3.3 DESCRIPCIÓN DEL MBR

The synthesis and subsequent microbiological testing of NO-releasing silica nanoparticles and dendrimers with different chemical and physical properties was described. Chapter 1 provided an overview of specific oral health ailments (i.e., dental caries and periodontal disease), current research into treating oral diseases, and the need for alternative therapies to effectively combat oral infections. The introduction established the potential advantages to using NO-release vehicles as dental therapeutics, with an emphasis on tuning their physical and chemical properties to maximize bactericidal efficacy while minimizing cytotoxicity. In Chapter 2, similarly sized (~150 nm), monodisperse NO-releasing silica particles with tunable NO storage and release kinetics were synthesized via the Stöber method. Optimization of N-diazeniumdiolate modification solution parameters (i.e., sodium methoxide concentration) was performed to maximize silica particle NO storage while limiting the formation of unwanted byproducts. Alteration of synthetic parameters provided 70 mol% MAP3 silica particles with increased size (~400 nm), but with similar NO- release totals (~1 µmol/mg) and release kinetics (half-life of ~15 min) to the 150 nm MAP3 particles. Additional N-diazeniumdiolate modification studies afforded ~150 nm NO-releasing silica particles with different NO-release half-lives allowing the effect of NO-release kinetics on bacterial killing, independent of both particle size and 2 h NO-release totals, to be studied. The bactericidal efficacy and cytotoxicity for a portion of the NO-releasing nanoparticles developed in Chapter 2 was described in subsequent chapters.

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In Chapter 3, the antibacterial activity of NO-releasing silica nanoparticles, dendrimers, and proline (PROLI/NO) was evaluated against pathogenic oral bacteria. Despite storing less NO, the bactericidal efficacy of macromolecular NO-release vehicles (i.e., dendrimers and silica) was greater than that of the small molecule NO donor PROLI/NO. The stabilization of the N- diazeniumdiolate donor (i.e., longer half-lives) by macromolecular NO-release scaffolds provided more efficient biocidal NO delivery to bacteria. Cariogenic bacteria (i.e., S. mutans and S. sanguinis) were less susceptible to macromolecular-based NO release (≥37.0 µmol NO/mL required to kill) than periodontopathogens (i.e., A. actinomycetemcomitans and P. gingivalis), which required ≤3.2 µmol NO/mL for eradication. The greatest concentrations of NO-releasing dendrimers and silica required to kill periodontopathogens (2 and 4 mg/mL, respectively) proved relatively non-toxic to human gingival fibroblasts (HGF-1) compared to untreated cells (viabilities ≥60%) and especially when compared to chlorhexidine (~20% cell viability). These results suggested a differential sensitivity between cariogenic bacteria and periodontopathogens to NO treatment and supported further investigation into macromolecular NO-release vehicles as potential periodontal disease therapeutics.

Chapter 4 described the bactericidal efficacy of NO-releasing silica particles with varied NO-release kinetics (i.e. half-lives) against oral bacteria. Extended NO-release kinetics (half-lives of ~130 min versus ~32 min) proved to be more effective at killing periodontal pathogens (i.e., A. actinomycetemcomitans and P. gingivalis) and reduced the bactericidal NO dose by at least half. Confocal microscopy using the intracellular NO-sensitive dye DAF-2 DA confirmed more efficient NO delivery to A. actinomycetemcomitans from silica particles with extended NO-release. Furthermore, the slower NO-release kinetics combined with the lower silica concentrations necessary to kill bacteria reduced HGF-1 cytotoxicity, supporting the use of extended NO-release

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vehicles to kill periodontopathogens. As observed previously, S. mutans was significantly less sensitive to NO treatment, with no observed reduction in bacterial viability up to 51.8 µmol NO/mL at pH 7.4. However, faster NO-release kinetics obtained under acidic conditions (pH 6.4) increased the bactericidal efficacy of NO-releasing silica particles and reduced the NO dose required to kill (30.4 µmol NO/mL). Confocal microscopy using DAF-2 DA confirmed more rapid intracellular NO accumulation and greater bacterial killing with faster NO-release kinetics at pH 6.4.

In Chapter 5, NO-releasing propyl-, butyl-, hexyl-, octyl-, and dodecyl-modified PAMAM dendrimers were synthesized to create dual-action antibacterial agents. The bactericidal efficacy and anti-biofilm activity of NO-releasing alkyl-modified dendrimers were evaluated against S. mutans with respect to pH, NO-release kinetics, and alkyl chain length. Greater bactericidal and anti-biofilm efficacy was observed for longer alkyl chain (i.e., hexyl, octyl, dodecyl) dendrimers versus shorter alkyl chain (i.e., propyl and butyl) modifications, attributed to more extensive bacterial damage from membrane intercalation of the longer hydrophobic chains. The addition of NO did not improve the bactericidal efficacy of longer alkyl chains, as the highly efficient membrane damage caused by the hydrophobic tails resulted in bacterial death prior to significant intracellular NO accumulation. At lower pH, confocal microscopy using RITC-modified butyl dendrimers confirmed greater dendrimer-bacteria association. The enhanced bactericidal efficacy of longer alkyl chain dendrimers at lower pH was thus due to increased pendant amine protonation, resulting in improved dendrimer-bacteria association and greater bacterial membrane damage. Since the shorter alkyl chain dendrimer scaffolds demonstrated minimal bactericidal efficacy at pH 6.4, the enhanced antibacterial and anti-biofilm activity at lower pH was attributed to faster NO-release kinetics. The greater instantaneous concentration of NO resulted in lower NO doses

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required to kill S. mutans. Short alkyl chain dendrimers proved to be non-toxic against L929 mouse fibroblasts, but were extremely toxic (0% viability relative to untreated cells) with the addition of NO release due to the large NO dose required to kill bacteria. Although the octyl- and dodecyl-modified dendrimer scaffolds were toxic to murine fibroblasts, the addition of NO release increased cell viability, partially mitigating the cytotoxicity of the scaffolds. The superior bactericidal efficacy of octyl- and dodecyl-modified dendrimers, combined with the reduced cytotoxicity observed with integrated NO release, supports the development of NO-releasing long alkyl chain-modified dendrimers as anti-biofilm dental caries therapeutics.