Liquid Crystalline Phases and Emulsifying Properties of Block

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Chapter 8

Liquid Crystalline Phases and Emulsifying Properties of Block Copolymer Hydrophobic Aliphatic and Hydrophilic Peptidic Chains 1

Bernard Gallot and Hussein Haj Hassan Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, 1A Avenue de la Recherche Scientifique, 45071 Orléans Cédex 2, France Amphiphilic lipopeptides with a hydrophobic paraffinic chain containing from 12 to 18 carbon atoms and a hydrophilic peptidic chain exhibit lyotropic mesophases and good emulsifying properties. The X-ray diffraction study of the mesophases and of dry lipopeptides showed the existence of three types of mesomorphic structures : lamellar, cylindrical hexagonal and body-centred cubic. Two types of polymorphism were also identified : one as a function of the length of the peptidic chain and the other as a function of the water content of the mesophases. The emulsifying properties of the lipopeptides in numerous pairs of immiscible liquids such as water/ hydrocarbons and water/base products of the cosmetic industry showed that small amounts of lipopeptides easily give three types of emulsions : simple emulsions, miniemulsions and microemulsions. Many surfactants have been used to formulate microemulsions ( 1 ) . They were o f three types : anionic surfactants such as petroleum sulfonates, sodium o c t y l benzene sulfonate, sodium dodecyl s u l f a t e , a l k a l i n e soaps ; c a t i o n i c surfactants such as dodecyl ammonium and hexadecyl ammonium chlorides or bromides ; and nonionic surfactants such as polyoxyethylene g l y c o l s . Furthermore, many e x h i b i t l i q u i d c r y s t a l l i n e properties (2) and i n some cases the structure o f the mesophases has been established ( 3 ) . Nevertheless, nearly nothing i s known about t h e i r compatibility with blood and t i s s u e s , and, from our own experience, some e x h i b i t a high l y t i c power f o r red c e l l s (4).

1Current address: Laboratoire des Matériaux Organiques, Centre National de la Recherche Scientifique, Bôite Postale 24, 69390 Vernaison, France 0097-6156/89/0384-0116$06.00y0 • 1989 American Chemical Society

El-Nokaly; Polymer Association Structures ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

8. GALLOT & HASSAN

Copolymers with Hydrophobic and Hydrophilic Chains 117

In order to obtain surfactants able to emulsify base products for cosmetic industry without presenting adverse side e f f e c t s to the skin and t i s s u e s , i t was necessary to synthesize new surfactants. We have chosen as new surfactants the amphiphilic lipopeptides (5-6). Amphiphilic lipopeptides C ( A A ) are formed by a hydrophobic l i p i d i c chain C containing from 12 to 18 carbon atoms l i n k e d through an amide bond to a hydrophilic p e p t i d i c chain (AA) with a number average degree o f polymerization ρ between 1 and 90. The repeating u n i t o f the peptidic chain i s an amino-acid residue and the general formula of the lipopeptides Cn(AA) i s : n

p

n

p

p

H-(CH ) -NH-(C0-ÇH-N) -H R R» 2

n

p

with R = Η (except f o r sarcosine where R = CH3) and R i s the side chain o f the amino-acid. The following are the various amino-acid residues : sarcosine (Sar) with R = Η ; l y s i n e bromhydrate (K) with R = (CH )^-NH ,HBr ; sodium s a l t of glutamic a c i d (E) with R = (CH )2-C00Na ; hydroxyethylglutamine (Eet) with R = (CH ) -C0-NH(CH ) 0H and hydroxypropylglutamine (Epro) with R = (CH ) -C0-NH(CH ) 0H. Lipopeptides present three main advantages. The two parts o f the lipopeptide molecules ( l i p i d i c and p e p t i d i c chains) are present i n many b i o l o g i c a l molecules and macromolecules and one can expect a good c o m p a t i b i l i t y with b i o l o g i c a l f l u i d s and tissues (4) and an absence of t o x i c i t y of t h e i r degradation products. The HLB of l i p o peptides can be e a s i l y adjusted by varying the degree of polymeriza t i o n ρ o f the p e p t i d i c chains and the nature of the amino-acid side chains R. The i n c o m p a t i b i l i t y between the hydrophobic p a r a f f i n i e chains and the hydrophilic peptidic chains leads to a phase separa t i o n at the molecular scale and to the existence of mesophases (7). In t h i s paper, we w i l l describe the l y o t r o p i c l i q u i d c r y s t a l l i n e and the emulsifying properties of lipopeptides with p e p t i d i c chains. f

1

2

2

2

2

2

2

3

2

2

2

2

EXPERIMENTAL METHODS Preparation of Mesophases. Lipopeptides are dissolved i n a small excess o f water and, when t o t a l homogeneity i s achieved, the desired concentration i s obtained by slow evaporation at room temperature. Then the sample i s l e f t at room temperature i n t i g h t c e l l s to reach equilibrium. X-ray D i f f r a c t i o n Studies. They are performed under vacuum with a Guinier type focussing camera equipped with a bent quartz monochromator g i v i n g a l i n e a r c o l l i m a t i o n of the CuK i (λ = 1.54 Â) r a d i a t i o n (8). a

Preparation of Emulsions. The mixture o i l - l i p o p e p t i d e i s heated to 70 C under a g i t a t i o n f o r complete homogèneization. I t i s then cooled to 45°C and water i s added. The a g i t a t i o n i s maintained throughout the preparation and u n t i l the system i s cooled to room temperature (9). Preparation of Miniemulsions. They are prepared by two methods :


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- In the f i r s t method, the i o n i c lipopeptide and c e t y l alcohol are mixed with water f o r an hour at 63°C (pre-emulsification stage). The o i l i s then added at 63°C and the a g i t a t i o n i s continued f o r an a d d i t i o n a l hour (9,10). - I n the second method, the mixture o i l / l i p o p e p t i d e / c e t y l alcohol i s homogeneized at 70°C f o r several minutes ; then i t i s cooled at 50°C and water i s added, a g i t a t i o n i s c a r r i e d on u n t i l complete homogeneization. The system i s then cooled to room temperature under a g i t a t i o n (9). Preparation o f Microemulsions. O i l , lipopeptide and water are mixed i n the same way as i n emulsion preparation ; the mixture i s then t i t r a t e d , at room temperature, with the cosurfactant u n t i l transparency i s obtained (9). S t a b i l i t y of Emulsions and Miniemulsions. The s t a b i l i t y of emulsions and miniemulsions i s determined by following, as a function o f time, the v a r i a t i o n o f the emulsified volume at f i x e d temperatures between - 10°C and + 50°C. RESULTS AND DISCUSSION L i q u i d - C r y s t a l l i n e Properties Structure and Polymorphism of Lipopeptides. Amphiphilic lipopeptides C (AA)p e x h i b i t mesophases i n aqueous s o l u t i o n f o r water concentrations smaller than about 60 %. The structure o f the mesophases and o f the dry lipopeptides obtained by evaporation of the mesophase water at a slow rate was determined by X-ray d i f f r a c t i o n . Lipopeptides X-ray diagrams obtained are s i m i l a r to those exhibited by c l a s s i c a l amphiphiles (11). They have allowed us to e s t a b l i s h the existence of three types of l i q u i d - c r y s t a l l i n e structures : lamell a r , hexagonal and cubic. The lamellar structure consists o f plane, p a r a l l e l equidistant sheets ; each sheet o f thickness d r e s u l t s from the superposition of two layers : one o f thickness d^ contains the hydrophilic p e p t i d i c chains and the water, while the other layer of thickness dg contains the hydrophobic p a r a f f i n i e chains. The hexagonal structure consists o f long and p a r a l l e l cylinders of diameter 2RJJ, f i l l e d with the hydrophobic paraf f i n i e chains of the lipopeptides and assembled i n a hexagonal array of parameter D, while the space between the cylinders i s occupied by the hydrophilic p e p t i d i c chains and the water. The body-centred cubic structure consists o f spheres o f diameter 2Rq f i l l e d with the hydrophobic p a r a f f i n i c chains of lipopept i d e s and assembled on a body centred cubic l a t t i c e o f side a, while the space between the spheres i s occupied by the hydrophilic p e p t i dic chains and the water. The l a t t i c e parameters d, D and a are d i r e c t l y obtained from Xray patterns, while the other parameters : d^, dg, 2R and S (average surface occupied by a chain at the interface between the hydrophilic and hydrophobic domains) are calculated using formulae based on simple geometrical considerations (11,12 ). The type of structure adopted by the lipopeptides i s determined by the r a t i o o f the volumes o f the hydrophilic domains (containing n


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the p e p t i d i c c h a i n s and the w a t e r ) and the h y d r o p h o b i c domains ( c o n t a i n i n g the p a r a f f i n i e c h a i n s ) . T h e r e f o r e the l i p o p e p t i d e s e x h i b i t two types o f polymorphism : one as a f u n c t i o n o f the l e n g t h o f the p e p t i d i c c h a i n s and the o t h e r as a f u n c t i o n o f the w a t e r c o n t e n t o f the mesophases. When the degree o f p o l y m e r i z a t i o n ρ o f the p e p t i d i c c h a i n s i n c r e a s e s d r y l i p o p e p t i d e s ( o b t a i n e d by evaporation of the w a t e r ) e x h i b i t s u c c e s s i v e l y l a m e l l a r , hexagonal and c u b i c s t r u c t u r e s i n the case o f l i p o s a r c o s i n e (12) and l a m e l l a r and hexagonal s t r u c t u r e i n the case o f l i p o l y s i n e and l i p o ( g l u t a m i c a c i d ) (13). Furthermore the a d d i t i o n o f w a t e r t o l i p o p e p t i d e s i s a b l e t o trans form the l a m e l l a r s t r u c t u r e i n t o a hexagonal one (12-13) o r a hexa g o n a l s t r u c t u r e i n t o a b o d y - c e n t r e d c u b i c one ( 1 2 ) . F a c t o r s G o v e r n i n g the G e o m e t r i c a l Parameters o f the Mesophase. The f a c t o r s g o v e r n i n g the g e o m e t r i c a l parameters o f the liquid-crystal l i n e s t r u c t u r e s are the water c o n t e n t o f the mesophases, the l e n g t h o f the p a r a f f i n i e c h a i n s and the l e n g t h o f the p e p t i d i c c h a i n s . I n f l u e n c e o f the Water C o n c e n t r a t i o n . When the w a t e r concen t r a t i o n increases : - The l a t t i c e parameter : d f o r the l a m e l l a r s t r u c t u r e , D f o r the hexagonal s t r u c t u r e and a f o r the c u b i c s t r u c t u r e i n c r e a s e s . - The c h a r a c t e r i s t i c parameter o f the h y d r o p h o b i c domains : dg f o r the l a m e l l a r s t r u c t u r e , 2Rjj f o r the hexagonal s t r u c t u r e and 2Rç f o r the c u b i c s t r u c t u r e d e c r e a s e s . - The c h a r a c t e r i s t i c parameter o f the h y d r o p h i l i c domains : d^ f o r the l a m e l l a r s t r u c t u r e , D-2R^ f o r the h e x a g o n a l s t r u c t u r e and a-2Rç f o r the c u b i c s t r u c t u r e i n c r e a s e s . - The average s u r f a c e S a v a i l a b l e f o r a m o l e c u l e a t the i n t e r f a c e i n c r e a s e s f o r the 3 t y p e s o f s t r u c t u r e . The f i g u r e s 1 and 2 i l l u s t r a t e such a b e h a v i o u r i n the case of the l a m e l l a r and hexagonal s t r u c t u r e s o f C18K2 » and o f the bodyc e n t r e d c u b i c s t r u c t u r e o f C^ySarô r e s p e c t i v e l y . Influence o f the Length o f the P a r a f f i n i e C h a i n s . Sets of l i p o p e p t i d e s C ( A A ) , w i t h the same degree o f p o l y m e r i z a t i o n ρ f o r the p e p t i d i c c h a i n s , but w i t h a number o f carbon atoms η o f their p a r a f f i n i e c h a i n s e q u a l t o 12, 14, 16, 17 and 18 have been s t u d i e d . When the number η o f carbon atoms i n c r e a s e s , d, D, dg and 2RJJ increase, while the c h a r a c t e r i s t i c parameter o f the hydrophilic p e p t i d i c domains and S b o t h remain c o n s t a n t (9,12). I n f l u e n c e o f the Length o f the P e p t i d i c C h a i n s . Three s e t s o f l i p o p e p t i d e s C^ySarp, 0^8 ρ l 8 p w i t h a c o n s t a n t l e n g t h o f the p a r a f f i n i c c h a i n s and d i f f e r e n t v a l u e s o f the degree o f polymeriza t i o n ρ o f the p e p t i d i c c h a i n s have been s t u d i e d . F o r the 3 t y p e s o f structures ( l a m e l l a r , hexagonal and b o d y - c e n t r e d c u b i c ) when ρ i n c r e a s e s the l a t t i c e parameter, the c h a r a c t e r i s t i c parameter o f the h y d r o p h i l i c p e p t i d i c c h a i n s and the s p e c i f i c s u r f a c e a l l i n c r e a s e . The c h a r a c t e r i s t i c parameter o f the h y d r o p h o b i c p a r a f f i n i c chains d e c r e a s e s (12,13). n

p

κ

Emulsifying

a n d

c

E

Properties

The emulsifying properties o f l i p o p e p t i d e s were t e s t e d i n many o i l / w a t e r systems. The o i l was a r o m a t i c such as t o l u e n e and s t y r e n e , p a r a f f i n i c such as decane and dodecane, o r a base p r o d u c t o f the c o s m e t i c i n d u s t r y . L i p o p e p t i d e s g i v e 3 t y p e s o f e m u l s i o n s : macro-


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Figure 1. V a r i a t i o n of the parameters of the lamellar and hexa gonal structures of lipopeptide 0 ^ 8 2 versus water concentration. Κ

H0 Figure 2. V a r i a t i o n of the parameters of the body-centered cubic structure of lipopeptide C^Sar.-^ versus water concentration. 2


8. GALLOT& HASSAN


e m u l s i o n s w i t h d r o p l e t d i a m e t e r s h i g h e r than 1000 nm, miniemulsions w i t h d r o p l e t d i a m e t e r s between 100 and 400 nm, and microemulsions w i t h d r o p l e t d i a m e t e r s between 10 and 100 nm. We w i l l sum up the main r e s u l t s o b t a i n e d w i t h the 3 types o f e m u l s i o n s . Type o f E m u l s i o n s . The e m u l s i o n s v a r y from a f l u i d m i l k l i k e t o a t h i c k cream, depending upon the n a t u r e o f the o i l , the r a t i o o i l / w a t e r , the n a t u r e and the c o n c e n t r a t i o n o f the l i p o p e p t i d e s . All the e m u l s i o n s are o f the o i l i n water (O/W) type as shown by the d i l u t i o n method, the s e l e c t i v e dyes method and the c o n d u c t i v i t y method (9). Such a r e s u l t i s i n agreement w i t h the HLB v a l u e s (between 8 and 15) o f the l i p o p e p t i d e s (9). S t a b i l i t y o f Emulsions. S t a b i l i t i e s v a r y i n g from 2 months t o more than 24 months were found. The main f a c t o r s g o v e r n i n g the s t a b i l i t y o f the e m u l s i o n s are : the l e n g t h o f the p a r a f f i n i c c h a i n s , the n a t u r e , the degree o f p o l y m e r i z a t i o n and the end group o f the p e p t i d i c c h a i n and the n a t u r e o f the o i l . I n f l u e n c e o f the Length o f the P a r a f f i n i c C h a i n s . The comparat i v e s t u d y o f l i p o p e p t i d e s w i t h the same p e p t i d i c c h a i n s but with p a r a f f i n i c c h a i n s c o n t a i n i n g from 12 t o 18 carbon atoms has shown that emulsions obtained w i t h l i p o p e p t i d e s w i t h a p a r a f f i n i c chain c o n t a i n i n g 12 o r 14 c a r b o n atoms are s t a b l e f o r l e s s than 4 days. Emulsions o b t a i n e d u s i n g l i p o p e p t i d e s w i t h p a r a f f i n i c c h a i n s c o n t a i n i n g 16 o r 18 c a r b o n atoms are s t a b l e f o r l o n g e r than 3 months. Nevertheless, the domain o f s t a b i l i t y o f the e m u l s i o n s i s s l i g h t l y h i g h e r f o r the 18 c a r b o n atoms p a r a f f i n i c c h a i n s than f o r the 16. C^2 and C^4 l i p o p e p t i d e s are more s o l u b l e i n w a t e r t h a n t h e i r C i 6 and Cê c o u n t e r p a r t s and can be more e a s i l y c a r r i e d i n t o the aqueous phase d e s t r o y i n g the h y d r o p h i l i c - h y d r o p h o b i c equilibrium between the l i p o p e p t i d e s and the water and o i l phases. I n f l u e n c e o f the P e p t i d i c C h a i n End Group. The i n f l u e n c e o f the n a t u r e o f the p e p t i d i c c h a i n end group has been s t u d i e d f o r 4 types o f l i p o p e p t i d e s : l i p o s a r c o s i n e , l i p o l y s i n e b r o m h y d r a t e , l i p o glutamic a c i d sodium s a l t and l i p o h y d r o x y e t h y l g l u t a m i n e ; similar r e s u l t s were o b t a i n e d . F i g u r e 3 i l l u s t r a t e s the r e s u l t s o b t a i n e d i n the e m u l s i f i c a t i o n o f the 0/W i s o p r o p y l m y r i s t a t e / w a t e r system by a l i p o s a r c o s i n e w i t h a degree o f p o l y m e r i z a t i o n o f 2 and a 18 c a r b o n atoms paraffinic c h a i n . The domain o f s t a b i l i t y o f the e m u l s i o n s d e c r e a s e s from the l i p o s a r c o s i n e c h l o r h y d r a t e ( d o t t e d l i n e ) , t o the l i p o s a r c o s i n e ( f u l l l i n e ) and t o the l i p o s a r c o s i n e whose t e r m i n a l amine f u n c t i o n has been a c e t y l a t e d ( p o i n t s ) . When the p o l a r i t y o f the end group o f the p e p t i d i c c h a i n dec r e a s e s , the e m u l s i f y i n g power o f the l i p o p e p t i d e d e c r e a s e s . I n f l u e n c e o f the Nature o f the A m i n o - a c i d S i d e C h a i n . The i n f l u e n c e o f the n a t u r e o f the a m i n o - a c i d s i d e c h a i n on the e m u l s i f y i n g p r o p e r t i e s o f l i p o p e p t i d e s i s i l l u s t r a t e d i n the f i g u r e 4 f o r the system O/W i s o p r o p y l m y r i s t a t e / w a t e r , and f o r 4 l i p o p e p t i d e s w i t h a p a r a f f i n i c c h a i n c o n t a i n i n g 18 carbon atoms and w i t h a degree o f p o l y m e r i z a t i o n o f 2 f o r the p e p t i d i c c h a i n s . The e m u l s i o n s s t a b i l i t y r e g i o n d e c r e a s e s from l i p o h y d r o x y e t h y l g l u t a m i n e ( f u l l l i n e ) t o lipolysine ( d o t t e d l i n e ) , t o l i p o g l u t a m i c a c i d ( p o i n t s ) and to hydroxypropylglutamine ( c r o s s e s ) . T h i s b e h a v i o u r i s r e l a t e d t o the


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L.P

W Ο Figure 3. Influence of the nature of the end group of the p e p t i dic chains on the domain of s t a b i l i t y of emulsions. L.P.

Figure 4. Influence of the nature o f the amino-acid side on the domain of s t a b i l i t y of emulsions.


chain

8. GALLOT & HASSAN


number of h y d r o p h i l i c s i t e s per amino-acid residue and to the hydrop h i l i c i t y of the amino-acid residue. For l e s s polar o i l s such as dodecane s i m i l a r r e s u l t s were obtained. The h y d r o p h i l i c i t y of the p e p t i d i c chain plays an impor tant part i n the emulsifying properties of lipopeptides f o r common oils. Influence of the Degree of Polymerization of the P e p t i d i c Chain. The influence of the degree of polymerization ρ of the p e p t i d i c chains of lipopeptides has been studied f o r 3 types of lipopeptides : l i p o s a r c o s i n e , l i p o l y s i n e bromhydrate and lipoglutamic a c i d sodium s a l t with p a r a f f i n i c chains containing 18 carbon atoms. The degree of polymerization ρ of the p e p t i d i c chains was between 1 and 10. Lipopeptides with ρ ^ 5 give emulsions stable f o r l e s s than 3 days. Lipopeptides with ρ = 1, 2 or 3 give stable emulsions. Figure 5 i l l u s t r a t e s the r e s u l t s obtained i n the case of the system 0/W isopropyl myristate/water and of l i p o l y s i n e bromhydrates. One can see that the domain of s t a b i l i t y of the emulsions decreases when ρ increases from 1 to 3. For lipoglutamic a c i d sodium s a l t s s i m i l a r r e s u l t s were obtained. For l i p o s a r c o s i n e s , i n contrast, the domain of s t a b i l i t y of emulsions i s nearly the same f o r ρ = 1,2 and 3. Influence of the O i l Nature. The influence of the o i l ' s nature on the domain of s t a b i l i t y of emulsions 0/W has been studied f o r 3 types o f lipopeptides with a p a r a f f i n i c chain containing 18 carbon atoms : l i p o l y s i n e bromhydrate, liposarcosine chlorhydrate and l i p o sarcosine. The r e s u l t s obtained were s i m i l a r f o r the 3 types of lipopeptides. The figure 6 i l l u s t r a t e s the r e s u l t obtained i n the case of liposarcosine with a degree of polymerization of 2. The domain of s t a b i l i t y of the 0/W emulsions decreases from isopropyl myristate and b u t y l stéarate ( f u l l l i n e ) to M y g l i o l (doted l i n e ) , to Cosbiol ( c r o s s ) , to dodecane (points) and to styrene. Polar o i l s are easier to emulsify than non polar and aromatic ones. Miniemulsions Lipopeptides emulsify with d i f f i c u l t y aromatic o i l s such as styrene or toluene. Furthermore they are not able to emulsify some o i l s such as v a s e l i n e , r i c i n , wheat germ and s i l i c o n o i l s . To emulsify such o i l s we have used a binary emulsifying system c o n s i s t i n g of a mixture of a f a t t y alcohol ( c e t y l alcohol) and a i o n i c lipopeptide (liposarcosine chlorhydrate, l i p o l y s i n e bromhydrate or lipoglutamic acid sodium s a l t ) . With concentrations of lipopeptide and c e t y l alcohol of 1 to 3 % we have obtained miniemulsions s i m i l a r to those obtained by E l Aasser and a l . with sodium l a u r y l s u l f a t e and c e t y l alcohol ( 1 0 ) . Type of Miniemulsions. the 0/W type.

A l l the miniemulsions were found to be of

S t a b i l i t y of the Miniemulsions. The main factors governing the s t a b i l i t y of the miniemulsions are : the length of the p a r a f f i n i c chains, the nature of the peptidic chains, the degree of polymerizat i o n of the p e p t i d i c chains, the mixed e m u l s i f i e r concentration, the molar r a t i o l i p o p e p t i d e / c e t y l a l c o h o l , the nature of the o i l and the method of preparation of the miniemulsions.


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Figure 5. Influence o f the degree of polymerization of the pept i d i c chains on the domain of s t a b i l i t y ef emulsions. C18 S a r

2

Figure 6. Influence o f the nature of the emulsified o i l on the domain of s t a b i l i t y of emulsions.


8.

GALLOT & HASSAN


Influence of the Length of the P a r a f f i n i c Chains. Lipopeptides with p a r a f f i n i c chains containing 18 or 16 carbon atoms give with o i l s very d i f f i c u l t to emulsify (styrene, r i c i n o i l , vaseline o i l , s i l i c o n o i l . . ) miniemulsions stable f o r more than 2 months. In contrast, lipopeptides with p a r a f f i n i c chains containing 14 or 12 carbon atoms give miniemulsions stable for less than 20 days. Influence of the Nature of the P e p t i d i c Chain. The s t a b i l i t y of the miniemulsions decreases when liposarcosine i s replaced by l i p o l y s i n e and lipoglutamic a c i d . This r e s u l t i s i l l u s t r a t e d i n Table I f o r two d i f f e r e n t o i l s (styrene and wheat germ o i l ) f o r a mass r a t i o 0/0+W of 0.2. Although the amounts of lipopeptide and c e t y l alcohol used to obtain miniemulsions have been increased from liposarcosine to lipoglutamic acid and to l i p o l y s i n e , the s t a b i l i t y of the miniemulsions decreases from more than 60 days for l i p o s a r c o sine, to 12 days f o r l i p o l y s i n e and 10 days f o r lipoglutamic a c i d . Table I . Influence of the Nature of the Lipopeptide. Wheat Germ O i l

Styrene LP % C!60H % C 8Sar ,HCl C (K,HBr) C (E,Na) 1

2

l8

l8

2

2

2

1

4

1.3

3

1.2

Stab. 60 12 10

LP %

CisOH %

Stab. 60 12 10

1.5

2.5 4.5

2.0

4.0

1.8

LP : lipopeptide ; Ο^ΟΗ : c e t y l alcohol ; Stab.: s t a b i l i t y i n days. Influence of the Degree of Polymerization. The amounts of lipopeptide and c e t y l alcohol necessary to s t a b i l i z e miniemulsions increases with the degree of polymerization ρ of the p e p t i d i c chains of lipopeptides. The Table I I i l l u s t r a t e s t h i s behaviour f o r the systems s t y r e n e / l i p o l y s i n e bromhydrate and wheat germ o i l / l i p o g l u t a mic a c i d sodium s a l t . Table I I . Influence of the Degree of Polymerization of the Peptidic Chains. 0/0+W =0.2 Styrene

C (K,HBr) C 0H l8

l6

Wheat germ o i l

C (E,Na) C 0H 12

l6

p

p

Ρ = 2

Ρ »3

4.5 % 2.0 %

7.0 % 3.0 %

4.0 % 1.5 %

6.0 % 2.5 %

Influence of the Mixed E m u l s i f i e r Concentration, The s t a b i l i t y of the miniemulsions increases with the concentration of the mixed e m u l s i f i e r . Table I I I i l l u s t r a t e s these r e s u l t s f o r the e m u l s i f i cation of two d i f f e r e n t o i l s : styrene and s i l i c o n o i l by l i p o s a r c o sine chlorhydrate with a degree of polymerization of 2.


126

POLYMER ASSOCIATION STRUCTURES Table I I I . Influence of the Concentration i n Weight of Mixed Emulsifier on the S t a b i l i t y of Miniemulsions. LP = C 8Sar ,HCl 1

0

LP

2

C 0H l6

S t a b i l i t y i n days

%

= η? 0+W

0+W

0+W

Styrene

2.0 4.0

1.0 2.0

100 > 180

Silicon o i l

2.5 5.0

1.5 3.0

70 > 120

Influence of the Molar Ratio Lipopeptide/Cetyl Alcohol. As already shown by d i f f e r e n t authors i n the case of c l a s s i c a l emulsif i e r s such as sodium l a u r y l s u l f a t e (10,14,15), the mixed e m u l s i f i e r system l i p o p e p t i d e / c e t y l alcohol gives stable miniemulsions f o r molar r a t i o s LP/CifcOH between 2/1 and 1/3. Influence of the Ratio Oil/Water. The mass r a t i o 0/0+W cannot exceed 60 % f o r aromatic o i l s and 50 % f o r cosmetic o i l s . The amounts o f lipopeptide and c e t y l alcohol necessary to obtain minie mulsions vary only s l i g h t l y with the amount of o i l i n the system oil/water. Influence of the O i l Nature. Aromatic o i l s (toluene and s t y rene) are easier to miniemulsify than cosmetic o i l s . They require smaller amounts of lipopeptide and c e t y l alcohol to give stable miniemulsions. Influence of the Method of Emulsification. Miniemulsions pre pared by the second method are more stable than those prepared with the p r e e m u l s i f i c a t i o n method. The Table IV gives 2 examples of the influence of the method of e m u l s i f i c a t i o n on the s t a b i l i t y of miniemulsions prepared with the same amounts of lipopeptide (LP) and c e t y l alcohol (CiÔH). When the o i l i s styrene the s t a b i l i t y o f the miniemulsion increases from 60 to 240 days. When the o i l i s vaseline o i l , the s t a b i l i t y o f the miniemulsion increases from 20 to 70 days. To understand the difference of s t a b i l i t y of the miniemulsions prepared by the two methods we have studied the miniemulsions by freeze fracture and electron microscopy (9) and measured the size of the p a r t i c l e s . For a l l the systems studied, the dimensions o f the p a r t i c l e s are smaller f o r the miniemulsions prepared by the second method ; f o r instance i n the case of styrene (Table IV) the diameter 0 of the p a r t i c l e s i s 310 nm against 840 nm. Such a difference i n the p a r t i c l e size explains the difference of s t a b i l i t y of the mini emulsions prepared by the two methods. We have also found by freeze fracture and electron microscopy that the size o f the p a r t i c l e s increases when the amount of emulsi f i e d o i l increases, and decreases when the concentration of the mixed e m u l s i f i e r increases. As an example, f o r the system C i 8 S a r , HCl/cetyl alcohol/styrene/water, the average diameter 0 of the par2


8. GALLOT & HASSAN


tides i n c r e a s e s from 700 nm t o 900 nm when t h e mass r a t i o 0/(0+W) i n c r e a s e s from 0.2 t o 0.5, b u t d e c r e a s e s from 900 nm t o 750 nm when the s u r f a c t a n t c o n c e n t r a t i o n i n c r e a s e s from 2 % t o 4 %. Table IV.

I n f l u e n c e o f t h e Method o f P r e p a r a t i o n o f M i n i e m u l s i o n . LP = C 8 S a r , H C l . 1

0/0+W = 0.2

2

Method 2

Method 1

Styrene

LP C 0H Stability 0

Vaseline o i l

LP C 0H Stability

2 1 60 840

l 6

2 1 240 310

% % days nm

2.5 % 1.5 % 70 days

2.5 % 1.5 % 20 days

l 6

% % days nm

Microemulsions I o n i c and n o n i o n i c l i p o p e p t i d e s g i v e m i c r o e m u l s i o n s when an a l i p h a t i c a l c o h o l o r amine w i t h l e s s than 7 carbon atoms i s used as c o s u r f a c t a n t . Nevertheless the best c o s u r f a c t a n t s are butanol, prop a n o l , b u t y l a m i n e and p r o p y l a m i n e . The enlargement o f t h e m i c r o e m u l s i o n r e g i o n i s i n f l u e n c e d by the f o l l o w i n g f a c t o r s : - I n c r e a s e i n t h e h y d r o p h i l i c i t y and HLB o f t h e l i p o p e p t i d e - Decrease o f t h e p a r a f f i n i c c h a i n l e n g t h - I n c r e a s e i n t h e p o l a r i t y o f t h e p e p t i d i c c h a i n s end group. An example i s t h e case o f l i p o s a r c o s i n e / n - b u t y l a m i n e / i s o p r o p y l myristate/water system. Microemulsions region increased from C i 8 S a r 2 t o C^Sarô as t h e h y d r o p h i l i c i t y o f t h e p e p t i d i c chain i n c r e a s e d , from C i 8 a r 2 t o C i 8 a r 2 , H C l as t h e p o l a r i t y o f t h e p e p t i dic c h a i n i n c r e a s e d , and from C i 8 S a r 2 , H C l t o C i 2 a r 2 , H C l as t h e p a r a f f i n i c chain length decreased. s

s

s

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Langevin, D. Mol. Cryst. L i q . Cryst. 1986, 138, 259. Ekwall, P. Advances i n Liquid Crystals 1975, 1, 1. Hendricks, Y.; Charvolin, J . J . Phys. 1981, 42, 1427. G a l l o t , B.; Haj Hassan, Η., unpublished r e s u l t s . G a l l o t , B.; Douy, A. French Patent 82 159 76, 1982 ; Chem. Abstr. 1984, 171762h. Gallot, B.; Douy, A. U.S. Patent 4 600 526, 1986. Gallot, B. In Liquid C r y s t a l l i n e Order i n Polymers; Blumstein, Α., Ed.; Academic: New York, 1978; Chapter 6, 191. Douy, A.; Mayer, R.; Rossi, J . ; Gallot, B. Mol. Cryst. L i q . Cryst. 1969, 7, 103. Haj Hassan, H. Ph.D. Thesis, Orléans University, Orléans, France, 1987. El-Aasser, M.S.; Lack, C.D.; Choi, Y.T.; Min, T.I.; Vanderhoff, J.W.; Fawkes, F.M. Colloids and Surfaces 1984, 12, 79.


128


11. L u z z a t i , V.; Mustacchi, H.; Skoulios, Α.; Husson, F.; Acta C r y s t a l l o g . 1960, 13, 660. 12. Douy, Α.; G a l l o t , B. Makromol. Chem. 1986, 187, 465. 13. G a l l o t , B.; Douy, A.; Haj Hassan, H. Mol. Cryst. L i q . Cryst. 1987,

153,

347.

14. Grimm, W.L.; Min, T.I.; El-Aasser, M.S.; Vanderhoff, J.W. J. C o l l o i d Interface Sci. 1983, 94, 531. 15. Brouwer, W.M.; El-Aasser, M.S.; Vanderoff, J.W. C o l l o i d and Surfaces 1986, 21, 69. RECEIVED

August 10, 1988


Liquid Crystalline Phases and Emulsifying Properties of Block

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