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Mechanistic investigation on fluorescence instability of AIE polymeric nanoparticles with a susceptible AIEgen prepared in miniemulsions
Dyes and Pigments 160 (2019) 572–578
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Mechanistic investigation on fluorescence instability of AIE polymeric nanoparticles with a susceptible AIEgen prepared in miniemulsions
T
Xiaoqin Lianga, Yaxin Hua, Junjie Loua, Zhihai Caoa,∗, Lin Liua, Yijia Wanga, Zujin Zhaob,∗∗, Dongming Qia, Qinmin Cuic,∗∗∗ a Key Laboratory of Advanced Textile Materials and Manufacturing Technology and Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou, 310018, China b Center for Aggregation-Induced Emission, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, 510640, China c School of Pharmacy, Hangzhou Medical College, 481 Binwen Road, Hangzhou, 310053, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluorescence stability Aggregation-induced emission Hydrolysis Miniemulsion polymerization AIE polymeric nanoparticles
Fluorescence stability is a key index for application of aggregation-induced emission (AIE) polymeric nanoparticles (NPs) as fluorescent probes. In this work, the temperature-dependent fluorescence stability of AIE polystyrene (PSt) NPs with a susceptible AIE luminogens (1-methyl-1,2,3,4,5-pentaphenylsilole, MPPS), which was named as PSt/MPPS NPs, was thoroughly investigated. The PSt/MPPS NPs in aqueous emulsions showed an abnormally fast fluorescence loss at the temperatures close to or above the glass transition temperature of the matrix polymer. The poor fluorescence stability of PSt/MPPS NPs at elevated temperatures could be ascribed to the continuous hydrolysis of MPPS. Firstly, the MPPS molecules at the surface of NPs undergo hydrolysis. At elevated temperatures, the incorporated MPPS inside the NPs can freely diffuse to the particle/water interface to undergo hydrolysis under the drive of MPPS concentration difference between the interior and surface of NPs, leading to an almost complete fluorescence quenching. Based on the fluorescence loss mechanism of the PSt/ MPPS NPs, several effective strategies were designed to improve fluorescence stability of AIE polymer/MPPS NPs, for example through using matrix polymers with a relatively less hydrophobicity or a higher glass transition temperature to replace PSt, or fabrication of a less hydrophobic polymer shell on the PSt/MPPS NPs.
1. Introduction Since proposed by Tang et al., in 2001, aggregation-induced emission (AIE) luminogens (AIEgens) have been extensively investigated due to their unique emission mechanism and wide applications in biology, disease diagnostics, optoelectronic devices, sensors, and advanced coatings [1–8]. For biological applications, it is a commonlyused strategy to incorporate AIEgens in a nanoscale polymeric matrix to fabricate AIE polymeric nanoparticles (NPs) [9–11]. Compared with nano-aggregates of pristine AIEgens, AIE polymeric NPs may embody better aqueous dispersibility, more tunable brightness, more controllable particle size, and more flexible surface functionalization [12–14]. Thus, the AIE polymeric NPs may display good biocompatibility, high-quality cell imaging, and targeting ability to specific cells or tissues [15,16]. In recent years, many innovative fabrication techniques, such as
solvent evaporation technique [17], self-assembly of amphiphilic copolymers with AIEgens [18,19], flash nanoprecipitation of block copolymers with AIEgens [20], surface functionalization of NPs with AIEgens [21], semi-continuous polymerization [22], and water-borne heterophase polymerization techniques, such as emulsion and miniemulsion polymerization techniques [23–26], have been devised to prepare versatile AIE polymeric NPs. In water-borne miniemulsion polymerization systems, many hydrophobic monomer droplets are homogenously dispersed in an aqueous continuous phase [27]. The polymeric NPs are formed mainly through monomer droplet nucleation, and each monomer droplet can be regarded as one separated nanoreactor [27]. Versatile functional NPs could be conveniently prepared through in situ encapsulation of functional compounds by polymers during the miniemulsion polymerization process [28,29]. In our previous work, AIE polymeric NPs were prepared through miniemulsion copolymerization of common monomers, like styrene (St) and methyl
∗
Corresponding author. Corresponding author. ∗∗∗ Corresponding author. E-mail addresses: [email protected] (Z. Cao), [email protected] (Z. Zhao), [email protected] (Q. Cui). ∗∗
https://doi.org/10.1016/j.dyepig.2018.08.051 Received 5 August 2018; Received in revised form 26 August 2018; Accepted 27 August 2018 Available online 30 August 2018 0143-7208/ © 2018 Elsevier Ltd. All rights reserved.
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addition of KPS (0.05 g), the monomer miniemulsion was poured into a 250 mL four-necked reactor, and the reactor was kept in a preheated oil bath at 70 °C. The polymerization ran for 3 h under a magnetic agitation of 400 rpm in a nitrogen atmosphere to obtain various polymer/MPPS NPs.
methacrylate (MMA), and a silole-based AIE functional monomer 1allyl-1-methyl-2,3,4,5,-tetraphenylsilole (AMTPS) [23,24]. The photoluminescence (PL) intensity of AIE polymeric NPs could be accurately tuned by the AMTPS content. The synthesized AIE poly (methyl methacrylate) (PMMA) NPs displaying high brightness, low cytotoxicity, and efficient cellar uptake, are of high potential in biological applications. It is well accepted that the fluorescence stability at various circumstances is an important index for AIE probes. In our previous work, the fluorescence stability of AIE PMMA NPs under continuous UV light irradiation was evaluated [23]. However, for AIE polymeric NPs with susceptible AIEgens, like silole-based AIEgens, their fluorescence stability may also been influenced by many external factors, such as temperature, type of the dispersed media and matrix polymers, and pH value of the dispersed media [30]. In this work, AIE polymeric NPs were prepared through miniemulsion polymerization using 1-methyl-1,2,3,4,5-pentaphenylsilole (MPPS) as the model susceptible AIEgen. The temperature-dependent fluorescence stability of polystyrene (PSt)/MPPS NPs was thoroughly investigated. Although the MPPS molecules were encapsulated in the PSt matrix, the PSt/MPPS NPs in aqueous emulsions displayed an abnormally faster fluorescence loss than the pristine MPPS particles at elevated temperatures. A possible fluorescence loss mechanism of the PSt/ MPPS NPs based on the continuous hydrolysis of MPPS was proposed. On the basis of this mechanism, the fluorescence stability of AIE polymeric NPs with a susceptible AIEgen was successfully enhanced by using matrix polymers with a less hydrophobicity or a higher glass transition temperature (Tg) to replace PSt, or fabrication of a less hydrophobic polymeric shell on the PSt/MPPS NPs to suppress the continuous hydrolysis of MPPS.
2.3. Fluorescence stability of polymer/MPPS NPs at various temperatures Ten gram of the original polymer/MPPS NP emulsions were used for each temperature-dependent fluorescence stability measurement. The polymer/MPPS NP emulsions were heated at various temperatures. A small amount of samples were periodically withdrawn in the heating process for fluorescence measurements. The typical PL spectra of PSt/ MPPS NP emulsion heated at 70 °C for various time lengths are shown in Fig. S3. The peak PL intensity was used to evaluate the time-dependent fluorescence loss of polymer/MPPS NPs at various temperatures. 2.4. Fluorescence stability of MPPS NPs in water MPPS (0.01 g) was dissolved in 2 g ethanol, and then mixed with 25 g deionized water. The mixed solution was equally divided into 4 parts, and they were put into oil baths preheated at 30 °C, 50 °C, 60 °C, and 70 °C, respectively. The samples were periodically withdrawn, and their PL spectra were recorded on a Hitachi F-4600 spectrofluorometer. The peak PL intensity was used to evaluate the time-dependent fluorescence loss of MPPS in water at various temperatures. 2.5. Preparation and fluorescence stability of PSt/MPPS blended films
2. Experimental section
Firstly, MPPS and PSt were completely dissolved in THF. Afterwards, uniform thin PSt/MPPS films were fabricated by spincoating and evaporation of THF. The PL spectra of the PSt/MPPS films heated at 70 °C for various time lengths were recorded on the Hitachi F4600 fluorescence spectrophotometer. The peak PL intensity was used to evaluate the time-dependent fluorescence loss of the PSt/MPPS blended films.
2.1. Materials MPPS was synthesized according to the Curtis method [31]. 1H NMR, UV–vis absorption spectrum, and PL spectra of MPPS in the mixed THF/water solvent with various water fractions are provided in supporting information (Fig. S1 and S2). MMA (AR, Tianjin Kermel Chemical Reagent Co., Ltd.) and St (AR, Tianjin Yongda Chemical Reagent Co., Ltd.) were purified through reduced distillation, and stored in a refrigerator before use. Butyl methacrylate (BMA, 99%, Shanghai Aladdin Chemistry Co., Ltd.) was purified through passing an aluminum oxide column. Hexadecane (HD, 99%, Acros Organics), isopar M (a C12eC14 isoparaffinic mixture, Exxon Mobil), sodium dodecyl sulfate (SDS, CP, Shanghai Aladdin Chemistry Co., Ltd.), potassium persulfate (KPS, AR, Shanghai Aladdin Chemistry Co., Ltd.), toluene (AR, Zhejiang Sanying Chemical Reagent Co., Ltd.), and ethanol (AR, Hangzhou Gaojing Fine Chemical Co., Ltd.) were used as received. Tetrahydrofuran (THF, 99%, Sinopharm Chemical Reagent Co., Ltd.) was purified through distillation in the presence of sodium benzophenone ketyl immediately prior to use. Self-made deionized water was used in all experiments.
2.6. Preparation and fluorescence stability of PSt/MPPS NP emulsion in isopar M The solid sample of PSt/MPPS NPs was obtained through drying the aqueous emulsion of PSt/MPPS NPs at 45 °C for 3 h by a rotary evaporator. Isopar M was added to the PSt/MPPS solid sample, and then the dispersion was stirred at room temperature until it became a uniform milky-white emulsion. For evaluation of the fluorescence stability of the PSt/MPPS NP emulsion in isopar M, the emulsion was heated in an oil bath at 70 °C for 19 h under magnetic agitation of 400 rpm, and the PL spectrum of the heated powder sample collected from the emulsion was recorded on the Hitachi F-4600 fluorescence spectrophotometer.
2.2. Preparation of AIE polymeric NPs
2.7. Preparation and fluorescence stability of core–shell PMMA@PSt/ MPPS NPs
A series of MPPS-doped polymeric NPs, including PSt/MPPS NPs, PMMA/MPPS NPs, and poly (butyl methacrylate) (PBMA)/MPPS NPs were prepared through miniemulsion polymerization. Firstly, 0.05 g MPPS was dissolved in a mixed solution of monomer (10 g) and HD (0.6 g) to form a hydrophobic solution, which was used as the oil phase. Secondly, 0.2 g SDS was dissolved in 125 g deionized water to form an aqueous solution, which was used as the water phase. Thirdly, both solutions were mixed and preemulsified with a magnetic agitation of 700 rpm at 40 °C for 15 min to obtain a crude emulsion. Subsequently, the crude emulsion was sonicated at 400 W for 9 min by using a pulse mode (work 12 s, break 6 s) to form a monomer miniemulsion. After
The core PSt/MPPS NPs were prepared as follows: 0.05 g MPPS was dissolved in 7 g St and 0.6 g HD to form an oil solution; 0.2 g SDS was dissolved in 125 g water to form an aqueous solution; the monomer miniemulsion was prepared with the same conditions of preemulsification and sonication as the preparation of other polymer/ MPPS NPs; 0.05 g KPS was added into the monomer miniemulsion to start the reaction; the polymerization ran at 70 °C; After 2 h, 3 g MMA was dropwise added into the polymerization system, and the polymerization was continued to run for another 3 h to obtain PMMA@PSt/ MPPS NPs. The emulsion of PMMA@PSt/MPPS NPs was heated at 70 °C for 573
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19 h. The samples were periodically withdrawn for fluorescence measurements; The peak PL intensity was used to evaluate the time-dependent fluorescence loss of the PMMA@PSt/MPPS NPs. 2.8. Characterization 2.8.1. Measurement of monomer conversion The final conversions of monomers were measured by gravimetric method. A certain amount of as-synthesized emulsions were dried at 80 °C for 24 h. Monomer conversions were calculated through the following equation:
C=
mt ×
m2 m1
mn
− ms
× 100%
in which mn, mt, ms, m1, and m2 indicate the feeding amount of monomers, the total feeding amount of materials, the amount of nonvolatile components, the mass of the withdrawn emulsions, and the mass of the dried samples, respectively. Fig. 1. Time-dependent fluorescence loss of the PSt/MPPS NPs in the aqueous emulsion heated at various temperatures. Photos of the heated PSt/MPPS NP emulsions under irradiation of UV light (inset).
2.8.2. Dynamic light scattering (DLS) Z-average particle sizes and polydispersibility (PDI) values of the polymer/MPPS NPs were measured by DLS (Zetasizer Nanoseries) at 25 °C under a scattering angle of 90°. A drop of emulsion was diluted with 2 mL of distilled water. Particle sizes were given as the average of three measurements.
superb ability to stably emit fluorescence under various circumstances [32]. In this work, the temperature-dependent fluorescence stability of the PSt/MPPS NPs was evaluated. In details, the aqueous emulsions of PSt/MPPS NPs were heated at various temperatures, and the samples were periodically withdrawn for the visual and PL spectroscopic observations. All the visual and PL spectroscopic results are shown in Fig. 1. When the heating temperature was below 55 °C, the heated PSt/ MPPS NP emulsions could still emit strong blue-green fluorescence under irradiation of 365 nm UV light, displaying a good fluorescence stability (Fig. 1 inset). The fluorescence of heated emulsions obviously decreased when the heating temperature was increased from 55 °C to 65 °C. Furthermore, the fluorescence of emulsions that were heated at higher temperatures was completely quenched. The PL spectroscopic results indicated that the peak PL intensity of PSt/MPPS NPs heated below 55 °C only slightly decreased with the increase of the heating time (Fig. 1), consistent with the visual observation. In the temperature range of 55 °C–85 °C, the fluorescence loss was significantly accelerated. The fluorescence of the PSt/MPPS NPs was almost completely quenched within 19 h when the heating temperature was higher than 65 °C. Moreover, the quenching time was obviously shortened with the increase of the heating temperature. For comparison, the time-dependent fluorescence loss of pristine MPPS particles dispersed in water was also monitored at various temperatures, and the results are shown in Fig. 2. Generally, the peak PL intensity of MPPS particles would decrease along with the heating time at various temperatures. Moreover, with the increase of heating temperature, the rate of fluorescence loss was accelerated, and the decreased extent of fluorescence was increased. Due to the incorporation of MPPS in the PSt matrix, the fluorescence stability of PSt/MPPS NPs was better than that of the pristine MPPS particles when the heating temperature was 30 or 50 °C (Fig. 2). Surprisingly, the fluorescence of PSt/MPPS NPs decreased much faster than the pristine MPPS particles did when the heating temperature was 70 °C (Fig. 2). Furthermore, less than 10% of the fluorescence of PSt/MPPS NPs was remained after heated for 14 h at 70 °C, while ∼20% of fluorescence of MPPS particles was still remained after heated for 19 h at the same temperature. The reasons for the worse fluorescence stability of PSt/MPPS NPs than that of pristine MPPS particles at 70 °C will be discussed in the next section.
2.8.3. Transmission electron microscopy (TEM) Particle morphologies were observed by a Hitachi HT-7700 transmission electron microscope operated at 80 kV. The preparation of TEM samples was as follows: a drop of emulsion was diluted with 2 mL of distilled water; a drop of the diluted sample was dropped on a 400mesh carbon-coated copper grid and dried at room temperature. 2.8.4. Fluorescence spectroscopy Aqueous emulsions of various polymer/MPPS NPs (5 μL) were diluted with 2 mL distilled water. The PL spectra of the diluted emulsions of polymer/MPPS NPs were recorded on the Hitachi F-4600 spectrofluorometer in a spectral range of 400–700 nm at the excitation wavelength of 385 nm. For dried powder samples, they were put into a sample tank, and then, the sample tank was inserted in the sample holder of the spectrofluorometer. The blended PSt/MPPS films were directly inserted in the sample holder. The PL spectra of the solid samples were recorded on the same spectrofluorometer in a spectral range of 400–700 nm at the excitation wavelength of 385 nm. 2.8.5. Differential scanning calorimetry (DSC) The emulsions of polymer/MPPS NPs were dialyzed in the deionized water to remove the surfactant. Subsequently, the dialyzed emulsions were dried at 50 °C for 24 h to obtain solid samples. The Tgs of the dried samples were measured by heating from −50 to 150 °C on a Q2000 differential scanning calorimeter (TA Instruments, USA) under a nitrogen atmosphere. The heating rate was set at 20 °C min−1. 3. Results and discussion 3.1. Temperature-dependent fluorescence loss of the PSt/MPPS NPs in the aqueous emulsion In this work, the PSt/MPPS NPs were prepared through water-borne miniemulsion polymerization. The final monomer conversion of the miniemulsion polymerization achieved 90%. The Z-average particle size and PDI of the PSt/MPPS NPs were 136.2 nm and 0.049, respectively. Normally, fluorescent NPs used as long-term probes must have 574
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observed for the sample even after being heated for 22 h. Furthermore, the PL spectra of the PSt/MPPS blended films heated for various time lengths were monitored by fluorescence spectroscopy, and the results are shown in Fig. 3B. The PSt/MPPS blended films displayed an obvious emission band in the spectral range of 400–600 nm, and the peak PL intensity remained almost constant during the heating process (Fig. 3C). All these results demonstrated the good fluorescence stability of the PSt/MPPS blended films at 70 °C. It means that the weak fluorescence stability of the PSt/MPPS NPs in the aqueous emulsion at elevated temperatures is not caused by the interaction between PSt and MPPS.
3.2.2. Hydrolysis of MPPS The pristine MPPS particles in water displayed an almost linear decrement in the PL intensity, and the fluorescence loss was ∼80% after being heated for 19 h at 70 °C (Fig. 2). MPPS, as a silole-based AIEgen, may undergo hydrolysis when contacting with water [31]. Therefore, the relative weak fluorescence stability of MPPS particles might be reasonably ascribed to the hydrolysis of MPPS. Normally, being encapsulated within a polymer matrix may protect AIEgens from oxygen, water, or other chemical species that may take part in photo-degradation reactions to acquire superb photo- and physical stability [11]. For the PSt/MPPS NPs, the MPPS molecules were encapsulated in the PSt matrix, and therefore, the contact between MPPS and water could be reduced in a certain extent. However, it should be pointed out that the MPPS molecules at the surface of NPs could still contact with water, and possibly hydrolyze. Moreover, the MPPS molecules inside the PSt/MPPS NPs may diffuse to the surface under the drive of concentration difference that is caused by the hydrolysis of MPPS at the particle/water interface. In order to clarify the influence of hydrolysis of MPPS on the fluorescence stability of PSt/MPPS NPs, both water and isopar M were selected as the dispersed media of emulsions. The PSt/MPPS emulsions in water and isopar M were heated at 70 °C for 19 h, respectively. The photos of the solid PSt/MPPS NP samples obtained from the aqueous emulsion (PM-W) and the isopar M emulsion (PM-I) are shown in Fig. 4
Fig. 2. Time-dependent fluorescence loss of the MPPS particles and PSt/MPPS NPs heated at various temperatures. The MPPS particles were dispersed in a mixed solvent of water and ethanol.
3.2. Discussion on the poor fluorescence stability of PSt/MPPS NPs at elevated temperatures 3.2.1. Interaction between PSt and MPPS Both MPPS and PSt contain many phenyl rings, and therefore, the reduced fluorescence stability of PSt/MPPS NPs may be caused by the interaction between PSt and MPPS, such as π–π stacking interaction. In order to clarify this issue, a physically blended film of PSt and MPPS was prepared through evaporation of solvent from a THF solution of PSt and MPPS. The fluorescence stability of PSt/MPPS blended films at 70 °C was also evaluated, and the results are shown in Fig. 3. The visual observation of the PSt/MPPS blended film irradiated with UV light indicated that all the heated films could still emit strong blue-green fluorescence (Fig. 3A). No obvious reduction in the PL intensity was
Fig. 3. Photos of the PSt/MPPS blended films heated at 70 °C for various time lengths under irradiation of UV light (A). PL spectra of the PSt/MPPS blended films heated at 70 °C for various time lengths (B). Time-dependent fluorescence loss of the PSt/MPPS blended films and PSt/MPPS NPs at 70 °C (C). 575
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MPPS molecules inside the PSt/MPPS NPs may diffuse from the interior to the surface of NPs to undergo the hydrolysis reaction at an elevated temperature, like 70 °C (Scheme 1 II). As a result, the fluorescence of PSt/MPPS NPs gradually decreased with the continuous hydrolysis of MPPS. In a word, the poor fluorescence stability of PSt/MPPS NPs especially at elevated external temperatures is mainly caused by the continuous hydrolysis of MPPS. The diffusion of MPPS molecules from the interior to the surface of NPs is crucial to the continuous hydrolysis of MPPS. The main factors which may significantly influence the diffusion of MPPS could be the movement ability of polymer chain segments (kinetic factor), the difference in the MPPS concentration between the interior and the surface of NPs, and the difference in the hydrophobicity of MPPS and matrix polymers (thermodynamic factor). Based on these analyses, we will present some effective means to improve the fluorescence stability of AIE polymeric NPs. 3.4. Means to improve the fluorescence stability of AIE polymeric NPs Fig. 4. PL spectra and photos (inset) of the solid PM-W and PM-I samples.
3.4.1. Type of matrix polymer According to the possible mechanism proposed in the previous section, it is important to suppress the diffusion of MPPS from the interior to the surface of NPs for improving the fluorescence stability of polymer/MPPS NPs. Therefore, PMMA was chosen as the matrix polymer, because the hydrophobicity of PMMA is less than that of PSt (Fig. S5). From the thermodynamic point of view, the distribution of MPPS molecules inside the PMMA/MPPS NPs may be the thermodynamically favorable state because of the relatively higher hydrophobicity of MPPS. Under this circumstance, instead of distributing onto the surface of NPs, more MPPS molecules would locate inside the as-synthesized PMMA/MPPS NPs to avoid contacting water. Thus, it is expected that the hydrolysis of MPPS can be effectively suppressed. As described in the experimental section, the PMMA/MPPS NPs were also prepared through miniemulsion polymerization, and the final monomer conversion of this system was 96%. As expected, the PMMA/MPPS NPs displayed a much better fluorescence stability, compared with the PSt/ MPPS NPs (Fig. 5). Only ∼10% of fluorescence was lost after heated at 70 °C for 19 h. The slight fluorescence loss may be ascribed to the hydrolysis of MPPS located at or close to the surface of NPs. It should be pointed out the hydrolysis of MPPS at the particle/ water interface may also build a concentration difference between the interior and the surface of NPs. The concentration difference may also drive the diffusion of MPPS to the surface of NPs. However, in this case, on one hand, the thermodynamic driving force may counteract or even overpass the concentration difference, leading to the distribution of MPPS inside the NPs; on the other hand, the external temperature (70 °C) was much lower than the Tg of dried PMMA/MPPS NPs (109.6 °C), and thus the polymer chain segments and MPPS molecules might be still frozen in this case. Therefore, the MPPS molecules inside the PMMA/MPPS NPs would not diffuse to the surface of NPs to undergo continuous hydrolysis. Only the MPPS molecules on the surface of NPs took part in the hydrolysis reaction (Scheme 1 I). Consequently, the majority of the fluorescence of PMMA/MPPS NPs in the heated emulsion could still be maintained. In order to clarify the role of the thermodynamic factor in the fluorescence stability of polymer/MPPS NPs, polymers with a low Tg (PBMA) was selected as the matrix polymer. The PBMA/MPPS NPs were prepared through miniemulsion polymerization again, and the final monomer conversion of this system was 93%. The Tg of dried PBMA/ MPPS NPs was only 22.6 °C (Fig. S4), far below 70 °C. Therefore, the influence of the kinetic factor on the diffusion of MPPS could be completely excluded, as the MPPS molecules inside the PBMA/MPPS NPs could freely diffuse under the rubbery state. As shown in Fig. 5, the PBMA/MPPS NPs showed a similar high fluorescence stability as the PMMA/MPPS NPs. PBMA is less hydrophobic than St (Fig. S5), and thus, the MPPS molecules may be inclined to partitioning inside the
inset. Under the irradiation of UV light, the solid PM-I sample could still emit strong blue-green fluorescence, while the solid PM-W sample could not emit obvious fluorescence. The PL spectroscopic results in Fig. 4 indicated that the solid PM-I sample displayed an obvious emission band in the spectral range of 400–600 nm. In the same spectral range, the PL spectrum of the solid PM-W sample was a line parallel to the abscissa, indicating that it was almost non-emissive (Fig. 4). All the results indicated that water is a vital factor that impacts the hydrolysis of MPPS and in turn the fluorescence stability of PSt/MPPS NPs. 3.3. Possible mechanism for the fluorescence loss of PSt/MPPS NPs in aqueous emulsions As demonstrated in the previous section, the hydrolysis of MPPS is the main reason for the fluorescence loss of PSt/MPPS NPs. The precondition to the hydrolysis of MPPS is that the MPPS molecules must contact with water. Therefore, the MPPS molecules inside the PSt/ MPPS NPs are required to diffuse to the particle/water interface prior to participation in the hydrolysis reaction. As shown in Fig. S4, the Tg of dried PSt/MPPS NPs was 74.3 °C. Under an external temperature (≤50 °C) far below the Tg of matrix polymer, the movement of polymer chain segments is frozen, and simultaneously, the MPPS molecules are also entrapped in the frozen polymer matrix. Under this circumstance, the diffusion of MPPS in the polymer matrix is restricted by the kinetic factors. Thus, the MPPS molecules inside the PSt/MPPS NPs cannot diffuse to the particle/water interface. As a result, only the MPPS molecules at or close to the particle/water interface can undergo the hydrolysis reaction at a relatively low external temperature (Scheme 1 I). Therefore, the fluorescence loss of PSt/MPPS NPs is less than 20% when the external temperature was below 50 °C (Fig. 1). At a higher external temperature (closed to the Tg of matrix polymer), the polymer chain segments may be gradually unfrozen, and their movement is enhanced. Meanwhile, the MPPS molecules inside the PSt/MPPS NPs are unfrozen too, and they may diffuse from the interior to the surface of NPs to participate in the hydrolysis reaction (Scheme 1 II). The driving force of the MPPS diffusion could be the concentration difference between the interior and surface of NPs due to the consumption of MPPS at the surface through hydrolysis. It should be pointed out that the distribution of MPPS in the PSt/MPPS NPs is also determined by the thermodynamic factors. Although we cannot accurately evaluate the difference in the hydrophobicity of PSt and MPPS, we assume that their hydrophobicity may be similar because both of them contain many phenyl groups. Therefore, the diffusion direction of MPPS in the PSt/MPPS NPs is mainly determined by the concentration difference between the interior and surface of NPs. Consequently, the 576
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Scheme 1. Possible mechanism of the fluorescence loss of polymer/MPPS NPs in aqueous emulsions.
could not be well distinguished, the number-average particle size of PMMA@PSt/MPPS NPs determined by TEM increased about 3 nm after the growth of the PMMA shell. According to the results of time-dependent fluorescence loss shown in Fig. 6C, the PMMA@PSt/MPPS NPs display a much better fluorescence stability than the PSt/MPPS NPs. This result point to the successful formation of PMMA shell on the PSt/MPPS NPs and furthermore the successfully suppressed continuous hydrolysis of MPPS by the formed PMMA shell.
4. Conclusions AIE polymeric NPs with a susceptible AIEgen, MPPS, were prepared through miniemulsion polymerization. The temperature-dependent fluorescence stability of PSt/MPPS NPs was thoroughly investigated. Compared with the pristine MPPS particles, the PSt/MPPS NPs displayed a better fluorescence stability when the external temperature was much below the Tg of the matrix polymer. However, the fluorescence stability of PSt/MPPS NPs became much worse than that of the MPPS particles at an external temperature close to or above the Tg of the matrix polymer. The interaction between PSt and MPPS did not show an obvious influence on the fluorescence stability of PSt/MPPS NPs. The worse fluorescence stability of PSt/MPPS NPs at elevated temperatures was mainly caused by the continuous hydrolysis of MPPS. The temperature and water are two key factors that may influence the hydrolysis of MPPS and furthermore the fluorescence stability of PSt/ MPPS NPs. Firstly, the MPPS molecules at the particle/water interface undergo hydrolysis to build a concentration difference between the interior and the surface of NPs. Under the drive of the concentration difference, the MPPS molecules inside the NPs may continuously diffuse to the interface to undergo hydrolysis. Consequently, the fluorescence was almost completely quenched due to the entire consumption of MPPS molecules in the PSt/MPPS NPs. From the kinetic and thermodynamic points of view, the fluorescence stability of AIE polymeric NPs may be enhanced by using matrix polymers with a less hydrophobicity or with a higher Tg, or fabrication of a less hydrophobic polymeric shell on the PSt/MPPS NPs. The results of this work may contribute to understanding the fluorescence instability of AIE polymeric NPs in aqueous emulsions, and guide the synthesis of AIE polymeric NPs with a long-term fluorescence stability through carefully choosing matrix polymers or particle morphology.
Fig. 5. Time-dependent fluorescence loss of polymer/MPPS NPs with various matrix polymers at 70 °C.
NPs. Although some MPPS molecules at or close to the surface of NPs still underwent hydrolysis, leading to the reduction in the MPPS concentration at the surface, the MPPS molecules inside the NPs may not diffuse to the surface of NPs under the control of thermodynamic forces (Scheme 1 III). Consequently, the continuous hydrolysis of MPPS molecules may not happen in the PBMA/MPPS NPs, resulting in an excellent fluorescence stability. All the results indicate that the diffusion of MPPS in the polymer/ MPPS NPs is simultaneously determined by the kinetic and thermodynamic factors. Therefore, the fluorescence stability of polymer/MPPS NPs may be efficiently enhanced through adopting a matrix polymer with a less hydrophobicity or a relatively higher Tg than that of PSt. 3.4.2. Particle morphology In previous sections, we demonstrated that the continuous hydrolysis of MPPS molecules was the major reason for the poor fluorescence stability of PSt/MPPS NPs and some matrix polymers, such as PMMA and PBMA, can suppress the diffusion of MPPS molecules to the particle/water interface to undergo hydrolysis. In order to improve the fluorescence stability of PSt/MPPS NPs, an additional PMMA shell was formed on the surface of PSt/MPPS NPs. The TEM images of PSt/MPPS NPs and PMMA@PSt/MPPS NPs are shown in Fig. 6A and B, respectively. Although the core–shell structure of PMMA@PSt/MPPS NPs 577
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Fig. 6. TEM images of PSt/MPPS NPs (A) and PMMA@PSt/MPPS core–shell NPs (B). Time-dependent fluorescence loss of PSt/MPPS NPs and PMMA@PSt/MPPS NPs at 70 °C (C).
Conflicts of interest
photoacoustic imaging. Chem Soc Rev 2014;43:6570–97. [12] Qi Y, Xu C, Nizam MN, Li Y, Yu BR, Xu F-J. Functionalized PGMA nanoparticles with aggregation-induced emission characteristics for gene delivery systems. Polym Chem 2016;7:5630–40. [13] Lv QL, Wang K, Xu DZ, Liu MY, Wan Q, Huang HY, et al. Synthesis of amphiphilic hyperbranched AIE-active fluorescent organic nanoparticles and their application in biological application. Macromol Biosci 2016;16:223–30. [14] Jin GR, Feng GX, QIn W, Tang BZ, Liu B, Li K. Multifunctional organic nanoparticles with aggregation-induced emission (AIE) characteristics for targeted photodynamic therapy and RNA interference therapy. Chem Commun 2016;52:2752–5. [15] Zhang XY, Wang K, Liu MY, Tao L, Chen YW, Wei Y. Polymeric AIE-based nanoprobes for biomedical applications: recent advances and perspectives. Nanoscale 2015;7:11486–508. [16] Yan LL, Zhang Y, Xu B, Tian WJ. Fluorescent nanoparticles based on AIE fluorogens for bioimaging. Nanoscale 2016;8:2471–87. [17] Qin W, Ding D, Liu JZ, Yuan WZ, Hu Y, Liu B, et al. Biocompatible nanoparticles with aggregation-induced emission characteristics as far-red/near-infrared fluorescent bioprobes for in vitro and in vivo imaging applications. Adv Funct Mater 2012;22:771–9. [18] Zhang XQ, Liu MY, Yang B, Zhang XY, Chi ZG, Liu SW, et al. Cross-linkable aggregation induced emission dye based red fluorescent organic nanoparticles and their cell imaging applications. Polym Chem 2013;4:5060–4. [19] Long Z, Liu MY, Wan Q, Mao LC, Huang HY, Zeng GJ, et al. Ultrafast preparation of AIE-active fluorescent organic nanoparticles via a “one-Pot” microwave-assisted kabachnik-fields reaction. Macromol Rapid Commun 2016;37:1754–9. [20] Wang MW, Yang N, Guo ZQ, Gu KZ, Shao AD, Zhu WH, et al. Facile preparation of AIE-active fluorescent nanoparticles through flash nanoprecipitation. Ind Eng Chem Res 2015;54:4683–8. [21] Xu SY, Bai XL, Ma JW, Xu MM, Hu GF, James TD, et al. Ultrasmall organic nanoparticles with aggregation-induced emission and enhanced quantum yield for fluorescence cell imaging. Anal Chem 2016;88:7853–7. [22] Li X, Li CP, Wang S, Dong H, Ma X, Cao DR. Synthesis and properties of photochromic spirooxazine with aggregation-induced emission fluorophores polymeric nanoparticles. Dyes Pigments 2017;142:481–90. [23] Cao ZH, Liang XQ, Chen HN, Gao M, Zhao ZJ, Chen XL, et al. Bright and biocompatible AIE polymeric nanoparticles prepared from miniemulsion for fluorescence cell imaging. Polym Chem 2016;7:5571–8. [24] Cao ZH, Xu C, Liang LH, Zhao ZJ, Chen B, Chen ZJ, et al. A green miniemulsionbased synthesis of polymeric aggregation-induced emission nanoparticles. Polym Chem 2015;6:6378–85. [25] Liu MY, Zhang XQ, Yang B, Deng FJ, Li Z, Wei JC, et al. Water dispersible, noncytotoxic, cross-linked luminescent AIE dots: facile preparation and bioimaging applications. Appl Surf Sci 2014;322:155–61. [26] Zhang XY, Zhang XQ, Yang B, Liu MY, Liu WY, Chen YW, et al. Fabrication of aggregation induced emission dye-based fluorescent organic nanoparticles via emulsion polymerization and their cell imaging applications. Polym Chem 2014;5:399–404. [27] Schork FJ, Luo YW, Smulders W, Russum JP, Butté A, Fontenot K. Miniemulsion polymerization. Adv Polym Sci 2005;175:129–255. [28] Qi DM, Cao ZH, Ziener U. Recent advances in the preparation of hybrid nanoparticles in miniemulsions. Adv Colloid Interface Sci 2014;211:47–62. [29] Landfester K. Miniemulsion polymerization and the structure of polymer and hybrid nanoparticles. Angew Chem Int Ed 2009;48:4488–507. [30] Zhao ZJ, He BR, Tang BZ. Aggregation-induced emission of siloles. Chem Sci 2015;6:5347–65. [31] Curtis MD. Synthesis and reactions of some functionally substituted sila- and germacyclopentadienes. J Am Chem Soc 1969;91:6011–8. [32] Li K, QIn W, DIng D, Tomczak N, Geng JL, Liu RR, et al. Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive longterm cell tracing. Sci Rep 2013;3:1150.
The authors declare no conflict of interest. Acknowledgements Financial supports from the National Natural Science Foundation of China project (51573168 and U1609205), the Zhejiang Provincial Natural Science Foundation (LY16E030006 and LZ18E030002), the Science Foundation of Zhejiang Sci-Tech University (14012208-Y), the Excellent Young Researchers Foundation (CETT2015001) of Zhejiang Provincial Top Key Academic Discipline of Chemical Engineering and Technology, the Young Researchers Foundation of Zhejiang Provincial Top Key Academic Discipline of Textile Science and Engineering (2015YXQN03), Zhejiang Province's Xinmiao Talent Plan (2017R406020 and 2017R406022) and National Undergraduate Training Programs for Innovation and Entrepreneurship of China (201710338021) are gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.dyepig.2018.08.051. References [1] Ding D, Li K, Liu B, Tang BZ. Bioprobes based on AIE fluorogens. Acc Chem Res 2013;46:2441–53. [2] Hong YN, Lam JWY, Tang BZ. Aggregation-induced emission. Chem Soc Rev 2011;40:5361–88. [3] Hong YN, Lam JWY, Tang BZ. Aggregation-induced emission: phenomenon, mechanism and applications. Chem Commun 2009:4332–53. [4] Feng GX, Kwok RTK, Tang BZ, Liu B. Functionality and versatility of aggregationinduced emission luminogens. Appl Phys Rev 2017;4:021307. [5] Dong H, Luo M, Wang S, Ma X. Synthesis and properties of tetraphenylethylene derivative diarylethene with photochromism and aggregation-induced emission. Dyes Pigments 2017;139:118–28. [6] Gu XG, Kwok RTK, Lam JWY, Tang BZ. AIEgens for biological process monitoring and disease theranostics. Biomaterials 2017;146:115–35. [7] Gu XG, Zhao EG, Lam JWY, Peng Q, Xie YJ, Zhang YL, et al. Mitochondrion-specific live-cell bioprobe operated in a fluorescence turn-on manner and a well-designed photoactivatable mechanism. Adv Mater 2015;27:7093–100. [8] Gu XG, Zhao EG, Zhao T, Kang MM, Gui C, Lam JWY, et al. A mitochondrionspecific photoactivatable fluorescence turn-on AIE-based bioprobe for localization super-resolution microscope. Adv Mater 2016;28:5064–71. [9] Zhao YM, Zhu W, Ren LX, Zhang K. Aggregation-induced emission polymer nanoparticles with pH-responsive fluorescence. Polym Chem 2016;7:5386–95. [10] Wan Q, Zeng GJ, He ZY, Mao LC, Liu MY, Huang HY, et al. Fabrication and biomedical applications of AIE active nanotheranostics through the combination of a ring-opening reaction and formation of dynamic hydrazones. J Mater Chem B 2016;4:5692–9. [11] Li K, Liu B. Polymer-encapsulated organic nanoparticles for fluorescence and
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Mechanistic investigation on fluorescence instability of AIE polymeric nanoparticles with a susceptible AIEgen prepared in miniemulsions
T
Xiaoqin Lianga, Yaxin Hua, Junjie Loua, Zhihai Caoa,∗, Lin Liua, Yijia Wanga, Zujin Zhaob,∗∗, Dongming Qia, Qinmin Cuic,∗∗∗ a Key Laboratory of Advanced Textile Materials and Manufacturing Technology and Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou, 310018, China b Center for Aggregation-Induced Emission, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, 510640, China c School of Pharmacy, Hangzhou Medical College, 481 Binwen Road, Hangzhou, 310053, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluorescence stability Aggregation-induced emission Hydrolysis Miniemulsion polymerization AIE polymeric nanoparticles
Fluorescence stability is a key index for application of aggregation-induced emission (AIE) polymeric nanoparticles (NPs) as fluorescent probes. In this work, the temperature-dependent fluorescence stability of AIE polystyrene (PSt) NPs with a susceptible AIE luminogens (1-methyl-1,2,3,4,5-pentaphenylsilole, MPPS), which was named as PSt/MPPS NPs, was thoroughly investigated. The PSt/MPPS NPs in aqueous emulsions showed an abnormally fast fluorescence loss at the temperatures close to or above the glass transition temperature of the matrix polymer. The poor fluorescence stability of PSt/MPPS NPs at elevated temperatures could be ascribed to the continuous hydrolysis of MPPS. Firstly, the MPPS molecules at the surface of NPs undergo hydrolysis. At elevated temperatures, the incorporated MPPS inside the NPs can freely diffuse to the particle/water interface to undergo hydrolysis under the drive of MPPS concentration difference between the interior and surface of NPs, leading to an almost complete fluorescence quenching. Based on the fluorescence loss mechanism of the PSt/ MPPS NPs, several effective strategies were designed to improve fluorescence stability of AIE polymer/MPPS NPs, for example through using matrix polymers with a relatively less hydrophobicity or a higher glass transition temperature to replace PSt, or fabrication of a less hydrophobic polymer shell on the PSt/MPPS NPs.
1. Introduction Since proposed by Tang et al., in 2001, aggregation-induced emission (AIE) luminogens (AIEgens) have been extensively investigated due to their unique emission mechanism and wide applications in biology, disease diagnostics, optoelectronic devices, sensors, and advanced coatings [1–8]. For biological applications, it is a commonlyused strategy to incorporate AIEgens in a nanoscale polymeric matrix to fabricate AIE polymeric nanoparticles (NPs) [9–11]. Compared with nano-aggregates of pristine AIEgens, AIE polymeric NPs may embody better aqueous dispersibility, more tunable brightness, more controllable particle size, and more flexible surface functionalization [12–14]. Thus, the AIE polymeric NPs may display good biocompatibility, high-quality cell imaging, and targeting ability to specific cells or tissues [15,16]. In recent years, many innovative fabrication techniques, such as
solvent evaporation technique [17], self-assembly of amphiphilic copolymers with AIEgens [18,19], flash nanoprecipitation of block copolymers with AIEgens [20], surface functionalization of NPs with AIEgens [21], semi-continuous polymerization [22], and water-borne heterophase polymerization techniques, such as emulsion and miniemulsion polymerization techniques [23–26], have been devised to prepare versatile AIE polymeric NPs. In water-borne miniemulsion polymerization systems, many hydrophobic monomer droplets are homogenously dispersed in an aqueous continuous phase [27]. The polymeric NPs are formed mainly through monomer droplet nucleation, and each monomer droplet can be regarded as one separated nanoreactor [27]. Versatile functional NPs could be conveniently prepared through in situ encapsulation of functional compounds by polymers during the miniemulsion polymerization process [28,29]. In our previous work, AIE polymeric NPs were prepared through miniemulsion copolymerization of common monomers, like styrene (St) and methyl
∗
Corresponding author. Corresponding author. ∗∗∗ Corresponding author. E-mail addresses: [email protected] (Z. Cao), [email protected] (Z. Zhao), [email protected] (Q. Cui). ∗∗
https://doi.org/10.1016/j.dyepig.2018.08.051 Received 5 August 2018; Received in revised form 26 August 2018; Accepted 27 August 2018 Available online 30 August 2018 0143-7208/ © 2018 Elsevier Ltd. All rights reserved.
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addition of KPS (0.05 g), the monomer miniemulsion was poured into a 250 mL four-necked reactor, and the reactor was kept in a preheated oil bath at 70 °C. The polymerization ran for 3 h under a magnetic agitation of 400 rpm in a nitrogen atmosphere to obtain various polymer/MPPS NPs.
methacrylate (MMA), and a silole-based AIE functional monomer 1allyl-1-methyl-2,3,4,5,-tetraphenylsilole (AMTPS) [23,24]. The photoluminescence (PL) intensity of AIE polymeric NPs could be accurately tuned by the AMTPS content. The synthesized AIE poly (methyl methacrylate) (PMMA) NPs displaying high brightness, low cytotoxicity, and efficient cellar uptake, are of high potential in biological applications. It is well accepted that the fluorescence stability at various circumstances is an important index for AIE probes. In our previous work, the fluorescence stability of AIE PMMA NPs under continuous UV light irradiation was evaluated [23]. However, for AIE polymeric NPs with susceptible AIEgens, like silole-based AIEgens, their fluorescence stability may also been influenced by many external factors, such as temperature, type of the dispersed media and matrix polymers, and pH value of the dispersed media [30]. In this work, AIE polymeric NPs were prepared through miniemulsion polymerization using 1-methyl-1,2,3,4,5-pentaphenylsilole (MPPS) as the model susceptible AIEgen. The temperature-dependent fluorescence stability of polystyrene (PSt)/MPPS NPs was thoroughly investigated. Although the MPPS molecules were encapsulated in the PSt matrix, the PSt/MPPS NPs in aqueous emulsions displayed an abnormally faster fluorescence loss than the pristine MPPS particles at elevated temperatures. A possible fluorescence loss mechanism of the PSt/ MPPS NPs based on the continuous hydrolysis of MPPS was proposed. On the basis of this mechanism, the fluorescence stability of AIE polymeric NPs with a susceptible AIEgen was successfully enhanced by using matrix polymers with a less hydrophobicity or a higher glass transition temperature (Tg) to replace PSt, or fabrication of a less hydrophobic polymeric shell on the PSt/MPPS NPs to suppress the continuous hydrolysis of MPPS.
2.3. Fluorescence stability of polymer/MPPS NPs at various temperatures Ten gram of the original polymer/MPPS NP emulsions were used for each temperature-dependent fluorescence stability measurement. The polymer/MPPS NP emulsions were heated at various temperatures. A small amount of samples were periodically withdrawn in the heating process for fluorescence measurements. The typical PL spectra of PSt/ MPPS NP emulsion heated at 70 °C for various time lengths are shown in Fig. S3. The peak PL intensity was used to evaluate the time-dependent fluorescence loss of polymer/MPPS NPs at various temperatures. 2.4. Fluorescence stability of MPPS NPs in water MPPS (0.01 g) was dissolved in 2 g ethanol, and then mixed with 25 g deionized water. The mixed solution was equally divided into 4 parts, and they were put into oil baths preheated at 30 °C, 50 °C, 60 °C, and 70 °C, respectively. The samples were periodically withdrawn, and their PL spectra were recorded on a Hitachi F-4600 spectrofluorometer. The peak PL intensity was used to evaluate the time-dependent fluorescence loss of MPPS in water at various temperatures. 2.5. Preparation and fluorescence stability of PSt/MPPS blended films
2. Experimental section
Firstly, MPPS and PSt were completely dissolved in THF. Afterwards, uniform thin PSt/MPPS films were fabricated by spincoating and evaporation of THF. The PL spectra of the PSt/MPPS films heated at 70 °C for various time lengths were recorded on the Hitachi F4600 fluorescence spectrophotometer. The peak PL intensity was used to evaluate the time-dependent fluorescence loss of the PSt/MPPS blended films.
2.1. Materials MPPS was synthesized according to the Curtis method [31]. 1H NMR, UV–vis absorption spectrum, and PL spectra of MPPS in the mixed THF/water solvent with various water fractions are provided in supporting information (Fig. S1 and S2). MMA (AR, Tianjin Kermel Chemical Reagent Co., Ltd.) and St (AR, Tianjin Yongda Chemical Reagent Co., Ltd.) were purified through reduced distillation, and stored in a refrigerator before use. Butyl methacrylate (BMA, 99%, Shanghai Aladdin Chemistry Co., Ltd.) was purified through passing an aluminum oxide column. Hexadecane (HD, 99%, Acros Organics), isopar M (a C12eC14 isoparaffinic mixture, Exxon Mobil), sodium dodecyl sulfate (SDS, CP, Shanghai Aladdin Chemistry Co., Ltd.), potassium persulfate (KPS, AR, Shanghai Aladdin Chemistry Co., Ltd.), toluene (AR, Zhejiang Sanying Chemical Reagent Co., Ltd.), and ethanol (AR, Hangzhou Gaojing Fine Chemical Co., Ltd.) were used as received. Tetrahydrofuran (THF, 99%, Sinopharm Chemical Reagent Co., Ltd.) was purified through distillation in the presence of sodium benzophenone ketyl immediately prior to use. Self-made deionized water was used in all experiments.
2.6. Preparation and fluorescence stability of PSt/MPPS NP emulsion in isopar M The solid sample of PSt/MPPS NPs was obtained through drying the aqueous emulsion of PSt/MPPS NPs at 45 °C for 3 h by a rotary evaporator. Isopar M was added to the PSt/MPPS solid sample, and then the dispersion was stirred at room temperature until it became a uniform milky-white emulsion. For evaluation of the fluorescence stability of the PSt/MPPS NP emulsion in isopar M, the emulsion was heated in an oil bath at 70 °C for 19 h under magnetic agitation of 400 rpm, and the PL spectrum of the heated powder sample collected from the emulsion was recorded on the Hitachi F-4600 fluorescence spectrophotometer.
2.2. Preparation of AIE polymeric NPs
2.7. Preparation and fluorescence stability of core–shell PMMA@PSt/ MPPS NPs
A series of MPPS-doped polymeric NPs, including PSt/MPPS NPs, PMMA/MPPS NPs, and poly (butyl methacrylate) (PBMA)/MPPS NPs were prepared through miniemulsion polymerization. Firstly, 0.05 g MPPS was dissolved in a mixed solution of monomer (10 g) and HD (0.6 g) to form a hydrophobic solution, which was used as the oil phase. Secondly, 0.2 g SDS was dissolved in 125 g deionized water to form an aqueous solution, which was used as the water phase. Thirdly, both solutions were mixed and preemulsified with a magnetic agitation of 700 rpm at 40 °C for 15 min to obtain a crude emulsion. Subsequently, the crude emulsion was sonicated at 400 W for 9 min by using a pulse mode (work 12 s, break 6 s) to form a monomer miniemulsion. After
The core PSt/MPPS NPs were prepared as follows: 0.05 g MPPS was dissolved in 7 g St and 0.6 g HD to form an oil solution; 0.2 g SDS was dissolved in 125 g water to form an aqueous solution; the monomer miniemulsion was prepared with the same conditions of preemulsification and sonication as the preparation of other polymer/ MPPS NPs; 0.05 g KPS was added into the monomer miniemulsion to start the reaction; the polymerization ran at 70 °C; After 2 h, 3 g MMA was dropwise added into the polymerization system, and the polymerization was continued to run for another 3 h to obtain PMMA@PSt/ MPPS NPs. The emulsion of PMMA@PSt/MPPS NPs was heated at 70 °C for 573
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19 h. The samples were periodically withdrawn for fluorescence measurements; The peak PL intensity was used to evaluate the time-dependent fluorescence loss of the PMMA@PSt/MPPS NPs. 2.8. Characterization 2.8.1. Measurement of monomer conversion The final conversions of monomers were measured by gravimetric method. A certain amount of as-synthesized emulsions were dried at 80 °C for 24 h. Monomer conversions were calculated through the following equation:
C=
mt ×
m2 m1
mn
− ms
× 100%
in which mn, mt, ms, m1, and m2 indicate the feeding amount of monomers, the total feeding amount of materials, the amount of nonvolatile components, the mass of the withdrawn emulsions, and the mass of the dried samples, respectively. Fig. 1. Time-dependent fluorescence loss of the PSt/MPPS NPs in the aqueous emulsion heated at various temperatures. Photos of the heated PSt/MPPS NP emulsions under irradiation of UV light (inset).
2.8.2. Dynamic light scattering (DLS) Z-average particle sizes and polydispersibility (PDI) values of the polymer/MPPS NPs were measured by DLS (Zetasizer Nanoseries) at 25 °C under a scattering angle of 90°. A drop of emulsion was diluted with 2 mL of distilled water. Particle sizes were given as the average of three measurements.
superb ability to stably emit fluorescence under various circumstances [32]. In this work, the temperature-dependent fluorescence stability of the PSt/MPPS NPs was evaluated. In details, the aqueous emulsions of PSt/MPPS NPs were heated at various temperatures, and the samples were periodically withdrawn for the visual and PL spectroscopic observations. All the visual and PL spectroscopic results are shown in Fig. 1. When the heating temperature was below 55 °C, the heated PSt/ MPPS NP emulsions could still emit strong blue-green fluorescence under irradiation of 365 nm UV light, displaying a good fluorescence stability (Fig. 1 inset). The fluorescence of heated emulsions obviously decreased when the heating temperature was increased from 55 °C to 65 °C. Furthermore, the fluorescence of emulsions that were heated at higher temperatures was completely quenched. The PL spectroscopic results indicated that the peak PL intensity of PSt/MPPS NPs heated below 55 °C only slightly decreased with the increase of the heating time (Fig. 1), consistent with the visual observation. In the temperature range of 55 °C–85 °C, the fluorescence loss was significantly accelerated. The fluorescence of the PSt/MPPS NPs was almost completely quenched within 19 h when the heating temperature was higher than 65 °C. Moreover, the quenching time was obviously shortened with the increase of the heating temperature. For comparison, the time-dependent fluorescence loss of pristine MPPS particles dispersed in water was also monitored at various temperatures, and the results are shown in Fig. 2. Generally, the peak PL intensity of MPPS particles would decrease along with the heating time at various temperatures. Moreover, with the increase of heating temperature, the rate of fluorescence loss was accelerated, and the decreased extent of fluorescence was increased. Due to the incorporation of MPPS in the PSt matrix, the fluorescence stability of PSt/MPPS NPs was better than that of the pristine MPPS particles when the heating temperature was 30 or 50 °C (Fig. 2). Surprisingly, the fluorescence of PSt/MPPS NPs decreased much faster than the pristine MPPS particles did when the heating temperature was 70 °C (Fig. 2). Furthermore, less than 10% of the fluorescence of PSt/MPPS NPs was remained after heated for 14 h at 70 °C, while ∼20% of fluorescence of MPPS particles was still remained after heated for 19 h at the same temperature. The reasons for the worse fluorescence stability of PSt/MPPS NPs than that of pristine MPPS particles at 70 °C will be discussed in the next section.
2.8.3. Transmission electron microscopy (TEM) Particle morphologies were observed by a Hitachi HT-7700 transmission electron microscope operated at 80 kV. The preparation of TEM samples was as follows: a drop of emulsion was diluted with 2 mL of distilled water; a drop of the diluted sample was dropped on a 400mesh carbon-coated copper grid and dried at room temperature. 2.8.4. Fluorescence spectroscopy Aqueous emulsions of various polymer/MPPS NPs (5 μL) were diluted with 2 mL distilled water. The PL spectra of the diluted emulsions of polymer/MPPS NPs were recorded on the Hitachi F-4600 spectrofluorometer in a spectral range of 400–700 nm at the excitation wavelength of 385 nm. For dried powder samples, they were put into a sample tank, and then, the sample tank was inserted in the sample holder of the spectrofluorometer. The blended PSt/MPPS films were directly inserted in the sample holder. The PL spectra of the solid samples were recorded on the same spectrofluorometer in a spectral range of 400–700 nm at the excitation wavelength of 385 nm. 2.8.5. Differential scanning calorimetry (DSC) The emulsions of polymer/MPPS NPs were dialyzed in the deionized water to remove the surfactant. Subsequently, the dialyzed emulsions were dried at 50 °C for 24 h to obtain solid samples. The Tgs of the dried samples were measured by heating from −50 to 150 °C on a Q2000 differential scanning calorimeter (TA Instruments, USA) under a nitrogen atmosphere. The heating rate was set at 20 °C min−1. 3. Results and discussion 3.1. Temperature-dependent fluorescence loss of the PSt/MPPS NPs in the aqueous emulsion In this work, the PSt/MPPS NPs were prepared through water-borne miniemulsion polymerization. The final monomer conversion of the miniemulsion polymerization achieved 90%. The Z-average particle size and PDI of the PSt/MPPS NPs were 136.2 nm and 0.049, respectively. Normally, fluorescent NPs used as long-term probes must have 574
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observed for the sample even after being heated for 22 h. Furthermore, the PL spectra of the PSt/MPPS blended films heated for various time lengths were monitored by fluorescence spectroscopy, and the results are shown in Fig. 3B. The PSt/MPPS blended films displayed an obvious emission band in the spectral range of 400–600 nm, and the peak PL intensity remained almost constant during the heating process (Fig. 3C). All these results demonstrated the good fluorescence stability of the PSt/MPPS blended films at 70 °C. It means that the weak fluorescence stability of the PSt/MPPS NPs in the aqueous emulsion at elevated temperatures is not caused by the interaction between PSt and MPPS.
3.2.2. Hydrolysis of MPPS The pristine MPPS particles in water displayed an almost linear decrement in the PL intensity, and the fluorescence loss was ∼80% after being heated for 19 h at 70 °C (Fig. 2). MPPS, as a silole-based AIEgen, may undergo hydrolysis when contacting with water [31]. Therefore, the relative weak fluorescence stability of MPPS particles might be reasonably ascribed to the hydrolysis of MPPS. Normally, being encapsulated within a polymer matrix may protect AIEgens from oxygen, water, or other chemical species that may take part in photo-degradation reactions to acquire superb photo- and physical stability [11]. For the PSt/MPPS NPs, the MPPS molecules were encapsulated in the PSt matrix, and therefore, the contact between MPPS and water could be reduced in a certain extent. However, it should be pointed out that the MPPS molecules at the surface of NPs could still contact with water, and possibly hydrolyze. Moreover, the MPPS molecules inside the PSt/MPPS NPs may diffuse to the surface under the drive of concentration difference that is caused by the hydrolysis of MPPS at the particle/water interface. In order to clarify the influence of hydrolysis of MPPS on the fluorescence stability of PSt/MPPS NPs, both water and isopar M were selected as the dispersed media of emulsions. The PSt/MPPS emulsions in water and isopar M were heated at 70 °C for 19 h, respectively. The photos of the solid PSt/MPPS NP samples obtained from the aqueous emulsion (PM-W) and the isopar M emulsion (PM-I) are shown in Fig. 4
Fig. 2. Time-dependent fluorescence loss of the MPPS particles and PSt/MPPS NPs heated at various temperatures. The MPPS particles were dispersed in a mixed solvent of water and ethanol.
3.2. Discussion on the poor fluorescence stability of PSt/MPPS NPs at elevated temperatures 3.2.1. Interaction between PSt and MPPS Both MPPS and PSt contain many phenyl rings, and therefore, the reduced fluorescence stability of PSt/MPPS NPs may be caused by the interaction between PSt and MPPS, such as π–π stacking interaction. In order to clarify this issue, a physically blended film of PSt and MPPS was prepared through evaporation of solvent from a THF solution of PSt and MPPS. The fluorescence stability of PSt/MPPS blended films at 70 °C was also evaluated, and the results are shown in Fig. 3. The visual observation of the PSt/MPPS blended film irradiated with UV light indicated that all the heated films could still emit strong blue-green fluorescence (Fig. 3A). No obvious reduction in the PL intensity was
Fig. 3. Photos of the PSt/MPPS blended films heated at 70 °C for various time lengths under irradiation of UV light (A). PL spectra of the PSt/MPPS blended films heated at 70 °C for various time lengths (B). Time-dependent fluorescence loss of the PSt/MPPS blended films and PSt/MPPS NPs at 70 °C (C). 575
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MPPS molecules inside the PSt/MPPS NPs may diffuse from the interior to the surface of NPs to undergo the hydrolysis reaction at an elevated temperature, like 70 °C (Scheme 1 II). As a result, the fluorescence of PSt/MPPS NPs gradually decreased with the continuous hydrolysis of MPPS. In a word, the poor fluorescence stability of PSt/MPPS NPs especially at elevated external temperatures is mainly caused by the continuous hydrolysis of MPPS. The diffusion of MPPS molecules from the interior to the surface of NPs is crucial to the continuous hydrolysis of MPPS. The main factors which may significantly influence the diffusion of MPPS could be the movement ability of polymer chain segments (kinetic factor), the difference in the MPPS concentration between the interior and the surface of NPs, and the difference in the hydrophobicity of MPPS and matrix polymers (thermodynamic factor). Based on these analyses, we will present some effective means to improve the fluorescence stability of AIE polymeric NPs. 3.4. Means to improve the fluorescence stability of AIE polymeric NPs Fig. 4. PL spectra and photos (inset) of the solid PM-W and PM-I samples.
3.4.1. Type of matrix polymer According to the possible mechanism proposed in the previous section, it is important to suppress the diffusion of MPPS from the interior to the surface of NPs for improving the fluorescence stability of polymer/MPPS NPs. Therefore, PMMA was chosen as the matrix polymer, because the hydrophobicity of PMMA is less than that of PSt (Fig. S5). From the thermodynamic point of view, the distribution of MPPS molecules inside the PMMA/MPPS NPs may be the thermodynamically favorable state because of the relatively higher hydrophobicity of MPPS. Under this circumstance, instead of distributing onto the surface of NPs, more MPPS molecules would locate inside the as-synthesized PMMA/MPPS NPs to avoid contacting water. Thus, it is expected that the hydrolysis of MPPS can be effectively suppressed. As described in the experimental section, the PMMA/MPPS NPs were also prepared through miniemulsion polymerization, and the final monomer conversion of this system was 96%. As expected, the PMMA/MPPS NPs displayed a much better fluorescence stability, compared with the PSt/ MPPS NPs (Fig. 5). Only ∼10% of fluorescence was lost after heated at 70 °C for 19 h. The slight fluorescence loss may be ascribed to the hydrolysis of MPPS located at or close to the surface of NPs. It should be pointed out the hydrolysis of MPPS at the particle/ water interface may also build a concentration difference between the interior and the surface of NPs. The concentration difference may also drive the diffusion of MPPS to the surface of NPs. However, in this case, on one hand, the thermodynamic driving force may counteract or even overpass the concentration difference, leading to the distribution of MPPS inside the NPs; on the other hand, the external temperature (70 °C) was much lower than the Tg of dried PMMA/MPPS NPs (109.6 °C), and thus the polymer chain segments and MPPS molecules might be still frozen in this case. Therefore, the MPPS molecules inside the PMMA/MPPS NPs would not diffuse to the surface of NPs to undergo continuous hydrolysis. Only the MPPS molecules on the surface of NPs took part in the hydrolysis reaction (Scheme 1 I). Consequently, the majority of the fluorescence of PMMA/MPPS NPs in the heated emulsion could still be maintained. In order to clarify the role of the thermodynamic factor in the fluorescence stability of polymer/MPPS NPs, polymers with a low Tg (PBMA) was selected as the matrix polymer. The PBMA/MPPS NPs were prepared through miniemulsion polymerization again, and the final monomer conversion of this system was 93%. The Tg of dried PBMA/ MPPS NPs was only 22.6 °C (Fig. S4), far below 70 °C. Therefore, the influence of the kinetic factor on the diffusion of MPPS could be completely excluded, as the MPPS molecules inside the PBMA/MPPS NPs could freely diffuse under the rubbery state. As shown in Fig. 5, the PBMA/MPPS NPs showed a similar high fluorescence stability as the PMMA/MPPS NPs. PBMA is less hydrophobic than St (Fig. S5), and thus, the MPPS molecules may be inclined to partitioning inside the
inset. Under the irradiation of UV light, the solid PM-I sample could still emit strong blue-green fluorescence, while the solid PM-W sample could not emit obvious fluorescence. The PL spectroscopic results in Fig. 4 indicated that the solid PM-I sample displayed an obvious emission band in the spectral range of 400–600 nm. In the same spectral range, the PL spectrum of the solid PM-W sample was a line parallel to the abscissa, indicating that it was almost non-emissive (Fig. 4). All the results indicated that water is a vital factor that impacts the hydrolysis of MPPS and in turn the fluorescence stability of PSt/MPPS NPs. 3.3. Possible mechanism for the fluorescence loss of PSt/MPPS NPs in aqueous emulsions As demonstrated in the previous section, the hydrolysis of MPPS is the main reason for the fluorescence loss of PSt/MPPS NPs. The precondition to the hydrolysis of MPPS is that the MPPS molecules must contact with water. Therefore, the MPPS molecules inside the PSt/ MPPS NPs are required to diffuse to the particle/water interface prior to participation in the hydrolysis reaction. As shown in Fig. S4, the Tg of dried PSt/MPPS NPs was 74.3 °C. Under an external temperature (≤50 °C) far below the Tg of matrix polymer, the movement of polymer chain segments is frozen, and simultaneously, the MPPS molecules are also entrapped in the frozen polymer matrix. Under this circumstance, the diffusion of MPPS in the polymer matrix is restricted by the kinetic factors. Thus, the MPPS molecules inside the PSt/MPPS NPs cannot diffuse to the particle/water interface. As a result, only the MPPS molecules at or close to the particle/water interface can undergo the hydrolysis reaction at a relatively low external temperature (Scheme 1 I). Therefore, the fluorescence loss of PSt/MPPS NPs is less than 20% when the external temperature was below 50 °C (Fig. 1). At a higher external temperature (closed to the Tg of matrix polymer), the polymer chain segments may be gradually unfrozen, and their movement is enhanced. Meanwhile, the MPPS molecules inside the PSt/MPPS NPs are unfrozen too, and they may diffuse from the interior to the surface of NPs to participate in the hydrolysis reaction (Scheme 1 II). The driving force of the MPPS diffusion could be the concentration difference between the interior and surface of NPs due to the consumption of MPPS at the surface through hydrolysis. It should be pointed out that the distribution of MPPS in the PSt/MPPS NPs is also determined by the thermodynamic factors. Although we cannot accurately evaluate the difference in the hydrophobicity of PSt and MPPS, we assume that their hydrophobicity may be similar because both of them contain many phenyl groups. Therefore, the diffusion direction of MPPS in the PSt/MPPS NPs is mainly determined by the concentration difference between the interior and surface of NPs. Consequently, the 576
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Scheme 1. Possible mechanism of the fluorescence loss of polymer/MPPS NPs in aqueous emulsions.
could not be well distinguished, the number-average particle size of PMMA@PSt/MPPS NPs determined by TEM increased about 3 nm after the growth of the PMMA shell. According to the results of time-dependent fluorescence loss shown in Fig. 6C, the PMMA@PSt/MPPS NPs display a much better fluorescence stability than the PSt/MPPS NPs. This result point to the successful formation of PMMA shell on the PSt/MPPS NPs and furthermore the successfully suppressed continuous hydrolysis of MPPS by the formed PMMA shell.
4. Conclusions AIE polymeric NPs with a susceptible AIEgen, MPPS, were prepared through miniemulsion polymerization. The temperature-dependent fluorescence stability of PSt/MPPS NPs was thoroughly investigated. Compared with the pristine MPPS particles, the PSt/MPPS NPs displayed a better fluorescence stability when the external temperature was much below the Tg of the matrix polymer. However, the fluorescence stability of PSt/MPPS NPs became much worse than that of the MPPS particles at an external temperature close to or above the Tg of the matrix polymer. The interaction between PSt and MPPS did not show an obvious influence on the fluorescence stability of PSt/MPPS NPs. The worse fluorescence stability of PSt/MPPS NPs at elevated temperatures was mainly caused by the continuous hydrolysis of MPPS. The temperature and water are two key factors that may influence the hydrolysis of MPPS and furthermore the fluorescence stability of PSt/ MPPS NPs. Firstly, the MPPS molecules at the particle/water interface undergo hydrolysis to build a concentration difference between the interior and the surface of NPs. Under the drive of the concentration difference, the MPPS molecules inside the NPs may continuously diffuse to the interface to undergo hydrolysis. Consequently, the fluorescence was almost completely quenched due to the entire consumption of MPPS molecules in the PSt/MPPS NPs. From the kinetic and thermodynamic points of view, the fluorescence stability of AIE polymeric NPs may be enhanced by using matrix polymers with a less hydrophobicity or with a higher Tg, or fabrication of a less hydrophobic polymeric shell on the PSt/MPPS NPs. The results of this work may contribute to understanding the fluorescence instability of AIE polymeric NPs in aqueous emulsions, and guide the synthesis of AIE polymeric NPs with a long-term fluorescence stability through carefully choosing matrix polymers or particle morphology.
Fig. 5. Time-dependent fluorescence loss of polymer/MPPS NPs with various matrix polymers at 70 °C.
NPs. Although some MPPS molecules at or close to the surface of NPs still underwent hydrolysis, leading to the reduction in the MPPS concentration at the surface, the MPPS molecules inside the NPs may not diffuse to the surface of NPs under the control of thermodynamic forces (Scheme 1 III). Consequently, the continuous hydrolysis of MPPS molecules may not happen in the PBMA/MPPS NPs, resulting in an excellent fluorescence stability. All the results indicate that the diffusion of MPPS in the polymer/ MPPS NPs is simultaneously determined by the kinetic and thermodynamic factors. Therefore, the fluorescence stability of polymer/MPPS NPs may be efficiently enhanced through adopting a matrix polymer with a less hydrophobicity or a relatively higher Tg than that of PSt. 3.4.2. Particle morphology In previous sections, we demonstrated that the continuous hydrolysis of MPPS molecules was the major reason for the poor fluorescence stability of PSt/MPPS NPs and some matrix polymers, such as PMMA and PBMA, can suppress the diffusion of MPPS molecules to the particle/water interface to undergo hydrolysis. In order to improve the fluorescence stability of PSt/MPPS NPs, an additional PMMA shell was formed on the surface of PSt/MPPS NPs. The TEM images of PSt/MPPS NPs and PMMA@PSt/MPPS NPs are shown in Fig. 6A and B, respectively. Although the core–shell structure of PMMA@PSt/MPPS NPs 577
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Fig. 6. TEM images of PSt/MPPS NPs (A) and PMMA@PSt/MPPS core–shell NPs (B). Time-dependent fluorescence loss of PSt/MPPS NPs and PMMA@PSt/MPPS NPs at 70 °C (C).
Conflicts of interest
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The authors declare no conflict of interest. Acknowledgements Financial supports from the National Natural Science Foundation of China project (51573168 and U1609205), the Zhejiang Provincial Natural Science Foundation (LY16E030006 and LZ18E030002), the Science Foundation of Zhejiang Sci-Tech University (14012208-Y), the Excellent Young Researchers Foundation (CETT2015001) of Zhejiang Provincial Top Key Academic Discipline of Chemical Engineering and Technology, the Young Researchers Foundation of Zhejiang Provincial Top Key Academic Discipline of Textile Science and Engineering (2015YXQN03), Zhejiang Province's Xinmiao Talent Plan (2017R406020 and 2017R406022) and National Undergraduate Training Programs for Innovation and Entrepreneurship of China (201710338021) are gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.dyepig.2018.08.051. References [1] Ding D, Li K, Liu B, Tang BZ. Bioprobes based on AIE fluorogens. Acc Chem Res 2013;46:2441–53. [2] Hong YN, Lam JWY, Tang BZ. Aggregation-induced emission. Chem Soc Rev 2011;40:5361–88. [3] Hong YN, Lam JWY, Tang BZ. Aggregation-induced emission: phenomenon, mechanism and applications. Chem Commun 2009:4332–53. [4] Feng GX, Kwok RTK, Tang BZ, Liu B. Functionality and versatility of aggregationinduced emission luminogens. Appl Phys Rev 2017;4:021307. [5] Dong H, Luo M, Wang S, Ma X. Synthesis and properties of tetraphenylethylene derivative diarylethene with photochromism and aggregation-induced emission. Dyes Pigments 2017;139:118–28. [6] Gu XG, Kwok RTK, Lam JWY, Tang BZ. AIEgens for biological process monitoring and disease theranostics. Biomaterials 2017;146:115–35. [7] Gu XG, Zhao EG, Lam JWY, Peng Q, Xie YJ, Zhang YL, et al. Mitochondrion-specific live-cell bioprobe operated in a fluorescence turn-on manner and a well-designed photoactivatable mechanism. Adv Mater 2015;27:7093–100. [8] Gu XG, Zhao EG, Zhao T, Kang MM, Gui C, Lam JWY, et al. A mitochondrionspecific photoactivatable fluorescence turn-on AIE-based bioprobe for localization super-resolution microscope. Adv Mater 2016;28:5064–71. [9] Zhao YM, Zhu W, Ren LX, Zhang K. Aggregation-induced emission polymer nanoparticles with pH-responsive fluorescence. Polym Chem 2016;7:5386–95. [10] Wan Q, Zeng GJ, He ZY, Mao LC, Liu MY, Huang HY, et al. Fabrication and biomedical applications of AIE active nanotheranostics through the combination of a ring-opening reaction and formation of dynamic hydrazones. J Mater Chem B 2016;4:5692–9. [11] Li K, Liu B. Polymer-encapsulated organic nanoparticles for fluorescence and
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