Specific photodamage on HT-29 cancer cells leads to endolysosomal failure and autophagy blockage by cathepsin depletion.

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.

Static Magnetic Field Reduces Intracellular ROS Levels and Protects Cells Against Peroxide-Induced Damage: Suggested Roles for Catalase.

A feature in neurodegenerative disorders is the loss of neurons, caused by several factors including oxidative stress induced by reactive oxygen species (ROS). In this work, static magnetic field (SMF) was applied in vitro to evaluate its effect on the viability, proliferation, and migration of human neuroblastoma SH-SY5Y cells, and on the toxicity induced by hydrogen peroxide (H2O2), tert-butyl hydroperoxide (tBHP), H2O2/sodium azide (NaN3) and photosensitized oxidations by photodynamic therapy (PDT) photosensitizers. The SMF increased almost twofold the cell expression of the proliferation biomarker Ki-67 compared to control cells after 7 days of exposure. Exposure to SMF accelerated the wound healing of scratched cell monolayers and significantly reduced the H2O2-induced and the tBHP-induced cell deaths. Interestingly, SMF was able to revert the effects of NaN3 (a catalase inhibitor), suggesting an increased activity of catalase under the influence of the magnetic field. In agreement with this hypothesis, SMF significantly reduced the oxidation of DCF-H2, indicating a lower level of intracellular ROS. When the redox imbalance was triggered through photosensitized oxidation, no protection was observed. This observation aligns with the proposed role of catalase in cellular proctetion under SMF.  Exposition to SMF should be further validated in vitro and in vivo as a potential therapeutic approach for neurodegenerative disorders.

Endogenous Photosensitizers in Human Skin.

Endogenous photosensitizers play a critical role in both beneficial and harmful light-induced transformations in biological systems. Understanding their mode of action is essential for advancing fields such as photomedicine, photoredox catalysis, environmental science, and the development of sun care products. This review offers a comprehensive analysis of endogenous photosensitizers in human skin, investigating the connections between their electronic excitation and the subsequent activation or damage of organic biomolecules. We gather the physicochemical and photochemical properties of key endogenous photosensitizers and examine the relationships between their chemical reactivity, location within the skin, and the primary biochemical events following solar radiation exposure, along with their influence on skin physiology and pathology. An important take-home message of this review is that photosensitization allows visible light and UV-A radiation to have large effects on skin. The analysis presented here unveils potential causes for the continuous increase in global skin cancer cases and emphasizes the limitations of current sun protection approaches.

Red Wine Inspired Chromophores as Photodynamic Therapy Sensitizers.

Hydroxypyranoflavylium (HPF) cations are synthetic analogs possessing the same basic chromophore as the pyranoanthocyanins that form during the maturation of red wine. HPF cations absorb strongly in the visible spectral region, and most are fluorescent, triplet-sensitize singlet oxygen formation in solution and are strong photooxidants, properties that are desirable in a sensitizer for photodynamic therapy (PDT). The results of this study demonstrate that several simple HPF dyes can indeed function as PDT sensitizers. Of the eight HPF cations investigated in this work, four were phototoxic to a human cervical adenocarcinoma cell line (HeLa) at the 1-10 µmol dm-3 level, while only one of the eight compounds showed noticeable cytotoxicity in the dark. Neither a Type I nor a Type II mechanism can adequately rationalize the differences in phototoxicity of the compounds. Colocalization experiments with the most phototoxic compound demonstrated the affinity of the dye for both the mitochondria and lysosomes of HeLa cells. The fact that relatively modest structural differences, e.g., the exchange of an electron-donating substituent for an electron-withdrawing substituent, can cause profound differences in the phototoxicity, together with the relatively facile synthesis of substituted HPF cations, makes them interesting candidates for further evaluation as PDT sensitizers.

Necroptosis activation is associated with greater methylene blue-photodynamic therapy-induced cytotoxicity in human pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinomas (PDAC) are the fourth leading cause of death due to neoplasms. In view of the urgent need of effective treatments for PDAC, photodynamic therapy (PDT) appears as a promising alternative. However, its efficacy against PDAC and the mechanisms involved in cell death induction remain unclear. In this study, we set out to evaluate PDT's cytotoxicity using methylene blue (MB) as a photosensitizer (PS) (MB-PDT) and to evaluate the contribution of necroptosis in its effect in human PDAC cells. Our results demonstrated that MB-PDT induced significant death of different human PDAC models presenting two different susceptibility profiles. This effect was independent of MB uptake or its subcellular localization. We found that the ability of triggering necroptosis was determinant to increase the treatment efficiency. Analysis of single cell RNA-seq data from normal and neoplastic human pancreatic tissues showed that specific necroptosis proteins RIPK1, RIPK3 and MLKL presented significant higher expression levels in cells displaying a transformed phenotype providing further support to the use of approaches that activate necroptosis, like MB-PDT, as useful adjunct to surgery of PDAC to tackle the problem of microscopic residual disease as well as to minimize the chance of local and metastatic recurrence.

Co-Encapsulation of Methylene Blue and PARP-Inhibitor into Poly(Lactic-Co-Glycolic Acid) Nanoparticles for Enhanced PDT of Cancer.

The development of resistance against photodamage triggered by photodynamic therapy (PDT) is ascribed mainly to the cellular redox defenses and repair. If the tumor tissue is not promptly eliminated by the first few PDT sessions, PDT-resistance can be favored, challenging the efficacy of the treatment. Although the mechanism of PDT resistance is still unclear, in vitro assays have evidenced that it can be developed through the PARP damage-repair signaling pathway. Therefore, inhibition of poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) has the potential to increase PDT efficacy. This work reports on the synthesis of a controlled release system of a photosensitizer, methylene blue (MB) and a PARP-inhibitor, the veliparib. MB and veliparib were co-encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (VMB-NPs). A colloidal stable aqueous suspension of nanoparticles was obtained. The average hydrodynamic diameter was 90 nm and a narrow size distribution was obtained, with a polydispersity index (PDI) of 0.08. The release kinetics of MB and veliparib from VMB-NPs showed an initial burst of 8.7% and 58.3% release of the total amounts of MB and veliparib respectively, in the first 6 h, and a delayed release of up to 11.3% and 70%, in 19 days, for MB and veliparib, respectively. The VMB-NPs showed no cytotoxicity in the dark but the viability of B16F10-Nex2 cells decreased by 36% when the cells were irradiated (102 J/cm2, 660 nm) and treated with VMB-NPs containing 1.0 µM of MB and 8.3 µM of veliparib. Considering the increased photoactivity even at low MB and veliparib concentrations and the absence of cytotoxicity in dark, the co-encapsulation of MB and veliparib was shown to be a promising strategy to improve the PDT efficacy.

Methylene blue-mediated antimicrobial photodynamic therapy can be a novel non-antibiotic platform for bovine digital dermatitis.

BACKGROUND: Bovine digital dermatitis (BDD) is one of the most important diseases that effect dairy cows. Methylene blue-mediated antimicrobial photodynamic therapy (MB-APDT) emerges as a promising technique to treat superficial infections in bovines. METHODS: Twenty BDD lesions located at the skin horn transition of the claw of pelvic limbs of 16 cows were treated by MB-APDT, using a red LED cluster (λ = 660 nm, irradiance =60 mW/cm2, exposure time = 40 s) combined with topical application of MB at 0.01 %; or by topical application of OXY (500 mg in 20 % solution). Each lesion was treated twice with an interval of 14 days. Lesions were weekly evaluated until day 28 by clinical analysis and by histological examination on days 0 and 28. RESULTS: Both treatments led to a similar reduction of lesions area. At day 28, three lesions treated by OXY did not present completely recovery, whereas no lesions were observed in MB-APDT group. OXY resulted in a slight increase in type I and III collagen levels, while MB-APDT led to a significant increase in the total area of both collagen types. An abundant number of spirochetes were histologically observed in all lesions before treatments. On the 28th day, five lesions treated by OXY still presented a slight number of spirochetes, whereas in MB-APDT group no spirochetes were evidenced. CONCLUSION: Our findings suggest that MB-APDT is more effective than OXY and could be used in Veterinary practice to fight BDD.

Glutaminolysis dynamics during astrocytoma progression correlates with tumor aggressiveness.

BACKGROUND: Glioblastoma is the most frequent and high-grade adult malignant central nervous system tumor. The prognosis is still poor despite the use of combined therapy involving maximal surgical resection, radiotherapy, and chemotherapy. Metabolic reprogramming currently is recognized as one of the hallmarks of cancer. Glutamine metabolism through glutaminolysis has been associated with tumor cell maintenance and survival, and with antioxidative stress through glutathione (GSH) synthesis. METHODS: In the present study, we analyzed the glutaminolysis-related gene expression levels in our cohort of 153 astrocytomas of different malignant grades and 22 non-neoplastic brain samples through qRT-PCR. Additionally, we investigated the protein expression profile of the key regulator of glutaminolysis (GLS), glutamate dehydrogenase (GLUD1), and glutamate pyruvate transaminase (GPT2) in these samples. We also investigated the glutathione synthase (GS) protein profile and the GSH levels in different grades of astrocytomas. The differential gene expressions were validated in silico on the TCGA database. RESULTS: We found an increase of glutaminase isoform 2 gene (GLSiso2) expression in all grades of astrocytoma compared to non-neoplastic brain tissue, with a gradual expression increment in parallel to malignancy. Genes coding for GLUD1 and GPT2 expression levels varied according to the grade of malignancy, being downregulated in glioblastoma, and upregulated in lower grades of astrocytoma (AGII-AGIII). Significant low GLUD1 and GPT2 protein levels were observed in the mesenchymal subtype of GBM. CONCLUSIONS: In glioblastoma, particularly in the mesenchymal subtype, the downregulation of both genes and proteins (GLUD1 and GPT2) increases the source of glutamate for GSH synthesis and enhances tumor cell fitness due to increased antioxidative capacity. In contrast, in lower-grade astrocytoma, mainly in those harboring the IDH1 mutation, the gene expression profile indicates that tumor cells might be sensitized to oxidative stress due to reduced GSH synthesis. The measurement of GLUD1 and GPT2 metabolic substrates, ammonia, and alanine, by noninvasive MR spectroscopy, may potentially allow the identification of IDH1mut AGII and AGIII progression towards secondary GBM.

Cytotoxicity of Methotrexate Conjugated to Glycerol Phosphate Modified Superparamagnetic Iron Oxide Nanoparticles.

A systematic study was carried out to evaluate the uptake and cytotoxicity of methotrexate (MTX) conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) modified with glycerol phosphate (Glyc) and phosphorylethanolamine (PEA), using MCF-7 cancer cell line as model. The ligand shell composition was controlled in such a way to get SPIONs with nine different surface functionalization and up to three co-conjugated ligands but the very iron oxide core, in order to test and compare uptake and cytotoxicity, and verify possible additive effects. Folic acid (FA), the non-toxic analogue of MTX, was also explored as ligand for SPIONs. Glyc was shown to enhance dramatically the cellular uptake despite the high negative zeta potentials, whereas PEA, FA and MTX was found to have a much lower effect on the cellular uptake. Also, a significant ten times lowering of IC50 was observed for the co-conjugated MTX in the SPION-Glyc/PEA/MTX as compared to the free drug, whereas the analogue SPION-Glyc/PEA/FA nanoparticles exhibited no significant cytotoxicity. In short, the conjugation of MTX to SPIONs enhanced dramatically its cytotoxicity and decreased the IC50 value against MCF-7 tumor cells as compared to the free drug, probably due to the enhanced uptake of SPIONs as a result of their surface modification with Glyc/PEA, demonstrating that SPION-Glyc/PEA is a good nanocarrier for co-conjugated methotrexate.

The Endogenous Tryptophan-derived Photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo.

UV-chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan-derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol-1  cm-1 ), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC-25, HaCaT-ras II-4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm-2 ) and FICZ (≥10 nm), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg-sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 "high-risk" mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV-induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.

Cellular compartments challenged by membrane photo-oxidation.

The lipid composition impacts directly on the structure and function of the cytoplasmic as well as organelle membranes. Depending on the type of membrane, specific lipids are required to accommodate, intercalate, or pack membrane proteins to the proper functioning of the cells/organelles. Rather than being only a physical barrier that separates the inner from the outer spaces, membranes are responsible for many biochemical events such as cell-to-cell communication, protein-lipid interaction, intracellular signaling, and energy storage. Photochemical reactions occur naturally in many biological membranes and are responsible for diverse processes such as photosynthesis and vision/phototaxis. However, excessive exposure to light in the presence of absorbing molecules produces excited states and other oxidant species that may cause cell aging/death, mutations and innumerable diseases including cancer. At the same time, targeting key compartments of diseased cells with light can be a promising strategy to treat many diseases in a clinical procedure called Photodynamic Therapy. Here we analyze the relationships between membrane alterations induced by photo-oxidation and the biochemical responses in mammalian cells. We specifically address the impact of photosensitization reactions in membranes of different organelles such as mitochondria, lysosome, endoplasmic reticulum, and plasma membrane, and the subsequent responses of eukaryotic cells.

Distinct photo-oxidation-induced cell death pathways lead to selective killing of human breast cancer cells.

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

Antimicrobial blue light and photodynamic therapy inhibit clinically relevant ß-lactamases with extended-spectrum (ESBL) and carbapenemase activity.

INTRODUCTION: The production of ß-lactamases by Gram-negative bacteria is among the most important factors of resistance to antibiotics, which has contributed to therapeutic failures that currently threaten human and veterinary medicine worldwide. Antimicrobial photodynamic therapy and antimicrobial blue light have a broad-spectrum antibacterial activity against multidrug-resistant and hypervirulent pathogens. OBJECTIVE: To investigate the ability of antimicrobial blue light to inhibit the hydrolytic activity of clinically relevant ß-lactamase enzymes (i.e., KPC, IMP, OXA, CTX-M, and SHV), with further comparison of the inhibitory effects of antimicrobial blue light with methylene blue-mediated antimicrobial photodynamic therapy. METHODS: Blue LED light (λ = 410 ± 10 nm) alone or red LED light (λ = 660 ± 10 nm) in combination with methylene blue were used to inactivate, in vitro, suspensions of Klebsiella pneumoniae strains producing clinically important ß-lactamase enzymes assigned to the A, B and D Ambler molecular classes. Furthermore, ß-lactamase activity inhibition mediated by antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy was measured by using the chromogenic ß-lactam substrate nitrocefin. RESULTS: ß-lactamase activities were effectively inactivated by both visible light-based approaches. In this regard, antimicrobial blue light and methylene blue-antimicrobial photodynamic therapy led to a significant reduction in the hydrolysis of nitrocefin (81-98 %). CONCLUSION: Sublethal doses of antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy are equally effective to inhibit clinically significant ß-lactamases, including extended-spectrum ß-lactamases and carbapenemases.

Nitric oxide inhibition of lipopolysaccharide-stimulated RAW 247.6 cells by ibuprofen-conjugated iron oxide nanoparticles.

Aim: To develop a series of superparamagnetic iron oxide nanoparticles (SPIONs) by coconjugating them with ibuprofen (ibu) and glycerol phosphate (glycerol) or ibu and glucose-1-phosphate and to assess capacity of these conjugates to inhibit the release of nitric oxide (NO) in macrophages, even at low concentrations. Materials & methods: The SPION conjugates were characterized and their properties evaluated showing the influence of those ligands on colloidal stability and inhibition of NO-release demonstrated. The cytotoxicity and possible anti-inflammatory activity were evaluated using murine macrophages (RAW 247.6). Results: SPION-glycerol phosphate/ibu conjugates inhibited the NO production induced by lipopolysaccharides, indicating a potential anti-inflammatory activity. Conclusion: SPION conjugated with ibu was shown to inhibit NO-release even at very low concentrations, suggesting possible action against inflammatory diseases.

Light-based technologies for management of COVID-19 pandemic crisis.

The global dissemination of the novel coronavirus disease (COVID-19) has accelerated the need for the implementation of effective antimicrobial strategies to target the causative agent SARS-CoV-2. Light-based technologies have a demonstrable broad range of activity over standard chemotherapeutic antimicrobials and conventional disinfectants, negligible emergence of resistance, and the capability to modulate the host immune response. This perspective article identifies the benefits, challenges, and pitfalls of repurposing light-based strategies to combat the emergence of COVID-19 pandemic.

Lipofuscin in keratinocytes: Production, properties, and consequences of the photosensitization with visible light.

A dysfunction in the mitochondrial-lysosomal axis of cellular homeostasis is proposed to cause cells to age quicker and to accumulate lipofuscin. Typical protocols to mediate lipofuscinogenesis are based on the induction of the senescent phenotype either by allowing many consecutive cycles of cell division or by treating cells with physical/chemical agents such as ultraviolet (UV) light or hydrogen peroxide. Due to a direct connection with the physiopathology of age-related macular degeneration, lipofuscin that accumulates in retinal pigment epithelium (RPE) cells have been extensively studied, and the photochemical properties of RPE lipofuscin are considered as standard for this pigment. Yet, many other tissues such as the brain and the skin may prompt lipofuscinogenesis, and the properties of lipofuscin granules accumulated in these tissues are not necessarily the same as those of RPE lipofuscin. Here, we present a light-induced protocol that accelerates cell aging as judged by the maximization of lipofuscinogenesis. Photosensitization of cells previously incubated with nanomolar concentrations of 1,9-dimethyl methylene blue (DMMB), severely and specifically damages mitochondria and lysosomes, leading to a lipofuscin-related senescent phenotype. By applying this protocol in human immortalized non-malignant keratinocytes (HaCaT) cells, we observed a 2.5-fold higher level of lipofuscin accumulation compared to the level of lipofuscin accumulation in cells treated with a typical UV protocol. Lipofuscin accumulated in keratinocytes exhibited the typical red light emission, with excitation maximum in the blue wavelength region (~450 nm). Fluorescence lifetime image microscopy data showed that the keratinocyte lipofuscin has an emission lifetime of ~1.7 ns. Lipofuscin-loaded cells (but not control cells) generated a substantial amount of singlet oxygen (1O2) when irradiated with blue light (420 nm), but there was no 1O2 generation when excitation was performed with a green light (532 nm). These characteristics were compared with those of RPE cells, considering that keratinocyte lipofuscin lacks the bisretinoids derivatives present in RPE lipofuscin. Additionally, we showed that lipofuscin-loaded keratinocytes irradiated with visible light presented critical DNA damages, such as double-strand breaks and Fpg-sensitive sites. We propose that the DMMB protocol is an efficient way to disturb the mitochondrial-lysosomal axis of cellular homeostasis, and consequently, it can be used to accelerate aging and to induce lipofuscinogenesis. We also discuss the consequences of the lipofuscin-induced genotoxicity of visible light in keratinocytes.

Porphyrin-Loaded TyroSpheres for the Intracellular Delivery of Drugs and Photoinduced Oxidant Species.

In order to understand the intracellular delivery of drugs and to improve the cell killing efficiency of photosensitizers (PSs) used in photodynamic therapy (PDT), we prepared TyroSphere nanoparticles, which are triblock polymer [poly(ethylene glycol)-block-oligo(desaminotyrosyltyrosine octyl ester suberate)-block-poly(ethylene glycol)] aggregates, loaded with amphiphilic porphyrins with either positive (CisDiMPyP) or negative (TPPS2a) charges. Their physicochemical and photochemical properties were investigated, as well as the efficiency and mechanism of PDT death in a cervical cancer cell line (HeLa). The photophysical properties of both PSs were improved when loaded in the nanocarrier, with a decrease in aggregation as well as an increase in the yield of singlet oxygen generation. The physical and chemical stability of TyroSphere nanoparticles allows them to enter cells and to promote the slow intracellular delivery of part of the PSs. Confocal steady-state and lifetime-resolved fluorescence imaging microscopy data showed that the released PSs are free to target their natural intracellular targets, which are mitochondria and lysosomes for CisDiMPyP and TPPS2a, respectively. The photodynamic efficiency of cell killing was increased considerably compared with the free PSs (∼3×), but the mechanism of cell death was the same as that of the free PSs, which are acute necro-apoptosis for CisDiMPyP and autophagy malfunction for TPPS2a, reflecting the specific damage in mitochondria and lysosomes, respectively. We are confident that TyroSpheres provide a novel and efficient platform to administrate PDT photosensitizers, as well as other drugs with intracellular targets.

Beyond electrostatic interactions: Ligand shell modulated uptake of bis-conjugated iron oxide nanoparticles by cells.

The effect of the ligand shell on the cellular uptake efficiency was evaluated by a systematic study using fully dispersed 6 nm diameter superparamagnetic iron oxide nanoparticles (SPIONs), mono and bis-conjugated with glycerol phosphate (glyc), dopamine (dopa), 4,5-dihydroxy-1,3-benzenedisulfonic acid (tiron) and phosphorylethanolamine (pea). Negatively charged SPION-glyc was more efficiently incorporated than positively charged SPION-pea and SPION-dopa clearly evidencing that there are strong enough short-range interactions in addition to the long-range electrostatic interactions, as measured by the zeta potential, to reverse our expectation on cellular uptake. Those effects were pursued by correlating the nanoparticles incorporation efficiency as a function of the respective zeta potentials and the molar fractions of glyc and pea ligands co-conjugated on the SPION surface. The possibility of associating different ligands to modulate the physicochemical properties and biological events was demonstrated, showing promising perspectives for the development of multifunctional nanosystems for biomedical applications.

Fluence Rate Determines PDT Efficiency in Breast Cancer Cells Displaying Different GSH Levels.

Photodynamic therapy (PDT) appears as a promising alternative in the treatment of breast cancer since it can be highly effective in curing cancer while preserving normal tissue. However, predicting outcomes in PDT still constitutes a great challenge. One of the parameters that are usually empirically determined is the rate of photon flux delivered to the tissue (light fluence rate). In the present study, we intended to understand why monolayers of human cells derived from mammary adenocarcinomas (MDA-MB-231 and MCF-7) respond quite differently to fluence rates (cells were irradiated either for 6 or for 16 min) at a fixed light dose (4.5 J cm-2 ) delivered with an array of LEDs in a typical methylene blue PDT protocol. While death rates of MDA-MB-231 cells were insensitive to the fluence rate, MCF-7 cells showed a quite impressive (three times) decrease in cell death levels in the shorter irradiation protocol. Independent on cell type cell death was invariably correlated with the depletion of reduced glutathione intracellular levels and consequently with widespread redox misbalance. Our data show the potential to optimize fluence rates to provide exhaustion of the cell antioxidant responses in order to circumvent therapy resistance of breast tumors.

Autophagy Regulation and Photodynamic Therapy: Insights to Improve Outcomes of Cancer Treatment.

Cancer is considered an age-related disease that, over the next 10 years, will become the most prevalent health problem worldwide. Although cancer therapy has remarkably improved in the last few decades, novel treatment concepts are needed to defeat this disease. Photodynamic Therapy (PDT) signalize a pathway to treat and manage several types of cancer. Over the past three decades, new light sources and photosensitizers (PS) have been developed to be applied in PDT. Nevertheless, there is a lack of knowledge to explain the main biochemical routes needed to trigger regulated cell death mechanisms, affecting, considerably, the scope of the PDT. Although autophagy modulation is being raised as an interesting strategy to be used in cancer therapy, the main aspects referring to the autophagy role over cell succumbing PDT-photoinduced damage remain elusive. Several reports emphasize cytoprotective autophagy, as an ultimate attempt of cells to cope with the photo-induced stress and to survive. Moreover, other underlying molecular mechanisms that evoke PDT-resistance of tumor cells were considered. We reviewed the paradigm about the PDT-regulated cell death mechanisms that involve autophagic impairment or boosted activation. To comprise the autophagy-targeted PDT-protocols to treat cancer, it was underlined those that alleviate or intensify PDT-resistance of tumor cells. Thereby, this review provides insights into the mechanisms by which PDT can be used to modulate autophagy and emphasizes how this field represents a promising therapeutic strategy for cancer treatment.