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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
A soft and supple skin has a marked cosmetic appeal and is an attribute of
normal-functioning epidermis. The outer layer of the epidermis, or stratum
corneum, can become brittle, dry, and flaky, due to loss of water.
Emollients, such as fats, phospholipids, sterols, and the like were at one
time thought to be substances essential to the maintenance or restoration
of softness and flexibility of the skin. More recently however,
investigators have become aware that dry-appearing and flaky skin is the
result of loss of water-soluble natural substances, mostly humectants,
from the skin. Loss of water-soluble substances from the skin can occur
when the skin is exposed to adverse conditions. When this happens the
stratum corneum, although it is somewhat hygroscopic and absorbs water
vapor, cannot retain moisture, and soon loses it if the atmosphere becomes
drier. In any event, the stratum corneum which has lost humectants does
not ordinarily absorb and retain sufficient moisture for restoration to a
normal condition, and the need is evident for an artificial moisturizing
system for persons who suffer from dry, chapped and flaky skin, or from
dry and brittle hair and nails.
2. Discussion of the Prior Art
I. H. Blank, in J. Invest. Dermatol., vol. 18, 433 (1952), presented
evidence that water is the only plasticizer of skin, and that a diminished
water content results in a cornified epithelium having the undesirable
properties of dryness, hardness, and brittleness.
Largely as a result of the work of Blank, mentioned above, there has been
investigated many factors relating to the relationship of the water
content of the stratum corneum to skin flexibility and plasticity.
Information obtained during the two decades following Blank's work has
been summarized by B. Idson, in Drug, Cosm. Ind. vol. 104(6), page 44,
vol. 105(1) page 48, and vol. 105(2), page 48, all published in 1969.
The ability of normal healthy stratum corneum to retain water has been
ascribed to the presence therein of water-soluble substances having strong
hygroscopic properties. Many such substances occur together in the stratum
corneum and collectively have been termed the "natural moisturizing
factor" by O. K. Jacobi, in Proc. Sci. Sect. T.G.A. vol. 31, 22(1959) and
in J. Soc. Cosm. Chem. vol. 18, 149 (1967).
Most of the water-soluble substances comprising the natural moisturizing
factor have been identified by H. W. Spier and G. Pascher in the text
"Aktuelle Probleme der Dermatologie", vol. I, pp. 1-46, published by S.
Karger AG, Basel/New York, 1959.
More recently, G. Smeenk and A. M. Rijnbeek in Acta derm-venereol., vol.
49, 476 (1969) have published information indicating that the hygroscopic
properties of the water-soluble corneum fraction are due to the
simultaneous presence, in physiological proportions, of the components
reported by Spier and Pascher, mentioned above.
It is known that a lipid membrane or covering in the stratum corneum
hinders the extraction of the water-soluble substances therefrom by water,
as reported by E. J. Singer and L. J. Vinson in Proc. Sci. Sect. T.G.A.
vol. 46, 29 (1966), and by I. H. Blank, J. Invest. Dermatol., vol. 21, 259
(1953).
G. Sessa and H. Weissmann, in J. Lipid Res., vol. 9, 310 (1968), describe
liposomes and discuss the rate of diffusion of ions across the lipid
layers thereof.
U.S. Pat. No. 3,231,472 discloses the use of a synthetic skin moisturizer
prepared by condensing an amino acid with a reducing sugar to provide an
N-glycoside and thereafter contacting the N-glycoside with a proton donor.
SUMMARY OF THE INVENTION
The common dry skin condition is attributable to various factors including
(a) the external atmospheric relative humidity, (b) disorganization of
lipid membranes, and (c) disruption of lipid membranes plus loss of
water-soluble hygroscopic substances. When the external relative humidity
is low, there is rapid water loss from the skin to external environment
although the moisturizing system of the skin exists in its normal physical
state. Water is lost from the skin at a faster rate than it can be
replaced by migration from the underlying dermal tissues. More commonly,
dry skin is a consequence of exposure to environmental agents capable of
disrupting the physical state of the moisturizing system. For example,
non-aqueous solvents and aqueous solutions of strong washing products
contribute to a dry skin condition by damaging the lipid membranes,
allowing the leaching out of water-soluble substances when the skin is
immersed in water.
For alleviation of the dry skin condition, it is necessary to restore water
to the skin. If the physical state of the skin's moisturizing system is
normal, or only the lipid membranes damaged, then water retention can be
increased by applying an occlusive film to the skin surface. The occlusive
film retards the rate of water vapor diffusion from the skin to the
outside environment. The use of water-soluble hygroscopic compounds, per
se, for moisturizing skin is not of practical value since such agents lack
the property of skin substantivity by virtue of their high degree of water
solubility.
Dry, rough, chapped and scaly skin areas are substantially devoid of
natural humectants, and although water vapor can migrate to these areas
from the underlying skin it is not retained, but is lost to the
atmosphere. If a lipid layer is superimposed over these areas, water may
still escape, since it is practically impossible to maintain an unbroken
film of lipid.
The application to the skin of humectants alone is unsatisfactory since
these are not skin-substantive, and are readily rinsed off.
The problems of non-substantivity and loss of water migrating from the
underlying skin are solved by the use of the liposomes described herein.
The liposomes are substantive to keratinous matter and moreover do not
depend upon absorption of water from the skin alone but function to absorb
water from the atmosphere in addition to providing their own water
content, and share the water with the keratinous matter to which they are
applied.
When applied to keratinous matter, moisturization, softness and flexibility
are imparted or maintained by the liposomes, which comprise a matrix of a
lipid mixture, having a plurality of cavities therein containing a
humectant in aqueous solution.
It is therefore an object of the invention to provide compositions capable
of moisturizing keratinous matter.
It is another object of the invention to provide compositions capable of
improving the softness, plasticity, and flexibility of water-deficient
stratum corneum.
It is still another object of the invention to provide a process for
moisturizing and improving or maintaining the flexibility of living
keratinous matter by applying to the keratinous matter a multiplicity of
the liposomes described hereinbelow.
It is a further object of the invention to provide a composition containing
a moisturizing humectant for keratinous matter and an occlusive substance
to retard the rate of water evaporation.
Accordingly the invention provides a composition having a moisturizing
humectant to supply water to, or replace water lost by keratinous matter,
and an occlusive substance to retard the rate of water loss. The
composition is a liposome comprising a matrix of a ternary lipid mixture
of lecithin, dicetyl phosphate, and a sterol, and having cavities disposed
internally of said liposome, said cavities containing a humectant
dissolved in an aqueous medium. The surface of the liposome is
substantially continuous.
DETAILED DESCRIPTION OF THE INVENTION
The sizes of the liposomes with which the present invention is concerned
vary somewhat, depending upon the species of lipid, species of humectant,
and type and intensity of the mechanical agitation used to form the
liposomes.
Liposomes may be prepared by the following procedure.
A desired weight of a mixture of soybean lecithin, dicetyl phosphate, and a
sterol, as described hereinafter, in predetermined molar ratios is
dissolved in chloroform in a round bottom flask of suitable size. The
chloroform is removed by evaporation, and there are added for each mole of
residue, about 60-80 moles of a humectant in the form of a 0.145 molar
aqueous solution at pH 5.3. The flask is shaken for 6 hours at 40 strokes
per minute on a shaker bath at a temperature of 25.degree.C whereupon the
lipids swell in the aqueous solution to form liposomes having structures
described hereinbelow. To induce additional swelling of the liposomes the
suspension is held at 5.degree.C for 18 hours without shaking. The
suspension is then dialyzed for 5 hours against four 800-ml changes of
0.145M NaCl to remove any excess humectant not incorporated into the
liposomes.
Suitable humectants for use in the present invention are for example
glycerol, urea, sodium pyroglutamate (2-pyrrolidone-5-carboxylic acid,
sodium salt), ornithine [H.sub.2 N(CH.sub.2).sub.3 CH(NH.sub.2)COOH, m.w.
132.16], and the Spier-Pascher water solubles consisting of a mixture of
the following substances in the indicated proportions.
______________________________________
mM/100g corneum
______________________________________
alanine 8.4
arginine.sup.. HCl 1.7
aspartic acid 4.8
citrulline 7.3
glutamic acid 1.9
glyocoll 11.5
histidine.sup.. HCl 4.4
leucine 3.1
lysine.sup.. HCl 1.2
ornithine.sup.. HCl 1.5
phenylalanine 1.1
proline 1.7
serine 23.9
threonine 4.3
tryptophan 0.5
tyrosine 1.7
valine 2.3
calcium chloride 10.1
citric acid 0.095
creatinine 0.1
glucosamine 0.1
glucose 1.1
lactic acid 17.8
magnesium chloride 1.9
______________________________________
mM/100g corneum
______________________________________
magnesium chloride 1.9
sodium phosphate (dibasic)
0.3
ammonium chloride 1.1
2-pyrrolidone-5-carboxylic acid
14.2
ribose 0.3
urea 9.0
uric acid 0.1
urocanic acid 4.8
______________________________________
The lecithin used in the examples described herein is commercial lecithin
derived from soybean oil and contains cephalin, choline lecithin, and
inositol phosphatides. The use of lecithin in cosmetics is discussed by G.
T. Walker in American Perfumer and Cosmetics, vol. 82, pages 73-76,
October, 1967.
Suitable sterols for use in the lipid mixture are cholesterol, phytosterol,
sitosterol, sitosterol pyroglutamate, 7-dehydrocholesterol, and
lanosterol. It is also possible to use caprolactum.
The lipid membranes preferably are ternary mixtures of lecithin, dicetyl
phosphate, and a sterol selected from the group listed hereinabove, in the
preferred molar ratios of 70:20:10, respectively. The molar percentage of
lecithin may range from about 50% to about 80%, the dicetyl phosphate from
about 10% to about 30%, and the sterol from about 10% to about 30%, basis
for ternary lipid mixture. Lecithin is employed to take advantage of its
property of swelling in salt solutions. Dicetyl phosphate has the property
of imparting a negative charge to the lipid membranes so that the mutual
repulsive action of opposing channel surfaces widens the channels. The
sterol functions as an occlusive film when the liposomes are applied to
skin or hair.
The gross appearance of the liposomes useful in the practice of the present
invention is that of an opalescent suspension having a milky while color.
The components which constitute the lipid matrix, or membrane, and the
aqueous humectant phase are commercially available or may readily be
prepared. L-pyroglutamic acid may be purchased from Nutritional
Biochemicals Corporation, Cleveland, Ohio; dicetyl phosphate and
commercial grade soybean L-alpha-lecithin may be obtained from Sigma
Chemical Company, St. Louis, Mo.; beta-sitosterols are supplied by Upjohn
Co., Kalamazoo, Mich.; cholesterol is purchased from Mann Research
Laboratories, Inc., New York, N.Y.; caprolactam is supplied by
Pfaltz-Bauer, Inc., Flushing, N.Y.; lanosterol, 7-dehydrocholesterol, and
ornithine are obtained from Calbiochem, Los Angeles, Calif.
Sitosterol pyroglutamate may be made by the following procedure. One
hundred grams, about 0.25 mole, of sitosterol are dissolved in 500 ml of
benzene together with 5 grams of paratoluenesulfonic acid as catalyst, and
65 grams, 0.5 mole, of pyroglutamic acid. The mixture is refluxed for 10
hours, and under azeotropic conditions 5 ml of water are collected. The
reaction mixture is filtered to remove unreacted pyroglutamic acid, which
is then washed with hot benzene and the washings are added to the main
filtrate. The benzene is evaporated to dryness on a steam bath. The
residue is crystallized from 1500 ml of acetone, producing a tan-colored
powder. A volume of 1500 ml methanol is added to the powder and heated to
boiling. The powder is allowed to settle and the methanol decanted. The
powder is treated with four additional portions of methanol, 400 ml each,
decanting each time, and filtering after the last treatment, yielding 27.7
grams of tan-colored powder. Analysis for nitrogen shows 2.49% N
(theoretical 2.67%). Some impurities are indicated by NMR analysis.
As demonstrated in Example 13 below, the amphoteric and anionic detergents
are suitable for use in the shampoos useful as a cosmetically acceptable
vehicle for applying the liposomes to the hair. Suitable amphoteric
detergents include
N-lauryl-N'-carboxymethyl-N'-(2-hydroxyethyl)ethylenediamine,
coco-beta-alanine, the alkali-metal salts of protein-coconut fatty acid
condensates, aminopropionates such as alkyl beta-iminodipropionates
represented by RN(CH.sub.2 CH.sub.2 COOM).sub.2 and alkyl
beta-iminopropionates represented by the formula RNHCH.sub.2 CH.sub.2
COOM, wherein R is an aliphatic hydrocarbon radical having about 8 to
about 18 carbon atoms and M is a cation to neutralize the charge on the
anion and to render the detergent compound water soluble. Suitable anionic
detergents are the water-soluble anionic sulfate, sulfonate and
carboxylate foaming detergents mentioned in the literature, such as the
texts "Surface Active Agents" by Schwartz and Perry, and "Surface Active
Agents and Detergents" by Swartz, Perry and Berch, both Interscience
Publishers, New York, the disclosures of which are incorporated herein by
reference.
The liposomes may be applied in the form of an aqueous suspension as
prepared, or may be applied in a composition comprising a physiologically
acceptable vehicle into which the liposomes have been incorporated. The
physiologically acceptable vehicle may be a skin lotion or cream, such as
a cold cream, vanishing cream, cleansing cream, etc., for application to
the skin. The vehicle may be a shampoo or brilliantine for application to
the hair.
The above-mentioned compositions may be prepared by the usual procedures
known to those skilled in the art, using however the precaution to
maintain the temperature of the mixture below the melting point of the
liposomes from the time the liposomes are incorporated.
The term "moisturize" or derivatives thereof, relates to the conservation
or enhancement of the water content of the keratinous matter of living
mammals, with particular reference to the stratum corneum, hair, and nails
of human beings.
The term "humectant" is used herein in its usual sense, and particularly
refers to water-soluble, physiologically acceptable, substances which are
hygroscopic and capable of spontaneously absorbing water vapor.
"Occlusion" is a term used herein to indicate the trapping of water in the
keratinous matter by a layer of a water-impervious fatty substance.
By "skin cream" is meant a spreadable composition in either paste or liquid
form, adaptable to be spread over the skin with the object of imparting or
maintaining a desirable characteristic.
The invention may be more fully understood by reference to the following
Examples.
EXAMPLE 1
A weight of 0.0602 gram of a ternary mixture of 70 molar proportions
(0.0455 gram) of soybean lecithin, 20 molar proportions (0.0109 gram) of
dicetyl phosphate, and 10 molar proportions (0.0038 gram) of cholesterol
is placed in a one-liter round bottom flask and dissolved in 10 ml of
chloroform. The chloroform is evaporated and 50 ml of a 0.145 molar
aqueous sodium pyroglutamate solution having a pH of 5.3 are added to film
of lipid residue remaining after evaporation of the chloroform. The flask
is shaken until the lipids swell in the aqueous medium and the flim is
removed from the walls of the flask, whereby liposomes are formed. The
flask and contents are then held for 18 hours at a temperature of
5.degree.C without shaking to permit additional swelling of the liposomes.
The liposome suspension is then dialyzed for five hours against four
800-ml changes of 0.145M NaCl to remove any sodium pyroglutamate not bound
in the liposomes.
Light microscopy of stained wet mounts of liposomes prepared as above shows
that the liposomes are in the smectic mesophase or wet liquid crystal
state. Structurally the liposomes are observed to be microscopic spherules
bound by a phospholipid-cholesterol membrane.
The water-binding characteristics of the above-described liposomes are the
same as those of the humectant employed, i.e., sodium pyroglutamate.
The above-described liposomes restore the in vitro water-binding capacity
of damaged corneum membranes as shown by the following test.
Ten neonate rat corneum membranes having dry weights from 8.2 to 18.8
milligrams are damaged by exposure to an aqueous 40% by volume solution of
tetrahydrofuran. This treatment removes 1.7 to 4.9 milligrams of the
corneum membranes, dry basis, and greatly reduces the water-binding
capacities of the membranes, as shown in Table I, below. The
above-described liposomes are applied uniformly over the membranes in
amounts equal to the weight of membrane removed by the tetrahydrofuran
treatment, dried over phosphorus pentoxide at 25.degree.C, and allowed to
remain 24 hours in a closed vessel at 81% relative humidity at
25.degree.C. The membranes are then weighed, and the amount of imbibed
water calculated.
Table I, below, shows the extent to which the water-binding capacity of the
animal membranes is reduced when the membrane is damaged by
tetrahydrofuran (column 3) and the extent to which the water-binding
capacity is restored by the liposome treatment described above (column 4).
TABLE I
______________________________________
MILLIGRAMS WATER BOUND PER 100 MILLIGRAMS
OF DRY MEMBRANE
Column 4
Damaged Fol-
Column 1 Column 2 Column 3 lowed by Lipo-
Membrane No.
Before Damage Damaged some Treatment
______________________________________
1 33.9 0.9 54.8
2 33.3 6.1 54.1
3 30.5 5.1 22.8
4 27.8 3.9 68.3
5 22.6 6.3 56.2
6 29.4 4.2 44.3
7 30.4 3.0 34.2
8 29.0 8.2 56.2
9 29.5 5.3 47.4
10 29.2 4.2 32.0
______________________________________
The foregoing data show that the water-binding capacity of normal corneum
membranes is substantially constant among the 10 specimens tested (column
2). Damaging the membranes by treatment with tetrahydrofuran sharply
reduces the water-binding capacity, as seen in column 3. The figures in
column 4 show the water-binding capacities of the membranes after treating
the damaged membranes with liposomes prepared as described above. For 9
out of the 10 specimens, the water-binding capacities are improved to
levels above the initial capacities, i.e., from about 110% to about 240%
of the initial values.
EXAMPLES 2-10
Liposomes having the lipid and aqueous components shown below are prepared
by the procedure described in Example 1. The lipid components in each
instance are a ternary mixture of lecithin, dicetyl phosphate, and sterol
in the molar ratios of 70:20:10, respectively. In Example 8, the sterol
component is a mixture.
______________________________________
Example
No. Sterol Humectant
______________________________________
2 cholesterol ornithine
3 cholesterol 1:1 weight ratio of
sodium pyroglutamate
and ornithine
4 cholesterol Spier-Pascher water-
solubles
5 sitosterol pyro- sodium pyroglutamate
glutamate
6 sitosterol sodium pyroglutamate
7 caprolactam sodium pyroglutamate
8 1:1 weight ratio of
sodium pyroglutamate
caprolactam and
cholesterol
9 7-dehydrocholesterol
sodium pyroglutamate
10 lanosterol sodium pyroglutamate
______________________________________
All of the liposomes prepared as in Examples 1-10 above are spherules
variously ranging in size from about 0.5 to about 12 microns in diameter.
The liposomes of Example 4, containing Spier-Pascher water-solubles, tend
to be the smallest spherules in this range, the liposomes of Example 1 are
in general intermediate in size, while the remaining are in the upper
region of the aforesaid range.
All of the aforementioned liposomes are characterized by their ability to
absorb water, a property determined by the following test procedure.
One ml of the prepared liposome suspensions is placed in each of several
aluminum weighing pans and dried to constant weight over phosphorus
pentoxide at 25.degree.C. Three pans from each of Examples 1-10 are placed
in closed containers having the relative humidities at 25.degree.C therein
regulated at 52% by a saturated aqueous sodium dichromate solution, 81% by
a saturated aqueous ammonium sulfate solution, and 98% by a saturated
aqueous lead nitrate solution. After exposure for 24 hours, the pans are
again weighed and the amount of water imbibed calculated. The results are
presented in Table II below.
TABLE II
______________________________________
WATER-BINDING CAPACITY OF LIPOSOMES
Weight Increase in Percent of Initial Liposome
Weight (Approximate)
Example
No. 52%RH 81%RH 98%RH
______________________________________
1 100 253 350
2 100 231 352
3 100 268 472
4 100 280 536
5 100 246 379
6 100 318 435
7 100 239 398
8 100 244 418
9 100 241 413
10 100 261 427
______________________________________
A comparison of the water-binding capacities at 98% relative humidity shows
that the liposomes having the mixture of Spier-Pascher water-solubles are
the most effective humectants of the group tested, holding 5.5 times their
weight of water, as compared with the remaining nine liposome compositions
which held about 4 times their weight of water.
The function of the sterol component has been explained hereinabove as
being that of an occlusive film which forms when the liposomes are applied
to the skin or hair. Inspection of the data at 98% relative humidity shows
that, among the liposomes having in common sodium pyroglutamate as the
humectant, namely those of Examples 1 and 5 through 10, the most effective
occlusive agents are sitosterol, 7-dehydrocholesterol, and lanosterol.
EXAMPLES 11-14
Examples 11 through 14 further illustrate the efficacy of liposomes of the
present invention as moisturizing agents. Example 11 shows that the
liposomes are effective moisturizers when deposited in in vitro tests on
animal stratum corneum from an aqueous medium at a temperature of
40.degree.C, this being the temperature approximating the 105.degree.F
reported by Suskind and Whitehouse, (Arch. Dermat. Vol. 88, 66 (1963)) as
being the temperature commonly used in the bath and ordinary household
chores.
Example 12 shows that the liposomes can be deposited on animal stratum
corneum membranes that have been damaged by treatment with
tetrahydrofuran, and that the deposited liposomes enhance the
water-binding capacity of the damaged corneum.
EXAMPLE 11
Fifteen membranes of rat stratum corneum are immersed in a 1% suspension of
liposomes, prepared as described in Example 1, at a temperature of
40.degree.C for a time of 30 minutes with agitation. The membranes are
then rinsed in distilled water at room temperature for 10 minutes. Each
individual membrane is pretreated in the foregoing manner, but without
liposome deposition, to serve as a control for determining the extent of
liposome deposition and the increase in water-binding capacity.
Table III, below, shows the increase in dry weight of the corneum
membranes, indicating deposition of the liposome spherules on the
membrane. The water-binding data indicate that the liposome deposition
increases the water-binding capacity of the corneum membranes.
The procedure for determining the moisturizing effect is described in
Example 1.
TABLE III
______________________________________
IN VITRO ENHANCEMENT OF THE WATER-BINDING
CAPACITY OF STRATUM CORNEUM MEMBRANES
Untreated Liposome Treated
mg. water mg. mg. water water bound
bound per material bound per in percent
membrane deposited membrane of control
______________________________________
2.65 0.65 3.65 137.7
3.25 0.85 4.40 135.3
2.30 1.35 2.60 113.0
2.10 1.45 2.50 119.0
2.60 1.75 2.40 109.0
1.90 1.10 1.60 110.0
2.60 0.75 2.35 109.3
1.75 1.65 2.10 120.0
2.90 2.70 3.40 117.2
2.45 1.25 2.95 120.4
2.80 0.80 3.35 119.6
2.15 0.95 2.35 109.3
2.60 1.25 3.50 134.6
2.90 0.80 3.15 108.6
2.35 0.95 2.70 114.8
______________________________________
The foregoing figures represent the percentage of water bound in each
membrane relative to its own control.
EXAMPLE 12
This example shows the beneficial action of liposomes on damaged stratum
corneum.
Rat stratum corneum membranes having known water-binding capacities are
damaged to varying degrees by (a) immersion in 37% aqueous tetrahydrofuran
for 2 hours, producing virtual barrier destruction, (b) immersion in the
same medium for 30 minutes, resulting in an intermediate grade of barrier
destruction, and (c) immersion in an 8% solution of a commercial bar soap
for 30 minutes at 40.degree.C, causing a relatively mild degree of barrier
damage. Following these treatments, the membranes are rinsed in distilled
water for 10 minutes, dried, and the extent of reduction in water-binding
capacity at 81% relative humidity and 25.degree.C ascertained.
The membranes treated as above are again dried, and then immersed in a 1%
suspension of liposomes prepared as in Example 1 for 30 minutes at
25.degree.C with agitation and rinsed for 10 minutes. The treated
membranes are dried, weighed, and subjected for 24 hours to an atmosphere
having a temperature of 25.degree.C and 81% relative humidity, then
reweighed, as described in Example 1.
The results are recorded in Table IV. The liposomes effect partial barrier
restoration on the damaged membranes as shown by the decreased rate of
water diffusion through the liposome-treated damaged membranes as compared
with diffusion through the damaged membrane before the liposome treatment.
The figures in the first two columns of Table IV show that the liposomes
have no effect on the barrier properties of undamaged stratum corneum.
The rate of water diffusion is determined by the following procedure, using
the Sage Moisture Meter, described by Baker and Kligman in Arch. Derm.,
Vol. 96, page 441 (1967). The membrane (skin) is stretched tightly in
sealed position over the chamber, which contains water. A current of dry
air flowing at the rate of 0.025 liter per minute is swept over the
membrane and is directed through a series of hygrometers which measure the
relative humidity of the air that has passed over the membrane. Using the
relative humidity data, the amount of water vapor that has diffused
through the membrane in a selected time interval is calculated.
TABLE IV
__________________________________________________________________________
WATER DIFFUSION RATES.sup.(a)
UNDAMAGED DAMAGED DAMAGED DAMAGED
(a)2-hour tetrahy-
(b)30-minute tetrahy-
(c)Soap
drofuran treatment
drofuran treatment
treatment
without with without
with without
with without
with
liposomes
liposomes
liposomes
liposomes
liposomes
liposomes
liposomes
liposomes
__________________________________________________________________________
A 0.20 0.20 12.5 9.36 8.74 3.47 2.00 0.86
B 0.23 0.29 12.8 -- 9.30 3.56 2.26 1.47
C 0.33 0.29 11.4 9.47 8.34 3.30 2.67 1.06
D 0.26 0.31 11.8 -- 8.90 3.60 3.18 1.16
E 0.19 -- 12.5 9.11 -- -- -- 1.26
Mean
0.20 0.24 12.2 9.31 8.82 3.48 2.52 1.16
__________________________________________________________________________
.sup.(a) Figures are milligrams of water diffused through one square
centimeter of corneum per hour.
EXAMPLE 13
This example shows the effect of a representative amphoteric, anionic,
nonionic, and quaternary ammonium surfactant on the barrier properties of
the lipid matrix of the liposomes within the invention.
The data below are obtained by a modification of the procedure described by
Weissmann et al in Nature, Vol. 208, 649 (1965). In the present study, the
release of radioactive glutamic acid from the liposomes on exposure to
solutions of the surfactants is measured to determine the extent of
membrane damage. One-ml portions of dialyzed liposomes containing as the
aqueous component a solution of .sup.14 C-glutamic acid, and otherwise
prepared as described in Example 1, are placed in several small dialysis
sacs. A solution of 0.6 gm. of each test surfactant in 100 ml of distilled
water is prepared and 0.2 ml of each solution is dispensed into separate
sacs. The final surfactant concentration is 0.1% (weight volume).
Following the addition of the surfactant, the contents of the sacs are
tied, placed in small narrow bore test tubes containing 5.0 ml of a 0.145
molar solution of equimolar NaCl and KCl, and held at 37.degree.C. At
15-minute intervals, the sacs are transferred to tubes containing fresh
NaCl/KCl solutions of the above strength. The .sup.14 C-glutamic acid
which diffuses into the salt solution in each period is determined by
liquid scintillation counting.
The results are presented in Table V below. It will be noted that the
amphoteric and anionic surfactants do not disrupt the liposomal membrane
since the leakage rate of the .sup.14 C-glutamic acid is comparable to
that of the control tested without a surfactant. The nonionic causes
slight membrane damage, and the quaternary produces marked damage.
The amphoteric detergent tested is Miranol C-2M, a trademark of the Miranol
Chemical Company, Inc., Irvington, N.J. The compound is described as
lauroylcycloimidinium-1-ethoxyethionic acid -2-ethionic acid, disodium
salt, by the manufacturer, and is a compound described in U.S. Pat. No.
2,773,068.
The anionic detergent is secondary alkylbenzene sulfonate wherein the
benzene ring is randomly positioned, except terminally, along a linear
alkyl chain of about 13 carbon atoms.
The nonionic is Pluronic L-64, a trademark of the Wyandotte Chemical
Company. Pluronic L-64 is a compound having the empirical formula
HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.8 O).sub.b (C.sub.2 H.sub.4
O).sub.c H prepared by condensing ethylene oxide with a hydrophobic base
formed by hhe condensation of propylene oxide with propylene glycol where
b is 26-30. The molecular weight of the base unit (C.sub.3 H.sub.8
O).sub.b is about 1501 to about 1800, and a plus c is an integer such that
the molecule contains 40% to 50% ethylene oxide.
The quaternary is Arquad 2C-75, a trademark of Armour and Company. The
compound is dicoco-dimethyl ammonium chloride, the term "dicoco"
indicating two long-chain alkyl groups having the carbon chain
distribution of coconut oil.
TABLE V
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THE EFFECTS OF SURFACTANTS ON THE STABILITY OF LIPOSOMAL MEMBRANES AT
37.degree.C.sup.(a)
Miranol C-2M.sup.(c)
LAS Pluronic L-64
Arquad 2C-75
Time(min)
Control.sup.(b)
Amphoteric Anionic
Nonionic Quaternary
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15 432 460 382 572 1899
30 826 803 718 1122 3551
45 1109 1065 1077 1492 4750
60 1351 1323 1319 1802 5747
75 1552 1564 1522 2123 6622
90 1739 1748 1711 2371 7305
105 1912 1902 1872 2569 7875
120 2048 2030 2013 2743 8358
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.sup.(a) Surfactant damage to the liposomal membranes was determined by
monitoring the release of .sup.14 C-glutamic acid over the time intervals
indicated.
.sup.(b) Controls consisted of liposomes suspended in 0.145M NaCl/KCl
solution.
.sup.(c) Liposome suspensions (0.145M NaCl/KCl) contained test surfactant
at a final concentration of 0.1%.
EXAMPLE 14
This example shows the beneficial action of the liposomes of the present
invention on the skin of human subjects.
Liposomes prepared as in Example 1 are applied to the rough, red, dry,
cracked skin of one hand of each of two human subjects. A cooling
sensation is noted upon the application of the liposomes. The treated
hands are rinsed, blotted dry, and the condition of the skin observed
about 18 hours following treatment. In both instances, the skin of the
treated hand feels softer and visually appears less cracked and scaly than
the skin of the untreated hand of the same person.
EXAMPLE 15
Following is an example of a shampoo within the invention.
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Percent by Weight
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Triethanolammonium lauryl sulfate
20
Ethanol 20
Bis(2-hydroxyethyl)alkylamine oxide
5
wherein the alkyl group is a
mixture of predominantly
C.sub.12 and C.sub.14 chain lengths
Liposomes in aqueous medium.sup.(a)
55
100
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.sup.(a) prepared as in Example 1, but omitting the dialysis against NaCl
The liposomes thus prepared are at a 0.12% weight/volume concentration in
0.145 molar sodium pyroglutamate solution.
The shampoo is prepared by merely mixing the four components together at
room temperature, maintaining the pH between 5 and 6, until the solid
compon | | |
