Precision imaging of superoxide anion radicals in vivo using a bicyclic dioxetane chemiluminescent nanoprobe

Abstract

Accurate detection of disease-related reactive oxygen species (ROS), such as superoxide anion radicals (O₂˙⁻), is essential for the diagnosis and treatment of inflammation or cancer. Herein, we report the development of a novel chemiluminescent nanoprobe, C10-SPN, based on chemiluminescence resonance energy transfer (CRET) for highly sensitive and specific detection of O₂˙⁻in vitro and in vivo. The obtained nanoprobe exhibited efficient energy transfer, a red-shifted emission peak at 680 nm, and an extended chemiluminescence half-life of up to 7 min. C10-SPN demonstrated excellent selectivity toward O₂˙⁻ over other biologically relevant species, low cytotoxicity, and strong biocompatibility. In vivo imaging in mouse models of peritonitis and breast cancer confirmed the capability of C10-SPN to visualize O₂˙⁻ levels with high contrast, enabling discrimination between normal and diseased tissues. This study highlights C10-SPN as a promising tool for O₂˙⁻-responsive imaging and early disease diagnosis.

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Reactive oxygen species (ROS) play a critical role in various physiological and pathological processes, including cellular signaling, immune response, and disease progression.¹ Among ROS, the superoxide anion radical (O₂˙⁻) is one of the most significant, acting as a primary ROS that can further generate other reactive species such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (˙OH).² The precise detection and imaging of O₂˙⁻in vivo are crucial for understanding its biological functions and its involvement in diseases such as cancer, neurodegenerative disorders, and inflammatory conditions.³ However, achieving accurate and real-time imaging of O₂˙⁻ in living systems remains a significant challenge due to its short lifespan, low concentration, and high reactivity.⁴

Traditional methods for detecting O₂˙⁻, such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based probes, often suffer from limitations in sensitivity, specificity, and spatial–temporal resolution.⁵ Moreover, many existing probes are unsuitable for in vivo applications due to issues such as limited penetration depth and interference from other ROS. Therefore, there is an urgent need for advanced nanoprobes that can overcome these limitations and enable precise imaging of O₂˙⁻ in complex biological environments.⁶

In recent years, chemiluminescent (CL) probes have emerged as a promising tool for ROS detection due to their high sensitivity, low background noise, and ability to operate without external light excitation.⁷ Among these, dioxetane-based chemiluminescent probes have garnered significant attention because of their unique mechanism of light emission upon reaction with specific ROS.⁸ The conventional chemiluminescence imaging methods for detecting O₂˙⁻ still face significant application challenges due to drawbacks such as low emission intensity, short emission wavelength, and brief chemiluminescence duration.⁹ Chemiluminescence based on resonance energy transfer (CRET) offers an opportunity to overcome these issues. CRET can effectively extend the luminescence duration and induce a redshift in the emission wavelength.¹⁰ It is, therefore, imperative to develop a chemiluminescence system based on the CRET principle with high sensitivity and strong specificity for the in vitro and in vivo detection of O₂˙⁻. In this study, we developed a novel chemiluminescence system based on CRET by modifying CL probes using a nanoprecipitation method. This system was applied for the in vitro and in vivo imaging and detection of O₂˙⁻ due to its high sensitivity and specificity.

The research commenced with the synthesis of a bicyclic dioxetane chemiluminescent probe featuring an O₂˙⁻-responsive aryl triflate group (Fig. 1a). The high nucleophilicity of O₂˙⁻ enables it to attack the electron-deficient aryl triflate moiety, inducing a nucleophilic aromatic substitution that cleaves the triflate group and subsequently activates the chemiluminescent dioxetane scaffold.^11,12 Starting from compound 1, an efficient ortho-carbonylation reaction, mediated by MgCl₂, produced compound 2. Subsequently, a Wittig reaction was employed to convert compound 2 into compound 3, where the phenolic hydroxyl group was protected by a triflyl group to generate 4. The final step involved the oxidation of the olefin on the bicyclic ring of 4, yielding the target dioxetane probe 5. The chemiluminescent nanoprobe (C10-SPN) was then formed via nano-precipitation, combining probe 5 with a PFODBT material in the presence of an amphiphilic triblock copolymer (PEG-b-PPG-b-PEG) (Fig. 1b). This nanoprobe was designed for precise in vivo imaging of disease lesions, leveraging the high sensitivity of the triflyl group to O₂˙⁻ and its potential for chemiluminescence resonance energy transfer (Fig. 1c).

Fig. 1 Design of the C10-SPN probe. (a) Synthetic route of chemiluminescent probe 5; conditions: a. MgCl₂, NEt₃, (CH₂O)_n, THF, reflux; b. Ph₃PCHCO₂Me, DCM, rt; c. trifluoromethanesulfonic anhydride, Et₃N, DCM, 0 °C–rt; d. TPP, O₂, yellow light, DCM, 0 °C; e. THF/H₂O. (b) Preparing the bicyclic dioxetane chemiluminescent nanoprobe with probe 5. (c) Highly sensitive in vivo disease lesion imaging.

We used the amphiphilic substance PEG as a stabilizer to encapsulate probe 5 and PFODBT, rendering the material hydrophilic. To verify that PFODBT, used for resonance energy transfer, was indeed incorporated into the chemiluminescent nanoprobe, we first performed UV characterization of the material. The UV absorption spectrum results showed that the C10-SPN chemiluminescent material exhibited the characteristic absorption peaks of PFODBT at 385 and 550 nm (Fig. 2a), confirming that PEG effectively encapsulated probe 5 and PFODBT, indicating the successful construction of the desired nanoprobe system (Fig. S1).

Fig. 2 Basic properties of C10-SPN. (a) The ultraviolet absorption spectrum of C10-SPN. (b) The fluorescence emission spectra of C10-SPN. (c) The DLS spectrum of C10-SPN (illustration is the image obtained using a transmission electron microscope). (d) Specificity of C10-SPN against O₂˙⁻.

Fig. 2b demonstrates that the C10-SPN chemiluminescent material emits a strong luminescence signal upon the introduction of O₂˙⁻. Owing to the effective incorporation of PFODBT, the chemiluminescence generated by the reaction between O₂˙⁻ and the C10 probe efficiently excites PFODBT fluorescence, facilitating energy transfer within the system. This energy relay markedly boosts the overall luminescence intensity and induces a red-shift in the emission peak to 680 nm, thereby enhancing the imaging capability of the nanoprobe.

The morphology and hydrated particle size of the C10-SPN nanoparticles were subsequently characterized in detail. The prepared C10-SPN dispersion was diluted to an appropriate concentration and examined using transmission electron microscopy (TEM). As shown in the inset of Fig. 2c, the nanoparticles displayed a spherical shape with an average diameter of approximately 70 nm. Dynamic light scattering (DLS) analysis further confirmed a hydrated diameter of around 82 nm, indicating good dispersion and colloidal stability in aqueous solution.

To evaluate the selectivity of C10-SPN, a panel of representative cations and reactive species was tested, including Mg²⁺, Na⁺, Cu²⁺, K⁺, Ca²⁺, Fe³⁺, Fe²⁺, H₂O₂, O₂˙⁻ and other biologically relevant species (Fig. 2d and Fig. S2). In phosphate-buffered saline (PBS, pH 7.4), the C10-SPN nanoparticles exhibited a pronounced chemiluminescence response selectively to O₂˙⁻, while showing minimal or no response to the other analytes tested. These results highlight the high specificity of C10-SPN toward superoxide anions in a complex chemical environment.

To evaluate the relative chemiluminescence intensity of C10-SPN, the material was incubated with O₂˙⁻ in a black 96-well plate. Quantitative analysis using the IVIS imaging system revealed a significant increase in luminescence intensity upon O₂˙⁻ exposure, compared to the untreated control (Fig. 3a). These results confirm the strong and specific chemiluminescence response of C10-SPN to O₂˙⁻.

Fig. 3 Chemiluminescence properties of C10-SPN. (a) Relative chemiluminescence intensity. (b) Half-life time of C10-SPN. (c) Cytotoxicity of the prepared C10-SPN probe.

To further evaluate the luminescence performance of C10-SPN, its CL half-life was measured. As shown in Fig. 3b, the C10-SPN system exhibited a prolonged half-life of up to 7 min, indicating excellent signal persistence. This extended emission duration provides a sufficient time window for real-time in vivo imaging, highlighting its potential use as a chemiluminescent probe for O₂˙⁻ detection.

The cytotoxicity of C10-SPN nanoparticles toward 4T1 cells was evaluated using the CCK-8 assay. As shown in Fig. 3c, cells were incubated with increasing concentrations of C10-SPN (0, 3.75, 7.5, 15, 30, 60, 120, 240, 480, and 960 µg mL⁻¹) for 24 h. Even at the highest concentration of 960 µg mL⁻¹, the cell viability remained largely unaffected. These results indicate that C10-SPN exhibits low cytotoxicity and excellent biocompatibility, supporting its suitability for safe in vivo applications. Building on the excellent performance of the C10-SPN nanoprobe observed in vitro, we conducted in vivo imaging experiments to assess its ability to detect O₂˙⁻ in a mouse model of peritonitis. Four mice were prepared for imaging experiments: the first one received saline injection as a background control, the second one received saline and C10-SPN, the third one was injected with lipopolysaccharide (LPS for inducing inflammation)^13,14 and C10-SPN, and the fourth one was injected with LPS, glutathione (GSH), and C10-SPN, with GSH being used to scavenge reactive oxygen species (ROS). Luminescence signals in the abdominal region were then detected using the IVIS imaging system. As shown in Fig. 4a, a strong luminescence signal was observed in the abdominal region in the third mouse, indicating successful induction of inflammation and elevated O₂˙⁻ levels. In contrast, no noticeable signal was detected in the second mouse, confirming the specificity of C10-SPN for O₂˙⁻. Notably, although inflammation was also induced in the fourth mouse, the presence of GSH reduced the ROS levels, resulting in markedly weaker luminescence compared to that of the third one (Fig. 4b). These findings demonstrate that the C10-SPN nanoprobe enables specific in vivo imaging of O₂˙⁻ and holds great potential for the visual diagnosis of inflammation-related pathologies.

Fig. 4 In vivo imaging of inflammation and tumors. (a) Imaging of LPS-induced peritonitis. (b) Statistical comparison of the imaging ability of C10-SPN in different mice. (c) Imaging of the 4T1 cell xenografted tumor and statistical comparison of the imaging ability of C10-SPN and PBS. (d) Comparison of the imaging ability of C10-SPN in tumor and healthy areas.

To further evaluate the ability of the C10-SPN nanoprobe to detect tumors in vivo, we established two 4T1 breast cancer xenograft mouse models. In the first mouse, PBS was injected into the tumor site as a control, while in the second mouse, C10-SPN was administered at the tumor site. Subsequently, full-spectrum chemiluminescence signals from both tumor regions were detected using the IVIS imaging system. As shown in Fig. 4c, the tumor site of the C10-SPN-injected mouse exhibited strong chemiluminescence, indicating effective detection of O₂˙⁻ within the tumor. Quantitative analysis revealed that the CL intensity in the C10-SPN group was approximately 34 times higher than that of the PBS control, demonstrating a substantial enhancement in signal contrast and detection sensitivity. These findings highlight the strong potential of C10-SPN for tumor-targeted O₂˙⁻ imaging in vivo.

Notably, to investigate whether the C10-SPN nanoprobe can distinguish tumor tissue from normal tissue in vivo, we simultaneously injected PBS or C10-SPN into both the normal and tumor regions of the same mouse. As shown in Fig. 4d, mice injected with PBS exhibited negligible luminescence, whereas those injected with C10-SPN showed significantly stronger chemiluminescence signals at the tumor site compared to normal tissue. These results demonstrate that the C10-SPN nanoprobe can effectively differentiate between healthy and tumor tissues, highlighting its potential for tumor-specific imaging.

In summary, we have developed a novel chemiluminescent nanoprobe, C10-SPN, based on a bicyclic dioxetane scaffold and chemiluminescence resonance energy transfer (CRET) for the selective and sensitive detection of superoxide anion radicals (O₂˙⁻) in vitro and in vivo. The nanoprobe exhibits excellent physicochemical properties, including efficient energy transfer, red-shifted emission at 680 nm, prolonged luminescence duration, and high stability in aqueous environments. C10-SPN shows remarkable specificity toward O₂˙⁻ over other reactive oxygen species and ions, along with low cytotoxicity and favorable biocompatibility. In vivo studies in mouse models of peritonitis and breast cancer confirmed the nanoprobe's ability to visualize O₂˙⁻ with high spatial resolution and signal contrast, enabling precise differentiation between inflamed or tumor tissues and normal tissues. Importantly, C10-SPN demonstrated excellent performance in distinguishing tumor regions from healthy tissues within the same animal, further validating its potential for accurate tumor-targeted imaging.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: the general procedures of probe 5 synthesis; the characterization of CL probe 5; ¹H NMR and ¹³C NMR spectra of CL probe 5 and in vitro and in vivo imaging. See DOI: https://doi.org/10.1039/d5qm00620a.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2023YFC3402900), Zhejiang Provincial Science and Technology Plan Project (No. 2024SDYXS0002), and The Independent Deployment Project of the Hangzhou Institute of Medicine, CAS (No. 2024ZZBS03).

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Footnote

† These authors contributed equally.

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