We thank Arthur Henrique Gomes de Oliveira and Lucas Julian Cruz Gomes for helping with the filming process. This project was supported by the Brazilian Research Council (CNPq, grant numbers 400953/2016-1-404286/2021-6) and Funda&#231;&#227;o Arauc&#225;ria-PPSUS 2020/2021 (SUS2020131000003). This study was also financed in part by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES)-Finance Code 001.

NOTE: See the Table of Materials for details related to all materials, reagents, and software used in this protocol.
1. Device construction
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Paint all the internal surfaces with black ink to promote homogeneous light incidence (Figure 1D).</li>
	<li>Attach, in parallel, three LED tapes with 10 LEDs each at the upper interior surface of the smaller box (Figure 1E).</li>
	<li>Install a brightness sensor at the center of the bottom interior surface of the smaller box (Figure 1F).</li>
	<li>Print the structure of the control unit (Figure 1G) using the 3D printing file (Supplemental File 2).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Attach four rubber foot pads at the bottom of the prototype to ensure more stability during the operations (Figure 1K).</li>
</ol>
2. Cell lines: cultivation, seeding, and treatment
<ol>
	<li>Chemicals
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Cell lines
	<ol>
		<li>Cultivate the HeLa cell line in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) low-glucose medium supplemented with 10% fetal bovine serum and 1% gentamicin.</li>
		<li>Keep the culture flasks in a 5% CO2 cell culture incubator at 37 &#176;C.</li>
		<li>Manage and inspect the cells until they reach 80%-90% confluence.</li>
	</ol>
	</li>
	<li>Seeding process
	<ol>
		<li>Remove the culture medium from the flask.</li>
		<li>Wash the cell monolayer with PBS.</li>
		<li>Detach the confluent cell culture with trypsin-EDTA (0.5%) 1x for 5 min at 37 &#176;C. Stop the action of trypsin by resuspending the cells with culture medium supplemented with 10% fetal bovine serum and 1% gentamicin.</li>
		<li>Count the resuspended cells with a hemocytometer and seed them into a 96-well plate at 2.0 &#215; 104 cells/well.</li>
		<li>Prepare two plates for dark and light conditions.</li>
		<li>Incubate the plates for 24 h for cell attachment.</li>
	</ol>
	</li>
	<li>Treatment process
	<ol>
		<li>Remove the medium from both 96-well plates.</li>
		<li>Treat the cells with 100 &#181;L of increasing concentrations of verteporfin (0.045 to 24 &#181;M, serial dilution).</li>
		<li>Incubate the cells with drug treatment for 24 h to allow verteporfin internalization.</li>
		<li>After 24 h, discard the medium containing the drug, wash the monolayer of cells with PBS (100 &#181;L), and add drug-free medium (100 &#181;L).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Device operation
<ol>
	<li>Plug the PDT device into the electrical outlet and turn it on by pressing the power button.</li>
	<li>Place the other microplate (light condition) into the PDT device and close the equipment by fastening the front door with the velcro tapes.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Check the setup information at the display.</li>
	<li>Press the Start button to initiate the assay. Ensure that a one-beep buzzer is heard at the beginning of the assay.</li>
	<li>In the course of the experiment, observe real-time information on the display, such as irradiance and time left.</li>
	<li>Do not open the front door or change any configuration during the PDT assay.</li>
	<li>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): 
	<img alt="Equation 1" src="/files/ftp_upload/64391/64391eq01.jpg" style="margin: auto;" /> (1) 
	Where F&#160;equals to J/cm2 and I&#160;equals to mW/cm2 or mJ/s&#183;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]): 
	<img alt="Equation 2" src="/files/ftp_upload/64391/64391eq02.jpg" style="margin: auto;" /> (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) &#215; operation time (s) (3)</li>
</ol>
4. Cell viability assay
<ol>
	<li>After the PDT assay, cover the microplate that was exposed to light and incubate for 24 h.</li>
	<li>After the incubation period, remove the culture medium from both plates, wash the monolayer of cells with PBS (100 &#181;L), and add MTT solution (0.5 mg/ml, 100 &#181;L). Incubate both plates-dark and light conditions-for 4 h to allow formazan crystal formation.</li>
	<li>Remove the MTT solution carefully and dissolve the purple crystals with a DMSO/ethanol (1:1) solution.</li>
	<li>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.</li>
</ol>

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The authors declare no competing interests.

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 other machines.
Certain critical steps of both construction and operation protocols gave rise to improvement opportunities during project conception. As a PDT chamber requires homogeneous light emission and consistent energy measurement, the internal and external boxes were sealed to avoid both exterior light interference and emitted light loss. Additional velcro tapes were fixed sideways on the frontal door to reinforce the chamber closure and ensure uninterrupted experiments. A representative LED was installed at the control unit to certify the required RGB configuration, indicating the color and intensity of the emitted light during the assay. Finally, the programming code underwent several upgrades to refine instant density measurements and final amounts of energy evaluations, endorsing reproducibility and mathematical consistency. Some other major details that require extra attention include: (i) homogeneous distribution of LEDs and central positioning of the brightness sensor to obtain representative and balanced results, (ii) installation of all components according to the electronic diagram (Figure 2) and programming code (Supplemental File 3, Supplemental File 4, and Supplemental File 5) to ensure correct operation, and (iii) the setup (keeping the same RGB and time configuration) before running an experiment to ensure consistent replicates with reliable results. Although the RGB system provides multiple visible color compositions with specific wavelengths, non-visible light experiments would require specific protocol upgrades. A decision flowchart is presented in Figure 5 to provide a systematic problem-solving approach to find and correct problems or errors during the operation.
Designed to meet the demands of in vitro experimentation with validated results obtained with the therapeutic activation of verteporfin to induce cytotoxicity in 2D HeLa cells (Figure 4), the PhotoACT can be recommended for universities, schools, industries, and other research centers. This device should extend the benefits of PDT to scientific research exploring the mechanism of action of photosensitizers and their clinical applications.

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 chemotherapy8,9.
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 culture11,12. 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 apoptosis15,16. 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 molecules7,17.
Regarding the light source for PDT, the classical diode laser systems are usually expensive, technically complicated, oversized, and thus unportable18,19. 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 unfeasible20,21. 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 capability24,25,26,27.
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.

<table><tbody><tr><td>0.5% Trypsin-EDTA (10x)</td><td>Gibco</td><td>15400054</td><td>Mammalian cell culture dissociation reagent</td></tr><tr><td>3D printer</td><td>Flashforge</td><td></td><td>Finder model</td></tr><tr><td>96-well plates</td><td></td><td></td><td>Non-sterile, polystyrene, and high-binding surface plates with flat bottom wells used for 2D cell culture</td></tr><tr><td>Arduino</td><td></td><td></td><td></td></tr><tr><td>Brightness sensor</td><td></td><td></td><td>TSL2561 model with 0.1-40.000+ lux detection levels and I2C interface</td></tr><tr><td>Buttons</td><td></td><td></td><td></td></tr><tr><td>Buzzer</td><td></td><td></td><td></td></tr><tr><td>Cell culture Flasks</td><td></td><td></td><td>Sterile, polystyrene, rectangular bottom flask with Tissue Culture (TC)-treated surface, canted neck and vent cap (sizes)</td></tr><tr><td>Centrifuge Tubes</td><td></td><td></td><td>Sterile, polypropylene tubes with 15/50 mL capacity used for cell culture dilution at seeding step of the assay</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td></td><td></td><td></td></tr><tr><td>Controller board</td><td></td><td></td><td>ESP32</td></tr><tr><td>Design Software</td><td>Trimble</td><td></td><td>SketchUp</td></tr><tr><td>DMEM High Glucose</td><td>Gibco</td><td>11965092</td><td>DMEM (Dulbecco&#039;s Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells.</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D4540-500ML</td><td>Dimethyl sulfoxide, &amp;ge;99.5% (GC), suitable for plant cell culture</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>12657029</td><td>FBS provides the best value by delivering consistency of cell growth over time and passages.</td></tr><tr><td>Gentamicin (50 mg/mL)</td><td>Gibco</td><td>15750060</td><td>Water-soluble antibiotic drug originally purified from the fungus&amp;nbsp;Micromonospora purpurea. Gentamicin acts by preventing cell culture contamination</td></tr><tr><td>Hemocytometer</td><td></td><td></td><td>Neubauer patterned chamber used for cell counting at seeding step of the assay</td></tr><tr><td>Inverted Laboratory Microscope</td><td>Leica</td><td></td><td>DM IL LED</td></tr><tr><td>Laminar Flow Hood</td><td></td><td></td><td>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</td></tr><tr><td>LCD display</td><td></td><td></td><td></td></tr><tr><td>LED RGB WS2812</td><td></td><td></td><td>5050 RGB SMD model with a built-in processor. Tape with 30 LEDs, 1 meter length and 9 watts</td></tr><tr><td>MDF fiberboards</td><td></td><td></td><td>3mm thickness medium-density fiberboards</td></tr><tr><td>Microcentrifuge Tubes</td><td></td><td></td><td>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</td></tr><tr><td>Microplate reader</td><td>ThermoFischer</td><td></td><td>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.</td></tr><tr><td>MTT Reagent</td><td>Invitrogen</td><td>M6494</td><td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Used for cell viability assays</td></tr><tr><td>Operational System</td><td>Real Time Engineers ltd.</td><td></td><td>FreeRTOS</td></tr><tr><td>P10 micripipette</td><td></td><td></td><td>Non-electronic, single-channel, 1-10 &amp;mu;L capacity</td></tr><tr><td>P1000 micropipette</td><td></td><td></td><td>Non-electronic, single-channel, 10-1000 &amp;mu;L capacity</td></tr><tr><td>P200 micropipette</td><td></td><td></td><td>Non-electronic, single-channel, 20-200 &amp;mu;L capacity</td></tr><tr><td>PDT Equipment</td><td>LumaCare</td><td></td><td>Model LC-122</td></tr><tr><td>Phosphate-Buffered Saline pH 7.4</td><td>Gibco</td><td>10010031</td><td>Balanced salt formulation used for washing cells during cultivation and assay procedures</td></tr><tr><td>Potentiometers</td><td></td><td></td><td></td></tr><tr><td>Tips</td><td></td><td></td><td>Non-sterile, universal fit, 10/200/1000 &amp;mu;L maximum volumes</td></tr><tr><td>Verteporfin</td><td>Sigma-Aldrich</td><td>SML0534-5MG</td><td>Verteporfin, &amp;ge;94% (HPLC)</td></tr></tbody></table>

an in-house-built and light-emitting-diode-based photodynamic therapy device for enhancing verteporfin cytotoxicity in a 2d cell culture model

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 &#177; 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&#39;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.

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 (Figure 1B). The internal box was painted black (Figure 1D) and composed of a tape model of LED RGB WS2812 (or WS281B) with 30 LEDs and of 1 m length (which was later cut into three pieces) and 9 watts, which holds a built-in processor. This apparatus allowed a more controlled utilization and, consequently, more accurate color emission. The 30 LEDs were equally distributed over the entire upper interior surface of the PDT chamber by three parallel tapes with 10 LEDs each (Figure 1E). A homogeneous light incidence was established due to the low reflectivity of the black interior surfaces and the uniform distribution of LED configuration. A TSL2561 brightness sensor was also incorporated in the bottom center of the internal box to indicate the light incidence and serve as a quality control tool, guaranteeing irradiation uniformity inside the dark chamber (Supplemental Table S1) and monitoring the power of the LED system, which is known to have a certain number of hours of useful life (Figure 1F). The external box is a structure that covers the internal box, composed of six MDF parts, painted gray, and laser-cut for a perfect fit (Figures 1A,B). The control unit was designed using online software31, 3D printed with polylactic acid as a single part using a 3D printer, and painted gray. It holds all the electronics components, including a display, potentiometers, buttons, and a buzzer (Figure 1I and Figure&#160;2).
To enable independent light incidence adjustments, the ESP32 controller board was selected to integrate the control unit (Figure 2). This configuration should allow a USB interface for programming, bluetooth, Wi-Fi, dual core processor, numerous ports, and the ability to communicate with an inter-integrated circuit (I2C), a serial peripheral interface (SPI), and other interfaces. To operate the system, a program was written in C language through an integrated development environment (IDE). The code structure32 is based on FreeRTOS, a free, real-time operational system supported by ESP32 controller board. The referred logic allows independent applications programming, which can be processed by ESP32 in echeloned or parallel approaches, making the project and its maintenance, improvement, and update processes much more versatile and secure.
The interface between the machine and the operator is user-friendly and consisted of a liquid crystal display (LCD), potentiometers, buttons, and a buzzer (Figure 3 and Supplemental File 6). The small, 16 mm x 2 mm (columns vs. lines) LCD had a built-in HD44780 controller with an I2C communication protocol, which confers the ease of installation and transmission versatility, respectively. The preliminary setup includes RGB and time adjustments by the operator. The light magnitude and color configuration can be adjusted using the potentiometers, which modify the intensity (0 to 255) of the three basic color components-RGB. These adjustments can result in several color compositions addressed in the international RGB colors table33. Each RGB composition owns a specific wavelength, which must be adjusted according to the PS used in the PDT experiment; the absorbance curve of the PS and the wavelength of the irradiation must be overlapped for the photoactivation to occur satisfactorily (Supplemental Figure S1). Along the process, the operation status with the time left and light incidence information can be accessed on the LCD.
At the end of the programmed time, the electronics system turned all the LEDs off, emitted an audible warning, and showed on the display the total amount of energy per area (J/cm&#178;), which was calculated by multiplying the constant value of irradiance by the operation time in seconds. This digital model presented a broad range of detection levels (limits between 0.1 and 40,000+ lux), an I2C interface, and a low intensity of electric current (0.5 mA and 15 &#181;A in operation and standby status, respectively).
As a proof of concept, the device was used to enhance the cytotoxic effect of verteporfin in 2D HeLa cell culture after light exposure of 1 h (49.1 &#177; 0.6 J/cm2). As shown in Figure 4A, the GI50 value was 3.1 &#181;M for the light condition and 13.8 &#181;M for the dark condition. Thus, the 4.4-fold shift comparing the conditions validated the use of verteporfin as a PS and the applicability of PhotoACT to PDT assays. To validate the use of the prototype described in this work, a commercial PDT device was used under the same experimental conditions, including a PS, cells, and fluence, and the results compared. As shown in Figure 4B, both devices photoactivated verteporfin equally, enhancing the cytotoxic effect. These results confirmed the applicability of this in house-built PDT device (Figure 4A,B). Finally, the ROS-mediated cell death triggered by verteporfin after light exposure was confirmed by flow cytometry using DCFDA assay (Figure 4C,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64391/64391fig01.jpg" /> 
Figure 1: PhotoACT construction and assembly instructions. Detailed illustration of the construction of, drilling, painting, mounting, and accessorizing components into the device. The panel shows a step-by-step guide to build the device. Abbreviation: LED = light-emitting diode. <a href="https://www.jove.com/files/ftp_upload/64391/64391fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64391/64391fig02.jpg" /> 
Figure 2: The control unit construction and electronic installation instructions. Zoomed-in view drawing of exploded control unit and detailed scheme of electronic connections at ESP32 controller board ports with connections and components used in the prototype. <a href="https://www.jove.com/files/ftp_upload/64391/64391fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64391/64391fig03.jpg" /> 
Figure 3: PhotoACT representation. Assembly drawing of the final product design with bill of materials, labelling balloons, and detailed view of the upper interior LEDs. The figure allows to identify parts and components of the prototype. Abbreviations: MDF = medium-density fiberboard; LED = light-emitting diode. <a href="https://www.jove.com/files/ftp_upload/64391/64391fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64391/64391fig04.jpg" /> 
Figure 4: In vitro phototoxicity of verteporfin and ROS generation. (A,B) Cell viability assay performed using the MTT method: the assays were conducted to evaluate the cytotoxic profile of the photosensitizer verteporfin at different concentrations.HeLa cells were treated for 24 h with different concentrations of verteporfin (0.045-24 &#181;M), exposed to light (PhotoACT (A) or commercial PDT equipment (B)) or dark conditions, and then subjected to the MTT assay. Both conditions showed a decreased cell viability at higher concentrations of verteporfin, but the light exposure-obtained from PDT assays-enhanced the cytotoxic profile of the PS, which implies that PDT enhances the cytotoxicity of verteporfin. The viability curves were fitted using GraphPad Prism 6 software. (C,D) HeLa cells were treated for 24 h with low and high concentrations of verteporfin (0.187 &#181;M and 6 &#181;M) and then exposed to light (PhotoACT) or dark conditions. Intracellular ROS levels were measured after irradiation by flow cytometry using DCFDA probe (incubation for 30 min at 1 &#181;M). A right shift among the histograms implied higher fluorescence intensity because of a higher intracellular accumulation of DCF, and thus, greater ROS levels. The results showed no relevant difference in ROS levels in the dark condition (C) but showed a dose-response increase in ROS levels after verteporfin photoactivation (D). Abbreviations: ROS = reactive oxygen species; DCF = 2&#39;,7&#39;-dichlorofluorescein; DCFDA = 2&#39;,7&#39;-dichlorohydrofluorescein diacetate. <a href="https://www.jove.com/files/ftp_upload/64391/64391fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64391/64391fig05.jpg" /> 
Figure 5: The device&#39;s decision flowchart: preliminary setup and operation troubleshooting. <a href="https://www.jove.com/files/ftp_upload/64391/64391fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Overlap between the absorbance curve of verteporfin and the LED emission spectrum of the device. Absorption spectrum of verteporfin with its specific absorbance peaks (x,y&#39;) and LED emission spectrum of RGB 255, 255, 255-white color (3,700K-5,000K CCT) utilized to photoactivate the photosensitizer (x,y&#39;&#39;). The overlap between the different absorbance peaks of verteporfin and the white irradiating light corroborates the photoactivation of the photosensitizer, which was also confirmed by the biological results. <a href="https://www.jove.com/files/ftp_upload/64391/Supplemental Fig S1 - Overlap between verteporfin X LED.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Table S1: Irradiation uniformity tests. Instant luminosity (lumen) measured by the brightness sensor at different points of the irradiated area. The presented results show no expressive variation, which attest the uniform irradiation of the device. <a href="https://www.jove.com/files/ftp_upload/64391/Supplemental Table S1 - Irradiation uniformity tests.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental File 1: Vector file for cutting. Drawing (DWG) file for MDF board cutting. The file must be used for computer numerical control (CNC) cutting. <a href="https://www.jove.com/files/ftp_upload/64391/Supplemental File 1 - Vector File for Cutting.dwg" target="_blank">Please click here to download this File.</a>
Supplemental File 2: 3D printing file of the unit control&#39;s. Stereolithography (STL) file to 3D print the control unit of the device. <a href="https://www.jove.com/files/ftp_upload/64391/renamed_ed6a2.stl" target="_blank">Please click here to download this File.</a>
Supplemental File 3: C programming code. Programming code developed in C language for configuring the control unit of the equipment. <a href="https://www.jove.com/files/ftp_upload/64391/renamed_0e9a1.c" target="_blank">Please click here to download this File.</a>
Supplemental File 4: INO programming code. Programming code developed in INO language for configuring the control unit of the equipment. <a href="https://www.jove.com/files/ftp_upload/64391/renamed_a891c.ino" target="_blank">Please click here to download this File.</a>
Supplemental File 5: Compilation instructions. Markdown (MD) &#34;READ ME&#34; file with additional instructions for programming code compilation. <a href="https://www.jove.com/files/ftp_upload/64391/Supplemental File 5 - Compilation instructions.md" target="_blank">Please click here to download this File.</a>
Supplemental File 6: Design model of the device. Preview of the Standard for the Exchange of Product Data (STEP) tridimensional model file for general visualization of the prototype. <a href="https://www.jove.com/files/ftp_upload/64391/renamed_2882b.stp" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model at JoVE.com

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

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.

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 ...

an-house-built-light-emitting-diode-based-photodynamic-therapy-device

Nanyang Technological University

Research

JoVE Journal

Biochemistry

3.0K Views.  Federal University of Parana. 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.

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

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Cancer Research

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

Chemistry

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

Bioengineering

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Medicine

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

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

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.

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.

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

Immunology and Infection

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

Source: Chang, J. E., et al. <a href="https://www.jove.com/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a>&#160;J. Vis. Exp.&#160;(2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

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

Summary

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An In Vitro Approach to Photodynamic Therapy

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells