- Email: [email protected]
The combination of controlled living polymerization and multicomponent reactions to prepare tetraphenylethylene-containing fluorescent block copolymers
Dyes and Pigments 171 (2019) 107673
Contents lists available at ScienceDirect
Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
The combination of controlled living polymerization and multicomponent reactions to prepare tetraphenylethylene-containing fluorescent block copolymers
T
Jianwen Tiana, Zhongxu Chena, Ruming Jianga, Liucheng Maoa, Meiying Liua, Fengjie Denga, Hongye Huanga, Liangji Liub,∗∗∗, Xiaoyong Zhanga,∗, Yen Weic,d,∗∗ a
Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang, 330031, China Jiangxi University of Traditional Chinese Medicine, 56 Yangming Road, Jiangxi, Nanchang, 330006, China Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, China d Department of Chemistry and Center for Nanotechnology, Chung-Yuan Christian University, Chung-Li, 32023, Taiwan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: RAFT polymerization Multicomponent reactions Aggregation-induced emission Fluorescent organic nanoparticles Cell imaging
High-efficiency multicomponent reactions (MCRs) have received increasing attention and widely used to prepare functional materials because of many prominent advantages such as simple “one-pot” operation, clear product structure, atom economic and environmentally friendly. Combining conventional reversible addition-fragmentation chain-transfer (RAFT) polymerization and new MCRs to prepare multifunctional polymer nanomaterials should be an interesting research direction for polymer chemistry and materials science. In this research, aggregation-induced emission (AIE)-active fluorescent amphiphilic block copolymers were designed and prepared by the combination of reversible RAFT polymerization and mercaptoacetic acid locking imine (MALI) method. These amphiphilic block copolymers that contain the hydrophobic tetraphenylethylene (TPE) derivative and hydrophilic polyethylene glycol (PEG) could be self-assembled into structure with spherical morphology and small particle size. Owing to the aggregation of hydrophobic TPE derivative, these self-assembles display obvious AIE feature. On the other hand, thus-obtained fluorescent organic nanoparticles (FONs) could also be well dispersed in aqueous solution because of the existence of hydrophilic PEG. All the characterization results demonstrated that we could facilely synthesized these fluorescent amphiphiles through the strategy described above. The preliminary results from biological assays suggested that these AIE-active FONs are of low cytotoxicity and could be potentially used for living cell imaging. Taken together, we reported a one-pot strategy for synthesis of TPE-containing FONs through the combination of RAFT polymerization and MALI reaction for the first time. This strategy could also be extended for fabrication of many other functional composites owing to their unique features. We trust this work should be great advance the biomedical applications of AIE-active functional materials.
1. Introduction In recent years, considerable attention has been paid to study fluorescent organic nanoparticles (FONs) in biological imaging owing to their intensive fluorescence, good photostability and outstanding optical properties [1–2]. Furthermore, biological imaging with high sensitivity can be applied to observe the biological processes and improve the veracity and decreasing the operation simultaneously [4,5]. To expand the scope of biological imaging applications, many novel
fluorescent materials have been reported, especially their synthetic methods. In the past few years, fluorescent inorganic nanoparticles (FINs), such as semiconductor quantum dots, [6] silicon quantum dots, [7,8] carbon quantum dots [9–11] and metal nanoclusters, [12,13] which were regarded as ideal fluorescent probes. However, the biomedical applications of FINs were largely limited because of their toxicity and poor degradability [5,14–16]. In contrast, FONs can be regarded as the ideal fluorescent agents to replace FINs for their excellent biodegradability, low toxicity and well designability.
∗
Corresponding author. Corresponding author. Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, China. ∗∗∗ Corresponding author. E-mail address: [email protected] (X. Zhang). ∗∗
https://doi.org/10.1016/j.dyepig.2019.107673 Received 22 May 2019; Received in revised form 25 June 2019; Accepted 25 June 2019 Available online 28 June 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
smaller size, great water dispersibility and intensive fluorescence emission at aggregation. Following, based on these outstanding features, the cell toxicity and cell staining performance of these AIE-active FONs have been carried out. Results suggest low cytotoxicity, good biocompatibility and stained cells ability of synthetic AIE-active FONs in this contribution [35,36]. Compared with the traditional methods via step-by-step or post-modification approaches, [37–39] we can see that the method that combination of MALI MCR strategy and RAFT polymerization is more convenient to prepare functional polymeric materials with well-defined structure, multifunctional interaction and desirable polymeric properties [35]. In a word, we expect the synthetic FONs with AIE effect are helpful in further biomedical applications, and new fabricated tactics may provide inspiration for the preparation of multifunctional polymer materials.
Unfortunately, most of conventional organic dyes still have a serious problem, which is the vicious aggregation-caused quenching (ACQ) effect. In other word, the fluorescence intensity of conventional organic molecules would be sharply decreased or even completely quenched at high concentration or aggregation state, this fatal defect had prevented them from being further applied in the field of bioimaging owing to the aggregation of these hydrophobic organic molecules in aqueous solution [5,17–20]. Therefore, many researchers have spent a great deal of efforts to solve this issue. Fortunately, Tang et al. first promoted the aggregation-induced emission (AIE) concept in 2001 [21]. They found that some fluorescent organic molecules can emit obvious enhanced fluorescence in their aggregated state. The phenomenon is opposite to the ACQ effect, and these FONs with AIE feature are regarded as the most promising candidates for bioimaging to overcome the ACQ effect of FONs based on conventional organic molecules. Although there were many strategies have been reported to prepare AIE-active FONs in recent years, there is still rarely studied on synthesis of AIE-active FONs through the combination of controlled living radical polymerization and multicomponent reactions (MCRs). As compared with the traditional methods for preparation of AIE-active FONs, the MCRs possess the following advantages, such as atom economy, molecular diversity, facile operation, mild reaction conditions and introduction of new structure and functions. Therefore, some efforts in designing new MCRs to obtain AIE-active FONs have been developed. For example, our group previously reported functional AIE-active fluorescent polysaccharide nanoparticles to label cells by the MCR. But the unicity of monomer limits its development for the preparation of multifunctional materials. So far, our group has still been exploiting new synthetic strategies to prepare AIE-active FONs over the past several years and achieve some effects [22–26]. As we all know, MCRs are one-pot procedures, which can obtain a product with various archetypical functional groups of each reactants and easy to operate for single-first discovered by Passerini in 1921. From then on, the research interest in the MCRs increases rapidly for their unique advantages, such as higher atomic economy, molecular diversity and high yield [27]. In addition, compared with other strategies for the preparation of AIE-active FONs, MCRs are easier to achieve commercialization for their efficiency synthetic steps. Over the past few decades, previous MCRs such as the Passerini reaction, Mannich reaction (a three-component reaction) and MALI reaction [28–30] were deeply studied because they play a vital role in polymer chemistry and material science. Although MCRs possess advantages in organic synthesis, the polymer synthesis based on MCRs are still faced huge challenges such as narrow molecular weight distribution, high molecular weight, lesser polymerizable monomers and divinable polymeric structure. For example, AIE-active polymeric assemblies prepared by controlled RAFT polymerization were developed by Yuan's group, this method possesses various advantages of abundant polymeric monomers, narrow polymer distribution and process control et al. Therefore, integrating the advantages of MCRs and reversible addition-fragmentation chain-transfer (RAFT) to prepare functional polymeric materials should be of special interesting and valuable. Previously, Tao et al. have made considerable efforts for the synthesis of functional polymers or polymeric materials by various MCRs and controlled living polymerization in a one-pot route. However, the preparation of fluorescent polymeric self-assemblies with AIE feature has still largely unexplored. In this contribution, a new fabricated tactic for synthesis of AIEactive FONs was reported via the combination of mercaptoacetic acid locking imine (MALI) reaction and RAFT polymerization in a “one-pot” route [31–33]. In the MALI reaction (a three-component reaction), we use mercaptoacetic acid as the "lock" to combine the amino-containing chromophore TPE-NH2 and the aldehyde-terminated polymerizable monomer of 4-formylphenyl acrylate (FBA), which can avoid the complicated operation [34]. The formation final product PEG-TPE polymer with amphiphilic property can self-assemble into nanoparticles in aqueous solution. Thus-prepared AIE-active nanoparticles possess
2. Experimental sections 2.1. Materials and measurements All of the reagents and solvents were purchased from commercial sources of analytical grade and used directly without further purification, and distilled water was used in the experiment. Mercaptoacetic acid (98%, MW: 92.12 Da), methacryloyl chloride (95%, MW: 104.53 Da), and triethylamine (99%, MW: 101.19 Da), 4-hydroxybenzaldehyde (98%, MW: 122.12 Da), 2,2′-azobis(2-methylpropionitrile) (AIBN, 99%, MW: 164.21 Da) and poly(ethylene glycol) methacrylate (PEGMA, MW: 500 Da) were obtained from the Aladdin company. The AIE dye TPE-NH2 and 4-cyano-4-(ethylthiocarbonothioylthio)pentanoic acid as chain transfer agent (CTA) were synthesized and characterized in our group's previous work. 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance400 spectrometer with DMSO‑d6 as the solvent. The synthetic materials were characterized by Fourier transform infrared spectroscopy (FT-IR) using KBr pellets. The FT-IR spectra were obtained from a Nicolet5700 (Thermo Nicolet corporation). Transmission electron microscopy (TEM) images were recorded on a Hitachi 7650B microscope operated at 80 kV; the TEM specimens were obtained by placing a drop of ethanol nanoparticle suspension on a carbon-coated copper grid. The fluorescence data was obtained from a fluorescence spectrophotometer (FSP, model: C11367-11), which was purchased from Hamamatsu (A Japan company). 2.2. Preparation of FBA The preparation route of FBA monomer was showed in Scheme S1. Triethylamine (10 mmol, 1.01 g) and 4-hydroxybenzaldehyde (10 mmol, 1.22 g) in 100 mL of anhydrous dichloromethane were stirred at 0 °C. Then acryloyl chloride (6 mmol, 540 mg) was added dropwise into the mixture and reacting at room temperature for 1 h. The obtained crude product was purified by silica-gel column chromatography using ethyl acetate/petroleum ether (1/50) as eluent to give FBA. (3.9 g, yield 89%) 2.3. One-pot synthesis of the Poly(PEGMA-co-FBA-TPE-NH2) The AIE-active copolymer Poly(PEGMA-co-FBA-TPE-NH2) was prepared by a simple one-pot process as depicted follows. First, PEGMA (1 mmol, 500 mg), TPE-NH2 (0.1 mmol, 47 mg), mercaptoacetic acid (0.2 mmol, 18 mg), FBA (0.1 mmol, 19 mg), AIBN (0.01 mmol, 1.6 mg) and CTA (0.05 mmol, 12 mg) were dissolved in dry THF solvent (3 mL) and put into a Schlenk tube. The Schlenk tube was sealed and filled with N2 for 30 min to remove oxygen. After that, the reaction mixture was continuous stirred at 70 °C for 12 h. The crude product was purified by dissolved in THF and precipitated into diethyl ether for three times, and then dried under vacuum for further characterization. 2
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
Scheme 1. Schematic showing the one-pot synthesis of PEG-TPE copolymers through the MALI reaction and RAFT polymerization. The advantages of the MCRs have also displayed in the Scheme.
2.4. Cytotoxicity evaluation of the Poly(PEGMA-co-FBA-TPE-NH2) FONs The biocompatibility of Poly(PEGMA-co-FBA-TPE-NH2) FONs was evaluated through a normal assay based on the CCK-8 assay. The detailed procedures were described in the supporting information. 2.5. Confocal microscopic imaging of the Poly(PEGMA-co-FBA-TPE-NH2) FONs The detailed experimental procedures for cell uptake behavior of were evaluated by confocal laser scanning microscopy (CLSM) were described in the supporting information. 3. Results and discussion 3.1. Characterization of Poly(PEGMA-co-FBA-TPE-NH2) The AIE-active Poly(PEGMA-co-FBA-TPE-NH2) copolymers were prepared by a simple one-pot process between PEGMA, TPE-NH2, mercaptoacetic acid and FBA based on the RAFT polymerization and MALI reaction (Scheme 1). As we can see, the fluorescent polymer Poly (PEGMA-co-FBA-TPE-NH2) introduced the functional groups of each reactant through the polymerization, which makes it with many outstanding properties such as excellent dispersion in water, good biodegradability and intensive fluorescence emission. Detailed synthesis and characterizations of the intermediates and final compounds are shown in the experimental sections, Scheme S1, and Fig. S1−S3. In order to confirm its good properties for biomedical applications, many characterization techniques such as 1H NMR, FT-IR, UV–vis and CLSM were performed. Fig. 1 shows the 1H NMR spectra of FBA and Poly(PEGMAco-FBA-TPE-NH2). Two chemical shifts at 5.53 ppm and 6.09 ppm can be found in sample of FBA, which can be ascribed to the protons connected with the C=C bond. After reaction, some characteristic chemical shifts of PEGMA units between 3.0 and 4.0 ppm are retained, and the peaks of C=C bond protons are disappeared. Moreover, multiple peaks located at 6.9–8.1 ppm derived from the aromatic rings are appeared in the spectrum of Poly(PEGMA-co-FBA-TPE-NH2) (Inset figure), providing powerful evidences of successful preparation of the Poly (PEGMA-co-FBA-TPE-NH2) polymer. Other than NMR spectra, FT-IR spectra were also used to verify whether successful synthesis of Poly(PEGMA-co-FBA-TPE-NH2). Fig. 2 shows the IR spectra of TPE-NH2, FBA and Poly(PEGMA-co-FBA-TPENH2). Obviously, two sharp peaks at 3480 and 3379 cm−1 could be
Fig. 1. The 1H NMR spectra of FBA (blue) and Poly(PEGMA-co-FBA-TPE-NH2) (red) measured in DMSO‑d6 at room temperature. The inset figure shows that the aromatic rings have been included in the copolymers. All the above results implied that the AIE-active FONs have been successfully synthesized through the RAFT polymerization and MALI reaction. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
found in TPE-NH2, which should be attributed to the stretching vibration of amino group. In the sample of FBA monomer, the stretching vibration of C=C bond was located at 1637 cm−1. Compared with the spectra of FBA monomer and TPE-NH2, some characteristic peaks (i.e., C-H stretching vibration of the aromatic rings, C-O stretching vibration) are retained in the Poly(PEGMA-co-FBA-TPE-NH2), and the C=O stretching vibration of the ester and amide group at 1727 cm−1, O-H stretching vibration at 3479 cm−1, C-H stretching vibration at 2862 cm−1, C-S stretching vibration at 617 cm−1 are appeared, accompanied by the disappearance of both of the stretching vibration of amino group and the C=C stretching vibration of alkene, further demonstrating successful synthesis of these fluorescent copolymers through the one-pot process. In addition, the number-average molecular weight (Mn) of Poly(PEGMA-co-FBA-TPE-NH2) was also obtained by GPC and is 11130 g mol−1 with a relatively narrow polydisperse index (PDI) of 1.14 (Fig. S4). In fact, the self-assembly of amphiphilic block copolymers is highly 3
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
properties and good water dispersibility. As shown in Fig. 4A, the Poly (PEGMA-co-FBA-TPE-NH2) FONs display an absorption and emission at 386 and 528 nm, respectively. The excitation curve shows the maximum intensity at 377 nm (Fig. S5). The inset of Fig. 4A shows the photographs of Poly(PEGMA-co-FBA-TPE-NH2) in water and bright yellow-green fluorescence when the aqueous solution of Poly(PEGMAco-FBA-TPE-NH2) under UV lamp irradiation at 365 nm. It's worth nothing that no any suspended matter can be found in the solution, and the background can be intuitively observed. This also indicate that the Poly(PEGMA-co-FBA-TPE-NH2) with great water dispersibility. Compared with the absorption spectra of FBA and TPE-NH2, the absorption wavelength at 386 nm in Poly(PEGMA-co-FBA-TPE-NH2) should be attributed to the introduction of TPE-NH2 (Fig. S6). As presented in Fig. 4B, the photoluminescence (PL) intensity proportionally increases with increase of the concentrations of Poly(PEGMA-co-FBA-TPE-NH2) FONs, indicating Poly(PEGMA-co-FBA-TPE-NH2) FONs with the antiACQ property. In generally, the key to improve the stability of FONs relied on reducing CMC value according to the previous literatures [40,41]. Therefore, the CMC value of FONs should be measured. Fig. 4C shows that Poly(PEGMA-co-FBA-TPE-NH2) has very low CMC value of 23.2 μg mL−1, which even lower than the previous reported AIE-active dye-based FPNs [42,43]. the low CMC value demonstrates that these FONs can stably exist in highly dilute aqueous solution. For biomedical applications, the good stability is critically important. As shown in Fig. 4D, only a slight decrease in the PL intensity (< 5%) is observed after continuously irradiation at 405 nm wavelength for 1 h, indicating that Poly(PEGMA-co-FBA-TPE-NH2) FONs possess desirable photostability. In addition, after storage at room temperature for 24 h, the average diameter nearly has not changed. It indicated that these AIEactive FONs possess great colloidal stability (Fig. S7).
Fig. 2. The FT-IR spectra of TPE-NH2 (A), FBA (B) and Poly(PEGMA-co-FBATPE-NH2) (C). The obvious increase of IR intensity at 2862 cm−1 indicate that the PEGMA has been copolymerized in the copolymers. The introduction of C=O and C-S functional groups was also found by FT-IR spectra. All the above results further suggested that the successful preparation of these fluorescent copolymers.
3.2. Biocompatibility of the Poly(PEGMA-co-FBA-TPE-NH2) FONs Biocompatibility of fluorescent nanoparticles determines whether it can be widely applied in biomedicine. In the study of their biocompatibility, L929 cells were incubated with different concentrations of Poly(PEGMA-co-FBA-TPE-NH2) FONs for 24 h. The cell viability was evaluated using the CCK-8 assay. As shown in Fig. 5, L929 cells can grow well even incubating with high concentration of Poly(PEGMA-coFBA-TPE-NH2) FONs for 24 h. These results demonstrated their excellent biocompatibility. Moreover, owing to these fluorescent polymeric nanoparticles are composited with the biocompatible and hydrophilic PEG and chemical inert AIE dye. These AIE-active FONs should be more promising for biomedical applications as compared with the inorganic fluorescent materials, which have been demonstrated to have significant toxicity to living organisms. In generally, based on their outstanding features, such as excellent biocompatibility, biodegradability, AIE fluorescence, low toxicity and great water dispersibility, Poly(PEGMA-co-FBA-TPE-NH2) FONs could serve as the ideal candidates for various biomedical applications. Furthermore, due to the advantages of this synthetic method with MCR and RAFT polymerization such as simple and highly efficient, ease of operation and high yields, this method is also very helpful in producing other functional polymeric materials to extend their biomedical applications.
Fig. 3. The size distribution of Poly(PEGMA-co-FBA-TPE-NH2) FONs determined by DLS. The inset shows TEM image of self-assembled nanoparticles for Poly(PEGMA-co-FBA-TPE-NH2) FONs, the scale bar is 500 nm. The TEM and DLS results clearly evidence the formation of nanoparticles and imply the successful preparation of the amphiphilic fluorescent copolymers.
related to their hydrophilic segments and hydrophobic portions. Thus, FONs were prepared by the self-assembly of amphiphilic Poly(PEGMAco-FBA-TPE-NH2) copolymers. The size, morphology of these self-assemblies was studied by TEM and DLS. The inset of Fig. 3 shows the TEM images of Poly(PEGMA-co-FBA-TPE-NH2) FONs after dispersion in water, many spherical nanoparticles with diameters between 100 and 150 nm could be observed. The formation of nanoparticles indicated that the amphiphilic copolymers are successfully synthesized and can self-assemble into nanoparticles in aqueous solution. In the process, the hydrophobic segments of the AIE dyes were aggregated in the inner as the core, while the hydrophilic PEGMA will dissolve in water that was regarded as the shell. DLS results display the hydrodynamic diameter of the Poly(PEGMA-co-FBA-TPE-NH2) FONs dispersed in water is 168.8 nm with the polydispersity index (PDI) of 0.273. This means that Poly(PEGMA-co-FBA-TPE-NH2) FONs possess better water dispersibility and small size. Therefore, these particles can easily get inside the cell via the endocytosis of cells, making them ideal candidates for biomedical applications. Owing to the self-assembly of AIE-active Poly(PEGMA-co-FBA-TPENH2) copolymers, the obtained FONs show significant optical
3.3. Biological imaging evaluation of Poly(PEGMA-co-FBA-TPE-NH2) FONs The in vitro cell uptake behavior of Poly(PEGMA-co-FBA-TPE-NH2) FONs were evaluated by using CLSM observation. As show in Fig. 6A, strong green fluorescence could be observed in the cytoplasm of the cells when L929 cells were incubated in culture dish with 40 μg mL−1 of Poly(PEGMA-co-FBA-TPE-NH2) FONs for 3 h at 37 °C. In contrast, the center of the cell where is supposed to be the nucleus of cells could only view the weak fluorescence. Fig. 6B is the merged image of Fig. 6A and C, which clearly showed that the Poly(PEGMA-co-FBA-TPE-NH2) FONs 4
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
Fig. 4. (A) Absorption and emission spectra of Poly(PEGMA-co-FBA-TPE-NH2) FONs dispersed in water, the inset shows the photographs of Poly(PEGMA-co-FBATPE-NH2) and bright fluorescence of Poly(PEGMA-co-FBA-TPE-NH2) under UV lamp irradiation at 365 nm (B) PL intensity of Poly(PEGMA-co-FBA-TPE-NH2) in water at different concentrations, the inset shows bright fluorescence of Poly(PEGMA-co-FBA-TPE-NH2) at different concentrations under UV lamp irradiation at 365 nm. (C) The relationship of the PL intensity and logarithm of the concentration of Poly(PEGMA-co-FBA-TPE-NH2), the CMC value obtained as 23.2 μg mL−1. (D) Photostability of Poly(PEGMA-co-FBA-TPE-NH2) FONs in different media with UV light irradiation for 1 h.
Fig. 5. Cell viability of L929 cells incubated with Poly(PEGMA-co-FBA-TPENH2) FONs at different concentrations for 24 h. No obvious cell viability decrease was found based on the CCK-8 assay. This is likely ascribed to the surface coating of biocompatible PEG segments.
were mainly entered into the cytoplasm through the endocytosis of cells but cannot enter cell nuclei. Fig. 6C shows the cells still maintained their normal growth morphology, further demonstrating their excellent biocompatibility. Furthermore, as increase of incubation time, fluorescence signals increased, displayed time-dependence (Fig. 6D). The above results showed that this AIE-active FONs could be applied perfectly for bioimaging applications. The Poly(PEGMA-co-FBA-TPE-NH2) FONs with unique AIE-active fluorescence feature can be used in biomedical fields for their excellent cell stain performance. In addition, we
Fig. 6. CLSM images of L929 cells. (A) Excited with a 405 nm laser, (B) bright field and (C) merged image of A and C. L929 cells incubated with PEG-TPE FONs (40 μg mL−1) for 3 h. (D) different incubated time, Ι. 30 min, П. 1 h, Ш. 3 h, Ⅳ. 6 h. Scale bar = 100 μm.
5
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
can replace the monomers of the “one-pot” reaction to synthesize other fluorescent materials. In some ways, this method provides a wider platform to synthesize more AIE-active functional materials for various biological applications.
[13] Lin CAJ, Lee CH, Hsieh JT, Wang HH, Li JK, Shen JL, et al. Synthesis of fluorescent metallic nanoclusters toward biomedical application: recent progress and present challenges. J Med Biol Eng 2009;29(6):276–83. [14] Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, et al. Renal clearance of nanoparticles. Nat Biotechnol 2007;25(10):1165. [15] Liu J, Hu R, Liu J, Zhang B, Wang Y, Liu X, et al. Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models. Mater Sci Eng C-Mater 2015;57:222–31. [16] Yi C, Liu L, Li C-W, Zhang J, Yang M. Synthesis, characterization and biomedical application of multifunctional luminomagnetic core–shell nanoparticles. Mater Sci Eng C-Mater 2015;46:32–40. [17] Long Z, Liu M, Mao L, Zeng G, Huang Q, Huang H, et al. One-step synthesis, selfassembly and bioimaging applications of adenosine triphosphate containing amphiphilies with aggregation-induced emission feature. Mater Sci Eng C-Mater 2017;73:252–6. [18] Thomas SW, Joly GD, Swager TM. Chemical sensors based on amplifying fluorescent conjugated polymers. Chem Rev 2007;107(4):1339–86. [19] Kumar M, George SJ. Green fluorescent organic nanoparticles by self-assembly induced enhanced emission of a naphthalene diimide bolaamphiphile. Nanoscale 2011;3(5):2130–3. [20] Li K, Liu B. Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging. Chem Soc Rev 2014;43(18):6570–97. [21] Luo J, Xie Z, Lam JW, Cheng L, Chen H, Qiu C, et al. Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem Commun 2001(18):1740–1. [22] Wan Q, Huang Q, Liu M, Xu D, Huang H, Zhang X, et al. Aggregation-induced emission active luminescent polymeric nanoparticles: non-covalent fabrication methodologies and biomedical applications. Appl Mater Today 2017;9:145–60. [23] Zhang X, Zhang X, Yang B, Hui J, Liu M, Liu W, et al. PEGylation and cell imaging applications of AIE based fluorescent organic nanoparticles via ring-opening reaction. Polym Chem 2014;5(3):689–93. [24] Zhang X, Zhang X, Yang B, Liu M, Liu W, Chen Y, et al. Facile fabrication and cell imaging applications of aggregation induced emission dye based fluorescent organic nanoparticles. Polym Chem 2013;4(16):4317–21. [25] Zhang X, Zhang X, Yang B, Liu M, Liu W, Chen Y, et al. Polymerizable aggregation induced emission dye based fluorescent nanoparticles for cell imaging applications. Polym Chem 2014;5(2):356–60. [26] Zhang X, Zhang X, Yang B, Liu M, Liu W, Chen Y, et al. Fabrication of aggregation induced emission dye-based fluorescent organic nanoparticles via emulsion polymerization and their cell imaging applications. Polym Chem 2014;5(2):399–404. [27] Wang S, Fu C, Zhang Y, Tao L, Li S, Wei Y. One-pot cascade synthetic strategy: a smart combination of chemoenzymatic transesterification and raft polymerization. ACS Macro Lett 2012;1(10):1224–7. [28] Banfi L, Riva R. The passerini reaction. Organic Reactions; 2005. [29] Erlenmeyer H, Oberlin V. Zur Kenntnis der Thiazolidone‐(4). Helv Chim Acta 1947;30(5):1329–35. [30] Arend M, Westermann B, Risch N. Modern variants of the Mannich reaction. Angew Chem Int Ed 1998;37(8):1044–70. [31] Moad G, Rizzardo E, Thang SH. Radical addition-fragmentation chemistry in polymer synthesis. Polymer 2008;49(5):1079–131. [32] Toure BB, Hall DG. Natural product synthesis using multicomponent reaction strategies. Chem Rev 2009;109(9):4439–86. [33] Zhang Y, Fu C, Zhu C, Wang S, Tao L, Wei Y. A multicomponent polymerization system: click–chemoenzymatic–ATRP in one-pot for polymer synthesis. Polym Chem 2013;4(3):466–9. [34] Zhao Y, Yang B, Zhu C, Zhang Y, Wang S, Fu C, et al. Introducing mercaptoacetic acid locking imine reaction into polymer chemistry as a green click reaction. Polym Chem-Uk 2014;5(8):2695–9. [35] Wan Q, Liu MY, Xu DZ, Mao LC, Huang HY, Gao P, et al. Fabrication of amphiphilic fluorescent nanoparticles with an AIE feature via a one-pot clickable mercaptoacetic acid locking imine reaction: synthesis, self-assembly and bioimaging. Polym Chem-Uk 2016;7(27):4559–66. [36] Wang D, Chen L. Temperature and pH-responsive single-walled carbon nanotube dispersions. Nano Lett 2007;7(6):1480–4. [37] Kabanov AV, Nazarova IR, Astafieva IV, Batrakova EV, Alakhov VY, Yaroslavov AA, et al. Micelle formation and solubilization of fluorescent-probes in poly (Oxyethylene-B-Oxypropylene-B-Oxyethylene) solutions. Macromolecules 1995;28(7):2303–14. [38] Hu J, Zhang X, Wang D, Hu X, Liu T, Zhang G, et al. Ultrasensitive ratiometric fluorescent pH and temperature probes constructed from dye-labeled thermoresponsive double hydrophilic block copolymers. J Mater Chem 2011;21(47):19030–8. [39] Long Z, Liu MY, Jiang RM, Wan Q, Mao LC, Wan YQ, et al. Preparation of water soluble and biocompatible AIE-active fluorescent organic nanoparticles via multicomponent reaction and their biological imaging capability. Chem Eng J 2017;308:527–34. [40] Xie G, Ma C, Zhang X, Liu H, Yang L, Li Y, et al. Chitosan-based cross-linked fluorescent polymer containing aggregation-induced emission fluorogen for cell imaging. Dyes Pigments 2017;143:276–83. [41] Xie G, Ma C, Zhang X, Liu H, Gong J, Zhao S, et al. An amphiphilic fluorescent polymer combining aggregation-induced emission monomer and ε-polylysine for cell imaging. Dyes Pigments 2017;145:174–80. [42] Li H, Zhang X, Zhang X, Yang B, Yang Y, Huang Z, et al. Zwitterionic red fluorescent polymeric nanoparticles for cell imaging. Macromol Biosci 2014;14(10):1361–7. [43] Li HY, Zhang XQ, Zhang XY, Yang B, Yang Y, Wei Y. Ultra-stable biocompatible cross-linked fluorescent polymeric nanoparticles using AIE chain transfer agent. Polym Chem 2014;5(12):3758–62.
4. Conclusions In summary, the AIE-active Poly(PEGMA-co-FBA-TPE-NH2) FONs could be successfully prepared via the combination of MCRs and RAFT polymerization, which could be applied to various biological applications such as bio-imaging, cell tracking and monitoring for their advantages. Moreover, the fluorescent polymers can self-assemble into nanoparticles for the hydrophobic fluorogen FBA are gather in the core of the sphere while the hydrophilic polymers covering the surface as the shell, which makes the FONs can overcome the notorious ACQ effect. The characterization results indicated the AIE-active functional polymers have many advantages such as excellent biocompatibility, biodegradability, low toxicity and strong fluorescence, which endow great potential for the biomedical applications of these AIE-active functional polymers. More than that, the strategy of the synthesis of AIE-active polymers could be used for the construction of other AIE-active functional materials, owing to the fungibility of components with amino groups or aldehyde groups in this multicomponent reaction. Compared with other previous strategies, the strategy is more environmentally friendly, has higher-efficiency and operated more conveniently. Finally, we expect that the AIE-active FONs could be applied to wider biomedical applications. Acknowledgements This research was supported by the National Natural Science Foundation of China (Nos. 21788102, 21865012, 51363016, 21474057, 21564006, 21561022, and 21644014). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107673. References [1] Tong L, Wei QS, Wei A, Cheng JX. Gold nanorods as contrast agents for biological imaging: optical properties, surface conjugation and photothermal effects. Photochem Photobiol 2009;85(1):21–32. [2] Zhang X, Wang K, Liu M, Zhang X, Tao L, Chen Y, et al. Polymeric AIE-based nanoprobes for biomedical applications: recent advances and perspectives. Nanoscale 2015;7(27):11486–508. [4] Zhang X, Zhang X, Tao L, Chi Z, Xu J, Wei Y. Aggregation induced emission-based fluorescent nanoparticles: fabrication methodologies and biomedical applications. J Mater Chem B 2014;2(28):4398–414. [5] Wan Q, Jiang R, Mao L, Xu D, Zeng G, Shi Y, et al. A powerful “one-pot” tool for fabrication of AIE-active luminescent organic nanoparticles through the combination of RAFT polymerization and multicomponent reactions. Mater Chem Front 2017;1(6):1051–8. [6] Freeman R, Willner I. Optical molecular sensing with semiconductor quantum dots (QDs). Chem Soc Rev 2012;41(10):4067–85. [7] Weber B, Tan YH, Mahapatra S, Watson TF, Ryu H, Rahman R, et al. Spin blockade and exchange in Coulomb-confined silicon double quantum dots. Nat Nanotechnol 2014;9(6):430–5. [8] Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science 1998;281(5385):2013–6. [9] Zhu SJ, Zhou N, Hao ZY, Maharjan S, Zhao XH, Song YB, et al. Photoluminescent graphene quantum dots for in vitro and in vivo bioimaging using long wavelength emission. RSC Adv 2015;5(49):39399–403. [10] Dong YQ, Wang RX, Li GL, Chen CQ, Chi YW, Chen GN. Polyamine-Functionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions. Anal Chem 2012;84(14):6220–4. [11] Yang ST, Wang X, Wang HF, Lu FS, Luo PJG, Cao L, et al. Carbon dots as nontoxic and high-performance fluorescence imaging agents. J Phys Chem C 2009;113(42):18110–4. [12] Qiao ZA, Zhang P, Chai SH, Chi M, Veith GM, Gallego NC, et al. Lab-in-a-shell: encapsulating metal clusters for size sieving catalysis. J Am Chem Soc 2014;136(32):11260–3.
6
Contents lists available at ScienceDirect
Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
The combination of controlled living polymerization and multicomponent reactions to prepare tetraphenylethylene-containing fluorescent block copolymers
T
Jianwen Tiana, Zhongxu Chena, Ruming Jianga, Liucheng Maoa, Meiying Liua, Fengjie Denga, Hongye Huanga, Liangji Liub,∗∗∗, Xiaoyong Zhanga,∗, Yen Weic,d,∗∗ a
Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang, 330031, China Jiangxi University of Traditional Chinese Medicine, 56 Yangming Road, Jiangxi, Nanchang, 330006, China Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, China d Department of Chemistry and Center for Nanotechnology, Chung-Yuan Christian University, Chung-Li, 32023, Taiwan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: RAFT polymerization Multicomponent reactions Aggregation-induced emission Fluorescent organic nanoparticles Cell imaging
High-efficiency multicomponent reactions (MCRs) have received increasing attention and widely used to prepare functional materials because of many prominent advantages such as simple “one-pot” operation, clear product structure, atom economic and environmentally friendly. Combining conventional reversible addition-fragmentation chain-transfer (RAFT) polymerization and new MCRs to prepare multifunctional polymer nanomaterials should be an interesting research direction for polymer chemistry and materials science. In this research, aggregation-induced emission (AIE)-active fluorescent amphiphilic block copolymers were designed and prepared by the combination of reversible RAFT polymerization and mercaptoacetic acid locking imine (MALI) method. These amphiphilic block copolymers that contain the hydrophobic tetraphenylethylene (TPE) derivative and hydrophilic polyethylene glycol (PEG) could be self-assembled into structure with spherical morphology and small particle size. Owing to the aggregation of hydrophobic TPE derivative, these self-assembles display obvious AIE feature. On the other hand, thus-obtained fluorescent organic nanoparticles (FONs) could also be well dispersed in aqueous solution because of the existence of hydrophilic PEG. All the characterization results demonstrated that we could facilely synthesized these fluorescent amphiphiles through the strategy described above. The preliminary results from biological assays suggested that these AIE-active FONs are of low cytotoxicity and could be potentially used for living cell imaging. Taken together, we reported a one-pot strategy for synthesis of TPE-containing FONs through the combination of RAFT polymerization and MALI reaction for the first time. This strategy could also be extended for fabrication of many other functional composites owing to their unique features. We trust this work should be great advance the biomedical applications of AIE-active functional materials.
1. Introduction In recent years, considerable attention has been paid to study fluorescent organic nanoparticles (FONs) in biological imaging owing to their intensive fluorescence, good photostability and outstanding optical properties [1–2]. Furthermore, biological imaging with high sensitivity can be applied to observe the biological processes and improve the veracity and decreasing the operation simultaneously [4,5]. To expand the scope of biological imaging applications, many novel
fluorescent materials have been reported, especially their synthetic methods. In the past few years, fluorescent inorganic nanoparticles (FINs), such as semiconductor quantum dots, [6] silicon quantum dots, [7,8] carbon quantum dots [9–11] and metal nanoclusters, [12,13] which were regarded as ideal fluorescent probes. However, the biomedical applications of FINs were largely limited because of their toxicity and poor degradability [5,14–16]. In contrast, FONs can be regarded as the ideal fluorescent agents to replace FINs for their excellent biodegradability, low toxicity and well designability.
∗
Corresponding author. Corresponding author. Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, China. ∗∗∗ Corresponding author. E-mail address: [email protected] (X. Zhang). ∗∗
https://doi.org/10.1016/j.dyepig.2019.107673 Received 22 May 2019; Received in revised form 25 June 2019; Accepted 25 June 2019 Available online 28 June 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
smaller size, great water dispersibility and intensive fluorescence emission at aggregation. Following, based on these outstanding features, the cell toxicity and cell staining performance of these AIE-active FONs have been carried out. Results suggest low cytotoxicity, good biocompatibility and stained cells ability of synthetic AIE-active FONs in this contribution [35,36]. Compared with the traditional methods via step-by-step or post-modification approaches, [37–39] we can see that the method that combination of MALI MCR strategy and RAFT polymerization is more convenient to prepare functional polymeric materials with well-defined structure, multifunctional interaction and desirable polymeric properties [35]. In a word, we expect the synthetic FONs with AIE effect are helpful in further biomedical applications, and new fabricated tactics may provide inspiration for the preparation of multifunctional polymer materials.
Unfortunately, most of conventional organic dyes still have a serious problem, which is the vicious aggregation-caused quenching (ACQ) effect. In other word, the fluorescence intensity of conventional organic molecules would be sharply decreased or even completely quenched at high concentration or aggregation state, this fatal defect had prevented them from being further applied in the field of bioimaging owing to the aggregation of these hydrophobic organic molecules in aqueous solution [5,17–20]. Therefore, many researchers have spent a great deal of efforts to solve this issue. Fortunately, Tang et al. first promoted the aggregation-induced emission (AIE) concept in 2001 [21]. They found that some fluorescent organic molecules can emit obvious enhanced fluorescence in their aggregated state. The phenomenon is opposite to the ACQ effect, and these FONs with AIE feature are regarded as the most promising candidates for bioimaging to overcome the ACQ effect of FONs based on conventional organic molecules. Although there were many strategies have been reported to prepare AIE-active FONs in recent years, there is still rarely studied on synthesis of AIE-active FONs through the combination of controlled living radical polymerization and multicomponent reactions (MCRs). As compared with the traditional methods for preparation of AIE-active FONs, the MCRs possess the following advantages, such as atom economy, molecular diversity, facile operation, mild reaction conditions and introduction of new structure and functions. Therefore, some efforts in designing new MCRs to obtain AIE-active FONs have been developed. For example, our group previously reported functional AIE-active fluorescent polysaccharide nanoparticles to label cells by the MCR. But the unicity of monomer limits its development for the preparation of multifunctional materials. So far, our group has still been exploiting new synthetic strategies to prepare AIE-active FONs over the past several years and achieve some effects [22–26]. As we all know, MCRs are one-pot procedures, which can obtain a product with various archetypical functional groups of each reactants and easy to operate for single-first discovered by Passerini in 1921. From then on, the research interest in the MCRs increases rapidly for their unique advantages, such as higher atomic economy, molecular diversity and high yield [27]. In addition, compared with other strategies for the preparation of AIE-active FONs, MCRs are easier to achieve commercialization for their efficiency synthetic steps. Over the past few decades, previous MCRs such as the Passerini reaction, Mannich reaction (a three-component reaction) and MALI reaction [28–30] were deeply studied because they play a vital role in polymer chemistry and material science. Although MCRs possess advantages in organic synthesis, the polymer synthesis based on MCRs are still faced huge challenges such as narrow molecular weight distribution, high molecular weight, lesser polymerizable monomers and divinable polymeric structure. For example, AIE-active polymeric assemblies prepared by controlled RAFT polymerization were developed by Yuan's group, this method possesses various advantages of abundant polymeric monomers, narrow polymer distribution and process control et al. Therefore, integrating the advantages of MCRs and reversible addition-fragmentation chain-transfer (RAFT) to prepare functional polymeric materials should be of special interesting and valuable. Previously, Tao et al. have made considerable efforts for the synthesis of functional polymers or polymeric materials by various MCRs and controlled living polymerization in a one-pot route. However, the preparation of fluorescent polymeric self-assemblies with AIE feature has still largely unexplored. In this contribution, a new fabricated tactic for synthesis of AIEactive FONs was reported via the combination of mercaptoacetic acid locking imine (MALI) reaction and RAFT polymerization in a “one-pot” route [31–33]. In the MALI reaction (a three-component reaction), we use mercaptoacetic acid as the "lock" to combine the amino-containing chromophore TPE-NH2 and the aldehyde-terminated polymerizable monomer of 4-formylphenyl acrylate (FBA), which can avoid the complicated operation [34]. The formation final product PEG-TPE polymer with amphiphilic property can self-assemble into nanoparticles in aqueous solution. Thus-prepared AIE-active nanoparticles possess
2. Experimental sections 2.1. Materials and measurements All of the reagents and solvents were purchased from commercial sources of analytical grade and used directly without further purification, and distilled water was used in the experiment. Mercaptoacetic acid (98%, MW: 92.12 Da), methacryloyl chloride (95%, MW: 104.53 Da), and triethylamine (99%, MW: 101.19 Da), 4-hydroxybenzaldehyde (98%, MW: 122.12 Da), 2,2′-azobis(2-methylpropionitrile) (AIBN, 99%, MW: 164.21 Da) and poly(ethylene glycol) methacrylate (PEGMA, MW: 500 Da) were obtained from the Aladdin company. The AIE dye TPE-NH2 and 4-cyano-4-(ethylthiocarbonothioylthio)pentanoic acid as chain transfer agent (CTA) were synthesized and characterized in our group's previous work. 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance400 spectrometer with DMSO‑d6 as the solvent. The synthetic materials were characterized by Fourier transform infrared spectroscopy (FT-IR) using KBr pellets. The FT-IR spectra were obtained from a Nicolet5700 (Thermo Nicolet corporation). Transmission electron microscopy (TEM) images were recorded on a Hitachi 7650B microscope operated at 80 kV; the TEM specimens were obtained by placing a drop of ethanol nanoparticle suspension on a carbon-coated copper grid. The fluorescence data was obtained from a fluorescence spectrophotometer (FSP, model: C11367-11), which was purchased from Hamamatsu (A Japan company). 2.2. Preparation of FBA The preparation route of FBA monomer was showed in Scheme S1. Triethylamine (10 mmol, 1.01 g) and 4-hydroxybenzaldehyde (10 mmol, 1.22 g) in 100 mL of anhydrous dichloromethane were stirred at 0 °C. Then acryloyl chloride (6 mmol, 540 mg) was added dropwise into the mixture and reacting at room temperature for 1 h. The obtained crude product was purified by silica-gel column chromatography using ethyl acetate/petroleum ether (1/50) as eluent to give FBA. (3.9 g, yield 89%) 2.3. One-pot synthesis of the Poly(PEGMA-co-FBA-TPE-NH2) The AIE-active copolymer Poly(PEGMA-co-FBA-TPE-NH2) was prepared by a simple one-pot process as depicted follows. First, PEGMA (1 mmol, 500 mg), TPE-NH2 (0.1 mmol, 47 mg), mercaptoacetic acid (0.2 mmol, 18 mg), FBA (0.1 mmol, 19 mg), AIBN (0.01 mmol, 1.6 mg) and CTA (0.05 mmol, 12 mg) were dissolved in dry THF solvent (3 mL) and put into a Schlenk tube. The Schlenk tube was sealed and filled with N2 for 30 min to remove oxygen. After that, the reaction mixture was continuous stirred at 70 °C for 12 h. The crude product was purified by dissolved in THF and precipitated into diethyl ether for three times, and then dried under vacuum for further characterization. 2
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
Scheme 1. Schematic showing the one-pot synthesis of PEG-TPE copolymers through the MALI reaction and RAFT polymerization. The advantages of the MCRs have also displayed in the Scheme.
2.4. Cytotoxicity evaluation of the Poly(PEGMA-co-FBA-TPE-NH2) FONs The biocompatibility of Poly(PEGMA-co-FBA-TPE-NH2) FONs was evaluated through a normal assay based on the CCK-8 assay. The detailed procedures were described in the supporting information. 2.5. Confocal microscopic imaging of the Poly(PEGMA-co-FBA-TPE-NH2) FONs The detailed experimental procedures for cell uptake behavior of were evaluated by confocal laser scanning microscopy (CLSM) were described in the supporting information. 3. Results and discussion 3.1. Characterization of Poly(PEGMA-co-FBA-TPE-NH2) The AIE-active Poly(PEGMA-co-FBA-TPE-NH2) copolymers were prepared by a simple one-pot process between PEGMA, TPE-NH2, mercaptoacetic acid and FBA based on the RAFT polymerization and MALI reaction (Scheme 1). As we can see, the fluorescent polymer Poly (PEGMA-co-FBA-TPE-NH2) introduced the functional groups of each reactant through the polymerization, which makes it with many outstanding properties such as excellent dispersion in water, good biodegradability and intensive fluorescence emission. Detailed synthesis and characterizations of the intermediates and final compounds are shown in the experimental sections, Scheme S1, and Fig. S1−S3. In order to confirm its good properties for biomedical applications, many characterization techniques such as 1H NMR, FT-IR, UV–vis and CLSM were performed. Fig. 1 shows the 1H NMR spectra of FBA and Poly(PEGMAco-FBA-TPE-NH2). Two chemical shifts at 5.53 ppm and 6.09 ppm can be found in sample of FBA, which can be ascribed to the protons connected with the C=C bond. After reaction, some characteristic chemical shifts of PEGMA units between 3.0 and 4.0 ppm are retained, and the peaks of C=C bond protons are disappeared. Moreover, multiple peaks located at 6.9–8.1 ppm derived from the aromatic rings are appeared in the spectrum of Poly(PEGMA-co-FBA-TPE-NH2) (Inset figure), providing powerful evidences of successful preparation of the Poly (PEGMA-co-FBA-TPE-NH2) polymer. Other than NMR spectra, FT-IR spectra were also used to verify whether successful synthesis of Poly(PEGMA-co-FBA-TPE-NH2). Fig. 2 shows the IR spectra of TPE-NH2, FBA and Poly(PEGMA-co-FBA-TPENH2). Obviously, two sharp peaks at 3480 and 3379 cm−1 could be
Fig. 1. The 1H NMR spectra of FBA (blue) and Poly(PEGMA-co-FBA-TPE-NH2) (red) measured in DMSO‑d6 at room temperature. The inset figure shows that the aromatic rings have been included in the copolymers. All the above results implied that the AIE-active FONs have been successfully synthesized through the RAFT polymerization and MALI reaction. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
found in TPE-NH2, which should be attributed to the stretching vibration of amino group. In the sample of FBA monomer, the stretching vibration of C=C bond was located at 1637 cm−1. Compared with the spectra of FBA monomer and TPE-NH2, some characteristic peaks (i.e., C-H stretching vibration of the aromatic rings, C-O stretching vibration) are retained in the Poly(PEGMA-co-FBA-TPE-NH2), and the C=O stretching vibration of the ester and amide group at 1727 cm−1, O-H stretching vibration at 3479 cm−1, C-H stretching vibration at 2862 cm−1, C-S stretching vibration at 617 cm−1 are appeared, accompanied by the disappearance of both of the stretching vibration of amino group and the C=C stretching vibration of alkene, further demonstrating successful synthesis of these fluorescent copolymers through the one-pot process. In addition, the number-average molecular weight (Mn) of Poly(PEGMA-co-FBA-TPE-NH2) was also obtained by GPC and is 11130 g mol−1 with a relatively narrow polydisperse index (PDI) of 1.14 (Fig. S4). In fact, the self-assembly of amphiphilic block copolymers is highly 3
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
properties and good water dispersibility. As shown in Fig. 4A, the Poly (PEGMA-co-FBA-TPE-NH2) FONs display an absorption and emission at 386 and 528 nm, respectively. The excitation curve shows the maximum intensity at 377 nm (Fig. S5). The inset of Fig. 4A shows the photographs of Poly(PEGMA-co-FBA-TPE-NH2) in water and bright yellow-green fluorescence when the aqueous solution of Poly(PEGMAco-FBA-TPE-NH2) under UV lamp irradiation at 365 nm. It's worth nothing that no any suspended matter can be found in the solution, and the background can be intuitively observed. This also indicate that the Poly(PEGMA-co-FBA-TPE-NH2) with great water dispersibility. Compared with the absorption spectra of FBA and TPE-NH2, the absorption wavelength at 386 nm in Poly(PEGMA-co-FBA-TPE-NH2) should be attributed to the introduction of TPE-NH2 (Fig. S6). As presented in Fig. 4B, the photoluminescence (PL) intensity proportionally increases with increase of the concentrations of Poly(PEGMA-co-FBA-TPE-NH2) FONs, indicating Poly(PEGMA-co-FBA-TPE-NH2) FONs with the antiACQ property. In generally, the key to improve the stability of FONs relied on reducing CMC value according to the previous literatures [40,41]. Therefore, the CMC value of FONs should be measured. Fig. 4C shows that Poly(PEGMA-co-FBA-TPE-NH2) has very low CMC value of 23.2 μg mL−1, which even lower than the previous reported AIE-active dye-based FPNs [42,43]. the low CMC value demonstrates that these FONs can stably exist in highly dilute aqueous solution. For biomedical applications, the good stability is critically important. As shown in Fig. 4D, only a slight decrease in the PL intensity (< 5%) is observed after continuously irradiation at 405 nm wavelength for 1 h, indicating that Poly(PEGMA-co-FBA-TPE-NH2) FONs possess desirable photostability. In addition, after storage at room temperature for 24 h, the average diameter nearly has not changed. It indicated that these AIEactive FONs possess great colloidal stability (Fig. S7).
Fig. 2. The FT-IR spectra of TPE-NH2 (A), FBA (B) and Poly(PEGMA-co-FBATPE-NH2) (C). The obvious increase of IR intensity at 2862 cm−1 indicate that the PEGMA has been copolymerized in the copolymers. The introduction of C=O and C-S functional groups was also found by FT-IR spectra. All the above results further suggested that the successful preparation of these fluorescent copolymers.
3.2. Biocompatibility of the Poly(PEGMA-co-FBA-TPE-NH2) FONs Biocompatibility of fluorescent nanoparticles determines whether it can be widely applied in biomedicine. In the study of their biocompatibility, L929 cells were incubated with different concentrations of Poly(PEGMA-co-FBA-TPE-NH2) FONs for 24 h. The cell viability was evaluated using the CCK-8 assay. As shown in Fig. 5, L929 cells can grow well even incubating with high concentration of Poly(PEGMA-coFBA-TPE-NH2) FONs for 24 h. These results demonstrated their excellent biocompatibility. Moreover, owing to these fluorescent polymeric nanoparticles are composited with the biocompatible and hydrophilic PEG and chemical inert AIE dye. These AIE-active FONs should be more promising for biomedical applications as compared with the inorganic fluorescent materials, which have been demonstrated to have significant toxicity to living organisms. In generally, based on their outstanding features, such as excellent biocompatibility, biodegradability, AIE fluorescence, low toxicity and great water dispersibility, Poly(PEGMA-co-FBA-TPE-NH2) FONs could serve as the ideal candidates for various biomedical applications. Furthermore, due to the advantages of this synthetic method with MCR and RAFT polymerization such as simple and highly efficient, ease of operation and high yields, this method is also very helpful in producing other functional polymeric materials to extend their biomedical applications.
Fig. 3. The size distribution of Poly(PEGMA-co-FBA-TPE-NH2) FONs determined by DLS. The inset shows TEM image of self-assembled nanoparticles for Poly(PEGMA-co-FBA-TPE-NH2) FONs, the scale bar is 500 nm. The TEM and DLS results clearly evidence the formation of nanoparticles and imply the successful preparation of the amphiphilic fluorescent copolymers.
related to their hydrophilic segments and hydrophobic portions. Thus, FONs were prepared by the self-assembly of amphiphilic Poly(PEGMAco-FBA-TPE-NH2) copolymers. The size, morphology of these self-assemblies was studied by TEM and DLS. The inset of Fig. 3 shows the TEM images of Poly(PEGMA-co-FBA-TPE-NH2) FONs after dispersion in water, many spherical nanoparticles with diameters between 100 and 150 nm could be observed. The formation of nanoparticles indicated that the amphiphilic copolymers are successfully synthesized and can self-assemble into nanoparticles in aqueous solution. In the process, the hydrophobic segments of the AIE dyes were aggregated in the inner as the core, while the hydrophilic PEGMA will dissolve in water that was regarded as the shell. DLS results display the hydrodynamic diameter of the Poly(PEGMA-co-FBA-TPE-NH2) FONs dispersed in water is 168.8 nm with the polydispersity index (PDI) of 0.273. This means that Poly(PEGMA-co-FBA-TPE-NH2) FONs possess better water dispersibility and small size. Therefore, these particles can easily get inside the cell via the endocytosis of cells, making them ideal candidates for biomedical applications. Owing to the self-assembly of AIE-active Poly(PEGMA-co-FBA-TPENH2) copolymers, the obtained FONs show significant optical
3.3. Biological imaging evaluation of Poly(PEGMA-co-FBA-TPE-NH2) FONs The in vitro cell uptake behavior of Poly(PEGMA-co-FBA-TPE-NH2) FONs were evaluated by using CLSM observation. As show in Fig. 6A, strong green fluorescence could be observed in the cytoplasm of the cells when L929 cells were incubated in culture dish with 40 μg mL−1 of Poly(PEGMA-co-FBA-TPE-NH2) FONs for 3 h at 37 °C. In contrast, the center of the cell where is supposed to be the nucleus of cells could only view the weak fluorescence. Fig. 6B is the merged image of Fig. 6A and C, which clearly showed that the Poly(PEGMA-co-FBA-TPE-NH2) FONs 4
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
Fig. 4. (A) Absorption and emission spectra of Poly(PEGMA-co-FBA-TPE-NH2) FONs dispersed in water, the inset shows the photographs of Poly(PEGMA-co-FBATPE-NH2) and bright fluorescence of Poly(PEGMA-co-FBA-TPE-NH2) under UV lamp irradiation at 365 nm (B) PL intensity of Poly(PEGMA-co-FBA-TPE-NH2) in water at different concentrations, the inset shows bright fluorescence of Poly(PEGMA-co-FBA-TPE-NH2) at different concentrations under UV lamp irradiation at 365 nm. (C) The relationship of the PL intensity and logarithm of the concentration of Poly(PEGMA-co-FBA-TPE-NH2), the CMC value obtained as 23.2 μg mL−1. (D) Photostability of Poly(PEGMA-co-FBA-TPE-NH2) FONs in different media with UV light irradiation for 1 h.
Fig. 5. Cell viability of L929 cells incubated with Poly(PEGMA-co-FBA-TPENH2) FONs at different concentrations for 24 h. No obvious cell viability decrease was found based on the CCK-8 assay. This is likely ascribed to the surface coating of biocompatible PEG segments.
were mainly entered into the cytoplasm through the endocytosis of cells but cannot enter cell nuclei. Fig. 6C shows the cells still maintained their normal growth morphology, further demonstrating their excellent biocompatibility. Furthermore, as increase of incubation time, fluorescence signals increased, displayed time-dependence (Fig. 6D). The above results showed that this AIE-active FONs could be applied perfectly for bioimaging applications. The Poly(PEGMA-co-FBA-TPE-NH2) FONs with unique AIE-active fluorescence feature can be used in biomedical fields for their excellent cell stain performance. In addition, we
Fig. 6. CLSM images of L929 cells. (A) Excited with a 405 nm laser, (B) bright field and (C) merged image of A and C. L929 cells incubated with PEG-TPE FONs (40 μg mL−1) for 3 h. (D) different incubated time, Ι. 30 min, П. 1 h, Ш. 3 h, Ⅳ. 6 h. Scale bar = 100 μm.
5
Dyes and Pigments 171 (2019) 107673
J. Tian, et al.
can replace the monomers of the “one-pot” reaction to synthesize other fluorescent materials. In some ways, this method provides a wider platform to synthesize more AIE-active functional materials for various biological applications.
[13] Lin CAJ, Lee CH, Hsieh JT, Wang HH, Li JK, Shen JL, et al. Synthesis of fluorescent metallic nanoclusters toward biomedical application: recent progress and present challenges. J Med Biol Eng 2009;29(6):276–83. [14] Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, et al. Renal clearance of nanoparticles. Nat Biotechnol 2007;25(10):1165. [15] Liu J, Hu R, Liu J, Zhang B, Wang Y, Liu X, et al. Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models. Mater Sci Eng C-Mater 2015;57:222–31. [16] Yi C, Liu L, Li C-W, Zhang J, Yang M. Synthesis, characterization and biomedical application of multifunctional luminomagnetic core–shell nanoparticles. Mater Sci Eng C-Mater 2015;46:32–40. [17] Long Z, Liu M, Mao L, Zeng G, Huang Q, Huang H, et al. One-step synthesis, selfassembly and bioimaging applications of adenosine triphosphate containing amphiphilies with aggregation-induced emission feature. Mater Sci Eng C-Mater 2017;73:252–6. [18] Thomas SW, Joly GD, Swager TM. Chemical sensors based on amplifying fluorescent conjugated polymers. Chem Rev 2007;107(4):1339–86. [19] Kumar M, George SJ. Green fluorescent organic nanoparticles by self-assembly induced enhanced emission of a naphthalene diimide bolaamphiphile. Nanoscale 2011;3(5):2130–3. [20] Li K, Liu B. Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging. Chem Soc Rev 2014;43(18):6570–97. [21] Luo J, Xie Z, Lam JW, Cheng L, Chen H, Qiu C, et al. Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem Commun 2001(18):1740–1. [22] Wan Q, Huang Q, Liu M, Xu D, Huang H, Zhang X, et al. Aggregation-induced emission active luminescent polymeric nanoparticles: non-covalent fabrication methodologies and biomedical applications. Appl Mater Today 2017;9:145–60. [23] Zhang X, Zhang X, Yang B, Hui J, Liu M, Liu W, et al. PEGylation and cell imaging applications of AIE based fluorescent organic nanoparticles via ring-opening reaction. Polym Chem 2014;5(3):689–93. [24] Zhang X, Zhang X, Yang B, Liu M, Liu W, Chen Y, et al. Facile fabrication and cell imaging applications of aggregation induced emission dye based fluorescent organic nanoparticles. Polym Chem 2013;4(16):4317–21. [25] Zhang X, Zhang X, Yang B, Liu M, Liu W, Chen Y, et al. Polymerizable aggregation induced emission dye based fluorescent nanoparticles for cell imaging applications. Polym Chem 2014;5(2):356–60. [26] Zhang X, Zhang X, Yang B, Liu M, Liu W, Chen Y, et al. Fabrication of aggregation induced emission dye-based fluorescent organic nanoparticles via emulsion polymerization and their cell imaging applications. Polym Chem 2014;5(2):399–404. [27] Wang S, Fu C, Zhang Y, Tao L, Li S, Wei Y. One-pot cascade synthetic strategy: a smart combination of chemoenzymatic transesterification and raft polymerization. ACS Macro Lett 2012;1(10):1224–7. [28] Banfi L, Riva R. The passerini reaction. Organic Reactions; 2005. [29] Erlenmeyer H, Oberlin V. Zur Kenntnis der Thiazolidone‐(4). Helv Chim Acta 1947;30(5):1329–35. [30] Arend M, Westermann B, Risch N. Modern variants of the Mannich reaction. Angew Chem Int Ed 1998;37(8):1044–70. [31] Moad G, Rizzardo E, Thang SH. Radical addition-fragmentation chemistry in polymer synthesis. Polymer 2008;49(5):1079–131. [32] Toure BB, Hall DG. Natural product synthesis using multicomponent reaction strategies. Chem Rev 2009;109(9):4439–86. [33] Zhang Y, Fu C, Zhu C, Wang S, Tao L, Wei Y. A multicomponent polymerization system: click–chemoenzymatic–ATRP in one-pot for polymer synthesis. Polym Chem 2013;4(3):466–9. [34] Zhao Y, Yang B, Zhu C, Zhang Y, Wang S, Fu C, et al. Introducing mercaptoacetic acid locking imine reaction into polymer chemistry as a green click reaction. Polym Chem-Uk 2014;5(8):2695–9. [35] Wan Q, Liu MY, Xu DZ, Mao LC, Huang HY, Gao P, et al. Fabrication of amphiphilic fluorescent nanoparticles with an AIE feature via a one-pot clickable mercaptoacetic acid locking imine reaction: synthesis, self-assembly and bioimaging. Polym Chem-Uk 2016;7(27):4559–66. [36] Wang D, Chen L. Temperature and pH-responsive single-walled carbon nanotube dispersions. Nano Lett 2007;7(6):1480–4. [37] Kabanov AV, Nazarova IR, Astafieva IV, Batrakova EV, Alakhov VY, Yaroslavov AA, et al. Micelle formation and solubilization of fluorescent-probes in poly (Oxyethylene-B-Oxypropylene-B-Oxyethylene) solutions. Macromolecules 1995;28(7):2303–14. [38] Hu J, Zhang X, Wang D, Hu X, Liu T, Zhang G, et al. Ultrasensitive ratiometric fluorescent pH and temperature probes constructed from dye-labeled thermoresponsive double hydrophilic block copolymers. J Mater Chem 2011;21(47):19030–8. [39] Long Z, Liu MY, Jiang RM, Wan Q, Mao LC, Wan YQ, et al. Preparation of water soluble and biocompatible AIE-active fluorescent organic nanoparticles via multicomponent reaction and their biological imaging capability. Chem Eng J 2017;308:527–34. [40] Xie G, Ma C, Zhang X, Liu H, Yang L, Li Y, et al. Chitosan-based cross-linked fluorescent polymer containing aggregation-induced emission fluorogen for cell imaging. Dyes Pigments 2017;143:276–83. [41] Xie G, Ma C, Zhang X, Liu H, Gong J, Zhao S, et al. An amphiphilic fluorescent polymer combining aggregation-induced emission monomer and ε-polylysine for cell imaging. Dyes Pigments 2017;145:174–80. [42] Li H, Zhang X, Zhang X, Yang B, Yang Y, Huang Z, et al. Zwitterionic red fluorescent polymeric nanoparticles for cell imaging. Macromol Biosci 2014;14(10):1361–7. [43] Li HY, Zhang XQ, Zhang XY, Yang B, Yang Y, Wei Y. Ultra-stable biocompatible cross-linked fluorescent polymeric nanoparticles using AIE chain transfer agent. Polym Chem 2014;5(12):3758–62.
4. Conclusions In summary, the AIE-active Poly(PEGMA-co-FBA-TPE-NH2) FONs could be successfully prepared via the combination of MCRs and RAFT polymerization, which could be applied to various biological applications such as bio-imaging, cell tracking and monitoring for their advantages. Moreover, the fluorescent polymers can self-assemble into nanoparticles for the hydrophobic fluorogen FBA are gather in the core of the sphere while the hydrophilic polymers covering the surface as the shell, which makes the FONs can overcome the notorious ACQ effect. The characterization results indicated the AIE-active functional polymers have many advantages such as excellent biocompatibility, biodegradability, low toxicity and strong fluorescence, which endow great potential for the biomedical applications of these AIE-active functional polymers. More than that, the strategy of the synthesis of AIE-active polymers could be used for the construction of other AIE-active functional materials, owing to the fungibility of components with amino groups or aldehyde groups in this multicomponent reaction. Compared with other previous strategies, the strategy is more environmentally friendly, has higher-efficiency and operated more conveniently. Finally, we expect that the AIE-active FONs could be applied to wider biomedical applications. Acknowledgements This research was supported by the National Natural Science Foundation of China (Nos. 21788102, 21865012, 51363016, 21474057, 21564006, 21561022, and 21644014). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107673. References [1] Tong L, Wei QS, Wei A, Cheng JX. Gold nanorods as contrast agents for biological imaging: optical properties, surface conjugation and photothermal effects. Photochem Photobiol 2009;85(1):21–32. [2] Zhang X, Wang K, Liu M, Zhang X, Tao L, Chen Y, et al. Polymeric AIE-based nanoprobes for biomedical applications: recent advances and perspectives. Nanoscale 2015;7(27):11486–508. [4] Zhang X, Zhang X, Tao L, Chi Z, Xu J, Wei Y. Aggregation induced emission-based fluorescent nanoparticles: fabrication methodologies and biomedical applications. J Mater Chem B 2014;2(28):4398–414. [5] Wan Q, Jiang R, Mao L, Xu D, Zeng G, Shi Y, et al. A powerful “one-pot” tool for fabrication of AIE-active luminescent organic nanoparticles through the combination of RAFT polymerization and multicomponent reactions. Mater Chem Front 2017;1(6):1051–8. [6] Freeman R, Willner I. Optical molecular sensing with semiconductor quantum dots (QDs). Chem Soc Rev 2012;41(10):4067–85. [7] Weber B, Tan YH, Mahapatra S, Watson TF, Ryu H, Rahman R, et al. Spin blockade and exchange in Coulomb-confined silicon double quantum dots. Nat Nanotechnol 2014;9(6):430–5. [8] Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science 1998;281(5385):2013–6. [9] Zhu SJ, Zhou N, Hao ZY, Maharjan S, Zhao XH, Song YB, et al. Photoluminescent graphene quantum dots for in vitro and in vivo bioimaging using long wavelength emission. RSC Adv 2015;5(49):39399–403. [10] Dong YQ, Wang RX, Li GL, Chen CQ, Chi YW, Chen GN. Polyamine-Functionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions. Anal Chem 2012;84(14):6220–4. [11] Yang ST, Wang X, Wang HF, Lu FS, Luo PJG, Cao L, et al. Carbon dots as nontoxic and high-performance fluorescence imaging agents. J Phys Chem C 2009;113(42):18110–4. [12] Qiao ZA, Zhang P, Chai SH, Chi M, Veith GM, Gallego NC, et al. Lab-in-a-shell: encapsulating metal clusters for size sieving catalysis. J Am Chem Soc 2014;136(32):11260–3.
6