Black Foam Films: Application in Medicine

738 CHAPTER 11

11.

BLACK FOAM FILMS: APPLICATION IN MEDICINE The alveolar surface represents a thin liquid film formed at the interface between the

alveolar gas phase and a liquid hypophase covering the epithelium. This film is stabilised by the alveolar surfactant (AS), consisting mainly of phospholipids and proteins. AS plays an important role in alveolar stabilisation in the process of breathing. It is known that AS components exist as individual molecules and as various lipid and protein/lipid micellar structures present in the so-called hypophase and, according to some researchers, form a continuous lipid monolayer at the water/air interface [e.g. 1-4]. AS can be studied in human or animal lung extracts as well as in the amniotic fluid (AF) where AS molecules are present [2,4-6]. Along with the biochemical, cytochemical and other techniques of AS investigation, very useful information can be derived from the model studies of lipid and protein/lipid monolayers at liquid substrates. These models provide important data about lung mechanics [e.g. 7-11 ]. It is already known that the maximum (on inhaling) and minimum (on exhaling) alveolar surface area is determined by measuring the surface pressure (zr = A t r ) with a Langmuir balance, i.e. at minimum and maximum compression of the monolayer. When the amount of phospholipids in the amniotic fluid is not enough, there appears an AS deficiency which is considered to be the main reason for lung immaturity, leading to development of a respiratory distress syndrome (RDS) in neonates. The timely detection of foetal lung maturity with respect of the potential risk of RDS development is a problem of major importance in prenatal medicine. Gluck et al. [6] have shown that the concentration of surface active components in the amniotic fluid sharply increases between the 32-th and 35-th gestation weeks. One of the largely applied until recently test for assessment of lung maturity is based on the determination of lecithin/sphingomyelin (L/S) ratio in the amniotic fluid.

11.1.

BLACKFILM METHOD FOR ASSESSMENT OF FOETAL LUNG MATURITY

The creation of sensitive methods for assessment of foetal lung maturity are needed for prophylactics and neonate treatment of RDS. Numerous methods for prediction of lung

Black Foam Films: Application in Medicine

739

maturity by examination of the amniotic fluid [e.g. 5,6,12-16] and lung surfactants [e.g. 17,18] have been developed. The air/solution interface is essential in the methods involving the monolayer [17], bubble [18] and foam [5]. Each of these methods exploits a certain surface parameter for determination of the properties of amniotic fluid. The microscopic foam film is convenient for investigation of microheterogeneous systems. It enables the formation of foam bilayers at very low amphiphile concentrations and the investigation of fluctuation phenomena. The physical parameters related to formation and stability of microscopic foam bilayers are very sensitive to the amphiphile concentration [19] which makes these bilayers very useful for assessment of the foetal lung maturity [20]. The microscopic foam bilayer proved to be an appropriate model for investigation of alveolar surface and alveolar stability as well [21]. This approach is in agreement with the findings of Scarpelli that at birth the lung surfactant takes the form of intraalveolar bubbles with formation of foam films [e.g. 2,22,23]. The results on formation and stability of black foam films, on the first place those on bilayer foam films (NBF) (see Sections 3.4.1.2 and 3.4.4) have promoted the development of methods which enable lung maturity evaluation. The research on stability of amphiphile bilayers and probability for their observation in the grey foam films laid the grounds of the method for assessment of foetal lung maturity created by Exerowa et al. [20,24]. Cordova et al. [25] named it Exerowa Black Film Method. It involves formation of films from amniotic

fluid to which 47% ethanol and 7.10 -2 mol dm 3 NaC1 are added [20,24]. In the presence of alcohol the surface tension of the solution is 29 mN m l and the adsorption of proteins from the amniotic fluid at the solution/air interface is suppressed, while that of phospholipids predominates. On introducing alcohol, the CMC increases [26], so that the phospholipids are present also as monomers in the solution. The electrolyte reduces the electrostatic disjoining pressure thus providing formation of black foam lipid films (see Sections 3.4.1.2 and 3.4.4). In order to apply the hole-nucleation theory of bilayer stability of Kashchiev-Exerowa [27] involving quantitative interpretation of the W(C) dependence (probability for observation of black films vs. surfactant concentration), the black films from amniotic fluid should be bilayer films. This is proved experimentally by two dependences: 1-I(hw) (Fig. 11.1) and

hw(Cel) (Fig. 11.2). As it can be seen in Fig. 11.1, the equivalent film thickness is 8 nm and does not change with the increase in rI (which is the difference between the pressures in the

740

Chapter 11

liquid and gas p h a s e s , Ap = H). T h i s c o n f i r m s c o n c l u s i v e l y the b i l a y e r structure o f the f i l m in w h i c h there is no free a q u e o u s c o r e b e t w e e n the p h o s p h o l i p i d a d s o r p t i o n layers.

o"~I

13.. 0

1.0

0.5 It

0

5

10

15

20 hw, nm

Fig. 11.1. Disjoining pressure FI vs. equivalent thickness hw of an AF film; solution containing 47% ethanol; curve 1 - 2.26.10 .2 mol dm -3 NaCI; curve 2 - 2.69-10 .2 mol dm -3 NaCI; curve 3 - 3.3.10 .2 mol dm -3 NaCI; curve 4 - 5.00-50.0.10 .2 mol dm 3 NaCI; r = 200 ~tm; t = 25~ [28].

2O

2ils 10

S

1 0 -2

' 10 -1

...... 1.0 Cei, mol din-3

Fig. 11.2. Equivalent thickness hw of AF film vs. electrolyte (NaCI) concentration 47% ethanol; Ap = 29 Pa; r = 200 lxm; t = 25~ [28].

S i m i l a r l y , the

hw(Cel)

dependence

(Fig.

Cet;solution containing

11.2) indicates the s a m e e q u i v a l e n t f i l m

t h i c k n e s s o f 8 n m w h i c h d o e s not c h a n g e with f u r t h e r i n c r e a s e in

Cet ( p l a t e a u

in the curve). As

Black Foam Films: Application in Medicine

741

discussed in Section 3.4 the equivalent thickness

hw of a foam bilayer is higher than its real

thickness. The difference between them can be estimated by using the three-layer model of film structure. Calculating the real film thickness from Eq. (2.2) with properly chosen h~ yields a value of ca. 5.5 nm. As it was shown the stability of bilayer foam films (NBFs) and, respectively, the probability W for their observation of the thicker (grey) films depends considerably on the concentration of the surface active molecules (see Sections 3.4.3.2 and 3.4.4.3). Fig. 11.3 plots such dependences for various individual phospholipids such as phosphatidylglycerol, egg lecithin, DPPC, phosphatidylinosytol and their mixture (amniotic fluid).

W 1,0 . qD q 0.5

\1 i 2,

3

50

14

j5

100

300 C, ~g cm-3

Fig. 1 1 . 3 . Dependence of the probability for observation of a foam bilayer on the total lipid concentration for foam films in the presence of 47% ethyl alcohol and 7-10.2 mol dm-3 NaC1 in the solution: curve 1 - data for AF solutions; curve 2 - for PG; curve 3 - for EL; curve 4 - for DPPC; curve 5 - for PI; for the AF C is referred to Cot,Pc,and all curves are drawn to guide the eye); t = 25~ [28]. Fig. 11.4 shows separately curve 1 from Fig. 11.3 which is the dependence of W on the DPPC concentrations in the AF. The W(C) curves allow to determine the threshold concentration

Ct, i.e. the minimum phospholipid concentration at which there is a 100%

probability of observation of black films (see Eq. (3.130)). At concentrations lower than

Ct

NBFs are no more observed, since W sharply decreases to zero (films rupture). At concentrations higher than

Ct (W = 1), NBFs always form. Special studies with phospholipid

analysis of amniotic fluid indicate that of all phospholipids in the AF, it is the DPPC that stabilises the foam bilayers. This analysis gives grounds to conclude that the concentration of each phospholipid (except DPPC) in the native AF is of an order lower than the corresponding

742

Chapter 11

individual Ct. The only substance the content of which in the native AF is close to Ct is DPPC, i.e. the phospholipid forming the bilayer foam film. W 1.0 I

05

11

10

I ..........

!

..............

Ct 20 Ctr~, ~g cm-3

Fig. 1 1 . 4 . Dependenceof the probability for observation of a foam bilayer on the total DPPC concentration for foam films obtained from 3.5 times diluted solution of AF in the presence of 47% ethyl alcohol and 7-10.2 mol dm3 NaCI; black circles - experimentaldata; the curve is drawn to guide the eye; t = 25~ [28]. In order to determine the statistical distribution of amniotic fluid samples taken at different gestation weeks, two relations are studied: rupture of foam films (W = 0) and development of RDS, and formation of a bilayer foam film (W = 1) and normal respiratory status of neonates. These correlations allowed to develop a new diagnostic method for estimation of lung maturity [20]. The function of the threshold dilution of various amniotic fluid samples (corresponding to Ct) on the gestation age and the clinical results (i.e. yes/no RDS in neonates) is given in Fig. 11.5. The respiratory status of the neonates is studied with the screening system of Masson et aL [26], modified by Hobel et al. [29]. The linear dependence between the threshold dilution and the initial total phospholipid concentration (respectively, DPPC) found allows to determine the threshold dilution for a 100% probability for formation of NBF instead of Ct. Fig. 11.5 shows that if a sample dilution of 3.1 times is applied, then it is possible to detect almost all cases with a developed RDS. Therefore, the threshold dilution of 3.1 times allows to distinguish the mature from immature AF samples which gives a good reason to employ it in diagnosing of RDS, and respectively, to estimate the lung surfactant deficiency. Hence, the formation of black foam films from AF samples taken at different gestation weeks and diluted 3.1 times, indicates that there is no risk of RDS, while film rupture predicts an eventual RDS development.

Black Foam Films: Application in Medicine

743

12.5 0

-~9 1o.o

0

~3

0

0 0

oOo~n~8 ~

..~ 7.5

7=

'~ s.o

d~

2,5 ~AA 0

AAA

A

AAAA~A/~eIAA

'.,

I

I

I

I

20

25

30

35

40

gestation weeks

Fig. 1 1 . 5 . Dependenceof the threshold dilution dt of amniotic fluid on its gestation age; clinical results from 420 cases: A - developmentof RDS in neonates; C) - no RDS in neonates; the line at dt 2.5 indicates the dilution separating the mature from immatureAF samples. =

This diagnostic method has several advantages compared to the other methods used: high reliability (-- 95%), prompt testing (-- 30 min), easy performance, small sample quantity (~- 1 cm3). It should also be noted that the comparison of the method for prediction of RDS by means of NBF with the largely used L/S method reveals a good conformity. In the range of L/S ratio from 1.5 to 2.0, the black film method exhibits better diagnostic abilities. This is an important fact since in this range the L/S method does not provide reliable clinical results. More details about the black foam film diagnostic method can be found in [20,24,30,31 ] and the latest results in [32]. The clinical data accumulated during the last years indicate a decrease in the threshold dilution down about 2.5. The scope of the method for assessment of foetal lung maturity has been extended for evaluation of the risk for development of RDS of new-borns by testing AF with blood and mecomium, obtained from women with normal pregnancy [33]. An experimental study of corticosteroid action on lung surfactant [34] as well as the effect of betamethasone (Celestone) on foetal lung development in rats [35] has been also carried out. The method for assessment of foetal lung maturity is very perspective and can be developed in the following directions: for quantitative estimation of lung maturity/immaturity at pathological pregnancy cases; for seeking of the most effective therapeutic agents ensuring lung maturation; RDS diagnosing in samples of tracheal neonate aspirate.

744 11.2.

Chapter 11 PHASESTATE OF FOAM BILAYER (NBF) FROM AMNIOTIC FLUID

As it is well known [36,37], the natural lipid/protein mixtures (such as amniotic fluid) can undergo different phase transitions due to variation in temperature or composition. Of special importance for the natural bilayer lipid membranes is the so-called main phase transition between the lipid crystalline and gel states at which a melting of the hydrocarbon tails of the lipid molecules occurs. For example, it has been demonstrated [36] that there exists an upper limit of the gel phase content in membranes above which the membrane morphology and permeability change dramatically thus making the execution of the physiological functions of the membrane impossible. The phase state of AF foam bilayers has been studied from the effect which the temperature exerts on the threshold dilution (i.e. threshold concentration) for formation of foam bilayers (NBFs) from AF [38]. Samples of AF were collected during the 39th gestation week. Microscopic foam films were formed from diluted AF in the temperature range from The values of dt were found from the dependence probability W for observation

10~ to 30~

of foam bilayer on dilution d at different temperatures. Such a dependence is shown in Fig. 11.6,a for one of the samples at 15~

W

O

1.0

. . . .

-

?.

A sharp drop in W value at dt is seen in the figure.

-

].5

5

0

15

d

10

15 C..~i9

crn-3

Fig. 1 1 . 6 . ProbabilityW for observation of AF foam bilayer (sample 1 at 15~ 9(a) experimental dependence of the dilution d; (b) experimentaldependence on the phosphatidylcholine concentration C. The equivalent dependence calculated with respect to the total concentration C of phosphatidylcholines in the AF solutions is shown in Fig. 11.6,b. This concentration is reversibly proportional to the dilution d

Black Foam Films: Application in Medicine

745

(11.1)

C- Cs / d

where Cs is the total concentration of phosphatidylcholines in the AF samples before dilution. The value of Cs was determined for each sample from the linear dependence of the total phosphtidylcholine concentration of AF samples on dt at 25~ ( C s = 13.3d t ). The values of the threshold dilution dt for different temperatures are presented in Fig. 11.7 in Arrhenius co-ordinates for the five samples investigated. As it is seen, linear dependences of similar slope were obtained within the temperature range from 10~ to 30~ The temperature dependences of dt were determined as concentrations higher than the critical electrolyte concentration

Cel, c r

for formation of foam bilayers from amniotic fluid and it was

found that dt is not a function of the electrolyte concentration. ~,*c 3

2

30 ,

20 ~

~

10 ,

20 3

1

15

10 5

0

I

3.3

i

_

I

i

1000/T, 3.4

_1_

3.5

I

3.6

K-~

Fig. 11.7. Arrhenius dependence of the threshold dilution d t for AF foam bilayers (sample 1 to 5); symbols - experimental data; straight lines - theoretical dependences according to Eq. (11.2).

The experimental curves W(d) and W(C), shown in Fig. 11.6, are very informative with respect to formation of foam bilayers from amniotic fluid because these dependences are very steep. This fact allows precise determination of d t and Ct. The clearly pronounced value of Cc (W = 0) practically coincides with the threshold concentration Ct for observation of foam bilayer with W = 1, which was defined in the method for assessment of foetal lung maturity.

746

Chapter 11 The systematic study of foam bilayers from phospholipids [28,38-40] reveals that they

do not rupture spontaneously at any concentration allowing their formation. That is why in the case of phospholipid foam bilayer the dependence of their mean lifetime on the bulk amphiphile concentration cannot be measured in contrast to foam bilayer from common surfactants [41,42]. This infinite stability of phospholipid foam bilayers is the cause for the steep W(d) and W(C) dependences. In the case of AF foam bilayers this high stability was confirmed by a very sensitive method [19,43] consisting of or-particle irradiation of foam bilayers. As discussed in Sections 2.1.6 and 3.4.2.2, the o~-particle irradiation substantially shortens the mean lifetime of foam bilayers. The experiments showed that at all temperatures and dilutions studied (even at dt), the foam bilayers from AF did not rupture even at the highest intensity of irradiation applied, 700 ~tCi. This extreme stability of AF foam bilayers allowed to assume that Ct = Ce and to use the data for the temperature dependence of threshold dilution (shown in Fig. 11.7) for determination of Q (binding energy) for each sample of amniotic fluid. For this reason Eq. (11.2) resulting from Eq. (11.1) and Eq. (3.115) can be used

In d, = K" + Q / 2kT

(11.2)

where K ' = ln(Cs/Co).

The above mentioned threshold dilution dt and critical concentration for formation of a bilayer are used as measures for bilayer stability [ 19] being determined by the first neighbour lateral and normal interactions in the foam bilayer. This is the difference of the parameter dt from the change in the free surface energy which is usually used as a measure of the surface activity. Thus, the parameters dt and Ct are proposed as new characteristics of the surface activity of an amphiphile molecule, evaluated with high accuracy from the sharp W(d) and W(C) dependences, respectively. The results for the binding energy Q of an amphiphile molecule in an AF foam bilayer, obtained from the slopes of the straight lines in Fig. 11.7 by using Eq. (11.2) are shown in Table 11.1 for the five AF samples studied. One should bear in mind that these values of Q are effective ones as far as they hold for foam bilayers from a complex natural mixture. These

Black Foam Films: Application in Medicine

747

values are physically relevant (in all cases Q > 8kT) and in agreement with the hole-nucleation theory provided that the condensed state of the monolayers composing the foam bilayers is accepted, which is, as it is well known, a necessary condition for foam bilayer formation [44]. It is seen from the Table that Q-values vary from 5.3.10 -20 to 9.5.10 .20 J, which is explicable if one recognises the different lipid/protein composition of the AF samples. The mean value of Q is 7.6.10 .20 J (--- 19kT at 20~

the standard deviation being 1.8.10 .20 J.

TABLE 11.1 Values of the binding energy Q of an amphiphile molecule in foam bilayers from AF (samples 1 to 5) obtained from the best fit of Eq. (11.2) to the data for temperature dependence of the threshold dilution dt 1

Q. 10-2~ J Q, kT (20~

9.4 23

2

3

4

5

7.2 18

5.3 13

9.4 23

6.9 17

A comparison of the lnC(1/T) dependence for AF and DPPC foam bilayers is shown in Fig. 11.8. It is seen that the slope of the linear ln(Ct) vs. 1/T dependence for the liquid crystalline DPPC foam bilayers (curve 1) is very close to that for AF foam bilayers (curve 3), which corresponds to similar Q values for AF and liquid crystalline DPPC foam bilayers. The Q value of AF foam bilayers is also very close to those for the liquid crystalline NaDoS and DMPC foam bilayers (see Section 3.4.4). Hence, the interaction between lipid molecules is very similar in these foam bilayers and it can be supposed that the AF foam bilayers are in the liquid crystalline state within the temperature range studied. This assumption is in agreement with the fact that amniotic fluid contains substantial amount of unsaturated phospholipids, which as known [45], lower considerably the temperature of the chain-melting phase transition. Bearing in mind the similarity of the phase behaviour of a phosphatidylcholine aqueous dispersion and foam bilayers [38-40], it can be supposed that at the temperatures which are important for in vivo systems, the foam bilayers are in the liquid crystalline state. This assumption allows to determine the critical concentration of phosphatidylcholines in amniotic fluid, necessary for formation of a foam bilayer by extrapolation of the Arrhenius dependence of Ct for AF foam bilayers to 37~

Thus, at 37~

Ct

=

19.9 ~g cm -3 and dt = 1.47. This value of

Ct

at

37~

corresponds to the lower limit (found by other methods [46,47]) of phosphatidylcholine concentration which permits to classify as mature a sample of amniotic fluid. The above value

748

Chapter 11

should be regarded as a tentative one as far as it is determined for foam bilayers from a complex lipid/protein mixture and the accuracy corresponds to that of the determination of the threshold dilution in samples of both normal and pathological pregnancies.

50

/,0

Is *C :30

20

10

i

i

I

i

1

200

1

150

i 100

50 E u

2

2

3.0

!

3.1

o'1

r~

3.2 3.3 3./, 10001 T,K I

3.5

56

Fig. 1 1 . 8 .

Arrheniusdependence of the critical concentration Ct for formation of AF foam bilayer (C, in experimental data for DPPC foam bilayers; 9 - experimental data for AF foam bilayers; O - Ct value for AF foam bilayers at 37~ calculated by extrapolation according to Eq. (3.115) under the assumption Ct = C,; curves - best fit of Eq. (3.115) to experimental data under the assumption C, = Ce; curve 1 - DPPC liquidcrystalline foam bilayers; curve 2 - DPPC gel foam bilayers" curve 3 - AF foam bilayers. m g cm-3):

9 -

The studies discussed expand the use of the method for assessment of foetal lung maturity with the aid of microscopic foam bilayers [20]. It is important to make a clear distinction between this method [20] and the foam test [5]. The disperse system "foam" is not a mere sum of single foam films. Up to this point in the book, it has been repeatedly shown that the different types of foam films (common thin, common black and bilayer films) play a role in the formation and stability of foams (see Chapter 7). The difference between thin and bilayer foam films [19,48] results from the transition from long- to short-range molecular interactions. The type of the foam film depends considerably also on the capillary pressure of the liquid phase of the foam. That is why the stability of a foam consisting of thin films, and a foam consisting of foam bilayers (NBF) is different and the physical parameters related to this stability

are

also

different.

Furthermore,

if the

structural

properties

(e.g.

drainage,

polydispersity) of the disperse system "foam" are accounted for it becomes clear that the foam and foam film are different physical objects and their stability is described by different physical parameters.

Black Foam Films: Application in Medicine

749

The above considerations indicate that the phase state of AF foam bilayers is liquidcrystalline and the mean value of the binding energy Q of an amphiphile molecule in the bilayer is 7.6.10 -20 J within the temperature range studied. These results are valuable also in relation to the application of foam bilayers for assessment of lung maturity in clinical practice. Lateral diffusion in a foam film from lung surfactant samples. Lalchev et al. [49] have recently reported a study of the lateral diffusion D in foam films stabilised by lung surfactant samples. In order to estimate the contribution of lung surfactant ingredients to the lateral mobility in the foam film plane, several fractions of natural lung surfactants were obtained [49]: crude lung surface active material (LSAM-1), purified lung surface active material (LSAM-2) and three different hydrophobic fractions of lung surfactants (HLFS) obtained by extraction with organic solvents of LSAM-2. In summary, the results show that lateral molecular diffusion at the surfaces of foam films composed of LSAM samples is observed at temperatures above 30~ temperature, up to and above 37~

With increasing

D increases differently for LSAM-1 and LSAM-2,

depending upon the composition of the preparations and the film characteristics as defined by ionic strength and its effect on foam film thickness. Another factor important for the lateral diffusion is the influence of the proteins (non-specific or specific lipid-binding) on the lipid phase state in the bulk phase and at the film interfaces. The results reveal that the increased content of non-specific surfactant proteins in the LSAM-1 sample compared with the LSAM-2 sample correlates with a relatively large decrease in D within the temperature range from 25~ to 55 ~ and with a slight increase in the temperature where measurable diffusion is first observed (the onset of pure diffusive mobility). So, a connection is found between increased content of non-specific surfactant proteins with decreased diffusion in films. The influence of electrolyte concentration on the thickness of foam films formed from HFLS samples was also investigated [49]. In the absence of added electrolyte, samples of HFLS in distilled water produced thick equilibrium films with a characteristic thickness of 88.3 nm. The presence of 0.14 mol dm -3 NaC1 resulted in formation of CBF with characteristic equivalent water thickness of 21.7 nm. Having established that under these conditions the foam films of HFLS were of the CBF type, the surface characterisation of these fractions by measuring the molecular diffusion in the films was extended using FRAP techniques (see Chapter 2). The heating-cooling curves obtained from a single temperature circle show that D in the HFLS film displays a significantly higher increase during heating

750

Chapter 11

than those observed in the LSAM-1 stabilised foam films from which the hydrophobic fraction was extracted. Furthermore, the cooling curve lays above the heating curve, i.e. hysteresis phenomena are observed. The temperature dependence of D for the two different black film types (CBF and NBF) stabilised with LSAM-2 was also determined [49]. Measurable diffusion was again detected above 30~

in films of both types but all D values in the temperature range studied

are lower for the NBFs than for the corresponding CBFs. This effect could be due to the lack of a free liquid core between the interfaces of NBF. This coincides with earlier observations of D in CBF and NBF stabilised by pure phospholipids [50,51 ]. The change in D as a function of temperature for HFI.S foam films before and after the addition of a lung surfactant: the specific protein SP-A, has been studied [49] to estimate the role of this hydrophilic protein. The temperature dependence of D in CBF stabilised by DPPC, the major HFLS phospholipid component (curve 5), by HLFS alone (curves 1 and 2) and by the reconstituted surfactant, comprised of SP-A added to HFI_S (curve 3 and 4) are shown in Fig. 11.9. In the DPPC films D remains at the immobile level up to 45~

and higher

temperatures only induced small increase in D. In contrast, D in HFLS foam films is much higher. Although the HFLS films were all CBFs, an electrolyte dependence of D was detected, which could be due to the change in film thickness. The higher D associated with the HFLS films compared to those of DPPC was explained by a number of effects: (i) the presence of other phospholipids in HFI_S some of which may be charged, have shorter acyl chain lengths and/or smaller head group size, unsaturation, etc. [50,51]; (ii) a shift in the phase transition temperature to a lower temperature with resultant fluidisation of the film surfaces or (iii) the presence of hydrophobic surfactant apoproteins in HFLS. The films from HFLS and SP-A mixtures were characterised by D values lower than those of HFLS but higher than those of DPPC (curves 3 and 4, Fig. 11.9). This effect could be attributed to the association of the large, lipid-binding SP-A molecules with adsorbed lipid at the interface resulting in reduced lateral diffusion. It is important to note that foam films formed from mixtures of HFLS and SP-A give D values in the range, similar to that observed with LSAM 1 and 2 (from which HFLS and SP-a are extracted). One could conclude, therefore, that the surface diffusion characteristics of LSAM are governed by HFLS and SP-A, and that LSAM can be reconstituted by mixing these components.

Black Foam Films: Application in Medicine

751

The quantitative comparison of the data about lateral diffusion in foam films from surface active materials with their phase states is worth to be done. 12-

A1

"T, 10 E o

o

8-

6-

3

4

4

2 0 20

m--+m~

30

40

m.../.m-"

Obile levlel

50

60

j

70

__,

t,~

80

Fig. 11.9. Temperature dependence of the diffuse coefficient D of surface-adsorbed 5-N(octanoyl) aminofluorescein in black foam films stabilised by hydrophobic fraction of the lung surfactant alone (curves 1 and 2) and in the presence of SP-A (curves 3 and 4); curve 1 - 0.8 mg cm -3 HFSL (F) in 0.14 mol dm 3 NaCI solution; curve 4 - same as curve 1 plus SP-A(3:I w/w); curve 2 - 0.8 mg cm 3 HFLS (F) in 0.5 tool dm "3 NaCl solution; curve 3 - same as 2 plus SP-A (3:1 w/w); curve 5 - the main component of HFLS, DPPC, in 0.125 mol dm -3 NaCI" solution; the type of film is CBF; p . = 30 Pa; r = 200 lam.

11.3.

A N E W H Y P O T H E S I S OF THE S T R U C T U R E AND S T A B I L I T Y OF A L V E O L A R S U R F A C E

The relation between the probability for observation of a black foam film from AF and the neonate lung maturity indicates that the bilayer film is an appropriate model for the study of the alveolar surface and its stability. This is confirmed by the fact that the threshold concentrations Ct used to estimate the foetal lung maturity corresponds to the DPPC concentration in AF, Ct = 44 ~g dm -3, respectively, to dt = 3.1 and Ct = 33 mg dm -3, respectively, to

dt =

2.5.

These values correspond to the critical DPPC concentration

determining the foetal lung maturity [e.g. 46,47]. The black foam films from amniotic fluid being a complex phospholipid mixture, are formed by DPPC. It has been proved that this phospholipid plays a major role in the maturation of the lung and its normal functioning [e.g. 2,4-6,22,52]. The dependence of the threshold dilution of the amniotic fluid on the gestation age indicates that the DPPC concentration increases with time. Exerowa and Lalchev [21] have reported a I-l(hw) isotherm of multilayer foam films from AS sample solutions containing 47% ethyl alcohol and 7.10 -2 mol dm -3 NaC1, which provides very important information. It is shown in Fig. 11.10. This isotherm has a stepwise

752

Chapter 11

course, i.e. the equivalent thickness hw decreases by a step of about 5.5 nm. At hw = 40 nm and 1-I = 5.102 Pa, flat lamellar structures exist between the two lipid monolayers. In these structures the lipid molecule orientation is probably similar to that in the protein/lipid membranes. It can be assumed that with the increase in 1-I a lamella of thickness 5.5 nm is "pushed out" from the multilayer film into the solution and a new equilibrium state is reached with a smaller film thickness. As shown in Section 3.4.2.6, the stratification process is spontaneous. These studies of AS films reveal that this is true also for phospholipid films formed at constant Ap. Moreover, the Fl(h) isotherm plotted on applying pressure to stratified films proves the multilayer structure of films from amniotic fluid. When there is an excess of phospholipids, similar multilayer structures can be formed in vivo in the lung hypophase (between the membrane of epithelial cells and the lipid monolayer at the solution/air interface).

J

~k~a~aum

o

a. 25

~au~a~Ram

,.r o

r"

rupture

n

2.0

~

1.5

1.0 05 0

J

10

I

20

I

30

"" -'-'-'---Q

~0 h w . flY~

Fig. 11.10. Isotherm of disjoining pressure of AS films, proving the multilayer film structure; the initial solution contains 47% ethanol and 7.10-2 mol dm-3 NaCI; 150 ~tg cm -3 DPPC; r = 200 lam; t = 25~

These studies of AS multilayer films as well as of lipid bilayers give reason to propose a new hypothesis of the structure of the alveolar surface. According to it the continuous lipid monolayer of the alveolar surface is in contact with the multilayer or with the membrane of epithelial cells situated under it, i.e. at the contact sites as well as between the individual

Black Foam Films: Application in Medicine

753

lamellae there is no free aqueous layer. This hypothesis is close to the idea of Hills [53] and Bangham et al. [54] that there is no free aqueous layer between the alveolar epithelial cells and the continuous surface monolayer. The morphological studies of AF confirm the new hypothesis since multilayer structure [55] and crystal dislocation defects [56] have been observed. The most essential feature of the proposed model of alveolar surface is that the stability of a foam film (bilayer and multilayer) is determined by the lateral molecular interactions in the monolayer as well as by the normal interactions with the molecules in the ordered layer underneath (see Section 3.4.4). The experimental criteria of alveolar stability account only for the surface tension, i.e. the lateral interaction between monolayer molecules in the unordered surface of the aqueous phase. According to the authors of this monograph this is a necessary but insufficient condition of alveolar stability. Another configuration of the black film position, proposed by Scarpelli and Mautone [57] is also possible (Fig. 11.11) where it apposes the gas phase. According to Scarpelli and Mautone this configuration is consistent with the surfactant film structure and function in situ. .......

Type II Cell

=..~.-~---_-. Type l C e l l

/ X ' ~ . . ~...~~~. ".~.:3~ ~s'j:. :: .,..k~,~~"

r.,.

.

'~'~.%~"x.

,

.5ta n ~

~...-~ 't "

"-'- c k

dg::. ~..#~.

Macrophage " ' - ~ Fig. 11.11.

.N

Positionof the foam film in the "mouth" of the alveolus, by Scarpelli and Mautone [57].

However, though very important, the physical differences between "open" and "closed" alveolar lining layer will not be discussed here since they need a separate consideration. Our present aim is to emphasise on the microscopic foam film as an effective

754

Chapter 11

model for the study of the alveolar surface and stability. Most probably, however, they exist in vivo in the lung.

Thus, the new hypothesis of the structure and stability of the alveolar surface is built on the basis of the method for assessment of foetal lung maturity employing the microscopic black foam film model of Exerowa and Lalchev [24] proposed in 1981. Its further development stands on the theoretical and experimental investigations of the reasons for formation and stability of the bilayer foam films involving short-range molecular interactions between the first neighbour molecules [e.g. 20,21,28,30,31,58]. The "response" of the in vivo situation gives reason to believe that the black foam film has an alveolar analogue in vivo which is a very important fact [e.g. 21,59]. Scarpelli [22] was the first to introduce in 1978 the hypothesis of an intraalveolar bubble film formation in vivo and excised lungs. Its further development [e.g. 2,60,61 ] leaded to confirmation of a bubble/bubble interface configuration in 1994 [57], i.e. there exists a foam film. This corresponds to the idea of black foam films as a model for the study of alveolar surface and stability and its existence in vivo. It is anticipated that the combination of the physical meaning of this phenomenon with the physiological reality would omit the word "hypothesis" with respect to the structure and alveolar stability. The clarification of the factors of alveolar stability can provide more successful diagnosing and treatment of respiratory disorders. It would be interesting to find the relationship between the parameters describing the stability of bilayers and multilayers (based on short-range molecular interaction in lateral and normal directions) as well as their surface properties (viscosity, elasticity modulus, etc.) given in literature [e.g. 62].

11.4.

BLACK FILM METHOD FOR ASSESSMENT OF THERAPEUTIC SURFACTANTS

The use of the black film method for characterising the action of therapeutic surfactants has been stimulated, as discussed above, by Scarpelli et al. [2,22,23,60,61,63,64], who define the formation of intraavleolar bubble and foam film formations as a normal condition in normally hydrated and laboratory-dried lungs both at the onset of breathing at birth and during normal breathing thereafter. This confirms the principal advantage of the black film method, i.e. the black film has an alveolar analogue in vivo [21,59].

Black Foam Films: Application in Medicine

755

Let us summarise the conditions of formation of a microscopic foam film in order to "serve" the in vivo situation. These are: film radius r from 100 to 400 ~tm; capillary pressure p~ = 0.3 - 2.5.102 Pa; electrolyte (NaC1) concentration Cel ~ 0.1 mol dm -3, ensuring formation of black films (see Section 3.4) and close to the physiological electrolyte concentration; sufficient time for surfactant adsorption at both film surfaces. Under such conditions it is possible also to study the suitable dependences for foam films and to use parameters related to formation and stability of black foam films, including bilayer films (see Section 3.4.4). For example, the threshold concentration Ct is a very important parameter to characterise stability and is based on the hole-nucleation theory of bilayer stability of Kashchiev-Exerowa. As discussed in Section 3.4.4, the main reason for the stability of amphiphile bilayers are the short-range interactions between the first neighbour molecules in lateral and normal direction with respect to the film plane. The binding energy Q of a lipid molecule in the foam bilayer has been estimated in Section 11.2.

1.0 - 0.5 rJ/ I

iJ!

I

i!.

Ilii

0.5

A

// m

llll

IN I0 20

EX

C

30 40 50 60 t, min

Fig. 11.12. Dependenceof probability Ws for black spot formation on adsorption time at phospholipid concentration of (A) 65 ~g cm-3, (B) 130 l.tg cm3 and (C) 170 ~tg cm-3; black spot formation (I4I,.= 1) by IN and SU required about 10 min at each concentration; EX required adsorption times of about 40 min at the lowest concentration (A) and about 12 rain 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 always ruptured; t = 22~ Foam film formation by three preparations used as surfactant replacement therapy by injection into the lungs in neonatal infants with surfactant insufficiency (RDS) has been

756

Chapter 11

studied [65]. The preparations contained 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, DPPC (Exosurf Neonatal (EX)). The therapeutic surfactants IN, SU and EX were characterised by the drainage time of the films up to the appearance of black spots at hcr,bl (see Section 3.2.) and by the threshold concentration Ct under properly chosen conditions. Fig.

11.12

demonstrates the probability for formation of black spots Ws vs. adsorption time for the three surfactants at 22~

and 37~

Thus it is possible to estimate the time for surfactant adsorption required for the formation of black spots. Table 11.2 presents the clinical and threshold concentrations for total phospholipids (PL) and for disaturated phosphatidylcholine (DSPC) in each preparation. The most abundant PL of the lung surfactant system is DSPC, principally the DPPC species, which is believed the essential determinant of surfactant function in vivo [2]. While DPPC 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. TABLE 11.2 Preparation

Clinical concentration

Threshold concentration,

Ct

Concentration above C,

22~ 37~ PL DSPC* PL DSPC* PL DSPC* PL DSPC* ~tg cm-3 ~tg cm-3 gg cm-3 gg cm3 ~tg cm-3 [xg cm-3 t-tg cm-3 ~g cm-3 IN 35 22.4 190 122 210 134 220 141 EX "* 13.5 "* 220 ** 250 ** 310 SU 25 12.5 450 225 490 245 600 300 Clinical concentration, threshold concentration (Ct) and concentration above Ct of IN, EX and SU. PL, phospholipids; *DSPC, disaturated phosphatidylcholine, namely, dipalmitoylphosphatidylcholine (DPPC) in IN and EX, and DPPC plus other disaturated PCs in SU; for EX, PL = DSPC = DPPC. **

These data show that the clinical concentration of DSPC is the 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, Ct for SU is more than twice that for IN. Temperature had little effect on Ct, which tended to be higher at 37~

and no effect on

the 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 EU.

Black Foam Films: Application in Medicine

757

The clinical concentration exceeds the actual intraalveolar concentration that might be expected during therapy, because the material is diluted in situ by the liquid in the air spaces and their surfaces

[65]. Other information gives some indication

of the surfactant

concentration in the normal lungs. The concentration in normal foetal pulmonary liquid [66] and the concentration required to restore alveolar function to immature neonatal infants and lambs [67] change from about 0.5 to 1.8 mg PL cm -3. These concentrations are close but slightly higher than both Ct and just above Ct. From the comparison of the parameters (Ct, drainage time, etc.), characterising the three preparations used it can be concluded than IN fits best 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 the lung surfactant in human amniotic fluid [20,28] and in lung lavage from normal rabbits [25]. EX, which is an incomplete surfactant and p~ 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 reological films of SU can function as such in vivo requires exploration, given the dynamic nature of the intraalveolar bubble films.

The comparison, reported by Cordova et al. [25], between the new in vitro tests for assessing structural and functional extracts of IN and SU and aqueous extracts from rabbit lung lavage is most informative. Shake test, click test, Pattle's stability test, bubble generation by gas dispersion from a single capillary and Exerowa black film method were used. It was shown that the Black Film Method exhibits a number of advantages providing unique information about the formation and stability of black films and clearly defines differences relative to the nature and concentration of the preparations. This is a good reason to employ it in clinical practice. Furthermore, it can give an insight of the physical causes of formation and stability of the alveolar surface, i.e. its most probable natural configuration. Thus, the processes related to respiratory disorders, determined by the "surfactant systems" of the lung might be better understood. Undoubtedly, it is better to strive for such a complete understanding of these phenomena, than to dispair of the human mind. With these words of belief we put the end of this book.

758

Chapter 11

REFERENCES

1. R.E. Pattie, Nature, 175 (1955) 1125. 2. E. M. Scarpelli, Surfactants and the Lining of the Lung, The Jonhs Hopkins University Press, Baltimore, MD, 1988. 3. R.J. King and J.A. Clements, Am. J. Physiol., 223 (1972) 715. 4. V.A. Berezovskii and V.Yu. Gorchakov, Poverkhnostno-aktivnye veshchestva legkogo, Naukova dumka, Kiev, 1982 (in Russian). 5. J.A. Clements, G.G. Platzker, D.F. Tierney, C.J. Hobel, R.K. Creasy, A.J. Margolis, D.W. Thiebault and W.H. Tooley, N. Eng. J. Med., 286 (1972) 1077. 6. L. Gluck, M. Kulovich, R. Borer, P. Brenner, G. Anderson and N. Spellacy, Am. J. Obstet. Ginecol., 109 (1971 ) 440. 7. G. Colacicco, Biochem. Biophys. Acta, 266 (1971) 313. 8. L. Horn and I. Gershfeld, Biophys. J., 18 (1977) 301. 9. A. Mueller-Tyl, Geburder und Frauenheilk, 37 (1977) 718. 10. I. Panaiotov, Ann. Univ. Sofia, Fac. Chem., 72 (1977/78) 95. 11. I. Panaiotov, Proc. 2nd Int. Symp. Lung Lipid Metabolism, Mechanisms of its Regulation and Alveolar Surfactants, Varna, 1980, p. 67. 12. C.M. Tiwary and J. Goldkrand, Obstet. Gynecol., 48 (1976) 191. 13. J. Goldkrand, A. Varki and S. McClurg, Am. J. Obstet. Gynecol., 128 (1977) 591. 14. S. Golde and G. Mosley, Am. J. Obstet. Gynecol., 136 (1980) 222. 15. A. Aberg and L. Gislen, Am. J. Obstet. Gynecol., 154 (1986) 68. 16. T.J. Garite, Clin. Obstet. Gynecol., 30 (1987) 985. 17. S. Schurch, J. Georke and J. Clements, Proc. Natl. Acad. Sci., U.S.A., 73 (1976) 4698. 18. J. Enhorning, J. Appli. Physiol. 43 (1977) 198. 19. D. Exerowa, D. Kashchiev and D. Platikanov, Adv. Coll. Interface Sci., 40 (1992) 201. 20. D. Exerowa, Z. Lalchev, B. Marinov and K. Ognyanov, Langmuir, 2 (1986) 664. 21. D. Exerowa and Z. Lalchev, Langmuir, 2 (1986) 668. 22. E.M. Scarpelli, Pediat. Res., 12 (1978) 1070. 23. E.M. Scarpelli, A. J. Mautone, D.O. DeFouw and B.C. Clutario, Anat. Res., 246 (1996) 245. 24. D. Exerowa, Z. Lalchev, B. Marinov and K. Ognyanov, BG Invention Certificate

Black Foam Films: Application in Medicine

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No 31659 (Reg. No 50291/1981), in: Bull. Inst. Inv. Razio., Bulgaria, No 3, 1982. 25. M. Cordova, A. Mautone and E. Scarpelli, Pediatr. Pulmonol., 21 (1996) 373. 26. D. Masson, K. Diedrich and G. Rehm, Geburde und Frauenheilk, 37 (1977) 579. 27. D. Kashchiev and D. Exerowa, J. Coll. Interface Sci., 77 (1980) 501. 28. D. Exerowa, Z. Lalchev and D. Kashchiev, Colloids & Surfaces, 10 (1984) 113. 29. C.J. Hobel, Pediatr. (St. Louis), 81 (1972) 78. 30. D. Exerowa, Z. Lalchev, B. Marinov and K. Ognyanov, Akush. i ginekol., 23 (1984) 457. 31. D. Exerowa, Z. Lalchev, B. Marinov and K. Ognyanov, Physiol. Zh., 33 (1987) 101. 32. D. Exerowa and Z. Lalchev, Abst. 9th Intern. Conference on Surface and Colloid Science, Sofia, 1997. 33. G. Georgiev, S. Avgarska, B. Marinov, Z. Lalchev and D. Exerowa, Akush. i ginekol., 3 (1986) 28. 34. G. Georgiev, S. Avgarska and D. Exerowa, Akush. i ginekol., 1 (1989) 41. 35. G. Georgiev, S. Avgarska, I. Savov and D. Exerowa, Akush. i ginekol., 6 (1989) 6. 36. M.N. Jones, Stad. Mod. Thermodyn., 1 (1979) 185. 37. P. Yeagle, The Membranes of Cells, Academic Press, New York, 1987. 38. D. Exerowa and A. Nikolova, Langmuir, 8 (1992) 3102. 39. A. Nikolova, D. Exerowa, Z. Lalchev and L. Tsonev, Eur. Biophys. J., 23 (1994) 145. 40. A. Nikolova and D. Exerowa, J. Stat. Phys., 78 (1995) 147. 41. D. Exerowa, B. Balinov and D. Kashchiev, J. Coll. Interface Sci., 94 (1983) 45. 42. A. Nikolova, D. Kashchiev and D. Exerowa, Colloids & Surfaces, 36 (1989) 339. 43. I. Penev, D. Exerowa and D. Kashchiev, Colloids & Surfaces, 25 (1987) 67. 44. D. Exerowa, R. Cohen and A. Nikolova, Colloids & Surfaces, 24 (1987) 43. 45. A. Lee, Biochim. Biophys. Acta, 472 (1977) 285. 46. D. Freer and B. Statland, Clin. Chem., 27 (1981) 1629. 47. J. Amenta and J. Silverman, Am. J. Clin. Pathol., 79 (1983) 52. 48. P.M. Kruglyakov and D. Exerowa, Pena i pennye plenki, Khimiya, Moscow, 1990 (in Russian). 49. Z. Lalchev, R. Todorov, Y. Christova, P. Wilde, A. Mackie and D. Clark, Biophys. J., 71 (1996) 2591. 50. Z. Lalchev, P. Wilde and D. Clark, J. Coll. Interface Sci., 167 (1994) 80. 51. Z. Lalchev, P. Wilde, A. Mackie and D. Clark, J. Coll. Interface Sci., 168 (1995)201.

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52. V.V. Erokhin and L.N. Filippenko, Priroda, 10 (1981) 32. 53. B. Hills, Thorax, 37 (1982) 713. 54. A. Bangham, N. Miller and R. Davies, Colloids & Surface, 10 (1984) 337. 55. E.R. Weibel, Morphometry of the Human Lung, Springer-Verlag, Heidelberg, 1963. 56. V.V. Erokhin, Funktsionalnaya morfologiya legkikh, Meditsina, Moscow, 1987 (in Russian). 57. E.M. Scarpelli and A.J. Mautone, Biophys. J., 67 (1994) 1080. 58. A. Nikolova and D. Exerowa, Langmuir, 12 (1996) 1846. 59. E.M. Scarpelli, Proc. Intern. Symp. Perinatal Medicine and Human Reproduction, Monduzzi Editore, Bologna, 1995, Chapter 2. 60. E.M. Scarpelli, B.C. Clutario, A.J. Mautone and J.Baum, Pflugers Arch. Eur. J. Physiol., 401 (1984) 287. 61. E.M. Scarpelli, B.C. Clutario and D. Traver, Pediatr. Res., 13 (1979) 1285. 62. M.J. Blank, J. Coll. Interface Sci., 75 (1980) 435. 63. E.M. Scarpelli, A.J. Mautone and M.R. Chinoy, Anat. Res., 244 (1996) 344. 64. E.M. Scarpelli, A.J. Mautone, M.R. Chinoy, D.O. DeFouw and B.C. Clutario, Anat. Res., (1997) submitted. 65. E.M. Scarpelli, A.J. Mautone, Z. Lalchev and D. Exerowa, Colloids & Surfaces B, 8 (1997) 133. 66. W. Tooley, J.A. Clements, K. Muramatsu, C. Brown and M. Schleuter, AM. Rev. Resp. Dis., 136 (1987) 651. 67. E.A. Egan, R.H. Notter, M.S. Kwong and D.L. Shapiro, J. Appl. Physiol., 55 (1983) 875.