Swift and remarkable advances in nanotechnology have made nanomaterials available for many applications in pharmacology, including transport and targeting of drug molecules, development of diagnostic imaging systems, design and production of controlled release systems, and support of plant nutrition (1–5). Nanoparticles can penetrate cells at a high rate thanks to their high surface area/ volume ratio (6), and these properties provide nanoparticles with different biological activities such as antioxidant (7), antimicrobial (8, 9), and anticancer (10). Metallic nanoparticle systems have attracted particular attention in recent years, as, unlike polymeric or lipid nanoparticles, they can be used in antimicrobial treatment, especially due to their surface plasmon resonances (11–14). In this respect silver nanoparticles (AgNPs) stand out (15, 16).
Since chemical nanoparticle synthesis may involve many toxic agents, and physical synthesis is costly (17, 18), more and more attention is being given to the so called green nanoparticle synthesis, which relies on plants, microorganisms, algae, and DNA templates to reduce and stabilise nanoparticle metal ions (19–23). This is particularly true for AgNPs synthesised using plant extracts because of their broad antimicrobial spectrum (24, 25).
In this study, we wanted to see how extracts of immortelle (
In this respect, high-efficiency extraction of plant components is the most crucial step in the green synthesis of AgNPs, and microwave-assisted extraction (MAE), which has earned it popularity in recent years, is more efficient and quicker than traditional extraction methods. In addition, MAE is a suitable for large-scale and small-scale systems (38).
To sum up, the aims of our study were to: (i) to synthesise and characterise two different AgNPs using the microwave-assisted aqueous extracts of
Silver nitrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Müller Hinton broth, Müller Hinton agar, Sabouraud dextrose broth, and Sabouraud dextrose agar were purchased from Merck (Darmstadt, Germany). The plants
We opted for a fast, efficient, and environmentally friendly MAE method using water, a non-toxic and reliable solvent, instead of chemical solvents (39). To prepare the aqueous extract, 4.5 g of powdered
The synthesis of G-AgNPs and H-AgNPs was optimised by trial and error, using different parameters such as pH (6–10), temperature (25–80 °C), AgNO3 concentration (0.1–2 mmol/L), and reaction time (0–240 min). The optimum green synthesis parameters for G-AgNPs were pH 8, temperature 70 °C, AgNO3 concentration 1 mmol/L, and reaction time 4 h. Interestingly, the optimum synthesis parameters for H-AgNPs were different: pH 9, temperature 60 °C, AgNO3 concentration 1 mmol/L (as in G-AgNPs), and reaction time 3 h. G-AgNPs and H-AgNPs analysed in this study were synthesised under these optimal conditions for biological activity.
To synthesise H-AgNPs 1 mL of aqueous
The reaction was followed with a UV-Vis spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). The produced particles were collected by centrifugation at 9056
The formation of nanoparticles was first confirmed visually based on the change in the colour of the reaction solution from yellow to brown. Analytically it was confirmed from the intensity of surface plasmon band absorption of nanoparticle solutions measured in the wavelength range of 200–800 nm at 25±0.1 °C using a UV-Vis spectrophotometer (UV-1700, Shimadzu). All measurements were made at a 1:10 dilution of the samples. For the blank we used ultrapure water.
The average size (hydrodynamic diameter) and polydispersity index (PDI) of nanoparticles were measured with dynamic light scattering (DLS), and the zeta potential with electrophoretic light scattering (ELS) using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a 4.0 mV He–Ne laser (633 nm) (7). Measurements were carried out in triplicate, at 25±0.1 °C, using the 0.8872 cP viscosity and 1.330 refractive indexes for the solutions and dielectric constant of 79 f(ka) 1.50 (Smoluchowski value). The number of runs and run durations were chosen automatically. All samples were diluted to a 1:10 ratio with distilled water before measurement.
The functional groups involved in both plant powders (
The surface morphology, topography, size, and size distribution of AgNPs were determined with both atomic force microscopy (AFM) (Shimadzu SPM 9600) and scanning electron microscopy (SEM) (FEI QUANTA 450 FEG, Thermo Fisher Scientific, Waltham, MA, USA). AFM images were taken with the force constant of 0.02–0.77 N/m, tip height 10–15 nm, and silicon lever in contact mode (40). Freeze-dried powdered nanoparticles were fixed on metallic studs with double-sided conductive tape, then coated with gold under vacuum and analysed with SEM (7).
The antimicrobial activity of G-AgNPs and H-AgNPs alone and in nanoformulations (NF-1, NF-2, and NF-3) was tested against
We prepared stock solutions of AgNPs and nanoformulations at a concentration of 2 mg/mL and serial dilutions in the range of 1–0.03125 mg/mL. 100 µL of the prepared samples were added to six wells each of the 96-well microplate. Microorganism suspension (106 CFU/mL) was added to each well to adjust the final volume in the wells to 200 µL. Microplates containing bacteria were incubated at 37 °C and microplates containing fungi at 28 °C for 16–18 h. After incubation, we determined minimum inhibition concentrations (MIC) and minimum bactericidal or fungicidal concentrations (MBC or MFC, respectively) by spectrophotometry (OD600 nm) and standard plate counting.
For statistical analysis we used the SPPS software version 22 (SPPS Inc., Chicago, IL, USA). All data are expressed as means ± standard deviations (SD) of three independent measurements. Statistical differences in MIC and colony counts between control and treated groups were analysed with the two-way analysis of variance (ANOVA) and significance set to p<0.05.
Figure 1 shows colour changes in the solution colour, which is attributed to the localised surface plasmon resonance (SPR) of metal nanoparticles (43–46). UV-Vis spectroscopy confirmed the formation of AgNPs with the characteristic SPR peak at 418 nm and 406 nm wavelengths for G-AgNPs and H-AgNPs, respectively.
UV–Vis absorption spectra of a) G-AgNPs and b) H-AgNPs with different incubation times (0–240 min)
Figure 2 shows that the particles were nanosized and highly monodisperse. Table 1 shows the average particle size, zeta potential, and PDIs of H-AgNPs and G-AgNPs. Because of excessive negative or positive zeta potential values (±30 mV), the electrostatic force between the nanoparticles prevents agglomeration of the NPs (47). These negative zeta potential values may be due to the presence of the stabilising agents (bioorganic components in the extract) acting as silver-reducing agents. Figure 3 shows the range of potentials for both H-AgNPs and G-AgNPs.
Particle size distribution of a) G-AgNPs and b) H-AgNPs
Zeta potential of a) G-AgNPs and b) H-AgNPs
Physicochemical properties of H-AgNPs and G-AgNPs
NPs | Particle Size (nm) | Zeta Potential (mV) | Polydispersity Index |
---|---|---|---|
H-AgNP | 23.9±1.0 | −21.3±2.7 | 0.285±0.034 |
G-AgNP | 52.0±10.9 | −17.9±0.9 | 0.280±0.032 |
Figures 4a and 4c show the FT-IR spectra of
FT-IR spectrum of a)
The band at 1650 cm-1 in the spectrum of
Figures 5 and 6 show that most of the synthesised AgNPs have spherical and monodisperse distribution. The sizes of G-AgNPs are between 40 and 60 nm (Figures 5a and 6a), and the sizes of H-AgNPs are between 20 and 30 nm (Figures 5b and 6b). These values correlate with DLS findings.
2-D Atomic force microscope images of a) G-AgNPs and b) H-AgNPs. Scan scale: 1×1 µm
Scanning electron microscope images of a) G-AgNPs and b) H-AgNPs at 200,000× magnification
Table 2 shows the minimum inhibitory, bactericidal, and fungicidal concentrations of G-AgNPs, H-AgNPs, and their three combinations against
MBC/MFC and MIC values of samples according to the broth microdilution method
Test sample | AgNP concentrations used in nanoformulations (μg/mL) | MIC μ/mL) by microorganisms | MBC/MFC μ/mL) by microorganisms | ||||||
---|---|---|---|---|---|---|---|---|---|
G-AgNPs | 62.5 | − | − | 2000 | 125 | − | − | − | |
H-AgNPs | − | 125 | 500 | − | − | − | − | − | |
NF-1 | 1000 G-AgNP + 1000 H-AgNP | 31.25* | 125 | 500 | − | 125 | 250 | − | − |
NF-2 | 500 G-AgNP + 1500 H-AgNP | 31.25* | 62.5 | 500 | − | 125 | 125 | − | − |
NF-3 | 1500 G-AgNP + 500 H-AgNP | 31.25* | 62.5 | 500 | − | 125 | 125 | − | − |
p<0.05, NFs vs AgNPs. MBC – minimum bactericidal concentration; MFC – minimum fungicidal concentration; MIC – minimum inhibitory concentration
Petri dish images obtained by standard plate counting related to respective MICs. The dilution coefficient for
In turn, all nanoformulations combining H-AgNPs and G-AgNPs were significantly more effective against
Cell uptake of nanoparticles depends on particle size and so does their antimicrobial activity, which increases with smaller nanoparticles, as reported by Manoslava et al. (50) and Agnihotri et al. (51). The antimicrobial activity of G-AgNPs and combined nanoformulations against
There are two interesting findings that we cannot yet explain. The first is that the combinations of G-AgNPs and H-AgNPs produced the same synergistic antimicrobial effect against
Despite these limitations of our study, our findings of the synergistic effects of the two synthesised nanoparticles when combined seem to support some earlier reports of improved antimicrobial effects by combinations of AgNPs with tomato, onion, acacia, neem,
In addition, we know that
Our study is the first to report the antimicrobial activity of nanoformulations containing both G-AgNPs and H-AgNPs and the first to identify their synergism against