Purpose

Fluorine ¹⁹F MR represents a promising tool for diagnostic imaging due to its high specificity caused by negligible abundance of fluorine in the tissues. Here we present a novel pH and temperature responsive ¹⁹F MR contrast agent based on fluorinated polymer systems. This agent allows phase transition from liquid into solid (gel-like) state at the temperature above 20 ºC. This forms an agent depot after injection into the living system, what could be very potential for tumor treatment after the drug incorporation. In this study, we have characterized the probe sensitivity and relaxivity dependence on temperature in the phantoms. Moreover, we have showed the depot in animal models in vivo for a long term.

Methods

MR properties and responsivity to temperature of the fluorinated polymer systems (poly(2,2-difluoroethylacrylamide-co-1-imidazolylpropylacrylamide)) were tested. The temperature dependent MR properties of the probe were assessed by ¹H and ¹⁹F relaxometry, ¹⁹F MR spectroscopy and ¹⁹F MR imaging. ¹H relaxometry of the probe was performed on a 0.5T NMR relaxometer (r1 - saturation recovery sequence, recycle delay 12s; r2 – CMPG sequence, recycle delay 10s). Temperature-dependent ¹⁹F MR spectroscopy and imaging were carried out on a 4.7T scanner (MRS: single pulse, repetition time TR: 1000ms, number of acquisitions NA: 64, acquisition time TA: 1min; MRI: RARE sequence, TR: 2000ms, echo time TE: 7ms, turbo factor TF: 16, NA: 128, TA: 17min, resolution: 0.64x0.64x3 mm³). The sensitivity of the probe was assessed by ¹⁹F MR imaging at 25°C on a phantom containing various concentration of the probe (4.5 mM – 1.3 M). To check the feasibility of the ¹⁹F MR probe in vivo, four healthy female rats (Lewis, 250-300g) were scanned after the contrast agent administration (1.3 M, 300ml injection into the muscle of the right hind leg and into the subcutaneous area (left hind leg) for two months. Then we have administrated the agent (1.8 M, 200 mL) to the subcutaneous solid tumor (HUH7 cell line) and to the muscle of a CD-1 nude mouse. In animal experiments the same MR sequence as in the phantom study was used.

Results

The relaxometry showed a strong relaxivity dependence on temperature, both relaxivity r1 and r2 were lower with increasing temperature (Fig. 1). Temperature-varied MRS and MRI experiment confirmed that dependence; we observed markedly lower MR signal in the phantoms with increasing temperature (Fig. 2). The minimal concentration of the agent at 25°C detected within reasonable measurement time for in vivo experiments (17 min) was 40 mM (Fig. 3). Strong ¹⁹F MR signal was detected at both injection sites in all animals; the ¹⁹F MR signal was detected two months after contrast agent administration and it was comparable with the signal detected in the first week (Fig.4). Signal to noise ratio (SNR) and volume of ¹⁹F MR signal after depot formulations within the first day slowly increased (Fig.5). No adverse effects of the probe to the animals were observed (no change of the body weight).

Discussion

Good sensitivity and temperature-dependent nature of the novel ¹⁹F probe was confirmed. In the in vivo measurement, ¹⁹F MR signal enhancement after two hours following contrast agent administration reflects creation of a depot upon temperature change by overreaching the lower critical solution temperature. At temperatures above 20°C and pH = 7.4 contrast could change liquid phase to solid reversibly. Slow degradation confirmed also the result from volumetry; volume enlargement of depots between day 4 and day 57 after contrast agent application is probably due to slow diffusion of polymer depot to surrounding tissue. This feature, and the size of the nanoparticles (less than 200 nm) allowing spontaneous accumulation in solid tumors (enhanced permeation and retention (EPR) effect) might be attractive for the use as a theranostic tumor probe after drug incorporation.

Conclusion

Here, we presented a novel efficient ¹⁹F MR probe which forms depot as a response to physiological parameters (pH and temperature) and can be detected in animals in vivo for a long term. Sufficient MR sensitivity of the probe and its slow degradation in animals suggest potential for theranostic use of this contrast agent.

Acknowledgements

Supported by Czech Science Foundation GACR (P205-16-03156S), MH CZ-DRO (Institute for Clinical and Experimental Medicine–IKEM, IN 00023001).

References

No reference found.