Highly fluorinated systems for oxygen transport, diagnosis and drug delivery

Colloids and Surfaces A: Physicochemical and Engineering Aspects, 84 (1994) 33-48 0927-7757/94/$07.00 0 1994 - Elsevier Science B.V. All rights reserved.

33

Highly fluorinated systems for oxygen transport, diagnosis and drug delivery Jean G. Riess Laboratoire de Chimie Moldculaire, Unit4 de Recherche Associke au C.N.R.S., Universik Antipolis, Facult6 des Sciences, 06108 Nice Cedex 02, France (Received

30 July 1993; accepted

15 October

de Nice-Sophia

1993)

Abstract This review summarizes the basic physical and chemical characteristics of fluorocarbons and fluorinated materials (especially surfactants), in neat or dispersed form (emulsions, vesicles), which determine their potential in medicine. These properties are discussed in relation to each major area of application (oxygen delivery, liquid ventilation, diagnosis, drug delivery). Specific topics are emphasized including, in particular, the lipophilic character of certain fluorocarbons, the stability of emulsions, and the stability, permeability and intravascular persistence of fluorinated vesicles. Key words: Blood substitute; Contrast agent; Diagnosis; Drug delivery; Drug targeting; Fluorocarbon; Liposome; Liquid ventilation; Oxygen carrier; Vesicle

1. Introduction: not only “blood substitutes” Fluorocarbons in medicine inevitably evoke thoughts of “blood substitutes”, the application that has received the most publicity by far. This has unfairly relegated to a position of secondary importance the numerous other potential biomedical applications that have germinated in the fertile minds of inspired scientists and physicians since the early days when Clark and Gollan [l] demonstrated that a mouse could survive while breathing an oxygenated liquid fluorocarbon. Although it may be strange to imagine such alien things as fluorocarbon droplets “replacing” red blood cells in one’s arteries, clinical evaluation of such oxygen carriers is now underway. Similarly, the ventilation of patients with liquid fluorocarbons was, until recently, just a science fiction movie theme; however, the fiction of Hollywood is now being rivalled by reality. Numerous medical applications involving fluorocarbons and highly fluorinated compounds are ,SSoIO927-7757(93)02696-C

Emulsion;

Fluorinated

surfactants;

now being investigated [2-S]. Oxygen-carrying fluorocarbon emulsions may be used as temporary blood substitutes as part of blood conservation strategies which, in conjunction with blood predonation and perioperative hemodilution, are aimed at reducing the need for allogeneic blood transfusion. They may be used during conditions of restricted blood flow such as occurs in myocardial infarction or stroke; for the prevention of ischemia during the percutaneous transluminal coronary angioplasty procedure; in cardioplegic solutions to protect the heart during cardiopulmonary bypass surgery; during extracorporeal circulation; as reperfusion solutions; to improve oxygen delivery to certain tumors, to make them more responsive to radiation and chemotherapy; for organ and tissue preservation, etc. A brominated fluorocarbon, perfluorooctyl bromide (perflubron), constitutes a most versatile and universal contrast agent for diagnosis using X-rays, magnetic resonance and ultrasound. Fluorocarbons may also be used as a tamponade during retinal repair and are being

J.G. Riess!‘Co/loids Swfaces A: Phpicochenr.

34

investigated

as oxygen

delivery

facilitators

in cell

cultures. More recently, highly fluorinated materials, especially amphiphiles and the vesicles they

represent

huge potential

a huge clinical

potential,

hence

a

market.

In the light of this diversity array of tailor-made products

2. Perfluorochemicals 2.1.

Attributes o~f[uouocurho~~s uwful

in biomedical

applicati0n.s

form, were shown to hold promise in drug formulation and delivery. Needless to say, all these applications

Eng. Asprcts 84 ( 1994 ) 33-48

of applications, an is desirable, which

may be based on various neat fluorocarbons, fluorinated amphiphiles, and diversely formulated emulsions, vesicles and other dispersed systems (Fig. 1). The intended biomedical applications impose severe constraints on the selection, purity, definition and bioacceptability of the fluorinated materials used, as well as on the formulation, processing conditions, characteristics and control of the emulsions, vesicles and other preparations of which they are components. The magnitude of these constraints depends, of course, on the dosage and administration route; they are obviously of lower magnitude for topical use than for high-dose intravenous injection. This review will briefly present the principal liquid and colloidal fluorinated materials at present under investigation, as well as their major present and potential applications in medicine. Because of the extent of the subject matter covered, only a limited number of references are given; these are mainly intended to give access to further publications in the various more-specific areas of interest.

(or perfluorochemicals) Fluorocarbons organic compounds in which all or nearly the hydrogen atoms are replaced The attributes which make selected liquid fluorocarbons

are all of

by fluorine atoms. certain properly

so attractive

for medi-

cal applications are numerous. They are water insoluble, chemically and biochemically inert, and have high dissolution capacities for oxygen (40-50 vol.%, i.e. 20-25 times greater than water). carbon dioxide and other gases [9]. Their inertness results from their strong C-F bonds (about 116 kcal mol-‘. i.e. 20 kcal mol-’ more than a standard C-H bond) and a dense coating of electron-rich, repellent fluorine atoms which protects the carbon backbone. They have a large-scale production feasibility using well-established technology [lo]. They are also stable. sterilizable and carry no risks of infection. The oxygen they transport, not being chemically bound to the carrier, is readily available to tissues, resulting in higher extraction rates and ratios than from hemoglobin. Likewise, there is no possibility of chelating and sequestrating nitric oxide. and therefore no possibility of interference [ 111 with the functioning of this important mediator. Fluorocarbons, being insoluble in water, do not diffuse into interstitial fluids. They are not metabolized and therefore do not present any metabolite-related toxicity. Compared with hydrogenated molecules of similar structure, they have lower surface tensions, lower kinematic viscosities, higher densities and higher compressibilities. Some have a positive spreading coefficient on an aqueous saline solution. These various characteristics have all proven to be beneficial for one or more applications. The absence of hydrogen, the presence of numerous 19F probes, the low acoustic velocity, and the possibility of making them opaque to X-rays by introducing a heavy halogen atom in their structure, form the basis of the unmatched potential of fluorocarbons

J.G. RieqJColloids

Swfaces

A: Physicochem.

Eng. Aspects 84 (1994)

in diagnosis. A certain degree of lipophilicity can be built into perfluorochemicals by appropriate substitutions and, as we see later (section 4.1), this has turned out to be essential in many applications. Conversely, the introduction of a perfluoroalkylated chain into a molecule enhances both lipophobic and hydrophobic interactions and can profoundly alter the molecule’s structure and properties, thus generating new behavior and potential for additional applications (sections 3 and 5). 2.2. Liquid ventilation Preclinical work and initial investigational clinical studies are underway to assess the usefulness of fluorocarbon liquid ventilation as a treatment for respiratory distress syndrome. This is a severe lung condition associated with high mortality and high morbidity for which only minimally effective treatment exists at present. Liquid ventilation with n-1-perfluorooctyl bromide, C,F,,Br, allowed efficient and homogeneous gas exchange at low inflating pressures. It also permitted the removal of meconium or edema fluid and the effective uniform administration of drugs by the pulmonary route [ 12-161. For such applications the most important properties of lluorocarbons include, in addition to inertness, immiscibility with water, low kinematic viscosity and low surface tension. The surface tension of fluorocarbons is typically 12-20 mN m -l, i.e. within the 5530 mN m-l range found in the presence of lung surfactant. Lipophilic fluorocarbons with positive spreading coefficients on saline and moderate vapor pressures, such as perfluorooctyl bromide or perfluorooctylethane, C,F,,C,H,, appear to be ideal for pulmonary applications since the fluorocarbon tends to disperse spontaneously over the pulmonary membrane and evenly reduces the surface tension. 2.3. Diagnosis Neat fluorocarbons are now either commercially available or in an advanced stage of clinical evaluation [17] for diagnostic use.

33-48

Neat hydrogen;

35

liquid when

fluorocarbons ingested,

do they

not

contain

will darken

the

image of the bowel by lack of signal when examined using standard magnetic resonance imaging techniques. This facilitates the distinction of the bowel from adjacent tissues and the detection of possible pathology [ 181. This application, which fills a well-documented need for a bowel marker, has now been approved for medical use by the US Food and Drug Administration. Immiscibility with water, density, fluidity, rapid spreading and transit time are again valuable attributes of the fluorocarbon used for this application, perfluorooctyl bromide. Perfluorooctyl bromide, being radiopaque, is also being investigated for use with X-rays in gastroenteropathy when a bowel obstruction is suspected [ 191. Instillation of neat perfluorooctyl bromide in the trachea has been proposed as a means of assessing lung structure and condition using X-rays [20]. The absence of a magnetic susceptibility difference between fluorocarbons and tissues led to the development of fluorocarbon-filled pads which are applied externally on the patient’s body to improve magnetic homogeneity when fat saturation techniques are employed during magnetic resonance imaging [21]. The removal of the interface between skin and air (which has a large magnetic susceptibility difference vs. tissues) leads to images with significantly increased accuracy. 2.4. Miscellaneous fluorocarbons

applications

of neat

The possibility of administering neat oxygenated fluorocarbons transperitoneally to improve arterial oxygenation [22], or enterally to treat intestinal ischemia [23], has been explored in animal experiments. The high specific gravity of fluorocarbons, typiis an asset when retinal cally 1.8-2.0 g ml-‘, manipulation is required during eye surgery. Fluorocarbons have also been proposed for replacement of vitreous fluid and for increasing

J.G. RiessiColloids

36

Surfaces A: Physicochem.

oxygen delivery to the eye 124-261. The use of fluorocarbons to regulate 0, and CO, supply to

carbon

high density cell cultures and to assure nontraumatic agitation has also been investigated

fluorophilic

c271.

emulsions.

Because

the surfactants

mine

external

appearance

3. Modular F-alkylated

amphiphiles

emulsions,

Et~g. Aspects 84 ( 1994 ) 33-48

vesicles

microemulsions

[30P331.

F-surfactants,

extremity,

droplets

or vesicles

drug delivery, vehicles

Surfactants are widely employed in the biomedical and pharmaceutical areas as dispersants, solubilizers, detergents, extractants for proteins, emulsifiers, constituents of liposomes and other drug delivery systems, and in the formulation of numerous drug products for human and animal uses [ 28,291. The introduction of a perfluoroalkylated chain in the hydrophobic tail of a surfactant greatly increases its amphiphilic character, resulting in considerably enhanced surface activity. The amounts of fluorinated surfactant (F-surfactant) needed to reach the CMC are significantly less (which indicates higher efficiency) and the surface tension reached at the CMC is also significantly lower (therefore having higher effectiveness) than for hydrogenated analogs. The huge tension that normally exists at the interface between water and a fluorocarbon (typically over 50 mN m-‘) can be reduced to a few milliNewtons per meter with minimal amounts of an F-surfactant. F-alkyl tails differ from hydrogenated ones with respect to their volume, their rigidity and, most importantly, by the stronger hydrophobic interactions they develop. As a result, fluorinated surfactants exhibit a stronger tendency to collect and organize at interfaces, or to self-associate into supramolecular assemblies, two ways of avoiding the thermodynamically unfavorable contact between their highly fluorinated part and water. The synthesis and evaluation of many series of well-defined F-surfactants intended for biomedical uses were triggered in the 1980s for the purpose of developing injectable oxygen carriers, contrast agents and drug delivery systems based on fluoro-

eventually,

to be

largely

of the

deter-

emulsion

they are part.

to help control

the fate of such particles tion, intravascular

to their

expected

fluorocarbon-in-water

of which

were also expected 3.1. Components for colloidal

owing

were indeed

ideally suited for stabilizing the

and fluorinated

they

and modulate

in vivo, i.e. their recogni-

persistence,

the extent

biodistribution

of their

effects

and,

and

side-

effects. Figure 2 presents some typical examples of F-alkylated amphiphiles (l-24) synthesized in our laboratory now

and illustrates

available.

the variety

A versatile

design was adopted

of structures

modular

structural

which allows stepwise modifi-

cation of the surfactant’s size, shape, charge, hydrophilic, lipophilic and fluorophilic character, nature of the head, number

of tails (identical

or different),

spacers,

units,

sites, etc. in

order

connecting

connection

to allow manipulation

biological

characteristics

of the physical

of the emulsions,

and

vesicles

and other colloidal systems in which they are involved. This work involved the controlled synthesis of numerous

families heads

of fluorinated

whose

polar

derive

amino

acids, phosphatides,

ties, trishydroxymethyl

from

and biological

moie-

and combinations

given to their purity

assessment.

Specific features to meet certain introduction

sugars.

polyoxyethylene

moieties,

thereof, with special attention

can be built into F-surfactants

objectives;

of a relatively

such examples

are the

hidden

charge

as in 23 or 24, or of a polymerizable group as in 15, or the possibility increase

surfactants

polyols,

in size and hydrophilic

negative

terminal vinyl of incremental character

of the

polar head, as in 3, 4, 12 or 13, or of varying the distance between charges, as in 17 and 18, etc. Finally,

the amphiphiles

groups susceptible

can be fitted with head

to recognition

by specific recep-

tors. They can then be used for targeting

particles

for specific tissues. This is the case, for example, with polar heads derived from glycosides such as

J.G. RiesslColloids 1

2

3

Surfaces A: Physicochem.

Eng. Aspects 84 ( 1994 ) 33-48

CnFLn+,(CHz)mOP(0)-*OCHICHIN’(CH,),

la

CnFzn+1(CHz),C00

2a. ” = 4. m = IO Zb.n=h,rn=ll 2c.n=X.m=5 2d:n=h,m=10 Ze,n=R,m=4

CnF~n+,Ktl~),,,COO

IL-

OPl(~)-OCHICH,F:‘(CH~),

/OH

n=X.m=2

/OH

14 OH

OH

C,F,,+,~CH~~O~O~IO(C,H4O~~H31, Cn~2n+l(C~2)mC00

4

37

15 Ol~O~IO~CH~Cll,~)pCH,l,

C,F2,+tKH,),CO0

16

6

HOCH~(CHOH),CH20CH2CH=CH=CH

7

HOCttZ(CHOH),CH*OC(0)(CH

C,F?,+, 1) m C n F*n+,

OH 22 COK:H2~Cnt’2n+,

10

23 i)H

(XI tt

Ho$(&o+

(CH,IpCII,

24 OH 12

,KH&C,FL,+,

0(CH2)2CnF2n+, i)H

Na+ lb I, = 6. m = 5 12b n=x,m=7

C,F2,,+,C,H,SICH,FHl~l UOWHUCH

zOH)z

2s

C&n+,(Ctld~\ 13

,CHNHC(O)C,tl,S[CH,CllC~O~~llC~CH~Otll~l~H CH,KW,,,, C,F2,+,(CtI=CH-Ctt2).,ICll~~~-

Fig. 2. Typical

examples

of F-alkylated

amphiphiles

9, 10 or 24 which can be recognized lectins [33(a)-351.

(l-29,

illustrating Refs. 30-33).

by membrane

Such F-surfactants allow the stabilization of fluorocarbon emulsions, either when used alone or in combination with other surfactants. The reduction of the fluorocarbon-water interfacial tension, although it does not guarantee emulsion stability, usually facilitates emulsification, reduces the dispersion’s free energy, can contribute to strengthening the surfactant film and, most importantly, reduces the rate of molecular diffusion of the fluorocarbon, thereby increasing the stability of their emulsions [36]. The combination of two surfactants, one of which (for example 7 or 11) acts primarily by reducing the interfacial tension and the other by steric stabilization (for example

the variety

of structures

c

O\’

now

available

(obtained

from

a poloxamer of the Pluronic F-68 type), and which are capable of hydrogen bonding, provides interesting synergistic emulsion stabilization effects [ 321. The fact that the presence of F-alkylated tails inside the surfactant film can influence particle recognition is shown by the in vitro phagocytic uptake, by mouse peritoneal macrophages, of polystyrene microspheres coated with the fluorinated surfactant 12b, in the presence of serum, being lower than when the microspheres were coated with its hydrogenated analog [36(a)]. Fluorinated amphiphiles were also found to form membranes, vesicles and other colloidal systems readily. Moreover, the properties of these systems are significantly different from those formed by their hydrogenated analogs (Section 5).

.J.G. Riess/Coiloids

38

Preliminary

biocompatibility

ing. High intravenous

tests are encourag-

LD,, (or maximum

Thus, for example, MTDs of around 4 g kg-’ and 8 g kg-’ body weight were found for dispersions of compounds

2a

and

fluorinated

12a,

respectively

surfactants

[33].

are also dra-

matically less hemolytic than their hydrogenated analogs, in spite of their significantly higher surface activity, indicating a lesser tendency nated tails to penetrate the membrane cells [ 371.

4. Fluorocarbon emulsions

tolerated

doses (MTDs)) were found in mice, especially for some of the double-tailed fluorinated amphiphiles.

Interestingly,

SurfacesA: Ph~sicoclten~ Eng. Aspecrs X4 ( 1994 ) 33- 4N

of the fluoriof red blood

3.2. Ampkipkilic drug carriers. Pro-drugs Surfactants are often used as components of colloidal drug vehicles. In some cases they can also be covalently bound to the drug, thereby facilitating its incorporation into an appropriate delivery system. Amphiphiles such as 12, 13 or 24, for example, can be employed to bind drugs, using one or more of their alcoholic hydroxyl groups or the remaining acid phosphate function, and constitute pro-drugs which may then be carried and delivered in vivo in the form of emulsions, vesicles or other dispersions. The same amphiphiles can simultaneously carry a targeting group. Delivery of metal-based cytotoxic drugs such as platinum and palladium complexes using perfluoroalkylated amphiphilic ligands such as 25 has been investigated [ 381. The metal-ligand complexes were incorporated into EYP liposomes or fluorocarbon emulsions. The cytotoxic activity of the liposome-entrapped drugs was preserved in in vitro experiments on head and neck carcinoma cells. Use of fluorocarbon emulsions as the delivery system should allow the simultaneous delivery of the cytotoxic agent and of oxygen to the tumor, which could result in beneficial synergistic effects. For all these reasons, well-defined fluorinated surfactants are expected to find a specific role as components of membranes, emulsions, vesicles and other colloidal systems useful for the delivery and targeting of drugs and other material.

4.1. Selection Of,%Uoroc(IrboMS,f01.injectuble emulsions: lipophilic,pzro~ocurbons

True

fluorocarbons

Some lipophilic duced terminal

are essentially

character

by appropriate positions,

lipophobic.

can, however,

substitution,

and can impact

be intro-

especially profoundly

in -

and favorably - on some of the key requirements cf injectable fluorocarbon emulsions. i.e. rapid excretion, prolonged stability and minimal sideeffects. 4.1 .l. Higher excretion rutes Intravenously injected fluorocarbon droplets are progressively cleared from circulation by phagocytosis by macrophages located in the liver, spleen and bone marrow (the reticuloendothelial system (RES)) where the fluorocarbon is temporarily stored. Eventually it is excreted unchanged through the expired air. The half-life of the fluorocarbon in the body should not exceed a few weeks and should be preferably a few days [39]. Lipophilicity has been suspected for years to be important in this excretion process [40]. One of the steps in the excretion of fluorocarbons was indeed found to involve their transport in the blood, from the above storage organs to the lungs, via lipid carriers such as high density lipoproteins [41]. Hence the importance of the solubility of fluorocarbons in lipidic carriers, cell membranes and other fatty compartments. Measuring critical solution temperatures (CSTs) of fluorocarbons in hexane has been proposed as a means of assaying their lipophilic character, thereby predicting organ excretion rates [40,42]. However, none of the fluorocarbons investigated at that time were significantly lipophilic, and their excretion rates correlated as well with molecular weights as with CSTs [39]. It is only when the “deviant” behavior of perfluorooctyl bromide [ 391 (it has a faster excretion rate and its EYP-based emulsions are more stable) was recognized as resulting from the lipophilic character introduced

J.G. RiesslColloids

Surfaces A: Physicochem.

by the terminal

bromine

atom

Eng. Aspects 84 ( 1994) 33-48

[43,44]

that

the

importance of using lipophilic compounds was substantiated. It has been shown since then that the organ retention time of a fluorocarbon can be reduced

by subsequent

of a lipid emulsion rier capacity

intravenous

which provides

to the blood

administration additional

car-

for the fluorocarbon

products,

39

such

as Fluosol

(Green

Cross

Corp.,

Osaka, Japan) and Perftoran (Perftoran Co., Pushchino, Russia), have to be shipped and stored frozen. The former product

needs to be reconstitu-

ted after thawing by admixing annex solutions prior to administration. The dilemma, at the time they were developed,

was that those fluorocarbons

[45]. Likewise, it was found that the saturation of liver microsomal membranes with perfluorochem-

from which stable emulsions could be produced were retained in the RES organs for an overly long

icals was a linear

duration, while those with acceptable excretion rates yielded only poorly stable emulsions [39]. This problem has now been overcome [36,47]. The primary cause of particle size increase over time in fluorocarbon emulsions, including concentrated ones, has been identified as resulting from Ostwald ripening (molecular diffusion of the fluorocarbon from small droplets to larger ones where its chemical potential is lower) [ 36,471. It has been known for a long time that, in accordance with the Lifshitz-SlezovWagner theory, Ostwald ripening can be slowed down by adding small amounts of a heavier compound that is less soluble and less diffusive than the compound being emulsified [ 48,491. However, heavy fluorocarbons usually have long organ retention times. It is only recently that Ostwald ripening suppression agents with short retention times in the reticuloendothelial system have been identified, and these consist again of lipophilic fluorocarbons [47,50]. Emulsions of perfluorooctyl bromide have, for example, been stabilized significantly by the addition of a few per cent of its higher homolog, perfluorodecyl bromide, C,,F2,Br. The latter compound has lower water solubility, and the lipophilic character induced by its terminal bromine atom results in faster excretion (half-organ retention time around 25 days [95]) than its higher molecular weight would this approach predict, making otherwise practicable.

function

of the CST, and also

that a lipophilic fluorocarbon can be extracted from the membranes by a less lipophilic one [46]. Significantly, lipophilic Auorocarbons have now been identified which have short organ retention in spite of relatively high molecular weights (Fig. 3). Such compounds have been investigated as oxygen carriers; this is the case, for example, with C8F1,Br, C,F1,C,HS and C8F,,CH,-CH= CH-C,H, [S]; others have found uses as emulsion stabilizers (see below). 4.1.2. Improved emulsion stability Lack of stability of fluorocarbon emulsions designed for intravascular injection has been a serious problem until recently. First-generation

-s

2 -40 .z v -60

1

300

, 400

500

600 Molecular

700 800 900 Weight (dalton)

Fig. 3. Critical solution temperatures (CSTs) of perfluorochemicals in hexane (as a measure of their lipophilic character) as a function of molecular weight (M,): A, acyclic; 0, monocyclic; 0, bicyclic; x , tricyclic; A, lipophilic. The larger, filled triangles correspond to exceptions to the linear CST vs. M, relationship, and are not included in the linear regression calculation (R= 0.72 for linear fluorocarbons; R = 0.83 for cyclic fluorocarbons). (Data obtained from Refs. 43, 52, 94 and 95.)

4.1.3. Surfactantfilm structuration eflects Small amounts of highly lipophilic additives consisting of partially fluorinated, partially hydrogenated linear alkanes and alkenes, R,R,, such as, for example, C,F,,C,,H,, (26) or C,F,,CH=

J.G. Riess.!Colloidu SU&CPS A: Ph_rsicochern. Eng. Aspects 84 ( 1994 1 33-48

40

CHC,H,,

phospholipids

(27), sometimes called “dowel” molealso led to dramatic stabilization of

cules, fluorocarbon-EYP

emulsions

[36,.51]

and,

to a

at the surface

is reduced

from about

is added,

indicating

of the fluorocarbon

85 f 1 to 73 + 1 w when 27 tighter

packing

[54].

This

F-68 [ 521

effect, which is not seen when C10F2rBr is used as

emulsions. Little or no particle size increase was, for example, found when a C,F,,Br-EYP emulsion stabilized with 26, taken in the same molar propor-

a stabilizing agent, implies that the dowel molecules are present in or near the phospholipid film. rather than dispersed throughout the continuous

tion as EYP, was stored for 9 months

fluorocarbon

lesser extent,

of fluorocarbon-Pluronic

at 40°C. This

approach also allowed easy control of particle sizes over a large range of sizes (Fig. 4) [ 531. The

4.1.4. hhimnl

EYP-dowel system can be regarded as a complementary combination of a hydrophilic/lipophilic amphiphile with a lipophilic/fluorophilic one. The stabilizing effect is probably the result of improved adhesion between the classical hydrogenated surfactant film and the fluorocarbon droplet it surrounds. There are indeed strong indications that the added material not only acts by lowering the solubility and diffusibility of the dispersed phase and of the interfacial tension but also has an effect on the structure of the interfacial film. This is demonstrated, surface area

for example, occupied by

by the fact that the the polar heads of

(pm)

0.40 0 30

phase. side-ej&Ts

During the course of the removal of the emulsion droplets from circulation by RES macrophages a number of biological effects can be observed which are characteristic of this normal host-defense process. These effects consist of cutaneous flushing and flu-like symptoms. They are reversible and probably result from a stimulation of the macrophages and subsequent release of intracellular products, in particular, metabolites of the arachidonic acid cascade and cytokines [SS]. Constant particle sizes over time and reproducible narrow particle size distributions are also important from a biocompatibility standpoint

0.14 006

J

m I

.1

I

I,

1

l/EYP

I

I

(c/o w/v)

I

I,,

to

Fig. 4. Control over particle sizes in fluorocarbon emulsions using a mixed fluorocarbon’hydrocarbon “dowel” molecule. C,F,XH= CH&H,, (from Ref. 53). The particle size histograms represented were measured after 3 months of storage at 40‘ C and reflect shelf stability.

J.G. RiesslColloids

because

Surfaces A: Physicochem.

some of the above

be related

to emulsion

size is also essential

side-effects

particle

smaller, the better, usually.

Eng. Aspects 84 (1994)

size

Maintenance

simply

appear

to

[56];

the

of particle

for maintaining

con-

stant pharmacodynamic behavior, intravascular persistence and a consistent side-effect profile throughout Another

the entire shelf-life of the product. side-effect,

increased

pulmonary

33-48

gas transport and delivery function when blood transfusion is undesired, ineffective, or contraindicated.

They are primarily

or reverse temporary

destined

to prevent

tissue hypoxia.

The road to the development of an effective injectable oxygen carrier has not been a smooth one.

resid-

41

Hemoglobin-based

extensively

investigated

products

have

been

since the 1930s [ 601. The

ual volume (IPRV), observed with certain fluorocarbons in certain animal species, appears to be

pioneering work of Clark, Sloviter and Geyer in the late 1960s raised tremendous hope of rapidly

related to their low boiling point (hence high vapor pressure) [ 57,581. It has not been observed in man either with Fluosol (over 1500 patients) or with perflubron emulsions (over 100 subjects and patients). IPRV occurs during elimination of the fluorocarbon when alveolar microbubbles grow as a result of a gas osmosis effect, in which fluorocarbon vapors dilute the gases inside the bubbles, and causes the diffusion of gases out of the bubbles to be slower than their diffusion into the bubbles [ 55,581. Adding a lipophilic termination to a fluorocarbon normally results in lower vapor pressure, which lowers the IPVR effect. Generally speaking, higher lipophilicity, by allowing the use of less volatile compounds without compromising organ excretion rate, reduces those side-effects which are related to vapor pressure.

achieving an oxygen carrier based on fluorocarbons, which could be used at least temporarily as a universal substitute for red blood cells [61]. Unfortunately, the first generation of commercial products, developed in the late 1970s did not meet expectations. Their greatest single drawback is certainly their lack of stability, resultant stringent storage requirements, complex reconstitution procedure, and overall lack of user-friendliness. The assessment of their insufficiencies helped, however, to set the conditions for progress [39,44]. A decade later the development of significantly more stable, more effective fluorocarbon emulsions clearly demonstrated that the limitations of the often-challenged first-generation products can be overcome [ 6,441. A second generation of products, which incorporates significant breakthroughs and improved knowledge and technology, has been developed [62,63] and is now undergoing clinical trials. The one point on which the least progress has

4.2. “Blood substitutes”

and other oxygen-delivering

products

The need for a blood

substitute

is now undis-

puted. Over the past decade the attitude of both the public and the physicians vis-a-vis blood transfusion has changed radically as awareness has increased that this treatment is not entirely free of risk, although our blood supply has never been as safe as it is today. The American College of Physicians has gone so far as to invite the medical community to “regard elective transfusion with allogeneic blood as an outcome to be avoided” [59]. It must be clear, however, that fluorocarbon emulsions are not artificial blood. They are “simply” 0, and CO, carriers expected to serve as temporary substitutes for red blood cells in their

been achieved thus far is intravascular persistence. The (dose-dependent) half-life of the present emulsions in the circulation is typically 4-12 h. This is usually sufficient for use during surgical procedures (which alone represent about two-thirds of the situations where red blood cells are transfused) or in diagnosis. However, it is not sufficient for cases of trauma if the objective is to avoid the eventual transfusion of allogeneic blood, let alone for chronic anemia, for which the product’s efficacy should extend over several weeks [IS]. Prolonging and controlling intravascular persistence by devising emulsions which evade premature phagocytosis is a challenging objective for the

42

J.G. Riess/Colloids

colloid chemist. This objective considers injectable preparations containing particulates in general, whether

fluorocarbon

droplets

or drug-carrying

Surfaces A: Ph_rsicochem. Eng. X.spects 84 ( 1994 ) 33-48

with minimal accumulate and injured

side-effects.

their improved

recognition, opsonization and phagocytosis of such particles when injected in the blood stream [Xl.

been developed

“Blood substitutes” and injectable oxygen carriers for uses indicated in Section 1 will not be further reviewed here. The reader is referred to the numerous recent articles in which state of development is summarized [2-S]. 4.3. Multi-modality

their present and analyzed

contrast ugents

Diagnosing pathologies with confidence often requires that an agent be administered that enhances the contrast between normal and abnormal tissues. When conventional X-ray or computerized X-ray tomography (CT) are used as the diagnosis modality, contrast can be created by agents containing heavy atoms, such as iodine, that are opaque to X-rays. Most of the radiopaque agents used at present are iodinated and hydrosoluble. These hydrosoluble agents have, however, a very short intravascular persistence owing to their rapid diffusion in the tissues. The time available for examination is therefore short and repeated injections are often necessary. Moreover, watersoluble iodinated contrast agents are not adapted to every type of diagnosis; for example, they are inefficient for hepatic diagnosis. These limitations could, in principle, be surmounted by using an emulsion of an oily contrast agent, but the lipidic iodinated contrast agent emulsions developed so far (Lipiodol, EOE-13) have stability and toxicity problems, and do not allow the distinction between tumors and blood vessels [ 171. These problems have finally been overcome by using concentrated emulsions of perfluorooctyl bromide. This agent allows optimal imaging of the blood pool and the normal Kupffer cells, resulting in high sensitivity liver metastasis detection using CT, with ample time for imaging, and is associated

droplets

also

in macrophages in tumors, abcesses or infarcted tissues, thus leading to

capsules or vesicles, and implies that appropriate surface coatings be found which prevent premature

Another

Perflubron

imaging

radiopaque

[ 64,651. fluorocarbon

emulsion

has

which allows lymph node imaging

[ 66,671. Accurate staging of lymph node involvement following diagnosis of a primary cancer is essential for the determination of the appropriate treatment. The emulsion particles. administered subcutaneously, are taken up by the afferent lymphatic vessels or phagocytozed by macrophages and accumulate in the regional lymph nodes. thereby enhancing the nodal interior on CT. The pattern and degree of contrast should allow the distinction of normal, hyperplastic and neoplastic nodes [ 17,671. Efforts have been devoted to finding more effective fluorocarbons with higher radiopacity. The commercially available perfluoroalkyl iodides, with their iodine atom in the terminal position, are too reactive, and hence too toxic for this use. More recently, however, a series of iodinated, partially fluorinated molecules such as C,F,3CH=CIC,F,, (28) have been prepared, which appear to have the required biological inertness. Compound 28 is about four times more radiopaque than perfluorooctyl bromide, gives stable sterilizable water-in-oil emulsions and appears to bc well tolerated in mice and rabbits [68]. Fluorocarbon emulsions are also effective as contrast agents in sonography because of the presence of reflective particles and of the intrinsic echogenicity of perfluorochemicals (due to high density and compressibility). Long-lasting venous contrast, related to long intravascular persistence, is obtained and allows imaging of the vascular tree, assessment of tissue perfusion and organ function, detection of liver tumors, renal infarction, ureteral obstruction, etc. [ 17,691. Because T, and T, relaxation times are shortened when the paramagnetic 0, molecule is present, 19F NMR can also be used to evaluate tissue po, [70], for example in the myocardium or in tumors [71].

J.G. RiessfColloids

4.4.

Topical

content

fluorocarbons,

Eng. Aspects 84 ( 1994 ) 33-48

applications

Viscoelastic water

Surfaces A: Physicochem.

43

the availability of appropriate used as membrane constituents,

transparent

gels

with

very

high

(up to 98%) were

obtained

from

water and mixtures

of hydrophobic

modifiers

and targeting

Bilayer genated

properties surfactants

amphiphiles to be stabilizers, surface

devices. of fluorinated

and

have been recognized,

hydroin partic-

non-ionic fluorinated surfactants. These materials were identified as consisting of water-in-(water-inoil microemulsions) emulsions. Such gels were sug-

ular by Kunitake [79] and Ringsdorf et al. [SO], to be significantly different. Fluorinated bilayers are less permeable than the hydrogenated ones

gested to have potential

toward

pharmaceutical

in topical

and cosmetic

preparations

for

uses [ 721.

Formulations composed of lecithin-stabilized fluorocarbon emulsions and collagen have been proposed for improvement of wound healing [ 733. Their study also led to better knowledge of how proteins adsorb onto emulsion droplets and which interaction forces are at work. Fluorocarbon gels for cosmetic applications and wound healing have also been obtained by concentrating emulsions via centrifugation [ 741 or by adding a thickener [ 751. 5. Fluorinated vesicles Liposomes, vesicles and other organized supramolecular systems are being investigated extensively as carriers for delivering and targeting drugs. contrast agents, genetic material, hemoglobin etc. The main objectives are to avoid premature breakdown of their contents, reduce tissue toxicity, target specific cells, and control drug release (see, for example, Refs. 76678). Progress in this field requires, however, that the shortcomings of the present liposomes (such as poor chemical and physical stability, rapid clearance from the circulation, poor retention of encapsulated materials in biological fluids, limited control of drug release, and difficulties in large-scale preparation), which severely restrict their applications in the pharmaceutical area, be overcome. This ideally would require an understanding of and control over membrane structure and permeability, particle size and particle size distribution, surface charge, stability on the shelf and in the blood, in vivo recognition, and hence intravascular persistence, biodistribution, pharmacology of entrapped drugs and vesicle components, etc. These objectives again presume

ions and small molecules,

and when mixed

tend to form separate domains which can be clearly visualized by electron microscopy [ 8 11. Their shelf stability, encapsulation stability in biological fluids, in vivo circulation time, etc. were, however, not documented. It was timely to investigate fluorinated systems from the viewpoint of drug delivery and in particular to evaluate what the newly synthesized fluorinated amphiphiles could contribute, for example, in terms of vesicle stability, drug entrapment stability, permeability and intravascular persistence. The recent availability of several series of welldefined perfluoroalkylated surfactants suitable, a priori, for in vivo use, with modular structures and properties, has made this undertaking almost inescapable. 5.1.

Vesicle formation

and physical

stability

The presence of a fluorinated chain in an amphiphilic molecule strongly enhances its tendency to form vesicles (F-vesicles) and other supramolecular assemblies. Various neutral and cationic amphiphilit compounds with one, two or three F-alkylated chains have been found to form stable bilayer membranes and vesicles [79,80]. It was shown that the fluidity of fluorinated liquid crystalline bilayers, as assessed by fluorescence depolarization experiments, was lower than those of hydrogenated vesicles [ 821. The F-vesicles were also reported to undergo less fusion and/or component exchange with other vesicles, fluorinated or not, than do classical liposomes [ 81-831. Stable F-vesicles have now been obtained from zwitterionic F-alkylated phosphocholines such as 1 [ 851, phospholipids such as 2 [ 841, from neutral

J.G. RiessiCol1oid.s Surfaces A: Physicochem.

44

glycolipids

such as 15 [34]

phosphates may

such

infer

stability,

as 24 [35].

different physical

such

(particle

distributions)

stability

to encapsulated

facets

The term

meanings stability

with respect multiple

or from anionic

as chemical

fluids, stability

material,

now

stability

sizes and size

in biological

are

sugar

etc. These

being

extensively

The strong

proclivity

vesicles is strikingly short

pounds

of F-amphiphiles

demonstrated

single-chain

such as la form stable assemblies,

ules and

while

com-

vesicles and other

including

analogs uted

amphiphiles

with respect to hydrogenated

of la, which form only micelles, was attrib-

primarily

actions chains.

to the strong

generated

The F-alkylated i.e. saturated

analogs

with long room-temperature months at room temperature) [ 841. Depending

ribbon-like

The thermotropic phatidylcholines the fluorinated total

length

tail

such as 2,

of the main com-

readily form highly unilamellar vesicles shelf life (at least 3 without the need for on chain length,

phase was also obtained behavior

inter-

fluorinated

phosphatidylcholines

fluorinated

any additive

hydrophobic

by the highly

ponents of natural membranes, stable, heat sterilizable small

unusual

of F-alkylated

an

[ 861.

of the

hydrophobic

chain.

and rigidity,

which usually

single-

and

sugar phosphates,

such as

24, readily form vesicles, as well. These vesicles are significantly more stable when one of the hydrophobic tails of the amphiphile is fluorinated. Thus those

formed

from 24 were stable

at 40°C

hydrogenated

while

analogs

those

for at least 3

made

precipitated

from

totally

after 12 h [35].

results in higher critical phase-transition double-tailed

5.2. Vesicle permeability membrane core concept

-- thejuorinated

One possible way of reducing the vesicle membrane’s permeability to both hydrophilic and lipophilic material was to create an impermeable repellent fluorinated sheet inside the membrane’s lipidic film (Fig. 5). As seen above, such F-vesicles could be produced from a variety of fluorinated amphiphiles. These vesicles were expected to protect the encapsulated material from external agents, including serum and other biological fluids, and to slow down its release. The internal fluorinated core was also likely to have repercussions on the packing and possibly the conformation of the polar heads, and hence, again, on the interaction of the vesicle’s membrane with serum and other components. The lower permeability of F-vesicles to ions and small molecules had been recognized early on [ 831. Different ways of fine tuning the membrane perme-

jluorophilic

I ~$F~~~~~ic} core of variable thickness

Longer

tails cause enhanced hydrophobic hence increased membrane packing

crystal-to-liquid crystal atures (69°C for 2~).

double-tailed

super-

phos-

is very sensitive to the length of tail and, to a lesser extent, to the

fluorinated interactions,

Neutral

Anionic

and helical

[ 341.

disks, glob-

hydrogenated

do not, unless rigid rod-like segments, hydrogen bonding, ion pairing, polymerization or rigid additives (cholesterol) are involved [SS]. This drastic difference in behavior

or tubules

were obtained

by the fact that

perfluoroalkylated

supramolecular fibers,

to form

structures

structures

months

investigated.

even

disk-like

Eng. Aspects 84 ( 1994) 33-4X

-

temperglycolipids

such as 14 and 15 were also found to form supramolecular assemblies readily. For double-tailed compounds, depending on the peptide spacer utilized, unilamellar and multilamellar vesicles or

Fig. 5. Formation, from perfluoroalkylated glycerophosphatidylcholines, of fluorinated vesicles (F-vesicles) and membranes with a hydrophobic and lipophobic core within their bilayer membrane (taken from Ref. X9).

J.G. RiesslColloids

Surf&es

A: Physicochem.

Eng. Aspects 84 (I 994) 33-48

45 1.5

ability can be envisioned. One can increase the length of the fluorinated segment in the amphiphile, modify

the

R,R, ratio in the hydrophobic

will be significantly different, depending on whether one uses amphiphiles with one fluorinated chain chain

present

1

tails,

have both fluorinated and hydrogenated chains present, and dose them. In the last case the result

and one hydrogenated

,,--

-

-

.-

DMPC F-WC

/

2a

2 r-c----eDPPC

II

c

in the same

molecule, or mixtures of two surfactants having either two fluorinated chains or two hydrogenated ones. Indeed, fluorinated chains do not tend to pack with hydrogenated chains unless forced to do so. When not forced, they will tend to segregate to form separate fluorinated or hydrogenated domains [79,80] which could be exploited to control membrane permeation and reactivity and to simulate biological processes. Miscibility can be forced by adding mixed fluorinated/hydrogenated amphiphiles to mixtures of totally fluorinated and totally hydrogenated amphiphiles [ 831. Vesicles with an outer fluorinated monolayer and an inner hydrogenated one have been prepared using bolaamphiphiles having one hydrocarbon and one fluorocarbon segment [ 873. In contrast, polymerized vesicles with clearly separate hydrogenated and cleavable fluorinated domains were designed to simulate the hole formation process in a tumor cell attacked by a macrophage [SO]. The presence of a sufficiently long F-alkyl tail in a saturated phosphatidylcholine was shown to decrease dramatically the membrane’s lipophilicity and permeability. Thus, when the partitioning of the lipophilic/hydrophilic probe TEMPO (2,2,6,6tetramethyl-l-piperidyloxy) between aqueous and lipidic phases of dispersions of various fluorinated and hydrogenated phosphatidylcholines was investigated by electron spin resonance, it was found that the probe did not penetrate the membrane of the vesicles formed from F-alkylated phospholipids of type 2 (with the exception of 2a which has short R, and long RH segments). The sudden change in vesicle permeability observed for hydrogenated lipids at their transition temperature was not detected with the phospholipids with the longer fluorinated chains (Fig. 6) [ 86,881.

0.0-r

2X0

,

300

,

320

I

,

340

360

Temperature

,

,

380

( K)

Fig. 6. The permeability of fluorinated vesicles, compared with hydrogenated vesicles, to the paramagnetic probe TEMPO. (a) ESR spectrum showing the peak height ratio, hip, for TEMPO partitioned between hydrophilic and hydrophobic regions. (b) The ratio h/p vs. temperature for vesicles made from dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DMPC) and from various fluorinated phosphatidylcholines (F-GPC, see Fig. 2; taken from Ref. 86).

Similarly, the release of entrapped 5(6)carboxyfluorescein (CF) from such fluorinated vesicles, in a buffer or in human serum, is considerably slower than from hydrogenated liposomes, whether the membrane is in the fluid or in the gel state. Leakage half-life in serum, at 37°C from vesicles in the gel state made of compound 2b was 165 h with 35 h for classical compared egg phospholipid-cholesterol liposomes [ 891. CF leakage was less affected by serum for F-vesicles than for hydrogenated ones, indicating that the fluorinated core inside the membrane protects the vesicles against the destabilization effects of serum components. The probe entrapment stability of the fluorinated vesicles was highly dependent on the RF/R,ratio in the hydrophobic tail; it increased much more strongly with the length of the fluorinated segment than with that of the hydrogenated segment. The vesicles formed from double-chained anionic sugar phosphates, such as 24, and analogs with glucose (grafted in position 3) or galactose instead of mannose, all encapsulate CF. The permeability of the membrane to CF was highly dependent both on the length of the fluorinated chain and on the nature of the sugar which constitutes the surfactant’s polar head [ 351.

J.G. RiessjColloids

46

Swfaces

hydrogenated

A: Phq’sicochrm. Eng. Aspects 84 ( 1994) 33-48

compounds

such as C,F,C,,H,,

are

Another means of creating an internal fluorinated barrier to membrane permeation was devised mixed consists of incorporating which

incorporated in the vesicles’ membrane [92]. The repercussion of the F-terminations on the

fluorocarbon/hydrocarbon cles made from classical

vesicle’s surface is also strikingly demonstrated by the significantly prolonged intravascular persis-

[90]. Thus, incorporated (DMPC) significant

compounds hydrogenated

into vesisurfactants

when C,F,CH=CHC,,H,, (28) was into dimyristoylphosphatidylcholine

made from perfluoro-

liposomes

in a 2 : 1 molar

proportion,

alkylated phospholipids larly sized conventional

reduction

of the leakage

of carboxy-

some of the more recent “stealth”

fluorescein and calcein was obtained. For example, the half-leakage time of CF from DMPC28 vesicles in Hepes buffer at 37°C was 15 h, compared with 55 min only for DMPC vesicles. 5.3. Other efects on surface

tence in mice of liposomes

offluorinated

properties

tails; repercussions

and in vice recognition

There is growing evidence that the presence of a fluorinated core within the membrane has a significant impact on the properties of the vesicle’s surface and in vivo behavior. The more organized, more rigid, more impermeable fluorinated layer can induce order in the rest of the bilayer, cause changes in thermotropic behavior, and reduce the membrane’s defects. It can also have repercussions on the structural organization (conformation, orientation) of the polar heads of the amphiphiles, their dynamics, hydration, etc. Such effects could hinder the adsorption and anchorage of plasma proteins onto a surfactant film and, consequently, lower their recognition and phagocytosis when injected. In addition, tighter packing can improve chemical stability. Experimental observations of such effects include changes in the area occupied by individual phosphocholine headgroups at the water-air interface when the hydrophobic chains are fluorinated; this area is usually larger than with hydrogenated analogs [91]. Tighter packing of the surfactant film at the surface of fluorocarbon emulsion droplets is indicated by a reduction of the surface area occupied by the polar head of phospholipids when fluorinated material is introduced in the film [54]. The action of pig phospholipase A2 on DMPC vesicles is slowed down when mixed fluorinated/

when compared with similiposomes and even with liposomes.

Thus,

for example, circulation half-lives, for similar doses. were measured to be 6 h for F-liposomes made from 2b ( 170 k 50 nm; 8.6 h for 110 & 30 nm) compared with 0.5 h for DSPC (about 150 nm). 1.54 h for DSPC-cholesterol 1 : 1 (about 120 nm) and 8.4 h for certain DSPC-PEG5000 9: 1 (about 190 nm) liposomes [ 931. Further work needs to be done along these lines. New possibilities may also emerge from combining the unique properties induced by fluorinated components with other means of protecting, carrying, targeting and delivering of therapeutic interest.

drugs and other material

Acknowledgments I wish to thank Valerie Briet for her patience with this manuscript, my collegues and co-workers, whose names appear in the references, for their patience with me, Gwen Rosenberg for patient nitpicking, and the Centre National de la Recherche Scientifique and ATTA for constant support. References L.C. Clark and F. Gollan, Science, 152 (1966) 1755. T.M.S. Chang (Ed.), Blood Substitutes and Oxygen Carriers. Marcel Dekker, New York, 1993. T.M.S. Chang, J.G. Riess and R.M. Winslow (Eds.), Biomater., Artif. Cells, Immobil. Biotechnol.. 22 ( 1993), in press. G.P. Biro, Transfusion Med. Rev., 7 ( 1993) 84. N.S. Faithfull. Adv. Exp. Med. Biol.. 317 (1992) 55. J.G. Riess, VOX Sang.. 61 (1991) 225. J.G. Riess, Biomater., Artif. Cells. Immobil. Biotechnol.. 20 (1992) 183. J.G. Riess. Biomater.. Artif. Cells. Immobil. Biotechnol., 22 (1993).in press.

J.G. RiesslColloids 9 10

11

12

13 14 15 16 17 18

19 20

21 22 23 24

2s

26

21

28

29

30

Surfaces A: Physicochem.

Eng. Aspects 84 (1994 ) 33-48

J.G. Riess and M. Le Blanc, Pure Appl. Chem., 54 (1982) 2383. J.G. Riess and M. Le Blanc, in KC. Lowe (Ed.), Blood Substitutes: Preparation, Physiology and Medical Applications, Ellis Horwood, Chichester, 1988, Chapter 5, p. 94. A.I. Alayash, B.A. Brockner Ryan, J.C. Fratantoni and R.E. Cashon, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993) in press. T.H. Shaffer, M.R. Wolfson, J.S. Greenspan, SD. Rubenstein and R.G. Stern, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993), in press. B.P. Fuhrman, P.R. Paczan and M. DeFrancisis, Crit. Care Med., 19 (1991) 712. B. Lachmann, AS. Tutuncu, J.A.H. Bos, N.S. Faithful1 and W. Erdmann, Adv. Exp. Med. Biol., 317 (1992) 409. J.S. Greenspan, M.R. Wolfson, SD. Rubenstein and T.H. Shaffer, J. Pediatr., 117 (1990) 106. M.R. Wolfson, J.S. Greenspan and T.H. Shaffer, Pediatr. Res., 29 (1991) 336A. R.F. Mattrey, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993) in press. D.L. Rubin, H.H. Muller, M. Nino-Murcia, M. Shidu, V. Christy and S.W. Young, J. Magn. Reson. Imag., 1 (1991) 371. S.L. Wooton, B.D. Coley, Sv.W. Hilton. D.K. Edwards and R.F. Mattrey, Am. J. Radiol., in press. M.R. Wolfson, R.G. Stern, N. Kechner, K.M. Sekins and T.H. Shaffer, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993), in press. S.S. Eilenberg, V.M. Tartar and R.F. Mattrey, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993). in press. J. Klein, N.S. Faithfull, P.J. Salt and A. Trouwborst, Anesth. Analg., 65 (1986) 734. J.L. Ricci, H.A. Sloviter and M.M. Ziegler, Am. J. Surg., 149 (1985) 84. S. Chang, V. Reppucci, N.L. Zimmerman, M.H. Heinemann and D.J. Coleman. Ophthalmology, 96 (1989) 785. G.A. Peyman, M.D. Conway, K.F. Soike and L.C. Clark, Ophthalmic Surg., 22 (1991) 657. K.J. Blinder, G.A. Peyman, U.R. Desai, N.C. Nelson, W. Alturki and CL. Paris, Br. J. Ophthalmol., 76 (1992) 525. M. Flores-Aguilar, J.A. Crapotta, D. Munguia, G. Bergeron-Lynn, D. Long, CA. Wiley, J.G. Weers and W.R. Freeman, in press. A.T. King, B.J. Mulligan and K.C. Lowe, in J. Piiper, T.K. Goldstick and M. Meyer (Eds.), Oxygen Transport to Tissue, Vol. 12, Plenum, New York, 1990. p. 283. D. Attwood and A.T. Florence, Surfactant Systems, Their Chemistry, Pharmacy and Biology, Chapman and Hall, London, 1983. F. Puisieux and M. Seiller (Eds.), Galenica, les Systemes Disperses: I. Agents de Surface et Emulsions, Technique et Documentation Lavoisier, Paris, 1983. J.G. Riess, Proc. 2nd World Surfactant Congr., Paris, May 1988, Vol. 4, ASPA Ed., Paris, 1988, p. 256.

31

32 33

33 34 35 36 36 37 38 39 40

41

42 43 44 45 46

41 48 49 50

51 52

53 54

41

J.G. Riess, C. Arlen, J. Greiner, M. Le Blanc, A. Manfredi, S. Pace, C. Varescon and L. Zarif, in T.M.S. Chang and R.P. Geyer (Eds.), Blood Substitutes, Marcel Dekker, New York. 1989, p. 421. J.G. Riess, J. Greiner, S. Abouhilale and A. Milius, Prog. Colloid Polym. Sci., 88 (1992) 123. J. Greiner, J.G. Riess and P. Vierling, in R. Filler, Y. Kobayashi and L.M. Yagupolski (Eds.), Organofluorine Compounds in Medicinal Chemistry and Biomedical Applications, Elsevier, Amsterdam, 1993, p. 339. (a)Carbohydrate Recognition Cellular Function, Ciba Foundation Symp. 145, Wiley, Chichester, 1989. L. Zarif, T. Gulik-Krzywicki, J.G. Riess, B. Pucci, C. Guedj and A.A. Pavia, Colloids Surfaces, in press. F. Guillod, J. Greiner, A. Milius and J.G. Riess, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993) in press. J.G. Riess and M. Postel, Biomater., Artif. Cells, Immobil. Biotechnol., 20 (1992) 183. (a)N. Privitera, R. Naon and J.G. Riess, Int. J. Pharm. in press. J.G. Riess. S. Pace and L. Zarif, Adv. Mater., 3 (1991) 249. N. Garelli and P. Vierling, Biophys. Biochim. Acta, 1127 (1992) 41. J.G. Riess. Artif. Organs, 8 (1984) 44. R.E. Moore and L.C. Clark, in R. Frey, H. Beisbarth and K. Stosseck (Eds.), Oxygen Carrying Colloidal Blood Substitutes, W. Zuckschwerdt Verlag, Mtinchen, 1981, p. 50. Y. Tsuda, K. Nakura, K. Yamanouchi, K. Yokoyama, M. Watanabe, H. Ohyanagi and Y. Saitoh, Artif. Organs, 13 (1989) 197. R.E. Moore and L.C. Clark, Int. Anesthesiol. Clin., 23 (1985) 11. J.G. Riess, Trasfus. Sangue, 32 (1987) 316. J.G. Riess, Curr. Surg., 45 (1988) 365. V.V. Obratzsov, A.S. Kabalnov and K.N. Makarov, J. Fluorine Chem., 54 (1991) 376. U. Gross, Colloid and Surface Chemistry of Fluorocarbons and Highly Fluorinated Amphiphiles, ACS Symp., Denver, March 1993. M. Postel, J.G. Riess and J.G. Weers, Biomater., Artif. Cells. Immobil. Biotechnol., 22 (1993) in press. S.S. Davis, H.P. Round and T.S. Purewal, J. Colloid Interface Sci., 80 (1981) 508. W.I. Higushi and J. Misra, J. Pharm. Sci., 51 (1962) 459. J.G. Weers, J. Liu, B.A. Arlauskas, P. Resch, T. Fields and J. Cavin, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993), in press. J.G. Riess, L. Sole-Violan and M. Postel, J. Dispersion Sci. Technol., 13 (1992) 349. H. Meinert, R. Fackler, A. Knoblich, J. Mader, P. Reuter and W. Riihlke, Biomater., Artif. Cells, Immobil. Biotechnol., 20 (1992) 95. C. Cornelus, M.P. Krafft and J.G. Riess, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993), in press. M.-P. Krafft, F. Giulieri and J.G. Riess, Colloids Surfaces A: Physicochem. Eng. Aspects, 84 (1994) 113.

48 55 56

57 58

59 60 61 62

63

64 65

66 67

68

69 70

71

J.G. RiesslColloids S.F. Flaim, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993),in press. K. Yokoyama, T. Suyama and R. Naito. in Filler and Kobayashi (Eds.). Biomedicinal Aspects of Fluorine Chemistry, Elsevier Biomedical, Amsterdam, 1982. p. 191. P.E. Keipert, S. Otto, SF. Flaim, J.G. Weers, E.A. Schutt, T.J. Pelura, D.H. Klein and T.L. Yaksh, Biomater.. Artif. Cells, Immobil. Biotechnol., 22 (1993), in press. L.C. Clark, R.E. Hoffmann and S.L. Davis, Biomater., Artif. Cells, Immobil. Biotechnol., 20 (1992) 1085. E. Schutt, P. Barber, T. Fields, S. Flaim. J. Horodniak. P. Keipert, R. Kinner, L. Kornbrust, T. Leakakos, T. Pelura, J. Weers, R. Houmes and B. Lachmann, Biomater., Artif. Cells, Immobil. Biotechnol., 22 ( 1993). in press. American College of Physicians, Ann. Intern. Med., 116 (1992) 403. R.M. Winslow, Hemoglobin-Based Red Cell Substitutes, Johns Hopkins University Press, Baltimore, MD. 1992. J.G. Riess and M. Le Blanc. Angew. Chem., Int. Ed. Engl., 17 (1978) 621. D.C. Long, D.M. Long, J.G. Riess, R. Follana, A. Burgan and R.F. Mattrey, in T.M.S. Chang and R.P. Geyer (Eds.), Blood Substitutes. Marcel Dekker. New York. 1989, p. 441. J.G. Riess, J.L. Dalfors, G.K. Hanna, D.H. Klein, M.P. Krafft, T.J. Pelura and E.G. Schutt, Biomater., Artif. Cells, Immobil. Biotechnol., 20 (1992) 839. R.F. Mattrey, Radiology, 170 (1989) 18. J.N. Bruneton, M.N. Falewee, E. Franqois, P. Cambon, C. Philip, J.G. Riess, C. Balu-Maestro and A. Rogopoulos, Radiology. 170 ( 1989) 179. G. Wolf, D.M. Long and J.G. Riess. Radiology, 177 ( 1990) 366. G. Hanna, D. Saewert, J. Shorr, K. Flaim. P. Leese, M. Kopperman and G. Wolf, Biomater., Artif. Cells. Immobil. Biotechnol., 22 (1993). in press. V. Sanchez, L. Zarif, J. Greiner, J.G. Riess, S. Cippolini and J.N. Bruneton, Biomater., Artif. Cells, Immobil. Biotechnol.. 22 (1993), in press. M. Behan, D. O’Connell, R.F. Mattrey and D.N. Carney, Am. J. Radio]., 160 (1993) 399. S.R. Thomas, in C.L. Pastain, R.R. Price, J.A. Patton et al. (Eds.), Magnetic Resonance Imaging. 2nd edn., Vol. II, 1988, p. 1536. S.R. Thomas, R.W. Millard, R.G. Pratt, Y. Shiferow and R.C. Samatunga, Biomater., Artif. Cells, Immobil. Biotechnol., 22 (1993), in press. R.P. Mason, F.M. Jeffrey, C.R. Malloy, E.E. Babcock and P.P. Antich, Magn. Reson. Med., 27 (1992) 310. R.P. Mason. Biomater., Artif. Cells. Immobil. Biotechnol., 22 (1993), in press.

72 73 74 75

76 77

78

79 80 81 82 83 84 85 86

87 88 89

90 91

92 93 94

95

Surfaces A: Ph~sicockerll.

Eq.

Aspects 84 ( 1994 ) 33-48

J.C. Ravey and M.J. St&b&. Prog. Colloid Polym. Sci., 82 (1990) 218. S. Magdassi. M. Royz and S. Shoshan, Int. J. Pharm.. 88 (1992) 171. RX. Moore. US Pat. 4 569 784. October 20, 1981. O.E. Oxynoid, D.P. Sydliarov. Yu.D. Aprosin and V.V. Obraztsov. Biomater.. Arrif. Cells. Immobil. Biotechnol.. 22 (1993), in press. G. Gregoriadis, Liposomes as Drug Carriers. Recent Trends and Progress, Wiley. Chichester. 1988. G. LopeL-Berestein and LJ. Fidler, Liposomes in the Therapy of Infectious Diseases and Cancer, Alan R. Liss. New York, 1989. R.H. Muller, Colloidal Carriers for Controlled Drug Delivery and Targeting: Modification. Characterization and In Vivo Distribution. CRC Press, Boca Raton. FL. 1991. T. Kunitake, Angew. Chem., Int. Ed. Engl., 31 (1992) 709. H. Ringsdorf. B. Schlarb and J. Venzmer. Angew-. Chem., Int. Ed. Engl., 30 ( 1992) 113. R. Elbert, T. Folda and H. Ringsdorf, J. Am. Chcm. Sot.. 106 (1984) 7687. T. Kunitake. Y. Okahata and S. Yasunami. J. Am. Chem. Sot., 104 (1982) 5547. T. Kunitake and N. Higashi. Makromol. Chem.. Suppl., 14 (1985) 81. C. Santaella. P. Vierling and J.G. Riess. Angew. Chem.. Int. Ed. Engl., 30 (1991) 567. M.P. Krafft, F. Guilieri and J.G. Riess, Angew. Chem.. Int. Ed. Engl., 32 (1993) 741. J.G. Riess, C. Santaella and P. Vierling. Proc. 22nd CED Meet. on Surfactants, Palma de Mallorca, Comite Espanol Detergencia, Tensioactivos y Afines. Barcelona, 1991. p. 157. K. Liang and Y. Hui, J. Am. Chem. Sot., 114 ( 1992) 6588. C. Santaella, F. Frtzard. P. Vierling and J.G. RICSS. Biochim. Biophys. Acta. in press. F. Frezard, C. Santaella. P. Vierling and J.G. Riess, Biomater.. Artif. Cells. Immobil. Biotechnol.. 22 (1993), in press. L. Trevino. F. FrCzard, M. Postel and J.G. Riess. J. Liposome Res.. in press. S.W. Barton, A. Goudot. 0. Bouloussa. F. Rondelez. B. Lin, F. Novak, A. Acero and S.A. Rice. J. Chem. Phys., 96 (1992) 1343: N. Collazo. S. Shin and S..4. Rice. J. Chem. Phys., 96 (1992) 4735. N. Privitera, R. Naon and J.G. Riess, unpublished results. F. Frezard, C. Santaella. P. Vierling and J.G. Riess. FEBS Lett.. in press. V.V. Obraztsov, D.G. Schekhman. A.N. Sklifas and K.N. Makarov, Biokhimiya, 53 (1988) 613 (in English]. AS. Kabalnov, K.N. Makarov and E.O. Shchukin. Colloids Surfaces, 62 ( 1992) 101. J.G. Weers. personal communication, 1993.