Chemical Synthesis of Silver Nanoparticles as a Dynamic Oncoming Antiviral and Antibacterial Therapy

INTRODUCTION 
The persistent issue of chemotherapy is the ability of microorganisms and viruses to develop drug resistance [1]. Therefore, it requires the development of new drugs and therapeutic approaches. Nanotechnologies provide numerous opportunities for novel treatment strategies [2] and have recently garnered global attention as potential antimicrobial drugs. The significance of nanotechnology arises from its ability to produce and employ materials with a nanoscale of 1-100 nm. This feature facilitates entry into many practical and applied fields, including science (energy and electronics), health, medicines, chemical applications, and various other fields. [3].
Silver nanoparticles (AgNPs) possess numerous physical and chemical properties, such as low toxicity, ease of preparation, ultra‐small size, diverse synthesis methods and high capacity to interact with formulated treatments in host cells [4].These unique characteristics and their broad-spectrum activity against bacteria, fungi, and viruses make AgNPs a promising antimicrobial agent [5]. AgNPs have been extensively examined in virology, bacteriology, and oncology, encouraging their application in medicine, their veridical properties have been reported against different viruses [6,7]. AgNPs have been extensively studied against clinical infections; they exhibit an excellent solubility profile, targeted distribution to the infection site, and sustained release with minimal side effects. On 22 February 2024, the World Health Organization (WHO) announced that more than half of the countries in the world anticipate critical measles outbreaks this year. The measles virus (MeV) is a member of the Morbillivirus genus related to the Paramyxoviridae family [8], causing highly acute febrile disease and possibly leading to death. Many programmers have been implemented to eradicate measles, but outbreaks of the virus persist in many countries. Antibiotic resistance is recognized in bacteriology as a critical public health concern worldwide. 
The hazard of antimicrobial resistance has resulted from inappropriate use, misuse of marketable antibiotics and the lack of new antibiotics with novel mechanisms of action [9]. Moreover, the rapid proliferation of antibiotic resistance is due to the transfer of resistance genes from one bacterium to other bacterial species [10,11]. In this study, we tested the antiviral and antibacterial efficacy of AgNPs to inhibit certain bacterial isolates and the vaccinated strain of the MeV, thereby addressing the issue pf microorganism resistance to antibiotics. 

MATERIALS AND METHODS 
The study was conducted in the laboratories of the College of Science and the Iraqi Center of Cancer and Medical Genetics Research (ICCMGR), Mustansiriyah University, Iraq.
The chemicals employed in the experiment were silver chloride (AgCl), polyvinylpyrrolidone (PVP) with an average molecular weight of 40,000 and hydrazine hydrate (N2H4 50%–60%) obtained from (Sigma-Aldrich) as well as ammonium hydroxide (NH4OH 28%–30%) from BDH.The reagents were used in their pristine condition without any purification. The glassware was immersed in a solution of HNO3 and H2SO4 at a molar ratio of 1:3 for 30 min, subjected to sonication for 3 min, washed with double distilled water (DDW), dried in an oven for 1 h at 140 °C and exposed to a mixture of (95% H2SO4) and (30 wt.% of H2O2) in a 3:1 proportion for 30 min. subsequently, the glassware was subjected to a thorough cleaning process by immersed in DDW and sterilized in the oven at 100 ℃ for 1 h [12]. The whole procedure was iterated three times.

AgNPs synthesis (modified approach) 
Silver chloride is a precursor for the synthesis of Ag NPs. Adding 71.66 mg of AgCl and 50 ml of ammonium hydroxide to the solution resulted in the complete dissolution of AgCl, yielding a clear solution devoid of solid particles. A specific quantity of polyvinyl pyrrolidone was included as a surfactant to stabilize the colloidal solution. The liquid was gently stirred to achieve a molar of Ag (NH3)2+: The PVP of 1:16, followed by the addition of 0.1 M hydrazine hydrate to the resultant solution. The solution was allowed to sit for 1 h via magnetic stirring at room temperature (RT). The equations below depict the successful generation of AgNPs through the reduction of [Ag (NH3)2] + with hydrazine at RT [13,14].

Characterization
Analysis using Spectral Examination Techniques
The absorption spectrum of the AgNPs solution was analysed by ultraviolet–visible double optical spectrophotometry (UV-M90, BEL, Italy) at two separate time points: immediately after manufacture and 1 month later. The result aligned with the spectral characteristics of AgNPs nanoparticles in their aqueous solution. 

Zeta Potential Evaluation
The SHIMADZU decomposer, designed for nano-sized zeta molecules, was used to examine colloidal AgNPs and assess their stability. The stability of the NPs was measured by measuring the surface charge density.

Characterization of AgNPs by atomic force microscopy (AFM)
The morphology, size and prevalence of the synthesized AgNPs were determined by atomic force microscope (SHIMADZU, AA3000, JAPAN). To obtain images via AFM, we deposited a small volume of colloidal AgNPs solution on high-grade mica and allowed to dry at RT in a sterile laminar flow environment.

Analyzing AgNPs using Flame Atomic Absorption Spectrometry (FAAS)
The concentration of AgNPs was quantified using a Flame Atomic Absorption Spectrometer (FAAS) AA-7000 model by SHIMADZU, a renowned Japanese manufacturer. The measurement was conducted at a precise wavelength of 328.1 nm.

AgNPs were analysed using transmission electron microscopy (TEM)
The morphology and dimensions of AgNPs were determined via transmission electron microscopy (TEM) using a JEOL JEM-2100 microscope from Japan.

Analysis using Dynamic Light Scattering
The dimensions and distribution of particles in the colloids were quantified using a dynamic light scattering (DLS) instrument manufactured by Brookhaven Instruments (USA). The measurement conditions were as follows: a laser with a wavelength of 659 nm (He-Ne), a fixed scattering angle of 173∘, a temperature of 25∘C, a viscosity of 0.890 Cp and a medium refractive index of 1.330. The specimen was inserted into a quartz microcuvette, and measurements yielded the average result. 

Cell line
The cell bank of the Iraqi Center for Cancer and Medical Genetics Research provided us with a VEROhSLAM cell line. These cells result from the transfection of Vero cells with pCXN2, a vector plasmid holding the gene of neomycin resistance, and an expression plasmid (pCAG-hSLAM), which encodes the receptor for the MeV [15]. 

Preservation of cell line
The cells were grown and maintained in minimum essential medium (MEM), supplemented with 10% fetal calf serum and an antibiotic /antimycotic Solution. When the cell density exceeded 80%, the growth medium was removed. Thereafter, 3 ml of trypsin- EDTA solution was added to T25 flasks, and after 1–2 min., the cells detached, and 10% fetal calf serum (FCS)- MEM was introduced. The cells were redistributed at an appropriate concentration in the tissue culture flasks (sub-culture) and incubated at 37°C with 5% CO2 [16].

Mesleas virus 
This study utilised a vaccination strain of Live attenuated MeV (India). The virus propagated in VERO-hSLAM cells line using MEM; when the cytopathic effect (syncytia) exceeded 80%, the cells underwent three freeze–thaw cycles. The virus was aliquoted into sterile Eppendorf tubes and stored at –70°C.

Virus titration 
The MeV tissue culture infected dose 50% (TCID50%) was used on the VERO-hSLAM cell line, with cell suspensions sub-cultured and inoculated in a 96-well microtitration plate containing 104 cells in 100 μL per well. After overnight cultivation, cell growth reached 80%, and the medium was removed to prepare a serial tenfold dilution of MeV (10-1– 10-9), which was allocated to the wells (50 μL/well) at a ratio of 4 wells per dilution. Meanwhile, the maintenance medium (200 µL) was injected into each control well, and the plate was incubated for 1 h at 37°C to facilitate viral adsorption. Subsequently, maintenance media with 3%–5% FCS was added (200 μL/each well) after washing the cells with PBS to eliminate the non-associated virus. The plate was analysed for 5-6 days, and TCID50 was determined using the equation of Reed and Muench [17].

Cytotoxicity of MeV
The ERO-hSLAM cell line was cultured in a 96-well micro-titer plate (1 x 104 cells/well). Cells were exposed to the virus via discarded medium. The cells were exposed to multiplicities of infections (MOI) with MeV at 1, 3,6 and 10 by adding 50 µL of each viral MOI; three replicates were used for each MOI, whereas control cells remained untreated. The plate was incubated at 37°C for 1 h, with shaking every 10 min to facilitate the distribution and attachment of the virus to the cells. Subsequently, the cells were rinsed with PBS, and 0.1 mL of maintenance medium containing 5% FCS was added. After 72 h, the cells were rinsed with PBS, and 5 μg/mL MTT solution was introduced for a duration of up to 2 h. After discarding the supernatant and drying the cells, 100 μL of DMSO was added to each well. The extinction values at 570 nm were determined photometrically via spectrophotometric analysis [18].

Antiviral effect of AgNPs 
The AgNPs were evaluated for antiviral activity. Cells were seeded onto 96-well plates at a density of 1×104 cells per well and incubated for 24 h at 37 °C. Cells monolayer were incubated with different MOIs (1, 3, 6 and 10) of MeV (three wells for each MOI) for 1 h to facilitate viral adsorption. The virus was removed, and various concentrations of AgNPs (100, 50, 25 and 12.5) were introduced, which were dissolved in MEM-free serum. For the positive control, cells were infected with the same concentrations of the virus but without AgNPs. The negative control consisted only of MEM supplemented with 5% FBS. On the third day after infection, MTT assay was performed in brief, the medium was discarded, and cells in each well were incubated with 50 μL of MTT solution (5 mg/mL) for 2 h. at 37°C. MTT solution was discarded, and 100μL dimethyl sulfoxide (DMSO) was added to dissolve the insoluble formazan crystal. Optical density was measured at a wavelength of 492 nm with an ELISA reader.

Pathogenic isolates
Sixteen pathogenic isolates, four from each bacterial species, were obtained from different clinical samples. They were Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aerogenosa, and Escherichia coli. The isolates were obtained from wound infections that exhibited antibiotic resistance. The identification of isolates was conducted using Gram staining, biochemical tests and a VITEK-2 compact system. 

AgNPs antibacterial activity
The antimicrobial efficacy was tested against each bacterial isolate, adjusted to 0.6 OD and uniformly applied onto Mueller–Hinton agar plate by using sterile cotton pads. Five wells, each 6 mm in diameter, were stamped with sterilized micropipette tips on the prepared plates, into which synthesized AgNPs were introduced at with varying concentrations of 100, 50, 25, and 12.5 µL (15 µL in each well); sterilized physiological saline served as the control well [19]. The plates were incubated at 37°C for 24 h, and the width of the inhibition zones was recorded in millimeters (mm) using an antibiotic zone scale referenced in [20]. All the trials were conducted in triplicate. 

Statistical Analysis
The research findings were subjected to statistical analysis using the Statistical Package for Social Science (SPSS) version 26 to determine the significance of variation. Statistical significance was defined as p ≤ 0.05 for significant or p ≤ 0.0 for highly significant. All statistically assessed samples were run in triplicate, unless stated otherwise. Data were expressed as mean ±standard deviation and approved using Graph Pad Prism version 8 (Graph Pad Software Inc.). To analyse the efficacy of the antiviral, we used CompuSyn software (ComboSyn Inc., Paramus, USA) to compute the Chou-Talalay antagonism indices (CIs) and variable ratios of MeV and chemotherapeutics. Additionally, we employed alternative precise formulae to obtain the CIs. A confidence interval (CI) ranging from 0.9 to 1.1 indicates additivity. A CI of 0.9 indicates synergism, whereas a CI of 1.1 indicates antagonism.

RESULTS AND DISCUSSION
Size distribution of NPs studied using UV/Vis spectroscopy
The synthesis of AgNPs included the reaction between silver ions as oxidizing factors and hydrazine hydrate as reducing agents. AgNPs were synthesized by reacting silver ions as oxidising agents with hydrazine hydrate as reducing agents. The first observed occurrence was a color change. The solution underwent a colour change from a light-yellow hue to a reddish-brown shade toward the conclusion of the reaction (Fig. 1a). The UV-Vis spectra of colloidal AgNPs (Fig. 1b) exhibited a distinct surface plasmon resonance (SPR) band at 423 nm. This peak confirmed the creation of AgNPs [21].
To evaluate the stability of colloidal AgNPs, we immediately recorded the spectra after production and again following 1 month without stirring or ultrasonication. Fig. 1a demonstrates no significant alterations in the location or form of the SPR band. However, we found a modest modification in the absorption intensities of the colloidal AgNPs, which shifted from 223 nm to 228 nm. Furthermore, the artificially produced AgNPs were stable at RT, with no clumping observed over 1 month. Our findings were aligned with the results reported in the reference [22].
Zeta potential of AgNPs
Fig. 2 displays the zeta potential values of colloidal AgNPs. The surface charge was +45.89 mV. Significant values were achieved, indicating exceptional durability. Therefore, this silver liquid was ready for use.

AFM Analysis
The primary data were extracted from AFM images of the shape and size of the NPs [23]. Figs. 3 and 4 show the AFM images and size distribution histogram of AgNPs, respectively. The particles exhibited a spherical morphology with an average diameter of 20.56 nm, which was consistent with previously reported values [24]. 

Concentration of colloidal AgNPs in the stock solution
Before measuring their concentration, the AgNPs were sterilised using a Millipore with a diameter of 0.45 μm. The concentration of silver ions was determined using a flame atomic absorption instrument, employing the standard curve methodology shown in Fig. 5. The result yielded a concentration of around 100 µg/mL.

Analysis of AgNPs via TEM
Fig. 6 displays the TEM images of Ag NPs. It was also used to determine the mean dimensions of AgNPs. The TEM observations were obtained at a distance of about 20 nm.

Analysis of AgNPs via DLS
The DLS study yielded the polydisperse system, which offered the particles’ half-width, diameters, and size. The results demonstrated remarkable monodispersity, as illustrated in Fig. 7, revealing an effective hydrodynamic diameter of 23.06 ± 0.6 nm and a polydispersity of 0.245 ± 0.06 nm for the synthesized AgNPs.

Using produced AgNPs as an antiviral tool
Virus titration (tissue culture infective dose 50%)
Vero/hSLAM cells were used to determine the TCID50 of MeV. This method involves infecting cultured cells with the tenfold dilution of the virus and observing the cytopathic effect in the cells, this effect is characterized by round cells, aggregations, small vacuoles in the cytoplasm of the infected cells and formation of syncytia (large multinucleated cells), resulting from the fusion of infected cells, which produce plaques. The endpoint at 50% was 2× 106.5 TCID50 / 100 Μl.

Cytotoxic effect of MeV
The hallmark of MeV infection in Vero/hSLAM cells was the development of syncytia. This phenomenon was a characteristic cytopathic effect caused by this virus. It led to tissue damage and a viral transmission mechanism compared with the control, which showed no pathological changes.
 The results in Fig. 8 showed that the most significant cellular damage occurred at MOI of 10 after 72 h of infection with inhibition percentage of 93.29 ± 1.73. The cell mortality rate declined when the MOI decreased (88.83 ± 1.52, 60.99 ± 0.72 and 57.86 ± 1.36 for MOIs of 6, 3 and 1, respectively). We found significant differences among the four virus concentrations (p.= 0.02).

Antiviral activity of AgNPs
The Vero/hSLAM cell line facilitated the proliferation of MeV within 24 h, with the cytopathic effects manifesting as rounding of infected cells and the formation of small, numerous cytoplasmic vacuoles, which became noticeable with time until 72 h of infection. The exposure of cells infected with different MOIs of the MeV to various concentrations of AgNPs resulted in the inhibition of the pathogenic effect of the virus on the cells. The inhibition percentages declined to 82.71 ± 1.52, 80 and 82 ± 1.53 in the first two concentrations used (MOI=10+100 µg/mL and MOI=6+50µg/mL). However, when the MOI of the virus declined, the effect of Ag NPs decreased (40.99 ± 1.52 and 27.72 ± 1.52) for the combination (MOI= 3+ 25µg/mL and MOI=1+12.µg/mL) as shown in Table 1 and Fig. 8.
In addition, Table 2 and Fig. 9 show that AgNPs excreted an antagonism effect with MeV in the Vero/hSLAM cell line after 72 h of exposure period at all concentrations used in this study
Antibacterial Inhibitory Activity of AgNPs In-vitro
The formulated AgNPs efficiently inhibited the growth of Gram‑positive and Gram‑negative pathogens. The maximum growth inhibition zones (mean ± SD = 18.7± 0.2 mm) were observed at 100 μg of AgNPs against P. aerogenosa, thereafter decreasing to 16.6 ± 0.7, 15.9 ± 0.1 and 13.9 ± 0.4 for concentrations of 50, 25 and 12.5 µg/mL, respectively. A highly significant difference was found among the effect of the four dilutions (P ≤ 0.0002). The strains of E. coli resulted in less inhibition (Mean ± SD = 16.6 ± 0.4, 15.9 ± 0.6, 15 ± 0.5, 13± 0.1) at the same concentrations with high significant difference (P ≤ 0.0001) as shown in Table 3.
When comparing the effect of Ag NPs on negative bacteria, we found that P. aerogenosa was affected more than E. coli, especially in the two highest concentrations of 100 and 50 µg/mL, as shown in Fig. 10 and Table 4. 
The results in Fig. 11 and Table 5 showed a highly significant inhibition effect for Ag NPs (P ≤ 0.009 and 0.007) on the growth of the S. aureus and S. epidermidis strains, respectively, at the four dilutions.
Against S. aureus, the maximum growth inhibition zones (mean ± SD = 17.2± 0.52 mm) were found at a concentration of 100 μg of Ag NPs, then decreased to (Mean ± SD = 16.4 ± 0.51, 14 ± 0.2, 12.8 ± 0.73) for concentrations of (50, 25, 12.5, respectively). 
Whereas the minimal zone of growth inhibition by Ag NPs (Mean ± SD = 12.2 ± 0.6 mm) was found at 12.5 μg/mL against S. epidermidis, followed by 13.6 ± 0.7, 15.5 ± 0.1 and 16.5 ± 0.3. 
The results indicate that the inhibition zone increased with the concentration of AgNPs, and AgNPs exhibited antimicrobial activity, even at low concentrations. However, the effect was more pronounced in Gram-negative strains than in Gram-positive strains as show in Fig. 12 
The incidence of infections caused by antibiotic-resistant bacteria is rising worldwide; such infections exacerbate patient disease and mortality and pose economic challenges for the health system. Nanotechnology has introduce promising agents for combating viruses and bacteria, mainly silver nanoparticles AgNPs [25]. 
However, Ag NPs are widely used as they are inexpensive and have definite antibacterial activity, limited resistance progress, negligible immunological response and cytotoxicity [26-29]. 
The features of AgNPs render them suitable for use in medical and healthcare products for the treatment or prevention of infections [30,31]. The optimal mechanism of interaction that makes AgNPs highly effective remains unclear due to the different conflicting processes influencing their activity [31]. 
UV/Vis spectroscopy analysis revealed that AgNPs produced UV-visible spectra via the phenomenon known as surface plasmon resonance (SPR), resulting from the interaction among light, electromagnetic radiation and free electrons, The presence of AgNPs was verified by detecting a peak at 423 nm and a colour transition from yellow to yellowish brown. The colour shift was most evident within the wavelength range of 350-450 nm, corresponding to the SPR of AgNPs [32].Furthermore, the stability of colloidal AgNPs was assessed by UV/vis spectroscopy by examining the convergence of the peak in the UV spectrum, which shifted slightly from 423 nm immediately after production to 428 nm after 1 month. This phenomenon validated the stability of the colloidal solution. The results of the zeta analysis confirmed the stability of the manufactured colloidal AgNPs. This test quantified the surface charge, providing accurate insights into the properties of the majority of the molecules in the suspension. As a result of electrostatic repulsion, the stability of the dispersion improves when the zeta value exceeded +30 or–30 mV. The zeta potential +54.89 mV indicated that the manufactured particles were stable in the liquid [33].
To determine the shape and size of the nanoparticles (morphology), we used AFM through a direct surface image of the sample. The prepared AgNPs exhibited a spherical morphology, systematically positioned on the silicon surface, with an average size of 20 nm. This result was consistent with the findings from TEM and DLS analysis [34].
TEM operates by depositing a droplet of AgNPs solution onto a conventional 200-mesh grid to obtain information regarding the shape and size. The AgNPs exhibited a spherical morphology, with an average diameter of 20 nm or less. This analysis provided an extensive understanding of the dimensions and dispersion patterns of the NPs. The findings were consistent with the outcomes of AFM and aligned with the results of prior research [35].The size disparity and stability of AgNPs are crucial for their utilization in medical and other fields. Confirmation was achieved by DLS, which provides significant and beneficial details. The analysis revealed an average particle size distribution of 23.06± 0.6 nm, which closely corresponded to the measurements obtained from AFM and TEM. In this examination, the size distribution was determined by analysing the Brownian motion of the suspended NPs. This motion, referred to as the diffusion coefficient, is related to the speed and is quantified by the polydispersion index (PDI). The PDI is between 0 and 1, with values close to zero indicating a narrower size distribution. A value of 0 implies a high level of homogeneity; conversely, a value close to 1 indicates the opposite. In this case, the result of 0.245 ± 0.06 nm suggested that the suspension was homogenous and stable. This finding was in agreement with the results of the zeta test. In the current study, the NPs were tested on an MeV isolate. This study concluded that the Vero/hSLAM cell line supported a measles-attenuated viral strain with a high inhibition percentage after 72 h of infection compared with the typical morphology of the cell control [36].Cytotoxicity was assessed through (A) modification of normal cell morphology and (B) cell culture viability via MTT assay. The cultured cells were checked daily for signs of cytopathic effect. The results of our research highlighted that AgNPs demonstrated good antiviral activity against MeV in its suitable cells; several studies have revealed that AgNPs primarily act by physically interacting with the free or bound viral particle. Thus, AgNPs can function as virucidal agents [37]. The volume and size of the AgNPs influenced the antiviral action of the complex system, which improved with increased concentration and reduced sizes of AgNPs. Antimicrobial substitutes such as NPs are favorable for treating bacterial infections, mainly due to the emerging risk of antibiotic resistance [38]. Li, Y. et al. Revealed that varying incubation times of AgNPs with the virus allowed for the observation of viral morphology by TEM. Their findings demonstrated that AgNPs can directly interact with the virus and damage the viral morphology [39]. A recent study described the antimicrobial potential of PLAL-AgNPs created by an inventive physical method named PLAL. The new PLAL-AgNPs showed no toxic properties on the classically used cells. Antiviral activity was assessed against the enclosed HSV-1 and the non-enclosed ked PV-1. the PLAL-AgNPs inhibited viral units during the early stage of viral infection [40,41]. The results proved that AgNPs exhibited remarkable antimicrobial activity against pathogenic bacterial strains, even at low concentrations. Different concentrations of AgNPs inhibited all bacterial species, and the inhibition zone was amplified with an increase in the concentration of NPs, consistent with previous studies [42,43]. The development of microbial resistance to NPs is limited by different mechanisms of action of AgNPs, which influence various aspects of bacterial growth and metabolism [43]. The possibility of AgNPs as antibiotics is linked to their numerous mechanisms of action, which attack microorganisms in multiple structures at a time, enabling them to kill diverse bacterial species [44]. The critical property of an antibacterial agent is its capability to interact with and alter the bacterial outer membrane, facilitating internalization [45].In addition to its ability to disrupt and penetrate the cell membrane by modifying its structure and permeability, leading to rupture and loss of the cytoplasmic content, it can similarly enter the cell and modify DNA and protein structures and functions through interaction with phosphorus or sulfur groups. AgNPs may adjust the respiratory chain in the inner membrane by interaction with thiol groups in the enzymes; inhibiting adenosine triphosphate production; generating free radicals and reactive oxygen species; causing damage to intracellular structures; leading to the oxidation of proteins, lipids and DNA breaks; and triggering the apoptosis pathway. Ag NPs disrupt cell division, leading to cell death [46-48].The release of positively charged ions upon suspension, their oxidation dissolution abilities and the ability to replace ligands facilitate their binding to the negatively electric bacterial cell wall, thereby enhancing their bactericidal efficacy [49]. Tang, S. et al. demonstrated that Ag NPs have broad antibacterial activity against E. coli and S. aureus. Given their diminutive size, Ag NPs rapidly penetrate cell membranes and disrupt intracellular processes [50]. Hasan, K. M. F. et al and Trzcińska-Wencel, J. et al. reported that the most dominant mechanism in Gram-negative bacteria may involve pit formation and membrane disruption. By contrast, in Gram-positive bacteria, the most dominant mechanism may be the damage of the thick peptidoglycan layer, and Gram-positive bacteria are less sensitive to the action of AgNPs than Gram-negative bacteria [51,52]. 
 Although the cell wall of E. coli is more damaging than that of S. aureus, which could appeal to AgNPs with increased intensity, this fact might not produce a significant influence as Gram-negative and positive bacteria possess negatively charged cell wall, and membrane disruption produced by metal NPs is more injurious for Gram-negative bacteria than for Gram-positive bacteria [53].
Similarly, Nath H. et al. reported that P. aeruginosa is sensitive to AgNPs [54]. Yuan, Y.-G. et al. found that AgNPs caused decreased lactate dehydrogenase (LDH) activity and decreased adenosine triphosphate levels in P. aeruginosa and S. aureus [55], Li, W.-R. et al. proved that the AgNPs inhibits the respiratory chain dehydrogenase by converting various enzymes, such as glycerol-3-phosphate dehydrogenase, into dihydroxyacetone in S. aureus, thereby interfering with the metabolism and growth of the bacteria cells. TEM images have illustrated that AgNPs affect the integrity of the E. coli membrane by depolarization and destabilization [56]. Vazquez-Muñoz, R. et al. discovered that oxidative stress of AgNPs causes alteration in kynurenine protein and activation of kynurenine pathways, thereby inhibiting the bacterial growth of P. aerogenosa, E. coli and S. aureus [46].

CONCLUSION
In conclusion, this research demonstrated that AgNPs successfully targeted the MeV vaccine strain and exerted a remarkable effect on pathogenic strains. Additional experiments may elucidate viral mortality pathways and parameters activated by this approach to support its antiviral and antibacterial properties.

ACKNOWLEDGMENT
We are thankful to Mustansiriyah University, Iraqi Center for Cancer and Medical Genetic Research and Department of Chemistry (IBS/UPM) for their technical assistance.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript