Perfluorocarbons-Based ¹⁹F Magnetic Resonance Imaging in Biomedicine

Introduction

Fluorine-19 magnetic resonance (¹⁹F MR) molecular imaging is a noninvasive tool widely exploited for in vivo applications due to low background signals. Similar to ¹H, ¹⁹F has a spin of one-half nucleus and no quadrupolar moment, thereby greatly simplifying spectral analysis. The ¹⁹F nucleus has a high gyromagnetic ratio and a sensitivity of 83%, which is comparable to that of ¹H and significantly higher than the sensitivity of other typically investigated MR receptive nuclei, such as ³¹P, ¹³C, and ¹⁵N. The natural abundance of ¹⁹F is 100%. Endogenous ¹⁹F is found primarily in bone marrow and teeth as solid fluorides, whereas soft tissues have undetectable ¹⁹F MR signals.^{1 19}F is virtually absent from tissues without endogenous background signal.² Finally, in addition to its high sensitivity, ¹⁹F nucleus exhibits a wide range of chemical shifts (>350 ppm) and it is extremely sensitive to relaxation changes, which can provide higher resolution than ¹H MRI.³ Thus, administered ¹⁹F-containing compounds have optimal properties for specifically and selectively assessing tissue physiology and pathology in vivo.

Perfluorocarbons (PFCs) are biologically inert, highly stable, non-toxic, non-carcinogenic, non-mutagenic, non-teratogenic compounds that can generate ¹⁹F signal and they are not metabolized in the human body.^3,4 PFC nanoparticles (NPs) have therefore been extensively used as ¹⁹F MRI agents in research applications. The strong C-F bond in PFCs is resistant to cleavage by endogenous enzymes, and the dense, electron-repelling sheath that coats F-chains allows only extremely weak intermolecular interactions.⁵ Due to their relatively large size (~250 nm), intravenous administration of PFC NPs is not susceptible to glomerular filtration and is instead removed from the circulation via the reticuloendothelial system and exhalation through respiration.^6,7 Thus, PFCs have excellent biosafety and exhibit no renal toxicity in animals or human, which do not increase kidney burden.^8,9 The frequently used PFCs are perfluorooctyl bromide (PFOB), perfluoro-15-crown-5-ether (PFCE) and perfluoropolyether (PFPE). When compared to iodinated or gadolinium-based contrast agents, PFC NPs are better suited for patients with kidney disease. Research shows that the blood clearance half-life of PFCs ranges from three to 42 hours,¹⁰ and tissue clearance occurs within four (PFOB) to 65 days (perfluorotripropylamine),¹¹ thereby allowing sufficient time for MR detection. A practical concern persists regarding the application of probes in patients with potentially impaired renal function. Gadolinium agent as a paramagnetic contrast agent for MRI scans is thought to be associated with susceptibility to diseases such as nephrogenic systemic fibrosis.^12–14

PFCs also have very good respiratory gas- (oxygen and carbon dioxide) carrying capacity and were first proposed as oxygen carriers in 1966.¹⁵ Over the last decade, PFCs have been widely used as oxygen-carrying blood substitutes¹⁶ and for liquid ventilation in respiratory diseases. Besides their low viscosity, liquid PFCs generally have 10–20 times more oxygen solubility than water or blood plasma,¹⁷ and they were shown to maintain oxygen transport and support life for several hours in rats lacking erythrocytes.¹⁸ Thus, PFCs are excellent candidates for protecting tissue from hypoxia and preventing irreversible tissue damage.

PFCs exchange oxygen with the surrounding medium through free diffusion on a millisecond timescale.¹⁹ The oxygen solubility of PFCs varies inversely with temperature, and the amount of oxygen dissolved in any type of PFC increases linearly with oxygen partial pressure (pO₂). To measure tissue oxygenation noninvasively, calibration curves can be used to illustrate the relationship between ¹⁹F concentration and pO₂.^20,21 Mechanistically, since O₂ is a paramagnetic substance, its partial pressure affects the longitudinal relaxation rate (R₁) of PFC in a linear way as follows: R₁=A+B*pO₂.²² Hence, pO₂ corresponds to R₁ and ultimately to longitudinal relaxation time T₁ (1/R₁). As a result, by measuring the ¹⁹F T₁ value of different tumor areas (acquiring a T₁ map), we can obtain the corresponding pO_2.^23–25 Due to these advantages, ¹⁹F MRI has increasingly being utilized in physiological molecular imaging and therapeutics.^26–28

The smart stimuli-responsive ¹⁹F MRI platform using PFC-based nanoprobes with exceptional sensitivity and off/on-switching is a powerful tool for visualizing in vivo enzymatic activity, redox-potential difference, and lower pH values.²⁹ By combined various stimuli-responsive with theranostic nanoplatforms for sensitive ¹⁹F MRI of biological and pathological situations, stimuli-responsive nanoparticles will eventually take advantage of specific tumor microenvironmental changes enable early accurate diagnosis and therapeutics. This novel technology is considered to have the potential to clarify the biomolecular networks in animals using the latest molecular imaging techniques.

¹⁹F MRI Techniques

Negligible signal background enables high contrast-to-noise ratios and improved quantification potential in ¹⁹F MRI. Challenges and future perspectives regarding routine ¹⁹F imaging for clinical translation of these techniques to patients are specialized coils and hardware for acquisition of ¹⁹F MR images. To meet these challenges, a variety of ¹⁹F MR relevant equipment, techniques, and sequences have been developed in recent years. Below we discuss some of the lessons that can be drawn from these advancements in ¹⁹F MRI technology.

As ¹⁹F MRI can only trace exogenous fluorinated materials, it is necessary to conduct ¹H MRI simultaneously to collect anatomical information. Dual-tuned ¹⁹F/¹H radiofrequency (RF) coils for ¹⁹F MRI have been developed to address this issue and are currently widely used. As the signal-to-noise ratio (SNR) can be maximized by tuning the frequency to either ¹⁹F or ¹H, this combined approach has significantly improved ¹⁹F imaging technology.^30,31 Villavalverde et al designed a multi-tuned RF coil based on the high B₁ homogeneity of birdcage coils to obtain high-quality images with good uniformity and sensitivity for ¹⁹F.³² Another study showed that the birdcage coil increased the B₁ homogeneity, which allowed estimation of the minimum detectable ¹⁹F atoms number and ¹⁹F content of the NP. The minimum detectable PLGA-PFCE concentration was 2.5 mg/mL using the birdcage coil in NP solution phantoms imaging and MRS. In addition, due to the advantage of increased B₁ detection sensitivity and field uniformity, the butterfly coil could be used in animal experiments.³³ This dual-tuned ¹⁹F/¹H RF coils have been successfully applied in preclinical studies. For instance, a ¹⁹F/¹H RF coil improved the B₁ field uniformity without reducing sensitivity in MRI of arthritic knee in rabbits.³⁴ More recently, an eight-channel transceiver ¹⁹F/¹H RF coil employed to locate and quantify administrated fluorinated materials in the knee at 7.0 T showed high sensitivity with an in-plane spatial resolution of 1.5 × 1.5 mm² and slice thickness of 5 mm, revealing great translation potential to clinical applications. Further technological developments are necessary to promote real-time bioavailability studies and quantification of ¹⁹F-containing medicinal compounds in vivo.³⁵

Several MR sequences have been designed to improve SNR and the quantification accuracy of ¹⁹F MRI.³⁰ PFCE has been widely investigated as ¹⁹F MRI contrast agent with a strong single peak resonance spectrum at −91.8 ppm related to CFCl₃ without any chemical shift artifact.³⁶ For ¹⁹F compounds with unique spectral properties, fast spin echo/rapid acquisition with relaxation enhancement (FSE/RARE) is the most useful sequence.³⁷ Unlike PFCE, many PFCs have multiple NMR spectral peaks with ¹⁹F MRI SNR decreased. PFPE has two major chemical shifts at −90.7 and −90.9 ppm used for cellular and molecular MRI.³⁸ PFOB (CF₃-(CF₂)₆-CF₂Br) is a blood substitute used for ¹⁹F MRI that exhibits a multi-peak resonance spectrum and complex ¹⁹F resonances and multiple relaxation conditions with single ¹⁹F resonance peaks for CF₂Br and CF₃ groups and five proximate chemical shift components of the CF₂ group.^39,40 The challenge for multispectral compounds with chemical shift artifacts in MRI is that the SNR is reduced if only the signal of a single spectrum is acquired.^38–40 To meet this challenge, Fluorine ultra-fast Turbo Spectroscopic Imaging (F-uTSI)⁴¹ has also been developed to improve SNR without sacrificing sensitivity or increasing scan time. Furthermore, F-uTSI can distinguish between various ¹⁹F compounds based on chemical shift differences, thereby allowing for “multi-color” imaging. Another approach of meeting this challenge is to adopt a novel pulse sequence, ¹⁹F/¹H 3D-balanced steady-state free precession (bSSFP) to avoid the chemical shift artifacts of PFC with multi-resonance spectra⁴⁰ Notably, the bSSFP sequence implemented on a clinical 3 T scanner enabled the detection of PFOB labels with higher sensitivity than traditional techniques. Additionally, by using a ¹⁹F chemical shift encoding (CSE) approach, Van Heeswijk et al demonstrated that CSE–bSSFP has higher sensitivity than bSSFP-UTE sequences at 3 T.⁴² The T₁ and T₂ relaxation time of three PFC emulsions commonly used in ¹⁹F MRI were measured at 3 T, including PFOB, PFCE and PFPE at different temperatures. By means of relaxation time for each PFC phantom, the optimized parameters repetition time (TR) and echo train length (ETL) with longitudinal magnetization restoration (LMR) (a −90 “flip-back” pulse) off and on in the turbo spin echo (TSE) pulse sequence and the optimal flip angle for the bSSFP pulse sequence were determined for PFCs.⁴³ Mastropietro et al proposed a procedure for optimizing the SNR in RARE sequences, which improved sensitivity of ¹⁹F MR. In this work the optimized RARE parameters (TR, number of echoes, flip back pulse) provided a method of improved SNR according to measured relaxation time (T₁, T₂) values at 7 T, which might be encountered in vivo and in vitro molecular imaging experiments.³⁷ Theoretical and experimental comparisons of spoiled-gradient echo (SPGR), RARE, and SSFP pulse sequences were conducted under phantom conditions using ¹⁹F MRI/MRS at 9.4 T. SSFP yielded the highest mean SNR higher than SPGR and RARE using the homogeneous birdcage coil, whereas there was no additional improvement of ¹⁹F signals for NP loading concentration beyond 7.5 mg/mL per million cells. In this work, SPGR yielded maximal SNR at long TRs and it is recommended to use it in combination with appropriate flip angle. A detectable limitation of cardiac stem cells was approximately 500k (10k cells/voxel) in fast 2D acquisitions spanning (3–5 min) achieved by the butterfly coil. This method of fast and quantitative in vivo cardiac ¹⁹F MRI of PFCE-labeled progenitor stem cells using SPGR/SSFP and MRS acquisitions with a butterfly coil provided evidence for preclinical work.³³ In another study, the authors developed a class of novel ¹⁹F chemical exchange saturation transfer (CEST) imaging probes, which detected multiple metal ions (Mg²⁺, Ca²⁺, Zn²⁺) with a single ¹⁹F NMR peak from multiple fluorines.⁴⁴ These imaging probes exhibited high sensitivity and specificity for detecting metal ions at low concentrations. Schoormans et al introduced an iterative sparse deconvolution method to discriminate different ¹⁹F compounds and remove chemical shift artifacts arising from multiple resonances at 7 T that was applicable for in vivo imaging.⁴⁵ In addition, studies have shown that lanthanide gadolinium ions can increase the relaxation rate of ¹⁹F. Recently, the influence of gadolinium-functionalized PFCE emulsions on ¹⁹F R₁ and ¹⁹F R₂ at different magnetic field strengths was studied. The results indicated that gadolinium-functionalized perfluorocarbon emulsions were suitable for ¹⁹F MRI at current clinical field strengths (1.5–3.0 T) as gadolinium ions increased the value of ¹⁹F R₁. However, for the emulsions without gadolinium, higher field strengths (6.3–14 T) are favorable for ¹⁹F measurements due to the fact that gadolinium does not increases ¹⁹F R₁, but leads to significantly increasing ¹⁹F R₂.⁴⁶

The Development of PFC Nanoparticles

PFC nanoparticles consist of a PFC core surrounded by a lipid monolayer that can be functionalized with a variety of agents. Different types of PFCs can be used as a core, including perfluorodichlorooctane (PFDCO), perfluorotributylamine (PFTB), perfluorodecalin (PFDC), PFCE, and PFOB. Although PFCE exhibits optimal MRI properties, its long retention time in the human body makes it unsuitable for repetitive clinical applications.⁴⁷ On the other hand, a variety of other PFCs, including PFOB, are quickly cleared from the body through exhalation by the lungs. Furthermore, while these compounds may induce chemical shift artifacts when ¹⁹F images are acquired in a conventional manner due to signal splitting resulting from ¹⁹F nuclei of different magnetism, this problem can be overcome by advanced detection methods based on fast chemical shift imaging techniques.^48,49 Thus, the ¹⁹F nucleus in the core of PFC NPs offers many favorable magnetic properties and provides a very high intrinsic signal level.

The surface of nanoparticles can be covalently or noncovalently linked to many types of imaging agents for molecular imaging and therapy, such as florescent material for fluorescence imaging and histology, radionuclides for nuclear imaging, iodine for computed tomography (CT) and paramagnetic metals (Gd, Fe, Mn) for MRI. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) have high sensitivity to detect, visualize, and quantitatively measure molecular targets in the tumor microenvironment. The limitations of PET and SPECT are their poor spatial resolution and relatively high doses of radiation.⁵⁰ CT is an imaging technique that offers great advantages such as high spatial and density resolution. However, CT generally has low sensitivity, specificity and temporal resolution visualization of the internal structure of soft tissues.⁵¹ MRI has many advantages such as no ionizing radiation, high-sensitivity, high spatial resolution and high image contrast. MRI is commonly used in anatomic, functional, and molecular imaging.⁵² In most MR molecular imaging researches, it is necessary to compare series of pre-/post-contrast images to distinguish superparamagnetic (eg, iron oxide) or paramagnetic metal (eg, gadolinium) contrast from background signals, which can be avoided due to the advantage of negligible ¹⁹F background signal. Gadolinium agent as a paramagnetic contrast agent for MRI scans is thought to be associated with susceptibility to diseases such as nephrogenic systemic fibrosis and acute complement activation found in clinical trials.^13,53,54 A practical concern persists regarding the application of probes in patients with impaired renal function and potential risk of systemic side effects, which calls for great caution. Superparamagnetic iron oxide (SPIO) particles are very sensitive to cell labeling and have been widely used to label NSCs in preclinical studies.⁵⁵ However, due to superparamagnetic bloom artifacts, iron oxide tends to reduce the resolution of soft tissue. Due to the lack of magnetic artifacts of ¹⁹F, these artifacts can be avoided and the details of tissue and cellular boundary images can be preserved.²⁸ In addition, in vivo cell apoptosis or cell lysis can liberate the iron, which can be engulfed by microglia or macrophages surrounding the transplanted stem cells, leading to false positive signals.⁵⁶ More recently, increasing attention has been paid to PFC labeling for MR cell tracking. Furthermore, PFCs can be used as ultrasound molecular imaging agents due to their composition, which allows detection by mechanical waves at clinically relevant imaging frequencies.^57,58 These intrinsic properties of PFCs, therefore, enable the unambiguous detection of exogenously administered PFC NPs and the quantitative monitoring of their biodistribution in vivo with different imaging modalities.^59,60 Finally, the surface of nanoparticles can also be linked with different molecular ligands or drugs for targeted molecular imaging, targeted therapy and other applications (Figure 1).⁶¹

Figure 1 Schematic representation of the contact-facilitated drug delivery mechanism. Reprinted with permission from Zhou H-F, Yan H, Senpan A et al Suppression of inflammation in a mouse model of rheumatoid arthritis using targeted lipase-labile fumagillin prodrug nanoparticles. Biomaterials. 2012;33(33):8632–8640.Copyright © 2012 Elsevier Ltd.61

The Applications of Perfluorocarbons-Based ¹⁹F MRI in Oncology

¹⁹F MR has many applications in targeted imaging, measurement of tumor oxygenation, monitoring of drug delivery, cell therapy, treatment evaluation, and stimuli-responsive imaging^26,62-70 which will be introduced in detail as follows. Table 1 is a summary of representative studies using ¹⁹F MR molecular imaging in these applications.

Table 1 Applications of 19F MR in Molecular Imaging

Ligand-Targeted Tumor Imaging

In recent years, targeted paramagnetic PFC NPs have been widely used in ¹⁹F MRI for the recognition of pathological biomarkers, for the detection of changes in expression levels due to disease development, in therapeutic interventions, or in the monitoring of disease recurrence. A variety of ¹⁹F-based imaging tracers have been introduced, including micelles,⁷¹ liposomes,⁷² and emulsions.⁷³ The physicochemical properties of PFC NPs, combined with their high surface area, which can incorporate 50 to 500 targeting ligands, allow the detection of sparse concentrations of cell surface biomarkers. Furthermore, the incorporation of large amounts of paramagnetic materials into the nanoparticles was shown to increase the contrast of molecular epitopes occurring in very small quantities in vivo.^74,75

PFC NPs or nanoemulsions covered with tumor-specific ligands have been extensively explored in noninvasive imaging and drug delivery. Most early research on MRI using targeted PFC materials was focused on ¹H MRI. For example, in a study with Vx-2 rabbit tumor models, the ¹H MR signal intensity increased by 56% in the α_vβ₃-integrin-targeted PFC NPs injected group, and this signal was significantly reduced by competitive blocking with α_vβ₃-integrin nonparamagnetic nanoparticles.⁷⁶ Diou et al modified the capsule morphology of PFOB nanocapsules for detecting tumors with ¹⁹F MRI, and were able to distinguish between passive and active targeting after decorating the particles with 600–950 integrin-binding RGDS peptide.⁷⁷ In another study, the dependence of neovascular molecular MRI on the relaxation time (R₁) of α_vβ₃-integrin-targeted paramagnetic PFC NPs was determined. The authors traced the temporal-spatial consistency of angiogenesis assessments in a rabbit Vx2 tumor model and compared the neovascular contrast enhancement obtained with α_vβ₃-integrin-targeted Gd-DOTA-PE and α_vβ₃-integrin-targeted Gd-DTPA-BOA nanoparticles.⁷⁸ MR neovascular contrast maps of Vx2 tumors at various time points after implantation revealed that surface enhancement is progressive and temporally consistent.

Despite the recent advances in ¹⁹FMR targeted imaging with PFC NPs, most studies focus on α_vβ₃-integrin,^77,79,80 folate receptor (FR)^81–85 and vascular endothelial growth factor receptor 2 (VEGFR2)^86,87 as molecular targets to detect tumor angiogenesis, proliferation and certain cardiovascular disease (Figure 2). ¹⁹F MRI of brain tumor angiogenesis with integrin-targeted PFC NPs in mice carrying U87 glioblastoma achieved a 50% increase in signal in the targeted group.⁷⁹ Waters et al used ¹⁹F diffusion weighted MR spectroscopy (DWS) at 11.7 T to detect angiogenesis in vivo with α_vβ₃-integrin-targeted PFC NPs in an epidermal squamous carcinomas mouse model (K14-HPV16). Progressive decay of the ¹⁹F signal with increased diffusion weighting at b-values below 1500 s/mm² was observed in both K14-HPV16 and control mice, showing that background ¹⁹F signal from unbound nanoparticles is suppressed in the blood. Whereas K14-HPV16 mice maintained a stationary ¹⁹F signal at high b-values in the ears, indicating profuse binding of PFC NPs to angiogenesis, the ¹⁹F signal in controls decayed completely at high b values (>1500 s/mm²) due to absence of binding. These results show that in vivo ¹⁹F DWS is useful for specifically detecting bound PFC NPs by selectively suppressing background ¹⁹F signal from unbound nanoparticles flowing in blood.⁸⁸ Angiogenesis-targeted PFOB nanoparticles in a rabbit Vx2 tumor model revealed heterogeneous areas of neovasculature at the tumor rim, as expected, whereas the resultant ¹⁹F signal overlaid with the ¹H MR signal clearly elucidated the anatomical colocalization of the heterogeneous distribution of the nanoparticles (Figure 2).⁸⁹ Bae et al synthesized folate-targeting PFC/rhodamine nanoemulsions for MR and optical imaging and showed that the nanoprobe was successfully delivered into FR-positive tumor xenograft models of nasopharyngeal carcinoma, with significantly enhanced ¹⁹F signal intensities in the tumor region in MR and fluorescence imaging. Folate-PFC/rhodamine nanoprobes therefore have excellent tumor-targeting ability and stability in vivo.⁹⁰

Figure 2 (A) Illustration shows the position of RF coil in the MRI scan. In vivo MRI of mice administered with PLGA-PEG PFOB/ICG (B) or PLGA-PEG-folate PFOB/ICG (C) 1H: anatomical images, 19F: 19F MRI SNR map, merge: 1H and 19F merged, T: tumor region, SPL: spleen, K: kidney.

Although EGFR is highly expressed in a variety of malignant tumor cells, including non-small cell lung cancer (NSCLC),⁹¹ head and neck cancer,⁹² and ovarian cancer,⁹³ fewer studies have explored the potential of targeting EGFR in ¹⁹F MRI to detect tumors cells. EGFR is an important transduction signal pathway involved in tumor cell growth, proliferation, angiogenesis, adhesion, invasion, metastasis and apoptosis. Thus, the non-invasive detection of EGFR in tumors is critical for early diagnosis and successful cancer treatment. For instance, PFC NPs carrying oxygen and the EGFR tyrosine kinase inhibitor (EGFR-TKI) erlotinib showed an important anti-tumor effect, revealing that PFC NP therapy targeting EGFR is a promising therapeutic strategy.⁹⁴ Thus, EGFR-targeted PFC NPs that can simultaneously assess pO₂ show great potential for tumor detection, monitoring and therapy.

Measurement of Tumor Oxygenation

Regional hypoxia is common in solid tumors due to their poorly organized vasculature network and high oxygen demands of proliferating tumor cells. Growing evidence shows that hypoxia promotes tumor angiogenesis, recurrence, progression, metastasis,⁹⁵ and sensitivity to radiotherapy. Currently, a wide variety of techniques are available to measure tumor oxygenation.⁹⁶ Methods to measure absolute pO₂ include polarographic oxygen electrodes and fluorescence quenching fiberoptic probes, as well as electron paramagnetic resonance oximetry and ¹⁹F relaxometry.⁹⁵ However, many of these approaches are highly invasive or cannot be applied to longitudinal studies of oxygen dynamics.

Figure 3 Anatomic images of PFC uptake in an HM-7 xenograft tumor at 9.4 T with a 1H/19F 10-mm surface coil.

¹⁹F MRI can be used to assess pO₂ in tissues quantitatively, which greatly increases the accuracy of tumor detection. As PFC has a high capacity of dissolving O₂, when a PFC emulsion injected intravenously reaches tumor tissues the O₂ rapidly interchanges between PFC and the surrounding tissue by free diffusion, until equilibrium is achieved.^25,97 Thus, the pO₂ of different tumor areas can be assessed by measuring the pO₂ of the PFC in the corresponding tissue.²² For given magnetic field and temperature, the dissolved O₂ has paramagnetic properties that affect the relaxation rate (R₁) of PFC in linear proportion to pO₂.⁹⁸ Thus, many studies use ¹⁹F MR relaxometry based on the linear relationship between the R₁ of ¹⁹F-spins in PFCs and pO₂ to noninvasively measure tissue oxygenation locally or globally.^99–101

The noninvasive measurement of pO₂ in tissues plays a key role in the treatment of tumors. Currently, the treatment efficacy of conventional radiotherapy and chemotherapy for malignant tumors is not satisfactory, largely because prevalent hypoxic areas in the tumor may cause resistance to these treatments. Thus, to some extent the noninvasive detection of pO₂ in tumor tissue can play a guiding role in cancer treatment. Furthermore, oxygen content in tumors may be improved, ultimately leading to a beneficial therapeutic effect. Specifically, as PFC has a high capacity to dissolve and carry oxygen to tumors, it can change local pO₂ and assist in treatment. In addition, ¹⁹F MRI can reveal changes in pO₂ in tumors before and after application of PFC, thereby allowing the monitoring of its therapeutic effects. Thus, the noninvasive quantification of tumor oxygenation not only provides unique insights into tumor biology and pathobiology, but it may also be important for developing new treatments¹⁰² and monitoring tumor response.¹⁰³ It is critical to develop dynamic methods for direct oxygen mapping.

Quantitative ¹⁹F MRI is the most extensively explored imaging method for in vivo mapping of tumor oxygenation due to the excellent oxygen-carrying capacities of PFCs and fast gas-exchange rate with surrounding tissues.⁹⁷ Hypoxia-responsive ¹⁹F MRI probes with improved redox properties and biocompatibility have been synthesized to detect hypoxia.¹⁰⁴ Furthermore, as mentioned above, the ¹⁹F NMR spin-lattice relaxation rates R₁ of PFC emulsions are highly sensitive to pO₂.¹⁰⁵ PFCs can be administered intravenously or directly by intratumoral injection of PFC droplets or emulsions. Finally, tumor hypoxia measured with PFC NPs strongly correlates with tumor size,^106–108 and generally larger tumors possess a lower baseline pO₂.⁸¹

The method of intravenous administration may affect the relative spatial distribution of PFC and produce the accumulation of PFC in other tissues, such as the liver and spleen. In addition, intravenous administration often results in lower tissue concentrations of PFC in areas of poor vascularization. SNR of these areas is too low to measure pO₂ accurately. This technique may bias pO₂ measurements toward regions close to the blood vessels and areas with higher perfusion and oxygenation, resulting in erroneous overestimation of tumor pO₂, which may be a major challenge for clinical translation (Figure 3).^109–111 Fast ¹⁹F MRI techniques, such as fluorocarbon relaxometry using echo planar imaging, have been developed for dynamic mapping of tumor pO₂ distributions within tumors, with 5–10% of tumor volume being observed with typical signal.^{112 19}F MR dynamic monitoring of tumor oxygenation with intratumoral-injected hexafluorobenzene is an important tool to characterize tumor acute hypoxia.^113,114 Kadayakkara et al successfully measured intracellular pO₂ ex vivo in labeled glioma cells using ¹⁹F MRI. This pO₂ measurement procedure only required 8 min and had a precision of 1–3 torr at 30–100 individual regions across a tumor. In addition, the authors showed that cellular oximetry may be used to monitor the efficacy of chemotherapy in CNS glioma.¹¹⁵

In summary, ¹⁹F MRI-based pO₂ mapping with PFCs has been successfully performed in animal models using clinically achievable field strengths (<7 T). The future translation of this technique to humans will significantly advance tumor-targeted noninvasive imaging and ultimately improve prognosis and the prediction of patient response to therapy.

Cell Tracking

Cell tracking permits the visualization and monitoring of cells labeled noninvasively ex vivo or in situ. Recently, ¹⁹F MRI has been utilized to monitor and quantify the in vivo biodistribution of immune cells labeled ex vivo with PFC NPs.^{60,70,116-119} For in vivo imaging, systematic intravenous injection of relatively large volumes of PFC NPs is performed. PFC NPs are engulfed by various subgroups of leukocytes, predominantly monocytes, macrophages, neutrophils, and dendritic cells (DCs), as part of their natural clearance from the body. ¹⁹F-labeled leukocytes, often resident in the spleen, are recruited by cytokines into inflamed areas and can be monitored via ¹⁹F MRI. This method has been used in different animal models to visualize myocardial infarction and myocarditis,^120,121 pneumonia,¹²² atherosclerosis,¹²³ arthritis,¹²⁴ and tumors infiltrated by macrophages.^125–127

Constantinides et al demonstrated the ability to in vivo track and detect intra-cardiac injections of PFCE-NP-labeled cardiac progenitor stem cells (CPCs) at 9.4 T PFCE-NP label uptake in CPCs are maximized for murine cardiac CPC ¹⁹F MRI by employing DNA transfection (FuGENE), which could be translatable to the clinic.¹²⁸ In addition, they further used Medium-chain length polyhydroxyalkanoates (MCL-PHAs)/polycaprolactone (PCL) blend scaffolds for controlled release of seeded CPCs in cardiac tissue engineering (CTE) applications. They found that PFCE-labeled CPCs ¹⁹F MRI signal and visibility could be improved in the double-layered scaffolds.¹²⁶ Ramos et al investigated the time course of inflammatory cell recruitment using PFPE and gadolinium-based elastin-specific magnetic resonance contrast agent (Gd-ESMA) in vivo in a murine model after post-myocardial infarction (MI) using a 3 T clinical scanner. In this study, they noninvasively assess and quantify cardiac inflammation extracellular matrix (ECM) remodeling of the myocardium at the molecular level.¹²⁹ Another study demonstrated the ability to use ¹⁹F-MRI cell tracking to detect, quantify and track human mesenchymal stem cells (hMSC) labeled with Cell Sense in vivo on 3D images at 9.4 T after grafting. In this study they showed strong linear between the number of labeled cells implanted and the real cell number by ¹⁹F-MRI.¹³⁰

Noninvasive monitoring of administered T cells labeled ex vivo could potentially allow the prediction of patient response to immunotherapy. Furthermore, as T cells can recognize tumor antigens and migrate to and infiltrate tumor tissue,¹³¹ they may also be used as a probe to detect tumor cells at metastatic sites. Gonzales et al labeled splenocytes and ovalbumin (OVA)-specific T cells with PFCs in vitro for ¹⁹F-MRS/MRI detection in liver, lung, and spleen in control and B16 OVA melanoma-bearing mice. The authors concluded that non-dividing ¹⁹F-labeled cells appear most promising for ¹⁹F MRS/MRI-based cell tracking.¹³² Clinical ¹⁹F MRI cell tracking using PFPE nanoparticle labeling was conducted in patients with colorectal adenocarcinoma to detect autologous immunotherapeutic mature dendritic cells.¹¹⁷ PFPE was incorporated into non-phagocytic cells without adjunctive cationic lipids or causing changes in cellular phenotype. Notably, only viable cells were labeled with PFPE. This is therefore a promising technique for detecting tumor cells in vivo and, importantly, for monitoring adopted cell transfer cancer therapies noninvasively. Furthermore, a recent study using ¹⁹F MRI to detect tumor-associated macrophages (TAM)⁴⁹ revealed that ¹⁹F-based cell tracking approaches represent TAM density more reliably and provide more information than other methods.¹²⁹ The commercially available PFC-based agents include Cell Sense and V-Sense (Celsense, Inc., Pittsburgh, USA) used for cell tracking in MRI. These types of labelling agents include fluorinated emulsions (CS-1000 and VS-1000H) and other fluorescently tagged formulations (CS-ATM DM Red, Green, NIR) for ex vivo and in situ cell labels.^133,134 A recent study showed that it is possible to effectively label hMSCs with cell sense without affecting cell viability, function or differentiation. The bSSFP sequence was used to detect and quantify the signal of Cell Sense-labeled hMSC cells in vitro and in intramuscular implantation.¹³⁵ Tennstaedt  et al also showed that transgenic hNSCs with stable expression of reporter genes Luciferase and GFP were further to be labeled with PFPE. This strategy provided a new multimodal imaging approach for in vivo application of transgenic hNSCs in deep brain implantation studies.⁵⁵ Neural stem cells (NSCs) have labelled with CS-1000 or CS green and luciferase expression were implanted in the striatum. This study investigated the viability of NSCs in stroke animals that underwent focal cerebral ischemia compared to healthy one using ¹⁹F MRI in combination with bioluminescence imaging.¹³⁶

Fink et al reported ¹⁹F MRI as a non-invasive imaging method capable of detecting and quantifying PFC-labeled DC migration at both 9.4 T and 3 T and suitable for therapeutic cell tracking in tumor-bearing mouse models. In addition, viability, phenotype, and function of more than 90% of DC labeled with PFC remained unchanged.¹³⁷ A detection limit of,10,000 cells for PFOB-labeled (bearing 17 fluorine atoms) cells with ¹⁹F MRS (11.7 T) was found in vitro. For in situ imaging at 1.5 T, the injection of 4 million PFCE-labeled stem cells could be detected a strong fluorine signal within 7 min. PFCE contains 20 equivalent fluorine atoms per molecule that allow a detection limit of approximately 2000 PFCE-labeled cells with ¹⁹F MRS and approximately 6000 PFCE-labeled cells/voxel in vitro with ¹⁹F MRI (11.7 T) within 7 min.¹³⁸ Boehmsturm et al found that human neural stem cells (hNSCs) can be labeled with a PFC marker as well as detected and measured the number of transplanted stem cells in vitro and in vivo after transplantation in the striatum of mouse brain. Related measurements suggested a detection limit of 1000 PFPE-labeled (containing more than 40 fluorine atoms per molecule) cells/voxel was found in vitro and 10,000 cells/voxel to generate significant SNR in vivo at 11.7 T.¹¹⁸

Isoflurane (ISO) is a fluorinated anesthetic commonly used in animal models owing to its minimal cardio-depressive effects.¹³⁹ Constantinides et al reported that isoflurane exhibited two resonances of ¹⁹F atoms correspond to the -CF₃ and -OCHF₂ moieties with chemical shifts of −4 and −10.3 ppm with respect to the trifluoroacetic acid (TFA) resonance at 0 ppm in the NMR spectrum. These peaks are close to the resonances of PFCs with chemical shift range of −50–86 ppm relative to TFA due to potential spectral overlaps. PFCE-labeled cells exhibited a single spectral peak at −16.25 ppm with respect to TFA in vitro that did not overlap with the ISO resonances. ISO effects on PFCE labels are minimal but may have more prominent effects on PFPE or PFOB.^140,141 A study investigated that an efficient imaging technique will also minimize any potential ¹⁹F signal from the use of inhalational isoflurane anesthesia by using helpful image acquisition parameters. They use narrow 1.5 kHz sinc excitation pulse and shorter TE (eg, TE 1.8 ms), which is simultaneously beneficial for detecting signals from both cells and isoflurane.¹³⁰ Thus, the use of ISO still has a primary choice for ¹H and multinuclear imaging studies.

The advantage of ¹⁹F MRI in ex vivo-labeled cell tracking is the complete absence of background signal due to the negligible amount of ¹⁹F in the body. Finally, ¹⁹F MRI has strong potential as an accurate quantification method of local cell numbers.

Drug Delivery and Therapy Efficacy Monitoring

The high ¹⁹F signal of PFCs allows the noninvasive quantification of ligand-bound PFC NPs, which in turn enables clinicians to confirm tissue drug concentrations during targeted therapy. Nanoparticles can be engineered to carry highly potent drugs and deliver them to specific cell populations displaying biosignatures of particular diseases. For instance, Rapoport et al recently reported novel drug-loaded PFC emulsions stabilized by biodegradable amphiphilic block copolymers.^142–145 PFC emulsions can deliver lipophilic therapeutic agents to solid tumors while simultaneously allowing the monitoring of their in vivo biodistribution. Furthermore, anti-angiogenic agents have played a critical role in the treatment of various types of tumor, including solid tumors.^146,147 α_vβ₃-integrin-targeted fumagillin-loaded nanoparticles suppressed neovasculature and inhibited tumor growth in Vx2 adenocarcinoma models without causing organ toxicity or neurocognitive dysfunction.¹⁴⁸ Notably, therapeutic efficacy for these targeted nanoparticles occurred at systemic doses about 1000-fold lower than those used in previous animal studies, and 60-fold lower than doses tested clinically for related anti-angiogenic compounds (TNP-470).

¹⁹F MRI has been used as a platform for guiding high-intensity focused ultrasound (HIFU) tumor ablation by quantitatively tracking the accumulation of PFC nanoemulsions (PFCNE).¹⁴⁹ PFCNE accumulation in the tumor periphery was clearly visible and quantifiable, and was confirmed by fluorescence imaging. Encapsulated PFCs can also be used for image-guided HIFU ablation and indeed, it was shown that PFOB can increase HIFU effectiveness.¹⁵⁰ Docetaxel-loaded PFCE nanodroplets (Doc-nd) developed by Gupta et al favored passive accumulation into most tumors due to their small particle size, thereby potentially increasing localized drug concentration.¹⁵¹ A high encapsulation efficiency of 93.70% was obtained for Doc-nd, and Docetaxel was released in a three-stage release kinetic pattern, with an initial release of 30% within an hour, followed by a 50% release within 12 hours and a 85% release after three days. This study suggests that Doc-nd combined with MR-guided focused ultrasound has great potential for treating prostate cancer.

Immune cell therapy has become an effective method to treat cancer, and NK cells are among the immune cell types used in this treatment.¹⁵² Recently, Bouchlaka et al had labelled human NK cells with PFC and injected them into tumors.¹⁵³ Strikingly, the PFC remained in the tumor up to 8 days after injections, as detected by ¹⁹F MRI. Another study on the application of poly(D,L-lactic-co-glycolic acid) (PLGA) entrapping PFCE and indocyanine green (ICG) focused on ¹⁹F MRI, fluorescence imaging and photoacoustic imaging (PAI), which could be used for detection of metastasis in melanoma patients.¹⁵⁴ This work showed the potential of labeled primary human dendritic cells for cell imaging and lymph node detection with PAI and ¹⁹F MRI.¹⁵⁵ Thus, ¹⁹F MRI is an effective method of monitoring immune cell therapy.

Stimuli-Responsive ¹⁹F MRI

Recently, the development of smart stimuli-responsive nanoparticles characterized by the off/on regulation of ¹⁹F MRI signals has attracted much attention. These ¹⁹F MRI probes are suitable for noninvasive visualization of enzymatic activity, redox-potential difference, and pH.^104,156,157 PFC NPs have been used to monitor specific biological events in living animals with an off/on ¹⁹F MRI switch for detecting enzymatic activity based on the paramagnetic relaxation enhancement effect (PRE) for spin-spin relaxation (T₂) of ¹⁹F MRI signal without endogenous background signals.¹⁵⁸ Because of the large electron spin quantum number, Gd³⁺ and Mn²⁺ have a very strong PRE effect on the MRI signal of ¹⁹F. The shielded ¹⁹F MRI signal of PFC by the adjacent Gd³⁺ was triggered to turn on because of the cleavage of enzyme (Figure 4).¹⁵⁹ Guo et al reported PFCE NP for in vivo turn-on ¹⁹F MRI sensing the activity of phospholipase A2 (PLA2) with low background (Figure 5).¹⁶⁰ In addition, this off/on ¹⁹F MRI switching strategy broadly applied to detect the activity of various enzymes, such as caspase-1, caspase-3/-7, and matrix metalloproteinases.^29,161,162

Figure 4 Schematic illustration of intracellular GSH-controlled self-assembly followed by Lgmn-controlled disassembly of 1-NPs, showing respective “off” and “on” 19F NMR signals for Lgmn detection, and Lgmn-controlled self-assembly of 2-NPs results in 19F NMR signals “off” inside cells. Reprinted with permission from Yuan Y, Ge S, Sun H, et al. Intracellular self-assembly and disassemblyof 19F nanoparticles confer respective “off” and “on” 19FNMR/MRI signals for legumain activity detection in zebrafish. ACSNano. 2015;9(5):5117–5124. Copyright © 2015, American Chemical Society.159

Figure 5 In vivo 1H, 19F MRI, and merged images of 1H and 19F MRI for (A) a tumor-bearing mouse and (B) a healthy mouse. Adapted from Guo C, Zhang Y, Li Y et al 19F MRI nanoprobes for the turn-on detection of phospholipase A2 with a low background. Anal. Chem. 2019;91(13):8147–8153. Copyright © 2019 American Chemical Society.160

Reducing microenvironment plays an important role in biological processes and abnormal redox reactions. Nakamura et al reported an activatable reduction-responsive PFC-encapsulated NP, FLAME-SS-Gd³⁺ (FSG), that can be used for in vivo ¹⁹F MRI.¹⁶³ In the presence of the disulfide of FSG, Gd³⁺ was removed from the reduced FSG surface and the subsequent ¹⁹F NMR/MRI signal intensity of the encapsulated PFCE would be increased. The highly sensitive detection and visualization of reducing microenvironment in vivo could provide effective information about biological functions for monitoring the process of disease and evaluating the effect of therapy.^164–166

Solid tumors have a slightly lower extracellular pH (pHe) than the normal tissue environment due to fast growing tumor cells, which causes increased glycolysis and accumulation of lactic acid as an intrinsic feature of the tumor phenotype.^167,168 Acidic environments with low pH have been used as a trigger for environment-responsive tumor imaging. These highly sensitive pH-responsive ¹⁹F probes are a potential smart platform for precise and specific detection of tumors.^169,170 Zhang et al developed a novel pH responsive ¹⁹F probes, Mn-LDH@PFPE NP, which is activated specifically within the acidic tumor environment.¹⁷¹ The ¹⁹F MRI signals from NPs are at physiological pH 7.4, but activated at extracellular pH 6.5. In vivo experiments reveal that an intense ¹⁹F MR signal can be detected in the tumor after injection of NPs. pH activated NPs are a potential smart ¹⁹F MRI agent for recognizing subtle pH differences.^172,173

Perfluorocarbons-Based ¹⁹F MRI in Non-Oncological Applications

Molecular Imaging of Thrombus

The use of ¹⁹F MRI for thrombus diagnosis dates back more than a decade. Thrombus is rich in molecular epitopes for targeting, in particular fibrin, thrombin, and in some instances, platelets. More recently, noninvasive detection of deep venous thrombi and subsequent pulmonary thromboembolism using ¹⁹F MRI and α2-antiplasmin peptide (α2AP)–targeted PFC nanoemulsions were reported.¹⁷⁴ In this study, developing thrombi with a diameter <0.8 mm could be visualized unequivocally in vivo as hot spots in the murine inferior vena cava, via the simultaneous acquisition of anatomic matching ¹H and ¹⁹F MR images at 9.4 T, with excellent signal-to-noise and contrast-to-noise ratios (71±22 and 17±5, respectively). Furthermore, α2AP-PFCs were successfully utilized in the diagnosis of experimentally induced pulmonary thromboembolism.

¹⁹F Angiogenesis Imaging

Angiogenesis is a critical process in some tissues, including the endometrium, bone growth plates, and wound healing, but also in pathologies such as rheumatoid arthritis, atherosclerosis, and asthma. In addition, abnormal angiogenesis is one of the hallmarks of cancer. High-resolution ¹⁹F imaging of angiogenesis was first used to detect and quantify neovasculature in a rabbit model of aortic valve disease with α_νβ₃-PFC nanoparticles.¹⁷⁵ Notably, the valves of rabbits treated with targeted PFC NPs had 220% more fluorine signal than those of rabbits treated with untargeted PFC NPs (p < 0.001). Pretreatment of the rabbits with targeted oil-based nonsignaling nanoparticles reduced the fluorine signal by 42% due to competitive inhibition. Finally, integrin-targeted PFC NPs specifically detected early angiogenesis in sclerotic aortic valves of cholesterol fed rabbits.

¹⁹F MR Imaging in the Lung

Whereas ¹⁹F MRI with PFC NPs has been widely used to study a variety of diseases, the application of ¹H MRI in the lung is limited, mainly due to the intrinsically low proton density, respiratory motion, and magnetic susceptibility artifacts of the air-tissue interfaces. Early neovascular expansion in the lungs is very difficult to assess noninvasively in patients, in particular quantitatively. However, the addition of an exogenous contrast agent makes the visualization of pulmonary structure and function possible. PFC materials have the distinct advantages of high ¹⁹F MRI sensitivity,⁶⁹ excellent oxygen-carrying capacities, lower cost, and the lack of a ¹⁹F background signal within the body. Numerous studies have successfully used ¹⁹F MR with PFC materials to image morphology and function in animal and human lungs.^176–178 For instance, Schmieder et al conducted simultaneous ¹⁹F/¹H MR molecular imaging with α_vβ₃-targeted PFC NPs to quantitatively assess neovascular expansion of the bronchial arteries following pulmonary artery ligation.¹⁷⁸ The authors demonstrated that ¹⁹F/¹H MR molecular imaging with α_vβ₃-targeted PFC NPs provides a means to assess the extent of systemic neovascularization in the lung. ¹⁹F MRI may also be used to quantitate pulmonary inflammation by tracking infiltrating PFC-loaded monocytes.¹²² However, a practical concern/limitation relates to the application of fluorinated anesthesia gases in animal MRI experiments. Isoflurane (CF₃CH₂ClOCHF₂) potentially affects signal from PFC-labeled cells due to accumulation predominantly in subcutaneous fat regions and potentially from within the lungs after a period of gaseous anesthesia. However, to avoid possible false positive ¹⁹F signals from isoflurane, an option is to deliver injectable liquid anesthesia via mechanical pump and intraperitoneal catheter rather than inhaled anesthetics such as isoflurane.¹⁷⁹ Another option is the use of injectable anesthetics such as sodium pentobarbital, ketamine, xylazine, and thiopental in order to avoid the ¹⁹F signals due to isoflurane for preclinical cell tracking and ¹⁹F lung imaging.⁷⁰

Hyperpolarized (HP) helium-3 (³He) and xenon-129 (¹²⁹Xe) MRI of the lungs are a noninvasive imaging technique capable of measuring lung ventilation, gas exchange, and lung microstructure in both animals and humans.¹⁸⁰ HP gas MR also provides functional information about respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis.¹⁸¹ Although ¹²⁹Xe is cheaper than ³He, both ³He and ¹²⁹Xe needs to prepare and process the gases. Thus, an attractive and economical alternative to HP gas MRI is functional MR imaging using inert fluorinated gases, such as sulfur hexafluoride (SF₆), hexafluoroethane (C₂F₆), and perfluoropropane (PFP) (C₃F₈), which are nontoxic and abundant.¹⁸² Couch et al studied the feasibility of ¹⁹F 3D UTE for lung imaging of healthy volunteer with inert mixture of 79% PFP and 21% O₂.¹⁸³ Inert fluorinated gas MRI is a feasible pulmonary imaging technique with the potential of clinical transformation.

In ¹⁹F MR molecular imaging, nontargeted PFC agents have been designed for blood pool imaging and perfusion,^74,184 cellular labeling and tracking^7,185 cellular and tissue uptake imaging in inflammation, allograft rejection monitoring, among others.^59,186

Conclusion

Molecular imaging is a rapidly developing method that allows early tumor detection noninvasively and with high specificity. PFC NPs have been widely used in ¹⁹F MR molecular imaging because of their dense fluorine content and relatively bio-inert properties. In this article, we focused on the applications of ¹⁹F MRI in tumor molecular imaging. In addition to ligand-targeted imaging and tumor oxygenation quantification, PFC NPs have been explored for cell tracking, stimuli-responsive imaging and therapeutic drug delivery, as well as for the monitoring of therapy efficacy. We believe that ¹⁹F MRI will be widely used in research and clinical applications in the near future.