Formulation and Biological Evaluation of Mesoporous Silica Nanoparticles Loaded with Combinations of Sortase A Inhibitors and Antimicrobial Peptides

This study aimed to develop synergistic therapies to treat superbug infections through the encapsulation of sortase A inhibitors (SrtAIs; trans-chalcone (TC), curcumin (CUR), quercetin (QC), or berberine chloride (BR)) into MCM-41 mesoporous silica nanoparticles (MSNs) or a phosphonate-modified analogue (MCM-41-PO3−) to overcome their poor aqueous solubility. A resazurin-modified minimum inhibitory concentration (MIC) and checkerboard assays, to measure SrtAI synergy in combination with leading antimicrobial peptides (AMPs; pexiganan (PEX), indolicidin (INDO), and [I5, R8] mastoparan (MASTO)), were determined against methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The results demonstrated that the MCM-41 and MCM-41-PO3− formulations significantly improved the aqueous solubility of each SrtAI. The MICs for SrtAI/MCM-41-PO3− formulations were lower compared to the SrtAI/MCM-41 formulations against tested bacterial strains, except for the cases of BR/MCM-41 and QC/MCM-41 against P. aeruginosa. Furthermore, the following combinations demonstrated synergy: PEX with TC/MCM-41 (against all strains) or TC/MCM-41-PO3− (against all strains except P. aeruginosa); PEX with BR/MCM-41 or BR/MCM-41-PO3− (against MSSA and MRSA); INDO with QC/MCM-41 or QC/MCM-41-PO3− (against MRSA); and MASTO with CUR/MCM-41 (against E. coli). These combinations also reduced each components’ toxicity against human embryonic kidney cells. In conclusion, MCM-41 MSNs provide a platform to enhance SrtAI solubility and demonstrated antimicrobial synergy with AMPs and reduced toxicity, providing novel superbug treatment opportunities.


Introduction
Antimicrobial-resistant pathogens represent a serious global threat to human health and economies. Owing to the global spread of resistant pathogens, current antimicrobial agents are losing their efficacy to treat or prevent important infections. Consequently, the morbidity and mortality associated with infectious diseases is increasing due to treatment failures [1]. The development of alternative therapies, which target virulence factors in antimicrobial-resistant bacteria, is an area of increasing interest. These so-called antivirulence approaches block virulence-associated pathways, without affecting pathogen viability, and thus apply less pressure for the development of antimicrobial resistance [2].

Preparation of MCM-41 MSNs
MCM-41 nanoparticles were prepared according to a previous report [21], with slight modifications (Scheme 1). Briefly, 1 g of CTAB was dissolved in 480 mL of 18 MΩ water and vigorously stirred (700 RPM) at room temperature (RT) to obtain a clear solution. Subsequently, 3.5 mL of 2 M NaOH was added, and the mixture was heated to 80 • C with a silicon oil bath, followed by the dropwise addition of TEOS (6.7 mL) to the mixture and stirring (700 RPM) for 2 h. The MCM-41 particles were harvested by vacuum filtration using filter paper (Whatman™, 150 mm) and washed with 200 mL of 18 MΩ water and then 200 mL of EtOH. The particles were dried (60 • C) overnight, and calcination was then conducted in a muffle furnace (2 h to reach 550 • C, 5 h at 550 • C; the temperature slowly decreased overnight to reach RT). loading capacity of MCM-41 and MCM-41-PO3 − MSNs. Transmission electron microscopy (TEM) was performed on a Hitachi HT7700B at 100 kV to analyse the shape and size of the particles. Micromeritics Tri-StarII ® (Norcross, GA, US) was used for N2-physisorption isothermal analysis of the surface area and porosity. A Perkin Elmer Spectrum TWO (Liantrisant, UK) Fourier-transform infrared spectrometer (FT-IR) was used to investigate interactions between MSNs and SrtAIs.

Preparation of MCM-41 MSNs
MCM-41 nanoparticles were prepared according to a previous report [21], with slight modifications (Scheme 1). Briefly, 1 g of CTAB was dissolved in 480 mL of 18 MΩ water and vigorously stirred (700 RPM) at room temperature (RT) to obtain a clear solution. Subsequently, 3.5 mL of 2 M NaOH was added, and the mixture was heated to 80 °C with a silicon oil bath, followed by the dropwise addition of TEOS (6.7 mL) to the mixture and stirring (700 RPM) for 2 h. The MCM-41 particles were harvested by vacuum filtration using filter paper (Whatman™, 150 mm) and washed with 200 mL of 18 MΩ water and then 200 mL of EtOH. The particles were dried (60 °C) overnight, and calcination was then conducted in a muffle furnace (2 h to reach 550 °C, 5 h at 550 °C; the temperature slowly decreased overnight to reach RT). Scheme 1. Process for MSN fabrication, phosphonate surface modification, and loading of SrtAIs.

Functionalization of MCM-41 with PO3 −
MCM-41 nanoparticles were modified with a phosphonate functional group (PO3 − ) (Scheme 1) based on a reported procedure [19]. Briefly, 400 mg of MCM-41 was suspended in 18 MΩ reverse osmosis water (65 mL) plus THMP (400 μL) at pH 5 (pH measured with pH test strips and adjusted with 1 M HCl) and refluxed at 100 °C overnight. The particles were then collected by centrifugation (16,000× g, 15 min, RT), and the obtained pellet was washed twice with 10 mL of 18 MΩ water and then twice with 10 mL of EtOH. MCM-41-PO3 − MSNs were then dried in an oven at 60 °C overnight and stored in a vacuum desiccator at RT.

Functionalization of MCM-41 with PO 3
− MCM-41 nanoparticles were modified with a phosphonate functional group (PO 3 − ) (Scheme 1) based on a reported procedure [19]. Briefly, 400 mg of MCM-41 was suspended in 18 MΩ reverse osmosis water (65 mL) plus THMP (400 µL) at pH 5 (pH measured with pH test strips and adjusted with 1 M HCl) and refluxed at 100 • C overnight. The particles were then collected by centrifugation (16,000× g, 15 min, RT), and the obtained pellet was washed twice with 10 mL of 18 MΩ water and then twice with 10 mL of EtOH. MCM-41-PO 3 − MSNs were then dried in an oven at 60 • C overnight and stored in a vacuum desiccator at RT.

Loading MCM-41 and MCM-41-PO 3 − MSNs with SrtAIs
SrtAIs (TC, CUR, BR, and QC) were individually loaded into MCM-41 and MCM-41-PO 3 − (Scheme 1) by rotary evaporation, as previously described [22], with modifications. For this purpose, individual SrtAIs (10 mg) were dissolved in MeOH (8 mL) using sonication (60 W; Branson CPX2800H-E; Branson Ultrasonics, Brookfield, CT, USA), into which MCM-41 or MCM-41-PO 3 − (90 mg) were dispersed, sonicated (60 W; 5 min), and stirred (400 RPM) overnight at RT. MeOH was then evaporated (40 • C) using a rotary evaporator, and the obtained powders were dried at RT and stored in the dark at 4 • C. (1 mg) were individually dispersed in EtOH (1 mL) and sonicated (2 min). One drop was then cast on a carbon-coated copper grid and allowed to air dry before imaging. DLS was also performed to determine SrtAI-loaded and non-loaded MCM-41 and MCM-41-PO 3 − Z-Ave, PDI, intensity mean, and Zeta-potential. For this purpose, samples (1 mg) were dispersed in PBS (1 mL; pH 7.4), bath sonicated (5 min), and measured in disposable folded capillary zeta cells (DTS1070, Malvern Panalytical, Malvern, UK). TGA and DSC measurements were performed to investigate the thermal degradation of each component and to determine the loading capacity. For each SrtAI, 5 mg was deposited into a TGA 70 µL alumina crucible. A temperature range of 50 (25 for TC) to 900 • C, a heating rate of 10 • C min −1 , and an airflow of 20 mL/min were used. N 2 -Physisorption isothermal analysis was performed to determine MSN pore volume, size, and surface area. The Brunauer-Emmeyt-Teller (BET) theory was used to determine surface area. The pore size distribution was measured using the BJH model from the isotherm adsorption branch. Prior to analysis, SrtAI-loaded and unloaded MCM-41 and MCM-41-PO 3 − (70 mg) were degassed for 10 h at 50 • C. FT-IR spectra were obtained for 2 mg of each sample (SrtAIs, MSNs, and SrtAI-loaded MSNs) over a 4000 to 400 cm −1 wavenumber range to investigate interactions between MSNs and SrtAIs (data provided in Figure S21).
The solubility of unformulated SrtAIs and MCM-41 and MCM-41-PO 3 − formulated SrtAIs were assessed using an established protocol [21], with modifications. Briefly, saturated solutions of SrtAIs and SrtAI-loaded MCM-41 and MCM-41-PO 3 − were prepared by dispersing SrtAIs and SrtAI-loaded MCM-41 and MCM-41-PO 3 − individually in 18 MΩ water at a concentration of 2 mg/mL, followed by stirring for 24 h in the dark at 37 • C. The solutions were then centrifuged for 10 min at 150 RPM at RT. The resulting supernatant was collected, and the area under the curve (AUC) was determined at 280 nm for TC, CUR QC, and BR using RP-HPLC. The concentrations were then calculated based on a calibration curve. All measurements were performed in triplicate, and the mean value was calculated.
Preparative HPLC was used to purify the crude peptides, and pure fractions were combined and freeze-dried to obtain purified peptides. To evaluate the purity and identity of the peptides, RP-HPLC and mass spectrometry were used. For each peptide, two RP-HPLC gradients were applied: 0-70% solvent B as a wide gradient for all peptides and 10-50, 25-45, and 30-50% solvent B as narrow gradients for PEX, INDO, and MASTO, respectively.

Preparation of Bacterial Inoculums
Bacterial inoculums were prepared according to the Clinical and Laboratory Standards Institute (CLSI) [23]. From overnight S. aureus (MSSA) ATCC25923, MRSA ATCC43300, E. coli ATCC25922, and P. aeruginosa ATCC27853 agar cultures, two colonies were suspended in individual vials containing sterile normal saline (2 mL). The turbidity of each bacterial suspension stock was then adjusted to equivalence with the 0.5 McFarland standard (1 × 10 8 colony-forming units/mL; CFU/mL) using sterile normal saline, then further diluted to − formulation against four bacterial strains (S. aureus (MSSA) ATCC25923, MRSA ATCC43300, E. coli ATCC25922, and P. aeruginosa ATCC27853) were determined using the CLSI BMD method [24] with a resazurin colorimetric readout [20]. For this method, quantities of SrtAI-loaded MCM-41 or MCM-41-PO 3 − containing 1 mg of individual SrtAIs were suspended in MHB (to 1 mL) to yield a 1 mg/mL final SrtAI concentration. From these stocks, 200 µL/well was dispensed into column one of a sterile 96-well clear, round-bottomed polystyrene microplate. In columns two-to-eleven, 100 µL/well of MHB was dispensed, and in column twelve, 200 µL/well of MHB was dispensed. Subsequently, a 2-fold serial dilution of the SrtAI-loaded MCM-41 or MCM-41-PO 3 − MSNs was prepared by transferring 100 µL from the first column, mixing this with the next column to the right, and repeating this process to column ten, with 100 mL of the mixture from column ten then discarded. Subsequently, 100 µL of the diluted standardized inoculum (1 × 10 6 CFU/mL) was added into the wells in columns one-to-eleven to obtain the final volume of 200 µL (5 × 10 5 CFU/mL) and final SrtAI concentrations ranging from 500 to 1 µg/mL. Column eleven (+) contained no SrtAI formulation and served as a control for bacterial growth. Column twelve (−) contained only MHB as a sterility control. The plates were then incubated at 37 • C for 24 h, and the wells visibly investigated for turbidity. Resazurin solution (30 µL; 0.15 mg/mL in sterile water) [25] was added to each well, and the plates were incubated for 2 h at 37 • C. The MIC values were visually determined as the lowest concentration that retained the dark blue resazurin colour [25].

Checkerboard Test
To investigate the potential for synergistic, additive, or antagonistic effects when combining SrtAI-loaded MCM-41 or MCM-41-PO 3 − with AMPs, a checkerboard test was performed to determine the fractional inhibitory concentration index (ΣFIC) according to a previously reported protocol [26], with modifications. For this purpose, SrtAI-loaded MCM-41 or MCM-41-PO 3 − equivalent to 2 mg of each SrtAI were suspended in MHB (to 1 mL), and PEX (200 µg), INDO (600 µg), and MASTO (400 µg) were individually mixed with 0.01% (v/v) DMSO-MHB (to 1 mL) to produce antimicrobial stocks. From each stock, six additional concentrations were prepared using a 2-fold serial dilution. To prepare the checkerboard assay, 50 µL/well of the highest concentration of each AMP was transferred into column one of individual sterile 96-well round-bottomed polystyrene plates, followed by their 2-fold dilution series in columns two-to-seven. In column eight, 50 µL/well of 0.01% (v/v) DMSO-MHB was added. Subsequently, 50 µL/well of the highest concentration SrtAI-loaded MCM-41 or MCM-41-PO 3 − MSNs was added to row A, followed by their 2-fold dilution series in rows B-to-G. In row H, 50 µL/well of MHB was added. Subsequently, 100 µL of the bacterial inoculum (1 × 10 6 CFU/mL) was added to each well to obtain a 5 × 10 5 CFU/mL final concentration. The plates were then incubated at 37 • C for 24 h, followed by the addition of resazurin solution (30 µL; 0.15 mg/mL in sterile water) to each well. The plates were incubated for 2 h at 37 • C. MIC values were visually determined as the lowest concentration that retained the dark blue resazurin colour in column eight for SrtAIs and in row H for AMPs. To determine if combinations provide additive, synergistic, or antagonistic effects, FIC index (ΣFIC) values were calculated using the following formula: The combination used to calculate the ΣFIC value represents the well/s where the largest change from the MIC value of one of the antimicrobial agents was observed. Combinations are defined as synergistic where ΣFIC is ≤ 0.5, additive where ΣFIC is > 0.5 to 1, indifferent where ΣFIC is > 1 to 4, and antagonistic where ΣFIC is > 4 [27].

Cell Viability
The effects of each SrtAI-loaded MCM-41 or MCM-41-PO 3 − MSN, AMP, and their combinations on HEK-293 cell viability were assessed using a resazurin reduction assay [28]. The concentrations of each antimicrobial used in these assays was selected based on their MIC and ΣFIC data from the BMD and checkerboard assays against MSSA, respectively. For this purpose, HEK-293 cells (5000 cells/well) were seeded in DMEM media supplemented with 10% (v/v) FBS and 100 U/mL penicillin G-100 µg/mL streptomycin (complete media) in 96 well flat-bottomed, black polystyrene plates. The cells were then incubated (37 • C, 5% CO 2 ) for 48 h to reach 80% confluency. The media was then discarded, and the cells were individually treated with each SrtAI-loaded MSN, AMPs, or their combinations, at different concentrations in 100 µL of complete media containing 0.01% (v/v) DMSO. Cells treated with complete media containing 0.01% (v/v) DMSO or with 10% (v/v) SDS and 0.1 M HCl were used as negative and positive toxicity controls, respectively. Wells containing complete media with 0.01% (v/v) DMSO were used as a background control. The plates were then incubated (37 • C, 5% CO 2 ) for 24 h, the supernatant was removed, 50 µL of 100 µM resazurin in PBS was added into each well, and the plates were incubated (37 • C, 5% CO 2 ) for 4 h. Resorufin fluorescence (Fl) was read at 590 nm, with excitation at 560 nm [28]. The percentage of the viable cells was calculated according to the following formula: Experiments were performed in triplicate to demonstrate reproducibility.

Statistical Analysis
For statistical analyses, GraphPad Prism software version 9.3 was used. Cell viability data and solubility analysis are presented as the mean ± standard deviation (SD, n = 3). One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used to analyse for statistical differences.

Characterization of SrtAI-loaded MCM-41 and MCM-41-PO 3 − MSNs
DLS was used to measure the particle size (Z-Ave, intensity mean), polydispersity (PDI), and surface charge (Zeta-potential) of the MCM-41 and MCM-41-PO 3 − MSNs before and after loading with SrtAIs, with the results presented in Table 1. No significant difference with respect to Z-Ave (120.8 ± 2.30 and 128.8 ± 3.20 nm, respectively) and intensity mean (143.9 ± 2.60 and 138.9 ± 3.80, respectively) were observed between the unloaded MCM-41 and MCM-41-PO 3 − MSNs. However, the MCM-41-PO 3 − MSNs exhibited a lower PDI (0.07 ± 0.01) and a more negative Zeta-potential (−49.0 ± 1.90 mV) compared to the MCM-41 MSNs (0.16 ± 0.03 and −41.1 ± 0.90 mV, respectively).  Figure 1). The polydispersity index (PDI) was also observed to increase with SrtAI-loading, with the MCM-41-PO 3 − MSNs continuing to demonstrate lower PDI values (0.16-0.22) compared to MCM-41 (0.19-0.29) after SrtAI loading (Table 1). Despite this, the observed PDI values for the SrtAI-loaded MSNs were similar (0.16-0.22), except for CUR-loaded MCM-41 (0.29 ± 0.01), which exhibited a higher variation in particle size. However, all formulations remained dispersed without large aggregates (PDI < 0.3) [29]. Finally, SrtAI-loading was observed to reduce the negative Zeta-potential associated with each MSN, with TC and BR having the greatest effect. For BR, this was hypothesized to be due in part to ionic interactions between its positively charged quaternary ammonium group and the negatively charged MSN silanol and phosphonate groups. The morphology of each MSN nanoformulation was also visualised by TEM before and after (Figure 2) SrtAI loading. The nanoparticles were approximately spherical in shape. The morphology was similar between the MCM-41 and MCM-41-PO3 − MSNs, suggesting that phosphonate modification did not affect the particle shape. The MSN size was also evaluated by TEM (Figure 2), with particle sizes appearing similar in magnitude to the DLS sizing data (Table 1). that phosphonate modification did not affect the particle shape. The MSN size was also evaluated by TEM (Figure 2), with particle sizes appearing similar in magnitude to the DLS sizing data (Table 1). The morphology of each MSN nanoformulation was also visualised by TEM before and after (Figure 2) SrtAI loading. The nanoparticles were approximately spherical in shape. The morphology was similar between the MCM-41 and MCM-41-PO3 − MSNs, suggesting that phosphonate modification did not affect the particle shape. The MSN size was also evaluated by TEM (Figure 2), with particle sizes appearing similar in magnitude to the DLS sizing data (Table 1).  − compared to MCM-41. In agreement with this study, other studies [19,30] have demonstrated the capacity of the rotary evaporation loading method to effectively encapsulate both hydrophilic and hydrophobic drugs within the extensive porous network of MSNs.
To further characterise the crystallinity of each SrtAI when loaded within MSNs, DSC was performed [31]. The investigation of each individual SrtAI by DSC revealed endothermic peaks at 61 (TC), 177 (CUR), 317 (QC), and 190 • C (BR) (Figure 3), which correspond to reported melting points for the crystalline form of each SrtAI [32]. Physical mixtures of non-loaded MSNs with each individual SrtAI, with the exception of QC, demonstrated these same peaks. In comparison, after loading the SrtAIs into MSNs, these peaks were greatly reduced or disappeared (Figure 3), suggesting that the SrtAIs were incorporated into the nanoparticles in an amorphous form [33].
To characterize the surface area, pore size, and volume of prepared MSNs, N 2adsorption-desorption isotherms were acquired for SrtAI-loaded and non-loaded MSNs. The results (Table 2 and Figure S2) were characteristic of an IV isotherm with a steep capillary condensation at a relative pressure (P/Po) range of 0.2-0.4, in loaded and unloaded particles, which is a characteristic of MCM-41 [34,35]. The surface area for MCM-41 was calculated as 892.6 m 2 /g, with a calculated pore volume of 0.9 cm 3 /g and pore size of 1.9 nm. After phosphonate modification, a decrease in the surface area (381.6 m 2 /g) and pore volume (0.6 cm 3 /g) was observed, with no effect on pore size (1.9 nm) for unloaded particles. After loading with SrtAIs, the surface area was also observed to decrease to 459.7, 421.6, 500.6, and 559.3 m 2 /g for TC, CUR, QC, and BR-loaded MCM-41, respectively, while the effects on the surface area of MCM-41-PO 3 − were more variable, with TC and QC-loaded particles showing surface area reductions (215.2 and 215.2 m 2 /g, respectively). In contrast, CUR and BR-loaded MCM-41-PO 3 − showed no effect or an increase in surface area (399.6 and 439.7 m 2 /g), respectively. Finally, the pore volume for TC, CUR, QC, and BR-loaded MCM-41 and MCM-41-PO 3 − was reduced ( Table 2). This reduction in pore volume, in combination with the reductions in surface area observed after SrtAI-loading for most formulations and TGA/DSC data (Figure 3) demonstrating the presence of each SrtAI and a loss of crystallinity for most formulations, means that it is highly likely that the SrtAIs were loaded within the mesopores.    SrtAI solubility was assessed in water to evaluate if SrtAI-loaded MSN formulations improved the dissolution of SrtAIs in water. The amount of each SrtAI that dissolved in water was significantly increased for each SrtAI MSN formulation (Figure 4), except for the QC-loaded MCM-41 formulation. The largest improvements in solubility were observed for the CUR (8.5-fold compared to CUR alone) and TC-loaded (34.7-fold compared to TC alone) MSNs, followed by BR-loaded MSNs (3.3-fold compared to BR alone). In comparison, minimal improvement was observed for the QC-loaded MSN formulations. Furthermore, no statistically significant differences in solubility were observed in the case of each SrtAI between their MCM-41 and MCM-41-PO 3 − formulations. The enhanced solubility profile for each SrtAI-loaded MSN formulation is likely explained by the conversion of the compounds from a crystalline to an amorphous state upon MSN encapsulation, which has previously been demonstrated to improve the aqueous solubility of hydrophobic crystalline compounds [36].

Checkerboard Assay
A checkerboard assay was performed to investigate the possibility of antimicrobial synergy between SrtAI-loaded MSNs and AMPs (PEX, INDO, and MASTO) against S. aureus (MSSA) ATCC25923, MRSA ATCC43300, E. coli ATCC25922, and P. aeruginosa ATCC27853. Such synergy would decrease the amount of AMP and/or SrtAI required for antimicrobial efficacy, potentially reducing the cost, adverse effects, and opportunities for antimicrobial resistance to develop. The results of this assay (Table 4 and Figures S5-S19, with S. aureus ATCC25923 examples presented in Figure 6) are presented after the calculation of ΣFIC values from the well/s that demonstrated the largest change in the MIC values of one of the antimicrobial agents (FIC wells are indicated by orange and green boxes in Figure 6, with individual component MIC values indicated by yellow boxes). ΣFIC values ≤ 0.5 are representative of synergistic interactions, whereas values greater than 0.5 to 1 are additive and values greater than 1 to 4 are indifferent. From these results, the best synergy data was obtained where PEX was combined with SrtAI-loaded MSNs. Of these, the most synergistic combinations were observed for PEX with TC-loaded MCM-41 MSNs, which demonstrated synergy against all tested bacterial strains, with the MCM-41-PO 3 − formulation displaying similar activity, although with additive rather than synergistic activity against P. aeruginosa (Table 4 and Figures S5-S8). Similarly, BR-loaded MSNs demonstrated synergy against MSSA ( Figure 6 and Table 4) and MRSA, with additive activity towards E. coli; however, this combination was borderline indifferent against P. aeruginosa (Table 4 and Figures S17-S19). The combination of CUR and QC-loaded MSNs with PEX demonstrated further reductions in benefit (Table 4), with the CUR-loaded MSNs demonstrating additive effects against MSSA, MRSA, E. coli, and P. aeruginosa when formulated with MCM-41 ( Figures S9-S12), with indifferent effects against P. aeruginosa when the MCM-41-PO 3 − formulation was used ( Figure S12). In contrast, the QC-loaded MSNs demonstrated little benefit when combined with PEX (Table 4), with additive effects seen against MSSA (Figure 6), MRSA, and E. coli for the MCM-41-PO 3 − formulation (Figures S13-S15), but reduced to indifferent effects against E. coli for the MCM-41 formulation ( Figure S15), and indifferent effects against P. aeruginosa for both MSN formulations ( Figure S16) were observed. Synergy was also observed for the combination of MASTO with CUR-loaded MCM-41 MSNs against E. coli ( Figure S11), with additive effects against all other assessed bacterial strains (Table 4 and Figures S9, S10 and S12) and in the cases of INDO with QC-loaded MCM-41 and MCM-41-PO 3 − MSNs against MRSA (Table 4 and Figure S14). In this second case, additive effects were observed against MSSA (Figure 6), with indifferent effects against E. coli and P. aeruginosa (Table 4 and Figures S15 and S16). Overall, many of the assessed combinations were synergistic or additive against the assessed bacterial library (Table 4), with the combination of BR-loaded MCM-41 MSNs with PEX providing the greatest synergy against the assessed bacterial library. This combination (Figure 6) reduced the MIC of BR towards MSSA by 16-(yellow box; 7.81 mg/mL) or 8-fold (orange box; 15.6 mg/mL) and PEX by 4-(yellow box; 1.57 mg/mL) or 8-fold (orange box; 0.79 mg/mL), depending on which synergistic combination was selected. 41-PO3 − formulation displaying similar activity, although with additive rather than synergistic activity against P. aeruginosa (Table 4 and Figures S5-S8). Similarly, BR-loaded MSNs demonstrated synergy against MSSA ( Figure 6 and Table 4) and MRSA, with additive activity towards E. coli; however, this combination was borderline indifferent against P. aeruginosa (Table 4 and Figures S17-S19). The combination of CUR and QC-loaded MSNs with PEX demonstrated further reductions in benefit (Table 4), with the CURloaded MSNs demonstrating additive effects against MSSA, MRSA, E. coli, and P. aeruginosa when formulated with MCM-41 ( Figures S9-S12), with indifferent effects against P. aeruginosa when the MCM-41-PO3 − formulation was used ( Figure S12). In contrast, the QCloaded MSNs demonstrated little benefit when combined with PEX (Table 4), with additive effects seen against MSSA (Figure 6), MRSA, and E. coli for the MCM-41-PO3 − formulation ( Figures S13-S15), but reduced to indifferent effects against E. coli for the MCM-41 formulation ( Figure S15), and indifferent effects against P. aeruginosa for both MSN formulations ( Figure S16) were observed. Synergy was also observed for the combination of MASTO with CUR-loaded MCM-41 MSNs against E. coli ( Figure S11), with additive effects against all other assessed bacterial strains (Table 4 and Figures S9, S10 and S12) and in the cases of INDO with QC-loaded MCM-41 and MCM-41-PO3 − MSNs against MRSA (Table 4 and Figure S14). In this second case, additive effects were observed against MSSA ( Figure 6), with indifferent effects against E. coli and P. aeruginosa (Table 4 and Figures S15 and S16). Overall, many of the assessed combinations were synergistic or additive against the assessed bacterial library (Table 4)

Cell Viability
The effects of SrtAI-loaded and non-loaded MSNs, AMPs (PEX, INDO, and MASTO), and their combinations on HEK-293 cell viability were evaluated using a resazurin-based assay [37] to determine if their synergistic combinations could reduce toxicity associated with individual components and if the phosphonate modification of MCM-41 MSNs affects toxicity. Concentrations (Table S1) based on a two-fold dilution series, which ensure two concentrations below and three concentrations above the MIC (Table 3) or synergistic concentrations determined by ΣFIC (Table 4)  In contrast, the CUR and QC-loaded MSNs demonstrated minimal effects on cell viability across the tested concentration ranges. This data suggests that CUR and QC-loaded MSNs may provide a less toxic means of inhibiting SrtA, with combinations of BR and TC-loaded MSNs that provide synergy providing a means to reduce these toxicities by allowing for delivery of lower SrtAI doses. Finally, combinations of SrtAI-loaded MSNs with AMPs, which demonstrated synergy in the checkerboard assay, were assessed for their effects on HEK-293 cell viability. Based on this data (Figure 8), all the assessed combinations demonstrated no effects on cell viability at concentrations corresponding to the checkerboard assay well that was antimicrobial synergies towards future in vivo and in vitro applications. Of these, the TCand BR-loaded MCM-41-PO3 − MSNs with PEX demonstrated synergistic or additive effects towards all the assessed bacterial strains, and thus may provide more broad-spectrum efficacy compared to the QC-loaded MCM-41-PO3 − MSNs with INDO, which only demonstrated synergistic or additive effects towards MSSA and MRSA.

Conclusions
Sortase A inhibitors have demonstrated significant promise for the treatment antibiotic resistant microorganisms, with their combination with other antimicrobials offering opportunities for antimicrobial synergy and improved efficacy, in addition to reductions in adverse effects and the development of antimicrobial resistance. However, relatively few SrtAIs have been identified, and of these, many exhibit poor pharmaceutical characteristics and are expensive. The naturally derived SrtAIs studied herein (TC, CUR, QC, and BR) are inexpensive, safe, and readily available, with many used as dietary supplements, but they display poor aqueous solubility. Herein, the use of MCM-41 and a phosphonate-modified analogue, MCM-41-PO3 − , were demonstrated to improve the aqueous solubility of these compounds, affording improved opportunities for their use as SrtAIs.  Figure 8A). Similarly, a greater than 60% reduction in cell viability was observed at 8-fold above the FIC concentrations for BR-loaded MCM-41 MSNs with PEX ( Figure 8A). Overall, this suggests that the MCM-41-PO 3 − MSN synergistic formulations may provide a less toxic means of translating the observed antimicrobial synergies towards future in vivo and in vitro applications. Of these, the TC-and BR-loaded MCM-41-PO 3 − MSNs with PEX demonstrated synergistic or additive effects towards all the assessed bacterial strains, and thus may provide more broad-spectrum efficacy compared to the QC-loaded MCM-41-PO 3 − MSNs with INDO, which only demonstrated synergistic or additive effects towards MSSA and MRSA.

Conclusions
Sortase A inhibitors have demonstrated significant promise for the treatment antibiotic resistant microorganisms, with their combination with other antimicrobials offering opportunities for antimicrobial synergy and improved efficacy, in addition to reductions in adverse effects and the development of antimicrobial resistance. However, relatively few SrtAIs have been identified, and of these, many exhibit poor pharmaceutical characteristics and are expensive. The naturally derived SrtAIs studied herein (TC, CUR, QC, and BR) are inexpensive, safe, and readily available, with many used as dietary supplements, but they display poor aqueous solubility. Herein, the use of MCM-41 and a phosphonatemodified analogue, MCM-41-PO 3 − , were demonstrated to improve the aqueous solubility of these compounds, affording improved opportunities for their use as SrtAIs. Using 5% (v/v) DMSO in media as a solvent, these formulations improved their MIC values against medically relevant Gram-positive (S. aureus), -negative (E. coli and P. aeruginosa), and antibiotic-resistant (MRSA) bacteria compared to non-encapsulated formulations, with the phosphonate-modified MSNs generally demonstrating slight improvements in antimicrobial potency and reduced effects on HEK-293 cell viability compared to MCM-41 MSNs. These formulations were also assessed for their capacity to elicit synergistic improvements in antimicrobial efficacy when combined with a library of leading antimicrobial peptides (PEX, MASTO, INDO), with synergistic improvements in antimicrobial efficacy against all tested bacteria observed with TC-loaded MCM-41 when combined with PEX, and similar results, albeit with additive effects on P. aeruginosa, observed with the TC-loaded MCM-41-PO 3 − formulation. Furthermore, antimicrobial synergy against MSSA and MRSA was observed for both BR-loaded MSNs when combined with PEX, while synergy against MRSA was observed for both QC-loaded MSNs combined with INDO. These findings suggest that such formulations can significantly improve the antimicrobial potency of both SrtAIs and AMPs, with PEX demonstrating the most synergistic combinations of all the assessed AMPs against the broadest range of bacterial strains. Such improvements will be of significant benefit for the translation of AMPs to the clinic, with the enhanced potency reducing the amount of an AMP required, along with their cost. The combinations also reduced toxicity towards HEK-293 cells, with the MCM-41-PO 3 − formulations demonstrating minimal effects on cell viability. In conclusion, this work demonstrates that MSNs provide a useful strategy for improving the aqueous solubility of the assessed SrtAIs, which provides opportunities for their translation to the market and for the identification of formulations/combinations with beneficial effects on antimicrobial activity and toxicity. and MASTO) against S. aureus ATCC25923 using checkerboard assay. Figure S6. Fractional inhibitory