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Stable Cross-linked Fluorescent Polymeric Nanoparticles for Cell Imaging Haiyin Li, Xiqi Zhang,* Xiaoyong Zhang, Bin Yang, Yang Yang, Yen Wei*
Aggregation-induced emission (AIE) dye-based cross-linked fluorescent polymeric nanoparticles (FPNs) are facilely prepared via a two-step polymerization process including emulsion polymerization and subsequent anhydride cross-linking. Then, a variety of characterization methods are carried out to determine the performance of the FPNs, which show high dispersibility and strong fluorescence in an aqueous solution due to the hydrophilic carboxyl groups on the surfaces and the AIE components as the cores. Biocompatibility evaluation and cell imaging results suggest that these FPNs are biocompatible for cell imaging. More importantly, this cross-linking strategy is proven to overcome the issue of critical micelle concentration and opens the opportunity to develop more robust fluorescent bioprobes.
1. Introduction Cellular fluorescence imaging is a useful tool to monitor biological targets within the complicated intracellular systems presenting a highly sensitive and noninvasive technology, and has been successfully exploited as a versatile visualization way for medical diagnosis, drug development, and clinical study.[1–3] Therefore, various fluorescent bioprobes including green fluorescent protein, semiconductor quantum dots, carbon based dots, rare earth-based nanoparticles, fluorescent-conjugated polymers, and fluorescent organic nanoparticles (FONs) have been reported and extensively studied for bioimaging applications over
Dr. H. Li College of Chemistry and Pharmaceutical Sciences, Qingdao Agriculture University, Qingdao 266109, China Dr. X. Zhang, Dr. X. Zhang, B. Yang, Y. Yang, Prof. Y. Wei Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084 , China E-mail: [email protected]; [email protected] Macromol. Rapid Commun. 2014, 35, 1661−1667 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the past 10 years.[4–10] However, some inherent drawbacks hamper them in practical application. Green fluorescent protein, for instance, suffers from easy enzyme degradation, poor photostability, small stokes shifts, and a tedious transfection process,[11] while semiconductor quantum dots (e.g., CdSe and CdTe) are highly cytotoxic and show susceptibility to aggregate in cellular environments.[12] Weak luminescence, nonbiodegradability, and nonfunctionalized hydrophobic features often hinder carbon and rare earth-based nanoparticles for biomedical applications.[13] Water-dispersible hydrophilic fluoridated hydroxyapatite:Ln3+ (Ln = Eu or Tb) nanoparticles have been prepared via hydrophobic/hydrophilic transformation with surfactants (Pluronic F127) in the literature.[14] These noncovalent polymeric nanoparticles show bright luminescent properties and excellent biocompatibility for cell imaging applications. Other fluorescent-conjugated polymers with intense fluorescence that could be utilized in cell imaging have also been reported.[15] Moreover, most of the conventional FONs show hydrophobic planar structures, which show strong intermolecular π–π interactions, inevitably causing aggregation-induced fluorescence
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DOI: 10.1002/marc.201400309
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quenching at high concentration.[16] In addition, low resistance to photobleaching is another drawback for most FONs, which makes them not ideal for application as bioprobes. In recent years, aggregation-induced emission (AIE) materials with an antiquenching effect, which emit intense fluorescence in an aggregated state, have been synthesized and widely investigated for potential bio-/ chemosensors and bioimaging applications.[17–19] A wide spectrum of different molecular structures of AIE fluorogens has been developed, including siloles,[20–22] tetraphenylethene,[23–27] cyano-substituted diarylethylene,[28,29] triphenylethene,[30–32] distyrylanthracene,[33–35] and so on, which offer an alternative material platform. Recently, various tactics for fabricating AIE-based fluorescent nanoparticles including noncovalent and covalent strategies have been developed. The previous reported noncovalent strategies include silica nanoparticles encapsulation,[36] amphiphilic block copolymers modification,[37] BSA encapsulation,[38] surfactant modification,[39] while the covalent strategies comprise AIE-functionalized siloxanes sol–gel reactions,[40] AIE molecules grafted amphiphilic polymer,[41] conjugated polyelectrolytes,[42] Schiff base condensation,[43] EP,[44] reversible addition– fragmentation chain transfer (RAFT) polymerizaion,[45] anhydride ring-opening polymerization.[46] Along with the great advances in the fabrication technology, AIE dyebased fluorescent nanoparticles with high water dispersibility, uniform size, strong fluorescence, excellent biocompatibility, and multi-functionality have been prepared, making them promising for cell imaging applications. Compared with the previous non-AIE fluorophores-based polymeric nanoparticles (covalently or noncovalently), these AIE systems are richly endowed by nature with an antiquenching effect and facile designability. Despite many impressive advances in constructing AIE-based nanoparticles, more versatile and robust fabricating strategies are still highly demanded, such as stable dispersion in physiological solution even below the critical micelle concentration (CMC). This is because many self-assembled structures are often unstable in dilute solution below the CMC, which limits their real biomedical application.[47] The previous reported examples of cross-linked polymeric nanoparticles have been demonstrated more stable than those non-cross-linked ones, however, the existing crosslinked strategy is rare.[48] Thus, the development of robust synthetic routes to prepare novel stable cross-linked fluorescent polymeric nanoparticles (FPNs) is of great scientific interest. Here, a novel way of preparing AIE-based cross-linked FPNs was developed (Scheme 1) via the two-step polymerization including emulsion polymerization (EP) and subsequent cross-linking (CL). A previous reported AIE monomer (PhENH2)[49] has been utilized to combine with
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Scheme 1. Preparation of EP-CL FPNs through emulsion polymerization and then cross-linking for cell imaging applications.
acrylic acid, styrene, and 4,4′-(hexafluoroisopropylidene)diphthalic anhydride (HFDA) for the construction of the cross-linked structure. The obtained amphiphilic crosslinked copolymers (EP-CL) are prone to self-assemble into stable FPNs and can be highly dispersed in physiological solution. A variety of characterization methods, including 1 H NMR spectrum, UV–vis absorption spectrum, fluorescence spectrum, Fourier transform infrared spectroscopy (FT-IR) spectrum, transmission electron microscopy (TEM), gel permeation chromatography (GPC), and dynamic light scattering, were carried out to determine the performance of EP-CL, furthermore, the biocompatibility and cell uptake behavior of EP-CL FPNs were studied to evaluate their potential applications for cell imaging.
2. Results and Discussion The AIE monomer (PhENH2) was prepared according to the previous literature. To prepare EP-CL FPNs, the hydrophobic PhENH2, and styrene were copolymerized with hydrophilic monomer acrylic acid via the EP to obtain a linear amphiphilic polymer (EP) (Scheme 2). The feed molar ratio of three monomers was 1:20:10 for PhENH2, styrene, and acrylic acid, respectively. The numberaverage molecular weight (Mn ) values of EP were determined by GPC. Result showed that the Mn value of EP was 5000 Da. Then EP was facilely cross-linked with HFDA at room temperature under air atmosphere to afford the resulting cross-linked amphiphilic copolymers (EP-CL). The GPC result of EP-CL showed that the Mn value was 22 000. The 1H NMR spectrum of EP-CL in d6-DMSO was conducted and shown in Figure 1A, although it was hard to calculate the molecular weight of the resulting polymers from the spectrum, the proportion of different alkyl hydrogen was approximately consistent with the design feed molar ratio, confirming the successful synthesis of EP-CL. The 1H NMR spectrum of EP in d6-DMSO was also shown in Figure 1A for comparison. It was found that a new peak around 8.0 of chemical shift emerged in EP-CL, which indicated the
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Scheme 2. Synthetic routes of EP-CL: emulsion polymerization of PhENH2 with acrylic acid and styrene to afford EP, then through crosslinking by HFDA to obtain EP-CL.
successful introduction of HFDA. Due to the amphiphilic properties, when EP-CL was dispersed in aqueous solution, it tended to self-assemble into polymeric nanoparticles
with surfaces covered with hydrophilic carboxyl groups, while the aromatic groups were aggregated into the hydrophobic cores. With the AIE component aggregated in the
Figure 1. A) 1H NMR spectra of EP-EL and EP dissolved in d6-DMSO; B) FT-IR spectra of PhENH2, HFDA, and EP-CL, strong stretching vibration bands of located at 1684 cm−1 and C F stretching vibration bands located at 1265 and 1109 cm−1 are observed in the sample of EP-CL FPNs, suggesting the successful preparation of EP-CL FPNs; C) UV–vis spectrum of EP-EL FPNs, inset is the visible image of EP-EL FPNs in water; D) fluorescence excitation and emission spectra of EP-CL FPNs, inset is the fluorescent image of EP-CL FPNs taken at 365 nm UV light.
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cores of the nanodots, such obtained FPNs are expected to emit bright fluorescence and show high dispersibility in an aqueous environment. Fourier transform infrared spectroscopy was further determined to confirm the successful synthesis of EP-CL copolymers. As shown in Figure 1B, two characteristic peaks located at 2919 and 2846 cm−1 were observed in the sample of PhENH2, which evidenced the stretching vibration of CH2 group. Meanwhile, a series of peaks distributed between 1450 and 1600 cm−1 could be ascribed to the stretching vibration of aromatic rings. After the twostep polymerization, strong stretching vibration of C O band of amide and carboxyl groups located at 1684 cm−1 was observed in EP-CL FPNs. Further, two characteristic peaks located at 1265 and 1109 cm−1 were also observed for C F stretching vibration band, and the stretching vibration bands of N H and O H were also found as a broad peak around 3430 cm−1. Moreover, some aromatic C H stretch and bending vibration bands also appeared in the EP-CL FPNs located at 3010, 1584, 1540, 1503, and 786 cm−1, also confirming successful formation of the cross-linked copolymers. The UV absorption spectrum of EP-CL FPNs dispersed in water was shown in Figure 1C, it could be found that there was no absorption peak ranged from 800 to 300 nm. It is noteworthy that the entire spectrum started to increase from 800 nm, which is ascribed to the Mie effect, indicating the existence of nanoparticles in the solution. The inset of Figure 1C demonstrated the EP-CL FPNs were readily dispersed in water after the two-step polymerization. Due to the aggregation and self-assembly of the amphiphilic copolymers, EP-CL FPNs showed strong yellow-green fluorescence in water (inset of Figure 1D). The PL spectra of EP-CL FPNs in water were shown in Figure 1D. The maximum emission wavelength was located at 510 nm, while the fluorescence excitation wavelength was around 417 nm. To quantitatively evaluate the fluorescence quantum yield and the PL lifetime of the EP-CL FPNs and its variation in the polymer matrix, another two cross-linked amphiphilic copolymers (EP-CL-1 and EP-CL-2) were prepared with different feed molar ratios of monomers. For the synthesis of EP-CL-1, the feed molar ratio of three monomers was 1:20:20 for PhENH2, styrene, and acrylic acid, respectively; while for the synthesis of EP-CL-2, the feed molar ratio of three monomers was 1:20:40 for PhENH2, styrene, and acrylic acid, respectively. The Mn values of EP-CL-1 and EP-CL-2 have been determined, which showed 28 100 and 32 400 for EP-CL-1 and EP-CL-2, respectively. The numberaverage molecular weight of these three as-prepared polymers demonstrated an increasing trend as increased the ratio of acrylic acid. The UV–vis spectra and fluorescent emission spectra of EP-EL, EP-EL-1, and EP-EL-2 FPNs in water were shown in Figures S1 and S2 (Supporting
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Information). The UV spectra results showed that light scattering effect was weaken as the proportion of acrylic acid increased, suggesting the increase of water solubility of the FPNs. Furthermore, the absorption peak around 440 nm was more obvious when increasing the proportion of acrylic acid. Noticeable red shift could be found in the PL spectra of the FPNs with higher proportion of acrylic acid, which might be due to the polarity effect. The fluorescence quantum yield values of the FPNs were estimated using quinine sulfate as the reference dye, which showed 41%, 28%, and 21% for EP-EL, EP-EL-1, and EP-EL-2 FPNs, respectively. The quantum yield of PhENH2 in the state of nanoparticles has also been determined in DMF/ ether mixture (1:9, volume ratio) for comparison, which showed 53%. Fluorescence lifetimes of EP-EL, EP-EL-1, and EP-EL-2 FPNs in water were shown in Figure S3 and Table S1 (Supporting Information). Two or three relaxation pathways were found in the fluorescence decays, indicating that the time-resolved PL spectra of the compound included independent emissions from the segments with different π-conjugation extent according to the detected multiple lifetimes. The fluorescence lifetime of EP-EL-1 FPNs showed the highest weighted mean lifetimes , while the of EP-EL-2 FPNs was the smallest one. In short, the bright fluorescence of these FPNs is greatly beneficial to the potential cell imaging applications. Self-assembly of polymeric materials to form nanoscale morphologies such as spherical nanoparticles is a particularly promising strategy for various biomedical applications. However, the CMC problem often hampers the use of nanoparticles at low concentration. Therefore, the cross-linking strategy has been employed in this work and determined whether could solve the issue of CMC. The response of the AIE-active fluorescence behavior toward the concentration of the aqueous FPNs solutions was used to locate the CMC.[50] The maximum fluorescent emission (510 nm) due to the aggregated cross-linked polymers was used to track the CMC values. Thus, the intensity of the aggregate emission versus the logarithm of the concentration of EP-CL was carried out to evaluate the CMC. At low concentrations below CMC, the detected intensity of aggregate emission is very low, but at concentrations above CMC, the aggregate emission increases abruptly. In this case, the EP-CL FPNs showed 0.020 mg mL−1 for the CMC (Figure 2A). Meanwhile, the size distribution of EP-CL FPNs in phosphate buffer solution (PBS) was determined using a zeta Plus particle size analyzer, showed that the size distribution was 302.9 ± 6.6 nm, with a polydispersity index (PDI) of 0.055. The zeta potential measurement of EP-CL FPNs in PBS was also determined and showed the value of zeta-potential of −29.7 ± 1.3 mV. What was surprising was that when we determined the size distribution of EP-CL FPNs below CMC, the results showed
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Stable Cross-linked Fluorescent Polymeric Nanoparticles for Cell Imaging
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no cell viability decrease was found when the cells were incubated with 10–120 μg mL−1 of EP-CL FPNs for 10 and 24 h. Even when the concentration was up to 120 μg mL−1, the cell viability value was still greater than 90%, suggesting that these FPNs were highly potential for biomedical applications. Cell imaging applications of EPCL FPNs were further explored. The cell uptake behavior of EP-CL FPNs Figure 2. A) Intensity of the aggregate emission versus the logarithm of the concentration of EP-CL (λex = 400 nm, λem = 510 nm); B) TEM image of EP-CL FPNs dispersed in was evaluated by confocal laser scanning microscope (CLSM) observation. water, scale bar = 200 nm. As shown in Figure 4, strong yellow fluorescence could be observed at the cell location after they were only incubated with that the nanoparticles were still stable in the solution, 10 μg mL−1 of EP-CL FPNs. Furthermore, many areas even when the concentration of the FPNs was as low as 0.001 mg mL−1. The CMC of the linear amphiphilic polymer with relative weak fluorescence intensity were found in the cells, which might be the possible location of cell (EP) was also determined for comparison, which showed nucleus (Figure 4B). EP-CL FPNs could be facilely uptaken 0.091 mg mL−1 for the CMC (Figure S4, Supporting Inforby cells and mainly located at cytoplasm. As comparing mation); however, the size distribution of EP FPNs could with the size of FPNs and nucleus pore, these FPNs be detected only when the concentration of the FPNs was were considered uptaken by endocytosis of the cells. As higher than 0.032 mg mL−1. Therefore, this cross-linking other reported chromophores may suffer fluorescence method is considered as a promising strategy to construct quenching at high concentration due to their strong highly robust FPNs for potential cell imaging applications. intermolecular π–π interactions, the content of the dyes The TEM images further confirmed the formation of the in the polymer block must be low enough to prevent selfconjugated EP-CL nanodots (Figure 2B). Many spherical quenching of the dyes.[19] Due to the intense fluorescence nanoparticles with diameters ranged from 100 to 200 nm could be clearly identified. As compared with the above originated from the AIE group of PhENH2, strong signal size distribution in PBS, the size characterized by TEM was somewhat smaller, which might be due to the dryingcausing shrinkage of the micelle. The TEM images shown here gave us direct evidence that the resulting amphiphilic cross-linked copolymers were self-assembled into nanoparticles in an aqueous solution. The biocompatibility was carried out to evaluate the potential biomedical applications of EP-CL FPNs. The influences of EP-CL FPNs to A549 cells were firstly examined by optical microscopy after the cells were incubated with different concentrations of EP-CL FPNs for 24 h (Figure 3A–C). The result showed that the cells grew well when incubated with 10 and 80 μg mL−1 of EP-CL FPNs, suggesting that the FPNs were biocompatible with cells. To further confirm the cytocompatibility of EP-CL FPNs, cell viability of EP-CL FPNs to A549 cells Figure 3. Biocompatibility evaluations of EP-CL FPNs. A–C) optical microscopy images of was determined by cell counting kit-8 A549 cells incubated with different concentrations of EP-CL FPNs for 24 h: A) control cells, (CCK-8) assay. As shown in Figure 3D, B) 10 μg mL−1, C) 80 μg mL−1; D) cell viability of EP-CL FPNs for 10 h and 24 h.
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Figure 4. CLSM images of A549 cells incubated with 10 μg mL−1 of EP-CL FPNs for 3 h. A) bright field, B) excited with 405 nm laser, C) merged image of A and B. Scale bar = 20 μm.
can be detected from EP-CL FPNs, which is great beneficial to cell imaging. The CLSM images of A549 cells incubated with 10 μg mL−1 of EP FPNs was also conducted and shown in Figure S5 (Supporting Information). Although the EP FPNs showed less stability in dilute solution than EP-CL FPNs, the EP FPNs also showed good result in cell imaging. In previous reports, biocompatible AIE FONs were fabricated via a covalent strategy such as Schiff base reaction, EP, RAFT polymerization, and anhydride ringopening polymerization for cell imaging, but most of them were non-cross-linking polymer. Compared with previous FONs, the EP-CL FPNs fabricated via the twostep polymerization in this work have more obvious advantages. First and most importantly, the EP-CL FPNs could be stable dispersed in physiological solution even below the CMC due to their inherent cross-linking structures. Second, other molecules such as drugs and nucleic acid could be further integrated into these FPNs taking advantage of the peripheral carboxyl groups. In addition, other functional monomers can be introduced into the copolymers via various polymerization strategies to afford multifunctional materials for biomedical applications. It also should be mentioned that, in this work, the resulting cross-linked FPNs have some drawbacks like inadequate biocompatibility due to the lack of stealthy elements (for example, polyethylene glycol, PEG), and the unsatisfactory metabolic behavior which can be predicted according to its nonbiodegradable structure. In this case, the next step of our research is to develop more biocompatible materials by incorporating PEG or polysaccharide into the system, and to construct more biodegradable structure through introducing metastable connection like disulfide bond into these cross-linked polymers.
3. Conclusions EP-CL FPNs are facilely prepared through EP and subsequent cross-linking based on an AIE monomer (PhENH2),
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acrylic acid, styrene, and a dianhydride (HFDA). These FPNs show a uniform morphology, high dispersibility, and intense fluorescence in an aqueous solution. Such FPNs are proven biocompatible for cell imaging according to the biocompatibility evaluation. Moreover, this cross-linking strategy is expected to provide an alternative path to construct various robust fluorescent bioprobes for biomedical applications.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: This research was supported by the National Science Foundation of China (Nos. 21134004, 21201108, and 51363016), and the National 973 Project (No. 2011CB935700), China Postdoctoral Science Foundation (Nos. 2012M520243 and 2013T60100), High-level Science Foundation of Qingdao Agriculture University (No. 6631334). Received: May 30, 2014; Revised: June 30, 2014; Published online: August 27, 2014; DOI: 10.1002/marc.201400309 Keywords: aggregation-induced emission; anhydride crosslinking; cell imaging; cross-linked fluorescent polymeric nanoparticles; emulsion polymerization
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Communication
Stable Cross-linked Fluorescent Polymeric Nanoparticles for Cell Imaging Haiyin Li, Xiqi Zhang,* Xiaoyong Zhang, Bin Yang, Yang Yang, Yen Wei*
Aggregation-induced emission (AIE) dye-based cross-linked fluorescent polymeric nanoparticles (FPNs) are facilely prepared via a two-step polymerization process including emulsion polymerization and subsequent anhydride cross-linking. Then, a variety of characterization methods are carried out to determine the performance of the FPNs, which show high dispersibility and strong fluorescence in an aqueous solution due to the hydrophilic carboxyl groups on the surfaces and the AIE components as the cores. Biocompatibility evaluation and cell imaging results suggest that these FPNs are biocompatible for cell imaging. More importantly, this cross-linking strategy is proven to overcome the issue of critical micelle concentration and opens the opportunity to develop more robust fluorescent bioprobes.
1. Introduction Cellular fluorescence imaging is a useful tool to monitor biological targets within the complicated intracellular systems presenting a highly sensitive and noninvasive technology, and has been successfully exploited as a versatile visualization way for medical diagnosis, drug development, and clinical study.[1–3] Therefore, various fluorescent bioprobes including green fluorescent protein, semiconductor quantum dots, carbon based dots, rare earth-based nanoparticles, fluorescent-conjugated polymers, and fluorescent organic nanoparticles (FONs) have been reported and extensively studied for bioimaging applications over
Dr. H. Li College of Chemistry and Pharmaceutical Sciences, Qingdao Agriculture University, Qingdao 266109, China Dr. X. Zhang, Dr. X. Zhang, B. Yang, Y. Yang, Prof. Y. Wei Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084 , China E-mail: [email protected]; [email protected] Macromol. Rapid Commun. 2014, 35, 1661−1667 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the past 10 years.[4–10] However, some inherent drawbacks hamper them in practical application. Green fluorescent protein, for instance, suffers from easy enzyme degradation, poor photostability, small stokes shifts, and a tedious transfection process,[11] while semiconductor quantum dots (e.g., CdSe and CdTe) are highly cytotoxic and show susceptibility to aggregate in cellular environments.[12] Weak luminescence, nonbiodegradability, and nonfunctionalized hydrophobic features often hinder carbon and rare earth-based nanoparticles for biomedical applications.[13] Water-dispersible hydrophilic fluoridated hydroxyapatite:Ln3+ (Ln = Eu or Tb) nanoparticles have been prepared via hydrophobic/hydrophilic transformation with surfactants (Pluronic F127) in the literature.[14] These noncovalent polymeric nanoparticles show bright luminescent properties and excellent biocompatibility for cell imaging applications. Other fluorescent-conjugated polymers with intense fluorescence that could be utilized in cell imaging have also been reported.[15] Moreover, most of the conventional FONs show hydrophobic planar structures, which show strong intermolecular π–π interactions, inevitably causing aggregation-induced fluorescence
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quenching at high concentration.[16] In addition, low resistance to photobleaching is another drawback for most FONs, which makes them not ideal for application as bioprobes. In recent years, aggregation-induced emission (AIE) materials with an antiquenching effect, which emit intense fluorescence in an aggregated state, have been synthesized and widely investigated for potential bio-/ chemosensors and bioimaging applications.[17–19] A wide spectrum of different molecular structures of AIE fluorogens has been developed, including siloles,[20–22] tetraphenylethene,[23–27] cyano-substituted diarylethylene,[28,29] triphenylethene,[30–32] distyrylanthracene,[33–35] and so on, which offer an alternative material platform. Recently, various tactics for fabricating AIE-based fluorescent nanoparticles including noncovalent and covalent strategies have been developed. The previous reported noncovalent strategies include silica nanoparticles encapsulation,[36] amphiphilic block copolymers modification,[37] BSA encapsulation,[38] surfactant modification,[39] while the covalent strategies comprise AIE-functionalized siloxanes sol–gel reactions,[40] AIE molecules grafted amphiphilic polymer,[41] conjugated polyelectrolytes,[42] Schiff base condensation,[43] EP,[44] reversible addition– fragmentation chain transfer (RAFT) polymerizaion,[45] anhydride ring-opening polymerization.[46] Along with the great advances in the fabrication technology, AIE dyebased fluorescent nanoparticles with high water dispersibility, uniform size, strong fluorescence, excellent biocompatibility, and multi-functionality have been prepared, making them promising for cell imaging applications. Compared with the previous non-AIE fluorophores-based polymeric nanoparticles (covalently or noncovalently), these AIE systems are richly endowed by nature with an antiquenching effect and facile designability. Despite many impressive advances in constructing AIE-based nanoparticles, more versatile and robust fabricating strategies are still highly demanded, such as stable dispersion in physiological solution even below the critical micelle concentration (CMC). This is because many self-assembled structures are often unstable in dilute solution below the CMC, which limits their real biomedical application.[47] The previous reported examples of cross-linked polymeric nanoparticles have been demonstrated more stable than those non-cross-linked ones, however, the existing crosslinked strategy is rare.[48] Thus, the development of robust synthetic routes to prepare novel stable cross-linked fluorescent polymeric nanoparticles (FPNs) is of great scientific interest. Here, a novel way of preparing AIE-based cross-linked FPNs was developed (Scheme 1) via the two-step polymerization including emulsion polymerization (EP) and subsequent cross-linking (CL). A previous reported AIE monomer (PhENH2)[49] has been utilized to combine with
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Scheme 1. Preparation of EP-CL FPNs through emulsion polymerization and then cross-linking for cell imaging applications.
acrylic acid, styrene, and 4,4′-(hexafluoroisopropylidene)diphthalic anhydride (HFDA) for the construction of the cross-linked structure. The obtained amphiphilic crosslinked copolymers (EP-CL) are prone to self-assemble into stable FPNs and can be highly dispersed in physiological solution. A variety of characterization methods, including 1 H NMR spectrum, UV–vis absorption spectrum, fluorescence spectrum, Fourier transform infrared spectroscopy (FT-IR) spectrum, transmission electron microscopy (TEM), gel permeation chromatography (GPC), and dynamic light scattering, were carried out to determine the performance of EP-CL, furthermore, the biocompatibility and cell uptake behavior of EP-CL FPNs were studied to evaluate their potential applications for cell imaging.
2. Results and Discussion The AIE monomer (PhENH2) was prepared according to the previous literature. To prepare EP-CL FPNs, the hydrophobic PhENH2, and styrene were copolymerized with hydrophilic monomer acrylic acid via the EP to obtain a linear amphiphilic polymer (EP) (Scheme 2). The feed molar ratio of three monomers was 1:20:10 for PhENH2, styrene, and acrylic acid, respectively. The numberaverage molecular weight (Mn ) values of EP were determined by GPC. Result showed that the Mn value of EP was 5000 Da. Then EP was facilely cross-linked with HFDA at room temperature under air atmosphere to afford the resulting cross-linked amphiphilic copolymers (EP-CL). The GPC result of EP-CL showed that the Mn value was 22 000. The 1H NMR spectrum of EP-CL in d6-DMSO was conducted and shown in Figure 1A, although it was hard to calculate the molecular weight of the resulting polymers from the spectrum, the proportion of different alkyl hydrogen was approximately consistent with the design feed molar ratio, confirming the successful synthesis of EP-CL. The 1H NMR spectrum of EP in d6-DMSO was also shown in Figure 1A for comparison. It was found that a new peak around 8.0 of chemical shift emerged in EP-CL, which indicated the
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Scheme 2. Synthetic routes of EP-CL: emulsion polymerization of PhENH2 with acrylic acid and styrene to afford EP, then through crosslinking by HFDA to obtain EP-CL.
successful introduction of HFDA. Due to the amphiphilic properties, when EP-CL was dispersed in aqueous solution, it tended to self-assemble into polymeric nanoparticles
with surfaces covered with hydrophilic carboxyl groups, while the aromatic groups were aggregated into the hydrophobic cores. With the AIE component aggregated in the
Figure 1. A) 1H NMR spectra of EP-EL and EP dissolved in d6-DMSO; B) FT-IR spectra of PhENH2, HFDA, and EP-CL, strong stretching vibration bands of located at 1684 cm−1 and C F stretching vibration bands located at 1265 and 1109 cm−1 are observed in the sample of EP-CL FPNs, suggesting the successful preparation of EP-CL FPNs; C) UV–vis spectrum of EP-EL FPNs, inset is the visible image of EP-EL FPNs in water; D) fluorescence excitation and emission spectra of EP-CL FPNs, inset is the fluorescent image of EP-CL FPNs taken at 365 nm UV light.
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cores of the nanodots, such obtained FPNs are expected to emit bright fluorescence and show high dispersibility in an aqueous environment. Fourier transform infrared spectroscopy was further determined to confirm the successful synthesis of EP-CL copolymers. As shown in Figure 1B, two characteristic peaks located at 2919 and 2846 cm−1 were observed in the sample of PhENH2, which evidenced the stretching vibration of CH2 group. Meanwhile, a series of peaks distributed between 1450 and 1600 cm−1 could be ascribed to the stretching vibration of aromatic rings. After the twostep polymerization, strong stretching vibration of C O band of amide and carboxyl groups located at 1684 cm−1 was observed in EP-CL FPNs. Further, two characteristic peaks located at 1265 and 1109 cm−1 were also observed for C F stretching vibration band, and the stretching vibration bands of N H and O H were also found as a broad peak around 3430 cm−1. Moreover, some aromatic C H stretch and bending vibration bands also appeared in the EP-CL FPNs located at 3010, 1584, 1540, 1503, and 786 cm−1, also confirming successful formation of the cross-linked copolymers. The UV absorption spectrum of EP-CL FPNs dispersed in water was shown in Figure 1C, it could be found that there was no absorption peak ranged from 800 to 300 nm. It is noteworthy that the entire spectrum started to increase from 800 nm, which is ascribed to the Mie effect, indicating the existence of nanoparticles in the solution. The inset of Figure 1C demonstrated the EP-CL FPNs were readily dispersed in water after the two-step polymerization. Due to the aggregation and self-assembly of the amphiphilic copolymers, EP-CL FPNs showed strong yellow-green fluorescence in water (inset of Figure 1D). The PL spectra of EP-CL FPNs in water were shown in Figure 1D. The maximum emission wavelength was located at 510 nm, while the fluorescence excitation wavelength was around 417 nm. To quantitatively evaluate the fluorescence quantum yield and the PL lifetime of the EP-CL FPNs and its variation in the polymer matrix, another two cross-linked amphiphilic copolymers (EP-CL-1 and EP-CL-2) were prepared with different feed molar ratios of monomers. For the synthesis of EP-CL-1, the feed molar ratio of three monomers was 1:20:20 for PhENH2, styrene, and acrylic acid, respectively; while for the synthesis of EP-CL-2, the feed molar ratio of three monomers was 1:20:40 for PhENH2, styrene, and acrylic acid, respectively. The Mn values of EP-CL-1 and EP-CL-2 have been determined, which showed 28 100 and 32 400 for EP-CL-1 and EP-CL-2, respectively. The numberaverage molecular weight of these three as-prepared polymers demonstrated an increasing trend as increased the ratio of acrylic acid. The UV–vis spectra and fluorescent emission spectra of EP-EL, EP-EL-1, and EP-EL-2 FPNs in water were shown in Figures S1 and S2 (Supporting
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Information). The UV spectra results showed that light scattering effect was weaken as the proportion of acrylic acid increased, suggesting the increase of water solubility of the FPNs. Furthermore, the absorption peak around 440 nm was more obvious when increasing the proportion of acrylic acid. Noticeable red shift could be found in the PL spectra of the FPNs with higher proportion of acrylic acid, which might be due to the polarity effect. The fluorescence quantum yield values of the FPNs were estimated using quinine sulfate as the reference dye, which showed 41%, 28%, and 21% for EP-EL, EP-EL-1, and EP-EL-2 FPNs, respectively. The quantum yield of PhENH2 in the state of nanoparticles has also been determined in DMF/ ether mixture (1:9, volume ratio) for comparison, which showed 53%. Fluorescence lifetimes of EP-EL, EP-EL-1, and EP-EL-2 FPNs in water were shown in Figure S3 and Table S1 (Supporting Information). Two or three relaxation pathways were found in the fluorescence decays, indicating that the time-resolved PL spectra of the compound included independent emissions from the segments with different π-conjugation extent according to the detected multiple lifetimes. The fluorescence lifetime of EP-EL-1 FPNs showed the highest weighted mean lifetimes , while the of EP-EL-2 FPNs was the smallest one. In short, the bright fluorescence of these FPNs is greatly beneficial to the potential cell imaging applications. Self-assembly of polymeric materials to form nanoscale morphologies such as spherical nanoparticles is a particularly promising strategy for various biomedical applications. However, the CMC problem often hampers the use of nanoparticles at low concentration. Therefore, the cross-linking strategy has been employed in this work and determined whether could solve the issue of CMC. The response of the AIE-active fluorescence behavior toward the concentration of the aqueous FPNs solutions was used to locate the CMC.[50] The maximum fluorescent emission (510 nm) due to the aggregated cross-linked polymers was used to track the CMC values. Thus, the intensity of the aggregate emission versus the logarithm of the concentration of EP-CL was carried out to evaluate the CMC. At low concentrations below CMC, the detected intensity of aggregate emission is very low, but at concentrations above CMC, the aggregate emission increases abruptly. In this case, the EP-CL FPNs showed 0.020 mg mL−1 for the CMC (Figure 2A). Meanwhile, the size distribution of EP-CL FPNs in phosphate buffer solution (PBS) was determined using a zeta Plus particle size analyzer, showed that the size distribution was 302.9 ± 6.6 nm, with a polydispersity index (PDI) of 0.055. The zeta potential measurement of EP-CL FPNs in PBS was also determined and showed the value of zeta-potential of −29.7 ± 1.3 mV. What was surprising was that when we determined the size distribution of EP-CL FPNs below CMC, the results showed
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no cell viability decrease was found when the cells were incubated with 10–120 μg mL−1 of EP-CL FPNs for 10 and 24 h. Even when the concentration was up to 120 μg mL−1, the cell viability value was still greater than 90%, suggesting that these FPNs were highly potential for biomedical applications. Cell imaging applications of EPCL FPNs were further explored. The cell uptake behavior of EP-CL FPNs Figure 2. A) Intensity of the aggregate emission versus the logarithm of the concentration of EP-CL (λex = 400 nm, λem = 510 nm); B) TEM image of EP-CL FPNs dispersed in was evaluated by confocal laser scanning microscope (CLSM) observation. water, scale bar = 200 nm. As shown in Figure 4, strong yellow fluorescence could be observed at the cell location after they were only incubated with that the nanoparticles were still stable in the solution, 10 μg mL−1 of EP-CL FPNs. Furthermore, many areas even when the concentration of the FPNs was as low as 0.001 mg mL−1. The CMC of the linear amphiphilic polymer with relative weak fluorescence intensity were found in the cells, which might be the possible location of cell (EP) was also determined for comparison, which showed nucleus (Figure 4B). EP-CL FPNs could be facilely uptaken 0.091 mg mL−1 for the CMC (Figure S4, Supporting Inforby cells and mainly located at cytoplasm. As comparing mation); however, the size distribution of EP FPNs could with the size of FPNs and nucleus pore, these FPNs be detected only when the concentration of the FPNs was were considered uptaken by endocytosis of the cells. As higher than 0.032 mg mL−1. Therefore, this cross-linking other reported chromophores may suffer fluorescence method is considered as a promising strategy to construct quenching at high concentration due to their strong highly robust FPNs for potential cell imaging applications. intermolecular π–π interactions, the content of the dyes The TEM images further confirmed the formation of the in the polymer block must be low enough to prevent selfconjugated EP-CL nanodots (Figure 2B). Many spherical quenching of the dyes.[19] Due to the intense fluorescence nanoparticles with diameters ranged from 100 to 200 nm could be clearly identified. As compared with the above originated from the AIE group of PhENH2, strong signal size distribution in PBS, the size characterized by TEM was somewhat smaller, which might be due to the dryingcausing shrinkage of the micelle. The TEM images shown here gave us direct evidence that the resulting amphiphilic cross-linked copolymers were self-assembled into nanoparticles in an aqueous solution. The biocompatibility was carried out to evaluate the potential biomedical applications of EP-CL FPNs. The influences of EP-CL FPNs to A549 cells were firstly examined by optical microscopy after the cells were incubated with different concentrations of EP-CL FPNs for 24 h (Figure 3A–C). The result showed that the cells grew well when incubated with 10 and 80 μg mL−1 of EP-CL FPNs, suggesting that the FPNs were biocompatible with cells. To further confirm the cytocompatibility of EP-CL FPNs, cell viability of EP-CL FPNs to A549 cells Figure 3. Biocompatibility evaluations of EP-CL FPNs. A–C) optical microscopy images of was determined by cell counting kit-8 A549 cells incubated with different concentrations of EP-CL FPNs for 24 h: A) control cells, (CCK-8) assay. As shown in Figure 3D, B) 10 μg mL−1, C) 80 μg mL−1; D) cell viability of EP-CL FPNs for 10 h and 24 h.
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Figure 4. CLSM images of A549 cells incubated with 10 μg mL−1 of EP-CL FPNs for 3 h. A) bright field, B) excited with 405 nm laser, C) merged image of A and B. Scale bar = 20 μm.
can be detected from EP-CL FPNs, which is great beneficial to cell imaging. The CLSM images of A549 cells incubated with 10 μg mL−1 of EP FPNs was also conducted and shown in Figure S5 (Supporting Information). Although the EP FPNs showed less stability in dilute solution than EP-CL FPNs, the EP FPNs also showed good result in cell imaging. In previous reports, biocompatible AIE FONs were fabricated via a covalent strategy such as Schiff base reaction, EP, RAFT polymerization, and anhydride ringopening polymerization for cell imaging, but most of them were non-cross-linking polymer. Compared with previous FONs, the EP-CL FPNs fabricated via the twostep polymerization in this work have more obvious advantages. First and most importantly, the EP-CL FPNs could be stable dispersed in physiological solution even below the CMC due to their inherent cross-linking structures. Second, other molecules such as drugs and nucleic acid could be further integrated into these FPNs taking advantage of the peripheral carboxyl groups. In addition, other functional monomers can be introduced into the copolymers via various polymerization strategies to afford multifunctional materials for biomedical applications. It also should be mentioned that, in this work, the resulting cross-linked FPNs have some drawbacks like inadequate biocompatibility due to the lack of stealthy elements (for example, polyethylene glycol, PEG), and the unsatisfactory metabolic behavior which can be predicted according to its nonbiodegradable structure. In this case, the next step of our research is to develop more biocompatible materials by incorporating PEG or polysaccharide into the system, and to construct more biodegradable structure through introducing metastable connection like disulfide bond into these cross-linked polymers.
3. Conclusions EP-CL FPNs are facilely prepared through EP and subsequent cross-linking based on an AIE monomer (PhENH2),
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acrylic acid, styrene, and a dianhydride (HFDA). These FPNs show a uniform morphology, high dispersibility, and intense fluorescence in an aqueous solution. Such FPNs are proven biocompatible for cell imaging according to the biocompatibility evaluation. Moreover, this cross-linking strategy is expected to provide an alternative path to construct various robust fluorescent bioprobes for biomedical applications.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: This research was supported by the National Science Foundation of China (Nos. 21134004, 21201108, and 51363016), and the National 973 Project (No. 2011CB935700), China Postdoctoral Science Foundation (Nos. 2012M520243 and 2013T60100), High-level Science Foundation of Qingdao Agriculture University (No. 6631334). Received: May 30, 2014; Revised: June 30, 2014; Published online: August 27, 2014; DOI: 10.1002/marc.201400309 Keywords: aggregation-induced emission; anhydride crosslinking; cell imaging; cross-linked fluorescent polymeric nanoparticles; emulsion polymerization
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