Auxochrome Dimethyl-Dihydroacridine Improves Fluorophores for Prolonged Live-Cell Super-Resolution Imaging.

Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.

Three birds one stone: an enzyme-activatable theragnostic agent for fluorescence diagnosis, photodynamic and inhibitor therapies.

AX11890, an inhibitor of overexpressed enzyme, KIAA1363, in some breast cancers, was conjugated with a benzo[a]phenothiazinium photosensitizer to develop a tumor micro-environment-responsive photosensitizer NBS-L-AX. In normal cells, the special geometry of NBS-L-AX causes the fluorescence and photodynamic therapeutic (PDT) effect of NBS-L to be quenched. In cancer cells, when allowed to interact with the enzyme KIAA1363, the geometry of NBS-L-AX changes such that it becomes fluorescent and photodynamically active. Thus, the material of NBS-L-AX serves as an activated imaging and PDT treatment agent for breast cancers. In addition, NBS-L-AX also shows a selective inhibition effect against breast cancer cells.

Unimolecular Photodynamic O2-Economizer To Overcome Hypoxia Resistance in Phototherapeutics.

Tumor hypoxia has proven to be the major bottleneck of photodynamic therapy (PDT) to clinical transformation. Different from traditional O2 delivery approaches, here we describe an innovative binary photodynamic O2-economizer (PDOE) tactic to reverse hypoxia-driven resistance by designing a superoxide radical (O2•-) generator targeting mitochondria respiration, termed SORgenTAM. This PDOE system is able to block intracellular O2 consumption and down-regulate HIF-1α expression, which successfully rescues cancer cells from becoming hypoxic and relieves the intrinsic hypoxia burden of tumors in vivo, thereby sparing sufficient endogenous O2 for the PDT process. Photosensitization mechanism studies demonstrate that SORgenTAM has an ideal intersystem crossing rate and triplet excited state lifetime for generating O2•- through type-I photochemistry, and the generated O2•- can further trigger a biocascade to reduce the PDT's demand for O2 in an O2-recycble manner. Furthermore, SORgenTAM also serves to activate the AMPK metabolism signaling pathway to inhibit cell repair and promote cell death. Consequently, using this two-step O2-economical strategy, under relatively low light dose irradiation, excellent therapeutic responses toward hypoxic tumors are achieved. This study offers a conceptual while practical paradigm for overcoming the pitfalls of phototherapeutics.

Superoxide Radical Photogenerator with Amplification Effect: Surmounting the Achilles' Heels of Photodynamic Oncotherapy.

Strong oxygen dependence, poor tumor targeting, and limited treatment depth have been considered as the "Achilles' heels" facing the clinical usage of photodynamic therapy (PDT). Different from common approaches, here, we propose an innovative tactic by using photon-initiated dyad cationic superoxide radical (O2-•) generator (ENBOS) featuring "0 + 1 > 1" amplification effect to simultaneously overcome these drawbacks. In particular, by taking advantage of the Förster resonance energy transfer theory, the energy donor successfully endows ENBOS with significantly enhanced NIR absorbance and photon utility, which in turn lead to ENBOS more easily activated and generating more O2-• in deep tissues, that thus dramatically intensifies the type I PDT against hypoxic deep tumors. Moreover, benefiting from the dyad cationic feature, ENBOS achieves superior "structure-inherent targeting" abilities with the signal-to-background ratio as high as 25.2 at 48 h post intravenous injection, offering opportunities for accurate imaging-guided tumor treatment. Meanwhile, the intratumoral accumulation and retention performance are also markedly improved (>120 h). On the basis of these unique merits, ENBOS selectively inhibits the deep-seated hypoxic tumor proliferation at a low light-dose irradiation. Therefore, this delicate design may open new horizons and cause a paradigm change for PDT in future cancer therapy.

Near-Infrared Light-Initiated Molecular Superoxide Radical Generator: Rejuvenating Photodynamic Therapy against Hypoxic Tumors.

Hypoxia, a quite universal feature in most solid tumors, has been considered as the "Achilles' heel" of traditional photodynamic therapy (PDT) and substantially impairs the overall therapeutic efficacy. Herein, we develop a near-infrared (NIR) light-triggered molecular superoxide radical (O2-•) generator (ENBS-B) to surmount this intractable issue, also reveal its detailed O2-• action mechanism underlying the antihypoxia effects, and confirm its application for in vivo targeted hypoxic solid tumor ablation. Photomediated radical generation mechanism study shows that, even under severe hypoxic environment (2% O2), ENBS-B can generate considerable O2-• through type I photoreactions, and partial O2-• is transformed to high toxic OH· through SOD-mediated cascade reactions. These radicals synergistically damage the intracellular lysosomes, which subsequently trigger cancer cell apoptosis, presenting a robust hypoxic PDT potency. In vitro coculture model shows that, benefiting from biotin ligand, ENBS-B achieves 87-fold higher cellular uptake in cancer cells than normal cells, offering opportunities for personalized medicine. Following intravenous administration, ENBS-B is able to specifically target to neoplastic tissues and completely suppresses the tumor growth at a low light-dose irradiation. As such, we postulated this work will extend the options of excellent agents for clinical cancer therapy.

Ratiometric Fluorescent Probe for Lysosomal pH Measurement and Imaging in Living Cells Using Single-Wavelength Excitation.

A novel lysosome-targeting ratiometric fluorescent probe (CQ-Lyso) based on the chromenoquinoline chromorphore has been developed for the selective and sensitive detection of intracellular pH in living cells. In acidic media, the protonation of the quinoline ring of CQ-Lyso induces an enhanced intramolecular charge transfer (ICT) process, which results in large red-shifts in both the absorption (104 nm) and emission (53 nm) spectra which forms the basis of a new ratiometric fluorescence pH sensor. This probe efficiently stains lysosomes with high Pearson's colocalization coefficients using LysoTrackerDeep Red (0.97) and LysoTrackerBlue DND-22 (0.95) as references. Importantly, we show that CQ-Lyso quantitatively measures and images lysosomal pH values in a ratiometric manner using single-wavelength excitation.

Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo.

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aqueous solution and living cells.

Antimicrobial photodynamic efficacy of side-chain functionalized benzo[a]phenothiazinium dyes.

5-(Ethylamino)-9-diethylaminobenzo[a]phenothiazinium chloride (EtNBS) is a photosensitizer (PS) with broad antimicrobial photodynamic activity. The objective of this study was to determine the antimicrobial photodynamic effect of side chain/end group modifications of EtNBS on two representative bacterial Gram-type-specific strains. Two EtNBS derivatives were synthesized, each functionalized with a different side-chain end-group, alcohol or carboxylic acid. In solution, both exhibited photochemical properties consistent with those of the EtNBS parent molecule. In vitro photodynamic therapy experiments revealed an initial Gram-type-specificity with two representative strains; both derivatives were phototoxic to Staphylococcus aureus 29,213 but the carboxylic acid derivative was nontoxic to Escherichia coli 25,922. This difference in photodynamic efficacy was not due to a difference in the binding of the two molecules to the bacteria as the amount of both derivatives bound by bacteria was identical. Interestingly, the carboxylic acid derivative produced no fluorescence emission when observed in cultures of E. coli via fluorescence microscopy. These early findings suggest that the addition of small functional groups could achieve Gram-type-specific phototoxicity through altering the photodynamic activity of PSs and deserve further exploration in a larger number of representative strains of each Gram type.

5,9-Diaminodibenzo[a,j]phenoxazinium chloride: a rediscovered efficient long wavelength fluorescent dye.

We have evaluated the chemical, photophysical and photostability properties of 5,9-diaminodibenzo[a,j]phenoxazinium chloride, 3, and its bis-5,9-ethylamino analogue, 4, with the goal of determining if they have characteristics that are compatible with the requirements of a useful fluorescent probe. In order to gauge the potential utility of these fluorophores in biological and non-biological applications, these data were compared to those obtained for Oxazine 118, 1, and Cresyl Violet, 2, two well known fluorescent dyes that differ in molecular structure from the title dye 3 by having two or one fewer benzo moieties fused to a generic oxazine ring structure, respectively. The findings of this investigation show that 3, as well as bis-ethylamino analogue, 4, have fluorescent lifetimes, quantum yields and photostabilities that compare favorably with the lower order benchmark fluorophores 1 and 2. Moreover, both dibenzo dyes have the highly desirable properties of absorbing and emitting further in the red and far red /near infrared spectral region, respectively, than do their less conjugated analogues. Taken together, these results suggest that 3 constitutes an archetype upon which a new class of long wavelength fluorescent reporters might be based.

Real-time fluorescence monitoring of phenothiazinium photosensitizers and their anti-mycobacterial photodynamic activity against Mycobacterium bovis BCG in in vitro and in vivo models of localized infection.

An objective was to explore the photodynamic activity of two cationic photosensitizers (PS) (benzo[a]phenothiazinium chloride and benzo[a]phenoselenazinium chloride) against Mycobacterium bovis BCG both in vitro and in a murine model of BCG-granuloma. The hypothesis being tested in this study was that cationic molecules could best interact with the negatively charged membrane of BCG as a model for mycobacterial infection. Cells in culture were incubated with various concentrations of PS and subsequently illuminated using a 635 nm diode laser. Dark- and light-induced killing profiles were generated as a function of fluence and dye concentration. In vivo, local injection of the PS into subcutaneous Mycobacterium-induced granuloma sites in murine model was followed by red light illumination of the same area. A special microscope was fabricated for real-time in vivo fluorescent microscopy to monitor EtNBS delivery to subcutaneous murine granulomata. Both PS demonstrated good in vitro antimycobacterial photodynamic activity with varying degrees of toxicity under dark conditions. Real time in vivo monitoring of benzophenothiazine chloride in the mouse model indicated that this fluorescent photosensitizer was delivered rapidly to the subcutaneous granuloma site. In vivo, photosensitizer specific dark- and photo-toxicities depended on the structure, concentration of the photosensitizer and the light dose utilized. Cationic phenothiazine photosensitizers are promising candidates for use in anti-mycobacterial PDT for localized diseases such as cutaneous and pulmonary granulomata.

Synthesis and properties of benzo[a]phenoxazinium chalcogen analogues as novel broad-spectrum antimicrobial photosensitizers.

The goal of this investigation was to develop improved photosensitizers for use as antimicrobial drugs in photodynamic therapy of localized infections. Replacement of the oxygen atom in 5-(ethylamino)-9-diethylaminobenzo[a]phenoxazinium chloride (1) with sulfur and selenium afforded thiazinium and selenazinium analogues 2 and 3, respectively. All three dyes are water soluble, lipophilic, and red light absorbers. The relative photodynamic activities of the chalcogen series were evaluated against a panel of prototypical pathogenic microorganisms: the Gram-positive Enterococcus faecalis, the Gram-negative Escherichia coli, and the fungus Candida albicans. Selenium dye 3 was highly effective as a broad-spectrum antimicrobial photosensitizer with fluences of 4-32 J/cm2 killing 2-5 more logs of all cell types than sulfur dye 2, which was slightly more effective than oxygen analogue 1. These data, taken with the findings of uptake and retention studies, suggest that the superior activity of selenium derivative 3 can be attributed to its much higher triplet quantum yield.

The role of photosensitizer molecular charge and structure on the efficacy of photodynamic therapy against Leishmania parasites.

Photodynamic therapy (PDT) is emerging as a potential therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). In order to establish a rationale for effective PDT of CL, we investigated the impact of the molecular charge and structure of photosensitizers on the parasitic phototoxic response. Two photosensitizers from the benzophenoxazine family that bear an overall cationic charge and two anionic porphyrinoid molecules were evaluated. The photodynamic activity of the photosensitizers decreases in the following order: EtNBSe > EtNBS > BpD > PpIX. The studies suggest that compared to hydrophobic anionic photosensitizers, the hydrophilic cationic benzophenoxazine analogs provide high effectiveness of PDT possibly due to (1) their strong attraction to the negatively charged parasitic membrane, (2) their hydrophilicity, (3) their high singlet oxygen quantum yield, and (4) their efficacy in targeting intracellular organelles.