Kinetic-dependent Killing of Oral Pathogens with Nitric Oxide

Abstract

Nitric oxide (NO)-releasing silica nanoparticles were synthesized via the co-condensation of tetramethyl orthosilicate with aminosilanes and subsequent conversion of secondary amines to N-diazeniumdiolate NO donors. A series of ~150 nm NO-releasing particles with different NO totals and release kinetics (i.e., half-lives) were achieved by altering both the identity and mol% composition of the aminosilane precursors. Independent of identical 2 h NO-release totals, enhanced antibacterial action was observed against the periodontopathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis with extended NO-release kinetics at pH 7.4. Negligible bactericidal effect was observed against cariogenic Streptococcus mutans at pH 7.4, even when using NO-releasing silica particles with greater NO-release totals. However, antibacterial activity was observed against S. mutans at lower pH (6.4). This result was attributed to more rapid proton-initiated decomposition of the N-diazeniumdiolate NO donors and greater NO-release payloads. The data suggest a differential sensitivity to NO between cariogenic and periodontopathogenic bacteria with implications for the future development of NO-releasing oral care therapeutics.

Keywords: Aggregatibacter actinomycetemcomitans; N-diazeniumdiolate; Porphyromonas gingivalis; dental caries; periodontal disease; silica.

Figure 1.

Bactericidal efficacy of (A) 50 mol% 3-methylaminopropyltrimethoxysilane (MAP3) particles, (B) 60 mol% N-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3) particles, and (C) 80 mol% N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3) particles against Aggregatibacter actinomycetemcomitans in Tris–phosphate-buffered saline (PBS) (pH 7.4) after 2 h. Bactericidal efficacy of (D) 50 mol% MAP3 particles, (E) 60 mol% AHAP3 particles, and (F) 80 mol% AEAP3 particles against Porphyromonas gingivalis in Tris-PBS (pH 7.4) after 2 h. Nitric oxide (NO)–releasing material denoted by rectangles (■) and non-NO-releasing controls denoted by circles (●). Error bars signify standard deviation of the mean bacterial viability (colony-forming units [CFU]/mL). For all measurements, n = 3 or more pooled experiments.

Figure 2.

Cytotoxicity of nitric oxide (NO)–releasing and control silica particles against HGF-1 human gingival fibroblasts at the greatest MBC2h values to kill periodontopathogens. The concentrations to kill Aggregatibacter actinomycetemcomitans were 8, 32, and 48 mg/mL for 80 mol% N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3), 60 mol% N-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3), and 50 mol% 3-methylaminopropyltrimethoxysilane (MAP3), respectively. Viability measured as metabolic activity versus untreated cells. Error bars represent standard deviation of the mean. For all values, n = 4 replicate measurements. Asterisk indicates P < 0.05 using 2-tailed Student’s t test.

Figure 3.

Confocal microscopy images of Aggregatibacter actinomycetemcomitans exposed to 1 mg/mL nitric oxide (NO)–releasing silica nanoparticles (50 mol% 3-methylaminopropyltrimethoxysilane [MAP3], 60 mol% N-(6-aminohexyl)aminopropyltrimethoxysilane [AHAP3], and 80 mol% N-(2-aminoethyl)-3-aminopropyltrimethoxysilane [AEAP3]) after (A) 30 min, (B) 60 min, and (C) 120 min of particle exposure. DAF-2 fluorescence is depicted as black in this image for clarity and represents intracellular NO concentrations.

Figure 4.

Confocal microscopy images of Streptococcus mutans exposed to 0.25 mg/mL 70 mol% 3-methylaminopropyltrimethoxysilane (MAP3) nitric oxide–releasing silica nanoparticles at (A) 40 min, (B) 60 min, and (C) 90 min after particle exposure. DAF-2 fluorescence is depicted as black in the images for clarity. Numbers in images represent the relative summed signal intensities normalized to the fluorescence intensity at 0 min and pH 7.4.