Single Atom Stabilization of Phosphinate Ester-Containing Rhodamines Yields Cell Permeable Probes for Turn-On Photoacoustic Imaging.

Photoacoustic imaging (PAI) is an emerging imaging technique that uses pulsed laser excitation with near-infrared (NIR) light to elicit local temperature increases through non-radiative relaxation events, ultimately leading to the production of ultrasound waves. The classical xanthene dye scaffold has found numerous applications in fluorescence imaging, however, xanthenes are rarely utilized for PAI since they do not typically display NIR absorbance. Herein, we report the ability of Nebraska Red (NR) xanthene dyes to produce photoacoustic (PA) signal and provide a rational design approach to reduce the hydrolysis rate of ester containing dyes, affording cell permeable probes. To demonstrate the utility of this approach, we construct the first cell permeable rhodamine-based, turn-on PAI imaging probe for hypochlorous acid (HOCl) with maximal absorbance within the range of commercial PA instrumentation. This probe, termed SNR700 -HOCl, is capable of detecting exogenous HOCl in mice. This work provides a new set of rhodamine-based PAI agents as well as a rational design approach to stabilize esterified versions of NR dyes with desirable properties for PAI. In the long term, the reagents described herein could be utilized to enable non-invasive imaging of HOCl in disease-relevant model systems.

Xanthene-Based Dyes for Photoacoustic Imaging and their Use as Analyte-Responsive Probes.

Developing imaging tools that can report on the presence of disease-relevant analytes in multicellular organisms can provide insight into fundamental disease mechanisms as well as provide diagnostic tools for the clinic. Photoacoustic imaging (PAI) is a light-in, sound-out imaging technique that allows for high resolution, deep-tissue imaging with applications in pre-clinical and point-of-care settings. The continued development of near-infrared (NIR) absorbing small-molecule dyes promises to improve the capabilities of this emerging imaging modality. For example, new dye scaffolds bearing chemoselective functionalities are enabling the detection and quantification of disease-relevant analytes through activity-based sensing (ABS) approaches. Recently described strategies to engineer NIR absorbing xanthenes have enabled development of analyte-responsive PAI probes using this classic dye scaffold. Herein, we present current strategies for red-shifting the spectral properties of xanthenes via bridging heteroatom or auxochrome modifications. Additionally, we explore how these strategies, coupled with chemoselective spiroring-opening approaches, have been employed to create ABS probes for in vivo detection of hypochlorous acid, nitric oxide, copper (II), human NAD(P)H: quinone oxidoreductase isozyme 1, and carbon monoxide. Given the versatility of the xanthene scaffold, we anticipate continued growth and development of analyte-responsive PAI imaging probes based on this dye class.

Azaphosphinate Dyes: A Low Molecular Weight Near-Infrared Scaffold for Development of Photoacoustic or Fluorescence Imaging Probes.

Near-infrared (NIR) dyes are desirable for biological imaging applications including photoacoustic (PA) and fluorescence imaging. Nonetheless, current NIR dyes are often plagued by relatively large molecular weights, poor water solubility, and limited photostability. Herein, we provide the first examples of azaphosphinate dyes which display desirable properties such as low molecular weight, absorption/emission above 750 nm, and remarkable water solubility. In PA imaging, an azaphosphinate dye exhibited a 4.1-fold enhancement in intensity compared to commonly used standards, the ability to multiplex with existing dyes in whole blood, imaging depths of 2.75 cm in a tissue model, and contrast in mice. An improved derivative for fluorescence imaging displayed a >10-fold reduction in photobleaching in water compared to the FDA-approved indocyanine green dye and could be visualized in mice. This new dye class provides a robust scaffold for the development of photoacoustic or NIR fluorescence imaging agents.

Photoluminescent Properties and Mechanism of Novel Cyanine-Borondifluoride Curcuminoid Hybrids as Red-to-Near Infrared and Endoplasmic Reticulum-Targeting Dyes.

Synthesis-oriented design led us to the discovery of a series of novel cyanine-borondifluoride curcuminoid hybrids called Nanchang Red (NCR) dyes that overcome the intrinsic low synthetic yields of symmetrical cyanine-difluoroboronate (BF2 )-hybridized NIR dyes. The hybridization endows NCR dyes with high molar extinction coefficients, efficient red-to-NIR emission, and enlarged Stokes shifts. Quantum chemical calculations revealed that the asymmetrical layout of the three key electron-withdrawing and electron-donating fragments results in a special pattern of partial charge separation and inconsistent degrees of charge delocalization on their π-conjugated backbones. While the nature of the hemicyanine fragment exerts significant influence on the excitation modes of NCR dyes, the borondifluoride hemicurcuminoid fragment is the major contributor to the enlarged Stokes shifts. Cell imaging experiments illustrated that a subtle change in the N-heterocycle of the hemicyanine fragment has a remarkable effect on the subcellular localization of NCR dyes. Unlike other previously reported cyanine-BF2 hybridized dyes, which mainly target mitochondria, the benzothiazole and indole-based NCR dyes accumulate in both the endoplasmic reticulum (ER) and lipid droplets of HeLa cells, whereas the benzoxazole and quinoline-based NCR dyes stain the ER specifically.

Near-Infrared Boron Difluoride Formazanate Dyes.

Near-infrared (NIR) dyes are sought after for their utility in light harvesting, bioimaging, and light-mediated therapies. Since long-wavelength photoluminescence typically involves extensive π-conjugated systems of double bonds and aromatic rings, it is often assumed that NIR dyes have to be large molecules that require complex syntheses. We challenge this assumption by demonstrating that facile incorporation of tertiary amine groups into readily available 3-cyanoformazans affords efficient production of relatively simple NIR-active BF2 formazanate dyes (λabs =691-760 nm, λPL =834-904 nm in toluene). Cyclic voltammetry experiments on these compounds reveal multiple reversible redox waves linked to the interplay between the tertiary amine and BF2 formazanate moieties. Density-functional calculations indicate that the NIR electronic transitions in BF2 formazanates are of π→π*-type, but do not always involve strong charge transfer.

Recent Advances in Polymeric Nanoparticles for Enhanced Fluorescence and Photoacoustic Imaging.

In recent years, polymeric nanoparticles (NPs) have become increasingly popular for in vitro and in vivo bioimaging because of their excellent versatility and exceptional optical properties. Following a brief introduction on fluorescence and photoacoustic imaging, as well as the merits of using polymeric NPs over other types of fluorescent probes, this Minireview gives a concise summary of key advances made in recent years (2017-2020) in the use of polymeric NPs for fluorescence and photoacoustic imaging. A particular focus is placed on various strategies used for enhanced bioimaging applications, including polymeric NPs that encapsulate near-infrared dyes, aggregation-induced-emission fluorogens, cationic dyes doped with bulky hydrophobic counterions, and semiconducting polymers. Next, the current limitations in some of these polymeric NP systems are summarized and potential solutions offered to overcome them. Finally, some critical considerations in regard to the design of polymeric NPs are given for future bioimaging and sensing applications.

Near-Infrared Fluorescence Hydrogen Peroxide Assay for Versatile Metabolite Biosensing in Whole Blood.

In emergency medicine, blood lactate levels are commonly measured to assess the severity and response to treatment of hypoperfusion-related diseases (e.g., sepsis, trauma, cardiac arrest). Clinical blood lactate testing is conducted with laboratory analyzers, leading to a delay of 3 h between triage and lactate result. Here, a fluorescence-based blood lactate assay, which can be utilized for bedside testing, based on measuring the hydrogen peroxide generated by the enzymatic oxidation of lactate is described. To establish a hydrogen peroxide assay, near-infrared cyanine derivatives are screened and sulfo-cyanine 7 is identified as a new horseradish peroxidase (HRP) substrate, which loses its fluorescence in presence of HRP and hydrogen peroxide. As hydrogen peroxide is rapidly cleared by erythrocytic catalase and glutathione peroxidase, sulfo-cyanine 7, HRP, and lactate oxidase are encapsulated in a liposomal reaction compartment. In lactate-spiked bovine whole blood, the newly developed lactate assay exhibits a linear response in a clinically relevant range after 10 min. Substituting lactate oxidase with glucose and alcohol oxidase allows for blood glucose, ethanol, and methanol biosensing, respectively. This easy-to-use, rapid, and versatile assay may be useful for the quantification of a variety of enzymatically oxidizable metabolites, drugs, and toxic substances in blood and potentially other biological fluids.

Reversible Charge Transfer with Single-Walled Carbon Nanotubes Upon Harvesting the Low Energy Part of the Solar Spectrum.

Here, the ability of a novel near-infrared dye to noncovalently self-assemble onto the surface of single-walled carbon nanotubes (SWCNTs) driven by charge-transfer interactions is demonstrated. Steady-state, Raman, and transient absorption spectroscopies corroborate the electron donating character of the near-infrared dye when combined with SWCNTs, in the form of fluorescence quenching of the excited state of the dye, n-doping of SWCNTs, and reversible charge transfer, respectively. Formation of the one-electron oxidized dye as a result of interactions with SWCNTs is supported by spectroelectrochemical measurements. The ultrafast electronic process in the near-infrared dye, once immobilized onto SWCNTs, starts with the formation of excited states, which decay to the ground state via the intermediate population of a fully charge-separated state, with characteristic time constants for the charge separation of 1.5 ps and charge recombination of 25 ps, as derived from the multiwavelength global analysis. Of great relevance is the fact that charge-transfer occurs from the hot excited state of the near-infrared dye to SWCNTs.

Impact of Ion-Pairing Effects on Linear and Nonlinear Photophysical Properties of Polymethine Dyes*.

The two-photon absorption (2PA) and photophysics of heptamethine dyes featuring cyanine or dipolar electronic structures have been compared for the first time. The perfectly delocalized cyanine system is classically characterized by a two-photon transition matching the vibronic component of its lower energy absorption band. The dipolar species is generated by ion-pairing with a hard counterion in a non-dissociating solvent and displays significant modifications oft he optical properties, including a significant hypochromic shift of absorption, weaker emission and 2PA matching the lower energy transition, thus revealing symmetry breaking within the polymethine electronic structure.

Near-infrared imaging of diseases: A nanocarrier approach.

Developments in fluorescence imaging, brought popularity to near infrared (NIR) imaging with far-red and NIR fluorophores applied for biosensing and bioimaging in living systems. Noninvasive NIR imaging gained popularity with the use of effective NIR dyes to obtain macroscopic fluorescence images. Several attributes of NIR dyes make them desirable agents, including high specificity, high sensitivity, minimized background interference, and the ability to easily conjugate with drug delivery systems. However, NIR dyes have some drawbacks and limitations, such as low solubility, low stability, and degradation. To overcome these issues, NIR dyes can be encapsulated in appropriate nanocarriers to achieve effective diagnosis, imaging, and therapy monitoring during surgery. Moreover, the vast majority of NIR dyes have photosensitizer features that can effectuate cancer treatment referred to as photodynamic therapy (PDT). In the near future, by combining NIR dyes with appropriate nanocarriers such as liposomes, polymeric micelles, polymeric nanoparticles, dendrimers, quantum dots, carbon nanotubes, or ceramic/silica based nanoparticles, the limitations of NIR dyes can be minimized or even effectively eliminated to form potential effective agents for imaging, therapy, and therapy monitoring of several diseases, particularly cancer.

Toward Stable Superbenzoquinone Diradicaloids.

Superbenzoquinone (SBQ) is a quinone derived from a classic polycyclic aromatic hydrocarbon (PAH), hexa-peri-hexabenzocoronene (so-called "superbenzene"), and is a challenging synthetic target. Herein we report the successful synthesis and characterization of its derivatives. We reveal that the high reactivity of SBQ is due to its intrinsic open-shell diradical character. Thus, two kinetically blocked SBQs, SBQ-Me and SBQ-Ph, were prepared by different synthetic strategies. 4-tert-Butylphenyl-substituted SBQ-Ph demonstrated good stability and could be isolated in crystalline form. Both compounds have an open-shell singlet ground state and show thermally populated paramagnetic activity. Our studies provide effective strategies toward stable quinone-based diradicaloids.

Remarkable In Vivo Nonlinear Photoacoustic Imaging Based on Near-Infrared Organic Dyes.

Two near-infrared dyes featuring good dispersion and light-harvesting property present a remarkable nonlinear photoacoustic response in vitro and in vivo comparing with conventional gold nanorods. This study benefits the fabrication of drug delivery platforms with accurate targeting and control effect under photoacoustic image guidance.

Fused perylene-phthalocyanine macrocycles: a new family of NIR-dyes with pronounced basicity.

The synthesis and characterization of a new type of chromophore, namely PePc consisting of a central phthalocyanine core and four fused perylene-bisimide (PBI) units is described for the first time. The entire architecture represents a highly extended conjugated heterocyclic π-system with C4h symmetry. In order to guarantee pronounced solubility in organic solvents the corresponding PBI units were bay-functionalized with tert-butylphenoxy substituents. Next to the metal-free macrocycle, PePcH2 , also metallated macrocycles PePcM (M=Zn, Ni, Pb, Ru, Fe) were synthesized. The extensive fusion of the corresponding aromatic building blocks to the very large extended π-system leads to a very narrow HOMO-LUMO gap and as a consequence to transparency in the visible but light absorption in the NIR region. Significantly, the azomethine N-atoms N1N4 of PePcM and PePcH2 are highly basic. The corresponding tetraprotonated systems can only be deprotonated with very strong non-nucleophilic bases such as phosphazene bases. In the protonated forms PePcMH4 (4+) and PePcMH6 (4+) the absorption maximum is shifted back to the visible region due to the loss of conjugation. The experimental findings were corroborated with quantum mechanical calculations.

Near-Infrared Absorbing Aza-BODIPY Dyes for Optoelectronic Applications.

Organic dyes that absorb light in the visible to near-infrared region have garnered significant interest, owing to their extensive utility in organic photovoltaics and various biomedical applications. Aza-boron-dipyrromethene (Aza-BODIPY) dyes are a class of chromophores with impressive photophysical properties such as tunable absorption from the visible region towards near infrared (NIR) region, high molar absorptivity, and fluorescence quantum yield. In this review, we discuss the developments in the aza-BODIPYs, related to their synthetic routes, photophysical properties and their applications. Their design strategies, modifications in chemical structures, mode/position of attachment, and their impact on photo-physical properties are reviewed. The potential applications of aza-BODIPY derivatives such as organic solar cells, photodynamic therapy, boron-neutron capture therapy, fluorescence sensors, photo-redox catalysis, photoacoustic probes and optoelectronic devices are explained.

Characterization of Near Infrared-Dye Colored Fabrics Using Hyperspectral Imaging.

Near-infrared (NIR) dyes have a unique ability to interact favorably with light in the NIR region, which is particularly interesting where stealth and camouflage are paramount, such as in military uniforms. Characterization of cotton fabric dyed with NIR-absorbing dyes using visible-NIR (Vis-NIR) and short-wave infrared (SWIR) hyperspectral imaging was done. The aim of the study was to discern spectral changes caused by variations in dye concentration and dyeing temperature as these parameters directly influence color- and crocking-fastness of fabrics impacting the camouflage effect. The fabric was dyed at three concentrations (2.5, 5, and 10%) and two dyeing temperatures (55 °C and 85 °C) and principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed on the spectra to discriminate the fabrics based on dye concentrations. The PCA models successfully segregated the fabrics based on the dye concentration and dyeing temperature, while PLS-DA models demonstrated classification accuracies between 75 and 100% in the Vis-NIR range. Spectra in the SWIR region could not be used to detect the differences in the concentrations of the NIR dyes. This finding is promising, as it aligns with the objective of creating NIR-dyed camouflage fabrics that remain indistinguishable under varying dye concentrations. These results open possibilities for further exploration in enhancing the stealth capabilities of textiles in military applications.

Clinical Consensus Statement on the Use of Indocyanine Green Fluorescence-guided Surgery in Pediatric Patients.

BACKGROUND AND AIMS: Indocyanine Green Fluorescence (ICG-F)- guided surgery is becoming an increasingly helpful tool in pediatric surgical care. This consensus statement investigates the utility of ICG-F in various pediatric surgical applications, primarily focusing on its evidence base, safety, indications, use across different surgical specialties and dosing strategies. The aim is to establish an international consensus for ICG-F use in pediatric surgery. METHODS: An international panel of 15 pediatric surgeons from 9 countries was assembled. The structured process consisted of a rapid scoping review, iterative discussion sessions, mixed-methods studies with key stakeholders, and voting rounds on individual statements to create draft consensus statements. RESULTS: 100 articles were identified during the review and summarized by application. Based on this condensed evidence, consensus statements were generated after 3 iterative rounds of anonymous voting. Key areas of agreement were quality of evidence, the safety of ICG, pediatric surgical indications, utilization per surgical specialty, and dosing of ICG. CONCLUSION: This consensus statement aims to guide healthcare professionals in managing ICG-F use in pediatric surgical cases based on the best available evidence, key stakeholder consultation, and expert opinions. Despite ICG-F's promising potential, the need for higher-quality evidence, prospective trials, and safety studies is underscored. The consensus also provides a framework for pediatric surgeons to utilize ICG-F effectively. LEVEL OF EVIDENCE: III.

Bright NIR-Emitting Styryl Pyridinium Dyes with Large Stokes' Shift for Sensing Applications.

Two NIR-emitting donor-π-acceptor (D-π-A) type regioisomeric styryl pyridinium dyes (1a-1b) were synthesized and studied for their photophysical performance and environment sensitivity. The two regioisomers, 1a and 1b, exhibited interesting photophysical properties including, longer wavelength excitation (λex ≈ 530-560 nm), bright near-infrared emission (λem ≈ 690-720 nm), high-fluorescence quantum yields (ϕfl ≈ 0.24-0.72) large Stokes' shift (∆λ ≈ 150-240 nm) and high-environmental sensitivity. Probe's photophysical properties were studied in different environmental conditions such as polarity, viscosity, temperature, and concentration. Probes (1a-1b) exhibited noticeable changes in absorbance, emission and Stokes' shift while responding to the changes in physical environment. Probe 1b exhibited a significant bathochromic shift in optical spectra (∆λ ≈ 20-40 nm) compared to its isomer 1a, due to the regio-effect. Probes (1a-1b) exhibited an excellent ability to visualize bacteria (Bacillus megaterium, Escherichia coli), and yeast (Saccharomyces cerevisiae) via fluorescence microscopy.

Fluorescence-based methods for studying activity and drug-drug interactions of hepatic solute carrier and ATP binding cassette proteins involved in ADME-Tox.

In humans, approximately 70% of drugs are eliminated through the liver. This process is governed by the concerted action of membrane transporters and metabolic enzymes. Transporters mediating hepatocellular uptake of drugs belong to the SLC (Solute carrier) superfamily of transporters. Drug efflux either toward the portal vein or into the bile is mainly mediated by active transporters of the ABC (ATP Binding Cassette) family. Alteration in the function and/or expression of liver transporters due to mutations, disease conditions, or co-administration of drugs or food components can result in altered pharmacokinetics. On the other hand, drugs or food components interacting with liver transporters may also interfere with liver function (e.g., bile acid homeostasis) and may even cause liver toxicity. Accordingly, certain transporters of the liver should be investigated already at an early stage of drug development. Most frequently radioactive probes are applied in these drug-transporter interaction tests. However, fluorescent probes are cost-effective and sensitive alternatives to radioligands, and are gaining wider application in drug-transporter interaction tests. In our review, we summarize our current understanding about hepatocyte ABC and SLC transporters affected by drug interactions. We provide an update of the available fluorescent and fluorogenic/activable probes applicable in in vitro or in vivo testing of these ABC and SLC transporters, including near-infrared transporter probes especially suitable for in vivo imaging. Furthermore, our review gives a comprehensive overview of the available fluorescence-based methods, not directly relying on the transport of the probe, suitable for the investigation of hepatic ABC or SLC-type drug transporters.

Naphthalimide-Fused Dipyrrins: Tunable Halochromic Switches and Photothermal NIR-II Dyes.

A family of tunable halochromic switches is developed using a naphthalimide-fused dipyrrin as the core π-conjugated motif. Electronic properties of these dipyrrins are tuned by substitution of their alpha and meso positions with aryl groups of variable donor-acceptor strength. The first protonation results in a conformational change that enhances electronic coupling between the dipyrrin chromophore and the meso substituent, leading to halochromic effects that occasionally exceed 200 nm and switch the absorption between the near-infrared (NIR)-I and NIR-II ranges. A NIR-II photothermal effect, switchable by acid-base chemistry is demonstrated for selected dipyrrins. Further protonation is possible for derivatives bearing additional amino groups, leading to up to four halochromic switching step. The most electron-rich dipyrrins are also susceptible to chemical oxidation, yielding NIR-absorbing radical cations and closed-shell dications.

In Vitro and In Vivo Evaluation of Targeted Fluorescent Imaging Agents for Diagnosis and Resection of Cancer.

Local re-occurrence of cancer in patients with solid tumors is currently the most common reason for failure of treatment strategies. This fact indicates that prevailing approaches for tumor resection can cure only 50% of patients. A major cause of failure in tumor resection is off-target drug cytotoxicity and lack of sensitivity in tumor detection methods. These disadvantages are addressed with the development of targeted therapy and diagnostics, which significantly aid treatment strategies. Targeted diagnostics exploit properties of tumor cells that show significant up-regulation of tumor biomarkers. These biomarkers are targeted by a homing ligand attached to a fluorophore for visual inspection during surgery. However, these approaches suffer from disadvantages like high autofluorescence from background tissues, tissue absorption, and scattering, resulting in decreased image sensitivity and resolution. The use of near-infrared (NIR) fluorophores to overcome these drawbacks has generated unprecedented interest among researchers. The NIR window lies within the range of 650 to 1,700 nm, which results in reduced absorption and scattering by the tissues, thereby providing deeper tissue penetration and reduced autofluorescence. NIR fluorophores can be designed to target tumor biomarkers such as prostate specific membrane antigen (PSMA) or folate receptors found over-expressed on cancer tissues. These targeted fluorophores consist of small-molecule ligands conjugated with NIR dyes that bind with high specificity to PSMA and folic acid receptors. In this protocol, we have extensively described the methodology for the synthesis of targeted NIR agents for PSMA (DUPA-NIR bioconjugate) and folic acid (folate-NIR bioconjugate), along with detailed steps for preclinical evaluation. Procedures to calculate the binding affinity to cancer cells in vitro are described, along with uptake and biodistribution in different mice models in vivo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and purification of DUPA and folate-peptide linkers via a SPPS strategy Basic Protocol 2: Conjugation, purification, and characterization of targeted bioconjugates with NIR probe for deep-tissue imaging applications Basic Protocol 3: In vitro evaluation of binding affinity of targeted DUPA-NIR and folate-NIR bioconjugates using a spectrophotometer Basic Protocol 4: Induction of tumor in mice to develop CDX or metastatic tumor models Basic Protocol 5: Intravenous administration of targeted DUPA-NIR and folate-NIR bioconjugates in mouse CDX or metastatic tumor models for deep-tissue NIR imaging and tumor resection.