Matrix-Mediated Delivery of Silver Nanoparticles for Prevention of Staphylococcus aureus and Pseudomonas aeruginosa Biofilm Formation in Chronic Rhinosinusitis

Abstract

Chronic rhinosinusitis (CRS) is a chronic health condition affecting the sinonasal cavity. CRS-associated mucosal inflammation leads to sinonasal epithelial cell death and epithelial cell barrier disruption, which may result in recurrent bacterial infections and biofilm formation. For patients who fail medical management and elect endoscopic sinus surgery for disease control, bacterial biofilm formation is particularly detrimental, as it reduces the efficacy of surgical intervention. Effective treatments that prevent biofilm formation in post-operative patients in CRS are currently limited. To address this unmet need, we report the controlled release of silver nanoparticles (AgNps) with silk-elastinlike protein-based polymers (SELPs) to prevent bacterial biofilm formation in CRS. This polymeric network is liquid at room temperature and forms a hydrogel at body temperature, and is hence, capable of conforming to the sinonasal cavity upon administration. SELP hydrogels demonstrated sustained AgNp and silver ion release for the studied period of three days, potent in vitro antibacterial activity against Pseudomonas aeruginosa (**** p < 0.0001) and Staphylococcus aureus (**** p < 0.0001), two of the most commonly virulent bacterial strains observed in patients with post-operative CRS, and high cytocompatibility with human nasal epithelial cells. Antibacterial controlled release platform shows promise for treating patients suffering from prolonged sinonasal cavity infections due to biofilms.

Keywords: SELPs; biofilm; chronic rhinosinusitis; in situ gel; silver nanoparticle; sustained release.

Figure 1

(A) AgNps features after characterization with TEM, DLS, spectrophotometry, and ICP/MS. (B) UV−Vis surface plasmon resonance spectrum indicates that AgNPs are colloidally stable upon receipt from the manufacturer and after 2 months in storage at 4 °C, (C) representative TEM images of AgNps, and (D) DLS size distribution demonstrate an average hydrodynamic radius of 10 nm for AgNps. TEM images were produced by nanocomposix. silver nanoparticles (AgNps), dynamic light scattering (DLS), transmission electron microscopy (TEM), and inductively coupled plasma mass spectroscopy (ICP/MS).

Figure 2

Effect of HA on the viability and pro-inflammatory cytokine release from LPS-stimulated THP-1 cells. HA significantly increased the (A) cell viability and decreased the secretion of (B) IL-6 and (C) IL-8. n = 3. Data are represented as mean ± SD. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Lipopolysaccharide (LPS), hyaluronic acid (HA).

Figure 3

The cytocompatibility of hydrogel formulations in the presence of HNEpCs. (A) A schematic illustrating the cell viability experimental setup; (B) the percentage of viable cells after exposure to different free AgNps and hydrogel formulations compared to non-treated control cells, measured using the CCK-8 cell viability assay. Biological replicates (N = 2) and technical replicates (n = 3) for each experiment were performed. Data are represented as the mean ± SD. (* p < 0.05; *** p < 0.001; **** p < 0.0001). Human nasal epithelial cells (HNEpCs), cell counting kit-8 (CCK-8), silk-elastinlike protein polymers (SELPs), hyaluronic acid (HA), silver nanoparticles (AgNps). Numbers in parentheses are micrograms per mL.

Figure 4

SELP/HA/AgNPs (625 µg/mL) showed a bactericidal effect against S. aureus and P. aeruginosa. (A) Schematic illustration of the cell viability assay. (B) The antibacterial effect is represented as log CFU/mL reduction in bacterial counts by treatment with the hydrogels compared to wide-spectrum antibiotic ciprofloxacin. Biological replicates (N = 2) and technical replicates (n = 3) for each experiment were performed. Data are represented as the mean ± SD. (**** p < 0.0001). Colony forming units (CFU), silk-elastinlike protein polymers (SELPs), hyaluronic acid (HA), silver nanoparticles (AgNps).

Figure 5

SELP/HA/AgNps (625 µg/mL) significantly prevented biofilm formation. (A) A schematic illustration of the colony biofilm experiment setup. (B) Images of polycarbonate filters after 24 h incubation with different treatments. The dark brown color observed in SELP/HA/AgNps (625 µg/mL) is due to the incorporation of AgNps. Notably, the AgNps stock solution is naturally dark in color. (C) Influence on CFU/filter due to treatment with different formulations. Biological replicates (N = 2) and technical replicates (n = 3) for each experiment were performed. Data are represented as the mean ± SD (**** p < 0.0001). Colony forming units (CFUs), silk-elastinlike protein polymers (SELPs), hyaluronic acid (HA), silver nanoparticles (AgNps), scanning electron microscopy (SEM).

Figure 6

SEM images of polycarbonate filters showing the presence of biofilm in control and SELP/HA treatment. The treatment with SELP/HA/AgNps and ciprofloxacin showed complete biofilm prevention after a 24-h incubation. Silk-elastinlike protein polymers (SELPs), hyaluronic acid (HA), silver nanoparticles (AgNps), scanning electron microscopy (SEM).

Figure 7

Release kinetics of Ag+ ions from SELP/AgNps (625 µg/mL) and SELP/HA/AgNps (625 µg/mL) in simulated nasal fluid. (* p < 0.05; ** p < 0.01). Silk-elastinlike protein polymers (SELPs), hyaluronic acid (HA), silver nanoparticles (AgNps).

Figure 8

Changes to AgNps in the presence of SNF. (A) Surface plasmon resonance spectrum of AgNps after incubation in SNF, (B) TEM image of AgNp after 24 h incubation in SNF, (C) SEM image of SELP/HA/AgNps (625 µg/mL) after 24 h incubation in SNF. Silver nanoparticle (AgNps), simulated nasal fluid (SNF), silk-elastinlike protein polymers (SELPs), hyaluronic acid (HA), transmission electron microscopy (TEM), scanning electron microscopy (SEM).

Figure 9

The thermoresponsive properties of SELP/HA/AgNPs (625 µg/mL). (A) Viscosity as a function of temperature, (B) viscosity at different time points, (C) storage modulus as a function of time, (D) storage modulus at different time points.