Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization.

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by 1H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate its efficacy for imaging animal models.

Supramolecular Encapsulation of a Neurotransmitter Serotonin by Cucurbit[7]uril.

pH-dependent host-guest complexation of a monoamine neurotransmitter, Serotonin, with cucurbit[7]uril has been thoroughly investigated. The binding phenomena were explored using steady-state and time-resolved fluorescence spectroscopy at different pH values. At lower pH, i.e., protonated Serotonin, the binding affinity with cucurbit[7]uril was significantly higher compared to higher pH. Furthermore, detailed NMR titration experiments depicted the solution structure of the host-guest complex through the complexation induced chemical shift values. A competitive binding assay with cesium ions at pD 2.8 was subsequently performed for the further manifestation of the binding. Finally, the molecular docking studies provided well-documented proof of the 1:1 inclusion complex and the geometry of the complex. We believe that understanding from such studies can be important for pH-controlled delivery of serotonin for biological applications.

Cellular metabolic activity marker via selective turn-ON detection of transporter protein using nitrobenzoxadiazole-based fluorescent reporter.

A nitrobenzoxadiazole-based fluoroprobe (NBD-Bu) is designed to probe cellular metabolic activity in cancer and normal cells. NBD-Bu shows a significant fluorescence enhancement upon selective binding to the transport protein serum albumin in PBS buffer at ambient conditions. Encouraged by this finding, the site- specificity of NBD-Bu has been explored through a competitive displacement assay in the presence of site-specific markers such as warfarin and ibuprofen. Notably, even at micromolar concentrations, the probe possesses the ability to displace the site marker drug ibuprofen, efficiently. Subsequently, high-resolution fluorescence imaging results consolidated the potential of NBD-Bu for detection of abnormal cellular metabolic activity.

Synthesis of Two-Photon Active Tricomponent Fluorescent Probe for Distinguishment of Biotin Receptor Positive and Negative Cells and Imaging 3D-Spheroid.

A fluorescence microscopy-based distinguishment between biotin receptor (BiR) positive and negative cell lines via receptor-mediated endocytosis has been demonstrated. A water-soluble, three-component, two-photon (2P) active solvatofluorochromic probe has been designed and synthesized. The applicability of the probe for 2P microscopy and 3D-spheroid was also assessed.

Encapsulation and modulation of protolytic equilibrium of ß-carboline-based norharmane drug by cucurbit[7]uril and micellar environments for enhanced cellular uptake.

The effect of supramolecular nanocavity on photophysical and acid-dissociation properties of Norharmane (NHM), a physiologically important, anxiety control and memory-enhancing ß-carboline-based drug, has been investigated using steady-state absorption and fluorescence spectroscopy. Self-assembled organization derived from surfactants and rigid water-soluble macrocyclic host Cucurbit[7]uril (CB7) have been selected for this investigation. The confined-space offered by the supramolecular assemblies modulates the pKa value of NHM (up to 3 units) as it can exist in two protolytic forms at near neutral pH. Therefore, the pH-dependent binding properties, modulation of pKa value and its consequences on the photophysical, chemical and solubility properties are investigated in detail. This investigation shows a large shift in the protolytic equilibrium which in turn causes ca. 15 times solubility-enhancement at near neutral pH. Moreover, the effect of enhanced solubility has been further investigated by the augmentation in the cellular uptake of NHM entrapped inside CB7. Thus, the modulation of the acid-base properties and solubility of ß-carboline-based drugs will have immense potential for their formulation, cellular uptake and bioavailability.

Large Stokes-shifted NIR-emission from nanospace-induced aggregation of perylenemonoimide-doped polymer nanoparticles: imaging of folate receptor expression.

The development of a water-soluble, perylenemonoimide (PMI) dye-doped polymer nanoparticle (PNP) with NIR emission for live-cell imaging is demonstrated. The large Stokes-shifted NIR emission is due to confined nanospace-induced aggregation offered by the polymer matrix. Later, folic acid functionalised PNP (PNP-FA) is successfully employed to differentiate folate receptor positive and negative cancer cells.

A rapid, naked-eye detection of hypochlorite and bisulfite using a robust and highly-photostable indicator dye Quinaldine Red in aqueous medium.

A "naked-eye" detection of health hazardous bisulfite (HSO3-) and hypochlorite (ClO-) using an indicator dye (Quinaldine Red, QR) in a wide range of pH is demonstrated. The molecule contains a quinoline moiety linked to an N,N-dimethylaniline moiety with a conjugated double bond. Treatment of QR with HSO3- and ClO-, in aqueous solution at near-neutral pH, resulted in a colorless product with high selectivity and sensitivity. The detection limit was 47.8µM and 0.2µM for HSO3- and ClO- respectively. However, ClO- was 50 times more sensitive and with 2 times faster response compared to HSO3-. The detail characterization and related analysis demonstrate the potential of QR for a rapid, robust and highly efficient colorimetric sensor for the practical applications to detect hypochlorite in water samples.

Differentiation of Folate-Receptor-Positive and -Negative Cells Using a Substrate-Mimicking Fluorescent Probe.

Diagnosis and therapy exploiting overexpressed receptors on the cell surface is one important strategy in medicine. Determination of the over expression level of a particular receptor is prerequisite for it to be of clinical use. Differentiation between FR-positive (FR=folate receptor) and -negative cells via fluorescence microscopy using a substrate mimetic fluorophore is presented in this work. The strategy adopted here is not the classical FA-conjugated (FA=folic acid) fluorescent probe but a small and environment-sensitive pterin-based (pterin is part of folate, i.e., vitamin B9) fluorescent probe. Electronically diverse pterin-based fluorescent probes have been designed and synthesized to understand the effect of the binding environment on the receptor-substrate interactions. By utilizing steady-state UV/Vis and fluorescence along with time-resolved fluorescence spectroscopy, the effects on the electronic and acid-base properties of the substrate were investigated. Evidently, one synthesized probe showed FA-mimicking behavior with strong binding interaction with FR.

Long-Lived Polypyridyl Based Mononuclear Ruthenium Complexes: Synthesis, Structure, and Azo Dye Decomposition.

Two mononuclear ruthenium complexes [(bpy)2RuIIL1/L2](ClO4)2 ([1]2+/[2]2+) (bpy-2,2' bipyridine, L1 = 2,3-di(pyridin-2-yl)pyrazino[2,3-f][1,10]phenanthroline) and L2 = 2,3-di(thiophen-2-yl)pyrazino[2,3-f][1,10]phenanthroline have been synthesized. The complexes have been characterized using various analytical techniques. The complex [1]2+ has further been characterized by its single crystal X-ray structure suggesting ruthenium is coordinating through the N donors of phenanthroline end. Theoretical investigation suggests that the HOMOs of both complexes are composed of pyridine and pyrazine unit of ligands L1 and L2 whereas the LUMOs are formed by the contribution of bipyridine units. The low energy bands at ∼480 nm of the complexes can be assigned as MLCT with partial contribution from ligand transitions, whereas the rest are ligand centered. The complexes have shown RuII/RuIII oxidation couples at E1/2 at 1.26 (70 mV) V and 1.28 (62 mV) V for [1]2+ and [2]2+ vs Ag/AgCl, respectively, suggesting no significant role of distal thiophene or pyridine units of the ligands. The complexes are emissive and display solvent dependent emission properties. Both complexes have shown highest emission quantum yield and lifetime in DMSO (ϕ = 0.05 and τavg = 460 ns and λmaxem at 620 nm for [1]2+; ϕ = 0.043 and τavg = 425 ns and λmaxem at 635 nm for [2]2+). Further, the long luminescent lifetime of these complexes has been utilized to generate reactive oxygen species for efficient azo dye decomposition.

Customized tuning of aggregation-induced emission of a napthalimide dye by surfactants and cyclodextrin.

The aggregation-induced emission behaviour of an environment sensitive 2,3-substituted napthalimide dye has been investigated at neutral pH in presence of three different surfactants i.e., cationic, anionic, and neutral. The changes observed in the excitation spectrum of the dye compared to its absorption spectra in water in presence of the surfactants above their micellar concentration reveals the transformation of the H-aggregates of the dye to the monomeric form. The alteration of the dye aggregates to its monomeric form and its consequence on emission properties has been utilized to estimate the surfactant concentration parameter for pre-micellar to micellar transformation. The aggregation of the dye molecules has been made reversible by removal of the surfactant molecules from the system upon host-guest complexation with α-cyclodextrin. This switchable aggregation-deaggregation phenomenon of DMN-Bu by employing surfactants and α-cyclodextrin at neutral pH in water is utilized for determining their critical micellar concentration.

Spectroscopic investigation of bio-mimetic solvolysis of 6-(N,N-dimethylamino)-2,3-naphthalic anhydride in confined nanocavities.

Enzymes are biological catalysts that can vastly accelerate the reaction rate of a substrate by accommodating it within the active site. The local environment provided by the active site of a natural catalyst causes a significant rate-enhancement of the reaction as compared to that without catalyst. The solvolysis reaction of a 6-(N,N-dimethylamino)-2,3-naphthalic anhydride probe is investigated using UV-Vis and fluorescence spectroscopy in pure alcohols and in bio-mimetic nano-sized environments like surfactants, macrocyclic hosts and protein nanocavities. The solvolysis rate in alcohols is found to be regulated directly by the alkyl chain length and follows Arrhenius dependence. The hydrolysis rate of the probe in water under physiological conditions (pH 7.4, at 25 °C) is very slow. However, under identical conditions, the rate can be accelerated significantly by protein and supramolecular nanocavities. Therefore, such fundamental kinetic analysis of the understanding of this bio-mimetic solvolysis will allow us to design a novel probe-drug conjugate with efficient controlled-release and function.

Investigation of the effect of cucurbit[7]uril complexation on the photophysical and acid-base properties of the antimalarial drug quinine.

Host-guest complexation of mono and dicationic quinine with cucurbit[7]uril (CB7), a water-soluble macrocyclic host molecule, has been investigated. Job's plot, time-resolved anisotropy as well as concentration dependent NMR titration confirm the binding of two CB7 macrocycles with one quinine molecule. The binding affinity of dicationic quinine with CB7 is one order of magnitude higher than the binding constant of mono-cationic quinine. Such preferential binding results in one unit pKa shift in the ground-state of the quinoline ring. However, using fluorescence spectroscopy we have obtained two acid-dissociation constants, one for quinoline ring nitrogen and the other for the nitrogen of the quinuclidine moiety. In the excited state, CB7 complexation causes one unit pKa shift for the quinoline ring and 1.9 unit shift for the quinuclidine moiety. Interestingly, a large enhancement of fluorescence lifetime and anisotropy of quinine at pH 2.7 and pH 9.0 upon CB7 complexation was observed due to the restriction of conformational flexibility. Moreover, at pH 3.0, a large fluorescence enhancement of quinine due to CB7 complexation was observed and it was quite significant as compared to that of quinine in 0.1 (M) HCl without CB7. We believe that this study of quinine complexation with CB7 will reduce phototoxicity, increase bioavailability and offer an alternative standard for quantum yield measurements in an amiable condition.

Supramolecular guest relay using host-protein nanocavities: an application of host-induced guest protonation.

Small drug molecules and other important metabolites are delivered via a suitable carrier protein-mediated transport through a specific receptor. The process is highly coordinated and associated with complexation induced properties of deliverable molecules. To get a molecular insight, in this report, we tried to mimic the delivery process to know how the carrier protein relocates the drug molecule from the macrocyclic host cavity to its binding pocket and how the electronic and the chemical properties of the guest get altered. Bovine and human serum albumin (BSA and HSA) were used as the model carrier proteins which can snatch out 6-propanoyl-2-(N,N-dimethylamino)naphthalene (PRO), dye used as a drug model (known to bind at the drug-binding pocket of the carrier protein), from the cucurbit[7]uril (CB7) cavity, a potential drug delivery carrier. Prior to performing the fluorescence-based bio-supramolecular relocation assay using BSA and HSA, CB7 and PRO, we have investigated the effect of CB7 encapsulation and protonation on the fluorescence properties of PRO. A significant shift in the pKa value from 3.4 to 6.6 (ca. 3.2 logarithmic units) of PRO was observed upon encapsulation with CB7, which causes a huge fluorescence quenching even at neutral pH. The binding affinity of protonated and neutral PRO for CB7 also confirms a 3.2 unit shift in the acid-dissociation constant. A displacement assay using a strong CB7 binder, viz., 1,6-diaminohexane, confirms encapsulation of PRO in the CB7 cavity. Encapsulation of neutral PRO by CB7 shows a significant fluorescence enhancement accompanied by a ∼35 nm blue shift in the emission maxima.

Iridium Complexes as a Roadblock for DNA Polymerase during Amplification.

Iridium-based metal complexes containing polypyridyl-pyrazine ligands show properties of DNA intercalation. They serve as roadblocks to DNA polymerase activity, thereby inhibiting the polymerization process. Upon the addition of increasing concentrations of these iridium complexes, a rapid polymerase chain reaction (PCR)-based assay reveals the selective inhibition of the DNA polymerization process. This label-free approach to study the inhibition of fundamental cellular processes via physical roadblock can offer an alternative route toward cancer therapy.

A ratiometric fluorescent probe for detection of biogenic primary amines with nanomolar sensitivity.

An ultrasensitive ratiometric fluorescent sensor made of an N,N-dimethylaminonaphthalene anhydride moiety for detection of aliphatic primary amines is reported. Biogenic amines at nanomolar concentration is detected with the additional ability to discriminate between primary, secondary and tertiary amines by using both UV-Visible and fluorescence spectroscopy.

Probing microenvironment of micelle and albumin using diethyl 6-(dimethylamino)naphthalene-2,3-dicarboxylate: An electroneutral solvatochromic fluorescent probe.

Synthesis, single-crystal X-ray characterization, and spectroscopic investigation of small, non-charged diethyl 6-(dimethylamino)naphthalene-2,3-dicarboxylate (DMNDC) by UV-Visible, steady-state, and time-resolved fluorescence reveal a series of interesting photophysical properties originating from the intrinsic intramolecular charge transfer (ICT) state, leading to diverse applications. Stokes shift, lifetime, and emission maxima of DMNDC show a very good correlation with ET(30) solvent polarity scale for a series of different polarity solvents, confirms that it has very good environment sensitivity. Furthermore, this dye has been found to be an exceptionally suitable probe for determining Critical Micelle Concentration (CMC) and probing self-organization processes of five different type of surfactant with structural diversity. A 20-60nm blue shift in emission maxima accompanied by a large fluorescence lifetime enhancement (ca. 23ns) was observed upon relocation of DMNDC into a hydrophobic microenvironment. Along with this, the small size, electroneutrality, pH stability, and excellent solvatochromic fluorescent properties are employed for deciphering the number of hydrophobic binding pockets with strong affinity and their local microenvironment present in Bovine Serum Albumin (BSA).

Complexation induced fluorescence and acid-base properties of dapoxyl dye with γ-cyclodextrin: a drug-binding application using displacement assays.

Host-guest complexation of dapoxyl sodium sulphonate (DSS), an intramolecular charge transfer dye with water-soluble and non-toxic macrocycle γ-cyclodextrin (γ-CD), has been investigated in a wide pH range. Steady-state absorption, fluorescence and time-resolved fluorescence measurements confirm the positioning of DSS into the hydrophobic cavity of γ-CD. A large fluorescence enhancement ca. 30 times, due to 1 : 2 complex formation and host-assisted guest-protonation have been utilised for developing a method for the utilisation of CD based drug-delivery applications. A simple fluorescence-displacement based approach is implemented at physiological pH for the assessment of binding strength of pharmaceutically useful small drug molecules (ibuprofen, paracetamol, methyl salicylate, salicylic acid, aspirin, and piroxicam) and six important antibiotic drugs (resazurin, thiamphenicol, chloramphenicol, ampicillin, kanamycin, and sorbic acid) with γ-CD.

Hydroxy-terminated conjugated polymer nanoparticles have near-unity bright fraction and reveal cholesterol-dependence of IGF1R nanodomains.

Fluorescent nanoparticles have enabled many discoveries regarding how molecular machines function. Quantum dots have been the dominant class of fluorescent nanoparticles but suffer from blinking and from a substantial dark fraction--particles where the fluorescence is never seen--complicating any analysis of biological function. Nanoparticles composed of conjugated fluorescent polymers (Pdots) have recently been shown to have high brightness and no blinking. Here we develop a robust and efficient means to measure the dark fraction of Pdots, conjugating Atto dyes to the nanoparticles and testing fluorescence colocalization of dye and Pdot puncta. This established that the Pdots we generated had minimal dark fraction: ∼3%. The application of nanoparticles in biological environments is highly sensitive to surface functionalization. For Pdots we found that passivation with uncharged hydroxy-terminated polyethylene glycol caused a dramatic reduction in nonspecific cell binding and aggregation compared to a charged coating. Using carbonyl di-imidazole the hydroxy-Pdots were functionalized efficiently with streptavidin for high stability targeting, allowing specific labeling of mammalian cells. Type I insulin-like growth factor receptor (IGF1R) regulates cell survival and development, with roles in aging, heart disease, and cancer. We used hydroxy-Pdots to track the dynamics of IGF1R on a breast cancer cell-line, determining the diffusion characteristics and showing cholesterol-containing membrane nanodomains were important for receptor mobility at the plasma membrane. The near-unity bright fraction and low nonspecific binding of hydroxy-Pdots, combined with Pdot photostability and lack of blinking, provides many advantages for investigations at the single molecule level.

Mechanisms for size-dependent protein segregation at immune synapses assessed with molecular rulers.

Immunological synapses are specialized intercellular contacts formed by several types of immune cells in contact with target cells or antigen-presenting cells. A late-stage immune synapse is commonly a bulls-eye pattern of immune cell receptor-ligand pairs surrounded by integrin complexes. Based on crystal structures, the intermembrane distance would be ∼15 nm for many immune cell receptor-ligand pairs, but ∼40 nm for integrin-ligand pairs. Close proximity of these two classes of intermembrane bonds would require significant membrane bending and such proteins can segregate according to their size, which may be key for receptor triggering. However, tools available to evaluate the intermembrane organization of the synapse are limited. Here, we present what we believe to be a novel approach to test the importance of size in the intercellular organization of proteins, using live-cell microscopy of a size-series of fluorescently-labeled molecules and quantum dots to act as molecular rulers. Small particles readily colocalized at the synapse with MHC class I bound to its cognate natural killer cell receptor, whereas particles larger than 15 nm were increasingly segregated from this interaction. Combined with modeling of the partitioning of the particles by scaled-particle adsorption theory, these molecular rulers show how membrane-bending elasticity can drive size-dependent exclusion of proteins within immune synapses.

How the biotin-streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer.

The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in Nature. SA is a widely used tool and a paradigm for protein-ligand interactions. We previously developed a SA mutant, termed Tr (traptavidin), possessing a 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. In the present study, we determined the crystal structures of apo-Tr and biotin-Tr at 1.5 Å resolution. In apo-SA the loop (L3/4), near biotin's valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, L3/4 was shut in both apo-Tr and biotin-Tr. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for Tr's reduced biotin off- and on-rates. L3/4 includes Ser45, which forms a hydrogen bond to biotin consistently in Tr, but erratically in SA. Reduced breakage of the biotin-Ser45 hydrogen bond in Tr is likely to inhibit the initiating event in biotin's dissociation pathway. We generated a Tr with a single biotin-binding site rather than four, which showed a simi-larly low off-rate, demonstrating that Tr's low off-rate was governed by intrasubunit effects. Understanding the structural features of this tenacious interaction may assist the design of even stronger affinity tags and inhibitors.