A Hydrogen Peroxide Responsive Biotin-Guided Near-Infrared Hemicyanine-Based Fluorescent Probe for Early Cancer Diagnosis

Abstract

H₂O₂ plays an important role in oxidative damage and redox signaling. Studies have shown that abnormal levels of H₂O₂ are closely related to the development of cancer. The levels of H₂O₂ in tumor cells are higher than in normal cells. Thus, it is of great importance to develop a fluorescent probe to monitor the level of H₂O₂ in vivo. This work reports a new biotin-guided NIR fluorescent probe, Bio-B-Cy, consisting of boronic acid ester as a H₂O₂-recognition site and biotin as a tumor binding site, which accelerates the fluorescence response to H₂O₂ in vivo. Bio-B-Cy exhibits good sensitivity and selectivity toward H₂O₂. In addition, Bio-B-Cy shows a dose-dependent response to H₂O₂ and the detection limit is 0.14 μM. We further demonstrate that Bio-B-Cy could successfully detect the H₂O₂ in biotin receptor-positive cancer cells and tumor tissues. Based on the results, Bio-B-Cy has the potential to serve as an efficient tool for early diagnosis of cancer.

1. Introduction

Hydrogen peroxide (H₂O₂) is one of the reactive oxygen species (ROS) and plays an important role in diverse pathological and physiological processes such as cell growth, poliferation, migration, and apoptosis [1]. H₂O₂ affects the regulatory function of proteins and the associated cascades by selectively reacting with the thiol group of cystine residues of proteins. Thus, abnormal expression levels of H₂O₂ can cause cell damage, apoptosis, and autophagy, thereby inducing diseases such as inflammation [2], Alzheimer’s disease [3], and cancers [4]. It is noteworthy that although H₂O₂ is ubiquitous in various cells, H₂O₂ is usually upregulated in cancer cells (~5.0 μM to 1.0 mM), while the concentration of H₂O₂ in normal cells is significantly lower (less than 0.7 μM), which makes it a promising biomarker for cancer diagnosis [5]. Furthermore, due to the widespread presence of H₂O₂ in vivo, the development of H₂O₂-targeted diagnostic strategies for tumors is particularly crucial to avoid false positives.

Compared with other clinical imaging technologies, fluorescence imaging technologies are attractive in biomedical applications due to their high selectivity, non-invasiveness, and high-resolution imaging [6,7,8,9,10]. Fluorescent probes play an important role in fluorescence imaging. Fluorescent probes refer to a type of fluorescent molecule that has a characteristic fluorescence response in the ultraviolet-visible-near-infrared (UV-Vis-NIR) region [11]. To date, many traditional fluorescent dyes have been applied to form H₂O₂-sensitive probes in biological imaging. For instance, 2,7-dichlorofluorescein (DCF) derivative probes have been widely used to detect gas, such as H₂S, ROS, and NO [12]. Fluorescein derivative probes are developed for sensitive detection of H₂O₂ in living cells [13]. However, the application in vivo of these reported H₂O₂ probes is limited due to its short analytical wavelengths. Near-infrared (NIR) fluorescent probes have long excitation/emission wavelengths, enabling deeper tissue penetration, and lower background fluorescence of proteins, which are beneficial to apply in biological imaging [14,15,16]. For instance, Lin reported an approach for near-infrared fluorescent imaging in vivo [17]. Wang et al. developed an NIR probe DHX-1 for imaging H₂O₂ in kidney injury caused by environmental hazards of physiological factors [18]. Zan et al. presented an NIR probe for the visualization of endogenous levels of H₂O₂ changes in different disease models [19]. However, as mentioned above, H₂O₂ is not exclusively expressed in tumor cells but is also present in other cell types, such as inflammatory cells. Therefore, non-targeted H₂O₂ fluorescent probes may produce strong signals in non-tumor cells, leading to false-positive results. Considering this, the development of fluorescent probes with tumor-cell-specific targeting is of great importance. To improve the selectivity of tumor cells in vivo, installing a recognition group for specific cell membrane receptors in cancer cells is a classical strategy [20]. Biotin receptors are overexpressed in various tumor cells including lung, breast, ovarian, and cervical cancer cells [21]. Therefore, biotin can be used as an NIR probe ligand to enhance tumor targeting in vivo due to its high affinity for biotin receptors [22]. However, biotin was also found to affect a range of metabolic processes in the liver [23], such as participating in liver lipid metabolism [24], and regulating liver glucokinase [25], which indicates that the excretion and degradation of biotin in organisms is delayed. Therefore, when designing the molecular probe, carefully considering the incorporation and removal of biotin is crucial, as it can impact the probe’s specificity and metabolic performance.

Herein, we present a tumor-cell-targeted NIR fluorescent probe (Bio-B-Cy), which contains a biotin group as the tumor cells targeting part, a cyanine dye as NIR fluorophore [26], and a boronic acid ester [27] as the H₂O₂-responsive unit. The dye is a stable hemicyanine skeleton and possesses an NIR feature. The conjugation of biotin in Bio-B-Cy facilitates its great tumor-targeting ability [22]. The fluorescence of cyanine is quenched by the boronic acid ester group. Upon reacting with H₂O₂, Bio-B-Cy quickly cleaves the boronic acid ester group and releases a strong NIR fluorescence. Bio-B-Cy exhibits high sensitivity and selectivity towards H₂O₂ in solution with a relatively low limit of detection of 0.14 μM. Meanwhile, Bio-B-Cy can quickly target biotin-receptor-positive cancer cells and tissues, which has been successfully utilized for detecting H₂O₂ in cancer cells and tumor-bearing mice, confirming its potential to be a promising agent for cancer diagnosis. Additionally, previous similar probe retained the biotin moiety even after the H₂O₂ response [28], which may potentially accumulate in the liver during metabolism. In contrast, molecular designed in this work incorporates the biotin group as a removable component. The clever molecular design work ensures that the biotin is cleaved from the NIR fluorescent dye upon the reaction with H₂O₂, thereby avoiding metabolic issues and false-positive concerns. This work designed a near-infrared fluorescent probe that responds to H₂O₂ and targets tumors, highlighting its potential applications in cancer imaging.

2. Materials and Methods

2.1. Instrumentation

Commercial analytical grade chemicals were purchased and used without purification. Cy-OH was synthesized according to the previously described method [26]. NMR spectra were collected on a 600 MHz spectrometer (Bruker AVANCE III HD 600, Bremen, Germany) using chloroform-d or Dimethyl sulfoxide-d as the solvent. Mass spectra were obtained by using MALDI-TOF-MS (Bruker Autoflex III, Bremen, Germany).

2.2. Synthesis of Bio-B-Cy

Scheme 1 illustrates the chemical structure of Bio-B-Cy and its synthesis routine. Compound 1 was treated with Di-tert-butyl decarbonate to protect the amino. Then, NaBH₄ converted the carbonyl group of compound 2 to the alcohol group, which was then treated with Bis(pinacolato)diboron to obtain compound 4. The protecting group left and the amino was linked to Biotin-yielding compound 6. The NIR fluorescent substrate (Cy-OH) was synthesized according to the method reported in the literature. Subsequently, Cy-OH was conjugated with compound 6 to yield the Bio-B-Cy.

2.2.1. Synthesis of Compound Cy-OH

Resorcinol (248 mg, 2.3 mmol) and K₂CO₃ (311 mg, 2.3 mmol) were dissolved in 5 mL acetonitrile under an argon atmosphere. The mixture was stirred for 10 min at room temperature. IR 780 (600 mg, 0.9 mmol) was dissolved in 5 mL acetonitrile and was added to the mixture with vigorous stirring. The reaction mixture was then warmed to 50 °C and stirred for 2 h. The desired product came at methanol and dichloromethane in the volume ratio of 1:60, yielding the product as a blue solid (231 mg, 38.5%). ¹H NMR (600 MHz, Chloroform-d) δ 8.66 (d, 1H), 7.53 (d, 1H), 7.47–7.42 (m, 1H), 7.38–7.32 (m,3H), 7.14 (d, 2H), 6.23 (d, 1H), 4.20 (s, 1H), 3.69 (s, 1H), 2.77 (t, 2H), 2.70 (t, 2H), 1.96 (d, 2H), 1.85 (s, 8H), 1.12 (t, 3H). HRMS (MALDI): m/z, 412.23 [M]⁺, calcd for C₂₈H₃₀NO₂⁺, 412.23.

2.2.2. Synthesis of Compound 2

In total, 7.5 mL Triethylamine in 50 mL tetrahydrofuran (THF) was added to a THF solution (100 mL) of compound 1 (5 g, 23.3 mmol) and Di-tert-butyl decarbonate (5.6 g, 25.7 mmol) at 0 °C. The mixture was heated to 40 °C and stirred for 2 h. Reaction mixture was filtrated, and the solvent was removed under reduced pressure. In total, 200 mL ethyl acetate (EtOAc) was added to dissolve the residues. The organic solution was washed with NH₄Cl aqueous solution, NaHCO₃ aqueous, and brine, then dried over Na₂SO₄. After removal of the solvent, the residues were purified by silica gel affording compound 2 a white solid (6.6 g, yield:90%). ¹H NMR (500 MHz, Chloroform-d) δ 7.92–7.86 (m, 2H), 7.44–7.37 (m, 2H), 4.02 (t, J = 8.6 Hz, 1H), 3.54 (t, J = 8.1 Hz, 1H), 1.38 (s, 9H). HRMS (MALDI): m/z, 336.02 [M + Na]⁺, calcd for C₁₃H₁₆BrNNaO₃⁺, 336.02.

2.2.3. Synthesis of Compound 3

Compound 2 (5 g, 15.9 mmol) was dissolved in 100 mL methanol. NaBH₄ (3 g, 79.3 mmol) was added to the solution and stirred for 30 min. The reaction solution was diluted with water and methanol was removed under reduced pressure. The aqueous solution was extracted with EtOAc 150 mL × 3. The organic solution was washed with brine and then dried over Na₂SO₄. After removal of the solvent, the residues were purified by silica gel affording compound 3 a white solid (4.6 g, yield:92%). ¹H NMR (600 MHz, CDCl₃) δ 7.53–7.48 (m, 2H), 7.27 (d, J = 8.5 Hz, 2H), 4.87–4.80 (m, 1H), 3.48 (d, J = 14.5 Hz, 1H), 3.24 (dd, J = 14.1, 7.8 Hz, 1H), 1.47 (s, 9H). HRMS (MALDI): m/z, 338.04 [M + Na]⁺, calcd for C₁₃H₁₈BrNNaO₃⁺, 338.04.

2.2.4. Synthesis of Compound 4

1,4-dioxane solution of compound 3 (2 g, 9.5 mmol), Bis(pinacolato)diboron (5.6 g, 22.1 mmol), Potassium Acetate (3.3,33 mmol), Pd(dppf)Cl₂·DCM (0.42 g, 0.52 mmol) was heated to 90 °C and stirred under a nitrogen atmosphere for 12 h. The reaction mixture was filtrated, and the solvent was removed under reduced pressure. In total, 200 mL ethyl acetate (EtOAc) was added to dissolve the residues, and washed by HCl (1 M), and brine, then dried over Na₂SO₄. After removal of the solvent, the residues were purified by silica gel affording compound 4 a white solid (2.8 g, yield:80%). ¹H NMR (600 MHz, CDCl₃) δ 7.83 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.7 Hz, 2H), 4.90–4.86 (m, 1H), 3.52 (s, 1H), 3.29–3.23 (m, 1H), 1.47 (s, 9H), 1.37 (s, 12H). HRMS (MALDI): m/z, 386.21 [M + Na]⁺, calcd for C₁₉H₃₀BNNaO₅⁺, 386.21.

2.2.5. Synthesis of Compound 6

Compound 4 (0.5 g, 1.36 mmol) was dissolved in 4 mL Methylene Chloride (CH₂Cl₂) and 2 mL Trifluoroacetic Acid (TFA). After stirring for 12 h, the solvent was removed under reduced pressure and the residues were dissolved in CH₂Cl₂. In total, 3 mL Triethylamine and Biotin-NHS ester (0.75 g, 2.20 mmol) were added to the solution and stirred for 24 h. The reaction mixture was filtrated, and the solvent was removed under reduced pressure. In total, 200 mL CH₂Cl₂ was added to dissolve the residues, washed with HCl (1 M) and brine and then dried over Na₂SO₄. After removal of the solvent, the residues were purified by silica gel affording compound 6 a yellow solid (2.7 g, yield:40%). ¹H NMR (600 MHz, DMSO-d₆) δ 7.55–7.48 (m, 2H), 7.35–7.30 (m, 2H), 4.66 (dd, J = 9.0, 3.5 Hz, 1H), 4.31 (dd, J = 7.7, 5.1 Hz, 1H), 4.13 (ddd, J = 7.5, 4.4, 1.7 Hz, 1H), 3.10 (ddd, J = 8.6, 6.2, 4.4 Hz, 1H), 2.84 (ddd, J = 25.9, 12.7, 4.4 Hz, 2H), 2.66 (dd, J = 12.8, 9.0 Hz, 1H), 2.58 (d, J = 12.4 Hz, 1H), 2.11 (t, J = 7.4 Hz, 2H), 1.62 (ddt, J = 12.2, 9.8, 6.2 Hz, 1H), 1.52–1.44 (m, 3H), 1.33 (dq, J = 9.5, 7.1 Hz, 2H), 1.17 (s, 12H). ¹³C NMR (151 MHz, DMSO-d₆) δ 176.48, 163.27, 142.87, 131.47, 128.64, 120.68, 83.30, 70.58, 61.56, 59.70, 55.94, 47.48, 35.60, 28.80, 28.54, 25.64, 25.30. HRMS (ESI): m/z, 490.27 [M + H]⁺, calcd for C₂₄H₃₇BN₃O₅S⁺, 490.25.

2.2.6. Synthesis of Compound Bio-B-Cy

Compound Cy-OH (50 mg, 0.125 mmol) and DIPEA (43.1 μL, 0.24 mmol) were dissolved in 10 mL acetone. Bis(trichloromethyl) carbonate (80 mg, 0.155 mmol) was added to the solution under an ice bath. After stirring for 4 h under an ice bath, 10 mL acetone of compound 6 (195 mg, 0.275 mmol) was added to the solution and stirred for 24 h under room temperature. The reaction mixture was filtrated, and the solvent was removed under reduced pressure. In total, 200 mL CH₂Cl₂ was added to dissolve the residues, and washed by HCl (1 M), and brine, then dried over Na₂SO₄. After removal of the solvent, the residues were purified by silica gel affording compound 6 a purple solid (34.7 mg, yield:30%). ¹H NMR (600 MHz, DMSO-d₆) δ 7.60 (s, 1H), 7.55 (dd, J = 23.5, 7.8 Hz, 4H), 7.44–7.28 (m, 6H), 7.25–7.08 (m, 3H), 6.70–6.62 (m, 1H), 6.50 (d, J = 16.2 Hz, 2H), 6.44 (s, 1H), 6.27–6.19 (m, 1H), 6.06 (d, J = 14.1 Hz, 1H), 4.33 (dd, J = 7.8, 5.0 Hz, 1H), 4.18–4.13 (m, 2H), 4.07 (d, J = 7.2 Hz, 1H), 3.12 (dd, J = 8.9, 4.8 Hz, 2H), 3.04 (dd, J = 12.8, 3.3 Hz, 1H), 2.87–2.81 (m, 2H), 2.63 (dd, J = 23.2, 15.2 Hz, 4H), 2.19 (t, J = 7.4 Hz, 2H), 1.83–1.76 (m, 2H), 1.65 (d, J = 16.0 Hz, 6H), 1.58–1.47 (m, 6H), 1.34 (t, J = 7.9 Hz, 3H), 1.17 (s, 15H). ¹³C NMR (151 MHz, DMSO-d₆) δ 175.08, 163.29, 158.93, 141.73, 131.68, 131.31, 130.09, 128.90, 128.72, 128.42, 128.09, 122.87, 121.98, 121.23, 106.66, 102.99, 81.81, 78.68, 72.71, 69.05, 61.57, 59.71, 55.88, 45.95, 45.20, 43.30, 34.15, 29.71, 28.62, 28.53, 28.39, 25.53, 25.41, 25.08, 24.95, 19.63, 11.85, 11.64. HRMS (MALDI): m/z, 927.63 [M]⁺, calcd for C53H64BN4O8S⁺, 927.45.

2.3. Fluorescence Response of Bio-B-Cy Toward H₂O₂

Bio-B-Cy was dissolved in DMSO to make a 5 mM stock solution. For fluorescence measurements, H₂O₂ with different concentrations was added into 2 mL PBS buffer solution containing 10 μM Bio-B-Cy. The mixture solution was incubated at 37 °C for 30 min. The fluorescence signal was recorded with the excitation at 680 nm and the emission at 700–710 nm. The slit was 5 nm.

2.4. The Selectivity of Bio-B-Cy Toward H₂O₂

For fluorescence measurements, 10 μM Bio-B-Cy was incubated with PBS buffer (blank), other ROS (O₂⁻, ClO⁻, ¹O₂, ·OH, TBHP, ⋅NO, ONOO⁻, ⋅O^tBu) and H₂O_2, respectively. The mixture solution was incubated at 37 °C for 20 min. The fluorescence signal was recorded with the excitation at 680 nm and the emission at 700–710 nm. The slit was 5 nm.

2.5. Theoretical Calculations

Density functional theory (DFT) calculations were used to understand the electronic excitation and photophysical properties of Bio-B-Cy and Cy-OH. Geometry optimizations on the ground were carried out with the B3LYP function in combination with the 6-31G(d) basis set. All calculations were carried out with Gaussian 16 W.

2.6. Cell Viability

The 4T1 cells and L929 cells were seeded at a density of 1 × 10⁴/well in 96 wells and grown overnight at 37 °C. The next day, the cells were incubated with different concentrations of Bio-B-Cy for 24 h. The viability of cells was evaluated by the CCK 8 kit.

2.7. Flow Cytometric Analyses

4T1 cells and L929 cells were seeded in six-well plates at a density of 2 × 10⁶ cells/well and cultured overnight at 37 °C. After 24 h of incubation, the cells were treated with 10 μM Bio-B-Cy. Following incubation, cells were harvested by scraping, resuspended in PBS, and transferred to flow cytometry tubes. Cellular analysis was performed using a flow cytometer with red RL2 fluorescence detection parameters (excitation: 680 nm, emission: 720/30 nm). A minimum of 10,000 cells were analyzed for each sample. All experiments were conducted three times.

2.8. In Vitro Cell Imaging

4T1 cells and L929 cells were plated on glass-bottomed dishes at a density of 2 × 10⁶ cells, respectively, and grown overnight. The next day, the cells were incubated with 10 μM Bio-B-Cy in 30 min. The cells were washed with PBS three times after incubation. Then, the cells were incubated with calcein-AM for 30 min to confirm the cell viability. Fluorescence images were acquired from CLSM using a 680 nm laser and a 707 nm emission filter.

2.9. In Vivo Animal Imaging

Four-week-old female BALB/C mice were subcutaneously injected with 4T1 cells (5 × 10⁵ cells/mouse) to establish a tumor model. The mice were used before the tumor grew to 10 mm in average diameter. The mice were separated into two groups. Bio-B-Cy: the mice were injected intravenously with 100 μL saline containing 500 μM Bio-B-Cy. B-Cy: the mice were injected intravenously with 100 μL saline containing 500 μM B-Cy. After intravenous injection, in vivo, fluorescence images were acquired at 1, 2, 4, and 8 h using an optical system with the excitation at 680 nm and the emission at 707 nm. The mice were sacrificed at 4 h after the injection and the fluorescence images of tumors and major organs were recorded with the excitation at 680 nm and the emission at 707 nm.

3. Results

3.1. Design and Synthesis of Bio-B-Cy

To enable reliable detection of the H₂O₂ produced from cancer cells, it is required that the probe should own a rapid response toward H₂O₂ associated with great tumor-targeting ability. The H₂O₂-activatable NIR probe Bio-B-Cy is designed to be composed of an NIR fluorescent substrate (Cy-OH), a H₂O₂-sensitive unit (Boronic acid ester), and a tumor-targeting group (biotin). Cy-OH is used because it showed strong fluorescence at 707 nm, which ensures great tissue penetration, allowing the application of Bio-B-Cy in vivo [26]. Considering the diffusion of H₂O₂ in tumor cells, we choose Boronic acid ester as the detection group, which exhibits fast oxidation kinetic towards H₂O₂ [29,30]. The Boronic acid ester is linked to the NIR fluorescent substrate via a carbamate bond, resulting in the quenching of the fluorescence. Different from normal cells, cancer cells overexpress different kinds of proteins on the surface. As reported, some types of tumor cells overexpressed biotin receptors [22,31]. Bio-B-Cy could immediately target tumor cells with the advantage of conjunction with Biotin. Figure 1 illustrates the general mechanism for NIR FL of the H₂O₂ in the tumor site. After intravenously injected into 4T1-bearing mice, Bio-B-Cy moved towards tumor cells with the help of biotin and quickly responded to H₂O₂ overexpressed by tumor cells, simultaneously turning on NIR FL signals, while the probe without biotin showed weaker NIR FL. Therefore, Bio-B-Cy was capable of tumor diagnosis via sensitive NIR FL of H₂O₂ in the tumor site. Scheme 1 showed the synthesis routine of Bio-B-Cy and the characterization (¹H NMR, HRMS and ¹³C NMR) of the probe Bio-B-Cy was presented in the Supporting Information (Figures S1–S14).

3.2. Photophysical Properties of Bio-B-Cy

We performed a spectral study in solutions to evaluate the sensitivity of Bio-B-Cy for H₂O₂ detection. First, we tested whether Bio-B-Cy could quickly react with H₂O₂ to recover fluorescence. As shown in Figure S15, there were no fluorescence emissions when Bio-B-Cy did not react with H₂O₂. We added 50 μM H₂O₂ to 10 μM Bio-B-Cy in PBS, which led to the increase in emission intensity at 707 nm in 20 min, indicating that boronic acid ester reacted with H₂O₂ and recovered the fluorescence from the NIR dye efficiently. As depicted in Figure 2A, Bio-B-Cy has a maximum absorption wavelength of 544 nm. Upon the addition of H₂O₂, the maximum absorption wavelength performed a red shift from 544 nm to 680 nm. The color of the solution changed from purple to blue. Next, we evaluated the correlation between the concentration of H₂O₂ and Bio-B-Cy. After incubation with H₂O₂ from 0 μM to 100 μM for 30 min, a strong fluorescence peak at 707 nm appeared, and the fluorescence intensity increased with the increasing concentrations of H₂O₂ (Figure 2B). In addition, when the concentration of H₂O₂ was 0–100 μM, the fluorescence intensity at 707 nm showed a linear relationship with the concentration of H₂O₂ (Figure 2C). Based on a regression analysis, the detection limit of Bio-B-Cy was found to be 0.14 μM. These results indicated that Bio-B-Cy could be used for the quantitative determination of H₂O₂ in PBS.

The reaction of Bio-B-Cy and H₂O₂ in serum was also investigated. As depicted in Figure S16, after reacting with H₂O₂, fluorescence intensity at 707 nm increased with increasing concentration of H₂O_2. This was consistent with the Bio-B-Cy in PBS. The results represented that Bio-B-Cy can sensitively respond to H₂O₂ with the change in fluorescence intensity. Second, we tested the reaction of Bio-B-Cy toward H₂O₂ at different pH. As shown in Figure 2D, Bio-B-Cy performed the maximal fluorescence intensity at pH 7. Meanwhile, to verify the selectivity of Bio-B-Cy toward H₂O₂, we also tested the fluorescence response of Bio-B-Cy to other ROS including O₂⁻, ClO⁻, ¹O₂, ·OH, TBHP, ⋅NO, ONOO⁻, ⋅O^tBu. There was no obvious fluorescence change at 707 nm after incubating with other ROS. This result suggested that the influence of other ROS on the fluorescence spectrum was negligible (Figure 2E). The equilibrium of fluorescence intensity was reached within 20 min with the addition of H₂O₂ at 37 °C (Figure S17). The fluorescence intensity of Bio-B-Cy stayed stable in PBS over 4 h (Figure S18). The fluorescence imaging of the Bio-B-Cy (10 μM) with varying concentrations of H₂O₂ (0, 10, 20, 30, 40, and 50 μM) was also investigated (Figure 2F), which was consistent with the spectral study. These results indicated that Bio-B-Cy was suitable for the selective detection of H₂O₂ in biological systems.

3.3. Mechanism Study

Based on the spectral results, the proposed mechanism is shown in Figure 3A. The H₂O₂ oxidized the p-(Boronic acid ester) benzyl group and induced the subsequent 1,4-elimination reaction, resulting in a self-cleavage of the linker to release Cy-OH. The released Cy-OH showed fluorescence emission at 707 nm. To confirm the presumption, the reaction of Bio-B-Cy and H₂O₂ was carried out and analyzed by HPLC. As depicted in Figure S19, the peak of Bio-B-Cy disappeared after incubation with H₂O₂ for 20 min at 37 °C, and the peak of the main product appeared at the same retention time as Cy-OH. Furthermore, density functional theory (DFT) calculations were performed to analyze the optical properties of Bio-B-Cy. All calculations of DFT were performed by using Gaussian 16. B3LYP with the 6-31G(d) level of DFT was used for estimating the molecular orbitals (highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO)). Frontier molecular orbitals diagrams and data on energy levels are given in Figure 3B. The computed energy gap between HOMO and LUMO between Bio-B-Cy and Cy-OH were 2.52 and 2.47 eV, respectively, which show the red shift of the absorption. Notably, previous reports showed that a red-shift of absorption appeared due to reducing the energy gap Eg (E_g = E_LUMO − E_HOMO) [32]. The above result confirmed the generation of Cy-OH.

3.4. In Vitro Probe Activation and Imaging of 4T1 Cells

Encouraged by the good sensitivity and selectivity of Bio-B-Cy toward H₂O₂ in PBS, we applied the Bio-B-Cy to H₂O₂ imaging in living cells. We chose biotin receptor-positive 4T1 cells [33,34] and biotin receptor-negative L929 mouse fibroblast cells to explore the detecting ability of the probe Bio-B-Cy to H₂O₂ secreted from cancer cells. Before the bioimaging application, the cytotoxicity of Bio-B-Cy was evaluated by a CCK 8 kit. Cytotoxicity was an important issue of whether Bio-B-Cy could be applied in vivo. After incubation with 0–50 μM Bio-B-Cy for 24 h, the viabilities of 4T1 and L929 cells were above 90%, which meant Bio-B-Cy was low cytotoxicity and biocompatibility (Figure S20).

Then, we chose 4T1 cells as a cancer cell model to explore the targeting ability of Bio-B-Cy to biotin receptor-positive cancer cells. 4T1 cells were treated with Bio-B-Cy, B-Cy, B-Cy+biotin, respectively, and fluorescence images of cells were acquired. As depicted in Figure 4A, each group showed red fluorescence, but the fluorescence intensity of the Bio-B-Cy group was the greatest. This was because the biotin of Bio-B-Cy helped the probe enter cells via biotin overexpressed on the surface of cancer cells, which increased the uptake rate of the Bio-B-Cy. The fluorescence intensity of the B-Cy+Biotin group was similar to the B-Cy group, just showing a weak red fluorescence. Meanwhile, we have also stained the cells of calcein-AM to ensure the viability of 4T1 cells after incubating with probes. As shown in Figure 4A, 4T1 cells treated with different groups exhibited great cell viability after incubation, which was consistent with the results of CCK8. This phenomenon indicated free biotin cannot increase the uptake rate of the probe. Based on the results obtained, it can be summarized that Bio-B-Cy can target biotin receptor-positive cancer cells.

We also explored the detecting ability of Bio-B-Cy to cellular H₂O₂. 4T1 cells and L929 cells were incubated with Bio-B-Cy for 30 min at 37 °C. As illustrated in Figure 4B, because of the low concentration of intracellular H₂O₂, L929 cells showed only weak fluorescence at 707 nm. In contrast, the fluorescence increased significantly in 4T1 cells, indicating the detection of cellular H₂O₂. To quantitatively examine whether Bio-B-Cy could respond towards H₂O₂, flow cytometric analysis was performed. The Bio-B-Cy showed greater fluorescence in 4T1 cells than in L929 cells, which was consistent with LSCM (Figure 4C). The results indicated that Bio-B-Cy could quickly respond to cellular H₂O₂ and it was suitable for the detection of tumor cells.

3.5. In Vivo Image of Tumor-Bearing Mice

With the excellent performance of Bio-B-Cy being firmly established in 4T1 cells, we next assessed its targeted imaging of H₂O₂ in a 4T1-bearing mouse model. We first investigated whether Bio-B-Cy could be delivered and retained in the 4T1-bearing mice. Longitudinal FL imaging showed that the fluorescence intensity of Bio-B-Cy and B-Cy at 707 nm gradually increased after intravenous injection, which peaked at 2 h (Figure 5A). However, due to the lack of biotin, the maximum tumor fluorescence intensity was ~3-fold higher than in mice treated with B-Cy. To further prove the fluorescence generation in the tumor site, we sacrificed the injecting mice after a 2 h injection. After sacrifice, organs and tumor tissues were harvested from tumor-bearing mice and their fluorescence intensity was detected individually. Compared with B-Cy, Bio-B-Cy exhibited less liver accumulation and the fluorescence intensity of Bio-B-Cy is mainly distributed in tumor tissue (Figure 5C). This indicated that Bio-B-Cy was able to achieve a specific distribution within the tumor through the targeting ability of biotin groups, resulting in a high signal-to-noise ratio. Quantification of the data revealed a 1.5-fold higher fluorescence intensity for Bio-B-Cy in tumor sites than B-Cy. The results demonstrated that the biotin-bearing probe Bio-B-Cy could be more efficient than B-Cy to enter and accumulate in 4T1 tumors assisted by the biotin-mediated targeting, generating fluorescence intensity to figure out the tumors.

4. Conclusions

In summary, we have designed and synthesized a new NIR fluorescent probe Bio-B-Cy that can discriminate between cancer cells and normal cells, selectively enter the former ones, and react with intracellular H₂O₂. With the addition of H₂O₂, Bio-B-Cy emitted intense fluorescence. The results demonstrated that Bio-B-Cy owns favorable properties, like well selectivity, stability, and low toxicity. Moreover, Bio-B-Cy was applied to selectively monitor the high level of H₂O₂ in biotin-receptor positive 4T1 cells. Most importantly, Bio-B-Cy not only can target the tumor of 4T1-bearing mice but also has a good screening ability for tumor cells in animal models. Our findings suggest that Bio-B-Cy has the potential to improve early diagnosis and monitoring of TNBC, giving a new approach to personalized treatment strategies for this challenging disease. We also expect that Bio-B-Cy could be used in preclinical and clinical studies, which will validate the efficacy and safety of this promising imaging tool.