A Dual-Functional Fluorescence Probe for Simultaneous in Vivo Imaging of Aβ Aggregates and Hydrogen Peroxide in the Brain of Mice with Alzheimer’s Disease

Open AccessCCS ChemistryRESEARCH ARTICLES22 Jan 2025A Dual-Functional Fluorescence Probe for Simultaneous in Vivo Imaging of Aβ Aggregates and Hydrogen Peroxide in the Brain of Mice with Alzheimer's Disease Jiajia Lv, Hongyu Li, Jie Gao, Nan Dong, Wen Shi, Huimin Ma and Zeli Yuan Jiajia Lv College of Pharmacy, Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou 563003 Guizhou International Scientific and Technological Cooperation Base for Medical Photo-Theranostics Technology and Innovative Drug Development, Zunyi, Guizhou 563003 , Hongyu Li College of Pharmacy, Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou 563003 Guizhou International Scientific and Technological Cooperation Base for Medical Photo-Theranostics Technology and Innovative Drug Development, Zunyi, Guizhou 563003 , Jie Gao College of Pharmacy, Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou 563003 Guizhou International Scientific and Technological Cooperation Base for Medical Photo-Theranostics Technology and Innovative Drug Development, Zunyi, Guizhou 563003 , Nan Dong *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] School of Chemistry and Chemical Engineering, Guizhou University, Guiyang, Guizhou 550025 , Wen Shi Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Huimin Ma *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 and Zeli Yuan *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] College of Pharmacy, Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou 563003 Guizhou International Scientific and Technological Cooperation Base for Medical Photo-Theranostics Technology and Innovative Drug Development, Zunyi, Guizhou 563003 https://doi.org/10.31635/ccschem.024.202404927 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookXLinked InEmail Amyloid-β (Aβ) plaques and reactive oxygen species (ROS) are two important and highly correlated pathological markers of Alzheimer's disease (AD). Therefore, the simultaneous reliable detection of the two markers is essential for elucidating their pathological roles in AD. In this study, a dual-functional fluorescence probe (NPBZ) was developed for simultaneous in vivo imaging of Aβ plaques and hydrogen peroxide (H2O2). NPBZ, composed of a rotatable D–π–A structural fluorophore and a H2O2-responsive p-pinacolborylbenzyl group, showed a large fluorescence increase at 708 nm upon interaction with Aβ aggregates; however, after reacting with H2O2, the emission of NPBZ was blueshifted to 618 nm. The large spectral shift (90 nm) enabled the independent recognition of H2O2 and Aβ aggregates in two channels, thus achieving dual-functional detection. Moreover, NPBZ can target mitochondria and monitor the in situ production of H2O2 induced by Aβ aggregates in neuronal cells. Most notably, NPBZ has the ability to penetrate the blood-brain barrier and to simultaneously image Aβ plaques and H2O2 in the brain of AD mice of different ages via the two independent channels in vivo. The dual-functional detection performance makes NPBZ useful in the diagnostic study of AD, which may provide direct elucidations for the roles of Aβ plaques and H2O2 in the pathogenesis of AD. Download figure Download PowerPoint Introduction Alzheimer's disease (AD) is a highly prevalent senile neurodegenerative disease, with patients' memory loss and cognitive impairment as the main clinical manifestations, which would seriously threaten the health of the elderly. Especially with the acceleration of the global aging process, the incidence of the disease has also increased, and it has become a major global public health problem.1–3 Despite a century of global study on AD, there is still a lack of sufficient understanding of its pathogenesis and effective treatment for the disease. The existing treatments can only temporarily relieve the symptoms rather than prevent or reverse the neurodegenerative process.4–7 Studies have shown that some pathological changes, for example, β-amyloid protein deposition (Aβ plaques), Tau protein aggregation, reactive oxygen species (ROS) and metal level increase, have already appeared in the AD brain before the onset of clinical symptoms.8–15 Therefore, the development of reliable monitoring methods for these pathological changes may offer an opportunity for early AD diagnosis and treatment. Fluorescence imaging with tailored probes has become an important method for the study of AD diagnosis because of its high sensitivity, temporal-spatial resolution, and noninvasiveness.16–23 Nevertheless, the existing fluorescence probes for AD imaging are mainly used to monitor Aβ aggregates.24–28 Compared with the traditional single-function probes, dual-function probes for two AD pathological changes offer advantages such as more reliable results, and simplified analytical procedures.29–31 Oxidative stress in the AD brain is another crucial pathological marker of AD that is highly correlated with Aβ proteins.32,33 There has been evidence that Aβ proteins can promote the generation of ROS, such as hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and superoxide anion (O2•−), by coordinating with the elevated transition metals (e.g., Fe and Cu) in AD brain; the overproduced ROS in turn accelerates the aggregation of Aβ proteins.34–39 Unfortunately, there have been few reports on fluorescence probes for the dual-function detection of ROS and Aβ aggregates, and to date, the probe capable of simultaneous in vivo imaging of Aβ aggregates and ROS within the AD brain is still lacking. Herein, we developed a dual-functional p-pinacolborylbenzyl fluorescence probe (NPBZ) that meets the above requirement. As shown in Scheme 1, the designed probe NPBZ comprised a rotatable D–π–A structural fluorophore and a H2O2-responsive p-pinacolborylbenzyl group. Upon the addition of Aβ aggregates, NPBZ exhibited a large fluorescence enhancement at 708 nm; however, after reacting with H2O2, the emission of NPBZ blueshifted to 618 nm. The large spectral shift (90 nm) enabled an independent assay of H2O2 and Aβ aggregated at different analytical wavelengths, thereby achieving a dual-functional detection. NPBZ was also mitochondria-targetable and could be used to monitor the in situ generation of H2O2 in Aβ aggregate-treated neuronal cells. Most importantly, the NPBZ is capable of simultaneous in vivo imaging of Aβ plaques and H2O2 in the brain of the AD mice via two independent channels, which might provide direct implications for these two crucial and correlated pathological markers in AD progression. Scheme 1 | Design and response mechanism of NPBZ. Download figure Download PowerPoint Experimental Methods Synthesis and characterization of NPBZ (E)-N,N-Dimethyl-4-(5-(2-(pyridin-4-yl)vinyl)thiophen-2-yl) aniline (NPT), as shown in Supporting Information Scheme S1, was prepared according to a known method.40 Then 153 mg of NPT (0.5 mmol), 149 mg of 4-bromomethylphenylboronic acid pinacol ester (0.5 mmol), and 10 mL of acetonitrile were added and placed in a two-neck round bottom flask. The above mixture was refluxed for 8 h under a nitrogen atmosphere and the reaction was monitored by thin-layer chromatography. Next, the solvent was removed under reduced pressure. The residues were purified by silica gel column chromatography with the eluent of CH2Cl2/CH3OH (v/v, 10:1), and further washed with diethyl ether, affording NPBZ as a dark red solid (230 mg, 76% yield). 1H NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6, δ): 8.92–8.89 (d, J = 6.4 Hz, 2H), 8.16–8.12 (d, J = 6.3 Hz, 2H), 7.74–7.70 (d, J = 7.6 Hz, 2H), 7.57–7.53 (d, J = 8.2 Hz, 2H), 7.51–7.45 (m, 3H), 7.42–7.39 (d, J = 4.2 Hz, 1H), 7.08–7.00 (d, J = 15.8 Hz, 2H), 6.78–6.74 (d, J = 8.2 Hz, 2H), 5.71 (s, 2H), 2.96 (s, 6H), and 1.27 (s, 12H). 13C NMR (101 MHz, DMSO-d6, δ): 153.6, 151.0, 149.8, 144.3, 137.5, 136.8, 135.8, 135.3, 130.9, 127.9, 127.2, 123.7, 123.0, 120.8, 120.5, 112.7, 84.3, 62.4, 56.0, and 25.1. high-resolution electrospray ionization mass spectrometry (HR-ESI-MS): m/z calcd for NPBZ (C32H36BN2O2S+, [M]+), 523.2585; found, 523.2585. Cell culture and fluorescence imaging of H2O2 in living cells The rat pheochromocytoma cells (PC12) were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2 and 95% air atmosphere. After culturing to 70% confluence, the cells were harvested with trypsin-ethylenediaminetetraacetic acid (EDTA) solution to prepare a cell suspension and reseeded in confocal glass bottom dishes to adhere for 24 h for the following experiments. The PC12 cells seeded in culture flasks at 5 × 104 cells/mL density were divided into six groups (n = 3). (1) without any treatment; (2) 10 μM NPBZ for 30 min; (3) 1 mM N-acetylcysteine (NAC) for 1 h, followed by 10 μM NPBZ for 30 min; (4) 100 μM H2O2 for 30 min, followed by 10 μM NPBZ for 30 min; (5) 5 μg/mL phorbol-12-myristate-13-acetate (PMA) for 1 h, followed by 10 μM NPBZ for 30 min; (6) 5 μg/mL PMA for 1 h, 1 mM NAC for 1 h, finally 10 μM NPBZ for 30 min. After that, the cells were washed three times with phosphate-buffered saline (PBS), then subjected to imaging with a 405 nm excitation, and collected the fluorescence data were collected from 420 to 620 nm using a Stellaris 5 WLL confocal laser scanning microscope (Leica, Germany). Imaging of H2O2 generated following Aβ aggregate-treatment in PC12 cells The PC12 cells seeded in confocal glass bottom dishes (5 × 104 cells/dish, n = 3) were incubated with 30 μM Aβ42 aggregates for 12, 24, 48, or 72 h, and then stained with 10 μM NPBZ for 30 min. After washing, the cells were subjected to fluorescence imaging at 405 nm excitation for the H2O2 channel (420–620 nm) and 520 nm excitation for the Aβ channel (670–760 nm) using fluorescence microscopy (Leica, Germany). In vivo blood-brain barrier (BBB) permeability study Wild-type (WT) C57BL/6 mice were intravenously injected with 100 μL NPBZ (10 mg/kg in 5% DMSO, 2% Tween-80, and 93% stroke-physiological saline solution) and euthanized (euthanized with pentobarbital sodium at a dose of 150 mg/Kg) 15 min after injection, followed by quick dissection of the brain organs. The harvested brains were rinsed with saline and blotted dry with filter paper, then 1.0 g of each brain tissue was weighed precisely and homogenized with precooled saline to obtain brain homogenate, followed by adding 1 mL of methanol. After vortexing for 5 min, the above mixture was subjected to a 10-min centrifugation (13,000 g, 4 °C). After being transferred to an Eppendorf tube, the supernatant was evaporated under a gentle nitrogen flow. The residue was resolubilized with 70 μL methanol, and 3 μL of the resulting solution was injected into the liquid Chromatography with tandem mass spectrometry (LC-MS/MS) system (Thermo Fisher Scientific, USA) for analysis. In vivo and ex vivo imaging of AD and WT mice brain with NPBZ The scalp hairs of 3/6/14 month-old APP/PS1 double transgenic C57BL6 mice model for AD (male, n = 3) and WT C57BL6 mice (male, n = 3) were removed and the mice were intravenously injected with NPBZ (10 mg/kg). In vivo fluorescence imaging was made at the indicated postinjection time points on a Nightowl II LB 983 imaging system (Berthold Technologies, Bad Wildbad, Germany) equipped with appropriate filter sets (Aβ channel: λex = 520 ± 10 nm and λem = 700 ± 20 nm; H2O2 channel: λex = 420 ± 10 nm and λem = 600 ± 20 nm) and each image was acquired with a 1s exposure time. The mice were anesthetized with isoflurane gas (2.5%) at an oxygen flow rate (1.5 L/min) throughout the imaging process. The acquired imaging data were processed using Living Image software 4.1 (https://living-image.software.informer.com/4.1/). For ex vivo imaging of mouse brains, 3/6/14 month-old AD mice (male, n = 3) and WT mice (male, n = 3) were intravenously injected with NPBZ (10 mg/kg). For the Aβ channel, the mice were euthanized after injection for 15 min; for the H2O2 channel, the mice were euthanized after injection for 120 min. After euthanasia, the mouse brains were collected and ex vivo imaging was conducted, as indicated above. Imaging of Aβ plaques and H2O2 in AD mouse brain slices 3/6/14 month-old AD mice (male, n = 3) and WT mice (male, n = 3) were intravenously injected with NPBZ (10 mg/kg). For Aβ plaque imaging, the mice were euthanized after a 15-minute injection. Then the brains were excised and serially cut into 10 μm slices using a cryosectioner at −20 °C. The brain slices were further stained with 20 μL thioflavin S (ThS) (10 mg/mL), washed with 75% ethanol and then PBS (1 mL × 3). The remaining liquid was removed using dust-free paper, followed by dropwise adding of antifade mounting medium and then covering with a slip. The fluorescence images were then recorded with an Olympus BX43 microscope (Tokyo, Japan). Similarly, for H2O2 imaging, the mice were euthanized after injection with NPBZ for 120 min. The brain slices were then prepared, as indicated above, and used for fluorescence imaging on an Olympus BX43 microscope. Results and Discussion Design and synthesis of NPBZ The designed dual-functional probe NPBZ for Aβ aggregates and H2O2 is presented in Scheme 1. NPBZ shares a similar structural moiety to the gold standard Aβ plaque staining probe thioflavin T (ThT), that is, N,N-dimethylaminophenyl group, which is a commonly used recognition unit that facilitates the binding of the probe to Aβ aggregates.41 A positively charged pyridinium, acting as a strong electron acceptor (A), is connected with the N,N-dimethylaminophenyl-thiophene building block (electron donor, D) to form a rotatable D–π–A structure with active intramolecular charge transfer (ICT).42 Such a structural feature is not only conducive to a near-infrared (NIR) emission and a large Stokes shift but also responsible for the fluorescence response to Aβ aggregates. Upon embedded within the hydrophobic and plugged gaps of Aβ aggregates, the conformational freedom of NPBZ is greatly restricted, thereby suppressing the nonradiative process and resulting in a large fluorescence enhancement at 708 nm; in addition, the hydrophobic microenvironment within Aβ aggregates may also enhance the fluorescence of NPBZ with ICT property. On the other hand, a p-pinacolborylbenzyl group is introduced as the recognition unit for H2O2 due to its unique reactivity with H2O2. After reacting with H2O2, the release of NPT with weaker ICT leads to a significant fluorescence increase at 618 nm. The large spectral shift (90 nm) is beneficial to the independent detection of H2O2 and Aβ aggregates in two channels, thereby achieving the dual-functional assay. The probe and related intermediates were facilely prepared ( Supporting Information Scheme S1), and characterized by NMR spectroscopy and mass spectral analyses ( Supporting Information Figures S1–S6). In addition, the purity of NPBZ was further determined to be >98% by high-performance liquid chromatography (HPLC) assay ( Supporting Information Figure S7). Spectroscopic detection of H2O2 The spectroscopic properties of NPBZ were examined in phosphate buffer (pH = 7.4). As depicted in Figure 1a, NPBZ displayed an absorption peak at approximately 500 nm, which, however, was blueshifted to 410 nm upon the addition of H2O2, consistent with that of NPT; the solution color changed from red to yellow (insert of Figure 1a). The transformation of absorption spectra and color in response to H2O2 strongly implied the release of NPT. The absorption spectra of NPBZ with various concentrations of H2O2 were also tested, showing a gradual decrease at 500 nm and the emergence of a new peak at 410 nm ( Supporting Information Figure S8). For fluorescence analysis, NPBZ itself exhibited weak fluorescence; however, with the addition of H2O2, a large fluorescence increase (26-fold) was found at 618 nm ( Supporting Information Figure S9), which resulted from the release of the highly fluorescent NPT. This fluorescence off-on response of NPBZ is favorable for the sensitive detection of H2O2. Moreover, it is worth noting that the fluorescence response of NPBZ exhibited a linear increase with the H2O2 concentration from 0–70 μM (Figure 1b,c). Based on such a linear relationship, the limit of detection (LOD) was determined to be 0.37 μM H2O2, much lower than the concentration of H2O2 in vivo under pathological conditions (e.g., 10–100 μM at inflammation sites).43 Figure 1 | (a) Absorption spectra of 10 μM NPT (gray), 10 μM NPBZ without (red) and with (blue) H2O2 (100 μM) in PBS/DMSO (7:3 v/v, pH 7.4). (b) Fluorescence spectra of NPBZ (10 μM) with different concentrations of H2O2 (0–120 μM) in PBS/DMSO (7:3 v/v, pH 7.4) for 90 min. (c) The linear relationship of the fluorescence intensity and the H2O2 concentration (0–70 μM); (d) Fluorescence intensity of NPBZ (10 μM) with (red) or without (black) H2O2 (100 μM) at different periods in PBS/DMSO (7:3 v/v, pH 7.4). (e) Fluorescence responses of NPBZ to various substances: 1, blank; 2–14, 100 μM metal ions (Fe2+, Cu2+, Zn2+, Ba2+, Ca2+, K+, Mg2+, Mn2+, Na+, Ni2+, Fe3+, Al3+, Ag+); 15–16, 100 μM anions (SO42−, CO32−); 17–26, 100 μM amino acids (Ala, Arg, Asp, Glu, His, Ile, Leu, Lys, Met, Phe); 27–29, biothiols (1 mM Cys, 100 μM Hcy, 5 mM GSH); 30–34, 100 μM ROS (TBHP, O2•−, OCl–, ONOO–, •OH); 35, 10 μM Aβ42 aggregates; and 36, 100 μM H2O2. λex/em = 410/618 nm. Download figure Download PowerPoint The fluorescence response time of NPBZ (10 μM) with or without H2O2 (100 μM) was then examined. As depicted in Figure 1d, no obvious response was observed without H2O2, indicating good stability of NPBZ; by contrast, with the addition of H2O2, a gradually ascending fluorescence signal at 618 nm was produced, and reached a plateau at around 90 min. In addition, it specifically responded well in the physiological pH range ( Supporting Information Figure S10). The analytical selectivity was studied by comparing the fluorescence response of NPBZ to H2O2 over other common biological interferents, including metal ions, amino acids, biothiols, Aβ aggregates, and ROS. The results showed that no apparent response was caused by these interferents in the H2O2 detection channel (Figure 1e), indicating the high selectivity of NPBZ for H2O2. To better understand the spectral response mechanism, the reaction product NPT was directly prepared from NPBZ and H2O2, and the reaction solution was studied via HPLC and HR-ESI-MS ( Supporting Information Figure S11). NPBZ itself had a retention time of about 2.2 min; after reacting with H2O2, a new peak appeared at about 8.1 min, similar to that of NPT (8.0 min). In the HR-ESI-MS analysis, a new peak at m/z = 307.1273 was found, in accordance with NPT, further demonstrating the release of NPT from NPBZ and H2O2. The above results suggest that NPBZ can act as an efficient tool for the detection of H2O2. Spectroscopic detection of Aβ aggregates The analytical performances of NPBZ for Aβ aggregates were then studied. The Aβ aggregates were prepared from human Aβ 1-42 peptide (Aβ42) based on the known protocol.26 Transmission electron microscopy assay verified the formation of Aβ aggregates ( Supporting Information Figure S12). It was found that the fluorescence of NPBZ itself was rather weak in PBS buffer; however, a large fluorescence enhancement (47-fold) at 708 nm was observed after the addition of Aβ42 aggregates (Figure 2a). To explain the high contrast response of NPBZ to Aβ aggregates, the fluorescence response of NPBZ to environmental polarity and viscosity was examined. As depicted in Supporting Information Figures S13–S14 and Table S1, NPBZ shows a higher fluorescence intensity in low-polarity and high-viscosity environments. In other words, NPBZ may exhibit very low background fluorescence in the high-polarity and low-viscosity aqueous solutions; however, the hydrophobic, low-polarity, and crowded microenvironment after binding to Aβ aggregates would result in a high contrast fluorescence enhancement response of the probe, conductive to increase the sensitivity for Aβ aggregates. Notably, NPBZ showed a rapid response to Aβ aggregates (Figure 2b), taking only about 1 min to reach a plateau (60 min for the commercial Aβ aggregate probe ThT as a comparison).44 In the presence of different amounts of Aβ aggregates, the fluorescence of NPBZ showed a concentration-dependent enhancement, with a good linear relationship and a LOD of 22 nM, indicating high sensitivity for Aβ aggregates ( Supporting Information Figure S15). Figure 2 | (a) Fluorescence spectra of NPBZ (2 μM) in PBS buffer (pH 7.4) without (black) or with (red) 10 μM Aβ42 aggregates. (b) Fluorescence intensity of NPBZ (2 μM) at 708 nm without (black) or with (red) Aβ42 aggregates (10 μM) for different periods in PBS (pH 7.4). (c) Fluorescence intensity of different amounts of NPBZ with 10 μM Aβ42 aggregates. (d) Competitive displacement with NPBZ (0–1 μM) to ThT from the ThT/Aβ aggregate complex. (e) Fluorescence responses of NPBZ to various potential interferents: 1, blank; 2–11, 100 μM of metal ions (Cu2+, Zn2+, Ba2+, Ca2+, K+, Mg2+, Mn2+, Na+, Fe3+, Al3+); 12–13, 100 μM anions (SO42−, CO32−); 14–23, 100 μM amino acids (Ala, Arg, Asp, Glu, His, Ile, Leu, Lys, Met, Phe); 24–26, biothiols (1 mM Cys, 100 μM Hcy, 5 mM GSH); 27–30, 100 μM ROS (O2•−, OCl–, ONOO–, •OH); 31, 10 μM Aβ42 monomers; 32–34, 10 μM proteins (lysozyme, BSA, α-synuclein); 35, 10 μM Aβ42 aggregates; and 36, 100 μM H2O2. λex/em = 520/708 nm. Download figure Download PowerPoint The binding affinity of NPBZ with Aβ aggregates was then evaluated using fluorescence saturation binding. The dissociation constant (Kd) of NPBZ with Aβ aggregates was calculated through nonlinear curve fitting to be 32.67 nM (Figure 2c), which was about 27.2-fold lower than ThT (Kd = 890 nM).45 The performance of NPBZ was also much better than that of most other reported probes. The competitive displacement of NPBZ toward ThT-bound Aβ aggregates was investigated. As expected, with the addition of NPBZ, the fluorescence intensity of ThT at 485 nm decreased rapidly, while the fluorescence at 708 nm of NPBZ increased sharply, with a 97% displacement efficiency (Figure 2d). These results suggested that NPBZ had a stronger binding ability than ThT, and might compete with ThT for the same binding site, consistent with the above finding of dissociation constant study. Having confirmed the excellent binding affinity of NPBZ for Aβ aggregates, we further evaluated the specificity of NPBZ to Aβ aggregates, revealing that it was virtually unaffected by other potential interferents (Figure 2e). The interference between H2O2 and Aβ aggregates might be another potential problem for a dual-functional probe, thus the fluorescence response of NPBZ in the respective signal channels was investigated when H2O2 and Aβ aggregates coexisted. Most notably, the results in Supporting Information Figure S16 suggested that NPBZ was able to detect H2O2 and Aβ aggregates in the two independent channels without cross-interference. To further reveal the interaction mechanism between NPBZ and Aβ aggregates, molecular docking and kinetic simulation were performed. The reported cryo-electron microscopy structure of Aβ42 (PDB ID: 5OQV) with near-atomic resolution was used as the binding scaffold.46 Two possible binding sites for NPBZ (sites a and b) were predicted ( Supporting Information Figure S17). Among them, one site was a tunnel hydrophobic pocket along the fibril axis, consisting of the amino acid residues of Asn27, Lys28, Gly29, Ala30, Phe19, and Ile31; site b was located on the groove along the fiber axis surface, adjacent to the Glu22 and Phe20 residues. It was worth noting that the binding energy values of NPBZ at site a and site b were −11.4 and −8.9 kcal/mol, respectively, much lower than −8.8 and −6.8 kcal/mol from the commercial probe ThT ( Supporting Information Table S2), further validating that NPBZ has superior binding affinity to Aβ aggregates. Moreover, in molecular dynamics simulations, the fluctuations were stable and there were no major deviations during the simulation period of 50 ns (5000 frames, Supporting Information Figure S18a). The interactions such as hydrogen bonds, hydrophobic, ionic, and water bridges were summarized and categorized by type ( Supporting Information Figure S18b). In the 5OQV binding pocket, NPBZ displayed crucial interactions with Phe20, Glu22, and Val 24 ( Supporting Information Figure S18c). From the interaction heatmap, it can be seen that NPBZ can form high-density interactions with Aβ aggregates throughout the simulation, especially with Phe20, suggesting that this residue was necessary for NPBZ binding ( Supporting Information Figure S18d). Fluorescence imaging of exogenous and endogenous H2O2 in PC12 cells Further, NPBZ was investigated for detecting and imaging H2O2 in living cells. First of all, the cytotoxicity of NPBZ was determined in PC12 cells by MTT assay [MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], and no obvious toxicity was found at concentrations up to 60 μM ( Supporting Information Figure S19). Next, colocalization imaging was performed to investigate whether NPBZ could be effectively localized in mitochondria to detect H2O2 in situ, as mitochondria are not only the main organelles that produce ROS, but also the main reservoirs of ROS. As shown in Figure 3a,b and Supporting Information Figure S20, the fluorescence of NPBZ in the red channel merges well with the green fluorescence of the commercial mitochondria-targeting probe Mito-Tracker Green (MTG), with a Pearson's coefficient of 0.88. Whereas, the red fluorescence of NPBZ shows a poor overlap with the lysosome-targeting probe LysoTracker Green (LTG) and the nucleus-targeting probe Hoechst 33342, with low Pearson's coefficient of 0.42 and 0.10, respectively. Obviously, these results demonstrated that NPBZ was mainly localized in mitochondria benefiting from the positively charged pyridinium, and thus, able to selectively image mitochondrial H2O2. Figure 3 | Colocalization imaging of 10 μM NPBZ, 5.0 μg/mL Hoechst 33342 with (a) 50 nM MTG or (b) 50 nM LTG in PC12 cells. Hoechst 33342: λex = 405 nm, λem = 420–460 nm; MTG/LTG: λex = 488 nm, λem = 510–560 nm; and NPBZ: λex = 520 nm, λem = 650–750 nm. Scale bar = 10 μm. Download figure Download PowerPoint Next, the ability of NPBZ to image exogenous and endogenous H2O2 was examined in PC12 cells. As shown in Figure 4a, only a dim fluorescence was observed with a 30-minute staining of NPBZ (10 μM) alone. In contrast, in the cells pretreated with exogenous H2O2 (100 μM) and then with NPBZ (10 μM), the fluorescence signal showed a large enhancement. Furthermore, in the presence of different levels of exogenous H2O2, a concentration-dependent fluorescence increase was found ( Supporting Information Figure S21), implying that NPBZ was capable of quantitative monitoring of intracellular H2O2 levels independently. On the other hand, it has been reported that PMA stimulation could induce the production of endogenous H2O2.47 Thus, with the cells that underwent a 1-h stimulation of PMA, the fluorescence signal was approximately 2.5-fold higher than that in the untreated cells, indicating an increase in endogenous H2O2 (Figure 4b). Subsequently, efficient elimination of the fluorescence was found when NAC (scavenger