Intracellular Modulation of Excited‐State Dynamics in a Chromophore Dyad: Differential Enhancement of Photocytotoxicity Targeting Cancer Cells

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O₂(a¹Δ_g)], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer‐related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.     

Oxygen‐Dependent Photochemistry and Photophysics of “MiniSOG,” a Protein‐Encased Flavin

Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so‐called “mini Singlet Oxygen Generator” (miniSOG), an FMN‐containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (?_Δ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron‐transfer reactions (Type‐I chemistry) at the expense of photosensitized singlet oxygen production (Type‐II chemistry) and results in a comparatively poor yield of singlet oxygen (?_Δ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well‐characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.     

TYPE I AND TYPE II MECHANISMS IN THE PHOTOSENSITIZED LYSIS OF PHOSPHATIDYLCHOLINE LIPOSOMES BY HEMATOPORPHYRIN

Abstract The lysis of phosphatidylcholine (PC) liposomes was sensitized to visible light (>500nm) by hematoporphyrin (HP) incorporated in the liposomes (0.09-1.5%, wt/wt) or in the external buffer (1-15 μM). The lytic mechanism changed from the Type II pathway mediated by singlet oxygen (¹O₂) at low HP concentrations to the anoxic, Type I pathway at high HP concentrations. Spectral measurements of HP in aqueous and organic solvents indicate that the HP was not aggregated (monomers and/or dimers) for Type II sensitization and aggregated for Type I conditions. High concentrations of azide (>0.1 M) or DABCO (>0.5 M) were protective with high HP concentration under oxic and anoxic conditions, which cannot involve the scavenging of ¹O₂. Feasible protective mechanisms are quenching of the HP triplet state by high azide and repair of the damaged membrane by DABCO via an electron transfer process. There was significant protection against lysis under Type I conditions by low concentrations of ferricyanide (>1 mM), indicative of an electron transfer mechanism. The incorporation of 22 mol % cholesterol in PC liposomes with 1% HP had no effect on the lytic efficiency for oxic and anoxic conditions. Dipalmitoylphosphatidylcholine liposomes incorporating 1% HP showed negligible photosensitized lysis at 50°C compared with PC liposomes with 1% HP at 25°C. The promotion of photosensitized lysis by hydrodynamic agitation observed in prior work with methylene blue (Grossweiner and Grossweiner, 1982) was significant with HP sensitization for both Type I and Type II conditions. Actinometry with PC liposomes incorporating 1% HP indicated that photosensitized lysis was very inefficient, requiring many absorbed quanta per lysed liposome. Preliminary experiments with crude hematoporphyrin derivative (Hpd) showed similar concentration effects on lytic efficiency, where PC liposomes incorporating 0.1% (wt/wt) Hpd were strongly sensitized by oxygen, whereas sensitization by oxygen was insignificant with 3.1% Hpd. The results with HP and crude Hpd indicate that lytic damage in a biomembrane does not necessarily require oxygenation.     

The Effect of Photodynamic Action on Leakage of Ions Through Liposomal Membranes that Contain Oxidatively Modified Lipids

Singlet oxygen, created in photosensitization, peroxidizes unsaturated fatty acids of the membrane's lipids. This generates alcoholic or aldehyde groups at double bonds' breakage points. In a previous study, we examined the leakage of a K⁺‐induced cross‐membrane electric potential of liposomes that undergo photosensitization. The question remains to what extent peroxidized lipids can compromise the stability of the membrane. In this study, we studied the effect of the oxidatively modified lipids PGPC and ALDOPC in the membrane on its stability, by monitoring the membrane electric potential with the potentiometric dye DiSC₂(5). As the content of the modified lipids increases the membrane becomes less stable, and even at just 2% of the modified lipids the membrane's integrity is affected, in respect to the leakage of ions through it. When the liposomes that contain the modified lipids undergo photosensitization by hematoporphyrin, the lipid bilayer becomes even more unstable and passage of ions is accelerated. We conclude that the existence of lipids with a shortened fatty acid that is terminated by a carboxylic acid or an aldehyde and more so when photosensitized damage occurs to unsaturated fatty acids in lecithin, add up to a critical alteration of the membrane, which becomes leaky to ions.     

Multifunctional Core–Shell Upconverting Nanoparticles for Imaging and Photodynamic Therapy of Liver Cancer Cells

Lanthanide‐doped upconversion nanoparticles (UCNPs) have attracted considerable attention for their application in biomedicine. Here, silica‐coated NaGdF₄:Yb,Er/NaGdF₄ nanoparticles with a tetrasubstituted carboxy aluminum phthalocyanine (AlC₄Pc) photosensitizer covalently incorporated inside the silica shells were prepared and applied in the photodynamic therapy (PDT) and magnetic resonance imaging (MRI) of cancer cells. These UCNP@SiO₂(AlC₄Pc) nanoparticles were uniform in size, stable against photosensitizer leaching, and highly efficient in photogenerating cytotoxic singlet oxygen under near‐infrared (NIR) light. In vitro studies indicated that these nanoparticles could effectively kill cancer cells upon NIR irradiation. Moreover, the nanoparticles also demonstrated good MR contrast, both in aqueous solution and inside cells. This is the first time that NaGdF₄:Yb,Er/NaGdF₄ upconversion‐nanocrystal‐based multifunctional nanomaterials have been synthesized and applied in PDT. Our results show that these multifunctional nanoparticles are very promising for applications in versatile imaging diagnosis and as a therapy tool in biomedical engineering.     

Unraveling the Degradation Mechanism of Purine Nucleotides Photosensitized by Pterins: The Role of Charge‐Transfer Steps

Photosensitized reactions contribute to the development of skin cancer and are used in many applications. Photosensitizers can act through different mechanisms. It is currently accepted that if the photosensitizer generates singlet molecular oxygen (¹O₂) upon irradiation, the target molecule can undergo oxidation by this reactive oxygen species and the reaction needs dissolved O₂ to proceed, therefore the reaction is classified as ¹O₂‐mediated oxidation (type II mechanism). However, this assumption is not always correct, and as an example, a study on the degradation of 2′‐deoxyguanosine 5′‐monophosphate photosensitized by pterin is presented. A general mechanism is proposed to explain how the degradation of biological targets, such as nucleotides, photosensitized by pterins, naturally occurring ¹O₂ photosensitizers, takes place through an electron‐transfer‐initiated process (type I mechanism), whereas the contribution of the ¹O₂‐mediated oxidation is almost negligible.     

Photodynamic Therapy Efficacy Enhanced by Dynamics: The Role of Charge Transfer and Photostability in the Selection of Photosensitizers

Progress in the photodynamic therapy (PDT) of cancer should benefit from a rationale to predict the most efficient of a series of photosensitizers that strongly absorb light in the phototherapeutic window (650–800 nm) and efficiently generate reactive oxygen species (ROS=singlet oxygen and oxygen‐centered radicals). We show that the ratios between the triplet photosensitizer–O₂ interaction rate constant (k_D) and the photosensitizer decomposition rate constant (k_d), k_D/k_d, determine the relative photodynamic activities of photosensitizers against various cancer cells. The same efficacy trend is observed in vivo with DBA/2 mice bearing S91 melanoma tumors. The PDT efficacy intimately depends on the dynamics of photosensitizer–oxygen interactions: charge transfer to molecular oxygen with generation of both singlet oxygen and superoxide ion (high k_D) must be tempered by photostability (low k_d). These properties depend on the oxidation potential of the photosensitizer and are suitably combined in a new fluorinated sulfonamide bacteriochlorin, motivated by the rationale.     

Photodegradation of Lecithin Liposomes by Nanoparticulate Titanium Dioxide

Abstract

Lecithin liposomes were studied by transmission electron microscopy (TEM), selected‐area electron diffraction (SAED), IR, and GC‐MS. Results indicate that titanium dioxide (TiO₂) nanoparticles can gain access into lecithin liposomes during sonication and the lecithin liposomes can be effectively decomposed upon illumination with near‐UV light.     

介孔钛纳米复合团簇应用于光热-光动力疗法

在本文,采用水热法合成了一种新型的介孔二氧化钛/碳/亚甲蓝复合纳米团簇(TiO₂@C-MB),并应用于肿瘤细胞的光动力(PDT)和光热治疗(PTT)。系统中介孔二氧化钛作为有效的光敏剂,MB作为重要的光敏添加剂以改善二氧化钛纳米晶的光化学效应,并将其光响应区域拓宽至光动力学疗法的理想治疗窗(650~900 nm)。柠檬酸在水热条件下被还原成碳并裹覆在二氧化钛表面。碳层表现出良好的光热效果,也充当多功能的电子受体以加速生成单线态氧。该纳米团簇不仅可以保持肿瘤细胞内部高浓度的MB和二氧化钛以产生大量的单线态氧杀死肿瘤细胞,而且可以避免MB退化失活。     

Kinetics and Yield of Singlet Oxygen Photosensitized by Hypericin in Organic and Biological Media

The spectroscopy and photophysics of the photosensitizer hypericin when in homogeneous solutions and when bound to liposomes were studied. Hypericin was found to partition efficiently into DMPC liposomes, with a binding constant of 58 (mg lipid/mL)^?1. In these liposomes the singlet oxygen production quantum yield was 0.43 ± 0.09. To determine the deactivation constant of singlet oxygen in lipid bilayers for the first time, we calculated extrapolated values from its quenching by DMPC and lecithin in homogeneous solutions and obtained decay times of 36.4 and 12.2 μs, respectively. We also measured the quenching of singlet oxygen, sensitized by hypericin in DMPC liposomes, by NaN₃, diphenyl isobenzofuran and H₂,O: D₂O mixtures and explained the results on the basis of singlet oxygen diffusing rapidly out of the lipid bilayer into the aqueous medium. The observed temperature effect on the lifetime of singlet oxygen of about 50% over a 15°C range in liposome suspension contrasts with a 3% change in a homogeneous solution in 1-nonanol and is explained by the temperature effect on the diffusion out of the liposome. A strong pH effect was observed, indicating that the deprotonated species formed above about pH 10 is a much weaker photosensitizer of singlet oxygen than the native, protonated species.     

介孔钛纳米复合团簇应用于光热-光动力疗法

采用水热法合成了一种新型的介孔二氧化钛/碳/亚甲蓝复合纳米团簇(TiO_2@C-MB),并应用于肿瘤细胞的光动力(PDT)和光热治疗(PTT)。系统中介孔二氧化钛作为有效的光敏剂,MB作为重要的光敏添加剂以改善二氧化钛纳米晶的光化学效应,并将其光响应区域拓宽至光动力学疗法的理想治疗窗(650~900 nm)。柠檬酸在水热条件下被还原成碳并裹覆在二氧化钛表面。碳层表现出良好的光热效果,也充当多功能的电子受体以加速生成单线态氧。该纳米团簇不仅可以保持肿瘤细胞内部高浓度的MB和二氧化钛以产生大量的单线态氧杀死肿瘤细胞,而且可以避免MB退化失活。     

SINGLET OXYGEN GENERATION BY FUROCOUMARINS: EFFECT OF DNA AND LIPOSOMES

The quantum yield of singlet oxygen generation by aqueous furocoumarins was measured at 365 nm using the photosensitized inactivation of subtilisin Carlsberg as the probe with the following results: psoralen (0.18), 5-methoxypsoralen (0.013), and 8-methoxypsoralen (0.035). Singlet oxygen formation was significant for dark complexes of 8-MOP with calf thymus DNA and the covalent DNA photoadducts. Incorporation of 8-MOP in sonicated egg phosphatidylcholine liposomes did not inhibit photosensitization of subtilisin Carlsberg and also led to lipid peroxidation, with positive tests for the involvement of singlet oxygen. Peroxidation of the liposomes was inhibited by the presence of α-tocopherol and promoted by the presence of cholesterol in the membranes.     

Azide Quenching of Singlet Oxygen in Suspensions of Microenvironments of Neutral and Surface Charged Liposomes and Micelles

The azide anion is often used as a physical quencher of singlet oxygen, the important active intermediate in photosensitized oxidation. An observed effect of azide on the rate of a reaction is considered an indication to the involvement of singlet oxygen. In most biological photosensitizations, the light‐absorbing sensitizer is located in a membrane or in an intracellular organelle, whereas azide is water soluble. The quenching it causes relies on a physical encounter with singlet oxygen during the latter's short lifetime. This can happen either if azide penetrates into the membrane's lipid phase or if singlet oxygen is intercepted when diffusing in the aqueous phase. We demonstrate in this article the difference, in liposomes’ suspension, between the effect of azide when using a water‐soluble and membrane‐bound chemical targets of singlet oxygen, whereas this difference does not exist when micelles are used. We explain the difference on the population of sensitizer and target in the liposome vs micelle. We also show the effect that exists on azide quenching of singlet oxygen by electrically charged lipids in liposomes. This is a result of the accumulation or dilution of azide in the debye layer near the membranes’ surface, due to the surface Gouy–Chapman potential.     

Photosynthetic Tumor Oxygenation by Photosensitizer‐Containing Cyanobacteria for Enhanced Photodynamic Therapy

Sustained tumor oxygenation is of critical importance during type‐II photodynamic therapy (PDT), which depends on the intratumoral oxygen level for the generation of reactive oxygen species. Herein, the modification of photosynthetic cyanobacteria with the photosensitizer chlorin e6 (ce6) to form ce6‐integrated photosensitive cells, termed ceCyan, is reported. Upon 660 nm laser irradiation, sustained photosynthetic O₂ evolution by the cyanobacteria and the immediate generation of reactive singlet oxygen species (¹O₂) by the integrated photosensitizer could be almost simultaneously achieved for tumor therapy using type‐II PDT both in vitro and in vivo. This work contributes a conceptual while practical paradigm for biocompatible and effective PDT using hybrid microorganisms, displaying a bright future in clinical PDT by microbiotic nanomedicine.     

Specific Generation of Singlet Oxygen through the Russell Mechanism in Hypoxic Tumors and GSH Depletion by Cu‐TCPP Nanosheets for Cancer Therapy

The generation of singlet oxygen (¹O₂) during photodynamic therapy is limited by the precise cooperation of light, photosensitizer, and oxygen, and the therapeutic efficiency is restricted by the elevated glutathione (GSH) levels in cancer cells. Herein, we report that an ultrathin two‐dimensional metal–organic framework of Cu‐TCPP nanosheets (TCPP=tetrakis(4‐carboxyphenyl)porphyrin) can selectively generate ¹O₂ in a tumor microenvironment. This process is based on the peroxidation of the TCPP ligand by acidic H₂O₂ followed by reduction to peroxyl radicals under the action of the peroxidase‐like nanosheets and Cu²⁺, and their spontaneous recombination reaction by the Russell mechanism. In addition, the nanosheets can also deplete GSH. Consequently, the Cu‐TCPP nanosheets can selectively destroy tumor cells with high efficiency, constituting an attractive way to overcome current limitations of photodynamic therapy.     

Compatibilization effect of TiO2 nanoparticles on the phase structure of PET/PP/TiO2 nanocomposites

Polyethylene terephthalate (PET)/Polypropylene (PP)/TiO₂ nanocomposites were prepared by compounding a PP/TiO₂ nanocomposite premix with PET in absence and presence (up to 6 vol %) of maleic anhydride grafted polypropylene (PP‐g‐MA). In absence of PP‐g‐MA, the TiO₂ nanoparticles were mainly located at the PET/PP interface and to a lesser extent in the dispersed PET droplets. As the TiO₂ nanoparticles were coated by polyalcohol their surface could react with PP‐g‐MA and thus improving the compatibilization with PP. Therefore in presence of PP‐g‐MA the TiO₂ nanoparticles were preferentially located in the PP. The incorporated TiO₂ nanoparticles exerted a compatibilization effect on the PET/PP blend. Depending on the location of TiO₂ three different compatibilization mechanisms were proposed to be at work: (1) Locating at the interface, the TiO₂ nanoparticles decrease the free energy of mixing, and thus increase the thermodynamic stability of the nanocomposites; (2) The TiO₂ nanoparticles at the interface also prevent the coalescence of PET droplets; (3) Preferentially located in the PP matrix, the TiO₂ nanoparticles decreased the viscosity ratio which facilitated the droplet breakup of PET. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 1616–1624, 2009     

Type I Photosensitized Oxidation of Methionine†

Methionine (Met) is an essential sulfur‐containing amino acid, sensitive to oxidation. The oxidation of Met can occur by numerous pathways, including enzymatic modifications and oxidative stress, being able to cause relevant alterations in protein functionality. Under UV radiation, Met may be oxidized by direct absorption (below 250 nm) or by photosensitized reactions. Herein, kinetics of the reaction and identification of products during photosensitized oxidation were analyzed to elucidate the mechanism for the degradation of Met under UV‐A irradiation using pterins, pterin (Ptr) and 6‐methylpterin (Mep), as sensitizers. The process begins with an electron transfer from Met to the triplet‐excited state of the photosensitizer (Ptr or Mep), to yield the corresponding pair of radicals, Met radical cation (Met^?+) and the radical anion of the sensitizer (Sens^??). In air‐equilibrated solutions, Met^?+ incorporates one or two atoms of oxygen to yield methionine sulfoxide (MetO) and methionine sulfone (MetO₂), whereas Sens^?? reacts with O₂ to recover the photosensitizer and generate superoxide anion (O₂^??). In anaerobic conditions, further free‐radical reactions lead to the formation of the corresponding dihydropterin derivatives (H₂Ptr or H₂Mep).     

Hybrid TiO2–Ruthenium Nano‐photosensitizer Synergistically Produces Reactive Oxygen Species in both Hypoxic and Normoxic Conditions

Photodynamic therapy (PDT) is widely used to treat diverse diseases, but its dependence on oxygen to produce cytotoxic reactive oxygen species (ROS) diminishes the therapeutic effect in a hypoxic environment, such as solid tumors. Herein, we developed a ROS‐producing hybrid nanoparticle‐based photosensitizer capable of maintaining high levels of ROS under both normoxic and hypoxic conditions. Conjugation of a ruthenium complex (N3) to a TiO₂ nanoparticle afforded TiO₂‐N3. Upon exposure of TiO₂‐N3 to light, the N3 injected electrons into TiO₂ to produce three‐ and four‐fold more hydroxyl radicals and hydrogen peroxide, respectively, than TiO₂ at 160 mmHg. TiO₂‐N3 maintained three‐fold higher hydroxyl radicals than TiO₂ under hypoxic conditions via N3‐facilitated electron–hole reduction of adsorbed water molecules. The incorporation of N3 transformed TiO₂ from a dual type I and II PDT agent to a predominantly type I photosensitizer, irrespective of the oxygen content.     

Type I Photosensitization of 2′‐deoxyadenosine 5′‐monophosphate (5′‐dAMP) by Biopterin and its Photoproduct Formylpterin

Biopterin (Bip) and its photoproducts 6‐formylpterin (Fop) and 6‐carboxypterin (Cap) accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder where the protection against UV radiation fails because of the lack of melanin. These compounds absorb in the UV‐A inducing a potential photosensitizing action that can cause damage to DNA and other biomolecules. In this work, we have investigated the capability of these pterin derivatives (Pt) to act as photosensitizers under UV‐A irradiation for the degradation of 2′‐deoxyadenosine 5′‐monophosphate (5′‐dAMP) in aqueous solutions, as model DNA target. Steady‐state and time‐resolved experiments were performed and the effect of pH was evaluated. The results showed that photosensitized degradation of 5′‐dAMP was only observed under acidic conditions, and a mechanistic analysis revealed the participation of the triplet excited state of the pterin derivatives (³Pt*) by electron transfer yielding the corresponding pair of radical ions (Pt^?? and 5′‐dAMP^?+), with successive photosensitizer recovery by electron transfer from Pt^?? to O₂. Finally, 5′‐dAMP^?+ participates in subsequent reactions to yield degradation products.     

A Smart Photosensitizer–Manganese Dioxide Nanosystem for Enhanced Photodynamic Therapy by Reducing Glutathione Levels in Cancer Cells

Photodynamic therapy (PDT) has been applied in cancer treatment by utilizing reactive oxygen species to kill cancer cells. However, a high concentration of glutathione (GSH) is present in cancer cells and can consume reactive oxygen species. To address this problem, we report the development of a photosensitizer–MnO₂ nanosystem for highly efficient PDT. In our design, MnO₂ nanosheets adsorb photosensitizer chlorin e6 (Ce6), protect it from self‐destruction upon light irradiation, and efficiently deliver it into cells. The nanosystem also inhibits extracellular singlet oxygen generation by Ce6, leading to fewer side effects. Once endocytosed, the MnO₂ nanosheets are reduced by intracellular GSH. As a result, the nanosystem is disintegrated, simultaneously releasing Ce6 and decreasing the level of GSH for highly efficient PDT. Moreover, fluorescence recovery, accompanied by the dissolution of MnO₂ nanosheets, can provide a fluorescence signal for monitoring the efficacy of delivery.