A Cascade Targeted and Activatable NIR-II Nanoprobe for Highly Sensitive Detection of Acute Myeloid Leukemia in an Orthotopic Model

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Mar 2021A Cascade Targeted and Activatable NIR-II Nanoprobe for Highly Sensitive Detection of Acute Myeloid Leukemia in an Orthotopic Model Xiaohu Yang†, Xinyi An†, Sisi Ling, Haoying Huang, Yejun Zhang, Guangcun Chen, Chunyan Li and Qiangbin Wang Xiaohu Yang† CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 , Xinyi An† CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 , Sisi Ling CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 University of Science and Technology of China, Hefei 230036 , Haoying Huang CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 University of Science and Technology of China, Hefei 230036 , Yejun Zhang CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 University of Science and Technology of China, Hefei 230036 , Guangcun Chen CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 University of Science and Technology of China, Hefei 230036 , Chunyan Li *Corresponding author: E-mail Address: [email protected] CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 University of Science and Technology of China, Hefei 230036 and Qiangbin Wang CAS Key Laboratory of Nano-Bio Interface, Suzhou Key Laboratory of Functional Molecular Imaging Technology, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 University of Science and Technology of China, Hefei 230036 College of Materials Sciences and Opto-Electronic Technology, University of Chinese Academy of Sciences, Beijing 100049. https://doi.org/10.31635/ccschem.020.202000228 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Acute myeloid leukemia (AML) remains a significant concern in modern medicine. Early diagnosis is the key to improving the therapeutic effects of AML. In the present work, a cascade-targeted and activatable NIR-II nanoprobe (Ald&[email protected]2S) was developed for early detection of AML in an orthotopic model. Upon intravenous injection, Ald&[email protected]2S effectively accumulated in bone tissue due to its high affinity for alendronate (Ald) to the bone. Thereafter, the AML microenvironment allowed for the membrane-penetrating peptide TAT (cell-penetrating peptide (CGRRRQRRKKRG)) to be exposed via pH-sensitive hydrazone bond-mediated detaching of bone-targeted ligands, resulting in efficient internalization of nanoprobes in HL60 cells. Endogenous peroxynitrite (ONOO–) in HL60 cells further activated NIR-II fluorescence of Ag2S QDs via A1094 oxidation, thereby inhibiting fluorescence resonance energy transfer (FRET). Such a unique cascade-targeted and activatable strategy enables the nanoprobes to only light up the AML lesion region in the bone marrow with negligible background effects, which holds great potential for clinical applications in the future. Download figure Download PowerPoint Introduction Acute myeloid leukemia (AML) is a highly heterogeneous disease derived from malignant proliferation of hematopoietic stem cells, with abundant inhibition of primitive and immature cells in the bone marrow with hemopoietic functionality. Despite intense research efforts in AML therapeutics, 70% of patients with advanced-stage AML still will die within 1 year.1–5 Thus, early diagnosis is the key to improving AML survival. Current AML diagnosis mainly relies on bone marrow puncture and peripheral blood tests.6,7 A drawback of these approaches lies in a limited sample amount, which may not provide a complete profile of AML and may result in a false diagnosis. Therefore, it is critical to develop new diagnosis technologies with high sensitivity for in situ, real-time detection of AML at its earliest stages. Noninvasive intravital imaging techniques hold great potential for AML diagnoses. In particular, fluorescence imaging technology is preferred owing to its intrinsic safety, rapid feedback, high sensitivity, and low cost.8,9 However, the spectral wavelength of classical fluorescence imaging is mainly located in the visible (400–700 nm) and NIR-I (700–950 nm) regions, where high absorption, scattering effects, and strong autofluorescence from biological tissues greatly compromise its tissue penetration depth (ca. mm), spatial resolution (ca. mm), and sensitivity. With its greatly decreased absorption and scattering effects, as well as transparent background, near-Infrared-II (NIR-II, 1000–1700 nm) fluorescence imaging featured with high penetration depth and high spatiotemporal resolution offers a favorable imaging strategy.10–23 To facilitate the early diagnosis of AML using this advanced NIR-II imaging modality, it is vital to identify specific biomarkers and develop highly selective and sensitive probes. Previous studies have showed that disordered redox has been found to be highly correlated with the occurrence and development of AML.24–27 Abundant endogenous reactive oxygen species (ROS)/reactive nitrogen species (RNS) are occupied in AML cells and disease-relevant microenvironments.28–31 Particularly, peroxynitrite (ONOO–) plays an important role in the progress of AML, which can serve as a typical biomarker of AML. Herein, we present a novel strategy to design a cascade-targeted and activatable NIR-II nanoprobe (Ald&[email protected]2S) for real-time detection of early-stage AML in vivo. As shown in Scheme 1, the Ald&[email protected]2S nanoprobe contains three functional components: (1) alendronate (Ald), conjugated with a long-chain PEG amphiphilic polymer (PCL2000-Hyd-PEG2000-Ald), serves as the bone-affinity ligand for nanoprobe assistance for the first targeting of bone tissues; (2) a membrane-penetrating peptide TAT, functionalized onto a short-chain PEG amphiphilic polymer (PCL2000-PEG600-TAT), works as the cellular membrane transportation ligand for secondary targeting to AML cells. The pH-sensitive hydrazone bond in PCL2000-Hyd-PEG2000 will break down under weakly acidic conditions in AML, and the PEG2000-Ald will detach from the nanoprobe surface, resulting in exposure of TAT to achieve the secondary targeting to AML cells; (3) a fluorescence resonance energy transfer (FRET) system of NIR-II-emitting Ag2S QDs and a NIR absorber A1094 enables the diagnosis of AML by turning "on" the Ag2S QDs fluorescence from the initial fluorescence "off" state. The A1094 is oxidized by ONOO–, the AML biomarker, which leads to fluorescence recovery and illuminates the AML cells. In the present study, a series of in vitro and in vivo experiments have been comprehensively conducted, demonstrating the feasibility for real-time detection of early-stage AML with high sensitivity with cascade targeting and a NIR-II fluorescence activation strategy. Scheme 1 | Schematic illustration of the construction of an Ald&[email protected]2S probe and mechanism for AML detection in the bone marrow. First, alendronate (Ald) assists the probes to target the bone tissue. Then, the AML-acidic microenvironment enables the membrane penetrating peptide TAT to be exposed via a pH-sensitive hydrazone bond-mediated detaching of Ald to achieve HL60 cell internalization of the probes. Finally, the endogenous peroxynitrite (ONOO–) in HL60 cells activates NIR-II fluorescence of Ag2S QDs via oxidation of A1094, thereby inhibiting FRET. Download figure Download PowerPoint Experimental Methods Materials PCL2000-PEG600-MAL and PCL2000-Hyd-PEG2000-COOH were purchased from Xi'an ruixi Biological Technology Co., Ltd (China). TAT peptide and alendronate (Ald) were purchased from Sangon Biotech (China). N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich. AgNO3, 1-dodecanethiol (DT), (C2H5)2NCS2Na•3H2O [Na(DDTC)], sodium borohydride (>96%), sodium dodecyl sulfonate (SDS), chloroform, dichloromethane, and tetrahydrofuran (THF) were purchased from Aladdin and Sinopharm Chemical Reagent Co., Ltd. (China). All chemicals were obtained from commercial suppliers and used without further purification. Water with a resistivity value of 18 MΩ cm−1 was obtained from a Milli-Q ion-exchange system (Waters-Millipore Ltd.). Characterizations Transmission electron microscopy (TEM) images of DT-Ag2S QDs and Ald&[email protected]2S were acquired via a Tecnai G2 F20S-Twin TEM (FEI, USA) operated at 200 kV. The absorption spectra of Ald&[email protected]2S and A1094 were measured with a PerkinElmer Lambda 25 UV–Vis spectrometer. The NIR-II fluorescence spectra of Ald&[email protected]2S were collected on an applied nanofluorescence spectrometer (USA) at room temperature with an excitation laser source of 785 nm. Hydrodynamic diameters (HDs) and zeta potentials of Ald&[email protected]2S were measured by using a Malvern Nanosizer. Synthesis of A1094 The A1094 compound was synthesized according to previously reported methods.32 Briefly, phloroglucinol and diisobutylamine (1∶1, molar ratio) were mixed to synthesize 5-(N,N-diisobutylamino)-1,3-benzenediol under azeotropic reflux in dry nitrogen atmosphere for 6 h (yield 92%). Then the column chromatograph-purified 5-(N,N-diisobutylamino)-1,3-benzenediol was condensed with croconic acid (2∶1, molar ratio) to generate A1094 (yield 7.5%). 1H NMR (400 MHz, CDCl3, δ): 13.92 (s, 2H, OH), 6.10 (s, 2H, Ar H), 5.91 (s, 2H, Ar H), 3.39 (s, 8H, NCH2), 2.17 (m, 4H, CH), 1.00 (d, J = 6.58 Hz, 24H, CH3). 13C NMR (100 MHz, CDCl3) δ: 165.55 (s, C–O−) 159.71 (s, C=O), 157.04 (s, Carom–O), 156.91 (s, Carom–N), 119.99 (s, =C), 95.96 (s, 2C, Carom), 90.56 (s, 4C, Carom), 61.19 (s, NCH2), 28.53 (s, CH), 20.17 (s, CH3). Synthesis of DT-coated Ag2S QDs (DT-Ag2S) A mixture of 0.1 mmol of (C2H5)2NCS2Ag and 10 g of DT was added into a three-necked flask. After oxygen was removed from the flask, the solution was heated to 160 °C and kept at this temperature for 3 min under a N2 atmosphere. When the mixture was cooled to room temperature (RT), an excess of ethanol was added and the precipitates were collected by centrifugation (15,000 rpm, 10 min). DT-Ag2S QDs were obtained and redispersed in chloroform. Synthesis of PCL-PEG-TAT Briefly, 2 mg PCL-PEG-MAL and 2 mg peptide TAT were dissolved in a buffer solution (50 mM triethanolamine hydrochloride, 50 mM sodium phosphate, 150 mM NaCl, and 1 mM EDTA, pH 8). PCL-PEG(600) maleimide was gently mixed with an equal volume of TAT peptide solution overnight in a glass bottle at 4 °C. The synthesized product was obtained by freeze-drying. Preparation of targeted PEGylated Ag2S QDs (TAT-Ag2S) A THF solution (1 mL) containing DT-Ag2S QDs (3 mg), PCL-Hyd-PEG (2.0 mg), PCL-PEG-TAT (2.0 mg), and SDS (2.0 mg) was poured into water (10 mL) under ultrasonic conditions at a 240 W output for 30 min. The organic solvent was further removed through ultrafiltration using the 100 kDa MW cutoff centrifugal filter unit at 4000 rpm. The QDs concentrated solution was redispersed with PBS buffer. Synthesis of Ald-Ag2S Ald was covalently conjugated onto carboxylic-functionalized Ag2S QDs using the standard EDC/NHS reaction. 0.3 mg EDC and 0.06 mg NHS were added into 5 mL TAT-Ag2S, and the mixture was then stirred at room temperature for 30 min to activate the carboxylic group of the QDs. Excess EDC and NHS were removed by washing the QDs with PBS. Subsequently, 0.65 mg Ald was added to the aforementioned solution and stirred at room temperature for 24 h. The product was washed twice with PBS to remove excess Ald, and then, Ald-Ag2S QDs were obtained. Attachment of molecular A1094 to Ald-Ag2S A1094 molecule can be immobilized on the nanoparticle surfaces via strong ionic bonding between quaternary ammonium salt and sulfonate groups, which has been verified according to a previous report.33 A1094 (0.1 mg/mL) was added into the as-prepared nanoparticles in PBS buffer (pH 7.4) and stirred at room temperature for 30 min. Finally, the end product was obtained by ultrafiltration and washed with PBS buffer three times, and the Ald&[email protected]2S was obtained. Preparation of ROS/RNS solution (1) ONOO–: Three kinds of solutions, including the mixture of hydrogen peroxide (0.7 M, 1.5 mL) and hydrochloric acid (0.6 M, 1.5 mL), a solution of sodium nitrite (0.6 M, 3 mL), and a solution of sodium hydroxide (1.5 M, 3 mL) were simultaneously added within 1 s to make the ONOO– stock solution.34 The resulting solution was stored at –20 °C. (2) •OH: It was generated in the Fenton system from ferrous chloride (FeCl2) and hydrogen peroxide (H2O2). (3) H2O2: The H2O2 solution was purchased from Sigma-Aldrich. The concentration of the H2O2 stock solution was determined by measuring the absorbance at 240 nm. (4) O2–: The O2– solution was generated from a mixed reaction of NaMoO4 (10 mM) and H2O2 (10 mM). Cell culture Human promyelocytic leukemia cells (HL60), mouse fibroblast cells (L929), and murine macrophages (RAW264.7) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. HL60 and L929 cells were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (P/S). RAW264.7 cells were grown in DMEM high-glucose medium (Hyclone) supplemented with 10% FBS and 1% P/S. All cells were cultured at 37 °C in a 5% CO2 humidified chamber. Cytotoxicity assay The cytotoxicity of nanoprobes toward HL60, RAW264.7, and L929 cells were evaluated using the MTT assay. Cells were cultured in 96-well plates at 1×105 cells/well overnight and then incubated with Ald&[email protected]2S (final concentration of Ald& [email protected] from 0 μg/mL to 60 μg/mL) for 24 h. Thereafter, the MTT solution (5 mg/mL 20 μL) was added to each well and incubated for an additional 4 h. The typical absorbance was measured by an enzyme-linked immunosorbent assay (ELISA) reader (infinite M200, Tecan, Austria). The cell viability was calculated based on measuring the UV–vis absorption at 570 nm using the following equation: Viability (%) = (mean of absorbance value of treatment group/mean absorbance value of control) × 100%. Fluorescent microscopic imaging HL60 cells were first treated with 0.1 mM SIN-1 for 30 min to induce endogenous ONOO– formation. Thereafter, Ald&[email protected]2S (30 μg/mL) was added into the cell suspension for another 30 min. After washing and centrifugation, the cells were resuspended into the fresh culture media containing no FBS. The cells were stained with Hoechst 33342 before imaging. Blood biochemistry and hematology analysis After intravenous injection of 200 μL of PBS solution and 0.5 or 1 mg/kg Ald&[email protected]2S solutions, blood samples were obtained from the mice at day 1 and day 7. A 0.2 mL of blood containing anticoagulant was collected for hematology analysis. About 0.8 mL of blood was centrifuged at 3000 rpm for 20 min at 4 °C after resting at room temperature for 30 min for biochemistry analysis. All samples were conducted at the Shanghai Research Center for Biomodel Organism. Animal model All animal procedures were performed in accordance with the national guidelines for the care and use of laboratory animals, China (GB/T 35892-2018), and were approved by the Animal Ethics Committee of Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences (Suzhou, China). A 6-week-old female nude mice were obtained from Shanghai SLRC Laboratory Animal Co., Ltd. and fed a standard diet at room temperature. AML orthotopic models were established by knee-joint injection of 1 × 105 HL60 cells suspended in 20 µL PBS (1X) into nude mice under anesthesia using isoflurane. Then the mice were placed in an incubator and returned to their home cages. In vivo fluorescence imaging of AML mice The AML nude mice were intravenously injected with Ald&[email protected]2S. About 5% chloral hydrate solution was used for anesthesia of the mice during injection and imaging. The mice were imaged at different time points postinjection Ald&[email protected]2S using the NIR-II fluorescence imaging system (Series II 900/1700, Suzhou NIR-Optics Technologies Co., Ltd., China). The excitation light was provided by an 808-nm diode laser with a power density at an imaging plane of ∼ 45 mW/cm2. A 1000-nm long-pass (LP 1000) filter was employed for fluorescent signal collection of Ag2S QDs. Results and Discussion Characterization of novel targeted activatable NIR-II nanoprobe Ald&[email protected] As shown in Figure 1a, the TEM image of Ag2S and Ald&[email protected]2S showed that the nanoparticles were well dispersed with an average size of about 3.3 and 110 nm in diameter, respectively. Dynamic light scattering (DLS) of Ald&[email protected]2S was consistent with its TEM results (Figure 1b). The zeta potential displayed a remarkable surface charge change from 0.3 to –23.9 mV before and after decoration with Ald (Figure 1c). The optical properties of the nanoprobes were also investigated as shown in Figure 1d. A1094 showed the broad absorbance peak centered at ∼ 1094 nm and Ag2S QDS displayed an intense fluorescence emission peak at ∼ 1050 nm, which verified the efficient FRET process achieving initial NIR-II fluorescence quenching (85%) ( Supporting Information Figures S1–S3). Ald&[email protected]2S displayed favorable colloidal stability and fluorescence stability after dispersing in PBS or FBS for 48 h using DLS measurements and spectral analysis ( Supporting Information Figures S4 and S5). These data demonstrated the successful preparation of Ald&[email protected]2S. Figure 1 | (a) TEM images of DT-Ag2S QDs and Ald&[email protected]2S. (b) Hydrodynamic diameter (HD) and (c) zeta potential analysis of [email protected]2S, [email protected]2S, and Ald&[email protected]2S in aqueous solution. (d) Absorbance and fluorescence spectra of [email protected]2S and Ald&[email protected]2S. Download figure Download PowerPoint Evaluation of cascade targeting capability of Ald&[email protected]2S in vitro To detect AML cells in bone marrow, the first step is to achieve efficient bone tissue targeting of nanoprobes by crossing the blood–bone marrow barrier (BMB). To validate the bone-specific affinity of Ald&[email protected]2S in vitro, bone affinity assays were performed by coincubating hydroxyapatite (HA) and calf bone slices with Ald&[email protected]2S labeled with Cy7.5 and further examined by fluorescence microscopy. As shown in Figure 2a, a concentration-dependent binding feature was displayed, in which intense fluorescence of HA was observed after adding Cy7.5-labeled Ald&[email protected]2S for 1 h. By contrast, negligible fluorescence was detected in HA after incubation with various of [email protected]2S without targeting ligand a consistent result was obtained in calf bone slices in serum (Figure All these data that Ald&[email protected]2S can achieve favorable bone-targeted Figure 2 | (a) and fluorescence images of hydroxyapatite (HA) binding of [email protected]2S and Ald&[email protected]2S at different (b) images of calf bone slices incubated with [email protected]2S and Ald&[email protected]2S for 1 h. (c) fluorescence images of HL60 cells treated with Ald&[email protected]2S under different pH 25 (d) fluorescence the in Download figure Download PowerPoint concern is the specific cellular of Ald&[email protected]2S after bone marrow The acidic AML microenvironment be to achieve specific of nanoprobes into AML cells. In the present a pH-sensitive hydrazone bond was to in the secondary targeting ligand of TAT as displayed in Scheme To a strategy was to the nanoprobes into the AML cells, and cellular imaging were investigated under different pH conditions (pH and the of bone marrow and the AML respectively. The Ald&[email protected]2S were measured by DLS at different it showed diameters (HDs) nanoprobes were dispersed into the pH aqueous from ∼ 110 nm to a ( Supporting Information Figures and can be to the of the hydrazone bond in which the detaching of PEG2000-Ald and in the of Subsequently, cellular imaging further that the nanoprobes be in the HL60 cells after at pH by measuring the fluorescence signal of Cy7.5 (Figure The fluorescence profile showed that the nanoprobes were mainly in the (Figure By contrast, it showed negligible Cy7.5 fluorescence in HL60 cells at a pH of All of these data that a strategy can greatly the of nanoprobes in HL60 cells. Ald&[email protected]2S to endogenous in HL60 cells by the aforementioned we further investigated the capability of Ald&[email protected]2S in ROS/RNS in HL60 cells. Previous studies showed that a generate to a disordered redox in The HL60 cells were first cultured in the of 0.1 mM SIN-1 and then further incubated with Ald&[email protected]2S at a pH of for 30 min. As shown in Figure the cells with SIN-1 displayed a significant fluorescence in NIR-II In contrast, in the of NIR-II fluorescence after 30 min of the fluorescence of the cells a after SIN-1 treatment (Figure further investigated the of Ald&[email protected]2S by the between the probes with various ROS/RNS and As a the NIR-II fluorescence signal of Ald&[email protected]2S be activated by peroxynitrite (ONOO–) (Figure The detection of Ald&[email protected]2S is based on the 3 is the standard of and is the of Supporting Information Figure These results demonstrated that Ald&[email protected]2S is to detect endogenous ONOO– in HL60 cells. Figure 3 | microscopy images (a) and analysis (b) of and HL60 cells incubated with Ald&[email protected]2S (30 μg/mL) for 30 min at 37 °C. 50 NIR-II fluorescence of Ald&[email protected]2S the of various ROS/RNS (c) and [email Ald&[email protected]2S in (d) spectra of A1094 of ONOO– 0 to of absorption at A1094 nm as a of ONOO– Download figure Download PowerPoint of Ald&[email protected]2S probe To the cytotoxicity of the HL60, and RAW264.7 cells were evaluated after treated with Ald&[email protected]2S, by using a standard MTT assay. As shown in Supporting Information Figure in the of Ald&[email protected]2S of cellular was evaluated to be after 24 h treatment in HL60 and L929 cells. the cellular of murine macrophages RAW264.7 treated with Ald&[email protected]2S was which was at a of 70% after incubation at a concentration of 60 μg/mL for 24 h. be to its to further the of a mice were for blood samples at 1 and after a injection of Ald&[email protected]2S at a of 1 As a various with the group ( Supporting Information Figure biochemistry showed negligible to the group ( Supporting Information Figure on the aforementioned we that was negligible of Ald&[email protected]2S at of 1 In vivo detection of AML Finally, to its feasibility for the detection of AML in an orthotopic model was established by HL60 cell in the of the in nude mice for Subsequently, Ald&[email protected]2S in PBS 0.1 100 was injected into the mice via the and [email protected]2S A1094, [email protected]2S Ald, as the As shown in Figure the fluorescence recovery process of Ald&[email protected]2S was in by an in vivo NIR-II imaging system Technology Co., Ltd., China). Such a targeted activatable NIR-II probe Ald&[email protected]2S and light up the of mice with HL60 cells, a negligible NIR-II fluorescence signal be detected in The process was also by NIR-II fluorescence imaging of mice at different time analysis of signal in the lesion region a in NIR-II fluorescence signal resulting in a from at 10 min to at min injection (Figure great potential for rapid diagnosis of AML. By contrast, the of the mice after of the probe [email protected]2S. It is to the AML region from tissue due to high background in the of targeting ligands, a probe [email protected]2S not effectively in the bone marrow and no remarkable signal was In the mice not a NIR-II fluorescence signal were no of peroxynitrite (ONOO–) to activate the FRET of Ald&[email protected]2S to on Ag2S QDs. All these data that a cascade-targeted and activatable NIR-II nanoprobe Ald&[email protected]2S achieve AML detection in an orthotopic model. Figure 4 | (a) images of NIR-II fluorescence in AML model and mice after injection of [email protected]2S, Ald&[email protected]2S, and [email protected]2S, respectively. (b)