Synopsis

Exact localization of acute thrombi is still a serious problem in the clinical setting. Here we show the feasibility of artefact-free ¹⁹F MR-imaging using clinically relevant PFOB-nanoemulsions functionalized with an α2-antiplasmin peptide, specific for early thrombi. Utilizing the isolated CF₃-peak of PFOB in combination with a conventional ¹⁹F RARE sequence is suitable for specific and artefact-free imaging of acute thrombi in vitro and also in vivo.

Background

Exact localization of acute thrombi is still a serious problem in the clinical setting and has significant implications for antithrombotic treatment regiments, since chronic thrombi are resistant towards thrombolysis. Detection of thrombi by conventional ¹H MR methods, like angiography or T1/T2-weighted techniques can be challenging, inparticular if small, non-occlusive thrombi have only minor impact on blood flow and therefore may not be detected in weighted images.

To overcome this limitation, we have recently used ¹⁹F MRI for detection of early thrombi utilizing a targeted perfluorocarbon nanoemulsion (perfluoro-15-crown-5 ether = PFCE) functionalized with a peptide derived from α2-antiplasmin (α2AP) which specifically accumulates in early thrombi (1). Due to the fact that 1⁹F is nearly absent from biological tissue, this appraoch enabled the unequivocal detection of early thrombi without any background signal.

PFCE is advantageous for imaging purposes due to its 20 magnetically identical ¹⁹F atoms, but has an extremly long biological half-life and is therefore not suitable for any clinical application (2). An alternative is perfluorooctyl bromide (PFOB) which has a more complex spectrum but – due to its low vapor pressure – has a short tissue retention time (2). PFOB has also been used in clinical trials as blood substitute. Normally, complex ¹⁹F spectra are not suitable for conventional ¹⁹F imaging sequences due to the occurence of chemical shift artefacts. However, the CF³-peak within the ¹⁹F spectrum of PFOB is clearly separated might suitable for artefact-free ¹⁹F MRI imaging using a conventional RARE sequence (Fig.1).

In the present study, we aimed to investigate whether the combination of α2^AP targeted PFOB emulsions with imaging of the CF₃-peak by a conventional ¹⁹F RARE sequence is suitable for the detection of acute thrombi by ¹⁹F MRI.

Methods

Using the semifluorinated diblock F6H10 as stabilizer, we generated stable PFOB nanoemulsions with a high fluorine content (40% w/w). To generate targeting PFCs, we used a 14 aa peptide derived from α2-antiplasmin (α2^AP) which is cross-linked to the developing fibrin network by the active factor XIII (restricted to the very early phase of the thrombus formation). First, α2^AP as well as Q3A-peptides (with 10-fold lower affinity for FXIIIa) were coupled to a maleimide-chlesterol-PEG2000-anchor. Resulting α2_AP/Q3A-PEG₂₀₀₀-cholesterol conjugates were incubated with preformed PFOB-emulsions which leads to the spontaneous insertion of the cholesterol moiety into the lipid layer of the PFC (sterol-based post-insertion = SPIT).

Ex vivo experiments were carried out with thrombi obtained by platelet rich plasma of human blood which were incubated with targeted PFCs about 5 min after induction of thrombus formation. For in vivo experiments, a murine model of deep venous thrombosis was was applied which resulted in non-occlusive thrombi. Combined ¹H/¹⁹F MRI was performed at a 9.4T Bruker AvanceIII WideBore NMR spectrometer using a microimaging unit (Micro 2.5) with actively shielded 40-mm gradients (1 T/m maximum gradient strength, 110 µs rise time at 100 % gradient switching). Mice were placed in a 25-mm ¹H/¹⁹F birdcage resonator and after acquisition of anatomical 1H reference images, a ¹⁹F RARE sequence with the following parameters was used: 2.56´2.56 cm2 FoV, 64×64 matrix, 1 mm slice thickness, TR 4000 ms, RARE factor 32, 256 averages, 34 min acquisition time.

Results and Discussion

To test the suitability of these emulsion for thrombus-labelling, we first incubated the α2^AP-/Q3A-emulsions with acute thrombi derived from human platelet rich plasma and analyzed the samples by fluorecence imaging and ¹⁹F MRI. For ¹⁹F MRI, we utilized a conventional RARE sequence applied to the CF₃-group of the PFOB. To prevent any chemical-shift artefacts by the neighbouring CF₂-Br peak due to line-broadening in vivo, we reduced the excitation bandwidth to 10.000 Hz. Imaging of α2^AP-/Q3A-PFOB labelled ex vivo generated human thrombi from several volunteers revealed a artefact-free ¹⁹F images in the α2^AP-labelled thrombi, whereas the thrombi incubated with Q3A-PFOB showed only low signal (Fig. 2A). This was also reflected by measurement of the fluorescence signal (Fig.2B), showing a specific accumulation of α2^AP-PFOBs in acute thrombi. In contrast, chronic thrombi were neither labelled with α2^AP-PFOB no Q3A-PFOB.

To validate this approach also in vivo, we used a model of acute deep venous thrombosis, by induction of vessel injury due to topical application of FeCl₃. Intravenous injection of α2^AP-PFOB emulsions shortly before thrombus induction resulted in a highly specific artefact-free ¹⁹F signals in acute thrombi without any ¹⁹F background from surrounding tissue.

Conclusions

Acknowledgements

References

1: Temme S, Grapentin C, Quast C, Jacoby C, Grandoch M, Ding Z, Owenier C, Mayenfels F, Fischer JW, Schubert R, Schrader J, Flögel U. Noninvasive Imaging of Early Venous Thrombosis by 19F Magnetic Resonance Imaging With Targeted Perfluorocarbon Nanoemulsions. Circulation. 2015 Apr 21;131(16):1405-14.

2: Jacoby C, Temme S, Mayenfels F, Benoit N, Krafft MP, Schubert R, Schrader J, Flögel U. Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half-lives and sensitivity. NMR Biomed. 2014 Mar;27(3):261-71.