SMALL MOLECULE-BASED FLUORESCENT MOLECULAR PROBES FOR FACILITATING BIOMEDICAL RESEARCH: RATIONAL DESIGN AND BIOIMAGING APPLICATIONS

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.

Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

Review: 29 refs.

The development of new fluorescent probes as molecular sensors is a critical step for the understanding of molecular mechanisms. BODIPY-based probes offer versatility due to their high fluorescence quantum yields, photostability, and tunable absorption/emission wavelengths. Here, we report the synthesis and evaluation of a novel 7-azaindole-BODIPY derivative to probe hydrophobic proteins as well as protein misfolding and aggregation. In organic solvents, this compound shows two efficiently interconverting emissive excited states. In aqueous environments, it forms molecular aggregates with unique photophysical properties. The complex photophysics of the 7-azaindole-BODIPY derivative was explored for sensing applications. In the presence of albumin, the compound is stabilized in hydrophobic protein regions, significantly increasing its fluorescence emission intensity and lifetime. Similar effects occur in the presence of protein aggregates but not with other macromolecules like pepsin, DNA, Ficoll 40, and coconut oil. Fluorescence lifetime imaging microscopy (FLIM) and two-photon fluorescence microscopy on breast (MCF-7) and lung (A549) cancer cells incubated with this compound display longer fluorescence lifetimes and higher emission intensity under oxidative stress. Synchrotron FTIR micro spectroscopy confirmed that the photophysical changes observed were due to protein misfolding and aggregation caused by the oxidative stress. These findings demonstrate that this compound can serve as a fluorescent probe to monitor protein misfolding and aggregation triggered by oxidative stress.

Fluorescent probes are widely used in membrane biophysical studies to investigate the properties of the molecular environment of cell membrane and biochemical processes related to it. These probes provide information of the molecular environment trough changes of their photophysical properties. While these probes can provide valuable information of the membrane molecular properties, a clear structural description, at the molecular scale, of the membrane properties surrounding the fluorescent molecular probe, is essential for the interpretation of the experimental fluorescent signal. In this work, we studied the membrane permeation properties of four benzo[a]phenoxazinium fluorescent probes with different alkyl chains in the tetracyclic ring in DPPC membranes using molecular modelling techniques . We describe the molecular mechanism of interaction between the molecular probes and the DPPC lipids. We show that probes can be preferentially located at the membrane water interface interaction with the lipid polar groups, while others can be cross the membrane in a passive way. This processes is dependent on the tetracyclic substitutions and alkyl chain present in the fluorescent probe. These findings can provide a detailed rational of the structural properties surrounding the molecular probes that could be correlated with the experimentally measured fluorescence.

The bipyridine-based constructs 4-(pyridine-2-yl)-3H-pyrrolo[2,3-c]quinoline (PPQ) and [6-(3H-pyrrolo[2,3-c]quinolin-4-yl)pyridin-2-yl]methanol (PPQ-OH) and their assemblies with surfactants are evaluated as turn-on fluorescent sensors for Zn2+ ions in aqueous solution. This study strives to overcome the problem of low water solubility of the hydrophobic PPQ and PPQ-OH by using micelles. Whereas the ligands show selective sensing behavior for Zn2+ over important biological cations including Na+ , K+ , Ca2+ , Mg2+ in anionic sodium dodecyl sulfate and non-ionic Tween 80 micelles, no Zn2+ sensing is observed in cationic cetyltrimethylammonium bromide micelles. Unlike in DMF, Cd2+ interference is observed in aqueous conditions, which can be avoided either by performing the study at pH≥9 or by carrying out a time-resolved fluorescence study. Analysis of the Job plot data, the fluorescence lifetimes, and experiments on varying micellar shape and pH, confirms that the coordination volume of the resulting octahedral metal complex and formation of a five-membered chelate ring are critical factors for Cd2+ interference. The described sensing systems are capable of detecting Zn2+ ions at the micromolar level. Additionally, it is shown that PPQ and PPQ-OH can be used to detect Zn2+ in HeLa cells under physiological conditions in bioimaging studies.

Two fluorochromes, ER-TrackerTM Blue-White DPX dye and the fluorescent brefeldin A (BFA) derivative, BODIPY-BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular-vacuole system at the light microscope level in living cells. The ER-Tracker dye labels a reticulate network similar in distribution to ER as seen in electron micrographs of freeze-substituted hyphae. BODIPY-BFA stains a thicker axially aligned structure with an expanded region at the apex, which is similar to that seen when hyphae are stained with ER-Tracker dye in the presence of unconjugated BFA. This structure is considered to be ER modified by BFA, a view supported by ultrastructural observations of the effect of BFA on the fungal ER. Both fluorescent probes also stain punctate structures, which are most likely to be Golgi bodies. Neither probe labels the tubular-vacuole system.

Multi-dimensional imaging of endogenous leucine aminopeptidase via fast response fluorescent read-out probe

The discovery of a near-infrared (NIR, 650-900 nm) fluorescent chromophore hemicyanine dye with high structural tailorability is of great significance in the field of detection, bioimaging, and medical therapeutic applications. It exhibits many outstanding advantages including absorption and emission in the NIR region, tunable spectral properties, high photostability as well as a large Stokes shift. These properties are superior to those of conventional fluorogens, such as coumarin, fluorescein, naphthalimides, rhodamine, and cyanine. Researchers have made remarkable progress in developing activity-based multifunctional fluorescent probes based on hemicyanine skeletons for monitoring vital biomolecules in living systems through the output of fluorescence/photoacoustic signals, and integration of diagnosis and treatment of diseases using chemotherapy or photothermal/photodynamic therapy or combination therapy. These achievements prompted researchers to develop more smart fluorescent probes using a hemicyanine fluorogen as a template. In this review, we begin by describing the brief history of the discovery of hemicyanine dyes, synthetic approaches, and design strategies for activity-based functional fluorescent probes. Then, many selected hemicyanine-based probes that can detect ions, small biomolecules, overexpressed enzymes and diagnostic reagents for diseases are systematically highlighted. Finally, potential drawbacks and the outlook for future investigation and clinical medicine transformation of hemicyanine-based activatable functional probes are also discussed.

Cell staining is a fascinating research area where monitoring and visualizing different cell organelles can be done using fluorescence techniques. However, the design and synthesis of organelle‐targeting fluorophores is still a challenge for several specific organelles. Herein, a platform for synthesizing efficient red‐emitting aggregation‐induced emission luminogens (AIEgens) with donor–acceptor characteristics is reported. The core molecule can be easily functionalized in order to modulate organelle targeting. The three synthesized AIEgens exhibit quantum yields of up to 39.3% and two‐photon absorption cross‐section values of up to 162 GM. The two zwitterionic AIEgens, CDPP‐3SO3 and CDPP‐4SO3, with the sulfonate function group, are successfully utilized for specific one‐photon and two‐photon imaging of the endoplasmic reticulum (ER) in live human cells. Substituting the zwitterionic nature with a singly positive charge group, one‐photon and two‐photon imaging of CDPP‐BzBr shows mitochondrial specificity, indicating the importance of the zwitterionic group for ER‐targeting. Owing to the good in vitro photostability, cell viability, and high efficiency, these red dyes serve as a good potential candidate for specific organelle targeting, as well as illustrate how such a platform can easily aid in the study of structure–property relationships for designing such probes.

The thesis describes the development of a novel reversible H2O2 fluorescent probe comprising the ternary complex of europium(III), tetracycline and hydrogen peroxide; and its application in the assays of H2O2, of H2O2 producing oxidases and their substrates, and of H2O2 consuming catalase and its inhibitors. The probe is applied in steady-state intensity-based, time-resolved �gated�, or lifetime-based detection modes both for microplate fluorescence measurement and imaging. The fluorescent probe�s advantages include the reversibility of the EuTc-HP system, the possibility of a kinetic real-time detection of the production and the consumption of H2O2, and the system works best at pH 6.9 - 7.0. It also exhibits the typical spectral characteristics of a ligand-to-europium energy transfer system which include a Stokes shift of ~210 nm, line-like emission, excitation at 380-420 nm (e.g. by the 405-nm blue diode laser), and a µs decay time (~60 µs) facilitating time-resolved fluorometry and imaging. For the probe study, Chapter 1 gives a overview of the state of art of the H2O2 measurements. In the following first part of Chapter 2 is presented the characterization of the fluorescent EuTc-HP probe for its absorbance, circular dichroism and fluorescence spectra, fluorescence lifetime and decay profile, optimal pH and stability, and the influence of temperature, buffers, quenchers and interferents. In the last part of Chapter 6, the peculiar molar ratio of the EuTc-HP probe is further investigated for its possible structure and a com-binatorial approach for discovery of new lanthanide probes is preliminarily proceeded as well. Different assays have been developed for H2O2, glucose, glucose oxidase and catalase, as examples for the detection of enzyme substrates and enzymes. Furthermore, different fluorometric schemes have been applied, such as the steady-state intensity-based detection (Chapter 5, catalase), the time-resolved gated detections (Chapter 3, glucose), the rapid lifetime determination method (Chapter 2, H2O2, novel on microplate) and the time-correlated single photon counting method of the lifetime-based detection (Chapter 2, H2O2), for both microplates and cuvettes, compatible with high-throughput screening. The µs range lifetime of the EuTc-HP probe has greatly facilitated fluorescence imaging, a means for visualization and mapping of the analyte with multiple chemical information. Four schemes of imaging, those are the conventional fluorescence intensity imaging (FII), the time-resolved ("gated") imaging (TRI), the phase delay ratioing imaging (PDI), and the rapid lifetime determination imaging (RLI), have been tested for the quantitative analysis. Hydrogen peroxide, glucose, and glucose oxidase have been determined by the fluorescent imaging system, with Chapter 4 highlighting the fluorescence imaging of glucose oxidase. There are possible further applications in perspective for the EuTc-HP probe. Chapter 6 summarizes the initial attempts, such as glucose oxidase based ELISA, the coupled catalase/glucose oxidase system as a platform for screening, and the construction of microplate arrays and sensors.

A colorimetric and turn-on NIR fluorescent probe based on xanthene system for sensitive detection of thiophenol and its application in bioimaging

The 2008 Nobel Prize in chemistry was awarded to three distinguished scientists who discovered and developed the green fluorescent protein (GFP), which has proved to be a powerful and versatile fluorescent probe for biological and biomedical researches [1]. Highperformance fluorescent biological probes are expected to possess several important properties including excellent water-dispersibility, high photoluminescent quantum yield (PLQY), robust photostability, and favorable biocompatibility. A consensus has been reached that the GFP and organic dyes, recognized as the well-established fluorescent probes, are to some extent inappropriate for long-time in bioimaging because of their photobleaching property (Figure 1). In this respect, II-VI QDs have attracted intensive attentions due to their unique optical and electronic properties (e.g. size-tunable emission, broad photoexcitation, narrow and symmetrical emission spectra, strong fluorescence, and robust photostability). Indeed, the QDs are currently considered as most promising fluorescent probes, which open new opportunities for real-time and long-term monitoring and imaging both in vitro and in vivo [2].

Small molecule fluorochromes (synonyms: biosensors, chemosensors, fluorescent probes, vital stains) are widely used to investigate the structure, composition, physicochemical properties and biological functions of living cells, tissues and organisms. Selective entry and accumulation within particular cells and cellular structures are key processes for achieving these diverse objectives. Despite the complexities, probes routinely are applied using standard protocols, often without experimenter awareness of what factors that control accumulation and localization. The mechanisms of many such selective accumulations, however, now are known. Moreover, the influence of physicochemical properties of probes on their uptake and localization often can be defined numerically, hence predicted, using quantitative structure activity relations (QSAR) models with its required numerical structure parameters (or “descriptors”). The state of the art of this approach is described. Available QSAR models are summarized for uptake into cells and localization in the cytosol, endoplasmic reticulum, generic biomembranes, Golgi apparatus, lipid droplets, lysosomes/endosomes, mitochondria, eukaryotic nuclei (histones and DNA), plasma membrane, and ribosomal RNA (cytoplasmic and nucleolar). Integration of such core models to both aid understanding and troubleshooting of current fluorescent probes and to assist the design of novel probes is outlined and illustrated using case examples. Limitations and generic problems arising with this approach and comments on application of such approaches to xenobiotics other than probes, e.g., drugs and herbicides, together with a brief note about an alternative approach to prediction, are given.

COPII proteins are essential for exporting most cargo molecules from the endoplasmic reticulum. The membrane-facing surface of the COPII proteins (especially SEC23-SEC24) interacts directly or indirectly with the cargo molecules destined for exit. As we characterized the SEC23A mutations at the SEC31 binding site identified from patients with cranio-lenticulo-sutural dysplasia, we discovered that the SEC23-SEC31 interface can also influence cargo selection. Remarkably, M702V SEC23A does not compromise COPII assembly, vesicle size, and packaging of cargo molecules into COPII vesicles that we have tested but induces accumulation of procollagen in the endoplasmic reticulum when expressed in normal fibroblasts. We observed that M702V SEC23A activates SAR1B GTPase more than wild-type SEC23A when SEC13-SEC31 is present, indicating that M702V SEC23A causes premature dissociation of COPII from the membrane. Our results indicate that a longer stay of COPII proteins on the membrane is required to cargo procollagen than other molecules and suggest that the SEC23-SEC31 interface plays a critical role in capturing various cargo molecules.