Chemical synthesis and surface properties of an analog of the pulmonary surfactant dipalmitoyl phosphatidylcholine

235

Biochimica et Biophysics Actu, 488 (1977) 235-248 @ ElsevieriNorth-Holland Biomedical Press

BBA 57027

CHEMICAL SYNTHESIS AND SURFACE PROPERTIES OF THE PULMONARY SURFACTANT DIPALMITOYL PHOSPHAT~DYLCHOLINE *

J.G. TURCOTTE

OF AN ANALOG

a, A.M. SACCO a, J.M. STEIM b, S.A. TABAK c and R.H. NOTTER =***

a Department of Medicinal Chemistry, College of Pharmacy, University of Rhode Island, Kingston, R.I. 02881, b Department of Chemistry, Brown Uniuersity, Providence, R.I. 02912 and c Department of ChemicalEngineering, The Pennsylvania State University, University Park, Pa. 16802 (U.S.A.) (Received February 14th, 1977)

Summary An analog, (S)-[3-[ [2,3-bis(hexadecyloxy)propoxyl]hydroxyphosphi~yl]propyl] t~methylammonium hydroxide, inner salt, hydrate (DPPC-analog), of dipalmitoyl phosphatidylcholine (DPPC) was synthesized. The analog differs from pulmonary DPPC in that it: (1) is a dipalmityl diether rather than a dipalmitoyl diester; (2) has a trimethylammonium propylene phosphono polar head instead of a trimethylammonium ethyleneoxy phosphate head; and (3) has an absolute configuration opposite that of pulmonary DPPC. The dynamic surface pressure-area (T-A) characteristics of the DPPC-analog were determined on a Wilhelmy surface balance and compared with those of DPPC at both 23°C and 37°C. The respreading of both surfactants upon compression past collapse was measured quantitatively by a collapse plateau ratio criterion on successive cycles. The post-collapse respreading ability of the DPPC-analog was found to be si~ifi~~tly better than that of DPPC at both 23°C and 37°C as a function of several surface initial conditions. To complement the dynamic surface pressure-area determinations, differential scanning calorimetry and dilatometry measurements were carried out on DPPC and DPPC-analog water dispersions. The results showed that the liquid-crystalline transition temperature of the DPPC-analog is 45” C, slightly higher than the 41°C found for DPPC. Thus, the superior interfacial respreading found for the DPPC-analog at 23” and 37°C indicates that its bulk phase liquid-crystalline transition temperature is not as directly related to its surface properties as it is in the case of DPPC.

* Correspondence **

Present

address:

concerning University

this paper of

Rochester.

should

be sent

School

to J.G.

of Medicine.

Turcotte Rochester,

or R.H. N.Y.

Netter. 14642,

U.S.A.

236

Introduction The fascinating and complex pulmonary surfactant system has been the object of intense research since its discovery two decades ago. Much of the work has dealt with the dynamic surface tension of films of dipalmitoyl phosphatidylcholine (DPPC), the primary phosphoglyceride component of pulmonary surfactant [ 11. However, when surfaces tensions of DPPC are compared with those of mammalian lung surfactants, an important difference appears. Although DPPC very effectively lowers surface tension it responds poorly if squeezed from the surface during monolayer collapse [l-4]. This fact could affect the efficacy of aerosolized DPPC as a possible treatment for in vivo states of surfactant insufficiency, such as in the Respiratory Distress Syndrome of premature infants. The use of DPPC aerosols to reverse the surface tension-mediated effects of distress syndrome [5-91 in both human infants [6,8] and experimental animals [5,7-g] has achieved limited success [l]. One problem with any aerosol treatment is the delivery system, since it is necessary to generate aerosols of the proper particle size distribution to ensure that sufficient surfactant is deposited in the lung alveoli. This consideration is particularly relevant for the early studies of Robillard and co-workers [ 5,6], although recent advances in aerosol generator technology coupled with a better understanding of pulmonary deposition have partially alleviated the problem. By using radioactively labeled aerosolized DPPC, both Chu et al. [S] and Geiger et al. ilO] found that a small but measurable fraction (
237

n

H

A

4

i CH3(CHZ),4

B

4 CH,(CH,),,CO

II

0

I

CH$3POCH,CH,N(CH,), / t

OL

H

CH,OCH,,.,,

0 t 0 (CH,)JdCHZCH2CH2POCH~ * I ‘_

0

C’

Fig. 1. Molecular structures of pulmonary DPPC (I) and DPPC-analog (II). The DPPC-analog c.11) has a methylene (CH1) substituent at sites A’, B’, and C’ as compared with the two carbonyl functions (sites A and B) and the oxygen atom (site C) of the DPPC (I) molecule; 1 can be described simply as a diestrr phosphate arId II as a diether phosphonate, rrsprctlvely. The number of intervening atoms between the phosphorus and nitrogen atoms of I and 11 are the same. affording similar inter-atomic distances at these poiar ends of the molecules; the mrthylene substituent (site C’) of II can be considered bioisosteric with the oxygen atom (site C) of I. The absolute configuration (5’) of the DPPC-analog is opposite to that CR) of pulmonary DPPC; the dipalmitoyl phosphatidylcholine used in this work is racemic and is always termed ‘DPPC” in contrast to the term “pulmonary DPPC,” which should be understood to mean optically active dipalmitoyl phosphatidylcholine having the R-configuration, The effect of “replacing” the atoms at sites A. B. and C of DPPC with methylene substituents results in a molecule (II). which is more hvdrophobic than DPPC. This is characterized by differences in the surface properties of the diether phosphonate, its solubility in organic solvents. and chromatographic behavior. etc.. in comparison with DPPC. In addition. significant differences in the catabolism of 1 and II can be anticipated. DPPC, a naturally occurring phosphoglyceride. is susceptible to enzymic cleavage in viva by phospholipases. lipases. POSsibly other esterases. and chemical hydrolysis; the DPPC-analog would be expected t
the present paper, in which we report the chemical synthesis of a DPPC-analog, and compare its surface tension lowering and respreading properties with those of DPPC. The molecular structures of pulmonary DPPC (I) and the DPPCanalog (II) are shown in Fig. 1. Materials and Methods Analytical reagent grade or ACS-reagent grade chemicals and solvents were used for synthetic procedures and surface studies. Dipalmitoyl phosphatidylcholine (DPPC, Fig. 1) was obtained from the Sigma Chemical Company, St. Louis, MO. The DPPC gave a single spot by thin-layer chromatography on silica gel H with chloroform/methanol/water/satd. NH,OH (70 : 30 : 4 : 1, by vol.) [ 111. The RF values for of 300 and 700 pg samples were the same as that of a DPPC standard (Applied Science Laboratories, State College, Pa.). The fatty acid chain purity of the DPPC was analyzed by gas-liquid chromatography as 99’% palmitic acid. Synthetics were analyzed by thin-layer chromatography on pre-coated plates (silica gel 60F-254, Brinkman Instruments, Inc.) and visualized by exposure to I2 vapor, or spraying with concentrated H2S04 acid and heating at 120-150°C for 3-10 min, or spraying with molybdenum blue reagent. Infrared data were determined with a Beckman IR-8 spectrophotometer, and NMR data with a Varian A-60 spectrometer or a Joel C-60 spectrometer. Tetramethylsilane was the internal NMR standard, with chemical shift values (6, ppm) reported as the centers of proton resonances. Optical rotations were measured with a Perkin-Elmer model 141 Polarimeter. Elemental analyses water were performed by Micro-Analysis, Inc., Wilmington, Del. Triply-distilled was used to form all of the film subphases and in all of the cleaning procedures.

238

The surfactants studied were spread from hexane/ethanol (95 : 5, v/v) solutions, which were stored at -4°C. All experiments were carried out with solutions less than 3 days old, since aging effects occurred if solutions were kept for longer periods. (R)-2,3-Bis(hexadecyloxy)-1-propanol was synthesized following the general procedure of Baumann and Mangold [ 121. 3-(Bromopropyl)phosphonochloridic acid was prepared by a method similar to that of Baer and Stanacev [ 131 for the synthesis of 2-(bromoethyl)phosphonochloridic acid.

of DPPC-analog (S)-[ 3-[ [ 2,3-Bis( hexadecylocy)propoxyl]

Synthesis

hydroxyphosohinyllpropyl] trimethylammonium hydroxide, inner salt, hydrate (II). (R)-2,3-Bis(hexadecyloxy)-l-propanol, 2.70 g (5.0 mmol), was dissolved in 20 ml of chloroform. The solution was added (dropwise) to an ice-cold solution of approx. 10 mmol of (3-bromopropyl)phosphonochloridic acid dissolved in 20 ml of chloroform. Simultaneously, 1.01 g (10 mmol) of triethylamine dissolved in 20 ml of chloroform was added (dropwise) to the ice-cold mixture. The mixture then was stirred at room temp. under anhydrous conditions for 48 h. Water (2 ml) was added and the mixture was stirred vigorously for 1 h. The mixture was concentrated (rotoevaporator) under reduced pressure to obtain a red-brown-colored residue, which was then dissolved in 42 ml of chloroform/methanol/water (20 : 20 : 2, v/v/v); 30 ml of Amberlite IR-120 resin was added and the mixture was stirred for 1 h. The mixture was filtered and the resin was washed with 42 ml of the same solvent mixture. The filtrates were combined and concentrated to yield 6.29 g of crude product which was dissolved in a minimal amount of chloroform and chromatographed on a column (3.3 X 40 cm) of 130 g of silica gel (70-230 mesh, ASTM, particle size 0.062-9.200 mm, Brinkman Instruments, Inc.). The column (packed in chloroform) was eluted successively with chloroform (1 l), chloroform/methanol (99 : 1, v/v, 1 1) and chloroform/methanol (98 : 2, v/v, 1 1). The product was collected in fractions (50 ml each) 5OE-66, which were evaporated (4045°C). Crystallization of the product from acetone at room temp. gave 3.22 g (88%) of the bromophosphonate intermediate [ (S)-2,3-bis(hexadecyloxy jpropyl hydrogen (3-bromopropyl)phosphonate] as a fine white amorphous solid: m.p. 40-42°C; [a]‘,’ + 3.0 (c, 0.51, n-hexane), + 1.51 (c, 5.2, CHCl,); RF (thin layer chromatography) 0.67, chloroform/methanol (10 : 4, v/v); infrared (KBr, cm-‘) 2920 and 2860 (CH*, CH3 stretching), 2280 (PO-H stretching), 1470 (CH, bending), 1380 (P = 0 stretching), 1120 and 1050 (C-O-C stretching), 1000 (P-OH stretching), 720 (CH, rocking); NMR, 60 MHz (C’HCl,, 6) 0.89 (triplet, 6, CH,), 1.28 (singlet, 56, CH,), 1.992.5 (multiplet, 4, PCH,CH,), 3.3-3.8 (multiplet, 9, CH,O, CH2Br, CH), 3.9-4.3 (multiplet, 2, CH,OP). Analysis:

C38H7805BrP Calculated : C, 62.79; Found : C, 62.99;

H, 10.82; Br, 11.13; P, 4.26% H, 10.63; Br, 10.99; P, 4.48%

The bromophosphonate intermediate, 1.20 g (1.65 mmol), was dissolved in 50 ml of methylethylketone. The solution was heated slowly to 45°C and then anhydrous trimethylamine was bubbled into the solution for approximately

239

30 min; the mixture then was stirred at 50°C (oil bath) for 72 h, cooled to room temp. (fine white ppt.), then to 0°C (ice bath), and filtered to yield 1.06 g of crude solid. The solid was dissolved in 21 ml of chloroform/methanol/ water (10 : 10 : 1, v/v/v), 550 mg (2 mmol) of Ag,C03 was added, and the mixture was stirred vigorously for 1 h. The mixture was filtered and the residue was washed with 21 ml of the same solvent mixture. The filtrates were combined and 25 ml of Amberlite IR-120 resin was added. The resin mixture was stirred vigorously for 1 h, filtered, and the resin was washed with 42 ml of the same solvent mixture. The combined filtrates were concentrated (rotoevaporator) to yield a crude solid. Crystallization from chloroform/acetone (1 : 2, v/v) yielded 854 mg (72.0%) of the DPPC-analog (II) as a light-yellow-colored (decomposition); [a]: - 6.0 (c, 0.46, crystalline solid : m.p. 205-206°C CHCl,), -5.1 (c, 0.51, CHCl,); RE. (thin-layer chromatography) 0.6 chloroform/methanol/water (6 : 4 : 1, v/v/v);infrared (KBr, cm-‘) 3390 and 3280 (HOH 2600 (PO-H stretching), stretching), 2920 and 2860 (CH,, CH, stretching), 1670, 1470 (CH, bending), 1190 and 1170 (P = 0 stretching), 1075 (C-O-C stretching), 718 (CH2 rocking); NMR (C2HC13, 6) 0.88 (singlet, 6,CH,), 1.28 (singlet, 56, CH2), 1.72 (multiplet, 4, PCH2CH2), 3.25 (singlet, 9, N(CH,)i), 3.60 (multiplet, 11, CHPN’, CH20 and CH). Analysis:

Surface

C4,HS605NP * Hz0 Calculated : C, 68.18; H, 12.30; N, 1.94; P, 4.29% Found : C, 68.06; H, 12.21; N, 1.87; P, 4.57% pressure-area

(n-A) methods

All T-A behavior was investigated with a custom-designed Wilhelmy balance [ 14,151. The balance permitted constant temperature and humidity operation, and cycling speed could be varied from 0.01 to 10 cycles/min. For all experiments, a moderate cycling rate of 0.25 cycles/min was used to display dynamic 7-A behavior without the complication of vibrations induced by high cycling rates. The surface balance permitted the use of both a recessed teflon dam-type barrier and a teflon ribbon barrier which completely contained the surfactant film. The teflon ribbon barrier was particularly important for DPPC-analog T-A films at body te”mperature (37”(Z), which showed a high degree of leakage past the recessed teflon dam barrier. Such leakage was greater for DPPC-analog films than for DPPC films, and suggest a relatively lower surface viscosity for the former. However, this surface property was not measured quantitatively. Calorimetry

and dila tome try methods

The DPPC-analog was thermally characterized with a modified Perkin-Elmer DSC2 scanning calorimeter [16]. Samples of 2--3 mg in lo-12 ~1 of water or Ringer’s solution were scanned in both ascending and descending modes at rates of 5 and 10 degrees/min. The standard for calibration was stearic acid. To eliminate the possibility of kinetic effects at the rapid scan rates used in the differential scanning calorimeter, the thermal behavior of the DPPC-analog was also defined by scanning dilatometry. The instrument, based upon the principle of buoyant density, is operated at a scan rate of 5 degrees/h [ 171. Dry weights for both calorimetry and dilatometry were determined with a Cahn recording vacuum electrobalance.

Results

and Discussion

The interfacial behavior of DPPC and DPPC-analog films was characterized at both 23°C and 37°C. At each temperature, experiments were carried out at both low and high initial surface concentrations. The low concentrations were chosen to be small enough to that no measurable effect upon surface tension was apparent before film compression. The higher concentration films were initially applied in amounts in excess of that required to cover the surface with a monomolecular film.

Interfacial

properties

at low initial film concentratiolk9

Initial surface concentrations of 200 A2/molecule for DPPC and 150 A2/molecule for the DPPC-analog were chosen. An important parameter of films formed under initial conditions of low surface concentration is “lift off” behavior, characterized by the surface concentration at which the monolayer first generates a measurable surface tension lowering. This concentration depends upon the accuracy of the surface tension measuring technique. Essentially all insoluble surfactants display gaseous film behavior at very large surface areas, generating surface pressures of about 1 dyne/cm2 or less (e.g. ref. 18). To characterize this gaseous film region accurately, however, requires an extremely sensitive millidyne surface balance such as that developed by Pegano and Gershfeld [ 191. Since our modified Wilhelmy balance lacks such sensitivity, we characterize an apparent film “lift-off” directly into the expanded film state, a procedure that has been followed by other investigators for DPPC films [ 11. The “lift-off” behavior of DPPC and DPPC-analog films at 23°C and 37°C is shown in Figs. 2 and 3. At 23”C, the DPPC-analog film is seen to be signifi-

70-

-

70-

60-

-

60-

250 :. 0

_

50-

y403 ae

_

40-

.a; \"

30-

a3O:

_

5 *no-

IO-

Area Fig.

2. n-A

per Molr’_de

behavior

of

(i?/Molec a DPPC

Area

1 film

at 23 and

37

per Molecule C: initial

(

i2 /Molec)

concentration

200

A2 /molwulr.

241

0 0

15 30

45

60

75 Area per Molecule (i”/

Fig. 3. n-A behavior

90 105 120 135 Molec.)

of a DPPC-analog

0

0

I5 30

45

60

75 90

Area per Molecule (i2/

film at 23 and 37’C;

105 120 135 I50

Molec.)

initial concentration

150 ~*/mOkcule.

cantly more expanded than DPPC at large areas, with an apparent “lift-off” of about 125 AZ/molecule compared to about 102 ~*/molecule for DPPC. As compression proceeds to film collapse, however, this initial difference between the DPPC and DPPC-analog films disappears, and both monolayers undergo a sharp collapse at essentially the same area of 39-40 A2/molecule. This 23°C surface behavior seems consistent with the structural differences (Fig. 1) between the DPPC and DPPC-analog molecules. The two molecules have similar cross-sectional areas, and thus their limiting areas at monolayer collapse should also be similar. At these small surface areas the molecules are packed tightly together, and differences in the freedom of chain rotation and interaction become secondary to the tight geometrical packing imposed on the film molecules. Any intermolecular force differences in surfact~t-surfac~nt or su~act~t-subph~e inte~ctions may affect the surface pressure generated at collapse, but in terms of collapse area the molecular geometry is dominant. Interestingly, the 23°C results of Figs. 2 and 3 show that the collapse surface pressures and the collapse areas of DPPC and DPPC-analog are equal. This surface pressure equality is not required, however, and the 37°C results (Figs. 2 and 3) show that at this temperature DPPC-analog collapses at a surface pressure 18 dynes/cm’ less than DPPC, although the collapse areas are still similar. However, very high surface pressures for phosphoglyceride films such as those of DPPC has been shown to be a dynamic compression effect [14,20]. Consequently, it is possible that DPPC-analog “lift-off” films at 37°C could reach higher collapse pressures comparable to DPPC if cycling rates were greater than the moderate 0.25 cycles/min used here. For the purposes of this work, one of the most relevant comparisons between the n-A behavior of DPPC and DPPC-analog films is the respreading of film molecules after compression past monolayer collapse. No uniformly

242

valid criteria for the qu~titative characterization of dynamic respreading behavior in surfactant films on successive cycles past collapse are available. However, it is clear that respreading behavior is directly related to the “‘squeeze out” or ejection of film molecules from the monolayer on compression and their subsequent re-entry to the interface upon film expansion [ 11. Such ejection may occur by surfactant molecules being pushed up into multilayered collapse structures or by the forced expulsion of surfactant into subphase lamellae. Regardless of mechanism, the collapse process is directly related to the loss of surfactant from the monolayer, and for this reason we have adopted a relatively easily applicable collapse plateau criterion as a characterization of respreading behavior. The length of the collapse plateau generated for a given insoluble surfactant film is at least an indirect measure of molecules ejected from the most condensed surface monolayer possible for that surfactant. Consequently, if successive compression-expansion cycles are carried out between the same end points on the surface balance, the reproducibility of collapse plateau lengths for successive cycles gives a measure of how effectively ejected molecules are able to respread or re-enter the interface. For example, if the lengths of the collapse plateaus observed for the first and second compressions of a given surfactant are the same, then all the molecules ejected from the monolayer on the first compression were able to re-enter the interface to be available to lower surface tension on the second compression. In general, however, some fraction of the molecules ejected on the first compression may be irrevocably lost from surface by incorporation into subphase lamellae or by the formation of irreversibly stable surface collapse structures. If this occurs, then fewer surfactant molecules will be available for effective monolayer coverage on the second compression, and the collapse point of this compression will be displaced to a smaller surface area. Thus, if compression is carried to the same endpoint, the length of the collapse plateau will decrease on successive cycles of the surface. Aside from collapse plateau lrngth and reproducibility on successive cycles, several other n-A parameters may give indications of respreading behavior. For example, T-A compression-expansion hysteresis and n-A “lift-off” on successive cycles are also related to respreading phenomena. However, the collapse plateau criterion seems the most free from misinterpretation since it directly reflects the phenomena which necessitates respreading: the ejection of molecules from the monolayer upon compression of the monolayer past collapse. Moreover, the length of the collapse plateau also directly indicates the range over which maximal surface tension lowering occurs, and this information is physiologically relevant for films related to pulmonary surfactant. The major difficulty in basing a respreading criterion upon collapse plateau lengths is the determination of the point on a T-A curve that actually represents the onset of collapse. Several collapse criteria have been suggested in the literature [18]. Since no single method has been agreed upon, we have arbitrarily selected the following method of delineating compression past collapse. On a given r-A compression, a line is drawn tangent to the point of steepest slope (i.e., the most incompressible film) on the curve. Then, another line is drawn tangent to the z-A curve at end compression and extended until it crosses the first line. The distance from end compression to this point of intersection is

243

chosen as the length of the collapse plateau. Since this same criterion is applied to characterize collapse plateau lengths on successive cycles of a given film, its arbitrary nature is somewhat alleviated, and a meaningful measure of respreading efficiency should result. This collapse plateau length criterion is more meaningful for some surfactants than for others, and can be uncertain if n-A compression curves change their slopes very gradually with changing area. The results of analyzing Figs. 2 and 3 in terms of collapse plateau lengths are shown in Table I. All these collapse plateau results are from cycles with compression ratios (initial surface area : final surface area) chosen at 8 : 1 in order to allow a considerable portion of the compression to be in the collapse regime. The data of Table I clearly show the superior respreading of DPPC-analog films as compared to those of DPPC. For example, at 23°C the collapse plateau ratios for cycle B/cycle 1 and cycle 7/cycle 1 were 0.73 and 0.60 for DPPCanalog films and 0.25 and 0.22 for DPPC. Thus, after seven compression-expansion cycles past collapse, a significant fraction of the originally spread DPPCanalog remains at the interface in a state that allows maximal surface tension lowering. However, this is not the case for DPPC films, and after seven surface cycles a major portion of the DPPC ejected from the interface during monolayer collapse on previous cycles has apparently not re-entered the surface. In terfacial properties at high initial film concentrations Most experiments at high initial concentrations were carried out at concentrations, chosen from monolayer collapse points, of 25 AZ/molecule and 15 AZ/molecule. These concentrations were beyond those required for monolayer coverage, and allowed the bulk of the first compression to be in the collapse regime. Hence respreading efficiency becomes a dominant factor on successive cycle T-A behavior. In addition, some experiments were carried out with an initial concentration of 50 A*/molecule, which is just prior to the approx. 40 A*/molecule monolayer collapse point of saturated phosphoglycerides such as DPPC.

TABLE

I

COLLAPSE Surfactant

AND

RESPREADING Initial

spreading

condition (A*

CHARACTERISTICS Collapse (arbitraw

OF

plateau

THE

DPPC

AND

DPPC-ANALOG Collapse

length

plateau

units) --

/molecule) Cycle

1

Cycle

2

Cycle

7

2/l

7/I

DPPC-analog

150

at 23OC

0.78

0.57

0.47

0.73

0.60

DPPC

200

at 23’C

0.69

0.17

0.15

0.25

0.22

DPPC-analog

150

at 37Oc

1.0

0.63

0.31

0.63

0.31

DPPC

200

at 37Oc

0.75

0.14

0.05

0.19

0.07

DPPC-analog

25

at 23’C

3.7

1.8

1.55

0.49

0.42

DPPC

25

at 23°C

4.0

1.5

0.94

0.38

0.24

DPPC-analog

25

at 37’C

2.8

1.2

0.9

0.43

0.32

DPPC

25

at 37°C

2.3

0.38

0

0.17

0

DPPC-analog

50 at 23OC

3.1

1.4

1.1

0.45

0.35

50 at 37OC

2.8

1.2

0.9

0.43

0.32

15

at 23°C

3.7

2.3

1.8

0.62

0.49

15

at 37Oc

2.8

1.7

1.35

0.61

0.48

ratios

244

Fig. 4 shows the first, second, and seventh cycles of DPPC and DPPC-analog films spread to initial surface concentrations of 25 AZ[molecule at 23°C. Although this surface concentration is beyond that required for monolayer coverage, the initial surface pressure is significantly less than the -70 dynes/ cm’ collapse maximum for both these surfactants at 23°C (Figs. 2,3). This apparent anomaly can be accounted for by differences between the dynamic and static surface pressures and is discussed in detail elsewhere [20]. Both DPPC and DPPC-analog films spread by means of a spreading solvent do not reach initial surface pressures greater then approx. 50 dynes/cm2, even though surface pressures reached under dynamic compression are considerably higher. In the surface excess case at 23°C (Fig, 4), DPPC-analog respreading after collapse is again superior to that of DPPC. However, the respreading differences between these two surfactants are not its pronounced for initial conditions of surface excess as they are for low initial surface concentrations. This behavior is shown quantitatively by the collapse plateau ratios of Table I. The DPPCanalog film gives cycle B/cycle 1 and cycle ‘I/cycle 1 ratios of 0.49 and 0.42, respectively, while the DPPC film gives corresponding ratios of 0.38 and 0.24. Thus, both films show an initially large irreversible ejection on the first compression followed by a more reversible ejection on succeeding cycles. This enhancement of respreading of DPPC at high initial concentrations and room temperature has been noted previously by Berg [21] and Tabak and Netter [3]. The mechanism for this effect is not known, but a possible explanation is that the large number of molecules squeezed from the interface on the first compression hinder ejection of molecules on subsequent cycles, or at least

60

"E

50

9 I &? f. 40

f f 30 e (L z ,o 20 t; In

0 0

IO

20

30

40

50

60

70

80

SO

100

0

IO

20

Percent Trough Areo - = OPPC --- = DPPC ANALOG Fig. 4. B’irst second and seventh x--e cvcles 25 A2 imolecule. Fig. 5. First, second 25 ;t~,im&e&?.

and seventh

n-A

30

40

50

60

70

80

90

Percent Trough .4reo

cycles

of DPPC arid

DPPC-analog

of DPPC and DPPC-analog

= DPPC fihs

at

--- = DPPC ANALOG 23” c: iriitial cancentration

films at 37°C; initial concentration

100

245

modify the ejection process to make it more reversible at room temperature. Fig. 5 shows the first, second, and seventh compression-expansion cycles of DPPC and DPPC-analog surface excess films at 37°C. At body temperature so much DPPC is irreversibly ejected from the interface that by the seventh cycle essentially all of the excess DPPC has been lost through collapse ejection, and the end compression point is just prior to collapse. The DPPC-analog film maintains a concentration in excess of that necessary to reach collapse at end compression on the seventh cycle at 37°C (Fig. 5). This superior respreading is shown quantitatively in Table I. Another property of surface excess DPPCanalog films (Fig. 5) is its ability to generate surface tension of approx. 1 dyne/ cm* on a water subphase. This was not the case for DPPC-analog films spread to low initial concentrations at 37”C, as shown in Fig. 3. Apparently, the larger number of molecules initially present in the surface excess experiment creates a surface condition that generates lower surface tensions. Because of the physiological relevance of maximal surface tension lowering for pulmonary surfactants, further work should be aimed at obtaining a mechanistic description of the interfacial conditions leading to very low surface tension. Initial conditions of 15 and 50 A2/molecule were also investigated for DPPC-analog films at 23°C and 37°C. These results are shown in Figs. 6 and 7 for the second and seventh compression-expansion cycles, and respreading is analyzed quantitatively in Table I. 11 all cases the DPPC-analog film shows good respreading. Again, apparently enough material is ejected from the interface during the first conlpression to hinder irreversible ejection on subsequent compressions past collapse. Thermodynamic properties Calorimeter scans of DPPC and the DPPC-analog show that both materials exhibit a well-defined transition above body temperature at 41” C in DPPC and

70-

60-

“E

soE E

Q40-

-

E : zl &30z z

_

520-

IO-

0

IO

20

30

40

Percent

Fig. 6. Second K= ~molecule.

50

60

70

60

90

100

0

IO

20

Trough Area

and seventh

n-A cycles

30

40

Percent

of a DPPd-analog

50 Trough

60

70

80

90

100

Area

film at 23OC; initial concentrations

15 and 50

0 Fig.

7.

IO

20

Second

30 40 Percent and

50 60 70 Trough Area

seventh

n-A

80

cycles

90

of

100

0

IO

a DPPC-analog

20

30 40 Percent film

at

50 60 70 Trough Area 37’C:

initial

80

93 loo

concentrations

15

and

50

R*/moleeule.

45°C in the analog. This change of state has been extensively characterized for DPPC, and is known to occur in biological membranes [ZZ]. It is a general property of phospholipids in the lamellar nonformation which they assume when suspended in aqueous media. The transition temperature depends upon the fatty acid composition, and ascends from 23°C to 41°C to 60°C a~ fatty acid chain lengths progress from C,4 in dimyristoyl to C,, in dipalmitoyl to Cl8 in distearoyl phosphatidylcholines, respectively. The smaller precursor peak produced by DPPC, which is characteristic of all true phosphatidylcholines, was not observed with the DPPC-analog. The transitions in DPPC and the DPPC-analog trace the same thermal course in the dilatometer as in the calorimeter, although the dilatometer is operated at a scan rate of only 5 degrees/h. Thus: the event seen in the calorimeter is not kinetically determined, and has significance for the surface balance studies. The heats of transition were found to be 8.5 calimol for DPPC and 8.3 calimol for the analog, while the volume changes through the transition were 3.5% for DPPC and 3.1% for the analog. In view of the similarities in these physical properties of DPPC and the anaLg, the ability of the latter to spread at the air-water interface is unexpected. The behavior of DPPC at the air-water interface at 37”C, 4°C below its transition temperature, is characteristic of ordinary saturated phosphatidylcholines in the crystalline state. Such saturated phosphatidylcholines spread very poorly from the subphase onto the surface at temperatures immediately below their transitions, but more rapidly at temperatures above. This change in spreading kinetics occurs abruptly at the transition temperature, which is 41°C for DPPC. This is convention~ly ascribed to the change in state of the lamellar structure in the hypophase from a solid to a liquid, from which unfolding at the surface can occur more easily. It is interesting to note that films of both

247

DPPC and the analog spread more rapidly at 23°C than 37”C, although this effect is far less pronounced for the analog than for the DPPC. In general, both the liquid-~~st~line transition temperatures of phosphatidylcholines in the bulk phase, and their ability to lower surface tension when spread from a solvent at the air-water interface in surface studies, are found to increase with increasing saturation and chain length. Thus, a conventional pure component phosphatidylcholine to be used as a lung surfactant substitute must meet two apparently contradictory requirements. First, it should have long saturated fatty acid chains to enable it to develop high surface pressures when spread at the interface. Howe rer, it should also have a low liquid-crystalline transition temperature to facilitate spreading, particularly after collapse into surface multilayers or subphase lamellae. One way to achieve these properties is to combine other components with phosphatidylcholines in mixed films. However, the DPPC-analog synthesized here is found to behave in a remarkable way. Its long saturated chains, which ensure high surface pressure upon compression, also give rise to a transition at 45”C, well above body temperature. Thus, in terms of bulk phase behavior when dispersed in water at 23°C or 37” C, the DPPC-analog is crystalline and, were it to behave as ordinary phosphatidylcholine, would hardly be expected to spread at all. Nevertheless, its respreading after collapse is reasonably rapid, and is in fact superior to ordinary DPPC which has a lower liquid-c~st~line transition temperature. Thus, the analog presents a new kind of behavior which is an extremely desirable one for a surfactant to have: it permits the use of saturated hydrocarbon chains, but at the same time its spreading does not seem to be seriously affected by its crystallinity. The present work has shown that it is possible to synthesize a DPPC-related compound which exhibits effective surface tension lowering together with enhanced post-collapse respreading characteristics compared to DPYC itself. Because the present work deals primarily with respreading properties, no attempt has been made to characterize such important properties as subphase ion effects or equilibrium surface pressure behavior. Moreover, mixed film studies with DPPC-DPPC-an~og binary films are not presented, nor are the properties of DPPC and DPPC-analog aerosols. All of these important parameters are currently under investigation. Even without these further studies, however, the present results validate the approach of synthesizing surfactants tailored to yield specific kinds of surface properties. Such an approach has in fact been very productive in many en~neering and industrial applications, and apparently has great potential for surfactant related problems in living systems. For applications to living systems, however, the pharmacology, toxicity, and metabolism of any pulmonary surfactant analog must be assessed in suitable animal models before its ultimate clinical potential can be judged,

This work was supported in part by NIH Grants HL-16207, HL-17120, and GM-20545. The technical assistance of Mary T. Walsh and Francis J. Scavitto at Brown University is gratefully acknowledged.

248

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