The characterization of the synthesized silver nanoparticles (Ag NPs) was carried out by using UV–vis absorption spectroscopy at ambient temperature. Figure 2 shows the UV–vis absorption measurement results of different Ag NPs, which are either covered by only chitosan (Ch-Ag NPs) or chitosan with quercetin and caffeic acid as co-capping agents (Ch/Q- and Ch/CA-Ag NPs), and pictures of the nanoparticle solutions under daylight.

Figure 2: UV–vis absorption spectra of (a) chitosan-only (Ch-), (b) chitosan/quercetin (Ch/Q-), and (c) chitosan/caffeic acid (Ch/CA-) capped Ag NPs. (d) The picture of the synthesized Ch-, Ch/Q-, and Ch/CA-Ag NPs under daylight.

As seen in Figure 2a, the absorption curve of the Ch-Ag NPs ends at around 600 nm, and the maximum for the characteristic surface plasmon resonance (SPR) absorption peak, which is due to the collective oscillation of free surface electrons in resonance with the electric field component of incoming photons, is located at 404 nm. However, the evolution of the absorption curve exhibits changes after the introduction of quercetin (or caffeic acid) into the structure of chitosan surrounding the Ag NPs. For instance, it is seen in Figure 2b and Figure 2c that the SPR peaks shift to 417 and 424 nm for Ch/Q- and Ch/CA-Ag NPs, respectively, while their absorption curves broaden to the near-IR region of the spectrum. The redshift of the SPR peak implies an increase in the size of Ag NPs. In addition, the broadening of the spectrum can be explained by the increasing size and shape distribution arising from excess co-capping agents, which are present in the reaction medium during the reduction of Ag ions. The broadening in the absorption spectra is also reflected in the color of nanoparticle solutions (see Figure 2d). Compared to the reference sample (i.e., Ch-Ag NPs), new absorption shoulders appeared at the higher energy side of the spectra (i.e., at about 200 and 345 nm) in the modified chitosan structure with quercetin or caffeic acid. These new shoulders indicate the presence of quercetin and caffeic acid in the modified chitosan surrounding Ag NPs. As seen in Figure 2b, the peaks at around 200, 250, and 370 nm in the absorption curve of quercetin match well with the shoulders that appear due to the contribution of quercetin in the absorption spectrum of Ch/Q-Ag NPs. A similar behavior can also be seen in the absorption spectrum of the Ch/CA-Ag NPs with a small shift compared to that of caffeic acid. The absorption peak at around 325 nm of the caffeic acid does not fully match with the shoulder appearing at the higher energy side of the SPR peak of the Ch/CA-Ag NPs while its absorption tail overlaps with that of the SPR peak. These partial matches in the absorption features might be due to the molecular interactions between caffeic acid and chitosan/silver nanostructures [63].

FTIR spectroscopy measurements were performed to confirm the formation of Ch/Q- and Ch/CA-Ag NPs and to obtain information about the interaction in the synthesized materials using characteristic bands of functional groups. Figure 3 depicts the FTIR spectra of the synthesized Ag NPs and the pure materials (quercetin, caffeic acid, and chitosan) used in the structure of the shell layers.

Figure 3: FTIR spectra of (a) the chitosan/quercetin (Ch/Q-)-, and (b) the chitosan/caffeic acid (Ch/CA-)-capped Ag NPs, including the spectra of chitosan, quercetin, and caffeic acid used in the synthesis of the NPs.

In the FTIR spectrum of pure quercetin (Figure 3a) the bands observed at about 3408 and 3292 cm−1 correspond to phenolic O–H stretching [64]. The peak at 1671 cm−1 stems from the stretching of the C=O carbonyl functional group. The characteristic C=C aromatic ring stretching bands were observed at 1614, 1562, and 1514 cm−1. The peaks at 1355, 1314, and 1265 cm−1 were attributed to aromatic C–H and O–H bending vibrations in quercetin. The FTIR spectrum of pure caffeic acid (Figure 3b) shows intense bands at 3401 and 3220 cm−1 indicating O–H stretching. The very strong band at 1640 cm−1 corresponds to a conjugated C=O stretching vibration. The characteristic bands at 1600, 1523, and 1447 cm−1 were assigned to both aromatic and olefinic C=C stretching modes. The strong band at 1273 cm−1 is due to the in-plane bending of C–H bonds. The FTIR spectrum of pure chitosan exhibit overlapping O–H and N–H stretching bands showing a broad band between 3500 and 3000 cm−1. C–H stretching is evident at 2873 cm−1. The bands at 1655, 1579, and 1320 cm−1 were assigned to C=O stretching (amide I), N–H bending (amide II), and C–N stretching (amide III) modes, respectively, due to the presence of residual N-acetyl groups [63,65]. The peaks at 1421 and 1379 cm−1 are due to C–H bending and symmetrical deformation modes. In the FTIR spectra of Ch/Q- and Ch/CA-Ag NPs, a decrease in the intensity of broad bands at around 3500–3000 cm−1 were attributed to the hydrogen bonding interactions between amino groups of chitosan and phenol groups of quercetin and caffeic acid [63,66]. Moreover, it was observed that the position and shape of some peaks, such as 1671 and 1514 cm−1 for quercetin, 1421 and 1379 cm−1 for chitosan, and 1640 and 1447 cm−1 for caffeic acid, also changed due to the formation of links between quercetin–chitosan–Ag and caffeic acid–chitosan–Ag in the synthesized NPs.

TEM was utilized to investigate the size and morphological features of the synthesized Ag NPs. Figure 4 shows TEM images of Ch/Q- and Ch/CA-Ag NPs. As can be seen from the TEM images in Figure 4a and Figure 4b, the Ag NPs covered by chitosan layers comprising quercetin or caffeic acid have mostly spherical shapes. However, both samples also include nanostructures with different shapes, such as rods and triangles, in smaller numbers than the spherical particles. This also resulted in broadening the absorption curve to the lower-energy side (i.e., longer wavelength) of the UV–vis spectrum for both samples (see Figure 2b,c). The size analysis of both Ch/Q- and Ch/CA-Ag NPs was performed using ImageJ software, and their average size distributions were determined using Gaussian distribution fitting. Figure 4c indicates the size distribution of both samples. The average particle size of the Ch/Q-Ag NPs was calculated as 11.2 ± 2.4 nm, whereas it was found to be 10.3 ± 2.4 nm for the Ch/CA-Ag NPs. To visualize the organic shell structure that covers the Ag NPs, which is difficult to display in TEM due to energetic electron bombardment causing burn, negative staining using uranyl acetate was performed. Figure 4d shows the TEM image of the Ch/Q-Ag NPs exposed by negative staining. It is seen that the chitosan/quercetin shell structure uniformly covers the core Ag NPs (see insets of Figure 4d).

Figure 4: TEM images of (a) the chitosan/quercetin- (Ch/Q-) and (b) the chitosan/caffeic acid (Ch/CA-)-capped Ag NPs. (c) Size distribution analysis of the synthesized Ch/Q- and Ch/CA-Ag NPs. (d) TEM image of the Ch/Q-Ag NPs taken by the negative staining method using uranyl acetate. The insets of (d) show the images of the stained shell structure of the Ch/Q-Ag NPs.

In addition, the amount of the capping agents (quercetin and caffeic acid) that may be of high importance for potential applications of NPs has been determined. As known, quercetin is one of the main reagents used as a reference standard for the quantification of flavonoids in a sample [67]. The total flavonoid content of a sample can be determined by the AlCl3 colorimetric method using a UV–vis spectrophotometer [67-69]. In this method, quercetin, which gives a light yellow solution, forms a quercetin–AlCl3 complex after mixing with aqueous AlCl3 solution, yielding an intensely yellow solution [67]. Because of the formation of the quercetin–AlCl3 complex, a red shift is observed in the absorption peaks of the quercetin. Since the absorption band observed between 400 and 450 nm does not have any overlap with pure quercetin, it is generally used to draw the calibration curve. Considering that the total amount of flavonoids in pure quercetin is proportional to the quercetin concentration, it was aimed to reveal the amount of quercetin in the Ch/Q-Ag NPs with the same method [70]. In the absorption spectrum in Figure 5a (inset graph), two absorption bands were observed at 256 and 373 nm for pure quercetin, while the quercetin–AlCl3 complex gave absorption bands at 268 and 433 nm (Figure 5a). The observed absorption band at 433 nm is the most suitable region to determine the total flavonoid content. Therefore, the wavelength of maximum absorbance (λmax) at 433 nm was chosen to obtain the calibration curve (Figure 5b). From this calibration curve and the absorbance of the Ch/Q-Ag sample measured at 433 nm, the amount of quercetin in the Ch/Q-Ag sample was determined to be approximately 31.0 ± 0.8 μg/mL. Similarly, the amount of caffeic acid, which is a phenolic compound, in the Ch/CA-Ag NPs has been determined from the phenolic content assay. To this end, the Folin–Ciocalteau (FC) assay, which is a method frequently used in the literature to determine the phenolic content of the samples, has been used [67-70]. Here, because of the reaction that takes place in the basic environment, caffeic acid is oxidized, while the FC reagent is reduced, giving the solution a blue color (λ ≈ 760 nm), that is, a colorimetric reaction [67,69,71,72]. This reaction can also be monitored by a UV–vis spectrophotometer, and the observed broad absorption band varies depending on the concentration of phenolics. Figure 5c shows the absorption spectra of pure caffeic acid (inset graph) and caffeic acid–FC–Na2CO3 mixtures. The calibration curve was obtained using absorbance values at 762 nm (Figure 5d). Since the phenolic content will be proportional to the amount of caffeic acid present in the medium, the amount of caffeic acid has been determined from the calibration curve and the absorbance of the Ch/CA-Ag sample measured at 762 nm for the synthesized Ch/CA-Ag sample to be 28.8 ± 0.4 μg/mL.

Figure 5: (a) UV–vis absorption spectra of the quercetin–AlCl3 complex and quercetin only (inset figure). (b) A calibration curve (r2 = 0.9994) was obtained for the quercetin–AlCl3 complex at λmax = 433 nm. (c) UV–vis absorption spectra of caffeic acid–FC–Na2CO3 mixture and caffeic acid only (inset figure). (d) A calibration curve (r2 = 0.9977) was obtained for the caffeic acid–FC–Na2CO3 mixtures at λmax = 762 nm.

Anticancer activity studies were also conducted for the synthesized Ch/Q- and Ch/CA-Ag NPs using XTT assay. As known, XTT and MTT, tetrazolium salts, are used to evaluate cell viability by a colorimetric method based on their reduction to colored formazan compounds by living cells. Actively respiring cells convert water-soluble XTT to a water-soluble, orange-colored formazan product, while water-soluble MTT converts to an insoluble purple formazan. Therefore, MTT requires solubilization of insoluble formazan to determine its concentration by absorbance, whereas XTT does not require any solubilization. Therefore, the use of XTT is an excellent solution for the quantification of cells and their viability, as it greatly simplifies the procedure for measuring proliferation over MTT, reduces assay time, and increases the sensitivity of the assay [73]. In this study, the dose-dependent cell viabilities of human brain glioblastoma (U-118 MG) and human retinal pigment epithelium (ARPE-19) cell lines after administration of Ch/Q- and Ch/CA-Ag NPs were measured using XTT assay (Figure 6). Another consideration in determining cell viability is the wavelength chosen for absorbance measurements of XTT (450 nm). Since XTT is in the same region with Ag NPs that can absorb (or scatter) light at this wavelength (450 nm), necessary washings were made after incubation to prevent misleading effects of Ag NPs on absorbance, and then the XTT test was applied (see section “Cell viability assay (XTT)”). Thus, the absorbance contribution that may arise from Ag NPs is eliminated. As seen in Figure 6a and Figure 6b, the absorbance values obtained for Ch/Q-Ag NPs after XTT application in U-118 MG and ARPE-19 cell lines at 6.9 mg/L (1/5 dilution) were approximately 0.45 and 0.55, respectively. If Ch/Q-Ag NPs had any effect on the absorbance measured at 450 nm, the absorbance at the highest dose of 41.4 mg/L (1/1 dilution) would be expected to be much higher than the other measured doses (1/2, 1/3, 1/4, and 1/5 dilutions). In addition, after the application of Ch/Q-Ag NPs to the U-118 MG cell line, it was observed from the microscope images that the cell viability decreased as the dose increased compared to the control (see Supporting Information File 1, Figure S1). Especially at low doses (Supporting Information File 1, Figure S1b), cell viability seemed to be very close to the control (Supporting Information File 1, Figure S1a). As a result, it is concluded that there is no interference of Ag NPs with XTT, so the absorbance values obtained are specific results showing only cell viability.

Figure 6: Concentration (dose)-dependent viability of U-118 MG and ARPE-19 cell lines for (a, b) the chitosan/quercetin (Ch/Q-)-, and (c, d) the chitosan/caffeic acid (Ch/CA-)-capped Ag NPs, respectively.

As seen in Figure 6, the cell viability values for all Ch/Q- and Ch/CA-Ag NPs doses were lower than those of the control group according to the Dunnett t post hoc test results (all p-values were below 0.001 for all doses). When Ch/Q- and Ch/CA-Ag NPs were used in the U-118 MG cell line, the highest cell viabilities were found at 6.9 and 11.4 mg/L Ag concentration (1/5 dilution) for quercetin and caffeic acid, respectively (Figure 6a and Figure 6c). For Ch/Q-Ag NPs, the lowest cell viability in the U-118 MG cell line was seen at 41.4 mg/L (1/1 dilution) and 13.8 mg/L (1/2 dilution) with p = 0.176. Also, the cell viability for Ch/Q-Ag NPs in U-118 MG was 78% at 6.9 mg/L (1/5 dilution), whereas it reduced to 27% at 41.4 mg/L (1/1 dilution) (Figure 6a, see also Supporting Information File 1, Figure S1). The lowest viability of Ch/CA-Ag NPs in the U-118 MG cell line was observed at 68.1 mg/L (1/1 dilution), and there were also no significant differences between 22.7 mg/L (1/2 dilution with p = 0.863) and 17.0 mg/L (1/3 dilution with p = 0.236) (Figure 6c). Consequently, for Ch/CA-Ag NPs, the cell viability of U-118 MG cell lines was 37% at 11.4 mg/L (1/5 dilution), while it reduced to 19% at 68.2 mg/L (1/1 dilution) (Figure 6c). When the effect of Ch/Q- and Ch/CA-Ag NPs in the U-118 MG cell line was compared at similar Ag concentrations, cell viability was found to be higher for Ch/Q-Ag NPs. For instance, cell viability was 33% at 13.8 mg/L (1/2 dilution) for quercetin, while it was 27% at 13.6 mg/L (1/4 dilution) for caffeic acid. Besides, cell viability was 59% at 10.4 mg/L (1/3 dilution) for quercetin, while it was 37% at 11.4 mg/L (1/5 dilution) for caffeic acid (Figure 6a and Figure 6c). These results show that the capping agent surrounding the silver nanoparticles have considerable dose-dependent effects against the cancer cell line (U-118 MG), and that the cell viability has constantly decreased by increasing the concentration of all synthesized nanoparticles. These findings are consistent with the results of other studies with silver and chitosan nanoparticles. For instance, Prema and Thangapandiyan synthesized and searched chitosan-stabilized Ag NPs for their cytotoxic effects against MCF-7 and HepG-2 cancer cell lines [57]. The results of the study showed an enhanced activity of chitosan-stabilized Ag NPs in comparison to colloidal Ag NPs, and the cytotoxicity was inversely proportional to the size of chitosan-stabilized Ag NPs, but directly proportional to their concentrations. In another recent study, antioxidants–chitosan–silver nanoparticles have been prepared and exhibited good and dose-dependent cytotoxicity against MCF-7 breast cancer cell lines [58].

When adding Ch/Q- and Ch/CA-Ag NPs to ARPE-19 cells, the viability values were also lower than those of the control group, especially for caffeic acid Ag NPs (Figure 6b and Figure 6d). When Ch/Q-Ag NPs doses were compared, the highest cell viabilities (95–98%) were observed at 6.9 mg/L (1/5 dilution) and 8.3 mg/L (1/4 dilution), respectively (Figure 6b). For Ch/Q-Ag NPs, the lowest cell viabilities (51–52%) were observed at 41.4 mg/L (1/1 dilution) and 13.8 mg/L (1/2 dilution), respectively, and there were no significant differences between these doses (p = 0.999). Besides, cell viabilities (73–89%) for 10.4 mg/L (1/3 dilution) and 8.3 mg/L (1/4 dilution) concentrations were found to be statistically significant (p = 0.002). Moreover, the viabilities of the ARPE-19 cells found for all concentrations of Ch/CA-Ag NPs were lower than the ARPE-19 cell viability in the control group according to the Dunnett t post hoc test results (all p-values were below 0.001 for all doses). The highest cell viability (31%) was observed at 11.4 mg/L (1/5 dilution), and this was higher than those all other doses (all p-values were below 0.001 for all doses). Conversely, the lowest viability (19%) was observed at 68.2 mg/L (1/1 dilution ratio), while there was no significant difference between 22.7 mg/L (1/2 dilution) (p = 0.976) and 17.0 mg/L (1/3 dilution) (p = 0.542), however, it was lower than those for concentrations of 13.6 mg/L (1/4 dilution) (p < 0.001) and 11.4 mg/L (1/5 dilution) (p < 0.001).

When Ch/Q- and Ch/CA-Ag NPs treatments against ARPE-19 cell lines were compared at similar Ag concentrations, cells viabilities were also found to be higher for quercetin NPs. For instance, cell viability was 52% at 13.8 mg/L (1/2 dilution) for quercetin, while it was 26% at 13.6 mg/L (1/4 dilution) for caffeic acid. Also, the cell viability was 73% at 10.4 mg/L (1/3 dilution) for quercetin, while it was 31% at 11.4 mg/L (1/5 dilution) for caffeic acid (Figure 6b and Figure 6d). Considering all the results for Ch/Q-Ag NPs, the cell viabilities of U-118 MG and ARPE-19 were 78% and 95% at 6.9 mg/L (1/5 dilution), while they reduced to 27% and 51% at 41.4 mg/L (1/1 dilution), respectively. On the other hand, for Ch/CA-Ag NPs, the cell viabilities of U-118 MG and ARPE-19 were 37% and 31% at 11.4 mg/L (1/5 dilution), even though it reduced to 19% at 68.2 mg/L (1/1 dilution) for both cells, respectively.

In summary, the concentration-dependent cell death in the U-118 MG human glioblastoma cells by Ch/Q-Ag NPs differs from cell death in the healthy ARPE-19 cells. The percentage of cell death caused by Ch/Q-Ag NPs is higher in U-118 MG cells than in ARPE-19 cells at the same concentrations. The quercetin-containing Ag nanoparticles (Ch/Q-Ag NPs) may be accepted as a candidate for use in cancer treatment. Furthermore, the concentration-dependent cell death caused by Ch/CA-Ag NPs in U-118 MG cells is comparable to that in the healthy ARPE-19 cells, suggesting that the effect may not be tissue specific.

Another issue that may affect the results of biological applications of NPs in general and should be considered is how the colloidal distribution and stability of the NPs are affected in the cell culture medium [74]. Since NPs are exposed to various forces that affect their stability and size in such environments (containing electrolytes, proteins and lipids), they may tend to collapse and aggregate. Although no aggregation and turbidity were observed during the preparation stage with the naked eye and in the microscope images of Ch/Q- and Ch/CA-Ag NPs in cell medium (see Supporting Information File 1, Figure S1), the stability of the synthesized NPs and how the size changes in cell culture medium compared to water were also evaluated by UV–vis and zeta potential measurements. An increase in the size of a metal nanoparticle gives rise to a decrease in the surface-to-volume ratio, which results in a decrease in the concentration of free surface electrons. Then, the required energy to polarize the electrons decreases, which leads to a redshift in the peak corresponding to the SPR absorption band. In other words, in the presence of the medium for cell culture (i.e., DMEM without phenol red), the possible formation of larger structures through the aggregation of Ag NPs will cause a shift in the position of the SPR peak toward the longer-wavelength side of the spectrum. Therefore, to get an insight into the aggregation and the stability of the Ch/Q- and Ch/CA-Ag NPs in the cell culture medium, UV–vis absorption measurements were carried out (see Supporting Information File 1, Figure S2). As can be seen in Figure S2 (Supporting Information File 1), the SPR peak emerges at 428 and 440 nm for Ch/Q- and Ch/CA-Ag NPs, respectively, in the cell culture medium. Compared with the NPs in water (see Figure 2b and Figure 2c), a redshift by about 11 and 16 nm of the SPR absorption peak for Ch/Q- and Ch/CA-Ag NPs, respectively, was found. In addition, a broadening was observed in the absorption curve for both Ag NPs in the medium. The redshift in the SPR peak wavelength and the broadening in the absorption curve towards the lower-energy region of the spectrum in the cell culture medium can be an indication of the formation of the aggregated structures [75]. UV–vis absorption measurements were also performed as a function of time to determine the stability of both Ch/Q- and Ch/CA-Ag NPs, which are highly stable in water, in the cell culture medium (see Figure S3, Supporting Information File 1). The measurements indicate that the absorbance of both Ag NPs decreases with time in the cell culture medium. This reveals the decreasing stability for both Ch/Q- and Ch/CA-Ag NPs in the cell culture medium compared to water, which can be explained by the formation of aggregated structures in the medium. In addition, the stability of the NPs was also determined by zeta potential measurements for both water and cell culture medium (with 1/10 dilution). The zeta potential values of Ch/Q- and Ch/CA-Ag NPs in water were found to be 37.1 ± 1.2 and 28.4 ± 2.2 mV, whereas they were −5.58 ± 0.47 and −2.08 ± 0.16 mV in medium (i.e., DMEM without phenol red), respectively. The variation in the zeta potentials, both regarding sign and numbers, clearly reveals that the stability of the NPs has changed significantly. Various studies have reported that in cell culture medium, there are pH-induced changes of NP conformation, particle size, stability, and functionality. Therefore, the nature of the cell culture medium should be kept in mind [74]. There are some approaches to provide electrostatic, steric, or electro-steric stabilization by adding electrolytes or polymeric substances to prevent aggregation of NPs [74]. However, in this study, no extra substance was added to the cell culture medium, and the direct interaction of Ag NPs with the cell lines was examined. In future studies, it will be valuable to examine the effect of additives that increase the stability of NPs and prevent their aggregation.

Moreover, the antibacterial activity of synthesized nanoparticles, Ch/Q- and Ch/CA-Ag NPs, were studied against Gram-negative (P. aeruginosa and E. coli) and Gram-positive (S. aureus and S. epidermidis) bacteria using the disc diffusion method. The inhibition zone diameters obtained after adding Ch/Q- and Ch/CA-Ag NPs in three different concentrations (1/1, 1/3, and 1/5 dilutions) are presented in Table 1.

Table 1: Antibacterial activity of Ch/Q- and Ch/CA-Ag NPs and standard antibiotics (ampicillin and amikacin) in terms of inhibition zone diameters by disc diffusion. Values in parentheses indicate the dilution ratios from the stock NPs and the measured diameters. A p-value of 0.05 was considered a statistical significance level.

inhibition zone diameter [mm] standard bacterial strains Ch/Q-Ag NPs [mg/L]   Ch/CA-Ag NPs [mg/L]     41.4
(1/1) 10.4
(1/3) 6.9
(1/5) p   68.2
(1/1) 17.0
(1/3) 11.4
(1/5) p ampicillin amikacin P. aeruginosa 9
(8–9) 7
(7–8) 7
(6–7) 0.047   9
(8–10) 7
(6–7) 6
(6–7) 0.050 — 16 E. coli 8
(7–8) 7
(6.5–7) 6
(6–6.5) 0.040   7
(7–8) 6
(6–6.5) 6
(5.5–6) 0.038 15 — S. epidermidis 8
(8–9) 8
(7–8) 7
(7–7.5) 0.085   8
(8–9) 8
(7–8) 6
(6–7) 0.047 18 — S. aureus 8
(8–9) 7
(7–7.5) 6
(6–7) 0.033   8
(8–9) 8
(7–8) 7
(6–7) 0.057 19 —

Almost all NPs are generally effective against both Gram-negative and Gram-positive bacteria. The median disc diameters of the Ch/Q- and Ch/CA-Ag NPs for all bacterial strains decreased as the dilution rate increased. According to the post hoc test results, these differences of Ch/Q-Ag NPs were statistically significant for P. aeruginosa (p = 0.047), E. coli (p = 0.040), and S. aureus (p = 0.033), but not for S. epidermidis (p = 0.085). The median disc diameters of Ch/CA-Ag NPs were statistically significant for P. aeruginosa (p = 0.050), E. coli (p = 0.038), and S. epidermidis (p = 0.047), but not for S. aureus (p = 0.057). A graphical evaluation of Table 1 is given in Figure S3 (Supporting Information File 1). In this study, inhibition zones of control antibiotics (ampicillin and amikacin) were also evaluated according to the European committee on antimicrobial susceptibility testing (EUCAST) (see section “Antibacterial activity”). In comparison with ampicillin and amikacin, the median disc diameters of the NPs were generally lower (Table 1). These findings can be compared or correlated with the results of some recent antibacterial studies. For instance, in a study by Alavi et al., quercetin–Ag NPs of similar size (11 nm) displayed good inhibition against P. aeruginosa and S. aureus at 2 and 4 μg/mL [76]. In another comparative antibacterial study, quercetin-loaded and unloaded Ag NPs did not exhibit any inhibition against the Gram-positive bacterium S. aureus [77]. While unloaded Ag NPs exhibited inhibition against the Gram-negative bacterium E. coli at low concentrations (1–2 µg/mL), quercetin loaded-Ag NPs showed no antibacterial effect. In contrast to these finding, quercetin-decorated Ag NPs at concentrations of 5 and 10 µg/mL exhibited stronger antibacterial and antibiofilm activities against multidrug-resistant E. coli strains when compared to Ag NPs or quercetin treatments [78]. Here, factors such as low NP concentrations, high bacterial concentration, nanoparticle size, and ambient pH may have played a role in obtaining such low antibacterial inhibition results. Furthermore, it may be possible that the NPs could not diffuse from the impregnated disc to the agar medium compared to a chemical antibacterial agent.