Magnetic Resonance Imaging Signals to

Feb 22, 2016 - Development of noninvasive approach for sensing their activity with high ... Mechanic properties and internal fibrous networks of the h...
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Using “On/Off” 19F NMR/Magnetic Resonance Imaging Signals to Sense Tyrosine Kinase/Phosphatase Activity in Vitro and in Cell Lysates Zhen Zheng,† Hongbin Sun,‡ Chen Hu,‡ Gongyu Li,† Xiaomei Liu,† Peiyao Chen,† Yusi Cui,† Jing Liu,‡ Junfeng Wang,‡ and Gaolin Liang*,† †

CAS Key Laboratory of Soft Matter Chemistry, Hefei Science Center CAS, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China ‡ High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China S Supporting Information *

ABSTRACT: Tyrosine kinase and phosphatase are two important, antagonistic enzymes in organisms. Development of noninvasive approach for sensing their activity with high spatial and temporal resolution remains challenging. Herein, we rationally designed a hydrogelator Nap-Phe-Phe(CF3)-GluTyr-Ile-OH (1a) whose supramolecular hydrogel (i.e., Gel 1a) can be subjected to tyrosine kinase-directed disassembly, and its phosphate precursor Nap-Phe-Phe(CF3)-Glu-Tyr(H2PO3)Ile-OH (1b), which can be subjected to alkaline phosphatase (ALP)-instructed self-assembly to form supramolecular hydrogel Gel 1b, respectively. Mechanic properties and internal fibrous networks of the hydrogels were characterized with rheology and cryo transmission electron microscopy (cryo-TEM). Disassembly/self-assembly of their corresponding supramolecular hydrogels conferring respective “On/Off” 19F NMR/MRI signals were employed to sense the activity of these two important enzymes in vitro and in cell lysates for the first time. We anticipate that our new 19F NMR/magnetic resonance imaging (MRI) method would facilitate pharmaceutical researchers to screen new inhibitors for these two enzymes without steric hindrance.

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two enzymes.17 Compared to 1H NMR/MRI, 19F NMR/MRI possess similar sensitivity (83% relative to 1H) but have much higher selectivity for practical applications. This is because, unlike the tremendous 1H NMR/MRI background signals from the bulk water in solutions (or bodies), there are almost no 19F NMR/MRI background signals in human bodies.18 To date, a lot of functional 19F probes have been developed for 19F NMR/ MRI assessment of biomarkers or biological events.19,20 However, to the best of our knowledge, there has been no report of using 19F NMR/MRI to sense the activity of tyrosine kinase and phosphatase. Similar to 1H, 19F NMR/MRI requires a high concentration of probe (several millimolar) to generate enough NMR/MRI signals at the region of interest. Supramolecular hydrogels, one type of biocompatible materials capable of gelling a large amount of water (more than 95 wt %) owing to the self-assembly of small molecular hydrogelators,21,22 have been extensively explored and found wide applications in drug delivery,23−25 tissue engineering,26 wound healing,27 three-dimensional cell culture,28 metal ion absorption,29 biomarker sensing,30−33 and functional materials

yrosine kinase and phosphatase are two classic antagonistic enzymes that play key roles in gene expression, signal transmission, cell cycle, and cell apoptosis by reversible phosphorylation and dephosphorylation of proteins.1−3 Overexpression or dysregulation of either tyrosine kinase or phosphatase can induce significant development of cancers or inflammatory diseases.4 Therefore, sensing the activity of these two enzymes in situ is of high importance for not only the identification of their inhibitors but also the early diagnoses of related diseases. Traditional methods for assay of the activity of tyrosine kinase or phosphatase are confined to radioactive or immunofluorescence-based approaches using costly biological reagents such as phosphopeptide-specific antibodies.5−8 Nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) are powerful noninvasive techniques and have been widely used in research and the clinic.9−12 Among the biologically relevant nuclei, 1H has the highest gyromagnetic ratio (γ value) and thus 1H NMR/MRI have the highest sensitivity. Some 1H MRI-based biosensors have been applied for the sensing of tyrosine kinase or phosphatase activity.13−16 For example, Maltzahn et al. employed 1H MRI to quantitate the T 2 relaxation changes of superparamagnetic Fe 3 O 4 nanoparticles during their self-assembly directed by antagonistic kinase and phosphatase, thus for the activity detection of these © 2016 American Chemical Society

Received: January 5, 2016 Accepted: February 22, 2016 Published: February 22, 2016 3363

DOI: 10.1021/acs.analchem.6b00036 Anal. Chem. 2016, 88, 3363−3368

Article

Analytical Chemistry design.34 Among paths to induce hydrogelation in vitro (including light, native ions, and other biologically inert means), enzyme-triggered ones are more biologically compatible and can be coupled to cellular processes.35 Yang et al. utilized this enzymatic pair (i.e., phosphatase and tyrosine kinase) as a switch to reversibly self-assemble/disassemble a supramolecular hydrogel and in turn used the gel−sol transition and high-performance liquid chromatography (HPLC) analyses to qualitatively and quantitatively sense these two enzymes, respectively.36 A characteristic property for hydrogelation process is that it also needs a high concentration of hydrogelator (usually more than several millimolar) to gel water to form a hydrogel. Inspired by the above-mentioned pioneering studies and considering the complementary advantages of 19F NMR/MRI and the hydrogelation process, as shown in Figure 1A, we

BaoMan Inc. (Shanghai, China) (one unit is the enzyme activity that cleaves 1 μmol of the standard substrate per minute at 37 °C). Recombinant Epidermal Growth Factor Receptor (EGFR) and H1975 cells were kindly supplied by Dr. Qingsong Liu’s lab at High Magnetic Field Laboratory. The Bioyotime RIPA lysis buffer P0013D which contains 1% NP-40 and 0.25% deoxycholate as the main component was used for the cell lysis experiments. General Methods. Rheological measurements in Figure 2A,B were performed on a Haake RheoStress 6000 rheometer

Figure 2. Frequency dependence of the dynamic storage moduli (G′) and the loss moduli (G″) of Gel 1a (A) and Gel 1b (B) at 1.0 wt % in phosphate buffer (0.2 M, pH 7.4), respectively. All rheological measurements were conducted at 25 °C and strain of 1.0%. Insets: photographs of 1a (A) or 1b (B) in phosphate buffer in vials before (left) and after (right) hydrogelation. Cryo-TEM images of Gel 1a (C) and Gel 1b (D) at 1.0 wt % in phosphate buffer (0.2 M, pH 7.4), respectively.

Figure 1. (A) Schematic illustration of tyrosine kinase-controlled disassembly and ALP-controlled self-assembly of 19F nanofibers in hydrogel to confer 19F NMR signals “on” and “off”. (B) Chemical structures of hydrogelator 1a and its phosphate precursor 1b.

developed a reversible disassembly/self-assembly system of supramolecular hydrogels, catalyzed by epidermal growth factor receptor (EGFR, one type of tyrosine kinase) and alkaline phosphatase (ALP), which displays respective “On/Off” 19F NMR (or MRI) signals for sensing EGFR/ALP activity in vitro and in cell lysates. In details, after being added into the hydrogel, the enzyme EGFR catalyzes the phosphorylation of the hydrogelator and thereafter disassembles the hydrogel into a solution. Transition of “Gel” to “Sol” induces the change of 19 F NMR/MRI signals from “Off” to “On”. Reversibly, the 19F phosphate precursor in sol has “On” 19F NMR/MRI signals. Upon the catalysis of ALP, the phosphate group on the precursor is removed to yield the hydrogelator which selfassembles into nanofibers to form the supramolecular hydrogel. Self-assembly of the hydrogelator induces fast transverse relaxation among the 19F magnetic spins which turns the 19F NMR/MRI signals “Off”. Thus, in turn, we could utilize the disassembly/self-assembly induced, reversible “On/Off” 19F NMR/MRI signals for the detection of EGFR/ALP activity.

(Thermo Scientific), with cone-and-plate geometry of 1° and 20 mm at the gap of 300 μm. Cryo transmission electron microscopy (cryo-TEM) images in Figure 2C,D were obtained on a Tecnai F20 transmission electron microscope from FEI Company, operating at 200 kV. The samples are prepared as follows: 3 μL of gel sample was dropped on a copper mesh and then quickly frozen under liquid nitrogen in a FEI Vitrobot Cyro sampling machine. The resolutions of this cryo-TEM machine are 0.27 nm for point resolution, 0.144 nm for line resolution, and 0.16 nm for information resolution. The spectrum of electrospray ionization-mass spectrometry (ESIMS) and secondary mass spectrum were recorded on a LTQ Orbitrap mass spectrometer (Thermo Fisher). Electrospray ionization-time-of-flight (ESI-TOF) mass spectra were obtained on an Agilent Technologies 6224 mass spectrometer. HPLC purifications were performed on a Shimadzu UFLC system equipped with two LC-20AP pumps and a SPD-20A UV−vis detector using a Shimadzu PRC-ODS column. HPLC analyses were performed on an Agilent 1200 HPLC system equipped with a G1322A pump and in-line diode array UV detector using an Agilent Zorbax 300SB-C18 RP column with CH3CN (0.1% of trifluoroacetic acid (TFA)) and water (0.1% of TFA) as the eluent. 1H NMR and 13C NMR spectra were obtained on a Bruker AV-300 MHz. 19F NMR spectra were recorded on a Bruker AVIII WB-600 MHz spectrometer.

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EXPERIMENTAL SECTION Materials. All chemicals were analytical reagent grade or better. The phosphatase inhibitor complex II was bought from Sangong Biotech Inc. (Shanghai, China). Recombinant Intestinal Alkaline Phosphatase (ALP), was obtained from 3364

DOI: 10.1021/acs.analchem.6b00036 Anal. Chem. 2016, 88, 3363−3368

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Analytical Chemistry Synthesis of 1a and 1b. Compound 1a or 1b was synthesized with solid phase peptide synthesis (SPPS), followed by the deprotection of tBu groups with dichloromethane (DCM, 300 μL) and triisopropylsilane (TIPS, 200 μL) in TFA (9.5 mL) for 3 h, purified with HPLC using water− acetonitrile added with 0.1% TFA as the eluent (from 6:4 to 2:8) and sent for high-resolution (HR) mass spectrometric analysis. Characterization of 1a. 1H NMR of compound 1a (d6DMSO, 300 MHz, Figure S1) δ (ppm): 9.14 (s, 1 H) 8.26 (d, J = 8.1 Hz, 1 H), 8.25 (d, J = 8.3 Hz, 1 H), 8.17 (d, J = 8.1 Hz, 1 H), 8.09 (d, J = 7.9 Hz, 1 H), 7.85 (d, J = 7.9 Hz, 1 H), 7.75 (s, 1 H), 7.72 (s, 1 H), 7.57 (s, 1 H), 7.53 (s, 1 H), 7.51 (s, 1 H), 7.47−7.43 (m, 2 H), 7.41 (s, 1 H), 7.39 (s, 1 H), 7.18−7.10 (m, 6 H), 7.04 (s, 1 H), 7.02 (s, 1 H), 6.62 (s, 1 H), 6.60 (s, 1 H), 4.61−4.48 (m, 3 H), 4.29 (m, 1 H), 4.18 (m, 1 H), 3.52 (m, 2 H), 2.89 (m, 2 H), 2.68 (m, 2 H), 2.21 (m, 2 H), 1.90−1.68 (m, 3 H), 1.44−1.13 (m, 2 H), 0.86−0.80 (m, 6 H). 13C NMR of 1a (75 MHz, d6-DMSO) δ (ppm): 174.5, 173.2, 171.7, 171.6, 171.1, 170.8, 170.3, 156.2, 143.0, 138.2, 134.3, 133.3, 132.2, 130.6, 130.5, 130.1, 129.7, 128.4, 128.0, 128.0, 127.9, 127.8, 127.7, 127.2, 126.8, 126.7, 126.6, 126.4, 126.2, 125.9, 125.2, 125.2, 123.1, 115.2, 56.7, 54.2, 53.7, 52.2, 42.6, 37.8, 37.6, 37.0, 36.9, 31.7, 29.5, 25.1, 15.9, 11.7 (Figure S2). MS: calculated for 1a (C51H53F3N5O10) [(M − H)−], 952.3744; obsvd. HR-ESI-MS, m/z 952.3761 (Figure S4). Characterization of 1b. 1H NMR of compound 1b (d6DMSO, 300 MHz, Figure S5) δ (ppm): 8.29 (S, 1 H), 8.26 (S, 1 H), 8.17 (d, J = 3.2 Hz, 1 H), 8.14 (d, J = 4.1 Hz, 1 H), 8.05 (d, J = 7.7 Hz, 1 H), 7.84 (d, J = 8.8 Hz, 1 H), 7.74 (d, J = 8.7 Hz,1 H), 7.58 (m, 1 H), 7.55 (s, 1 H), 7.52 (s, 1 H), 7.48−7.41 (m, 4 H), 7.23−7.14 (m, 8 H), 7.04 (d, J = 5.1 Hz, 1 H), 7.02 (s, 1 H), 4.66−4.50 (m, 3 H), 4.35−4.27 (m, 1 H), 4.22−4.16 (m, 1 H), 3.62−3.42 (m, 2 H), 3.09−2.68 (m, 6 H), 2.22 (m, 2 H), 1.90−1.68 (m, 3 H), 1.44−1.15 (m, 2 H), 0.86−0.80 (m, 6 H). 13C NMR of 1b (75 MHz, d6-DMSO) δ (ppm): 174.0, 172.7, 171.2, 170.8, 170.8, 170.4, 169.7, 150.0, 149.9204, 142.5, 137.7, 133.8, 133.0, 132.9, 131.7, 130.2, 130.0, 129.1, 127.8, 127.5, 127.4, 127.3, 127.2, 126.1, 125.9, 125.4, 124.7, 119.6, 119.5, 56.3, 56.0, 53.7, 53.2, 51.8, 42.1, 36.3, 31.2, 30.0, 29.0, 27.7, 24.6, 18.5, 15.4, 11.2 (Figure S6). 31PNMR (122 MHz, d6DMSO) δ (ppm): −6.21 (Figure S8). MS: calculated for 1b (C51H54F3N5O12P) [(M − H)−], 1032.3409; obsvd. HR-ESIMS, m/z 1032.3429 (Figure S9). 19 F Nuclear Magnetic Resonance Methods. 19F NMR experiments were performed using a 4 mm broad-band N−C/ F/H MAS probe. Accumulated scans for one-dimensional (1D) 19 F spectra are 16 scans for in vitro tests (Figure 3A), 128 and 64 scans for cell lysate tests of 1a and 1b (Figure 4), respectively. 1D 19F spectra was acquired with one pulse program with a 90 pulse width of 5.5 μs. 19F chemical shifts were referenced to trifluoroacetic acid (TFA, −75.6 ppm). All the data were acquired and analyzed with Bruker’s Topspin 3.1 software. All the experiments were performed at 25 °C. 19 F Magnetic Resonance Imaging. MR images were acquired on a Bruker Ascend WB 600 MHz spectrometer using a Bruker Micro 5 imaging probe with triple axis gradients (maximum strength 200 G/cm) and an 8 mm diameter rf saddle coil. 19F magnetic resonance images of phantom samples in Figure 3B were obtained by fast spin echo with repetition time/echo time 2 000/7.9 ms using flash imaging pulse sequence, field of view 3 cm × 3 cm without slice selection, matrix size 64 × 64, and the number of accumulations 256. The

Figure 3. (A) (Left) 19F NMR spectra of Gel 1a at 10 mM in phosphate buffer (0.2 M, pH 7.4) in the absence (top) or presence (bottom) of 1 mg/mL EGFR at 37 °C for 12 h. (Right) 19F NMR spectra of solution 1b at 10 mM in phosphate buffer (0.2 M, pH 7.4) in the absence (top) or presence (bottom) of 200 U/mL ALP at 37 °C for 12 h. Number of accumulations: 16. Note, the inset photos are only for demonstration while the corresponding 19F NMR experiments were carried out in NMR tubes. (B) (Left) 19F MR images of Gel 1a at 10 mM in the absence (top) or presence (bottom) of 1 mg/mL EGFR at 37 °C for 12 h. (Right) 19F MR images of Solution 1b at 10 mM in the absence (top) or presence (bottom) of 200 U/mL ALP at 37 °C for 12 h. Number of accumulations: 256. Slice thickness: 5.5 mm.

Figure 4. (A) (Left panel) The normalized 19F NMR signals of Gel 1a at 500 μM (left), Gel 1a incubated with H1975 cell lysate at 37 °C for 6 h (middle), and Gel 1a incubated with EGFR-inhibitor-pretreated H1975 cell lysate at 37 °C for 6 h (right), respectively. (Right panel) corresponding relative integrals for 19F NMR peaks in the left panel. Number of accumulations: 128. (B) (Left panel) The normalized 19F NMR signals of solution 1b at 500 μM (left), solution 1b incubated with LoVo cell lysate at 37 °C for 6 h (middle), solution 1b incubated with ALP-inhibitor-pretreated LoVo cell lysate at 37 °C for 6 h (right), respectively. (Right panel) Corresponding relative integrals for 19F NMR peaks in the left panel. Number of accumulations: 64. All the spectra were normalized according to that of 500 μM NaF as the internal standard.

excitation pulse width was 2 740 Hz. All images were acquired and analyzed using Bruker’s Para Vision 5.1 software. Cell Culture. LoVo cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hycolon) supplemented with 10% fetal bovine serum at 37 °C, 5% CO2, and humid atmosphere. H1975 cells were routinely cultured in Roswell Park Memorial Institute (RPMI-1640, Hycolon) supplemented with 10% fetal bovine serum at 37 °C, 5% CO2, and humid atmosphere. Cell Experiment for 19F NMR. For tyrosine kinase assay, live H1975 cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer, then the supernatants were collected and incubated with 500 μM Gel 1a in a NMR tube for 6 h at 37 °C for 19F NMR spectrometric analysis (middle spectrum in 3365

DOI: 10.1021/acs.analchem.6b00036 Anal. Chem. 2016, 88, 3363−3368

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Analytical Chemistry

1a, Gel 1a treated with EGFR (i.e., disassembly of Gel 1a), 1b solution, 1b solution treated with ALP (i.e., formation of Gel 1b) are shown in Figure 3A. We first dissolved 1.0 wt % (10 mM) 1a in phosphate buffer (0.2 M, pH 7.4) containing 10 mM adenosine triphosphate (ATP) in a NMR tube and then used the heat up-cool down method to obtain Gel 1a in the tube. As proposed, the 19F NMR spectrum of Gel 1a did not show detectable signal (top left panel in Figure 3A). This was ascribed to the fast transverse relaxation among the 19F magnetic spins in Gel 1a which severely broadens the 19F NMR signal. After 12 h-incubation of as-formed Gel 1a with 1 mg/ mL EGFR at 37 °C, we found that the Gel 1a was disassembled and the 19F NMR signal at −62.2 ppm on the spectrum was restored (bottom left panel in Figure 3A). HPLC traces clearly indicated that 21.3% of 1a was converted to 1b by EGFR (blue and red traces in Figure S12), which is responsible for the disassembly of Gel 1a and the recovery of the 19F NMR signal. Reversibly, 19F NMR spectrum of 1.0 wt % (10 mM) 1b in PBS buffer in a NMR tube showed a sharp peak at −62.2 ppm (top right panel in Figure 3A). After 12 h-incubation of the solution with 200 U/mL ALP at 37 °C, we found that the clear solution in the tube turned into a transparent gel and the 19F NMR peak at −62.2 ppm disappeared from the spectrum (bottom right panel in Figure 3A). HPLC traces indicated that most of 1b was converted into 1a by ALP (black and green traces in Figure S12), which is responsible for the self-assembly of Gel 1b and disappearance of the 19F NMR signal. All signal-to-noise (S/N) values of above 19F NMR spectra for in vitro study were measured and summarized in Table S2. To test the feasibility of direct 19F MR imaging of EGFR (or ALP) with 1a (or 1b), the 19 F MR images of Gel 1a before/after EGFR treatment and those of 1b solution before/after ALP treatment were recorded on a Bruker Ascend WB 600 MHz spectrometer equipped with a Bruker Micro 5 imaging probe and shown in Figure 3B. Clearly we observed the “Off/On” 19F MRI signals of Gel 1a before/after EGFR treatment and “On/Off” 19F MRI signals of 1b solution before/after ALP addition. These results suggested that EGFR-controlled disassembly (or ALP-controlled selfassembly) of the nanofibers confer 19F NMR/MRI signals of 1a (or 1b) “On” (or “Off”), respectively, which in turn could be applied for 19F NMR/MRI detections of EGFR and ALP activity in vitro. “On/Off” 19F NMR Signals for Sensing EGFR and ALP Activity in Cell Lysates. After in vitro studies, we also applied 1a and 1b for 19F NMR detection of tyrosine kinase and phosphatase activity in cell lysates of H1975 cells (overexpress EGFR)38 and LoVo cells (overexpress phosphatase), 31 respectively. NaF was used as the internal standard. The 19F NMR spectra are shown in Figures S14 and S15 (with internal standard). The 19F NMR signals (normalized to that of NaF) were presented in the left panels while their corresponding relative integrals of the peaks at −62.2 ppm were presented in the right panels in Figure 4 and summarized in Table S3. For EGFR assay, as shown in the left panel in Figure 4A, 500 μM Gel 1a in phosphate buffer (50 mM, pH 7.4) in the tube only showed relatively weak 19F NMR signal at −62.2 ppm (relative peak integral of 1.82, left column in the right panel in Figure 4A). After incubation with H1975 cell lysate at 37 °C for 6 h, the hydrogel collapsed and its 19F NMR peak on the spectrum rose (relative peak integral of 4.25, middle column in the right panel in Figure 4A), suggesting part of the hydrogelator 1a was phosphorylated to yield 1b by the enzyme EGFR in the cells. HPLC analyses indicated that 22.6% of 1a was converted to its

Figure 4A). An EGFR inhibitor, WZ4002 (Haoyuan Inc. China), was also utilized to pretreat the H1975 cells and then the cell lysates were incubated with 500 μM Gel 1a in a NMR tube as control for 19F NMR spectrometric analysis (right spectrum in Figure 4A). For phosphatase assay, the collected LoVo cells were lysed with RIPA buffer and incubated with 500 μM 1b solution in a NMR tube for 6 h at 37 °C for 19F NMR spectrometric analysis (middle spectrum in Figure 4B). Besides, we also used an ALP inhibitor complex II (Sangon Biotech, China) to pretreat the LoVo cells and then incubated the cell lysates with 500 μM 1b solution in a NMR tube as another control for 19F NMR spectrometric analysis (right spectrum in Figure 4B).

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RESULTS AND DISCUSSION Rationale of the Design. As shown in Figure 1B, we designed a hydrogelator Nap-Phe-Phe(CF3)-Glu-Tyr-Ile-OH (1a) and its phosphate precursor Nap-Phe-Phe(CF3)-GluTyr(H2PO3)-Ile-OH (1b). Hydrogelator 1a can form supramolecular hydrogels upon physical adjustment (careful pH adjustment from 8.0 to 7.4 or vigorous heat up to 80 °C and cool down to room temperature, Table S1). We denote this physically adjusted hydrogel as Gel 1a. Since the Glu-Tyr-Ile (EYI) residue on 1a is the specific substrate for EGFR phosphorylation,37 addition of EGFR to Gel 1a will induce the phosphorylation of the hydrogelator to yield the hydrophilic phosphate 1b, rendering the disassembly of the hydrogel. Interestingly, the phosphate precursor 1b is the substrate for ALP. Upon ALP-catalyzed dephosphorylation, 1b is converted back to its corresponding hydrogelator 1a, which self-assembles into supramolecular hydrogel. We denote here the enzymatic hydrogel as Gel 1b. Moreover, each of 1a and 1b was designed to contain an L-4-trifluoromethylphenylalanine moiety to generate the 19F NMR/MRI signal. Rheological Characterization and Morphological Study of the Gels. As shown in Figure 2A,B, both Gel 1a and Gel 1b at 1.0 wt % in phosphate buffer (0.2 M, pH 7.4) are transparent. To evaluate the viscoelastic properties of the gels, we first conducted dynamic strain sweep to determine the proper condition for the dynamic frequency sweep. As shown in Figure S10, the storage modulus (G′) and the loss modulus (G″) values of the two hydrogels exhibit a weak dependence of strain from 0.5% to 10% (with G′ dominating G″), indicating the samples are hydrogels. After setting the strain amplitude at 1.0% (within the linear response region of strain amplitude), we used dynamic frequency sweep to study the hydrogels. Figure 2A,B shows that G′ and G″ values of the two hydrogels slightly increase with the increase of frequency from 0.1 to 10 Hz. The values of G′ are several times larger than those of G″ in the whole range (0.1−10 Hz), suggesting that the hydrogels are fairly tolerant to external force. For comparison, temperaturecontrolled Gel 1a shows better tolerance to external force than its corresponding enzymatic Gel 1b. After rheology tests, we conducted cryo transmission electron microscopy (cryo-TEM) to study the internal networks in the hydrogels. Figure 2C,D indicates that both hydrogels are composed of fibrous networks and their nanofibers have a close average diameter of about 2.8 nm (Table S1). As proposed above, treatment of Gel 1a with 1 mg/mL EGFR at 37 °C for 12 h did result in the collapses of the hydrogel and the disassembly of the nanofibers, as shown by the dry-TEM image in Figure S11. “On/Off” 19F NMR/MRI Signals for EGFR and ALP Detection in Vitro. Corresponding 19F NMR spectra of Gel 3366

DOI: 10.1021/acs.analchem.6b00036 Anal. Chem. 2016, 88, 3363−3368

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Analytical Chemistry

quantitate the efficiency of these two enzymes in cell lysates. Inhibitory study indicated that the “On”/“Off” 19F NMR signals of Gel 1a/solution 1b are actually induced by the action of intracellular EGFR/ALP. Although there have been a large number of fluorescence assays for the detection of these two enzymes’ activity in cell lysates or blood, they suffered from internal interference from the autofluorescence of the biological samples or their inherent low penetrability. We anticipate that our new 19F NMR/MRI method would facilitate pharmaceutical researchers to screen new inhibitors for these two enzymes without steric hindrance.

phosphorylated product 1b (Figure S16). When Gel 1a was incubated with EGFR-inhibitor-pretreated H1975 cell lysate, its 19 F NMR signal (relative peak integral of 3.01, right column in the right panel in Figure 4A) was obviously lower than that directly incubated with the cell lysate (middle column) but higher than that of Gel 1a (left column). These results suggested that above increase of 19F NMR signals of H1975 cell lysate-treated Gel 1a (middle column) was indeed induced by EGFR-catalyzed phosphorylation of the hydrogelator. HPLC analyses reaffirmed that, due to strong inhibition of EGFR by its inhibitor WZ4002, only 1.10% of 1a in Gel 1a incubated with the EGFR-inhibitor-treated H1975 cell lysate was converted to 1b (Figure S16). Here for EGFR assay, if we denote a threshold value 3.10 of relative integral for “On” 19F NMR signals, clearly we could observe “Off”, “On”, and “Off” signals for Gel 1a, H1975 cell lysate-treated Gel 1a, and EGFRinhibitor-H1975 cell lysate-treated Gel 1a, respectively (right panel in Figure 4A). For phosphatase assay, as shown in the left panel in Figure 4B, solution of 500 μM 1b in phosphate buffer (50 mM, pH 7.4) showed very sharp 19F NMR signal (relative peak integral of 7.94, left column in the right panel in Figure 4B). After incubation with LoVo cell lysate at 37 °C for 6 h, the solution turned into a hydrogel and its 19F NMR peak on the spectrum declined obviously (relative peak integral of 4.33, middle column in the right panel in Figure 4B), suggesting the precursor phosphate 1b was dephosphorylated to yield the hydrogelator 1a. HPLC analyses demonstrated that 1b was almost converted to its dephosphorylated product 1a (Figure S16). Interestingly, when solution 1b was incubated with ALPinhibitor-pretreated LoVo cell lysate, we found the 19F NMR signal (relative peak integral of 5.55, right column in the right panel in Figure 4B) was weaker than that of 1b solution (left column) but obviously stronger than that of 1b treated with LoVo cell lysate (middle column). This suggests that the above disappearance of 19F NMR signal of 1b is actually induced by ALP-catalyzed dephosphorylation of the phosphate. HPLC analyses unanimously indicated that only 44.0% of 1b in the ALP-inhibitor-treated LoVo cell lysates was dephosphorylated to yield hydrogelator 1a (Figure S16). Here for ALP assay, if we denote a threshold value 4.40 of relative integral for “On” 19F NMR signals, clearly we could observe “On”, “Off”, and “On” signals for solution 1b, LoVo cell lysate-treated solution 1b, and ALP-inhibitor-LoVo cell lysate-treated solution 1b, respectively (right panel in Figure 4B). These above results indicate that our functional short peptides 1a and 1b could be applied for 19F NMR sensing EGFR and phosphatase activity in cell lysates, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00036. Synthesis route, HPLC conditions, Scheme S1, Figures S1−S16, and Tables S1−S5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Prof. Yi Cao for his assistance in the rheology study. This work was supported by the Collaborative Innovation Center of Suzhou Nano Science and Technology, Hefei Science Center CAS (Grant 2015HSC-UP012), and the National Natural Science Foundation of China (Grants U1332142 and 21375121).

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REFERENCES

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CONCLUSIONS In summary, by rational design of a 19F-fluorinated hydrogelator (i.e., 1a) and its phosphate precursor (i.e., 1b), we have developed a new reversible method of tyrosine kinase/ phosphatase-instructed disassembly/self-assembly of their corresponding hydrogels rendering respective “On/Off” 19F NMR/MRI signals for sensing the activity of these two important enzymes in vitro and in cell lysates. Using Gel 1a and solution 1b as the substrates for EGFR or ALP, we obtained respective “On” and “Off” 19F NMR/MRI signals for sensing the activity of these two enzymes in vitro. HPLC analyses were employed to quantitate the phosphorylation (or dephosphorylation) efficiency of the enzymes. Similarly, we also conducted “On/Off” 19F NMR studies on Gel 1a/solution 1b incubated with cell lysates of H1975/LoVo and employed HPLC to 3367

DOI: 10.1021/acs.analchem.6b00036 Anal. Chem. 2016, 88, 3363−3368

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DOI: 10.1021/acs.analchem.6b00036 Anal. Chem. 2016, 88, 3363−3368