Aggregation-Induced Emission Fluorophore-Based Molecular Beacon

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Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3618−3630

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Aggregation-Induced Emission Fluorophore-Based Molecular Beacon for Differentiating Tumor and Normal Cells by Detecting the Specific and False-Positive Signals Qinghua Guan,†,§ Nan Li,∥ Leilei Shi,‡ Chunyang Yu,‡ Xihui Gao,‡ Jiapei Yang,‡ Yuanyuan Guo,‡ Peiyong Li,*,§ and Xinyuan Zhu*,‡ †

School of Biomedical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, 197 Ruijin Second Road, Shanghai 200025, China ‡ School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China ∥ Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, 270 Dong’an Road, Shanghai 200032, China

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ABSTRACT: Accurate and nondestructive detection of tumor-related mRNA in living cells is of great significance for tumor diagnosis. The universal technique for imaging mRNA in living cells is nucleic-acid-based fluorescent probes. However, the majority of developed nucleic-acid-based fluorescent probes were only designed to detect the targeted mRNA but could not avoid the interference arising from nuclease or other biological matrices, which results in inevitable false-positive signals. To overcome this dilemma, a new aggregation-induced emission (AIE) fluorophore and the fluorescence resonance energy transfer (FRET) principle were used to establish a novel AIE fluorophore-based molecular beacon (AIE-MB). The AIE fluorophore tetraphenylethylene-quinoxaline (TPEQ) was designed by incorporating quinoxalinone with one typical AIE active luminogen tetraphenylethene (TPE), which could acquire a wide range of excitation wavelength. On this basis, the AIE-MB was designed by labeling two fluorophores: the TPEQ acceptor and an aggregation-caused quenching (ACQ) fluorophore 7-amino-4methylcoumarin acid (AMCA) donor. On the basis of these two fluorophores, the AIE-MB could exhibit three states: weak fluorescence at primary stage, blue fluorescence (specific signal) generated by pairing with target mRNA in tumor cells, and both blue and green fluorescence (false-positive signal) due to the endogenous degradation in normal cells. Obviously, the specific imaging for target mRNA in tumor cells and the false-positive signal resulting from endogenous degradation in normal cells could be accurately distinguished through the different fluorescence emission. As a result, in contrast to traditional nucleicacid-based fluorescent probes, the AIE-MB could improve the accuracy of the tumor detection by efficiently differentiating both specific and false-positive signals, which showed potential application value in tumor diagnosis and biomedical research. KEYWORDS: AIE, fluorescence imaging, tumor, mRNA, false-positive signal

1. INTRODUCTION

which are not suitable for directly monitoring the spatial and temporal information on mRNA at the cellular level. Therefore, the search for powerful techniques that can detect mRNA with real-time, in situ monitoring capability, and high sensitivity in living cells is quite important. Nucleic-acid-based fluorescent probes based on traditional aggregation-caused quenching (ACQ) fluorophores should be

Messenger RNA (mRNA) is a blueprint for the production of protein in cells, which translates genetic information stored in DNA into protein.1−3 Aberrant mRNA plays significant roles in tumor progression and prognosis.4 Hence, detection of mRNA alteration holds great promise for identifying tumor cells and further assists in tumor diagnosis and therapy.5−7 Current well-known methods for identifying and characterizing mRNA, including in situ hybridization (ISH),8,9 reverse transcription polymerase chain reaction (RT-PCR),10,11 microarray analysis,12,13 and Northern blots14,15 use lysed cells, © 2019 American Chemical Society

Received: May 5, 2019 Accepted: May 20, 2019 Published: May 22, 2019 3618

DOI: 10.1021/acsbiomaterials.9b00627 ACS Biomater. Sci. Eng. 2019, 5, 3618−3630

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ACS Biomaterials Science & Engineering Scheme 1. Imaging of AIE-MB in Tumor and Normal Cellsa

a (A) A new AIE fluorophore TPEQ was designed and used to establish a novel AIE-MB. The AIE fluorophore (TPEQ, acceptor) has a FRET effect with the ACQ fluorophore (AMCA, donor). (B) AIE-MB was encapsulated by an amphiphilic pH-responsive polymer to form a nanoparticle. After it transports the AIE-MB-loaded nanoparticle into cells, it will disassemble and release the AIE-MB because the amphiphilic polymer converts to a hydrophilic polymer when pH is less than pKa in the lysosome. The AIE-MB emits strong blue fluorescence of AMCA (specific signal) after it pairs with targeted mRNA in tumor cells. In normal cells, part of the AIE-MB is degraded into fragments, and the two fluorophores are separated. A false-positive signal is produced, in which AMCA fluorescence is weaker than that in tumor cells, whereas TPEQ aggregates together and green fluorescence is “turn on”. (C) In tumor and normal cells, the AIE-MB emits different fluorescence.

developed: DNA tetrahedron nanotweezer (DTNT),22 nanoflares (an oligonucleotide gold nanoparticle conjugate),23,24 and multiplexed fluorescent in situ hybridization imaging.25 Although these techniques enhance the specific imaging of target mRNA to some extent, they still cannot completely avoid false-positive signal caused by chemical or biological interferences in living cells. Differentiating the specific and false-positive signals of a nucleic-acid-based fluorescent probe in living cells is still a challenge so far. To overcome this challenge, an ideal method is to discover a fluorescent probe that can directly image the false-positive

the most universal technique for detecting mRNA in living cells. For example, a molecular beacon (MB) is one common nucleic-acid-based fluorescent probe for imaging DNA and RNA in a purified environment, in which a hairpin structure and fluorophore-quencher pair make it directly visualize target DNA or RNA with excellent sensitivity and selectivity.16−18 However, with the development of nucleic-acid-based fluorescent probes, two pivotal problems need to be addressed: effective delivery of probes and avoiding intrinsic interferences due to cellular components.19−21 To overcome these challenges, various novel fluorescent probes have been 3619

DOI: 10.1021/acsbiomaterials.9b00627 ACS Biomater. Sci. Eng. 2019, 5, 3618−3630

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ACS Biomaterials Science & Engineering signal with different fluorescence from the specific signal. However, all ACQ fluorophores at nucleic-acid-based fluorescent probes are powerless for resolving the problem because the emission fluorescence of them is consistent for both specific pairing and nonspecifically degrading. Fortunately, with the discovery of novel aggregation-induced emission (AIE) fluorophores, the problem is expected to be resolved.26−34 In contrast to the ACQ fluorophore, the AIE fluorophore is faintly luminous or dim in dilute solutions, and bright fluorescence is emitted when they are in a state of aggregation or solid state. Therefore, fluorescence of AIE fluorophore conjugating on hydrophilic cargo will remain “off” in aqueous solution because it is water-soluble. After it is cleaved from its hydrophilic cargo, the AIE fluorophore will aggregate and emit fluorescence.35−42 Obviously, the unique photophysical phenomenon is very suitable for detecting the degradation of the nucleic-acid-based fluorescent probe due to nuclease or other cellular components. On this basis, a novel AIE fluorophore-based MB (AIE-MB) was designed with a new AIE fluorophore and a traditional ACQ fluorophore (Scheme 1). In order to prepare the AIEMB, an AIE fluorophore, tetraphenylethylene-quinoxaline (TPEQ), was first synthesized and characterized, which displayed remarkable AIE property and a wide range of excitation wavelength. After that, TPEQ was conjugated at one end of the AIE-MB by reacting with the P−S bonds. In the meantime, an ACQ fluorophore, 7-amino-4-methylcoumarin acid (AMCA), was conjugated on the other end. Specifically, TPEQ (acceptor) and AMCA (donor) had fluorescence resonance energy transfer (FRET) property and could be excited by the same wavelength laser. After obtaining the AIEMB, the imaging properties of AIE-MB to target DNA and DNase I were determined in aqueous solution, in which AIEMB expressed excellent fluorescence response. Subsequently, the AIE-MB was encapsulated into a nanoparticle by selfassembly with an amphiphilic pH-responsive polymer to transport into cells, and further, the intracellular imaging of the AIE-MB in tumor and normal cells was performed. In the initial state, AIE-MB fluorescence remained “off” because TPEQ and AMCA were in close proximity, which induced high FRET efficiency, and the TPEQ was almost nonfluorescent when it was labeled on AIE-MB and dissolved in aqueous solution. However, in tumor cells, the target mRNA was at a high level and could pair with AIE-MB to emit AMCA fluorescence (specific signal), because two fluorophores on AIE-MB were separated with low FRET efficiency. In contrast, in normal cells, the level of target mRNA was very low, and AIE-MB could not be paired with target mRNA. Meanwhile, a part of AIE-MB would be cleaved into pieces by cellular nuclease, and two fluorophores were separated from the hydrophilic cargo. Both AMCA and TPEQ fluorescence were “turn on”, which represented the degradation signal (falsepositive signal). Comparing specific signal (AMCA fluorescence) and false-positive signal (TPEQ fluorescence), the tumor could be effectively distinguished from normal cells. Generally, the AIE-MB could efficiently differentiate the specific signal by pairing with target mRNA in tumor cells and the false-positive signal due to the endogenous degradation in normal cells with two different colored fluorescent signals, which obviously improved the accuracy of the tumor detection.

2. EXPERIMENTAL SECTION Materials. 4-(Bromomethyl)phenylacetic acid (BPAc), o-phenylenediamine, ethyl pyruvate, N,N-dicyclohexylcarbodiimide (DCC), 4dimethylaminopyridine (DMAP), methyl-2-bromoacetate, and Nhydroxysuccinimide (NHS) were purchased from Tokyo Chemical Industry Co., Ltd. 6-Amino-1-hexanol, O-(7-azabenzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), N-succinimidyl tetradecanoate (C14 NHS ester), and pyridine were purchased from J&K Scientific Ltd. 4-(1,2,2-Triphenylethenyl)benzaldehyde was acquired (TPE-CHO) from Zhengzhou Alfachem Co., Ltd. K2CO3, MgSO4, KHSO4, KOH, dichloromethane (DCM), ethyl acetate (EA), petroleum ether (PE), N,N-dimethylformamide (DMF) tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO) were purchased from Adamas. The DNA sequences (MB-AMCA) were as follows: 5′-(AMCA) CGC GTC TCT TTG GCA TAC TTG ATC ACC GAC GCG T*T*T*T*T (*Phosphorothioate). Synthesis of 3-Methylquinoxalin-2(1H)-one (1). First, 150 mL of anhydrous ethanol and o-phenylenediamine (10.8 g) were added into a beaker. After the temperature was reduced to 0 °C, ethyl pyruvate (13.92 g) dissolved in 10 mL of anhydrous ethanol was dripped under stirring. As the reaction temperature gradually returned to room temperature, the mixture continued to react for 10 h, and the solid product was precipitated gradually in the solution. Then the solid was filtered and washed with anhydrous ethanol. After the removal of ethanol in vacuum, the product was obtained as a white solid (13.6 g, 85%). 1H NMR and mass spectrometry (MS) spectra were shown in Figure S1. Liquid chromatograph mass spectrometry (LC-MS) m/z: 161.0832 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ): 12.33 (s, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.49−7.45 (m, 1H), 7.27 (t, J = 7.4 Hz, 2H), 2.41 (s, 3H). Synthesis of Methyl-2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetate (2). First, compound (1) (3.2 g) and methyl-2-bromoacetate (3.67 g) were suspended in acetone (100 mL) and stirred vigorously. After K2CO3 (3.31 g) was added into the solution, the mixture was refluxed at 63 °C for 10 h. After the reaction was completed, K2CO3 was removed by filtering, and then the solvent was removed via rotary evaporators. The residue was dissolved in EA (50 mL) and washed with distilled water (15 mL × 3). Subsequently, the EA layer was dried over anhydrous MgSO4. After MgSO4 was removed by filtering, DCM was concentrated in vacuum, and the crude solid product was obtained. Finally, a white solid was produced by recrystallizing from EA/PE (3.0 g, 54%). 1H NMR and MS spectra were demonstrated in Figure S2. LC-MS m/z: 233.0985 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ): 7.80 (d, J = 7.9 Hz, 1H), 7.57−7.55 (m, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 7.2 Hz, 1H), 5.12 (s, 2H), 3.71 (s, 3H), 2.46 (s, 3H). Synthesis of (E)-Methyl-2-(2-oxo-3-(4-(1,2,2-triphenylvinyl)styryl)quinoxaline-1(2H)-yl)acetate (3, TPEQ). Compound (2) (500 mg) and 4-(1,2,2-triphenylethenyl)benzaldehyde (FTPE, 1.08 g) were suspended in acetic acid (10 mL). Then catalytic concentrated sulfuric acid (100 μL) was dripped into the solution. The resulting solution was reacted at 65 °C for 8 h. After it was allowed to return to room temperature, the mixture was dissolved by 60 mL of DCM and washed with distilled water (15 mL × 3). Subsequently, the DCM layer was dried over anhydrous MgSO4. After MgSO4 was removed by filtering, DCM was concentrated in vacuum, and the crude solid product was obtained. Finally, the solid was purified by silica chromatography (PE:EA = 4:1) to obtain a pure yellow solid (1.1 g, 76%). The NMR and MS spectra were shown in Figure S3. LC-MS m/z: 575.2339 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ): 7.98 (d, J = 16.2 Hz, 1H), 7.86 (dd, J = 8.0, 1.5 Hz, 1H), 7.56−7.51 (m, 5H), 7.41 (t, J = 1.3 Hz, 1H), 7.16−7.12 (m, 9H), 7.03−7.01 (m, 8H), 5.76 (s, 2H), 3.71 (s, 3H). 13C NMR (100 MHz, DMSO-d6, δ): 167.93, 153.92, 151.24, 144.48, 142.95, 142.92, 142.82, 141.15, 139.96, 137.05, 133.85, 132.55, 132.03, 131.25, 130.64, 130.58, 130.54, 130.18, 129.29, 127.87, 127.83, 127.71, 127.22, 126.7, 126.59, 126.54, 124.02, 121.54, 114.48, 54.81, 52.38, 43.66. Synthesis of (E)-2-(2-Oxo-3-(4-(1,2,2-triphenylvinyl)styryl)quinoxaline-1(2H)-yl)acetic acid (4). Compound (3) (574 mg) 3620

DOI: 10.1021/acsbiomaterials.9b00627 ACS Biomater. Sci. Eng. 2019, 5, 3618−3630

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ACS Biomaterials Science & Engineering

polydispersity index (PDI) of 1.24. Moreover, pKa and pH responsiveness of the polymer was determined using the protocol from our earlier work.43 1H NMR (400 MHz, DMSO-d6, δ): 4.20 (H18), 3.85 (H-10), 3.67 (H-2, 3), 3.38 (H-1), 3.00 (H-12), 2.8−2.61 (H-11, 19−27), 1.91−1.81 (H-6, 8), 1.61 (H-28), 1.25 (H-29, 30), 1.15−0.91 (H-4, 5, 7, 9, 13, 14, 16, 17, 31). Spectra Characterization of the TPEQ and MB-AMCA. First, the ultravoilet-visible spectrophotometer (UV−vis) absorption and photoluminescence spectra of the TPEQ solid were measured on a UV−vis spectrophotometer and a fluorescence spectrometer, respectively. Furthermore, the excitation and emission spectra of TPEQ in pure THF (1 × 10−5 M) and MB-AMCA in distilled water (1 × 10−6 M) were investigated on a fluorescence spectrometer. AIE Property. The AIE property of TPEQ in THF/H2O solvent mixtures was investigated. The THF solution of TPEQ (1 × 10−4 M, 200 μL) was added into 1800, 1600, 1200, 800, 400, 200, 0 μL of THF, respectively. Then different volumes of distilled water were added into the above THF solutions to ensure the total volume of all mixed solutions was 2 mL. Finally, a series of THF/H2O solvent mixtures (1 × 10−5 M) were prepared, and the proportion of water was 0, 10%, 30%, 50%, 70%, 80%, 90%, respectively. The emission spectra were measured with λex of 353 nm. Furthermore, the AIE property of TPEQ in pure THF was investigated. TPEQ was dissolved in pure THF to prepare a series of solutions with the concentrations of 1 × 10−2, 1 × 10−3, 1 × 10−4, 1 × 10−5, 1 × 10−6, 1 × 10−7 M, respectively. The emission spectra were measured with λex of 353 nm. Moreover, the quantum yields of the TPEQ solid and TPEQ in pure THF were also measured with λex of 353 nm. Theoretical Investigations. To better demonstrate the AIE property of TPEQ, density functional theory calculation was performed. The calculation reported here was carried out with the Gaussian09 program.44 Geometry optimization of TPEQ structure was calculated at B3LYP/6-31G* without any symmetry restriction.45−47 After that, at the same level, analytical vibration frequencies were performed to determine the nature of the located stationary point. Synthesis and Characterization of AIE-MB. The TPEQ-Br DMSO solution (238 nM) and the MB-AMCA aqueous solution (4 OD) were combined in a brown centrifuge tube, in which the molecular ratio of TPEQ-Br with the P−S bonds of the DNA was 5:1. The mixture was vibrated at 50 °C for 20 h. Then the resulting solution was transferred into a centrifuge tube, and 7 mL of water was added. The solution was continuously extracted by EA to remove the unreacted TPEQ-Br. After that, the water layer was concentrated to 500 μL with n-butanol. Finally, the resulting solution was further dried by a rotator and purified by high-performance liquid chromatography (HPLC) (CH3CN:H2O = from 30:70 to 10:90 with 0.1 M triethylammonium acetate aqueous buffer (TEAA)) to obtain a pure product (1.2 OD, 30%). In order to determine the successful synthesis of the AIE-MB, the product was characterized by LC-MS and denaturing polyacrylamide gel electrophoresis (PAGE). LC-MS analysis was performed by Sangon Biotech Co., Ltd.. The denaturing PAGE gel analysis was prepared by using 20% acrylamide solution containing 8.3 M urea, and the electrophoretic buffer was 1× TBE buffer. The AIE-MB aqueous solution (5 μL, 0.2 OD) was mixed with loading buffer (15 μL, containing 0.03% bromophenol blue and 0.03% xylene cyano) and added into the gel well. After the gel was allowed to run for 2 h, the PAGE gel was immersed into ethidium bromide (EB) solution for the DNA staining, and the result was observed by BioRad imaging system. Fluorescence Response of AIE-MB To Target DNA and DNase I. First, the fluorescence response of the AIE-MB paired with target DNA was investigated. A series of different concentrations (0, 4, 10, 20, 30, 50, 70, 100, 150, 200, 300 nM) of target DNA Tris-HCl buffer solution (pH 7.4) were prepared. After that, the AIE-MB solution (100 μM, 2 μL) was added into 998 μL of Tris-HCl buffer, and the solution was added into 1 mL of each of the above target DNA solutions, respectively. After 30 min, the emission spectra were measured with λex of 353 nm.

was suspended in ethanol (60 mL). Then KOH (1.35 g) was dissolved in distilled water (60 mL) and was dripped into the above solution and reacted at 45 °C. After the solution was allowed to react for 24 h, it became clear. Ethanol was removed via rotary evaporators, and 50 mL of distilled water was added. The solid product precipitated gradually in the solution after the aqueous HCl (1 M, 25 mL) was dripped. The solid product was filtered and purified by silica chromatography (DCM:MeOH = 50:1) to obtain a pure yellow solid (439 mg, 78%). 1H NMR and MS spectra were demonstrated in Figure S4. LC-MS m/z: 561.2206 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ): 13.34 (s, 1H), 7.98 (d, J = 16.1 Hz, 1H), 7.85 (dd, J = 8.0, 1.5 Hz, 1H), 7.57−7.51 (m, 5H), 7.40 (m, 1H), 7.16−7.13 (m, 9H), 7.03−7.01 (m, 8H), 5.05 (s, 2H). Synthesis of (E)-N-(6-Hydroxyhexyl)-2-(2-oxo-3-(4-(1,2,2triphenylvinyl)styryl)-quinoxaline-1(2H)-yl)acetamide (5). In a glovebox, compound (4) (560 mg), HATU (570 mg), and pyridine (240 mg) were suspended in DMF (22 mL) and reacted for 30 min. Then 6-amino-1-hexanol (176 mg) was added, and the mixture was sealed and taken out of the glovebox to react for 24 h. The resulting solution was added into 100 mL of DCM and then washed with KHSO4 solution and distilled water, respectively. The DCM layer was dried over anhydrous MgSO4. After MgSO4 was removed by filtering, DCM was concentrated in vacuum, and the crude solid product was obtained. Finally, the solid was purified by silica chromatography (DCM:EA = 5:1) to obtain a yellow solid (573 mg, 87%). 1H NMR and MS spectra were shown in Figure S5. LC-MS m/z: 661.3331 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ): 8.23 (s, 1H), 7.95 (d, J = 16.2 Hz, 1H), 7.83 (dd, J = 8.0, 1.5 Hz, 1H), 7.53−7.51 (m, 4H), 7.37 (t, 1H), 7.31 (dd, J = 8.5, 1.1 Hz, 1H), 7.18−7.13 (m, 9H), 7.03−6.97 (m, 8H), 4.90 (s, 2H), 3.37 (q, J = 5.2 Hz, 2H), 3.06 (q, J = 6.3 Hz, 2H), 1.39 (t, J = 6.8 Hz, 4H), 1.25 (m, 4H). Synthesis of (E)-6-(2-(2-Oxo-3-(4-(1,2,2-triphenylvinyl)styryl)quinoxaline-1(2H)-yl)acetamido)hexyl-2-(4(bromomethyl)phenyl)acetate (6, TPEQ-Br). In a glovebox, compound (5) (659 mg) and 4-(bromomethyl)phenylacetic acid (345 mg) were suspended in DCM (10 mL). Then DCC (309 mg) and DMAP (12 mg) were added into the solution. The solution was sealed and taken out of the glovebox to react for 24 h. The resulting solution was added into 50 mL of DCM and then washed with NaHCO3 solution and deionized water. Then the DCM layer was dried over anhydrous MgSO4. After MgSO4 was removed by filtering, DCM was concentrated in vacuum, and the crude solid product was obtained. Finally, the solid was purified by silica chromatography (DCM:EA = 20:1) to obtain a pure yellow solid (713 mg, 82%). The NMR and MS spectra were shown in Figure S6. LC-MS m/z: 870.2898 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ): 8.23 (t, J = 5.6 Hz, 1H), 7.97 (d, J = 16.2 Hz, 1H), 7.82 (dd, J = 8.0, 1.6 Hz, 1H), 7.59−7.51 (m, 4H), 7.44−7.37 (m, 3H), 7.31−7.25 (m, 3H), 7.18− 7.13 (m, 9H), 7.03−7.01 (m, 8H), 4.90 (s, 2H), 4.73 (d, J = 26.0 Hz, 2H), 4.04−4.01 (m, 2H), 3.66 (s, 2H), 3.07 (q, J = 6.6 Hz, 2H), 1.54 (t, J = 7.1 Hz, 2H), 1.39 (t, J = 6.9 Hz, 2H), 1.24−1.22 (m, 4H). 13C NMR (100 MHz, DMSO-d6, δ): 170.93, 165.56, 154.12, 151.53, 144.37, 142.96, 142.93, 142.84, 141.12, 139.97, 136.63, 136.43, 136.06, 134.57, 133.94, 132.61, 132.55, 131.66, 131.25, 130.64, 130.58, 130.54, 130.37, 129.84, 129.54, 129.5, 129.18, 129.12, 128.76, 127.87, 127.82, 127.71, 127.16, 126.69, 126.59, 123.65, 121.84, 114.38, 64.08, 59.65, 54.81, 45.84, 44.73, 34.24, 28.7, 27.91, 25.75, 24.84, 20.66, 13.98. Synthesis of mPEG-b-P(DPA-co-GMA-TEPA-C14) (7). To transport the AIE-MB into cells, an amphiphilic pH-responsive polymer (7) was synthesized using the protocol from our earlier work.43 The polymer material was also synthesized and characterized in our other work.43 Briefly, C14 NHS ester (1 mg) and mPEG-bP(DPA-co-GMA-TEPA) (0.15 g) were suspended in DMF (6 mL) and reacted for 24 h in a glovebox. Subsequently, the solution was dialyzed against DMSO and distilled water. After the solution was freeze-dried under vacuum, polymer (7) was obtained as a white powder (0.14 g, yield 93%). The 1H NMR and Fourier transform infrared (FTIR) spectra were shown in Figure S7 (Mw = 2.02 × 104 Da). Gel permeation chromatography (GPC): 1.95 × 104 Da with 3621

DOI: 10.1021/acsbiomaterials.9b00627 ACS Biomater. Sci. Eng. 2019, 5, 3618−3630

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ACS Biomaterials Science & Engineering

Figure 1. Synthesis route of compounds TPEQ and TPEQ-Br. BPAc and TPE-CHO are the abbreviations for 4-(bromomethyl)phenylacetic acid and 4-(1,2,2-triphenylethenyl)benzaldehyde. After that, the fluorescence response of the AIE-MB degraded by DNase I was studied. Next, 20 μL of the AIE-MB (100 μM) and 10 μL of the 10× DNase I buffer were mixed with 70 μL of distilled water. Then, a series of DNase I solutions (0, 4, 10, 20, 40, 80, 120, 160, 200, 300, 400 U/L) were prepared, and 1 mL of the solution was added into the above AIE-MB solution, respectively. Afterward, 900 μL of distilled water was added. The fluorescence spectra were measured with λex of 353 nm after the solutions were incubated for 0.5 h at 37 °C. Preparation and Characterization of the Nanoparticle. Polymer (7) was dissolved in pure THF (4 mg/mL) to prepare the transfection carrier solution. Subsequently, the AIE-MB (100 μM, 20 μL) and the polymer solution (600 μL) were mixed (N/P = 40:1). Under stirring (1000 rpm), the mixture was added into 6 mL of distilled water to form the AIE-MB-loaded nanoparticle. Subsequently, to remove organic solvent and free polymer, the nanoparticle dispersion was centrifuged at 8000 rpm in an ultrafiltration device (MWCO 100 kDa). After that, the residual solution was washed with distilled water and finally dispersed in 1 mL of distilled water. The size distribution and zeta potential were measured by dynamic light scattering (DLS), while the morphology was visualized on a transmission electron microscope (TEM). Moreover, the AIE-MBloaded nanoparticle stability in plasma was studied by the method described in our earlier work.43 In Vitro Cytotoxicity Study and Cellular Uptake Study. In 96-well plates, HepG2, MCF-7, L02, and MCF-10A cells were incubated for 12 h. Then the cells were treated with TPEQ or AIEMB-loaded nanoparticle, respectively. The concentration of TPEQ was from 0 to 20 μg/mL, while the concentration of the AIE-MBloaded nanoparticle was from 0 to 100 μg/mL. After the cells were incubated for 48 h, MTT stock solution was added for another 4 h treatment. Finally, the entire medium was removed, and the plate was shaken for 15 min after 200 μL of DMSO was added to each well. The absorbance of the solution was measured at a wavelength of 570 nm. After that, the ability of cellular uptake was studied by flow cytometry. In 6-well plates, HepG2 and L02 cells were incubated for 12 h. Then the cells were treated with nanoparticles (containing 1 μM Cy5.5) for 0.5, 1, 2, 4, and 6 h, respectively. After the medium was removed, PBS was added to wash the cells. Finally, the cells were

measured by flow cytometry after they were treated with trypsin and collected into a flow tube. Intracellular Imaging of the AIE-MB-Loaded Nanoparticle. In order to investigate the intracellular imaging, TK1 mRNA was detected as target mRNA by flow cytometry and confocal laser scanning microscopy (CLSM) in HepG2, MCF-7 cells (positive control) and L02, MCF-10A cells (negative control). First, the cells (5 × 105) were seeded in 6-well plates for flow cytometry analysis. After the old medium was removed, fresh Opti-MEM containing the AIE-MB-loaded nanoparticle (CAIE‑MB = 1 μΜ) was added. After the cells were incubated for 4 h, fresh PBS replaced the Opti-MEM to wash the cells. Finally, cells were digested by trypsin and measured by flow cytometry. After that, four kinds of cells (3 × 105) were seeded in four confocal dishes for CLSM analysis, respectively. The cells were cultured for 12 h in culture medium. Then the original medium was replaced with fresh Opti-MEM containing AIE-MB-loaded nanoparticle (CAIE‑MB = 1 μΜ). After the cells were incubated for another 4 h, fresh PBS was added to replace Opti-MEM to wash the cells. Finally, PBS (1 mL) was added before imaging. The resulting dishes were observed by CLSM at a 405 nm laser, in which the emission collected at 430−500 nm and 510−600 nm. Statistical Analysis. The results are illustrated as the mean ± standard deviation (SD). The difference was evaluated by Student’s t test and considered statistically significant and very significant if p < 0.05 (*) and p < 0.01 (**), respectively.

3. RESULTS AND DISCUSSION Preparation and Characterization of AIE Fluorophore and Polymer. First, a novel AIE fluorophore (TPEQ) and TPEQ-Br were prepared as illustrated in Figure 1. Compound TPEQ was synthesized through aldol reaction of FTPE and quinoxalinone (QX), in which QX was obtained through the method previously reported by our group.48 On this basis, compound TPEQ-Br was further synthesized for labeling on the AIE-MB. The 1H NMR, 13C NMR, and LCMS analysis demonstrated that the compounds were successfully synthesized (Figures S1−S6). Apart from that, in order to transport 3622

DOI: 10.1021/acsbiomaterials.9b00627 ACS Biomater. Sci. Eng. 2019, 5, 3618−3630

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ACS Biomaterials Science & Engineering

Figure 2. (A) UV−vis absorption spectrum of TPEQ solid. (B) Fluorescent excitation and emission spectra of TPEQ solid. (C) The fluorescent emission spectra of TPEQ solid and TPEQ THF solution with λex of 353 nm. (D) The fluorescent excitation and emission spectra of TPEQ and MB-AMCA.

Figure 3. (A) Emission spectra of TPEQ in the mixture of THF and distilled water. (B) Emission spectra of TPEQ with different concentrations in pure THF. λex of 353 nm.

resulted from the π−π* transition of the TPEQ (Figure S9). Moreover, the fluorescent spectra exhibited a wide range of excitation wavelength from 300 to 490 nm and the emission maxima at 545 nm, in which the quantum yield (Φ) was 37.1%. In contrast, when TPEQ (1 × 10−5 M) was dissolved in pure THF, the emission maxima were shifted to 533 nm, and the Φ was 9.2% (Figure 2C, Supporting Information, Figure S10). This was attributed to the intramolecular charge-transfer process in TPEQ, which was widespread in donor−acceptor system AIE fluorophores.49−54 Finally, in order to determine the FRET effect between TPEQ and AMCA, the excitation and emission spectra of TPEQ and MB-AMCA were determined (Figure 2D, Supporting Information, Figure S11). Interestingly, both excitation and emission spectra of AMCA were completely overlapped with the excitation spectrum of TPEQ, which meant that the two fluorophores exhibited a significant FRET effect and could be excited by one laser. AIE Property. The majority of TPE-derived molecules exhibited AIE characteristics. To obtain insight into the AIE

the AIE-MB into cells, an amphiphilic pH-responsive polymer (7) was synthesized according to our previous work (Figure S7).43 The amphiphilic polymer could self-assemble into the nanoparticle and encapsulate the AIE-MB. The numberaverage molecular weight (Mn = 1.95 × 104 Da) of the polymer measured by GPC was consistent with NMR characterization (Mw = 2.02 × 104 Da), in which the PDI was 1.24. Finally, as a pH-responsive amphiphilic polymer, the pKa of polymer was also measured via acid−base titration (6.24), while the evaluation of pH responsiveness also demonstrated that the polymer was ultrasensitive to pH variation (Figure S8). After that, the photophysical properties of compound TPEQ were studied, including UV−vis absorption and fluorescent spectra. Because TPEQ was an AIE fluorophore, the spectra of its solid powder were first measured. UV−vis absorption spectrum and fluorescent excitation and emission spectra were shown in panels A and B, respectively, of Figure 2. In the UV− vis absorption spectrum, TPEQ exhibited strong UV−vis absorption from 300 to 450 nm. The higher energy absorption 3623

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Figure 4. Optimized structures and frontier molecular orbitals for TPEQ. Calculations were carried out by density functional theory calculations at the B3LYP/6-31G* level.

Figure 5. (A) Imaging of AIE-MB and MB-AMCA stripes on the PAGE gel. (B) The fluorescence intensity changes of AIE-MB upon the increase of the concentration of target DNA with λex of 353 nm. (C) The approximate first-order linear correlation of AIE-MB with different concentration of target DNA. (D) AIE-MB fluorescence changes of the different concentrations of DNase I with λex of 353 nm. (E) The approximate first-order linear correlation of AIE-MB with the different concentration of DNase I. F was the fluorescence intensity of different concentration of target DNA or DNase I. F0 was the fluorescence intensity when the concentration of target DNA was 0 nM or the concentration of DNase I was 0 U/L.

fluorophore prefer to relax via a radiative-decay pathway. To further confirm the AIE property, the emissive spectra of TPEQ with different concentrations were measured with λex of 353 nm in pure THF (Figure 3B). The fluorescent feature of TPEQ was insensitive at the concentrations of 1 × 10−7, 1 × 10−6, and 1 × 10−5 M. However, with the increase of concentration (1 × 10−4, 1 × 10−3, and 1 × 10−2 M), the emission intensity enhanced significantly. The above results indicated that TPEQ exhibited obvious AIE properties. Theoretical Investigations. To better investigate the optical behaviors of TPEQ molecule, quantum chemical calculations were performed using the B3LYP/6-31G* method via the Gaussian 09 program. Figure 4 showed the frontier molecular orbitals and the optimized structures of TPEQ molecule. The HOMO was localized on the TPE unit, and the quinoxalinone unit had less contribution. In contrast, the LUMO was mainly localized on the quinoxalinone unit, but the phenyl ring of the TPE unit also had considerable

property of TPEQ, the fluorescent spectra in THF/water solvent mixture and in pure THF were investigated (Figure 3). Because of the wide excitation wavelength of TPEQ and finally application of AIE-MB, the excitation wavelength of 353 nm was selected, which was the same as the excitation maxima of AMCA. TPEQ in pure THF solution (1 × 10−5 M) emitted weak fluorescence (533 nm, Figure 3A). With the increase of water fractions (f w) from 10% to 70%, the fluorescent intensities of TPEQ were insensitive but progressively redshifted to 545 nm. In dilute solutions, the weak emissive property of TPEQ resulted from the molecular free rotations, which enhanced nonradiative decay. Moreover, the polarity of the solvent medium was increased with the addition of water, which induced the emission of TPEQ (with donor−acceptor structure) red-shift.38 However, upon further increase of f w to 80% and 90%, the fluorescent intensities enhanced rapidly. The reason was that the aggregation of TPEQ restricted the molecular free rotations, which made the photoexcited 3624

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Figure 6. Characterization of the nanoparticle (NP) and AIE-MB-loaded nanoparticle (AIE-MB-NP). (A) Size distribution of two nanoparticles. (B) Zeta potential of two nanoparticles. (C) TEM image of the nanoparticle. (D) TEM image of AIE-MB-loaded nanoparticle.

contribution. Moreover, HOMO−1 was localized on the whole molecule while LUMO+1 was TPE centered, and the quinoxalinone had less contribution. The localized frontier molecular orbitals of TPEQ show that the charge of the molecule will redistribute upon photoexcitation. Meanwhile, the TPE group will dissipate the absorbed energy through rotating around the central double bond. Thus, the TPEQ molecule shows high luminescent properties when the rotating motion is restricted through aggregation. Preparation and Characterization of AIE-MB. MB was a common nucleic-acid-based fluorescent probe for imaging DNA and RNA in a purified environment. The stem-loop hairpin structure could hybridize with target DNA or RNA and cause the hairpin to open, which separated the fluorophore and quencher. As a result, the target recognition could effectively convert into a fluorescence signal. In contrast to the traditional MB, herein, the quencher was replaced by TPEQ, and a new AIE-MB was produced, in which the AIE fluorophore (TPEQ, acceptor) performed the FRET effect with the ACQ fluorophore (AMCA, donor). In order to achieve this purpose, TPEQ should be first connected onto the AIE-MB by suitable method. After trying different methods, site-specific labeling by phosphorothioate diesters on DNA sequences was chosen to connect TPEQ onto the MB-AMCA (Figure S13). The reaction was vibrated in DMSO at 50 °C for 20 h and treated by EA and n-butanol before it was dried by a rotator. The crude product was further purified by HPLC to obtain a pure product. In order to confirm the successful preparation of target AIE-MB, the product was characterized by LC-MS and denaturing PAGE gel. The theoretical mass of the AIE-MB was 12939.18, in which the observed mass was 12941.30 (Figure S14). This error was within the allowable range. In addition, the PAGE gel was immersed into ethidium bromide (EB) solution for the DNA staining, and the result was observed by a BioRad imaging system, in which the AIE-MB and MB-AMCA stripes

were obviously differentiated (Figure 5A, Supporting Information, Figure S15). These results confirmed that TPEQ was successfully conjugated with the MB-AMCA. Fluorescence Response of AIE-MB To Target DNA and DNase I in Vitro. The pivotal problem of nucleic-acidbased fluorescent probes is intrinsic interferences due to cellular components. To overcome this challenge, the AIE-MB containing both AIE and ACQ fluorophores was designed for detecting specific and false-positive signals in tumor and normal cells, respectively. Herein, TK1 mRNA was chosen as the targeted signal because it was associated with cell division and had been considered as a biomarker for tumor growth.55 In order to investigate the characteristic of AIE-MB, the fluorescence response to the target DNA and DNase I was investigated, in which the target DNA was a synthetic TK1 DNA and the sequence was the same with TK1 mRNA. First, to investigate the behavior of AIE-MB for detecting specific mRNA, the fluorescence intensity of AIE-MB in PBS solution (pH 7.4) was monitored upon changing the target DNA concentration. The fluorescence of the solution was excited with the maximum excitation wavelength of AMCA. Without any target DNA, the solution emitted weak fluorescence because of the water-solubility of the AIE-MB and the FRET effect between AMCA (donor) and TPEQ (acceptor). However, when the AIE-MB was incubated with the different concentration of synthetic target DNA for 30 min in PBS solution, the stem-loop hairpin of AIE-MB could hybridize with target DNA and open the hairpin, which determined that the fluorescent labels (TPEQ and AMCA) of AIE-MB were separated and the FRET effect was eliminated. As a result, the fluorescence intensity of AMCA increased gradually upon the increase of the concentration of target DNA (0−150 nM, Figure 5B). Figure 5C demonstrated the relationship between the concentration of target DNA and the fluorescence intensity of AMCA. The inset of Figure 5C described an approximate first-order linear correlation from 0 3625

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Figure 7. (A−D) Cell viability of four kinds of cells with different concentrations of TPEQ (A, C) and AIE-MB-loaded nanoparticle (B, D) (n = 5). (E, F) Time-dependent flow cytometry of L02 (E), HepG2 (F) cells treated with Cy5.5-loaded nanoparticle (1 μM).

fluorescent probes is effective delivery of probes. In order to deliver AIE-MB into cells, an amphiphilic pH-responsive copolymer was synthesized according to our early work.43 After that, the nanoparticle was assembled by dripping the THF solution of polymer into distilled water. Moreover, the pH responsiveness of the nanoparticle had been determined in our early work.43 The AIE-MB-loaded nanoparticle was prepared by dripping the mixture of polymer and AIE-MB into distilled water. Then, to characterize the two nanoparticles, size and zeta potential were measured by DLS, while TEM was used to visualize the morphology. As a result, the average hydrodynamic diameters were approximately 56 and 121 nm for two nanoparticles (Figure 6A). In the meantime, spherical nanoparticles were observed on TEM imaging with average diameters of approximate 39 and 92 nm, respectively (Figure 6C,D). The results demonstrated that the average diameters on TEM were slightly smaller than the average hydrodynamic diameters measured by DLS. Moreover, the results of both TEM and DLS showed that the average diameters of AIE-MB-loaded nanoparticle were bigger than that of the nanoparticle, because AIE-MB was successfully wrapped into the nanoparticle. After that, the zeta potential results of the nanoparticle and AIE-MB-loaded nanoparticle also illustrated that AIE-MB was successfully encapsulated into the nanoparticle. As shown in Figure 6B, the positive charge surface of the nanoparticle (zeta potential 16.3 ± 1.0 mV) resulted from the TEPA (tetraethylenepentamine, composition on the polymer mPEG-b-P(DPA-co-GMA-TEPA-C14)). Moreover, the positive charge was beneficial for encapsulating AIE-MB. In contrast, after the negatively charged AIE-MB was successfully encapsulated into the nanoparticle, zeta potential decreased to 4.3 ± 1.0 mV (Figure 6B). Finally, for determining the stability in plasma, culture medium (containing FBS) was used to disperse the AIE-MB-loaded nanoparticle. The size was measured by DLS at 0 and 48 h (Figure S17). The result demonstrated that the AIE-MB-loaded

to 50 nM (R = 0.9982), and the limit of detection for target DNA of 0.96 nM (3σ/slope rule) could be deduced.22 Consequently, the fluorescence of AMCA represented the specific response to target DNA and indicated that AIE-MB could effectively recognize the target DNA. After that, to investigate the behavior of AIE-MB for detecting nonspecific degradation, the fluorescence intensity change of AIE-MB in PBS solution was monitored upon changing the concentration of DNase I. Without any DNase I, the solution emitted weak fluorescence. However, when the AIE-MB was incubated with the different concentration of DNase I (0−200 U/L, Figure 5D) at 37 °C for 30 min, the AIE-MB was degraded into fragments, which caused the fluorescent labels (TPEQ and AMCA) dispersing into the solution. With the addition of DNase I, the amount of TPEQ separating from hydrophilic DNA increased gradually, in which TPEQ was insoluble in water. As a result, TPEQ was aggregated and emitted strong green fluorescence. Meanwhile, the fluorescence intensity of AMCA also increased gradually. Figure 5D demonstrated the relationship between the concentration of DNase I and the fluorescence intensity. The inset of Figure 5E described an approximate first-order linear correlation from 0 to 80 U/L (R = 0.9991), and the limit of detection for DNase I of 1.6 U/L (3σ/slope rule) could be deduced. Consequently, the fluorescence of TPEQ represented the degradation of nuclease and indicated that AIE-MB could effectively identify the nonspecific degradation of AIE-MB. Comparing the fluorescence response of AIE-MB to target DNA and DNase I, the fluorescence of AMCA and TPEQ could represent the specific and nonspecific response of AIEMB respectively (Figure S16). Through the different fluorescence, the specific signal to target DNA was efficiently distinguished from the false-positive signal arising from nuclease degradation. Preparation and Characterization of the Nanoparticle. Another important problem of nucleic-acid-based 3626

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Figure 8. Flow cytometry analysis in HepG2, MCF-7, MCF-10A, and L02 cells after treatment with the AIE-MB-loaded nanoparticle (1 μM) for 4 h. (A) Flow cytometry analysis of AMCA for pairing with TK1 mRNA in HepG2 and L02 cells. (B) Analysis of TPEQ for nonspecific degradation in HepG2 and L02 cells. (C) Fluorescence intensity of AIE-MB in HepG2 and L02 cells (n = 5). (D) Flow cytometry analysis of AMCA for pairing with TK1 mRNA in MCF-7 and MCF-10A cells. (E) Analysis of TPEQ for nonspecific degradation in MCF-7 and MCF-10A cells. (F) Fluorescence intensity of AIE-MB in MCF-7 and MCF-10A cells (n = 5).

nanoparticle was stable in plasma because the size at 0 h (118 nm) and at 48 h (120 nm) remained unchanged. In Vitro Cytotoxicity Study and Cellular Uptake Study. Since the TPEQ and AIE-MB-loaded nanoparticles were utilized in living cells, biocompatibility was a vital factor and should be first considered.56−59 Hence, the potential cytotoxicity of TPEQ and AIE-MB-loaded nanoparticles was evaluated by a standard MTT assay. According to previous reports,60 MCF-10A, L02 cells and MCF-7, HepG2 cells were chosen as the negative control and the model target cells because TK1 mRNA was overexpressed in MCF-7 and HepG2 cells but not in MCF-10A and L02 cells. Therefore, the four kinds of cells were treated for 48 h with TPEQ and AIE-MBloaded nanoparticle at maximum concentrations of 20 and 100 μM, respectively. Cell viability of four kinds of cells remained high (approximate 90%, Figure 7A−D), which demonstrated that both TPEQ and AIE-MB-loaded nanoparticles were low cytotoxicity. In order to determine the time it took for a nanoparticle to completely enter cells, the Cy5.5-loaded nanoparticle was prepared to investigate the property of cellular uptake. The cells were first incubated with the Cy5.5-loaded nanoparticle for 0.5, 1, 2, 4, and 6 h, and the fluorescence in the cells was measured (Figure 7E,F). The results illustrated that the fluorescence intensity gradually increased with time before reaching its maximum at 4 h. Therefore, the nanoparticle could completely enter cells within 4 h. In subsequent experiments, the cells were incubated with the AIE-MB-loaded nanoparticle for 4 h before it was detected. Intracellular Imaging of the AIE-MB-Loaded Nanoparticle. Having demonstrated its success in responding to target DNA and degradation of nuclease in vitro, the AIE-MB

was further investigated for the ability of imaging specific target mRNA and nonspecific degradation in living cells. HepG2, MCF-7 cells and L02, MCF-10A cells were incubated for experiments as the model target cells and the negative control, respectively. Four kinds of cells were scanned by flow cytometry and CLSM to study the behavior of AIE-MB in living cells after treatment with the AIE-MB-loaded nanoparticle. First, HepG2, MCF-7, MCF-10A, and L02 cells were treated with AIE-MB-loaded nanoparticle for 4 h. The results were measured by flow cytometry (Figure 8). Figure 8A,D demonstrated that strong blue fluorescence (specific signal) of AMCA was detected in HepG2 and MCF-7 cells, which was due to the AIE-MB pairing with overexpressed TK1 mRNA. This specific signal illustrated that AIE-MB could efficiently detect the target mRNA in tumor cells. However, weak fluorescence of AMCA was also detected in L02 and MCF-10A cells. This resulted from the degraded part of AIE-MB by nuclease degradation or other cellular components. In the meantime, in Figure 8B,E, the strong green fluorescence of TPEQ was detected in L02 and MCF-10A cells, and very weak green fluorescence was observed in HepG2 and MCF-7 cells. The green fluorescence was emitted from aggregated TPEQ which was separated from hydrophilic DNA and aggregated together in the cytoplasm. As a result, the TPEQ green fluorescence (false-positive signal) could represent the nonspecific degradation of AIE-MB, which meant that the AIE-MB degradation by nuclease or other cellular components could be effectively detected. Finally, the specific signal for pairing with TK1 mRNA and the false-positive signal arising from nonspecific degradation could be efficiently distinguished by the different fluorescence in cells. 3627

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Figure 9. CLSM images of HepG2, MCF-7, MCF-10A, and L02 after treatment with the AIE-MB-loaded nanoparticle (1 μM) for 4 h. The fluorescence of AIE-MB is displayed in blue and green by excitation with a 405 nm laser, in which the emission was collected at 430−500 nm for AMCA and 510−600 nm for TPEQ.

in tumor cells by pairing with it and emitting a specific signal (blue AMCA fluorescence signal). In contrast, in normal cells, part of the AIE-MB was degraded by nuclease or other cellular components, which could be represented by a different color signal (green TPEQ fluorescence signal). Obviously, compared with the prevailing nucleic-acid-based fluorescent probes, the greatest advantage of the AIE-MB was that it could image both the specific pairing signal with target mRNA and the endogenous degradation signal by different color fluorescence, which could distinguish the specific and false-positive signals and improve the detection accuracy. The experimental results show that we successfully synthesized a new AIE fluorophore with remarkable AIE property, and further, we built an AIEMB for improving the accuracy of the tumor detection result. We hope that our new AIE-MB design that cooperates with AIE and ACQ fluorophores can be a potential tool for tumor diagnosis in the near future.

For more comprehensive imaging of AIE-MB in living cells, the cells were observed with CLSM. HepG2, MCF-7, MCF10A, and L02 were incubated with the AIE-MB-loaded nanoparticle for 4 h in confocal dishes and then observed with CLSM directly (Figure 9). As shown in Figure 9A,C,E,G, I,K, strong blue fluorescence and weak green fluorescence in HepG2 and MCF-7 cells were observed. These results illustrated that AIE-MB could image the TK1 mRNA specifically and a fraction of AIE-MB was degraded in cells. In contrast, bright green and blue fluorescence were observed in L02 and MCF-10A cells, which resulted from nonspecific degradation (Figure 9B,D,F,H,J,L). The blue AMCA fluorescence was just dimmer than the specific signal in HepG2 and MCF-7 cells, but the green TPEQ fluorescence (falsepositive signal) arising from aggregated TPEQ was very different from that in HepG2 and MCF-7 cells. Comparing the imaging of the four kinds of cells, AIE-MB could efficiently distinguish the tumor and normal cells by the different blue and green fluorescence.

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4. CONCLUSIONS In summary, we designed a new AIE fluorophore (TPEQ) and then proposed an AIE-MB to improve the accuracy of the tumor detection result by efficiently detecting both specific and false-positive signals of the AIE-MB in living cells. In aqueous solution, TPEQ showed a wide range of excitation wavelength and exhibited splendid AIE characteristics, while the AIE-MB expressed excellent fluorescence response to target DNA and DNase I. In vitro, the AIE-MB could image the target mRNA

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00627. NMR, MS, FTIR, scheme showing synthetic route, fluorescent spectra, and DLS data (PDF) 3628

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for P.L.: [email protected], Tel.: +86-21-34188822, Fax: +86-21-34188822. *E-mail for X.Z.: [email protected], Tel.: +86-21-34206899, Fax: +86-21-54741297. ORCID

Chunyang Yu: 0000-0003-1175-8362 Xinyuan Zhu: 0000-0002-2891-837X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (51690151, 21805131, 21774077), the National Basic Research Program (2015CB931801), the National Postdoctoral Program for Innovative Talents (BX201700152), Shanghai Municipal Science and Technology Commission 2019 “Science and Technology Innovation Action Plan” Funded Project (19441905900), the China Postdoctoral Science Foundation Funded Project (2017M621485), and the Interdisciplinary Program of Shanghai Jiao Tong university (YG2016QN56).

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DOI: 10.1021/acsbiomaterials.9b00627 ACS Biomater. Sci. Eng. 2019, 5, 3618−3630

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DOI: 10.1021/acsbiomaterials.9b00627 ACS Biomater. Sci. Eng. 2019, 5, 3618−3630