LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

Summary

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

Abstract

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

Introduction

Photodynamic therapy (PDT) is a minimally invasive therapeutic approach that has shown promising results in recent years, particularly in dermatological clinical treatment¹. One particularly interesting area of application of this therapeutic modality is the treatment of microbial infections, a process known as antimicrobial photodynamic inactivation (aPDI)². Although initially overlooked, mostly because of the remarkable efficiency of antibiotics, light-triggered eradication of bacterial growth underwent a renewed interest over the recent years, driven by the emergence of antimicrobial multidrug resistance (AMR) and the need to find alternative and efficient strategies to address this public-health concern^3,4. Significant results can be achieved within seconds or minutes, with spatial and temporal precision offered by specifically irradiating the area of interest and the on/off feature of the procedure, respectively.

The mechanism behind the photodynamic principle relies on the combination of light-sensitive molecules (photosensitizer), molecular oxygen, and an external light source. Although these three components are harmless, when combined, they might become lethal to the target cells due to the photoactivation of the photosensitizer. Light-triggered reactions result in a massive generation of reactive oxygen species (ROS), which ultimately causes severe and irreversible damage in crucial cellular components, leading to cell death^5,6. Also, the combination of aPDI with other conventional antimicrobial treatments has shown promising therapeutic outcomes, including higher treatment efficacy, reduced treatment time, and lower drug dosages⁷.

Moreover, the topical administration of photosensitizers (PS) in PDT allows for targeted treatment with minimal damage to surrounding tissues and low systemic effects, making it a well-appreciated option for treating superficial skin infections⁸. The convenience of topical treatment and the superficial nature of many skin infections and lesions make it an appealing target for PDT. Studies have supported the effectiveness of aPDI in treating burn infections, surgical wound infections, ulcerated lesions, and other superficial ailments such as acne and impetigo^9,10,11.

Numerous molecules, both natural and synthetic, have been tested and proposed for aPDI. However, the use of varying and often unspecified parameters in experimental protocols makes it challenging to compare results, which affects the clarity of scientific findings. Developing simple and reproducible tests can facilitate the swift screening of numerous compounds, expediting the identification of promising candidates. Straightforward, reproducible, and cost-effective protocols are especially valuable in early-stage testing, as they enable comparison of results across different research facilities. Efficient screening processes also make it easier to explore a wider range of molecules, potentially leading to the discovery and selection of new photosensitizers for further testing in more advanced, complex, and costly methodologies.

This protocol outlines a straightforward method for evaluating the phototoxicity of potential photosensitizers on bacteria using an LED panel with a light intensity of 25 mW/cm². A representative bacterial strain, S. aureus, was used, and the white light channel was selected, offering full coverage of the visible spectrum. The procedure involved a 30 min pre-incubation period followed by a 15 min exposure to light (22.5 J/cm²). While the results are based on these specific experimental conditions, they can be tailored according to individual protocols. It is essential to clearly define these settings and include them in the procedural description.

Protocol

An overall description of the protocol is illustrated in Figure 1. The details of the reagents and equipment used are listed in the Table of Materials.

1. Bacterial culture preparation

1. Grow S. aureus (ATCC 29213) bacteria on Mueller-Hinton agar (MH) from stock cultures. Incubate the MH plates at 37 °C for 24 h prior to the assay to obtain fresh cultures.
2. Inoculate bacteria: Collect 2-3 fresh colonies from the agar plate and dilute in 6 mL of pre-warmed phosphate-buffered saline (PBS) pH 7.4, sterile filtered.
3. Vortex (1200 rpm) the inoculum 3-4 times to ensure proper homogenization of the sample.Withdraw 3 mL to a disposable cuvette.
4. Measure OD₆₀₀ and adjust it to 0.1 (approximately 1 x 10⁸ CFU/mL) using PBS.
   NOTE: The exact OD to CFU conversion can vary based on the strain of S. aureus. One can establish a standard growth curve for the specific strain and lab experimental conditions working with.

2. Photosensitizer preparation

1. Stock solution: Prepare a stock solution of the compound of interest photosensitizer (see Results section for details) in an appropriate solvent (e.g., DMSO or PBS; Figure 2).
2. Working solution: Dilute the stock solution in PBS. The use of PBS rather than bacterial culture media ensures that the components of the media do not interfere with light absorption during aPDI experiments.
3. Vortex the sample to ensure complete homogenization. Use an ultrasonic bath if necessary to guarantee complete solubilization and achieve a clear solution (in that case, carefully control. For hydrophobic compounds, adjustment of the solvent system might be required (e.g., adding a % of DMSO or ethanol). Ensure that the effect of the solvent on cellular viability is always checked.
   NOTE: The conditions for sonication will vary depending on the type of compound, concentration, and system solvent; shorter times (e.g., 5 min) might be sufficient for easily solubilized compounds, while longer times (20-30 min) may be required for highly hydrophobic compounds.

3. Plate setup

1. Prepare two separate 96-well plates: one to undergo light exposure and another to be kept in the dark (dark control).
2. In each of the plates, mix 100 µL of the photosensitizer solution with 100 µL of the bacterial suspension. Pipette up and down 5-10 times.
3. Incubate the plates at 37 °C for a pre-determined interval (in this study, a period of 30 min was used) to allow the photosensitizer to interact with the bacterial cells.

4. Irradiation

1. Remove the plates from the incubator and expose one of them to the light emitted from the LED panel (the device used in this experiment consisted of a rectangular LED projector of 50W power; IP65; input voltage: AC 85-265 V), keeping the other one protected from light exposure.
2. Place one of the plates above the LED panel. To ensure precise adjustment of the plate through several protocol repetitions, mark the edges of the 96-well plate directly on the LED surface.
3. Expose the plate for a suitable irradiation time (in this study, a period of 15 min was used).
   NOTE: Ensure the light dose is sufficient to activate the photosensitizer without compromising cellular viability. For that reason, light control must be included (bacteria exposed to light in the absence of photosensitizer).

5. Post-irradiation and agar plate sampling

1. Use another 96-well plate to prepare the serial dilutions of the samples (controls, non-irradiated, and irradiated wells).
2. Add 180 µL of PBS/well according to the number of samples. To properly define the layout, take into consideration that a row of 6 wells is needed for the serial dilution of one sample (10-fold dilution, from 10⁻¹ to 10⁻⁶).
3. Aliquot 20 µL from each well (from the irradiated and non-irradiated 96-well plate) to the first column of the plate previously filled up with PBS.
4. With the multi-channel micropipette, perform a simple serial dilution from well 1 to 6 (10^-1 to 10^-6). NOTE: Sample homogenization is critical at this point to ensure a uniform and consistent mixture. At every dilution step, pipette up and down 5-10 times.
5. Divide the agar plate into six equal parts, each section corresponding to one of the dilutions (see Figure 3).
6. Aliquot 10 µL from each well onto the corresponding section on the surface of the agar plate (for each sample, include at least 3 replicates). Avoid placing aliquots too close to each other. Gently transport the agar plates to the incubator and wait for 18-20 h.
   NOTE: Take into consideration that the morphology of CFUs varies significantly depending on the bacteria species. For instance, while S. aureus colonies are relatively easy to count, P. aeruginosa tends to form more diffuse-type colonies. The number of replicates might be adjusted considering this.

6. Cellular viability determination (colony-forming units counting)

1. Remove the agar plates from the incubator and select the areas suitable for cell counting.
   NOTE: Avoid counting within sections with abundant, overlapped, and non-well-defined colonies.
2. Within each section, count the number of colony-forming units (CFUs) and determine the average of the 3 replicates
3. Convert the number of counted CFU to CFU/mL, multiplying the average counting by the dilution factor and splitting by the volume of culture plated in mL.

7. Data analysis

1. Convert the data of CFU/mL to the logarithmic scale and represent cellular viability in log₁₀|CFU/mL| as a function of the concentration tested.

Results

Amino-based flavylium compounds (Figure 2): 7-diethylamino-4′-dimethylaminoflavylium (7NEt₂4′NMe₂), 7-diethylamino-2-(dimethylaminostyryl)-1-benzopyrylium (7NEt₂st4′NMe₂), 7-diethylamino-4′-aminoflavylium(7NEt₂4′NH₂) and 7-diethylamino-4′-hydroxyflavylium (7NEt₂4′OH), whose light responsive nature was previously discussed^12,

Discussion

Although this protocol serves as an initial stage testing platform, it is important to consider more meaningful testing conditions for the treatment of microbial infections in a more advanced study phase. When assessing the effectiveness of aPDI, experiments on biofilm-type bacterial organizations rather than planktonic bacteria should be conducted. Many clinical cases involve microorganisms organized in this more resistant conformation, such as chronic wounds, cystic fibrosis, and on implanted medical devices

Materials

Name	Company	Catalog Number	Comments
Cary 60 UV-Vis Spectrophotometer	Agilent	G6860A
Dimethyl Sulfoxide	Sigma-Aldrich	D8418
Disposable cuvettes PMMA	BRAND GMBH + CO KG	759030
Eppendorfs (500 mL)	Fisher Scientific	15625367
Falcon tubes (15 mL)	Corning	430791
Falcon tubes (50 mL)	Corning	430291
LED panel IP65 50W
Micropippete (100 uL)	Transferpette S	705874
Micropippete (1000 uL)	Transferpette S	705880
Mueller Hinton agar	Oxoid	CM0405
Multichannel pippete 12-channel	Transferpette S
Nunc MicroWell 96-Well Microplates	Thermo Scientific	260844
Phospate Buffered Saline tablets pH 7.4	Panreac Applichem	A9177
Serological Pipets (10 mL)	Thermo Scientific	170356N
Serological Pipets (5 mL)	Thermo Scientific	170366N
Tissue Culture Dish	TPP Techno Plastic Products AG	93150