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.

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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 Ca and PO 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.

This study reports a strategy to temporarily mask arginine residues within antimicrobial peptides (AMPs) with methoxy poly(ethylene glycol) (mPEG). PEGylation protects AMPs from serum proteases, and can be released at a pharmaceutically-relevant rate. Fully active and unmodified (i.e., native) AMPs are released with time.

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.