Photodynamic Inactivation of Bacteria with Porphyrin Derivatives: Effect of Charge, Lipophilicity, ROS Generation, and Cellular Uptake on Their Biological Activity In Vitro

Abstract

Resistance of microorganisms to antibiotics has led to research on various therapeutic strategies with different mechanisms of action, including photodynamic inactivation (PDI). In this work, we evaluated a cationic, neutral, and anionic meso-tetraphenylporphyrin derivative's ability to inactivate the Gram-negative and Gram-positive bacteria in a planktonic suspension under blue light irradiation. The spectroscopic, physicochemical, redox properties, as well as reactive oxygen species (ROS) generation capacity by a set of photosensitizers varying in lipophilicity were investigated. The theoretical calculations were performed to explain the distribution of the molecular charges in the evaluated compounds. Moreover, logP partition coefficients, cellular uptake, and phototoxicity of the photosensitizers towards bacteria were determined. The role of a specific microbial efflux pump inhibitor, verapamil hydrochloride, in PDI was also studied. The results showed that E. coli exhibited higher resistance to PDI than S. aureus (3-5 logs) with low light doses (1-10 J/cm²). In turn, the prolongation of irradiation (up to 100 J/cm²) remarkably improved the inactivation of pathogens (up to 7 logs) and revealed the importance of photosensitizer photostability. The PDI potentiation occurs after the addition of KI (more than 3 logs extra killing). Verapamil increased the uptake of photosensitizers (especially in E. coli) due to efflux pump inhibition. This effect suggests that PDI is mediated by ROS, the electrostatic charge interaction, and the efflux of photosensitizers (PSs) regulated by multidrug-resistance (MDR) systems. Thus, MDR inhibition combined with PDI gives opportunities to treat more resistant bacteria.

Keywords: antimicrobial activity; efflux pumps; multidrug resistance (MDR); photodynamic inactivation (PDI); porphyrins; reactive oxygen species (ROS); singlet oxygen.

Figure 1

The chemical structures of the investigated porphyrins.

Figure 2

Normalized electronic absorption and emission spectra of the studied porphyrins measured in DMSO solution at room temperature.

Figure 3

Electronic density maps of the photosensitizers from the total self-consistent field density mapped with the electrostatic potential, isovalue = 0.0004, at the B3LYP/6-31G(d) level in the atomic units.

Figure 3

Electronic density maps of the photosensitizers from the total self-consistent field density mapped with the electrostatic potential, isovalue = 0.0004, at the B3LYP/6-31G(d) level in the atomic units.

Figure 4

The cyclic voltammograms of ClTPP (concentration 0.5 mM) in DMSO in 0.2 M TBAP 20 mV/s with a vitreous carbon working electrode (A), and the absorption spectra of the ClTPP recorded for different applied electrode potentials (B).

Figure 5

The comparison of the HOMO–LUMO level of porphyrins with water, oxygen, and iodine redox potential. The blue lines represent the potentials determined from the electrochemical measurements and the purple lines indicate values from the photoelectrochemical measurements.

Figure 6

Singlet oxygen quantum yield determination using 9,10-dimethylanthracene (DMA) as a singlet oxygen quencher. There is a decrease in DMA absorption measured in PBS (with 0.5% DMSO) in the presence of porphyrins under the irradiation with visible light using a 420 nm (±20 nm) diode as the light source.

Figure 7

Photogeneration of ROS: the fluorescence intensity from ROS probes (10 µM), namely, APF (A), HPF (B), SOSG (C), and DHE (D), during irradiation of a porphyrin solution with a 420 nm LED.

Figure 8

The absorbance of aliquots of photosensitizers (PSs) with 100 mM KI after irradiation with a 420 nm LED with various light doses and added to a starch indicator reagent.

Figure 9

The photostability of porphyrins: TPP, ClTPP, Cl₂TPP, TPPS, Cl₂TPPS, and TMPyP in PBS solution (DMSO content < 1%) with an absorbance ca. 1. Irradiation of the porphyrin solution was carried out using a 420 nm LED with various light doses.

Figure 10

Time-dependent cellular uptake of PS with the initial 20 μM concentration by E. coli without (A) and with verapamil (B) and S. aureus without (C) and with verapamil (D), for various incubation times.

Figure 11

Uptake of PSs (with the initial 20 μM concentration) by E. coli (A) and S. aureus (B) with verapamil and KI addition.

Figure 12

Laser scanning confocal microscopy imaging of porphyrin accumulation with different overall charges (ClTPP, Cl₂TPPS, and TMPyP) and with the addition of verapamil (Ver) in E. coli and S. aureus.

Figure 13

Cellular uptake was determined in E. coli (upper) and S. aureus (lower panel) based on the red fluorescence of the selected porphyrin (ClTPP, Cl₂TPPS, and TMPyP) using flow cytometry. Histograms of the fluorescence intensities of the unstained control (black line), a photosensitizer (red), and PS uptake after the addition of verapamil (blue).

Figure 14

Flow cytometry analysis of reactive oxygen species (ROS) generation in vitro in (A) E. coli and (B) S. aureus. After incubation of bacteria with PS (2 h) and APF (2 h), the cells were irradiated with a sublethal light dose 10 J/cm². The APF fluorescence signal monitored the level of ROS in the green channel, and the cellular uptake (red porphyrin fluorescence) was detected as an increased red fluorescence signal.

Figure 15

The surviving fraction of bacteria following PDI of E. coli and S. aureus with TPP, ClTPP, Cl₂TPP, TPPS, Cl₂TPPS, and TMPyP as the PS without (A,D) and with EPI (B,E) and KI addition (C,F) after 2 h of incubation with a low light dose up to 10 J/cm².

Figure 16

The surviving fraction of bacteria following PDI of E. coli and S. aureus with TPP, ClTPP, Cl₂TPP, TPPS, Cl₂TPPS, and TMPyP as the PS without (A,C) and with EPI (B,D) after 2 h of incubation and application of light doses up to 100 J/cm².

Figure 17

The effect of the EPI on high-dose PDI. The colonies of E. coli grew on agar plates after PDI mediated by a Cl₂TPPS of 40 J/cm² and TMPyP of 60 J/cm², with and without verapamil addition.

Figure 18

Scanning electron microscopy images of the control and after the PDI-treated bacteria E. coli (upper part) and S. aureus (lower part) with and without the addition of verapamil (Ver) and potassium iodide (KI) at a 10 J/cm² light dose.

Figure 19

Laser scanning confocal microscopy images of the non-treated (upper panels), PDI-treated (middle), and PDI+KI-treated (bottom panels) E. coli stained with Hoechst3342 and PI. The yellows squares on the top panels indicate the zoom area displayed below.

Figure 19

Laser scanning confocal microscopy images of the non-treated (upper panels), PDI-treated (middle), and PDI+KI-treated (bottom panels) E. coli stained with Hoechst3342 and PI. The yellows squares on the top panels indicate the zoom area displayed below.

Figure 20

Regulation of drug efflux in the photosensitizer-treated bacteria: (A) E. coli and (B) S. aureus. After the treatment of cells with 20 µM PS for 2 h, 0.25 µM calcein AM was added to the cells, and the calcein AM retention was examined. Greater numbers suggest a more significant inhibitory effect on MDR activity.

Figure 21

A summary of the factors influencing the efficacy of photodynamic photoinactivation. The structural modification of porphyrins and their influence on photophysical and biological activity in the performed experiments. Abbrev. In order: -/+/++/+++ or ↑/↑↑/↑↑↑ = no effect or the worst/moderate/most potent.