*3.1. Characterization of Ag7NO11:SiO2*

Synthesis of higher oxidation states of metals, such as silver oxynitrate, may proceed through the addition of a soluble metal to a strong oxidizing agen<sup>t</sup> [30]. Aqueous oxidization of silver nitrate by potassium persulfate, as per Equation (3), results in the formation or deposition of Ag7NO11.

$$\begin{aligned} \text{7AgNO}\_{3\text{ (aq)}} + \text{K}\_{2}\text{S}\_{2}\text{O}\_{8\text{ (aq)}} + 8\text{H}\_{2}\text{O}\_{(l)} &\to \\ \text{Ag}\_{7}\text{O}\_{8}\text{NO}\_{3\text{ (s)}} + 6\text{HNO}\_{3\text{ (aq)}} + \text{H}\_{2}\text{SO}\_{4\text{ (aq)}} + \text{K}\_{2}\text{SO}\_{4\text{ (aq)}} + 4\text{H}\_{2}\text{(g)} \end{aligned} \tag{3}$$

Based on the methods described herein, a series of silver oxynitrate-silica co-deposition products were prepared through the addition of an alkaline potassium silicate (K2SiO3) to a potassium persulfate solution prior to silver nitrate addition. Within the series of co-deposition products, the molar equivalents of silicon dioxide to silver were incrementally augmented following the series: 0:1, 0.1:1, 0.25:1, and 0.5:1 molar equivalents SiO2:Ag; these co-deposition products are herein referred to as Ag7NO11:SiO2. Subsequent to their isolation, the relative solid-state composition of the powders was determined by X-ray di ffractometry (XRD). Principal di ffraction patterns collected from each the powders, shown in Figure 1, were in agreemen<sup>t</sup> with the crystallographic parameters for Ag7NO11 [29]. Minor di ffraction patterns were identified in each of the powder samples in agreemen<sup>t</sup> with the crystallographic parameters for silver sulfate. No additional di ffraction patterns were identified in any of the powder samples. Silica gels formed from alkali silicates are commonly amorphous and poorly crystalline and therefore are expected to have limited or indiscernible di ffraction patterns [31]. The relative di ffraction peak area for silver sulfate Ag2SO4 (28.1 ◦2Θ) versus silver oxynitrate (36.3 ◦2Θ) was identified to increase with the relative molar ratio of SiO2:Ag, as shown in Figure S1. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to observe the structural composition and distribution of silver oxynitrate and silica within the isolated products. SEM images of silver oxynitrate control and Ag7NO11:SiO2 prepared at di fferent silica concentrations ranging from 0:1 to 0.5:1 molar equivalents SiO2:Ag are shown in Figure 2. It is observed from these images that distinct cuboctahedron structure of silver oxynitrate is conserved from 0:1 to 0.25:1 SiO2:Ag, where the silica is observed as amorphous structures. At 0.5:1, SiO2:Ag solid silica gel is the primary structure observed and the geometric silver oxynitrate structures are not visibly detected. Upon increasing the relative concentration of silica, it is also observed that the relative crystalline size of the silver oxynitrate decreases from 0:1 to 0.5:1 SiO2:Ag. This reduction in crystalline size upon increasing silica concentration was also corroborated by XRD, employing the Debye-Scherrer equation, as shown in Figure S2 [32]. Complimentary to the SEM images, TEM provides a spatial orientation of the silver within the silica framework as the density disparity between silver oxynitrate and silica a ffords clear demarcation between the materials as shown in Figure 3. At a low relative concentration of silica, 0.1:1, molar equivalents SiO2:Ag, the distinct cuboctahedra structure of silver oxynitrate is observed as a black silhouette, Figure 3A–C. Surrounding silver oxynitrate, the less electron dense structure of silica is observed on the structural faces of silver oxynitrate, Figure 3B, and encasing the silver oxynitrate particles in a framework of silica gel, Figure 3A. Increasing the relative concentration of silica to 0.5:1 molar equivalents SiO2:Ag, as shown in Figure 3D–F, the distinct cuboctahedra structure of silver oxynitrate is partly conserved. Additionally, sub-micron sized areas of high electron density within the silica gel framework are observed, Figure 3D,E. Presence of silver in these areas of high electron density in Ag7NO11:SiO2 containing 0.5:1 molar equivalents SiO2:Ag was confirmed using energy dispersive X-ray spectroscopy as shown in Figure S3. Confirming that the silica framework contains 55 to 65 wt/wt% silver, in good agreemen<sup>t</sup> with quantitative silver titration results shown in Figure S4.

### *3.2. Ambient and Accelerated Thermal Stability of Ag7NO11:SiO2*

Di ffraction patterns of silver oxynitrate and Ag7NO11:SiO2 containing 0.1:1 SiO2: were evaluated over a period of four months (16 weeks) under both ambient and elevated temperature storage conditions (40 ◦C). Within this timeframe, degradation of solid-state silver oxynitrate was evaluated by monitoring the depletion of the standard Ag7NO11 di ffraction peaks: 31.3, 36.3, and 39.7 ◦2Θ and the onset of the first thermal decomposition product argentic oxide, Equation (4), following standard AgO di ffraction patterns peaks: 32.2, 32.4, 37.3, and 38.6 ◦2Θ [21,23]. For the purposes of this study, the silver oxynitrate peak at 36.3 ◦2Θ peak was utilized to distinguish the presence/absence of silver oxynitrate as there are no overlapping impurity or degradation product di ffraction peaks at this di ffraction angle. At the study commencement, minor silver sulfate (Ag2SO4) impurities were additionally identified using standard di ffraction patterns peaks: 28.1, 31.2, 33.9 ◦2Θ.

$$\text{Ag}\_7\text{NO}\_{11} \rightarrow \text{6AgO} + \text{AgNO}\_3 + \text{O}\_2 \tag{4}$$

Following one month of silver oxynitrate storage under ambient conditions, Figure 4A, the primary solid-state compound observed was Ag7NO11 with minor peaks attributed to AgO and Ag2SO4. Following four months of storage at room temperature, AgO and Ag2SO4 alone were observed, consistent with the first thermal decomposition step shown in Equation (4). In parallel, the stability of Ag7NO11:SiO2 containing 0.1:1 SiO2:Ag was evaluated under ambient conditions as shown in Figure 4B. Silver oxynitrate was identified as the primary solid-state compound following one month of storage under ambient conditions with minor peaks attributed to AgO and Ag2SO4. Similarly, at four months of storage under ambient conditions, the primary solid-state compound observed was Ag7NO11 with only minor peaks attributed to AgO and Ag2SO4.

**Figure 1.** Powder X-ray diffraction of Ag7NO11:SiO2 (0.0:1 to 0.5:1 molar equivalents SiO2:Ag). Solid-state silver compounds were identified as silver oxynitrate (Ag7NO11, inverted red triangles) and silver sulfate (Ag2SO4, green circles).

Storage of silver oxynitrate under elevated temperatures, Figure 5A, was observed to result in multi-stage thermal decomposition processes as shown in Equations (5) to (7) [21,23]. Within one week of storage under 40 ◦C, the predominant solid-state compound was identified as silver nitrate, in good agreemen<sup>t</sup> with standard AgNO3 diffraction peaks: 29.7, 32.8, and 35.3 ◦2Θ. Secondary solid-state compounds were identified as Ag7NO11, AgO, and Ag2SO4. Following 16 weeks of storage at elevated temperatures, the diffraction peak at 36.3 ◦2Θ for Ag7NO11 was not observed nor were any standard diffraction patterns for AgO. The primary solid-state diffraction patterns identified in the XRD pattern at 16 weeks were in agreemen<sup>t</sup> with AgNO3, Ag2O, and Ag (metallic silver), indicative of advanced thermal decomposition pathways as per Equations (5) to (7). In contrast, decomposition of Ag7NO11:SiO2, containing 0.1:1 SiO2:Ag, under elevated temperatures was less severe. Retaining Ag7NO11 as the predominant solid-state compound with only minor peaks attributed to AgO and Ag2SO4 after one week and conversion to the first thermal decomposition product, AgO, following 16 weeks of storage under 40 ◦C.

**Figure 2.** Scanning electron microscopy images of Ag7NO11:SiO2 (0.0:1 to 0.5:1 molar equivalents SiO2:Ag). (**A**,**B**) Silver oxynitrate (Ag7NO11); (**C**,**D**) Ag7NO11:SiO2 containing 0.10:1, SiO2:Ag; (**E**,**F**) Ag7NO11:SiO2 containing 0.25:1, SiO2:Ag; (**G**,**H**) Ag7NO11:SiO2containing 0.50:1, SiO2:Ag.

**Figure 3.** Transmission electron microscopy images of Ag7NO11:SiO2 (0.0:1 to 0.5:1 molar equivalents SiO2:Ag) prepared by the co-deposition synthetic process. (**A**–**C**) Ag7NO11:SiO2 containing 0.10:1, SiO2:Ag; (**D**–**F**) Ag7NO11:SiO2 containing 0.50:1, SiO2:Ag.

**Figure 4.** Powder X-ray diffraction patterns of (**A**) silver oxynitrate (Ag7NO11) and; (**B**) Ag7NO11:SiO2 containing 0.10:1, SiO2:Ag, equivalents silica over a four month period under storage at ambient room temperature (RT) conditions. Solid-state silver compounds were identified as silver oxynitrate (Ag7NO11, inverted red triangles), silver sulfate (Ag2SO4, green circles), and argentic oxide (AgO, blue squares).

$$\rm{3-AgO} + \rm{AgNO\_3} \rightarrow \rm{3Ag\_2O} + \rm{AgNO\_3} + 1.5O\_2 \tag{5}$$

$$\rm{3Ag\_2O + AgNO\_3 \to 3.5Ag\_2O + NO\_2 + 0.25O\_2} \tag{6}$$

$$\text{C:} \text{5Ag}\_2\text{O} \to \text{7Ag} + \text{1.75O}\_2 \tag{7}$$

**Figure 5.** Powder X-ray diffraction patterns of (**A**) silver oxynitrate (Ag7NO11) and; (**B**) Ag7NO11:SiO2 containing 0.10:1, SiO2:Ag, equivalents silica over a four month period under accelerated storage in an incubator at 40 ◦C. Solid-state silver compounds were identified as silver oxynitrate (Ag7NO11, inverted red triangles), silver sulfate (Ag2SO4, green circles), argentic oxide (AgO, blue squares), silver nitrate (AgNO3, orange diamonds), silver oxide (Ag2O, blue triangle), and metallic silver (Ag, grey arrows).

### *3.3. Aqueous Decomposition of Ag7NO11:SiO2*

The aqueous decomposition profiles of silver oxynitrate and Ag7NO11:SiO2 containing 0.1:1 SiO2:Ag were also investigated. In brief, the solid powder products were dispersed into aqueous media at room temperature and, at set time intervals over seven days, were evaluated by XRD and SEM. Following dispersion into aqueous media it was observed that, with increasing relative ratios of silica, the Ag7NO11:SiO2 powders had an increased time of suspension as shown in Figure S5. The aqueous decomposition profiles were observed to be similar for silver oxynitrate and Ag7NO11:SiO2 as seen in Figure 6. Within two hours of exposure to water, silver oxynitrate was confirmed as the primary solid-state silver species, with minor components attributed to AgO and Ag2SO4 confirmed by means of their respective standard diffraction patterns. Following 24 h in aqueous media, silver oxynitrate remained the primary solid-state compound, with minor components attributed to AgO and Ag2SO4. Relative counts for AgO versus Ag7NO11 were proportionally higher in Ag7NO11:SiO2. At the terminus of the seven-day study, AgO was identified as the primary solid-state species for both silver oxynitrate and Ag7NO11:SiO2, with secondary diffraction patterns attributed to Ag2SO4. The recovered solids at each time point were imaged by SEM. Typical geometric crystalline structures of silver oxynitrate were observed in Figure 7A,D for both silver oxynitrate and Ag7NO11:SiO2. Subsequent to aqueous exposure, a crystalline morphology transformation was observed in Figure 7C,F previously identified to be associated with the formation of argentic oxide, AgO [22]. Retention of the silica framework in the co-deposition product was observed over the seven-day evaluation period as seen in Figure 7E,F.

**Figure 6.** Powder X-ray diffraction patterns of (**A**) silver oxynitrate (Ag7NO11) and; (**B**) Ag7NO11:SiO2 containing 0.10:1, SiO2:Ag, equivalents silica over a seven-day period in an aqueous solution at room temperature. Solid-state silver compounds were identified as silver oxynitrate (Ag7NO11, inverted red triangles), silver sulfate (Ag2SO4, green circles), and argentic oxide (AgO, blue squares).

**Figure 7.** Scanning electron microscopy images of silver oxynitrate and Ag7NO11:SiO2 containing 0.1:1, SiO2:Ag, exposed to aqueous media for a period of seven days. Silver oxynitrate (Ag7NO11) (**A**) prior to aqueous exposure; (**B**) after 24 h of exposure to aqueous media; and (**C**) after 168 h of exposure to aqueous media. Ag7NO11:SiO2 containing 0.1:1, SiO2:Ag (**D**) prior to aqueous exposure; (**E**) after 24 h of exposure to aqueous media; and (**F**) after 168 h of exposure to aqueous media.

### *3.4. Antimicrobial <sup>E</sup>*ffi*cacy Evaluation of Ag7NO11:SiO2*

Silver oxynitrate is known as a potent antimicrobial agen<sup>t</sup> against planktonic, biofilm, and drug resistant bacterium [16–18]. Modification of the form or function of silver oxynitrate may result in an impairment of this antimicrobial activity. Therefore, it is imperative to determine the impact of synthetic modifications on the efficacy of silver oxynitrate. Towards this, a series of antimicrobial methodologies were employed to evaluate the efficacy of silver oxynitrate and Ag7NO11:SiO2 against both planktonic and biofilm states of *Staphylococcus aureus* (ATCC 6538) and *Pseudomonas aeruginosa* (ATCC 9027).

Single time point log reduction of Gram-positive *S. aureus* (4 h) and Gram-negative *P. aeruginosa* (1 h) were evaluated in Mueller Hinton Broth (MHB) inoculated to a concentration of 1 × 10<sup>6</sup> CFU/mL. The efficacy of silver oxynitrate and Ag7NO11:SiO2 containing 0.1:1 SiO2:Ag were evaluated at normalized silver concentration. Following treatment, the remaining bacteria were numerated and log reduction quantified versus untreated control. The efficacy of Ag7NO11:SiO2 was compared with silver oxynitrate and was found to be equivalent for both organisms tested (*p* value > 0.05), as shown in Figure 8A.

**Figure 8.** Antimicrobial efficacy of silver oxynitrate (Ag7NO11) and Ag7NO11:SiO2 containing 0.1:1, SiO2:Ag. (**A**) Single-time log reduction for 1-h treatment period against *P. aeruginosa* and 4-h treatment period against *S. aureus* challenged at 1 × 10<sup>6</sup> CFU/mL; (**B**) Biofilm log reduction values for 2-h treatment period against *P. aeruginosa* and 4-h treatment period against *S. aureus* established 3D biofilm. Results representing the average of triplicate data (*n* = 3), error bars indicated represent standard deviations of the triplicate measurements.

The role of biofilm in infection and delayed wound healing is becoming increasingly evident [33–35]. Using a clinically relevant biofilm model, adapted from literature, we evaluated the capacity of silver oxynitrate and Ag7NO11:SiO2 containing 0.1:1 SiO2:Ag to target and reduce biofilm [17,36]. In brief, established biofilms of *P. aeruginosa* and *S. aureus* were treated with silver oxynitrate or Ag7NO11:SiO2. Following a 2-h and 4-h treatment period for *P. aeruginosa* and *S. aureus* respectively, viable cells were recovered and numerated versus untreated control to determine log reduction values. Silver oxynitrate and Ag7NO11:SiO2 were found to equivalently reduce viable biofilm counts (*p* value > 0.05), as shown in Figure 8B. In comparison with silver oxynitrate, no hindrance of antibacterial or antibiofilm activity was observed with the incorporation of silver oxynitrate to the silicon dioxide framework through the described co-deposition procedures.
