Ultrastable Near-Infrared Aggregation-Induced Emission Nanoparticles as a Fluorescent Probe: Long-Term Tumor Monitoring and Lipid Droplet Tracking

Open AccessCCS ChemistryRESEARCH ARTICLE1 Jun 2021Ultrastable Near-Infrared Aggregation-Induced Emission Nanoparticles as a Fluorescent Probe: Long-Term Tumor Monitoring and Lipid Droplet Tracking Haijun Ma, Danning Hu, Jiajia Zhao, Mei Tian, Jinying Yuan Yen Wei Ma MOE Key Laboratory of Bioorganic Phosphorus Chemistry Chemical Biology, Department Chemistry, Tsinghua University, Beijing 100084 Organic Optoelectronics Molecular Engineering, Google Scholar More articles by this author , Hu Zhao Nanostructure Nanotechnology, Institute Chinese Academy Sciences, 100190 Tian Nuclear Medicine Medical PET Center, The Second Hospital Zhejiang University School Medicine, Hangzhou 310009 *Corresponding authors: E-mail Address: [email protected] Center for Biomedical Technology, Chung Christian Taoyuan 32023 https://doi.org/10.31635/ccschem.020.202000383 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Effective real-time tumor monitoring cell tracking are great importance precise diagnosis therapy tumors, also the surveillance biological processes. In study, new organic fluorescent nanoprobe (named TPATBT NPs) with unique aggregation-induced emission (AIE) characteristics has been obtained first time via facile synthesis achieve long-term in living cells. advantages NPs include small size (∼80 nm), large Stokes shift (∼150 high stability, good dispersibility aqueous media, biocompatibility. addition, such have showed excellent bioimaging performance unusual properties. red fluorescence signals inside MDA-MB-231 cells last longer than 10 generations (18 days). Moreover, cellular uptake found highly rely on energy-dependent endocytosis clathrin-mediated endocytosis, primarily accumulate lipid droplets (LDs), which can lead targeted LD imaging therapy. Thus, work an malignant growth dynamic Download figure PowerPoint Introduction Cancer is one most important problems human health world. reliable real crucial early diagnosis, surgical navigation, evaluation cancer therapeutics, well recognition invasion metastasis.1–3 Noninvasive indispensable versatile approach biomedical research, provides physiological pathological details bioorganisms monitors complicated processes.2,4–7 So far, technologies methods widely investigated diagnoses, positron tomography,8,9 computed tomography,10,11 photoacoustic tomography,12–14 magnetic resonance imaging,15,16 imaging, single-photon tomography.17–19 Among them, beneficial providing vital information, serve extraordinary method precancer diagnosing postcancer monitoring, due its direct visualization, resolution sensitivity, low cost fast key information collecting at subcellular level, so forth.20–24 recent years, vast number materials (e.g., dyes,25 inorganic quantum dots,26,27 carbon dots,28,29 proteins,30,31 upconversion NPs,32 semiconducting polymer NPs33,34) extensively studied applied bioimaging. However, these dyes still face many severe challenges, biotoxicity, lower molar absorptivity, limited sensitivity resolution, easy photobleaching photoquenching concentrations or aggregation state, forth. Hence, their application bioimaging, biomedicine, clinical medicine hindered.35–38 are: (1) aggregation-caused quenching (ACQ) effect, that is, significant decrease when dye concentration increased because intermolecular π–π stacking nonradiative pathways, (2) diminished upon long exposure light.39,40 phenomenon was discovered Tang’s group 2001 explored ever since.41–43 contrast ACQ, AIE only show nonluminous weak diluted solution, while it demonstrates strong solid state. hydrophobic, hindering applications. To overcome limitations, nanoprecipitation strategy developed synthesize nanoparticles (NPs) encapsulating molecules into amphiphilic polymers.44 Modified displayed water solubility biocompatibility, medical visualization systems largely based fixed static specimens using formaldehyde fixing agents), makes hard precisely elucidate dynamical Therefore, construction preservation specimen activity facilitating enhanced understanding processes.45,46 although several designed achieved effects, necessary develop types properties near-infrared (NIR) systematically investigate absorption–distribution–metabolism mechanism, effectively monitor processes, expand applications biomedicine.47–49 we present AIE-active system duration up 18 days time. This constructed triphenylamine (TPA) units electron donor (D) 4,7-bis(5-bromothiophen-2-yl)benzo[c][1,2,5]thiadiazole (TBT) acceptor (A) through typical Suzuki reaction. followed subsequent reactions lipid-PEG derivative 1,2-distearoyl-snglycero-3-phosphoethanol-amine-N-[methoxyl(polyethylene glycol)-2000] (DSPE-PEG2000). product named D-A-D structure, induces red-shifted wavelength NIR region Stoke shifts. Such dispersive ultrapure water, phosphate-buffered saline (PBS), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), exhibiting It maintain uniform hydrodynamic diameter various media 15 days, relative intensity above 90% continuously exposed light 1 h, suggesting stability ability resist photobleach. These superior photophysical shown remain bright over (80 nm) be readily internalized mechanism accumulated (LDs) act promising tracking/therapeutic agent provide effective tool Experimental Methods Materials reagents TBT palladium acetate [Pd(OAc)2] were from J&K Scientific Ltd (Beijing, China). 4-(Diphenylamino)phenylboronic acid purchased Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, DSPE-PEG2000 ToYongBio Tech. Inc. China) Sodium carbonate [Na2(CO)3] toluene Reagents Co. Calcien AM, propidium iodide (PI), MitoTracker Green, LysoTracker BODIPY 493/503 Thermo Fisher (Waltham, MA). All solvents commercially used without further purification. Measurements characterizations 1H NMR spectra measured JEOL JNM-ECA400 spectrometer (JEOL Ltd., Tokyo, Japan). UV captured Perkin Elmer LAMBDA 750 UV/VIS/NIR (PerkinElmer, USA) spectrometer. Photoluminescence (PL) performed spectrophotometer (SHIMADZU, model: RF-6000, SHIMADZU, Dynamic scattering (DLS) ζ potential carried out Malvern Instruments Zetasizer Nano ZSI (Malvern Instruments, Worcestershire, U.K.) 90° angles. Transmission microscopy (TEM) images recorded Hitachi 7650B (Hitachi, Japan) microscope operating acceleration voltage 80 kV. Water purified Millipore Milli-Q Synthesis purifier (18.0 MΩ cm, Barnstead, MERCK, USA). (249.00 mg, 0.50 mmol), 4-(diphenylamino)phenylboronic (434.00 1.50 Na2CO3 (530.00 5.00 Pd(OAc)2 (22.40 0.10 mmol) placed 250 mL two-neck flask equipped condenser, (30.0 mL) added. reaction stirred overnight under nitrogen 100 °C 12 h. After mixture cooled room temperature, (80.00 chloroform (200.00 layer separated washed brine, dried anhydrous MgSO4, evaporated dryness reduced pressure. crude column chromatography silica gel petrol ether/dichloromethane (v/v = 20/1) eluent obtain orange-red solid. (400 MHz, CDCl3, δ): 8.09–8.10 (d, 2H), 7.85 (s, 7.53–7.55 4H), 7.31–7.34 7.25–7.29 (t, 3H), 7.24 7.10–7.15 8H), 7.00–7.10 (m, 8H). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (m/z): (M+) calcd C50H34N4S3, 786.63; found, 786.65. prepared method. Simply, compounds (5 mg) (10 dissolved 5 tetrahydrofuran (THF) solution sonicated min ultrasonic device W. solvent totally removed rotary evaporation. residual sample poured 30 temperature. dialysis, freeze-dried powder. Cell culture DU-145, L-929, Beina Chuanglian Biotechnology cultured DMEM (Invitrogen, Gibco, Grand Island, containing 1% penicillin streptomycin (100.00 μg mL−1; NY) 10% FBS (Gibco), maintained 37 humidified atmosphere 5% CO2 experiments. Cytotoxicity For cytotoxicity assay, plated 96-well plates 24 then incubated samples different (10, 20, 40, 80, 160 mL−1) dark h (36, 48, 72 h). Then viability determined Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, absorption value 450 nm read plate reader (iMark microplate reader; Bio-RAD, determine viability. detailed experimental procedures characterization data available Supporting Information. Results Discussion molecular structures preparation route illustrated Scheme 1. desired generated TPA D core A, Pd(PPh3)4 catalyst reflux solution. chemical structure composition final products characterized MALDI-TOF MS ( Information Figures S1 S2). lifetime (7.3 ns) yield (9.4%) transient steady-state Figure S3). Furthermore, highest occupied orbital (HOMO; −4.69 eV) unoccupied (LUMO; −2.53 distribution calculated time-dependent density functional theory (TD-DFT; S4). Aiming exploit deciphering optical single crystals grown dichloromethane–hexane mixtures slow twisted geometry segment distance (>3.2 Å) between two parallel planes, significantly decreasing preventing interactions aggregated increasing restriction motions S5 S6 Tables S1–S4). On basis results, suggested synthesized compound possessed potentially Considering state cannot directly poor would choice make fabricated encapsulation matrix. UV–Vis PL spectroscopy. absorbance peak exhibited range 280–600 maximum about 520 nm. appeared 670 intense tail extending 850 nm, (Figures 1a 1b). Meanwhile, signal affected concentration, gradually raised 50 µg mL−1 S7). detected TEM DLS. quasi-spherical morphologies distribution. sizes approximately dry diameters 108 ± 1c 1d). slightly larger DLS attributed formation hydrated media.50 general, exhibit sizes, shift, solubility, they easily endocytosed | Syntheses NPs. Stability confirm biocompatibility ensure systems, average (ultrapure PBS, FBS, DMEM) intervals 2a 2b). relatively (around polydispersity indexes (PDI) 0.2 diverse intervals, results no differences. spectrum evaluate change initial retained 2c–2e). intensities consistent S8 S9). photostabilities (water, FBS) continuous irradiation remained 85%. demonstrated dispersible biomedia, photostability, probe tracking. (a) Absorption spectra, (b) excitation spectrum, (c) diameter, (d) 2 Hydrodynamic DMEM, FBS. PDI, respectively. (e) FBS). critical indicator determining whether nanomaterials systems. experiments, DU-145 L-929 viabilities DU145 CCK-8 essay 95% (Figure 3a), 0–160 mL−1. selected biosecurity incubation times 24, 36, 95%, had bioactivity. effects cells, flow cytometry detect proportion survival, apoptosis, necrosis. periods (24, h), where greater (95.7%, 97.6%, 92.4%, 96.0%; 3b). Simultaneously, coincubated (80, 160, 240, 320 survival rates (94%, 93%, 91.7%, 90.4%; 3c). firmly reveal could 3 tested apoptosis necrosis concentration. right area (Annexin V-FITC-positive, PI-negative cells) indicates apoptotic upper PI-positive necrotic Cellular Numerous research studies indicated HePG2, imaged confocal scanning (CLSM; 4a S10). There little observed after 0.5 worth noting 6 mean labeled 500-fold higher clearly suggests likely stained grew healthy normal morphology, indicating owned cytotoxicity. intercellular quantitatively analyze internalization 4b). prone able probes interestingly, executed substituted fresh medium, daughter divided second, fourth, sixth, eighth, 10th, respectively 5). TPATB possess metastasis monitoring. 4 Confocal 0.5, 1, 3, cytometry. Colocalization study carefully accumulating sites brief colocalization fluorescently Hoechst 33258, nucleus, lysosome, mitochondria, LDs, blue nucleus-selective marker (Hoechst 33258) did not overlap Green overlap, Green. colocalized green (87.8%; 6a–6c). demonstrate absorbed neither surface membrane, nor entered nucleus. Interestingly, mainly concentrated LDs few them lysosomes mitochondria. targeting agents treat kinds inflammation diseases caused LDs. organelle Blue labels nuclei. (LysoTracker 493/503) lysosomes, LD, Red emitted Scale bar: 20 μm. (b c) Overlapping commercial marked white lines correlations. At present, endocytic pathways reported, including caveolin-dependent micropinocytosis, forth.51 contribution, chose initially Temperature, amiloride, cytisine, Chlorpromazine (CPZ) inhibitors energy, Qualitative toward comparing changes intracellular inhibitory conditions. decreased °C, relevant pathway temperature impede adenosine 5′-triphosphate (ATP) 7a). Compared control group, CPZ, another inhibitory, weakened. When amiloride (200 mL−1), cytisine minimal unrelated micropinocytosis endocytosis. quantitative assessment 7b), untreated group. We regarding strongly uptaken process relied rather 7 inhibitors: temperatures (4 °C); (+Cytisine, 200 mL−1); CPZ (+Chlorpromazine, (+Amiloride, mL−1). analyzed Conclusions presented NPs, numerous advantages,