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.     

Eradication of Gram‐Positive and Gram‐Negative Bacteria by Photosensitizers Immobilized in Polystyrene

Immobilization of photosensitizers in polymers opens prospects for their continuous and reusable application. Methylene blue (MB) and Rose Bengal were immobilized in polystyrene by mixing solutions of the photosensitizers in chloroform with a polymer solution, followed by air evaporation of the solvent. This procedure yielded 15–140 μm polymer films with a porous surface structure. The method chosen for immobilization ensured 99% enclosure of the photosensitizer in the polymer. The antimicrobial activity of the immobilized photosensitizers was tested against Gram‐positive and Gram‐negative bacteria. It was found that both immobilized photosensitizers exhibited high antimicrobial properties, and caused by a 1.5–3 log₁₀ reduction in the bacterial concentrations to their total eradication. The bactericidal effect of the immobilized photosensitizers depended on the cell concentration and on the illumination conditions. Scanning electron microscopy was used to prove that immobilized photosensitizers excited by white light caused irreversible damage to microbial cells. Photosensitizers immobilized on a solid phase can be applied for continuous disinfection of wastewater bacteria.     

Self-Assembly of Mitochondria-Targeted Photosensitizer to Increase Photostability and Photodynamic Therapeutic Efficacy in Hypoxia

The development of photosensitizers for cancer photodynamic therapy has been challenging due to their low photostability and therapeutic inefficacy in hypoxic tumor microenvironments. To overcome these issues, we have developed a mitochondria-targeted photosensitizer consisting of an indocyanine moiety with triphenylphosphonium arms, which can self-assemble into spherical micelles directed to mitochondria. Self-assembly of the photosensitizer resulted in a higher photostability by preventing free rotation of the indoline ring of the indocyanine moiety. The mitochondria targeting capability of the photosensitizer allowed it to utilize intramitochondrial oxygen. We found that the mitochondria-targeted photosensitizer localized to mitochondria and induced apoptosis of cancer cells both normoxic and hypoxic conditions through generation of ROS. The micellar self-assemblies of the photosensitizer were further confirmed to selectively localize to tumor tissues in a xenograft tumor mouse model through passive targeting and showed efficient tumor growth inhibition.     

A Comparison of Functional Bladder Damage after Intravesical Photodynamic Therapy with Three Different Photosensitizers

Abstract— The influence of type of photosensitizer, drug and light dose, and time interval between photosensitizer and illumination on the extent of photodynamic therapy (PDT)-induced bladder damage and recovery was investigated using a mouse model. The three photosensitizers studied were Photofrin, meso-tetrahydroxyphenylchlorin (m-THPC) and bacteriochlorin a (BCA). Functional bladder damage was quantitatively assessed from increases in urination frequency index (FI) at 1-35 weeks after illumination and histological damage was qualitatively assessed at 1 day, 1, 2 and 12 weeks. Photofrin-mediated PDT caused an acute increase in FI at 1 week, with recovery within 2-8 weeks after light doses of 2.7-8.2 J/cm². After higher light doses there was only partial recovery. Previous results indicated that the acute response and rate of recovery was the same whether Photofrin was given at 1 day or up to 7 days before illumination. The m-THPC-mediated PDT at drug doses of 0.3 mg/kg also resulted in a marked acute response with good recovery, even after 10.8 J/cm². Lower drug doses in combination with 5.4 J/cm² did not result in acute or late damage. There was no significant difference in acute response when m-THPC was given 1, 3 or 7 days before illumination, although recovery was faster for the longer illumination intervals (3 or 7 days). Illumination at 1 h after 20 mg/kg BCA induced an acute response within 2 days after illumination, with recovery within 4-8 weeks. Lower drug doses did not result in damage. The most prominent histological changes during the acute period with all three photosensitizers were submucosal edema and vessel dilation, with epithelial denudation (depending on drug/light dose). We conclude that BCA and m-THPC are both potent new photosensitizers. They can induce a moderate to severe acute bladder response with complete healing over a period of a few weeks. The photosensitizer m-THPC is very effective with low doses of photosensitizer and light, whereas relatively high doses of BCA and light are required to obtain equivalent functional bladder damage in our mouse model.     

DYNAMIC CAPILLAROSCOPY: A MINIMALLY INVASIVE TECHNIQUE FOR ASSESSING PHOTODYNAMIC EFFECTS in vivo

Abstract A noninvasive method for visualizing the microvasculature in the mouse tail is described, consisting of a custom-built microscope with through-lens illumination. The microscope is fitted with a television camera and images can be recorded on videotape and displayed on a television monitor. Blood vessels are imaged as columns of red blood cells, in which flow is clearly observed. Administration of photosensitizers and illumination with the standard light source produces no observable photodynamic effect on blood flow. The combination of photosensitizer and a more intense light source (either broadband light from a filtered mercury arc or red light from a laser) causes photodynamic cessation of flow within a few minutes. The magnitude of the effect is dependent on the dose and nature of the photosensitizer, the delay after photosensitization and the match between the laser light and the absorption spectra of the photosensitizers in the red region. We conclude that the technique yields results consistent with the known photodynamic effects of the photosensitizers in tumors and propose its use as an initial screening method in YWO , as a means of conducting pharmacokinetic experiments and as an assay of prolonged cutaneous photosensitivity.     

Cationic fullerenes are effective and selective antimicrobial photosensitizers

Fullerenes are soccer ball-shaped molecules composed of carbon atoms, and, when derivatized with functional groups, they become soluble and can act as photosensitizers. Antimicrobial photodynamic therapy combines a nontoxic photosensitizer with harmless visible light to generate reactive oxygen species that kill microbial cells. We have compared the antimicrobial activity of six functionalized C(60) compounds with one, two, or three hydrophilic or cationic groups in combination with white light against gram-positive bacteria, gram-negative bacteria, and fungi. After a 10 min incubation, the bis- and tris-cationic fullerenes were highly active in killing all tested microbes (4-6 logs) under conditions in which mammalian cells were comparatively unharmed. These compounds performed significantly better than a widely used antimicrobial photosensitizer, toluidine blue O. The high selectivity and efficacy exhibited by these photosensitizers encourage further testing for antimicrobial applications.     

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.     

A Genetically Encoded RNA Photosensitizer for Targeted Cell Regulation

Genetically encoded RNA devices have emerged for various cellular applications in imaging and biosensing, but their functions as precise regulators in living systems are still limited. Inspired by protein photosensitizers, we propose here a genetically encoded RNA aptamer based photosensitizer (GRAP). Upon illumination, the RNA photosensitizer can controllably generate reactive oxygen species for targeted cell regulation. The GRAP system can be selectively activated by endogenous stimuli and light of different wavelengths. Compared with their protein analogues, GRAP is highly programmable and exhibits reduced off-target effects. These results indicate that GRAP enables efficient noninvasive target cell ablation with high temporal and spatial precision. This new RNA regulator system will be widely used for optogenetics, targeted cell ablation, subcellular manipulation, and imaging.     

Research advances in the use of tetrapyrrolic photosensitizers for photodynamic therapy

Photodynamic therapy (PDT) is a promising new treatment modality for several diseases, most notably cancer. In PDT, light, O2, and a photosensitizing drug are combined to produce a selective therapeutic effect. Lately, there has been active research on new photosensitizer candidates, because the most commonly used porphyrin photosensitizers are far from ideal with respect to PDT. Finding a suitable photosensitizer is crucial in improving the efficacy of PDT. Recent synthetic activity has created such a great number of potential photosensitizers for PDT that it is difficult to decide which ones are suitable for which pathological conditions, such as various cancer species. To facilitate the choice of photosensitizer, this review presents a thorough survey of the photophysical and chemical properties of the developed tetrapyrrolic photosensitizers. Special attention is paid to the singlet-oxygen yield (PhiDelta) of each photosensitizer, because it is one of the most important photodynamic parameters in PDT. Also, in the survey, emphasis is placed on those photosensitizers that can easily be prepared by partial syntheses starting from the abundant natural precursors, protoheme and the chlorophylls. Such emphasis is justified by economical and environmental reasons. Several of the most promising photosensitizer candidates are chlorins or bacteriochlorins. Consequently, chlorophyll-related chlorins, whose PhiDelta have been determined, are discussed in detail as potential photosensitizers for PDT. Finally, PDT is briefly discussed as a treatment modality, including its clinical aspects, light sources, targeting of the photosensitizer, and opportunities.     

A high-swelling reagent scaffold suitable for use in aqueous and organic solvents

A polymeric scaffold with excellent swelling properties in organic and aqueous environments is highly desirable for the medicinal chemist. Here, we demonstrate that a cross-linked polyacrylamide hydrogel that displays large swelling properties in both organic solvent and water can serve as a scaffold for the photosensitizer hematoporphyrin. Upon exposure to light, the resulting resin efficiently generates singlet oxygen which can then react with appropriate substrates.     

Synthesis of a Photostable Near‐Infrared‐Absorbing Photosensitizer for Selective Photodamage to Cancer Cells

A new class of near‐infrared (NIR)‐absorptive (>900 nm) photosensitizer based on a phenothiazinium scaffold is reported. The stable solid compound, o‐DAP, the oxidative form of 3,7‐bis(4‐methylaminophenyl)‐10H‐phenothiazine, can generate reactive oxygen species (ROS, singlet oxygen and superoxide) under appropriate irradiation conditions. After biologically evaluating the intracellular uptake, localization, and phototoxicity of this compound, it was concluded that o‐DAP is photostable and a potential selective photodynamic therapy (PDT) agent under either NIR or white light irradiation because its photodamage is more efficient in cancer cells than in normal cells and is without significant dark toxicity. This is very rare for photosensitizers in PDT applications.     

Theoretical and experimental analysis of the luminescence signal of singlet oxygen for different photosensitizers

After the generation by different photosensitizers, the direct detection of singlet oxygen is performed by measuring its luminescence at 1270 nm. Using an infrared sensitive photomultiplier, the complete rise and decay time of singlet oxygen luminescence is measured at different concentrations of a photosensitizer, quencher, or oxygen. This allows the extraction of important information about the photosensitized generation of singlet oxygen and its decay, in particular at different oxygen concentrations. Based on theoretical considerations all important relaxation rates and rate constants were determined for the triplet T(1) states of the photosensitizers and for singlet oxygen. In particular, depending on the oxygen or quencher concentration, the rise or the decay time of the luminescence signal exhibit different meanings regarding the lifetime of singlet oxygen or triplet T(1)-state. To compare with theory, singlet oxygen was generated by nine different photosensitizers dissolved in either H2O, D2O or EtOD. When using H2O as solvent, the decaying part of the luminescence signal is frequently not the lifetime of singlet oxygen, in particular at low oxygen concentration. Since cells show low oxygen concentrations, this must have an impact when looking at singlet oxygen detection in vitro or in vivo.     

Photodynamic therapy: A special emphasis on nanocarrier-mediated delivery of photosensitizers in antimicrobial therapy

Resistance to antimicrobial drugs is an impending healthcare problem of growing significance. In the post-antibiotic era, there is a huge push to develop new tools for effectively treating bacterial infections. Photodynamic therapy involves the use of a photosensitizer that is activated by the use of light of an appropriate wavelength in the presence of oxygen. This results in the generation of singlet oxygen molecules that can kill the target cells, including cancerous cells and microbial cells. Photodynamic therapy is shown to be effective against parasites, viruses, algae, and bacteria. To achieve high antimicrobial activity, a sufficient concentration of photosensitizer should enter the microbial cells. Generally, photosensitizers tend to aggregate in aqueous environments resulting in the weakening of photochemical activity and lowering their uptake into cells. Nanocarrier systems are shown to be efficient in targeting photosensitizers into microbial cells and improve their therapeutic efficiency by enhancing the internalization of photosensitizers into microbial cells. This review aims to highlight the basic principles of photodynamic therapy with a special emphasis on the use of nanosystems in delivering photosensitizers for improving antimicrobial photodynamic therapy.     

A Closer Look at Dark Toxicity of the Photosensitizer TMPyP in Bacteria

Photodynamic inactivation of bacteria (PIB ) is based on photosensitizers which absorb light and generate reactive oxygen species (ROS ), killing cells via oxidation. PIB is evaluated by comparing viability with and without irradiation, where reduction of viability in the presence of the photosensitizer without irradiation is considered as dark toxicity. This effect is controversially discussed for photosensitizers like TMP yP (5,10,15,20‐Tetrakis(1‐methyl‐4‐pyridinio)porphyrin tetra(p‐toluensulfonate). TMP yP shows a high absorption coefficient for blue light and a high yield of ROS production, especially singlet oxygen. Escherichia coli and Bacillus atrophaeus were incubated with TMP yP and irradiated with different light sources at low radiant exposures (μW per cm²), reflecting laboratory conditions of dark toxicity evaluation. Inactivation of E. coli occurs for blue light, while no effect was detectable for wavelengths >450 nm. Being more susceptible toward PIB , growth of B. atrophaeus is even reduced for light with emission >450 nm. Decreasing the light intensities to nW per cm² for B. atrophaeus , application of TMP yP still caused bacterial killing. Toxic effects of TMP yP disappeared after addition of histidine, quenching residual ROS . Our experiments demonstrate that the evaluation of dark toxicity of a powerful photosensitizer like TMP yP requires low light intensities and if necessary additional application of substances quenching any residual ROS .     

Dithiaporphyrin derivatives as photosensitizers in membranes and cells

We synthesized a series of analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-21,23-dithiaporphyrin (I) as potential photosensitizers for photodynamic therapy (PDT). The photosensitizers differ in the length of the side chains that bind the carboxyl to the phenol at positions 10 and 15 of the thiaporphyrin. The spectroscopic, photophysical, and biophysical properties of these photosensitizers are reported. The structural changes have almost no effect on the excitation/emission spectra with respect to I's spectra or on singlet oxygen generation in MeOH. All of the photosensitizers have a very high, close to 1.00, singlet oxygen quantum yield in MeOH. On the contrary, singlet oxygen generation in liposomes was considerably affected by the structural change in the photosensitizers. The photosensitizers possessing short side chains (one and three carbons) showed high quantum yields of around 0.7, whereas the photosensitizers possessing longer side chains showed smaller quantum yield, down to 0.14 for compound X (possessing side-chain length of 10 carbons), all at 1 microM. Moreover a self-quenching process of singlet oxygen was observed, and the quantum yield decreased as the photosensitizer's concentration increased. We measured the binding constant of I to liposomes and found Kb = 23.3 +/- 1.6 (mg/mL)-1. All the other photosensitizers with longer side chains exhibited very slow binding to liposomes, which prevented us from assessing their Kb's. We carried out fluorescence resonance energy transfer (FRET) measurements to determine the relative depth in which each photosensitizer is intercalated in the liposome bilayer. We found that the longer the side chain the deeper the photosensitizer core is embedded in the bilayer. This finding suggests that the photosensitizers are bound to the bilayer with their acid ends close to the aqueous medium interface and their core inside the bilayer. We performed PDT with the dithiaporphyrins on U937 cells and R3230AC cells. We found that the dark toxicity of the photosensitizers with the longer side chain (X, VI, V) is significantly higher than the dark toxicity of sensitizers with shorter side chains (I, III, IV). Phototoxicity measurements showed the opposite direction; the photosensitizers with shorter side chains were found to be more phototoxic than those with longer side chains. These differences are attributed to the relationship between diffusion and endocytosis in each photosensitizer, which determines the location of the photosensitizer in the cell and hence its phototoxicity.     

新型绿色农药——光活化农药

光活化农药包括光活化杀虫剂和光活化除草剂, 与传统农药相比具有廉价、高效、无污染等优越性。光活化农药的关键是光敏剂, 在有光和氧存在条件下光敏剂催化产生单重态氧, 杀灭害虫。光敏剂效果取决于其单重态氧的量子产率, 其分子本身只起催化作用并不介入毒性反应, 并且易被降解, 因此对环境无污染。由于单重态氧在细胞上的生物化学作用点多, 使害虫不易对其产生抗药性。这类绿色农药正在逐步走向实用化, 它的发展将会大大改善因长期使用传统农药而造成的危害, 对农业发展将会产生巨大的促进作用。     

GSH and H2O2 Co‐Activatable Mitochondria‐Targeted Photodynamic Therapy under Normoxia and Hypoxia

Currently, photosensitizers (PSs) that are microenvironment responsive and hypoxia active are scarcely available and urgently desired for antitumor photodynamic therapy (PDT). Presented herein is the design of a redox stimuli activatable metal‐free photosensitizer (aPS), also functioning as a pre‐photosensitizer as it is converted to a PS by the mutual presence of glutathione (GSH) and hydrogen peroxide (H₂O₂) with high specificity on a basis of domino reactions on the benzothiadiazole ring. Superior to traditional PSs, the activated aPS contributed to efficient generation of reactive oxygen species including singlet oxygen and superoxide ion through both type 1 and type 2 pathways, alleviating the aerobic requirement for PDT. Equipped with a triphenylphosphine ligand for mitochondria targeting, ^mito aPS showed excellent phototoxicity to tumor cells with low light fluence under both normoxic and hypoxic conditions, after activation by intracellular GSH and H₂O₂. The ^mito aPS was also compatible to near infrared PDT with two photon excitation (800 nm) for extensive bioapplications.     

First-Row d6 Metal Complex Enables Photon Upconversion and Initiates Blue Light-Dependent Polymerization with Red Light

Photosensitizers for sensitized triplet-triplet annihilation upconversion (sTTA-UC) often rely on precious heavy metals, whereas coordination complexes based on abundant first-row transition metals are less common. This is mainly because long-lived triplet excited states are more difficult to obtain for 3d metals, particularly when the d-subshell is only partially filled. Here, we report the first example of sTTA-UC based on a 3d⁶ metal photosensitizer yielding an upconversion performance competitive with precious metal-based analogues. Using a newly developed Cr⁰ photosensitizer featuring equally good photophysical properties as an Os^II benchmark complex in combination with an acetylene-decorated anthracene annihilator, red-to-blue upconversion is achievable. The upconversion efficiency under optimized conditions is 1.8 %, and the excitation power density threshold to reach the strong annihilation limit is 5.9 W/cm². These performance factors, along with high photostability, permit the initiation of acrylamide polymerization by red light, based on radiative energy transfer between delayed annihilator fluorescence and a blue light absorbing photo-initiator. Our study provides the proof-of-concept for photon upconversion with elusive first-row analogues of widely employed precious d⁶ metal photosensitizers, and for their application in photochemical reactions triggered by excitation wavelengths close to near-infrared.     

A new spirofluorene-based nonplanar PBI-dyad and its utilization in the film-based photo-production of singlet oxygen

Traditional photosensitizers are predominantly based on various types of polypyrrole macrocycles, which are generally used in homogeneous and/or suspension states. In the present study, a new non-polypyrrole-based photosensitizer(LW-PBI) was developed via the introduction of a nonplanar spirofluorene into a derivative of perylene bisimides(PBI) containing two longalkyl chains. Photophysical studies demonstrated that the compound shows good solubility in common organic solvents, great photochemical stability, and high absorption efficiency in the visible light region. Due to containing of two energetically matchable, independent fluorescent units, the compound as prepared displays strong tendency to form non-fluorescent chargeseparated states under light irradiation in polar solvents. Based on the merits, LW-PBI was examined for its catalytic property in the photo-production of singlet oxygen in film state. Luckily, the compound is an effective photosensitizer in the generation of the active oxygen species as verified by its unique reaction with uric acid(UA). Further studies revealed that the effective photoproduction of singlet oxygen can be also realized via the utilization of a tiny and low-price LED lamp as a light source and as a film support. Detailed studies on the application of the conceptual device as a medical instrument for photodynamic therapy(PDT) are in progress.