Note: Descriptions are shown in the official language in which they were submitted.
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PERSONAL CARE COMPOSITIONS CONTAINING SUBTILISIN ENZYMES BOUND
TO WATER INSOLUBLE SUBSTRATES
TECHNICAL FIELD
The present invention relates to personal care compositions comprising
singularly
substituted subtilisin enzymes. bound to the wipe substrate. Embodiments of
the personal care
compositions include a personal care wipe and a personal care skin mask. The
compositions
provide improved cleansing and skin conditioning due to the activity of the
active proteins, with
minimized risk of allergic reaction to the active protein by the user.
BACKGROUND OF THE INVENT10N
An increasing number of commercial products containing active proteins are
becoming
available. The majority of these products utilize an enzyme, as the active
protein. Enzymes are
proteins which react with a compound, or substrate, to break down that
compound. Enzymes are
divided into numerous classes based on the class of substrate they react upon.
Each class of
enzyme generally catalyzes the severing of different chemical bonds resulting
in the specific
selection of activity. The lipase class of enzymes are known for their ability
to hydrolyze ester
bonds created between, but n,ot limited to, hydrocarbons and polyalcohol
backbone substrates.
Examples of these substrates are mono-, di-, and triglyceride polyglycerol
esters. The protease
class of enzymes are known for their ability to hydrolyze proteins. Naturally
occurring and bio-
engineered protease enzymes are incorporated into household cleaning
detergents to hydrolyze
proteinaceous dirt and stains, into personal care products to remove dirt and
dead skin, into oral
cleansing products to facilitate plaque removal in the mouth, and into
medicines to affect
undesired proteins in the body.
It is known that current commercial cleansing products are made more effective
by the
incorporation of protease enzymes. U.S. Patent Number 4,261,868 (flora et
al.), U.S. Patent
Number 4,404,115 (Tai), U.S. Patent Number 4,318,818 {Letton et al.), European
Patent
Application 130,756 (published Jan. 9, 1985) and U.S. Patent 5,030,378
(Venegas) all disclose
the use of protease enzymes in cleansing or detergent products.
It is also realized, however, that many active proteins, including enzymes,
are potential
antigens, and may cause allergic reactions in humans under certain conditions.
The human
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immune system can produce specific antibodies upon exposure to active
proteins. This process
of producing specific antibodies is referred to as "immunization" when a
clinically beneficial
response is obtained. When the response leads to hypersensitivity, however, it
is refen ed to as
"sensitization". Allergenic sensitization to active proteins has been observed
in environments
where humans are regularly exposed to the protein. Such environments include
manufacturing .
facilities, where workers can be exposed to uncontrolled dust or aerosol
containing an active
protein, or the marketplace, where consumers' repeated use of products
containing active proteins
has, on occasion, caused an allergic reaction.
Presently, allergic responses to active proteins can be minimized by limiting
the selection
of those proteins used in products to those of human origin. While this
approach minimizes
allergenicity problems, it is not a complete solution since it is often not
possible to find such an
active protein which also has the activity properties desired.
Another way of diminishing allergic response has been to reduce the size of
the protein
molecules (see JP Patent Publication Number 4,112,753). However, size
reduction may also
cause a significant reduction in enzyme activity.
A third proposition for decreasing allergenicity is through epitope mapping
and alteration
of the protein amino acid sequence to deliver a protein with reduced
allergenicity, This approach
usually requires a large investment of development time and money.
In the medical field, suggestions have been made to diminish the
immunogenicity of
proteins through yet another method. This method involves attaching unreactive
polymers to the
protein. U.S. Patent No. 4,179,337 (Davis, et al.) relates to enzymes coupled
to substantially
straight chain polyethylene glycol (PEG) or polypropylene glycol (PPG) polymer
moieties.
While PEG/PPG coupling was found to mitigate the allergenicity of the enzyme,
only IS% of the
physiological activity was maintained. PCT Application WO 96/17929 (Olsen, et
al., published
June 13, 1996) relates to the modification of enzymes by conjugating them with
suitable
polymers. The Olsen application describes modified enzymes which demonstrate a
reduction in
allergenicity of from 25% to 66% compared to the parent enzyme, while
maintaining from 39%
to 100% of the activity of the ~aarent.
The U. S. Patent Application, Serial Number 08/903,298 discloses the use of
enzymes
modified by the addition of twin polyethylene glycol polymer moieties to
reduce allergenicity
while delivering high enzymatic activity. The modified enzyme therein is used
in combination
with a fibrous substrate in a wipe application. The modified enzymes are not
attached to the
substrate. Reduced allergenicty is achieved via the modification of the
enzyme.
The U. S. Patent Application, Serial Number 09/088,912 disclosed polymeric
chemical
modification of subtilisin enzymes at one or more of three specific epitope
regions which was
found to mask the immunogenic determinants of the enzyme.
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Another approach to reduce the allergenicity of active proteins has been by
granulating,
coating or dissolving the active proteins to avoid their becoming airborne.
U.S. Patent 4,556,554
(Calvo) discloses cosmetic compositions which comprise enzymes which have been
immobilized
by attachment to particles of polymeric support. The particles with attached
enzymes are
dispersed in the cosmetic vehicle. Upon application of the vehicle to the
skin, the enzyme is
released from the support and. is therefore reactivated. Methods such as this
address consumer
exposure to airborne proteins, however they still leave the substantial risks
associated with
extended tissue contact with the released enzyme which are deposited on the
skin.
Canadian Patent 1,229,808, issued December 1, 1987 teach the immobilization of
enzymes,
specifically (i-galactosidase and (3-glucosidase, on cellulosic substrates
wherein the enzyme is
immobilized by absorption into a agarose gel coating the substrate.
UK Patent Application GB 2,240,040, published July 24, 1991 also teaches
immobilized
enzymes on substrates. Enzymes, therein as covalently bonded to substrates to
provide a
medicated dressing.
The activity of enzymes used in biological equipment such as biosensors,
bioseparators,
and bioreactors has been enhanced by the use of site-specific attachment of
enzymes to
equipment surfaces. See Huang et al., "Improving the Activity of Immobilized
Subtilisin by Site-
specific Attachment to Surface", Analytical Chemistry, 69(22), November 15,
1997. Huang
teaches the immobilization of subtilisin enzymes via mutation of serine249 or
serine145 to
cysteine, and bonding to silica beads functionalized with amino groups.
It would be highly desirable to develop a composition which would provide
improved
levels of protein activity while maintaining low allergenic responses from
exposure to the active
proteins. If this were accomplished it would provide consumers with safer ways
to utilize the
benefits of protein technology.
It is an object of the prcaent invention to provide a wipe composition which
delivers this
improved activity while maintaining reduced stimulation of and resulting
activation of the
immune system.
SUMMARY OF THE INVENTION
The present invention relates to personal care compositions comprising a water
insoluble
substrate, a plurality of singularly substituted subtilisin BPN' enzyme
variants and a binding
means, permanently attaching each of the enzymes to the substrate, wherein the
personal care
compositions comprise from about 0.01 pg/emz to about 1000 Ng/cm' of the
enzyme on 'the
substrate.
DETAILED DESCRIPTION OF THE INVENTION
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The personal care compositions of the present invention comprise subtilisin
BPN' enzymes
and derivatives modified by a single substitution of a cysteine amino acid
group permanently
bound to a water insoluble substrate. The compositions provide a convenient
means to utilize the
specialized activity of subtilisin BPN' enzyme and its derivatives, where the
enzymes are bound
to the substrate, thereby minimizing any risk of allergic reaction. Preferred
embodiments of the
compositions are highly efficacious for cleaning sweat, sebum, dead skin
cells, fats and oils from
the skin and for moisturizing of the skin.
Without being limited by theory, it is believed that by permanently binding
the enzymes to
the substrate, they may be brought into contact with the skin for use,
allowing them to act on the
surface. Then as the wipe or mask is removed all of the enzymes are lifted
from the skin surface.
and removed and disposed of with the used wipe, thereby eliminating the risk
of aerosolization
and extended dermal exposure°. The active protein reacts with the
compounds it has specific
reactivity for while in contact with the skin and none remain on the skin
after use to stimulate the
immune system and subsequently form antibodies responsible for allergic
reaction.
As used herein, the phrase "amino acid sequence" refers to a specific
configuration of the
amino acids comprising a protein. The following is a list of abbreviations
used herein to describe
amino acids:
Amino Acid Three-letter AbbreviationOne-letter Symbol
Alanine Ala A
Arginine Arg
Asparagine Asn N
Aspartic Acid Asp p
Cysteine Cys C
Glutamine Gln
Glutamic Acid Glu
Glycine Gly G
Histidine His H
Isoleucine Ile (
Leucine Leu L
Lysine Lys IC
Methionine Met M
Phenylalanine Phe p
Proline Pro p
Serine Ser S
Threonine Thr T
Tryptophan Trp
Tyrosine Tyr y
Valine Val V
No amino acid Xaa
a~osition
As used herein, the term "mutation" refers to the genetic alteration of an
organism, which
in turn alters the amino acid sequence of the enzyme produced by that
organism. The mutation of
an organism has been often found to alter the properties of the enzyme.
As used herein, the term "wild-type" refers to an enzyme produced by unmutated
hosts.
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As used herein, the term "variant", means an enzyme having an amino acid
sequence which
differs from that of the wild-type enzyme due to the genetic mutation of the
host producing that
enzyme.
All percentages and ratios used herein, unless otherwise indicated, are by
weight and all
measurements made are at 25°C, unless otherwise designated. The
invention hereof can
comprise, consist of, or consist essentially of, the essential as well as
optional ingredients and
components described therein.
The essential components of the personal care compositions of the present
invention, as
well as a non-exclusive list of preferred and optional ingredients, are
described in detail below.
WATER INSOLUBLE SUBSTRATE
The products of the present invention comprise a water insoluble substrate. By
"water
insoluble" is meant that the substrate does not dissolve in or readily break
apart upon immersion
in water. The water insoluble substrate is the implement or vehicle for
delivering the active
proteins of the present invention to the skin to be cleansed and moisturized,
and for removing
substantially all of the proteins from the skin.
A wide variety of materials can be used as the substrate. The following
nonlimiting
characteristics are desirable: (i) sufficient wet strength for use, (ii)
sufficient abrasivity, (iii)
sufficient loft and porosity, (iv) sufficient thickness, and (v) appropriate
size.
Nonlimiting examples of suitable insoluble substrates which meet the above
criteria
include nonwoven substrates, woven substrates, hydroentangled substrates, air
entangled
substrates, natural sponges, s~mthetic sponges, polymeric netted meshes, and
the like. Preferred
embodiments employ nonwoven substrates since they are economical and readily
available in a
variety of materials. By noncvoven is meant that the layer is comprised of
fibers which are not
woven into a fabric but rather are formed into a sheet, mat, or pad layer. The
fibers can either be
random (i.e., randomly aligned) or they can be carded (i.e. combed to be
oriented in primarily one
direction). Furthermore, the nonwoven substrate can be composed of a
combination of layers of
random and carded fibers.
Nonwoven substrates may be comprised of a variety of materials both natural
and
synthetic. By natural is meant that the materials are derived from plants,
animals, insects or
byproducts of plants, animals, and insects. By synthetic is meant that the
materials are obtained
primarily from various man-made materials or from natural materials which have
been further
altered. The conventional base starting material is usually a fibrous web
comprising any of the
common synthetic or natural textile-length fibers, or mixtures thereof.
Nonlimiting examples of natural materials useful in the present invention are
silk fibers,
keratin fibers and cellulosic fibers. Nonlimiting examples of keratin fibers
include those selected
from the group consisting of wool fibers, camel hair fibers, and the like.
Nonlimiting examples
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of cellulosic fibers include those selected from the group consisting of wood
pulp fibers, cotton
fibers, hemp fibers, jute fibers, flax fibers, and mixtures thereof.
Nonlimiting examples oil synthetic materials useful in the present invention
include those
selected from the group consisting of acetate fibers, acrylic fibers,
cellulose ester fibers,
modacrylic fibers, polyamide fibers, polyester fibers, polyolefm fibers,
polyvinyl aicohol-fibers,
rayon fibers, polyurethane foam, and mixtures thereof. Examples of some of
these synthetic
materials include acrylics such as acrilan, creslan, and the acrylonitrile-
based fiber, orlon;
cellulose ester fibers such as cellulose acetate, arnel, and acele; polyamides
such as nylons (e.g.,
nylon 6, nylon 66, nylon 610, a,nd the like); polyesters such as fornel,
kodeI, and the polyethylene
terephthalate fiber, dacron; polyolefins such as polypropylene, polyethylene;
polyvinyl acetate
fibers; polyurethane foams avnd mixtures thereof. These and other suitable
fibers and the
nonwoven materials prepared therefrom are generally described in Riedel,
"Nonwoven Bonding
Methods and Materials," Nonwoven World (1987); The Encyclopedia Americana,
vol. l l, pp.
147-153, and vol. 26, pp. 566-581 (1984); U.S. Patent No. 4,891,227, to Thaman
et al., issued
January 2, 1990; and U.S. Patewt No. 4,891,228 which are all incorporated by
reference herein in
their entirety.
Nonwoven substrates made from natural materials consist of webs or sheets most
commonly formed on a fine mire screen from a liquid suspension of the fibers.
See C.A. Hampel
et al., The Encyclonedia of Chemis , third edition, 1973, pp. 793-795 ( 1973);
The Encyclopedia
Americana, vol. 21, pp. 376-:38:3 (1984); and G.A. Smook, Handbook of Pulp and
Paper
Technologies, Technical As::ociation for the Pulp and Paper Industry (1986);
which are
incorporated by reference herein in their entirety.
Substrates made from nadural materials useful in the present invention can be
obtained from
a wide variety of commercial sources. Nonlimiting examples of suitable
commercially available
paper layers useful herein include Airtex~, an embossed airlaid cellulosic
layer having a base
weight of about 71 gsy, available from James River, Green Bay, WI; and
Walkisoft~, an
embossed airlaid cellulosic having a base weight of about 75 gsy, available
from Walkisoft
U.S.A., Mount Holly, NC.
Methods of making nonwoven substrates are well known in the art. Generally,
these
nonwoven substrates can b~e made by air-laying, water-laying, meltblowing,
coforming,
spunbonding, or carding processes in which the fibers or filaments are first
cut to desired lengths
from long strands, passed into a water or air stream, and then deposited onto
a screen through
which the fiber-laden air or water is passed. The resulting layer, regardless
of its method of
production or composition, is then subjected to at least one of several types
of bonding operations
to anchor the individual fibers together to form a self sustaining web. In the
present invention the
nonwoven layer can be prepared by a variety of processes including
hydroentanglement,
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thermally bonding ar thetmo-bonding, and combinations of these processes.
Moreover, the
substrates of the present invention can consist of a single layer or multiple
layers. In addition, a
multilayered substrate can include films and other nonfibrous materials.
Nonwoven substrates made from synthetic materials useful in the present
invention can
also be obtained from a wide variety of commercial sources. Nonlimiting
examples of suitable
nonwoven layer materials useful herein include HEF 40-047, an apertured
hydroentangled
material containing about 50% rayon and 50% polyester, and having a basis
weight of about 43
grams per square yard (gsy), available from Veratec, lnc., Walpole, MA; HEF
140-102, an
apertured hydroentangled material containing about SO% rayon and SO%
polyester, and having a
basis weight of about 56 gsy, available from Veratec, Inc., Walpole, MA;
Novonet~ 149-616, a
thermo-bonded grid patterned material containing about 100% polypropylene, and
having a basis
weight of about 50 gsy, available from Veratec, Inc., Walpole, MA; Novonet~
149-801, a
thermo-bonded grid patterned material containing about 69% rayon, about 25%
polypropylene,
and about 6% cotton, and having a basis weight of about 75 gsy, available from
Veratec, Inc.
Walpole, MA; Novonet~ 149-191, a thermo-bonded grid patterned material
containing about
69% rayon, about 25% polypropylene, and about 6% cotton, and having a basis
weight of about
100 gsy, available from Veratec, lnc. Walpole, MA; HEF Nubtex~ 149-801, a
nubbed, apertured
hydroentangled material, containing about 100% polyester, and having a basis
weight of about 70
gsy, available from Veratec, Inc. Walpole, MA; Keybak~ 951 V, a dry formed
apertured material,
containing about 75% rayon, about 25% acrylic fibers, and having a basis
weight of about 43 gsy,
available from Chicopee, New Brunswick, NJ; Keybak~ 1368, an apertured
material, containing
about 75% rayon, about 25°/. polyester, and having a basis weight of
about 39 gsy, available from
Chicopee, New Brunswick, NJ; Duralace~ 1236, an apertured, hydroentangled
material,
containing about 100% rayon, and having a basis weight from about 40 gsy to
about 11 S gsy,
available from Chicopee, New Brunswick, NJ; Duralace~ 5904, an apertured,
hydroentangled
material, containing about 100% polyester, and having a basis weight from
about 40 gsy to about
115 gsy, available from Chicopee, New Brunswick, NJ; Sontaro 8868, a
hydroentangled material,
containing about SO% cellulose and about 50% polyester, and having a basis
weight of about 60
gsy, available from Dupont Chemical Corp.
Alternatively, the water insoluble substrate can be a polymeric mesh sponge as
described in
European Patent No. EP 7025:50 Al published March 27, 1996, incorporated by
reference herein
in its entirety. The polymeric sponge comprises a plurality of plies of an
extruded tubular netting
mesh prepared from a strong flexible polymer, such as addition polymers of
olefin monomers and
polyamides of polycarboxylic acids. Although these polymeric sponges are
designed to be used
in conjunction with a liquid cleanser, these types of sponges can be used as
the water insoluble
substrate in the present invention.
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The substrate can be made into a wide variety of shapes and forms including
flat pads,
thick pads, thin sheets, ball-shaped implements, irregularly shaped
implements, and having sizes
ranging from a surface area of about a square inch to about hundreds of square
inches. The exact
size will depend upon the desired use and product characteristics. Especially
convenient are
square, circular, rectangular, or oval pads having a surface area of from
about 1 in2 to about 144
in2, preferably from about 10 ir~2 to about 120 in2, and more preferably from
about 30 in2 to about
80 in2, and a thickness of from about 1 mil to about 500 mil, preferably from
about 5 mil to about
250 mil, and more preferably from about 10 mil to about 100 mil.
The water insoluble substrates of the present invention can comprise two or
more layers,
each having different textures and abrasiveness. The differing textures can
result from the use of
different combinations of materials or from the use of different manufacturing
processes or a
combination thereof. A dual textured substrate can be made to provide the
advantage of having a
more abrasive side for exfoliation and a softer, absorbent side for gentle
cleansing. In addition,
separate layers of the substrate can be manufactured to have different colors,
thereby helping the
user to further distinguish the surfaces.
SINGULARLY SUBSTITUTED SUBTILISIN BPN' ENZYME - "PROTEASE G"
An essential component of the present invention is a plurality of singularly
substituted
subtilisin BPN' enzymes (hereinafter referred to as Protease G). The Protease
G is present on the
surface of the water insoluble substrate at a level ranging from about 0.01
pg/cm'' to about 1000
Irg/cm', preferably from about 0..05 itg/cm'' to about 100 ~g/cm'', and most
preferably from about
0.1 pg/cm- to about 10 ~g/cm''.
in general, protease enzynes are classified under the Enzyme Classification
number E.C.
3.4 (Carboxylic Ester Hydrolases) in accordance with the Recommendations
(1992) of the
International Union of Biochemistry and Molecular Biology (fUBMB). Related
proteases are
also described in PCT publications: WO 95130010 published November 9, 1995 by
The Procter
& Gamble Company; WO 95/30011 published November 9, 1995 by The Procter &
Gamble
Company; WO 95/29979 published November 9, 1995 by The Procter & Gamble
Company.
Subtilisin enzymes are protease enzymes which are naturally produced by
Bacillus
alcalophilus, Bacillus amyloliguefaciens, Bacillus amylosaccharicus, Bacillus
licheniformis,
Bacillus lentus and Bacillus sulitilis microorganisms. One known subtilisin
enzyme is BPN'. The
wild-type BPN' from Bacillus a~nn~loliquefaciens is characterized by the amino
acid sequence:
1 10 20
Ala Gln Ser Val Pro Tyr Gly Vai Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln
Gly
30 40
Tyr Thr Gly Ser Asn Val Lys Va1 Ala Val Ile Asp Ser Gly Ile Asp Ser Ser His
Pro
50 60
Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln
Asp
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70 80
Asn Asn Ser His Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile
Gly
90 100
Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp
Gly
I10 120
Ser Gly G1n Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn Asn Met
Asp
130 140
Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val
Asp
150 160
Lys Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser
Gly
170 180
Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Va! Gly Ala
Val
190 200
Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Scr Val Gly Pro Glu Leu Asp Vat Met
Ala
210 220
Pro Gly Val Ser tle Gln Ser Thr Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly
Thr
230 240
Ser Met Ala Ser Pro His V'al Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro
Asn
250 260
Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp
Ser
270 275
Phe Tyr Tyr Gly Lys Lys Gly Leu lle Asn Asn Val Gln Ala Ala Ala Gin
Several variants of BPN' also are known. Several related variants, all
hereafter referred to
as "Protease A", are disclosed in U.S. Patent 5,030,378 (issued to Venegas,
July 9, 1991) as
characterized by the BPN' amino acid sequence with the following mutations:
a.) the Gly at position. G1y166 is replaced with Asn, Ser, Lys, Arg, His, Gln,
Ala or Glu;
the Gly at position G1y1169 is replaced with Ser; the Met at position Met222
is replaced
with Gln, Phe, Cys, His, Asn, Glu, Ala or Thr; or
b.) the Gly at position G1y166 is replaced with Lys and the Met at position
Met222 is
replaced with Cys; or
c.) the Gly at position GIy160 is replaced with Ala and the Met at position
Met222 is
replaced with Ala.
Additional variants of :BPN', heretoforth referred to as "Protease B", are
disclosed by
Genencor International, Inc. (:>an Francisco, California) European Patent EP-B-
251,446 {granted
December 28, 1994 and published 3anuary 7, 1988) as characterized by the wild-
type BPN' amino
acid with the mutations in one or more of the following amino acids: Tyr2l,
Thr22, Ser24,
Asp36, Ala 45, A1a48, Ser49, Met50, His67, Ser87, Lys94, Va195, G1y97, Ser101,
G1y102,
G1y103, Ilel07, G1y110, Met 124, G1y127, G1y128, Pro129, Leu135, Lys170,
Tyr171, Pro172,
Asp 197, Met 199, Ser 204, Lys213, Tyr214, G1y215, and Ser221; or two or more
of the amino
acids listed above and Asp32, Ser33, Tyr104, A1a152, Asn155, G1u156, Glyl66,
G1y169, Phe189,
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Tyr217, and Met222 wherein both mutations cannot be made on the Asp32, Ser33,
Tyr104,
Ala 152, Asn 1 S5, G1u156, Gly 166, G1y169, Phe 189, Tyr217, and Met222 amino
acids.
Another BPN' variant protease, hereafter referred to as "Protease D", is
described in WO
95/10615 published April 20, 1995 by Genencor International as characterized
by the wild-type
BPN' amino acid with mutation to position Asn76, in combination with mutations
in one or more
other amino acid positions selected from the group consisting of Asp99,
Ser101, GIn103, Tyr104,
Ser105, I1e107, Asn109, Asn123, Leu126, G1y127, G1y128, Leu135, G1u156,
G1y166, G1u195,
Aspl97, Ser204, G1n206, Pro210, A1a216, Tyr217, Asn218, Met222, Ser260,
Lys265, and/or
A1a274.
Another BPN' variant protease, hereafter referred to as "Protease F", is
described in U.S.
Patent Number 4,760,025, issued to Estell, et al. on July 26, 1988 as
characterized by the wild-
type BPN' amino acid with mutation to one or more amino acid positions
selected from the group
consisting of Asp32, Ser33, Hfis64, Tyr104, Asn155, G1u156, G1y166, Glyl69,
Phel89, Tyr217,
and Met222.
The enzyme used in the personal care compositions of the present invention,
Protease G,
comprises any of the BPN' enzymes and their variants listed above with a
singular substitution or
insertion of a cysteine amino acid in the amino acid sequence. Cysteine is the
most preferred
substituting amino acid for substitution in the desired epitope region since
it does not occur in
wild-type subtilisin BPN' or its derivatives. The substituted or inserted
amino acid provides a
moiety suitable for attachment to the substrate of the present invention at a
specific site within
the enzyme.
Preferably the substitution or insertion should be made at a position in an
epitope region
which falls at a point in the protein away from the active site of the
protein. In subtilisin BPN'
and derivatives this active site is spacially defined by the triad Asn32,
His64, and Ser 221. In a
Protease G enzyme, a cysteine is substituted at a point away from that triad.
Preferable epitope
regions for substitution are the of Asp140-Va1150 and A1a230-Leu250 region.
Non-limiting
examples of possible cysteine substitutions at either Ser145, Asn 240 or
Ser249. Cysteine is the
most preferred substituting amino acid for substitution in the desired epitope
region since it does
not occur in wild-type subtilisin BPN' or its derivatives.
BINDING MEANS
The active proteins are bound to the water insoluble substrate by any suitable
binding
means. Binding means include any physical or chemical method of permanently
binding an
active protein to a substrate. Many such means are lrnown in the art.
Physical Entrapment
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One means of binding the active protein of the present invention to the
substrate is to
physically trap the protein within the body of the substrate. A preferred
means of entrapment is
to seal the protein in a coating crn the surface of the substrate. Any
adhesive or polymeric lrnown
in the art may be used to seal the protein to the substrate. A preferred
coating is poly-2-
hydroxyethyl acrylate which is. formed by the separate application of 2-
hydroxyethyl acrylate
monomer and an iron (II) sulfate: heptahydrate initiator.
A solution of either a) active protein and adhesive or b) active protein and a
monomer/initiator combination is uniformly sprayed onto the surface of the
substrate. A second
coating of adhesive or monomer/initiator or a separate initiator may be
required to achieve
sufficient binding. The substraoe is then dried to allow the polymer to set.
The substrate is then
fully rinsed to remove any &ee protein.
Protein Tethered to Polymeric Ciel Coating on Substrate
The protein may be bound to the substrate by a chemical tether bound to a
polymeric
coating on the substrate. The protein is bonded to a polymeric tether which is
covalently attached
to the polymer coating. One embodiment of a polymeric tether is Polyethylene
glycol (PEG)-
Maleimide, sold by Shearwvater Polymers, Inc. PEG-Maleimide must be used with
a cysteine
amino acid, therefore it may be used when the active protein is Protease G.
PEG-Maleimide may
also be used in conjunction with a poly-2-hydroxyethyl acrylate coating. A
generic structure of a
preferred acrylate-PEG-Maleim;ide tether can be represented by the formula:
Protease G
y
S
~O
N
C
H
~I =O
PolyEthyleneGlycol
X
R-~CH-CH2, CH-CH2---CH-CH2 --~CH-CH~R
m ~ P n
CCH-CH2~R
~X
Yet another embodiment of a cysteine containing protein tethered to a
polymeric coating on
the wipe substrate comprises a PEG-Maleimid covalently bonded to a
polyethyleneimine coating
by N-hydroxysuccinimide, as represented by the formula:
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lz
Protease G
S
~O
O N
H
O
Pal yLthyleneGlycol~
O
NH, NH NHa
C;H--CH, x CH= CH, CH, CH, y
Binding means comprisinf_=. tethered proteins are preferred over physical
entrapment since
tethered proteins are more mobile and are less covered by the polymer coating,
both of which
provide more activity of the proteins.
Protein Covalentl~Linked to Activated Implement Surface
Another means for binding the protein of the present invention is a covalem
link to an
activated site on the surface. For this means of binding the substrate is
preferrably a cellulosic
material. The chemical link can be any di-functional compound which will react
with the
substrate and the protein. The preferred chemical link is ethylenediamine/N-y-
maleimidobutyryloxysuccinimide ester (GMBS).
A generic structure of a preferred ethylenediamine/GMBS linkage can be
represented by
the formula:
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13
Protease G
S
~O
O N
O
H
H
'O~~
HO HO p
CE HO rl0 O'"
GLULOSICS(IgSTRATL HO
Protein Covalentlv Linked to Activated Implement Surface Via Polvmeric Tether
Yet another means for binding the active protein to the substrate of the
personal care wipe
of the present invention is a polymeric tether covalently bonded to an
activated site on the surface
of the substrate. The preferred) tether is ethylenediamine/polyethylene glycol-
Maleimide. A
generic structure of a directly bonded ethylenediamine/PEG-Maleimide tether
can be represented
by the formula: '
Protease G
~O
O N
O
HN
,PolyEthyleneGlycol
~~,/O
H
HO\
l 0 HO
O HN
i~« HO O
C~LLUI,pSICSU$ST~T HO
E
OPTIONAL INGREDIENTS
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14
The wipe compositions of the present invention can Comprise a wide range of
optional
ingredients. The CTFA International Cosmetic Ingredient Dictionary, Sixth
Edition, 1995, which
is incorporated by reference herein in its entirety, describes a wide variety
of nonlimiting
cosmetic and pharmaceutical ingredients commonly used in the skin care
industry, which are
suitable for use in the compositions of the present invention. Nonlimiting
examples of functional
classes of ingredients are described at page 537 of this reference. Examples
of these functional
classes include: abrasives, anti-acne agents, anticaking agents, anti-
microbial agents,
antioxidants, binders, biological additives, bulking agents, chelating agents,
chemical additives,
colorants, cosmetic astringents, cosmetic biocides, denaturants, drug
astringents, emulsifiers,
external analgesics, film forrners, fragrance components, humectants, mildness
enhancers
(cationic and nonionic polymers, co-surfactants, lipid moisturizers,
hydrocarbon oils, silicone
oils, waxes), opacifying agents., plasticizers, preservatives, propellants,
reducing agents, skin
bleaching agents, skin-conditioning agents (emollient, humectants,
miscellaneous, and occlusive),
skin protectants, solvents, foam boosters, hydrotropes, solubilizing agents,
stabilizers, suspending
agents, sunscreen agents, surfactants (anionic, cationic, amphoteric,
zwitterionic), ultraviolet light
absorbers, and viscosity increasing agents (aqueous and nonaqueous). Examples
of other
functional classes of materials useful herein that are well known to one of
ordinary skill in the art
include solubilizing agents, sequestrants, and keratolytics, and the like.
METHODS OF USE
The personal care compo<.~itions of the present invention are useful for
personal cleansing,
cosmetic skin treatment, and/or skin conditioning. The present invention may
take the form of a
personal care wipe or a personal care skin mask. Typically, the wipe is used
to expose the area to
be cleansed to the active enzyrnes for a relatively short period of time. For
use, the wipe is
contacted with or wiped skin which needs treatment and then removed. Typical
quantities of the
present wipes useful for cleansing, range from about 1 to about 4 wipes per
use, preferably from
about 1 to about 2 wipes per use. The skin mask is used to expose the area to
be treated for a
relatively longer period of time. Typical quantities of the present skin masks
useful for cleansing,
range from about 1 to about 2 masks per use, preferably 1 mask per use.
EXAMPLES AND METHODS OF MANUFACTURE
The following examples further describe and demonstrate embodiments within the
scope
of the present invention. In the following examples, all ingredients are
listed at an active level.
The examples are given solely for the purpose of illustration and are not to
be construed as
limitations of the present invention, as many variations thereof are possible
without departing
from the spirit and scope of the invention. Ingredients are identified by
chemical or CTFA name.
The following are nonlimiting examples of the wipes with bound active proteins
of the
present invention.
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EXAMPLE 1 - Protease G entrapped in acr l~gel to a rayon/PET substrate.
Purify and concentrate Protease G in IOmM monopotassium phosphate (KH~POa)
buffer,
pH 5.5, to a concentration of 3 mg/mL. Add 2-hydroxyethyl acrylate (HEA) to a
final molar
concentration of 1.3M. Add SOrnM hydrogen peroxide (H~Oz) to achieve a final
molar
concentration of 2.SmM H,OZ. lJniformly spray Protease F/HEA/HzO~ solution
onta rayon/PET
sheet until sheet is saturated. Uniformly spray a solution of iron (II)
sulfate heptahydrate
(FeSO,~7H,0) onto sheet. Allow sheet to sit for 5 minutes. Rinse sheet through
successive baths
of 0.01 M KH~PO, until all free protein has been removed. Dry sheets.
EXAMPLE 2 - Protease G tethered to polymeric coating
Purify and concentrate Protease G to approximately 2.5 mg/mL in 10 mM KH,PO,
buffer,
pH 5.5. Add Acrylate-PEGS,°o-Maleiminde (Shearwater Polymers, Inc.) in
a I5:1 molar excess.
Raise the solution pH to 7 with dilute sodium hydroxide (NaOH). Let Protease G
and Acrylate-
PEG-Maleimide react for 1-2 hours at room temperature. Drop the solution pH to
S.5 with dilute
phosphoric acid (H3PO.,). Add 2-hydroxyethyl acrylate (HEA) to the Protease G-
PEG-Maleimide
solution to achieve a final molar concentration of 1.3 M. Add 50 mM HBO,
solution to the
Protease G-PEG/HEA solution such that a final molar concentration of 2.5 mM
H,O, exists.
Spray the solution onto a rayon/1'I:T sheet until the sheet has become
saturated. Separately spray
50 mM iron (II) sulfate heptahydrate (FeSO.,~7H~0) solution onto the sheet
such that the entire
surface area is sprayed uniformly. Allow the sheet to sit for 5 minutes. Then
rinse the sheet
through successive 0.01 M KH,P~Oa baths until all free Protease G has been
removed. Dry sheets.
EXAMPLE 3 - Protease G Directly Covalently Linked to Activated Implement
Surface
Soak rayon/PET sheets in .an aqueous 10% (w/v) NaOH solution for 10-15 minutes
with
shaking on auto-shaker, using 1 n~I~ solution per 1 cmz sheet. Wash sheets
three times with
deionized water under suction, with approxiamately 1 ml water per cm'' sheet
each wash. Wash
sheets five times with acetone under suction. Allow sheets to soak in acetone
for I minute
between washes. React sheets in. a 10% (w/v) p-toluenesulfonyl chloride in
acetone solution for
25-30 minutes with shaking (~ 1 ml/cm2). Rinse sheets 3 times with acetone
under suction to
remove excess p-toluenesulfonyl chloride. React sheets in 2.2M ethylenediamine
in acetone
solution, pH 13-14, for 2 hours with shaking. Rinse sheets 3 times with
acetone under suction to
remove excess EDA. Rinse sheets 5 times with dry N,N dirnethylformamide (DMF)
under
suction. Allow sheets to soak in DMF for 1 minute between washes. React sheets
in 10% (w/v)
N y maleimidobutyryloxysuccinimide ester (GMBS) overnight with shaking
(~lml/cmz). Rinse
sheets 3 times with dry DMF under suction. Rinse sheets 5 times with 0.01 M
KHZP04 buffer, pH
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16
7 under suction. Let sheets soak for 1 minute in buffer between rinses. React
sheets in 3 mg/ml
Protease G in IOmM KHZP04 buffer, pH 7 (~lml/cmz) for approximately 1-2 hours.
Rinse sheets
3 times with 0.01 M KH~POa buffer, pH 5.5. Dry sheets.
EXAMPLE 4 - Protease G Covalently Linked to Activated Imulement Surface via
Polymer
Tether
Soak rayon/PET sheets in an aqueous 10% (w/v) NaOH solution for 10-1 S minutes
with
shaking on auto-shaker, using 1 mL solution per 1 cmz sheet. Wash sheets three
times with
deionized water under suction (--Iml/cm2/wash). Wash sheets five times with
acetone under
suction. Allow sheets to soak in acetone for 1 minute between washes. React
sheets in a 10%
(w/v) p-toluenesulfonyl chloride in acetone solution for 25-30 minutes with
shaking (~lml/cm2).
Rinse sheets 3 times with acetone under suction to remove excess p-
toluenesulfonyl chloride.
React sheets in 2.2M ethylenediamine in acetone solution, pH 13-14, for 2
hours with shaking.
Rinse sheets 3 times with acetone under suction to remove excess EDA. Rinse
sheets 5 times
with 0.2M sodium borate buffer, pH 8.5 under suction. Let sheets soak for I
minute in buffer
between rinses. React sheets im 3-5% (w/v) Maliemide-PEG34oo-NHS (Shearwater
Polymers,
Inc.) in 0.2M sodium borate buffer, pH 8.5 for I-2 hours with shaking
(~Iml/cm''). Rinse sheets
times with 10 mM KH,PO., buffer, pH 7, under suction to remove excess PEG.
React sheets in
3 mg/ml Protease G in IOmM :KH,P04 buffer, pH 7 (~lml/cm2) for approximately I-
2 hours.
Rinse sheets 3 times with 0.01 M KH,PO.~ buffer, pH 5.5. Dry sheets.
EXAMPLE 5 - Protease G Cova;lently Linked to Coated Implement Surface via
Polymer Tether
Soak rayon/PET sheets in :500 ppm bath of polyethyleneimine for I hour with
shaking at
room temperature (~l-2ml/cm'' sl~teet). Rinse rayon/PET sheets in 2 successive
0.2M sodium
borate buffer, pH 12, baths (~5-10 ml/em''). Rinse sheets in 2 successive 0.2M
sodium borate
buffer, pH 8.5, baths. React shecas in 5 mg/mL NHS-PEG34oo-Maleimide in 0.2M
sodium borate
buffer, pH 8.5, with shaking at room temperature for 1 hour. Rinse cloths
through 4 deionized
water baths (-S-10 ml/cm2). Rinse cloths through 2 0.01 M KH,POa buffer, pH 7-
7.5, baths.
Separately, purify and concentrate Protease G to 1-2 mg/ml in 0.01 M KH,PO.,
buffer, pH 7-7.5.
React "PEGylated" sheets in Protease G solution, pH 7-7.5, for 1 hour at room
temperature with
shaking. Rinse sheets through S successive O.OI M KH~P04, pH 5.5, baths to
remove unreacted,
unbound Protease G. Let sheets dry.
Exat~les 6-9
A personal care wipe composition product is prepared as follows:
Ingredients Weight Percent
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17
Example 6 Example 7 Example 8 Example 9
Phase A
Water QS 100 QS 100 QS 100 QS 100
Glycerin 10.00 10.00 10.00 10.00
Disodium Lauroamphodiacetate 4.00 ---- ----
(and) 4.00
Sodium Trideceth Sulfate
Sodium Lauroamphoacetate ---- ---- 2.40 2.40
Sodium Lauroyl Sarcosinate 4.004.00 ---- ----
Ammonium Laureth Sulfate ---- ---- 4.20 4.20
Ammonium Lauryl Sulfate ---- ---- 1.40 1.40
Polyquarternium-10 0.25 0.25 0.25 0.25
Disodium EDTA 0.10 0.10 0.10 0.10
Phase B
Sucrose Ester Fatty Acid Cottonate3.00 3.00 3.00
3.00
Petrolatum ---- 1.50 ---- ----
Cetyl Dimethicone ---- ---- ---- 2.00
Phase C
Butylene Glycol 2.00 2.00 2.00 2.00
DMDM Hydantoin (and) 0.20 0.20 0.20 0.20
Iodopropynyl Carbamate
Water Insoluble Substrate
A hydroapertured, nonwoven substratebasis of about sy comprising
having a weight 60 g 50%
rayon and SO% polyester approximately7.6 in. out 20 mil
6 in. by and having a
a thiclmess
of ab
bound active protein per Examples
1-7.
In a suitable vessel., they Phase A ingredients are mixed at room temperature
to form a
dispersion and heated with stirring to 65°C. The Phase B ingredients
are mixed in a separate
suitable vessel and heated to 65°'C.'. Once the temperatures are the
same, the Phase B ingredients
are mixed into the vessel containing the Phase A ingredients and then cooled
to 45°C. 'The Phase
C ingredients are then mixed together in a separate vessel at room
temperature. Next, the Phase
C mixture is added into the v~asel containing the combination of Phases A and
B at room
temperature. I .S grams of the resulting solution is sprayed onto each
substrate. Alternatively, the
substrate can be dipped into the resulting solution. The treated substrate is
then dried in an oven
to constant weight. Alternatively, the treated substrate is dried in a
convection oven at 45°C to
constant weight.
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18
In alternative embodirr~ents, other substrates such as woven substrates,
hydroentangled
substrates, natural sponges, synthetic sponges, or polymeric netted meshes.
Alternative
embodiments may be in the from of person care skin masks.
Examples 10-13
A personal care wipe is prepared as follows:
Ingredients Weight Percent
Example 10 Example 11 Example 12 Example 13
Phase A
Water QS 100 QS 100 QS 100 QS 100
Glycerin 10.00 10.00 10.00 10.00
Panthenol 0.50 ---- 0.50 0.50
Sodium Lauroamphoacetate2.40 2.40 2.40 2.40
Ammonium Lauryl Sulfate1.40 1.40 1.40 I .40
Polyquarternium-10 0.25 0.25 0.25 0.25
Disodium EDTA 0.10 0.10 0.10 0.10
Phase B
Sucrose Ester Fatty 3.00 3.00 3.00 3.00
Acid Cottonate
Petrolatum ---- ---- ---- 0.50
Cetyl Dimethicone ---- ---- ---- 0.50
Cetyl Ricinoleate ---- 2.00 2.00 1.00
Phase C
Butylene Glycol 2.00 2.00 2.00 2.00
DMDM Hydantoin (and) 0.20 0.20 0.20 0.20
Iodopropynyl Carbamate
Water Insoluble Substrate
A hydroapertured, nonwoven basis of about sy comprising
substrate having a weight 60 g 50%
rayon and 50% polyester 7.6 in. out 20 mil
approximately 6 in. and a having a
by thickness
of ab
bound active protein
per Examples 1-7.
In a suitable vessel., the Phase A ingredients are mixed at room temperature
to fonm a
dispersion and heated with stirring to 65°C. The Phase B ingredients
are mixed in a separate
suitable vessel and heated to 65°C. Once the temperatures are the same,
the Phase B ingredients
are mixed into the vessel containing the Phase A ingredients and then cooled
to 45°C. Next, the
Phase C mixture is added into t:he vessel containing the combination of Phases
A and B at room
temperature. 1.5 grams of the resulting solution is sprayed onto each
substrate. Alternatively, the
substrate can be dipped into the resulting solution. The treated substrate is
then dried in an oven
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19
to constant weight. Alternatively, the treated substrate is dried in a
convection oven at 45°C to
constant weight.
In alternative embodiments, other substrates such as woven substrates,
hydroentangled
substrates, natural sponges, synthetic sponges, or polymeric netted meshes.
Alternative
embodiments may lae in the from of person care skin masks.
Examples 14-17
Ingredients Weight Percent
Example 14 Example 15 Example 16 Example 17
Phase A
Water QS 100 QS 100 QS 100 QS 100
Disodium Lauroamphodiacetate 4.00 ---- ----
I;and) 4.00
Sodium Trideceth Sulfate
Sodium Lauroamphoacetate ---- ---- 2.40 2.40
Sodium Lauroyl Sarcosinate 4.00 ---- ----
4.00
Ammonium Laureth Sulfate ---- ---- 4.20 4.20
Ammonium Lauryl Sulfate ---- ---- 1.40 1.4U
Disodium EDTA 0.10 0.10 0.10 0.10
Phase B
Sucrose Ester Fatty Acid Cottonate3.00 3.00 3.00
3.00
Petrolatum ____ 1.50 ____ ___-
Cetyl Dimethicone ---- ---- ---- 2.00
Phase C
DMDM Hydantoin (and) 0.20 0.20 0.20 0.20
Iodopropynyl Carbamate
Water Insoluble Substrate
A hydroapertured, nonwoven basis of about
sulbstrate having a weight 60 gsy
comprising
50%
rayon and 50% polyester approxiimately7.6 in.
6 in. by and a
thickness
of about
20 mil
having
a
bound active protein per Exampifs
I-7.
In a suitable vessel., the Phase A ingredients are mixed at room temperature
to form a
dispersion and heated with stirring to 65°C. The Phase B ingredients
are mixed in a separate
suitable vessel and heated to 65°C. Once the temperatures are the same,
the Phase B ingredients
are mixed into the vessel containing the Phase A ingredients and then cooled
to 45°C. The Phase
C ingredients are then mixed together in a separate vessel at room
temperature. Next, the Phase
C mixture is added into the vessel containing the combination of Phases A and
B at room
temperature. 1.5 grams of the resulting solution is sprayed each substrate.
Alternatively, the
CA 02345127 2001-03-22
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substrate can be dipped into the solution. The treated substrate is then dried
in an oven to
constant weight. Alternatively, the treated substrate is dried in a convection
oven at about 45°C
to constant weight.
In alternative embodiments, other substrates such as woven substrates,
hydroentangled
substrates, natural sponges, syrothetic sponges, or polymeric netted meshes.
Alternative
embodiments may be in the from of person care skin masks.
Examples 18-21
Ingredients Weight Percent
Example 18 Example 19 Example 20 Example 21
Phase A
Water QS 100 QS 100 QS 100 QS 100
Sodium Lauroamphoacetate 2.40 2.40 2.40 2.40
Ammonium Laureth Sulfate 4.20 4.20 4.20 4.20
Ammonium Lauryl Sulfate 1.40 1.40 1.40 1.40
Disodium EDTA 0.10 0.10 0.10 0.10
Phase B
Sucrose Ester Fatty Acid Cottonate3.00 3.00 3.00
3.00
Petrolatum ---- 0.50 1.00 ----
Cetyl Dimethicone ---- 0.50 ---- 1.00
Cetyl Ricinoleate 2.00 0.50 1.00 1.00
Phase C
DMDM Hydantoin (and) 0.20 0.20 0.20 0.20
Iodopropynyl Carbamate
Water Insoluble Substrate
A hydroapertured, nonwoven substrate of about
having a basis weight 60 gsy
comprising
50%
rayon and 50% polyester approximately hickness
6 in. by 7.6 in, and a t of about
20 mil
having
a
bound active protein according
to Examples I-7.
In a suitable vessel., the Phase A ingredients are mixed at room temperature
to form a
dispersion and heated with stirring to 65°C. The Phase B ingredients
are mixed in a separate
suitable vessel and heated to 65°C. Once the temperatures are the same,
the Phase B ingredients
are mixed into the vessel containing the Phase A ingredients and then cooled
to 45°C. Next, the
Phase C mixture is added into the vessel containing the combination of Phases
A and B at room
temperature. 1.5 grams of the resulting solution is sprayed onto each
substrate. Alternatively, the
substrate can be dipped into the solution. The treated substrate is then dried
in an oven to
CA 02345127 2001-03-22
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21
- constant weight. Alternatively, the treated substrate is dried in a
convection oven at about 45°C
to constant weight.
In alternative embodiments, other substrates such as woven substrates,
hydroentangled
substrates, natural sponges, synthetic sponges, or polymeric netted meshes.
Alternative
embodiments may be in the from of person care skin masks.
