Ratiometric Fluorescent Probes for the Detection of Reactive Oxygen Species.

Reactive oxygen species (ROS) are endogenously produced oxidants with various functions ranging from host defense to signaling. These transient species can cause severe damage to the body when their production is dysregulated or when environmental factors elevate their concentrations. To study their effects and prevent oxidative harm, tools capable of monitoring ROS in cells and tissue in a sensitive and selective fashion are required. In this Review, a summary of existing ratiometric probes is provided, together with a critical discussion of selected examples.

Development of a Modular Ratiometric Fluorescent Probe for the Detection of Extracellular Superoxide.

Extracellular detection of endogeneous analytes (e.g., superoxide) can provide important insights into mechanisms of homeostasis and diseases, such as tumorigenesis. A ratiometric probe with a fluorescent reference and an analyte-specific switch-on dye was developed. Detection of ROS in the extracellular milieu was ensured by connecting the two fluorophores with a modular peptide-nucleic-acid-based linker. The ROS-sensing ability was assessed and validated in cell-free assays and in cell culture.

Quantitative measurement of lymphatic function in mice by noninvasive near-infrared imaging of a peripheral vein.

Optical imaging methods have been developed to measure lymphatic function in skin; however, the lymphatic system of many organs is not accessible to this technology. Since lymphatic transport of macromolecules from any organ proceeds to the blood circulation, we aimed to develop a method that can measure lymphatic function by monitoring the fluorescence in a superficial vein of an interstitially injected tracer. We selected a 40-kDa PEGylated near-infrared dye conjugate, as it showed lymphatic system-specific uptake and extended circulation in blood. Lymphatic transport to blood from subcutaneous tissue required a transit time before signal enhancement was seen in blood followed by a steady rise in signal over time. Increased lymphatic transport was apparent in awake mice compared with those under continuous anesthesia. The methods were validated in K14-VEGFR-3-Fc and K14-VEGF-C transgenic mice with loss and gain of lymphatic function, respectively. Reduced lymphatic transport to blood was also found in aged mice. The technique was also able to measure lymphatic transport from the peritoneal cavity, a location not suitable for optical imaging. The method is a promising, simple approach for assessment of lymphatic function and for monitoring of therapeutic regimens in mouse models of disease and may have potential for clinical translation.

Characterization of Calcium Phosphate Nanoparticles Based on a PEGylated Chelator for Gene Delivery.

Calcium phosphate (CaP) nanoparticles are promising gene delivery carriers due to their bioresorbability, ease of preparation, high gene loading efficacy, and endosomal escape properties. However, the rapid aggregation of the particles needs to be addressed in order to have potential in vivo. In addition, there is a need to better understand the relationship between CaP nanoparticle properties and their interactions with cells. Here, a new synthesis route involving click chemistry was developed to prepare the PEGylated chelator PEG-inositol 1,3,4,5,6-pentakisphosphate (PEG-IP5) that can coat and stabilize CaP nanoparticles. Two methods (1 and 2) differing on the time of addition of the PEGylated chelator were employed to produce stabilized particles. Method 1 yielded amorphous aggregated spheres with a particle size of about 200 nm, whereas method 2 yielded 40 nm amorphous loose aggregates of clusters, which were quickly turned into needle bundle-like crystals of about 80 nm in a few hours. Nanoparticles prepared by method 1 were internalized with significantly higher efficiency in HepG2 cells than those prepared by method 2, and the uptake was dramatically influenced by the reaction time of Ca2+ and PO43- and sedimentation of the particles. Interestingly, morphological transformations were observed for both types of particles after different storage times, but this barely influenced their in vitro cellular uptake. The transfection efficiency of the particles prepared by method 1 was significantly higher, and none of the formulations tested showed signs of cytotoxicity. This study provides a better understanding of the properties (e.g., size, morphology, and crystallinity) of PEGylated CaP nanoparticles and how these influence the particles' in vitro uptake and transfection efficiency.