Preparation, characterization and release properties of
hydrogels based on hyaluronan for pharmaceutical and biomedical
use
Preparation, characterization and release properties of
hydrogels based on hyaluronan for pharmaceutical and biomedical
use
Ferran Roig-Roig1, Conxita Solans2,3, Jordi Esquena2,3, Mª. José
García-Celma1,3
1Pharmacy and Pharmaceutical Technology Dpt., R+D Associated
Unit to CSIC, Faculty of Pharmacy, University of Barcelona (UB),
Joan XXIII s/n, 08028 Barcelona, Spain.
2Institute of Advanced Chemistry of Catalonia (IQAC), Spanish
Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034
Barcelona, Spain
3CIBER de Bioingeniería, Biomateriales y Nanomedicina
(CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain
Corresponding autor: M.J. García-Celma. Email:
[email protected]
ABSTRACT
Hyaluronic Acid (HA) is a natural polysaccharide widely
distributed into human body. Its physico-chemical properties and
the high biocompatibility make it a good candidate for biomedical
and pharmaceutical use. HA based hydrogels have been developed to
be applied as drug delivery systems or as implants for treatment of
joint diseases. The preparation of HA hydrogels has been carried
out by chemical crosslinking using butanediol diglycidyl ether
(BDDE). A model drug, ketoprofen, has been incorporated to HA
hydrogels. The release of ketoprofen from HA hydrogels was studied
by using a new dissolution tester. Homogeneous and transparent
hydrogels consisting of HA chemically crosslinked with high
strength and viscosity were obtained. Its swelling ratio depends on
the crosslinker concentration and pH media. The release studies
showed differences between the release profile of ketoprofen from
swollen and unswollen hydrogels. The characteristics and the
differences in ketoprofen release profiles depending on the
swelling ratio suggest the possibility of obtaining a controlled
release from HA based hydrogels.
KEYWORDS: Crosslinker, Dissolution Tester, Drug Delivery,
Hyaluronan, Hydrogels
1
INTRODUCTION
Hyaluronic acid (HA), also called Hyaluronan, is a
polysaccharide formed by repeating units of disaccharides. It
belongs to the family of glycosaminoglycan consisting of repeating
disaccharides with the general formula: (sugar acid-amino sugar)n.
HA consists of N-acetyl-D-glucosamine (GlcNAc) as amino sugar, and
D-glucuronic acid (GlcA) as sugar acid linked by a β[1-4]
glycosidic bonds. The disaccharides are linked by β[1-3] bonds to
form the HA chain. This polymer is an important component of the
extracellular matrix of connective tissue and is found in human
skin, cartilage, vitreous humour and intra-articular joint fluid.
Therefore, this polymer is an excellent candidate for biomedical
and pharmaceutical uses.1,2 HA plays an important role in cartilage
matrix stabilization, cell proliferation, control of morphogenesis,
cancer metastases, inflammation process and wound healing.3,4 HA
molecules are not recognized as foreign by the immune system, and
their administration do not cause inflammatory or toxic reactions.
Besides its biocompatibility, the physicochemical properties of HA
make it a good candidate in drug delivery field and tissues
engineering and viscosupplementation in treating of
osteoarthritis.5-9
Under normal physiological conditions, HA pervades the surface
of particular tissues and diffuses into the synovial space to
lubricate the joint and to prevent mechanical damage by its
shock-absorbing properties. The viscoelastic properties are
responsible for protecting, lubricating and stabilizing cells and
tissues layers while walking. Due to the depolymerization of HA
chains by reactive radicals that are generated during the
inflammatory process of arthritis, the rheological properties of
endogenous HA alters tremendously. HA may benefit patient with
arthritis by supplementing the lubricating characteristics of
synovial fluids. HA plays a role in tissue reconstruction, because
the first few days of tissue repair, endogenous HA is the
predominant glycosaminoglycan present in wound and form the
template necessary for reconstructions following injury.
HA is degradable in vivo by enzymes such as hyaluronidase
present in human tissues. A useful approach to solve this problem
could be the preparation of hydrogels based on HA chemically
crosslinked that shows an increase in its resistance against
hyaluronidase.10,11 Hydrogels are crosslinked networks of
hydrophilic polymers. They can be classified based on a variety of
characteristics: the nature of side group, physical structure,
method of preparation, and responsiveness to physiologic
environment stimuli such as pH or temperature.12
Hydrogels possess the ability to absorb a large amount of water
and swell, while maintaining their three-dimensional structure. The
equilibrium swelling ratio (i.e. weight ratio of swollen hydrogel
over the dry hydrogel) is an important parameter because influence
in solute diffusion coefficient, surface wettability and mobility,
and the optical and mechanical properties of the
hydrogels.13,14
The hydrogels are very similar to biological tissues in terms of
its physical properties due to their high water content and soft
consistency. Furthermore, the ability of hydrogels to contain
molecules of different size enables the use of these as drug
delivery systems for oral, nasal, buccal, rectal, vaginal, ocular
and parenteral routes of administration.15 The development of HA
hydrogels could be a good approach for improving the conventional
treatments (for example, HA local injections) because HA chemically
crosslinked increase in vivo resistance against hyaluronidase. The
most common chemical crosslinking agents for HA are diglycidyl
ethers,16 divinylsulfone,17,18 and glutaraldehyde.19
In this work butanediol diglycidyl ether (BDDE) was selected
because it is more cytocompatible than other agents. The epoxy
groups of BDDE react with the -OH radicals presents in the
HA.11
Chemically crosslinked Hyaluronan based hydrogels have been
studied as controlled drug delivery systems and the main factors
influencing a model drug release have been identified.
Biocompatibility and biodegradability properties of hyaluronan
hydrogels and the tunable ketoprofen release profiles obtained as a
function of swelling ratio suggest new approaches for the design of
new dosage forms for lipophilic drugs through several routes of
administration.
EXPERIMENTAL
Materials
Hyaluronic acid sodium salt from Streptococcus equi of molecular
1.6 x 106 Da with 97% purity was obtained from Sigma-Aldrich
(Madrid, Spain). The chemical crosslinker butanediol diglycidyl
ether (BDDE) with a molecular weight 202.25g mol-1 with 95% purity
was obtained from Sigma-Aldrich (Madrid, Spain). Hyaluronidase from
bovine testes 801U mg-1 was obtained from Sigma-Aldrich (Madrid,
Spain). Ketoprofen (C16H14O3), (KP), used as a model drug, has been
purchased from Fagron with 99.8% purity. Phosphate buffer solution
pH 7.4, (PBS) was prepared from: KH2PO4 from Fagron Iberica S.A.V,
Na2HPO4 from Probus S.A, NaCl from Acofarma®, and Milli-Q®
deionized water. Cellulose tubular membrane was purchased from
Orange Scientific. Its properties are a 12.000-14.000 nominal MWCO,
and 20μm wall thickness. Mobile phase for HPLC (pH 3.0) was
prepared from 45% of aqueous phase comprising: Citric acid from
Acofarma® with 99.5% purity, NaCl from Acofarma®, NaOH from
Acofarma®, and Milli-Q® deionized water; and 55% of organic phase
acetonitrile obtained from Carlo Erba Reagents, with 99.9% purity.
A commercial gel used as a reference, FastumGelTM (Menarini),
contains 2.5% of ketoprofen and the excipients are: carbomer,
ethanol (96%), diethanolamine, methyl parahydroxybenzoate,
propylparahydroxybenzoate, essence and water. To study the release
of ketoprofen a dissolution tester Elite 8TM manufactured by Hanson
Research Corporation (USA) was used. To determine the concentration
of ketoprofen a HPLC Shimadzu equipped with a column Kromasil®
100-5C18 purchased from Akzo Nobel with dimensions of 250x4.6mm and
pore size of 5μm and a UV detector has been used.
Methods
Preparation of chemically crosslinked hydrogels
For hydrogels preparation, 50mg of sodium hyaluronate were
introduced into test tubes of 12x75mm. A 500μL volume of
crosslinking solution, consisted on Butanediol Diglycidyl Ether
(BDDE) in 0.2M NaOH solution, was added to the test tube with
sodium hyaluronate. The HA and the crosslinking solution were
stirred with a vortex at 2500rpm, and then incubated into an oven
at 40ºC for 8h in order that the crosslinking reaction takes place.
Crosslinker concentration in the hydrogels will be indicated as µL
BDDE/g HA.
Hydrogels swelling ratio measurements
After preparation, hydrogels were swollen to equilibrium in
water (pH 5.5) at room temperature for one week. The hydrogels were
carefully weighted before and after water addition and the swelling
ratio (SR) was calculated according to equation (1)
(1)
WS (mg) is the weight of the swollen hydrogel and WP (mg) is the
weight of HA polymer for each hydrogel.
Swelling ratio in different pH media measurements
The swelling ratio in different pH as a function of time has
been studied. The growth media used was Britton-Robinson buffer. It
consists of a mixture of 0.04M H3BO3, 0.04M H3PO4 and 0.04 M
CH3COOH that has been tight to the desired pH with 0.2M NaOH. The
pHs selected for the swelling ratio study have been: 1.5, 3.0, 7.0,
and 12.0.
Rheological measurements
The rheological properties of the HA hydrogels were quantified
in terms of G’ (elastic modulus), and G’’ (viscous modulus), both
of these parameters indicates the stiffness of the hydrogel
represented with the complex modulus G, as shown in the equation
(2).
(2)
Dynamic viscoelastic measurements were performed with Thermo
Haake Rheo Stress 300 (Department of Chemical Engineering,
Universitat de Barcelona) using parallel serrated plate geometry
with a plate diameter of 35mm and gap size of 1mm. The moduli were
measured with a frequency sweep of 10-0.01Hz at 25ºC.
Samples were swollen in water and all mesurements were performed
in cuadriplicate at room temperature.
Determination of Ketoprofen solubility in PBS and crosslinking
solution
The determination of maximum solubility of ketoprofen in PBS is
needed to ensure sink conditions in the release experiments.
Solubility of ketoprofen in crosslinking solution is needed to
ensure that 2.5%w/w of ketoprofen can be added to the hydrogels,
that is the concentration of ketoprofen in commercial
formulations.
The maximum solubility of ketoprofen in PBS and in crosslinking
solution was determined by adding an excess of the drug to a small
amount of solution, thermostated at 25ºC. The samples were
thoroughly stirred and sonicated. Afterwards they were allowed to
stabilize overnight in a water bath at 25ºC. The supernatant was
analyzed for ketoprofen quantification.
Hyaluronan hydrogels degradation by Hyaluronidase
With the objective to determine the amount of ketoprofen
existing in the hydrogels, hyaluronidase has been used for
hydrogels degradation. Hydrogels were treated with 1000 U mL-1
hyaluronidase at 37ºC in PBS. Degradation was assessed by
completely weigh loss of hydrogels after 48 hours. Then, ketoprofen
in the remaining solution was analyzed. Also, to test the strength
of the hydrogels against hyaluronidase, these were immersed in a
solution of hyaluronidase 1000U mL-1 PBS at 37ºC. We determined the
degradation profile by representing the remaining weight (%) of the
hydrogels as a function of time at different crosslinker
concentrations.
Determination of ketoprofen concentration by HPLC
Ketoprofen was analyzed by HPLC. The chromatographic system
consisted of Shimadzu equipment with a Kromasil® 100-5C18 column,
and a UV detector set at 233nm for ketoprofen determination.
Separation was carried out at room temperature using 55%
acetonitrile and 45% aqueous phase (pH 3.0), as a mobile phase,
with flow rate of 1mL/min, and an injection volume of 20μL. The
ketoprofen retention time was about 7 minutes. This HPLC methode
was previously validated.
Release studies of Ketoprofen from hydrogels
In vitro release studies were carried out in a dissolution
tester. Dissolution testing is well-established and standardized
test in Pharmacopoeias for evaluating solid and semisolid dosage
forms. Dissolution testing allows one to examine the dissolution
behaviour of pharmaceutical dosage forms in vitro in order to
differentiate formulations types and perhaps give an estimate of a
dissolution behavoir in vivo.20,21
The dissolution apparatus used was an Elite 8TM dissolution
tester from Hanson Research Corporation (USA). It consists of 8
dissolution vessels inmersed in a thermostatized bath. Each
dissolution vessel consisted of a 150mL glass vessel with a setup
for semisolid formulations called Ointment cell, as described in
Figure 1. A cellulose membrane was placed in each ointment cell to
separate the hydrogel from the receptor solution. The receptor
solution consisted of 150mL of PBS (pH 7.4). The temperature of the
receptor solution was 37°C. The stirring speed of the paddles in
each dissolution vessel was 25rpm.
Around 550mg of hydrogel were placed in the ointment cell. Then,
150mL of receptor solution (PBS) were placed into the glass vessel.
In order to determine the amount of drug released as a function of
time, 0.5mL of receptor solution has been removed for analysis and
the same amount of virgin PBS solution was replaced. The release
study lasted 24 hours. The first sample was withdrawn after 10
minutes from the beginning of the study. The following samples were
obtained in intervals of 15 minutes for a total period of two
hours. The following samples were obtained in intervals of one hour
until a time of 8 hours was reached. The last samples were obtained
after 22 hours from the beginning of the study and with intervals
of one hour till concluding the 24 hours of the length of the
study.
RESULTS
Hyaluronan hydrogels formation
Hyaluronan hydrogels, using BDDE as chemical crosslinker, have
been obtained. The epoxy groups of BDDE reacts with -OH radicals
presents in the HA polymer. Hydrogels with different crosslinker
concentration have been prepared. The HA hydrogels selected for
further assays due to its concistency was those with 250μL BDDE/g
HA. The pH of the crosslinking solution used was 12.3.
HA hydrogels loaded with 2.5%w/w of ketoprofen were also
prepared. Ketoprofen was added to the crosslinking solution just
before hydrogel formation. The hydrogels were obtained in the same
manner described previously, but the crosslinking solution
contained 2.5%w/w of ketoprofen.
Characterization of hyaluronan hydrogels
Crosslinked HA hydrogels exhibited an ability to absorb a large
amount of water and swell (Fig. 2). The swelling ratio as a
function of crosslinker concentration and temperature incubation
was determined (Fig. 3). The ability to absorb water depends on the
crosslinker concentration and temperature incubation. The
crosslinking reaction shows higher efficiency at 40oC, because the
swelling of the hydrogels was lower at 40ºC than at room
temperature for a given crosslinker concentration, specially at
BDDE concentration lower than 500µL/g HA. Also, the relationship of
swelling ratio depending on the pH media was studied. Figure 4 show
that the swelling ratio for HA hydrogels is higher for a value of
pH above of pKa value of the polymer (pKa = 3.0). Rheological
characterisation of the chemically crosslinked hydrogels was
performed to know the hydrogel stiffness. All rheological
measurements were carried out on swollen hydrogels. The HA
hydrogels with a crosslinker concentration of 250µL BDDE/g HA
exhibit a storage modulus about 1500Pa and their loss modulus about
300Pa (Fig. 5).
Degradation of hyaluronan hydrogels with hyaluronidase
The resistance against hyaluronidase increase with the
concentration of crosslinker (Fig. 6). For a crosslinker
concentration of 250μL BDDE/g HA the complete degradation of
hydrogels occurs after 48 hours. However, for a concentration of
500μL BDDE/g HA the total degradation is estimated at around three
days. This method was also used to ensure a homogeneous
concentration of drug in the hydrogels, prerequisite to the release
study.
Release studies of ketoprofen from hydrogels
Figure 7 shows that the release profiles of ketoprofen from
unswollen hydrogels, swollen hydrogels, and FastumGelTM, took place
with no lag-time but revealed slight differences. Both unswollen
and swollen hydrogels showed a fast initial release which slows
down progressively after several hours, but the maximum ketoprofen
released arose around 100% with unswollen hydrogels and 85% with
swollen hydrogels.
In order to determine the influence of the cellulose membrane in
the release profile, a release study without membrane was carried
out. The release profile was compared with that obtained with
membrane (Fig. 8). The study was carried out only for unswollen
hydrogels due to the impossibility to guarantee the integrity of
the swollen hydrogels during the experiment.
DISCUSSION
Biocompatible hydrogels based on HA have been developed and
several key factors of the polymerization process have been
identified, such as BDDE concentration, temperature and time
incubation. The stability of the hydrogels was determined by visual
observation of appearance of phase separation and/or fluency at
room temperature. It is important to remark that the stirring
process was carried out immediately after the mixture of
crosslinker solution and HA. Otherwise, the jellification is not
homogeneous and it occurs in different phases, and a slightly
viscous fluid was obtained after incubation in the oven. It is also
important to note that if the mixture of HA with crosslinking
solution are kept in the oven, more time or temperature that the
indicated previously, the result was a very fluid yellow solution,
that cannot be considered as a hydrogel. This result is due to the
increased exposure to heat, which destroys the chemical
crosslink.
In order to determine the equilibrium swelling ratio of the
hydrogels, these were immersed in a large amount of water in a
Petri dish. Their affinity to absorb water is attributed to the
presence of hydrophilic groups such as -OH. Due to contribution of
this group and domains in the network, the polymer is thus hydrated
to different degrees, depending on the nature of the aqueous
environment and polymer composition. The decrease of hydrogels
swelling based on crosslinker concentration may be due to the
stiffness of hydrogel and the presence of free -OH groups of
hyaluronan. The higher concentration of crosslinker results on the
presence of less free -OH groups in hyaluronan because the epoxy
groups of crosslinker reacts with -OH. This results in less
hydrogen bridges between -OH of hyaluronan and water, and therefore
less swelling. Swelling ratio depends also on the pH media. When
the pH is above the pKa value of Hyaluronan, this is ionized. In
these circumstances the stiffness of the polymer network is lower
so that water can penetrate more easily. Therefore the swelling is
higher.
It is interesting to note that once the hydrogel has been
formed, the shape remains if they will submit to swelling
processes. This result may be interesting in the field of tissue
engineering, because the prosthesis can be implanted within the
body with a small incision and then in vivo make it grow until it
reaches the desired shape and size of the defective tissue. The
swollen hydrogels exhibits high stability. Hydrogels have been left
in a closed Petri dish for more than 2 years. After this time their
physical properties remain unchanged.
In the present work, the release behaviour of ketoprofen
incorporated to HA hydrogels has been studied in order to
understand the factors influencing the release of this model drug
from unswollen and swollen hydrogels. HA unswollen hydrogels with
2.5%w/w of ketoprofen were prepared. The amount to be incorporated
in the hydrogels was chosen taking into account the solubility of
the drug in the crosslinking solution, the therapeutic dose and
also the accomplishment of sink conditions in the receptor solution
during the release experiments. Some of the HA hydrogels with
2.5%w/w of ketoprofen, were immersed in water to obtain swollen
hydrogels. Then, the concentration of ketoprofen in swollen
hydrogels (0.2%w/w) was lower than in unswollen hydrogels. To
determine the amount of ketoprofen in the samples an HPLC method
has been used. The original setup used, designed specifically for
semisolid formulations, allowed to determine the amount of
ketoprofen that can be released from HA hydrogels to a PBS receptor
solution. The release of unswollen and swollen hydrogels show
differences; while about 100% of drug can be released from
unswollen hydrogels after 24h, around 85% is released from swollen
hydrogels. It seems that swollen hydrogels, a fraction of drug is
retained into the hydrogel after 24h, while with unswollen
hydrogels, the tendency to absorption of water by the hydrogels
could facilitate the complete release of ketoprofen from the
formulations. Quantification of ketoprofen in the hydrogels has
been determined by using hyaluronidase for hydrogels degradation.
The concentration of hyaluronidase employed was higher than the
concentration of this enzime in physiologic conditions.
Dialysis membrane can influence the release behavior of
molecules,22,23 in this study no effect would be expected due to
the cellulose membrane, as the molecular weight cut-off (MWCO) of
the membrane was far above the molecular weight of ketoprofen.
However the passage of the molecule through the membrane seems to
be a rate-limiting step, both for unswollen and swollen hydrogels.
The effect of the membrane has been a delay in the ketoprofen
release. So while the unswollen hydrogel reach their maximum
release after 3 hours, with membrane it was after 20 hours.
Overestimation of the concentration of ketoprofen has been
considered, because part of the receptor solution has been absorbed
by the hydrogel for swelling.
CONCLUSIONS
Biocompatible hydrogels based on HA have been developed and
several key factors of the polymerization process have been
identified, such as HA and BDDE concentration, temperature and time
incubation.
The swelling ratio of hydrogels depends on the crosslinker
concentration and the pH media. It was observed that after swelling
the hydrogels remains with their original shape. This fact and the
long stability of the hydrogels make them interesting for use as
implants.
Biocompatibility and biodegradability properties of hyaluronan
hydrogels, their characteristic properties and the tunable
ketoprofen release profiles obtained as a function of the swelling
ratio suggest new approaches for the design and development of
dosage forms based on hyaluronan hydrogels for lipophilic drugs
delivery through several routes of administration.
ACKNOWLEDGEMENTS
The authors acknowledge financial support of the Ministerio de
Ciencia e Innovación with the project CTQ
2011-29336-C03-03/PPQ.
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FIGURES
(Figure 1. Schematic diagram of the dissolution vessel. (i)
adapter, (ii) dissolution vessels (150mL glass vessel flat-bottom),
(iii) paddle, (iv) ointment cell. Right, the different parts of
ointment cell are represented in detail.)
(Figure 2. a) Unswollen hydrogel with a crosslinker
concentration of 250μL BDDE/g HA, b) Swollen hydrogel after 48h
immersed in water.)
(Figure 3. The ability to absorb water depends on the
crosslinker concentration and temperature incubation. )
(Figure 4. SR of hydrogels with 250µL BDDE/g HA vs time for
different pH media.)
(Figure 5. Viscoelasticity of the HA hydrogels chemically
crosslinked with BDDE. The storage modulus (G’) and the loss
modulus (G’’) are plotted as a function of frequency. Data points
are the mean ± standard deviation of four measurements.)
(Figure 6. Degradation of HA hydrogels with different
concentration of crosslinker, BDDE, as a function of time.)
(Figure 7. Comparative profile of cumulative release of
ketoprofen as a function of time from unswollen hydrogels, swollen
hydrogels and FastumGelTM. These results have been obtained by
using a cellulose membrane in the ointment cell.)
(Figure 8. Cumulative release of ketoprofen as a function of
time from unswollen hydrogels with and without membrane.)
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