Targeting Early Apoptosis in Acute Ischemic Stroke with a Small

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Imaging and Diagnostics

Targeting Early Apoptosis in Acute Ischemic Stroke with a Small-molecule Probe Cheng Qian, Dong-Fang Liu, Congxiao Wang, Jie Ding, Yan-Li An, Pei-Cheng Li, and Gao-Jun Teng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00213 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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ACS Biomaterials Science & Engineering

Targeting early apoptosis in acute ischemic stroke with a small-molecule probe Cheng Qian1, Dong-Fang Liu1, Cong-Xiao Wang 1, Jie Ding1, Yan-Li An1, Pei-Cheng Li 1,2, Gao-Jun Teng1* 1

Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of

Radiology, Medical School, Zhongda Hospital, Southeast University, 87 Dingjiaqiao Rd., 210009 Nanjing, China. 2

Department of Interventional Radiology, First Affiliated Hospital of Soochow

University, 188 Shizi St., 215006 Suzhou, China.

* Corresponding author: Gao-Jun Teng Fax: +86-25-83272541; Tel: +86-25-83272121 Email: [email protected]

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Abstract Inhibiting apoptotic cells helps ameliorate ischemic injury. Actually, only the apoptotic cells in early stage could be rescued. Molecular imaging of the early apoptosis would make sense in ischemic stroke, however, few of apoptosis molecular probes could specifically target early apoptosis. This study developed a small-molecule early-apoptosis targeting probe CYS-F, which was synthesized by cystine with fluorescein isothiocyanate dyes. And the final molecular weight of CYS-F was only 1013 Da, which was much smaller than the traditional apoptosis marker annexin V. CYS-F showed excellent early-apoptosis targeting ability both in vitro and in vivo. And CYS-F was cleared rapidly from the circulation with a blood half-life of 1.325 h. A favorable match was obtained between the images in fluorescence imaging and magnetic resonance imaging in stroke models. The target-to-background ratio of the lesions on 0 hour was negative, which reflected the decreased blood flow. Multimodal molecular imaging showed the therapeutic effect of cystamine was dose dependence and CYS-F could also predict the outcome of ischemic stroke at an early stage. The versatility of CYS-F provides a comprehensive and convenient route for clinical decision-making in acute ischemic stroke. Key words: early apoptosis; acute ischemic stroke; small-molecule probe; multimodal molecular imaging

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Introduction Stroke is among the top three causes of death worldwide1. Unfortunately, few neuroprotective strategies have apparent effect on stroke. The penumbra, an area of salvageable tissue in stroke, is closely associated with the improvement in outcomes2. And the main manner of cell death in the penumbra is suggested to be the apoptosis3-4. It has been proven that attenuating apoptosis could ameliorate ischemic injury5-6. Apoptosis is a process of programmed cell death that involves three phases, and only apoptotic cells in the early phase could be rescued7. Therefore, detection of early apoptosis is of great significance in the treatment of stroke8. Among various apoptosis-detecting molecular probes, the annexin V probes are mostly studied. These probes target the phosphatidylserine (PS) exposed on the cell surface9. Different imaging methods, including magnetic resonance imaging (MRI)10, optical imaging11 and positron emission tomography (PET)12, have been developed based on annexin V. However, these annexin V probes could not precisely identify early apoptosis. Even distinguishing apoptosis from necrosis seems difficult for annexin V probes since PS is also presented on the plasma membrane of necrotic cells. In addition to annexin V, other proteins, such as anti-apoptotic protein gsa6, also bind to PS13. For these proteins, PS binding is mediated by γ-carboxyglutamic-acid (Gla)13. Based on this feature, Reshef et al has developed a series of small-molecule apoptosis probes named ApoSense. The ApoSense family includes fluorescent probes, such as didansyl-L- cystine (DDC)14 and NST-73215, and a radionuclide probe 3



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(18F-ML-1016). These probes have been introduced in variant diseases, such as ischemic stroke14, melanoma tumors16 and acute renal tubular necrosis15. The studies above demonstrated that these probes specifically targeted the cells undergoing apoptosis. Furthermore, the molecular weights of these probes are low, which has obvious advantages compared with large-molecule probes17-18, including safety profiles, rapid diffusion rates, and a satisfactory blood clearance rate9,

17

.

Unfortunately, the fluorescent probes were unable to detect apoptosis in vivo, and the synthesis of the radionuclide probe was relatively complicated19. All

of

these

insufficiencies

limit

the

clinical

translation

of

current

apoptosis-targeting molecular probes. Therefore, developing a novel small-molecule probe that specially images the early apoptosis in vivo will assist in breaking the limitations and contributes to the in-depth study of stroke. Besides, using the multimodal molecular imaging to evaluate and guide the anti-apoptosis therapy will make molecular imaging play a greater role in stroke. Taken together, this study aims to develop a small-molecule probe to detect the early apoptosis in ischemic stroke models, as well as to monitor and guide the anti-apoptosis therapy. Materials and Methods Synthesis of the probes. The small-molecule probe CYS-F was synthesized by the conjugation of fluorescent dye (fluorescein isothiocyanate, FITC) to cystine. Briefly, 16 mg of FITC (Sigma, St. Louis, MO, USA) and 40 mg of cysteine (Sigma, St. Louis, MO, USA) were dissolved in water and dimethylsulfoxide (DMSO) solution with 45 4


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mg of sodium carbonate. The aqueous mixture reacted for 3 hours at room temperature; then, the products were analyzed and collected using high-performance liquid chromatography (HPLC, Waters, USA). Briefly, chromatographic separation was performed on an XBridgeTM BEH300 C18 5 µm column (4.6 mm × 150 mm) maintained at 35 °C. The mobile phase consisted of 0.1% acetic acid in water (A) and acetonitrile (B) (Tedia, Fairfield, USA) at a flow rate of 1 mL/min (A / B = 6 / 4). The total program time was 50 minutes. Cystine served as a Gla-derived domain that targeted the early apoptosis cells and carried the fluorescent reporter. FITC was conjugated for fluorescent imaging. Cysteine (Sigma, St. Louis, MO, USA) was used for a control probe, and the reaction was performed under the protection of argon gas. The final molecular weight was measured by liquid chromatography-mass spectrometry (LC-MS, Agilent, USA). Cell culture and apoptosis induction. HeLa cells were obtained from ATCC (Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high-glucose medium (Biological Industries, Beit Haemek, Israel) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum. Cells were maintained in 12-well plates at 37 °C in a humidified atmosphere containing 5% CO2. Apoptosis was induced in the HeLa cells by incubating with doxorubicin (DOX, 100 µM, Sigma St. Louis, MO, USA) for two hours. Paclitaxel (0.1 µM, Jiangsu Aosaikang Pharmaceutical Co., Ltd., China) was used for 12 hours as another induction method to further study the universality of the apoptosis targeting ability. 5



Apoptosis phase assay. The DOX-treated cells and normal cells (106 cells/mL) were incubated with CYS-F (50 µM) or the control probe for 15 minutes at room temperature. The redundant probes were removed, and the cells were washed three times with PBS. Then, the cells were photographed by a fluorescence microscope (IX71, Olympus Optical Co., London, UK). The paclitaxel-treated cells were also incubated with CYS-F (50 µM) for 15 minutes at room temperature; then, the cells were detected by two kits, Annexin V-PE/ 7-ADD (KeyGEN BioTECH Co., Jiangsu, China) and Annexin V-PE /PI (eBioscience Inc., San Diego, CA). Afterward, the cells treated with the Annexin V-PE / propidium iodide (PI) kit were photographed using a fluorescence microscope (IX71, Olympus Optical Co., London, UK). A confocal microscope (FV1000; Olympus) was used to photograph the cells treated with the Annexin V-PE/ 7-ADD kit. Cytotoxicity. The cytotoxicity of the CYS-F probe was detected with a Cell Counting Kit-8 (CCK-8) assay. Briefly, the HeLa cells were cultured in 96-well plates (104 cells per well) in 200 µL of culture medium and then maintained for 24 hours. Five different concentrations of CYS-F probe (25, 250, 625, 1250, and 2500 µg/ml) were studied, and six duplicate wells were used for each concentration. The cells were then incubated with the probes for 24 hours. Then, the redundant probes were removed, and 10 µL of CCK-8 was added to each well; the samples were incubated at 37°C for two hours and then read immediately at 450 nm with a microplate spectrophotometer (Thermo Fisher Scientific, USA). Blood half-life. All animal experiments were approved by the Institutional Animal 6


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Care and Use Committee of Southeast University. Forty-four male C57BL/6J mice (22-25g, Yangzhou University, China) were evaluated in this study. Mice were anesthetized by intraperitoneal injection with pentobarbital (60 mg/kg; Sigma-Aldrich, USA). To study the blood half-life of the CYS-F, healthy C57BL/6J mice (n=6) were used in our study. The probe was administered to mice via tail vein at a dose of 1 mg/ml. Then, 0.1 mL of blood was collected at each scheduled time point by retro-orbital puncture. Serum was obtained by centrifugation of the extracted blood samples. The optical densities in the serum samples were measured with a microplate spectrophotometer (Thermo Fisher Scientific, USA). Animal models. Two stroke models were involved in this study, the procedure details were described as following: Photothrombotic

cortex

stroke

model.

Focal

stroke

was

induced

by

photothrombosis as described previously5. After the hair was removed, Rose Bengal solution (100 mg/kg) was administered intraperitoneally 5 minutes before illumination. Then a 4-mm-diameter cold light source (KL1500 LCD, Zeiss, Germany) was placed 2 mm lateral from Bregma. The illumination was maintained within 15 minutes. Photothrombotic MCA stroke model. The proximal middle cerebral artery (pMCA) was occluded in this model, as previously described20. Briefly, after the pMCA was revealed, Rose Bengal (25mg/kg; Sigma-Aldrich, USA) was injected by tail vein. pMCA was illuminated immediately for 2 minutes by a 532nm green laser (35 mW, 7



GL532TA-100FC, Shanghai Laser & Optics Century, China). The surgical wound was covered with lidocaine gel. Afterward, the mouse was allowed to return to its cage. Anti-apoptosis therapy. Cystamine (Sigma, St. Louis, MO, USA) was introduced as an anti-apoptosis drug. Three groups were set as follows (n = 6 per group): vehicle, low dose of cystamine (50 mg/kg), and high dose of cystamine (100 mg/kg) groups. Cystamine was dissolved in 0.9% saline. Both saline and cystamine were administered intraperitoneally. Mice were killed after imaging. Imaging process. Magnetic resonance imaging (MRI): A 7.0 Tesla small animal magnetic resonance scanner (Bruker PharmaScan, Germany) was used. Mice were anesthetized with 1% isoflurane. The respiratory rate and the body temperature were monitored by a unit. T2 weighted imaging (T2WI) were performed by a two-dimensional turbo spin-echo sequence with the following parameters: repetition time (TR), 2800ms; echo time (TE), 50 ms; rapid acquisition with relaxation enhancement (RARE) factor, 8 with 3 averages; field of view (FOV), 20×20 mm; thickness, 1 mm; and matrix, 256×256. The final relative lesion volume ratio was calculated as previous described20. Near-infrared fluorescence imaging (NIRF): NIRF imaging was performed using the In Vivo Imaging System Spectrum (PerkinElmer, USA). The skull of each mouse was exposed for in vivo imaging after the mouse was anesthetized. NIRF images were collected by the Spectral Unmixing/Filter Scan technique (excitation wavelength from 465 nm to 500 nm and emission wavelength from 520 nm to 600 nm). After imaging, the mice were sacrificed and the organs were excised for ex vivo imaging. The signal 8


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intensity was quantiﬁed using Living Image software. Region of interests (ROIs) with equal size were placed on the contralateral and ipsilateral hemispheres. Target-to-background ratio (TBR) = (average radiant efficiency value from the ipsilateral hemisphere) / (average radiant efficiency value from the contralateral hemisphere). Immunostaining. The mice were euthanized with pentobarbital and transcardially perfused with phosphate buffer solution (PBS). Then, the brains were immersed in 4% paraformaldehyde. The brain tissues were dehydrated and cryostat-sectioned to10 mm. The slices were incubated with rabbit anti-mouse MAP-2 (Abcam, Hong Kong, China), annexin V (Abcam, Hong Kong, China) and NeuN (Abcam, Hong Kong, China) antibodies, which was followed by staining with Alexa Fluor 546 goat anti-rabbit antibody (Thermo Fisher Scientific, Carlsbad, USA). Nuclei were counterstained with DAPI. Then, the sections were scanned by fluorescence microscope (Scope.A1, Carl Zeiss Shanghai Co., Ltd., China). Statistical analysis. All statistical analyses were performed with SPSS software (version 19). Numerical data were expressed as the mean ± standard deviation (SD). Multiple comparisons were made by one-way ANOVA followed by Bonferroni post-tests. Correlations between NIR imaging and MRI imaging were analyzed by Pearson's correlation coefficients. P < 0.05 was considered statistically significant.

Results Synthesis and characterization of the probes. The chemical structures of the early

9



apoptosis-targeting probe CYS-F and the control probe are showed in Figure 1. As the two carboxyl groups were proved to be the key roles in PS-targeting ability, cystine was employed as a scaffold. Then two FITC dyes was linked to the each amino group which gives the probe fluorescence property (Fig. 1a). Cysteine with only one carboxyl group was chosen as the control probe (Fig. 1a). The probes were purified by HPLC. The longitudinal HPLC traces of CYS-F (red line) and FITC (green line) were showed in Fig. 1b. Compared with the retention time of FITC, the retention time of CYS-F was apparent shorter (30 minutes vs 16 minutes). The final molecular weight of CYS-F was 1 013 Da according to LC-MS, which was much smaller than the 36 000 Da of annexin V.

Figure 1. Synthesis and Characterization of the Probes. (a) The chemical structures of CYS-F and control probe. (b) The longitudinal HPLC traces of CYS-F (red) and FITC (green).

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Cytotoxicity and specificity of CYS-F. The cytotoxicity of CYS-F was examined. Compared with the control group treated with PBS, the cell viability of the cells incubated with different concentrations of CYS-F (25, 250, 625, 1250, and 2500 µg/ml) showed no significant toxicity (Fig. 2a). The targeting ability of CYS-F was first studied in HeLa cells treated with DOX (Fig. 2b). After incubating with the target probe CYS-F for 15 minutes, The CYS-F-positive cells (green) were co-localized with the DOX-treated cells (red). The weak fluorescence in PBS-treated cells indicated the low uptake of CYS-F in viable cells. DOX-treated cells showed significantly brighter signals compared to the PBS-treated cells (Fig. 2b), which means the apoptotic cells showed greater cellular uptake of the small-molecule probe. On the other hand, after incubating with the control probe, the viable cells and the apoptotic cells showed consistent fluorescent signals, which validated the targeting ability from the reverse side. The specificity of CYS-F was further demonstrated in another apoptosis-inducing method with paclitaxel. After incubating with CYS-F for 15 minutes, the cells were then co-labeled with annexin V, and 7-ADD or PI. Annexin V is an established classical marker of early apoptosis, 7-ADD and PI are markers of necrosis. As shown in Fig. 2c and Fig. S1, the targeted small-molecular probe CYS-F (green) co-localized with the annexin V-positive cells (red), while few 7-ADD- and PI-positive cells (red) were labeled with CYS-F. The in vitro results unambiguously demonstrated that CYS-F specifically targeted the early apoptosis cells and distinguished apoptosis from necrosis. And the specificity of CYS-F was independent 11



of the apoptosis-induced methods. In addition, the in vitro results also confirmed that annexin V was mostly attached to the cell membrane, while CYS-F extensively accumulated in the cytoplasm of cells.

Figure 2. Cytotoxicity and Specificity of CYS-F. (a) CCK-8 was used to study the cytotoxicity of CYS-F. (b) The specificity of CYS-F was firstly studied in the apoptotic cells treated with DOX. Scar bar, 100µm. (c) The upper and lower showed the 12


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CYS-F-positive apoptotic cells (green) were incubated with annexin V (red) /PI (red) kit. Scar bar, 50µm.

Probe biodistribution assay. To confirm the optimized imaging concentration, five different concentrations of CYS-F were studied with PBS as a control. As shown in Fig. 3a, CYS-F at 1 mg/mL showed significantly higher signal intensity than other concentrations and PBS. Then the concentration of 1 mg/mL was used in the subsequent studies. To study the biodistribution of CYS-F, the heart, liver, spleen, lung, brain and kidney of the mice (n=6) were resected for ex vivo fluorescence imaging. A high kidney uptake with little accumulation in other organs indicated predominantly renal clearance of CYS-F (Fig. 3b). The quantitative data of the target-to-background ratio (TBR) also verified this finding (Fig. 3c). And in healthy mice (n=6), CYS-F was cleared rapidly from the circulation with a blood half-life of 1.325 h. Nearly complete elimination of CYS-F from the blood circulation occurred 12 hours after administration (Fig. 3d).

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Figure 3. Biodistribution of the probes. (a) CYS-F at 1mg/mL showed a stronger signal compared with other concentrations. (b) Representative NIRF images showed significantly higher signals in the lesion of the brain and the kidney. (c) Quantitative data of the target-to-background ratio (TBR) in different organs. (d) Blood retention study of CYS-F in healthy mice.

Non-invasive monitoring of early apoptosis in acute ischemia stroke. The in vivo specificity of CYS-F was further investigated in the photothrombotic cortex stroke model. Compared with the control probe (n=4), CYS-F (n=4) presented brighter signals both in vivo and ex vivo at 2 hours after administration (Fig. 4a). To 14


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dynamically evaluate early apoptosis in stroke, whole-body fluorescence was longitudinally monitored using an in vivo imaging system spectrum (IVIS) imager. CYS-F was injected intravenous (i.v.) immediately after the mice were subjected to the photothrombotic MCA stroke model (n=6). Subsequently, the NIRF images were collected at 0, 1, 3, 6, 8, 12, 24, 72 hours and on Day 7 after CYS-F administration. As shown in Figure 4b, CYS-F showed significantly bright signals in the lesion in vivo, with the maximum fluorescence at six hours. Similarly, the TBR also indicated that early apoptosis peaked around six hours after stroke (Fig. 4c). Notably, the TBR on 0 hour was -0.15±0.15, which suggested there were fewer probes in the ischemic lesion than those in the tissue around the lesion. The lower TBR in the ipsilateral hemisphere at early stage was related to the decreased blood flow. The results revealed that CYS-F could also be used to reflect the tissue blood perfusion. Moreover, the NIRF images were also compared with each slice on MRI to investigate the distribution of the probes in the photothrombotic MCA stroke model. A favorable match was obtained between the images on NIRF and MRI (Fig. 4d). According to the lesions presented on MRI, the areas with brighter fluorescence were peri-infarct areas (red squares) and on the edge of (red circles) the core infarct lesions, which represented the ischemic penumbra. This finding demonstrated that early apoptosis is relevant to the penumbra.

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Figure 4. Non-invasive imaging of early apoptosis in acute ischemia stroke. (a) Representative color-coded NIRF images of control probe and CYS-F in photothrombotic cortex stroke mice are shown. (b) Longitudinal near-infrared fluorescence imaging with CYS-F in stroke mice. (c) The TBR peaked around 6 hours after stroke. (d) Representative images of MR and NIRF in photothrombotic cortex stroke mice were presented with a favorable match.

The brain was excised for immunofluorescence staining after multimodal imaging. The small-molecule probe CYS-F co-localized with the annexin V-positive cells (Fig. 16


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5a). And compared with annexin V, CYS-F accumulated in the cytoplasm of apoptotic cells (arrows), which was in good agreement with the in vitro data. Different areas in the in vivo imaging were chosen for MAP-2 staining in Figure 5b. The red square represented the areas on the edge of the core infarct lesions, lots of CYS-F-positive cells (green) are around the infarct lesions (red represented the normal tissue) (Figure 5c). The red circle represented the peri-infarct areas, a few CYS-F positive cells were also detected in this area (Figure 5d and 5e). The peri-infarct areas and the areas on the edge of lesions were regarded as the penumbra. These findings further verified the in vivo results. The red pentacle represented the area in the contralateral hemisphere, while no CYS-F positive cells could be observed (Figure 5f). Additionally, NeuN staining also indicated some apoptotic cells were neurons, which was in agreement with the MAP-2 staining (Figure S2).

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Figure 5. Microphotographs of brain sections stained with annexin V and MAP-2. (a) Immunofluorescence annexin V staining confirmed that CYS-F co-localized with annexin V. Upper scale bar, 100 µm; lower scale bar, 50µm. (b) Different areas in the in vivo imaging were chosen for MAP-2 staining. (c) It shows that lots of CYS-F-positive cells (green) are around the infarct lesions (red represented the normal tissue). Scale bar, 200 µm. (d) It shows the peri-infarct area with a few CYS-F positive cells. Scale bar, 100µm. (e) The amplified images were presented. Scale bar = 50µm. (f) It represents the area in the contralateral hemisphere. Scale bar, 50µm.

Imaging-guided anti-apoptosis therapy. Thus CYS-F-positive cells would be a target for future therapies. As shown in our previous study5, cystamine decreased the number of terminal-deoxynucleotidyl-transferase mediated dUTP nick end labeling (TUNEL)-positive cells in the peri-infarct zone. It is convincing that benefits would be achieved with image-guided anti-apoptosis treatment. The capability of CYS-F to guide anti-apoptosis therapy was demonstrated in the photothrombotic MCA stroke model. Cystamine (100 mg/kg or 50 mg/kg) was used as an anti-apoptosis drug and was injected intraperitoneally (i.p.) after stroke. CYS-F was administered i.v. immediately after stroke, and brain fluorescence was imaged at 6 hours after administration (Figure 6a). Treatment with 50 mg/kg cystamine caused a significant decrease in the TBR (Figure 6b), but the decrease was slighter compared with that of the group treated with 100 mg/kg cystamine (n = 6, P < 0.001). Similarly, the MR images presented that the relative lesion volume ratios of final lesions on 24 hours 19



decreased with cystamine treatment (Figure6c and 6d). The multimodal molecular imaging substantiated the dose dependence of the therapeutic effect. A strong linear correlation (R2 = 0.875, P < 0.001) was shown between the TBR at 6 hours and the final lesion volume ratio on the MR images at 24 hours (Figure 6e). These findings demonstrated that CYS-F helps monitor the responses to anti-apoptosis therapy and predict the outcome of ischemic stroke at an early stage.

Figure 6. Imaging analysis of cystamine anti-apoptosis therapy in ischemic stroke mice. (a) Representative NIR images of the mice treated with cystamine or saline at 6 hours after stroke. (b) The TBR in the mice treated with saline was significantly higher than those treated with cystamine. (c) Representative MR images of the mice treated with cystamine or saline at 24 hours after stroke. (d) In agree with NIR 20


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findings, cystamine therapy caused a remarkable decrease of the relative lesion volume ratio in stroke mice (n=6). (e) A linear correlation was identified between the TBR on the NIR images at 6 hours after stroke and the relative lesion volume ratio on the MR images at 24 hours after stroke (R2 = 0.875, P < 0.001). ** P < 0.001.

Discussions It has been reported that apoptosis is correlated with the outcomes of stroke patients7. Apoptosis is a major mechanism for cell death in the penumbra4. Anti-apoptosis therapy is promising treatment method for stroke 8. Apoptosis progression contains three phases, and only cells in the early phase can be rescued. Therefore, a non-invasive, clinically practicable and cost-effective method for evaluating early apoptosis remains to be explored. In this study, a small-molecule probe CYS-F was employed to evaluate early apoptosis in ischemic stroke. In addition to detecting apoptosis, CYS-F also reflected the blood perfusion status and predicted the outcome at early stage of administration. It is well documented that molecular imaging of the apoptosis cells with annexin V, which could be achieved by NIRF imaging21, MRI22, single-photon emission computed tomography (SPECT)23 and PET24. However, several issues restrict their further clinical application. First, these probes are unable to differentiate between apoptosis and necrosis because PS also exposes on the disrupted plasma membrane during necrosis. Secondly, annexin V is a relatively large molecule protein with 36-kDa. The large molecule results in suboptimal pharmacokinetics and slow blood 21



clearance. Of note, most apoptosis probes are based on large-molecule proteins or peptides, such as the C2A domain25 and caspase substrates26-27. In an effort to break these limitations, a novel, small-molecule and early-apoptosis probe was developed in this study. In addition to the annexin-V and C2A domain, another group of proteins can also target the anionic membrane, namely, the Gla-domain proteins. They include the clotting factors, anticoagulant proteins28, and anti-apoptotic protein gas613. In fact, the alkyl-malonate group of these proteins mediates binding to the anionic phospholipid surfaces. ApoSense is a family of small-molecule apoptosis probes based on this structure. The family contains some fluorescent probes, such as DDC14 and NST-73215, and nuclear probe ML-1016. Those fluorescent probes were introduced in various diseases, such as traumatic brain injuries (TBI), stroke and tumor. However, the probes in these studies could only be detected ex vivo. The lack of in vivo images makes it difficult to perform further clinical study. The nuclear probe is not widely adopted because of its high cost, short available time and risk of exposure to radioactivity. As shown in the present study, CYS-F has several small-molecule features, such as more extensive biodistribution and more rapid blood clearance rates. As managing the time is really important in acute ischemic disease29, these features make CYS-F a reasonable apoptotic detector in acute ischemic stroke. Apoptosis induction is associated with a significant increase of CYS-F in cytoplasm. In vitro and in vivo CYS-F uptake occurs in parallel to the apoptotic hallmark of annexin-V binding. The 22


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true mechanism by which CYS-F targets early apoptosis remains unknown. The uptake of CYS-F occurs in the early phase of apoptosis when the membrane integrity is preserved. With membrane disruption, the CYS-F is released into the extracellular environment. As membrane disruption is also a hallmark of necrosis, CYS-F could distinguish between apoptosis and necrosis, which is verified by the lack of 7-ADD or PI in the CYS-F positive cells. The malonate motif is also suggested to play a key functional role. The membrane potential and cellular pH contributed to membrane target and intracellular accumulation16. Briefly, the acidification of the membrane in apoptotic cells facilitates the proton capture by the two carboxyl groups. As a result, hydrophobicity is increased and charge is dispersed, which favor the penetration into the membrane. The depolarization of apoptotic cells promotes the trans-membrane passage. And the cytosolic acidification and the membrane integrity sequestrated CYS-F in the cytoplasm. As viable cells highly regulate the potential and cellular pH, the CYS-F uptake by the viable cells is only transient. As demonstrated in the present study, an ideal performance of CYS-F in detecting apoptosis was manifested with different apoptotic inducers, i.e., treatment with DOX or paclitaxel. With these pro-apoptotic triggers, CYS-F excluded apoptotic cells from viable and necrotic cells. Intracellular accumulation has another advantage in that it may enhance the signal/background ratios. These results indicated the universality of the probe as a detector of early apoptosis. Notably, using neurons to examine the specificity would be more reasonable than using HeLa cells in this study. The purpose of this experiment, however, was to verify the early-apoptosis targeting ability of CYS-F. 23



Tumor cells were used in various studies to investigate the specificity of apoptosis probes16-17, and the culture of HeLa cells is more convenient and economical than neurons. Although NIR imaging could detect the lesion with a high sensibility, the spatial resolution is poor. Multimodal molecular imaging would get rid of this limitation. The penetration depth of fluorescence dyes was controversial which also limited its applications beyond animal studies, however, intra-arterial catheter imaging technology would make optical imaging applicable in stroke patients30.

Conclusions This study suggested that the small-molecule probe can be used to specifically evaluate early-apoptosis in murine ischemic stroke models. Furthermore, CYS-F also reflected the blood perfusion after administration and predicted the outcome of ischemic stroke at early stage. The versatility of this small-molecule probe facilitates clinical decision-making in apoptosis-related diseases.

Supporting Information Available: The following files are available free of charge. Figure S1. The specificity of CYS-F tested by annexin V (yellow) /7-ADD (red) kit. Figure S2. Representative images of NeuN staining.

Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the National Key Basic Research Program of China (973 Program; 2013CB733800, 2013CB733803), the National Natural Science Foundation 24


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Targeting early apoptosis in acute ischemic stroke with a small-molecule probe Cheng Qian, Dong-Fang Liu, Cong-Xiao Wang, Jie Ding, Yan-Li An, Pei-Cheng Li, Gao-Jun Teng*

Table of Contents graphic

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31



Graphical abstract 39x19mm (300 x 300 DPI)


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Figure 1. Synthesis and Characterization of the Probes. 83x98mm (300 x 300 DPI)



Figure 2. Cytotoxicity and Specificity of CYS-F. 177x378mm (300 x 300 DPI)


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Figure 3. Biodistribution for probes. 187x175mm (300 x 300 DPI)



Figure 4. Non-invasive imaging of early apoptosis in acute ischemia stroke. 198x189mm (300 x 300 DPI)


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Figure 5. Microphotographs of brain sections stained with annexin V and MAP-2. 242x455mm (300 x 300 DPI)



Figure 6. Imaging analysis of cystamine anti-apoptosis therapy in ischemic stroke mice. 182x152mm (300 x 300 DPI)


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Targeting Early Apoptosis in Acute Ischemic Stroke with a Small

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