Theranostic Fluorescent Probes.

The ability to gain spatiotemporal information, and in some cases achieve spatiotemporal control, in the context of drug delivery makes theranostic fluorescent probes an attractive and intensely investigated research topic. This interest is reflected in the steep rise in publications on the topic that have appeared over the past decade. Theranostic fluorescent probes, in their various incarnations, generally comprise a fluorophore linked to a masked drug, in which the drug is released as the result of certain stimuli, with both intrinsic and extrinsic stimuli being reported. This release is then signaled by the emergence of a fluorescent signal. Importantly, the use of appropriate fluorophores has enabled not only this emerging fluorescence as a spatiotemporal marker for drug delivery but also has provided modalities useful in photodynamic, photothermal, and sonodynamic therapeutic applications. In this review we highlight recent work on theranostic fluorescent probes with a particular focus on probes that are activated in tumor microenvironments. We also summarize efforts to develop probes for other applications, such as neurodegenerative diseases and antibacterials. This review celebrates the diversity of designs reported to date, from discrete small-molecule systems to nanomaterials. Our aim is to provide insights into the potential clinical impact of this still-emerging research direction.

Fluorescent probes for the detection of chemical warfare agents.

Chemical warfare agents (CWAs) are toxic chemicals that have been intentionally developed for targeted and deadly use on humans. Although intended for military targets, the use of CWAs more often than not results in mass civilian casualties. To prevent further atrocities from occurring during conflicts, a global ban was implemented through the chemical weapons convention, with the aim of eliminating the development, stockpiling, and use of CWAs. Unfortunately, because of their relatively low cost, ease of manufacture and effectiveness on mass populations, CWAs still exist in today's world. CWAs have been used in several recent terrorist-related incidents and conflicts (e.g., Syria). Therefore, they continue to remain serious threats to public health and safety and to global peace and stability. Analytical methods that can accurately detect CWAs are essential to global security measures and for forensic analysis. Small molecule fluorescent probes have emerged as attractive chemical tools for CWA detection, due to their simplicity, ease of use, excellent selectivity and high sensitivity, as well as their ability to be translated into handheld devices. This includes the ability to non-invasively image CWA distribution within living systems (in vitro and in vivo) to permit in-depth evaluation of their biological interactions and allow potential identification of therapeutic countermeasures. In this review, we provide an overview of the various reported fluorescent probes that have been designed for the detection of CWAs. The mechanism for CWA detection, change in optical output and application for each fluorescent probe are described in detail. The limitations and challenges of currently developed fluorescent probes are discussed providing insight into the future development of this research area. We hope the information provided in this review will give readers a clear understanding of how to design a fluorescent probe for the detection of a specific CWA. We anticipate that this will advance our security systems and provide new tools for environmental and toxicology monitoring.

The design of small-molecule prodrugs and activatable phototherapeutics for cancer therapy.

Cancer remains as one of the most significant health problems, with approximately 19 million people diagnosed worldwide each year. Chemotherapy is a routinely used method to treat cancer patients. However, current treatment options lack the appropriate selectivity for cancer cells, are prone to resistance mechanisms, and are plagued with dose-limiting toxicities. As such, researchers have devoted their attention to developing prodrug-based strategies that have the potential to overcome these limitations. This tutorial review highlights recently developed prodrug strategies for cancer therapy. Prodrug examples that provide an integrated diagnostic (fluorescent, photoacoustic, and magnetic resonance imaging) response, which are referred to as theranostics, are also discussed. Owing to the non-invasive nature of light (and X-rays), we have discussed external excitation prodrug strategies as well as examples of activatable photosensitizers that enhance the precision of photodynamic therapy/photothermal therapy. Activatable photosensitizers/photothermal agents can be seen as analogous to prodrugs, with their phototherapeutic properties at a specific wavelength activated in the presence of disease-related biomarkers. We discuss each design strategy and illustrate the importance of targeting biomarkers specific to the tumour microenvironment and biomarkers that are known to be overexpressed within cancer cells. Moreover, we discuss the advantages of each approach and highlight their inherent limitations. We hope in doing so, the reader will appreciate the current challenges and available opportunities in the field and inspire subsequent generations to pursue this crucial area of cancer research.

Phenotyping of Methicillin-Resistant Staphylococcus aureus Using a Ratiometric Sensor Array.

Chemical tools capable of classifying multidrug-resistant bacteria (superbugs) can facilitate early-stage disease diagnosis and help guide precision therapy. Here, we report a sensor array that permits the facile phenotyping of methicillin-resistant Staphylococcus aureus (MRSA), a clinically common superbug. The array consists of a panel of eight separate ratiometric fluorescent probes that provide characteristic vibration-induced emission (VIE) profiles. These probes bear a pair of quaternary ammonium salts in different substitution positions around a known VIEgen core. The differences in the substituents result in varying interactions with the negatively charged cell walls of bacteria. This, in turn, dictates the molecular conformation of the probes and affects their blue-to-red fluorescence intensity ratios (ratiometric changes). Within the sensor array, the differences in the ratiometric changes for the probes result in "fingerprints" for MRSA of different genotypes. This allows them to be identified using principal component analysis (PCA) without the need for cell lysis and nucleic acid isolation. The results obtained with the present sensor array agree well with those obtained using polymerase chain reaction (PCR) analysis.

Orthogonally Engineered Albumin with Attenuated Macrophage Phagocytosis for the Targeted Visualization and Phototherapy of Liver Cancer.

The five-year survival rate of hepatocellular carcinoma (HCC) remains unsatisfactory. This reflects, in part, the paucity of effective methods that allow the target-specific diagnosis and therapy of HCC. Here, we report a strategy based on engineered human serum albumin (HSA) that permits the HCC-targeted delivery of diagnostic and therapeutic agents. Covalent cysteine conjugation combined with the exploitation of host-guest chemistry was used to effect the orthogonal functionalization of HSA with two functionally independent peptides. One of these peptides targets glypican-3 (GPC-3), an HCC-specific biomarker, while the second reduces macrophage phagocytosis through immune-checkpoint stimulation. This orthogonally engineered HSA proved effective for the GPC-3-targeted delivery of near-infrared fluorescent and phototherapeutic agents, thus permitting target-specific optical visualization and photodynamic ablation of HCC in vivo. This study thus offers new insights into specificity-enhanced fluorescence-guided surgery and phototherapy of HCC through the orthogonal engineering of biocompatible proteins.

A Highly Sensitive and Selective Near-Infrared Fluorescent Probe for Imaging Peroxynitrite in Living Cells and Drug-Induced Liver Injury Mice.

Drug-induced liver injury (DILI) is a major clinical issue associated with the majority of commercial drugs. During DILI, the peroxynitrite (ONOO-) level is upregulated in the liver. However, traditional methods are unable to timely monitor the dynamic changes of the ONOO- level during DILI in vivo. Therefore, ONOO--activated near-infrared (NIR) fluorescent probes with high sensitivity and selectivity are key to the early diagnosis of DILI in situ. Herein, we report a novel ONOO--responsive NIR fluorescent probe, QCy7-DP, which incorporates a donor-dual-acceptor π-electron cyanine skeleton with diphenyl phosphinate. The ONOO--mediated highly selective hydrolytic cleavage via an addition-elimination pathway of diphenyl phosphinate produced the deprotonated form of QCy7 in physiological conditions with a distinctive extended conjugated π-electron system and ∼200-fold enhancement in NIR fluorescence emission at 710 nm. Moreover, the probe QCy7-DP was successfully used for the imaging of the endogenous and exogenous ONOO- concentration changes in living cells. Importantly, in vivo fluorescence imaging tests demonstrated that the probe can effectively detect the endogenous generation of ONOO- in an acetaminophen (APAP)-induced liver injury mouse model. This study provides insight into the design of highly selective NIR fluorescent probes suitable for spatiotemporal monitoring of ONOO- under different pathological conditions.

Human serum albumin-based supramolecular host-guest boronate probe for enhanced peroxynitrite sensing.

Peroxynitrite (ONOO-) is an important oxygen/nitrogen reactive species implicated in a number of physiological and pathological processes. However, due to the complexity of the cellular micro-environment, the sensitive and accurate detection of ONOO- remains a challenging task. Here, we developed a long-wavelength fluorescent probe based on the conjugation between a TCF scaffold and phenylboronate; the resulting conjugate is capable of supramolecular host-guest assembly with human serum albumin (HSA) for the fluorogenic sensing of ONOO-. The probe exhibited an enhanced fluorescence over a low concentration range of ONOO- (0-9.6 µM), whist the fluorescence was quenched when the concentration of ONOO- exceeded 9.6 µM. In addition, when human serum albumin (HSA) was added, the initial fluorescence of the probe was significantly enhanced, which enabled the more sensitive detection of low-concentrations of ONOO- in aqueous buffer solution and in cells. The molecular structure of the supramolecular host-guest ensemble was determined using small-angle X-ray scattering.

Tuning the Solid- and Solution-State Fluorescence of the Iron-Chelator Deferasirox.

Deferasirox, an FDA-approved iron chelator, has gained increasing attention for use in anticancer and antimicrobial applications. Recent efforts by our group led to the identification of this core as an easy-to-visualize aggregation-induced emission platform, or AIEgen, that provides a therapeutic effect equivalent to deferasirox (J. Am. Chem. Soc. 2021, 143, 3, 1278-1283). However, the emission wavelength of the first-generation system overlapped with that of Syto9, a green emissive dye used to indicate live cells. Here, we report a library of deferasirox derivatives with various fluorescence emission profiles designed to overcome this limitation. We propose referring to systems that show promise as both therapeutic and optical imaging agents as "illuminoceuticals". The color differences between the derivatives were observable to the unaided eye (solid- and solution-state) and were in accord with the Commission Internationale de L'Eclairage (CIE) chromaticity diagram 1913. Each fluorescent derivative successfully imaged the respective spherical and rod shapes of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. They also displayed iron-dependent antibiotic activity. Three derivatives, ExNMe2 (3), ExTrisT (11), and ExDCM (13), display emission features that are sufficiently distinct so as to permit the multiplex (triplex) imaging of both MRSA and P. aeruginosa via stimulated emission depletion microscopy. The present deferasirox derivatives allowed for the construction of a multi-fluorophore sensor array. This array enabled the successful discrimination between Gram-positive/Gram-negative and drug-sensitive/drug-resistant bacteria. Antibiotic sensitivity and drug-resistant mutants from clinically isolated strains could also be identified and differentiated.

Dual-Channel Fluorescent Probe for the Simultaneous Monitoring of Peroxynitrite and Adenosine-5'-triphosphate in Cellular Applications.

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO-) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO- and ATP. ONOO- selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO- and ATP products, ATP-LW allows ONOO- levels to be monitored in the green channel (λex = 488 nm, λem = 500-575 nm) and ATP concentrations in the red channel (λex = 514 nm, λem = 575-650 nm). The use of ATP-LW as a combined ONOO- and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO- donor).

Small-molecule fluorescence-based probes for interrogating major organ diseases.

Chemical tools that allow the real-time monitoring of organ function and the visualisation of organ-related processes at the cellular level are of great importance in biological research. The upregulation/downregulation of specific biomarkers is often associated with the development of organ related diseases. Small-molecule fluorescent probes have the potential to create advances in our understanding of these disorders. Viable probes should be endowed with a number of key features that include high biomarker sensitivity, low limit of detection, fast response times and appropriate in vitro and in vivo biocompatibility. In this tutorial review, we discuss the development of probes that allow the targeting of organ related processes in vitro and in vivo. We highlight the design strategy that underlies the preparation of various promising probes, their optical response to key biomarkers, and proof-of-concept biological studies. The inherent drawbacks and limitations are discussed as are the current challenges and opportunities in the field. The hope is that this tutorial review will inspire the further development of small-molecule fluorescent probes that could aid the study of pathogenic conditions that contribute to organ-related diseases.

Correction: Fluorescent glycoconjugates and their applications.

Correction for 'Fluorescent glycoconjugates and their applications' by Baptiste Thomas et al., Chem. Soc. Rev., 2020, 49, 593-641, DOI: 10.1039/C8CS00118A.

Deferasirox (ExJade): An FDA-Approved AIEgen Platform with Unique Photophysical Properties.

Deferasirox, ExJade, is an FDA-approved iron chelator used for the treatment of iron overload. In this work, we report several fluorescent deferasirox derivatives that display unique photophysical properties, i.e., aggregation-induced emission (AIE), excited state intramolecular proton transfer, charge transfer, and through-bond and through-space conjugation characteristics in aqueous media. Functionalization of the phenol units on the deferasirox scaffold afforded the fluorescent responsive pro-chelator ExPhos, which enabled the detection of the disease-based biomarker alkaline phosphatase (ALP). The diagnostic potential of these deferasirox derivatives was supported by bacterial biofilm studies.

Fluorescent glycoconjugates and their applications.

Glycoconjugates and their applications as lectin ligands in biology have been thoroughly investigated in the past decades. Meanwhile, the intrinsic properties of such multivalent molecules were limited essentially to their ability to bind to their receptors with high selectivity and/or avidity. The present review will focus on multivalent glycoconjugates displaying an additional capability such as fluorescence properties not only for applications toward imaging of cancer cells and detection of proteins or pathogens but also for drug delivery systems toward targeted cancer therapy. This review is a collection of research articles discussed in the context of the structural features of fluorescent glycoconjugates organized according to their fluorescent core scaffold and with their representative applications.

Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents.

In this tutorial review, we will explore recent advances in the construction and application of Förster resonance energy transfer (FRET)-based small-molecule fluorescent probes. The advantages of FRET-based fluorescent probes include: a large Stokes shift, ratiometric sensing and dual/multi-analyte responsive systems. We discuss the underlying energy donor-acceptor dye combinations and emphasise their applications for the detection or imaging of cations, anions, small neutral molecules, biomacromolecules, cellular microenvionments and dual/multi-analyte responsive systems.

Metal-based imaging agents: progress towards interrogating neurodegenerative disease.

Central nervous system (CNS) neurodegeneration is defined by a complex series of pathological processes that ultimately lead to death. The precise etiology of these disorders remains unknown. Recent efforts show that a mechanistic understanding of the malfunctions underpinning disease progression will prove requisite in developing new treatments and cures. Transition metals and lanthanide ions display unique characteristics (i.e., magnetism, radioactivity, and luminescence), often with biological relevance, allowing for direct application in CNS focused imaging modalities. These techniques include positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and luminescent-based imaging (LumI). In this Tutorial Review, we have aimed to highlight the various metal-based imaging techniques developed in the effort to understand the pathophysiological processes associated with neurodegeneration. Each section has been divided so as to include an introduction to the particular imaging technique in question. This is then followed by a summary of key demonstrations that have enabled visualization of a specific neuropathological biomarker. These strategies have either exploited the high binding affinity of a receptor for its corresponding biomarker or a specific molecular transformation caused by a target species, all of which produce a concomitant change in diagnostic signal. Advantages and disadvantages of each method with perspectives on the utility of molecular imaging agents for understanding the complexities of neurodegenerative disease are discussed.

Transition metal chelators, pro-chelators, and ionophores as small molecule cancer chemotherapeutic agents.

Cancer is among the leading causes of death worldwide. Although a number of new treatment options have been developed in recent years, there remains a need for improved chemotherapies. The primary challenges facing new cancer drugs include: (1) improving patient quality of life, (2) overcoming drug resistance and (3) lowering reoccurrence rates. Major drawbacks of current chemotherapeutics arise from poor selectivity towards cancer cells, dose limiting toxicities, compliance-reducing side effects, and an inability to address resistance mechanisms. Chemotherapeutics that fail to achieve complete eradication of the disease can also lead to relapse and promote treatment resistance. New strategies to overcome these drawbacks include the use of transition metal chelators and ionophores to alter selectively the concentrations of iron, copper, and zinc in cancer cells. A number of metal chelators have successfully demonstrated cytotoxicity and targeted activity against drug-resistant cancer cells; several have proved effective against cancer stem cells, a significant cause of tumour reoccurrence. However, problems with formulation and targeting have been noted. Recent efforts have thus focused on the design of pro-chelators, inactive versions of chelators that are designed to be activated in the tumour. This is an appealing strategy that may potentially increase efficacy towards cancer-resistant malignant cells. This Tutorial Review summarizes recent progress involving transition metal chelators, pro-chelators, and ionophores as potential cancer chemotherapeutics. We will focus on the reported agents that are able to coordinate iron, copper, and zinc.

Ferrocene-Labelled Electroactive Aptamer-Based Sensors (Aptasensors) for Glycated Haemoglobin.

Glycated haemoglobin (HbA1c) is a diagnostic biomarker for type 2 diabetes. Traditional analytical methods for haemoglobin (Hb) detection rely on chromatography, which requires significant instrumentation and is labour-intensive; consequently, miniaturized devices that can rapidly sense HbA1c are urgently required. With this research, we report on an aptamer-based sensor (aptasensor) for the rapid and selective electrochemical detection of HbA1c. Aptamers that specifically bind HbA1c and Hb were modified with a sulfhydryl and ferrocene group at the 3' and 5'-end, respectively. The modified aptamers were coated through sulfhydryl-gold self-assembly onto screen printed electrodes, producing aptasensors with built in electroactivity. When haemoglobin was added to the electrodes, the current intensity of the ferrocene in the sensor system was reduced in a concentration-dependent manner as determined by differential pulse voltammetry. In addition, electrochemical impedance spectroscopy confirmed selective binding of the analytes to the aptamer-coated electrode. This research offers new insight into the development of portable electrochemical sensors for the detection of HbA1c.

Manganese(II) Texaphyrin: A Paramagnetic Photoacoustic Contrast Agent Activated by Near-IR Light.

The NIR absorptivity of the metallotexaphyrin derivatives MMn, MGd, and MLu for photoacoustic (PA)-based imaging is explored in this study. All three complexes demonstrated excellent photostabilities; however, MMn provided the greatest PA signal intensities in both doubly distilled water and RAW 264.7 cells. In vivo experiments using a prostate tumor mouse model were performed. MMn displayed no adverse toxicity to major organs as inferred from hematoxylin and eosin (H&E) staining and cell blood count testing. MMn also allowed for PA-based imaging of tumors with excellent in vivo stability to provide 3D tumor diagnostic information. Based on the present findings and previous magnetic resonance imaging (MRI) studies, we believe MMn may have a role to play either as a stand-alone PA contrast agent or as a single molecule dual modal (PA and MR) imaging agent for tumor diagnosis.

Cyclodextrin-Based Peptide Self-Assemblies (Spds) That Enhance Peptide-Based Fluorescence Imaging and Antimicrobial Efficacy.

As a result of their high specificity for their corresponding biological targets, peptides have shown significant potential in a range of diagnostic and therapeutic applications. However, their widespread use has been limited by their minimal cell permeability and stability in biological milieus. We describe here a hepta-dicyanomethylene-4H-pyran appended ß-cyclodextrin (DCM7-ß-CD) that acts as a delivery enhancing "host" for 1-bromonaphthalene-modified peptides, as demonstrated with peptide probes P1-P4. Interaction between the fluorescent peptides P1-P3 and DCM7-ß-CD results in the hierarchical formation of unique supramolecular architectures, which we term supramolecular-peptide-dots (Spds). Each Spd (Spd-1, Spd-2, and Spd-3) was found to facilitate the intracellular delivery of the constituent fluorescent probes (P1-P3), thus allowing spatiotemporal imaging of an apoptosis biomarker (caspase-3) and mitosis. Spd-4, incorporating the antimicrobial peptide P4, was found to provide an enhanced therapeutic benefit against both Gram-positive and Gram-negative bacteria relative to P4 alone. In addition, a fluorescent Spd-4 was prepared, which revealed greater bacterial cellular uptake compared to the peptide alone (P4-FITC) in E. coli. (ATCC 25922) and S. aureus (ATCC 25923). This latter observation supports the suggestion that the Spd platform reported here has the ability to facilitate the delivery of a therapeutic peptide and provides an easy-to-implement strategy for enhancing the antimicrobial efficacy of known therapeutic peptides. The present findings thus serve to highlight a new and effective supramolecular delivery approach that is potentially generalizable to overcome limitations associated with functional peptides.

Photochromic Fluorescent Probe Strategy for the Super-resolution Imaging of Biologically Important Biomarkers.

Here, we report a ß-galactosidase (ß-Gal)-responsive photochromic fluorescent probe, NpG, that was designed to prebind to human serum albumin (HSA) to form the probe/protein hybrid, NpG@HSA. The formation of NpG@HSA led to an increase in fluorescence emission (520 nm) corresponding to the binding of the fluorescent naphthalimide unit with HSA. In addition, this enabled visualization of the spiropyran fluorescence emission in aqueous media. Our probe/protein hybrid approach afforded a unique imaging platform with enhanced cell permeability and solubility that was capable of visualizing the cellular uptake of NpG@HSA before its activation by ß-Gal. The ß-Gal-mediated cleavage of the galactose unit within the NpG@HSA hybrid resulted in the formation of NpM@HSA and an increase in red fluorescence emission (620 nm). The resultant merocyanine unit was then able to undergo photoisomerization (merocyanine ↔ spiropyran) to facilitate STORM (i.e., stochastic optical reconstruction microscopy) imaging with minimal phototoxicity and excellent photostability/reversibility. Using STORM, NpG@HSA was able to determine the subcellular distribution of ß-Gal activity between cell lines with nanoscale precision. We believe that this system represents a versatile imaging platform for the design of photochromic fluorescent probes suitable for illuminating the precise location of disease-specific biomarkers in various cellular processes.