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Singlet molecular oxygen, O 2 (a 1 Δ g ), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O 2 (a 1 Δ g ) in this regard is acknowledged, an understanding of the targets and mechanisms of O 2 (a 1 Δ g ) action is inadequate. Thus, methods that better facilitate studies of O 2 (a 1 Δ g ) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O 2 (a 1 Δ g ) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O 2 (a 1 Δ g ) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O 2 (a 1 Δ g ) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O 2 (a 1 Δ g ) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O 2 (a 1 Δ g ) at the expense of other ROS. We also comment on our recent efforts to monitor O 2 (a 1 Δ g ) in cells and to monitor the cell's response to O 2 (a 1 Δ g )., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Photodynamic therapy (PDT) is a minimally invasive treatment for several diseases. It combines light energy with a photosensitizer (PS) to destroy the targeted cells or tissues. A PS itself is a non-toxic substance, but it becomes toxic to the target cells through the activation of light at a specific wavelength. There are some limitations of PDT, although it has been used in clinical studies for a long time. Two-photon excitation (TPE) and upconversion (UC) for PDT have been recently developed. A TPE nanoparticle-based PS combines the advantages of TPE and nanotechnology that has emerged as an attractive therapeutic agent for near-infrared red (NIR) light-excited PDT, whilst UC is also used for the NIR light-triggered drug release, activation of 'caged' imaging, or therapeutic molecules during PDT process for the diagnosis, imaging, and treatment of cancers. Methods: Nine electronic databases were searched, including WanFang Data, PubMed, Science Direct, Scopus, Web of Science, Springer Link, SciFinder, and China National Knowledge Infrastructure (CNKI), without any language constraints. TPE and UCNP were evaluated to determine if they had different effects from PDT on cancers. All eligible studies were analyzed and summarized in this review. Results: TPE-PDT and UCNP-PDT have a high cell or tissue penetration ability through the excitation of NIR light to activate PS molecules. This is much better than the conventional PDT induced by visible or ultraviolet (UV) light. These studies showed a greater PDT efficacy, which was determined by enhanced generation of reactive oxygen species (ROS) and reduced cell viability, as well as inhibited abnormal cell growth for the treatment of cancers. Conclusions: Conventional PDT involves Type I and Type II reactions for the generation of ROS in the treatment of cancer cells, but there are some limitations. Recently, TPE-PDT and UCNP-PDT have been developed to overcome these problems with the help of nanotechnology in in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

Background: In addition to their great optical properties, nanodiamonds (NDs) have recently proved useful for two-photon-excited photodynamic therapy (TPE-PDT) applications. Indeed, they are able to produce reactive oxygen species (ROS) directly upon two-photon excitation but not with one-photon excitation; Methods: Fluorescent NDs (FNDs) with a 100 nm diameter and detonation NDs (DNDs) of 30 nm were compared. In order to use the gems for cancer-cell theranostics, they were encapsulated in a bis(triethoxysilyl)ethylene-based (ENE) periodic mesoporous organosilica (PMO) shell, and the surface of the formed nanoparticles (NPs) was modified by the direct grafting of polyethylene glycol (PEG) and amino groups using PEG-hexyltriethoxysilane and aminoundecyltriethoxysilane during the sol–gel process. The NPs' phototoxicity and interaction with MDA-MB-231 breast cancer cells were evaluated afterwards; Results: Transmission electronic microscopy images showed the formation of core–shell NPs. Infrared spectra and zeta-potential measurements confirmed the grafting of PEG and NH2 groups. The encapsulation of the NDs allowed for the imaging of cancer cells with NDs and for the performance of TPE-PDT of MDA-MB-231 cancer cells with significant mortality. Conclusions: Multifunctional ND@PMO core–shell nanosystems were successfully prepared. The NPs demonstrated high biocompatibility and TPE-PDT efficiency in vitro in the cancer cell model. Such systems hold good potential for two-photon-excited PDT applications. [ABSTRACT FROM AUTHOR]

Facile tailoring of photosensitizers (PSs) with advanced and synergetic properties is highly expected to broaden and deepen photodynamic therapy (PDT) applications. Herein, a catalyst‐free thiol–yne click reaction was employed to develop the sulfur atom‐based PSs by using the in situ formed sulfur "heavy atom effect" to enhance the intersystem crossing (ISC), while such an effect can be remarkably magnified by the polymerization. The introduction of a tetraphenylpyrazine‐based aggregation‐induced emission (AIE) unit was also advantageous in PS design by suppressing their non‐radiative decay to facilitate the ISC in the aggregated state. Besides, the resulting sulfur atom electron donor, together with a double‐bond π bridge and AIE electron acceptor, created a donor‐π‐acceptor (D‐π‐A) molecular system with good two‐photon excitation properties. Combined with the high singlet oxygen generation efficiency, the fabricated polymer nanoparticles exhibited an excellent in vitro two‐photon‐excited PDT towards cancer cells, therefore possessing a huge potential for the deep‐tissue disease therapy. [ABSTRACT FROM AUTHOR]

Abstract We successfully prepared water-soluble fullerenol [C60(OH)46] that exhibited a high singlet oxygen quantum yield and efficiently generated reactive oxygen species. Additionally, the water-soluble C60(OH)46 with a higher composition of exposed hydroxyl groups had superior two-photon stability and characteristics compared with that with a lower composition of such groups. Therefore, the prepared fullerenol can be an effective two-photon photosensitizer. The water-soluble C60(OH)46 had favorable two-photon properties. During two-photon photodynamic therapy, the water-soluble C60(OH)46 had substantial antimicrobial activity against Escherichia coli at an ultralow-energy level of 211.2 nJ pixel−1 with 800 scans and a photoexcited wavelength of 760 nm.

Treatment in combination with real-time monitoring of dynamic organelle changes is of great significance for evaluating therapeutic efficacy. However, it remains highly challenging to realize such specific treatment and monitoring in organelles. Herein, we developed one kind of red emissive two-photon carbon dots (TP-CDs) via an N doping strategy for nucleolus-targeted treatment combined with real-time monitoring of dynamic changes in the nucleolus. In the TP-CD system, N doping endows TP-CDs with intrinsic two-photon excitation, red emission at 605 nm, and singlet oxygen (1O 2) production under 638 nm laser irradiation. More importantly, N heterocycles induced by doping offers significant affinity for TP-CDs toward RNA to realize specific self-targeting toward nucleolus and fluorescence variation during the photodynamic therapy (PDT) via the cleaving of RNA chain by 1O 2 to dissociate TP-CDs@RNA complex. By combining 1O 2 production, fluorescent variation during the PDT process, long-wavelength excitation and emission characteristics, as well as specific self-targetability of nucleolus, TP-CDs can be considered as a kind of "smart" CDs to realize treatment in combination of real-time dynamic changes in nucleolus for the first time. [Display omitted] [ABSTRACT FROM AUTHOR]

Developing novel photosensitizers for deep tissue imaging and efficient photodynamic therapy (PDT) remains a challenge because of the poor water solubility, low reactive oxygen species (ROS) generation efficiency, serve dark cytotoxicity, and weak absorption in the NIR region of conventional photosensitizers. Herein, cyclometalated iridium (III) complexes (Ir) with aggregation-induced emission (AIE) feature, high photoinduced ROS generation efficiency, two-photon excitation, and mitochondria-targeting capability were designed and further encapsulated into biocompatible nanoparticles (NPs). The Ir-NPs can be used to disturb redox homeostasis in vitro, result in mitochondrial dysfunction and cell apoptosis. Importantly, in vivo experiments demonstrated that the Ir-NPs presented obviously tumor-targeting ability, excellent antitumor effect, and low systematic dark-toxicity. Moreover, the Ir-NPs could serve as a two-photon imaging agent for deep tissue bioimaging with a penetration depth of up to 300 μm. This work presents a promising strategy for designing a clinical application of multifunctional Ir-NPs toward bioimaging and PDT. [ABSTRACT FROM AUTHOR]

We successfully prepared water-soluble fullerenol [C60(OH)46] that exhibited a high singlet oxygen quantum yield and efficiently generated reactive oxygen species. Additionally, the water-soluble C60(OH)46 with a higher composition of exposed hydroxyl groups had superior two-photon stability and characteristics compared with that with a lower composition of such groups. Therefore, the prepared fullerenol can be an effective two-photon photosensitizer. The water-soluble C60(OH)46 had favorable two-photon properties. During two-photon photodynamic therapy, the water-soluble C60(OH)46 had substantial antimicrobial activity against Escherichia coli at an ultralow-energy level of 211.2 nJ pixel−1 with 800 scans and a photoexcited wavelength of 760 nm. [ABSTRACT FROM AUTHOR]

Singlet oxygen (1O2) detection remains an active area of research in the field of biological and biomedical science. In this work, we reported a novel Off‐On type FRET‐based fluorescent 1O2 nanoprobe comprising the energy‐donating conjugated polymer, poly(9,9‐dioctylfluorenyl‐2,7‐diyl) (PFO), and the energy‐accepting 1O2 indicator, 3‐diphenylisobenzofuran (DPBF). Prior to the reaction with 1O2, the nanoprobe was in Off‐state because fluorescence of PFO (λem=441 nm) was completely quenched by loaded DPBF; in the presence of 1O2, it switched to On‐state due to the depletion of DPBF. The as‐prepared 1O2 nanoprobe had a particle size of ∼59 nm, good biocompatibility, high specificity and sensitivity with a limit of detection of 353 nM. Importantly, the fluorescent nanoprobes were highly sensitive to 1O2 under two‐photon excitation (λex=800 nm). Using a multiphoton confocal microscope, generation of intracellular 1O2 during photodynamic therapy was sensitively detected by recording fluorescence intensity of the nanoprobes. [ABSTRACT FROM AUTHOR]

Reactive Oxygen Species, ROS, are small molecules ( e.g. , hydroxyl radical, superoxide radical anion, hydrogen peroxide, nitric oxide and singlet oxygen) that play key signaling roles in mammalian cells. They are generated as part of normal cell function, but also play roles in a cell’s response to perturbation ( e.g. , disease) and in many disease treatments ( e.g. , drugs). Although the importance of ROS is acknowledged, a general understanding of the mechanisms of ROS action and their biological effects is inadequate. Thus, new experimental methods that better facilitate studies of ROS behavior in mammalian cells are highly desired. In this feature article, we focus on one ROS in particular: singlet oxygen, O 2 (a 1 Δ g ). We summarize our recent efforts to selectively produce singlet oxygen in sub-cellular, spatially-resolved experiments performed on single mammalian cells. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to initially create a localized femtoliter volume of singlet oxygen, (2) protein-encapsulated photosensitizers that can be localized in cells using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments. We also provide a brief overview of our recent efforts to monitor singlet oxygen in cells ( e.g. , direct time-resolved optical detection, fluorescent probes) and to monitor the cell’s response to singlet oxygen ( e.g. , the use of rapid super-resolution microscopy). In all cases, we discuss the advantages and disadvantages of that particular approach/tool. [ABSTRACT FROM AUTHOR]

[Display omitted] • A two-photon photodynamic photosensitizer with mitochondrion-targeting and AIE characteristics was prepared. • The integration of Ir make 50 times enhancement of ROS production compared to its counterpart molecule. • TPABP-Ir@BSA NPs exhibits satisfactory biosafety. • A 90% tumor suppression was achieved by type-I&II synergetic photochemical reactions under hypoxic environment. In recent years, two-photon photodynamic therapy (TP-PDT) has received extensive attention in cancer treatment owing to its unique advantages of deep tissue penetration and high spatial–temporal controls. However, the high oxygen dependency of photosensitizers still remained as the major obstacle which undermined its overall therapeutic efficacy, especially in hypoxic-tolerant tumors. Exploration of new TP-PDT strategy with less-oxygen-dependency and subcellular organelle-targeting ability is an appealing yet seriously challenging task. In this work, a mitochondrion-targeting TP-PDT protocol based on type-I PSs with aggregation-induced emission (AIE) characteristics is proposed for the first time. Of the two synthesized AIE-active PSs, TPABP-Ir is demonstrated superior reactive oxygen species (ROS) production (both type I&II) owing to the doping of Ir(III). After encapsulation into bovine serum albumin (BSA) matrix, TPABP-Ir@BSA nanoparticles (Ir-NPs) are capable of realizing mitochondrion-targeting, high ROS production, and suppression of tumor growth under 880 nm femtosecond laser excitation. This study provides a novel treatment strategy which maximizes the PDT efficacy and offers a conceptual but practical paradigm for cancer treatment in translational nanomedicine. [ABSTRACT FROM AUTHOR]

Hydroxyl-terminated fourth generation poly(amido amine) dendrimer and folic acid were chemically bound on graphene oxide. The resultant hybrids exhibited one-photon and two-photon fluorescence emission, since the excitation irradiation at 390 and 780 nm on the hybrids brought a fluorescence emission in the visible region around 450 nm. In addition, the photocytotoxicity study revealed that under the two-photon excitation at 780 nm, the hybrids can absorb near-infrared light and generate reactive oxygen species which can oxidize the HeLa cells and cause their death, suggesting the phototherapeutic behavior. Cytotoxicity measurement revealed the high biocompatibility of the hybrids toward HeLa cells. Thus, the present biocompatible hybrids consisting of only dendrimer, folic acid and graphene oxide have potentials as photodynamic therapeutic agents for medical treatment. [ABSTRACT FROM AUTHOR]

Phototherapy techniques such as photodynamic therapy (PDT) and photothermal therapy (PTT) have aroused extensive interest as powerful cancer therapy techniques because of their advantages of minimal invasiveness. However, conventional PDT/PTT agents usually suffer from low efficiency and drawback of no imaging capability. Metal nanoparticle conjugates have been recently demonstrated to display a new type of aggregation induced emission, making them attractive multifunctional therapeutic agents. Herein, we designed Chlorin e6 (Ce6) conjugated gold nanoparticles (Au NPs) that displayed significantly enhanced one- and two-photon excitation fluorescence, singlet oxygen (1O 2) generation and photothermal effects. The photoactivity of Ce6-Au NPs was suppressed in the isolated form and became significantly enhanced in the aggregated form, together with significantly improved photostability owing to shortened emission lifetime. By forming aggregate in-situ inside cancer cells, aggregated Ce6-Au NPs were utilized to act as the fluorescence imaging probes under one- and two-photon excitation using red and near-infrared light, respectively. The aggregated Ce6-Au NPs also displayed excellent PDT and PTT performance and can effectively eradicate cancer cells by using red light continuous wave lasers with power density below the skin tolerance threshold. This plasmonic nano-system offers a promising potential for imaging guided PDT/PTT synergetic therapy, which might open a new venue to explore safe, and noninvasive theranostics. [Display omitted] • Aggregated Ce6-Au NPs display enhanced 1PE and 2PE fluorescence, 1O 2 generation and photothermal effects. • Both brightness and photostability of aggregated Ce6-Au NPs are significantly improved compared to free Ce6. • Ce6-Au NPs can act as fluorescence imaging probe and synergistic phototherapy agent upon forming aggregates in-situ. • Aggregated Ce6-Au NPs can effectively eradicate cancer cells by using red light with power below skin tolerance threshold. [ABSTRACT FROM AUTHOR]

The response of HeLa cells to subcellular spatially localized two-photon irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. Upon irradiation under these conditions, a localized population of PpIX excited states can be produced with meaningful intracellular spatial resolution; the dimensions of the domain where the incident light flux is high enough for PpIX two-photon absorption are defined by the microscope optics and by the diffraction of light (spot diameter at beam waist of ˜0.5-1.0 μm). In turn, the dimensions of the intracellular domain containing cytotoxic PpIX-sensitized singlet oxygen will likewise be confined. Most importantly, cell response (e.g., morphological signs of cell death) correlates with the light dose delivered and the intracellular domain irradiated. Thus, controlling light delivery can complement other techniques used to impart intracellular spatial localization in mechanistic studies of photoinitiated reactive oxygen species. Such controlled light delivery is also expected to be a particularly useful tool to study the so-called bystander effect in which a selectively-perturbed cell can influence a neighboring cell through intercellular signaling mechanisms. [ABSTRACT FROM AUTHOR]

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur. [ABSTRACT FROM AUTHOR]

Keywords: Singlet oxygen; nanoprobe; FRET; two-photon excitation; fluorescence quenching Under visible excitation or even NIR two-photon excitation, the initially non-fluorescent nanoprobe lit up in the presence of SP 1 sp O SB 2 sb . Singlet oxygen, nanoprobe, FRET, two-photon excitation, fluorescence quenching. [Extracted from the article]