Photodynamic Therapy: Critical PDT Theory†

Photodynamic therapy can be useful for the eradication of malignant cells at sites that are accessible to light delivery. There are few adverse effects, with many clinical reports indicating that PDT has curative potential. Patients with minimal disease, where success is more likely, are also sought by those promoting other protocols. New photosensitizing agents that initiate light-catalyzed reactions continue to be discovered. Reports describing advances in understanding fundamental aspects of photobiology are always of interest. But, implications for treatment of neoplasia and other diseases are not always justified, especially when poorly penetrating wavelengths of light are employed, often at very high light doses. Efficacy is sometimes estimated by protocols that may not accurately measure photokilling. Many reports claiming potential clinical relevance for in vitro observations are based on a limited understanding of the determinants of clinical efficacy. The future of photodynamic therapy depends on an appreciation of what can be accomplished, especially when used with other modalities, but will also depend on the goals and interests of granting agencies, pharmaceutical groups, and clinical personnel. This commentary is intended to provide some thoughts on current research efforts, especially where clinical implications are suggested, hinted at or otherwise implied.     

The Biochemical Mechanisms of Antimicrobial Photodynamic Therapy†

The unbridled dissemination of multidrug-resistant pathogens is a major threat to global health and urgently demands novel therapeutic alternatives. Antimicrobial photodynamic therapy (aPDT) has been developed as a promising approach to treat localized infections regardless of drug resistance profile or taxonomy. Even though this technique has been known for more than a century, discussions and speculations regarding the biochemical mechanisms of microbial inactivation have never reached a consensus on what is the primary cause of cell death. Since photochemically generated oxidants promote ubiquitous reactions with various biomolecules, researchers simply assumed that all cellular structures are equally damaged. In this study, biochemical, molecular, biological and advanced microscopy techniques were employed to investigate whether protein, membrane or DNA damage correlates better with dose-dependent microbial inactivation kinetics. We showed that although mild membrane permeabilization and late DNA damage occur, no correlation with inactivation kinetics was found. On the other hand, protein degradation was analyzed by three different methods and showed a dose-dependent trend that matches microbial inactivation kinetics. Our results provide a deeper mechanistic understanding of aPDT that can guide the scientific community toward the development of optimized photosensitizing drugs and also rationally propose synergistic combinations with antimicrobial chemotherapy.     

Redaporfin Development for Photodynamic Therapy and its Combination with Glycolysis Inhibitors†

Photodynamic therapy (PDT) remains an underutilized treatment modality in oncology. Many efforts have been dedicated to the development of better photosensitizers, better formulations and delivery methods, rigorous planning of light dose distribution in tissues, mechanistic insight, improvement of treatment protocols and combinations with other therapeutic agents. Hopefully, progress in all these fields will eventually expand the use of PDT. Here we offer a brief review of our own contribution to the development of a photosensitizer for PDT – redaporfin – currently in Phase II clinical trials, and present data on its combination with two glycolysis inhibitors: 2-deoxyglucose and 3-bromopyruvate. We show that 3-bromopyruvate is more cytotoxic to a carcinoma cell line (CT26) than to a normal fibroblast (3T3) cell line, and that this selectivity is maintained in the in vitro combination with redaporfin-PDT. This combination was investigated in BALB/c mice with large subcutaneous CT26 tumors and it is shown that the cure rate in the combination is higher (33% cures) than in PDT (11% cures) or in 3-bromopyruvate (no cures) alone. The combination of redaporfin-PDT with 3-bromopyruvate illustrates the potential of combination therapies and how PDT benefits can be enhanced by systemic drugs with complementary targets.     

Photosensitizer-Mesoporous Silica Nanoparticles Combination for Enhanced Photodynamic Therapy†

Mesoporous silica nanoparticles (MSNs) are widely known for their versatile applications. One of the most extended is as drug delivery systems for the treatment of cancer and other diseases. This review compiles the most representative examples in the last years of functionalized MSNs as photosensitizer carriers for photodynamic therapy (PDT) against cancer. Several commercially available photosensitizers (PSs) demonstrated poor solubility in an aqueous medium and insufficient selectivity for cancer tissues. The tumor specificity of PSs is a key factor for enhancing the PDT effect and at the same time reducing side effects. The use of nanoparticles and particularly MSNs, in which PS is covalently anchored or physically embedded, can overcome these limitations. For that, PS-MSNs can be externally decorated with compounds of interest in order to act as an active target for certain cancer cells, demonstrating enhanced phototoxicity in vitro and in vivo. The objective of this review is to collect and compare different nanosystems based on PS-MSNs pointing out their advantages in PDT against diverse types of cancers.     

Red Wine Inspired Chromophores as Photodynamic Therapy Sensitizers†

Hydroxypyranoflavylium (HPF) cations are synthetic analogs possessing the same basic chromophore as the pyranoanthocyanins that form during the maturation of red wine. HPF cations absorb strongly in the visible spectral region, and most are fluorescent, triplet-sensitize singlet oxygen formation in solution and are strong photooxidants, properties that are desirable in a sensitizer for photodynamic therapy (PDT). The results of this study demonstrate that several simple HPF dyes can indeed function as PDT sensitizers. Of the eight HPF cations investigated in this work, four were phototoxic to a human cervical adenocarcinoma cell line (HeLa) at the 1–10 μmol dm⁻³ level, while only one of the eight compounds showed noticeable cytotoxicity in the dark. Neither a Type I nor a Type II mechanism can adequately rationalize the differences in phototoxicity of the compounds. Colocalization experiments with the most phototoxic compound demonstrated the affinity of the dye for both the mitochondria and lysosomes of HeLa cells. The fact that relatively modest structural differences, e.g., the exchange of an electron-donating substituent for an electron-withdrawing substituent, can cause profound differences in the phototoxicity, together with the relatively facile synthesis of substituted HPF cations, makes them interesting candidates for further evaluation as PDT sensitizers.     

Recent Photosensitizer Developments,Delivery Strategies and Combination-based Approaches for Photodynamic Therapy†

Photodynamic therapy of cancer (PDT) is a therapeutic technique, minimally invasive, which is currently used to treat cancerous lesions and tumors that have been in the spotlight for its potential over the recent decades. Nonetheless, PDT still needs further development to become a first-option treatment for patients. This review compiles recent progress in several aspects of the current research in the constantly growing area of PDT to overcome the main challenges as an attempt to serve as a guide and reference for newcomers into this research area. This review has been prepared to highlight the use of chemical modifications on photosensitizers to improve their solubility, photostability, selectivity and phototoxicity. Additionally, the use of liposomes and cavitands as drug delivery systems to aid in the biodistribution and bioaccumulation of photosensitizers is presented. Also, the combination of PDT with chemotherapy or immunotherapy as an option to boost and improve treatment outcomes is discussed. Finally, the inhibition of antioxidant enzymes as a strategy for a synergistic effect to ameliorate the performance of the photosensitizers in PDT is presented as an alternative for future researchers.     

Photodynamic Therapy for Bladder Cancers,A Focused Review†

Bladder cancer is the first cancer for which PDT was clinically approved in 1993. Unfortunately, it was unsuccessful due to side effects like bladder contraction. Here, we summarized the recent progress of PDT for bladder cancers, focusing on photosensitizers and formulations. General strategies to minimize side effects are intravesical administration of photosensitizers, use of targeting strategies for photosensitizers and better control of light. Non-muscle invasive bladder cancers are more suitable for PDT than muscle invasive and metastatic bladder cancers. In 2010, the FDA approved blue light cystoscopy, using PpIX fluorescence, for photodynamic diagnosis of non-muscle invasive bladder cancer. PpIX produced from HAL was also used in PDT but was not successful due to low therapeutic efficacy. To enhance the efficacy of PpIX-PDT, we have been working on combining it with singlet oxygen-activatable prodrugs. The use of these prodrugs increases the therapeutic efficacy of the PpIX-PDT. It also improves tumor selectivity of the prodrugs due to the preferential formation of PpIX in cancer cells resulting in decreased off-target toxicity. Future challenges include improving prodrugs and light delivery across the bladder barrier to deeper tumor tissue and generating an effective therapeutic response in an In vivo setting without causing collateral damage to bladder function.     

Photophysical Insights of Halogenated Dipyrrolonaphthyridine-Diones as Potential Photodynamic Therapy Agents†

We report the synthesis and photophysical characterization of novel halogenated dipyrrolonaphthyridine-diones (X₂–DPNDs, X = Cl, Br, and I), as candidates for photodynamic therapy (PDT) application. Apart from the heavy atom-induced spin-orbit coupling (SOC) dynamics in the investigated X₂–DPNDs, it was found that the position of the halogen atom (relative to the nitrogen of the pyrrole ring) also influenced the triplet excited state behavior. Interestingly, the faster/efficiency sensitization of ³O₂ to ¹O₂ using X₂–DPND correlates with the rate of triplet population, k_ISC >1.6 × 10⁸ s⁻¹ for I₂–DPND vs k_ISC >2.9 × 10⁹ s⁻¹ for Cl₂–DPND and Br₂–DPND (where τ_ISC = 343 ± 3 ps for I₂–DPND and τ_ISC = 5–6 ns for Cl₂–DPND and Br₂–DPND are the lowest time constants/values for ISC). Furthermore, the heavy atom-induced SOC in Cl₂–DPND and Br₂–DPND did not lead to a reduction of the corresponding fluorescence (ca 75% vs 67% for the parent DPND). The attractive photophysical characteristics of Cl₂/Br₂–DPND put them on the landscape as not only promising PDT agents but also as fluorescence probes. The present study is a stepping stone in the development of novel organic photosystems for synergistic photomedicinal applications.     

Combination of 5-Fluorouracil with Photodynamic Therapy: Enhancement of Innate and Adaptive Immune Responses in a Murine Model of Actinic Keratosis†

We previously showed that a combination of differentiation-inducing agents (5-fluorouracil [5FU], vitamin D3 or methotrexate) and aminolevulinate-based photodynamic therapy (PDT) improves clinical responses by enhancing protoporphyrin IX (PpIX) photosensitizer levels and cell death. Here, we show that in addition to its previously known effects, 5FU enhances PDT-induced tumor-regressing immunity. Murine actinic keratoses were treated with topical 5FU or vehicle for 3 days prior to aminolevulinic acid application, followed by blue light illumination (~417 nm). Lesions were harvested for time-course analyses of innate immune cell recruitment into lesions, i.e. neutrophils (Ly6G+) and macrophages (F4/80+), which peaked at 72 h and 1 week post-PDT, respectively, and were greater in 5FU-treated lesions. Enhanced infiltration of activated T cells (CD3+) throughout the time course, and of cytotoxic T cells (CD8+) at 1–2 weeks post-PDT, also occurred in 5FU-treated lesions. 5FU pretreatment reduced the presence of cells expressing the immune checkpoint marker PD-1 at ~72 h post-PDT, favoring cytotoxic T cell activity. A combination of 5FU and PDT, each individually known to induce long-term tumor-targeting immune responses in addition to their more immediate effects on cancer cells, may synergize to provide better management of squamous precancers.     

Evaluation of Detector Position and Light Fluence Distribution Using an Infrared Navigation System during Pleural Photodynamic Therapy†

Photodynamic therapy (PDT) has been used to treat malignant pleural mesothelioma. Current practice involves delivering light to a prescribed light fluence with a point source, monitored by eight isotropic detectors inside the pleural cavity. An infrared (IR) navigation system was used to track the location of the point source throughout the treatment. The recorded data were used to reconstruct the pleural cavity and calculate the light fluence to the whole cavity. An automatic algorithm was developed recently to calculate the detector positions based on recorded data within an hour. This algorithm was applied to patient case studies and the calculated results were compared to the measured positions, with an average difference of 2.5 cm. Calculated light fluence at calculated positions were compared to measured values. The differences between the calculated and measured light fluence were within 14% for all cases, with a fixed scattering constant and a dual correction method. Fluence-surface histogram (FSH) was calculated for photofrin-mediated PDT to be able to cover 80% of pleural surface area to 50 J cm⁻²(83.3% of 60 J cm⁻²). The study demonstrates that it will be possible to eliminate the manual measurement of the detector positions, reducing the patient's time under anesthesia.     

Effect of Gut Microbiota-Derived Metabolites on Immune Checkpoint Inhibitor Therapy: Enemy or Friend?

The human gut is inhabited by hundreds of billions of commensal microbiota that collectively produce thousands of small molecules and metabolites with local and systemic effects on the physiology of the host. Much evidence from preclinical to clinical studies has gradually confirmed that the gut microbiota can regulate anti-tumor immunity and affect the efficacy of cancer immune checkpoint inhibitors (ICIs) therapy. In particular, one of the main modes of gut microbiota regulating anti-tumor immunity is through metabolites, which are small molecules that can be transported in the body and act on local and systemic anti-tumor immune responses to promote ICIs immunotherapy efficacy. We discuss the functions of microbial metabolites in humans, focusing on the effects and mechanisms of microbial metabolites on immunotherapy, and analyze their potential applications as immune adjuvants and therapeutic targets to regulate immunity and enhance ICIs. In summary, this review provides the basis for the rational design of microbiota and microbial metabolite-based strategies of enhancing ICIs.     

Photodynamic Inactivation of Bacteria in Ionic Environments Using the Photosensitizer SAPYR and the Chelator Citrate†

Many studies show that photodynamic inactivation (PDI) is a powerful tool for the fight against pathogenic, multiresistant bacteria and the closing of hygiene gaps. However, PDI studies have been frequently performed under standardized in vitro conditions comprising artificial laboratory settings. Under real-life conditions, however, PDI encounters substances like ions, proteins, amino acids and fatty acids, potentially hampering the efficacy of PDI to an unpredictable extent. Thus, we investigated PDI with the phenalene-1-one-based photosensitizer SAPYR against Escherichia coli and Staphylococcus aureus in the presence of calcium or magnesium ions, which are ubiquitous in potential fields of PDI applications like in tap water or on tissue surfaces. The addition of citrate should elucidate the potential as a chelator. The results indicate that PDI is clearly affected by such ubiquitous ions depending on its concentration and the type of bacteria. The application of citrate enhanced PDI, especially for Gram-negative bacteria at certain ionic concentrations (e.g. CaCl₂ or MgCl₂: 7.5 to 75 mmol L⁻¹). Citrate also improved PDI efficacy in tap water (especially for Gram-negative bacteria) and synthetic sweat solution (especially for Gram-positive bacteria). In conclusion, the use of chelating agents like citrate may facilitate the application of PDI under real-life conditions.     

A Multi-action and Multi-target RuII–PtIV Conjugate Combining Cancer-Activated Chemotherapy and Photodynamic Therapy to Overcome Drug Resistant Cancers

Pt^II complexes are commonly used to treat cancer. To reduce their side effects and improve their pharmacological properties, Pt^IV complexes are being developed as prodrug candidates that are activated by reduction in cancer cells. Concomitantly, Ru^II polypyridine complexes have gained much attention as photosensitizers for use in photodynamic therapy due to their attractive characteristics. In this article, a novel Pt^IV–Ru^II conjugate, which combines cancer activated chemotherapy with PDT, is presented. Upon entering the cancer cell, the Pt^IV centre is reduced to Pt^II and the axial ligands including the Ru^II complex and phenylbutyrate are released. As each component has its individual targets, the conjugate exerts a multi-target and multi-action effect with (photo-)cytotoxicity values upon irradiation up to 595 nm in the low nanomolar range in various (drug resistant) 2D monolayer cancer cells and 3D multicellular tumour spheroids.     

Photodynamic Priming Overcomes Per- and Polyfluoroalkyl Substance (PFAS)-Induced Platinum Resistance in Ovarian Cancer†

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants linked to adverse outcomes, including for female reproductive biology and related cancers. We recently reported, for the first time, that PFAS induce platinum resistance in ovarian cancer, potentially through altered mitochondrial function. Platinum resistance is a major barrier in the management of ovarian cancer, necessitating complementary therapeutic approaches. Photodynamic therapy (PDT) is a light-based treatment modality that reverses platinum resistance and synergizes with platinum-based chemotherapy. The present study is the first to demonstrate the ability of photodynamic priming (PDP), a low-dose, sub-cytotoxic variant of PDT, to overcome PFAS-induced platinum resistance. Comparative studies of PDP efficacy using either benzoporphyrin derivative (BPD) or 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) were conducted in two human ovarian cancer cell lines (NIH:OVCAR-3 and Caov-3). BPD and PpIX are clinically approved photosensitizers that preferentially localize to, or are partly synthesized in, mitochondria. PDP overcomes carboplatin resistance in PFAS-exposed ovarian cancer cells, demonstrating the feasibility of this approach to target the deleterious effects of environmental contaminants. Decreased survival fraction in PDP + carboplatin treated cells was accompanied by decreased mitochondrial membrane potential, suggesting that PDP modulates the mitochondrial membrane, reducing membrane potential and re-sensitizing ovarian cancer cells to carboplatin.     

BODIPY@Ir(Ⅲ) Complexes Assembling Organic Nanoparticles for Enhanced Photodynamic Therapy

We present a new cyclometalated Ir(Ⅲ) complexes Ir BDP, which could self-assemble into organic nanoparticles(IrBDP NPs). IrBDP NPs show enhanced photodynamic effect and can be engulfed by He La cells for cell imaging as well as photodynamic therapy(PDT) upon low energy irradiation.     

Sub-lethal Photodynamic Damage to ARPE-19 Cells Transiently Inhibits Their Phagocytic Activity†

Efficient phagocytosis of photoreceptor outer segments (POS) membranes by retinal pigment epithelium (RPE) plays a key role in biological renewal of these highly peroxidizable structures. Here, we tested whether photodynamic treatment, mediated by merocyanine 540 (MC 540), rose Bengal or a zinc-substituted chlorophyllide inhibited phagocytic activity of ARPE-19 cells in vitro. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS isolated from cow retinas and nonspecific phagocytosis of fluorescent polystyrene beads were measured by flow cytometry. Photodynamic treatment, mediated by all three photosensitizers with sub-threshold doses, induced significant inhibition of the cell-specific phagocytosis. The nonspecific phagocytosis was inhibited by photodynamic treatment mediated only by MC 540. The inhibition of phagocytosis was a reversible phenomenon and after 24 h, the photodynamically treated cells exhibited phagocytic activity that was comparable with that of untreated cells. This study provides proof of principle that sub-threshold photodynamic treatment of ARPE-19 cells with appropriate photosensitizers is a convenient experimental approach for in vitro study of the effects of oxidative stress on specific phagocytic activity of RPE cells. We postulate that oxidative damage to key components of the cell phagocytic machinery may be responsible for severe impairment of its activity, which can lead to retinal degeneration.     

A Nanoscale Metal–Organic Framework to Mediate Photodynamic Therapy and Deliver CpG Oligodeoxynucleotides to Enhance Antigen Presentation and Cancer Immunotherapy

Checkpoint blockade immunotherapy (CBI) awakes a host innate immune system and reactivates cytotoxic T cells to elicit durable response in some cancer patients. Now, a cationic nanoscale metal–organic framework, W-TBP, is used to facilitate tumor antigen presentation by enabling immunogenic photodynamic therapy (PDT) and promoting the maturation of dendritic cells (DCs). Comprised of dinuclear W^VI secondary building units and photosensitizing 5,10,15,20-tetra(p-benzoato)porphyrin (TBP) ligands, cationic W-TBP mediates PDT to release tumor associated antigens and delivers immunostimulatory CpG oligodeoxynucleotides to DCs. The enhanced antigen presentation synergizes with CBI to expand and reinvigorate cytotoxic T cells, leading to superb anticancer efficacy and robust abscopal effects with >97 % tumor regression in a bilateral breast cancer model.     

In Situ Self-Assembled J-Aggregate Nanofibers of Glycosylated Aza-BODIPY for Synergetic Cell Membrane Disruption and Type I Photodynamic Therapy

The in situ self-assembly of exogenous molecules is a powerful strategy for manipulating cellular behavior. However, the direct self-assembly of photochemically inert constituents into supramolecular nano-photosensitizers (PSs) within cancer cells for precise photodynamic therapy (PDT) remains a challenge. Herein, we developed a glycosylated Aza-BODIPY compound ( LMBP ) capable of self-assembling into J-aggregate nanofibers in situ for cell membrane destruction and type I PDT. LMBP selectively entered human hepatocellular carcinoma HepG2 cells and subsequently self-assembled into intracellular J-aggregate nanovesicles and nanofibers through supramolecular interactions. Detailed studies revealed that these J-aggregate nanostructures generated superoxide radicals (O₂⁻⋅) exclusively through photoinduced electron transfer, thus enabling effective PDT. Furthermore, the intracellular nanofibers exhibited an aggregation-induced retention effect, which resulted in selective toxicity to HepG2 cells by disrupting their cellular membranes and synergizing with PDT for powerful tumor suppression efficacy in vivo.     

Photocatalytic Generation of Hydrogen Radical (H⋅) with GSH for Photodynamic Therapy

As a reactive hydrogen species, the hydrogen radical (H⋅) scarcely sees applications in tumor biological therapy due to the very limited bio-friendly sources of H⋅. In this work, we report that TAF can act as an organic photosensitizer as well as an efficient photocatalytic H⋅ generator with reduced glutathione (GSH) as a fuel. The photoactivation of TAF leads to cell death in two ways including triple amplification of oxidative stress via ferroptosis-apoptosis under normoxia and apoptosis through biological reductions under hypoxia. TAF presents excellent biosafety with ultrahigh photocytotoxicity index at an order of magnitude of 10²–10³ on both normoxic and hypoxic cells. The in vitro data suggest that H⋅ therapy is promising to overcome the challenge of tumor hypoxia at low doses of both photocatalyst and light. In addition, the capability of near-infrared two-photon excitation would benefit broad biological applications.