International Journal of Biological Macromolecules 123 (2019) 1204–1210

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Evaluation of modified hyaluronic acid in terms of rheology, enzymatic

degradation and mucoadhesion
Flavia Laffleur a,b,⁎, Kesinee Netsomboon a,c, Liliana Erman a, Alexandra Partenhauser a
a
University of Innsbruck, Institute of Pharmacy, Department of Pharmaceutical Technology, Innrain 20-82, 6020 Innsbruck, Austria
b
Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research at MIT, Langer Lab, 77 Massachussets Ave, Cambridge, MA 02139, USA
c
Division of Pharmaceutical Sciences, Faculty of Pharmacy, Thammasat University, Rangsit campus, Phahonyothin Rd., Khlong Luang, Pathumthani 12120, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: This study aimed to investigate the properties of modified hyaluronic acid in terms of rheological prop-
Received 31 July 2018 erties, enzymatic degradation and mucoadhesiveness.
Received in revised form 30 October 2018 Methods: Hyaluronic acid (HA) was chemically modified with sulfhydryl ligand cysteine ethyl ester (C) in order
Accepted 18 November 2018 to immobilize sulfhydryl groups on the polymeric backbone. MTT assay was performed to evaluate the safety of
Available online 19 November 2018
hyaluronic acid-cysteine ethyl ester (HAC). Rheological and enzymatic degradation studies were accomplished
by preparing hydrogels of HA and HAC, respectively. HA served as control. Enzymes such as lysozyme, amylase
Keywords:
Amylase
and hyaluronidase were chosen to perform degradation studies. To study mucoadhesiveness, hydrogels of HA
Hyaluronic acid and HAC, respectively, were mixed with mucus and evaluated by rheology.
Hyaluronidase Results: MTT assay indicated no toxicity at all. The rheological assay showed 2.2-fold increase in gelling properties
Lysozyme in case of HAC in comparison to HA. Furthermore, it could be shown that HAC was degraded by amylase to a
Mucoadhesion lesser extent of 11.5-fold than HA. After 2 h, HA showed a higher degradation by lysozyme with 67.97% than
Rheology HAC. Adhesion studies exhibited a 2.17-fold higher mucoadhesion of HAC with mucus compared to HA.
Conclusion: These results will open the door for high efficient drug delivery systems based on hydrogels for mu-
cosal application.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction
Hyaluronic acid (HA) is a well-investigated biocompatible and bio-
degradable natural polymer [1]. HA consists of alternating disaccharide
units of D-glucuronic acid and N-acetyl-D-glucosamine with beta 1,4-
inter-glycosidic linkage [2]. HA exhibits distinctive properties of visco-
elasticity and its lubrication function is tremendous [3]. Therefore, HA
with its various functions and physicochemical properties is a well-
established representative in the pharmaceutical and medical research
field [4]. HA is widely used in the cosmetic field, tissue engineering as
well as drug delivery [5]. The latter is intending in developing novel de-
livery systems for various applications. The non-invasive application is
most favorable for patients. Benefits associated with non-invasive path-
ways are painlessness, patient-friendliness and low costs. Non-invasive
delivery comprises oral, dermal and mucosal delivery. Mucosal delivery
is attractive as mucosa is found all over our body cavities such as eyes,
mouth, and respiratory, reproductive as well as gastrointestinal tract
[6]. But targeting the mucosa, mucoadhesion is a key challenge as adhe-
sion is a complex phenomenon and an interplay of many factors [7].
⁎ Corresponding author at: University of Innsbruck, Institute of Pharmacy, Department
of Pharmaceutical Technology, Innrain 20-82, 6020 Innsbruck, Austria.
E-mail addresses: Flavia.Laffleur@uibk.ac.at, Laffleur@mit.edu (F. Laffleur).
Water up take capacity, in situ gelling properties and prolonged resi-
dence time are the main achievements when mucoadhesion is aimed.
One major drawback of current mucosal formulations in form of
hydrogels are their low mucoadhesion resulting in frequent application
[8,9]. But with a prolonged residence time on the mucosal site, this
application frequency can be reduced, leading to improved patient
compliance.
Following that, it was aimed to modify the well-known HA in order
to improve its mucoadhesiveness and water uptake capacity. The nov-
elty in this study, lies in comparative studies of modified HA highlight-
ing rheological properties in terms of mucoadhesion.
Enzymatic degradation was investigated by different enzymes; rep-
resentatives of our body fluids, e.g. amylase and lysozyme were chosen
to determine the degradation of the polymers viscosity.
2. Materials and methods
2.1. Materials
Hyaluronic acid sodium salt from Streptococcus equi (1.5 to 1.8
∗ 106 Da), L-cysteine ethyl ester hydrochloride, N-hydroxysuccinimide
(NHS) and 1–ethyl–3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDAC), disodiumhydrogenphosphate (Na2HPO4), potassium

https://doi.org/10.1016/j.ijbiomac.2018.11.186
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 F. Laffleur et al. / International Journal of Biological Macromolecules 123 (2019) 1204–1210
phosphate monobasic (KH2PO4), potassium phosphate dibasic
(K2HPO4), lysozyme, amylase, hyaluronidase, iodine, sodium borohy-
dride (NaBH4), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent),
L-cysteine HCl, Hanks' balanced salt solution (HBSS), Triton® X-100
and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) were purchased from Sigma-Aldrich (Vienna, Austria). All
other chemicals were of analytical grade and obtained from commercial
sources.
2.2. Methods
2.2.1. Synthesis of modified hyaluronic acid cysteine ethyl ester
The modification of the polymeric backbone was previously pub-
lished by Laffleur et al. [10]. Briefly, a 0.4% (m/V) solution of hyaluronic
acid (HA) in demineralized water was prepared and the solution's pH
was set to 5.5 with 1 M HCl. In the second step, EDAC and NHS in a
final concentration of 50 mM, respectively, were added to HA solution.
In the following step, L-cysteine ethyl ester (C) was subjoined to the
polymer and stirred for 6 h at pH 6 in the dark. For purification purpose,
modified HAC was added into dialysis tubing (Cellulose hydrate, cut-off
12.000 Da, Carl Roth, Karlsruhe, Germany) and dialyzed against
demineralized water containing 1% NaCl. HA served as controls in the
study. After purification step, HAC and HA were lyophilized (Christ,
Fig. 1. Schematic illustration of the synthesis of modified hyaluronic acid (HAC). Hyaluronic acid was covalently attached with L-cysteine ethyl ester by amide bond formation.
1205
Gamma 1-16 LSC, Osterode am Harz, Germany) and kept stored at 4
°C until further use.
2.2.2. Characterization of HAC via thiol group detection, FT-IR and TGA
2.2.2.1. Ellman's assay and disulphide bond assay. The colorimetric assay
was performed to determine the modification on the polymeric back-
bone. For this, colorless DTNB reacted with thiolate anions in thiol-
disulphide exchange by forming new mixed disulphides bonds. This re-
action results in releasing of the yellow TNB (5-thio-2-nitrobenzoic
acid) which can be detected spectrophotometrically detection at
450 nm. Furthermore, disulphide bond assay was performed in order
to quantify the amount of disulphide bonds. After preparing Tris buffer
consisting of 0.788 g Tris-HCl dissolved in 100 mL purified water and
adjusted to ph 7.6, 0.5 mg of the HA and HAC were dissolved in 500 μL
Tris buffer. 1000 μL of freshly prepared 4% NaBH4-solution was added
to each of the sample in order to reduce the existing disulphide bonds
and incubated for 1 h at 37 °C. Afterwards the reaction was stopped
by addition of 200 μL of 5 M HCl, followed by a neutralization step
with 1 mL phosphate-buffer (1 M, ph 8). Immediately, 100 μL Ellman's
reagent (4 mg DTNB dissolved in 1 mL Ellman's buffer) was added to
each of the samples, the calibration reference solutions and the blank-
test followed by incubation in the dark at room temperature for
90 min and spectrophotometrical detection at 450 nm [11].
1206 F. Laffleur et al. / International Journal of Biological Macromolecules 123 (2019) 1204–1210

Fig. 2. IR-Spectra of modified hyaluronic acid (black) and corresponding unmodified hyaluronic acid (dotted).

2.2.2.2. Attenuated-total-reflectance Fourier transform infrared spectros-
copy (ATR-FTIR). A second method, namely attenuated-total-
reflectance Fourier transform infrared spectroscopy FT-IR analysis, was
used to analyze the synthesis success. Perkin Elmer Spectrum 100
ATR-FTIR spectrometer with a diamond crystal (Perkin Elmer, Waltham,
USA) was combined with a Spectrum software version 6.3.1.0134
(Perkin Elmer, Waltham, USA). The measurements of six subsamples
were taken 22 °C. The scan was recorded in a range from 4000 cm−1
to 600 cm−1 and a resolution of 4 cm−1, respectively [12].
hyaluronic acid-cysteine ethyl ester (HAC) 0.5% (w/v), Hank's balance
buffer as negative control and Triton™ X-100 2% (v/v) as positive con-
trol for 12 h, respectively. Cell viability was measured at 570 nm wave
length and calculated based on the following equation [13]:
cell viability ½% ¼ Average absorbance value of each sample
=Average absorbance value of negative control  100

2.2.3. Cell viability investigations 2.2.4. Rheological investigation

In order to determine the safety of modified HAC, MTT assay, was
used. MTT is a colorimetric assay based on mitochondrial activity. Cyto-
toxicity screening of testing substances was performed in Caco-2 cells
(European Collection of Cell Cultures, UK, passage number 30–40)
using MTT assay. Briefly, Caco-2 cells were seeded in 96-well plate at
density of 2 × 104 cells/well. After 48 h of incubation, cells were washed
twice with pre-warmed HBSS (pH 7.4). Therefore, colon carcinoma cell
(Caco-2) were treated with hyaluronic acid (HA) in a 0.5% (w/v),
2.2.4.1. Evaluation of gelling properties. Gelling properties of modified
and unmodified HA were investigated using a plate-plate combination
rheometer (Haake Mars Rheometer, 379-0200, Thermo Electron
GmbH, Karlsruhe, Germany; Rotor: PP35, D = 35 mm) based on a tech-
nique previously published by our research group [14]. During the ex-
periment, shear stress rate was set to a range of 0.5–1 Pa, with a
frequency of 1 Hz at a temperature of 37 ± 1 °C and the gap between

Fig. 3. Cell viability results of MTT assay on Caco-2 cells. The cells were treated with unmodified HA and HAC for 12 h incubation of the final concentration 0.5%, respectively. Depicted
values are means of at least three experiments ± SD.
 F. Laffleur et al. / International Journal of Biological Macromolecules 123 (2019) 1204–1210 1207

two plates was chosen about 0.5 mm. Mimicking physiological condi-
tions, rheological study was performed at pH 6.8 with 0.1 M phosphate
buffer. Therefore, 1% (w/v) test polymer solution and 500 μL of sample
per each measurement were prepared and incubated at room tempera-
ture, respectively. Each measurement was performed in triplicate after
0.5 h, 1 h, 2 h and 16 h, respectively.

2.2.4.2. Rheological assay with iodine. Iodine, as oxidizing agent with

the modified and unmodified polymer, respectively, was used to de-
termine the viscoelastic characteristics [15]. In brief, 800 μL of each
1% polymer sample was admixed with 400 μL of 10% w/v iodine solu-
tion in ethanol. Afterwards, samples were incubated at room tem-
perature for 2 h and 4 h, respectively. Dynamic oscillatory tests
within the linear viscoelasticity range were conducted with 1 mL al-
iquots of the samples in triplicate. Unmodified hyaluronic acid
served as control.
2.2.4.3. Enzymatic degradation studies with lysozyme, amylase and hyal-
uronidase. Physiological enzymes such as lysozyme, amylase and hyal-
uronidase were examined in terms of degrading activity of hyaluronic
acid and modified one, respectively. The enzymatic degradability of
these polymers was investigated by viscosity measurements with a
plate-plate viscometer [16]. In brief, HA and HAC 2% (w/v) mixtures
were prepared as stock mixtures, respectively. Amylase with a pH opti-
mum of 6.9, was dissolved in 20 mM phosphate buffer (pH 6.9). Lyso-
zyme shows its pH optimum at pH 6.2. Therefore, a 0.1 M phosphate
buffer (pH 6.2) was prepared. The third enzyme hyaluronidase was dis-
solved at pH 5.35 in 300 mM phosphate buffer (pH 5.35). The investi-
gated enzymes, amylase and lysozyme in a final concentration of 1.5
mg/mL and hyaluronidase 0.1 U, were added to each sample. In order
to measure the viscosity, 500 μL aliquots were taken at predetermined
time points of 0 h, 0.5 h, 1 h and 2 h. All experiments were performed
in triplicate. The degree of viscosity loss was calculated by using follow-
ing equation:
n1 ¼ ½ðn0−ntÞ=n0  100
where percent n1: percent viscosity lost after time t; nt: final viscos-
ity at time t and n0: initial viscosity of the gel.
Fig. 4. Rheological measurements. Increase in viscosity of a 1% (m/v) solution HAC (grey)
and HA (white), respectively, at physiological conditions, at 37 °C. Data are means ± SD of
at least three experiments.
3. Results and discussion
3.1. Synthesis and characterization of modified hyaluronic acid
As shown in Fig. 1, L-cysteine ethyl ester was covalently attached to
the polymeric backbone of hyaluronic acid via amide bond formation.
The immobilized thiol groups of the ligand on the polymeric backbone
were determined via Ellman's reagent resulting in 250.66 ± 0.39 μmol
per gram of hyaluronic acid. According to the disulfide bond assay, HA
showed no disulfide groups at all on the polymeric backbone in compar-
ison to HAC, leading to a success in the synthesis of HAC. The obtained
HAC powder was white colored and fibrous structure. Furthermore,
Fig. 2 showed the succeeded synthesis of HAC representing the specific
peaks such as 3161 cm−1 and 3159 cm−1 representing the C\\H stretch
as well as the N\\H bend and\\C_C\\were shown at 1653–1619 cm−1.
The amide bond at 1735–1700 cm−1 which was initial for the covalent
attachment to the polymeric backbone could be detected for HAC but
not for HA [18]. Taking these findings in consideration, the succeeded
synthesis could be proved.
3.2. Safety investigation

2.2.5. Mucoadhesive assay In vitro toxicity studies were performed on Caco-2 cell line by incu-
To determine the viscoelastic properties of polymer-mucus mix- bating cells with unmodified hyaluronic acid and HAC in concentrations
tures, a plate-plate viscometer was used. In brief, mucus collection of 0.5% for 12 h to determine their effect on cell viability, respectively.
was performed on fresh porcine intestine, which was carefully with Findings were depicted in Fig. 3. Owing to the results, no toxic effect
physiological saline. Following that washing process, mucus was
scrapped off the mucus layer from underlying tissue. Then, 1.0 mL of
each 1% polymer solution was admixed to 1.0 mL fresh intestinal
mucus and incubated at 37 °C. After predetermined time points of 2
and 4 h, viscoelastic properties were measured by adding 500 μL of
each mucus-polymer mixture onto the viscometer. Rheological investi-
gations were performed at a frequency of 1 Hz and a sheer stress range
of 0.5–500 Pa. Mucus samples without particle suspension served as ref-
erences. The rheological parameters were calculated using the following
formula:

Δη ¼ ηmix−ηmuc

where ηmix is the apparent viscosity of the polymer-mucin mixture

(Pa s) and nmuc is the apparent viscosity of each [17].

2.2.6. Statistical data analysis

Statistical data analysis was performed using the Student's t-test
with p b 0.05 as the minimal level of significance. Results are expressed Fig. 5. Effect of 10% w/v iodine solution on the viscosity determined at 37 °C for HAC (grey)
as means of at least three experiments ± SD. conjugate and HA (white) as control, respectively (means ± SD).
1208 F. Laffleur et al. / International Journal of Biological Macromolecules 123 (2019) 1204–1210

of all polymers was revealed 12 h after exposure. Triton® X-100 as pos-
itive control exhibited severe damage to the cells. After 12 h incubation
HA and HAC showed cell viability of 97.26 ± 3.22% and 93.98 ± 4.62%,
respectively. In conclusion, these results showed no harmful potential
to the cells, while HAC slightly reduced viable cells under the same ex-
perimental conditions.
3.3. Rheology investigations in terms of in situ gelation, iodine as oxidizing
agent and mucoadhesiveness
3.3.1. In situ gelation
As shown in Fig. 4, viscosity measurements were conducted in order
to study the comparison between HA and HAC in water and their ability
to build the intramolecular network of the hydrogel scaffold. Results re-
vealed that HAC showed per se due to the cross linking of the thiol
groups to disulfide bonds 2.2-fold increase compared to unmodified
HA. These results were in good agreement with the performed
disulphide bond assay proving that HA lacks of free and immobilized
thiol groups on its polymeric backbone. The in situ gelling effects are
caused due to the water uptake capacity of HA.
3.3.2. Iodine as oxidizing agent
The in situ gelling process for HAC and HA was accelerated by
addition of iodine solution. Iodine acts as oxidizing agents which
strengthens the intramolecular structure of the hydrogels. As illustrated
in Fig. 5, the viscosity of HAC was 5.1-fold increase in comparison to un-
modified polymer HA. Within this experiment, the gelling properties of
modified HAC with regard to their behavior after iodine incubation
were evaluated. Viscosity studies underpin the assumption, that an in-
crease in dynamic viscosity as well as a phase-shift strongly depends
on the total amount of free thiol groups covalently attached to the poly-
mer. The assumption that the degree of modification is in connection to
the viscoelastic properties has already been described previously [19].
This enhanced viscosity property is very important for a sustained

Fig. 6. A–F. Compendium of viscosity as well as enzymatic degradation. Effect of viscosity of hyaluronic derivate represented by white bars (HA) and grey bars (HAC) with amylase (A),
lysozyme (C) and hyaluronidase (E). Effect of polymeric modification on viscosity by enzymatic degradation with amylase (B) (cross represents HA and squares represents HAC), lysozyme
(D) (square represents HA and triangle represents HAC) and hyaluronidase (F) (square represents HA and triangle represents HAC). Indicated values of HA and HAC are means (±SD) of at
least three experiments.
 F. Laffleur et al. / International Journal of Biological Macromolecules 123 (2019) 1204–1210
delivery on mucosal membranes assuring not only contact with this
mucosal layer, but also prolonged residence time at the site of action
plays an prominent role.
3.3.3. Enzymatic degradation
Hydrogels of unmodified and modified hyaluronic acid were pre-
pared in order to study enzymatic degradation. Focusing on mucosal de-
livery, we studied amylase and lysozyme as representatives of several
body fluids such as tear fluid and salvia [20]. Viscosimetry was described
by Lee et al. as a very sensitive method to study enzymatic degradation
[21]. The viscosity was measured after certain incubation time of 2 h. In
Fig. 6A, it could be shown that HAC is exhibiting a mainly constant vis-
cosity in the range of 12–13 Pa s whereas HA showed decrease in viscos-
ity down to 1.3 Pas. Regarding, the viscosity with the incubation of
lysozyme, it could be detected that HA was to a 2.2-fold lesser pro-
nounced in comparison with HAC as shown in Fig. 6C. Amylase which
cleaves alpha 1, 4 linkages mainly known for starch and lysozyme
which cleaves β-1, 4 linkages are regarded to be able to degrade many
kinds of polysaccharides. From the results obtained it was found that
modified hyaluronic acid showed less susceptibility towards enzymatic
degradation as compared to unmodified one as shown in Fig. 6B for am-
ylase and Fig. 6D for lysozyme, respectively. One possible assumption
could be the interaction between the thiol groups and the aspartate
and glutamate of amylase. According to Hahn et al. hyaluronidase
(HAse) was used in order to prove the degradation of HA [22]. Unfortu-
nately, HA content decreases with age. The turnover of HA depends on
location and has an approximate half-life in the skin of only one day
or less. HA is degraded into smaller HA fragments by HAse via hydrolysis
of HA in the disaccharides at hexosaminidic β (1–4) linkages [23]. De-
pending on the cleavage site, HAC size results in either oligosaccharides
or other larger polymers. The viscosity was found to be lower in case of
HA compared to HAC according to the effect of HAse as shown in Fig. 6E.
HA was found to be rapid degraded by HAse within 0.5 h at 70% of loss in
degradation whereas HAC exhibited no loss in degradation at time point
0.5 h and finally showed a loss of 20% after 2 h of experiment as shown
in Fig. 6F. Taking the findings of enzymatic study in consideration, we
now know that the degree of HA modification impacts the sensitivity
of hydrogels due to enzymatic degradation. Furthermore, from the re-
sults we concluded, that the degradation rate of HA hydrogels is control-
lable by immobilizing functional groups to the polymeric backbones.
The determined viscosity loss resulted due to splitting the polymer net-
work in different sized fragments. Taken together, degradation studies
of unmodified and modified hyaluronic acid revealed significant differ-
ences in their biodegradability and susceptibility towards examined en-
zymes. These facts might be explained by the interactions between thiol
moieties of the modified HA as well as sterical topography of the HA
Fig. 7. Rheological comparison regarding mucoadhesion of HA and HAC, respectively. Data
is shown in mucus alone (black bars), mucus with HA (white bars) and mucus with HAC
(grey bars) Indicated values are means ± SD of at least three experiments.
1209
attached by the thiol agent might, however, represent another possibil-
ity for the lower degradation [24].
3.3.4. Mucoadhesiveness
Mucoadhesion is driven by key factors such as polymer entangle-
ments, inter-diffusion and chemical interactions. This phenomenon
can be observed while adding mucus to a polymeric hydrogel. The vis-
cosity increase of the polymer/mucus mixtures is directly in correlation
with the mucoadhesion of the concerned polymers [25]. Therefore, a so-
called rheological assay was performed to evaluate the force of interac-
tion involved in mucoadhesion. As illustrated in Fig. 7, rheological
synergy measurement was performed to evaluate the interaction be-
tween HA and HAC, respectively, with mucus.
The results demonstrated low viscous properties in the absence and
presence of mucin in case of HA, whereas the modified HAC provided sig-
nificantly pronounced viscosity values in combination with mucus. After
2 h, HA exhibited a value of 7 Pas whereas HAC showed 16 Pas of viscosity.
During 4 h of incubation with the mucus, HAC revealed 29.03 Pas viscos-
ity. HAC showed a 2.2-fold improved mucoadhesiveness. The modifica-
tion bears covalent attachment of cysteine residues to thiol groups of
cysteine rich substructure of mucus glycoproteins, leading to stronger co-
valent bonds in comparison to relatively weak hydrogen bonds in case of
HA.
4. Conclusion
This study proved that hyaluronic acid, being well established in food
and pharmaceutical industry, improved its gelling and mucoadhesive
properties according to the modification with cysteine ethyl ester. So
far, we found out, that due to modification of the polymeric backbone
with immobilized sulfhydryl groups enzymatic degradation was hindered
by stereo chemical hindrance. Furthermore, mucoadhesive properties
were enhanced as proved by the rheological measurements with freshly
collected mucus. Taken these findings in considerations, this might
bring us to the next level in medical and pharmaceutical research fields,
where high gelation and auspicious mucoadhesion is desired.
Acknowledgement
The authors wish to thank diploma student Liliana Erman for her
contribution to this work.
Declaration of interest
The authors have no other relevant affiliations or financial involve-
ment with any organization or entity with a financial interest in or fi-
nancial conflict with the subject matter or materials discussed in the
manuscript apart from those disclosed. No writing assistance was uti-
lized in the production of this manuscript. This research did not receive
any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
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