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. 2024 Mar 20;67(7):5924–5934. doi: 10.1021/acs.jmedchem.4c00179

Novel PET Imaging Probe for Quantitative Detection of Senescence In Vivo

Xin Xiang †, Chuning Dong †, Lianbo Zhou †, Jun Liu ‡, Zachary M Rabinowitz ‡, Yuzhao Zhang ‡, Honghui Guo †, Feng He †, Xingdou Chen †, Yunhua Wang †, Lina Cui ‡,*, Xiaowei Ma †,*
PMCID: PMC11017977  PMID: 38507820

Abstract

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Real-time detection of cellular senescence remains a clinical challenge. Here, we aimed to develop a positron emission tomography (PET) imaging probe targeting senescence-associated β-galactosidase (SA-β-Gal), the most widely used biomarker of cellular senescence, and investigate its performance for real-time in vivo quantitative detection of cellular senescence. A stable PET imaging agent [68Ga]Ga-BGal was obtained with a high labeling yield (90.0 ± 4.3%) and a radiochemical purity (>95%). [68Ga]Ga-BGal displayed high sensitivity and specificity for β-Gal both in vitro and in vivo. The reaction and uptake of the probe correlated with the β-Gal concentration and reaction time. In PET imaging, high β-Gal-expressing CT26.CL25 tumors and doxorubicin-treated HeLa tumors showed high signals from [68Ga]Ga-BGal, while a low signal was observed in CT26.WT and untreated HeLa tumors. In summary, we showcased successful PET imaging of senescence in preclinical models using probe [68Ga]Ga-BGal. This finding holds the potential for translating senescence imaging into clinical applications.

Introduction

Cellular senescence, a cellular state triggered by stress, is characterized by a prolonged and generally irreversible cell-cycle arrest with macromolecular damage, a distinct secretory phenotype (referred to as the senescence-associated secretory phenotype or SASP), and altered metabolism.1 Various endogenous and exogenous triggers have been found to induce cellular senescence.2,3 Generally, the main biological roles of senescent cells are to prevent damaged or stressed cells from proliferating and to recruit the immune system to clear themselves and surrounding damage.3−7 Additionally, cellular senescence is viewed as a tumor suppression mechanism.8 Thus, inducing cellular senescence has been considered as a potent anticancer therapeutic strategy. However, the accumulation and persistence of a senescent cell milieu and the secretion of inflammatory, growth-promoting, and extracellular matrix remodeling SASP factors are thought to contribute to adverse consequences.9−11 Furthermore, cellular senescence has been implicated in aging and age-related diseases such as neurodegenerative disorders, diabetes, and atherosclerosis.12,13

Due to the physiological and pathophysiological significance of cellular senescence, detecting senescent cells has become increasingly important.14 There are many different senescence-associated biomarkers such as G1/S cell-cycle checkpoint inhibitors (i.e., p16lnk4a, p21CIP1, p53), SASP factors (i.e., IL-6, IL-8, MMPs), and SA-β-Gal that can be used to in vitro or in vivo detect senescent cells.14−17 Importantly, methods to detect senescence in vivo hold great promise for the clinical evaluation of anticancer therapies and senolytic agents18 and are indispensable for deepening our understanding of its role in aging and age-related diseases in living subjects.

The irregular metabolism of senescent cells leads to significantly elevated activity of lysosomal β-galactosidase (β-Gal, encoded by GLB1), a glycoside hydrolase that catalyzes the hydrolysis of the glycosidic bond of β-galactosides.19,20 SA-β-Gal is the most commonly used senescence-associated biomarker and has been successfully used in small-molecular imaging to visualize senescent cells at the cellular and organismal levels.14 Staining of fixed cells or tissues20 with 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal), a β-Gal-targeting colorimetric-based small-molecule probe that releases an insoluble blue ingoid dye once activated by β-Gal, is the standard method for detecting senescent cells in vitro or ex vivo.21 Moreover, a wide variety of fluorescent-based small-molecule probes were developed for β-Gal activity imaging.14,22−26 Unfortunately, most of these molecules cannot be used for in vivo imaging due to the short fluorescence emission wavelength (<600 nm), limiting tissue penetration. However, fluorescent probes that emit longer wavelength light (far-red or near-infrared (NIR) range) have been utilized to detect β-Gal activity in vivo (i.e., in small animals such as mice).14,27 Using NIR-BG, a fluorogenic, NIR-based β-Gal probe, we demonstrated real-time imaging of senescence in xenograft human tumors in living mice for the first time.28 Additionally, we developed NIR-BG2, which features an activatable, self-immobilizing moiety on the fluorophore, to extend the intracellular residence time of the signaling moiety in the cell (i.e., the fluorophore), once activated by SA-β-Gal.29 NIR-BG2 was shown to be significantly more sensitive in detecting SA-β-Gal activity than NIR-BG in chemotherapy-treated HeLa cells and in chemotherapy-treated HeLa tumor xenografts in living mice.29 Optical imaging probes serve as useful tools for preclinical evaluation in vitro and in small animals; however, due to the penetration limitation of optical imaging, optical probes face challenges for clinical translation. We are therefore developing senescence probes adaptable to clinically translatable modalities such as PET.30

PET is a clinical nuclear medicine imaging technique that detects paired γ rays emitted from positron emission radionuclides on specific molecules to image their distribution in the human body and achieve the purpose of accurate evaluation of the disease.31 It has the advantages of high specificity and sensitivity as well as the ability to quantitatively study the expression, distribution, metabolism, and function of biomolecules in vivo. Currently, a clinical trial is planned to detect therapy-resistant tumor cells by the senescence-specific radiotracer [18F]FPyGal;32 however, no characterization or evaluation data has been reported for this tracer thus far. In this study, we developed a PET imaging probe, [68Ga]Ga-BGal, and successfully applied it to accurately visualize the activity of SA-β-Gal in living mice with genetically edited tumors (CT26.CL25, CT26 cells with the LacZ reporter gene to overexpress β-Gal) and chemotherapy-induced senescence in HeLa xenograft mice models.

Results and Discussion

Synthesis and Characterization of [68Ga]Ga-BGal

[68Ga]Ga-BGal was synthesized following the optimized reaction route (Figure 1). The reaction of compounds 1 with p-SCN-Bn-NOTA, a thioisocyanate (SCN)-modified macrocyclic metal-chelator, under basic conditions afforded BGal, which was characterized by NMR (Figure S1) and HRMS (Figure S2). The purity of BGal was greater than 95% (Figure S3). Then, BGal was labeled efficiently with 68Ga under mild conditions in 10 min to produce [68Ga]Ga-BGal. The product was purified using a radio-HPLC with a C18 cartridge and filtered through a 0.22 μm sterilized filter. The 68Ga labeling yield of [68Ga]Ga-BGal was 90.0 ± 4.3%, and the radiochemical purity was greater than 95%, as determined by radio-HPLC (Figure 2). The in vitro (Figure 2) and in vivo (Figure S4) stability of the probe for 4 h was higher than 95%.

Figure 1.

Figure 1

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Optimized reaction route to [68Ga]Ga-BGal.

Figure 2.

Figure 2

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Radio-HPLC analysis of [68Ga]Ga-BGal. Top, HPLC analysis of the probe before purification; middle, analysis of the probe after 4 h in serum; bottom, analysis of the probe after purification. The labeling yield was 90.0 ± 4.3%. The radiochemical purity was greater than 95%. The stability of the probe in serum for 4 h was above 95%.

Gel Electrophoresis for Investigating the Interaction of [68Ga]Ga-BGal with β-Gal

To demonstrate the specificity of [68Ga]Ga-BGal in the presence of β-Gal, the probe was incubated with proteins, and then, gel electrophoresis was performed, followed by a PET/CT scan to obtain the images. In the presence of active β-Gal, a new band was observed at 25–35 kDa, while no signal was observed in the inactive β-Gal, BSA, and PBS channels (Figure 3a). An anti-interference experiment was carried out (Figure S5). Furthermore, we examined the specificity of the probe against β-Gal in CT26.CL25 cells, which was the same as that with recombinant β-Gal, while no signal was observed in CT26.WT cells (Figure 4a). With longer incubation time or higher protein concentration, the signal gradually increased (Figures 3b,c and 4b,c). Quantitative analysis confirmed the results. The band at 70–100 kDa may be an intact probe trapped due to a sudden change in charge or polarity. The band at 15–25 kDa may be a noncovalent binding of the probe to some component in the loading buffer. The dynamic process of gel electrophoresis is shown in Online Resource 1. To further confirm the enzyme-triggered cleavage reaction underlying the response of [68Ga]Ga-BGal to β-Gal, radio-HPLC experiments were conducted. As shown in Figure S6, following the incubation of [68Ga]Ga-BGal with β-Gal, a new peak appeared, while there was no change after incubating with inactive β-Gal. The results confirmed that the β-glycosidic bond was cleaved by β-Gal.

Figure 3.

Figure 3

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PET images of [68Ga]Ga-BGal incubated with (a) β-Gal, inactive β-Gal, BSA, and PBS, (b) β-Gal for 1, 5, 15, 30, and 60 min, and (c) β-Gal for 0.01, 0.015, 0.022, 0.033, and 0.05 μg/μL and the corresponding quantitative analysis. (Percentage: the band accounts for the entire lane.)

Figure 4.

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PET images of [68Ga]Ga-BGal incubated with (a) the protein extracted from CT26.CL25 and CT26.WT cells, (b) CT26.CL25 for 1, 15, 30, 60, and 120 min, and (c) the protein of CT26.CL25 for 0.009, 0.0375, 0.15, 0.6, and 2.4 μg/μL and the corresponding quantitative analysis. (Percentage: the band accounts for the entire lane.)

Binding Mode of [68Ga]Ga-BGal with β-Gal

To reveal the binding mode with β-Gal derived from different sources, we performed a molecular docking study of [68Ga]Ga-BGal with E. coli and human β-Gal, respectively (Figure 5). In the binding mode with E. coli β-Gal (PDBID: 1JYX), the β-galactoside moiety of [68Ga]Ga-BGal positioned well in the binding pocket, forming hydrogen bonds with Glu461 and Glu537, the acid/base, and nucleophilic catalytic residues of E. coli β-Gal. The β-galactoside moiety also interacted with surrounding residues, including Asn102, Asp201, and His540 (Figure 5a). The other residues located within 5 Å of [68Ga]Ga-BGal are shown in sequence form and colored blue (Figure 5a). Notably, Glu461, the glutamic acid in its protonated form that protonates the glycosidic bond of its substrate, is within 3.5 Å of the glycosidic bond of [68Ga]Ga-BGal, providing evidence of the cleavage mechanism of the probe at the molecular level. The NOTA unit of [68Ga]Ga-BGal is exposed to the solvent space and does not participate in interactions with the protein target (Figure 5a). To reveal the binding and functional mechanism of [68Ga]Ga-BGal with human-derived β-Gal, we performed a parallel study using human β-Gal (PDBID: 3WEZ). Similarly, the β-galactoside moiety of the probe participates in binding to the enzymatic pocket of human β-Gal, forming polar interactions with the catalytic acid/base residue Glu188 and nucleophilic catalytic residue Glu268. The distance between Glu188, the acid/base that protonates the glycosidic bond, is within 2.6 Å of the glycosidic bond of [68Ga]Ga-BGal. In addition to the two catalytic residues, Ala128, Glu129, Tyr183, and Asn187 also form hydrogen bonds with the β-galactosides. Similar to E. coli β-Gal, the NOTA unit of [68Ga]Ga-BGal is exposed to solvent and does not participate in interactions with the residues within the enzymatic pocket. Residues within 5 Å of the probe are shown in sequence and colored lilac (Figure 5b).

Figure 5.

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Binding modes of [68Ga]Ga-BGal with (a) E. coli β-Gal and (b) human β-Gal.

Detection of β-Gal in Live Cells

The cytotoxicity of the probe was evaluated by using a CCK-8 kit. After incubation with different concentrations of BGal for 4 h, the cells maintained a survival rate of over 90% (Figure S7), suggesting that BGal exhibited almost no toxicity to cells at concentrations lower than 20 μM. Cell uptake assays were then performed to assess the uptake of [68Ga]Ga-BGal in CT26.CL25 and CT26.WT cells. Both cell lines were separately incubated with the probe at 0.5, 1, 2, and 4 h (Figure 6a). The results showed that the uptake of the probe was higher in CT26.CL25 cells than in CT26.WT cells at all of the indicated time points and exhibited a time-dependent effect. The maximum uptake was 2.97 ± 0.20%ID/mg at 4 h in CT26.CL25 cells and 1.15 ± 0.09%ID/mg in CT26.WT cells (Figure 6c). The expression of β-Gal was confirmed by X-Gal staining (Figure S8).

Figure 6.

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Uptake of [68Ga]Ga-BGal in CT26.CL25, CT26.WT, saline-treated HeLa, and DOX-treated HeLa cells. (a) Dynamic uptake of the probe in CT26.CL25 and CT26.WT cells with or without β-Gal at 0.5, 1, 2, and 4 h. (b) Dynamic uptake of the probe in saline-treated HeLa and DOX-treated HeLa cells at 0.5, 1, 2, and 4 h. (c) Quantitative analysis of the probe in CT26.CL25, CT26.WT, saline-treated HeLa, and DOX-treated HeLa cells at 4 h incubation. (d) Uptake of the probe in CT26.CL25 and CT26.WT with different quantities (0.125, 0.25, 0.5, and 1 μg) of β-Gal after 4 h of incubation (*p < 0.05, **p < 0.01, ***p < 0.001).

Furthermore, [68Ga]Ga-BGal with different quantities (0.125, 0.25, 0.5, and 1 μg) of recombinant β-Gal and different periods (0.5, 1, 2, and 4 h) was incubated in CT26.CL25 and CT26.WT cells (Figure 6a, d). The cell uptake of the probe was significantly reduced in CT26.CL25 cells when coincubated with recombinant β-Gal, compared to that in CT26.CL25 cells incubated with the probe without the addition of recombinant β-Gal. Decreased probe uptake in CT26.WT cells was also observed when coincubated with recombinant β-Gal but was found to be significantly lower than the uptake in CT26.CL25. Furthermore, the more recombinant β-Gal added, the less cellular uptake of the probe was observed (Figure 6d). Presumably, after the addition of recombinant β-Gal to the medium, the probe was cleaved by β-Gal and could no longer be taken up and retained by the cells after β-Gal activation, highlighting the probe’s high specificity for β-Gal.

Following the promising results in model cell lines, we verified the specificity of [68Ga]Ga-BGal for detecting β-Gal in chemotherapy-induced senescent cells. After treatment with DOX (50 nM) for 5 days, HeLa cells became flat, irregular, and enlarged, which was consistent with the morphological characteristics of senescent cells (Figure S9a,b). The overexpression of SA-β-Gal in senescent HeLa cells was first investigated using X-Gal staining, which was stained blue, while the saline-treated HeLa cells did not show significant staining (Figure S9c,d). The cellular uptake of [68Ga]Ga-BGal was higher in senescent HeLa cells than in saline-treated HeLa cells at all time points and showed an increasing trend over time (Figure 6b). The maximum uptake was 3.06 ± 0.24%ID/mg in DOX-treated cells, whereas the uptake in saline-treated HeLa cells was only 1.02 ± 0.23%ID/mg after 4 h incubation. (Figure 6d). Overall, the result suggests that [68Ga]Ga-BGal can enter cells via a β-galactose specific manner, reacts with SA-β-Gal, and is able to differentiate between senescent and nonsenescent cells.

Here, we introduce the principle of probe entry into cells. It was approved that galactose is a crucial carbohydrate for cellular metabolism and contributes to energy production and storage in several body tissues. Due to its chemical structure, solubility, and specific transport system that allows diffusion across membranes, galactose is a good choice for new biotechnology and pharmacological applications. Increasingly, research has been conducted to develop galactose-containing conjugates to improve drug delivery and absorption.33,34[68Ga]Ga-BGal thereby could diffuse into the cells. However, it cannot transport through cell membranes once it was cleaved by β-Gal. We further verified this hypothesis by adding recombinant β-Gal in a cell culture medium and incubating with [68Ga]Ga-BGal. The result indicated that [68Ga]Ga-BGal cell uptake was significantly reduced because the probe was cleaved by β-Gal. After all, it could not be transported into the cells once it was cleaved.

In Vivo PET Imaging of LacZ(+) Tumor-Bearing Mice

[68Ga]Ga-BGal was first evaluated in mice bearing CT26.CL25 and CT26.WT tumors. CT26.CL25 tumors were detected with high contrast after the injection compared with the contralateral background. Dynamic imaging showed continuous accumulation of the probe in CT26.CL25 tumors (Figure 7a,b). Tumor uptake and tumor-to-muscle ratio in CT26.CL25 tumors were 4.68 ± 0.56%ID/g and 7.48 ± 1.74, respectively, at 4 h postinjection (Figure 7c). On the other hand, the tumor uptake and tumor-to-muscle ratio in CT26.WT tumors were significantly lower, 1.38 ± 0.53%ID/g and 1.99 ± 0.32, respectively (Figure 7c). Dynamic and delayed static imaging showed that the biodistribution patterns of the probe in normal organs were similar in both mouse groups (Figure 7a–c). Biodistribution experiments were performed after the last PET scan, and the results confirmed those of PET imaging. The uptake of the probe was higher in CT26.CL25 tumors (3.13 ± 0.75%ID/g) than in CT26.WT tumors (0.69 ± 0.07%ID/g), which was consistent with the tumor-to-muscle ratio results (8.49 ± 0.83 vs 1.20 ± 0.12%ID/g) (Figure 7d). X-Gal staining of tumor slices confirmed the expression of β-Gal in the CT26.CL25 tumors (Figure S10a,b). The probe in liver was also analyzed by radio-HPLC (Figure S11).

Figure 7.

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(a, b) Representative PET images obtained at 10 min, 30 min, 1, 2, and 4 h after injection of [68Ga]Ga-BGal in (a) CT26.WT and (b) CT26.CL25 tumor-bearing mice (the red circle represents the tumors). (c) Quantitative analysis of the probe in the heart, liver, kidneys, tumor, muscle, and tumor/muscle ratio from (a, b). (d) Biodistribution of the probe in CT26.WT and CT26.CL25 tumor-bearing mice at 4 h after injection and analysis of the tumor-to-muscle ratio (**p < 0.01, ***p < 0.001).

In Vivo PET Imaging of Chemotherapy-Induced Tumor Senescence

Therapy-induced senescent cells lead to increased expression of stemness-like markers, and some senescent cells re-enter the cell cycle, activating antiapoptotic, dormancy, and recurrence mechanisms to promote tumor progression.35,36 Overexpression of SA-β-Gal is one of the most widely used markers of cellular senescence. Therefore, the detection of therapy-induced senescent cells via imaging of SA-β-Gal is feasible to assess the response of tumors to treatment. Therefore, the detection of therapy-induced senescence via imaging SA-β-Gal is feasible to assess the response of tumors to treatment.

In vivo PET imaging of HeLa xenografts was performed to evaluate the capability of [68Ga]Ga-BGal to visualize chemotherapy-induced senescence in human tumor xenograft models. HeLa tumor-bearing mice were treated with DOX to induce cellular senescence. The DOX-treated mice showed a significantly higher signal in the tumor compared to that in the control group receiving saline (Figure 8a,b). The quantification of the PET imaging showed that the signals at 4 h from the tumors with or without drug treatment were 2.58 ± 0.56 and 1.39 ± 0.40%ID/g, respectively (Figure 8c). Likewise, the tumor-to-muscle ratios were 4.50 ± 0.33 and 2.33 ± 0.28, respectively. The biodistribution test further verified the in vivo imaging results. [68Ga]Ga-BGal had high accumulation in DOX-treated HeLa tumors (3.59 ± 0.51%ID/g) at 4 h postinjection. On the contrary, the uptake was lower in saline-treated HeLa tumors (2.23 ± 0.61%ID/g) (Figure 8d). X-Gal staining of tumor slices confirmed the induction of SA-β-Gal in the tumor tissues from DOX-treated mice models (Figure S10c,d).

Figure 8.

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(a, b) Representative PET images obtained at 10 min, 30 min, 1, 2, and 4 h after injection of [68Ga]Ga-BGal in (a) saline-treated HeLa and (b) DOX-treated HeLa tumor-bearing mice (the red circle represents the tumors). (c) Quantitative analysis of the probe in the heart, liver, kidneys, tumor, muscle, and tumor/muscle ratio from (a, b). (d) Biodistribution of the probe in saline-treated HeLa and DOX-treated HeLa tumor-bearing mice at 4 h after injection and analysis of the tumor-to-muscle ratio (*p < 0.05, **p < 0.01).

Besides therapy-induced senescence, the occurrence and development of various age-related diseases, such as atherosclerosis,37 diabetes,38 and neurodegenerative diseases, are also linked to cellular senescence.39 The PET imaging probe [68Ga]Ga-BGal, therefore, has enormous potential for the detection of senescence-associated diseases in vivo. Currently, we are also working on the application of the probe to other senescence-related diseases and expect to contribute to the research, diagnosis, and treatment guidance of these diseases. In the meantime, we are also developing chemical strategies to obtain activity-based probes with enhanced cell retention and improved pharmacokinetics in vivo.

Conclusions

Unlike fluorescence optical imaging probes, the limitless depth of penetration and excellent sensitivity of the PET modality enable radionuclide-labeled small-molecule probes to be potentially translatable into clinical research to acquire whole-body quantitative data to study the expression, distribution, and function of biomolecules in vivo.40 In this work, we have developed a PET imaging probe, [68Ga]Ga-BGal, for noninvasive, real-time quantitative detection of cellular senescence in living animals. This probe demonstrated suitable stability in serum and was efficiently activated by β-Gal, as indicated by gel electrophoresis and radio-HPLC studies. Importantly, the probe was able to detect cellular senescence in vitro and in vivo. In mice, PET imaging showed clear uptake and retention of the probe in tumors expressing β-Gal activity after intravenous injection compared to controls. Due to the limitless penetration depth and very high sensitivity of the PET modality, this PET probe offers the ability to provide quantitative whole-body detection of cellular senescence in various contexts. We anticipate that this PET probe will have great promise for visualizing cellular senescence under various conditions in vivo and help to advance clinical translational research in senescence-related diseases and conditions.

Experimental Section

Cell Culture and Induction of Cellular Senescence

All cell lines were obtained from the American Type Culture Collection (ATCC). CT26.WT, mouse colon cancer cells, and CT26.CL25 (LacZ-encoded CT26 cell line for β-Gal expression) cells were cultured in RPMI-1640 medium (Gibco, C11875500BT) supplemented with 10% fetal bovine serum (FBS) (Excell, FSP500) and 1% penicillin–streptomycin (Gibco, 15070063) at 37 °C in a humidified atmosphere containing 5% CO2. HeLa cervical cancer cells (minimal β-Gal expression) were cultured in DMEM (Gibco, C11995500BT) supplemented with 10% FBS and 1% penicillin–streptomycin under the same conditions. HeLa cells were cultured for 5 days in a freshly prepared culture medium supplemented with 50 nM doxorubicin (DOX) (MedChemExpress, 23214–92–8) to induce cellular senescence.40

Animal Models

All animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the Department of Animal Resources, Second Xiangya Hospital of Central South University (No. 2022–071). CT26.WT and CT26.CL25 cells (1 × 105 cells in 120 μL of phosphate-buffered saline (PBS, Gibco, C10010500BT)) were inoculated subcutaneously into the left front flanks of female BALB/c mice. HeLa cells (1 × 105 cells in 120 μL of PBS) were inoculated subcutaneously into the left front flanks of female BALB/c nude mice.

For all in vivo experiments, mice bearing CT26.WT and CT26.CL25 tumors were utilized when the tumor size reached 300 mm3 (2–3 weeks after inoculation). HeLa tumor-bearing mice were randomly divided into two groups when the tumor size reached 300 mm3. One group was administered DOX by intraperitoneal injection (2.5 mg/kg, six times a fortnight, a total dose of 15 mg/kg), and another group was given saline as the control. On day 15, mice were subjected to in vivo PET imaging experiments.

Synthesis and Characterization of [68Ga]Ga-BGal

All chemicals were purchased from commercial sources, unless otherwise noted. High-performance liquid chromatography (HPLC) was performed on a Dionex Ultimate 300 HPLC System (Thermo Scientific) equipped with a GP50 gradient pump and an inline diode array UV–vis detector. Reverse-phase C18 columns were used with an acetonitrile (Supelco, 75–05–8)/water gradient mobile phase containing 0.1% trifluoroacetic acid (Aladdin, 76–05–1). High-resolution mass spectrometry (HRMS) spectra of all samples were obtained on an Agilent 6230 ESI-TOF. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker instruments (600 MHz for 1H NMR) and internally referenced to the residual solvent signals. NMR chemical shifts (δ) and the coupling constants (J) for 1H are reported in parts per million (ppm) and Hertz, respectively. The following conventions are used for multiplicities: s, singlet; d, doublet; t, triplet; m, multiplet; and dd, doublet of doublet.

Compound 1 was prepared following the literature procedure.29 It was dissolved in a mixture of DMF/NEt3 (10/1), and then p-SCN-Bn-NOTA (1 equiv) was added to the solution. The resulting mixture was stirred at room temperature and monitored by HPLC. Upon completion, it was purified by semipreparative HPLC. Then, its chemical purity was determined by HPLC. For 68Ga radiolabeling, 68GaCl3 (185–370 MBq) was eluted with 4 mL of HCl (0.05 M) into the reaction vial from a 68Ge–68Ga generator (ITM Isotopen Technologien München AG, Germany) and diluted with 276 μL of sodium acetate buffer (NaOAc, 1.0 M, pH 5.0). Subsequently, the BGal solution (10 μg, 1 μg/μL in ultrapure water) was added to the reaction vial, and the pH of the resulting mixture was checked as 4.5–5.0. After being incubated at 37 °C for 10 min, the resulting solution was first diluted 10-fold with sterilized water and injected through a Sep-Pak C18 Light Cartridge (Waters, USA) followed by a 10 mL water flush. The product trapped on C18 was slowly eluted with 1 mL ethanol and formulated with 10 mL saline. Before injection into the mice, the solution was passed through a 0.22 μm Millipore filter into a sterile vial and aliquoted into many vials for cell or in vivo study. The labeling yield and radiochemical purity of [68Ga]Ga-BGal were determined by radio-HPLC. All compounds were >95% pure by HPLC analysis.

To test the stability of the probe in serum, 3.7 MBq of [68Ga]Ga-BGal (37 MBq/mL in serum) was incubated with 0.5 mL of mouse serum for 4 h. According to radio-HPLC analysis, the stability results were determined as a percentage of the intact radiotracer. Moreover, radio-HPLC analysis of [68Ga]Ga-BGal in reaction with active or inactive recombinant β-Gal (Sigma-Aldrich, 9031–11–2) was obtained at 37 °C in PBS.

To measure the stability of the probe in vivo, the probe was injected into mice and circulated for 4 h. Then, blood samples were collected and centrifuged at 3000 rpm, and the plasma was filtered with a 10 kDa syringe filter. The filtrate was collected for radio-HPLC analysis.

Computational Study of [68Ga]Ga-BGal with E. coli and Human β-Gal To Characterize the Binding Mode

The molecular docking study of [68Ga]Ga-BGal with E. coli β-Gal was performed using the crystal structure obtained from the Protein Data Bank at https://www.rcsb.org.41,42 The crystal structure 1JYX,43 selected for its good structural quality, underwent a series of preparation procedures. The preparation procedures included removing water molecules and ions outside of the binding pocket and retaining only one unit of the tetramer. The protein was further prepared by following the preparation wizard workflow. The alternative residue positions in the protein were solved; cocrystallized small molecules that located outside of the binding pocket were removed. Hydrogens were added based on the protonation states of the protein and the ligand at a pH of 7.0 ± 2.0. The IPT in the binding pocket was used for grid box generation, with an inner grid box size of 15 Å × 15 Å × 15 Å, buried in an outer box of 30 Å × 30 Å × 30 Å. The OPLS 2005 force field was applied for grid box generation. A similar preparation procedure was followed for the human β-Gal crystal structure (PDBID: 3WEZ) to study the binding conformation of the probe with the target. The grid was generated with an inner grid box of 15 Å × 15 Å × 15 Å and an outer box of 30 Å × 30 Å × 30 Å. [68Ga]Ga-BGal was prepared using the Ligand Preparation module44 in Schrödinger Maestro, and the pKa values and protonation states were determined under a pH of 7.0 using Epik.45 Glide SP46 was used for generating binding conformations of the prepared ligands with a flexible ligand sampling method. The van der Waals radii were scaled with a scaling factor of 0.80, and a partial charge cutoff of 0.15 was applied. Additionally, nitrogen inversion and ring conformation sampling options were applied. Bias sampling of the torsions option was kept for all predefined functional groups. Epik state penalties were added to the docking score. Postdocking minimizations were performed following the OPLS2005 force field. The generated conformations were inspected and chosen for generating binding maps in PyMOL.47

Gel Electrophoresis for Investigating the Interaction of [68Ga]Ga-BGal with β-Gal

To evaluate the interaction of [68Ga]Ga-BGal with β-Gal, recombinant β-Gal was incubated with the probe at 37 °C for 30 min, and inactive recombinant β-Gal, BSA, and PBS were used as controls. A variety of interfering substances may be present in organisms. Therefore, other potential interfering components, such as Cys, vitamin C, and CaCl2, were added to the specificity assay experiments. The probe was incubated separately with the various interfering substances at 37 °C for 30 min. Furthermore, the probe was incubated with different concentrations (0.01, 0.015, 0.022, 0.033, and 0.05 μg/μL) of recombinant β-Gal at 37 °C for 30 min. To test the time dependence of the interaction of [68Ga]Ga-BGal with the enzyme, 0.022 μg/μL of recombinant β-Gal was incubated with 370 kBq of [68Ga]Ga-BGal at 37 °C for 1, 5, 15, 30, and 60 min. Then, all samples were loaded onto 10% Bis-Tris gels and run for 55 min at 150 V and scanned with a PET scanner (Biograph mCT, SIEMENS). Then, the gels were placed on a flat board and scanned. In addition, the probe was incubated with CT26.CL25 and CT26.WT cells for 1, 15, 30, 60, and 120 min to extract protein. And the protein extracted after the probe incubated with both cells for 120 min was configured as 0.009, 0.0375, 0.15, 0.6, and 2.4 μg/μL. Gel electrophoresis and PET/CT imaging were then performed.

Cell Viability Assays

Cell viability and cytotoxicity were assessed by the Cell Counting Kit-8 (CCK-8) (Abiwell, AWC0114a) assay. CT26.WT cells were seeded in a 96-well plate at a density of 1 × 104 cells/well and cultured overnight. Then, the cells were incubated with different concentrations (0–20 μM) of BGal for 4 h at 37 °C. 10 μL of CCK-8 reagent was added to each well for a 1 h incubation at 37 °C, and then the absorbance was recorded at 450 nm.

Cellular Uptake Assay in Live Cells

The expression of β-Gal in CT26.WT, CT26.CL25, saline-treated HeLa cells, and DOX-treated HeLa cells was verified by X-Gal staining. The cells were seeded in a 6-well plate at a density of 3 × 105 cells/well. One group of HeLa cells was treated with DOX to induce senescence, and the other group was treated with saline. Cells were stained with X-Gal (Beyotime, C0602) according to our previously reported method.28

The specificity of [68Ga]Ga-BGal against β-Gal was assessed in vitro. A total of 1 × 106 HeLa cells were cultured for 5 days in freshly prepared culture medium supplemented with 50 nM doxorubicin (DOX) to induce cellular senescence. Saline was used as the control group. One day before the cellular uptake test, 1 × 106 CT26.CL25 and CT26.WT cells were separately seeded in six-well plates and incubated overnight. Then, the cells were incubated with [68Ga]Ga-BGal (1110 kBq/well) at 37 °C for 0.5, 1, 2, and 4 h in Hank’s Buffer (Solarbio, H1025) with HEPES (Sigma-Aldrich, 7365–45–9). Cells were immediately washed with ice-cold PBS buffer (pH 7.4), which was used to remove the free probes. Then, each cell sample was lysed with 150 μL of RIPA buffer (Beyotime, P0013B) and collected in an Eppendorf tube. The radioactivity and protein concentrations were tested separately to determine the cell uptake. The results are shown as the percentage of added radioactivity per milligram of protein (%ID/mg).

Additionally, different quantities (0.125, 0.25, 0.5, and 1 μg) of recombinant β-Gal were added for 4 h or 1 μg of recombinant β-Gal was added for different times (0.5, 1, 2, and 4 h) before CT26.CL25 and CT26.WT cells were incubated with [68Ga]Ga-BGal. The specificity of the probe for β-Gal was verified similarly.

In Vivo PET Imaging

To evaluate the tumor uptake and in vivo distribution of [68Ga]Ga-BGal, PET imaging was performed on mice bearing CT26.CL25, CT26.WT, and DOX-treated HeLa or saline-treated HeLa tumors. Mice were subjected to gas anesthesia using isoflurane and were then placed on a PET/CT scanner (Super Nova PET/CT, PINGSENG) for CT imaging before intravenous (i.v.) injection of 3.7 MBq of [68Ga]Ga-BGal (n = 3 per group). Imaging acquisition was started immediately for 1 h of dynamic PET imaging and static PET scan at 2 and 4 h postinjection. By drawing volumes of interest (VOIs) on tumors and other tissues of interest, such as the heart, liver, kidneys, and muscle, PMOD 4.302 software (PMOD Technologies, Switzerland) was used to reconstruct and further analyze all PET images.

Biodistribution

To assess the distribution of the probe in tumors and major organs, all tumor-bearing mice were injected i.v. with 3.7 MBq of [68Ga]Ga-BGal (n = 3 per group). At 4 h after injection, the mice were sacrificed and dissected, and the liver, kidneys, tumors, and blood were collected and weighed. The radioactivity in the tissues was measured using a γ counter (CAPRAC-t well counter, USA). The results are presented as the percentage of the injected dose per gram of tissue (%ID/g).

Tissue Staining

After resection, CT26.WT, CT26.CL25, saline-treated HeLa, and DOX-treated HeLa tumors were immediately submerged in an optimal cutting temperature (OCT) solution and frozen on dry ice. The tissues were subsequently cryosectioned into 6 μm slides and stained with X-Gal (for β-Gal).

Statistical Analysis

Data are reported as the mean ± SEM. Origin 2023 (OriginLab) was used to determine the statistical significance of differences between groups by applying an unpaired Student’s t test. P values <0.05 were considered significant.

Acknowledgments

This work was supported by a major grant from the Research and Development Program of Hunan Province of China (2022SK2035), the Fundamental Research Funds for the Central Universities of Central South University (2022ZZTS0856), and NIH (R41AG081007).

Glossary

Abbreviations Used

CCK-8

cell counting Kit-8

DOX

doxorubicin

FBS

fetal bovine serum

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

NaOAc

sodium acetate

NMR

nuclear magnetic resonance

NIR

near-infrared

OCT

optimal cutting temperature

PET

positron emission tomography

ROS

reactive oxygen species

SA-β-Gal

senescence-associated β-galactosidase

SASP

senescence-associated secretory phenotype

VOIs

volumes of interest

X-Gal

5-bromo-4-chloro-3-indolyl-β-D-galactoside.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00179.

  • 1H NMR spectrum of BGal; HRMS of BGal; HPLC analysis of BGal; radio-HPLC analysis of [68Ga]Ga-BGal for 4 h after injection in vivo; radio-HPLC analysis of [68Ga]Ga-BGal that reacted with β-Gal or inactive β-Gal; PET images of [68Ga]Ga-BGal incubated with interfering substances; cell viability of CT26.WT cells upon incubation with BGal at different concentrations; X-Gal staining of CT26.WT and CT26.CL25; characterization of senescent HeLa cells; and X-Gal staining for the β-Gal activity of tumor slides (PDF)

  • Molecular formula strings (CSV)

  • E. coli model (PDB)

  • Human model (PDB)

  • Dynamic gel electrophoresis PET imaging of [68Ga]Ga-BGal incubated with β-Gal, inactive β-Gal, BSA, and PBS (MP4)

Author Contributions

X.X. and C.D. contributed equally. X.M. and L.C. developed the concept and supervised the research. All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by X.X. and C.D. J.L. and Z.M.R. performed the synthesis and chemical analysis of BGal. L.Z. and Y.Z. performed computational modeling. H.G. performed the PET imaging acquisition. F.H. performed radiolabeling of the probe. X.C. performed tumor xenograft preparation. Y.W. processed the PET imaging data analysis. All authors wrote and approved the final manuscript.

The authors declare the following competing financial interest(s): Prof. L. Cui is the founder of SenoTrac Biotechnology, a private company developing senescence imaging probes for various applications. The University of Florida has filed patent applications for a variety of senescence imaging probes.

Notes

All animal experiments were approved by the Animal Care and Use Committee of the Department of Animal Resources, The Second Xiangya Hospital of Central South University (No. 2022–071).

Supplementary Material

jm4c00179_si_001.pdf (817.6KB, pdf)
jm4c00179_si_002.csv (135B, csv)
jm4c00179_si_003.pdb (759KB, pdb)
jm4c00179_si_004.pdb (1.2MB, pdb)
jm4c00179_si_005.mp4 (1.4MB, mp4)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jm4c00179_si_001.pdf (817.6KB, pdf)
jm4c00179_si_002.csv (135B, csv)
jm4c00179_si_003.pdb (759KB, pdb)
jm4c00179_si_004.pdb (1.2MB, pdb)
jm4c00179_si_005.mp4 (1.4MB, mp4)

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