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. 2020 Jan 21;7:926.
doi: 10.3389/fchem.2019.00926. eCollection 2019.

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

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Free PMC article

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

Mohamed Y Mahmoud et al. Front Chem. .
Free PMC article

Abstract

Active agents targeting key bacterial interactions that initiate biofilm formation in the oral cavity, may alter periodontitis progression; however, to date, specifically-targeted prophylactic and treatment strategies have been limited. Previously we developed a peptide, BAR (SspB Adherence Region), that inhibits oral P. gingivalis/S. gordonii biofilm formation in vitro and in vivo, and BAR nanoparticles that increase BAR effectiveness via multivalency and prolonged delivery. However, limited BAR loading and nanoparticle retention in the oral cavity can result in inadequate release and efficaciousness. Given this, an effective delivery platform that can release concentrations of BAR suitable for twice-daily applications, may offer an alternative that enhances loading, ease of administration, and retention in the oral cavity. With this in mind, the study objectives were to develop and characterize a rapid-release platform, composed of polymeric electrospun fibers (EFs) that encapsulate BAR, and to evaluate fiber safety and functionality against P. gingivalis/S. gordonii biofilms in vitro. Poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic acid) (PLLA), and polycaprolactone (PCL) were electrospun alone or blended with polyethylene oxide (PEO), to provide high BAR loading and rapid-release. The most promising formulation, 10:90 PLGA:PEO EFs, provided 95% BAR release after 4 h, dose-dependent inhibition of biofilm formation (IC50 = 1.3 μM), disruption of established dual-species biofilms (IC50 = 2 μM), and maintained high cell viability. These results suggest that BAR-incorporated EFs may provide a safe and specifically-targeted rapid-release platform to inhibit and disrupt dual-species biofilms, that we envision may be applied twice-daily to exert prophylactic effect in the oral cavity.

Keywords: Porphyromonas gingivalis; Streptococcus gordonii; electrospun fibers; oral biofilm; peptide delivery; periodontal disease.

Figures

Figure 1
Figure 1
(A) SEM images of 1% w/w BAR PLGA, PLLA, and PCL non-blended fibers. (B) SEM images of 40:60, 20:80, and 10:90 1% w/w BAR blended PLGA:PEO, PLLA:PEO, and PCL:PEO fibers.
Figure 2
Figure 2
Average diameters of electrospun fibers measured from SEM images, using ImageJ. (A) Non-blended and blended (B) 40:60, (C) 20:80, and (D) 10:90 PLGA:PEO, PLLA:PEO, and PCL:PEO 1% w/w BAR fibers. Error bars represent the mean ± the standard deviation (n = 3) of three independent experiments.
Figure 3
Figure 3
The cumulative release of F-BAR from 1% w/w F-BAR non-blended (100:0) PLGA, PLLA, and PCL fibers. The cumulative release is reported as (A) μg F-BAR per mg of fiber, and (B) percent of total loaded F-BAR. PLGA showed the greatest release of incorporated BAR among the non-blended formulations at 24 h. Error bars represent the mean ± the standard deviation (n = 3) of three independent experiments.
Figure 4
Figure 4
The cumulative release of F-BAR from 1% w/w F-BAR blended PLGA:PEO, PLLA:PEO, and PCL:PEO fibers (A) 40:60, (B) 20:80, and (C) 10:90. The cumulative release is reported as the total quantity of F-BAR released on the left (μg F-BAR per mg of fiber), and as the percent of total loaded F-BAR on the right. Error bars represent the mean ± the standard deviation (n = 3) of three independent experiments.
Figure 5
Figure 5
The cumulative release of F-BAR from the non-blended and PEO-blended formulations as a function of hydrophobic polymer type (A) PLGA, (B) PLLA, or (C) PCL and PEO ratio in each blend. The release of encapsulated BAR increases with an increase in PEO fraction. PLGA and PEO blends exhibit the most significant and rapid F-BAR release, relative to PLLA and PCL blends. For all polymer types, the 10:90 blends show the greatest release of BAR as compared to the 20:80 and 40:60 formulations at any given time point. PLGA:PEO (10:90) fibers provide the highest amount of BAR release across formulations. Data represent the mean ± standard deviation (n = 3) of three independent experiments.
Figure 6
Figure 6
BAR-incorporated PLGA:PEO (10:90) EFs prevent P. gingivalis adherence to S. gordonii. Biofilms were visualized with confocal microscopy and the ratio of green (P. gingivalis) to red (S. gordonii) fluorescence in z-stack images was determined using Volocity image analysis software. Each grid represents 21 μm.
Figure 7
Figure 7
BAR-incorporated PLGA:PEO (10:90) EFs disrupt pre-established P. gingivalis-S. gordonii biofilms. Biofilms were visualized with confocal microscopy and the ratio of green (P. gingivalis) to red (S. gordonii) fluorescence in z-stack images was determined using Volocity image analysis software. Each grid represents 21 μm.
Figure 8
Figure 8
(A) Biofilm inhibition (prevention) and (B) disruption (treatment), as a function of different concentrations of BAR-incorporated PLGA:PEO (10:90) EFs and free BAR (3 μM). Data represent the mean ± standard deviation (n = 6) of six independent experiments.
Figure 9
Figure 9
(A) The hemolytic activity of free BAR or 10:90 PLGA:PEO BAR-EFs (1.3, 3.4 μM) was assessed after administration to sheep erythrocytes for 3 h. Free BAR and BAR-EFs showed negligible hemolysis for sheep erythrocyte relative to release from H2O-treated cells (****P ≤ 0.0001). (B) The effect of free BAR and BAR-EFs (1.3, 3.4 μM) on TIGK cell viability was assessed. Free BAR and BAR-EFs were non-toxic, relative to cells treated with DMSO (****P ≤ 0.0001). (C) Metabolic activity of cells treated with free BAR or BAR-EFs (1.3, 3.4 μM) was assessed. BAR-EF (3.4 μM) treated cells showed decreases in ATP levels relative to medium-only treated cells, while TIGK cells treated with staurosporine demonstrated lower ATP levels than the cells treated with medium-only, free BAR, and BAR-EFs (**P ≤ 0.01,****P ≤ 0.0001). (D) TIGK cell membrane integrity was assessed after administration of free BAR or BAR-EFs (1.3, 3.4 μM) by measuring LDH release levels. None of free BAR or BAR-EF (1.3, 3.4 μM) treated cells released a significant level of LDH relative to medium-only treated cells. Staurosporine-treated cells demonstrated significantly elevated LDH levels (****P ≤ 0.0001). Data represent the mean ± standard deviation (n = 5) of five independent experiments.
Figure 10
Figure 10
The number of AP sites per 100,000 base pairs of genomic DNA obtained from TIGK cells treated with free BAR or BAR-EFs (1.3, 3.4 μM). Negligible changes in the number of AP sites were observed in cells treated with free BAR or 10:90 PLGA:PEO BAR-EFs (1.3, 3.4 μM), relative to medium-only treated cells. However, TIGK cells treated with 2 mM H2O2 demonstrated significantly (***P ≤ 0.001) higher numbers of AP sites relative to the untreated control, free BAR and BAR-EF (1.3, 3.4 μM) treated cells. Data represent the mean ± standard deviation (n = 3) of three independent experiments.

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