AIE-Active Fluorescent Nonconjugated Polymer Dots for Dual

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Bioengineering

The AIE-Active Fluorescent Non-conjugated Polymer Dots for Dual-Alternating-Color Live Cell Imaging Xiaolin Guan, Baocui Lu, Qijun Jin, Zhifei Li, Lin Wang, Kailong Wang, Shoujun Lai, and Ziqiang Lei Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b03776 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Industrial & Engineering Chemistry Research

The AIE-Active Fluorescent Non-conjugated Polymer Dots for Dual-Alternating-Color Live Cell Imaging Xiaolin Guan,*, † Baocui Lu, † Qijun Jin, † Zhifei Li, † Lin Wang, † Kailong Wang, † Shoujun Lai, ‡ and Ziqiang Lei*, † † Key

Laboratory of Eco-Environment-Related Polymer Materials Ministry of Education, Key Laboratory of Polymer

Materials Ministry of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou, Gansu 730070, P.R. China. ‡ School

of Chemical Engineering, Lanzhou University of Arts and Science, Lanzhou, Gansu

730070, P.R. China. KEYWORD: dual-emission; AIE; Eu(III)-complex; Pdots; dual-color cell imaging

ABSTRACT: Fine-tuning emission fluorescent bioprobes are highly desirable but still scarce for dual-alternatingcolor live cell imaging. Herein, a facile strategy has been reported to develop a dual-color nanoprobe for targeted cancer cell imaging. A new kind of nonconjugated polymer dots (Pdots) are prepared by the solution selfassembly of an amphiphilic 4-arm star polymer TPEtetraPNIPAM-Eu(III) in aqueous media. The as-prepared Pdots show two well-separated emission peaks with a wavelength difference of 195 nm and possess fluorescent-thermo responsive property with a LCST at 37 °C. Furthermore, the efficient cellular uptake and low cytotoxicity of Pdots can be favourable for its applications in live cell imaging. The investigation of cellular imaging indicates that the photoswitchable dual-emission could be easily realized in targeted Hela cell with a fluorescent label of Pdots by merely turning the excitation wavelength. Therefore, this research may be of great help to open up a new and feasible path of developing AIE nonconjugated Pdots biomaterials for multi-color cell imaging applications. 1 ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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■ INTRODUCTION Fluorescence cell imaging as an efficient and versatile investigation approach in the field of biology has attracted increased attentions in the past few decades.1-3 Various organic dyes, fluorescent proteins and semiconductor quantum dots quantum dots (QDs) have been commercially available for cell imaging. As a member of cell imaging reagents, the dual-color fluorescent imaging reagents, which can image cell with two different colors, can distinguish sites of interest from false positive signals generated by adventitious fluorescent biomolecules in monitoring biological processes.4-6 So, the development and application of dual-color cell imaging reagents are advantageous for biological research. Fluorescent imaging requires brighter probes. QDs are famous fluorescent nanoparticles and have received immense popularity as fluorescent imaging probe in biotechnological applications due to its superior

brightness and photostability.

7-9

Unfortunately, the high

cytotoxicity of QDs remains a challenge for further applications in bioimaging due to the existence of heavy metal ions. 10,11 The green fluorescent protein (GFP) had been attempted to use as dual-colour imaging reagents in vivo.

12

But, the lack of susceptibility to proteolytic

enzymes and poor photostability of GFP have limited its broad application in bioimaging.13,14 Compared with QDs and GFP, organic fluorescent dyes show advantages of tunable optical properties, easily functional modification and higher fluorescence quantum efficiency for biological imaging.

15

However, the defects of low absorptivity, high biotoxicity and poor

photostability of conventional dyes have affected the application and development of dyes as cell imaging probe.

16

Therefore, development of novel fluorescent probes is of great importance for

the application of high-sensitivity imaging techniques. In recent years, polymer dots (Pdots) have gained interest as a novel fluorescent nanoprobe in bioimaging and biosensing because of their excellent biocompatibility, tunable optical 2 ACS Paragon Plus Environment

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properties, extraordinary fluorescence brightness and photostability. Pdots had been demonstrated in cell image applications.

21-23

17-20

Numerous functional

For example, Chiu et al reported a

simple method to build biomolecule-Pdots system for labeling cellular targets.

24

However, the

fluorescence weakening or quenching of Pdots due to the aggregation-caused quenching (ACQ) effect in the aggregation state limits its applications for bioimaging. Fortunately, Tang et al reported a novel fluorescence molecule with aggregation-induced emission (AIE) properties in 2001, which is exactly the opposite characteristic to ACQ. 25 The discovery of AIE molecules have intrinsically solved the ACQ problem and AIEbased materials exhibit exceptional potential to replace conventional fluorescent materials due to their high aggregation-state quantum efficiencies.

26-30

The aggregation-induced emission polymer dots (AIE Pdots) with different

surface functionalities show advanced features over quantum dots and small molecule dyes. So, AIE Pdots is an kind of ideal materials for biological imaging. For example, Xu et al reported an AIE Pdots by self-assembly of AIE-conjugated block copolymer for targeting HeLa cells. 31 And Chen et al constructed AIE Pdots from an AIEbased amphiphilic copolymer bearing photochromic spiropyran dye. The cell-permeable Pdots could be used as an excellent intracellular dual-color imaging probe with a rapid photo-responsiveness.

32

But, up to now,

reports about dual-color fluorescent AIE Pdots as live cell imaging probe are still small. Therefore, the development of novel dual-alternating-color fluorescent imaging reagents based on AIEbased Pdots is a really challenging task for biological applications. Recently, two kinds of novel tetraphenylethylene (TPE)-based radical initiators: TPE-BMP and TPE-AZO were synthesized in our previous work.

33, 34

After the initiation of vinyl

polymerization by TPE-BMP and TPE-AZO, all kinds of TPE-based polymers with specific properties were successfully prepared via a simple method. The temperature-sensitive TPEPNIPAM showed the fluorescence increasing effect on aggregation state, which was beneficial 3 ACS Paragon Plus Environment


for image applications in live cell.

35

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Moreover, it is well known that PNIPAM can coordinate

with Eu(III) due to the carbonyl group in polymer chains. The PNIPAM-Eu(III) complex has both characteristic red fluorescence of Eu(III) and temperature-sensitive property of PNIPAM. 35, 36

Therefore, there is an ideal method for preparing dual-color fluorescent Pdots imaging reagent

through coordination between Eu(III) and TPE-PNIPAM. Herein, we reported a facile method to fabricate a novel amphiphilic PNIPAM-derived nonconjugated AIE Pdots with Eu(III)-complex (TPE-tetraPNIPAM-Eu(III)) in aqueous media for targeted dual-color cancer cell imaging. The TPE-tetraPNIPAM-Eu(III) Pdots exhibits a finetuning dual emission fluorescence property, one from the hydrophobic TPE moieties in the inner core segmentation, and the other from the hydrophilic PNIPAM-Eu(III) complex in the shell. The combination of AIEbased Pdots and PNIPAM-Eu(III) complex offers the advantages of biocompatibility, thermosensitivity, photostability and dual-alternating-color fluorescent ability, which are important for multi-color monitoring cellular events. Therefore, this study opens up the door to new class of highly fluorescent, photostable, and low-toxic AIE nonconjugated Pdots biomaterial for multi-color cell imaging applications.

■ EXPERIMENTAL SECTION Materials.

4,4’-Dihydroxybenzophenone(BHBP,

99%),

Zn

powders(99%),

titanium

tetrachloride (TiCl4, 99%), N,N-dicyclohexylcarbodiimide (DCC, 98%), 4-(dimethylamino) pyridine-p-toluenesulfonate (DPTS), 4,4-azobis(4-cyanovaleric acid) (ACVA, 98%), NIPAM monomer (98%, stabilized by MEHQ), and Europium Oxide (Eu2O3, 99%) were obtained from J&K Scientific. Europium chloride (EuCl3) was prepared according to the reported method by M. D. Taylor et al.

37

N-isopropylacrylamide (NIPAM) monomer was processed purification by

acetone dissolution and hexane precipitation. 1, 1, 2, 2-tetrakis(4-hydroxyphenyl)-ethylene (TPE4 ACS Paragon Plus Environment

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OH) was synthesized by the procedure reported by Kato et al.

38

Ethanol (EtOH, AR) and

tetrahydrofuran (THF, AR) were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. and purified by distillation. Other chemicals and solvents were obtained from Beijing Chemical Works. Doubly distilled water was used throughout the experiments. Characterization. 1H NMR and 15N NMR spectroscopic measurements were performed on the Brucker AM 600 MHz spectrometer using d6-DMSO as the solvent. MS measurements were carried out with a Hewlett-Packard 5989A mass spectrometer. FTIR spectra were recorded with a Nicolet AVATAR 360FTIR spectrometer. The number-average molecular weight (Mn) and PDI were determined by GPCV2000 gel-permeation chromatograph using polystyrene as the calibration standard and tetrahydrofuran as eluent. X-ray photoelectron spectroscopy (XPS) were carried out with Thermo-Fishier Scientific EscaLab 250Xi XPS. Morphology measurement were carried out on a JEM-1200EX analytical high-resolution transmission electron microscope (HRTEM) with a 200 kV accelerating voltage. Energy Dispersive Spectrometer (EDS) measurements were carried out on a Scanning Electron Microscope (SEM) ULTRA Plus spectrometer. Fluorescence spectra of the samples were recorded by a F97 Pro spectrometer. Synthesis of tetraphenylethene-based hyperbranched-azo-initiator (TPE-azoinitiator). TPE-azo-initiator was synthesized according to our published procedure 34, which was shown in Scheme 1. An anhydrous THF solution (50 mL) of 4,4-azobis(4-cyanovaleric acid) (1.40 g, 5.0 mmol), TPE-OH (0.42 g, 1.1 mmol) and DPTS (0.13 g, 0.6 mmol) were stirred for 0.5 h at room temperature under an N2 atmosphere. DCC (1.23 g, 6.0 mmol) in anhydrous 20.0 mL THF was then slowly dropped into the mixed solution. The reaction mixture was stirred at ambient temperature. After 24 h, the reaction mixture was filtered, and the filtrate was concentrated. The crude product mixture was purified by column chromatography (ethyl acetate:ether = 1:3, v/v), affording 0.3 g of TPE-azo-initiator product. The molecular structure of 5 ACS Paragon Plus Environment


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TPE-azo-initiator could be confirmed by 1H NMR and IR spectra (Figure S1 and Figure S3 of Supporting Information). Synthesis

of

star-shaped

polymer

(TPE-tetraPNIPAM).

TPE-tetraPNIPAM

was

synthesized by conventional free radical polymerization of NIPAM initiated by TPE-azo-initiator (Scheme 1). NIPAM (5.0 g) and TPE-azo-initiator (0.05 g) were mixed in 10 mL of dry THF in a glass tube equipped with a magnetic stir bar. After the mixture was degassed by performing three freeze-pump-thaw cycles and then sealed under vacuum, the glass tube was immersed in an oil bath and the polymerization was carried out at 75 ºC for 24 h. After polymerization, the mixed solution was precipitated using 100 mL ether. A white flocculent precipitate formed immediately. The precipitate were separated by centrifugation and the product was dissolved in deionized water again. The redispersion-precipitation procedure was repeated thrice for removing unreacted residual monomers. The target product TPE-tetraPNIPAM was obtained as a white solid in 66% in yield by filtration and dried under vacuum at 30 °C for 24 h. 1H NMR (600 MHz, DMSO-d6, δ): 7.08-7.40 (s, -NH-C=O), 6.75-6.80 (d, aromatic backbone), 6.45-6.50 (d, aromatic backbone), 3.55-3.61 (s, -CH-NH-), 2.10-1.80 (m,-CH3-CH-CH2-), 1.81-1.72 (d, -CH3-CH-CH2-), 1.18-.098 (m, -CH3) (Figure S4 of Supporting Information). GPC: Mn = 4.2×103, Mw = 6.1×103, PDI = 1.45. (Table S1 of Supporting Information). Synthesis of Eu(III) complexes (TPE-tetraPNIPAM-Eu(III)). Ethanol solutions of EuCl3 and TPE-tetraPNIPAM (WEu3+: W

TPE-tetraPNIPAM

= 1 : 2) were added into a flask and then were

stirred with a magnetic stirring bar at room temperature for 20 h. The mixed solution was then precipitated in hexane and then dried under vacuum at 25 °C for 48 h, yielding the TPEtetraPNIPAM-Eu(III) complexes. Cytotoxicity Assay by MTT Method. The MTT assay was used to study the cytotoxicity of TPE-tetraPNIPAM-Eu(III) Pdots against living HeLa, A549 and HepG2 cells. All of the three 6 ACS Paragon Plus Environment

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cells were cultured in 96-well plates (1.0×105 cells per well) for 24 h of incubation to allow cells to attach. Then, a series of Pdots with different concentrations (25, 50, 100, 200, 300, 400 μg/mL) were cultured for 48 h. After the medium containing Pdots were pouring out, 10 μL of freshly MTT solution (5 mg/mL in PBS) was added to the cells in each well and cultured for another 4 h. Subsequently, the MTT medium solution was removed and the cells were lysed by adding 100 μL of DMSO. Then, the cell viability values (%) were estimated using the absorbance at 492 nm of each plate obtained using an RT-6100 microplate reader. Cellular Imaging Experiments. HeLa, A549 and HepG2 cells were seeded in confocal dishes in the solution containing 10 % (v/v) fetal bovine serum and 1% streptomycin at 37 °C for 24 h. Then, these cells were incubated with the TPE-tetraPNIPAM-Eu(III)(100 μg/mL) in PBS solution for 30 min. After removing the medium, three cells were washed once using PBS (pH 7.4). The cellular imaging of three cells were observed under an Olympus FV1000 confocal microscope. The fluorescent excitation wavelengths were fixed at 360 nm and 395 nm while the fluorescent emission wavelengths were recorded at 420 nm and 615 nm, respectively.

■ RESULTS AND DISCUSSION Design, Synthesis, and Characterization of TPE-tetraPNIPAM-Eu(III) Pdots The AIE nonconjugated Pdots was formed from amphiphilic polymer TPE-tetraPNIPAMEu(III) by self-assembling as illustrated in Scheme 1. Firstly, the TPE-tetraPNIPAM was synthesized through a polymerization process of TPE-AZO as the initiator. The detailed procedure can be found elsewhere but with modification. 34 It is well known that PNIPAM is easy to coordinate with rare earth ions owing to the amide group in the polymer side chain. Therefore, the complex of TPE-tetraPNIPAM-Eu(III) may possess excellent temperature responsive and fluorescence properties after the coordination between PNIPAM and Eu(III). 7 ACS Paragon Plus Environment


O

O O

O

O

O

HO

O

O

CN

OH

N

CN

N

O

NC

+

O

CN

O

N

HO

HO

O

CN

O

NIPAM

O

NC

OH

CN

N N

TPE-OH

N

O

O

N

DPTS, DCC

OH N

O

ACVC

O

O O

O NC

NC

N N

CN O

O

O

O

O

O

O

O

O

TPE-azo-initiator

n

HN

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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NH O

CN

NC O

O

O

O

O

self-assembling

Eu (III) coordination O O NC

O

O CN

n

n N H

O N H

O

TPE-tetraPNIPAM

Pdots

TPE-tetraPNIPAM-Eu(III) O

NC

O

n O

Eu(III)

N

Scheme 1. The schematic illustration of the synthesis of TPE-tetraPNIPAM-Eu(III) complex and the conformation of Pdots by self-assembly procedure

FT-IR spectra can confirm the complex formation of TPE-tetraPNIPAM with Eu(III). The FT-IR spectra of TPE-tetraPNIPAM (Figure S5 of Supporting Information) exhibited obviously the amino(-NH-) and carbonyl(C=O) peaks at 3300 cm-1 and 1650 cm-1 respectively, the 1,4substituted benzene peaks of TPE structure in fingerprint range of 700-600 cm-1, and the benzene carbon-ring frame appeared at 1580 cm-1, 1450 cm-1, accordingly. Compared with the spectrum 8 ACS Paragon Plus Environment

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of TPE-tetraPNIPAM, the carbonyl absorbance peaks of the complex TPE-tetraPNIPAM-Eu(III) moved to short wavenumber direction due to the coordination between Eu(III) and oxygen and nitrogen atom of amide group on the PNIPAM chains. Meanwhile, the amino peaks at 3300 cm-1 became wider at the presence of Eu(III). These results indicated that metal Eu(III) is coordinated by nitrogen and oxygen atoms in the polymer chain of TPE-tetraPNIPAM to form the TPEtetraPNIPAM-Eu(III) complex.

C1s

18000

TPE-tetraPNIPAM TPE-tetraPNIPAM-Eu(III)

35000

16000

30000


14000 Intensity

25000 20000 15000 10000

12000 10000 8000

5000 0

N1s

Intensity

40000

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6000 282

285

288 291 B.E.(eV)

294

297

390

395

400

405 410 B.E.(eV)

415

420

24000 22000 20000 18000 16000 14000 12000 10000 8000 6000

O1s

525


530

535 540 B.E. (eV)

545

Figure 1. The XPS spectra of of C1s, N1s, and O1s of TPE-tetraPNIPAM(black) and TPE-tetraPNIPAMEu(III)(red)

Table 1. The binding energies of C(1s), O(1s), N(1s) for TPE-tetraPNIPAM and TPE-tetraPNIPAM-Eu(III)

Condition

C(1s)/eV

N(1s)/eV

O(1s)/eV

TPE-tetraPNIPAM

284.6

399.5

531.2

TPE-tetraPNIPAM-Eu(III)

284.6

400.3

532.4

X-ray photoelectron spectroscopy(XPS) spectra has been used to provide precise information on the electronic structure and binding energies of molecules. 39 To further confirm the structure of the complex, XPS had been carried out to measure the binding energy change of 9 ACS Paragon Plus Environment

550


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coordinate bond of oxygen and nitrogen atoms in TPE-tetraPNIPAM and TPE-tetraPNIPAMEu(III). The XPS spectra of C1s, N1s, and O1s were recorded in Figure S6 and Figure 1. The electronic binding energy of C1s, N1s and O1s were listed in Table 1. The N1s binding energy for C-N bonds and O1s for C-O bonds are about 399.5 eV and 531.2 eV in the TPE-tetraPNIPAM. The two binding energies are mainly contributed from C-O bonds and C-N bonds, respectively. And, the C1s binding energy is 284.6 eV as a standard value. 35 It is obviously observed that the binding energy values of O(1s) and N(1s) in TPE-tetraPNIPAM-Eu(III) increased by 1.2 eV and 0.8eV due to the introduction of Eu(III) in Table 1. The results showed that Eu(III) was coordinated by oxygen and nitrogen atoms of amide group, which caused the decrease in electron density of the O and N atoms as well as the increasing for Eu(III). 35

TPE-tetraPNIPAM-Eu(Ш)

TPE-tetraPNIPAM


Figure 2. The EDS analysis of TPE-tetraPNIPAM and TPE-tetraPNIPAM-Eu(III)

Table 2. The weigh percentage of the corresponding elements in each substance

C

N

O

Eu

TPE-tetraPNIPAM

44.8%

25.8 %

19.0 %

/


42.3%

17.5 %

9.9 %

30.1 %

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To further indicate the Eu(III) complexation process, EDS comparison of TPEtetraPNIPAM and TPE-tetraPNIPAM-Eu(III) Pdots had been performed in Figure 2. The percentage of the corresponding elements were listed in Table 2. Obviously, Eu element appeared in TPE-tetraPNIPAM-Eu(III) Pdots after the Eu(III) was coordinates by oxygen and nitrogen atoms of amide group compared with TPE-tetraPNIPAM. Moreover, the existence of Eu(III) in TPE-tetraPNIPAM-Eu(III) reduced the weigh percentages of C, N and O elements in TPE-tetraPNIPAM. The changes of element analyses also illustrated the successful preparation of rare-earth complex TPE-tetraPNIPAM-Eu(III). Meanwhile, the distribution of elements of TPEtetraPNIPAM-Eu(III) was also studied with EDX elemental mappings, the bright points indicated the high concentration of the elements (Figure S7 of Supporting Information). The elemental mappings showed that C, N, O, and Eu element were homogeneously distributed throughout sample, suggesting that Eu(III) was successfully modified on TPE-tetraPNIPAM.

a

b

Figure 3. a) TEM image of the prepared TPE-tetraPNIPAM-Eu(III) Pdots. b) The particle size distribution histograms.

Preparation and microstructure of Pdots



AIE Pdots could be easily prepared when H2O was slowly added into a THF solution of polymers by taking advantage of the amphiphilic properties of TPE-tetraPNIPAM-Eu(III). The amphiphilic 4-arm star polymer TPE-tetraPNIPAM-Eu(III) formed Pdots consisted of hydrophobic AIEgens TPE core and hydrophilic PNIPAM shell as shown in Scheme 1. Compared with a weak physical adsorption, PNIPAM chains could be permanently anchored on the AIEgens surface by the method. PNIPAM chains created a biocompatible superhydrophilic nanostructured layer. Meanwhile, the functional amide group could easily coordinate with Eu(III) through oxygen and nitrogen atoms. Therefore, the hydrophobic fluorophore TPE could be safely applicated in a biocompatible environment due to the advantage of the Pdots. TPE-tetraPNIPAMEu(III) Pdots was a relatively uniform diameter around 80 nm and exhibited regular sphere morphology with narrow particle size distribution, as shown in Figure 3. Moreover, it is important to ensure Pdots possess excellent long-term stability. The TEM image of Pdots and its particle size distribution histograms at first day and tenth day were shown in Figure S8 in the Supporting Information. After being placed at room temperature for 10 days, Pdots can still exhibit regular sphere morphology with diameter around 100 nm and good dispersity. So, TPE-tetraPNIPAM-Eu(III) Pdots possessed good long-term stability.


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160 140

200

a Intensity

100 80 60 40

120 80 40

20 0

b

160

120

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


250 300 350 400 450 500 550 600 650

0

Wavelength (nm)

350 400 450 500 550 600 650

d

c

Wavelength (nm)

Figure 4. a) The fluorescence excitation under emission at 420 nm (left) and emission spectra under excitation at 360 nm (right) of TPE-tetraPNIPAM-Eu(III). (Inset: photograph of of aqueous solution of TPEtetraPNIPAM-Eu(III) under 365 nm ultraviolet irradiation). b) The fluorescence excitation under emission at 615 nm (left) and emission spectra under excitation at 395 nm (right) of TPE-tetraPNIPAM-Eu(III). (Inset: photographs of aqueous solution of TPE-tetraPNIPAM-Eu(III) under 395 nm ultraviolet irradiation). c) The normalized fluorescence intensity excited with corresponding excitation (top) and the FL image of TPEPNIPAM-Eu(III) excited at 325 nm, 360 nm, 370 nm, 390 nm, 395 nm from left to right, respectively (bottom). d) The CIE coordinate system marked by excitation points.

Dual-emission fluorescence properties of Pdots The photoluminescence (PL) of the TPE-tetraPNIPAM-Eu(III) Pdots in water was investigated in detail. The excitation and emission curves in Figure 4a and Figure 4b displayed two emission bands centered at about 420 and 615 nm obtained under optimum excitation at 360 13 ACS Paragon Plus Environment


and 395 nm, respectively. It indicated that the Pdots possessed unique dual-emitting property. Due to the covalent link between the TPE molecules and PNIPAM chains, the intramolecular motion of the TPE molecules decreases significantly. Accordingly, the immobilization-induced emission at 420 nm could be obtained (Figure 4a). Besides, the strong narrow-band luminescence at 615 nm could be interpreted as the characteristic emission of PNIPAM-Eu(III) complex outside the Pdots.

35

In fact, the Pdots showed two characteristic red emission peaks at 590 and

615 nm (Figure 4b), corresponding to the electronic transitions of 5D0→7F1 and 5D0→7F2 of Eu(III) ions.

40

Comparison between the two peaks, the most prominent peak at 615 nm

(5D0→7F2) is responsible for the red color emitted by TPE-tetraPNIPAM-Eu(III) Pdots. It is wellknown that the spectroscopic overlap between the emission of the donor and the absorption of the acceptor together is the prerequisite to achieve efficient FRET.

41

A very low degree of spectral

overlap between the emission of TPE and absorption of PNIPAM-Eu(III) indicated a low FRET efficiency from TPE to PNIPAM-Eu(III). Therefore, dual emission at 420 nm and 615 can be simultaneously observed. It is noteworthy that the two emission peaks were well separated by 195 nm, which is beneficial for high ratiometric detection and imaging analysis. Moreover, it is important to ensure materials possess excellent fluorescence stability in solution. As can be seen in Figure S8 in the Supporting Information, there is a weak fluorescence change in solution for ten days, which indicated TPE-tetraPNIPAM-Eu(III) possessed good photostability. Remarkably, the intensities of the two emission bands of TPE-tetraPNIPAM-Eu(III) Pdots could be tuned when monitored at different excitation wavelengths from 325 to 395 nm. The change of fluorescence intensity with different excitation was shown in Figure 4c. The blue highenergy emission of Pdots was much stronger than the red lower-energy emission band when excited by 325 nm. However, the relative intensity of the red emission band was gradually enhanced with the increase of the excitation wavelength, and TPE-tetraPNIPAM-Eu(III) Pdots 14 ACS Paragon Plus Environment

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showed a major red light when excited by 395 nm. Meanwhile, these changes of emission lights could be observed by our naked eyes (Figure 4c). Excitation-dependent color tunability from blue to red of Pdots could be probably assigned to the different adsorption bands of the TPEtetraPNIPAM-Eu(III) Pdots. As reflected in the chromaticity diagram (CIE) in Figure 4d, the emission color changed gradually from blue to red.

600 550 500 450 400 350 300 250 200 150 100 50 0

a

400

5 mg/mL 10 mg/mL 15 mg/mL 20 mg/mL 25 mg/mL 30 mg/mL 35 mg/mL 40 mg/mL 45 mg/mL 50 mg/mL

450

500

550

600

700 Fluorescence Intensity

Fluorescence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


650

wavelength /nm

b

600

5

50

Blue Emission Red Emission

500 400 300 200 100 5

10 15 20 25 30 35 40 45 50 Concentration /(mg/mL)

Figure 5. a) Emission spectra of fluorescent TPE-tetraPNIPAM-Eu(III) with different concentrations (λex = 360 nm). b) The Plot of emission intensity against concentrations of TPE-tetraPNIPAM-Eu(III) at 420 nm (blue emission) and 615 nm (red emission), respectively. (Inset: photographs of TPE-tetraPNIPAM-Eu(III) in 5 mg mL-1 and 50 mg mL-1 concentrations under 365 nm UV light irradiation, respectively).



a

CH2Cl2 fraction (vol%) 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

800 600 400 200 0

1000

400

450

500

550

Fluorescence Intensity


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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b

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Blue Emission Red Emission 0%

90%

800 600 400 200 0

Wavelength /nm

10%

30% 50% 70% CH2Cl2 fraction (vol%)

90%

Figure 6. a) Emission spectra of TPE-tetraPNIPAM-Eu(III) in CH2Cl2/ethanol mixed solvents (50 mg/mL) with different CH2Cl2 fractions (λex = 360 nm). b) The Plot of emission intensity against CH2Cl2 fraction at 420 nm (blue emission) and 615 nm (red emission) in CH2Cl2/ethanol mixed solvents, respectively. (Inset: photographs of TPE-tetraPNIPAM-Eu(III) in CH2Cl2/ethanol mixed solvents with 0% and 90% CH2Cl2 fractions under 365 nm UV light irradiation, respectively).

AIE behavior of Pdots An effective method to prove the AIE behavior was to obtain increased fluorescence intensity upon increasing the concentration of the TPE-tetraPNIPAM-Eu(III) Pdots. TPEtetraPNIPAM-Eu(III) showed good solubility in aqueous solution and its fluorescence spectra at different concentration were shown in Figure 5a. At low concentrations of 5 mg/mL, two feeble fluorescence emission signals at 420 nm and 615 nm were observed when the dilute solution of TPE-tetraPNIPAM-Eu(III) was excited at 360 nm. Meanwhile, both the two fluorescence intensity of TPE-tetraPNIPAM-Eu(III) increased as the concentration increased from 5 to 50 mg/mL, and the emission of TPE-tetraPNIPAM-Eu(III) had been lighted up in high concentration (as shown in Figure 5b). Compared with the fine nanoparticle dispersions of TPEtetraPNIPAM-Eu(III) Pdots solution in low concentration (Figure 3a), large aggregates could 16 ACS Paragon Plus Environment

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been observed in high concentration (Figure S10 of Supporting Information), which proves the aggregates of the Pdots are formed in solution at high concentration. Moreover, the photoluminescence of the TPE-tetraPNIPAM-Eu(III) in solid state was also investigated. A strong emission band at about 420 nm and weak emission band at 615 nm could be observed (Figure S11 of Supporting Information). While, a strong bright blue light under UV light could be easily observed by the naked eye, indicating that TPE-tetraPNIPAM-Eu(III) is induced to emit by aggregation. Based on the above results, we concluded that TPEtetraPNIPAM-Eu(III) Pdots possessed typical AIE property, which originated from the real collapse and aggregate of PNIPAM chains at high concentrations. To further examine whether TPE-tetraPNIPAM-Eu(III) was an AIE-active material, another experiment was arranged to investigated its fluorescence property using CH2Cl2 and ethanol solvent mixtures. As shown in Figure 6a, TPE-tetraPNIPAM-Eu(III) was weak emissive intensity in pure ethanol. Meanwhile, no visible luminescence was observed in photographs of Pdots under 365 nm UV light irradiation in pure ethanol. However, the fluorescence intensities of Pdots obviously increased after adding CH2Cl2 into ethanol solution. With the increase of CH2Cl2 content in ethanol/CH2Cl2 mixtures from 0 to 90%,

a gradual and sharp enhancement of

fluorescence intensity was observed in Figure 6b. The blue fluorescence intensity at 420 nm with 90% CH2Cl2 fraction was more 5-fold higher than that in the pure ethanol. This experimental phenomenon could been explained by a theory of aggregation-induced enhanced emission (AIEE). 42 Furthermore, a bright blue visible luminescence at 420 nm at 90 vol % CH2Cl2 content could be observed under 365 nm UV light irradiation (Figure 6b). However, the fluorescence intensity at 615 nm had no obvious change with the adding of dichloromethane. So, the above results also identified the high AIE property with emission maxima at 420 nm of TPEtetraPNIPAM-Eu(III) Pdots. 17 ACS Paragon Plus Environment


b Fluorescence Intensity

80 60 40

o

LCST ~37 C (body temperature)

20 0

o

T/ C

0 5 10 15 20 25 27.5 30 32.5 35 37.5 40 45 50 55

500 400 300 200

Blue Emission Red Emission


a 100 Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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440 330 220 110 0

0

10

20

30

40

50

o

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100

27 30 33 36 39 42 45 48 Temperature / oC

0

400 450 500 550 600 650 700 750 Wavelength /nm

Figure 7. a) Plot of transmittance of TPE-tetraPNIPAM-Eu(III) in aqueous solution (50 mg/mL) versus temperature from 25 to 50 °C. ( Inset: photos of aqueous solution of TPE-tetraPNIPAM-Eu(III) at 25 °C (left) and 40 °C (right) under visible light and UV light irradiation). b) Fluorescence spectra of aqueous solution of TPE-tetraPNIPAM-Eu(III) (50 mg/mL) at different temperatures from 0 to 55 °C excited at 360 nm. (Inset: Plot of fluorescence intensity at 420 nm and 615 nm changes of TPE-tetraPNIPAM-Eu(III) against temperatures from 25 to 50 °C., respectively.

Temperature-sensitive properties of Pdots PNIPAM is a well-known thermoresponsive polymer with a relatively low volume phase transition temperature at around 32 °C based on reversible hydrogen bonds in water. At room temperature, the linear PNIPAM is dissolved in water to form a homogeneous solution. However, the solution becomes a phase separated state when the temperature rises from 30 to 35 oC. The different phase state made significant distinguish absorbance in UV spectra, which can help confirm the temperature-dependent transmittance of aqueous solution. The plot of transmittance of Pdots versus temperature from 25 to 50 °C was showed in Figure 7a. It was exhibited that the transmittance of the aqueous solution reduced dramatically in a narrow interval of 36-38 oC, and the LCST of TPE-tetraPNIPAM-Eu(III) was around 37 oC. So, the Pdots has enormous potential 18 ACS Paragon Plus Environment

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in the application as stimuli-responsive materials in human body because the LCST was close to normal body temperature.

Furthermore, there was a simple method to confirmed the

temperature-sensitive property of TPE-tetraPNIPAM-Eu(III) by increasing the temperature of aqueous solution of TPE-tetraPNIPAM-Eu(III) as shown in the inset in Figure 7a and Figure S12 (Supporting Information). The transparent aqueous solution of Pdots at 25 °C became cloudy after being heated to 40 °C and further changed into transparent after cooling to 25°C. The reversible phase transition keeps after several cycles of the change of external temperature 25°C /40 °C. The result confirmed the reversible and thermosensitive soluble-to-insoluble phase transition of TPE-tetraPNIPAM-Eu(III) at LCST. Compared with the LCST (~34 °C) of similar TPE-PNIPAM reported in our previous work, 34

TPE-tetraPNIPAM-Eu(III) in this work shows the higher LCST of 37 °C. The reason for the

high LCST may be due to the existence of Eu(III) ion. PNIPAM undergoes a transition from hydrated coils to dehydrated granules in aqueous solution, which is caused by different types of H-bonding below and above the LCST. After metal Eu(III) is coordinated by parts of nitrogen and oxygen atoms in the PNIPAM chain, a higher temperature is needed for the H-bonding dissociation with water molecules and the formation of strong interchain H-bonding between C=O and N-H groups. Besides, the fluorescence thermal sensitivity character of Pdots was also studied. Figure 7b shows the fluorescence spectra of Pdots in water temperatures at different temperatures (from 0 to 55 °C). Obviously, changes in sensitive behaviors showed opposite trends in two temperature ranges of 0 to 37.5 and 37.5 to 55 °C, respectively. With increasing temperature from 0 to 37.5 °C, the emission intensity of Pdots at 420 nm gradually decreased. But on the contrary, the emission intensity increased from 37.5 to 55 °C. It was interesting that the phase transfer of PNIPAM could cause the fluorescent response of TPE-tetraPNIPAM-Eu(III). When the 19 ACS Paragon Plus Environment


temperature was below the LCST, TPE molecules in TPE-tetraPNIPAM-Eu(III) existed in an aggregated state because of the coil conformation of the polymer chain and the hydrate formed deriving from amide-water H-bonding. With the temperature increasing, there would be much more polymer chains in the stretched state because of the chain conformational transformations. So, the decrease of fluorescence intensity with increasing temperature from 0 to 37.5 °C was due to the low degree of aggregation of TPE within the TPE-tetraPNIPAM-Eu(III) polymer network. Furthermore, the fluorescence intensity began to increase due to the aggregation of TPE molecules at the LCST of 37 °C at the same time. 43 All of the results were found to agree with the works in previous reports.

44,45

However, the change of red emission intensity at 615 nm,

which is assigned to the emission of PNIPAM-Eu(III) complex, was not obvious with increasing temperature from 0 to 55 °C. It indicated that the red emission of Pdots is stable in a wide range of temperature.


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Figure 8. Cytotoxic effects of TPE-tetraPNIPAM-Eu(III) Pdots against Hela, A549 and HepG2 cells upon 48 h of incubation by MTT assay. Control: cells in the absence of the Pdots.

Photoswitchable dual-color live-cell imaging of Pdots TPE-tetraPNIPAM-Eu(III) Pdots emitted visible blue light at 360 nm excitation and red light at 395 nm excitation. Therefore, the dual-emission fluorescence property enable Pdots to alternate blue- and red-channel in live-cell images. Herein, we explored the ability of optically switchable dual-color cell imaging of the Pdots in live human normal liver cells. HeLa cells were chosen for this analysis as a model based on their ease of harvesting. In biomedical applications, cytotoxicity test of materials is essential. The cytotoxicity of Pdots was evaluated by the MTT-cell culture assay in HeLa cells. The results in Figure 8 clearly indicated that more than 90% of the cells were healthy after all of the three cells were treated with TPE-tetraPNIPAM-Eu(III) for 48 hours with different concentrations from 25 μg/mL to 400 μg/mL. So, the Pdots was very safe to be applicated in living cells.



Figure 9. Confocal laser scanning microscopy (CLSM) images of three cells (Hela, A549 and HepG2 ) stained with TPE-tetraPNIPAM-Eu(III) (100 μg/mL) for 24 h. The excitation laser wavelength for TPE-tetraPNIPAMEu(III) are 360 nm (blue channel) and 395 nm (red channel), respectively.

Besides, the photoswitchable capabilities of Pdos in three cells (Hela, A549 and HepG2 ) were investigated by using confocal laser scanning microscopy (CLSM). The CLSM images were shown in Figure 9. Strong fluorescence signals could be simultaneously observed in both blue and red channels in cells after all of the three cells were treated with TPE-tetraPNIPAM-Eu(III) Pdots for 24 h with the concentration of 100 μg/mL, which indicated the ability of Pdots to maintained its dual-emission characteristic. The regions with weak fluorescence intensity in three cells were probably the location of the nuclei, which indicated that polymer successfully escaped the endosomal compartment with intact aggregation and selectively stained the cytoplasmic area of cells. Under 360 nm light irradiation, the blue fluorescence observed from cells can be interpreted as the emission of AIEgens TPE dye inside the Pdots. After tuning the excitation light from 360 to 395 nm, the light red fluorescence from the characteristic emission of PNIPAMEu(III) complex outside the Pdots could be observed in cells, and the blue fluorescence in cells had disappeared as exhibited in Figure 9. Hence, the fine-tuning dual-emission could be quickly realized in a complex biological environment with the fluorescent label of TPEtetraPNIPAM-Eu(III) Pdos by only turning the excitation wavelength. The results indicated the potential application of TPE-tetraPNIPAM-Eu(III) Pdos as a kind of smart fluorescent labels in biological systems.

■ CONCLUSION


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We had designed and successfully developed a novel biocompatible dual-emission fluorescent Pdots through the assembly of a core hydrophobic AIEgens TPE with the shell hydrophilic PNIPAM-Eu(III) complex for targeted dual-modal cancer cell imaging. TPEtetraPNIPAM-Eu(III) can be synthesized by the free radical polymerization of monomer NIPAM with TPE-AZO as initiator. Meanwhile, the Pdots was prepared in aqueous media with regular sphere morphology around 80 nm and a good monodispersity. Such structure design provided a good vantage point to combine the AIE property with luminescence characteristic of responsive polymer-Eu(III) complex. The Pdots exhibited excellent dual-emission with a well-separated wavelength of 195 nm. The blue higher-energy emission at 420 nm should be interpreted as the emission of AIEgens TPE dye as the core of Pdots, and the red lower-energy emission at 615 nm was assigned to the characteristic emission of PNIPAM-Eu(III) complex as the shell of Pdots. Furthermore, fluorescence intensity of Pdots in aqueous solution increased rapidly when temperature was above LCST (~37 °C), which was close to human body temperature. In addition, the results of cellular imaging experiment showed that the fine-tuning dual-color imaging of Pdots in living cell could be easily realized by merely turning the excitation wavelength. Our results in this work developed practical applications of dual-emission Pdots in tracking and labeling components of complex biological systems.

■ ASSOCIATED CONTENT * Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 1H

NMR spectra, 15N NMR and FT-IR spectra of TPE-azo-initiator. 1H NMR spectra, FT-IR

spectra, XPS spectra and GPC data of TPE-tetraPNIPAM. FT-IR spectra, XPS spectra and EDX elemental mappings of TPE-tetraPNIPAM-Eu(III).



■ AUTHOR INFORMATION Corresponding Authors *E-mail:

[email protected]

*E-mail:

[email protected]

■ ACKNOWLEDGMENTS We acknowledge Prof. Dr. Qiaosheng Pu (School of chemistry and chemical engineering, Lanzhou university) for assistance with cell culture. The authors are grateful to the National Natural Science (51363019, 21761032).

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AIE-Active Fluorescent Nonconjugated Polymer Dots for Dual

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