Experimental evaluation of the effect of porphyrins in antimicrobial photodynamic inactivation of burn wounds

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Introduction

Antibiotic resistance is a global health problem¹. This threat is becoming especially pressing in the context of wound infection treatment, when traditional antimicrobial methods, such as antibiotic therapy, are no longer always effective. This increases the need to find new approaches to etiotropic treatment and overcoming antimicrobial resistance, such as shifting attention to alternative methods.

Antimicrobial photodynamic therapy is one of such methods, interest in which has increased significantly in recent years [1; 2]. This therapy is based on antimicrobial photodynamic inactivation (PDI). The principle of antimicrobial PDI is based on the dynamic interaction between a photosensitizer, light of a specific wavelength and molecular oxygen, which promotes the selective destruction of microbial cells. The method involves microorganisms accumulating specific photosensitizers, causing them to become sensitive to light of the corresponding wavelength. A photochemical reaction occurs in the sensitized cells, releasing singlet oxygen and free radicals—highly active biological oxidants that are cytotoxic to most microorganisms [2; 3].

The ability to overcome multiple drug resistance, minimize damage to healthy tissues, and the lack of convincing data on the development of insensitivity to this type of exposure are the advantages of the method [3; 4].

The recognition that PDI is a valuable tool for combating infectious disease pathogens has given new impetus to the development of photosensitizer drugs capable of effectively inactivating bacteria in planktonic and biofilm forms under conditions of low toxicity to mammalian cells. Several classes of new photosensitizers have been studied in recent years: the ones containing macrocyclic tetrapyrrole cores (such as porphyrins, phthalocyanines, chlorins and bacteriochlorins), texaphyrins, phenothiazines (methylene blue class), nanoparticles, fullerenes and others [3; 5; 6]. However, porphyrins are among the most widely used ones [5; 6].

Porphyrins have interesting features and advantages for antimicrobial PDI, namely low dark toxicity in vitro or in vivo, high efficiency for intracellular generation of reactive oxygen species, structural versatility allowing modulation of their characteristics, promoting bioavailability and interaction with cellular structures [6; 7].

To justify further study and application of monoheteryl-substituted porphyrin compounds for the treatment of infected wounds, an experiment was conducted on animals.

The aim of the study is to investigate and evaluate the effect of porphyrins on the microbial contamination of an infected burn wound in experimental animals with antimicrobial PDI.

Materials and Methods

The study is planned as a comprehensive, exploratory, multi-stage investigation. The experimental part presented is a continuation of laboratory microbiological studies [8], which determined the high antimicrobial activity of porphyrins against antibiotic-resistant gram-positive clinical strains, pathogens of wound infections.

Study bases:

1) synthesis of chemical compounds: Federal State Budgetary Scientific Institution G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, Ivanovo;

2) animal experiments: vivarium of the Research Institute of Experimental Oncology and Biomedical Technologies of the Nizhny Novgorod State Medical University of the Ministry of Health of the Russian Federation, Nizhny Novgorod;

3) Microbiological research: Bacteriology Laboratory of the University Clinic of the Federal State Budgetary Educational Institution of Higher Education "Privolzhsky Research Medical University" of the Ministry of Health of the Russian Federation, Nizhny Novgorod.

In a series of experiments, the main objects of study were chemical compounds of water-soluble asymmetric cationic porphyrins and animals with infected burn wounds.

Three different monoheteryl-substituted porphyrin compounds were studied:

1) triiodide: 5-[4¢-(1¢¢,3¢¢- benzothiazol-2¢¢-yl)phenyl]-10,15,20- tris (N- methylpyridin -3¢-yl) of porphyrin (S-por). PBS (7.4) M = 1176.73 C = 1·10^-5 mol/l;

2) triiodide: 5-[4¢-(1¢¢,3¢¢- benzoxazole -2¢¢-yl)phenyl]-10,15,20- tris (N- methylpyridine -3¢-yl) of porphyrin (O-por). PBS (7.4) M = 1160.67 C = 1·10^-5 mol/l;

3) triiodide: 5-[4-(N- methyl -1¢¢,3¢¢- benzimidazole -2¢¢- yl)phenyl]-10,15,20- tris (N- methylpyridine -3¢-yl)- of porphyrin (N-por). PBS (7.4) M = 1173.74 C = 1·10^-5 mol/l.

The study was performed on random bred white Wistar rats weighing 250–300 g (males, n = 20). General anesthesia was administered at each stage of the experiment using intramuscular injections of zoletil 100–40 mg/kg (250 mg tiletamine hydrochloride and 250 mg zolazepam hydrochloride) and rometar 4 mg/kg (20 mg xylazine). The drugs were administered in the required volume and frequency, taking into account the animal's weight, duration, and traumatic nature of the procedure.

In vivo experiment progress:

1. Burn wound modeling. Contact burns were created using the technique described in A. Orenstein's burn model [9]. A metal plate measuring 7 × 4 × 1 cm, heated to »150°С for 10 s, was applied to the shaved skin of all animals in the interscapular region. This simulated a third-degree burn on the animal's body. After the procedure and until the end of the experiment, the rats were kept in individual cages to reduce the risk of subsequent unplanned endogenous and exogenous wound contamination.
2. Necrectomy. Three days after the injury, a dense, light-brown scab formed in the contact burn area. This scab required fascial necrectomy, which resulted in an open burn wound on an animal.
3. Burn wound infection. Immediately after necrectomy, the wound became infected with a test culture of S. aureus ATCC 29213.

3.1. Preparation of test culture. Standardization of the bacterial suspension was adjusted in a microplate spectrophotometer to obtain an optical density corresponding to 0.5 McFarland standard, with a turbidity equivalent to an approximate bacterial concentration of 1.5 · 10⁸ CFU/mL.

3.2. 0.5 ml of microbial suspension was applied to the open wound, allowed to absorb for 5 minutes, the open wound was covered with a transparent hypoallergenic sterile fixing plaster made of polyurethane film, and a dressing made of veterinary self-fixing bandage was applied on top.

4. Photodynamic inactivation with porphyrins. Three days after infection, a microbiological examination of the wound and the first PDI session were performed. A second PDI session was performed 24 hours after the first session. After each session, after removing the fixing bandage and sterile adhesive tape, the wound was cleaned: detritus, necrotic matter, and pus were removed with a sterile gauze dressing.

4.1. Microbiological examination of wound fluid. Wound fluid was collected twice: immediately after dressing removal (to confirm infection and qualitatively and quantitatively assess wound contamination) and after photoirradiation (to qualitatively and quantitatively assess wound contamination after PDI). Thus, two swabs were collected from each animal during a single PDI session. Each time, the culture was collected in a standard, uniform manner, in accordance with the guidelines for collection and delivery of biomaterial using a sterile swab². The swab loaded with material was placed in a polystyrene tube containing Amies medium with charcoal and delivered to the microbiology laboratory within 1 hour.

Further examination of the wound swab was conducted in accordance with the standard operating procedure “Microbiological (cultural) quantitative study of burn wound contamination for aerobic and facultative-anaerobic microorganisms”. The results of the microbiological study in this case were qualitative (isolation of the infectious agent, its identification) and quantitative (determination of the concentration of the isolated microorganism (CFU/ml)). Species identification of microorganisms was carried out by MALDI-TOF mass spectrometry on the appropriate equipment (MALDI-TOF MS (Germany) and MALDI-TOF AUTOF MS1000 (Autobio, China)).

4.2. Photoirradiation. All animals were randomly divided into four groups depending on which photosensitizer was used. Group No.1 – PDI with O-por (n = 5); Group No. 2 – PDI with S-por (n = 5); Group No. 3 – PDI with N-por (n = 5); Group No. 4 (control) – PDI with 0.9 % sterile sodium chloride solution (n = 5). Light source: LED lamp (white light), maximum power 20 W, maximum luminous flux 1500–1800 lm. Photoirradiation time (exposure): 10 min. Distance from the light source to the animal: 22 cm.

After wound cleaning and wound swabbing, 400 µl of the appropriate photosensitizer was applied to the infected wound in animals from groups 1, 2, and 3. Animals from group 4 were given 400 µl of saline solution. The solution was then soaked for 15 minutes in a darkened box. All animals were then exposed to LED light alone, without additional natural or general artificial lighting, at room temperature. Following light exposure, samples were immediately collected for microbiological analysis.

The second FDI session was conducted in the same sequence as the first session. Thus, over the two FDI sessions, 80 swabs with smears were collected from each animal.

The effect of PDI with porphyrins was assessed based on quantitative changes in the concentration of microorganisms colonizing the wounds of animals. To summarize the data on the effectiveness of PDI after two sessions, we used the logarithmic reduction in colony-forming units (CFU) [10], which was calculated as minus the common logarithm of the ratio of the number of CFU in the control (group No. 4) to the number of CFU after exposure for each of the exposure groups. The obtained indicator could vary from 1 to 6, which was interpreted as the corresponding percentage reduction in CFU: 1 – 90 %; 2 – 99 %; 3 – 99,9 %; 4 – 99,99 %; 5 – 99,999 %; 6 – 99,9999 %.

Statistical data processing was performed in the R 4.2.1 environment (Rstudio 1.1.463)³. The nature of the distribution was tested using the Shapiro-Wilk test and plotting the quantile graph (QQ). In accordance with the distribution of quantitative continuous data and the type of samples (dependent or independent), we used the appropriate parametric and nonparametric criteria: for two dependent samples – Student's t-test or Wilcoxon's signed-rank test, respectively; for two independent samples (result for a specific porphyrin vs. control group) – Student's t-test with the required modification or Mann-Whitney test, respectively. To describe quantitative data whose distributions differ from normal, the median (Me) was used with the interquartile range presented (Q₂₅ – Q₇₅). Percentages are presented as p values, accompanied by 95% confidence intervals (95% CIs). Confidence intervals for frequencies (qualitative data) were calculated using the Wilson-Wald test with Agresti-Cole correction. The level of statistical significance for differences in hypothesis testing was set at p < 0.05.

Results and Discussion

The primary criterion for the effectiveness of any antimicrobial therapy is the elimination of the pathogen from the site of infection. The treatment of infected burn wounds is no exception. The results of our in vivo experiment present data on the microbial contamination of the wound before and after antimicrobial PDI.

When studying the microbial contamination of the wound after infection (one smear), it was revealed that in all wounds (n = 20) there was a purulent inflammatory process caused by S. aureus. The inoculation rate of this microorganism from wounds after infection on the 3rd day was 100%, the initial concentration of bacteria among all animals was from 5·10⁵ to 1·10⁶ CFU/ml in different groups (table). No statistically significant differences were found between groups No. 1–4 in the degree of wound contamination before exposure (comparison according to Me, p_1–3 = 0.065, p_2–3 = 0.178).

Throughout the experiment, no other microorganisms were isolated from the wounds except S. aureus, which was artificially introduced into the wound at the first stage. Exogenous and endogenous contamination of the wound with other foreign microorganisms was prevented by observing aseptic and antiseptic precautions at every stage of exposure to animals, keeping animals in separate boxes, and using fixative veterinary dressings on the wound defect area.

When studying the effect of PDI in the exposure groups, it was found that for all the compounds studied, there was a decrease in the titer of microorganisms after the first and second sessions of PDI (see table).

After the first PDI session, the concentration of microorganisms in groups 1–3 decreased by 5–20 times when compared by the median titer before and after exposure. The number of CFU did not change in group 4, where the photosensitizer was not used (see table). The change in wound contamination towards cleansing from the pathogen on the first day of the experiment was statistically significant for all tested porphyrin compounds (O-por, p = 0.0126; S-por, p = 0.0377; N-por, p = 0.0128) compared with the data of the control group.

 

Microbial contamination of burn wounds at different stages of PDI

PDI session number/ study duration	Group No.	The moment of biomaterial collection in relation to the PDI session	Median (Me) values Of CFU/ml	Level p*	Q25	Q75
First PDI session / 72 hours after infection	1	Before	1·106	0.0099	5·105	1·106
		After	5·104		5·104	5·104
	2	Before	1·106	0.0238	5·105	1·106
		After	1·105		1·105	1·105
	3	Before	5·105	0.0010	1·105	1·106
		After	1·105		1·105	5·105
	4	Before	1·106	-**	5·105	1·106
		After	1·106		5·105	1·106
Second PDI session / 96 hours after infection	1	Before	1·106	0.1666	1·106	5·106
		After	1·105		1·105	5·105
	2	Before	5·105	0.0192	1·105	5·105
		After	5·104		1·104	5·104
	3	Before	1·106	0.0181	1·106	5·105
		After	1·104		1·104	5·104
	4	Before	1·106	-*	1·105	1·106
		After	1·106		1·106	1·106

Note: * – p level for the Me value of CFU/ml (pre- and post-exposure comparison within one group); ** – It is impossible to compare, since the values ​​are identical.

 

After the second session of exposure to the photosensitizer and light, a decrease in the titer of microorganisms (according to Me) occurred in groups No. 1–3 by 10–100 times before and after the PDI. We observed the greatest effect for N-por – a decrease in concentration occurred from 1 10⁶ to 1 10⁴ CFU/ml (see table).

However, it remains important to understand whether phototherapy with different types of porphyrins is effective in the absence of a photosensitizer in the wound. When comparing the results of the bacteriological examination of the third smear after the second PDI session in groups No. 1–3 with the data from the control group, it was found that, in terms of the logarithmic reduction in colony-forming units, all three types of porphyrins demonstrated an effectiveness of 90 %–99 %, which corresponded to an index value of 1 to 2 (Figure).

 

Fig. Logarithmic reduction rate of CFU in three porphyrin exposure groups

 

Thus, after several applications of a photosensitizer to the wound and exposure to light, quantitative changes in microbial colonization occurred towards a decrease in the concentration of the etiologically significant microorganism compared to the initial level. An asymmetrical water-soluble porphyrin containing a heterocyclic benzoxazole fragment at the periphery of the porphyrin cycle (O-por, group No. 1) showed the effect of reducing CFU by 90 % (value 1, p = 0.4237) from the control level and, despite the fact that this indicator turned out to be the lowest in a series of similar indicators for the other two compounds (see figure), there is every reason to consider this result positive, since with such a result one can already speak of a significant effect of the intervention. In addition, in an in vivo experimental model, a more serious problem of determining the degree of activity of the antimicrobial effect is expected due to the complexity of the biological system in the wound, the action of the immune system of the animal's body, etc.

The results of the experiment demonstrate that monoheteryl-substituted porphyrin compounds have demonstrated antimicrobial activity in vivo, which is supported by microbiological studies. It should be noted that since the 1990s, the effectiveness of PDI in combating microorganisms has been extensively studied in numerous in vitro studies. Many of these studies have shown promising results, often achieving significant levels of microbial inactivation, but not all of the photosensitizer compounds and/or photoirradiators studied have reached the next stage – studying their action in living organisms. Therefore, the number of experimental studies in vivo remains limited⁴, which creates significant obstacles for the clinical implementation of PDI [11–13].

Local therapy for wound infections can be used alone or as part of a combination treatment. It should be emphasized that photodynamic therapy inactivates bacteria through oxidation, destroying microorganisms, or weakening their resistance to antibiotics. The latter effect could be extremely interesting and important for the development of a treatment option combined with systemic medications for wounds infected with resistant microorganisms.

Conclusions

When studying the effect of the PDI method with three compounds of monoheteryl-substituted porphyrins in a mono-mode, we discovered changes in the microbial colonization of burn wounds in experimental animals towards cleansing from the pathogen after the first session of photoirradiation. The greatest and statistically significant reduction in the number of microorganisms was observed in groups of animals that were treated with photosensitizers with heterocyclic fragments of benzimidazole (N-por) and benzothiazole (S-por), the logarithmic reduction in CFU was2 (р = 0.0065) and 1,3 (р = 0.0232) respectively. The data obtained identify promising further issues for studying these chemical compounds with the aim of expanding the arsenal of methods for combating wound infections and developing a comprehensive approach for photodynamic inactivation using porphyrins.

 

¹ World health statistics 2024: monitoring health for the SDGs, Sustainable Development Goals. Geneva, World Health Organization 2024. Licence: CC BY-NC-SA 3.0 IGO.

² MU 4.2.2039-05. Techniques for collecting and transporting biomaterials to microbiological laboratories: Approved and put into effect by the Chief State Sanitary Doctor of the Russian Federation on December 23, 2005.

³ R Core Team; R Foundation for Statistical Computing (Hrsg.): R: A Language and Environment for Statistical Computing. Vienna, Austria, 2022

⁴ Spokoiny, A.L. Optimization of photodynamic therapy of purulent wounds of soft tissues (experimental study): author's abstract. dis. ... candidate of medical sciences. M. 2017; 20.

About the authors

Darya D. Kvashnina

Privolzhsky Research Medical University

Author for correspondence.
Email: daria_tsariova@mail.ru
ORCID iD: 0000-0001-9317-4816

PhD (Medicine), Associate Professor of the Department of Epidemiology, Microbiology and Evidence-Based Medicine

Russian Federation, Nizhny Novgorod

Irina Yu. Shirokova

Privolzhsky Research Medical University

Email: shirokova_i@pimunn.net
ORCID iD: 0000-0002-8387-6344

PhD (Medicine), Bacteriologist, Head of the Bacteriological Laboratory of the University Clinic

Russian Federation, Nizhny Novgorod

Natalya А. Belyanina

Privolzhsky Research Medical University

Email: belyanina_n@pimunn.net
ORCID iD: 0000-0002-8578-3600

Biologist of the Bacteriological Laboratory of the University Clinic

Russian Federation, Nizhny Novgorod

Sergey А. Syrbu

G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences

Email: ssa@isc-ras.ru
ORCID iD: 0000-0003-1482-2809

DSc (Chemistry), Professor, Head of the Laboratory "New Materials Based on Macrocyclic Compounds"

Russian Federation, Ivanovo

Natalia Sh. Lebedeva

G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences

Email: nsl@isc-ras.ru
ORCID iD: 0000-0001-7260-3239

DSc (Chemistry), Associate Professor, Head of the Laboratory "Physical Chemistry of Supramolecular Systems Based on Macrocyclic Compounds and Polymers"

Russian Federation, Ivanovo

Zhanna V. Boeva

Privolzhsky Research Medical University

Email: zhnn_boeva@mail.ru
ORCID iD: 0009-0008-6594-4471

Laboratory Assistant of the Research Department

Russian Federation, Nizhny Novgorod

Anastasiia А. Burashnikova

Privolzhsky Research Medical University

Email: burashnikova.nastasya@mail.ru
ORCID iD: 0009-0009-7102-4791

Laboratory Assistant of the Research Department

Russian Federation, Nizhny Novgorod

Olga V. Kovalishena

Privolzhsky Research Medical University

Email: kovalishena_o@pimunn.net
ORCID iD: 0000-0002-9595-547X

DSc (Medicine), Head of the Department of Epidemiology, Microbiology and Evidence-Based Medicine

Russian Federation, Nizhny Novgorod

Nikolai V. Saperkin

Privolzhsky Research Medical University

Email: saperkinnv@mail.ru
ORCID iD: 0000-0002-3629-4712

PhD (Medicine), Associate Professor, Associate Professor of the Department of Epidemiology, Microbiology and Evidence-Based Medicine

Russian Federation, Nizhny Novgorod

Daniil K. Lazarev

Privolzhsky Research Medical University

Email: danilazar@yandex.ru
ORCID iD: 0009-0002-2069-5367

Student

Russian Federation, Nizhny Novgorod

References

Supplementary files