Tumor-Targeted Graphitic Carbon Nitride Nanoassembly for

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Tumor-targeted Graphitic Carbon Nitride Nanoassembly for Activatable Two-photon Fluorescence Imaging Jin-Wen Liu, Yu-Min Wang, Chong-Hua Zhang, Lu-Ying Duan, Zheng Li, Ru-Qin Yu, and Jian-Hui Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05192 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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Analytical Chemistry

Tumor-targeted Graphitic Carbon Nitride Nanoassembly for Activatable Two-photon Fluorescence Imaging ‡

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Jin-Wen Liu , Yu-Min Wang , Chong-Hua Zhang, Lu-Ying Duan, Zheng Li*, Ru-Qin Yu and Jian-Hui Jiang* Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, P. R. China ABSTRACT: Unique physicochemical characteristics of graphitic carbon nitride (g-CN) nanosheets suit them a useful tool for two-photon fluorescence bioimaging. Current g-CN nanosheets based imaging probes typically use the “always-on” design strategies, which may suffer from increased fluorescence background and limited contrast. To advance corresponding applications, g-CN nanosheets based activatable two-photon fluorescence probes remain to be explored. For the first time, we developed an activatable two-photon fluorescence probe, constructed from a nanoassembly of g-CN nanosheets and hyaluronic acid (HA)-gold nanoparticles (HA-AuNPs), for detection and imaging of hyaluronidase (HAase) in cancer cells. The deliberately introduced HA in our design not only function as the buffering layer for stabilizing AuNPs and inducing corresponding self-assembly on g-CN nanosheets, but also as a pilot for targeting HA receptors overexpressed on cancer cell surfaces. Our results show that the developed nanoassembly enables specific detection and activatable imaging of HAase in cancer cells and deep tissues, with superb signal-tobackground ratio and high sensitivity. This nanoassembly can afford a promising platform for highly specific and sensitive imaging of HAase and for related cancer diagnosis.

Graphitic carbon nitride (g-CN) nanosheets, made from conjugated C-N layers with weak interlayer Van der Waals' force, possess unique physicochemical properties, such as high surface area, facilely accessible functional sites on the basal planes, and inertness to the environmental influences.1-5 Recently, g-CN nanosheets have been demonstrated with excellent performances for two-photon fluorescence related applications, based on following criteria. First, large two-photon absorption cross section (δ). The simultaneous absorption of two photon and corresponding interaction with electron at the ground state is a three-particle process, which has much lower probability compared to one photon spectroscopy, especially at low photon flux. To increase the efficiency of two photon spectroscopy, compounds with large δ are evidently preferred. The δ of g-CN is on the order of 28000 - 40000 GM at 750 nm, much larger than organic dyes and fluorescent proteins (tens to thousands GM scale), and is among the best in the mainstream fluorophores.6-8 Second, minimum photobleaching. Since the probability of two photon absorption depends upon the square of the intensity of incident light source, high photon flux with short pulse are generally applied. The irreversible photochemical reaction induced by intense photon bombarding would ruin original optical properties of the absorbing compounds. In consequence, the negligible photobleaching of gCN under prolonged light irradiation precludes potential photo-stability issues. Third, reasonable quantum yield.9-11 In addition, g-CN nanosheets are highly biocompatible with low phototoxicity.12-14 Combined with the unique advantages of two-photon fluorescence imaging, such as improved penetration depth, low background interferences, high spatial precision and so on, g-CN nanosheets have emerged as invaluable functional materials for biosensors and bioimaging.15-17 Singlelayered g-CN quantum dots was developed with the capability of effectively passing through the nuclear pore complex and penetrating into the nuclei, enabling two-photon fluorescence

imaging of cellular nucleus.10 Enhanced photocurrent from two-photon excitation of g-CN nanosheets were employed to stimulate cytosolic Ca2+ accumulation, and corresponding therapeutic potential in bone regeneration and fracture healing were demonstrated.18 Multifunctional nanocarrier of g-CN nanosheets with mesoporous silica were also introduced for tumor-targeted imaging and drug delivery.19 Despite those successes, activatable two-photon fluorescence imaging based on g-CN nanosheets have not been explored. Current approaches are typically realized through the “always-on” design, i.e., the g-CN nanosheets remain fluorescent under excitation and reveal the areas where the nanosheets are accumulated. This “always-on” design may suffer from issues such as increased fluorescence background and limited contrast for molecular imaging.20-22 Herein, we develop a novel activatable two-photon fluorescence probe using a g-CN nanosheets/hyaluronic acid-gold nanoparticles (HA-AuNPs) nanoassembly, for detection and imaging of hyaluronidase (HAase) in cancer cells. Hyaluronidase, a family of enzymes that catalyze the degradation of hyaluronic acid, is known to be implicated in the progression of many solid tumor types, and their utility as diagnostic and therapeutic biomarkers has been extensively validated and exploited.23-25 To design an activatable two-photon fluorescence imaging probe for HAase, we exploit their natural substrate, hyaluronic acid (HA), and then engineer a functional nanoassembly using g-CN nanosheets with HA and AuNPs, as illustrated in Scheme 1. The nanoassembly is prepared using a facile two-step procedure: First, a nanocomplex of HA-AuNPs is synthesized using HA as the stabilizer, with hydroxyl groups from HA coordinated around AuNPs, after corresponding reduction of HAuCl4 by NaBH4; Second, the g-CN/HA-AuNPs nanoassembly is formed by the self-assembly of HA-AuNPs nanocomplex on the g-CN nanosheets, through the electrostatic and

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hydrophobic interactions between HA and the g-CN nanosheets. In the g-CN/HA-AuNPs nanoassembly, HA is not only used as the buffering layer for stabilizing AuNPs and promoting corresponding self-assembly on g-CN nanosheets, but also plays a role as functional ligand for targeting cell surface receptors of HA, such as CD44, which is known to be overexpressed in many tumors.26-28 Based on those designs, the g-CN/HA-AuNPs nanoassembly acts as an activatable two-photon fluorescence probe for specific detection of HAase in cancer cells, as illustrated in Scheme 1B. The nanoassembly itself merely delivers a negligible fluorescence signal, since the intrinsic fluorescence of g-CN nanosheets was efficiently quenched by AuNPs through the energy transfer from excited nanosheets to AuNPs. Upon incubating with cancer cells, the nanoassembly is delivered into cancer cells due to its selective targeting of HA receptors on cell surface, and then by a receptor-mediated endocytosis pathway. Inside the cells, HA is degraded by HAase,23,24 which destroys the intermediate layer in the nanaoassembly and causes dissociation of AuNPs from the nanoassembly. As a result, the fluorescence of g-CN nanosheets is recovered, with an enhanced fluorescence signal directly correlated with the activity of HAase. To our knowledge, this is the first time that an activatable two-photon fluorescence probe has been demonstrated with g-CN nanosheets, at the same time, the first two-photon fluorescence probe for detection and imaging of HAase. Due to the superb biocompatibility of nanoassembly, efficient two-photon fluorescence of g-CN nanosheets, high fluorescence quenching efficiency of AuNPs, and effective fluorescence recovery by HAase, this nanoassembly probe should deliver a useful platform for highly specific and sensitive imaging of HAase and for related cancer diagnosis.

Scheme 1. (A) The schematic illustration of the synthetic process of HA-AuNPs and (B) the application of prepared HA-AuNPs/gCN nanoassembly as an efficient platform for activatable twophoton fluorescence imaging.

EXPERIMENTAL SECTION Reagents and Materials. All the information can be found in the Supporting Information. Instrument and Characterization. All the experimental details are supplied in the Supporting Information. Synthesis of Graphitic Carbon Nitride Nanoassembly. The detailed experimental procedure for preparing g-CN nanosheets is provided in the Supporting Information.

The HA-AuNPs were synthesized according to the previous reports with minor modifications.29,30 In a typical synthesis, 1.2 mL HAuCl4 (0.1 mol/L) mixed with 5 mL 0.1% (w/v) sodium hyaluronate aqueous solution were vigorously stirred at room temperature for 20 minutes. Then, 1 mL NaBH4 (4 mg/mL) was slowly injected, followed with a color change of the solution from yellow to red purple, indicating the formation of AuNPs. The reaction was kept under vigorous stirring for another 10 min. To remove small molecules and ions, the product was purified via ultra-filtration (10 kDa ultrafiltration centrifuge tube) and redissolved in deionized water. The purified HA-AuNPs solution was stored at 4 °C for further research. The tumor-targeted graphitic carbon nitride nanoassembly was prepared as following: 20 µL g-CN nanosheets (0.21 mg/mL) and 20 µL as-prepared HA-AuNPs were mixed together in 10 mM NaH2PO4-Na2HPO4 buffer solution (pH 6.0) under vigorous stirring at room temperature for 45 min. The mixture was centrifuged twice at 10,000 rpm for 10 min to remove excessive HA-Au NPs. The nanoassembly sediments were resuspended in the NaH2PO4-Na2HPO4 buffer solution for further use. In Vitro Detection of HAase Target. In 100 µL of 5 µg/mL nanoassembly solution, 20 µL varied concentration of HAase or control samples was mixed with the nanoassembly in a calibrated centrifuge tube and incubated at 37 °C for 2 h. And corresponding fluorescence and UV-Vis absorption spectra were recorded for quantitative analysis. Two-photon Fluorescence Microscopy Imaging. The cells were cultured in RPMI 1640 medium supplemented with 15% FBS, 100 IU∕mL penicillin and 100 IU∕mL streptomycin at 37 °C in a humidified incubator containing 5% wt/vol CO2. The cell number was determined using a hemocytometer. MDA-MB-231 cells, MCF-7 cells and HEK-293 cells were centrifuged at 1000 rpm for 4 min and washed twice with the washing buffer, and then re-dispersed in the binding buffer. HA-AuNPs/g-CN nanoassembly was incubated with the cells at 37 °C for a controlled time. The cells were then applied under two-photon fluorescence microscope for further observation. Tissue imaging was also used to monitor intracellular HAase levels in nude mice slice with a similar procedure. Cell Cytotoxicity. The cells were seeded in 96-well plates and incubated at 37 °C in 5% CO2 atmosphere for 48 h. The cells were then incubated for another 48 h with a given dosage of HA-AuNPs/g-CN nanoassembly in RPMI 1640 medium supplemented with 15% FBS. And the cytotoxicity was assessed using CellTiter 96® AQueous One Solution Cell Proliferation Assay against different cell lines. After the removal of cell medium, 20 µL CellTiter Reagent diluted with 100 µL fresh medium was added to every well and incubated with the cells in a humidified incubator (37 °C, 5% CO2) for 2 h. Then, the absorbance at 490 nm for each well were acquired on a Thermo Scientific Multiskan Microplate Reader (Thermo Fisher, USA). To avoid the interference from nanoparticles, control experiments in the absence of the cells were implemented, and the corrected absorbance values after the elimination of background were applied to assess the cell viability. All the cell viability was calculated per specification provided by the manufacturer. RESULTS AND DISCUSSION Characterization of Two-Photon Fluorescent g-CN Nanosheets. The ultrathin g-CN nanosheets were prepared


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Analytical Chemistry with methods modified from our previous reports.31,32 The obtained g-CN nanosheets were milk-like under sunlight and exhibited blue emission under the UV excitation (Figure 1A). The fluorescent g-CN nanosheets were stable for more than four months, while sealed under dark environment at room temperature. As expected, the g-CN nanosheets exhibit typical two-photon absorption (TPA) characteristic, which could simultaneously absorb two near-infrared photons and emit bright fluorescence in the visible light region. The maximum TPA cross section of g-CN nanosheets reached 29623 GM at 760 nm, which is beneficial for spatial locating and deep-tissue imaging (Figure 1B). This value is orders of magnitude higher than that of previously reported organic dyes,6,33-36 and comparable to the well-known graphene QDs/carbon dots,37-39 and heavy-metal containing semiconductor QDs.40-43 As shown in Figure 1, the emission profile of g-CN nanosheets are nicely overlapped while under one-photon (355 nm) or two-photon (760 nm) excitation conditions, with an intense peak centered at around 438 nm, indicating that the fluorescence excited at the near-infrared region is two-photon induced emission.

Formation and Characterization of the HA-AuNPs/gCN Nanoassembly. To fabricate the tumor-targeted and activatable fluorescent probe for bioimaging, HA-AuNPs was used as the highly efficient energy transfer acceptor of g-CN nanosheets. Herein, we directly used HA as a template, to prepare multifunctional AuNPs with targeting and fluorescence activation capabilities, via a simple one-step synthesis. As-prepared HA-AuNPs shows a spherical shape, with a diameter of around 11 nm (Figures 2A). The AuNPs prepared from HA exhibited an UV-vis absorption peak at 521 nm, in accordance with the Au surface plasmon resonance peak position. The appearance of characteristic IR bands from HA on the HA-AuNPs (Figure S5), suggests the successful immobilization of HA on the AuNPs surface. Moreover, a ζ-potential of around -54.2 mV was observed for the HA-AuNPs, indicating a negatively charged surface for the HA-AuNPs, which maintained corresponding colloidal dispersibility of Au nanoparticles in water (Figure S6). While HA-AuNPs is used as an acceptor, we expected that strong adsorption of HA-AuNPs on g-CN nanosheets could be achieved not only via electrostatic attraction between g-CN nanosheets and HA-AuNPs, but also via hydrophobic interaction between g-CN nanosheets and HA backbones.47 More importantly, the g-CN nanosheets exhibits a high planar surface, which enables an increased number of HA-AuNPs adsorbed to the surfaces of g-CN nanosheets.

Figure 1. (A) UV-vis absorption (black) and one photon emission spectra (blue) of g-CN nanosheets solution, λex = 355 nm. Inset shows the color change of g-CN nanosheets solution under sunlight and UV light irradiation, respectively. (B) Two-photon absorption cross sections (δ) of g-CN nanosheets in PB buffer solution. The inset shows two photon excited fluorescence spectrum of g-CN nanosheets in PB buffer solution, λex = 760 nm.

The g-CN nanosheets possessed a ζ-potential value of around +25.7 mV, with hydrodynamic radius of ~120 nm (Figure S1). Elemental analysis revealed three major chemical elements (C, N, H) with a C/N/H molar ratio of around 0.435/0.520/0.034, where H mainly comes from few terminal ones that remained due to incomplete condensation. The surface composition was further identified by X-ray photoelectron spectroscopy (Figure S2), mainly C and N were observed, although oxygen signal was also detected, which could be due to the acidification by HCl during sample preparation. The XRD pattern (Figure S3) presented a strong peak at 27.4°, a characteristic stacking peak of π-conjugated layers with an interlayer distance of d = 0.326 nm, furthermore, compared to the bulk g-CN, the disappearance of in-plane (100) peak at 13.3° and weaker inter-layer (002) peak at 27.4° in the protonated g-CN, indicates that the bulk g-CN was successfully exfoliated into nanoscale layered structures.9,44 The morphology and thickness of the g-CN nanosheets were scanned under atomic force microscopy (AFM). As shown in Figure S4, asprepared g-CN nanosheets exhibited smooth thickness of ~1.62.0 nm. As a result, the large basal planes and high specific surface area of g-CN nanosheets could contribute an increased number of binding sites for biomolecules or drugs.45,46 In addition, the quantum yield of the g-CN nanosheets was measured to be ~15.6%. All those results imply that the as-prepared gCN nanosheets could be superior energy donor candidates in energy transfer process.

Figure 2. TEM images of (A) HA-AuNPs and (B) nanoassembly. (C) High-resolution Au 4f XPS spectra of g-CN nanosheets and nanoassembly. (D) Fluorescence spectra of g-CN nanosheets (a) before and (b) after assembly with HA-AuNPs.

The successful formation of nanoassembly between HAAuNPs and g-CN nanosheets was directly confirmed by TEM. As shown in Figure 2B, the HA-AuNPs were well dispersed on the surface of g-CN nanosheets, neither individual HAAuNPs nor isolated g-CN nanosheets were observed. Elemental analysis of the nanoassembly by energy dispersive Xray (EDS) showed the signals from C, N, O, and Au (Figure S7), the vibrational modes of molecules made from those elements were also observed on corresponding IR spectrum in Figure S5. The high-resolution XPS of Au 4f in Figure 2C showed no obvious signals from the Au elements in the g-CN nanosheets (black line in Figure 2C), while strong Au 4f peak was observed in the spectrum of HA-AuNPs/g-CN nanoas-



sembly (red line in Figure 2C), indicating the metallic state of the gold nanoparticles attached on the g-CN nanosheets. Moreover, the splitting of the 4f doublet of Au is deduced to be 4.0 ev, which agrees well with the theoretical value of the metallic gold.48 It should be noted that, compared to the value of 84.0 eV from bulk metallic gold, the binding energy of Au 4f7/2 shown in Figure 2C displays a down-shift to 83.2 eV, indicating the strong electronic interaction between AuNPs and g-CN nanosheets.

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nanoassembly were observed to exhibit very weak fluorescence at the emission peak of 438 nm (curve a in Figure S8). After incubation with 0.04 U/mL HAase, the mixture showed a very strong fluorescence signal at the emission peak of 438 nm (curve f in Figure S8). The dramatic recovery of the fluorescence signal from g-CN nanosheets in the nanoassembly, originates from the catalytic degradation of glycosidic linkages in HA-AuNPs by HAase, and the simultaneous release of AuNPs from g-CN nanosheets. A further investigation was performed to understand the mechanism of the sensing strategy.

Figure 4. (A) Two-photon fluorescence images of HEK-293, MCF-7, and MDA-MB-231 cells with/out free HA, after incubating with nanoassembly for 4 h. (B) The bar graph is the quantification of relative fluorescence intensity of these cells treated with nanoassembly, normalized to the HEK-293 cells. Figure 3. (A) Fluorescence spectral responses of nanoassembly to HAase with varied concentrations. (B) Fluorescence peak intensity versus the concentration of HAase. Inset is the linear plot of the fluorescence intensity against the HAase concentration at the lower end.

In addition, the loading amount of AuNPs on g-CN nanosheets was determined to be 15.5 µg/µg (Wloaded AuNPs/WgCN) by ICP-MS (See details in SI). This relatively high AuNPs loading in the nanoassembly may be conducive to the efficient quenching of intrinsic fluorescence from g-CN nanosheets. As expected, the strong fluorescence of g-CN nanosheets was effciently quenched (∼95%) after the loading of HA-AuNPs on the surface of g-CN nanosheets (Figure 2D). All those results consistently confirm the successful assembly of HAAuNPs on g-CN nanosheets. Performance of HA-AuNPs/g-CN Nanoassembly. Having successfully constructed the HA-AuNPs/g-CN nanoassembly, we then explored the usage of the nanoassembly for sensing HAase activity. Figure S8 reveals the typical fluorescence responses of the nanoassembly in the assay of HAase. The

As shown in Figure S9, TEM image of HA-AuNPs/g-CN nanoassembly after incubation with HAase (0.04 U/mL) exhibited considerable decrease of AuNPs attached to the g-CN nanosheets (compare Figure S9 to Figure 2B). ICP-MS characterization demonstrated that when the loading amount of AuNPs on nanoassembly decreased by ~6.7%, the recovered fluorescence approached a signal/background ratio (≥ 2) appropriate for confocal microscope imaging. At the same time, the decreased number of attached AuNPs per g-CN nanosheet, namely, the reduced AuNPs density on g-CN nanosheet, would greatly reduce the amount of light scattered. This trend was umambiguously observed in Figure S10, the introduction of HAase promoted segregation between AuNPs and g-CN nanosheets, and the light scattering from aggregated AuNPs on the g-CN nanosheets gradually waned with the increase of HAase concentration. Consequently, the disentanglement of nanoassembly sever the interaction paths (possibly FRETbased) between AuNPs and g-CN nanosheets, inducing the gradual fluorescence recovery of g-CN nanosheets. In contrast, when 50 mg/mL human serum albumin (HSA) or 10% fetal bovine serum was added to the reaction system to replace


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Analytical Chemistry

Figure 5. Depth fluorescence images of nanoassembly (15 µg/mL) in MDA-MB-231 cancer tissue slices. The change of fluorescence intensity with scan depth were determined by spectral confocal multiphoton microscopy in the z-scan mode (step size, 5 µm). TP images were collected in the range of 400-500 nm.

HAase, or the HAase was denatured by heating at 90 °C for 10 min, neither experiment showed any obvious fluorescence increase at the emission peak of 438 nm (curves b, c and d in Figure S8), verifying that the fluorescence recovery was specifically mediated by active HAase. In addition, PAA, a common inhibitor of HAase,49 was added together with HAase into the nanoassembly, only a weak fluorescence peak was detected (curve e in Figure S8), further indicating the effective inhibition of enzyme activity from HAase by PAA. All those results demonstrated that the fluorescence response indeed arises from the enzyme-specific cleavage action, and this designed platform should have excellent tolerance to environment interference, i.e., promising for the efficient detection of HAase. The ability of the nanoassembly for quantitative analysis of the activity of HAase was then investigated. Figure 3 displays typical fluorescence spectra of the nanoassembly after incubating with varied concentrations of HAase. The intensity of the fluorescence peak at 438 nm increased linearly with the HAase concentration in the range of 0-0.015 U/mL, and the detection limit was estimated to be as low as 0.6 mU/mL, which was comparable or better than the majority of existing fluorescence methods for HAase detection.50-52 Such a detection limit corresponds to around 842 cells, after applying our method for quantifying the MDA-MA-231 cell (with high expression level of HAase) concentration. This low detection limit further reveals the high sensitivity of developed nanoassembly for tumor cell detection, also, demonstrates that this nanoassembly can afford a promising platform for highly specific and sensitive detection of HAase, and for related cancer diagnosis. Cytotoxicity Investigation of the Nanoassembly. Before the application of nanoassembly in the living cells, the cytotoxicity was assessed with an MTT assay in MDA-MB-231 cell lines. We chose the cells only incubated with culture medium as blank sample, and the ratio of the cells incubated with the nanoassembly to the blank sample was used to assess the cell viability. As shown in Figure S11, both g-CN nanosheets and HA-AuNPs/g-CN nanoassembly barely show any cytotoxicity. As a type of emerging graphene-like nanomaterial, the gCN nanosheets show excellent biocompatibility and nontoxicity even with a concentration as high as 40 µg/ml, and HAAuNPs/g-CN nanoassembly with varied concentration (5-40 µg/mL) also barely showed any cytotoxicity in living cells,

confirming that the nanoassembly could be used for HAase detection in living cells. Two-photon Imaging of Nanoassembly in Cells and Tissues. To assess the prepared activatable nanoassembly for two-photon imaging of HAase in living cells, two different human cancer cell lines (MDA-MB-231 with high expression level of CD44 and MCF-7 with low expression level of CD44) and one normal human cell line (HEK-293) with different levels of CD44 expression were used for cell imaging. As expected, significant fluorescence recovery of nanoassembly was observed after 4 h incubation with MDA-MB-231 cells. This was due to the fact that the nanoassembly delivered in the cytosol reacted with HAase in the cytosol of MDA-MB-231 cells, resulting in the removal of AuNPs from g-CN nanosheets, then accompanied by the enhancement of blue fluorescence from the original g-CN substrate. In contrast, after incubating nanoassembly with either MCF-7 or normal HEK-293 cells, both of which express low levels of CD44, no obvious fluorescence enhancement was observed (Figure 4). Moreover, a competitive inhibition test was carried out in the presence of free HA to evaluate their CD44-targeting capability. We observed that the presence of free HA significantly decreased the fluorescence intensity, this result implied that the nanoassembly enabled specific targeting at CD44 through HA, which may promote the entry of nanoassembly into cell and then induce activatable fluorescence imaging. In addition, a further localization study revealed that the nanoassembly was not colocalized with lysosomes, confirming cytosolic delivery of the nanoassembly (Figure S12). This may be due to the environment of endo/lysosomal acid organelles, the g-CN nanosheets would be easily protonated and then escape from endo/lysosomal. Moreover, real-time two-photon imaging analysis for nanoassembly showed that the intracellular two-photon fluorescence gradually increased with time (Figure S13), and reached a plateau after 4 h, indicating that the nanoassembly could be adopted for monitoring the fluorescence activation events and shedding light on the related kinetics in living cells. Then, we assessed the ability of the enzyme-activatable nanoassembly for two-photon fluorescence imaging in tissues. The normal nude mice slice, nude mice bearing MCF-7, or MDA-MB-231 tumor slice were incubated with 15 µg/mL nanoassembly for 4 h, respectively. Subsequently, the twophoton fluorescence images of the nanoassembly in each tissue was collected with a two-photon laser scanning confocal



microscope. As shown in Figure S14, the normal nude mice slice or nude mice slice bearing MCF-7 tumor after treating with nanoassembly did not show obvious two-photon fluorescence. However, the slice bearing MDA-MB-231 tumor exhibited strong fluorescence, after incubating with the nanoassembly for 4 h. In addition, the depth fluorescence tissue imaging results of the nanoassembly was shown in Figure 5, by collecting fluorescence signal in the wavelength range of 400-500 nm upon excitation at 760 nm. The nanoassembly could realize tissue imaging at depth range of 0180 µm, which demonstrated that the nanoassembly was capable of effective visualization of HAase levels in cancer tissues down to a depth of 180 µm by two-photon fluorescence imaging. All those results thoroughly demonstrated that our nanoassembly has excellent staining ability, spatial location capability, decent detection proficiency with low background interference, and in-depth imaging, the combination of which make them suitable means for effective and activatable twophoton fluorescence imaging at tissue level. CONCLUSIONS

AUTHOR INFORMATION

ACKNOWLEDGMENT This study was financially supported by the National Natural Science Foundation of China (21527810, 21605044, 21705041, 21705040) and National Key Basic Research Program (2011CB911000).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website It includes additional experimental details; DLS, zeta potential, XPS, XRD and AFM for the characterization of g-CN nanosheets; FT-IR, zeta potential and EDS for the characterization of nanoassembly; Enzymatic activity evaluation of HAase; Cell viability assay; Two-photon confocal fluorescence microscope image.

REFERENCES (1)

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In summary, we developed an activatable nanoassembly for two-photon fluorescence imaging of hyaluronidase in living cells and tissues. In this nanoplatform, the positively charged g-CN nanosheets effectively assembled with negatively charged HA-AuNPs through electrostatic and hydrophobic interactions, leading to an efficient interactions between the g-CN nanosheets and HA-AuNPs. And the dramatically quenched fluorescence from g-CN nanosheets could be successfully recovered in the presence of HAase, which plays the role of degrading HA into low molecular weight fragments, separating the g-CN nanosheets from HAAuNPs, and inducing the reappearance of g-CN nanosheets fluorescence. More importantly, the nanoassembly has the specific targeting at CD44, which may assist the entry of nanoassembly into tumor cell/tissues, and promote corresponding activatable fluorescence imaging. By taking advantages of two-photon fluorescence imaging technology, such as high spatial localization, low background interference, decent temporal resolution, in-depth imaging, as well as excellent permeability and good biocompatibility of the nanoassembly, our method was applied for two-photon fluorescence activatable imaging in living cells and nude mice tissues, both of which were demonstrated with satisfactory results. We expect this activatable two-photon fluorescence imaging strategy should provide a unique platform for earlystage disease diagnosis and related biomedical applications.

ASSOCIATED CONTENT

The authors declare no competing financial interest.

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Corresponding Author *Fax: +86-731-88821916. Email: [email protected].

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*Email: [email protected].

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ORCID Zheng Li: 0000-0001-5281-8101 Jian-Hui Jiang: 0000-0003-1594-4023

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Author Contributions

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‡ These authors contributed equally.

Notes

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