Intracellular Self-Assembly of Cyclic d-Luciferin ... - ACS Publications

Jun 27, 2016 - amino-D-luciferin (NH2-Luc) as control BL probes, we validated that the persistent BL of 1 from luciferase-expressing cells or tumors w...
0 downloads 0 Views 3MB Size
Download PDF
Intracellular Self-Assembly of Cyclic D‑Luciferin Nanoparticles for Persistent Bioluminescence Imaging of Fatty Acid Amide Hydrolase Yue Yuan,†,§ Fuqiang Wang,‡,§ Wei Tang,† Zhanling Ding,† Lin Wang,⊥ Lili Liang,† Zhen Zheng,† Huafeng Zhang,⊥ and Gaolin Liang*,† †

CAS Key Laboratory of Soft Matter Chemistry, Hefei Science Center CAS, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ‡ State Key Laboratory of Reproductive Medicine, Analysis Center, Nanjing Medical University, Nanjing, Jiangsu 210093, China ⊥ School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China S Supporting Information *

ABSTRACT: Fatty acid amide hydrolase (FAAH) overexpression induces several disorder symptoms in nerve systems, and therefore long-term tracing of FAAH activity in vivo is of high importance but remains challenging. Current bioluminescence (BL) methods are limited in detecting FAAH activity within 5 h. Herein, by rational design of a latent BL probe (D-Cys-Lys-CBT)2 (1), we developed a “smart” method of intracellular reduction-controlled self-assembly and FAAHdirected disassembly of its cyclic D-luciferin-based nanoparticles (i.e., 1-NPs) for persistent BL imaging of FAAH activity in vitro, in cells, and in vivo. Using aminoluciferin methyl amide (AMA), Lys-amino-D-luciferin (Lys-Luc), and amino-D-luciferin (NH2-Luc) as control BL probes, we validated that the persistent BL of 1 from luciferase-expressing cells or tumors was controlled by the activity of intracellular FAAH. With the property of long-term tracing of FAAH activity in vivo of 1, we envision that our BL precursor 1 could probably be applied for in vivo screening of FAAH inhibitors and the diagnosis of their related diseases (or disorders) in the future. KEYWORDS: bioluminescence, self-assembly, cyclic D-luciferin, disassembly, fatty acid amide hydrolase

F

quantities of mice to evaluate the inhibitors; thus they cannot give longitudinal data from each individual animal.12 Noninvasive molecular imaging strategies for sensing enzyme activity have attracted researchers’ great attention, and a number of positron emission tomography (PET) radiotracers for FAAH imaging have been reported.13 However, PET imaging needs specialized and expensive instruments with low throughput and signal-to-noise ratio. Moreover, probes for PET imaging are mostly labeled with the short-lived radionuclide carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.7 min).14 Whole animal bioluminescence imaging (BLI) is more widely applied by investigators from diverse backgrounds due to its ease of operation, high throughput, and low cost in visualizing a wide variety of intracellular and in vivo events.15−19 On the basis of the wide range of saturated and unsaturated fatty acid amide substrates of FAAH, Miller and co-workers developed a

atty acid amide hydrolase (FAAH) belongs to the serine hydrolase enzyme family. It degrades many fatty acid amides, including the endogenous cannabinoid Narachidonoylethanolamine (anandamide), to maintain the normal functionalization of nerve systems.1−4 However, FAAH overexpression will induce several nerve system disorder symptoms (e.g., inflammation, anxiety, and cannabinoid dependence).5−7 Therefore, a potential therapeutic approach of FAAH inhibitor development has been extensively explored.8,9 To date, many inhibitors of FAAH have been synthesized and developed as potential therapeutics since 1994, and their reversibility, potency, and specificity for FAAH have been reviewed.10 Since neurological disorders are usually chronic, long-term follow-up evaluation of FAAH inhibitor efficacy is of high importance. This makes the long-period detection of FAAH activity in cells and in vivo of great interest.9,11 Current primary assays for FAAH activity detection in mice require sacrificing the mice, homogenizing the tissues, and chromatographic analyses of the hydrolyzed products by FAAH. They are costly and labor-intensive and require © 2016 American Chemical Society

Received: May 23, 2016 Accepted: June 27, 2016 Published: June 27, 2016 7147

DOI: 10.1021/acsnano.6b03412 ACS Nano 2016, 10, 7147−7153

Article

www.acsnano.org

Article

ACS Nano

Figure 1. Chemical structure of 1 and schematic illustration of intracellular reduction-controlled self-assembly and FAAH-directed disassembly of cyclic D-luciferin-based 1-NPs for persistent bioluminescence imaging of FAAH.

Figure 2. (a) Chemical structures of AMA, Lys-Luc, and NH2-Luc. (b) TEM image of 1-NPs formed by 500 μM 1 treated with 1 mM TCEP for 30 min at 37 °C and pH 7.4. (c) Bioluminescence images of 500 μM 1, 1 mM AMA, 1 mM Lys-Luc, and 1 mM NH2-Luc in 1 mM TCEP incubated with (left) or without (middle) FAAH or co-incubated with FAAH and UBR597 (right). (d) Quantified total photon output in c. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant by t test. L-cysteine (L-Cys), we reported a “smart” strategy of intracellular self-assembly/disassembly of Taxol or 19F nanoparticles for controlled release of Taxol to overcome multidrug resistance or long-term 19F magnetic resonance imaging of tumors in vivo.26−28 Interestingly, if the L-Cys is replaced with D-Cys, the above condensation between CBT and D-Cys will yield D-luciferin-based nanoparticles. Thus, we could propose a strategy of intracellular glutathione (GSH)-triggered selfassembly of cyclic D-luciferin nanoparticles, which could be hydrolyzed by FAAH to slowly release D-luciferin or D-luciferin derivatives (Figure 1 and Scheme S1 in the Supporting Information). Under the catalysis of luciferase, this slow release process ensures persistent BL, realizing the long-term tracing of FAAH activity or evaluating the effect of FAAH inhibitors in vitro and in vivo. As shown in Figure 1, we rationally designed a water-soluble precursor, (D-Cys)-Lys-CBT)2 (1). After entering cells, the disulfide bond on 1 is reduced by intracellular GSH to yield the active intermediate 1-Red, which instantly condenses with each other to form amphiphilic cyclic dimer 1-Dimer and selfassembles into cyclic D-luciferin-based nanoparticles (1-NPs), as schematically illustrated in Scheme S1 (Supporting Information). Due to their hydrophobicity and nanoscale sizes, as-formed 1-NPs are difficult to pump out,and therefore

BL method for FAAH activity detection.2 They synthesized luciferin amides for exquisitely selective and sensitive imaging of endogenous FAAH activity in living cells and mice. As we know, D-lucferin has a short half-life of less than 30 min, and the luciferin amide monomers they developed were also easily degraded and quickly secreted out from the animal, causing in vivo detection of FAAH activity to be transient and changeful and also making long-term monitoring of the reversibility and potency of FAAH inhibitors even more difficult.19−21 There are many strategies of using nanocarriers to encapsulate D-luciferin for prolonged bioluminescence. For example, Singh et al. used a lipid nanocarrier system, Trewyn et al. used mesoporous silicon nanoparticles, and Zare et al. used poly(lactic acid) particles to encapsulate D-luciferin and showed prolonged bioluminescence in vitro, in cells, or in vivo.22−24 Many synthetic luciferins have also been developed for this purpose. For example, cyclic alkylaminoluciferin CycLuc1 prolonged the BL half-life to 50 min, and the complicated PEGylated probes were used for BLI of tumors in vivo with an extended half-life of 3−4 h after the initial injection.21,25 However, to the best of our knowledge, there has been no report of BLI half-life for more than 5 h by a synthetic D-luciferin. Recently, by employing a biocompatible condensation reaction between 2-cyano-6-aminobenzothiazole (CBT) and 7148

DOI: 10.1021/acsnano.6b03412 ACS Nano 2016, 10, 7147−7153

Article

ACS Nano

Figure 3. (a) Bioluminescence images of MDA-MB-231-fLuc cells incubated with 250 μM 1 or 100 μM URB597 with 250 μM 1, 500 μM AMA, 500 μM Lys-Luc, or 500 μM NH2-Luc for 0.5, 0.75, 1, 2, 3, 4.5, or 6 h, respectively. (b) Quantified total photon output of 1 and URB597 with 1, AMA, and Lys-Luc in a. (c) Quantified total photon output of NH2-Luc in a. Each error bar represents the standard deviation of three independent experiments.

structures are shown in Figure 2a. Characterizations of these three compounds are shown in Figures S9−S14. Note, Lys-Luc is the direct substrate for fLuc.29 As shown in Figure 2c and d, in the presence of FAAH, both AMA and Lys-Luc at 1 mM showed stronger BL signals than that of 1-NPs, and similar to 1 that containing an amide bond for FAAH cleavage, these two compounds showed decreased BL signal in the absence of FAAH or in the presence of both FAAH and its inhibitor URB597. Because NH2-Luc does not contain an amide bond for FAAH cleavage, the presence of FAAH or FAAH inhibitor did not affect its BL signal, which further verified that the BL response of 1-NPs was induced by FAAH. Persistent BLI of FAAH Activity in Cells. After the verification of FAAH cleavage of 1-NPs in solution, our next plan was to explore whether Lys-Luc or NH2-Luc was slowly released to maintain a persistent BL signal in cells incubated with 1, as proposed in Figure 1. Human breast cancer cell lines MDA-MB-231 and MCF-7 cells are reported to express FAAH, while human cervical cancer cell line HeLa cells are reported to be FAAH-deficient.2,30−34 Our experimental results indicated that, incubated with 1 and in the presence of fLuc, MDA-MB231 and MCF-7 cells showed approximately 12-fold BL intensity of that of HeLa cells (Figure S15). Thus, we transferred fLuc to MDA-MB-231 cells and used the cells for the following BL experiments. Cytoxicity of 1 on parental MDA-MB-231 cells was evaluated with methyl thiazolyl tetrazolium assay, and the results are shown in Figure S16. The results indicate that 92.5% of the cells survived after 48 h incubation with 1 mM 1, suggesting a concentration of 1 below 1 mM is safe for cell imaging. A 250 μM concentration of 1, 500 μM AMA, 500 μM Lys-Luc, or 500 μM NH2-Luc was respectively incubated with 1 × 105 MDA-MB-231-fLuc cells in one well on a 96-well plate, and the BL images of the cells were monitored in real time. As Figure 3a shows, the BL signal from cells incubated with 1 increased with time (top row). Although the BL signal from cells that were sequentially treated with URB597 and 1 also increased with time (the second row in Figure 3a), its intensity was obviously lower than that of cells without inhibitor treatment (top row), suggesting FAAH activity in cells could be irreversibly inhibited by URB597.35 Since NH2-Luc is the direct substrate for fLuc, it has the shortest BL half-life among the tested compounds (Figure 3). AMA and Lys-Luc are substrates for FAAH cleavage, and they did show longer half-lives in cells (BL signal peak of AMA was at 1 h, while that of Lys-Luc was at 3 h, respectively) than that of NH2-Luc. However, neither AMA nor Lys-Luc showed a similar persistent BL signal to that of 1 in cells. This indirectly

their circulation time inside cells is effectively prolonged. Upon FAAH cleavage of the cyclic D-luciferin amides of 1-Dimer in 1NPs, the nanoparticles are slowly disassembled to release the enzymatic products of Lys-amino-D-luciferin (Lys-Luc) or amino-D-luciferin (NH2-Luc) (Figure 2a). In the presence of firefly luciferase (fLuc), they bring persistent BL, which in turn can be employed for long-term monitoring of FAAH activity.

RESULTS AND DISCUSSION Reduction-Controlled Self-Assembly and FAAH-Instructed Disassembly of 1-NPs in Vitro. After the syntheses and characterizations of 1 (Schemes S2 and S3 and Figures S1− S4), we first validated the reduction-controlled condensation and self-assembly of the cyclic D-luciferin-based nanoparticles 1NPs. 1 was dissolved in 100 μL of FAAH buffer at pH 7.4 to a final concentration of 500 μM; then 1 mM tris(2carboxyethyl)phosphine (TCEP) was added and incubated for 30 min at 37 °C. The disulfide bond of 1 was reduced by TCEP, initiating the condensation reaction to yield 1-Dimer, which instantly self-assembles into 1-NPs. High-performance liquid chromatography (HPLC) and high-resolution matrixassisted laser desorption/ionization mass spectroscopic (HRMALDI/MS) analyses of the incubation mixture indicated that 1 was almost converted to 1-Dimer in this condition (Figures S5 and S6). Transmission electron microscope (TEM) images clearly indicated the formation of 1-NPs in the mixture with an average diameter of 216 ± 35 nm (Figure 2b). Time course disassembly of 1-NPs in the presence of FAAH was directly observed with TEM images (Figure S7). We then verified the FAAH cleavage of 1-NPs to generate the BL signal in the presence of fLuc. After 500 μM 1-NPs (calculated in 1) were incubated with 2 μM FAAH at 37 °C for 6 h and a further incubation with 1 μM fLuc and 1 mM adenosine triphosphate (ATP) for 5 min, a BL image of the mixture was taken and measured with a small animal imaging system. As shown in Figure 2c and d, the BL intensity of FAAH-treated 1-NPs was obviously higher than that of 1-NPs without FAAH treatment. As expected, addition of 50 μM FAAH inhibitor URB597 to the FAAH-treated 1-NPs’ incubation resulted in an obvious decrease of the BL intensity, suggesting a BL signal was induced by FAAH cleavage of 1-NPs. A specificity test indicated that only FAAH, but no other robust proteases (e.g., trpsin or proteinase K), was active in the 1-NPs’ BL generation (Figure S8). To further verify the above-mentioned mechanism, we synthesized a control compound, aminoluciferin methyl amide (AMA), and the two FAAH-cleaved products of 1-Dimer (i.e., Lys-Luc and NH2-Luc), whose 7149

DOI: 10.1021/acsnano.6b03412 ACS Nano 2016, 10, 7147−7153

Article

ACS Nano

Figure 4. (a) Bioluminescence images of MDA-MB-231-fLuc tumor-bearing mice after being intraperitoneally injected with 0.1 g/kg 1 (123 μmol/kg) or 3 mg/kg URB597 with 0.1 g/kg 1, 0.07 g/kg AMA (246 μmol/kg), 0.1 g/kg Lys-Luc (246 μmol/kg), or 0.07 g/kg NH2-Luc (246 μmol/kg) for 0.5, 1, 2, 4, 7, 11, and 16 h, respectively. (b) Quantified total photon output of the top three rows in a. (c) Quantified total photon output of the bottom two rows in a. Each error bar represents the standard deviation of three independent experiments.

indicated that the above persistent BL of 1 was induced by FAAH-directed slow disassembly of 1-NPs, which continuously releases the fLuc substrate Lys-Luc or NH2-Luc for BL generation. To chemically verify the self-assembly and disassembly of 1-NPs in the cells, we incubated parental MDA-MB-231 cells or URB597-pretreated parental cells with 2 mM 1 at 37 °C for 2 h and lysed the cells for HPLC and TEM analyses. As shown in Figure S17, after 2 h of incubation with 1, both lysates of parental MDA-MB-231 cells and URB597pretreated parental cells showed the disappearance of the HPLC peak of 1 but the appearance of a main HPLC peak of 1Dimer on the traces. This suggested that 1 was effectively reduced by intracellular GSH and underwent CBT-Cys condensation to yield the cyclic 1-Dimer for the self-assembly of 1-NPs. By comparing the HPLC traces, we confirmed the existence of Lys-Luc and NH2-Luc in the lysate of 1-treated parental MDA-MB-231 cells but barely in the URB597pretreated parental cells (Figure S17). This indicated that 1Dimer of 1-NPs in parental cells was slowly cleaved by FAAH to yield the enzymatic products Lys-Luc and NH2-Luc, which are responsible for the BL in the presence of fLuc. TEM images of the lysates of URB597-treated parental MDA-MB-231 cells (control) and the URB597-pretreated parental MDA-MB-231 cells incubated with 1 are shown in Figure S18a. A TEM image of 500 μM 1 treated with 1 mM TCEP for 30 min at 37 °C and pH 6.0 in vitro is shown in Figure S18b. From Figure S18a, we could find small nanoparticles with an average diameter of 4.2 ± 1.4 nm scattered in the lysates of 1-treated cells but not in the control cells. The sizes of the intracellular nanoparticles are close to those of 1-NPs in vitro formed at pH 6.0 (i.e., 4.0 ± 0.5 nm, Figure S18b). Thus, we concluded that the above intracellular 1-NPs were formed in endosomes or lysosomes with pH values around 6.0, which also have enough GSH

concentration for reduction, but not in the cytoplasm, with a pH value around 7.4.36−38 FAAH-Directed Persistent BL in Vivo. To evaluate the persistent BL of 1 in vivo, we applied 1 for long-term BLI of FAAH activity in MDA-MB-231-fLuc tumors in nude mice. Each 5-week-old BALB/c nude mouse was xenografted with MDA-MB-231-fLuc tumor in the right thigh. After intraperitoneal (i.p.) injection of 0.1 g/kg 1 (123 μmol/kg), 0.07 g/ kg AMA (246 μmol/kg), 0.1 g/kg Lys-Luc (246 μmol/kg), or 0.07 g/kg NH2-Luc (246 μmol/kg) into the mice, respectively, BL images of each mouse were monitored until 16 h by a smallanimal imaging system. As shown in Figure 4, from 0.5 to 16 h postinjection, the BL signal from tumors in mice injected with 1 gradually increased with time (top row in Figure 4a and b). Although the BL signal from the tumors in sequentially URB597 and 1-treated mice also increased with time (the second row in Figure 4a), its intensity was much weaker (Figure 4b), suggesting the above persistent BL of 1 was controlled by FAAH activity. AMA-injected mice (the third row in Figure 4a) showed comparable BL intensity in the tumors to that in mice injected with 1. However, the in vivo BL signal of AMA reached its peak at 1 h and decreased quickly after (Figure 4b), suggesting a quick cleavage of the amide bond on AMA by FAAH in the tumors. Surprisingly, compared with NH2-Luc at the same dose, Lys-Luc showed a 4.5-fold higher photon flux in the tumors (Figure 4c). Nevertheless, both LysLuc- and NH2-Luc-injected mice showed the highest BL signal in the tumors at 0.5 h, which decreased quickly afterward, suggesting that they could not be applied for long-term tracing of FAAH activity in vivo. To validate that the persistent BL signal of 1 in vivo was due to the formation of 1-NPs in tumors, we synthesized a fluorescent analogue probe of 1, CBTLys(Alexa 488)-(D-Cys)2-Lys-CBT (1-Alexa 488) (Scheme S7 and Figure S19) and used a three-dimensional structural 7150

DOI: 10.1021/acsnano.6b03412 ACS Nano 2016, 10, 7147−7153

Article

ACS Nano

at 0 °C for 30 min, pulse-sonicated four times in 30 s pulses, and centrifuged at 16 000 rpm for 20 min. Next, the lysate supernatant was poured into a column (with stopcock) with 1 mL of GST-Sefinose resin (BBI Life Sciences) and rocked for 2 h to let the GST-FAAH fully bind to the resin. Then protein-bound resin was sequentially washed with 50 mL of wash buffer 2 (wash buffer 1, 1 mM MgSO4, 0.1 mM ATP, protease and phosphatase inhibitors, 1 mM EGTA), 5 mL of wash buffer 3 (wash buffer 1 and 300 mM potassium glutamate), and 50 mL of wash buffer 1. Finally, to cleave GST off GST-FAAH, protein-bound resin was resuspended in cleavage buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 2.5 mM CaCl2, 0.1% β-mercaptoethanol) with 2 U/μL thrombin (BBI Life Sciences) and rocked overnight at 4 °C. Then the flow-through, which is mainly FAAH, was collected and buffer exchanged into protein storage buffer (50 mM Tris at pH 7.4, 1 mM DTT, 2 mM MgCl2, 100 μM ATP, 25% sucrose), aliquoted, and stored at −80 °C for future experiment. The recombinant FAAH was characterized in Figure S24. BL Signal Test in Vitro. 1 was dissolved in 100 μL of FAAH buffer (20 mM Tris [pH 7.4], 0.1 mM EDTA, 4 mM MgSO4, and 0.4 mg/ mL BSA) to a final concentration of 500 μM in a black 96-well plate. Then 1 mM TCEP and 2 μM FAAH were added, and the mixture was incubated at 37 °C for 6 h. After the addition of 1 μM fLuc and 1 mM ATP and incubation for 5 min, a BL image of the mixture was immediately monitored under a Xenogen IVIS Lumina II small-animal imaging system. For FAAH inhibitor experiment, 50 μM URB597 was preincubated with 500 μM 1 for 15 min; then the mixture was incubated with 1 mM TCEP and 2 μM FAAH at 37 °C for 6 h. The following BLI method with luciferase was as described above. Persistent BLI of FAAH in Living Cells. A total of 1 × 105 living MDA-MB-231-fLuc cells were transferred into one well of a black 96well plate; then 250 μM 1, 500 μM AMA, 500 μM NH2-Luc, or 500 μM Lys-Luc was added into each well, respectively. Then the BL images of the cells were monitored under the Xenogen IVIS Lumina II small-animal imaging system in real time. For FAAH inhibitor experiment, 100 μM URB597 was preincubated with cells for 30 min; then 250 μM 1 was treated and the cells were imaged as mentioned above. HPLC Analyses and TEM Sample Preparation for Cell Experiments. First, 6 × 106 living parental MDA-MB-231 cells were trypsinized and collected into a 5 mL centrifuge tube. After the incubation of 2 mM 1 with cells in serum-free culture medium at 37 °C for 2 h, the cells were centrifuged (1000 rpm, 5 min) and washed three times with PBS to remove the probe. Then the cells were resuspended in 300 μL of PBS buffer, pulse-sonicated on ice at 40% power for 20 min, and centrifuged at 16, 000 rpm for 2 min to collect the lysate supernatant. A 50 μL amount of lysate supernatant was injected into an HPLC system for analysis, as shown in Figure S17. For FAAH inhibitor-pretreated cells, after 6 × 106 living parental MDA-MB-231 cells were trypsinized and collected into a 5 mL centrifuge tube, the cells were pretreated with 100 μM URB597 for 30 min; then 2 mM 1 was added as described above. Similarly, 50 μL of lysate supernatant was injected into the HPLC system for analysis, as shown in Figure S17. For TEM observations, copper grids with carbon mesh were dipped into the above lysate supernatants of FAAHinhibitor-pretreated cells to prepare the TEM samples for observation. Persistent BLI of FAAH in Vivo. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. A total of 2 ×106 MDA-MB-231-fLuc breast cancer cells were subcutaneously implanted into the right thigh of 5-week-old BALB/c mice. All probes were dissolved in saline for injection. Then 0.1 g/kg 1 (123 μmol/kg), 0.07 g/kg AMA (246 μmol/kg), 0.1 g/kg Lys-Luc (246 μmol/kg), or 0.07 g/kg NH2-Luc (246 μmol/kg) was injected i.p. Imaging was performed at 0.5, 1, 2, 4, 7, 11, and 16 h post probe injection. For inhibitor experiment, 3 mg/kg URB597 dissolved in DMSO was i.p. preinjected into mice for 30 min; then 0.1 g/kg 1 in saline was injected and the mice were imaged as mentioned above. 3D-SIM Imaging of 1-Alexa 488 Nanoparticles in Tumors. Using the same i.p. injection strategy, 45 nmol of 1-Alexa 488 was

illumination microscope (3D-SIM) to image the tumors in mice i.p. injected with 1-Alexa 488. The results indicated that the fluorescent nanoparticles of 1-Alexa 488, whose morphology was similar to that of 1-NPs in cells or in vitro at pH 6.0 (Figure S18), accumulated in the tumor (Figures S20 and S21, Videos S1−S3). To further validate the persistent tumor BL of 1 was induced by the disassembly of 1-NPs but not the increased concentration of free 1, we i.p. injected the tumorbearing mice with 1-Alexa 488 and measured the fluorescence emission and UV−vis absorbance intensity from the fluorophore Alexa 488 in resected tumors with time. The results indicated that both the fluorescence emission and UV− vis absorbance in tumors reached their peaks around 4 h, but the BLI in tumors continuously increased at 16 h postinjection (Figure S22). Long-time BLI of mice indicated that BL intensity in tumors of mice injected with 1 reached its peak at 24 h and sustained a detectable signal for at least 2.5 days (Figure S23). The p values between the BL intensities after 3 h postinjection and those of 0 h of the experimental mice were calculated to be ≤0.001, while the corresponding p values of the control mice were calculated to be ≤0.194, suggesting that, under the action of FAAH, 1 induces obvious bioluminescence signal in vivo. All the above results indicated that only 1 in these compounds could be applied for long-term tracing of FAAH activity in vivo.

CONCLUSIONS In summary, by the rational design of the latent BL probe 1, we have developed a “smart” method of intracellular self-assembly and FAAH-directed disassembly of cyclic D-luciferin nanoparticles (i.e., 1-NPs) for persistent BLI of FAAH activity in vitro, in cells, and in vivo. Reduction-controlled self-assembly of 1-NPs in vitro and in cells was characterized with TEM images, and intracellular FAAH-directed cleavage of 1-NPs was validated by HPLC analyses. The results of control probes AMA, Lys-Luc, and NH2-Luc further validated that the persistent BL of 1 from MDA-MB-231-fLuc cells or tumors was controlled by the activity of intracellular FAAH. The enzymatic hydrolysis rate for nanoparticles should be slower than that for small molecules, as we demonstrated before.39 Thus, the direct substrate for fLuc in 1-NPs can be slowly released under the action FAAH, which results in persistent bioluminescence. Due to its property of long-term tracing of FAAH activity in vivo, we envision that our BL precursor 1 could probably be applied for in vivo screening of FAAH inhibitors and the diagnosis of their related diseases (or disorders) in the future. EXPERIMENTAL SECTION FAAH Expression and Purification. Rat FAAH gene was purchased from the Mammalian Gene Collection of Thermo Fisher Scientific (clone ID: rFAAH 7370226). Residues 30-579 of the rat FAAH gene were PCR-amplified and cloned into the BamHI and NotI sites of pGEX-4T1. Then rat FAAH (30-579) was expressed as GSTfusion proteins using the pGEX-4T1 vector in the BL21(DE3)pLysS competent E. coli (Vazyme Biotech). Cells were grown at 37 °C until the OD 600 reached 0.6, induced with 0.3 mM isopropyl β-D-1thiogalactopyranoside, and incubated at 20 °C for 5 h. For FAAH purification, E. coli pellets from a 2 L culture were thawed on ice after washing with cold wash buffer 1 (1× PBS and 0.5 mM DTT, pH 7.4), then resuspended in lysis buffer (20 mM Tris [pH 8.0], 137 mM NaCl, 1 mM EGTA, 1% Triton X-100, 10% sucrose, 1.5 mM MgCl2, 1 mM DTT, 0.3 mM PMSF, 1:100 protease inhibitor mix (Sigma), and 1:500 phosphatase inhibitor (Sigma)) at 5 mL lysis buffer/g wet pellet 7151

DOI: 10.1021/acsnano.6b03412 ACS Nano 2016, 10, 7147−7153

Article

ACS Nano injected into each nude mouse xenografted with MDA-MB-231 tumor. Two hours later, mice were euthanized and the tumors were resected. Tumors were fixed in buffered formalin overnight and then in 30% (w/v) sucrose solution overnight at 4 °C. Then, tumors were bisected and frozen in optical cutting temperature (OCT) medium. Sections (10 mm) were cut and mounted on poly-L-lysine-coated coverslips. The OCT medium was removed by washing with PBS (1×) three times, mounting medium that contained DAPI was applied, and the samples were mounted on microscope slides and sealed. The superresolution images were acquired by 3D-SIM using a DeltaVision OMX imaging system (Applied Precision) with simultaneous excitation at 408 nm for DAPI and 488 nm for Alexa 488, according to the standard procedure in the manufacturer’s instructions, and analyzed with API DeltaVision OMX softWoRx image-processing software. Finally, the 3D-SIM images were processed with Imaris software (Bitplane, Inc.).

(5) Aso, E.; Ferrer, I. Cannabinoids for Treatment of Alzheimer’s Disease: Moving Toward the Clinic. Front. Pharmacol. 2014, 5, 37. (6) Campos, A. C.; Moreira, F. A.; Gomes, F. V.; Del Bel, E. A.; Guimaraes, F. S. Multiple Mechanisms Involved in the Large-spectrum Therapeutic Potential of Cannabidiol in Psychiatric Disorders. Philos. Trans. R. Soc., B 2012, 367, 3364−3378. (7) Rubino, T.; Realini, N.; Castiglioni, C.; Guidali, C.; Vigano, D.; Marras, E.; Petrosino, S.; Perletti, G.; Maccarrone, M.; Di Marzo, V.; et al. Role in Anxiety Behavior of the Endocannabinoid System in the Prefrontal Cortex. Cereb. Cortex 2008, 18, 1292−1301. (8) Lichtman, A. H.; Leung, D.; Shelton, C. C.; Saghatelian, A.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Reversible Inhibitors of Fatty Acid Amide Hydrolase That Promote Analgesia: Evidence for an Unprecedented Combination of Potency and Selectivity. J. Pharmacol. Exp. Ther. 2004, 311, 441−448. (9) Otrubova, K.; Ezzili, C.; Boger, D. L. The Discovery and Development of Inhibitors of Fatty Acid Amide Hydrolase (FAAH). Bioorg. Med. Chem. Lett. 2011, 21, 4674−4685. (10) Deutsch, D. G.; Ueda, N.; Yamamoto, S. The Fatty Acid Amide Hydrolase (FAAH). Prostaglandins, Leukotrienes Essent. Fatty Acids 2002, 66, 201−210. (11) Seierstad, M.; Breitenbucher, J. G. Discovery and Development of Fatty Acid Amide Hydrolase (FAAH) Inhibitors. J. Med. Chem. 2008, 51, 7327−7343. (12) Huang, H.; Nishi, K.; Tsai, H. J.; Hammock, B. D. Development of Highly Sensitive Fluorescent Assays for Fatty Acid Amide Hydrolase. Anal. Biochem. 2007, 363, 12−21. (13) Shoup, T. M.; Bonab, A. A.; Wilson, A. A.; Vasdev, N. Synthesis and Preclinical Evaluation of [18F]FCHC for Neuroimaging of Fatty Acid Amide Hydrolase. Mol. Imaging Biol. 2015, 17, 257−263. (14) Skaddan, M. B.; Zhang, L.; Johnson, D. S.; Zhu, A.; Zasadny, K. R.; Coelho, R. V.; Kuszpit, K.; Currier, G.; Fan, K. H.; Beck, E. M.; et al. The Synthesis and In Vivo Evaluation of [18F]PF-9811: a Novel PET Ligand for Imaging Brain Fatty Acid Amide Hydrolase (FAAH). Nucl. Med. Biol. 2012, 39, 1058−1067. (15) Baker, M. Whole-Animal Imaging: The Whole Picture. Nature 2010, 463, 977−980. (16) Contag, P. R.; Olomu, I. N.; Stevenson, D. K.; Contag, C. H. Bioluminescent Indicators in Living Mammals. Nat. Med. 1998, 4, 245−247. (17) Tsien, R. Y. Imagining Imaging’s Future. Nat. Rev. Mol. Cell Biol. 2003, No. Suppl, SS16−SS21. (18) Yang, Y. M.; Shao, Q.; Deng, R.; Wang, C.; Xue, T.; Chen, K.; Cheng, Z.; Huang, L.; Liu, X.; Xing, B. G. In Vitro and In Vivo Uncaging and Bioluminescent Imaging through Photocaged Upconversion Nanoparticles. Angew. Chem., Int. Ed. 2012, 51, 3125−3127. (19) Van de Bittner, G. C.; Bertozzi, C. R.; Chang, C. J. Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation. J. Am. Chem. Soc. 2013, 135, 1783−1795. (20) Yao, H.; So, M. K.; Rao, J. A Bioluminogenic Substrate for In Vivo Imaging of Beta-Lactamase Activity. Angew. Chem., Int. Ed. 2007, 46, 7031−7034. (21) Evans, M. S.; Chaurette, J. P.; Adams, S. T.; Reddy, G. R.; Paley, M. A.; Aronin, N.; Prescher, J. A.; Miller, S. C. A Synthetic Luciferin Improves Bioluminescence Imaging in Live Mice. Nat. Methods 2014, 11, 393−394. (22) Patel, A. R.; Lim, E.; Francis, K. P.; Singh, M. Opening Up the Optical Imaging Window Using Nano-Luciferin. Pharm. Res. 2014, 31, 3073−3084. (23) Sun, X. X.; Zhao, Y. N.; Lin, V. S. Y.; Slowing, I. I.; Trewyn, B. G. Luciferase and Luciferin Co-immobilized Mesoporous Silica Nanoparticle Materials for Intracellular Biocatalysis. J. Am. Chem. Soc. 2011, 133, 18554−18557. (24) Jacobson, G. B.; Shinde, R.; Contag, C. H.; Zare, R. N. Sustained Release of Drugs Dispersed in Polymer Nanoparticles. Angew. Chem., Int. Ed. 2008, 47, 7880−7882.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b03412. General methods; syntheses and characterizations of 1, AMA, Lys-Luc, NH2-Luc, and 1-Alexa 488; Schemes S1− S7; Figures S1−S24; Table S1 (PDF) Fluorescence video (AVI) Fluorescence video (AVI) Fluorescence video (AVI)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (G. Liang). Author Contributions §

Y. Yuan and F. Wang contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors are grateful to Shuoguo Li (Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences) for her assistance in 3D-SIM imaging. This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the Major Program of Development Foundation of Hefei Center for Physical Science and Technology (2014FXZY005), Hefei Science Center CAS (2015HSC-UP012), the China Postdoctoral Science Foundation (Grant 2016M590571), the Fundamental Research Funds for the Central Universities (WK2060190054), and the National Natural Science Foundation of China (Grants 31400727, U1532144, and 21375121). REFERENCES (1) Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.; Lerner, R. A.; Gilula, N. B. Molecular Characterization of an Enzyme that Degrades Neuromodulatory Fatty-Acid Amides. Nature 1996, 384, 83−87. (2) Mofford, D. M.; Adams, S. T.; Reddy, G. S. K. K.; Reddy, G. R.; Miller, S. C. Luciferin Amides Enable In Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity. J. Am. Chem. Soc. 2015, 137, 8684−8687. (3) Blankman, J. L.; Cravatt, B. F. Chemical Probes of Endocannabinoid Metabolism. Pharmacol. Rev. 2013, 65, 849−871. (4) Cravatt, B. F.; Saghatelian, A.; Hawkins, E. G.; Clement, A. B.; Bracey, M. H.; Lichtman, A. H. Functional Disassociation of the Central and Peripheral Fatty Acid Amide Signaling Systems. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 10821−10826. 7152

DOI: 10.1021/acsnano.6b03412 ACS Nano 2016, 10, 7147−7153

Article

ACS Nano (25) Chandran, S. S.; Williams, S. A.; Denmeade, S. R. ExtendedRelease PEG-Luciferin Allows for Long-Term Imaging of Firefly Luciferase Activity. Luminescence 2009, 24, 35−38. (26) Yuan, Y.; Wang, L.; Du, W.; Ding, Z. L.; Zhang, J.; Han, T.; An, L. N.; Zhang, H. F.; Liang, G. L. Intracellular Self-Assembly of Taxol Nanoparticles for Overcoming Multidrug Resistance. Angew. Chem., Int. Ed. 2015, 54, 9700−9704. (27) Yuan, Y.; Sun, H.; Ge, S.; Wang, M.; Zhao, H.; Wang, L.; An, L.; Zhang, J.; Zhang, H.; Hu, B.; et al. Controlled Intracellular SelfAssembly and Disassembly of 19F Nanoparticles for MR Imaging of Caspase 3/7 in Zebrafish. ACS Nano 2015, 9, 761−768. (28) Yuan, Y.; Ge, S.; Sun, H.; Dong, X.; Zhao, H.; An, L.; Zhang, J.; Wang, J.; Hu, B.; Liang, G. Intracellular Self-Assembly and Disassembly of 19F Nanoparticles Confer Respective“ Off ” and “On” 19F NMR/MRI Signals for Legumain Activity Detection in Zebrafish. ACS Nano 2015, 9, 5117−5124. (29) Kojima, R.; Takakura, H.; Ozawa, T.; Tada, Y.; Nagano, T.; Urano, Y. Rational Design and Development of Near-InfraredEmitting Firefly Luciferins Available In Vivo. Angew. Chem., Int. Ed. 2013, 52, 1175−1179. (30) Li, H.; Wood, J. T.; Whitten, K. M.; Vadivel, S. K.; Seng, S.; Makriyannis, A.; Avraham, H. K. Inhibition of Fatty Acid Amide Hydrolase Activates Nrf2 Signalling and Induces Heme Oxygenase 1 Transcription in Breast Cancer Cells. Br. J. Pharmacol. 2013, 170, 489−505. (31) Chakravarti, B.; Ravi, J.; Ganju, R. K. Cannabinoids as Therapeutic Agents in Cancer: Current Status and Future Implications. Oncotarget 2014, 5, 5852−5872. (32) Wei, B. Q.; Mikkelsen, T. S.; McKinney, M. K.; Lander, E. S.; Cravatt, B. F. A Second Fatty Acid Amide Hydrolase with Variable Distribution among Placental Mammals. J. Biol. Chem. 2006, 281, 36569−36578. (33) Moore, S. A.; Nomikos, G. G.; Dickason-Chesterfield, A. K.; Schober, D. A.; Schaus, J. M.; Ying, B. P.; Xu, Y. C.; Phebus, L.; Simmons, R. M. A.; Li, D.; et al. Identification of a High-Affinity Binding Site Involved in the Transport of Endocannabinoids. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 17852−17857. (34) Day, T. A.; Rakhshan, F.; Deutsch, D. G.; Barker, E. L. Role of Fatty Acid Amide Hydrolase in the Transport of the Endogenous Cannabinoid Anandamide. Mol. Pharmacol. 2001, 59, 1369−1375. (35) Kathuria, S.; Gaetani, S.; Fegley, D.; Valino, F.; Duranti, A.; Tontini, A.; Mor, M.; Tarzia, G.; La Rana, G.; Calignano, A.; et al. Modulation of Anxiety Through Blockade of Anandamide Hydrolysis. Nat. Med. 2003, 9, 76−81. (36) Sorkin, A.; von Zastrow, M. Signal Transduction and Endocytosis: Close Encounters of Many Kinds. Nat. Rev. Mol. Cell Biol. 2002, 3, 600−614. (37) Li, Q. L.; Sun, Y. F.; Sun, Y. L.; Wen, J. J.; Zhou, Y.; Bing, Q. M.; Isaacs, L. D.; Jin, Y. H.; Gao, H.; Yang, Y. W. Mesoporous Silica Nanoparticles Coated by Layer-by-Layer Self-assembly Using Cucurbit[7]uril for in Vitro and in Vivo Anticancer Drug Release. Chem. Mater. 2014, 26, 6418−6431. (38) Huang, L.; Liu, Y. In Vivo Delivery of RNAi with Lipid-based Nanoparticles. Annu. Rev. Biomed. Eng. 2011, 13, 507−530. (39) Yuan, Y.; Zhang, J.; Cao, Q. J. W.; An, L. N.; Liang, G. L. Intracellular Disassembly of Self-Quenched Nanoparticles Turns NIR Fluorescence on for Sensing Furin Activity in Cells and in Tumors. Anal. Chem. 2015, 87, 6180−6185.

7153

DOI: 10.1021/acsnano.6b03412 ACS Nano 2016, 10, 7147−7153