Combined Black Phosphorus Nanosheets with ICG/aPDT is an Effective Anti-Inflammatory Treatment for Periodontal Disorders

Abstract

Introduction

Antibacterial photodynamic treatment (aPDT) has indispensable significance as a means of treating periodontal disorders because of its extraordinary potential for killing pathogenic bacteria by generating an overpowering amount of reactive oxygen species (ROS). The elevated ROS that may result from the antibacterial treatment procedure, however, could exert oxidative pressure inside periodontal pockets, causing irreparable damage to surrounding tissue, an issue that has severely restricted its medicinal applications. Accordingly, herein, we report the use of black phosphorus nanosheets (BPNSs) that can eliminate the side effects of ROS-based aPDT as well as scavenge ROS to produce an antibacterial effect.

Methods

The antibacterial effect of ICG/aPDT was observed by direct microscopic colony counting. A microplate reader and confocal microscope enabled measurements of cell viability and the quantification of ROS fluorescence. BPNS administration regulated the oxidative environment. IL-1β, IL-6, TNF-α, IL-10, TGF-β, and Arg-1 mRNA expression levels were used to assess the inflammatory response after BPNS treatment. In vivo, the efficacy of the combination of BPNSs and ICG/aPDT was evaluated in rats with periodontal disease by histomorphometric and immunohistochemical analyses.

Results

The CFU assay results verified the antibacterial effect of ICG/aPDT treatment, and ROS fluorescence quantification by CLSM indicated the antioxidative ability of the BPNSs. IL-1β, IL-6, TNF-α, IL-10, TGF-β, and Arg-1 mRNA expression levels were significantly decreased after BPNS treatment, confirming the in vitro anti-inflammatory effect of this nanomaterial. The histomorphometric and immunohistochemical analyses showed that the levels of proinflammatory factors decreased, suggesting that the BPNSs had anti-inflammatory effects in vivo.

Conclusion

Treatment with antioxidative BPNSs gives new insights into future anti-inflammatory therapies for periodontal disease and other infection-related inflammatory illnesses and provides an approach to combat the flaws of aPDT.

Graphical Abstract

Keywords:

• periodontitis
• black phosphorus nanosheets
• reactive oxygen species
• antibacterial photodynamic therapy
• macrophage polarization

Abbreviations

aPDT, antibacterial photodynamic treatment; ROS, receptive oxygen species; BPNSs, black phosphorus nanosheets; ICG, indocyanine green; FDA, fluorescein diacetate; PI, propidium iodide; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; DLS, dynamic light scattering; ESR, electron spin resonance; UV-Vis, ultraviolet-visible; HGF-1 cells, human gingival fibroblast cells; P.g, P.gingivalis; LPS, lipopolysaccharide; qPCR, quantitative real time polymerase chain reaction; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; IL-10, interleukin-10; TGF-β, transforming growth factor-β; Arg-1, arginine-; SD rat, Sprague–Dawley rat; TEM, transmission electron microscopy; CLSM, confocal laser scanning microscopy; H&E, hematoxylin and eosin; IHC, immunohistochemical staining.

Ethics Approval

Sprague-Dawley rats (6-week-old, male, 160–180 g, certificate no. SCXK- (Zhejiang) 2019-0001) were obtained from Vital River Laboratories (Zhejiang, China). All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of the Nanjing University (IACUC-2003040). Moreover, approval was received prior to beginning this research.

Data Sharing Statement

The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no conflicts of interest in this work.

Additional information

Funding

This work was supported by the Natural Science Foundation of Jiangsu Province (Grant No. BK 20200665, BK20221177, SKB2022020532), Key Laboratory in Science and Technology Development Foundation, Nanjing Department of Health (Grant No. ZKX19031, ZKX22055), Nanjing Clinical Research Center for Oral Diseases (No.2019060009), and “2015” Cultivation Program for Reserve Talents for Academic Leaders of Nanjing Stomatological School, Medical School of Nanjing University (No.0223A203).