Electron diffraction spectroscopy analysis (EDS)

The EDS spectrum given in Fig. 4.6 clearly confirmed the formation of silver nanoparticles without any impurity. The presence of copper comes from the copper grid used for the EDS sample preparation.

Figure 4.6: EDS spectrum of dextrose reduced gelatin -capped Ag-NPs.

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Inhibition zone values were obtained for the as- synthesized Ag-NPs tested against two Gram negative bacteria (E.coli and P. aeruginosa). Fresh silver nitrate solution was used as control. On incubation, the plates in which disc diffusion was carried out were checked for the presence of inhibition zone around the antibiotic discs and those discs loaded with nanoparticles. Figure 4.7 illustrate the images of each inhibition zones. The diameter of each inhibition zones measured and the results of the finding are shown in Table 4.1. The results show that, there was zone of growth inhibition around all the discs tested against the E. coli except for the ciprofloxacin disc. In case of P. aeruginosa, there was zone of inhibition around all the discs except the two antibiotic discs.

The preliminary disc diffusion method indicated that all the as-synthesized Ag-NPs showed antibacterial activity against the two bacteria under study. At the same time both bacteria were shown to be resistant to the present drug of choice ciprofloxacin. E coli were found to be sensitive to imipenem, whereas P.aeruginosa was resistant to the antibiotic highlighting its drug resistant nature. The MIC of the samples were evaluated using the microtube broth dilution technique and the results of the finding are shown in Table 4.2.

The MIC of the silver nanoparticles at different reaction times was found to be between 10 - 12.5 µg/mL against E.coli and between 6-18.37 µg/mL against P.aeruginosa. The MBC were between 12.5-18.37 for both E.coli and P. aeruginosa respectively. The MIC of the antibiotics was also evaluated and was found to be 16 µg/mL for both E.coli and P.aeruginosa in case of imipenem. For the ciprofloxacin, the MIC was found to be 10 and 20 µg/mL respectively for E.coli and P.aeruginosa. The as-synthesized Ag-NPs at

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different reaction times show better efficacy against the two bacteria than imipenem and silver nitrate, and a better efficacy against P.aeruginosa than ciprofloxacin. However, the antibiotic, ciprofloxacin showed a stronger antibacterial activity against E.coli than most of the as-synthesized Ag-NPs. Contrary to the reports that, the smaller the Ag-NPs the higher the antimicrobial activity, the results of this analysis showed that, the smaller Ag- NPs produced at higher reaction time showed lower antibacterial activity. This anomaly has been attributed to the effective passivation of the smaller Ag-NPs surface by the gelatin, thus reducing its antimicrobial activity as well as its toxicity. However, the antimicrobial activity is still stronger than silver nitrate except for Ag-NPs produced at 30 h reaction time. Furthermore, the as-synthesised Ag-NPs also have bactericidal effects resulting not only in inhibition of bacterial growth but also in killing bacteria. This irreversible inhibition of bacterial growth has been reported to be desirable to prevent bacterial colonization of silver-containing medical devices, such as catheters (Rupp et al., 2004; Samuel & Guggenbichler, 2004) where bacteria-killing activity is required. The MBC of the as-synthesised Ag-NPs against Gram-negative bacteria were the same as MIC for some of the samples (Table 4.2) and were higher than the value obtained for silver nitrate except for 30 h sample. These results clearly show that the as-synthesised dextrose reduced, gelatin capped-Ag-NPs can inhibit the growth and multiplication of the tested bacteria.

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Figure 4.7: Comparison of the inhibition zone test for Gram-negative bacteria (a) E.coli and (b) P.aeruginosa

Table 4.1: Average diameter of inhibition zone for dextrose reduced gelatin capped Ag-NPs against E.coli and P aeruginosa.

Sl

No Sample

Diameter of zone of inhibition (mm) E. coli P. aeruginosa 1 1 hour 8 12.5 2 5 hour 11 13 3 18 hour 12 13 4 24 hour 13 12 5 30 hour 12 14 6 AgNO3 11 13

7 Imipenem (10mg) 15 Not Appearing 8 Ciprofloxacin

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Table 4.2: MIC and MBC values of the dextrose reduced gelatin capped Ag-NPs against E.coli and P.aeruginosa

Sl No Sample Code

E coli ATCC E coli ATCC P.aeruginosa P.aeruginosa

MIC (µg/ml) MBC (µg/ml) MIC (µg/ml) MBC (µg/ml) 1 1 hour 10 12.5 6 12.5 2 5 hour 12.5 12.5 6 12.5 3 18 hour 12.5 12.5 12.5 12.5 4 24 hour 12.5 12.5 12.5 12.5 5 30 hour 12.5 18.37 18.37 18.37 6 AgNO3(fresh) 18.37 18.37 18.37 18.37 7 Imipenem 16 - 16 - 8 Ciprofloxacin 10 - 20 - 4.3.1.4 Toxicity study

The cell viability study for the gelatin capped-Ag-NPs at different concentration is given in Figure 4.8. Compared to the control, the as-synthesised Ag-NPs are less toxic at lower concentrations (less than 10 µg/ml). The presence of gelatin layer over the nanoparticles protect its environment and reduces the release of silver ions thereby reducing the toxicity. It has been reported that the interaction of metal ions with cells can cause a phenomena bacterial aggregation, inhibiting microbial colonization and invasion (Ryan et al., 2006). The metal ions get attached to the negative cell wall and further depleting it which causes the bacteria to aggregate. Silver in its nano size possess large surface area for the microbe to be exposed to. Though silver nanoparticles find use in many antibacterial applications, the action of this metal on microbes is not fully known. It has

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been hypothesized that silver nanoparticles can cause cell lysis or inhibit cell transduction.

Silver nanoparticles have the ability to penetrate into the bacterial cell wall thereby forming ‘pits’ which cause structural changes in the cell membrane and accumulation of the nanoparticles on the cell surface (Sondi & Salopek, 2004). The formation of free radicals by the silver nanoparticles when in contact with the bacteria have the ability to damage the cell membrane and make it porous which can ultimately lead to cell death (Danilcauk et al., 2006; Kim et al., 2007). It has also been proposed that there can be release of silver ions by the nanoparticles, and these ions can interact with the thiol groups of many vital enzymes and inactivate them (Matsumura et al., 2003). The bacterial cells in contact with silver take in silver ions, which inhibit several functions in the cell and damage the cells.

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Figure 4.8: Relative cell viability of the dextrose reduced gelatin capped Ag-NPs against human HTP-1 cell line.

Summary: Green synthesis of highly monodispersed, water soluble, stable and smaller sized dextrose reduced gelatin -capped-silver nanoparticles (Ag-NPs) via an eco-friendly, completely green method is discussed. By varying the reaction time, the temporal evolution of the growth, optical and antimicrobial properties of the as-synthesised Ag- NPs were investigated. The nanoparticles were characterized using UV-vis absorption spectroscopy, Fourier transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HR-TEM). The absorption maxima of the as-synthesized materials at different reaction time showed characteristic silver surface plasmon resonance (SPR) peak. The antimicrobial properties of the gelatin capped Ag-NPs at different reaction times where tested against Escherichia coli and Pseudomonas aeruginosa.

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