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

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 Qiangbin Wang Yang† CAS Key Laboratory Nano-Bio Interface, Suzhou Functional Molecular Imaging Technology, Division Nanobiomedicine i-Lab, Institute Nano-Tech Nano-Bionics, Chinese Academy Sciences, 215123 †X. Yang X. An contributed equally to this work.Google Scholar More articles by author , An† Ling University Science Technology China, Hefei 230036 Google Huang Zhang Chen *Corresponding author: E-mail Address: [email protected] College Materials Sciences Opto-Electronic Beijing 100049. https://doi.org/10.31635/ccschem.020.202000228 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd favoritesTrack Citations ShareFacebookTwitterLinked InEmail myeloid leukemia (AML) remains a significant concern modern medicine. Early diagnosis is the key improving therapeutic effects AML. In present work, cascade-targeted activatable nanoprobe (Ald&[email protected]2S) was developed early detection AML orthotopic model. Upon intravenous injection, Ald&[email protected]2S effectively accumulated bone tissue due its high affinity alendronate (Ald) bone. Thereafter, microenvironment allowed membrane-penetrating peptide TAT (cell-penetrating (CGRRRQRRKKRG)) be exposed via pH-sensitive hydrazone bond-mediated detaching bone-targeted ligands, resulting efficient internalization nanoprobes HL60 cells. Endogenous peroxynitrite (ONOO–) cells further activated fluorescence Ag2S QDs A1094 oxidation, thereby inhibiting resonance energy transfer (FRET). Such unique strategy enables only light up lesion region marrow with negligible background effects, which holds great potential clinical applications future. Download figure PowerPoint Introduction highly heterogeneous disease derived from malignant proliferation hematopoietic stem cells, abundant inhibition primitive immature hemopoietic functionality. Despite intense research efforts therapeutics, 70% patients advanced-stage still will die within 1 year.1–5 Thus, survival. Current mainly relies on puncture peripheral blood tests.6,7 A drawback these approaches lies limited sample amount, may not provide complete profile result false diagnosis. Therefore, it critical develop new technologies sensitivity situ, real-time at earliest stages. Noninvasive intravital imaging techniques hold diagnoses. particular, technology preferred owing intrinsic safety, rapid feedback, sensitivity, low cost.8,9 However, spectral wavelength classical located visible (400–700 nm) NIR-I (700–950 regions, where absorption, scattering strong autofluorescence biological tissues greatly compromise penetration depth (ca. mm), spatial resolution sensitivity. With decreased absorption as well transparent background, near-Infrared-II (NIR-II, 1000–1700 featured spatiotemporal offers favorable strategy.10–23 To facilitate using advanced modality, vital identify specific biomarkers selective sensitive probes. Previous studies have showed that disordered redox has been found correlated occurrence development AML.24–27 Abundant endogenous reactive oxygen species (ROS)/reactive nitrogen (RNS) are occupied disease-relevant microenvironments.28–31 Particularly, plays important role progress AML, can serve typical biomarker Herein, we novel design early-stage vivo. As shown Scheme 1, contains three functional components: (1) (Ald), conjugated long-chain PEG amphiphilic polymer (PCL2000-Hyd-PEG2000-Ald), serves bone-affinity ligand assistance first targeting tissues; (2) TAT, functionalized onto short-chain (PCL2000-PEG600-TAT), works cellular membrane transportation secondary The bond PCL2000-Hyd-PEG2000 break down under weakly acidic conditions PEG2000-Ald detach surface, exposure achieve cells; (3) (FRET) system NIR-II-emitting NIR absorber turning “on” initial “off” state. oxidized ONOO–, biomarker, leads recovery illuminates study, series vitro vivo experiments comprehensively conducted, demonstrating feasibility cascade activation strategy. | Schematic illustration construction probe mechanism marrow. First, assists probes target tissue. Then, AML-acidic penetrating Ald cell Finally, activates oxidation A1094, FRET. Experimental Methods PCL2000-PEG600-MAL PCL2000-Hyd-PEG2000-COOH were purchased Xi’an ruixi Biological Co., Ltd (China). Sangon Biotech N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC) N-hydroxysuccinimide (NHS) obtained Sigma-Aldrich. AgNO3, 1-dodecanethiol (DT), (C2H5)2NCS2Na•3H2O [Na(DDTC)], sodium borohydride (>96%), dodecyl sulfonate (SDS), chloroform, dichloromethane, tetrahydrofuran (THF) Aladdin Sinopharm Chemical Reagent Ltd. All chemicals commercial suppliers used without purification. Water resistivity value 18 MΩ cm−1 Milli-Q ion-exchange (Waters-Millipore Ltd.). Characterizations Transmission electron microscopy (TEM) images DT-Ag2S acquired Tecnai G2 F20S-Twin TEM (FEI, USA) operated 200 kV. spectra measured PerkinElmer Lambda 25 UV–Vis spectrometer. collected applied nanofluorescence spectrometer (USA) room temperature excitation laser source 785 nm. Hydrodynamic diameters (HDs) zeta potentials Malvern Nanosizer. Synthesis compound synthesized according previously reported methods.32 Briefly, phloroglucinol diisobutylamine (1∶1, molar ratio) mixed synthesize 5-(N,N-diisobutylamino)-1,3-benzenediol azeotropic reflux dry atmosphere 6 h (yield 92%). Then column chromatograph-purified condensed croconic acid (2∶1, generate 7.5%). 1H NMR (400 MHz, CDCl3, δ): 13.92 (s, 2H, OH), 6.10 Ar H), 5.91 3.39 8H, NCH2), 2.17 (m, 4H, CH), 1.00 (d, J = 6.58 Hz, 24H, CH3). 13C (100 CDCl3) δ: 165.55 C–O−) 159.71 C=O), 157.04 Carom–O), 156.91 Carom–N), 119.99 =C), 95.96 2C, Carom), 90.56 4C, 61.19 28.53 20.17 DT-coated (DT-Ag2S) mixture 0.1 mmol (C2H5)2NCS2Ag 10 g DT added into three-necked flask. After removed flask, solution heated 160 °C kept 3 min N2 atmosphere. When cooled (RT), excess ethanol precipitates centrifugation (15,000 rpm, min). redispersed chloroform. PCL-PEG-TAT 2 mg PCL-PEG-MAL dissolved buffer (50 mM triethanolamine hydrochloride, 50 phosphate, 150 NaCl, EDTA, pH 8). PCL-PEG(600) maleimide gently equal volume overnight glass bottle 4 °C. product freeze-drying. Preparation targeted PEGylated (TAT-Ag2S) THF (1 mL) containing (3 mg), PCL-Hyd-PEG (2.0 SDS mg) poured water (10 ultrasonic 240 W output 30 min. organic solvent through ultrafiltration 100 kDa MW cutoff centrifugal filter unit 4000 rpm. concentrated PBS buffer. Ald-Ag2S covalently carboxylic-functionalized standard EDC/NHS reaction. 0.3 EDC 0.06 NHS 5 mL TAT-Ag2S, then stirred activate carboxylic group QDs. Excess washing PBS. Subsequently, 0.65 aforementioned 24 h. washed twice remove Ald, then, obtained. Attachment molecular molecule immobilized nanoparticle surfaces ionic bonding between quaternary ammonium salt groups, verified previous report.33 (0.1 mg/mL) as-prepared nanoparticles (pH 7.4) end times, ROS/RNS ONOO–: Three kinds solutions, including hydrogen peroxide (0.7 M, 1.5 hydrochloric (0.6 mL), nitrite hydroxide (1.5 simultaneously s make ONOO– stock solution.34 stored –20 •OH: It generated Fenton ferrous chloride (FeCl2) (H2O2). H2O2: H2O2 concentration determined measuring absorbance (4) O2–: O2– reaction NaMoO4 mM) mM). Cell culture Human promyelocytic (HL60), mouse fibroblast (L929), murine macrophages (RAW264.7) Bank Type Culture Collection Sciences. L929 cultured RPMI 1640 medium (Hyclone) supplemented 10% fetal bovine serum (FBS, Hyclone) 1% penicillin/streptomycin (P/S). RAW264.7 grown DMEM high-glucose FBS P/S. 37 5% CO2 humidified chamber. Cytotoxicity assay cytotoxicity toward HL60, RAW264.7, evaluated MTT assay. Cells 96-well plates 1×105 cells/well incubated (final Ald& 0 μg/mL 60 μg/mL) (5 mg/mL 20 μL) each additional enzyme-linked immunosorbent (ELISA) reader (infinite M200, Tecan, Austria). viability calculated based UV–vis 570 nm following equation: Viability (%) (mean treatment group/mean control) × 100%. Fluorescent microscopic treated SIN-1 induce formation. (30 suspension another centrifugation, resuspended fresh media no FBS. stained Hoechst 33342 before imaging. Blood biochemistry hematology analysis injection μL 0.5 or mg/kg samples mice day 7. 0.2 anticoagulant analysis. About 0.8 centrifuged 3000 rpm after resting conducted Shanghai Research Center Biomodel Organism. Animal model animal procedures performed accordance national guidelines care use laboratory animals, China (GB/T 35892-2018), approved Ethics Committee (Suzhou, China). 6-week-old female nude SLRC fed diet temperature. models established knee-joint 105 suspended µL (1X) anesthesia isoflurane. placed incubator returned their home cages. intravenously injected protected]2S. chloral hydrate during imaged different time points postinjection (Series II 900/1700, NIR-Optics Technologies Ltd., provided 808-nm diode power density plane ∼ 45 mW/cm2. 1000-nm long-pass (LP 1000) employed fluorescent signal collection Results Discussion Characterization Figure 1a, image dispersed average size about 3.3 110 diameter, respectively. Dynamic (DLS) consistent results (Figure 1b). displayed remarkable surface charge change –23.9 mV decoration 1c). optical properties also investigated 1d. broad peak centered 1094 QDS emission 1050 nm, FRET process achieving quenching (85%) ( Supporting Information Figures S1–S3). colloidal stability dispersing 48 DLS measurements S4 S5). These data demonstrated successful preparation (a) (b) diameter (HD) (c) protected]2S, aqueous solution. (d) Absorbance Evaluation capability detect marrow, step crossing blood–bone barrier (BMB). validate bone-specific vitro, assays coincubating hydroxyapatite (HA) calf slices labeled Cy7.5 examined microscopy. 2a, concentration-dependent binding feature displayed, HA observed adding Cy7.5-labeled By contrast, detected incubation various concentrations Ald. Similarly, even more complex physiological solution-containing 2b). indicate capability. Bright field Cy7.5-modified concentrations. Representative (left) (right) Microscopic conditions. Scale bar: μm. Cross-sectional intensity profiles across red lines (c). Another recognition targeting. could harnessed uptake nanoprobe, introduced assist activating 1. assess whether such pH-activated able transport morphology characterization carefully 7.4 5.5) mimicking environments normal microenvironment, sizes pHs. Interestingly, significantly-enhanced hydrodynamic when 5.5 solution, and, correspondingly, changed micrometer scale S6 S7). This phenomenon attributed breakage PCL2000-Hyd-PEG2000-Ald, initiated resulted aggregation nanoprobes.35 confirmed efficiently internalized pretreatment 2c). distributed cytoplasm 2d). 7.4. cascade-targeting improve accumulation response RNS Encouraged results, detecting endogenously produced SIN-1, nitric oxide donor, extensive oxidative/nitrogen stress mimic environment AML.36 presence pretreated 3a, stimulated increase region. absence very weak appeared incubation. Quantification revealed than six fold improvement 3b). We specificity assessing interaction predominant ions. result, specifically 3c–3e). limit 113 nM Sb/k criterion (Sb deviation 11 blank replicate determinations, k slope calibration curve; S8). experimental Fluorescence quantitative SIN-1-treated nontreated changes upon addition analytes ions (d). Ctrl1: protected]2S; Ctrl2: H2O. Absorption (40 μM) gradual (from 38.4 μM). Inset: Plot function concentration. Toxicity assessment examine nanoprobes, L929, being respectively, S9, 0–60 μg/mL, vitality over 93% both slightly suppressed, maintained level natural endocytosic properties. Moreover, evaluate biosafety sacrificed collecting 7 days tail dosage mg/kg. hematological parameters remained steady control S10). all indexes compared S11). Based concluded there toxicity dosages noninvasive investigate vivo, xenografting proximal tibia right-back legs (see “Experimental Methods” section details). (Ag2S QDs: mg/mL, µL) vein (without “always-on” probe), while nontargeted probe) served group. 4a, dynamically monitored situ employing (Suzhou Near-Infrared Optical rapidly right transplanted any other dynamic living points. Quantitative continuous gradually improved tumor-to-background ratio (TBR) 2.5 10.9 90 post 4b), providing whole bodies glowed administration hard distinguish extremely noise. accumulate detected. addition, healthy did report since trigger molecules switch