A two-photon AIEgen for simultaneous dual-color imaging of atherosclerotic plaques

Bo Situab, Meng Gaoc, Xiaojing Heab, Shiwu Lid, Bairong Heab, Fengxia Guoab, Chunmin Kangab, Shan Liue, Lei Yange, Meijuan Jiangfg, Yanwei Huab, Ben Zhong Tang*dfg and Lei Zheng*ab
aDepartment of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China. E-mail: nfyyzhenglei@smu.edu.cn
bGuangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
cNational Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, 510006, China
dCenter for Aggregation-Induced Emission, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, 510640, China
eDepartment of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
fDepartment of Chemistry and Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China. E-mail: tangbenz@ust.hk
gHKUST-Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen, 518057, China

Received 13th October 2018 , Accepted 22nd November 2018

First published on 23rd November 2018

High-resolution imaging of lipids within the artery is of great value to obtain fundamental knowledge about their roles in atherosclerosis, but ideal probes are still lacking. In this study, we report a smart probe, namely, IND with aggregation-induced emission property for two-photon visualization of lipids within cell lines and arteries. Taking advantage of high lipid specificity, deep tissue penetrability, and excellent two-photon imaging performance, IND is capable of high-resolution imaging of lipids in cultured cells and in situ mapping of three-dimensional lipid distributions in mouse atherosclerotic plaques without tedious histological preparations. Importantly, we find that IND exhibits distinct emissive properties in the molecular monomer and dimer states; also, we uncover its remarkable applications for single-excitation dual-color imaging of lipid/water in the tissue microenvironment to reveal the ultra-structure of atherosclerotic plaques. The findings described herein provide a simple tool with minimal disruptive procedures for studying atherosclerosis at the tissue level and a powerful strategy for the development of imaging-based diagnostic techniques with color switching capability.

Conceptual insights

Visualizing lipids within the arteries is of great scientific value to study atherosclerosis. Currently, this process usually requires tedious histological preparations to obtain tissue sections followed by staining with chemical dyes. Here, we develop a smart color-switchable bioprobe named IND for high-resolution imaging of atherosclerotic plaques in situ. Unlike traditional excimer systems that mostly exhibit fluorescence quenching in the aggregated state, IND can pack as unique pairwise dimers with aggregation-induced emission (AIE) activity and an uncommon large red-shifted fluorescence. These distinct characteristics together with its advantages of high penetrability, good biocompatibility, large two-photon absorption, and convenient operation allow two-photon dual-color imaging of lipid and water distributions in mouse atherosclerotic plaques. The unique functions of IND show merit over existing lipid dyes in terms of advantages that include simple, function-transformable, and deep-tissue imaging without tissue sectioning. This study provides a novel strategy to rationally guide the design of new color-switchable fluorescent materials for biosensing and bioimaging.


Atherosclerosis is a chronic disease caused by plaque formation on the artery walls.1 It is the major pathogenesis of strokes and heart attacks and is responsible for about 50% of all deaths in western countries.2,3 Aberrant accumulation of lipids in the arteries is a sign of atherosclerosis, and many studies have focused on the critical roles of lipids in the development of atherosclerosis due to their potential significance in disease prevention, treatment, and reversal.4–6 To gain direct insights into lipid functions within atherosclerosis, it is necessary to observe their amounts, localizations, and distributions in the arteries.7 In current clinical pathology, imaging and quantifying lipids are mainly inferred from staining tissues by histological dyes such as Oil Red O and Sudan Black B.8,9 Their performance, however, is far from satisfactory. Besides low specificity and resolution, they require tedious histological procedures including tissue fixation, dehydration, clearing, cutting, staining, second dehydration, and mounting to obtain thin vessel sections for observation10 due to their inability to be imaged deeply. Even so, analysis of isolated tissue sections can neither reveal the shape, size, and spatial microstructures of the whole specimen nor help to precisely measure the overall lipid distribution within tissues. These procedures can disrupt the original tissue structures and components in varying degrees.11 Especially, lipids are susceptible to removal from the solvents utilized during routine histological preparations.12 Given these limitations, the development of ideal strategies with minimal disruption to tissues for lipid imaging is highly desired.

Fluorescence imaging is a powerful tool for observing the microstructures and biological processes with high resolution.13 A number of fluorescence-based methods have been developed to study atherosclerosis by imaging various targets such as fibrin,14 matrix metalloproteinases (MMP),15 cathepsin K,16 lipoprotein,17 and vascular cell adhesion molecule 1 (VCAM1).18 In tissue imaging, two-photon fluorescence microscopy offers noticeable advantages over traditional fluorescence techniques such as deep penetration, high 3D resolution, and in situ visualization with simple operation.19 Nevertheless, unlike proteins that can be readily imaged by engineered fluorescent proteins or fluorescent antibodies, ideal probes for tissue lipids have lagged far behind.20 Currently, the most widely used lipid probes are BODIPY 493/503 and Nile Red.21,22 Their applications, however, are mostly restricted in cultured cells. Besides limited specificity, they suffer from problems of poor penetrability at the tissue level as structural barriers such as tight junctions between cells and extracellular matrix in blood vessels are usually impenetrable to these dyes. Moreover, simultaneous imaging of both lipid accumulation sites and small hydrophilic cavities is highly desirable for biomedical studies as it is valuable for revealing aqueous/lipid interfaces, tissue microstructure, and for using one of the fluorescence signals as a reference for quantitative analysis or position correction.23,24 However, the conventional method using two different dyes for the respective imaging of lipids and water-containing cavities requires two different excitation and emission filters, which is usually not feasible in multi-photon microscopy because adding one more expensive titanium–sapphire (Ti:sapphire) laser source is unusual and it is difficult to control multiple ultrashort pulse lasers at the same time.25 An ideal strategy is to develop single fluorophores with separable emissions in lipids and aqueous microenvironments that can be excited with the same wavelength so that different signals can be collected simultaneously.26 However, lipophilicity and hydrophilicity are usually competitive natures of an organic molecule. Conventional fluorophores can undergo serious aggregation-caused quenching (ACQ) effect in certain tissue regions with dye accumulation.27 Development of smart materials that emit strongly in both lipid and aqueous media is challenging in addition to dual-emission sensing capacity.

Luminogens with aggregation-induced emission (AIEgens) are a new class of molecules, which are non-emissive when dissolved but fluoresce strongly in the aggregated state.28 AIE-based materials have shown great potential for biosensing and bioimaging with remarkable performance due to their intriguing working mechanisms and noticeable advantages.29–32 Herein, we report a smart AIEgen named IND that can specifically target lipids within culture cells and tissues. We uncover that IND may serve as a simple and facile two-photon probe for staining lipids within atherosclerotic plaques with good performance, which allows for high-resolution imaging of tissue lipids and quantification without tissue sectioning. Notably, IND molecules pack as special dimers with enhanced and red-shifted emission in the aggregated state. This enables the achievement of dual-color imaging of lipids and water with single excitation to reveal the ultra-structure of atherosclerotic plaques.

Result and discussion

Synthesis and photophysical properties

IND was synthesized by condensation of 4-(N,N-dibutylamino)benzaldehyde with 1,3-indandione via a one-step Knoevenagel reaction with a high yield of 72% (Fig. 1a). The structure of IND was characterized and verified by NMR and HRMS measurements (Fig. S1–S3, ESI). The photophysical properties of IND were then investigated. IND showed an absorption maximum at 491 nm and emitted green fluorescence with an emission peak at 526 nm in THF (Fig. 1b). The photoluminescence spectrum of IND was then investigated in THF/water mixtures. Upon increasing the water fractions (fw) from 0% to 80%, the emission maximum gradually red-shifted to 546 nm accompanied with decreased emission intensity (Fig. 1c and d); this can be ascribed to the twisted intramolecular charge transfer (TICT) effect caused by the electron donor–acceptor (D–A) structural feature. Since water is a solvent of high polarity, IND in the TICT state can be largely stabilized in high polarity environment and it can return to the ground state via red-shifted emission or non-radiative pathways.33 However, further increase in the water fractions (from 80% to 99%) led to significant emission enhancements. Nanoparticle tracking analysis (NTA) suggested that IND in an aqueous solution could form water-dispersible nanoaggregates with mean particle size of 52.3 nm (Fig. S4, ESI), which was consistent with the transmission electron microscopy (TEM) image (Fig. S5, ESI). These results indicated that IND exhibited aggregation-induced emission (AIE). Unusually, the enhanced emission was accompanied with a large red-shift to 639 nm when fw reached 99 vol% (Fig. 1c and d), which might be due to special intermolecular interactions in the aggregated state.
image file: c8mh01293h-f1.tif
Fig. 1 Synthesis, properties and structure of IND. (a) Synthetic route to IND. (b) Normalized absorption and photoluminescence (PL) spectra of IND (10 μM) in THF. (c) PL spectra of IND in THF/water mixtures with different water fractions (fw). (d) Plot of maximum PL intensity and peak emission wavelength of IND versus the composition of THF/water mixtures with various water fractions. (e) Geometric structure, (f) unit cell, and (g) pairwise packing mode of IND in single crystal.

Crystal structures

To gain in-depth understanding of the underlying mechanisms, we further analyzed the stacking mode of IND in the single crystal state. As revealed by the crystallographic data, there are four molecules in a unit cell and IND is almost planar with a dihedral angle of only 6.46° between two aryl rings (Fig. 1e and f). Multiple intermolecular interactions can be found between adjacent IND molecules, including C–H⋯O (2.533–2.706 Å), C[double bond, length as m-dash]O⋯π (3.194 Å), and π–π (3.558 Å) interactions (Fig. S6, ESI). Notably, a pair of IND molecules can co-overlap and exhibit π–π interactions (3.558 Å) in an antiparallel manner, forming a special molecular dimer. The neighboring dimers are assembled in a step-like architecture via C–H⋯O (2.700 Å) interactions (Fig. 1g and Fig. S7, ESI). Additionally, the solid-state fluorescence shows a clear red-shifted emission (109 nm) with longer fluorescence lifetime (τ = 11.1 ns) and higher emission efficiency (ΦF = 11.7%) than those in THF solution (τ = 0.97 ns, ΦF = 0.6%) (Table S1, ESI). All these data suggest that the distinct photophysical behaviors of IND in the aggregated state result from the formation of excimers. Unlike the traditional excimer systems that mostly exhibit fluorescence quenching,34 the emission enhancement of IND can be ascribed to two reasons: on the one hand, molecular rigidification and intramolecular motions are restricted by π–π stacking and multiple intermolecular interactions, which block their non-radiative relaxations and switch on the AIE effect. On the other hand, one-sided dibutylamino substituents of the molecules can serve as steric spacers to isolate dimers from each other, which facilitates efficient excimer emission with red-shifted fluorescence and also prevents strong π–π interactions between the aromatic rings that usually lead to fluorescence quenching. This unique pairwise-packing mode of IND brings about the uncommon AIE effect with large fluorescence red-shift; such a simple structure can potentially be used as a model to rationally guide the design of new color-switchable AIEgens.

Fluorescent differentiation of lipid/water

We next sought to investigate the performance of IND in lipids as its two alkyl tails were designed to be lipid-targeting. As expected, IND was completely soluble in sunflower oil with a green fluorescence peak at 522 nm. We then added an equal volume of water into the oil and mixed them gently. Interestingly, a remarkable difference in fluorescence color can be observed by the naked eye under UV-light illumination within minutes: The oil layer on the top shows greenish emission, whereas the lower water layer gradually emits red fluorescence with a peak at 640 nm (Fig. 2a, b and Movie S1, ESI). Their PL spectra were well-separated with a large shift of 108 nm, which can be ascribed to the aforementioned different packing modes of IND as monomers in lipids and as pairwise dimers in aqueous solution (Fig. 2c). These results suggest that IND can serve as a simple, rapid and sensitive probe for lipid and water sensing under single-excitation.
image file: c8mh01293h-f2.tif
Fig. 2 (a) Photograph of IND (10 μM) in lipid/water mixture taken under 365 nm UV illumination. (b) Normalized PL spectra of IND (10 μM) in lipid and in water, respectively. Excitation wavelength: 491 nm. (c) Illustration of the transformable photophysical processes of IND in lipid and water.

Lipid-specific cell imaging

The distinct properties of IND prompted us to further explore its applications in bioimaging. Prior to live cell imaging, the cytotoxicity of IND was evaluated by a Cell Counting Kit-8 (CCK-8) cell proliferation assay. As shown in Fig. S8 (ESI), cell growth was not significantly affected when up to 20 μM IND was added, indicating its low toxicity. We next investigated whether IND could target lipids on the cell level. Foam cells, a special type of macrophages, contain large amounts of lipids usually found in atherosclerosis;35 they were selected as model cells and were studied by a well-accepted method.36 Briefly, PMA (phorbol 12-myristate 13-acetate) was used to induce differentiation of human monocytic cell line THP-1 into macrophages. Oxidized low-density lipoprotein (oxLDL), a lipid-induced agent, was then incubated with the macrophages to mediate foam cell transformation. The successful formation of foam cells was confirmed by the cell morphology and their accumulated reddish lipid droplets in cytoplasm stained by lipid-specific histochemical dye Oil Red O (ORO). Fixed cell co-staining with IND showed that the punctate green fluorescence in the cytoplasm localizes in the same area of ORO, demonstrating the selective targeting of IND towards intracellular lipids (Fig. 3a). The performance of IND was then studied with two-photon fluorescence microscopy. IND exhibits a two-photon absorption cross section of 33.4 GM (Göeppert-Mayer) at 900 nm (Fig. S9, ESI); it is ideal as a small molecule probe and is suitable for two-photon imaging. As expected, under two-photon excitation, the fluorescent lipids within the foam cells could also be visualized. The emission contrast and imaging quality were superior to that of the one-photon images, which allowed intracellular lipid droplets to be visualized at high resolution on the nanoscale (Fig. 3b) and analyzed in 3D (Movie S2, ESI). In addition, IND could easily penetrate through the cell membrane and was also suitable for live cell imaging. As shown in Fig. 3c, the quantity and size of the fluorescent lipids within the live foam cells significantly increased with increase in oxLDL concentrations from 0 to 75 μM. Subsequently, the fluorescence signal of the labelled cells was analyzed by flow cytometry. Consistent with the live-cell imaging data, the cellular fluorescence increased significantly after oxLDL treatment and exhibited a dose-dependent manner (Fig. 3d). The lipid-targeting capability of IND was further verified by another lipid-laden cell model, in which oleic acid was used to induce lipid generation in HeLa cells.27 Similarly, the results obtained from two-photon microscopy and flow cytometry showed that cellular fluorescence also increased proportionally with oleic acid concentration (Fig. S10, ESI). These data demonstrate that IND is a lipid-targeting probe well-suited for two-photon bioimaging and quantitative analysis of lipids at the cell level.
image file: c8mh01293h-f3.tif
Fig. 3 (a) Bright-field and one-photon images of fixed foam cells co-stained with 0.6% Oil Red O and IND (20 μM). (b) Two-photon images of lipids within fixed foam cells labeled with IND (20 μM). (c) Two-photon and (d) flow cytometry analysis of live foam cells in the absence or presence of oxLDL pre-treatment at various concentrations (25 μM, 50 μM and 75 μM) for 12 hours.

Two-photon dual-emission imaging of lipid/water in tissues

We next systematically tested IND using an animal model of atherosclerosis induced from a genetically engineered apolipoprotein E-deficient (ApoE−/−) mouse fed on a high-fat diet for 6 months. The main aortic tree of the mouse was carefully separated and unfolded longitudinally. From the gross examination, elevated and pale lesions within the arterial lumen could be observed (Fig. 4a, left panel). After en face staining of the whole artery with IND, we could clearly observe that these areas emit strong green fluorescence under UV irradiation (Fig. 4a, lower right panel). Further analysis of their histological sections stained by hematoxylin and eosin showed that the non-emissive area (Fig. 4a, upper right panel) reveals a normal arterial structure consisting of three layers of the intima, media, and adventitia (Fig. S11a, ESI). However, in the green emissive regions, we may find thick depositions of lipids, fibrous tissues, and cell debris between the intima and media (Fig. S11b, ESI). These features imply that they were typical atherosclerotic plaques37 and the diagnosis was further confirmed by a pathologist. These results clearly demonstrate that IND exhibits high specificity to lipids and can target atherosclerotic plaque selectively.
image file: c8mh01293h-f4.tif
Fig. 4 Fluorescent images of lipid accumulation in the aorta of an ApoE gene knockout mouse labeled with IND. (a) En face photograph of the opened aorta taken under daylight (left). Fluorescent images at high magnification taken under 365 nm UV illumination corresponding to the upper and lower boxed areas (right). (b) Two-photon image of the atherosclerotic plaques. Zoom-in (boxed region) is shown to the right. (c) Two-photon images of the atherosclerotic plaques at various imaging depths. (d) Simultaneous dual-color imaging of the microstructures within lumen with separated emission ranges of 495–540 nm and 575–630 nm under single two-photon excitation (900 nm). (e) Schematic illustration of IND distributions in lipids and water-containing grooves.

It is noteworthy that it only takes 1 minute for IND to effectively illuminate the plaque regions, which may be due to its lipophilic property and small size with a low molecular weight of only [M + Na]+ 384.19. The excellent tissue penetrability and biodistribution of IND imply that it can be well-suited for in situ two-photon imaging at the tissue level. We observe the following phenomena: under two-photon excitation at 900 nm, IND could clearly light up the boundary, location and shape of each atherosclerotic plaque in the artery wall (Fig. 4b). Despite the artery being rich in collagen, a structural protein with strong autofluorescence, no clear background fluorescence was observed, which could be ascribed to the bright emission of IND excited at 900 nm and the non-lipid areas having less absorption in the near-infrared (NIR) region. Sequential scanning of the plaques at various depths was then performed. As shown in Fig. 4c, the high spatial distribution and deep tissue penetration of IND allow lipids within the intact atherosclerotic plaques to be visualized from various depths with a high-contrast view of the sample up to ∼400 μm deep. In addition, the ultra-structures of lipid deposits, which are formed as parallel fatty lines of around 20 μm width, could also be precisely resolved (Fig. 4b, right panel). Interestingly, red emission signals with distinct distributions can also be simultaneously observed upon excitation with the same two-photon laser. Close inspection of the images shows that red fluorescence is emitted from small aggregates that align between the fatty lines, exhibiting a distinctive red and green stripe pattern around the plaques (Fig. 4d). The two-photon images were taken with a water immersion objective lens, that is, drops of water were added on the surface of the IND stained arterial lumen. For this reason, the green fatty lines were filled with trace amounts of water. Some of the IND molecules could thus diffuse from the lipid deposits to the adjacent small aqueous grooves and induce in situ generation of red-emissive IND aggregates in these hydrophilic microenvironments (Fig. 4e). Unlike traditional fluorophores that usually suffer from self-quenching when accumulated, due to the AIE feature of IND, it emits brightly in both local lipid and water regions. The microstructure and components of the tissue can thus be precisely visualized and differentiated from the dual-color images. Such a unique function can be useful in visualizing lipid/water contents, interactions, and interfaces in the biological microenvironment as well as for motion correction in tissue imaging with either of the channels as a reference. In addition, its dual-emission imaging capacity with a single laser can also be especially suitable for newly developed super-resolution imaging techniques that are usually difficult to use for different excitations simultaneously, such as adaptive optics,38 structured illumination,39 optical lattices,40 and Bessel beam plane illumination,41 due to the technical limitations in hardware controlling and signal analysis.

We further quantitatively measured the lipid distributions within an atherosclerotic plaque at the early stage. We observed that the excellent two-photon imaging performance of IND allows a high-definition 3D image of the plaque to be successfully reconstructed (Fig. 5). From the image, we could clearly observe that the plaque is protruding from the artery with a large amount of fluorescent lipids (Fig. 5a). The high brightness and the negligible background signal of IND under two-photon excitation enable lipid localization, distribution, and morphology in the plaque to be precisely visualized in situ and quantitatively analyzed at specific views of interest. We found that lipids within the atherosclerotic plaque are morphologically heterogeneous with various shapes and sizes (Fig. 5a and b). For example, the numbers of lipids at four depths of the plaque (5, 10, 20, 25 μm) are 64, 228, 405, and 662, respectively, which is positively related to the plaque area. The lipid regions mostly range from 0.1 μm2 to 30 μm2, with a maximal lipid accumulation area of 241.6 μm2 located at a depth of 15 μm (Fig. 5b and c). The normal arterial intima adjacent to the plaque was also imaged. A lower and narrower distribution range of lipid signal was detected than that in the plaque region. These results suggest that IND may serve as a powerful tool for high-resolution imaging of both whole tissue dimensions and local microstructures in situ. Compared with histological preparations that usually require around 1 day, the one-step procedure with a staining time of 1 min is much faster and simpler. Moreover, no disruptive procedure is required compared to routine tissue sectioning, thus greatly avoiding tissue deformation and removal of lipids by solvents.

image file: c8mh01293h-f5.tif
Fig. 5 In situ mapping of lipid distributions within an early stage of plaque. (a) 3D reconstructed two-photon image of the plaque. (b) Slices of views (z-stack) of the plaque (left boxes area) at different imaging depths (i–iv). (v) The zoomed image of the adjacent normal arterial intima corresponding to the red boxed area in (a). (c) Quantitative measurement of fluorescence signals in (b). i–iv are two-photon images at four depths of 5, 10, 15, and 25 μm.


In summary, we have developed a two-photon excited AIE-active probe for bioimaging in cells/tissues. IND exhibits high specificity to lipids and can visualize and quantify lipids in cells with excellent performance. At the tissue level, with the advantages of high penetrability, good biocompatibility, large two-photon absorption, and convenient operation, IND allows lipids within atherosclerotic plaques to be morphologically visualized and analyzed with high resolution. Importantly, distinct from traditional excimer systems that usually lead to fluorescence quenching, IND can pack as special molecular dimers with AIE feature and exhibit red-shifted emission in the aggregated state. Its two distinct emissive properties in monomer and dimer states allow two-photon dual-color imaging of lipid and water with a single excitation to reveal microstructures of mouse atherosclerotic plaques in situ. Such a smart bioprobe with unique functions shows merits over existing lipid dyes in terms of its advantages that include simple, function-transformable, and deep-tissue imaging without tissue sectioning. This can be well-applicable to other advanced microscopy techniques in which it is difficult to use different excitation wavelengths simultaneously, for the in situ analysis of lipid/water within other tissues (such as liver, muscle, and adipose tissue), and for parallel tracking of multiple targets (such as proteins, nucleic acids, and small molecules) to observe their subcellular localizations and dynamic interactions.

Experimental section

Materials and methods

Fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM), and RPMI 1640 medium were purchased from Gibco. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo. Oxidized low-density lipoprotein (oxLDL) was obtained from Guangzhou Yiyuan Biotechnologies Inc. Oil red O was purchased from Macklin. Tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) and all other chemicals and solvents in this study were obtained from Sigma-Aldrich or Aladdin and used as received without further purification.

NMR spectra were measured on a Bruker Avance 500 MHz spectrometer with tetramethylsilane as internal standard. Mass spectra were recorded on a Thermo Fisher ITQ1100 mass spectrometer. UV absorption spectra were analyzed on a Shimadzu UV-2600 spectrophotometer. Particle sizes were measured on a Malvern NS300 nanoparticle tracking analysis system. TEM images were acquired using a JEOL JEM1400-Plus transmission electron microscope. Photoluminescence (PL) spectra were recorded on a Perkin-Elmer spectrofluorometer LS 55. A single crystal of IND was grown from ethanol via solute solution diffusion method. The Single crystal X-ray diffraction intensity data were collected on a Bruker–Nonices Smart Apex CCD diffractometer with graphite monochromated MoKα radiation. Processing of the intensity data was carried out using the SAINT and SADABS routines, and the structure and refinement were conducted using the SHELTL suite of X-ray programs (version 6.10).

Synthesis of IND

4-(N,N-Dibutylamino)benzaldehyde (233 mg, 1.0 mmol), 1,3-indandione (146 mg, 1.0 mmol) and morpholine (85 mg, 1.0 mmol) were first dissolved in ethanol (5 mL); the mixture was then heated at reflux for 3 h under nitrogen protection. After completion of the reaction, the mixture was cooled to room temperature and evaporated under reduced pressure. The residue was washed by petroleum ether and further recrystallized with ethanol to afford IND as a red solid (260 mg, 72% yield).

1H NMR (DMSO-d6, 500 MHz): δ 8.51 (d, J = 8.0 Hz, 2H), 7.93–7.90 (m, 2H), 7.78 (s, 1H), 7.77–7.70 (m, 2H), 6.73 (d, J = 8.5 Hz, 2H), 3.41 (t, J = 8.0 Hz, 4H), 1.66–1.62 (m, 4H), 1.42–1.37 (m, 4H), 0.99 (t, J = 7.0 Hz, 6H); 13C NMR (CD3Cl, 125 MHz): δ 191.9, 190.1, 147.3, 142.2, 139.9, 138.2, 134.3, 134.0, 122.4, 111.5, 51.1, 29.4, 20.2, 13.9; HRMS (ESI): m/z [M + Na]+ calcd for C24H27NNaO2, 384.1939; found 384.1937.

Cell imaging and flow cytometry

Human monocytic THP-1 cells and cervical cancer HeLa cells were obtained from American Type Culture Collection (ATCC). THP-1 cells were cultured at 37 °C with 5% CO2 in RMPI 1640 medium containing 10% FBS and macrophage differentiation was induced by incubation with 100 nM mL−1 phorbol 12-myristate 13-acetate (PMA) for 72 h. Macrophages were then transformed into foam cells by treatment with various concentrations of oxLDL (0, 25, 50 and 75 μM) in serum-free RPMI 1640 medium containing 0.3% BSA for 12 h. Cultured HeLa cells were grown in DMEM at 37 °C with 5% CO2 and lipid generation was induced by treatment with different concentrations of oleic acid (0, 50, 100 and 200 μM) for 12 h. For Oil red O staining experiment, cells were fixed for 10 min in 60% isopropanol and stained with 0.6% Oil Red O in 60% isopropanol for 30 min and subsequently washed with 60% isopropanol. For lipid fluorescence imaging, cells were stained with 20 μM of IND for 30 min, followed by washing three times with phosphate buffered saline (PBS) buffer before observation. Cells were imaged under a fluorescence microscope (Olympus BX41) or a two-photon microscope (Olympus FV1200MPE). In the flow cytometry experiment, cells after treating with different concentrations of oxLDL or oleic acid were harvested and washed three times with PBS. Analysis was then performed on a flow cytometer (BD LSRFortessa) with a 488-nm excitation laser.

Cell viability evaluation

HeLa cells were cultured in a 96 well-plate at a density of 5000 cells per well. After overnight culture, DMEM with various concentrations of IND was added into the plate and incubated for 4 h. After washing with PBS, 100 μL DMEM containing 10% CCK-8 solution was added into each well. After 1 hour incubation at 37 °C, the absorbance of each well at 450 nm was recorded by a microplate reader (Perkin-Elmer Victor3TM).

Mouse atherosclerotic plaque imaging

All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Animal Experimental Committee of Nanfang Hospital, Southern Medical University. Male six-week-old apolipoprotein E gene knockout (ApoE−/−) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd and fed on a high-fat diet containing 15% fat and 0.25% cholesterol, equaling 5 mg kg−1 body weight per day for 6 months. Mice were anaesthetized and sacrificed; the entire arterial tree was carefully isolated, opened longitudinally, followed by en face staining with 10 mM IND for 1 min. Tissue photographs were taken under UV light or daylight after washing with PBS, and the atherosclerotic plaque areas were imaged by a two-photon microscope. Formalin-fixed paraffin-embedded sections of the artery were stained with hematoxylin and eosin (H&E) and the histopathological results were confirmed by a pathologist.

Conflicts of interest

There are no conflicts to declare.


The authors thank Chenglin Xiang, Manna Lin, and Yufang Cheng for their assistance in two-photon imaging. They also thank Prof. Tingting Li for helpful discussions. This work was partially supported by the National Natural Science Foundation of China (81672076, 81802116, 81871735, 21788102, 51620105009, 21877040, 21602063), the Medical Science and Technology Research Foundation of Guangdong Province (A2017326), the Science and Technology Program of Guangzhou (201510010097), the Major Program of Health Care and Innovation of Guangzhou Project (201704020213, 201604020015), the Pearl River S&T Nova Program of Guangzhou (201806010152), the Natural Science Foundation of Guangdong Province (2016A030313852), and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2018J002).


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Electronic supplementary information (ESI) available. CCDC 1863840. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8mh01293h
These authors contributed equally to this work.

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