Recent advances for enhanced photodynamic therapy: from new mechanisms to innovative strategies.

Photodynamic therapy (PDT) has been developed as a potential cancer treatment approach owing to its non-invasiveness, spatiotemporal control and limited side effects. Currently, great efforts have been made to improve the PDT effect in terms of safety and efficiency. In this review, we highlight recent advances in innovative strategies for enhanced PDT, including (1) the development of novel radicals, (2) design of activatable photosensitizers based on the TME and light, and (3) photocatalytic NADH oxidation to damage the mitochondrial electron transport chain. Additionally, the new mechanisms for PDT are also presented as an inspiration for the design of novel PSs. Finally, we discuss the current challenges and future prospects in the clinical practice of these innovative strategies. It is hoped that this review will provide a new angle for understanding the relationship between the intratumoural redox environment and PDT mechanisms, and new ideas for the future development of smart PDT systems.

Polymer-Drug Conjugates Codeliver a Temozolomide Intermediate and Nitric Oxide for Enhanced Chemotherapy against Glioblastoma Multiforme.

Polymer-drug conjugates (PDCs) provide possibilities for the development of multiresponsive drug delivery and release platforms utilized in cancer therapy. The delivery of Temozolomide (TMZ, a DNA methylation agent) by PDCs has been developed to improve TMZ stability under physiological conditions for the treatment of glioblastoma multiforme (GBM); however, with inefficient chemotherapeutic efficacy. In this work, we synthesized an amphiphilic triblock copolymer (P1-SNO) with four pendant functionalities, including (1) a TMZ intermediate (named MTIC) as a prodrug moiety, (2) a disulfide bond as a redox-responsive trigger to cage MTIC, (3) S-nitrosothiol as a light/heat-responsive donor of nitric oxide (NO), and (4) a poly(ethylene glycol) chain to enable self-assembly in aqueous media. P1-SNO was demonstrated to liberate MTIC in the presence of reduced glutathione and release gaseous NO upon exposure to light or heat. The in vitro results revealed a synergistic effect of released MTIC and NO on both TMZ-sensitive and TMZ-resistant GBM cells. The environment-responsive PDC system for codelivery of MTIC and NO is promising to overcome the efficacy issue in TMZ-based cancer therapy.

Reactive Reductive Species Participating Photodynamic Therapy for Cancer Treatment.

Although the development of oxidative photodynamic therapy (O-PDT) based on reactive oxygen species (ROS) has led to great progress in cancer treatment, tumor hypoxia, cellular adaptation and intrinsic antioxidant defenses are still obstacles at this stage. Fortunately, with the discovery and development of reactive reductive species (RRS) in the PDT process, reductive PDT (R-PDT) is receiving increasing research interest. R-PDT with oxygen-independence is an effective reduction therapy that promises excellent therapeutic efficacy in extremely hypoxic or even anaerobic environments. In the concept, we introduce representative strategies to boost the type-I photosensitizing pathway, and then focus on the most recent R-PDT involving hydrogen radical (H⋅) and the single electron transfer (SET) process.

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.

Dithiol-Activated Bioorthogonal Chemistry for Endoplasmic Reticulum-Targeted Synergistic Chemophototherapy.

The controlled intracellular release of nitrite is still an unmet challenge due to the lack of bio-friendly donors, and the antitumor effect of nitrite is limited by its physiologically inert activity. Herein, we designed benzothiadiazole-based organic nitrite donors that are stable against bio-relevant species but selectively respond to dithiol species through SN Ar/intramolecular cyclization tandem reactions in the aqueous media. The bioorthogonal system was established to target the endoplasmic reticulum (ER) of liver cancer HepG2 cells. The nitrite and nonivamide were coupled to induce elevation of intracellular levels of calcium ions as well as reactive oxygen/nitrogen species, which resulted in ER stress and mitochondrial dysfunction. We demonstrated that a combination of photoactivation and "click to release" strategy could enhance antitumor effect in cellular level and show good potential for cancer precision therapy.

Intrinsic Mitochondrial Reactive Oxygen Species (ROS) Activate the In†Situ Synthesis of Trimethine Cyanines in Cancer Cells.

Environment-responsive in situ synthesis of molecular fluorescent dyes is challenging. Herein, we develop a photoextension strategy to make trimethine cyanines with decent conversion efficiency (up to 81 %) using 1-butyl 2,3,3-trimethyl 3H-indole derivatives as the sole precursors, and demonstrate a free radical mechanism. In the inducer-extension stage, free radicals and reactive oxygen species (ROS) were able to mediate similar reactions with no assistance of light. We explored a Mito-extension strategy to in situ synthesize trimethine cyanines in the living cells. The cellular ROS-dependence provided a foundation for preferential cyanine expression in cancer cells. Finally, we applied an iodized precursor as an intrinsic ROS-activated theranostic agent that integrated mitochondria-targeted cyanine synthesis, cell imaging and phototherapy.

Visible Light and Glutathione Dually Responsive Delivery of a Polymer-Conjugated Temozolomide Intermediate for Glioblastoma Chemotherapy.

Temozolomide (TMZ) is a prodrug of 5-(3-methyltriazene-1-yl)imidazole-4-carboxamide (MTIC, short-lived) and used as a first-line therapy drug for glioblastoma multiforme (GBM). However, little progress has been made in regulating the kinetics of TMZ to MTIC degradation to improve the therapeutic effect, particularly in the case of TMZ-resistant GBM. In this work, we introduced a strategy to cage MTIC by N-acylation of the triazene moiety to boost the MTIC stability, designed a diblock copolymer-based MTIC prodrug installed with a disulfide linkage, and achieved self-assembled polymer micelles without the concern of MTIC leakage under physiological conditions. Polymer micelles could be induced to disassemble by stimuli factors such as glutathione (GSH) and visible light irradiation through thiol/sulfide exchange and homolytic sulfide scission mechanisms, which contributed to MTIC release in GSH-dependent and GSH-independent pathways. The in vitro results demonstrated that microenvironment-responsive polymeric micelles benefited the suppression of both TMZ-sensitive and TMZ-resistant GBM cells. The chemistry of polymer-MTIC prodrug provided a new option for TMZ-based glioma treatment.

Biomimetic Polymer-Templated Copper Nanoparticles Stabilize a Temozolomide Intermediate for Chemotherapy against Glioblastoma Multiforme.

The short half-life of temozolomide (TMZ) limits its therapeutic effect on highly aggressive glioblastoma (GBM). Few approaches attempting to intervene the metabolic kinetics of TMZ are successful. Herein, we designed anionic copolymers via radical polymerization to prepare polymer-coated small copper nanoclusters, taking advantage of the role of pendent thymine groups as a template. The active and key intermediate of TMZ, typically called 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC), was stabilized by copper under physiological (slightly alkaline) conditions, alleviating concerns associated with spontaneous drug degradation and nonspecific drug activation. Importantly, the complexes formed by MTIC and copper nanoclusters could catalyze the Fenton reaction to generate hydroxyl radicals and also respond to pH and glutathione to release therapeutic MTIC, which allows combined chemotherapy and chemodynamic therapy against GBM cells and paves a way for circumventing the complication of TMZ resistance.

Temozolomide-Doxorubicin Conjugate as a Double Intercalating Agent and Delivery by Apoferritin for Glioblastoma Chemotherapy.

We designed a conjugated compound by coupling temozolomide (TMZ) with doxorubicin (DOX) via an acylhydrazone linkage as a potential prodrug used for glioblastoma multiforme (GBM) treatment. Viscosity and spectroscopic studies revealed that the drug conjugate could act as a nonclassical double intercalating agent. Although free TMZ is an inefficient DNA binder in comparison to DOX, the TMZ moiety interacted with DNA as an induced intercalator, arising from the synergistic effect of DOX moiety that mediated conformational changes of the DNA helix. Two binding modes were proposed to interpret the double intercalating effect of the drug conjugate on intra- and inter-DNA interactions that could cause DNA cross-linking and fibril aggregates. We also developed a delivery nanoplatform with a loading efficiency of 83% using copper-bound apoferritin as a nanocarrier. In sharp contrast to the short half-life of free TMZ, the nanocomposite was stable under physiological conditions without detectable drug decomposition after a 2 week storage, and drug release was activatable in the presence of glutathione at millimolar levels. The antitumor effect of the drug conjugate and nanocomposite against GBM cells was reported to demonstrate the potential therapeutic applications of double intercalating materials.

Folate-Modified Photoelectric Responsive Polymer Microarray as Bionic Artificial Retina to Restore Visual Function.

A high-optical-resolution artificial retina system that accurately communicates with the optic nerve is the main challenge in the modern biological science and bionic field. Here, we developed a bionic artificial retina possessing phototransduction "cells" with measurements even smaller than that of the neural cells. Using the technique of micrometer processing, we constructed a pyramid-shape periodic microarray of a photoreceptor. Each "sensing cell" took advantage of polythiophene derivative/fullerene derivative (PCBM) as a photoelectric converter. Because folic acid played an essential role in eye growth, we particularly modified the polythiophene derivatives with folic acid tags. Therefore, the artificial retina could enlarge the contact area and even recognize the nerve cells to improve the consequence of nerve stimulation. We implanted the artificial retina into blinded rats' eyes. Electrophysiological analysis revealed its recovery of photosensitive function 3 months after surgery. Our work provides an innovative idea for fabricating a high-resolution bionic artificial retina system. It shows great potential in artificial intelligence and biomedicine.

GSH and H2 O2 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 (H2 O2 ) 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 H2 O2 . The mito aPS was also compatible to near infrared PDT with two photon excitation (800 nm) for extensive bioapplications.

Apoferritin as a Carrier of Cu(II) Diethyldithiocarbamate and Biomedical Application for Glutathione-Responsive Combination Chemotherapy.

The antialcoholic drug disulfiram (DSF) has attractive biomedical interests for its anticancer effects, particularly in combination use with copper. CuET, the complex of copper and diethyldithiocarbamate (DTC) that is derived from disulfiram, exhibits high redox stability against glutathione, cysteine, and hydrogen peroxide, but appears reactive to ascorbic acid and superoxide ions. By virtue of the copper binding property, apoferritin is developed as a carrier of CuET to alleviate concerns of poor water solubility and nonspecific toxicity and shows superior stability and protection over human serum albumin in nanocomposite manipulation. CuET and doxorubicin are codelivered by apoferritin with excellent efficiency (>97%). The glutathione-responsive system demonstrates enhanced antitumor effect and potentials for combination tumor therapy.

Inhibition of AIM2 inflammasome-mediated pyroptosis by Andrographolide contributes to amelioration of radiation-induced lung inflammation and fibrosis.

Radiation-induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy, whereas no effective interventions are available. Andrographolide, an active component extracted from Andrographis paniculate, is prescribed as a treatment for upper respiratory tract infection. Here we report the potential radioprotective effect and mechanism of Andrographolide on RILI. C57BL/6 mice were exposed to 18 Gy of whole thorax irradiation, followed by intraperitoneal injection of Andrographolide every other day for 4 weeks. Andrographolide significantly ameliorated radiation-induced lung tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine release in the early phase and progressive fibrosis in the late phase. Moreover, Andrographolide markedly hampered radiation-induced activation of the AIM2 inflammasome and pyroptosis in vivo. Furthermore, bone marrow-derived macrophages (BMDMs) were exposed to 8 Gy of X-ray radiation in vitro and Andrographolide significantly inhibited AIM2 inflammasome mediated-pyroptosis in BMDMs. Mechanistically, Andrographolide effectively prevented AIM2 from translocating into the nucleus to sense DNA damage induced by radiation or chemotherapeutic agents in BMDMs. Taken together, Andrographolide ameliorates RILI by suppressing AIM2 inflammasome mediated-pyroptosis in macrophage, identifying Andrographolide as a novel potential protective agent for RILI.

Enhanced Copper-Temozolomide Interactions by Protein for Chemotherapy against Glioblastoma Multiforme.

Current treatment of recurrent glioblastoma multiforme (GBM) demands dose-intense temozolomide (TMZ), a prodrug of 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), based on the spontaneous hydrolysis of TMZ at basic pH. However, how to control the activity of MTIC remains unknown, which poses a particular challenge to search a reliable MTIC receptor. We reported that copper, for the first time, is found to recognize and bind MTIC in the process of TMZ degradation, which means copper can play an important role in enhancing the bioavailability of MTIC derived from TMZ. Using apoferritin as a model copper-bound protein, we studied the copper-TMZ interaction in protein and observed efficient MTIC immobilization with high binding efficiency (up to 92.9% based on original TMZ) and capacity (up to 185 MTIC moieties per protein). The system was stable against both alkaline and acidic pH and could be activated by glutathione to liberate MTIC, which paves a way to deliver a DNA-alkylating agent for both TMZ-sensitive and TMZ-resistant GBM chemotherapy. Our study provides a new insight for understanding the potential relationship between the special GBM microenvironment (specific copper accumulation) and the therapeutic effect of TMZ.

Light-Controlled in Vitro Gene Delivery Using Polymer-Tethered Spiropyran as a Photoswitchable Photosensitizer.

A gene delivery system using spiropyran as a photoswitchable photosensitizer for the controlled photochemical internalization effect was developed by engineering the outer coating of a polyethylenimine/DNA complex with a small amount of spiropyran-containing cationic copolymers. The successful binding of cationic polymers by the polyethylenimine coating was detected by the distance-sensitive fluorescence resonance energy-transfer technique that evidenced the occurrence of energy transfer between fluorescein-labeled cationic copolymers and polyethylenimine-condensed rhodamine-labeled DNA. The ternary polyplexes feature reversible controllability of singlet oxygen generation based on the dual effect of spiropyrans in photochromism and aggregation-induced enhanced photosensitization, allowing significant light-induced amplification of bPEI-mediated in vitro transgene efficiency (from original 15% to final 91%) at a low DNA dose, with the integrity of supercoiled DNA structure unaffected. The use of spiropyran without the need of other photosensitizers circumvents the issue of uncontrolled long-lasting photocytotoxicity in gene delivery.

Iron sulfur clusters in protein nanocages for photocatalytic hydrogen generation in acidic aqueous solutions.

We took advantage of the iron binding affinity of apoferritin to immobilize iron-sulfur clusters into apoferritin up to 312 moieties per protein, with a loading rate as high as 25 wt%. The photocatalytic hydrogen generation activity in acidic aqueous solutions was achieved with TONs up to 31 (based on a single catalyst moiety) or 8.3 × 103 (based on a single protein) upon 3 h of visible light irradiation. The present study provides a versatile strategy to construct uniform protein/photocatalyst supramolecular systems with FeFe-H2ase activity.

A water soluble donor-acceptor-donor conjugated oligomer as a photosensitizer for mitochondria-targeted photodynamic therapy.

We designed a water soluble benzotriazole-cored donor-acceptor-donor molecule bearing four triphenylphosphonium moieties for mitochondria targeting. The organic molecule showed strong singlet oxygen sensitizing capability (ΦΔ 0.64), minimal dark cytotoxicity and high photocytotoxicity. The molecule is efficient in photodynamic killing of tumour cells under normal and hypoxia conditions using a very low light flux, and has potential in near-infrared two-photon imaging and photodynamic therapy.

Spiropyran in nanoassemblies as a photosensitizer for photoswitchable ROS generation in living cells.

Reversibly controlled generation of singlet oxygen from photosensitizing nanosystems has the benefits of selective cell killing and controllable effect time, but is a challenging option for photodynamic therapies. We report a strategy for integrating photochromic spiropyrans into biocompatible cationic polymers, which involved assembling nucleic acids into functional nanoparticles without introducing additional photosensitizers and imaging agents. We found that spiropyran-containing nanoparticles have photoswitching properties for both fluorescence (with a quantum yield of up to 0.27) and singlet oxygen generation (with a quantum yield of up to 0.22) in aqueous solutions and cells, and demonstrated that spiropyrans in nanoassemblies featuring aggregation-induced enhanced photosensitization and emission could be potentially applied in photodynamic therapy studies on tumor cells.

Remarkable Amplification of Polyethylenimine-Mediated Gene Delivery Using Cationic Poly(phenylene ethynylene)s as Photosensitizers.

Conjugated polymers can serve as good photosensitizers in biomedical applications. However, it remains unknown whether they are phototoxic to the supercoiled structure of DNA in improving gene delivery by the photochemical internalization (PCI) strategy, which complicates the application of conjugated polymers in gene delivery. In this work, we introduced a trace amount of cationic poly(phenylene ethynylene)s (cPPEs) into the polymeric shell of branched polyethylenimine (BPEI)/DNA complexes, studied the photosensitization of singlet oxygen by cPPEs, and confirmed that the supercoiled DNA is undamaged by the singlet oxygen generated by the photoexcitation of cPPEs. By taking advantage of the cPPE-mediated PCI effect, we report that the addition of the trace amount of cPPEs to the outer shell of the BPEI/DNA polyplexes could greatly amplify the transfection of gene green fluorescent protein on tumor cells with the efficiency from 14 to 86% without decreasing the cell viabilities, well solving the problem with a poor transfection capability of BPEI under low DNA-loading conditions. Our strategy to employ conjugated polymers as photosensitizing agents in gene delivery systems is simple, safe, efficient, and promising for broad applications in gene delivery areas.

Synthesis of Structurally Defined Cationic Polythiophenes for DNA Binding and Gene Delivery.

Water-soluble conjugated polymers (WCPs) have prospective applications in the field of bioimaging, disease diagnosis, and therapy. However, the use of WCPs with controllability and regioregularity for bioapplications have scarcely been reported. In this work, we synthesized polythiophenes containing ester side chains (P3ET) via Kumada catalyst-transfer polycondensation (KCTP) and confirmed a quasi-"living" chain-growth mechanism. In addition, we obtained cationic regioregular polythiophenes (cPTs) by aminolysis of P3ET with varied chain lengths, and studied DNA binding capability and gene delivery performance. Benefiting from photocontrolled generation of intracellular reactive oxygen species (ROS), the cationic polythiophenes successfully delivered DNA into tumor cells without additional polymer species.