Surfactant liquid and black foam film formation and stability in vitro and correlative conditions in vivo

COLLOIDS AND SURFACES ELSEVIER

B

Colloids and Surfaces B: Biointerfaces 8 (1997) 133 145

Surfactant liquid and black foam film formation and stability in vitro and correlative conditions in vivo Emile M. Scarpelli a,, Alan J. Mautone

b, Zdravko

Lalchev c, Dotchi Exerowa d

a Perinatology Center, Cornell University Medical College, New York, NY, USA b Departments of Anesthesiology, Pediatrics and Physiology, UMD-New Jersey Medical School, Newark, N J, USA ° Department of Biochemistry, University of Sqfia, 1421 Sofia, Bulgaria Institute of Physical Chemistry, Bulgarian Academy of Sciences, i113 Sofia, Bulgaria Received 22 April 1996; accepted 2 August 1996

Abstract We studied foam film formation by three preparations that are used as surfactant replacement therapy by injection into the lungs of neonatal infants with surfactant insufficiency ("respiratory distress syndrome"). The preparations contain either all putative hydrophobic components of the normal surfactant system (prepared from bovine lung lavage, Infasurf (IN); prepared from minced bovine lung tissue, Survanta (SU)) or a single component of the system, dipalmitoylphosphatidylcholine (Exosurf Neonatal (EX)). Foam film formation is relevant directly to normal surfactant structure and function in vivo in the neonatal period (Pediatr. Res. 12 (1978) 1070-1076). We assessed morphology and stability of liquid to black film formation and its dependence on substrate concentration C, adsorption time, temperature T, "capillary pressure" Pc, film drainage time ~o-1 and black film formation times ~1 2. We found that (1) in contrast with the other preparations, IN regularly formed stable black films at the lowest C ("threshold C" Ct) under all conditions of T (22 and 37°C) and Pc (0.3, 0.4, 1.2 and 2.4 x 103 dyn cm 2), (2) EX also formed black films but differed from IN in that Ct was higher, film integrity required a longer adsorption time and black film formation was blocked (instability) at Pc values of 1.2 and 2.4 x 103 dyn c m - 2 and (3) SU did not form black films but instead formed slowly (long r0-1 and ~1-2), at relatively high Ct, viscosity-dependent inhomogeneous atypical ("rheological") films containing aggregates of material in the preparation. Extrapolation to in-vivo conditions of C, T and Pc indicates that both IN and SU (but not EX) may form stable foam films in situ, that film formation by IN is the more efficient process and that stable surfactant foam films, formed naturally in vivo, are consistent with both the in-vitro characteristics described here and correlative lung function in vivo. © 1997 Elsevier Science B.V.

Keywords: Surfactant black films; Surfactant liquid films; Surfactant rheological films; Foam film formation; Surfactant in situ; Surfactant therapy

1. Introduction The microscopic foam film m e t h o d for formation of black films has been developed by Exerowa et al. for the study of film m o r p h o l o g y and biophysics in general (see for example [1,2]) and lung * Corresponding author: EMS: 133 Constitution Drive, Orangeburg, NY 10962, USA. 0927-7765/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PI1 S0927-7765 (96)01315-X

surfactant films in particular [-3 5]. It is especially useful in the latter instance because it fits the studies that have identified the ubiquitous presence of intraalveolar bubble and foam film formations as a normal condition in normally hydrated and laboratory-dried lungs both at the onset of breathing at birth [ 6 ] and during normal breathing thereafter [ 7 - 1 2 ] . The foam film m e t h o d offers a n u m b e r of advan-

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tages [4,5,13] over conventional in-vitro methods such as the surface balance [ 14] and the surfactometer [15], in which pulmonary surfactant films are evaluated as open monolayers, and the more recent "captive bubble" method [16], in which a monolayer or multilayer film is formed at the airliquid interface of a gas pocket in a liquid-filled chamber. Advantages of the foam film method include high sensitivity; evaluation of film dynamics such as drainage time and "capillary pressure" Pc, direct visualization of surface film architecture, and both short-term and long-term analysis of molecular interactions that bear on film orientation and stability [17-20]. From the perspective of this report, the principal advantage is that the surfactant foam film has an alveolar analogue in vivo [6,7,21]. The relevance of surfactant foam films in vitro to normal intraalveolar and intraductal bubbles and bubble films in vivo is clear: ( 1) Foam films in vitro are essentially phospholipid (PL) in equilibrium with both the gas phase and the bulk liquid phase [3-5] as are surfactant films in vivo [6-10,12]. (2) Studies of human amniotic fluid, which contains pulmonary surfactant, by Exerowa and Lalchev [3] and Exerowa et al. [4,5] showed a direct correlation between the rapid formation of stable black films and the concentration of phosphatidylcholine (PC) in amniotic fluid. The probability of black film formation rested on the threshold concentration Ct, and failure to obtain stable black films indicated, correctly, high risk to respiratory distress syndrome at birth. These observations are consistent with the discoveries that normal pulmonary function at birth is established by intraalveolar foam film formation, in which pulmonary surfactants, primarily PC, are the essential components of the films [6], that surfactant deficiency inhibits the process, thereby preventing normal adaptation and survival at birth [6,8,9] and that therapeutic administration of surfactants to deficient lungs restores foam film formation and normal pulmonary function [8,9]. The proposed alveolar surface network [7,21,12] is formed at birth and throughout life by contiguous intraalveolar and intraductal surfactant bubble (foam) films which function to establish alveolar stability,

prevent alveolar or ductal collapse and modulate alveolar surface liquid balance. (3) Surfactant foam film thickness may vary from less than 500 nm ("liquid films") to less than 10-30 nm ("black films") depending on the volume and state of the film's aqueous phase [5]. These dimensions are consistent with those of alveolar surface films as indicated by electron microscopy [22], as calculated from studies of alveolar surface liquid volume [23] and as estimated from analysis of normal foam film formation in vivo [12]. Film stability is the fundamental requirement for normal function of pulmonary alveolar surfactants in vivo, albeit molecular configuration has been debated (see [7] for review). The hypothesized continuous monolayer of surfactant extending from alveolus to alveolus as an open film has not received morphological confirmation [22] and has been shown to be operationally unstable under in-vitro conditions that simulate normal breathing movements [24,25]. In contrast, visible alveolar surfactant bubbles are formed naturally and spontaneously in vivo at all ages [6-10,12]. When freed from the alveolar space into an aqueous medium, bubble film, i.e. foam film, integrity is maintained and bubble volume is virtually constant for hours. These characteristics are in agreement with the criteria of Pattle [26,27], who concluded from his studies (which mark the discovery of surfactant) that bubbles formed from lung surfactant are stable and reduce surface tension 7 to near 0 dyn cm 1. However, surfactant bubbles had been considered laboratory artifacts produced by experimental manipulation until the discovery of their natural intraalveolar position in vivo [6,7] and their role in establishing alveolar stability throughout life [8-10,12]. In the present study, the foam film properties of three surfactant preparations, Exosurf Neonatal (EX), Infasurf (IN) and Survanta (SU), are evaluated. These preparations were chosen as representative of pulmonary surfactants in current use as replacement therapy by intratracheal instillation for neonatal respiratory distress syndrome [28], a condition of surfactant insufficiency. Our study seeks to define the architecture, concentration dependence and dynamic properties of films

E.M. Scarpelli et al. / Colloids Surfaces B: Biointerfaces 8 (1997) 133-145

formed by each preparation and their relevance to film formation, stability and function in vivo.

2. Materials and methods

2.1. Surfactant preparations The three substrates were commercially available: EX from Burroughs Welcome Co., Research Triangle Park, NC, SU from Ross Laboratories, Columbus OH; and IN from ONY, Inc., Buffalo, NY. The following compositions were obtained from the suppliers. IN is a suspension containing 90-94% PL, of which 79% is PC, 6% is phosphatidylglycerol (PG), 15% other lipids and about 1% protein. The PC fraction is 80% dipalmitoylphosphatidylcholine (DPPC). The preparation is obtained from hydrophobic extracts of lavage liquid from neonatal calf lungs. The extract is dried, sterilized, dispersed in normal saline and packaged in vials to be administered by intratracheal injection. The concentration ("clinical concentration") of PL is 35 mg ml- 1 and of DPPC is about 22.1 mg m1-1. EX is a synthetic preparation supplied as a sterile lyophilized powder. When reconstituted with sterile water for intratracheal injection, the resulting suspension contains 13.5 mg DPPC ml- 1 (the "clinical concentration"), 1.5 mg ml- 1 of hexadecanol (cetyl alcohol) and 1.0 mg ml- 1 of formaldehyde polymer with oxirane and tetramethylbutylphenol Ctyloxapol"), in 0.1 N NaC1. We tested EX within 1-8 h of reconstitution. The reconstituted material is stable for 12 h. SU is the hydrophobic extract from saline extracts of minced bovine lungs. It contains 88% PL, of which about 50% is disaturated PC (DSPC), i.e. DPPC and other disaturated PC species. It also contains about 1.1% protein. Lipids are extracted after differential centrifugation and acetone precipitation. Synthetic DPPC and PG are added to the preparation. The material is dried, sterilized, dispersed in normal saline and packaged in vials as a suspension for intratracheal injection. The concentration ("clinical concentration") of PL is 25 mg ml -~ and of DSPC is about 12.5 mg ml -~.

135

2.2. Foam.film system Formation and assessment of foam films have been described [2,29-32]. The apparatus used in the present study, shown schematically in Fig. 1, is contained in a thermoregulated water jacket. Briefly, individual cylinders were loaded by immersion in the test preparation. Upon withdrawal, the sample within the cylinder was viewed through an inverted microscope with eyepiece graticule. We have found that film formation and film characteristics are the same when samples are loaded in cylinders of inside diameter 0.4, 0.3, 0.1 and 0.05 cm [-33]. For simplicity, we now describe experiments with a cylinder of inside diameter 0.4cm. The cylinder was connected by a side-arm glass capillary to a mercury precision pump, manually controlled for adjustment of bulk liquid at either 22 or 37°C. Initially, the sample formed a biconcave drop with two surfactant monolayers and a bulk

!

E

Fig. 1. Schematic diagram of apparatus used to assess liquid and black film formation. Surfactant preparation in the form of a biconcave drop A in the glass cylinder B after emersion in and withdrawal from the surfactant bulk liquid solution C. Optically flat glass D through which the film is viewed from an inverted microscope. Glass capillary E containing the surfactant preparation with attachment for regulation of liquid volume of the film by adjusting the mercury precision pump F. Temperature control is established by a thermoregulated water jacket surrounding the apparatus. See text and Fig. 2.

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liquid layer between them, as shown in Fig. 2(a). The volume of the drop was about 0.1 ml and distance between the two air-liquid interfaces was about 0.3 cm. Foam film formation was controlled by withdrawal of bulk liquid with the precision pump. Initial contact between the two surfaces occurred when they were aligned as shown in Fig. 2(b). The parallel surfaces are suspended and held by the menisci. This is the liquid film configuration (about 500 nm thickness upon formation); its surface appearance by microscopy is that of a colored disc with concentric (newtonian) rings. The size of the film was set to a constant diameter by adjusting the bulk liquid volume with the precision pump. The liquid film then thinned spontaneously (a function of film drainage) and black spots appeared on the surface in places where film thickness decreased to about 30 nm, the "critical film thickness" (Fig. 2(c)). If the film did not rupture, the black spots fused and expanded to encompass the entire film area; this is the so-called "transition to black foam film" (Fig. 2(e)). The bulk phase of the black foam film is free liquid, the c o m m o n black film, or water hydrated to molecules of the surface monolayers, the newtonian black film (Fig. 2(d)). The two types of black foam film have been reviewed (see for example [2,29,31,34,35].

A

B

.:~!t.:,'."; ~ ' ~

C

D

2.3. Indices of film stability Foam films have been studied in relation to their thermodynamic and kinetic (including hydrodynamic) stability [2,29,35,36]. For the present study, we assessed the following parameters. (1) Thefilm drainage time ro-a is the time from initial formation of the liquid film (Fig. 2(b)) to the first appearance of a black spot (Fig. 2(c)). This transformation occurs spontaneously as bulk liquid drains toward peripheral menisci and film thickness decreases from about 500 nm (liquid film) to about 30 nm (black spot) [32,36,37]. The drainage time r o 1 and "capillary pressure" Pc are inversely related. The "capillary pressure" in dynes per square centimetre (roughly equivalent to the pressure in centimetres of H 2 0 x 10 -3) is the pressure difference between the bulk liquid phase at the menisci and the air phase, i.e. the pressure difference across each monolayer. We calculated Pc

E

Fig. 2. Schematicdiagrams of typical configuration of the foam film during its evolution: (a) Biconcave drop with two phospholipid monolayers and macroscopicliquid layer between them. (b) By reducing the liquid volume, the central surfaces become ordered in parallel, thus forming a liquid foam film. (c) Foam film following drainage to formation of a "black spot". (d) Cross-section of foam film, either a black spot as in (c) or a black film as in (e) (scale increased), presented as apposed monolayers of phospholipid surfactants (~). Common black films (CBFs) and newtonian black films (NBFs) are much thinner than liquid films; the CBFs have a free liquid layer between two monolayers, while NBFs have no free liquid between the monolayers (see text). (e) Expansion and coalescence of black spots leads to the formation of a black film.

E.M. Scarpelli et al. / Colloids Surfaces B: Biointerfaces 8 (1997) 133 145

from the Laplace equation Pc = 2y/r; where r is the radius of curvature of the surface and y is the surface tension at the air-liquid interface. The radius was determined from the diameter of the cylinder and ~ was measured by both the surface balance and the capillary rise methods. We found that y for each preparation was 30 + 0.3 dyn cm 1 under equilibrium conditions and used 30 dyn c m - ~ to calculate pc. (2) The time rl 2 for formation of the black film was measured from initial appearance of a black spot to its expansion and coalescence with other spots to form a continuous black film of definitive radius (Fig. 2(e)). The formation and stability of black spots with respect to black film formation has been evaluated for various types of surfactants [2,29,35]. The total film formation time %-2, from the phenomenologic perspective, is the sum of two dynamic events, film drainage and black spot to black film formation.

2.4. Additional fundamental parameters Characterization of the film includes assessment of time available for adsorption of surfactants from the bulk liquid phase to the interface and requisite concentrations of substrate material. Black spot formation could be identified to the lower limits of microscopic resolution, i.e. 1-10 ~tm. The black film area in our model' ranged from about 3 x 104 to 50 x 10 4 gm 2 at black film radii from 100 to 400 gm. The film thickness is a function of surface molecular interactions, i.e. the disjoining pressure in the film [17 20,30]. The adsorption time is the time in the biconcave drop configuration (Fig. 2(a)) during which molecular adsorption takes place from bulk liquid to both air-liquid interfaces. It is set by the experimenter. The threshold concentration Ct is the minimum concentration of surfactant in micrograms of PL per millilitre or micrograms of DSPC per millilitre at which black films always form [ 1 ]. For IN and EX, DSPC is the same as DPPC, since D P P C is the only disaturated species reported for IN and the only PL in EX. For SU, DSPC was reported

137

as such. The probability Wf of black film formation was determined as Wf = AN/N, where N is the total number of trials and AN is the number of trials in which black films were formed. The limits are Wf= 1 (films formed in each trial) and Wf=0 (no film formation). Assessment of the probability W~ of black spot formation was handled in an analogous manner. At Ct, therefore, Wf=l and W~=I.

2.5. Experimental protocols We first determined Wf and W~of clinical concentrations of each preparation. Each preparation wz then diluted serially with normal saline solution in order to establish Ct. Trials were done at both 22 and 37°C with Pc=0.3 x 103 d y n c m and an adsorption time of 5 min. We also studied each preparation (22°C) at different concentrations below Ct to assess the effect, if any, of time available for adsorption on film formation; Pc was 0.3 x 103 dyn cm 2. To assess the effect of Pc, each preparation was studied at both the clinical concentration and at a concentration just above Ct [29] at each of three Pc values: 0 . 4 x 103 , 1.2 x 103 and 2.4 x 103 dyn cm 2. Trials were conducted at both 22 and 37°C; the adsorption time was 5 min in each case. All films were monitored and photographed through the inverted microscope, and times were recorded for each phase of film formation. Ten trials were conducted for each determination.

3. Results

3.1. Clinical and threshold concentrations The most abundant PL of the lung surfactant system is DSPC, principally the D P P C species, which is believed to be the essential determinant of surfactant function in vivo [7]. While D P P C is the only PL in EX, both IN and SU contain other PLs and small quantities of hydrophobic surfactant-associated proteins that may add to the desired functional properties of the material in situ.

E.M. Scarpelli et al. / Colloids Surfaces B: Biointerfaces 8 (1997) 133 145

138

The clinical concentration in milligrams per millilitre, and Ct and just above Ct in micrograms per millilitre are given for total PL and for DSPC of each preparation in Table 1. These data show that the clinical concentration of DSPC is highest in IN, about 1.7 times higher than in EX and SU, but that the Ct of DSPC in both EX and SU is about 1.8 times higher than in IN. When the total PL is compared, the Ct for SU is more than twice that for IN. Temperature had little effect on Ct, which tended to be higher at 37°C, and no effect on relative Ct values. At each temperature, therefore, black film formation by IN requires less of the material than does black film formation by EX and atypical film formation by SU (see following sections).

IN

,

EX

.

$U

A

B

C

3.2. Black film formation Photographs of the surface of films from clinical concentrations of IN, EX and SU are shown in Fig. 3 (22°C; Pc=0.3 × 103 dyn cm-2). The stages of black film formation are evident for IN and EX, namely liquid film (line A in Fig. 3, comparable with Fig. 2(b)); black spot formation and growth (line B in Fig. 3, comparable with Fig. 2(c)), and black film formation (line C in Fig. 3, comparable with Fig. 2(e)). In general, IN and EX films appeared homogeneous in thickness; particles and aggregates were absent. Occasionally, white spots ("iceberg complexes") were seen in 1N black films, probably representing lipid-protein complexes

Fig. 3. Stages of foam film formation by IN, EX and SU. For IN and EX columns, line A shows liquid films comparable with Fig. 2(b); line B shows black spot formation and growth comparable with Fig. 2(c); line C shows black film formation comparable with Fig. 2(e). The SU column shows the stages of atypical slow film formation by this preparation; homogeneous black films never formed (see text).

and/or surface separation of components of the monolayer akin to "island formation" [29]. Once formed, black films persisted for hours.

Table 1

Preparation

Clinical concentration

Threshold concentration (Ct) 22°C

IN EX SU

PL (mg/mL)

DSPC* (mg/mL)

35 ** 25

22.4 13.5 12.5

Concentration above Ct [>Ct]

37°C

PL (~g/mL)

DSPC* (~g/mL)

PL (~g/mL)

DSPC* (~g/mL)

PL (~g/mL)

DSPC* (~g/mL)

190 ** 450

122 220 225

210 ** 490

134 250 245

220 ** 600

141 310 300

Clinical concentration, threshold concentration (Ct) and concentration above Ct [ > Ct] of Infasurf (IN), Exosurf Neonatal (EX), and Survanta (SU). PL, phospholipoids; DSPC*, disaturated phosphotidylcholine, namely, dipalmitoylphosphatidylcholine (DPPC) in IN and EX, and DPPC plus other disaturated PCs in SU; **for EX, PL = DSPC = DPPC.

K M.

Scarpelli et al. / Colloids Surfaces B: Biointerjhces 8 (1997) 133-145

In contrast, SU p r o d u c e d atypical transformations very slowly. Black spots formed primarily at the periphery (line A in Fig. 3); their growth was very slow, up to hours (line B in Fig. 3); films contained m a n y particles and aggregates (line C in Fig. 3); the film thickness was heterogeneous. When films of IN, EX and SU were formed at Ct and just above Ct, the transformations of film architecture were analogous to those described.

3.3. Foam film stability T w o p a r a m e t e r s of film stability, namely the film integrity during drainage and black film formation, are presented as functions of time and Pc in Figs. 4-6, in which I N and EX are c o m p a r e d at two temperatures at each of two concentrations: clinical concentration and just above Ct. The film drainage time to-1 (Fig. 4) was shortened, i.e. the drainage rate increased, as Pc increased. The largest change occurred from 0 . 4 x 103 to 1.2x 1 0 3 d y n c m -2 in all cases. Temperature had relatively little effect on EX film drainage at both concentrations. T e m p e r a t u r e

500

400 ~- [7

Clinical Concentration

80

[> Ct]

effect on I N films was small at the two higher Pc values; the drainage time at the lowest Pc was eight times higher at 22°C than at 37°C (clinical concentration). The black film formation time zi 2 (Fig. 5) was shortened, i.e. the formation time decreased, as Pc increased. For IN, the largest change occurred from 0.4 x 1 0 3 to 1.2 x 1 0 3 dyn c m - 2 at clinical concentr~ition. A striking feature was the failure of EX just above Ct to form black films, and hence to maintain film integrity, at the two higher Pc at both 22 and 37°C. In contrast, I N produced black films under all conditions. Film formation times were shorter at 37°C than at 22°C, except for EX at the lowest Pc just above Ct. The largest difference was recorded for I N at clinical concentration and lowest Pc where formation time was 3.5 times longer at 22cC than at 37°C. The total film formation time %-2 is shown in Fig. 6. Several features of spontaneous film transformation from liquid to black film are put into perspective: ( 1 ) the effect of temperature was greatest for clinical concentration of I N at the lowest Pc; (2) the shortest formation times were recorded

200 ~ [-7 80,

~, I

Clinical Concentration

[> Ct]

I

122°C 37°C

%"

139

122°C 37°C

60

60 E

[..,

E

40

40

20 20

"<--IN--> <-EX-->

~---IN--'-> <-'-EX --~

"~--IN--> <--EX--->

Fig. 4. Film drainage times 1-o-1for IN and EX at "capillary pressures" of 0.4 x 103, 1.2 x 103 and 2.4 x 103 dyn cm -2 (left to right between arrows) at 22°C (open bars) and 37°C (shaded bars). Clinical concentration and above-threshold concentration, I-> Ct] for each preparation are given in Table 1. See text for discussion.

~--IN--~_ <-EX--->

Fig. 5. Black film formation times zl 2 for IN and EX at "capillary pressures" of 0.4 x 103, 1.2 x 103 and 2.4 x 103 dyncm 2 (left to right between arrows) at 22°C (open bars) and 37°C (shaded bars). Clinical concentration and above4hreshold concentration, [> Ct] for each preparation are given in Table 1. **. EX failed to form black films. See text for discussion.

E.M. Scarpelli et al. / Colloids Surfaces B." Biointerfaces 8 (1997) 133-145

140 650 ~-

Clinical Concentration

160 7,

[ > Ct]

IN SU

n

'

1'

EX

q~-~.

1.0

, :

,

l

,

A

#l o.s ° ,

-dd

o

loo

'

1.0 -

///

80 o.5

8

///

60 ~

40

!

IN SUEX

!

t

?

T

IN SUEX

1.o

<--IN----> <--EN-->

<-,-IN---><--EX--->

Fig. 6. Total film formation times to : for IN and EX at "capillary pressures" of 0.4 × 10 3, 1.2 x 103 and 2.4 x 103 dyncm -z (left to right between arrows) at 22°C (open bars) and 37°C (shaded bars). Clinical concentration and above-threshold concentration, [> Ct] for each preparation are given in Table 1. **, EX failed to form black films. See text for discussion.

at 37°C for IN at clinical concentration, 10 s, and just above Ct, 17 s; (3) for EX just above Ct, the liquid film drainage rate increased and black film formation ceased as Pc was increased above 0.4 x 103 dyn cm -2. SU produced the atypical films described above ("black film formation") (Fig. 3). The shortest formation times, 150 s and 85 s, were rec.orded at Pc values of 1.2 x 103 dyn cm -2 and 2.4x 1 0 3 d y n c m -2 respectively, at 37°C just above Ct. The formation time was greater than 1000 s under all other conditions. No black films were formed. The many particles and aggregates apparent in SU films (Fig. 3) suggest its classification as a "rheological film" comparable with certain protein films reported by Platikanov et al. [38] and Clark et al. [39]. Predictably, the viscosity of these substrates is high. We have determined in a series of studies not reported here that the drainage times of IN, EX and SU are directly proportional to the bulk viscosities; the bulk viscosity in SU is significantly higher than in either IN or EX.

o.s

:::

/// / / ...' ~ L T

lo

t

t

IN

EX

I

~

I

2.0

:30

40

C t

50

.6o

t, min

Fig. 7. Dependence of probability W~for black spot formation (22°C) on adsorption time at PL concentrations of (a) 65 p.g ml-x, (b) 130 ~tgml-1 and (c) 170 ~tgml -l. Black spot formation (W~=I) by IN and SU required about 10min at each concentration. EX required adsorption times of about 40 rain at the lowest concentration (a) and about 12 min at higher concentrations. Black films were formed only by IN and EX at the highest concentration below Ct when adsorption times were increased to longer than 30 min for IN and longer than 40 min for EX (arrows in (c)). Films of SU alwaysruptured.

3.4. Effect of adsorption time on film formation Whether or not adsorption from bulk phase to interface was a limiting factor in our study was assessed by extending adsorption time to 60 min at three concentrations below Ct. Fig. 7 shows the probability W~ of black spot formation plotted against adsorption time as the concentration was increased towards Ct. Black spot formation (W~= 1) by I N and SU required adsorption times up to about 10min at each concentration, while EX required about 40 min at the lowest concentration and about 12 min at the higher concentrations. Black films were formed ( W f = l ) by I N and EX only at the highest concentration below Ct, when the adsorption time had been increased to greater than 30 min for I N and greater than 40 min for

E.M. Scarpelli et al. / Colloids Surfaces B. Biointerfaces 8 (1997) 133 145

EX (arrows in Fig. 7(c)). SU films always ruptured (Wf=0) during the time of observation.

4. Discussion

4.1. Foam film formation in vitro Both IN and EX formed black foam films. There were, however, notable differences between the two preparations. (1) The Ct of EX was higher than that of IN, i.e. a substantially greater amount of DPPC was required to form stable black films with EX. (2) The stability of the liquid films of EX required longer adsorption times than did IN, as shown by the studies below Ct. (3) Black film formation by EX was blocked just above Ct by increasing Pc to 1.2 x 103 and 2.4x 103 dyncm -2, whereas formation by IN was not. These undesirable properties of EX may be related to its overall formulation. First, EX lacks the hydrophobic components of the surfactant system that are found in IN. For example, both PG and surfactant-associated proteins may be required to facilitate adsorption, spreading and stabilization of DPPC at the air-liquid interface I-14]. The effect of PG to enhance molecular lateral diffusion at the interface has been demonstrated 1-39-42]. Second, inability of DPPC in EX to form stable films at relatively high Pc values was due to film rupture upon formation of black spots, i.e. when the critical film thickness is approached (Fig. 2). This suggests that the relatively high flow rates of bulk liquid further impaired formation of a stable film by facilitated transport of DPPC away from or out of the interface. The combination of factors, namely relatively poor adsorption and spreading properties on the one hand and facilitated transport on the other hand, establish nonequilibrium conditions in which the film area increases, Vincreases and the film ruptures 1-32,43]. The non-biological additives in EX may be another factor to mitigate against stable film formation. There is no quantitative information about the effect of these additives on the surface-hypophase dynamics of components of the surfactant system.

141

IN produced stable black films under virtually all conditions of our study, including concentration (109 ~tg to 22.4 mg DSPC ml-1), temperature (22 and 37°C), and Pc (0.3 × 103-2.4 × 103 dyn cm-Z). Whereas the lower temperature was generally associated with slightly longer drainage and formation times, the effect on IN was marked only when the highest concentration was tested at low Pc. It is well known that each of the factors that influenced film drainage and formation [44] is affected by temperature. Our data permit only limited speculation with regard to IN films. For example, pure DPPC has a sharp phase transition at 41.5°C, while mixtures of DPPC With other lipidic components, as in IN, have lower and/or broader transition temperatures. It has been shown that complex surfactant fractions from rabbit [45] and cow [46] lavage liquid melt over a broad range of 20-39°C, i.e. including the temperature range of our study. Thus the expected influence of temperature on interfacial and bulk phase fluidity of mixtures such as IN is consistent with the facilitated drainage and film formation times observed at 37°C, particularly at the highest concentration (Fig. 6). In contrast, little temperature effect is expected for preparations in which DPPC with high transition temperature is the only PL (as in EX). Three additional factors are relevant to temperature effect on film formation by IN at high concentration and low Pc. First, the approximately 160-fold increase in concentration from just above Ct to clinical concentration is consistent per se with an associated increase in bulk liquid viscosity. Second, as Pc approaches zero, e.g. as Pc was lowered to 0.4 × 103 dyncm -2, the viscosity becomes the principal determinant of flow in any given system. Third, we have found that lowering temperature from 37 to 22 °C significantly increases the bulk viscosity of surfactant mixtures [47]. Therefore, under conditions of low temperature, low Pc and high concentration, the expected effects on viscosity and flow are consistent with the extended times required for black film formation by IN films (Figs. 4-6). Atypical films of SU contained particles and aggregates, had higher viscosity than both IN and EX and were slow moving with very long formation times. When contrasted with IN and EX,

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formation times of 85 and 150 s at 37°C, high Pc and low concentration, and longer than 1000 s at lower temperatures and Pc values and higher concentrations, reveal the exceptional dependence on viscosity and thus the "rheological" nature of SU films. These fluid-mechanical forces are principal determinants of film structure and stability of rheological films [38,39]. When compared with IN in other studies that tested bubble formation and stability under dynamic conditions, i.e. bubble generation by gas dispersion at a flow rate of 0.3 mlmin -1, we found that SU did not form stable bubbles, whereas IN did [13]. We also found that SU was significantly less efficient at forming stable bubbles following dilution with ethanol and vortex agitation. These findings further demonstrated the labile characteristics of viscositydependent rheological films.

4.2. Foam film formation in vivo In this section, we relate the findings in vitro to conditions in vivo. The correlation is of fundamental importance, given that normal pulmonary alveolar infrastructure is a function of intraalveolar bubble and foam film formation [6,7,9-12,21,47,48] and that the goal of therapy with EX, IN and SU is to restore this normal condition to deficient lungs [ 13,26-28,49-51].

4.2.1. Composition of surfactants Hydrophobic extracts are generally accepted as the fundamental operational comi~onents of the intraalveolar surfactant system [ 16]. The distributions of lipids in IN and SU are in agreement with the composition of surfactant reported by many investigators (see [52] for a review). Hydrophobic surfactant-associated proteins in IN and SU are considered natural components of the system that may enhance function at the alveolar level. EX, which contains a single biological surfactant, DPPC, permits comparison with the surfactant system. 4.2.2. Concentration Are the concentratiorrs at which foam films were formed relevant to conditions in vivo? Clinical concentrations exceed actual intraalveolar concen-

tration that might be expected during therapy, because the material is diluted in situ by liquid in the air spaces and at their surfaces. Estimation of dilution is difficult but may be approached roughly for administration prior to breathing at birth, when potential air spaces are filled with liquid, about 30mlkg -1 body weight [53]. If the surfactant, given at the recommended dose [28], is distributed evenly into this volume, the resultant concentration in milligrams of PL per millilitre of pulmonary liquid would be 3.3 (IN and SU) and 2.3 (EX), i.e. well above Ct. The concentration either could be higher when liquid volume is reduced, as expected after birth, or could be lower, if the surfactant is either distributed unevenly or deposited in air spaces proximal to terminal lung units. Other information gives some indication of surfactant concentration in normal lungs; the concentration in normal fetal pulmonary liquid [51] and concentration required to restore alveolar function to immature neonatal infants and lambs [49] range from about 0.5 to 1.8 mg PL m1-1. These concentrations are close to but higher than both Ct and just above Ct.

4.2.3. Adsorption Black films were formed from both IN and EX below Ct when the adsorption time was increased to longer than 30min and longer than 40min respectively. This reinforces the validity of our Ct determinations at the fixed adsorption time of 5 min. Whereas we did not test the lower limits of adsorption time for IN and EX, we would note the contrasting rapid adsorption times reported for surfactant monolayer on plane surfaces in vitro, measured in seconds [50,54] and for surfactant bubble film formation in situ, measured in milliseconds or less [7]. Adsorption was enhanced in the monolayer studies by agitation of the liquid which contained a large reservoir of surfactant substrate. Adsorption is facilitated further by several factors during bubble formation, including agitation of the liquid [43], rapid preferential incorporation into bubble films of the more highly surface-active PLs [6], rapid film closure or "pinch-off" [47] and spontaneous reduction in film area to increase surface concentration or packing [8,27]. It is therefore reasonable to expect very

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rapid adsorption in vivo, with consequent formation of liquid and black films determined by the surfactant composition, the concentration, Pc and the intrinsic stability of the film.

4.2.4. "Capillary pressure" Pc The driving force for bulk liquid flow in foam films in vivo and in vitro is essentially determined by the curvature and ~, at the film's Plateau borders [12,47]. Movement of liquid from the alveolar space, e.g. Plateau borders, to interstices is modulated by interstitial, lymphatic and vascular (pulmonary capillary) pressures (see review in [55]). Net drainage pressure in vivo, therefore, is analogous to Pc as measured in our study. Taylor and Parker [56] provide measurements of alveolar surface liquid and interstitial pressures which indicate that driving pressure at functional residual capacity is of the order of (1 2) x 103 dyncm -2, i.e. within the range of Pc of our study. Ordinarily, lymphatic and pulmonary capillary pressures (both hydrostatic and oncotic) modulate liquid exchange to maintain physiological liquid balance. We found that film formation rates generally increased as Pc (driving pressure) was increased, suggesting that accelerated formation might be expected in vivo during inflation when both the alveolar surface liquid and the interstitial (negative) pressures increase [56].

4.2.5. Black films The microscopic resolution that permits clear visualization of bubbles and bubble film contacts in fresh lung tissue [6,7,10,12] is quite insufficient for black films. Gil [22] has noted that films in general are rarely, if ever, seen in conventional light and electron micrograph studies of prepared tissue. The reason is that foam films are quickly disrupted by the tissue-preparative processes [-11]. However, when lungs are air dried so that stable films are protected and chemical dehydration and embedding of the tissue are omitted, intact films spanning the alveolar air spaces are visible by three-dimensional scanning electron microscopy [7]. The present study is consistent with the finding of film formation in vivo and reinforces the possibility of formation of black films. Black films, which are generally stable, can be

143

disrupted by extrinsic factors, such as agitation, particle impaction, physical stress and chemical insult (see for example [29,57]). We have shown [7,10,12] that, following disruption in vivo, bubble films may be re-formed through a number of mechanisms, including formation as alveolar mouths approach or achieve closure, as first suggested by Macklin [58], and during inflation, as air entry leads to formation by dispersion [10,12,48], the so-called "sparger" phenomenon [433. The functional significance of black films per se is conjectural. Recent studies show that bubble films are intact and stable during inflation of the lung from functional residual capacity to maximal volume and subsequent deflation to minimal volume [12]. Since film contacts are sustained throughout the volume history of the lung (albeit that the bubble size and film thickness vary with volume change), it is clear that the effect of intraalveolar bubbles on liquid drainage (above) and on structural integrity of the alveolus [-8,10,12] (see also Fig. 8 in [24]) is also sustained throughout the volume history. In addition, Exerowa and Lalchev [3] have suggested the presence of bilayer and multilayer formations with structures such as foam films in the alveolar hypophase. If present in vivo, determination of their function (e.g. hydrodynamic, film-stabilizing, metabolic exchange) requires additional study.

4.3. Comparison of surfactants Of the three preparations tested, IN best fits the structural and operational characteristics of normal intraalveolar bubbles as described above. IN formed stable films under all experimental conditions relevant to lung function in vivo. Films from IN are consistent with those from lung surfactant in human amniotic fluid [4,5] and in lung lavage from normal rabbits [ 13]. EX, which is an incomplete surfactant and Pc dependent for black film formation, may have a limited range of usefulness as a therapeutic agent. Films of SU are problematic because of the long time constants and inhomogeneity of the films. The issue as to whether or not the rheological

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films of SU can function as such in vivo requires exploration given the dynamic nature of intraalveolar bubble films as described above.

Acknowledgements This work was supported, in part, by PHS Grant HL-38303, and a Grant from the Eastern European Cooperative of the Fogarty Institute of the National Institutes of Health to A.J.M.

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