Preparation and Characterization of Silver Nanoparticles and its Medical Application against Pathogenic Bacteria

: The fabrication of Solid and Hollow silver nanoparticles (Ag NPs) has been achieved and their characterization was performed using transmission electron microscopy (TEM), zeta potential, UV–VIS spectroscopy, and X-ray diffraction (XRD). A TEM image revealed a quasispherical form for both Solid and Hollow Ag NPs. The measurement of surface charge revealed that although Hollow Ag NPs have a zeta potential of -43 mV, Solid Ag NPs have a zeta potential of -33 mV. According to UV-VIS spectroscopy measurement Solid and Hollow Ag NPs both showed absorption peaks at wavelengths of 436 nm and 412 nm, respectively. XRD pattern demonstrates that the samples' crystal structure is cubic, similar to that of the bulk materials, with average particle sizes of 28 nm and 27 nm for Solid and Hollow Ag NPs, respectively. The antimicrobial activity of synthesized Ag NPs was tested on some pathogenic bacterial strains which were isolated from urinary tract infection (UTI) and burn infection. The experiment results showed positive bactericidal activity against isolated bacteria with Solid Ag NPs which were most effective against both G-ve and G+ve bacteria. In addition, solid nanoparticles showed time and concentration dependent antibacterial activity.


Introduction:
Nanotechnology is the area of research to fabricate and restructure materials on the order of nanometers with a novel properties and functions. There have been ample revolutionary developments in physics, biology, and chemistry that have confirmed the idea of manipulating materials at extremely small scale 1 . The variations in physicochemical characteristics such as light absorption, scattering, electrical and thermal conductivity, melting point, and mechanical properties, which result in enhanced performance over their bulk counterparts, are the factors that lead to a greater contribution in fields of research with a great distinction in technology 2 . The outstanding features of nanoparticles are due to the larger surface area to volume 3,4 . Michael Faraday synthesize a ruby-colored colloidal gold fluid in 1857 5 , in which a 50 nm gold solution revealed a wine-red color 6 . The nanoscale size range of nanoparticles is appropriate for circulating in the bloodstream, penetrating cells, and traversing tissues in biological systems 7 .
Traditional medicine has gained popularity as an alternative health system over time, not only in underdeveloped countries but also in developed ones. This alternative medical system has made major inroads into the health-care sector of wealthier countries, where conventional medicine has a stronghold. The traditional knowledge and understanding involved in medication production by certain people have grown in value as traditional medicine has become more widely used around the world. China was the first to use gold-based medical remedies to treat illness, maybe as early as 2500 BC 8 . Bhasma is an herbo-metallic drug (metals such as gold, silver, zinc, calcium, iron, copper, mercury, etc.) that has been utilized in India since ancient times, where they found that the size of this medicine is usually between 10-50 nm 9 . Mental illnesses and syphilis were treated with colloidal gold during the Middle Ages in Europe 10 . Additionally, nanoparticles can significantly enhance the delivery of drugs due to their potential to enhance blood-brain barrier permeability 11 . Nanotechnology is now one of the most significant technologies in all fields of research.
Silver nanoparticles (Ag NPs) have been studied for a diverse range of applications due to their unique physical and chemical properties at nanoscale, i.e. electrical and thermal conductivities, chemical stability, catalytic and antibacterial activity 12,13 . Ag NPs can disturb functions of cell membranes such as permeability and respiration. Moreover, the penetrated Ag NPs into the bacteria cell can react with Sulfur containing proteins and phosphorus-containing compounds such as deoxyribonucleic acid (DNA) which cause a disturbance in bacteria cells 14,15 . Pathogenic bacteria are bacteria which are capable of causing disease and inflammation when enters the biological system. The traditional technique to treat bacterial infection in the biological system is to use chemical drug (antibiotics). Excessive use of antibiotics can cause bacteria to become increasingly antibiotic resistant 16 . Thus, developing potential and effective medicines with a nanotechnology became an exciting subject especially, when the previous researches showed that difficult for bacterial cells to become resistant to NPs. Hasan and Al-Niaame utilized the rosemary leaves (Rosmarinus officinalis) aqueous extract for the biosynthesis of silver nanoparticles and it was efficient against Pseudomonas aeruginosa bacteria isolated from wounds 17 . Shareef et al. used Carthamus oxyacantha M. Bieb. aqueous extract to fabricate silver nanoparticles with particle sizes between 50-80 nm. They investigated their antibacterial effectiveness against bacteria that were multi-drug resistant (MDR), extremely antibiotic resistant (XDR), and pan drug-resistant (PAN), the examined microorganisms had notable variances in their sensitivity to Ag NPs 18 . Marinescu, et. al. used two methods of synthesis chemical reduction at room temperature (RT) and the solvothermal procedure at high temperature (HT). The particle size of RT method was 1-20 nm and the particle size of HT method was more than 50 nm. HT sample showed relatively better antimicrobial activity and lower agglomeration tendency than Ag NPs prepared at RT 19 . The aim of this study is to fabricate and characterize two different shapes of silver nanoparticles and to assess their antibacterial activity against selected pathogens by in vitro susceptibility tests.

Fabrication of solid Ag NPs
In order to synthesis a solid Ag nanoparticle, silver nitrate (AgNO3) was reduced using sodium citrate (Na3C6H5O7). Under constant stirring, 250 ml of 0.001M AgNO3 aqueous solution was heated to boiling. Then 2.5 ml of a 1% Na3C6H5O7 aqueous solution was added drop-wise. After being heated to a full boil for one hour the colorless solution changed to an olive color, the reaction solution was cooled to room temperature 20 . Using this method, the mechanism of the reaction could be described as follows: 12AgNO3 + 4Na3C6H5O7 + 6H2O → 12Ag + 4C6H8O7 +12NaNO3 + 3O2 The nanoparticles were centrifuged, cleaned several times with DI water to eliminate any remaining chemicals, and then they were stored for further characterizations.

Fabrication of Hollow Ag NPs
First, 52 ml of ice-cold water are continuously stirred while aqueous solutions of AgNO3 (3 ml, 0.01 M), and glutathione (C10H17N3O6S) (0.36 ml, 0.01 M) are also added as a capping agent. This produces silver oxide (Ag2O) nanoparticles, which are the precursor to Hollow Ag nanoparticles. The mechanism of the reaction could be described as follows: 2AgNO3 + H2O → Ag2O + 2HNO3 Then, 3.6 ml of freshly prepared 0.01 M Sodium borohydride (NaBH4) solution was added all at once to the generated pale yellowish solution of Ag2O nanoparticles. The reaction mixture turns orange as Hollow Ag nanostructures are formed from Ag2O nanoparticles 21 . The mechanism of the reaction could be described as follows: 3Ag2O + NaBH4 → 6Ag + NaO3 + BH4 The nanoparticles were centrifuged, cleaned several times with DI water to eliminate any remaining chemicals, and then they were stored for further characterizations.

Characterization of silver nanoparticles
A double beam ultraviolet-visible (UV-Vis) spectrophotometer (T92+ UV Spectrophotometer, PG INSTRUMENTS) was used to measure the formation of Ag NPs in order to assess the effectiveness of the chemical reduction of silver nitrate. A cuvette with a 1 cm distance was filled with materials for analysis. Readings from 350 to 750 nm were scanned at intervals of 2 nm for the UV-Vis spectrophotometric measurements. Using a ZEISS (EM10C) Transmission Electron Microscope (TEM) (100Kv), the morphologies of the synthesized Ag NPs were examined. With the use of the DX-2700BH Multipurpose X-ray Diffractometer, patterns of the produced Ag NPs were acquired using X-ray Diffraction (XRD). The Zeta Potential of Ag NPs measured by HORIBA SZ-100 to determine the stability and surface charge of the particles. The EC/TDS and temperature meters HI 99300 device was used to determine the concentration of Solid and Hollow Ag NPs.

Concentration of synthesized Ag NPs
A serial of concentrations was prepared for Solid and Hollow silver nanoparticles. Then, the standard solution was diluted with deionized water by following Eq.

Sample collection
Bacterial samples were collected from patients with urinary tract infection (UTI) and burn infections in Kirkuk and Azadi Hospital / Kirkuk Governorate from 1/12/2021 to 1/5 of 2022. The samples were examined under light microscope and differentiated by using Gram stain then stored in nutrient agar media.

Maintenance of bacterial samples
The isolated samples were maintained on nutrient agar culture. The medium was prepared as recommended by manufactured company by dissolving 28g of nutrient agar in 1000 ml of distilled water, pH was adjusted to 7-7.2. and sterilized using autoclave at 121°C, 1.25kg/cm 2 for 15 min. Bacterial strains were activated 24 hours before use and incubated at 37°C . For long time maintenance, 85 ml of Nutrient broth was prepared and 15 ml of glycerol were added to the medium. The samples were grown and incubated at a of 37°C for 24 hours, and then kept in the freezer at -20°C until use.

Preparation of bacterial suspension for sensitivity test
A sufficient quantity of pure bacterial colonies was transferred by a sterile loop to a test tube containing 5 ml of sodium chloride sterile normal saline to suspend cells. The bacterial suspension was compared with 0.5 standard fixed McFarland's solution to give an approximate number for bacterial growth of 1.5*10 8 cells/ml 22 .

Antimicrobial activity of Ag NPs (sensitivity test): Disk diffusion methods (Kirby-Bauer disk)
The antibacterial activity of chemically synthesized Ag NPs was analyzed by agar-disk diffusion method against both Gram-positive and Gram-negative bacterial strains. The bacterial suspension was incubated for 24-48 hours to test. Isolated bacteria were inoculated on the sterilized Muller-Hinton agar plate and allowed to dry for 5 min. A sterilized disk was loaded with specific concentration of Solid or Hollow Ag NPs. The disk was pressed down tightly to make contact with the surface of the plate. The inoculated plate was incubated at 37°C for 24 hours. Later, the inhibition zone around each disk was measured by ruler expressed by millimeter (mm) and recorded.

Agar well diffusion method
Antimicrobial activity of Solid and Hollow Ag NPs was also evaluated by agar well diffusion method. The activated bacteria were inoculated on sterilized Muller-Hinton agar plates and allowed to dry for 5 min. Then a hole with a diameter of 6 mm is punched using sterilized tip and a volume of 100µl at desired concentration from Solid and Hollow Ag NPs introduced into the well. The plate was inoculated at 37°C for 24 hours. Later, the inhibition zone around each hole was measured and recorded.

Results and Discussion: Visual observation of nanoparticles formations
The visual observation of the solution's color changing from colorless to olive for Solid Ag NPs and orange for Hollow Ag NPs upon heating provided as visual confirmation of the production of silver nanoparticles (the reduction process of Ag + to Ag 0 nanoparticles) as shown in Fig. 2.

Solid Ag NPs
Hollow Ag NPs Figure 2. Visual identification of nanoparticles formation.

TEM analysis
The interaction between a high-energy electron beam and a solid material is the basis for Transmission Electron Microscope (TEM) images. Fig.3 shows a TEM image for analyzing the particle size and shape of the solid and Hollow Ag NPs.
From Fig.3a quasispherical shape can be observed with average size of ~ 30 nm for solid Ag NPs, while for Hollow Ag NPs the outer diameter is ~30 nm and the inner diameter is ~20 nm as can be seen in Fig.3b which is comparable to Abdulrahman's 21 results..

Zeta potential analysis
The effective electric charge on the surface of a nanoparticle is measured by its zeta potential. The zeta potential's magnitude gives information on the stability of the particles 23 . Values between 30 and 40 mV (positive or negative) exhibit moderate stability; however, values between 40 and 60 mV may be indications of strong stability 24 . Low zeta potential nanoparticles will eventually aggregate due to Vander Waal inter-particle interactions 25 . Therefore, Hollow Ag NPs have a Zeta Potential value of -43 mV, indicating high stability, compared to solid Ag NPs, which have a Zeta Potential value of -33 mV Fig. 4. The Zeta Potential value is influenced by fabrication techniques and nanoparticles environment 26 .

UV-Vis spectra analysis
One of the most commonly used methods for characterizing the optical properties of silver nanoparticles is UV-Visible spectroscopy. Considering that silver nanoparticles have a high absorption peak as a result of surface plasmon excitation, it is quite sensitive to the presence of silver colloids 27 . The absorption spectra of the solid and hollow silver nanoparticles are shown in Fig. 5. The plasmon resonance absorption peak of the solid and hollow nanoparticles is observed at wavelength 436 nm and 412 nm, respectively. The surface plasmon resonance (SPR) absorption peak is developed by the simultaneous vibration of the free electrons in metal nanoparticles (NPs) in resonance with the light wave, and in contrast to semiconductor or insulator nanomaterials, the optical properties of metal nanostructures are more sensitive to shape and less responsive to size 27 . For solid metal NPs, the absorption peak shifts to the red because the particle becomes more oblate according to the previous study 28 , as observed in TEM images Fig. 3.

Figure 5. Absorbance spectrum of Solid and Hollow Ag NPs.
According to Tauc's plot, which is depicted in Fig. 6, the direct band gap of silver nanoparticles was estimated to be 2.28 eV for Solid Ag NPs and 2.34 eV for Hollow Ag NPs. This result is quite similar to the previously reported band gap for silver nanoparticles, which is approximately 2.20 eV 29 . The synthesis method influences the band gap of silver nanoparticles since the band gap increases as the particle size reduces. As the grain size increases, the particles' optical band gap reduces. The rise in the barrier height at the grain boundary brought on by an increase in grain size causes the band gap of the particles to reduce 30 .

XRD analysis
The crystal planes of thin films and nanostructures that are parallel to the substrate surface can be identified using the XRD technique.   31 . With the use of the Scherrer Equation 32 (Eq. 2), D = 0.9λ / β cosθ 2 D is the crystallite size, θ is the Bragg angle, λ is the X-ray wavelength, and β is full width at half maximum (FWHM).
According to Eq. 2 the average crystallite size for Solid and Hollow Ag NPs, respectively, was determined to be about 22 nm and 25 nm, which is quite consistent with the average particle diameter observed in Fig. 3's TEM images, which is close to the nanoparticles fabricated previously 21 . Table 1 lists the fitting results for all samples.

Antibacterial activity analysis
The sensitivity test was conducted to evaluate the antibacterial activity of synthesized Ag NPs. Disk and well diffusion in vitro antimicrobial screening methods are the basic, well known and most applied procedures in microbiology lab. The antimicrobial activity of both Solid and Hollow Ag NPs was tested by applying serial of nanoparticles concentration. Generally, Solid Ag NPs showed efficient antibacterial activity than Hollow Ag NPs with most concentrations used in this study and were active against both Gve and G + ve bacterial strains. In addition to, the Solid Ag NPs showed clear inhibition zone in both sensitivity tests used in experimental part.
The well respond bacterial strain to solid nanoparticles in well diffusion agar method are E.coli , from burn infection as well as S. aureus, Klebsiella, and Proteus from UTI infection which gave a clear inhibition zone of 12 mm in Fig. 8 ( E. coli No.6, S. aureus No.10, Klebsiella No.11, Proteus No.12) after 24 hours when exposed to highest concentration 380 µg/ml Ag NPs Fig. 9a. The nanotoxicity of Solid Ag NPs showed a direct relationship with concentration, the antibacterial activity increased as the concentration of nanoparticles increased. When low concentrations (50 and 10 µg/ml) applied, no inhibition zone appeared with most bacterial isolates except for E.coli No. 6 in Fig. 9a which isolated from burn infection showed an inhibition zone of 6 mm appeared after 24 hours of incubation. On the other hands, Klebsiella No. 11 in Fig. 9b which isolated from UTI infection also responded to low concentrations of nanoparticles after 48 hours of incubation.  Figure 8. The inhibition zone by agar well diffusion method. bacteria exposed to 380μg/ml of solid Ag NPs for 24 hours (a) (b) Figure 9.

Zone of inhibition for Solid Ag NPs after (a) 24 hours and (b) 48 hours by agar well diffusion method.
Similar results were proved with disk diffusion method Fig. 10 E. coli No.1 from burn infection well responded to high concentrations of AgNPs (380 and 300 µg/ml) which showed the largest inhibition zone of 19 and 16 mm respectively after 24 hours of incubation Fig. 11a while the zone diameter increased which became 22 and 20 mm after 48 hours Fig. 11b for both concentrations. E. coli from UTI infection also showed positive response to high concentration of solid AgNPs. Nevertheless, G-ve bacterial strains were more sensitive to solid Ag NPs than G+ve bacterial strains. The inhibition zone of Solid Ag NPs for all samples was tabulated in Table 2 (agar well diffusion method) and Table 3 (agar disc diffusion method) which is in a good agreement with previous studies 13 .      Hollow Ag NPs showed no antibacterial activity against all bacterial strains and all concentrations used in both tests Table 4 and Table  5. Hollow silver nanoparticles appeared to have no bactericidal effect on the tested bacterial strain because bacterial cell surfaces have a negative electrostatic charge 33 and Hollow Ag NPs have a larger negative surface charge (-43 mV), which will repel 34 bacteria cells with a greater electrostatic force (Coulomb force) than Solid Ag NPs. Previous studies proved that surface of Ag NPs plays a critical role in their antimicrobial activity, positively charged exhibit the highest antibacterial activity 35 . Ag NPs are preferred in medical applications because their small size that helps to reach vital areas within the living microorganism add to it, Ag NPs exert more efficient antimicrobial activity rather than silver salt 36 . In addition, silver ions have high toxic effect against microbes rather than mammalian cells 37 .
Ag NPs' mechanism of action against bacteria is still not well understood. However, a number of theories are offered to account for silver nanoparticle's antibacterial properties: (1) Nanoparticles are known to have an enormous surface area which either penetrates the bacteria cell or connects to the cell wall, disrupting the membrane's permeability and makes it porous, which causes further leaking of cell content 38 . Additionally, the development of membrane pores causes the flow of nanoparticles into the cell, where they connect with proteins that contain sulfur and phosphorus, inactivating proteins and DNA 39 . (2) Since Ag+ ions are released during the oxidation reduction process, Ag NPs have an antimicrobial effect. The primary interactions between the oxidized silver ions from Ag NP and the thiol groups of several enzymes and proteins are what breakdown the bacterial cell wall and interfere with the respiratory chain 39 . (3) Reactive oxygen species (ROS), which are regarded to be the main cause of the majority of bacterial cell deaths, are made impossible to produce by silver ions due to the suppression of ATP production and DNA replication 18,39 . Previous report proved the action of Ag NPs on the morphology of S. aureus and C. albicans, incubation of bacteria with Ag NPs for 12 hours lead to cell lysis, breakdown of bacterial cell wall compares to control sample as revealed by transmission electron microscope (TEM) these series of events lead to cell death after releasing inside components to surrounding environment 14 .

Conclusion:
Transmission electron microscopy (TEM), Zeta potential, UV-VIS spectroscopy, and X-ray diffraction were used to characterize the Solid and Hollow silver nanoparticles (Ag NPs) that were synthesized by chemical reduction of Silver Nitrate. Both Hollow and Solid Ag NPs have a quasispherical shape, according to a TEM image. Although Hollow Ag NPs have a zeta potential of -43 mV, Solid Ag NPs have a zeta potential of -33 mV, according to the Zeta potential measurement. UV-VIS spectroscopy measurements revealed that both Solid and Hollow Ag NPs showed a sharp peak at wavelengths of 436 nm and 412 nm, respectively. According to XRD results, the samples' cubic crystal structure is identical to that of the bulk materials, and their typical particle sizes, as determined by Scherrer equation for Solid and Hollow Ag NPs, respectively, are approximately 28 nm and 27 nm. The Solid Ag NPs showed clear inhibition zone in sensitivity tests which proves their antibacterial activity reveled broad spectrum antimicrobial activity against G-ve and G+ve bacteria compared to Hollow particles. in addition to, the antimicrobial activity of synthesized silver nanoparticles was stimulated in time and concentration-dependent manner. We showed, based on the findings of this study, that the surface charge of nanoparticles is crucial for antimicrobial sensitivity tests.