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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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