Diamino acid derivatives of PpIX as potential photosensitizers for photodynamic therapy of squamous cell carcinoma and prostate cancer: In vitro studies

Photodynamic therapy (PDT) is an alternative treatment modality involving light activated drugs, called photosensitizers (PSs), to treat cancer and non-cancerous conditions. The search for new compounds which might become effective PSs is the major direction for PDT development. In the present work we have studied the dark toxicity, intracellular localization and photodynamic properties of four potential, water soluble, second generation PSs – PP(Arg)₂, PP(Ser)₂Arg₂, PP(Ala)₂Arg₂, PP(Phe)₂Arg₂, all diamino acid derivatives of protoporphyrin IX. Human prostate cancer (DU-145) and squamous carcinoma (A431) cells were used as experimental model.Among investigated compounds PP(Ser)₂Arg₂ exhibited the lowest dark toxicity and the highest PDT effectiveness towards both cell lines. Fluorescence microscopy revealed the time-dependent changes in intracellular localization of the PS which were related to the phototoxicity. The results show that PP(Ser)₂Arg₂ may be a potential PS for PDT.     

Development of Biotechnological Photosensitizers for Photodynamic Therapy: Cancer Research and Treatment—From Benchtop to Clinical Practice

Photodynamic therapy (PDT) is a noninvasive therapeutic approach that has been applied in studies for the treatment of various diseases. In this context, PDT has been suggested as a new therapy or adjuvant therapy to traditional cancer therapy. The mode of action of PDT consists of the generation of singlet oxygen (¹O₂) and reactive oxygen species (ROS) through the administration of a compound called photosensitizer (PS), a light source, and molecular oxygen (³O₂). This combination generates controlled photochemical reactions (photodynamic mechanisms) that produce ROS, such as singlet oxygen (¹O₂), which can induce apoptosis and/or cell death induced by necrosis, degeneration of the tumor vasculature, stimulation of the antitumor immune response, and induction of inflammatory reactions in the illuminated region. However, the traditional compounds used in PDT limit its application. In this context, compounds of biotechnological origin with photosensitizing activity in association with nanotechnology are being used in PDT, aiming at its application in several types of cancer but with less toxicity toward neighboring tissues and better absorption of light for more aggressive types of cancer. In this review, we present studies involving innovatively developed PS that aimed to improve the efficiency of PDT in cancer treatment. Specifically, we focused on the clinical translation and application of PS of natural origin on cancer.     

Spin–Orbit Charge-Transfer Intersystem Crossing (ISC) in Compact Electron Donor–Acceptor Dyads: ISC Mechanism and Application as Novel and Potent Photodynamic Therapy Reagents

Spin–orbit charge-transfer intersystem crossing (SOCT-ISC) is useful for the preparation of heavy atom-free triplet photosensitisers (PSs). Herein, a series of perylene-Bodipy compact electron donor/acceptor dyads showing efficient SOCT-ISC is prepared. The photophysical properties of the dyads were studied with steady-state and time-resolved spectroscopies. Efficient triplet state formation (quantum yield Φ_T=60 %) was observed, with a triplet state lifetime (τ_T=436 μs) much longer than that accessed with the conventional heavy atom effect (τ_T=62 μs). The SOCT-ISC mechanism was unambiguously confirmed by direct excitation of the charge transfer (CT) absorption band by using nanosecond transient absorption spectroscopy and time-resolved electron paramagnetic resonance (TREPR) spectroscopy. The factors affecting the SOCT-ISC efficiency include the geometry, the potential energy surface of the torsion, the spin density for the atoms of the linker, solvent polarity, and the energy matching of the ¹CT/³LE states. Remarkably, these heavy atom-free triplet PSs were demonstrated as a new type of efficient photodynamic therapy (PDT) reagents (phototoxicity, EC₅₀=75 nm ), with a negligible dark toxicity (EC₅₀=78.1 μm ) compared with the conventional heavy atom PSs (dark toxicity, EC₅₀=6.0 μm, light toxicity, EC₅₀=4.0 nm ). This study provides in-depth understanding of the SOCT-ISC, unveils the design principles of triplet PSs based on SOCT-ISC, and underlines their application as a new generation of potent PDT reagents.     

Synthesis of Sn nanocluster@carbon dots for photodynamic therapy application

Recently, photodynamic therapy (PDT) has been extensively applied in clinical and coadjuvant treatment of various kinds of tumors. However, the photosensitizer (PS) of PDT still lack of high production of singlet oxygen (¹O₂), low cytotoxicity and high biocompatibility. Herein, we propose a facile method for establishing a new core-shell structured Sn nanocluster@carbon dots (CDs) PS. Firstly, Sn⁴⁺@S-CDs complex is synthesized using the sulfur-doped CDs (S-CDs) and SnCl₄ as raw materials, and subsequently the new PS (Sn nanocluster@CDs) is obtained after vaporization of Sn⁴⁺@S-CDs solution. Remarkably, the obtained Sn nanocluster@CDs show an enhanced fluorescence as well as a higher ¹O₂ quantum yield (QY) than S-CDs. The high ¹O₂ QY (58.3%) irradiated by the LED light (400–700 nm, 40 mW/cm²), induce the reduction of 4T1 cancer cells viability by 25%. More intriguingly, no visible damage happens to healthy cells, with little impact on liver tissue due to renal excretion, both in vitro and in vivo experiments demonstrate that Sn nanocluster@CDs may become a promising PS, owning a high potential for application in PDT.     

Synthesis and effect on SMMC-7721 cells of new benzo[c,d]indole rhodanine complex merocyanines as PDT photosensitizers

Photodynamic therapy (PDT) represents a modern and noninvasive therapeutic approach, however, it relies on the development of photosensitizers. Here five new benzo[c,d]indole rhodamine complex merocyanines (BIRCM) D1-D5, displaying low dark toxicity and significant photo toxicity, were synthesized as PDT photosensitizers, and characterized by ¹H NMR, IR, UV–Vis and HRMS. The investigation of their absorption spectra in different solvents showed that the absorption maxima and molar extinction coefficient were in the region 507–679 nm and 0.21 × 10⁴–1.27 × 10⁵ L · mol^?1cm^?1, respectively. The evaluation of PDT activity showed that only irradiation could not kill SMMC-7721 cells, and the cell survival rate and inhibition rate at the application dose and duration was 92%–87% and 78%–49%, respectively. Especially, using D2, absorbed in the red zone, as photosensitizer for PDT analyzed its effect on SMMC-7721 cells survival, it could be found that the cell survival rate was 92% without irradiating and the cell inhibited rate was 78% under irradiating at concentrations of 2.5 × 10^?6 mol/L, displaying low dark toxicity and high photo toxicity, which was valuable for PDT of some microvascular diseases or other superficial diseases.     

Design and synthesis of novel water-soluble amino acid derivatives of chlorin p6 ethers as photosensitizer

Eight new water-soluble amino acid derivatives of chlorin p₆ ethers 6a-h were designed and synthesized using purpurin-18 (2) as key intermediate. All target compounds exhibited better phototoxicity than talaporfin and the most phototoxic compound 6d showed IC₅₀ values of 0.20 μmol/L against A549 cell and 0.41 μmol/L against B16-F10 cell, which represented 31- and 24-fold increase of PDT antitumor efficacy compared to talaporfin.     

A mini review of nanomaterials on photodynamic therapy

In this account, the reactive oxygen species (ROS) in photodynamic therapy (PDT) were deliberately reviewed. First, the specific definition of ROS and PDT were readily clarified. Afterward, this review focuses on the fundamental principles and applications of PDT. Due to strong oxidation ability of radicals (e.g., •OH and O₂^•-) and non-radical (e.g., ¹O₂ and H₂O₂), these ROS would attack the in vitro and in vivo tumor cells, thus achieving the goal of cancer treatment. Then, ROS in PDT for cancer treatment was thoroughly reviewed, including the mechanism and photosensitizer (PS) selection (i.e., nanomaterials). Ultimately, emphasis was made on the challenges, research gap, and prospects of ROS in cancer treatment and critically discussed. Hopefully, this review can offer detailed theoretical guidance for the researchers who participate in the study regarding ROS in PDT.     

Type I Photosensitizer Targeting G-Quadruplex RNA Elicits Augmented Immunity for Cancer Ablation

Type I photodynamic therapy (PDT) represents a promising treatment modality for tumors with intrinsic hypoxia. However, type I photosensitizers (PSs), especially ones with near infrared (NIR) absorption, are limited and their efficacy needs improvement via new targeting tactics. We develop a NIR type I PS by engineering acridinium derived donor-π-acceptor systems. The PS exhibits an exclusive type I PDT mechanism due to effective intersystem crossing and disfavored energy transfer to O₂, and shows selective binding to G-quadruplexes (G4s) via hydrogen bonds identified by a molecular docking study. Moreover, it enables fluorogenic detection of G4s and efficient O₂⋅⁻ production in hypoxic conditions, leading to immunogenic cell death and substantial variations of gene expression in RNA sequencing. Our strategy demonstrates augmented antitumor immunity for effective ablation of immunogenic cold tumor, highlighting its potential of RNA-targeted type I PDT in precision cancer therapy.     

Measuring the Physiologic Properties of Oral Lesions Receiving Fractionated Photodynamic Therapy

Photodynamic therapy (PDT) can treat superficial, early‐stage disease with minimal damage to underlying tissues and without cumulative dose‐limiting toxicity. Treatment efficacy is affected by disease physiologic properties, but these properties are not routinely measured. We assessed diffuse reflectance spectroscopy (DRS) for the noninvasive, contact measurement of tissue hemoglobin oxygen saturation (S_tO₂) and total hemoglobin concentration ([tHb]) in the premalignant or superficial microinvasive oral lesions of patients treated with 5‐aminolevulinic acid (ALA)‐PDT. Patients were enrolled on a Phase 1 study of ALA‐PDT that evaluated fluences of 50, 100, 150 or 200 J cm^?2 delivered at 100 mW cm^?2. To test the feasibility of incorporating DRS measurements within the illumination period, studies were performed in patients who received fractionated (two‐part) illumination that included a dark interval of 90–180 s. Using DRS, tissue oxygenation at different depths within the lesion could also be assessed. DRS could be performed concurrently with contact measurements of photosensitizer levels by fluorescence spectroscopy, but a separate noncontact fluorescence spectroscopy system provided continuous assessment of photobleaching during illumination to greater tissue depths. Results establish that the integration of DRS into PDT of early‐stage oral disease is feasible, and motivates further studies to evaluate its predictive and dosimetric value.     

Universal Scaffold for an Activatable Photosensitizer with Completely Inhibited Photosensitivity

Activatable photosensitizers (aPSs) sensitive to specific stimuli hold potential for targeting multiple disease biomarkers and are desirable for photodynamic therapy (PDT). Presented herein is the design of aPSs that can be activated and fully recover from an inhibited state in the presence of specific biomarker. A designed long‐wavelength D‐π‐A photosensitizer, PS_Se‐I , with highly efficient photosensitivity for generation of ¹O₂ was used. Caging of the phenolate donor of PS_Se‐I with a biomarker‐sensitive group provided ^ALP PS , and the drastic activation of its photosensitivity was demonstrated intracellularly. To enhance the flexibility of the design strategy, a clickable azide group was introduced into the scaffold to allow integration of more functionality. Modularly derived ^mito‐PN PS , equipped with a mitochondria‐targeting group and a specific peroxynitrite‐reactive trigger, was synthesized and demonstrated superior performance in cells. This strategy could lead to customization of aPSs applicable to a specific PDT.     

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.     

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.     

A Phototheranostic Strategy to Continuously Deliver Singlet Oxygen in the Dark and Hypoxic Tumor Microenvironment

Continuous irradiation during photodynamic therapy (PDT) inevitably induces tumor hypoxia, thereby weakening the PDT effect. In PDT‐induced hypoxia, providing singlet oxygen from stored chemical energy may enhance the cell‐killing effect and boost the therapeutic effect. Herein, we present a phototheranostic (DPPTPE@PEG‐Py NPs) prepared by using a 2‐pyridone‐based diblock polymer (PEG‐Py) to encapsulate a semiconducting, heavy‐atom‐free pyrrolopyrrolidone‐tetraphenylethylene (DPPTPE) with high singlet‐oxygen‐generation ability both in dichloromethane and water. The PEG‐Py can trap the ¹O₂ generated from DPPTPE under laser irradiation and form a stable intermediate of endoperoxide, which can then release ¹O₂ in the dark, hypoxic tumor microenvironment. Furthermore, fluorescence‐imaging‐guided phototherapy demonstrates that this phototheranostic could completely inhibit tumor growth with the help of laser irradiation.     

Paramagnetic Oxygen as Contrast Agent for a Potential PDT Treatment MRI Monitoring

As a proof of concept, we demonstrate in this preliminary study that ³O₂ could be used as magnetic Contrast Agent (CA) to monitor photodynamic treatments (PDT). In this purpose, and using a well-studied Photo-Sensitizer (PS) from our team ( Ruteg ), we highlighted that the combined action of the PS and irradiation led to significant changes in T₁ (and R₁) values of the protons of the water molecules. We assume that with these conclusive results using relaxometric measurements, transposition to Magnetic Resonance Imaging (MRI) in pertinent conditions (oxygen concentration, magnetic field) should be achievable.     

Multifunctional Logic in a Photosensitizer with Triple‐Mode Fluorescent and Photodynamic Activity

Herein we describe a photosensitizer (PS) with the capacity to perform multiple logic operations based on a pyrene‐containing phthalocyanine (Pc) derivative. The system presents three output signals (fluorescence at 377 and 683 nm, and singlet oxygen (¹O₂) production), which are dependent on three inputs: two chemical (concentration of dithiothreitol (DTT) and acidic pH) and one physical (visible light above 530 nm for ¹O₂ sensitization). The multi‐input/multioutput nature of this PS leads to single‐, double‐, and triple‐mode activation pathways of its fluorescent and photodynamic functions, through the interplay of various interrelated AND, ID, and INHIBIT gates. Dual fluorescence emissions are potentially useful for orthogonal optical imaging protocols while ¹O₂ is the main reactive species in photodynamic therapy (PDT). We thus expect that this kind of PS logic system will be of great interest for multimodal cellular imaging and therapeutic applications.     

Smart Magnetic and Fluorogenic Photosensitizer Nanoassemblies Enable Redox-Driven Disassembly for Photodynamic Therapy

Stimuli-responsive smart photosensitizer (PS) nanoassemblies that allow enhanced delivery and controlled release of PSs are promising for imaging-guided photodynamic therapy (PDT) of tumors. However, the lack of high-sensitivity and spatial-resolution signals and fast washout of released PSs from tumor tissues have impeded PDT efficacy in vivo. Herein, we report tumor targeting, redox-responsive magnetic and fluorogenic PS nanoassemblies ( NP-RGD ) synthesized via self-assembly of a cRGD- and disulfide-containing fluorogenic and paramagnetic small molecule ( 1-RGD ) for fluorescence/magnetic resonance bimodal imaging-guided tumor PDT. NP-RGD show high r₁ relaxivity but quenched fluorescence and PDT activity; disulfide reduction by glutathione (GSH) promotes efficient disassembly into a small-molecule probe ( 2-RGD ) and an organic PS (PPa-SH), which could further bind with intracellular albumin, allowing prolonged retention and cascade activation of fluorescence and PDT to ablate tumors.     

Activatable Type I Photosensitizer with Quenched Photosensitization Pre and Post Photodynamic Therapy

The phototoxicity of photosensitizers (PSs) pre and post photodynamic therapy (PDT), and the hypoxic tumor microenvironment are two major problems limiting the application of PDT. While activatable PSs can successfully address the PS phototoxicity pre PDT, and type I PS can generate reactive oxygen species (ROS) effectively in hypoxic environment, very limited approaches are available for addressing the phototoxicity post PDT. There is virtually no solution available to address all these issues using a single design. Herein, we propose a proof-of-concept on-demand switchable photosensitizer with quenched photosensitization pre and post PDT, which could be activated only in tumor hypoxic environment. Particularly, a hypoxia-normoxia cycling responsive type I PS TPFN-AzoCF₃ was designed to demonstrate the concept, which was further formulated into TPFN-AzoCF₃ nanoparticles (NPs) using DSPE-PEG-2000 as the encapsulation matrix. The NPs could be activated only in hypoxic tumors to generate type I ROS during PDT treatment, but remain non-toxic in normal tissues, pre or after PDT, thus minimizing side effects and improving the therapeutic effect. With promising results in in vitro and in vivo tumor treatment, this presented strategy will pave the way for the design of more on-demand switchable photosensitizers with minimized side effects in the future.     

Design of Photosensitizing Agents for Targeted Antimicrobial Photodynamic Therapy

Photodynamic inactivation of microorganisms has gained substantial attention due to its unique mode of action, in which pathogens are unable to generate resistance, and due to the fact that it can be applied in a minimally invasive manner. In photodynamic therapy (PDT), a non-toxic photosensitizer (PS) is activated by a specific wavelength of light and generates highly cytotoxic reactive oxygen species (ROS) such as superoxide (O²⁻, type-I mechanism) or singlet oxygen (¹O₂*, type-II mechanism). Although it offers many advantages over conventional treatment methods, ROS-mediated microbial killing is often faced with the issues of accessibility, poor selectivity and off-target damage. Thus, several strategies have been employed to develop target-specific antimicrobial PDT (aPDT). This includes conjugation of known PS building-blocks to either non-specific cationic moieties or target-specific antibiotics and antimicrobial peptides, or combining them with targeting nanomaterials. In this review, we summarise these general strategies and related challenges, and highlight recent developments in targeted aPDT.     

Establishing a Robust and Reliable Response from a Potent Osmium-Based Photosensitizer Via Lipid Nanoformulation†

Osmium (Os) based photosensitizers (PSs) are a unique class of nontetrapyrrolic metal-containing PSs that absorb red light. We recently reported a highly potent Os(II) PS, rac-[Os(phen)₂(IP-4T)](Cl)₂, referred to as ML18J03 herein, with light EC₅₀ values as low as 20 pm . ML18J03 also exhibits low dark toxicity and submicromolar light EC₅₀ values in hypoxia in some cell lines. However, owing to its longer oligothiophene chain, ML18J03 is not completely water soluble and forms 1–2 μm sized aggregates in PBS containing 1% DMSO. This aggregation causes variability in PDT efficacy between assays and thus unreliable and irreproducible reports of in vitro activity. To that end, we utilized PEG-modified DPPC liposomes (138 nm diameter) and DSPE-mPEG₂₀₀₀ micelles (10.2 nm diameter) as lipid nanoformulation vehicles to mitigate aggregation of ML18J03 and found that the spectroscopic properties important to biological activity were maintained or improved. Importantly, the lipid formulations decreased the interassay variance between the EC₅₀ values by almost 20-fold, with respect to the unformulated ML18J03 when using broadband visible light excitation (P = 0.0276). Herein, lipid formulations are presented as reliable platforms for more accurate in vitro photocytotoxicity quantification for PSs prone to aggregation (such as ML18J03) and will be useful for assessing their in vivo PDT effects.     

An azo dye for photodynamic therapy that is activated selectively by two-photon excitation

Two-photon photodynamic therapy (TP-PDT) is a promising approach for the treatment of cancer because of its better penetration depth and superior spatial selectivity. Here, we describe an azo group containing cyclized-cyanine derivatives (ACC1 and ACC2) as a two-photon activated, type I based photosensitizer (PS). These small-molecule and heavy atom-free organic dyes showed marked reactive oxygen species (ROS)-generating ability under physiological conditions, as well as fast loading ability into the cells and negligible dark toxicity. Live cell analyses with one- and two-photon microscopy revealed that these dyes showed higher ROS generation ability upon two-photon excitation than upon one-photon excitation via the type I process. The PSs have superior PDT properties compared to conventional Visudyne and 5-ALA under mild conditions. These characteristics allowed for precise PDT at the target region in mimic tumor spheroids, demonstrating that the developed TP PS could be useful in efficient PDT applications and in designing various PSs.

Azo containing dyes as a two-photon selective and type I based photosensitizers (PSs) were developed that exhibit excellent photodynamic therapy properties under mild condition.