Development of a novel laboratory photodynamic therapy device: automated multi-mode LED system for optimum well-plate irradiation.

Photodynamic therapy (PDT) is a targeted treatment method that utilizes a photosensitizer (PS) to induce cytotoxicity in malignant and non-malignant tumors. Optimization of PDT requires investigation of the selectivity of PS for the target tissues, irradiating light source, irradiation wavelengths, fluence rate, fluence, illumination mode, and overall treatment plan. In this study, we developed the Multi-mode Automatized Well-plate PDT LED Laboratory Irradiation System (MAWPLIS), an innovative device that automates time-consuming well plate light dosage/PS dose measurement experiment. The careful control of LED current and temperature stabilization in the LED module allowed the system to achieve high optical output stability. The MAWPLIS was designed by integrating a 3-axis moving system and motion controller, a quick-switching LED controller unit equipped with interchangeable LED modules capable of employing multiple wavelengths, and a TEC system. The proposed system achieved high optical output stability (1 mW) within the range of 0-500 mW, high wavelength stability (5 nm) at 635 nm, and high temperature stability (0.2 °C) across all radiation modes. The system's validation involved in vitro analysis using 5-ALA across varying concentrations, incubation periods, light exposures, and wavelengths in HT-29 colon cancer and WI-38 human lung fibroblast cell lines. Specifically, a combination of 405 nm and 635 nm wavelengths was selected to demonstrate enhanced strategies for colon cancer cell eradication and system validation. The MAWPLIS system represents a significant advancement in photodynamic therapy (PDT) research, offering automation and standardization of time-intensive experiments, high stability and precision, and improved PDT efficacy through dual-wavelength integration.

Comparison of the anti-cancer activity of 5-aminolevulinic acid-mediated photodynamic therapy after continuous wave and pulse irradiation in different histological types of canine mammary sarcoma tumors.

Canine mammary sarcoma tumors (CMST) are the most aggressive tumors with poor prognosis in dogs. Due to inadequate treatment options for CMST, recent studies have focused on alternative treatment strategies. We previously determined the optimized protocol of 5-ALA-based photodynamic therapy (PDT) in canine liposarcoma. However, its molecular mechanisms in the treatment of different histological types of CMST remain unclear.In this context, we, for the first time, assessed 5-aminolevulinic acid (5-ALA)-PDT-mediated anti-cancer activity and its molecular mechanism after continuous wave (CW) and pulse radiation (PR) on three different histological types (liposarcoma, chondrosarcoma, and osteosarcoma) of CMST cells by WST-1, Annexin V, ROS, acridine orange/propidium iodide staining, RT-PCR, and western blot analysis.Our findings showed that 5-ALA/PDT significantly suppressed the proliferation of CMST cells (p < 0.01) and induced apoptosis via increased ROS level and overexpression of Caspase-9 and Caspase-3 mRNA and cleaved protein levels in especially liposarcoma and chondrosarcoma cells following CW and PR irradiation at 9 J/cm2. However, the response of CMST cells to 5-ALA was different upon CW and PR irradiation due to differences in their origin.Collectively, our findings provided the first evidence that 5-ALA-based PDT could be used as an alternative treatment strategy, especially liposarcoma and chondrosarcoma. However, further in vitro and in vivo studies are required to elucidate the underlying molecular mechanism of the efficacy of 5-ALA in CMST cells at the molecular level.

Efficacy of 5-aminolevulinic acid-based photodynamic therapy in different subtypes of canine mammary gland cancer cells.

Canine mammary gland tumors (CMGTs) are heterogeneous disease and subclassified [sarcomas (S), carcinomas (C), and carcinosarcomas (CS)] according to histopathological differentiation. Photodynamic therapy (PDT) is a promising treatment strategy based on the use of a photosensitizer (PS) activated by light. However, the therapeutic potential of PDT in the treatment of CMGTs has not been investigated, yet. Therefore, the aim of this study was to determine the in vitro protocol of 5-ALA-based-PDT for the treatment of three subtypes of CMGTs, for the first time. The intracellular PpIX florescence intensity was measured for 5-ALA (0.5 and 1 mM). After irradiation with different light doses (6, 9, 12, 18, and 24 J/cm2) for two different modes [continuous wave (CW) and pulse radiation (PR)], the cytotoxic effects of 5-ALA (0.5 and 1 mM) on the subtypes (C, S, and CS) of CMGTs were analyzed by WST-1. Finally, the optimal PDT treatment protocol was validated through Annexin V and AO/EtBr staining. Our results showed that 1 mM 5-ALA for 4-h incubation was the best treatment condition in all subtypes of CMGTs due to higher intracellular PpIX level. After irradiation with different light doses, PR mode was more effective in S primary cells at 9 J/cm2. However, a significant decrease in the viability of C and CS cells was detected at 12 /cm2 in CW mode (p < 0.05). Additionally, 1 mM 5-ALA induced apoptotic cell death in each subtype of CMGTs. Our preliminary findings suggest that (i) each subtype of CMGTs differentially responds to PDT and (ii) the light dose and mode could play an important role in the effective PDT treatment. However, further studies are needed to investigate the role of the different light sources and PDT-based apoptotic cell death in CMGTs cells.

Radiation mode and tissue thickness impact on singlet oxygen dosimetry methods for antimicrobial photodynamic therapy.

The target of the presented study is to evaluate the performances of illumination modes on Photodynamic therapy (PDT) for different tissue depths. For this purpose, radiation-based super pulse and pulse illumination modes were investigated for antimicrobial PDT (AmPDT). Singlet oxygen luminescence level was measured from two different points. The first one was to appraise the light penetration depth effect on singlet oxygen luminescence level for various radiation modes. The second one explored the singlet oxygen luminescence dosimetry (SOLD) method from deeper photosensitizer accumulated tissue levels. Two main experiments were performed in this study. The singlet oxygen concentration was calculated with singlet oxygen explicit dosimetry (SOED) and SOLD methods for various tissue depths in these experiments. According to the results of the experiments, super pulse mode (SPM) provided relatively high Staphylococcus Aureus (S. aureus) cell death by 5-12%. The penetration depth was increased between 0.2 mm and 0.7 mm during the experiments. SOLD-based singlet oxygen detection system was utilized to detect singlet oxygen production levels from various tissue thicknesses to evaluate the system's usefulness for deeper infected tissues. It was observed that SPM was more effective than pulse mode radiation after a certain tissue depth (≤ 2 mm).

Investigation of LED-based photodynamic therapy efficiency on breast cancer cells.

Photodynamic therapy (PDT) is based on special light source, photosensitizer (PS), and in the presence of oxygen. Different light sources have been used for PDT applications. Recent studies have focused on LED light sources for PDT applications due to reducing the cost of laser-based PDT and providing easy access for research laboratory or clinic facilities. LED-mediated PDT applications have shown promising results for the treatment of different types of disease. However, few studies have determined the effects of LED-based PDT on cancer cells. For the first time, the aim of this study was to explore the therapeutic effects of 5-aminolevulinic acid (5-ALA)-mediated PDT after LED irradiation on two sub-types (a poorly aggressive MCF-7 and a highly aggressive MDA-MB-231) of breast cancer cell lines. The effectiveness of 5-ALA PDT treatment was evaluated by WST-1, annexin V, and acridine orange staining with different energy levels. The LED system was specially developed with optical power and wavelength stability techniques. The system consists of user interface and embedded LED controller with real-time optic power output calibration by photodiode feedback. Our results demonstrated that the cell viability of breast cancer cells was considerably decreased a LED dose-dependent manner (P < 0.05). Additionally, a significant increase in the percentage of apoptotic cells was detected in breast cancer cells after irradiation with LED at a density of 18 and 30 J/cm2 energy. Consequently, the LED system could be effectively used for irradiation of 5-ALA in the treatment of breast cancer cells.

Optimization of 5-aminolevulinic acid-based photodynamic therapy protocol for breast cancer cells.

BACKGROUND: Photodynamic therapy (PDT) is a therapeutic strategy for the treatment of cancer. 5-aminolevulinic acid (5-ALA) as a precursor of the protoporphyrin IX (PpIX) has a great potential for PDT application. Although 5-ALA-based PDT has been studied in many pre-clinical and clinical studies for breast cancer, there are different PDT application protocols in the literature. Therefore, the aim of this study was to determine the optimal in vitro protocol for 5-ALA-based PDT in breast cancer treatment. METHODS: The therapeutic effects of 5-ALA (1 and 2.5 mM) on two different subtypes of breast cancer cell line (MCF-7 and MDA-MB-231) were evaluated by PpIX-fluorescence accumulation and WST-1 analysis. Then, the cells were irradiated with diode laser at different doses (1.5, 3, 6, 9 and 12 J/cm2). After irradiation, the anticancer effects of 5-ALA were analyzed through cell viability and cell death analysis. RESULTS: Our results showed that 5-ALA exhibited a higher PpIX fluorescence in both breast cancer cells for 4 h incubation. After irradiation, 1 mM 5-ALA significantly reduced the proliferation of breast cancer cells in a laser dose-dependent manner and induced apoptotic cell death upon 24 h incubation (p < 0.05). However, MDA-MB-231 cells were more sensitive to 5-ALA-based PDT than MCF-7 cells in a dose of 9 J/cm2 and 12 J/cm2. CONCLUSION: Our preliminary findings proposed an optimal in vitro protocol of 5-ALA-based PDT by using a laser diode for breast cancer. However, there is a need to investigate the underlying molecular mechanisms of 5-ALA/PDT sensitivity among the subtypes of breast cancer.

Comparison of pulse and super pulse radiation modes' singlet oxygen production effect in antimicrobial photodynamic therapy (AmPDT).

Photodynamic Therapy (PDT) is a treatment method in which a target region is irradiated with a light source of an appropriate wavelength to activate an introduced photosensitizer to ideally ablate the target by creation of highly toxic singlet oxygen. Due to the increment of antibiotic resistant bacteria, PDT has also become a salient method for infection treatments. The amount and the location of singlet oxygen gives information about the effectiveness of PDT. The quantitative evolution of singlet oxygen is a gold standard for the real time monitoring of the treatment efficiency during PDT. In the proposed study, the effect of radiation modes on PDT is investigated with singlet oxygen explicit dosimetry (SOED) and singlet oxygen luminescence dosimetry (SOLD) methods. For this purpose, super pulse and pulse radiation modes are applied for antimicrobial PDT (AmPDT). Five in vitro experiments were carried out to investigate the effect of radiation mode. According to the achieved results, super pulse mode provides 3-10 % more singlet oxygen concentration and 2-5 % more bacteria (Staphylococcus Aureus) death (necrosis and apoptosis) than pulse mode. Furthermore, radiation mode effect on instantaneous and cumulative singlet oxygen concentration is considered in the experiments. It is demonstrated that the singlet oxygen concertation measured by SOED and SOLD methods are coherent. Thus, the SOED method can be used for real-time singlet oxygen measurements during PDT.

A New handheld singlet oxygen detection system (SODS) and NIR light source based phantom environment for photodynamic therapy applications.

Photodynamic therapy (PDT) is an emerging treatment modality in various areas such as cancer treatment and disinfection. The photosensitizer and oxygen have crucial roles for effective PDT treatment. The quantitative evaluation of singlet oxygen, which is a gold standard for monitoring effective treatment, remains as an important problem for PDT. However, low quantum yield and low life span of the singlet oxygen make the system expensive, unnecessarily large and unadaptable for clinical usage. In our study, a new mobile singlet oxygen detection system (SODS) was designed to detect singlet oxygen illumination during PDT and a new singlet oxygen phantom environment was constituted to test the designed SODS system. The singlet oxygen phantom environment composed of fast switching led driver & microcontroller and led light source (1200-1300 nm radiation). The elements of the singlet oxygen detection system are optic filter and collimation, avalanche photodiode transimpedance amplifier, differential amplifier and a signal processing block. According to the performance evaluation of the system on the phantom environment, the presented SODS can measure the illuminations at 1270 nm wavelength between 10 ns and 15 µs timespans. The results showed that the proposed system might be a good candidate for clinical PDT applications.