Lipid rafts display a lateral heterogeneity forming membrane microdomains that hold a fundamental role on biological membranes and are indispensable to physiological functions of cells. Oxidative ...stress in cellular environments may cause lipid oxidation, changing membrane composition and organization, thus implying in effects in cell signaling and even loss of homeostasis. The individual contribution of oxidized lipid species to the formation or disruption of lipid rafts in membranes still remains unknown. Here, we investigate the role of different structures of oxidized phospholipids on rafts microdomains by carefully controlling the membrane composition. Our experimental approach based on fluorescence microscopy of giant unilamellar vesicles (GUV) enables the direct visualization of the impact of hydroperoxidized POPC lipid (referred to as POPCOOH) and shortened chain lipid PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine) on phase separation. We found that the molecular structure of oxidized lipid is of paramount importance on lipid mixing and/or demixing. The hydrophobic mismatch promoted by POPCOOH coupled to its cylindrical molecular shape favor microdomains formation. In contrast, the conical shape of PazePC causes disarrangement of lipid 2D organized platforms. Our findings contribute to better unraveling how oxidized phospholipids can trigger formation or disruption of lipid rafts. As a consequence, phospholipid oxidation may indirectly affect association or dissociation of key biomolecules in the rafts thus altering cell signaling and homeostasis.
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•Phospholipid oxidation directly impacts on lipid rafts formation and/or disruption.•Hydroperoxidized phospholipid triggers to membrane microdomains formation.•Shortened chain oxidized lipid causes disarrangement of lipid microdomains.•Lipid mixing and/or demixing depends on the molecular geometry of oxidized phospholipid.
Mobilization of specific mechanisms of regulated cell death is a promising alternative to treat challenging illness such as neurodegenerative disease and cancer. The use of light to activate these ...mechanisms may provide a route for target-specific therapies. Two asymmetric porphyrins with opposite charges, the negatively charged TPPS
and the positively charged CisDiMPyP were compared in terms of their properties in membrane mimics and in cells. CisDiMPyP interacts to a larger extent with model membranes and with cells than TPPS
, due to a favorable electrostatic interaction. CisDiMPyP is also more effective than TPPS
in damaging membranes. Surprisingly, TPPS
is more efficient in causing photoinduced cell death. The lethal concentration on cell viability of 50% (LC
) found for TPPS
was ~3.5 (raw data) and ~5 (considering photosensitizer incorporation) times smaller than for CisDiMPyP. CisDiMPyP damaged mainly mitochondria and triggered short-term phototoxicity by necro-apoptotic cell death. Photoexcitation of TPPS
promotes mainly lysosomal damage leading to autophagy-associated cell death. Our data shows that an exact damage in lysosome is more effective to diminish proliferation of HeLa cells than a similar damage in mitochondria. Precisely targeting organelles and specifically triggering regulated cell death mechanisms shall help in the development of new organelle-target therapies.
Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy to either survive or die. However, the use of macroautophagy/autophagy to improve the efficiency of ...photosensitizers, in terms of inducing cell death, remains unexplored. Here, we addressed the concept that a parallel damage in the membranes of mitochondria and lysosomes leads to a scenario of autophagy malfunction that can greatly improve the efficiency of the photosensitizer to cause cell death. Specific damage to these organelles was induced by irradiation of cells pretreated with 2 phenothiazinium salts, methylene blue (MB) and 1,9-dimethyl methylene blue (DMMB). At a low concentration level (10 nM), only DMMB could induce mitochondrial damage, leading to mitophagy activation, which did not progress to completion because of the parallel damage in lysosome, triggering cell death. MB-induced photodamage was perceived almost instantaneously after irradiation, in response to a massive and nonspecific oxidative stress at a higher concentration range (2 µM). We showed that the parallel damage in mitochondria and lysosomes activates and inhibits mitophagy, leading to a late and more efficient cell death, offering significant advantage (2 orders of magnitude) over photosensitizers that cause unspecific oxidative stress. We are confident that this concept can be used to develop better light-activated drugs.
Abbreviations: ΔΨm: mitochondrial transmembrane inner potential; AAU: autophagy arbitrary units; ATG5, autophagy related 5; ATG7: autophagy related 7; BAF: bafilomycin A
1
; BSA: bovine serum albumin; CASP3: caspase 3; CF: carboxyfluorescein; CTSB: cathepsin B; CVS: crystal violet staining; DCF: dichlorofluorescein; DCFH
2
: 2ʹ,7ʹ-dichlorodihydrofluorescein; DMMB: 1,9-dimethyl methylene blue; ER: endoplasmic reticulum; HaCaT: non-malignant immortal keratinocyte cell line from adult human skin; HP: hydrogen peroxide; LC3B-II: microtubule associated protein 1 light chain 3 beta-II; LMP: lysosomal membrane permeabilization; LTG: LysoTracker™ Green DND-26; LTR: LysoTracker™ Red DND-99; 3-MA: 3-methyladenine; MB: methylene blue; mtDNA: mitochondrial DNA; MitoSOX™: red mitochondrial superoxide probe; MTDR: MitoTracker™ Deep Red FM; MTO: MitoTracker™ Orange CMTMRos; MT-ND1: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; MTT: methylthiazolyldiphenyl-tetrazolium bromide;
1
O
2
: singlet oxygen; OH
.
hydroxil radical; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PBS: phosphate-buffered saline; PI: propidium iodide; PDT: photodynamic therapy; PS: photosensitizer; QPCR: gene-specific quantitative PCR-based; Rh123: rhodamine 123; ROS: reactive oxygen species RTN: rotenone; SQSTM1/p62: sequestosome 1; SUVs: small unilamellar vesicles; TBS: Tris-buffered saline
Hypericin (Hyp) is considered a promising photosensitizer for Photodynamic Therapy (PDT), due to its high hydrophobicity, affinity for cell membranes, low toxicity and high photooxidation activity. ...In this study, Hyp photophysical properties and photodynamic activity against melanoma B16-F10 cells were optimized using DPPC liposomes (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) as a drug delivery system. This nanoparticle is used as a cell membrane biomimetic model and solubilizes hydrophobic drugs. Hyp oxygen singlet lifetime (τ) in DPPC was approximately two-fold larger than that in P-123 micelles (Pluronic™ surfactants), reflecting a more hydrophobic environment provided by the DPPC liposome. On the other hand, singlet oxygen quantum yield values (Φ
O
) in DPPC and P-123 were similar; Hyp molecules were preserved as monomers. The Hyp/DPPC liposome aqueous dispersion was stable during fluorescence emission and the liposome diameter remained stable for at least five days at 30 °C. However, the liposomes collapsed after the lyophilization/rehydration process, which was resolved by adding the lyoprotectant Trehalose to the liposome dispersion before lyophilization. Cell viability of the Hyp/DPPC formulation was assessed against healthy HaCat cells and high-metastatic melanoma B16-F10 cells. Hyp incorporated into the DPPC carrier presented a higher selectivity index than the Hyp sample previously solubilized in ethanol under the illumination effect. Moreover, the IC
was lower for Hyp in DPPC than for Hyp pre-solubilized in ethanol. These results indicate the potential of the formulation of Hyp/DPPC for future biomedical applications in PDT treatment.
In this review, we describe how photooxidation changes membrane properties that can ultimately lead to permanent membrane damage. Lipid photooxidation occurs in the presence of reactive oxygen ...species such as singlet oxygen and by direct reactions of lipids with a photosensitizer in the excited state. Indeed, lipid oxidation triggers chemical transformations that can alter lipid packing; change the membrane surface area, thickness and elastic modulus; and induce pore formation and phase separation. Here, we highlight how lipid hydroperoxides promote membrane remodelling and phase separation. Further, we emphasize the alterations caused by truncated oxidized lipids that lead to increased membrane permeability. Finally, the consequences of lipid photooxidation on cell functions are also discussed.
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•Photosensitization reactions cause membrane oxidation.•Unsaturated lipids are photooxidation targets.•Photooxidation causes biophysical and biochemical changes in membrane properties.•Lipid oxidation has a role in membrane permeability and membrane microdomains.•Cellular dysfunctions are mediated by lipid oxidation.
The xanthene dye erythrosin B (ERY) has high molar absorptivity and singlet oxygen quantum yield (ΦΔO21), which make it a potential photosensitizer for photodynamic applications. However, ERY is very ...hydrophilic in water at physiological pH due to its two negative charges, which results in low interaction with biological membranes. In this work we synthesized ester derivatives of ERY: methyl (ERYMET), butyl (ERYBUT) and decyl (ERYDEC) esters. The light absorption process was not greatly affected by the insertion of the alkyl group. The ester hydrophilic–lipophilic balance was evaluated by partition in a 1-octanol/water mixture. The insertion of the alkyl chain and the loss of one charge increased the hydrophobicity and, as expected, ERYDEC was the most hydrophobic dye. The interaction of the esters with the biological membranes was simulated in biomimetic models, micelles of: CTAB (cationic), SDS (anionic) and the polymeric surfactant pluronic F-127® and P-123® (non-charged). The order of the binding constants was ERY < ERYMET < ERYBUT < ERYDEC in all micelles investigated, equal to the hydrophobicity sequence. The localization of the dyes in micelles determined by quenching fluorescence measurements using iodide as a suppressor was consistent with their charge and hydrophobic characteristics. CTAB micelles showed a biphasic interaction due to the association of the cationic monomers of the surfactant and the anionic dyes. The fluorescence quantum yields of the esters in buffered water were equal to those of ERY, with low values of ∼0.02 and a slight increase to ∼0.1 in micellar media, the value of which was still small. The quantum yields of singlet oxygen of all xanthenes investigated were high in micellar systems and similar to each other, independent of the alkyl group (0.6–0.7). ERYDEC showed low ΦΔO21 of 0.38 in buffered water due to the self-aggregation process, but high values in micellar systems. These results in micelles are very important for formulated medications in photodynamic therapy and photodynamic inactivation of microorganisms. Ester derivatives of erythrosin show a potential as photosensitizers in photodynamic clinical applications, especially those formulated with biocompatible surfactants, such as polymeric pluronics.
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•HLB parameter influences the interaction between erythrosin esters and micelles.•Photophysical properties are modulated by charge and hydrophobic character of dye.•Erythrosin esters have shown good properties to be applied in photodynamic treatments.
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.
Rhenium complexes show great promise as anticancer drug candidates. Specifically, compounds with a Re(CO)3(NN)(py)+ core in their architecture have shown cytotoxicity equal to or greater than that of ...well-established anticancer drugs based on platinum or organic molecules. This study aimed to evaluate how the strength of the interaction between rhenium(I) tricarbonyl complexes fac-Re(CO)3(NN)(py)+, NN = 1,10-phenanthroline (phen), dipyrido3,2-f:2′,3′-hquinoxaline (dpq) or dipyrido3,2-a:2′3’-cphenazine (dppz) and biomolecules (protein, lipid and DNA) impacted the corresponding cytotoxic effect in cells. Results showed that fac-Re(CO)3(dppz)(py)+ has higher Log Po/w and binding constant (Kb) with biomolecules (protein, lipid and DNA) compared to complexes of fac-Re(CO)3(phen)(py)+ and fac-Re(CO)3(dpq)(py)+. As consequence, fac-Re(CO)3(dppz)(py)+ exhibited the highest cytotoxicity (IC50 = 8.5 μM for HeLa cells) for fac-Re(CO)3(dppz)(py)+ among the studied compounds (IC50 > 15 μM). This highest cytotoxicity of fac-Re(CO)3(dppz)(py)+ are probably related to its lipophilicity, higher permeation of the lipid bilayers of cells, and a more potent interaction of the dppz ligand with biomolecules (protein and DNA). Our findings open novel avenues for rational drug design and highlight the importance of considering the chemical structures of rhenium complexes that strongly interact with biomolecules (proteins, lipids, and DNA).
This study evaluated the impact of biomolecule interactions on the cytotoxic effects of rhenium(I) tricarbonyl complexes and their interactions with proteins, lipids, and DNA. Our findings revealed that more hydrophobic complex exhibited the highest cytotoxicity among the studied compounds, emphasizing the significance of its lipophilicity and strong interaction with biomolecules. Display omitted
•Rhenium complexes showed potent cytotoxicity than platinum based anticancer drugs.•The most hydrophobic complex rhenium displayed highest cytotoxicity.•Lipophilicity contributes to increased cytotoxicity.•The strength of interaction with biomolecules impacted cytotoxicity.•Understanding biomolecule interactions is vital for rational drug design.
The search for conditions that maximize the outcome of Photodynamic Therapy (PDT) continues. Recent data indicate that PDT-induced cell death depends more on the specific intracellular location of ...the photosensitizer (PS) than on any other parameter. Indeed, knowledge of the PS intracellular location allows the establishment of clear relationships between the mechanism of cell death and the PDT efficacy. In order to determine the intracellular localization sites of a given PS, classical co-localization protocols, which are based in the comparison of the emissive profiles of organelle-specific probes to those of the PS, are usually performed. Since PSs are usually not efficient fluorophores, co-localization protocols require relatively high PS concentrations (micromolar range), distorting the whole proposal of the experiment, as high PS concentration means accumulation in many low-affinity sites. To overcome this difficulty, herein we describe a method that identifies PS intracellular localization by recognizing and quantifying the photodamage at intracellular organelles. We propose that irradiation protocols and characterization of major sites of photodamage results from many cycles of photosensitized oxidations, furnishing an integrated picture of the PS location. By comparing the results of protocols based in either method, we showed that the analysis of the damaged organelles can be conducted at optimal conditions (low PS concentrations), providing clear correlations with cell death mechanisms, which is not the case for the results obtained with co-localization protocols. Experiments using PSs that target either mitochondria or lysosomes were described and investigated in detail, showing that evaluating organelle damage is as simple as performing co-localization protocols.
It was evaluated the properties of the xanthene dyes Erythrosin B, Eosin Y and theirs Methyl, Butyl and Decyl ester derivatives as possible photosensitizers (PS) for photodynamic treatments. The more ...hydrophobic dyes self‐aggregate in water/ethanol solutions above 70% water (vol/vol) in the mixture. In buffered water, these PS were encapsulated in Pluronic polymeric surfactants of P‐123 and F‐127 by two methodologies: direct addition and the thin‐film solid dispersion methods. The thin‐film solid method provided formulations with higher stabilities besides effective encapsulation of the PS as monomers. Size measurements demonstrated that Pluronic forms self‐assembled micelles with uniform size, which present slightly negative surface potential and a spherical form detected by TEM microscopy. The ester length modulates xanthene localization in the micelle, which is deeper with the increase in the alkyl chain. Moreover, some PS are distributed into two populations: one on the corona micelle interface shell (PEO layer) and the other into the core (PPO region). Although all PS formulations show high singlet oxygen quantum yield, promising results were obtained for Erythrosin B esters with the hydrophobic P‐123, which ensures their potential as drug for clinical photodynamic applications.
Photodynamic therapy (PDT) has been applied as a promising modality in the treatment of tumor cells and against a series of microorganisms, a technique also known as photodynamic inactivation of microorganisms (PDIMO). Halogenated xanthene derivatives are one of the cheapest dye molecules that have been proposed as possible photosensitizing agents due to their high light absorption and high quantum yield of singlet oxygen. Examples of halogenated xanthenes are Eosin Y (EOS) and Erythrosin B (ERY). Xanthenes can be applied on body surface diseases or against microorganisms.
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