Tailored Supramolecular Cage for Efficient Bio-Labeling

Abstract

Fluorescent chemosensors are powerful imaging tools used in a broad range of biomedical fields. However, the application of fluorescent dyes in bioimaging still remains challenging, with small Stokes shifts, interfering signals, background noise, and self-quenching on current microscope configurations. In this work, we reported a supramolecular cage (C_A) by coordination-driven self-assembly of benzothiadiazole derivatives and Eu(OTf)₃. The C_A exhibited high fluorescence with a quantum yield (QY) of 38.57%, good photoluminescence (PL) stability, and a large Stokes shift (153 nm). Furthermore, the CCK-8 assay against U87 glioblastoma cells verified the low cytotoxicity of C_A. We revealed that the designed probes could be used as U87 cells targeting bioimaging.

1. Introduction

Self-assembly allows for the efficient construction of supramolecular structures from relatively simple components. As a class of such architectures, metal-organic cages (MOCs) have gained significant attention in recent years [1,2,3,4]. Numerous MOCs exhibit astonishing multifunctional capabilities that have been created by regulating and controlling the coordination environment surrounding the core metal ions, as well as the suitable choice of organic ligands. Consequently, they have found applications in areas such as catalysis [5,6,7,8,9,10], molecular recognition [11,12,13,14,15,16,17], biological imitation [18,19,20,21,22], guest sequestration [23,24,25], reactive species stabilization [26], drug delivery [27,28,29], and membrane transportation [30,31], among others.

The diagnosis and treatment of disease require better and faster pathological analysis, as well as highly contrasting real-time biological images. Optical emission probes combined with disease diagnosis and treatment are becoming strong candidates, owing to their only slight disturbance of the systems and organs being researched. When the appropriate wavelength is used, the penetration depth of optical emission probes can be substantial, and then the light can reach regions of complex molecular structures that are inaccessible to other molecular probes. As a consequence, building supramolecular structures, which have strong fluorescence to achieve deeper tissue penetration for imaging or cell tracking studies to monitor biological processes, has recently attracted increasing attention [32,33]. Significantly, lanthanide ions with special optical and magnetic properties serve as functional centers on which to build the supramolecular structures, and which have found uses across various fields [34,35], such as luminous probes [36,37,38,39], analyte sensing [40,41], and tissue [42,43]. Furthermore, the pharmaceutical properties of lanthanide (III) ionic have also been demonstrated as antibacterial agents (anticancer, anticoagulant, anti-inflammatory, etc.), and play a crucial role in medicine, especially cancer diagnosis and treatment [44]. However, the difficulty of predicting the coordination numbers of lanthanides and coordination geometry makes the controlled self-assembly of predetermined structures quite challenging. Most studies have been limited to small systems, such as single-core beams or two-core helixes; the design and the application of three-dimensional lanthanide components have not been thoroughly studied.

In this context, we report the synthesis of three-dimensional (3D) dimetallic lanthanide complexes with saturated coordination spheres. Two-armed tri-toothed benzothiadiazole derivatives acted as organic ligands and coordination-driven self-assembly with Eu(OTf)₃ in a ratio of 2:3 to form a single structure. Trivalent lanthanides display a tendency to adopt nine-coordinate tricapped trigonal prismatic (TTP) arrangements around the metal ion in the solid state, and the 3D molecular buildings are capable of great fluorescence. Well-defined solid clusters enable this supramolecular cage structure to efficiently target the staining of living cancer cells with low biotoxicity (Figure 1).

2. Results and Discussion

2.1. The Preparation and Characterization of C_A

Highly predisposed O-N-O tridentate chelators with amide groups were introduced into linear shape molecules N²,N^2’-(benzo[c][1,2,5]thiadiazole-4,7-diylbis(4,1-phenylene))bis(N⁶,N⁶-diethylpyridine-2, 6-dicarboxamide) (L-B), to control the coordination of lanthanide ions and obtain highly ordered architectures [45,46,47]. The L-B was synthesized by amide condensation of freshly prepared 6-(diethylcarbamoyl)picolinic acid with 4, 4’-(benzo[c][1,2,5]thiadiazole-4, 7-diyl)dianiline. The formation of the supramolecular cage was quantitatively carried out, L-B (3 equiv.) and Eu(OTf)₃ (2 equiv.) were dissolved in 600 μL CH₃CN/CH₃OH (v/v, 1/1), respectively, and stirred in a 10 mL flask at room temperature for 5 h. After the reaction, 5 mL diethyl ether was added and centrifuged to obtain C_A as an orange-red solid (Scheme 1 and Figures S1–S9).

Multinuclear NMR (¹H, DOSY, COSY, and NOSEY) analysis (Figure 2 and Figures S10–S14), IR spectrum, and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) confirmed the formation of C_A. The ¹H NMR spectrum of C_A displayed only one set of ligand signals (Figure 2c), showing the formation of faces and vertices with a single stereochemical orientation. The shift in NMR signal could be observed after the formation of the supramolecular cage. As evidenced by the results, upfield chemical shifts were observed for the β-pyridyl protons He (from 8.066 to 7.146 ppm) and Hg (from 7.74 to 6.61 ppm) and γ-pyridyl protons Hf (from 8.324 to 7.629 ppm). Chemical displacement changes could also be observed in amide hydrogen Hd (from 10.052 to 7.225 ppm). Moreover, aromatic protons Ha, Hb, and Hc, belonging to 4,4’-(benzo[c][1,2,5]thiadiazole-4,7-diyl)dianiline, shifted upfield as well (Figure 2b,c). All of these chemical shift changes pointed to the formation of C_A. 2D DOSY NMR also revealed the formation of a single species in solution, with a diffusion coefficient of 4.79 × 10⁻¹⁰ m²/s. The infrared spectrum also confirmed the successful coordination-driven self-assembly of C_A (Figures S15 and S16). According to the results, the characteristic peaks of C=O (from 1686 to 1601 cm⁻¹) and N-H (from 1625 to 1575 cm⁻¹) were significantly changed.

Additionally, electrospray ionization-mass spectrometry (ESI-MS) indicated the successful assembly of C_A, and the detected ESI-MS data of the resultant cage was nearly identical to the theoretical data. The spectra showed a series of peaks with charge states ranging from 3⁺ to 6⁺, resulting from the sequential loss of OTf^- counterions or HOTf molecules. For example, peaks with m/z = 414.11 correspond to [Eu₂L₃(OTf)₆-6OTf^-]⁶⁺ charged species. (Figure 2d). The most abundant peak at m/z = 496.84 exhibited typical m/z splitting for a 5⁺ charged species and was assigned to [Eu₂L₃(OTf)₆-6OTf^--HOTf]⁵⁺. The average molar mass of the self-assembled cage C_A after deconvolution was 3378 Da, which was in good agreement with their predicted chemical compositions [C₁₂₆H₁₁₄N₂₄O₃₀F₁₈S₉Eu₂] and ruled out the formation of any subsequent undesirable complexes (Figures S17–S19).

The complete data for single crystal X-ray analysis could not be obtained despite numerous attempts. Consequently, we developed a computerized molecular model of the C_A (Figure 3a,b). Two europium ions were nine-coordinated by tridentate O-N-O chelating moieties from three distinct ligands in the energy-minimized structure. The metal-metal distance between europium ion centers in the energy-minimized structure was 19.17 Å (as measured by Materials Studio.) Contrary to L-B, C_A had a geometry-optimized structure that revealed one of the ligands to be severely twisted out of the ligand plane, which prevented a cavity from forming in the supramolecular cage. Scanning electron microscopy (SEM) revealed that C_A was in a good state of aggregation, and showed a uniform spherical morphology (Figure 3c). This matched the molecular structure that we modeled.

2.2. Optical Properties

The enormous Stokes shift and the outstanding PL QY are the distinguishing characteristics of C_A. Figure 4 shows the optical properties of C_A in acetonitrile. The UV-Vis absorption spectra (Figure S20) showed that the C_A possessed continuous absorption from the UV region to about 475 nm. The absorption bands at 210–250 nm could be ascribed to π-π* transition from aromatic C=C bonds; a peak at 275–340 nm was also observed, which may be attributed to the n-π* transition of C=O on the surface of C_A. Absorbance significantly increased compared with L-B, and an M-shaped peak appeared, which was attributed to the interaction between C=O bonds and Eu (III) ions within the process of self-assembly. The absorption peak located at 375–450 nm was assigned to the C=N absorption of pyridine-N in the carbon core and the polycyclic aromatic units. The excitation spectrum of C_A showed the best excitation wavelength of 400 nm, and the PL emission peak was centered at 553 nm (Figure 4a). Due to the C_A having a large Stokes shift reaching 153 nm, the small self-absorption of C_A could be expected, which enabled efficient PL emission. Under the optimum excitation wavelength, the absolute PL quantum yield of C_A was 38.57% (Figure S21). As illustrated, the decay profiles fit single exponential functions, giving lifetimes of 7.93 ns for C_A (Figure 4b). The emission spectrum of C_A with different concentrations demonstrated that the change of concentration did not affect maximum emission (Figure 4c). Additionally, we explored C_A’s PL stability (Figure 4d) under the influence of a 400 nm blue light for 400 s; the PL intensity of the C_A solution remained constant. Therefore, the supramolecular cage structure showed surprising advantages in terms of a significant Stokes shift, an extremely high PL QY, and good fluorescence stability, all of which are advantageous for applications.

2.3. In Vitro Cytotoxicity Profiles and CLSM Images

After the addition of DMEM medium, it was found that the characteristic peaks of C_A did not change (Figure S22). Therefore, the structure of C_A remained stable during the process of biological experiments. The cytotoxic activity of the C_A against U87 cells was evaluated. Cultured cancer cells were treated with concentrations of 0, 12.5, 25, 50, and 100 μg/mL of C_A in basal medium. The percentage of cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. In the U87 cytotoxicity assay, the C_A presented 84.95, 82.97, 87.27, and 77.43% cell viability at increasing concentrations (Figure 5a), and the results showed good biosafety. Then, we used confocal laser scanning microscopy to evaluate the suitability of low-toxicity C_A (25 μg/mL, and 50 μg/mL) for bioimaging. An intracellular fluorescence signal from C_A was clearly observed (Figure 5b), the fluorescence intensity gradually increased in line with the increase in concentration of C_A, while no emission was found in the control group. Therefore, C_A with 50 μg/mL concentration was selected for cell imaging to achieve accurate labeling of U87 glioblastoma cells.

3. Materials and Methods

3.1. Materials

All reagents were purchased from Sigma-Aldrich, Shanghai, China, Fisher, Shanghai, China, Acros, Shanghai, China, Bide Pharmatech Ltd., Shanghai, China, and, Alfa Aesar, Tianjin, China, and were used without further purification. All solvents were dried according to standard procedures and all of them were degassed under N₂ for 30 min before use.

3.2. Measurements

¹H NMR and ¹³C NMR spectra were measured on a Bruker AV 600 spectrometer. ESI-MS was recorded with a Waters Synapt G2-Si mass spectrometer, USA, in ESI mode. The UV–Vis spectra were recorded on a dual-beam UV–Vis spectrophotometer (TU-1901), PERSEE, Beijing, China. Fluorescent spectra were collected on a HORIBA FluoroLog-3 fluorescence spectrometer and Horiba-FluoroMax-4 spectrofluorometer, HORIBA, Edison, NJ, USA. The luminescence lifetimes were measured on an Edinburgh FLS 980 fluorescence spectrometer, Edinburgh Instruments, UK. The photoluminescence quantum yield (PLQY) was measured using an integrating sphere on a HORIBA FluoroLog-3 fluorescence spectrometer, HORIBA, Edison, NJ, USA. The Fourier Transform Infrared FT-IR spectra were recorded on a Spectrum TWO FT-IR spectrophotometer, PerkinElmer, Llantrisant, UK.

3.3. Materials Synthesis

3.3.1. Preparation of 2-Ethoxycarbonyl-carboxypyridine

Dipicolinic acid (5.00 g, 32.9 mmol) was refluxed for 4 h in methanol: water mixture (50 mL/50 mL) containing concentrated H₂SO₄ (5 mL). The cooled solution was poured onto saturated NaHCO₃ (500 mL) and the aq. phase was extracted with dichloromethane (4 × 100 mL). Acidification of the resulting aq. phase (pH = 2) with concentrated hydrochloric acid (pH = 2) followed by extraction with dichloromethane (4 × 100 mL) provided a second organic phase, which was dried (anhydrous Na₂SO₄) and evaporated to dryness to afford 2-ethoxycarbonyl-carboxypyridine (3.51 g, 59% 19.4 mmol) as a white powder. ¹H NMR (600 MHz, CD₃OD) δ 8.33 (dd, J = 18.5, 7.5 Hz, 2H), 8.17 (t, J = 7.8 Hz, 1H), 4.02 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 164.2, 163.5, 146.8, 146.5, 139.6, 128.8, 126.9, 53.2.

3.3.2. Preparation of L-A

Oxalyl chloride (1.68 g, 13.25 mmol) and 2-ethoxycarbonyl-carboxypyridine (2.00 g, 11.04 mmol) were dissolved in DMF (0.1 mL) and dry dichloromethane (100 mL) at 0 °C for 30 min; the whole reaction mixture was stirred at room temperature for 12 h. The resulting mixture was evaporated to dryness, and dissolved in dry dichloromethane (50 mL), then, N, N-diethylamine (17.10 mL, 16.50 mmol), and stoichiometric excess Et₃N were added dropwise at room temperature and the solution was stirred for 5 h. The solution was washed with portions of 1 N HCl, followed by 1 N NaOH, and, finally, water. The organic layer was isolated, dried over MgSO₄, and concentrated by rotary evaporation. Diethyl ether was added to precipitate the product as white precipitates, which were collected by filtration and dried. The compound obtained above was then dissolved into a solution of 1 N KOH and stirred for 1 h at room temperature. After washing with dichloromethane (2 × 50 mL), the aq. phase was neutralized (pH = 2) with concentrated hydrochloric acid and kept at 4 °C for 12 h. The mixture was extracted with dichloromethane (3 × 50 mL), dried over anhydrous Na₂SO₄, filtered, and then concentrated under reduced pressure to afford 6-(N,N-diethylcarbamoyl)pyridine-2-carboxylic acid (L-A, 1.69 g, 69% 7.62 mmol) as a white powder. ¹H NMR (600 MHz, CD₃OD) δ 8.21 (d, J = 7.8 Hz, 1H), 8.10 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 3.58 (q, J = 7.1 Hz, 2H), 3.33 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H), 1.19 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 166.8, 163.6, 154.0, 145.0, 140.0, 127.2, 124.4, 43.2, 40.3, 14.5, 12.8.

3.3.3. Preparation of L-B

L-A (111.05 mg, 0.50 mmol) and Oxalyl chloride (76.15 mg, 0.60 mmol) were dissolved in DMF (0.1 mL) and dry dichloromethane (30 mL) at 0 °C for 30 min; the whole reaction mixture was stirred at room temperature for 12 h, and then concentrated by rotary evaporation. Then, 4,4’-(benzo[1,2,5]thiadiazole-4,7-diyl)dianiline (53.10 mg, 0.17 mmol) was added dropwise at room temperature and the solution was stirred for 5 h. The solution was washed with portions of 1 N HCl, followed by 1 N NaOH and, finally, saturated sodium chloride solution. The organic layer was isolated, dried over MgSO₄, and concentrated to 5 mL by rotary evaporation. The mixture was poured into diethyl ether (50 mL) to give a precipitate, which was collected by centrifugation to afford L-B (65.40 mg, 54% 0.09 mmol) as a yellow powder. ¹H NMR (600 MHz, CDCl₃/CD₃CN = 1:1) δ 10.05 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.11 (t, J = 7.7 Hz, 1H), 8.07 (d, J = 8.6 Hz, 2H), 7.98 (d, J = 8.6 Hz, 2H), 7.88 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 3.62 (q, J = 7.0 Hz, 3H), 3.37 (q, J = 6.9 Hz, 2H), 1.48–1.12 (m, 7H). ¹³C NMR (151 MHz, CDCl₃) δ 167.6, 161.5, 154.1, 153.7, 148.5, 138.9, 137.7, 133.6, 132.5, 130.0, 127.8, 126.0, 123.1, 119.8, 43.2, 40.2, 14.7, 12.8.

3.3.4. Preparation of C_A

L-B (13.10 mg, 0.018 mmol) and Eu(OTf)₃ (7.19 mg, 0.012 mmol) were added to a solution of methanol: acetonitrile mixture (600 μL/600 μL); the solution was stirred at room temperature for 5 h. The reaction mixture was poured into diethyl ether (10 mL) to give a precipitate, which was collected by centrifugation to afford C_A (16.40 mg, 81% 0.0048 mmol) as an orange-red solid. ¹H NMR (600 MHz, CD₃CN) δ 7.86 (s, 2H), 7.66 (t, J = 7.7 Hz, 1H), 7.37 (d, J = 6.6 Hz, 2H), 7.26 (s, 1H), 7.18 (d, J = 7.7 Hz, 1H), 6.64 (d, J = 7.9 Hz, 1H), 6.13 (s, 1H), 3.66 (d, J = 6.7 Hz, 2H), 3.07 (d, J = 51.0 Hz, 2H), 1.40 (t, J = 6.7 Hz, 3H), 0.66 (s, 3H).

3.4. Fluorescence Lifetime

The fluorescence lifetime is measured by the single exponential fitting method; the formula is as follows:

$$\mathrm{y}=\mathrm{A}+\mathrm{B}1\exp \left(-t/t1\right)$$

(1)

3.5. Fluorescence Quantum Yield

The absolute fluorescence quantum yield is measured using the four-curve method; the formula is as follows:

$$\mathrm{QY}=\left\{Ea-\left[Ec\left(Lc/La\right)\right]\right\}/\left(Lc-La\right)$$

(2)

Ea is sample emission area, Ec is blank emission area, La is sample excitation area, Lc is blank excitation area. (Scatter Range: 395.00 to 415.00 nm. Emission Range: 465.00 to 749.00 nm).

3.6. Cell Variability Assay

U87 glioblastoma cells were purchased from Stem Cell Bank, Chinese Academy of Sciences. The cell line was authenticated and tested free of mycoplasma. The U87 cells were grown in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂. For this, 5000 cells in 100 µL per well were seeded in a 96-well plate and incubated for 24 h, then the cells were treated with different concentrations of C_A in basal medium, in triplicate. Following 24-h incubation, the cells were incubated with 10% CCK-8 containing DMEM high-glucose medium at 100 µL per well at 37 °C in a humidified atmosphere with 5% CO₂. CCK-8 could selectively stain the viable cells in 1 h, read by a spectrometer at 450 nm.

3.7. Live Cell Imaging

U87 cancer cells were plated on 35 mm plates (1 × 10⁵ cells in 1 mL media per plate) and maintained at 37 °C in a humidified atmosphere with 5% CO₂ for 24 h. Subsequently, the medium was discarded and the disks were rinsed with PBS twice, before 1 mL of DMEM high-glucose containing 25 μg/mL and 50 μg/mL of C_A were replaced. Following 4-h incubation, forming fluorescent of C_A (λex = 405 nm, λem = 546 nm) could be observed by confocal laser scanning microscopy (LSM 980 ZEISS).

4. Conclusions

In conclusion, we designed and synthesized a coordination-driven self-assembly supramolecular for cell imaging of live U87 glioblastoma cells. This supramolecular cage structure showed surprising advantages in terms of its unique low biotoxicity, high targeting, a large Stokes shift, high fluorescence efficiency, and strong photostability. This study will provide some guidance for the design of abundant organelle targeting probes and provide new ideas for further cancer diagnosis and treatment.