Ruthenium(II) Polypyridyl Complexes for Antimicrobial Photodynamic Therapy: Prospects for Application in Cystic Fibrosis Lung Airways

Abstract

Antimicrobial photodynamic therapy (aPDT) depends on a variety of parameters notably related to the photosensitizers used, the pathogens to target and the environment to operate. In a previous study using a series of Ruthenium(II) polypyridyl ([Ru(II)]) complexes, we reported the importance of the chemical structure on both their photo-physical/physico-chemical properties and their efficacy for aPDT. By employing standard in vitro conditions, effective [Ru(II)]-mediated aPDT was demonstrated against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus strains notably isolated from the airways of Cystic Fibrosis (CF) patients. CF lung disease is characterized with many pathophysiological disorders that can compromise the effectiveness of antimicrobials. Taking this into account, the present study is an extension of our previous work, with the aim of further investigating [Ru(II)]-mediated aPDT under in vitro experimental settings approaching the conditions of infected airways in CF patients. Thus, we herein studied the isolated influence of a series of parameters (including increased osmotic strength, acidic pH, lower oxygen availability, artificial sputum medium and biofilm formation) on the properties of two selected [Ru(II)] complexes. Furthermore, these compounds were used to evaluate the possibility to photoinactivate P. aeruginosa while preserving an underlying epithelium of human bronchial epithelial cells. Altogether, our results provide substantial evidence for the relevance of [Ru(II)]-based aPDT in CF lung airways. Besides optimized nano-complexes, this study also highlights the various needs for translating such a challenging perspective into clinical practice.

Keywords: antimicrobial photodynamic therapy; antimicrobial resistance; benchmark analysis; biofilm; cystic fibrosis; micro-environment; ruthenium complexes.

Figure 1

Chemical structure and relevant features of [Ru(II)] used in this study. The partitioning index LogP is a measure of the difference in solubility of the compound in 1-butanol and in water. I₀/I is a measure of the singlet oxygen production efficiency, as determined using a Stern–Volmer analysis. LogP and I₀/I were calculated and reported in our previous study (in which the counter-ion was hexafluorophosphate for both compounds) [23]. “Phen”: 1,10-phenanthroline.

Figure 2

Illumination set-up. Schematic representation (a) showing the two LED panels (① and ②) placed above and underneath an in-between plate (③) used to hold materials placed in a central exposure area (④). The picture in (b) shows the illumination of a 96-well plate containing [Ru(II)] samples. Fluorescence was clearly visible to the naked eyes. Light transmittances was measured through various samples with increasing thicknesses, including ASM and its individual components (c) as well as several bacterial inoculums prepared in saline (d). The dashed lines correspond to 50 and 70% transmittances, respectively. See Figure S1 for more technical details about this setup.

Figure 3

Photophysical properties of [Ru(II)] in water or in saline solutions. Absorption in the UV-visible spectrum (a,b) and fluorescence (c,d) were determined with [Ru(II)]1 (a,c) and [Ru(II)]2 (b,d). In every test, [Ru(II)] concentration was 50 µM in 200 µL. Fluorescence measurements were performed before (OFF) then after (ON) light treatment. Mean ± SD with n = 3. The fluorescence background is ~0.05 RFU in every case. “RFU”: relative fluorescence unit.

Figure 4

Production of singlet oxygen and intracellular ROS upon light treatment of [Ru(II)]. Results were obtained using SOSG (a) and DCFH-DA (b), respectively; S. aureus and P. aeruginosa were RN4220 and PA19660, respectively. [Ru(II)] = 25 µM in Tris-HCl for SOSG and 1X NaCl for DCFH-DA. Mean ± SD with n = 3 (representative results of N = 2). ***, p-value ≤ 0.001; **, p-value ≤ 0.01; *, p-value ≤ 0.05. See Figure S3 for more data.

Figure 5

Interaction assay between [Ru(II)] and bacteria when mixed either in water or in 1X NaCl. Results were obtained using [Ru(II)]1 (a,b) or [Ru(II)]2 (c,d) with S. aureus RN4220 (a,c) and P. aeruginosa PA19660 (b,d). See Figure S4 for results obtained with other bacterial strains. [Ru(II)] = 25 µM. Mean ± SD with n = 3 and N ≥ 2. **, p-value ≤ 0.01.

Figure 6

PDT effects of [Ru(II)] at various pH. Results were obtained using [Ru(II)]1 (a,b) or [Ru(II)]2 (c,d) with S. aureus RN4220 (a,c) and P. aeruginosa PA19660 (b,d). [Ru(II)] concentration was adapted to each strain evaluated: [Ru(II)]1 was 40 and 10 µM, whereas [Ru(II)]2 was 0.1 and 1 µM in acetate buffers, when assaying S. aureus and P. aeruginosa, respectively. Mean ± SD with n = 3 and N = 2. ***, p-value ≤ 0.001; “ns”: non-significant.

Figure 7

PDT effects of [Ru(II)] in normal or hypoxic media. Results were obtained with S. aureus RN4220 (a) and P. aeruginosa PA19660 (b). [Ru(II)]1 was 50 and 25 µM, whereas [Ru(II)]2 was 0.1 and 1.25 µM in 0.5X NaCl, when assaying S. aureus and P. aeruginosa, respectively. Mean ± SD with n = 3 (representative data of N = 4). ***, p-value ≤ 0.001; **, p-value ≤ 0.01; *, p-value ≤ 0.05. “Ud”: undetectable bacteria.

Figure 8

PDT effects of [Ru(II)] in ASM. Results were obtained with S. aureus RN4220 (a) and P. aeruginosa PA19660 (b) for prolonged irradiation. See Figure S8 for results obtained when using standard light treatment. [Ru(II)] concentration = 25 µM in 1X ASM. Mean ± SD with n = 3 and N = 2. **, p-value ≤ 0.01; “ns”: non-significant.

Figure 9

PDT effect of [Ru(II)] towards bacteria in biofilms (“pre-delivery antibiofilm assay”). The workflow of the experimentation is presented (a) with the results obtained when assaying S. aureus RN4220 (b) and P. aeruginosa PA19660 (c). Mean ± SD with n = 3 and N = 3. ***, p-value ≤ 0.001; **, p-value ≤ 0.01. “Cip”: ciprofloxacin.

Figure 10

PDT effects of [Ru(II)] in the presence of eukaryotic cells cultured in different conditions. Results were obtained when assaying bacteria (B) with CFBE-Luc cells, with the latter being cultivated either at the air–liquid interface (a,c) or under submerged conditions (b,d). Luminescence measurements ((a,b); expressed in the unit of RLU/mg of total protein) and CFU enumeration (c,d) were used to assess cell viability and antibacterial effects, respectively. Mean ± SD with n = 3 and N = 2. ***, p-value ≤ 0.001; “ns”: non-significant. “Ud”: undetectable.