The Positively Charged Hyperbranched Polymers with Tunable

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The Positively Charged Hyperbranched Polymers with Tunable Fluorescence and the Cell Imaging Application Hengchang Ma, Yanfang Qin, Zenming Yang, Manyi Yang, Yucheng Ma, Pei Yin, Yuan Yang, Tao Wang, Ziqiang Lei, and Xiaoqiang Yao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05073 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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ACS Applied Materials & Interfaces

The Positively Charged Hyperbranched Polymers with Tunable Fluorescence and the Cell Imaging Application Hengchang Ma,* Yanfang Qin, Zengming Yang, Manyi Yang, Yucheng Ma, Pei Yin, Yuan Yang, Tao Wang, Ziqiang Lei and Xiaoqiang Yao ABSTRACT: Fluorescence-tunable materials are becoming increasingly attractive for their potential application in optics, electronics, and biomedical technology. Herein, a multi-color molecular pixel system is realized using simple copolymerization method. Bleeding both of complementary colors from blue and yellow fluorescence segments, reproduced a serious multicolor fluorescence materials. Interestingly, the emission colors of the polymers can be fine-tuned in solid state, solution phase, and in hydrogel state. More importantly, the positive fluorescent polymers exhibited cell-membrane permeable ability, and were found to accumulate on the cell nucleus, exhibiting remarkable selectivity to give bright fluorescence. The DNA/RNA selectivity experiments in vitro and in vivo verified that [tris(4-(pyridin-4-yl)phenyl)amine]-[1,8-dibromooctane] (TPPA-DBO) has prominent selectivity to DNA over RNA inside cells.

KEYWORDS: aggregation-induced emission (AIE), hyperbranched polymers, tunable fluorescence, cell Imaging, DNA sensing

1. INTRODUCTION Recent research progress in fluorescent materials has demonstrated that the fluorescence matters with bio-application is an very interesting but also an great challenging topic.1-4 The great demand for fluorescent materials requires that the fluorescent materials possess a high fluorescence efficiency, tunable emissions, long lifetime, low synthesis cost and mass production feasibility.5-9 Generally, fluorescent materials can be divided into two categories: inorganic and organic. The inorganic materials mainly include quantum dots (e.g., CdS, InP, ZnS)10-13 and lanthanide (Ln)-doped nanoparticles.14 It was well known, most of them are toxic to humans to some extents.15,16 Meanwhile,

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the low fluorescence efficiency is always inevitable due to quenching at a high concentration.17,18 Organic fluorescent materials are consisted of organic segments in solution or as aggregates that is of good fluorescent properties.19,20 However, the conventional organic fluorescent molecules undergo fluorescence quenching due to the aggregation caused quenching (ACQ), which restricts their applications in many cases.21,22 In 2001, Tang's group have observed a unique photophysical process termed aggregation-induced emission (AIE)23,24 which is opposite to the common ACQ effect. Among of AIE fluorogens, triphenylamine (TPA)25-27 and tetraphenylethene (TPE)28-30 are two archetypical AIE fluorogens. In our previous works, we synthesized a serious AIE-active materials, all of them demonstrated excellent sensing performances, especially in the cell imaging applications.31-33 For the representative instance, we explored the hyperbranched fluorescent material of tris(4-(pyridin-4-yl)phenyl)amine][1,8-dibromooctane] (named as TPPA-DBO),32 which is synthesized simply by nucleophilic reactions. And also, the multiple cations charged TPPA-DBO demonstrated nucleolus-specificity imaging behavior. We also found that furnishing the AIE-active luminogens with positive charges composes a special class of AIE materials.34 The net charges could help the aromatic AIE luminogens to acquire hydrophilicity or dissolve in aqueous media, and on the other hand, are able to the formation of electrostatic complex with oppositely charged species in the aqueous media.35,36 Following this research step, we look forward to fluorescent material with colorful emissions in solid, solution, and gel states. And their emission performances are able to be tuned through chemical modifications rather than physical mixture.37,38 And thus, the following research works have been extended. Herein, apart from the orange color emissive tris(4-(pyridin-4-yl)phenyl)amine (TPPA), we synthesized the blue emissive tris(4-(1H-imidazol-1-yl)phenyl)amine (TIPA). Then, a set of positively hyperbranched polymers were prepared by chemical structures modifications with different TPPA, TIPA, and 1,8-dibromooctane

(DBO)

ratios.

(Fig.

1,

named

as

[tris(4-(pyridin-4-yl)phenyl)amine]-

[1,8-dibromooctane]tris(4-(1H-imidazol-1-yl)phenyl)amine ([TPPA-DBO]TIPA), [tris(4-(pyridin-4-yl)phenyl)amine]1[1,8-dibromooctane]-[tris(4-(1H-imidazol-1-yl)phenyl)amine]1

([TPPA1-DBO-TIPA1]),

[tris(4-(pyridin-4-yl)phenyl)amine]1-[1,8-dibromooctane]-[tris(4-(1H-imidazol-1-yl)phenyl)amine]2([T PPA1-DBO-TIPA2]),

[tris(4-(pyridin-4-yl)phenyl)amine]1-

[1,8-dibromooctane]-[tris(4-(1H-imidazol-1-yl)phenyl)amine]5

([TPPA1-DBO-TIPA5]),

and

[tris(4-(1H-imidazol-1-yl)phenyl)amine]-[1,8-dibromooctane]tris(4-(pyridin-4-yl)phenyl)amine ([TIPA-DBO]TPPA). Afterwards, it was demonstrated that the positive charges on the materials are helpful for the probe molecules to be internalized by living cells thus they are good candidates for cell imaging agents.39,40 Then, in order to further improve these materials' applicability, the electrostatic complexes formed between the hyperbranched fluorescent materials with sodium polyacrylates (PAAS),41 which carries multi-negative charges and also is a water soluble polymer. The resulted materials exhibited more colorful emissions and hydrogel formation ability. For the first time, we present a feasible and simple way to control the emission colors. In addition, benefiting from the appealing AIE feature that prevents effectively quenching in the solid-state, which is able to facilitate the cell staining with high image resolution.


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Fig. 1 Schematic illustration of the hyperbranched cationic polymers and the corresponding photographic images under UV light illumination at 365 nm (top) and day light (bottom).

2. MATERIALS AND METHODS 2.1 Materials. Tris(4-iodophenyl)amine (98%), tris(4-bromophenyl)amine (98%), pyridine-4-boronic acid (99%), tetrakis(triphenylphosphine)palladium(0) (Pb(PPh3)4 98%), potassium carbonate (K2CO3, 99%), imidazole (98%), CuSO4 (99%), Cu2O (98%), 1,8-dibromooctane (DBO, 99%) were purchased from Aladdin Co. Sodium polyacrylates (Mw>106) was purchased from Tianjin Guangfu Fine Chemical Research Institute and the average Mw is millions. Nitrogen with a purity of 99.99% was provided from commercial source. Other reagents, such as N, N-dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), methanol, ethanol, tetrahydrofuran (THF) and chloroform were of analytical grade and was purchased from Energy Chemical Company.1H NMR (600 MHz),

13

C NMR (150 MHz)

spectra was recorded on MERCURY spectrometer at 25 °C, and all NMR spectra were referenced to the solvent. UV-visible absorption spectra (UV) were recorded on a TU-1901 spectrometer. Fluorescence spectra and fluorescence quantum yields were measured using a Fluoro Sens 9003 Fluorescence Spectrophotometer. Time-resolved fluorescence (TRF) measurements were carried out using a C11367-11 Quantaurus-Tau system. Fluorescent microscope images of HepG-2 cells were taken on DM5000B (Leica, Germany), Co-staining fluorescent microscope images of HepG-2 cells were taken on IX71 (Leica, Germany). All the samples were prepared according to the standard methods. Fourier transform infrared (FT-IR) spectra were recorded on a DIGIL FTS3000 spectrophotometer using KBr tablets. The xerogel was produced by solvent evaporation by LGJ-10E multi-manifold freeze-drying machine. 2.2 Synthesis and characterization of tris(4-(pyridin-4-yl)phenyl)amine (TPPA).32 TPPA was synthesized according the literature. A dried 250 mL round-bottom flask was charged with Tris(4-iodophenyl)amine (3 mmol, 623 g/mol, 1.869 g), pyridine-4-boronic acid (9.5 mmol, 215 g/mol, 2.042 g), potassium carbonate (9 mmol, 138 g/mol, 1.242 g) and tetrakis(triphenylphosphine)palladium (0) (0.3 mmol, 1155 g/mol, 5% mol, 0.346 g). Then the mixture of THF (40 mL) and MeOH (40 mL)



were added. And the reaction was refluxed at 120 °C for 48 h under N2 atmosphere. The reaction mixture was concentrated by rotary evaporation. Then, the crude product purified by column chromatography on silica gel (300-400 mesh) with a mixture of petroleum ether and ethyl acetate as eluent (1:1 by volume) to give a yellow solid (1.014 g, 71% yield). (Scheme S1, Figure S1, Figure S10, Figure S17, ESI†) 1H NMR (600 MHz, CDCl3) δ (TMS, ppm):14.01-13.05 (m, 2H), 8.65 (s, 6H), 7.61 (d, J = 8.6 Hz, 6H), 7.51 (d, J = 4.2 Hz, 6H), 7.27 (d, J = 8.5 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ (TMS, ppm):150.17 (s), 147.85 (s), 147.44 (s), 132.86 (s), 128.07 (s), 124.69 (s), 121.04 (s). IR (KBr): 3423, 1719, 1645, 1584, 1490, 1288, 1166, 1038, 823, 666 cm-1. 2.3 Synthesis and characterization of TPPA-DBO.32 TPPA-DBO was synthesized according the literature. TPPA (1 mmol, 476 g/mol, 0.476 g), 1,8-dibromooctane (2 mmol, 270 g/mol, 0.54 g) and DMF (50 mL), were introduced into a clean round-bottom flask with a magnetic stirrer. Subsequently, the mixture was stirred at 100 °C for 48 h. During this period a white solid precipitated. After cooling the reaction mixture to ambient temperature, the white solid was isolated by filtration, washed with diethylether, and dried in vacuo to give an orange solid (0.749 g, 85% yield). (Scheme S1, Figure S3, Figure S10, Figure S17, ESI†) 1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm): 8.92 (s, 1H), 8.62 (s, 1H), 8.39 (s, 1H), 7.95 (d, J = 157.9 Hz, 1H), 7.39 (s, 1H), 4.60 (s, 1H), 4.15 (s, 1H), 3.00 (s, 1H), 2.88 (s, 1H), 2.71 (s, 1H), 2.06 (s, 1H), 1.44 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ (TMS, ppm):161.72, 148.61, 144.18, 129.6, 128.25, 125.67, 123.7, 63.52, 60.32, 30.85, 28.19, 25.21. IR (KBr): 3437, 1591, 1479, 1402, 1328, 1270, 1018, 802, 748, 668, 560 cm-1. 2.4 Synthesis and characterization of tris(4-(1H-imidazol-1-yl)phenyl)amine (TIPA).42 TIPA was synthesized according the literature. Tris(4-iodophenyl)amine (4.2 mmol, 623 g/mol, 2.616 g), imidazole (1.261 g, 18.163 mmol), potassium carbonate (9 mmol, 138 g/mol, 1.242 g) and tetrakis(triphenylphosphine)palladium (0) (0.3 mmol, 1155 g/mol, 5% mol, 0.346 g). Then the mixture of THF (40 mL) and MeOH (40 mL) were added. And the reaction was refluxed at 120 °C for 48 h under N2 atmosphere. The reaction mixture was concentrated by rotary evaporation. Then, the crude product purified by column chromatography on silica gel (300-400 mesh) with a mixture of petroleum ether and ethyl acetate as eluent (1:1 by volume) to give a white solid (1.612 g, 87%). (Scheme S2, Figure S2, Figure S11, Figure S18, ESI†) 1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm):8.21 (s, 1H), 7.71 (s, 1H), 7.61 (s, 2H), 7.20 (s, 2H), 7.11 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ (TMS, ppm): 145.75 (s), 135.96 (s), 132.77 (s), 130.18 (s), 125.25 (s), 122.31 (s), 118.57 (s). IR (KBr): 3086, 1503, 1268, 1105, 1046, 958, 897, 823, 735, 661, 527 cm-1. 2.5 Synthesis and characterization of TIPA-DBO. TIPA (1 mmol, 445 g/mol, 0.445 g), 1,8-dibromooctane (2 mmol, 270 g/mol, 0.54 g) and DMF (50 mL) respectively, using procedures similar to those for TPPA-DBO. White solid, yield 85%. (Scheme S2, Figure S5, Figure S11, Figure S18, ESI†)1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm): 8.34 (s, 1H), 7.71 (s, 1H), 7.28 (s, 1H), 4.26 (s, 1H), 3.64 (s, 1H), 1.93 (d, J = 7.3 Hz, 1H), 1.38 (s, 2H).

13

C NMR (150 MHz, DMSO-d6) δ (TMS,

ppm): 168.59, 155.83, 135.69, 132.75, 126.02, 123.59, 122.22, 121.56, 97.81, 65.31, 57.48, 33.73, 31.49, 29.70, 28.36, 26.13, 9.12. IR (KBr): 3429, 3032, 2931, 1517, 1304, 1187, 1052, 951, 809, 722, 620 cm-1.


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2.6 Synthesis and characterization of [TPPA-DBO]TIPA. TPPA-DBO (0.300 g), TIPA (0.100 g) and DMF (50 mL), were introduced into a clean round-bottom flask with a magnetic stirrer. Subsequently, the mixture was stirred at 100 °C for 48 h. During this period a white solid precipitated. After cooling the reaction mixture to ambient temperature, the orange solid was isolated by filtration, washed with diethylether, and dried in vacuo to give was an orange solid (0.34 g, 85% yield). (Scheme S1, Figure S4, Figure S12, Figure S19, ESI†) 1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm): 9.04 (s, 1H), 8.46 (s, 1H), 8.11 (s, 1H), 7.62 (s, 1H), 7.17 (s, 1H), 4.54 (s, 1H), 3.29 (s, 1H), 1.91 (s, 1H), 1.25 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ (TMS, ppm): 150.76, 145.07, 125.25, 122.65, 61.58, 32.50, 28.96, 25.34, 20.10. IR (KBr): 3403, 2917, 1583, 1492, 1289, 1154, 826, 723, 532 cm-1. 2.7 Synthesis and characterization of [TIPA-DBO]TPPA. TIPA-DBO (0.300g), TPPA (0.100 g) and DMF (50 mL), respectively, using procedures similar to those for [TPPA-DBO]TIPA. Brilliant yellow solid, yield 87%. (Scheme S2, Figure S6, Figure S13, Figure S20, ESI†) 1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm): 8.59 (s, 1H), 8.30 (s, 1H), 7.72 (d, J = 78.4 Hz, 1H), 7.20 (s, 1H), 4.24 (s, 1H), 1.86 (s, 1H), 1.31 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ (TMS, ppm): 150.49, 147.80, 128.57, 124.36, 122.28, 120.81, 109.52, 49.30,28.61. IR (KBr): 3424, 2931, 1591, 1496, 1261, 1052, 802, 722, 607 cm-1. 2.8 Synthesis and characterization of [TPPA1-DBO-TIPA1]. TPPA (0.4mmol, 476 g/mol, 0.188 g), TIPA (0.4 mmol, 445 g/mol, 0.176 g), 1,8-dibromooctane (1.2 mmol, 270 g/mol, 0.324 g) and DMF (50 mL) respectively, using similar procedures as described above. Orange solid, yield 83%.(Figure S7, Figure S14, Figure S21, ESI†) 1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm): 9.89 (s, 1H), 9.06 (s, 1H), 8.29 (d, J = 223.3 Hz, 1H), 7.69 (s, 1H), 7.29 (s, 1H), 4.39 (d, J = 176.4 Hz, 1H), 1.87 (s, 1H), 1.32 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ (TMS, ppm): 157.18, 152.25, 150.41, 135.57, 124.11, 122.21, 120.94, 116.18, 97.35, 94.85, 53.75, 36.07, 22.18. IR (KBr): 3410, 3032, 2911, 1638, 1584, 1496, 1274, 1160, 1059, 823, 729, 540 cm-1. 2.9 Synthesis and characterization of [TPPA1-DBO-TIPA2]. TPPA (0.25 mmol, 476 g/mol, 0.119 g), TIPA (0.5 mmol, 445 g/mol, 0.223 mg), 1,8-dibromooctane (1.2 mmol, 270 g/mol, 0.324 g) and DMF (50 mL) respectively, using similar procedures as described above. Yellow solid, yield 82%. (Figure S8, Figure S15, Figure S22, ESI†)1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm): 8.29 (s, 1H), 7.73 (s, 1H), 7.29 (s, 1H), 4.22 (s, 1H), 1.86 (s, 1H), 1.29 (s, 1H).

13

C NMR (150 MHz, DMSO-d6) δ (TMS,

ppm): 156.28, 136.52, 133.17, 126.49, 121.97, 110.27, 97.18, 80.26, 55.47, 21.59, 3.96. IR (KBr): 3410, 3026, 2925, 1645, 1584, 1490, 1285, 1173, 1052, 816, 708, 527 cm-1. 2.10 Synthesis and characterization of [TPPA1-DBO-TIPA5]. TPPA (0.15 mmol, 476 g/mol, 0.072 g), TIPA (0.75 mmol, 445 g/mol, 0.334 g), 1,8-dibromooctane (1.35 mmol, 270 g/mol, 0.365 g) and DMF (50 mL) respectively, using similar procedures as described above. Yellow solid, yield 88%.(Figure S8, Figure S15, Figure S22, ESI†) 1H NMR (600 MHz, DMSO-d6) δ (TMS, ppm): 8.28 (s, 1H), 7.69 (s, 1H), 7.26 (s, 1H), 4.23 (s, 1H), 3.56 (s, 1H), 1.72 (s, 1H), 1.29 (s, 1H).

13

C NMR (150

MHz, DMSO-d6) δ (TMS, ppm): 140.32, 136.30, 120.01, 110.75, 110.08, 49.22, 21.33. IR (KBr):3443, 2931, 1618, 1490, 1294, 1072, 816, 722, 614, 519 cm-1. 2.11 Preparation of a serious of electrostatic complexes. Sodium polyacrylates (500 mg) were



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dissolved into 20 mL twice distilled water. And TPPA-DBO, [TPPA-DBO]TIPA, [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO (5 mg), respectively, were dissolved in 5 mL of ethanol and added in flask, and stirred at 50 °C for 2 h. After cooling at room temperature to give colorful hydrogel. Then, the xerogels were produced by solvent evaporation. 2.12 Fluorescence quantum yield (ΦF) measurement. Fluorescence quantum yield measurements were measured using a Fluoro Sens 9003 Fluorescence Spectrophotometer. The fluorescence quantum yields were Calculated using integrated sphere. The fluorescence quantum yields were collected at the excitation wavelengths as 425 and 320 nm (TPPA-DBO, [TPPA-DBO]TIPA were 425nm, [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO were 320nm). And each emission spectrum was collected from 450 to 750 nm with 0.5 nm step and 100 ms dwell time. 2.13 Time-resolved fluorescence measurement. Time-resolved fluorescence measurements were carried out using a C11367-11 Quantaurus-Tau system. The time resolution was 20 ps. The laser energy level for excitation is 2 mW. Decay of the fluorescence intensity (I) with time (t) was fitted by a double-exponential function as shown in Equation (1): (1)

in which t1 and t2 are the lifetimes of the shorter- and longer-lived species, respectively, and A1 and A2 are their respective amplitudes. The weighed mean lifetime () was calculated according to Equation (2):

(2)

2.14 Dynamic light scattering (DLS). Particle size measurements process were performed on a Zetasizer Nano ZS dynamic light scattering (DLS) system. This system determined the particle size distribution of particles in solution, with measurement capability from 0.3 nm to 10 µm. All the samples were tested in ethanol and H2O solution. 2.15 Cell viability. Cell viability assay was conducted using HepG-2 cells. Firstly, TPPA-DBO, [TPPA-DBO]TIPA,

[TPPA1-DBO-TIPA1],

[TPPA1-DBO-TIPA2],

[TPPA1-DBO-TIPA5],

[TIPA-DBO]TPPA and TIPA-DBO were sterilized with ultraviolet light and dissolved in DMSO. Then, the solution was diluted with PBS buffer (pH = 7.4) to different concentrations (0, 5, 10, 15, 20, and 25 µg/mL). To obtain complete cell culture medium, 10% PBS, 100 units mL−1 penicillin, and 100 units mL−1 streptomycin were added in the mixture. HepG-2 human Hepatic cancer cells (purchased from Gansu Provincial Cancer Hospital, Gansu, China) were cultured in the conditioned medium at 37 °C in a humidified environment of 95% O2 and 5% CO2. After 96 h of incubation, cell viability was


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determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. 2.16 Cell fluorescence imaging. Living human Hepatic cancer (HepG-2) cells (1.0 × 105/well) were cultured overnight and then stained in a Petri dish at 50% confluence with TPPA-DBO, [TPPA-DBO]TIPA,

[TPPA1-DBO-TIPA1],

[TPPA1-DBO-TIPA2],

[TPPA1-DBO-TIPA5],

[TIPA-DBO]TPPA and TIPA-DBO solution (10 µg/mL). An image was taken after 1 h incubation.

3. RESULTS AND DISCUSSION 3.1 Polymer synthesis and structure characterization. TPPA-DBO and TIPA-DBO were prepared by only two steps, as shown Scheme 1. Initially, TPPA and TIPA were synthesized simply from a Suzuki coupling reaction. Then 1,8-dibromooctane (DBO) is employed to cross couple TPPA and TIPA cores, leading to the formation of polymeric pyridine quaternary ammonium salts (TPPA-DBO) and imidazolium salts (TIPA-DBO), both of them could be produced in grams scale, and even stored to open air at room temperature for years. And due to the tertiary ionized TPPA cores and the more soft linkers of DBO, both of polymers have moderate to excellent solubility in polar solvents, such as DMF, DMSO and water. Correspondingly, [TPPA-DBO]TIPA and [TIPA-DBO]TPPA were prepared from the precursors of TPPA-DBO and TIPA-DBO, which then terminated by TIPA and TPPA respectively, thus leading to the powders of [TPPA-DBO]TIPA

and

[TIPA-DBO]TPPA.

(Scheme

1)

Finally,

[TPPA1-DBO-TIPA1],

[TPPA1-DBO-TIPA2] and [TPPA1-DBO-TIPA5] were obtained from reaction of different TPPA, TIPA and DBO ratios. After stirring in DMF for 48 h, all of hyperbranched fluorescent materials could be precipitated from solvents with more than 85% yields. The experiment details and structure characterization data were described in the Experimental section and the Supplementary Information.

Scheme 1 The synthesis procedures of TPPA, TIPA, TIPA-DBO and [TIPA-DBO]TPPA. 3.2 Fluorescence property of the positively charged polymers in solid state. In general, the manipulation of color output follows various routes. Recently, the popular way toward



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the goal of multicolor emission was based on stringent control over various experimental conditions, such as preparation method and delicate chemical modification.43,44 However, most of the non-rational design involved processes are both time-, resource-consuming. Therefore, we look forward to the fluorescent material with colorful emissions, which could be tuned through simple chemical modifications rather than physical mixture. Then, a set of fluorescent polymers were prepared by three partners of TPPA, TIPA and DBO via different synthetic methods. Correspondingly, their solid fluorescence was estimated. From the CIE diagram (Fig. 2), we can see that TPPA-DBO, [TPPA-DBO]TIPA, [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5] are located in the orange-yellow region. Reasonably, TIPA-DBO and [TIPA-DBO]TPPA are ranged in the blue region. Their fluorescence quantum yields were tested as 4.597, 4.023, 1.788, 7.250, 3.395, 3.810 and 2.840, respectively. Apart from the time-averaging processes of the steady-state spectra measurements, time-resolved PL spectra can offer more valuable information. Fig. 3 shown the PL decay curves of TPPA-DBO, [TPPA-DBO]TIPA, [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO, their fluorescence lifetimes were measured as 2.62, 5.44, 2.95, 4.35, 3.86, 3.73, 3.43 nanoseconds, respectively. It was clear from our observation that the life time of the cationic polymers (especially [TPPA-DBO]TIPA) in this study are significantly longer than most organic fluorophores. Their long lifetimes make these materials particularly appealing for the visualization of cellular processes in time-resolved confocal measurements, which can eliminate unwanted interfering background for high-sensitivity assays. Herein, we believed that after copolymerization taking place between TPPA, TIPA and 1,8-dibromooctane(DBO), the fluorescence cores are stayed at the more fixed places, which keeps their emission sources independent and free from energy transfer crosstalk through spatial separation of them. So, we concluded that the different color's fluorescence generation of our hyperbranced polymers is due to the blending two complementary colors (blue and yellow).

Fig. 2 The CIE chromaticity diagram of hyperbranched polymers in solid state and the corresponding fluorescence quantum yields(%).(The excitation wavelength of TPPA-DBO, [TPPA-DBO]TIPA were 425

nm

and

the

excitation

wavelength

of

[TPPA1-DBO-TIPA1],

[TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO were 320 nm.)


[TPPA1-DBO-TIPA2],

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Fig. 3 Time-resolved fluorescence decays of the hyperbranched polymers. 3.3 Fluorescence property of the positively charged polymers in solution phase. Additionally, we really intend to understand the different emission performances under the solution phase conditions. Then, we tested the fluorescence spectrum in the good solvent of EtOH with the same mass concentration. (10 µg/mL) (Fig. 4) Interestingly, except for the samples of TPPA-DBO, [TPPA-DBO]TIPA, most of them settled in the blue or near-blue area. This results exhibited the obvious polarity

effect

of

solvent

in

the

case

of

[TPPA1-DBO-TIPA1],

[TPPA1-DBO-TIPA2],

[TPPA1-DBO-TIPA5]. Meanwhile, a near white-light emission with color coordinates of (0.2584, 0.3031) was obtained with quantum yields as 2.489. With a very sharp contrast, [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5] emitted the colors within the orange-yellow region in the solid state. However, in the solution phase, three of them moved to the short emission length region around 480 nm. The totally different emission behaviors in the different states are possibly of application interests in the field of smart materials.45 Therefore, we can conclude that our concepts to manipulation of color output takes advantages of simple synthetic procedures, multi-colors outputs, and excellent material stability.

Fig. 4 The CIE chromaticity diagram of the hyperbranched polymer in ethanol solution (conc. 10 ug/mL) and fluorescence quantum yield (%). The corresponding photographs taken under illumination at 365 nm.(The excitation wavelength of TPPA-DBO, [TPPA-DBO]TIPA were 425 nm and the excitation wavelength of [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO were 320 nm.)



3.4 Fluorescence property of the hyperbranched polymers dispersed in the PAAS matrix. What should be noted that incorporating the more toxic and water insoluble fluorescent units into biocompatible polymer matrix has been verified as an ideal proposal to deal with some undesirable problems in the field of bio-related applications.46 Accordingly, we dispersed the positively charged polymers into the negative polymer matrix of PAAS. In order to obtaining the highly homogeneous electrostatic complexes, we firstly dissolved both polymers into the mixture of water and EtOH, then freeze drying was employed to produce the sponge-like solid. That solid then was grind into fine powders. Afterwards, the powders fluorescence property was estimated. (Fig. 5) Although the corresponding fluorescence quantum yields decreased slightly. More attractively, the electrostatic complexes exhibited more colorful emissions, which ranged over a wide area of the visible spectrum (from blue to yellow). Meanwhile, their fluorescence lifetimes were measured as long as 2.38, 1.89, 4.29, 4.26, 3.96, 3.72, 4.27 nanoseconds, respectively. (Fig. 6) Therefore, we can concern that this combined strategy not only overcomes the water insolubility of the fluorescent units, but also endows the electrostatic complexes with tunable emissions.

Fig. 5 The CIE chromaticity diagram of the electrostatic complexes in solid state and the corresponding fluorescence quantum yield (%). (The excitation wavelength of TPPA-DBO, [TPPA-DBO]TIPA were 425 nm and the excitation wavelength of [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO were 320 nm.)

Fig. 6 Time-resolved fluorescence decays of the electrostatic complexes. Reasonably, the water solubility of all electrostatic complexes is able to supply the opportunity to


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hydrogel formation. Additionally, as we disclosed previously that TPPA-DBO is an AIE-active material, its emission behaviors could be adjusted by different aggregate states. Therefore, we prepared the fluorescent hydrogels by inversion method. From CIE diagram, (Fig. 7) we clearly see that multi-color outputs are displayed. Compared to the CIE chromaticity diagram of the solid state samples, the hydrogels demonstrated more wide color's locations. More importantly, an very near white light with color coordinates of (0.3046, 0.3606) could be seen. With the almost same postulations, this tunable emissions were possibly due to the AIE phenomenon. Furthermore, the potential of the fluorescent hydrogels for practical multi-light emission application has been demonstrated. (Fig. 8) A rough WLED was fabricated by simply coating the hydrogels solid on the surface of a commercially available ultraviolet LED chip (5 mm, 365 nm).The resultant WLED device emitted bright light at a voltage of 3.3 V, as illustrated in Fig. 8. Therefore, the fluorescent hydrogels, serving as novel single-phase multi-light materials, are potential for practical lighting application.

Fig. 7 The CIE chromaticity diagram of the electrostatic complexes in gel states and the fluorescence quantum yield (%). The photographs taken under illumination at 365 nm. (The excitation wavelength of TPPA-DBO, [TPPA-DBO]TIPA were 425 nm and the excitation wavelength of [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO were 320 nm.)

Fig. 8 Image of commercial available UV-LED (λem = 365 nm, 3.2-3.4 V, 20 mA) coated with hydrogels when the LED turned on. 3.5 The different cell imaging world of the positively charged polymers. In order to carry out the application for cell imaging, the cytotoxicity of TPPA-DBO, [TPPA-DBO]TIPA, [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results exhibited that HepG-2 liver cancer cells viability is not obviously varied when 0, 5, 10, 15, 20,



and 25 µg/mL probes are charged to the culture medium after long incubating time of 96 h. Then, HepG-2 liver cancer cells were stained with TPPA-DBO, [TPPA-DBO]TIPA, [TPPA1-DBO-TIPA1], [TPPA1-DBO-TIPA2], [TPPA1-DBO-TIPA5], [TIPA-DBO]TPPA and TIPA-DBO and examined under a fluorescence microscope. The cellular uptake and distribution profiles were displayed in Fig. 9. Clearly, all of multiple cations charged polymers were very cell-membrane permeable, and found to accumulate on the cell nucleus, exhibiting remarkable selectivity to give bright blue and green fluorescence. Taking TPPA-DBO as an example, the nuclear staining was confirmed by co-staining with DAPI. As shown in Fig. 9, the cell nucleus were clearly visualized with a very high signal-to-noise ratio. The fluorescence from TPPA-DBO overlapped perfectly with DAPI. Therefore, we can conclude that the specific cell nucleus staining is possibly due to the multi-cationic charges. Correspondingly, the complex of polymers of [TPPA-DBO]/PAAS could not be internalized into cells, which was easily washed away by PBS buffer solution. Furthermore, we measured Zeta Potential of complex of [TPPA-DBO]/PAAS, the result clearly displayed the negative value (-0.195 mV), so we can conclude that electrostatic repulsion is the mean reason for the no-staining behavior.

Fig. 9 Fluorescence micrographs of HeGP-2 cells stained with hyperbranched polymers at the concentration 10 µg/mL for 60 min in different magnifications, and co-staining of nucleus-specific dye DAPI with TPPA-DBO and the overlay image. 3.6 DNA/RNA selectivity experiments in vitro and in vivo. It was well know that the cationic fluorescence probes are able to contact with negatively charged biomacromolecules, such as DNA, RNA. Due to the different analyte' conformations and the contact models, cationic probes could be employed as DNA or RNA specific sensors. Herein, based on the fluorescence emission changes, the sensitivity and selectivity of TPPA-DBO to dsDNA over RNA were detected (Table 1 and Fig. 10).At the highest concentration of DNA&RNA tested (0.35 eqv. toward TPPA-DBO), the fluorescence emission reached up to 30 (with dsDNA) 15 (with RNA) folds higher than that of the free compound under the same analysis conditions. (Fig. 10). A blue shift of 25 nm in the emission wavelength upon DNA addition was observed. And in the case of RNA, less than


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15 nm blue shift could be seen. The detailed spectroscopic properties of TPPA-DBO with and without DNA&RNA are summarized in Table 1. The Stokes shifts were higher than 140 nm in both of cases, and the absorbance were red-shifted to a slightly shorter wavelength (less than 10 nm). The fluorescence quantum yield was increased from 6.213 to 14.41, 7.847 upon binding to dsDNA and RNA respectively. And the quantum yield of TPPA-DBO was 2 times higher than that of the binding with RNA. From these results, we confirmed that TPPA-DBO is of better sensitivity and selectivity toward dsDNA rather than RNA. Compare to several nucleic acid dyes, such as PicoGreen, YOYO, and SYBR dyes,47-49 TPPA-DBO demonstrated the absolute high DNA selectivity. Reasonably, the electrostatic complexes of [TPPA-DBO]/PSSA exhibited negligible sensitivity and selectivity toward dsDNA and RNA, which was due to the surface negative charges of the complexes of [TPPA-DBO]/PSSA. Table 1. Spectroscopic properties of TPPA-DBO and [TPPA-DBO]/PSSA responding to DNA and RNA. λabsa/nm

λema/nm

Stokes shift/nm

ΦFb/%

TPPA-DBO

425

600

175

6.213

[TPPA-DBO]-DNA

431

576

145

14.41

[TPPA-DBO]-RNA

435

577

142

7.847

[TPPA-DBO]/PSSA

326

554

228

2.377

[TPPA-DBO]/PSSA-DNA

325

550

225

1.125

[TPPA-DBO]/PSSA -RNA

327

549

222

0.581

Compound

a

λabs and emission λmax of TPPA-DBO were obtained by TU-1901 spectrophotometer and Hitachi

F-2500 FL fluorophotometer in PBS buffer (10 mM, pH = 7.4). b The fluorescence quantum yields (ΦF) were Calculated using integrated sphere. The final DNA/RNA solution concentration was 10 µg/mL.

Fig. 10 Plots of maximum emission intensity (I/I0) of TPPA-DBO (black lines) and [TPPA-DBO]/PAAS (blue lines) with the addition of DNA and RNA. With this distinctive selectivity to dsDNA in vitro, other important biological properties of TPPA-DBO were further investigated in live cells (Fig. 11). The in vivo selectivity of DNA over RNA was evaluated by deoxyribonuclease (DNase) and ribonuclease (RNase) digest experiments. The fluorescence signal of TPPA-DBO diminished dramatically after the DNase digestion, but was not influenced by RNase digestion. These results supported that TPPA-DBO has prominent selectivity to



DNA over RNA inside cells.

Fig. 11 Fluorescence images of the digest experiments for TPPA-DBO (10 µg/mL) with fixed A549 cells. (a) Control, cells incubated with TPPA-DBO; (b) cells incubated with TPPA-DBO and DNase (30 µg/mL); (c) cells incubated with TPPA-DBO and RNase (25 µg/mL). (Probe concentration:10 µg/mL, λex = 425 nm).

4. CONCLUSIONS In summary, we have successfully synthesized a series of positively charged hyperbranched polymers via a facile synthetic method. The multicolor tunable emission, including an very near-white emission light with color coordinates of (0.3046, 0.3606), could be realized in the solid state, solution phase, and hydrogel state. As we postulated that the multicolor fluorescence generations are originated from the bleeding two complementary colors of blue and yellow, which are emitted from TIPA and TPPA fluorescence cores. Also, the different aggregate states and the polarity of solvent exert great influence on the emission behaviors. The further cell staining experiments verified that the multiple cations charged polymers are very cell-membrane permeable, which is possibly due to the multiple cationic charges. And also, the positively charged hyperbranched polymers could specifically accumulate on the cell nucleus, exhibiting remarkable selectivity to give bright blue, green, and yellow fluorescence, and the nucleus staining was confirmed by co-staining with DAPI. The cationic fluorescence materials are able to be applied as probes to detect negatively charged biomacromolecules, such as DNA, RNA. Then, DNA/RNA selectivity experiments verified that one of representative cationic polymer of TPPA-DBO has prominent selectivity to DNA over RNA in vitro and in vivo.

■

ASSOCIATED CONTENT Supporting Information 1

H, 13C spectrum of TPPA, TIPA, 1H spectrum of all hyperbranched polymers, all of UV and PL

analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.


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■

ASSOCIATED CONTENT This work is supported by the National Natural Science Foundation of China (nos. 1764012). We thank the Key Laboratory of Eco-Environment-Related Polymer Materials (Northwest Normal University) and the Ministry of Education Scholars Innovation Team (IRT 1177) for financial support.

■

REFERENCES

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The Positively Charged Hyperbranched Polymers with Tunable

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