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. This probe enables single-molecule localization microscopy (SMLM) under physiological conditions without harmful additives or intense lasers and exhibits spontaneous blinking orchestrated by biological nucleophiles, such as glutathione or hydroxide anion. With a low duty cycle (∼0.1%) and high-emission rate (∼6 × 104 photons/s under 400 W/cm2), the squaraine probe surpasses the benchmark Cy5 dye by 4-fold and Si-rhodamine by a factor of 1.7 times. Live-cell SMLM with the probe reveals intricate structural details of cell membranes, which demonstrates the high potential of squaraine dyes for next-generation 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). Enzymatic reduction of a 4-nitrobenzyl group on the Cy7 component induces a large increase in Cy7/Cy5 fluorescence ratio. The deep penetration of near-infrared light enables 3D optical sectioning of intact tumor spheroids, and visualization of individual hypoxic cells (i.e., cells with raised Cy7/Cy5 ratio) as a new way to study tumor spheroids. Beyond preclinical imaging, the near-infrared fluorescent molecular probe has high potential for ratiometric imaging of hypoxic tissue in living subjects.

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. Polyphosphates can disrupt the CMP/CalixPyr complex and alter the fluorescence intensity (responsive signal). CalixPyr has no effect on the fluorescence emission of cationic pentamethine cyanine fluorophore, cCy5, which emits at 665 nm and acts as a non-responsive reference signal. The continuous ratiometric fluorescent assay for alkaline phosphatase monitored hydrolytic consumption of adenosine triphosphate (ATP). The continuous ratiometric fluorescent assay for phytase activity monitored hydrolytic consumption of phytate. With further development this latter assay may be useful for high throughput assessment of phytase activity in individual batches of fortified animal feed. It is likely that the three-molecule mixture (CMP, CalixPyr, cCy5) can become a general assay platform for other enzymes that catalyse addition/removal of phosphate groups from appropriate molecular substrates.

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. Five new cyanine dye cassettes were created by covalently attaching a pyrene, tetraphenylethylene, or biphenyl(ethynylene) component as the ultraviolet-absorbing energy donor directly to the pentamethine chain of a deep-red cyanine (Cy5) energy acceptor. Fluorescence emission studies showed that these Cy5-cassettes exhibited large pseudo-Stokes shifts and high through-bond energy transfer efficiencies upon excitation with ultraviolet light. Practical potential was demonstrated with two examples of ratiometric fluorescence sensing using a single ultraviolet excitation wavelength. One example mixed a Cy5-cassette with a pyrene-based fluorescent indicator that responded to changes in Cu2+ concentration, and the other example mixed a Cy5-cassette with the fluorescent pH sensing dye, pyranine.

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 Ka =1010  m-1 ) and improved selectivities. Binding can be followed through changes to squaraine fluorescence and absorbance. The TOP units are easy to prepare and potentially variable, while the TOP-based receptor shows improved photostability, both in itself and in complex with squaraines. The results suggest that this system could prove valuable in the further development of practical "synthavidin" chemistry.

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. Subsequent sections describe methods to anchor a three-component synthetic construct that is composed of a lipophilic membrane anchor, hydrophilic linker, and exposed functional component. The surface exposed payload can be a fluorophore, aptamer, oligonucleotide, polypeptide, peptide nucleic acid, polysaccharide, branched dendrimer, or linear polymer. Hydrocarbon chains are commonly used as the membrane anchor, and a general experimental trend is that a two chain lipid anchor has higher membrane affinity than a cholesteryl or single chain lipid anchor. Amphiphilic fluorescent dyes are effective molecular probes for cell membrane imaging and a zwitterionic linker between the fluorophore and the lipid anchor promotes high persistence in the plasma membrane of living cells. A relatively new advance is the development of switchable membrane anchors as molecular tools for fundamental studies or as technology platforms for applied biomaterials.

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. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1, with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.

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

This report describes cucurbit[7]uril (CB7) complexation of azobenzene dyes that have a 4-(N,N'-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 trans azo conformation and substantial quinoid resonance character. Because CB7 complexation stabilizes the dye conjugate acid, there is an upward shift in its pKa, and in one specific case, the pKa of the protonated azobenzene is increased from 3.09 to 4.47. Molecular modeling indicates that the CB7/azobenzene complex is stabilized by three major noncovalent factors: (i) ion-dipole interactions between the partially cationic 4-(N,N'-dimethylamino) or 4-amino group on the encapsulated protonated azobenzene and the electronegative carbonyl oxygens on CB7, (ii) inclusion of the upper aryl ring of the azobenzene within the hydrophobic CB7 cavity, and (iii) a hydrogen bond between the proton on the azo nitrogen and CB7 carbonyls. CB7 complexation enhances azobenzene stability and increases azobenzene hydrophilicity; thus, it is a promising way to improve azobenzene performance as a pigment or prodrug. In addition, the striking yellow/pink color change that accompanies CB7 complexation can be exploited to create azobenzene dye displacement assays with naked eye detection.

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). Liposome treatment with PdCl2 removes the chemical cage, liberates membrane destabilizing dioleoylphosphoethanolamine (DOPE), and triggers liposome leakage of encapsulated aqueous contents. The results indicate a path towards liposomal drug delivery technologies that exploit transition metal triggered leakage.

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. Moreover, transfer of ICG from liposome membranes to external albumin reduces light-activated leakage from thermosensitive liposomes with membrane-embedded ICG. A survey of lipophilic liposome additives discovered that the presence of clinically approved antioxidant, α-tocopherol, greatly increases ICG retention in the liposomes (presumably by forming favorable aromatic stacking interactions), inhibits ICG photobleaching and prevents albumin-induced reduction of light-triggered liposome leakage. This new insight will help researchers with the specific task of optimizing ICG-containing liposomes for fluorescence imaging or phototherapeutics. More broadly, the results suggest a broader design concept concerning light triggered liposome leakage, that is, proximity of the light absorbing dye to the bilayer membrane is a critical design feature that impacts the extent of liposome leakage.

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-(N,N-dimethylamino)azobenzene component of each azobenzene-cyanine conjugate and produces a distinctive new absorption band at 534 nm. The complex is quite hydrophilic, which suggests that CB7 can be used as a supramolecular additive to solubilize this new family of NIR azobenzene-cyanine conjugates for future biomedical applications. Since many azobenzene compounds are themselves potential drug candidates or theranostic agents, it should be possible to formulate many of them as CB7 inclusion complexes with improved solubility, stability, and pharmaceutical profile.

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. In mice, the dye cleared from the blood primarily through the renal pathway rather than the biliary pathway for ICG. This change in biodistribution reflects the much smaller hydrodynamic diameter of the shielded hydrophilic ICG analog compared to the 67 kDa size of the ICG/albumin complex. An attractive feature of versatile synthetic chemistry is the capability to systematically alter the dye's hydrodynamic diameter. The sterically shielded hydrophilic ICG dye platform is well-suited for immediate incorporation into dynamic contrast-enhanced (DCE) spectroscopy or imaging protocols using the same cameras and detectors that have been optimized for ICG.

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. For two sets of dyes, supramolecular location of the surrounding CB7 over the center of the dye favors a nonpolar ground state, with a symmetric π-electron distribution that produces a sharpened absorption band with enhanced fluorescence brightness. The opposite supramolecular effect (i.e., broadened absorption and partially quenched fluorescence) is observed with a third set of dyes because the surrounding CB7 is located at one end of the encapsulated cyanine chromophore. From the perspective of enhanced near-infrared bioimaging and sensing in water, the results show how that the principles of host/guest chemistry can be employed to mitigate the "cyanine limit" problem.

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

N-Acetyl-ß-d-hexosaminidases (EC 3.2.1.52) are exo-acting glycosyl hydrolases that remove N-acetyl-ß-d-glucosamine (Glc-NAc) or N-acetyl-ß-d-galactosamine (Gal-NAc) from the nonreducing ends of various biomolecules including oligosaccharides, glycoproteins, and glycolipids. The same enzymes are sometimes called N-acetyl-ß-d-glucosaminidases, and this review article employs the shorthand descriptor HEX(NAG) to indicate that the terms HEX or NAG are used interchangeably in the literature. The wide distribution of HEX(NAG) throughout the biosphere and its intracellular location in lysosomes combine to make it an important enzyme in food science, agriculture, cell biology, medical diagnostics, and chemotherapy. For more than 50 years, researchers have employed chromogenic derivatives of N-acetyl-ß-d-glucosaminide in basic assays for biomedical research and clinical chemistry. Recent conceptual and synthetic innovations in molecular fluorescence sensors, along with concurrent technical improvements in instrumentation, have produced a growing number of new fluorescent imaging and diagnostics methods. A systematic summary of the recent advances in optical sensors for HEX(NAG) is provided under the following headings: assessing kidney health, detection and treatment of infectious disease, fluorescence imaging of cancer, treatment of lysosomal disorders, and reactive probes for chemical biology. The article concludes with some comments on likely future directions.

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 ( o-MPN) and 2-mercaptopyridine-5-carbonitrile ( p-MPN) 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-1. p-MPN is a more sensitive reporter and can be detected at low nanomolar concentrations. An assay validation study synthesized two novel substrate molecules, Glc-o-MPN and Glc-p-MPN, and showed that they can be cleaved efficiently by ß-glucosidase (K m = 228 and 162 µM, respectively), an enzyme with broad industrial and biomedical utility. Moreover, SERS detection of the released reporters ( o-MPN or p-MPN) enabled sensing of ß-glucosidase activity and ß-glucosidase inhibition. Comparative experiments using a crude almond flour extract showed that the presence of ß-glucosidase activity could be confirmed by SERS detection in a much shorter time period (>10 time shorter) than by UV-vis absorption detection. It is likely that a wide range of enzyme assays and diagnostic tests can be developed using 2-mercaptopyridine-carbonitriles as SERS reporter molecules.

Structural Engineering of Fluorescent Self-Threaded Peptide Probes for Targeted Cell Imaging.

Squaraine figure-eight (SF8) molecules are a new class of deep-red fluorescent probes that are well suited for fluorescence cell microscopy due to their very high fluorescence brightness and excellent stability. Three homologous SF8 probes, with peptidyl loops that differ by very minor changes in the peptide sequence, were synthesized and assessed for probe uptake by cancer cells. One of probes included the RGD motif that is recognized by many classes of integrin receptors that reside on the surface of the cancer cells, and it permeated the cells by receptor-mediated endocytosis. In contrast, cell microscopy showed that there was negligible cell uptake of the two homologous SF8 probes indicating differences in probe targeting capability. The synthetic method allows for easy alteration of the peptide sequence; thus, it is straightforward to develop new classes of peptidyl SF8 probes with loop sequences that target other cancer biomarkers.

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. The chemical structures have either terminal benzoindolinenes or propargyloxy auxochromes, which provide favorable red-shifted absorption/emission wavelengths and a hyperchromic effect that enhances the photon output when excited by 808 nm light. A fluorescent probe with terminal propargyloxy-indolenines exhibited less self-aggregation and was rapidly activated by nitroreductase with large "turn on" fluorescence; thus, it is the preferred choice for translation towards in vivo applications.

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 A2.

Comparison of cRGDfK Peptide Probes with Appended Shielded Heptamethine Cyanine Dye (s775z) for Near Infrared Fluorescence Imaging of Cancer.

Previous work has shown that the sterically shielded near-infrared (NIR) fluorescent heptamethine cyanine dye, s775z, 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 s775z 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. A set of cancer cell microscopy and mouse tumor imaging experiments showed that all three probes were very effective at targeting cancer cells and tumors; however, the change in the linker structure produced a statistically significant difference in some aspects of the mouse biodistribution. The mouse studies included a mock surgical procedure that excised the subcutaneous tumors. A paired-agent fluorescence imaging experiment co-injected a binary mixture of targeted probe with 850 nm emission, an untargeted probe with 710 nm emission and determined the targeted probe's binding potential in the tumor tissue. A comparison of pixelated maps of binding potential for each excised tumor indicated a tumor-to-tumor variation of integrin expression levels, and a heterogeneous spatial distribution of integrin receptors within each tumor.