Superhydrophobic Photosensitizers: Airborne 1O2 Killing of an in Vitro Oral Biofilm at the Plastron Interface

Abstract

Singlet oxygen is a potent agent for the selective killing of a wide range of harmful cells; however, current delivery methods pose significant obstacles to its widespread use as a treatment agent. Limitations include the need for photosensitizer proximity to tissue because of the short (3.5 μs) lifetime of singlet oxygen in contact with water; the strong optical absorption of the photosensitizer, which limits the penetration depth; and hypoxic environments that restrict the concentration of available oxygen. In this article, we describe a novel superhydrophobic singlet oxygen delivery device for the selective inactivation of bacterial biofilms. The device addresses the current limitations by: immobilizing photosensitizer molecules onto inert silica particles; embedding the photosensitizer-containing particles into the plastron (i.e. the fluid-free space within a superhydrophobic surface between the solid substrate and fluid layer); distributing the particles along an optically transparent substrate such that they can be uniformly illuminated; enabling the penetration of oxygen via the contiguous vapor space defined by the plastron; and stabilizing the superhydrophobic state while avoiding the direct contact of the sensitizer to biomaterials. In this way, singlet oxygen generated on the sensitizer-containing particles can diffuse across the plastron and kill bacteria even deep within the hypoxic periodontal pockets. For the first time, we demonstrate complete biofilm inactivation (>5 log killing) of Porphyromonas gingivalis, a bacterium implicated in periodontal disease using the superhydrophobic singlet oxygen delivery device. The biofilms were cultured on hydroxyapatite disks and exposed to active and control surfaces to assess the killing efficiency as monitored by colony counting and confocal microscopy. Two sensitizer particle types, a silicon phthalocyanine sol-gel and a chlorin e6 derivative covalently bound to fluorinated silica, were evaluated; the biofilm killing efficiency was found to correlate with the amount of singlet oxygen detected in separate trapping studies. Finally, we discuss the applications of such devices in the treatment of periodontitis.

Keywords: biofilm eradication; dentistry; photodynamic therapy; singlet oxygen; superhydrophobic device.

Figure 1.

A schematic of the superhydrophobic device: a red diode laser (669 nm) is coupled to an optical fiber; the output SMA ferrule is mounted such that the light is directed downward. The superhydrophobic (SH) surface, printed on a 130 μm thick coverslip, is placed tip-face down on the bacterial biofilm. SiO₂ nanoparticles are used to cap the SH surface. Singlet oxygen traverses the plastron to reach the biofilm, where inactivation then takes place.

Figure 2.

Two types of sensitizing particles were examined (particles Si-Pc and e6). Particle Si-Pc has bis-amino Si-phthalocyanine incorporated in a sol-gel. Particle e6 has chlorin covalently bound to fluorinated silica.

Figure 3.

Photooxidation of the anthracene 1 and alkene 3 traps in a particle/solution dispersion via 669-nm irradiation of Si-Pc or e6 particles in D₂O. The particle surface is wetted and solution-phase ¹O₂ is generated at the sensitizer particle surface. Evidence for a reaction of ¹O₂ with 1 is the formation of endoperoxide 2 (quantified by the disappearance of a peak at 378 nm), and with 3 is the formation of hydroperoxide 4 (quantified by the appearance of the hydroperoxide peaks at H_b, H_c, and H_d by ¹H NMR).

Figure 4.

Singlet oxygen production by Si-Pc and e6 particles (20 mg) in D₂O solution (pre-saturated with O₂) plotted as a function of fluence. The singlet oxygen concentrations were estimated from the photooxidation of anthracene 1. The Si-Pc or e6 particles were irradiated for 6, 12, 18, and 24 min at constant irradiance of 0.25 W/cm²

Figure 5.

Photobleaching evaluation: Singlet oxygen production based on the formation of endoperoxide 2 by the oxidation of anthracene 1 by Si-Pc and e6 particles dispersed in D₂O solution at 0.25 W/cm² for 15 min (fluence = 270 J/cm²). The left hand (solid) columns indicate values using particles without pre-irradiation, whereas the right hand columns (cross-hatched) were from particles pre-irradiated at 0.25 W/cm² for 45 min in air (fluence = 675 J/cm²), prior to the singlet oxygen trapping measurement.

Figure 6.

A water droplet poised on superhydrophobic surfaces a. low magnification view of water on a SH surface with chlorin e6 particles without PDMS-silica caps. b. a high magnification view showing the water partially wetting the upper chlorin e6 particles. c. high magnification view of a Si-Pc SH surface capped with PDMS-silica nanoparticles; water penetration is limited due to the high surface area low surface energy nanoparticle coating.

Figure 7.

a. P. gingivalis inactivation by superhydrophobic surfaces as a function of fluence (J/cm²), measured by CFUs after exposure. Biofilm only controls without light (SH- L- S-) were incubated for 7.5 and 15 min. respectively. Sensitizer-less SH surfaces (SH+ L+ S-) and SH surfaces with Si-Pc (SH+ L+ S+) were exposed to fluence values of 45, 90, 180, 270 and 315 J/cm². Data is expressed as Mean ± SEM of three independent experiments, n=3. *p<0.05. The numbers in the parentheses refer to irradiance values of 0, 0.1, 0.2, 0.3 or 0.35 W/cm² at either 7.5 or 15 minutes. b. Representative 3D images of 72-hr grown P. gingivalis biofilms following treatment with a Si-Pc SH surface. The photoinactivation effect on biofilms as rendered by Si-Pc SHS at variable light doses, compared to biofilm only control without light treatment. Green signal represents viable cells (Syto 9), red signal indicates damaged/dead cells (propidium iodide). Panels are of xyz-stacks of biofilm growth.

Figure 8.

Inactivation of P. gingivalis biofims by Si-Pc SH surfaces and controls at a fluence of a. 270 J/cm² (*p<0.006) and b. 315 J/cm² (*p<0.003). The inactivation of bacterial biofilms is shown as log viable count and percentage killing. Data is expressed as Mean ± SEM of three independent experiments, n=3.

Figure 9.

Representative images of P. gingivalis biofilms following each treatment group with Si-Pc SHS and controls. Green signal represents viable live cell (Syto 9), red signal indicates damaged/dead cells (propidium iodide). Image panels; Live, Dead, and Merged (Live + Dead) are x-y plane images.

Figure 10.

Inactivation of P. gingivalis biofims by e6 SH surfaces and controls at a fluence of a. 270 J/cm² (*p<0.0002) and b. 315 J/cm² (*p<0.006). The inactivation of bacterial biofilms is shown as log viable count and percentage killing. Data is expressed as Mean ± SEM of three independent experiments, n=3.