An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

Summary

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

Abstract

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.

As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 ± 0.6 J/cm²). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug's cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Introduction

Among the most lethal noncommunicable diseases, cancer represents a global leading cause of premature death. It accounted for nearly 10 million deaths in 2020, representing about one in six deaths worldwide¹. Additionally, the multidrug resistance (MDR) phenomenon represents a tremendous public health threat, as approved chemotherapeutic protocols fail to reach remission stages for this clinical condition². Cancer cells can develop resistance to chemotherapy through several mechanisms; however, the overexpression of some ATP-binding cassette (ABC) transporters - ATP-dependent efflux pumps - is considered the main cause of MDR development within a tumor microenvironment³. In addition to MDR, other cancer complications, such as recurrence and metastasis, reinforce the urgent demand to develop and improve therapeutic approaches to overcome this oncological challenge.

The curative utilization of light has been practiced for centuries⁴, and photodynamic therapy (PDT) represents a clinically approved therapeutic approach for solid tumors. PDT combines the administration of a photosensitizer (PS) followed by light irradiation to generate reactive oxygen species (ROS) to exert selective cytotoxicity in tumor cells. This therapeutic approach is superior to conventional methods, including surgery, radiation, and chemotherapy⁵; it is a minimally invasive technique showing lower cytotoxicity in connective tissues⁶. The light application and PS accumulation directly in the tumor or its microenvironment ensure precise targeting and, consequently, minor, undesirable systemic side effects⁷ and the possibility of repeated treatment at the same site. Moreover, the cost is lower than that of other approaches. Owing to its promising features, PDT can be considered an appropriate option for both single, especially in the case of inoperable tumors, or adjuvant cancer treatment⁷, and represents an alternative for MDR related to chemotherapy^8,9.

The first report showing a high objective response rate using PDT was described in 1975 in a mouse and rat model¹⁰. Since then, studies have been conducted using PDT with positive outcomes⁷ both in vivo and in vitro with human tumor cell lines in 2D cell culture^11,12. Considering the broad applicability of clinically approved PS, regardless of their specific accumulation pathways and wavelength ranges of absorption peaks, the general process is as follows: (i) PS uptake, (ii) peaking of PS concentration at the tumor or its microenvironment, (iii) light application, (iv) PS-light interaction, (v) transfer of PS excited-state energy to either tissue substrate or surrounding oxygen molecules, (vi) ROS production involving singlet oxygen or superoxide anion, (vii) tumor cell death via, essentially, necrosis or apoptosis (direct death), autophagy (cytoprotective mechanism), tissue ischemia (vascular damage), immune modulation, or an overlap of these mechanisms⁷. In this final stage, the activation of a specific cell death pathway depends on many factors, such as cell characteristics, experimental design and, most importantly, PS intracellular localization and PDT-related targeted damage¹³.

Verteporfin is a second-generation PS, approved by regulatory agencies for clinical use in Norway and China to treat age-related macular degeneration⁷. After dose delivery, this prodrug was reported to partially accumulate in mitochondria¹⁴ and induce cellular protein tyrosine phosphorylation and DNA fragmentation, leading to tumor cell apoptosis^15,16. After 24 h incubation for verteporfin internalization, a PDT protocol using a 690 nm wavelength setup is recommended to achieve effective levels of electromagnetic radiation transfer to adjacent molecules^7,17.

Regarding the light source for PDT, the classical diode laser systems are usually expensive, technically complicated, oversized, and thus unportable^18,19. As a consequence of its single-wavelength profile, which can also be observed in LED-based PDT equipment, the demand for independent units for each photosensitizer application makes the utilization of diode laser systems even more complex and economically unfeasible^20,21. Therefore, the utilization of LED machinery is considered the most promising alternative to solve not only costs²² and maintenance issues, but also to provide high power output and less harmful²³ and wider illuminating capability^24,25,26,27.

Despite the potential contribution that LED-based equipment can offer to PDT experiments²⁸, most commercial options still possess drawbacks such as a lack of portability, high cost, and complex construction projects and operation²⁹. The main objective of this work was to offer a simple and reliable tool for in vitro PDT assays. This paper describes PhotoACT, an in-house-built LED-based PDT device, which is inexpensive, user-friendly, and portable. As a proof of concept, this device is shown to enhance the cytotoxicity of verteporfin in a 2D cell culture model and, therefore, can be used as a research tool in PDT experiments.

Protocol

NOTE: See the Table of Materials for details related to all materials, reagents, and software used in this protocol.

1. Device construction

1. Saw 3 mm thickn medium-density fiberboards (MDF) to obtain pieces with the dimensions shown in Figure 1A.
   NOTE: Use the vector file (Supplemental File 1) for computer numerical control (CNC) cutting.
2. Build two boxes with the following dimensions (length x width x height ): 330 mm x 235 mm x 225 mm for the bigger boxes, and 300 mm x 220 mm x 150 m for the smaller boxes (Figure 1B).
3. Drill the back of the bigger box to install a barrel jack connector. Drill the top of the bigger box andk the top and bottom of the smaller box to provide a passage for electric cables (Figure 1C).
4. Paint all the internal surfaces with black ink to promote homogeneous light incidence (Figure 1D).
5. Attach, in parallel, three LED tapes with 10 LEDs each at the upper interior surface of the smaller box (Figure 1E).
6. Install a brightness sensor at the center of the bottom interior surface of the smaller box (Figure 1F).
7. Print the structure of the control unit (Figure 1G) using the 3D printing file (Supplemental File 2).
8. Install all the components (power button, potentiometers, time/start touchpad, LEDs, brightness sensor, LCD, buzzer, and power supply) at the ports of an ESP32 control board mounted at the control unit interior (Figure 2).
9. Upload the programming code (Supplemental File 3, Supplemental File 4, and Supplemental File 5) and run a test to check that all the connections are working (Figure 1H).
10. Assemble the boxes and fix them together to avoid gaps, and consequently external light interference and emitted light loss. Attach the mounted control unit to the drilled area at the top of the prototype (Figure 1I).
11. Fix a front door of the same material and 330 mm x 225 mm (length x width) dimensions on the bigger box with two small hinges. Also, attach velcro tapes sideways to the bigger box to reinforce the prototype closure (Figure 1J). Install a handle to manipulate the front door of the equipment.
12. Attach four rubber foot pads at the bottom of the prototype to ensure more stability during the operations (Figure 1K).

2. Cell lines: cultivation, seeding, and treatment

1. Chemicals
   1. Dissolve the porphyrin in dimethyl sulfoxide (DMSO) to achieve a concentration of 100 mM.
      CAUTION: DMSO must be manipulated carefully (handling with the use of personal protective equipment and in a ventilated area). Manipulate both stock and diluted solutions carefully to avoid excessive light exposure.
2. Cell lines
   1. Cultivate the HeLa cell line in Dulbecco's Modified Eagle's Medium (DMEM) low-glucose medium supplemented with 10% fetal bovine serum and 1% gentamicin.
   2. Keep the culture flasks in a 5% CO₂ cell culture incubator at 37 °C.
   3. Manage and inspect the cells until they reach 80%-90% confluence.
3. Seeding process
   1. Remove the culture medium from the flask.
   2. Wash the cell monolayer with PBS.
   3. Detach the confluent cell culture with trypsin-EDTA (0.5%) 1x for 5 min at 37 °C. Stop the action of trypsin by resuspending the cells with culture medium supplemented with 10% fetal bovine serum and 1% gentamicin.
   4. Count the resuspended cells with a hemocytometer and seed them into a 96-well plate at 2.0 × 10⁴ cells/well.
   5. Prepare two plates for dark and light conditions.
   6. Incubate the plates for 24 h for cell attachment.
4. Treatment process
   1. Remove the medium from both 96-well plates.
   2. Treat the cells with 100 µL of increasing concentrations of verteporfin (0.045 to 24 µM, serial dilution).
   3. Incubate the cells with drug treatment for 24 h to allow verteporfin internalization.
   4. After 24 h, discard the medium containing the drug, wash the monolayer of cells with PBS (100 µL), and add drug-free medium (100 µL).
   5. Cover one microplate with aluminum foil to protect it from light exposure and incubate it for 24 h. This plate will provide control data for PDT results (dark condition). The other microplate will be utilized on the light exposure condition in the device.

3. Device operation

1. Plug the PDT device into the electrical outlet and turn it on by pressing the power button.
2. Place the other microplate (light condition) into the PDT device and close the equipment by fastening the front door with the velcro tapes.
3. Use the potentiometers to adjust the RGB configuration (a RGB 255, 255, 255 experiment here) and set the color of light emission.
   NOTE: Each RGB combination has a specific emission spectrum, which must be adjusted for experiments with different photosensitizers and, consequently, different absorbance curves.
4. Press the (+)/(-) touchpad to adjust the time configuration (a 60 min experiment here) and set the duration of the assay.
   NOTE: The time configuration, in association with the irradiance value, will determine the fluence of the process-the light dose applied in the assay.
5. Check the setup information at the display.
6. Press the Start button to initiate the assay. Ensure that a one-beep buzzer is heard at the beginning of the assay.
7. In the course of the experiment, observe real-time information on the display, such as irradiance and time left.
8. Do not open the front door or change any configuration during the PDT assay.
9. At the end of the assay, wait for a four-beep buzzer and for the electronic system to turn all the LEDs off. Observe a Finished message and the final amount of energy expended-fluence-during the experiment in the display.
   NOTE: The fluence final value is calculated using equation (1):
   (1)
   Where F equals to J/cm² and I equals to mW/cm² or mJ/s·cm². The irradiance value considers the potency of the LEDs (emitting source) and the uniformly irradiated area of the dark chamber (660 cm²) (equation [2]):
   (2)
   The theorical irradiance value is shown on the device display throughout the entire PDT assay. At the end of the operation, use equation (3) to calculate the fluence:
   Fluence (F) = irradiance (I, constant value) × operation time (s) (3)

4. Cell viability assay

1. After the PDT assay, cover the microplate that was exposed to light and incubate for 24 h.
2. After the incubation period, remove the culture medium from both plates, wash the monolayer of cells with PBS (100 µL), and add MTT solution (0.5 mg/ml, 100 µL). Incubate both plates-dark and light conditions-for 4 h to allow formazan crystal formation.
3. Remove the MTT solution carefully and dissolve the purple crystals with a DMSO/ethanol (1:1) solution.
4. After complete dissolution of the crystals, carry out the absorbance measurement using a microplate reader at 595 nm.
   NOTE: The device can be used in other important experiments such as ROS-mediated cell death triggered by photosensitizers after light exposure by flow cytometry³⁰.

Results

The final PDT device, named the PhotoACT, included a dark chamber to allocate up to four multiwell microplates, with its upper interior surface equipped with a set of 30 scattered LEDs programmed to emit distinct spectrums of visible light (Figure 3 and Supplemental File 6). The device was built using two associated boxes: an internal box designed as a dark chamber for the PDT assays, and an external box to cover the internal chamber and hold the control unit (

Discussion

The final PhotoACT device was convenient to construct with commercially available, low-cost components at a total cost of less than $50. Additional advantages include low maintenance demands, the capacity to irradiate multiple types of culture plates, the simultaneous use of up to four units per assay, low weight (2 kg)/size (44 cm³) that allows portability, accurate and reproducible irradiation (data not shown), and a user-friendly and simple setup interface that does not require connection to computers or ot...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5% Trypsin-EDTA (10x)	Gibco	15400054	Mammalian cell culture dissociation reagent
3D printer	Flashforge		Finder model
96-well plates			Non-sterile, polystyrene, and high-binding surface plates with flat bottom wells used for 2D cell culture
Arduino
Brightness sensor			TSL2561 model with 0.1-40.000+ lux detection levels and I2C interface
Buttons
Buzzer
Cell culture Flasks			Sterile, polystyrene, rectangular bottom flask with Tissue Culture (TC)-treated surface, canted neck and vent cap (sizes)
Centrifuge Tubes			Sterile, polypropylene tubes with 15/50 mL capacity used for cell culture dilution at seeding step of the assay
CO2 Incubator
Controller board			ESP32
Design Software	Trimble		SketchUp
DMEM High Glucose	Gibco	11965092	DMEM (Dulbecco's Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells.
DMSO	Sigma-Aldrich	D4540-500ML	Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Fetal Bovine Serum	Gibco	12657029	FBS provides the best value by delivering consistency of cell growth over time and passages.
Gentamicin (50 mg/mL)	Gibco	15750060	Water-soluble antibiotic drug originally purified from the fungus Micromonospora purpurea. Gentamicin acts by preventing cell culture contamination
Hemocytometer			Neubauer patterned chamber used for cell counting at seeding step of the assay
Inverted Laboratory Microscope	Leica		DM IL LED
Laminar Flow Hood			Cabin designed to protect the working environment from contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. Used at several steps of cell cultivation and treatment procedures
LCD display
LED RGB WS2812			5050 RGB SMD model with a built-in processor. Tape with 30 LEDs, 1 meter length and 9 watts
MDF fiberboards			3mm thickness medium-density fiberboards
Microcentrifuge Tubes			Sterile, polypropylene tubes with safety lid and 1.5/2.0 mL capacity. Convenient tools for manipulating small volumes at treatment step of the assay
Microplate reader	ThermoFischer		Multiskan FC Microplate Photometer designed to detect a broad wavelength range of absorbance (340-850 nm). The equipment was used to evaluate cell viability after MTT incubation.
MTT Reagent	Invitrogen	M6494	3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Used for cell viability assays
Operational System	Real Time Engineers ltd.		FreeRTOS
P10 micripipette			Non-electronic, single-channel, 1-10 μL capacity
P1000 micropipette			Non-electronic, single-channel, 10-1000 μL capacity
P200 micropipette			Non-electronic, single-channel, 20-200 μL capacity
PDT Equipment	LumaCare		Model LC-122
Phosphate-Buffered Saline pH 7.4	Gibco	10010031	Balanced salt formulation used for washing cells during cultivation and assay procedures
Potentiometers
Tips			Non-sterile, universal fit, 10/200/1000 μL maximum volumes
Verteporfin	Sigma-Aldrich	SML0534-5MG	Verteporfin, ≥94% (HPLC)