Synthesis of the PVP-AgNPs: AgNPs were synthesized by the chemical reduction of the silver ions. The following stock solutions were prepared using Milli-Q water: 15 mM AgNO3, 3 mM PVP K-10, and NaBH4 at different concentrations from 4 to 50 mM. The PVP is the coating agent, while the NaBH4 is the reducing agent. A detailed description for the PVP K-10 and the NaBH4 characteristics is provided at the supplementary materials section.
The following is a step-by-step description of the synthesis protocol. First, 30 mL of the 15 mM AgNO3 stock solution, under vigorous stirring, were warmed at 70 ±5 °C. Second, 5 mL of 30 mM PVP were added to the AgNO3 solution, while maintaining under vigorous stirring. Third, immediately after, 300 µL NaBH4 were added dropwise to the colorless solution until it turned to a brown or a grayish color (depending on the NaBH4 concentration). The NaBH4 solutions were prepared fresh immediately before their use to avoid loss of activity. It is important to add the NaBH4 slowly since it induces an exothermic reaction that can generate a lot of heat and increase the temperature of the solution. The color change is associated with the AgNPs formation, while the turbidity can be used to conjecture if the nanoparticles are highly concentrated. Fourth, the suspension was vigorously stirred for an additional 15 minutes at 70 ± 5 °C. Finally, the AgNPs were transferred to a light-protected (i.e. wrapped in aluminum foil) Falcon® plastic tube and left to cool down at room temperature, and then stored at 4 °C.
AgNPs are synthesized regardless of the concentration range of NaBH4 used in this work. Yet, concentration, size, and shape of AgNPs vary according to the molar ratio of NaBH4:Ag. The different synthesis displayed similar antimicrobial activity, as seen in other works (19,20). For the subsequent physicochemical characterization described in the following sections, the evaluated PVP-AgNPs were those synthesized with the lowest NaBH4 concentration (4 mM).
Characterization of the resulting PVP-AgNPs: UV-Vis spectrophotometry: the absorbance profile had a single peak with a maximum absorbance at ʎ=401 nm, typical for small spheroid AgNPs (Figure 2A). The AgNO3 absorbance profile indicated its conversion to AgNPs.
HR-TEM analysis: HR-TEM images reveal that the majority of AgNPs exhibit aspect ratio close to 1. (Figure 2B). Defined shapes, such as trunked pyramids, were observed in significantly low numbers. The AgNPs have an average size of 6.18 ± 5 nm, ranging from 1 to 75 nm, and >90% fell within the 1 to 10 nm range (n=1,025 counted nanoparticles) (Figure 2B, insert). Few large particles (>100 nm) and conglomerates were sporadically observed. The EDS chemical composition analysis detected the characteristic X-ray energy bands from silver at 2.984 KeV (Lα), and at 22.163 (Kα) (Figure 2C). HR-TEM unveiled the detailed lattice of AgNPs (Fig. 3A). The SAED analysis displayed the d-spacing of 2.36, 2.04, and 1.44 nm, corresponding to the hkl planes {202, 200, 111}, respectively (Fig. 3B), according to the standard powder diffraction card of the JCPDS, silver file No. 04–0783. The distance between fringes is 2.9 Å, confirming a face-centered cubic (fcc) crystalline structure (Fig. 3C).
Dynamic Light Scattering: The hydrodynamic size (HS) of the PVP-AgNPs was 35.18 ± nm and their Zeta potential -16.2 mV. The HS is greater than their metallic core size (35.18 nm and 6.18 nm, respectively), revealing that most of the particle volume (>65%) is in the extended PVP-chains coating. The Zeta potential revealed that AgNPs have a negative surface charge and a low stability score (stability for colloids is achieved at >|30| mV). Our Zeta value is similar to the values reported in other studies: -18.4 mV by Silva et al (21), and +13.4 by Romero-Urbina et al (10). Also, The Zeta potential of commercial nanoparticles are close to ours: for Sigma-Aldrich AgNPs (cat. No. 484059) is +0.91 mV, according to Park et al (22), and for Vector Vita PVP-AgNPs is -14.1 mV (19). Higher Zeta potential values have been reported: -31 mV by Orlowski et al (23) and -45 mV by Sondi et al (24).
Antimicrobial activity of the synthesized AgNPs: PVP-AgNPs exhibited potent antimicrobial activity against both S. aureus and C. albicans. S. aureus is a leading cause of hospital bacterial infections (25) while C. albicans is an opportunistic pathogenic fungus and the main causative agent of candidiasis, affecting an increasing number of immunosuppressed and medically compromised patients, with unacceptably high mortality rates (26,27). The MIC values for these PVP-AgNPs were 3 µg mL-1 and 1.5 µg mL-1 for S. aureus and C. albicans, respectively. The antimicrobial activity of our synthesized PVP-AgNPs is comparable to other reported in different studies, using similar culture conditions. MIC values in the literature range from 0.8 µg mL-1 to 13.5 µg mL-1 for S. aureus (Table S1, supplementary material) (13,28), and from 0.4 µg mL-1 to 40 µg mL-1 for C. albicans (Table S2, supplementary material) (29,30). The physicochemical properties of AgNPs depend upon their morphology and capping agent; yet different AgNPs with different traits may display close antimicrobial activity (19). The MBC for S. aureus was 3 µg mL-1, while the MFC for C. albicans was 1.5 µg mL-1. For our AgNPs, the Microbicidal and the Inhibitory Concentrations were the same for each strain.
Stability and antimicrobial activity of the synthesized silver nanoparticles after storage. AgNPs surface plasmon changed at week 6, showing a difference of 11.9 % on their absorbance spectra when compared to the original profile (Figure 3). The AgNPs absorbance profile showed another change at week 18, yet the antimicrobial activity remained unchanged since the initial synthesis.