The impact of biomolecule interactions on the cytotoxic effects of rhenium(I) tricarbonyl complexes.

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), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq) or dipyrido[3,2-a:2'3'-c]phenazine (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).

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.

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.

Hypericin photodynamic activity in DPPC liposomes - part II: stability and application in melanoma B16-F10 cancer cells.

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 (ΦΔ1O2) 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 IC50 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.

Understanding membrane remodelling initiated by photosensitized lipid oxidation.

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.

Identifying Specific Subcellular Organelle Damage by Photosensitized Oxidations.

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.

Contrasting roles of oxidized lipids in modulating membrane microdomains.

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.

Parallel damage in mitochondria and lysosomes is an efficient way to photoinduce cell death.

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 A1; BSA: bovine serum albumin; CASP3: caspase 3; CF: carboxyfluorescein; CTSB: cathepsin B; CVS: crystal violet staining; DCF: dichlorofluorescein; DCFH2: 2',7'-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; 1O2: 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.

Enhanced efficiency of cell death by lysosome-specific photodamage.

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 TPPS2a 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 TPPS2a, due to a favorable electrostatic interaction. CisDiMPyP is also more effective than TPPS2a in damaging membranes. Surprisingly, TPPS2a is more efficient in causing photoinduced cell death. The lethal concentration on cell viability of 50% (LC50) found for TPPS2a 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 TPPS2a 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.

Nanostructured Polymeric Micelles Carrying Xanthene Dyes for Photodynamic Evaluation.

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.