Triphenylquinoline (TPQ)-Based Dual-State Emissive Probe for Cell

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Triphenylquinoline (TPQ)-based dual-state emissive probe for cell imaging in multicellular tumor spheroids Wenbo Dai, Pai Liu, Shuai Guo, Zhiqi Liu, Mengni Wang, Jianbing Shi, Bin Tong, Tianqing Liu, Zhengxu Cai, and Yuping Dong ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00596 • Publication Date (Web): 21 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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ACS Applied Bio Materials

Triphenylquinoline (TPQ)-based dual-state emissive probe for cell imaging in multicellular tumor spheroids Wenbo Dai,† Pai Liu,† Shuai Guo,† Zhiqi Liu,† Mengni Wang,† Jianbing Shi,† Bin Tong,† Tianqing Liu,*‡ Zhengxu Cai,*† and Yuping Dong† †

Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green

Applications, School of Materials Science & Engineering, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing, 100081, China. ‡

QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane, QLD 4006, Australia.

KEYWORDS: multicellular tumor spheroids; quinoline-based fluorophores; dual-state emission; one-pot reaction; twisted intramolecular charge transfer (TICT) effect.

ABSTRACT: Insufficient intratumoral penetration and limited stroma distribution of the imaging probes or theranostics can lead to poor quality diagnosis or therapeutic resistance. Multicellular tumor spheroids can recapitulate the physiological environment of tumor tissues with the extracellular matrix, and thus a better in vitro tumor model to evaluate the imaging performance and barrier penetration capability of an advanced cancer imaging probes. In this paper, we designed and synthesized a series of quinoline-based fluorophores with strong emissions in both solution

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and solid states. The quinoline core can be constructed via a one-pot iron-catalysis reaction. Optical properties and single crystal structures of these quinoline derivatives were tuned by varying the substitutes at 6-position of the quinoline core. The twisted intramolecular charge transfer effect can enhance the fluorescent efficiency, resulting in high quantum yield of TPQ-TPA in both solution (70%) and solid (48%) states. In addition, TPQ-TPA exhibited a good biocompatibility and can deeply penetrate into 3D tumor spheroids within 12 h. The results indicated quinoline can be a new fluorescent scaffold and the employment of quinoline-based probes will provide a new platform for biological application.

INTRODUCTION Insufficient intratumoral penetration and limited stroma distribution of the imaging probes or theranostics can lead to poor quality diagnosis or therapeutic resistance. 1 The penetration is normally impeded by the densely packed cancer cells and the extracellular matrix. 2,3 Hence, there is a need to develop advanced imaging probes with superior imaging performance, great barrier penetration capability and biocompatibility to improve the imaging quality of tumor tissues. 4 However, conventional monolayer cancer cell culture does not contain tissue-like extracellular matrix network and physical mass transfer gradient, leading to poor predictive power for the screening of novel probes.5 Multicellular tumor spheroids can closely recapitulate the microenvironment of avascular tumor tissues, such as cell-cell and cell-matrix interactions, spatial architecture, hypoxia, physiological responses, gene expression patterns and drug penetration mechanisms.6-9 Therefore, 3D tumor models are being increasingly used in cancer research and used as a screening tool for the development of imaging probes or drug molecules. 10-12 The detailed evaluation of cancer imaging probes within multicellular activity can add valuable information to


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their preclinical studies and may have immediate implications for fluorescence-guided surgical approach to improve clinical outcome. 13 Most of traditional fluorescence probes showed poor performance due to the presence of aggregation-caused quenching (ACQ) effect.14,15 In addition, another type of molecules, namely aggregation-induced emission (AIE) molecules could exhibit non-radiative decay via intramolecular rotation in dilute solution whereas becoming highly emissive in aggregated or solid state.16-20 Nevertheless, the dual-state emission (DSE) materials with a strong emission in both solution and solid state showed the advantage 3D spheroid models staining. Since the fluorescent probe need to penetrate into the spheroids, large concentration gradient existed between the outer cells and inner cells. AIE materials suffered a weak emission in inner cells due to the lack of aggregation. While ACQ materials suffered a fluorescence quenching in outer cells due to the high concentration. Therefore, the DSE fluorescent molecules fill the substantial gap between AIE and ACQ materials.21-25 Recently, some works on DSE fluorescent materials have been reported. For example, Wan and his co-workers reported a series of arch-bridge-type fluorophore with a high dual emissive efficiency.26 Tang and his co-workers synthesized triphenylamine-based compounds with intense emission in both solution and solid states on the basis of conjugation-induced rigidity strategy.27 Our group also presented a pioneering work on the investigation of DSE materials. 28 A simple molecule, 2, 3, 4, 5-tetraphenyl-1H-pyrrole was designed and synthesized. It showed a strong emission in both solution and solid states with high quantum yields. However, within the context of biological systems, the highly emissive probes are still in high demand. To address this limitations, new fluorescent materials are needed, especially the effort for pursuing the new fluorescent scaffolds.29


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Amount all the new fluorescent scaffolds, quinoline-based fluorophores are highly attractive for their synthetic versatility.30-32 In addition, quinoline is widely observed in natural products and drugs, indicating its low cytotoxicity and high cell permeability. 33 Very recently, Chenoweth et al developed a series of quinoline-based probes to live-cell imaging.34 Tang et al also reported isoquinolinium derivatives with an AIE character for mitochondrial and bacterial imaging.35,36 Despite considerable efforts to develop new synthetic quinoline scaffold, the development of quinoline-based fluorophores with desired structural and biological properties is still a challenging task.34 In this paper, we introduce a small, yet tunable quinoline-based scaffold in two steps. The photophysical properties of triphenylquinoline (TPQ) molecules can be tuned via functionalizing with different electron donating and withdrawing substitutes. All the molecules showed a dualstate emissive property. The density functional theory (DFT) and X-ray structural analysis were used to deduct the emission mechanism. We also studied the biocompatibility of TPQ-TPA by using 2D cellular and 3D spheroid models. We have demonstrated that TPQ-TPA has high cell permeability throughout 3D spheroids. All the results indicated that quinoline material has great potentials to be used as fluorescent probe for intratumoral analysis. Our scaffold can be easily functionalized and optimized to present potential advances over classic and contemporary dyes.


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Scheme 1. Synthetic routes and molecular structures of TPQ-based molecules. RESULTS AND DISCUSSION Scheme 1 shows the molecular structures and synthetic route of four TPQ-based molecules. Compound 4 was synthesized via a three components reaction of corresponding benzaldehyde, phenyl acetylene, and 4-bromoaniline under the catalysis of FeCl3 with a high yield of 63%.37 Then compound 4 was further functionalized with different electron donating or withdrawing groups via Pd mediated Suzuki coupling reaction (Details see Supporting Information, SI). All the molecular structures were confirmed with mass spectra (MS) and nuclear magnetic resonance (NMR) spectra (SI). The purities of TPQ-based molecules were checked with elemental analysis. Single crystals of all target compounds were analyzed by single crystal X-ray diffraction (Figure 2 and Figure 3). Thermal gravity analysis (TGA) results were shown in Figure S1 (SI). The temperature for 5% weight loss was higher than 300 ℃. The good thermal stability will be favored for many applications.


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Absorption spectra of TPQ-based molecules were recorded in THF solution. TPQ-TPA and TPQ-OCH3 exhibited three absorption bands in the region of 250-450 nm, as depicted in Figure 1a, while TPQ and TPQ-CN showed two absorption bands with a shoulder peak in the same region. Since the quinoline core is a weak electron-withdrawing unit, the attachment of electron donating groups (TPA and 4-methoxyphenyl) formed a donor-acceptor (D-A) structure, thus redshifting the wavelengths of absorption to long wavelength region. The phenyl and benzonitrile groups in TPQ and TPQ-CN, respectively, only extended the conjugation of the quinoline core. The lack of D-A interaction resulted in the short absorption wavelengths. The emission spectra of TPQ-based compounds were shown in Figure 1b. TPQ and TPQ-CN exhibited almost identical emission spectrum, while the maximum emissive wavelength of TPQOCH3 and TPQ-TPA gradually red-shift due to intramolecular D-A interaction. The maximum emissive wavelength in solid showed a slight bathochromic emission shift in comparison to the emission in THF solution except TPQ-TPA. The maximum emissive wavelength of TPQ-TPA in solid showed shorter wavelength than that of TPQ-TPA in solution state, likely due to the twisted intramolecular charge transfer (TICT) effect of these D-A molecules.38-42


6

a

1.0

TPQ TPQ-CN TPQ-OCH3

0.8

Normalized Emission

1.0

Normalized Absorbance

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


TPQ-TPA 0.6 0.4 0.2 0.0 250

300

350

400

450

0.8

TPQ-TPA

0.6 0.4 0.2 0.0 350

500

TPQ TPQ-CN TPQ-OCH3

b

70%

0%

TPQ

TPQ TPQ-CN TPQ-OCH3

0.8

TPQ-TPA 0.6 0.4 0.2

450

500

550

600

650

400

Wavelength (nm)

0%

c

0.0 400

Wavelength (nm)

d

1.0

Normalized Emission

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70%

TPQ-OCH 3

0%

450

500

550

600

Wavelength (nm)

80%

TPQ-TPA

0%

70%

TPQ-CN

Figure 1. (a) UV-vis absorption spectra and (b) Emission spectra of TPQ derivatives in THF solution, concentration: 1 × 10-5 mol/L (excitation wavelength: TPQ: 320 nm; TPQ-CN: 335 nm; TPQ-OCH3: 330 nm; TPQ-TPA: 377 nm). (c) Emission spectra of TPQ derivatives in solid state (excitation wavelength: TPQ: 310 nm; TPQ-CN: 320 nm; TPQ-OCH3: 310 nm; TPQ-TPA: 350 nm). (d) Images of TPQ derivatives in THF/water mixtures with different water volume fraction (fw) and solid state taken under 365 nm UV illumination. To check photophysics properties of TPQ-based aggregates, emission spectra were also recorded in the water-THF mixtures with different water volume fraction (fw). For TPQ-TPA, adding water with fw up to 60% causes an obvious bathochromic emission shift, due to the intramolecular charge transfer of TPQ-TPA. When fw is larger than 70%, the emission spectrum


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is blueshifted (Figure S3, SI). Similar trends were observed in TPQ and TPQ-OCH3 compounds. However, the emission wavelength of TPQ-CN compound lacking D-A interaction showed a redshift when the aggregates formed. The fluorescent intensity of TPQ-TPA decreased gradually when the fw increase to 70%, while the fluorescent intensity of the other three compounds gradually increase until the aggregates formed (Figure S3, SI). The size of the aggregates were further confirmed by dynamic light scattering (DLS, Figure S5). The results indicated that large aggregates of all TPQ derivatives were formed as the water contents were higher than 70%. Generally, the compounds with multiple rotatable phenyl rings, such as the multiple phenyl pyrrole, triphenyl-isoquinolinium compound, may enable the compounds with AIE characteristics, because the fast rotations of phenyl rings can serve as non-radiative pathways to deactivate the excited states in solutions and such rotational motions can be restricted in the condensed state.16,17,43-45 However, our TPQ-based molecules showed a strong emission both in solution and in solid state. All the molecules showed the quantum yields (Q. Y.) higher than 10% both in solution and in solid state. It worth to mention that TICT effect always weaken the luminescent efficiency in D-A molecules.46 However, TPQ-TPA with the highest TICT effect showed the highest Q. Y. of 70% in solution and 48% in solid, respectively (Table 1). This highly emissive material is favorable for 3D tumor spheroids. Table 1. Photophysical properties of the TPQ-based compounds. Abs/THF

E m /THF

Stoke's shift

E m /solid

Ф F /THF

Ф F /solid

/solid

nm

nm

nm

nm

%

%

ns

TPQ

274, 323

399

76

402

25

11

0.83

TPQ-OCH 3

279, 332, 351

424

92

425

56

30

1.65

TPQ-TPA

263, 302, 377

492

115

481

70

48

4.24

Compounds


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TPQ-CN

279, 331

398

67

413

13

28

1.31

To further confirm the TICT effect in TPQ-based molecules, the absorption and emission spectra of TPQ-based molecules were measured in a variety of solvents covering a wide polarity range (Figure S6 and Figure S7, SI). The Lippert-Mataga polarity parameter ∆f is correlated to the polarity of solvents.28,47 Solvent polarity showed a dramatic effect on the fluorescence spectra of the large dipole molecules. This is because the more polar excited state resulting from a charge transfer is more stable than the less polar ground state in a polar solvent. Emission spectrum redshifts to a longer wavelength by increasing the polarity of the solvents. Therefore, the larger dipole moments of the excited state lead to a larger solvating effect. Figure S8 (SI) showed the Mataga-Lippert plot for the Stokes shift between the absorption and fluorescence spectra of TPQbased molecules. As shown in Figure S8, Stokes shift of TPQ-OCH3 and TPQ-TPA showed a linear relationship with increasing the polarity of the solvents, indicating the existence of charge transfer state in TPQ-OCH3 and TPQ-TPA molecules. However, the increases in the Stokes shift with increasing solvent polarity are rather small in TPQ and TPQ-CN molecules. In addition, although the solid line reproduces the experimental results in the solvent polarity parameter, large deviation was observed, suggesting that the charge transfer state in TPQ and TPQ-CN molecules was very weak. DFT calculations were carried out on the basis set of B3LYP/6-31G* via Gaussian 09. The frontier molecular orbitals of TPQ-based molecules were shown in Figure 2. The electron cloud of both the highest occupied molecular orbital (HOMO) energy levels and lowest unoccupied molecular orbital (LUMO) distributed throughout the conjugation backbones of TPQ-based molecules except TPQ-TPA molecule. As shown in Figure 2, the higher density of electron cloud is distributed on electron-donating TPA unit in HOMO level of the TPQ-TPA molecule, while


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the distribution tends to be denser around electron-withdrawing quinoline part in the LUMO level. This result indicates the occurrence of intramolecular charge transfer in TPQ-TPA upon excitation, which is responsible for its large solvent effect and redshift THF-water titration measurements.

Compounds

TPQ

TPQ-OCH3

58.7°

Crystal Structure

36.9° 19.9°

TPQ-TPA 52.6° 22.6° 14.0°

63.1° 19.7°

TPQ-CN 52.7° 34.8° 26.5°

31.9°

-2.00 eV

-1.94 eV

-1.95 eV

-2.37 eV

△E=3.98 eV

△E=3.78 eV

△E=3.27 eV

△E=3.95 eV

-5.98 eV

-5.72 eV

LUMO

HOMO -5.22 eV

-6.32 eV

Figure 2. Crystal structures and HOMO/LUMO energy levels of the TPQ derivatives. All the TPQ-based compounds can be crystallized via slow evaporation of CHCl3/MeOH (1:1) under ambient conditions. All the compounds belonged to the triclinic system except TPQ-CN compound. TPQ-CN showed a monoclinic molecular packing system. The conjugated backbone in TPQ is not fully coplanar. The twisted angles between the quinoline core and the adjacent phenyl rings were listed in Figure 2. The angles between phenyl rings at 2, 6-positions and quinoline core were typically smaller than the torsion angle between phenyl ring at 4-position and quinoline core. The smaller twisted conformation of the backbone can effective extend the


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conjugation and enhance the emissive intensity. In addition, the phenyl ring at 4-position with dihedral angles larger than 50° can enhance the fluorescence quantum yields in solid state, because the twisted conformation are preventing their molecules from close packing that forms detrimental species. Intermolecular arrangement of TPQ-based molecules is confirmed this result. As it shown in Figure 3, molecules were assembled to form layers via intermolecular interactions. The closest intermolecular distance between the quinoline centers of two juxtaposed molecules (Marked in red in Figure 3) was 4.48 Å (TPQ-CN in Figure 3d), which was larger than normal π-π interaction ( ＞3.6 Å). No effective π-π surface overlap was observed in all of the crystal structure of TPQ molecules. In addition, weak C-H···π interactions in TPQ-based molecules were observed (Marked in green in Figure 3). Therefore, all TPQ-based molecules showed a strong fluorescence in solid state.

a

b 5.603

5.592

7.527

7.254

c

d 4.479

4.479

Figure 3. Schematic intermolecular interactions in the crystal of (a) TPQ (b) TPQ-OCH3 (c)


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TPQ-TPA (d) TPQ-CN. The interaction distances of C-H···π center is green, and π-π center is red (carbon, gray; hydrogen, white; nitrogen, yellow). Since the quinoline derivatives showed low cytotoxicity and high cell permeability, compound TPQ-TPA was selected to stain the HeLa cells. The results showed TPQ-TPA can be taken up by HeLa cell and visualized by live cell imaging (Figure 4) on the basis of hydrophobic interactions and dual-state emissive property (Experimental details see SI). Cell apoptosis experiments were conducted with flow cytometer. Cell viability rates were above 95% after 72 h incubation at the working concentration of 1 × 10 -5 mol/L (Figure S9, SI). The results indicated TPQ-TPA showed a limited cytotoxicity, which can be a new probe for cell tracking.

a

b

c

75 μm

Figure 4. Confocal images of HeLa cells stained with TPQ-TPA (excitation wavelength: 380 nm): (a) Bright field, (b) Fluorescent field, and (c) Merged image of (a) and (b). The biocompatibility of TPQ-TPA at 3D tumor spheroid level was also carried out (Figure 5). In comparison with monolayer culture, 3D tumor spheroids, which consist of cells with different growth phases and extracellular matrix, are a better mimic of avascular tumor microenvironment. We established 3D HeLa tumor spheroids using microfabricated concave microwell devices. Histological analysis of H&E staining was used to show the cell condition in the spheroids. Figure


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5 showed that 3D tumor spheroid models have the uniform spherical morphology with size around 500 µm, and closely packed cell structure and cell-cell contacts. After the treatment of TPQ-TPA up to 12 h, no significant morphological and cellular structural changes have been observed. The size of the tumor spheroids is slightly increased after 12 h, suggesting the cells are proliferating within the 3D tissue and TPQ-TPA did not affect spheroid growth. The images showed no clear signs of necrosis after the treatment, suggesting the good biocompatibility of this fluorescent probe to the 3D tutor spheroid model. In addition, the tumor spheroids are suitable for studying the penetration behavior of the fluorescent probes.

Figure 5. Histochemical H&E staining on 3D tumor spheroids treated with control or TPQ-TPA at different time points. Scale bar: 200 μm. To confirm the fluorescent probe with good penetration capacity, we evaluated the transport of TPQ-TPA inside tumor spheroids at different time points (Figure 6). The middle layer of tumor spheroids was focused along the Z-axis based on both fluorescent signals and bright field channel. A time-dependent penetration through tumor spheroids was observed after the incubation for 2 h, 6 h, or 12 h. At 2 h post-treatment, green fluorescent spots in the confocal images indicated a heterogeneous distribution of TPQ-TPA in the out region of the 3D model. As the time increases, there was a clear uniform distribution formed in the outer region, suggesting more homogenous uptake at 6 h treatment, while TPQ-TPA penetrated to the center of the 3D spheroids after 12 h


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incubation. This indicated that even with the extracellular matrix as a transport barrier, TPQ-TPA can penetration around 500 μm tissue over 12 h. This suggests that TPQ-TPA has great potentials to be used as fluorescent probe for intratumoral analysis.

Figure 6. Penetration of TPQ-TPA within the 3D tumor spheroids over 2 h, 6 h, or 12 h. Scale bar: 200 μm, excitation wavelength: 405 nm. CONCLUSIONS In conclusion, our results demonstrated iron-catalysis synthetic strategy to develop the quinoline as a fluorescent scaffold “core”. The quinoline core can be easily functionalized with electron donating and withdrawing groups to tune the optical properties, which provides a great opportunity for the exploration and optimization of TPQ-based materials for a wide range of applications. In addition, all the quinoline-based molecules showed DSE property, and their structure-


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photophysical relationship of the TPQ scaffold were well investigated by DFT calculation and single crystal analysis. The results provided a full demonstration to reveal the diverse potential of quinoline-based fluorogens. In addition, TPQ-TPA was a useful imaging probe for 3D tumor spheroids with a low cytotoxicity. These TPQ-based fluorophores with dual-state emissive property will provide a new platform for biological and biomedical applications. ASSOCIATED CONTENT Supporting Information (SI). Experimental details, TGA date, photophysical properties, 1H NMR and

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C NMR spectra and

mass spectra, single crystal data. CCDC 1853700, 1853702, 1853708, 1853710. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Z.X. Cai); [email protected] (T.Q. Liu). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Scientific Foundation of China (Grant Nos. 51803009, 51673024, 21474009, 51328302), Graduate Technological Innovation Project of Beijing Institute of Technology (2018CX10005), Beijing Institute of Technology Research Fund Program for Young Scholars. Dr Tianqing Liu is supported by the National Health and Medical Research Council (NHMRC) Early Career Fellowship (Grant No. 1112258).


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Table of Contents Graphic

1.0

Hexane Toluene EA THF DCM Acetone DMSO MeCN

TPQ-TPA

0.8

Normalized Emission

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 TPQ23 24 TPQ 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


0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 -0.8 400

450

500

550

600

650

700

Wavelength (nm) TPQ

TPQ-OCH3

TPQ-TPA

TPQ-CN

3D tumour spheroids

DSE

TPQ

TPQ-OCH3

TPQ

TPQ

TPQ-CN TPQ TPQ-TPA TPQ-TPA TPQ-OCH TPQ-TPA 3 TPQ-TPA TPQ-OCH TPQ-CN 3 TPQ-OCH3

TPQ3 TPQ-OCH

TPQ-CN

TPQ 3TPQ-TPA TPQ-OCH3 TPQ-TPA TPQ-OCHTPQ-CN TPQ-TPA TPQ-OCH 3 TPQ-TPA TPQ-CN

TPQ-CN

TPQ-CN

TPQ-CN


22

Triphenylquinoline (TPQ)-Based Dual-State Emissive Probe for Cell

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