Introduction

³¹P-MR is used mainly for assessing various metabolites in vivo. Its further potential for imaging is tested, but due to high physiological background signal, it requires a specific exogenous probe¹. Presented polymer is a metal-free phosphorus-containing probe for a specific ³¹P-MR, as phosphorothioate group (P=S) in its structure creates a high chemical shift from biological signal (P=O). Moreover, the P=S groups are prone to metabolic oxidation to phosphoester groups P=O, when exposed to reactive oxygen species (ROS), represented in greater amounts in cancer tissue. This feature can serve as a promising functional in situ sensor of oxidative stress. Here, we provide the results confirming ROS production in the cell line using fluorescent imaging and the polymer measurement using MR in both in vitro (³¹P-MRS) and in vivo (³¹P-MRS/¹⁹F-MRI) testing of the oxidation-triggered chemical shift.

Methods

The polymer (Poly[O-(2-(methacryloyloxy)ethyl)O-(2(trimethylamoniumyl)ethyl)phosphorothioate]: pTMPC) was synthesized by controlled radical polymerization technique of the corresponding zwitterionic monomer. MR spectroscopy and imaging were performed on 4.7T scanner using custom dual ¹H/³¹P solenoid (in vitro) and ¹H/³¹P/¹⁹F surface (in vivo) coils. The fluorescence measurement was used to test 4T1 cell line (96-well plate; 0.1x10⁶ mL⁻¹) ROS production and was performed on optical imaging system using OxyBurst fluorescent dye (OB) and with additional 20 and 60 µL of H₂O₂, as a control. The OB is a non-fluorescent dye, which is oxidised by ROS to fluorescein derivate. In the first step of MR measurements, the in vitro oxidation (conversion from P=S to P=O) was tested using singlepulse ³¹P-MRS (TR=500 ms, ST=5 min, BW=200 ppm) of the pTMPC in two conditions: i) the polymer (c^P=10 mmol L⁻¹) mixed with the hydrogen peroxide (c=20 mmol L⁻¹; mimicking hypoxic tumor tissue), ii) the polymer (c^P=2 mmol L⁻¹ ) added to 4T1 cancer cell line suspension (40×10⁶ mL⁻¹ cells). Finally, 4T1 cells suspension (0.3×10⁶ in PBS) was injected subcutaneously to Balb/C mouse, which, at tumor volume ~0.3 mL, underwent intratumoral injection of the dual ³¹P/¹⁹F pTMPC (c^P=100 mmol L⁻¹; c^F=112.5 mmol L⁻¹; 0.2 mL in PBS). The animal was monitored using several MR methods: ¹H-MRI (localization), ³¹P/¹⁹F-MRS single pulse (TR=200/500 ms, ST=10/5 min, BW=200/40 ppm) and ¹⁹F-MRI (CSI; TR=200 ms, ST=1h). Isoflurane was used during the in vivo MR. The ³¹P-MRS signal quantification and ¹H/¹⁹F-MR image overlay were carried out in ImageJ and Matlab software. The ³¹P-MRS signal amplitude was averaged from each hour and is presented as a percentage change from the experiment onset. For quantification the P=S signal was used, because when the phosphorus signal is converted by oxidation it overlay with the signal originating from the organism. The ¹⁹F-MRI was used as an on-site signal to track the polymer in vivo.

Results/Discussion

The fluorescent measurement confirm that 4T1 cells alone can produce ROS. In the first phase of the experiment, the fluorescence signal from the cells was comparable to the control (with additional H₂O₂; Fig.1). The MR signal conversion from P=S to P=O accompanied by a significant frequency shift in the ³¹P-MR spectrum was observed in vitro. The polymer underwent oxidation and the P=S amplitude was reduced by 91.1% (H₂O₂) and 36.0% (4T1 cells; Fig.2). The structural modifications ensured chemical shift in in vivo ³¹P and ¹⁹F-MR, making it easily distinguishable from the biological background (³¹P-MRS: ∆δ=60 ppm) and isoflurane-based anaesthesia (¹⁹F-MRS: ∆δ=20 ppm). The in vivo oxidation of the polymer in tumor resulted in 43% signal conversion of the ³¹P-MRS signal in first 6 hours and the ¹H/¹⁹F-MRI measured 24h post-injection confirm that the dual pTMPC is localized in the tumor, assuring that the ³¹P-MR signal conversion was triggered by hypoxic tumor tissue (Fig.3).

Conclusion

Phosphorus-containing polymer showed both a high sensitivity for ³¹P-MR and a chemical shift from biological phosphorus signal. Furthermore, it can serve as a sensitive sensor of pathological conditions, as it undergoes oxidation-induced structural changes in the presence of ROS. With favourable MR properties and biocompatibility, the probe represents a conceptually new approach for ³¹P-MR. The ¹⁹F-MR signal can provide an on-site information on the polymer distribution in the organism during the P=S to P=O conversion in the ³¹P-MRS.

Acknowledgements

The project was supported by the Ministry of Health of the Czech Republic [NU20-08-00095], SGS project no. 2022/3002 of the Technical University of Liberec and by MH CR-DRO (Institute for Clinical and Experimental Medicine IKEM, IN00023001).

References

1. Kracíková, L., Ziółkowska, N., Androvič, L., Klimánková, I., Červený, D., Vít, M., Jirak, D. Phosphorus‐containing Polymeric Zwitterion: A pioneering bioresponsive probe for ³¹P‐magnetic resonance imaging. Macromolecular Bioscience. 2022; 2100523. doi:10.1002/mabi.202100523.