The Effect of NF-κB Deactivation on Cancer Cell Response to ALA Mediated Photodynamic Therapy.

OBJECTIVE: Breast cancer is one of the most widespread tumors among women worldwide, which is difficult to treat due to the presence of chemoresistance and the risk of tumor recurrence and metastasis. There is a pressing necessity to develop efficient treatments to improve response for treatment and increase prolong survival of breast cancer patients. Photodynamic therapy (PDT) has attracted interest for its features as a noninvasive and relatively selective cancer treatment. This method relies on light-activated photosensitizers that, upon absorbing light, generate reactive oxygen species (ROS) with powerful cell-killing outcomes. Nuclear factor kappa B (NF-κB), a transcription factor, plays a key role in cancer development by regulating cell proliferation, differentiation, and survival. Inhibiting NF-κB can sensitize tumor cells to chemotherapeutic agents. Dimethyl fumarate (DMF), an NF-κB inhibitor approved by the FDA for multiple sclerosis treatment, has further shown promise in suppressing breast cancer cell growth in vitro. We hypothesized that combining PDT with Dimethyl fumarate (DMF) could further enhance therapeutic efficacy for both treatment modalities. METHODS: In the current study, we explored the PDT effect of 1 and 2 mM aminolaevulinic acid (ALA) and low-power He-Ne laser irradiation combined with different concentrations of DMF (2.5, 1.25, or 0.652 µg/ml) against hormone nonresponsive AMJ13 breast cancer cell line that is derived from Iraqi patient. RESULTS: Our results demonstrated that co-administration with all tested DMF concentrations significantly enhanced the cytotoxicity of PDT antitumor effect. The combination index analysis showed presence of synergism in combining PDT with DMF. CONCLUSION: This finding suggests that the combination of PDT with DMF could be a promising novel strategy against triple negative breast cancer that could be applied clinically due to the fact that both of these treatments are already clinically approved therapies.

Enhancing 5-ALA-PDT efficacy against resistant tumor cells: Strategies and advances.

As a precursor of protoporphyrin IX (PpIX), an endogenous pro-apoptotic and fluorescent molecule, 5-Aminolevulinic acid (5-ALA) has gained substantial attention for its potential in fluorescence-guided surgery as well as photodynamic therapy (PDT). Moreover, 5-ALA-PDT has been suggested as a promising chemo-radio sensitization therapy for various cancers. However, insufficient 5-ALA-induced PpIX fluorescence and the induction of multiple resistance mechanisms may hinder the 5-ALA-PDT clinical outcome. Reduced efficacy and resistance to 5-ALA-PDT can result from genomic alterations, tumor heterogeneity, hypoxia, activation of pathways related to cell surveillance, production of nitric oxide, and most importantly, deregulated 5-ALA transporter proteins and heme biosynthesis enzymes. Understanding the resistance regulatory mechanisms of 5-ALA-PDT may allow the development of effective personalized cancer therapy. Here, we described the mechanisms underlying resistance to 5-ALA-PTD across various tumor types and explored potential strategies to overcome this resistance. Furthermore, we discussed future approaches that may enhance the efficacy of treatments using 5-ALA-PDT.

Tumor Cell Membrane-Encapsulated MLA Solid Lipid Nanoparticles for Targeted Diagnosis and Radiosensitization Therapy of Cutaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is a common nonmelanoma skin cancer. Radiotherapy plays an integral role in treating SCC due to its characteristics, such as diminished intercellular adhesion, heightened cell migration and invasion capabilities, and immune evasion. These problems lead to inaccurate tumor boundary positioning and radiotherapy tolerance in SCC treatment. Thus, accurate localization and enhanced radiotherapy sensitivity are imperative for effective SCC treatment. To address the existing limitations in SCC therapy, we developed monoglyceride solid lipid nanoparticles (MG SLNs) and enveloped them with the A431 cell membrane (A431 CM) to create A431@MG. The characterization results showed that A431@MG was spherical. Furthermore, A431@MG had specific targeting for A431 cells. In A431 tumor-bearing mice, A431@MG demonstrated prolonged accumulation within tumors, ensuring precise boundary localization of SCC. We further advanced the approach by preparing MG SLNs encapsulating 5-aminolevulinic acid methyl ester (MLA) and desferrioxamine (DFO) with an A431 CM coating to yield A431@MG-MLA/DFO. Several studies have revealed that DFO effectively reduced iron content, impeding protoporphyrin IX (PpIX) biotransformation and promoting PpIX accumulation. Simultaneously, MLA was metabolized into PpIX upon cellular entry. During radiotherapy, the heightened PpIX levels enhanced reactive oxygen species (ROS) generation, inducing DNA and mitochondrial damage and leading to cell apoptosis. In A431 tumor-bearing mice, the A431@MG-MLA/DFO group exhibited notable radiotherapy sensitization, displaying superior tumor growth inhibition. Combining A431@MG-MLA/DFO with radiotherapy significantly improved anticancer efficacy, highlighting its potential to serve as an integrated diagnostic and therapeutic strategy for SCC.

Battery-free optoelectronic patch for photodynamic and light therapies in treating bacteria-infected wounds.

Light therapy is an effective approach for the treatment of a variety of challenging dermatological conditions. In contrast to existing methods involving high doses and large areas of illumination, alternative strategies based on wearable designs that utilize a low light dose over an extended period provide a precise and convenient treatment. In this study, we present a battery-free, skin-integrated optoelectronic patch that incorporates a coil-powered circuit, an array of microscale violet and red light emitting diodes (LEDs), and polymer microneedles (MNs) loaded with 5-aminolevulinic acid (5-ALA). These polymer MNs, based on the biodegradable composite materials of polyvinyl alcohol (PVA) and hyaluronic acid (HA), serve as light waveguides for optical access and a medium for drug release into deeper skin layers. Unlike conventional clinical photomedical appliances with a rigid and fixed light source, this flexible design allows for a conformable light source that can be applied directly to the skin. In animal models with bacterial-infected wounds, the experimental group with the combination treatment of metronomic photodynamic and light therapies reduced 2.48 log10 CFU mL-1 in bactericidal level compared to the control group, indicating an effective anti-infective response. Furthermore, post-treatment analysis revealed the activation of proregenerative genes in monocyte and macrophage cell populations, suggesting enhanced tissue regeneration, neovascularization, and dermal recovery. Overall, this optoelectronic patch design broadens the scope for targeting deep skin lesions, and provides an alternative with the functionality of standard clinical light therapy methods.

Effect of 5-aminolevulinic acid-mediated photodynamic therapy against Fusobacterium nucleatum in periodontitis prevention.

Periodontitis, a chronic infectious disease leading to gingival atrophy and potential tooth loss through alveolar bone resorption, is closely linked to the oral microbiome. Fusobacterium nucleatum, known to facilitate late-stage bacterial colonization in the oral microbiome, plays a crucial role in the onset of periodontitis. Controlling F. nucleatum abundance is vital for preventing and treating periodontal disease. Photodynamic therapy combined with 5-aminolevulinic acid (ALA-PDT) has been reported to be bactericidal against Pseudomonas aeruginosa and Staphylococcus aureus. We aimed to investigate the bactericidal potential of ALA-PDT against F. nucleatum, which was evaluated by examining the impact of varying 5-ALA concentrations, culture time, and light intensity. After ALA-PDT treatment, DNA was extracted from interdental plaque samples collected from 10 volunteers and sequenced using the Illumina MiSeq platform. To further elucidate the bactericidal mechanism of ALA-PDT, porphyrins were extracted from F. nucleatum following cultivation with 5-ALA and subsequently analyzed using fluorescence spectra. ALA-PDT showed a significant bactericidal effect against F. nucleatum. Its bactericidal activity demonstrated a positive correlation with culture time and light intensity. Microbiota analysis revealed no significant alteration in α-diversity within the ALA-PDT group, although there was a noteworthy reduction in the proportion of the genus Fusobacterium. Furthermore, fluorescence spectral analysis indicated that F. nucleatum produced an excitable photosensitive substance following the addition of 5-ALA. Overall, if further studies confirm these results, this combined therapy could be an effective strategy for reducing the prevalence of periodontitis.

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.

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.

Therapeutic and fluorescence evaluation of 20% 5-aminolevulinic acid-mediated photodynamic therapy in actinic keratosis.

BACKGROUND: Actinic keratosis (AK) is a precancerous lesion that occurs in areas that are chronically exposed to sunlight and has the potential to develop into invasive cutaneous squamous cell carcinoma (cSCC). We investigated the efficacy of 20 % 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) with LED red light for the treatment of AK in Chinese patients by examining changes in dermoscopic features, histopathology and fluorescence after treatment. METHODS: Twenty-eight patients with fourty-six AK lesions from March 2022 to September 2023 were treated with 20 % ALA, and 3 h later, they were irradiated with LED red light (80-100 mW/cm2) for 20 min. A session of 20 % ALA-PDT was performed once a week for three consecutive weeks, and the dermoscopic, histopathological, fluorescent and photoaging outcomes were measured one week after the treatment. RESULTS: One week after ALA-PDT, complete remission (CR) was reached in 53.6 % of patients. The CR of Grade I AK lesions was 100 %, that of Grade II lesions was 71.4 %, and that of Grade III lesions was 38.1 %. There was a significant improvement in the dermoscopic features, epidermal thickness and fluorescence of the AK lesions. The presence of red fluorescence decreased, and there was an association between CR and post-PDT fluorescence intensity. ALA-PDT also exhibited efficacy in treating photoaging, including fine lines, sallowness, mottled pigmentation, erythema, and telangiectasias, and improved the global score for photoaging. There were no serious adverse effects during or after ALA-PDT, and 82.1 % of the patients were satisfied with the treatment. CONCLUSION: AK lesions can be safely and effectively treated with 20 % ALA-PDT with LED red light, which also alleviates photoaging in Chinese patients, including those with multiple AKs. This study highlights the possibility that fluorescence could be used to diagnose AK with peripheral field cancerization and evaluate the efficacy of ALA-PDT.

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.

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.

In vitro modeling of recurrent Dermatofibrosarcoma Protuberans: Assessment of 5-aminolevulinic acid photodynamic therapy efficacy.

BACKGROUND: Dermatofibrosarcoma Protuberans (DFSP) is a rare, low-grade malignant tumor of the dermis with a high recurrence rate post-surgery. Current treatments, including surgery, radiotherapy, and targeted therapy, have limitations. Photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA) is a promising non-invasive approach, but its efficacy in DFSP treatment remains underexplored. METHODS: This study aimed to evaluate the anti-tumor efficacy of 5-ALA PDT using an in vitro model derived from a recurrent DFSP patient. The cells were treated with varying concentrations of 5-ALA and exposed to red light, followed by assessments of cell viability, proliferation, apoptosis, migration, invasion, angiogenesis, and expression of DFSP-related genes and proteins. RESULTS: 5-ALA PDT significantly reduced DFSP cell viability in a dose-dependent manner and induced apoptosis. It also effectively inhibited cell proliferation, migration, and invasion, as well as suppressed angiogenic activity in conditioned media. Furthermore, 5-ALA PDT downregulated the expression of COL1A1 and PDGFRB, key genes in DFSP pathogenesis. CONCLUSIONS: The findings provide the first evidence of 5-ALA PDT's in vitro anti-tumor efficacy against DFSP, suggesting its potential as a novel therapeutic approach for DFSP. Further studies are warranted to explore the clinical utility of 5-ALA PDT in preventing DFSP recurrence.

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.

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.

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.

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.

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.

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.

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.

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.