Reactivity Differences Enable ROS for Selective Ablation of Bacteria.

An effective strategy to engineer selective photodynamic agents to surmount bacterial-infected diseases, especially Gram-positive bacteria remains a great challenge. Herein, we developed two examples of compounds for a proof-of-concept study where reactive differences in reactive oxygen species (ROS) can induce selective ablation of Gram-positive bacteria. Sulfur-replaced phenoxazinium (NBS-N) mainly generates a superoxide anion radical capable of selectively killing Gram-positive bacteria, while selenium-substituted phenoxazinium (NBSe-N) has a higher generation of singlet oxygen that can kill both Gram-positive and Gram-negative bacteria. This difference was further evidenced by bacterial fluorescence imaging and morphological changes. Moreover, NBS-N can also successfully heal the Gram-positive bacteria-infected wounds in mice. We believe that such reactive differences may pave a general way to design selective photodynamic agents for ablating Gram-positive bacteria-infected diseases.

Molecular Design of Highly Efficient Heavy-Atom-Free Triplet BODIPY Derivatives for Photodynamic Therapy and Bioimaging.

Novel BODIPY photosensitizers were developed for imaging-guided photodynamic therapy. The introduction of a strong electron donor to the BODIPY core through a phenyl linker combined with the twisted arrangement between the donor and the BODIPY acceptor is essential for reducing the energy gap between the lowest singlet excited state and the lowest triplet state (ΔEST ), leading to a significant enhancement in the intersystem crossing (ISC) of the BODIPYs. Remarkably, the BDP-5 with the smallest ΔEST (ca. 0.44 eV) exhibited excellent singlet oxygen generation capabilities in both organic and aqueous solutions. BDP-5 also displayed bright emission in the far-red/near-infrared region in the condensed states. More importantly, both in vitro and in vivo studies demonstrated that BDP-5 NPs displayed a high potential for photodynamic cancer therapy and bioimaging.

An Emerging Molecular Design Approach to Heavy-Atom-Free Photosensitizers for Enhanced Photodynamic Therapy under Hypoxia.

A novel strategy for designing highly efficient and activatable photosensitizers that can effectively generate reactive oxygen species (ROS) under both normoxia and hypoxia is proposed. Replacing both oxygen atoms in conventional naphthalimides (RNI-O) with sulfur atoms led to dramatic changes in the photophysical properties. The remarkable fluorescence quenching (ΦPL ≈ 0) of the resulting thionaphthalimides (RNI-S) suggested that the intersystem crossing from the singlet excited state to the reactive triplet state was enhanced by the sulfur substitution. Surprisingly, the singlet oxygen quantum yield of RNI-S gradually increased with increasing electron-donating ability of the 4-R substituents (MANI-S, ΦΔ ≈ 1.00, in air-saturated acetonitrile). Theoretical studies revealed that small singlet-triplet energy gaps and large spin-orbit coupling could be responsible for the efficient population of the triplet state of RNI-S. In particular, the ROS generation ability of MANI-S was suppressed under physiological conditions due to their self-assembly and was significantly recovered in cancer cells. More importantly, cellular experiments showed that MANI-S still produced a considerable amount of ROS even under severely hypoxic conditions (1% O2) through a type-I mechanism.

In Vivo Albumin Traps Photosensitizer Monomers from Self-Assembled Phthalocyanine Nanovesicles: A Facile and Switchable Theranostic Approach.

Albumin is a promising candidate as a biomarker for potential disease diagnostics and has been extensively used as a drug delivery carrier for decades. In these two directions, many albumin-detecting probes and exogenous albumin-based nanocomposite delivery systems have been developed. However, there are only a few cases demonstrating the specific interactions of exogenous probes with albumin in vivo, and nanocomposite delivery systems usually suffer from tedious fabrication processes and potential toxicity of the complexes. Herein, we demonstrate a facile "one-for-all" switchable nanotheranostic (NanoPcS) for both albumin detection and cancer treatment. In particular, the in vivo specific binding between albumin and PcS, arising from the disassembly of injected NanoPcS, is confirmed using an inducible transgenic mouse system. Fluorescence imaging and antitumor tests on different tumor models suggest that NanoPcS has superior tumor-targeting ability and the potential for time-modulated, activatable photodynamic therapy.

Nanostructured Phthalocyanine Assemblies with Protein-Driven Switchable Photoactivities for Biophotonic Imaging and Therapy.

Switchable phototheranostic nanomaterials are of particular interest for specific biosensing, high-quality imaging, and targeted therapy in the field of precision nanomedicine. Here, we develop a "one-for-all" nanomaterial that self-assembles from flexible and versatile phthalocyanine building blocks. The nanostructured phthalocyanine assemblies (NanoPcTBs) display intrinsically unique photothermal and photoacoustic properties. Fluorescence and reactive oxygen species generation can be triggered depending on a targeted, protein-induced, partial disassembly mechanism, which creates opportunities for low-background fluorescence imaging and activatable photodynamic therapy. In vitro evaluations indicate that NanoPcTB has a high selectivity for biotin receptor-positive cancer cells (e.g., A549) compared to biotin receptor-negative cells (e.g., WI38-VA13) and permits a combined photodynamic and photothermal therapeutic effect. Following systemic administration, the NanoPcTBs accumulate in A549 tumors of xenograft-bearing mice, and laser irradiation clearly induces the inhibition of tumor growth.

Highly Selective and Sensitive Two-Photon Fluorescence Probe for Endogenous Peroxynitrite Detection and Its Applications in Living Cells and Tissues.

A new two-photon fluorescence probe for endogenous peroxynitrite (ONOO-) detection was designed and synthesized. The probe exhibits good selectivity and sensitivity for ONOO- in phosphate-buffered saline solution with a low detection limit (3.5 × 10-8 M). Furthermore, the probe displays good performance in detecting endogenous ONOO-, not only in RAW 264.7 cells but also in rat hippocampal tissue, with a high two-photon cross-section value (δ ≈ 100 GM) at a deep depth of 120 µm.

Mitochondria-Targeted Reaction-Based Fluorescent Probe for Hydrogen Sulfide.

In this study, we developed a turn-on mitochondria-targeting hydrogen sulfide, "probe 1", based on the selective thiolysis of 7-nitro-1,2,3-benzoxadiazole amine moiety attached to the piperazine-based naphthalimide scaffold. Probe 1 exhibited excellent properties with 68-fold fluorescence enhancement, a low detection limit (2.46 µM), a low cytotoxicity, and a good selectivity toward hydrogen sulfide. The success of intracellular imaging indicated that probe 1 could be used in further applications for the investigation of biological functions and pathological roles of H2S in living systems.

A Selective Imidazoline-2-thione-Bearing Two-Photon Fluorescent Probe for Hypochlorous Acid in Mitochondria.

Hypochlorite (OCl(-)) plays a key role in the immune system and is involved in various diseases. Accordingly, direct detection of endogenous OCl(-) at the subcellular level is important for understanding inflammation and cellular apoptosis. In the current study, a two-photon fluorescent off/on probe (PNIS) bearing imidazoline-2-thione as an OCl(-) recognition unit and triphenylphosphine (TPP) as a mitochondrial-targeting group was synthesized and examined for its ability to image mitochondrial OCl(-) in situ. This probe, based on the specific reaction between imidazoline-2-thione and OCl(-), displayed a selective fluorescent off/on response to OCl(-) with the various reactive oxygen species in a physiological medium. PNIS was successfully applied to image of endogenously produced mitochondrial OCl(-) in live RAW 264.7 cells via two-photon microscopy.

One-photon and two-photon sensing of biothiols using a bis-pyrene-Cu(II) ensemble and its application to image GSH in the cells and tissues.

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are three major biothiols, which play key roles in various biological systems. Accordingly, the development of imaging probes has been actively studied. We report a new pyrene derivative 1, which showed large fluorescence quenching with Cu(2+) at pH 7.4. The ensemble 1-Cu(2+) was applied to detect biothiols. Among the various amino acids, GSH, Cys, and Hcy induced distinct turn-on fluorescence changes. The 1-Cu(2+) ensemble was further applied for GSH detection in living cells.

Development of imidazoline-2-thiones based two-photon fluorescence probes for imaging hypochlorite generation in a co-culture system.

We designed and prepared the imidazoline-2-thione containing OCl(-) probes, PIS and NIS, which operate through specific reactions with OCl(-) that yield corresponding fluorescent imidazolium ions. Importantly, we demonstrated that PIS can be employed to image OCl(-) generation in macrophages in a co-culture system. We have also employed two-photon microscopy and PIS to image OCl(-) in live cells and tissues, indicating that this probe could have wide biological applications.

Cyanine-based fluorescent probe for highly selective detection of glutathione in cell cultures and live mouse tissues.

Glutathione (GSH) plays a crucial role in human pathologies. Near-infrared fluorescence-based sensors capable of detecting intracellular GSH in vivo would be useful tools to understand the mechanisms of diseases. In this work, two cyanine-based fluorescent probes, 1 and 2, containing sulfonamide groups were prepared. Evaluation of the fluorescence changes displayed by probe 1, which contains a 2,4-dinitrobenzenesulfonamide group, shows that it is cell-membrane-permeable and can selectively detect thiols such as GSH, cysteine (Cys), and homocysteine (Hcy) in living cells. The response of 1 to thiols can be reversed by treatment with N-methylmaleimide (NMM). Probe 2, which possesses a 5-(dimethylamino)naphthalenesulfonamide group, displays high selectivity for GSH over Cys and Hcy, and its response can be reversed using NMM. The potential biological utility of 2 was shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can determine changes in the intracellular levels of GSH modualated by H2O2. The properties of 2 enable its use in monitoring GSH in vivo in a mouse model. The results showed that intravenous injection of 2 into a mouse generates a dramatic image in which strong fluorescence is emitted from various tissues, including the liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in mouse tissue cells promoted by excessive administration of the painkiller acetaminophen. The combined results coming from this effort suggest that the new probe will serve as an efficient tool for detecting cellular GSH in animals.

Charge Transfer-Promoted Excited State of a Heavy-Atom-Free Photosensitizer for Efficient Application of Mitochondria-Targeted Fluorescence Imaging and Hypoxia Photodynamic Therapy.

Conventional photosensitizers (PSs) used in photodynamic therapy (PDT) have shown preliminary success; however, they are often associated with several limitations including potential dark toxicity in healthy tissues, limited efficacy under acidic and hypoxic conditions, suboptimal fluorescence imaging capabilities, and nonspecific targeting during treatment. In response to these challenges, we developed a heavy-atom-free PS, denoted as Cz-SB, by incorporating ethyl carbazole into a thiophene-fused BODIPY core. A comprehensive investigation into the photophysical properties of Cz-SB was conducted through a synergistic approach involving experimental and computational investigations. The enhancement of intersystem crossing (kISC) and fluorescence emission (kfl) rate constants was achieved through a donor-acceptor pair-mediated charge transfer mechanism. Consequently, Cz-SB demonstrated remarkable efficiency in generating reactive oxygen species (ROS) under acidic and low-oxygen conditions, making it particularly effective for hypoxic cancer PDT. Furthermore, Cz-SB exhibited good biocompatibility, fluorescence imaging capabilities, and a high degree of localization within the mitochondria of living cells. We posit that Cz-SB holds substantial prospects as a versatile PS with innovative molecular design, representing a potential "one-for-all" solution in the realm of cancer phototheranostics.

Rhodamine-thiourea Linked Naphthalimide Derivative to Image ATP in Mitochondria using Two Channels.

Adenosine 5'-triphosphate (ATP), synthesized in mitochondria, is an energy molecule in all living things. ATP not only serves as an energy source for protein synthesis and muscle contraction, but also as an important indicator for various diseases, such as Parkinson's disease, cardiovascular disease, and others. Accordingly, detection and sensing of ATP, especially in mitochondria, are important. In this study, a unique ring-opening process of rhodamine was coupled to recognition of ATP via introduction of a thiourea moiety, which was further linked to a naphthalimide group. A strong fluorescent emission at ∼580 nm was accompanied by a color change from colorless to pink upon addition of ATP at pH 7.4. Fluorescent probe 1 successfully imaged mitochondrial ATP with a Pearson's coefficient of 0.8. In addition, green emission from the naphthalimide moiety at ∼530 nm was observed without any change upon addition of ATP. This emission can be considered equivalent to an internal standard to utilize probe 1 as a dual-channel probe for ATP. Furthermore, probe 1 showed negligible cytotoxicity based on MTT assays.

Albumin-mediated "Unlocking" of supramolecular prodrug-like nanozymes toward selective imaging-guided phototherapy.

Construction of an activatable photosensitizer and integration into an adaptive nanozyme during phototherapy without producing off-target toxicity remains a challenge. Herein, we have fabricated a prodrug-like supramolecular nanozyme based on a metallic-curcumin and cyanine co-assembly. The albumin-mediated phenol AOH group transformation of nanozyme changes its adjustable oxygen stress from negative superoxide dismutase-like activity of ROS-scavenging to positive photo oxidase activity with an ROS-amplifying capacity. It further increases the depth penetration of a nanozyme in a tumor spheroid, selectively targeting tumorous phototherapy. It also triggers a signal in targeted tumor cells and helps increase cancer cell ablation. This work suggests new options for development of activatable supramolecular nanozymes and provides a synergetic prodrug-like nanozyme strategy for early diagnosis and preclinical phototherapeutics.

A J-aggregated nanoporphyrin overcoming phototoxic side effects in superior phototherapy with two-pronged effects.

Phototherapy has been a promising therapeutic modality for pathological tissue due to its spatiotemporal selectivity and non-invasive characteristics. However, as a core component of phototherapy, a single photosensitizer (PS) nanoplatform integrating excellent therapeutic efficiency and minimal side effects remains an urgent but unmet need. Here, we construct a J-aggregated nano-porphyrin termed MTE based on the self-assembly of methyl-pheophorbide a derivative MPa-TEG (MT) and natural polyphenolic compound epigallocatechin gallate (EGCG). Due to the synergistic interaction between similar large π-conjugated structural EGCG and MT, MTE with small and uniform size is obtained by effectively hindering Ostwald ripening of MT. Noteworthily, MTE not only effectively avoids the inadvertent side effects of phototoxicity during transport thank to the ability of reactive oxygen species (ROS) scavenging, but also achieves two-pathway augmented superior phototherapy: (1) enhancing photodynamic therapy (PDT) via inhibiting the expression of anti-apoptosis protein surviving; (2) achieving adjuvant mild-temperature laser interstitial thermal therapy (LITT) via reducing the tumor thermoresistance on account that MTE inhibits the overexpression of HSP 70 and HSP 90. This research not only offers a facile strategy to construct multicomponent nanoplatforms but also provides a new pathway for efficient and low-toxicity phototherapy, which is beneficial to the future clinical application.

Activation of apoptosis by rationally constructing NIR amphiphilic AIEgens: surmounting the shackle of mitochondrial membrane potential for amplified tumor ablation.

In recent years, the use of aggregation-induced emission luminogens (AIEgens) for biological imaging and phototherapy has become an area of intense research. However, most traditional AIEgens suffer from undesired aggregation in aqueous media with "always on" fluorescence, or their targeting effects cannot be maintained accurately in live cells with the microenvironment changes. These drawbacks seriously impede their application in the fields of bio-imaging and antitumor therapy, which require a high signal-to-noise ratio. Herein, we propose a molecular design strategy to tune the dispersity of AIEgens in both lipophilic and hydrophilic systems to obtain the novel near-infrared (NIR, ∼737 nm) amphiphilic AIE photosensitizer (named TPA-S-TPP) with two positive charges as well as a triplet lifetime of 11.43 µs. The synergistic effects of lipophilicity, electrostatic interaction, and structure-anchoring enable the wider dynamic range of AIEgen TPA-S-TPP for mitochondrial targeting with tolerance to the changes of mitochondrial membrane potential (ΔΨ m). Intriguingly, TPA-S-TPP was difficult for normal cells to be taken up, indicative of low inherent toxicity for normal cells and tissues. Deeper insight into the changes of mitochondrial membrane potential and cleaved caspase 3 levels further revealed the mechanism of tumor cell apoptosis activated by AIEgen TPA-S-TPP under light irradiation. With its advantages of low dark toxicity and good biocompatibility, acting as an efficient theranostic agent, TPA-S-TPP was successfully applied to kill cancer cells and to efficiently inhibit tumor growth in mice. This study will provide a new avenue for researchers to design more ideal amphiphilic AIE photosensitizers with NIR fluorescence.

A Simple Route toward Next-Generation Thiobase-Based Photosensitizers for Cancer Theranostics.

Sulfur-substituted biocompatible carbonyl fluorophores have been recognized as effective heavy-atom-free photosensitizers (PSs) for cancer therapy due to their remarkable phototherapeutic properties. However, guidelines on their molecular design are still a substantial challenge. Most of the existing thiocarbonyl-based PSs are nonemissive in both the solution and restricted states, which hinders their further biomedical applications. Herein, we report the interesting finding that sulfur-substituted coumarins exhibit an uncommon phenomenon, aggregation-induced emission. More intriguingly, we also found that the introduction of a strong electron-accepting trifluoromethyl group is crucial to facilitate the mitochondrial-targeting ability of neutral coumarin fluorophores. The resulting CMS-2 PS displayed selective imaging of mitochondria and exhibited much higher photodynamic therapy efficiency toward cancer cells than that of the commercial PS erythrosine B. This work provides deep insight into the molecular design of heavy-atom-free thiobase-based PSs and simultaneously offers a great opportunity to develop novel mitochondrial-targeting fluorescent indicators with neutral bioinspired platforms.

A boronic acid-functionalized phthalocyanine with an aggregation-enhanced photodynamic effect for combating antibiotic-resistant bacteria.

Most existing photosensitizers (e.g., porphyrins) are often aggregated in aqueous solution because of their large conjugated molecular structures. This aggregation usually results in a lack or low levels of reactive oxygen species (ROS) generation due to aggregation-caused quenching, which severely hampers the application of photosensitizers in photodynamic therapy (PDT). Herein, we make an interesting finding that a boronic acid-functionalized phthalocyanine (PcN4-BA) displays an uncommon phenomenon, an aggregation-enhanced photodynamic effect. The combination of the ability to form uniform nanostructured self-assemblies in water, highly efficient ROS generation and boronic acid-induced targeting give PcN4-BA excellent performances in antimicrobial PDT.

Highly Efficient Aggregation-Induced Red-Emissive Organic Thermally Activated Delayed Fluorescence Materials with Prolonged Fluorescence Lifetime for Time-Resolved Luminescence Bioimaging.

Organic thermally activated delayed fluorescence (TADF) materials are emerging as potential candidates for time-resolved fluorescence imaging in biological systems. However, the development of purely organic TADF materials with bright aggregated-state emissions in the red/near-infrared (NIR) region remains challenging. Here, we report three donor-acceptor-type TADF molecules as promising candidates for time-resolved fluorescence imaging, which are engineered by direct connection of electron-donating moieties (phenoxazine or phenothiazine) and an electron-acceptor 1,8-naphthalimide (NI). Theoretically and experimentally, we elucidate that three TADF materials possessed remarkably small ΔEST to promote the occurrence of reverse intersystem crossing (RISC). Moreover, they all exhibit aggregation-induced red emissions and long delayed fluorescence lifetimes without the influence of molecular oxygen. More importantly, these long-lived and biocompatible TADF materials, especially the phenoxazine-substituted NI fluorophores, show great potential for high-contrast fluorescence lifetime imaging in living cells. This study provides further a molecular design strategy for purely organic TADF materials and expands the versatile biological application of long-lived fluorescence research in time-resolved luminescence imaging.