Bio-fabrication of silver nanoparticles by phycocyanin, characterization, in vitro anticancer activity against breast cancer cell line and in vivo cytotxicity

In recent decades, researchers were attracted towards cyanobacterial components which are potential low-cost biological reagents for silver nanoparticle biosynthesis. This article describes the biological synthesis of silver nanoparticles using a proteinaceous pigment phycocyanin extracted from Nostoc linckia as reducing agent. The synthesized silver nanoparticles have a surface plasmon resonance band centered at 425 nm. Face-centered central composite design used for optimization of silver nanoparticles (AgNPs) biosynthesis using phycocyanin. The maximum AgNPs biosynthesis obtained using the optimized four variables, initial pH level (10), AgNO3 concentration (5 mM), phycocyanin pigment concentration (1 mg/mL) and incubation period (24 h) was 1100.025 µg/mL. The TEM analysis of AgNPs showed spherical nanoparticles with mean size between 9.39 to 25.89 nm. FTIR spectra showed major peaks of proteins involved in AgNPs biosynthesis by identifying different functional groups involved in effective capping of AgNPs. The biosynthesized AgNPs significantly inhibited the growth of medically important pathogenic Gram-positive (Staphylococcus aureus), Gram-negative bacteria (Pseudomonas aeruginosa, E. coli and Klebsiella pneumonia). The synthesized AgNPs exhibited effective cytotoxic activity against MCF-7 and the inhibitory concentration (IC50) was recorded at 27.79 ± 2.3 µg/mL. The in vivo studies clearly indicated that AgNPs has a capacity to inhibit the growth of tumor in Ehrlich ascites carcinoma bearing mice.


Evaluation of silver nanoparticles biosynthesis by phycocyanin extracted from cyanobacterium
Nostoc linckia. Silver nanoparticles biosynthesis was carried out by using 19 ml of phycocyanin extracted from cyanobacterium Nostoc linckia treated with 1 mL (5 mM) aqueous AgNO 3. The reaction mixture was kept in light. The phycocyanin colored reaction mixture converted to brown color indicating the formation of AgNPs ( Fig. 2A,B). Conversely, no change in color observed in aqueous AgNO 3 incubated without phycocyanin under the same conditions. Formation of color is depending on the excitation of surface Plasmon vibrations of silver nanoparticles 23 . Light-assisted methods for nanoparticles synthesis are established on reducing the cation of the metal M n+ to M 0 either by direct or indirect (photosensitized) photolysis, which largely developed since 18 th century 24 . In this study, the phycocyanin chromophores which responsible for conformational change of the phycocyanin blue color initiate the photosynthesis in Nostoc linckia by the absorption of light. Chromophores can excite the molecules from the ground state to an electronic excited state after absorbing light 25 . Thus under illumination condition, the electrons which jump between energy levels can also reduce AgNO 3 in the medium to form silver nanoparticles.
UV-Visible spectral analysis. This investigation focused on the formation of AgNPs was proven using scanning UV-visible spectrophotometry in the range of 200-900 nm, over the sequence of time interval. The specific surface plasmon resonance (SPR) spectrum of silver nanoparticles produced by phycocyanin pigment revealed an absorption peak at 425 nm (Fig. 2C), this result was compatible with the result of Yadav et al. 26 . AgNPs biosynthesis using algal and cyanobacterial cells were followed by change in UV-vis absorbance peak associated with surface plasmon resonance of the AgNO 3 solution 16 . The plasmon absorption size and shape of silver nanoclusters are strongly depend on particle size, stabilizing molecules or surface adsorbed particles as well as the dielectric constant of the medium 17 . With increasing in size of silver nanoparticles in aqueous solution, surface plasmon resonance peak shifts to longer wavelengths.

Response surface methodology for optimization of silver nanoparticles biosynthesis and model
analyzing. Response surface methodology (RSM) is used to determine the optimum variables concentrations affecting silver nanoparticles biosynthesis. In this study, the face-centered central composite design (FCCD) was applied to design the experiments and evaluate the interactive effects of the four selected variables. The experiments were designed using the software, Design Expert ® 7.0 for Windows. Table 1 presents the face-cantered central composite design matrix of four independent variables X 1 (initial medium pH), X 2 (AgNO 3 concentration), X 3 (phycocyanin concentration) and X 4 (incubation period) with their actual and coded levels. Thirty experiments were performed contain 18 factorial, 6 axial and 6 central points. The six central points are found in runs order 5, 14, 19, 21, 24 and 29. A maximum biosynthesis of silver nanoparticles value was (1100.025 µg/mL) which is the mean value of runs order 5, 14, 19, 21, 24 and 29. The maximum biosynthesis of silver nanoparticles was achieved under the experimental conditions of initial medium pH level (10), AgNO 3 concentration (5 mM), phycocyanin pigment concentration (1 mg/mL) and incubation period (24 h). However, a minimum biosynthesis of silver nanoparticles values (330.046 µg/mL) was found in experiment number run order 10 under the conditions of initial pH level (12), AgNO 3 concentration (1 mM), phycocyanin pigment concentration (2 mg/mL) and incubation period (2 h). The experimental and predicted values of yields of silver nanoparticles biosynthesis are also given in Table 2.
Multiple regression analysis and ANOVA. The relationship between a set of independent variables and silver nanoparticles biosynthesis is determined by a mathematical model called multiple-regression model using Design Expert 7.0 for Windows. Statistical analysis of the response was performed, summarized and presented in Tables 2 and 3 and Supplementary Table S1. In Table 2 the positive coefficients for X 1 , X 2 , X 4 , X 1 X 2 , X 1 X 4 , X 4 2 indicate that linear effect of X 1 , X 2 , X 4 interaction effects for X 1 X 2 , X 1 X 4 and the quadratic effect of X 4 2 increase silver nanoparticles biosynthesis, while other negative coefficients indicate decrease in silver nanoparticles biosynthesis.
In order to evaluate the relationship between dependent and independent variables and to determine the maximum silver nanoparticles biosynthesis to the optimum levels of the four independent variables X 1 (initial medium pH), X 2 (AgNO 3 concentration), X 3 (phycocyanin concentration) and X 4 (incubation period), a second-order polynomial mathematical model (Eq. 1) was proposed to calculate the optimum levels of these variables and defines predicted response (Y) in terms of the independent process variables: where, Y is the predicted response (silver nanoparticles biosynthesis), X 1 is initial medium pH, X 2 is the AgNO 3 concentration, X 3 is the phycocyanin pigment concentration and X 4 is the incubation period. The coefficient of determination R 2 is a widely accepted parameter for verification of the model adequacy 27 . The adequate of the model was also expressed by the coefficient of determination (R 2 ) which was found to be 0.9736 (Table 3) indicating that 97.36% of variability in the silver nanoparticles biosynthesis could explained by the model and only 2.64% of the total variance could not be explained by the model. Box et al. 28 suggested calculating the adjusted R 2 , because it can corrects the R 2 value for the sample size and the numbers of terms of the model. The "Pred R-Squared" of 0.8250 is in reasonable agreement with the "Adj R-Squared" of 0.9489. This indicated a good adjustment between the observed and predicted values.
The analysis of variance (ANOVA) of the experimental design was calculated, and the sum of square, mean square, F-value, P-value and confidence level are shown in Table 3. The Model F-value of 39.45 implies that the model is significant where Model P-value (Prob > F) is very low which less than (0.0001). Also the probability values (P values) were used to check the significance of each of the coefficients, which, in turn, are necessary to understand the pattern of the mutual interactions between the test variables. The corresponding P values, along with the coefficient estimate, are given in Table 3. The smaller P values the more significant is the corresponding coefficient 29 . Some investigators have found that confidence levels greater than 70% are acceptable 30 . Variables at confidence levels greater than 90% (P < 0.1) were considered significant 31 . Thus, in the current experiment, variables evidencing P-values of less than 0.1 (confidence levels exceeding 90%) were considered to have significant effects on silver nanoparticles biosynthesis. In this study, the linear coefficient terms X 1 (initial medium pH) and X 4 (incubation period) affect significantly (P < 0.1) on the silver nanopaticles biosynthesis. Where the linear coefficients of X 3 (initial medium pH) is significant (P value 0.0012), indicating that 99.88% of the model affected by the level of pH and the linear coefficients terms of X 4 (incubation period) is significant (P value 0.0822), indicating that 91.78% of the model affected by the incubation period. The mutual interaction coefficient terms between X 1 (initial medium pH) and X 3 (phycocyanin concentration) with (P value 0.0641) also indicating that 93.95% of the model significantly affected by the interaction of these variables. The quadratic coefficient term of X 3 2 (phycocyanin concentration) showed (P value 0.0293) indicating that 97.07% of the model affected by the phycocyanin pigment concentration and X 2 (AgNO 3 concentration) showed (P value ˃ 0.0001) indicating that 99.99% of the model affected by AgNO 3 concentration. However, the linear coefficient terms (X 2 and X 3 ), the interaction coefficient terms between (X 1 X 2 , X 1 X 4 , X 2 X 3 , X 2 X 4 and X 3 X 4 ) and the quadric coefficient terms (X 1 2 and X 4 2 ) were found to be insignificant. "Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. "Adeq Precision" ratio of 17.5286 indicates an adequate signal to noise ratio. Also in Table 3, the value of PRESS is 503061.47; standard deviation values is 71.17 and mean value is 693.89 with C.V. value equals 10.26, which was very low value, indicating that there is an accuracy and fitness in this experimental execution 28 . The fit summary results are presented in Supplementary Table S1.  The normal probability plot is important a diagnostic tool that indicates whether the residuals follow a normal distribution, in which case the points will follow a straight line expect some scatter even with normal data. Figure 3A represents as the residuals from the fitted model were normally distributed along the silver nanoparticles biosynthesis straight line, indicating that the model had been validated. Figure 3B presents a plot of predicted versus experimental values of silver nanoparticles biosynthesis as a visual diagnostic plot indicated that, there is a close agreement between the experimental results and theoretical values predicted by the model equation, which confirms the adequacy of the model. As observed from Fig. 3C, the blue line indicates the current transformation (Lambda = 1) and the green line indicates the best lambda value (Lambda = 0.84), while the red lines indicate the minimum and maximum 95% confidence interval values (−0.08 and 1.68 respectively). Therefore, the model needs no transformation, as current value of confidence interval (λ = 1) is very close to model design value (best = 0.84) and the model is in the optimal zone since the blue line falls within the red lines. So that the model is well fit to the experimental data obtained and well satisfies the assumptions of the analysis of variance. Three-dimensional graphs were generated for the pair-wise combination of the four factors, the silver nanoparticles biosynthesis on z-axis against two independent variables while keeping the other variables are held at zero level. Figure 4A represents the effect X 1 (initial medium pH level) and X 2 (AgNO 3 concentration), while X 3 (phycocyanin concentration) and X 4 (incubation period) were held at their zero levels (1 mg/ mL and 24 h; respectively). It showed that the maximum silver nanoparticles yield appeared at alkaline initial pH. The silver nanoparticles biosynthesis increases rapidly by increasing initial medium pH level with the increase of AgNO 3 concentration until middle point, but further increase in the pH concentration led to the gradual decrease in the silver nanoparticles biosynthesis due to denaturation of the phycocyanin pigment. Heydari and Rashidipour 32 reported that the rate of AgNPs synthesis increases with increasing pH up to 9 pH then decrease. The physical factor (pH) affects on the size and the distribution of the nanoparticles. Some authers reported that the basic pH make a rapid growth rate 33 . Perfect yield and mono dispersity, size of AgNPs produced by casein milk protein were at pH > 7 ranged between 3 to 18 nm and in pH < 6 ranged between 60 to 80 nm 34 . By increasing the pH of the mixture, small spherical nanoparticles were formed under conditions of pH = 11using starch as capping agent 35 . Spherical size (7-20 nm) of AgNPs were observed in crude hot water soluble polysaccharide extracted from different marine algae with 0.1 mM of AgNO 3 in pH = 10 36 . However, at higher pH (>11 pH), silver ions will aggregated and unstable AgNPs will appear 37 . However, face-centered cubic structure with spherical shape (5-25 nm) in size with filtered aqueous extract of Caulerpa racemosa marine algae as reducing agent 38 , crystal structure with spherical shape ranged between 28-41 nm in size using ethyl acetate extract of Ulva fasciata as reducing agent 39 were obtained at neutral pH. Acidic medium also have been used in biosynthesis of AgNPs but in a little manner. Finally, alkaline pH is the perfect medium for AgNPs synthesis like in our research pH = 10 that was ratified by El-Rafie et al. 36 .
The reduction of Ag+ ions to Ag° nanoparticles was indicated by changing the color of any given solution mixture to yellowish-brown gradually increasing to deep brown color depending on both concentration of silver nitrate and the reducing agent. Authors use a wide range of AgNO 3 concentration in the biosynthesis on AgNPs ranging from 1 mM to 10 mM. 1 mM of AgNO 3 is the most common used concentration for AgNPs biosynthesis. Kathiraven et al. 38 used 1 mM of silver nitrate with algal extracts for production AgNPs. El-Naggar and Abdelwahed 40 and El-Naggar et al. 5 used 1 mM AgNO 3 in AgNPs biosynthesis by a nanofactory Streptomyces viridochromogenes and Streptomyces sp. SSHH-1E. While Bankura et al. 41 used 10 mM of AgNO 3 in AgNPs preparation using dextran as reducing agent. Figure 4B represents the effect of X 1 (initial medium pH level) and X 3 (phycocyanin concentration), while X 2 (AgNO 3 concentration) and X 4 (incubation period) were held at their zero levels (5 mM and 24 h; respectively). Maximum silver nanoparticles yield appeared at alkaline pH. The silver nanoparticles biosynthesis increases gradually by increasing phycocyanin concentration with increasing of initial medium pH. Then AgNPs gradually decreased at higher phycocyanin concentration.
AgNPs can obtained neither by grams of wet weight biomass of algal or cyanobacterial culture or cell-free culture liquid that suspended in mL(s) of 1 mM aqueous AgNO 3 , but in case of extracts like polysaccharides or phycobiliproteins, dry weight dissolved in mL (s) of deionized water then suspended in mL (s) of 1 mM aqueous AgNO 3 16 . Figure 4C represents the effect of X 1 (initial medium pH level) and X 4 (incubation period), while X 2 (AgNO 3 concentration) and X 3 (phycocyanin concentration) were kept at their zero levels (5 mM and 1 mg/mL; respectively). It showed that the maximum silver nanoparticles yield appeared at alkaline medium pH. AgNPs biosynthesis increase with increasing incubation period and the initial medium pH, then decreasing of AgNPs occurred due to agglomeration of AgNPs at increased.
Biosynthesis of silver nanoparticles using Ulva fasciata ethyl acetate extract starts immediately after 2 min 39 42 . Figure 4D represents the effect of X 3 (phycocyanin concentration) X 2 and (AgNO 3 concentration), while X 1 (initial pH level) and X 4 (incubation period) were kept at their zero levels (10 and 24 h; respectively). It showed that the maximum silver nanoparticles yield clearly situated close to the central point of both AgNO 3 concentration and phycocyanin concentration. Figure 4E describes the effects of X 4 (incubation period) and X 2 (AgNO 3 concentration) on AgNPs biosynthesis using phycocyanin pigment, when X 1 (initial medium pH level) and X 3 (phycocyanin concentration) were kept at their zero levels (10 pH and 1 mg/mL; respectively). It showed that the maximum silver nanoparticles yield clearly situated close to the central point of both incubation period and AgNO 3 concentration. Shameli et al. 43 reported that the increasing in the time of reaction resulted in gradual increase in the intensity of the surface plasmon resonance peak until 24 h but after 48 h the surface plasmon resonance peak change to broad shape and intensity decreased, this phenomenon is related to the increased size and also agglomeration of silver nano-crystals. Figure 4F the effect of X 3 (phycocyanin concentration) and X 4 (incubation period) on AgNPs biosynthesis when X 1 (initial medium pH level) and X 2 (AgNO 3 concentration) were kept at their zero levels (10 pH and 5 mM; respectively). Analysis of Fig. 4F clearly suggests that the increasing of phycocyanin concentration with increasing incubation period, AgNPs biosynthesis increase till middle levels, however increase in phycocyanin concentration leads to decrease in AgNPs biosynthesis. Verification of the model. For determination accuracy of the model and verifying the optimal concentrations of the factors obtained from the optimization experiment, experiments were repeated in triplicates under the optimized conditions and compared with the predicted data. The experimental AgNPs was 1100.025 µg/mL, where the predicted value by the polynomial model was 1064.032 µg/mL. The verification revealed a high degree of accuracy of the model of more than 96.72%, indicating the model validation under the tested conditions. The optimized conditions of the variables for silver nanoparticles biosynthesis by using phycocyanin obtained from fitted model were initial pH level (10), AgNO 3 concentration (5 mM), phycocyanin pigment concentration (1 mg/ mL) and incubation period (24 h).
Characterization of AgNPs synthesized using phycocyanin pigment extracted from Nostoc linckia. Energy-dispersive X-ray (EDX) spectroscopy. The sample powder of AgNPs was compressed to form tablets before analysis with EDX that assured that silver is the basic constituent element (Fig. 5). The crystal silver SCIEntIFIC REPoRTS | 7: 10844 | DOI:10.1038/s41598-017-11121-3 nanoparticles show typical optical observation peak approximately at 3 keV due to surface plasmon resonance and this result was compatible with Hebeish et al. 44 . Additionally, O and C elements peaks were observed. Peaks for Na, P, K, CL, Si, Cu and Zn were also observed.
X-Ray Diffraction (X-RD). X-ray diffraction is now a common technique for the study of crystal structures and atomic spacing. The interaction of the incident rays with the sample produces constructive interference and a diffracted ray when conditions satisfy Bragg's Law (nλ = 2d sin θ). The freeze-dried AgNPs was drop-coated using an X-ray powder diffractometer (Philips X'pert Pro, Panalytical) onto silica plate by applying many layer of small amount of samples on the plate with intermittent drying. The average size of AgNPs was estimated by the use of full width at half maximum (FWHM) of face-centered cubic (111) using the Debye-Scherrer equation, K λ/β cos θ, where K is the Scherrer constant with value from 0.9, λ is the wavelength of the X-ray, β is the full width at half maximum and θ is the Bragg angle in radians. XRD pattern for the silver nanoparticles is shown in Fig. 6, four well resolved diffraction peaks are observed. Diffraction features appearing at 2 theta (degree) as 37.9°, 44.3°, 64.5° and 77.3°, which correspond to the 111, 200, 220, and 311 planes of face centered cubic silver corresponding to the standard fcc structure; respectively, of silver is observed and compared with the standard powder diffraction card of Joint Committee on Powder Diffraction Standards (JCPDS), silver file No. 04-0783. Presence of these four intense peaks corresponding to the nanoparticles was in agreement with the Bragg's reflections of silver identified with the diffraction pattern. The pattern of XRD suggests that the silver nanoparticles were essentially in the face centered cubic (fcc) structure and crystal in nature. The diffraction peaks observed for the silver nanoparticles indicates that these are crystalline in this size range. The XRD pattern of AgNPs (Fig. 6) comprised  an amorphous peak and several crystallization peaks. AgNPs crystallite size determined from the highest intense refection (111) was found to be 3.24 nm.
Transmission electron microscopy. Transmission electron microscopy (TEM) is a direct and effective approach to determine the particle size and morphology of the biosynthesized AgNPs, besides their dispersion uniformity. TEM was used for getting important information on primary nanoparticles size and morphology 45 . Transmission electron microscopy analysis of the biosynthesized AgNPs revealed the formation of spherical nanoparticles with a size range of 9.39 to 25.89 nm (Fig. 7). AgNPs is predominantly spherical in shape through microbial mediated synthesis 46 .
Fourier transformed infrared (FTIR) spectroscopy analysis. FTIR spectrum used for investigation of the functional groups and Ag nanoparticles formation mechanism particularly to identify possible interaction between silver precursor salt and protein molecules, leading the reduction of silver ions and stabilization of silver nanoparticles 47 . FTIR spectrum (Fig. 8) shows different 14 peaks positions at 3474, 3417, 3304, 3121, 1654, 1262, 1102, 985, 861, 769, 660, 599, 513 and 420 cm −1 . The band at 3474 cm −1 could be assigned to N-H stretch found in aromatic amines, primary amines and amides 48 , and the peak at 3417 cm −1 attributed to O-H stretching vibrations of phenol and alcohol 49 . The band at 3304 cm −1 attributed to -NH stretch present in polysaccharide and protein, ≡CH-H stretch in acetylene and O-H in oximes. The band at 3121 cm −1 assigned to NH 3 + antisym stretch in amino acids present in extract 50 . The FTIR band at 1654 cm −1 assigned to C=O stretch with N-H deformation found in primary amides II, C=O stretch in carbonyl groups 51 . The peak at 1262 cm −1 assigned to the presence of C-N stretching vibration of aromatic amines 52 . In addition, the presence of bands at 1102 cm −1 assigned to C=S    stretching in thiocarbonyl compounds. It was shown a peak in the range of 985 cm −1 relating to C-N stretching vibration of primary amines that referring to possible involvement of primary amines during nanoparticles synthesis 53 The peaks at 599 and 513 cm −1 assigned to C-C-CN nitrites, C-C=O bend ketones respectively. The FTIR band at 420 cm −1 assigned to CL-C=O in plane deformation of acid chlorides 50 . Thus, these biomolecules may be responsible for capping and efficient stabilization of synthesized nanoparticles. Theivasanthi et al. 47 reported that proteins can bind to AgNPs through free amine groups of proteins which is compatible with our results.
FTIR spectrum supports the presence of a type of a protein on the surface of biosynthesized AgNPs, confirming that metabolically produced proteins acted as capping agents during production and prevented the reduced silver particles agglomeration. It has been suggested that stability of the AgNPs could be due to the presence of a proteinaceous capping agent that encapping and encapsulating them and forms a layer which protects the nanoparticles from agglomeration 54 .
The surface zeta potential distribution of silver nanoparticles. In the research under study, zeta potential measurement ( Fig. 9) is −31.8 mV at standard deviation 5.37 mV with conductivity 0.420 mS/cm. The biosynthesized AgNPs are consider strongly anionic which is important in biological application according to Aiad et al. 55 . Typically, nanoparticles with zeta potentials greater than 20 mV or less than −20 mV have sufficient electrostatic repulsion to remain stable in solution. Salehi et al. 56 suggested that the greater negative surface charge potential (−31 mV) value may be attributed to the effective functional constituents as capping agents present in the ethanol extract of Artemisia marschalliana Sprengel aerial part extract.

Applications of the synthesized AgNPs. Antimicrobial activity of synthesized AgNPs. The antimicrobial
activity of silver nanoparticles produced by using phycocyanin pigment was evaluated against bacterial species: Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumonia and E. coli) by the disc-diffusion method (Fig. 10). A control (5 mM AgNO 3 ) was also maintained in each plate. The diameter of inhibition zones around each disc is measured. The highest antibacterial activity was observed against E. coli which was similar to the antibacterial activity to Pseudomonas aeruginosa (10 mm) whereas a low activity was found against Klebsiella pneumonia (9 mm), which also similar to the antibacterial activity against Staphylococcus aureus. This result is compatible with previous studies that examined the antimicrobial activity of AgNPs against Staphylococcus aureus 57 . Biosynthesized silver nanoparticles showed significant antibacterial effect on both Gram classes of bacteria. There are many ways for AgNPs to interact with microorganisms and damage them. Silver ions released from silver nanoparticles when come in contact with bacterial cells may deactivate the production of some enzymes and cellular proteins necessary for adenosine tri-phosphate (ATP) synthesis or influence on the bacterial DNA replication functions 58 . In other researches, silver ions may disrupt the working of membrane-bound enzymes of the respiratory chain 59 . The antibacterial activity of silver nanoparticles are affected by the particles size and the smaller the particles, the greater the antibacterial efficacy 60 . Other studies showed that the bacterial damages by rupture of plasma membrane or by blocking respiration in association with oxygen and sulfhydryl (S H) groups on the cell wall to form R S S R bonds thereby, leading to exhaust of intercellular ATP and cell death 61 . It is reported that the electrostatic interaction between the positive Anti-hemolytic assay. Erythrocyte hemolysis is the disruption of the integrity of its membrane that causes releasing of haemoglobin into the surrounding blood plasma. Erythrocytes are oversensitive to oxidative disruption due to membrane high poly-unsaturated fatty acid content and high concentration of oxygen and hemoglobin which are important promoter of oxidative processes 63 . The free radical can produce lipid peroxyl radicals which attack the lipids membrane and converted into lipid hidroperoxides leading to adverse effects on membrane structure and function 64 . AAPH is consider a soluble hydrophilic azo-compound at which generates 2-amidinopropyl radicals called C-radicals through a process depending on specific temperature. These C-radicals reacts with oxygen molecules (if present) and generate peroxyl radicals and hemolysis increases by time with increasing the incubation period of erythrocyte with AAPH 65 .
The addition of AgNPs to erythrocyte exposed to the free radicals of AAPH exhibited low erythrocyte hemolysis of 7.8% (i.e AgNPs exhibit anti-hemolytic activity with 92.2% inhibition) which was very low as compared with complete (100%) erythrocyte hemolysis which occurs at the incubation of erythrocyte and AAPH without AgNPs. AgNPs can prevent the oxidative damage in healthy erythrocytes. Standard ascorbic acid has showed percentage erythrocyte hemolysis of 4.0%. The proposal anti hemolytic activity of AgNPs is due to the presence of O-H group present in phenolic compounds or Ar-OH or -NH 2 in aromatic amines compounds surrounded the Ag ion possess an important antioxidant activity which prevent oxidation by the free radicals 66 . Mallesha et al. 67 proposed that the anti-hemolytic activity of AgNPs is that AgNPs include -NH 2 in aromatic amines, primary amines and amides which is the functional group of amine compound that exhibit anti-hemolytic activity with 58% inhibition compared to standard quericitin with 74% inhibition in the synthetic compound (4-nitro-benzylidene)-pyridin-3-ylmethyl amine.
In vitro anti-cancer activity. Silver nanoparticles have earned a great reputation in the field of nanomedicine due to their unique properties which obvious therapeutic potential in the diagnosis and treatment of some human cancer types 68 . In the research under study, the cytotoxic effect of silver nanoparticles against mammary gland   Table 4. Effect of AgNPs and 5-FU on hematological parameters, tumor parameters (volume and cell count) and body weight of EAC bearing mice.
breast cancer (MCF-7) cell line and cytotoxicity (%) was carried out in vitro by MTT assay and compared with the standard 5-5-5-fluorouracil that commercially available as anticancer drug. Comparison of the cytotoxicity of synthesized AgNPs (1.56, 3.12, 6.25, 12.5, 25, 50, 100 µg/mL) and similar concentration in 5-FU disclosed similar mortality rate. Cytotoxicity rate against MCF-7 cell lines increases with increase in concentration of silver nanoparticles (Fig. 11). The inhibitory effect was observed after 48 h of incubation. The results were exhibited as growth inhibitory concentration (IC 50 ) values, which represent the compounds concentrations required to produce a 50% inhibition of cell growth after 48 h of incubation, compared to untreated controls. The IC 50 of cell inhibition of silver nanoparticles was observed at 27.79 ± 2.3 µg/mL of AgNPs against the breast cancer MCF-7 cell lines. The IC 50 of the cell inhibition of silver nanoparticles against human lung fibroblast (WI38) and human amnion (WISH) cell lines were observed at 31.78 ± 2.2 and 32.97 ± 1.7 µg/mL respectively. So the produced AgNPs exhibit stronger cytotoxic effect on the breast cancer MCF-7 than WI38 and WISH normal cell lines. Fig. 12. AgNPs uptaken by tumor cells (MCF-7) are catabolized forming amino acids and Ag ions 69 . The cytotoxic properties of the nanoparticles due to the release of Ag+ cations which interact with cells and intracellular macromolecules like proteins and DNA. AgNPs have shown to cause DNA damage 70 and increased mitochondrial membrane permeability 71 .Cellular uptake of nanoparticles which subsequently captured free electrons and increased the synthesis and accumulation of intercellular reactive oxygen species (ROS) which reacts with protein and cause oxidative stress 72 . This process leads to the partial or permanent loss of structure and/or function of cellular protein. It further reduced adenosine triphosphate (ATP) production 73 . In addition to enhancing the generation of potentially damaging radicals, AgNPs have also been shown to negatively regulate the activity of DNA-dependent protein kinase, a key enzyme involved in DNA damage repair via nonhomologous end joining 74 . Cell damage by silver nanoparticles may be due to loss of cell membrane integrity, apoptosis and oxidative stress.

Proposed mode of action of AgNPs on MCF-7 cells. Schematic representation of the proposed mode of action of AgNPs on MCF-7 cells was demonstrated in
The differential response of breast cancer cells to AgNPs induced hyperthermia, which implies AgNPs to be effective photothermal agents 75 . Apoptosis could be activated through mitochondrial dysfunction, which potentially inhibits the proliferation of MCF-7 cells 76 . Ranjitham et al. 77 studied in vitro cytotoxicity of the AgNPs against MCF-7 breast cancer cell line at different concentrations. The samples showed a considerable cytotoxicity against the MCF-7cell line. It was reported that the toxicity of AgNPs increase with increase in concentration.
The results clearly indicate that AgNPs has a capacity to inhibit the growth of tumor induced by EAC cell line in the experimental animals. The morphology of AgNPs formed by phycocyanin pigment extracted from Nostoc linckia showed that the size range is 9.39 to 25.89 nm. These small sizes obtained silver particles have capability of distribution and accumulation in tumor cells after its injection at the site of tumor. Devi et al. 78 reported that the cytotoxic effect is inversely proportional to the size of AgNPs.
Once a tumor grows to a certain size, it needs nutrients and oxygen from the blood to grow and spread. The tumor sends chemical signals that stimulate the growth of new blood vessels that carry the blood to it, growth and metastasis of tumor depend on angiogenesis 79 . Cancer treatments with AgNPs block angiogenesis, limited angiogenesis occurs after AgNPs treatment. The tumor starved and dies if it cannot get enough nutrients and oxygen. Martins et al. 80 reported that compounds having anti-angiogenic properties are known for their ability to block abnormally expressing signaling proteins. Antony et al. 79 reported that anti-angiogenesis occurs after AgNPs treatment of DAL tumor cells. Also, AgNPs have a cytotoxic effect on EAC bearing mice by production of lipid peroxidation and free radicals in tumor tissues that proved by El Bialy et al. 81 .
Treatment with AgNPs at the dose of 5 mg/kg body weight significantly inhibited the tumor cells volume, tumor cells count and tumor weight. In case of control group, a regular rapid increase in ascetic tumor volume was observed. Ascitic fluid is the direct nutritional source for tumor cells and a rapid increase in ascetic fluid with tumor growth would be a means to meet the nutritional requirement. It may be concluded that AgNPs decreases the nutritional fluid volume and arresting the tumor growth. Thus AgNPs possess antitumor activity against EAC bearing mice. Body weight of Ehrlich ascites carcinoma bearing mice inoculated with AgNPs was observed to be decreased compared to Ehrlich ascites carcinoma bearing mice which could be positively correlated with the ascites fluid volume 79 . Anemia is the main problems in cancer treatment which encountered in tumor bearing mice is mainly due to reduction in RBC or haemoglobin percentage, and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions 82 .

Materials and Methods
Organism and culture conditions. The investigating cyanobacterium, Nostoc linckia, was previously collected from garden soil sample in Dakahlia, Egypt. One inoculum of alga was cultured in BG-11 medium 83 . The fresh culture of the alga was prepared in 500 mL Erlenmeyer flasks containing 250 mL of BG-11 medium and inoculated with 10 mL of 14 days old culture. The flasks were incubated at 25 ± 2 °C for 28 days under illumination (600-800 lux) by 36 W white fluorescent lamp to achieve a maximum grow rate. Collecting all the fresh biomass from the medium for being freeze drying by Freeze dryer (SIM international, USA, FD8-8T).

Extraction of phycocyanin.
Phycocyanin is pigment-protein complexes from the light-harvesting pycobiliprotein. It was extracted by adding 100 mg of freeze-drying biomass to a solution of 100 mL phosphate buffer (pH7) and successive freezing and thawing process was applied for about one week to complete extraction of the pigment. The extract was filtered to remove cell depresis. Extracellular synthesis, precipitation and centrifugation of AgNPs. 1 mL of AgNO 3 solution (5 mM) was added to 19 mL of phycocyanin pigment extracted from Nostoc linckia. pH was adjusted to pH 10 using NaOH (1 M) and incubated within closed system under direct illumination (2400-2600 Lux) for 24 h at room temperature. The biosynthesis of AgNPs was indicated by color change to dark brown. After incubation period, the bio reduction reaction was monitored by visual color change and UV-visible absorbance of the reaction mixture in the range of 200-800 nm on UV-Vis spectrophotometer (ATI Unicam 5625 UV/VIS Vision Software V3.20). AgNO 3 solution was maintained as a control. AgNPs were obtained by centrifugation (MIKRO 120 Hettich Zentrifugen D-78532 Tuttlingen Germany) at 10,000 g for 10 min. The supernatant was discarded and then the precipitated AgNPs were washed with sterile deionised water for 10 times for complete purification. After washing, the precipitated AgNPs were freeze-dried to obtain the dry AgNPs used for both characterization and applications.

Face-centered central composite design (FCCD).
In this study, the effect of four process variables namely initial pH level (X 1 ), AgNO 3 concentration (X 2 ), phycocyanin pigment concentration (X 3 ) and incubation period (X 4 ) on biosynthesis of silver nanoparticles was studied and optimized using face-centered central composite design (FCCD). FCCD is an effective design that is used for sequential experimentation and provides reasonable amount of information for testing the goodness of fit and does not require large number of design points thereby reducing the overall cost associated with the experiment 84 . In this study, the experimental plan consisted of 30 trials and the independent variables were studied at three different levels, low (−1), middle (0) and high (+1). The center point was repeated six times in order to evaluate the curvature and the experiment replication facilitated the pure error estimation, so that the significant lack of fit of the models could be predicted. All the experiments were done in duplicate and the average of AgNPs obtained was taken as the dependent variable or response (Y). The experimental results of FCCD were fitted via the response surface regression procedure using the following second order polynomial equation: In which Y is the predicted response, β 0 is the regression coefficients, β i is the linear coefficient, β ii is the quadratic coefficients, β ij is the interaction coefficients), and X i is the coded levels of independent variables.
However, in this study, the independent variables were coded as X 1 , X 2 , X 3 and X 4 . Thus, the second order polynomial equation can be presented as follows: Energy-dispersive X-ray (EDX) spectroscopy analysis. Energy dispersive X-ray analysis (EDX) was carried out with the scanning electron microscope (Oxford X-Max 20) with secondary electron detectors at an operating voltage of 20 kV at Electron Microscope Unit, Mansoura University, Mansoura, Egypt.
X-ray diffraction analysis (XRD). X-ray diffraction analysis was carried out by using an X-ray diffractometer (Philips X'pert Pro, Panalytical) having CuKa (k = 1.54 A°) radiation and a programmable divergence slit. The voltage and current of the X-ray source were 40 kV and 20 mA; respectively. The sample was drop-coated onto silica plate by applying many layer of small amount of biosynthesized AgNPs on the plate with intermittent drying. This leads to a thick coat of AgNPs to be examined with monitoring the diffraction angle from 5° to 80° (2θ).
Transmission electron microscopy (TEM) analysis. TEM produce a high-resolution, two-dimensional image which utilizes energetic electrons to provide morphologic, compositional and crystallographic information on the biosynthesized AgNPs. The morphology and size of the AgNPs synthesized using phycocyanin extracted from Nostoc linckia was visualized using ultra high resolution transmission electron microscope (JEOL-JEM-100 CXII instrument) operating at an accelerating voltage of 200 kV. Samples were prepared by drying a drop of the washed AgNPs dispersion onto the carbon-coated copper grid and dried under infrared lamp prior to examination at Electron-Microscope-Unit of Mansoura University, Egypt.
Fourier-Transform Infra-Red (FTIR) spectroscopy analysis. The synthesized AgNPs sample was freeze dried and diluted with potassium bromide (in the ratio of 1:100) to make a pellet. The FTIR spectrum of sample was recorded on a FTIR instrument (Thermo Scientific Nicolet iS10 FT-IR spectrometer) At Spectrum Unit of Faculty of Science, Mansoura University, Mansoura, Egypt. The measurement was carried out in the range of 500-4000 cm −1 at a resolution of 4 cm −1 .
The surface zeta potential distribution of silver nanoparticles. Laser Doppler Micro-electrophoresis is the technique used to measure zeta potential and zeta size. The particle size distribution and surface charge of AgNPs were determined using particle size analyzer (Zeta sizer nano ZS90, Malvern Instruments Ltd., U.K.) at 25 °C with 90° detection angle at Electron Microscope Unit of Mansoura University, Mansoura, Egypt.

Applications of the synthesized AgNPs. Antimicrobial activity of AgNPs by disk-diffusion
method. Antimicrobial activity of AgNPs biosynthesized by phycocyanin was tested against bacterial pathogens of Gram-positive (Staphylococcus aureus), Gram-negative bacteria (Pseudomonas aeruginosa, E. coli and Klebsiella pneumonia) using disk-diffusion method on nutrient agar plates. Sterile filter paper discs (6 mm in diameter) were saturated with 20 µl (1000 µg/mL) AgNPs solution, disc saturated with 5 mM AgNO 3 solution was used as control. The plates were incubated at 37 °C for 24 h and then were examined for the presence of inhibition zones. The diameters of inhibition zones around the discs were measured and the mean value for each organism was recorded and expressed in millimeter.
Anti-hemolytic activity of AgNPs. The anti-hemolytic activity of AgNPs was determined using blood obtained from rats by cardiac puncture and collected in heparinized tubes. Erythrocytes were detached from the buffy coat and plasma and washed three times with 10 volumes of 0.15 M NaCl. During the last washing, the erythrocytes were centrifuged at 2500 Xg for 10 min to obtain a constantly packed cell preparation. Erythrocyte hemolysis was mediated by peroxyl radicals in this assay system 85 . A suspension (about 10%) of erythrocytes in pH 7.4 phosphate-buffered saline (PBS) was added to the same volume of 200 mM 2,2ʹ-azobis (2-amidinopropane) dihydrochloride (AAPH) solution (in PBS) containing samples of AgNPs to be tested. While the reaction mixture has incubated at 37 °C for approximately 1 h, it was shacked gently. The reaction mixture was then removed, diluted with eight volumes of PBS and centrifuged at 2500 Xg for 10 min. The absorbance A (AgNPs) of the supernatant was read at 540 nm. Similarly, the reaction mixture was treated with eight volumes of distilled water to achieve complete hemolysis, and the absorbance B (dist. H 2 O) of the supernatant obtained after centrifugation was measured at 540 nm. The hemolysis percentage was calculated by equation (1−A/B) X100%. The data were displayed as mean standard deviation. Vitamin C (L-ascrobic) was used as a positive control.
In vitro cytotoxic effect of AgNPs on cancer and normal cell lines. The cytotoxic effect of AgNPs on Mammary gland breast cancer (MCF-7), human lung fibroblast (WI38) and human amnion (WISH) the cell lines which were obtained from ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt was determined using in vitro colorimetric technique 3-(4, 5-Dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay which based on being split by NAD-dependent mitochondrial dehydrogenase of viable cells, resulting in purple color of the formazan product. This formazan production is directly proportional to the number of viable cell and inversely proportional to the cytotoxicity degree 86 . The cells were cultured in RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C in a 5% CO 2 incubator. The monolayer cells were detached with trypsin-ethylene diamine tetra acetic acid (EDTA) to make single cell suspensions. The viable cells were counted using a hemocytometer. The cell suspensions (100 µL/ well) were seeded onto 96-well plates, maintaining the plating density as 10,000 cells/well, and then incubated for 48 h at 37 °C, 5% CO 2 , 95% air and 100% relative humidity for cell, attachment to the bottom of the wells. The cells were treated with serial concentrations of the AgNPs for 24 h. The AgNPs were previously passed through a 0.45 µm filter syringe. Serial dilutions were made to provide a total of seven samples concentrations. Various concentrations (1.56, 3.125, 6.52, 12.5, 25, 50, 100 µg/mL) of silver nanoparticles were inoculated into grown cell containing 100 µL medium. The plates were incubated for 24 h at 37 °C, 5% CO 2 , 95% air and100% relative humidity. Various concentrations (1.56, 3.125, 6.52, 12.5, 25, 50, 100 µM) of 5-fluorouracil (5-FU) were used as standard. The study was run in duplicate to ensure accuracy of the results. The yellow solution of 3-[4, 5-dimethylthiazol-2-yl] 2, 5-diphenyltetrazolium bromide (MTT) (20 µL) was added to phosphate buffered saline (5 mg/mL) in each well. The plates were incubated at 37 °C for 4 h for the reduction of MTT. The resulting purple formazan crystals were solubilized in 100 µL of DMSO and the colorimetric assay is measured and recorded at absorbance of 570 nm using a plate reader (EXL 800, USA). Cytotoxicity percentage was determined using the following equation: Viability % (Test OD/Control OD) 100 Cytotoxicity % 100 Viability% (4) In vivo cytotxicity of AgNPs on Ehrlich ascites tumor. Ethics statement. All experimental protocols were approved by Research Ethics Committee, Faculty of science, Mansoura University, Mansoura, Egypt. All the experiments were performed in accordance with the relevant guidelines and regulations. Albino adult Swiss male mice (20-25 g) were obtained from Pharmacology Department, Mansoura University, Egypt and used throughout the study. They are housed in a controlled environment (temperature 25 ± 2 °C) and 12 hours dark/light cycle) with standard laboratory diet and water 'ad libitum' . The Ehrlich ascites carcinoma (EAC) cells were provided by National Cancer Institute (NCI), Cairo, Egypt. EAC cells were diluted with normal saline (0.9% NaCl) to reach the desired concentration (2 × 10 6 cells/0.2 mL).
The mice were divided into four groups comprising five animals in each group. Group I represents negative control -received vehicle (normal saline) with no EAC cells; group II served as tumor control injected with EAC (2 × 10 6 cells per mouse); group III represents EAC bearing mice inoculated with 5-FU; group IV represents EAC bearing mice inoculated with AgNPs. So all groups were injected intraperitoneally with 2 × 10 6 cells per mouse except group I. After 24 h, mice were injected with the anti cancer 5-FU and AgNPs (5 mg/kg body weight). The inoculation process was repeated for 10 days. After 24 h of the last dose including 18 h of fasting half of the mice in each group were weighed and sacrificed. Blood samples were collected from each group of sacrificed mice for the estimation of hemoglobin (Hb) content and counts for red (RBC) and white blood cells (WBC) were performed. Tumor cells volume and their count were determined by collecting the ascetic fluid from the peritoneal cavity of the mice. The count of viable cells and non-viable cells was estimated by taking a part of the ascetic fluid then centrifugated and stained with trypan blue (0.4% in normal saline) and the other part was centrifuged in a graduate centrifuge tube at 1,000 rpm for 5 min and the packed cell volume was measured. The increase of mice body weights were recorded both in the treated and control groups at the start of the experiment to the end (0 to 11 days).

Conclusion
A biological method has been reported for the synthesis of silver nanoparticles using the proteinaceous pigment phycocyanin which is plentiful component of cyanobacterial cells. Phycocyanin extracted from Nostoc linckia was successfully used for biosynthesis of AgNPs. The face-centered central composite design has been used for the optimization of factors affecting AgNPs biosynthesis for attaining the maximum production of AgNPs. After statistical optimization by face-centered central composite design, the biosynthesis of silver nanoparticles by using phycocyanin extracted from Nostoc linckia was improved (1100.025 µg/mL) with fold of increase 2.6 as compared with that of unoptimized conditions (508.346 µg/mL). The physicochemical characterization viz. UV-Vis