17 Zuo Review

Biochimica et Biophysica Acta 1778 (2008) 1947–1977

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Biochimica et Biophysica Acta

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Review

Current perspectives inpulmonary surfactant— Inhibition, enhancement andevaluation

Yi Y. Zuo a,b,c, Ruud A.W. Veldhuizen d,e, A. Wilhelm Neumann f, Nils O. Petersen a,b,g, Fred Possmayer a,c,⁎a Department of Biochemistry, University of Western Ontario, London, Ontario, Canadab Department of Chemistry, University of Western Ontario, Canadac Department of Obstetrics/Gynaecology, Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, University of Western Ontario, Canadad Department of Physiology and Pharmacology, University of Western Ontario, Canadae Department of Medicine, University of Western Ontario, Canadaf Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canadag National Institute for Nanotechnology, National Research Council Canada, Edmonton, Alberta, Canada

Abbreviations: ADSA, axisymmetric drop shape analyssurfactant; CBS, captive bubble surfactometer; CSD, constrLangmuir–Wilhelmy balance; PBS, pulsating bubble surfaPMPC, palmitoyl–myristoyl phosphatidylcholine; POPC, pdistress syndrome; SP, surfactant protein; TC, tilted-condepressure; γ, surface tension(s); γeq, equilibrium surface te⁎ Corresponding author. Department of Obstetrics/Gyn

ON, Canada N6A 5C1. Tel.: +1 519 661 2111x80972; fax:E-mail address: [email protected] (F. Possmayer).

0005-2736/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.bbamem.2008.03.021

a b s t r a c t
a r t i c l e i n f o
Article history:
Pulmonary surfactant (PS) i Received 6 December 2007Received in revised form 26 March 2008Accepted 26 March 2008Available online 8 April 2008
Keywords:Pulmonary surfactantPhospholipidSurface activitySurfactant inhibitionWater-soluble polymerTensiometry

s a complicated mixture of approximately 90% lipids and 10% proteins. It plays animportant role in maintaining normal respiratorymechanics by reducing alveolar surface tension to near-zerovalues. Supplementing exogenous surfactant to newborns suffering from respiratory distress syndrome (RDS),a leading cause of perinatal mortality, has completely altered neonatal care in industrialized countries.Surfactant therapy has also been applied to the acute respiratory distress syndrome (ARDS) but with onlylimited success. Biophysical studies suggest that surfactant inhibition is partially responsible for thisunsatisfactory performance. This paper reviews the biophysical properties of functional and dysfunctional PS.The biophysical properties of PS are further limited to surface activity, i.e., properties related to highly dynamicand very low surface tensions. Three main perspectives are reviewed. (1) How does PS permit both rapidadsorption and the ability to reach very low surface tensions? (2) How is PS inactivated by different inhibitorysubstances and how can this inhibition be counteracted? A recent research focus of using water-solublepolymers as additives to enhance the surface activity of clinical PS and to overcome inhibition is extensivelydiscussed. (3) Which in vivo, in situ, and in vitro methods are available for evaluating the surface activity of PSand what are their relative merits? A better understanding of the biophysical properties of functional anddysfunctional PS is important for the further development of surfactant therapy, especially for its potentialapplication in ARDS.

© 2008 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19482. Fundamentals of pulmonary surfactant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1949

2.1. A brief history of the discovery of surfactant and its clinical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19492.2. Alveolar surfactant metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19492.3. Surfactant composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19502.4. Phospholipid phase behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1950

2.4.1. Bilayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19502.4.2. Monolayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19512.4.3. Nonbilayer phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1952

is; AFM, atomic force microscopy; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BLES, bovine lipid extractained sessile drop; DPPC, dipalmitoyl phosphatidylcholine; DPPG, dipalmitoyl phosphatidylglycerol; LE, liquid-expanded; LWB,ctometer; PC, phosphatidylcholine; PD, pendant drop; PEG, polyethylene glycol; PG, phosphatidylglycerol; PL, phospholipid(s);almitoyl–oleoyl phosphatidylcholine; POPG, palmitoyl–oleoyl phosphatidylglycerol; PS, pulmonary surfactant; RDS, respiratorynsed; TEM, transmission electronmicroscopy; Tm, main transition temperature; π, surface pressure(s); πe, equilibrium spreadingnsion; γmin and γmax, minimum and maximum surface tensions (upon film oscillation), respectivelyaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, Dental Sciences Building 5009, London,+1 519 661 3175.

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1948 Y.Y. Zuo et al. / Biochimica et Biophysica Acta 1778 (2008) 1947–1977

2.5. Biophysical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19532.5.1. Rapid adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19532.5.2. Film stability at low surface tensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19532.5.3. Surface-associated surfactant reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1955

3. Inhibition of clinical surfactants and reversal thereof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19563.1. Different types of clinical surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19563.2. Surfactant inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1957

3.2.1. Surfactant inhibition by plasma proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19573.2.2. Surfactant inhibition by lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1958

3.3. Overcoming surfactant inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19593.3.1. Optimizing lipid and protein contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19593.3.2. Using polymeric additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19593.3.3. Beyond surface activity enhancement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1961

4. Methods for evaluating pulmonary surfactant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19634.1. In vivo methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19634.2. In situ methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19644.3. In vitro methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1964

4.3.1. Langmuir–Wilhelmy balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19644.3.2. Pulsating bubble surfactometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19654.3.3. Captive bubble surfactometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19664.3.4. Constrained sessile drop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19684.3.5. Other in vitro methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1969

5. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1970Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1970References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1970

1. Introduction

The entire alveolar surface of mammalian lungs is lined with a thinfluid continuum, called the alveolar lining layer [1]. This liningconsists of an aqueous hypophase covered by a film of pulmonarysurfactant (PS) [2]. The major function of this PS film is to reduce thesurface tension of the alveolar surface, thereby contributing signifi-cantly towards maintaining the normal mechanics of respiration [3].First by lowering alveolar surface tension, PS reduces the energyrequired to inflate the lungs thereby increasing pulmonary compli-ance (i.e., the ratio of lung volume change to an applied distendingpressure). Second by decreasing elastic recoil, PS reduces the like-lihood of alveolar collapse during expiration. As a result, the lungs caneasily maintain patency with a small transpulmonary pressure of lessthan 10 cm H2O [4].

PS exists not only in alveoli, but also in bronchioles and smallairways. Themain function of airway surfactant is to maintain patencyof the conducting airways, i.e., to prevent cohesion of bronchiolarwalls by keeping thewater lining spread out and by decreasing surfacetension of the airway mucus lining [5,6]. This latter function isparticularly important in diving mammals (e.g., seals) and in reptilesbecause these animals often collapse part, or all, of their lungs as partof their diving and expiratory cycles [7,8]. When it comes to humans,PS can play an important role in protecting the epithelial cells fromdamage by air thrusts during reopening of collapsed airways, acommon condition in mechanical ventilation [9]. In addition to the

Table 1Physiological functions of pulmonary surfactant

Surface tension related Non-surface tension related

Maintaining a large gas transfer area Specific and non-specific host defenseIncreasing lung compliance on inspiration Pathogen barrierStabilizing alveoli on expiration Antibacterial/antiviral activityAirway stabilization Smooth muscle relaxationAnti-edema effectsAnti-adhesion agentProtecting epithelial cells in airway reopeningFacilitating mucociliary transportFluid dispersalParticle removal

classical functions relevant to surface tension reduction, PS possessesa number of other functions which make it physiologically indispens-able. These functions are summarized in Table 1 [10–13]. Nevertheless,this review will be restricted to the surface tension-relevant proper-ties of alveolar PS.

Deficiency or dysfunction of PS causes severe respiratory disease.Neonatal respiratory distress syndrome (RDS), the major disease of PSdeficiency worldwide, is due to prematurity [14]. Premature infantssuffering from RDS exhibit increased work of breathing, decreased lungcompliance, prominent atelectasis (alveolar collapse) with reducedfunctional residual capacity (FRC, i.e., the lung volume remaining at theendof expiration), impaired gas exchange, and diffuse interstitial edema[14]. The actual incidence of RDS in premature infants declines greatlywith increasing gestational age. It is estimated that RDS generally affects10% of all premature infants in developed countries [15]. In 2002 RDSaffected an estimated 24,000 newborns in the USA alone [15].

Displaying symptoms similar to RDS, acute lung injury (ALI) and itsmore severe form, the acute respiratory distress syndrome (ARDS),occur as a rapid onset of respiratory failure that can affect patients ofany age [16]. ARDS affects approximately 150,000 people per year inthe USA and has a case fatality rate of approximately 30–40% [17]. Thepathogenesis of ARDS is still not fully understood but surfactantinhibition, due to leakage of a variety of inhibitory substances (e.g.,serumproteins, hemoglobin, and certain lipids) into the alveolar space,is believed to be an operative cause induced by primary pathogenesissuch as extensive lung inflammation, trauma, severe pulmonaryinfection, near-drowning, oxygen toxicity, or radiation damage [16,17].

Exogenous surfactant replacement therapy, in which eithersynthetic or modified natural PS (extracted from bovine or porcinesources) is delivered into the patients' lungs, has been establishedas a standard therapeutic intervention for patients with RDS [18].Surfactant therapy has also shown limited positive effects with ARDSpatients [11,19–21].

Therefore, the study of PS has not only physiological but alsoclinical significance. This paper reviews the history and recentprogress in PS research. Due to the interdisciplinary nature of thesestudies, a large body of literature in a variety of areas includingbiochemistry, biophysics, physiology, colloid and interface science,biomedical and clinical applications, is available. This reviewwill focuson the biophysical properties, especially surface activity, of PS. Other

1949Y.Y. Zuo et al. / Biochimica et Biophysica Acta 1778 (2008) 1947–1977

areas closely related to the biophysical studies are also covered, albeitto a lesser extent. The remainder of this paper is organized into thefollowing three parts: (1) Fundamentals of PS, including a brief historyof the discovery of PS and its clinical applications, alveolar surfactantmetabolism, component functionality, and interfacial properties. Thefocus of this part is on the biophysical properties of functional PS.(2) Clinically used PS preparations and the challenge of ARDS treat-ment. We will emphasize biophysical inhibition of PS and the reversalof this inhibition by water-soluble polymeric additives, a recentresearch focus in developing inactivation-resistant surfactant formu-lations. (3) Methods for evaluating the surface activity of PS. Althoughin vivo, in situ and in vitro methods will be discussed, focus will be onthe in vitro methodologies.

2. Fundamentals of pulmonary surfactant

2.1. A brief history of the discovery of surfactant and its clinical applications

The initial indication for PS can be traced to 1929 when vonNeergaard first demonstrated the importance of interfacial forces forlung mechanics [22]. By comparing the recoil pressure (i.e., thetendency to collapse) of lungs filled with air and lungs filled withaqueous solution, von Neergaard demonstrated that alveolar surfacetension contributed significantly to lung recoil. He also hypothesizedthat the alveoli might contain a special surface active material capableof lowering alveolar surface tension. However, he was not able todemonstrate that surface active materials were present in the lungs,using the methods available to him [23].

von Neergaard's findings drew little attention. In 1955, Pattlereported in his classic paper, published in Nature [24], that smallbubbles generated in lung extracts initially decreased in size but thenstabilized at 40–50 μm diameter. Pattle concluded that the surfacetension of these microbubbles likely fell to near 0 mN/m. Otherwisethe pressure difference across the bubble interface, predicted by theLaplace equation of capillarity (see Section 4), would cause thebubbles to deflate rapidly. Initially, Pattle erroneously concluded thatthe film responsible for the zero surface tension was composed of aninsoluble protein. Pattle later corrected this error by identifying thefilm as a lipid–protein complex [25].

In 1957, Mead et al. reported the dependence of lung complianceon surface tension forces [26]. They observed that, when an excisedlung was filled with air, the quasi-static (step-by-step) transpulmon-ary pressure observed during inflation was higher than that observedduring deflation, a phenomenon they called “pulmonary volume–pressure hysteresis”. The hysteresis largely disappeared with lungsfilled with saline. They attributed this difference to the effect ofsurface tension forces operating at the air–water interface of the lung.Interestingly, they noted a large discrepancy (∼10-fold) between thesurface area predicted by their measurements (assuming alveolarsurface tension to be 60 mN/m as with serum) and the surface areacalculated by histology.

Also in 1957, Clements published the first direct surface tensionmeasurements on lung extracts using his modified Langmuir balance(see Section 4) [27]. He found that the surface tension of salineextracts from animals' lungs decreased from 46 to 10 mN/mwhen thesurface area was reduced. He also calculated the compressibility ofthese films to be as low as 0.01 (mN/m)−1, consistent with a strongability to stabilize the lungs against collapse at the end of expiration.This in vitro surface activity of PS, i.e., its ability to decrease surfacetension to remarkably low values upon film compression, was laterconfirmed by Schurch et al. in situ through direct surface tensionmeasurements in excised lungs [28].

The clinical correlation between PS deficiency and RDS was firstestablished by Avery andMead in 1959 using a Clements-type Langmuirbalance [29]. They reported that very little surface active material couldbe recovered from the lungs of infants who died of hyaline membrane

disease, now known as RDS. However, surfactant was found in lungextracts of infantswhodied fromnonpulmonary diseases, provided thattheir birthweight wasmore than 1000 g. Avery andMead hypothesizedthat the lack of surfactant due to prematurity was responsible for RDS.The pathological relationship between prematurity and surfactantdeficiency was later confirmed by Brumley et al. who found that thePS system does not mature until late in gestation [30].

In 1980, Fujiwara et al. reported the first successful trial of sur-factant replacement in RDS infants using a surfactant extractedfrom bovine lungs [31]. Since that discovery, surfactant replacementtherapy has become a standard therapeutic intervention for RDSpatients. Partially owing to surfactant therapy, the mortality rate ofpremature infants in the USA due to RDS fell by 24% in 1990 and hascontinued to decrease thereafter [32,33].

In 1967, Ashbaugh et al. first described a syndrome of acuterespiratory distress in adults which displayed some symptoms similarto RDS in premature infants [34]. This syndrome is now referred to asARDS. Inspired by the symptomatic similarity between ARDS and RDS,surfactant replacement therapy has been tested in treating ARDS [35].It was found that surfactant therapy generally showed a positive effecton the respiratory aspects of ARDS [19,20,36]. Gaseous exchange isnormally improved in the patients, although the effects on the diseaseas a whole can be variable and are largely dependent on dosing,administration, timing and the actual surfactant preparation used[37,38]. However, ARDS is a complicated disease which can affect non-respiratory systems, such as liver and kidney, leading to multiorganfailure [37,38]. With the exception of two trials conducted withpediatric patients suffering from a selected etiology consistent withdirect lung involvement and using a highly surface active, modifiednatural surfactant (Infasurf) [39,40], surfactant treatment has notdemonstrated a significant impact onmortality fromARDS. As a result,clear indications of a distinct surfactant-mediated decrease inmortality or improvement in ventilatory care of ARDS patients arestill lacking and further definitive clinical trials, possibly using“designer” surfactant preparations (see Section 3), are required.

Excellent reviews on the history of the discovery of PS and itsclinical applications can be found elsewhere [23,41–46].

2.2. Alveolar surfactant metabolism

Using electron microscopy, Weibel and coworkers [2,47,48] haveconvincingly demonstrated that a substantial area of the alveolar sur-face is lined with a thin aqueous layer. The overall continuity of thislining layer was definitively established by studies on rat lungs usinglow-temperature electron microscopy [49]. It was found that theaqueous layer has an average thickness of 0.14 μm over the alveolarsurface, 0.89 μm in alveolar corners, with an overall area-weightedaverage thickness of 0.2 μm [49]. Although very thin, this aqueouslayer is considerably larger than that required to accommodate a bulkphase comparable to the surfactant film on top, which is in moleculardimensions. This aqueous subphase is essential for PS metabolismbecause it provides a medium for surfactant secretion, morphologicaltransformation, adsorption, desorption and recycling [14]. These eventsare commonly referred to as the life cycle or alveolar metabolism ofPS [3].

PS is synthesized by pulmonary type II epithelial cells, processedand packed into lamellar bodies, structures consisting of closelypacked multiple bilayers. Subsequently, PS is secreted into theaqueous subphase of the alveoli, where the lamellar bodies undergotransformation into a morphological form called tubular myelin. Theformation of tubular myelin is dependent on the presence of sur-factant apoproteins and calcium [50–52]. Tubular myelin is composedof large square elongated tubes, constituted primarily of phospholi-pids and proteins, ranging in size from nanometers tomicrons [53,54].It appears that the surfactant components are subsequently releasedfrom tubular myelin to form a surface active film at the air–water


interface of alveoli by rapid adsorption. Traditionally, the PS film hasbeen considered to be a monomolecular film, i.e., a monolayer [55,56].However, accumulated evidence from studies using electron micro-scopy [57–59], captive bubble tensiometry [59–61], autoradiography[62], atomic force microscopy [63,64], and neutron reflection [65] allsuggest that at least part of the PS film is thicker than a singlemonolayer. It consists of a surface monolayer plus, in places, one ormore lipid bilayers, closely and apparently functionally associatedwith the interfacial monolayer. These multilayers form the so-calledsurface-associated “surfactant reservoir” (detailed later) [60].

After de novo adsorption, the surfactant film is periodically com-pressed and expanded during breathing. This action modulatessurface tension in the lungs. After performing its physiologicalfunctions, surfactant is released as small, unilamellar vesicles. Someof this spent surfactant is taken up by the alveolar macrophageswhile most of the remainder is cleared from the alveolar space byendocytosis back into type II cells. These cells recycle part of thesurfactant components into lamellar bodies. Some surfactant appar-ently flows into the tracheae and is eventually swallowed. Theestimated turnover period of PS is surprisingly short, ranging from4 to11 h [66]. Reviews on the metabolism of PS can be found in [67,68].

2.3. Surfactant composition

Comparative biological studies suggest that PS exists in all air-breathing vertebrates, although with somewhat differing composi-tions [69–71]. However, the composition of mammalian PS isremarkably similar among diverse species, i.e., approximately 90%lipids and 10% proteins by weight [72]. The composition of bovinesurfactant, as shown in Fig. 1, is representative of most species [73].

The lipids consist mainly of phospholipids (PL, ∼90–95 wt.%) with asmall amount of neutral lipids (∼5–10 wt.%), primarily cholesterol[68,74]. Among the PL components, phosphatidylcholine (PC) is themost prevalent class, accounting for ∼80% of the total PL. Althoughnotable exceptions exist [70], in most cases about 30–60% of the PC isdipalmitoyl phosphatidylcholine (DPPC) (16:0/16:0 PC), a long-chained,disaturated, zwitterionic PL. Most species also contain significant levelsof palmitoyl–myristoyl phosphatidylcholine (PMPC) (16:0/14:0 PC) [70].The other prominent PC components are mainly unsaturated, e.g.,palmitoyl–oleoyl phosphatidylcholine (POPC) (16:0/18:1 PC). Apartfrom PC, other classes of PL which constitute the remaining 20% areprimarily unsaturated, including mainly anionic PL (e.g., phosphatidyl-glycerol (PG), phosphatidylinositol (PI), and lyso-bis-phosphatidic acid)but also including non-PC zwitterionic PL (e.g., phosphatidylethanola-mine (PE) and sphingomyelin). Anionic PL as a group accounts for ∼15%of the total PL. Small amounts of lyso-PC are also present.

Fig. 1. The composition of bovine pulmonary surfactant. DPPC: dipalmitoyl phosphati-dylcholine; PC: phosphatidylcholine; PG: phosphatidylglycerol; PI: phosphatidylinositol;PE: phosphatidylethanolamine; Lyso-bis-PA: lyso-bis-phosphatidic acid; SPM: sphingo-myelin; DG: diacylglycerol; Chol: Cholesterol. Adapted from Yu et al. [73].

Four proteins have been found associated with PS [75]. They arenamed surfactant protein (SP)-A, -B, -C and -D, based on the nomen-clature proposed by Possmayer [76,77]. SP-A (monomeric MW 26–38 kDa) and SP-D (43 kDa) are large, multimeric, hydrophilicglycoproteins. They are members of the Ca2+-dependent carbohy-drate-binding collectin (collagen-like lectin) family. In contrast, SP-B(MW 8.7 kDa) and SP-C (4.2 kDa) are smaller and extremelyhydrophobic. Among these surfactant associated proteins, SP-A isthe most abundant by mass, accounting for about 5 wt.% of PS, withSP-D, accounting for about 0.5%. SP-B and SP-C together constituteapproximately 2% (∼1:3 in bovine surfactant).

SP-A is the most abundant surfactant protein by mass, but not interms of molar ratio. Taking bovine surfactant as an example, for everySP-A octadecamer (composed of six trimers) there are approximately3 SP-B dimers, 45 palmitoylated SP-C and 12,500 PL molecules. Thereare about half as many SP-D dodecamers (composed of four trimers)as SP-A octadecamers in the alveolar space, but SP-D is soluble andonly a small proportion of this collectin will be associated withsurfactant.

Reviews on the PL and protein compositions of PS and itscomponent functionality can be found elsewhere [10,14,74,75,78–84].

2.4. Phospholipid phase behavior

Discussion of the biophysical properties of PS requires some basicknowledge of PL phase behavior. The phase behavior of PL iscommonly studied with monolayers, bilayers, or vesicles [85]. Thephase behavior of PL can be profoundly varied by the addition ofcholesterol. Here, we briefly review the PL and PL-cholesterol phasebehavior and lipid polymorphism relevant to PS study. Recent reviewson the PL phase behavior of bilayers [84], monolayers [86], vesicles[87], and lipid polymorphism [88,89] can be found elsewhere.

2.4.1. BilayersWhen dispersed in aqueous phase, at room temperature and

atmospheric pressure, most PLmolecules spontaneously form bilayers(Fig. 2(a)), where the hydrophobic PL fatty acid groups shield eachother from being exposed to water. The formation of closed vesicles(i.e., liposomes, Fig. 2(b)–(d)) completes this water-avoidance process,resulting in energetically optimized structures. With increasingtemperature, PL bilayers without cholesterol “melt” from an orderedgel (Lβ) phase to a disordered liquid–crystalline (Lα) phase at the maintransition temperature (Tm). This fatty acid disorder permits diffusionof individual PL molecules within each leaflet of the bilayer, a propertyreferred to as “fluidity”.

Addition of a sterol such as cholesterol transforms PL bilayers in Lβand Lα phases into a liquid-ordered (Lo) phase (sometimes referred toas β phase) and a liquid-disordered (Ld) phase, respectively. Theoverall fluidity of the bilayers in the above mentioned four phaseswould be LβbLobLdbLα. This is because when intercalated betweenthe closely packed Lβ PL molecules, cholesterol disrupts (fluidizes) theordered structures. On the other hand, when associated with mobileLα PL molecules, cholesterol condenses and increases the packingdensity of the disordered structure [90]. Thus the overall effect ofcholesterol is to have a fluidizing effect on lipids in the gel state(because it disrupts packing) and a condensing effect on fluidmembranes (because it interacts with disordered chains and stabilizesthem). However, the actual phase behavior of PL-cholesterol mixturesis complex and depends greatly on both the temperature andcholesterol mol fraction [91].

The readers are cautioned that some authors have not yet adoptedthe above nomenclature and may, for example, refer to plasmamembrane lipids, which contain cholesterol, as being in the Lα phase.It should also be noted that the actual phase behavior of PL bilayerscan be more complicated and intermediate phases, such as the ripplephase, can exist between the main Lβ and Lα phases [85].

Fig. 2. Different phospholipid (PL) morphology in the aqueous phase. The formation of bilayer and nonbilayer structures is largely dependent on the molecular shape of the PL.(a)–(d) show bilayer structures and (e)–(g) show a nonbilayer structure. (a) Cross section of a PL bilayer, where the fatty acids interact and the hydrophilic headgroups areclose together to help keep the acyl moieties “dry”. There is an inherent positive curvature with bilayers which is exaggerated here; (b) Unilamellar vesicle, which is similar inform to a tennis ball; (c) Multilamellar vesicle, which is similar to an onion; (d) Multivesicular body, where small vesicles are trapped within a larger vesicle; (e)–(g) Invertedhexagonal (HII) phase. (e) Cross section of a HII phase cylinder. Due to the small size of the PE headgroup, with a planar bilayer the mobile acyl groups would be exposed to water.Moving the headgroups together results in negative curvature of the resulting cluster. As a consequence the PE headgroups enclose a water pocket and the acyl groups extendoutwards. (f) A HII phase cylinder, where the PE headgroups interact with the long column of water extending down the middle of the cylinder. Note that only two methyl groupsper PE molecule are exposed to the exterior. (g) HII phase, where a number of the hydrophobic cylinders depicted in (f) interact together in a hexagonal pattern, thus minimizingwater–fatty acid interactions.


2.4.2. MonolayersWith PLmonolayers, water avoidance is achieved through extension

of the fatty acid groups into the air (which is much more hydrophobicthan water). The behavior of PL in interfacial monolayers can becorrelated at least in part with the phase behavior of hydrated bilayers.Deposition of a small amount of a saturated PL, such as DPPC, on asurface balance will result in a monolayer in the dilute gaseous phase.Because van derWaals attractive forces areweak and the fatty acids can“wave” in air, theDPPCmolecules can collidewith one another, althoughthey tend to remain separated. Reducing the surface area available toeach lipid molecule by moving a barrier across the surface or bydepositing additional DPPC will increase the surface concentration. At acertain point, all of the molecules will touch each other. A furthercompression will then lead to a detectable increase in surface pressure,which can be measured either as back pressure on the barriers of asurface balance or, as explained later, as a decrease in surface tension.Here PLmolecules will no longer be in the dilute gaseous state but are inthe more concentrated lipid-expanded (LE) phase where they interact,but the fatty acids remainmobile due to the presence of kinked gauche–gauche (also known as trans-gauche) configurations.

As the film is further compressed, some of the PL molecules willbecome more closely packed to generate the more ordered tilted-condensed (TC) phase (traditionally called the liquid-condensed (LC)phase) where all of the acyl groups are in the extended all-trans(straight) configuration [86]. At this point, further decreases in surfaceareawill generate LE-TC coexistence where the decreased surface areais taken up by forming a greater proportion of TC phase [86,92–94].Nucleation and growth of the TC phase in PL monolayers can bedirectly visualized as “domain” formation using fluorescence micro-

scopy or atomic force microscopy (AFM) (detailed later). Within the TCdomains, PL molecules interact side by side and the fatty acids tilt in aparallel fashion with a tilting angle smaller than the PL molecules inthe LE phase. (See Fig. 3(b) for a schematic of the different chain tiltingin the LE and TC phases.) Withmonolayers composed of free saturatedfatty acids, further pressure (area reduction) can lead to a solid phase,also known as the untilted-condensed phase [86], in which the fattyacid chains are nearly perpendicular to the surface. Theoretically, thiscould also occur with DPPC, possibly by dehydration and reorientationof the phosphorylcholine headgroups, but to our knowledge, there isno direct experimental evidence for the formation of the solid phasewith either DPPC or PS [93,94].

The domain formation occurring during LE-TC phase transition duetomonolayer compression can be considered as a process of pressure-induced crystallization. This raises a distinction between phase transi-tion in monolayers and bilayers. While (bulk) pressure has only asmall effect on bilayers, phase transition in monolayers is dependentnot only on temperature but also on the surface pressure of themonolayers. With a monolayer in the LE-TC coexistence state, in-creasing the temperature will lead to a reduction in the size andpossibly the number of the TC domains. However, simply increasingthe surface pressure will restore the domains. This is true fortemperatures up to and, in practice, somewhat above the correspond-ing Tm for bilayers of that particular PL [92,94–98]. For example, it hasbeen observed that highly compressed DPPC monolayers only meltedat temperatures of 48–55 °C, well above the DPPC bilayer Tm of 41.5 °C[97]. In essence, this means that compared to bilayers, monolayers donot possess a characteristic Tm. Rather, the “Tm” for monolayers ishighly dependent on the surface pressure.

Fig. 3. Schematic representation of pulmonary surfactant adsorption. (a) Diffusion of a surfactant vesicle/bilayer towards the air–water interface. In this schematic, the surfactantbilayers consistmainly of DPPC,fluid PC (such as POPC), PG (DPPG and POPG) and PE, in similar proportion to BLES. (b) Fusion of the vesicle/bilayerwith the interface stabilized by SP-B.Note that PE molecules could help stabilize the negative curvature generated at the fusion pore/neck monolayer and fusion pore bilayer interfaces. The monolayer contains tilted-condensed (TC) and liquid-expanded (LE) domains composed of saturated and unsaturated phospholipids, respectively. (c) Fusion of the vesicle/bilayerwith the interface stabilized bySP-C. Note that the bilayer can be also connected to the interfacial monolayer by a SP-C molecule. Such SP-B and SP-C stabilized fusion necks/pores can occur simultaneously andindependently during the adsorption of surfactant. Note that SP-B and SP-C are not drawn to scale and the proportions of these hydrophobic proteins to PL are not representative.


Analogous to bilayers, incorporating cholesterol into a DPPCmonolayer changes the TC phase to a liquid-ordered (LO) phase,whereas adding this sterol to an unsaturated POPC monolayerconverts it to a liquid-disordered (LD) phase. The LO phase is morefluid than the TC phase and the LD phase is less fluid than the LE phase,leading to an order of fluidity: TCbLObLDbLE.

2.4.3. Nonbilayer phasesIn addition to bilayer structures, certain PL molecules upon

hydration can self-assemble into some nonbilayer structures, suchas the inverted hexagonal (HII) phase (Fig. 2(e)–(g)) and cubic phases

[88,89,99]. These nonbilayer phases have a curved morphology whichcontrasts with the planar morphology of lamellar phases. As shown inFig. 2(g), in HII phase, PL molecules are arranged cylindrically with thepolar headgroups facing toward the interior of the structure. Theformation of nonbilayer phases arises due to the molecular shape ofthe PL [88,89,99]. Typical bilayer-forming molecules, such as PC, bothsolid and fluid, have a cylindrical shape with nearly equal headgroupand hydrocarbon areas. Such molecules pack into sheets which can fitreadily into bilayers. In contrast, nonbilayer-forming lipids, such asfluid PE, exhibit a cone-shape with a small headgroup area and largehydrocarbon area. This molecular shape prevents them from packing


into a planar bilayer arrangement without allowing water to slip inbetween the headgroups and wetting the acyl groups. Consequently,monolayers comprised of such lipids have a spontaneous tendency toform structures with negative curvature [100].

Formation of HII phase is promoted by increasing the temperature,introducing acyl chain unsaturation, and decreasing pH [101,102].Unsaturated PE is found to be particularly effective in forming HII

phase [89]. In addition, certain proteins or peptides are found tomodulate the tendency of lipids to form HII phase [88]. The nonbilayerphases, such as the HII phase, have found particular interest in thestudy of membrane fusion [89,103].

2.5. Biophysical properties

At least three biophysical properties of PS are considered essentialfor normal respiratory physiology, especially in the neonatal period.These are: (1) rapid adsorption, (2) very low surface tension upon filmcompression, and (3) effective film replenishment upon film expan-sion [3,84,104,105].

2.5.1. Rapid adsorptionA functional PS should adsorb rapidly to form a film at the air–

water interface of the lungs, probably within a few seconds or faster.Analogous to bilayer fusion, the adsorption of surfactant aggregatesto an air–water interface likely consists of two sequential steps[106–109]. As shown in Fig. 3(a), the surfactant aggregates must firstdiffuse to the subphase closely adjacent to the interface. This diffusionstep is highly dependent on the surfactant concentration in thesubphase [110,111]. In the second step, as shown in Fig. 3(b) and (c),the surfactant aggregates need to fuse with the interface. This fusionstep involves “unzipping” and spreading of the surfactant PL vesiclesat the interface. Although PL vesicles are held together by weak vander Waals forces, the major cohesive force arises from the so-calledhydrophobic effect, because considerable energy is conserved throughthe formation of vesicles. Therefore, unzipping of PL vesicles needs toovercome an energy barrier, which arises during unravelling of the PLvesicles in water, before the hydrophobic fatty acid chains becomeexposed to the atmosphere [106,107]. Not surprisingly, adding fluid PLto DPPC decreases the energy barrier as they increase overall disorder.Anionic PL such as fluid PG has been shown to be particularly effective.However, recent studies indicate that with either native or modifiedPS, or the lipids derived from surfactant, the factors mentioned aboveplay only a small role. Rather, it appears that the formation of a fusionneck or pore, perhaps similar to those observed with viral membranecell fusion or bilayer fusion, contributes even more to the adsorptionprocess [101,102]. For example, addition of lyso-PC, which shouldincrease disorder but would hinder the formation of negatively curvedstructures with concave hydrophilic faces as present in fusion necks,markedly prolongs adsorption times [112].

Addition of SP-B and SP-C counteracts the energy barrier limitingadsorption, especially in the presence of anionic PL (such as PG),possibly by introducing electrostatic interactions [107,109,113]. SP-Band SP-C significantly promote adsorption [107,109,113] and reinser-tion of PL vesicles from the surfactant multilayers into the interfacialmonolayer during film expansion [114]. Perhaps the most effectiveway for SP-B or SP-C to promote adsorptionwould be to stabilize neckstructures (as shown in Fig. 3(b) and (c)) [101,115]. However, to date,direct evidence, for example by microscopy or by demonstrating thepresence of negatively curved structures such as the HII phase, is stilllacking [112].

During adsorption, films of a physiologically competent surfactantcan decrease the surface tension of the air–water interface from∼70 mN/m to ∼20–25 mN/m at 37 °C, but no further [84]. At this finalsurface tension range, the PLmolecules at the air–water interface are inthermodynamic equilibriumwith themolecules in the bulk phase [84].This equilibrium surface tension (γeq) corresponds to the equilibrium

spreading pressure (πe) of PL, which is the highest surface pressureobtained by spreading excess saturated or unsaturated PL at the air–water interfacewith organic solvent or by placing dry crystals of the PLat the interface [84]. Increases in surface pressure beyond πe can onlybe achieved at a fixed temperature by lateral film compression.

It should be noted that surface pressure (π) and surface tension (γ)are linearly correlated by

p = g0−g ð1Þ

where, γ0 is the surface tension of a clean air–water interface at theexperimental temperature, approximately equal to 70 mN/m at 37 °C.Thus, π corresponds to the extent that a film decreases γ. Surfacetension, γ, with quantitatively equivalent units of mN/m or mJ/m2, iscommonly used in lung physiology and biology as a measure of theenergy used to create a unit area of a new interface in air. The γ of aliquid can be directly measured by monitoring the shape of a sessile orpendant drop of the liquid (detailed in Section 4). Alternatively, γ canbe measured from the additional force exerted on a suspended plateor other object due to capillary rise, upon contact with the liquidsurface as, for example, with the Wilhelmy plate or the Du Noüy ringmethod [116].

Surface pressure, π, is the concept usually used in the physicalchemistry of monolayers where surface energy is interpreted in termsof intermolecular forces [117]. The original Langmuir balancemeasured π directly using a barrier connected to a pressure transducer[118]. π is a more fundamental concept under this circumstance as itoffers the direct 2D monolayer analog of the bulk phase (3D) pressure.As a result, many equations of state derived for the bulk phase can bedirectly applied tomonolayers without significant reformatting. In theremaining part of this review, both γ and πwill be used dependent onconvenience and clarity, with the corresponding value included,where deemed helpful to the reader.

2.5.2. Film stability at low surface tensionsAlthough limited, the available experimental evidence indicates

that γ of the alveolar surface falls to very low values during expiration[28,119–121]. Accordingly, upon compression, competent PS filmsshould lower γ to near-zero values [119], in other words, increase π toa value close to 70mN/m at 37 °C. Equally important, this low γ shouldbe achieved by only a slight film compression, i.e., no more than 20–30% area reduction, corresponding to the maximum variation of thealveolar surface area during normal tidal breathing [121,122]. Hence,the film should have a low compressibility (dlnA /dγ) of less than 0.01(mN/m)−1. Upon expansion, the PS film should only moderatelyincrease γ and remain close to γeq [120]. The complete compression–expansion loop should have limited hysteresis, reflecting minimizedfilm collapse and efficient film replenishment.

These premier biophysical properties of PS, however, seeminglycreate a paradox. Rapid adsorption requires fluid PL molecules withhighly mobile amphiphilic structures [116]. However, physicochemicalstudies have established that monolayers formed by fluid amphi-philic molecules generally collapse rapidly when π is increased beyondπe, for example, in a Langmuir balance [123]. To sustain stable (ormetastable) π higher than πe, a monolayer must consist of highly rigid,insoluble surfactant molecules [117,124]. Experiments with Langmuirmonolayers demonstrate that only such stiff molecules allow theformation of highly ordered, tightly packed, solid-like films in a con-densed phase, thus being capable of increasing π higher than πe withoutmonolayer collapse. Fully hydrated DPPC bilayers have a Tm near 41 °C.Hence, DPPC is traditionally taken to be the only significant componentof PS capable of reaching high π at physiological temperatures. This ledto the classical model of surfactant function which suggested that thealveoli were stabilized by a monolayer highly enriched in DPPC [125–127]. Note that such a monolayer is in a metastable state, confined bythe πe (∼45 mN/m) and the collapse pressure (∼70 mN/m) of DPPC.


However, adsorption of vesicles of stiff molecules, such as hydratedDPPC bilayers below 41 °C, is very slow. (Actually, dry DPPC will notadsorb below Tm. This is because PL molecules do not hydrate wellunless the fatty acids of the PL are mobile and this occurs at Tm.) Nosingle component in endogenous PS is able to both adsorb rapidly andwithstand π higher than πe at 37 °C for a time period long enough toavoid alveolar collapse. This dilemma has been rationalized by the so-called “squeeze-out” hypothesis [125–127]. The squeeze-out modelpredicts a rapid cooperative adsorption of DPPC and other unsaturatedPL components to the air–water interface in which the unsatisfactoryadsorption of DPPC is compensated for by the presence of unsaturatedPL, neutral lipids, and, most importantly, the surfactant associatedproteins. SP-B and SP-C independently promote rapid adsorption of PL[75,81]. SP-A further enhances adsorption in the presence of SP-B andcalcium ions [128–131]. These proteins enhance adsorption muchmore than the effects of unsaturated PL. The squeeze-out hypothesisproposed that after adsorption, the fluid non-DPPC components,which are less effective in lowering γ, are selectively squeezed out ofthe film during film compression. This likely occurs with the help ofSP-B and SP-C [63,64,132–140]. This selective squeeze-out processwould result in a condensed, highly DPPC-enriched film that would beresponsible for the further reduction of γ to near-zero values uponfurther film compression [125–127].

Although theoretically plausible, both the classical model and thesqueeze-out hypothesis have been challenged by recent studies onmonolayer phase transition/separation using direct film imaging[141–149]. As shown in Fig. 4, when examined in a Langmuir balanceat room temperature, pure DPPC monolayers show a plateau of LE-TCphase coexistence at π of 8–13 mN/m [86]. The TC phase consists ofdomains which exclude fluorescent dyes (detected by epifluorescencemicroscopy) [86,150] and extend ∼1 nm higher than the LE phase

Fig. 4. Compression isotherms and characteristic film structures for monolayers of bovine lipAFM at room temperature. The AFM scan areas for BLES films are 20×20 μm and for DPPC filand for DPPC (10×10 μm) show the nanometer-sized TC domains. The AFM images of BLES cle20 to 40mN/m. Nanodomains are observed at 30mN/m and increase in number up to 40mN/to 40 mN/m. At 50 mN/m, multilayers that contain stacks of PL bilayers appear (see the surfareflected by the plateau at 40–50 mN/m in the compression isotherm of BLES. In contrast to B(The kink shown in DPPC isotherm at ∼50 mN/m is likely an artefact due to film leakage to tnanodomains. After this transition region, indicated by the rising plateau in the DPPC isotheadapted from Zuo et al. [149]. AFM images of DPPC are courtesy of Dr. Eleonora Keating, Un

(detected by AFM) [141,151]. At π beyond the plateau region, pureDPPC monolayers consist of a nearly homogeneous TC phase [86,141].In contrast, as shown in Fig. 4, LE-TC coexistence in PS monolayersclearly persists to a π of at least 40 mN/m [141–143,149]. The resultsfrom recent fluorescence microscopy studies have demonstrated thatLE-TC phase coexistence in the PL fraction of PS persists even at πapproaching 70 mN/m (i.e., at near-zero γ) [146]. The TC domainscontain mostly DPPC and small amounts of other disaturated PL suchas DPPG (16:0/16:0 PG) [152] and perhaps PMPC [153], while the LEdomains contain mostly unsaturated components. As revealed byfluorescence microscopy, the TC domains account for a total areafraction of 30–40% of the PS films, roughly corresponding to thepercentage of disaturated PL [146,147]. These findings clearlydemonstrate that a TC film composed of almost pure DPPC is notrequired for reaching high π. This obviously contradicts the classicalmodel, which contends that a monolayer highly enriched in DPPCstabilizes the alveoli at end-expiration [125–127].

The question as to how surfactant films with mixed LE and TCdomains attain π near 70mN/m remains. The TC domains are enrichedin DPPC and hence are intrinsically stable at high π. However, how theLE domains, which account for most of the surface area, persist withhigh π remains unknown. The most obvious explanation is that withappropriate Langmuir balances and experimental conditions, the PLfraction of PS can be compressed into a metastable film that acts like a“solid”, despite being predominantly in a LE phase [146].

A potential explanation for this seemingly paradoxical situation isthat these LE domains may form a sort of matrix in which the TCdomains are uniformly distributed [149,154,155]. This can beenvisioned as a kind of composite material or be analogous to analloy [75]. This suggestion is supported by recent studies using AFM.With a higher resolution than fluorescence microscopy, AFM has

id extract surfactant (BLES) and DPPC, studied by the Langmuir–Wilhelmy balance andms are 50×50 μm. The enlargements of the AFM images for BLES at 30 mN/m (1×1 μm)arly show LE-TC phase separations and transitions as the surface pressure increases fromm; in contrast, microdomains increase in area from 20 to 30mN/m but decrease from 30ce plot of the AFM image). The transition from a monolayer to multilayers is apparentlyLES, DPPC monolayers only show LE-TC phase coexistence and transition at 8–13 mN/m.he Teflon ribbon.) The TC phase in DPPC monolayers also consists of microdomains andrm, DPPC monolayers consist of nearly homogenous TC phase. AFM images of BLES areiversity of Western Ontario.


revealed numerous nanometer-sized TC domains embedded withinthe LE phase, in DPPC and DPPC/DPPG monolayers [155]. The numberand size of these nanodomains depend on the presence andconcentration of surfactant proteins [155]. Recently, such nanodo-mains have been demonstrated in a modified natural surfactant(bovine lipid extract surfactant, BLES) with and without SP-A [149]. Itwas found that when π of BLES films was increased, the originalmicrometer-sized TC domains (i.e., microdomains) dissociated intomany nanodomains (Fig. 4). As a result, at 40 mN/m the BLESmonolayers contained only a few microdomains but many nanodo-mains uniformly embedded within the LE phase. The total area ofthesemicroscale and nanoscale TC domains accounted for ∼40% of thesurface area [149], approximately equal to the total fraction ofdisaturated PL in BLES [153]. Topographic analysis also suggestedthat these nanodomains, similar to their microscale counterparts, arecomposed of disaturated PL [149,155]. Due to the composite structure,such surfactant monolayers could be both flexible and stable.

A second mechanism for attaining high π (low γ) arises from the3D multilayer structures formed when the composite monolayers arefurther compressed beyond a π close to the πe of PL (i.e., 40–45mN/m)[149]. Such a 3D architecture consists of the interfacial monolayer plusinterconnected bilayers closely associated with the interface (see thesurface plot at 50 mN/m in Fig. 4) [63,149,156,157]. The monolayer-to-multilayer transition is reflected in the compression isotherm of PLfilms by a plateau at 40–50 mN/m, as shown in Fig. 4. This 3Darchitecture could provide additional stability by acting as a skeletonor scaffold to resist further compression and hence allow the films toattain π above πe [149,154]. This would be particularly true if, as hasbeen proposed, the excluded multilayers are bridged to the interfacialmonolayer through SP-B and SP-C (see Fig. 3(b) and (c) for schematics)[136,157–159].

It should be noted that the compression-driven monolayer-to-multilayer transition generally occurs at a π close to the πe of PL atwhich multilayers are formed during adsorption, i.e., the surfactantreservoir (detailed later). This coincidencemay indicate that these twomultilayer structures are formed due to the same molecular basis, i.e.,accumulation of excess material at the air–water interface beyondsaturation. In other words, if there is insufficient free surface area toaccommodate the PL in the interfacial monolayer, the excess materialbecomes surface-associated multilayers [62,84]. Actually, theoreticalstudies have suggested that there may be a slight deviation betweenthe πe at which 3D nuclei coexist with a 2D monolayer in an adsorbedfilm and the π at which 3D nucleation initiates in a Langmuirmonolayer during compression [160]. However, such a deviation maynot be distinguishable for a complicated biological system like PS.Recent molecular dynamics simulations suggested that the compres-sion-driven multilayer structures could protrude into the aqueoussubphase, as folding bilayers, instead of extending into the air [161].This further indicated that the multilayer structures formed duringcompression appear to be similar, if not identical, to the surface-associated surfactant reservoir formed during adsorption, both func-tionally and structurally.

Here, it is important to distinguish the monolayer-to-multilayertransition occurring at πe from the two other phenomena/hypotheses.First, it is different from the “true” collapse of PS films near π of 70 mN/m. During the film collapse at 70 mN/m, the PL molecules are primarilyirreversibly lost into the bulk phase in the form of small aggregates/vesicles [162]. Hence further film compression causes no increase in πabove the collapse pressure. In contrast, the monolayer-to-multilayertransition appears to represent a reversible, partial collapse of theinterfacial monolayer occurring near πe. The resultant multilayerstructures apparently remain closely attached to the interfacial mono-layer as they can readily re-spread to the interfacial monolayer duringfilm expansion [63,149]. Second, themonolayer-to-multilayer transitionis intrinsically different fromthe “squeeze-out”predictedby the classicalmodel [125–127]. The 40–45 mN/m squeeze-out model mentioned

above predicts that PS films maintain stability by selectively excludingfluid non-DPPC components from the interfacial monolayers. Conse-quently, the interfacial monolayer after formation of the multilayeredprotrusions would be expected to be purified with DPPC. However,recent AFM and Kelvin probe force microscopy studies have found thatthe topography and electrical surface potential of the interfacialmonolayer appear to be heterogeneous even after the multilayerformation [157,163]. Hence, the multilayers would appear to show noabsolute differences from the interfacial monolayer in terms ofcomposition. A similar conclusion has been derived from time-of-flightsecondary ion mass spectrometry (ToF-SIMS) studies of compressedDPPC/DPPG/SP-C films [156]. Autoradiographic studies also indicatedthat the lipid composition of multilayers formed during adsorption issimilar to the interfacial monolayer [62].

In addition to the above indicated PL phase transition andmultilayer model based on microscopic studies, another hypothesishas been proposed by Hall and coworkers to explain how PSmonolayers could reach low surface tensions [147,148,164]. Thisgroup found that monolayers of the PL of PS or a single-componentfluid PL can be transformed to a metastable structure when themonolayers are compressed to π higher than πe (actually higher than∼55 mN/m) using a sufficiently rapid compression, the so-called“supercompression”, in a captive bubble surfactometer (CBS)[164,165]. Once transformed by a supercompression, the physico-chemical properties of the initial fluid films are markedly altered.These films (for example a POPC film that usually collapses at πe whencompressed slowly in a Langmuir balance) can withstand high π forprolonged periods, even if the films are expanded back to π below πe[164,165]. These workers proposed that these films are compressed soquickly that they do not have enough time to collapse near πe, butrather form an amorphous, non-crystalline structure (termed a “jam”

phase). This can be considered similar to 3D liquids supercooledtowards a glass transition [147,148]. A recent study of the meltingbehavior of supercompressed PS, DPPC and POPC monolayers clearlysuggested that a supercompressed PS film is different from either apure DPPC or a pure POPC monolayer [97,98]. So far, no detailedmicroscopic examination of the film structure after supercompressionis available, probably due to technical difficulties.

It should be noted that the PL phase transition model and thesupercompressionmodel are not necessarily conflicting. A recent AFMstudy of BLES monolayers, conducted in a Langmuir balance at roomtemperature, showed that after a rapid compression (close to the rateof supercompression for extracted surfactants, defined by Hall et al.,i.e., ∼4% area per second [147]) the monolayers consisted of mainlynanometer-sized TC domains [149]. Although only a low π of 30mN/mwas examined [149], this observation suggested an alternativeinterpretation of the supercompression model: the rapid compres-sions likely facilitate formation of TC nanodomains rather thanmicrodomains because the latter structures require longer times forassembling. Monolayers containing evenly distributed TC nanodo-mains could be stable despite containing a large proportion of LE phasesince they could act as an alloy or a composite material, as explainedabove. Upon compression, suchmonolayerswould be expected to havelittle chance of fracture. Further, should fractures occur at extremecompression, they would not be readily propagated because theywould have to proceed around the intervening TC nanodomains.

2.5.3. Surface-associated surfactant reservoirIn order to function appropriately in the lung, a competent PS must

be able to maintain a near equilibrium γ during inspiration [28,120].Dynamic compression–expansion cycling studies conducted with thepulsating bubble surfactometer (PBS) or the CBS indicate that PL uptakeinto the interface occurs too quickly to be explained by de novoadsorption of PL vesicles from the subphase. Hence this can be bestexplained by re-spreading from a surface-associated surfactant reser-voir, formed during adsorption and/or film compression, and


functionally associated with the interfacial monolayer. The initialevidence for the surfactant reservoir came from CBS studies where itwas shown that although bulk phase PS was removed by washing, theremaining surfacefilmcontainedhighly surface activematerial in excessof the interfacial monolayer [60]. This concept has been reinforced bynumerous studies using electron microscopy, autoradiography, fluores-cence microscopy, and AFM, all of which revealed excessive PL materialassociated with the surface monolayer [61–65,136,158,166,167].

Although notable exceptions exist, as a general rule, PS preparationsthat adsorb well also tend to function well to reduce γ to low valuesduring film compression and to re-spread effectively during filmexpansion. This correlation likely exists because the surfactant reservoircan be created during adsorption and situations promoting rapidadsorption (e.g., high surfactant concentration, presence of SP-A, lack ofinhibitors) favor formation of the multilayered reservoir. Once estab-lished, the interfacial surfactant monolayer can exchange surface activematerial with the surfactant reservoir during film compression and ex-pansion. Although not fully understood, it is evident that SP-B and SP-Ccontribute to reservoir formation (as shown in Fig. 3(b) and (c)), mostlikely at independent sites, as does SP-A in the presence of SP-B andcalcium [60,62,131,168,169].

In closing Section 2, we would like to emphasize that functional PSfilms may reach low γ (high π) by forming kinetically-controllednanoscale TC domains. These nanodomains are uniformly distributedin the LE phase, thus forming a 2D alloy-type structure which impartsboth flexibility and stability to the monolayers. Upon further compres-sion, such an alloy structure also facilitates partial collapse of surfactantmonolayers from the domain boundaries [170,171]. The resultantmultilayer structures could provide additional stability to PSmonolayers,thereby allowing the attainment of very low γ. It should be evident thatthebiophysical properties of PS, althoughdependenton surfactant PL, aredirected by the surfactant apoproteins SP-B, SP-C, and to a lesser extentby SP-A. These surfactant proteins influence the extent of nanodomainformation. They also facilitate formation of multilayers which act as thesurfactant reservoir. The biophysical performance of PS monolayers issignificantly enhanced by the surfactant reservoir. A complete under-standing of the biophysical properties of PS on a mechanistic level,however, is still unavailable. Both the classical and the modern models

Table 2Surfactant preparations used clinically and preclinically

Trade name Source Manufacturer

Human surfactant containing all surfactant proteins– Human amniotic fluid –

Modified natural surfactant containing hydrophobic proteins SP-B and SP-CAlveofact Bovine lung lavage Boehringer Ingelhei

Ingelheim, GermanyBLES Bovine lung lavage BLES Biochemicals,

Ontario, CanadaInfasurf Calf lung lavage Forest PharmaceuticSurfacten Bovine lung mince+DPPC+palmitic acid+tripalmitin Tokyo Tanabe Co., ToSurvanta Bovine lung mince+DPPC+palmitic acid+tripalmitin Abbott LaboratoriesCurosurf Porcine lung mince Chiesi Farmaceutici,

Synthetic surfactant containing simplified peptides or protein analogsSurfaxin SP-B-like peptide (KL4)+ lipids Discovery Laborator

Warrington, PA

Venticute Recombinant SP-C (rSP-C)+lipids Altana PharmaceutiKonstanz, Germany

Synthetic surfactant free of proteinsALEC DPPC+PG Britannia Pharmace

Surrey, UKExosurf DPPC+hexadecanol+tyloxapol Burroughs Wellcom

Triangle Park, NC

ALEC: artificial lung-expanding compound; BLES: bovine lipid extract surfactant.

have limitations [147]. Recent reviews on themolecular interactions of PScomponents and their contributions to the biophysical properties of PSfilms can be found elsewhere [105,147,154,172].

3. Inhibition of clinical surfactants and reversal thereof

3.1. Different types of clinical surfactants

A number of exogenous surfactants have been tested in clinical orpreclinical trials for treating RDS and ARDS. As summarized in Table 2,these therapeutic preparations generally fall into four categoriesbased on surfactant apoprotein content [11,14,36,173]. They are: (1)whole surfactant from human amniotic fluid, containing both thehydrophobic and hydrophilic proteins; (2) modified natural surfac-tants derived from either bovine or porcine sources, which containonly the hydrophobic surfactant proteins SP-B and SP-C (e.g., BLES,Curosurf, Infasurf and Survanta); (3) synthetic surfactants that containsimplified peptides or recombinant surfactant protein analogs (e.g.,Surfaxin and Venticute); and (4) protein-free synthetic surfactantsthat consist of only PL components (mainly DPPC) and additives (e.g.,ALEC and Exosurf). Among these surfactant preparations, humanamniotic fluid lacks commercial capacity due to its source limitation.The synthetic surfactants devoid of proteins have become unpopulardue to their relatively poor clinical performance [174]. ALEC, whichwas licensed in the UK, has been withdrawn from the market. Exosurfis no longer available in the USA.

Both preclinical animal experiments and clinical practice suggestthat the animal-derived surfactant preparations are superior to thesynthetic preparations [174–176]. However, these modified naturalsurfactants also have some limitations. The general concerns ofanimal-derived products are batch-to-batch variation in compositionand potential risk of transmission of microbes. In addition, thesurfactant protein contents in the modified natural surfactantscurrently available for clinical use can be very low compared withthe endogenous surfactant. Because of immunological considerations,none of the modified natural surfactants currently available containsthe hydrophilic proteins (SP-A and SP-D), which are removed duringthe purification processes used in manufacturing the products. This is

Advantages Disadvantages

• High resistance to inhibition • Not readily available

m Co., • Good biophysical properties • Transmission risk

London, • High resistance to inhibition • Batch-to-batch variation

als, St. Louis, MOkyo, Japan, Abbott Park, ILParma, Italy

ies, • Completely defined formulation • Under development• Good biophysical properties • Current preparations are

less effective than modifiednatural surfactants

cals, • High resistance to inhibition

uticals, Redhill, • No risk of disease transmission • Poor biophysical properties

e, Research • Less immunological rejection • Low resistance to inhibition


because, unlike the low molecular weight hydrophobic proteins, SP-Aand SP-D are multimeric glycoproteins and consequently imposepotential immunologic hazards. Economically synthesizing or reco-vering human SP-A from natural sources is difficult. Thus, the hostprotective benefits of these proteins are lost. The content of thehydrophobic proteins (SP-B and SP-C) in different surfactant prepara-tions varies but can be significantly lower than the endogenoussurfactant. For instance, Survanta contains only ∼1/8 SP-B and ∼1/2SP-C found in the endogenous bovine surfactant [177]. Curosurfcontains only ∼1/3 SP-B and ∼1/2 SP-C found in the endogenousporcine surfactant [177]. The in vitro surface activity of differentanimal-derived surfactants and their sensitivity to inhibition varysignificantly [177,178], presumably related to the different proteincontents of these products.

As a result of different animal sources (bovine or porcine) anddifferent production procedures (bronchoalveolar lavage or lungmincing), the modified natural surfactants differ not only in theirprotein contents but also in the compositions of PL (especially DPPC,PG and PI), neutral lipids (mainly cholesterol), and additives (such asthe palmitic acid and triacylglycerol supplemented in Survanta). Adetailed comparison of lipid and protein compositions of the modifiednatural preparations can be found in a recent review by Blanco andPerez-Gil [179]. The different PL contents can result in a significantdifference in the fluidity of different preparations and hence differentbehavior of adsorption. The neutral lipids and additives likely play animportant role in altering the surface rheological properties ofdifferent clinical preparations [180,181]. It should be noted thatdespite the evident biophysical differential and clinical superiority ofsome animal-derived surfactants over others, statistical differences inmortality or days in neonatal intensive care units related to differentsurfactant preparations have not been demonstrated [174–176].

Another limitation of animal-derived surfactants is the relativelyhigh cost, at approximately US$500 per dose for premature infants[182]. These costs are mainly associated with the high expense ofconducting quality control with relatively small batches of biologi-cally-derived products and recovery costs for the very expensiveclinical trials that must be conducted. When treating ARDS patients,large surfactant amounts, multiple doses and a continuous supply willbe required. This will further increase the cost of surfactant therapy.Socioeconomic analysis shows that surfactant replacement therapy isonly cost-effective in developed countries [182]. In the developingcountries, surfactant therapy is used only sparingly due to the lack ofan affordable clinical surfactant and the costs associated withintensive care facilities. For instance, RDS affects 7–12% of newbornsin India and is associated with a high mortality rate due to the lack oftertiary care, including surfactant treatment [183]. In China, surfactanttherapy has been introduced into clinical practice but only limited tothe highly developed regions of the country [184]. The high cost ofsurfactant has prevented its worldwide distribution for neonates andconsiderably more so for adult therapy. Thus, there is an urgent needfor developing an inexpensive surfactant preparation.

A new generation of clinical surfactants is under development[185–189]. These preparations are fully synthetic and containsynthesized surfactant protein analogs. The long-term goal of thisresearch is to develop synthetic surfactant preparations that fully orclosely mimic the biophysical properties of the endogenous surfactantand highly resist inactivation. Mass production could result in a morereasonable price. Both preclinical and clinical trials have beenconducted using synthetic surfactants with the addition of simplifiedSP-B/C-like peptides.

The initial surfactant protein mimic studied was KL4, a shortamphipathic peptide consisting of leucine amino acids with a lysinemoiety introduced at every fifth residue. This structure is based on apositively charged helical segment of SP-B [190]. KL4 enhances theadsorption of DPPC/PG/palmitic acid mixtures and interestingly, lowlevels of SP-A further augment this adsorption [191]. Recent studies

have demonstrated that KL4 can promote formation of lipid protru-sions with DPPC/DPPG mixtures [192]. Although KL4 exhibits anumber of interesting SP-B-like properties, it is still considered afirst generation protein mimic. The manner in which this peptidereplicates SP-B properties is still being investigated [192–194].Nevertheless, KL4 is being used as the basis of a wholly syntheticsurfactant, Surfaxin, which has been reported to be efficacious withpremature infants and in preliminary studies with ARDS [195–197].

Another wholly synthetic surfactant is Venticute, a preparationbased on recombinant human SP-C (rSP-C), which has been utilized ina number of clinical trials. Venticute is presently being investigated ina trial involving direct lung insult-induced ARDS [198–201].

While the advantages of synthetic surfactant peptides are obvious,synthesizing fully functional analogs of hydrophobic surfactantproteins is unfortunately not a trivial task. The SP-B molecule is toolarge and structurally complex to be easily synthesized. Folding thishydrophobic protein to generate the three intra- and one inter-disulphide bonds has proven particularly arduous. The palmitoylatedcysteine residues in SP-C have been replaced by phenylalanines.However, SP-C molecules are structurally unstable in pure form andtend to aggregate. A major goal of such research is to generate“designer” surfactants with properties particularly well suited forARDS and other specific lung diseases. So far, none of the syntheticpreparations available have proven more efficacious than the naturalpreparations [176,189]. In addition, the presence of potentially danger-ous reaction side-products and the possibility of adverse metaboliceffects related to intracellular processing of synthetic compoundsmust be considered.

Nevertheless, it is important to note that significant progress isbeing made in this direction. Waring and associates have generateda number of simplified SP-B analogs such as mini-SP-B (containsN-terminal and C-terminal sections of SP-B) which mimic thebiophysical and physiological properties of the whole hydrophobicprotein [202,203]. Barron and coworkers have produced a number ofpeptoids with SP-B and SP-C-like properties [204,205]. Such com-pounds would have the advantage of being protease resistant. Anumber of recent reviews offer in-depth discussion of these novelapproaches [185–189,206,207].

3.2. Surfactant inhibition

Surfactant inhibition, or inactivation, refers to those processes thatdecrease or abolish the normal surface activity of PS. Such processesmay interfere with the PL adsorption to form a functional surfactantfilm, prevent the film from reaching low γ upon compression, or affectre-spreading of PL during expansion. A number of substances havebeen reported to inhibit PS. The major inhibitory factors includeplasma proteins, unsaturated membrane PL, lysophospholipids, freefatty acids, meconium (fetal feces expelled during stress), andsupraphysiological levels of cholesterol [11,14]. Surfactant inhibitioncan also arise from degradation of surfactant lipids by phospholipasesor of surfactant proteins by proteases. These degradative agents,normally present in the alveolus at very low levels, can be increasedduringmicrobial infection andmore importantly through secretion byleukocytes and type II cells with pulmonary inflammation [208–211].In addition to the above, surfactant can be compromised by reactiveoxygen species [212,213] and by pollutants [214,215] but these lattereffects will not be covered here.

3.2.1. Surfactant inhibition by plasma proteinsLeakage of plasma proteins into the alveolar space due to an

impaired alveolar-capillary barrier is an early event in the pathogen-esis of ARDS [208]. The mechanism by which PS is inactivated byplasma proteins is not yet fully understood. Evidence is availabledemonstrating that albumin, fibrinogen, and hemoglobin can inhibitPS by competitive adsorption [216–221]. These protein inhibitors are


surface active. When mixed with PS they compete with PL for the air–water interface by spontaneous adsorption. Although larger thanindividual PL molecules, these protein molecules are water solubleand hence can quickly reach the interface by molecular diffusion. Incontrast, the insoluble PL of PS adsorb to the air–water interface bycooperative diffusion of large molecular aggregates (essentiallyvesicles), followed by vesicle “unzipping” and monolayer spreading(see Section 2 for detail). Consequently, the protein molecules canadsorb to the air–water interface quicker than the surfactant PL. Onceadsorbed, the protein film excludes the PL from entering the interfaceby creating a steric and/or electrostatic energy barrier [222,223]. Inkeeping with this view, surfactant injected under preformed proteinfilms adsorbs slowly and takes longer to reach low γ duringcompression–expansion cycling [218]. Surfactant inhibition due tocompetitive adsorption can be largely overcome in vitro by increasingthe surfactant concentration [220] or adding SP-A [130,224] becausethese approaches enhance adsorption kinetics of PL.

Not surprisingly, surfactant inhibition depends on the plasmaprotein/surfactant ratio [220]. The alveolar hypophase normally con-tains only small amounts of soluble proteins [225–227]. For instance,Rennard et al. [228] and Ishizaka et al. [229] have estimated the albuminconcentration in the epithelial lining layer to be 3.7 to 4.9 mg/mL,corresponding to∼10%of theplasmavalues of albumin (i.e.,∼40mg/mL).The concentration of PS in the alveolar hypophase has been estimatedto be 30 mg/mL in rats and 100 mg/mL in rabbits [230], calculated onthe basis of surfactant PL recovery by lavage [231] and an averagealveolar lining layer thickness of 0.2 μm [49,232]. Since inhibitionnormally requires a much larger amount of protein than surfactant[220], it appears that normal lungsmaintain a considerable safety factor.

However, in ALI/ARDS, the alveolar space experiences markedlyincreased concentrations of plasma proteins, due to capillary leakage,with average levels of 25mg/mL and individual values of over 100mg/mL being reported [229]. (The high values, above plasma levels,probably arise during epithelial recovery when ions and water areeliminated more rapidly than proteins [225]). In contrast, the concen-trations of surfactant PL and/or surfactant associated proteins decreasedue to inflammation, oxidation and simple dilution [208,233]. Conse-quently, competitive adsorption may play a role in inactivating surfac-tant under these circumstances.

Although considerable effort has been directed toward studying theeffects of plasma proteins on surfactant adsorption, it should bestressed that these proteins can inhibit surfactant function in otherways. Warriner et al. demonstrated that albumin interfered with re-spreading of a model surfactant PL mixture, DPPC/POPG/palmitic acid[221]. This inhibition occurswhenγ of 50mN/mor higher are attained,where 50 mN/m corresponds to the γeq of albumin. This implies thatonly when γ rises to the equilibrium for albumin can this proteinadsorb and interfere with PL re-adsorption/re-spreading. This is con-sistent with the inability of albumin to inhibit high levels of surfactant[220] and with the ability of DPPC, spread onto albumin films to a lowγ, to displace this protein from the interface [234,235]. Nevertheless, itshould be noted that themodel PL systemdescribed above [221] did notcontain SP-B or SP-C, either of which tends to maintain the surfactantfilms near a γ of 25 mN/m, i.e., the γeq of PL. Unless inactivated, thehydrophobic surfactant proteins would protect the film againstattaining the high γ required for this inhibition mechanism [236,237].

In addition to the above example, recent work has provided furtherevidence that albumin may inhibit surfactant by mechanisms otherthanonly interferingwith surfactant PL adsorption. BothNMR [238] andX-ray diffraction [239] studies suggested that albumin directly inter-acted with PL bilayers of BLES. Such interactions thinned the PL bilayersand altered the distribution and motion of PL in bilayers/monolayers.AFM studies demonstrated that serum altered the microstructures ofBLES monolayers [240,241]. As recently revealed by AFM and confocalfluorescence microscopy, albumin can remain within the LE phase ofthe PL of BLES up to a π of 40 mN/m, significantly higher than the πe of

albumin (i.e., ∼20mN/m) [241]. By remaining at the surface andmixingwith the surfactant films, albumin increases the film compressibilityand may also disturb the normal PL phase transition and separation[241]. Such inhibitoryeffects cannot be effectivelymitigated by repeatedcompression–expansion cycles in a Langmuir balance [241]. Thesestudies suggest new biophysical mechanisms for surfactant inhibitiondue to plasma proteins, and possibly also indicate that surfactant–protein interaction/binding may play a role in the inhibition process.

Investigations of surfactant inhibition have emphasized serumalbumin because at 35–50 mg/mL, it is the predominant plasmaprotein, accounting for about half of the plasma proteins [242]. Theavailability of cheap, highly purified albumin is a likely consideration.However, it must be mentioned that albumin is a very weak inhibitorcompared to a number of other plasma proteins. For example, earlystudies by Seeger's group demonstrated that fibrin generated fromfibrinogen by a variety of methods was considerably more potent thanalbumin in inhibiting a variety of surfactant preparations [230]. Thismechanism appears to involve coagulation of surfactant PL andprotein molecules, resulting in the hyaline membranes for which RDSwas originally named [23].

Surfactant function is alsomarkedly hampered through interactionwith C-reactive protein (CRP) [243,244]. CRPwas originally discoveredthrough its ability to bind the polar phosphorylcholine groups of PC.This binding and the corresponding inhibition can be blocked bywater-soluble phosphorylcholine. Simultaneous increases in CRP anddecreases in SP-A in the alveolar space are conditions associated withALI [245]. In contrast to albumin, CRP markedly inhibits surfactantfunction at 50 wt/wt.% of the surfactant and this inhibitory effectcannot be relieved by repeated compression–expansion cycling.Recent studies suggested that CRP inhibits PS by fluidizing the surfac-tant PL films, i.e., a completely different mechanism from the com-petitive adsorption of albumin [246,247]. As will be discussed shortly,this mechanism is commonly associated with surfactant inhibition byunsaturated lipids. As in the case of albumin and fibrinogen, addingSP-A at low levels relative to surfactant blocks the inhibitory effects ata fraction of the inhibitory plasma proteins' concentration [246,247].The mechanism by which SP-A reverses CRP-inhibition appears to bealso different from the case of albumin. It was suggested that directinteraction between SP-A and CRP was responsible for preventinginhibition due to this serum protein [246,247]. As discussed above,however, SP-A overcomes albumin-induced inhibition mainly byenhancing surfactant adsorption.

3.2.2. Surfactant inhibition by lipidsAlthough not nearly as well studied as protein inhibition, a second

inhibition mechanism involving unsaturated membrane PL, lysopho-spholipids, free unsaturated fatty acids (such as oleic acid), bile acids,diacylglycerol and cholesterol has been identified [248–251]. Some ofthese lipids have detergent properties and can be referred to assoluble amphipathic lipids because they form micelles [252]. None ofthese lipids are bilayer formers, although they can be incorporatedinto bilayers to some degree. Lysophospholipids are PL containing asingle fatty acid chain per molecule and are generated by phospho-lipase A2 secreted by white blood cells and likely type II cells,particularly during ALI and ARDS [253]. Bile acids, which are strongdetergents, are present in meconium, along with a number of otherlipids, mucous glycoproteins [254], and phospholipases [255]. Suchlipid substances can be considered diluents of the specific surfactantlipid assembly. Thus, insertion and mixing of these unsaturatedamphipathic lipid and fatty acid molecules with the surfactant PLmolecules would significantly fluidize the PL monolayers and couldpromote early collapse, thus preventing low γ from being reached.The inactivation due to lipid penetration cannot be effectively over-come by raising surfactant concentration [220,248,249].

Cholesterol is a special case of lipid inhibition since it is not veryamphipathic. Cholesterol appears to be an inherent component of


natural surfactant [74,256,257]. Most isolated mammalian surfactantswhich have been examined contain 5–10 wt.% (10–20 mol%)cholesterol [74,256–258]. Cholesterol is also present in premamma-lian vertebrates where it can reach very high levels [256]. Althoughcholesterol has been associated with surfactants for a very long time,phylogenetically, and its presence in mammalian surfactants has longbeen recognized, the precise physiological role of this steroid insurfactant remains somewhat ambiguous.

Early studies on model membranes and PS extracts revealed thatthe presence of cholesterol tended to decrease film stability at low γ[73,128], presumably due to the increased fluidity of the LO phase (seeSection 2 for the description of PL-cholesterol phase behavior). For thisreason, methods were developed for removing cholesterol from BLESand Curosurf [73,84]. These observations were apparently related tothe use of the Langmuir balance and the PBS, which will be describedin Section 4. More recently, studies with the CBS have demonstratedthat the levels of cholesterol in natural surfactant and its lipid extractsdo not inhibit surfactant biophysical activity when assayed with thisdevice [251,259,260]. This difference is attributed to the lack of edgeeffects (i.e., barriers, walls, and capillary) which make the CBS lesssusceptible to the loss of PL from the air–water interface (i.e., so-called“film leakage”, detailed later in Section 4).

While cholesterol at 5–10 wt.% had no negative effects on surfactantfunction with the CBS, a large number of studies have demonstratedsupraphysiological levels are detrimental [163,251,258,260,261]. Eleva-tions in surfactant cholesterol have been reported in a number of animalmodels of ARDS such ashigh-stretch ventilation-inducedALI [262], oleicacid-induced ALI [263] and oxidant-induced ARDS [264]. Elevatedcholesterol has also been detected with ARDS patients due to fatembolism syndrome [265]. A recent randomized, controlled clinical trialfound a remarkably elevated level of neutral lipids, including diacylgly-cerol, triacylglycerol, and cholesterol, in the bronchoalveolar lavagefluids of patients with ARDS arising from various predisposing factors[201].

Although the mechanism of cholesterol-induced inhibition has notbeen fully elucidated, several groups, including our own, have demon-strated alterations of surface-derived films using AFM [163,260,261].In particular, the ability to generate DPPC-rich condensed domainswithin the LO/LD phase and to generate PL multilayers (surfactantreservoirs) appears impaired with the addition of high levels ofcholesterol. Whether these two surface aspects of surfactant functionare related has not been clearly established.

The detrimental effects of high cholesterol on surfactant func-tion raise the question as to whether it should be included in thesynthetic and semi-synthetic clinical preparations currently beingformulated, or removed from those modified natural surfactantswhich contain this steroid. Bernardino de la Serna et al. [266] sug-gested that small amounts of cholesterol may play a crucial role inphase separation in porcine surfactant bilayers by inducing theformation of Lo and Ld phases. Malcharek et al. [261] found phy-siological levels of cholesterol (10 mol%) stabilize DPPC/DPPG/SP-Cfilms by strengthening the surface-associated multilayer structures.Nevertheless, to date, the authors know of no negative effects ofomitting cholesterol from clinical surfactants, although admittedlythe verdict is clearly still out. Since high levels of cholesterol areclearly deleterious, it would appear judicious to avoid includingcholesterol in clinical preparations. This would build in a potentiallyimportant safety factor by providing a sink for the elevated endoge-nous sterol.

3.3. Overcoming surfactant inhibition

Overcoming inhibition will play a key role in developing newsurfactant formulations for ARDS treatment. Two general approachesto be discussed here are (1) optimizing lipid and protein contents, and(2) using water-soluble polymers as surfactant additives.

3.3.1. Optimizing lipid and protein contentsSimply increasing the PL concentration of surfactant preparations

can effectively reverse plasma protein-induced inactivation [220]. Itwas reported that increasing surfactant concentration also mitigatedmeconium-induced inactivation [267]. As a new direction in optimiz-ing the lipid components of surfactant, Notter and coworkers havedeveloped a novel surfactant preparationwhich consists of a syntheticC16:0 diether phosphonolipid (DEPN-8) in combination with 1.5 wt.%purified bovine SP-B/C [268] or mini-SP-B [269]. Compared to theglycerophospholipids in other surfactant preparations, DEPN-8 isstructurally resistant to phospholipase degradation [268]. Thissynthetic surfactant was found to be highly resistant to inhibitiondue to serum proteins, phospholipase A2, and lyso-PC, both in vitro[268] and in excised rat lungs [270]. A potential advantage of thispreparation is that, being resistant to phospholipases (A1, A2, and D),DEPN-8 could be cycled into lamellar bodies and secreted back into thealveolus without degradation. This would greatly prolong the effec-tiveness of this synthetic preparation. A potential problem might bethat this synthetic lipidmay not be routed in the samemanner as DPPC.However, the presence of significant quantities of 1-alkyl ether PCs inmarsupial surfactants, particularly the Tasmanian devil [70], indicatesthat ether lipids are compatiblewith normal processing in type II cells.

Optimizing the content of surfactant proteins is another effectivemeans to reverse inactivation. The superior performance of modifiednatural surfactants over protein-free synthetic surfactants clearlydemonstrates the importance of SP-B and SP-C in overcoming surfac-tant inactivation. Addition of peptide analogs of SP-B and/or SP-C alsoimproves the resistance to surfactant inactivation [202,203,271,272].Adding SP-A to lipid extract surfactants increased their resistance toinactivation due to blood proteins [130,224] and meconium [273]. Invitro experiments showed that small amounts of SP-A can signifi-cantly enhance adsorption and dynamic surface activity of lipidextract surfactants, thereby increasing their effectiveness at reducedPL concentrations [129,131,274]. In animal experiments, surfactantscontaining SP-A [275,276] showed higher resistance to inactivationthan the surfactants without SP-A.

3.3.2. Using polymeric additivesTaeusch et al. [277] and Kobayashi et al. [278] first introduced the

concept of using low-cost, water-soluble, nonionic polymers, such asdextran and polyethylene glycol (PEG), as additives to clinical PS. Thebenefits from these polymeric additives were found to be twofold[279–281]. First, they can improve the surface activity of dilutesurfactant preparations (mainly by enhancing adsorption) and henceare capable of decreasing the cost of surfactant therapy. Second, theycan effectively counteract surfactant inactivation due to a variety ofinhibitory substances (such as plasma proteins and meconium), thushaving a potential to enhance the clinical efficacy of surfactant therapyin treating ARDS.

The polymers tested so far cover a surprisingly broad range, includ-ing nonionic (e.g., PEG [277] and dextran [278]), anionic (e.g., hyal-uronan [282]) and cationic (e.g., chitosan [283]) polymers. Many invitro [222,277,278,282–290] and in vivo animal studies [278,291–297]have shown that these polymers can significantly improve the surfaceactivity of different clinical surfactants and reverse surfactantinactivation. The encouraging results from these preclinical studiesmake these polymers very promising for the development of low-costand inactivation-resistant surfactant formulations.

The increasing experimental success led to exploration of theunderlying mechanism responsible for the enhancement. These poly-mers are hydrophilic. Alone they have no or only poor surface activity[283,288].Whenmixedwith the inhibitory substances, such as albumin,they do not alter the interfacial properties of the inhibitors significantly[283]. Therefore, it is unlikely that these polymers enhance surfaceactivity by directly accumulating at the interface or reverse inactivationby directly interacting with the inhibitors. Yu et al. [288] found that


nonionic polymers, such as PEG, improved the adsorption of dilutesurfactant preparations as a function of both polymer concentration andmolecular weight. These experimental results fit very well with apolymer-induced depletion–attraction model.

Depletion–attraction is a non-specific, polymer-induced entropicforce dependent on both the concentration andmolecular weight of thepolymer [298,299]. Depletion–attraction forces are routinely used topromote fusion of cells in culture likely by the same mechanism thatimproves surfactant function. The simplified model described here willbe followed byamore rigorousmathematical formulation. Consider twosurfactant vesicles approachingeachother. At a certainpoint the smaller,non-deformable polymer molecules will no longer fit in the spaceavailable between the vesicles and so will be excluded (i.e., depleted)from this space. The resulting enrichment in areas other than the regionbetween the two vesicles produces an increase in osmotic pressure inthose regions. This increased osmotic pressure draws water from thepolymer-depleted region, thus forcing the two vesicles together (i.e.,attraction). The initial effect will be to promote membrane–membrane(bilayer) fusion or aggregation. The resulting larger vesicles/aggregateswill be even further subject to depletion–attraction forces because theyare more effective in eliminating the polymer between them. A similarprocess will occur at the air–water interface and the resultant depletionforces will drive the vesicle towards the interface, thus decreasing theenergy barrier for surfactant adsorption. It should be noted that somenonionic polymers such as PEG (10 kDa) may adsorb to the air–waterinterface due to a weak surface activity (γeq of ∼60 mN/m [288]). Thismay weaken the depletion forces near the surface region (personalcommunication with Dr. Tonya Kuhl, UC Davis).

In physicochemical terms, the depletion forces described above arisewhen two surfaces approach each other closely in a solution of non-adsorbing polymers. As shown in Fig. 5, when the distance between the

Fig. 5. Schematic representation of the polymer-induced depletion–attraction mechanismapplies to non-adsorbing polymers such as low and mediummolecular weight polyethylenea vesicle approaches the air–water interface, at a surface-to-surface distance nomore than twmolecules are depleted since otherwise the polymer coils lose configuration entropy. Condepletion zone. The osmotic effect will drawwater away from the depletion zone, thereby gen“pushes” the lipid aggregates to the air–water interface. As indicated by the interaction potedepletion can be as large as the van der Waals force in the absence of the polymer. The pro

two surfaces/vesicles decreases to approximately two times the radius ofgyration of thepolymer chains (2Rg), thepolymermolecules in the regionbetween these two surfaces are depleted since otherwise the polymercoils lose configuration entropy, i.e., they will be deformed or involved inother randomchanges inmorphology. Consequently, anosmoticpressureappears between the bulk solution that contains the polymer and thepolymer depletion zone of radius Rg between the two surfaces. Theosmotic effect generates a net force of attraction between these twosurfaces and hence causes flocculation. Kuhl et al. [300,301] haveprovided solid evidence that depletion–attraction is responsible for thefusion of pure PC vesicles in PEG solutions by direct force measurementsusing a surface force apparatus. They also found a change from attractionto repulsion with increasing molecular weight of PEG [300,302]. Thisturnabout was due to adsorption of PEG molecules onto the surfaces ofthe PC vesicles, which eliminates depletion–attraction but raises stericrepulsion between the vesicles [302]. A similar turnabout has beenreported when BLES was mixed with a high molecular weight PEG(300 kDa), where surfactant adsorptionwas completely abolished [288].

The depletion force can be quantitatively estimated from theclassical model first developed by Asakura and Oosawa [303]. Whenconsidering the depletion force between two flat surfaces, this modelcan be simplified as follows [304].

The depletion pressure (P) between two flat surfaces is:

P = −qkT ð2Þ

where ρ is the molecular density of the polymer, i.e., the number ofmolecules per unit volume (m−3); ρ=6.022 ×1029 CW/MW, where Cwand Mw are the polymer concentration (wt.%) and molecular weight,respectively; k is the Boltzmann constant, 1.38×10−23 (J/K); T is theabsolute temperature (K).

in promoting surfactant aggregation and adsorption. Note that this mechanism onlyglycol (PEG). The depletion forces arise when two vesicles approach each other, or wheno times the radius of gyration of the polymer chains (2Rg). In these regions, the polymersequently, an osmotic pressure appears between the bulk solution and the polymererating a net force of attraction between these two vesicles. This causes flocculation andntial — vesicle-to-vesicle distance (E–D) curve, the attraction force due to PEG-inducedfiles of interaction potentials with/without PEG are adapted from Kuhl et al. [300].


The net attractive interaction potential (E) over the depletionregion D at Db2Rg is:

E Dð Þ = −Z 2Rg

DqkTdD = −qkT 2Rg−D

� �= E0 1−D= 2Rg

� �� : ð3Þ

At contact of the two surfaces (i.e., at D=0)

E0 = E 0ð Þ = −2RgqkT : ð4Þ

To relate the interaction potential between two flat surfaces to theadhesion force (F) between two identical spheres of radius R, one canuse the Derjaguin approximation, which gives:

F Dð Þ = pRE Dð Þ: ð5Þ

Substituting Eq. (4) into Eq. (5), gives

F = pRE0 = −2pRRgqkT = −1:2� 1030pRRgCWkT

MW: ð6Þ

Eq. (6) predicts the polymer-induced depletion–attraction force (F)between twovesicleswith a similar size to beproportional to the vesicleradius (R), polymer size (Rg), polymer concentration (Cw), and tempera-ture (T), and inversely proportional to the molecular weight of thepolymer (Mw). Strictly speaking,Rg in Eq. (6) should be replacedwith thethickness of the depletion layer, which can be experimentally estimatedfrom the distance between two approaching vesicles when they startexperiencing the attractive force [300]. In addition, Cw in Eq. (6) shouldbe replaced by the activity at high polymer concentrations.

Using Eq. (6), it is estimated that PEG (Mw=8 kDa, Rg=5 nm) at aconcentration of 50mg/mL (Cw=5wt.%) can induce a depletion force of∼50 pN in a colloidal system of vesicles with an average radius of100 nm at 37 °C. This force is in the same order of magnitude as thecontribution of van derWaals attraction that is always present even inan aqueous suspension free of polymers [300]. Although being anextremely simplifiedmodel, the depletion forces predicted fromEq. (6)were found to correspond very closely to those directly measuredusing a surface force apparatus [300]. More complicated calculationsresulted in no significant deviation from this simplified model [300].

Zasadzinski et al. [223] have developed a more detailed thermo-dynamic model to study the mechanisms of surfactant inactivationdue to serum proteins and to explain how polymers such as PEGovercome this inactivation by depletion–attraction. Using the classicalSmolukowski analysis of colloid stability, this theoretical model con-firmed that the polymer-induced depletion force promotes adsorptionof PL vesicles/aggregates of PS, thereby overcoming competitive ad-sorption of serum proteins. This model predicts that the adsorptionrate of PL vesicles/aggregates increases only linearly with the bulksurfactant concentration but exponentially with decreasing theenergy barrier for adsorption [223]. Hence, the use of water-solublepolymers appears to provide a very effective and inexpensive way toovercome surfactant inactivation.

The depletion–attraction model predicts that adding nonionicpolymers would alter the surfactant morphology in the bulk phase,i.e., inducing larger surfactant vesicles/aggregates with the depletionforces. Morphological alterations of surfactant due to the presence ofinhibitors and/or polymers were evident under optical microscopy[223,283] and transmission electron microscope (TEM) [267,305,306].Optical microscopy detected that PEG added to different clinical sur-factant preparations induced large aggregated structures [223]. UsingCryo-TEM, Gross et al. [305] found thatmeconium altered themorphol-ogy of Curosurf from spherical unilamellar andmultilamellar vesicles tosmaller and elongated structures with nonuniform curvatures. At highmagnifications, these workers observed disruption of PL vesicles whenCurosurf was mixed with meconium or taurocholic acid, a bile acid in

meconium. This may indicate that meconium inhibits surfactant in partby penetrating the bilayer structures of the PL vesicles. Ochs et al. [306]found that adding dextran into meconium-inhibited Curosurf reversedthe surfactant morphology from small, irregular structures to largelamellar body-like, unilamellar and multilamellar structures. Togetherwith the reversal of surfactant morphology, the surface activity ofCurosurf was also restored [306]. All of these observations suggest acorrelation between morphology of surfactants and their surfaceactivity, which is consistent with the biophysical analysis of surfactantsubfractions (detailed in Section 4) [307–309].

It should be stressed that the surfactant morphology – surfaceactivity correlation discussed above appears to be only valid forsurfactants mixed with nonionic polymers, where depletion–attrac-tion is the major driving force for flocculation of PL vesicles.Surfactants with the addition of ionic polymers such as hyaluronando not necessarily follow this rule. Both freeze-fracture TEM [310] andturbidity [290] studies found that low-concentration hyaluronandid not induce as much aggregation as high-concentration PEG butenhanced surface activity more. This indicates that ionic polymersenhance surface activity of surfactants by a different mechanism thanthe nonionic polymers (detailed later).

3.3.3. Beyond surface activity enhancementThe use of water-soluble polymers as additives to clinical PS could

potentially break new ground in the development of cost-effective,inactivation-resistant formulations that are highly favorable for ARDStreatment. The early in vitro tests mainly focused on the improvementin surface activity, i.e., rapid adsorption, low γmin upon compression,and high resistance to surfactant inactivation. However, recent studiessuggest that a number of further considerations beyond surfaceactivity enhancement will be necessary for further development.

First, the efficacy of different polymers seems to be dependent onthe clinical surfactant to which the polymer is added. Lu et al. [292]found that both in vitro and in vivo dextran was more effective whenmixed with Curosurf while PEG was more effective when mixed withSurvanta. The specificity of a polymer for a certain surfactantpreparation may imply a specific molecular interaction between thepolymeric additive and certain PL/protein components which arevaried in different surfactant preparations. If such a specific interac-tion does exist, the depletion–attraction mechanism needs to beamended as, theoretically, depletion–attraction ought to be non-specific. In addition, this means that the experimental results obtainedfrom one polymer-surfactant combination may not necessarily begeneralizable to the others.

Second, the performance of a polymer may depend on the specificexperimental approach used for evaluation. It has been shown thatdifferent lung injury models yield different alveolar environmentsthat can play a critical role in determining the overall efficacy of thesurfactant therapy [311]. Lu et al. found that PEG (10 kDa) significantlyimproved the efficacy of surfactant therapy in meconium-induced[295], acid-induced [294], and endotoxin-induced [294] animal ARDSmodels. However, Campbell et al. [312] reported an adverse effect ofPEG in a saline lung lavage model. With these latter studies, rabbitstreated with mixtures of BLES and PEG showed greater hypoxemia,lower lung compliance, and higher hypercapnia compared to thoseadministered only BLES. The deleterious effects of PEG in the salinelung lavage model were confirmed by a different group using adifferent animal (rats) and a different surfactant (Survanta) [313].These deleterious effects were attributed to increased pulmonaryedema due to the addition of PEG which has a high capacity to bindwater. In the saline lung lavage model, this side-effect of PEG canoverwhelm its beneficial effects on enhancing surface activity. Thesenegative reports constitute a serious caveat to the use of polymers as asurfactant additive.

In addition, it should be noted that most previous in vitro studiesinvolving polymers used serum proteins (usually albumin), whole


serum, or meconium as model inhibitors. A recent in vitro study hasshown that PEG cannot effectively overcome surfactant inhibition dueto supraphysiological levels of cholesterol [314]. These experimentalresults, on the one hand, are consistent with the argument that PEGovercomes surfactant inhibition mainly by enhancing surfactantadsorption, which has been compromised in the presence of plasmaproteins. On the other hand, these results also show the inability ofPEG to deal with surfactant inhibition by lipids, such as cholesterol,which inhibit surfactant by a completely different mechanism. Giventhe fact that cholesterol may play a significant role in ARDS [201,315],the clinical potential of the polymeric additives in treating ARDS is stillto be confirmed.

Third, polymer dosing has not always been optimized. A PEG(10 kDa) concentration of 50 mg/mL was recommended in a numberof early studies [277,294,295]. However, it has been reported that amuch lower concentration near 20 mg/mL was sufficient to render asatisfactory surface activity in vitro [285,288]. Further increasing PEGconcentration yielded no apparent improvement in surfactantadsorption [288]. Similarly, a much lower concentration of hyaluronanthan previously used [282,293] was found to be sufficient to producesatisfactory performance both in vitro and in vivo [291]. High polymerconcentrations can introduce difficulty in drug delivery due toincreased viscosity [283,297], increased osmotic stress in the lungs[313], and possible impaired pulmonary gas exchange due to reducedgas diffusivity in the alveolar lining layer [316]. Hence, it is necessaryto optimize the polymer dosing relative to the particular in vivosituation.

The difficulty in endotracheal instillation due to viscous surfactantpreparations may be mitigated to some degree by using a newapproach of drug delivery, i.e., bronchoalveolar lavage with a largevolume of dilute surfactant. A recent study has shown that a mixtureof 10 mg/mL Curosurf and 30 mg/mL dextran (69 kDa) improvedmeconium clearance and lung function in rabbits with meconiumaspiration [296]. Compared to bolus delivery, bronchoalveolar lavagewith dilute surfactant–polymer mixtures may help to directly removeinhibitors from the alveolar space and facilitate drug delivery. Anotherstudy showed that dextran improved the efficacy of Curosurf given ata low dose but not at a high dose in a lung injury model induced bytracheal instillation of albumin [297]. This further demonstrates thenegative effect of increased viscosity on the efficacy of surfactantformulations.

Finally, the use of ionic polymers with PS clearly requiresconsiderably more investigation. Ionic polymers are superior tononionic polymers in requiring lower effective concentrations.Hyaluronan (1240 kDa), an anionic biopolymer inherently existingin the alveolar fluid, was able to prevent serum-induced surfactantinactivation at a concentration 40 times lower than that of PEG(10 kDa) (1.25 mg/mL vs. 50 mg/mL) [222]. Chitosan (612 kDa), acationic biopolymer derived from fully or partially deacetylated chitin,was shown to enhance the surface activity of dilute BLES and toreverse albumin-induced inactivation at a concentration 1000 timeslower than PEG (10 kDa) (0.05 mg/mL vs. 50 mg/mL) [283,289]. Thereduced polymer concentration facilitates drug delivery and mini-mizes osmotic stress in vivo. In spite of these advantages themechanisms by which the ionic polymers interact with PL of PS arestill vague. When the added polymer moieties are polyelectrolytes, acombination of electrostatic interactions and polymeric effects (suchas depletion–attraction) is expected [283,289,290]. At extremely lowconcentrations, however, the polymer-induced depletion force (aspredicted by Eq. (6)) would be weak and only secondary to theelectrostatic interactions. Moreover, if the ionic polymers adsorb tothe surface of PL vesicles by non-specific electrostatic binding, thedepletion force would be more or less eliminated. Although directexperimental evidence for PS is still lacking, studies of pure PL vesicles[317,318] and monolayers [319,320] both suggested that chitosaninteracted with PL mainly through electrostatic interactions.

When using ionic polymers as surfactant additives, it is alsoimportant to keep in mind that the introduced electrostatic interac-tions may disturb the charge balance in surfactant. It was found thatthe effects of chitosan and hyaluronan on the surface activity of BLESwere critically dependent on the polymer-to-PL ratio [283,289]. Abovethe optimal range of this ratio, i.e., at higher polymer concentration, aninhibitory effect on surface activity was observed [283,289]. Thesefindings are consistent with previous reports that addition ofpolycations could inactivate surfactant as a function of the cationicadditives-to-surfactant PL ratio [321,322]. Determination of theoptimal ratio in vitro appears to require characterization of the pHand ionic strength of the electrolytes, especially the calciumconcentration [289]. The ratio dependence of the ionic polymerscomplicates the surfactant formulation and would be best, if not only,tested using in vivo animal models (see Section 4).

To conclude Section 3, it has become apparent that, compared toALI and ARDS, surfactant treatment of premature infants with RDS isrelatively straightforward. One must simply recognize the signs thatsignificant RDS is imminent and administer surfactant before seriouslung injury occurs. This explains why the relatively poor protein-freesynthetic surfactants employed in early trials were beneficial. Theymerely had to provide sufficient surfactant PL substrate to mix withendogenous alveolar pools of surfactant and/or cycle through thedeveloping type II cells to maintain infants until they could generatesufficient endogenous surfactant of their own. Nevertheless, establish-ing surfactant therapy for neonates with basically healthy lungs tookmany years.

ARDS has clearly proven much more difficult. Part of the reasonthat ALI and ARDS are so difficult clinically is the lack of appropriateearly markers for identifying those patients whose disease will notresolve spontaneously. Further difficulty arises from the multipleinsults involved in lung injury and surfactant inhibition. Early pilottrials with infants clearly demonstrated the futility of introducingsurfactant into lungs filled with surfactant inhibitors and this led toearly treatment protocols with RDS. For a number of reasons,including the lack of dependable markers and expense, pulmonolo-gists do not have the option of treating ARDS early and in practice arealmost always faced with multiple inhibitory mechanisms arisingfrom the original insult and including membrane and lysopho-spholipids as well as various plasma proteins. In addition there isthe release of proteases, phospholipases and other disruptive agentsfrom lung tissue. To give one example, it has been shown that lyso-PC,at levels which do not affect the biophysical activity of surfactant,sensitizes surfactant to protein inhibition [249]. Another example ismeconium aspiration syndrome, which involves many inhibitoryagents including bile acids, fatty acids, proteins and other substances[14,173,250,323]. Under these circumstances, surfactant function willbe inhibited by both competitive adsorption and film penetration andlikely some other mechanisms. Consequently, the actual mechanismof inhibition in the case of ARDS can be expected to be quitecomplicated due to additive and synergistic effects of differentinhibitors [324]. Different biophysical mechanisms of surfactantinhibition have been summarized in Fig. 6.

As it stands, overcoming inhibition will play a crucial role in thedevelopment of clinical surfactants for ARDS treatment. The use oflow-cost, water-soluble polymers as surfactant additives has provento be a promising approach in this direction. The early studies focusedon the nonionic polymers, such as dextran and PEG. These polymerslikely contribute to improving surface activity and overcominginhibition by plasma proteins through a depletion–attractionmechan-ism. However, generating a large enough depletion force to enhancesurfactant adsorption requires high concentrations of these polymers,thus introducing difficulties in tracheal drug delivery and causingincreased osmotic stress in the lungs. More recent studies exploit theionic polymers, such as hyaluronan and chitosan, which require amuch lower effective concentration than the nonionic polymers.

Fig. 6. Biophysical mechanisms of surfactant inhibition by different inhibitors. Differentinhibitors, including plasma proteins and certain lipids, can (A) interfere with the PLadsorption to form a functional surfactant film at γeq of ∼23mN/m; (B) prevent the filmfrom reaching low γmin of ∼0 mN/m or increase film compressibility upon filmcompression; and (C) affect PL re-spreading from surfactant reservoirs to obtain γmax of∼30 mN/m upon film expansion. (A) Soluble proteins such as fibrinogen, fibrinogenmonomer, hemoglobin, and albumin interfere with de novo surfactant PL adsorption toequilibrium [112,130,178,201,217–220,222–224,230,249,272,282,324–330]. Surfactantadsorption is also markedly impaired by lyso-PC [283,314,112,220,327–329] and lyso-PG [249,324,329]. However, supraphysiological levels of neutral lipids such asmonoacylglycerol, diacylglycerol, unsaturated fatty acids, cholesterol, and cholesterolester show little or no effect on adsorption [201,251,327]. (B) The ability of surfactant PLto attain low surface tensions during compression is hampered by total serum proteins,fibrinogen, fibrinogen monomer, CRP, and to a lesser extent, by albumin [241,244,314].Surfactant compressibility is markedly increased by supraphysiological levels ofcholesterol [163,251,260]. Surface tension reduction is also affected by high mono-acylglycerol, diacylglycerol, unsaturated fatty acids, and cholesterol esters [201]. (C) Re-spreading of surfactant PL from surface-associated reservoirs is inhibited by CRP and, athigh surface tensions, by albumin [221,244]. Supraphysiological levels of cholesterolalso have a deleterious effect on surfactant re-spreading [163,260]. The re-spread filmsare thought to be very similar, if not identical, to de novo adsorbed films described in(A). Only small amounts of the surfactant films are lost during each cycle and equivalentamounts of PL are taken up through de novo adsorption.


Nevertheless the mechanism by which these ionic polymers benefitsurfactant function and resistance to inhibition is still unclear.Considerable attention should be given to the negative reports ofusing polymers in animal trials. A number of in vitro studies have alsodemonstrated adverse effects of adding polyelectrolytes to surfac-tants, depending on the polymer-to-PL ratio. Taking into account thedifficulty in controlling a fixed polymer-to-PL ratio, before and afterdrug delivery, we suggest that in vivo animal trials are crucial fortesting the polymeric additives, especially the ionic polymers.

4. Methods for evaluating pulmonary surfactant

Methods for assessing the surface activity of PS fall into threecategories: in vivo, in situ and in vitro techniques.

4.1. In vivo methods

As discussed in Section 2, a functional surfactant must be able torapidly form a surface film at an air–liquid interface and to reduce γ tovery low values under dynamic compression. When exogenoussurfactant is administered to a surfactant-deficient lung, thesebiophysical characteristics should translate into lung complianceand, as a consequence, improved blood oxygenation. However, the in

vivo situation is more complicated since the physiological response isnot only determined by the biophysical properties of the surfactant,but also by how this material is delivered to the terminal airspaces andhow it is metabolized once deposited [331]. For this reason evaluationof newly developed exogenous surfactants utilizing in vivo experi-mental models is critical prior to clinical usage of such preparations.These in vivo methods contribute first-hand physiologically relevantinformation on the factors influencing surfactant therapy, such as theefficacy of different surfactant preparations, the effects of differentmeans of administration, dosing and timing, etc.

Various in vivo models have been developed to evaluate theefficacy of surfactant replacement therapy on preterm and termanimals. Accordingly, the commonly used in vivo models can begenerally divided into those primarily involving surfactant deficiency,such as the premature animal model and the saline lung lavage modelin adult animals, and those primarily involving surfactant dysfunctionor inactivation, such as the meconium aspiration, high-stretch ven-tilation, and acid aspiration models [332,333]. The overall objective ofthe latter models is to investigate the therapeutic potential for thespecific lung injury rather than an examination of the biophysicalproperties in vivo. As such, a detailed discussion of those studiesis beyond the scope of this review and we will focus solely on thesurfactant-deficient models.

Evaluation of surfactant efficacy in premature animals has beenperformed mainly with premature rabbits [273,334–336]. In theseexperiments the rabbit fetuses are delivered by caesarean section at agestational age of 27 or 28 days, at which time the lungs are stillsurfactant-deficient. The premature rabbits are connected to a pres-sure controlled ventilator in a 37 °C plethysmography setup in whichthe peak pressure at which each rabbit is ventilated can be controlledindividually. Surfactant can be administered directly into the lungs ofthe animals though a bolus injection via an endotracheal tube. Themain outcome of surfactant efficacy in this model is dynamic lungcompliance during ventilation.

The two main advantages of the preterm rabbit model are the sizeof the preterm rabbit lung which ensures optimal surfactantdistribution throughout the lung with a relatively simple instillationprocedure, and the ability to rapidly performmeasurements on a largenumber of animals simultaneously. Disadvantages include the rela-tively short period of observation which does not allow for detailedmetabolic assessments of the exogenous surfactant, and limitedflexibility in terms of ventilation strategy during the analysis. Thismodel has been utilized extensively to test exogenous surfactant,including studies comparing the efficacy of different surfactants,testing protein inhibition and examining the physiological activity ofdifferent surfactant subtypes [273,334–336].

A second preterm animal model that has been utilized is thepremature lamb [337,338]. Although conceptually similar to thepreterm rabbit model, this model has the advantage of a larger animalsizewhichmore closelymimics the premature infant observed in RDS.This model allows for more extensive measurements on blood gases,surfactant distribution, and metabolism. On the other hand, thepremature lamb is considerably more labour-intensive and expensive.In general, this model is suitable for more extensive preclinical studiesfor exogenous surfactant therapies.

The saline lavage model in adult animals has also been utilizedextensively for testing surfactant preparations [309,339–343]. Ingeneral, this method involves connecting the animal to a mechanicalventilator and subsequently lavaging the lung with saline to removethe endogenous surfactant. One response to the lavage procedure willbe the secretion of intracellular surfactant; thus the lavage procedureis repeated several times at regular intervals. Depletion of surfactant isgenerally reflected by low blood oxygenation, and can be confirmed byanalysis of the amount of surfactant removed by the final lavageprocedure. Subsequently, exogenous surfactant can be administeredto the surfactant-deficient animal. The most relevant physiological

Fig. 7. Schematic of a Langmuir–Wilhelmy balance (LWB). The surfactant film is usuallyformed by spreading at the air–water interface of the aqueous subphase filling theLangmuir trough. Moving the barrier to the right decreases the area of the spread film,thereby increasing surface pressure (i.e., decreasing surface tension, according to Eq. (1)).The surface tension is determined by measuring the change in the vertical pull on theWilhelmy plate.


outcome of surfactant function in these studies will be bloodoxygenation. The animals can be monitored for several hours whichmay reflect how the surfactant is handled in the airspace.

The adult saline lavage model has been employed in a large varietyof species including rats, rabbits, pigs and sheep. In general, smallerspecies are utilized for simple comparisons between differentsurfactant preparations. Larger animal models may also test efficacyof surfactant preparations, but more often are utilized for otheraspects that influence the surfactant therapy such as the deliverymethod or the effects of ventilation strategy after treatment.

In general, most in vivo studies examining the activity of surfactanthave confirmed the previous in vitro data. For example, in vitroanalysis of the biophysical activity of the different surfactant sub-fractions, i.e., the large aggregates (lamellar bodies, multilamellarvesicles, tubular myelin) and small aggregates (small unilamellarvesicles), indicated that the large aggregates represented the func-tional component of surfactant [307]. This was subsequently con-firmed in vivo utilizing both the premature rabbit and the lavage ratmodels [309,335]. In another study Bailey et al. utilized lavaged rats tocompare the physiological responses to an exogenous surfactant(BLES) with and without exposure to oxidation [339]. This studyconfirmed previous in vitro data that had demonstrated that oxidationimpaired the surfactant function [153,212]. Importantly however, notall in vivo studies correlate with the in vitro biophysical activity. Forexample, as part of the study mentioned above, Bailey et al. [344]found that SP-A addition improved the biophysical activity of oxidizedBLES in vitro, but this combinationwas not as effective as control BLESin vivo. Also, based on investigations in which the addition of PEG tosurfactant was able to improve the exogenous surfactant properties invitro, Campbell et al. [312] investigated this in the lavaged rabbitmodel. In contrast to the in vitro data, animals receiving surfactantwith PEG had an inferior physiological response compared to animalsreceiving just surfactant. A subsequent study suggested that theadministration of PEG in this model led to increased extracellular lungwater thereby limiting its functionality (see also Section 3) [313].

4.2. In situ methods

Two in situ methods have been developed to estimate alveolar γ inexcised lungs. One is the pressure–volume (P–V) method initially usedby von Neergaard [22] in his early discovery of the importance ofalveolar γ forces. This procedure was also used by Mead et al. [26],followed by standardization by Fisher et al. [345], Bachofen et al.[121,346] and Wilson [347,348]. This method indirectly determinesalveolar γ by analyzing the P–V isotherms of excised lungs ventilatedin a quasi-static manner. The principle of this method is as follows:inflating or maintaining the lungs at a fixed volume with air requireswork against both tissue forces and γ forces. Filling the lungs withsaline annihilates the air–liquid interface, thus leaving only the lungtissue forces. Hence, the difference between the P–V isotherms of air-filled and saline-filled lungs reflects the contribution of γ forces. Inpractice, this procedure is complicated by the need to remove all airfrom the lungs. This can be achieved by allowing the lungs to collapsecompletely. For instance, this can be aided by brief ventilation withpure O2 which, in contrast to N2-containing air, can be completelyabsorbed by the vasculature.

Another in situ method is the microdroplet technique firstdeveloped by Schurch and coworkers [28,119,120,349] in the 1970s.This is so far the onlymethod truly capable of direct γmeasurement inthe lung. With this method, a droplet of a water-immiscible liquid(e.g., fluorocarbon) with known γ is deposited onto the alveolarsurface of excised lungs using a micropipette. The droplet will spreadon the surfactant lining layer of the alveoli to form a liquid lens.Theoretically, the equilibrium shape of this lens (its contact angle) isdetermined by the balance of γ of the three adjacent interfaces, i.e.,air–lens liquid, air–lining layer, and lens liquid–lining layer interfaces,

as predicted by the classical Neumann triangle relation [116]. Hence,the shape of the liquid lens can be used to estimate γ of the surfactantlining layer. In practice, the alveolar γ is estimated from the diameterof the liquid lens, monitored by amicroscope. A calibration curve (γ vs.diameter of the lens) for the immiscible fluid is determined in aseparate in vitro experiment using a Langmuir balance. It should benoted that this calibration procedure may introduce errors into the insitu γ measurements [350]. This is because the shape and diameter ofthe liquid lens is determined by the surface tension balance only for asufficiently deep subphase. If the thickness of the subphase is similarto the penetration depth of the liquid lens, the lens can be significantlydistorted by a disjoining pressure [351]. Consequently, the γ-diametercurve calibrated in a Langmuir balance, with a macroscopic subphase,could deviate from the in vivo situation, where the aqueoushypophase of alveoli is extremely thin (i.e., ∼0.2 μm, see Section 2).

The microdroplet method has also been successful applied to mea-sure γ in vivo, for example, in the trachea of non-anesthetised horses[352].

4.3. In vitro methods

Because of their convenience, in vitro methods are most commonlyused for examining the surface activity of PS. A variety of techniqueshave been developed for this purpose. Three widely used methods arethe Langmuir–Wilhelmy balance, pulsating bubble surfactometer, andcaptive bubble surfactometer. In addition to these three, amethod calledthe constrained sessile drop was recently developed for the in vitroassessment of PS. These four methods will be extensively discussed andcompared.Other invitromethodswill alsobe introduced, albeit inmuchlesser detail. Other reviews on the three traditionally used in vitromethods are also available [14,84,104,105,353,354].

4.3.1. Langmuir–Wilhelmy balanceThe classical Langmuir balance was introduced to surface science

in the early years of the 20th century [118]. An insoluble surfactantmonolayer, usually in organic solvent, is spread on top of a liquidsubphase, usually aqueous, filling a trough. The film is confined by twobarriers that move symmetrically, or by a fixed one on one side and amovable one on the other side. The film can be slowly compressed andexpanded in a quasi-static fashion by the relative movement of thetwo barriers. The force acting on the floating barrier is measured by ahorizontal force transducer, which directly indicates π. Clements[27,355–357] modified Langmuir's original design to introduce theLangmuir–Wilhelmy balance (LWB) to the study of PS. As shown inFig. 7, the LWB consists of a Langmuir trough constructed of Teflon andaWilhelmy plate for measuring γ of the film-covered subphase. Recallthat π and γ are coupled by Eq. (1) (see Section 2).

Fig. 8. Schematic of a pulsating bubble surfactometer (PBS). The polyacrylamide chamberfilled with the sample (∼20 μL) to be tested is positioned on the pulsating unit. Sufficientfluid is withdrawn by the pulsator such that the sample water level moves down throughthe capillary and a bubblewith Rmin is created. The pressure difference across the bubble isrecorded for 10 s to monitor surfactant adsorption, and this is reported as surface tension(according to Eq. (7)). The bubble is then pulsated between Rmin and Rmax at 20 cycles/minwhile surface tension is recorded. The inset illustrates film leakage, i.e., spreading of thesurfactant film to the water layer remaining inside the capillary during bubble formation.The leakage can beminimized by keeping the capillary dry. However, the effect of leakagecan be augmented when studying surfactant inhibition due to plasma proteins as theseproteins can adsorb onto the capillary during bubble formation and these proteins, beinghydrophilic, retain water, thus impeding drainage.


This early tensiometric apparatus not only initiated a virtualexplosion in surfactant research (Section 2), but also remains populareven now. The LWB is well suited for recording π-area isotherms ofspread monolayers as the surface area per molecule can be preciselydetermined by controlling the amount of surfactant spread and thesurface area available for spreading. This feature is particularly useful tocharacterize the surface rheological properties of individual PScomponents and their simple mixtures. Another primary advantage ofthe LWB is the capability for easy combination with a variety ofmicroscopic and spectroscopic techniques [358], which are not readilyamenable to the other in vitro methods. Such assemblies allow directexamination of molecular structure, orientation, domain formation,topography, electrical surface potential, and localized chemical compo-sition of either surfactant films at the air–water interface or filmstransferred to a solid substrate using the Langmuir–Blodgett technique.The LWB has been used in conjunctionwith Brewster angle microscopy[143,146,359], fluorescence microscopy [63,142,143,146,167,359], con-focal microscopy [87,360], scanning near-field optical microscopy[361,362], AFM [63,149,157,166,241,363–365], grazing incidence X-raydiffraction [364,366], infrared spectroscopy [367,368], sum-frequencygeneration spectroscopy [369,370], and ToF-SIMS [152,156,260]. Appli-cation of these film imaging/analysis techniques to PS studies hasprovided valuable information that complements the traditionaltensiometry techniques.

In spite of the above merits, the LWB has a number of drawbacks forstudying PS. First, it is not ideal for studying surfactant adsorption fromthe subphase as it requires a relatively large amount of liquid sample, i.e.,usually no less than several tens of millilitres. Second, the LWB onlyallows relatively slow compression–expansion cycles as fast cyclingcreates waves at the air–water interface, which interfere with the γmeasurement. Together with the difficulties in precisely controlling thephysiological temperature and humidity, the LWB does not directlysimulate respiration. Third, there can be a problem with the measure-ment accuracy of the Wilhelmy plate as it requires a 0° contact anglebetween thevertical dippingplate and theadherentfluid layer [116]. Thiscondition can be difficult to maintain, especially during film expansion,since the PLmolecules tend to adsorb onto the plate during the previousfilm compression [371]. While these concerns can be overcome withcareful work, they make the LWB a challenging apparatus to operateaccurately. Finally, a less obvious but serious problem, the LWB can sufferfrom a phenomenon known as “film leakage”. Film leakage is driven bythermodynamics: at sufficiently low γ, surface active material tends tospread fromtheair–water interface onto the solid framework supportingthe film, i.e., wetting of the solid barriers and walls, as this processdecreases the total free energy of the system [372]. Leakage of PL at theair–Teflon surface usually starts at γ of ∼18 mN/m [353,372]. Due to theloss offilmmaterial from theair–water interface, theapparentmoleculararea and film compression ratio can be erroneous. Also because of filmleakage the ability to reach low γ can be greatly compromised.

With the LWB, film leakage may occur both at the trough walls andat the barriers, either above the water level (at the air–solid interface)or below the water level (at the liquid–solid interface) [353]. Leakageat the barriers can be reduced using tightly fitted barriers [357] orcontinuous Teflon ribbons [355,373]. Leakage at the trough walls canbe reduced by priming the Teflon walls with an alcoholic solution oflanthanum chloride and long-chain saturated PC [96]. Using thesetreatments, near-zero γ can be achieved by extreme film compression.A constant π, however, can normally only be maintained by pro-gressively compressing the film, demonstrating that the LWB fails toreproduce the extraordinary film stability found in situ [119]. Theproblems in film stability are particularly evident at physiologicaltemperature and humidity [96,374,375].

4.3.2. Pulsating bubble surfactometerThe pulsating bubble surfactometer (PBS) was first introduced by

Enhorning in 1977 [376]. As shown in Fig. 8, the currently commer-

cially available PBS consists of an air bubble formed in a disposablepolyacrylamide chamber. The chamber contains only 20 μL of the testliquid and is immersed in a temperature-controlled bath. The bubbleis formed by drawing air from the atmosphere through a capillary(bubble-on-a-tube model). Adsorption is monitored for 10 s. Subse-quently, the bubble is oscillated by a pulsator between two fixedpositions: a minimum radius of 0.4 mm and a maximum radius of0.55 mm, which produces a maximum 50% reduction in surface area.The cycling frequency usually used is 20 cycles/min to mimicbreathing. However, it can be changed from 0.02 to 80 cycles/min.

During oscillation, the maximum and minimum radii (R) of thebubble are monitored by a microscope. The pressure gradient acrossthe bubble (ΔP) is measured by a pressure transducer. Since thebubble communicates with the ambient atmosphere, the pressuregradient measured by the transducer corresponds to the negativepressure across the liquid phase. γ is calculated using the Laplaceequation for a spherical interface

DP =2gR

: ð7Þ

The PBS is highly efficient and time-effective. One measurementcan be completed within 5 min and takes only ∼20 μL of sample.Therefore the PBS has been widely used for the quality control ofclinical surfactants. In addition, it is superbly useful for monitoringand comparing the surface activity of different surfactant sampleswhen many animal or clinical samples are generated. The rapidadsorption during expansion is indicated by the γ of the film at themaximum bubble radius and the ability to reach low γ is examinedwhen the film is compressed to the minimum bubble radius. Anotheradvantage of the PBS, which makes it superior to the LWB, is the fastpulsating rate that permits direct simulation of breathing.

Nevertheless, the PBS has some drawbacks. First, for a number ofreasons, leakage is a serious problem. At low γ leakage can occur atboth the inner surface (air–solid) and outer surface (liquid–solid) of

Fig. 9. Schematic of a captive bubble surfactometer (CBS). The captive bubble floatsagainst the hydrophilic ceiling but is separated from it by a thin wetting film. Thisprevents the film leakage normally encountered at low surface tension with othersurface balances. The inset shows multilayer structures of the adsorbed pulmonarysurfactant at the air–water interface of the bubble. The surface tension is determined bybubble shape analysis (according to Eq. (8)). Surface area manipulations are conductedby altering the hydraulic pressure in the chamber.


the capillary. The original PBS reported by Enhorning used home-made chambers with a Teflon capillary which reduced leakage ontothe air–solid interface [376]. With the present polyacrylamidechamber, a thin film of water can be retained inside the capillarywhen the bubble is formed, thus leading to a much greater film areathan anticipated (see the inset in Fig. 8). During pulsation, the waterfilm can drain from the capillary but this can be variable [377]. Forexample, plasma proteins can adsorb onto the capillary during bubbleformation and the proteins, being hydrophilic, retain water, thusimpeding drainage. Hence, it is particularly problematic with studiesof surfactant inhibition by plasma proteins where the apparentdeleterious effects of these proteins can be augmented in the PBS dueto leakage. Also because of leakage, the PBS is not suitable for studyingfilm stability at γmin in a non-pulsating mode.

A secondproblemwith the commercial PBS is the lack offlexibility inassessing the surface activity. For example, the time for adsorption toequilibrium is set at 10 s. While this can be changed, it cannot be doneeasily. As a result, animal and clinical samples are subject to expansionand contraction, often before γeq is attained. This obviously has animpact on γmin and γmax, as well as γeq recorded for that particularsample. Lengthening the adsorption time would also help to minimizethe amount of water remaining inside the capillary and therefore filmleakage. In addition, the way that the current commercial apparatusfunctions is to initiate pulsation by expanding the bubble from theminimum radius at the end of the 10 s adsorption period. Thus, even ifthe sample has attainedγeq, an additional 50% of area is imposed in 1.5 s(for pulsating at 20 cycles/min), making it impossible to relate initiationof compression to γeq. Moreover, although additional information canbe accessed, the PBS usually records only the γ of the bubble at themaximum and minimum radii, to be the γmax and γmin, in a pulsatingmode. The maximum reduction of bubble area, i.e., the compressionratio, is fixed at 50%, which prevents detailed study of surface rheology.

Finally, γmeasurements at low values using Eq. (7) are not reliable.Even for a small bubble less than 1 μL (∼1 mm in diameter), theassumption of spherical shape does not hold true when the γ falls tovalues near 1 mN/m [378]. The effect of gravity on bubble deformationat low γ and other effects of altering the bubble shape such as thehydrodynamic effects due to rapid pulsating have been extensivelystudied by Franses and coworkers [379–381]. Nevertheless, it shouldbe stressed that in most situations where this apparatus is used tocompare functional vs. non-functional surfactants, the estimations ofγmin are more than adequate [378].

A recent development by Seurynck et al. [382] contributes toimproving the current PBS. A real-time image acquisition system hasbeen integrated into the traditional PBS, which allows the determina-tion of γ and surface area at any point of pulsating. In addition, insteadof simply assuming a spherical shape, these workers fit the bubbleprofile to an ellipse. This yields enhanced accuracy compared tospherical fitting. Although still incapable of preventing film leakage,the real-time imaging facilitates visual detection of film leakage intothe capillary. Those images with leakage can be discarded in dataprocessing.

4.3.3. Captive bubble surfactometerThe captive bubble surfactometer (CBS) was first introduced by

Schurch et al. in 1989 [383]. As shown in Fig. 9, an air bubble (2–7 mmin diameter) is introduced into a chamber where it floats against aceiling coated with 1% agar gel. The agar coating renders the ceilingcompletely hydrophilic. Consequently, the bubble is separated fromthe ceiling by a thin wetting film of the surrounding aqueous liquid,thus avoiding adhesion to any solid support and apparently eliminat-ing all potential pathways for film leakage [384]. It should be notedthat the use of the agar coating is not always necessary provided thatthe ceiling is smooth and hydrophilic. It has been shown that achamber made of stainless steel without the agar coating is alsocapable of maintaining a leakage-proof environment [385].

The original CBS chamber was constructed from a gastight syringemaking it very difficult to spreadfilms so that only adsorbedfilmswerestudied [383,384]. More recently, different film spreading techniqueshave been developed for the CBS [386,387]. With one spreadingtechnique developed by Putz et al. [386],films are spread fromorganic-extracted surfactants onto the bubble surface with a microsyringe,followed by exhaustive subphase replacement to remove the organicsolvent vapor. Another type of spreading technique was developed bySchurch et al. [387]. Films are spread/adsorbed at the bubble surfacefrom an aqueous suspension of high-concentration surfactant using amicrosyringe. To ensure the surfactantfloats around the bubble surfaceto enhance spreading, the saline subphase can contain 10wt.% sucrose,which increases the density of the subphase.

Afterfilm formation, the bubble can be compressed and expanded ineither quasi-static or dynamic fashion by varying the hydraulic pressurein the chamber. The pressure can be changed by varying the chambervolume [383] or by regulating liquid flow between the chamber and anexternal reservoir (with the manufactured plastic chamber) [388]. Thefrequency of cycling can be varied from extremely slow (i.e., in the caseof quasi-static cycling) to faster than 60 cycles/min.

In contrast to the PBS, the shape of the bubble in the CBS cannot beassumed to be spherical due to its relatively large size. (See Fig. 10(a)and (b) for two typical captive bubble imageswith different γ.) Instead,the bubble shape is controlled by the mechanical balance betweenthe γ forces and local gravity, according to the Laplace equation ofcapillarity:

g1R1

+1R2

� �=2gR0

+Dqgz ð8Þ

where R1 and R2 are the two principal radii of curvature at the studiedpoint on the surface, which reflect the shape of the bubble; R0 is theradius of curvature at the apexof the bubble;Δρ is the density differenceacross the interface; g is the local gravitational acceleration; z is thevertical distance from the apex to the studied point. Normalizing Eq. (8)yields a single nondimensional characteristic parameter, called the Bondnumber (B)

B =DqgR2

0

g: ð9Þ

The Bond number reflects the relative effects of gravity andcapillary forces on the shape of a drop/bubble. Hence, it is possible todetermine γ from the bubble shape since gravity is known. Oneway ofcharacterizing γ from the bubble shape is to use the height-to-diam-eter ratio of the bubble, as formulated by Malcolm and Elliott [389].Bubble area and volume can be calculated from polynomial functions

Fig. 10. Typical images of captive bubble (CB), constrained sessile drop (CSD) and pendant drop (PD) at different surface tensions. (a) CB at 21 mN/m; (b) CB at 1.3 mN/m; (c) CSD at47 mN/m; (d) CSD at 0.68 mN/m; (e) PD at 42 mN/m; (f) PD at 16 mN/m. The black particles in the CB images are insoluble surfactant aggregates suspended in the subphase, whichintroduce optical noise to the images. The arrow in (a) points at a satellite bubble formed during the experimental manipulations, which can have a very deleterious effect on thesurface tensionmeasurement. Arrows in the CSD and PD images point at the three-phase contact line. The three-phase contact line in (f) is not discernable due to film leakage. The PDand CSD images are courtesy of Ms. Zdenka Policova, University of Toronto.


regressed from measurements of a series of sample bubbles with awide range of height-to-diameter ratios [390].

A more accurate and automatic way to determine γ, surface areaand volume from a bubble image is to use axisymmetric drop shapeanalysis (ADSA). ADSAwas first developed byNeumann and associatesin the 1980s [391]. ADSA has been continuously improved during thelast two and a half decades [392–394]. Conceptually, ADSA determinesγ by numerically fitting the shape of experimental drops or bubbles tothe theoretical profiles given by the Laplace equation of capillarity(Fig. 11). The input parameters of ADSA are g, Δρ, and a number ofcoordinates of the experimental drop/bubble profile automaticallydetected by digital image analysis. ADSA was first applied to analyzecaptive bubble images by Prokop et al. [385]. A new generation ofADSA has been recently developed [394–396]. By using advancedimage analysis, this new ADSA is capable of automatic γ measure-ments of turbid fluids, such as PS in which optical noise is introducedby insoluble surfactant aggregates and sometimes satellite bubbles(see Fig. 10(a) for an example). This new ADSA was found to beparticularly useful for studying surfactants with polymeric additiveswhere system turbidity increases (see Section 3 for detail) [394–396].

Strictly speaking, determination ofγ from the shape of a drop/bubbleis only applicable to an equilibrium condition since the Laplace equation(Eq. (8)) considers only the balance between the gravity and capillaryforces. Under a highly dynamic condition, such as dynamic cycling in theCBS, the bubble profile may deviate from the theoretical profilepredicted by the Laplace equation due to hydrodynamic effects (such

as inertial and/or viscous effects caused by fluid flow). This may intro-duceerrors inγmeasurementusingADSA. Liaoet al. have systematicallystudied the hydrodynamic effects on the measurement of dynamicγ using an oscillating bubble [380,381]. Results from the numericalsimulation suggested that the hydrodynamic effects were negligibleexcept at some extreme conditions, such as a highly viscous liquidoscillated at an extremely high frequency. For a bubble in aqueousmedia, only if the oscillatory frequency is slower than 10 Hz (i.e.,600 cycles/min) will the bubble profiles predicted from ADSA and fromthe model considering the hydrodynamic effects be in good agreement[381]. The effects of viscous forces on ADSA measurement were alsostudied by Freer et al. [397]. Similarly itwas found that the viscous forceswere only significant enough to alter the ADSAmeasurement for highlyviscous liquid upon highly rapid dynamic oscillation.

The CBS has been used to measure adsorption rate (time toequilibrium), γmin, and γmax, during quasi-static or dynamic compres-sion–expansion cycling, and the percent of area reduction required toattain γmin from γmax. The percentage of area reduction has beenshown to be a more sensitive parameter than γmin in evaluatingsurface activity of PS, especially in the study of surfactant inhibition. Inaddition, owing to its leakage-proof capacity, the CBS is well suited forstudying mechanisms of surfactant films, such as film compressibility,stability, and collapse at extreme π. For films adsorbed at low surfac-tant concentrations (0.2–0.5 mg/mL), near-zero γ can be readily ob-tained by a moderate compression ratio (i.e.,b20% area reduction).This is in good agreement with the in situ measurements. Studies of

Fig. 11. Principle of axisymmetric drop shape analysis (ADSA). ADSA measures surfacetension from the shape of pendant drops, sessile drops or captive bubbles by comparingthe experimental drop/bubble profiles (dots in the X–Z coordinate) with the theoreticalLaplacian curves (curve in the x–z coordinate). The experimental profiles areautomatically extracted from the drop/bubble images using advanced image analysis.The theoretical Laplacian curves are generated by numerical integration of the Laplaceequation of capillarity (Eq. (8)). ADSAdetermines surface tensionby iterativelyfitting theexperimental profile to a family of theoretical curves until the best match is found. Thebest matched theoretical curve represents the experimental drop/bubble. ADSA solvesthis optimization problem by minimizing an objective function that consists of the sumof least squares of the minimum distance (i.e., normal distance, di) between theexperimental points and the theoretical profiles.With the input of the local gravitationalacceleration (g) and the density difference across the interface (Δρ) (see Eq. (9)), fiveparameters can be simultaneously optimized: the surface tension (γ), the curvature atthe apex (1/R0), the coordinates of the apex of the experimental profile (x0, z0), and theverticalmisalignment (α). The surface area and volumeof the drop/bubble are calculatedfrom the surface of revolution of the best matched theoretical profile. Adapted from delRío and Neumann [393].


film stability in the CBS are commonly performed in two differentways. Stability can be examined by compressing the bubble and hold-ing the volume at a minimum value. A stable film is able to maintainthe γmin (in an isovolumetric fashion) without returning towardsequilibrium for a prolonged period [307,374]. Similarly, stability can beexamined by studying the rate of bubble area reduction required tomaintain a constant γ, i.e., in an isobaric fashion [97,145]. Alterna-tively, stability can be examined by studying “bubble clicks”, i.e., asudden decrease in bubble area associated with an increase in γ,which indicates partial collapse of the surfactant film coating on thebubble surface [374,383]. Bubbles coated with a stable film have lesstendency to click. The CBS is also flexible in terms of being able to varyindependently the amount of film compression (i.e., compressionratio) and the speed of the compression (i.e., compression rate) [377].Moreover, in conjunction with other techniques, the CBS is able toscrutinize a variety of surfactant characteristics, such as the surface-associated surfactant reservoir, using a subphase depletion technique[60] and a subphase solidification technique (solidification of thehypophase–surfactant lining complex in a sodium alginate solution byadding calcium ions) [59]. In addition to γmeasurements, the CBS hasbeen broadly expanded to other investigations, e.g., gas transfer prop-erties of PS films [398,399], dissolution characteristics of anestheticvapors and gases [400].

A major advantage of the CBS is the ability to conduct investiga-tions at physiological and higher temperatures. For example, with theLWB it is difficult to demonstrate that DPPCmonolayers can attain π of70 mN/m (i.e., near-zero γ) at 37 °C [95,96] and relatively few studiesusing this apparatus show π higher than 50 mN/m even at roomtemperature, when the films are compressed in a quasi-static fashion.In fact, results from a number of LWB studies have concludedthat DPPC monolayers become unstable somewhat above 41 °C[92–94,401], which of course corresponds to the main transitiontemperature Tm for DPPC bilayers (detailed in Section 2). Theseexperimental studies suggested that PL monolayers might possess acritical point. A critical point can be defined as the temperature andpressure above which the properties of two phases no longer remaindistinct. For example, above 31 °C and 73 atm, CO2 becomessupercritical CO2, a fluid which has the characteristics of both a liquid

and a gas. However, more recent investigations using the CBS haveshown that spread DPPC monolayers melt between 48 and 53 °C andfound no evidence for critical behavior [97,145]. While the case forcritical behavior of PL monolayers depends on several arguments,these melting data make this phenomenon considerably less certain.In any event, such CBS studies emphasize the advantage of employingthis apparatus for experimentation under extreme conditions.

Nevertheless, the CBS also has weaknesses. First, in contrast to theLWB, the γ and surface area (i.e., the bubble area) in the CBS is coupled.This is due to the fact that the area of a bubble is a function of not only itsvolume but also its shape as determined by the γ forces. When asurfactant-coated bubble is compressed, the bubble volume decreases,which tends to decrease the bubble area, and meanwhile, the γdecreases, which tends to increase the bubble area [374]. Hence, it ismore difficult to precisely control the bubble area than its volume in theCBS. Nevertheless, in most cases this does not pose a serious concernon the recorded γ-area isotherms because the apparent area at eachtime is still calculated based on the same bubble image fromwhich γ isestimated. Second, compared to the PBS, operation and data processingof the CBS is relatively time-consuming. The problem of tedious dataprocessing can be largely solved using ADSA [394–396]. Third, foradsorption studies, the maximum surfactant concentration is usuallyrestricted to no more than 3 mg/mL [58]. This restriction arises fromoptical limitations since surfactant suspensions become murky andeventually opaque at increased concentrations. High-concentrationsurfactants can be studied using spreading techniques. Finally, the CBSmay not ensure full humidification automatically. This concern ariseswhen the CBS is used to study surfactant adsorption inwhich the rapidlyadsorbed surfactant films may present a barrier to water evaporation,thus slowing down the process of humidification [374]. Although directhumidity measurements in the bubble are not yet available, there wasclearly a difference in the adsorption kinetics and film stability of BLESfilms formed on bubbles of ambient air and prehumidified air [374,402].

4.3.4. Constrained sessile dropThe prototype of the constrained sessile drop (CSD) was first

developed by Wulf et al. [403] for simultaneously measuring γ andthe density of polymer melts. It was later modified by Yu et al. [404] fordetermining theγ of PS. As shown in Fig.12, a sessile drop (4∼8 μL) of PSrests onapedestal,whichemploys a sharpknife-edge toprevent thedropfrom spreading, thus eliminating film leakage. The pedestal is machinedfrom stainless steel with a diameter of 2.5 to 4 mm. The angle betweenthe horizontal and the lateral surfaces of the pedestal is 45° to 60°. Thepedestal has a central hole of 0.5 mm, through which the drop isconnected to a surfactant reservoir of 0.5–0.75 mL. The surfactantreservoir is continuously stirred by a magnetic stir bar. Formationand oscillation of the sessile drop is performed by a programmablemotor-driven syringe connected to the surfactant reservoir.

Film formation in the CSD can be conducted by adsorption orspreading. For adsorption studies, a drop can be formed within 0.5 s,which ensures adsorption at a fresh, clean air–water interface.Spreading on the CSD from either aqueous solution or organic solventis also straightforward. After film formation, the drop can be oscillatedat a predetermined compression ratio and compression rate, fromvery slow to very fast.

The CSD facilitates rigorous atmospheric control by means of anenvironmental control chamber. Full humidification, monitored by ahygrometer, is ensured by awater reservoir enclosed in the chamber. Agas manifold system (to produce different gas compositions) and a gaschromatography system (to analyze the gas composition) are underdevelopment. This development aims at studying the effect of differentgas compositions on the surface activity of PS films. It has beenreported that different gases, such as CO2 [405] and fluorocarbon gases[406,407], affected the surface activity of PS and PL films.

So far, no apparent serious limitations have been found with theCSD. This device eliminates both the problems of film leakage, as in

Fig. 12. Schematic of a constrained sessile drop (CSD). The sessile drop is formed on apedestal that employs a sharp knife-edge (as shown in the inset) to confine the dropfrom spreading over, even at low surface tensions, thus preventing film leakage. Thesessile drop is connected to a surfactant reservoir under constant stirring. Dropformation and oscillation are performed by regulating outflow of the surfactant samplein the reservoir using a computer-controlled motor-driven syringe. Surface tension isdetermined by shape analysis (according to Eq. (8)). The entire setup is enclosed in anenvironmental control chamber.


the LWB and PBS, and of concentration restriction, as in the CBS.Compared to the CBS, the CSD is much simpler and easier to operate,and requires a much smaller amount of liquid sample (i.e., only a fewmicrolitres for each measurement). The γ, surface area and dropvolume can be automatically computed by ADSA with a simplifiedimage analysis scheme taking advantage of the high-contrast edge of asessile drop image (Fig. 10(c) and (d)).

The CSD is very suitable for measuring the low γ of PS films formedby adsorption from a surfactant subphase at physiologically relevantconcentrations. Preliminary tests showed a good agreement between

Table 3Comparison of four in vitro methods for assessing the surface activity of pulmonary surfact

Methods Advantages Disadvantages

LWB • Precisely-controlled molecular areas for mixtures ofphospholipids

• Not readily adaptab

• Easy assembly with different surface analysis techniques • Difficult to control

• Commercially available (e.g., Kibron Inc., Espoo, Finland;KSV Instruments Inc., Helsinki Finland; Nima TechnologyLtd, Coventry, UK)

• Not capable of dire

• γ measured from a• Film leakage at low

PBS • Highly efficient in both assay time and sampleconsumption

• Commercial producactivity

• Direct simulation of breathing • Film leakage at low

• Commercially available (General Transco, Inc., Largo, FL) • γ measured at low

CBS • Close simulation of alveolar environment • Difficult to operate• Leakage-proof capacity • γ and surface area

• Capable of studying both adsorbed and spread films • Limitation on the mstudying adsorbed fi

• Accurate and automatic γ measurement in conjunctionwith ADSA

• Uncontrolled humid

• Commercially available (Department of Physiology andBiophysics, University of Calgary)

CSD • Precisely-controlled experimental environment • γ and surface area

• Little sample consumption (in μL range) • Not yet commercia

• Leakage-proof capacity

• Capable of studying both adsorbed and spread films• Accurate and automatic γ measurement in conjunctionwith ADSA• Easy to operate and clean• No limitation in surfactant concentration

LWB: Langmuir–Wilhelmy balance; PBS: pulsating bubble surfactometer; CBS: captive bubbleγ: surface tension.

CSD and CBS measurements [404]. The CSD has been applied to avariety of studies, such as the effect of humidity on film formation andcycling [375,402], and the development of polymeric additives forclinical surfactants [283,289].

4.3.5. Other in vitro methodsIn addition to the above four techniques, other in vitromethods are

available for assessing the surface activity of surfactants. For instance,the surface activity can be examined by studyingmicrobubble stability[408–410]. Enhorning and Holm developed a capillary surfactometer,which is especially suitable for examining the role of surfactant inmaintaining airway patency [411]. This method has proven especiallyuseful for research involving asthma. Meier et al. [412] developed anoscillating drop surfactometer in which a 1 μL pendant drop isoscillated in resonance with an exciter. This method simultaneouslymeasures γ and energy dissipation at the surface, thus providing anapproach to evaluate surface viscosity. The γ is calculated from theexperimentally determined oscillation period by considering theRayleigh instability. However, this method is restricted to γmin of∼15 mN/m, at which point the drop is released. Bertocchi et al. [413]developed an inverted interface technique in which γ is estimatedfrom the curvature of a submicron-sized pendant drop. Combinedwith fluorescence microscopy, this method permits the study ofadsorption of single surfactant aggregates. However, this method doesnot allow compression and expansion of the surfactant films.

Neumann et al. [414] have developed a pendant drop (PD)tensiometry system, which has been used in conjunction with ADSA

ant

Main applications

le for studying adsorbed films • Study of surface rheological propertiesof surfactant monolayers

the environment • Study of phospholipid phase behaviorsin conjunction with microscopic andspectrometric methods

ct simulation of breathing

Wilhelmy plate may not be very accurateγ

t lacks flexibility in assessing surface • Quality control of clinical samples

γ • Rapid comparison of the surface activityof many different samples

values are inaccurate

and clean • Study of surfactant mechanismsare correlated • Comprehensive and accurate evaluation

of surfactant propertiesax. surfactant concentration whenlms

• Miniaturized Langmuir balance forstudying spread monolayers

ity when studying adsorbed films • Study of gas transfer properties ofsurfactant films

are correlated • Study of high surfactant concentrationsat very low γ

lly available • Study of environmental effects (humidity,gas composition, etc.) on surfactant• Miniaturized Langmuir balance forstudying spread monolayers

surfactometer; CSD: constrained sessile drop; ADSA: axisymmetric drop shape analysis;


for measuring surface activity of PS [288,402,415–417]. The PDmethod features advantages of simplicity and high accuracy. However,it suffers from film leakage when γ of the surfactant films is decreasedto ∼18 mN/m [385]. (See Fig. 10(e) and (f) for PD images at different γ.Film leakage is shown in Fig. 10(f).) The PD is well suited for the studyof surfactant adsorption as the limitation of film leakage is relativelyminor in such a case. As mentioned in Section 2, the γeq of PS films isapproximately 22–25 mN/m, which is well above the threshold valueat which leakage may occur. Inspired by the CSD, a new PD method isunder development in which the traditional capillary is replaced by apedestal with a sharp knife-edge to prevent film leakage. The pedestalis similar to that used in the CSD but upside-down. Cabrerizo-Vilchezet al. have further developed the ADSA-based PD technique byintroducing a novel coaxial capillary design for rapid subphaseexchange in a pendant drop [418]. With this development, the PDcan be used as a fully functional miniaturized alternative to thetraditional Langmuir balance for the study of insoluble PL films.

In summary of Section 4, different in vivo, in situ, and in vitromethods have been developed for the evaluation of biophysicalproperties of PS. The in vitro methods outperform the others in termsof convenience of operation and efficiency in collecting data. Amongthe in vitro methods available, four tensiometry techniques, LWB, PBS,CBS and CSD have exhibited full capacity to simulate and measure allthree important biophysical properties of PS, i.e., rapid adsorption, lowγmin upon film compression, and limited γmax upon film expansion.The relative merits and disadvantages of these four methods aresummarized in Table 3. Selection of a specific method depends on themeasurements of interest and the desired accuracy.

5. Concluding remarks

After more than a half century of research, PS proves itself toremain a “super extraordinary juice” [42] full of surprises. The mostattractive motivation for studying PS is perhaps its clinical applica-tions. Though there is still room for improvement, such as the ongoingdevelopment of fully synthesized designer surfactants, surfactanttherapy has been very successful in treating premature infants withRDS. However, the applicability to ARDS, and other pulmonarydiseases such as asthma, has not clearly been established. Inactivationof PS undoubtedly contributes to its unsatisfactory performance inARDS. In vitro biophysical studies led to two distinct inhibitionmechanisms: inhibition due to competitive adsorption of plasmaproteins, and inhibition due to mixing and fluidizing an otherwisestable PS film by lipids. This simple classification implies that theformer mechanism mainly influences surfactant adsorption while thelatter prevents the films from reaching low γ. Recent studies usingdirect film imaging, however, have provided new evidence that theactual inhibition mechanisms due to both proteins and lipids, such ascholesterol, could be much more complicated. In addition, the exactrelevance of these inhibitionmechanisms to ARDS is still undefined. InARDS, the endogenous and exogenous surfactants are likely to beinactivated by multiple mechanisms. The predominant mechanismsby which surfactant is inhibited in vivo are still unestablished.

We would suggest that overcoming surfactant inhibition plays acentral role in resolving the clinical constraints on surfactant therapyin ARDS. The use of polymers as surfactant additives is an intriguingapproach due to its potent preclinical performance in resistinginhibition and the simplicity in formulation, i.e., by simply mixingwater-soluble polymers with currently available surfactant prepara-tions. However, it should be noted that the selection of a polymericadditive and the determination of its optimal concentration requireconsideration of physiological factors and not just surface activity.Selection of an appropriate evaluation model representing clinicalpractice, examination of the pulmonary fluid balance and theinterfacial gas transfer properties, plus the ease of tracheal instillationall need to be kept in mind for the preclinical tests. Some of these

concerns can beminimized by using ionic polymers, which seem to beeffective at much lower concentrations than nonionic polymers.However, introducing ionic polymers may disturb the electrostaticbalance in the PS system, which must be carefully considered.

Since Clements conducted the first direct γ measurements of PSusing his home-made LWB half a century ago, many more in vitrotensiometric techniques have been developed. These methods havetheir own relative merits and disadvantages. Selection of a particularmethod depends on the application and the desired accuracy. Duringthe last decade, in combination with the LWB, more and more surfaceanalysis techniques have been applied to studies of PS. Application ofthese techniques opens new horizons to the study of PS. These toolsenable direct film imaging on domain formation, topography,molecular orientation, electrical surface potential, film thickness,and even localized chemical compositions. Combining γ measure-ments with information obtained from direct film imaging hasprovided unprecedented insight into the behavior of PS, such as thefinding that pressure-driven phase transition/separation occurs insurfactant monolayers, and that the formation of multilayer structuresfrom monolayers occurs at high π.

Progress has also beenmade in combining film imaging techniqueswith γ measurements using drop shape methods. It has provenpossible to directly apply fluorescence microscopy [419] and evenscanning force microscopy [420] at the air–water interface of a bubblewith an adjustable size. Although limitations still prevent thesemethods from acting as a fully functional miniaturized Langmuirbalance with in situ film imaging capacity, it is not unreasonable toforesee that such amini-balance-imaging assembly may be developedsoon. The CSD could provide a promising drop configuration in thisdirection as the air–water interface in the CSD is readily accessible tomicroscopic facilities. With the aid of new techniques more insightwould be gained towards the biophysical understanding of PS andeventually for the development of new inhibition-resistant surfactantformulations for ARDS treatment.

Acknowledgements

We gratefully thank Drs. Cristina Casals, Jesus Perez-Gil, MatthiasAmrein and Stephen Hall for helpful discussions. This work wassupported by Operating Grants (MOP-64406 (NOP), FRN 15462 (FP),MOP-42556 (RAWV) and MOP-38037 (AWN)) from the Canadian Insti-tutes of Health Research (CIHR). YYZ is grateful for the PostdoctoralFellowships (PDF-328777-2006) from the Natural Sciences and Engi-neering Research Council of Canada (NSERC).

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