Article pubs.acs.org/cm
Supramolecular Hybrids of AIEgen with Carbon Dots for Noninvasive Long-Term Bioimaging Jianxu Zhang,†,‡ Min Zheng,§ Fengli Zhang,∥ Bin Xu,*,∥ Wenjing Tian,∥ and Zhigang Xie*,† †
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China ‡ University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, P. R. China § School of Chemistry and life Science, Advanced Institute of Materials Science, Changchun University of Technology, 2055 Yannan Street, Changchun, Jilin 130012, P. R. China ∥ State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, 130012 Jilin P. R. China S Supporting Information *
ABSTRACT: Fluorescent bioprobes have been regarded as promising tools for bioimaging in recent years due to their excellent properties. However, the aggregationcaused quenching of emissions is a big limitation in practice for this strategy. Organic dyes with aggregation-induced emission (AIE) feature can effectively solve this problem. Herein, stable fluorescent nanoparticles were prepared by supramolecular assembling of carbon dots (CDs) and hydrophobic AIEgen. The formulated CDsG-AIE 1 exhibits superior physical and photo stability than AIE self-assembling nanoparticles in various physiology conditions. On the other hand, the formulated CDsG-AIE 1 also showed advanced features such as large Stokes shift, good biocompatibility, high emission efficiency, and strong photobleaching resistance. More importantly, the CDsG-AIE 1 can be readily internalized by HeLa cells, and strong red fluorescence from the nanoparticles can still be clearly observed after six generations over 15 days. In addition, the CDsG-AIE 1 also exhibits superior long-term imaging ability in vivo. These incredible features make the AIE nanoparticles to be an ideal fluorescent probe for noninvasive long-term tracing and imaging applications. This work highlights the potential of using carbon dots to assemble AIEgen for the preparation of nanoscale AIEgen-contained particles for desirable bioimaging and diagnostic. for this strategy.16 The emergence of fluorescent materials with aggregation-induced emission (AIE) characteristics provide a promising solution for the problem of ACQ.17 After Tang’s group first discovered the peculiar photophysical process,18 many AIE fluorophores have been synthesized and explored in chemical sensing and bioimaging.19−27 For bioimaging, AIE nanoparticles showed more emissive, high cellular retention, resistant to photobleaching, flexible surface functionalization, robust biocompatibility, and long-term imaging.22,24,28−32 Many kinds of AIE-based nanoparticles have been developed through many strategies, such as physical cladding and adsorption and covalent AIE binding.32−37 For example, 1,2-distearoyl-sn-glycero-3-phosphoethan-olamine-N-[methoxy(polyethylene glycol) (DSPEPEG5000) was widely used as the encapsulation matrix for preparing AIEgen-containing nanoparticles.37 Although great progress has been made, it is still worth developing universal, efficient, and alternative methods to fabricate AIEgen nanoparticles.33
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ioimaging is a highly important and powerful tool in life science and biological research today because it makes the process of diagnosis easy and offers a unique approach to visualize the morphological details of cells and tissues.1−3 In recent years, researchers have paid increasing attention to the use of various imaging techniques, such as magnetic resonance imaging, positron emission tomography, computed tomography, single photon emission computed tomography, fluorescence imaging, and ultrasound.4 Among these imaging techniques, fluorescence imaging possesses numerous advantages, for example, minimal invasiveness, high contrast, good temporal resolution, high sensitivity, large in vitro and in vivo throughputs, and ease of use.5−8 In the past decade, fluorescent proteins, quantum dots, carbon dots, organic fluorescent dyes, and lanthanide chelates have been extensively studied and used as imaging agents.6,9−11 However, intrinsic drawbacks such as poor stability, short Stokes shift, poor biocompatibility, and degradation under repeated excitation have limited their application in bioimaging.9−11 In recent years, fluorescent organic nanoparticles based on organic dye molecules have acquired great promise for bioimaging due to their unique size, ease to prepare, and abilities to be modified with biological recognition ligand.12−15 However, the aggregation-caused quenching (ACQ) of emissions is a big limitation in practice © 2016 American Chemical Society
Received: November 16, 2016 Published: November 18, 2016 8825
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Scheme 1. (a) Chemical Structures of AIE dyes 1, 2, and 3 and (b) Schematic Illustration of CDsG-AIE Preparation and Cellular Imaging
Figure 1. (a) DLS intensity-weighted diameter of CDsG-AIE 1. Insets: TEM image of CDsG-AIE 1. Scale bar in TEM image: 200 nm. (b) Absorbance spectra and (c) PL (excited at 450 nm) spectra of AIE 1, CDsG, and CDsG-AIE 1. (d and e) Photographs of AIE 1 in THF, CDsG in water, and CDsG-AIE 1 in water under room light (left picture) and UV light (right picture), respectively.
application in bioimaging,48,51,52 drug delivery,53−56 pH responsive materials,53 and analytical investigations.57 In our previous work, we have reported CDs as fluorescent probe and theranostic agent.47,48 Inspired by recent supramolecular hybrid of CDs with drug and protein,58,59 we anticipate that CDs also could assemble with hydrophobic AIE molecules into fluorescent nanoparticles through supramolecular interactions.
Carbon nanomaterials have been extensively studied and used in sensors, electronics, renewable energy, photonics, and biomedicine.38−44 It is worth mentioning that fluorescent nanodiamonds as diagnostic and therapeutic agents have been widely studied due to their high chemical inertness and biocompatibility. However, the major drawback of them have limited their applications, such as the difficult fabrication, forming large aggregates in aqueous solution and the lack of any bright intrinsic PL.45,46 Among another various carbon nanomaterials, carbon dots (CDs) have attracted tremendous attention due to favorable biocompatibility, high solubility in water, high photostability, and flexibility in synthesis and modification.47−50 Until now, CDs have extensive potential
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RESULTS AND DISCUSSION Preparation and Characterization of CDsG-AIE NPs. Herein, carbon dots were used to assemble with AIEgen to form stable fluorescent nanoparticles. Three AIE molecules were synthesized, and corresponding fluorescent nanoparticles 8826
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Figure 2. Stability of size distribution of (a) CDsG-AIE 1 and (b) AIE 1 NPs during 8 days. (c) The stability of absorbance intensity and (d) fluorescence intensity of CDsG-AIE 1 and AIE1 NPs for 7 days, respectively. Data represent mean values ± standard deviation, n = 4.
were prepared as shown in Scheme 1. We first prepared the CDs named CDsG (made from glutamic acid and glucose) according to our previous work,48,58,59 and then added AIE molecules into water solution containing CDsG under vigorous stirring at room temperature to form the hybrid of CDsG and AIE dyes (CDsG-AIE). As revealed by TEM and dynamic light scattering (DLS) analysis (Figure S1a), the as-synthesized CDsG had an average size of about 3.6 nm. The absorbance and emission spectra of CDsG in aqueous dispersion were shown in Figure S1b, its emission peak was at 550 nm. As shown in Figures 1a and S2, all three CDsG-AIE nanoparticles were spherical and possessed average sizes of 105.7 nm (CDsGAIE 1), 164.2 nm (CDsG-AIE 2), and 190.4 nm (CDsG-AIE 3), respectively. This result indicated the assembly between CDs and AIEgen with various structure is universal. Then we used X-ray photoelectron spectroscopy (XPS) to explore the elemental composition and chemical bonds of CDsG and CDsG-AIE 3. As shown in Figure S3, three peaks at 530.9, 399.4, and 284.6 eV were attributed to O 1s, N 1s, and C 1s XPS characteristic peaks, respectively. The ratio of C/O was about 0.985 in CDsG, while that of CDsG-AIE 3 was about 0.84, indicating the successfully assembly between CDsG with AIE 3. The high-resolution XPS spectra of C 1s in CDsG (Figure S3b) could be deconvolved into three components, corresponding to sp2 carbons in CC/C−C at 284.6 eV, sp3 atoms in C−N/C-O at 285.9 eV, and a carboxyl group at 288.6 eV. These carbon atoms are good for CDsG to assembly with AIEgens by noncovalent interactions. As shown in Figure S 3d, after assembly with AIE 3, the ratio of these carbon atoms were changed, further confirming the successfully assembly between CDsG with AIE 3. We also used Fourier transform infrared (FT-IR) spectra to further study the assembly between CDs and AIEgen. As shown in Figure S4, the spectrum of the CDsG showed strong bands at 1407, 1596, and 3405 cm−1, which are
attributed to the presence of the respective aromatic amine and the aromatic skeleton of CC, hydroxyl, and carboxyl groups, respectively. Compared with CDsG, new peaks at 1507 and 1493 cm−1 and other vibration bands in the fingerprint region were assigned to stretching vibration of AIE 3, showing the successful assembly between CDsG and AIE 3. In addition, the bands at 1596 and 3405 cm−1 in CDsG shifted to 1584 and 3392 cm−1, respectively, in the case of the assembly between CDsG and AIE 3. This red shift indicates strong interactions between CDsG and AIE 3, presumably as a result of synergy of noncovalent supramolecular interactions including π−π stacking and hydrophobic interactions. Compared to green fluorescence, red fluorescence has lower photodamage to living cells, has more deep penetration in tissue, and can reduce interference from the background autofluorescence. Moreover, CDsG-AIE 1 showed smaller diameter than CDsG-AIE 2 and CDsG-AIE 3. Therefore, CDsG-AIE 1 was used for further study of bioimaging in vitro and in vivo. Next, we compared the absorbance and emission spectra of AIE 1 in THF, CDsG in water and CDsG-AIE 1 in water to further validate the successful synthesis of CDsG-AIE 1. As shown in Figure 1b, the CDsG-AIE 1 showed an additional absorption around 440 nm compared to the spectra of CDsG, which should be from the AIE 1 in CDsG-AIE 1. In Figure 1c, CDsG-AIE 1 gave stronger fluorescence intensity than AIE 1 in THF. The emission maximum of CDsG-AIE 1 was at 640 nm with an intense emission tail extending to 800 nm, and they have a large Stokes shift which is beneficial for in vivo fluorescence imaging. Photographs of AIE 1 in THF, CDsG in water, and CDsG-AIE 1 in water under room light and UV light were shown in Figure 1d,e and exhibited a similar result with Figure 1c. These results suggested that both AIE 1 and CDsG were present in CDsGAIE 1 nanoparticles. 8827
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Figure 3. (a) Photostability of CDsG-AIE 1 under continuous scanning at 488 nm. Insets show confocal images of the cells stained by CDsG-AIE 1 before (0 min, left) and after the laser irradiation for 30 min (right). I0 is the initial PL intensity, while I is that of the corresponding sample after a designated time interval. (b) Cell viability of HeLa cells after incubation with various concentrations of CDsG for 24 h. (c) Cell viability of CDsGAIE 1 in HeLa cells at various concentrations of AIE 1 after 24 h of incubation. (d) Cell viability of CDsG-AIE 1 in HeLa cells after different hours of incubation. Data represent mean values ± standard deviation, n = 4.
Study of the Stability of CDsG-AIE 1. In order to demonstrate the excellent stability of CDsG-AIE 1 nanoparticles, we used the reprecipitation method to directly prepare nanoparticles from AIE 1 (AIE 1 NPs) as control. The AIE 1 NPs were spherical and had an average size of 141.7 nm (Figure S5a), and the absorbance and emission spectra of AIE 1 NPs were shown in Figure S5b. The loading efficiency of AIE 1 in CDsG-AIE 1 was about 80%, which was higher than that of AIE 1 NPs (73%), as determined by UV−vis standard curves (Figure S6). We also detected the fluorescence excitation spectra of CDsG-AIE 1 and AIE 1 NPs. As shown in Figure S7, their spectra were different from 300 to 400 nm, due to the CDsG in the CDsG-AIE 1, so we chose 450 nm as the excitation wavelength for the following study. The fluorescence efficiency was recorded for AIE 1 in different conditions, including AIE 1 in THF/water mixture solutions (f water = 70, 80, and 90 vol %), AIE 1 NPs in water, and CDsGAIE 1. As shown in Figure S8a, little emission from AIE 1 was observed when the fractions of water were 70% and 80%, but the fluorescence intensity dramatically increased when the water fraction reached 90%, and AIE 1 NPs showed the strongest fluorescence intensity. As far as we know, the emission efficiency of AIE dyes is proportional to their aggregation degree. As shown in Figure S8b, the particle size decreased with an increase of the water content. CDsG-AIE 1 possessed a smaller size possibly because of hybriding of CDsG with AIE 1. Furthermore, the fluorescence lifetime and quantum yield (QY) was collected. As shown in Figure S9, the fluorescence lifetime of CDsG-AIE 1 was almost the same as that of AIE 1 NPs. The QY of CDsG-AIE 1 was about 17%, and that of AIE 1 NPs was about 21%. These results illustrated
that hybriding with CDsG showed no obvious influence on optical properties of AIE 1. To study stability of CDsG-AIE 1, we monitored the size distribution, absorbance, and fluorescence spectra of CDsGAIE 1 and AIE 1 NPs for 7 days. As shown in Figure 2a, CDsGAIE 1 maintained the initial hydrodynamic diameter and size distribution in water for 7 days. While the size of AIE 1 NPs changed from 141 to 400 nm, and their PDI value increased to 0.4 (shown in Figure 2b) for 1 day. Then we used TEM to observe the morphologies of nanoparticles. After 36 h, AIE 1 NPs aggregated but the CDsG-AIE 1 kept unchanged (Figure S10). Furthermore, we collected the absorbance and fluorescence spectra of CDsG-AIE 1 and AIE 1 NPs within 7 days. As depicted in Figures 2c and S11a,b, the absorbance of CDsG-AIE 1 decreased slightly and retained more than 88% of the original value. However, that of AIE 1 NPs decreased significantly and attenuated to 34.3% after 7 days. In addition, changes of fluorescence intensity gave the similar results (Figures 2d and S11c,d). These results illustrated that the hybrids of CDs and AIEgen possess excellent physical stability and photostability. In consideration of the microenvironment of tumor tissue, we evaluated the stabilities of CDsG-AIE 1 in solutions with different pH. As shown in Figure S12a,b, there is little attenuated in absorbance and fluorescence when pH value changed from 7 to 2, but decreased remarkably when pH value was 1. DLS revealed no significant size changes when pH value changed from 7 to 4 (Figure S12c), and increased with the pH from 4 to 1. Above results indicated that CDsG-AIE 1 kept stable nanostructure under pH value from 7 to 4. We further evaluated the fluorescence stability of the CDsG-AIE 1 in 8828
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Figure 4. CLSM images of HeLa cells incubated with CDsG-AIE 1 for 1 or 3 h at 37 °C. Cells are viewed in the blue channel for DAPI and the red channel for AIE 1. Scale bars represent 20 μm in all images.
Figure 5. Long-term cell tracing images of the CDsG-AIE 1 at 37 °C for 6 h and then subcultured for designated time intervals including (a) day 0; (b) day 3; (c) day 6; (d) day 9; (e) day 12; and (f) day 15. Scale bars represent 20 μm in all images.
with the CDsG-AIE 1 under laser irradiation for 30 min. There was no obvious bleaching in fluorescence and the CDsG-AIE 1 in HeLa still exhibited a bright red fluorescence signal after 30 min of laser irradiation. For direct comparison, we also study the photostability of AIE 1 NPs, fluorescein sodium, and indocyanine green (ICG) in the same conditions. As shown in Figure S14 (upper pictures), the fluorescence intensity of AIE 1 NPs maintained only about 60% of their initial value after continuous laser irradiation, while the fluorescence intensity of fluorescein sodium (middle pictures) and ICG (lower pictures) rapidly diminished and became negligible due to severe photobleaching. The biocompatibility is very important for fluorescent nanoparticles as bioimaging agents. First, we studied the potential cytotoxicity of CDsG toward human cervical carcinoma (HeLa) cells by MTT (3-[4,5-dimethylthiazol-2-
different conditions including solutions with pH 4−6, PBS (pH 7.4), and DMEM with 10% serum. As depicted in Figure S13, the fluorescence intensity of CDsG-AIE 1 kept unchanged in DMEM with 10% serum and decreased partly in other conditions. All these results revealed that CDsG-AIE 1 could maintain their photoluminescent function in a variety of bioenvironments, which is beneficial for bioimaging applications. Photostability and Biocompatibility of CDsG-AIE 1. The photostability of CDsG-AIE 1 was investigated by monitoring the fluorescence intensity changes upon continuous laser irradiation. As depicted in Figure 3a, after continuous laser irradiation at 488 nm for 30 min, the fluorescence intensity of CDsG-AIE 1 showed a mild intensity decrease and maintained about 90% of their initial value. For further studies, we monitored the red fluorescence signals from HeLa cells labeled 8829
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Figure 6. (a−g) Representative time-dependent in vivo fluorescence images of the tumor-bearing mouse that was subcutaneously injected with CDsG-AIE 1 from day 0 to day 21. (h) Time-dependent fluorescence intensity changes for the tumors. Data represent mean values ± standard deviation, n = 5.
yl]-2,5-diphenyltetrazolium bromide) assay. Figure 3b showed CDsG have low cytotoxicity toward HeLa cells at different concentrations. As shown in Figure 3c,d, with the increase of concentration and time, the cell viabilities slightly decreased, but still maintained above 90%, respectively. Cellular Uptake of CDsG-AIE. We next studied the cellular uptake and imaging of CDsG-AIE 1 by confocal laser scanning microscopy. HeLa cells were incubated with CDsG-AIE 1 for 1 and 3 h, and 4, 6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei. As shown in Figure 4, after incubation with CDsG-AIE 1, an intense cytoplasmic red fluorescence around nuclei can be clearly observed under laser excitation at 488 nm, demonstrating that CDsG-AIE 1 can pass across the cell membrane into the cytoplasm. The fluorescence intensity increased obviously from 1 to 3 h, indicating that the CDsGAIE 1 had a sustained cellular uptake and accumulation in HeLa cells. CDsG-AIE 2 and CDsG-AIE 3 indicated similar cellular uptake as validated in Figures S15 and S16. These results validated that the as-prepared CDsG-AIE nanoparticles could be applied for cellular imaging. To study the influence of
stability of nanoparticles on cellular uptake, we studied the cellular uptake of AIE 1 NPs in two conditions: freshly made and storing 36 h. As shown in Figure S17, the freshly made AIE 1 NPs could pass into cells, but after 36 h storing, the AIE 1 NPs were difficult to enter cancer cells. Long-Term Imaging in Vitro and in Vivo. The applications of the CDsG-AIE 1 for in vitro and in vivo longterm imaging were subsequently investigated. As demonstrated in Figure 5, we captured fluorescence images of HeLa cells treated with CDsG-AIE 1 at 37 °C for 6 h and then subcultured for different days. Strong and bright red fluorescence was observed in Figure 5a (day 0) and gradually decreased with time. After 15 days subculture, red fluorescence from CDsGAIE 1 was still clearly observed in HeLa cells (Figure 5f), and the fluorescence intensity was kept at 40% of the initial intensity (Figure S18). As a control, MitoTracker Green FM (commercial dye) can only track the living cells for three passages or two passages.23 All results suggest that CDsG-AIE 1 may act as a fluorescent probe for long-term imaging. 8830
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AIE 1 NPs were prepared using a reprecipitation method. In a typical procedure, the AIE 1 solution (400 μL) was quickly dropwise dispersed into 4 mL of Milli-Q water with vigorous stirring at room temperature for 30 min. Then the solutions were dialyzed against Milli-Q water for 24 h to remove THF, the cutoff molecular weight of the dialysis bags is 3500. Cell Experiments. The experimental procedure was given in our previous work.13,14,36,47,48 The experimental procedure in detail is shown in the Supporting Information. Long-Term Cellular Imaging. Cells harvested in a logarithmic growth phase were seeded in 6-well plates at a density of 2.5 × 105 cells/well and incubated in DMEM for 24 h. The medium was then replaced by 2 mL of DMEM containing CDsG-AIE 1 with 8 μg mL−1 of AIE 1 and incubated for 6 h at 37 °C (Day 0). Then the cells were diluted and subcultured in 6-well plates containing cell culture coverslips for designated days (from day 0 to day 15). Upon reaching the designated day, the cells were washed with PBS buffer and then fixed with 4% of paraformaldehyde solution for 10 min. After that, DAPI was added for another 5 min incubation to locate the nucleus. Later, the cells were washed with PBS and observed using confocal laser scanning microscopy (CLSM, Zeiss LSM 700). Animal Experiments. All animal experiments were performed complying with the NIH guidelines for the care and use of laboratory animals. HeLa cells were administered by subcutaneous injection into the armpit or the right flank region of the male BALB/c mice. In Vivo Imaging in Living Mice. We choose the mice to bear the tumor in the armpit to carry out this study. In order to detect the imaging capacity, CDsG-AIE 1 (1 mg kg−1 AIE 1) was administrated into the mice via intratumor injection and subcutaneous injection. Then, under anesthesia, the in vivo imaging was performed using an in vivo imaging system at 0, 1, 3, 6, 10, 14, and 21 days postinjection, respectively. Maestro software was used to remove the mouse background fluorescence.
Moreover, this long-term imaging strategy only needed one shot rather than addition of imaging agents every time. In order to study the in vivo imaging capacity of CDsG-AIE 1, a U14 tumor-bearing BALB/c mouse was administered by subcutaneously injection with CDsG-AIE 1 near the tumor and then imaged by an in vivo optical imaging system. As shown in Figure 6a−g, fluorescence emission from the site injected CDsG-AIE 1 could be readily detected in 21 days. We used spectral unmixing with the Maestro software to get rid of the mouse background fluorescence. At the initial injection time (day 0), CDsG-AIE 1 formed a highlight point at the injection site. As time goes on, the fluorescence intensity gradually decreased, but still maintained relative strong signals after 21 days, implying CDsG-AIE 1 still remained at the injection site. Quantitative analysis of the fluorescent signals at the injection site revealed negligible change of fluorescence intensity over time (Figure 6h). Figure S19a−g shows optical images of the mouse at each time point. The body weight of the mouse did not decrease and gradually increased over time (Figure S19h), which suggested that CDsG-AIE 1 had no distinct systemic toxicity. We also evaluated the in vivo long-term imaging capacity of CDsG-AIE 1 in tumor-bearing mice by intratumorally injection during 14 days (Figures S20 and S21). The results were almost the same as that of the subcutaneously injection samples (Figures 6 and S19).
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CONCLUSIONS In summary, stable fluorescent nanoparticles were prepared by supramolecular assembling of CDs and AIEgen and used in noninvasive long-term bioimaging. The formulated hybrids exhibit superior physical and photo stability in physiology conditions. Significant aggregation-induced emission properties, large Stokes shift, and good biocompatibility are in favor of the excellent long-term imaging in cells and animals. The superior performance of CDs-AIE hybrids makes them promising for various unique biomedical applications as long-term cellular tracers for monitoring drugs therapeutic effects, biological processes, pathologic evolution, and so on. This supramolecular assembling strategy may open up a new route for the preparation of nanoscale AIEgen-containing particles for desirable bioimaging and diagnostics.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b04894. Experiments methods in detail, characterization of CDsG, standard absorbance curve of AIE 1, stability of CDsG-AIE 1 and AIE 1 NPs in different conditions, in vitro cell imaging of CDsG-AIE 2 and CDsG-AIE 3 in HeLa cells, representative time-dependent in vivo fluorescence images of the tumor-bearing mouse that was intratumorally injected with CDsG-AIE 1 from day 0 to day 14 (PDF)
MATERIALS AND METHODS
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Materials and Characterization. Carbon dots (CDsG) were prepared following the protocol that has been reported.48 The detailed description of the protocol was shown in the Supporting Information. Milli-Q water was collected from a Milli-Q system (Millipore, USA). AIE luminogens used in this article were synthesized in Tian’s group. Chemicals and reagents were acquired from commercial sources without further purification. The instruments used here for characterization have been provided in our previous works,13,14,36,47,48 and the detailed description of the instruments was shown in the Supporting Information. Preparation of CDsG-AIE Nanoparticles and AIE 1 NPs. CDsG-AIE nanoparticles were achieved following the protocol as follows: CDsG was dissolved in Milli-Q water at a certain concentration (100 μg/mL). AIE molecules were dissolved in THF at a certain concentration (200 μg/mL). The AIE solution (400 μL) was quickly dropwise dispersed into 4 mL of Milli-Q water containing CDsG with vigorous stirring at room temperature for 30 min. Then the solutions were dialyzed against Milli-Q water for 24 h to remove THF, the cutoff molecular weight of the dialysis bags is 3500. The dialyzed solution was then centrifuged at 5000 r/min for 10 min to remove the excess CDsG.
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Zhigang Xie: 0000-0003-2974-1825 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The project was supported by the National Natural Science Foundation of China (Project No. 51522307) and 973 Program (2013CB834701). 8831
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