Steric protection of near-infrared fluorescent dyes for enhanced bioimaging.

Near-fluorescent (NIR) dyes that absorb and emit light in the wavelength range of 650-1700 nm are well-suited for bioimaging due to the improved image contrast and increased penetration of the long-wavelength light through biological tissue. However, the imaging performance of NIR fluorescent dyes is limited by several inherent photophysical and physicochemical properties including, low fluorescence quantum yield, high chemical and photochemical reactivity, propensity to self-aggregate in water, non-specific association with off-target biological sites, and non-optimal pharmacokinetic profiles in living subjects. In principle, all these drawbacks can be alleviated by steric protection which is a structural process that surrounds the fluorophore with bulky groups that block undesired intermolecular interactions.

Fluorescence Imaging Using Deep-Red Indocyanine Blue, a Complementary Partner for Near-Infrared Indocyanine Green.

Indocyanine Blue (ICB) is the deep-red pentamethine analogue of the widely used clinical near-infrared heptamethine cyanine dye Indocyanine Green (ICG). The two fluorophores have the same number of functional groups and molecular charge and vary only by a single vinylene unit in the polymethine chain, which produces a predictable difference in spectral and physicochemical properties. We find that the two dyes can be employed as a complementary pair in diverse types of fundamental and applied fluorescence imaging experiments.

Miniature wireless LED-device for photodynamic-induced cell pyroptosis.

The inability of visible light to penetrate far through biological tissue limits its use for phototherapy and photodiagnosis of deep-tissue sites of disease. This is unfortunate because many visible dyes are excellent photosensitizers and photocatalysts that can induce a wide range of photochemical processes, including photogeneration of reactive oxygen species. One potential solution is to bring the light source closer to the site of disease by using a miniature implantable LED.

Squaraine Dyes Exhibit Spontaneous Fluorescence Blinking That Enables Live-Cell Nanoscopy.

Hampered by their susceptibility to nucleophilic attack and chemical bleaching, electron-deficient squaraine dyes have long been considered unsuitable for biological imaging. This study unveils a surprising twist: in aqueous environments, bleaching is not irreversible but rather a reversible spontaneous quenching process. Leveraging this new discovery, we introduce a novel deep-red squaraine probe tailored for live-cell super-resolution imaging.

Ratiometric near-infrared fluorescent probe for nitroreductase activity enables 3D imaging of hypoxic cells within intact tumor spheroids.

Fluorescent molecular probes that report nitroreductase activity have promise as imaging tools to elucidate the biology of hypoxic cells and report the past hypoxic history of biomedical tissue. This study describes the synthesis and validation of a "first-in-class" ratiometric, hydrophilic near-infrared fluorescent molecular probe for imaging hypoxia-induced nitroreductase activity in 2D cell culture monolayers and 3D multicellular tumor spheroids. The probe's molecular structure is charge-balanced and the change in ratiometric signal is based on Förster Resonance Energy Transfer (FRET) from a deep-red, pentamethine cyanine donor dye (Cy5, emits ∼660 nm) to a linked near-infrared, heptamethine cyanine acceptor dye (Cy7, emits ∼780 nm).

Cy5 Dye Cassettes Exhibit Through-Bond Energy Transfer and Enable Ratiometric Fluorescence Sensing.

The chemosensor literature contains many reports of fluorescence sensing using polyaromatic hydrocarbon fluorophores such as pyrene, tetraphenylethylene, or polyaryl(ethynylene), where the fluorophore is excited with ultraviolet light (<400 nm) and emits in the visible region of 400-500 nm. There is a need for general methods that convert these "turn-on" hydrocarbon fluorescent sensors into ratiometric sensing paradigms. One simple strategy is to mix the responsive hydrocarbon sensor with a second non-responsive dye that is excited by ultraviolet light but emits at a distinctly longer wavelength and thus acts as a reference signal.

Fluorescent ratiometric supramolecular tandem assays for phosphatase and phytase enzymes.

Ratiometric fluorescent assays have a built-in correction factor which enhances assay accuracy and reliability. We have developed fluorescent ratiometric supramolecular tandem assays for phosphatase and phytase enzymes using a mixture of three molecular components. One of the molecules is a tetra-cationic fluorescence quencher called CalixPyr which can bind and quench the polyanionic pyrene fluorophore, CMP, that emits at 430 nm.

A High-Affinity "Synthavidin" Receptor for Squaraine Dyes.

Strong-binding host-guest pairings in aqueous media have potential as "supramolecular glues" in biomedical techniques, complementing the widely-used (strept)avidin-biotin combination. We have previously found that squaraine dyes are bound very strongly by tetralactam macrocycles possessing anthracenyl units as cavity walls. Here we show that replacing the anthracenes with pentacyclic 5,7,12,14-tetrahydro-5,7,12,14-tetraoxapentacene (TOP) units generates receptors which bind squaraines with increased affinities (around K =10  m ) and improved selectivities.

Lipophilic Anchors that Embed Bioconjugates in Bilayer Membranes: A Review.

A wide range of biomaterials and engineered cell surfaces are composed of bioconjugates embedded in liposome membranes, surface-immobilized bilayers, or the plasma membranes of living cells. This review article summarizes the various ways that Nature anchors integral and peripheral proteins in a cell membrane and describes the strategies devised by chemical biologists to label a membrane protein in living cells. Also discussed are modern synthetic and semisynthetic methods to produce lipidated proteins.

Supramolecular Complexation of Azobenzene Dyes by Cucurbit[7]uril.

This report describes cucurbit[7]uril (CB7) complexation of azobenzene dyes that have a 4-(,'-dimethylamino) or 4-amino substituent. Absorption and NMR data show that CB7 encapsulates the protonated form of the azobenzene and that the complexed dye exists as its azonium tautomer with a azo conformation and substantial quinoid resonance character. Because CB7 complexation stabilizes the dye conjugate acid, there is an upward shift in its p, and in one specific case, the p of the protonated azobenzene is increased from 3.

Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging.

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4'-O-aryl linker is susceptible to cleavage by biological nucleophiles.

Palladium responsive liposomes for triggered release of aqueous contents.

Palladium (Pd) is a promising metal catalyst for novel bioorthogonal chemistry and prodrug activation. This report describes the first example of palladium responsive liposomes. The key molecule is a new caged phospholipid called Alloc-PE that forms stable liposomes (large unilamellar vesicles, ∼220 nm diameter).

Silica nanoparticle remodeling under mild conditions: versatile one step conversion of mesoporous to hollow nanoparticles with simultaneous payload loading.

A binary mixture of mesoporous silica nanoparticles plus organic polyammonium additive (dye or drug) is cleanly converted upon mild heating into hollow nanoparticles. The remodeled nanoparticle shell is an organized nanoscale assembly of globular additive/silica subunits and cancer cell assays show that a loaded drug additive is bioavailable.

Spontaneous Transfer of Indocyanine Green from Liposomes to Albumin Is Inhibited by the Antioxidant α-Tocopherol.

Indocyanine Green (ICG) is a clinically approved organic dye with near-infrared absorption and fluorescence. Over the years, many efforts to improve the photophysical and pharmacokinetic properties of ICG have investigated numerous nanoparticle formulations, especially liposomes with membrane-embedded ICG. A series of systematic absorption and fluorescence experiments, including FRET experiments using ICG as a fluorescence energy acceptor, found that ICG transfers spontaneously from liposomes to albumin protein residing in the external solution with a half-life of ∼10 min at 37 °C.

Cucurbit[7]uril Complexation of Near-Infrared Fluorescent Azobenzene-Cyanine Conjugates.

Two new azobenzene heptamethine cyanine conjugates exist as dispersed monomeric molecules in methanol solution and exhibit near-infrared (NIR) cyanine absorption and fluorescence. Both conjugates form non-emissive cyanine H-aggregates in water, but the addition of cucurbit[7]uril (CB7) induces dye deaggregation and a large increase in cyanine NIR fluorescence emission intensity. CB7 encapsulates the protonated azonium tautomer of the 4-(,-dimethylamino)azobenzene component of each azobenzene-cyanine conjugate and produces a distinctive new absorption band at 534 nm.

Sterically Shielded Hydrophilic Analogs of Indocyanine Green.

A modular synthetic process enables two or four shielding arms to be appended strategically over the fluorochromes of near-infrared cyanine heptamethine dyes to create hydrophilic analogs of clinically approved indocyanine green. A key synthetic step is the facile substitution of a heptamethine 4'-Cl atom by a phenol bearing two triethylene glycol chains. The lead compound is a heptamethine dye with four shielding arms, and a series of comparative spectroscopy studies showed that the shielding arms (a) increased dye photostability and chemical stability and (b) inhibited dye self-aggregation and association with albumin protein.

Structure-Activity Studies of Nitroreductase-Responsive Near-Infrared Heptamethine Cyanine Fluorescent Probes.

Two new classes of near-infrared molecular probes were prepared and shown to exhibit "turn on" fluorescence when cleaved by the nitroreductase enzyme, a well-known biomarker of cell hypoxia. The fluorescent probes are heptamethine cyanine dyes with a central 4'-carboxylic ester group on the heptamethine chain that is converted by a self-immolative fragmentation mechanism to a 4'-caboxylate group that greatly enhances the fluorescence brightness. Each compound was prepared by ring opening of a Zincke salt.

Supramolecular Mitigation of the Cyanine Limit Problem.

Currently, there is a substantial research effort to develop near-infrared fluorescent polymethine cyanine dyes for biological imaging and sensing. In water, cyanine dyes with extended conjugation are known to cross over the "cyanine limit" and undergo a symmetry breaking Peierls transition that favors an unsymmetric distribution of π-electron density and produces a broad absorption profile and low fluorescence brightness. This study shows how supramolecular encapsulation of a newly designed series of cationic, cyanine dyes by cucurbit[7]uril (CB7) can be used to alter the π-electron distribution within the cyanine chromophore.

Advances in Optical Sensors of -Acetyl-β-d-hexosaminidase (-Acetyl-β-d-glucosaminidase).

-Acetyl-β-d-hexosaminidases (EC 3.2.1.

Enzyme Sensing Using 2-Mercaptopyridine-Carbonitrile Reporters and Surface-Enhanced Raman Scattering.

The high sensitivity and functional group selectivity of surface-enhanced Raman scattering (SERS) make it an attractive method for enzyme sensing, but there is currently a severe lack of enzyme substrates that release SERS reporter molecules with favorable detection properties. We find that 2-mercaptopyridine-3-carbonitrile ( ) and 2-mercaptopyridine-5-carbonitrile ( ) are highly effective as SERS reporter molecules that can be captured by silver or gold nanoparticles to give intense SERS spectra, each with a distinctive nitrile peak at 2230 cm. is a more sensitive reporter and can be detected at low nanomolar concentrations.

Supramolecular capture of highly polar amidosquaraine dye in water with nanomolar affinity and large turn-on fluorescence.

A supramolecular dye-capture system comprising anionic amidosquaraine guest and macrocyclic tetralactam host exhibits nanomolar affinity and "turn on" visible fluorescence. Utility is demonstrated with a new fluorescent assay for liposome leakage induced by the biomedically important enzyme phospholipase A.

Comparison of cRGDfK Peptide Probes with Appended Shielded Heptamethine Cyanine Dye () for Near Infrared Fluorescence Imaging of Cancer.

Previous work has shown that the sterically shielded near-infrared (NIR) fluorescent heptamethine cyanine dye, , with a reactive carboxyl group produces fluorescent bioconjugates with an unsurpassed combination of high photostability and fluorescence brightness. This present contribution reports two new reactive homologues of with either a maleimide group for reaction with a thiol or a strained alkyne group for reaction with an azide. Three cancer-targeting NIR fluorescent probes were synthesized, each with an appended cRGDfK peptide to provide selective affinity for integrin receptors that are overexpressed on the surface of many cancer cells including the A549 lung adenocarcinoma cells used in this study.

Deuterated Indocyanine Green (ICG) with Extended Aqueous Storage Shelf-Life: Chemical and Clinical Implications.

Indocyanine Green (ICG) is a clinically approved near-infrared fluorescent dye that is used extensively for various imaging and diagnostic procedures. One drawback with ICG is its instability in water, which means that reconstituted clinical doses have to be used very shortly after preparation. Two deuterated versions of ICG were prepared with deuterium atoms on the heptamethine chain, and the spectral, physiochemical, and photostability properties were quantified.

Intracellular fluorescence competition assay for inhibitor engagement of histone deacetylase.

An intracellular fluorescence competition assay was developed to assess the capability of inhibitor candidates to engage histone deacetylase (HDAC) inside living cells and thus diminish cell uptake and staining by the HDAC-targeted fluorescent probe APS. Fluorescence cell microscopy and flow cytometry showed that pre-incubation of living cells with candidate inhibitors led to diminished cell uptake of the fluorescent probe. The assay was effective because the fluorescent probe (APS) possessed the required performance properties, including bright fluorescence, ready membrane diffusion, selective intracellular HDAC affinity, and negligible acute cytotoxicity.

Generalizable synthesis of bioresponsive near-infrared fluorescent probes: sulfonated heptamethine cyanine prototype for imaging cell hypoxia.

Continued advancement in bioresponsive fluorescence imaging requires new classes of activatable fluorescent probes that emit near-infrared fluorescence with wavelengths above 740 nm. Heptamethine cyanine dyes (Cy7) have suitable fluorescence properties but it is challenging to create activatable probes because Cy7 dyes have a propensity for self-aggregation and fluorescence quenching. A new synthetic strategy is employed to create a generalizable class of hydrophilic bioresponsive near-infrared fluorescent probes with appended sulfonates that provide excellent physiochemical properties.