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Volume 1 • Issue 1 • 1000103Int J Chem Res.
ISSN: 0000-0000
International
Journal of Chemistry and Research
Research Article Open Access
Hyaluronic Acid: Evaluation of Efficacy with Different
Molecular Weights
Antonio Mazzucco*
Department for Life Quality Studies, University of Bologna, Italy
Article Info
*Corresponding author:
Antonio Mazzucco
Adjunct Professor
Department for Life Quality Studies
University of Bologna
Italy
E-mail: antonio.mazzucco@unibo.it
Received: February 9, 2019
Accepted: February 11, 2019
Published: February 15, 2019
Citation: Mazzucco A. Hyaluronic Acid:
Evaluation of Efficacy with Different Molecular
Weights. Int J Chem Res. 2018; 1(1): 13-18.
doi: 10.18689/ijcr-1000103
Copyright: © 2019 The Author(s). This work
is licensed under a Creative Commons
Attribution 4.0 International License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the
original work is properly cited.
Published by Madridge Publishers
Abstract
Hyaluronic acids are compared to different molecular weights, initially evaluating
their chemical stability and microbiological compliance and - subsequently - by means
of tests in vitro, the difference in efficacy between two classes of hyaluronic acid. In
particular, we will try to understand how the molecular weight can influence the activity
of hyaluronic acid and its possible anti-wrinkle effect in order to obtain highly performing
cosmetic products.
The affinity of the CD44 receptor in the in vitro tests represents an interesting
method for the evaluation of the biological characteristics of cosmetic ingredients, in
accordance with the current regulations that require cosmetic producers to verify the
claimed activity.
Keywords: Hyaluronic Acid; Proteoglycans; Fibroblasts; D-glucuronic acid.
Introduction
Discovered inside a vitreous body in 1934 and then synthesized in 1964, Hyaluronic
Acid (HA) is one of the main components of connective tissues. It gives the skin its
particular properties of resistance and maintenance of the shape and supports the
preservation of the natural degree of hydration of the skin cells; its concentration in the
body tends to decrease with the advancing of age and a lack of it leads to a weakening
of the skin promoting the formation of wrinkles and imperfections. Today with the
NASHA (Non Animal Stabilized HA) technology, it is possible to stabilize a synthetic
hyaluronic acid of non-animal origin, but bacterial, in order to obtain a product of high
purity, similar to that produced by the human body, eliminating the risk of anaphylactic
reactions present in other products. In particular, it is possible to obtain it conveniently
from bacterial cultures of Streptococcus equis var, zooepidemicus or from recombinant
strains of Bacillus subtilis by a fermentation process. This progress has led to greater
availability of HA on the international market with the consequent possibility of a wider
use in the cosmetic sector. Since hyaluronic acid is a natural substance that is already
present in our body, once applied it is slowly and gradually reabsorbed by the body
itself.
Hyaluronic Acid
Hyaluronic acid is a complex carbohydrate, belonging to the category of
mucopolysaccharides, more modernly called glycosaminoglycans (GAGs). Unlike all the
other compounds of the same class, which possess sulphate groups, they are of limited size
and form glycoprotein complexes (proteoglycans); hyaluronic acid contains no sulfur, does
not have a polypeptide component and reaches very high molecular weights. It is therefore
a non-sulphured glycosaminoglycan with no protein core, from the non-branched
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polysaccharide chain produced by the aggregation of thousands
of disaccharide units (Figure 1) formed in turn by D-glucuronic
acid and N- acetyl-Dglucosamine combined together through
alternating glycosidic bonds (β1→4, β1→3 glycosidic bonds as
well as intramolecular hydrogen bonds that stabilize their
conformation) [1].
In the beta configuration it is admissible for all of its
cumbersome groups to be in an equatorial position (sterically
favorable), while all hydrogen atoms occupy small sterically
less favorable axial positions, which makes the structure of
the disaccharide energetically very stable.
Figure 1. Structure of repeated disaccharide units of hyaluronic
acid.
D-Glucuronic acid is an organic compound belonging to
the class of alduronic acids; it derives from the carboxyl group
oxidation of the primary alcohol group (OH bonded to C6) of
D-glucose. N- acetylglucosamine is a monosaccharide
deriving from the modification of 2-glucosamine which
undergoes an N-acetylation reaction. These substances are
both negatively charged and when they are joined together,
the strong repulsion gives rise to a linear, flexible and
extremely polar molecule [2].
In aqueous solution, the polyelectrolyte character of the
macromolecule (mutual repulsions of the negative charges on
the carboxyl groups) and the intra-molecular hydrogen bonds
cause the HA to appear as a ball and occupy a high hydrodynamic
volume, whose size depends on the concentration of the polymer
and the presence of fillers in the solvent.
At a physiological pH the carboxyl groups of the glucuronic
units are therefore ionized, giving the hyaluronate molecule a
high polarity, and consequently a high solubility in water.
Thanks to this property, hyaluronate is able to mix with many
molecules of water reaching a high degree of hydration [3].
In addition to these properties, hyaluronic acid can also
interact with membrane proteins or surface cell receptors
belonging to three main classes: CD44, RHAMM and ICAM-1
[4]. The interaction with these receptors triggers cellular
responses and different metabolic processes in relation to the
respective molecular weights. About one third of cutaneous
HA is contained in the epidermis where, by binding to the
CD44 receptor (Figure 2), it ensures not only the adhesion
between the individual keratinocytes, but also [5] intervenes
in the regulation of gene expression [6]. It has been shown
how HA has a positive effect on the proliferation of skin
fibroblasts and how CD44 plays a fundamental role in this.
New research has confirmed that HA is produced dynamically
by most skin cells, not only by fibroblasts but also by
keratinocytes in the outer protective layer (epidermis). For
both fibroblasts and keratinocytes, HA plays a regulatory role
in the control of cell physiology through the interaction of
extracellular HA with the cell surface receptor, CD44. This
interaction mediates intracellular signaling both directly and
indirectly, through CD44 interactions with the cytoskeleton
and with EGF receptors (Epidermal growth factor: or epidermal
growth factor receptors) and TGFβ (Transforming growth
factor: Transforming growth factor beta) [7].
Figure 2. Structure of the CD44 protein.
CD44 (Cluster of Differentiation 44) is a transmembrane
glycoprotein of type I, monomeric and ubiquitous, encoded
by a single gene and expressed in different isoforms all
endowed with the interaction site for hyaluronic acid; it is
mainly a molecule of intercellular adhesion localized on
lymphocytes and leukocytes. The CD44 is composed of 177
AA and is divided into 4 domains among which an extracellular
called HBD (Hyaluronan Binding Domain) that represents the
site for [8] hyaluronic acid, a transmembrane with anchor
function, an intermediate domain between the two, which
varies in length according to the exons, and an intracellular
one with various functions (Figure 3) [9]. The HBD domain is
157 amino acids long and has 3 disulfide bridges and
numerous O- and N-glycosylation points to which up to two
hundred glucidic residues of various types are linked and with
various bonds (xylose, N-acetylglucosamine, galactose, etc.),
many to almost completely mask the basic protein and modify
its affinity for the various ligands; furthermore, every cell type
produces a protein with a specific glycosylation sequence.
Another important aspect that significantly modifies affinity is
the possibility of having up to 10 alternative splicing, the
basic protein is called CD44H (H: hematopoietic) or CD44s,
while isoforms are indicated with CD44v x (where instead of x
indicates one or more numbers from 1 to 10), these show
lower affinity for hyaluronic acid.
The binding with this receptor does not have simple
anchoring function but causes the protein to transduce
signals inside the cell with variable consequences depending
on the cell. The interaction between hyaluronic acid and CD44
has been implicated in a variety of physiological events, which
include cell-cell adhesion, cell- substrate adhesion, migration,
cell proliferation and activation, up-take and hyaluronic acid
degradation. Recent studies suggest that the two main
functions of CD44 in the skin are the regulation of keratinocyte
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proliferation in response to extracellular stimuli and the
maintenance of local hyaluronic acid homeostasis [10].
Hyaluronic acid is a component of the so-called
fundamental substance of the dermis and, to date is the main
substance of anti-aging research thanks to its ability to
hydrate, lubricate and elasticize the skin, maintain the shape
of the tissues and reinforce the tone. Furthermore, the
application of topical hyaluronic acid helps to support the
synthesis of collagen.
Hydration, turgidity, plasticity and all the particular properties
of the dermis are therefore influenced by its determining role, as
well as the protection against free radicals with scavenger
antioxidant action, the collagen neosynthesis and other
important biological activities mainly dependent on its weight
molecular. It has also been shown that the fragments of the anti-
aging molecule par excellence stimulate the activity of fibroblasts,
thereby favoring the synthesis of hyaluronic acid. In the
epidermis, hyaluronic acid, thanks to its negative charge, is
located in the extracellular region where it forms a hydrophilic
network capable of transporting nutrients and metabolites to
the different cell types through a percolation mechanism.
Hyaluronic acid with different molecular weights
Approximately half of our body’s hyaluronic acid is
distributed in the cutaneous region where molecules of
different molecular weight are synthesized by fibroblasts,
keratinocytes and endothelial cells that produce hyaluronic
acid families of weights between 50 kDa and 3000 kDa.
The hyaluronic acid with different molecular weights has the
peculiar characteristic of channeling its intervention towards
different skin levels:
a) HA with high molecular weight: ceases on the surface
of the epidermis to create an invisible film coating
capable of blocking outgoing water and limiting
evaporation thanks to the interactions it establishes
with the main molecules present in this compartment
(proteoglycans, glycoproteins, elastin and seven
types of collagen). This thin protective film coating
helps keep the skin soft, smooth and hydrated. In
practice, the topical application of this macromolecule
determines the formation of an external film coating
that reduces perspiratio insensibilis and improves,
indirectly, skin hydration. Their size, however, does
not allow a good interaction with the receptors due
to the steric encumbrance (Figure 3).
b) HA with a medium molecular weight: it overcomes
the skin barrier and provides the skin with all the
water necessary to preserve the principal sum of
beauty and turgor intact. Its dimensions are optimal
to guarantee the best interactions with surface
receptors (Figure 3).
c) HA with low molecular weight: it boasts the unique
peculiarity of knowing how to penetrate into the
epidermis until reaching the subcutaneous layers and
favoring the physiological production of collagen, a
protein that is responsible for preserving the density
and compactness of the skin. The low molecular
weight increases the possibilities of penetration
through the skin ensuring a better moisturizing effect,
it also determines a temporary filling of tissues and
smoothing of small wrinkles. It has, however, low
affinity for receptors [11].
Figure 3. Interaction between cells and hyaluronic acid with
different weights molecular.
The aim of the work is to compare the effectiveness of a
“standard” hyaluronic acid (1,000-1,800 kDa) compared to a
so-called “second generation” hyaluronic acid, ie with a wider
range of molecular weights (50-2,500 kDa). The second
generation hyaluronic acids have been designed to take
advantage of the moisturizing and anti-wrinkle properties; in
fact, aesthetic medicine aims to exploit the nutritive-reshaping
action of hyaluronic acid deriving from the synergy of the
activities connected with its molecular weight.
Materials and Methods
Solutions A and B containing respectively hyaluronic acid
with weight molecular ranging from 1,000- 1,800 kDa and
hyaluronic acid with weight molecular comprised between
50-2,500 kDa were prepared according to the GLP (Good
Laboratory Practice) as required by European Regulation No.
1223 of 2009. The solutions (sol A and sol B) are exactly the
same (Table 1). In this regard, aqueous solutions for external
use containing the two different hyaluronic acids have been
prepared.
A: hyaluronic acid weighing 1,000-1,800 kDa
B: hyaluronic acid weighing between 50-2,500 kDa
After verifying the stability of the following solutions and
having carried out the microbiological analyses, tests were
carried out in vitro in order to verify and compare their anti-
wrinkle efficacy. The solutions are prepared with a cold
process by combining the phase 1 components, carefully
mixed with turbo emulser, to those of phase 2. The two phases
are continuously stirred until complete mixing. Attention
must be paid to the solubility of hyaluronic acid, which has a
solubilization rate dependent on its molecular weight; the
higher molecular weight molecules dissolve slower. In the
case of solution B, in fact, having hyaluronic acid with a
molecular weight up to 2,500 kDa, it was dissolved in glycerol
and then joined to the aqueous phase where sodium benzoate
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and tetracylic ethylenediamine acid had previously been
solubilized. In the case of solution A, instead, hyaluronic acid
is directly dissolved in demineralized water together with the
other components of step 1.
The entire dissolution process is facilitated by the use of a
benchtop turboemulser, which allows reducin g considerably
the time that would be required for normal stirring. Finally,
the pH is measured which must be included in the range 4.5-
5.5 and optionally corrected with citric acid.
Table 1. Formula Solution A and B. INCI (International nomenclature of
cosmetics ingredients): it is an international name used to indicate on
the label the different ingredients present in a cosmetic product; it is
used in all EU member states.
Description: Hyaluronic Acid Solution
Ingredients INCI %Function
Water purified Aqua 86,3 EXCIPIENT
P. 1
Sodium Benzoate Sodium benzoate 0,5 Preservative
Etilendiammino
tetracetic acid,
disodium salt
Disodium EDTA 0,1 Chelating
Gliceryn vegetale Glycerin 11 Humectant
Hyaluronic acid (A or B) Hyaluronic Acid 0,5 Active
Ingredients
Phenoxyethanol Phenoxyethanol 0,8 Preservative
P.2Geogard 221 Benzyl Alcohol,
Dehydroacetic acid, aqua 0,8 Preservative
Citric acid Citric Acid q.b. pH modifier
As required by the European Regulation n. 1223/2009,
“cosmetic products made available on the market must be
safe for human health when used under normal or reasonably
foreseeable conditions of use”. This Regulation prohibits
experimentation on animals both of finished cosmetic
products and of ingredients (or combinations of ingredients)
intended to be contained in cosmetic products. This restriction
has favored the development of alternative methods in vitro
to protect companies and consumers and that, unlike the
animal model, allow the experiment to be carried out without
numerical limits and under conditions of greater control and
standardization, as well as to apply quantitative and objective
measurement systems. In addition to being an alternative to
animal testing, as they lack prejudicial ethics and potentially
quicker and less expensive, tests in vitro have other
advantages; in particular [11], allow the use of normal human
cells and precise control of the doses of the substances used.
Tests in vitro are generally carried out to highlight the
performances provided by ingredients or finished products but
also to be able to be compared and quantified and, for this, must
be reliable and reproducible. These tests can therefore be used
as screening tools during the development of the product but
also to illustrate the mode of action of the components. After
verifying the stability of the solutions and compliance with
microbiological analyses, Tests in vitro were carried out in order
to evaluate their efficacy and safety. These tests are carried out
on specific skin cell lines by professionals in the sector at
specialized laboratories and university facilities.
Cytotoxicity test
As far as Solutions A and B are concerned, a preliminary
evaluation was carried out through in vitro cytotoxicity tests
on cellulary cultures of fibroblasts. This test allows to evaluate
in vitro the ability of an agent to inhibit cell growth. The MTT
assay was used to determine the possible cytotoxicity of
Solutions A and B. The cell viability test (or MTT assay) is a
colorimetric test (Figure 4) that allows estimating the number
of live cells present in culture and therefore evaluating the
effect of treatment with an external agent on the viability of
the cell population.
Figure 4. MTT colorimetric assay.
The average cell viability at different concentrations was
expressed as a percentage relative to the mean value of the
negative controls. A reduction in cell viability greater than
30% is considered a cytotoxic effect.
Results
The samples Sol A and Sol B at 3 different concentrations
(0.62; 0.31; 0.16) under the test conditions described herein n
or n cause cytotoxic effects with an IC50 of 3.92 mg/ml (Table
2). All the criteria defined for the checks for the acceptability
of the test are respected. The standard deviation among the
six replicates of the samples is well within the defined limits of
18%. After verifying that Solutions A and B do not give
cytotoxicity problems, a quantitative analysis was performed
using Western Blot to determine the presence of CD44.
Table 2. IC50 of Solutions A and B.
Solution A
Concentration 0,6 0,31 0,16
Average Cell Vitality % 113 109,6 113,5
7,0 2,8 4,1
IC50=3,92 mg/ml
Solution B
Concentration 0,62 0,31 0,16
Average Cell Vitality % 117,2 117,8 113,5
7,0 3,5 4,1
IC50=3,92 mg/ml
Western blot quantitative analysis
As previously noted CD44 is the major cell surface
receptor for hyaluronic acid and is expressed in many cell
types including fibroblasts. The terminal amino group shares
the homologous sequence with hyaluronans.
The cell extracts were then treated with solutions A and B
and western blot was used to highlight changes in protein
levels of CD44. Western blot is a biochemical technique that
allows to identify a particular protein in a mixture of proteins,
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through the recognition of specific antibodies; in general, to
facilitate recognition, the protein mixture is first separated
based on their size using a polyacrylamide gel; subsequently,
the proteins are transferred onto a support, which is commonly
a nitrocellulose membrane, and then the protein is recognized
by using a specific antibody.
The cells were first sown and incubated. Subsequently,
the cell monolayers were detached from incubation culture
plates with 16 mmol of EDTA in PBS for 20 minutes at 37°C
and centrifuged for 10 minutes at 1000 rpm. After
centrifugation, the precipitate containing the cells was
resuspended in a suitable volume of lysis buffer (300 mM
NaCl, 50 mM Tris HCl pH 7-6, 0/5% Triton X-100, 2 mM of
phenyl methylsulfonylfluoride) for 45 minutes in ice. The cells
were lysed and the protein lysate was recovered by
centrifugation. 10% sodium thiosulfate and 10% sodium
dodecyl sulfate were added to the supernatant up to a final
concentration of 0.2%. The supernatant was dosed into its
protein content through the analysis of the bicinconic acid
(BCA) (Pierce, United Kingdom). The protein dosage by means
of the Bicynonic Acid (BCA) assay allows to determine the
total protein quantity by means of a colorimetric reaction
which involves the reduction of Cu2+ copper by proteins
which produce an alkaline intermediate containing the Cu1+
ion to which the Bicinconic acid (BCA, Pierce). Following the
chelation of a Cu1+ ion by two molecules of BCA a violet-
colored water-soluble complex is formed. The absorbance at
570 nm is then measured, which is proportional to the protein
concentration.
Samples of supernatant with balanced amounts of protein
for each sample were transferred to 5-7% polyacrylamide gel
in non-reducing conditions; this allows separating proteins
on the basis of their mass differences by electrophoresis. The
separated proteins were then transferred onto a nitrocellulose
membrane. Immunolabeling was performed with a CD44
antibody using Mabs F. 10.44.2, F. 10.62.1 and A3D8. The
secondary antibody was a goat anti-mouse Ig conjugated
with peroxidase (Southern Biotechnology, USA). The antigen
was visualized by chemiluminescence by first applying the
chemiluminescent reagent (ECL) and then an autoradiographic
plate (Sigma, UK). The intensity of staining of the CD44 bands
was measured by densitometry. The bands were scanned
using a Hewlett Packard Scanjet 2C scanner connected to an
Apple Macintosh IIfx with NIH Image software with gel
plotting macros.
Figure 5. The bands A1 and B1 represent the expression of CD44
on cells before treatment with hyaluronic acid; the bands A2 and
B2, instead, after treatment with Solutions A and B.
The bands represent the expression of CD44 before and
after exposure with solutions A and B. As can be seen from
the image (Figure 5), the band B2 is the most extensive. Thus,
a higher expression of CD44 can be correlated in cells treated
with solution B, i.e. with hyaluronic acid of molecular weight
between 50- 2500 kDa.
Discussion
As can be seen from the tables, Solutions A and B over time
do not show significant changes. The comparison between
the second generation hyaluronic acids compared to the
standard ones did not show any significant differences with
regard to stability tests and microbiological checks; both
solutions are in fact stable over time and secure. Furthermore,
they did not show cytotoxicity by the MTT assay. In contrast,
quantitative analysis using Western Blot has shown substantial
differences between the two solutions; from the reading of
the obtained bands a greater expression of CD44 protein is
visible in the tissues treated with second generation hyaluronic
acid (Sol. B). The comparison between the two hyaluronic
acids by means of tests in vitro thus confirms the initial
hypothesis of greater anti-wrinkle efficacy of Solution B
containing hyaluronic acid with a molecular weight ranging
from 50-2,500 kDa. The greater efficacy of hyaluronic acid
with a molecular weight between 50-2,500 kDa is an
expression of the synergistic action deriving from the different
dimensions of the molecules, which, when combined, create
the optimal conditions for preventing and combating the
aging process, guaranteeing the corrective result of the
wrinkle. Therefore, the anti-wrinkle effect of solution B can be
considered as resulting from the different activities of:
a) HA low PM that can penetrate deeply ensuring
interaction with receptors and in particular with CD44
located on cell membranes. This interaction induces a
reactivation of cellular metabolism to help fight
environmental stress through mitosis and cell
proliferation.
b) It has a medium PM that penetrates through the skin
and provides the water necessary to preserve turgidity
and firmness of the skin.
c) HA with high PM that ceases on the surface of the
epidermis and forms an invisible film coating capable of
blocking the evaporation of water, thus counteracting
dehydration.
Conclusion
The use of a second generation hyaluronic acid showed a
higher affinity with the CD44 receptor. The interaction with
these receptors trigger a better cellular response and different
metabolic process in relation to the respective molecular
weights. The in vitro test showed that although, one third of
cutaneous HA is contained in the epidermis where, by binding
to the CD44 receptor, it ensures not only the adhesion
between the individual keratinocytes, but also intervenes in
the regulation of gene expression, the II Generation Hyaluronic
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Acid plays an important and better role than single hyaluronic
acids at different molecular weights both at the fibroblast and
keratinocyte levels.
Although a higher expression of CD44 in tissues treated
with second generation hyaluronic acids has been shown by
Western Blot, this should be further verified with tests in vitro
and, possibly, with tests in vivo that can confirm the greater
anti-wrinkle efficacy of hyaluronic acid. weight between
5-2,500 kDa. It must also be considered that products
containing HA are in continuous development; as proof of
this, cosmetics with cross-linked hyaluronic acid have recently
been marketed. The use of hyaluronic acid in the
dermocosmetic field is acquiring growing interest as a valid
alternative to the much more invasive techniques of aesthetic
medicine.
Role of Sponsor
The funding organisations had no role in the design,
collection, management, analysis and interpretation of the
data; preparation, review or approval of the manuscript; and
decision to submit the manuscript for publication.
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