Near Infrared Light‐Activatable Platelet‐Mimicking NIR‐II NO Nano‐Prodrug for Precise Atherosclerosis Theranostics

Abstract Atherosclerosis is a chronic inflammatory disease that affects arteries and is the main cause of cardiovascular disease. Atherosclerotic plaque formation is usually asymptomatic and does not manifest until the occurrence of clinical events. Therefore, early diagnosis and treatment of atherosclerotic plaques is particularly important. Here, a series of NIR‐II fluorescent dyes (RBT‐NH) are developed for three photoresponsive NO prodrugs (RBT‐NO), which can be controllably triggered by 808 nm laser to release NO and turn on the NIR‐II emission in the clinical medicine “therapeutic window”. Notably, RBT3‐NO is selected for its exhibited high NO releasing efficiency and superior fluorescence signal enhancement. Subsequently, a platelet‐mimicking nano‐prodrug system (RBT3‐NO‐PEG@PM) is constructed by DSPE‐mPEG5k and platelet membrane (PM) for effectively targeted diagnosis and therapy of atherosclerosis in mice. The results indicate that this platelet‐mimicking NO nano‐prodrug system can reduce the accumulation of lipids at the sites of atherosclerotic plaques, improve the inflammatory response at the lesion sites, and promote endothelial cell migration, thereby slowing the progression of plaques.


Materials and Instrumentals
The chemical reagents used in the synthesis of these compounds were purchased from China Reagent Network.The 1 H-NMR and 13 C-NMR data were obtained either through Bruker 300 MHz or Bruker 400 MHz.Mass spectroscopy data was collected on Waters Q-TOF MicroTM.High performance liquid chromatography (HPLC) data were obtained via Thermo Scientific dionex ultimate 3000.UV-Vis and fluorescence spectra were recorded on a Shimadzu UV-Vis spectrophotometer, UV-3600 Plus and Edinburgh Instruments Spectrofluorometer, FLS-1000, respectively.Fluorescence imaging of cells were carried out by a confocal laser canning microscopy (CLSM, LSM800, Zeiss, Germany).Fluorescence imaging of mice was performed on an small animal optical in vivo imaging system (Dali-IGS 600, China).

Phototriggered and Control Release Assay
Dissolve RBT-NO (10 μM) and RBT3-NO-PEG@PM (10 μM) in dichloromethane and aqueous solution respectively.Then, exposed to NIR laser (808 nm, 0.5 W cm -2 ), record UV absorption spectrum and fluorescence emission spectrum respectively (λex = 808 nm).The same procedure shall be applied to the controlled release measurement, and the NO donor shall be conducted alternately between the laser on and the laser off environment for 10 minutes.

In vitro NO Detection Assay
RBT-NO (10 μM) were dissolved in 3 mL of analytically pure methanol and purified water (v/v, 1:1), RhBs (10 μM) were added to the quartz cuvette in liquid form that is pre-dissolved in DMSO.Then, the two compounds were exposed to light irritation (808 nm, 0.5 W cm -2 ) for 70 min while the fluorescent turn-on was recorded with fluorescent spectra (λex = 545 nm).

Cell Culture
Cells were cultured in DMEM with 10% FBS.Generally, cells were maintained under a humidified atmosphere containing 5% CO2 at 37 ℃.Experiments were performed either in 96-well plates and confocal dish.

Cytotoxicity Assay
RAW and HUVECs cells were seeded in 96-well plates for 24 h at 37 ℃.Further, the media was removed from the plate and added fresh media 190 μL.Meanwhile, RBT3-NO-PEG@PM diluted by media and added to per well 10 μL with final concentration of 25 μg mL -1 , 20 μg mL -1 , 15 μg mL -1 , 10 μg mL -1 and 5 μg mL -1 .96-well plate was incubated for 24 h and 48 h carefully wrapped with silver paper.A solution of MTT was added to each well 10 μL before incubated for another 4 h. the media was removed from the 96-well and replaced with 100 μL DMSO.Cytotoxicity and proliferation were acquired by comparing the absorbance of each well with the absorbance of the control wells at 490 nm.The cytotoxic experiment of RBT3-NO-PEG@PM was completed as described above.In the same way, the MTT assay of RBT3-NO and RBT3 co-cultured for 24 hours in RAW cells and HUVECs cells was performed.

Intracellular NO Release Assay
RAW and HUVECs cells (5x10 4 mL -1 ) were inoculated in confocal culture dishes for 24 h.The media in the dish were replaced with fresh one, meanwhile, RBT3-NO-PEG@PM were added in the dish with the final concentration of 15 μg mL -1 and incubated in the dark for 3 h.The NO indicator DAF-FM-DA was dissolved in DMSO with the final concentration of 20 μM.Then, the two were co-incubated in the same dish for another 0.5 h in the dark.After that, the dish was rinsed with PBS for three times before added 100 μL polyoxymethylene for cells fixation.Then, the cells were ready for the irritation of NIR laser light (808 nm, 0.5 W cm -2 ) and the detection of intracellular NO release, which was acquired with confocal laser canning microscopy (CLSM, LSM800, Zeiss, Germany) equipped with a 63×oil objective lens.
The green fluorescence (FITC channel) was excited with 492 nm.For the image acquisition and statistical analysis, the Zen 2008 software was used.
RAW and HUVECs cells (5x10 4 mL -1 ) were inoculated in confocal culture dishes for 24 h.The media in the dish were replaced with fresh one, meanwhile, RBT3-NO-PEG@PM were added in the dish with the final concentration of 15 μg mL -1 and incubated in the dark for 3 h.After that, the cells were washed three times with PBS.The NO indicator DAF-FM-DA was dissolved in DMSO with the final concentration of 20 μM.Then, the two were co-incubated in the same dish for another 0.5 h in the dark and the cells were ready for the irritation of NIR laser light (808 nm, 0.5 W cm -2 ).After that, the dish was rinsed with PBS for three times before added pancreatin.Finally, the collected cells were washed and centrifuged with cold PBS buffer, and cells were resuspended with 500 μL of cold PBS buffer and signals were collected using flow cytometry.

Animal Model
ApoE knockout homozygous mice (ApoE -/-) were purchased from Qinglongshan, and at the age of 5-6 weeks the atherosclerosis model was establish by ligation of the right carotid artery combined with high-fat diet.After feeding a high-fat diet for one month, the mice were injected intravenously with RBT3-NO-PEG@PM (5 mg kg -1 ), and fluorescence imaging was performed at different points of laser irradiation (808 nm, 0.5 W cm -2 ) for 0 min, 10 min, and 30 min.All the animal experiments were approved by the ethics committee of China Pharmaceutical University and conducted with the "guide for the care and use of laboratory animals" of the institute of laboratory animal resources.

Pharmacokinetic Experiments
ApoE knockout homozygous mice (ApoE -/-) were used to induce atherosclerosis by ligation of the right carotid artery combined with high-fat diet.After feeding a high-fat diet for one month, the mice were divided into two groups, with 3 mice in each group, and injected RBT3-NO (0.8 mg kg -1 ) and RBT3-NO-PEG@PM (5 mg kg -1 , calculated as RBT3-NO-PEG) into the tail vein respectively.30 μL blood samples were taken from the tail of mice at different time points, stored in a centrifuge tube coated with heparin sodium solution, and 0.3% triton X-100 was added for ultrasound for 3 minutes.Then add 300 μL DMSO solution and centrifuge at 10000 rpm for 15 minutes.Finally, the supernatant was taken and its drug concentration was determined by HPLC.

Chemical synthesis of RBT-NH and RBT-NO
Scheme S1.Synthetic route for RBT-NH and RBT-NO.
[a] Photophysical properties in dichloromethane.[b] For determination of the fluorescence quantum effificiency, IR26 in dichloroethane (Ф = 0.05%) was used as a fluorescence standard.

Figure S1 .
Figure S1.Working curve of Griess NO quantification methods.

Figure S10 .
Figure S10.Quantitative analysis of fluorescence intensity at different times.

Figure S11 .
Figure S11.(a) MTT assay of RBT3-NO-PEG@PM co-cultured for 24 hours in RAW cells and HUVECs cells.(b) MTT assay of RBT3-NO-PEG@PM co-cultured for 48 hours in RAW cells and HUVECs cells.(c) MTT assay of RBT3-NO co-cultured for 24 hours in RAW cells and HUVECs cells.(d) MTT assay of RBT3 co-cultured for 24 hours in RAW cells and HUVECs cells.

Figure S14 .
Figure S14.Protein content analysis of PM and RBT3-NO-PEG@PM using

Figure S15 .
Figure S15.Quantitative analysis of fluorescence intensity at different times.

Figure S16 .
Figure S16.(a) NIR-II Fluorescence image of RBT3-NO-PEG@PM and RBT3-NO-PEG arriving at the plaque with blood circulation after injection through the tail vein of mice.After 5 minutes, the plaque was irradiated with an 808 nm laser at a fluence rate of 0.5 W cm −2 .(b) Quantitative analysis of fluorescence intensity at different times.

Figure S17 .
Figure S17.(a) NIR-II Fluorescence image of RBT3-NO-PEG@PM arriving at the plaque with blood circulation after injection through the tail vein of mice.After 5 minutes, the plaque was irradiated with an 808 nm laser at a fluence rate of 0.5 W cm −2 .(b) Quantitative analysis of fluorescence intensity at different times.

Figure S18 .
Figure S18.Pharmacokinetic curve and half-life of RBT3-NO and

Figure S20 .
Figure S20.Whole blood cell count analysis in treatment groups with different doses of medication (RBC: red blood cell; WBC: white blood cell; PLT: platelet; HGB: Hemoglobin).

Figure
Figure S26. 1 H NMR spectrum of compound RBT-Br in CDCl3.