Flow cytometric analyses on the damage pattern of phenalen-1-one-mediated antimicrobial photodynamic therapy towards biofilms in vitro

鉴于耐药微生物的日益增多，限制传统抗生素和抗菌剂的使用，促进替代抗菌方法的研究，是牙科医学的主要目标，抗菌光动力疗法（aPDT）可能是一种替代抗菌方法。aPDT的原理是基于将细菌与本身无毒的染料（光敏剂; PS）一起孵育，随后在分子氧的存在下通过适当波长的光照射。由于辐射过程中活性氧或单线态氧的出现，细菌结构被氧化爆发非选择性地破坏。基于非那烯-1-酮结构的PS似乎是特别有利的，这是由于它们的高单线态氧量子产率和随之而来的显著的抗微生物功效。然而，基本的机制和损害模式仍然几乎是未知的。由于秒范围内的处理时间足以灭活嗜热菌，因此提出细菌细胞质膜可能是目标结构，然而，这在申请人先前的DFG项目中已被证明是错误的。然而，精确理解特定抗菌方法的作用机制和靶结构对于优化该方法和评估耐药诱导的风险至关重要，因此，本建议的目的是通过多参数流式细胞术研究aPDT与非那烯-1-酮PS对生物膜中组织的细菌的损伤模式和靶结构。首先，应评估aPDT与两种非那仑-1-酮衍生物对内氏放线菌和大肠杆菌的单种生物膜的适当条件。然后，应建立与不同荧光染色相结合的流式细胞术方法，以评估aPDT治疗后单细胞水平上的以下重要参数：膜完整性、膜电位、代谢活性和细胞内ROS水平。此外，通过扫描电子显微镜和透射电子显微镜观察aPDT介导的损伤。此外，aPDT与两种PS联合应用对牙周膜细胞的细胞毒性作用有待进一步研究，为将来此类PS的临床应用和进一步开发提供了依据。这里开发的流式细胞术方法可以进一步应用于研究其他抗菌方法的损伤模式，因此，对于医学领域内外的其他问题是有趣的。

In view of increasing numbers of resistant microorganisms, it should be major goals in dentistry, where usually no life-threatening diseases need to be treated, to restrict the use of conventional antibiotics and antiseptics and to promote research on alternative antimicrobial approaches.In this light, the antimicrobial photodynamic therapy (aPDT) may be an alternative antimicrobial approach. The principle of aPDT is based on incubation of bacteria with a per se non-toxic dye (photosensitizer; PS) and subsequent irradiation by light of an appropriate wavelength in the presence of molecular oxygen. Due to the emergence of reactive oxygen species or singlet oxygen during irradiation bacterial structures are destroyed non-selectively by an oxidative burst. PS based on phenalen-1-one structure seem to be particularly advantageous due to their high singlet oxygen quantum yield and consequential pronounced antimicrobial efficacy. However, the underlying mechanisms and damage patterns still are virtually unknown. Since treatment periods in the seconds range are sufficient for inactivation of planktonic bacteria, it was proposed that bacterial cytoplasmic membranes may be the target structure, which, however, has been disproved within the applicant’s preceding DFG-project. Nevertheless, precise understanding of the mechanism of action and the target structures of a given antimicrobial approach are of vital importance for optimization of this approach and evaluation of the risk of resistance induction.Therefore, the aim of this proposal is to investigate the damage pattern and the target structures of aPDT with phenalen-1-one PS towards bacteria organized in biofilms by means of multi-parameter flow cytometry. First, appropriate conditions shall be evaluated for aPDT with two phenalen-1-one derivates towards monospecies biofilms of Actinomyces naeslundii and Escherichia coli. Then, the method of flow cytometry combined with distinct fluorescent stains shall be established in order to assess the following vital parameters on the single-cell level following aPDT-treatment: membrane integrity, membrane potential, metabolic activity and the level of intracellular ROS. Furthermore, the damage mediated by aPDT shall be visualized by means of scanning electron microscopy and transmission electron microscopy. Furthermore, cytotoxic effects of aPDT with both PS on periodontal ligament cells shall be evaluated.These findings provide the basis for a future clinical application and further development of this class of PS. The flow cytometric method developed here can further be applied in order to investigate the damage pattern of other antimicrobial approaches and, thus, is interesting for other questions in and beyond the field of medicine.