Synthesis and Characterization of Silver Nanoparticles Using Prodigiosin Pigment and Evaluation of Their Antibacterial and Anti-Inflammatory Activities

This study focused on the biological synthesis of silver nanoparticles (AgNPs), using prodigiosin pigment produced by Serratia marcescens. The effect of parameters such as pH, temperature, time, with various concentrations of silver nitrate (AgNO3) and prodigiosin on the synthesis of AgNPs were also studied. Optimized results of the biosynthesis process revealed an increase in the intensity of Surface Plasmon Resonance (SPR) bands of nanoparticles with shifting at the wavelength of 400 nm. In addition, optimum synthesis of AgNPs was achieved at pH 12, temperature 55°C, and reaction time 24 h, with concentrations of prodigiosin, as a reducing agent, of 12.5 μg/ml and silver ion concentration of 1 mM. Measurement of the size of silver nanoparticles by SEM diffraction revealed a value of 30 nm. Finally, the minimum inhibitory concentration of AgNPs against pathogenic bacteria was 32 μg/ml for Staphylococcus aureus and Pseudomonas aeruginosa. The results of anti-inflammatory effects of Ag NPs obviously demonstrated that the infections of test animals treated with AgNPs were completely healed after 4 days of treatment, while the animals treated with fucidin (as control) did not exhibit any healing.

removed, the cell pellet was suspended in 95 percent methanol, and the extract was treated for 3 minutes using an ultrasonic homogenizer until the solution became colorless. The mixture was vortexed and centrifuged for 15 min at 10,000 rpm. By using a Whatman filter paper of 45 μm, the extract then was filtered and transferred to a conical flask for methanol portion evaporation using a rotary evaporator. The dry weight was determined and the extract was stored in a glass tube covered with aluminum foil. Synthesis of silver nanoparticles using prodigiosin A concentration of 5 μg/ml of prodigiosin powder was mixed with deionized water (DI), then the aqueous silver nitrate (AgNO3) solution (1 mM) was added. The volume was ultimately completed to 20 ml with DI. The reaction took place in 24 hrs. at 25 ° C.

Optimized conditions for nanoparticles synthesis Initial pH
Silver nanoparticles were synthesized in different pH values (5, 7, 9, 11 and 12). The synthesis was performed using prodigiosin pigment with AgNO 3 particles as mentioned above. The pH value was set with 0.1N HCl and 0.1N NaOH.

Temperature
The effects of different temperature degrees on AgNPs synthesis were tested. The temperature values were 15, 25, 35, 45, and 55℃. The production of silver nanoparticles was performed at an optimum pH as shown in a previous step.

Effect of reaction time on synthesis of AgNPs
The effect of reaction time for AgNPs synthesis was studied for 24 hrs. at regular intervals of 30 min. The shifting of color of the reaction mixture to dark brown indicated the formation of AgNPs, which was supported by UV -VIS spectrophotometry [14].

Effect of silver nitrate concentration
Various concentrations of AgNO 3 , ranging from 1 to 1000 mM, in test tubes containing 5 μg / ml prodigiosin were used to determine the effect of AgNO 3 concentration on the synthesis of AgNPs. The reactions were incubated at optimal temperature and pH for an optimal time that was determined from the previous steps. Formation of AgNPs was confirmed by UV-visible spectrophotometry [15].

Effect of prodigiosin concentration
The effect of different concentrations of prodigiosin (1, 3, 6, 12.5, 25, 60, 125, 250, and 500 μg / ml) on AgNP output was evaluated using AgNO 3 at its optimum concentration and at the optimum pH, temperature, and reaction time, as calculated in the previous experimental steps.
Characterization of AgNPs UV -visible spectroscopy was used to confirm the formation of silver nanoparticles. Furthermore, sample patterns were determined by using XRD. FT -IR measurements were recorded to identify the major functional groups. Atomic force microscopy (AFM) for the determination of AgNPs surface morphology.

Determination of minimum inhibitory concentration (MIC)
MIC is the lowest concentration which inhibits the bacteria's visible growth. AgNPs MIC values were determined against two antibiotic resistant isolates, Staphylococcus aureus and Pseudomonas aeruginosa. These isolates were tested against many antibiotics and their resistance to many antibiotics was observed. A stock solution was prepared using different concentrations of AgNPs, ranging from 2-1024 μg / ml. AgNPs were dissolved in Mueller-Hinton broth and transferred into a microtiter plate. Overnight bacterial culture (50 μl) was performed and the size of bacterial culture was adjusted to 0.5. McFarland standard was added to each well with the exception of the negative control well which contained Mueller-Hinton broth. Wells that contained only bacterial cultures were considered as positive control. The microtiter plate was incubated overnight at 37°C. After that, 10 μl of Alamar blue added to each well and the culture was incubated again for 24h. The wells which showed no color change were considered as having the MIC that inhibits bacterial growth [16].

Anti-inflammatory activity of AgNPs Preparation of AgNPs cream
The cream was prepared according to Atwan and Hayder [17] using the following ingredients: 25g of vaseline to collect components and protect the mixture from drying, 25mg of methyl paraben (0.1%) as preservative, 1.25ml of glycerol (5%) as a moisturizer and to prevent the skin from becoming dry, rough, scaly, itchy, and irritated. All the above components were mixed with the synthesized AgNPs at the concentration of MIC determined from the previous step. These ingredients were mixed and kept in the refrigerator until use.

Anti-inflammatory activity test in vivo
Laboratory animals (Rabbits) were used for the in vivo anti-inflammatory test. The animals were divided into two groups: control (A) and test (B) groups. For infecting the animals, both groups were subcutaneously injected with 1 ml of the overnight activated clinical pathogen S. aureus and P. aeruginosa, with OD = 0.5. The animals were left for 48 hrs. for the appearance of pus, cracking, and finally inflammation of the skin. Thereafter, the inflammation test carried out by the daily wiping of the tested animals with the synthesized AgNPs cream until the healing of inflammation. Control animals were treated using Fucidin ointment (LEO pharma, Ireland) instead of the synthesized AgNPs cream.

Results and Discussion Identification of the prodigiosin producing isolate
In this analysis carried out by the presence of intracellular shiny red pigmentation, the morphological characteristics of Serratia isolates appeared with colonies ( Figure-1). Many marked signs appeared, such as the rod shape of cells under microscopic inspection and Gram negative staining. The ability of the bacterial isolate for prodigiosin production was confirmed by using VITEK2 compact system based on biochemical tests. The isolate exhibited 95% similarity with the genus Serratia marcescens.

Production and quantification of prodigiosin
In this test, the optimized physical factors which are used to increase the output of prodigiosin were modified according to [18]. These included temperature (30 rpm) pH (7) and shaking rate in the shaker incubator (150 rpm). The incubation was performed in dark since S. Marcescens typically develops in a dark environment and produces more intracellular prodigiosin to avoid phototoxicity. The maximum prodigiosin concentration that was produced from the isolate was 0.51 g/L, which occurred at 48 hrs. of incubation, when the cells reached their early stationary phase.
Cang et al. [19] notified that the maximum prodigiosin manufacturing was observed at 48 h and that it was completed by 72 hrs. Also, another study noticed that growth and manufacturing of prodigiosin starts after 48hrs. of incubation and thereafter the production starts to decrease [20]

UV-Visible analysis of AgNPs
The synthesis of AgNPs was monitored by color change and UV-Vis spectroscopy. The formation of AgNPs was confirmed by the change in the solution color from colorless to brownish yellow ( Figure-2). El-Batal et al. study [21] found that the AgNps synthesized by S. Marcescens collect WSE found to have maximum suction at 412 nm, their thickness allocation was set by dynamic light scattering (DLS) and a medium particle size of 11.5 nm. The peak enlargement is the best signal used to monitor nanoparticles size. As particle size increases, the plasmon resonance peak shifts to longer wavelengths. As the particle size increases, the suction peak converts to the red wavelength, indicating the output of larger nanoparticles [22,23]. The absorption spectrum of spherical silver nanoparticles was reported to exhibit a maximum of 420 nm to 450 nm, with a blue or red change when the particle size is decreased or increased, respectively [24,25]. The silver nanoparticles generated by S are for

Alaa and Hassan
Iraqi Journal of Science, 2021, Vol. 62, No. 4, pp: 1103-1120 0011 this purpose. WSE marcescens isolate is presenting a blue shifted plasmon peak is the most potent strain to silver nanoparticles produced. It is well known that a plasmon surface band of spherical silver nanoparticles exists about the 420 nm region [26,27]. In order to achieve the optimum conditions for AgNP synthesis, different parameters were studied in this procedure, such as pH, temperature, reaction time, and concentrations of silver nitrate and prodigiosin. In each step of the synthesis of ideal nanoparticles, samples are examined by using the UV-Vis spectroscopy in order to determine the optimum conditions in the synthesis and production of silver nanoparticles.

Optimum conditions for AgNPs Synthesis The Effect of pH on AgNPs Synthesis
PH plays an important role in the creation of a mixture of nanoparticles by modifying their charges. Green synthesis of silver nanoparticles could achieve their stabilization when the natural material of concentrated prodigiosin (5 μg / ml) was used as a reduction and stabilization agent. The observed results indicated that the optimum pH for AgNP synthesis at a wavelength of 430 nm was 12, as shown in Table-1. The relation between the average size of silver nanoparticles and the pH values of the solution was examined. The surface plasmon peak was shown to move to the left as the pH value increases, indicating a reduction in the size of the prepared nanoparticles (Figure-3). The observed findings agree with those established earlier [28]. El-Batal et al., [29] reported the synthesis of AgNPs using prodigiosin pigment as a reduction agent. They found that the absorption spectrum of AgNPs with SPR band was blue-shifted due to a pH rise from 9 to 12 and showed a maximal absorption at pH 12.

Effects of temperature
Temperature elevation normally increases reaction rate and the efficiency of synthesis. Most experiments are carried out at room temperature because it is the easiest and most efficient method of synthesizing nanoparticles. When the temperature increases, the reaction time reduces and the conversion to nanoparticles is completed by 95 percent within a limited period. The mixture was exposed to different temperatures (15, 25, 35, 45, and 55 ° C), as shown in Table-2, with an ideal interaction condition that was obtained from the results of previous steps. The result showed that 55℃ was the optimum temperature for synthesis of AgNPs at the wavelength of 413 nm (Figure-4). The results are in agreement with that mention by Li et al. [30]. Also, approximately similar results were obtained in the study conducted by El-Batal et al. [31]. Due to the increase in reaction temperature from 20 ° C to 70 ° C, they showed a red shift of the SPR band. However, the increase from 80 ° C to 100 ° C resulted in a decrease in the wavelength of the absorbance peaks with a small blue shift for the synthesized AgNPs.

Effects of reaction time on the synthesis of AgNPs
Time is one of the important parameters which directly affect nanoparticles biosynthesis. The reduction reaction for the production of nanoparticles started when reducing agents were instantly applied to the silver nitrate solution, which is demonstrated by the color transition from white to brown. Nevertheless, it is shown that particle size decreases with increasing time, and at a given time, it stabilizes. The mixture was subjected to various time intervals for AgNPs synthesis (0.5 , 1, 2, 3 and 24 hrs.), as seen in Table-3, taking into account the effects of optimal conditions in the previous steps. Maximum synthesis of AgNPs was observed at 24 hours at a wavelength of 410 nm (Figure-5). Increasing the reaction time provides the opportunity to complete the formation of nanoscale silver [32].
Aslan et al. [33] and E-Batal et al. [34] performed UV -Vis spectral analysis to validate synthesis of silver nanoparticles, after 24 hrs., using prodigiosin as a reducing agent. The results showed that the standard absorption spectrum of AgNPs is between 350 nm and 450 nm.

Effects of AgNO 3 concentration on the synthesis of AgNPs
The concentration of the silver nitrate solution used also affects the formation of AgNPs. At first, neutral silver atoms react with each other, forming stable nuclei and creating nanoparticles, until all metal ions are consumed. Thus, an introduction of protective agents must be sought to stop aggregation by their interactions with small particles [35]. Different concentrations of silver nitrate (1, 10, 25, 50, 100, 200, 400, 800 and 1000 mM), as shown in Table-4, were added to solutions of prodigiosin (5 μg/ml) using the above described optimum conditions (pH 12, temperature 55℃, and reaction time 24 hrs.). The results showed that the best concentration of silver ion was 1 mM for the synthesis of nanoparticles at the wavelength of 415 nm, as shown in Figure-6. The same results were observed by Vanaja et al., [36], who found that the concentration of AgNO 3 of 1 mM was typical for the maximum reducing reaction to form AgNPs. El-Batal et al. [37] also observed a red shift in the SPR due to the gradual increase in AgNO 3 concentration (from 1 to 10 mM), using prodigiosin as a capping agent. TEM images show spherical nanoparticles at a concentration of 1mM AgNO3, with a mean diameter of 14.11 nm.  2  10  497  3  25  411  4  50  385  5  100  371  6  200  374  7  400  378  8  800  369  9 1000 220

Effects of prodigiosin concentration on synthesis of AgNPs
One of the values of developing environmentally sustainable materials that are relevant to metallic nanoparticles synthesis is the application of prodigiosin as a caping agent. The concentration of prodigiosin applied to the mixture has an impact on the formation of silver nanoparticles, simultaneously acting as an agent for reduction and stabilization. The effect of partially purified prodigiosin concentration on the AgNPs synthesis was studied by adding different levels of prodigiosin (3, 6, 12.5, 25, 50 , 100 , 200, 400 and 500 μg / ml) based on the previously tested optimum conditions, as shown in Table-5. The results clearly show that the prodigiosin concentration of 12.5 μg / ml was ideal for AgNPs synthesis at 425 nm wavelength, as shown in Figure-7. The results The results of the current study are in accordance with the findings of El-Batal et al. [38]. Table 5-Effects of different concentrations of prodigiosin on AgNPs synthesis

FT-IR analysis
FT-IR measurements were conducted to identify the major functional groups of prodigiosin and examine their potential involvement in AgNPs synthesis and capping. The FTIR spectra of the red pigment revealed many degrees of similarity to the typical prodigiosin spectra [39]. FT-IR spectrum of prodigiosin shows different bands positioned at 3421, 2858, 1045, and 779 cm−1 (Figure-8). The presence of 3421 cm−1 bands contributes to the N -H stretch. The peaks of 2851 cm−1 are typical of symmetrical stretching of aromatic methylene groups (CH) [40]. The C-O stretching was due to the 1045 cm−1 peak. The value assigned to prodigiosin carbon-carbon double bond was observed at about 779 cm−1 [41,42]. FT-IR spectrum was tested for the AgNO3 synthesis (Figure-1). The band attached at 1380cm−1 was for the (N-O) stretching. FT-IR spectrum analyses for AgNPs aqueous solution and the emerged functional prodigiosin group bands after washing were detected. The peaks at 1674cm−1 was found only due to the combination of AgNPs with the feature groups of -NH prodigiosin [43]. This indicates that prodigiosin was formed by AgNPs as a capping agent for synthesis. However, the AgNPs band at 1380 cm−1 moved to a different location and appeared at 1364 cm−1 band (Figure-1), which is another confirmation of AgNPs synthesis.

Atomic Force Microscope (AFM)
Atomic force microscope was used to determine topography and surface morphology. The AFM, at an atomic level, provides two-dimensional and three-dimensional representations of the nanoparticles surface [45], as displayed in Figure -10. The average diameter of particles measured was found to be on the nanoscale. The scale of silver nanoparticles was measured using AFM-SPM, and the results show that the AgNPs average size was 69 nm, as illustrated in Figure-

Scanning electron microscope analysis of silver nanoparticles
The results of the morphology and size distribution of the synthesized AgNPs by using prodigiosin are demonstrated through the SEM images in Figure-12. The surface of nanoparticles is smooth with good crystallinity and spherical shape [46]. The average particles size and distribution are determined randomly on the SEM. The most prevalent particles size range of the AgNPs is about 30 to 70 nm. Ullah et al. [47] showed that NPs studied by SEM were dispersed on the surfaces, preserved a good uniformity, and seemed to be less agglomerated. The cause of minimal aggregation of the nano-silver material might be because of its encapsulation with bio-molecules.

Determination of minimal inhibitory concentration
In the current study, different concentrations of AgNPs (2-1024 μg / ml) dissolved in Mueller-Hinton broth were transferred against pathogenic bacteria, P. aeruginosa and S. aureus, within micro titer plates. For this experiment, the violet pigment was used to confirm MIC values for the bacterial

Alaa and Hassan
Iraqi Journal of Science, 2021, Vol. 62, No. 4, pp: 1103-1120 0001 isolated, as illustrated by the change of pigment color from blue to pink. The pink color of the well refers to bacterial growth, while the MIC value is that of the well that is located directly before the first pink well. The results suggested that the MIC was 32 μg / ml for both isolates, as shown in Figure-13. Silver nanoparticles destroy bacteria by interfering with their outer membrane, causing structural changes that lead to degradation of the microbes and eventually death [48]. Silver nanoparticles adhere to microbial cells. Then, the most popular antimicrobial mode of action was recognized as cell membrane penetration and ROS and free radical generation [49]. To metabolize oxygen, all bacteria use an enzyme as a part of the "chemic lung." The silver ions disable the enzyme and stop oxygen acquisition [50]. Synthesized AgNPs have a stronger antibacterial effect on Pseudomonas spp, followed by E. coli. Silver ions are well known for their strong bacteriostatic and bactericidal effects [51,52].

Table 5-Effects of different concentrations of prodigiosin on AgNPs synthesis
The release of K + ions from bacteria is caused by silver ions. The bacterial plasma membrane is associated with many important enzymes and DNA is an essential target site for silver ions [53]. Due to their size, AgNPs can easily enter the bacteria's nuclear content. AgNPs show a remarkably large surface area and , therefore, have increased interactions with bacteria. Silver nanoparticles synthesized by chemical means were reported to show equal antibacterial activity against S. typhimuium (Gram -ve ) and S. aureus (Gram +ve ) [54].

In vivo challenge test
The results of the in vivo biological activity of AgNPs cream showed potent anti-inflammatory effects against the infected skin of laboratory animals (rabbits) compared with control groups (Figure-14). After the injection of pathogens, the infected skin with pathogenic bacteria showed acute inflammation with abscess formation and sever congestion in different parts of the infected skin. The infection appeared after 48 hrs. The treatment of the infected animals with AgNPs cream (by wiping the infections twice daily) started at the time of the appearing of the infection and continued for 4 days until healing. As compared to the control group that was treated with fucidin ointments, the results obviously showed that the healing was beginning after the first day of treatment and completely achieved after 4 days. While the control group exhibited severe infection and congestion throughout the experiment time, in spite of treatment with fucidin. The healing condition was delayed due to severe infection (Figure-15). Additionally, it can be suggested that the AgNPs have antimicrobial effects against pathogenic bacteria. It was also concluded that small sized AgNPs (30 nm) mainly have bacteriostatic effects against S. aureus, possibly by adding the AgNPs to the bacteria's cell wall, where they can disturb the plasma membrane 's shape and permeability [55].

Alaa and Hassan
Iraqi Journal of Science, 2021, Vol. 62, No. 4, pp: 1103-1120 0001 Tian et al. [56] demonstrated the potential role of AgNPs in animal model's wound healing, with dose -dependent fast healing and a better cosmetic appearance [57]. In addition, AgNPs showed positive effects through their antimicrobial potential, reducing wound inflammation through decreasing infiltration of lymphocytes and mast cells and modification of fibrogenic cytokines [58]. Similarly, Liu et al. [59] investigated the effects of AgNPs on dermal contraction and epidermal reepithelialisation during wound healing. They indicated that AgNPs may increase the rate of wound closure. This property was interpreted by promoting reproduction and migration of keratinocytes. Additionally, AgNPs could increase the differentiation of fibroblasts into myo-fibroblasts and thus induce wound contraction [60]. The structure of cream used in this study is similar to that described by Atwan and Hayder [61].
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