NANO PARTICLE

                                             

                        

INTRODUCTION

Medicinal and preservative properties of silver have been known for over 2,000 years. The ancient Greek and Roman civilizations used silver vessels to keep water potable. Since the nineteenth century, silver-based compounds have been widely used in bactericidal applications, in burns and in wound therapy, etc.

Over the last decades silver has been engineered into nanoparticles , structures from 1 to 100 nm in size. Owing to their small size, the total surface area of the nanoparticles is maximized, leading to the highest values of the activity to weight ratio. Due to this property being distinctly different from that of the bulk metal, silver nanoparticles have attracted much attention and have found applications in diverse areas, including medicine, catalysis, textile engineering, biotechnology and bioengineering, water treatment ,electronics and optics . Furthermore, currently silver nanoparticle used as antibacterial/antifungal agents in a diverse range of consumer products: air sanitizer sprays, socks, pillows, slippers, respirators, wet wipes, detergents, soaps, shampoos, toothpastes, air filters, coatings of refrigerators, vacuum cleaners, washing machines, food storage container , cellular phones.

 Numerous synthesis approaches were developed to obtain silver nanoparticles of various shapes and sizes, including laser ablation , gamma irradiation, electron ,chemical reduction by inorganic and organic reducing agents, photochemical methods, microwave processing ,and thermal decomposition of silver oxalate in water and in ethylene glycol . Having compared minimum inhibitory concentration (MIC) values for bacterial cultures, one can see that the antimicrobial activity of silver nanoparticles strongly depends on the method of their synthesis.

This paper deals with the authors’ research in the field of antimicrobial properties of silver nanoparticles obtained by our recently suggested electrochemical technique, which provides extremely low minimum inhibitory concentration (MIC) values as well as a high efficacy of nano silver as antimicrobial agent against a range of microbes on the surface of paints and fabrics . This paper  also provides a review of the most recent scientific publications regarding the possible toxic effects of silver nanoparticles to the environment and human health.

 REVIEW OF LITERATURE

                               Silver nanoparticles have been extensively used in health care, food, environment and biomedical sectors. Application of silver nanoparticles includes anti-bacterial, anti-viral, anti-fungal and anti-cancer activities. Silver nanoparticles are the alternative anti-bacterial agents which show the ability to overcome the resistance of bacteria against the antibiotics. A study reported the anti-microbial activity of silver nanoparticles against the E.coli, in which E.coli cells were treated with silver nanoparticles and these nanoparticles gets accumulated in cell wall and forms the pits in the cell walls which lead to the death of the cells . Another study reported the role of silver nanoparticles against E.coli, yeast and Staphylococcus aureus. In this study, it was suggested that at a lower silver nanoparticles concentration inhibits the complete growth of E.coli and yeast whereas mild effect was observed in S. aureus .Silver nanoparticles synthesized from biological method shows significant role against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis and Streptococcus pyogenes, whereas in case of Salmonella typhi and Klebsiella pneumoniae it shows moderate .  Khurana et al.  investigated the physical and surface properties of silver nanoparticles against Fungal infections are more common in patients who are immune-suppressed and overcoming this problem is not an easy process due to the less number of anti-fungal drugs. Therefore there is a requirement to develop antifungal agents which is non-toxic, biocompatible and eco-friendly in nature. Silver nanoparticles play an important role in various fungi. Silver nanoparticles showed potential against clinical isolates and ATCC strains of Candida species and Trichophyton mentagrophytes. Silver nanoparticles synthesized from biological method showed increased antifungal activity with fluconazole against Phoma herbarum, Phoma glomerata, Fusarium semitectum, Candida albicans and Trichoderma sp. .  Another study also reported the role of silver nanoparticles against several phytopathogenic fungi which include Alternaria alternata, Macrophomina phaseolina, Sclerotinia sclerotiorum,   Botrytis cinerea, Rhizoctonia solani, and Curvularia lunata  Silver nanoparticles not only inhibits human and plant pathogenic fungi, but also able to inhibit other fungal species such as Aspergillus fumigates, Penicillium brevicompactum, Chaetomium globosum,Cladosporium cladosporoides, Stachybotrys chartarum, and ortierellal

Viral diseases are frequent and becoming more prominent all over the world, therefore, there is a requirement of developing new antiviral agents. Silver nanoparticles show unique interactions with bacteria and viruses due to its certain size and shapes. Anti-viral activity of silver nanoparticles incorporated into the poly sulfone ultrafiltration membranes was evaluated against the MS2 bacteriophage and it showed the significant antiviral activity . Lara et al.   demonstrated the anti-HIV activity at an early stage of viral replication. Polyvinyl pyrrolidone coated silver nanoparticles blocks the transmission of cell-associated HIV-1 and cell-free HIV-1 isolates . Another study also reported the role of silver nanoparticles against HIV and hepatitis B virus .

 Cancer is one of the prominent diseases which infect both developing as well as the developed country. Therefore, there is a requirement to develop new techniques which are able to reduce the systemic side effects. Gopinath et al.  investigated the molecular mechanism of silver nanoparticles and found that programmed cell death was concentration-dependent. Further, the synergistic effect on apoptosis using uracil phosphoribosyl transferase expressing cells and non- uracil phosphoribosyl transferase expressing cells in the presence of fluorouracil was observed. In these conditions, it was observed that silver nanoparticles not only induce apoptosis but also sensitize cancer cells. also reported that silver embedded magnetic nanoparticles showed significant activity against breast-cancer cells and floating leukemia cells. Plant extract synthesized silver nanoparticles showed a toxic effect on the human lung carcinoma cells (A549) which indicates that silver nanoparticles could target cell-specific toxicity .

ABSTRACT

                Recent advances in nanoscience and nanotechnology radically changed the way we diagnose, treat, and prevent various diseases in all aspects of human life. Silver nanoparticles (AgNPs) are one of the most vital and fascinating nanomaterials among several metallic nanoparticles that are involved in biomedical applications. AgNPs play an important role in nanoscience and nanotechnology, particularly in nanomedicine. Although several noble          metals

have been used for various purposes, AgNPs have been focused on potential applications in cancer diagnosis and therapy. In this review, we discuss the synthesis of AgNPs using physical, chemical, and biological methods. We also discuss the properties of AgNPs and methods for their characterization. More importantly, we extensively discuss the multifunctional bio-applications of AgNPs; for example, as antibacterial, antifungal, antiviral, anti-inflammatory, anti-angiogenic, and anti-cancer agents, and the mechanism of the anti-cancer activity of AgNPs. In addition, we discuss therapeutic approaches and challenges for cancer therapy using AgNPs. Finally, we conclude by discussing the future perspective of AgNPs.

 METHODOLOGY

                                         The process of electrochemical synthesis of silver nanoparticles [10] is based on using an inexpensive two-electrode setup in which the anode and the cathode made from the bulk Ag are placed vertically, face-to-face, 10 mm apart. The electrodes are immersed into an electrochemical cell filled with 500 ml of distilled water obtained with water distiller (DE-25, Russia). In the tests reported here, the electrolysis was performed during 1 h at the temperature range of 325–340 K with a constant voltage of 20 V. Periodical changing the polarity of the direct current between the electrodes with a period of 4 min and vigorous stirring during the process of electrolysis were applied in order to reduce the agglomeration of particles. Synthesized silver nanoparticle solutions were stored under ambient conditions in glass containers. The morphology of the silver nanoparticles/ powders obtained was studied using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS) measurements. The concentration of silver nanoparticles in solutions was determined by neutron activation analysis. To evaluate the antibacterial and fungicidal properties of Ag nanoparticles Escherichia coli was used as a representative Gram-negative bacterium; Staphylococcus aureus was used as a Gram-positive bacterium; As per gillusniger andPenicillium phoeniceum were used to represent cosmopolitan saprotrophic fungi. To assay the antimicrobial activity of silver nanoparticles in aqueous solution against E. coli on solid media, the agar disk diffusion method was used. Bacteria in were prepared from a log-phase culture of E. coli K12 grown in LB-media on a rotary shaker (120 rpm) at 37°C. The in ocula were diluted with 0.9% NaCl to the 0.5 McFarland standard and 100 ml were applied onto 9 cm Mueller-Hinton agar plates with a depth of approximately 5 mm. Disks of absorbent paper (5 mm in diameter) were impregnated with 10 ml of silver nanoparticle solutions (47.5, 42.5, 22.6 and 11.3 ppm). For comparison, disks of the same diameter with 10 ml Tetracycline, Penicillin G and Ampicillin (1 g/l each) were used, leading to a concentration of the respective substance of 10

mg/disk. The freshly prepared disks were placed on the surface of the inoculated agar plates. After incubation at 37°C for 18 h the zones of bacterial inhibition were measured optically. In order to impregnate a cotton fabric with silver nanoparticles, the simple padding procedure was used. In a separate exercise, commercially available water paint was mixed with silver nanoparticles solutions in a ratio of 7:1 in order to impart antimicrobial properties to the paint. To evaluate the antibacterial and fungicidal properties of Ag nanoparticles added to a cotton fabric and a water paint, samples (1.5 × 1.5 cm) treated by different compositions of Ag nanoparticles as well as control samples were immersed in a thin layer of beef-extract agar. A 1 ml of suspension of approximately 10⁵ CFU/ml density of the microorganisms to be tested were distributed uniformly on agar surface and incubated at 28°C (CFU = colony forming units). Antimicrobial activity was evaluated according to the presence or absence of microbial growth just above the sample after a 24-h incubation for bacteria and a 72-h incubation for fungi. All microbiological tests were performed in triplicate.

MICs of silver nanoparticle solutions for various microbes were determined using the macro dilution broth susceptibility test. Nutrient broth used in the macro dilution method contained peptic digest of animal tissue 50.00 g/l; beef extract 1.5 g/l; sodium chloride 5.00 g/l; glucose 5 g/l; pH 7.4 ± 0.2. A standardized suspension of approximately 10⁶ CFU/ml density was obtained by inoculating the culture in nutrient broth (Hi-Media) and incubating the tubes at 37°C for 3 h. Ten milliliter of the standardized culture suspension was then inoculated and tubes were incubated at 37°C for 24 h. MIC was defined as the lowest concentration of the inhibiting agent that completely inhibited bacterial growth, the unit for MIC was chosen as mg (Ag)/l. MIC was examined visually, by checking theturbidity of the tubes.                          

FINDING

                                     It was shown by DLS measurements that a typical sample of silver nanoparticles solution obtained by the two-electrode setup described above contains not only nanoparticles, but also a small amount of large (>100 nm) colloidal silver particles. In order to remove these coarse particles and to provide reduction of silver ions present in the solution, we used filtration of the solution through a 3-μm pore size paper filter. The filter narrows the range of size distributions of synthesized silver nanoparticles while providing additional reduction of Ag ions according to the following reaction: Ag+1 + e → Ag0. As a result, the ratio of the concentrations of silver ions and silver nanoparticles suspended in the solution is reduced. A final stage of Ag nanoparticle synthesis involves additional treatment of the smallest-size fraction of silver nanoparticles remaining in solution after the filtering stage. It consists of adding hydrogen peroxide to a level of up to 0.005% concentration of H2O2 to the solution. Due to the reaction Ag2O + H2O2 → 2Ag + H2O + O2 silver oxide is reduced to Ag which is released in the solution. By this process the size of the silver nanoparticles is reduced, while new Ag nanoparticles may be forming as well. Examination of TEM images taken 2 weeks after the addition of H2O2 revealed that silver nanoparticles suspended in water solution were nearly spherical and that their size distribution fell in the range of 2–20 nm, the average size being about 7 nm, cf. Figure 1.

                                  

                                  In this figure antibacterial effect of silver colloids with the concentrations of 47.5, 42.5, 22.6 and 11.3 ppm is presented vis-à-vis to that of known antibiotics. The concentrations of silver were selected in such a way as to correspond to maximum Ag concentrations used in consumer nanoproducts which are currently available on the market. Comparing zones of growth inhibition around the disks impregnated with various antibiotics and Ag nanoparticles, one can see that silver nanoparticle solution demonstrates a certain antimicrobial effect. The intensity of the effect is increased with the concentration of the solution. Figure 3 demonstrates zones of growth inhibition around the disks impregnated with various antibiotics and the disk with the largest Ag nanoparticles concentrations that we have used. Considering that the Ag concentration used in the experiment was approximately 20 times lower than that of the antibiotics, one can expect that silver nanoparticles would outperform Ampicillin, Penicillin and Tetracycline antibiotics of the same concentration.

        In order to reveal an effect of the size of silver nanoparticles on their bactericidal efficiency the minimum inhibitory concentration (MIC) assays were conducted against the gram-negative bacterium E. coli and the gram-positive bacteria S. aureus and B. subtilis. The results for MIC assays shown in Table 1 demonstrate that smaller silver nanoparticles had a greater antibacterial efficacy. The conducted MIC assays have also shown clearly that the proposed electrochemical technique provides very high antimicrobial activity of synthesized silver nanoparticles.

 

Zones of growth in habitation around disks impregnated with silver nano particles and various antibiotics.

provides very high antimicrobial activity of synthesized silver nanoparticle. For example, al .have recently proposed a method synthesis of Ag nanoparticles that provided the same MIC values for E. coli and S. aureus varieties as in the present study. Sarkar and coauthors claimed that “such a low value of MIC showed by silver nano particles is unprecedented”. The results obtained for larger nanoparticles (with a mean size of 70 nm) are in good agreement with

assays for E. coli for colloidal silver Stabilized by sodium oleate. On the other hand, MIC values for the same bacteria obtained by rupareli et. Al are higher than those presented in Table 1, although they studied smaller silver nanoparticles (3.32 ± 1.129 nm). We suppose that it is mainly connected with the high purity of nanoparticles obtained by our electrochemical technique without surfactants. Unfortunately, existing studies on nanotoxicity were concentrated on empirical evaluation of the toxicity of various nanoparticles, with less regard given to the relationship between          nanoparticle properties and toxicity.                            .

CONCLUSION

Silver nanoparticles have attracted the attention of researchers because of their unique properties, and proven applicability in diverse are such as medicine, catalysis, textile engineering, biotechnology, nanobiotechnology, bioengineering sciences, electronics, optics, and water treatment. Moreover, silver nanoparticles have significant inhibitory effects against microbial pathogens, and are widely used as antimicrobial agents in a diverse range of consumer products, including air sanitizer sprays, socks, pillows, slippers, respirators, wet wipes, cosmetics, detergents, soaps, shampoos, toothpastes, air and water filters, coatings of refrigerators, vacuum cleaners, bone cement, wound dressings, surgical dressings, washing machines, food storage packaging, and cell phones. The flexibility of silver nanoparticle synthetic methods and facile incorporation of silver nanoparticles into different media have interested researchers to further investigate the mechanistic aspects of antimicrobial, antiviral and anti-inflammatory effects of these nanoparticles. In brief, there are limited well-controlled investigations on potential toxicities of nano-silver particles, and it seems that additional long-term studies (preferably using multiple particle sizes) are needed to better characterize and understand the risk of using these particles. Various chemical, physical and biological synthetic methods have been developed to obtain silver nanoparticles of various shapes and sizes, including laser ablation, gamma irradiation, electron irradiation, chemical reduction, photochemical methods, microwave processing, and thermal decomposition of silver oxalate in water and in ethylene glycol, and biological synthetic methods. Most of these methods are still in the development stages and the problems experienced involve the stability and aggregation of nanoparticles, control of crystal growth, morphology, size and size distribution, and occasional difficulty in the management of the synthesis, as in the case of the radiolysis technique. Moreover, the separation of produced nanoparticles for further applications is still an important issue. By using different reducing agents and stabilizers, the particle size and morphology of silver nanoparticles have been controlled. Selection of solvent medium and selection of ecofriendly nontoxic reducing and stabilizing agents are the most important issues which must be considered in green synthesis of silver nanoparticles. In biological synthetic methods, it was shown that the silver.