Two-Photon Supramolecular Nanoplatform for Ratiometric Bioimaging

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Two-photon Supramolecular Nanoplatform for Ratiometric Bioimaging Cheng Zhang, Peng Wang, Xia Yin, Hong-Wen Liu, Yue Yang, Liang Cheng, Guosheng Song, and XiaoBing Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01455 • Publication Date (Web): 08 Apr 2019 Downloaded from http://pubs.acs.org on April 8, 2019

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Analytical Chemistry

Two-photon

Supramolecular

Nanoplatform

for

Ratiometric

Bioimaging Cheng Zhang†‡, Peng Wang†‡, Xia Yin†*, Hong-Wen Liu†, Yue Yang†, Liang Cheng§, Guosheng Song†*, Xiao-Bing Zhang† †State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082, P. R. China §Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University Suzhou 215123, China E-mail: [email protected]; [email protected]

ABSTRACT: Two-photon fluorescent imaging that utilizes near-infrared two photons as excitation source is characteristic with the higher penetration depth of tissue for biomedical research, compared with one-photon fluorescent imaging. However, the high laser power levels of excitation source may induce photobleaching of two-photon dyes and photodamage to biosamples, which hampers its wide application for in vivo imaging. Inspired by the supramolecular chemistry, we have developed two-photon excited nanoprobe (TPFN) via host-guest interaction with excellent sensitivity, selectivity, biocompatibility, water solubility and imaging penetration depth. Notably, such supramolecular assembly can significantly amplify the fluorescence intensities of guest molecules (21-fold increase), and thereby affording a detection limit of 0.127 μM for sensing H2O2, which is greatly lower than that of free guest molecules (11.98 μM). In particular, the ratiometric fluorescent imaging provides more accurate analysis of intracellular H2O2 via the built-in correction of internal reference. Importantly, TPFN excited by two-photon laser provides higher penetration depth for visualizing H2O2 in deeper liver tissues, compared with that of one-photon excitation. Thus, TPFN can serve as powerful nanoplatform for ratiometric imaging of various species via the facile supramolecular self-assembly strategy. INTRODUCTION

Two-photon fluorescent imaging, which utilizes near-infrared two photons as excitation source, has emerged as an indispensable imaging tool for biomedical research.1,2 Compared with one-photon excitation, two-photon fluorescent imaging possesses several advantages such as lower background fluorescence, increased tissue penetration and better three-

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dimensional spatial resolution in living cells and tissues.3-5 Because of the much less brightness of fluorescent molecules excited by two-photon than one-photon, the high powered laser is necessary for two-photon imaging.2,6 Such high-energy excitation may induce the damage to biosamples and the photobleaching of fluorescent molecules.7,8 Although abundant efforts have been devoted to developing efficient two-photon probes, most of them were not optimized for two-photon imaging. For example, they were usually suffered from less twophoton fluorescent brightness, poor water solubility, less specificity for sensing targeting species and low stability for long-term imaging. Besides, although ratiometric two-photon probes are more useful for quantitative measurements, those are often subject to the complicated designs and tedious synthetic procedures.9,10 To facilitate the utilization of twophoton imaging in biomedical application, there are strong desires for developing a variety of facile, efficient, specific two-photon probes. Hydrogen peroxide (H2O2) is one of the important members of the reactive oxygen species (ROS) that mediate various physiological and pathological processes.11-13 So far, it is known that aberrant production of H2O2 is involved in many diseases such as cancers,14 Alzheimer,15 Parkinson,16 and diabetes etc.17 Thus, the precise monitoring H2O2 level in live cells and tissues is very crucial to deep comprehension of the pathogenesis of those diseases. For this purpose, lots of fluorescent probes for sensing H2O2 have been reported with various recognizing mechanisms.18-35 Unfortunately, most of them were limited by the short excitation wavelength that may result in poor tissue penetration and strong autofluorescence during realtime imaging of cells or tissues. Inspired by unique performance of functional supramolecular nanostructures that are self-assembled by small building blocks,36-41 herein, we have developed two-photon excited fluorescent nanoprobe (TPFN) via host-guest interaction. Such two-photon nanoprobe possessed several advantages, such as: the amplified fluorescent intensity for potentially reducing photodamage, improved water solubility and stability, enhanced biological compatibility and flexible synthesis/ assembly, which is superior to the free molecules. To utilize such advantages, we further developed TPFN for sensing H2O2 via two-photon excitation. This probe exhibited 97-fold increase in sensitivity, as well as a high selectivity for H2O2 over other competing species. Moreover, TPFN provided the ratiomatric two-photon imaging of H2O2 in living cells and ex vivo liver tissues with deeper tissue penetration and more accurate analysis via internal reference. EXPERIMENTAL SECTION

Materials and Instruments. The chemical and biological reagents were obtained from commercialized companies. NMR spectra was measured by Bruker-400 spectrometer. Mass spectra was conducted in LCQ advantage ion trap mass spectrometer (Thermo Finnigan). The


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morphology and size of nanoparticles were characterized with field emission scanning electron microscope (JSM-6700F, JEOL) and dynamic light scattering (Zetasizer 3000Hs, Malvern). The optical properties of nanoprobes were measured with fluorescence spectrophotometer (HITACHI F7000) and UV-1800 spectrophotometer (Shimadzu Corporation, Japan). Two-photon fluorescent bioimaging of living cancer cells and ex vivo tissues were performed through a multiphoton laser scanning confocal microscopy (Nikon A1plus). Synthesis of β-cyclodextrin polymer (Poly-β-CD). Poly-β-CD was synthesized basing on the previous literature.42 Briefly, 5 g of β-cyclodextrins (β-CD) (4.405 mmol) was added into 10 mL of NaOH aqueous solution and stirred for 3 h at 35 °C. Then, the mixture solution was added with 345 μL of epichlorohydrin (4.405 mmol) and stirred for another 3 h. Subsequently, the product was precipitated by adding 100 mL isopropanol, filtered, neutralized by hydrochloric acid, and further dialyzed for 7 days, successively. The purified Poly-β-CD was lyophilized to afford white powder (1.25 g, ~ 25 %). Synthesis of adamantane- labelled naphthalene derivative (NpRh-Ad, Np-Ad, α-NpAd). NpRh-Ad was synthesized according to the previous literature.9 For synthesis of Np-Ad, 2 2-(6-aminonaphthalen-2-yl)benzo[d]oxazole-6-carboxylic acid (304 mg, 1.0 mmol), 1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium

3-oxid

hexafluorophosphate (HATU) (570 mg, 1.5 mmol) and N,N-Diisopropylethylamine (DIPEA) (0.35 mL, 2.0 mmol) were dissolved in dry dichloromethane (200 mL). The mixture solution was stirred at 0 °C for 1 h, and then added with 1-adamantanamin (181 mg, 1.2 mmol). After stirring at room temperature for overnight, the mixture solution was evaporated and purified via chromatography (Dichloromethane / ethyl acetate = 5:1, v/ v). Np-Ad was obtained as a yellow solid (180 mg, 41 %). For synthesis of α-Np-Ad, 4-nitrophenylglyoxylic acid (195 mg, 1.0 mmol) and HATU (579 mg, 1.5 mmol) were dissolved in dry dichloromethane (50 mL). Next, the mixture solution was added with triethylamine dropwise (1.0 mL) under stirring at 0 °C for 1 h in N2atmosphere, and then added with Np-Ad (437 mg, 1.0 mmol) under stirring at room temperature for overnight. After reaction, the solution mixture was evaporated and purified via chromatography (dichloromethane/ ethyl acetate = 100:1, v/ v). Finally, α-Np-Ad was obtained as an orange solid (200 mg, 32.6 %). Preparation two-photon excited fluorescent nanoprobe (TPFN). NpRh-Ad, α-Np-Ad was pre-dissolved into DMSO as stock solutions. In typical preparation, 200 μL of NpRh-Ad/ DMSO and α-Np-Ad/ DMSO was added in 3.8 mL of PBS solution containing Poly-β-CD and subjected to vibration at 300 rpm for 4 h (25 °C). The ultimate concentration for NpRhAd, α-Np-Ad and Poly-β-CD was 5 μM, 10 μM, and 10 mM, respectively. After vibration, the



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resulted nanoprobes were purified by ultrafiltration (10K MWCO) to remove the free guest molecules. Characterization of TPFN. For agarose gel electrophoresis experiments, nanoprobes were placed on agarose gelelectrophoresis (3 % agarose) and conducted in 1 × TBE for 20 min. Subsequently, the gelelectrophoresis was excited at 365 nm and imaged by digital camera. For measurement of binding constants, nanoprobe was prepared by adding various concentration of Poly-β-CD (the molarity of β-CD) and (NpRh-Ad or Np-Ad) (5 μM). Then, fluorescence spectra was measured to calculate the binding constants (K) of NpRh-Ad or NpAd with Poly-β-CD by the Benesi-Hildebrand plot as given in follow43:

1 /( F  F 0)  1 /( F ' F 0)  1 /{K ( F ' F 0)[Poly - β - CD]}

(1)

Where K is the binding constant, F0 is the original fluorescence intensity of free guest molecules, F is the fluorescence intensity of the nanoprobes, and F’ is the observed fluorescence intensity at its maximum. If [1/ (F-F0)] vs [1/ Poly-β-CD concentration] is a linear relationship, it indicates the formation of 1: 1 between the guest molecules with Poly-βCD, and the ratio of slope to intercept is the K value of guest molecules with Poly-β-CD.

For measurement of the fluorescence quantum yield (Φf), coumarin 102 in EtOH at 25 °C was used as a standard (Φf = 0.93).44 The quantum yield was calculated according to the following equation45: ΦF(X) = ΦF(S) (AS FX/ AX FS) (nX/ nS)2

(2)

Where A is the absorbance at the excitation wavelength (A < 0.05), F is the area under the corrected emission curve, and n is the refractive index of the solvents used. Subscripts S and X is the standard and tested sample, respectively. Measurement of the sensing ability of TPFN toward H2O2. For optimizing sensitivity, different molar ratios of α-Np-Ad and NpRh-Ad (e.g. 2: 1; 1: 1 and 1: 2) were used to prepare TPFN. These three kinds of TPFN were incubated with different concentration of H2O2 (0 200 μM) for 2 h. Then, fluorescence spectra were recorded to calculate the ratio of F475/ F595, λex = 420 nm. For testing selectivity, various species were incubated with TPFN for 2 h, such as Vc (100 μM); GSH (1 mM); Hcy (1 mM); Cys (1 mM); S2O32- (100 μM); SO32- (100 μM); (8) HS(100 μM); (9) Ca2+ (100 μM); (10) Mg2+ (100 μM); (11) K+ (100 μM); (12) Na+ (100 μM); (13) O2.- (100 μM); (14) NO (100 μM); (15).OH (100 μM); (16) ClO- (100 μM); (17) TBHP (100 μM); (18) SIN-1(SIN-1 as the donor of ONOO-, 100 μM); (19) H2O2 (200 μM). Cellular experiments. Cervical cancer HeLa cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % antibiotics


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(penicillin-streptomycin) at 37 °C in a humidified incubator containing 5 % CO2. The entire process of cell culture followed ATCC instructions. For testing cytotoxicity, HeLa cells pre-plated in 96-well plates were incubated with various concentration of nanoprobe for 24 h. The relative cellular viabilities were measured via a standard MTS assay (Promega) to evaluate cytotoxicity.

Two-photon fluorescence Imaging of H2O2 in Living Cells. For two-photon imaging of exogenous H2O2, HeLa cells were seeded in optical culture dish and then incubated with TPFN (10 μM of α-Np-Ad) for 2 h at 37 °C. After washing with DPBS for three times, those HeLa cells were further incubated with different concentrations of H2O2 (20, 50, 100,150 μM, respectively) for another 2 h at 37 °C. Then, those HeLa cells was observed by two-photon excited confocal fluorescence microscopy (λex = 780 nm; green channel, λem = 425 - 475 nm; red channel, λem = 570 - 620 nm). For two-photon imaging endogenous H2O2, HeLa cells firstly received different treatement: (1) no treatment; (2) incubated with 100 μM of TEMPO (2,2,6,6tetramethylpiperidine-1-oxyl) for 1 h; (3) incubated with 100 μM of TEMPO for 1 h and then 5mg/mL of phorbol myristate acetate (PMA) for 1 h. Next, those HeLa cells were further incubated with TPFN (10 μM of α-Np-Ad) for another 2 h at 37 °C. Subsequently, those cells were washed by DPBS buffer three times before two-photon excited confocal fluorescence imaging (λex = 780 nm; green channel, λem = 425 - 475 nm; red channel, λem = 570 - 620 nm). Two-photon fluorescence imaging of H2O2 in liver tissue. The fresh livers from the nude mice were cut into slices by using a vibrating-blade microtome. The fresh liver slices were incubated with TPFN (10 μM of α-Np-Ad, 37 °C, 2 h), washed with DPBS (three times), and then treated with H2O2 (0 μM or 150 μM, 37 °C, 2 h). Finally, those slices were scanned via confocal fluorescence microscope with Z-scanning mode under excitation of one-photon (405 nm) or two-photon (780 nm), respectively (green channel, λem = 425 - 475 nm; red channel, λem = 570 - 620 nm). Measurement of H2O2 in liver tissue of mice with nonalcoholic fatty liver disease. To prepare murine model with nonalcoholic fatty liver disease, the mice were fed with high-fat diet for eight-week. Subsequently, those liver tissues from mice with nonalcoholic fatty liver disease were dissected and homogenized in DPBS (liver tissue, 100 mg/ mL) by ultrasonic cell smash (300 W, 30 min). After centrifugation of tissue homogenates (4000 r/min for 10 min at 4 °C), the supernatants were collected and incubated with TPFN (10 μM of α-Np-Ad). The fluorescent F475/ F595 values of TPFN were measured to calculate H2O2 concentration in liver tissues, according to the standard curve of F475/ F595 toward H2O2 concertation. As a control experiment, the H2O2 concentrations in supernatants were also measured by a commercial H2O2 kits (Nanjing Jiancheng Bioengineering Institute).



RESULTS AND DISCUSSION

Preparation and Characterization of two-photon excited fluorescent nanoprobe. Two-photon excited fluorescent nanoprobe (TPFN) was constructed by the following three main steps (Figure 1): (a) Synthesis of two-photo excited fluorescent molecule as internal reference: adamantine-labelled naphthalene combined with rhodamine derivative (NpRh-Ad); (b) Synthesis of another two-photon excited fluorescent molecule as responsive unit: adamantine-labelled naphthalene derivative (α-Np-Ad). After the cleavage of α-ketoamide moiety to form Np-Ad via H2O2, Np-Ad exhibits the fluorescence at 475 nm; (c) Selfassembly of NpRh-Ad, α-Np-Ad and β-cyclodextrin polymer (Poly-β-CD) toward TPFN via host-guest interaction. In the presence of H2O2, TPFN showed the enhanced fluorescence at 475 nm under excitation of two-photon laser at 780 nm, while the fluorescence at 595 nm remained constant, affording the ratiomatric sensing of H2O2. The successful synthesis of NpRh-Ad, Np-Ad, α-Np-Ad and Poly-β-CD was confirmed by 1H NMR, 13C NMR and mass spectrometry (see Supporting Information, Figure S1-S7).

Figure 1. Scheme showed the preparation of two-photon excited fluorescent nanoprobe (TPNF). (a) The synthetic routes of NpRh-Ad; (b) The synthetic routes of α-Np-Ad and H2O2 response of α-Np-Ad; (c) Self-assembly of NpRh-Ad, α-Np-Ad and Poly-β-CD into TPFN via host-guest interaction and illustration of TPFN for ratiometric detection of H2O2.

In order to investigate the host-guest interaction between Poly-β-CD and our prepared guest molecules, we firstly prepared Poly-β-CD nanoparticles via selfassembly of Poly-β-CD in PBS solution (Figure S8), and then separately or coincorporated various guest molecules (e.g. Np-Ad, α-Np-Ad, NpRh-Ad) into Poly-β-CD


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nanoparticles via host-guest interaction. The successful incorporation of various guest molecules into Poly-β-CD was verified by the retention in the agarose gel electrophoresis, compared with the free guest molecules (Figure S9). Furthermore, the binding constants of Np-Ad/ Poly-β-CD and NpRh-Ad/ Poly-β-CD were measured to be 2.25×103 M-1 and 2.35×103 M-1, respectively (Figure S10). Such strong binding force may reduce the potential release of guest molecules from host nanoparticle, as so to avoid the background signals. Besides, Poly-β-CD nanoparticles incorporating various guest molecules exhibited their typical absorption and fluorescence spectra of their corresponding molecules (Figure 2e & S11). Notably, after incorporation into Poly-β-CD nanoparticles, the fluorescent intensity of Np-Ad or NpRh-Ad was increased 4.5- and 21.1- fold, respectively, compared with that of free guest molecules (Figure 2c and 2d), which indicated the supramolecular assembly could significantly amplify the fluorescent intensities of guest molecules and the possibility of using lower power of laser for two-photon imaging. In a typical preparation of TPFN (5 μM of NpRh-Ad, 10 μM of α-Np-Ad, and 10 mM of Poly-βCD), the obtained TPFN exhibited spherical morphology with uniform diameter of ~ 90 nm (Figure 2a), which is similar to Poly-β-CD nanoparticles. TPFN could be well dispersed into water with DLS size of ~140 nm (Figure 2b), demonstrating Poly-β-CD nanostructure could improve the hydrophilicity of guest molecules. Next, we tested the stability of TPFN in buffer solutions and found that TPFN could keep the original fluorescence ratio values (F475/ F595) in PBS (pH = 7.4) and blood serum buffer (15 % of FBS) for various times and 1.38 % of guest molecules were released from TPFN after 12 h incubation, indicating its good stability (Figure 2f, S12 and S13). We also studied the stability of TPFN in different pH condition (Figure S14) and found the good stability of TPFN in pH = 4 - 9.

Figure 2. Characterization of TPFN. (a) SEM image of TPFN; (b) Size distribution of TPFN by DLS; (c) Fluorescent spectra of Np-Ad and Poly-β-CD nanoparticle incorporating Np-Ad; (d) Fluorescent



spectra of NpRh-Ad and Poly-β-CD nanoparticle incorporating NpRh-Ad; (e) Absorption spectra of Poly-β-CD nanoparticle incorporating indicated guest molecules; (f) Stability of the TPFN in PBS (pH = 7.4) and blood serum-contained solution (15 % of FBS).

TPFN sensing H2O2 in solution. Next, we investigated the sensing ability of TPFN by incubation of TPFN with various concentrations of H2O2 for 120 min and then measured the fluorescent spectra under excitation of 420 nm to determine the ratio of F475/ F595. We found that the fluorescent intensities at 475 nm was gradually enhanced with the concentration of H2O2 increasing, while the fluorescent intensity at 595 nm showed no obvious change (Figure 3a), resulting in the F475/ F595 values increased as the increasing concentration of H2O2. To study the effect of α-Np-Ad/ NpRh-Ad ratio on sensing ability, three kinds of TPFN were prepared with various molar ratios of guest molecules (e. g. α-Np-Ad: NpRh-Ad = 2: 1; 1: 1; 1: 2). After incubation with different concentrations of H2O2, the F475/ F595 values were linear to H2O2 concentrations for all three kinds of TPFN (Figuere 3b & S15). Importantly, the responsivity to H2O2 was enhanced as the ratios of α-Np-Ad / NpRh-Ad increased. In view of the sensitivity, this molar ratio (α-Np-Ad: NpRh-Ad = 2: 1) was chosen for preparation of TPFN in later studies. Next, we compared the sensing ability of our optimized TPFN with free guest molecules (Figure S16). Notably, the F475/ F595 values of TPFN increased 5.9-fold with H2O2 concentration from 0 to 50 µM, while the F475/ F595 values of corresponding free guest molecules only increased 1.06 fold (Figure 3c). The linear correlation between F475/ F595 and H2O2 concentrations in range of 0 to 50 µM was 0.994 for TPFN, which is higher than that of free guest molecules (R2 = 0.894). Importantly, based on signal/ noise = 3, the detection limit was calculated to be as low as 0.127 μM for TPFN, while the detection limit reached 11.98 μM for free guest molecules (Figure 3d), demonstrating the sensitivity of TPFN was improved 94-fold compared with free guest molecules. Besides, the fluorescence quantum yields (Φf) of TPFN were determined to be 0.027 and 0.41 before and after activation, respectively. Thus, supramolecular assembly via host-guest interaction could significantly enhance the responsivity, sensitivity and linear correlation of TPFN. Furthermore, we tested the selectivity of TPFN, and found the F475/ F595 values exhibited no obvious increase after incubation with a panel of ROS, reactive nitrogen specie, reactive sulfur species, or other biological relevant species, except H2O2, demonstrating the high selectivity of TPFN toward H2O2 (Fig 3e). Besides, the dynamic measurement of TPFN to H2O2 showed the F475/ F595 value reached platform after 2 h of incubation (Figure S17). Moreover, TPFN exhibited high repeatability and recovery (more than 96 %) in sensing complex samples, such as measuring the H2O2 concentration in contaminated water (Figure S18).


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Figure 3. TPFN for sensing H2O2. (a) Fluorescent spectra of TPFN (10 μM of α-Np-Ad) toward varied concentrations of H2O2 (0 - 200 μM) in PBS buffer (pH 7.4, 5 % of DMSO) after 2 h of incubation; (b) Fluorescent F475/ F595 values of TPFN (e.g. α-Np-Ad: NpRh-Ad = 2: 1; 1: 1; 1: 2) toward varied concentrations H2O2; (c) Fluorescent F475/ F595 values of TPFN (α-Np-Ad: NpRh-Ad = 2: 1) and corresponding mixture of guest molecule toward varied concentrations H2O2; (d) Detection limit of TPFN (α-Np-Ad: NpRh-Ad = 2: 1) and corresponding mixture of guest molecules; (e) Fluorescent F475/ F595 values of TPFN (10 μM of α-Np-Ad) toward indicated species (λex = 420 nm); (f) Cell viability (%) of HeLa cells incubated with TPFN (α-Np-Ad: NpRh-Ad = 2:1) of various αNp-Ad concentration for 24 h, measured by MTS assay. The error bars indicated the standard deviations of three experiments.

TPFN for two-photon imaging of intracellular H2O2. Due to the high sensitivity and selectivity of TPFN toward H2O2 in solution, TPFN was applied to two-photo excited confocal fluorescence imaging of living cells. Before doing intracellular sensing, we firstly investigated the cytotoxicity of TPFN and found no obvious cytotoxicity induced by TPFN toward Hela cells after 24 h incubation (Figure 3f). The colocalization experiments demonstrated TPFN mainly localized in lysosomes after phagocytosis (Figure S19). Because of the acidic condition of lysosomes, the fluorescence response of TPFN in pH = 5.0 was investigated (Figure S20). It was found that TPFN was still able to sense H2O2 well in acidic condition. Next, we further performed two-photon excited fluorescence imaging. Hela cells were pretreated with different concentrations of exogenous H2O2 (e.g. 20, 50, 100 and 150 μM). The confocal images showed the green fluorescence gradually enhanced with the concentration of H2O2 increasing from 20 to 150 μM, meanwhile the red fluorescence were almost unchanged, indicating the excellent intracellular response of TPFN (Figure 4a). In particular, the fluorescence green/ red values of TPFN were



linear to the concentration of H2O2 with good linear relationship (R2 = 0.99), while the green signal values to concentration of H2O2 had poor linear correlation (R2 = 0.87) (Figure 4b, 4c). Thus, owing to the internal reference of NpRh-Ad as built-in correction, the ratiometric fluorescence from TPFN could avoid some interference (e.g. probe concentration and distribution) and thereby provide more accurate measurement of H2O2.

Figure 4. TPFN for two-photon imaging of intracellular H2O2. (a) The two-photon excited confocal fluorescent images of Hela cells incubated with TPFN and different concentrations of exogenous H2O2 (e.g. 20, 50, 100 and 150 μM); (b) The fluorescent intensities of green channel in (a) toward H2O2 concentrations; (c) The ratio values of fluorescent intensity (Fgreen/ Fred) in (a) toward H2O2 concentrations; (d) The two-photon excited confocal fluorescent images of Hela cells pretreated with no treatment, TEMPO, TEMPO + PMA, then incubated with TPFN; (e) The fluorescent intensities of green channel from each group in (d); (f) The fluorescent intensity ratios (Fgreen/ Fred) from each group in (d). λex = 780 nm; λem of green channel: 425 - 475 nm; λem of red channel: 570 - 620 nm; Scale bar: 20 μm.

Subsequently, we explored whether TPFN could detect endogenous H2O2. 2,2,6,6tetramethylpiperidine-1-oxyl (TEMPO, 100 μM) was used as a ROS scavenger to eliminate H2O2 in living cells,20 and Phorbol myristate acetate (PMA) employed for inducing inflammation to increase cellular H2O2 level.22 Two-photon excited confocal images showed those cells treated with TEMPO exhibited weaker green signals and lower fluorescence green/ red values, while those cells treated with PMA exhibited stronger green signals and higher fluorescence green/ red values, compared with that of control group, which indicated our


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TPFN was sensitive enough for monitoring the endogenous H2O2 fluctuation in living cells (Figure 4d - 4f). TPFN for two-photon imaging of H2O2 in ex vivo tissue. Next, we promoted TPFN to sense H2O2 in ex vivo tissue. The fresh liver slices were incubated with TPFN and H2O2 successively, and then scanned via confocal fluorescence microscope with Zscanning mode under excitation of one-photon (405 nm) or two-photon (780 nm), respectively (Figure 5a). Notably, the fluorescence from two-photon excitation was obviously brighter than that from one-photon excitation at the various imaging depth (Figure S21). Specifically, the two-photon excited imaging penetration depth was determined to be 130 μm, which was higher that of one-photon excitation (70 μm) for both green and red channel. Next, we performed two-photon excited fluorescence images of a rat liver frozen slice at depth of 50 μm. We found the normal liver tissue exhibited lower green/ red value, suggesting the low concentration of endogenous H2O2. After incubation of 150 μM of H2O2 in liver tissue, the green/ red value was drastically increased, indicating that TPFN could perform the ratiometric sensing H2O2 in deeper tissues (Figure 5b). It is known that high-fat diet could increase the H2O2 level in liver of mice.28 The accurate measurement of H2O2 concentration in liver tissue would be benefited to evaluating the progress of nonalcoholic fatty liver disease. A murine model with nonalcoholic fatty liver was established by feeding high-fat diet for eight-week. The liver tissue was dissected from the mice with nonalcoholic fatty liver, homogenized, and measured via the fluorescence F475/F595 values of TPFN to determine H2O2 concentration. Meanwhile, a commercial H2O2 kits was used as a control experiment. It is found the H2O2 concentration of liver tissue from mice with nonalcoholic fatty liver was higher than that with normal mice (Figure 5c). Moreover, the results from TPFN were well matching to the commercial kits (Figure 5d), indicating TPFN provided the momentum to quantitatively detect the concentrations of H2O2 in ex vivo liver tissue.



Figure 5. (a - b) TPFN for two-photon imaging of H2O2 in ex vivo tissue. (a) Confocal fluorescent images of fresh liver slices incubated with TPFN and H2O2, and scanned via Z-scanning mode under excitation of one-photon (λex = 405 nm) or two-photon laser (λex = 780 nm), respectively; (b) Twophoton (λex = 780 nm) excited confocal fluorescent images of fresh liver slices incubated with TPFN and H2O2 (e.g. 0, 150 μM); λem of green channel: 425 - 475 nm; λem of red channel: 570 - 620 nm; Scale bar: 50 μm. (c - d) Qualitative analysis of H2O2 level in ex vivo dissected liver tissue of normal mice and mice with nonalcoholic fatty liver disease, respectively. The liver tissues were dissected and homogenized, and the supernatants were collected and incubated with TPFN. (c) The ratio values of fluorescent intensity (Fgreen/ Fred) of TPFN for normal mice and mice with nonalcoholic fatty liver disease (λex = 405 nm). (d) H2O2 concentration of ex vivo liver tissue from normal mice and mice with nonalcoholic fatty liver disease, measured by TPFN and a commercial H2O2 kits.

CONCLUSIONS

In summary, we have successfully developed a supramolecular strategy to construct twophoton fluorescent nanoprobe that has several advantages: (i) Supramolecular self-assembly provided significant amplification of fluorescence intensities to obtain brightness images at low probe concentrations and 94-fold improved sensitivity, which may reduce the photodamage to probes and biosamples; (ii) High water solubility and appreciable biocompatibility so as to stain cells and tissues; (iii) High specificity for targeting H2O2 over other species; (iv) Ratiometric fluorescence imaging offered more accurate analysis owing to the built-in correction to avoid interferences; (v) Two-photon excited imaging provided higher penetration depth in tissue. Considering the facile integration of guest molecules in


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host, in the future, such powerful strategy may be extended to integrate a variety of guest molecules to develop various ratiometric two-photon or photoacoustic nanoprobes for sensing multiple species. ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Characterization of compounds and nanoprobe; Stability of nanoprobe; Fluorescence spectra; Confocal fluorescent images of fresh liver slices. AUTHOR INFORMATION Corresponding Authors

E-mail: [email protected] E-mail: [email protected] ‡ Those

authors were contributed equally to this work.

Notes The authors declare no competing financial interest ACKNOWLEDGMENT

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Two-Photon Supramolecular Nanoplatform for Ratiometric Bioimaging

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