FEN1-IN-4 | HSP Signal

Facile Method for Speciﬁcally Sensing Sphingomyelinase in Cells and Human Urine Based on a Ratiometric Fluorescent Nanoliposome Probe

Yangyang Zhang, Wenjing Wu, Junjie Zhang, Zhao Li, Huimin Ma, and Zhenwen Zhao*

ABSTRACT

Sphingomyelinase (SMase) is closely related to diseases like Niemann−Pick disease and atherosclerosis, and the development of a simple method for the assay of SMase activity is very useful to screen new potential inhibitors or stimulators of SMase or biomarkers of disease. Fluorophore-encapsulated nano- liposomes (FENs) are emerging as a new ﬂuorescent probe for sensing the enzymatic activity. In this work, two ﬂuorochromes (cy7 and IR780) were encapsulated into the liposome of sphingomyelin, and therefore, a sphingomyelin-based ratiometric FEN probe for the SMase activity assay was constructed. The probe shows high selectivity and sensitivity to acid SMase with a detection limit of 4.8 × 10−4 U/mL. Sphingomyelin is the natural substrate of SMase; therefore, the probe has native ability for all kinds of SMase activity assays. Moreover, the probe has been successfully applied to the analysis of acid SMase activity in cells and urine samples. As far as we know, this is the ﬁrst example of a nanoliposome ﬂuorescence method for assaying acid SMase, and the method is biocompatible and much simpler than the existing ones, which might provide a new strategy for developing new methods for other important esterases.

INTRODUCTION

Sphingomyelinase (SMase, EC 3.1.4.12), a glycoprotein, is a kind of hydrolytic enzyme, which catalyzes the degradation of sphingomyelin to ceramide and phosphorylcholine.1,2 In mammals, ﬁve types of SMase have been identiﬁed based on their cation dependence and optimal pH of action: lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium- dependent neutral SMase, magnesium-independent neutral SMase, and alkaline SMase. Among those ﬁve types, lysosomal acid SMase and magnesium-dependent neutral SMase are considered to be the major factors for the production of ceramide in cellular stress responses.3,4
Recent ﬁndings indicated that both lysosomal acid SMase and secreted zinc-dependent acid SMase were encoded by a single Smpd1 gene and underwent diﬀerential posttranslational modiﬁcation.4−9 Lysosomal acid SMase was a lysosomal protein, and the genetic mutation causes Niemann−Pick disease types A and B with the characteristic of sphingomyelin accumulation in lysosomes.3 Secreted zinc-dependent acid
SMase was a plasma protein secreted from cells, and its function was related to inﬂammation and pathophysiologically linked to atherosclerosis.1,4 These acid SMase also played an important role in CD95-induced apoptosis.10 Given the importance of acid SMase, developing a simple approach to monitor acid SMase activity was of great signiﬁcance for the prevention and treatment of related diseases.
Classically, 14C-labeled sphingomyelin as a substrate combined with thin-layer chromatography (TLC) was used for the SMase activity assay. The method was tedious and unfriendly to people and the environment due to the hazard of radioactive elements.11 Further, ﬂuorescent substrates such as BODIPY-SM and NBD-SM, combined with TLC or ultra- performance liquid chromatography (UPLC), had been applied in SMase assays, which proved that the acid SMase/ ceramide signaling pathway regulated the endoplasmic reticulum stress to prevent oXidized low-density lipoprotein- induced macrophage apoptosis.12−14 In addition, small- molecule ﬂuorescent probes composed of a sphingomyelin analogue with two ﬂuorophores, Nile red (NR)-nitrobenzoX- adiazole (NBD)15 or FAM-BODIPY,16 were synthesized for measuring acid SMase activity in live cells. In these methods, the substrates used usually required complex organic synthesis operation; moreover, these substrates were not natural substrates of SMase, which might lead to a questionable assay result.
Recently, liposomes have been applied in the ﬁeld of biochemistry detection.17−34 For example, Xue et al. developed a target-controlled gating liposome for sensing phospholipase D activity in breast cancer cells.30 We once constructed a ﬂuorophore-encapsulated nanoliposome (FEN) probe for sensing intracellular pH values.17 Inspired by these works, considering that sphingomyelin, like phosphatidylcholine, was also the main component of the cell membrane, a FEN probe composed of sphingomyelin, not phosphatidylcholine, might be capable of assaying SMase activity. Since sphingomyelin is the natural substrate of SMase, theoretically, the enzyme
SMase would hydrolyze sphingomyelin, and therefore, the liposome membrane would be destroyed. Accordingly, the ﬂuorochromes encapsulated by the liposome would be released, and ultimately, the spectral properties of the FEN probe would be aﬀected. These changes in the spectral properties of the FEN probe might be used for determining the activity of SMase.
Based on the above assumptions, in this work, two ﬂuorochromes (cy7 and IR780) were encapsulated into the liposome composed of sphingomyelin to construct a sphingomyelin-based FEN probe. The feasibility of the FEN probe for the SMase activity assay was further investigated. The probe was further applied to the acid SMase activity assay in cells and urine samples of patients with peritonitis.

EXPERIMENTAL SECTION
Materials and Reagents. Cholesterol, IR780 {2-[2-[2- chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2- ylidene)ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl- 1-propylindolium iodide}, neutral SMase (CAS number: 9031- 54-3), carboXylesterase, phospholipase 2, and 3-(amino-
propyl)-1-hydroXy-3-isopropyl-2-oXo-1-triazene (NOC 5) PBS buﬀer (pH 7.4) containing 1 mM IR780 and 2 mM cy7 at 40 °C with the ﬂask rotated vigorously for 1 h and then sonicated for 10 min using a bath-type sonicator. Finally, the resulting liposome was dialyzed to remove the unencapsulated ﬂuorochromes using a membrane with a molecular weight cutoﬀ of 3500 (MD34-3500, Viskase, Lombard, IL) in PBS (0.2 M, pH 7.4) for 24 h at room temperature. After dialysis to remove the unencapsulated ﬂuorochromes, the liposomes were lyophilized using a freezing dryer (FD-1C-80, Beijing Biocool), and then a certain amount of PBS (0.2 M, pH 7.4) was added to prepare a stock solution of 5 mg/mL, which was stored at −20 °C for subsequent use. Cryo-transmission electron microscopy (cryo-TEM) images of the FEN probe were taken on a JEM-2011F microscope. The size distribution and ζ-potential of the FEN probe were measured using a dynamic light scattering (DLS) instrument (ZEN3600, Zetasizer Nano ZS, Malvern, England).
General Procedure for the SMase Activity Assay. Unless otherwise stated, all of the ﬂuorescence measurements were performed in the PBS solution (10 mM, pH 5.0 for acid SMase or pH 7.4 for neutral SMase) according to the following procedure. In a 10 mL tube, 5 mL of PBS and 100 μL of the stock solution of the FEN probe (5 mg/mL) were miXed, followed by addition of an appropriate volume of the sample solution (commercial acid SMase or neutral SMase, cell lysate, or urine). The ﬁnal volume was adjusted to 10 mL with PBS and the reaction solution was miXed rapidly. After incubation for 90 min at 37 °C in a thermostat, a 3 mL portion of the reaction solution was transferred to a quartz cell to measure ﬂuorescence intensity/spectrum (λex = 700 nm) with both excitation and emission slit widths of 10 nm using a Hitachi F- 4600 spectrophotometer in 10 mm × 10 mm quartz cells (Tokyo, Japan). In the meantime, a blank solution containing PBS (10 mM) was prepared and measured under the same conditions for comparison.
Cytotoxicity Assay. The cytotoXicity of the probe was were purchased from Sigma-Aldrich (St. Louis, MO). Acid SMase (product number: 50749-M08B) was purchased from Sino Biological Incorporation (Beijing, China). Sulfo-cyanine7 carboXylic acid (cy7) was purchased from Resenbio (Xi’an, China). N-Palmitoyl-D-erythro-sphingosylphosphorylcholine (d18:1/16:0 sphingomyelin) and N-heptadecanoyl-D-erythro- sphingosine (17:0 ceramide) were purchased from Avanti Polar Lipids (Birmingham, AL). RNA oligo was purchased from GenePharma (Shanghai, China). The transfection reagent was purchased from Engreen (Beijing, China). Fetal bovine serum (FBS), Dulbecco’s modiﬁed Eagle medium (DMEM), streptomycin, penicillin, and phosphate-buﬀered saline (PBS) solution were purchased from Invitrogen Corporation (Pittsburgh, PA). 3-(4,5-Dimethylthiazol-2-yl)-evaluated by the standard MTT assay according to the previous report.17
Cell Culture and Fluorescence Detection by the FEN Probe. The MCF-7 cells were plated on glass-bottom culture dishes (MatTek Co.) in DMEM supplemented with 10% (v/v) FBS, and 1% (v/v) penicillin−streptomycin in a 5% CO2 incubator at 37 °C. All siRNAs were synthesized by GenePharma (Shanghai, China). The target sequences are listed in Table 1. siRNAs were transfected into MCF-7 cells
Table 1. Sequences of siRNA Transfected into MCF-7 Cells
5′-AUCCAUUGUCCACCUCUUUGAGGTT-3′ ASM-2Si
5′CGGAUACUGGGGCGAAUACAGCTT-3′ NCSi
5′-UUCUCCGAACGUGUCACGU-3′
2,5-diphenyltetrazolium bromide (MTT) was purchased from Serva Electrophoresis GmbH (Heidelberg, Germany). Ultra pure water with resistivity over 18 MΩ·cm was obtained from a Milli-Q equipment (Millipore Corp., Burlington, MA).
Preparation of the FEN Probe. The FEN probe was prepared on the basis of a previous report with modiﬁcations.17 First, d18:1/16:0 sphingomyelin and cholesterol with a molar ratio of 1:1 (30 μmol in total, dissolved in 4 mL of chloroform) were miXed and then sonicated for 5 min. The solution was then subjected to rotary evaporation under reduced pressure at 40 °C to remove the solvents. This produced a thin lipid ﬁlm on the inside wall of the round-bottom ﬂask. The FEN probe was obtained via hydration of the ﬁlm using 2 mL of 0.2 M with Lipofectamine 2000 (Invitrogen), according to the protocol supplied by the manufacturer. The siRNA concen- tration for transfection was about 100 nM. In each siRNA transfection experiment, nonspeciﬁc siRNA (NCSi) was used as a normal control.
For ﬂuorescence detection, the MCF-7 cells were incubated with the FEN probe (50 μg/mL) for 30 min at 37 °C and then washed three times by PBS (pH 7.4). The probe-loaded MCF- 7 cells were digested, collected by centrifugation, resuspended by PBS (1 mL, pH 5.0), and then lysed ﬁve times with the ultrasonic method for 5 s a time, with an interval of 5 s.
Figure 1. (A) DLS and cyro-TEM analyzing the size distribution and the morphology of the FEN probe. Scale bar: 100 nm. (B) The stability of the FEN probe after storing for 30 days, measured by DLS and cyro-TEM. Scale bar: 100 nm. (C) The ﬂuorescence spectrum of cy7 (λex = 700 nm, red line) and IR780 (λex = 780 nm, light blue line), as well as the absorption spectrum of cy7 (purple line) and IR780 (blue line). (D) Fluorescence intensity and their ratio (I750/I830) of cy7 and IR780 in the FEN probe stored in 0.2 M PBS buﬀer (pH 5.0) during diﬀerent time frames.
Fluorescence detection by the FEN probe was performed in accordance with the “General procedure for SMase activity assay”. Western Blot Analyses. Cells with siRNA transfection were lysed in RIPA buﬀer for 30−60 min. After centrifugation (13 500 g, 30 min) at 4 °C, the supernatant was collected as a cell lysate. The proteins in the cell lysate were quantiﬁed by the BCA assay (Micro BCA; Pierce Biotechnology). Protein samples were further separated by SDS-PAGE and analyzed by the western blot as described previously.35
Extraction of Lipids. For cell pellet lipid assays, the methanol method was used to extract lipids.36 In brief, a pellet containing 0.04 mg of the total protein was resuspended in 50 μL of water before it was transferred into 450 μL of methanol containing 50 pmol of 17:0 ceramide (used for quality control). After vortexing and centrifugation (10 000g, 5 min, room temperature), 300 μL of the supernatant was directly used for the analysis of d18:1/16:0 sphingomyelin and 16:0 ceramide by mass spectrometry (MS). Two microliters of the supernatant were loaded into a mass spectrometer for the analysis of these lipids.
Mass Spectrometry. MS analysis was performed using a QTRAP 4500 mass spectrometer (SCIEX) with the Analyst data acquisition system. Both the nebulizer and desolvation gases were nitrogen. Typical operating parameters were set as follows: curtain gas (CUR) 25, collision gas (CAD) medium, ion source gas 1 (GS1) 45, ion source gas 2 (GS2) 50, electrospray voltage 5500 with positive ion MRM mode, and a temperature of 500 °C. Samples were loaded through an LC system (I-class Acquity ultra-performance liquid chromatog- raphy, Waters) with an autosampler. Mobile phase A was isopropanol/acetonitrile/formic acid (90:10:0.1, v/v/v) con- taining 10 mM ammonium formate; mobile phase B was acetonitrile/water/formic acid (70:30:0.1, v/v/v) containing 10 mM ammonium formate. A CSH C18 column (1.7 μm, 2.1 mmID × 100 mm, Waters) was used for the separation of sphingomyelin and ceramide. The column was maintained at 55 °C. The separations were 20 min/sample using the following scheme: (1) 0 min, 70% B; (2) 2 min, 57% B; (3) 2.1 min, 50% B; (4) 12 min, 46% B; (5) 12.1 min, 30% B; (6) 18 min, 1% B; (7) 18.1 min, 70% B; and (8) 20 min, 70% B. All of the changes are linear, and the ﬂow rate was set to 400 μL/min.
Urine Acid SMase Determination. Urine samples of peritonitis were provided by Dr. Zhao Li, including ﬁve patients with peritonitis and ﬁve unaﬀected controls enrolled at the Peking University People’s Hospital. Urine samples (10 mL) were collected in 15 mL centrifuge tubes (Corning Life Sciences, Corning, NY) and frozen at −80 °C until use. Urine samples (10 mL) were clariﬁed by centrifugation for 5 min at 10 000g at room temperature, and the supernatant aliquots were dried via lyophilization and dissolved in 1 mL of the PBS
Scheme 1. (A) Construction of the Sphingomyelin-Based FEN Probe and (B) the FEN Probe for the SMase Activity Assay solution (10 mM, pH 5.0). Then, the FEN probe (10 μL, 5 mg/mL) was added, and the acid SMase activity was determined.

RESULTS AND DISCUSSION

Construction of the Sphingomyelin-Based Fluorophore-Encapsulated Nanoliposome (FEN) Probe for the SMase Activity Assay. In our previous work, nanoliposomes were ﬁrst applied to encapsulate two ﬂuorophores, ﬂuorescein and cresyl violet, to construct a novel ﬂuorescence resonance energy transfer (FRET)-based ratiometric ﬂuorescent probe (we called it the ﬂuorophore-encapsulated nanoliposome (FEN) probe), for sensing intracellular pH values. In this work, a similar FRET-based ratiometric ﬂuorescent probe was constructed using nanoliposomes for encapsulating two ﬂuorochromes, cy7 and IR780. The diﬀerence is that the nanoliposomes in this work were composed of sphingomyelin and cholesterol, unlike the traditional nanoliposomes, which were mainly composed of phosphatidylcholine and cholesterol. Sphingomyelin, like phosphatidylcholine, was also the main component of the cell membrane, and therefore, sphingomye- lin could also be used to construct liposomes.
The FEN probe was successfully prepared by the thin-ﬁlm dispersion method (see the EXperimental Section for details) and the molar ratio of cy7/IR780 was 2:1. Under this condition, the average hydrodynamic diameter of the FEN probe was about 100 nm (Figures 1A and S1), the ζ-potential was −5.72 mV (Figure S2), and the morphology was orbicular (inset of Figure 1A). The encapsulation eﬃciency was evaluated by the ratio of the amount of the ﬂuorochromes in the nanoliposomes to the total amount of the ﬂuorochromes added, in which the encapsulated cy7 and IR780 were released
from nanoliposomes with 1% Triton X-100. On the basis of the calibration curves of standard cy7 and IR780, the encapsula- tion eﬃciencies of cy7 and IR780 were about 50 and 61%, respectively. The FEN probe (5 mg/mL) was stored in 0.2 M PBS (pH 7.4) at −20 °C for further use, and after 30 days of storage, the DLS analysis and cryo-TEM images showed that the diameter and morphology of the FEN probe were pretty much the same (Figures 1B and S1), indicating that the FEN probe was stable for at least 1 month.
The main absorption peaks of cy7 (solvent: methanol) and IR780 (solvent: methanol) were at 650−750 nm (Figure 1C, purple line) and 750−800 nm (Figure 1C, blue line), respectively. Using 700 nm as ﬂuorescence excitation wave- length, the ﬂuorescence emission peak of cy7 was at 700−80
Figure 2. (A) Eﬀects of pH on the I750/I830 of the FEN probe reacting with acid SMase (0.30 U/mL). The reaction was performed in a 10 mM Na2HPO4−NaH2PO4 solution with diﬀerent pH values adjusted by HCl and NaOH. (B) The change in the ﬂuorescence intensity of I750/I830 of the FEN probe reacting with acid SMase (0.30 U/mL) as a function of incubating time at diﬀerent temperatures (20, 30, and 37 °C). (C) Fluorescence emission spectra of the FEN probe (50 μg/mL) in 10 mM Na2HPO4/NaH2PO4 buﬀer for 90 min at diﬀerent acid SMase concentrations (0−0.5 U/mL). (D) Plot of I750/I830 versus acid SMase concentration in the range of 0−0.5 U/mL. λex = 700 nm was used to obtain the ﬂuorescence emission spectra.
nm (Figure 1C, red line) and was overlapped with the absorption peak of IR780 (Figure 1C). In this case, cy7 and IR780 inside the nanoliposomes could undergo eﬃcient FRET. As a matter of fact, just as we expected, when using 700 nm as the ﬂuorescence excitation wavelength, the sphingomyelin- based FEN probe demonstrated the ﬂuorescence emission peaks of cy7 and IR780 at 725−775 and 800−850 nm, respectively. It should be noted that IR780 alone has almost no ﬂuorescence emission peak using an excitation wavelength of 700 nm. Thus, the ﬂuorescence emission peak of the probe’s IR780 is indeed generated by the FRET eﬀect.
Because sphingomyelin is the natural substrate of SMase, theoretically, the enzyme SMase would catalyze the hydrolysis of sphingomyelin. Liposomes that we constructed were composed of sphingomyelin (amphoteric molecules), and the hydrolysis would remove the hydrophilic head of the sphingomyelin and generate ceramides (nonamphoteric molecules). In this case, the liposome membrane would be theoretically destroyed; therefore, the ﬂuorochromes encapsulated by the liposome would be released, and the FRET of the FEN probe would be ultimately aﬀected. To be speciﬁc, without SMase, cy7 in the probe would absorb the energy of the excitation light at 700 nm and emit the light wave at 750 nm. Meanwhile, IR780 in the probe would absorb this 750 nm light wave energy (FRET eﬀect) and emit a wavelength of 830 nm. In this case, the emitted light wave energy of cy7 would become weaker, and the emitted light wave of IR780 would become stronger. In the other case, in the presence of SMase, SMase would destroy liposomes, releasing cy7 and IR780 in liposomes and disrupting the FRET eﬀect between them. In this case, cy7 would continue to absorb the energy of the excited light with a wavelength of 700 nm and emit the light wave with a wavelength of 750 nm. However, free IR780 was unable to absorb 750 nm energy (because the FRET eﬀect was disrupted), and as a result, the emitted energy of cy7 increased and the emitted energy of IR780 decreased. The process is shown in Scheme 1, and these changes in the spectral properties of the FEN probe reﬂected the activity of SMase, which would be discussed in the next section in detail.
The stability of the spectral properties of the FEN probe was investigated. Using 700 nm as the ﬂuorescence excitation wavelength, after 30 days of storage, the ﬂuorescence intensities of cy7 (750 nm) and IR780 (830 nm) slightly decrease by less than 7%, and their ratio was barely changed (Figure 1D), indicating that the FEN probe could be used directly for 1 month.
Fluorescence Response of the FEN Probe to Acid SMase. The FEN probe was prepared using sphingomyelin as a nanoliposome membrane for SMase recognition. Since sphingomyelin is the natural substrate of SMase, theoretically, the FEN probe could respond with any kind of SMase. Meanwhile, the action of SMase needed a speciﬁc cation and pH environment. Therefore, to measure the activity of one kind of SMase, a speciﬁc environment was necessary. For example, for measurement of secreted zinc-dependent acid SMase activity, Zn2+ and an acidic environment might be necessary. Hence, by regulating the detection environment, diﬀerent types of SMase activities in the sample could be detected. Since acid SMase was considered to be major SMase and closely related to diseases like Niemann−Pick disease and atherosclerosis, the conditions and quantitative feasibility of acid SMase detection were studied in detail.
As shown in Figure 2A, the FEN probe showed a good ratiometric response to acid SMase (commercial acid SMase, product number: 50749-M08B) with pH = 5.0, whereas nearly no change was detected when the FEN probe reacted with acid SMase with pH = 7.4 and 9.0. This suggests that acid SMase was not able to cleave the FEN probe or the activity was very low in neutral and alkaline environments. The eﬀects of temperature on the ﬂuorescence intensity of the reaction system were examined. The results showed that temperatures or lysosomal acid SMase was the major acid SMase in this standard (data not shown).
Moreover, the feasibility of the probe for the neutral SMase activity assay was investigated. The FEN probe was miXed with neutral SMase (commercial product) in pH = 7.4 PBS solution with 10 mM Mg2+, the ﬂuorescence intensity ratio (R) of cy7 at 750 nm versus IR780 at 830 nm increased with the increase of neutral SMase concentration, showing the ability of the probe for the neutral SMase activity assay (Figure 3).
Figure 3. Fluorescence changes of the FEN probe toward the concentrations of neutral SMase from 0.1 to 0.5 U/mL.
Selectivity of the FEN Probe for the Acid SMase Activity Assay. The selectivity of the FEN probe was examined for acid SMase over other potential interfering species in cells, such as inorganic salts (KCl, MgCl2, CaCl2, lower than 37 °C result in a slower enzymatic cleavage reaction NaNO3, and CuSO4), reactive oXygen species (•OH and and less ﬂuorescence enhancement (Figure 2B).
As depicted in Figure 2C, the increase of acid SMase concentration increased the ﬂuorescence (750 nm) of cy7 vigorously, while that of IR780 (830 nm) was decreased. The increase of acid SMase concentration released a large amount of cy7 and IR780 in nanoliposomes, thus destructing the FRET eﬀect, which led to the changes in the spectral properties of the FEN probe. To prove that SMase could destruct the liposome by cleaving the head group of sphingomyelin, we performed the nanoparticle tracking analysis (NTA) before and after the liposomes reacted with SMase. The results (Figure S3) showed that the size of liposomes did not change (116 nm); however, the amount of liposomes decreased from 5.3 × 106 (Figure S3A) to 3.5 × 106 particles/mL (Figure S3B), which indicated that the liposomes were really disrupted by SMase. The ﬂuorescence intensity ratio (R) of cy7 at 750 nm versus IR780 at 830 nm increased with the increase of acid SMase concentration. Most notably, good linearity is found in a concentration range of 0−0.5 U/mL acid SMase (Figure 2D).
The detection limit (3S/m, in which S is the standard deviation of blank measurements, n = 11, and m is the slope of the linear equation) is determined to be 4.8 × 10−4 U/mL acid SMase. Further, we added 10 mM Zn2+ to the PBS solution to measure secreted zinc-dependent acid SMase activity; however, no obvious signal enhancement was found when the FEN probe reacted with this acid SMase with pH = 5.0, probably because the low concentration of zinc-dependent acid SMase H2O2), glucose, reduced glutathione (GSH), cysteine, BSA, HSA, and two main enzymes existing in cells (carboXylesterase and phospholipase 2 (PLA2)). This FEN, unlike conventional liposomes (synthesized by phosphatidylcholine with choles- terol), avoided hydrolysis from PLA2. As shown in Figure 4A, the probe displayed high selectivity for acid SMase over the other species tested.
To measure the acid SMase activity in human urine, the common potential interferents present in urine, including urea, uric acid, L-histidine, glycine, and oXalic acid, were examined as well. As shown in Figure 4B, the FEN probe displayed the most sensitive ﬂuorescence response to acid SMase compared to the other substances even with considerable concentrations. To further conﬁrm that the FRET-based reaction (the changes in the spectral properties) was caused by acid SMase, two common inhibitors of acid SMase, NO37 and desipramine (DES),38 were utilized. Acid SMase was preincubated with diﬀerent concentrations of NOC 5, a NO donor, which was usually used in experiments directly for providing NO,39 at 37°C for 30 min, then the pretreated enzyme solution was miXed with the solution containing the FEN probe, and ﬁnally the ﬂuorescence changes were monitored. As shown in Figure 5A, with the increase of NOC 5 concentration, the ratio gradually decreased, meaning the acid SMase activity became lower and lower. For example, addition of 0.05 mM NOC 5 caused a ﬂuorescence decrease of 14%, whereas higher concentrations of NOC 5 such as 0.1 or 0.2 mM could lead to a larger ﬂuorescence decrease of 29 or 57%, suggesting that the activity
Figure 4. (A) Fluorescence changes of the FEN probe to diﬀerent species in cells: (1) acid SMase (0.3 U/mL), (2) MgCl2 (2 mM), (3) CaCl2 (2 mM), (4) KCl (150 mM), (5) NaNO3 (100 μM), (6) CuSO4 (100 μM), (7) H2O2 (100 μM), (8) •OH (100 μM), (9) glucose (10 mM), (10) GSH (1 mM), (11) cysteine (1 mM), (12) bovine serum albumin (BSA, 100 mM), (13) human serum albumin (HSA, 100 mM), (14) carboXylesterase (0.20 U/mL), (15) PLA2 (3.2 ng/mL), and (16) blank (buﬀer control sample). (B) Fluorescence changes of the FEN probe to diﬀerent species in urine: (1) urea (20 mM), (2) uric acid (0.3 mM), (3) L-histidine (0.1 mM), (4) glycine (0.2 mM), (5) oXalic acid (0.05 mM),m(6) acid SMase (0.3 U/mL), and (7) blank (buﬀer control sample). Data were acquired in 10 mM PBS (pH 5.0) with λex = 700 nm.
Figure 5. Change in the ﬂuorescence intensity of I750/I830 of the FEN probe reacting with acid SMase in the presence of diﬀerent concentrations of NO (0, 0.05, 0.1, and 0.2 mM) (A) and DES (0, 2, 5, and 10 μM) (B). Buﬀer control sample was shown as blank. of acid SMase was greatly suppressed. Similarly, 2−10 μM DES could also play an inhibitory role (Figure 5B). This indicated that the FRET-based reaction indeed raised from acid SMase. Obviously, such FEN probe might be useful to screen new potential inhibitors or stimulators for acid SMase.
Acid SMase Activity in MCF-7 Cells and siRNA Transfection MCF-7 Cells. Prior to cell experiments, the cytotoXicity of the FEN probe was evaluated by the standard MTT cytotoXicity assay. As shown in Figure 6A, when the dosage used was not more than 100 μg/mL, about 90% cell viability was retained. Therefore, in this work, a 100 μg/mL FEN probe was employed in cell culture medium for the cell acid SMase activity assay.

To validate the feasibility of the FEN probe for the cell acid
SMase activity assay, cells with diﬀerent acid SMase expression levels, including wild-type (WT) breast cancer cell MCF-7 and siRNA (ASM-1Si and ASM-2Si) transfection cells, were ﬁrst cultured. In Figure 6B, the western blot result clearly showed that acid SMase was low expressed in cells transfected with speciﬁc siRNA (ASM-1Si and ASM-2Si). Further, the acid SMase activity assay results showed that transient transfection of MCF-7 cells displayed a lower ﬂuorescence ratio value (I750/ I830) compared to untransfected MCF-7 cells (Figure 6C), indicating the lower acid SMase activity of siRNA transfection (ASM-1Si and ASM-2Si) MCF-7 cells.
In the meantime, since acid SMase could catalyze the hydrolysis of sphingomyelin to ceramide, based on the ultrahigh-performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/ MS), d18:1/16:0 sphingomyelin and its hydrolytic product 16:0 ceramide were measured and compared in MCF-7 cells and siRNA transfection MCF-7 cells. The results (Figure 6D) disclosed a higher d18:1/16:0 sphingomyelin level and a lower d18:1/16:0 ceramide level in siRNA transfection (ASM-1Si
Figure 6. (A) Cell viability of MCF-7 treated with the FEN probe at diﬀerent concentrations (1−100 μg/mL). The results are expressed as the mean ± SD of ﬁve separate measurements. (B) Western blot analysis of MCF-7 cells. The MCF-7 cells were transfected with speciﬁc siRNA (ASM- 1Si and ASM-2Si) or nonspeciﬁc siRNA (NCSi). β-Actin was used as a protein standard. (C) The change in the ﬂuorescence intensity of I750/I830 of the FEN probe reacting with acid SMase in MCF-7 cells, ASM-1Si MCF-7 cells, and ASM-2Si MCF-7 cells. **P < 0.01. (D) Quantiﬁcation of d18:1/16:0 sphingomyelin and 16:0 ceramide by mass spectrometry in MCF-7 cells and siRNA transfection MCF-7 cells. *P < 0.05 and **P < 0.01. and ASM-2Si) MCF-7 cells due to the lower acid SMase activity. The results further conﬁrmed the accuracy of the FEN probe in measuring acid SMase activity.
Acid SMase Activity in Human Urine. Finally, the FEN probe was used for measuring acid SMase activity in human urine. The results obtained are shown in Figure 7. The acid SMase activity in the urine of peritonitis patients was signiﬁcantly higher than that in normal humans, whereas nearly no acid SMase activity was detected in the urine of normal humans. Sandhoﬀ reported that the amount of acid SMase was elevated in the urine of peritonitis patients 200 times more than that in normal humans.40 Our results also supported this conclusion. Our approach, by detecting acid SMase activity in urine with the FEN probe, might provide a clinical diagnosis of peritonitis.

CONCLUSIONS

In summary, nanoliposomes were ﬁrst used to encapsulate two ﬂuorochromes, cy7 and IR780, to construct a novel FRET- based ratiometric ﬂuorescent nanoliposome probe, for speciﬁc
Figure 7. Changes in the ﬂuorescence intensity of I750/I830 of the FEN probe reacting with acid SMase in the urine of peritonitis patients and normal humans. ***P < 0.001. detection of acid SMase in cells and urine samples. The probe shows high selectivity and sensitivity with a detection limit of 4.8 × 10−4 U/mL acid SMase and has been applied to evaluating the downregulation of acid SMase levels in MCF-7 cells by siRNA transfection, mimicking the phenotype of Niemann−Pick patient cells, as further evidenced by western blot analysis. Moreover, the applicability of the method has been demonstrated by detecting acid SMase in the urine of peritonitis patients. As far as we know, this is the ﬁrst example of a nanoliposome biosensing method for assaying SMase, and the method is simpler than the existing ones, which may provide a helpful tool for screening SMase inhibitors or stimulators in some complex biosystems, and the present strategy may be useful for developing new methods for other important esterases.

ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.analchem.1c02197.

Size histogram from the cyro-TEM images of the FEN probe and the FEN probe stored for 30 days; ζ-potential distribution graph measured by dynamic light scattering; and nanoparticle tracking analysis of liposomes before and after the liposomes reacted with SMase (PDF)

AUTHOR INFORMATION
Corresponding Author
Zhenwen Zhao − Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of Sciences, Beijing 100190, China; University of Chinese Academy of Sciences, Beijing 100049, China; orcid.org/ 0000-0001-6127-808X; Phone: +86-10-62561239;
Email: [email protected]; Fax: +86-10-62561285

Authors
Yangyang Zhang − Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of Sciences, Beijing 100190, China; University of Chinese Academy of Sciences, Beijing 100049, China
Wenjing Wu − Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of
Sciences, Beijing 100190, China; University of Chinese Academy of Sciences, Beijing 100049, China
Junjie Zhang − College of Life Sciences, Beijing Normal University, Beijing 100875, China
Zhao Li − Department of Hepatobiliary Surgery, Peking University People’s Hospital, Beijing 100044, China Huimin Ma − Beijing National Laboratory for Molecular
Sciences, Key Laboratory of Analytical Chemistry for Living
Biosystems, Institute of Chemistry Chinese Academy of Sciences, Beijing 100190, China; University of Chinese Academy of Sciences, Beijing 100049, China; orcid.org/ 0000-0001-6155-9076
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.analchem.1c02197

Notes
The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS
This research was supported by grants from the National
Natural Science Foundation of China (Nos. 21874142 and 21635008) and the National Key R&D Program of China (No. 2018YFA0800900).

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