Luminescence-Resonance-Energy-Transfer-Based Luminescence

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A LRET-based luminescence nanoprobe for in situ imaging of CD36 activation and CD36-oxLDL binding in atherogenesis Yuhui Sun, Wen Gao, Zhenhua Liu, Huazhen Yang, Wenhua Cao, Lili Tong, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01398 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 22, 2019

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Analytical Chemistry

A LRET-based luminescence nanoprobe for in situ imaging of CD36 activation and CD36-oxLDL binding in atherogenesis Yuhui Sun, Wen Gao,* Zhenhua Liu, Huazhen Yang, Wenhua Cao, Lili Tong, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Biomedical Sciences, Shandong Normal University, Jinan 250014, P.R. China. ABSTRACT: Macrophage foam cell formation mediated by CD36 receptor-dependent internalization of oxidized low-density lipoprotein (oxLDL) is an important hallmark of early atherosclerosis. Activation of CD36 and its binding to oxLDL are the key points in foam cell formation. Herein, we develop a site-specific luminescence resonance energy transfer (LRET) system for the simultaneous imaging of CD36 activity and CD36-oxLDL binding on the cell surface. The system utilizes CD36 antibody modified SiO2-coated upconversion luminescent nanoparticles (UCNPs) as an energy donor to target the plasma membrane of macrophages, and the DiI-oxLDL (energy acceptor) binds to CD36 and passes through the membrane during macrophage foam cell formation. Upon excitation at 980 nm, the LRET signal can be obtained because of the short distance between DiI-oxLDL and the nanoprobe. Additionally, the very specific fluorescence can be used to visualize distinct features of CD36. The nanoprobe also exhibits high sensitivity, good stability, simplicity and low cost for the accurate detection and evaluation of macrophage foam cell formation. Moreover, using this novel nanoprobe, we also investigate the mechanism whereby reactive oxygen species (ROS) signaling enhances the binding of oxLDL to CD36. ROS, especially O2•− alter endothelial permeability and facilitate CD36 clustering, ultimately promoting the entry and internalization of oxLDL. Because of these advantages, this nanoprobe may provide a versatile platform for monitoring the progression of atherogenesis and elucidating atherogenesis signaling at the cellular level.

Atherosclerosis is a chronic inflammatory disease of medium- and large-sized arteries, which associated with high morbidity and mortality worldwide.1, 2 The hallmark event in early atherosclerosis is the formation of foam cells caused by excessive lipoprotein accumulation in macrophages.3-5 CD36, a transmembrane glycoprotein receptor expressed on the surface of macrophages, mediates oxidized low-density lipoprotein (oxLDL) uptake.6-8 The activation of CD36 triggered by its clustering is the key step of CD36-oxLDL binding and internalization, often defining the point of no return in macrophage foam cell formation.9-11 CD36 clustering has recently been shown to be relevant to atherosclerosis risk factors that lead to excessive reactive oxygen species (ROS) production, such as hypertension, hypercholesterolemia, or cigarette smoking.12-16 We hypothesize that ROS may play an important role in CD36 clustering and impact the uptake of oxLDL. Therefore, monitoring CD36 activation and CD36oxLDL binding and elucidating the signaling pathways modulated by ROS in living cells are essential parts of atherogenesis research. Imaging tools enabling direct visualization of CD36 activation and CD36-oxLDL binding in living macrophage cells have rarely been explored. Existing technologies for CD36 clustering analysis are based on CD36 immunolabeling with an anti-CD36 Fab fragment followed by single-molecule imaging.9, 10, 17 CD36-oxLDL binding assays are implemented via immunoprecipitation (IP)/western blot with cell extracts or immunocytochemistry with immobilized cells.18, 19 However, all of these techniques require tedious operations to derive whether CD36 exists as monomers or as clusters and indirect

complicated colocalization analysis of CD36 with oxLDL. Most importantly, they are inherently not suitable for living cell detection and are inadequate for simultaneously imaging CD36 activation and CD36-oxLDL binding. Recently, some imaging methods based on fluorescence resonance energy transfer (FRET) have offered an attractive solution for imaging protein-protein interactions or protein-nucleic acid interactions.20-22 However, these strategies rely on the respective labeling of proteins or nucleic acids with donors and acceptors, such as organic dyes and QDs, causing difficulties in FRET analysis owing to their strong background fluorescence and susceptibility to photobleaching. To monitor CD36 activation and CD36-oxLDL binding in situ and perform a more accurate evaluation of the occurrence of macrophage foam cells, one-step FRET-based imaging tools with small background, water-dispersibility and biocompatibility urgently need to be developed. In comparison with organic dyes and QDs, upconversion luminescent nanoparticles (UCNPs) with the sharp emission peaks, improved quantum yield, tunable multicolor emission and negligible scattering light hold great promise for developing such an imaging tool.23-25 To make the nanoprobe water-dispersible and suitable for conjugation,26 a silicon dioxide (SiO2) layer is considered to be an ideal candidate.27 Herein, we use SiO2-coated UCNPs to construct a site-specific luminescence resonance energy transfer (LRET, FRET was termed as LRET when rare earth materials were involved) system on the macrophage cell surface for imaging CD36oxLDL binding, and further define the features of CD36, as illustrated in Scheme 1. The luminescence nanoprobe donor

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consists of NaYF4:Yb, Er@SiO2 core-shell nanocomposites conjugated with a CD36 antibody (UCNPs@SiO2-CD36), which enables specific binding to CD36 antigen on the plasma membrane of macrophage RAW 264.7. During macrophage foam cell formation, DiI-oxLDL binds to CD36 and passes through the membrane. This process will bring DiI-oxLDL and the nanoprobe in close proximity, which can then produce fluorescent signals through LRET upon continuous-wave excitation at 980 nm. Since excess DiI-oxLDL in the cytoplasm is distant from the nanoprobe, fluorescence is not emitted. Meanwhile, the nanoprobe exhibits very specific fluorescence for the effective detection of the signaling changes associated with CD36 clustering. This is the first time that a LRET-based luminescence nanoprobe simultaneously enables in situ visualization of CD36 activation and CD36oxLDL binding on the surface of a living macrophage. Furthermore, based on the advantages of this nanoprobe, the oxLDL internalization pathway modulated by ROS in atherogenesis was demonstrated for the first time.

Schemes 1. The site-specific LRET method for imaging CD36 activation and CD36-oxLDL binding on a living macrophage cell surface. EXPERIMENTAL SECTION Synthesis of NaYF4:Yb, Er UCNPs. The NaYF4:Yb, Er UCNPs were prepared via a solvothermal method. YCl3•6H2O (0.2420 g, 0.798 mmol), YbCl3•6H2O (0.0774 g, 0.20 mmol), and ErCl3•6H2O (0.0007 g, 0.002 mmol) were dispersed in oleic acid (OA, 8 mL) and 1-octadecene (ODE, 18 mL) The mixture was gradually heated to 110 °C under vacuum (30-40 min) and the water removed by distillation. The reaction was then kept at 160 °C for another 30 min under argon, forming a homogeneous solution. After the mixture was cooled to room temperature, NaOH (0.1 g, 2.5 mmol) and NH4F (0.148 g, 4 mmol) in methanol solution (10 mL) were added dropwise under vigorous stirring for 30 min. The temperature was gradually heated to 110 °C to evaporate methanol, then raised to 300 °C in an argon atmosphere for 90 min and finally cooled to room temperature naturally. The resulting NaYF4:Yb, Er UCNPs were precipitated by adding 20 mL ethanol and then were centrifuged and washed with ethanol and cyclohexane for several times. The UCNPs were redispersed in hexamethylene for further use. Synthesis of UCNPs@SiO2. First, 5 mg of as-synthesized UCNPs were dispersed in a 10 mL hexamethylene solution and then 0.48 g Igepal CO-520 was added. After the mixture

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was sonicated for 30 min, an ammonium hydroxide solution (80 μL, 30%) was added and gently stirred for 1 h. Subsequently, tetraethyl orthosilicate (TEOS, 32 μL, 0.143 mmol) was introduced to the mixture dropwise, and then (3aminopropyl) triethoxysilane (APTES, 5 μL, 0.02 mmol) was added dropwise to the mixture to generate an aminoterminated surface. Finally, the mixture was allowed to stir for 48 h and then centrifuged and washed 3 times with ethanol. The resulting dried products (amino-modified core-shell UCNPs@SiO2) were weighed and stored under dry conditions. In Vitro LRET Assay. The amino-modified UCNPs@SiO2 (10 mg) were stirred with propargyl bromide (2.04 mmol) and potassium carbonate (3.4 mmol) for 5 h, the resulting nanoparticles were centrifuged and washed with ethanol and water. The prepared nanoparticles were then redispersed in 10 mL PBS buffer (10 × phosphate, pH 7.4) for further use. In addition, an octadecanol aqueous solution (0.27 g, 0.998 mmol) was mixed with tosyl chloride (TsCl, 0.286 g, 2.03 mmol) and triethylamine (280 μL) at room temperature and stirred for 2 h. Following recrystallization in water, the precipitate was dried at 60 °C under vacuum, then the intermediate product octadecanol-OTs was obtained. To obtain octadecanol-N3, octadecanol-OTs (0.1 g, 0.077 mmol) and sodium azide (0.025 g, 0.387 mmol) were mixed and stirred constantly in water for 12 h. DiI-oxLDL was then labeled using octadecanol-N3 (37 °C, 16 h). Different amounts (2.5, 5, 10, 20, 40, 60, 80, 100, 150 and 200 μg) of N3 labeled DiI-oxLDL were added to 1 mL UCNPs@SiO2-alkyne (0.1 mg) in a micro-tube, and the solutions were stirred for 30 min at room temperature. Upon excitation at 980 nm, DiI-oxLDL-N3 clicked with UCNPs@SiO2-alkyne showed a new LRET peak at approximately 585 nm. Preparation and Characterization of UCNPs@SiO2CD36. UCNPs@SiO2 (1 mg mL-1) were cross-linked with CD36 antibody (4 μg) under stirring for 12 h, which involved a simple condensation reaction between the carboxyl groups of antibody and amino groups on the surfaces of the NPs with the aid of nhydroxysuccinimide (NHS, 8.4 μmol) and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 8.4 μmol). The prepared UCNPs@SiO2-CD36 were washed with PBS several times and redispersed in PBS (pH = 7.4) for further use. For gel electrophoresis analysis, samples were run using a Mini-PROTEAN Tetra cell (Bio-Rad, Hercules, CA, USA). The Fourier transform infrared (FT-IR) spectrum was also collected on a Nicolet Impact 410 FTIR spectrometer in the range of 400-4000 cm−1. Localization of UCNPs@SiO2-CD36 on Macrophage RAW 264.7 Cell Membranes. The intracellular localization of UCNPs@SiO2-CD36 was primarily monitored by using confocal laser scanning microscope (CLSM). Macrophages RAW 264.7 were seeded at an initial density of 5 × 104 cells/dish in 20-mm glass bottom dishes and incubated for 24 h. Then the cells were incubated with sterilized UCNPs@SiO2-CD36 (0.1 mg mL−1) or UCNPs@SiO2 (0.1 mg mL−1) for 1 h. The cells were then washed three times with PBS buffer to remove the excess nanoparticles. Subsequently, the cells were incubated with the cell membrane marker DiD (red, 5 μM) in serum-free DMEM for 20 min at 37 °C. After that, the cells were washed with PBS for 3 times and immediately observed using LSM 880 confocal laser scanning microscope and the cell fluorescence images were captured with 980 nm excitation for UCNPs@SiO2-CD36 and


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Analytical Chemistry UCNPs@SiO2 (emission = 480-545 nm) and 633 nm excitation for DiD (emission = 640-745 nm). A Ti: sapphire laser was used to excite the specimens at 980 nm with a laser power of 100 mW. The colocalization ratio was quantified using Image-Pro Plus Imaging software. LRET Imaging of CD36-oxLDL Binding. RAW 264.7 cells were seeded at a density of 5 × 104 cells/dish in 20-mm glass bottom dishes and cultured for 24 h. Then, fresh medium containing sterilized UCNPs@SiO2-CD36 (0.1 mg mL−1) was added to each well and incubated for 1 h. After washing, the cells were incubated with serum-free DMEM containing DiIoxLDL (35 μg mL−1) for an additional 90 min at 37 °C. Cells treated with UCNPs@SiO2-CD36 (0.1 mg mL−1, 1 h) and DiIoxLDL (35 μg mL−1, 90 min) were used as controls. Then, the cells were observed using LSM 880 confocal laser scanning microscope and fluorescence images were captured with 980 nm excitation and collected from 565 to 600 nm. A Ti: sapphire laser was used to excite the specimen at 980 nm with a laser power of 100 mW. Monitoring of CD36-oxLDL Binding With ROS Treatment. A HUVECs and RAW 264.7 double-layered cell coculture model was established in a transwell chamber 12well dishes (Corning, Corning, NY, USA) with a membrane pore size of 0.4 μm. HUVECs were seeded at an initial density of 2 × 104 cells/insert on the upper compartment, and RAW 264.7 cells were seeded on glass slides (5 × 104 cells) and cultured in the bottom compartment overnight. HUVECs were incubated with different amounts of ROS (H2O2, NO, •OH, and O2•−, 5-250 μM) in serum-free DMEM for 15 min prior to incubation with the DiI-oxLDL (35 μg mL−1) for further 90 min. Simultaneously, the RAW 264.7 cells in the bottom compartment were incubated with UCNPs@SiO2-CD36 (0.1 mg mL−1). CD36-oxLDL binding was captured at 980 nm excitation with an LSM 880 confocal laser scanning microscope and the emission was collected from 565 to 600 nm. A Ti: sapphire laser was used to excite the specimen at 980 nm with a laser power of 100 mW. RESULTS AND DISCUSSION Synthesis and characterization of the nanoparticles. The NaYF4:Yb, Er UCNPs were first prepared via a solvothermal method. Dynamic light scattering (DLS) and high resolution transmission electron microscopy (HRTEM) showed that the UCNPs exhibited uniform morphology with a size of 18 ± 3.25 nm and the pattern was hexagonal (β-) phase (Figure S1a, Figure 1a). These as-synthesized oleic acid (OA)-conjugated UCNPs were first coated with a silica shell to improve their biocompatibility and then functionalized with APTES to form an amino-terminated surface. The size of the UCNPs@SiO2 was 42 nm (42 ± 5.3 nm), and they appeared regularly spherical in shape (Figure S1a, Figure 1a). X-ray photoelectron spectroscopy (XPS) was carried out to characterize the SiO2 coating. As shown in Figure 1b, silicon and oxygen signals could be attributed to the silica shell, and were detected for only the UCNPs@SiO2 and not for the UCNPs. Zeta potential measurements were employed to verify the surface modification of the UCNPs, and values of -1.45 ± 0.29 mV and 11.7 ± 0.75 mV were obtained before and after modification, respectively. (Figure S1b). The FT-IR spectra further confirmed the successful surface modification, as the strong transmission band in the region around 1021 cm-1 could be attributed to the symmetrical stretching vibration of the SiO bonds, moreover, the stretching and bending vibration bands

of the amine group appeared at 1613 cm-1 in the spectrum (Figure S1c). After being coated with SiO2, the UCNPs@SiO2 could be well-dispersed in water and still emitted strong upconversion luminescence efficiency. As shown in Figure 1c, the luminescence spectrum of the UCNPs@SiO2 exhibited strong emission peaks at 543 nm, which corresponded to 4S3/2 → 4I15/2 transformation of Er3+ ion, whereas the two weak Er3+ ion emission peaks at 524 nm and 661 nm were assigned to the 2H 4 4 4 11/2→ I15/2 and F9/2→ I15/2 transitions, respectively.

Figure 1. Characterization of the UCNPs@SiO2. (a) HRTEM and (b) XPS of UCNPs (black) and UCNPs@SiO2 (red) (c) Upconversion luminescence (UCL) spectra of UCNPs (0.1 mg mL-1, black) and UCNPs@SiO2 (0.1 mg mL-1, red) under 980 nm laser. Scale bar = 50 nm.

In vitro LRET assay. Firstly, the feasibility of the proposed UCNPs@SiO2 nanoprobe for generating the LRET effect was evaluated in vitro. The fluorescent dye DiI labeled oxLDL used in this work displayed a strong absorption band at approximately 549 nm, thus there was a suitable spectral overlap between the UCNPs@SiO2 emission and the DiIoxLDL absorption in the spectra (Figure 2a). Then the UCNPs@SiO2 and DiI-oxLDL were labeled by alkyne groups and azide groups, respectively, to shorten the donor-acceptor pair distance using a click reaction (Figure S2). Successful modification was confirmed by FT-IR (Figure S3). UCNPs@SiO2-alkyne showed no obvious morphological change and still emitted strong upconversion luminescence. Accordingly, as shown in Figure 2b, 2c and 2d, upon excitation at 980 nm, the luminescence intensity around 543 nm decreased along with the enhancive addition of N3 labelled DiI-oxLDL, and simultaneously a new observable LRET emission band around 585 nm gradually increased. The ratio of the emission intensities at 585 and 543 nm (I585/I543) also exhibited a drastic change from 0.0026 to 1.39 (Figure 2e). In contrast, the luminescence spectra of the mixture of UCNPs@SiO2 and DiI-oxLDL did not give appreciable characteristic peaks under 980 nm excitation. This result verified that LRET cannot occur unless the donor and acceptor are close enough, validating the tiny LRET background for LRET imaging (Figure S4). Notably, DiI-oxLDL did not exhibit an observable signal under 980 nm excitation even at a high concentration of 200 μg mL−1 (Figure S4, burgundy), indicating a unique advantage of using UCNPs as the donor in preventing acceptor spectral bleed-through. Taken together, these results revealed that the designed UCNPs@SiO2 can



successfully generate LRET and produce a highly sensitive signal when the nanoparticles and DiI-oxLDL were in proximity, demonstrating the potential utility of this nanoparticle for in situ imaging of oxLDL internalization in living cells.

Figure 2. In vitro LRET assay. (a) Photophysical properties of UCNPs@SiO2 (donor) and DiI-oxLDL (acceptor). UCL spectrum of UCNPs@SiO2 excited at 980 nm (0.1 mg mL-1, green); Excitation and emission spectra of DiI-oxLDL (20 μg mL −1, red). The yellow areas indicated the overlap between the donor emission spectrum and acceptor excitation spectra. (b) Emission spectra of UCNPs@SiO2-alkyne (0.1 mg mL-1) after a click reaction with various concentrations of N3 labeled DiI-oxLDL (2.5 to 200 μg mL-1) under 980 nm laser. (c) Magnified image of the curves in the black dashed box shown in b. (d) Scatter plot derived from the luminescence intensity at 585 nm and the N3 labeled DiI-oxLDL concentration. (e) Enhanced luminescence intensity ratio (I585/I543) of UCNPs@SiO2-alkyne (0.1 mg mL-1) upon adding of increasing amounts of N3 labeled DiI-oxLDL (2.5200 μg mL-1). All of the spectra were acquired in PBS buffer (10 × phosphate, pH = 7.4). Data are shown as the mean ± S.D. of three independent experiments.

In situ imaging of CD36-oxLDL binding on the macrophage surface by LRET. Prior to the fluorescence imaging of CD36-oxLDL binding on living macrophage cell membranes, UCNPs@SiO2-CD36 were first obtained by conjugation of amino-modified UCNPs@SiO2 with a CD36 antibody, which enabled specific binding with CD36 antigen on the plasma membrane of macrophages RAW 264.7. The successful conjugation of UCNPs@SiO2-CD36 was confirmed by FT-IR spectroscopy (Figure S5a) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Figure S5b). The UCL spectrum and HRTEM image also showed that the modification with CD36 antibody had no significant effect on the luminescence efficiency and morphology of the UCNPs@SiO2 (Figure S5c). The targeting capability of UCNPs@SiO2-CD36 was evaluated based on CLSM and imaging flow cytometry (IFC). Figure 3a and Figure S6 showed that a high density of UCNPs@SiO2-CD36 was evenly attached to the cell membranes after incubation for 1 h. The fluorescence of the UCNPs@SiO2-CD36 overlapped well with that of the cell membrane marker DiD, as evidenced by the clear yellow signals. The above observations were also verified by quantifying the fluorescence intensity of the line scanning profiles. In contrast, uncoated UCNPs@SiO2 were

internalized by cells and did not show specific localization on the RAW 264.7 cell membranes. To investigate the binding affinity of the UCNPs@SiO2-CD36, a competitive binding study was developed. Importantly, with a blocking dose (0.5 mg mL-1) of CD36 antibody, only a weak green fluorescence signal was observed on the surface of these cells. Although UCNPs@SiO2-CD36 displayed an increased internalization into RAW 264.7 cells with dramatically weakened CD36binding affinity, only 9.03-fold increase of fluorescence intensity was found in cytoplasm until 24 h incubation (Figure S7). These results demonstrated that UCNPs@SiO2-CD36 had a specific and strong binding affinity with CD36 on RAW 264.7 cell membrans, and that the stability of these nanoprobe was sufficient for application in live cell imaging. Cell viability measurements showed no obvious cytotoxicity after incubation for 16 h at 37 °C, even with the UCNPs@SiO2CD36 concentration up to 0.4 mg mL-1 (Figure S8), suggesting that the nanoprobe has fairly high biocompatibility and low cytotoxicity. We therefore sought to visualize CD36-oxLDL binding on live cells using the LRET strategy. As expected, after the cells were tagged with UCNPs@SiO2-CD36 and subsequently treated with DiI-oxLDL for 90 min, an obvious red fluorescence signal was observed on the RAW 264.7 cell surface under 980 nm excitation (Figure 3b), which could be attributed to the LRET between UCNPs@SiO2-CD36 and DiIoxLDL bound with CD36. No visible fluorescence signal was observed in cytoplasm due to the nanoprobe and DiI-oxLDL suffered from the distance separation and hence insufficient energy transfer. In the absence of UCNPs@SiO2-CD36, the DiI-oxLDL-treated cells exhibited an invisible signal, indicating no bleed-through signal even when a large excess of acceptor was presented. Additionally, the fluorescence signal from 565 to 600 nm on the surface of the cells incubated with only UCNPs@SiO2-CD36 appeared as a straight line. This result further demonstrated that the LRET-based nanoprobe could be used as a practical tool for the visualization of CD36oxLDL binding on living cell surface. The bright red fluorescence was uniformly distributed in the region of the cell membrane, suggesting a diffusion-like distribution of CD36 monomers. Consistency with previous findings, oxLDL could be bound and internalized into macrophages RAW 264.7 (Figure S9). But because UCNPs@SiO2-CD36 maintained high levels of CD36-targeting activity in this situation, it was important to determine whether DiI-oxLDL was only binding CD36 at the plasma membrane or whether DiI-oxLDL was being internalized by the RAW 264.7. The CLSM imaging indeed revealed a new interesting finding: the UCNPs@SiO2CD36 did not affect the function of oxLDL internalization, indicating that DiI-oxLDL binding to CD36 was followed by uptake even in the presence of UCNPs@SiO2-CD36 (Figure S10). This result implied that the nanoprobe might be a viable platform for elucidating signaling pathways in atherogenesis. ROS increase CD36 clustering and accelerate CD36oxLDL binding. Both the initiation and progression of cardiovascular dysfunction involved the same initial signaling paradigms, all of which involved ROS.28 ROS were the initial small molecule encountered by macrophages, and the precise role of ROS in foam cell formation has yet to be defined. Hence, the nanoprobe was utilized as a valuable platform for monitoring the dynamic change in CD36-oxLDL binding against ROS signaling. To simulate the atherosclerotic


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Analytical Chemistry

Figure 3. In situ imaging of CD36-oxLDL binding. (a) Targeting of UCNPs@SiO2-CD36 to macrophage RAW 264.7 cell membranes. Representative CLSM images of RAW 264.7 cells incubated with 0.1 mg mL-1 UCNPs@SiO2-CD36 or UCNPs@SiO2 for 1 h and 5 μM cell membrane marker DiD for 20 min at 37 °C. Quantification of fluorescent intensity of the line scanning profiles in the corresponding images (right). The images were obtained with 980 nm excitation for UCNPs@SiO2-CD36 and UCNPs@SiO2 (emission = 480-545 nm) and 633 nm excitation for DiD (emission = 640-745 nm). Scale bar = 50 μm. (b) In situ fluorescence imaging of CD36-oxLDL binding on the RAW 264.7 surface by LRET. i: untreated group; iv: RAW 264.7 cells incubated with 0.1 mg mL-1 UCNPs@SiO2-CD36 for 1 h followed by treatment with 35 μg mL-1 DiI-oxLDL for 90 min at 37 °C. RAW 264.7 cells incubated with DiI-oxLDL (ii, 35 μg mL-1, 90 min) and UCNPs@SiO2 (iii, 0.1 mg mL-1, 1 h) were used as controls. The images were captured under 980 nm excitation and collected from 565 to 600 nm. Scale bar = 20 μm.

environment, a human umbilical vein endothelial cells (HUVECs) and RAW 264.7 double-layered cell coculture model was established using a transwell chamber in vitro (Figure 4a). HUVECs were plated onto the insert of the transwell chamber, and RAW 264.7 cells were placed on the bottom. We chose four of the most important ROS in the vasculature: hydrogen peroxide (H2O2), nitric oxide (NO), hydroxyl radical (•OH) and superoxide (O2•−).29, 30 Figure S11 showed that the UCNPs@SiO2-CD36 were stable in DMEM containing ROS, retaining their strong upconversion luminescence efficiency and emission peaks. Then, the HUVECs in the upper compartment were treated with different kinds of ROS prior to DiI-oxLDL (35 μg mL-1) incubation. Subsequently, the change in CD36-oxLDL binding on the RAW 264.7 cell surface in the lower compartment was monitored using UCNPs@SiO2-CD36 (Figure 4b, Figure S12, Figure S13). After the HUVECs were subsequently treated with H2O2, NO, •OH, and O2•− at different concentrations (50, 100, 150, 200 and 250 μM) for 15 min, gradually enhanced fluorescence signals were observed in the RAW 264.7 cell membrane region under excitation at 980 nm, which could be attributed to the LRET from the UCNPs@SiO2-CD36 to DiIoxLDL. A significant binding of DiI-oxLDL to CD36 was observed when •OH and O2•− were present. To determine which kind of ROS play a major role in CD36-oxLDL binding, we then incubated HUVECs with •OH, O2•− and •OH+O2•−

under low-concentration conditions (5, 10, 20 and 40 μM) for 15 min. Figure S14 and Figure S15 showed that increasingly bright red fluorescence were observed in the images with increasing •OH or O2•− concentrations. Specifically, the fluorescence signal caused by O2•− was significantly higher than cells exposed to •OH, suggesting that O2•− played a significant role in oxLDL uptake. As the fluorescence intensity increased with the extent of CD36-oxLDL binding, the occurrence of foam cell formation could be evaluated. In addition, the red fluorescence image showed large aggregated spots morphology, indicating the clustering of CD36. We then investigated the possible mechanism whereby ROS signaling enhanced the binding and internalization of oxLDL. It was conceivable that ROS, especially O2•− promote the entry of oxLDL and alter CD36 activities, which ultimately increased the binding of CD36 and oxLDL. To assess this possibility, we first confirmed that O2•− in fact induced endothelial injury that further resulted in endothelial loss. Annexin V-FITC/PI staining was used to visualize the destruction of HUVECs treated with O2•− (Figure S16). Bright green fluorescence indicated Annexin V-FITC staining of membrane disruption, and red fluorescence indicated the PI staining of necrotic cells with leaky membranes. Additionally, upon treatment with O2•−, the cell spacing became larger, changing from neatly and tightly packed turned to loosely



Figure 4. ROS increase CD36-oxLDL binding. (a) Schematic diagram of the in vitro double-layered cell coculture model. HUVECs were plated onto the insert, and RAW 264.7 cells were placed at the bottom of the transwell chamber. (b) The upper HUVECs were incubated with 50 μM H2O2, NO, •OH, and O2 •− for 15 min followed by 35 μg mL −1 DiI-oxLDL for 90 min. Simultaneously, the RAW 264.7 cells in the lower compartment were incubated with UCNPs@SiO2-CD36 (0.1 mg mL − 1). CD36-oxLDL binding signals on the RAW 264.7 surface were captured under 980 nm excitation and collected from 565 to 600 nm. Scale bar = 20 μm. Right: the relative fluorescence intensity of LRET in the corresponding confocal images in left. Data are shown as the mean ± S.D. of three independent experiments. binding and evaluation of CD36 activity on living cell surface. arranged. After being traeted with O2•− for 15 min, the The nanoprobe specifically bound to CD36 and could be used HUVECs were incubated with DiI-oxLDL for an additional 90 as an energy donor. CD36 internalized DiI-oxLDL (as energy min. Cell culture mediums in the bottom were then collected acceptors) and in turn served as a bridge to make the distance to assess the changes in DiI-oxLDL and O2•− levels. As between the donors and the acceptors short enough for LRET anticipated, compared with those in the control group, the DiIto occur. To the best of our knowledge, this nanoprobe with oxLDL and O2•− levels seemed to be significantly increased good stability and high sensitivity, eliminated the need for (Figure S17). The results indicated exposed to O2•− induced immunocytochemical analysis and for the first time realized in apoptosis in HUVECs, which led to HUVECs loss and situ fluorescence imaging of CD36-oxLDL binding as well as resulted in the entry of DiI-oxLDL and O2•−. On the other monitoring of dynamic changes in CD36 activation. It was hand, the clustering of CD36 prime the macrophages to also found that the nanoprobe did not affect the downstream internalize oxLDL. Therefore, we hypothesized that exposing function of oxLDL internalization. Using this novel macrophages RAW 264.7 to O2•− might promote CD36 nanoprobe, we discovered that ROS, especially O2•−, could clustering and alter its ability to bind oxLDL. Hence, CD36 alter endothelial permeability, facilitated CD36 clustering and clustering on the RAW 264.7 surface in the bottom compartment was characterized using scanning electron ultimately promote the entry and internalization of oxLDL. Because of these advantages and capabilities, this approach microscopy (SEM). In the absence of O2•−, UCNPs@SiO2might provide a versatile platform for monitoring the CD36 remained dispersed on cell membranes. However, the progression of atherogenesis and elucidating atherogenesis initially evenly distributed UCNPs@SiO2-CD36 became signaling at the cellular level. densely populated aggregates after the treatment of HUVECs in the upper compartment with O2•− (Figure S18). This finding ASSOCIATED CONTENT was in good agreement with the previous fluorescence imaging results in Figure 4b, Figure S12 and Figure S14. Supporting Information These observations revealed that O2•− facilitated CD36 Materials and Reagents, characterizations, cell cultures, in vitro clustering, enhanced its affinity for oxLDL, and ultimately cytotoxicity assay, IFC imaging of the localization of promoted the formation of macrophage foam cells. UCNPs@SiO2-CD36, binding affinity of UCNPs@SiO2-CD36, CONCLUSIONS CLSM imaging of the internalization of DiI-oxLDL, the preparation of various ROS, HUVECs loss analysis, CD36 In conclusion, this work designed a LRET-based clustering study and supporting figures are included in the luminescence nanoprobe for in situ imaging of CD36-oxLDL


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Analytical Chemistry supporting information. The supporting information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21535004, 91753111, 21775092, 21605097, 21755099), the Natural Science Foundation of Shandong Province of China (ZR2018JL008), the China Postdoctoral Science Foundation (2019M650168).

REFERENCES

Michalakis, S. Front Neurosci. 2016, 10, 356. (22). Linden, S.; Singh, M. K.; Wegner, K. D.; Regairaz, M.; Dautry, F.; Treussart, F.; Hildebrandt, N. Dalton Trans. 2015, 44, 4994-5003. (23). Su, Q.; Han, S.; Xie, X.; Zhu, H.; Chen, H.; Chen, C. K.; Liu, R. S.; Chen, X.; Wang, F.; Liu, X. J. Am. Chem. Soc. 2012, 134, 20849-20857. (24) Wu, Y. M.; Cen, Y.; Huang, L. J.; Yu, R. Q.; Chu, X. Chem. Commun. 2014, 50, 4759-4762. (25). Haase, M.; Schafer, H. Angew. Chem. Int. Ed. 2011, 50, 5808-5829. (26). Wang, F.; Deng, R.; Wang, J.; Wang, Q.; Han, Y.; Zhu, H.; Chen, X.; Liu, X. Nat. Mater. 2011, 10, 968-973. (27) Li, Z.; Zhang, Y.; Jiang, S. Adv. Mater. 2008, 20, 4765-4769. (28) Taniyama, Y.; Griendling, K. K. Hypertension 2003, 42, 1075-1081. (29) Craige, S. M.; Kant, S.; Keaney, J. F., Jr. Circ J. 2015, 79, 1145-1155. (30) Griendling, K. K.; Sorescu, D.; Ushio-Fukai, M. Circ. Res. 2000, 86, 494-501.

(1). Libby, P.; Ridker, P. M.; Hansson, G. K. Nature 2011, 473, 317-325. (2). Weber, C.; Noels, H. Nat. Med. 2011, 17, 1410-1422. (3). Aryal, B.; Rotllan, N.; Araldi, E.; Ramirez, C. M.; He, S.; Chousterman, B. G.; Fenn, A. M.; Wanschel, A.; Madrigal-Matute, J.; Warrier, N.; Martin-Ventura, J. L.; Swirski, F. K.; Suarez, Y.; Fernandez-Hernando, C. Nat. Commun. 2016, 7, 12313. (4). Sergin, I.; Evans, T. D.; Zhang, X.; Bhattacharya, S.; Stokes, C. J.; Song, E.; Ali, S.; Dehestani, B.; Holloway, K. B.; Micevych, P. S.; Javaheri, A.; Crowley, J. R.; Ballabio, A.; Schilling, J. D.; Epelman, S.; Weihl, C. C.; Diwan, A.; Fan, D.; Zayed, M. A.; Razani, B. Nat. Commun. 2017, 8, 15750. (5). Bobryshev, Y. V. Micron. 2006, 37, 208-222. (6). Park, Y. M. Exp Mol Med. 2014, 46, e99. (7). Rahaman, S. O.; Lennon, D. J.; Febbraio, M.; Podrez, E. A.; Hazen, S. L.; Silverstein, R. L. Cell. Metab. 2006, 4, 211-221. (8). Que, X.; Hung, M. Y.; Yeang, C.; Gonen, A.; Prohaska, T. A.; Sun, X.; Diehl, C.; Maatta, A.; Gaddis, D. E.; Bowden, K.; Pattison, J.; MacDonald, J. G.; Yla-Herttuala, S.; Mellon, P. L.; Hedrick, C. C.; Ley, K.; Miller, Y. I.; Glass, C. K.; Peterson, K. L.; Binder, C. J.; Tsimikas, S.; Witztum, J. L. Nature 2018, 558, 301-306. (9). Jaqaman, K.; Kuwata, H.; Touret, N.; Collins, R.; Trimble, W. S.; Danuser, G.; Grinstein, S. Cell 2011, 146, 593-606. (10). Wong, H. S.; Jaumouille, V.; Freeman, S. A.; Doodnauth, S. A.; Schlam, D.; Canton, J.; Mukovozov, I. M.; Saric, A.; Grinstein, S.; Robinson, L. A. Cell. Rep. 2016, 14, 2859-2871. (11). Moore, K. J.; Freeman, M. W. Arterioscler. Thromb. Vasc. Biol. 2006, 26, 1702-1711. (12). Ramos-Arellano, L. E.; Munoz-Valle, J. F.; De la CruzMosso, U.; Salgado-Bernabe, A. B.; Castro-Alarcon, N.; Parra-Rojas, I. BMC Cardiovasc Disord. 2014, 14, 54. (13). Incalza, M. A.; D'Oria, R.; Natalicchio, A.; Perrini, S.; Laviola, L.; Giorgino, F. Vascul Pharmacol. 2018, 100, 1-19. (14). Li, H.; Horke, S.; Forstermann, U. Atherosclerosis 2014, 237, 208-219. (15). Ambrose, J. A.; Barua, R. S. J. Am. Coll. Cardiol. 2004, 43, 1731-1737. (16). Perlstein, T. S.; Lee, R. T. Arterioscler. Thromb. Vasc. Biol. 2006, 26, 250-256. (17). Jaqaman, K.; Loerke, D.; Mettlen, M.; Kuwata, H.; Grinstein, S.; Schmid, S. L.; Danuser, G. Nat. Methods. 2008, 5, 695-702. (18). Jay, A. G.; Chen, A. N.; Paz, M. A.; Hung, J. P.; Hamilton, J. A. J. Biol. Chem. 2015, 290, 4590-4603. (19). Chen, Y.; Kennedy, D. J.; Ramakrishnan, D. P.; Yang, M.; Huang, W.; Li, Z.; Xie, Z.; Chadwick, A. C.; Sahoo, D.; Silverstein, R. L. Sci Signal. 2015, 8, ra91. (20). Park, H.; Kang, H.; Ko, W.; Lee, W.; Jo, K.; Lee, H. S. Amino Acids. 2015, 47, 729-734. (21). Becirovic, E.; Bohm, S.; Nguyen, O. N.; Riedmayr, L. M.; Hammelmann, V.; Schon, C.; Butz, E. S.; Wahl-Schott, C.; Biel, M.;



For TOC only


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Luminescence-Resonance-Energy-Transfer-Based Luminescence

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