Osmotic pressure studies of some phospholipid sols

Chem. Phys. Lipids 4 (1970) 261-268 © North-Holland Publ. Co., Amsterdam.

OSMOTIC

PRESSURE

STUDIES OF SOME

PHOSPHOLIPID

SOLS

I. W. KELLAWAY and L. SAUNDERS Department of Pharmaceutical Chemistry, The School of Pharmacy, University of London, England

The number average molecular weight for ultrasonicated phosphatidyl choline (PC) in water and lysophosphatidyl choline (LPC) in water agreed with values previously determined by light-scattering measurements, indicating a narrow molecular weight distribution. The second virial (solvent interaction) coefficients, As, were also of the order indicated by light scattering measurements. In 25 per cent (w/w) n-propanol/water, a solvent system selected from a consideration of the ternary phase diagram for PC/n-alkanol/water, aggregates were found to contain only 3 to 4 monomers, for both PC and LPC. A maximum molecular weight was observed for the mixed phospholipids when they were present in weight proportions of 3 LPC to 2 PC, corresponding to molar proportions of approximately 2:1.

Introduction A l t h o u g h o s m o m e t r y was a technique e m p l o y e d by T h o m a s I in 1915 to m e a s u r e the micellar size o f P C sols o f d u b i o u s purity, very few o s m o t i c pressure d e t e r m i n a t i o n s have been recorded for a q u e o u s p h o s p h o l i p i d dispersions. Recently T i n k e r a n d Saunders 2 d e t e r m i n e d the micellar weight o f the i s o t r o p i c solution f o r m e d by dissolving P C in 25 per cent (w/w) n - p r o p a n o l / water. The present w o r k extends the osmotic studies o f P C a n d L P C in b o t h water a n d w a t e r - p r o p a n o l solvents.

Experimental Materials P C a n d L P C were p r e p a r e d , purified a n d their p u r i t y assessed with thin layer c h r o m a t o g r a p h y b y m e t h o d s previously described 8). A n a l y s i s figures : P C : N 1.78%, P. 3.79%, iodine value 71. L P C : N 2.66%, P 5.91% M e t h a n o l a n d E t h a n o l were A n a l a R grade. 261

262

I. W. KELLAWAY AND L. SAUNDERS TABLE 1

Results for PC, LPC and mixtures (by weight) of the two natural lipids Sol

Concentration C (g/l)

n(cm of solvent)

n/C

Mn

A2(cm of s°lvent/c2)

Coefficient of linear correlation

32.28 64.56 96.84 156.0

0.41 0.87 1.33 2.28

0.0127 0.0135 0.0137 0.0146

2.04 x 106

1 x 10-5

PC/ LPC 4:1

25.0 50.0 75.0 100.0

0.30 0.60 0.92 1.23

0.0120 0.0120 0.0123 0.0123

2.13 x 106

1.19 x 10-~

0.925

PC/ LPC 3 :2

25.0 50.0 75.0 100.0

0.26 0.51 0.74 0.94

0.0104 0.0102 0.0098 0.0094

2.34 x 106 -1.08 x 10 2

--0.990

PC/ LPC 1:4

25.0 50.0 75.0 100.0

1.77 4.97 9.81 16.54

0.0708 0.0994 0.1308 0.1654

667 000

1.26 x 10-s

0.999

2.67 5.49 8.60 11.92

0.2660 0.2734 0.2855 0.2968

98 800

1.04 x 10-z

PC

LPC

10.04 20.08 30.12 40.16

0.983

0.995

No measurements were possible with a sol containing PC/Lyso PC 2: 3, the high viscosity of the sol preventing the filling of the solution chamber.

n - p r o p a n o l was o f r e a g e n t g r a d e p u r i f i e d by d i s t i l l a t i o n o v e r m a g n e s i u m propoxide.

Instrumentation M e c h r o l a b ( H e w l e t t - P a c k a r d ) 503 H i g h - S p e e d M e m b r a n e O s m o m e t e r .

Membranes C e l l u l o s e ester m e m b r a n e s f o r a q u e o u s s o l v e n t s as s u p p l i e d b y S c h l e i c h e r a n d S c h u e l l w e r e used. T h e m e m b r a n e s , w h i c h w e r e s u p p l i e d in a d i l u t e a q u e o u s b a c t e r i c i d e , w e r e w a s h e d t h o r o u g h l y in w a t e r b e f o r e s t o r a g e in w a t e r o r 25~o n - p r o p a n o l / w a t e r .

Measurements All m e a s u r e m e n t s w e r e c a r r i e d o u t in d u p l i c a t e at 25 ° w i t h t h e g a i n c o n t r o l

263

OSMOTIC PRESSURE STUDIES OF SOME PHOSPHOLIPID SOLS TABLE 2

Results for 1 Cla.o LPC and 1 Cls.~ LPC dispersed in 0.9 ~o NaCl Compound

1 C18.0 LPC

1 C18.2 LPC

Concentration C (g/l)

z~

zr/C

0.667 1.000

0.55 1.18

0.83 1.18

1.430 1.667 2.000

1.91 2.42 3.16

1.34 1.45 1.58

0.667 1.429 1.667 2.000

1.285 3.09 3.76 4.70

1.927 2.162 2.26 2.35

Mn

A2

Coefficient o f linear correlation

65 500

0.533

0.974

14 600

0.321

0.999

of the osmometer at the maximum setting without causing oscillations of the solvent reservoir. Solvent equilibrium was assessed by changing the solvent three times, the readings taken after 30 min agreeing to _+0.01 cm of solvent.

Osmometry of aqueous phospholipid systems Bovine albumin powder Fraction V from bovine serum plasma (Armour) without further purification was selected as a sample to give test solutions for a non-permeating solute. The unopened sample had been stored at 5°C and the solutions were made immediately before use to reduce solute association. The number average molecular weight obtained was 6.9 × 104 in agreement with the literature values by light scattering4), and by osmotic pressure ~). All sols containing PC were prepared by the ultrasonic dispersion technique, the high molecular weights of PC in these sols gave rise to the difficulty of dispersing sufficient solute to obtain reasonable osmotic pressures. This was overcome by dissolving excess PC in the minimum quantity of ether, adding distilled water and removing the ether by the application of reduced pressure. Pure nitrogen (white spot) was bubbled through the coarse dispersion for ten minutes to displace air, before sonicating for two hours at 20 kHz using a 60 watt Mullard ultrasonic generator and a titanium probe. During sonication, the sample was kept cool by a surrounding ice bath. This period of sonication under the conditions stated, did not cause any hydrolysis of pure PC to lyso PC, detectable by thin layer chromatography. The excess PC and fragments of titanium from the probe were removed by centrifugation for two hours at 27 000 #. The concentration of the PC in this master solution was determined by weighing the residue remaining after evaporation of the water from a known weight of sol in a vacuum oven.

264

I . W . KELLAWAY AND L. SAUNDERS TABLE 3

Results for PC, LPC and mixtures of the two lipids, in 25 ~o n-propanol/75 ~ water system Sol

PC

PC/ LPC 4:1 PC/ PC 3:2

PC/ LPC 2:3 PC/ LPC 1:4

LPC

Concentration C (g/l)

n

n/C

Mn

A2

Az

0.503 1.007 1.796 2.012 2.515 5.030

4.78 8.97 14.313 14.88 17.944 19.493

9.505 8.911 7.968 7.397 7.135 3.875

2600

-- 1.243

-

0.419 0.837 1.675 3.349

3.391 6.317 10.919 14.728

8.101 7.545 6.521 4.398

3000

-- 1.260

0.253 0.507 1.014 2.028 3.041

1.790 3.218 5.150 7.162 8.870

7.063 6.349 5.080 3.532 2.917

3300

--3.185

+0.518

0.993 1.985 2.978 3.970

6.350 11.100 14.606 16.559

6.398 5.592 4.905 4.171

3700

-- 0.742

-

0.421 0.843 1.264 1.897 3.793

3.797 6.146 7.587 8.648 10.635

9.010 7.292 6.001 4.560 2.804

2400

-- 4.494

0.286 0.536 0.857 1.071

3.540 6.270 9.420 11.071

12.392 11.707 10.992 10.334

2000

-- 2.564

T h e h i g h v i s c o s i t y o f the m i x e d P C / L P C

Coefficient of linear correlation

-- 0.998

--

1.000

-- 0.999

+ 0.635

-

-- 0.998

sols 6) c a u s e d c o n s i d e r a b l e re-

sistance in the s y p h o n s y s t e m u s e d to b r i n g the sols i n t o t h e s o l u t i o n c h a m b e r o f the o s m o m e t e r . A n y t r a p p e d air b u b b l e s w e r e r e m o v e d f r o m the sols b e f o r e filling the s o l u t i o n c h a m b e r , by the r a p i d a p p l i c a t i o n o f a n e g a t i v e pressure.

Treatment of results G r a p h s o f n o n c e n t i m e t r e s o f s o l v e n t ) a g a i n s t C ( c o n c e n t r a t i o n in g/l) a n d n/C a g a i n s t C w e r e p l o t t e d ; the first to e n s u r e t h a t the p l o t passes t h r o u g h t h e o r i g i n a n d t h e s e c o n d f o r e x t r a p o l a t i o n to e v a l u a t e ( n / C ) c ~ 0 . R e g r e s s i o n

2.155 3.695 4.300 6.304 7.080 7.894

0.099 0.198 0.248 0.495 0.660 0.792

R a p i d solute p e r m e a t i o n of the m e m b r a n e suggesting presence o f m o n o m e r s

25.0

50.0

100.0

21.763 18.659 17.374 12.734 10.727 9.967

9.505 8.911 7.968 7.397 7.135 3.875

1100

2600

1.24

-- 34.88

--

- - 2 . 6 x 10 -2

4.780 8.970 14.313 14.880 17.944 19.493

32000

0.503 1.007 1.796 2.012 2.515 5.030

0.775 0.669 0.579 0.562

1.70 4.21 5.02 6.04

1 x 10 -5

A2

2.195 6.294 8.676 10.758

2.04 x l0 s

Mn

21.09

0.0127 0.0135 0.0137 0.0146

n/C

0.41 0.87 1.33 2.28

Z~

32.28 64.56 96.84 156.00

of PC (g/l)

Concentration

0 (ultrasonicated P C sol)

%w/w n-propanol

TABLE 4

Results for P C in solvent s y s t e m s o f varying n - p r o p a n o l c o n c e n t r a t i o n

20.03

A3

-- 0.998

-- 0.987

0.983

Coefficient of linear correlation

o

¢3

266

1. W. KELLAWAY AND L. SAUNDERS

analysis was applied to linear plots using an Olivetti Programma 101. Any plots deviating from linearity were fitted with a polynomial function using the P lO00 programme form the library of the University of London Computing Centre. The number average micellar weight (M.) was obtained form the Van 't Hoff equation Mn=

RT (zt/c)c~o"

The PC/n-alkanol/water systems As PC does not form a true solution in water, studies have been made with a monophasic solvent system in which PC remains soluble over a wide concentration range and which contains a high percentage of water. Thus, the PC/nalkanol/water system has been investigated using methanol and n-propanol. Ternary phase diagrams were constructed after the method of Tinker and Saunders2), (fig. 1) which resulted in the selection of the solvent system 25 per cent (w/w) n-propanol/water for further osmotic studies of PC; LPC was also freely soluble in this solvent. Recent osmotic pressure studies 2) of PC in 25 per cent (w/w) n-propanol/ water have shown that solute permeation of the membrane occurs. Errors are known to occur when the osmotic pressure is extrapolated to zero time, but with the rapid attainment of equilibrium which is achieved in the Mechrolab 500 series osmometers, the error is small. In a molecular weight determination with a sample of silicotungstic acid (B.D.H.) in water, experimental values of rc recorded between 20 and 40 min were extrapolated to zero time. These values were then used for molecular weight calculations giving a result with an error less than 3~o. This technique was therefore adopted for systems with membrane permeating solutes.

Aqueous phospholipid dispersions The results for the micellar weight of PC ultrasonicated in water for 2 hr at 20 kHz agree with the value obtained by light scattering measurements v), and with the sedimentation value obtained by Huang s) for a homogeneous fraction obtained by chromatography on an agarose gel, of a sonicated PC dispersion, indicating a reasonably monodispersed system. Agreement is also obtained with light scattering in the evaluation of the interaction coefficient, A2 in c.g.s, units, which has a low, positive magnitude expressing the small degree of solute interaction. The presence of a critical micelle concentration (c.m.c.) for aqueous LPC necessitated an alternative procedure in the recording of osmotic pressure. LPC was applied to the solute side of the membrane until the c.m.c, of LPC was established on the solvent side by the diffusion of monomers across the membrane. The micellar average molecular weight recorded therefore re-

OSMOTIC PRESSURE STIDIES OF SOME PHOSPHOLIPID SOLS

267

presents an upper limiting value. Robison and Saunders 9 measured the c.m.c, of LPC by surface tension studies to be 0.00044-0.00180% w/v at 25 °C, this value is so low that corrections of it applied to the concentrations of the sols used in the osmotic pressure measurements had a negligible effect on the calculated micellar weight. The solvent interaction constant for LPC when expressed in the same units, is of a similar order to that reported by Robinson 10) for light scattering by LPC in electrolyte solutions. The negative value of A 2 for PC/LPC 3 : 2 sol may indicate some instability of the large aggregates on dilution. It is interesting to note that the micellar weight increases on the addition of LPC to the aqueous PC system in a manner analogous to the rise in the limiting viscosity number noted by Perrin 6), which was explained by Saunders 11 in terms of the asymmetric mixed micelles existing in the form of highly hydrated helices. The micellar weight of the saturated (1 C, s.o) LPC was lower than expected in view of the results obtained with natural LPC and the fact that the determination was carried out in the presence of an electrolyte. The unsaturated (1 C18.2) LPC produced micelles of lower molecular weight than the saturated (1 Cls.O) LPC, presumably due to steric hindrance in the packing of the unsaturated hydrocarbon chains into a compact micellar form. The interaction coefficients were considerably greater for the two synthetic LPCs dissolved in 0.9% NaC1 than for natural LPC dissolved in water illustrating the more stable aggregation obtained with the natural hydrocarbon chain mixture.

Aqueous-alkanolie phospholipid dispersions The phase diagrams for the systems PC/n-alkanol/water are similar to those obtained by Tinker and Saunders2), although non-linear plots were obtained for methanol and ethanol. The concentrations of alkanol necessary to disrupt the PC aggregate were found to be 71.5% methanol, 48% ethanol and 19% n-propanol. The osmotic pressure determinations in 25% w/w n-propanol demonstrated the presence of small micelles in comparison with the ultrasonicated lipids in water. The number of monomers associating in the micelle is between 3 and 4 for both PC and LPC and the micellar weight of 2600 for PC is identical to that obtained by Tinker and Saunders 2). Mixtures of the two lipids show an increase in micellar weight with a maximum occurring around 60% LPC, 40% PC; a result similar to that obtained with ultrasonicated lipids in water. The large negative interaction coefficients indicate strong interaction between the n-propanol and the phospholipids and hence the unsuitability of the

268

I . W . K E L L A W A Y A N D L. S A U N D E R S

solvent system for accurate o s m o t i c pressure measurements. The micellar weights q u o t e d only serve as a guide to the size o f lipid aggregates present in 25% (w/w) n - p r o p a n o l . W h e n the ratio o f n - p r o p a n o l to water was v a r i e d in the solvent system (table 4), it was observed t h a t only a r o u n d the solvent c o m p o s i t i o n at which PC does n o t f o r m isotropic solutions, does the size o f the m o l e c u l a r aggregates increase to any great extent. T h e r e c o r d e d micellar weights were n o t in a g r e e m e n t with the dielectric c o n s t a n t rule p r o p o s e d by E l w o r t h y a n d Macintosh12).

Acknowledgements T h e a u t h o r s t h a n k Professor L. L. M. van Deenen a n d F. C. R e m a n o f the University o f U t r e c h t for the gift o f synthetic L P C ; a n d the Science Research C o u n c i l for a g r a n t for the p u r c h a s e o f the m e m b r a n e o s m o m e t e r ; I. W. K. t h a n k s the M e d i c a l Research C o u n c i l for a g r a n t which enabled him to c a r r y o u t this work.

References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12)

A. Thomas, J. Biol, Chem. 23 (1915) 359 D. O. Tinker and L. Saunders, Chem. Phys. Lipids 2 (1968) 316 I. W. Kellaway and L. Saunders, J. Pharm. Pharmacol. 21 (1969) 1898 W. B. Dandliker, J. Am. Chem. Soc. 76 (1954) 6036 E. J. Cohn, W. L. Hughes and J. H. Weare, J. Am. Chem. Soc. 69 (1947) 1753 J. H. Perrin, Ph.D. thesis, University of London (1962) 92a D. Attwood and L. Saunders, Biochem. Biophys. Acta 98 (1965) 344 C. Huang, Biochemistry 8 (1969) 344 N. Robinson and L. Saunders, J. Pharm. Pharmacol. 10 (1958) 755 N. Robinson, Ph.D. thesis, University of London (1959) 106a L. Saunders, Biochim. Biophys. Acta 125 (1966) 70 P. H. Elworthy and D. S. Macintosh, Koll. Z. Z. Polymere 195 (1964) 27