Cho et al.

Biomaterials Research (2018) 22:30

https://doi.org/10.1186/s40824-018-0137-7

RESEARCH ARTICLE Open Access

Synthesis and characterization of thiolated

hexanoyl glycol chitosan as a
mucoadhesive thermogelling polymer
Ik Sung Cho1, Hye Min Oh1, Myeong Ok Cho1,2, Bo Seul Jang1, Jung-Kyo Cho3, Kyoung Hwan Park1,2,
Sun-Woong Kang2,4*† and Kang Moo Huh1*†

Abstract
Background: Mucoadhesive polymers, which may increase the contact time between the polymer and the tissue,
have been widely investigated for pharmaceutical formulations. In this study, we developed a new polysaccharide-
based mucoadhesive polymer with thermogelling properties.
Methods: Hexanoyl glycol chitosan (HGC), a new thermogelling polymer, was synthesized by the chemical
modification of glycol chitosan using hexanoic anhydride. The HGC was further modified to include thiol groups to
improve the mucoadhesive property of thermogelling HGC. The degree of thiolation of the thiolated HGCs (SH-
HGCs) was controlled in the range of 5–10% by adjusting the feed molar ratio. The structure of the chemically
modified polymers was characterized by 1H NMR and ATR-FTIR. The sol-gel transition, mucoadhesiveness, and
biocompatibility of the polymers were determined by a tube inverting method, rheological measurements, and in
vitro cytotoxicity tests, respectively.
Results: The aqueous solution (4 wt%) of HGC with approximately 33% substitution showed a sol-gel transition
temperature of approximately 41 °C. SH-HGCs demonstrated lower sol-gel transition temperatures (34 ± 1 and 31 ±
1 °С) compared to that of HGC due to the introduction of thiol groups. Rheological studies of aqueous mixture
solutions of SH-HGCs and mucin showed that SH-HGCs had stronger mucoadhesiveness than HGC due to the
interaction between the thiol groups of SH-HGCs and mucin. Additionally, we confirmed that the thermogelling
properties might improve the mucoadhesive force of polymers. Several in vitro cytotoxicity tests showed that SH-
HGCs showed little toxicity at concentrations of 0.1–1.0 wt%, indicating good biocompatibility of the polymers.
Conclusions: The resultant thiolated hexanoyl glycol chitosans may play a crucial role in mucoadhesive
applications in biomedical areas.
Keywords: Hexanoyl glycol chitosan, Chemical modification, Mucoadhesive polymer, Rheological measurements,
Thermogelling property, Thiolation

Background and reduced administration frequency [1]. Therefore,

Mucoadhesive polymers have been extensively investi- many researchers have developed mucoadhesive poly-
gated as pharmaceutical formulations for drug delivery mers as drug delivery carriers via various administration
systems due to their many potential advantages, such as routes, including ocular, nasal, gastrointestinal and vagi-
prolonged residence time, improved drug bioavailability, nal routes [2–5].
Recently, thermogelling polymers that show a thermo-
* Correspondence: [email protected]; [email protected] sensitive sol-gel transition in aqueous media have re-
†
2
Sun-Woong Kang and Kang Moo Huh contributed equally to this work. ceived much attention for mucoadhesive drug delivery
Predictive Model Research Center, Korea Institute of Toxicology, 141,
Gajeong-ro, Yuseong-gu, Daejeon 34114, Republic of Korea
due to their potential for easy administration and pro-
1
Department of Organic Materials Engineering, Chungnam National longed active residence time on the mucosal surface [6].
University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea A sol-gel transition property may allow administration
Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Cho et al. Biomaterials Research (2018) 22:30 Page 2 of 10

in a solution formulation (by spraying, dropping, inject- further chemical modifications. Additionally, its low tox-
ing, etc.) below the transition temperature, ensuring icity and good biocompatibility makes it more suitable
complete spreading on the mucous layer. After applica- for biomedical applications [25].
tion, the body temperature causes the solution to Recently, new classes of polysaccharide-based thermo-
undergo quick gelation, which can subsequently stabilize gelling polymers, acyl glycol chitosans that were pre-
the formulation and overcome the early removal mech- pared by the N-acylation of glycol chitosan, have been
anism of the formulation from the mucosa, prolonging reported by our group, and their basic properties have
the residence time of the loaded drug at the administra- been studied for various biomedical applications. Their
tion site [7]. promising properties, such as biocompatibility, bio-
Most typical thermogelling polymers, such as PEG/ degradability, and thermoreversible sol-gel transition be-
PPG and PEG/PLA block copolymers, demonstrate poor havior (even at the low range of concentrations from 3
bioadhesion and low physical stability although they re- to 7 wt%), could make the acyl glycol chitosans useful as
quire a high concentration of polymer for thermogela- new potential biomaterials for various biomedical appli-
tion, limiting their practical application as mucoadhesive cations, including injectable drug delivery systems and
formulations. To overcome their limitations, Yuan et al. cell/tissue engineering. One of the acyl glycol chitosans,
developed a rectal gel formulation based on a mixture of acetylated glycol chitosan, was evaluated as a mucoadhe-
poloxamer 407 and sodium alginate/hydroxypropyl sive thermogelling polymer to develop a vaginal delivery
methylcellulose (HPMC) as a thermogelling component hydrogel formulation of progesterone [26]. The results
and a mucoadhesive component, respectively [8]. Wu et showed that the hydrogel formulation retained many
al. prepared a thermosensitive hydrogel as a nasal drug characteristics useful for an effective vaginal delivery sys-
delivery system using a mucoadhesive polysaccharide tem and could be a promising alternative to current
such as chitosan [9]. However, since the polymer alone mucoadhesive formulations. However, these acyl glycol
cannot demonstrate any thermosensitivity, the chemical chitosans have mucoadhesive properties based on only the
modification and the additional use of salts or additives noncovalent bond formation such as hydrogen bonds,
such as α,β-glycerophosphate (α,β-GP) were needed for ionic interaction, and polymer chain entanglement be-
thermogelation. tween the polymer and the mucous layer, and thus may
A new generation of mucoadhesive polymers, desig- provide a limited range of mucoadhesiveness [27].
nated thiolated polymers, that contain thiol functional The objective of the present study is to develop a glycol
moieties has been developed. Due to the presence of thiol chitosan-based thermogelling polymer with enhanced
groups on the polymer backbone, thiolated polymers have mucoadhesive properties via thiolation. Here, new
the ability to form a covalent disulfide bond with the mu- mucoadhesive thermogelling polymers, thiolated hexanoyl
cous layer, leading to enhanced mucoadhesive properties glycol chitosans (SH-HGCs), were synthesized by a series
[10, 11]. The mechanism is based on thiol/disulfide ex- of N-hexanoylation and N-thiolation reactions of glycol
change reactions and an oxidation process between the re- chitosans. SH-HGCs with different degrees of thiolation
active thiol groups of the mucoadhesive polymer and the were synthesized and characterized by 1H NMR and
cysteine-rich subdomains of the mucin glycoproteins [12]. ATR-FTIR measurements. Their thermogelling and
Therefore, various thiomers as mucoadhesive polymers mucoadhesive properties were evaluated and compared
have been developed, such as thiolated xyloglucan [13], with HGC by rheological measurements. Three kinds of
alginate-cysteine conjugate [14], thiolated chitosan [15], in vitro cytotoxicity tests were performed to investigate
thiolated gelatin [16], thiolated poly(aspartic acid) [17], the potential of SH-HGCs for biomaterials application by
and thiolated silicone oil [18]. However, these polymers do an MTT assay using HeLa cells and human fibroblasts, a
not have thermogelling properties, and they require a long direct contact method using epithelial cells, and a live and
time or the addition of chemicals for stable hydrogel for- dead assay using epithelial cell aggregates.
mation [17, 19].
Chitosan, one of the polysaccharides, is known to be Methods
biocompatible, biodegradable, and mucoadhesive [20– Materials
22]. Because of its many advantages, chitosan has been Glycol chitosan (GC, DP ≥ 200) and hexanoic anhydride
extensively investigated for pharmaceutical, cosmetics, (97%) were purchased from WAKO (Japan) and
biomedical, and biotechnological applications [23]. How- Sigma-Aldrich (India), respectively. 3-Mercaptopropionic
ever, one of the major disadvantages for its use as a bio- acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
material is the poor water solubility in physiological chloride (EDC), N-hydroxysuccinimide (NHS), and mucin
conditions [24]. Glycol chitosan, a water-soluble chito- from porcine were purchased from Sigma-Aldrich (St. Louis,
san derivative, is easily soluble in aqueous media regard- MO, USA). Acetone, methanol and ethanol were supplied
less of the pH and has free amine groups available for from Samchun Chemical (Korea).
Cho et al. Biomaterials Research (2018) 22:30 Page 3 of 10

Synthesis of hexanoyl glycol chitosan phosphate-buffered saline (PBS, pH 7.4) at 4 °С in a 5-mL

Hexanoyl glycol chitosan (HGC) was synthesized by the vial. The sol–gel transition temperature was determined
N-acylation of GC under mild conditions. Briefly, 3 g of over 1 min in the inverted tube [28]. Each data point is an
GC was dissolved in 375 mL of distilled water and di- average of three measurements with the standard deviation
luted with 375 mL of methanol. A predetermined (mean ± SD). The sol–gel transition phase diagram obtained
amount (1.029 mL) of hexanoic anhydride was added to from this method is known to have a precision of ±1 °С
a GC solution under magnetic stirring. After continuous [29]. Sol–gel transition temperatures of the aqueous HGC
stirring at room temperature for 24 h, the polymer was and SH-HGC solutions were also investigated by a rotating
collected by precipitation in acetone. The polymer was rheometer (TA Instruments, AR 1500ex). The aqueous solu-
then dialyzed against distilled water for 2 days using a tions of GC, HGC and SH-HGCs were placed between par-
dialysis membrane (molecular weight cut-off, 12–14 allel plates with a 20 mm diameter and a gap of 1 mm. The
KDa), followed by lyophilization. Lyophilization proced- frequency was optimized to 1 Hz, as determined using a fre-
ure is as follows: the obtained aqueous polymer solution quency sweep experiment. A constant stress of 25 Pa was
was frozen in liquid nitrogen for 15 min. Subsequently, a used for the measurement.
flask containing the solution was submitted to lyophi-
lizer (ISFD-8512, iSBio, South Korea) for 3 days. The Mucoadhesion analysis
temperature of the condenser was − 85 °C and the Rheological measurement is one of the most common
pressure was 5 mTorr. The lyophilized polymers were methods to evaluate the mucoadhesion of polymers [30]. To
finally collected and kept in the refrigerator (below determine the mucoadhesive properties of HGC and
4 °C) until use. SH-HGCs, an HGC-mucin mixture and an SH-HGC-mucin
mixture were prepared in PBS (pH 7.4). The final concentra-
Synthesis of thiolated hexanoyl glycol chitosans (SH-HGCs) tion of mucin, HGC, or SH-HGCs in the PBS solutions was
Thiolated hexanoyl glycol chitosans (SH-HGCs) were syn- 4 wt%. Rheological evaluations were carried out at 25 °С and
thesized by thiolation of HGC. Briefly, 0.5 g of HGC was 37 °С using a MARS-40 (Thermo Scientific, USA) with a
dissolved in 100 mL of distilled water. To activate the car- parallel plate (20 mm) and a gap of 1 mm. Polymer samples
boxylic acid of 3-mercaptopropionic acid, predetermined were loaded on the rheometer platform and equilibrated at
amounts of 3-mercaptopropionic acid (0.009–0.019 mL), 25 °С or 37 °С for 1 min before rheological measurement. A
EDC (60 mg), and NHS (60 mg) were added to 10 mL frequency sweep analysis was performed from 0.1 to 10 Hz
MES buffer, and the pH was adjusted to 5.2 by adding to determine the storage modulus (G`). All samples were pre-
0.1 M HCl. After 2 h, the activated 3-mercaptopropionic pared in triplicate (n = 3). The mean values of the storage
acid solution was added to an HGC solution. The reaction modulus for each sample were taken from the frequency
was then carried out at room temperature under stirring sweep spectra. The absolute synergism parameter (△G`) is a
for 24 h. The resultant polymers were then dialyzed storage modulus component showing the interactions among
against distilled water for 2 days using a dialysis mem- polymers and mucins [31]. The following equation was used
brane (molecular weight cut-off, 12–14 KDa) and lyophi- to calculate △G` [32]:
lized with the same conditions as previously mentioned.
ΔG‘ ¼ G‘mix −G‘p −G‘m ;
Characterization of SH-HGCs
where G’mix, G’p, and G’m are the storage moduli of the
SH-HGCs were characterized by 1H NMR spectroscopy
mixture, polymers, and mucin, respectively. The elastic
using an AVANCE III 600 spectrometer (BRUCKER,
modulus of the mucin solution can be removed from the
Germany) operating at 600 MHz. The polymer samples
equation since the elastic modulus of the mucin disper-
were dissolved in D2O at 1.0 wt%. The D2O peak at δ
sion was negligible [30, 31]. Therefore, △G` was deter-
4.65 was used as a reference peak. To confirm the poly-
mined from the following equation:
mer composition, ATR-FTIR spectra of GC and HGCs
were recorded using a Nicolet iS 5 (Thermo Scientific, ΔG‘ ¼ G‘mix −G‘p :
USA). The ATR-FTIR analysis was performed with 16
scans at a resolution of 4 cm− 1 over a frequency range
of 4000–750 cm− 1. Cytotoxicity tests by MTT and direct contact method
The in vitro cytotoxicity of SH-HGCs was evaluated
Thermogelling properties using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
The sol–gel transition temperatures of HGC and SH-HGCs lium bromide (MTT, sigma) assay. HeLa cells and hu-
were determined using a tube inverting method with a man lung fibroblasts were seeded on 96-well tissue
temperature increase of 0.5 °С/min. The polymer solutions culture plates (Corning, Saint Louis, MO, USA) at a
(4 wt%, 1 mL) were prepared by dissolving HGC in density of 5 × 103 cells/well and incubated for 24 h at 37
Cho et al. Biomaterials Research (2018) 22:30 Page 4 of 10

°С in the presence of 5% CO2. The culture medium was to determine the significant difference. The experimental
then replaced by various concentrations of SH-HGCs (0, data are presented as the mean ± standard deviation and
0.1, 0.5, and 1.0 wt% in culture medium). After incuba- were analyzed with one-way analysis of variance (one--
tion for 24 h, the MTT solution (20 μl, 5 mg/mL in PBS) way ANOVA). A value of *p < 0.05 was considered sta-
was added to each well, and the cells were incubated for tistically significant.
2 h at 37 °С. After removing the MTT solution, 150 μL
of dimethyl sulfoxide (DMSO, Sigma) was added to dis- Results
solve the formazan crystals. The absorbance of each well Synthesis and characterization of SH-HGCs
was measured by a microplate reader (SpectraMax M3, Various SH-HGCs with different degrees of thiolation
Molecular devices, Sunnyvale, CA, USA) at 540 nm. were synthesized from glycol chitosan using a two-step
For the direct contact test, the cultured cells (human reaction procedure under mild conditions (Fig. 1). In the
conjunctiva epithelial cells; Korean Cell Line Bank, first step of the reaction, the amino groups of glycol chi-
Seoul, Korea) were plated at 2 × 105 cells/well and prein- tosan were reacted with hexanoic anhydride to form
cubated for 24 h. After 24 h, specimen films (1 × 1 cm2) HGC. In the second step, the obtained HGC was further
were placed on the centers of the wells where a conflu- modified by reacting the residual amine groups with the
ent monolayer of the cells had formed, and the cells carboxylic acids of 3-mercaptopropionic acid to form
were cultured for 24 h. After removing the specimens SH-HGCs. The chemical compositions of the HGC and
from each well, the wells were washed with PBS and SH-HGCs were confirmed by 1H NMR measurements.
stained with 0.2% crystal violet solution. The percentage The 1H NMR spectra of GC, HGC, and SH-HGCs are
of the detached area affected by the cytotoxic specimen shown in Fig. 2a. The D2O peak was used as a reference
was measured using an image analysis system (ImageJ, a peak at 4.65 ppm. The overlapped peaks at 3.2–4.0 ppm
public domain, Java-based, image-processing software contributed to the protons of the glucopyranosyl rings at
program developed by the National Institutes of Health). positions 2–8 (H-2 through H-8). The peak at 2.65 ppm
The results were interpreted by the grade of reactivity arose from the protons of the primary amine residues.
zone (Table 1). The PU-ZDEC film (Hatano Research In- The peak at 2.0 ppm was assigned to the methyl protons
stitute, Kanagawa, Japan) served as the positive control. of the acetyl group in GC. The new proton peaks at 0.8,
1.2, 1.5, and 2.2 ppm were assigned to -CH3, -CH2–
Viability assay of multicellular epithelial cells on SH-HGCs CH2–CH3, -CO-CH2-CH2-, and -CO-CH2- of the hexa-
For the formation of multicellular aggregates of epithe- noyl groups, respectively. Based on these assignments,
lial cells (human conjunctiva epithelial cells), the cells the degree of hexanoylation (DH) of HGC was calcu-
were plated at densities of 5 × 104 cells/well (12-well lated as approximately 33% by comparing the integrated
dish; ULA plate, Corning, Saint Louis, MO, USA) and signal area of the protons of the glucopyranosyl ring
incubated for 1 day. Next, epithelial cell aggregates were with that of the hexanoyl groups. As shown in Fig. 2a,
transferred to SH-HGC coated plates and cultured for thiolation was successfully achieved, as evidenced by the
1 day. The viability of multicellular aggregates was deter- peak arising at 3.1 and 2.5 ppm due to methylene pro-
mined with a live/dead assay kit (Abcam, Cambridge, tons of 3-mercaptopropionic acid residues in SH-HGCs.
United Kingdom). The culture medium was exchanged The degree of thiolation was calculated from the relative
with a staining solution, and the samples were incubated integration area of the methyl protons at 2.5 ppm com-
for 15 min at 37 °C and examined via fluorescence mi- pared with that of the glucopyranosyl ring protons at
croscopy (DMi8; Leica, Heerbrugg, Germany). 3.2–4.0 ppm.
The chemical structures of GC, HGC, and SH-HGCs
Statistical analysis were also confirmed by ATR-FTIR (Fig. 2b). The broad
Statistical analysis was performed using an Origin pro band at 3400 cm cm− 1 was assigned to the stretching vi-
version 8 software package (OriginLab Corp., MA, USA) bration of the hydroxyl groups, which overlapped with
the N-H stretching vibrations in the same region. The
Table 1 Reactivity grades for direct contact cytotoxicity [39] absorption peak at 2900 cm− 1 was attributed to the –
Grade Reactivity Description of reactivity zone CH2- groups. The absorption peak at 1596 cm− 1 was as-
0 None No detectable zone around or under specimen cribed to the amino bending vibration of GC. The pres-
1 Slight Zone limited to area under specimen ence of absorption bands at 1655 cm cm− 1 and
1555 cm cm− 1 corresponded to the carbonyl stretching
2 Mild Zone extends less than 0.5 cm beyond specimen
and the amide II bending vibration of HGCs, respect-
3 Moderate Zone extends 0.5–1.0 cm beyond specimen
ively. The disappearance of the amino vibration band at
4 Severe Zone extends greater than 1.0 cm beyond 1596 cm cm− 1 and the appearance of the amide II band
specimen but does not involve entire dish
at 1555 cm cm− 1 after hexanoylation indicated that the
Cho et al. Biomaterials Research (2018) 22:30 Page 5 of 10

Fig. 1 Synthetic scheme of thiolated hexanoyl glycol chitosan

HGCs were successfully synthesized [33]. Additionally, the initial temperature range (below Tgel), G` was lower
the increase in the peak intensity at 1555 cm− 1 relative than G``; however, as the temperature increased above
to the peak at 1655 cm− 1 indirectly supports the thiola- Tgel, the G` of HGC and SH-HGCs increased rapidly,
tion of HGCs. The above 1H NMR and ATR-FTIR re- leading to a crossover with G`` at a certain temperature,
sults confirmed that the chemical modifications, Tgel, indicating the sol-gel phase transition of the aqueous
hexanoylation and thiolation, of GC were successfully polymer solution. Among the GC derivatives, SH10-HGC
accomplished. SH-HGCs with two different degrees of showed the largest increase in the G` values.
thiolation were synthesized by adjusting the feed molar
ratio of 3-mercaptopropionic acid as shown in Table 2 Mucoadhesion evaluation of thermogel
(yield: 79–85%). The degree of thiolation (DT) of the By a rheological analysis, mucoadhesion of theromogels
SH-HGC could be tuned from 5.1 to 10.9% by control- was evaluated. As shown in Fig. 5, the introduction of
ling the feed molar ratio of the 3-mercaptopropionic mucins affected the △G` values of the HGC, SH5-HGC,
acid (Table 2). and SH10-HGC depending on temperature (25 °С, 37
°С). Rheological analysis of each polymer demonstrated
Thermosensitive sol-gel transition a positive rheological synergism parameter (△G` > 0).
The thermosensitive sol-gel transition properties of The SH-HGCs showed a higher △G` relative to the
HGC and SH-HGCs were investigated by a tube invert- HGC. The highest △G` was observed from SH10-HGC.
ing method. An aqueous solution of 4 wt% HGC under- We also observed temperature-dependent mucoadhesive
went a phase transition from a flowing liquid (sol) to a properties of the hydrogels when comparing △G’s at 25
non-flowing (gel) as the temperature increased. The and 37 °С. As shown in Fig. 5, almost polymeric solu-
SH-HGCs also showed a sol-gel phase transition, but tions showed higher △G’s at 37 °С than at 27 °С.
their gelation temperatures (Tgel) were observed at a
lower temperature range relative to that of HGC. As In vitro biocompatibility
shown in Fig. 3, the Tgel values of HGC, SH5-HGC, and The cytotoxicity of the SH-HGCs was estimated by an
SH10-HGC were observed at 41 ± 0.5, 34 ± 1, and 31 ± 1 MTT assay using HeLa cells and human fibroblasts as
°С, respectively. shown in Fig. 6. The cytotoxicity was determined with
Rheological studies were also carried out to determine various concentrations of the polymers and degrees of
the viscoelastic properties of HGC and SH-HGCs as a thiolation in the SH-HGCs after 24 h of incubation. The
function of temperature. Fig. 4 shows the changes in the SH5-HGC and SH10-HGC showed low cytotoxicity at
storage modulus (G`) and loss modulus (G``) of GC, various concentrations (0.1–1.0 mg/ml) for HeLa cells
HGC, SH5-HGC, and SH10-HGC (4 wt%, PBS) as a and human fibroblasts.
function of temperature. In case of GC, G`` was continu- Additionally, the cytotoxicity of SH-HGCs was evalu-
ously higher than G` in the experimental temperature ated by the direct contact method using epithelial cells.
range from 10 to 50 °С. In case of HGC and SH-HGCs, at In the direct contact test, the size of the reactivity zone
Cho et al. Biomaterials Research (2018) 22:30 Page 6 of 10

Fig. 2 (a) 1H NMR spectroscopy and (b) ATR-FTIR spectra of GC, HGC, and SH-HGC

in the well was observed. The cells cultured under and

around the PU-ZDEC were detached from the culture
dish and produced a round-shaped cell-free zone on the
plate by the PU-ZDEC film. In contrast, no cytotoxic re-
gions were detected for the SH5-HGC and SH10-HGC
hydrogels or the non-treatment group (Fig. 7).
To determine whether multicellular aggregates adher-
ing on SH-HGCs hydrogels were viable, we performed
live and dead assays through staining with a live/dead

Table 2 Chemical data for the SH-HGC

Samples Feed molar ratioa) DTb) (%) Yield (%)
SH5-HGC 0.10 5.1 ± 0.5 85
SH10-HGC 0.20 10.9 ± 0.4 79
Fig. 3 Sol-gel transition of HGC and SH-HGCs measured by the tube
a)
Feed molar ratio of 3-mercaptopropionic acid to glucosamine residue of GC inverting method (n = 3, *p < 0.05)
b)
Degree of thiolation (DT) determined by the peak integration of 1H NMR
Cho et al. Biomaterials Research (2018) 22:30 Page 7 of 10

Fig. 4 Temperature-dependent rheological behavior of the aqueous solutions (4 wt%) of (a) GC, (b) HGC, (c) SH5-HGC, and (d) SH10-HGC

assay kit on day 1. The epithelial cells were plated on a reactions to give SH-HGCs. In our previous studies, the
ULA culture dish. The multicellular aggregates were thermosensitive HGC was found to be useful for ocular
formed within 24 h on the ULA dish. When multicellu- delivery formulation by prolonging the retention time on
lar aggregates in the ULA dish for 1 day were transferred the preocular surface and thus enhancing ocular bioavai-
to the SH-HGC coated dish, the multicellular aggregates lablility [33]. Based on the promising physicochemical
adhered on the surface of hydrogels. In addition, reason- properties of HGC, we here synthesized thiolated HGCs
able numbers of cells in aggregate were viable (Fig. 8). to enhance the mucoadhesive property of HGC. The 1H
We could not find a difference between the cells on the NMR and ATR-FTIR results confirmed that the chem-
SH5-HGC and SH10-HGC hydrogels. These results in- ical modifications, hexanoylation and thiolation of GC
dicate that SH-HGCs may not affect the viability of the were successfully accomplished. The degree of thiolation
mucosa. (DT) of the SH-HGCs could be easily controlled by
adjusting the feed molar ratio of 3-mercaptopropionic
Discussion acid.
To develop a new mucoadhesive thermogelling polymer The obtained SH-HGC polymers were stored in a re-
for potential biomedical applications, glycol chitosan frigerator (below 4 °С) until use. In case that the samples
was modified by a series of hexanoylation and thiolation were stored at room temperature for a long time, gel
particles were sometimes observed after dispersion in
water, presumably due to disulfide bond crosslinking for-
mation. Bernkop-Schnürch et al. synthesized two kinds
of thiolated polymers based on polycarbophil and chito-
san and studied their stability at several different storage
conditions for 6 months [34]. They reported that thiol
groups of the thiolated polymers which were stored at
room temperature (20 °С) only decreased. Therefore, the
thiolated polymers should be stored under adequate
conditions (below 4 °С and low humidity) to preserve
the thiol groups intact.
Sol-gel transition behaviors of HGC and SH-HGCs
were studied by the tube inverting method and rheo-
logical measurements. The obtained SH-HGCs had
Fig. 5 △G` of HCG and SH-HGC at 25 and 37 °C. The mucoadhesive lower Tgel values compared to HGC since the residual
properties of the gels are estimated by measuring △G`
free amine groups of HGC were substituted with more
(n = 3, *p < 0.05)
hydrophobic thiol-propyl amide groups, which may
Cho et al. Biomaterials Research (2018) 22:30 Page 8 of 10

Fig. 6 Viability of (a) HeLa cells and (b) human fibroblasts at various concentrations of SH5-HGC and SH10-HGC dilution medium

enhance the hydrophobic interaction for thermogelation. between polymer and mucin causes a rheological change
In the same way, the SH-HGC having a higher DT [30]. The SH-HGCs showed higher △G` values relative
(SH10-HGC) showed a lower Tgel value relative to one to that of the HGC since SH-HGCs demonstrated a
having a lower DT (SH5-HGC). Accordingly, the ther- stronger interaction between gel and mucin compared
mosensitive properties of SH-HGCs could be tuned by with the HGC. SH-HGCs have both free amine groups
controlling the DT of SH-HGC. Rheological study as a and thiol groups on their polymer backbone; thus, these
function of temperature showed similar results as the functional groups of SH-HGC might interact with those
above one. GC did not show thermosensitivity while of mucins by ionic interaction and disulfide covalent
HGC and SH-HGCs showed thermogelling behaviors in bonding [27]. However, the HGC only exhibits ionic in-
the experimental temperature range from 10 to 50 °С. teractions, so this noncovalent bond provides only weak
Considering the change of G’ values as a function of interactions with mucin. SH10-HGC had the highest
temperature, SH10-HGC represented the strongest ther- △G`, indicating that more thiol groups in the polymer
mosensitivity among the GC derivatives. might cause more efficacious polymer-mucin interac-
Hassan and Gallo first reported a simple rheological tions. Gelation of a polymeric solution might affect the
method to assess in vitro mucin-polymer bioadhesive interaction between polymer and mucin. When the
bond strength. They suggested that the interaction aqueous polymeric solutions were gelated, higher △G’s
were observed relative to their aqueous solutions (Fig.
5). This supports the idea that the thermosensitive prop-
erty seems to strengthen the interactions between mucin
and the thermogelling polymer by hydrogel formation.
Therefore, the obtained SH-HGCs which contain thiol
groups and exhibit a thermogelation property have great
potential for mucoadhesive applications [35, 36]. Add-
itionally, optimization of the synthesis of SH-HGCs
would be required for the applications.
To investigate the potential of such a thermogelling
polymer for biomaterial applications, three kinds of in
vitro biocompatibility test were accomplished: an MTT
assay using HeLa cells and human fibroblasts, the direct
contact method using epithelial cells, and live and dead
assays using epithelial cell aggregates. In this live and dead
assay, we used epithelial cell aggregates to provide an en-
vironment similar to the mucosa, which consists of one or
more layers of epithelial cells. Generally, a greater number
of dead cells are shown in the central region of aggregates
because nutrient and oxygen uptake by cells in the inner
Fig. 7 Direct contact cytotoxicity assay: (a) non-treatment, core of aggregates may be reduced because of the limita-
(b) PU-ZDEC, (c) SH5-HGC (5 wt%), (d) SH10-HGC (5 wt%)
tion of diffusion [37, 38]. In the case of the SH-HGC, it
Cho et al. Biomaterials Research (2018) 22:30 Page 9 of 10

Fig. 8 (a) Schematic illustration of aggregates forming process for live/dead assay and (b) live/dead assay of epithelial cell aggregates on
HS5-HGC and HS10-HGC hydrogel at day 1

was observed that reasonable numbers of cells in aggre- Abbreviations

gate were viable. This indicates that the SH-HGCs have DMSO: Dimethyl sulfoxide; DT: Degree of thiolation; GC: glycol chitosan;
HGC: hexanoyl glycol chitosan; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-
little cytotoxicity. The biocompatibility study show that diphenyltetrazolium bromide; SH-HGC: thiolated hexanoyl glycol chitosan
the resultant thiolated polymers are not cytotoxic to sev-
eral cell lines, including HeLa cells, human fibroblasts, Funding
and epithelial cells. Accordingly, the polymers have the This research was supported by the Bio & Medical Technology Development
potential for mucoadhesive applications. Program of the National Research Foundation (NRF-2016 M3A9B4919616)
funded by the Korean government.

Availability of data and materials

Conclusion For data requests, please contact the authors.
In this study, new polysaccharide-based mucoadhesive
thermogelling polymers were successfully synthesized by Authors’ contributions
the sequential reactions of N-hexanoylation and N-thio- ISC performed most of the experiments. HMO, MOC, BSJ, and KHP
lation of glycol chitosan. An aqueous solution of HGC participated in polymer synthesis and analysis. SWK and KMH designed the
experiments, analyzed the data, and drafted the manuscript. JKC wrote the
and SH-HGCs demonstrated thermosensitive sol-gel discussion part of the manuscript and revised the manuscript critically. All
transition properties at 4 wt%. SH-HGCs showed a authors read and approved the final draft of manuscript.
lower transition temperature range compared to HGC
due to the hydrophobic thiol group. The rheological Ethics approval and consent to participate
mucoadhesion method proved that the covalent attach- Not applicable.
ment of a thiol group to HGC endowed polymers with
improved mucoadhesive properties. Cell viability tests Consent for publication
All authors have consented to the submission of this manuscript for
showed good biocompatibility of SH-HGCs. Due to their publication.
thermogelling property, mucoadhesive property, and low
cytotoxicity, SH-HGCs have great potential for biomed- Competing interests
ical applications. The authors declare that they have no competing interests.
Cho et al. Biomaterials Research (2018) 22:30 Page 10 of 10

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