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A Nanoscale Metal-Organic Framework Based TwoPhoton Sensing Platform for Bioimaging in Live Tissue Chan Yang, Kun Chen, Mei Chen, XiaoXiao Hu, Shuangyan Huan, Lanlan Chen, Guosheng Song, and Xiao-Bing Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04405 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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Analytical Chemistry

A Nanoscale Metal-Organic Framework Based Two-Photon Sensing Platform for Bioimaging in Live Tissue Chan Yang,† Kun Chen,† Mei Chen,§ Xiaoxiao Hu,† Shuang-Yan Huan,*,† Lanlan Chen,‡ Guosheng Song,† Xiao-Bing Zhang*,† †Molecular

Science and Biomedicine Laboratory, College of Chemistry and Chemical

Engineering and College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, People’s Republic of China §College

of Materials Science and Engineering, Hunan University, Changsha 410082,

People’s Republic of China ‡ Shandong

Provincial Key Laboratory of Detection Technology for Tumour Markers,

College of Chemistry and Chemical Engineering, Linyi University, Linyi, Shandong 276005, People’s Republic of China *To whom correspondence should be addressed. E-mail: [email protected] [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Nanoscale metal-organic frameworks (NMOFs) have been applied for biomedical sensing in recent years. However, it is still a great challenge to construct high efficient NMOFs fluorescent probe for sensing in biological system, with high signal-to-noise ratio, photostability, and deep tissue penetration. Herein, for the first time, we report the two-photon metal-organic framework (TP-MOF) as a sensing platform. The design of TP-MOF is based on NMOFs incorporated a target-responsive two-photon organic moiety through click chemistry. PCN-58, as a model building block, was covalently modified with small-molecule probe for H2S or Zn2+ as model analytes. TP-MOF probes retain the fluorescence-responsive properties of the TP organic moiety and possess excellent photostability, selectivity, as well as biocompatibility. Benefitting from the near-infrared (~820 nm) excited two-photon fluorophore, TP-MOF probes serve to sensing and imaging their respective targets in live cells and tissue slices with a penetration of 130 µm. The molecular design presented here bodes well for the extension to other MOFs displaying sensing components for other analytes of interest.

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INTRODUCTION Metal organic frameworks (MOFs), composed of metal ions and bridging organic ligand, have emerged as a promising class of functional materials that have invigorated extensive interest in recent years thanks to their highly ordered porosity, pore tunability, as well as structural diversity1-4. In particular, the nanoscale MOFs with tunable size, tailorable surface chemistry, good biocompatibility and biodegradability, have been widely applied in biomedical imaging5-8. Very recently, fluorophore integrated MOFs have been developed for fluorescent biosensing9-15. For example, Lin’s group developed a MOF nanoprobe for imaging pH changes in live cells16, demonstrating that MOFs based nanoprobe were efficiently internalized by the cells and remained structurally intact inside endosomes. Deng and coworkers reported the first example of detecting ATP in live cells through incorporating ZIF-90 with RhB as fluorescent signal reporter17. Besides, amino-functionalized MOFs for DNA sensing18 were reported. In addition, some MOF-based sensors were built gas molecules, such as sulfur dioxide or hydrogen sulfide12, 15. And redox-based probes using MOFs as building blocks were constructed for highly selective detection of ions in live cells19. The efficient sensing performance of these probes benefitted from the highly porous and oriented structures, which accommodate efficient loading of diverse imaging cargoes as well as filtering out large-sized biological interferent components. Thus, MOF-based fluorescent probes offer some interesting and distinct advantages over other nanoprobes in intracellular imaging. However, most of these fluorescent sensing probes are realized with onephoton (OP) excitation, resulting in low signal-to-background ratios or photo 3



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bleaching20, which largely restricts the applicability of MOF-based sensors in biological samples. To address these issues, fluorescence imaging technology with TP excitation was developed to improve the intracellular sensing and imaging performance. The two photon fluorescent probes exhibits many advantageous features when compared with conventional

one-photon

fluorescence

probes

including

minimal

tissue

autofluorescence, deeper penetration depth, and lower photo induced damage21-25. Integrating the strengths of TP fluorophore and MOF-based probes, the TP-MOF-based probes would be a desired fluorescent sensing platform for more effective intracellular sensing as well as deep tissue imaging. Among the many conjugation reactions at our disposal, the Cu(I)-catalyzed azidealkyne cycloaddition (CuAAC) was demonstrated a great opportunity to functionalize MOFs26-31. The click reaction shows many advantages such as, the mild reaction conditions, high yields, and well-retained framework and crystallinity of the functionalized MOFs. Such critical features prompted us to use it for the functionalization of well-structured MOFs. Herein, for the first time, we reported a TP-MOF sensing platform employing click chemistry for sensing and imaging in biological systems. We chose PCN-5830, an azideappended MOFs, as a model building block, which was assembled with zirconium and elongated organic linkers to ensure large enough cavities for loading fluorophore. The MOFs then served to covalently cross link with TP fluorescent organic probes via CuAAC without any cross reactivity towards the MOF structure itself or other 4


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functional groups present on the organic probes. And then we constructed TP-MOF probe 1 and TP-MOF probe 2 (Scheme 1A) for sensing H2S and Zn2+ respectively as model analytes, which were involved in various physiological processes or various diseases such as Alzheimer’s disease32-41. Our TP-MOF probes retained the fluorescence-responsive properties of the TP organic moiety and they served to detect their respective targets in live cells and tissue using two-photon microscopy (TPM) (Scheme 1B), with a tissue penetration depth of 130 μm.

Scheme 1. Schematic illustration for the construction of TP-MOF sensing platform (A) and the process of intracellular sensing (B).

EXPERIMENTAL SECTION 5



Reagents and Instruments: Both the silica powder (100-200 mesh) for column chromatography and the thin layer chromatographic sheets (60 F254) for TLC were purchased from Qingdao Ocean Chemicals (Qingdao, China). NMR spectra were conducted by using Bruker DRX-400 spectrometer with tetramethylsilane (TMS) as an internal standard. All chemical shifts were reported in the standard δ notation of parts per million. Powder X-ray diffraction (PXRD) data was collected on Rigaku Miniflex II diffactometer at 30 kV, 15 mA for Cu Ka (l = 1.5418 Å), with a scan speed of 5°/min and a step size of 0.05° in 2θ at room temperature. One-photon fluorescent experiments were conducted on a Hitachi-F4500 fluorometer with both excitation and emission slits of 5.0 nm. Two-photon fluorescence studies were carried out by employing a mode-locked Ti:sapphire pulsed laser (Chameleon Ultra II, Coherent Inc.) as excitation and a DCS200PC single photon counting (Beijing Zolix Instruments Co., Ltd.) as collector. Two-photon fluorescence images were obtained using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). Preparation of TP-MOF Probes ! CAUTION: Azide compounds are potentially explosive, and also display significant human toxicity. Be extremely careful when heating of these compounds. Wear goggles and gloves, and perform the experiment in a fume hood. PCN-58 was synthesized according to literature protocols30 with only minor modifications. In brief, ZrCl4 (24 mg) and TPDC derivative dicarboxylic acid ligand (synthesis details in SI) (30 mg) were ultrasonically dissolved in DMF (3.0 mL) and 6


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heated to 100 °C for 48 h in an oil-bath pan. After cooling down to room temperature, the resulting light yellow solid was centrifuged and stirred with absolute ethanol for 3 days at room temperature before being centrifuged again, then washed with ethanol three times, before being dried under vacuum. Next, the synthesized MOFs were modified with the organic probes through the Cu(I)-catalyzed azide-alkyne cycloaddition protocol (CuAAC). PCN-58 (10 mg) in DMF (5 mL) was reacted with organic moiety for H2S, named as alkynyl-BR-NH2 (52 mg) (synthesis details in Supporting Information) under nitrogen and in the presence of CuI (5 mg) at 60 °C for 30 h. The product was centrifuged and washed with DMF and ethanol, and then dried in vacuum. The amino group of this material (PCN-58-BRNH2) was then turned into azide group by use of t-BuONO and TMSN3 to approach the synthesis of probe 1. Probe 2 was prepared by reacting PCN-58 (10 mg) in DMF (5 mL) with organic moiety for Zn2+, named as alkynyl-DL (70 mg) (synthesis details in Supporting Information) under nitrogen and in the presence of CuI (3 mg) at 60 °C for 30 h. The product was centrifuged and washed with DMF and ethanol, and then dried in vacuum, thus obtaining probe 2. Part of two probes were separately dispersed in water with HF and kept at room temperature for 1 h to decompose. The suspension was filtered and washed with water (200 mL), and then the residue was dried in vacuum for NMR spectra record. In vitro detecting of H2S and Zn2+ To study the performance of TP-MOF as nanoprobes for fluorescent molecular sensing, the probe was dispersed in 1×HEPES (10 mM, 150 mM NaCl, pH 7.4). A 7



series of NaHS or Zn2+ solutions at rising concentrations were added to dispersions of probe 1 or probe 2 (100 μg/mL) accordingly. The resulting mixtures were then incubated at 37 °C for 1 h before being analyzed by fluorescence spectroscopy. Biostability in serum/cell culture The TP-MOF probe was dispersed in fetal bovine serum (FBS, HyClone, UT) or high-glucose Dulbecco’s modified eagle’s medium (DMEM, HyClone, UT), with a final concentration of 100 μg/mL. These dispersions were kept at room temperature for 72 h. Subsequently, the probe was collected as a powder via centrifugation (13000 rpm, 5 min, RT) and washed with water 3 times, then dried under vacuum, before being stored at room temperature in a desiccator prior to PXRD analysis. Cytotoxicity assay An MTS assay was performed. First, HeLa cells were seeded in a 96-well plate at a density of 5× 103 per well and incubated with the probe at concentrations ranging from 10 to 200 μg/mL for 48 h. After the incubation, the medium containing probes was removed, then fresh culture medium (100 μL) was supplied. The cell viability was measured by adding 20 μL of MTS solution to each well followed by incubation of 0.5h, then the UV absorbance of MTS at 490 nm was recorded with a microplate reader. Intracellular sensing of H2S and Zn2+ For intracellular sensing of H2S, HeLa cells were first seeded on glass-bottom culture dishes (Nest, China) for 24 h. Probe 1 (10 μL, 10 mg/mL) was mixed with 990 μL DMEM and then incubated with HeLa cells for 6 h. The cells were then washed 3 times with DPBS and used for TPM imaging at 820 nm excitation wavelength. HeLa cells 8


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were first incubated with probe 1, then further incubated with NaHS (200 μM) for 30 min. These same experimental conditions were also applied to incubation with A549 and HepG2 cells. In the case of probe 2, TPM imaging for presence of Zn2+ in live cells was investigated. HeLa cells were seeded on glass-bottom culture dishes, and first incubated with probe 2 (100 μg/mL) for 6 h, then with Zn2+ (20 μM) and pyrithione (40 μM) for 30 min. Subsequently, the cells were washed with DPBS and then incubated with DMEM for TPM imaging at 820 nm excitation wavelength. Imaging of H2S and Zn2+ in tissue slice For imaging in tissue slices of TP-MOF probes, frozen rat liver or lung tissue slices were thawed at 37°C with wet cotton, then incubated with probe 1 (100 μg/mL) for 6 h, respectively. They were further incubated with NaHS (200 μM). Imaging of Zn2+ in rat liver tissue slices was explored by first incubating with probe 2 (100 μg/mL) for 6 h, then further incubating with Zn2+ (20 μM). Subsequently, the tissue slices were washed with DPBS for TPM imaging at 820 nm excitation wavelength.

RESULTS AND DISCUSSION Characterization of TP-MOF probes Morphology of TP-MOF probes were confirmed through transmission electron microscope (TEM) with results shown in Figure 1A and 1D, presenting a structured morphology with an average size of 200 nm. The structural identity of TP-MOF probes with naked MOFs were confirmed through powder X-ray diffraction (PXRD) (Figure 1B and Figure 1E). Similar peaks of PXRD pattern between TP-MOF probes and naked MOFs demonstrated the well preserved morphologies and crystalline structures 9



of TP-MOFs inherited from MOF building blocks without significant changes as a consequence of their chemical modifications. Besides, UV-vis absorbance spectra (Figure 1C and 1F) demonstrate successful conjugation of TP organic probes with MOFs.

Figure 1. (A) TEM image of the probe 1 nanoparticles. (B) PXRD pattern of probe 1 nanoparticles. (C) UV-vis absorbance of MOF (black curve), organic probe for H2S (red curve), and TP-MOF probe 1 (blue curve). (D) TEM image of the probe 2 nanoparticles. (E) PXRD pattern of probe 2 nanoparticles. (F) UV-vis absorbance of MOF (black curve), organic probe for Zn2+ (red curve), and TP-MOF probe 2 (blue curve). Two-photon properties of TP-MOF probes The TP action cross-section values (Φδ)42-46 excited at 820 nm were measured to be 137.8 GM for probe 1 reacted with NaHS (Figure 2A), and 99.4 GM for probe 2 reacted with Zn2+ (Figure 2C), and the reaction processes between TP-MOF and target analytes were illustrated in Scheme 2. Figure 2B and Figure 2D displayed the maximum 10


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fluorescence emission of reacted probe 1 and probe 2 were 535 nm and 475 nm, respectively. Besides, the TPA cross section value of unreacted probe 1 and probe 2 were measured to be 32.9 GM (Figure 2A) and 13.2 GM (Figure 2C), respectively. These data suggested that the probes well retained the TP fluorescence properties of the TP fluorophore with negligible fluorescent background from the unreacted probes. Photostability of reacted TP-MOF probes were also investigated under sustaining excitation at 820 nm, and within 2 hours, as shown in Figure S1, only 9.5% and 7.5% decreases were observed for probe 1 and probe 2 respectively, which suggested satisfying photostability, avoiding false positive signal resulted from photobleaching.

Figure 2. (A) TP action cross-section values of probe 1 (100 μg/ml) (red) and probe 1 (100 μg/ml) with NaHS (200 μM) (black) in HEPES buffer (pH 7.4). (B) TP excited fluorescence spectrum of probe 1 (100 μg/ml) and NaHS (200 μM) in buffer (pH 7.4); the excitation wavelength was 820 nm. (C) TP action cross-section values of probe 2 (100 μg/ml) (red) and probe 2 (100 μg/ml) with Zn2+ (20 μM) (black) in HEPES buffer (pH 7.4). (D) TP excited fluorescence spectrum of probe 2 (100 μg/ml) and Zn2+ (20 11



μM) in buffer (pH 7.4); the excitation wavelength was 820 nm.

Scheme 2. Illustration of the sensing process between TP-MOF probes and target molecules. In vitro sensing and biostability of TP-MOF probes Sensing performance of TP-MOF probes in vitro was investigated through fluorescence spectra recorded in the absence and presence of target analytes. First, time dependent fluorescence spectrum (Figure S2) displayed that the emission intensity reached the maximum after 1 h. Furthermore, fluorescent intensity of probe 1 increased gradually with growing concentration of NaHS, and a maximum fluorescent enhancement of 18-fold upon incubating with 1 mM NaHS was observed in Figure 3. The detection limit was calculated to be 26.6 μM. Comparatively, equal amount of TPMSN probe reacted with increasing amount of NaHS exhibited a maximum fluorescent enhancement of about 10-fold upon incubating with 1 mM NaHS (Figure S3). The LOD of TP-MOF probe for H2S is lower than that of TP-MSN probe, demonstrating 12


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higher sensitivity of TP-MOF, and this is because of the larger specific surface area of MOF30, which permit higher TP organic moiety loading capacity than MSN47. Likewise, the sensing performance of probe 2 was presented in Figure S4. An approximately 5fold fluorescence enhancement is observed when probe 2 is exposed to saturated amount of Zn2+. The detection limit was calculated to be 0.4 μM. These results preliminarily demonstrate the superior sensing performance of TP-MOF sensing platform in vitro. Besides, selectivity was measured in the presence of selected biologically relevant ions (Figure 3D and Figure S5). Furthermore, organic probe for H2S exhibited fluorescent signal intensity increment in different concentration of cell lysate and a 2.3-fold increment (Figure S6) while TP-MOF probe 1 showed tiny changes even though in high concentration of cell lysate. The organic probe for H2S is vulnerable to proteins in cell lysates while our TP-MOF probe resists to them. This demonstrates the bigger shielding effect of TP-MOF probe in cell lysate, which benefits cellular sensing. These data demonstrate the favorable selectivity of our TP-MOF probes, which greatly supports the applicability of this probe in complex samples.

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Figure 3. (A) The fluorescence emission spectra of probe 1 (100 μg/mL) in the presence of different concentrations of NaHS (0, 30, 50, 100, 150, 200, 250, 400, 500, 600, 1000, 1200 μM) in 1×HEPES buffer (pH 7.4). (B) Calibration curve of probe 1 to NaHS. The curve was plotted with the fluorescence intensity vs NaHS concentration after incubation of them for 60 min. (C) The linear relationship at low concentration. Error bar was calculated from three parallel samples. (D) Fluorescence responses of the TPMOF probe 1 (100 μg/mL) (black bar) and organic probe for H2S (red bar) to testing species in buffer: 1, 1 mM ClO−; 2, 1 mM HCO3−; 3, 1 mM Cl−; 4, 1 mM Br−; 5, 1 mM I−; 6, 100 μM SO32−; 7, 1 mM SO42−;

8, 100 μM HSO3−; 9, 100 μM S2O32−; 10, 100

μM S2O42−;11, 100 μM S2O52−; 12, 100 μM H2O2; 13, 1 mM citrate; 14, 1 mM GSH; 15, 1 mM Cys; 16, 1 mM OAc−; 17, 1 mM HPO42−; 18, 1 mM NaHS.

Biostability is a critical character for bio-sensors. In Figure S7, rather similar peaks of PXRD pattern among TP-MOF probe, TP-MOF probe in serum, and TP-MOF probe 14


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in DMEM were observed. These results suggest TP-MOF probes maintain their structure even though they are sank in biological media for 72 hours, which demonstrate good biostability. Intracellular sensing by TP-MOF probes Biocompatibility of the TP-MOF probe was demonstrated by an MTS assay in contact with HeLa cells (Figure 4). The cells exhibited more than 90% viability even at high probe concentration (200 μg/mL). The minimal cytotoxicity suggests the good biocompatibility of these TP-MOF probes, which contributes largely to their biological applications.

Figure 4. Cytotoxicity assay of HeLa cells treated with different concentration of probe 1 (black bar) and probe 2 (red bar).

In Figure S8, strong green fluorescence was observed, indicating high cell uptake effect. Incubation of live HeLa cells with probe 1 and NaHS led to easily detectable green fluorescence (Figure 5A) through TPM while incubation of HeLa cells with only probe 1 resulted in negligible fluorescence. In addition, images collected through OPM exhibited much lower green fluorescence than that of through TPM, demonstrating the 15



advantages of TP imaging in living cells. In Figure 5B, the pixel signals of each fluorescent images were presented to further validate the fluorescent intensity. Besides of HeLa cells, we further explored the intracellular sensing performance of probe 1 with two other cells, A549 and HepG2 cells. Rather similar sensing images were obtained, as shown in Figure S9. Our second TP-MOF probe 2 serves to detect Zn2+ in live HeLa cells (Figure S10), as can be deduced from the remarkable fluorescence enhancement. From these images, we conclude that our TP-MOF probes could be used to detect intracellular analytes without causing damage to the tumor cells.

Figure 5. (A) OP and TP confocal microscopy images of H2S in HeLa cells incubated with probe 1 (100 μg/ml) for 6 h and then NaHS (200 μM) for 30 min. (B) Relative pixel intensity of OPM (1) and TPM images (2). Scale bar = 40 μm. Imaging of H2S and Zn2+ in tissue slice Further evaluation of TP-MOF probes was performed in rat tissue slice. While only weak fluorescence occurs in slices of rat liver and lung incubated solely with probe 1 16


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(Figures S11A and Figure S11C), much stronger fluorescence is observed in both tissues if incubated with probe 1 and NaHS (Figures S11B, S11D), suggesting the general feasibility for detection of analytes in live tissues. The penetrating ability of this probe was investigated by using a z-scan technique to image the tissues at various depths, with a penetration depth up to 130 μm for liver tissue slice (Figure 6A) and lung tissue slice (Figure S12). For comparison, the corresponding OP excited fluorescence images at different tissue penetration depths were also recorded through z-scan technique, with a penetration depth of only 40 μm (Figure 6B), indicating deeper penetration depths of TP-MOF probe with TPM. Probe 2 generates strong green fluorescence in a frozen rat liver slice, which was pre-incubated with Zn2+ (Figure S13), with a penetration depth up to 130 μm; this also suggests the feasibility for imaging Zn2+ in liver tissue. In addition to the excellent sensing performance, the incorporating of TP organic moiety into metal-organic frameworks also improves the depth fluorescent imaging ability.

Figure 6. Depth fluorescence images of probe 1 (100 μg/mL) in rat liver tissue. The change of fluorescence intensity with scan depth was determined by spectral confocal multiphoton microscopy (A) or one-photon microscopy (B) in the z-scan mode. Scale bar = 50 μm. 17



CONCLUSION In summary, we reported the first TP-MOF sensing platform for intracellular sensing and deep tissue imaging. The design of the TP-MOF was based on PCN-58 incorporated a target-responsive two-photon organic moiety for H2S or Zn2+ through click chemistry. The TP-MOF probes exhibited good photostability, high selectivity, negligible cytotoxicity, and excellent sensing performance in live cells. Through incorporating TP organic moiety into MOFs, not only intracellular sensing, but also the depth imaging capability was achieved. The TP excited fluorescence imaging in tissues were recorded, with a penetration depth up to 130 μm. In a word, The TP-MOF sensing platform presents satisfying sensing and imaging performance in sophisticated biological system. Additionally, the molecular design of MOF-based fluorescent nanoprobe will allow the construction of various probes for intracellular targets as well as regulating specific functions in biological systems.

SUPPORTING INFORMATION AVAILABLE The Supporting Information is available free of charge on the ACS Publications website. Synthesis of ligand and fluorophores, photostability spectrum, reaction time of TPMOF probes, the fluorescence emission spectra of probe 2, selectivity of probe 2, PXRD pattern for biostability of TP-MOF probes, confocal microscopy images, reference, NMR spectra characterization

AUTHOR INFORMATION Corresponding authors 18


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*E-mail: [email protected] *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (NSFC) (Grants 21675043, 21804039, 51872088, 21705037), and the science and technology project of Hunan Province (2016RS2009, 2016WK2002).

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