Synergy Between Cell‐Penetrating Peptides and Singlet Oxygen Generators Leads to Efficient Photolysis of Membranes

Cell‐penetrating peptides such as TAT or R9 labeled with small organic fluorophores can lyse endosomes upon light irradiation. The photoendosomolytic activity of these compounds can in turn be used to deliver proteins and nucleic acids to the cytosol of live cells with spatial and temporal control. In this report, we examine the mechanisms by which such fluorescent peptides exert a photolytic activity using red blood cells as a membrane model. We show that the peptides TAT and R9 labeled with tetramethylrhodamine photolyze red blood cells by promoting the formation of singlet oxygen in the vicinity of the cells' membranes. In addition, unlabeled TAT and R9 accelerate the photolytic activity of the membrane‐bound photosensitizer Rose bengal in trans, suggesting that the cell‐penetrating peptides participate in the destabilization of photo‐oxidized membranes. Peptides and singlet oxygen generators therefore act in synergy to destroy membranes upon irradiation.     

H2S‐Activable MOF Nanoparticle Photosensitizer for Effective Photodynamic Therapy against Cancer with Controllable Singlet‐Oxygen Release

Photodynamic therapy (PDT) has emerged as an important minimally invasive tumor treatment technology. The search for an effective photosensitizer to realize selective cancer treatment has become one of the major foci in recent developments of PDT technology. Controllable singlet‐oxygen release based on specific cancer‐associated events, as another major layer of selectivity mode, has attracted great attention in recent years. Here, for the first time, we demonstrated that a novel mixed‐metal metal–organic framework nanoparticle (MOF NP) photosensitizer can be activated by a hydrogen sulfide (H₂S) signaling molecule in a specific tumor microenvironment for PDT against cancer with controllable singlet‐oxygen release in living cells. The effective removal of tumors in vivo further confirmed the satisfactory treatment effect of the MOF NP photosensitizer.     

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.     

Mass Spectrometric Study of Acoustically Levitated Droplets Illuminates Molecular‐Level Mechanism of Photodynamic Therapy for Cancer involving Lipid Oxidation

Even though the general mechanism of photodynamic cancer therapy is known, the details and consequences of the reactions between the photosensitizer‐generated singlet oxygen and substrate molecules remain elusive at the molecular level. Using temoporfin as the photosensitizer, here we combine field‐induced droplet ionization mass spectrometry and acoustic levitation techniques to study the “wall‐less” oxidation reactions of 18:1 cardiolipin and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) (POPG) mediated by singlet oxygen at the air–water interface of levitated water droplets. For both cardiolipin and POPG, every unsaturated oleyl chain is oxidized to an allyl hydroperoxide, which surprisingly is immune to further oxidation. This is attributed to the increased hydrophilicity of the oxidized chain, which attracts it toward the water phase, thereby increasing membrane permeability and eventually triggering cell death.     

Control of the photocatalytic activity of bimetallic complexes of pyropheophorbide-a by nucleic acids

Photocatalytic activity of a photosensitizer (PS) in an oligodeoxyribonucleotide duplex 5'-PS~ODN1/ODN2~Q-3' is inhibited because of close proximity of a quencher Q. The ODN2 in this duplex is selected to be longer than the ODN1. Therefore, in the presence of a nucleic acid (analyte), which is fully complementary to the ODN2 strand, the duplex is decomposed with formation of an analyte/ODN2~Q duplex and a catalytically active, single stranded PS~ODN1. In this way the catalytic activity of the PS can be controlled by the specific nucleic acids. We applied this reaction earlier for the amplified detection of ribonucleic acids in live cells (Arian, D.; Cló, E.; Gothelf, K.; Mokhir, A. Chem.-Eur. J.2010, 16(1), 288). As a photosensitizer (PS) we used In(3+)(pyropheophorbide-a)chloride and as a quencher (Q)--Black-Hole-Quencher-3 (BHQ-3). The In(3+) complex is a highly active photocatalyst in aqueous solution. However, it can coordinate additional ligands containing thiols (e.g., proteins, peptides, and aminoacids), that modulate properties of the complex itself and of the corresponding bio- molecules. These possible interactions can lead to undesired side effects of nucleic acid controlled photocatalysts (PS~ODN1/ODN2～Q) in live cells. In this work we explored the possibility to substitute the In(3+) complex for those ones of divalent metal ions, Zn(2+) and Pd(2+), which exhibit lower or no tendency to coordinate the fifth ligand. We found that one of the compounds tested (Pd(pyropheophorbide-a) is as potent and as stable photosensitizer as its In(3+) analogue, but does not coordinate additional ligands that makes it more suitable for cellular applications. When the Pd complex was introduced in the duplex PS~ODN1/ODN2~Q as a PS, its photocatalytic activity could be controlled by nucleic acids as efficiently as that of the corresponding In(3+) complex.     

Selective Photooxidation of a Mustard‐Gas Simulant Catalyzed by a Porphyrinic Metal–Organic Framework

The photooxidation of a mustard‐gas simulant, 2‐chloroethyl ethyl sulfide (CEES), is studied using a porphyrin‐based metal–organic framework (MOF) catalyst. At room temperature and neutral pH value, singlet oxygen is generated by PCN‐222/MOF‐545 using an inexpensive and commercially available light‐emitting diode. The singlet oxygen produced by PCN‐222/MOF‐545 selectively oxidizes CEES to the comparatively nontoxic product 2‐chloroethyl ethyl sulfoxide (CEESO) without formation of the highly toxic sulfone product. In comparison to current methods, which utilize hydrogen peroxide as an oxidizing agent, this is a more realistic, convenient, and effective method for mustard‐gas detoxification.     

Nucleobase‐Modified PNA Suppresses Translation by Forming a Triple Helix with a Hairpin Structure in mRNA In Vitro and in Cells

Compounds that bind specifically to double‐stranded regions of RNA have potential as regulators of structure‐based RNA function; however, sequence‐selective recognition of double‐stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA–RNA triplexes. Herein, we demonstrate that a 9‐mer PNA forms a sequence‐specific PNA–RNA triplex with a dissociation constant of less than 1 nm at physiological pH. The triplex formed within the 5′ untranslated region of an mRNA reduces the protein expression levels both in vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex‐forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double‐stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein–RNA interactions.     

Peripheral RAFT Polymerization on a Covalent Organic Polymer with Enhanced Aqueous Compatibility for Controlled Generation of Singlet Oxygen

A covalent organic polymer (COP) is prepared by crosslinking the photosensitizer 4,4′,4′′,4′′′‐(porphyrin‐5,10,15,20‐tetrayl)tetraaniline (TAPP) with 4,4′‐(anthracene‐9,10‐diyl)dibenzoic acid (ADDA) via 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide/4‐dimethylaminopyridine coupling. The COP is further modified with a hydrophilic polymer, poly(poly(ethylene glycol) methyl ether methacrylate) by grafting‐from reversible‐addition‐fragmentation chain transfer (RAFT) polymerization to enhance its solubility in various solvents. The modified COP can bind singlet oxygen through the formation of endoperoxide by ADDA upon the exposure to red light irradiation. Singlet oxygen can be then released via the photodynamic mechanism or the cycloreversion by endoperoxide when heated at 110 °C. These results open new possibilities for simultaneous generation of singlet oxygen by the photodynamic route and singlet oxygen carriers, demonstrating promise for treating hypoxic tumors.     

Lysosome‐Targeting Amplifiers of Reactive Oxygen Species as Anticancer Prodrugs

Cancer cells produce elevated levels of reactive oxygen species, which has been used to design cancer specific prodrugs. Their activation relies on at least a bimolecular process, in which a prodrug reacts with ROS. However, at low micromolar concentrations of the prodrugs and ROS, the activation is usually inefficient. Herein, we propose and validate a potentially general approach for solving this intrinsic problem of ROS‐dependent prodrugs. In particular, known prodrug 4‐(N ‐ferrocenyl‐N ‐benzylaminocarbonyloxymethyl)phenylboronic acid pinacol ester was converted into its lysosome‐specific analogue. Since lysosomes contain a higher concentration of active ROS than the cytoplasm, activation of the prodrug was facilitated with respect to the parent compound. Moreover, it was found to exhibit high anticancer activity in a variety of cancer cell lines (IC₅₀=3.5–7.2 μm ) and in vivo (40 mg kg⁻¹, NK/Ly murine model) but remained weakly toxic towards non‐malignant cells (IC₅₀=15–30 μm ).     

RNA Interference Controlled by Light of Variable Wavelength

Known molecular, “caged” siRNAs are activated by UV light. Since the light of this type is toxic to cells, the uncaging can cause undesired side effects. A modular, molecular system for designing siRNAs is reported, which can be activated by non‐toxic light in live cells. For example, siRNAs responsive to green and red light are described. The uncaging is mediated by ¹O₂ photogenerated on a photosensitizer, which is attached to the 3′‐terminus of the lagging strand. The 5′‐terminus of the guide strand is alkylated (“caged”) with a 9‐anthracenyl residue. The latter fragment reacts with the ¹O₂ with formation of the free (uncaged) 5′‐OH terminus. Simultaneously with the uncaging the photosensitizer is bleached and no more ¹O₂ is generated after this process is completed. The photoactivation of the siRNAs described here is not toxic to cells.     

Membrane Damage Efficiency of Phenothiazinium Photosensitizers

Structure–activity relationships have been widely reported for porphyrin and phthalocyanine photosensitizers, but not for phenothiazinium derivatives. Here, four phenothiazinium salts (methylene blue, toluidine blue O, 1,9‐dimethyl methylene blue and the pentacyclic derivative DO15) were used to investigate how the ability to damage membranes is affected by membrane/solution partition, photophysical properties and tendency to aggregation of the photosensitizer. These two latter aspects were studied both in isotropic solutions and in membranes. Membrane damage was assessed by leakage of a fluorescent probe entrapped in liposomes and by generation of thiobarbituric acid‐reactive species (TBARS), while structural changes at the lipid bilayer were detected by small‐angle X‐ray scattering. We observed that all compounds had similar singlet‐oxygen quantum yields in ethanol, but only the photosensitizers that had higher membrane/solution partition (1,9‐dimethyl methylene blue and DO15, the latter having the higher value) could permeabilize the lipid bilayer. Moreover, of these two photosensitizers, only DO15 altered membrane structure, a result that was attributed to its destabilization of higher order aggregates, generation of higher amounts of singlet oxygen within the membranes and effective electron‐transfer reaction within its dimers. We concluded that membrane‐based protocols can provide a better insight on the photodynamic efficiency of the photosensitizer.     

NIR‐Light‐Activated Combination Therapy with a Precise Ratio of Photosensitizer and Prodrug Using a Host–Guest Strategy

The co‐delivery of photosensitizers with prodrugs sensitive to reactive oxygen species (ROS) for light‐triggered ROS generation and cascaded prodrug activation has drawn tremendous attention. However, the absence of a feasible method to deliver the two components at a precise ratio has impaired the application potential. Herein, we report an efficient method to produce a nanosized platform for the delivery of an optimized ratio of the two components by the means of host–guest strategy for maximizing the combination therapy efficacy of cancer treatment. The key features of this host–guest strategy for the combination therapy are that the ratio between photosensitizer and ROS‐sensitive prodrug can be easily tuned, near‐infrared (NIR) irradiation can sensitize the photosensitizer and activate the paclitaxel prodrug for its release, and the accumulation process can be tracked by NIR imaging to maximize the efficacy of photodynamic and chemotherapy.     

DNA-programmed control of photosensitized singlet oxygen production

DNA sequence-controlled on-and-off switching of a singlet oxygen sensitizer has been developed and demonstrated. The singlet oxygen photosensitizer pyropheophorbide-a (P) was attached to a 15-mer nucleotide sequence. A molecule that could quench the sensitizer, the so-called "black hole quencher 3" (Q), was attached to a complementary nucleotide strand. Upon hybridization of the two conjugates, singlet oxygen production from P was completely shut down. Upon the addition of a third DNA sequence that can displace and release the P-DNA conjugate from the P-Q pair, up to 85% of the singlet oxygen production was recovered. This system is a model for a benign drug that becomes active only in the presence of a specific targeted nucleotide sequence.     

Site-specific prodrug release using visible light

Using a photosensitization-singlet oxygenation-dioxetane cleavage strategy, a photodynamic prodrug system has been developed, whereby drugs bearing carbonyl groups can first be attached to a photosensitizer to give a photosensitizer-drug complex and then released from the complex upon visible light irradiation. Visible light, which has good penetration through tissue, generates singlet oxygen via the photosensitizer, which then releases the prodrug when and where required. With this system, drug mimics and methyl esters of NSAIDs have been successfully incorporated with photosensitizers related to verteporfin and then released by visible light illumination in high to quantitative yields within minutes.     

Advanced Photosensitizer Activation Strategies for Smarter Photodynamic Therapy Beacons

Photodynamic therapy (PDT) is a clinical treatment in which a light‐absorbing drug called a photosensitizer (PS) is combined with light and molecular oxygen to generate cytotoxic singlet oxygen. PDT provides additional tissue selectivity compared to conventional chemotherapy as singlet oxygen is generated only in areas in which PS accumulates and that are simultaneously illuminated by a light source with sufficient irradiance and dose. Early PDT beacons built on this concept by adding an analyte‐responsive element that simultaneously turns on PDT and fluorescence, providing both an additional layer of selectivity and real‐time feedback of the PS′s activation state. More recent PDT beacons have expanded this idea, with new methods now available for sensing analytes, generating singlet oxygen, and reporting treatment status. In this Minireview, we consider developments in advanced activation strategies implemented in therapeutic and theranostic beacons.     

Oxygen scavengers and sensitizers for reduced oxygen inhibition in radical photopolymerization

Oxygen inhibition in the free‐radical photopolymerization of (meth)acrylates is one of the most challenging problems in thin film application. Apart from the utilization of an inert gas atmosphere, additives reducing oxygen inhibition due to production of new propagating centers are used. In the present study, a more straightforward approach to reduce oxygen inhibition by photosensitized generation of singlet oxygen and subsequent scavenging of these species by selective singlet oxygen trappers was investigated. The potential of 1,2‐dions conventionally used as type‐II photoinitiators for visible light polymerization to function as singlet oxygen generators was verified in sensitized steady state photooxidation experiments in solution. A set of furan and anthracene derivatives were tested as oxygen scavengers and their corresponding relative reaction rates were determined. The ability of these sensitizer/scavenger systems to reduce oxygen inhibition in practical applications was studied in photo‐DSC‐experiments. In thin film polymerization (investigated by ATR‐FTIR), the formation of insufficiently cured surfaces could be prevented by the usage of singlet oxygen trappers. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6916–6927, 2008     

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.     

Photosensitized Oxidation of Tetrabromobisphenol A by Humic Acid in Aqueous Solution†

The brominated flame retardant 3,3′,5,5′‐tetrabromobisphenol A (TBBPA) may accumulate in the environment, including surface waters, and degrade there to potentially toxic products. We have previously shown that singlet oxygen (¹O₂), produced by irradiation of rose bengal with visible light, oxidizes Triton X‐100‐solubilized TBBPA to yield the 2,6‐dibromo‐p‐benzosemiquinone anion radical while consuming oxygen (Environ. Sci. Technol. 42 , 166, 2008). Here, we report that a similar ¹ O ₂ ‐induced oxidation can be initiated in aqueous solutions by the irradiation of TBBPA dissolved in a humic acid (HA) solution. HA is a known weak ¹ O ₂ photosensitizer and we indeed detected the infrared ¹ O ₂ phosphorescence from HA preparations in D ₂ O. When an aqueous preparation of HA was irradiated (λ > 400 nm) in the presence of TBBPA, oxygen was consumed, and the 2,6‐dibromo‐ p ‐benzosemiquinone anion radical was generated and detected using electron paramagnetic resonance. Radical formation and oxygen consumption were inhibited by sodium azide, a singlet oxygen quencher. Our results suggest that solar radiation, in the presence of HA, may play an important role in the photodegradation of TBBPA in the aquatic environment.     

Site-directed photoproteolysis of 8-oxoguanine DNA glycosylase 1 (OGG1) by specific porphyrin-protein probe conjugates: a strategy to improve the effectiveness of photodynamic therapy for cancer

The specific light-induced, non-enzymatic photolysis of mOGG1 by porphyrin-conjugated or rose bengal-conjugated streptavidin and porphyrin-conjugated or rose bengal-conjugated first specific or secondary anti-IgG antibodies is reported. The porphyrin chlorin e6 and rose bengal were conjugated to either streptavidin, rabbit anti-mOGG1 primary specific antibody fractions or goat anti-rabbit IgG secondary antibody fractions. Under our experimental conditions, visible light of wavelengths greater than 600 nm induced the non-enzymatic degradation of mOGG1 when this DNA repair enzyme either directly formed a complex with chlorin e6-conjugated anti-mOGG1 primary specific antibodies or indirectly formed complexes with either streptavidin-chlorin e6 conjugates and biotinylated first specific anti-mOGG1 antibodies or first specific anti-mOGG1 antibodies and chlorin e6-conjugated anti-rabbit IgG secondary antibodies. Similar results were obtained when rose bengal was used as photosensitizer instead of chlorin e6. The rate of the photochemical reaction of mOGG1 site-directed by all three chlorin e6 antibody complexes was not affected by the presence of the singlet oxygen scavenger sodium azide. Site-directed photoactivatable probes having the capacity to generate reactive oxygen species (ROS) while destroying the DNA repair system in malignant cells and tumors may represent a powerful strategy to boost selectivity, penetration and efficacy of current photodynamic (PDT) therapy methodologies.