| Peroxynitrite (ONOO-), formed in vivo from a diffusion-controlled reaction between nitric oxide (·NO) and superoxide (O2(·-)), is a highly reactive species implicated in the pathogenesis of numerous diseases, including inflammatory processes, ischemic reperfusion injury, multiple sclerosis, and neurodegenerative disorders. This peroxide easily crosses biological membranes, and despite a relatively short half-life (1 s) under physiological conditions, it can interact with various biological molecules, such as DNA, lipids, proteins, thiols, and metalloenzymes, which may result in serious damage to living cells. In order to fully understand the biological effects of ONOO-, a major focus of research is to exploit highly selective and sensitive methods to monitor the concentration changes of ONOO- in biosystems. Among these methods, spectrofluorimetry has become a powerful tool due to its high sensitivity and simplicity in data collection in imaging techniques.Polymeric micelles, self-assembled nanoparticles from amphiphilic block copolymers, have many advantages in constructing multifunctional fluorescent nanoprobe. Micelle structures can incorporate different hydrophobic dyes in a single nanoparticle without complicated synthetic and separated procedures, so they offer a fascinating strategy for constructing ratiometric probes. The nanoparticles can be excited at the near-infrared (NIR) range by encapsulating NIR dyes, which leads to minimize photo damage and cell autofluorescence, and also have stability in aqueous media and surface functionalization to act as biological probes. Moreover, such nanoparticles exhibit higher brightness and better photostability than small-molecule dyes, owing to large numbers of chromophores per particle as well as the protective matrix. As ONOO- is a highly permeable ROS, another appealing feature of using micelle structure is that the nanomatrix can improve selectivity of the embedded molecular probe toward ONOO- over other biologically relevant substances. Such induced selectivity can be achieved by a delicate complementarity of properties between the nanomatrix and the embedded molecular probe.Based on the above, we attempt to develop a polymeric micelle-based nanoprobe that is highly specific for ONOO-. A hybrid polypeptide-polymer-dye nanoprobe for ONOO- has been designed and synthesized. The nanoarchitectures are composed of two key components: 1) a polymeric micelle that has a hydrophobic core made of poly (D, L-lactic acid) (PLA) and a hydrophilic shell consisting of polyethylene glycol (PEG) conjugated to CPPs; 2) benzylselenide- tricarbocyanine (BzSe-Cy) as ONOO- indicator dye and isopropylrhodamine B (IRhB) as reference dye that are simultaneously encapsulated in the hydrophobic core of micelle for ratiometric ONOO- measurement. Because of its high diffusibility, ONOO- can rapidly diffuse into the core of nanomatrix that blocks the entry of interferential bioanalytes and react with BzSe-Cy, resulting dramatic fluorescence decrease, whereas the fluorescence of IRhB does not change, leading to the selectively ratiometric detection of ONOO-. However, other ROS such as O2-· and ClO- with relatively low diffusibility are hard to permeate the nanomatrix. ·OH has the shortest lifetime (nanoseconds or shorter) among all the ROS, which has no enough time to penetrate into the nanomartrix. The nanomatrix resistance to HRP interference is due to the large size of the HRP, being too large to penetrate into the nanomatrix. It is quite evident that the nanomatrix is beneficial to improve the specificity of BzSe-Cy for ONOO-. In this paper, the spectral characteristics of the nanoprobe were investigated under a simulated physiological environment, and the selectivity of free BzSe-Cy and the nanoprobe toward ONOO- were evaluated, respectively. Finally, the nanoprobe was applied to imaging of ONOO- in living macrophages (RAW 264.7), normal human liver cells (HL-7702) and human hepatoma cells (HepG2).
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