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.

Evaluation of ALA-capped silver, copper, and silver-copper nanoparticles for controlling fungal plant pathogens.

Phytopathogenic fungi significantly threaten global food security, causing substantial yield and quality losses. Sustainable solutions are urgently needed to combat these agricultural pathogens. This study explored the potential of silver (Ag), copper (Cu), and combined Ag/Cu nanoparticles capped with aminolevulinic acid (ALA) as antifungal agents. The nanoparticles (ALAAg, ALACu, and ALAAgCu) were synthesized via photoreduction and characterized using various techniques (UV-Vis, TEM, XRD, Zeta potential). Their antifungal activity against four key plant pathogens (Alternaria grandis, Colletotrichum truncatum, Corynespora cassiicola, and Fusarium oxysporum) was evaluated using poisoned food techniques. Notably, ALAAgCuNPs demonstrated superior antifungal activity compared to a conventional fungicide against two fungal strains. Even at lower concentrations, ALAAgCuNPs exhibited fungistatic effects comparable to those of the control. These promising results suggest the potential of ALAAgCu NPs as a broad-spectrum, potentially eco-friendly alternative for fungal control in plants and seeds. This approach is crucial for ensuring crop health, harvest quality, and food safety.

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.

A dew-responsive pectin-based herbicide for enhanced photodynamic inactivation.

5-aminolevulinic acid (5-ALA) has been fully demonstrated as a biodegradable, without resistance, and pollution-free pesticide. However, the lack of targeting and the poor adhesion result in a low utilization rate, limiting its practical application. Herein, a dew-responsive polymer pro-pesticide Pec-hyd-ALA was successfully synthesized by grafting 5-ALA onto the pectin (PEC) backbone via acid-sensitive acylhydrazone bonds. When the pro-pesticide is exposed to acid dew on plant surfaces at night, 5-ALA is released and subsequently converted to photosensitize (Protoporphyrin IX, PpIX)in plant cells, leading to its accumulation and promoting photodynamic inactivation (PDI). An inverted fluorescence microscope has verified the accumulation of tetrapyrrole in plant cells. In addition, the highly bio-adhesive PEC backbone effectively improved the wetting and retention of 5-ALA on leaves. The pot experiment also demonstrated the system's control effect on barnyard grass. This work provides a promising approach to improving the herbicidal efficacy of 5-ALA.

Exogenous 5-Aminolevulinic acid improved low-temperature tolerance tomato seedling by regulating starch content and phenylalanine metabolism.

Tomato is an important horticultural cash crop, and low-temperature stress has seriously affected the yield and quality of tomato. 5-Aminolevulinic acid (ALA) is widely used in agriculture as an efficient and harmless growth regulator. It is currently unclear whether exogenous ALA can cope with low-temperature stress by regulating tomato starch content and phenylalanine metabolism. In this study, exogenous ALA remarkably improved the low-temperature tolerance of tomato seedlings. RNA-sequencing results showed that exogenous ALA affected starch metabolism and phenylalanine metabolism in tomato seedling leaves under low-temperature stress. Subsequently, we used histochemical staining, observation of chloroplast microstructure, substance content determination, and qRT-PCR analysis to demonstrate that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism (SlPAL, SlPOD1, and SlPOD2). Simultaneously, we found that exogenous ALA induced the expression of SlMYBs and SlWRKYs under low-temperature stress. In addition, dual luciferase, yeast one hybrid, and electrophoretic mobility shift assays indicate that SlMYB4 and SlMYB88 could regulate the expression of SlPOD2 in phenylalanine metabolism. We demonstrated that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism.

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.

Ionic Liquid Pretreatment Enhances Skin Penetration of 5-Aminolevulinic Acid: A Promising Scheme for Photodynamic Therapy for Acne Vulgaris.

Acne vulgaris is one of the most prevalent skin disorders; it affects up to 85% of adolescents and often persists into adulthood. Topical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) provides an alternative treatment for acne; however, its efficacy is greatly undermined by the limited skin permeability of ALA. Herein, biocompatible ionic liquids (ILs) based on aliphatic acid/choline were employed to enhance the dermal delivery of ALA, thereby improving the efficacy of PDT. In addition to the one-step delivery of ALA by utilizing ILs as carriers, a two-step strategy of pretreating the skin with blank ILs, followed by the administration of free ALA, was employed to test the IL-facilitated dermal delivery of ALA in vitro. The cumulative permeation of ALA through the excised rat skin after IL pretreatment was significantly greater than that in the untreated group, the 20% dimethyl sulfoxide (DMSO) penetration enhancer group, and the one-step group. The penetration efficiency was influenced by formulation and treatment factors, including the type of IL, pretreatment duration, water content in the ILs, and concentration of ALA. In rats, IL pretreatment facilitated faster, greater, and deeper ALA-induced protoporphyrin IX (PpIX) accumulation. Moreover, the IL pretreatment regimen significantly improved the efficacy of ALA-based PDT against acne vulgaris in a rat ear model. The model IL choline citrate ([Ch]3[Cit]1) had a moderate effect on the skin barrier. Trans-epidermal water loss could be recovered 1 h after IL treatment, but no irritation to the rat skin was detected after 7 days of consecutive treatment. It was concluded that biocompatible IL pretreatment enhances the penetration of ALA and thus facilitates the transformation of PpIX and improves the efficacy of PDT against acne vulgaris.

On the Possibility of Using 5-Aminolevulinic Acid in the Light-Induced Destruction of Microorganisms.

Antimicrobial photodynamic inactivation (aPDI) is a method that specifically kills target cells by combining a photosensitizer and irradiation with light at the appropriate wavelength. The natural amino acid, 5-aminolevulinic acid (5-ALA), is the precursor of endogenous porphyrins in the heme biosynthesis pathway. This review summarizes the recent progress in understanding the biosynthetic pathways and regulatory mechanisms of 5-ALA synthesis in biological hosts. The effectiveness of 5-ALA-aPDI in destroying various groups of pathogens (viruses, fungi, yeasts, parasites) was presented, but greater attention was focused on the antibacterial activity of this technique. Finally, the clinical applications of 5-ALA in therapies using 5-ALA and visible light (treatment of ulcers and disinfection of dental canals) were described.

Correlation of in vitro cell viability and cumulative singlet oxygen luminescence from protoporphyrin IX in mitochondria and plasma membrane.

SIGNIFICANCE: Photodynamic therapy (PDT) can be targeted toward different subcellular localizations, and it is proposed that different subcellular targets vary in their sensitivity to photobiological damage. Since singlet oxygen (1O2) has a very short lifetime with a limited diffusion length in cellular environments, measurement of cumulative 1O2 luminescence is the most direct approach to compare the PDT sensitivity of mitochondria and plasma membrane. APPROACH: PDT-generated near-infrared 1O2 luminescence at 1270 nm was measured together with cell viability for 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and exogenous PpIX, at different incubation times. Confocal fluorescence microscopy indicated that ALA-induced PpIX (2 h) localized in the mitochondria, whereas exogenous PpIX (1 h) mainly localized to the plasma membrane. Cell viability was determined at several time points during PDT treatments using colony-forming assays, and the surviving fraction correlated well with cumulative 1O2 luminescence counts from PpIX in mitochondria and plasmas membrane, respectively. RESULTS: The mitochondria are more sensitive than the plasma membrane by a factor of 1.7. CONCLUSIONS: Direct 1O2 luminescence dosimetry's potential value for comparing the PDT sensitivity of different subcellular organelles was demonstrated. This could be useful for developing subcellular targeted novel photosensitizers to enhance PDT efficiency.

Effect of photodynamic therapy with 5-aminolevulinic acid and EDTA-2Na against mixed infection of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

BACKGROUND: The increasing abundance of drug-resistant bacteria is a global threat. Photodynamic therapy is an entirely new, non-invasive method for treating infections caused by antibiotic-resistant strains. We previously described the bactericidal effect of photodynamic therapy on infections caused by a single type of bacterium. We showed that gram-positive and gram-negative bacteria could be killed with 5-aminolevulic acid and 410 nm light, respectively. However, clinically, mixed infections are common and difficult to treat. OBJECTIVE: We investigated the bactericidal effects of photodynamic therapy on mixed infections of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. METHODS: We compared bacterial growth with and without photodynamic therapy in vitro. Then, in vivo, we studied mixed infections in a mouse skin ulcer model. We evaluated the rates of ulcer area reduction and transitions to healing in treated and untreated mice. In addition, a comparison was made between PDT and existing topical drugs. RESULTS: We found that photodynamic therapy markedly reduced the growth of both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, in culture, and it reduced the skin ulcer areas in mice. PDT was also more effective than existing topical medicines. CONCLUSION: This study showed that photodynamic therapy had antibacterial effects against a mixed infection of gram-positive and gram-negative bacteria, and it promoted skin ulcer healing. These results suggested that photodynamic therapy could be effective in both single- and mixed-bacterial infections.

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.

Evaluation of the potential of Delta-aminolevulinic acid for simultaneous detection of bioburden and anti-microbial photodynamic therapy of MRSA infected wounds in Swiss albino mice.

BACKGROUND: The dramatic increase of drug-resistant bacteria necessitates urgent development of platforms to simultaneously detect and inactivate bacteria causing wound infections, but are confronted with various challenges. Delta amino levulinic acid (ALA) induced protoporphyrin IX (PpIX) can be a promising modality for simultaneous bioburden diagnostics and therapeutics. Herein, we report utility of ALA induced protoporphyrin (PpIX) based simultaneous bioburden detection, photoinactivation and therapeutic outcome assessment in methicillin resistant Staphylococcus aureus (MRSA) infected wounds of mice. METHODS: MRSA infected wounds treated with 10% ALA were imaged with help of a blue LED (∼405 nm) based, USB powered, hand held device integrated with a modular graphic user interface (GUI). Effect of ALA application time, bacteria load, post bacteria application time points on wound fluorescence studied. PpIX fluorescence observed after excitation with blue LEDs was used to detect bioburden, start red light mediated antimicrobial photodynamic therapy (aPDT), determine aPDT effectiveness and assess selectivity of the approach. RESULTS: ALA-PpIX fluorescence of wound bed discriminates infected from uninfected wounds and detects clinically relevant load. While wound fluorescence pattern changes as a function of ALA incubation and post infection time, intra-wound inhomogeneity in fluorescence correlates with the Gram staining data on presence of biofilms foci. Lack of red fluorescence from wound granulation tissue treated with ALA suggests selectivity of the approach. Further, significant reduction (∼50%) in red fluorescence, quantified using the GUI, relates well with bacteria load reduction observed post topical aPDT. CONCLUSION: The potential of ALA induced PpIX for simultaneous detection of bioburden, photodynamic inactivation and "florescence-guided aPDT assessment" is demonstrated in MRSA infected wounds of mice.

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.

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.

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.

ALA-PDT promotes IL-1ß secretion from human SZ95 sebocytes via activation of the NLRP3 inflammasome.

BACKGROUND: 5-Aminolevulinic acid photodynamic therapy (ALA-PDT) is an effective treatment for pilosebaceous inflammatory diseases, such as acne vulgaris. In this study, we explored ALA-PDT's mechanisms against acne in vitro. METHODS: We treated human SZ95 sebocytes with ALA (0.2 mM) and subjected them to varied PDT doses (0, 5, 10, 20 J/cm²) over 12 h. We assessed cell viability post-treatment using the Annexin V FITC/PI apoptosis kit. ROS accumulation in the sebocytes was detected with a DCFDA probe. We quantified NLRP3 and caspase-1 mRNA via quantitative PCR and determined IL-1ß release following ALA-PDT by ELISA. Western blotting helped identify the levels of proteins associated with pyroptosis (NLRP3, caspase-1, and IL-1ß). To elucidate the mechanisms, we re-evaluated these parameters after administering various concentrations of NAC antioxidants (0, 0.4, 2, 10 mM) and the caspase inhibitor Z-VAD-FMK (0, 5, 10, 20 µM). RESULTS: Increasing PDT dose inversely affected SZ95 sebocyte survival, with a corresponding rise in ROS and pyroptosis-related proteins (NLRP3, caspase-1, and IL-1ß). Furthermore, NAC and Z-VAD-FMK modulated the expression and secretion of these molecules in a dose-responsive manner. CONCLUSION: Our findings suggest ALA-PDT's potential mechanism of action on sebaceous glands could involve ROS induction, leading to NLRP3 inflammasome assembly, thereby heightening caspase-1 activation and IL-1ß secretion. This cascade may amplify the local inflammatory response to break chronic inflammation in acne vulgaris treatment.

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.

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.