Date on Master's Thesis/Doctoral Dissertation

5-2019

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Steinbach-Rankins, Jill M.

Committee Co-Chair (if applicable)

Demuth, Donald R.

Committee Member

Hein, David W.

Committee Member

Palmer, Kenneth E.

Committee Member

El-Baz, Ayman

Author's Keywords

polymer nanoparticle; peptide delivery; periodontal disease; BAR peptide; drug delivery

Abstract

Background: Periodontal diseases are globally prevalent inflammatory disorders that affect ~47% of U.S adults. Porphyromonas gingivalis (Pg) has been identified as a “keystone” pathogen that disrupts host-microbe homeostasis and contributes to the initiation and progression of periodontitis. Pg associates with oral streptococci in supragingival plaque and this interaction represents a potential target for therapeutic intervention. Previously our group developed a peptide (designated BAR), that potently inhibits Pg/Streptococcus gordonii (Sg) adherence in vitro and Pg virulence in a murine model of periodontitis. While efficacious, BAR (SspB Adherence Region) provided transient inhibition and required higher concentrations of BAR to disrupt established biofilms. Hypothesis and Aims: To address these challenges, we hypothesized that BAR-surface modified and BAR-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) may more potently inhibit and disrupt biofilms in vitro and in vivo, relative to free BAR. In addition, a new rapid-release platform, composed of polymeric electrospun fibers (EFs) that encapsulate BAR peptide, was developed. Given this, our objectives were to evaluate BAR-surface modified NPs in a murine model of periodontitis; to fabricate and assess the ability of BAR-encapsulated NPs to inhibit and disrupt in vitro oral biofilm formation, and to evaluate a new dosage form, electrospun fibers, to inhibit andformation, and to evaluate a new dosage form, electrospun fibers, to inhibit and disrupt in vitro oral biofilm formation. In addition, the safety of all platforms was determined via viability, apoptosis, adenosine triphosphate (ATP), lactate dehydrogenase (LDH) and oxidative DNA assays using telomerase immortalized gingival keratinocytes (TIGKs). Methods: BAR-encapsulated and BAR-surface modified PLGA NPs were synthesized using adapted double- and single-emulsion techniques, respectively. Electrospun fibers were formed using a uniaxial approach, with different hydrophobic polymers (PLGA, polycaprolactone, poly(L-lactic acid)); each blended with different polyethylene oxide ratios (PEO: 0, 10, 20, or 40% w/w) to achieve maximal release of BAR. Both BAR-encapsulated NPs and EFs were assessed for inhibition of two-species biofilm formation and for disruption of pre-existing biofilms, against an equimolar free BAR concentration. In vivo efficacy of BAR-surface modified NPs was assessed using a murine model of periodontitis by measuring alveolar bone resorption and gingival IL-17 expression as outcomes of Pg-induced inflammation. Results: BAR-encapsulated NPs and EFs inhibited biofilm formation (IC50s = 0.7 and 1.3 μM, respectively) in a dose-dependent manner, relative to free BAR (IC50 = 1.3 µM). In addition, BAR-encapsulated NPs and EFs efficiently disrupted established dual-species biofilms (IC50s = 1.3 and 2 μM, respectively). Treatment of Pg/Sg infected mice with BAR-surface modified NPs reduced alveolar bone loss and IL-17 expression almost to the levels of sham-infected mice and to a greater extent than treatment with an equimolar amount of free BAR. The in vitro cytotoxicity studies, which utilized the maximum concentration of BAR-encapsulated NPs, BAR-surface modified NPs, BAR EFs, and free BAR (1.3 and 3.4 μM) demonstrated > 90% viability for all samples and showed no significant lysis or apoptosis relative to untreated cells. In addition, all tested formulations exhibited a lack of hemolytic activity. Conclusion: These data suggested that BAR NPs and EFs provide novel and potent platforms to inhibit and disrupt dual-species biofilms. All formulations exhibited minimal cellular toxicity or hemolytic activity, highlighting the potential of NPs and EFs as a biocompatible platform for translatable oral biofilm applications.

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