Photoinactivation Eciency of Cationic Porphyrin Derivatives Against Multidrug-Resistant Wound Pathogens

The photoinactivation eciency of antimicrobial photodynamic therapy (aPDT) with cationic porphyrin derivatives (CPDs) against multidrug-resistant (MDR) bacterial strain was assessed. MDR bacterial strains including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae were used. The CPDs named PM, PE, PN, and PL were synthesized as a photosensitizer (PS). A diode laser with a wavelength of 655 nm was used as a light source. Photoinactivation eciency of the combinations formed with different energy density (50, 100, and 150 J/cm²) and PS concentrations (ranging from 3.125 µM and 600 µM) on each bacterial strain were evaluated. Toxicity of the aPDT combinations that showed a strong photoinactivation on the bacterial strains and dark toxicity of PSs and were evaluated on broblasts cells. In the aPDT experiments, survival reductions of up to 5.80 log(cid:0)(cid:0) on E. coli, 5.90 log(cid:0)(cid:0) on P. aeruginosa, 6.11 log(cid:0)(cid:0) on K. pneumoniae and 6.78 log(cid:0)(cid:0) on A. baumannii were obtained. There was an increase in the photoinactivation eciency in parallel with increasing the energy density, and the best effect seen at an energy density of 150 J/cm2. PL did not show any toxic effect on broblasts. However, other PSs were toxic in broblasts at high concentrations. In this research, which reected the results of in vitro experiments, aPDT provided potent photoinactivation against MDR clinical isolates. The research results lead to an in vivo wound model study of aPDT with CPD infected with an MDR clinical isolate. calculated 5 CFU/mL) was added to each well of the plates and, the plates were allowed to incubate at 37°C for 16–18 h. After the incubation, the MIC values of the CPDs were determined by calculating the lowest compound concentrations that prevent the growth of bacteria. All experiments were each repeated at least three times. each of 96 well plates identied as PS, aPDT, L, and C. 50 µL PS from the stock suspensions at specic concentrations was added to wells of the PS and aPDT group plates containing bacteria. 50 µL of PBS was added to the wells of the L and C group plates with bacteria. All four groups were incubated for 15 min at room temperature. The aPDT and L group plates were exposed to light. After light exposure, bacterial suspensions in all groups were diluted by serial dilution using PBS. 100 µL of aliquot was taken from the dilutions and plated on tryptic soy agar and left for an overnight incubation in the dark area at 37 ºC. After incubation, bacterial survival was calculated at CFU/mL. Each experiment was repeated three times in triplicate. environment and allowed to incubate at 37°C incubate for 15 min. Phototoxicity or L (50 J/cm², 100 J/cm², and 150 J/cm²) and aPDT groups were irradiated at an appropriate energy density with a diode laser. Control and PS groups were taken from the incubator but not exposed to ambient light or laser. After the light application to aPDT or light groups was nished, all the plates wrapped with aluminum foil again and allowed to incubate at 37°C incubate for 15 min or 24 h. and a maximal level of antimicrobial activity in a wide spectrum (Klausen, Ucuncu, and Bradley 2020; Taslı et al. 2018). In this current study, MIC evaluation results showed that the antimicrobial activities of the CPDs without light were negligible. CPDs provided a strong antimicrobial activity against MDR clinical isolates at concentrations well below the MIC values. With this feature, CPDs met one of the important conditions to be a PS.


Introductions
Critical colonization or infection is a common problem in chronic wounds such as surgical site infections, burns, diabetic foot, venous leg, and pressure ulcers. Similarly, critical colonization (10 5  Healing of the infected wounds is directly related to the reduction of microbial colonization on the wound. Today, antiseptics and antimicrobials are recommended to reduce the critical colonization or eliminate the infection (Nasir et al. 2016; National Pressure Ulcer Advisory Panel 2014). However, there are some problems that limit the use of these antimicrobials. Some antimicrobials are not selective to microorganisms and can damage healthy tissue.
Using for more than two weeks may harm the granulation and epithelialization process. Some antimicrobials may have limited penetration to the wound tissue and bio lm layer and remain insu cient to heal the deep wound infection (Daeschlein et al. 2010;Norman et al. 2016). Therefore, the search for novel methods to accelerate wound healing has become inevitable.
Antimicrobial photodynamic therapy (aPDT) is a treatment method based on the principle of killing microbial cells using a non-toxic dye or a photosensitizer (PS) and a harmless visible light that stimulates PS. aPDT shows its antimicrobial e cacy with the reactive species formed as a result of chemical and physical reactions that occur in two different ways including Type I and Type II. The reactive species formed cause damage to bacterial DNA, cytoplasmic membrane, enzyme, and transport systems and provide antimicrobial activity ( nontoxic, light-activated, broad-spectrum photoinactivation effect and high singlet oxygen quantum yield are ongoing. Porphyrin derivatives have the potential to be an ideal PS. Porphyrins are macro-cyclic aromatic molecules formed by connecting four pyrrole rings together with methylene (-HC=) bridges (Nitzan and Ashkenazi 2001). Porphyrins, which originated from protoporphyrin IX (PPIX), are isolated from natural environments such as body uids and feces of animals, eggshells, and feathers of birds. Porphyrins are of vital importance for living, including bacteria. Heme (Fe² PPIX)/hemin (Fe³ PPIX) compound in the structure of hemoglobin and myoglobin is involved in important biological processes, such as oxygen binding, oxygen transfer, nitric oxide synthetase and transfer of electrons in cytochromes. Most bacteria meet their iron needs from Fe² PPIX. Gram-positive and Gram-negative bacteria have Fe² + PPIX uptake mechanisms, including TomB and ExbBD proteins (Stojiljkovic 2001). Natural or porphyrin derivatives using conjugates such as porphyrincellulose-nanocrystals or antibiotics can easily penetrate in bacteria by Fe² PPIX acquisition mechanisms and demonstrate through several chemical processes such as transferring electrons, catalyzing peroxidase and oxidase reactions, absorbing photons, production of reactive oxygen species (Carpenter et al. 2012;Lippert et al. 2017).
Cationic porphyrins show their antimicrobial activity through type II reaction mechanism. Single oxygen (¹O ) formed by the transfer of the energies of light-induced cationic porphyrins to molecular oxygen reacts with structures such as phospholipids, peptides, and sterols in the cell wall and cell membrane and cause cell death  (Dosselli et al. 2014). In this current study, different from previous studies, the aim was to obtain a broad spectrum photoinactivation e ciency on MDR clinical isolates with the CPDs synthesized by us. In our previous study, a strong photoinactivation was obtained on methicillin-resistant Staphylococcus aureus (MRSA) (Taslı et al. 2018). The goals in this present study in which multi-drug resistant Gram negative bacteria were used can be listed as follows: To determine the light energy density and PS concentration ranges that create photoinactivation on the strain selected representing each species; To investigate the photoinactivation of the combinations of energy density and PS concentration providing strong photoinactivation on other strains of each species.
To investigate the toxicity of all aPDT combinations with photoinactivation effect on broblasts.  Fig. 1a. CPDs can absorb a wavelength in the broad spectrum varying from 250 to 800 nm and, the maximum light absorption was at 422 ± 3 nm (Fig. 1b).
Chemistry: The infrared (IR) spectra of the compounds were monitored by attenuated total re ectance (ATR) incubated in a humidi ed environment containing 95% air and 5% CO until they form a con uent culture in a single layer. The cells reaching 80% con uence were washed with PBS and trypsinized using 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) (Biological Industries, Israel). 2×10 4 broblast cells were seeded into each well of 96-well plates and allowed to incubate at 37°C for 24 h so that the cells adhere to the wells of the plate. Then the cell culture medium was discarded and the experimental process continued as described in the groups below.
The dark toxicity of the concentrations ranging from 25 to 600 µM for PM, 3.125 to 400 µM for PE, PN, and PL on the broblast was performed using 15 min and 24 h incubation. In the experiment, 100 µL PS suspension from stock suspension dissolved at speci c concentrations in cell culture medium for dark toxicity groups was transferred to plate wells containing broblasts. 100 µL cell culture medium without PS was transferred to control group plate wells. The plates wrapped with aluminum foil to create a dark environment and allowed to incubate at 37°C incubate for 15 min or 24 h.
The toxicity of aPDT on the broblast cells was performed using 15 min and 24 h incubation. 100 µL from PS suspension dissolved at speci c concentrations in cell culture medium was transferred to plate wells containing broblasts cells (Combinations were as in Fig. 7b). 100 ml cell culture medium without PS was placed in the control and light group plate wells containing broblast cells. The plates wrapped with aluminum foil to create a dark environment and allowed to incubate at 37°C incubate for 15 min. Phototoxicity or L (50 J/cm², 100 J/cm², and 150 J/cm²) and aPDT groups were irradiated at an appropriate energy density with a diode laser. Control and PS groups were taken from the incubator but not exposed to ambient light or laser. After the light application to aPDT or light groups was nished, all the plates wrapped with aluminum foil again and allowed to incubate at 37°C incubate for 15 min or 24 h.
Following, cell culture medium or PSs added to the plate wells were removed. Cells in the wells were washed with PBS. 100 µL MTT (4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (5 mg/mL) (Sigma, St. Louis, MO, USA) was added to each well. After 2 h of incubation, the formazan crystals were dissolved with 100 µL of DMSO, and the absorbance was measured at 570 nm with a microplate reader (iMark, Bio-Rad Lab., USA.). The absorbance values were used to determine the change in survival in broblast cells. Control groups were used for each experiment. Each experiment was repeated three times in triplicate.

Data analysis and Evaluation
In the photoinactivation experiments, the calculations were done as described below: First, bacterial survival in CFU/mL for each plate was calculated according to formula 1.

Formula 1
The control group was taken as a reference for determining survival reduction of the aPDT, PS, or L groups.
Survival reductions were calculated as logarithmic as shown in formula 2.

Reduction = log 10 NumberofcoloniespermLinthecontrolgroup NumberofcoloniespermLintheapplicationgroup
For the toxicity on the broblast cells, the calculations were done as follows. The control group was taken as a reference for determining the toxic effect of dark toxicity, phototoxicity, or aPDT applications on broblast cells. Percentage changes of the broblast cell survival based on the absorbance values of the groups were calculated according to formula 3. Formula 3 Cellviablility(%) = (Absorbancevalueofcontrolgroup − Absorbancevalueoftheapplicationgroup)x100 Absorbancevalueofthecontrolgroup SPSS 16.0 was used for data analysis. "Paired sample t-test" was used to determine the differences of laser or aPDT groups compared to the control. In the comparisons, differences with p < 0.05 were accepted as statistically signi cant.

Antibacterial activity
The MIC values of CPDs and cipro oxacin for bacterial strains ranged from 850.40 to > 7365.51 µM ( Table 1).

Photoinactivation of the bacterial strains
In the experiments, the photoinactivation e ciency of the combinations (energy density and PS concentration) on the clinical isolates representing the species for each PS was determined. In these preliminary experiments, the combinations with low PS concentration that provided strong photoinactivation on the clinical isolates were selected. In secondary experiments, the photoinactivation e ciency of combination selected by pioneering experiments was examined on other clinical isolates of the species.   (Fig. 3a). As shown in Fig. 3b, [100 (Fig. 4a). For all four PSs, these same combinations provided reductions ranging from 3.92 to 6.11 log in K. pneumoniae-2 (Fig. 4b).

Toxicity on the broblast cells
The toxicity of the concentrations for each PS used on clinical isolates was investigated on broblast cells. Any of PM concentrations at 15 min incubation did not show dark toxic effects on broblast cells. 200 µM and above of PM concentration at 24 h incubation resulted in a decrease in survival ranging from 9.52 to 41.99% (Fig. 6a). PE, which did not show dark toxic effects after 15 min of incubation, caused reductions varying between 18.15 and 32.70% at 24 h incubation (Fig. 6b). PN was toxic at 50 µM and above concentrations at 15 min of incubation. All PN concentrations were toxic at 24 h incubation, and the survival decline ranged from 18.5 to 57.80% (Fig. 6c). At the 15 min of incubation, the toxic effect for PL began at concentrations above 200 µM. It caused broblast cell reductions ranging from 15.38 to 31.95% at 24 h incubation (Fig. 6c).  (Fig. 7a). For PE and PN, [50 j/cm²-50µM] at both incubation times did not cause a signi cant reduction in broblast cells compared to other combinations ( Fig. 7b and Fig. 7c). There was no signi cant reduction in any combination of broblast cells at both 15 min and 24 h incubations for PL (Fig. 7d).
No phototoxicity was observed at any of the incubation periods. On the contrary, proliferation was seen and signi cant, especially at 100 J/cm² and 150 J/cm².

Discussions
In the aPDT application, it is among the primary priorities that the PS can only be activated by light, has a high wavelength absorption capacity, and a maximal level of antimicrobial activity in a wide spectrum ( In pioneering photoinactivation experiments on clinical isolates, PS concentrations ranging from 50-600 µM were required for 3 log and above bacterial reduction. On the other hand, 300 µM and above for PM and 200 µM and above for PE, PN, and PL at 24 h incubation caused dark toxicity on the broblast cells. This result will limit the use of CPD concentrations of 200 and above in aPDT combinations. However, the nding of increased dark toxicity due to increased CPD concentration was consistent with the literature ( Fig. 6a and Fig. 6d), at 24 h incubation, aPDT combinations containing concentrations of 200 µM for PL, 300 µM and above for PM did not damage broblast cells (Fig. 7a and Fig. 7d). The reason for these discordant results may be related to the proliferation effect of light on broblast cells. Thus, a signi cant increase on broblast cells was observed at 100 J/cm² and 150 J/cm² energy density (Fig. 7e).

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