ChapterPDF Available

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


Abstract and Figures Structure and selected physical-chemical properties of hyaluronan Preparation of hyaluronan fragments, isolation and characterization thereof, characterization of degradation products of hyaluronan Preparation of chemical derivatives of hyaluronan, characterization thereof Hyaluronan penetration into the stratum corneum and into the skin Moisturizing properties of native high-molecular hyaluronan and how the moisturizing properties change as the molecular weight is reduced Cosmetic application for various molecular weights of hyaluronan
Content may be subject to copyright.
Daniela Smejkalova, Gloria Huerta-Angeles, Tereza Ehlova
Contipro Pharma, Doln Dobrouc 401, 561 02, Czech Republic
TABLE OF CONTENTS Structure and selected physical-chemical properties of hyaluronan 606 P reparation of hyaluronan fragments, isolation and characterization
thereof, characterization of degradation products of hyaluronan 607 P reparation of chemical derivatives of hyaluronan,
characterization thereof 610 Hyaluronan penetration into the stratum corneum and into the skin 612 M oisturizing properties of native high-molecular hyaluronan
and how the moisturizing properties change as the molecular
weight is reduced 613 S elected Derivatives of Hyaluronan and their Effect on Skin
Moisturizing 616 Cosmetic application for various molecular weights of hyaluronan 617
References 618 Glossary 621
PART 4.1.3
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
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
TABLE OF CONTENTS S tructure and selected physical-chemical
properties of hyaluronan 606 P reparation of hyaluronan fragments, isolation
and characterization thereof, characterization
of degradation products of hyaluronan 607 P reparation of chemical derivatives of
hyaluronan, characterization thereof 610 H yaluronan penetration into the stratum corneum
and into the skin 612 M oisturizing properties of native high-molecular
hyaluronan and how the moisturizing properties
change as the molecular weight is reduced 613
606 S elected Derivatives of Hyaluronan and their Effect on
Skin Moisturizing 616 C osmetic application for various molecular weights of hyaluronan
References 618
Glossary 621
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.
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
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
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. PREPARATION OF HYALURONAN FRAGMENTS,
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
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
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
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
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-
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
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
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
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
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
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
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,
and biological properties of the polysaccharide, as well as possible degradation
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
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
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. 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
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. MOISTURIZING PROPERTIES OF NATIVE HIGH-
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
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.
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
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.
free water
free water
non-freezable bound water
non-freezable bound water
freezable bound water
freezable bound water
time (days)
Figure 5.2. Changes in hydration capacity of skin after the treatment with 0.005%
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
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)
kDa HA
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. 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. COSMETIC APPLICATIONS FOR VARIOUS
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
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.
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
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
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.
1. Lapčik, L.; De Smedt, S.; Demeester, J.; Chabreček, P., Hyaluronan:
Preparation, Structure, Properties, and Applications. Chemical Reviews
1998, 98, (8), 26632684.
2. Stern, R.; Maibach, H. I., Hyaluronan in skin: aspects of aging and its
pharmacologic modulation. Clinics in Dermatology 2008, 26, (2), 106
3. Cowman, M. K.; Matsuoka, S., Experimental approaches to hyaluronan
structure. Carbohydrate Research 2005, 340, (5), 791809.
4. Cowman, M. K.; Spagnoli, C.; Kudasheva, D.; Li, M.; Dyal, A.; Kanai,
S.; Balazs, E. A., Extended, Relaxed, and Condensed Conformations of
Hyaluronan Observed by Atomic Force Microscopy. Biophysical
Journal 2005, 88, (1), 590602.
5. Schante, C.; Zuber, G.; Herlin, C.; Vandamme, T. F., Synthesis of N-
alanyl-hyaluronamide with high degree of substitution for enhanced
resistance to hyaluronidase-mediated digestion. Carbohydrate
Polymers 2011, 86, (2), 747752.
6. Schante, C. E.; Zuber, G.; Herlin, C.; Vandamme, T. F., Improvement
of hyaluronic acid enzymatic stability by the grafting of amino-acids.
Carbohydrate Polymers 2012, 87, (3), 22112216.
7. Šoltes, L.; Kogan, G.; Stankovska, M.; Mendichi, R.; Schiller, J.;
Gemeiner, P., Degradation of High-Molar-Mass Hyaluronan and
Characterization of Fragments. Biomacromolecules 2007, 8, (9), 2697
8. Blundell, C. D.; Almond, A., Enzymatic and chemical methods for the
generation of pure hyaluronan oligosaccharides with both odd and even
numbers of monosaccharide units. Analytical Biochemistry 2006, 353,
(2), 236247.
9. Stern, R.; Kogan, G.; Jedrzejas, M. J.; Šoltés, L., The many ways to
cleave hyaluronan. Biotechnology Advances 2007, 25, (6), 537557.
10. Šmejkalová, D.; Hermannová, M.; Buffa, R.; Čožíková, D.; Vištejnová,
L.; Matulková, Z.; Hrabica, J.; Velebný, V., Structural characterization
and biological properties of degradation byproducts from hyaluronan
after acid hydrolysis. Carbohydrate Polymers 2012, 88, (4), 14251434.
11. Stern, R.; Asari, A. A.; Sugahara, K. N., Hyaluronan fragments: An
information-rich system. European Journal of Cell Biology 2006, 85,
(8), 699715.
12. Kuo, M. T.; Yu, D.; Hung, M.-C., Roles of Multidrug Resistance Genes
in Breast Cancer Chemoresistance Breast Cancer Chemosensitivity. In
Springer New York: 2007; Vol. 608, pp 2330.
13. Hosono, K.; Nishida, Y.; Knudson, W.; Knudson, C. B.; Naruse, T.;
Suzuki, Y.; Ishiguro, N., Hyaluronan Oligosaccharides Inhibit
Tumorigenicity of Osteosarcoma Cell Lines MG-63 and LM-8 in Vitro
and in Vivo via Perturbation of Hyaluronan-Rich Pericellular Matrix of
the Cells. The American Journal of Pathology 2007, 171, (1), 274286.
14. Toole, B. P.; Ghatak, S.; Misra, S., Hyaluronan Oligosaccharides as a
Potential Anticancer Therapeutic. Current Pharmaceutical
Biotechnology 2008, 9, (4), 249252.
15. Mahoney, D. J.; Aplin, R. T.; Calabro, A.; Hascall, V. C.; Day, A. J.,
Novel methods for the preparation and characterization of hyaluronan
oligosaccharides of defined length. Glycobiology 2001, 11, (12), 1025
16. Volpi, N., On-Line HPLC/ESI-MS Separation and Characterization of
Hyaluronan Oligosaccharides from 2-mers to 40-mers. Analytical
Chemistry 2007, 79, (16), 63906397.
17. Sakai, S.; Hirano, K.; Toyoda, H.; Linhardt, R. J.; Toida, T., Matrix
assisted laser desorption ionization-time of flight mass spectrometry
analysis of hyaluronan oligosaccharides. Analytica Chimica Acta 2007,
593, (2), 207213.
18. Tranchepain, F. d. r.; Deschrevel, B.; Courel, M.-N. l.; Levasseur, N.;
Le Cerf, D.; Loutelier-Bourhis, C.; Vincent, J.-C., A complete set of
hyaluronan fragments obtained from hydrolysis catalyzed by
hyaluronidase: Application to studies of hyaluronan mass distribution
by simple HPLC devices. Analytical Biochemistry 2006, 348, (2), 232
19. Schanté, C. E.; Zuber, G.; Herlin, C.; Vandamme, T. F., Chemical
modifications of hyaluronic acid for the synthesis of derivatives for a
broad range of biomedical applications. Carbohydrate Polymers 2011,
85, (3), 469489.
20. Banerji, S.; Wright, A. J.; Noble, M.; Mahoney, D. J.; Campbell, I. D.;
Day, A. J.; Jackson, D. G., Structures of the Cd44-hyaluronan complex
provide insight into a fundamental carbohydrate-protein interaction.
Nat Struct Mol Biol 2007, 14, (3), 234239.
21. Zhong, S. P.; Campoccia, D.; Doherty, P. J.; Williams, R. L.; Benedetti,
L.; Williams, D. F., Biodegradation of hyaluronic acid derivatives by
hyaluronidase. Biomaterials 1994, 15, (5), 359365.
22. Kong, M.; Chen, X.; Park, H., Design and investigation of
nanoemulsified carrier based on amphiphile-modified hyaluronic acid.
Carbohydrate Polymers 2011, 83, (2), 462469.
23. Young, J.-J.; Cheng, K.-M.; Tsou, T.-L.; Liu, H.-W.; Wang, H.-J.,
Preparation of cross-linked hyaluronic acid film using 2-chloro-1-
methylpyridinium iodide or water-soluble 1-ethyl-(3,3-
dimethylaminopropyl) carbodiimide. Journal of Biomaterials Science,
Polymer Edition 2004, 15, (6), 767780.
24. Bergman, K.; Elvingson, C.; Hilborn, J. n.; Svensk, G. r.; Bowden, T.,
Hyaluronic Acid Derivatives Prepared in Aqueous Media by
TriazineActivated Amidation. Biomacromolecules 2007, 8, (7), 2190
25. Huerta-Angeles, G.; Šmejkalová, D.; Chládková, D.; Ehlová, T.; Buffa,
R.; Velebný, V., Synthesis of highly substituted amide hyaluronan
derivatives with tailored degree of substitution and their crosslinking via
click chemistry. Carbohydrate Polymers 2011, 84, (4), 12931300.
26. Zhao, X., Synthesis and characterization of a novel hyaluronic acid
hydrogel. Journal of Biomaterials Science, Polymer Edition 2006, 17,
(4), 419433.
27. Mlčochová, P.; Bystrický, S.; Steiner, B.; Machová, E.; Koóš, M.;
Velebný, V.; Krčmář, M., Synthesis and characterization of new
biodegradable hyaluronan alkyl derivatives. Biopolymers 2006, 82, (1),
28. Ruhela, D.; Riviere, K.; Szoka, F. C., Efficient Synthesis of an
Aldehyde Functionalized Hyaluronic Acid and Its Application in the
Preparation of Hyaluronan-Lipid Conjugates. Bioconjugate Chemistry
2006, 17, (5), 13601363.
29. Matarasso, S. L.; Herwick, R., Hypersensitivity reaction to nonanimal
stabilized hyaluronic acid. Journal of the American Academy of
Dermatology 2006, 55, (1), 128131.
30. Ibrahim, S.; Kang, Q. K.; Ramamurthi, A., The impact of hyaluronic
acid oligomer content on physical, mechanical, and biologic properties
of divinyl sulfone-crosslinked hyaluronic acid hydrogels. Journal of
Biomedical Materials Research Part A 2010, 94A, (2), 355370.
31. Podzimek, S.; Hermannova, M.; Bilerova, H.; Bezakova, Z.; Velebny,
V., Solution properties of hyaluronic acid and comparison of
SECMALS-VIS data with off-line capillary viscometry. Journal of
Applied Polymer Science 2010, 116, (5), 30133020.
32. Draelos, Z. D., New treatments for restoring impaired epidermal barrier
permeability: Skin barrier repair creams. Clinics in Dermatology 2012,
30, (3), 345348.
33. Harding, S. G.; Wik, O.; Helander, A.; Ahnfelt, N. O.; Kenne, L., NMR
velocity imaging of the flow behaviour of hyaluronan solutions.
Carbohydrate Polymers 2002, 47, (2), 109119.
34. Liu, J.; Cowman, M., Thermal Analysis of Semi-Dilute Hyaluronan
Solutions. Journal of Thermal Analysis and Calorimetry 2000, 59, (1-
2), 547557.
35. Kučerík, J.; Průšová, A.; Rotaru, A.; Flimel, K.; Janeček, J.; Conte, P.,
DSC study on hyaluronan drying and hydration. Thermochimica Acta
2011, 523, 245249.
36. Prušová, A.; Šmejkalová, D.; Chytil, M.; Velebný, V.; Kučerík, J., An
alternative DSC approach to study hydration of hyaluronan.
Carbohydrate Polymers 2010, 82, (2), 498503.
37. Brown, M. B.; Jones, S. A., Hyaluronic acid: a unique topical vehicle
for the localized delivery of drugs to the skin. Journal of the European
Academy of Dermatology and Venereology 2005, 19, (3), 308318.
38. Prow, T. W.; Grice, J. E.; Lin, L. L.; Faye, R.; Butler, M.; Becker, W.;
Wurm, E. M. T.; Yoong, C.; Robertson, T. A.; Soyer, H. P.; Roberts, M.
S., Nanoparticles and microparticles for skin drug delivery. Advanced
Drug Delivery Reviews 2011, 63, (6), 470491.
Angiogenesis: The development of new blood vessels.
Anti-tumorigenic: Tending to suppress tumors.
BTH: Bovine testicular hyaluronidase, EC
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
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
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
MALS: Multi-Angle Light Scattering. An analytical technique for
determining absolute molar masses, sizes and conformation of all types of
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. ...
Full-text available
Spontaneously formed hydrogels are attracting increasing interest as injectable or wound dressing materials because they do not require additional reactions or toxic crosslinking reagents. Highly valuable properties such as low viscosity before external application, adequate filmogenic capacity, rapid gelation and tissue adhesion are required in order to use them for those therapeutic applications. In addition, biocompatibility and biodegradability are also mandatory. Accordingly, biopolymers, such as hyaluronic acid (HA) and chitosan (CHI), that have shown great potential for wound healing applications are excellent candidates due to their unique physiochemical and biological properties, such as moisturizing and antimicrobial ability, respectively. In this study, both biopolymers were modified by covalent anchoring of catechol groups, and the obtained hydrogels were characterized by studying, in particular, their tissue adhesiveness and film forming capacity for potential skin wound healing applications. Tissue adhesiveness was related to o-quinone formation over time and monitored by visible spectroscopy. Consequently, an opposite effect was observed for both polysaccharides. As gelation advances for HA-CA, it becomes more adhesive, while competitive reactions of quinone in CHI-CA slow down tissue adhesiveness and induce a detriment of the filmogenic properties.
... HA fragments can also be used for treating and preventing wrinkles, expression lines, fibroblastic depletion, and scars. HA oligosaccharides (with molecular weight <5 × 10 3 Da) contribute to the suppression of wrinkles in both layers of the skin (Smejkalova et al., 2015). At the epidermis, they stimulate the endogenous high molecular HA, which has a positive effect on hydration. ...
... HA is a natural moisturizing factor substance (Guarise et al., 2019). The more important advantage of HA compared with other moisturizers (polyethylene glycol, glycerol, ethylene glycol, propylene, and sorbitol) is that it is not affected by relative humidity (Smejkalova et al., 2015). Dissimilar to the other moisturizers, HA has good water retention capability both at high and low relative humidity. ...
Full-text available
Hyaluronic acid (HA) is a large non-sulfated glycosaminoglycan that is the main component of the extracellular matrix (ECM). Because of its strong and diversified functions applied in broad fields, HA has been widely studied and reported previously. The molecular properties of HA and its derivatives, including a wide range of molecular weights but distinct effects on cells, moisture retention and anti-aging, and CD44 targeting, promised its role as a popular participant in tissue engineering, wound healing, cancer treatment, ophthalmology, and cosmetics. In recent years, HA and its derivatives have played an increasingly important role in the aforementioned biomedical fields in the formulation of coatings, nanoparticles, and hydrogels. This article highlights recent efforts in converting HA to smart formulation, such as multifunctional coatings, targeted nanoparticles, or injectable hydrogels, which are used in advanced biomedical application.
... Botanical substances are also used in the formulation of moisturizers [6,88,89]. However, the use of herbal extracts in moisturizers has not always been justified by clinical trials [90,91]. ...
Full-text available
Moisturizers are one of the most widely used preparations in cosmetics and have been extensively used to soften the skin for consumers. Moisturizers work effectively in combating dry skin which may cause pain, tightness, itch, stinging, and/or tingling. The aim of this review is to evaluate published studies on the history, ingredients, preparation processes, characteristics, uses, and applications of moisturizers. Moisturizers bridge the gap between medicine and consumer goods by being used to make the skin more beautiful and healthy. In the future, in moisturizer therapy, the capacity to adapt specific agents to specific dermatological demands will be crucial. Cosmetically, moisturizers make the skin smooth by the mechanism of increasing the water content in the stratum corneum, hence exerting its most vital action, which is moisturizing action and maintaining a normal skin pH.
... which is adequate to make the preparations very smooth and viscous. 80 ...
Full-text available
Antimicrobial resistance is the leading cause of burden on healthcare sector. There is scientific challenge of developing new functional material as a platform to prevent and treat viral and microbial infection and cosmetic utility. In material chemistry, there is progress in the development of functional material with advent of nanotechnology with aid of synthetic organic chemistry. The properties and application of material can be changed significantly by modification of the surface functional groups (namely, COOH, HO, NH2OH, SO4,), formation of composite with inorganic material and incorporation of active pharmaceutical agents. In antibacterial application functional material of copper, silver, gold, platinum, tin, iron, cobalt, ruthenium, zinc and pharmaceutical antimicrobial agents found utility in the treatment of bacterial and hospital acquired infection with different resistant strains of microorganisms. In antiviral application many functional materials have been shown to possess remarkable antiviral ability like quantum dots, gold and silver nanoparticles, nanoclusters, carbon dots, graphene oxide and silicon materials. The polymers and dendrimers functionalized with USFDA approved antiviral agent also has potential therapeutic outcomes. Despite their difference in antiviral mechanism and inhibition efficacy, these functional material structures have unique features as potential antiviral candidates. In cosmetic applications functional material based on mica, sericite, fullerene, charcoal, peptides, mineral, lipids, glucocorticoid, nanocellulose hybrid material are extensively used. In this review, we have highlighted early promise and prospects of functional material for cosmetics, antibacterial and antiviral applications, advantages and disadvantages, Patent scenario, current challenges for translation into commercial products.
... HA is available in many different molecular weights (ranging from ten thousands to millions of Daltons) and the molecular weight of HA needs to be considered when formulating a cosmetic product. Where high molecular weight (HMW) HA works as a film forming polymer and thereby contributes to the moisture content of the skin, and the possible decrease of the transepidermal water loss, Low molecular weight (LMW) HA is primarily utilized to improve skin penetration to restore a sustained physiological and hydrated micro environment for optimize skin rejuvenation and tissue repair [9][10][11][12][13]. Thus, the larger the molecular weight of HA, the more dominant the physical chemical properties of the vehicle (e.g. ...
Full-text available
In this work, we underline the importance of the molecular weight of hyaluronic acid on the elongational properties of concentrated emulsions. The filament formation properties, e.g. the stringiness, of an emulsion is a key determinant of a product liking and repeat purchase. Here, we find that high molecular weight hyaluronic acid and a high stretching speed are the control parameters affecting the filament formation of an emulsion.
... HA is available in many different molecular weights (ranging from ten thousands to mil-lions of Daltons) and the molecular weight of HA needs to be considered when formulating a cosmetic product. Where high molecular weight (HMW) HA works as a film forming poly-mer and thereby contributes to the moisture content of the skin, and the possible decrease of the transepidermal water loss, Low molecular weight (LMW) HA is primarily utilized to im-prove skin penetration to restore a sustained physiological and hydrated micro environment for optimize skin rejuvenation and tissue repair [9][10][11][12][13]. Thus, the larger the molecular weight of HA, the more dominant the physical chemical properties of the vehicle (e.g. ...
Full-text available
Objective Cosmetic emulsions containing hyaluronic acid are ubiquitous in the cosmetic industry. However, the addition of (different molecular weight) hyaluronic acid can affect the filament stretching properties of concentrated emulsions. This property is often related to the “stringiness” of an emulsion, which can affect the consumer’s choice for a product. It is thus very important to investigate and predict the effect of hyaluronic acid on the filament stretching properties of cosmetic emulsions. Methods Model emulsions and emulsions with low and high molecular weights are pre- pared and their filament stretching properties are studied by the use of an extensional rheometer. Two different stretching speeds are employed during the stretching of the emul- sions, a low speed at 10 µm/s and a high speed at 10 mm/s. The shear rheology of the samples is measured by rotational rheology. Results We find that filament formation only occurs at high stretching speeds when the emulsion contains high molecular weight hyaluronic acid. The formation of this filament, which happens at intermediate states of the break-up, coincides with an exponential decay in the break-up dynamics. The beginning and end of the break-up of high molecular weight hyaluronic acid emulsions show a power-law behaviour, where the exponent depends on the initial stretching rate. At a lower stretching speed no filament is observed for both high molecular weight and low molecular weight hyaluronic acid emulsions, and the model emulsion. The emulsions show a power-law behavior over the whole break-up range, where the exponent also depends on the stretching rate. No significant difference is observed between the shear flow properties of the emulsions containing different molecular weights hyaluronic acid. Conclusion In this work, we underline the importance of the molecular weight of hyaluronic acid on the elongational properties of concentrated emulsions. The filament formation properties, e.g. the stringiness, of an emulsion is a key determinant of a product liking and repeat purchase. Here, we find that high molecular weight hyaluronic acid and a high stretching speed are the control parameters affecting the filament formation of an emulsion.
Background: In esthetic medicine, different techniques have been used against the aging of the human skin especially in the facial area. Hyaluronic acid is used for improving the quantity of water and extracellular matrix molecule. The aim of this study is a clinical and histological evaluation of the effect of low-molecular-weight hyaluronic acid fragments mixed with amino acid (HAAM) on the rejuvenation the face skin treated with intradermal microinjections. Methods: Twenty women with mean age 45 range from 35 to 64 were studied, thereof 8 in menopause and 12 of childbearing age. The patients were treated with the HAAM products by mesotherapy technique; before and after 3 months of the therapeutic procedure, each patient underwent small biopsies with a circular punch biopsy. Results: The clinical results of the present study showed that the administration of the dermal filler containing fragments of hyaluronic acid between 20 and 38 monomers and amino acid via dermis injection technique produces an esthetic improvement in the faces of the treated patients, while the histological evaluation shows an increased fibroblast activity with the production of type III reticular collagen and increased number of vessels and epidermis thickness. Conclusions: The clinical and histological assessment showed that subcutaneous HAAM infiltration has a significant impact on the dermis and clinical aspects of the face.
Full-text available
A sample of high-molar mass hyaluronan was oxidized by seven oxidative systems involving hydrogen peroxide, cupric chloride, ascorbic acid, and sodium hypochlorite in different concentrations and combinations. The process of the oxidative degradation of hyaluronan was monitored by rotational viscometry, while the fragments produced were investigated by size-exclusion chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and non-isothermal chemiluminometry. The results obtained imply that the degradation of hyaluronan by these oxidative systems, some of which resemble the chemical combinations present in ViVo in the inflamed joint, proceeds predominantly via hydroxyl radicals. The hyaluronan fragmentation occurred randomly and produced species with rather narrow and unimodal distribution of molar mass. Oxidative degradation not only reduces the molecular size of hyaluronan but also modifies its component monosaccharides, generating polymer fragments that may have properties substantially different from those of the original macromolecule.
Hyaluronan (HA) based hydrogels with well defined 3D-molecular architecture have been synthesized combining chemical modification of hyaluronic acid and ‘click chemistry’. The high degree of substitution of HA was obtained after activation of the carboxyl group with ethyl chloroformate and subsequent functionalization of the carboxylic group with primary amine containing either a terminal azido or alkynyl groups. The degree of amide substitution could be controlled by reaction conditions. The chemical modification probed to be highly chemo-selective providing only amide modified HA derivates. The prepared derivates showed higher resistance towards thermal degradation than starting hyaluronan material. The crosslinking reaction of azido- and alkynyl-amide derivates of HA led to the formation of highly organized and porous networks, which due to their high stability against degradation are potential candidates for application as drug delivery systems, or scaffolds in tissue engineering.
The processes of hyaluronan (HYA) drying and hydration were studied using differential scanning calorimetry. In the first approach the isoconversional Kissinger–Akahita–Sunose (KAS) method was applied in order to determine actual activation energies of evaporation of pure water and water from concentrated HYA solutions. Since the evaporation is a single-step process, the activation energies for pure water provided results consistent with tabulated values of evaporation enthalpies. In the course of water evaporation from hyaluronan solution a break in increasing enthalpy followed by a decrease below 0.34 g of water per 1 g of HYA was observed. This result confirmed earlier observation that at this particular water content evaporation from hyaluronan is compensated by heat evolution associated with the formation of new bonds in hyaluronan supramolecular structure. Subtraction of water evaporation enthalpy from enthalpies obtained for HYA concentrated solution provided a possibility to extrapolate the evaporation enthalpies to the concentration (approximately 2 g of water per 1 g of HYA) at which free water is not present any longer and only bound water starts being evaporated from the HYA solution. Similar results were obtained in the second approach in which using slightly modified “traditional” freezing/thawing experiment, melting enthalpy of ice was plotted against water fraction in HYA. It was found out that the melting enthalpy of ice exponentially increases from 0.8 up to 2 g of water per g of hyaluronan where it reaches and keeps the melting enthalpy of hexagonal ice. It was shown that both approaches can serve as alternatives providing an additional insight into the state of water and biopolymers in highly concentrated solutions.
Extensive study of solution properties of sodium hyaluronate including about 140 samples and covering broad molar mass range was carried out by classical viscometry using an Ubbelohde capillary viscometer and by combination of size-exclusion chromatography with a multi-angle light scattering detector and an on-line viscometer. The study also involved critical overview of literature data of Mark-Houwink relation for sodium hyaluronate. Continuous decrease of the Mark-Houwink exponent with increasing molar mass to the values markedly lower than those typical of linear random coils in thermodynamically good solvents was observed. This fact was attributed to branched molecular structure of hyaluronic acid as a result of unknown side reactions during the manufacturing process. The molar mass dependence of the second virial coefficient was determined and proved aqueous salt solutions to be thermodynamically good solvents for sodium hyaluronate. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010
The freezing and melting of water in semi-dilute (0.5–3.0%) solutions of the polysaccharide hyaluronanhave been investigated by modulated differential scanning calorimetry. High molecular weight hyaluronan inhibited nucleation of ice and significantly depressed thefreezing temperature in a dynamic scan conducted at −3.0°C min−1. Low molecular weight hyaluronan had a weaker and more variable effect on nucleation. Theeffects on nucleation, especially by the high molecular weight hyaluronan, are attributed tothe influence of a hyaluronan network on the formation of critical ice nuclei. Both high and low molecular weight hyaluronan reduced the melting temperature of ice by 0.4–1.1°C, depending on concentration. The enthalpy change associated with this transitionwas significantly reduced. If all of the enthalpy difference is attributed to the presence of non-freezing water, approximately 3.65 g water/g hyaluronan would be non-freezing. This result appears incompatible with published studies on hyaluronan samples of low water content. An alternative hypothesis and quantitative approach to analysis of the data are suggested. The data are interpreted in terms of a small amount of non-freezing water, and amuch larger boundary layer of water surrounding hyaluronan chains, which has slightly altered thermodynamic properties relative to those of bulk water. The boundary layer water behaves similarly to water trapped in small pores in solid materials and hydrogels.
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.