Chemical modification of hyaluronic acid for intraoral application

Article abstract of DOI:10.1002/jps.24060

This study was aimed to investigate chemical preactivated thiomers for their potential use in mucosal drug delivery. Thiomers - thiolated polymers - are mucoadhesive polymers with sulfhydryl group-bearing side chains. Thiomers are synthesized by covalent attachment of low molecular mass compounds bearing sulfhydryl group to the polymeric backbone of well-established polymers. Hyaluronic acid-cysteine ethyl ester-mercaptonicotinamide conjugates (HA-CYS-MNA) were synthesized by the oxidative S-S coupling of HA-CYS with 6-MNA. Conjugates were compressed into test discs to investigate cohesive properties, cytotoxicity assays, and mucoadhesion studies. Because of the immobilization of MNA, the HA-CYS-MNA conjugates exhibit comparatively higher swelling properties and cohesive properties corresponding unmodified HA. On the rotating cylinder, discs based on HA-CYS-MNA conjugates displayed fourfold improved mucoadhesion time compared with thiolated polymers. Tensile study results were found in good agreement with rotating cylinder results. Moreover, preactivated thiomers showed higher stability. All polymers were found nontoxic over Caco-2 cells. On the basis of achieved results, the preactivated thiomeric therapeutic agent seems to represent a promising generation of mucoadhesive polymers that are safe to use for a prolonged residence time to target the mucosa requested for intraoral application. 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2414-2423, 2014

Full text of DOI:10.1002/jps.24060

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Chemical Modification of Hyaluronic Acid for Intraoral Application
1
1
1
2
¨
FLAVIA LAFFLEUR, JULIA ROGGLA, MUNEEB AHMAD IDREES, JULIA GRIESSINGER
1Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck,
Innsbruck 6020, Austria
2
ThioMatrix GmbH, Research Center Innsbruck, Innsbruck 6020, Austria
Received 15 April 2014; revised 14 May 2014; accepted 29 May 2014
Published online 1 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24060
ABSTRACT: This study was aimed to investigate chemical preactivated thiomers for their potential use in mucosal drug delivery. Thiomers—
thiolated polymers—are mucoadhesive polymers with sulfhydryl group-bearing side chains. Thiomers are synthesized by covalent attach-
ment of low molecular mass compounds bearing sulfhydryl group to the polymeric backbone of well-established polymers. Hyaluronic
acid–cysteine ethyl ester–mercaptonicotinamide conjugates (HA–CYS–MNA) were synthesized by the oxidative S–S coupling of HA–CYS
with 6-MNA. Conjugates were compressed into test discs to investigate cohesive properties, cytotoxicity assays, and mucoadhesion stud-
ies. Because of the immobilization of MNA, the HA–CYS-MNA conjugates exhibit comparatively higher swelling properties and cohesive
properties corresponding unmodified HA. On the rotating cylinder, discs based on HA–CYS–MNA conjugates displayed fourfold improved
mucoadhesion time compared with thiolated polymers. Tensile study results were found in good agreement with rotating cylinder results.
Moreover, preactivated thiomers showed higher stability. All polymers were found nontoxic over Caco-2 cells. On the basis of achieved
results, the preactivated thiomeric therapeutic agent seems to represent a promising generation of mucoadhesive polymers that are safe
to use for a prolonged residence time to target the mucosa requested for intraoral application. ꢀC 2014 Wiley Periodicals, Inc. and the
American Pharmacists Association J Pharm Sci 103:2414–2423, 2014
Keywords: thiomers; hyaluronic acid; mercaptonicotinamide; preactivated thiomers; intraoral application; biomaterials; buccal; mucosal
delivery; polymers; polymeric drug carrier
and $-1-4 glycosides bonds.10,11 HA was investigated as a pos-
sible intraoral drug delivery agent.
According to this, it is the aim of this study to evaluate the
impact of thiolated HA and especially preactivated HA on mu-
coadhesion for their potential as intraoral therapeutic agents.
INTRODUCTION
Currently, the employ of multifunctional polymeric excipients
for drug delivery is considerably researched. Promising candi-
dates among multifunctional polymers are thiolated polymers
1–3
or so-called thiomers. Because of the immobilization of thiol
groups on already well-established polymeric excipients such as
polyacrylates, hyaluronic acid (HA), or chitosan, features such
MATERIALS AND METHODS
Materials
4
5
6
as mucoadhesive, in situ gelling, efflux pump inhibiting, and
7
permeation-enhancing properties are strongly improved.
Nevertheless, besides all these advantages, shortcomings of
thiomers having been developed so far are their instability to-
ward oxidation and pH-dependent reactivity. The reactive form
of thiomers is the thiolate anion, whereas the pKa of alkyl
thiols is in the range of 8–10, meaning thiomers will be most
reactive in a pH range slightly above the physiological pH.
As a consequence, their full potential is not extended in mu-
cosal delivery. To overwhelm these limitations, preactivated
thiomers bearing an additional ligand with a heteroaromatic
structure reacting in a pH-independent manner were designed Synthesis of HA–L-Cysteine Ethyl Ester Conjugates
Hyaluronic acid sodium salt from Streptococcus equi (1.5–
6
1
.8 × 10 Da), 6-chloronicotinamide, thiourea, hydrogen
peroxide, L-cysteine ethyl ester hydrochloride, reduced glu-
tathione (GSH), N-hydroxysuccinimide (NHS) and 1-ethyl-3-
(
3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC),
Resazurin salt, and lactate dehydrogenase (LDH) cytotoxicity
kit were purchased from Sigma–Aldrich (St. Louis, Missouri).
All other reagents used were of analytical grade.
8
and evaluated within this study.
Hyaluronic acid–L-cysteine ethyl ester (HA–CYS) was synthe-
sized as described previously by our research group. Briefly,
So far, a synthetic high-molecular-weight poly acrylic acid
12
9
was preactivated. However, this study presents the novelty in
mediated by EDAC and NHS, CYS ethyl ester hydrochloride
was covalently attached to HA. After dialyzing, the obtained
a biomaterial HA as one of the most versatile biomaterials and
common natural substances to be preactivated. HA being also
known as hyaluronan or hyaluronate, is a viscoelastic linear
polysaccharide consisting of alternating disaccharide units of
◦
solution was frozen, freeze-dried, and kept stored at 4 C until
further use.
D-glucuronic acid and N-acetyl–D-glucosamine linked by $-1-3 Synthesis of Monomer and Dimer
Synthesis and Purification of 6-Mercaptonicotinamide
Correspondence to: Flavia Laffleur (Telephone: +43-512-507-58608; Fax: +43-
A two-step reaction is necessary.13 In brief, 6-chlornicotinamide
and thiourea were suspended in ethanol and refluxed for 6 h at
boiling temperature. The yellow-turned mixture was allowed
5
12-507-58699; E-mail: Flavia.Laffleur@uibk.ac.at)
Journal of Pharmaceutical Sciences, Vol. 103, 2414–2423 (2014)
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
ꢀ
2
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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Figure 2. Modification of thiolated HA obtaining preactivated thio-
lated HA. 6-Chloronicotinamide reacts with thiourea to 6-MNA. Follow-
ꢁ
ing oxidation of 6-MNA to 6,6 -DTNA. Receiving preactivated thiomer
ꢁ
by adding 6-MNA and 6,6 -DTNA.
Figure 1. Synthetic pathway of the modification of HA with CYS
ethyl ester hydrochloride.
to cool down to room temperature and stirred overnight. As a
result of the first step, the gained salt, namely, S-(5-carbamyl-
Figure 3. FTIR spectra of 6-MNA and 6,6´–DTNA.
2
-pyridyl) thiuronium chloride was separated by filtration and
brought to dryness. In a second step, the dry thiouronium salt
was decayed by adding 2 M NaOH and stirred for 45 min at
room temperature. Afterwards, acetic acid was added to ad-
just the pH to 4; during this step, the reaction color turned
into a dark yellow. The resulting mercaptonicotinamide (MNA)
was purified by filtration, washed with acetone, and brought to
dryness.
was isolated by filtration, washed with water, and brought to
dryness.
Synthesis of HA–CYS–MNA
To synthesize preactivated HA–CYS, 0.200 g of the conjugate
was dissolved in 200 mL of a dimethyl sulfoxide (DMSO)–water
mixture with a ratio of 7:3. The dimer of MNA was dissolved
ꢀ
Synthesis and Purification of the Dimer 6,6 Dithionicotinamide
Avoiding any oxidation of 6-MNA itself to use oxidizing agents in DMSO. The molecular ratio for preactivation of HA–CYS
for disulfide bond formation with thiolated polymers, a dimeric with MNA was 1:2. The MNA solution was carefully added in
reaction was accomplished.14 In brief, 6-MNA was suspended aliquots of 100 :L and stirred for 24 h. Afterwards, the solution
◦
in water and the pH was adjusted to 7 prior to adding H
2
O
2
was dialyzed in the dark at 10 C in tubings with a MWCO of
solution. The reaction was allowed to take place for 1 h at 12,000–20,000 Da. Dialysis process was conducted first three
room temperature under continuous stirring, whereby the dark times against 3 L of demineralized water and 0.5 L DMSO. Af-
yellow color turned into white because of the resulting dimer terwards, dialysis was performed against demineralized water
ꢁ
ꢁ
ꢁ
6
,6 dithionicotinamide (6,6 -DTNA). The obtained 6,6 -DTNA until no DMSO could be detected spectrophotometrically. The
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Figure 4. NMR-spectrum of 6-MNA and 6,6´-DTNA.
Figure 6. Lactate dehydrogenase assay on Caco-2 cells as a percent-
age of total cell LDH activity. Cells were incubated with indicated test
compound, and the percentage of LDH release was determined after
3
h (white bars) and 24 h (gray bars) of incubation period. Each bar
represents the mean ± SD of three experiments.
a microplate reader (Tecan; Groedig, Salzburg, Austria). The
amount of thiol groups immobilized on the polymer was calcu-
16
lated by using CYS standards.
Characterization of the Synthesized Monomer 6-MNA and Dimer
ꢀ
6
,6 -DTNA
The synthesized monomer and dimer exhibit solubility in
DMSO. To control all further reactions, both aromatic reagents
were analyzed by UV spectrometer. The monomer showed a
Figure 5. Cytotoxicity potential of HA–CYS–MNA conjugate in com-
parison to respective thiomers and unmodified polymers. Cytotoxicity
was tested on Caco-2 cells using Resazurin assay and expressed in
percent. White and gray bars express the influence on cell availability
after 3 and 24 h, respectively. Indicated values are the means of at least
three experiments (± SD).
17
peak at 307 nm, and the dimer displayed an expressive peak
at 297 nm and a minor at 253 nm. Correlating with the liter-
ꢁ
ature values, the monomer 6-MNA and the dimer 6,6 -DTNA
◦
◦
expressed a melting point at around 266 C and 264 C, respec-
tively.
Fourier Transform Infrared Characterization of the Synthesized
Monomer 6-MNA and Dimer 6,6 -DTNA
◦
resulting product was freeze-dried and kept stored at 4 C until
further use.
ꢀ
Fourier transform infrared (FTIR) analysis of the novel syn-
ꢁ
Characterization of Thiolated and Preactivated Conjugates
thesized dimer 6,6 -DTNA for the following synthesis step
of the preactivated polymer was performed. Attenuated to-
Determination of the Thiol Groups
tal reflectance FTIR (ATR-FTIR) was utilized to characterize
ꢁ
The degree of modification, that is, the amount of thiol groups 6-MNA and 6,6 -DTNA. A PerkinElmer Spectrum 100 ATR-
immobilized on the HA–CYS ethyl ester conjugate was deter- FTIR spectrometer (PerkinElmer, Waltham, Massachusetts)
mined photometrically by Ellman’s reagent quantifying free in combination with a Spectrum software version 6.3.1.0134
15
18
thiol groups. First, 0.5 mg of each of the conjugates and cor- (PerkinElmer) was deployed to obtain the spectra. Samples
responding controls was hydrated in 500 :L of 0.5 M phos- were divided into six subsamples, and every subsample was
−1
phate buffer pH 8.0, and afterwards, 500 :L of Ellman’s reagent recorded with eight scans in a range from 4000 to 600 cm and
ꢁ
−1
◦
[
3 mg of 5,5 -dithiobis (2-nitrobenzoic acid)] dissolved in 10 mL a resolution of 4 cm . Measurements were carried out at 22 C.
of 0.5 M phosphate buffer pH 8.0 were added. Samples were
incubated for 120 min at room temperature in the dark. There-
1
ꢀ
H NMR Characterization of the Synthesized Dimer 6,6 -DTNA
after, 300 :L of each sample was transferred into a microplate, 1H NMR spectra was recorded on a Bruker DRX 300 MHz or
and absorbency was measured at a wavelength of 450 nm with Avance II + 600 MHz instrument (Billerica, Massachusetts).
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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Figure 7. Swelling behavior of unmodified HA (black square), HA–
CYS (black dot), and preactivated HA–CYS–MNA (black triangle) test
◦
discs in artificial saliva pH 6.75 at 37 C. Indicated values are means
(
± SD) of at least five experiments.
Figure 9. Comparison of the adhesion time of unmodified HA, HA–
CYS, and preactivated HA on the rotating cylinder. Indicated values
are means (± SD) of at least five experiments.
The chemical shifts are referenced to the residual proton signal
1
of the deuterated solvents d
6
-DMSO (2.49 ppm) for H NMR
1
19
spectra. H-assignments was based on COSY experiments.
Quantification of the Conjugated Stabilizing Ligand
The quantification of the amount of conjugated aromatic lig-
and was determined by measuring a 0.1% (m/v) stabilized
thiomer solution with UV spectrophotometer at a wavelength of
3
07 nm. Calculations were carried out by interpolation from a
standard curve obtained with increasing amounts of 6-MNA.
The consequent addition of reduced GSH 0.1% exchanged the
aromatic ligand, whereby the released monomer was quanti-
Figure 8. Comparison of the disintegration behavior of unmodified,
thiolated, and preactivated HA test discs (30 mg). Studies were per-
formed with a disintegration apparatus according to the European
◦
Pharmacopoeia in artificial saliva pH 6.75 at 37 C. The indicated dis- fied photometrically at 307 nm according to previous analyses.
integration time represents an average of at least five experiments The amount of remaining free thiol groups immobilized on the
20
(
± SD).
stabilized thiomer was determined by Ellman’s reagent.
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Subsequently, cells were incubated in a humidified chamber at
◦
3
7 C, 5% CO
2
. The cytotoxicity of tested polymers was eval-
uated at a concentration of 0.5% (m/v). Minimum essential
22
medium (MEM) without phenol red served as control. After
incubating the cells with polymer solutions for 3 and 24 h, the
medium was replaced from each well with 0.5 mL of Resazurin
solution. Subsequently, the plate was incubated at room tem-
perature for 3 h and fluorescence was measured at 540 nm
excitation and 590 emission wavelengths with a microplate flu-
orescence using a spectrophotometer TECAN (Tecan Austria,
Groedig, Ssalzburg, Austria). Viability of cells was calculated
based on the following equation:
Average absorbance value of each sample
Cell viability(%) =
Average absorbance value of low control
×100
LDH Assay
In vitro cytotoxicity of the polymers was determined by mea-
suring the release of the enzyme LDH from nonviable Caco-2
cells as a result of interaction with 0.5% of polymer solution in
MEM without FCS and phenol red. For this purpose, approxi-
mately 100,000 cells/well were seeded to a 24-well plate and in-
◦
cubated for 24 h at 37 C in 5% CO
2
environment. After 24 h, the
cultured cells were first washed once with phosphate-buffered
◦
saline (prewarmed at 37 C). Testing solutions of HA, HA–CYS,
and HA–CYS–MNA 0.5% (m/v), negative control MEM with-
out phenol red and positive control 2% (m/v) Triton-X 100 were
added in triplicate to the cell in 1 mL quantity. Samples of
100 :L at time point zero were taken from supernatant and
Figure 10. In vitro wash-off test of unmodified HA, HA–CYS, and
preactivated HA. Indicated values are means (± SD) of at least five
experiments. Adhesion studies were performed in saliva fluid buffer
◦
pH 6.75 at 37 C.
◦
stored at 4 C. The cells with medium were incubated again at
◦
23
3
2
7 C in 5% CO environment for 3 and 24 h. One-hundred
Safety Screening
microliter samples were withdrawn and centrifuged for 2 min
at 1677g to remove detached cells. The LDH content of 50 :L
supernatant was measured at 492 nm with spectrophotometer
Cell Viability Studies
Cell viability studies were performed by working on Resazurin TECAN using a commercial test kit (Roche Diagnostics) after
21
assay. To evaluate the toxicity of tested polymers in Caco-2 incubation for 30 min at room temperature in the dark. The
5
cells, 1 × 10 Caco-2 cells were seeded per well in 24-well plates. apparent cytotoxicity was calculated according to the following
Figure 11. In vitro shear strength comparison of the mucoadhesive properties of the test discs (30 mg) based on unmodified, thiolated, and
preactivated HA was determined by tensile studies. Indicated values are the means (± SD) of at least five experiments.
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
2419
Figure 12. In vitro peel strength comparison of the mucoadhesive properties of the test discs (30 mg) based on unmodified, thiolated, and
preactivated HA was determined by tensile studies. Indicated values are the means (± SD) of at least five experiments.
equation:
Cytotoxicity (%).
Avarage absorbance value of each triplicate − low control
=
High control − low control
×100
Test Discs Manufacture
Lyophilized stabilized thiomers, corresponding chitosan conju-
gates, and unmodified chitosan were compressed into a 30-mg,
5
.0 mm diameter flat-faced tablet with a single-punch eccentric
press (Paul Weber, Remshalden, Germany). The compaction
pressure of 12 kN was kept constant during the preparation of
all discs.
Evaluation of Water-Binding Properties
To investigate the water-absorbing capacity of HA–CYS–MNA
conjugates, corresponding HA–CYS and the unmodified HA
were prepared as test discs and were determined in simulated
9,24
saliva fluid by a gravimetric method as described previously.
Briefly, polymer test discs were accurately weighted and fixed
on the tip of a needle by applying a little force with thumb. The
test discs were then placed in a beaker containing simulated
saliva solution that consisted of phosphate-buffered saline so-
2 4 2 4
lution (2.38 g Na HPO , 0.19 g KH PO , and 8.0 g NaCl per
liter of demineralized water) adjusted with phosphoric acid to
Figure 13. Comparison of the mucoadhesive properties of the test
◦
pH 6.75 at 37 ± 0.5 C (according to the USP). At various time discs (30 mg) based on unmodified, thiolated, and preactivated HA was
intervals, hydrated test discs were taken out of the incubation determined by tensile studies. TWA is represented in black and MDF
medium, and excess water was removed by gentle soaking with is displayed in white. Indicated values are the means (± SD) of at least
five experiments.
a tissue paper. The test discs were weighted again and the
amount (%) of water uptake was determined gravimetrically as
following:
Evaluation of Cohesive Properties
Water uptake (%) = ((Wt − W0) /Wt) × 100
Disintegration behavior of the test discs comprising HA–CYS–
MNA conjugates corresponding HA–CYS and the unmodified
where Wt is the weight of the swollen polymer at a given time lyophilized HA was evaluated in simulated saliva fluid at 37
◦
and W0 is the weight in the dry state.
± 0.5 C with a disintegration test apparatus according to the
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
European Pharmacopoeia. The constant frequency rate was thread (15 cm). The porcine mucosa was fixed to a glass plat-
adjusted between 29 and 32 cycles per minute.25
form using a cyanoacrylate adhesive and was placed in a beaker
filled with simulated saliva fluid. Afterwards, the beaker was
placed on a balance and carefully raised by a mobile platform
until the mucus contacts the test disc. The contact was deter-
mined when the nylon thread holding the test discs became
bent. By manual turning of a regulating knob provided with
the mobile platform, the mucosa was pulled down from the test
Mucoadhesive Strength Assays
Rotating Cylinder Assay
The mucoadhesion time of HA–CYS–MNA conjugates corre-
sponding HA–CYS and the unmodified HA was determined ac-
cording to a method described previously. Briefly, freshly ex-
cised porcine buccal tissue was provided from a local slaughter
house. After necessary cleaning, the buccal mucosa was cut off
the cheek. The polymer test discs were attached to the thin
buccal mucosa being fixed on a stainless steel cylinder (diame-
ter: 4.4 cm; height: 5.1 cm; apparatus 4-cylinder). The cylinders
were placed in the dissolution apparatus according to the Eu-
ropean Pharmacopoeia containing 900 mL of artificial saliva
◦
disc at a rate of 0.1 mm/s after 20 min incubation at 25 C. This
regulating knob was precisely marked and validated via length
measurements to ensure the exact displacement of 0.1 mm at
each turn. Data points were collected every second by computer
software (SartoCollect V 1.0; Sartorius AG, Germany) linked to
the balance with integrated interface. Data were transferred
to EXCEL 2007 (Microsoft), and the force versus displacement
curves were analyzed to calculate the maximum detachment of
force (MDF) and the total work of adhesion (TWA) as the area
◦
fluid at 37 ± 0.5 C. The fully immersed cylinders were agitated
with 100 rpm. The detachment of the test discs was monitored
15,18
under the curve in accordance with the trapezoidal rule.
26
during an observation period of 1 week.
Statistical Data Analysis
In Vitro Wash-Off Assay
For statistical analysis, analysis of variance and Student’s t-test
were used where appropriate. A probability of less than 0.05 (p
Mucoadhesive property of the test discs was evaluated by wash-
27
off in vitro method. Freshly excised pieces of buccal mucosa
<
0.05) was considered statistically significant. All results are
2
(
1
2 × 2 cm ) from porcine were mounted onto glass slides (3 ×
presented as mean ± SD.
inch) with cyanoacrylate glue. Approximately five test discs
were spread onto each wet-rinsed tissue specimen, and imme-
diately the slide was hung onto the arm of a USP tablet dis- RESULTS AND DISCUSSION
integrating test apparatus. The tissue specimen was given a
slow, regular up-and-down movement in the test fluid at 37 C
Synthesis and Characterization of Thiolated HA
◦
contained in a 1-L vessel. Adhesion studies were performed in The synthesis of thiolated HA has been described by our re-
◦
12
saliva fluid buffer pH 6.75 at 37 C.
search group previously. The synthesis of thiolated HA was
achieved by the formation of an amide bond between the car-
boxylic group of the HA and the amine of the CYS ethyl ester
as shown in Figure 1. By using EDAC and NHS for the cou-
pling reaction, the grade of thiolation was enhanced. It could
be shown in a previous study that EDAC or NHS being used
alone is not as effective as being used together. Determination
of the thiol group content by using Ellman’s test showed that
an average of 191.49 ± 16.26 :mol of thiol groups was immo-
bilized per gram of HA. The synthesized polymer was a white
fibrous powder.
Shear Strength Assay
The shear strength of the adhesive test discs was calculated
by measuring the force required for parallel detachment from
freshly excised porcine buccal mucosa using a specially de-
signed construction. The back of the test disc was fixed to
a movable plastic strip with synthetic adhesive. The other side
of the test disc was pressed over excised porcine mucosa for 5
min. After 5 min, the weight required to detach the test disc
from the mucosa was determined. The force of adhesion and
the bond strength were calculated with following equations:
28
Synthesis of Monomer and Dimer
Weight (g)
In Figure 2, the synthesis of the monomer 6-MNA was achieved
according to a method evolved by Forrest and Walker. The
Force of adhesion (N) =
× 9.81
31
1
000
obtained product could be characterized as a fine yellow powder.
ꢀ
ꢁ
ꢁ
N
m2
Force of adhesion (N)
Surface area of tablet m2
The dimer 6,6 -DTNA was emerged as a result of added H
2
O
2
to
Bond strength
=
ꢂ
ꢃ
ꢁ
the monomer 6-MNA. 6,6 -DTNA showed white color and hold
a structure as a fine white powder.
Peel Strength Assay
Elemental, FTIR, and H NMR Characterization of Monomer and
Dimer
A similar experiment was performed to evaluate the force re-
quired for tangential detachment of adhesive test discs from
freshly excised porcine mucosa. According to the equations for
the force of adhesion and bond strength, the resulting was
calculated.29
Two peaks were significant in the UV analyses in the case of
ꢁ
6
,6 -DTNA showing a significant peak at 297 nm and a minor
one at 253 nm. 6-MNA, however, exhibited a well-pronounced
peak at 307 nm. For the reason to verify the achievement of the
desired product, DSC thermograms were conducted to char-
Tensile Assay
acterize the obtained monomer and dimer. 6-MNA showed a
◦
Tensile studies were performed using excised porcine buccal melting point at around 264 C being in good correlation with
mucosa according to a method as described previously.30 Briefly, literature values (266 C–268 C).
polymer test disc was glued to a stainless steel flat disc (10 mm Moreover, the ATR-FTIR exhibits typical signals such as
in diameter), hanging from a laboratory stand with a nylon 3161 and 3159 cm (C–H stretch, aromatic), 1653–1619 cm
◦
◦
−1
−1
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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N–H bend and –C=C– stretch) as well as 840 cm− (C–H bend, adhesiveness.34 Therefore, slow-swelling behavior of preacti-
1
(
para, and aromatic) for monomeric and dimeric components, re- vated thiomers might be advantageous to circumvent overhy-
spectively, as shown in Figure 3. The thiol function was found dration and loss of adhesiveness.
in the monomeric structure with 2515 cm weak signal of the
thiol function as shown in Figure 3.
−1
Additionally, the NMR spectrum of the novel-synthesized
dimer 6,6 -DTNA was in good agreement with the FTIR of the
Cohesive Properties
ꢁ
dimer as seen in Figure 4. Typical Peaks could be identified Another essential parameter for the cohesiveness of polymeric
35
as following: 7.50 NH
-pyridine. The dimeric compound was taken for the following havior of the test discs comprising HA–CYS–MNA conjugates
2
primary amide; 7.64, 8.02, and 8.62 CH test discs is their disintegration behavior. Disintegration be-
2
synthesis of the preactivated thiomer.
corresponding HA–CYS and the unmodified lyophilized HA was
evaluated in simulated saliva fluid at 37 ± 0.5 C with a dis-
◦
Synthesis and Characterization of Preactivated HA–CYS–MNA
integration test apparatus according to the European Phar-
macopoeia. As expected, test discs comprising unmodified HA
were completely disintegrated within 1 h. Disintegration stud-
ies based either on HA–CYS or HA–CYS–MNA revealed much
higher stability than test discs comprising the unmodified poly-
mer. HA–CYS–MNA showed a 24-fold more pronounced sta-
bility over unmodified polymer. These observations might be
explained by the particulate nature of MNA together with the
higher amount of inter- and/or intramolecular disulfide bonds
within the conjugate being responsible for increased stability
and hardness of HA–CYS–MNA. The results of the disintegra-
tion studies are depicted in Figure 8.
The formation of disulfide bonds between thiol groups of
thiomer and aromatic ligand was achieved as depicted in
Figure 2. The best molar ratio of thiol groups on thiomer and
aromatic ligand was found to be 1:2. The resulting preactiva-
tion ratio for the preactivated thiomer was shown to be 65.59
±
11.29%. The coupling reaction was achieved by using the
monomer with an oxygen donating compound (H ) and by
2
O
2
adding the dimer. In case of 6-MNA, it is assumed that thiol
groups convert into deprotonated form thiolated anions. Nucle-
ophilic attack of an activated thiol group leads to a disulfide
bond with another thiol group. 6,6 -DTNA reacts with the
thiomer to form a mixed disulfide under release of 6-MNA.
32
ꢁ
Safety Screening
In Vitro Mucoadhesion Studies
Resazurin (as depicted in Fig. 5) and LDH assay (results shown
in Fig. 6) were performed on Caco-2 cell lines to determine cy-
totoxicity of thiomers. The cells were incubated with HA–CYS–
MNA, HA–CYS, and unmodified HA to evaluate their in vitro
cytotoxic effect. Cell viability of more than 92% and 86% in the
presence of HA–CYS–MNA after 3 and 24 h, respectively, indi-
cating that these polymers are not at all harmful for the cells.
All polymers were tested in a final concentration of 0.5% (m/v).
Moreover, the chosen concentration allows comparing cytotox-
icity of the preactivated polymers to that of well-established
thiomers. LDH results were in good agreement with the Re-
sazurin assay.
Mucoadhesion studies were carried out with test discs com-
prising the control polymer, the conjugate HA–CYS, and pre-
activated HA–CYS–MNA, respectively. The simulation of the
physical conditions was performed by using simulated saliva
◦
fluid and the temperature was set to 37 ± 0.5 C. Results seen
Figure 9 are determined out of rotating cylinder method to
examine the in vitro mucoadhesive behavior. Preactivated HA
showed fourfold improved mucoadhesion compared with thio-
lated HA. These findings were in correlation with the in vitro
wash-off test depicted in Figure 10. Shear, peel, and tensile
strength of adhesive test discs were measured to evaluate their
mucoadhesiveness as depicted in Figures 11 (shear strength)
and 12 (peel strength). The results are presented in Figure 13
as TWA and MDF. In the present study, the MDF and the TWA
Water-Binding Properties
The water-binding behavior of mucoadhesive polymers is a ma- were low for HA compared with HA–CYS and preactivated HA–
jor part of mechanisms, which are responsible for adhesive and CYS–MNA, demonstrating the high affinity of the thiolated and
cohesive properties, stability during disintegration, and drug preactivated HA for mucosal tissue. TWA of preactivated HA–
release. Absorbing, swelling, and capillarity processes explain CYS was increased 18-fold compared with unmodified HA. In
33
the adhesion between the polymer and the mucus layer. As contrary, thiolated HA–CYS showed a 2.8-fold increase of TWA
depicted in Figure 7, water uptake studies revealed that the compared with the control. The MDF of all tested polymers cor-
covalent attachment of thiol ligand had a great impact on the related well with TWA. Furthermore, the in vitro wash-off test
water-absorbing behavior. In contrary, incorporating an aro- on porcine buccal mucosa confirmed the mucoadhesive findings
matic ligand, the swelling behavior of the preactivated poly- as shown in Figure 12. The obtained results are explained by
mer test discs was reduced compared with their corresponding the evidential theory of enhanced mucoadhesive properties of
thiomers, but still higher than unmodified HA. The weight of thiolated HA in view of the formation of covalent bonds between
thiolated HA increased rapidly and steadily over 1 h of the ex- thiol groups of the polymer and CYS-rich subdomains of glyco-
36,37
periment. Test discs comprising HA–CYS reached a maximum proteins in the mucus layer.
Freshly excised porcine mucosa
of 752 mg, resulting in a 25.4-fold increase to the initial weight. was used as the substrate to assess the in vitro mucoadhesive
In contrast, preactivated polymer test discs gained a maximum strength. A prolonged residence time also leads to a better pa-
weight of 180 mg, which is around 75% lower than thiolated tient compliance because the interval of drug application can
33
polymer test discs. Nevertheless, Mortazavi and Smart high- be reduced and an increased concentration of the drug at the
lighted the advantageous effect of a low-swelling behavior for target site can be achieved by this close attachment. Therefore,
prolonged mucoadhesion. An excessive water uptake leads to the new derivate can be expected to represent considerable po-
overhydration-forming slippery mucilage and results in less tential as a dosage form for intraoral application.
DOI 10.1002/jps.24060
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2
422
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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work of adhesion in tensile studies. Because of this slightly
chemical modification, the swelling behaviour and the cohesive
properties of thiolated HA showed improvement in comparison
with corresponding unmodified polymer. Moreover, the newly
synthesized preactivated thiolated polymer (HA–CYS–MNA)
revealed excellent stability properties. This property might be
1
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1
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ACKNOWLEDGMENTS
terface: FTIR/ATR—The tool of choice. Adv Colloid Interface Sci 93(1–
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This work was supported by the FWF (Fonds zur F o¨ rderung der
wissenschaftlichen Forschung) project no. ZFP 235150. The au-
thors wish to thank the slaughterhouse Josef Mayr in Natters
for providing buccal porcine mucosa.
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