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