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Understanding the Fundamentals of
Perfluorocarbons and Perfluorocarbon EmulsionsRelevant to In Vivo Oxygen Delivery
Jean G. Riess
MRI Institute, University of California at San Diego and AlliancePharmaceutical Corp. San Diego, California, USA
Abstract: The unique behavior of perfluorocarbons (PFCs), including their highoxygen dissolving capacity, hydrophobic and lipophobic character, and extremeinertness, derive directly, in a predictable manner, from the electronic structureand spatial requirements of the fluorine atom. Their low water solubility is keyto the prolonged in vivo persistence of the now commercially available injectable
microbubbles that serve as contrast agents for diagnostic ultrasound imaging.OxygentTM, a stable, small-sized emulsion of a slightly lipophilic, rapidly excretedPFC, perfluorooctyl bromide (perflubron), has been engineered. Significant oxy-gen delivery has been established in animal models and through Phase II and IIIhuman clinical trials. However, an inappropriate testing protocol and the lack offunding led to temporary suspension of the trials.
UNIQUE, BUT NOT MYSTERIOUS
Last year, when we met in Stockholm, a distinguished colleague of ours,highly competent in hemoglobin matters, told me that fluorocarbons(or perfluorocarbons, PFCs) were for him a total mystery (his words).However, when questioned about what he had read about PFCs, headmitted frankly that although he was intrigued by them, he had neverhad time to really read any paper about PFCs.
This is one reason why I choose, in this presentation, to return tobasics about PFCs and PFC emulsions, in case there still were a few
colleagues who hadnt yet had a chance to learn about PFCs and withthe hope of solving some of the perceived mysteries. The topic is alsoi l b f Alli Ph i l C (S Di CA) h i
Artificial Cells, Blood Substitutes, and Biotechnology, 33: 4763, 2005
CopyrightQTaylor & Francis, Inc.
ISSN: 1073-1199 print/1532-4184 online
DOI: 10.1081/BIO-200046659
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voluntarily interrupted a clinical trial of its emulsion, OxygentTM, in a
cardio pulmonary bypass surgery setting involving augmented acute
normovolemic hemodilution.My message is There are no mysteries, just specific molecules with
their rather unique attributes and performances. These attributes derive
directly, can be understood and can be predicted from the specific elec-
tronic structure and spatial requirement of the constituent atoms,
especially the fluorine atom.
Back to basics implies back to chemistry. There should be little won-
der that replacing all the hydrogen atoms by fluorines in an organic mol-
ecule should bring about some substantial changes in behavior.[14]
Fluorine has 9 electrons (and 9 protons and 10 neutrons) as comparedto only one electron (and one proton) for hydrogen. These 9 electrons
are packed inproportionallyless space, hence in a more compact
way (Fig. 1). In other words, fluorine has a much denser electron cloud.
Fluorine also has a higher ionization potential than hydrogen (just after
the inert gases He and Ne), a considerably larger electron affinity, the
highest electronegativity of all atoms, and a lower polarizability than
hydrogen, second only to Ne.
As a result, perfluoroalkyl chains (F-chains) are structurally quite
different from standard alkyl chains (Fig. 2). The fluorine atom, beingmore space demanding (CF3 is only marginally smaller than C(CH3)3),
forces the CC skeleton to adopt a helical arrangement rather thanthe usual planar zig-zag configuration found in hydrocarbon chains
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(H-chains). F-chains are also bulkier thanH-chains (cross section of 30
vs. 20 A, respectively). The larger trans=gauche interchange energy bar-rier (4.6 vs. 2.0 kJ mol1, respectively) makes them more rigid (it takes
more energy to twist them) and allows for fewer kinks. Finally, the larger,electronically more dense fluorine atoms cover and protect the CCbackbone much more effectively than hydrogen atoms do.
Fluorocarbons: Among the Most Stable, Most Inert Chemicals
Known to Man
Why are PFCs more stable (in the thermodynamic sense) and chemically
more inert (in the kinetic sense) than their hydrocarbon (HC) counter-parts? A better match between carbon and fluorine orbitals as compared
to that between carbon and hydrogen leads to the strongest single bond
found in molecular compounds (e.g. 530 kJ mol1 for CF in C2F6 vs.439 kJ mol1 for CH in CH4). Moreover, the extreme electron attract-ing character of fluorine enhances the CC bond energy in the skeletonby shrinking the orbitals of the carbons (e.g. 413 kJ mol1 in CF3CF3vs. 376 kJ mol1 in CH3CH3).
From the reactivity standpoint, there simply exist no low energy mol-
ecular orbitals accessible for binding O2, CO, NO. The fluorine atomsshield the CC skeleton sterically. Additionally, the dense electronicsheath repels approaching reagent i e exercises some sort of a Scotch
Figure 2. A schematic, comparative view showing the structural differences
between fluorocarbon and hydrocarbon chains, with cross-sections on the right.
Understanding the Fundamentals of Perfluorocarbons 49
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octane) are highly flammable, PFCs (e.g. F-octane) are not, and could
even serve as fire extinguishers! Typical n-CnF2n2 compounds resist
heating to 400
C; n-CnF2n1Br withstands 300
C for 24 h; cooking with5 N H2SO4 at 105
C for 10 days; exposure to 300 nm UV (ICH test),
etc. Polytetrafluoroethylene (PTFE, well-known under the brand name
Teflon1) is one of the most inert organic materials known. Expanded
PTFE (Gore-Tex1) is used in body implant devices and allows natural
tissues to grow in its pores. Fluorinated surfactants can resist highly
aggressive media, including strong acids, alkalies and oxidants, even at
high temperatures, e.g. can withstand contact with 98% sulfuric acid con-
taining 10 g=L chromic acid for 28 days at 90C; no non-fluorinated sur-
factant is known that resists such harsh conditions. WhichH-surfactantcould possibly have a hydrophobic moiety that is also lipophobic?
Finally, PFCs are not metabolized; since Mother Nature did not exploit
the PFC route, she did not develop the enzymes that would have been
needed to recycle them. Pure PFCs have no effects on cell cultures either,
other than the benefits that result from their capacity to provide O2 or
CO2. One can drink PFCs by the liter without side effects other than
wet pants.
Figure 3 relates to the difference in behavior of F-chains and H-
chains at interfaces. It compares the difference in the contribution tothe free energy of adsorption of CF2 and CH2 segments from water
to air=water or HC=water or PFC=water interfaces. It shows that thesefree energy differences are roughly twice as large for CF2 as compared
to CH2. This reflects the higher interfacial activity of CF2s (lower sur-
face tension) and their higher affinity for (and alikeness to) gases as
compared to CH2s. For example, the surface tension of F-n-octane
is 13.6 mN m1 vs. 21.1 mN m1 for n-octane (as compared to
72.8 mN m1 for water).
Highly Hydrophobic and Lipophobic, as Well
PFCs are the most hydrophobic organic substances ever invented. They
are considerably more hydrophobic than HC oils. Increased hydrophobi-
city is primarily a matter of low polarizability and, for a given PFC mol-
ecular structure, of increased surface area exposed to the surrounding
medium, as compared to the parent HC. On a polarity scale (where
water would be on the high polarity side, Fig. 4), PFCs are locatedfurther out than HCs with respect to water: The PFC=water interfacialtension (which opposes the dispersion of PFCs in water) can reach
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The well-known hydrophobic effect is the basis for the self-associ-
ation of lipids into bilayer membranes, such as cell membranes. Being
both extremely hydrophobic and substantially lipophobic as well, PFCs
and F-chains tend to keep to themselves and do not tend to mix with
either aqueous phases or lipids.
Figure 3. A quantitative illustration of the difference in behavior of fluorocar-
bons and hydrocarbons at interfaces. The numbers represent incremental free
energies (cal mol1 at 25C) of adsorption per CF2 or CH2 groups to various
interfaces (see Mukerjee and Handa, J. Phys. Chem. 85: 2238 (1981)).
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The extreme hydrophobicity of PFCs translates into very low water
solubility (typically one order of magnitude lower than HCs). These low
water solubilities, lower than those of any volatile HC, combined withhigh volatility (that also results from low intermolecular forces), are the
basis for the stabilization of injectable dispersions of micron-size PFC-
containing gas bubbles that serve as in vivo reflectors for contrast ultra-
sound imaging. Gas bubbles, small enough to pass the capillary beds,
are indeed ideal sound wave scatterers. However, plain air bubbles, when
injected in the circulation, dissolve within seconds under the combined
effect of blood pressure and surface tension pressure (Fig. 5a). Rapid
microbubble dissolution could be prevented by introducing a volatile
PFC inside the bubbles. The water-soluble gases, i.e. O2, N2 and CO2,equilibrate then with the gases present in the plasma, while the very
poorly water-soluble PFC stays in the bubbles and compensates for
blood pressure and Laplace pressure (Fig. 5b).[67]
Several such perfluorochemical-based contrast agents, namely
Optison1 (Amersham Health Corp.), SonoVue1 (Bracco), Definity1
(Bristol Myers Squib) and Imagent1 (Alliance Pharmaceutical Corp.),
have been licensed by the FDA in the United States or EMEA in Europe
in recent years and are now commercially available.
The same stabilization phenomenon is likely to play a key role instabilizing the O2microbubbles that are being investigated as O2carriers
by Lundgren and Tyssebotn.
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Oxygen Dissolving and Delivery CapacityGas-Like Liquids
Why PFCs dissolve gases better than any other liquid is one of the mostfrequently asked questions. Remember that there exists no possibility
for PFCs to bind gases chemically; PFCs dissolve them, as water does,
or HCs. The exceptionally high gas-dissolving capacity of PFCs derives
from fluorines extremely low polarizability: low polarizability translates
into low van der Waals interactions between PFC molecules, as van der
Waals interactions depend directly on fluctuations in polarity of the elec-
tronic cloud. Since van der Waals interactions are the onlyintermolecular
forces that keep together non-polar molecules, the intermolecular forces
in PFCs are very feeble, in sharp contrast with their strongintramolecularbonds. Consequently, liquid PFCs behave like nearly ideal, gas-like
fluids. They easily dissolve other substances of similarly low cohesivity,
namely gases, including O2, CO2, N2, NO, etc. Birds of a feather flock
together. The low cohesivity of PFCs is also reflected by their low boiling
points and high volatility relative to their molecular weight (MW). If one
compares the Hildebrandt parameters (which express the cohesive energy
density of fluids, hence their aptitude for mutual solubility) of oxygen,
typical PFCs and HCs, and water:
dO2 5:7 dPFC 6 < dHC 7 9
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exceptional biological inertness that creates the potential. The fact that
PFCs do not tend to mix with either water or lipids certainly contributes
to their biological inertness.In summary, the properties and behavior of PFCs (and of perfluoro-
alkylated (F-alkylated) compounds) are in essence of the same nature as
those of regular organic (HC-derived) compounds. However, the excep-
tionally strong intramolecular binding and uniquely low intermolecular
cohesiveness of liquid PFCs related to the low polarizability of fluorine
Figure 6. It takes less energy to make a hole in a less cohesive material, e.g. a
fluorocarbon (left) and host a guest molecule of similarly low cohesiveness (a
gas) than in a more cohesive material, e.g. a hydrocarbon (right).
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result in properties that can become substantially different (exceeding the
usual range) from those ofH-analogs and, in practice, unique (but in no
way mysterious). ManyF-compounds can reach a level of effectiveness in
their performances that cannot be attained by HC compounds, leading totechnological feats that just cannot be achieved with non-fluorinated
materials. Compared to HCs, PFCs are typically much more inert, have
higher densities, compressibilities, fluidity, spreading coefficients and
gas-dissolving capacities, and lower refraction indexes, surface tensions,
dielectric constants and water solubilities, and magnetic susceptibilities
comparable to that of water. Moreover,F-compounds offer uniquecom-
binations of properties that can make them irreplaceable and constitute
the basis for further potential biomedical applications.
SELECTING A PFC FOR IN VIVO OXYGEN TRANSPORT
Figure 8. Oxygen solubility in fluorocarbons follows Henrys law, i.e. is directly
proportional to the gas partial pressure, as expected in the absence of chemicalbonding, while hemoglobin binds O2 through a strong covalent (coordination)
bond to its iron atoms, with consequent saturation at pO2 exceeding that of O2in the earths atmosphere. Oxygen extraction from a PFC emulsion can reach
90% of O2 content (see Riess, Chem. Rev. 101: 2797 (2001).
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are to decide which PFC[14] to use and to develop a stable injectable
emulsion of that PFC. It has been determined that rapid excretion of
the PFC requires a touch of lipid solubility, meaning that the PFC should
not be too heavy in terms of MW. On the other hand, emulsion stability
requires low water solubility, hence that the PFC not be too light. A good
candidate PFC should thus have relatively high lipid solubility and the
lowest possible water solubility, two conditions that are difficult to satisfy
simultaneously. Vapor pressure, which also depends on MW, is animportant parameter; too light a PFC can favor retention of air in the
alveoli, resulting in increased pulmonary residual volume. In order to
avoid this phenomenon, the vapor pressure of the PFC phase should
not exceed about 10 torr.
There are, a priori, many PFCs to choose from since, in principle,
almost any molecular structure can be synthesized. Figure 9 displays
some of the PFCs that have been investigated as candidate O2 carriers.
Actually, there are very few candidate PFCs acceptable for parenteral
use, i.e. PFCs that optimally combine rapid excretion with the capabilityof producing stable emulsions. Table 1 displays some characteristics of
PFC emulsions that are directly related to the PFC that constitutes its
dispersed phase.
Figure 9. Fluorocarbons that have been most investigated for use as O2 carriers.
Table 1. Characteristics of PFC emulsions related to the dispersed PFC(s)
Dissolved O2 readily and immediately available
high extraction ratio
Linear O2 vs. pO2 uptake no saturationPassive delivery, no binding of CO, NO
O2 dissolution increases when temperature decreases
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Figure 10 reminds us that organ retention of PFCs is primarily an
exponential function of MW, with cyclization, branching and the pres-
ence of heteroatoms within their structure having little effect on excretionrate other than through their effect on MW. There are, however, a few
interesting exceptions to this rule, i.e. PFCs that are excreted more rap-
idly than would be predicted on the sole basis of their MW. This is the
case ofF-octyl bromide (PFOB, perflubron).F-octyl bromide is slightly
more lipophilic than a standard PFC of the same MW, due to its
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covered by a monomolecular film of the phospholipids. Practical PFC
emulsions need to remain stable for several years without significant
changes in particle sizes and particle size distribution. Frozen storage,thawing and reconstitution are clearly impractical and not acceptable.
Over time, submicronic PFC droplets grow, not through droplet
coalescence, but as a result of molecular diffusion (also known as
Figure 13. Producing a fluorocarbon emulsion requires use of a pharmaceutically
Figure 12. Industrial access to F-octyl bromide (perflubron): one step from a
pivotal perfluorochemical, F-octyl iodide.
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Ostwald ripening). In this process, individual PFC molecules leave the
smaller droplets, where the chemical potential is higher, to join larger
droplets, where curvature and, consequently, chemical potential is smal-ler (Fig. 14a). Molecular diffusion is characterized by a linear increase of
the average droplet volume over time and by a time-invariant droplet size
distribution function.
Droplet growth by molecular diffusion follows the Lifshitz-Slezov
equation:
drr3
dt x
8VmCDci9RT
fu
which says that the average droplet volume rr3 in a given emulsion
increases over timetproportionally to the water=PFC interfacial tensionciand to the solubility and diffusibility,Cand D, of the PFC in the aque-
ous phase. What can one do about slowing down molecular diffusion? By
chance, phospholipids (the emulsifier used in Intralipidand other phar-
maceuticals, including liposome preparations) are particularly apt at
reducing ci. Additionally, the solubility of the PFC phase in water
diminishes rapidly when a heavier (higher MW) PFC is added. The
longer organ retention of higher MW PFCs can be mitigated by using
a somewhat lipophilic PFC. This is the case when F-decyl bromide isselected as the heavier PFC. Figure 15 shows that the droplet growth
in anF-octyl bromide emulsion can be very effectively reduced by adding
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just a small percentage of its higher homologue,F-decyl bromide. Table 3
summarizes the properties of PFC emulsions that are related to their
being a dispersion of droplets.
Manufacturing of practical, small-sized, narrowly dispersed emul-sions requires a good deal of know-how. Extensive formulation and pro-
cess optimization led to OxygentTM AF0144, a 60% w=v concentratedPFC emulsion, that is heat sterilized, has an average droplet size of
0.16 mm after terminal heat sterilization, and a viscosity around 4 cP,
i.e. slightly above that of water. Its pH and osmolarity were adjusted
to 7.1 and 304 mOsm, respectively. It is stable for 2 years at 510C
and is ready for use.
PROSPECTS
Figure 15. Droplet growth by molecular diffusion in an F-octyl bromide emulsion
can be effectively repressed by addition of a small amount of a heavier PFC; effect
on organ retention can be limited by using F-decyl bromide, which is slightly lipophi-lic and benefits from faster excretion than non-lipophilic PFCs of similar molecular
weight (see Weers et al.,Artif. Cells, Blood Subst., Immob. Biotech.22: 1175 (1994)).
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Where do we presently stand in terms of using PFCs as O2 carriers?
There is no doubt that PFCs dissolve, transport and deliver O2in vivo.[3,5,7,8] A randomized, multicenter, European, Phase III clinical
evaluation ofOxygentin general surgery patients has established the abil-
ity of the emulsion to significantly reduce and avoid red blood cell trans-
fusion. The trial was conducted using an augmented acute normovolemic
hemodilution with PFC emulsion protocol. In the protocol-defined target
population (330 subjects with blood loss 20 mL=kg body weight) sig-nificantly greater avoidance of any red blood cell transfusion, as com-
pared to controls, was maintained through day 21 or day of hospital
discharge (P< 0.05). There was also a significant reduction in the num-ber of units of blood transfused (P< 0.001). From the clinical datacollected, the hemoglobin equivalency, in terms of added O2-delivering
Table 2. Some physical properties of F-octyl bromide (PFOB) and F-decalin
(FDC) compared
Property (units) Symbol PFOB
FDC
(cis trans)
molecular formula C8F17Br C10F18molecular weight (g mol1) Mw 499 462
melting point (C) m.p 5 10vapor pressure (torr, 37C) v.p 10.5 14
kinematic viscosity (centistokes, 25C) V 1.0 2.9
interfacial tension vs. saline (mN m1) ci 51.3 60
spreading coefficient (mN m1
) S (o=w) 2.7 1.5O2 solubility (vol.%, 25
C) [O2] 50 40
CO2 solubility (vol.%, 25C) [CO2] 210 140
critical solution temperature
(n-hexane, C)
CST (hexane) 20 22
solubility in water (mol L1) 5.109 10.109
solubility in olive oil (mmol L1) 37 4.6
Table 3. PFC emulsion characteristics related to their particulate nature
Small sizes=RBC (0:150:2 mm vs: 7 mm) yet no extravasationNumerous particles - facilitates O2 diffusion
Adjustable viscosity, close to bloodMechanical resistance (pumps, filters)
Foreign particles RES clearance
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