Reversible redox ¹⁹F magnetic resonance imaging nanoprobes for monitoring the redox state in vivo

Abstract

Redox processes are indispensable for physiology, and dysregulated redox balance is critical in various metabolic diseases. The development of imaging diagnosis tools for real-time monitoring of the redox state in vivo is of great importance yet highly challenging. Here, we designed trifluoromethyl (–CF₃) grafted selenide polymer nanoprobes for reversible redox sensing in vivo. Based on the reversible shift of the ¹⁹F-nuclear magnetic resonance (NMR) peak between oxidation and reduction states of the nanoprobes exposed to different redox species, the ¹⁹F-magnetic resonance imaging (MRI) signal ratio of S_Ox/(S_Ox + S_Red) was successfully applied to monitor the redox state in a tumor. These nanoprobes demonstrated good biocompatibility and great potential for exploring physiological and pathological redox processes in deep tissues. We envision that this work will enable the rational design of ¹⁹F-MRI nanoprobes with excellent redox response for the real-time monitoring of the redox state at the lesion location.

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Introduction

Redox processes play a vital role in many biological and physiological functions.^1–3 Dysregulated redox status is associated with many diseases, including cancer^4,5 and cardiological⁶ and neurodegenerative diseases,^7–9 but how the redox status impacts diseases is largely unknown. Developing bioimaging probes for dynamic monitoring of redox status in deep tissue is critical for understanding and studying various diseases.^10,11

Recently, various fluorescent,¹² photoacoustic,^13,14 and proton magnetic resonance imaging (¹H-MRI)^15–20 probes have significantly progressed in monitoring redox processes. However, small-molecule fluorescent and photoacoustic probes are only employed to evaluate the redox status within cells or superficial tissues due to the limited penetration depth of light propagation in tissues, and sometimes fluorescence imaging suffers from strong autofluorescence interference of tissue. ¹H-MRI allows non-invasive visualization of deep tissues, but the heterogeneous background of tissues makes the interpretation of ¹H-MR images more difficult.^{21 19}F-MRI is a splendid “hot spot” in vivo imaging technology due to its zero-background in living organisms.^22–27 Most redox-responsive ¹⁹F-MRI probes^28–30 are based on the breakable disulfide bond, which is generally irreversible and unsuitable for monitoring redox cycles. The other interesting strategy^31–34 relies on the metal center switched between paramagnetic and diamagnetic states by redox status to reversibly modulate the ¹⁹F-signal. Despite great progress, it is still highly challenging to fabricate an ideal imaging probe for in vivo redox sensing with advantages. Specifically, achieving non-invasive and radiation-free imaging with high penetration depth and ultra-low backgrounds is particularly difficult in current techniques. Additionally, ensuring a reversible response for dynamically measuring the redox state and excellent biocompatibility of the probe itself further complicates the development of an ideal imaging probe for redox sensing in vivo.

Herein, we designed a PIBAM–FSeN nanoprobe, based on trifluoromethyl (–CF₃, as the ¹⁹F tag)-grafted selenide (as the redox-recognition site), for reversible redox status ¹⁹F-MRI visualization in deep tissue. As depicted in Scheme 1, when exposed to oxidizing species, PIBAM–FSeN was readily oxidized to PIBAM–FSeON; as a result, the ¹⁹F-NMR signal at −64.2 ppm decreased and a new ¹⁹F-NMR peak emerged at −58.7 ppm. With the increment of the oxidation degree, the signal at −64.2 ppm (S_Red) decreased step by step and the signal at −58.7 ppm (S_Ox) simultaneously increased. This phenomenon would reverse in the presence of reductive species. Therefore, the ¹⁹F-MRI signal ratio S_Ox/(S_Ox + S_Red) could be utilized to monitor reversible redox processes in deep tissue.

Scheme 1 Schematic illustration of the nanoprobes for ¹⁹F-MRI monitoring of reversible redox processes.

Results and discussion

Preparation and characterization of PIBAM–FSeON and PIBAM–FSeN NPs

Before the synthesis of reversible ¹⁹F-MRI nanoprobes, the precursor, 2-(2,2,2-trifluoroethyl)selanylethan-1-amine (FSeN), was first synthesized and characterized (Fig. S1–S7†). Then, poly(isobutylene-maleic anhydride) (PIBAM) was modified with FSeN to get PIBAM–FSeN through a ring-opening reaction (Fig. S1†). The selenide ether group in PIBAM–FSeN was further oxidized to the selenoxide group, termed PIBAM–FSeON (Fig. S8 and S9†). Notably, compared with most reported fluoropolymers (fluorine content is generally below 5 wt%), both PIBAM–FSeN and PIBAM–FSeON had a high fluorine content of 16 wt% and 15 wt%, respectively (Fig. S10†), which were highly desirable for ¹⁹F-MRI benefiting from their high ¹⁹F content and thus a strong MRI signal. Next, the PIBAM–FSeN NPs and PIBAM–FSeON NPs were obtained through self-assembly under ultrasonic emulsification (Fig. S11†). The good stability of these NPs in aqueous solution was confirmed by the hardly changed values in the ¹⁹F-NMR signal (Fig. S12†) and dynamic light scattering (DLS) size (Fig. S13†) even after 50 days.

In vitro detection of redox species by ¹⁹F-NMR

Then the redox responsive ability of these NPs was carefully checked. As shown in Fig. 1A, in the presence of Na₂S, the ¹⁹F-NMR spectrum of PIBAM–FSeON NPs shifted significantly from −58.7 to −64.2 ppm, stemming from the reduction of selenoxide. With the increment of Na₂S content, the ¹⁹F-NMR signal at −64.2 ppm was enhanced step by step, and a linear relationship between the ratio of A_Ox/(A_Ox + A_Red) and Na₂S content was constructed (Fig. 1B). Here, A_Ox and A_Red are integral areas of the ¹⁹F-NMR peak located at −58.7 and −64.2 ppm, respectively, which are proportional to the fluorine atom content. Meanwhile, the reduction state (PIBAM–FSeN) was easily returned to the oxidation state (PIBAM–FSeON) in the presence of H₂O₂ (Fig. 1C), and the linear relationship between A_Ox/(A_Ox + A_Red) and H₂O₂ content was also established (Fig. 1D). The reversibility of the nanoprobes was further verified by alternately exposing nanoprobes to H₂O₂ and Na₂S (Fig. 1E and F), which makes it possible to use them for real-time monitoring of the redox state.

Fig. 1 (A) ¹⁹F-NMR spectra of PIBAM–FSeON NPs (10 mg mL⁻¹, in H₂O) after adding Na₂S. (B) Linear correlation between the A_Ox/(A_Ox + A_Red) ratio and Na₂S content (n = 3, error bars represent standard deviation). The band in the graph is the 95% confidence interval band. (C) ¹⁹F-NMR spectra of PIBAM–FSeN NPs (10 mg mL⁻¹, in H₂O) after adding H₂O₂. (D) Linear relationship between the A_Ox/(A_Ox + A_Red) ratio and H₂O₂ content (n = 3, error bars represent standard deviation). The band in the graph is the 95% confidence interval band. (E) ¹⁹F-NMR spectra of PIBAM–FSeN NPs in 20 mM PBS buffer (pH 7.4) with repeated oxidation and reduction using H₂O₂ (1.1 eq.) and Na₂S (1.1 eq.). (F) Reversibility tests of 10 redox cycles. A_Ox and A_Red represent the integral area of peaks at −58.7 and −64.2 ppm, respectively.

Next, the redox selectivity of these nanoprobes was studied via recording the ¹⁹F-NMR responses after treatment with various analytes (Fig. S14†). Redox species caused obvious effects on ¹⁹F-NMR signal intensity, whereas non-redox analytes had negligible influence. Moreover, the ¹⁹F longitudinal (T₁) and transverse (T₂) relaxation times of PIBAM–FSeN NPs (T₁ = 0.96 s, T₂ = 0.59 s) and PIBAM–FSeON NPs (T₁ = 1.01 s, T₂ = 0.45 s) were measured (Table S1†), which is suitable for ¹⁹F-MRI with a refocused echo (RARE) sequence.

In vitro detection of redox species by ¹⁹F-MRI

Phantom studies were conducted to evaluate the ¹⁹F-MRI potential of these nanoprobes (Fig. 2). The center frequency in ¹⁹F-NMR of PIBAM–FSeON (δ = −58.7 ppm, red channel) and PIBAM–FSeN (δ = −64.2 ppm, blue channel) was chosen for radio frequency (RF) output. As shown in Fig. 2A, with the increment of Na₂S content, the ¹⁹F-MRI signal of PIBAM–FSeON NPs in the red channel decreased step by step; meanwhile that in the blue channel gradually increased. A good linear relationship between the ¹⁹F-MRI signal ratio of S_Ox/(S_Ox + S_Red) and Na₂S content was observed (Fig. 2B). Compared with the signal ratio in the absence of Na₂S, it showed a 12.5-fold decrease after exposure to 0.8 eq. of Na₂S. Following the treatment with H₂O_2, the signal ratio of PIBAM–FSeN NPs linearly increased (Fig. 2C and D), and the signal ratio showed a 10.5-fold increase after exposure to 0.8 eq. of H₂O₂, compared with that without H₂O₂. The reversibility of PIBAM–FSeN NPs for redox response was also validated using the ¹⁹F-MRI results shown in Fig. 2E and F.

Fig. 2 (A) ¹⁹F-MRI of PIBAM–FSeON NPs after adding Na₂S. (B) Linear correlation between the S_Ox/(S_Ox + S_Red) ratio and Na₂S content. The band in the graph is the 95% confidence interval band. (C) ¹⁹F-MRI of PIBAM–FSeN NPs after adding H₂O₂. (D) Linear correlation between the S_Ox/(S_Ox + S_Red) ratio and H₂O₂ content. The band in the graph is the 95% confidence interval band. (E) ¹⁹F-MRI of PIBAM–FSeN NPs in the presence of alternate adding H₂O₂ (1.1 eq.) and Na₂S (1.1 eq.), respectively. (F) The corresponding S_Ox/(S_Ox + S_Red) ratio. S_Ox and S_Red represent the imaging signal intensity in red (−58.7 ppm) and blue (−64.2 ppm) channels, respectively. The nanoprobes were dispersed in PBS (20 mM, pH 7.4) for tests.

Preparation and characterization of the semioxidized PIBAM–FSeN NPs

To ensure that the signal of the probes in both channels (−58.7 ppm and −64.2 ppm) is higher than the detection limit of the instrument, achieving the observation of slight changes from the ratio images, we prepared the semioxidized PIBAM–FSeN NPs for further use. Thus, by controlling the oxidation conditions, the semioxidized PIBAM–FSeN polymer (Fig. 3A) was synthesized, where the content ratio of the selenide ether to the selenoxide group was 1 : 1 (Fig. 3B). Thereafter, the semioxidized PIBAM–FSeN NPs with an average size of 82 ± 25 nm were obtained by the same ultrasonic emulsification method for PIBAM–FSeN NPs (Fig. 3C and D). The semioxidized PIBAM–FSeN NPs also exhibited excellent relaxation performance, as evidenced by the longitudinal relaxation time (T₁) and transverse relaxation time (T₂) values (Table S1†). It was found that no significant variations of T₁ and T₂ were observed over a concentration range from 20 to 140 mg mL⁻¹, implying that fluorine atoms on the nanoparticles retain excellent relaxation properties (Fig. S15†). The critical aggregation concentration (CAC) of semioxidized PIBAM–FSeN NPs is 4.2 mg L⁻¹ (Fig. S16†), which falls between the CAC of PIBAM–FSeN NPs (1.6 mg L⁻¹) and PIBAM–FSeON NPs (750 mg L⁻¹). Subsequently, their good stability was confirmed by the results of ¹⁹F-NMR signals, DLS size distribution tests, and actual photos (Fig. S17–S19 and Table S4†). It is important to note that semioxidized PIBAM–FSeN nanoparticles (NPs) demonstrate enhanced stability under acidic conditions (pH 6.0–6.5) compared to PIBAM–FSeN NPs. This improved stability may be attributed to their lower pK_a value of 5.52, in contrast to the pK_a of PIBAM–FSeN NPs, which is 6.31 (Fig. S20†).

Fig. 3 (A) Chemical structure, (B) ¹⁹F-NMR spectrum, (C) TEM image, and (D) DLS size distribution of semioxidized PIBAM–FSeN NPs (10 mg mL⁻¹, in H₂O). (E) The selectivity of semioxidized PIBAM–FSeN NPs to different analytes (n = 3, error bars represent standard deviation).

The influence of some coexisting substances on the ¹⁹F-NMR signal ratio of A_Ox/(A_Ox + A_Red) was further investigated. As shown in Fig. 3E, no change in the signal ratio was observed in the presence of non-redox analytes including BSA, Na⁺, K⁺, Ca²⁺, Mg²⁺ and serum. However, oxidants such as H₂O₂, ClO⁻, ¹O₂, ONOO⁻ and ·OH would greatly enhance the signal ratio, and conversely reductants including Vc, L-Cys, GSH, Na₂S and DTT depressed the signal ratio. The moderate oxidation (0.41 V) and reduction (0.31 V) potentials make it easy to oxidize or reduce the semioxidized PIBAM–FSeN NPs in vivo by the endogenous redox species (Fig. S21 and Tables S2 and S3†). The redox process was further verified by checking the redox state of Se in the nanoprobes through X-ray photoelectron spectroscopy (XPS, Fig. S22†).

In vitro ¹⁹F-MRI of semioxidized PIBAM–FSeN NPs

To investigate the imaging benefits of semioxidized nanoprobes for low-concentration analytes, the imaging results of semioxidized PIBAM–FSeN NPs and PIBAM–FSeN NPs were compared. Fig. 4A and C illustrate an increase in the signal at −58.7 ppm and a decrease in the signal at −64.2 ppm as the oxidation degree of semioxidized PIBAM–FSeN NPs deepens. Thus, a significantly elevated signal is obtained in the ratio channel, even though the increasing oxidation degree of the probe is only 3%. However, Fig. 4B and D show that with PIBAM–FSeN NPs, only the signal at −64.2 ppm decreases gradually with an increased oxidation degree. At low oxidation degrees (3% and 7%), the signal at −58.7 ppm is nearly undetectable, but as oxidation levels increase (22%), changes in the signal can be observed due to it reaching the detection limit of the instrument. These results have confirmed the benefits of using semioxidized PIBAM–FSeN NPs.

Fig. 4 ¹⁹F-MRI of semioxidized PIBAM–FSeN NPs (A) and PIBAM–FSeN NPs (B) with different increased oxidation degrees (Δ(oxidation degree)). The ¹⁹F-MRI signal ratio of semioxidized PIBAM–FSeN NPs (C) and PIBAM–FSeN NPs (D) at different increased oxidation degrees (Δ(oxidation degree)). *, p < 0.05; **, p < 0.01; ***, p < 0.001, n = 3, error bars represent standard deviation (SD). (E) ¹⁹F-MRI of semioxidized PIBAM–FSeN NPs (7.2 mg mL⁻¹) after adding H₂O₂. (F) Linear correlation between the S_Ox/(S_Ox + S_Red) ratio and H₂O₂ concentration. (n = 3, error bars represent standard deviation). The band in the graph is the 95% confidence interval band. The limit of detection (LOD) was 0.18 mM, which was calculated by using the method of 3σ/k.

Subsequently, we investigated the ¹⁹F-MRI signal response of semioxidized PIBAM–FSeN NPs toward H₂O₂. As shown in Fig. 4E, the intensity of their ¹⁹F-MRI signal ratio gradually enhanced with the increase of the H₂O₂ concentration. A linear relationship was observed between the ratio and the concentration of H₂O₂. Furthermore, the ¹⁹F-MRI signal ratio of S_Ox/(S_Ox + S_Red) showed a good linear relationship with the H₂O₂ concentration with the linear equation: y = 0.046 (±0.001) x + 0.500, R² = 0.999 (Fig. 4F). The limit of detection (LOD) was detected as 0.18 mM (LOD = 3σ/k, n = 3). Taken together, semioxidized PIBAM–FSeN NPs could be used to quantify H₂O₂ by ¹⁹F-MRI in vitro.

In vivo ¹⁹F-MRI of semioxidized PIBAM–FSeN NPs

Encouraged by these promising results, we explored the potential of utilizing these semioxidized PIBAM–FSeN NPs for ¹⁹F-MRI visualization of the redox state in vivo. The cytotoxicity and hemolysis tests were first carried out, and the results suggested their favorable biocompatibility and biosafety, making them suitable for in vivo applications (Fig. S23–S27†). A Balb/c mouse model with a 4T1 tumor was constructed to serve as a proof of concept and showcase the ability of the nanoprobes to monitor localized redox state in vivo. As depicted in Fig. 5A and B, the same amounts of semioxidized PIBAM–FSeN NPs colloidal solution were injected into the tumor and healthy tissue of the same mouse, respectively. After injection, ¹⁹F-MRI at time points of 0.5 and 4 h post-injection was captured at the center frequencies (−58.7 ppm, red channel) and (−64.2 ppm, blue channel). ¹H-MRI was also provided to show the mouse and tumor boundaries in the overlaid ¹⁹F-MRI. As shown in Fig. 5C, stronger ¹⁹F-MRI signals in the red channel and weaker ¹⁹F-MRI signals in the blue channel were observed at the tumor site 4 h post-injection compared to those in normal tissues. The signal ratio was significantly increased (**p < 0.01, n = 3), implying that it is more oxidative in the tumor in this period (Fig. 5D).

Fig. 5 (A) Scheme for MRI of the redox environment in the tumor and normal tissue. (B) Diagram of the injection location. (C) Representative ¹H/¹⁹F-MR images of 4T1 tumor-bearing mice after intratumoral injection of nanoprobe colloidal solution at different times. (D) S_Ox/(S_Ox + S_Red) ratio at 0.5 and 4 h in normal tissue or the tumor. **, p < 0.01; n = 3, error bars represent standard deviation (SD). (E) Scheme for MRI of the redox environment in the 4T1 tumor with or without NAC treatment. (F) Representative ¹⁹F-MR ratio images of 4T1 tumor-bearing mice with or without NAC treatment at 1, 3 and 6 h. NAC (0.1 M, 100 μL) was paratumorally injected at 4 h. (G) The ¹⁹F-MR signal ratio in the tumor at different time points. *, p < 0.05; n = 3, error bars represent standard deviation (SD).

Moreover, we conducted the ¹⁹F-MRI tests to monitor the redox cycle in the tumor of live mice with N-acetyl cysteine (NAC) as the anti-inflammation drug (Fig. 5E). As shown in Fig. 5F and G, the ¹⁹F-MRI signal ratio of semioxidized PIBAM–FSeN NPs in the tumor increased at 3 h after intratumoral injection. When pretreated with NAC at 4 h post-injection, the ¹⁹F-MRI signal ratio remarkably decreased compared to that without NAC treatment (*p < 0.05, n = 3). These results demonstrated that the nanoprobes could be used for reversible redox sensing in vivo. In addition, the blood biochemical analysis (Fig. S28†) and histological hematoxylin and eosin (H&E) staining (Fig. S29†) further confirmed that no apparent toxicity was induced by the nanoprobes.

Conclusions

In conclusion, we designed selenide polymer nanoprobes with favorable biocompatibility and stability for in situ reversible monitoring of the redox process in vivo. Based on the reversible shift of the ¹⁹F-NMR peak between oxidation (FSeON, −58.7 ppm) and reduction (FSeN, −64.2 ppm) states of the nanoprobes exposed to different redox species, the ¹⁹F-MRI signal ratio of S_Ox/(S_Ox + S_Red) was successfully applied to visualize the redox state in a tumor. Importantly, the nanoprobe demonstrates excellent water solubility and good biocompatibility, making it promising for intravenous injection. Considering the wide range of biomolecules, such as proteins, saccharides and aptamers, that can be potentially labeled with the trifluoromethyl-grafted selenide structure, it might be possible to adapt this strategy to redox sensing in other specific regions. This work paves a new way to develop powerful tools for reversible sensing and imaging of redox the state in vivo with high penetration depth.

Ethical statement

All experiments involving animals were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the China-Japan Friendship Hospital and Beijing University of Chemical Technology.

Data availability

Experimental procedures and all relevant data are available in the ESI† and from the authors.

Author contributions

The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript. L. Y. W. and C. G. supervised the project. L. Y. W., C. G., S. Y. X. and X. Y. X. designed the experiments. X. Y. X., S. J. L. and Y. M. L. conducted the experiments and analyzed the data. L. Y. W., C. G. and X. Y. X. wrote the manuscript. All authors discussed and commented on the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was partially supported by the National Natural Science Foundation of China (22334002, 22322402, and 22306010), the Beijing Municipal Natural Science Foundation (Z231100002723006), and the Fundamental Research Funds for the Central Universities (XK2023-19 and JD2308).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4sc08297d

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