the concentration of silver NPs High reduction of the viable cell count was achieved by using 10 mgL -1 of the biosynthesized silver NPs.The percentages of reduction of viable cells reached % 92±2.0,% 96±3.0 and % 98±2.0 for the silver NPs produced by S. platensis, C. vulgaris and Sc.obliquus, respectively.

Effect of silver NPs on chlorophyll content
The reduction levels in chlorophyll content of the tumor promoting cyanbacterium cells exposed to different concentrations of the biosynthesized silver NPs for 24 hours are presented in Figure 7

Discussion
Due to their unique physical and chemical properties, nanomaterials are extensively studied.Natural nanoparticles (NP) were investigated for their 2006; Novak and Bucheli, 2007), but less for human health and environmental consequences with the exception of small-sized dust particles (Yin et al., 2013).
As observed in this study one of the possible roles of the microorganisms is providing a multitude of nucleation centers; establishing conditions for obtaining highly dispersed nanoparticle systems.In addition, using microorganisms in green biosynthesis of nanoparticles slow down aggregation, or entirely prevent it by immobilizing the particles and providing viscous medium (Sun and Xia, 2002).Thus the biosynthesized nanoparticles have highly intricate architectures and are ordered during assembly.
The biosynthesized silver NPs were monitored by UV-Vis spectrophotometer at various nm-s.The surface Plasmon band in the silver NPs solution remained close to 400 nm throughout the reaction period indicating that the particles are dispersed in the aqueous solution, with no evidence for aggregation.The silver ions are reduced by the extracellular reductase enzymes produced by the microorganisms to silver metal in nanometer range (Sosa et al., 2003;Fu et al., 2006).The exact mechanism leading to the formation of silver NPs by the algal biomass is not fully understood; there are still several possible mechanisms involved in the process.It is thought that surface of algal cells, possibly via electrostatic interaction between the ions and negatively charged carboxylate groups present in the cell surface.Thereafter, the ions are reduced by the enzymes, leading to the formation of nuclei, which subsequently grow through the further reduction of metal ions and accumulation of these nuclei (Mandal et al., 2006).
TEM images of the different silver NPs reveal that the silver NPs seem to be spherical in morphology and well distributed with an average size of 20.8±4.0;8.2±3.0 and 8.8±2.0 nm for S. platensis and C. vulgaris as well as Sc.obliquus, respectively.Similar results were reported by The EDX spectrum of the biosynthesized silver NPs of an optical absorption band at 3 KeV reveals the presence of pure metallic silver NPs.The other peaks may be due et al., 2011).
FTIR measurements were carried out to identify the biomolecules which may be responsible for synthesis and stabilization of silver NPs.FTIR analyses revealed the presence of molecules like proteins, lipids and carbohydrates.Stretching vibrations of proteins were observed by strong bands in the spectrum of silver NPs synthesized by the three algae species.In accordance with these results, Sable et al. (2012) stated that representative spectra of biosynthesized silver NPs manifest absorption peaks of respective functional groups and indicated the presence of stabilized protein molecules.Moreover, Govindaraju et al. (2008) suggested the interaction of single cell protein of S. platensis with aqueous silver nitrate (AgNO 3 ) for the synthesis of silver NPs.The conformation of protein molecules plays an important role in silver NPs synthesis and stabilization.The results suggested that the capping ligand of the silver NPs may be an aromatic compound.It was reported that the extract of unicellular green algae C. vulgaris was used to synthesize single-crystalline silver nanoplates at room temperature (Xie et al., 2007).Proteins in the extract provide dual function of Ag+ reduction and shape-control in the synthesis of the silver NPs.Up to the current knowledge, silver NPs by Sc. obliquus.
It was concluded from protein assay of microorganisms that the preparation of silver NPs is a NADH-dependent reductase.The reductase enzyme gains electrons from NADH and oxidizes it to NAD+.The enzyme is then oxidized by the simultaneous reduction of silver ions forming silver metal in nano form.In some cases a nitrate-dependent reductase is responsible for the bioreduction process, therefore a complex electron shuttle materials may be involved in the biosynthesis process (Moghaddam, 2010).Moreover, (-COO-) of carboxylate ions is responsible for stabilizing the silver NPs.It was reported that extracts from microbes act both as reducing and capping agents in metal synthesis of nanoparticles.Reduction of metal ions by combinations of biomolecules such as enzymes or proteins, amino acids, polysaccharides, and vitamins (Collera-Zuniga et al., 2005) which may be used as reductants to react with silver ions, leading to silver NPs synthesis in solutions (Li et al., 2007) is environmentally benign, yet chemically complex.The overall suggestions are that proteins and polysaccharides were involved in the reduction of AgNO 3 to form silver NPs and the capping agents may be the aromatic compounds.
The cytotoxicity of silver NPs is expected to be mediated mainly by the release of silver ions (Ag+) from the particle surface.Consequently, silver NPs can be considered as a source of toxic Ag+, which are adsorbed to particle surfaces or formed upon oxidative dissolution in presence of oxygen, ligands, or organisms (Navarro et al., 2008;Xiu et al., 2011).
In the present study, the cultures of the cancer promoter cyanobateruim, M. aeruginosa exposed to different concentrations of the biosynthesized silver NPs for 24h showed formation of cell aggregates comparing to control cultures.Large aggregates were observed in the algal cultures under the stress of 10 mgL -1 after administration of all biosynthesized silver NPs.In this respect, it was reported that metal nanoparticles like TiO 2 NPs were adsorption to the cell walls (Sadiq et al., 2011).The pores across the cell wall have diameters ranging from 5 to20 nm that determines its sieving properties, the biosynthesized silver NPs have diameters within the mentioned range.It was suggested that the formation of aggregates might reduction of viable cells count and chlorophyll content a reduction in viable cell number after 24 h treatment.As the concentration of the biosynthesized silver NPs was The percent reduction of viable cells reached 98 % for the silver NPs produced by Sc. obliquus.
The chlorophyll concentration is often used as a measure for the viability of cyanobacteria cultures (Schulze et al., 2011).The data of the present study have shown an expected dose dependent decrease of the chlorophyll concentrations for all the samples along with decreased numbers of viable cells due to silver NPs administration.It was established that chlorophyll auto method for differentiation between living and dead cells.cytoplasm appeared to be useful for vitality assessment, because chlorophyll fluorescence was lost and did not mask cytoplasmic fluorescence (Pouneva 1997).Interestingly, increasing the concentrations of silver NPs have caused more decreasing in the chlorophyll content of the studied Cyanobacterium.The chlorophyll concentration also matched the results of the viability with those of Schulze et al., (2011).
In line with the present results, many studies have investigated the cytotoxic effects of metal nanoparticles against different human cancer cell lines (Rosarin et al. 2013).El-Kassas and Attia, (2014) reported the cytotoxic activity of biosynthesized silver nanoparticles with an extract of the red seaweed Pterocladiella capillacea on the HepG2 cell line.In addition El-Kassas and El-Sheekh, (2014), studied the cytotoxic activity of gold nanoparticles of on human breast cancer (MCF-7) cell line.Other investigations are necessary to improve the particle size and other necessary features of the biosynthesized nanoparticles.The results of this work provided strong evidence for the consideration of silver nanoparticles (AgNPs) as antialgal agent against the liver inducing cancer M. aeruginosa.Such positive environmental and toxicological applications will be imperative to ensure the nanomaterials design process yields both effective and safe technologies for prevention of liver cancer in human as results of the toxin excreted by M. aeruginosa.
of the biosynthesized silver NPs using S. platensis, a) C vulgaris; b) and Sc.obliquus; c) respectively EDX Monographs of the Biosynthesized Silver NPs Showing Elemental Silver in High Signals NPs Synthesized using S. platensis.a) C. vulgaris; b) and Sc.obliquus; c) respectively a b c ketone amide and the peak at 544 cm -1 may be due to C-I stretching vibration presence of Iodo compounds.Figures 4(b) and 4(c) show FTIR spectrum of silver NPs synthesized using C. vulgaris and Sc.obliquus.Stretching vibrations of proteins were observed by two clear bands.The peaks at 3390 and 3407 cm -1 correspond to Protein v (N-H) stretching (amide A), for C. vulgaris and Sc.obliquus, respectively.The other peak at 1638 cm -1 presented in the 2 spectra is due to Protein amide I band mainly v(C=O) stretching.However the peak at 2073 cm -1 may be assigned to Transition metal carbonyls.The band at 1047 cm -1 is due to Symmetric C-H Stretching vibration, presence of Antioxidant enzyme, Carbohydrate v(C-O-C) of polysaccharides.However, the peak observed at 1268 cm -1 may be assigned to the presence of C-O asymmetric C-O-C stretching presence of esters.The band at 1055 cm -1 corresponds to Carbohydrate v(C-O-C) of polysaccharides.Peaks at 560 cm -1 and 563 cm -1 represents C-I stretching vibration presence of Iodo compounds.
. The results show that using silver NPs produced by S. platensis, C. vulgaris and Sc.obliquus, have reduced the chlorophyll content of cyanobacterium by % 20.3±0.4,% 34.7±0.3, and % 41.1±0.5 respectively.Increasing the silver NPs Morphological Changes of M. aeruginosa Colonies Showing Control Cells (A) and Images of Cells Exposed to 10 mgL -1 of Silver NPs Synthesized using S. platensis, a; and C. vulgaris, b; as well as Sc.Obliquus A) Images of Cells Showing Control M. aeruginosa Cells (A) and Images of Cells Exposed to 10 mgL -1 of Silver NPs Synthesized using S. platensis, a; C. vulgaris, b; and Sc.obliquus aeruginosa Under the Stress of Different concentrations have caused more pronounced decrease in the chlorophyll content.The application of silver NPs (10.0 mgL -1 ) produced by Sc. obliquus led to the highest reduction of the total chlorophyll content of M. aeruginosa by 47.3%±0.3.