State of the art

Perfluorinated blood substitutes and artificialoxygen carriers

K. C. Lowe

Blood transfusion is a remarkably safe, routine clinical procedure. However, the need for sophisticatedblood processing, storage and cross-matching, coupled with increasing concerns about the safety ofblood products, has fuelled the search for safe and efficacious substitutes. Candidate materials based onmodified haemoglobin (including recombinant molecules) or highly inert, respiratory gas-dissolvingperfluorinated liquids (perfluorochemicals) have been developed. The latter are immiscible in aqueoussystems and must, therefore, be injected as emulsions. Second-generation perfluorochemical emulsionsare available and in clinical trials as temporary intravascular oxygen carriers during surgery, therebyreducing patient exposure to donor blood. One commercial product is currently under Phase III clinicalevaluation, with regulatory approval expected within 1–2 years. Other biomedical applications forperfluorochemicals and their emulsions include their use as pump-priming fluids for cardiopulmonarybypass, lung ventilation fluids, anti-cancer agents, organ perfusates and cell culture media supplements,diagnostic imaging agents and ophthalmologic tools. Novel applications for perfluorochemicals asimmunomodulating agents are also being explored. © 1999 Harcourt Publishers Ltd

Blood Reviews (1999) 13, 171–184© 1999 Harcourt Publishers Ltd

INTRODUCTION

Blood or concentrated red cells are routinely trans-fused following extensive blood loss or as therapy forchronic anaemia. The increasing sophistication ofblood fractionation technology means that specificblood constituents, such as platelets or haema-topoietic progenitor cells, can be administered topatients with life-threatening haematological disor-ders.1 Plasma is routinely separated from blood cellsand, for example, the extracted coagulation factorsare used to treat haemophiliacs, despite the increasing

171

K.C. Lowe PhD, Reader in Biotechnology, School of BiologicalSciences, University of Nottingham, University Park, NottinghamNG7 2RD, UK. Tel.: +44 (0)115 951 3311;Fax: +44 (0)115 951 3251

Correspondence to: K.C. Lowe

availability of alternative recombinant molecules.2 Inparallel with the evolution of modern transfusion andthe ongoing improvements in blood processing andstorage3 has been increasing interest in so-called‘blood substitutes’. This is illustrated by consideringsome shortcomings of blood transfusion. The usefulshelf-life of blood or red cells is only a maximum of42 days (35 days in the UK), even with the use ofmodern nutrient additives (e.g. mannitol, glucose,adenine). Inevitably, blood donated for transfusion iswasted. Another problem is the need to type andcross-match blood for donor–recipient compatibility.Whilst the occurrence of life-threatening reactionscaused by incompatible red cells are relatively rare,immune-mediated transfusion reactions can stilloccur due to alloimmunization to antigens on redcells, white cells, platelets and plasma proteins.1

The recent UK Government decisions to suspendthe fractionation of UK plasma and to filter blood to

172 Blood Reviews

Table 1 Clinical targets for blood substitutes

1. To replace acute blood loss during surgery or following trauma2. Adjuncts to angioplasty3. Adjuncts to ionising radiation and chemotherapy in cancer

treatment

Table 2 Candidate blood substitutes

1. Haemoglobin-based materials

• modified haemoglobin solutions

• recombinant haemoglobin

• nanoencapsulated haemoglobin2. Perfluorochemical-based materials

remove leucocytes (leucodepletion) as precautionarymeasures to minimize the possible risk of transmittingthe agent thought to cause new variant Creutzfeldt-Jakob disease (nvCJD)4,5 further illustrate increasingconcerns about the safety of blood products.

Exposure to donated (allogeneic) blood can bereduced by using the patient’s own, pre-deposited orsalvaged (autologous) blood. Whilst pre-deposited,autologous blood is becoming an increasingly attrac-tive option in the UK, which has significant safetybenefits, it is not suitable for all patients, especiallythose who cannot pre-donate, and its use is associatedwith additional costs and logistical problems.6,7

All the problems outlined above could, in principle,be solved by the development of a volume-expanding,synthetic substitute fluid that had the same osmolarityas blood and could bind to O2 in the lungs and deliverit to the tissues. Such a blood substitute should ideallybe stable for long periods at room temperature, needno cross-matching, and produce no adverse cardiovas-cular or immunological reactions when infused intohumans. Isotonic electrolyte solutions containing anoncotically-active agent, such as gelatin or dextran,are now widely used in medicine, primarily as bloodvolume expanders or haemodiluents.8 However, suchfluids have a very limited capacity to carry O2, due tothe low solubility of the gas in aqueous systems(ca. 2–3 ml 100 ml–1). A blood replacement fluidcapable of making a significant contribution tosystemic O2 transport could be used, for example, asan alternative to transfusing red cells. Such productscould be administered to patients who otherwizerefuse blood transfusion on religious grounds (e.g.Jehovah’s Witnesses). Furthermore, suitable substi-tute fluids would significantly underpin transfusionservices in emerging countries where the blood supplyis frequently contaminated with viruses or parasites.

CANDIDATE BLOOD SUBSTITUTES

Almost 5 years ago, the US Food and DrugAdministration identified specific clinical targets forpotential blood substitutes (Table 1), including theiruse to replace acute blood loss during surgery orfollowing trauma and as adjuncts to angioplasty andcancer therapy.9

Whilst used in the title of this paper, primarily toplace the present discussion in context, the term‘blood substitute’ is strictly misleading since it impliesthat candidate materials can replace, in toto, theproperties of blood. Clearly, this is not the case.Available materials have, therefore, variously beendescribed as ‘red cell substitutes’,1 ‘blood supple-ments’,10 ‘O2 therapeutics’ or ‘anti-hypoxic drugs’.11

This is because fluids currently under evaluation areprincipally replacing the O2-transporting properties oferythrocytes (CO2 homeostasis in patients receivingsuch substitutes has generally been ignored). Whencoupled with the use, wherever possible, of auto-logous blood, such substitutes will underpin futureblood-conserving strategies to avoid patient exposureto allogeneic blood, thereby reducing the risk ofinducing transfusion-mediated adverse reactions or ofcontracting blood transmissible diseases.

There are two current approaches for developingclinically useful substitutes for blood. These are (1)fluids based on haemoglobin (Hb), including theproducts of recombinant technology, and (2) emul-sions of inert, perfluorinated compounds called per-fluorochemicals (PFCs), often described simply asfluorocarbons (Table 2).

Several Hb solutions have been developed commer-cially, including materials based on human (e.g.HemAssist, Baxter International) and cows blood(e.g. Hemopure, Biopure Corporation). Whilst clinicaltrials with Hb O2 carriers are at an advanced stage, itis doubtful that a bovine Hb-based product will beacceptable to the UK public, primarily because ofconcerns about the transmission of the infective agentcausing Bovine Spongiform Encephalopathy. There isalso a risk that such animal Hb may adversely affectthe human immune system, especially on repeat infu-sion.1 In mid-1998, Baxter International announcedthat it had suspended clinical trials with HemAssist inEurope because of concerns that the efficacy of theproduct. However, a bovine Hb-based O2 carrier,Oxyglobin (Biopure), was licensed in the USA inJanuary 1998 for veterinary use in the treatment ofchronic anaemia in dogs; the product currently costsca. £100 per unit.

Human Hb has also been produced from geneti-cally modified microorganisms12 and plants,13 anapproach that avoids using human or animal blood as

Perfluorinated blood substitutes and artificial oxygen carriers 173

Fig. 1 Chemical structures of (A) a bicyclic PFC,perfluorodecalin, and (B) a linear compound, perflubron.

Fig. 2 Relationship between oxygen content and oxygen partialpressure (pO2) for haemoglobin and emulsions containing 20% or60% (w/v) of PFC.23

starting material, thereby overcoming cultural andbioethical objections to animal or donor blood-derived human Hb and the problem of transmittinginfections. Such recombinant molecules are, inprinciple, virus free and contain no residual red cellmembrane contaminants. The most advanced mate-rial in this category is Optro (Somatogen, recentlypurchased by Baxter), which contains cross-linked Hb (rHb 1.1) and is produced in Escherichia colithrough the expression of a modified human Hbgene.12 Phase II trials with Optro in surgical patientsare in progress.

Recombinant Hbs are a logical way forward fortherapeutic O2 carriers since ‘tailor-made’ moleculescan, in principle, be produced. However, future workmust take account of the discovery that nitric oxide(NO) can bind not only to iron in the haem portion ofHb, but also to reactive sulphydryl groups of cysteineresidues in the molecule.14 When Hb delivers O2 totissues, it also releases NO from its cysteine residuesand this, in turn, causes vasodilation of capillaries tofacilitate oxygenation. Recombinant technology hasbeen used to produce Hb mutants which have alteredaffinity for NO,15 thereby overcoming the transient,but unwanted, hypertensive side-effects that have beenconsidered as a major flaw in the ultimate usefulnessof Hb-based intravascular (i.v.) O2 carriers.1

A further, novel approach to the development ofi.v. O2 carriers is the production of nanoencapsulatedHb (Table 2), in which modified Hb is incorporatedwith, for example, 2,3-DPG or O2 radical-scavengingenzymes, such as superoxide dismutase, within lipid orbiodegradable polymeric vesicles to create so-called‘artificial red cells’.16,17 The target size of suchnanocapsules is <0.15 µm. Whilst there are many

exciting possibilities presented by nanoencapsulation,relevant not only to i.v. O2 transport but also totargetted drug delivery, much further work is neededto assess the efficacy and biocompatibility of theresultant materials. For further particulars onprogress in the development and biomedical applica-tions of Hb-based blood substitute materials thereader is referred to recent relevant reviews.16–22

PFCs AS BLOOD SUBSTITUTES ANDARTIFICIAL OXYGEN CARRIERS

General properties

PFCs are chemically inert, fluorine-substituted hydro-carbons that can dissolve large volumes of non-polargases. A typical value for O2 solubility in PFC liquids(STP) is 45 ml 100 ml–1; the corresponding value forCO2 can be over 200 ml 100 ml–1.23 Gas solubility inPFC liquids decreases in the order CO2 >> O2 > CO >N2 which correlates with the decrease in molecularvolume of the solute.23 Linear PFCs, such as per-fluoro-octyl bromide (also known by the genericname, perflubron; Fig. 1), dissolve O2 more effectivelythan cyclic molecules (e.g. perfluorodecalin; Fig. 1). Ingeneral, O2 solubility in PFCs is inversely propor-tional to the molecular weight (m.w.) and directlyrelated to the number of fluorine atoms present.24

Oxygen solubility in PFC liquids is about 20–25 timesgreater than in either water or blood plasma under thesame conditions. Unlike the chemical binding of O2 tothe porphyrin-iron sites of Hb, O2 dissolution in PFCsis a simple passive process, in which gas moleculesoccupy so-called ‘cavities’ within the PFC liquid. In

174 Blood Reviews

Table 3 Properties and biomedical uses of PFCs

Compound Molecular Molecular Actual/proposed use(s)formula weight

Perfluoro-ethane C2F6 138 Ophthalmologic agentPerfluoro-propane C3F8 188 Diagnostic imaging agent

Ophthalmologic agentPerfluoro-butane C4F10 238 Diagnostic imaging agentPerfluoro-pentane C5F12 288 Diagnostic imaging agentPerfluoro-hexane C6F14 338 Diagnostic imaging agentPerfluoro-octane C8F18 438 Ophthalmologic agentPerfluoro-decalin C10F18 462 i.v. O2 carrier

Ophthalmologic agentCell culture media supplement

Bis(perfluoro-butyl)ethene C4F9CH=CHC4F9 464 i.v. O2 carrierPerfluoro-dichlorooctane C8F16Cl2 471 i.v. O2 carrier

Diagnostic imaging agentPerfluoro-methylisoquinoline C10F19N 495 i.v. O2 carrierPerflubron C8F17Br 499 i.v. O2 carrier

Liquid ventilation fluidDiagnostic imaging agentCell culture media supplement

Perfluoro-tripropylamine C9F21N 521 i.v. O2 carrier*Perfluoro-methylcyclopiperidine C12F23N 595 i.v. O2 carrier*Perfluoro-decyl bromide C10F21Br 599 i.v. O2 carrier*Perfluoro-dimorpholinopropane C11F22N2O2 610 i.v. O2 carrier*Bis(perfluoro-hexyl)ethene C6F13CH = CHC6F13 664 i.v. O2 carrierPerfluoro-tributylamine C12F27N 671 i.v. O2 carrierPerfluoro-perhydrophenanthrene C16F26 686 Ophthalmologic agent

i.v. O2 carrier*

* Minor constituents only.

contrast to the characteristic sigmoid binding curve ofO2 to Hb, O2 solubility in PFCs and their emulsionsincreases linearly with partial pressure essentiallyfollowing Henry’s Law (Fig. 2). Details of the syn-thesis and physico-chemical properties of PFCsrelevant to their uses in biological systems have beenreviewed elsewhere.10,23–29

PFC liquids are immiscible with aqueous systems,including blood and other body fluids, but can beinjected i.v. in an emulsified form. PFC emulsionshave been evaluated clinically as, for example,temporary respiratory gas-carrying fluids for i.v.administration (including their use to augment thetumour-killing effects of ionizing radiation orchemotherapeutic drugs), priming fluids for cardio-pulmonary bypass machines and perfusates forisolated organs10,23–29 (Table 3). In the blood vascu-lature, PFC emulsions provide their main benefit byincreasing the O2 solubility in the plasma compart-ment,24 as described later. Neat PFC liquids are alsobeing assessed clinically as respiratory tract infiltrates,in both babies and adults, for the treatment of acuterespiratory failure through liquid ventilation, andsurgical tools in ophthalmology (Table 3), as dis-cussed later.

This paper gives an overview on current andpredicted biomedical uses of PFCs focusing, in par-ticular, on their use as i.v. respiratory gas carriers.Because recent research indicates that PFC emulsionsmay have novel, clinically relevant, applications, asimmunomodulating agents, this aspect is also dis-cussed.

First-generation injectable PFC emulsions

The first commercial injectable PFC emulsion wasFluosol (Green Cross, Japan) which consisted of14.0% (w/v) perfluorodecalin, 6% (w/v) perfluoro-tripropylamine and the surfactants, Pluronic F-68(poloxamer 188), egg yolk phospholipids (EYP) andpotassium oleate (Table 4). Fluosol had an O2-carry-ing capacity of approximately 40% of that of redblood cells (at 37°C). The emulsion received regula-tory approval in the USA and Europe in 1989–1990for clinical use as an O2-carrying adjunct to percuta-neous transluminal coronary artery balloon angio-plasty (PTCA).10,23–25,30,31 Fluosol was not, however,widely adopted by cardiologists, principally becauseimprovements in angioplasty technology made itsuse redundant and, therefore, its manufacture was

Perfluorinated blood substitutes and artificial oxygen carriers 175

Table 4 Composition and characteristics of injectable PFC emulsion oxygen carriers

Emulsion Perfluorocarbon(s) Perfluorocarbon Surfactant(s)/ Typical storageconcentration stabilizer(s) conditions(% w/v)

1. ‘First-Generation’ emulsionsFluosol Perfluorodecalin 14.0 Pluronic F-68 Frozen*

Perfluorotripropylamine 6.0 EYP**Potassium oleate

Perftoran Perfluorodecalin 14.0 Proxanol FrozenPerfluoromethylcyclopiperidine 6.0

Oxypherol Perfluorotributylamine 20.0 Pluronic F-68 Refrigeration***2. ‘Second-Generation’ emulsionsOxygent Perflubron 58.0 EYP Refrigeration

Perfluorodecyl bromide 2.0Oxyfluor Perfluorodichlorooctane 76.0 Safflower oil Refrigeration

EYPTherOx Bis(perfluorobutyl)ethene 83.0 EYP Refrigeration

* Stem emulsion stored frozen; annex solutions stored under refrigeration (ca. 4°C); ** EYP = egg yolk phospholipids; *** Emulsionideally stored at <10°C.

terminated in 1994. Limitations of Fluosol includedthe need for the stem emulsion to be stored frozen, theneed to thaw and re-constitute before use, and, cru-cially, its relatively poor efficacy as an O2 carrier.10,23

Intravenous use of Fluosol was also associated, insome patients, with side-effects (albeit transient),including complement activation and inhibition ofleucocytes, that were attributed mainly to the syn-thetic Pluronic F-68 surfactant component.10,23,31

Nevertheless, Fluosol provided an important mile-stone in the development of improved, ‘second-gener-ation’ PFC emulsions for i.v. applications.

Concurrent with the development of Fluosol wasthe production of other, first-generation, PFC emul-sions, including Emulsion No. II and Perftoran fromChina and Russia, respectively.10,23–26,32 As for Fluosol,Perftoran also consists of 14% (w/v) perfluorodecalin,but contains 6% (w/v) perfluoromethylcyclopiperidinein place of perfluorotripropylamine32 (Table 4).Emulsion No. II is a 20% (w/v) emulsion with thesame PFC constituents as Fluosol. For both emul-sions, a poloxamer-type compound is incorporated assole or principal surfactant. Following clinical evalua-tion in >500 patients, Perftoran was approved (in1995–1996) by the Russian health regulatory authori-ties for clinical use as a temporary i.v. O2 carrier forhaemorrhagic shock and perfusate for human organsex-vitro. Emulsion No. II has been administered to atleast 340 patients, some of them war casualties,33 butlittle else is known about its clinical status. A furthercommercial emulsion, Oxypherol, containing 20%(w/v) of perfluorotributylamine (Table 4) was alsoproduced by Green Cross, but was not intended forclinical use because of its high m.w. (Table 3) and

consequent prolonged retention half-time (>500 days)in the body.23

Second-generation injectable PFC emulsions

Research and development objectives

The main objectives of the research and developmenteffort to produce superior PFC emulsions to super-sede Fluosol and other first-generation emulsionswere to (1) identify highly purified PFCs with biocom-patibility and excretion properties acceptable for invivo use, (2) improve stability characteristics andhence, shelf-life, through the use of non-poloxamersurfactants (especially EYP) and perfluorinated(‘fluorophilic’) stabilisers, and (3) develop concen-trated emulsions having significantly increased PFCcontent conferring superior O2-carrying capacity,23 asreflected by the steeper slope of the O2 content/pO2

relationship (Fig. 2). However, it has been necessaryto achieve a compromise between producing highlyconcentrated PFC emulsions with increased O2-carry-ing potential, whilst avoiding formulations withviscosity’s that are too high for use in the vasculature.Such physico-chemical developments were also linkedwith a quest for further understanding of thebehaviour and efficacy, not only of the wholeemulsion(s), but also of their constitutive PFCs andsurfactant components in biological systems.

Selection of biocompatible PFCs

The two PFCs most widely studied as core con-stituents of injectable emulsions are perflubron and

176 Blood Reviews

Table 5 Composition and characteristics of the commercial PFCemulsion temporary oxygen carrier, Oxygent (AlliancePharmaceutical Corporation, USA)

PFC content (% w/v) 60.20 ± 0.06Mean droplet diameter (µm) 0.17 ± 0.01pH 7.10 ± 0.04Viscosity (cP) 4.0 ± 0.1Osmolarity (mOsm kg–1 304 ± 6Heat sterilized, non-pyrogenicStable for >1 year at 5–10°C

perfluorodecalin (Fig. 1). The m.w.’s of both com-pounds fall within the range 460–500, which is recog-nised as that giving acceptable tissue retentiontimes10,23–28 (Table 3). The excretion of PFCs from thebody, principally by exhalation, as discussed later,depends primarily on m.w., with molecular structureand the presence of cycles or heteroatoms having min-imal influence.34 Both perflubron and perfluorodecalincan be synthesised to a very high degree of purity,thereby avoiding unwanted side-effects that havefrequently been attributed to partially-fluorinatedcontaminants.25–28

An important variable that governs the selection ofPFCs for in vivo applications is the critical tempera-ture (°C) of solubility of the compound in n-hexane(CTSH).35 The CTSH of a PFC is a measure of itsrelative solubility in lipids and characterizes the rateof transfer of individual compounds through alveolarmembranes. PFCs with CTSH values of <28°C, suchas perfluorodecalin, are considered as lipophilic,35

whereas compounds with CTSH values of >42°C,such as perfluorotributylamine, have prolongedretention times in the body, as noted already. Bothperflubron and perfluorodecalin, the former becauseof its molecular composition, fall into the formercategory and are thus considered suitable for in vivoapplications.

Stabilization of emulsions

As mentioned already, a major objective in the pro-duction of second-generation, injectable emulsionswas to improve stability and, thus significantly extendshelf-life. Emulsions are thermodynamically unstablesystems and, in PFC-based formulations, the prin-cipal mechanism by which droplets grow is throughmolecular diffusion (Ostwald Ripening). During thisprocess, PFC molecules from smaller droplets diffusethrough the continuous phase to the larger dropletswhich progressively increase in size at the expenseof the former.36,37 Ostwald Ripening in emulsions ofperfluorodecalin can be suppressed by adding a smallamount of a perfluorinated, high m.w., high boilingpoint oil (HBPO) additive, such as perfluoroperhy-drophenanthrene38,39 (Table 3). This strategy wasused in the production of both Fluosol, in whichemulsified perfluorodecalin was stabilized withperfluorotripropylamine, and Perftoran, whereperfluoromethylcyclopiperidine was used as theHBPO10,23,25–29 (Table 4). Second-generation emulsionsbased on perflubron or perfluorodecalin have simi-larly been stabilized against Ostwald Ripening usingsmall quantities of an appropriate HBPO, as dis-cussed later. Effective stabilization of PFC emulsionsagainst Ostwald Ripening-mediated ageing has been a

major obstacle for researchers to overcome in thedevelopment of injectable room temperature-stableformulations.

Perflubron-based emulsions

Perflubron has one of the highest respiratory gas-dissolving capacities (ca. 50 ml 100 ml–1) of any of thePFCs commonly used for biomedical applications. Itis also an attractive compound for in vivo use becauseof its excellent imaging properties,40 as noted later.Perflubron can be readily emulsified with EYP andshows exceptionally fast excretion characteris-tics.10,23,25–29 This latter property arises because of itsvery high lipophilicity endowed by the presence of asingle bromine atom on the terminal carbon25–29

(Fig. 1). Studies in animals have shown that the body retention half-time (t1/2

) of perflubron is ca.4 days.23

Perflubron is the main PFC component in a com-mercial O2-carrying emulsion (Oxygent; Tables 4 & 5)developed by the Alliance PharmaceuticalCorporation, San Diego, USA, initially in collabora-tion with the Johnson and Johnson subsidiaries, theRobert Wood Johnson Pharmaceutical ResearchInstitute and Ortho Biotech Inc., USA. The currentOxygent formulation (AF0144) consists of 58% (w/v)perflubron and 2% (w/v) of its higher homologue, per-fluoro-decyl bromide (Table 3), to stabilize againstOstwald Ripening27–29 (Table 4). The emulsion has ashelf-life at 5–10°C of >1 year.

Oxygent is stabilized with EYP (3.6% w/v; Table 4)which are an obvious choice for emulsifying PFCssince they are widely used in injectable lipid emulsionsfor parenteral nutrition. EYP are excellent stabilizersof PFC emulsions, as reflected in their ability toreduce the PFC/water interfacial tension.36,37 EYP aresensitive to slow oxidative degradation but, in somePFC-based emulsions, this has been inhibited byadding an antioxidant, such as α-tocopherol.41

Indeed, α-tocopherol is routinely added (0.1–0.2%w/v) to some commercial phospholipid formulations(e.g. Lipoid E100, Lipoid GmbH, Germany) that havebeen used in other PFC emulsions, as described later.

Perfluorinated blood substitutes and artificial oxygen carriers 177

Perfluorodecalin-based emulsions

A further commonly used PFC in second-generationemulsions is perfluorodecalin. This is because itsacceptable tissue retention time outweighs its rela-tively poor emulsifying properties.10,23,26–28

Perfluorodecalin dissolves ca. 40 ml 100 ml–1 of O2

and ca. 140 ml 100 ml–1 of CO2 and has a bodyclearance t1/2

of ca. 7 days.23 A novel series of perfluo-rodecalin-based emulsions, stabilized with up to 2.5%(w/v) of lecithin (Lipoid E100) have been producedrecently by a European consortium.42 Some of theemulsions also contained 1.0% (w/v) of perfluorodi-morpholinopropane (Table 3) to retard dropletgrowth by Ostwald Ripening, analogous with earlierrelated studies.38,39 Perfluorodimorpholinopropanehas a m.w. of 610, a boiling point of 182°C and candissolve ca. 43 ml 100 ml–1 of O2. It has an estimatedbody clearance t1/2

of 55 days.43 This comparesfavourably with corresponding values of 60 days forthe perfluorotripropylamine and perfluoromethylcy-clopiperidine constituents of Fluosol and Perftoran,respectively.23 The novel emulsions were prepared byhomogenization and had a total PFC content of20–40% (w/v). Emulsions were steam sterilizable andshowed no significant changes in droplet diameter(ca. 0.2–0.3 µm) during >300 days’ storage at roomtemperature. Experiments are in progress to assess theefficacy of the novel emulsions as O2-carrying per-fusates of animal organs, including the dog heart andpig liver.

Other concentrated emulsions

A further, second-generation PFC emulsion isOxyfluor, developed in the USA by Hemagen-PFC ofSt. Louis. Oxyfluor consists of 76% (w/v) of a linearcompound, perfluoro-dichlorooctane (Table 3),safflower oil and EYP as surfactant (Table 4). Theemulsion has an average droplet diameter of0.22–0.25 µm. Oxyfluor has been evaluated as an i.v.O2 carrier in preclinical studies,44 and as a perfusatefor cardiopulmonary bypass (CPB) machines,45 butfurther information on its current commercial orclinical status is limited. Other, concentrated, experi-mental second-generation emulsions, now discontin-ued, include TherOx, containing 83% (w/v)bis(perfluorobutyl)ethene (Tables 3 & 4),26,46 emulsi-fied perfluoromethylisoquinoline (FMIQ; Table 3),and an emulsion consisting of 54% (w/v) bis(perfluo-rohexyl)ethene (Table 3),26,46 all of which have beenexperimentally for preclinical assessments of efficacy.

Safety and body tolerance

PFCs are biochemically unreactive and excreted fromthe body primarily as a vapour by exhalation.

Intravenous-administered PFC droplets are alsocleared from the circulation by phagocytic cells of themonocyte-macrophage system (MMS), principallyliver Kupffer cells and spleen macrophages. PFCs sub-sequently diffuse back into the blood where they arecarried in plasma lipids to the lungs and exhaled.47

Recent studies in rabbits have shown that the pul-monary elimination of perflubron is not associatedwith any significant changes in the functional residualcapacity, vital capacity, lung compliance or thedynamic behaviour of endogenous surfactant.48,49

Temporary increases in the weights of liver andspleen coupled, in some instances, with transientalterations in cellular enzymes (e.g. cytochromesP-450) following injection of PFCs, have been welldocumented.10,23 It is also well established that theduration and magnitude of such responses are highlyspecies-specific and dependent on dose and type ofPFC injected.10,23 It has recently been claimed31 thatmost of the side-effects attributed to second-genera-tion PFC emulsions (e.g. delayed febrile reactions,flu-like symptoms) in some patients can be attributedto the normal activity of the phagocytic cells of theMMS removing PFC droplets from the circulation.Further detailed discussion on the fate and effects ofPFCs and other emulsion constituents in the body canbe found in recent reviews.10,23,26

Oxygen transport and delivery

Oxygen dissolves in PFC droplets as they pass throughthe lungs. The total amount of O2 dissolved in a PFCemulsion depends on the concentration of PFC and thesolubility coefficient for the gas. The alveolar O2 load-ing in PFCs is linearly related to the pO2 and this can besignificantly enhanced when the recipient breathes sup-plementary O2. For example, a typical physiologicalpO2 gradient between alveoli and tissues during airbreathing would be about 60 torr. Under such condi-tions, normal blood (haematocrit ca. 45%, Hb 15 g100 ml–1) would release about 5 ml O2 100 ml–1, repre-senting an extraction rate of approximately 25%. Incontrast, the corresponding figure for a 60% (w/v)perflubron emulsion under ambient pO2 would beabout 2 ml O2 100 ml–1. However, if an atmospherecontaining 90–100% O2 were breathed (FiO2 = 0.9–1.0),which would raise the arterial pO2 to >400 torr, thisvalue would rise to about 10 ml O2 100 ml–1,50,51

representing an extraction rate of about 90%. Thisillustrates why, in clinical studies in which i.v. PFCemulsion was used to enhance tissue oxygenation,efficacy was maximized when patients breathed anO2-enriched atmosphere.11,31

Oxygen extraction from PFCs is significantlyenhanced by the lack of chemical fixation and by the

178 Blood Reviews

Table 6 Potential benefits of an intravenous PFC O2 carrier31

1. Increased plasma O2 concentration2. Increased systemic O2 delivery3. Increased uptake of dissolved O2

4. Increased venous drainage pO2

5. Increased tissue oxygenation6. Increased mixed venous O2

large surface area provided by the emulsion dropletswhich, typically, are only 2–3% of the diameter of thenormal erythrocyte (ca. 7–8 µm). O2 delivery by PFCis much simpler than the release of O2 from Hb, wherethe gas has to cross the red cell membrane, passthrough the plasma, and then diffuse through boththe membranes of the endothelial cells and those ofthe tissues it is supplying.

Oxygen delivery by PFCs appears to be morecomplex than simple ‘bulk’ transport: it has been sug-gested50–53 that PFCs may facilitate the transfer of O2

into tissues by acting as ‘stepping stones’ between redcells and blood vessel walls. In vessels with rapid flow(e.g. arterioles), PFC emulsion droplets in the circula-tion are postulated to flow mainly in the plasma layerthat forms close to the vessel walls as a result oferythrocyte streaming.31 In the microcirculation, PFCdroplets will occupy the plasma gaps between redblood cells and thereby perfuse even the smallestcapillaries. Some perfusion by PFCs will be expectedto occur in vessels that effectively exclude red cells, asa result of local vasoconstriction or ischaemia. Thus,under such conditions, a PFC emulsion will make asignificant contribution to overall tissue O2 delivery.

Recently, Patel and Mehra54,55 assessed aspects of O2

transport in mathematical models of uniform and non-uniform blood-PFC systems in relation to O2 uptakefrom both large (> 300 µm diameter) and smaller (<300µm) vessels into tissues. Their modelling approach wasbased on an earlier study which used engineering masstransfer principles to compare O2 uptake in PFC emul-sions and blood.56 They compared their theoreticalfindings with the experimental observations on PFC-mediated O2 supply reported by Braun et al.57 andVaslef and Goldstick.58 At high O2 tensions, it wasfound that PFC emulsion significantly increased O2 fluxat the vessel wall, leading to enhanced tissue oxygena-tion. This increased O2 flux was greater for non-uni-form dispersions where the erythrocytes were predictedto migrate towards the central, low shear regions of thevessel.54,55 Significantly, they found that a near-wallexcess of PFC droplets was not essential to cause thisincrease but, where it occurred, the overall plasma O2

concentration achieved was even greater. Further sup-port for PFC-enhanced increased transfer of O2 fromred cells to tissues also comes from the related mod-elling studies of Perevedentseva et al.59 Overall, suchtheoretical analyses assist in predicting the precisemechanisms by which PFCs enhance tissue oxygena-tion and form a baseline for the interpretation of invivo studies.

PFC emulsions as transfusion alternatives

Faithfull and others60 described a sophisticated com-puter programme which predicted that the infusion of

ca. 1.5 ml 100 ml–1 of Oxygent to haemodilutedpatients breathing supplementary O2 during surgerywould temporarily maintain adequate tissue oxygena-tion, as measured by the mixed venous O2 tension(PvO2), thereby delaying the indication for transfusionof allogeneic (donor) blood. Thus, pre-donated bloodcould be kept in reserve until needed. The programmehas subsequently been validated in animal studies andin ongoing clinical trials.11,31,61–64 In one investigation byBatra and colleagues,62 anaesthetized dogs were sub-jected to moderate haemodilution (final Hb concentra-tion 8 g 100 ml–1) using a proprietary hydroxyethylstarch solution. Following haemodilution, and duringconcurrent 100% O2 breathing, animals received asingle dose of 1.8 g kg–1 Oxygent (60% w/v perflubron)which raised the mean pO2 in skeletal muscle, brainparietal cortex and gut serosa by 64%, 33% and 29%,respectively. Infusion of Oxygent also elevated the PvO2

by 16%, confirming this variable to be a reliable indica-tor of PFC-induced increased tissue pO2, in accordwith predictions arising from the computer modellingof Faithfull and colleagues.60 The potential oxygena-tion benefits from a PFC-based O2 carrier (assumingstable O2 consumption) are listed in Table 6.

Oxygent is currently being evaluated in advancedclinical trials as a temporary oxygenation fluid for usein patients undergoing potentially high blood loss(typically >3 units) surgeries. Indeed, it is this contextthat PFC emulsions are initially expected to enter theclinical therapeutic arena. Multiple-site Phase IIaefficacy trials with Oxygent in surgical patients wereinitiated in the USA and Europe during 1995–1996.For example, in one pilot study, patients were initiallysubjected to acute normovolaemic haemodilution(ANH) with a colloidal plasma expander to collect anaverage of 2 units of fresh autologous blood fromeach individual immediately prior to surgery. A singlebolus dose of a 90% (w/v) perflubron emulsion (0.9 gPFC kg–1 body weight) was infused into O2-breathing(FiO2 = 1.0) patients as an alternative to blood.64 Theinfusion of the PFC emulsion caused a 17% increasein mean PvO2, with no concommitant change in eithercardiac index or total O2 consumption. Such use of arelatively low dose of PFC emulsion to maintaintissue oxygenation means that autologous blood canbe conserved and re-infused into the patient towards

Perfluorinated blood substitutes and artificial oxygen carriers 179

the end of surgery, or in the postoperative period, asneeded. Thus, the use of PFC emulsion in conjunctionwith ANH not only reduces or eliminates the needto infuse allogeneic blood, but also allows surgery tobe initiated at a lowered haematocrit, thereby reduc-ing the number of red cells lost during subsequentbleeding.11,31

In September 1997, it was reported65 that over 250surgical patients had received Oxygent during thePhase II clinical trials and by November 1998, thenumber had risen to >340.66 Phase III studies withthe product were initiated at the end of 1998 andshould last for about 1 year. Regulatory approval forOxygent as a tissue oxygenation fluid is expectedwithin 2 years. The anticipated cost of the emulsion islikely to be comparable to that of blood.

It has been noted67 that one problem with theperioperative use of current PFC O2 carriers is that oftheir short i.v. persistence compared to transfused redblood cells. The same authors also emphasized thecurrent controversial questions of whether periopera-tive ANH, coupled with autologous blood transfu-sion, are desirable and cost-effective strategies forblood conservation and limiting the use of bankedblood.68–70 Future research should be directed atresolving these outstanding issues.

Priming fluids for cardiopulmonary bypass systems

It has been suggested that PFC emulsions may beuseful for priming CPB machines during heartsurgery.71 PFCs can deliver O2 under the hypothermicconditions associated with CPB. This would avoid thesignificant dilution of the patient’s blood by the largevolume (ca. 2 L) of the (typically crystalloid) fluidneeded to fill the extracorporeal circuit. Animalstudies have shown that PFC emulsions, such asOxygent, are an effective replacement when used inconjunction with membrane oxygenators.71 Exposureof human blood to emulsified perfluoro-dichlorooc-tane (Oxyfluor) in a simulated CPB procedurerevealed no significant effects on complement activa-tion.45 As noted already, N2 is also highly soluble inPFCs and one further use for these compoundsduring CPB would be as scavengers of air emboli thatcan occur during surgery.71 In addition to respiratorygas-related applications, PFC emulsions may bedesirable during CPB because of the downregulatoryeffects of emulsion droplets, as a result of phago-cytosis, on the neutrophil-mediated inflammatoryresponse which can be associated with this proce-dure.71 This has led to speculation about the use ofPFCs as immunomodulating agents, as discussedlater.

Liquid ventilation with PFCs

Over 30 years ago, Leland Clark and Frank Gollanreported that mice could survive whilst ‘breathing’from an O2-saturated PFC liquid in which they weresubmerged.72 Their pioneering experiments providedthe first demonstration that PFCs could be valuable asrespiratory gas carriers in biological systems.Subsequent studies using several compounds haveindicated that total or partial ventilation with neatliquid PFC may be useful for the treatment of respira-tory distress syndrome (RDS), a common cause ofdeath in premature infants due to a lack of naturalsurfactant.73,74 The extremely low surface tensions ofPFCs23 ensures that the liquid spreads rapidly anduniformly throughout the respiratory tree, opening upcollapsed alveoli and improving respiratory compli-ance, thereby facilitating gas exchange.75 Clark et al.76

emphasized that the choice of PFCs suitable for liquidventilation should be restricted to compounds withboiling points >150°C, to avoid complications arisingfrom chronic lung hyperinflation.

A ‘neat’ perflubron liquid-based product,LiquiVent, has been produced by the AlliancePharmaceutical Corporation specifically for intratra-cheal administration. Studies in full-term, newbornlambs have shown that tidal liquid ventilation (TLV)with LiquiVent is effective for up to 24 h,77 signifi-cantly longer than the 4 h period reported in previousanimal studies.74

Clinical studies with LiquiVent have focused onpartial liquid ventilation (PLV), in which the lung isfilled with a volume of PFC approximating to thefunctional residual capacity, coupled with gas ventila-tion. This approach reduces the patient’s exposure tothe potentially harmful effects of conventional venti-lation while facilitating respiratory gas exchange. PLVusing perflubron in critically ill newborn babies withsurfactant deficiency and severe RDS producedsignificant improvements in gas exchange.78 There is,however, some controversy as to whether the effective-ness of PLV using PFC can be further enhanced byhigh frequency ventilation which improves pulmonaryfunction in respiratory distressed individuals.79 Smithet al.80 did not observe any significant change inpulmonary gas exchange in respiratory-distressednewborn piglets that received PLV with LiquiVentcoupled with high-frequency ventilation. In contrast,Sukumar et al.81 reported that high frequency PLVwith the same product in preterm lambs not onlyimproved respiratory gas exchange, but resulted in a78% decrease in pulmonary vascular resistance and a5-fold increase in pulmonary blood flow. These differ-ences could be due to variations in the high frequencyventilatory technology between the two studies or

180 Blood Reviews

simply reflect differing species responsiveness to PLVwith PFC.

Earlier related studies82–83 also suggest that PLVwith LiquiVent may a be a simple, but effective, treat-ment for respiratory disorders in adults. However, apreliminary, uncontrolled, study of PLV in adultswith acute RDS yielded inconclusive results.84

Nevertheless, more extensive Phase III clinical trials inthis novel area of therapeutics are expected to beginduring 1999. Whilst liquid ventilation studies may beregarded as somewhat peripheral to blood biology,they, nevertheless, reinforce the effectiveness of PFCsas biomedical respiratory gas carriers.

PFCs as anti-cancer agents

PFC emulsions, such as Oxygent, have been usedboth in vitro and in vivo, including clinical trials, toaugment the tumour cell-killing effects of ionizingradiation and chemotherapy through increased O2

supply to targetted tumours, as discussed in relevantreviews.85–87 In this respect, PFCs fulfill one of theclinical targets for blood substitutes referred to inTable 1. One particularly effective strategy has been touse PFC emulsion in combination with a drug such asnicotinamide, which increases tumour responsivenessto radiation.88

PFCs as organ perfusates and cell culture mediasupplements

Emulsified PFCs have been used as O2-carrying ex-vivo perfusates of organs from several species,including the brain, heart, kidney, liver, lungs, pan-creas and testis.23 This application of PFCs isespecially relevant in transplantation medicine, wheretheir use could significantly extend the donation-implantation time interval.

There is also growing interest in the use of PFCsfor regulating respiratory gas supply to cultured cells,and this aspect was recently reviewed in detail byLowe et al.89 Such use of PFCs in culture systems canreduce or eliminate cellular damage caused by conven-tional, and more vigorous, aeration methods (i.e.stirring), leading to improved cell growth. Thisapproach has been applied to studies with humancells, including neonatal foreskin fibroblasts andretinoblastoma cells, which were grown successfully atan interface formed between PFC liquid and aqueousculture media. McGregor et al.90 reported a novelculture system for human HeLa cells, in which amicro-layer of gelatin, produced by perfluoroalkyla-tion, had been deposited at an interface between per-fluorodecalin and aqueous medium. The cells dividednormally at the interface producing multi-cellular,

tissue-like colonies consisting of up to 19 cell layers.PFCs have enormous potential in cell biotechnologywhere, through their facilitation of gas supply, theystimulate cell division and biomass production, lead-ing, in some instances, to increased production ofvaluable cellular products.89 PFCs are especiallyadvantageous for use in cell and tissue culture systemssince they are heat stable, readily sterilized (e.g. byautoclaving) and can be recovered from aqueoussystems with the potential for recycling, thus makingthem economically viable.

Other biomedical applications for PFCs

Diagnostic imaging agents

There is a long-standing interest in the use of PFCs ascontrast-enhancing agents for non-invasive diagnosticprocedures. For example, perflubron is radiopaqueand the neat liquid is effective in humans for contrastenhancement of X-rays in gastroenterography andbronchography.10,40 Administration of perflubornwas found to be safe and effective in differentiatingintestinal obstruction and gastro-intestinal fistulas.The subsequent advent of modern computerizedtomographic scanners has enabled the use of per-flubron in radiography to be optimized.10,40

PFCs also have good acoustic impedance makingthem useful for the facilitation of ultrasound imag-ing.10,40 This echogenicity arises as a result of the highdensity of PFCs and their low acoustic velocity.Lanza et al.91 reported the development of a novel,biotinylated, lipid-encapsulated perfluoro-dichlorooc-tane emulsion for use as a target-specific ultrasoundcontrast imaging agent and its preliminary use inanimals for the detection of vascular thrombi. Theemulsion was administered at the end of a three-stepapproach, in which the first two phases involvedi.v. injection of a biotinylated, antifibrin antibody(ligand) to target thrombi, followed by avidin, toconjugate and cross-link the ligand. Injection of thePFC emulsion resulted in its binding to the anti-body-avidin complexes, thus visualizing the vascularabnormalities. The success of this technology willdepend, crucially, on the availability of specific mono-clonal antibodies for the desired molecular epitope tobe visualized. Nevertheless, it represents an importantstep in the development of effective, site-directedultrasonic contrast agents, which hitherto have eludedresearchers in this field.

Recent interest in the use of PFCs as ultrasoundcontrast imaging agents has also focused on low m.w.PFCs than are gaseous at body temperature27–29 andseveral products are at an advanced stage of develop-ment. Echogen (Sonus Pharmaceuticals, USA), isone example currently in clinical trials. It consists of

Perfluorinated blood substitutes and artificial oxygen carriers 181

emulsified perfluoro-pentane (Table 3) that is con-verted immediately prior to i.v. injection into highlyechogenic microbubbles (diameter <10 µm).92

Imagent US (Alliance Pharmaceutical, in conjunctionwith Schering AG, Germany), the active constituentof which is perfluoro-hexane (Table 3) microbubbles,is similarly in advanced clinical trials primarily for theassessment of cardiac function and myocardial perfu-sion.27–29 A product based on perfluoro-propane gas(Table 3), available commercially as Aerosomes(ImaRx Pharmaceutical Corporation, USA), has alsobeen used to study aspects of cardiac function inanimals.93 The advantage of using the more volatilePFCs in this context is that they persist in the circula-tion for a few minutes to produce their echo contrasteffect before being rapidly exhaled. However, oneoutstanding question is whether and to what extentany non-specific uptake of PFC by the MMS andother tissues, however transient, can distort theoverall echogenic signal. Clearly, the ultimate clinicaldiagnostic value of these products will depend onobtaining a very high degree of image precision.

Ophthalmologic agents

Several PFC liquids, including perfluoro-ethane,perfluoro-propane, perfluoro-octane, perfluorodecalinand perfluoroperhydrophenanthrene (Table 3), havebeen used successfully during ophthalmic surgery forhydrokinetic retinal manipulation (tamponade) and inthe treatment of severe proliferative vitreoretinopa-thy.94,95 The high densities and low viscosity’s of PFCsmake them more effective for intra-ocular use thanalternative materials, such as fluorosilicone oil orsodium hyaluronate. Low m.w. PFCs are preferred inthis context because their relatively high vapour pres-sure facilitates good evaporation of residual dropletsfrom the retinal surface.94,95 In one clinical study, a68% success rate in retinal reattachment was observedin 87 patients at 1 month after vitrectomy plus retinaltamponade with either perfluoro-ethane or perfluoro-propane.96 More recently, a major study on the surgi-cal management of traumatic retinal detachment in111 patients using perfluoroperhydrophenanthreneindicated that the compound was well tolerated bypatients, with >75% success in retinal reattachment.97

However, in a subsequent report, Batman and Cekic98

noted that in one patient examined several weeks afterintraocular tamponade with perfluoroperhydrop-henanthrene, some retinal damage had occurred.Similar evidence of corneal abnormalities has alsobeen seen in patients that had retained small amountsof perfluorodecalin in their eyes for up to 18 monthsafter its intraoperative use.99 Whilst there is substan-tial support for the use of PFCs in vitreoretinal

surgery, reinforcing the multiplicity of their clinicalapplications, it is nevertheless clear that further workon the longer-term effects of the retention of residualliquid in the eye is needed.

Immuno-modulating agents

Emulsified PFCs may be useful for protecting tissues(e.g. coronary vasculature) against inflammatoryreperfusion damage through their transient alter-ations in blood leucocyte functions, arising as a resultof the phagocytic uptake of emulsion droplets.100,101

Exposure of pig alveolar macrophages to perflubronin vitro decreases phorbol 12-myristate 13-acetate(PMA)-induced chemiluminescence102 and similarfindings have been observed with human neutro-phils.103 A novel, perfluorodecalin-based emulsionsimilarly produced a transient, dose-dependent,decrease in PMA-induced chemiluminescence inhuman peripheral blood leucocytes.42 Overall, thesefindings reinforce previous suggestions10,23,25 that, inaddition to their use as temporary i.v. O2 carriers,emulsified PFCs may also be valuable in ischaemictissues as immunomodulating agents, acting to tem-porarily suppress leucocyte-mediated inflammation.However, any potential anti-inflammatory effects ofPFC emulsions must be balanced against the possibleproblems of immunosuppression, especially on repeator prolonged administration and in high-risk patients.Injection of emulsified PFCs can alter immune systemfunction in vivo, with the responses depending on thedose, timing and route of administration relative toimmunological challenge.10,104 Therefore, future stud-ies in this area should assess the extent to which thecomposition and physical characteristics of PFCemulsions can alter leucocyte functions and determinethe time course and significance of any immunosup-pressive responses in the recipient.

CONCLUSIONS

This paper has given an overview of the developmentand applications of PFCs as i.v. O2 carriers, focusingon their potential clinical value as alternatives to thetransfusion of red blood cells, especially duringsurgery. In this context, PFC- and Hb-based i.v. O2

carriers should, ultimately, increase transfusionoptions, thereby conserving autologous blood andreducing patient exposure to donated blood. PFCsand their emulsions have additional, multi-facetedapplications in medicine and biotechnology and this isreflected in their use as pump-priming fluids forcardiopulmonary bypass machines, pulmonary venti-lation fluids, anti-cancer agents, organ perfusates and

182 Blood Reviews

cell culture media supplements. Such uses for PFCsshould not overshadow their further value as diag-nostic contrast imaging agents, ophthalmologicaltools and possible immuno-modulators, all of whichare being actively investigated.

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