Monitoring Lipid Peroxidation within Foam Cells by Lysosome

Jul 22, 2015 - Monitoring Lipid Peroxidation within Foam Cells by ... and clarified, which benefits the understanding in the initiation and control fa...
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Monitoring Lipid Peroxidation within Foam Cells by LysosomeTargetable and Ratiometric Probe Xinfu Zhang, Benlei Wang, Chao Wang, Lingcheng Chen, and Yi Xiao* State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116024, P.R. China S Supporting Information *

ABSTRACT: Lipid peroxidation (LPO) in lysosomes is a valuable analyte because it is close associated with the evolutions of some major diseases. As a typical example, in the start-up phase of atherosclerosis, lysosomes get as swollen as foams, by accumulating a large amount of lipoproteins, which facilitates the free-radical chain propagation of LPO. Despite the existences of several fluorescent LPO probes, they are not appropriate for reporting the local extents of lysosomal LPO, for their unspecific intracellular localizations. Here, Foam-LPO, the first fluorescent LPO probe specifically targeting lysosomes, has been developed through straightforward synthesis using low-cost reagents. A basic tertiary amine group enables it to selectively localize in acidic lysosomes; and the conjugated diene moiety within the BODIPY fluorophore will degrade in response to lipid peroxidation, which results in fluorescence maximum shifting from 586 to 512 nm. Thus, under a confocal fluorescence microscope, Foam-LPO is able not only to visualize dynamic morphological changes of lysosomes during the evolution of foam cells, but also to relatively quantify local LPO extents in single lysosomes through ratiometric imaging. In addition, Foam-LPO proves applicable for two-color flow cytometry (FCM) analysis to make quantitative and high-throughput evaluation of LPO levels in large quantity of cells at different stages during the induction to form foam cells. Also importantly, with the aid of this new probe, the different roles played by low-density lipoprotein (LDL) and its oxidized form (ox-LDL) for the LPO processes of foam cells are distinguished and clarified, which benefits the understanding in the initiation and control factors of atherosclerosis.

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cells until the AS plaques take shape. Thus, accurate and quantitative evaluation of the dynamic LPO levels in foam cells’ lysosomes should be very meaningful for the early diagnosis of AS and the related pathological or pharmacological studies. To our knowledge, there is no reliable tool for real-time monitoring the local LPO in lysosomes. Although fluorescence imaging has been frequently adopted in the determination of cellular LPO,12−14 there is no fluorescent LPO probe specifically localizing in lysosomes. Actually, almost all the existing LPO probes, including the common used and commercially available C11-BODIPY581/591 (shown in Chart 1), do not show targeting specificity to any organelles. They nonspecifically stain all the lipid-rich regions that cover very broad cellular areas, and thus, their fluorescence changes can hardly provide the direct and accurate LPO information for any certain organelles of interest. The only exception is MitoPerOx (shown in Chart 1) which is a mitochondria-targeting LPO probe developed by Murphy and co-workers in 2012.14 In spite the rare precedent like MitoPerOx, we are sure that the deficiency in specificity will be more and more recognized as

ipid peroxidation (LPO) is one of major pathological mechanisms involved in a lot of diseases, including cancers,1 neurodegenerative diseases,2,3 atherosclerosis,4,5 etc.6 Essentially, LPO is a kind of free-radical chain oxidation on unsaturated fatty acids.7 While LPO is usually initiated by reactive oxygen species (ROS), its pathogenic effects are beyond ROS’s direct oxidative damage of lipids in cellular membranes. LPO’s far-reaching consequences result from a self-amplification of the chain propagation which continuously releases unstable lipid peroxides that further undergo decompositions into a large amount of reactive and diffusible small molecule products, for example, malondialdehyde. Atherosclerosis (AS) is a clear example for such a damage amplified by LPO propagation. As has been well recognized, the transformation from macrophages into foam cells is the start-up phase of AS.5,8 During this evolution, a large excess of lipid proteins are accumulated in lysosomes. These swollen and foam-like lysosomes become the optimal places for LPO propagation owing to the high density and large quantity of lipids stored there.5,7,8 Many LPO-generated toxic substances will be leaked out of foam cells,7,9,10 and will amplify the immune responses, because they can act as proinflammatory mediators and the recruiters for other immune cells.8,11 For these reasons, severe and continuous inflammation will be induced and will speed up the death of vascular smooth muscle © XXXX American Chemical Society

Received: April 16, 2015 Accepted: July 22, 2015

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serum) in an atmosphere of 5% CO2 and 95% air at 37 °C. For FCM studies, macrophages in the exponential phase of growth were plated into 35 mm glass-bottom culture dishes (Φ 20 mm) containing 2 mL of RPMI 1640. After incubation at 37 °C with 5% CO2 for 1−2 days to reach 70−90% confluency, the media was removed. Then the cells were washed with 2 mL of PBS buffer, and 2 mL of fresh RPMI 1640 was added along with LDL or ox-LDL. Cells were stained sometime during the stimulation before FCM analysis. Samples were illuminated with a sapphire laser at 488 nm on a FACScan flowcytometer (BD Biosciences Pharmingen, USA). The fluorescence of the forward-scattered and side-scattered light from 10000 cells is detected at rate of 150 events/second. Flow cytometric data were analyzed with FlowJo software. MTT Assay. The cytotoxic effect of Foam-LPO is assessed using the MTT assay. Briefly, the cells in the exponential phase of growth are used in the experimentation. 1.5 × 103 cells/well are seeded onto 96-well plates and allowed to grow for 24 h prior to treatment with Foam-LPO. The incubation time of Foam-LPO is 5 min with a concentration of 1 μM. At the end of this time, the Foam-LPO containing medium is replaced with dye free medium. After 12 or 24 h, MTT is added to each well (final concentration 0.5 mg/mL) for 4 h at 37 °C, and formazan crystals formed through MTT metabolism by viable cells are dissolved in DMSO. Optical densities are measured at 570 nm. Procedure for the Synthesis of Foam-LPO. OH-BDP. OH-BDP is prepared according to literature.30 3-Chloro-1(N,N-dimethyl)-propylamine is prepared from its hydrochloride. Hydrochloride of 3-Chloro-1-(N,N-dimethyl)-propylamine is added to excessive NaOH aqueous solution, and 3chloro-1-(N,N-dimethyl)-propylamine extracted by using diethyl ether. Organic phase was dried over Mg2SO4 and then concentrated under vacuum. DMA-BDP. OH-BDP (500 mg, 1.47 mmol), freshly prepared 3-chloro-1-(N,N-dimethyl)-propylamine (550 μL, 4.41 mmol) and K2CO3 (202 mg, 1.47 mmol) were refluxed in acetone for 6h. After OH-BDP was consumed, the solution was evaporated off under vacuum. CH2Cl2 was added, and the organic phase was washed three times with water. Organic phase was dried over Mg2SO4. Crude product was then concentrated under vacuum and purified by silica gel column chromatography using 30:1 CH2Cl2/CH3OH as the eluent, which yielded the desired product DMA-BDP as an orange solid (393 mg, 63%). mp: 153−155 °C. 1H NMR (CDCl3, 400 MHz): δ 7.15 (2H, J = 8.0 Hz, d), 7.01 (2H, J = 8.0 Hz, d), 5.97 (2H, s), 4.07 (2H, J = 6.4 Hz, t), 2.55 (6H, s), 2.50 (2H, J = 8.0 Hz, t), 2.28 (6H, s), 2.00 (2H, m), 1.43 (6H, s). 13C NMR (CDCl3, 100 Hz): δ 159.7, 155.3, 143.2, 142.0, 131.9, 129.2, 127.0, 121.2, 115.2, 66.4, 56.4, 45.4, 30.4, 29.8, 27.6. m/z (ESI): Calcd [M + H]+ for C24H31BF2N3O: 426.2450. Found: 426.2514. Foam-LPO. DMA-BDP (500 mg, 1.18 mmol) and cinnamic aldehyde (297 μL, 2.36 mmol) were added to a 100 mL round bottomed flask containing 50 mL of toluene, and to this solution was added piperidine (1 mL) and acetic acid (1 mL). The mixture was heated under reflux by using a Dean−Stark trap, and the reaction is monitored by TLC 30:1 (v/v) CH2Cl2/CH3OH (Rf = 0.3). When all the starting material has been consumed, the mixture is cooled to room temperature and solvent is evaporated. Water (300 mL) is added to the residue, and the product is extracted into the CH2Cl2 (3 × 200 mL). The organic phase is dried over Mg2SO4 and evaporated, and

Chart 1. Structures of Reported LPO Probes and Foam-LPO

the key issue hampering the application of a LPO probe. Considering the important pathological roles of LPO in foam lysosomes for early AS lesion, we direct our efforts toward the development and application of the first lysosome-targeting LPO probe Foam-LPO (shown in Chart 1).

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EXPERIMENTAL SECTION General Methods. The 400 (1H) MHz NMR and 100 (13C) MHz NMR spectra are registered at room temperature using perdeuterated solvents as internal standard. Melting points were obtained with a capillary melting point apparatus in open-ended capillaries and are uncorrected. Chromatographic purification is conducted with silica gel. All solvent mixtures are given as volume/volume ratios. Culture of RAW 264.7 and Fluorescent Imaging. RAW 264.7 (Macrophages cells) are obtained from Institute of Basic Medical Sciences (IBMS) of Chinese Academy of Medical Sciences (CAMS). All cell lines are maintained under standard culture conditions (atmosphere of 5% CO2 and 95% air at 37 °C) in RPMI 1640 medium, supplemented with 10% FBS (fetal calf serum). Grow RAW 264.7 in the exponential phase of growth on 35 mm glass-bottom culture dishes (Φ 20 mm) for 1−2 days to reach 70−90% confluency. These cells are used in colocalization and stimulation experimentation. For colocalization study, the cells is washed three times with RPMI 1640, and then incubated with 2 mL RPMI 1640 containing Foam-LPO (1.0 μM) and DND-198 (1.0 μM) in an atmosphere of 5% CO2 and 95% air for 5 min at 37 °C. Wash cells twice with 1 mL PBS at room temperature, and then add 1 mL RPMI 1640 culture medium and observe under a confocal microscopy (Olympus FV1000). For stimulation study, macrophages in the exponential phase of growth were plated into 35 mm glassbottom culture dishes (Φ 20 mm) containing 2 mL of RPMI 1640. After incubation at 37 °C with 5% CO2 for 1−2 days to reach 70−90% confluency, the media was removed. Then the cells were washed with 2 mL of PBS buffer, and 2 mL of fresh RPMI 1640 containing LDL or ox-LDL was added. Cells were stained sometime during the stimulation. Flow Cytometry (FCM). RAW 264.7 (Macrophages cells) were obtained from Institute of Basic Medical Sciences (IBMS) of Chinese Academy of Medical Sciences (CAMS) and cultured in RPMI 1640 supplemented with 10% FBS (fetal bovine B

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for MitoPerOx, its total synthesis should be similarly challenging because it was derived from an analogue of C11BODIPY581/591.14 However, we adopt the easily obtained and stable 2, 4-dimethyl pyrrole to condense with 4-hydroxybenzadehyde which readily produce a symmetrical BODIPY derivative. Followed with a simple alkylation on phenolic hydroxyl, tertiary amino moiety is attached to the BODIPY core. Finally, with the active methylene groups, the key intermediate DMA-BDP smoothly undergoes Knoevenagel condensation18 with cinnamic aldehyde to form the target Foam-LPO possessing conjugated diene group. Our synthesis of Foam-LPO, avoiding use of costly and unstable pyrroles, suggests a new, facile and versatile approach that can be slightly modified for developing various functional LPO probes. Optic Properties. Independence on Acidic pH, Response to Peroxidation, and Photostability. It is well-known that pH values of different lysosomes or those within the single lysosomes do not always keep constant, but vary in the pH range from 4.5 to 6.5 according to their working or health statuses. Actually, it is a big challenge to follow the dynamic pH variation of lysosomes under pathological conditions. Especially, little has been known about the pH of foam cells’ lysosomes. Under this context, it is a precondition for a lysosome-targeting LPO probe to have stable fluorescence properties independent of such pH changes. As shown in Figure 1a, in a broad pH range 3.0−8.0, Foam-LPO shows an identically strong emission peaked at 586 nm. That means, no matter how lysosomal pH fluctuates in its reasonable weakly acidic range, Foam-LPO’s emission will not be affected. At higher pH than 8, there is a gradual decrease (by ∼50%) in the fluorescence intensity, which should be ascribed to quenching effect of the PET (photoinduced electron transfer)19 from the tertiary amino to the fluorophore. Obviously, the higher pKa of the tertiary amine is decisive that, if in acidic lysosomes, it will be protonated and positively charged and thus lose the electron-donating capability. As expected, Foam-LPO demonstrates sensitive spectral shift in response to the LPO. Analogue to C11-BODIPY581/591 and MitoPerOx,12,14 our LPO probe have a fluorophoric structure featured by a conjugated diene connecting BODIPY core and a phenyl moiety. Because of this extended conjugation, the intact Foam-LPO displays bright red fluorescence with an emission maximum at 586 nm. As reported, this diene structure is exclusively sensitive to free radical species formed from hydroperoxides, but not to hydroperoxides and superoxide.15 As shown in Figure S1, Foam-LPO is insensitive to regular ROS, such as H2O2, CumCOOH, HClO and so on. Under suitable induction by ROS, such conjugated diene is subject to peroxidation with one or both of two double bonds being degraded. Once this process happens, the conjugation system is shortened to the BODIPY core that emits green fluorescence at shorter wavelength. The cleavage of the diene probably generate hydroxylated or carboxylic acid derivatives as reported.20 And they were proved to maintain the BODIPY core, which emits green fluorescence. By employing LC/MS we tracked several oxidized products according to the mechanism. MS spectra of their possible structures are given in Supporting Information figure S3. The absorption spectrum of the above products shows typical absorption maximum of BODIPY at about 500 nm. We simulate the lipid peroxidation in PBS (containing 30% methanol, pH ∼5) by adding hemin and cumene hydroperoxide (CumOOH). Hemin will help to generate free radical species from CumOOH in solution,

the residue is purified by Al2O3 column chromatography using 30:1 CH2Cl2:CH3OH as the eluent, which yielded the desired product Foam-LPO as purple solid (95 mg, 15%). mp: 213− 215 °C. 1H NMR (CDCl3, 400 MHz): δ 7.48 (2H, J = 8.0 Hz, d), 7.35 (3H, J = 8.0 Hz, t), 7.20 (2H, m), 7.08 (2H, m), 7.01 (2H, J = 8.0 Hz, d), 6.77 (1H, s), 6.55 (1H, s), 6.01 (1H, s), 5.35 (1H, s), 4.12 (2H, s), 2.86 (2H, s), 2.59 (3H, s), 2.55 (6H, s), 2.22 (2H, s),1.47 (3H, s), 1.44 (3H, s). 13C NMR (CDCl3, 100 Hz): δ 158.3, 155.4, 152.2, 142.9, 142.3, 140.2, 136.9, 136.6, 136.0, 133.4, 132.4, 129.9, 129.5, 129.3, 128.8, 128.3, 127.4, 127.3, 126.8, 123.1, 121.4, 117.7, 115.0, 65.7, 56.2, 44.5, 35.8, 32.0, 29.7, 29.6, 29.3, 27.2, 26.3, 25.4. m/z (ESI): Calcd [M + H]+ for C33H37BF2N3O: 540.2919. Found: 540.2993

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RESULTS AND DISCUSSION Design and Synthesis of Foam-LPO. To monitor the local LPO in the lysosomes of foam cells, the fluorescent probe should meet two fundamental requirements of LPO sensitive signaling and the specific localization in lysosomes during the whole process of cell transformation. As one of the key design in Foam-LPO, a BODIPY derivative containing a conjugated diene group within its fluorophoric structure is chosen as the LPO signaling unit. Once this conjugated diene group mimicking the polyunsaturated fatty acids in lipids undergoes peroxidation, the fluorophore’s conjugation length will be shortened. Thus, Foam-LPO can respond LPO through a spectral shifting or ratiometric way. This signaling concept of diene-BODIPY is learnt from previous LPO probes C11BODIPY581/591 and MitoPerOx.12,14 The other advantages of this signaling unit include the high selectivity toward LPO against other oxidation and the insensitivity to environmental factors, etc.15 Another key design in Foam-LPO is a weakly alkaline tertiary amino group, which enables the probe to be protonated and then selectively accumulate in acidic lysosomes. This lysosome-targeting moiety is distantly and flexibly connected to the diene-BODIPY fluorophore, so that the former will not obstruct the latter of rigid, long and lipophilic framework to efficiently intercalate the accumulated lipids and sense local LPO. The synthesis of Foam-LPO is straightforward and efficient, as shown in Scheme 1. Although our probe possesses the Scheme 1. Synthetic Route of Foam-LPO

similar signaling unit (i.e., diene-BODIPY) to C11-BODIPY581/591 and MitoPerOx, our synthetic strategy is completely different from the previous ones.16,17 According to a U.S. patent,16 to prepare for C11-BODIPY581/591 as well as its analogues, an unsymmetrically condensation between a long fat acid substituted pyrrole formaldehyde and phenyl butadienyl pyrrole was employed. The challenges lied in obtaining the two pyrrole derivatives and overcoming their chemical instability. As C

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Figure 1. (a) Change of intensity plots of Foam-LPO at 580 nm vs different pH values. Inset: Change in fluorescence spectra of Foam-LPO in PBS (containing 30% methanol). (b) Changes of fluorescence wavelength of Foam-LPO in pH ∼5 PBS (containing 30% methanol) in the present of 1 μM hemin and 1 mM CumOOH. Excited at 488 nm. (c) Changes of absorption wavelength of Foam-LPO in pH ∼5 PBS (containing 30% methanol) in the present of 1 μM hemin and 1 mM CumOOH. (d) Fluorescence intensity decay of Foam-LPO in pH ∼5 PBS (containing 30% methanol) compared with fluorescein in pH ∼11 water. Samples are irradiation by 488 nm light from mercury lamp. F0 is the fluorescence maximum intensity before the irradiation. F is the fluorescence intensity after a fixed time of irradiation.

Figure 2. Fluorescence images of healthy macrophage cells (a, b, and c) and foam cells (e, f, and g) costained by DND-189 (a and e, green channel, λex = 405 nm, λem = 500−560 nm) and Foam-LPO (b and f, red channel, λex = 488 nm, λem = 570−630 nm). (c) Merged image of panels a and b and bright field. (g) Merged image of panels e and f and bright field. Panels d and h are amplified images of regions in panels b and f.

which is a similar situation as lipid peroxidation in living cells.15 As shown in Figure 1b, Foam-LPO exhibits evident spectral change from 586 to 512 nm. During the oxidation, there is a continuous increasing in fluorescence intensity at 512 nm and a decreasing at 586 nm. Finally, the intensity ratio512/586 changes from 0.16 to 8.91. Such a large ratiometric signal is welcomed for sensing and imaging in cells, for the improved accuracy and reliability in relatively quantitative detection. To our knowledge, there have been an increasing number of ratiometric probes reported in literatures.21−24 Chemosensors use the intensity enhancement of single emission as signals will be inevitably interfered by several uncertain factors such as the uneven concentrations of the fluorescent probes within highly inhomogeneous cellular microenvironment. Figure 1c is the absorption spectral change of Foam-LPO under the stimulation of hemin and CumOOH, which show a clear spectral change

from 567 to 500 nm. This result demonstrates a cleavage of the π-structure. Photodecomposition experiment proves that Foam-LPO has good photostability. Here, photostability of Foam-LPO has been compared with that of the well-known fluorophore fluorescein. The result shows Foam-LPO has superior photostability over fluorescein (Figure 1d). After irradiation by 488 nm light (the same wavelength used for fluorescence imaging) from mercury lamp for 60 min, the intensity fluorescence maximum (585 nm) of Foam-LPO remains as high as 89%, while fluorescein maintain less than 50%. Another intuitive experiment was carried out to demonstrate the performance of Foam-LPO in fluorescence imaging (Figure S2). Stained foam cells were induced to undergo LPO (detailed information see the next section). Upon subjecting the cells to laser irradiation 50 times in sequence, nether fluorescence intensity in two channels showed evident change. These results D

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Figure 3. Fluorescence images of stained (Foam-LPO, 1 μM for 5 min) macrophage cells being incubated with ox-LDL (20 mg/L) and LDL (100 mg/L) for 0, 6, 12, 18, and 24 h. Excitation wavelength: 488 nm. Green channel: 525 ± 25 nm. Red channel: 585 ± 25 nm.

Figure 4. (a). Fluorescence images of stained (Foam-LPO, 1 μM for 5 min) macrophage cells being incubated with ox-LDL (20 mg/L) and different amount of LDL (50−200 mg/L) for 24 h, cells are stained with Foam-LPO at the 12th hour. (b) Bar graph of mean ratio data of cells under different view. Excitation wavelength: 488 nm. Green green channel: 525 ± 25 nm. Red channel: 585 ± 25 nm.

first thing is to make sure that Foam-LPO can specifically and stably localize in lysosomes in the whole process. For this sake, we carry out the colocalization imaging experiments using Foam-LPO and DND-189 (one of most commonly used lysotrackers) together to stain the macrophage cells or foams cells. DND-189 is chosen as the colocalization reagent, because

demonstrate that Foam-LPO is photostable enough to give correct information through fluorescence imaging. Specifically Lysosome-targeting of Foam-LPO in Normal Macrophage Cells and Foam Cells. To verify the feasibility of real-time monitoring the local LPO during the complete evolution from normal macrophage cells to the foam cells, the E

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Analytical Chemistry it emits green fluorescence (with maximum at ∼520 nm) which can be easily distinguished from that of Foam-LPO. Although peroxidation of Foam-LPO will definitely generate green fluorescence, it will take a relatively long time for such a reaction, as indicated in Figure 1b. However, both Foam-LPO and DND-189 can stain the cells so fast that we could quickly finish the colocalization imaging in 5 min. During such a short staining and imaging time, the interference by the peroxidation of Foam-LPO is ruled out. The images of normal macrophage cells are recorded in Figure 2a−d. The red fluorescence from Foam-LPO are clearly distributed in some small punctuate organelles (diameter less than 1 μm). These organelles are also stained by the standard lysotracker DND-189. The fluorescence images of the two channels overlap very well, with high Pearson’s correlation factor of 0.93, revealing a satisfactory colocalization. These results confirm that Foam-LPO specifically stain lysosomes of normal macrophage cells. Another set of colocalization experiment is performed on foam cells derived from normal macrophages by a mixture of LDL (100 mg/mL) and ox-LDL (20 mg/mL). The combination use of LDL and ox-LDL is a well-known method to induce foam cells from macrophages and the rationality for such an optimized induction toward foam cells will be discussed later in detail. As shown in Figure 2e−f, the red fluorescence from Foam-LPO only appears in a few foam-like organelles of large sizes and irregular shapes. These foam regions are also specifically stained by DND-189. The Pearson’s correlation factor is high up to 0.94, unambiguously proving that the foam regions stained by Foam-LPO are exactly the swollen lysosomes. With the fluorescent staining of Foam-LPO, it is easy to distinguish the swollen lysosomes (foam regions) in sharp contrast to the surrounding areas. As illustrated in the partially enlarged image 2 h, one of the typical foam is a large sphere with diameter over 5 μm which is almost 5−10 folds that of normal lysosomes in Figure 2d. The dynamic changes of lysosomes observed during the formation of foam cell agree well with previous results obtained by scanning electron microscope.25 Subsequently, the localization stability Foam-LPO in lysosomes is confirmed through long-time fluorescence tracing. Twelve or twenty-four hours after staining by Foam-LPO (Figure S4), normal macrophage cells maintained almost the same fluorescence intensity as well as the cellular morphology. Similar to DND-189, Foam-LPO’s good stability of specific localization in lysosomes should be ascribed to the tertiary amino group. Even if the foam cells can be regarded as a kind of unhealthy macrophage cells, their foam-like lysosomes remained acidic environment favoring the retention of the basic probe. Such a stable lysosome-targeting ability benefits completely tracking the ultrastructural changes of lysosome during its lesion. In other words, Foam-LPO is applicable for tracking the evolution from normal macrophages to foam cells. In addition, as seen in Figure 3 and Figure 4a, the green fluorescence from oxidized Foam-LPO in foam cells shows good colocalization with red fluorescence from Foam-LPO. There is not any green fluorescence outside lysosomes in foam cells, which demonstrates the targeting ability of oxidized Foam-LPO. Ratiometric Images Visualizing of LPO in Developing Foam Cells. To this date, the direct association between LPO in lysosomes and the development of foam cells as well as their damage effects has hardly been established. Without any doubt,

severe LPO takes place in foam cells, as a large amount of products released by LPO have been captured.7,9,10 And theoretically, foam cells’ lysosomes filled up with an excess of lipid proteins are at much higher risk of LPO than other organelles. Even though, the key question whether or not LPO happens in lysosomes is still not answered. Using the commercial probe C11-BODIPY581/591 as most researchers have done can only reveal LPO levels of the whole cells, which is still difficult to clarify the contribution from lysosomes. Ones might argue that lysosomes in foam cells could just act as the temporary warehouses for lipids which could be transferred to other cellular places for LPO. For the first time, we achieve not only the visualization LPO in lysosomes but also the relative quantification of local LPO extent during the evolution from macrophage cells to foam cells. Under our optimized induction for 24 h, macrophage cells gradually transform into foam cells, producing apparent morphological changes without causing cell death. The cells are stained by Foam-LPO and a series of two-channel fluorescent images at different time points are recorded as shown in Figure 3. As induction time goes on, more and more swollen lysosomes are clearly observed, reflecting that the foam cells are forming. Also, the fluorescence intensity of the red channel decreases gradually, which is accompanied by an apparent increase in the fluorescence intensity of the green channel. Such a remarkable shift in fluorescence maximum is the evidence for the capture of LPO in lysosomes by FoamLPO. Generally, the total fluorescence intensity ratio of the green channel to red channel increase remarkably from less than 0.1 at the beginning to the final 1.1 after 24 h, indicating the great elevation of the LPO extent. However, these overall ratios at different induction times reflect just the average LPO levels for all the lysosomes at different stages of the developing foam cells. Fortunately, when overlapping the corresponding images of two channels, it was found the green-stained regions and the red ones are entirely identical. This means that the green fluorescent products generated by the peroxidation of FoamLPO keep the same localization specificity to lysosomes as the parent probe. This is, actually, a very important feature of Foam-LPO for practical applications. Only under the precondition of the good overlapping of two channels, it is feasible to accurately evaluate the local LPO extents of single lysosomes by comparing the local green/red ratios. As can be seen in Figure 3, at any time, the local LPO in different lysosomes are not the same. Different Effects of LDL and ox-LDL to the Formation of Foam Cells and LPO Process. Faom-LPO provides a new opportunity to clarify the roles of LDL and ox-LDL in the startup phase of AS. It has been well recognized that the initial AS lesion is mainly concerning the formation of foam cells and LPO propagation.5,8,26 But these two processes are always interwoven and reinforce each other. Thus, the contributions from LDL and ox-LDL as the major substances involved in these two processes can hardly be differentiated in previous pathological studies. Controversially, while many reports concluded that LDL had to be transformed to ox-LDL that played the key roles,27,28 some others supported that native LDL alone was effective.29 Since we have already confirmed Faom-LPO’s dual functions of visualizing the formation of foam cells and monitoring the local and overall LPO levels, we decide to apply this probe for a systematical study on mimicking early stage of AS induced by different doses of F

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Figure 5. (a) Flow cytometric analysis of healthy macrophage cells (red) and macrophage derived-foam cells (green). (b) FCM result of stained (Foam-LPO, 1 μM for 5 min) macrophage cells being incubated with ox-LDL (20 mg/L) and LDL (100 mg/L) for 0h red, 6 h blue, 12 h orange, 18 h green and 24 h pink. (c) Average intensity of green channel (FITC-A) and red channel (PE-A) in FCM analysis; the ratio between intensities of two channels. Excitation wavelength: 488 nm. FITC-A channel: 530 ± 30 nm. PE-A channel: 585 ± 42 nm.

LDL and ox-LDL. The two-channel fluorescence images and ratiometric images are recorded in Figure 4a, and the mean green/red ratios value of different cells are shown in Figure 4b. Such ratio imaging of cells under same stimulating condition is repeatable. Reproduced ratio imaging experiments of two stimulating condition are given in Supporting Information (Figure S5). These images demonstrate close ratio change in foam cells under same stimulating condition. First, macrophage cells are incubated with native LDL alone for 24 h. Fluorescence images demonstrate that lysosomes keep normal morphology without any sign of swelling, even if the dose of LDL is high up to 200 mg/mL. The intact morphology indicate that lysosomes will not uptake excess LDL. However, the green channel fluorescence intensity increases while that of red channel decreases, which results in apparently larger green/ red ratios than that (less than 0.1) of the control cells (normal macrophages without stimulation). And it is found that a higher does (200 mg/mL) LDL lead to a larger green/red ratio of 1.05 than lower does (0.29 for 100 mg/mL). These results indicate that native LDL cannot induce the transformation from macrophage cells into foam cells, but will effectively initiate LPO within lysosomes. Next, macrophage cells were incubated with lower dose (20 mg/mL) ox-LDL alone for 24 h. Very remarkable phenomenon of lysosomal swelling is observed, which indicates ox-LDL alone can induce formation of foam cells efficiently. However, in those foam regions (swollen lysosomes), only strong red channel fluorescence is detected, while green channel fluorescence is still very weak. This results in a very small green/red ratio close to that of the control cells, which means LPO in these foam regions is ignorable. We also try to increase a little the dose of ox-LDL, but find that large amount of cells are killed because of the high cytotoxicity of ox-LDL. Then, macrophage cells are incubated in different levels of LDL (50, 100, 200 mg/L) with a small share of ox-LDL (20 mg/L) for 24 h. Not only the very swollen lysosomes, but also the apparent spectral shifts in these foam regions are observed. In addition, with the increase in the dose of LDL, there is a gradient increase in green/red ratio from 0.90 (for 50 mg/mL LDL) to 3.21 (for 200 mg/mL LDL). This also reflects that in the swollen lysosomes there is not only ox-LDL, but also accumulates different amounts of LDL ready for LPO. Moreover, by comparing the foam cells formed under the stimulation by the mixture of LDL and ox-LDL and those normal macrophage cells incubated with LDL alone, it is clear that the former had much larger green/red ratio than the latter. This means ox-LDL promotes foam cells to take in more LDL (and thus, stronger LPO) than normal macrophages.

From all the above results, we can conclude that there is a clear division of labor for LDL and ox-LDL and their synergy promotes AS developing: The role of ox-LDL is to efficiently induce formation of foam cells that are capable of taking in much more amounts of native LDL than normal macrophages; and native LDL’s role is to provide unsaturated fatty acids for the free-radical chain propagation of LPO; once the uptake of ox-LDL reaches a threshold, macrophages begin to transform into foam cells and to unlimitedly take in native LDL; with accumulation of more and more native LDL, the LPO propagation becomes favored. Different from most previously reports that emphasized the AS initiation by either ox-LDL alone or LDL alone, our conclusion derived from fluorescent imaging reasonably points out that the indispensable condition is the combination of a larger amount of LDL and a smaller amount of ox-LDL. Flow Cytometry Study. For further demonstrating FoamLPO’s value, we try to use it in flow cytometry (FCM), another important fluorescent analytic tool widely applied in disease diagnosis and drug screening. FCM is suitable for highthroughput quantitative evaluation of overall change based on statistically analyzing the fluorescence data of large quantity of cells. This advantage of FCM just makes up for the deficiency of confocal fluorescence imaging that can only visualize small portion of cells appearing in the narrow field of a confocal microscope. On the one hand, the combination of Foam-LPO and FCM is a recommendable method distinguishing foam cells from normal macrophages. With the lysosomes swelling during pathological transformation, they simply uptake more probe molecules than they usually can. As is expected, FCM analysis in the PE-A channel (585 ± 42 nm) shows (Figure 5a) a shift of fluorescence signal from 3200 for normal macrophages to 9900 for foam cells. This demonstrates an evident increase of intracellular fluorescence in foam cells, which provides the basic and clear criterion for the formation of foam cells. On the other hand, two-color FCM analysis can relatively quantify the level of lipid peroxidation in foam cells on large cell populations. Lipid peroxidation level at different time is studied by FCM. Macrophage cells are treated with the same procedure like cells in Figure 3. The data dots (Figure 5b) for each group of cells (different stimulation time) distribute in different regions that means different fluorescence intensity ratio of two channels (channel FITC-A 530 ± 30 nm vs channel PE-A 585 ± 42 nm). The average values of intensities from two channels are plot in Figure 6c. In general, the values in red channel decreases from about 10000 to 6000, while the values in green channel increase from 500 to 7000. The ratio of G

DOI: 10.1021/acs.analchem.5b01428 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

benefits the understanding in the initiation and control factors of atherosclerosis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01428. Additional imaging results, FCM results, and 1H and 13C spectra (PDF)

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Figure 6. Viability of macrophages with Foam-LPO (1 μM) after treatment for 12 and 24 h.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. two intensities show continuous increase from 0.06 to 1.17 as stimulation time extends. Another group of FCM analyses corresponding to cells treatment in Figure 4 are also conducted. As shown in Figure S6, the data dots in the two-color FCM analysis shows different distribution that is higher channel FITC-A intensity versus channel PE-A ratio corresponding to higher LDL concentration. The average intensity plot for two channels demonstrates a clear trend: as the LDL concentration increases, intensity in red channel decreased, while intensity in green channel increases. Thus, the ratio of green and red channels shows an evident increase from 0.51 to 2.70. These FCM analyses give intuitive and reliable information that high level of LDL may increase the risk of vascular system. The cytotoxicity of Foam-LPO is low. MTT assay (Figure 6) has revealed that even incubated with 1 μM Foam-LPO continuously for 24 h, ∼81% of cells survives. This is a favorable characteristic of a practical lysosomal probe applied in living cells.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by National Natural Science Foundation of China (Nos. 21174022, 21376038, and 21421005), National Basic Research Program of China (No. 2013CB733702), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1213), and the Fundamental Research Funds for the Central Universities (No.DUT14YQ103).

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REFERENCES

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CONCLUSION In conclusion, Foam-LPO, the first fluorescent LPO probe specifically localizing in lysosomes, has been developed through straightforward synthesis using low-cost reagents. Our aim is to provide a reliable tool for the early diagnosis of atherosclerosis at cellular levels. The transformation from macrophages to foam cells and the aggravation of LPO accompanying this pathological change are two major early symptoms and factors initiating atherosclerosis. As we also know, within foam cells, lysosomes are the subcellular regions vulnerable to LPO, because these unhealthy and swollen organelles accumulate a large amount of lipoproteins to facilitate the free-radical chain propagation. Based on all these considerations, our probe is purposely designed to have a basic tertiary amino unit to target acidic lysosomes and to have a BODIPY fluorophore containing a conjugated diene to respond LPO by a remarkable fluorescence maximum shifting from 586 to 512 nm. Under a confocal fluorescence microscope, Foam-LPO is able not only to dynamically visualize morphological changes of lysosomes during the evolution of foam cells, but also to relatively quantify local LPO in single lysosome through ratiometric imaging. In addition, Foam-LPO proves applicable for two-color flow cytometry (FCM) analysis to show relatively quantitative and high-throughput evaluation of the extent of LPO in large quantity of cells at different stages during the induction to form foam cells. Also importantly, with the aid of this new probe, the different roles played by low-density lipoprotein (LDL) and its oxidized form (ox-LDL) for the LPO processes of foam cells are distinguished and clarified, which H

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DOI: 10.1021/acs.analchem.5b01428 Anal. Chem. XXXX, XXX, XXX−XXX