Evaluation of photosensitizer-containing superhydrophobic surfaces for the antibacterial treatment of periodontal biofilms

Highlights

• A superhydrophobic film delivered airborne singlet oxygen to multispecies biofilms.

• Biofilms associated with periodontitis were effectively eradicated by SH-aPDT.

• Killing was linear with increasing fluence and photosensitizer loading for all species.

• CFU reductions of >4.6 log₁₀ were achieved at 71.6 J/cm² using a red LED.

• Gram-negative and Gram-positive species were killed equivalently.

Abstract

Antimicrobial photodynamic therapy (aPDT) is a promising approach to control biofilms involved in periodontal diseases. However, certain challenges, such as staining of teeth, preferential interaction of photosensitizer (PS) with Gram-positive versus Gram-negative bacteria, and insufficient oxygen in hypoxic periodontal pockets have presented barriers to its use in the clinic. To overcome these challenges, a novel superhydrophobic (SH) film that generates airborne singlet oxygen has been developed. The SH-aPDT approach isolates the PS onto a topologically rough solid SH film on which channels allow air to diffuse to the PS surface, thus ensuring sufficient oxygen supply. Upon illumination, gas phase singlet oxygen (¹O₂) is produced and diffuses from the SH surface to the underlying biofilm. The killing efficacy was assessed as a function of transmitted fluence (17.9–89.5 J/cm²) and chorin e6 loading (96–1110 nmol/cm²) by counting of colony forming units, biofilm metabolism by XTT and confocal microscopy. The decrease in viability of both Gram-positive and Gram-negative bacteria in a multi-species biofilm was found to be linearly dependent on the fluence as well as the loading of the PS up to 71.6 J/cm² when 1110 nmols/cm² of chlorin e6 was used. A > 4.6 log bacterial reduction was observed under these conditions (p < 0.05). This novel SH-aPDT approach shows promise as an effective method to disinfect multi-species bacterial biofilms associated with periodontal disease and will be evaluated in animal models in future studies.

Introduction

Periodontitis is an inflammatory disease leading to the formation of periodontal pockets and progressive destruction of tissue, bone and supporting structures of dental elements [[1], [2], [3]]. Severe periodontitis (gingival pocket depth ≥ 6 mm) affects 9.8% of the global population with 796 million prevalent cases [4]. The prevalence of periodontitis in US adults aged ≥30 years is 42.2% overall with 7.8% exhibiting severe periodontitis, according to the National Health and Nutrition Examination Survey, 2009–2014 [5].

Bacterial species organized into biofilms play an important role in the initiation and progression of periodontal disease [6]. The disease manifests itself as a result of the imbalance in the biofilm-host-oral microenvironment triad [7]. If not treated, this condition can result in abscess, tooth loss, systemic complications, as well as the reduced quality of life of affected patients [[7], [8], [9]].

Considering the biofilm-dependent etiology of periodontitis, therapy includes elimination of supra and subgingival plaque in order to halt its progression [7,9,10]. Scaling and root planning (SRP) represent the “gold standard” non-surgical treatment for mechanical plaque control. However, SRP has limitations related to the difficulty of access for the mechanical instrumentation in interproximal and concave areas, curved roots, furcation regions and in deep periodontal pockets [11]. By itself, SRP is often not sufficient for complete elimination of bacterial plaque, especially the subgingival plaque [12], which can lead to poor treatment outcomes due to disease recurrence.

To achieve better results than what SRP can attain, complementary therapies including disinfection with a local antiseptic as well as systemic or local antibiotics are used [[13], [14], [15], [16]], but these adjunctive therapies are not without their own shortcomings. SRP combined with an antiseptic such as chlorhexidine (CHX) is commonly used, but CHX is associated with severe anaphylaxis and is known to stain teeth. Moreover, a recent systematic review found no evidence that full mouth disinfection with CHX provided any additional benefit over SRP alone [17].

SRP combined with antibiotics are more effective in treating periodontal disease than SRP alone [13,16,[18], [19], [20], [21], [22]]. However, this treatment suffers from effectiveness questions [23] risks such as gastrointestinal changes, hypersensitivity, and difficulty in delivering the desired concentration to the gingival fluid.[9] Most importantly, widespread antibiotic use can lead to the development of bacteria that exhibit resistance to antibiotics [9,24].

In an effort to minimize the use of antibiotics, antimicrobial photodynamic therapy (aPDT) is being explored to manage and treat chronic periodontitis [12]. aPDT utilizes a photosensitizer drug (PS), with a light source to activate molecular oxygen [[25], [26], [27]]. The reactive oxygen species (ROS) generated by aPDT form as oxygen radicals via a type I process and/or singlet oxygen (¹O₂) via a type II process [27,28]. Both types of ROS species have been shown to effectively inactivate microorganisms [29]. Notably, ROS including ¹O₂ can inactivate all types of microbial cells including Gram-negative bacteria, which contain an outer cell membrane with endotoxins that can block antibiotics, dyes, and detergents, protecting the sensitive inner membrane and cell wall.

However, there are also challenges that limit the use of aPDT in Dentistry such as: insufficient uptake of PS by certain bacterial species [30]; elimination of PSs by bacterial efflux pump mechanisms [31]; inadequate oxygen concentrations in deep hypoxic pockets; and staining of teeth, periodontal tissues as well as aesthetic restorative materials [[32], [33], [34], [35]]. In addition, the amount of light reaching deep into periodontal pockets can be occluded by intense PS absorption at the excitation wavelength, reducing the concentration of ROS generated and the effectiveness of bacterial inactivation in deep pockets. Thus, there is a need to develop a new aPDT strategy to kill pathogenic biofilms that meet the unique needs of Dentistry.

We report here an innovative approach for aPDT, using a superhydrophobic (SH) polydimethylsiloxane surface coated with chlorin e6 that generates airborne singlet oxygen (¹O₂) locally without causing staining or discoloration (Fig. 1). In addition, the SH properties of the device create channels for air to diffuse to the photosensitizer surface [[36], [37], [38], [39]], thus ensuring sufficient oxygen supply, even in deep periodontal pockets. A novel dual-size templating removal method was used to fabricate self-supporting superhydrophobic PDMS films that measure 200 μm thick. These films are much thinner than our previously reported superhydrophobic PDMS materials (>1.0 mm thick), fabricated by 3D printing, yet exhibit excellent water-repellent properties. The thinner superhydrophobic PDMS layer reported here is designed to facilitate use in future animal models of periodontitis.

The antibacterial activity of freestanding SH films was studied in vitro using a multi-species biofilm of Streptococcus mutans, Actinomyces naeslundi, and Porphyromonas gingivalis cultured on hydroxyapatite discs. We find that SH-aPDT treatment of the biofilm results in up to 5 log killing of bacteria, depending upon the light-dose and PS loading on the device surface. SH-aPDT provides a unique way to deliver ¹O₂ as a gaseous species from the SH surface to reach the biofilm bacteria for effective killing, as we will see below.