Rechargeable Afterglow Nanotorches for In Vivo Tracing of Cell-Based Microrobots.

As one of the self-luminescence imaging approaches that require pre-illumination instead of real-time light excitation, afterglow luminescence imaging has attracted increasing enthusiasm to circumvent tissue autofluorescence. In this work, we developed organic afterglow luminescent nanoprobe (nanotorch), which could emit persistent luminescence more than 10 days upon single light excitation. More importantly, the nanotorch could be remote charged by 660 nm light in a non-invasive manner, which showed great potential for real-time tracing the location of macrophage cell-based microrobots.

Image-Guided Enhanced PDT/PTT Combination Therapy Using Brominated Hemicyanine-Loaded Folate Receptor-Targeting Ag2S Quantum Dots.

Tumor-targeting nanoparticles and phototherapies are the two major trends in tumor-specific, local cancer therapy with minimal side effects. Organic photosensitizers (PSs) usually offer effective photodynamic therapy (PDT) but require enhanced solubility and tumor-targeting, which may be provided by a nanoparticle. Near-infrared (NIR)-emitting Ag2S quantum dots may act as a delivery vehicle for the PS, NIR tracking agent, and as a phototherapy (PTT) agent. A combination of the two provides luminescent dual-phototherapy agents with tumor-specificity and image-guided and enhanced cytotoxicity as a result of synergistic PDT and PTT. In this study, brominated hemicyanine (Hemi-Br), a photosensitizer, was loaded onto folic acid (FA)-tagged, glutathione (GSH)-coated Ag2S quantum dots (AS-GSH QDs) to provide enhanced phototoxicity via a photodynamic and mild photothermal effect in folate receptor(+) cancer cell lines at clinically relevant 640 nm irradiation. Final particles (AS-GSH-FA/Hemi-Br) had a hydrodynamic size of 75.5 nm, dual emission at both 705 and 910 nm, and a 93% light-to-heat conversion efficiency under 640 nm laser irradiation. In vitro cytotoxicity studies were conducted with folate receptor (FR)-positive HeLa and -negative A549 cell lines to differentiate receptor-mediated uptake. Enhanced phototoxicity on HeLa cells was observed with AS-GSH-FA/Hemi-Br compared to free Hemi-Br and AS-GSH-FA QDs due to increased uptake of the photosensitizer via active targeting and combination therapy, which is especially visible at the safe dose of single agents. Upon irradiation with a 640 nm (300 mW, 0.78 W/cm2) laser for 5 min, the viability of the HeLa cells decreased from 64% to 42 and 25% when treated with free Hemi-Br, AS-GSH-FA, and AS-GSH-FA/Hemi-Br, respectively. Overall, AS-GSH-FA/Hemi-Br provides image-guided enhanced PDT/PTT, which may be adopted for different FR(+) tumors.

Development of a cysteine responsive chlorinated hemicyanine for image-guided dual phototherapy.

A cysteine (Cys) activatable chlorinated hemicyanine (Cl-Cys) was introduced as a tumour selective image-guided dual phototherapy agent. Cl-Cys exhibited a significant turn on response in its near-IR emission signal and activated its singlet oxygen generation as well as photothermal conversion potentials upon reacting with Cys. The laser irradiation of Cl-Cys induced significant cell death in cancer cells with high Cys level, while it stayed deactivated and non-emissive in a healthy cell line. A profound synergistic PDT/PTT effect was observed at high doses. Remarkably, Cl-Cys marks the first ever example of Cys-responsive small organic-based therapeutic agent and holds a great promise to develop new activity-based photosensitizers for dual phototherapy action.

Molecular logic gates: the past, present and future.

The field of molecular logic gates originated 25 years ago, when A. P. de Silva published a seminal article in Nature. Stimulated by this ground breaking research, scientists were inspired to join the race to simulate the workings of the fundamental components of integrated circuits using molecules. The rules of this game of mimicry were flexible, and have evolved and morphed over the years. This tutorial review takes a look back on and provides an overview of the birth and growth of the field of molecular logics. Spinning-off from chemosensor research, molecular logic gates quickly proved themselves to be more than intellectual exercises and are now poised for many potential practical applications. The ultimate goal of this vein of research became clearer only recently - to "boldly go where no silicon-based logic gate has gone before" and seek out a new deeper understanding of life inside tissues and cells.

Thioether Coordination Chemistry for Molecular Imaging of Copper in Biological Systems.

Copper is an essential element in biological systems. Its potent redox activity renders it necessary for life, but at the same time, misregulation of its cellular pools can lead to oxidative stress implicated in aging and various disease states. Copper is commonly thought of as a static cofactor buried in protein active sites; however, evidence of a more loosely bound, labile pool of copper has emerged. To help identify and understand new roles for dynamic copper pools in biology, we have developed selective molecular imaging agents for this metal, drawing inspiration from both biological binding motifs and synthetic model complexes that reveal thioether coordination as a general design strategy for selective and sensitive copper recognition. In this review, we summarize some contributions, primarily from our own laboratory, on fluorescence- and magnetic resonance-based molecular-imaging probes for studying copper in living systems using thioether coordination chemistry.

Remote-Controlled Release of Singlet Oxygen by the Plasmonic Heating of Endoperoxide-Modified Gold Nanorods: Towards a Paradigm Change in Photodynamic Therapy.

The photodynamic therapy of cancer is contingent upon the sustained generation of singlet oxygen in the tumor region. However, tumors of the most metastatic cancer types develop a region of severe hypoxia, which puts them beyond the reach of most therapeutic protocols. More troublesome, photodynamic action generates acute hypoxia as the process itself diminishes cellular oxygen reserves, which makes it a self-limiting method. Herein, we describe a new concept that could eventually lead to a change in the 100 year old paradigm of photodynamic therapy and potentially offer solutions to some of the lingering problems. When gold nanorods with tethered endoperoxides are irradiated at 808 nm, the endoperoxides undergo thermal cycloreversion, resulting in the generation of singlet oxygen. We demonstrate that the amount of singlet oxygen produced in this way is sufficient for triggering apoptosis in cell cultures.

Intracellular modulation of excited-state dynamics in a chromophore dyad: differential enhancement of photocytotoxicity targeting cancer cells.

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O2 (a(1) Δg )], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer-related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.

Optical imaging probes for selective detection of butyrylcholinesterase.

Butyrylcholinesterase (BChE), a member of the human serine hydrolase family, is an essential enzyme for cholinergic neurotransmission as it catalyzes the hydrolysis of acetylcholine. It also plays central roles in apoptosis, lipid metabolism, and xenobiotic detoxification. On the other side, abnormal levels of BChE are directly associated with the formation of pathogenic states such as neurodegenerative diseases, psychiatric and cardiovascular disorders, liver damage, diabetes, and cancer. Thus, selective and sensitive detection of BChE level in living organisms is highly crucial and is of great importance to further understand the roles of BChE in both physiological and pathological processes. However, it is a very complicated task due to the potential interference of acetylcholinesterase (AChE), the other human cholinesterase, as these two enzymes share a very similar substrate scope. To this end, optical imaging probes have attracted immense attention in recent years as they have modular structures, which can be tuned precisely to satisfy high selectivity toward BChE, and at the same time they offer real time and nondestructive imaging opportunities with a high spatial and temporal resolution. Here, we summarize BChE selective imaging probes by discussing the critical milestones achieved during the development process of these molecular sensors over the years. We put a special emphasis on design principles and biological applications of highly promising new generation activity-based probes. We also give a comprehensive outlook for the future of BChE-responsive probes and highlight the ongoing challenges. This collection marks the first review article on BChE-responsive imaging agents.

Selective antibacterial and antibiofilm activity of chlorinated hemicyanine against gram-positive bacteria.

Antibiotic-free therapies are highly needed due to the limited success of conventional approaches especially against biofilm related infections. In this direction, antimicrobial phototherapy, either in the form of antimicrobial photothermal therapy (aPTT) or antimicrobial photodynamic therapy (aPDT), have appeared to be highly promising candidates in recent years. These are local and promising approaches for antibiotic resistant bacterial infections and biofilms. Organic small photosensitizers (PSs) are extensively preferred in antimicrobial phototherapy applications as they offer a great opportunity to combine therapeutic action (aPTT, aPDT or both) with fluorescence imaging on a single molecule. In this study, the bactericidal effect of cationic chlorinated hemicyanine (Cl-Hem)-based type I PS, which can function as a dual aPDT/aPTT agent, was investigated on both planktonic cells and biofilms of different gram-positive (E. faecalis and S. epidermidis) and gram-negative bacteria (P. aeruginosa and K. pneumoniae) with and without 640 nm laser irradiation. Cl-Hem was shown to induce a selective phototheranostic activity against gram-positive bacteria (E. faecalis and S. epidermidis). Cl-Hem exhibited both dose and laser irradiation time dependent bactericidal effect on planktonic and biofilms of S. epidermidis. These results clearly showed that highly potent Cl-Hem can treat resistant microbial infections, while allowing fluorescence detection at the same time. High biofilm reduction observed with combined aPDT/aPTT action of Cl-Hem together with its non-cytotoxic nature points out that Cl-Hem is a promising PS for antibacterial and antibiofilm treatments.

Hydrogen Sulfide Responsive Phototherapy Agents: Design Strategies and Biological Applications.

Hydrogen sulfide (H2S) is one of the critical gasotransmitters, which play important roles in regular physiological processes, especially in vital signaling pathways. However, fluctuations in endogenous H2S concentration can be linked to serious health problems, such as neurodegenerative diseases, cancer, diabetes, inflammation, cardiovascular diseases, and hypertension. Thus, it has attracted a great deal of attention in therapeutic applications, specifically in the field of phototherapy. Photodynamic therapy (PDT) and photothermal therapy (PTT) are two subclasses of phototherapy, which utilize either reactive oxygen species (ROS) or local temperature increase upon irradiation of a photosensitizer (PS) to realize the therapeutic action. Phototherapies offer unique advantages compared to conventional methods; thus, they are highly promising and popular. One of the design principles followed in new generation PSs is to build activity-based PSs, which stay inactive before getting activated by disease-associated stimuli. These activatable PSs dramatically improve the selectivity and efficacy of the therapy. In this review, we summarize small molecule and nanomaterial-based PDT and PTT agents that are activated selectively by H2S to initiate their cytotoxic effect. We incorporate single mode PDT and PTT agents along with synergistic and/or multimodal photosensitizers that can combine more than one therapeutic approach. Additionally, H2S-responsive theranostic agents, which offer therapy and imaging at the same time, are highlighted. Design approaches, working principles, and biological applications for each example are discussed in detail.

A mitochondria-targeted chemiluminescent probe for detection of hydrogen sulfide in cancer cells, human serum and in vivo.

Hydrogen sulfide (H2S) as a critical messenger molecule plays vital roles in regular cell function. However, abnormal levels of H2S, especially mitochondrial H2S, are directly correlated with the formation of pathological states including neurodegenerative diseases, cardiovascular disorders, and cancer. Thus, monitoring fluxes of mitochondrial H2S concentrations both in vitro and in vivo with high selectivity and sensitivity is crucial. In this direction, herein we developed the first ever example of a mitochondria-targeted and H2S-responsive new generation 1,2-dioxetane-based chemiluminescent probe (MCH). Chemiluminescent probes offer unique advantages compared to conventional fluorophores as they do not require external light irradiation to emit light. MCH exhibited a dramatic turn-on response in its luminescence signal upon reacting with H2S with high selectivity. It was used to detect H2S activity in different biological systems ranging from cancerous cells to human serum and tumor-bearing mice. We anticipate that MCH will pave the way for development of new organelle-targeted chemiluminescence agents towards imaging of different analytes in various biological models.

A hydrogen sulfide and tyrosinase responsive dual-locked fluorophore for selective imaging of melanoma cells.

A resorufin-based dual-locked fluorescent probe (RHT) was introduced to image melanoma cells selectively. RHT was shown to function as an AND molecular logic gate as it emitted a signal only in the presence of both hydrogen sulfide (H2S) and tyrosinase (Tyr), which are known to be overexpressed in melanoma cells. In vitro cell culture studies revealed that RHT can be activated with endogenous H2S and Tyr and allows selective imaging of B16-F10 cancer cells under confocal microscopy. RHT marks the first ever example of a fluorescent probe that is sequentially activated by H2S and Tyr.

Real-time visualization of butyrylcholinesterase activity using a highly selective and sensitive chemiluminescent probe.

Butyrylcholinesterase (BChE), one of the critical human cholinesterases, plays crucial roles in numerous physiological and pathological processes. Accordingly, it is a striking and at the same time challenging target for bioimaging studies. Herein, we developed the first ever example of a 1,2-dixoetane-based chemiluminescent probe (BCC) for monitoring BChE activity in native biological contexts such as living cells and animals. BCC was initially shown to exhibit a highly selective and sensitive turn-on response in its luminescence signal upon reacting with BChE in aqueous solutions. Later, BCC was utilized to image endogenous BChE activity in normal and cancer cell lines. It was also shown through inhibition experiments that BChE can detect fluctuations of BChE levels successfully. In vivo imaging ability of BCC was demonstrated in healthy and tumor-bearing mice models. BCC enabled us to visualize the BChE activity in different regions of the body. Furthermore, it was successfully employed to monitor tumors derived from neuroblastoma cells with a very high signal to noise ratio. Thus, BCC appears as a highly promising chemiluminescent probe, which can be used to further understand the contribution of BChE to regular cellular processes and the formation of diseased states.

Heavy atom free singlet oxygen generation: doubly substituted configurations dominate S1 states of bis-BODIPYs.

S(0), S(1), and T(1) states of various orthogonal 8,8' and 8,2'-bis-boradiaza-s-indacene (BODIPY) dyes, recently (Angew. Chem., Int. Ed.2011, 50, 11937) proposed as heavy atom free photosensitizers for O(2)((1)Δ(g)) generation, were studied by multireference quantum chemical approaches. S(0)→S(1) excitation characteristics of certain bis-BODIPYs are shown to be drastically different than the parent BODIPY chromophore. Whereas a simple HOMO→LUMO-type single substitution perfectly accounts for the BODIPY core, S(1) states of certain orthogonal bis-BODIPYs are described as linear combinations of doubly substituted (DS) configurations which overall yield four electrons in four singly occupied orbitals. Computed DS character of S(1), strongly correlated with facile (1)O(2) production, was presumed to occur via S(1)→T(1) intersystem crossing (ISC) of the sensitizer. Further confirmation of this relation was provided by newly synthesized BODIPY derivatives and comparison of spectroscopic properties of their dimers and monomers. Near-IR absorption, desired for potential photodynamic therapy applications, was not pursuable for bis-chromophores by the standard strategy of π-extension, as DS singlet states are destabilized. Decreased exchange coupling in π-extended cases appears to be responsible for this destabilization. Comparisons with iodine incorporated bis-BODIPYs suggest that the dynamics of (1)O(2) generation via DS S(1) states are qualitatively different from that via ISC originating from heavy atom spin-orbit coupling. Although red-shifting the absorption wavelength to enter the therapeutic window does not seem attainable for orthogonal bis-BODIPYs with DS S(1) states, modifications in the chromophore cores are shown to be promising in fine-tuning the excitation characteristics.

Activity-Based Photosensitizers with Optimized Triplet State Characteristics Toward Cancer Cell Selective and Image Guided Photodynamic Therapy.

Activity-based theranostic photosensitizers are highly attractive in photodynamic therapy as they offer enhanced therapeutic outcome on cancer cells with an imaging opportunity at the same time. However, photosensitizers (PS) cores that can be easily converted to activity-based photosensitizers (aPSs) are still quite limited in the literature. In this study, we modified the dicyanomethylene-4H-chromene (DCM) core with a heavy iodine atom to get two different PSs (DCMO-I, I-DCMO-Cl) that can be further converted to aPS after simple modifications. The effect of iodine positioning on singlet oxygen generation capacity was also evaluated through computational studies. DCMO-I showed better performance in solution experiments and further proved to be a promising phototheranostic scaffold via cell culture studies. Later, a cysteine (Cys) activatable PS based on the DCMO-I core (DCMO-I-Cys) was developed, which induced selective photocytotoxicity along with a fluorescence turn-on response in Cys rich cancer cells.

Locked and Loaded: ß-Galactosidase Activated Photodynamic Therapy Agent Enables Selective Imaging and Targeted Treatment of Glioblastoma Multiforme Cancer Cells.

Selective detection and effective therapy of brain cancer, specifically, the very aggressive glioblastoma multiforme (GBM), remains one of the paramount challenges in clinical settings. While radiotherapy combined surgery is proposed as the main treatment course, it has several drawbacks such as complexity of the operation and common development of recurrent tumors in this course of patient care. Unique opportunities presented by photodynamic therapy (PDT) offer promising, effective, and precise therapy against GBM cells along with simultaneous imaging opportunities. However, activatable, theranostic molecular systems in PDT modality for GBM remained scarce. Specifically, even though elevated ß-galactosidase (ß-gal) activity in glioblastoma cells is well-documented, targeted, activatable therapeutic PDT agents have not been realized. Herein, we report a ß-galactosidase (ß-gal) activatable phototheranostic agent based on an iodinated resorufin core (RB-1) which was realized in only three steps with commercial reagents in 29% overall yield. RB-1 showed very high singlet oxygen (1O2) quantum yield (54%) accompanied by a remarkable turn-on response in fluorescence upon enzymatic activation. RB-1 was tested in different cell lines and revealed selective photocytotoxicity in U-87MG glioblastoma cells. Additionally, thanks to almost 7% fluorescence quantum yield (ΦF) despite extremely high 1O2 generation yield, RB-1 was also demonstrated as a successful agent for fluorescence imaging of U-87MG cells. Due to significantly lower (ß-gal) activity in healthy cells (NIH/3T3), RB-1 stayed in a passive state and showed minimal photo and dark toxicity. RB-1 marks the first example of a ß-gal activatable phototheranostic agent toward effective treatment of glioblastoma.

Selective detection of carboxylesterase 2 activity in cancer cells using an activity-based chemiluminescent probe.

Carboxylesterase 2 (CES2) has crucial roles in both xenobiotic metabolism and formation of pathogenic states including cancer. Thus, it is highly critical to monitor intracellular CES2 activity in living cancer cells. Here, we report a CES2 activatable phenoxy 1,2-dioxetane based chemiluminescent agent (CL-CES2). The probe exhibited a selective turn-on response in the presence of CES2 enzyme and enabled detection of CES2 activity in three different cancer cells that possess varying enzyme concentrations with high signal to noise ratios. In contrast no signal was obtained with CES1, an isoform of CES2 enzyme. CL-CES2 marks the first ever example of a CES2-responsive chemiluminescent luminophore and holds a great potential in further understanding the roles of CES2 activity in tumorogenesis.

Selenophene-Modified Boron Dipyrromethene-Based Photosensitizers Exhibit Photodynamic Inhibition on a Broad Range of Bacteria.

Microorganisms are crucial for human survival in view of both mutualistic and pathogen interactions. The control of the balance could be achieved by use of the antibiotics. There is a continuous arms race that exists between the pathogen and the antibiotics. The emergence of multidrug-resistant (MDR) bacteria threatens health even for insignificant injuries. However, the discovery of new antibiotics is not a fast process, and the healthcare system will suffer if the evolution of MDR lingers in its current frequency. The cationic photosensitizers (PSs) provide a unique approach to develop novel, light-inducible antimicrobial drugs. Here, we examine the antimicrobial activity of innovative selenophene-modified boron dipyrromethene (BODIPY)-based PSs on a variety of Gram (+) and Gram (-) bacteria. The candidates demonstrate a level of confidence in both light-dependent and independent inhibition of bacterial growth. Among them, selenophene conjugated PS candidates (BOD-Se and BOD-Se-I) are promising agents to induce photodynamic inhibition (PDI) on all experimented bacteria: E. coli, S. aureus, B. cereus, and P. aeruginosa. Further characterizations revealed that photocleavage ability on DNA molecules could be potentially advantageous over extracellular DNA possessing biofilm-forming bacteria such as B. cereus and P. aeruginosa. Microscopy analysis with fluorescent BOD-H confirmed the colocalization on GFP expressing E. coli.

Recent Advances in Cyanine-Based Phototherapy Agents.

Phototherapies, in the form of photodynamic therapy (PDT) and photothermal therapy (PTT), are very promising treatment modalities for cancer since they provide locality and turn-on mechanism for toxicity, both of which are critical in reducing off-site toxicity. Irradiation of photosensitive agents demonstrated successful therapeutic outcomes; however, each approach has its limitations and needs to be improved for clinical success. The combination of PTT and PDT may work in a synergistic way to overcome the limitations of each method and indeed improve the treatment efficacy. The development of single photosensitive agents capable of inducing both PDT and PTT is, therefore, extremely advantageous and highly desired. Cyanine dyes are shown to have such potential, hence have been very popular in the recent years. Luminescence of cyanine dyes renders them as phototheranostic molecules, reporting the localization of the photosensitive agent prior to irradiation to induce phototoxicity, hence allowing image-guided phototherapy. In this review, we mainly focus on the cyanine dye-based phototherapy of different cancer cells, concentrating on the advancements achieved in the last ten years.

Balanced Intersystem Crossing in Iodinated Silicon-Fluoresceins Allows New Class of Red Shifted Theranostic Agents.

Iodination of the silicon-fluorescein core revealed a new class of highly cytotoxic, red-shifted and water-soluble photosensitizer (SF-I) which is also fairly emissive to serve as a theranostic agent. Singlet oxygen generation capacity of SF-I was evaluated chemically, and up to 45% singlet oxygen quantum yield was reported in aqueous solutions. SF-I was further tested in triple negative breast (MDA MB-231) and colon (HCT-116) cancer cell lines, which are known to have limited chemotherapy options as well as very poor prognosis. SF-I induced efficient singlet oxygen generation and consequent photocytotoxicity in both cell lines upon light irradiation with a negligible dark toxicity while allowing cell imaging at the same time. SF-I marks the first ever example of a silicon xanthene-based photosensitizer and holds a lot of promise as a small-molecule-based theranostic scaffold.