A biotin-conjugated highly stable cationic viologen derivative for live-cell imaging

Abstract

Traditional cancer imaging modalities cannot achieve ideal diagnostic results. Fluorescence imaging is an emerging modality for tumor imaging because of its high selectivity and sensitivity. However, conventional imaging agents have some drawbacks, including significant photobleaching, low fluorescence quantum yield, and inadequate targeting specificity. Therefore, there is an urgent need for the development of new imaging agents. Viologen derivatives have been widely used in the optical field owing to their excellent water solubility and good optical properties. However, because of its propensity to readily obtain electrons and form free radicals, its biological toxicity is significant, restricting its further application in the field of biological staining. To solve these problems, this study incorporated phenyl viologen as the primary fluorescent structure, which is capable of directly forming a quinone structure in a single step, thereby mitigating the impact of free radicals on cells. Furthermore, the introduction of biotin further enhanced the targeting of the imaging agent, ensuring that it was delivered more precisely to the desired cellular locations. This dual approach not only minimizes the harmful effects of free radicals but also improves the specificity and efficiency of cellular imaging. Experimental results demonstrate that the developer exhibits high photostability, excellent biosafety, and outstanding biocompatibility. This study investigated the application of cationic viologen derivatives in living cell imaging, laying a foundation for the advancement of cationic viologen derivatives in the biological field.

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Introduction

Cancer poses a grave threat to human life and health.^1,2 Currently, research on pertinent treatment methods is steadily progressing. Importantly, tumor identification is pivotal in the treatment continuum.^3,4 In the field of tumour detection and diagnosis, imaging technologies including X-ray, ultrasound imaging (USI), magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET) and ultrasound technologies all play a key role.^5–7 However, these conventional imaging techniques have obvious limitations, such as insufficient sensitivity and resolution, long imaging time, low signal-to-background ratio, high cost, and even harmful radiation, which limits their application in clinical practice.^8–10 Consequently, there is an urgent need to develop innovative live cell imaging techniques that can provide more accurate, efficient, and safe diagnostic capabilities.

In 2008, Weissleder and Pittet proposed the concept of molecular imaging, which opened a new chapter on the development of imaging technology.⁶ Subsequently, various imaging techniques have been developed and are widely used in medical research.^11–13 Among these imaging techniques, fluorescence imaging is distinguished by its high sensitivity, high temporal resolution, non-invasiveness, and real-time detection capability.^14–17 It can accurately demonstrate the targets and dynamic processes in biological systems, which contributes to an in-depth exploration of the complexity, diversity, and behavioural patterns of cancer.^18–20 The development of fluorescence imaging technology not only enhances the accuracy and reliability of cancer diagnosis but also provides a basis for developing more precise treatment plans.^21–23 As fluorescence imaging technology begins to reveal its remarkable ability to delve deeply into the intrinsic mechanisms of disease at the cellular and molecular levels, it holds great promise for the early detection, precise diagnosis, and surgical guidance of cancer.^24,25 Currently, fluorescence imaging technology, particularly the fluorescence probes used for live cell imaging, has become an indispensable and important tool in the domains of medical diagnosis and biological research.^26–29

The selection of fluorophores with desirable optical properties is crucial to achieve high-quality fluorescence imaging. While a wide variety of fluorescent probes are commercially available, including organic dyes, metal–ligand complexes, polymer nanostructures, and nanoparticles,^30–32 they often suffer from limitations, such as significant photobleaching, low fluorescence quantum yields, inadequate targeting specificity, and subpar pharmacokinetic performance.^33,34 These problems impose significant constraints on the advancement and utilization of fluorescence imaging technology. Therefore, there is an urgent need to develop novel fluorescent probes with superior optical and pharmacokinetic properties.^35,36

Cationic viologen compounds are renowned for their optoelectronic properties, making them valuable in photocatalysis, solar energy conversion, and electrochromism.^37,38 However, the inherent biotoxicity of viologens due to their engagement in redox processes and production of free radicals, poses a significant challenge for their use in bioimaging.^39–42 To address this issue, π-conjugated viologen derivatives have been designed to alter their redox characteristics and reduce toxicity.^43,44 Yet, these modified derivatives still retained some level of toxicity. A breakthrough has been discovered; the insertion of a benzene ring between two pyridine moieties enabled the molecule to acquire two electrons simultaneously, ultimately forming a quinone-like structure, which effectively prevented the formation of free radical cations.^45–47 This finding prompted us to consider incorporating this structure into imaging agents by bypassing the intermediate step involving free radicals, thereby significantly reducing the toxicity of the molecules. Furthermore, biotin, an indispensable nutrient for cell growth, and its receptors, are overexpressed on the surface of cancer cells, which can be harnessed for the precise targeting of cancer cells.^48–50 It is anticipated that the introduction of appropriate tumor-targeting functionalities into this framework will lead to the development of a highly effective imaging agent.

Based on these considerations, a novel imaging agent, Bio-C6, was synthesized by integrating a benzene ring into the 4,4′-bipyridine structure and attaching biotin with strong targeting properties. Cationic viologen was used as the fluorophore, with the benzene ring serving to curtail free radical production and reduce biotoxicity. The biotin component acted as a targeting unit, enabling precise homing-in ability on cancer cells and markedly enhancing the accuracy of imaging. And Bio-C6 was equipped with hydrophobic alkyl chains on both sides, which, due to the hydrophobic properties of the cell membrane phospholipid bilayer, can be precisely inserted into the interior of cancer cells through a hydrophobic effect.⁵¹ Moreover, Bio-C6 emitted bright fluorescence under visible light excitation, in particular, Bio-C6 could target cancer cells and illuminate them under 456 nm light excitation, resulting in clear and precise imaging. Both experimental and theoretical results showed that Bio-C6 was a stable and superior imaging agent. This study not only provided an innovative perspective for the design of imaging agents but also paved the way for more advanced tumor detection techniques.

Experimental section

Materials and instrumentation

All reactions were performed using standard Schlenk and glovebox (Vigor) techniques under an argon atmosphere. Acetone and toluene were distilled from sodium/benzophenone prior to use. All the chemicals used in the experiments were purchased from Energy Chemical Inc, Sigma, Thermo Fisher Scientific Inc. and Sangon Biotech. If no other treatment is indicated, reagents and solvents were used as commercially available without further purification. Column chromatographic purification of products was accomplished using 200–300 mesh silica gel. NMR spectra were measured on a Bruker Avance-400 and Avance-HD 600 MHz spectrometer in the solvents indicated; chemical shifts are reported in units (ppm) by assigning DMSO-d₆ resonance in the ¹H spectrum as 2.50 ppm, DMSO-d₆ resonance in the ¹³C spectrum as 39.50 ppm. Coupling constants are reported in Hz with multiplicities denoted as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). UV-Vis measurements were performed using a DH-2000-BAL Scan spectrophotometer. Fluorescence measurements were conducted on a FLS920 system (Edinburgh Instruments) and Hitachi F-7000. IR was collected on a Bruker ALPHA II PLATINUM-ATR. The cyclic voltammetry (CV) and differential pulse curve (DPV) in solution were measured using CHI660E B157216, with a glassy carbon electrode as the working electrode, a Pt-net as the counter electrode, and silver/silver chloride (Ag/Ag⁺) as the reference electrode. High-resolution mass spectra (HRMS) were collected on a Bruker maXis UHRTOF mass spectrometer in an ESI positive mode. Viability of cells was assessed under visible light via a Mejiro Genossen MVL-210 photoreactor supplied by Beijing Perfect Light Co. Ltd. EPR was measured using a Bruker EMX PLUS6/1 instrument at room temperature in dry degassed DMF. The EPR parameters for the experiments are as follows: modulation frequency = 100 kHz, modulation amplitude = 1.0 G, time constant = 0.01 ms, conversion time = 5.0 ms, center field = 3390 G, sweep width = 100 G, microwave attenuation = 30 dB, microwave power = 0.2 mW. The BOT455 455 nm continuous used for irradiation, was supplied by Xi’an Lei Ze Electronic Technology Co. Ltd. OD was measured using a microplate reader (HM-SY96A) supplied by Shandong Hengmei Electronic Technology Co. Ltd. Fluorescence microscopy was measured using a Laite LF200. CLSM characterization was conducted with a confocal laser scanning biological microscope (Leica TCS SP8 STED 3X). Live-imaging was performed on a PerkinElmer IVIS Spectrum. Photographs were taken using a Nikon D5100 digital camera.

Experimental conditions

Singlet oxygen measurements and the quantum yield of singlet oxygen. The process to determine the quantum yield of singlet oxygen was based on a literature procedure by light modification.⁵² Specifically, 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was used as the specific probe to trap singlet oxygen and methylene blue (MB) was employed as the reference (ΔΦ = 0.42 in air-saturated D₂O). ABDA solution (20 μg mL⁻¹) was added to tellurium viologens or MB in air-saturated D₂O. The UV-Vis absorption spectra were measured at 1.0 min intervals after the specimens were irradiated with white light (50 mW cm⁻²). Then, the absorption values of ABDA at 378 nm were recorded. The quantum yield of singlet oxygen generation was determined using the following equation:

Φ^S_Δ = Φ^R_Δ × (k^S/k^R)

where k is the slope of ABDA consumption by singlet oxygen. The superscript “S” and “R” represent the sample and the reference, respectively.

Stability testing. An aqueous solution of Bio-C6 was prepared at a concentration of 1 mM. The Bio-C6 solution was continuously irradiated with a benchtop UV-vis analyser for 60 min at a wavelength setting of 365 nm and a power of 6 W. The UV-vis absorbance of the Bio-C6 solution was tested with a UV-vis spectrophotometer at intervals of 10 min, in addition, the photostability of the commercial cellular nuclear stain, 4,6-diamidino-2-phenylindole (DAPI), was tested as a comparison. The photostability of Bio-C6 and DAPI was characterised by calculating the half-life from the degree of attenuation of the maximum absorbance of the UV-vis spectrum.

Acid environment stability test. The tumour microenvironment in the human body is a slightly acidic environment with a pH usually maintained in the range 6.5 to 6.9. Acidic solutions with a pH of 1 to 6 were prepared, as well as an ultrapure water solution with a pH of 7 as a control. And in each solution, the concentration of Bio-C6 molecules was controlled to be 1 mM.

These solutions were tested for UV-vis absorbance using a UV-vis spectrophotometer. Meanwhile, the fluorescence emission intensity of these solutions was tested using a fluorescence spectrometer. Again, the same experiment was performed using DAPI as a comparison.

Biological environmental stability testing. PBS (phosphate buffer solution) as well as 10% foetal bovine serum (FBS) were chosen as solvents to test the UV-vis absorbance and fluorescence emission intensity of pure solvents (i.e., PBS and 10% FBS), respectively, and the same tests were carried out by preparing Bio-C6 solutions with a concentration of 1 mM using water, PBS and 10% FBS as solvents. Again, the same experiment was performed using DAPI as a comparison.

Cell culture. Human liver cancer cell line HepG2 was purchased from the Shanghai Institute of Cell Biology in the Chinese Academy of Sciences. HepG2 cells were cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 IU per mL penicillin and 100 μg mL⁻¹ streptomycin). The cell lines were incubated in a humidified atmosphere of 5% CO₂ at 37 °C.

Cell viability assay. Cells were seeded in 96-well plates at 5000 cells per well in 180 μL of complete medium, and incubated in a 5% CO₂ atmosphere or 1% O₂ and 5% CO₂ hypoxic environment at 37 °C for 24 h. Then the culture medium was replaced with a freshly prepared culture medium containing 20 μL of different tellurium viologens at different concentrations. Then the mixture solutions were exposed to 50 mW cm⁻² white light for 10 min, or incubated in the dark for 10 min. The cells were further incubated for 24 h, and then the medium was replaced with fresh culture medium and the MTT solution (5 mg mL) was added. The cells were incubated for another 4 h to allow viable cells to reduce the yellow tetrazolium salt (MTT) into dark blue formazan crystals. Finally, 100 μL of lysis buffer was added to wells and incubated for another 4 h at 37 °C. The absorbance was measured at 490 nm using a Bio-Rad 680 microplate reader. The IC50 values were calculated using GraphPad Prism software (version 8.0) based on data from five parallel experiments.

Confocal laser scanning microscopy (CLSM) characterization. The HepG2 cells were seeded at a density of 1 × 10⁴ cells on a round cover slip (diameter 12 mm) in complete DMEM medium. After 24 h, they were treated with Bio-C6 at a concentration of 100 μM for 12 h and exposed to light. Cells were then washed twice with cold PBS. CLSM was used to observe the co-localization of mitochondria in cells. The data was analyzed using Image J.

Synthetic procedures.
Synthesis of 4. 60 mg of compound 3 (0.171 mmol) and 129 mg of biotin-N-succinimidyl ester (0.377 mmol) were added to the reaction vial under argon protection, 20 mL of ultra-dry N,N-dimethylformamide solution was added, the reaction vial was subjected to an ice bath, and ultra-dry triethylamine (35 mg, 0.342 mmol) was added to the reaction vial in the ice bath, the reaction was heated to 60 °C with stirring for 24 hours. At the end of the reaction, the solvent was cooled to room temperature, and then the solvent was removed to obtain a brown-yellow solid, which was purified using 200–300 mesh silica gel column chromatography using dichloromethane : methanol : ammonia = 15 : 1 : 0.1 (v/v/v) as the eluent to obtain a white solid, which was compound 4, with a yield of 60%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.62 (s, 4H), 7.94 (s, 2H), 7.67 (s, 4H), 7.22 (s, 2H), 6.44 (s, 2H), 6.36 (s, 2H), 4.28 (s, 2H), 4.07 (m, 6H), 3.30 (s, 4H), 3.03 (s, 2H), 2.77 (m, 2H), 2.66 (s, 2H), 2.05 (m, 4H), 1.45 (m, 12H). ¹³C NMR (101 MHz, DMSO-d₆) δ 174.3, 172.4, 162.8, 161.5, 149.8, 149.5, 144.6, 128.3, 124.1, 115.7, 67.5, 61.1, 59.2, 55.5, 36.8, 35.2, 34.9, 28.3, 25.2.
Synthesis of Bio-C6. 200.75 mg of compound 4 (0.25 mmol) was dissolved in 15 mL of DMF, 36.89 μL of iodohexane (2.5 mmol) was added, and the reaction was carried out at 70 °C for three days. Then the product was washed with 50 mL of ethyl acetate, three times to obtain a yellow solid, and separated by silica gel column chromatography (methanol : saturated aqueous ammonium chloride solution : nitromethane = 15 : 2 : 1). The yellowish-green solid was obtained as the target product Bio-C6, with a yield of 40%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.10 (d, J = 6.3 Hz, 4H), 8.47 (d, J = 6.3 Hz, 4H), 8.03 (d, J = 5.9 Hz, 2H), 7.54 (s, 2H), 6.39 (s, 4H), 4.64 (t, J = 7.1 Hz, 4H), 4.19 (tt, J = 37.4, 6.1 Hz, 8H), 3.17 (d, J = 4.5 Hz, 4H), 3.03 (d, J = 12.9 Hz, 2H), 2.77 (dd, J = 12.5, 5.0 Hz, 2H), 2.56 (s, 2H), 2.06 (d, J = 7.2 Hz, 4H), 1.98 (d, J = 8.9 Hz, 4H), 1.52–1.21 (m, 24H), 0.89 (t, J = 6.2 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.4, 162.7, 152.2, 150.4, 144.0, 127.9, 126.7, 67.9, 61.0, 60.2, 59.2, 55.4, 37.8, 35.3, 30.9, 30.7, 28.1, 25.2, 21.9, 13.9. ESI-MS calcd for C₅₂H₇₆N₈O₆S₂ (M-2I⁺): 486.26591, found: 486.26497.
Synthesis of 5. 100 mg of 4,4′-bipyridine (0.64 mmol) was weighed separately with 700.64 mg of bromopropylamine hydrobromide (3.2 mmol) and added to a 50 mL reaction vial under argon protection, followed by the addition of 20 mL of DMF as the reaction solvent to completely dissolve the solid. The reaction was carried out at 110 °C for 48 h. Then the product was washed with 20 mL of DMF, acetone and ether to give light yellow solid 5 with a yield of 80%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.45 (d, J = 4.0 Hz, 4H), 8.87 (d, J = 5.4 Hz, 4H), 4.85–4.70 (m, 4H), 2.93 (t, J = 7.4 Hz, 4H), 2.31 (t, J = 7.7 Hz, 4H).
Synthesis of Bio-DPY. 50 mg of compound 5 (0.18 mmol) and 309 mg of Biotin-NHS (0.9 mmol) were dissolved in 20 mL DMF, triethylamine (36.8 mg, 0.36 mmol) was added under an ice bath, and reaction was carried out at 70 °C for 24 h. The product was washed with 50 mL of ethyl acetate, three times to obtain brown solid Bio-DPY with a yield of 60%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.43 (d, J = 6.2 Hz, 4H), 8.83 (d, J = 6.3 Hz, 4H), 8.08–8.02 (m, 2H), 6.40 (d, J = 11.9 Hz, 4H), 4.72 (s, 4H), 4.32 (t, J = 6.4 Hz, 2H), 4.14 (s, 2H), 3.17–3.08 (m, 6H), 2.82 (dd, J = 12.4, 5.0 Hz, 2H), 2.58 (d, J = 12.6 Hz, 2H), 2.17–2.08 (m, 8H), 1.41 (d, J = 77.6 Hz, 12H). ESI-MS calcd for C₃₆H₅₂N₈O₄S₂ (M-2I⁺): 362.17710, found: 362.17791.

Results and discussion

Synthesis and structure characterization

Compounds 1, 2, 3, and 4 were synthesized according to previously reported methods.^53,54 Compound 4 was treated with iodohexane in DMF to produce the target molecule Bio-C6 as a yellow solid with 40% yield (Scheme 1). Compound 5 was synthesized from 4,4′-bipyridine with bromopropylamine hydrobromide as a yellow solid in 82% yield. Subsequently, the reaction of compound 5 with biotin-N-succinimidyl ester afforded brown solid Bio-DPY in 63% yield.

Scheme 1 Synthesis route of Bio-C6.

Photophysical and electrochemical properties

The photoelectrical properties of the desired molecules were investigated. Bio-C6 exhibited an absorption peak at approximately 400 nm in water, with a maximum emission wavelength of 512 nm, indicating that the molecule can be excited by visible light. In contrast, Bio-DPY showed a maximum absorption wavelength at 283 nm and a maximum emission wavelength at 555 nm. Notably, the absorption spectrum of Bio-DPY displayed a distinct tailing phenomenon, extending to around 600 nm, (Fig. 1a, b and Fig. S1). This could be due to the charge transfer state in the molecular structure. Although Bio-DPY had a wider absorption range and a more red-shifted wavelength, its weak absorption capacity significantly limited its application in visible light imaging. Therefore, Bio-C6 was more suitable for visible light imaging compared to Bio-DPY. As shown in Fig. S2, the fluorescence lifetime of Bio-C6 and Bio-DPY were 5.23 ns and 3.86 ns, respectively. Both molecules exhibited short fluorescence lifetimes, which can be attributed to their locally excited (LE) state emission. It is worth noting that the fluorescence quantum yield of Bio-C6 (32.15%) was nearly seven times higher than that of Bio-DPY (4.74%). This difference arises because Bio-DPY adopts an extended chain structure, allowing the molecules to rotate freely and resulting in energy loss. Conversely, Bio-C6 contains two ether oxygen bonds on its benzene ring, where the oxygen atoms can form hydrogen-bonding interactions with the hydrogen atoms on adjacent pyridine rings, restricting molecular rotation. Moreover, the steric hindrance between the two alkyl chains on the pyridinium nitrogen and the biotin side chain on the benzene ring further limits molecular rotation (Fig. S3). These structural features contribute to the higher quantum yield of Bio-C6, making it more promising for cellular imaging applications.

Fig. 1 (a) PL emission spectra of Bio-C6 and Bio-DPY in H₂O, c ∼ 10⁻⁵ M. (b) UV-vis spectra of Bio-C6 and Bio-DPY in H₂O (inset: photographs of Bio-C6 and Bio-DPY). The cyclic voltammogram of Bio-C6 (c) and Bio-DPY (d) at different scan rates in DMF solution with tetrabutylammonium hexafluorophosphate (0.05 M) as the supporting electrolyte, potential E referenced to Fc/Fc⁺, c ∼ 5 × 10⁻⁴ M. The DPV of Bio-C6 (e) and Bio-DPY (f).

Due to the intrinsic fluorescent properties of Bio-C6, its critical micelle concentration (CMC) was determined using the direct fluorescence method. The fluorescence emission spectra were recorded over a concentration range of 0.1 mM to 1 mM (Fig. S4a). At low Bio-C6 concentrations, the fluorescence intensity increased sharply, but the rate of increase gradually diminished at higher concentrations. This trend suggested structural changes of the molecules beyond a certain concentration, likely due to micelle formation. As illustrated in (Fig. S4b), a break point occurs at approximately 0.37 mM Bio-C6 concentration. This break point was most likely attributed to the aggregation of individual Bio-C6 molecules, which was indicative of the CMC of Bio-C6.

Further experiments were conducted to elucidate the redox properties of Bio-C6 and Bio-DPY. The electrochemical behavior of the Bio-C6 was assessed by fabricating an electrochromic device. As shown in Fig. S5, the UV-vis absorption spectrum revealed a distinct increase in the absorption peak at 559 nm, accompanied by a color change from yellow to violet in the device. This observation suggested that Bio-C6 can only undergo a single redox process involving the synchronous transfer of two electrons during electrical reduction. Meanwhile, the electrochemical properties of the two molecules were examined by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) (Fig. 1c–f). The oxidation potential of Bio-C6 was determined to be −0.84 V, and the reduction potential was −0.78 V. The electrochemical tests indicated that Bio-C6 exhibited only a single set of redox peaks, implying a singular and well-defined electron transfer process. A detailed examination of the DPV curve revealed a solitary peak at −0.89 V, which not only confirmed the stability of Bio-C6 during electrochemical processes but also suggested the presence of a single redox process. These findings indicated that Bio-C6 can acquire two electrons simultaneously under energized conditions, resulting in a neutral state. Notably, this transformation prevented the formation of free radicals within the molecule. This property rendered Bio-C6 particularly suitable for biological applications, while significantly enhancing its biosafety. In contrast, Bio-DPY exhibited two sets of redox peaks in the cyclic voltammetry curve and two peaks in the differential pulse curve. This indicated that Bio-DPY has two redox states under electrical stimulation, with the molecule first gaining one electron to form the single radical state, and then acquiring another electron to reach the neutral state. Thus, it was demonstrated that the introduction of the benzene ring had a pronounced impact on the molecular redox behavior, transforming the redox process of Bio-C6 into a simultaneous two-electron transfer.

Electron paramagnetic resonance (EPR) was performed to confirm the two-electron transfer mechanism of the Bio-C6. The investigation found that no EPR signals were detected for Bio-C6 in its reduced state, whether in the presence of zinc (Zn) or under continuous illumination (Fig. 2a). This absence of signals indicated that Bio-C6 did not produce free radicals during reduction but rather follows a two-electron transfer pathway. Consequently, it was confirmed that the reduced form of Bio-C6 was devoid of free radical species that were detrimental to the tissue cells. In contrast, Bio-DPY exhibited distinct EPR signals under both Zn-added and illumination conditions owing to free radical formation (Fig. 2b). In addition, the ¹O₂ can inflict cellular damage, so the biosafety of Bio-C6 and Bio-DPY was further validated by assessing their ¹O₂ generation capacities. The anthracene-9,10-dipropionic acid (ADPA) was selected as the probe for testing ¹O₂, and methylene blue (MB) was employed as the reference. As depicted in Fig. 2c and Fig. S6, under white light irradiation for different times, the absorption peak at 378 nm decreased to different degrees, revealing that Bio-C6 and Bio-DPY had low ¹O₂ yields, which proved that the ¹O₂ produced by light will have little effect on the cell.

Fig. 2 EPR spectra of the radical species of Bio-C6 (a) and Bio-DPY (b) after addition of Zn and light radiation. (c) The decay of absorbance of ABDA at 378 nm in the presence of 10 μM Bio-C6 and Bio-DPY as a function of irradiation using light with a fluence of 50 mW cm⁻². Transient absorption (d) 3D contour map and (e) 2D spectrum of Bio-C6 in H₂O (λ_ex = 375 nm). (f) Transient kinetic traces and fit curves of Bio-C6 probed at 500, 520, 540, 560, 580 and 600 nm.

Femtosecond transient absorption (fs-TA) was utilized to elucidate the electron transfer mechanism of Bio-C6. The contour map exhibited an absorption peak at 500 nm, which increased gradually under 375 nm light excitation, peaking at 50 ps, indicating the formation of excited state species Bio-C6* (Fig. 2d and e). Subsequently, the absorption peak at 500 nm gradually diminished, while the absorption intensity at 480 nm concurrently increased. This phenomenon was attributed to the formation of excited triplet state species, Bio-C6³*. The presence of an iso-absorption point near 590 nm in the fs-TA spectrum was characteristic of two-state transitions. Multi-exponential fitting was applied to the kinetic trajectories at 480 nm and 500 nm (Fig. 2f). The kinetic trajectories revealed that after the rapid formation of the excited states, a prolonged decay process occurred with a duration of up to 3000 ps. This finding revealed that Bio-C6 exhibited a long charge-separated state lifetime, which was favorable for its application in imaging.

Stability testing of photophysical properties

The stability of imaging agents is crucial for their effectiveness in biological applications. The photostability of Bio-C6 was assessed by exposing it to a 365 nm UV lamp with a power of 6 W, and monitoring the UV-visible absorption spectra at set intervals. As depicted in Fig. 3a and b, the UV absorption peak of Bio-C6 did not change significantly after 60 min of exposure, with no significant change in brightness between the photographs taken before and after exposure. Meanwhile, the absorption spectrum intensity of the commercial cell nuclear stain DAPI gradually decreased with prolonged exposure to light. The results demonstrated that Bio-C6 exhibited superior photostability compared to DAPI under continuous UV light exposure. The maximum absorbance values from the UV-visible spectra of both Bio-C6 and DAPI after UV irradiation were subjected to decay fitting, and their half-lives were calculated to be 1.2240 × 10⁵ s for Bio-C6 and 5.1832 × 10⁴ s for DAPI, respectively (Fig. S7). Notably, the half-life of Bio-C6 was significantly longer than that of DAPI, suggesting that Bio-C6 possessed greater photostability and was less prone to photobleaching. This made Bio-C6 a more reliable imaging agent.

Fig. 3 The Bio-C6 (a) and DAPI (b) changes in UV spectra with increasing light exposure time (inset: photographs of Bio-C6 and DAPI). Effects of pH on the fluorescence spectra of Bio-C6 (c) and DAPI (d). Effects of different solvents on the fluorescence spectra of Bio-C6 (e) and DAPI (f).

Given the acidic nature of tumor microenvironments, it is essential to evaluate the effect of pH on molecular stability. To this end, the UV-visible absorption and fluorescence emission spectra of Bio-C6 and the commercial nuclear stain DAPI were examined across a range of pH values. As shown in Fig. 3c and Fig. S8a, Bio-C6's UV-visible absorption and fluorescence emission peaks exhibited a notable decrease in acidic environments compared to neutral conditions. Despite this decrease, Bio-C6 displayed a comparatively stable profile in the acidic range, with only minor fluctuations in both the absorption and emission intensities as the pH decreased. In contrast, the UV-visible absorption of DAPI decreased significantly under acidic conditions, with its maximum absorbance almost halved, indicating that DAPI is very sensitive to acidic environments. DAPI's fluorescence emission intensity also varied with decreasing pH, showing a substantial reduction at pH = 1. These results indicated that the photophysical properties of Bio-C6 were more stable at different pH levels, especially in acidic conditions, compared to DAPI (Fig. 3d and Fig. S8b). This characteristic enabled Bio-C6 to maintain its fluorescence properties in the tumor micro-environment. Consequently, Bio-C6 can be utilized as a potential agent for live cell imaging. To further verify the stability of Bio-C6 in biological environments, its behavior was evaluated through UV-visible and fluorescence emission spectroscopy in a variety of biological milieus. Bio-C6 displayed prominent UV-visible absorption peaks in PBS solution and 10% fetal bovine serum solution, demonstrating its excellent absorption properties in complex biological environments However, the fluorescence intensity of Bio-C6 was observed to decrease in both PBS and 10% fetal bovine serum (Fig. 3e and Fig. S9a).

As a comparison, the UV-vis absorption peaks of DAPI showed obvious fluctuations in both solutions, generally being lower than their absorbance in aqueous solution. Moreover, the fluorescence emission intensity of DAPI in 10% fetal bovine serum was significantly higher than that in its aqueous solution, while the fluorescence emission intensity in PBS showed a slight increase compared with that in water (Fig. 3f and Fig. S9b). It is evident that the Bio-C6 fluorescence performance, although variable in different biological solvents, remained more stable overall compared to DAPI, which rendered it a promising candidate for imaging agents in biological imaging applications.

DFT calculations and transient absorption spectroscopy

To validate the experimental results, density functional theory (DFT) calculations were conducted to simulate the energy level, optical and electronic properties of Bio-C6 and Bio-DPY (Fig. S10). Compared with Bio-DPY, Bio-C6 showed a more negative reduction potential, indicating that it was less likely to gain electrons. The narrower bandgap of Bio-C6 suggested a higher propensity for charge transitions to the excited state, which are key traits for a luminescent imaging reagent. Furthermore, the UV absorption values calculated were located at 317 nm, which is in agreement with the experimental data (Fig. S11).

In vitro imaging characterisation

To comprehensively evaluate the cytotoxicity and biosafety of Bio-C6 under different conditions, mouse fibroblasts (L929) were selected as model cells for in-depth studies. The cells were exposed to light for 5 minutes at a concentration of 100 μM and the cell survival rates were compared between light and dark conditions (Fig. S12). The experimental results demonstrated that after a brief period of light exposure, there was no remarkable decline in cell survival rates when compared to dark conditions. This finding provided compelling evidence that the molecule still maintained high biosafety under illuminated conditions and did not induce substantial cellular damage, which provided strong support for its application in live cell imaging studies.

In order to explore the fluorescence imaging ability and cancer cell targeting ability of Bio-C6, human hepatoblastoma cells (HepG2) rich in biotin receptors were selected as the model system. Firstly, to verify the cell membrane imaging effect of the material, a commercial DiD cell membrane staining agent was compared with Bio-C6. As can be seen from Fig. 4a, DiD and Bio-C6 have a high coupling degree in cell membrane imaging. However, the lipophilicity of DiD caused a part of it to concentrate on the lipids within the cells, presenting punctate or block-like fluorescence in the cytoplasm, resulting in poor imaging effects. In contrast, Bio-C6 did not show similar situations, proving that our material has a good cell membrane imaging effect. At the same time, to prove that the material has broad-spectrum applicability in cell membrane staining, HeLa cells were also used for imaging. As shown in Fig. S13, our material also has a good cell membrane imaging effect on HeLa cells, proving that Bio-C6 has broad-spectrum applicability. To investigate the concentration-dependent effects of Bio-C6 on cellular imaging, we evaluated five different concentrations (20–100 μM) for staining HepG2 cells. As demonstrated in the results (Fig. 4b), distinct cell contours became visible at the lowest tested concentration of 20 μM. The imaging quality progressively improved with increasing concentration, reaching optimal cellular visualization at 100 μM. The experimental results showed that Bio-C6 maintains effective membrane staining capability across a wide concentration range. The biotin receptor is overexpressed in various cancer cells. Introducing biotin into the material can significantly enhance its uptake efficiency for cancer cells. To demonstrate the selectivity of Bio-C6 for cancer cells, the uptake of Bio-C6 by L929 cells and HepG2 cells was compared. The test was conducted using a flow cytometer to observe the staining conditions of Bio-C6 on HepG2 cells and L929 cells under the same incubation time (30 minutes). As shown in Fig. S14, after co-incubation, the fluorescence of HepG2 cells was slightly higher than that of L929 cells, which proved that Bio-C6 entered HepG2 cells more frequently within the same time period. This verified the design concept of the molecule. The imaging results demonstrated that Bio-C6 can adhere to cancer cells for live-cell imaging, facilitating the monitoring of cancer cells and enabling noninvasive cancer diagnosis.

Fig. 4 (a) Confocal laser scanning microscopy images of HepG2 cells incubated with DAPI, Bio-C6 and DiD. [Bio-C6] = 100 μM, Ex = 375 nm, Em = 450–650 nm; [DAPI] = 100 nM, Ex = 350 nm, Em = 380–600 nm; [DiD] = 10 μM, Ex = 633 nm, Em = 650–750 nm; (b) the concentration-dependent effects of Bio-C6 on cellular imaging by confocal laser scanning microscopy. [Bio-C6] = 20–100 μM, Ex = 375 nm, Em = 450–650 nm; [DAPI] = 100 nM, Ex = 350 nm, Em = 380–600 nm.

To further investigate the cellular uptake mechanism of Bio-C6, we conducted temperature-dependent uptake experiments.⁵⁵ At both 0.5 h and 2 h incubation time points, HepG2 cells exhibited significantly higher Bio-C6 fluorescence intensity (MFI) at 37 °C compared to 4 °C. While uptake at 37 °C progressively increased with time, indicating continuous internalization, the MFI at 4 °C remained markedly lower, suggesting a strong inhibition of uptake at reduced temperatures (Fig. S15 and S16). These findings collectively indicated that the primary mechanism for Bio-C6 cellular uptake is an active, energy-dependent endocytic pathway, rather than passive diffusion. Endocytosis is an active cellular process that requires energy and is sensitive to temperature. At 4 °C, cellular metabolic activity and membrane fluidity were significantly reduced, thereby inhibiting active endocytic processes. In this experiment, at both 0.5-hour and 2-hour time points, the Bio-C6 uptake in the 37 °C group was significantly higher than that in the 4 °C group, which is consistent with the characteristics of a typical endocytic process.

To address whether the internalization of the Bio-C6 probe is dependent on biotin receptors present on the cell surface, we conducted a biotin competitive inhibition experiment (Fig. S15 and S16). The flow cytometry data from the biotin competitive inhibition experiment demonstrated that Bio-C6 cellular internalization is significantly dependent on cell surface biotin receptors. Pre-treatment of HepG2 cells with excess free biotin markedly reduced Bio-C6 uptake at both 0.5-hour and 2-hour incubation time points compared to untreated controls (p < 0.001 for both time points). This competitive inhibition indicated that free biotin successfully occupies biotin receptor binding sites, thereby impeding Bio-C6 internalization. Cancer cells often overexpress biotin receptors on their surface, providing a pathway for biotin-conjugated molecules to precisely target cancer cells. The results of this experiment directly supported that the biotin moiety incorporated into Bio-C6 indeed functions as a targeting unit, facilitating its efficient cellular uptake through binding with overexpressed biotin receptors on the cell surface.

Conclusions

In conclusion, we have developed a stable imaging agent, Bio-C6, featuring a phenyl viologen skeleton. The distinctive structural modification strategy had effectively transformed the cationic viologens into a quinone-like structure under light, thereby preventing the generation of free radicals and markedly improving its biocompatibility. This feature endowed the agent with the capacity to emit bright and stable fluorescence upon excitation by visible light, even under prolonged exposure to ultraviolet light or in fluctuating micro-environments. Compared with commercial dye DAPI, Bio-C6 showed superior stability in several aspects. Most notably, Bio-C6 exhibited excellent targeting capabilities, enabling the imaging of cancer cell membranes in the cellular environment. This innovative design not only provides a new perspective for the advancement of cellular imaging, but also presents a novel option for tumor diagnosis.

Author contributions

Weijie Song: investigation, methodology, software, writing – original draft. Wenqiang Ma: writing – original draft, writing – review & editing. Qi Sun: methodology, writing – original draft, writing – review & editing. Yujing Gao: investigation, writing – original draft, software. Zengrong Wang: investigation, software. Chenjing Liu: software. HuiRuo Dong: methodology. Jinlu Ma: writing – review & editing. Suxia Han: writing – review & editing. Kun Zhou: writing – review & editing. Gang He: formal analysis, methodology, supervision, writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the SI. Supplementary information: Experimental details, structural characterization, photochemical and electrochemical measurements. See DOI: https://doi.org/10.1039/d5tb00830a.

Acknowledgements

Funding support was received from the Natural Science Foundation of China (grant no. 22405203, 22205172, 22175138, and 22201228), the Fundamental Research Funds for the Central Universities (xzy012024063), Shaanxi Province Postdoctoral Science Foundation (2024BSHSDZZ055), the China Postdoctoral Science Foundation (2022M712497, 2022M712530), Shaanxi Province Technological Innovation Guidance Special (2024ZC-YYDY-96, 2022QFY08-01), Guangdong Basic and Applied Basic Research Foundation (2023A1515110346), the Taihu Lake lnnovation Fund for the School of Future Technology of Xi’an Jiaotong University. We thank Dr Lu Bai and Dr Dan He at the Instrument Analysis Center of Xi’an Jiaotong University for HRMS, and photoluminescence measurements. The high-performance computing center at Xi'an Jiaotong University is especially acknowledged for providing the computational resources.

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Footnote

† These authors contributed equally to this work.

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