Peptide-modified nanoparticles inhibit formation of <em>Porphyromonas gingivalis</em> biofilms with <em>Streptococcus gordonii</em>

Abstract

Purpose The interaction of Porphyromonas g ingivalis with commensal streptococci promotes P. gingivalis colonization of the oral cavity. We previously showed that a synthetic peptide (BAR) derived from Streptococcus gordonii potently inhibited the formation of P. gingivalis/S. gordonii biofilms (IC 50 =1.3 µM) and reduced P. gingivalis virulence in a mouse model of periodontitis. Thus, BAR represents a novel therapeutic to control periodontitis by limiting P. gingivalis colonization of the oral cavity. Here, we sought to develop drug-delivery vehicles for potential use in the oral cavity that comprise BAR-modified poly(lactic-co-glycolic)acid (PLGA) nanoparticles (NPs). Methods PLGA-NPs were initially modified with palmitylated avidin and subsequently conjugated with biotinylated BAR. The extent of BAR modification was quantified using a fluorescent-labeled peptide. Inhibition of P. gingivalis adherence to S. gordonii by BAR-modified NPs was compared with free peptide using a two-species biofilm model. Results BAR-modified NPs exhibited an average size of 99±29 nm and a more positive surface charge than unmodified NPs (zeta potentials of −7 mV and −25 mV, respectively). Binding saturation occurred when 37 nmol BAR/mg of avidin-NPs was used, which resulted in a payload of 7.42 nmol BAR/mg NPs. BAR-modified NPs bound to P. gingivalis in a dose-dependent manner and more potently inhibited P. gingivalis/S. gordonii adherence and biofilm formation relative to an equimolar amount of free peptide (IC 50 of 0.2 µM versus 1.3 µM). BAR-modified NPs also disrupted the preformed P. gingivalis/S. gordonii biofilms more effectively than free peptide. Finally, we demonstrate that BAR-modified NPs promoted multivalent association with P. gingivalis , providing an explanation for the increased effectiveness of NPs. Conclusion These results indicate that BAR-modified NPs deliver a higher local dose of peptide and may represent a more effective therapeutic approach to limit P. gingivalis colonization of the oral cavity compared to treatment with formulations of free peptide.

Rapid Release Polymeric Fibers for Inhibition of Porphyromonas gingivalis Adherence to Streptococcus gordonii

“…It was obtained with purity <94% and comprised residues 1167-1193 of the SspB (Antigen I/II) protein sequence of S. gordonii. A fluorescent BAR peptide (F-BAR), synthesized by covalently attaching 6carboxyfluorescein (F-BAR) to the epsilon amine of the lysine residue underlined in the sequence above, was used to more easily characterize BAR loading and release from the fibers via fluorescence detection (Kalia et al, 2017).…”

“…The fiber solution was subsequently vortexed, sonicated for 5 min, and dissolved for 1 h in a dark room. The quantity of extracted F-BAR was determined by measuring the fluorescence using a spectrophotometer (488/518 nm excitation/emission), relative to an F-BAR standard (Kalia et al, 2017;Mahmoud et al, 2018Mahmoud et al, , 2019. A standard curve of F-BAR was obtained by adding 0.1 mg F-BAR to 1 mL of 1:9 DMSO:TE, and serially diluting in 1:9 DMSO:TE.…”

“…The in vitro release of F-BAR from fibers was measured by gentle agitation of EFs in phosphate buffered saline (PBS, pH 7.4) at 37 • C. At fixed time points (1, 2, 4, 8, 12, and 24 h), samples were collected and the amount of F-BAR released from the EFs was quantified via fluorescence spectroscopy, against an F-BAR standard in PBS (Kalia et al, 2017;Tyo et al, 2017;Mahmoud et al, 2018).…”

“…GraphPad InStat (La Jolla, CA) was used for data analysis and differences were considered to be statistically significant when P ≤ 0.05. The percent inhibition of P. gingivalis adherence was calculated with the following formula: GR sample/GR control (Kalia et al, 2017;Mahmoud et al, 2018).…”

“…We previously showed that BAR-modified and BARencapsulated nanoparticles inhibit P. gingivalis biofilm formation (Kalia et al, 2017;Mahmoud et al, 2018Mahmoud et al, , 2019. These vehicles were envisioned to serve in formulations such as an oral gel, varnish or mouthwash that require two to three daily applications.…”