pH- and Temperature-Sensitive Hydrogel Nanoparticles with Dual

May 27, 2016 - A red-emission rare-earth complex and a blue-emission quaternary ... Manish DebnathRakesh PaulDeepanjan PandaJyotirmayee Dash...
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pH- and Temperature-Sensitive Hydrogel Nanoparticles with Dual Photoluminescence for Bioprobes Yue Zhao,† Ce Shi,§ Xudong Yang,*,‡ Bowen Shen,† Yuanqing Sun,† Yang Chen,† Xiaowei Xu,§ Hongchen Sun,§ Kui Yu,⊥ Bai Yang,† and Quan Lin*,† †

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, People’s Republic of China ‡ School of Chemical Engineering, Changchun University of Technology, Changchun 130012, People’s Republic of China § School of Stomatology, Jilin University, Changchun 130041, People’s Republic of China ⊥ Institute of Atomic and Molecular Physics, Sichuan University, Chengdu 610065, People’s Republic of China S Supporting Information *

ABSTRACT: This study demonstrates high contrast and sensitivity by designing a dual-emissive hydrogel particle system, whose two emissions respond to pH and temperature strongly and independently. It describes the photoluminescence (PL) response of poly(N-isopropylacrylamide) (PNIPAM)-based core/shell hydrogel nanoparticles with dual emission, which is obtained by emulsion polymerization with potassium persulfate, consisting of the thermo- and pH-responsive copolymers of PNIPAM and poly(acrylic acid) (PAA). A red-emission rare-earth complex and a blue-emission quaternary ammonium tetraphenylethylene derivative (d-TPE) with similar excitation wavelengths are inserted into the core and shell of the hydrogel nanoparticles, respectively. The PL intensities of the nanoparticles exhibit a linear temperature response in the range from 10 to 80 °C with a change as large as a factor of 5. In addition, the blue emission from the shell exhibits a linear pH response between pH 6.5 and 7.6 with a resolution of 0.1 unit, while the red emission from the core is pH-independent. These stimuli-responsive PL nanoparticles have potential applications in biology and chemistry, including bio- and chemosensors, biological imaging, cancer diagnosis, and externally activated release of anticancer drugs. KEYWORDS: photoluminescence, hydrogels, nanoparticles, dual emission, pH and temperature responses, bioprobes attracted much interest due to desirable advantages.14−17 Compared to traditional single PL nanoparticles, dual-emission nanoparticles have two fluorescent moieties, one of which is protected from the external environment to provide a stable reference signal, while the other one can be activated only with external stimuli. The dual-emission nanoparticles do not suffer from limitations such as stoichiometric imbalance between fluorescent and room-temperature phosphorescence dyes, which will improve the detectability of different signals and minimize background signals.18,19 Thus, dual-emission nanoparticles display potential applications in biological imaging and detection.

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n recent decades, photoluminescent (PL) nanoparticles have acquired great promise for chemical and biological sensing due to their unique size, properties, and abilities to be functionalized with biological recognition elements.1−8 Fluorescence technology is now becoming one of the key detection methods in chemistry and cell biology due to the advantage of rapidity, sensitivity, and low background noise.9−11 Therefore, the development of a photoluminescent nanoparticles based sensing platform is of great significance. The fluorescence of the nanoparticles mostly possesses a single color, which usually tends to be influenced by the complex bioenvironment and probe concentration. These pharmacokinetic characteristics result in a high background signal and low efficiency of detection.12,13 Thus, it is necessary to construct a two-dimensional fluorescence coordinate system as a remarkable and stable detection method that facilitates discrimination of different signals. Dual-emission hydrogel nanoparticles have © 2016 American Chemical Society

Received: January 30, 2016 Accepted: May 27, 2016 Published: May 27, 2016 5856

DOI: 10.1021/acsnano.6b00770 ACS Nano 2016, 10, 5856−5863

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Scheme 1. Scheme of the synthetic route leading to the temperature- and pH-response with dual emission of Eu-doped PS-coPNIPAM/d-TPE-doped PNIPAM-co-PAA core/shell hydrogel nanoparticles.

nanoparticles and polymer brushes and formation of nanostructures by induced aggregation.48−53 Herein, we report the development of pH- and temperatureresponsive core/shell hydrogel nanoparticles with dual photoluminescence emission. The PL sources are europium organic complexes Eu(III)(TTA)3Phen doped in the PNIPAM-co-PS core and quaternary ammonium tetraphenyl ethylene derivatives (d-TPE) self-assembled on the PNIPAM-co-PAA shell, respectively. We designed the core to be Eu-doped PNIPAMco-PS and the shell to be d-TPE-doped PNIPAM-co-PAA. Thus, our PL hydrogel with dual thermo- and pH-response is denoted as Eu-doped PS-co-PNIAM/d-TPE-doped PNIPAMco-PAA core/shell nanoparticles. The red emission from the hydrophobic core is pH-independent and thus can be used to trace cells, while the blue emission from the hydrophilic shell responds to the pH. Therefore, our dual-emission hydrogel nanoparticles provide highly sensitive detection for cancer cells. These smart hydrogel nanoparticles have practical applications in PL nanogel thermometry and bioimaging and should inspire more exciting research in this emerging field for cancer diagnosis.

The development of a dual-emission nanoparticle based sensing platform is promising. The challenge still remains in developing a method for detecting or tracing cancer cells. The method to address this challenging issue is to achieve signal activation within the acidic microenvironment of cancer cells and construct a dual-emission fluorescent system with only one type of emission responding to a pH change. Especially, compared to the neutral surrounding of normal tissue with pH ∼7.4, the acidic microenvironment (pH < 6.5) is related to a fundamentally important character of various tumors, because it has been associated closely with the malignant phenotype of cancer.20−23 Thus, two kinds of fluorescent molecules in dualemission nanoparticles should show different emission colors under the same excitation light. One of them provides different fluorescence response to pH changes, and the other fluorescent molecule should be stable and nonresponsive to a pH change. Stimuli-responsive polymers change their structure and physical properties in response to changes of surrounding environments, such as temperature, pH, or light, and it is most desirable to have a sensor detecting simultaneously these properties.24−31 Recently, these materials have played an increasingly important part in various ranges of applications, such as diagnostics, drug delivery, and tissue engineering, as well as biosensors and bioseparation systems.24,32−38 In particular, the construction of multifunctional photoluminescent nanocomposites composed of pH- and temperatureresponsive polymers has been a research hot spot in the forefront of materials science.39 Lanthanide organic complexes, such as Eu(DBM)3Phen (DBM = dibenzoylmethide; Phen = 1,10-phenanthroline), with excellent emission and narrow bandwidths, have been widely used as PL probes and labels in optics and bioanalysis.40−43 In 2001, Professor Tang’s group discovered the “aggregationinduced emission” (AIE) system. Tetraphenylethene (TPE) and its derivatives (d-TPEs), as typical AIE molecules, have attracted much attention.44−48 As a family of compounds, they are nonemissive if dissolved in solution, but exhibit high PL in aggregated and in molecular immobilization states (immobilization-induced emission, IIE). The AIE and IIE phenomena are ascribed to the restricted intramolecular rotation (RIR) of the phenyl peripheries against the central double bond in the aggregate state.48 To implement the RIR process, several approaches have been developed, including combination with

RESULTS AND DISCUSSION The dual-emission hydrogel nanoparticles with pH and temperature response, Eu-doped PS-co-PNIPAM/d-TPE -doped PNIPAM-co-PAA, were synthesized according to the illustration in Scheme 1. The europium complex (Eu(TTA)3Phen) was introduced into a surfactant-free emulsion polymerization system to prepare the Eu-doped PS-coPNIPAM core. In addition, the core/shell hydrogel nanoparticles Eu-doped PS-co-PNIPAM/PNIPAM-co-PAA were synthesized through a seed emulsion polymerization. The water-soluble d-TPE molecule has two positively charged sites, which could be tightly bound with two carboxyl groups of AA repeat units via Coulomb attraction. 50,51 The detailed procedure can be found in our previous reports.51 The PL hydrogel nanoparticles exhibit two emission peaks at 613 and 468 nm, respectively, as a result of the incorporated Eu(TTA)3Phen and d-TPE molecules. The PL hydrogel nanoparticles exhibit two emission peaks at 613 and 468 nm from Eu(TTA)3Phen and d-TPE molecules, respectively. Due to the electrostatic interaction between the d-TPE molecules and the PNIPAM-co-PAA shell, the intramolecular motion of 5857

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Figure 1. (a) SEM images of Eu-doped PS-co-PNIPAM cores with narrow size distribution. (b) Eu-doped PS-co-PNIAM/PNIPAM-co-PAA hydrogel core/shell nanoparticles; the core diameter is 245 ± 3 nm, and the core/shell diameter is 278 ± 4 nm. (c) Zeta potential of the Eudoped PS-co-PNIPAM/PNIPAM-co-PAA nanoparticles in different pH buffer solution. (d) DLS measurements of the core and core/shell hydrogel nanoparticles in water, collected during the increase in temperature.

almost uncharged. In contrast, the surface of the nanoparticles is negatively charged at a pH value above the pKa due to the carboxylic acid ionization. During the temperature increase from 10 to 80 °C, the size of the core/shell hydrogel nanoparticles decreases with temperature, while the size of the core does not change (Figure 1d). As well-known, PNIPAM is a thermoresponsive polymer with a relatively low critical solution temperature (LCST) at around 32 °C due to its reversible hydrogen bonds with water.22 The hydrophilic groups on the surfaces of nanoparticles form hydrating layers with water, but the thickness of the hydrating layers decreases with the temperature increase because the hydrogen bonds are broken. When the temperature increases toward the LCST of PNIPAM-co-PAA, the thickness of the hydrating layer significantly decreases in size and the polymer chains of PNIPAM-co-PAA chains shrink considerably.54 The soft-shell PNIPAM-co-PAA with a majority of PNIPAM has been proved to show strong thermoresponsivity. The rigid PS-co-PNIPAM core is composed mostly of PS, and with very little PNIPAM it does not exhibit a thermoresponse; thus the size of the core does not change.55 Besides, the Eu-doped PS-co-PNIPAM/ PNIPAM-co-PAA core/shell hydrogel nanoparticles undergo a volume phase transition at around 34 °C, which is different from that of the homo-PNIPAM. This is because the LCST of PNIPAM has been altered by copolymerization with the low amount of AA.22,56 Fascinatingly, the colloidal and thermal stability of the core and core/shell hydrogel nanoparticles in water are high. In order to distinguish normal cells (pH ∼7.4) from cancer cells (pH ∼6.5), we design polymer hydrogel nanoparticles (PNIPAM-co-PAA) to be pH sensitive. As the ionization degree of Eu-doped PS-co-PNIPAM/PNIPAM-co-PAA nanoparticles increases with the pH values from 4.0 to 7.6, the electrostatic

the d-TPE molecules decreases significantly. Accordingly, immobilization-induced emission can be obtained (Figure S1). It is worth noting that the Eu(TTA)3Phen and d-TPE molecules have similar excitation spectra (Figure S2). Therefore, a single radiation source can excite two fluorescent molecules to obtain dual emission. Thus, we constructed a functional polymer platform of dual-emission hydrogel nanoparticles with a pH- and temperature-sensitive response. The SEM images of cores and core/shell nanoparticles are displayed in Figure 1a and b. Both the prepared cores and the core/shell nanoparticles exhibit good monodispersity. The diameter of the nanoparticle core is 245 ± 3 nm, and that of the core/shell nanoparticle is 278 ± 4 nm. The shell of the nanoparticles, PNIPAM-co-PAA providing pH- and temperature-responsive functions, also represents a polymer hydrogel with aqueous solubility and biocompatibility. Particularly, poly(acrylic acid) (PAA) was designed to activate negative charges, which could bind the fluorescent d-TPE molecules with two positive charges via Coulomb attraction. Dynamic light scattering (DLS) was used to examine the zeta potential at different pH values and the average hydrodynamic diameter of particles at different temperatures. As shown in Figure 1c, after the effective copolymerization of the PAA on the shell, it is obviously negatively charged. The zeta potential of Eu-doped PS-co-PNIPAM/PNIPAM-co-PAA nanoparticles increases with pH values from 4.0 to 7.6. The pH variations below or above its pKa lead to either protonation or deprotonation of the polymer chains of PNIPAM-co-PAA, which is accompanied by dramatic changes of the zeta potential of nanoparticles. The pKa of PNIPAM-co-PAA chains is about 5.5. The ionization degree of PNIPAM-co-PAA increases with pH. At a pH value below the pKa of the PNIPAM-co-PAA chains, most carboxylic acid groups are protonated and the surface of the nanoparticles is 5858

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in Figure 2b. In contrast, the hydrophilic and active shell of PNIPAM-co-PAA exhibits pH-responsive functions. The interaction between cooperative fluorescent molecules (dTPE) and the shell polymers changes, resulting in varying PL properties. The PL intensity at 468 nm increases gradually when the pH increases from pH ∼4 to 7.6 with a resolution of 0.1 unit (Figure 2c). Obviously, the dual-emission provides a sensitive fluorescence response following small environmental pH changes. With two fluorescent molecules separately localized in the core/shell, the nanoparticles are able to sensitively respond to temperature variations. Figure 3a shows the PL spectra of the smart dual-emission nanoparticles in aqueous solution upon increasing the temperature. The emission bands at 468 and 613 nm can be assigned to the d-TPE molecules and Eu(TTA)3Phen, respectively. The PL intensity of both emission types decreases linearly with the temperature increasing from

interactions between the PNIPAM-co-PAA shell and the positively charged d-TPE molecules strengthen gradually. At high pH values, the d-TPE molecules in the immobilized state are encompassed by the contracted PNIPAM-co-PAA shells, which results in a gradual increase of the PL emission intensity at 468 nm. Oppositely, at low pH values, the electrostatic interaction between d-TPE molecules and the PNIPAM-coPAA shell is weakened, which leads to the d-TPE molecules in the free state dissociated from the PNIPAM-co-PAA carboxylic groups. Thus, the PL emission intensity around 468 nm for the PNIPAM-co-PAA shell decreases with decreasing pH. As a result, the PL emission of the Eu-doped PS-co-PNIPAM/dTPE-doped PNIPAM-co-PAA core/shell hydrogel nanoparticles shows a strong pH dependence (Figure 2a). The hydrophobic and rigid PS prevents water from penetrating into the core and protects the emission of fluorescent molecules (Eu(TTA)3Phen) from being affected by the outside pH environment. The PL intensity at 613 nm is stable and negligible changes in the range of pH from ∼4 to 7.6, as shown

Figure 3. (a) Normalized PL spectra of Eu-doped PS-co-PNIPAM/ d-TPE-doped PNIPAM-co-PAA hydrogel nanoparticles in water, collected during the indicated increases in temperature (λex = 360 nm). (b) Apparent linear relationship of the PL intensity at 468 nm (left axis) and 613 nm (right axis) versus temperature. (c) Intensity ratio of two emission wavelengths (I613/I468) versus temperature; the concentration of the nanoparticles was ∼0.32 wt %.

Figure 2. (a) Normalized PL spectra of Eu-doped PS-co-PNIPAM/ d-TPE-doped PNIPAM-co-PAA hydrogel nanoparticles in buffer solution with different pH values (λex = 360 nm). (b) Plot of the PL intensity at 468 nm (left axis) and 613 nm (right axis) versus pH. (c) Intensity ratio of two emission intensities (I468/I613) versus pH values; the concentration of the nanoparticles was ∼0.32 wt %. 5859

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Figure 4. (a) Fluorescence color and PL spectra of Eu-doped PS-co-PNIPAM/d-TPE-doped PNIPAM-co-PAA hydrogel nanoparticle aqueous solutions at pH’s of ∼5.0, 6.0, 7.0, and 7.4, respectively. Confocal fluorescence images of MC3T3-E1 (b) and CAL-27 (c) cells incubated with nanoparticles for 24 h. Confocal fluorescence images of dissected normal tissue (d) and tumor tissue (e) of tumor-bearing mice sacrificed at 2 days postinjection.

10 to 80 °C, but the blue emission band decreases by a factor of 2 the red one by a factor of 5 (Figure 3b). Therefore, the ratio also depends on temperature (Figure 3c). This is understood as the PNIPAM-co-PAA shell shrinking with a temperature increase, which results in inhibition of intramolecular rotations and vibrations of d-TPE molecules. On the other hand, the dTPE molecules in the immobilized state are encompassed by the contracted PNIPAM-co-PAA shells at low temperatures. At the same time, the PL intensity of Eu(TTA)3Phen in the core is also quenched, the rare earth complex loses more energy from thermal vibrations upon increasing the temperature, and the absorbed energy transmitted to Eu3+ decreases as a result. These results support that the two kinds of emission intensity vary linearly with temperature,; however their variations are different. The dual emission also leads to different PL colors upon small environmental pH changes. We thus tried to build a fluorescence color map. For example, based on the pH value change from 7.4 to 5.0, the color of the nanoparticles changes from purple to pink (Figure 4a). This labeling technique producing clearly different fluorescence colors comparing tumor/normal tissue is critical to help surgeons accurately distinguish tumor tissue from normal tissue, which enables improved precision in surgical operations for removing tumors in the clinic. The as-prepared Eu-doped PS-co-PNIPAM/dTPE-doped PNIPAM-co-PAA hydrogel nanoparticles show good biocompatibility and low cytotoxicity. The methylthiazolyldiphenyltetrazolium (MTT) assay was adapted to investigate the cytotoxicity of nanoparticles by studying the metabolic viability of MC3T3-E1 normal cells (Figure S3a) and CAL-27 cancer cells (Figure S3b) after incubation with PL nanoparticles at various concentrations. The cell viability remains above 80% within 72 h under the experimental conditions, indicating the low cytotoxicity of Eu-doped PS-co-PNIPAM/d-TPE-doped PNIPAM-co-PAA nanoparticles, which will benefit both in vitro and in vivo PL imaging. The application of Eu-doped PS-coPNIPAM/d-TPE-doped PNIPAM-co-PAA hydrogel nanoparticles for in vitro cellular imaging was studied by confocal laser scanning microscopy with MC3T3-E1 normal cells in a neutral environment and CAL-27 cancer cells in an acidic environment.

The confocal images of the two different cells after incubation with PL nanoparticles are shown in Figure 4b and c, respectively. The MC3T3-E1 cells show a purple emission color, while the CAL-27 cells show a pink coloration due to the different pH environments of the two types of cells. The dualemission system can improve the contrast and the signal-tonoise ratio. Eu-doped PS-co-PNIPAM/d-TPE-doped PNIPAMco-PAA nanoparticles exhibit a potent functional system for detection and tracing methods to distinguish normal cells from cancer cells. To image the tumoral acidic pH microenvironment, nanoparticles with pH sensitivities were administered by local injection into CAL-27 tumor-bearing mice. The mice treated with Eu-doped PS-co-PNIPAM/d-TPE-doped PNIPAM-coPAA nanoparticles show an intense PL signal throughout the tumor tissue after 24 h, while in the control experiment the nonbearing mice do not show the PL signal (Figure S4). As the region of tumor tissue exhibits a red color, the low-pH microenvironment of the tumor tissue can be imaged and discriminated from the healthy tissue. This is because in the tumoral acidic microenvironment the d-TPE molecules are almost dissociated from the PNIPAM-co-PAA carboxylic groups, presenting the free state, which loses the IIE effect. The PL is primarily provided by the Eu(TTA)3Phen of the cores. Finally, we sliced the healthy tissue and tumor tissue, and the confocal images also showed the same results as the cell detection. Tumor tissue and normal tissue exhibit purple and pink coloration, respectively (Figure 4d and e). The Eu -doped PS-co-PNIPAM/d-TPE-doped PNIPAM-co-PAA nanoparticles exhibit excellent PL imaging in vivo of the tumoral acidic environment. It shows a most promising use in cancer diagnosis and follow-up therapy.

CONCLUSIONS In summary, biocompatible PL polymeric hydrogel Eu-doped PS-co-PNIPAM/d-TPE-doped PNIPAM-co-PAA nanoparticles presented here exhibit dual-emission responses to both temperature and pH in aqueous solution. The two emissions possess strong sensitivity, decreasing linearly with increasing temperature from 10 to 80 °C. The nanoparticles exhibit also a 5860

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PNIPAM-co-PAA hydrogel nanoparticles were obtained and purified three times by centrifugation. The purified nanoparticles were dispersed in water (≈25 mL), as a stock solution. Cellular Imaging. MC3T3-EI and CAL-27 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 °C using a humidified 5% CO2 incubator, respectively. Suspensions (20 μg/mL) of Eu-doped PS-co-PNIPAM/dTPE-doped PNIPAM-co-PAA nanoparticles from the stock solution were prepared with serum-containing DMEM. MC3T3-E1 and CAL27 cells were seeded in 96-well plates with a content of 1.5 × 105 cells/ well for uptake experiments. The growth medium was replaced by the Eu-doped PS-co-PNIPAM/d-TPE-doped PNIPAM-co-PAA nanoparticle suspension 24 h after seeding, and incubation was for another 24 h at 37 °C. The excess Eu-doped PS-co-PNIPAM/d-TPE-doped PNIPAM-co-PAA nanoparticles were removed by washing three times with warm phosphate-buffered saline (PBS). Animal Experiments. Animal experiments were approved by Jilin University Animal Care and Use Committee. Nine male Balb/c nude mice (CAnN.Cg-Foxnlnu/Crl) (4−6 weeks old) from Shanghai SLAC Laboratory Animal Co. (China) were used to create tumors by injecting CAL-27 cells (5.0 × 106 cells per mouse in 100 μL of HDMEM) subcutaneously into the right oxter. After the diameters of the tumors approached about 5 mm, Eu-doped PS-co-PNIPAM/dTPE-doped PNIPAM-co-PAA nanoparticles (2 mg/mL, 20 μL) were injected into both sides of the oxter, including the tumor and the normal epithelial tissue, using an insulin syringe (28 gauge; BD Biosciences). Twenty-four hours after the injection, mice were anesthetized with ketamine (60 mg/kg) and xylazine (8 mg/kg) intramuscularly for optical imaging. After imaging, the tumors and the normal epithelial tissues were cut, fixed in 10% formalin, and embedded in paraffin. Formalin-fixed paraffin-embedded tissues were cut into slices of about 3 μm thick, and LSCM was performed. Characterization. Photoluminescence experiments were performed with a Shimadzu RF-5301 PC spectrofluorimeter. SEM images were obtained on a JEOL JSM-6700F scanning electron microscope operated at an acceleration voltage of 3 kV. The size and zeta potential measurements were carried out on a Zetasizer-nanozs. The confocal microscopy images were taken with an Olympus Fluoview FV1000.

sensitive PL response to pH from 4 to 7.6 and show color changes with small pH variations. The PL nanoparticles could be endocytosed by cells, enabling imaging in living cells and tissue. Moreover, they exhibit different emission colors upon excitation by a single wavelength to distinguish cancer from normal cells. To further improve cell labeling techniques, some dyes with near-infrared fluorescent properties can be used in this system with high potential for tracing cancer cells in vivo. This promises applications in cancer diagnosis and real-time monitoring of therapy.

EXPERIMENTAL SECTION Synthesis of the Eu Complex [Eu(TTA)3Phen]. TTA (thenoyltrifluoroacetone) (0.006 mol) and Phen (1,10-phenanthroline) (0.002 mol) were dissolved in ethanol (50 mL). The aqueous solution was kept at pH ∼7.0−7.5 by dropwise addition of a NaOH solution (1 M) and stirred for 30 min under a N2 atmosphere. Then EuCl3 (0.002 mol) dissolved in ethanol (40 mL) was added slowly into the stock solution. The reaction was allowed to proceed for about 20 h (at 55 °C under N2 with stirring). The products were filtered and washed with ethanol three times. After the solvent was evaporated, the crude products were dried in a 40 °C vacuum oven overnight. Finally, highly pure Eu(III)(TTA)3Phen was obtained by three recrystallizations in chloroform.55 Preparation of Eu-Doped PS-co-PNIPAM. The Eu-doped PS-coPNIPAM nanoparticles were synthesized by surfactant-free emulsion polymerization.54,57 NIPAM (≈0.5 g, 4.4 mmol) was dissolved in deionized water (≈35 mL) in a 500 mL three-necked round-bottom flask equipped with a stirrer, a condenser, and a nitrogen inlet. Afterward, styrene (≈5.0 mL, 50.0 mmol), Eu(TTA)3Phen (≈0.01 g, 9.8 × 10−3 mmol), and deionized water (≈150 mL) were added to the reaction flask. The mixture was stirred under a N2 atmosphere at room temperature for 30 min to remove oxygen, and then the temperature was increased to 70 °C. An initiator solution of KPS (K2S2O8) (≈0.1 g) in water (≈10 mL) was swiftly injected into the reaction mixture. Originating from thermal decomposition of KPS, the sulfate radicals initiated the polymerization reaction of water-soluble NIPAM monomers first. The second step was chain propagation until NIPAM chains reached the critical length. Then, some colloidally unstable NIPAM chains agglomerated to form stable particles by interwinding. Styrene monomers entered into PNIPAM micelles to polymerize.54 Europium organic complexes dissolved in styrene are brought into the core.55 Without any other surfactant, the reaction was clean and allowed to proceed for 10 h (at 70 °C under N2 with stirring). The prepared Eu-doped PS-co-PNIPAM nanoparticles were purified three times by centrifugation and dispersion in deionized water. Preparation of the Eu-Doped PS-co-PNIPAM/PNIPAM-coPAA Core/Shell Nanoparticles. The PNIPAM-co-PAA shells were grafted on the purified PS-co-PNIPAM nanoparticles by seed emulsion polymerization.58 The purified Eu-doped PS-co-PNIPAM nanoparticles were dispersed in deionized water (≈185 mL) in a 500 mL three-necked round-bottom flask. NIPAM (≈4.5 g, 39 mmol) and acrylic acid (≈0.2 mL, 2.8 mmol) were added to the reaction flask. Similarly, the mixture was stirred at room temperature for ∼30 min to remove oxygen, and the temperature was increased to 70 °C. An initiator solution of KPS (≈0.2 g) in water (≈15 mL) was swiftly injected into the reaction mixture. The reaction was allowed to proceed for 10 h (at 70 °C under N2 with stirring). Again, the resulting Eu-doped PS-co-PNIPAM/PNIPAM-co-PAA hydrogel nanoparticles were purified three times by centrifugation and dispersion in deionized water (≈200 mL). Preparation of the Eu-Doped PS-co-PNIPAM/d-TPE-Doped PNIPAM-co-PAA Hydrogel Nanoparticles. Water-soluble fluorescent molecules of quaternary ammonium tetraphenyl ethylene derivatives (d-TPE) (≈3.75 mg ≈ 5.2 μmol) were mixed with the Eu-doped PS-co-PNIPAM nanoparticles (≈10 mL) in a 100 mL round-bottom flask.51 The reaction mixture was stirred at 35 °C under N2 for 3 h. Finally, Eu-doped PS-co-PNIPAM/d-TPE-doped

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b00770. PL intensity of d-TPE-doped nanospheres and d-TPE aqueous solution; PL excitation spectra of the Eu(TTA)3Phen and d-TPE; viability of MC3T3-E1 and CAL-27 cells after incubation with different concentrations of nanoparticles; confocal fluorescence images of MC3T3-E1 and CAL-27 cells incubated with nanoparticles; fluorescence images of tumor-bearing mouse and control mouse (PDF)

AUTHOR INFORMATION Corresponding Authors

*Tel (X. Yang): +86-431-85716328. Fax: +86 431-8571-6328. E-mail: [email protected]. *Tel (Q. Lin): +86 431-8516-8483. Fax: +86 431-8519-3423. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was financially supported by the National Nature Science Foundation of China (Grant Nos. 21174048, 5861

DOI: 10.1021/acsnano.6b00770 ACS Nano 2016, 10, 5856−5863

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51373061, and 21304090). We would like to thank Prof. Benzhong Tang and Prof. Jingzhi Sun from Zhejiang University, China, for kindly providing d-TPE molecules. We also thank Prof. Helmuth Möhwald from the Max Planck Institute of Colloids and Interfaces, Germany, and Prof. Myongsoo Lee from Jilin University, P.R. China, for useful discussions and helpful suggestions.

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DOI: 10.1021/acsnano.6b00770 ACS Nano 2016, 10, 5856−5863