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Hyaluronan (Hyaluronic Acid): a natural moisturizer for skin care

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4.1.3.1 Structure and selected physical-chemical properties of hyaluronan 4.1.3.2 Preparation of hyaluronan fragments, isolation and characterization thereof, characterization of degradation products of hyaluronan 4.1.3.3 Preparation of chemical derivatives of hyaluronan, characterization thereof 4.1.3.4 Hyaluronan penetration into the stratum corneum and into the skin 4.1.3.5 Moisturizing properties of native high-molecular hyaluronan and how the moisturizing properties change as the molecular weight is reduced 4.1.3.6 Cosmetic application for various molecular weights of hyaluronan
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HYALURONAN (HYALURONIC ACID)
A NATURAL MOISTURIZER FOR SKIN CARE
Authors:
Daniela Smejkalova, Gloria Huerta-Angeles, Tereza Ehlova
Contipro Pharma, Doln Dobrouc 401, 561 02, Czech Republic
TABLE OF CONTENTS
4.1.3.1 Structure and selected physical-chemical properties of hyaluronan 606
4.1.3.2 P reparation of hyaluronan fragments, isolation and characterization
thereof, characterization of degradation products of hyaluronan 607
4.1.3.3 P reparation of chemical derivatives of hyaluronan,
characterization thereof 610
4.1.3.4 Hyaluronan penetration into the stratum corneum and into the skin 612
4.1.3.5 M oisturizing properties of native high-molecular hyaluronan
and how the moisturizing properties change as the molecular
weight is reduced 613
4.1.3.6 S elected Derivatives of Hyaluronan and their Effect on Skin
Moisturizing 616
4.1.3.7 Cosmetic application for various molecular weights of hyaluronan 617
References 618 Glossary 621
PART 4.1.3
HYALURONAN (HYALURONIC ACID) A
NATURAL
MOISTURIZER SKIN CARE
Authors
Dr. Daniela Smejkalova Nano-carrier Development Group
Dr. Gloria Huerta-Angeles Biopolymers Modification Group
Tereza Ehlova Hyaluronan Fragments Group
Contipro Pharma, Dolní Dobrouč 401, 561 02, Czech Republic
ABSTRACT
This chapter describes the chemical and physical properties of hyaluronic acid and
its cleaved, lower-molecular-weight fragments as well as its derivatives relative to
their interesting and useful topical application to moisturize skin in anti-aging
products.
TABLE OF CONTENTS
4.1.3.1 S tructure and selected physical-chemical
properties of hyaluronan 606
4.1.3.2 P reparation of hyaluronan fragments, isolation
and characterization thereof, characterization
of degradation products of hyaluronan 607
4.1.3.3 P reparation of chemical derivatives of
hyaluronan, characterization thereof 610
4.1.3.4 H yaluronan penetration into the stratum corneum
and into the skin 612
4.1.3.5 M oisturizing properties of native high-molecular
hyaluronan and how the moisturizing properties
change as the molecular weight is reduced 613
606
4.1.3.6 S elected Derivatives of Hyaluronan and their Effect on
Skin Moisturizing 616
4.1.3.7 C osmetic application for various molecular weights of hyaluronan
617
References 618
Glossary 621
605
4.1.3.1 STRUCTURE AND SELECTED PHYSICAL-CHEMICAL PROPERTIES OF HYALURONAN
4.1.3.1 STRUCTURE AND SELECTED PHYSICAL-CHEMICAL
PROPERTIES OF HYALURONAN
Hyaluronan (HA) is a naturally occurring linear polysaccharide consisting of
alternating β-1,4-linked units of β-1,3-linked glucuronic acid and N-acetyl-D-
glucosamine (Figure 1.1). HA is a main component of the extracellular matrix in
connective, epithelial, and neural tissues and is known to play an important role in
the tissue hydration and water transport mainly due to its enormously high water-
binding capacity. The high water-binding capacity of HA can be attributed to high
density of fixed negative charges in HA chains from carboxyl groups, which causes
the osmotic pressure and draws the water molecules into the tissues containing
HA.1 For example, HA within skin layer holds up 1000 times its weight in water
molecules.2 Additional biological functions of HA include maintenance and
viscoelasticity of liquid connective tissues such as joint synovial fluid and eye
vitreous, supramolecular assembly of proteoglycans in extracellular matrix, and
numerous receptor-mediated roles in cells, mitosis, migration, tumor development,
and metastasis and inflammation.1
Figure 1.1 Hyaluronan (HA) structure.
CO
2
NA
H
3
C
OH
OH
OH
NH
HO
O
n
607
The extensive repertoire of HA biological functions suggests the existence of
different conformations and specific binding interactions.3 It is likely that the
conformation is affected by the local environment, including ionic strength and
specific ion interactions, and the presence of interacting species including proteins
and lipids. In the extracellular matrix, under normal physiological conditions and
ionic strength, HA is generally believed to exist as a crowded random coil
molecule.4 A change in HA conformation is suspected under specific pathological
conditions. Lowering the pH results in protonation of carboxyl groups and
increased solution viscosity. In contrary, there is a polyanion contraction with
rising ionic strength
4.1.3.2 PREPARATION OF HYALURONAN FRAGMENTS, ISOLATION AND CHARACTERIZATION
due to the increased electrostatic shielding of HA. This latter effect is accompanied
by a decreased solution viscosity. The HA solution viscosity is also temperature
dependent and is a function of the overlapping parameter, molecular weight and
concentration.
Some applications involving HA as an active ingredient require filtered HA
solutions. The question that arises is whether filtration changes the physical-
chemical properties of HA or not. According to our unpublished results, when
proper dilution is used, filtration of HA (from 15 kDa up to 1.7 MDa) solutions
through 0.22 µm nylon filter had no impact on molecular weight, polydispersity,
and solution viscosity. The filtration impact on biological properties has not yet
been established.
The major disadvantage of native HA is its low resistance against enzymatic
degradation and short half-life after internal administration due to natural scission
by enzymes and degradation by radicals. For this reason, at the present time the
main challenge is the design of new HA materials that retain or increase the
hydration properties of the polysaccharide and at the same time last longer in the
application point. In order to increase the biological resistance of HA towards
enzymatic degradation, modifications are being introduced in HA structure.5,6
Another challenge is to synthesize HA derivatives with altered and specific
physical-chemical and biological functions. Since the biological properties are
connected with the HA molecular weight, there is also a great interest in HA
degradation and evaluation of biological behavior of HA fragments.
4.1.3.2 PREPARATION OF HYALURONAN FRAGMENTS,
ISOLATION AND CHARACTERIZATION THEREOF,
CHARACTERIZATION OF DEGRADATION PRODUCTS
Disintegration of the HA β-linkages leads to formation of hyaluronan fragments of
different sizes, identified by a name that indicates the number of monosaccharide
608
units of which they are composed, e.g., a tetramer consists of four monosaccharide
unitstwo glucuronic acids and two N-acetyl-D-glucosamines (GlcNAc). The
name of the other fragments is created analogically. Fragments comprising more
units are characterized by average molecular weight in Daltons. Thus, an eicosamer
having 20 subunits is in literature specified as 20-mer, but it is also possible to say
HA of molecular weight 4 kDa.
Mechanisms of the HA cleavage into its smaller fragments comprise
enzymatic, free radical, thermal, ultrasonic, and chemical methods such as acid and
alkaline hydrolysis.7 Non-enzymatic reactions proceed randomly, and by this
means mixtures of HA saccharides with different molecular weight can be
obtained. By contrast, enzymatic cleavage is selective and allows preparation of
HA
4.1.3.2 PREPARATION OF HYALURONAN FRAGMENTS, ISOLATION AND CHARACTERIZATION
oligosaccharides with specific structure. Enzymatic digestion is catalyzed by two
main types of enzymeshyaluronate lyase and bovine testicular hyaluronidase
(BTH).8 Both enzymes produce fragments that contain an even number of
saccharide units with GlcNAc at the reducing end. While HA oligosaccharides
obtained by BTH are fully saturated, hyaluronate lyase produces oligosaccharides
with the double bond between C-4 and C-5 position of uronic acid at their
nonreducing end. Both saturated and unsaturated HA oligosaccharides lose under
alkaline conditions GlcNAc from the reducing terminus with formation of odd-
numbered oligosaccharides.9
During non-enzymatic cleavage, HA can be partially transformed into
undesired degradation by-products exhibiting unwanted biological properties like
carcinogenesis. Their identification is important so as to suggest ways of their
elimination and to offer a final fragmented HA product free of toxic impurities.
Until now only degradation by-products arising from acidic and alkaline hydrolysis
have been established. Cleavage of hyaluronan under alkaline conditions gives a
mixture of stereoisomers (E)-3-dehydroxy-3-en-D-GlcNAc and (E)-3-dehydroxy-
3-en-D-ManNAc,8 while acid hydrolysis of hyaluronan leads to creation of furan-
like derivatives and derivatives of cyclopentanone.10 These degradation by-
products can be removed from the reaction mixture by ultrafiltration through a
membrane of suitable cut-off.
Physical-chemical properties and biological activity of native HA significantly
differ from its low-molecular-weight fragments. While high-molecular-weight HA
is a space-occupying material and contributes to the structure of the extracellular
matrix and tissue integrity, smaller-sized HA fragments are involved in a wide
spectrum of complex biological mechanisms. From the cosmetic perspective, the
most important properties of HA fragments involve stimulation of angiogenesis
and supporting the proliferation of fibroblasts. Pro-angiogenic properties are
609
exhibited especially by short HA oligosaccharides comprising from 6 to 20
saccharide units. HA oligosaccharides are mitogenic for endothelial cells, support
their migration, and induce multiple signaling pathways.11 Their good
biocompatibility and low toxicity make them an ideal therapeutic agent for the
situations when stimulation of new blood vessel growth is needed. HA
oligosaccharides are also components of dermatological compositions for
combating hair loss and stimulating hair growth. In the field of reproductive
medicine, HA oligosaccharides may help fertilization.
Hyaluronan fragments can also be used for treating and preventing wrinkles,
expression lines, fibroblastic depletion, and scars. HA oligosaccharides (with
molecular weight <5 kDa) contribute to the suppression of wrinkles in both layers
of the skin. At the epidermis, they stimulate the endogenous synthesis of
highmolecular HA, which has a positive effect on hydration. At the level of the
dermis,
4.1.3.2 PREPARATION OF HYALURONAN FRAGMENTS, ISOLATION AND CHARACTERIZATION
HA fragments decrease the creation of pro-inflammatory interleukins, which are
responsible for generation of free radicals capable of damaging both components
of the extracellular matrix and the skin cells themselves. The most ambitious
application of HA oligosaccharides is treatment of cancer. Anti-tumorigenic
effects of HA oligosaccharides are based on their ability to sensitize cancer cells
to treatment. The binding of oligomers to the HA receptors also inhibits the pro-
tumorigenic signaling of high-molecular-size HA. Some low-molecular-weight
HA molecules are believed to release strong immune reaction to the cancer cell.12-
14
Analytical Techniques for Characterizing HA Molecular Weight
Since the length of HA polysaccharide chain is closely related to its biological
functions, it is necessary to determine its molecular weight properly. A range of
analytical techniques has been employed to assess the sizes of HA fragments.
These were originally determined from the ratio of total glucuronic acid
concentration to that of the reducing end GlcNAc group. More recently, other
methods including viscosimetry, electrophoresis, mass spectrometry, and liquid
chromatography have been used.15 Electrospray ion mass spectrometry (ESI-MS)
is mainly used for molecular weight characterization of short HA oligomers (up to
40-mer) derived from enzymatic digestions.16 Enzymatic degradation provides a
complex mixture of even-numbered HA oligomers, which must be fractionated
using suitable techniques such as electrophoresis or liquid chromatography before
ionization. ESIMS utilizes a flowing stream containing the analyte and provides
gentle ionization. Proper care must be taken in setting the cone voltage that
accelerates the ions into the mass analyzer because high values of the cone voltage
610
may result in an artifact of an increase of charged species. By contrast, low cone
voltage leads to insufficient ions. At lower cone voltage it is possible to observe
odd-numbered HA oligomers as artifacts in ionization.
As in the ESI-MS technique, it is not possible to avoid fragmentation of larger
HA oligomers; it is advisable to use MALDI-TOF. This technique is suitable for
characterization of polydisperse mixtures; it presents tolerance towards salts and
buffers and is highly sensitive.17 However, MALDI-TOF cannot be considered a
quantitative method because desorption of HA fragments differs according to their
size extensively. These complications can be overcome by using size-exclusion
chromatography (SEC) coupled with multi-angle light scattering detector (MALS)
and refractive index (RI), which provides detailed information of molecular
weight, molecular weight distribution, molecular size, and solution properties in a
wide range of HA fragments.18 A limitation of the technique is that an SEC column
cannot separate HA fragments sufficiently. Hence, two SEC analytical columns
packed with an appropriate resin and connected in series are the possible solution
for the separation of HA fragments. In this set, the length of the analysis is still
acceptable.
4.1.3.3 PREPARATION OF CHEMICAL DERIVATIVES OF HYALURONAN AND THEIR
CHARACTERIZATION
4.1.3.3 PREPARATION OF CHEMICAL DERIVATIVES OF
HYALURONAN AND THEIR CHARACTERIZATION
As it has been mentioned earlier, native HA suffers from its low resistance towards
enzymatic digestion. This means that native HA has a high rate of in vivo turnover,
which limits the period of time during which HA may act in the place of
application. For this and other reasons, chemical modifications of HA intended for
cosmetic or pharmaceutical applications are recommended. Chemically modified
HA derivatives are also usually more mechanically robust and thus they are more
suitable for final products manufactured using higher temperatures, pressures, or
shear stress. Modified HA derivatives usually have stronger binding capacity to the
skin, and as a result they are not washed off as easily as unmodified HA. An
extensive research has been developed for chemical modification of HA. The
strategies employed can be divided into two categories. The first one involves
chemical attachment of pendant groups to change the properties of the
polysaccharide and modulate the hydrophilic character of HA. The usual aim of
these modifications is to lower the hydrophilicity of HA, so that the modified
biopolymer will be able to blend with hydrophobic materials typically used in
cosmetic products.
The second strategy for chemical modification of HA involves cross-linking in
order to prepare insoluble derivatives of HA or hydrogels.19 In fact, almost all of
611
the commercially available HA products are stabilized by cross-linking to obtain
materials with longer residence times and increased mechanical robustness.
Regardless of the modification strategy, the new, modified HA materials
should remain biocompatible and biodegradable, as well as retaining the
moisturizing properties. A critical point in the synthesis of HA derivatives is the
preservation of the original chain length, since HA lubricant, shock-absorbent, and
space-filling properties are closely correlated to its high viscosity, which is directly
related to the initially high molecular weight of the biopolymer. From our personal
experience, degradation is always inevitable during chemical modification
processes.
Chemical modification of HA
Such modification can be carried out by the addition of the following functional
groups to the polysaccharide backbone: carboxyl, primary or secondary hydroxyl
groups, or the amino group after deacetylation. The chemical modification of
carboxyl group is often preferred because it is the recognition site for HA natural
receptors and hyaluronidase.20 This method of modification may decrease the HA
susceptibility towards enzymatic degradation.21 However, the biological behavior
of the polysaccharide, including its biodegradability, may change after such
derivatization. Thus, the effect of each modification on the biocompatibility of the
polysaccharide must be investigated in order to characterize the result on its
properties.
4.1.3.3 PREPARATION OF CHEMICAL DERIVATIVES OF HYALURONAN AND THEIR
CHARACTERIZATION
HA coupling via its carboxyl group requires its previous activation to make it
reactive to nucleophiles. The modification of carboxyl groups produces new amide
or ester bonds in HA backbone. The typical reagents are the same as those used in
common peptide synthesis, such as water-soluble carbodiimides (EDC and
derivatives), carbonyldiimidazole (CDI), carbonyltriazole (CDT), N-
hydroxysuccinimide (NHS), p-nitro-phenol, p-nitrophenyltrifluoracetate, and ethyl
chloroformate (ECF). Chemical modification of HA is primarily performed in
water or in organic solvent. To carry out modification in organic solvents such as
DMSO, DMF, or formamide, a previous conversion of sodium hyaluronate to its
acid form has to be done. However, this conversion drastically degrades the
polysaccharide. Thus, the modification in water is always preferred. Previously
enounced methodologies are in general patented and very well known by people
skilled in the art. These strategies have been used for the synthesis of nano-
emulsifiers,22 films,23 hydrophobized HA,24 or hydrogels.25
612
On the other hand, the modification of the hydroxyl groups may involve the
formation of ether bonds by reaction with bis-epoxides.26 An example is the
commercially available Juvederm®, a commonly used filler in cosmetic surgery.
This product is chemically cross-linked by divinyl sulfone. Hydrophobized HA can
be synthesized via formation of carbamoyl bonds after activation of hydroxyl
groups.27 The oxidation of the primary hydroxyl has been used for bioconjugation
with lipids and subsequent hydrophobization.28 However, some allergic reactions
have been recently reported for commercially available products,29 and so new
challenges have appeared for scientists devoted to HA chemistry.
In the case of the formation of insoluble derivatives of HA (hydrogels), the
capacity to retain water by the polysaccharide should be always determined by
measuring the degree of swelling. The denser the hydrogel is, the lower its water
uptake capacity and swelling ratio. In fact, regarding HA, most of the experimental
data indicate that high swelling ratio values correspond to the lowest modification
of the biopolymer. Low degree of substitution usually results in the formation of
hydrogels with low cross-linking density, resulting in larger absorption of water
and thus high swelling ratio. Conversely, high degree of substitution helps to form
homogenous and more compact hydrogel network with lower water uptake
capacity. Compression tests are often used to determine the smoothness of the
hydrogel. Additionally, rheological studies are essential to evaluate the effect of
derivatization or crosslinking.30 Typically, the rheological behavior of HA and its
derivatives is characterized by non-Newtonian behavior. The mechanical integrity
of HA hydrogels is usually determined by measuring shear modulus (G), the larger
the G value, the more resistant network towards deformation. The G value usually
increases with increasing HA modification. After chemical modification it is
always necessary to evaluate the effects of the derivatization on the physical,
chemical,
4.1.3.4 HYALURONAN PENETRATION INTO THE STRATUM CORNEUM AND INTO THE SKIN
and biological properties of the polysaccharide, as well as possible degradation
during the process.31
4.1.3.4 HYALURONAN PENETRATION INTO THE STRATUM
CORNEUM AND INTO THE SKIN
Skin is the largest reservoir of HA in our body, since it accounts for 50% of the
total body HA content. Under ideal conditions, HA is found in all the layers of the
epidermis and dermis. In the epidermis, HA is most prominent in the upper spinous
and granular layers, where most of it is extracellular. By contrast, the basal layer
of the skin also contains HA but it is predominantly intracellular. Presumably, basal
keratinocyte HA is involved in cell-cycling events, whereas secreted HA in the
upper outer layers of epidermis is part of the mechanism for dissociation and
613
eventual sloughing of cells. The HA content in the dermis is far greater than that
of the epidermis, and this accounts for most of the HA content in skin. The HA of
the dermis is in equilibrium with the lymphatic and vascular system, while
epidermal HA is not.2
Both epidermal and dermal cells are able to synthesize HA throughout our
lifetime. However, the skin cells lose their ability to produce optimal amounts of
HA during the aging process. That is why the skin of babies is very rich in HA and
is so soft, full, smooth, and supple, while with increasing age dehydration occurs;
the skin thins and wrinkles appear. The decline in HA production is also
accompanied by a decreased suppleness, reduced elasticity, and loss of skin tone.
The most dramatic histochemical change observed in senescent skin is the marked
decrease in epidermal HA. In senile skin, HA is still present in dermis, whereas the
HA of the epidermis has disappeared entirely. In fact, recent studies have indicated
that the total level of HA in dermis remains constant with aging. However, at the
same time, the proportion of total glycosaminoglycan synthesis devoted to HA is
greater in the epidermis than in the dermis, and the reasons for precipitous fall with
aging is unknown at the present time.
In order to retain the aesthetic appearance of the skin, and treat the signs of
“dermatological” aging, it is recommended to keep “refilling” skin with HA from
adolescent age onwards. It is already known that HA taken orally does not show
any benefit to skin, because skin cells are not able to extract HA from the
bloodstream.
The more reliable HA skin refill is by applying HA topically. Topical
application of high-molecular-weight native HA (>500 kDa) is not recommended,
because it does not efficiently penetrate the deeper skin layers mainly due to its
large size. Instead it forms films that act as barriers against moisture loss.
4.1.3.5 MOISTURIZING PROPERTIES OF NATIVE HIGH-MOLECULAR-WEIGHT HYALURONAN
Conversely, HA fragments sized smaller than 500 kDa are claimed to permeate
and moisturize the skin. An efficient uptake of HA (357 kDa) by epidermis is
indicated in Figure 4.1, where an isotopically labeled14 C-HA was incubated with
full thickness porcine ear skin using Franz diffusion cells at 37°C for 48 hours.
However, at the same time there was hardly any HA uptake deeper into dermis
layers. This in vitro penetration test into the porcine skin should resemble what
will, with high probability, occur in vivo in humans. The results suggest that HA
of 357 kDa applied topically is useful for the replenishment of HA levels in
epidermis but is still too large to efficiently penetrate into dermis. Smaller HA
fragments are then needed for more efficient penetration when dermis targeting is
required.
614
epidermis -> subcutis
Figure 4.1 Penetration of HA Fragments: 14C-HA activities within 200 µm skin
slices (pigs ear) after 48 hours penetration at 37°C.
4.1.3.5 MOISTURIZING PROPERTIES OF NATIVE HIGH-
MOLECULAR-WEIGHT HYALURONAN AND HOW THE
MOISTURIZING
WEIGHT IS REDUCED
PROPERTIES OF HYALURONAN CHANGE AS THE
MOLECULAR
Due to the unique hydration, viscoelastic, and biocompatibility properties of HA
and in view of a range of molecular-weight materials’ ability to penetrate skin, HA
is being extensively used in cosmetic products as moisturizer, thickener, and
stabilizer. Since HA is a water-soluble product, it can be easily incorporated in the
water phase of cosmetic formulas.
Due to its negative charge, HA is incompatible with cationic compounds or
proteins at certain concentrations. A typical concentration of HA in cosmetic
products varies between 0.01 and 0.2%32. HA is suitable for application in a wide
range of cosmetic compositions devoted to skin care and designed for topical
applications. Typical cosmetic products supplemented with HA molecules include
serum,
4.1.3.5 MOISTURIZING PROPERTIES OF NATIVE HIGH-MOLECULAR-WEIGHT HYALURONAN
moisturizers, creams, shampoos, conditioners, and bath oils. The benefits of HA
being in the formulation are usually connected with reduction of the dermatological
aging, reduction of the depth of lines and wrinkles (including fine lines), decrease
of skin fragility, amelioration of skin atrophy, improvement of skin firmness and
texture, decrease in pore size, restoration of skin brightening, and improvement of
skin barrier function.
14
C-HA
control
0
5
10
15
20
25
30
35
40
0-200
µ
m200-400
µ
m400-600
µ
m600-800
µ
m800-1000
µ
m1000-1200
µ
m
615
In hair-care and hair-treatment formulations, HA restructures the keratin
fibers “inside” the fibers, improves elasticity of hair, and increases the washing
resistance of colored hair.
The advantage of HA over other widely used moisturizers (glycerin, propylene
glycol, and sorbital, polyethylene glycol) resides in the fact that its moisturizing
properties are not significantly influenced by relative humidity. Unlike the other
moisturizers, HA is able to keep good water retention capacity both at low and high
relative humidity. In contrast, common moisturizers at low humidity and/or under
xerotic conditions (dry skin, deep wrinkles, and squamous skin) absorb moisture
from the inner skin rather than from the air, which in turn causes the skin to dry
out. Together with other substances, HA belongs to Natural Moisturizing Factor
(NMF) substances, which are hygroscopic, act as a surfactant, and buffer the skin
thereby preserving its aesthetic appearance.
The moisturizing properties of HA are related to its hydration, often referred
to as an average number of water molecules affected by the presence of HA anion
in aqueous solutions. HA hydration was studied using several approaches including
NMR,33 viscosimetry, ultrasonic velocimetry, and thermal analyses.34 Among
these, thermal analyses, namely differential scanning calorimetry (DSC), is able to
estimate i) non-freezable bound, ii) freezable bound, and iii) free water due to their
different thermodynamic properties caused by the presence of HA (Figure 5.1).
The non-freezable bound water is the fraction of total water content that is in close
contact with HA surface and it is supposed to be either hydrogen bonded to HA
molecules or is restricted by junction zones formed by HA chains. The freezable
bound water is the next layer, which interacts weakly with HA molecules and non-
freezable water layer. Free water is not influenced by HA and its properties
resemble pure water. Therefore, it is mainly the amount of non-freezable and
freezable bound water, which is considered as the number of water molecules
hydrating HA structure. The number of bound non-freezable water molecules in
650 kDa HA is 7.6 per disaccharide HA unit. In terms of freezable bound water,
the hydration increases up to 43 molecules per HA disaccharide unit.35 In terms of
grams, this means that 1g of 650 kDa HA can strongly hold from 0.8 to 2g of water.
Further DSC results indicate that the water-binding capacity is independent of
molecular weight from 30 kDa up to 750 kDa. Increased hydration (for about 0.12
g of water per g of HA) was detected for 1.4 MDa HA.36
4.1.3.5 MOISTURIZING PROPERTIES OF NATIVE HIGH-MOLECULAR-WEIGHT HYALURONAN
616
Figure 5.1 Depiction of freezable-bound, non-freezable-bound and free water
molecules in the presence of HA
To estimate the effect of different molecular-weight HA on skin hydration
properties, a group of volunteers (n = 8) applied for 60 days (1 time per day) an
o/w emulsion containing 0.005% native or fragmented HA. The hydration results
reported in Figure 5.2 indicate that in the case of smaller HA fragments, namely
4.6 and 17 kDa HA, there is a significant skin hydration capacity increase after the
first month of emulsion application. This result is in agreement with easier and
faster penetration of smaller-sized molecules into the skin and with the fact that at
epidermis small HA fragments stimulate the endogenous synthesis of high-
molecular HA and in this way positively affect skin hydration properties. However,
the more efficient hydration tendency of small-sized HA fragments was completely
suppressed after another two months of emulsion application, where the best results
were obtained for the native 1.66 MDa HA. As it was discussed above, the high-
molecular-weight HA did not penetrate the skin but formed an efficient barrier
against moisture loss. The results of the three-month study indicate that the skin
hydration capacity is both HA molecular size and application time dependent.
115
110
105
free water
free water
non-freezable bound water
non-freezable bound water
freezable bound water
freezable bound water
time (days)
0
28
56
placebo
4.6
kDa
17
kDa
310
kDa
1660
kDa
80
85
90
95
100
120
617
Figure 5.2. Changes in hydration capacity of skin after the treatment with 0.005%
HA in o/w emulsion.
4.1.3.6 SELECTED DERIVATIVES OF HYALURONAN AND THEIR EFFECT ON SKIN
MOISTURIZING
4.1.3.6 SELECTED DERIVATIVES OF HYALURONAN
AND THEIR EFFECT ON SKIN MOISTURIZING
Although a great number of HA derivatives have been synthesized in the last 15
years, there is a scarcity of literature devoted to the impact of HA modification on
its moisturizing properties.
In general, it is believed that increasing the hydrophobic character of HA will
enhance its penetration through the hydrophobic stratum corneum into the
epidermis. Then, due to the fact that the permeability of substances through skin is
not only related to their lipophilic character but is also inversely related to their
size, small-sized hydrophobic HA fragments should theoretically favor
transdermal transport.
To approve or disapprove the theoretical prediction, hydrophobized HA (50
kDa HA modified with 50% of hexyl functional groups) was tested in vivo for
moisturizing effect. Similarly as in previous case, hydrophobized HA was
integrated in o/w emulsion, which was daily applied by a group of eight volunteers.
The changes in skin hydration capacity were compared to other two groups of
volunteers using placebo and 17 kDa HA (Figure 6.1). In this case, the results
indicated that the hydrophobized HA enhanced skin hydration capacity comparably
to unmodified HA fragment. However, the effect on skin-moisturizing properties
of tailor-made hydrophobic HA derivatives is unknown and still remains to be
elucidated.
Figure 6.1. Changes in hydration capacity of skin after the treatment with 0.005%
unmodified and hydrophobized HA in o/w emulsion.
Brown and colleagues37 found that topical unmodified HA significantly
enhanced the partitioning of both diclofenac and ibuprofen into human skin when
compared to an aqueous control, pectin, and carboxymethylcellulose. This suggests
time (days)
0
28
56
80
85
90
95
100
105
110
115
120
placebo
kDa
17
hydrophobized
50
kDa HA
618
that glycosaminoglycans, when allowed to interact with water, can enhance the
percutaneous penetration of some drugs and cosmeceuticals useful in cosmetics
and personal care. The details of their interaction remain to be elucidated.
4.1.3.7 COSMETIC APPLICATIONS FOR VARIOUS MOLECULAR WEIGHTS OF HYALURONAN
In this matter, hydrophobized HA may represent even more efficient
transdermal drug delivery systems because of two main reasons. First, as it was
mentioned earlier, the partial hydrophobicity should increase the permeation rate
of HA through the stratum corneum. Second, the hydrophobized HA contains both
hydrophilic and hydrophobic functional groups, which under certain conditions
may enable formation of micro and nano vehicles. These are able to increase the
stability of poorly soluble lipophilic components and nutrients. The transformation
of HA molecules into liposomes, microemulsions, or polymeric micelles can be
used in transdermal research as a better alternative method to enhance permeation
of components through skin. In fact, penetration of small hydrophobic drugs from
such delivery systems into the epidermis can be increased more than two times.38
Unlike other polymeric based delivery systems, HA-based polymeric micro and
nano structures have major advantages due to excellent biocompatibility and
biodegradability of the backbone. Thus, the feasibility of native HA and its
derivatives for the development of new materials for applications in cosmetic,
biomedical, and pharmaceutical fields is still under review.
4.1.3.7 COSMETIC APPLICATIONS FOR VARIOUS
MOLECULAR WEIGHTS OF HYALURONAN
The skin hydration process proceeds through a very complex mechanism.
Regarding HA, the whole range of molecular weights of HA is related to skin
moisturizing. However, as it was described in details above, the exact moisturizing
mechanism differs with HA molecular weight. For this reason, the choice of HA
fragment for cosmetic composition should be always considered in context with
intended reason of use and application. Simplifying the problem of Mw choice, the
larger the Mw of hyaluronan the more predominant are physical-chemical
properties, while biological properties will prevail in case of smaller Mw
fragments.
For this reason, high-molecular-weight HA (about 1 MDa) is usually added in
cosmetic formulation in order to increase composition viscosity and improve the
stability of composition film when applied on skin. In this way, high-
molecularweight HA has positive effect on hydration of upper epidermis layers,
which is manifested by lower trans-epidermal water loss (TEWL). Penetration
properties (and thus anti-aging effect) of high-molecular-weight HA can be
improved by combination of HA with skin penetration enhancers.
619
HA with molecular weights around 250 kDa enables deeper skin hydration due
to its deeper penetration, where HA interacts with skin cells and extracellular
matrix components. It is efficient in skin texture enhancement and wrinkles
reduction.
Very small HA fragments (50 kDa and below) are very useful in anti-aging
because after their penetration into deeper skin layers they serve as signal
molecules for skin cells to synthetize new HA molecules. In addition, small HA
fragments are
620
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easily hydrophobized and in this way they penetrate deeper into epidermis and
dermis, enhancing skin hydration capacity and enabling deep transdermal transport
of complexed or bound hydrophobic actives from formulation.
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GLOSSARY
Angiogenesis: The development of new blood vessels.
Anti-tumorigenic: Tending to suppress tumors.
BTH: Bovine testicular hyaluronidase, EC 3.2.1.35.
DMF: N,N-Dimethylformamide
DMSO: Dimethyl sulfoxide
Endothelial cell: A thin, flattened cell, a layer of them lines the inside
surfaces of body cavities, blood vessels, and lymph vessels, making up the
endothelium.
624
ESI-MS: Electrospray Ionization Mass Spectrometry. An analytical
technique that can provide both qualitative (structure) and quantitative
(molecular mass or concentration) information on analyte molecules after
their conversion to ions.
GlcNAc: N-acetyl-D-glucosamine
GLOSSARY
HA: Hyaluronic acid, hyaluronan.
Inflammation: The end result of the different bodily processes, in response
to an injurious agent.
Interleukins: A variety of naturally occurring polypeptides modulating
inflammation and immunity.
MALDI-TOF: Matrix-Assisted Laser Desorption/Ionization Time Of
Flight. A method of mass spectrometry in which laser radiation of
analytematrix mixture results in the vaporization of the matrix which carries
the analyte with it. Ion´s mass to charge ratio is determined via a time
measurement.
MALS: Multi-Angle Light Scattering. An analytical technique for
determining absolute molar masses, sizes and conformation of all types of
macromolecules.
Mitosis: The process where a single cell divides resulting in generally two
identical cells, each containing the same number of chromosomes and
genetic content as that of the original cell.
Mitogenic: Causing mitosis.
NMF: Natural Moisturizing Factor
Pro-angiogenic: Tending to promote angiogenesis.
Pro-inflammatory: Tending to promote inflammation.
Proliferation: The reproduction or multiplication of cells.
SEC: Size exclusion chromatography. A method in which molecules in
solution are separated by their size.
... As it is observed in the stress graphs (Figure 10a), the adhesion of HA-CA is greater than that of CHI-CA As it is observed in the stress graphs (Figure 10a), the adhesion of HA-CA is greater than that of CHI-CA even though there is a greater content of quinone determined by VIS spectroscopy. In addition to this, it is known that CHI hydrates more slowly than HA [29,31], which has a negative influence on adhesion, as we have already mentioned. Indeed, hydration, which has the function of a lubricant, seems to increases the mobility of HA chains in contrast to CHI, which improves adhesion properties. ...
... Additionally, hydration is a key factor in adhesion, as it enhances the mobility of the polymer chains that promotes tissue adhesion [28]. In this sense, HA is one of the most hydrating polymers known [29,30], which can interestingly enhance the tissue adhesion of HA-CA gels. Figure 10 shows specifically the stress applied at the detachment (Figure 10a) and the displacement produced by the gels before breakage (Figure 10b) for both catechol modified polysaccharides after 4 and 24 h of oxidation by air exposure. ...
... As it is observed in the stress graphs (Figure 10a), the adhesion of HA-CA is greater than that of CHI-CA even though there is a greater content of quinone determined by VIS spectroscopy. In addition to this, it is known that CHI hydrates more slowly than HA [29,31], which has a negative influence on adhesion, as we have already mentioned. Indeed, hydration, which has the function of a lubricant, seems to increases the mobility of HA chains in contrast to CHI, which improves adhesion properties. ...
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Skin health depends on an intact barrier composed of protein-rich corneocytes surrounded by the lamellar intercellular lipids. This barrier provides waterproof protection for the body, preventing infection, regulating electrolyte balance, maintaining body temperature, and providing a mechanism for sensation. Damage to the skin barrier results in skin disease that can be treated by a variety of externally applied substances, such as ceramides, hyaluronic acid, licorice extracts, dimethicone, petrolatum, and paraffin wax. These substances are found in moisturizers that are sold as cosmetics and in prescriptions as 510(k) devices. This contribution examines the formulation and effect of skin barrier creams.