Selective detection of peroxynitrite using an isatin receptor and a naphthalimide fluorophore

Peroxynitrite is a reactive oxygen and nitrogen species that participates in various biological reactions. Therefore, it is important to readily detect and track peroxynitrite in biological systems. Here, a novel turn-on probe encapsulated in PEG DSPE-PEG/HN-I was used to fluorescently detect ONOO (cid:2) rapidly. The encapsulation of HN-I using DSPE-PEG2000 optimizes the sensing performances of the naphthalimide probe and avoids ACQ. Using DSPE-PEG/HN-I to detect changes in the levels of exogenous ONOO (cid:2) in HepG2 cells and endogenous ONOO (cid:2) induced by LPS in RAW 267.4 cells was demonstrated.

Peroxynitrite (ONOO À ) is a short-lived bioactive agent, belonging to the category of reactive oxygen and nitrogen species (ROS/ RNS). Peroxynitrite is an oxidant that generates free radicals, and acts as a Lewis base in living systems. 1 Upon exposure to peroxynitrite, biochemical cycles can be promoted while cellular function and viability may be adversely affected, depending on the concentrations of peroxynitrite. 2 Peroxynitrite at low concentrations can contribute to programmed cell death while higher concentrations of peroxynitrite promote a disruption of cellular energy production resulting in necrotic cell death. [3][4][5] Peroxynitrite is involved in mediating numerous reactions, including the promotion of mitochondrial dysfunction, the regulation of cellular signalling pathways, the breaking of cellular redox status, and the induction of pain response under inflammatory conditions. 6-10 While, Hooper and Padalko have reported that peroxynitrite can mediate the immune response to virus infection. 11,12 However, due to a lack of rapid measurement tools suitable to detect peroxynitrite and quantify its concentrations in vivo, most of the biological reactions peroxynitrite participates in have only been investigated in vitro. 2 As such, in order to further explore the role of peroxynitrite in disease pathogenesis, it is necessary to investigate and develop effective methods for peroxynitrite detection.
Fluorescent probes can detect analytes based on changes in spectroscopic properties promoted by targeted reactions. [13][14][15] In general, a fluorescent probe consists of three units: a fluorophore that produces spectroscopic signals; a receptor that is able to react with a specific analyte; a linker that is suitable for connecting the fluorophore with the receptor. 13,16,17 According to the reaction between the receptor and the targeted analyte, a fluorescent probe can exhibit specified fluorescence responsiveness to its targeted analyte. 18 In addition, turn-on fluorescent probes can exhibit enhanced monitoring capabilities against dark backgrounds, which results in reduced background interference. 18 Aryl boronate groups and a-ketoamide groups have been widely applied in the design of peroxynitrite targeting probes due to their good sensitivity and rapid response. 19 However, these receptors can also react with other ROS species, such as hydrogen peroxide and hypochlorite. [20][21][22][23][24] Significantly, isatin exhibits a sensitive, selective and rapid response to peroxynitrite. [25][26][27][28] Naphthalimide fluorophores are on the whole cell permeable and exhibit good photostabilities. 29 Due to the electronwithdrawing ability of its imide core, naphthalimide exhibits strong intramolecular charge transfer (ICT) in its solution state when the C-4 site of naphthalimide is modified by electron donors. [30][31][32] These properties make naphthalimide an excellent fluorophore candidate for the construction of chemiluminescent probes for analyte detection in biological systems. 30,[33][34][35][36][37] However, one of the main drawbacks of naphthalimide-based systems is low solubility. Poor solubility and the planar structure make naphthalimide undergo p-p stacking easily, which results in aggregation-caused quenching (ACQ). 29,38,39 In most cases, structural modification is used to solve solubility problems and overcome the ACQ of naphthalimidebased fluorophores. [40][41][42] However, this approach requires additional time-consuming synthesis. Therefore, we decided to explore a much simpler approach and use PEG encapsulation to improve solubility. 43,44 Polyethylene glycol (PEG) is widely used in the field of drug delivery as an excipient to improve molecular solubility. [45][46][47] In addition, DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly (ethylene glycol)) can reduce aggregation, improve the stability of nanoparticles and increase the circulation times of molecules in vivo. [47][48][49] Here, we selected 4-hydroxy-1,8-naphthalimide (HN) as the fluorophore and a 1-methylindoline-2,3-dione moiety as the receptor to construct a novel fluorescent probe HN-I for the detection of ONOO À . According to the specific redox reaction between ONOO À and 1-methylindoline-2,3-dione, the fluorophore HN is released (Scheme 1). 32,50 In order to ensure the improvement of water solubility and the reduction of ACQ effect, we used DSPE-PEG2000 to encapsulate the fluorescent probe HN-I (Scheme 1).
We were inspired by the group of Bruemmer who found that isatin-based groups could detect ONOO À with high selectivities without any interference from other ROS/RNS. 25 As such, we prepared HN where the C-5 position of a 1-methylindoline-2,3dione moiety was linked to a naphthalimide (Fig. S1, ESI †). Upon the addition of ONOO À , the fluorescence of HN-I can be turned on ( Fig. S2 and S3, ESI †). In addition, by using DSPE-PEG2000, the aqueous solubility of DSPE-PEG/HN-I can be improved. As we mentioned above, the improvement in solubility of HN-I when using DSPE-PEG2000 decreases the p-p stacking of HN-I, which reduces ACQ and contributes to the recovery of the fluorescence ( Fig. S4 and S5, ESI †). In other words, the sensing performances of DSPE-PEG/HN-I were optimized by the encapsulation of DSPE-PEG2000 ( Fig. S4 and S5, ESI † and Fig. 1a).
First, the absorption spectra of DSPE-PEG/HN-I were explored (Fig. 1a). Initially, the ICT effect of the probe was inhibited so that the probe was nonfluorescent. However, on the addition of ONOO À , the naphthalimide fluorophore of DSPE-PEG/HN was released, and the recovery of the ICT effect resulted in fluorescence enhancement. As such, when the solution was excited at 448 nm, the fluorescence emission of DSPE-PEG/HN at 563 nm was enhanced. The fluorescence intensity of DSPE-PEG/HN-I increased with increasing concentrations of ONOO À from 0 mM to 30 mM (Fig. 1b). For ONOO À from 0-15 mM, the fluorescence response was linear (Fig. 1c). The limit of the detection of DSPE-PEG/HN-I for ONOO À was calculated to be 22 nM (Fig. 1c). The reaction rate between DSPE-PEG/HN-I and ONOO À was rapid. From Fig. 1f (Fig. S7, ESI †). DSPE-PEG/HN-I was evaluated with and without SIN-1 (a typical ONOO À exogenous donor). As expected, without SIN-1, there was no fluorescence observed (Fig. 2a). However, with a concentration increase of SIN-1, the fluorescence intensity enhanced 5-fold (Fig. 2a-c). Then, DSPE-PEG/HN-I was also shown to detect exogenous ONOO À in SIN-1treated human cervical cancer cell line (HeLa). However, after pretreatment of cells with N-acetylcysteine (NAC, an ONOO À scavenger), it attenuated the increase in the fluorescence of DSPE-PEG/HN-I induced by SIN-1 (Fig. S8, ESI †). Furthermore, the possibility of using DSPE-PEG/HN-I for endogenous ONOO À detection was also investigated in a macrophage cell line (RAW 264.7). RAW 264.7 cells were incubated with lipopolysaccharide (LPS) which can promote inflammatory response and upregulate the concentration of ONOO À . [53][54][55][56] As shown in Fig. 2D, the fluorescence intensity of DSPE-PEG/HN-I exhibited a 1.5-fold increase for LPS-loaded RAW 264.7 cells. After RAW 264.7 cells were incubated with both LPS and NAC, weak fluorescence was observed. All these results indicated that DSPE-PEG/HN-I can be applied for the detection of both exogenous and endogenous ONOO À .
In summary, a novel turn-on probe was designed for the highly selective detection of ONOO À . To avoid the ACQ effect, a disadvantage of naphthalimide fluorophores, DSPE-PEG2000 was used to encapsulate HN-I to improve the sensing performances.

Conflicts of interest
There are no conflicts to declare.