the effect of concentration on the bulk adsorption of bovine lipid extract surfactant

The effect of concentration on the bulk adsorption of bovinelipid extract surfactant

J.J. Lu a, L.M.Y. Yu a, W.W.Y. Cheung b, Z. Policova a, D. Li a, M.L. Hair a,A.W. Neumann a,*

a Department of Mechanical and Industrial Engineering, University of Toronto, 5 King’s College Road, Toronto, Ont., Canada M5S 3G8b Division of Engineering Science, Faculty of Applied Science and Engineering, University of Toronto, 170 College Street, Toronto, Ont.,

Canada M5S 3E3

Received 22 January 2002; accepted 27 September 2002

Abstract

The film adsorption of bovine lipid extract surfactant (BLES) onto the air�/liquid interface was examined using

axisymmetric drop shape analysis. In combination with a pendant drop constellation, BLES concentrations as high as

10 mg/ml were studied, i.e. concentrations far higher than those accessible with the captive bubble set-ups. ‘Adsorption

clicks’, i.e. dynamic processes in which the interfacial tension of surfactant films decreases quickly in a stepwise fashion,

were studied at concentrations below 1 mg/ml. Adsorption clicks with high magnitudes up to approximately 35 mJ/m2

(within 0.2 s) were observed. The rate of adsorption was investigated as a function of surfactant concentration. At

concentrations below 1 mg/ml, the rate of adsorption is highly concentration dependent. Surfactant films formed on 1

mg/ml BLES solutions reached a surface tension of about 25 mJ/m2 in approximately 10 s, while 0.1 mg/ml BLES

required more than 100 s to reach a similar value.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Bovine lipid extract surfactant; Lung surfactant; Adsorption

1. Introduction

Pulmonary surfactant is a generic name given to

the surface active agent mixture that exists in the

lungs and plays an important role in the mechanics

of respiration. The main physiological function of

pulmonary surfactant is to reduce the surface

tension at the air�/liquid interface in the alveoli

during respiration. By reducing the surface ten-

sion, the energy required to inflate the lungs

during breathing is also reduced. It is crucial to

maintain a low surface tension in the alveoli

during breathing in order to avoid lung collapse

* Corresponding author. Tel.: �/1-416-978-1270; fax: �/1-

416-978-7753

E-mail address: [email protected] (A.W.

Neumann).

Colloids and Surfaces B: Biointerfaces 29 (2003) 119�/130

www.elsevier.com/locate/colsurfb

0927-7765/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 9 2 7 - 7 7 6 5 ( 0 2 ) 0 0 1 6 1 - 3

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under extremely high compressions. These appro-priate conditions can be maintained by pulmonary

surfactant.

Pulmonary surfactant is synthesized and se-

creted from alveolar type II cells [1]. The total

lipid composition of the isolated type II cells

reflects a large amount of intracellular surfactant

in lamellar bodies, indicating that type II cells

contain a high percentage of dipalmitoyl phospha-tidylcholine (DPPC) and phosphatidylglycerol

(PG). Pulmonary surfactant is a complex mixture

of various chemical materials, consisting mostly of

lipids and proteins. A typical pulmonary surfac-

tant consists of approximately 10% protein and

90% lipid. DPPC, the main contributor to low

surface tension, accounts for 35�/40% of the lipid

in pulmonary surfactant [2]. From the work ofPossmayer et al. [3�/5], it is known that, in addition

to DPPC, several other types of phospholipids

such as PG, phosphatidylethanolamine and phos-

phatidylinositol are also present in pulmonary

surfactant. Cholesterol is also found in small

amounts [5]. There are at least four surfactant-

associated proteins in a typical pulmonary surfac-

tant, denoted as SP-A, SP-B, SP-C and SP-D.Due to a shortage of surfactant in the lungs,

patients may experience a respiratory disease

named respiratory distress syndrome [6]. Without

sufficient pulmonary surfactant in the lungs, sur-

face tension in the alveoli can be quite high, and

more energy is required for breathing. Premature

babies are often not able to produce sufficient

quantities of surfactant and can encounter neona-tal respiratory distress syndrome [7], which is one

of the major contributors to infant mortality in

industrialized countries. Adults may suffer from

an acute respiratory distress syndrome due to lung

injury [8].

The focus of this paper is on the rate of

surfactant adsorption onto air�/liquid interfaces

as a function of surfactant concentration. Thework of Schurch et al. [9] has shown that the

adsorption process is concentration dependent.

Adsorption of surfactant is faster at higher con-

centrations. Due to the limitation (i.e. the edges of

the bubble become blurred at higher surfactant

concentrations) of the captive bubble technique

used in Schurch’s studies, the highest concentra-

tion of surfactant suspensions investigated was 3mg/ml. However, the concentration of surfactant

in the lungs can be much higher than 3 mg/ml.

Therefore, it is important to conduct studies at

higher concentrations in order to understand the

mechanism of film formation under in vivo con-

ditions. The pendant drop constellation described

in this paper was used to study high surfactant

concentrations. In the captive bubble set-up, theair bubble is surrounded by a surfactant solution,

which becomes opaque at high concentrations,

making observation and measurement impossible.

On the other hand, a pendant drop is surrounded

by a gas phase, and hence the concentration of

surfactant solution has no effect on the quality of

drop images.

Another objective of this paper is to investigate‘adsorption clicks’ in more detail. The adsorption

click is a highly dynamic process, in which the

surface tension of pulmonary surfactant films

decreases quickly in a stepwise fashion. Although

Schurch et al. also reported adsorption clicks in

their work [9], our study employed an image

acquisition technique that can acquire up to 30

images/s and hence can provide more detailedinformation.

2. Material and methodology

Bovine lipid extract surfactant (BLES) is a

therapeutic preparation of pulmonary surfactant.

BLES was provided by BLES Biochemicals Inc.

(London, ON, Canada) at a concentration of 27mg/ml and was used without further purification.

The major difference between a natural surfactant

and BLES is that BLES lacks the hydrophilic

proteins SP-A and SP-D. Upon arrival, the BLES

samples were divided and stored in vials at a

temperature of �/20 8C, until the day of the

experiment. Prior to the experiments, the samples

were warmed up to room temperature and dilutedto the desired concentrations, using a buffer

containing 0.6% NaCl and 1.5 mM CaCl2.

Although lipids form aggregates and micelles in

water so that their concentration cannot be

accurately deduced by diluting the bulk solution,

such dilution still provides a good approximate

J.J. Lu et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 119�/130120

measure of the lipid concentration in the solution.Furthermore, the experimental procedures docu-

mented in this paper were designed to minimize

any fluctuation of the lipid concentrations in the

drops. A broad range of concentrations (0.1�/10

mg/ml) was tested. The water used in the experi-

ments was demineralized and doubly distilled, with

a PH value between 5.0 and 5.3. All glassware and

Teflon components were cleaned by soaking inchromic acid for 8 h. They were then rinsed with

distilled water and dried under heat lamps. Syr-

inges and metal tubes were cleaned by repeated

sonication.

The effect of BLES bulk concentrations on the

rate of adsorption and film formation was inves-

tigated in the following fashion: a glass syringe (1.5

ml, Hamilton Co., USA) was used to hold BLESsuspension and was connected to a stepper motor

(Model 18705, Oriel Corp., USA). The end of the

glass syringe was connected to a Teflon capillary,

which has an internal diameter of 0.076 in. and an

external diameter of 0.1 in. At the beginning of

each experiment, the stepper motor was used to

form a surfactant drop at the end of the Teflon

capillary in less than 1.5 s, by pushing the plungerof the syringe. During each individual measure-

ment, time zero was defined as the time when the

quickly formed drop reached its final volume of

approximately 10 ml. Each individual adsorption

measurement was continued for 300 s. The surface

area of the drop had an average increase of about

18% throughout the 300 s adsorption. The reason

to choose 300 s as the time period to studysurfactant film formation was based on some

preliminary experiments. In the preliminary ex-

periments with a BLES concentration of 2 mg/ml,

the adsorption was measured for 2 h. However, the

surface tension values obtained after 300 s and 2 h

of adsorption were essentially identical, at ap-

proximately 22 mJ/m2. For all practical purposes,

any changes after 300 s are negligible, exceptperhaps for very low concentrations. Therefore,

the adsorption time was limited to 300 s.

Since the adsorption process is highly dynamic,

images of surfactant drops were acquired continu-

ously at a speed of 30 images/s. In order to

maintain a consistency in the measurements, after

each drop measurement, a new syringe set-up was

used and the solution was vortexed to eliminate

any surfactant precipitation.

In order to prevent evaporation and contamina-

tion, a quartz glass cuvette (Hellma Limited

Company, USA) was used to enclose the surfac-

tant drop. A Teflon stopper was used to seal the

cuvette. The cuvette was placed into a tempera-

ture/pressure cell (Rame-Hart, Inc., USA), which

was connected to a water bath (Model RTE-111,

Neslab Instruments Inc., USA) for temperature

control. Throughout the experiment, the tempera-

ture inside the cell was kept between 36.5 and

37 8C, in order to mimic in vivo conditions.

During each measurement, images of the sur-

factant drop were acquired through a microscope

(Apozoom, Leitz Wetzlar, Germany) and a CCD

camera (Model 4815-5000, Cohu Co., USA). A

frosted diffuser was used in front of a light source

(Model V-WLP-1000, Newport Corp., USA) to

provide a uniformly illuminated background dur-

ing image acquisition. All images were acquired

with a 480�/640 pixels resolution and 256 gray

levels for each pixel. The drop images were

digitized and stored on a workstation (Sparc

Station-10, Sun Microsystems Inc., USA). The

surface tension at the air�/liquid interface of the

surfactant drop during adsorption was examined

by axisymmetric drop shape analysis (ADSA).

ADSA is based on a rigorous integration of the

Laplace equation of capillarity for interfacial

profiles. ADSA extracted the profiles of surfactant

drops from the digitized images using an edge

detection algorithm based on a SOBEL 3�/3

pixels operator. After the profile of a surfactant

drop has been extracted, 20 random points were

selected ten times from each profile and run

through the ADSA algorithms. An image of a

calibration grid was used for calibration and to

minimize optical distortion. The ADSA output

includes surface tension at the air�/water interface,

surface area, volume and their corresponding 95%

confidence limits.

A commercial software (XMGR†) was used to

plot experimental graphs using the ADSA output.

The 95% confidence limits were calculated with

MICROSOFT EXCEL†, based on student t -distribu-

tion. More information about ADSA and the

J.J. Lu et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 119�/130 121

pendant drop set-up can be found in references

[10�/15].

3. Results and discussion

Four individual measurements showing the

adsorption of BLES at a concentration of 0.1

mg/ml are given in Fig. 1. It should be noted that,in order to improve graphical illustration, only the

trend lines of individual measurements are shown.

On the curve, only the points in the horizontal

direction are densely spaced, i.e. the jumps in

surface tension occurred in a very short time.

These sudden drops in surface tension have been

referred to as adsorption clicks. Because of these

adsorption clicks, the surface tensions obtained inindividual runs are quite random. For example, in

run b a surface tension of only approximately 55

mJ/m2 was reached after 300 s of adsorption. On

the other hand, run c went well below 30 mJ/m2 in

the first 100 s of adsorption, due to a big

adsorption click after about 30 s of adsorption.

Although the mechanism of adsorption clicks

remains unknown at this point, there are several

possible explanations. It is known that myelinic

and liposomal structures form in pulmonary

surfactant dispersions. During initial adsorption

at low surface pressures, these molecular structures

spontaneously unravel to form a dispersed low-

density monolayer at the interface [16]. The rate at

which these super-molecular structures form a

monolayer depends on the bulk concentration of

the suspension. The initial adsorption of the

monolayer at the air�/water interface causes the

surface tension to drop quickly to a first surface

tension plateau. After reaching this plateau, the

surface tension starts decreasing again, but more

slowly than in the first decrease, and approaches

its final equilibrium value. The study of self-

assembled films of DMPC by Lawrie et al.

demonstrated such a characteristic transitional

pattern in the adsorption isotherms of DMPC

films [16]. A liquid-expanded to liquid-condensed

phase transition was observed by Nag et al. along

the adsorption curves of a lung surfactant extract

[17]. At low surface pressures, the surfactant film

Fig. 1. Adsorption of 0.1 mg/ml BLES. Surface tensions of four individual runs are plotted as a function of time. For graphical

reasons, only the trend lines are shown instead of the actual data points.


at the interface exhibits an almost homogenousfluid phase, and more condensed domains start to

form at higher surface pressures. At a surface

tension (45 mJ/m2) close to the equilibrium value

of the surfactant extract, a significant portion of

the interface is covered by condensed domains of

lipid aggregates [17]. The occurrence of such

transitions from liquid-expanded to liquid-con-

densed domains depends on bulk concentrations.For example, when a surfactant at a concentration

of 0.06 mg/ml was used in their study, the surface

tension at the air�/liquid interface decreased to 25

mJ/m2 in a few minutes and most of the film was in

a liquid-condensed phase. On the other hand, if a

surfactant at much lower concentration (i.e. 0.006

mg/ml) was used, the adsorption time was much

longer and a significant portion of the filmremained in the liquid-expanded form. As a result,

the surface tension remained above 35 mJ/m2 at

this surfactant concentration even after 200 min of

adsorption. The adsorption clicks shown in Fig. 1

may be accompanied by a rapid two-dimensional

phase transition at the interfacial film. However, it

remains unknown if such a rapid phase transition

is the main reason of the adsorption click.On the other hand, adsorption clicks may be due

to a quick and cooperative movement of large

flakes of aggregated surfactant molecules (�/

1014�/1018 molecules/m2) into the air�/liquid inter-

face [9]. Since this movement of surfactant mole-

cules dramatically increases the surfactant film

concentration during a short period of time, it can

induce a sudden drop in surface tension, as shownin Fig. 1. The fact that both the magnitude and the

position of adsorption clicks are unpredictable

favorites this hypothesis. Furthermore, we found

that significant adsorption clicks usually occur at

surface tensions above 40 mJ/m2. Presumably, at

low surface tensions, the relatively high concentra-

tion of surfactant at the interface blocks further

adsorption of large aggregates. Nevertheless, theuse of imaging techniques such as fluorescence and

atomic force microscopy is expected to clarify the

mechanism of surfactant adsorption in future

studies.

In Fig. 1, initial surface tension at low concen-

trations is slightly higher than that of a pure air�/

water interface. An ADSA artifact due to the

initial near spherical shape of the drop cannot be

excluded. On the other hand, this effect may be

real: even in a system as simple as dextrose in

water, an increase in surface tension at low

concentrations can be observed [18]. In Fig. 1,

because the surfactant concentration is low (0.1

mg/ml), the effect of surfactant is not significant

enough to offset the surface tension elevating

effect by the solution and results in high surface

Fig. 2. Images show an example of an adsorption click. In (A),

the image was taken after 32.54 s of adsorption, and the drop

surface tension is 70.69/1.1 mJ/m2. A second image of the drop

was taken 0.05 s after (A) and is shown in (B). Due to the

vibration of the drop, the image could not be processed by

ADSA. In (C), the image was taken at the time of 32.75 s, and

the drop surface tension is 36.99/0.2 mJ/m2.


tension during the initial adsorption. Clearly, such

surface tension increases deserve further investiga-

tion.

Fig. 2 shows three images taken in an adsorp-

tion click. In Fig. 2A, the image shows a surfactant

drop after 32.54 s of adsorption, the drop volume

and surface tension are 9.22 ml and 70.69/1.1 mJ/

m2, respectively. An adsorption click occurred

between 32.54 and 32.75 s. (i.e. between Fig. 2A

and Fig. 2C). Fig. 2B shows an image of the drop

after 32.59 s of adsorption, which could not be

processed by ADSA. This is due to the fact that

the drop changed its shape significantly with a

drastic change in surface tension, and this dra-

matic change of the drop shape resulted in

vibration and hence blurring of the images. As

ADSA is based on equilibrium shapes given by the

Laplace equation, images of vibrating drops were

discarded. Fig. 2C shows an image of the same

drop after 32.75 s of adsorption, at which time the

drop has stabilized and the surface tension of the

drop could be determined by ADSA. The drop

volume and surface tension at this moment are

9.26 ml and 37.09/0.2 mJ/m2, respectively. During

the approximate 0.2 s interval between Fig. 2A and

Fig. 2C, a total of four images of the vibrating

drop were discarded by ADSA. The corresponding

plot of the drop surface tension vs. time is shown

in Fig. 3, with the corresponding 95% confidence

limits. The surface tension curve illustrates the

magnitude of the adsorption click. The larger

errors before the adsorption click are presumably

due to the fact that the initial drop is relatively

close to spherical, i.e. a situation in which ADSA

will be less effective. It other words, the large

errors are an artifact. The key points are that

adsorption clicks can be quite significant (i.e. with

a magnitude of more than 30 mJ/m2 in this case),

and that they can occur in a time interval as short

as 0.2 s.In Fig. 4, two more examples of adsorption

clicks were plotted. In one case (i.e. from point A

to B), the sudden decrease in surface tension (from

48.79/0.2 to 37.29/0.1 mJ/m2) occurred in 0.11 s.

During this adsorption click, three drop images

were acquired but rejected by ADSA due to the

drop vibration. In the other adsorption click, the

change of surface tension between point C and D

is moderate (i.e. from 59.79/0.3 to 56.19/0.3 mJ/

m2), and no data point was missed.

The surfactant adsorption at 0.2 and 0.8 mg/ml

was shown in Fig. 5 and Fig. 6, respectively. It

should be noted that although images of surfactant

drops were taken at a speed of 30 images/s, only

Fig. 3. Plot of the adsorption shown in Fig. 2. A and C represent the corresponding pictures shown in Fig. 2, respectively.


one data point per second was plotted, for

graphical reasons. In both figures, the effect of

concentration is apparent. First, at a concentra-

tion of 0.2 mg/ml, the surface tension is still above

30 mJ/m2 after 300 s of adsorption, while at 0.8

mg/ml, all four individual runs reached a surface

tension of around 25 mJ/m2 in the initial 200 s of

adsorption. Second, the initial speed of adsorption

is also strongly concentration dependent. At 0.2

mg/ml, film surface tension changes little in the

first 50 s. At 0.8 mg/ml, the surface tension started

to decrease immediately after the drop formation.

Furthermore, although adsorption clicks could

also be observed at 0.8 mg/ml, the frequency of

such adsorption clicks was less. The film surface

tension at 0.8 mg/ml decreased more gradually.

This may be explained by considering the surface

concentration of the film: The adsorption clicks

are expected to occur before the surface concen-

tration of the film reaches a certain threshold.

Once that threshold concentration is reached,

there is a sufficiently high coverage of lung

surfactant material on the aqueous surface to

prevent the adsorption of larger aggregates. This

threshold coverage will be reached the faster the

higher the bulk concentration. This does not

preclude adsorption clicks at higher concentra-

tions. If they occur, they cannot be followed by the

methodology employed here. From a physiological

perspective, such events, occurring on a time scale

much shorter than the frequency of human breath-

ing, would presumably be inconsequential.

The results of film adsorption at concentrations

of 1 and 10 mg/ml were plotted in Fig. 7 and Fig.

8, respectively. Again, in order to obtain a better

graphical illustration, the density of data points in

Fig. 4. Adsorption clicks observed in two individual runs, performed with BLES concentrations of 0.1 and 0.5 mg/ml, respectively.


the graphs was reduced. Adsorption clicks are

absent in both graphs, due to the fact that the

initial surface tension of the film was below 30 mJ/

m2 at the end of drop formation. Due to the

absence of adsorption clicks, the results of indivi-

dual runs are quite consistent. It can be seen that

the effect of concentration is less pronounced than

at lower concentrations, again within the above

Fig. 5. Adsorption of 0.2 mg/ml BLES. Three individual runs are shown.

Fig. 6. Adsorption of 0.8 mg/ml BLES. Four individual runs are plotted.


physiological time frame. For example, after 300 s

of adsorption, although the film formed on a 10

mg/ml BLES solution reached a value somewhat

lower than that obtained with 1 mg/ml BLES, the

difference is only about 1 mJ/m2. Furthermore, the

shape of the adsorption isotherms shown in Fig. 7

Fig. 7. Adsorption of 1 mg/ml BLES. Four individual runs are shown.

Fig. 8. Adsorption of 10 mg/ml BLES. Four individual runs are plotted.


and Fig. 8 is similar, i.e. the surface tension

decreased quickly in the first 50 s, and then

remained relatively constant.

Film adsorption experiments were also per-

formed at concentrations of 2, 3, 6, 8 and 10 mg/

ml. The results obtained in the initial 20 s of

Fig. 9. Surface tension as a function of time for the initial 20 s of adsorption, at BLES concentrations of 1, 2, 3, 6, 8 and 10 mg/ml,

respectively. Each curve is the average of four individual runs.

Fig. 10. Surface tension as a function of time for the initial 2 s of adsorption, at BLES concentrations of 1, 2, 3, 6, 8 and 10 mg/ml,

respectively. Each shown curve is the average of four individual runs.


adsorption were plotted and compared in Fig. 9.

Each curve is the average of four individual runs.

In order to illustrate in more detail the early stages

of the adsorption at concentrations above 1 mg/ml, the initial 2 s of adsorption shown in Fig. 9

were magnified and plotted in Fig. 10. It should be

noted that certain points on the curves were

missing due to vibrations of the surfactant drops

after their initial formation, which caused the

image to be blurry, causing failure of ADSA to

process these images. Similar to the conclusion

obtained from Fig. 9, the effect of concentration issmall. It can be seen from Fig. 10 that the initial

surface tension values were all in the range of 27�/

29 mJ/m2, indicating that a large amount of

surfactant molecules were already adsorbed onto

the interface during the time of drop formation

(�/1.5 s). It is of physiological interest that at

physiologically realistic concentrations, the surface

tension reaches a low surface tension value (e.g. �/

25 mJ/m2) in a time period comparable to the

frequency of breathing. Table 1 collects the

averaged surface tension values after 2, 20 and

300 s of adsorption, corresponding to the experi-

ments shown in Fig. 9 and Fig. 10.

4. Conclusions

Adsorption and film formation of a therapeutic

lung surfactant, BLES, were investigated as a

function of solution concentration by a pendant

drop-ADSA arrangement. At low concentrations

below 1 mg/ml, adsorption is slow and the effect of

concentration can be readily followed by the

methodology employed. At high concentrations

above 1 mg/ml, adsorption and film formation are

fast compared to the physiological time scale given

by the frequency of human breathing.Adsorption clicks were investigated and dis-

cussed in detail. The magnitude of adsorption

clicks can be either large (e.g. a surface tension

reduction of :/35 mJ/m2 in �/0.2 s) or quite

moderate (e.g. in a range of 1�/5 mJ/m2). When the

concentration of surfactant solution increases,

significant adsorption clicks tend to be replaced

by a series of moderate adsorption clicks, or defacto a continuous change.

Acknowledgements

This work is supported by the Medical Research

Council of Canada (grant MOP38037) and a

University of Toronto Open Fellowship (J.J. Lu).

We also thank Dr David Bjarneson of BLES

Biochemicals Inc. for his generous donation of

the BLES samples.

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Table 1

Surface tension after 2, 20 and 300 s of adsorption, for several BLES concentrations

Concentration (mg/

ml)

Surface tension (mJ/m2) (after 2 s

adsorption)

Surface tension (mJ/m2) (after 20 s

adsorption)

Surface tension (mJ/m2) (after 300 s of

adsorption)

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3 26.09/0.1 24.59/0.1 23.99/0.1

6 25.89/0.2 24.39/0.3 23.69/0.2

8 25.19/0.1 24.09/0.1 23.49/0.1

10 24.99/0.1 23.99/0.1 22.99/0.1

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the effect of concentration on the bulk adsorption of bovine lipid extract surfactant

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