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

Figures

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  Scheme 1. a) Comparison of emission wavelengths of several light triggered NO donors. b) Schematic diagram of nano‐prodrug RBT3‐NO‐PEG@PM for in vivo phototriggering treatment of atherosclerosis and allows simultaneous NIR‐II real‐time drug delivery monitoring.

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  Scheme 2. Synthetic route of RBT‐NH and RBT‐NO.

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  Figure 1. Spectral characteristics of RBT‐NH and RBT‐NO. a) Comparison of ultraviolet absorption spectra of RBT‐NH (10 µm) in dichloromethane. b) Comparison of fluorescence emission spectra of RBT‐NH (10 µm) in dichloromethane, λex = 808 nm. c) Photostability comparison of all fluorophores (10 µm) in dichloromethane under continuous‐wave laser exposure (808 nm) at a fluence rate of 1.4 W cm−2, λex = 808 nm. d–f) Ultraviolet absorption/fluorescence emission spectra of RBT‐NH and RBT‐NO (10 µm) in dichloromethane, λex = 808 nm.

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  Figure 2. Photoresponsive release properties of prodrugs RBT‐NO. a–c) Ultraviolet absorption spectrum changes of RBT‐NO (10 µm, a) RBT1‐NO, b) RBT2‐NO, c) RBT3‐NO) in dichloromethane irradiated by 808 nm laser at a fluence rate of 0.5 W cm−2, time = 0, 10, 20, 30, 40, 50, 60, 70, 80 min. d–f) Fluorescence emission spectrum changes of RBT‐NO (10 µm, d) RBT1‐NO, e) RBT2‐NO, f) RBT3‐NO) in dichloromethane irradiated by 808 nm laser at a fluence rate of 0.5 W cm−2, time = 0, 10, 20, 30, 40, 50, 60, 70, 80 min, λex = 808 nm. g) Comparison of fluorescence emission spectrum changes of RBT‐NO (10 µM) in dichloromethane, λex = 808 nm. h) Comparison of NO yield of RBT‐NO (50 µm). i) Controllability of NO release from RBT‐NO (10 µm) by switching 808 nm laser “on” (white) and “off” (grey).

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  Figure 3. a) Photolysis splitting mechanism of RBT3‐NO. b) Energy calculation of RBT3‐NO and RBT3. c) Fluorescence emission spectrum changes of RBT3‐NO (10 µm) with RhBs (10 µm) in methanol/water (v/v, 1:1) irradiated by 808 nm laser at a fluence rate of 0.5 W cm−2. d) EPR spectra of spin‐trapped NO released from RBT3‐NO.

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  Figure 4. a) Preparation of RBT3‐NO‐PEG@PM. b) TEM image, DLS and Zeta potential of RBT3‐NO‐PEG. c) TEM image, DLS and Zeta potential of RBT3‐NO‐PEG@PM. d) Ultraviolet absorption spectrum changes of RBT3‐NO‐PEG@PM (10 µm, calculated as RBT3‐NO) in aqueous solution irradiated by 808 nm laser at a fluence rate of 0.5 W cm−2, time = 0, 10, 20, 30, 40, 50, 60, 70, 80 min. e) Fluorescence emission spectrum changes of RBT3‐NO‐PEG@PM (10 µm) in aqueous solution irradiated by 808 nm laser at a fluence rate of 0.5 W cm−2, time = 0, 10, 20, 30, 40, 50, 60, 70, 80 min, λex = 808 nm. f) Controllability of NO release from RBT3‐NO‐PEG@PM (10 µm) in aqueous solution by switching 808 nm laser “on” (white) and “off” (grey). g) NIR‐II fluorescence imaging of RBT3‐NO‐PEG@PM (10 µm) in aqueous solution irradiated by 808 nm laser at a fluence rate of 0.5 W cm−2, time = 0, 10, 20, 30, 40, 50, 60, 70, 80 min. Excitation wavelength: 808 nm. Emitted signals were collected through passing 1000 nm LP filter.

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  Figure 5. a) Fluorescence imaging and flow cytometry analysis b) of proving gaseous NO release in RAW cells co‐incubated with RBT3‐NO‐PEG@PM (15 µg mL−1, calculated as RBT3‐NO‐PEG) and NO indicator DAF‐FM‐DA (20 µm) with laser exposure (808 nm, 0.5 W cm−2), scale bar: 50 µm. c) Fluorescence imaging and flow cytometry analysis d) of proving gaseous NO release in HUVECs cells co‐incubated with RBT3‐NO‐PEG@PM (15 µg mL−1) and NO indicator DAF‐FM‐DA (20 µm) with laser exposure (808 nm, 0.5 W cm−2), scale bar: 50 µm. e) The influence of NO on lipid storage in foam cells determined with Oil Red O staining, scale bar: 50 µm. f) The ratio of Oil Red O stained cells in total 1 × 107 cells. The results are showed as mean ± SD (n = 3). Statistical significance was assessed applying one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. a) Diagrammatic sketch of carotid ligation surgery model. The right carotid artery (RCA) was ligated, and the left carotid artery (LCA) was regarded as the normal group. b) NIR‐II fluorescence imaging of artery sections after NO therapy. 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 min, the plaque was irradiated with an 808 nm laser at a fluence rate of 0.5 W cm−2. The injection concentration of RBT3‐NO‐PEG@PM is 5 mg kg−1 (Calculated as RBT3‐NO‐PEG). c) H&E, Oil Red O staining and CD68 imaging of artery sections after NO therapy (scale bar: 100 µm). The results are presented as mean ± SD (n = 6). Statistical significance was assessed applying one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 7. Immunofluorescence imaging of TNF‐α and TGF‐β and the imaging of MCP‐1 and IL‐8 in vascular slices after NO therapy (scale bar: 100 µm). The injection concentration of RBT3‐NO‐PEG@PM is 5 mg kg−1 (Calculated as RBT3‐NO‐PEG). The results are presented as mean ± SD (n = 6). Statistical significance was assessed applying one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 8. a) Treatment schedule of atherosclerotic plaques in mice. RBT3‐NO‐PEG@PM with a concentration of 5 mg kg−1 (Calculated as RBT3‐NO‐PEG) was injected every other day from day 0, and laser irradiation was performed half an hour after injection. b) H&E staining of sections from various organs after different therapies (scale bar: 50 µm). c) Photos of heart, liver, spleen, lung, kidney, intestine, and bladder after different treatments. d) Mice body weights after different treatments during 14 d (mean ± SD, n = 6).