Small-Molecule Fluorophores for Near-Infrared IIb Imaging and Image-Guided Therapy of Vascular Diseases

Open AccessCCS ChemistryRESEARCH ARTICLE7 Dec 2022Small-Molecule Fluorophores for Near-Infrared IIb Imaging and Image-Guided Therapy of Vascular Diseases Yang Li†, Hua Zhu†, Xiaobo Wang†, Yan Cui, Lijuan Gu, Xiaowen Hou, Mengting Guan, Junzhu Wu, Yuling Xiao, Xiaoxing Xiong, Xianli Meng and Xuechuan Hong Yang Li† Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 College of Science, Research Center for Ecology, Laboratory of Extreme Environmental Biological Resources and Adaptive Evolution, Innovation Center for Traditional Tibetan Medicine Modernization and Quality Control, Medical College, Tibet University, Lhasa 850000 Center for Experimental Basic Medical Education, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of Developmentally Originated Disease, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan 430071 †Y. Li, H. Zhu, and X. Wang contributed equally to this work.Google Scholar More articles by this author , Hua Zhu† Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 †Y. Li, H. Zhu, and X. Wang contributed equally to this work.Google Scholar More articles by this author , Xiaobo Wang† State Key Laboratory of Southwestern Chinese Medicine Resources, Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137 †Y. Li, H. Zhu, and X. Wang contributed equally to this work.Google Scholar More articles by this author , Yan Cui Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 College of Science, Research Center for Ecology, Laboratory of Extreme Environmental Biological Resources and Adaptive Evolution, Innovation Center for Traditional Tibetan Medicine Modernization and Quality Control, Medical College, Tibet University, Lhasa 850000 Google Scholar More articles by this author , Lijuan Gu Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 Google Scholar More articles by this author , Xiaowen Hou Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 Center for Experimental Basic Medical Education, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of Developmentally Originated Disease, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan 430071 Google Scholar More articles by this author , Mengting Guan Center for Experimental Basic Medical Education, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of Developmentally Originated Disease, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan 430071 Google Scholar More articles by this author , Junzhu Wu Center for Experimental Basic Medical Education, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of Developmentally Originated Disease, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan 430071 Google Scholar More articles by this author , Yuling Xiao Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 Center for Experimental Basic Medical Education, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of Developmentally Originated Disease, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan 430071 Shenzhen Institute of Wuhan University, Shenzhen 518057 Google Scholar More articles by this author , Xiaoxing Xiong *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 Google Scholar More articles by this author , Xianli Meng *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Southwestern Chinese Medicine Resources, Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137 Google Scholar More articles by this author and Xuechuan Hong *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Department of Neurosurgery, Renmin Hospital of Wuhan University, State Key Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430060 College of Science, Research Center for Ecology, Laboratory of Extreme Environmental Biological Resources and Adaptive Evolution, Innovation Center for Traditional Tibetan Medicine Modernization and Quality Control, Medical College, Tibet University, Lhasa 850000 Center for Experimental Basic Medical Education, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of Developmentally Originated Disease, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan 430071 Shenzhen Institute of Wuhan University, Shenzhen 518057 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202101547 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Accurate and dynamic visualization of vascular diseases can contribute to restraining further deterioration from diseases in a timely manner. However, visualization is still unable to precisely determine whether and to what extent blood vessels or brain tissues are damaged. Here, we report novel benzo-bis(1,2,5-thiadiazole)-based second near-infrared region (NIR-II) fluorophores HY1–HY4 with highly twisted structures (55° at the S0 state), extremely strong aggregation-induced emission (AIE) characteristics (I/I0 > 13), and remarkably high fluorescence quantum yields (QYs) (up to 14.45%) in the NIR-II region (>1000 nm) and ∼0.27% in the near-infrared IIb window (NIR-IIb, >1500 nm) in aqueous solution. Using NIR-IIb AIE HY4 dots, high-resolution NIR-IIb fluorescence imaging of revascularization and thrombolysis, and real-time feedback of the therapeutic efficacy of Chinese medicine Dengzhan Xixin injection (DXI) on ischemic stroke, were achieved for the first time. In addition, results showed that DXI conferred neuroprotection against cerebral ischemia injury mediated via the angiogenesis pathway. These attractive results provide a new perspective for designing ultrabright NIR-IIb probes for vascular-related phenomena, disease assessment, and precise intraoperative image-guided therapy with a deeper tissue penetration depth and higher resolution. Download figure Download PowerPoint Introduction Vascular diseases, such as cerebrovascular accident, thrombosis, atherosclerosis, peripheral artery disease (PAD), coronary artery disease, and transplantation arteriopathy, cause tremendous loss of lives in developed countries worldwide.1 A previous report showed that ∼17.9 million people died from cardiovascular diseases related to stroke and heart attacks in 2019 alone.2 Ischemic stroke, a primary consequence of carotid and cerebrovascular disease, which is caused by middle cerebral artery occlusion (MCAO), narrowing of the internal carotid artery or vertebral artery, and thrombosis, results in neuronal damage and manifests with neurological symptoms characterized by the loss of blood, oxygen, and nutrient supply to the brain.3 Substantial research advances have been made in the past decade toward early diagnosis and development of first-line imaging strategies to minimize the impact of ischemic stroke. Early diagnosis and effective treatment for patients within 4.5 h of stroke onset are critical steps for the management of this disease.4 Currently, several measures, such as surgical mechanical thrombectomy or thrombolytic therapy with intravenous tissue plasminogen activator for recirculation of cerebral blood flow, have been proven to effectively treat ischemic stroke.5,6 However, due to the limited time window of thrombolytic therapy, only a few patients are eligible for this treatment. Therefore, prevention and treatment of ischemic stroke remain great challenges worldwide. PAD is a slow, progressive disorder of the lower-extremity arterial vessels caused by chronic narrowing of the peripheral arteries.7 Previous studies have shown that loosely adherent thrombus may dislodge without warning, thereby lodging into blood vessels of the limb and causing occlusion or loss of functionality of the heart, brain, arms, legs, pelvis, and kidneys.7,8 PAD patients have an increased risk of developing coronary artery disease, heart attack, and stroke. PAD affects more than 200 million people worldwide, who manifest with different symptoms.7 Traditional Chinese medicine (TCM) has become an alternative pharmacotherapy for the prevention and rehabilitation of ischemic stroke and PAD in China because of its curative efficacy and few side effects.9–11 Results from numerous clinical and preclinical trials have shown that most herbs used in TCM injection, such as Dengzhan Xixin, Mailuoning, and Danhong, can alleviate angiogenesis in the ischemic penumbra of the brain.12-15 To date, however, there is still a lack of visual imaging evidence on specific brain functional areas of neovascularization. Accurate and dynamic visualization of ischemic sites can help prevent further deterioration of patients from vascular diseases in a timely manner. At present, several tools, including positron emission tomography, computerized tomography (CT) scan, magnetic resonance imaging (MRI), and duplex ultrasound, are available for detection of ischemic stroke or PAD.16 However, their application in most patients has been limited by the inevitable exposure to ionizing radiation. Previous studies have also shown that indocyanine green (ICG) can be injected into a blood vessel to allow visualization of the arteries and veins and highlight blood flow.17 Nevertheless, the above-mentioned medical imaging methods fail to precisely determine the occurrence and extent of damage to blood vessels or brain tissues.18–20 Therefore, it is essential to develop a novel imaging procedure that allows the observation of the dynamic flow of vascular diseases within a limited treatment time window, with deeper penetration depth, higher clarity, and more accurate anatomic features. Fluorescent imaging in the second near-infrared region (NIR-II, 1000–1700 nm) has shown great potential during dynamic assessment of blood parameters, vascular anomalies, angiography of tumor pathology, ischemic stroke, and PAD because the light in this wavelength window can achieve high resolution and deeper tissue penetration.21–24 The first intravital NIR-II and NIR-IIb (1500–1700 nm) vascular imaging was successfully reported using single-wall carbon nanotubes (SWCNTs) in 2009 and 2015.25,26 In vivo, NIR-IIb imaging shows negligible background signals and ultrahigh resolution.27–43 Until now, few small-molecule NIR-IIb imaging agents, such as FD-1080J-aggregates, HQL2, HL3, 2TT-oC610B, and TT3-Ocb, have been employed in NIR-IIb vascular imaging.44–48 A practical strategy for development of a new type of small-molecule NIR-IIb probe is to utilize aggregation-induced emission luminogens (AIEgens) with highly twisted skeletons to promote radiative decay and enhance fluorescence emission in the NIR-IIb region.49 Nevertheless, organic small-molecule NIR-IIb optical imaging for revascularization, thrombolysis, and real-time feedback of therapeutic strategies have yet to be reported. In this study, we have rationally designed four new NIR-II fluorophores HY1–HY4 employing a donor–acceptor–donor (D–A–D) scaffold with benzo-bis(1,2,5-thiadiazole) (BBTD) as the electron acceptor with a 3,4-bis(alkyloxy)thiophene ring and N,N-diphenylnaphthalen-2-amine (BPN) as the electron donors. The 3,4-bis(2-ethylhexyloxy) chain substituent at both sides of thiophene in HY4 not only acted as a strong donor unit, but also dramatically enhanced the dihedral angle up to 52° between thiophene and BBTD for S0 geometries, thereby boosting its AIE property (I/I0 > 13). In addition, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-3400) (DSPE-PEG3.4k) encapsulated HY4 dots emitted NIR light in a broad range of 900–1600 nm with a high quantum yield (QY) of 14.45% in aqueous solution, ∼18 times enhancement compared to HY4 in tetrahydrofuran (THF) solution. The QY of HY4 dots in the NIR-IIb window (>1500 nm) was 0.27%, and this was significantly higher than that of 2TT- o C26B (∼0.12%). Moreover, for the first time, we achieved intravital fluorescence imaging over 1500 nm of ischemic stroke, thrombosis, PAD, and tumor angiogenesis, and real-time feedback of the therapeutic efficacy of the Chinese medicine Dengzhan Xixin injection (DXI) on ischemic stroke. Furthermore, we elucidated the therapeutic mechanism underlying this TCM’s effect against ischemic stroke. To the best of our knowledge, this is the first report describing the application of a NIR-IIb fluorophore for imaging central and peripheral ischemic disease and NIR-IIb image-guided TCM therapy with higher resolution and deeper tissue penetration. Experimental Methods Preparation of HY1–HY4 dots HY1–HY4 dots were synthesized through a nanoprecipitation method utilizing DSPE-PEG3.4k as an encapsulation matrix. The solution of HY1–HY4 in THF (1 mL) was slowly added into the aqueous solution of DSPE-PEG3.4k (9 mL) under constant sonication. Subsequently, THF was eliminated under inert gas flow. Cell cultures Human umbilical vein endothelial cells (HUVEC) and mouse brain microvascular endothelial cells (bEnd.3) were cultured in Dulbecco’s Modified Eagle Medium (Gibco), supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin, and maintained in a humidified incubator at 37 °C with 5% CO2. Cellular toxicity assay Cellular toxicity of HY4 dots was evaluated via a standard CCK-8 assay (DOJINDO CK04). Briefly, HUVEC and bEnd.3 cells were seeded in 96-well plates, incubated for 12 h, then treated with various concentrations of HY4 dots (0, 0.5, 1, 2.5, 5, 10, 25, 50, 100, and 200 μg/mL) followed by a 12-h incubation. Next, CCK-8 solution (10 μL) was added to sample wells, and the absorbance (at 450 nm) of each well was determined after 1 h. Cell viability was determined by calculating the ratio of absorbance from HY4 dots treated wells to that from wells incubated with culture medium only. Intravital NIR-II and NIR-IIb imaging HY4 dots were injected intravenously into normal and ischemic mice models, and the mice were intraperitoneally administered isoflurane. Next, the HY4 dots-injected mice were placed on the living optical NIR-II imaging system, in various postures, and the whole-body NIR-II fluorescent as well as halogen light images were taken at various time points postinjection. The instrument parameters of the intravital optical NIR-II fluorescent imaging system were different filters with various exposure times under 808-nm laser. Establishment of peripheral ischemic murine models Ligation of the femoral artery and vein was performed as follows: mice were first anesthetized with isoflurane by facemask, then ischemia of the left femoral artery and vein induced by ligating the vessels with a 6-0 silk suture at the proximal location near the lateral circumflex femoral artery. To establish FeCl3 induced thrombosis models, the whole abdomen and legs of anesthetized mice were first sterilized with 75% alcohol. Next, blunt separation of subcutaneous muscles and tissues was performed, then 5 mm of the abdominal vein and femoral artery and vein were dissected and covered with a 1 × 2 mm piece of filter paper r that had been fully soaked in 10% FeCl3 for 3 min. To establish a peripheral arterial disease (PAD) model, mice were first anesthetized with isoflurane, the femoral artery was separated from the femoral vein and nerve at the proximal location near the groin, and then a 7-0 silk suture was inserted beneath the proximal femoral artery to occlude the proximal femoral artery. Next, the femoral artery was isolated from the femoral vein in a position close to the knee, and the vessel occluded at the distal femoral artery proximal to the popliteal artery. Finally, the segment of the femoral artery between the distal and proximal ends was transected. Measurement of the infarction volume and neurological evaluation The brains were gathered after transcranial perfusion by saline and subsequent 4% paraformaldehyde. They were collected, cut into 2 mm coronal sections, and stained with 2% 2,3,5-triphenyltetrazolium chloride solution (TTC) (17779, Sigma-Aldrich). The infarction size was calculated by a blinded observer using Image–Pro Plus software version 6.0 (Media Cybernetics, Inc.). Neurological deficit scores were evaluated 24 h after MCAO. Hematoxylin and eosin staining Whole brains, from three independent mice, were fixed with 4% paraformaldehyde at room temperature for 24 h, then subjected to hematoxylin and eosin (H&E), Nissl, and immunofluorescence staining. Paraffin-embedded brain tissues were sectioned into 5-μm slices using a cryotome (RM2016; Leica Microsystems GmbH, Wetzlar, Germany), and baked at 60 °C overnight. Next, the sections were sequentially dewaxed with xylene I and II for 20 min, then stained with H&E for 30 and 5 min at 25 °C, respectively. The sections were then dehydrated, sealed with neutral balsam, and observed under an optical microscope (NIKON ECLIPSE TI-SR, Japan) at ×400 magnification for typical histopathological changes in neurons. Nissl staining Sections (5 μm thick) were incubated with 0.1% toluidine blue for 10 min at 25 °C, washed with double-distilled water, then sequentially treated with 70%, 95%, and 100% ethanol. Next, the sections were rinsed with xylene and coated with neutral resin. Finally, neuronal viabilities were evaluated under an optical microscope (NIKON ECLIPSE TI-SR, Japan) at ×400 magnification. TUNEL staining The 5 μm sections were processed using the TUNEL apoptosis assay kit (No. 11684817910, Roche) based on the manufacturer’s instructions, and TUNEL-positive apoptotic cells were detected under a fluorescence imaging microscope (NIKON ECLIPSE TI-SR, Japan). The rate of apoptosis was calculated by Image–Pro Plus. Immunofluorescence staining The 5-µm slices were incubated with primary antibodies against cleaved caspase-3 (1:100, AY0458, ABways), CD31 (1:500, ab182981, Abcam), CD34 (1:200, ab81289, Abcam), GFAP (1:200, BA0056, Boster Biological Technology Co. Ltd., Wuhan, China) and Neun (1:100, CY5515, ABways) at 4 °C overnight, and subsequently incubated with a fluorescent goat anti-rabbit secondary antibody (1:500, 111-165-003, Jackson) at 25 °C for 50 min, then stained with DAPI at 25 °C for 10 min. Finally, images were acquired using fluorescence imaging microscope (NIKON ECLIPSE TI-SR, Japan). And the fluorescence intensity of interesting proteins was assessed through Image-Pro Plus. Statistical analysis Statistical analyses were performed using GraphPad Prism 9.0 and presented as mean ± SD of the mean. Differences among groups were determined using ANOVA followed by a Tukey’s post hoc test. Data followed by P < 0.05 were considered significantly different. Results and Discussion Design, synthesis, and characterizations of HY1–HY4 The D–A–D architecture based on a BBTD core has been previously used to successfully construct small molecule-based NIR-II fluorophores.50 However, these NIR-II π-electron conjugated luminophores are prone to aggregation-caused quenching in the aggregation state, which subsequently suppresses fluorescence emission during bioimaging.51–53 In this study, we optimized QYs of small molecule-based NIR-II imaging agents to extend the emission wavelength of AIEgens in the NIR-IIb region. Consequently, we successfully constructed four BBTD-based fluorophores, namely HY1–HY4, using BPN as an electron donor a 3,4-bis (alkyloxy)thiophene ring as the π-spacer, and an electron donor (Figure 1a). Notably, the naphthyl group in BPN contributes to the twisting structures of fluorophores, thereby achieving high fluorescence enhancement compared with the widely used triphenylamine donor unit.54 In addition, the 2-ethylhexyloxy group on thiophene decreased the π–π stacking of HY4 and promoted intramolecular motion compared with HY1–HY3 (Figure 2b). Next, we executed the density functional theory (DFT) experiment using the B3LYP/6-31G (d) method in Gaussian 09 software to investigate the electronic properties and geometries of HY1–HY4 (Figures 1b and 1c). The S0 geometries revealed that dihedral angles between the BBTD and thiophene spacer of HY1–HY4 were ∼38.5°, 47.1°, 47.6°, and 55.0°, respectively. Moreover, the dihedral angle between the thiophene spacer and triphenylamine of HY1–HY4 were found to be ∼12.6°, 23.3°, 24.6°, and 29.1°, respectively. These results suggested that HY4 possessed the most distorted backbone. The lowest unoccupied molecular orbital (LUMO) was primarily localized on the BBTD core in HY1–HY4. Particularly, the energy gap (Egap) between the LUMO and the highest occupied molecular orbital (HOMO) of HY1–HY4 were 1.44, 1.39, 1.36, and 1.33 eV, respectively, which were lower than that of a typical NIR-II dye CH1055 (1.5 eV).55 Next, we successfully synthesized BBTD-based fluorophores HY1–HY4 through sequential Stille coupling, Zn-powder induced reduction, N-thionylaniline (PhNSO)-induced ring closure, and Suzuki coupling from compounds 1a–4a (Figures 2a). The overall yields ranged between 15% and 25%. The structures of all the intermediates and fluorescence molecules were confirmed by 1H NMR, 13C NMR, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (see Supporting Information Figures S1–S22). Figure 1 | DFT calculations for HY1–HY4. (a) The chemical structures of HY1–HY4. (b) Optimized ground-state geometries (S0) of the molecular fluorophores at the optimal B3LYP/6-31G(d) scrf methods using Gaussian 09. (c) HOMOs and LUMOs of HY1–HY4 using Gaussian 09 B3LYP/6-31G(d) methods of TD-DFT calculations. Download figure Download PowerPoint Figure 2 | (a) The synthetic routes for HY1–HY4. Reaction conditions: (a) Pd(PPh3)4, toluene, 110 °C, 65–70%; (b) Zn, NH4Cl, CH2Cl2/MeOH/H2O, 25 °C; (c) PhNSO, pyridine, TMSCl, 85 °C, two steps, 60–70%; (d) DMF/CH3CN, NBS, HBr; (e) PdCl2(dppf)2CH2Cl2, K2CO3, 75 °C, 20–40%. (b) Schematic illustration of the effect of AIE. Notably, AIE decreased π–π stacking and promoted intramolecular motion of organic dots. Download figure Download PowerPoint Profiles of the spectroscopic features of HY1–HY4 in THF are shown in Figures 3c and 3d. Summarily, HY1–HY4 had maximum absorbance peaks of ∼772, ∼735, ∼736, and ∼736 nm, respectively, with corresponding maximum emission peaks of ∼1080, ∼1034, ∼1058, and ∼1036 nm for HY1–HY4. QY values for HY1–HY4 in THF were ∼0.99%, ∼0.66%, ∼3.4%, and ∼2.82%, respectively (QYIR-26 = 0.5%) ( Supporting Information Figure S23 and S25). Results from analysis of the AIE effects of HY1–HY4 in a water/THF mixture with increasing water volume fractions (fw) revealed that HY2–HY4 had a significantly stronger fluorescent intensity than HY1, which exhibited extremely weak fluorescent intensity, in the water/THF mixed solution (90% fw) (Figure 3b). To investigate the AIE features of HY1–HY4, we measured fluorescent emission spectra using various fw at 808 nm excitation (Figures 3a and 3b and Supporting Information Figure S24). Results indicated that the fluorescence intensity ratio (I/I0) of HY2–HY4 diminished with the gradual increase of fw, from 0% to 40%, then sharply increased upon enhancement of fw, from 40% to 90%, thereby confirming a typical AIE effect. Strikingly, HY4’s I/I0 reached 13, which was much higher than those recorded in HY3 (I/I0 = 9) and HY2 (I/I0 = 3). Collectively, these results demonstrated that a large dihedral angle between BBTD and 2-ethylhexyl substituted thiophene enables the AIE effect for HY4. Figure 3 | The effects of AIE and optical spectra of HY1–HY4. (a) Fluorescence emission spectra of HY4 as fw increased from 0% to 90%. FL, fluorescence. (b) Fluorescent intensity ratios (I/I0) of HY1–HY4 at various fw, I and I0 indicate the fluorescent intensity of HY1–HY4 at various fwand the fluorescent intensity of HY1–HY4 in THF, respectively. (c) Absorption and (d) emission spectra of HY1–HY4 dots in water. PL, photoluminescence. Download figure Download PowerPoint Preparation and characterization of HY1–HY4 dots Water-soluble HY1–HY4 dots were prepared through a nanoprecipitation method using DSPE-PEG3.4k (Figure 2b). Transmission electron microscopy (TEM) images revealed that the obtained dots had a mean particle size of ∼80 nm, while dynamic light scattering (DLS) showed that they had a hydrodynamic diameter of ∼100 nm (Figure 4a). HY4 dots in water had a Zeta potential of −11.8 eV ( Supporting Information Figure S26) and an encapsulation efficiency of ∼85% ( Supporting Information Figure S27). UV–vis–NIR spectra of HY1–HY4 dots in water showed the absorption peaks at 740–770 nm, whereas the fluorescent emission spectra exhibited emission peaks that were centered at 1020–1090 nm with signals ranging between 900 and 1600 nm under 808 nm laser excitation (Figures 4b and 4c). HY1–HY4 dots in aqueous solution had QY values of ∼0.27%, ∼1.68%, ∼12.69%, and ∼14.45%, respectively (Figure 4d, QYIR-26 = 0.5%). In contrast, HY1–HY4 dots in water had QY ratios of 0.27, 2.54, 3.73, and 5.12, respectively, relative to the molecular fluorophores in THF, and these matched well with their AIE property (Figure 4e). The molar-extinction-coefficient (ε) of HY4 dots in water was 8707 L·mol−1·cm−1 ( Supporting Information Figure S28). The brightness of HY4 dots in aqueous solution was further compared with ICG (aqueous) and IR-26 (in dichloroethane (DCE)) at the same concentration. Notably, the fluorescent intensity of HY4 dots was ∼7.5 and ∼6 times higher than those of ICG and IR-26, respectively (Figure 4f). Most importantly, the QY beyond 1500 nm wavelength of HY4 dots in aqueous solution was ∼0.27%, which is much higher than the previously reported organic fluorophore HL3 dots (0.05%)45 and 2TT-oC26B NPs (0.12%)44 (Figure 4h, 808 nm laser, 1500 nm long-pass (LP) filter (150 ms, 100 mW/cm2). Furthermore, HY4 dots exhibited excellent photostability in water and plasma, under 808 nm laser irradiation, while ICG exhibited a marked reduction in fluorescent intensity based on identical measurement conditions (Figure 4g and Supporting Information Figure S29). The half-life in blood of HY4 dots was measured as ∼114 min by fluorescence intensity analysis ( Supporting Information Figure S30). Next, we evaluated the cellular toxicity of HY4 dots in HUVEC and bEnd.3 cells through a CCK-8 assay, and found no significant cellular toxicity, even at a concentration up to 200 μg/mL (Figure 4i). In addition, the safety profiles of HY4 dots administered to Institute of Cancer Research (ICR) mice were evaluated. The hemolytic features of HY4 dots were measured using an ASTM E2524-08 standard test. Results indicate that HY4 dots had no detectable hemolytic characteristic at a concentration up to 100 μg/mL ( Supporting Information Figure S31). The physiological toxicity was evaluated 30 days postinjection of HY4 dots to ICR mice by intravenous injection (i.v.) at the concentration of 5 mg/mL. There were no statistically significant differences in levels of biochemical blood biomarkers, including alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, serum creatinine, and total bilirubin, between HY4 dots-treated animals and those in the phosphate-buffered saline group ( Supporting Information Figure S32). Furthermore, w