Characterization of Silver Nanoparticles Obtained by a Green Route and Their Evaluation in the Bacterium of Pseudomonas aeruginosa

: Metal nanoparticles are widely used in di ﬀ erent areas such as biotechnology and biomedicine, for example in drug delivery, imaging and control of bacterial growth. The antimicrobial e ﬀ ect of silver has been identiﬁed as an alternative approach to the increasing bacterial resistance to antibiotics. Silver nanoparticles were synthesized by the green route using the Geranium extract as a reducing agent. The characterization was carried out by the techniques of UV-Vis spectrophotometry, transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), X-ray emitted photoelectron spectroscopy (XPS) and X-ray di ﬀ raction. Nanoparticle diameters between 15 and 50 nm were obtained and the interplanar spaces calculated from the electron di ﬀ raction pattern corresponding to a mixture of silver with 4H and FCC structures. To determine the minimum inhibitory concentration of silver nanoparticles (AgNPs) on the Pseudomonas aeruginosa bacteria (ATCC-27853), di ﬀ erent concentrations of colloidal solution 0.36, 0.18, 0.09 and 0.05 µ g / mL were evaluated as a function of the incubation time, measuring the inhibition halo and colony forming unit (CFU) during 0, 2 and 4 h of incubation. The minimum inhibitory AgNPs concentration (MIC) is 0.36 µ g / mL at 0 h while the concentration of 0.18 µ g / mL presents a total inhibition of the bacterium after 2 h. For the rest of the dilutions, gradual inhibitions as a function of time were observed. We evaluate the antibacterial e ﬀ ect of silver nanoparticles obtained by a green methodology in Pseudomonas aeruginosa bacteria. Finally, the colloidal nanoparticle solution can be an antibacterial alternative for di ﬀ erent biomedical approaches.


Introduction
Recently, nanotechnology has revolutionized several areas of science in the search for new alternatives to improve the living conditions of humans, both in health and environmental fields [1][2][3][4].

Strain and Growth Culture
Pseudomonas aeruginosa (ATCC-27853) was incubated in nutrient broth at 35 • C in a natural convection incubator (INB 500, Memmert, WI, USA) for 24 h until reaching an optical density equivalent to 0.5 on the McFarland scale.

Extract Preparation
Extract was prepared using fresh leaves of Geranium. Leaves were washed and mixed with distilled water up to boiling point for 5 min. Finally, the solution was filtered using a vacuum Kitasato filter (Büchner funnel, Busch R5 RA 0010C vacuum pump, Munich, Germany) and refrigerated at 4 • C.

Synthesis of Silver Nanoparticles (AgNPs)
Silver nanoparticle colloidal solution was synthesized by a green method, through the reduction of silver nitrate (AgNO 3 ) NC-0846 (Science company, Lakewood, CO, USA), using 90 mL at a concentration of 5 mM mixed with 10 mL of Geranium extract. The reaction was carried out at 85 • C for 2 h.

Nanoparticles Characterization
To determine the initial concentration of the colloidal solutions of AgNPs, the samples were analyzed by an accredited external laboratory (Laquimia lab) of the Federal Commission for the Protection Against Sanitary Risks (COFEPRIS, https://www.gob.mx/cofepris/documentos/relacion-deterceros-autorizados) using the EPA 6020A analytical method with an inductively coupled plasma mass spectrometry system (ICP-MS). UV-Vis absorption spectra were measured by a spectrophotomer (NanoDrop 2000 c, Thermo Fisher Scientific, Wilmington, USA) in the wavelength range from 200 to 900 nm with a resolution of 1 nm. Diameter of nanoparticles was determined by transmission electron microscopy, and 3 µL of the colloidal solution was collected in a copper mesh grid coated with Crystals 2020, 10, 395 3 of 13 carbon substrate Ted Pella, using a transmission electron microscope (JEOL JEM-1010, Tokyo, Japan) at 80 kV. XRD measurement was recorded in a diffractometer from Rigaku using an X-source of CuKα, operated with a step size 2 θ = 0.01 • and a step time of 1s. HRTEM micrographs were taken with a JEM-2100 (JEOL, Tokyo, Japan) microscope, equipped with a LaB6 source operated from 80 to 200 kV. X-ray photoelectron spectroscopy (XPS) was carried out using a K-alpha spectrometer (Thermo Fischer Scientific, Wilmington, USA) with a monochromatic Al Kαradiation (1486.6 eV) as an X-Ray source, and was micro-focused at the source to give a spot size on the sample of 400 microns in diameter. XPS assessment and high-resolution spectra were collected using analyzer pass energies of 120 and 40 eV, respectively. The samples remained in the pre-chamber for 15 h and later shifted to the analytical chamber with a base pressure of 1 × 10 −9 Torr. A quantity of 100 µL of each sample was added to nutritive (Nutrient Agar) agar plates during different incubation times (0, 2, and 4 h), distributing the sample on the plate with a Digralsky loop (Digralsky spreader). The plates were incubated at 35 • C in a natural convection incubator for 24 h and then the CFU count was performed.

Antibacterial Sensitivity
Dilutions of silver nanoparticles were made in distilled water at concentrations of 0.36, 0.18, 0.09 and 0.05 µg/mL. Sterile filter paper disks of approximately 5 mm in diameter with 1 µL of each concentration were dispensed into a sterile Petri dish. Sterile swabs were used to inoculate Mueller Hinton Agar plates, immersing the tip of the latter in the culture of P. aeruginosa and passing the wet swab over the entire surface of the plate. Using sterile forceps, the dry disc was taken, placing it on the inoculated plate and pressing gently. Three discs were placed per plate and incubated at 35 • C for 24 h.

Results and Discussion
The synthesis of nanoparticles is considered a complex, expensive and contaminating process due to the use of chemicals and reagents, so it is convenient to use green synthesis, using plants to obtain extracts with high antioxidant capacity. The extract reacts with a silver nitrate solution, initiating the reduction of the metal ions and inducing the formation of the nucleation centers and, thus, forming the nanoparticles [23,29]. Once the reaction was initiated, samples were taken at 0, 30, 60, 120 and 180 min to monitor the changes in color solution that indicate the formation of the nanoparticles. Due to the excitation of plasmon surface vibrations in the metallic nanoparticles, it is possible to observe that the solution turns a brown color from 20 min after the start of the reaction kinetics [43,44]. The results obtained by the authorized external laboratory on the concentration of colloidal silver in a 1:1 ratio was 0.72 × 10 −3 mg/mL, according to the ICP-MS analytical method applied.
In UV-Vis technique measurements, the absorption peak depends mainly on the morphology, structure and size of the particles that are obtained through some green or chemical synthesis methodology [45,46]. Using a sweep in the range of 200 to 900 nm, the absorption peak was obtained at 435 nm ( Figure 1A), which is very similar to those reported by different researchers. In the case of chemical synthesis methods, the absorption peak of AgNPs is within the range of 398-406 nm at 65 • C using NaBH4. In other research works, it has been reported that after 120 min of the reaction, the absorption peak is located at 434 nm, when the extract of M. wightiana is used as a reducing agent for a synthesis methodology by the green route [47][48][49]. 50 nm. Figure 1B to 1D shows a representative micrograph of the nanoparticles synthesized by the green route with a uniform spherical morphology. Unlike chemical methods, the average size ranges between 10 and 20 nm [48], while for M. wightiana it is from 15-65 nm with irregular and polydisperse morphologies [47]. The difference in morphology and size distribution can be explained by the different mechanisms of nucleation and growth, which depend on multiple conditions, including temperature and time [50,51]. At higher temperatures, the induction period is accelerated, and in both the nucleation stages a greater number of particles are formed, but this is smaller compared to lower temperature conditions [52,53]. Figure 2 shows the XRD pattern of silver nanoparticles. Small diffraction peaks belonging to the trigonal structure of AgNO3 (COD entry No. 96-210-5348) and cubic-Ag2O (JCPDS No. 761396) revealed that the reduction reaction of Ag-precursor was incomplete. In addition, a FCC-Ag structure was identified with high peaks centered around 2 θ = 38.202, 44.402, 64.602, 77.6 and 81.758°, corresponding to (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2), respectively (JCPDS No. 040783). However, the experimental pattern slightly shifted towards higher angles compared to JCPDS card (blue lines). This was evidence of cell contraction of Ag nanoparticles with FCC structure. Interestingly, weak diffractions at 2 θ = 35.89, 37.043 and 40.3° (red lines) were also observed and assigned to the (1 0 0), (1 0 1) and (1 0 2) planes of the hexagonal silver 4H structure (ICDS No. 064707). This result was unexpected, as 4H phase is hardly ever reported in nanoparticle synthesis by green routes. In order To document the morphological properties of the synthesized nanoparticles, transmission electron microscopy was performed. The average size of the nanoparticles obtained was from 15 to 50 nm. Figure 1B-D shows a representative micrograph of the nanoparticles synthesized by the green route with a uniform spherical morphology.
Unlike chemical methods, the average size ranges between 10 and 20 nm [48], while for M. wightiana it is from 15-65 nm with irregular and polydisperse morphologies [47]. The difference in morphology and size distribution can be explained by the different mechanisms of nucleation and growth, which depend on multiple conditions, including temperature and time [50,51]. At higher temperatures, the induction period is accelerated, and in both the nucleation stages a greater number of particles are formed, but this is smaller compared to lower temperature conditions [52,53]. Figure 2 shows the XRD pattern of silver nanoparticles. Small diffraction peaks belonging to the trigonal structure of AgNO 3 (COD entry No. 96-210-5348) and cubic-Ag 2 O (JCPDS No. 761396) revealed that the reduction reaction of Ag-precursor was incomplete. In addition, a FCC-Ag structure was identified with high peaks centered around 2 θ = 38.202, 44.402, 64.602, 77.6 and 81.758 • , corresponding to (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2), respectively (JCPDS No. 040783). However, the experimental pattern slightly shifted towards higher angles compared to JCPDS card (blue lines). This was evidence of cell contraction of Ag nanoparticles with FCC structure. Interestingly, weak diffractions at 2 θ = 35.89, 37.043 and 40.3 • (red lines) were also observed and assigned to the (1 0 0), (1 0 1) and (1 0 2) planes of the hexagonal silver 4H structure (ICDS No. 064707). This result was unexpected, as 4H phase is Crystals 2020, 10, 395 5 of 13 hardly ever reported in nanoparticle synthesis by green routes. In order to provide deeper knowledge of the crystal structure, HRTEM analyses were carried out for different nanoparticles of the sample.
Crystals 2020, 10, x FOR PEER REVIEW 5 of 13 to provide deeper knowledge of the crystal structure, HRTEM analyses were carried out for different nanoparticles of the sample.  Figure 3A and 3B show an example of HRTEM and IFFT acquired in some Ag nanoparticles; the interplanar spaces calculated from the electron diffraction pattern correspond to (1 0 1), (1 0 2) and (1 0 3) planes of a contracted hexagonal close-packed structure (HCP). This result was unexpected, but confirmed the XRD results already discussed, as a face-centered cubic structure (FCC) for metallic silver is typically found in synthesis reports of Ag nanoparticles [50][51][52][53][54][55][56]. However, recent studies have also reported the formation of HCP structures in silver thin films and nanoparticles obtained from chemical and green synthesis methods [57]. For instance, Chakraborty et al. [58] observed the presence of 2H and 4H hexagonal polytypes in Ag nanorods, and Leite et al. [59] reported the formation of a HCP structure from stacking faults in FCC crystals at a high temperature of synthesis using sodium borohydride as a reductive agent [58,59].
The lattice parameter contractions (Equation 1) obtained from interplanar distances were in the range of −1.97% to −1.39%, compared to the JCPDS-411402 value (a = 2.88 A). Similarly, Figure 3C,D show an example of a nanoparticle with FCC structure found in the same sample. The lattice contraction has an average of −0.83%, which was less than those found in 4H nanoparticles. The lattice contraction of pure silver nanoparticles with FCC structure has been predicted by theoretical calculations, and confirmed by experimental data; the lattice contraction found in the present study was similar to those predicted for Ag nanoparticles smaller than 20 nm (−0.8%) [60][61][62][63]. However, further analysis should be carried out to study the possible correlation between the type of structure and lattice expansion in the inhibitory activity of Ag.  Figure 3A,B show an example of HRTEM and IFFT acquired in some Ag nanoparticles; the interplanar spaces calculated from the electron diffraction pattern correspond to (1 0 1), (1 0 2) and (1 0 3) planes of a contracted hexagonal close-packed structure (HCP). This result was unexpected, but confirmed the XRD results already discussed, as a face-centered cubic structure (FCC) for metallic silver is typically found in synthesis reports of Ag nanoparticles [50][51][52][53][54][55][56]. However, recent studies have also reported the formation of HCP structures in silver thin films and nanoparticles obtained from chemical and green synthesis methods [57]. For instance, Chakraborty et al. [58] observed the presence of 2H and 4H hexagonal polytypes in Ag nanorods, and Leite et al. [59] reported the formation of a HCP structure from stacking faults in FCC crystals at a high temperature of synthesis using sodium borohydride as a reductive agent [58,59].
The lattice parameter contractions (Equation 1) obtained from interplanar distances were in the range of −1.97% to −1.39%, compared to the JCPDS-411402 value (a = 2.88 A). Similarly, Figure 3C,D show an example of a nanoparticle with FCC structure found in the same sample. The lattice contraction has an average of −0.83%, which was less than those found in 4H nanoparticles. The lattice contraction of pure silver nanoparticles with FCC structure has been predicted by theoretical calculations, and confirmed by experimental data; the lattice contraction found in the present study was similar to those predicted for Ag nanoparticles smaller than 20 nm (−0.8%) [60][61][62][63]. However, further analysis should be carried out to study the possible correlation between the type of structure and lattice expansion in the inhibitory activity of Ag. XPS analysis was carried out to study the composition and oxidation state of Ag nanoparticles. A survey scan showed the presence of different elements such as Ag, O, C, N, K, S, Cl, Mg and Na. Further detailed scans were performed to calculate the following concentration of each element in weight percent: 41.8%, 25.6%, 21.7%, 6.7%, 2.3%, 0.9%, 0.5%, 0.4% and 0.2% (in the same sequence). The absence of a cleaning process resulted in high content of elements different than Ag in the sample. Additionally, the deconvolution of detailed scans in the Ag3d region showed the presence of two oxidation states for the silver. Green peaks located at 373.8 and 367.8 eV correspond to the Ag +1 component (3d3/2 and 3d3/2), whereas blue signals sited at 374.3 and 368.3 eV belong to 3d3/2 and 3d3/2 photoemissions of metallic silver, Ag 0 ( Figure 4). The binding energies agreed with previous XPS reports [62]. Ag 0 and Ag +1 proportion results were very similar (51% and 49%, respectively). This result confirmed that synthesis parameters were not sufficient to completely reduce the silver from AgNO3 precursor. Considering that XRD diffractions for AgNO3 and Ag2O were very weak and the XPS concentration of Ag +1 species was high, it was concluded that part of silver from the precursor simply crystallized as a non-identified organometallic compound from geranium (see un-identified peaks with higher intensity at 2 θ = 31 and 26.5°). XPS analysis was carried out to study the composition and oxidation state of Ag nanoparticles. A survey scan showed the presence of different elements such as Ag, O, C, N, K, S, Cl, Mg and Na. Further detailed scans were performed to calculate the following concentration of each element in weight percent: 41.8%, 25.6%, 21.7%, 6.7%, 2.3%, 0.9%, 0.5%, 0.4% and 0.2% (in the same sequence). The absence of a cleaning process resulted in high content of elements different than Ag in the sample. Additionally, the deconvolution of detailed scans in the Ag3d region showed the presence of two oxidation states for the silver. Green peaks located at 373.8 and 367.8 eV correspond to the Ag +1 component (3d 3/2 and 3d 3/2 ), whereas blue signals sited at 374.3 and 368.3 eV belong to 3d 3/2 and 3d 3/2 photoemissions of metallic silver, Ag 0 ( Figure 4). The binding energies agreed with previous XPS reports [62]. Ag 0 and Ag +1 proportion results were very similar (51% and 49%, respectively). This result confirmed that synthesis parameters were not sufficient to completely reduce the silver from AgNO 3 precursor. Considering that XRD diffractions for AgNO 3 and Ag 2 O were very weak and the XPS concentration of Ag +1 species was high, it was concluded that part of silver from the precursor simply crystallized as a non-identified organometallic compound from geranium (see un-identified peaks with higher intensity at 2 θ = 31 and 26.5 • ).  For the analysis of antibacterial activity, P. aeruginosa was incubated until reaching turbidity of 0.5 (McFarland scale) corresponding to a concentration of 1.5 × 10 8 CFU/mL (  Tests were performed using nanoparticle concentrations of 0.36, 0.18, 0.09, 0.05 and 0.02 μg/mL. As shown in Figure 5, P. aeruginosa has a short latency phase and an exponential phase with a duration of 8 hours [65,66]; therefore, contact times of 0, 2 and 4 h were determined to ensure the culture was within the exponential phase. Figure 5 shows the plates for limit concentrations; for 0.02 μg/mL it is possible to observe abundant growth of colonies, while for the concentration 0.72 μg/mL there is near-zero growth.    Tests were performed using nanoparticle concentrations of 0.36, 0.18, 0.09, 0.05 and 0.02 µg/mL. As shown in Figure 5, P. aeruginosa has a short latency phase and an exponential phase with a duration of 8 h [65,66]; therefore, contact times of 0, 2 and 4 h were determined to ensure the culture was within the exponential phase.  For the analysis of antibacterial activity, P. aeruginosa was incubated until reaching turbidity of 0.5 (McFarland scale) corresponding to a concentration of 1.5 × 10 8 CFU/mL (Table 1) [64].  Tests were performed using nanoparticle concentrations of 0.36, 0.18, 0.09, 0.05 and 0.02 μg/mL. As shown in Figure 5, P. aeruginosa has a short latency phase and an exponential phase with a duration of 8 hours [65,66]; therefore, contact times of 0, 2 and 4 h were determined to ensure the culture was within the exponential phase. Figure 5 shows the plates for limit concentrations; for 0.02 μg/mL it is possible to observe abundant growth of colonies, while for the concentration 0.72 μg/mL there is near-zero growth.   Figure 5 shows the plates for limit concentrations; for 0.02 µg/mL it is possible to observe abundant growth of colonies, while for the concentration 0.72 µg/mL there is near-zero growth.
Subsequently, P. aeruginosa was incubated in nutrient broth at 35 • C with constant agitation promoting contact with the AgNPs. The colony-forming units (CFU) were counted on nutritive agar plates for each sample; the plates were allowed to incubate for 24 h at 35 • C. Figure 6 and Table 2 show, quantitatively and qualitatively, the variation of the growth of colonies according to the concentration of nanoparticles and incubation time. The highest concentration corresponds to 0.36 µg/mL, which does not present any growth at any incubation time, while for the concentration of 0.18 µg/mL, inhibitory activity is observed after 2 h. In the case of the lowest concentrations (0.09 and 0.05 µg/mL), a slight decrease in the growth of colonies is observed, presenting as inhibitory at 4 h. Subsequently, P. aeruginosa was incubated in nutrient broth at 35 °C with constant agitation promoting contact with the AgNPs. The colony-forming units (CFU) were counted on nutritive agar plates for each sample; the plates were allowed to incubate for 24 hrs at 35 °C. Figure 6 and Table 2 show, quantitatively and qualitatively, the variation of the growth of colonies according to the concentration of nanoparticles and incubation time. The highest concentration corresponds to 0.36 μg/mL, which does not present any growth at any incubation time, while for the concentration of 0.18 μg/mL, inhibitory activity is observed after 2 h. In the case of the lowest concentrations (0.09 and 0.05 μg/mL), a slight decrease in the growth of colonies is observed, presenting as inhibitory at 4 h.   Table 3 shows, in summary, the conditions used in cultures of P. aeruginosa with different sizes of AgNPs and the minimum inhibitory concentrations obtained. All experiments were always carried out under the same conditions. The minimum inhibitory concentration to eliminate bacteria depends mainly on the diameter and concentration stored by the nanoparticles in the colloidal solution.    Table 3 shows, in summary, the conditions used in cultures of P. aeruginosa with different sizes of AgNPs and the minimum inhibitory concentrations obtained. All experiments were always carried out under the same conditions. The minimum inhibitory concentration to eliminate bacteria depends mainly on the diameter and concentration stored by the nanoparticles in the colloidal solution.
Subsequently, a disk test was performed to analyze the antimicrobial activity of the AgNPs based on measurement of the inhibition zones, using concentrations of 0.72, 0.36, 0.18, 0.09, 0.05 µg/mL on Mueller-Hinton agar plates. Figure 7 shows the inhibition halos obtained; although the halos are not very evident, the decrease of these can be observed, as the concentration of AgNPs decreases. μg/mL on Mueller-Hinton agar plates. Figure 7 shows the inhibition halos obtained; although the halos are not very evident, the decrease of these can be observed, as the concentration of AgNPs decreases.  Table 4 shows the average values of the inhibition halos; the largest halo is 1.08 ± 0.2 cm, obtained at the concentration of 0.72 μg/mL; this value is close to 1.4 ± 0.32 cm at a concentration of 31 μg/mL [65] after 24 h. For the lowest concentrations, there is no significant variation between 0.09 and 0.05 μg/mL.

Conclusions
Silver nanoparticles were synthesized by a green method using Geranium extract as a reducing agent. The presence of AgNPs with a diameter between 15 and 50 nm was determined by UV-Vis measurements, HRTEM and TEM imaging. The antibacterial activity of AgNPs in solution against Pseudomonas aeruginosa (ATCC-27853) was determined with a minimum inhibitory AgNPs concentration of 0.36 μg/mL at 0 hours, while the concentration of 0.18 μg/mL presents a total inhibition of the bacterium after 2 h. We suggest that AgNPs can be an optimal antibacterial alternative for biomedical applications. Future work can be on cytotoxicity analysis in living cells,  Table 4 shows the average values of the inhibition halos; the largest halo is 1.08 ± 0.2 cm, obtained at the concentration of 0.72 µg/mL; this value is close to 1.4 ± 0.32 cm at a concentration of 31 µg/mL [65] after 24 h. For the lowest concentrations, there is no significant variation between 0.09 and 0.05 µg/mL. The mechanisms of action presented by the AgNPs against bacterial cultures depend on their size, shape, surface, surface charge, solubility, time of exposure and concentration. In studies on Pseudomonas aeruginosa cultures, it is due to the inhibition of several proteins present in the membrane related to the transport of ions, causing oxidative stress [70,71].

Conclusions
Silver nanoparticles were synthesized by a green method using Geranium extract as a reducing agent. The presence of AgNPs with a diameter between 15 and 50 nm was determined by UV-Vis measurements, HRTEM and TEM imaging. The antibacterial activity of AgNPs in solution against Pseudomonas aeruginosa (ATCC-27853) was determined with a minimum inhibitory AgNPs concentration of 0.36 µg/mL at 0 h, while the concentration of 0.18 µg/mL presents a total inhibition of the bacterium after 2 h. We suggest that AgNPs can be an optimal antibacterial alternative for biomedical applications. Future work can be on cytotoxicity analysis in living cells, using the minimum inhibitory AgNP concentrations or complex.