Aggregation-Induced Emission Probe for Light-Up and in Situ

7 days ago - The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b00952. NMR and ... Abstra...
5 downloads 4 Views 3MB Size
Download PDF
Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

www.acsami.org

Aggregation-Induced Emission Probe for Light-Up and in Situ Detection of Calcium Ions at High Concentration Meng Gao,*,†,‡,⊥ Yunxia Li,†,‡,⊥ Xiaohui Chen,†,‡ Shiwu Li,§ Li Ren,*,†,‡ and Ben Zhong Tang*,§,∥ †

National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, China ‡ School of Materials Science and Engineering and §Guangdong Innovative Research Team, Center for Aggregation-Induced Emission, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China ∥ Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China S Supporting Information *

ABSTRACT: The fluorescent probe for the detection of calcium ions is an indispensable tool in the biomedical field. The millimolar order of Ca(II) ions is associated with many physiological processes and diseases, such as hypercalcemia, soft tissue calcification, and bone microcracks. However, the conventional fluorescent probes are only suitable for imaging Ca(II) ions in the nanomolar to micromolar range, which can be because of their high affinities toward Ca(II) ions and aggregation-caused quenching drawbacks. To tackle this challenge, we herein develop an aggregation-induced emission (AIE) probe SA-4CO2Na for selective and light-up detection of Ca(II) ions in the millimolar range (0.6−3.0 mM), which can efficiently distinguish between hypercalcemic (1.4−3.0 mM) and normal (1.0−1.4 mM) Ca2+ ion levels. The formation of fibrillar aggregates between SA4CO2Na and Ca(II) ions was clearly verified by fluorescence, scanning electron microscopy, and transmission electron analysis. Moreover, this AIE-active probe can be used for wash-free and light-up imaging of a high concentration of Ca(II) ions even in the solid analytes, including calcium deposits in psammomatous meningioma slice, microcracks on bovine bone surface, and microdefects on hydroxyapatite-based scaffold. It is thus expected that this AIE-active probe would have broad biomedical applications through light-up imaging and sensing of Ca(II) ions at the millimolar level. KEYWORDS: aggregation-induced emission, calcium ions, light-up detection, hypercalcemia, bone microcracks fluorescent sensors based on genetically encoded fluorescent proteins and synthetic fluorophores have been developed for calcium sensing and imaging.9−13 However, these conventional fluorescent probes are only suitable for the study of intracellular Ca2+ in the nM to micromolar (μM) range, which can be because of their high affinities toward Ca2+ and self-quenching drawbacks at high concentration.14,15 For example, the squaraine-derived fluorescent probe will undergo serious selfquenching after binding with Ca2+ at the mM level.16,17 Despite mM order of Ca2+ being associated with many diseases, such as

1. INTRODUCTION Calcium ions play crucial roles in a variety of physiological and pathological processes.1,2 The concentration of Ca2+ ions in biological system differs greatly depending on the microenvironment. For example, the concentration of free Ca2+ ions in cytoplasm and extracellular fluids varied from nanomolar (nM) to millimolar (mM) range.3 Although various methods have been developed for the detection of Ca2+ ions, such as atomic absorption spectrometry,4 colorimetric titration,5 ionselective electrode,6 and 19F NMR spectroscopy,7,8 they suffer from expensive equipments and complicated procedures for sample pretreatment. Compared with these traditional methods, fluorescent sensing has advantages in terms of high sensitivity, low cost, and easy operation. To date, various © XXXX American Chemical Society

Received: January 18, 2018 Accepted: April 11, 2018

A

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces hypercalcemia,18 soft tissue calcification,19,20 and bone microcracks,21 the corresponding fluorescent probes have been much less explored.22−27 To develop the fluorescent probes suitable for in situ detection and imaging of Ca2+ at the mM level, the following challenges need to be tackled: (1) the probe should not undergo self-quenching after binding with Ca2+ at high concentration; (2) the balance needs to be adjusted between a low affinity toward Ca2+ to achieve mM order sensitivity and a high selectivity to avoid the disruption of other metal ions or biomolecules; and (3) the probe should have a strong in situ retention ability after binding with Ca2+ to guarantee the spatial imaging resolution and reduce background noise. Recently, aggregation-induced emission (AIE) fluorogens (AIEgens) have provided enormous opportunities for chemo-/ biosensing and imaging because they are featured with unique properties of poor emission in dilute solution but become highly emissive in the aggregate state.28−31 For example, a series of AIE probes based on the salicyladazine (SA) fluorogen have been developed for the biological study and are featured with advantages of simple preparation, easy decoration, and wide pH-range tolerance (pH = 4.0−10.0).32−35 Herein, we develop an AIE-active probe of SA-4CO2Na for the selective detection of Ca2+ by incorporating the negatively charged iminodiacetate groups as chelating ligands into the AIEgen of SA. In the absence of Ca2+ ion, the probe SA-4CO2Na can be welldispersed in aqueous solution and emitted only a very weak fluorescence, whereas in the presence of Ca2+ ion, the probe could form highly emissive fibrillar aggregates through electrostatic and chelating interactions between the iminodiacetate groups and Ca2+ (Figure 1). On the basis of selective

bone surface, and microdefects on hydroxyapatite (HA)-based scaffold.

2. RESULTS AND DISCUSSION 2.1. Preparation of SA-4CO2Na. The synthetic route of SA-4CO2Na is shown in Scheme 1. The reaction of 5(chloromethyl)-2-hydroxybenzaldehyde 1 with diethyl iminodiacetate 2 first generated compound 3, which further reacted with hydrazine monohydrate to afford SA-4CO2Et in 81% yield. Compound SA-4CO2Et then underwent hydrolysis reaction to afford SA-4CO2H in 55% yield. The following reaction with sodium methoxide afforded product SA-4CO2Na in 98% yield. All of these compounds have been well-characterized and verified by NMR and HRMS spectroscopies (Figures S1-4). 2.2. Photophysical Properties Characterization. We then studied the photophysical properties of SA-4CO2Na that can be well-dissolved in water, and a main absorption peak at 351 nm and a very weak emission peak at 541 nm were observed (Figure 2). To reduce its solubility in water, a watermiscible solvent of THF was added. The emission intensity first increased slowly with THF fractions from 0 to 90 vol % and then increased rapidly with THF fractions from 90 to 99 vol %. The fluorescence quantum yield increased from 0.23% in water solution to 5.2% in THF/H2O (99:1, v/v) mixture, and then increased to 10.6% in the solid state (Table 1). The excellent emission efficiency increase (46-fold) in the solid state can be ascribed to the restriction of intramolecular motion (RIM) and excited-state intramolecular proton transfer (ESIPT) mechanisms.36 Meanwhile, the fluorescence lifetime increased from 1.32 ns in aqueous solution to 2.48 ns in the solid state (Figure S5), and a large Stokes shift of ∼190 nm was observed. Moreover, the synthetic intermediate SA-4CO2Et also showed a typical AIE behavior (Figure S6). 2.3. Detection of Ca2+ in Solution. We then tested the detection ability and selectivity of SA-4CO2Na for Ca2+ in the PBS buffer solution (pH 7.4). Before addition of Ca2+, the probe emitted a weak fluorescence; upon addition of Ca2+, a gradually increased fluorescence was observed with a maximum emission wavelength at 560 nm (Figure 3A). As a result, a linear detection range of 0.6−3.0 mM and a light-up ratio up to 11.5-fold were observed (Figure 3B), which is promising to distinguish between hypercalcemic (1.4−3.0 mM) and normal (1.0−1.4 mM) Ca2+ ion levels (Figure S7).18 The light-up detection ability of SA-4CO2Na could be ascribed to the formation of aggregates by chelating with Ca2+ ions, which will efficiently restrict the intramolecular motion and protect the intramolecular hydrogen from polar water’s disruption to guarantee the smooth proceeding of ESIPT process.36 Moreover, the photoinduced electron transfer (PET) process from the electron-donating benzyl nitrogen atom to the SA

Figure 1. Illustration of the light-up detection of AIE-active probe SA4CO2Na for Ca2+.

binding with mM order of Ca2+ and formation of highly emissive aggregates, this AIE-active probe SA-4CO2Na can be used for wash-free and light-up imaging of a high concentration of Ca(II) ions even in the solid analytes, including calcium deposits in psammomatous meningioma slice, microcracks on Scheme 1. Synthetic Route of SA-4CO2Na

B

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 2. (A) Normalized UV−vis absorption spectra of SA-4CO2Na in water (dashed line); PL spectra of SA-4CO2Na in water and water/THF mixtures with different THF fractions ( f THF); [SA-4CO2Na] = 10 μM; λex = 351 nm. (B) Plot of relative PL intensity (I/I0) of SA-4CO2Na at 541 nm vs the solvent composition of water/THF mixture. Inset: Photographs of SA-4CO2Na in water and water/THF (1/99, v/v) taken under UV light (365 nm).

treating with biological molecules of bovine serum albumin (BSA), porcine hemoglobin (PHB), and fetal bovine serum (FBS), and the fluorescence spectra of SA-4CO2Na almost did not change in the presence of these biomolecules. These interference experiments clearly demonstrated the high selectivity of SA-4CO2Na toward Ca2+ over other metal ions and biomolecules. We then used confocal microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analysis to visualize the morphological changes of SA4CO2Na before and after chelating with Ca2+. The SA-4CO2Na formed irregular aggregates in the absence of Ca2+ (Figure 4A− C), whereas it quickly formed fibrillar aggregates in the presence of Ca2+ (Figure 4D−F). The direct observation through fluorescence, SEM, and TEM images clearly verified the chelating ability of SA-4CO2Na with Ca2+ to form highly emissive and fibrillar aggregates. 2.4. Detection of Calcium Deposits in Soft Tissue. Calcium deposits in soft tissue are related with many diseases, such as atherosclerosis, tumor, and bacterial infection.19,20 The fluorescent imaging of calcium deposits will greatly contribute to the precise diagnosis of related diseases. For example, calcification is a feature of benign meningiomas, and the detection of calcium deposits will help to differentiate the benign meningiomas from malignant brain neoplasms.37 Before conducting the imaging experiment for calcium deposits in psammomatous meningioma slice, we first verified whether the probe could be used for imaging Ca2+ in the solid state. By staining with embedded CaCl2 in agarose hydrogel, a strong fluorescence signal was observed for Ca2+-embedded region (Figure 5A−C), which clearly verified the detection ability of the probe for Ca(II) ions even in the solid state. We then used the SA-4CO2Na probe for imaging calcium deposits in the pathological slice of human psammomatous meningioma, which is a kind of benign brain tumors.38−40 Staining with SA4CO2Na showed a circle-like pattern of calcium deposits with a high contrast ratio (Figure 5D−F), whereas a similar circle-like pattern was exhibited by alizarin red S, which is a commonly used staining agent for calcium deposits (Figure S14). These results suggest that the SA-4CO2Na probe can selectively detect the calcium deposits and is promising for the histological analysis of soft tissue calcification with high signal-to-noise ratio.

Table 1. Photophysical Properties of SA-4CO2Na in Water/ THF Mixture with Different THF Fractions ( f THF)a f THF (%)

λem [nm]b

Φf [%]c

τ(ns)d

kr [107 S−1]e

knr [108 S−1]f

0 99 solid

544 541 539

0.23 5.20 10.6

1.32 2.24 2.48

0.17 2.32 4.27

7.56 4.23 3.60

a

Maximum absorption wavelength. bMaximum emission wavelength. Absolute quantum yield. dAverage fluorescence lifetime. eRadiative decay rate kr = Φ/τ. fNonradiative decay rate knr = (1−Φ)/τ. [SA4CO2Na] = 10 μM. λex = 351 nm. c

fluorogen could also be inhibited after binding with Ca2+, which would further contribute to the emission enhancement in the aggregate state (ΦF = 14.5%, Figure S8). The binding of the probe with Ca2+ in aqueous solution was also investigated by measurement of the UV−vis absorption and HRMS spectroscopy and isothermal titration calorimetry (ITC) (Figures S9−11). Although the UV−vis absorption spectra of SA-4CO2Na showed no obvious changes upon addition of Ca2+, the HRMS spectra clearly showed the existence of SA-4CO2-2Ca complex, which could be generated through binding with Ca2+ in a 1:2 molar ratio. However, because Ca(II) ion has six coordination sites, the dynamic binding equilibrium between the probe and Ca2+ tends to shift further to 1:1 molar ratio (Figure S12), which was verified by the ITC experiment (n = 0.803). A binding constant of 20.7 ± 2.69 M−1 was also obtained by fitting with the binding isotherm. To further verify the Ca2+-induced aggregation of the probe and the light-up fluorescence, excess of EDTA (3.0 mM) was added and a gradually decreased emission was observed along with the prolonged time (Figure S13), which indicated the redissolving of the probe by the removal of Ca2+. The selectivity of the probe was then investigated by using a variety of metal ions that might interfere with Ca2+ ion detection at their physiological concentrations, including monovalent ions (Li+, Na+, and K+), divalent ions (Zn2+, Mg2+, Co2+, Ni2+, and Cu2+), and trivalent ion (Fe3+). Among all of the tested ions (Figure 3C,D), only Ca2+ ion led to a significant light-up fluorescence, which indicates the high selectivity of the probe. Although Mg2+ usually severely interferes with the Ca2+ detection,25 the probe only showed a slightly light-up response for Mg2+ even at a high concentration (2.0 mM). Further interfering experiments were conducted by C

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 3. (A) PL spectra of SA-4CO2Na treated with CaCl2 at different concentrations in PBS buffer solution (pH = 7.4). (B) Relative fluorescence intensity changes of SA-4CO2Na at 560 nm upon addition of different concentrations of Ca2+ ions. (C) PL spectra of SA-4CO2Na in the PBS buffer upon addition of various metal ions and biological molecules: Ca2+ (3.0 mM), Li+ (1.0 mM), Na+ (150 mM), K+ (150 mM), Zn2+ (50 μM), Mg2+ (2.0 mM), Co2+ (50 μM), Ni2+ (50 μM) Cu2+ (50 μM), Fe3+ (50 μM), [BSA] = [PHB] = [FBS] = 0.05 mg/mL. (D) Relative fluorescence intensity change at 560 nm upon addition of different metal ions and biomolecules. [SA-4CO2Na] = 1.0 mM; λex = 351 nm.

Figure 4. Fluorescence, SEM, and TEM images of SA-4CO2Na before (A−C) and after (D−F) the addition of Ca2+. [SA-4CO2Na] = 1.0 mM; [Ca2+] = 3.0 mM; λex = 405 nm, λem = 460−750 nm.

Figure 5. CLSM images of CaCl2 embedded in agarose hydrogel (A− C) and calcium deposits in psammomatous meningioma slice (D−F) after incubation with SA-4CO2Na (0.5 mM) for 4.0 h: (A,D) the bright-field images; (B,E) the fluorescence images; and (C,F) the merged images. λex = 405 nm, λem = 460−750 nm.

2.5. Detection of Bone Microcracks. Bone provides structural support and enables body mobility, but bone microcracks caused by repetitive stress, strain, and shear force may lead to serious fracture and fragmentation.41 The sensitive detection of bone microcracks is critical for investigating its formation mechanism and prevent further damage.42 HA (Ca10(PO4)6(OH)2) is the mineral matrix of bone, and a high concentration of unsaturated Ca(II) binding sites will be exposed at the bone microcracks.43 Bone microcracks could

thus be detected by incorporating the fluorophores with selective binding ligands toward calcium ions.44−47 However, the previous fluorescent probes for detection of bone microcracks may suffer from self-quenching drawbacks and high background noise caused by their high-concentration accumulation and diffusion movement.48 For example, calcein D

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

nondestructive method is thus important to guarantee the clinical applications in bone repair.52 As shown in Figure 7, a

for staining of bone microcracks showed a poor selectivity for microcracks and suffers from high background noise (Figure 6A−F).49 The binding of aggregation-caused quenching

Figure 7. CLSM images of spot defects and microcracks on HA-based scaffold stained with SA-4CO2Na (0.5 mM) for 4.0 h: (A) bright-field image and (B) fluorescent image (the spot defects are indicated by white arrows. λex = 405 nm, λem = 460−750 nm. Scale bar = 200 μm.

strong fluorescence signal was observed at spot defects and microcracks of the HA-based scaffold after staining with SA4CO2Na, which demonstrates that this AIE-active probe can efficiently identify microdamages. The probe’s biocompatibility was also investigated.53 Mouse bone marrow stem cells (mBMSCs) were incubated with different concentrations of SA-4CO2Na, and a very low cytotoxicity was observed (Figure S17), which indicates the promising applications of the probe for quality inspection of the HA-based bone-repair scaffold.

Figure 6. CLSM images of bovine bone microcracks by staining with calcein and SA-4CO2Na. (A−C) Whole-projection images (z stack) and (D−F) 3D images of the microcrack stained with calcein. (G−I) Whole-projection images (z stack) and (J−L) 3D images of the microcrack stained with SA-4CO2Na. [Calcein] = [SA-4CO2Na] = 0.5 mM. For SA-4CO2Na, λex = 405 nm, λem = 460−750 nm. For calcein, λex = 488 nm, λem = 490−600 nm.

3. CONCLUSIONS In conclusion, we have developed an AIE-active probe of SA4CO2Na for selective and light-up detection of mM order Ca(II) ions by the formation of highly emissive fibrillar aggregates. On the basis of the wide linear-detection range for Ca(II) ions at a high concentration (0.6−3.0 mM), the probe is promising for various biomedical applications, such as diagnosis of hypercalcemia, histological analysis of calcium deposits in soft tissue, wash-free imaging of bone microcracks, and sensitive inspection of defects on HA-based bone scaffold. Compared with conventional fluorescent probes with ACQ drawbacks, this AIE-active probe has significant advantages in terms of free selfquenching, high signal-to-noise ratio, good water solubility, and strong in situ retention ability. This AIE-active probe is thus promising for biomedical studies of Ca(II) ions in the mM range.

(ACQ)-active calcein at the microcrack sites can only be observed by repeated washing to reduce the background noise. Moreover, because of the self-quenching drawbacks, the seriousness of bone microcracks might be underestimated because of the local high-concentration accumulation of ACQactive calcein at microcrack sites. It is thus highly desirable for a new type of fluorescent probe with wash-free and light-up imaging ability for the study of bone microcracks. Because the AIE-active probe SA-4CO2Na could selectively bind with Ca2+ ions in a light-up manner, we suppose it could be used for wash-free imaging of bone microcracks via chelation with the exposed Ca(II) ions at the microcrack sites. With artificial microcracks on bovine bone surface as an example, a strong fluorescence emission was observed at the bottom and walls of the microcrack sites after incubation with SA-4CO2Na, whereas healthy bone surfaces surrounding the microcracks remained nonfluorescent even without washing steps (Figure 6G−I). The formation of highly emissive aggregates from SA4CO2Na and Ca(II) ions at the microcrack sites will efficiently inhibit the diffusion movement and greatly improve the spatial imaging resolution. The 3D fluorescent images provided an indepth information to evaluate the seriousness of damage caused by bone microcracks (Figure 6J−L). Moreover, the SA4CO2Na probe is featured with excellent photostability (Figure S15) and can thus be used to visualize microcracks in a much more convenient and reliable manner than the ACQ-active calcein. The importance of the iminodiacetate group for binding with exposed Ca(II) ions at bone microcracks sites was confirmed by staining with SA-4CO2Et as a comparison, which showed almost no fluorescence signal (Figure S16). 2.6. Fluorescent Inspection of Microdefects on HABased Scaffold. HA is widely used as a scaffold material for bone repair.50 However, a potential risk in application of HAbased scaffold for bone repair is the defect caused by thermal treatment or external force during manufacture.51 The detection of microdefects on HA-based bone scaffold with a

4. EXPERIMENTAL SECTION 4.1. Synthesis of Compound 3. A mixture of 5-(chloromethyl)2-hydroxybenzaldehyde (340 mg, 2.0 mmol), diethyl iminodiacetate (378 mg, 2.0 mmol), and N,N-diisopropylethylamine (330 μL, 258 mg, 2.0 mmol) in 10 mL of MeCN was stirred at a reflux for 2 h under nitrogen. After cooling to room temperature, the reaction mixture was dried under reduced pressure and the residue was further separated by column chromatography (silica, petroleum ether/ethyl acetate = 5:1) to afford compound 3 as a colorless oil (288 mg, 89%). 1H NMR (CDCl3, 500 MHz): δ 10.79 (s, 1H), 9.72 (s, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.43 (dd, J1 = 8.5 Hz, J2 = 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 4.01 (q, J = 7.5 Hz, 4H), 3.74 (s, 2H), 3.39 (s, 4H), 1.11 (t, J = 7.5 Hz, 6H); 13C NMR (CDCl3, 125 MHz): δ 196.5, 170.9, 160.9, 137.8, 133.9, 129.9, 120.3, 117.5, 60.4, 56.5, 54.0, 14.1. 4.2. Synthesis of SA-4CO2Et. Compound 3 (332 mg, 1.0 mmol) was first dissolved in ethanol solution (10 mL), followed by the addition of hydrazine monohydrate (25 mg, 0.5 mmol) at room temperature. The mixture was then stirred at a reflux for 15 min. After completion of the reaction and further cooling to room temperature, the reaction mixture was filtered off and the obtained solid was washed with ethanol twice (5 mL × 2). After drying under vacuum, SAE

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

ACS Applied Materials & Interfaces

■

4CO2Et was obtained as a light green solid (235 mg, 81%). 1H NMR (CDCl3, 500 MHz): δ 11.34 (s, 2H), 8.70 (s, 2H), 7.49−7.36 (m, 4H), 7.00 (d, J = 8.5 Hz, 2H), 4.18 (q, J = 7.0 Hz, 8H), 3.88 (s, 4H), 3.55 (s, 8H), 1.28 (t, J = 7.0 Hz, 12H); 13C NMR (CDCl3, 125 MHz): δ 171.0, 164.7, 159.3, 134.5, 133.1, 129.4, 117.2, 117.1, 60.6, 56.9, 54.1, 14.3; HRMS (MALDI-TOF) m/z: [M + H]+ calcd for C32H43N4O10, 643.2979; found, 643.2973. 4.3. Synthesis of SA-4CO2H. SA-4CO2Et (128 mg, 0.2 mmol) was dissolved in THF (5 mL), followed by the addition of sodium hydroxide solution (5 mL, 0.8 M). The mixture was stirred at room temperature for 2 h. After completion of the reaction, the mixture was added to aq HCl solution (1 wt %) to afford yellow precipitation, which was filtered off and dried under vacuum to yield SA-4CO2H (57.9 mg, 55%). 1H NMR (500 MHz, DMSO-d6): δ 12.26 (s, 4H), 11.05 (s, 2H), 8.98 (s, 2H), 7.63 (d, J = 2.0 Hz, 2H), 7.39 (dd, J1 = 8.5 Hz, J2 = 2.0 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 3.78 (s, 4H), 3.42 (s, 8H); 13C NMR (DMSO-d6, 125 MHz): δ 172.4, 162.5, 157.9, 134.0, 130.7, 129.8, 118.0, 116.6, 56.4, 53.6; HRMS (ESI) m/z: [M + Na]+ calcd for C24H26N4NaO10, 553.1547; found, 553.1538. 4.4. Synthesis of SA-4CO2Na. A mixture of SA-4CO2H (26.5 mg, 0.05 mmol) and sodium methoxide (10.8 mg, 0.2 mmol) in 5 mL of MeOH was stirred at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was dried under reduced pressure to yield SA-4CO2Na as a light-yellow solid (30.3 mg, 98%). 1H NMR (500 MHz, D2O): δ 8.71 (s, 2H), 8.46 (s, 2H), 7.54 (d, J = 1.5 Hz, 2H), 7.38 (dd, J1 = 8.5, J2 = 2.0 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 4.22 (s, 4H), 3.70 (s, 8H); 13C NMR (D2O, 125 MHz): δ 170.1, 164.5, 159.2, 135.7, 135.3, 120.7, 117.9, 117.4, 57.8, 55.9. 4.5. Fluorescence Imaging of Calcium Deposits in Psammomatous Meningioma Slice. The psammomatous meningioma slice embedded in paraffin was obtained from clinical laboratory of Southern Medical University. The psammomatous meningioma slice was first deparaffinized by xylene and ethanol and then stained with SA-4CO2Na solution (0.5 mM) for 4.0 h. After washing by distilled water, the specimen was observed under CLSM. λex = 405 nm, λem = 460−750 nm. Alizarin red S staining was conducted according to the literature method,54 and the image was obtained by an inverted fluorescence microscope. 4.6. Fluorescence Imaging of Bone Microcracks. Fresh bovine tibiae were bought from a meat wholesaler, and the soft tissue was fully removed. Longitudinal sections of cortical bone from the middiaphysis were cut into beams using an electric saw blade and then polished with P1200 grit sandpaper to afford a mechanically smooth surface.44 Microcracks on the bone surface were made with a surgical scalpel, and the specimen was treated with an aqueous solution of SA4CO2Na (0.5 mM) or calcein (0.5 mM) for 4.0 h. The specimen was then observed under confocal laser-scanning microscopy (CLSM). For SA-4CO2Na, λex = 405 nm, λem = 460−750 nm; for calcein, λex = 488 nm, λem = 490−600 nm. 4.7. Fluorescent Inspection of Defects on HA-Based Bone Scaffold. The HA-based scaffold was prepared according to the literature method.55 Defects and microcracks on the surface of scaffold were made with a surgical scalpel. After staining with an aqueous solution of SA-4CO2Na (0.5 mM) for 4.0 h, the specimen was directly observed under CLSM. For SA-4CO2Na, λex = 405 nm, λem = 460− 750 nm. 4.8. SEM Experiment. The precipitation generated from the mixture of SA-4CO2Na (1.0 mM)/CaCl2 (3.0 mM) was collected by centrifugation and then dried under vacuum. The solid samples for SA4CO2Na and the precipitation of SA-4CO2Na/CaCl2 were put on substrates precoated with conductive glue and then were coated with gold for SEM observations. 4.9. TEM Experiment. The TEM samples for SA-4CO2Na (1.0 mM) and the mixture of SA-4CO2Na (1.0 mM)/CaCl2 (3.0 mM) were prepared by dropping the corresponding solutions onto carboncoated copper grids and dried under room temperature for further TEM observation.

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b00952. NMR and HRMS spectra, absorption spectra, fluorescence lifetime measurement, calcium deposits imaging, ITC titration curve, photostability measurement, and cell viability data (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.G.). *E-mail: [email protected] (L.R.). *E-mail: [email protected] (B.Z.T.). ORCID

Meng Gao: 0000-0001-8071-8079 Li Ren: 0000-0003-0604-9166 Ben Zhong Tang: 0000-0002-0293-964X Author Contributions ⊥

M.G. and Y.L. authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21788102, 51620105009, and 21602063); the National Key R&D Program of China (2017YFC1105004 and 2017YFC1103402); the Science and Technology Planning Project of Guangzhou (project no. 201607020015 and 201704030069); Pearl River S&T Nova Program of Guangzhou (201806010152); Natural Science Foundation of Guangdong Province (2016A030313852 and 2016A030312002); the Fundamental Research Funds for the Central Universities (2015ZY013 and 2015ZZ104); the Innovation and Technology Commission of Hong Kong (ITC-CNERC14SC01); and Guangdong Innovative Research Team Program (201101C0105067115).

■

REFERENCES

(1) de la Fuente, S.; Fonteriz, R. I.; Montero, M.; Alvarez, J. Ca2+ Homeostasis in the Endoplasmic Reticulum Measured with a New Low-Ca2+-Affinity Targeted Aequorin. Cell Calcium 2013, 54, 37−45. (2) Berridge, M. J.; Lipp, P.; Bootman, M. D. The Versatility and Universality of Calcium Signalling. Nat. Rev. Mol. Cell Biol. 2000, 1, 11−21. (3) Hofer, A. M. Another Dimension to Calcium Signaling: A Look at Extracellular Calcium. J. Cell Sci. 2005, 118, 855−862. (4) Pohl, P.; Prusisz, B. Fractionation of Calcium and Magnesium in Honeys, Juices and Tea Infusions by Ion Exchange and Flame Atomic Absorption Spectrometry. Talanta 2006, 69, 1227−1233. (5) Zhang, J.; Wang, Y.; Xu, X.; Yang, X. Specifically Colorimetric Recognition of Calcium, Strontium, and Barium Ions Using 2Mercaptosuccinic Acid-Functionalized Gold Nanoparticles and Its Use in Reliable Detection of Calcium Ion in Water. Analyst 2011, 136, 3865−3868. (6) Fu, X.; Xu, K.; Feng, X.; Chang, B.; Yang, F.; Huang, R. CaCO3 Nanoparticles as the Tags for Potentiometric Detection of a Cardiac Biomarker Using a Calcium Ion-Selective Electrode. Anal. Methods 2017, 9, 6609−6612. (7) Doi, T.; Nakagawa, Y.; Takegoshi, K. Solid-State Nuclear Magnetic Resonance Analysis Reveals a Possible Calcium Binding Site of Pradimicin A. Biochemistry 2017, 56, 468−472. F

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces (8) Yanagida, S.; Luo, C. S.; Balschi, J. A.; Pohost, G. M.; Pike, M. M. Simultaneous Multicompartment Intracellular Ca2+ Measurements in the Perfused Heart Using 19F NMR Spectroscopy. Magn. Reson. Med. 1996, 35, 640−647. (9) McCombs, J. E.; Palmer, A. E. Measuring Calcium Dynamics in Living Cells with Genetically Encodable Calcium Indicators. Methods 2008, 46, 152−159. (10) Suzuki, J.; Kanemaru, K.; Iino, M. Genetically Encoded Fluorescent Indicators for Organellar Calcium Imaging. Biophys. J. 2016, 111, 1119−1131. (11) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Creating New Fluorescent Probes for Cell Biology. Nat. Rev. Mol. Cell Biol. 2002, 3, 906−918. (12) Oheim, M.; van ’t Hoff, M.; Feltz, A.; Zamaleeva, A.; Mallet, J.M.; Collot, M. New Red-Fluorescent Calcium Indicators for Optogenetics, Photoactivation, and Multi-Color Imaging. Biochim. Biophys. Acta 2014, 1843, 2284−2306. (13) Rudolf, R.; Mongillo, M.; Rizzuto, R.; Pozzan, T. Looking Forward to Seeing Calcium. Nat. Rev. Mol. Cell Biol. 2003, 4, 579−586. (14) Johnson, I.; Spence, M. T. Z. Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11th ed.; Life Technologies: 2010. (15) Allen, T. M.; Cleland, L. G. Serum-Induced Leakage of Liposome Contents. Biochim. Biophys. Acta 1980, 597, 418−426. (16) Akkaya, E. U.; Turkyilmaz, S. A Squaraine-Based Near IR Fluorescent Chemosensor for Calcium. Tetrahedron Lett. 1997, 38, 4513−4516. (17) Arunkumar, E.; Ajayaghosh, A.; Daub, J. Selective Calcium Ion Sensing with a Bichromophoric Squaraine Foldamer. J. Am. Chem. Soc. 2005, 127, 3156−3164. (18) Carroll, M. F.; Schade, D. S. A Practical Approach to Hypercalcemia. Am. Fam. Physician 2003, 67, 1959−1966. (19) Korff, S.; Riechert, N.; Schoensiegel, F.; Weichenhan, D.; Autschbach, F.; Katus, H. A.; Ivandic, B. T. Calcification of Myocardial Necrosis Is Common in Mice. Virchows Arch. 2006, 448, 630−638. (20) Zaheer, A.; Murshed, M.; De Grand, A. M.; Morgan, T. G.; Karsenty, G.; Frangioni, J. V. Optical Imaging of Hydroxyapatite in the Calcified Vasculature of Transgenic Animals. Arterioscler., Thromb., Vasc. Biol. 2006, 26, 1132−1136. (21) Lee, T. C.; Mohsin, S.; Taylor, D.; Parkesh, R.; Gunnlaugsson, T.; O’Brien, F. J.; Giehl, M.; Gowin, W. Detecting Microdamage in Bone. J. Anat. 2003, 203, 161−172. (22) Moirangthem, M.; Arts, R.; Merkx, M.; Schenning, A. P. H. J. An Optical Sensor Based on a Photonic Polymer Film to Detect Calcium in Serum. Adv. Funct. Mater. 2016, 26, 1154−1160. (23) Ishiwari, F.; Hasebe, H.; Matsumura, S.; Hajjaj, F.; HoriiHayashi, N.; Nishi, M.; Someya, T.; Fukushima, T. Bioinspired Design of a Polymer Gel Sensor for the Realization of Extracellular Ca2+ Imaging. Sci. Rep. 2016, 6, 24275. (24) Ding, Y.; Ling, J.; Qiao, Y.; Li, Z.; Sun, Z.; Cai, J.; Guo, Y.; Wang, H. A High-Throughput Fluorimetric Microarray with Enhanced Fluorescence and Suppressed “Coffee-Ring” Effects for the Detection of Calcium Ions in Blood. Sci. Rep. 2016, 6, 38602. (25) Sui, B.; Liu, X.; Wang, M.; Belfield, K. D. A Highly Selective Fluorescence Turn-On Sensor for Extracellular Calcium Ion Detection. Chem.Eur. J. 2016, 22, 10351−10354. (26) Kim, S.; Kim, J.; Lee, N. H.; Jang, H. H.; Han, M. S. A Colorimetric Selective Sensing Probe for Calcium Ions with Tunable Dynamic Ranges Using Cytidine Triphosphate Stabilized Gold Nanoparticles. Chem. Commun. 2011, 47, 10299−10301. (27) He, H.; Jenkins, K.; Lin, C. A Fluorescent Chemosensor for Calcium with Excellent Storage Stability in Water. Anal. Chim. Acta 2008, 611, 197−204. (28) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718−11940. (29) Mei, J.; Huang, Y.; Tian, H. Progress and Trends in AIE-Based Bioprobes: A Brief Overview. ACS Appl. Mater. Interfaces 2018, 10, 12217−12261.

(30) Li, Q.; Li, Z. AIE Probes Towards Biomolecules: The Improved Selectivity with The Aid of Graphene Oxide. Sci. China: Chem. 2015, 58, 1800−1809. (31) Wang, C.; Li, Z. Molecular Conformation and Packing: Their Critical Roles in the Emission Performance of Mechanochromic Fluorescence Materials. Mater. Chem. Front. 2017, 1, 2174−2194. (32) Tang, W.; Xiang, Y.; Tong, A. Salicylaldehyde Azines as Fluorophores of Aggregation-Induced Emission Enhancement Characteristics. J. Org. Chem. 2009, 74, 2163−2166. (33) Gao, M.; Hu, Q.; Feng, G.; Tomczak, N.; Liu, R.; Xing, B.; Tang, B. Z.; Liu, B. A Multifunctional Probe with Aggregation-Induced Emission Characteristics for Selective Fluorescence Imaging and Photodynamic Killing of Bacteria over Mammalian Cells. Adv. Healthcare Mater. 2015, 4, 659−663. (34) Hu, Q.; Gao, M.; Feng, G.; Liu, B. Mitochondria-Targeted Cancer Therapy Using a Light-Up Probe with Aggregation-InducedEmission Characteristics. Angew. Chem., Int. Ed. 2014, 53, 14225− 14229. (35) Hu, Q.; Gao, M.; Feng, G.; Chen, X.; Liu, B. A Cell Apoptosis Probe Based on Fluorogen with Aggregation Induced Emission Characteristics. ACS Appl. Mater. Interfaces 2015, 7, 4875−4882. (36) Gao, M.; Wang, L.; Chen, J.; Li, S.; Lu, G.; Wang, L.; Wang, Y.; Ren, L.; Qin, A.; Tang, B. Z. Aggregation-Induced Emission Active Probe for Light-Up Detection of Anionic Surfactants and Wash-Free Bacterial Imaging. Chem.Eur. J. 2016, 22, 5107−5112. (37) Zeng, L.; Liang, P.; Jiao, J.; Lei, T.; Chen, J. Will an Asymptomatic Meningioma Grow or Not Grow? A Meta-analysis. J. Neurol. Surg. Cent. Eur. Neurosurg. 2015, 76, 341−347. (38) Whittle, I. R.; Smith, C.; Navoo, P.; Collie, D. Meningiomas. Lancet 2004, 363, 1535−1543. (39) Giachelli, C. M. Ectopic Calcification: New Concepts in Cellular Regulation. Z. Kardiol. 2001, 90, III31−III37. (40) Murakami, M.; Kuratsu, J.-I.; Mihara, Y.; Matsuno, K.; Ushio, Y. Histochemical Study of Alkaline Phosphatase in Primary Human Brain Tumors Diagnostic Implications for Meningiomas and Neurinomas. Neurosurgery 1993, 32, 180−184. (41) Zimmermann, E. A.; Ritchie, R. O. Bone as a Structural Material. Adv. Healthcare Mater. 2015, 4, 1287−1304. (42) Taylor, D.; Hazenberg, J. G.; Lee, T. C. Living With Cracks: Damage and Repair in Human Bone. Nat. Mater. 2007, 6, 263−268. (43) McMahon, B. K.; Mauer, P.; McCoy, C. P.; Lee, T. C.; Gunnlaugsson, T. Luminescent Terbium Contrast Agent for Bone Microdamage Detection. Aust. J. Chem. 2013, 64, 600−603. (44) Surender, E. M.; Comby, S.; Cavanagh, B. L.; Brennan, O.; Lee, T. C.; Gunnlaugsson, T. Two-Photon Luminescent Bone Imaging Using Europium Nanoagents. Chem 2016, 1, 438−455. (45) Krishna, A. S.; Radhakumary, C.; Antony, M.; Sreenivasan, K. Functionalized Carbon Dots Enable Simultaneous Bone Crack Detection and Drug Deposition. J. Mater. Chem. B 2014, 2, 8626− 8632. (46) Yadav, V.; Freedman, J. D.; Grinstaff, M.; Sen, A. Bone-Crack Detection, Targeting, and Repair Using Ion Gradients. Angew. Chem., Int. Ed. 2013, 52, 10997−11001. (47) Harmatys, K. M.; Cole, E. L.; Smith, B. D. In Vivo Imaging of Bone Using a Deep-Red Fluorescent Molecular Probe Bearing Multiple Iminodiacetate Groups. Mol. Pharm. 2013, 10, 4263−4271. (48) Yadav, V.; Pavlick, R. A.; Meckler, S. M.; Sen, A. Triggered Detection and Deposition: Toward the Repair of Microcracks. Chem. Mater. 2014, 26, 4647−4652. (49) O’Brien, F. J.; Taylor, D.; Lee, T. C. An Improved Labelling Technique for Monitoring Microcrack Growth in Compact Bone. J. Biomech. Eng. 2002, 35, 523−526. (50) Wei, G.; Ma, P. X. Structure and Properties of NanoHydroxyapatite/Polymer Composite Scaffolds for Bone Tissue Engineering. Biomaterials 2004, 25, 4749−4757. (51) Case, E. D.; Smith, I. O.; Baumann, M. J. Microcracking and Porosity in Calcium Phosphates and the Implications for Bone Tissue Engineering. Mater. Sci. Eng., A 2005, 390, 246−254. G

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces (52) Ren, F.; Smith, I. O.; Baumann, M. J.; Case, E. D. ThreeDimensional Microstructural Characterization of Porous Hydroxyapatite Using Confocal Laser Scanning Microscopy. Int. J. Appl. Ceram. Technol. 2005, 2, 200−211. (53) Ventura, M.; Boerman, O. C.; de Korte, C.; Rijpkema, M.; Heerschap, A.; Oosterwijk, E.; Jansen, J. A.; Walboomers, X. F. Preclinical Imaging in Bone Tissue Engineering. Tissue Eng., Part B 2014, 20, 578−595. (54) Mori, F.; Tanji, K.; Wakabayashi, K. Widespread Calcium Deposits, as Detected Using The Alizarin Red S Technique, in The Nervous System of Rats Treated with Dimethyl Mercury. Neuropathology 2000, 20, 210−215. (55) Yeong, K. C. B.; Wang, J.; Ng, S. C. Fabricating Densified Hydroxyapatite Ceramics from a Precipitated Precursor. Mater. Lett. 1999, 38, 208−213.

H

DOI: 10.1021/acsami.8b00952 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX