Article pubs.acs.org/cm
Tunable Ratiometric Fluorescence Sensing of Intracellular pH by Aggregation-Induced Emission-Active Hyperbranched Polymer Nanoparticles Yinyin Bao,† Herlinde De Keersmaecker,‡ Stijn Corneillie,† Feng Yu,† Hideaki Mizuno,‡ Guofeng Zhang,§ Johan Hofkens,§ Barbara Mendrek,⊥ Agnieszka Kowalczuk,⊥ and Mario Smet*,† †
Polymer Chemistry and Materials Unit, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium ‡ Biochemistry, Molecular and Structural Biology Unit, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200G, 3001 Leuven, Belgium § Molecular Imaging and Photonics Unit, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium ⊥ Centre of Polymer and Carbon Materials, Polish Academy of Sciences, ul. M. Curie-Sklodowskiej 34, Zabrze, Poland S Supporting Information *
ABSTRACT: Recently, ratiometric pH nanosensors have emerged as a robust tool for the fluorescence sensing and imaging, but there is no report of ratiometric sensors based on hyperbranched polymers for intracellular pH sensing. Herein, we describe the first example of hyperbranched polymer-based tunable fluorescent pH nanosensor with aggregation-induced emission activity, which exhibits great potential for ratiometric sensing of intracellular pH. These polymer nanoparticles can selectively accumulate in the acidic organelles of living cells by endocytosis process, and no obvious cytotoxicity was observed. The quantitative analysis of the intracellular pH values in HeLa cells was successfully conducted based on this new sensing platform. This platform provides a new choice for future developments of ratiometric fluorescent nanosensors, targeting not only protons but also a variety of other analytes of biological interest, such as metal ions, anions, and other biomolecules.
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INTRODUCTION
sensors only based on the intensity changes. This is because the pH sensing will not be influenced by many parameters such as optical path length, photobleaching, and leakage from the cells when using ratiometric methods, while the sensors only depending on the emission intensity could be significantly influenced by these parameters.1−3 Among these ratiometric nanosensors, polymer assemblies can allow for the noninvasive and real-time optical imaging of cellular dynamics and processes.14 For example, Wolfbeis and co-workers8 developed a polyurethane-based nanogel as a ratiometric fluorescent sensor for imaging intracellular pH in living epithelial normal rat kidney (NRK) cells. Gao and co-workers10 reported a series of pH activatable micellar nanoparticles from poly(tertiary amine methacrylate)-containing double-hydrophilic block copolymers, the fluorescence of which can be selectively and ultra sensitively activated in different endocytic compartments of
Intracellular pH plays many critical roles in cellular behaviors, such as proliferation and apoptosis, ion transport, enzyme activity, protein degradation, and endocytosis.1−3 In a typical mammalian cell, the pH in the mitochondria is about 8.0, the cytosol and the nucleus have a pH of 7.2−7.4, whereas the pH in the acidic organelles, such as endosomes and lysosomes, varies from 6.3 to 4.7 along the endocytic pathways.4−7 In view of many common diseases related to abnormal values of pH in the acidic organelles, it is of great importance to develop efficient platforms for sensing and monitoring pH changes inside living cells, which can help us better understand the cellular processes.1−7 A number of fluorescent sensors have been designed to quantify the intracellular pH values.1−3 Recently, ratiometric pH nanosensors have emerged as a robust tool for the pH sensing and imaging. Based on polymer assemblies,8−22 quantum dots,23−27 silica nanoparticles,28−30 gold nanoparticles,31 and carbon dots,32,33 these ratiometric nanosensors exhibit great advantages compared to the fluorescent pH © XXXX American Chemical Society
Received: March 5, 2015 Revised: April 21, 2015
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DOI: 10.1021/acs.chemmater.5b00858 Chem. Mater. XXXX, XXX, XXX−XXX
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aggregation-induced emission activity into the hyperbranched polymer nanoparticle systems(Chart 1), which exhibits great
human H2009 cells, such as early endosomes (pH 5.9−6.2) and lysosomes (5.0−5.5). However, there are only very few reports18−22 using polymer assemblies to do quantitative analysis of intracellular pH. To the best of our knowledge, there are no reports of ratiometric fluorescent sensors based on hyperbranched polymers for intracellular pH sensing, although several dendrimers have been used to construct pH probes.18,19,34,35 Hyperbranched polymers have received much attention because of their unique chemical and physical properties as well as their potential applications in drug and gene delivery, nanotechnology, and supramolecular science.36 Recently, benefiting from their highly branched topological structures and convenient synthetic procedures, hyperbranched polymers have been used to construct bioimaging probes and contrast agents through postfunctionalization.37 With a large number of end groups, hyperbranched polymers can be easily functionalized with various functional groups, which is an ideal platform to construct nanosensors with different functions. However, there are very few hyperbranched polymers used for fluorescence sensing and imaging, and most of the examples are based on hyperbranched conjugated polymers38,39 and hyperbranched polyamines,40,41 which are not convenient to design and synthesize compared to common hyperbranched polymers. In addition, the functional groups of the hyperbranched polymers for imaging by postfunctionalization are mainly based on commercial dyes, such as Rhodamine and Fluorescein.42−47 This may be because most of the synthetic fluorescent dyes are not soluble in water, the aggregation of which usually causes the emission of the functionalized hyperbranched polymers to be quenched in aqueous environment. All the above issues limit the development of hyperbranched polymer-based fluorescent sensors. To solve these problems, we envisaged that introducing aggregation-induced emission (AIE)-based fluorescent dyes into the hyperbranched polymer systems may be a smart strategy to afford them with strong emission in aqueous environment, resulting in a new platform for fluorescence sensing and imaging. Since the debut of the AIE concept in 2001, fluorescent materials with AIE phenomenon have attracted much interest.48,49 In contrast to common synthetic dyes, AIE-active dyes exhibit strong emission in aggregated state, which has been utilized for developing a number of fluorescent bio/chemosensors.50−55 If special AIE-active dyes are designed and functionalized to hyperbranched polymer systems, new fluorescent hyperbranched polymers for sensing/ imaging may be obtained. For example, if we introduce two different AIE-active dyes into the hyperbranched polymers, one pH-sensitive and the other acting as a reference, we can construct ratiometric sensors for pH sensing and imaging. 1,8-Naphthalimide-based dyes have recently emerged as potential fluorescent sensors with aggregation induced emission activity.56−61 In these dyes, there is always a twisted intramolecular charge transfer (TICT) process because of the intramolecular rotation, causing the emission quenched in dilute solutions, but in aggregated state the intramolecular rotation is inhibited then strong fluorescence can be recovered. In addition, the chemical structure of 1,8-naphthalimide-based dyes is very easy to modify, which helps to design various fluorescent sensors for metal ions, anions, proton, and other biomolecules.62−67 In this work, we describe the first example of hyperbranched polymer-based tunable fluorescent pH nanosensor, by introducing 1,8-naphthalimide-based dyes with
Chart 1. Ratiometric pH Nanosensors: Hyperbranched Polylactide Nanoparticles Functionalized with the Naphthalimide-Based Fluorophores N2 (green) as the pHSensitive Probe and N1 (blue) as the Referencea
a
For P1−P5, the seed molar ratios of N1 and N2 are 1:0, 3.3:1, 1:1, 1:2.5, and 0:1, respectively.
potential for ratiometric sensing of intracellular pH. These fluorescent polymer nanoparticles can selectively accumulate in the acidic organelles of living cells by endocytosis process, and the quantitative analysis of the intracellular pH values was successfully conducted based on this new sensing platform.
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RESULTS AND DISCUSSION As shown in Supporting Information, an amphiphilic polymer P0 with hyperbranched polylactide as the hydrophobic scaffold and PEG2000 as the hydrophilic outer layer was synthesized. The polylactide was chosen because it is commonly used in diverse biomedical devices, drug delivery systems, and tissue engineering benefiting from its good biocompatibility.68,69 We prepared the hyperbranched polylactide (PLA) by the polymerization of lactide with diethylene glycol monoglycidyl ether as a initiator, according to a modified literature method.70 The initiator was introduced with diethylene glycol to increase the biocompatibility of the polymer. Then the hyperbranched polylactide was grafted by poly(ethylene glycol) (PEG 2000) through esterification to give amphiphilic polymer P0, the structure of which was confirmed by 1H NMR. P0 was further functionalized by a green emissive naphthalimide-based dye N266 as pH probe and a blue emissive naphthalimide-based dye N160 as reference, with five different feed ratios (1:0, 3.3:1, 1:1, 1:2.5, 0:1), resulting in five fluorescent polymers P1−P5. The molecular weights (Mn) and polydispersity index (PDI) of all the polymers were determined by gel permeation chromatography (GPC) calibrated with polystyrene standards (Table S1 B
DOI: 10.1021/acs.chemmater.5b00858 Chem. Mater. XXXX, XXX, XXX−XXX
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spectra of N1 in DMF-H2O mixed solvents, a slight red-shift was observed upon increasing the water content (Figure S5 in the Supporting Information), which confirms the TICT effect.71 After admixing H2O to DMF solutions, the intramolecular rotations are inhibited gradually because of molecular aggregation, then the TICT process is also restricted, resulting in enhanced emission. Similar phenomenon can also be observed in other TICT-based AIE system.72 For N2, the AIE property can be explained with the same mechanism, although it is never reported previously. In addition, there is a photoinduced electron transfer (PET) occurs from the N,Ndimethyl group to the 1,8-naphthalimide moiety simultaneously, which provides the opportunity to detect proton.66 As we expected, the functionalized hyperbranched polymers P1-P5 also show excellent aggregation-induced emission properties. The emission of the polymers in aqueous solution is much stronger than in DMF (Figures S7−S11 in the Supporting Information). Take P1 for instance, the fluorescence intensity in DMF/H2O (1/9, v/v) exhibited 2.9-fold stronger than in DMF solution (Figure S7 in the Supporting Information), which is smaller than the value of N1. This can be due to the fact that there is already aggregation to some extent of the dye even in DMF solution, because of the hyperbranched polymeric strcture, and the TICT process is also more restricted compared to the free N1 dye, so the emission of P1 in DMF is not as weak as N1. The similar enhanced emissions were also observed for P2−P5. Overall, highly emissive hyperbranched polymers P1−P5 in aqueous were obtained by introducing 1,8-naphthalimide-based AIE dyes. For comparison, a naphthalimide-based dye N0 without AIE activity was synthesized and functionalized into P0. The resulted polymer P6 exhibits strong emission in DMF, but very weak fluorescence in aqueous solution (Figure S12 in the Supporting Information), like most of the synthetic organic dyes, which indicates that introducing AIE dyes to hyperbranched polymer systems is indeed an efficient strategy to prepare highly emissive hyperbranched polymers in aqueous solution. By this way, we can prepare fluorescent hyperbranched polymers without limitation to use commercial dyes. After obtaining the promising results above, the fluorescence emission spectra of the polymer nanoparticles prepared by nanoprecipitation were examined in K2HPO4/KH2PO4 buffer solutions (pH 7.0). As shown in Figure 1a, b, P1 possesses bright blue emission at 450 nm, whereas P5 exhibits strong green emission at 530 nm, which is similar to the fluorescence properties of the small molecular dyes N1 and N2, benefiting from the aggregation-induced emission activity. For P2−P4, along with increasing the feed ratios of N1 and N2, the intensity ratio I530/I450 gradually increases from 0.37 to 0.59 and 0.98, resulting in different emission colors of the polymers (Figure 1b). Therefore, thanks to the AIE activity, strong fluorescence emission of the hyperbranched polymer nanoparticles were obtained and it can be efficiently tuned by changing the feed ratio of N1 and N2, resulting in multicolor fluorescence. The result was also confirmed by UV−vis absorbance spectroscopy (Figure S2 and S3 in the Supporting Information). The pH-responsive properties of the polymer nanoparticles were further studied by fluorescence spectroscopy in K2HPO4/ KH2PO4 buffer solutions at various pH values. A pH range from 4.5 to 8.1 was chosen in consideration of that the intracellular pH value varies from 4.7 to 8.0 according to the literature.4 As an optimized example (see below), P3 prepared
in the Supporting Information). From the GPC results, we can see the Mn (4600) of the PLA is much higher than the theoretical molecular weight (1400), which indicates the formation of hyperbranched polylactide, according to reported structure-molecular weight relationships.70 After PEGylation, the Mn of P0 increased to 9300 that further confirms the hyperbranched structure. The conjugation was confirmed by FT-IR spectra (Figure S21 in the Supporting Information), and the dye loading was calculated based on the UV−vis absorbance method (Table S2 in the Supporting Information). The resulting polymers P1−P5 were dissolved in DMF, and nanoprecipitated in water to afford fluorescent nanoparticles. These nanoparticles were further characterized by dynamic light scattering (DLS) and scanning electronic microscope (SEM), which show good dispersion in aqueous solution with the hydrodynamic radius from 70 to 102 nm (Table S3 and Figure S1 in the Supporting Information and Figure 1c, d ).
Figure 1. (a) Normalized fluorescence emission spectra of P1−P5 in K2HPO4/KH2PO4 buffer solutions (pH 7.0); (b) visible emission of P1−P5 observed under UV lamp (365 nm); (c) SEM image of P2based nanoparticles; (d) SEM image of P3-based nanoparticles; (e) normalized (I450 nm) fluorescence emission spectra of P3 (20 μg mL−1) in 0.1 M K2HPO4/KH2PO4 buffer at various pH values; (f) pH reversibility study of P3 between pH 5.0 and 8.1. λex = 365 nm.
The fluorescence properties of N1 and N2 were first investigated (Figures S5 and S6 in the Supporting Information), both of which show obvious aggregation-induced emission activity. The fluorescence spectra of N1 and N2 were examined in DMF, which exhibit very weak emission at 435 and 525 nm, with large Stokes shift of 70 and 120 nm, respectively. However, when a large amount of distilled water is admixed with DMF, the two dyes display bright fluorescence. As seen from Figures S5 and S6 in the Supporting Information, the emission peaks of N1 and N2 gradually increase along with increasing the water content, finally at a water content of 90% with a 50-fold and 65-fold stronger than in DMF solution. This phenomenon should be attributed to the aggregation-induced emission effect.57,60 Take N1 for example, there is a TICT process because of the intramolecular rotations along the donor−acceptor system with push−pull interactions, which causes weak emission in DMF solution. From the fluorescence C
DOI: 10.1021/acs.chemmater.5b00858 Chem. Mater. XXXX, XXX, XXX−XXX
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To investigate the feasibility of intracellular pH sensing of these polymer nanoparticles, we selected the ratiometric sensors P2−P4 to introduce into the HeLa cells. As shown in Figure S18 in the Supporting Information, bright emissions of P2−P4 were observed in the living cells after 24 h incubation by confocal laser scanning microscope (CLSM). The fluorescence images of the blue channel and green channel show the separate fluorescence emission of the N1- and N2based fluorophores on the polymer nanoparticle, respectively. The strong green fluorescence can be ascribed to the protonation of the amino group in piperazine in the acidic environment of the lysosome. However, no obvious emission can be observed in the cells without polymer nanoparticles, which indicate all three nanosensors enter the cells and were localized in acidic regions. In addition, it was found that P3based nanosensor can also enter Chinese hamster ovary (CHO) cells (Figure S19 in the Supporting Information), which suggests these hyperbranched polymer nanoparticles can be used in various living cells. To further study the subcellular localization of these sensors in the HeLa cells, the commercially available lysosome specific staining probe LysoTracker Deep Red was used to costain the cells with the nanosensors. The HeLa cells were first incubated with 200 μg/mL nanosensors at 37 °C for 4 h, and then incubated with 50 nM LysoTracker Deep Red at 37 °C for another 30 min. Figure 3 (red channel for 650−700 nm, λex =
with a molar feed ratio of N1/N2 at 1:1 shows a distinct fluorescent response to pH. As depicted in Figure 1a and Figure S16 in the Supporting Information, P3 only shows a blue emission with a maximum around 450 nm at pH 8.1, however, upon decreasing the pH values, a new emission peak at 520 nm appears. As a result, the intensity ratio I520/I450 gradually increases from 0.54 to 0.99 with the decrease in pH. In this case, the TICT process has been inhibited because of the highly aggregated state, which cannot affect the fluorescence emission. The pH response is attributed to the modulation of the intramolecular PET process from the nitrogen lone electron pair to the naphthalimide fluorophore of dye N2 caused by protonation.66 Upon decreasing the pH values from 8.1 to 5.0, the nitrogen is protonated gradually, resulting in inhibition of the PET process, then the quenched emission is recovered steadily. Benefiting from that the fluorescence emission of N1 is not sensitive to pH changes in the range of 8.1−4.5 (Figure S13 in the Supporting Information), ratiometric pH response can be obtained. The pH response was also confirmed by UV−vis titration experiments (Figure S4 in the Supporting Information). Most notably, this pH nanosensor displays an excellent reversibility between pH 6 and pH 8, and the fluorescence color changes under UV lamp can be distinctly observed by naked eyes (Figure 1f and Figure 2, insert).
Figure 2. Plots of I520/I450 versus pH values for P1−P4 prepared with different molar feed ratios (N1/N2 = 1:0, 3.3:1, 1:1, and 1:2.5). I520/I450 is the ratio of the fluorescence intensity of P1−P4 at 520 and 450 nm. Insert: visible emission of P3 observed under UV lamp (365 nm). Figure 3. Confocal fluorescence microscopy images of HeLa cells incubated with (a, b) P2, (e, f) P3, and (i, j) P4-based nanosensors and (c, g, k) LysoTracker Deep Red. The fluorescence images of first, second, and third column were collected in blue channel (430−490 nm, λex = 405 nm), green channel (500−550 nm, λex = 405 nm), and red channel (650−700 nm, λex = 635 nm). (d, h, l) Composite images of blue, green, red channels.
The molar feed ratio of N1 to N2 is an important factor for the pH response range of the hyperbranched polymer nanoparticles. As shown in Figure 2, different ratios from 1:0 to 1:2.5 make the pH-responsive emission intensity ratios and colors of the nanosensor tunable from pH 5.0 to 8.1, during most of the physiological pH ranges. Among these nanosensors, P2 and P4 also exhibit ratiometric fluorescence response to pH with intensity ratio changes in the range of 0.41−0.56 and 0.83−1.91, respectively, whereas P5 shows OFF-ON pH fluorescence response based on only intensity (Figures S14 and S15 in the Supporting Information). We can conclude that the fluorescence sensing properties of the hyperbranched polymer nanosensors can be efficiently tuned through varying feed ratio of the dyes. It should be pointed out that various functional fluorescent probes not only for pH but also for other targets can be constructed by introducing different functional AIE dyes to this hyperbranched polymer platform, which generates an efficient strategy to prepare ratiometric fluorescent nanosensors.
635 nm) highlights the red fluorescence emission from LysoTracker Deep Red located in lysosomes, and the colocalization of the pH nanosensors is clearly demonstrated by the white spots in the merge image of blue, green, and red channels. Therefore, the lysosome localization result demonstrated that all the three nanosensors were taken up by the living cells and then accumulated in lysosomes, which indicate the hyperbranched polymer nanoparticles can be used as an pH nanosensor for lysosome imaging in living cells. The cytotoxicity of the nanosensors was investigated by a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetraD
DOI: 10.1021/acs.chemmater.5b00858 Chem. Mater. XXXX, XXX, XXX−XXX
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nanosensor. This is the first example to construct a ratiometric fluorescent sensor for intracellular pH, with AIE dye-conjugated hyperbranched polymer nanoparticles as the sensing platform. This platform provides a new choice for future developments of ratiometric fluorescent nanosensors, targeting not only protons but also a variety of other analytes of biological interest, such as metal ions, anions, and other biomolecules, by conveniently changing the conjugated fluorophores and probes.
zolium bromide) assay.73 As shown in Figure S17 in the Supporting Information, the cell viability was not significantly changed upon treatment with the nanosensors for 24 h, which also did not exhibit any concentration-dependent effect. This result clearly indicates the hyperbranched polymer nanoparticle-based sensors have low cytotoxicity and good biocompatibility. To demonstrate the applicability of the nanosensors for quantitative analysis of intracellular pH, the intracellular calibration experiment was first made in HeLa cells with H+/ K+ ionophore nigericin utilizing P3-based nanosensor, which is a standard approach for homogenizing the pH of cells and culture medium.32 As shown in Figure 4a, the fluorescence
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ASSOCIATED CONTENT
S Supporting Information *
Experimental details and supporting figures. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.5b00858.
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AUTHOR INFORMATION
Corresponding Author
* E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the financial support from the Tsinghua University-KU Leuven Bilateral Scientific Cooperation (bilateral grant BIL09/08). The authors express their thanks to Karel Duerinckx and Bert Demarsin for help with NMR and HRMS measurement, respectively.
Figure 4. (a) Titration of P3-based nanosensor in living cells. HeLa cells were clamped at pH 5.61, pH 6.51, and pH 7.98 with nigericin. (b) pH map (bottom) calculation for untreated HeLa cells based on the calibration curve obtained using nigericin (top).
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from the green channel in cells increases dramatically with reduction of pH values, whereas that from the blue channel slowly alters. The ratio channel, obtained based on the above two channels, displays a characteristic pH-dependent signal, which generates a calibration curve in the pH range from 5.6 to 8.0 (Figure 4b). On the basis of this curve, it is possible to estimate the averaged intracellular pH value of the organelles in the HeLa cells. As depicted in Figure 4b, the pH values of spot 1 and spot 2 were determined to be 6.0 ± 0.2, and 5.2 ± 0.2, respectively. These are the expected values for acidic organelles (endosomes and lysosomes), which are reported to have a pH between 4.7 and 6.3.4−7 Thus, the hyperbranched polymer nanoparticles can be used as an efficient sensing platform for intracellular pH in living cells, which is the first example utilizing hyperbranched polymer for quantitative detection of intracellular pH values. This platform also privides a new strategy to construct other ratiometric fluorescent nanosensors for sensing metal ions, anions, and other biomolecules in living cell by changing the functional AIE dyes.
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CONCLUSIONS In conclusion, a tunable ratiometric fluorescent pH nanosensor has been developed by incorporating aggregation-induced emission-active fluorophores to hyperbranched polymer nanoparticles, which shows strong emission in aqueous solutions and exhibits excellent sensing properties for intracellular pH. In addition, the fluorescence emission and sensing properties of the polymer nanosensors can be easily tuned by changing the feed ratio of the conjugated fluorophores. Cell imaging studies and MTT assay demonstrated its good biocompatibility and selective accumulation in acidic organelles of living cells. More importantly, quantitative analysis of intracellular pH in HeLa cells has been successfully performed with this ratiometric E
DOI: 10.1021/acs.chemmater.5b00858 Chem. Mater. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.chemmater.5b00858 Chem. Mater. XXXX, XXX, XXX−XXX
