Polymeric Micelle-based Nanoprobes For Ultrasensitive Detection And Imaging Of Reactive Oxygen Species In Living Cells

Reactive oxygen species (ROS) are generated continuously as a normal productduring oxidative metabolism. ROS are a class of active oxygen radicals (such as OH,O2, NO, ROO) and nonradical oxygen-containing species (such as H2O2). Numerousstudies have shown that ROS play very important roles in a wide range of physiologicaland pathological processes. ROS are endogenously generated from oxygen in themitochondrial respiration pathway. However, under stimulated by xenobiotics,infectious agents, and UV light, ROS can also be exogenously produced. In livingsystems, ROS are produced and cleared constantly so as to maintain at a very low level.Hydrogen peroxide (H2O2), one of the important ROS, acts as a signaling molecule in awide variety of signaling transduction processes, an oxidative stress marker in aging anddisease, and a defense agent in response to pathogen invasion. It is believed that H2O2isgenerated by activation of NADPH oxidase complexes during cellular stimulation withcytokines, peptide growth factors, and neurotransmitters. H2O2is involved in reversibleoxidation of proteins that ultimately regulate cellular processes ranging from proteinphosphorylation to gene expression. However, excessive H2O2production has beenimplicated with various diseases including neurodegenerative diseases, diabetes andcancer. Meanwhile nitric oxide (NO), as a prototypical reactive nitrogen species (RNS),is essential to many biological functions. NO is associated with immune response andcyclic nucleotide biosynthesis in organisms. With regard to the research on thegeneration, metabolism, physiological and pathological effects and detection of H2O2and NO has attracted widespread attention. In order to fully understand the biologicaleffects of H2O2and NO, a major focus of research is to exploit highly selective and sensitive methods to monitor the concentration changes of H2O2and NO in biosystems,which makes sense for the further in-depth study on pathological mechanism and thedevelopment of human health.Polymeric micelles have brought new opportunities for designing new fluorescentnanoprobes. The polymeric micelles are formed based on the supramolecularself–assembly of the amphiphilic block copolymers. Micelles with core-shell structurehave stability in aqueous media, good biocompatibility and low cytotoxicity, whichhave been widely applied in biological systems. Compared with other nanomaterials,micelles have a series of advantages: Firstly, they can act as excellent nanocarriers.Micelle structures can incorporate different hydrophobic drugs and fluorescent probes ina single nanoparticle to improve the efficiency of therapy and enhance the selectivity ofdetection; Secondly, they can exist stably in physiological environment because of thelow critical micellar concentration; Thirdly, due to the diversity of the core-shellstructure, it is free to choose appropriate micelle nanocarrier; Fourthly, they have goodbiocompatibility, low cytotoxicity and also facilitate the metabolism of the organism.Lastly, their surface functionalization by a variety of targeted biomolecules is especiallyapplicable for drug delivery system, which shows good development prospect.Based on the above, we attempt to develop polymeric micelle-based nanoprobes fordetecting H2O2and NO with high sensitivity and selectivity. We carried out two aspectsof the investigation:1. We developed an ultrasensitive polymeric micelle system for monitoring H2O2inliving cells and in vivo using a signal amplification strategy. First, a series of triblockamphiphilic copolymers were designed and synthesized with the hydrophilicpoly(ethylene glycol)(PEG) segment and two hydrophobic segments by atom–transferradical polymerization. The boronate–based group can act as one hydrophobiccomponent (M1) and recognition unit, which responds to H2O2specifically. AH2O2–insensitive dye, rhodamine B isothiocyanate, was employed as anotherhydrophobic component (M2) and luminescence unit. The polymeric micelles were formed based on the supramolecular self–assembly of the block copolymers. In thehydrophobic core of micelles, the fluorophores were in close proximity and thefluorescence can be quenched by homo fluorescence resonance energy transfer(homoFRET). In the presence of a very small amount of H2O2, the H2O2–responsivegroup could be oxidized and become hydrophilic, enabling the disassembly of themicelles. Significantly, one H2O2molecule could light thousands of fluorophores,thereby efficiently amplifying the fluorescence and enormously improving thesensitivity. Based on this, the detection limit of the micelle-based nanoprobe is as low as12pM, which is the most sensitive for H2O2detection to date. Moreover, the nanoprobeexhibits good biocompatibility, good stability and excellent specificity. Themicelle–based nanoprobe could signal the H2O2level without external stimuli in cancercells and normal cells, as well as xenograft tumor models.2. A polymeric micelle-based nanoprobe for detection of NO with high sensitivityand selectivity has been designed and synthetized. To begin with, triblock amphiphiliccopolymers were synthesized with the hydrophilic poly(ethylene glycol)(PEG) segmentand two hydrophobic segments by atom–transfer radical polymerization. Theo-phenylenediamine–based group can act as one hydrophobic component andrecognition unit, which responds to NO specifically. Rhodamine B isothiocyanate, wasemployed as another hydrophobic component and luminescence unit. The polymericmicelles were formed based on the supramolecular self–assembly of the blockcopolymers. In the hydrophobic core of micelles, the fluorophores can be quenched byhomo fluorescence resonance energy transfer (homoFRET). When encountered withNO, o-phenylenediamine groups are reactive towards NO, producing benzotriazolemoieties which gradually hydrolyze, and enabling the disassembly of the micelles.Moreover, the nanoprobe exhibits good biocompatibility, good stability and excellentspecificity.