Fluorine (¹⁹F) MRS and MRI in biomedicine

Abstract

Shortly after the introduction of ¹H MRI, fluorinated molecules were tested as MR-detectable tracers or contrast agents. Many fluorinated compounds, which are nontoxic and chemically inert, are now being used in a broad range of biomedical applications, including anesthetics, chemotherapeutic agents, and molecules with high oxygen solubility for respiration and blood substitution. These compounds can be monitored by fluorine (¹⁹F) MRI and/or MRS, providing a noninvasive means to interrogate associated functions in biological systems. As a result of the lack of endogenous fluorine in living organisms, ¹⁹F MRI of ‘hotspots’ of targeted fluorinated contrast agents has recently opened up new research avenues in molecular and cellular imaging. This includes the specific targeting and imaging of cellular surface epitopes, as well as MRI cell tracking of endogenous macrophages, injected immune cells and stem cell transplants.

INTRODUCTION

The ¹⁹F nucleus has a 100% natural abundance and resonates at a resonant frequency that is 94% of that of ¹H. Its NMR sensitivity is 83% of that of ¹H (with constant noise), so that its MRI signal-to-noise ratio (SNR) is about 89% of ¹H per nucleus, assuming sample-dominant noise (i.e. the noise increases linearly with frequency). There is negligible endogenous ¹⁹F MRI signal from the body, as the physiological concentrations of detectable mobile fluorine are below the detection limit (usually less than 10⁻³ μmol/g wet tissue weight). Fluorine exists at higher concentrations in the bone matrix and teeth, but, being immobilized, exhibits a very short spin–spin relaxation time (T₂) that is not visible to conventional MRI methods. This lack of background signal provides ¹⁹F MRI with a potentially extremely high contrast-to-noise ratio and specificity, if a fluorinated compound can be introduced as a contrast agent.

However, for ¹⁹F MRI to produce an image quality similar to that of ¹H MRI, whose signal derives from nearly two-thirds of all nuclei present in the body, the agent requires a very high density of ¹⁹F nuclei on the molecule in addition to a high tissue concentration. To achieve the former, perfluorination offers the attraction of providing a comparable density of ¹⁹F nuclei when all ¹H nuclei on a hydrocarbon chain are replaced.

Perfluorocarbons (PFCs) are molecules of similar structure to common organic compounds (e.g. alkanes), except that all of the hydrogen atoms are replaced by fluorine. These agents have a number of interesting properties that are well suited for medical applications. In addition, fluorine is the most electronegative element in the Periodic Table, and therefore the synthetic introduction of fluorine into contrast agents can produce dramatic electronic perturbations and modify sterical tertial structures.

HISTORICAL PERSPECTIVE

Some of the current potential applications of ¹⁹F imaging and spectroscopy for obtaining functional information in living systems were suggested in the 1970s (1), around the time of the first human ¹H MRI applications. The first ¹⁹F images of NaF, and a PFC, perfluorotributylamine (PFTA), in phantoms are shown in Fig. 1. ¹⁹F MRI of biocompatible fluorinated compounds thus represents one of the earliest examples of MRI contrast agents (2). Around the same time, fluorinated drugs appeared on the market and the properties of PFCs were being investigated. Several PFCs were developed during World War II as part of the Manhattan Project to isolate the isomers of uranium. One of their most important biological properties, their high oxygen affinity, was recognized in the 1960s, when mice and cats were shown to survive complete immersion in oxygenated PFCs, followed by long-term survival without apparent ill-effects (3). However, it was not until the 1980s that the chemical inertness of PFCs and, more specifically, their inability to be metabolized were fully documented. One problem with regard to the biological use of many PFC species is that they are insoluble in water. This is routinely addressed for a variety of medical applications by preparing PFCs as water emulsions (4).

Figure 1.

The first ¹⁹F images from 1977 (1). (a) ¹⁹F MRI of three tubes of 1.4 M NaF with resolution of about 0.13 T0.13 T3 mm³. (b, c) ¹⁹F MRI of a perfluorocarbon (PFC), perfluorotributylamine, in a star-shaped ‘Union Jack’ phantom made from 2-mm (diagonal) and 6-mm tubes. The resolution is halved to about 0.6 T0.6 mm². All images were acquired in about 400 s at 0.7 T using a steady-state free-precession (SSFP) MRI sequence (TR of several milliseconds). In (c), the image bandwidth per point is reduced, resulting in signal-to-noise ratio (SNR) loss and a ‘ghost’ image or ‘chemical shift artifact’ from the two chemically shifted moieties of this PFC, which are about 6 ppm apart. The two peaks are seen directly when the gradient in the vertical direction is switched off in the inset (d). Adapted, with permission, from Holland et al. (1).

The ¹⁹F MRI properties of many fluorinated compounds in pharmaceutical, anesthesiological and oncological applications have been discussed in other excellent reviews and book chapters (5–13). In this review, special emphasis is placed on emerging applications, including the use of PFCs for molecular imaging and cell tracking, as well as for studying lung function.

GENERAL PROPERTIES OF PFCs

As noted above, liquid PFCs have a low water solubility [e.g. perfluoro-octyl bromide (PFOB) has a maximal water solubility of 10⁻⁹ M], which leads to slow diffusion and a long tenancy at the target site of the compound in its natural form. Although PFCs are lipophobic, because the degree of lipophobicity is commonly less than the hydrophobicity, PFCs tend to partition into the lipid component of cellular membranes and, in some cases, affect the cellular response to certain stimulants and stressors. The depth of penetration and the penetration rate can be modulated as a function of the particle size and the lipid solubility of emulsions prepared with different PFCs. PFCs are also characterized by a very low surface tension, which make them attractive for certain applications (e.g. intra-alveolar). An effective fluidity (viscosity) and a positive coefficient of spreading allow these molecules to spread evenly over a surface. For example, they could spread homogeneously into the entire lung, and potentially form a barrier around target cells to seal and prevent further growth of a malignant disorder. The water–PFC interfacial tension is around 60 mN/m (14), which explains the difficulty in preparing stable emulsions. The stability of the emulsion is directly proportional to the water solubility. PFCs also have a high density and compressibility, which provide a natural cushion between opposing surfaces to reduce the contact force, as well as high volatility and low polarizability. The combination of these properties with their low water solubility makes them ideally suited for use as injectable emulsions of PFC-containing gas, for example, in contrast agents for ultrasound imaging, or for the intravenous delivery of hyperpolarized gases in perfusion and functional MRI studies (15).

The main applications for PFCs as exogenous contrast agents for ¹⁹F NMR can be divided into two categories. First, PFCs can be detected and quantified directly by ¹⁹F NMR. Second, PFCs can be used for the indirect observation of molecular level changes in biologically important molecules or environmental factors as a result of their influence on fluorine nuclei. The first group of applications, based on the direct detection of fluorinated molecules, includes functional lung imaging (using gases such as SF₆, C₂F₆, CF₄, C₃F₈ and liquid PFCs), cell tracking using PFC emulsions and in vivo monitoring of fluorinated drugs and their metabolites. An example is the use of ¹⁹F MRS techniques for the detection of 5-fluorouracil (5-FU), a chemotherapeutic agent (16). As a result of the low tissue concentration of 5-FU (in the μmol/g wet weight range) and fluorine-containing pharmaceuticals used at clinical doses, the sensitivity of ¹⁹F MRS and MRI depends primarily on the number of fluorine atoms present in the compound and the dose, in addition to the conventional factors that determine SNR, such as the magnetic field strength, detector design, etc.

The second group of applications includes examples in which the fluorine molecules respond to a specific parameter, such as the presence of ligands. Fluorinated compounds are capable of detecting changes in oxygen, H⁺ (pH), Na⁺, Ca²⁺ and Mg²⁺concentrations in biological tissues, and may therefore provide proxy measurements of these (17). Of particular interest are the paramagnetic relaxation effects imparted by oxygen on ¹⁹F nuclei, which cause changes in the spin–lattice relaxation rates (1/T₁), and can alter the chemical shift of one or more of the fluorine moieties. Changes associated with temperature and blood flow in the microenvironment may also affect the ¹⁹F signals. In addition to the limits imposed by the low in vivo ¹⁹F concentration of the agent being used, the utility of the ¹⁹F agent as a proxy largely depends on the magnitude and sensitivity of the changes that are elicited.

Also included in this second group is the use of fluorinated emulsions in ¹H MRI applications. One type of PFC, PFOB, has been shown to be an effective negative contrast agent for delineating the bowel and improving bowel wall visualization (18,19). The bowel lumina appear homogeneously black on T₁-and T₂-weighted MR images because of the insolubility of PFOB in water and intestinal secretions. Paramagnetic nanoparticles that combine gadolinium diethylenetriaminepentaacetate (Gd-DTPA) and PFC have also been developed for molecular imaging of fibrin (20), and as a platform for delivering a very large number of Gd³⁺ ions to targeted binding sites (21).

PREPARATION AND CHEMICAL STABILITY OF PFCs

The PFCs used in biomedical applications are chemically inert. They are derived synthetically, consist primarily of carbon and fluorine atoms, and are typically clear, colorless liquids that are insoluble in water. They must therefore be emulsified for clinically relevant applications involving intravenous injection, intraperitoneal injection, tissue intraparenchymal injection or administration in oxygen-permeable biodegradable and biocompatible capsules. The process is analogous to the routine preparation of lipid emulsions for parenteral nutrition. Despite the intrinsically low solubility, diffusivity, density and interfacial surface tension of PFCs, it is possible to generate stable nanoparticles of these compounds using a high-pressure micro-emulsifier. The latter deagglomerates and disperses submicrometer PFC particles uniformly in the fluid. This results in a smaller particle size compared with unpressurized emulsification, which, in turn, permits higher PFC concentrations – 40% and higher – to be achieved. The nanoparticles obtained with this procedure typically have a very small size (~ 200 nm). However, the stability of commercially available PFC preparations varies greatly, and there is a direct relationship between PFC stability and the clearance time from the body.

From a design standpoint, most clinical applications require stable preparations that are rapidly cleared from the body. For this reason, PFCs are commonly combined in commercial products to optimize the stability and clearance profile. For example, perfluorodecalin (PFDC) is rapidly cleared from the body, but forms emulsions that have poor stability. However, perfluorotripropylamine (PFTPA) forms stable emulsions that have a long retention time. By combining these two agents, emulsions such as Fluosol^® achieve both a clinically acceptable stability and clearance profile. Nevertheless, Fluosol^® remains stable for only 6 h after PFDC and PFTPA are mixed. Consequently, PFDC and PFTPA emulsions are stored frozen in separate solutions, and the solutions are thawed and mixed immediately prior to use. For practicality and other reasons, this version of the product was replaced in 1994 by a new PFDC/PFTPA preparation, Oxygent^®, that has proved to be much more stable, and does not require frozen storage.

As noted above, the most useful and versatile formulations are a compromise between body clearance, emulsion stability and, for MRI/MRS applications, the maximum number of chemically equivalent fluorine atoms. Perfluoropolyethers (PFPEs) (e.g. containing 12, 15 or 18 crown ethers) are excellent ¹⁹F MRI contrast agents as they provide a single sharp resonance, eliminating any chemical shift artifact, maximizing the SNR and allowing an unambiguous identification of the PFC. Nanoparticle preparations of some of these agents are thermodynamically stable (they do not coalesce) and can be prepared using several different types of emulsifying agent that form a film around the dispersed globules of PFC. Typical emulsifying agents are surface-active agents, adsorbing at oil–water interfaces to form monomolecular films that reduce the interfacial surface tension. A large variety of agents have been used to improve the stability (lecithin is one of the most commonly used) and to increase the effective encapsulation of PFCs. In practice, combinations of emulsifiers are commonly used, rather than a single agent (e.g. safflower oil and lecithin, cholesterol and lecithin, etc.) (22,23). This enables the modification and optimization of the balance between the hydrophilic and lipophilic parts of the emulsifier or mixture of emulsifiers.

The addition of other agents to a PFC emulsion can improve its performance from an MRI/MRS standpoint, but can also affect the stability of the preparation. For example, adding a paramagnetic element that reduces the ¹⁹F NMR T₁ values improves the signal intensity and SNR efficiency, by enabling the acquisition of a larger number of scans with a shorter pulse sequence repetition period (TR) in the same scan time. Adding fluorescent lipids, cationic transfection reagents (lipofectamine) or targeted ligands to PFCs provides a means to detect the agents by fluorescence microscopy, to enhance cellular labeling or to perform molecular imaging, respectively (21,23,24). A number of drugs, including antibacterial agents, vasoactive bronchodilators, mucolytic agents, glucocorticoids, antineoplastic agents and DNA, have also been incorporated into PFC emulsions without reducing their stability (25). These drugs offer a lot of ‘bang for the buck’ as the PFC phase can contain a high payload of hydrophobic drugs.

GAS SOLUBILITY OF PFCs

As mentioned earlier, emulsions of these molecules can dissolve significant quantities of relatively small molecules that have a low cohesivity, i.e. gases. Emulsified PFCs can transport and deliver oxygen, CO₂, N₂, NO, etc., with a solubility in the PFC that is independent of temperature. Unlike hemoglobin, which binds O₂ through a covalent bond to its iron atoms, oxygen is not bound but dissolved by the PFC carrier. The oxygen-carrying capacity of an emulsified PFC, such as PFDC/PFTPA, is around three times that of blood at 25°C, and the CO₂-carrying capacity is approximately four times greater than that for oxygen.

The dissolved oxygen has paramagnetic properties that affect the relaxation rate, R₁ = 1/T₁, of fluorine nuclei in linear proportion to the dissolved oxygen concentration, although the temperature and magnetic field also play a role (17). This allows the MRI monitoring of partial pressure of O₂ (pO₂) effects, including the possibility of using ¹⁹F as a proxy for determining the tissue oxygenation state, or even for generating a proxy image of the oxygen distribution in an organ. When perfluorinated compounds are used as contrast media in these applications, their distribution reflects the region of blood flow and tissue perfusion, providing selective imaging of influx or efflux analogous to that of a radiographic tracer. The selective imaging of blood flow and perfusion has potential applications for the identification of ischemic and/or nonperfused necrotic tissue. The high solubility of other inert gases, such as xenon (¹²⁹Xe), in PFC emulsions enables its use as a vehicle for the intravenous delivery of hyperpolarized gas for high-SNR, high-resolution imaging of the perfused tissue (15).

The high gas solubility in PFC emulsions has been used for other applications, such as an in vivo nitrous oxide (NO) sink (26). Certain PFC formulations have been Food and Drug Administration (FDA)-approved for human use in media that support microbubbles that reflect sound waves and serve as contrast agents for ultrasound imaging. These are currently in use for the diagnosis of cardiac wall motion abnormalities. The dissolved gas bubbles are small enough to pass through the capillary beds, and are not destroyed when injected into the circulation, as usually happens with nonemulsified air bubbles.

¹⁹F MRS OF PFCs

Most NMR applications in MRI and MRS use the ¹H signal from endogenous tissue water and mobile hydrocarbons, but other nuclei have also been employed for MRI. ¹⁹F MRI enjoys the advantages of the nucleus’ high gyromagnetic ratio (γ = 40.05 MHz/T), spin 1/2, 100% natural isotopic abundance and the fact that it is not a particularly rare (or expensive) element. The similarity of ¹⁹F’s γ to that of ¹H is another advantage, to the extent that measurements can often be performed with the same MRI scanner as used for conventional ¹H MRI, with the same receiver electronics but retuned radiofrequency coils. Fluorine also has a similar van de Waals’ radius to hydrogen (1.2 vs 1.35 A ^°). When a single hydrogen atom is replaced with fluorine, the change typically does not cause significant conformational changes, although fluorination usually increases the lipophilicity and thus enhances the bioavailability of new drugs. However, as noted above, the substitution of a large number of fluorine atoms in a specifically designed molecule influences the structure and function of this new molecule. Fortunately, for NMR, PFCs have magnetic susceptibilities comparable with that of water.

For ¹⁹F MRS, the most challenging aspect is quantification, for both technical and biological reasons (the concentrations of some drugs are prone to underestimation because some, such as fluoxetine, are highly bound to plasma proteins, and thus have very short T₂ values and are ‘MRS-invisible’). Most of the in vivo MRS data have been generated with surface coils, which have associated with them the problems of a rapid loss in SNR with distance from the surface coil, and nonuniform excitation fields, both of which hamper quantification. Some common methods that facilitate quantification are the use of adiabatic excitation pulses, coil sensitivity mapping techniques and the use of concentration references. Traditional decoupling and nuclear Overhauser enhancements have been applied for quantitative carbon (¹³C) and phosphorus (³¹P) MRS and can also be used to improve ¹⁹F SNR (27), although the differences between the gyromagnetic ratios of ¹H and ¹⁹F are small. All MRS methods are sensitive to local magnetic field variations because of tissue magnetic susceptibility. Magnetization transfer methods may also be applied to investigate the fraction of fluorinated compounds that are bound. The detection limits depend on the particular application. For in vivo studies, applications requiring high spatial localization of ¹⁹F signals from low-concentration fluoro compounds may not be feasible in practical or tolerable scan times.

¹⁹F MRS OF DRUG METABOLISM

¹⁹F MRS provides a highly specific tool for the investigation of drugs and their metabolic byproducts that contain fluorine atoms, which is also potentially suitable for quantification. The most commonly used drugs in ¹⁹F NMR are listed in Table 1. Their relative ¹⁹F SNRs, when present at a tissue concentration of 1 μmol/g wet weight, are also listed.

Table 1.

List of ¹⁹F drugs and therapeutic agents

Agent	Commercial name or acronym	Chemical formula	Modality	Application	Physical state	Relative SNRa (reference)
5,5′-Difluoro-1,2-bis (o-amino-phenoxy) ethane-N,N,N′,N′-tetraacetic acid	FBAPTA	C22H22F2N2O10	MRS	Calcium chelator	Solution	2.1 × 10−5 (46)
5-Fluorouracil	Efudex®	C4H3FN2O2	MRI, MRS	Antineoplastic	Saline solution	1.0 × 10−5 (8,12,27, 30,34,36–38,42,105)
Flurbiprofen	Adfeed, Ansaid	C15H13FO2	MRS	Anti-inflammatory	Gel, oral tablets	1.0 × 10−5 (106)
Fluorodeoxyglucose	FDG	C6H11FO5	MRS	Inhibitor	Solution	1.0 × 10−5 (17) b
Deoxyfluouridine (pro-drug)	Capecitabine (Xeloda®)	C15H22FN3O6	MRS	Prodrugs, antineoplastic	Oral tablets, injectable	1.0 × 10−5 (105,107)
Fluoxetine hydrochloride	Prozac®	C17H18F3NO, HCl	MRS	Antidepressant	Capsule, liquid solutions	3.1 × 10−5 (108)
Fluvoxamine	Dumirox	C15H21F3N2O2	MRS	Antidepressant	Oral tablet	3.1 × 10−5 (109,110)
Flurazepam	Dalmane, Felison	C21H23ClFN3O	MRS	Anti-anxiolytic	Capsule	1.0 × 10−5 (111)
Fluoexetine	Prozac®, Sarafem®	C17H18F3NO	MRS	Antidepressant	Capsule, syrup	3.1 × 10−5 (112,113)
Floxuridine	Fudr®	C9H11FN2O5	MRS	Antineoplastic	Injectable	1.0 × 10−5 (107)
Gefinitib	Iressa®	C22H24ClFN4O3	MRS	Antineoplastic	Oral tablet	1.0 × 10−5 (114)

^a19F signal-to-noise ratio (SNR) of the fluorinated agent at a concentration of 1 μmol/g wet tissue weight relative to the ¹H signal detected from an equivalent tissue volume with a detector coil of the same geometry. The calculation assumes a tissue water content of 76.5% (± 0.37 SD), which is an average of the water contents of brain, skeletal muscle and liver tissues (115), and sample-dominant noise resulting in a linear-dependent SNR with field strength.

^bAnd references therein.

The study of such drugs by ¹⁹F MRI/MRS tends to focus on their chemical structure, anabolism, catabolism, distribution and pharmacokinetics in vivo and in excised tissues. As an example, ¹⁹F MRS has been used widely in pharmacokinetic studies of the anticancer drug 5-FU. 5-FU was proposed as a prototypical antineoplastic agent (28), with the first MRS work using this drug conducted in the late 1970s (29) and early 1980s (30). Since then, numerous studies in both animal models and in humans, and in different tissues and biological fluids, including plasma and urine, have been presented (31). These studies have facilitated an understanding of the metabolic aspects and cytotoxic activity of this drug.

As an anticancer agent, 5-FU has been applied in concomitant radiotherapy and chemotherapy of different neoplastic diseases, particularly for neoplasms of the colorectal system, the head and neck, the trunk and some breast cancers (11,12,31–37). As a result of the intrinsic toxicity of 5-FU, different pro-drugs (a drug in its nonactive form) of the molecule have been designed to pass intact through the gastrointestinal tract, ultimately localizing and selectively converting to 5-FU in the malignant tissue, based on the higher activity of thymidine phosphorylase (38). Capecitabine is one such protodrug designed in an oral formulation to provide higher accumulation of 5-FU in the tumor, whilst reducing the exposure of healthy tissues to 5-FU.

¹⁹F MRS OF EXTRACELLULAR pH AND CATIONS IN CELLS AND TISSUES

¹⁹F MRS is also useful for observing biological processes in complex systems, such as the maintenance of intracellular pH in different cell lines (39), and intracellular free calcium and magnesium levels via fluorinated chelates. A good exogenous pH indicator must have favorable pharmacokinetics, i.e. an ionizable group with a pK value in the physiological range, good sensitivity and specificity, low toxicity, efficient cell penetration, fast exchange between acid and base forms, but slow exchange across cell membranes, and a large chemical shift range (10,40–43). The intracellular uptake and concentration must be high enough to provide adequate SNR from just the intracellular space. A number of pH indicators have now been investigated (42).

The intracellular concentration of different cations can also be obtained from the change in the ¹⁹F NMR spectrum of an indicator cation to which it is bound (44). Ca²⁺ plays an important role as a second messenger in living cells. ¹⁹F-based methods have been proposed for the determination of cytosolic calcium in cells and tissues. One of the more exciting approaches for the detection of this cellular cation is the use of 1,2-bis(o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (45). Here, the ¹⁹F-NMR Ca²⁺ indicator is derived from its symmetrically 5,5-substituted difluoro-derivative (FBAPTA) (46), which exhibits a chemical shift response on binding calcium. One of the critical issues for intracellular interrogation of any reporter molecule is the loading of the reporter molecule into cells. As tetracarboxylate does not penetrate the cell, a lipophilic agent, such as acetoxymethyl, is used. Other ¹⁹F-bearing ligands have been proposed for ions such as Na⁺, Mg²⁺, Zn²⁺. Pb²⁺, etc., as recently reviewed in ref. (17).

FUNCTIONAL LUNG 19F MRI USING GASES AND VOLATILE COMPOUNDS

¹⁹F MRI of the lungs was reported in the literature in the early 1980s (47). Recent interest may have been rekindled by new applications involving hyperpolarized noble gases. The possibility of imaging ¹⁹F gases directly, together with the unique properties of these compounds, has enabled the measurement of several functional parameters, such as airspace size (via the apparent diffusion coefficient), regional oxygen partial pressure, ventilation distribution, ventilation/perfusion ratios and gas exchange, including oxygen uptake (48–52).

Despite the fact that fluorinated gases offer much less SNR when compared with hyperpolarized gases, fluorinated gases are easy to manage, can be mixed with oxygen, do not involve a polarizing apparatus and are low in price, all of which are advantageous in the context of functional lung MRI performed with inert gases. In particular, fluorinated gases at thermal equilibrium polarization, such as sulfur hexafluoride (SF₆), hexafluoroethane (C₂F₆), tetrafluoromethane (CF₄), and fluoropropane (C₃F₈), have recently been presented as alternatives to hyper-polarized gases that can be mixed with 20% oxygen for continuous breathing without requiring a hyperpolarization apparatus, resulting in a considerable advantage in cost and effort (see Table 2).

Table 2.

List of volatile ¹⁹F agents

Agent	Commercial name or acronym	Chemical formula	Modality	Application	Physical state	Relative SNRa	Reference
Linear perfluorocarbons (e.g. tetrafluoromethane, hexafluoroethane, fluoropropane, etc.)	Optison™, Perflutren	CF4, C2F6, C3F8, etc.	MRI, US	Respiratory and cardiac imaging	Gas dispersion (lipid microsphere) in a injectable suspension	(4, 6, 8, etc.) ×10−5	(116,117)
Anesthetics (e.g. halothane, isoflurane, sevoflurane, desflurane, methoxyflurane)	Forane®, Ultane®, Suprane®, Analgizer, Inhalan	C2HBrClF3 (halothane)	MRI, MRS	General anesthesia	Volatile liquids for inhalation administered via vaporizer	3.1 ×10−5	(118–127)
		C3H2ClF5O (isofluorane)				5.2 ×10−5
		C4H3F7O (sevofluorane)				7.3 ×10−5
		C3H2F6O (desflurane)				6.3 ×10−5
		C3H4Cl2F2O (methoxyflurane)				2.1 ×10−5
Sulfur hexafluoride	SonoVue®	SF6	MRI, US	Vent, cardiac and microvasculature imaging	Gas: SonoVue® is a gas dispersion in a liquid (NaCl)	6.3 ×10−5	(128)

US, ultrasound.

^a19F signal-to-noise ratio (SNR) of the fluorinated agent at a concentration of 1 μmol/g wet tissue weight relative to the ¹H signal detected from an equivalent tissue volume with a detector coil of the same geometry. The calculation assumes a tissue water content of 76.5% (± 0.37 SD), which is an average of the water contents of brain, skeletal muscle and liver tissues (115), and sample-dominant noise resulting in a linear-dependent SNR with field strength.

SF₆ is one of the more widely used fluorinated molecules in the clinical setting, in addition to the anesthetic halothane [CF(3) CHBrCl]. It has been used in clinical trials as part of a multiple inert gas elimination technique, and as a microbubble contrast agent in ultrasound imaging (Sonovue^®). It is a heavy molecule (146.06 g/mol) with six chemically equivalent fluorine atoms. Its low solubility in water is similar to that of ³He (49). Although its higher fluorine count per molecule compared with the MRI-active nuclei on other inhaled gases is an advantage, its very short T₂ and T₁ relaxation times of around 2 ms, together with a much lower diffusivity than that of ³He, make ¹⁹F MRI applications employing SF₆ gas very challenging, especially with respect to system gradient and bandwidth requirements. Despite the short relaxation times, some dynamic (53) and diffusion-weighted (48,54) images have been successfully recorded from animal studies.

The use of fluoroethane (C₂F₆, Fig. 2) and fluoropropane (C₃F₈) gases, with relatively longer T₁ and T₂ values of 10 and 20 ms, respectively, seems more promising and more suitable than SF₆ for measurements of diffusion in human lungs with ¹⁹F MRI (50,55,56). Indeed, fluoropropane (Optison^®– an injectable liquid suspension) is currently used for ultrasound imaging of the internal organs and other anatomical structures in humans.

Figure 2.

Post-mortem ¹⁹F MRI of lung gas density using C₂F₆. Axial images (a) were acquired within 2 min and coronal images (b) were acquired within 3 min using a 4.7-T magnet. These images show excellent quality similar to that obtained with hyperpolarized gases, although with much more accumulation.

USE OF PFCs AS BLOOD SUBSTITUTES AND AEROSOLS

As noted above, physiologically important gases, such as oxygen and carbon dioxide, are highly soluble in many PFCs, and do not react with them. These physical properties, together with the reduced need for allogeneic blood transfusion and the facilitation of oxygen transfer from red cells to the tissue, are central to the utility of PFC emulsions as blood substitutes (see Table 3).

Table 3.

List of liquid ¹⁹F agents

Agent	Commercial name or acronym	Chemical formula	Modality	Application	Physical state in normal conditions	Relative SNRa	Reference
Perfluorotripropylamine, perfluorodecalin	Fluosol-DA®, PFTPA	C9F21N	MRI	BS, OS	Liquid emulsion (20%), Fluosol® discontinued	2.2 ×10−4	(4,129)
Perfluorodecalin	Perflunafene, PFDC	C10F18	MRI	BS, OS	Liquid emulsion (20%), longer blood half-life	1.9 ×10−4	(4,130)
Perfluorotributylamine	Oxypherol™, PFTA	C12F27N	MRI	BS, OS	Liquid emulsion	2.8 ×10−4	(131)
Perfluorodichlorooctane	PFDCO, Oxyfluor	C8F16Cl2	MRI, US	BS, OS, CL	Liquid emulsion (40%)	1.7 ×10−4	(132,133)
Perfluorooctylbromide, perflubron	LiquiVent®, Oxygent™, PFOB, Imagent™, Oxycyte™	C8F17Br	MRI, MRS, CT	BS, OS, CL, GI	Liquid emulsion (60%) Oral administration in some cases	1.8 ×10−4	(18,130, 134,135)
		C8F16O4				1.7 ×10−4
		(CF2CF2O)4				1.7 ×10−4
		C10F20O5				2.1 ×10−4
	12-crown-4-ether, 15-crown-5-ether, 18-crown-6-ether	(CF2CF2O)5				2.1 ×10−4
		C12F24O6				2.5 ×10−4
Perfluoro-poly-ethers		(CF2CF2O)6	MRI, MRS	OS, CL	Liquid emulsions (20–40%)	2.5 ×10−4	(22,67,69)
Fluorinated aromatics, e.g. hexafluorobenzene		C6F6	MRI, MRS	OS	Liquid	6.3 ×10−5	(136)

BS, blood substitute; OS, oxygen sensor; CL cell labeling; CT, computed tomography; GI, gastrointestinal; US, ultrasound.

^a19F signal-to-noise ratio (SNR) of the fluorinated agent at a concentration of 1 μmol/g wet tissue weight relative to the ¹H signal detected from an equivalent tissue volume with a detector coil of the same geometry. The calculation assumes a tissue water content of 76.5% (± 0.37 SD), which is an average of the water contents of brain, skeletal muscle and liver tissues (115), and sample-dominant noise resulting in a linear-dependent SNR with field strength.

High oxygen levels can also limit the use of PFC blood substitutes to the extent that their oxygen transport characteristics differ from those of blood (57,58). The ¹⁹F signals of a number of biological PFCs exhibit R₁ values that depend on oxygen content, thereby potentially providing a measure of the percentage oxygenation. Thus, targeted cell-specific PFC-bearing nanoparticles could potentially provide a means of assessing regional oxygen content, to the extent that the PFC is in contact with the oxygen and the nanoparticle itself does not interfere with the interaction.

PFCs, alone or in a mixture with surfactants, have been used for liquid ventilation (59,60) and drug delivery during liquid ventilation (61). These kinds of applications have been investigated for possible therapeutic use in the treatment of various respiratory diseases, and were the subject of a recent symposium on PFC applications (62,63). As mammals use gas rather than liquid ventilation, PFC vaporization has been developed for the respiratory circuit. Aerosolization appears to be an efficient way to facilitate pulmonary gas exchange in acute respiratory distress syndrome (64).

SENSITIVITY AND DETECTION LIMITS FOR ¹⁹F MRI OF PFCs

Organofluoro compounds are rare in nature, which makes the interpretation of studies on how exogenous organofluorides behave in vivo less complicated. However, the concentrations of PFCs normally introduced into biological systems are low, and thus typical ¹⁹F MRI SNR is low when compared with ¹H MRI (see Tables 1–3). Although the use of large quantities of highly fluorinated fluoro compounds can yield high-quality ¹⁹F images, high doses (more than 1–5 g/kg for PFCs) may be limited in humans by safety concerns. To date, MRS studies in a whole-body scanner suggest a minimum detectable limit of ¹⁹F at 1.5 T of about 30 μmol/g wet weight in a volume of about 33 mL in 6 min (65), based on surface coil measurements following the infusion of 5-FU chemotherapy. This concentration is difficult to achieve with systemic agents, or by passive accumulation of in vivo agents. Other authors have reported a detection threshold for spectroscopic 5-FU quantification of about 5 nmol/g in 0.5–1.2-g samples at 11.7 T, albeit with a 1-h data acquisition (38). Our own MRI experience with perfluoro-crown ether (PFCE), which contains 20 chemically equivalent ¹⁹F nuclei, indicates that the sensitivity of this compound in vivo in a 9.4-T magnet is around 30 μmol/g wet weight in a 4-min acquisition time in a volume of 60 μL (66). Partlow et al. (67) have made specific measurements of the detection limit of PFC-labeled cells using ¹⁹F MRS and MRI at high (11.7 T) and low (1.5 T) fields. The lower detection limit was found to be 2000 cells for PFCE- and 10,000 cells for PFOB-labeled cells for in vitro ¹⁹F MRS, and around 6000 PFCE-labeled cells for in vitro ¹⁹F MRI. Quantification of PFCs revealed intracellular concentrations of up to 3 pmol per cell for a 12-h incubation, without the help of any transfection agent.

Given the linear relationship between signal intensity and PFC concentration (as well as voxel size), it is possible to dramatically increase the final PFCE concentration using targeted nanoparticles containing multiple PFCE molecules. The specific binding of such targeted nanoparticles can increase the local concentration above the minimum detection limit, potentially enabling their use as contrast agents on clinical systems with clinically relevant scan times (23).

OPTIMAL ¹⁹F MRI PULSE SEQUENCES FOR PFCs

¹⁹F MRS and MRI have been demonstrated with compounds that exhibit several peaks in their NMR chemical shift spectra (e.g. PFTA, PFOB). For fluoro compounds, the ¹⁹F chemical shift dispersion (in Hz) is typically larger than that of ¹H. When the ¹⁹F signal is divided among several peaks, the ¹⁹F MRI SNR may be reduced when compared with a monochromatic spectrum. In addition, if the bandwidth per ¹⁹F image pixel is less than the chemical shift dispersion, chemical shift artifacts can arise, wherein the signal from each peak is shifted into a different voxel, creating a ‘ghost’ image, and/or the location of a selected slice may be displaced in space to a different extent for each moiety (Fig. 1). This is the case for PFOB and PFTA, thus complicating the use of these molecules. Chemically selective imaging pulse sequences can be employed to circumvent the chemical shift artifact problem by excluding the signals from the other peaks, but this does not compensate for the SNR loss. In addition, the relatively short T₂ and the further splitting of the resonances of each moiety in some of these molecules as a result of J-modulation (68,69) can further reduce SNR through signal decay and/or increased receiver bandwidth requirements, even at short TE.

Another problem for ¹⁹F MRI acquisition is the relatively long T₁ relaxation times of many fluorinated molecules (1–4 s), which necessitates lengthy TR intervals for the recovery of appreciable longitudinal magnetization (69,70). This makes time-efficient and clinically relevant ¹⁹F MRI challenging. ¹⁹F MRI pulse sequences are typically performed using ultrafast imaging methods, such as keyhole techniques or echo planar imaging (EPI). Ultrafast, low-angle, rapid acquisition and relaxation enhancement is utilized where short TEs are needed, including the use of linear PFPE molecules with small T₂/T₁ ratios (71), and is even useful when the T₂ relaxation time is not a problem (as is the case with PFCE or hexafluorobenzene) (69,72). These sequences can easily be combined with an inversion recovery pulse sequence for T₁ measurements to increase their acquisition efficiency. Although some investigators have proposed the use of paramagnetic contrast agents to shorten the ¹⁹F T₁ relaxation times of water-miscible ¹⁹F molecules (73,74), this approach is problematic because most PFCs are immiscible in water. Thus, when 15% w/w Gd-DTPA is incorporated into a 20% (w/v) PFCE nanoparticle emulsion (as the bisoleate acid Gd-DTPA-BOA), a small (~ 15%) reduction in T₁ is achievable, but is accompanied by a much larger reduction in T₂ (85%), such that the use of long-TE sequences is no longer an option. The implementation of parallel imaging techniques for ¹⁹F MRI also offers scope for improving the ¹⁹F acquisition efficiency in the coming years.

MOLECULAR AND CELLULAR 19F MRI

MRI is able to visualize cells in vivo in real time. When cells are imaged in living animals, it can provide new insights into the biology of cell trafficking and migration. An example is the homing of white blood and hematopoietic cells in cancer and immunological diseases. Because MRI methods are noninvasive, they can be applied repeatedly to monitor targeted cells and cellular processes. For cells to be visualized by MRI, they generally must be labeled to enable their discrimination from surrounding tissue.

For ¹H MRI, superparamagnetic iron oxides (SPIOs) offer one method of magnetic labeling that disturbs the local magnetic field near excited spins (75). The proton signals from water close to SPIO particles are seen as regions of hypointensity, acting as signal voids. However, it is sometimes difficult to discriminate between targeted molecules or cells and image artifacts arising from susceptibility and/or other effects associated with the MRI pulse sequences. Superparamagnetic contrast agents that can provide a positive signal also exist with some MRI techniques, such as on- (76) and off- (77,78) resonance saturation. However, similar problems can arise as those occurring with hypointense contrast.

Another alternative that generates positive contrast is the use of paramagnetic contrast agents, typically gadolinium chelates, that shorten the T₁ relaxation times. Paramagnetic contrast agents suffer from a relatively high threshold for detection. Much effort has been directed towards the development of intracellular labeling methods that allow sufficient uptake without affecting cellular proliferation or function. Fluorine agents and ¹⁹F imaging could present opportunities for labeling with minimal effects on cell proliferation, function and maturation, whilst permitting ¹⁹F label-based quantification. A recent article provides a comparative evaluation of the use of various fluorine-containing nanoemulsions for MRI cell tracking (79).

One important article demonstrated the first cellular ¹⁹F MRI of passively fluorine-labeled macrophages, which appeared as ‘hotspots’ in the central nervous system in experimental allergic encephalomyelitis (80). This is an animal model for multiple sclerosis which is characterized by infiltration of the macrophages into the inflamed brain. After induction of the disease, cells were observed after intravenous injection of a PFCE emulsion at 3 g/kg. PFCs have also been used to image macrophage infiltration in the infarcted myocardium (see Fig. 3) (81). This work is similar in concept to ¹H MRI studies of the uptake of ultra-small SPIO particles by macrophages in inflammatory diseases. ‘Hotspot’ ¹⁹F MRI of cells gained renewed interest after Ahrens and coworkers (22,71,82) presented an approach to ‘active’ cell labeling and MRI cell tracking of immune cells in vivo (Fig. 4).

Figure 3.

Infiltration of perfluorocarbons (PFCs) after myocardial infarction as detected by in vivo ¹⁹F MRI. (a) Anatomically corresponding ¹H and ¹⁹F images from the mouse thorax recorded 4 days after ligation of the left anterior descending coronary artery, showing accumulation of ¹⁹F signal near the infarcted region (I) and at the location of surgery where the thorax was opened (T). PFCs were injected at day 0 (2 h after infarction) via the tail vein. (b) Sections of ¹H images superimposed with the matching ¹⁹F images (red) acquired 1, 3 and 6 days after surgery (post OP) indicate a time-dependent infiltration of PFCs into injured areas of the heart and the adjacent region of the chest affected by thoracotomy. At day 4, an additional bolus of PFCs was injected to compensate for clearance of the particles from the bloodstream after 3 days. Subsequent histology demonstrated that the uptake of PFCs had occurred in cells of the monocyte/macrophage lineage. Reproduced, with permission, from Flogel et al. (81).

Figure 4.

In vivo ¹⁹F MRI of perfluoro-crown ether (PFCE)-labeled dendritic cells in a mouse. (a) Mouse quadriceps after intramuscular injection of PFCE-labeled cells. From left to right are ¹⁹F, ¹H and a composite ¹⁹F/¹H image. (b) Composite image of dendritic cell migration into the popliteal lymph node following a hind foot pad injection. (c) Composite image through the torso following intravenous inoculation with perfluoropolyether (PFPE)-labeled cells. Cells are apparent in the liver (L), spleen (S) and, sporadically, lungs (Lu). Electron micrograph of a labeled fetal skin-derived dendritic cell line at a low magnification (d) and a higher magnification (e). Particles (100–200 nm) appear as smooth spheroids. Arrows show a typical multiple-membrane compartment enclosing these particles. Adapted, with permission, from Ahrens et al. (22).

Recently, this approach has been extended further to the tracking of stem cells (66,67). Using different PFC preparations with different ¹⁹F spectral frequencies (as ‘signatures’), it was suggested that multiple cell populations, labeled differently, could be detected simultaneously (Fig. 5) (67). Hotspot imaging is analogous to the approaches of other imaging techniques that employ radionuclides, such as positron emission tomography or single-photon emission tomography, albeit without the associated hazards of ionizing radiation. Although hotspot ¹⁹F MRI images the labeled cells, anatomical ¹H MRI performed with the same scanner during the same MRI examination is superimposed without any image registration problems to precisely determine the biodistribution of the labeled cells. In this respect, MRI has clear advantages over conventional optical and nuclear imaging techniques.

Figure 5.

Localization of labeled cells after in situ injection. (a) To determine the utility for cell tracking stem/progenitor cells labeled with either perfluoro-octyl bromide (PFOB) (green) or perfluoro-crown ether (PFCE) (red), nanoparticles were locally injected into mouse thigh skeletal muscle. (b–d) At 11.7 T, spectral discrimination permits the imaging of the fluorine signal attributable to ~ 1 × 10⁶ PFOB-loaded (b) or PFCE-loaded (c) cells individually which, when overlaid onto a conventional ¹H image of the site (d), reveals PFOB- and PFCE-labeled cells localized to the left and right leg, respectively (broken line indicates 3 × 3-cm² field of view for ¹⁹F images). (e, f) Similarly, at 1.5 T, ¹⁹F image of ~ 4 × 10⁶ PFCE-loaded cells (e) locates to the mouse thigh in a ¹H image of the mouse cross-section (f). The absence of background signal in ¹⁹F images (b, c, e) enables unambiguous localization of PFCE-containing cells at both 11.7 and 1.5 T. Reproduced, with permission, from Partlow et al. (67). PFC, perfluorocarbon.

PFCE has been used recently in different mixtures of lipids to formulate emulsified cationic and anionic nanoparticles, with and without the lipophilic gadolinium derivative Gd-DTPA-BOA, and including fluorescent rhodamine to tag different cells (66). Cationic nanoparticles showed, in all cases, a greater and more rapid uptake than anionic PFCE preparations (Fig. 6) (66). Different amounts of PFCE-labeled cells were injected into each hemisphere of a mouse brain, and then imaged with ¹⁹F and ¹H MRI (Fig. 7). It was observed that the injected PFCE-labeled cells remained viable and were still rhodamine-positive, with no appreciable reduction in the fluorine signal, at least up to 2 weeks post-injection (66).

Figure 6.

Fluorescence microscopy of cationic (a, c) and anionic (b, d) perfluoro-crown ether (PFCE)-labeled C17.2 mouse neural stem cells after 4 h of incubation with 2.4 mM PFCE. (a, b) Rhodamine fluorescent (red) and phase contrast overlay image of cells immediately after 4 h of incubation. (c,d) Rhodamine fluorescent images of cells cultured for an additional 18 h after removal of PFCE at 4 h of incubation. Note the transport and intracellular redistribution of label between the two time points. Scale bars: 100 μm in (a, b) and 50 μm in (c, d). Reproduced, with permission, from Ruiz-Cabello et al. (66).

Figure 7.

In vivo MRI of transplanted C17.2 neural stem cells, with the ¹⁹F signal superimposed on the ¹H MR images. (a–c) MR images at 1 h (a), 3 days (b) and 7 days after injection of 4 × 10⁴ (left hemisphere, arrowhead in a) or 3 × 10⁵ (right hemisphere, arrow in a) cationic perfluoro-crown ether (PFCE)-labeled cells. (e, f) Corresponding histopathology at day 7 with phase contrast (e) and anti-β-gal immunohistochemistry (f) demonstrates that implanted cells remain viable and continue to produce the marker enzyme. In (f), the right arrow indicates cells migrating from the injection site into the brain parenchyma. (d) MR image of a different animal at 14 days after injection of equal amounts of 4 × 10⁵ C17.2 cells in both hemispheres, demonstrating the persistence of the ¹⁹F signal for 2 weeks. (g) Corresponding histopathology showing rhodamine fluorescence from PFCE-labeled cells co-localizing with the ¹⁹F signal. Scale bar, 500 μm. Reproduced, with permission, from Ruiz-Cabello et al. (66).

A different research area within molecular and cellular MRI is the use of transfected enzymes (reporter genes) that can convert a pro- (precursor) drug. In this technique, a gene with specific enzymatic activity is first introduced into tumor or other cells of interest. Then, a pro-drug is administered and, on internalization of the pro-drug into cells, it is converted by the transgene into an active drug. This method ensures that the drug will be active only in the target cells and will not affect other tissues. A similar example involves 5-FU, wherein yeast cytosine deaminase is introduced into an HT29 colon carcinoma cell line (83) to convert the precursor 5-fluorocytosine (5-FC) into 5-FU, and elicit a chemotherapeutic response. The formation of 5-FU, measured in xenografted tumors using ¹⁹F MRS, provides an indication of the efficacy of drug delivery. By this means, ¹⁹F MRS might provide a means of monitoring and optimizing the administration of such pro-drugs to patients for chemotherapy.

With regard to the use of ¹⁹F probes as ‘smart tracers’ or ‘molecular beacons’, ¹⁹F MRS has been employed to probe the enzymatic activity of a prototype reporter enzyme, β-galactosidase. This enzyme has been shown to liberate a glycone from the substrate 4-fluoro-2-nitrophenyl-β-D-galactopyranoside, resulting in a pH-dependent ¹⁹F chemical shift of 5–10 ppm that can be used to measure intracellular pH (84). Other enzymes can be probed as well. For example, certain proteases (caspase-3), which are overexpressed in tumors, are able to cleave paramagnetic chelates from fluorinated molecules, thereby modulating ¹⁹F relaxation times and signal intensity (85). Finally, temperature-sensitive liposomes containing ¹⁹F tracers have been developed as beacons for image-guided drug delivery (86).

In addition to enzymes, fluorinated metabolic substrates are also potential targets of study that are accessible to ¹⁹F MRI or MRS, when present in sufficient concentration. For example, the distribution of 2-fluoro-[¹⁸F]-2-deoxy-glucose, a widely used positron emission tomography probe for the measurement of abnormal glucose consumption in tumors and ischemia, has been monitored with ¹⁹F MRS after replacing the unstable ¹⁸F atom with ¹⁹F (87).

INCORPORATION OF PFCs INTO CELLULAR THERAPEUTIC BIOMATERIALS

The incorporation of PFCs into biomaterials is attractive for a number of reasons. By exploiting the various features of PFCs, fluorinated biomaterials can be used to create smart scaffolds that are capable of producing an oxygen-rich environment whilst permitting the noninvasive assessment of biological parameters, such as O₂ tension, pH and metabolite concentrations, with ¹⁹F MRI. Fluorinated biomaterials, in conjunction with ¹⁹F MRI, could thus provide important information on the delivery and long-term survival of cellular therapeutics. For these reasons, fluorinated biomaterials show promise for both the assessment and enhancement of the long-term viability of cellular therapeutics after transplantation.

To date, the use of fluorinated biomaterials has been limited to PFC-containing microcapsules. The use of microcapsules to provide the immuno-isolation of cellular therapeutics has clinical potential for a wide range of diseases that require enzyme or endocrine replacement therapy. A number of groups have explored the use of PFC-containing microcapsules. Barnett et al. (88) incorporated PFPE emulsions in alginate/poly-L-lysine (PLL) microcapsules to enable the assessment of the biodistribution and integrity of microcapsules with MRI. As PFCs are rapidly cleared from the body when they are no longer encapsulated, MRI could provide a means of assessing capsule rupture and loss of immunoprotection.

Other groups have used PFC-containing microcapsules for the determination of the local oxygen saturation in vivo. The paramagnetic properties of a polar oxygen affect the T₁ and chemical shift of ¹⁹F nuclei in proportion to pO₂, as noted earlier, potentially enabling the use of microcapsular PFCs for the serial assessment of pO₂ in vivo. The first study of this type, by Nöth et al. (89), utilized PFC-loaded alginate capsules in conjunction with quantitative ¹⁹F MRI to determine pO₂ inside capsules implanted in the peritoneal cavity and elsewhere in a rat (Fig. 8). Fraker et al. (90) later reported a related method using PFTA.

Figure 8.

¹⁹F (a) and ¹H (b) images of a 2-mm transaxial slice through the peritoneal cavity of a rat showing two alginate capsules loaded with pure trans-1,2-bis(perfluorobutyl)-ethylene (F-44E), and the calculated ¹⁹F pO₂ maps of the same slice whilst the animal is breathing oxygen (c) and air (d). The average pO₂ values in the alginate capsules are 9.0 ± 1.4% (left capsule) and 12.8 ± 2.0% (right capsule) in (c) and 3.2 ± 1.0% (left capsule) and 5.0 ± 1.4% (right capsule) in (d). Images were acquired on day 1 after implantation. The scale on the right of (a) and (b) gives the signal intensity in arbitrary units; the scale in (c) and (d) gives the pO₂ values in per cent. 100% corresponds to 760 mmHg. Reproduced, with permission, from Nöth et al. (89).

In addition to providing a means of assessing pO₂, PFCs can also increase local oxygen tension. The ability to increase oxygen availability provides added value for encapsulation therapy, where many studies have suggested that graft failure occurs because of the lack of vascular access for the encapsulated cells. This results in the gradual necrosis of implanted cells. For this reason, the work of Khattak et al. (91) is particularly relevant. They found that the incorporation of a PFC emulsion in alginate micro-capsules increased significantly the viability of an immortalized hepatocyte cell line. They attributed this enhanced viability to the ability of the PFC reservoir to ‘reload’ itself with O₂ from plasma perfusing through the matrix – analagous to the O₂ transfer in emulsified PFCs that are currently being used for surgical applications (25). By acting as oxygen sinks, PFC-containing micro-capsules may have broad applications for increasing the viability of many encapsulated cell types.

In addition to increasing local oxygen concentrations, PFCs may enhance the immuno-isolation properties of alginate micro-capsules by modulating the immune response. In vitro studies have suggested that PFOB reduces cytokine release by human alveolar macrophages and attenuates oxidative injury in rat pulmonary artery endothelial cells. Furthermore, neutrophil adhesion to lung epithelial cells and subsequent cytolysis are inhibited by PFOB during activation with a pro-inflammatory stimulus (92), and PFOB attenuates neutrophil adhesion to activated endothelial cells (93). For these reasons, PFC-loaded alginate capsules could attenuate the rejection of cellular therapeutics in immunocompetent hosts.

As the incorporation of PFCs is not limited to alginate micro-capsules, further exploration of fluorinated biomaterials may prove to be attractive in tissue engineering. The unique behavior of PFCs, including their hydrophobic and lipophobic character and extreme inertness, nevertheless presents some significant design obstacles for direct incorporation into polymers. For this reason, only emulsified PFCs have been incorporated into biomaterials so far. Nevertheless, the many potential advantages of directly fluorinated biomaterials warrant future research in this area.

PHARMACOKINETICS AND TOXICITY OF PFCs

PFCs are chemically inert and stable compounds that are not metabolized and are excreted by normal clearance mechanisms, such as phagocytosis by the reticuloendothelial system, with eventual elimination by exhalation and bowel excretion (94). Most fluorocarbons within the molecular weight range of 460–520 Da are biologically inactive, and exhibit no significant toxicological risks, carcinogenicity or mutagenicity (23,25,95,96). The inertness of most of these compounds is an attractive property in many applications. However, environmental monitoring suggests that some degradation of PFCs into other persistent fluorinated chemicals can occur, and may demand recovery procedures, particularly for applications such as partial liquid ventilation (97).

PFCs for medical applications must be consistently prepared with high purity on a cost-effective basis that is typically large scale (25). Ideally, they should be excreted rapidly, which depends on their lipid solubility – the higher the better. This property is important for PFCs intended for intravascular use. Fluorocarbons containing other chemical elements, such as a terminal bromine atom, tend to be excreted more rapidly than predicted solely on the basis of their molecular weight (14). PFOB has been the subject of much of the research into PFC emulsions to date. Another factor to consider is the ease with which stable emulsions can be formed and sustained, as preparations must be able to withstand heat sterilization when required by regulatory agencies, such as the FDA (25), although irradiation or sterile filtration may be suitable alternatives.

The toxicity of organofluorinated compounds follows the same general principles with regard to the toxicity of drugs: it depends on the mechanism of action of the drug and the molecules generated by its metabolism, if any. Although not providing evidence of nontoxicity, when PFTA, PFOB or PFCE is injected into an animal, there is no significant difference between the in vivo and in vitro ¹⁹F spectra of these molecules, consistent with their being resistant to metabolism, at least in the short term. However, another feature that is often attributed to organofluorine compounds is their activity on the P450 enzyme system, which is important, as it relates to the elimination of many other drugs (98). Organofluorinated compounds seem to be associated with greater liver damage and interfere with thyroid hormones, when compared with their corresponding nonfluorinated analogs.

Highly fluorinated (carbon) compounds are not miscible with either aqueous or organic solvents. The smaller molecules tend to be cleared rapidly from the circulation, with brief retention in the mononuclear phagocyte system, mainly in the liver, spleen and bone marrow. PFTPA forms stable emulsions, but has a long retention time. Emulsions have been prepared with PFDC, which is rapidly cleared from the body, to take advantage of both effects.

PFCE and PFOB have been emulsified with pure lecithin, forming stable emulsions with small particle sizes (0.1–0.2 μm in diameter). Lecithin-based (and pluronic-based) emulsions show moderate inhibition of endotoxin-induced cytokine production, although they also cause substantial cytotoxicity in phagocytic cells after 24 h of incubation. After intravenous administration, PFC particles are taken up by the reticuloendothelial system, and evaporate through the lung, an elimination process that does not involve the breakdown of its chemical structure. Reports from a phase I clinical safety trial in healthy volunteers receiving PFOB (Table 2) indicated that the PFC emulsion does not affect coagulation (99,100).

The degradation products of some widely used fluorine-containing household chemicals (wetting agents, lubricants, corrosion inhibitors, insecticides, cosmetics, fire retardants and surfactants), such as perfluoro-octanoic acid, are known to induce peroxisome proliferation in hepatocytes, as well as considerable changes in enzymatic activity (101). The C–F bond is essentially xenobiotic and has an extremely high stability, making it resistant to degradation, even in volatile forms such as Freon. Fluorocarbons can accumulate in human tissues when inhaled, ingested or given intravenously. The length of time they remain in the body depends on their molecular weight and vapor pressure (volatility): the more volatile they are, the shorter their half-life, which can range from minutes to years. Indeed, evidence exists that there is long-term retention of some PFCs by the reticuloendothelial system (102). The major predictors for the diffusion of small molecules across cellular membranes are lipophilicity and molecular weight. The incorporation of fluorine into a drug can increase its lipophilicity and enhance its absorption into biological membranes. The small covalent radius of fluorine can facilitate the docking of drugs with their receptors. When the molecular weight of the fluorinated version of one drug is not significantly altered, passive diffusion should be comparable with that of the nonfluorinated form.

One fluorinated drug, fluoxetine hydrochloride (Prozac^®), is a widely prescribed antidepressant in the world today. Fluoxetine is a potent inhibitor of serotonin. The brain tends to accumulate these agents at levels that are an order of magnitude higher than serum levels because of their lipophilicity and pH trapping in acidic vesicles. Based on data from 22 patients treated with fluoxetine, the brain concentration of the drug continues to increase long after its clinical effects are manifest, leveling off some 6–8 months after therapy commences (103). The drug accumulated to about 20 times the plasma level in these patients, although the brain concentration was not correlated with the clinical response. The study showed that, even 3.5 weeks after the end of drug therapy, a significant ¹⁹F fluoxetine signal could be observed in the brain, suggesting a very long washout time. In another study, a weak correlation of fluoxetine level with positive clinical response was found in patients with social phobia (104).

Fluoxetine metabolizes to the therapeutically active compound norfluoxetine in the brain. Because the ¹⁹F chemical shift of norfluoxetine is very close to that of fluoxetine, the two compounds are not resolved in vivo. In vitro ¹⁹F NMR studies of the extracts of both human and rat brain confirm that the in vivo signal arises roughly equally from fluoxetine and norfluoxetine.

CONCLUSIONS

As a result of their unique physicochemical properties, there is increasing interest in the use of fluorinated compounds in ¹⁹F MRI and MRS to serve as biomedical contrast agents, biomarkers for monitoring the local physiological environment and indicators of therapy delivery vehicles, ranging from chemotherapeutic agents to cells to nanoparticles, in both experimental animal models and human studies. Some of these applications are already being used in patients, whereas others are on the verge of introduction to the clinic. Most of the recent studies have been performed at higher magnetic fields (1.5 T and above), as the low SNR remains a challenge because of the intrinsically low ¹⁹F concentrations in vivo. The recent development of targeted and improved nanoparticle formulations with high payloads offers new promises for ¹⁹F MRS and MRI applications in molecular and cellular imaging.

Abbreviations used

BAPTA

1,2-bis(o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid

BOA

bis-oleate acid

DTPA

diethylenetriaminepentaacetate

EPI

echo planar imaging

FBAPTA

5,5′-difluoro-1,2-bis(o-amino-phenoxy) ethane-N,N,N′,N′-tetraacetic acid

5-FC

5-fluorocytosine

FDA

Food and Drug Administration

5-FU

5-fluorouracil

PFC

perfluorocarbon

PFCE

perfluoro-crown ether

PFDC

perfluorodecalin

PFOB

perfluoro-octyl bromide

PFPE

perfluoropolyether

PFTA

perfluorotributylamine

PFTPA

perfluorotripropylamine

pO₂

partial pressure of O2

SNR

signal-to-noise ratio

SPIO

super-paramagnetic iron oxide