U.S. patent application number 16/458080 was filed with the patent office on 2019-10-24 for apparatus and method for preparing cosmeceutical ingredients containing epi-dermal delivery mechanisms.
The applicant listed for this patent is PPP&C Inc., dba ROBIN MCGRAW REVELATION. Invention is credited to David A. Richard.
Application Number | 20190321270 16/458080 |
Document ID | / |
Family ID | 59064892 |
Filed Date | 2019-10-24 |
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United States Patent
Application |
20190321270 |
Kind Code |
A1 |
Richard; David A. |
October 24, 2019 |
APPARATUS AND METHOD FOR PREPARING COSMECEUTICAL INGREDIENTS
CONTAINING EPI-DERMAL DELIVERY MECHANISMS
Abstract
The skin serves as a barrier that protects the body from the
external environment and prevents water loss. This barrier function
also prevents most hydrophilic or hydrophobic and large molecular
weight ingredients (>500 kDa) from penetrating intact skin.
Until recently, methods to increase stratum corneum permeability
were generally not effective enough to make the stratum corneum so
permeable that the barrier posed by the viable epidermis mattered.
However, that has now changed with the development of the present
embodiment's physical methods and highly optimized chemical
formulations, such that we revisited the permeability of the full
epidermis with the example embodiment's constructs and not focus
only on the stratum corneum. This example embodiment therefore
tests the hypothesis that the viable epidermis offers a significant
permeability barrier to both small molecules and macromolecules
that becomes the rate limiting step.
Inventors: |
Richard; David A.; (Shingle
Springs, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PPP&C Inc., dba ROBIN MCGRAW REVELATION |
Burbank |
CA |
US |
|
|
Family ID: |
59064892 |
Appl. No.: |
16/458080 |
Filed: |
June 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14971320 |
Dec 16, 2015 |
10342747 |
|
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16458080 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61Q 19/00 20130101;
A61K 8/63 20130101; A61K 2800/70 20130101; A61K 8/14 20130101; A61K
8/416 20130101; A61Q 19/08 20130101; A61K 8/553 20130101; A61K 8/06
20130101; A61K 8/735 20130101; A61K 2800/805 20130101; A61K 8/9789
20170801 |
International
Class: |
A61K 8/06 20060101
A61K008/06; A61Q 19/00 20060101 A61Q019/00; A61K 8/55 20060101
A61K008/55; A61K 8/73 20060101 A61K008/73; A61Q 19/08 20060101
A61Q019/08; A61K 8/14 20060101 A61K008/14; A61K 8/41 20060101
A61K008/41; A61K 8/63 20060101 A61K008/63 |
Claims
1. A method for the construction of cosmeceutically bioactive
compositions including liposome vesicle epi-dermal delivery
vehicles without use of supercritical CO2 and in a continuously
operating process, the method comprising: forming a liposome
solution or mixture of cosmeceutically benevolent phospholipids,
the solution being hydrophobic or hydrophilic, the solution
including an ingredient from the group consisting of a natural
ingredient and a synthetic ingredient, the solution having an
aqueous phase; removing any constituent attributes of
water-insolubility from the solution, while operating under
conditions to preserve activity of labile biomolecules; loading the
liposome solution and desired hydrophobic bio-actives into an
organic solvent residing in a pressure reactor, the pressure
reactor having been previously driven to a pre-determined working
temperature; pressurizing the reactor with compressed CO2 until
reaching a pre-determined working pressure; and depressurizing the
resulting CO2-expanded solution over an aqueous phase to form
vesicular conjugates, the resulting solution containing water
soluble cationic or non-ionic surfactants and hydrophilic or
hydrophobic bio-actives.
2. The method of claim 1 further comprising producing liposomes,
niosomes, and plastic niosomes in bulk, wherein the phospholipids
are selected from the group consisting of: a natural phospholipid,
a synthetic phospholipid, a non-ionic compound, and an ionic
compound.
3. The method of claim 2 wherein the liposome approximate mean size
is between 25 to 200 nm.
4. The method of claim 1 wherein lipids are present in the pressure
reactor in an organic phase in an amount of 5 to 90 percent by
weight of the organic phase.
5. The method of claim 1 wherein the reactor maintaining a pressure
and temperature to prevent the CO2 from going supercritical.
6. The method of claim 1 wherein the lipid is selected from a
nonionic amphiphilic lipid, an ionic amphiphilic lipid, or a
mixture of a nonionic lipid and an ionic lipid.
7. The method of claim 1 including forming nano and macro carriers
as either unimolecular or multi-molecular carriers of beneficial
cosmeceutical ingredients.
8. The method of claim 1 further comprising providing a skin
penetration enhancing vesicle carrier for incorporating cholesterol
into a bilayer increasing entrapment efficiency by bio-actives
interaction with surfactant head groups and developing a charge
that creates mutual repulsion between surfactant bilayers and
increasing vesicle content.
9. The method of claim 2 wherein the liposomes, niosomes, and
plastic niosomes trap hydrophilic or hydrophobic compounds within
the aqueous interior of the vesicle.
10. The method of claim 8 including entrapping a compound.
11. The method of claim 8, wherein the skin penetration enhancing
vesicle carrier is a phospholipid membrane.
12. The method of claim 1, wherein the vesicle content is one of
natural or synthetic cosmetic ingredients used for treating or
correcting a therapeutic effect of the skin surface and epi-dermal
layers to be beneficial including hydration, skin lightening,
anti-wrinkle, skin smoothing, antioxidant activity/free radical
scavenger, anti-inflammatory/anti-irritant, collagen stimulation,
cell regeneration/stimulation, sebum regulation, anti-cellulite,
antimicrobial, acne(s) and antibacterial results.
13. The method of claim 1, wherein the vesicle contains naturally
derived cosmetic beneficial ingredients that originate from plants,
herbs, roots, flowers, fruits, leaves or seeds, aloe vera, almond
oil, avocado oil, coconut oil, hazelnut oil, jojoba oil, olive oil,
palm oil, pumpkin seed oil, sesame oil, sunflower oil, tamanu oil,
candeia oil, arnica, chamomile, oat extract, hibiscus flower,
boswellia serratta, cocoa powder, green and white tea, gotu kola,
chamomile extract, L-arginine, glutamine, pantothenic acid, white
willow bark extract, tetrahydrocurcuminoids, alpha-arbutin,
aloesin, alpha glucosyl hesperidin, niacinimide, fucoidan,
magnesium asorbyl phosphate, azelaic acid, N-acetyl-D-glucosamine,
glutathione, mulberry, pomegranate seed oil, cyprus rotund root
extract, licorice, licorice-glabrin root extract, kojic acid, panax
ginseng root extract, ginko bilbao, salicylic acid, Lauric acid,
glycerin, caffeine, tocopheryl acetate, copper peptide, retinyl
palmitate, asorbyl palmitate, wakame, dimethylethanolamine, beta
glucan, triglyceride as well as hyaluronic acid.
14. The method of claim 1, wherein a phospholipid ingredient
trapped in a membrane is present in an amount of about 0.05% to 5%
by weight per 100 unit's volume of the composition.
15. The method of claim 1, wherein the cosmetically bioactive
composition is a phospholipid or a mixture of phospholipid vesicles
and is present in an amount of 0.2 to 5% by weight per 100 units
volume of the composition.
16. The method of claim 1 wherein the vesicle size is controlled
between 0.2 and 0.3 .mu.m by the rate of depressurization.
17. The method of claim 1 including incorporating less than 25% of
cholesterol into the liposome solution to entrap hydrophobic or
hydrophilic compounds.
18. The method of claim 1 including obtaining homogeneous
nanovesicles composed of hyaluronic acid, cholesterol and a
cationic surfactant CTAB (cetyltrimethylammonium bromide).
19. The method of claim 1 wherein the extent and rate of
percutaneous absorption and transportation by non-systemic or
systemic routes are influenced by the process of vesicle
formulation.
Description
PRIORITY APPLICATIONS
[0001] This is a continuation patent application drawing priority
from U.S. patent application Ser. No. 14/971,320; filed Dec. 16,
2015. This present patent application draws priority from the
referenced patent applications. The entire disclosure of the
referenced patent applications is considered part of the disclosure
of the present application and is hereby incorporated by reference
herein in its entirety.
TECHNICAL FIELD
[0002] The application of innovative micro and nano vesicle forming
technologies to effect beneficial results through the application
of synthetic and natural ingredients to the skin has shown a great
potential to significantly benefit the cosmetic formulation
practice, offering solutions to many of the current limitations in
ingredients, treatment style and management of human skin effected
by environmental and physiological impact.
BACKGROUND
[0003] A liposome vesicle encapsulates a region of aqueous solution
inside a hydrophobic membrane; dissolved hydrophilic solutes cannot
readily pass through the lipids. Hydrophobic chemicals can be
dissolved into the membrane, and in this way liposome can carry
both hydrophobic molecules and hydrophilic molecules.
[0004] Several CFs (Compressed Fluid) methodologies have been used
to generate vesicles, some of them already existed and others were
developed for this specific application. Most of the methods
involve a mixture between the compressed CO2, the vesicle membrane
constituents and an organic solvent for producing the vesicles upon
contact with an aqueous phase.
[0005] Depending on the role of the compressed CO2 used in each
method, they can be classified as: Process involving the use of CO2
as a solvent (e.g. Supercritical Liposome Method and Rapid
Expansion of Supercritical Solutions), Processes involving the use
of CO2 as an anti-solvent (e.g. Gas Antisolvent Precipitation and
Aerosol Solvent Extraction System) and Processes involving the use
of CO2 as a co-solvent or a processing aid (e.g. Depressurization
of an Expanded Liquid Organic Solution-Suspension and Supercritical
Reverse Phase Evaporation).
[0006] Model hydrophilic and hydrophobic compounds, such as
fluorescent dyes, sugars and cholesterol, have been encapsulated
into vesicles using these methodologies whereas biomolecules like
proteins, anticancer drugs and antibiotic, have been integrated in
less extent.
[0007] Transdermal delivery systems (TDS) were introduced onto the
US market in the late 1970s), but transdermal delivery of drugs had
been around for a very long time. There have been previous reports
about the use of mustard plasters to alleviate chest congestion and
belladonna plasters used as analgesics. The mustard plasters were
homemade as well as available commercially where mustard seeds were
ground and mixed with water to form a paste, which was in turn used
to form a dispersion type of delivery system.
[0008] Once applied to the skin, enzymes activated by body heat led
to the formation of an active ingredient (allyl isothiocyanate).
Transport of the active drug component took place by passive
diffusion across the skin--the very basis of transdermal drug
delivery.
[0009] The epi-dermis undergoes changes in structure and function
which result in many of the characteristics of aged skin, including
loss of elasticity, formation of wrinkles, loss of water-holding
capacity, sagging, and poor microcirculation. At the molecular
level, these changes have been correlated with biochemical changes
in the content and structure of the extracellular matrix to which
the major cells of the epi-dermis (i.e., the fibroblasts) reside.
Collagen becomes highly cross-linked and inelastic, elastin is
reduced in amounts and is incorrectly distributed, which results in
reduced intercellular water for reduction and repair of these
changes. Nonsurgical options include chemical peels and chemicals
with minor irritant properties (e.g., topical retinoid, salicylic
acid, and alpha-hydroxy acids), are based on the principle of
wounding the stratum corneum--the skin's primary defense against
the transit of exogenous materials into the epidermis and
dermis--to allow the penetration of constituents through the
disrupted skin, which stimulates the desired response, typically
restorative healing. All of these techniques require a wound
healing response to the skins being intentionally wounded as a
method to initiate the rejuvenation process.
[0010] Owing to the selective nature of the skin barrier, only a
small pool of ingredients can be delivered non-systemically or
systemically at therapeutically relevant rates. Besides great
potency, the physicochemical ingredient characteristics often
evoked as favorable for percutaneous delivery include moderate
lipophilicity and low-molecular-weight. However, a large number of
skin damage mitigating active agents do not fulfill these
criteria.
[0011] Chemical permeation enhancers facilitate drug permeation
across the skin by increasing drug partitioning into the barrier
domain of the stratum corneum, increasing drug diffusivity in the
barrier domain of the stratum corneum or the combination of both
(2).
[0012] The heterogeneous stratum corneum is composed of keratin
`bricks` and intercellular continuous lipid `mortar` organized in
multilamellar strata (3)(4)(5). Depending on the nature of the drug
or ingredient, either of these two environments may be the
rate-limiting milieu (barrier domain) for the percutaneous
transport.
[0013] As a consequence, it is anticipated that the magnitude of
permeation improvement obtained with a given permeation enhancer
will vary between lipophilic and hydrophilic ingredients. Several
mechanisms of action are known: increasing fluidity of stratum
corneum lipid bilayers, extraction of intercellular lipids,
increase of ingredient's thermodynamic activity, increase in
stratum corneum hydration, alteration of proteinaceous corneocyte
components and others.
[0014] The stratum corneum is a formidable barrier to exogenous
agents including cosmeceutical ingredients. Therefore, it is often
necessary to add permeation-enhancing chemicals to aid beneficial
constituents in passing through the stratum corneum.
Permeation-enhancing chemicals include fatty acids, organic
solvents (i.e., acetone and ethanol), alcohols, esters and
surfactants.
[0015] It is generally understood that for enhancers, increased
potency is directly correlated with increased skin irritation.
Difficulty in reducing the irritation of these agents has been
expressed since the same mechanisms responsible for increasing
permeation cause irritation. While potent enhancers are effective
at transiently compromising the integrity of the stratum corneum
barrier, their action is not entirely limited to the stratum
corneum and the interaction with viable epidermis can cause
cytotoxicity and irritation. Published methods for reducing the
skin irritation of permeation enhancers include combining
permeation enhancers (synergistic mixtures) and manipulation of
their chemical structures.
[0016] Conventional lipid or niosome vesicle production techniques
have drawbacks such as complex and time consuming procedures
involving organic solvents. For liposomes, conventional methods can
involve harsh conditions that result in denaturation of the lipids
and active ingredients, and also cause poor ingredient
encapsulation efficiency.
[0017] Since the liposomes were first used as drug carriers in
1970s. Many methods, such as Supercritical fluids (SCFs), for
preparing liposomes have been developed, but these methods require
large amounts of organic solvents like chloroform, ether, freon,
methylenechloride and methanol that are harmful to the environment
and the human body, and very few methods have been developed that
yield liposomes that have a high trapping efficiency for water
soluble substances without using any organic solvent.
[0018] Additionally, all these methods are not suitable for mass
production of liposomes because they consist of many steps. With
the advent of Green Chemistry in the early 1990s, the surge of
supercritical fluids (SCFs) increased vastly.
[0019] The supercritical state of a fluid (SCF) is intermediate
between that of gas and liquids. The SCF has been used widely in
pharmaceutical industrial operations including crystallization,
particle size reduction, drug delivery preparation, coating and
product sterilization. In the pharmaceutical field, supercritical
carbon dioxide (scCO2) is by far the most commonly used gas, which
can become supercritical at conditions that are equal or exceed its
critical temperature of 31.1.degree. C. and its critical pressure
of 7.38 Megapascals (Mpa).
[0020] The encapsulation degree of any drug into vesicles is
influenced by several parameters related to the: a) vesicle
composition, b) the nature of the cosmeceutical ingredient and c)
the preparation methodology. Regarding the vesicle composition,
besides the selection of the lipids forming the membrane and the
presence of charges on it, the type of vesicle plays also an
important role. Thus, for hydrophilic drugs, such as proteins or
peptides, the encapsulation degree appears to increase in the
following order: MLV<SUV<LUV. (FIG. 1.0) Nevertheless in the
case of hydrophobic drugs, the size and type of liposomes do not
seem to play a major role.
[0021] Liposomes with a single bilayer are known as unilamellar
vesicles (UV). UVs may be made extremely small (SUVs) or large
(LUVs) (FIG. 3.0). Liposomes are prepared in the laboratory by
sonication, detergent dialysis, ethanol injection, French press
extrusion, ether infusion, and reverse phase evaporation.
[0022] These methods often leave residuals such as detergents or
organics with the final liposome. From a production standpoint, it
is clearly preferable to utilize procedures which do not use
organic solvents since these materials must be subsequently
removed.
[0023] Some of the methods impose harsh or extreme conditions which
can result in the denaturation of the phospholipid raw material and
encapsulated ingredients. These methods are not readily scalable
for mass production of large volumes of liposomes.
[0024] Several methods, such as energy input in the form of sonic
energy (sonication) or mechanical energy (extrusion), exist for
producing MLVs (multilamellar vesicles), LUVs and SUVs without the
use of organic solvents.
[0025] MLVs (multilamellar vesicles), free of organic solvents, are
usually prepared by agitating lipids in the presence of water. The
MLVs are then subjected to several cycles of freeze thawing in
order to increase the trapping efficiencies for water soluble
ingredients.
[0026] MLVs are also used as the starting materials for LUV and SUV
production. One approach of creating LUVs, free of organic
solvents, involves the high pressure extrusion of MLVs through
polycarbonate filters of controlled pore size. SUVs can be produced
from MLVs by sonication,
[0027] French press or high pressure homogenization techniques.
High pressure homogenization has certain limitations. High pressure
homogenization is useful only for the formation of SUVs. In
addition, high pressure homogenization may create excessively high
temperatures.
[0028] Contrary to the present embodiment, extremely high pressures
are associated with equipment failures. High pressure
homogenization does not insure end product sterility. High pressure
homogenization is associated with poor operability because of valve
plugging and poor solution recycling.
[0029] The use of liposomes for the delivery and controlled release
of therapeutic drugs requires relatively large supplies of
liposomes suitable for in vivo use (FIG. 6.0). Present laboratory
scale methods lack reproducibility, in terms of quantity and
quality of encapsulated ingredients, lipid content and integrity,
and liposome size distribution and captured volume.
[0030] The multidimensional characteristics of the ingredient and
the liposome, as well as potential raw material variability,
influence reproducibility. Present state-of-the-art liposome and
niosome products are not stable. It is desirable to have final
formulations which are stable for six months to two years at room
temperature or at refrigeration temperature.
[0031] Present liposome products are difficult to sterilize.
Sterility is currently accomplished by independently sterilizing
the component parts lipid, buffer, ingredient and watery autoclave
or filtration and then mixing in a sterile environment.
[0032] This sterilization process is difficult, time consuming and
expensive since the product must be demonstratively sterile after
several processing steps. Heat sterilization of the finished
product is not possible since heating liposomes or niosomes does
irreparable damage. Filtration through 0.22 micron filters may also
alter the features of multilayered liposomes and elastic
niosomes.
[0033] Gamma ray treatment, not commonly used in the pharmaceutical
industry, may disrupt liposome or elastic niosome membranes.
Picosecond laser sterilization is still experimental and has not
yet been applied to the sterilization of any commercial
pharmaceutical.
[0034] In the past two decades, several cosmetic formulations based
on ingredient delivery systems have been successfully introduced
for the treatment of skin disorders. Many problems exhibited by
free active cosmetic ingredients (ACIs), such as poor solubility,
toxicity, rapid in vivo breakdown, unfavorable pharmacokinetics,
poor bio distribution and lack of selectivity for target tissues
can be ameliorated by the use of a VDS (vesicle delivery system) as
offered by the current embodiment. Although a whole range of
delivery agents exist nowadays, the main components typically
include a nanocarrier, a targeting moiety conjugated to the
nanocarrier, and a cargo, such as the desired cosmeceutical
ingredient.
[0035] In 1846, Gobley separated phospholipids from egg yolk. The
term "lecithin" which is derived from the Greek lekithos was first
used to describe a sticky orange material isolated from egg yolk.
"Lecithin" refers to the lipids containing phosphorus isolated from
eggs and brains; (3) from a scientific point of view, "lecithin"
refers to PCs (phosphatidylcholine) the most common phospholipid,
egg yolks, liver, wheat germ and peanuts contain the phospholipid
lecithin.
[0036] Phospholipids (FIG. 3.0) have excellent biocompatibility. In
addition, phospholipids are renowned for their amphiphilic
structures. The amphiphilicity confers phospholipids with
self-assembly, emulsifying and wetting characteristics. When
introduced into aqueous milieu, phospholipids self-assembly
generates different super molecular structures which are dependent
on their specific properties and conditions.
[0037] In the need for synthetic analogs of natural phospholipids,
further synthetic phospholipids were for instance designed to
optimize the targeting properties of liposomes. Examples are the
PEG-ylated phospholipids and the cationic phospholipid
1,2-diacyl-P--O ethylphosphatidylcholine. Also attempts were made
to convert by organic chemical means phospholipids into
pharmacological active molecules (for instance ether phospholipids
or to make phospholipid pro-drugs.
[0038] DPPC is the major constituent of stratum corneum surfactants
which controls the dynamic surface tension (DST) and helps
maintaining the epi-dermis health. It is also one of the most
popular phospholipids used for preparing lipid or niosome bilayers
and model biological membranes.
SUMMARY
[0039] The present embodiment features methods and apparatus for
producing liposomes and niosomes containing hydrophobic and
hydrophilic ingredients know to be beneficial to the repair and
rejuvenation to the stratum corneum and underlying epi-dermis with
the ability to effect non-systemic drug absorption and
transportation are influenced by various factors. The methods and
apparatus are suitable for large scale production of pharmaceutical
grade liposomes which are sterile, of a predetermined size, and are
substantially free of organic solvents. The present embodiment
features a method of making liposomes and elastic niosomes using
low pressure fluids.
[0040] As constructed according to the present embodiment example,
nano and macro carriers can be either unimolecular (i.e.:
dendrimers, carbon nanotubes, polymer-conjugate drug/protein, etc.)
or multimolecular carries, based on molecular self-assemblies
(nanoshells, vesicles, etc.). Their major constituents are either
lipids or polymers and they all have in common that the final
arrangement is governed by the nature of the initial components and
the methodology used in their preparation. Some of the advantages
are the incorporation of ACIs (active cosmeceutical
ingredients).
[0041] One method of the example embodiment comprises the steps of
forming a solution or mixture of a phospholipid, a hydrophobic or
hydrophilic cosmeceutical ingredient, an aqueous phase and a low
pressure fluid. The solution or mixture is decompressed to separate
the low pressure, critical fluid, from the phospholipid and aqueous
medium, to form one or more liposomes. This method is referred to
as the decompression method of forming liposomes in the embodiment.
Preferably, the rate of depressurization influences the size of the
liposomes formed.
[0042] According to the procedure of the example embodiment,
schematically represented in FIG. 4.0, operating always under mild
conditions to preserve the activity of the labile biomolecules. The
general method consists in loading a solution of the membrane lipid
components and the desired hydrophobic bio-actives in an organic
solvent (e.g. ethanol), into the high-pressure reactor previously
driven to the preferred working temperature (FIG. 4.0 A). The
reactor is then pressurized, in a second stage, with a large amount
of compressed CO2 until reaching the working pressure (10 MPa)
(FIG. 4.0 B).
[0043] Finally in the third stage, the vesicular conjugates are
formed by depressurizing the resulting CO2-expanded solution over
an aqueous phase, which might contain water soluble surfactants and
hydrophilic bio-actives (FIG. 4.0 C). In this step a flow of N2 at
the working pressure is used in order to push down the CO2-expanded
solution and to keep constant the pressure inside the reactor. It
is worth to note that no further energy input is required for
achieving the desired SUVs (small unilamellar vesicles) structural
characteristics, neither for increasing the loading or
functionalization.
[0044] In applications utilizing the example embodiment with low
pressure fluids, the properties of the coating material and
particularly the interactions of coating materials with low
pressure low temperature fluids are especially important.
[0045] These interactions may be important for enabling the
incorporation of cosmeceutical essential oils into carrier
materials, for example by facilitating the diffusion of the
essential oil due to the swelling and opening of the pores of
carrier material particles.
[0046] One method comprises the steps of (1) forming a solution or
mixture of a phospholipid, (2) an aqueous phase and low pressure
low temperature methodologies. (3) The solution or mixture is
decompressed to separate the fluid, from the phospholipid and
aqueous media, to form one or more liposomes.
[0047] In some embodiments, the aqueous, or addition phase, has a
therapeutic cosmeceutical agent included. As used herein, the term
"therapeutic cosmeceutical agent" means a chemical or ingredient
capable of effecting a desirable response in an individual subject.
This embodiment is ideally suited for therapeutic cosmeceutical
agents which are not shear sensitive.
[0048] Preferably the compressed fluid is recycled. To the extent
that phospholipids and aqueous phase are carried over with the CF,
such components may also be recycled. For convenience, liposomes
formed with CF fluid in the current embodiment are referred to as
"LPLTVs."
[0049] An example embodiment features an apparatus for forming
liposomes/niosomes (non-ionic) vesicles. The apparatus comprises a
first vessel wherein a phospholipid, an aqueous phase and a CF are
combined to form a mixture or solution. The apparatus further
comprises a second vessel in communication with the first vessel
for expansion.
[0050] The apparatus of the embodiment further comprises a third
vessel for depressurization as a means capable of reducing the
pressure of the solution or mixture. Depressurization means may be
interposed between the first and second vessels or may be integral
with a third vessel. The third vessel receives the solution or
mixture of phospholipids and an aqueous phase which form liposomes
upon depressurization.
[0051] Preferably, the CF is removed from depressurization means
and/or the third vessel and recycled.
[0052] One example embodiment comprises the steps of forming a
solution or mixture of a phospholipid and a compressed fluid. The
solution or mixture is then decompressed through a tip or orifice
into an aqueous phase to form one or more liposomes. As a result of
the decompression, the CF is separated from the phospholipids and
the aqueous phase. The released CF is either vented or recycled to
form a solution or mixture of phospholipid.
[0053] A further example embodiment features a method of making
liposomes or niosomes comprising the steps of forming a solution or
mixture of a phospholipid and a CF. The solution or mixture is
injected into an aqueous phase to form one or more liposomes or
niosomes as the phospholipids and CFs are decompressed.
[0054] Preferably, the aqueous phase or phospholipids contain a
cosmeceutical therapeutic agent which is incorporated into the
liposome or niosomes.
[0055] Embodiments of the present method are ideally suited for
skin rejuvenating agents which are shear sensitive such as
botanicals, proteins and peptides. Embodiments of the present
method do not subject botanicals, proteins and peptides to extreme
shear forces or temperatures.
[0056] Example embodiments are ideally suited to form unilamellar
liposome or niosome vesicles. The size of the liposome or niosome
is determined by the rate of decompression.
[0057] A preferred method uses a CF selected from the group of
compositions capable of forming a critical fluid comprising carbon
dioxide; nitrous oxide; halo-hydrocarbons, such as FREON; alkanes
such as propane and ethane; and alkanes such as ethylene.
[0058] One example embodiment features an apparatus for forming
liposomes and niosomes. The apparatus comprises a first vessel for
containing a solution or mixture of a phospholipid and a compressed
fluid. The apparatus further comprises a second vessel for
containing an aqueous phase. The first vessel and the second vessel
are in communication by means of injection means for injecting the
phospholipid and CF fluid mixture into the aqueous phase. Upon
injection into the aqueous phase in the third vessel, liposomes are
formed.
[0059] Preferably, the aqueous phase contains a cosmeceutically
therapeutic agent which cosmeceutical therapeutic agent is
encapsulated within the liposome.
[0060] Conjugation of cosmeceutical bio-beneficial ingredients to
nano carriers can offer over the free ingredient the protection
from premature degradation, a higher stability, an enhance
permeability through biological membranes, a higher control of the
pharmacokinetics, a better ingredient tissue distribution profile,
and an improvement of intracellular, intercellular, and
intra-follicular penetration and the ability to control whether the
nano-carrier goes systemic or non-systemic.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] For a better understanding of the example embodiments,
reference should be made to the following detailed description
disclosed in conjunction with the accompanying drawings, in
which:
[0062] FIG. 1.0 illustrates the classification of vesicles
regarding size and lamellarity.
[0063] FIG. 2.0 illustrates the construction and composition of
phospholipids
[0064] FIG. 3.0 illustrates the major classifications of liposomes
as vesicular systems according to their size and membrane
lamellarity.
[0065] FIG. 4.0 (A, B, C) is a representation of the steps of
forming a solution or mixture of a phospholipid, an aqueous phase
and low pressure low temperature methodologies.
[0066] FIG. 5.0 is a TEM image of liposomes produced in the LPLTVs
process.
[0067] FIG. 6.0 is an image of the appearance of small spheres
aggregating into larger spheres or captured within larger spheres
in the LPLTVs liposomal forming process.
[0068] FIG. 7.0 shows rod or coffee-bean morphology observed in the
liposomes samples produced by the LPLTVs process.
[0069] FIG. 8.0 is a schematic representation of the LPLTV process
method.
[0070] FIG. 9.0 shows a solubility curve of hyaluronic acid and
cholesterol, in ethanol/CO2 at 10 MPa and 308 K.
[0071] FIG. 10.0 is a schematic illustration of the formation of
(a) the hyaluronic acid cholesterol/CTAB bimolecular amphiphile and
(b) their self-assembling into bilayer vesicles based on the
packing parameter concept.
[0072] FIG. 11.0 is a chart showing Hyaluronic Acid levels in
active and control samples.
DETAILED DESCRIPTION
[0073] The present embodiment features methods and apparatus for
producing cosmeceutically benevolent ingredient content liposomes
and niosomes. The methods and apparatus are suitable for large
scale production of pharmaceutical and cosmeceutical grade vesicles
for the treatment of skin anomalies created as a result of aging
skin or chronic environmental insult which are sterile, of a
predetermined size, and are substantially free of organic
solvents.
Definitions
[0074] As used herein, the word "hydrophilic" in relation to the
material means that that material is above 10% soluble in water by
weight at standard temperature and pressure (STP).
[0075] As used herein, the word "hydrophobic" as used in relation
to a material means that that material is less than 0.1% soluble in
water by weight at standard temperature and pressure (STP).
[0076] As used herein, the term (IDS) as used in relation to the
explanation of the current embodiment means Ingredient Delivery
Systems.
[0077] As used herein, the word "micelle" as used in relation to a
material means "molecules having both polar or charged groups and
non-polar regions (amphiphilic molecules) formed aggregates".
[0078] As used herein, the word "vesicle" as used in relation to
one prepared artificially, in which case they are called liposomes.
If there is only one phospholipid bilayer, they are called
unilamellar liposome vesicles; otherwise they are called
multilamellar.
[0079] As used herein, the word "niosome" as used in relation to a
non-ionic surfactant-based Vesicle formed mostly by non-ionic
surfactant and cholesterol incorporation as an excipient.
[0080] As used herein, the term "LPLTVs as used means Low Pressure
Low Temperature alternative construction of Vesicles based on
milder conditions of pressure (<10 MPa) and temperature (<308
K) than the previously described methodologies based on CFs
(Compressed Fluids).
[0081] As used herein, the term Active Cosmetic Ingredients (ACIs)
as used means but is not limited to such substances as synthetic or
natural skin rejuvenating ingredients, sunscreen ingredients,
skin-lightening agents, and anti-acne ingredients.
[0082] As used herein, the term CFs as used means such substances
made from compressed fluids based technologies to produce niosomes
or vesicles.
[0083] As used herein, the term phospholipids as used means lipids
containing phosphorus, a polar potion and non-polar potion in their
structures.
[0084] As used herein, the term niosomes are microscopic lamellar
vesicular structures, which are formed on the admixture of
non-ionic surfactant and cholesterol with subsequent hydration in
aqueous media.
[0085] One example embodiment features an apparatus for forming
liposomes and niosomes. The apparatus comprises a first vessel or
mixing the organic phase, a second vessel for containing a mixture
of multi-lamellar vesicles and a compressed fluid and a third
vessel for decompressing into the aqueous phase. The first vessel
is in communication with a second vessel which second vessel is in
communication with a third vessel capable of decompressing the
mixture to remove the compressed fluid. During decompression, one
or more liposomes or niosomes are formed.
[0086] Another embodiment further comprises a third vessel for
forming multilamellar vesicles by hydrating phospholipids in an
aqueous phase.
[0087] In the embodiment, the aqueous phase or the phospholipids
may contain a therapeutic agent to impart special qualities to the
liposome for beneficial partitioning of the stratum corneum to aid
in transiting cosmeceutically beneficial liposomes or niosomes to
the epi-dermis.
[0088] An embodiment further features control means for determining
the rate of decompression. The rate of decompression determines the
size of liposomes or niosomes.
[0089] Preferably, compressed fluid removed from the liposome
preparation in the decompression vessel is recycled to the first
vessel to form additional mixtures of multilamellar vesicles and
compressed fluid.
[0090] Contact with compressed fluid may cause destruction of the
cellular structures particularly upon rapid decompression. Thus,
embodiments are, for the most part, self-sterilizing.
[0091] Methods and apparatus of the example embodiment are capable
of forming liposomes or niosomes which carry a cosmeceutical
therapeutic agent. The cosmeceutical therapeutic agent can be
incorporated into ingredients which are used to form the liposome
or niosome or the liposome or niosome can be loaded with the
cosmeceutical therapeutic agent after the liposome or niosome is
formed.
[0092] Embodiments allow the recovery of raw materials, lipids and
solvents which are not incorporated into the final liposome or
niosome product. Example embodiments feature efficient
cosmeceutical ingredient entrapment and recovery of un-encapsulated
cosmeceutical ingredient. The operating parameters of the apparatus
and method are consistent with other industrially applied
processes. The method and apparatus are capable of operating
continuously.
[0093] These and other features, aspects, and advantages of the
embodiment will become evident to those of ordinary skill in the
art from a reading of the present disclosure.
[0094] During the depressurization step of the example embodiment,
the expanded organic solution experiences a large, abrupt and
extremely homogenous temperature decrease produced by the CO2
evaporation from the expanded solution. This is the reason that
explains the obtaining of homogenous vesicles regarding size,
lamellarity and morphology compared with the same system but
prepared by a conventional mixing method.
[0095] However, changes in the procedures and equipment, as in the
present embodiment, result in vesicular systems with differentiated
characteristics. The processes can also be distinguished by the
latter hydration step that can occur either during the
pressurization or the depressurization step.
[0096] These lipid or niosome vesicles of the present embodiment
allow the physicochemical properties of ingredient molecules, of a
higher molecular weight in excess of 700 kDa, in a liposomal system
to be changed, which facilitates crossing of the stratum corneum
barrier into the epi-dermis.
[0097] The size of the liposome can be controlled by the rate of
decompression to form liposomes or niosomes of predetermined size
to control the volume and depth of penetration.
[0098] Among the various approaches for exploiting developments in
nano and micro technology for cosmetic applications, ingredient
delivery systems (IDS) have already had an enormous impact on
cosmetic formulation technology, improving the performance of many
existing ingredients and enabling the use of entirely new
therapies. The fact that IDSs can protect sensitive molecules, such
as hormones, enzymes and proteins, from degradation and the in-vivo
attack of the immune system providing longer resident times, have
been used to improve the effectiveness and delivery of these
ingredients. Although nano and micro particulate carriers can be
made from a variety of organic and inorganic materials, vesicle and
polymer based-nanocarriers are perhaps the most widely used for
ingredient delivery purposes.
[0099] Particularly vesicles, liposome and noisome, have served as
convenient delivery vehicles for biologically active compounds
because they are non-toxic, biodegradable and non-immunogenic.
Contrary to products where the active substance is in simple
solution, the pharmacological properties of vesicle-based delivery
systems strongly depend on the structural characteristics of the
conjugates. Indeed, a high degree of structural homogeneity
regarding size, morphology and vesicle organization in the membrane
is crucial, for their optimum performance as functional
entities.
[0100] Liposomes and niosomes are vesicles in which, in the current
embodiment, cosmeceutical ingredients can be trapped and
administered more efficiently. However, these vesicles, micelle,
liposome and niosome, are not similar to each other. In a
comparison, micelles vs. liposomes, and or elastic niosomes, the
differences between the two are explained as; Micelles are
structures composed of a monolayer of amphipathic molecules. In a
biological system, the molecules tend to arrange themselves in such
a manner that the inner core of these structures are hydrophobic
and the outer layers are hydrophilic in nature.
[0101] Liposomes as in the present embodiment, are composed of a
bilayer of amphipathic molecules, the molecules are arranged in two
concentric circles, such that the hydrophilic heads of the outer
layer are exposed to the outer environment, and the hydrophilic
heads of the inner layer make the inner hydrophilic core. The
hydrophobic tails are tucked between the two layers.
[0102] In the present embodiment, elastic liposomes are microscopic
vesicles having single or multiple phospholipid bilayers which can
entrap hydrophilic compounds within their aqueous cores.
[0103] Elastic niosomes are composed of nonionic surfactants,
ethanol and water. They are superior to conventional niosomes
because they enhance penetration of a drug through intact skin by
passing through pores in the stratum corneum, which are smaller
than the vesicles. In fact, their elasticity allows them to pass
through channels that are less than one tenth of their own
diameter. Thus they can deliver ingredients or compounds of both
low and high molecular weight. Furthermore, they can provide
prolonged action and demonstrate superior biological activity
compared to conventional niosomes. The transport of these elastic
vesicles is concentration independent and driven by trans-epidermal
hydration.
[0104] To deliver the molecules to sites of action, the lipid or
niosome bilayer can fuse with other bilayers such as the cell
membrane, thus delivering the liposome contents. By making
liposomes in a solution of natural or synthetic ingredients that
can effect a beneficial change to the skin, (which would normally
be unable to diffuse through the membrane) they can be
(indiscriminately) delivered past the lipid bilayer. A liposome or
niosome vesicle does not necessarily have lipophobic contents, such
as water, although, in the case of the present embodiment, it
usually does.
[0105] The preferred phospholipid in the current process embodiment
is naturally derived, for example phospholipids obtained from plant
or animal sources. Natural phospholipids are purified from, e.g.,
soybean, rapeseed, and sunflower seed. The phospholipid may be
salted or desalted, hydrogenated or partially hydrogenated or
natural, semi-synthetic or synthetic.
[0106] Liposomes, niosomes and in general vesicles, are undoubtedly
one of the most promising carriers in nano and micro cosmeceutical
ingredient delivery. They are particularly important in the stratum
corneum percutaneous transit field due to their great versatility
respect to size, composition, surface characteristics,
biocompatibility, biodegradability, low toxicity, capacity for
entrapping and/or integrating hydrophilic and/or hydrophobic
molecules and possibility of surface functionalization. Vesicles of
the present process embodiment are spherical objects enclosing a
liquid compartment, with a diameter ranging from 20 nm to a few
thousand of nanometers, separated from its surroundings by at least
one thin membrane consisting of a bilayer (unilamellar) or several
layers (multilamellar) of amphiphilic molecules.
[0107] Sometimes the terms liposome, niosome and vesicle are used
interchangeably, although a liposome is a type of vesicle composed
mainly by phospholipids, a niosome as a non-ionic surfactant-based
vesicle formed mostly by non-ionic surfactant and cholesterol
incorporation as an excipient. Vesicles can be formed also by
non-lipid building blocks, such as block co-polymers or cationic or
non-ionic surfactants.
[0108] A liposome or niosome is an artificially-prepared vesicle
composed of a lipid bilayer. The liposome or niosome can be used as
a vehicle for administration of percutaneous skin nutrients and
pharmaceutical drugs. Liposomes and niosomes are composed of
natural phospholipids, and may also contain mixed lipid chains with
surfactant properties (e.g., egg phosphatidylethanolamine).
According to the present process embodiment, a liposome design may
employ surface ligands for attaching to unhealthy tissue.
[0109] In the present embodiment, phospholipids have a propensity
to form liposomes and niosomes, which can be employed as the
cosmetic ingredient carriers. Phospholipids have good emulsifying
property which can stabilize the cosmetic serum emulsions. In
addition, phospholipids as surface-active wetting agents which can
coat on the surface of crystals to enhance the hydrophilicity of
hydrophobic ingredients. The above properties are successfully
employed in the LPLTVs design.
[0110] As used herein, in the current embodiment example, the term
"phospholipid" refers to compositions which are esters of fatty
acids in which the alcohol component of the molecule contains a
phosphate group as an integral part (FIG. 2.0).
[0111] In order to extend LPLTVs (Low Pressure Low Temperature
alternative construction of Vesicles) to the preparation of other
kinds of vesicle systems taking full advantage of the possibilities
offered by this process were also undertaken. Phospholipids-based
formulations are widely used for delivery purposes and for this
reason 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was
selected as a membrane component for the liposome preparation with
LPLTVs.
[0112] Phospholipids comprise the glycerol-phosphatides, containing
glycerol, and the sphingomyelins containing sphingosine.
[0113] According to the alcohols contained in the phospholipids,
they can be divided into glycerophospholipids and
sphingomyelins.
[0114] For the present embodiment, the use of Glycerophospholipids,
which are the main phospholipids in eukaryotic cells, refer to the
phospholipids in which glycerol is the backbone are preferred. All
naturally occurring glycerophospholipids possess .alpha.-structure
and L-configuration.
[0115] Preferred phospholipids used in the embodiment comprise
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine
and sphingomyelin; and although not preferred, in the present
embodiment, synthetic phospholipids comprising dimyristoyl
phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl
phosphatidylcholine, distearoyl phosphatidylglycerol, dipalmitoyl
phosphatidylglycerol, dimyristoyl phosphatidylserine, distearoyl
phosphatidylserine, and dipalmitoyl serine.
[0116] In the case of the present method embodiment, liposomes or
niosomes are used as carriers for beneficial ingredients for the
treatment of skin anomalies. Liposomes and niosomes can be made
with different features can enhance an ingredients efficacy, reduce
an ingredients toxicity, restriction from going systemic and
prolong the ingredients therapeutic effect.
[0117] Niosomes are self-assembled vesicles composed primarily of
synthetic surfactants and cholesterol. They are analogous in
structure to the more widely studied liposomes formed from
biologically derived phospholipids.
[0118] The type of epi-dermal activity resulting from the
application of the current embodiment's content of natural or
synthetic ingredients to be beneficial includes: Hydration Skin
lightening Anti-wrinkle/skin smoothing Antioxidant activity/free
radical scavenger Anti-inflammatory/anti-irritant Collagen
stimulation Cell regeneration/stimulation Sebum regulation
Anti-cellulite Antimicrobial Antibacterial.
[0119] With chronological age and chronic exposure to adverse
environmental factors, (notably UVA, UVB, and IR radiation) the
visual appearance, physical properties, and physiological functions
of skin change in ways that are considered cosmetically
undesirable. The most notable and obvious changes include the
development of fine lines and wrinkles, loss of elasticity,
increased sagging, loss of firmness, loss of color evenness (tone),
coarse surface texture, and mottled pigmentation.
[0120] Less obvious, but measurable changes which occur as skin
ages or endures chronic environmental insult include a general
reduction in cellular and tissue vitality, reduction in cell
replication rates, reduced cutaneous blood flow, reduced moisture
content, accumulated errors in structure and function, and a
reduction in the skin's ability to remodel and repair itself.
[0121] Many of the above alterations in appearance and function are
caused by changes in the outer epidermal layer of the skin, while
others are caused by changes in the lower dermis.
[0122] Regardless of the stimulus for skin damage, when damage
occurs, numerous natural and complex biochemical mechanisms are set
into motion in attempts to repair the damage.
[0123] The present embodiment relates generally to construct a
process for a vesicle-driven treatment method and composition for
improving the skin's visual appearance, function, and
clinical/biophysical properties which have been changed by factors
such as chronological age, chronic sun exposure, adverse
environmental pollutants, household chemicals, disease pathologies,
smoking, and malnutrition. In particular, the present embodiment
relates to a process to create a method of treating skin by
increasing the skin's stratum corneum transit of known beneficial
ingredients through dynamic infusion of vesicles (DIV) generated
from natural and biocompatible phospholipids with an aqueous volume
enclosed within a lipid or niosome membrane.
[0124] The result of the present process embodiment is to deliver
larger molecular weight, longer lasting, beneficial ingredients to
areas of the epi-dermis depleted of needed vitamins, hydration,
nourishment and complimentary ingredients need for the rejuvenation
of elastin and collagen.
[0125] Now, in the current embodiment, comes the development of a
new, single process, ingredient vesicle methodology based on a Low
Pressure Low Temperature alternative construction of liposome or
niosome Vesicles process (LPLTVs) for the direct, robust and
scalable encapsulation of biomolecules in vesicles. The development
of reproducible and scalable methodologies in order to
functionalize those vesicles with targeting/protective units
enabling greater selectivity of the therapeutic epidermal targets
and therefore more effective treatments.
[0126] The use of the biomolecules-vesicles conjugates prepared by
LPLTVs can be used in the treatment of different skin anomalies.
The embodiments process uses milder conditions of pressure (<10
MPa) and temperature (<308 K) than previous methodologies based
on CFs, allowing the processing of heat labile compounds and
reducing the investment cost of a high pressure plant when the
process is scaled-up.
[0127] The present embodiment encompasses compressed fluid-based
methodologies (CF), also called dense gas technologies, for the
production of lipid-based ingredient carrier systems with
structural characteristics not reachable by already existing
procedures using liquid organic solvents. In the present
embodiment, we have improved the processing of vesicles and
niosomes because they provide the ability to reduce the amount of
organic solvent required by conventional methods and allow a better
control over the final vesicle structural characteristics. Moreover
compressed fluid processing offers sterile operating conditions and
the ability for one-step production processes, which is convenient
in transferring the technology to larger scale operations.
[0128] The present embodiment's compressed fluid technology was
developed as a platform for producing lipid and niosome-based
cosmetic ingredient carrier systems that can address most of the
limitations of conventional methods.
[0129] LPLTVs methodology allows an easy and direct preparation of
different liposome-biomolecule conjugates with micro and nano
scopic sizes and great degrees of unilamelarity.
[0130] The stability time of the liposome-based conjugates is
somewhat smaller than those of LPLT Vesicle-based conjugates. This
stability is improved by the addition of stabilizing/targeting
units to the formulation.
[0131] Bioactivity of the integrated biomolecules is unaffected
under the processing conditions with CO2-expanded solvents.
[0132] Liposomes and Niosomes prepared by the current embodiment's
process of LPLTVs, fulfill the structural and physio-chemical
requirements to be a platform for the percutaneous delivery of
synthetic or natural ACIs (active cosmeceutical ingredients).
[0133] Major advantages of the embodiment's application of CFs
technology are that sterile and stable liposomal and niosomal
formulations can be produced with minimum amounts of organic
solvents.
[0134] In the case of blemished or compromised complexion of the
skin the following properties could be desirable: Sebum regulating
Anti-bacterial Anti-inflammatory/anti-irritant Soothing/calming
Skin healing and regeneration uniform complexion lightening and
brightening.
[0135] These and other advantages will be apparent to individuals
skilled in the art in view of the drawings and detailed description
which follow.
[0136] Examples of some preferred preparation ingredients in the
present embodiment include natural botanicals, those ingredients
that that originates from plants, herbs, roots, flowers, fruits,
leaves or seeds such as: aloe vera, almond oil, avocado oil,
coconut oil, hazelnut oil, jojoba oil, olive oil, palm oil, pumpkin
seed oil, sesame oil, sunflower oil, tamanu oil, candeia oil,
arnica,
[0137] chamomile, oat extract, hibiscus flower, boswellia serratta,
cocoa powder, green and white tea, gotu kola, chamomile extract,
L-arginine, glutamine, pantothenic acid, white willow bark extract,
tetrahydrocurcuminoids, alpha-arbutin, aloesin, alpha glucosyl
hesperidin, niacinimide, fucoidan, magnesium asorbyl phosphate,
azelaic acid, N-acetyl-D-glucosamine, glutathione, mulberry,
pomegranate seed oil, cyprus rotund root extract, licorice,
licorice-glabrin root extract, kojic acid, panax ginseng root
extract, ginko bilbao, salicylic acid, Lauric acid, glycerin,
caffeine, tocopheryl acetate, copper peptide, retinyl palmitate,
asorbyl palmitate, wakame, dimethylethanolamine, beta glucan,
triglyceride as well as hyaluronic acid (Hyaluronic acid is a
natural and sugar-like biopolymer in the human body that
alternately consists of D-glucuronic acid and
N-acetyl-D-glucosamine-units).
[0138] Additionally, preferred natural polymers for the current
embodiment such as starch, starch, xanthan or guar gum,
carrageenan, alginates, polysaccharides, pectin, gelatin, agar, and
cellulose derivatives can be used to this end. On the synthetic
side, polyacrylate derivatives and polyacrylamide polymers can be
incorporated in to the carrier system of the present embodiment.
More recent developments include combining hydrophobic and
hydrophilic polymers into block and star copolymers and thermally
responsive systems.
[0139] Polymers are particularly susceptible to the construction of
vesicle that can physically entrap the active component, preserving
its biological stability, or the bioactive component can be
incorporated chemically into a polymer chain or pendant group, then
released through hydrolysis. For example, salicylic acid (an
anti-acne ingredient) can be incorporated into the main chain of
polyanhydride ester and released within a short time.
[0140] The current example embodiment also applies to the
construction of vesicle encapsulated polymers that are routinely
used in many personal care and cosmetic products.
[0141] The current embodiment takes advantage of the various
properties of these polymers to impart unique benefits to their
formulations. The range of properties is as varied as the class of
polymers that have been utilized. Using polymers, cosmetic chemists
can create high performance products. Broad spectrums of polymers;
natural polymers, synthetic polymers, organic polymers as well as
silicones are used in a wide range of cosmetic and personal care
products as film-formers, emulsifiers, thickeners, modifiers,
protective barriers, and as aesthetic enhancers.
[0142] A further embodiment features a method of making liposomes
comprising forming a mixture of multilamellar vesicles and a CF.
The mixture is decompressed to remove the CF to form one or more
liposomes or niosomes.
[0143] Preferably, multilamellar vesicles are made by hydrating
phospholipids in an aqueous phase. Preferably, the aqueous phase or
the phospholipids contain a cosmeceutical therapeutic agent.
EXAMPLE 1
Phase Behavior Studies for the Low Pressure Low Temperature
alternative construction of a Liposome and Niosome Vesicles
(LPLTVs)--CO2-Solvent System
[0144] Prior to liposome or niosome formation, the phase behavior
and solubility of the chosen lipid in dense CO2 were investigated
to verify the suitability of the lipid for dense gas processing
and, in particular LPLTVs processing.
[0145] Knowledge of the threshold pressure for precipitation of
lipid from solution is also a key factor for design of the LPLTVs
process in order to determine the maximum pressure for the
technique so that yield is enhanced and loss of lipid in the
expansion chamber minimized. The solid state of 1,2
distearoyl-sn-glycero-3-phosphocholine (DSPC) was maintained when
the lipid was exposed to CO2 below 350 bar at 50.degree. C. and 150
bar at 70.degree. C. The solubility of DSPC in pure CO2 at
50.degree. C. and pressures up to 280 bar was considered
negligible. The addition of 5 mol % ethanol co-solvent did not
significantly improve the solubility of DSPC in CO2 at 50.degree.
C. and 250 bar. Use of higher pressures or larger amounts of
organic solvent are undesirable, thus the results of the solubility
study are in agreement with the literature in concluding that
effects arising from poor solubility of lipids in dense CO2 are not
easily overcome.
[0146] In prior art, at 50.degree. C. and 250 bar, DSPC required
the addition of 4.8% v/v ethanol as well as the use of a recycling
system for homogeneous dissolution of the lipid in CO2. In the
current embodiment, the use of the LPLTVs process eliminates the
current limitations of dense gas techniques associated with
solubilizing lipids using a supercritical fluid and simply utilizes
a dense gas as an aerosolization aid. The threshold pressure for
the precipitation of DSPC from a 10 mg/mL ethanol solution at
22.degree. C. was 55 bar.
[0147] Precipitation was first observed at 58, 55, and 56 bar for
the 5, 10, and 20 mg/mL solutions of DSPC and cholesterol (70:30
lipid to cholesterol weight ratio) in ethanol at 22.degree. C.,
respectively. Therefore it can be seen that cholesterol had
negligible effect on the threshold pressure. When the
pressurization rate for the 5 mg/mL lipid/cholesterol solution was
dramatically increased, precipitation was not observed until 60 bar
was reached. A faster pressurization rate is preferable for the
embodiments LPLTVs process in order to minimize the time
requirement for each experiment. During this experiment, noticeable
expansion only started to occur after 50 bar was reached. Solution
expansion is desired to maximize the effect of utilizing CO2 as an
aerosolization aid to disperse the lipid solutions throughout the
aqueous phase. Therefore, the expansion pressure used in the LPLTVs
experiments to avoid solute precipitation and enhance the yield for
or niosome formation from ethanol solutions was between 50 and 55
bar at 22.degree. C.
[0148] The threshold pressure for the precipitation of a 20 mg/mL
DSPC/cholesterol chloroform solution (90:10 lipid/cholesterol
weight ratio) at 22.degree. C. was 41 bar. The solvent volume had
significantly expanded (doubled) by the time 40 bar was reached in
the chloroform experiments. Therefore, expansion pressures between
38 and 40 bar were used for the LPLTVs chloroform experiments to
achieve maximum expansion without lipid precipitation.
EXAMPLE 2
Effects of Process Variables on LPLTVs Operation
[0149] The effects of solute composition, solute concentration,
type of solvent, nozzle diameter, type of aqueous media,
temperature of vesicle formation chamber, and volume of dense gas
used for spraying on both the ease of operation of the embodiments
LPLTVs process and the product were investigated. The results
obtained for liposome formation are summarized in Table 1.
Preliminary trials were conducted to establish viable nozzle
options for the LPLTVs system. A variety of nozzles were tested
including 102, 178, 254, 508, and 1016 .mu.m i.d. stainless steel
tubing and 100 .mu.m i.d. Peeksil tubing (polymer tubing with fused
silica lining). The most suitable nozzle for the LPLTVs apparatus,
to control the flow rate and prevent blockages, was the 178 .mu.m
i.d. stainless steel tubing. The 254 .mu.m nozzle was used in Set 1
(Table 1); however, there were difficulties in controlling the flow
rate and maintaining constant pressure in the expansion chamber.
Other nozzle dimensions may be selected depending on the pump
capacity and vessel dimensions.
[0150] The LPLTVs process of the present example embodiment is
robust and, within the range examined, variation of solute
concentration and composition, type of solvent, type of aqueous
media, and volume of CO2 used for spraying had minimal effect on
the operation of the LPLTVs process. The temperature of the vesicle
formation chamber did, however, significantly affect the process
since a smaller amount of liposomal product was obtained at
90.degree. C. (Set 5) compared with 75.degree. C. The smaller
volume can be attributed to the aqueous medium being closer to its
boiling point at 90.degree. C., and thus some of the water was lost
to the solvent trap via evaporation.
EXAMPLE 3
Characterization of Liposomes Produced by the LPLTVs Process
[0151] The liposome morphology.
[0152] TEM (Transmission electron microscopy) was used to
investigate the morphology of the particles produced in the
embodiments LPLTVs process. At all conditions studied, submicron
spheres were observed that possessed a similar structure to
liposomes previously reported in the literature. The image shown in
FIG. 5.0 indicates that spherical particles, generally ranging in
size from 35 to 200 nm and more commonly 35-100 nm, were formed
using the LPLTVs process. Images collected suggest that the
liposomes were unilamellar. Not only were the spheres of a size
range common to unilamellar liposomes, but in many images a single,
thin wall can be seen at the edge of each particle. However, the
arguments against positive identification of lamellarity using
negative staining and TEM have been well documented in the
literature.18 Staining artifacts are difficult to identify and are
often interpreted as unexpected morphologies. Confirmation that the
particles formed were in fact liposomes was found by utilizing SANS
(Small-Angle Neutron Scattering) to identify an aqueous core, as
discussed below. The spherical particles shown in FIG. 5.0 are a
general indication of the liposomes formed; however, some other
features have also been observed. In several samples, a large
quantity of smaller spherical particles (10-20 nm) was observed,
which are at or below the lower size limit at which liposomes can
be formed and may be considered as micelles. In some samples, small
vesicles appear to be aggregated into or contained within a larger
liposome vesicle, as shown in FIG. 6.0.
[0153] A vesicle-in-vesicle structure may be formed in the last
stage of the LPLTVs process due to liposomes forming in the
presence of existing vesicles. However, the lipid vesicles are more
likely to have formed into aggregate structures during the negative
staining process in order to minimize any deleterious effects when
the aqueous phase was removed or to minimize the interactions of
the lipid with the stain. The artifact of these aggregated systems
could also result from a larger vesicle superimposed upon smaller
vesicles, which is a common feature in TEM analysis. The particle
size and morphology of the LPLTVs liposomes was not significantly
changed within the range of process parameters varied. However,
rods or coffee bean morphology (liposomes exhibiting a
characteristic `coffee-bean` appearance due to the presence of an
inner structure apparently separating the LUV into two sections)
appeared in a few samples in addition to spherical particles, as
shown in FIG. 7.0. It is suggested that the coffee bean morphology
was formed due to the collapse of vesicles, predominantly for the
smaller particles. This effect can be attributed to the lower
stability of small vesicles due to the high curvature of the
membrane.
[0154] The unilamellar liposomes produced using a conventional
technique were stained with ammonium molybdate with and without the
presence of protein. The images of the liposomes stained without
protein showed "cup-like structures" and vesicles consisting of two
lipid membranes. When protein was included in the staining process,
the images showed vesicles consisting of a single lipid
bilayer.
[0155] In the present embodiment, it is concluded that the
liposomes in both images were unilamellar and that the vesicles had
collapsed in the absence of protein. The double membrane feature
can therefore be explained by the thick edge of the collapsed
sphere, and the "cup-like structures" can be observed if the
collapsed spheres are rotated.
[0156] The correct choice of vesicle or niosome preparation method
in the current embodiment depends on the following parameters: the
physicochemical characteristics of the material to be entrapped and
those of the liposomal or niosomal ingredients; the nature of the
medium in which the vesicles are dispersed; the effective
concentration of the entrapped substance and its potential
toxicity; additional processes involved during application/delivery
of the vesicles; optimum size, polydispersity and shelf-life of the
vesicles for the intended application; and, batch-to-batch
reproducibility and possibility of large-scale production of safe
and efficient liposomal products.
TABLE-US-00001 TABLE 1 Summary of the Conditions Investigated and
the Results Obtained for Producing Liposomes via the embodiments of
the LPLTVs Process* set 1 2 3 4 5 6 7 8 9 nozzle diameter 254 178
178 178 178 178 178 178 178 (.mu.m) solute lipid content 70 70 90
90 90 90 90 90 90 (% w/w) solute conc. 20 20 20 5 20 20 20 20 20
(mg/mL) VFC temp. 75 75 75 75 90 75 75 75 75 (.degree. C. .+-. 2.5)
CO.sub.2 spraying vol. 200 200 200 200 200 50 200 200 200 (mL)
aqueous media RO H.sub.2O RO H.sub.2O RO H.sub.2O RO H.sub.2O RO
H.sub.2O RO H.sub.2O DI H.sub.2O TBS RO H.sub.2O organic solvent
EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH Chlfm effective diameter
156 .+-. 2 166 .+-. 5 121 .+-. 1 162 .+-. 23 207 .+-. 75 122 .+-. 2
119 .+-. 3 143 .+-. 5 387 .+-. 64 (nm) polydispersity 0.27 .+-.
0.01 0.27 .+-. 0.01 0.15 .+-. 0.01 0.19 .+-. 0.01 0.18 .+-. 0.01
0.15 .+-. 0.01 0.17 .+-. 0.02 0.18 .+-. 0.02 0.29 .+-. 0.03 product
lipid content 75.2 .+-. 1.9 77.3 .+-. 1.8 82.4 .+-. 0.4 76.1 .+-.
0.1 81.8 .+-. 0.5 80.5 .+-. 0.2 80.9 .+-. 0.3 80.1 .+-. 0.6 81.8
.+-. 1.3 (% w/w) residual solvent 3.1 .+-. 0.4 1.6 .+-. 0.8 1.4
.+-. 0.4 2.2 .+-. 0.3 1.9 .+-. 0.7 3.9 .+-. 0.2 1.8 .+-. 0.4 2.0
.+-. 0.8 0.4 .+-. 0.3 (% v/v) *VFC: vesicle formation chamber; RO
H.sub.2O: water purified via reverse osmosis; DI H.sub.2O:
deionized water; TBS: TRIS buffered saline; EtOH: ethanol. Chlfm:
chloroform.
[0157] Collapsed spheres were present in all LPLTVs samples;
however, rods or "coffee bean" particles were rare except in those
samples from Sets 1 and 2, where a higher proportion of cholesterol
was used compared with other samples.
TABLE-US-00002 TABLE 2 The calculated SLD for the components LPLT
Vs of the LPLT Vs liposome samples material SLD (.times.10.sup.-6
.ANG..sup.-2) H.sub.2O -0.56 D.sub.2O 6.33 hydrocarbon Chain
(CH.sub.2).sub.n -0.44 cholesterol (C.sub.27H.sub.45OH) 0.21 lipid
headgroup (C.sub.10H.sub.18O.sub.8NP) 1.12
TABLE-US-00003 TABLE 3 SANS Fitted parameters for the LPLT liposome
sample fitted parameter (.ANG..sup.-2) value Core SLD 6.30 .times.
10.sup.-6 Shell SLD 3.85 .times. 10.sup.-6 Solvent SLD 6.82 .times.
10.sup.-6
[0158] Cholesterol was incorporated in order to improve stability,
and it has been reported in the literature that the incorporation
of cholesterol causes larger liposomes or niosomes to form.
[0159] However, the rod-shaped particles were at the smaller end of
the size range for the LPLTVs. Comparison of images from a number
of samples indicated that the presence of rods may be promoted by
the level of stain as well as the size of the vesicles. It is
therefore also possible that the relative proportion of rods found
in Sets 1 and 2 was amplified by the staining process. Because of
the improved spherical morphology observed in Set 3, the
experiments were carried out using a preferred lipid/cholesterol
ratio of 90:10.
Advantages of the Current Embodiments LPLTVs Process for Bulk
Liposome or Niosome Vesicle Formation.
[0160] The LPLTVs process has many advantages over conventional
liposome or niosome formation techniques. These advantages include
the fact that it is a simple and rapid process for bulk production
of unilamellar liposomes or niosomes. A conventional liposome
standard was produced, and the formation process took almost 24h
and multiple stages to complete. The embodiments LPLTVs process
produced a greater volume of the same formulation in less than half
an hour, clearly demonstrating the dramatic reduction in processing
time.
[0161] The conventional ethanol and ether injection methods exhibit
some similarities to the embodiments LPLTVs process since they
involve the dissolution of a lipid into an organic phase, followed
by the injection of the lipid solution into aqueous media forming
liposomes. The drawbacks of the ethanol injection method as opposed
to the examples of the present embodiment, are the poor homogeneity
of the vesicles if there is not adequate mixing and the residual
solvent levels in the product.
[0162] Either injection method eliminates the residual solvent
issue by having a heated aqueous phase, but is a time- consuming
technique. It has been suggested that injecting the ether solution
at a rate faster than 0.2 mL/min can cause cooling of the aqueous
phase due to evaporation, and that pre-evaporation of ether can
cause nozzle blockages and the formation of multilamellar vesicles.
The LPLTVs process of the present embodiment for the formation of
liposomes or niosomes formed around cosmeceutically benevolent
ingredients has significant advantages over both the ethanol and
ether injection methods since the depressurization from a high
pressure environment creates outstanding dispersion of the lipid
solution and mixing with the aqueous environment. The incorporation
of both heating and dense gas washing enables the solvent to be
efficiently removed. The LPLTVs process can also produce an
equivalent volume of product in a significantly reduced time span.
Compared with other dense gas processes developed for liposome
formation, the LPLTVs process is beneficial due to its simplicity
and the incorporation of residual solvent removal measures into the
method. The LPLTVs process also operates at pressures generally
less than 60 bar and moderate temperatures, therefore making the
process more cost-effective and avoiding the concerns of
uncontrollable foam formation present in the low pressure liposome
method. A significant advantage of the LPLTVs process is that it
can be used to process a broad range of materials since there is no
requirement for the compound to be solubilized in the dense gas and
there are no high shear forces. Furthermore, time-consuming
solubility studies and recycling loops for lipid solubilization are
not needed. The only preliminary investigation required is the
determination of the threshold pressure for precipitation of the
solutes from expanded solution, such that the solution expansion
can be carried out without precipitation.
[0163] In the LPLTVs process of the current embodiment, the
entrapment of hydrophilic compounds may be achieved through the
dissolution of the target compound into the aqueous media prior to
release of the lipid solution. The liposomes or niosomes would then
form, entrapping the hydrophilic or hydrophobic compound within the
aqueous interior of the vesicle.
[0164] To entrap a hydrophobic, hydrophilic, lipophilic, or
amphipathic compound into liposomes or niosomes using the LPLTVs
process, the compound is dissolved along with the phospholipid and
other solutes in the liquid solvent.
[0165] The compound then becomes entrapped within the phospholipid
membrane as a result of the affinity of the compound for the
membrane rather than the aqueous phase.
[0166] The suitability of the LPLTVs process for entrapping
hydrophobic compounds has already been demonstrated through
incorporating up to 25% w/w cholesterol into the liposome
formulation. The LPLTVs technique can also be applied to the
formation of structures other than liposomes. Micro particles of
hydrophobic compounds could be produced through precipitation into
aqueous media in the LPLTVs process.
[0167] Liposomal Particle Size Distribution and Stability. Photon
correlation spectroscopy (PCS) was used to assess the particle size
distribution of the liposomal population using the Brookhaven
ZetaPlus. Each liposomal sample was diluted in RO or DI water and
placed in a disposable polypropylene cuvette. Ten runs, each of 1
min duration, were conducted at 23-25 oC for each sample. A laser
wavelength of 678 nm was used with a destination angle of 90 o. The
dust cutoff was set between 20 and 50 .mu.m. The instrument
calculates an effective diameter for each run and an overall
effective diameter for the 10 runs combined. The effective diameter
is the mean diameter that is calculated by the following
equation:
Effective diameter = ( 1 d k ) - 1 = i N i d i 6 P i i N i d i 5 P
i ##EQU00001##
[0168] Where Ni refers to the number per scattering volume of the
ith particle, and Pi accounts for angular scattering effect for
particles larger than .lamda./20. Pi is calculated using Mie theory
and requires the particle refractive index; however, for Rayleigh
scatters and at sufficiently low angles, Pi=1 is used in the
program.
EXAMPLE 4
A Formulation for the Treatment of Acne made Using the Current
LPLTVs Embodiment
[0169] A solution for treating Acne vulgaris or Propionibacterium
acnes containing lipids formed of the following ingredients
utilizing the science of the present embodiment may be formulated
using the constructed phospholipids of the following volumes;
[0170] D.I. water 50% to 95% (preferably 60 to 90%, ethanol 15 to
40% (preferably 25 to 30%), hyaluronic acid 5 to 50% (preferably 12
to 18%) propanediol 10 to 80% (preferably 20 to 25%), aloe vera 0.2
to 20% (preferably 0.5 to 5%), azelaic acid 2 to 50% (preferably 4
to 8%), salicylic acid 0.2 to 20% (preferably 0.5 to 5.0%), lauric
acid 0.2 to 20% (preferably 0.5 to 5.0%), asorbyl palmitate 0.1 to
20% (preferably 0.2 to 8%) niacinimide 0.2 to 20.0% (preferably 0.5
to 5%),lecithin 0.2 to 10% (preferably 0.5 to 5.0%), glycerin 0.5
to 25% (preferably 2 to 10%), caffeine 0.2 to 20% (preferably 0.5
to 10%)
EXAMPLE 5
A Formulation for the Enhanced Hydration and the Reduction of Fine
Lines and Wrinkles Made Using the Current Embodiment
[0171] A solution for treatment of lack of skin hydration and the
reduction of fine lines and wrinkles containing lipids formed of
the following ingredients utilizing the science of the present
embodiment may be formulated using the constructed phospholipids of
the following volumes; D.I. water 50% to 95% (preferably 60 to 90%
ethanol 15 to 40% (preferably 25 to 30%), hyaluronic acid 5 to 50%
(preferably 12 to 18%) propanediol 10 to 80% (preferably 20 to
25%), aloe vera 0.2 to 20% (preferably 0.5 to 5%), hexa-peptide 8
2% to 50% (preferably 5% to 20%), caffeine 0.2 to 20% (preferably
0.5 to 10%), glycerin 0.5 to 25% (preferably 2 to 10%), tocopheryl
acetate 0.1 to 10% (preferably 0.5 to 8%), retinyl palmitate 0.1 to
10% (preferably 0.5 to 8%), asorbyl palmitate 0.1 to 20%
(preferably 0.2 to 8%), Copper tri-peptide GHK-Cu 0.1 to 20%
(preferably 0.2 to 8%), hesperidin 0.1 to 20% (preferably 0.2 to
8%), dimethylethanolamine (DMAE) 0.05 to 20% (preferably 0.08 to
8%), sesame oil 2 to 50% (preferably 3 to 20%) beta glucan 0.1 to
20% (preferably 0.2 to 8%)
Test 1
LPLTVs Method for the Preparation of Hyaluronic Acid-Rich
Vesicles
[0172] The present embodiment is based on the use of compressed CO2
in a process called LPLTVs for the production of micron-sized and
submicron-sized crystalline particles from an organic solution. As
novelty the process used the CO2 as co-solvent being completely
miscible at a given pressure and temperature with a specific
solution of an organic solvent containing the solute to be
crystallized. In order to take full advantage of compressed fluid
processing without using severe working conditions a novel and
improved procedure based on the LPLTVs process was developed. This
method, named as LPLTVs (Low Pressure Low Temperature alternative
construction of Liposome Vesicles), enabled the preparation of
cholesterol rich-hyaluronic acid vesicles. The process uses milder
conditions of pressure (<10 MPa) and temperature (<308 K)
than the previously described methodologies based on CFs, allowing
the processing of heat labile compounds and reducing the investment
cost of a high pressure plant when the process is scale-up. Using
this procedure, homogeneous nanovesicles composed of hyaluronic
acid, cholesterol and the cationic surfactant CTAB
(cetyltrimethylammonium bromide, in a molar ratio 1:1, were
prepared by depressurizing a volumetric expanded organic solution
containing the cholesterol and hyaluronic acid over a flow of an
aqueous solution containing the CTAB surfactant (FIG. 5.0). An
alternate non-ionically formed elastic noisome can be constructed
using the same apparatus.
[0173] During the depressurization step, the expanded organic
solution experiences a large, abrupt and extremely homogenous
temperature decrease produced by the CO2 evaporation from the
expanded solution. This explains the obtaining of homogenous
vesicles regarding size, lamellarity and morphology.
[0174] In order to prepare any vesicular system using LPLTVs is
necessary that the lipids forming the membrane are completely
soluble in the CO2-expanded organic solvent , presenting one phase
at the working conditions of pressure, P w, temperature, T w and
CO2 molar fraction, X2. Therefore for the preparation of
cholesterol rich-hyaluronic acid vesicles by LPLTVs method is
always necessary to analyze the solubility behavior of the used
sterol in CO2-expanded solvents, by means of a detailed phase
diagram study, like the one showed in FIG. 6.0.
[0175] An important prerequisite for the effective use of vesicles
as a cosmeceutical ingredient carrier as described above is to
control their stability, which can be defined as the extent to
which the carrier retains its ingredient contents either in vitro
or in vivo studies. One of the major disadvantages when using
classical vesicles based on phospholipids, is the leakage of the
encapsulated ingredient during their storage. One variant that can
enhance the retention of drugs and promote the stability of
liposomes or niosomes is the presence of hyaluronic acid in the
formulation. Another variant is the preparation of liposomes from
non-phospholipid amphiphiles, such as surfactants or polymers.
[0176] This kind of vesicular formulations show low passive leakage
in comparison to liposomal systems based only on phospholipids and
therefore a higher retention of the encapsulated materials, as for
example epi-dermal therapeutically active molecules.
[0177] In the present example embodiment for the preparation of
positively charged vesicles composed by cholesterol, hyaluronic
acid and the cationic surfactant hexadecyltrimethylammonium bromide
(CTAB). More recently nanoscopic vesicles, composed by different
sterols and other quaternary ammonium surfactants have been also
successfully prepared. This is why it was decided to name this kind
of formulations as LPLTV (low pressure low temperature vesicles)
that are stable for periods as long as several years, their
morphology do not change upon rising the temperature or by dilution
and they show a great homogeneity regarding size and
morphology.
[0178] Studies at molecular level of the self-assembling of
cholesterol hyaluronic acid and CTAB molecule in aqueous medium
showed that a pure vesicular phase is only formed at equimolar
ratios of both components. Moreover molecular dynamic (MD)
simulations revealed that the cholesterol, hyaluronic acid and the
CTAB self-assemble in a unique bimolecular synthon that can be
considered as a single entity which further self- assembles in
particularly stable vesicles (FIG. 7.0) (FIG. 10). Moreover, MD
simulations have provided a theoretical support to justify the
experimental high thermal stability and the exceptional
morphological properties attributed to cholesterol, hyaluronic
acid/CTAB vesicles at 1:1 molar ratio.
Test 2
Analysis of Active and Placebo Tape Strips from an In-Vivo Study of
Skin Permeation of 800 KDa Hyaluronic Acid using the embodiments
LPLTVs Formation Process
Introduction:
[0179] The test was to extract and analyze tape strips and blanks
from an in vivo tape stripping study of 800KDa Hyaluronic Acid skin
penetration transport.
[0180] HA distribution in stratum corneum (SC) layer was
investigated. Distribution in SC was studied using a tape-stripping
method.
Methods:
[0181] Each tape sample will be extracted individually by
extraction solvent: 1.times.PBS with 0.2% NaN3/acetonitrile (50/50
v). Samples with extraction solvent were vortexed at high speed for
1 minute followed with centrifugation at 12,000 rpm for 10 minutes
(chill the samples on ice at 4 oC and then centrifuge). The
supernatant solution was then collected from each tube/container
and stored at 4 oC and ready for analysis.
Outcome:
[0182] See the chart in FIG. 11.0 showing Hyaluronic Acid levels in
active and control samples.
[0183] Abstract of the Disclosure is provided to allow the reader
to quickly ascertain the nature of the technical disclosure. It is
submitted with the understanding that it will not be used to
interpret or limit the scope or meaning of the claims. In addition,
in the foregoing Detailed Description, it can be seen that various
features are grouped together in a single embodiment for the
purpose of streamlining the disclosure. This method of disclosure
is not to be interpreted as reflecting an intention that the
claimed embodiments require more features than are expressly
recited in each claim. Rather, as the following claims reflect,
inventive subject matter lies in less than all features of a single
disclosed embodiment. Thus, the following claims are hereby
incorporated into the Detailed Description, with each claim
standing on its own as a separate embodiment.
* * * * *