Targeting The Early Apoptosis In Acute Ischemic Stroke By A Small-molecule Probe

Part I Construction and characterization of small-molecule probeObjective To establish a set of early apoptosis targeting small-molecule probe CYS-F synthesis and preparation methods,and to verify its cytotoxicity and specificity in vitro,then lay the foundation for further in vivo research.Materials and Methods An one-step method was used to react cystine with fluorescein isothiocyanate(FITC).The product was purified by high performance liquid chromatography(HPLC)and the molecular weight was determined by liquid chromatography-mass spectrometer(LC-MS).1 x 107 Hela Cells were incubated with six different concentrations of the small-molecule CYS-F probe(0 ug/mL,25 ug/mL,250 ug/mL,625 ug/mL,1250 ug/mL,2500 ug/mL)at 37? for 24 hours in the dark,and 6 wells were repeated for each probe concentration.Finally,the residual probe was washed off with phosphate-buffered saline(PBS).The cell activity assay kit CCK-8(Cell Counting Kit-8)was used for detection and analysis the cytotoxicity.And the targeting of small molecular probe was studied by different apoptosis inducing methods.The Hela cells was induced apoptosis by doxorubicin,and PBS was used as the control inducing drug.After two hours,the two groups were incubated with an appropriate concentration of CYS-F and control probe for 15 minutes,and washed three times with PBS.Then each groups were compared to study the targeting of CYS-F.In order to verify the universality of the probe,the Hela cells was also treated with paclitaxel.After 12 hours of drug intervention,the cellswere washed three times with PBS to remove the residual drugs,and appropriate concentration of the probe CYS-F was added to the medium and incubated for 15 minutes.Then the residual probe was removed by 3 times washing with PBS.Then cells were stained with annexin V/propidium iodide(PI),or 7-amino-actinomycin D(7-ADD)/annexin V.Annexin V was a marker of apoptosis,While PI and 7-ADD were markers of necrosis.The cells were observed under fluorescence microscopy to analyze the early apoptotic targeting of the probe.Results The molecular weight of the small-molecule probe CYS-F was 1013 Dalton(Da).And the cytotoxicity test showed that the six different concentrations of the probe did not produce significant cytotoxicity to the cells(P>0.05).Under the fluorescence microscope,doxorubicin-induced apoptotic cells showed red,and the probe-positive cells showed green,the two kind of cells showed a good co-localization.Whereas there was no significant fluorescence signals in normal cells.The results showed that the small-molecule probe CYS-F could specifically target the apoptotic cells without being engulfed by normal cells.After the apoptosis-inducing cells or the normal cells were incubated with control probe,all cells showed green.It proved that the control probe has no specific targeting ability.When the apoptosis was induced by paclitaxel,the probe-positive cells were well co-located with annexin V-positive cells.However,the annexin V mainly targeted on the cell surface,while the small-molecule probe CYS-F was concentrated in the cytoplasm.In addition,the CYS-F positive cells were not co-located with the PI-positive cells or the 7-ADD positive cells.The targeting ability of the small-molecule probe was verified again,and it also showed the targeting of the probe was universal.Conclusions The early apoptosis targeting small-molecule probe CYS-F was successfully synthesized by one-step method,and the probe was showed no significant cytotoxicity.Furthermore,with different apoptosis inducing methods,the small-molecule probe CYS-F showed excellent early apoptosis targeting.Compared with annexin V,the early apoptosis marker,the small molecular probe could concentrate in the cytoplasm,which would improve the imaging signal to noise ratio.This in vitro study provided an experimental basis for further in vivo imaging.Part ? Multimodal imaging of early apoptosis in acute ischemic strokeObjective To investigate the biodistribution of the small-molecule probe CYS-F and to study the evolution of early apoptosis in vivo in acute ischemia stroke by using 7.0T magnetic resonance imaging and near-infrared optical imaging in vivo based on the small-molecule probe CYS-F.Materials and Methods A total of 6 healthy male C57BL/6J mice were selected for the biodistribution study.CYS-F probe was administered to mice via the tail vein.O.1mL of blood samples were collected at each scheduled time points(0,0.5,1,2,3,6,12 hours)by retro-orbital puncture.Serum was obtained by centrifugation of the extracted blood samples.The small-molecule probe concentration in the plasma was measured.After the last time of blood collection,the mice were sacrificed by 1%pentobarbital sodium chloride solution.Then heart,spleen,lung,kidney and brain were examined ex vivo by near-infrared scanning to study the biodistribution of the CYS-F probe.The acute ischemic stroke mouse model was established by photothrombotic method.Once the mouse was subjected to stroke model,the CYS-F and the control probe were injected through the tail vein.The near-infrared imaging was performed at 0,1,3,6,8,12 and 24 hours after stroke to study the time-intensity curve of the fluorescence signal at the lesion.The final lesion volumes were evaluated by the magnetic resonance imaging at 24 hours.After imaging,the mice were sacrificed and the brains were obtained for immunofluorescence staining.Annexin V immunofluorescence staining was used to verify the early apoptosis targeting ability of the small-molecule probe.Meanwhile,the MAP-2 and NeuN immunofluorescence staining was also performed to determine the type of apoptotic cells.Results The small-molecule probe CYS-F was cleared rapidly from the circulation with a blood half-life of 1.3 h.And a high kidney uptake with little accumulation in other organs indicated predominantly renal clearance of CYS-F.Nearly complete elimination of CYS-F from the body had occurred by 12 hours after administration.In the acute ischemic stroke model,compared with the control probe,the CYS-F showed significantly brighter signals both in vivo and ex vivo at 2h after injection either probes.The in vivo near-infrared images indicated that the target-to-background ratio reached its peak on 6 hours after ischemia stroke.It reflected the early apoptosis reached the peak at six hours after acute ischemic stroke.What's more,the final near-infrared fluorescence images matched well with the magnetic resonance images.Interestingly,the fluorescence signal at the lesion was weaker than that at the surrounding tissues once after injection.Immunofluorescence staining showed that CYS-F-positive cells were co-located with annexin V-positive cells,and some of the probe-positive cells were co-located with MAP-2-positive and NeuN-positive cells.Conclusions The small molecule probe CYS-F rapidly distributed in the blood and was excreted through the kidneys.Furthermore,it reflected the evolution of early apoptosis after acute stroke.More importantly,CYS-F may reflect the blood perfusion in tissues at early time.Part III Multimodal molecular imaging to guide anti-apoptotic therapy in acute ischemic strokeObjective Based on the results from the multimodal molecular imaging,the anti-apoptotic effect of cystamine on acute ischemic stroke was evaluated by magnetic resonance imaging and near-infrared imaging in vivo.Materials and Methods A total of 18 healthy male C57BL/6J mice were randomly divided into three groups:control group,high-dose treatment group and low-dose treatment group.The acute ischemic stroke model of was established by photothrombotic the middle cerebral artery.Once the mouse was subjected to the stroke model,the mice in the three groups were treated with 0.9%normal saline(control group),50 mg cystamine(low dose treatment group)or 100 mg cystamine(high dose treatment group).At the same time,the CYS-F was injected via the tail vein.The near-infrared imaging was performed at 6 hours after injection.Magnetic resonance imaging was performed at 24 hours after the occlusion.The intensity of the fluorescence signal and the volume of the lesion were measured.Then the two imaging results were matched to analyze the correlation between them.Results 50mg/kg cystamine treatment caused a moderate decrease in the target-to-background ratio on the fluorescence images,but the decrease was weaker compared with the mice treated with 100mg/kg cystamine(n=6,P<0.001).In agreement with the fluorescence findings,the final lesion volumes measured by MRI was reduced in the cystamine-treated groups.And the therapeutic effect was dose-dependent.A linear correlation was detected between the TBR on the fluorescence images at 6 hours and the final lesion volume ratio on the MR images at 24 hours(R2=0.875,P<0.001).Conclusions Multimodal imaging based on the small-molecule early apoptosis probe CYS-F could guide and monitor cystamine therapy in acute ischemic stroke.Furthermore,the small-molecule probe CYS-F could predict the outcome at the early stage.