Perfluorocarbons as oxygen-transport fluids

Camp. Biochem. Physiol. Vol. 8lA, No. 4, pp. 825-g-838,1987

0300-9629/87$3.00+ 0.00 0 1987 Pergamon Journals Ltd

Printed in Great Britain

MINI REVIEW

PERFLUOROCARBONS

AS OXYGEN-TRANSPORT

FLUIDS

K. C. LOWE Mammalian Physiology Unit, Department of Zoology, University of Nottingham, Nottingham NG7 2RD, UK

University Park,

(Received 27 October 1986) Abstract-l.

An overview of the proposed biological applications of perfluorocarbons and their emulsions as oxygen-transport fluids is presented. 2. Aspects of the properties, preparation, composition and physiological assessment of perfluorocarbon emulsions are discussed. 3. The experimental basis for some of the potential therapeutic uses of PFCs in liquid ventilation, treatment of decompression sickness, organ perfusion, oxygenation of ischaemic and malignant tissues, and as contrast media for NMR imaging is described. 4. The extent to which emulsified perfluorocarbons may have value as substitutes for red blood cells is discussed in detail. Data from both animal and human studies with such emulsions is reviewed. Brief consideration is also given to the possible use of native and modified haemoglobin in blood replacement together with recent work on the preparation of so-called “synthetic red cells”.

INTRODUCTION Living tissues depend upon a continuous supply of O2 with a corresponding removal of CO,. The blood serves as a transportation medium for this essential gas exchange: O2 transport involves reversible binding to haemoglobin in the red cells while CO* is carried partly in solution but mainly in chemical combination as bicarbonate. Small quantities of CO2 also combine with the amino groups on haemoglobin and various plasma proteins. Haemoglobin is the respiratory pigment found in the blood of most vertebrates. With the exception of the cyclostomes, vertebrate haemoglobin is a tetramer with a molecular weight of ca 64,000. In some invertebrates, haemoglobin is replaced by other respiratory pigments, including chlorocruorin (polychaete worms), haemerythrin (some annelids) and haemocyanin (mollusts and crustaceans). Many invertebrates, however, do not have a respiratory pigment. In vertebrates which lack haemoglobin, such as the antarctic icefish, Chaenocephalus aceratus, 0, is absorbed through the skin and gills and transported through the vascular system in simple solution. By a combination of reduced metabolism and high circulation rate, icefishes can survive without haemoglobin. In addition to the transport of respiratory gases, blood also carries electrolytes, metabolites, clotting factors, hormones and enzymes. Blood is therefore a complex connective tissue, unique in that it exists in a liquid state; it is often and perhaps more correctly referred to as the largest single organ in the body. During the past 20 years, much research has been directed towards the development and assessment of fluids to replace the gas-transporting properties of blood. The purpose of this paper is to review progress in this field and consider some of the possible biological and clinical applications of putative O,-transport fluids. Broadly speaking, such fluids are those based c BP 87,4A--A

825

on (1) haemoglobin or similar molecules; perfluorochemicals (PFCs).

or (2)

HAEMOGLOBIN AND ITS DERIVATIVES It is perhaps inevitable that haemoglobin has been tested as the basis of many O,-carrying resuscitation fluids since a considerable and universally available source of raw material exists in out-dated banked blood. However, a number of problems have severely limited progress in this area and these include: rapid circulatory clearance of cell-free haemoglobin due to renal excretion and reticuloendothelial system (RES) uptake; unacceptably high O2 affinities of modified (polymerized) haemoglobin; and potential nephrotoxic and immunotoxic effects of different preparations. Detailed discussion of the potential use and physiological effects of both native or modified haemoglobin in 0,.transport fluids is, however, beyond the intended scope of this paper. The interested reader is therefore referred to several recent reviews of such work (Devenuto, 1983; Moss et al., 1984; Gould et al., 1985; Lowe, 1986). A related area of interest has been in the development of so-called synthetic red blood cells or “neohemocytes” in which purified human haemoglobin and 2,3-diphosphoglycerate were incorporated into microcapsules; these neohemocytes show a physiologically acceptable O,-affinity and have been tested in rats with encouraging results (Hunt et al., 1985). However, clinical tests with encapsulated haemoglobin have not been performed. It is important to emphasize that despite considerable interest in the potential use of haemoglobin-based O,-transport fluids, no single preparation is universally available for detailed clinical assessment. There have been a number of attempts to produce totally synthetic, iron-containing molecules which can reversibly bind 0, at room temperature (Baldwin,

826

K. C. LOWE F2

F

Table 2. Gas solubilities in liquid PFCs*

F2

F2C~c\~‘-c--CF2

Compound

I W,

water Pertluorodecalin Perfluorotripropylamine Perfluorotributvlamine

I I CF,--+‘.F CFz ,cF2

Perfluorodecal

in

Perfluorotripropylamine

FgcB PF9 ;: ‘39

butyl amine

Fig. 1. Chemical structure of perfluorochemicals.

1983; Herron et al., 1983). However, progress in this area has been slow and physiologically acceptable molecules are not available at present. PERFLUOROCHEMICALS

The second and more widely tested group of potential O,-transport fluids are those containing emulsified PFCs. These are cyclic or straight chain organic compounds in which hydrogen atoms have been replaced with fluorine. Because many of the commonly used PFCs in this field are perfluorinated hydrocarbons, the molecules are often referred to as perfluorocarbons or fluorocarbons. The structures of the most widely used PFCsand perffuorotripropylamine perfluorodecalin, perfluorotributylamine-are given in Fig. 1. Due to the strength of the carbon-fluorine bond (ca 116 kcal/mol), PFCs are recognized as being chemically inert (Sargent and Seffl, 1970; Riess and Le Blanc, 1982). They are unaffected by boiling in strong acids and show remarkable thermal stability. Some of the basic physico-chemical properties of PFCs are listed in Table 1. Gas solubility PFCs have already found many industrial applications (e.g. aerosol propellants, laser coolants, refrigeration fluids) but their most important property of relevance to biology is gas solubility: pure PFC liquids can dissolve over 40 vol.% O2 at 37°C compared to 2-3 vol.% for water or plasma (Riess and Le Blanc, 1982; Table 2). The solubilities of both CO1

Emulsification While the gas-dissolving properties of PFC liquids make them attractive for potential biological use, they are immiscible with aqueous systems and poor solvents for most physiological solutes. Intravascular injection of PFCs was shown to produce emboli and other circulatory abnormalities (Sass et al., 1976) but this problem was overcome by the development of 25 -

Molecular formula

,

11

I

I

I

10

20

30

40

50

PO,

60 (kPa)

70

80

90

100

Fig. 2. Relationship between oxygen partial pressure (p0,) and oxygen content for normal blood (haematocrit 45%) and the PFC emulsion, Fluosol-DA 20% (Green Cross, Japan). Note that the emulsion can deliver approximately 5ml 0, to tissues when the arterial p02 decreases from 70 KPa (Fi02 = 1.0) to 7 Kpa as indicated by the broken lines (Figure courtesy Dr N. S. Faithfull).

Table 1. Some physico-chemical Molecular weight

properties of PFCs Boiling point (0°C) 142 129 177

Vapour Pressure at 25°C (m.bar)

9.0 462 C,,F,s 15.0 521 C,F,,N 1.3 671 C,,FnN Data taken from Riess and Le Blanc (1982); Jeanneaux et al. (1984). *Relates to emulsions of individual PFCs prepared with Pluronic F-68 as emulsifying agent.

Perfluorodecalin Pertluorotripropylamine Perfluorotributylamine

N, 1.6 20.4 35.1 2X.6

and N2 in PFC liquids are also very high and, in the case of CO*, can exceed 160 vol.% (Table 2). This solubility in PFCs depends upon the molecular volume of the gas and decreases in the order: CO* $O,$ Nz (Riess and Le Blanc, 1982). In contrast the sigmoidal binding curve of oxygen to haemoglobin, O2 solubility in liquid PFCs and their emulsions increases linearly with partial pressure according to Henry’s Law (Fig. 2). The amount of gas dissolved therefore depends upon the concentration of PFC and its solubility coefficient for the gas. In addition, gas solubility in liquid PFCs is inversely related to temperature.

C3F7

Compound

CO, 65 148 166 140

*Values are ml/d1 per atmosphere at 37°C. Data taken from Riess and LeBlanc (1982); Jeanneaux er al. (1984); Lutz and Herrman (1984).

F’C3\ FF’ i

Pet% uorotri

0, 2.5 42.3 45.3 40.3

Stability of emulsions* Poor Good Excellent

PerAuorocarbons as oxygen-transport fluids fine emulsions of PFCs dispersed in isotonic electrolyte solutions: such preparations have subsequently been tested as O2 carriers in many clinical and biomedical contexts (see Lowe, 1983, 1984a,b, 1986; Bollands and Lowe, 1985). Clearly, the use of PFCs in an emulsified form allows simultaneous transport of 02, mineral salts and metabolites. PFCs are normally emulsified either by ultrasonic vibration (e.g. Dawe Soniprobe) or by high-pressure homogenization using the Manton-Gaulin apparatus (Riess and Le Blanc, 1982). While sonication is a more vigorous emulsification procedure, it is associated with PFC degradation and release of potentially toxic fluoride ions (Riess and Le Blanc, 1982); however, fluoride ion liberation is suppressed if sonication is performed under COZ atmosphere (Geyer, 1975). The most commonly used surfactant for PFC emulsification is the polyoxyethylene block copolymer, Pluronic F-68: when used in conjunction with egg yolk-phospholipids and glycerol, it can form an effective and relatively stable barrier between PFC and aqueous phase. Pluronic is claimed to be atoxic, inert and excretable and consequently, has been approved for human use (Naito and Yokoyama, 1978b). However, some concern has been expressed recently about possible toxic effects of pluronic or its derivatives when used both in vivo and in vitro in commercial PFC emulsions. This point is discussed in more detail below. Particle size and size distribution are of fundamental importance in PFC emulsions proposed for biological uses. These factors determine viscosity, surface area for gaseous exchange and intravascular persistance time. For example, RES blockade by emulsified PFCs increases considerably when particle size is about 0.4pm (Fujita et al., 1973) but diminishes to acceptable levels when particles of ca 0.1-0.2 pm are used. Available commercial emulsions are claimed to have an average particle diameter of ~0.1 pm (Naito and Yokoyama, 1978a,b) but this can vary according to storage time and temperature (Meinart et a/., 1985). Fate and excretion of PFCs

PFC particles are removed from the circulation by cells of the RES and are thus detectable in the liver, spleen and other lymphoid tissues as foamy vesicles (Nanney et al., 1984). When administered in very large doses, this accumulation of PFCs can lead to organ enlargement (Lutz and Metzenauer, 1980, Pfannkuch and Schnoy, 1983; Lowe and Bollands, 1985). Tissue retention of PFCs is highly variable and this depends upon the compound in question: for example, vacuolated cells containing PFCs have been seen throughout the RES in Macaque monkeys up to 6 years after injection of pertluorotributylamine whereas no corresponding accumulations were obat 8 years following injection of served pertluorodecalin (Rosenblum et al., 1985). PFCs are primarily excreted by expiration from the lungs and this depends upon the vapour pressure of the individual molecules (Naito and Yokoyama, 1978a); in addition, some PFCs are also transpired through the skin. There is no convincing evidence

827

that PFCs are metabolized within the body: this being further evidence for the inertness of these compounds. Where toxic effects have been observed, these have invariably been attributed to certain impurities such as fluoroamines, or in the case of emulsified PFCs, to other emulsion constituents (Le Blanc and Riess, 1982). It should, however, be noted that toxicological evaluation of PFCs’ emulsion components is an area currently attracting much interest and is discussed in more detail below. Expiration measurements have been used to determine the half-lives of individual PFCs in animals and these are highly variable: for example, pertluorodecalin has a body half-life in rats of approximately 7 days while for perfluorotripropylamine, the corresponding figure is about 65 days (Naito and Yokoyama, 1978a; Le Blanc and Riess, 1982). This difference is believed to be due to the presence of a N, atom in the latter molecule (see Fig. 1) which is invariably associated with a longer retention time in body tissues. Other factors which can influence the rate of elimination of different PFCs from the body include: molecular size, molecular structure and critical temperature coefficients. Before describing in detail the proposed uses of emulsified PFCs as red cell substitutes, other more general applications of PFCs and their emulsions will be outlined. Liquid ventilation

The O,-dissolving properties of PFCs were admirably demonstrated by Clark and Gollan when they showed that mice could survive for extended periods submerged in while completely oxygenated periluorobutyltetrahydrofuran liquid (Clark and Gollan, 1966). The impetus for these classical experiments was provided by the earlier paper of J. A. Kylastra and colleagues entitled “Of mice as fish”, in which it was reported that mice had been kept alive while “breathing” from oxygenated saline solutions (Kylastra et al., 1962). These initial experiments stimulated further work on the possible applications of PFCs for liquid ventilation with a view to providing a viable alternative to air-breathing in deep-sea diving. For example, the physiological effects of liquid ventilation with fluorocarbons have been studied in adult dogs (Matthews et al., 1978) and cats (Lowe et al., 1979; Shaffer et al., 1984a) although data on possible adverse changes in lung structure and function are inconclusive. Liquid PFCs have also been used to ventilate the lungs of immature fetal rabbits (Schwieler and Robertson, 1976) and sheep (Shaffer et al., 1983, 1984b) and thus improve fetal oxygenation. It has also been noted that in contrast to respiratory problems that occur during mechanical gas ventilation (Shaffer et al., 1976, 1978), preterm lambs ventilated with fluorocarbon liquid showed good gas exchange and stable blood gas tensions (Shaffer et al., 1983). These observations therefore raise the question of whether lung ventilation with liquid PFCs could have some therapeutic value in certain clinical situations (e.g. neonatal respiratory distress syndrome) although further work is needed to clarify this.

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K. C. Lows

Decompression sickness

Nitrogen is highly soluble in liquid PFCs (Riess and Le Blanc, 1982; Table 2) and possible applications of emulsified PFCs for therapeutic treatment of decompression sickness have been examined in both hamsters (Lynch et al., 1983) and rats (Lutz and Herrman, 1984). While no corresponding studies have been performed in humans, the potential value of PFCs for “mopping up” N, from the blood to alleviate life-threatening effects of decompression sickness is worthy of further investigation. Microcirculation and ischaemic tissues

The reduced viscosity and small particle size are properties which make PFC emulsions useful for infiltrating the microcirculation and carrying 0, to otherwise ischaemic tissues. Experiments in rats (Ricci et al., 1984), cats (Peerless et al., 1981; Sutherland et al., 1984; Peerless et al., 1985), dogs (Glogar et al., 1981; Nunn et al., 1983; Menasche et al., 1984; Forman et al., 1985; Kolodgie et al., 1985) and pigs (Rousou et al., 1982) together with perfusion studies using isolated rabbit hearts (Flaherty et al., 1984; Parrish et al., 1984; Mushlin et al., 1985) have provided evidence to support the proposed use of emulsified PFCs for improving tissue oxygenation and reducing the severity of infarcts arising from cerebral, myocardial or intestinal ischaemia. Emulsified PFCs have also been employed as a distal oxygen perfusate during experimental coronary artery angioplasty with encouraging results (Spears et al., 1983; Anderson et al., 1986). In some studies, PFCs have been tested in conjunction with other therapeutic agents, notably anti-oxidants (Suzuki et al., 1984) and glycerol (Naruse et al., 1984). It should, however, be emphasized that PFCs alone appear to be unable to prevent ischaemic damage from occurring but do offer protection against reperfusion injury (Menasche et al., 1985). Nevertheless, the use of PFC emulsions in combination with other therapies may have some clinical value in this area. Organ perfusion and preservation

Attention has been given to the possible applications of PFC emulsions for perfusion and preservation of vital organs prior to transplantation. The first such use of PFCs involved perfusion of the isolated rat brain (Sloviter and Kamimoto, 1967). They were subsequently used as perfusates for liver (Lutz et al., 1978; Guaitani et al., 1983; Skibba et al., 1985), kidneys (Franke et al., 1978) and heart (Tomera and Geyer, 1982; Deutschmann et al., 1984; Rahamathulla et al., 1985; Chemnitius et al., 1985). In each case, PFCs were able to maintain adequate oxygenation of the perfused tissue. More recent work has shown that emulsified PFCs supplemented with potassium and magnesium were able to maintain myocardial capillary endothelial integrity in the isolated perfused rabbit heart (Kyosola et al., 1986). Success with re-implantation of PFC-perfused rat livers and human cadaveric kidneys (Kamada et al., 1980; Honda et al., 1980) together with replantation of amputated extremities (Smith et al., 1985) has also been achieved. Recent related work has shown that emulsified pertluorotributylamine can maintain steroid secretion

from an isolated rat testis preparation (Chubb, 1985; Chubb and Draper, 1985). While not directly relevant to transplantation studies, those reports nevertheless described the first PFC perfusion of an endocrine tissue; the system also provided a convenient method for assessing toxicological effects of fluoride ion contaminants in the emulsion (Chubb, 1985). Pertluorotributylamine emulsion had additionally been used as perfusate for the isolated dog pancreas and its use for studying the role of blood components in modulating acute panceatitis (O’Malley et al., 1986). It has also been used to perfuse rat lungs in vitro (Lindgren et al., 1985). While many previous studies have shown that PFCs can have value in maintaining adequate oxygenation in isolated tissues, recent work has shown that perfusion of rat kidneys with Fluosol-DA emulsion produced nephrotoxic effects which were not seen when stroma-free haemoglobin solution was used as perfusate (Millis et al., 1985). However, the extent to which normal physiological function of tissues may be altered by PFC perfusion has not been studied in detail. Malignant tissues

Tumours generally contain hypoxic cells and this decreases their sensitivity to the cytotoxic effects of ionizing radiation (Moulder and Rockwell, 1984). Such conditions arise in solid tumours as a result of abnormal vasculatures and consequent inadequate blood flow (Peterson, 1979). Hyperbaric 0, has been used to augment the therapeutic effects of radiation in tumours in animals and man (Henk, 1981) but it has not been adopted for routine clinical use. The ability of emulsified PFC preparations to transport 0, has raised the possibility of them being used to enhance oxygenation of tumours and thus sensitize them to either radiation (Teicher and Rose, 1983; Rockwell, 1985; Song et al., 1985; Lustig and McIntosh, 1986) or chemotherapy (Kuwamura et al., 1982; Teicher et al., 1985). The potential therapeutic value of emulsified PFCs depends upon the ability of emulsion particles to penetrate inside tumour(s) and thereby oxygenate sites otherwise inaccessible to red cells. This work is presently at the developmental stage but it can be anticipated that PFC preparations could play an important therapeutic role in tumour management. However, such a use of emulsified PFCs will require careful studies of any effects on the progression of neoplastic disease together with their interactions with cytotoxic drugs and other therapeutic agents (Lowe, 1987). Contrast media: NMR imaging

PFCs have a high content of the natural fluorine isotope i9F and this makes them attractive for in viuo use as imaging agents in nuclear magnetic resonance (NMR) studies. In 1977, Holland and others reported the first NMR imaging of PFC liquids (Holland et al., 1977). Since that time, several reports of 19F NMR imaging of tissues in animals infused with emulsified PFCs have appeared (Clark et al., 1984; MaletMartin0 et al., 1984; Joseph et al., 1985). Various PFC liquids have additionally been employed for NMR imaging of lungs (Thomas et al., 1986). 19F is virtually ideal for use in NMR imaging since it is

Perfluorocarbons as oxygen-transport fluids Table 3. Composition of Fluosol-DA 20% and Fluosol-43 Cross Corporation, Japan) Fluosol-DA 20% Perlluorodecalin Pcrfluorotripropylamine Perfluorotributylamine Pluronic F-68 Yolk phospholipids Glycerol NaCl KC1 M&I, CaCI, NaHCO, Glucose Hydroxyethylstarch

14.0 6.0 2.1 0.4 0.8 0.600 0.034 0.020 0.028 0.210 0.180 3.0

(Green

Fluosol-43 (Oxypherol) 20.0 2.56

0% 0.034 0.020 0.028 0.210 0.180 3.0

All values are w/v (%).

normally found in only trace quantities in tissues. However, PFCs have also been used as contrast media for “P NMR studies of normal and ischaemic rat brain (Ackerman et al., 1984; Naruse et al., 1984). Emulsified PFC derivatives (i.e. containing one or more bromine atoms) have been effectively employed as blood-pool contrast agents in computed tomography (Mattrey et al., 1984) and also for enhancing X-ray imaging in liver, spleen and other RES tissues (Long et al., 1982). Moreover, the uptake of radiopaque fluorocarbons into macrophages of malignant tissues (Long et al., 1978) has provided a convenient and specific method for localizing tumours. It is tempting to speculate that emulsified PFCs could have some considerable value in oncology as dual diagnostic and therapeutic agents. Such a combination of the imaging and O,-transporting properties of PFC could make them potentially very powerful clinical tools.

829

glycerol while oncotic pressure is provided by hy droxyethyl starch. A second commercial preparation, Fluosol-43 (FC-43) or Oxypherol, is also available and consists of emulsified petlluorotributylamine (Table 3). In contrast to F-DA, FC-43 is not advocated for clinical use owing to the prolonged retention time of pertluorotributylamine in the body (Riess and Le Blanc, 1982). The intravascular half-life of F-DA is highly variable depending on dose administered. For example, in man the calculated half-life determined by gas chromatography was 7.5 hr at 10 ml/kg body weight rising to 14.5 hr after 20ml/kg and 22 hr after 30ml/kg (Yokoyama et al., 1982). While the 02-dissolving capacities of pure PFC liquids are substantial, these are less for available commercial emulsions and estimates of between 5.7 and 7.6 ml. 0, per 100ml per atmosphere at 37°C have been reported for F-DA (Naito and Yokoyama, 1978b; Rosen et al., 1982). Though this amount is small in relation to that of fully saturated normal arterial blood, it is nevertheless almost 2.5 times that of plasma (Table 2). Such O2 transport by F-DA is only achieved when the inspired 0, concentration (F,O,) is increased to 1.0: consequently, when F-DA has been used in haemodilution or exchangetransfusion studies in both animals and humans, the recipients invariably breathed supplementary 0,. Under such conditions, F-DA can deliver significant amounts of 0, to tissues (see Fig. 2). The fact that there is no chemical combination of 0, with PFC facilitates good 0, extraction: for example, liver perfusion studies have shown that under normal conditions, only 5060% of 0, available in blood is extracted by tissues whereas this increases to > 90% when PFCs are used (Lutz et al., 1978).

EMLJLSJFIED PFCs AS RED CELL SURSTITUTFS

The use of emulsified PFCs as a substitute for red blood cells has probably attracted more interest in the medical and scientific communities than all the other work discussed in this paper. The use of the term “artificial blood” to describe such emulsions has been widespread but this is misleading since as we have seen already, blood does much more than transport 0, and CO*. It is for this reason that PFC preparations should be more correctly described as “red cell substitutes”, or perhaps simply “0,-transport fluids”. The search for a safe and easily administered fluid capable of replacing the gas-transporting properties of the red cells together with the oncotic functions of plasma proteins resulted in the development of the first commercial PFC emulsion, Fluosol-DA 20% (F-DA), in Japan (Naito and Yokoyama, 1978b). F-DA is a first-generation preparation which has now been available for over 10 years; it is the only PFC emulsion to have been tested in clinical trials (Mitsuno et al., 1982; Tremper et al., 1982; Waxman et al., 1984; Stefaniszyn et al., 1985). F-DA contains perfluorodecalin and periluorotripropylamine emulsified with Pluronic F-68 in an isotonic electrolyte solution (Table 3). Pertluorotripropylamine is added because emulsions of perlluorodecalin alone are relatively unstable (Table 1). F-DA is stabilized by yolk-phospholipids and

Animal studies Initial experiments investigated aspects of physiological function following varying degrees of blood replacement with F-DA in anaesthetized animals: species studied included the rat (Watanabe et al., 1979), rabbit (Kohno et al., 1979), dog (Takiguchi et al., 1979) and Rhesus monkey (Ohyanagi et al., 1978). FC-43 has also been tested in a similar series of studies in the rat (Zucali et al., 1979; Schneeberger and Neary, 1982; Sylvia et al., 1982; Schneeberger and Hamelin, 1984; Piantadosi et al., 1985). The development of a convenient and easily reproducible technique for continuous, isovolaemic, exchange-transfusion in conscious rats enabled more thorough studies on the effects of blood replacement with F-DA to be performed in the absence of anaesthesia (Lowe et al., 1982, 1985; Hardy et al., 1983). For example, exchange-transfusion to very low haematocrits (ca 2% or less) produced an immediate increase in total circulating lymphocyte count (Lowe et al., 1982; Hardy et al., 1983) suggesting either recirculation of cells from extravascular stores (e.g. lymph nodes) or immunological disturbances. Moreover, blood replacement was followed by pronounced increases in plasma lactate dehydrogenase and alkaline phosphatase concentrations (Lowe and McNaughton, 1985, 1986) reflecting a progressive and

830

K.

C. Lowr

Table 4. Summary of lymphoid tissue weight changes in rats and mice after treatment with Fluosol-DA 20% Rats 5 ml/kg F-DA IO ml/kg F-DA Mice 5 ml/kg F-DA IO ml/kg F-DA

Liver

Spleen

Thymus

MLN

i.v. i.p. i.v. i.p.

Unc. Unc. UW UIIC.

TP < 0.05 tP < 0.05 tP < 0.05 tP < 0.05

Unc. Unc. Unc. Unc.

IP < 0.05

i.v. i.p. i.v. i.o.

TP < 0.005 tP < 0.005

JP < 0.05

UW. Unc. UIIC. UIIC.

JP < 0.05 1P < 0.05 JP < 0.05 IP < 0.05

tP < 0.005 TP < 0.005

Unc. Unc. Unc.

Unc. UIIC. UIIC.

t = increased, I= decreased, Unc. = unchanged compared to mean control values.

ultimately lethal deterioration of homeostatic control. These observations conflicted with previous results showing that animals could survive indefinitely when allowed to recover from almost complete blood replacement with PFC emulsion (Geyer, 1975, 1978). The requirement for animals to breathe supplementary O2 when perfused with F-DA or other PFC emulsion has raised the obvious question of whether 0, toxicity may be responsible for some of the observed homeostatic changes. While this possibility has not been studied in detail, Geyer (1978) noted that 0, toxicity did not, at least in the short-term, appear to be an important factor affecting survival of animals almost completely transfused with emulsified PFCs. Recent work has shown that the extent to which PFC emulsion particles accumulate in lymphoid tissues depends upon species used together with dose, route and composition of emulsion administered (Bollands and Lowe, 1986a,b,c, 1987a). For example, injection of 5 or 10 ml/kg F-DA either intravenously (i.v.) or intraperitoneally (i.p.) into rats or mice produces differential changes in liver, spleen, thymus and gut mesenteric lymph node weights measured after 8 days (Table 4). Such variability in tissue accumulation and retention of PFC emulsion particles has inevitably introduced complications into studies of the fate of PFCs in the body. In addition to morphological changes produced by emulsified PFCs in cells of reticuloendothelial and other tissues (Nanney et al., 1984) interest has also focussed on alterations in tissue function induced by these compounds. Lutz and others (Lutz et al., 1982b) observed a transient depression of RES phagocytic function following injection of F-DA stem emulsion in rats while Castro and colleagues reported similar findings in both rats and primates (Castro et al., 1983, 1984). This suppression of RES clearance function by F-DA would help to explain the decreased resistance to bacterial endotoxin seen in rats injected with the emulsion (Lutz et al., 1982a). It is likely that alterations in RES function induced by emulsified PFCs may persist for longer than was initially believed since increases in serum fi globulins have been measured in monkeys up to 8 years after exchange-transfusion with either F-DA or FC-43 (Rosenblum et al., 1985). There are inconsistencies in the literature regarding the effects of emulsified PFCs on immune system function and in particular, on the manifestation of

humoral responses to immunological challenge. In an initial preliminary report, Shah and colleagues (Shah et al., 1984) noted that pretreatment of Balb/c mice with F-DA or FC-43 iv. led to a decrease in the in vivo production of antibodies to sheep erythrocytes (SRBC). However, subsequent work has produced conflicting results with an increase in the haemagglutination response to SRBC being noted in rats or mice injected i.p. but not i.v. with comparable doses of F-DA (Bollands and Lowe, 1986a,b,c,1987a; Fig. 3). This difference may reflect strain variations in response but other factors, notably timing and route of administration of emulsion relative to the immune challenge, must also be considered. In this regard, Mitsuno and others (Mitsuno et al., 1984) reported that in rats injected with >20 ml/kg F-DA, the emulsion enhanced antibody production when given after immunization whereas it inhibited antibody generation when injected before immunization. More recently, Bollands and Lowe (1987b) demonstrated that lymphoid tissue responses and plasma antibody titres to SRBC varied markedly according to the time of a previous or subsequent injection of F-DA via the same route. It has been proposed that F-DA acts as an adjuvant when injected into the peritoneal cavity of either rats or mice 24 hr prior to SRBC (Bollands and Lowe, 1986b, 1987a). The possibility exists that PFCs may promote release of the intracellular mediator, interleukin-1 (IL-l), from macrophages in a similar manner to that already described for other adjuvants (Allison, 1983). Such IL-1 release would, in turn, potentiate a subsequent immune response. If this is the case, it would help to explain the observed immunopotentiating effects of F-DA and account for some of the variation in humoral immune responses seen in animals receiving the emulsion. Further evidence of potential deleterious effects of emulsified PFCs on immune system function is that near-total transfusion of rats with F-DA inhibited the afferent (induction) phase of a specific immune response but did not alter an ongoing efferent phase (Hodges et al., 1986). This latter effect was attributed to RES blockade by the emulsion as discussed above. Transfusion with F-DA also enhanced the subsequent early specific immune response to SRBC but the active component(s) involved were not identified (Hodges et al., 1986). Work is in progress to determine the mechanism(s) by which different PFC emulsions can affect the immune system and identify the active principle(s) involved.

Pemuorocarbons as oxygen-transport fluids 10 A. ulco

I

IV

v

l T

Jl-

I

II

Ill

IV

V

EXPERIMENTAL QAOUP

Fig. 3. The mean plasma haemagglutination response (expressed as log, titre) of either (A) mice (upper panel) or (B) rats (lower panel) at seven days after intraperitoneal injection of SRBC. Experimental pre-treatments were as follows: Group I: saline-injected controls (n = l&15); Group II: F-DA i.v. 5 ml/kg (n = 5-6); Group III: F-DA i.v. 10 ml/kg (n = 4-5); Group IV: F-DA i.p. 5 ml/kg (n = 5-6); Group V: F-DA i.p. lOml/kg (n = k-5). Vertical bars represent SEM. *P < 0.01 compared with corresponding mean control value. Adverse ejiects

In addition to effects of PFCs on the immune system, animal experiments have also revealed adverse reactions to PFC emulsions and some of their components. Reported changes include: coagulation defects characterized by prolongation of activated partial thromboplastin time together with decreases in blood concentrations of some clotting factors (Lau et af., 1975); haematological disturbances involving complement activation via the alternate pathway (Vercellotti et al., 1982); and inhibition of plasma and liver phospholipase A2 activities with possible alterations in prostaglandin (PG) metabolism (Shakir and Williams, 1982). However, regarding the latter work, any alterations in PG biosynthesis produced by PFCs were not of sufficient magnitude to offset the increase

831

in plasma immunoreactive thromboxane B2 which followed endotoxin injection in FC-43-transfused rats (Cook et al., 1985). A detailed discussion of these responses, which vary according to emulsion composition and species used, is beyond the scope of this paper and the interested reader is therefore referred to recent relevant reviews (Lowe and Bollands, 1985; Lowe, 1986; Hammerschmidt and Vercellotti, 1986; Faithful1 et al., 1987). There is growing concern that many of the reported adverse effects of commercial emulsions may be caused, at least in part, by the Pluronic F-68 surfactant. Pluronic is able to mimic the complementactivating effects of F-DA both in vivo and in vitro (Vercellotti et al., 1982). It can also account for the inhibitory effect of F-DA on both plasma and liver phospholipase A2 activities (Shakir and Williams, 1982). McCoy and others (McCoy et al., 1984) have suggested that peroxide derivatives of pluronic, formed during steam sterilization or long-term storage of the emulsion, could contribute to, but not fully account for, adverse effects of F-DA on arterial endothelial ultrastructure in rats. In addition, recent experiments have also shown that pluronic can damage tubular brush borders and enhance protein loss from isolated perfused rat kidneys (Gronow et al., 1986). The extent to which pluronic is responsible for other reported adverse reactions to commercial PFC emulsions (Lowe and Bollands, 1985) remains to be determined. However, this concern has prompted the search for suitable alternative emulsifiers for PFC emulsification and these should be available for assessment in the near future. Human studies

F-DA has been tested in preliminary clinical trials in over 200 human subjects, many of whom were Jehovah’s Witnesses or severely anaemic surgical patients (Mitsuno et al., 1982; Tremper et al., 1982; Waxman et al., 1984; Stefaniszyn et al., 1985). The emulsion was able to significantly improve tissue oxygenation providing that patients breathed supplementary 0,: observed increases in 0, consumption suggested that this effect was due to an enhancement of 0, distribution within the microcirculation (Tremper et al., 1982; Waxman et al., 1984). The O,-transporting properties of F-DA have more recently been employed clinically in the treatment of post-partum haemorrhage (Karn et al., 1985). While clinical interest in F-DA is undoubtedly increasing, its widespread acceptance in many areas has been severely hampered by the occurrence of adverse reactions, similar to those noted in animal experiments. In humans, F-DA can induce hypotension and pulmonary complications, which can occur in response to injection of only a small (cu 0.5 ml) test dose (Tremper et al., 1982, 1984). The reactions to F-DA were comparable to those accompanying intravascular complement activation and could be diminished by treatment with corticosteroids (Vercellotti et al., 1982). As with the individual (as well as species) variations seen in animals, there also appears to be much variability in the human responses to F-DA: while

832

K. C. Lowr

adverse reactions were noted in clinical studies with the emulsion in the USA (Vercellotti et al., 1982; Tremper et al., 1982, 1984; Police et al., 1985), no such responses were reported in initial clinical trials in Japanese patients (Mitsuno et al., 1982). The reasons for these differences are unclear but it has been suggested (Hammerschmidt and Vercellotti, 1986) that immunogenetic variability between races together with dietary factors may be involved. Cell culture studies Emulsified PFCs may have considerable value for oxygenating and maintaining viability of mammalian and non-mammalian cells in culture. Such a use for PFCs would inevitably be of value both in basic and applied research involving isolated mammalian cells together with applications in other cell and tissue culture systems. However, a pre-requisite to any use of emulsified PFCs as incubation or perfusion media additives is knowledge of their effects upon in vitro function of cells from different species. Because in uivo use of emulsified PFCs may lead to altered immunological competence, a number of studies have been concerned with analysis of their direct and indirect effects upon phagocytic and other immune cells. Incubation of human blood with either F-DA or FC-43 inhibited phagocytic activity of both neutrophils and monocytes, as assessed by their uptake of fluorescent polystyrene beads (Virmani et al., 1983, 1984). Other changes reported included cytoplasmic vacuolation, decreased chemotaxis and reduced aggregation, adherence and superoxide (0; ) release in the presence of phorbol myristate acetate (PMA). Reduced cellular adherence to the culture surface in the presence of F-DA or FC-43 has also been observed in cultures of mouse peritoneal macrophages (Bucala et al., 1983) and non-depleted splenocytes (Bollands and Lowe, unpublished observations). It is possible that this loss of adhesion was caused either by adverse effects upon the cells themselves or as a result of interference by the PFCs or other emulsion constituent(s) on the tissue culture plate coating. Results available at present demonstrate that in vitro cellular responses to PFC preparations and their components is largely dependent on the cell type under study and the species from which it originates (Lowe and Bollands, 1985). For example, FC-43 is selectively toxic to mouse macrophages but not lymphocytes (Bucala et al., 1983) and it was proposed that this selective toxicity was caused either by disruption of the phospholipid membrane or adverse effects on the intracellular O,-detoxification system or a combination of both. Further evidence of cytotoxic effects of F-DA is that incubation of rat peritoneal mast cells with a methanol-soluble extract of the emulsion was followed by an increase in histamine release into the incubation medium in proportion with the amount of extract added (Lowe et al., 1984); maximum histamine release in response to incubation of cell aliquots with a petrol-soluble extract of F-DA was only 30% of that which followed incubation with the methanol fraction. F-DA has also been shown to induce cytotoxic changes and growth inhibition in human embryonic lung fibroblasts in culture (Wake et al., 1985).

Fluosol emulsions appear to have differential effects on cultured mammalian cells since F-DA is more effective than FC-43 in promoting procoagulant generation from human mononuclear leucocytes (Janco et al., 1985). F-DA also significantly impaired stimulated oxidative metabolism (i.e. 0; generation) from monocytes whereas FC-43 was ineffective (Janco et al., 1985). It was also shown that Pluronic F-68 was at least partly responsible for some of the adverse cellular effects produced by F-DA although the results were not conclusive: pluronic itself was able to stimulate procoagulant release from monocytes but it did not affect stimulated 0; production (Janco et al., 1985). Pluronic does, however, appear to be almost entirely responsible for the inhibition of migration seen when cultured human polymorphonuclear cells are exposed to F-DA (Lane and Lamkin, 1985). It is tempting to speculate that pluronic contributes to the mast cell-degranulating effects attributable to F-DA (Lowe et al., 1984). However, it seems more likely that other components, especially the yolkphospholipid emulsion stabilizer, are also involved. A summary of the principal reported effects of Fluosol emulsions on mammalian cells in vitro is given in Table 5. It is perhaps paradoxical that cytotoxic effects of PFC emulsions could be of clinical value in transplantation studies for perfusion of donor tissue to remove macrophages. Since such cells appear to be important determinants of tissue antigenicity, perfusion with PFC prior to transplantation may help to reduce subsequent rejection by the recipient. Differential effects of PFCs on plasma-mediated deformability of human erythrocytes have been reported (Holloway et al., 1986). Thus, emulsified perfluorotributylamine (FC-43) decreased red cell deformability but neither F-DA nor its constituent PFCs, perfluorodecalin and perfluorotripropylamine, were similarly effective. While these results demonstrate the need for further studies on haematological effects of different PFCs, related experiments have shown that FC-43 reduced the incidence of sickling when cultured with red cells from patients with sickle cell anaemia (Reindorf et al., 1985). This raises the question of whether emulsified PFCs may have therapeutic value in certain blood rheological disorders. An additional potential use of PFCs and their emulsions is for oxygenation of microbial cultures. This is an area currently attracting much interest owing to its obvious relevance to the microbial biotechnology industry. Preliminary experiments have shown that microbial cell growth can be altered in the presence of PFCs (Mattiasson and Adlercreutz, 1984; Damiano and Wang, 1985; Chandler et al., 1986). However, further detailed studies of the effects of PFCs and their individual emulsion constituents on both structure and function of procaryotic and eucaryotic cells should be performed before any wider applications in this field can be recommended. Novel formulations It is now generally accepted that the Fluosol emulsions have provided a very useful “first generation” class of proprietary formulations with which an initial series of physiological studies could be performed, both in animals and humans. However,

833

Perfluorocarbons as oxygen-transport fluids Table 5. Cellular effects of petibtorocarbon

species

Cells

Mouse

Macrophages

FC-43’

Mouse Mouse

Splcnocytcs Splenocytes

FC-43 F-DAt

Rat

Peritoneal mast

F-DA

Cel1.S

Pig

Lymphocytes

F-DA

Rabbit

Monocytes Neutrophils Fibroblasts

FC-43

FC-43

Human

Monocytes Neutrophils Neutrophils

Human

Neutrophils

FC-43

Human

Neutrophils

F-DA

Human

Lymphocytes

F-DA

Human

Mononuclear leucocytes Mononuclear leucocytes

F-DA

Human Human

Human

F-DA

F-DA

FC-43

emulsions References

Response(s)

Emulsion

Cytoplasmic vacuolation Decreased LDH content No change in LDH content Inhibition of Con-Ainduced transformation Dcgranulation and histamine release Inhibition of Con-Ainduced transformation Cytoplasmic vacuolation Inhibition of phagocytosis Cytoplasmic vacuolation Inhibition of proliferation Cytoplasmic vacuolation Inhibition of phagocytosis Inhibition of adhesion, aggregation and phagocytosis Reduced superoxide generation Inhibition of adhesion, aggregation and phagocytosis Reduced superoxide generation Inhibition of adhesion, migration and chemotaxis No change in PHA-Pinduced transformation Increased procoagulant activity Reduced superoxide generation Increased procoagulant activity No change in superoxide generation

Bucala et al. (1983) Bucula et al. (1983) Lowe et al. (unpublished observations) Lowe et al. (1984) Lowe et al. (unpublished observations) Virmani et al. (1983) Wake

et al. (1985)

Virmani et al. (1983) Virmani et al. (1984)

Virmani

et al. (1984)

Lane and Lamkin (1984) Fujita et al. (1983) Janco

et al. (1985)

Janco

et al. (1985)

lFC43 = Fluosol-43/0xyphcrol (Green Cross, Japan). tF-DA = Fluosol-DA 20% (Green Cross, Japan). Table modified from Lowe and BolIands (1985).

more recent attention has been focussed on the development of second- and perhaps even thirdgeneration formulations which will supersede the Fluosol emulsions and to provide clinically acceptable preparations. Commercially available PFC emulsions show relatively poor storage stability resulting in increased particle size and therefore have to be stored either deep frozen or under refrigeration (Naito and Yokoyama, 1978b). Factors affecting emulsion stability are numerous and complex involving parameters such as surfactants, composition and emulsification procedures (Riess, 1984). Molecular weight (M.W.) of the PFCs themselves is also of prime importance since the stability of similarly prepared emulsions consistently decreases with decreasing M.W. (Riess, 1984). However, in vivo use of compounds with greater M.W.s is generally associated with longer tissue retention (Yamanouchi et al., 1985). Attempts to develop improved PFC emulsions for possible biological uses have been concerned with the testing of several series of new PFC compounds alicyclic amines and polycyclic including PFC/polytluorinated amine oxides (Clark et al., 1983; Yokoyama et al., 1983; Jeanneaux et al., 1984). There is also growing interest in the use of alternative emulsifiers to the pluronics but PFC preparations containing such surfactants are not yet available for routine biological assessment. The possibility that coating of emulsified PFC particles with either lecithin or a mixture of lecithin and cholesterol may alter intravascular persistence time and reduce the incidence of adverse haematopoietic effects in the recipient has also been explored (Sloviter and Mukherji, 1984).

More recent work has shown that emulsified perfluorodecalin can be stabilized by the addition of small quantities of pertluorinated high boiling point oils (Davis et al., 1986; Sharma et’ al., 1986). The addition of such oils retards the process of molecular diffusion known as Ostwald Ripening which can contribute to the destabilization of PFC emulsions (Riess, 1984). Preliminary biocompatibility tests with one such pertluorodecalin emulsion have been carried out in rats with encouraging results (Davis et al., 1986; Bollands et al., 1987). Work is now in progress to assess the effects of these novel formulations in different biological systems, including cell culture studies. CONCLUDING

REMARKI

This paper has given some insight into the many potential applications of PFCs and their emulsions as Or-transport fluids. Judging by the rate at which the number of published papers on this subject has increased during the past ten years, there can be no doubt that interest in biological applications of PFCs is growing. While progress in emulsion technology and biocompatibility testing has been substantial, we are still some way from achieving the goal of producing a safe, clinically acceptable formulation. Nevertheless, work completed to date suggests that this is a realistic objective and one which is achievable in the forseeable future. Acknowledgements-Some of the original work described in this paper was supported by research grants from I.S.C. Chemicals Ltd., Avonmouth. The assistance of the Green Cross Corporation is gratefully acknowledged.

834

K. C. LOWE REFERENCES

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