Synthesis and Characterization of New Carboxymethylcellulose Coated Ag Nanoparticle as Antibacterial

INTRODUCTION
Nanotechnology has recently been acknowledged as a crucial and fruitful technology that can contribute to a variety of fields including physics, chemistry, engineering, medicine, and biology. These attributes include high oxidation resistance, strong antibacterial activity and high thermal conductivity. It manifests a lot of potential for spawning a variety of innovations that will change the direction of technological advancements in the near future. [1, 2]. Silver is also thought to be particularly essential because it performs better in a variety of applications [3]. Aside from their numerous applications SERS, or surface-enhanced Raman scattering catalysis, electronics, as well as sterilization that is antimicrobial to lessen toxicity toward mammalian cells, silver nanoparticles may be produced in a variety of forms, such as cubes, spheres, discs, rods, wires, stars, and right bipyramids [4,5]. Various techniques for creating silver nanoparticles, including physical, chemical, and biological ones, have been established. Chemical reduction has some benefits, including producing nanoparticles without aggregation, a high yield, little preparatory costs, and a simple and gentle process. [6,7]. 
Since It has been discovered that silver nanoparticles have potent antibacterial and strong inhibitory influences and a wide range of antimicrobial activities, they are  utlized for centuries to both stop as well as treat illnesses, particularly infections. Numerous studies claim that the antifungal, antiviral, anti-inflammatory, and antiangiogenic properties of silver nanoparticles [8, 9]. Ag NPs’ adherence and penetration to bacterial cell membranes is one key mechanism of silver nanoparticles’ antibacterial properties [10]. The antimicrobial properties of the silver nanoparticles are effectively utilized in fields and applications such as materials for water filtration [11], textiles [12], as well as disinfection or for medical purposes. [13].
In this research is to prepared the new polymer coated nanoparticles (Ag) as antimicrobial polymers and test against different types of microorganisms for determination the biological activity including as Gram-positive microorganisms Pseudomonas aeurginosa, a gram-negative bacterium, and Staphylococcus aureus.

MATERIALS AND METHODS
Carboxy methylcellulose (MERCK), silver nanoparticles (MERCK). ciprofloxacin and amphotericin –B drugs (BDH).

Instruments 
FTIR TENSOR 27, a Fourier transform infrared spectroscope made by Braun, Germany the measurement was done at the University of AL Qadisiyah College of Engineering. The shape of the nanoparticles’ fracture surface and the distribution of the nanoparticles were determined using scanning electron microscopy (SEM). The in-strument was a FEI Nova Nano SEM 230 from Eindhoven, the Netherlands, and it was utilized at IRAN on the Electron Microscopy Unit at the setting. 

Synthesis of polymer-coated nanoparticles (Ag) 
A quick and inexpensive hydrothermal synthesis technique was used to create polymer-coated nanoparticles (NPs) under ambient air conditions. The synthesis techniques have been previously published with changes. The polymer coated nanoparticles were made by mixing 25 mL of ultrahigh quality water and 3.6 g of polymer (carboxymethylcellulose) at 80 °C for 10 minutes. The solution was swirled at 80°C. Ag Nano-particles (1.0 gm) were added to the solution and agitated for another 10 minutes at 80 °C. Sonication was used to re-distribute the suspension in water. For the experiment on oil removal, NPs solutions were kept.

Organisms tested
Gram-positive microorganisms The synthetic polymers’ antibacterial properties was evaluated using Staphylococcus aureus and the Gram-negative bacterium Pseudomonas aeurginosa, that came from the College of Science and Biology at AL-Qadisiyah University.

Antibacterial activities   
For antibacterial activity, pure cultures of the pathogenic bacteria Staphylococcus aureus, Salmonella typhi, used, as well as Pseudomonas aeurginosa. using a cup or well method has been utilized in antibacterial research. The bacteria were cultured on nutrient agar medium. The mixture was composed of distilled water (1000 ml), peptones (5.0 g), sodium chloride (5.0 g), agar-agar (15 g), as well as beef extract (3 g). Autoclaving was done with the nutrient agar medium. for 15 minutes at 15 psi as well as 121 °C. Petri dishes were sterilized and set on the laminar flow bench. Each petri dish’s lid was lifted at one end, and 15 to 20 ml of molten agar medium were then poured inside, where they were left to set. These were then inoculated using the spread plate method with a 0.2 ml suspension of the organismSix wells were made in the medium using a sterile borer—five on the periphery and one in the center—and the standard medication, ciprofloxacin, at the same concentration, was placed in the central well. Then, the filling of peripheral wells with a 500 ppm solution of synthesized carboxy methylcellulose coated Ag nanoparticles. Other petri dishes are paraffin-sealed and incubator-incubated at 37 °C. After 24-48 hours, the zone of inhibition within the petri dishes was investigated. The concentration of samples used to test for antibacterial activity was 500 g/ml. [14].

RESULTS AND DISCUSSION 
FTIR Spectrum of CMC- Ag
FTIR spectra of CMC-Ag (carboxymethyl cellulose-Ag) was analyzed Fig. 1. In the FTIR spectrum while the peaks were observed at 476cm-1 in CMC-Ag NPs spectrums agree with the occurrence of Ag+ ions. The vibrations of the adsorbed water, (OH), (CH2), and (O-CH) are seen at 3554 cm −1 and 3324 cm −1, 1675 cm −1, 1433 cm −1, and 1383 cm −1, correspondingly A visible band is present at 3434 cm-1 due to the presence of -OH groups, intermolecular hydrogen bonds, and intramolecular hydrogen bonds. Strong bands at 1595 cm-1 and 1186 cm-1 as well as 968 cm-1 were attributed to (COO) and (CH-O-CH2) stretch, respectively. [15-17].

SEM of Carboxymethylcellulose-Coated Ag Nanoparticles
The SEM image capture of Ag nanoparticles produced by CMC on drop-coated films. The size, shape, and morphologies of developing silver nanoparticles have been described using scanning electron microscopy. Fig. 2 displays the sample’s SEM image. The findings revealed a more pronounced morphology of Ag NPs, with circular, polydispersed particle shapes that ranged in size from 200 to 500 nm. Silver nanoparticles of different sizes as well as shapes might be synthesized by controlling the environment of nanoparticle synthesis, which resulted the entire structure is a transparent network with a smooth surface.    
According to Fig. 3, this test reveals the Ag nanoparticles’ size and shape at two different magnifications of 5 m and 2 m. The layers and structure of the Nano solution are depicted in these images.

Effects of Antimicrobials on Growth of Organism
Antimicrobials are chemicals which either eradicate or stop the development of microbial cells. Due to their selective activity, some antimicrobials, like penicillin, which are medicines made from microorganisms that treat illnesses brought on by microbial pathogens. In other words, they disrupt a pathogen’s metabolic process or factor while having little to no impact on the host. Selectivity is typically caused by the absence of the biggest component or activity in the host cell. [18].
 
Factors effected on antimicrobial activity based on synthetic polymers
Numerous groups have demonstrated successful replication of the biochemical activity of antimicrobial polymers. By carefully adjusting the hydrophobicity, specifics of membrane insertion, and charge density of the molecule, this has been made possible. [19] As a result of the interaction of numerous factors, polymeric synthetic mimics’ biological characteristics cannot yet be precisely predicted from their chemical structure. However, it has been shown here that the impact of specific design elements, like charge as well as hydrophobicity, onto the characteristics of a polymer series is known. The interactions of polymeric with membranes are relatively the interactions of small antibacterial molecules with membranes as well as cells are poorly understood in comparison to the known mechanistic information. [20].

Charge effected 
Due to the negatively charged phospholipids at the outside membrane of Gram-negative bacteria as well as the teichoic acids of Gram-positive bacteria, microbial cells typically have a negative net charge on the surface. In this way, polycations are drawn to one another, and if they are sufficiently amphiphilic, they can disrupt both the outer and cytoplasmic membranes of the cell, causing death to cell [21].

Hydrophilic/hydrophobic effected 
The concept of hydrophilicity as well as hydrophobicity, which determines how prepared polymers interact with bacteria in an antimicrobial manner, is also based on water. Most polymers, it was found to have an overall facially amphiphilic architecture, consisting of an aromatic backbone, cationic hydrophilic and hydrophobic groups arranged on the molecule’s opposing faces. [22]. Hydrogen bonds between molecules are also absent. The repeat units changed their functional groups to a facially amphiphilic conformation when they came into contact with the cell membrane or another hydrophilic-hydrophobic interface. by rotating around the single bonds of the backbone [23]. But as the hydrophobicity of the polymers rises, so do their toxicity to microbial cells.

Biological Activity    
A polymer called an antimicrobial polymer can get rid of microorganisms by providing sanitizing ions or molecules.
Usage of traditional antimicrobial substances is typically accompanied by issues with residual toxicity of these agents, that may result in higher serious environmental issues. When antimicrobial agents are used in food packaging, for instance, there is a chance that these agents will diffuse into the food as well as lead to a variety of issues [24]. Use of chlorine and other related chemicals is the most widely used treatment method for water disinfection and sterilization. However, because of the concentration of their residues in the environment and food chain, and the potential creation of halomethane similar that are thought to be carcinogenic, their use ought to be avoided. [25].
FTIR Spectra were used to confirm the chemical composition of the prepared polymers. Staphylococcus aureus and Pseudomonas aeruginosa are examples of gram-positive and gram-negative bacteria, correspondingly. are just a couple of the microorganisms that the activity that is antimicrobial of the prepared polymers have shown to be effective against. 
High antimicrobial activity in contradiction of gram-positive and gram-negative bacteria is found in polymers having the phenolic, Nitro, Chloro, Amine, Sulfonic, as well as Carbonyl groups [26]. In general, it was discovered that the tested microorganism and the microstructure of the polymer had an impact on the diameter of the inhibition zone. Because there are more and different types of functional groups in polymers, the diameter of the inhibition zone grew higher out of homo-polymer to coated-polymer. The lengthening of the distance between the function groups and polymer backbone also caused an increase in the reserve zone’s diameter for prepared polymers [26].

Antibacterial activities
The antibacterial activity was tested using Muller Hinton agar. of prepared polymers was investigated thru inoculating 50 ml of fresh culture broth (18 hrs.).
The made polymers were tested for 24 hours at 37 °C against gram positive and gram-negative bacteria, staphylococcus aureus, and pseudomonas aeruginosa. The disc technique was applied to measure the inhibition zones. Antibacterial activity of polymer-coated nanoparticles NPs.
Staphylococcus aureus, gram negative bacteria, and biogenic polymer CMC coated Ag NPs have all been tested for their antibacterial activity. Aeruginosa pseudomonas. The agar well diffusion method was utilized for detecting the antibacterial activity of biogenic polymer CMC coated NPs. Polymer CMC coated Ag NPS with different concentrations (1.0,1.2,1.4, and 1.6 µg/ml) showed inhibition activities against all tested bacteria. The highest inhibition zone of NPs observed in Gram positive bacteria was (34mm)    with concentration (1.6 µg/ml) polymer CMC-coated Ag, while the lower inhibition zone was (25mm) in with concentration (1.0 µg/ml) in polymer CMC-coated Ag NPs.  as in (Figs. 4 and 5) and (Table 1).  It was very similar to other reports where the NPs could inhibit the growing of bacteria at high concentration.  The findings demonstrated that together Gram positive as well as Gram negative bacteria can grow without being hampered by nanoparticles. Gram positive bacteria were additionally sensitive to biogenic polymer CMC-coated Ag NPs than Gram negative bacteria. This   agrees with reported Gram positive and negative bacteria are more susceptible to NPs as a result, interpreting bacterial exposure to NPs is difficult.

CONCLUSION
Through penetrating the membrane of the cell as well as interacting through amino acids, proteins, as well as nucleic acids, the CMC-coated Ag NPs increase the inhibition zone treated with Ag NP nanoparticles. Additionally, the outcomes support the development of interactive oxygen species and consider increased pressure as well as oxidative stress. Oxidative stress was one of the markers that makes it possible to monitor how toxic heavy metals affect bacteria. The toxic effect of silver ions binding within the bacterial cell wall and plasma membrane is the foundation for this., which results in bacterial respiration being inhibited and disrupting functions like permeability and breathing. It might be fair to say that the amount of particle surface area obtainable for interaction determines the relationship amid the particles as well as the bacteria. Additionally, it is discovered which smaller particles own a greater compared to larger ones, surface, which means that they will interact with bacteria more potently than larger particles.

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