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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Thermoresponsive Nanospheres with Entrapped Fluorescent Conjugated Polymers for Cellular Labeling Yang Sheng,†,§ Zongquan Duan,†,‡ Zheng Jia,†,‡ Yan Pan,∥ Yixin Sun,†,§ Jian Li,*,†,‡,§ Linhong Deng,∥ Mark Bradley,⊥ and Rong Zhang*,†,‡,§ †
School of Materials Science and Engineering, Changzhou University, Changzhou 213614, Jiangsu, China Jiangsu Collaboration Innovation Center of Photovoltaic Science and Engineering, Changzhou University, Changzhou 213164, Jiangsu, China § Jiangsu Key Laboratory of Environmentally Friendly Polymeric Materials, Changzhou University, Changzhou 213164, China ∥ Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou 213164, Jiangsu, China ⊥ School of Chemistry, EaStCHEM, University of Edinburgh, Joseph Black Building, West Mains Road, Edinburgh EH93JJ, U.K.
ACS Appl. Bio Mater. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/24/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Thermoresponsive fluorescent nanospheres were prepared by entrapping fluorescent conjugated polymers (FCP) into biocompatible copolymerized nanospheres comprised of methacryloyloxyethyltrimethylammonium chloride and n-isopropylacrylamide. A systematic study allowed monodisperse nanospheres ranging from 50 to 150 nm to be produced by precisely controlling the amount of monomer, initiator, and solvent during the dispersion polymerization process. The as-synthesized fluorescent nanospheres exhibited reversible temperature-dependent fluorescence with strong emission at lower temperature (20 °C) and weak emission at higher temperature (50 °C). Cell viability assays showed that the nanoshperes were nontoxic. The cellular imaging showed a rapid response in fluorescence intensity change upon variation of temperature in HeLa cells, indicating that our fluorescent probes are promising as in situ thermometers for labeling cells. KEYWORDS: fluorescence bioimaging, thermoresponsiveness, conjugated polymer, nanospheres, cell labeling
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opportunities.25−27 The FCP can be used directly to generate micelles due to their hydrophobic backbones and hydrophilic side chains, while they can also be a “payload” encapsulated “within” other materials.28 Such a “loaded vessel” strategy offers a versatile platform due to their colloidal stability and biocompatibility while offering a variety of surface functionalities.29In previous high-throughput screening studies we have demonstrated that the fluorescence of FCP can be enhanced by entrapment within specific polymeric beads, including copolymers of N-isopropylacrylamide (NiPAAm) and methacrylatoethyltrimethylammonium chloride (DMC).30 Indeed the NiPAAM/DMC matrix had the capability of bestowing thermoresponsive fluorescence behavior on FCP, offering the fluorophore unique opportunity as an in situ temperature indicator.31 NiPAAm and DMC are known to be nontoxic and biocompatible, making the copolymer matrix potentially safe for in vivo use.32 However, the large size of these micron beads
INTRODUCTION Fluorescence imaging is an attractive technique for the identification of biomarkers within cells or tissues, offering high sensitivity, multiplexing capabilities, and low cost.1−5 Due to an increasing interest in understanding biological and chemical processes at a cellular level, where temperature effects might well play critical roles, an emergent technological development is thermally responsive fluorescent probes that allow visualization of local temperature changes.6−12 In the development of thermoresponsive fluorescent probes, nanostructures based on quantum dots, carbon dots, and organic dyes have been reported with temperature changes inducing both wavelength and intensity changes.13−19 It is known that commonly used organic dyes suffer from photobleaching,20,21 while inorganic quantum dots and carbon dots have limitations such as potential toxicities, low quantum efficiencies, and lack of biodegradability.22−24 Among the possible fluorescent candidates, fluorescent conjugated polymers (FCP) display favorable merits of high absorption coefficients, high fluorescence quantum efficiencies, excellent photostability, and low toxicity, thus making FCP an interesting material for a wide range of fluorescence imaging © XXXX American Chemical Society
Received: July 11, 2018 Accepted: August 14, 2018 Published: August 14, 2018 A
DOI: 10.1021/acsabm.8b00311 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials inhibited the potential applications for fluorescence imaging at molecular and cellular levels. Herein we report the preparation of fluorescent nanoparticles comprised of a NiPAAm/DMC copolymer matrix with the entrapment of FCP via an oil-in-water microemulsion method. Due to the incorporation of NiPAAm, the fluorescent nanospheres showed significant thermoresponsive fluorescence. Subsequently we successfully visualized fluorescence changes of labeled live HeLa cells upon variations in temperature, proving the effectiveness of using our fluorescent nanospheres as thermoresponsive imaging probes.
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Table 1. Influence of SDS and Solvent (Water) on the Size of the Copolymer Nanospheres MBA (wt %)
SDS (wt %)
H2O (mL)
8 8 8 8 8 8
5 6 7 5 5 5
20 20 20 20 15 10
size (nm) 97 79 52 97 106 156
± ± ± ± ± ±
12 10 7 12 6 9
The water was changed every 12 h for 3 days, and the product was collected and diluted to a concentration of 0.02 g/mL. Fluorescence Analysis. A fluorospectrometer (PerkinElmer, LS 45) was used to analyze the fluorescent properties of the nanospheres and the influence of temperature on their fluorescence over multiple heating−cooling cycles under an excitation wavelength of 450 nm (emission = 572 nm). Cytotoxicity Assay and Cell Imaging. The cytotoxic effects of the fluorescent nanospheres were examined by incubating HeLa cells with the nanospheres at various concentrations and analysis via a CCK-8 assay. The HeLa cells were cultured in 200 μL of H-DMEM at 37 °C in a 5% CO2, humidified environment. The cells were then transferred into a 48-well cell culture plate with identical cell concentrations (∼104 cells/mL, 0.2 mL). After incubation for 12 h, 100 μL solutions of the nanospheres with various concentrations (from 8 mg/mL to 0.8 mg/mL) were added into the cell culture and incubated together for another 12 h. Subsequently, the culture medium containing extra nanospheres was removed, followed by the addition of fresh H-DMEM. Finally 10 μL of CCK-8 was added, followed by 4 h incubation at 37 °C in a 5% CO2 environment. Absorbance measurements were analyzed spectrophotometrically at 355 nm using BioTek Epoch. To demonstrate the cell imaging, fluorescent nanospheres (50 μL, 8 mg/mL) were injected into culture wells with HeLa and HepG2 cells, respectively, and 24 h incubation was allowed at 37 °C in a 5% CO2, humidified environment. The cells were then washed with fresh H-DMEM to remove excess nanspheres, followed by observation using a Zeiss LSM 710 laser scanning confocal microscope with excitation at 450 nm (emission = 572 nm). In order to visualize the fluorescence changes of the nanoparticles labeled live cells in vitro, the temperature of the cell medium was changed from 40 to 20 °C, and fluorescence images of the cells were captured as the temperature was changed. Fluorescence images of the cells were captured by using a laser scanning confocal microscopy with excitation at 450 nm (emission = 572 nm).
EXPERIMENTAL SECTION
Materials. Tetramethylethylenediamine (TEMED) and N-isopropylacrylamide (NiPAAm, 99%, MW 169.22) were purchased from Aladdin Chemicals (Shanghai, China). NiPAAm was recrystallized from n-hexane prior to use. Methacryloyloxyethyltrimethylammonium chloride (DMC, 99%, MW 207.70) was purchased from Aldrich chemicals and was purified to remove polymerization inhibitors before use. Ammonium persulfate (APS) and sodium dodecyl sulfate (SDS) were purchased from LingFeng Chemical Reagents (Shanghai, China) and were recrystallized in methanol prior to use. The water used was Milli-Q purity grade (18.25 MΩ·cm) produced using an ultrapure water machine (ULUP). The fluorescent conjugated polymers used in this study were prepared by Suzuki cross-coupling chemistry as detailed in our previous papers.33 The chemical structure of the fluorescent conjugated polymer is shown in Figure 1, and the corresponding molecular weight is shown in Table S1.
Figure 1. Structure of the fluorescently conjugated polymer used in this study. Synthesis of Fluorescent Nanospheres. The fluorescent conjugated polymer (FCP) was dissolved in chloroform at various concentrations (0.001, 0.0015, 0.002, 0.003, and 0.005 wt %). The fluorescent nanospheres were prepared by a modified oil-in-water microemulsion method.34 To produce 97 nm nanospheres, 0.4 g of a mixture of NiPAAm and DMC (mass ratios 3:1), 0.02 g of SDS (5 wt %), and 0.032 g of the cross-linker MBA (8 wt %) were dissolved in water (15 mL). Subsequently, 3 mL of the fluorescent dye solution was added into the mixture under vigorous stirring to give a homogeneous solution. The solution was heated to 30 °C followed by adding dropwise 5 mL of an aqueous solution of initiators (0.02 g of APS and 0.01 g of TEMED). The mixture was heated to 40 °C and kept for 6 h to complete the copolymerization reaction. Then the temperature was increased to 65 °C and stirred for 3 h to evaporate the chloroform to obtain a transparent aqueous solution of fluorescent nanospheres. To control the size of the fluorescent nanospheres, the amounts of SDS and the solvent volume were adjusted, as shown in Table 1. FCP solution in chloroform was used as a control in the temperature-dependent fluorescence analysis. To purify the fluorescent nanospheres, the obtained solution was dialyzed against distilled water. Prior to dialysis, the membrane (molecular weight cutoff at 8000) was boiled in 400 mL of aqueous solution (pH 8.0) of NaHCO3 (0.238M)/EDTA·2Na (1 mM) for 10 min. After that the membrane was washed with distilled water and boiled again for another 10 min in 500 mL of 1 M MEDTA·2Na (pH 8.0). Finally the dialysis membrane was placed in ethanol before use. Approximately 5 mL of the solution of fluorescent nanospheres was added into the treated dialysis membrane, which was sealed at both ends and placed into a beaker containing 300 mL of distilled water.
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RESULTS AND DISCUSSION Fluorescent nanospheres were produced by entrapment of the fluorescent conjugated polymer (FCP)30 (molecular weight ∼6000 Da, see Supporting Information Table S1) during the copolymerization of NiPAAm and DMC using an oil-in-water microemulsion technique. A chloroform solution containing the FCP was added into the solution containing monomers of NiPAAm and DMC. Due to the vigorous stirring and the presence of the surfactant SDS, chloroform droplets containing FCP and monomers were created and stabilized. The copolymerization was initiated by APS/TEMED at the interface between chloroform and H2O. Subsequent heating to 65 °C removed the chloroform, leaving the fluorescent cargo FCP entrapped within NiPAAm/DMC copolymer nanospheres. A systematic study of the experimental parameters which influenced the particle size, including the amount of SDS and solvent volume, was performed when NiPAAm to DMC molar ratio was fixed at 3:1. As shown in Table 1, when the amount B
DOI: 10.1021/acsabm.8b00311 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials of surfactant (SDS) was increased while other parameters were fixed, the average hydrodynamic size of the polymer nanospheres decreased probably because higher concentrations of surfactant produced larger number of smaller droplets. In addition, reduction in the solvent volume (water) also increased the average particle size (see Supporting Information Figure S1), which was probably because high viscosity of the solution produced larger chloroform droplets. SEM image (see Figure 2) shows monodisperse nanospheres with an average diameter of 97 ± 12 nm, prepared with 5 wt %
Figure 3. Photophysical analysis of nanospheres with entrapped FCP. (A) Absorption spectra of pure FCP dispersed in chloroform and fluorescent nanospheres suspended in H2O. (B) Images of the nanosphere solution (NiPAAm:DMC = 3:1, 0.005 wt % FCP) in H2O (left: bright field, right: under 365 nm excitation) and (C) image of FCP dispersed in chloroform (left: bright field, right: under 365 nm excitation).
Figure 4. (A) Fluorescence intensity of nanospheres N3D1, N2D2, and N1D3 (the concentration of nanoparticles is 8.0 mg/mL) and pure fluorescent conjugated polymer at 20 °C (all at 0.005 wt % FCP loading and equivalent FCP concentration). (B) The fluorescence intensity of nanospheres N3D1 with various FCP loadings (0.001, 0.0015, 0.002, 0.003, and 0.005 wt %) measured at 20 °C. Inset: the corresponding fluorescence spectra. (C) The fluorescence intensity of nanospheres N3D1 measured between 20 and 50 °C at 5 °C intervals. Inset: The fluorescence intensity maximum versus temperatures. (D) The hydrodynamic size of the fluorescent nanospheres N3D1 (0.005 wt % FCP) from 22 to 38 °C at 2 °C intervals.
Figure 2. Characterization of nanospheres. (A) SEM and TEM image (inset) of fluorescent nanospheres loaded with the fluorescent conjugated polymer. (B) Size distribution of the fluorescent nanspheres (synthesized with 5 wt % SDS and 20 mL of H2O) measured using a Zetasizer Nano (97 ± 12 nm, PDI = 0.229).
SDS in 20 mL of H2O, while TEM shows one typical nanosphere. No aggregation was observed, indicating the nanospheres were well dispersed in water, while FT-IR analysis showed characteristic vibration bands of its precursors DMC, NiPAAm, and FCP (see Supporting Information Figure S2). To study the optical property of the fluorescent nanospheres, the absorption and emission spectra were measured. The absorption peaks (see Figure 3A and Figure S3) of the nanospheres loaded with FCP demonstrated an approximately 30 nm red-shift from 420 to 450 nm compared to pure FCP. In the meantime, the emission peak showed a very slight blueshift (Δλ = 6 nm) from 578 nm of pure FCP in chloroform to 572 nm in polymer matrix (see Figure 4A), which is consistent with our previous study.30 As the SEM/TEM images and particle size analysis (see Figure 2) indicated little aggregation, the red-shifted absorption and blue-shifted emission were not
attributable to particle aggregation. Instead, the red-shifted absorption and blue-shifted emission indicate the FCP was probably strained during the entrapment process.30 It was also observed that the fluorescence intensity increased linearly with increased loading of FCP in the range from 0.001 to 0.005 wt %, suggesting that aggregation of FCP was not occurring in the polymer matrix (see Figure 4B and SI Figure S5). Therefore, in the following discussions the loading of FCP was kept at 0.005 wt %. To determine the influence of the composition of the polymer matrix on the fluorescence properties of the FCP, nanospheres with different NiPAAm to DMC molar ratio (e.g., 3:1, 2:2, and 3:1) were prepared and named as N3D1, N2D2, and N3D1, respectively, under the same experimental C
DOI: 10.1021/acsabm.8b00311 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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counting and a toxicity assay (CCK-8). The cytotoxicity assay in Figure S7 shows that no apparent difference in cell viability was observed for various FCP loadings. The cell viabilities were above 90% for all concentrations, indicating extremely low toxicity of all fluorescent nanospheres. To demonstrate the effectiveness of the fluorescence nanospheres for cellular labeling, the particles were incubated with HeLa and HepG2 cells and analyzed by laser confocal scanning microscopy (see Figure 6). The fluorescence images
conditions (8 wt % MBA, 5 wt % SDS, and 20 mL of H2O). It was found that the fluorescence intensity of the nanospheres increased with an increase in the NiPAAm to DMC molar ratio (see Figure 4A). The fluorescence of the FCP was almost quenched in sample N1D3, while it was enhanced for sample N3D1, consistent with previous findings.30 Due to the introduction of thermoresponsive segments based on NiPAAm, it was expected that the nanospheres would display thermoresponsive behavior. The emission spectra were measured at different temperatures (from 20 to 50 °C). It was found that the fluorescence intensity of FCP alone showed no change within the temperature range (see SI Figure S4). However, the fluorescence intensity of N1D3 showed a distinguishable decrease from 20 to 50 °C, while the intensity decrease for N2D2 within the same temperature change was more obvious. Among these three samples, N3D1 showed the most significant fluorescence decrease (see Figure 4C), suggesting strong correlation to the NiPAAm/DMC ratio in the nanospheres. To explain the temperature-dependent fluorescent intensity changes, the hydrodynamic size of fluorescent nanospheres N3D1 at different temperatures was monitored by dynamic light scattering (see Figure 4D). When the solution was heated from 22 to 38 °C, the hydrodynamic size of the fluorescent nanospheres decreased from ∼140 to ∼90 nm, consistent with the behavior of NiPAAm at elevated temperature. As the nanospheres shrank, the internal concentration of the FCP became larger (the volume change is ∼3.8-fold) which results in shorter distance and larger number of short-range interactions between the fluorophores of FCP, thus leading to enhanced self-quenching. Alternatively, it can be viewed as a change in the hydrophilicity of nanospheres, which changed the solvent content in the copolymer spheres, possibly leading to a variation in fluorescent intensity.35 However, the weakened fluorescence could be reversibly switched back “on” again by decreasing the temperature. The reversibility of the thermoresponsive fluorescence intensity of the nanospheres N3D1 was demonstrated by heating to 50 °C and then cooling to 20 °C over 10 cycles (see Figure 5). This robust reversibility suggests the nanospheres exhibited good thermal stability and photostability. To apply the fluorescent nanosphere to biomedical imaging, the biocompatibility and toxicity must be evaluated. The fluorescent nanospheres N3D1 loaded with different levels of FCP (from 0.001 to 0.005 wt %, see Supporting Information Figure S6) were incubated with HeLa cells for 24 h before cell
Figure 6. (A) Fluorescent, bright-field, and merged images of HeLa cells following incubation with fluorescent nanospheres. (B) Images of HepG2 cells incubated with the fluorescent nanospheres (N3D1, 0.005 wt % FCP, 8.0 mg/mL). Images were taken using a laser confocal microscope (λex = 450 nm, λem = 572 nm).
of the cells were captured after 24 h incubation, indicating good fluorescence stability of the FCP within the biological environment. Images of the HepG2 cells showed that the nanopsheres were internalized but did not enter the nuclei of the cells. To demonstrate the thermoresponsiveness of the fluorescence nanoprobes in live cells, a series of fluorescence images of HeLa cells were captured every 5 s (see Supporting Information Figure S7) during the temperature drop from 40 to 20 °C (room temperature). Figure 7 shows the fluorescence images of HeLa cells taken at 10 s intervals. Originally, the cells showed a bright yellow fluorescence at 20 °C which was decreased significantly when the medium was increased to 40 °C, shown in the image labeled as “0 s”. The diminished fluorescence was gradually recovered to its original intensity when the medium was allowed to cool back down to 20 °C. The differences between images obtained at 20 and 40 °C was consistent with our previous characterization that the fluorescence was strong at low temperature and weak at high temperature.
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CONCLUSIONS In summary, we have successfully developed a nanosized thermoresponsive fluorescent probe comprised of a biocompatible polymer matrix of copolymerized NiPAAm and DMC and a fluorescent cargo of conjugated polymer via an oil-inwater microemulsion method. The size of the fluorescent nanospheres was controlled by precisely adjusting the concentrations of SDS as well as the solvent volumes. The fluorescent nanospheres obtained displayed strong emission at 20 °C, and the fluorescence gradually diminished as the
Figure 5. Reversibility of the fluorescent intensity of the nanospheres with temperature switching. (A) The fluorescence spectra of N3D1 (0.005 wt % FCP) measured at 20 and 50 °C, respectively (λex = 455 nm). (B) Fluorescent intensity changes between 20 and 50 °C across 10 cycles of heating and cooling. D
DOI: 10.1021/acsabm.8b00311 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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project in Jiangsu Province (SWYY-CXTD-001), and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. We would also like to thank the start-up funding (ZMF15020107) from Changzhou University, Jiangsu Shuangchuang Program, and the Natural Science Foundation of Jiangsu Province (BK20160278) for supporting Dr. Yang Sheng and the Natural Science Foundation of Guangxi Province (2015GXNSFAA139255) for supporting Dr. Yixin Sun, and MBR thanks the ERC (ERC-2013-ADG 340469 ADREEM).
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Figure 7. Fluorescent intensity change of the nanospheres in HeLa cells with temperature decrease of the culture medium within 1 min. (A) Fluorescence images of HeLa cells taken by confocal laser scanning microscopy over time when cooling from 40 °C to room temperature (∼20 °C). (B) Calculated fluorescent intensity of the nanospheres over time upon cooling.
temperature was elevated in a fully reversible process. The thermoresponsive fluorescence of our probe resulted from this shrinking/swelling behavior upon heating/cooling; when the temperature increased, the nanospheres shrank, reducing the distance between FCP monomers in the nanospheres, leading to self-quenching as well as alteration in nanosphere hydrophilicity. As the portion of NiPAAm increased, the thermoresponsiveness was also enhanced. We demonstrated the use of our thermoresponsive fluorescence nanosensors in HeLa cells, proving their effectiveness as potential temperature indicators of local temperature changes for a variety of thermorelated biomedical applications.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00311. Detailed data of molecular weight, DLS measurement of nanospheres, FTIR of products, absorption and emission spectra of nanospheres at different conditions, cell viability of nanospheres with different FCP loadings, and fluorescence images of HeLa cells captured every 5 s during temperature decrease (PDF).
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REFERENCES
(1) Bradley, M.; Alexander, L.; Duncan, K.; Chennaoui, M.; Jones, A. C.; Sanchez-Martin, R. M. pH Sensing in Living Cells Using Fluorescent Microspheres. Bioorg. Med. Chem. Lett. 2008, 18, 313− 317. (2) Demchenko, A. P. Nanoparticles and Nanocomposites for Fluorescence Sensing and Imaging. Methods Appl. Fluoresc. 2013, 1, 022001. (3) Sánchez-Martín, R. M.; Cuttle, M.; Mittoo, S.; Bradley, M. Microsphere-Based Real-Time Calcium Sensing. Angew. Chem., Int. Ed. 2006, 45, 5472−5474. (4) Sheng, Y.; Tang, X.; Peng, E.; Xue, J. Graphene Oxide Based Fluorescent Nanocomposites for Cellular Imaging. J. Mater. Chem. B 2013, 1, 512−521. (5) Sheng, Y.; Liao, L.-D.; Bandla, A.; Liu, Y.-H.; Yuan, J.; Thakor, N.; Tan, M. C. Enhanced Near-Infrared Photoacoustic Imaging of Silica-Coated Rare-Earth Doped Nanoparticles. Mater. Sci. Eng., C 2017, 70, 340−346. (6) Menges, F.; Mensch, P.; Schmid, H.; Riel, H.; Stemmer, A.; Gotsmann, B. Temperature Mapping of Operating Nanoscale Devices by Scanning Probe Thermometry. Nat. Commun. 2016, 7, 10874. (7) Shang, L.; Stockmar, F.; Azadfar, N.; Nienhaus, G. U. Intracellular Thermometry by Using Fluorescent Gold Nanoclusters. Angew. Chem., Int. Ed. 2013, 52, 11154−11157. (8) Uchiyama, S.; Gota, C.; Tsuji, T.; Inada, N. Intracellular Temperature Measurements with Fluorescent Polymeric Thermometers. Chem. Commun. 2017, 53, 10976−10992. (9) Takei, Y.; Arai, S.; Murata, A.; Takabayashi, M.; Oyama, K.; Ishiwata, S. i.; Takeoka, S.; Suzuki, M. A Nanoparticle-Based Ratiometric and Self-Calibrated Fluorescent Thermometer for Single Living Cells. ACS Nano 2014, 8, 198−206. (10) Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D. Nanometre-Scale Thermometry in a Living Cell. Nature 2013, 500, 54−58. (11) Zhang, H.; Jiang, J.; Gao, P.; Yang, T.; Zhang, K. Y.; Chen, Z.; Liu, S.; Huang, W.; Zhao, Q. Dual-Emissive Phosphorescent Polymer Probe for Accurate Temperature Sensing in Living Cells and Zebrafish Using Ratiometric and Phosphorescence Lifetime Imaging Microscopy. ACS Appl. Mater. Interfaces 2018, 10, 17542−17550. (12) Chen, Z.; Zhang, K. Y.; Tong, X.; Liu, Y.; Hu, C.; Liu, S.; Yu, Q.; Zhao, Q.; Huang, W. Phosphorescent Polymeric Thermometers for In Vitro and In Vivo Temperature Sensing with Minimized Background Interference. Adv. Funct. Mater. 2016, 26, 4386−4396. (13) Dong, F.; Zheng, T.; Zhu, R.; Wang, S.; Tian, Y. An Engineered Thermo-Sensitive Nanohybrid Particle for Accurate Temperature Sensing at The Single-Cell Level And Biologically Controlled Thermal Therapy. J. Mater. Chem. B 2016, 4, 7681−7688. (14) Haro-González, P.; Ramsay, W. T.; Maestro, L. M.; del Rosal, B.; Santacruz-Gomez, K.; del Carmen Iglesias-de la Cruz, M.; SanzRodríguez, F.; Chooi, J. Y.; Sevilla, P. R.; Bettinelli, M.; Choudhury, D.; Kar, A. K.; Solé, J. G.; Jaque, D.; Paterson, L. Quantum Dot-Based Thermal Spectroscopy and Imaging of Optically Trapped Microspheres and Single Cells. Small 2013, 9, 2162−2170. (15) Maestro, L. M.; Jacinto, C.; Silva, U. R.; Vetrone, F.; Capobianco, J. A.; Jaque, D.; Solé, J. G. CdTe Quantum Dots as Nanothermometers: Towards Highly Sensitive Thermal Imaging. Small 2011, 7, 1774−1778.
AUTHOR INFORMATION
Corresponding Authors
*(J. L.) E-mail:
[email protected]. *(R. Z.) E-mail:
[email protected]. ORCID
Yang Sheng: 0000-0002-8985-4197 Mark Bradley: 0000-0001-7893-1575 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the support from the National Natural Science Foundation of China (21374012, 11532003, and 51563003), Jiangsu Province for support under the distinguished professorship program, “Six talent peaks” team E
DOI: 10.1021/acsabm.8b00311 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials (16) Qiao, J.; Chen, C.; Qi, L.; Liu, M.; Dong, P.; Jiang, Q.; Yang, X.; Mu, X.; Mao, L. Intracellular Temperature Sensing by A Ratiometric Fluorescent Polymer Thermometer. J. Mater. Chem. B 2014, 2, 7544−7550. (17) Wang, H.; Ke, F.; Mararenko, A.; Wei, Z.; Banerjee, P.; Zhou, S. Responsive Polymer-Fluorescent Carbon Nanoparticle Hybrid Nanogels for Optical Temperature Sensing, Near-Infrared LightResponsive Drug Release, and Tumor Cell Imaging. Nanoscale 2014, 6, 7443−7452. (18) Ferdinandus; Arai, S.; Takeoka, S.; Ishiwata, S. i.; Suzuki, M.; Sato, H. Facilely Fabricated Luminescent Nanoparticle Thermosensor for Real-Time Microthermography in Living Animals. ACS Sensors 2016, 1, 1222−1227. (19) Kalytchuk, S.; Poláková, K.; Wang, Y.; Froning, J. P.; Cepe, K.; Rogach, A. L.; Zbořil, R. Carbon Dot Nanothermometry: Intracellular Photoluminescence Lifetime Thermal Sensing. ACS Nano 2017, 11, 1432−1442. (20) Gavrilyuk, S.; Polyutov, S.; Jha, P. C.; Rinkevicius, Z.; Ågren, H.; Gel’mukhanov, F. Many-Photon Dynamics of Photobleaching. J. Phys. Chem. A 2007, 111, 11961−11975. (21) Shamirian, A.; Appelbe, O.; Zhang, Q.; Ganesh, B.; Kron, S. J.; Snee, P. T. A Toolkit for Bioimaging Using Near-Infrared AgInS2/ ZnS Quantum Dots. J. Mater. Chem. B 2015, 3, 8188−8196. (22) Bottrill, M.; Green, M. Some Aspects of Quantum Dot Toxicity. Chem. Commun. 2011, 47, 7039−7050. (23) Valizadeh, A.; Mikaeili, H.; Samiei, M.; Farkhani, S. M.; Zarghami, N.; kouhi, M.; Akbarzadeh, A.; Davaran, S. Quantum Dots: Synthesis, Bioapplications, and Toxicity. Nanoscale Res. Lett. 2012, 7, 480−494. (24) Shamsipur, M.; Barati, A.; Karami, S. Long-Wavelength, Multicolor, And White-Light Emitting Carbon-Based Dots: Achievements Made, Challenges Remaining, And Applications. Carbon 2017, 124, 429−472. (25) Chen, Z.; Wu, P.; Cong, R.; Xu, N.; Tan, Y.; Tan, C.; Jiang, Y. Sensitive Conjugated-Polymer-Based Fluorescent ATP Probes and Their Application in Cell Imaging. ACS Appl. Mater. Interfaces 2016, 8, 3567−3574. (26) Wu, C.; Szymanski, C.; Cain, Z.; McNeill, J. Conjugated Polymer Dots for Multiphoton Fluorescence Imaging. J. Am. Chem. Soc. 2007, 129, 12904−12905. (27) Zhan, R.; Tan, A. J. H.; Liu, B. Conjugated Polyelectrolyte as Signal Amplifier for Fluorogenic Probe Based Enzyme Activity Study. Polym. Chem. 2011, 2, 417−421. (28) Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy. Chem. Rev. 2012, 112, 4687−4735. (29) Peng, E.; Choo, E. S. G.; Tan, C. S. H.; Tang, X.; Sheng, Y.; Xue, J. Multifunctional PEGylated Nanoclusters for Biomedical Applications. Nanoscale 2013, 5, 5994−6005. (30) Wang, G.; Duan, Z.; Sheng, Y.; Neumann, K.; Deng, L.; Li, J.; Bradley, M.; Zhang, R. Tuning the Emission Properties of a Fluorescent Polymer Using a Polymer Microarray Approach Identification of an Optothermo Responsive Polymer. Chem. Commun. 2016, 52, 10521−10524. (31) Zhu, A.; Miao, K.; Deng, Y.; Ke, H.; He, H.; Yang, T.; Guo, M.; Li, Y.; Guo, Z.; Wang, Y.; Yang, X.; Zhao, Y.; Chen, H. Dually pH/ Reduction-Responsive Vesicles for Ultrahigh-Contrast Fluorescence Imaging and Thermo-Chemotherapy-Synergized Tumor Ablation. ACS Nano 2015, 9, 7874−7885. (32) Zhao, C.; Yuan, G.; Han, C. C. Stabilization, Aggregation, and Gelation of Microsphere Induced by Thermosensitive Microgel. Macromolecules 2012, 45, 9468−9474. (33) Potai, R.; Traiphol, R. Controlling Chain Organization and Photophysical Properties of Conjugated Polymer Nanoparticles Prepared by Reprecipitation Method: The Effect of Initial Solvent. J. Colloid Interface Sci. 2013, 403, 58−66. (34) Lu, J.; Peng, B.; Li, M.; Lin, M.; Dong, Z. Dispersion Polymerization of Anionic Polyacrylamide in an Aqueous Salt Medium. Pet. Sci. 2010, 7, 410−415.
(35) Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum Dots Versus Organic Dyes as Fluorescent Labels. Nat. Methods 2008, 5, 763−775.
F
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