Targeted photodynamic therapy technique of Janus nanoparticles on breast cancer.

Spherical gold/polyacrylic acid (Au/PAA) polymer-inorganic Janus nanoparticles (JNPs) with simultaneous therapeutic and targeting functions were fabricated. The obtained Au/PAA JNPs were further selectively functionalized with folic acid (FA) and thiol PEG amine (SH-PEG-NH2) on Au sides to provide superior biocompatibility and active targeting, while the other PAA sides were loaded with 5-aminolevulinic acid (5-ALA) to serve as a photosensitizer (PS) for photodynamic therapeutic (PDT) effects on MCF-7 cancer cells. The PS loading of 5-ALA was found to be 83% with an average hydrodynamic size and z-potential of 146 ± 0.8 nm and -6.40 mV respectively for FA-Au/PAA-ALA JNPs. The in vitro PDT study of the JNPs on MCF-7 breast cancer cells under 636 nm laser irradiation indicated the cell viability of 24.7% ± 0.5 for FA-Au/PAA-ALA JNPs at the IC50 value of 0.125 mM. In this regard, the actively targeted FA-Au/PAA-ALA JNPs treatment holds great potential for tumour therapy with high cancer cell-killing efficacy.

A Novel Photosensitizer Based 450-nm Blue Laser-Mediated Photodynamic Therapy Induces Apoptosis in Colorectal Cancer - in Vitro and in Vivo Study.

BACKGROUND: Due to its non-invasive and widely applicable features, photodynamic therapy (PDT) has been a prominent treatment approach against cancer in recent years. However, its widespread application in clinical practice is limited by the dark toxicity of photosensitizers and insufficient penetration of light sources. This study assessed the anticancer effects of a novel photosensitizer 5-(4-amino-phenyl)-10,15,20-triphenylporphyrin with diethylene-triaminopentaacetic acid (ATPP-DTPA)-mediated PDT (hereinafter referred to as ATPP-PDT) under the irradiation of a 450-nm blue laser on colorectal cancer (CRC) in vivo and in vitro. METHODS: After 450-nm blue laser-mediated ATPP-PDT and the traditional photosensitizer 5-aminolevulinic acid (5-ALA)-PDT treatment, cell viability was detected through Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays. Reactive oxygen species (ROS) generation was quantified by flow cytometry and fluorescence microscopy. Western blotting and transcriptome RNA sequencing and functional experiments were used to evaluate cell apoptosis and its potential mechanism. Anti-tumor experiment in vivo was performed in nude mice with subcutaneous tumors. RESULTS: ATPP-DTPA had a marvelous absorption in the blue spectrum. Compared with 5-ALA, ATPP-DTPA could achieve significant killing effects at a lower dose. Owing to generating an excessive amount of ROS, 450-nm blue laser-mediated PDT based on ATPP-DTPA resulted in evident growth inhibition and apoptosis in CRC cells in vitro. After transcriptome RNA sequencing and functional experiments, p38 MAPK signaling pathway was confirmed to be involved in the regulation of apoptosis induced by 450-nm blue laser-mediated ATPP-PDT. Additionally, animal studies using xenograft model confirmed that ATPP-PDT had excellent anti-tumor effect and reasonable biosafety in vivo. CONCLUSIONS: PDT mediated by 450-nm blue laser combined with ATPP-DTPA may be a novel and effective method for the treatment of CRC.

Differences in the response of normal oral mucosa, oral leukoplakia, oral squamous cell carcinoma-derived mesenchymal stem cells, and epithelial cells to photodynamic therapy.

OBJECTIVE: The objective of this study is to investigate the variances in transcriptome gene expression of normal oral mucosa-derived mesenchymal stem cell (OM-MSC), oral leukoplakia-derived MSC (OLK-MSC) and oral squamous cell carcinoma-derived MSC(OSCC-MSC). as Additionally, the study aims to compare the in vitro proliferation, migration, invasion ability, and response to photodynamic therapy (PDT) of these three MSC, HOK, DOK, leuk1, and Cal27 cell lines. METHODS: HOK, DOK, leuk1, Cal27 cells were cultured in vitro. 3 MSC cells were obtained from OM, OLK, OSCC tissue (n = 3) and identified through flow cytometry. They were also cultured in vitro for osteogenic and lipogenic-induced differentiation. Based on the Illumina HiSeq high-throughput sequencing platform, OM-MSC, OLK-MSC, OSCC-MSC (n = 3) were subjected to transcriptome sequencing, functional annotation, and enrichment analysis of differentially expressed genes and related genes. CCK8 assay, wound healing assay, and transwell assay were performed to compare the proliferation, migration, and invasion of the seven types of cells. The 7 cells were incubated with 0, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM of the photosensitizer (5-aminolevulinic acid, 5-ALA) in vitro. Subsequently, they were irradiated with a 150 mM, 635 nm laser for 1 min, and the cell activity was detected using the CCK8 assay after 24 h. The mitochondrial changes in the 7 cells before and after the treatment of PDT were detected using the JC-10 probe, and the changes in ATP content were measured before and after the PDT treatment. RESULTS: OM-MSC, OLK-MSC, and OSCC-MSC expressed positive MSC surface markers. After osteogenic and lipogenic-induced differentiation culture, stained calcium nodules and lipid droplets were visible, meeting the identification criteria of MSC. Pathway enrichment analysis revealed that the differentially expressed genes (DEGs) of OSCC-MSC compared to OLK-MSC were primarily associated with the PI3K-Akt signaling pathway and tumor-related pathways. OSCC-MSC exhibited stronger migratory and invasive abilities compared to Cal27. The IC50 values required for OM, OLK, and OSCC-derived MSC were lower than those required for epithelial cells treated with PDT, which were 1.396 mM, 0.9063 mM, and 2.924 mM, respectively. Cell membrane and mitochondrial disruption were observed in seven types of cells after 24 h of PDT treatment. However, HOK, DOK, leuk1, and Cal27 cells had an ATP content increased. CONCLUSIONS: OLK, OSCC epithelial cells require higher concentrations of 5-ALA for PDT treatment than MSC of the same tissue origin. The concentration of 5-ALA required increases with increasing cell malignancy. Differences in the response of epithelial cells and MSC to PDT treatment may have varying impacts on OLK recurrence and malignancy.

Photodynamic anticancer activity evaluation of novel 5-aminolevulinic acid and 3-hydroxypyridinone conjugates.

5-Aminolevulinic acid (ALA) and its derivatives, serving as the endogenous precursor of the photosensitizer (PS) protoporphyrin IX (PpIX), successfully applied in tumor imaging and photodynamic therapy (PDT). ALA and its derivatives have been used to treat actinic keratosis (AK), basal cell carcinoma (BCC), and improve the detection of superficial bladder cancer. However, the high hydrophilicity of ALA and the conversion of PpIX to heme have limited the accumulation of PpIX, hindering the efficiency and potential application of ALA-PDT. This study aims to evaluate the PDT activity of three rationally designed series of ALA-HPO prodrugs, which were based on enhancing the lipophilicity of the prodrugs and reducing the labile iron pool (LIP) through HPO iron chelators to promote PpIX accumulation. Twenty-four ALA-HPO conjugates, incorporating amide, amino acid, and ester linkages, were synthesized. Most of the conjugates, exhibited no dark-toxicity to cells, according to bioactivity evaluation. Ester conjugates 19a-g showed promoted phototoxicity when tested on tumor cell lines, and this increased phototoxicity was strongly correlated with elevated PpIX levels. Among them, conjugate 19c emerged as the most promising (HeLa, IC50 = 24.25 ± 1.43 µM; MCF-7, IC50 = 43.30 ± 1.76 µM; A375, IC50 = 28.03 ± 1.00 µM), displaying superior photodynamic anticancer activity to ALA (IC50 > 100 µM). At a concentration of 80 µM, the fluorescence intensity of PpIX induced by compound 19c in HeLa, MCF-7, and A375 cells was 18.9, 5.3, and 2.8 times higher, respectively, than that induced by ALA. In conclusion, cellular phototoxicity showed a strong correlation with intracellular PpIX fluorescence levels, indicating the potential application of ALA-HPO conjugates in ALA-PDT.

Comparative response to PDT with methyl-aminolevulinate and temoporfin in cutaneous and oral squamous cell carcinoma cells.

Cutaneous and Head and Neck squamous cell carcinoma (CSCC, HNSCC) are among the most prevalent cancers. Both types of cancer can be treated with photodynamic therapy (PDT) by using the photosensitizer Temoporfin in HNSCC and the prodrug methyl-aminolevulinate (MAL) in CSCC. However, PDT is not always effective. Therefore, it is mandatory to correctly approach the therapy according to the characteristics of the tumour cells. For this reason, we have used cell lines of CSCC (A431 and SCC13) and HNSCC (HN5 and SCC9). The results obtained indicated that the better response to MAL-PDT was related to its localization in the plasma membrane (A431 and HN5 cells). However, with Temoporfin all cell lines showed lysosome localization, even the most sensitive ones (HN5). The expression of mesenchymal markers and migratory capacity was greater in HNSCC lines compared to CSCC, but no correlation with PDT response was observed. The translocation to the nucleus of ß-catenin and GSK3ß and the activation of NF-κß is related to the poor response to PDT in the HNSCC lines. Therefore, we propose that intracellular localization of GSK3ß could be a good marker of response to PDT in HNSCC. Although the molecular mechanism of response to PDT needs further elucidation, this work shows that the most MAL-resistant line of CSCC is more sensitive to Temoporfin.

Globus Lucidus: A porcine study of an intracranial implant designed to deliver closed, repetitive photodynamic and photochemical therapy in glioblastoma.

OBJECTIVE: Herein we describe initial results in a porcine model of a fully implantable device designed to allow closed, repetitive photodynamic treatment of glioblastoma (GBM). METHODS: This implant, Globus Lucidus, is a transparent quartz glass sphere with light-emitting diodes releasing wavelengths of 630 nm (19.5 mW/cm2), 405 nm (5.0 mW/cm2) or 275 nm (0.9 mW/cm2). 5-aminolevulinic acid was the photosensitizing prodrug chosen for use with Globus Lucidus, hence the implants illuminated at 630 nm or 405 nm. An additional 275 nm wavelength-emittance was included to explore the effects of photochemical therapy (PCT) by ultraviolet (UV) light. Twenty healthy domestic pigs underwent right-frontal craniotomies. The Globus Lucidus device was inserted into a surgically created right-frontal lobe cavity. After postoperative recovery, irradiation for up to 30 min daily for up to 14 d, or continuous irradiation for up to 14.6 h was conducted. RESULTS: Surgery, implants, and repeated irradiations using the different wavelengths were generally well tolerated. Social behavior, wound healing, body weight, and temperature remained unaffected. Histopathological analyses revealed consistent leukocyte infiltration around the intracerebral implant sites with no significant differences between experimental and control groups. CONCLUSION: This Globus Lucidus porcine study prepares the groundwork for adjuvant, long-term, repeated PDT of the GBM infiltration zone. This is the first report of a fully implantable PDT/PCT device for the potential treatment of GBM. A preclinical effectivity study of Globus Lucidus PDT/PCT is warranted and in advanced stages of planning.

Protective autophagy enhances antistress ability through AMPK/ULK1 signaling pathway in human immortalized keratinocytes.

Keratinocytes, located in the outermost layer of human skin, are pivotal cells to resist environmental damage. Cellular autophagy plays a critical role in eliminating damaged organelles and maintaining skin cell homeostasis. Low-dose 5-Aminolevulinic acid photodynamic therapy (ALA-PDT) has been demonstrated to enhance skin's antistress ability; however, the regulatory mechanisms of autophagy in keratinocytes remain unclear. In this study, we treated immortalized human keratinocytes (HaCaT cells) with low-dose ALA-PDT (0.5 mmol/L, 3 J/cm2). Through RNA-sequencing analysis, we identified that low-dose ALA-PDT modulated autophagy-related pathways in keratinocytes and pinpointed Unc-51-like kinase 1 (ULK1) as a key gene involved. Western blot results revealed that low-dose ALA-PDT treatment upregulated the expression of autophagy-related proteins Beclin-1 and LC3-II/LC3-I ratio. Notably, low-dose ALA-PDT regulated autophagy by inducing an appropriate level of reactive oxygen species (ROS), transiently reducing mitochondrial membrane potential, and decreasing adenosine triphosphate production; all these processes functioned on the AMP-activated protein kinase (AMPK)/ULK1 pathway to activate autophagy. Finally, we simulated external environmental damage using ultraviolet B (UVB) at a dose of 60 mJ/cm2 and observed that low-dose ALA-PDT mitigated UVB-induced cell apoptosis; however, this protective effect was reversed when using the autophagy inhibitor 3-methyladenine. Overall, these findings highlight how low-dose ALA-PDT enhances antistress ability in HaCaT cells through controlling ROS generation and activating the AMPK/ULK1 pathway to arouse cellular autophagy.

Potential therapeutic efficacy of photodynamic therapy on female hormonal-dependent cancers in a hormonal simulated microenvironment.

BACKGROUND: Photodynamic Therapy (PDT) is a clinically approved cancer treatment. Sex hormones, the key drivers for the development of female hormonal dependent cancers, might affect cancer treatment. There are seldom studies to evaluate the effect of sex hormones mimicked the menstrual cycle on the PDT mediated by prodrug 5-aminolevulinic acid (ALA) and its ester derivatives to the hormonal dependent cancers. AIMS: To evaluate the efficacy of sex hormones on Hexyl-ALA-PDT in hormonal dependent cancers and the effect of the sex hormones on heme biosynthetic pathway. METHODS: Cell culture system that mimicked the fluctuation of sex hormones 17ß-estradiol (E2) and progesterone (PG) in the menstrual cycle was developed. Two pairs of hormonal-independent and hormonal dependent uterine sarcoma and breast cancer cell lines were used as cell models. Hexyl-ALA induced PpIX production and intracellular localization were examined. Key enzymes for PpIX synthesis were analysed. Hexyl-ALA-PDT mediated phototoxicity was evaluated. RESULTS: The PpIX generation was increased in the hormonal-dependent cells (28-50 %) when cultured in the hormonal microenvironment with long incubation of Hexyl-ALA for 15 and 24 h compared to that cultured without hormones; whereas only slight difference in PpIX generation in their hormonal-independent counterpart. The PpIX generation was in a time-dependent manner. The CPOX, PPOX and FECH expressions were significantly enhanced by Hexyl-ALA-PDT in uterine sarcoma cells in hormonal microenvironment. Hexyl-ALA-PDT triggered significant increase of PPOX expression in breast cancer cells in hormonal microenvironment. The Hexyl-ALA-PDT phototoxicity was enhanced by 18-40 % in cells cultured in the hormonal system in a dose-dependent manner. CONCLUSION: The PpIX generation and the efficacy of Hexyl-ALA-PDT in both uterine sarcoma and breast cancer cells was significantly enhanced by the sex hormones via cultured in the hormonal microenvironment.

The Effect of Photodynamic Therapy Using 5-Aminolevulinic Acid in Bone and Soft Tissue Sarcoma Cells.

BACKGROUND/AIM: 5-Aminolevulinic acid (5-ALA) is a natural amino acid and a precursor of protoporphyrin IX (PpIX). Following light irradiation, the PpIX generates reactive oxygen species (ROS) in the presence of oxygen. Increased ROS levels can cause apoptotic cell death and necrosis of targeted cancer cells. This study examined whether photodynamic therapy using 5ALA (5-ALA PDT) could be used as a potential adjuvant therapy for bone and soft tissue sarcomas. MATERIALS AND METHODS: The human osteosarcoma (143B), mouse osteosarcoma (LM8), human fibrosarcoma cell (HT1080) cell lines were used. In vitro, cultured cells were exposed to 5-ALA at various concentrations followed by strobe scope light irradiation for 10 min as 5-ALA PDT. Cell viability was then measured. In vivo, each tumor cell line was inoculated subcutaneously into the backs of mice. In the 5-ALA PDT group, 5-ALA (250 mg/kg) was administered intraperitoneally followed by light irradiation. Change in tumor volume by 5-ALA PDT were primarily evaluated. RESULTS: In vitro, treatment of sarcoma cells with 100 and 200 µg/ml 5-ALA PDT significantly inhibited cell proliferation at 24 and 48 h compared with the group treated with 0 and 10 µg/ml 5-ALA PDT. In vivo, in all cell lines, a significant inhibition of the tumor volume was observed in the 5-ALA-PDT group as compared to that in control, strobe scope light, and 5-ALA groups. CONCLUSION: 5-ALA PDT effectively inhibited proliferation of bone and soft tissue sarcoma cell lines. Further in vivo research using other subtypes of bone and soft tissue sarcoma is warranted to confirm the applicability in the clinical setting.

Modifications of DAMPs levels in extracellular environment induced by aminolevulinic acid-based photodynamic therapy of esophageal cancer cells.

PURPOSE: Immunogenic cell death plays an important role in anticancer treatment because it combines cell death with appearance of damage associated molecular patterns that have the potential to activate anticancer immunity. Effects of damage associated molecular patterns induced by aminolevulinic acid-based photodynamic therapy were studied mainly on dendritic cells. They have not been deeply studied on macrophages that constitute the essential component of the tumor microenvironment. The aim of this study was to analyze features of esophageal cancer cell death in relation to release capacity of damage associated molecular pattern species, and to test the effect of related extracellular environmental alterations on macrophages. MATERIAL AND METHODS: Esophageal Kyse 450 carcinoma cells were subjected to aminolevulinic acid-based photodynamic therapy at different concentrations of aminolevulinic acid. Resting, IFN/LPS and IL-4 macrophage subtypes were prepared from monocytic THP-1 cell line. Cell death features and macrophage modifications were analyzed by fluorescence-based live cell imaging. ATP and HMGB1 levels in cell culture media were determined by ELISA assays. The presence of lipid peroxidation products in culture media was assessed by spectrophotometric detection of thiobarbituric acid reactive substances. RESULTS: Aminolevulinic acid-based photodynamic therapy induced various death pathways in Kyse 450 cells that included features of apoptosis, necrosis and ferroptosis. ATP amounts in extracellular environment of treated Kyse 450 cells increased with increasing aminolevulinic acid concentration. Levels of HMGB1, detectable by ELISA assay in culture media, were decreased after the treatment. Aminolevulinic acid-based photodynamic therapy induced lipid peroxidation of cellular structures and increased levels of extracellular lipid peroxidation products. Incubation of resting and IL-4 macrophages in conditioned medium from Kyse 450 cells treated by aminolevulinic acid-based photodynamic therapy induced morphological changes in macrophages, however, comparable alterations were induced also by conditioned medium from untreated cancer cells. CONCLUSION: Aminolevulinic acid-based photodynamic therapy leads to alterations in local extracellular levels of damage associated molecular patterns, however, comprehensive studies are needed to find whether they can be responsible for macrophage phenotype modifications.

Blue light aminolevulinic acid photodynamic therapy downregulates cell division and proliferation pathways in cutaneous squamous cell carcinoma.

5-Aminolevulinic acid (5-ALA) photodynamic therapy (PDT) is a treatment for actinic keratosis (AK) and has been studied as a treatment for noninvasive cutaneous squamous cell carcinoma (cSCC). PDT induces apoptosis and necrosis in AKs and cSCC. 5-ALA blue light PDT may modulate gene expression and pathways in surviving cells. In this study, differential gene expression and pathway analysis of cSCC and human dermal fibroblasts were compared before and after 5-ALA blue light PDT using RNA sequencing. No genes were differentially expressed after correcting for multiple testing (false discovery rate < 0.05). As a result, transcription factor, gene enrichment, and pathway analysis were performed with genes identified before multiple testing (p < 0.05). Pathways associated with proliferation and carcinogenesis were downregulated. These findings using 5-ALA blue light PDT are similar to previously published studies using methyl-aminolevulinic and red light protocols, indicating that surviving residual cells may undergo changes consistent with a less aggressive cancerous phenotype.

Enhancement of the effect of novel targeted 5-aminolevulinic acid conjugated bismuth oxide nanoparticles-based photodynamic therapy by simultaneous radiotherapy on KB cells.

BACKGROUND: Selective accumulation of photosensitizers into cancerous cells is one of the most important factors affecting photodynamic therapy (PDT) efficacy. 5-aminolevulinic acid (5-ALA) is the precursor of a strong photosensitizer, protoporphyrin-IX; but it has poor permeability into the cells. Folate receptors are overexpressed on the surface of many tumor cells. In the present study, folic acid (FA) and 5-ALA conjugated bismuth oxide nanoparticles were synthesized; and used in PDT, radiotherapy (RT), and concurrent PDT & RT against nasopharyngeal carcinoma (KB cell line). METHODS: The KB cells were incubated with the synthesized nanoparticles (NPs) for 2 h; then illuminated using a custom-made LED lamp at the light dose of 26 J/cm2. Irradiation of the cells was carried out using X-ray 6 MV (2 Gy); and synergistic effect of the simultaneous RT and PDT treatments was evaluated using fractional product values. Efficacy of the treatments was determined using MTT and Caspase-3 enzyme activity assays. RESULTS: Targeting of folic acid receptors enables the selective endocytosis of the conjugated NPs. RT results in the presence of Bi2O3 NPs showed a significant radiosensitizer potential of these NPs. Fractional product values of 1.49±0.05, 1.36±0.06, and 1.05±0.06 obtained in the presence of FA-5-ALA conjugated NPs, 5-ALA conjugated NPs, and in the absence of the NPs, respectively. Therefore, simultaneous RT and PDT in the presence of these conjugated NPs is superior to RT in the presence of the NPs. CONCLUSION: Simultaneous PDT and RT in the presence of FA-5-ALA conjugated bismuth oxide NPs can be introduced as a promising therapeutic approach in controlling KB cancer cells.

Skin wound healing in diabetic rat model using low-dose photodynamic therapy.

Chronic wound is one of the major challenges in medicine and imposes a heavy financial burden on the healthcare of different countries. Diabetic foot ulcers as one of the important examples for chronic wounds can lead to lower limb amputation, disability, and death in diabetics. In this regard, novel technology with low side effects got attention in recent years. Low-dose photodynamic therapy (LDPDT) is one of the noninvasive techniques that can be considered for wound healing in diabetic wounds. In this experiment, we aim to study the effect of LDPDT on diabetic rats' wound healing and compare it to healthy rats. In this in vitro experimental study, 32 male rats were used. Rats in both normal and diabetic (streptozotocin injection) groups after being wounded (two wounds [0.8 × 0.8 cm]) on the back of each rat were randomly divided into four groups, including the control group (without treatment), radiation-only (660 nm-1 J/cm2) group, 5-ALA-only (1 µg/mL) group, and LDPDT-recipient group. The procedure has been done for 2 days, and at the end of Days 3, 7, 14, and 21, the wound sample was sent to the histopathology laboratory, and the wound size and tissue indices in these groups were evaluated by histology and microscopy techniques. The impact of low concentrations of 5-ALA and low irradiation energy density in both normal and diabetic rats were positive, which accelerated the wound-healing process as seen in the histology study. In diabetic rats treated with only radiation and LDPDT, the process of epithelial regeneration, collagen production, reduction of mast cells, and production of follicles was more as compared to the normal group. The results suggest that LDPDT can have a positive impact on the diabetic rat model wound healing.

In vitro evaluation of the efficacy of photodynamic therapy using 5-ALA on homologous feline mammary tumors in 2D and 3D culture conditions and a mouse subcutaneous model with 3D cultured cells.

BACKGROUND: Numerous studies have shown that photodynamic therapy (PDT) has a therapeutic effect on mammary tumor cells, with 5-aminolevulinic acid (5-ALA-HCL) being a commonly used photosensitizer for PDT. Feline mammary tumors (FMTs) are relatively common. However, the cytotoxic and antitumor effects of 5-ALA-PDT on FMTs have not been clarified. To this end, we evaluated the therapeutic effect of 5-ALA-PDT on FMTs through in vitro experiments using an FMT FKR cell line established for this study. METHODS: We performed 5-ALA-PDT in 2D-cultured FKR-A (adherent cells) and 3D-cultured FKR-S (spheroid cells) cells and performed a series of studies to evaluate the cell viability and determine the protoporphyrin IX (PpIX) content in the cells as well as the expression levels of mRNAs associated with PpIX production and release. An in vivo study was performed to assess the effectiveness of 5-ALA-PDT. RESULTS: There was a significant difference in the concentration of PpIX in FMT cells under different incubation culture modes (2D versus 3D culture). The concentration of PpIX in FMT cells was correlated with the differences in cell culture (2D and 3D) as well as the expression levels of genes such as PEPT1, PEPT2, FECH, and HO-1. CONCLUSIONS: In the in vitro study, 5-ALA-PDT had a stronger inhibitory effect on 3D-cultured FKR-S cells, which resemble the internal environment of organisms more closely. We also observed a significant inhibitory effect of 5-ALA-PDT on FMT cells in vivo. To our knowledge, this is the first study on 5-ALA-PDT for FMTs under both 2D and 3D conditions.

Combination of vitamin D and photodynamic therapy enhances immune responses in murine models of squamous cell skin cancer.

Improved treatment outcomes for non-melanoma skin cancers can be achieved if Vitamin D (Vit D) is used as a neoadjuvant prior to photodynamic therapy (PDT). However, the mechanisms for this effect are unclear. Vit D elevates protoporphyrin (PpIX) levels within tumor cells, but also exerts immune-modulatory effects. Here, two murine models, UVB-induced actinic keratoses (AK) and human squamous cell carcinoma (A431) xenografts, were used to analyze the time course of local and systemic immune responses after PDT ± Vit D. Fluorescence immunohistochemistry of tissues and flow analysis (FACS) of blood were employed. In tissue, damage-associated molecular patterns (DAMPs) were increased, and infiltration of neutrophils (Ly6G+), macrophages (F4/80+), and dendritic cells (CD11c+) were observed. In most cases, Vit D alone or PDT alone increased cell recruitment, but Vit D + PDT showed even greater recruitment effects. Similarly for T cells, increased infiltration of total (CD3+), cytotoxic (CD8+) and regulatory (FoxP3+) T-cells was observed after Vit D or PDT, but the increase was even greater with the combination. FACS analysis revealed a variety of interesting changes in circulating immune cell levels. In particular, neutrophils decreased in the blood after Vit D, consistent with migration of neutrophils into AK lesions. Levels of cells expressing the PD-1+ checkpoint receptor were reduced in AKs following Vit D, potentially counteracting PD-1+ elevations seen after PDT alone. In summary, Vit D and ALA-PDT, two treatments with individual immunogenic effects, may be advantageous in combination to improve treatment efficacy and management of AK in the dermatology clinic.

5-Aminolevulinic acid treatment mitigates pesticide stress in bean seedlings by regulating stress-related gene expression and retrotransposon movements.

Overdoses of pesticides lead to a decrease in the yield and quality of plants, such as beans. The unconscious use of deltamethrin, one of the synthetic insecticides, increases the amount of reactive oxygen species (ROS) by causing oxidative stress in plants. In this case, plants tolerate stress by activating the antioxidant defense mechanism and many genes. 5-Aminolevulinic acid (ALA) improves tolerance to stress by acting exogenously in low doses. There are many gene families that are effective in the regulation of this mechanism. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. In this study, the expression levels of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and stress-associated protein (SAP) genes were determined by Q-PCR in deltamethrin (0.5 ppm) and various doses (20, 40, and 80 mg/l) of ALA-treated bean seedlings. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. It was determined that deltamethrin increased the expression of SOD (1.8-fold), GPX (1.4-fold), CAT (2.7-fold), and SAP (2.5-fold) genes, while 20 and 40 mg/l ALA gradually increased the expression of these genes at levels close to control, but 80 mg/l ALA increased the expression of these genes almost to the same level as deltamethrin (2.1-fold, 1.4-fold, 2.6-fold, and 2.6-fold in SOD, GPX, CAT, and SAP genes, respectively). In addition, retrotransposon-microsatellite amplified polymorphism (REMAP) was performed to determine the polymorphism caused by retrotransposon movements. While deltamethrin treatment has caused a decrease in genomic template stability (GTS) (27%), ALA treatments have prevented this decline. At doses of 20, 40, and 80 mg/L of ALA treatments, the GTS ratios were determined to be 96.8%, 74.6%, and 58.7%, respectively. Collectively, these findings demonstrated that ALA has the utility of alleviating pesticide stress effects on beans.

Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn-porphyrins.

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn-porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.

Modified 5-aminolevulinic acid photodynamic therapy induces cutaneous squamous cell carcinoma cell pyroptosis via the JNK signaling pathway.

Modified 5-aminolevulinic acid photodynamic therapy (M-PDT) is a novel therapeutic modality for cutaneous squamous cell carcinoma (cSCC) that is reported to be effective and well tolerated. However, the mechanisms underlying its antitumor effects are not fully understood. In this research, we investigated the effects of M-PDT on pyroptosis, a form of programmed cell death characterized by cell swelling, ruptures of cell membrane, and inflammatory cytokine release, in two human cSCC cell lines, SCL-1 and HSC-5. We found that M-PDT triggered pyroptosis in a dose-dependent manner, as evidenced by increased lactate dehydrogenase release, propidium iodide staining, and expression of pyroptosis-related proteins, such as NLR family pyrin domain containing 3 (NLRP3), N-terminal of gasdermin D (N-GSDMD), cleaved caspase-1, and mature interleukin 1 beta (IL-1B) in both cell lines. This process was inhibited by treatment with MCC950, an NLRP3-specific inhibitor, suggesting the involvement of the NLRP3 inflammasome in M-PDT-induced pyroptosis. We also demonstrated that M-PDT activated c-Jun N-terminal kinase (JNK) signaling, which is required for pyroptosis induction, as treatment with SP600125, a JNK inhibitor, suppressed the expression of pyroptosis-related proteins after M-PDT. JNK activation enhanced M-PDT-induced pyroptosis, highlighting the significance of the JNK pathway in M-PDT. Moreover, M-PDT increased intracellular reactive oxygen species (ROS) levels, which are responsible for JNK activation and pyroptosis induction. In summary, our results revealed that M-PDT triggers pyroptosis through ROS-mediated JNK activation and subsequent NLRP3 inflammasome activation in cSCC cells, providing a better understanding of the molecular mechanism of M-PDT and promoting its clinical application.

The effects of 5-aminolevulinic acid photodynamic therapy on the local immune response of women with cervical intraepithelial neoplasia grade 2.

Objective: To evaluate and elucidate the effects and mechanism of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on the local immune response of women with cervical intraepithelial neoplasia grade 2 (CIN2). Materials and methods: Immunofluorescence staining was used to compare immune cells infiltration before and after ALA-PDT in 23 patients with CIN2. The infiltration of immune cells into the cervical tissues of patients with different outcomes was also compared at the 6-month follow-up period. Immune cell counts in samples collected before and after treatment were compared. Results: We found an increased number of CD8+ T cell infiltration, an increased proportion of CD8+ T cells expressing Granzyme B (GrB), Chemokine receptor 3 (CXCR3), and CD8+ tissue-resident memory T (TRM) cells, and a decreased proportion of CD8+ T cells expressing PD-1 in patients with CIN2 compared to that before ALA-PDT. Moreover, at the 6-month follow-up, there was higher infiltration of CD8+ T and CD8+ TRM cells, higher expression of GrB and CXCR3, and lower expression of PD-1 on CD8+ T cells in the HPV clearance and CIN2 disappearance groups than in the HPV-positive and CIN2 regression groups. However, no significant difference was observed in the number of CD8+ TSCM following ALA-PDT. Conclusion: ALA-PDT could activate CD8+ T cell responses by modulating the expression of CXCR3 and PD-1 in CD8+ T cells and increasing the infiltration of CD8+ TRM cells. And the infiltration of CD8+ T cells is correlated with the prognosis of CIN2.

Development and optimisation of in vitro sonodynamic therapy for glioblastoma.

Sonodynamic therapy (SDT) is currently on critical path for glioblastoma therapeutics. SDT is a non-invasive approach utilising focused ultrasound to activate photosensitisers like 5-ALA to impede tumour growth. Unfortunately, the molecular mechanisms underlying the therapeutic functions of SDT remain enigmatic. This is primarily due to the lack of intricately optimised instrumentation capable of modulating SDT delivery to glioma cells in vitro. Consequently, very little information is available on the effects of SDT on glioma stem cells which are key drivers of gliomagenesis and recurrence. To address this, the current study has developed and validated an automated in vitro SDT system to allow the application and mapping of focused ultrasound fields under varied exposure conditions and setup configurations. The study optimizes ultrasound frequency, intensity, plate base material, thermal effect, and the integration of live cells. Indeed, in the presence of 5-ALA, focused ultrasound induces apoptotic cell death in primary patient-derived glioma cells with concurrent upregulation of intracellular reactive oxygen species. Intriguingly, primary glioma stem neurospheres also exhibit remarkably reduced 3D growth upon SDT exposure. Taken together, the study reports an in vitro system for SDT applications on tissue culture-based disease models to potentially benchmark the novel approach to the current standard-of-care.