Latent Naphthalimide Bearing Water-Soluble Nanoprobes with

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Biological and Medical Applications of Materials and Interfaces

Latent Naphthalimide Bearing Water-Soluble Nanoprobes with CatecholFe(III) Cores for In Vivo Fluorescence Imaging of Intracellular Thiols Bing Li, Yunlong Yu, Fuqing Xiang, Shiyong Zhang, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02539 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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ACS Applied Materials & Interfaces

Latent Naphthalimide Bearing Water-Soluble Nanoprobes with Catechol-Fe(III) Cores for In Vivo Fluorescence Imaging of Intracellular Thiols Bing Li,†,# Yunlong Yu,†,# Fuqing Xiang,† Shiyong Zhang,†,‡,* and Zhongwei Gu† †

National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road,

Chengdu 610064, China. ‡

College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China.

*

To whom correspondence should be addressed. S. Zhang, E-mail: [email protected]; Phone: +86-28-

85411109. Fax: +86-28-85411109. #

These authors contributed equally to this work.

Keywords: Naphthalimide; Glutathione; Fe(III) Cross-linking; Nanoprobe; Tumor imaging

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ABSTRACT. Here a novel latent naphthalimide bearing water-soluble nanoprobe with catechol-Fe(III) cores (Fe@LNNPs) was designed, synthesized and evaluated for in vivo fluorescence imaging of intracellular thiols, as various diseases are associated with overexpression of cellular biothiols. The Fe@LNNPs are mainly composed of three components. The inner part constitutes pyrocatechol groups, which can coordinate with Fe(III) to form a cross-linked core for improving the stability in the complex biological environment. The naphthalimide group is linked by disulfide with the core to quench the probe fluorescence. The outer part is designed to be a hydrophilic glycol corona for prolonging blood circulation. Also a biotin group can be easily introduced into the nanoprobe for actively targeting the HepG2 cells. The fluorescence spectra reveal that the Fe@LNNPs can be reduced explicitly by glutathione (GSH) to trigger on the fluorescence emission. Confocal microscopic imaging and animal experiments manifest that the Fe@LNNPs, especially with biotin groups, have much better fluorescent signal imaging compared to the reported small molecule probe 1′′ both in vitro and in vivo (up to 24 h). The Fe@LNNPs thus feature great advantages on specificity, stability, biocompatibility and long retention time for thiol-recognition imaging, and hold potential application in clinical cancer diagnosis.


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INTRODUCTION The intracellular thiols, such as glutathione (GSH), cysteine (Cys), and thioredoxin (Trx) et al., play important roles in the regulation of redox-related behavior of biological organism, such as DNA synthesis, apoptosis inhibition, metastasis and angiogenesis.1-9 It is disclosed that various human diseases are tightly in relation to the perturbation of the intracellular thiol-disulfide equilibrium, i.e. redox homeostasis.1, 10-12 For example, due to the genetic alterations and fast growth of cancer cells, the produced reactive oxygen species (ROS) could cause a high level of oxidative stress locally. As a result, more and more GSH was secreted to maintain the antioxidant system for their survival and proliferation,13 which leads to a higher GSH concentration (~10 mM) in cancer cells than in normal tissue and plasma (20-40 µM).6, 8, 14-17 Accordingly, it is of great significance to find an efficient way to detect and track the intracellular thiols, especially in vivo, for understanding the biological processes and the early diagnosis of diseases.7 Fluorescence imaging is a very powerful method for monitoring biomolecules in living systems because of its facility, fast response as well as high sensitivity and selectivity. On the basis of the unique chemical reactivity of thiols, such as reducing capacity and nucleophilicity,18-19 various fluorescent probes have been designed in the last decade for the thiol detection.20 Among them, the recent reported small-molecule fluorescent probes based on the principle of intramolecular charge transfer (ICT) shows great prospects at the specific detection of certain thiols in living cells.21 However, due to low solubility and poor biocompatibility, the reported systems mainly stay at in vitro, which is difficult to apply in vivo.16, 18, 20, 22-23 This defection makes these systems difficult to be promoted to the clinic since no one would be willing to carry out an invasive biopsy, especially in the early stages of the disease. Recently, modification of small-molecule probes with water-soluble moieties and targetable ligands for in vivo intracellular thiol imaging has received attention.21-24 For example, Kim and coworkers reported a multifunctional naphthalimide based probe that was composed of a thiol cleavable disulfide bond, a latent naphthalamide fluorophore, and a specific hepatocyte targeting unit. Confocal ACS Paragon Plus Environment

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microscopic imaging results showed that this probe was preferentially taken up by HepG2 cells through specific targeting and endocytosis. The liver-specificity targeting probe was further evaluated in vivo by using of a rat model.22 However, the in vivo imaging of small-molecule probes suffer from drawbacks of fast metabolism, rapid elimination, low enrichment of the lesion tissue, and produce easily to a larger dose of toxicity, and extensive distribution into other organs and tissues, which are not economical and complicated owing to the nonspecific toxicity.25-28 Very recently, the nanomaterial based probes (i.e., nanoprobes) were fabricated to detect the intracellular thiols,17,

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which seemed holding the potential to overcome the above mentioned

disadvantages suffered by small molecular probes. However, most of the reported nanoprobes have still been studied in cellular level. The complex biological condition in vivo may cause different results as in vitro. As far as we know, there were only two examples for the in vivo detection of GSH. Either the body retention time was short (1.5 h) or a tedious fabrication was needed.17,31 On basis of our previous core cross-linking micelles,34-37 we herein developed a new water-soluble core cross-linked nanoprobe for in vivo fluorescence imaging of intracellular thiols. As detailed in Scheme 1, our strategy revolves around the use of a rationally designed multifunctional amphiphile 1, which features an activatable disulfide-linked naphthalimide fluorophore, an inner pyrocatechol group, and a surface oligo-ethylene glycol (OEG) corona. The OEG chains not only serve as the hydrophilic part, but also make the resulting nanoparticles stealth and long circulation in the blood. The inner pyrocatechol group aims to enhance the stability of nanoparticles by virtue of the coordination between catechol and Fe(III).38-41 This stabilization could avoid the dissociation of nanoparticles in the complex environment in vivo, which often happened in the general self-assemblies due to the dynamic structure.34,

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Taking advantage of the disulfide linkage, the latent naphthalimide buried in the

nanoparticles can be specifically triggered by thiols to activate the dormant fluorescence. The confocal laser scanning microscopy (CLSM) results revealed that our nanoprobe showed preferential imaging behavior as expected. Especially for introducing the targeting group, the nanoprobe behaved a much ACS Paragon Plus Environment

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specific and efficient cellular uptake to HepG2 cells, which is ~2 times stronger than that of probe 1′′ on the fluorescent intensity at the identical incubation condition. Moreover, the animal experiments further confirmed that the nanoprobe presented good fluorescence imaging on tumor tissue even after injection for 24 h, which was much longer than that of the small molecule probes25-28 and reported nanoprobe.31 Featured by simple preparation, highly adjustable functional structure, excellent biocompatibility, high sensitivity and selectivity, and most importantly the noninvasive in vivo imaging effect, the latent naphthalimide bearing water-soluble nanoprobes with catechol-Fe(III) cores (Fe@LNNPs) would have highly prospect in the clinical application of cancer diagnosis. Scheme 1. The schematic representation of the preparation of the water soluble nanoprobe Fe@LNNPs and in vivo fluorescence imaging of intracellular thiols.

MATERIALS AND EXPERIMENTAL SECTION Materials and measurements. m-PEG 750, PEG 800, biotin, DIPEA, p-toluene sulfonyl chloride, sodium azide, triphosgene, 4-amino-1,8-napthalic anhydride, 2,2′-dithiodiethanol, DMAP, HOBT, DIPEA, DCC and protocatechuic acid and triphenyl phosphine were purchased from commercial company, and used as received. All solvents were freshly distilled before added into the reaction system.


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In all aqueous experiments, deionized water was applied. HepG2 cell lines were bought from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and the animals for the experiments were purchased from Experimental Animal Center of Sichuan University and approved by the ethics committee of Sichuan University. As illustrated in Scheme 2, amphiphile 1 was prepared by several steps, and the details were shown in Supporting Information (SI). Also, a structural analogue 1′′ was synthesized as a control. 1H NMR and

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C NMR spectra were obtained on a Bruker Advance 400

spectrometer. Mass spectrometry (MS) was performed on a Thermo LTQ mass spectrometer. The fluorescence emission intensity was measured using a RF-5301PC (Shimadzu, Japan) fluorescence spectrometer. The UV-Visible absorption spectra were obtained using a UV-2600 (Shimadzu, Japan). Dynamic Light Scattering (DLS) Analyzer (Malvern NANO ZS 90) was applied to characterize the particle size and zeta potential. Transmission electron microscope (TEM) imagings were obtained using a FEI Tecnai GF20S-TWIN, at 120 kV. The TEM samples were prepared by dropping the particles solution on a carbon-coated copper grid and then stained with 2% phosphotungstic acid aqueous solution. Evaluation of the cell toxicity was carried out by a Cell Counting Kit-8 (CCK-8) viability assay. After incubated for 2 h, its absorbance at 450 nm was recorded by a microplate reader Varioscan Flash (Thermo Fisher SCIENTIFIC). The GSH detection was carried out by using CLSM (Leica TCS SP5). Typical preparation of the latent naphthalimide bearing nanoprobes (LNNPs). Compound 1 (11.3 mg, 0.01 mmol) was added to 10 mL of DI water, and then it was shaked at room temperature. After a few minutes’ micelle assembly, the nanoprobe was formed spontaneously with a strong Tyndall effect. Characterization of the critical aggregation concentration (CAC) of amphiphile 1 and compound 4. Pre-calculated amount of nile red was dissolved in 1 mL of CH2Cl2, after which the solution was moved into several vials and then evaporating the CH2Cl2. The solution of 1 was added into each vial and the deionized water was added to make the concentration of 1 ranging from 1 to 1000 ACS Paragon Plus Environment

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µM, simultaneously all the nile red had a concentration of 1 µM. The vials were kept shaking overnight at room temperature, and the fluorescence emission intensity, which was excited at 485 nm, was measured. The critical aggregation concentration was acquired as the node of the two linear lines on the graph. The CAC of compound 1 was ~ 60 µM and the same procedure was conducted to obtain the CAC of compound 4 as ~ 75 µM. Typical preparation of latent naphthalimide bearing nanoprobes with cross-linked catecholFe(III) cores (Fe@LNNPs). Fe@LNNPs were prepared through the dialysis method and simultaneous cross-linking process. An aqueous solution of FeCl3 (40 mM, pH 1.8) was firstly dropped into a stirred solution of LNNPs (1 mM) at the feed molar ratio of [1] : [Fe(III)] = 2:1. To form Fe(III)-catechol biscomplex formation, the solution pH was adjusted to 7.4. After stirred for 1 h, the solution was dialyzed (MWCO, 1 kDa) in PBS buffer to exclude the unreacted ionic species, then the supernatant solution was freeze-dried to get the Fe@LNNPs. Finally it was re-dissolved in water at the concentration of 1 mM. The concentration of Fe(III) was deemed to be half of the LNNPs because of the pH-controlled stoichiometry of catechol-Fe(III) complexes.38 For the construction of targeted Fe@LNNPs, a similar procedure was carried out except replacement of the micelle dispersion with amphiphile 2 contained micelle solution ([1] = 1 mM; [2] = 0.1 mM). Stability assay of Fe@LNNPs. The stability of Fe@LNNPs was measured through diluting 1 below its CAC (60 µM). First, the cross-linked micelles ([1] = 1 mM) was diluted to the concentrations of 500 µM, 250.0 µM, 125.0 µM, 62.5 µM, 32.0 µM, 16.0 µM, 8.0 µM, respectively. Afterwards, the samples of the above solutions were tested by DLS to evaluate the stability. The stability of LNNPs and Fe@LNNPs in fetal bovine serum (FBS) was experimented by incubating with 10% (V/V) FBS for at least 12 h. pH-dependent coordination between LNNPs and Fe(III) ions. . The aqueous mixtures of 1 (0.1 mM) and FeCl3 (the molar ratio of [1] : [Fe(III)] = 2:1) at various pH conditions (pH = 5.0, 7.4) were


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prepared. The variation in the absorption spectra was monitored from 380 to 700 nm by using ultraviolet spectrophotometer. Photo images of the aqueous mixture at various pH were obtained in 2 clear window cuvettes. GSH detection of Fe@LNNPs. . Parent stock solutions (10 mM) of Na+, K+, NH4+, Mg2+, Ca2+, Zn2+, Fe3+, Cu2+, Al3+, Ba2+, Ag+, Mn2+, Arg, Gly, Cys-Cys, Asp, Glu, Lys, Ser, H2O2 and GSH were prepared in DI water. Cys and Hcy were also implemented with concentrations at ~100 µM. Then Fe@LNNPs was added into each test tube to make its concentration as 10 µM. The fluorescence spectra were obtained after various analytes addition at 37 oC for 2 h (other than time-dependent experiments) by excitation at 430 nm. The excitation and emission slit widths were all 5 nm. For the detection of GSH in broken cells, about one million cancer cells were dispersed in PBS buffer (0.01 mM, 10 mL), and the optimal condition for ex traction was as following: when putting into liquid nitrogen cooling environment, the output power of ultrasonic cell breaker is 200W, radiation/intermission time 5 s / 5 s, total working time 10 min, then the supernatant was mixed with Fe@LNNPs (10 µM). The fluorescence spectra were obtained after analytes addition at 37 oC for different times by excitation at 430 nm. Cytotoxicity assay. Corresponding HepG2 cells (2000/well) were seeded in two 96-well plates and incubated in a cell-incubator at 37 oC under 5% CO2 atmosphere. After the incubation of 24 h, the culture media was totally removed and 200 µL media containing Fe@LNNPs, targeted Fe@LNNPs with a series concentrations ranging from 0 to 500 µM were added to the corresponding well, separately. Cells with fresh media were set as control. After 48 h, the added culture media were removed, following by adding 100 µL fresh media containing 10 µL CCK-8 to each well. The two plates were incubated at 37 oC for another 2 h. Then, each well was measured to get the absorbance at 450 nm via a microplate reader Varioscan Flash. In vitro GSH detection. Corresponding HepG2 cells (5 × 104 cells/mL) were seeded in a glass Petri dish (Φ = 35 mm) and incubated in a cell-incubator for 24 h. Afterwards, the cells were cultured


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with 1′′ (1 µM), 1 (10 µM), Fe@LNNPs (10 µM) and targeted Fe@LNNPs (10 µM) at 37 °C for 8 h respectively. The stale medium was then removed and PBS was added to wash the cells for three times. The cell imaging was observed under CLSM with an excitation at 405 nm and a long path emission filter (> 505 nm). In vivo GSH detection. To determine the biodistribution, HepG2 tumor-bearing nude mice (each rat weighed 20 g) were intravenously injected with 1′′ (50 µg mL−1, 100 µL), Fe@LNNPs (200 µg mL−1, 100 µL) and targeted Fe@LNNPs (200 µg mL−1, 100 µL) via the tail vein. After that, the fluorescence signals of nude mice after 1, 4, 12, and 24 h injection was obtained with fluorescence imaging system (CRi Maestro EX, USA), then the heart, liver, spleen, lungs, kidneys, and tumor from the experimental mice (n = 3) were collected for analysis.

RESULTS AND DISCUSSION Synthesis and Characterization of Latent Naphthalimide Bearing Nanoprobes with Crosslinked Catechol-Fe(III) Cores (Fe@LNNPs). The amphiphile 1 was prepared following the synthetic route in Scheme 2 and fully characterized by 1H NMR, 13C NMR, and high resolution MS spectra (see SI for details). With a hydrophobic naphthalimide-SS-catechol head and a hydrophilic OEG tail, the multifunctional amphiphile 1 was soluble in water and gave rise to a strong Tyndall effect, which was the characteristic of forming micelles. The hydrodynamic diameter of the latent naphthalimide bearing nanoprobes (LNNPs) was ~120.0 nm, which is shown in Figure 1a and the CAC was ~55.0 µM (see Figure S1). The morphology characterization using TEM further confirmed that the spherical particles were formed with mean size of ~106 nm (Figure 1a). The design of compound 1 incorporated numerous catechol groups in the core of the micelles, which were ready for coordination in the presence of Fe(III) to get the robust latent naphthalimide bearing nanoprobes with cross-linked catechol-Fe(III) cores (Fe@LNNPs, Scheme 1). The coordinated


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catechol-Fe(III) system was chosen for covalent capturing mainly based on the following considerations: (1) Iron ions are harmless and essential trace element found in nearly all living bodies. (2) The high coordination constant between Fe(III) and catechol is enough to stabilize the Fe@LNNPs to tolerate the complex internal environment.39-41 (3) The coordination reaction occurs at room temperature without auxiliary agents, which is cleaner and more environment-friendly compared to the other covalent capture systems.38 (4) The coordination between Fe(III) and catechol are pH-sensitive, which makes it broken easily in endosome so as to realize rapid biodegradation.39, 44 (5) It was reported recently that Fe(III) could react with the over expressed H2O2 in cancerous tissue to form the OH· for cancer therapy,41, 45-46 which reveals the potential of the nanoprobe for further theranostic application. Scheme 2. Synthesis of typical compounds.


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Figure 1. TEM and DLS characterization of (a) LNNPs and (b) Fe@LNNPs. (c) UV-vis absorption of 2:1 of Fe(III): [1] at pH 5 and 7.4, and (d) the corresponding color variation of the solution. Samples in (a) and (b) were stained by adding of 2% phosphotungstic acid aqueous solution, [1] = 7.9 × 10−4 M, and the scale bar is 100 nm. In (c) and (d), [Fe@LNNPs] = 7.9 × 10−4 M, and pH was adjust by HCl. The DLS measurement results showed that the particle size after cross-linking (~90.0 nm, Figure 1c) was a little decrease compared to the uncross-linked micelle, which was reasonable due to the shrinkage of the micelle core after cross-linking. This change was further confirmed by TEM characterization, which gave a diameter decrease from prior to cross-linking ~120.0 nm to after crosslinking ~85.0 nm (Figure 1b). The success of the coordination between Fe(III) and catechol was also verified by UV-vis spectroscopy. As shown in Figure 1c, the mono-complex of Fe(III)-catechol possessed an obvious absorption at 410 nm at pH = 5.0, while as pH was increased to pH = 7.4, two new absorption peaks appeared at 430 and 525 nm, and concomitantly, the solution color changed


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dramatically from colorless at pH = 5.0 to light brown at pH = 7.4 (Figure 1d), suggesting the biscomplex formed.37-38 Enhanced Stability of the nanoprobe Fe@LNNPs. The stability of Fe@LNNPs was firstly investigated through dilution method monitored by DLS. With gradually diluting the aqueous solution of amphiphile 1, the formed particle size kept relatively the same (Figure 2a). Even below the CAC, there was not much variation on the particle size. After that, 10% FBS was used to mimic the blood stream in vitro to evaluate the stability of Fe@LNNPs during circulation. After incubation for 12 h, there was almost no change of the particle size, as shown in Figure 2b. However, for uncross-linked LNNPs, almost no reasonable size could be detected by DLS under the same incubation condition, suggesting the tolerance of Fe@LNNPs to the blood circulation.

Figure 2. Stability test of LNNPs and Fe@LNNPs. (a) Size of Fe@LNNPs as a function of [1] in aqueous solution. (b) Particle sizes of LNNPs and Fe@LNNPs after incubation with 10% FBS at 37 °C for 0h and 12 h, [LNNPs] = [1] = 7.9 × 10−4 M. Specific response of Fe@LNNPs for GSH. Once the stable Fe@LNNPs was achieved, we turned to test their specific response to thiols. The UV-vis spectrometer was used to monitor the spectral change of Fe@LNNPs upon their reaction with thiol-containing compounds. As shown in Figure 3a, the


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Fe@LNNPs had a maximum absorption at 365 nm, and the spectrum underwent a red-shift to 430 nm upon addition of a GSH-containing solution under the physiological condition (at 37 oC in PBS buffer, pH 7.4) for ~2 h. Then, the dose response of GSH to Fe@LNNPs was tested by using fluorescence spectrophotometer and the data were shown in Figure S2. With increase of the GSH concentration, there was a linear increase of the fluorescence intensity at 540 nm before 5 mM. After that, the fluorescence intensity reached a plateau. For the following experiments, GSH with a concentration of 10 mM was chosen according to the environment in cancerous cells.22 Further the fluorescence change of Fe@LNNPs was monitored by varying the incubating time with GSH, which was shown in Figure 3b. Initially the Fe@LNNPs (10 µM) displayed a weak fluorescence mission at 540 nm. Upon incubation with GSH for various time, the fluorescence intensity increased gradually. Especially, after 100 minutes incubation, the emission intensity at 540 nm was enhanced about 7 times. The mechanism of GSHmediated fluorescence change could be referenced to the ICT.21 Before treated with GSH, the fluorescence of the modified probe was quenched. Upon GSH addition, the disulfide could be cleaved. Then the intramolecular cyclization was followed, which finally caused the cleavage of a neighboring carbamate bond. Then the released naphthalimide group could be triggered to give a bright green fluorescent signal. Besides the enhanced fluorescence, there was also a linear correlation between the fluorescence intensity of F540/F475 as a function of time (Figure S3). Since the disulfide bonds were entrapped in the core, a certain time was needed to reach the interchange equilibrium reaction with GSH47. Thus the ICT-based probe showed this linear relationship. To further confirm the GSH specificity on the Fe@LNNPs, a well-known thiol inhibitor N-ethylmaleimide (NEM) was preincubated with GSH for 1 h, and the spectrum was shown in Figure S4. At the presence of this agent, no obvious fluorescence change could be detected after culturing with Fe@LNNPs, which confirmed the GSH specific response. As reported, GSH is overexpressed in cancer cells,22 and the cell lysates should also have the function of GSH to reduce the disulfide. Here we used HepG2 cell lysates to culture with Fe@LNNPs. The time course of fluorescence intensity was detected and similar spectrum changes were


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shown in Figure S5, indicating the breakage of disulfide and release of fluorescent probe. In addition, the interferences from other biologically relevant analytes were also conducted to prove the specific GSH response of Fe@LNNPs. Various essential ions, thiol-free amino acids and thiols were incubated with the nanoprobe separately under simulated physiological conditions at a concentration of 10 mM. As shown in Figure S6, all the three thiols could strongly increase the fluorescence intensity of Fe@LNNPs at 540 nm. However, in the real tumor cell environment, the concentration of GSH was about 100 times higher than that of Cys and Hcy. Thus, Cys and Hcy were implemented with the concentrations of 100 µM according to the real concentration in human body.6, 21, 48

Figure 3c showed that only GSH could significantly enhance the fluorescence intensity of

Fe@LNNPs at 540 nm, suggesting a high specific choice. For determining the residues of Fe@LNNPs treated with GSH, the products after incubation were analyzed by MALDI-TOF mass spectroscopy. The predominantly detected peak at m/z 821.3924 was assigned to compound 4 (Figure S7), which could not be detected in the solution containing NEM. Thus, we could infer that the Fe@LNNPs showed a precise and selective response to GSH (Scheme 1). Considering the varied pH environment in vivo, the pH sensitivity of the GSH-mediated disulfide-cleavage was also investigated. As shown in Figure S8, the disulfide was stable from pH = 1 to 11 with the absence of GSH. When GSH was present in the system, the disulfide-cleavage showed a high activity within pH from 5 to 10, which was the biologically relevant pH range. All these findings above reveal that Fe@LNNPs have the potential application to detect cellular GSH without interference from analytes and pH of the circumstance.


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Figure 3. Specific response of Fe@LNNPs (10 µM) for GSH. (a) UV-visible spectra of Fe@LNNPs before (black) and after (red) GSH addition. (b) Time course of fluorescence changes of Fe@LNNPs in the presence of GSH (10 mM) at excitation of 430 nm from 0 to 100 minutes. (c) Fluorescence response of Fe@LNNPs in the presence of different ions and bioreductive thiols at concentration of 10 mM, except for Cys and Hcy at 100 µM. All the measurement condition is at 37 °C in PBS buffer (pH 7.4).

Intracellular GSH Detection. After investigating the specificity of Fe@LNNPs for GSH in solution, we turned towards the detection of intracellular GSH. As active targeting could increase the target efficiency through the recognition of specific groups on the surface of cells, lots of researches focused on the investigation of introducing targeting group on the probe.23,

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beginning, the Fe@LNNPs were composed of functional small-molecule amphiphile, which made introducing targeting groups on the nanoprobes easily. To show this superiority and endow the Fe@LNNPs with high recognition capability, the amphiphile 2 with biotin unit was synthesized ACS Paragon Plus Environment

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(Scheme 2). As well-known, biotin could specifically recognize HepG2 cells with a high expression of avidin. The DLS showed that the formed micelles containing 10% amphiphile 2 was not much difference with that of amphiphile 1 while the zeta potential increased to -11 mV from -18 mV (Figure S9), suggesting the successful incorporation of 2 into the nanoprobe.50 It should be mentioned that the Fe@LNNPs with or without targeting groups showed no cytotoxicity to HepG2 cells for 48 h incubation even up to 500 µM (Figure S10). For comparison of the cellular detection effect, the CLSM was conducted to characterize the intracellular fluorescence of LNNPs, Fe@LNNPs and targeted Fe@LNNPs by HepG2 cells, and a reported small molecule probe 1′′ with similar structure as the control. The cell uptake images after incubated for 8 h were shown in Figure 4a. The green fluorescence in the cytoplasm of HepG2 could be observed by incubation with all the various probes. Fe@LNNPs showed comparable fluorescence intensity as that of LNNPs, and both of them were a bit lower than that of probe 1′′. This might be ascribed to the fact that the lipophilic alkyl chain guided the probe 1′′ inserting to the cell membrane and subsequent easy diffusion across the membrane,20 while the OEG part of LNNPs and Fe@LNNPs owned the negative zeta potential, which relatively reduced the cell uptake efficiency. However, when biotins were introduced, the targeted Fe@LNNPs showed much stronger green fluorescence. The fluorescence intensity was almost twice than that of Fe@LNNPs and probe 1′′, which indicated the targeted Fe@LNNPs were taken up selectively into HepG2 cells through avidin-mediated endocytosis. In addition, as HepG2 cells were preincubated with NEM for one hour, there was nearly no fluorescence after further incubation with targeted Fe@LNNPs for 8 h. Moreover, the control experiment was also conducted by incubating targeted Fe@LNNPs with normal cells L929 as the same condition as that of HepG2 cells. As shown in Figure S11, the L929 cells showed negligible fluorescence, as there was less GSH containing inside compared to cancerous HepG2 cells.8, 22 All these results demonstrate that the Fe@LNNPs can be used to detect intracellular GSH in vitro as a result of thiol-induced disulfide cleave


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and producing lots of fluorophore 4 as proposed in Scheme 1.

Figure 4. Intracellular GSH detection by various probes. (a) Confocal microscopy images of HepG2 cells treated with probe 1′′ (1 µM), 1 (10 µM), Fe@LNNPs (10 µM), targeted Fe@LNNPs (10 µM), and the NEM (1 mM) primed cells incubated with targeted Fe@LNNPs (10 µM), for 8 h at 37 oC. (b) Relative pixel intensity of the HepG2 cells extracted from the confocal microscopy images. Scale bar = 20 µm. In Vivo GSH Detection. The in vitro evaluation showed that our Fe@LNNPs, especially with the targeting groups, had specific and effective GSH detection in HepG2 cells. To verify the tumor-marking effect, we investigated the in vivo GSH detection of Fe@LNNPs for nude mice bearing HepG2 tumors, and the results were shown in Figure 5a. At the first 4 h post injection, the fluorescence signal of probe 1′′ could be detected and gradually enhanced a bit with time. While no visible signal could be detected anymore after 12 h, which indicated that probe 1′′ was quickly cleared out due to the renal metabolism.51-52 In contrast, the fluorescence signals became gradually stronger as a function of time after injection of Fe@LNNPs even after 12 h, which showed that there were many accumulations of probes in tumor section. After 24 h, the signal intensity decreased a bit, but still much stronger than that of probe 1′′. There are two reasons to explain the long term tumor-marking effect of Fe@LNNPs. On one hand, the catechol-Fe(III) coordination improved the stability of nanoprobes, and thus enhanced the ACS Paragon Plus Environment

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passive targeting to tumor tissue.53 On the other hand, the OEG tails attached on the particle surface would prolong the blood circulation. Then amounts of Fe@LNNPs could accumulate in the tumor tissue for the GSH detection. More intriguingly, the targeted Fe@LNNPs presented much stronger fluorescence signals than that without targeting groups, which would be attributed to that the biotin groups afforded the nanoprobes with more active targeting. Notably, the in vivo fluorescence imaging time over 24 h was much longer than that of reported nanoprobe (1.5 h),31 demonstrating a superior diagnostic effect. For further investigating the biodistribution of these probes at major organs and tumor, the mice were sacrificed after intravenous injection for 24 h, and the images were shown in Figure 5b. The mice treated with small molecule 1′′ had a relatively high fluorescence signal in liver and kidney, which were 1.4 and 1.1 times separately compared to those with Fe@LNNPs injection (Figure 5c). However, their fluorescent signal at tumor section was relatively low, which indicated there was a small amount of tumor accumulation by injecting probe 1′′. For Fe@LNNPs, the fluorescent intensity at tumor tissue was about 2-fold stronger than that of 1′′. Especially for the targeted Fe@LNNPs, they possessed much stronger fluorescence at the tumor section, which increased 3.3-fold and 1.5-fold compared to probe 1′′ and Fe@LNNPs, respectively. This result suggested that the biotin modified nanoparticles could actively and efficiently trap into HepG2 cells to be reduced by GSH to release the fluorophore in vivo.


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Figure 5. In vivo biodistribution of probe 1′′ (50 µg mL−1, 100 µL), Fe@LNNPs and targeting Fe@LNNPs (200 µg mL−1, 100 µL), through intravenous injection in the HepG2 cancer mice model. (a) The fluorescence images of nude mice at 1, 4, 12 and 24 h post administration in vivo, (b) The fluorescence images of major organs and tumors of the nude mice after the probe injection for 24 h. (c) The fluorescence signals analysis of major organs and tumors after the injection of probe 1′′, Fe@LNNPs and targeting Fe@LNNPs for 24 h. Red circles indicate the position of tumors (*p < 0.01, **p < 0.05).

CONCLUSION In conclusion, a novel latent naphthalimide bearing water-soluble nanoprobes with catechol-Fe(III) cores for in vivo fluorescence imaging of intracellular thiols had been established, which successfully overcome the stability and rapid kidney clearance of traditional small molecule probe. The in vivo experiments disclosed that the novel Fe@LNNPs presented good fluorescence imaging on tumor tissue even after injection for 24 h, which was much better than that of the small molecule probes and reported nanoprobe (1.5 h).31 Thus a promising application would be expected in cancer diagnosis. In particular,


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as Fe(III) is involved in the system, the potential function of ferroptosis can be introduced for the theranostics.41, 45-46 The corresponding work is undergoing and will be reported later.

ASSOCIATED CONTENT Supporting Information. Additional 1H NMR spectra,

13

C NMR spectra of synthesized compounds,

fluorescence spectra and cytotoxicity assay are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author. *To whom correspondence should be addressed. S. Zhang, E-mail: [email protected]. Author Contributions. #

These authors contributed equally to this work.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 51673130 and 51703145), the Applied Basic Research Project of Sichuan Province (15JC0440), the Excellent Young Foundation of Sichuan Province (Nos. 2014JQ0032 and 2016JQ0028) and the Postdoc Research Foundation of Sichuan University (2017SCU12009) and China Postdoctoral Science Foundation (2017M620426). Dr. Nor Hakimin Bin Abdullah from Universiti Malaysia Kelantan was acknowledged for the grammar revision.

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Latent Naphthalimide Bearing Water-Soluble Nanoprobes with

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