Journal of Colloid and Interface Science 531 (2018) 261–268

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Journal of Colloid and Interface Science

journal homepage: www.elsevier.com/locate/jcis

Regular Article

S-protected thiolated cyclodextrins as mucoadhesive oligomers for

drug delivery
Mulazim Hussain Asim a,b, Ali Moghadam a,e, Muhammad Ijaz c, Arshad Mahmood d, Roman Xaver Götz a,
Barbara Matuszczak f, Andreas Bernkop-Schnürch a,⇑
a
Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria
b
Department of Pharmaceutics, Faculty of Pharmacy, University of Sargodha, 40100 Sargodha, Pakistan
c
Department of Pharmacy, COMSATS University Islamabad, Lahore Campus, 54000 Lahore, Pakistan
d
Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, 22060 Abbottabad, Pakistan
e
Institute of Biotechnology, College of Agriculture, Shiraz University, Shiraz, Iran
f
Center for Chemistry and Biomedicine, Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria

g r a p h i c a l a b s t r a c t

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

Article history: Aim: The purpose of this study was to develop a novel mucoadhesive thiolated and S-protected gamma
Received 18 May 2018 cyclodextrin (c-CD) with an intact ring backbone to assure a prolonged residence time at specific target
Revised 14 July 2018 sites.
Accepted 16 July 2018
Method: Thiolated c-CD was generated through bromine substitution of its hydroxyl groups followed by
Available online 17 July 2018
replacement to thiol groups using thiourea. In the second step, thiol groups were protected by disulfide
bond formation with 2-mercaptonicotinic acid (2-MNA).
Keywords:
Result: Thiolated c-CD displayed 1385 ± 84 mmol thiol groups per gram of oligomer and the amount of
Thiolated gamma cyclodextrin
Mucosal drug delivery
MNA determined in the S-protected oligomer was 1153 ± 41 mmol per gram of oligomer. In-vitro screening
Mucoadhesion of mucoadhesive properties of thiolated and S-protected c-CD was done by two methods. Rheological
S-protected thiomer investigation revealed the conjugates non-mucolytic with only a slight increase in viscosity of thiolated
and S-protected c-CD as compared to unmodified c-CD, whereas mucoadhesive properties of the new
thiolated and S-protected c-CD performed on freshly excised porcine intestinal mucosa showed
44.4- and 50.9-fold improvement in mucoadhesion, respectively. The new conjugates did not show any
cytotoxicity to Caco-2 cells even at a concentration of 1% (m/v) for 24 h. In addition, in-vitro studies of
a-amylase degradation of c-CD, c-CD-SH and c-CD-SS-MNA confirmed that all conjugates are biodegrad-
able.

⇑ Corresponding author.
E-mail address: andreas.bernkop@uibk.ac.at (A. Bernkop-Schnürch).

https://doi.org/10.1016/j.jcis.2018.07.062
0021-9797/Ó 2018 Elsevier Inc. All rights reserved.
262 M.H. Asim et al. / Journal of Colloid and Interface Science 531 (2018) 261–268
Conclusion: These outcomes predict that these new conjugates of c-CD might provide a new favorable tool
for drug delivery providing a prolonged residence time on mucosal surfaces.
1. Introduction
Cyclodextrins (CDs), first discovered in 1891 [1], are cyclic
oligosaccharides consisting of six (a-cyclodextrin), seven (b-
cyclodextrin), eight (c-cyclodextrin) or more glucopyranose units
linked by a- (1, 4) bonds [2]. CDs bear hydrophobic inner cavities
and a hydrophilic outer surfaces [3–5], which are accountable for
their ability to encapsulate hydrophilic guest molecules [6]. Due
to superior solubilizing and complexing abilities, cyclodextrins
are used as complexing agents to enhance the solubility of poorly
water-soluble drugs [7]. Furthermore, cyclodextrins are likely con-
sidered the smallest drug carriers possessing the ability to inter-
penetrate within mucus [8]. As they lack mucoadhesive
properties, their residence time on mucosal surfaces, however, is
comparatively short and in many cases insufficient to guarantee
a therapeutic effect of incorporated drugs [9].
In order to overcome this shortcoming, first generation thio-
lated cyclodextrin conjugates (b-CD-cysteamine) were introduced
to the scientific community as novel mucoadhesive delivery sys-
tem for intra-oral use [10]. This thiolated conjugates formed disul-
fide linkages with cysteine-rich subdomains of glycoproteins
covering mucosal membranes and provided significantly improved
mucoadhesive properties [11]. The thiolated conjugates, however,
were synthesized under comparatively harsh conditions via oxida-
tive opening of the glucose rings and importantly, significant cyto-
toxic effects were observed during in-vitro studies on Caco-2 cells
due to the cationic nature [10]. These shortcomings were overcome
via development of second generation non-ionic thiolated
cyclodextrins obtained by reductive amination without ring-
opening [12]. However, we believe that the full potential of this
novel technology has yet by far not been reached. On the one hand,
first and second generation thiolated conjugates were developed
with non-biodegradable b-CD [10,12] and on the other hand, free
thiol groups on CD backbone are highly susceptible towards oxida-
tion leading to inter- and intra-molecular disulfide bond formation
prior to interaction with mucus [14].
Therefore, the aim of this study was to synthesize the third gen-
eration of thiolated CDs of biodegradable and S-protected nature.
As CD, c-CD was chosen because of being biodegradable, better
water-soluble and less toxic [13]. Synthesis was performed by sub-
stituting hydroxyl groups for thiol groups on the glucose subunits
via bromination followed by reaction with thiourea [15]. S-
protection to thiolated conjugate was provided by disulfide bond
formation with an aromatic thiol bearing ligand namely 2-
mercaptonicotinic acid (2-MNA) [16]. The resulting S-protected
thiolated c-CD was evaluated for cytotoxicity, mucoadhesive prop-
erties and degradation studies with a-amylase.
2. Materials & methods
2.1. Materials
Gamma cyclodextrin (c-CD, 98%, molecular weight: 1297.12 Da),
lithium bromide (LiBr,
99%), triphenylphosphine (Ph3P,
95%), N,
N-dimethylacetamide (DMA,
99.5%), N-bromosuccinimide (NBS,

99%), thiourea (CS(NH2)2,
99%), 2-mercaptonicotinic acid (2-
MNA,
98%), 5,50 -dithiobis(2-nitrobenzoic acid) (Ellman’s reagent,

98%), Spectra/PorÒ dialysis tubing MWCO (Molecular weight cut-
off) 500–1000 Da, Hanks’ balanced salt solution (HBSS), resazurin
Ó 2018 Elsevier Inc. All rights reserved.
(7-hydroxy-3H-phenoxazin-3-one 10-oxide) sodium salt (dye con-
tent
75%), cysteine (
98%), sodium borohydride (NaBH4,
99%),
dimethyl sulfoxide-d6 (DMSO d6,
99.9%), fluorescein diacetate
(FDA,
98%,), a-amylase (Type II-A,
1500 units/mg protein), 3,5-
dinitrosalicylic acid (DNS,
98%), potassium sodium tartrate
tetrahydrate (
99%), sodium sulfite (Na2SO3,
99%), minimum
essential eagle medium (MEM), sodium chloride (NaCl), methanol
(
95–99%) and Triton-X 100 were all purchased from Sigma-
Aldrich, Vienna, Austria.
Cell culture medium was prepared by using MEM powder 9.66
g/L (modified with Earle’s salts, phenol red, 19 amino acids and the
non-essential amino acids L-asp, L-asn, L-glu, L-ser, L-ala, L-gly, and
L-pro), L-glutamine 2 mM, sodium bicarbonate 2.2 g/L, 1%
penicillin-streptomycin solution (10,000 U/mL) and 10% fetal
bovine serum (FBS). FBS and 100 mM phosphate-buffered saline
(PBS) pH 6.8 were purchased from Gibco (Invitrogen, Lofer,
Austria). All other materials were of analytical grade and received
from commercial sources.
2.2. Synthesis of thiolated c-CD
Primary and secondary hydroxyl groups of gamma cyclodextrin
were chosen for substitution with bromine moieties followed by
replacement with thiol groups. Thiolated c-CD was synthesized
by replacing the hydroxyl groups of gamma cyclodextrin with bro-
mine and then this bromine was replaced by thiol groups based on
a method described previously [17]. Bromination and thiolation as
outlined in Fig. 1 were performed under inert conditions (nitrogen
gas). c-CD, NBS, and Ph3P were dried in a desiccator under reduced
pressure in the presence of molecular sieves 4 Å. Before experi-
ment LiBr anhydrous was dried at 150 °C in oven.
First of all, 1 g of dried c-CD was dissolved in 100 mL of DMA,
the mixture was heated at 90 °C for one hour with constant stirring
and then 23 g of LiBr was slowly added. This LiBr-DMA mixture
gave homogeneous bromination of c-CD [17]. The reaction mixture
was kept at 90° C for one hour until LiBr was completely dissolved
and then maintained at 70 °C for 18–20 h until a clear solution was
obtained. After cooling the mixture at ice water, 100 mL of 3%
(m/v) NBS and 100 mL of 4% (m/v) Ph3P (both in DMA solution)
were added.
The final solution was kept at 70 °C for 3 h while stirring. In the
following, the brominated c-CD was precipitated by the addition of
400 mL of acetone (ratio 1:4). The precipitate was filtered with a
Whatman filter paper, washed three times with acetone, dried in
oven at 40 °C, dissolved in water, dialyzed using cellulose mem-
brane (molecular weight cutoff of 500–1000 Da) against water
and finally freeze-dried (51 °C, 0.01 mbar, Gamma LSC 1–16,
Martin Christ, Germany).
In the following, 1 g of bromo-c-CD was dissolved in a solution
of thiourea (0.5%; m/v) in 100 mL of DMA and the mixture was stir-
red at 70 °C for 16 h. After cooling the reaction mixture at ice
water, 12 mL of 3 M NaOH solution was added slowly to produce
thiolated c-CD. After stirring for 15 min at room temperature, the
mixture was neutralized with 18 mL of 3 M H2SO4 solution. The
final product was precipitated in acetone as described above,
dialyzed using cellulose membrane (molecular weight cutoff of
500–1000 Da) against water, freeze dried and stored at 4 °C until
further use.
 M.H. Asim et al. / Journal of Colloid and Interface Science 531 (2018) 261–268
Fig. 1. Presumptive chemical substructure of c-CD thiolation and S-protection: Bromination of c-CD with NBS and Ph3P in LiBr-DMA and substitution of thiol groups using
thiourea. 2-MNA dimer is covalently attached to thiolated c-CD via disulfide bond formation.
2.3. Synthesis of S-protected thiolated c-CD
After thiol groups conjugation to the oligomeric backbone,
S-protected thiolated c-CD was generated by disulfide bond forma-
tion between free thiol groups of thiolated c-CD and
2-mercaptonicotinamide (2-MNA) as shown in Fig. 1. 2-MNA
dimer (2,20 -dithiodinicotinic acid) was prepared by oxidation of
2-MNA with hydrogen peroxide under neutral pH conditions to
obtain 2-MNA modified oligomers [18].
Briefly, in the first step, 1 g of 2-MNA was dispersed in 12.5 mL
of demineralized water by ultrasonication for 30 min. Then pH of
the solution was adjusted to 8 using 5.0 M NaOH with constant
stirring until a slight yellowish clear solution was obtained. In
the following 1.25 mL of hydrogen peroxide (30%, w/v) was drop-
wise added to this solution till a colorless solution (2-MNA dimer;
2,20 -dithiodinicotinic acid) was obtained. This colorless 2-MNA
dimer solution was continuously stirred for 1 hr at room tempera-
ture and finally diluted with water to volume of 25 mL. The pro-
duct was lyophilized and stored at room temperature. The
formation of 2-MNA dimer was confirmed by comparing the UV
absorption spectra of the monomer and dimer at a wavelength
between 200 nm and 400 nm using a UV–VIS spectrophotometer
(UVmini-1240, Shimadzu Corp., Kyoto, Japan). UV-analyses in case
of 2,20 -DTNA showed a significant peak at 297 nm, whereas the
monomer 2-MNA displayed a well-pronounced peak at 307 nm.
In the second step, the thiolated cyclodextrin was coupled with
aromatic dimeric ligand through disulfide exchange reaction. One
gram of thiolated c-CD was hydrated in 100 mL of demineralized
water. 2 mL of 2-MNA solution was added drop-wise to thiomer
solution buffered to pH 8 using 5 M NaOH. The reaction mixture
was incubated overnight at room temperature under constant stir-
ring and then dialyzed for 3 days in the dark using Spectra/PorÒ
dialysis membrane (MWCO: 500–1000 Da) in 5 L of demineralized
water. Thereafter, the dialyzed product was freeze-dried at 80 °C
under reduce pressure and stored at 4 °C until use.
2.4. Evaluation of the thiol and disulfide content
The free thiol groups immobilized on oligomer backbone were
measured with Ellman’s reagent [19]. The amount of thiol groups
263
was calculated by using a standard curve of cysteine prepared in
exactly the same way as the samples, whereas the disulfide
contents were calculated after reduction with NaBH4 [20].
2.5. Quantification of conjugated 2-mercaptonicotinic acid
The degree of S-protected thiol groups was measured photo-
metrically using a method previously described by our research
group [18]. Samples of 0.5 mg were dissolved in 500 lL of 0.5 M
phosphate buffer solution at pH 8. To liberate 2-MNA, 500 lL of
a 2% (m/v) reduced glutathione solution were added and the mix-
ture was incubated for 1 hr under stirring. The absorbance of free
2-MNA was measured at 354 nm. To ensure that there is no
unbound 2-MNA in the sample a control without the addition of
GSH was measured at 354 nm.
2.6. Characterization of thiolated and S-protected c-CD structure
IR spectra were recorded on a Bruker ALPHA FT-IR apparatus
equipped with a Platinum ATR (attenuated total reflection) mod-
ule. The 1H NMR spectra were recorded on a Varian Gemini 200
spectrometer in D2O with DSS (4,4-dimethyl-4-silapentane-1-sulfo
nic acid sodium salt) as an internal reference.
2.6.1. Molecular weight determination of CD conjugates by LC-MS
Mass spectrometry detection was carried out using Chromaster
5610 MS detector (Hitachi) controlled by a computer running the
MSD system manager software (version 2.1).
Source conditions were as follows: ionization potential (V):
2000; ion injection (eV): 2.0; counter gas flow (L/min):1.0; AIF
temperature (°C):140 and ion source temperature (°C): 80. Sample
solutions were prepared by dissolving the c-CD and c-CD conju-
gates in the mobile phase (methanol, water; 85:15, v/v) to reach
the concentration of 500 ng/mL.
Mass spectral studies were performed in negative electrospray
ionization (ESI) mode in the mass range of m/z 600 ? 700 to calcu-
late molecular weight of CD and its conjugates. In order to get clear
mass spectrum without any background noise, the conjugates
were directly infused using a syringe pump with flow rate of
2 lL min1 into the mass spectrometer.
264 M.H. Asim et al. / Journal of Colloid and Interface Science 531 (2018) 261–268
2.7. Cytotoxicity studies
2.7.1. Resazurin assay
The effect of unmodified, thiolated and S-protected c-CD on cell
viability was evaluated by resazurin assay. For this purpose, Caco-2
cells were purchased from the European Collection of Cell Cultures
(ECACC, Health Protection Agency, Porton Down, Salisbury, Wilt-
shire, United Kingdom). Cells (passage number 25–33) were
seeded at a density of 2.5  104 cells/well in a 24-well plate for
10 days in a final volume of 500 mL of MEM with Earle’s balanced
salts supplemented with 2.0 mM L-glutamine, 10% FBS, and 1%
penicillin-streptomycin at 37 °C in 5% CO2 environment. The cul-
ture medium was refreshed every other day.
Resazurin assay was performed as an oxidation-reduction indi-
cator to determine in-vitro cytotoxicity of thiolated and
S-protected c-CD. When the cells were approximately 80% conflu-
ent (80% of surface of flask covered by cell monolayer after 8–10
days), they were washed twice with PBS pre-warmed at 37 °C. Test
solutions including unmodified c-CD, thiolated c-CD and
S-protected c-CD (1% m/v), positive control prepared in white
MEM and negative control (4% v/v Triton X-100) were added in
500 mL volume in triplicate to the cell culture. Then, the treated
cells were incubated at 37 °C in 5% CO2 environment for 3 and
24 h. Afterwards, test solutions were removed and cells were
washed twice with prewarmed phosphate buffer saline (PBS). A
diluted resazurin solution (2.2 mM) in 500 mL volume was added
to each well and cells were incubated for 3 h. Supernatant
(100 mL) was afterwards transferred to black 96-well plate and
fluorescence intensity was measured using microplate reader
(M200 spectrometer; Tecan infinite, Grödig, Austria) at a wave-
length of 540 nm with background subtraction at 590 nm [21]. Per-
centage of viable cells was calculated using the following equation:
Cell Viability ð%Þ
Experimental values  Negative control
¼  100
Positive control  Negative control
2.7.2. LDH assay
In parallel to the resazurin assay lactate dehydrogenase (LDH)
release assay was also performed. In principle, if test samples dam-
age cell membrane integrity, cytoplasmic LDH is released into cul-
ture medium [22]. The amount of LDH leakage is then measured
using a commercial test kit (Promega, Madison, WI). In detail, the
cells with approximately 80% confluence were washed with pre-
warmed HBSS before being treated with unmodified or modified
samples (1% m/v) as described above. HBSS was used as negative
control and Triton-X 100 (4% m/v) served as positive control. After
3 and 24 h of incubation at 37 °C in 5% CO2 environment, super-
natant was collected and LDH assay was performed according to
the manufacturer’s instruction. Fluorescence was recorded using
microplate reader (M200 spectrometer; Tecan infinite, Grödig,
Austria) with an emission wavelength of 590 nm and an excitation
wavelength of 560 nm. Percentage of cell toxicity was estimated
using the following equation:
Cell toxicity ð%Þ
Average fluorescence intensity of each sample
¼  100
Average fluorescence intensity of positive control
2.8. Enzymatic (a-amylase) degradation of c-CD
In-vitro a-amylase degradation of c-CD was investigated by
measuring the formation of reducing sugars (mainly maltose) via
a slightly modified method as described previously [13]. 5 mM
solutions of unmodified, thiolated and S-protected c-CD were
prepared in 20 mM sodium phosphate buffer (pH 6.9) containing
6.7 mM NaCl at 20 °C. Degradation reaction was started by the
addition of a-amylase in a final concentration of 1 unit/mL to
5 mM c-CD solution. The reaction was kept at 37 °C with continu-
ous shaking for 4 h. Degradation products resulting from this reac-
tion are mainly maltose and to a minor extent glucose. As the
degradation products have been investigated in detail previously
[23,24], the reaction was analyzed by quantification of recom-
mended degradation products. Aliquots (250 lL) were withdrawn
at predetermined time points and analyzed by reducing sugar
method with some changes [13].
Briefly, 250 lL of enzyme-c-CD solutions were transferred to
preheated tubes (95 °C) at predetermined time points in order to
denature the enzyme and consequently terminate the reaction.
Then 750 lL of a solution consisting of 96 mM 3,5-dinitrosalicylic
acid (DNS), 2 M NaOH, 20% (w/v) potassium sodium tartrate
tetrahydrate and 0.05% (m/v) Na2SO4 was added. The mixture
was kept at 95 °C for 15 min before cooling on ice and analyzed
using microplate reader (M200 spectrometer; Tecan infinite,
Grödig, Austria) at 540 nm. All reactions and analyses were per-
formed in triplicate.
2.9. In-vitro evaluation of mucoadhesive properties
Thiolated and S-protected c-CD, were fluorescent-labelled by
incorporation of FDA for the assessment of mucoadhesive proper-
ties. Briefly, 20 mg of CDs were dissolved in 20 mL of demineral-
ized water and pH of solutions was adjusted to 6.5 with 100 mM
HCl. Then 1 mg of FDA was dissolved in 5 mL of 96% ethanol and
1 mL of this solution was added to each CD solution. After 24 h
of stirring at room temperature, the suspensions were filtered in
order to eliminate free FDA and freeze dried.
For evaluation of mucoadhesive properties, freshly excised por-
cine intestinal mucosa was collected from a local slaughterhouse.
First, porcine small intestine was cleaned and rinsed with 100
mM phosphate buffered saline pH 6.8 [18]. Then, it was cut into
smaller pieces of 4  2 cm and fixed on half cut 50 mL falcon tubes
that were placed at an angle of 45° in an incubation chamber at 37
°C and 100% relative humidity. Afterwards, the mucosa was contin-
uously rinsed with phosphate buffer pH 6.8 for 5 min at a flow rate
of 1 mL/min. In the following, 20 mg of FDA-labelled c-CD samples
were separately placed on each mucosa and after 10 min the phos-
phate buffer flow was restarted. 30 mL of phosphate buffer flowing
down the mucosa was collected at following time points: 30, 60,
90, 120, 150 and 180 min. In parallel, reference samples containing
100% c-CD were prepared by rinsing the mucus with 30 mL of
phosphate buffer and dissolving 20 mg of c-CD in the collected
buffer.
To quantitatively hydrolyze FDA to sodium fluorescein, 1 mL of
NaOH 5 M was added to 1 mL of each collected samples and the
remaining intestine on the falcon tube. Samples were incubated
while shaking at 37 °C for 20 min and then centrifuged at 13400
rpm for 5 min at room temperature. Finally, 100 mL of each sample
was transferred to the microplate reader (M200 spectrometer;
Tecan infinite, Grödig, Austria) and fluorescence intensity was
measured at an emission wavelength of 535 nm and exciting
wavelength of 485 nm. All experiments were performed in
triplicate.
2.10. Viscosity measurement
The dynamic viscosity of unmodified, thiolated and S-protected
c-CD was measured in presence of freshly excised porcine intesti-
nal mucus. The mucus was collected and purified as described
before [18].
 M.H. Asim et al. / Journal of Colloid and Interface Science 531 (2018) 261–268
Briefly, porcine mucus was acquired from the small intestine of
a freshly slaughtered pig donated by a local slaughter house. To get
the mucus, the emptied intestine was cut into small pieces and
opened lengthwise. Mucus was isolated from the tissue using a
scraper. To decontaminate the collected mucus, it was dispersed
in NaCl solution (0.1 M) and stirred on ice for one hr. Afterwards
suspension was centrifuged at 4 °C and 13,000g for 2 h. The super-
natant and granular material at the bottom were removed. The
purified and homogenized mucus was used immediately.
Dynamic oscillatory tests were performed at a frequency of 1 Hz
on a plate–plate viscometer (Haake MARS Rheometer, 379–0200;
Thermo Electron GmBH, Karlsruhe, Germany). In detail, test sam-
ples (0.5%, w/v) in 200 mM phosphate buffer at pH 7 were mixed
with porcine intestinal mucus in a ratio of 1:4 in a petri dish using
a spatula. This mixture was incubated at 37 °C without stirring
until measurement. The oligomer-mucus mixture was incubated
for 0.5, 2, 4 and 6 h at 37 °C and then 500 mL was transferred to
rheometer plate for measurement. All experiments were repeated
three times.
2.11. Statistical analysis
The statistical difference among groups was compared by one-
way ANOVA and p < 0.05 was considered statistically significant
(Graph Pad Prism 5.01). In addition, Student’s t-test was applied
with a confidence interval (p < 0.05) for the analysis of two groups.
3. Results and discussion
3.1. Synthesis and characterization of thiolated c-CD
In comparison to the synthetic pathway previously used for CDs
[10], thiolated c-CD was synthesized without ring-opening. Immo-
bilization of thiol groups to c-CD was carried out via an intermedi-
ate brominated derivative. The hydroxyl groups on the
carbohydrate backbone were initially substituted with bromine
followed by its replacement with thiol groups. Hydroxyl groups
at terminal site of polysaccharide chain are likely favored for this
type of substitution, as primary hydroxyl groups display a higher
reactivity to nucleophilic substitution reactions than secondary
hydroxyl groups [25,26]. From stability point of view the interme-
diary step was important as bromination of carbohydrates is a
temperature dependent reaction and higher temperatures result
in degradation of carbohydrates with an insignificant degree of
substitution [27].The thermal degradation pattern of cyclodextrins
is similar to that of cellulose and temperature of decomposition
strongly depend on the degree of substitution [28]. Bromination
of carbohydrates was mostly proceeded at 90 °C but recoveries of
the products were much lower at this temperature as cellulose
was degraded [27]. In order to achieve a higher degree of bromina-
tion and at the same time to avoid degradation of CD, the reaction
was initially proceeded at 90 °C for just 1 hr and afterwards contin-
ued at 70 °C for 18–20 h [12].
Furthermore, bromine conjugation was found dependent on the
concentration of NBS and Ph3P during the reaction. Thiolated CDs
show an almost two times higher degree of substitution (DS) when
a twice as high concentration of Ph3P and NBS is applied in the
reaction. This effect might be explained by the higher degree of
bromination on the backbone of the oligomer [12]. However, an
increasing concentration of thiourea in the later step did not affect
the thiolation efficiency identifying the degree of bromination to
be controlling the degree of thiolation [15]. Thiolation of CDs in
LiBr-DMA mixture generates homogeneous bromination of CDs,
as shown by our previous research work [12]. An efficiently regios-
elective bromination was achieved using NBS/Ph3P in LiBr-DMA
265
Table 1
Amount of thiols (ASH), disulfide groups (ASASA) and MNA on c-CD-SH and c-CD-
SS-MNA. Indicated values are means ± standard deviation of three experiments.
Conjugates ASH (mmol/g) ASASA (mmol/g) MNA (mmol/g)
c-CD-SH 1385 ± 84 180 ± 48 –
c-CD-SS-MNA 20 ± 3 1124 ± 74 1153 ± 41
mixture. Additionally, LiBr-DMA mixture is useful for chemical
modification [29] and also produces high degrees of uniform sub-
stitution [30]. In bromination reaction, yield was 95.4% with 1.9
degree of substitution. Thiolated c-CD appeared as odorless, white,
fibrous structured oligomer that was soluble at physiological pH in
aqueous media. Thiolation reaction yield was almost 90%. The
amount of immobilized thiol groups and disulfide bonds formed
during the reaction were quantified by Ellman’s reagent as shown
in Table 1. Results showed that oxidation of thiol groups to disul-
fides could not be completely excluded during the preparation
process.
The successful immobilization of thiol groups to c-CD was fur-
ther confirmed via FT-IR spectroscopy. FT-IR spectra of c-CD-SH
describe significant differences from unmodified c-CD, as shown
in supplementary Fig. S1. The strong peak around 1140 cm1 and
moderate peak at 613 cm1 are interpreted as CS stretching vibra-
tions in case of c-CD-SH [31].
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.jcis.2018.07.062.
3.2. Synthesis and characterization of S-protected thiolated c-CD
Thiolated CDs (CD-SH) exhibit ASH groups that are less stable
and can be easily oxidized at pH
5 unless sealed under inert con-
ditions [14]. This too early oxidation of thiol groups before getting
into contact with the mucus layer might deteriorate the interac-
tions between thiolated CDs and mucus layer. Consequently, free
ASH groups cannot participate in thiol/disulfide exchange reac-
tions and this deteriorating factor reduces the efficiency of CDs-
SH, mainly in body regions where pH is raised. To overcome this
shortcoming, thiolated c-CDs were S-protected to enhance stabil-
ity of thiol groups and to subsequently improve their mucoadhe-
sive properties [32].
The formation of disulfide bonds between thiol groups of thio-
lated c-CD and the aromatic ligand 2-mercaptonicotinic acid
(2-MNA) was achieved as previously described by our research
group [16]. The suitable molar ratio of thiol groups on the thiomer
and aromatic ligand was found to be 1:2. The lyophilized c-CD-SS-
MNA was a water soluble, white and odorless powder of fibrous
structure. Reaction yield of S-protected thiolated CD was almost
90%. The degree of S-protection of thiol groups was quantified
spectrophotometrically using a method previously described by
our research group [18]. Chemical characterization of S-protected
thiolated c-CD showed high amount of MNA conjugated to CD
are shown in table 1.
Comparison the 1H NMR spectrum of c-CD-SS-MNA with those
of the unmodified c-CD and c-CD-SH indicates that in c-CD-SS-
MNA there are additional signals in the aromatic area between
7.6 and 8.6 ppm, which are characteristic for the protons of the
pyridine moiety of the 2-thionicotinic acid subunit (supplementary
Fig. S2).
3.2.1. Molecular weight determination of CD conjugates by LC-MS
Liquid chromatography-mass spectrometry under optimized
conditions with negative ESI mode provided a highly selective
method for determination of molecular weight of c-CD and its
derivatives. Direct infusion through a syringe pump was used to
266 M.H. Asim et al. / Journal of Colloid and Interface Science 531 (2018) 261–268
achieve most suitable mass spectrometry conditions. The solutions
were filtered through a 0.45 lm membrane filter prior to transfer-
ring into ESI-MS. All samples were operated in negative polarity
mode. Methanol and water (85:15 v/v) was identified as most
appropriate eluent. The doubly charged state of ions at m/z
647.0, 686.1 and 686.3 represented c-CD, c-CD-SH and c-CD-SS-
MNA, respectively. The spectrum of c-CD and its derivatives
showed base peak ions at mass-to-charge ratio (m/z) in range of
1.3 kDa as shown in supplementary Figs. S3–S5.
3.3. Cytotoxicity
The cell viability of both thiolated and S-protected c-CD on
Caco-2 cells was assessed by utilization of two methods: resazurin
and LDH assays. Cells were treated with c-CD-SH and c-CD-SS-
MNA at higher concentrations (1%) as compare to 0.5% concentra-
tion of cationic oligomers (b-CD) that were found toxic [12].
Results showed over 94% cell viability for the concentration tested
after 3 and 24 h of incubation (Fig. 2). The thiolated and
S-protected c-CD did not show significant toxicity (p < 0.05). The
newly synthesized thiolated and S-protected c-CD can be consid-
ered as relatively safe for in-vivo applications. Results of LDH assay
are shown in Fig. 3. Again both thiolated and S-protected c-CD
showed no significant difference in comparison to the negative
control after 3 and 24 h. These results are in good accordance with
the resazurin results.
Fig. 2. Histogram shows cell viability of S-protected thiolated gamma cyclodextrin
(c-CD-SS-MNA) compared to thiolated gamma cyclodextrin (c-CD-SH) and unmod-
ified gamma cyclodextrin (c-CD) after 3 and 24 h. Viability assays were performed
on Caco-2 cells using resazurin. As negative control MEM without phenol red was
used and the positive control was TritonTM X-100 (4% m/v). Indicated values
represent an average of at least three experiments (±SD).
Fig. 3. LDH test performed on Caco-2 cell line with c-CD (1%), c-CD-SH (1%) and c-
CD-SS-MNA (1%) for 3 and 24 h. The results are expressed as % cytotoxicity of cells
(means ± SD; n = 3).
3.4. Enzymatic (a-amylase) degradation of c-CD
The degradation products of a-amylase and c-CD reaction are
reducing sugars mainly maltose. It has already been demonstrated
that the rate-determining step in the degradation of c-CD is the
ring opening [28]. Hence, it can be expected that once the c-CD
ring is opened, the degradation reaction will lead to formation of
primarily maltose and, to a much less extent, glucose. Full degrada-
tion of c-CD will create 20 mM reducing products due to the for-
mation of 4 mM maltose per mM c-CD [13]. a-Amylase
hydrolyzes c-CD by a multiple attack mechanism [33–36]. Accord-
ing to this mechanism, an encounter between cyclodextrin and
a-amylase results in a ring-opening reaction to produce smaller
oligosaccharides with subsequent cleavage of the initial product.
Results showed the same degradation kinetic for c-CD, thio-
lated and S-protected c-CD (Fig. 4). The results of the reactions
are to a high extent compatible with previous results, which
showed about 72% degradation of c-CD after 4 h at 37 °C using
enzyme concentrations of 50 nM [23]. As quantitative hydrolyses
of c-cyclodextrin by a-amylase is independent of the cyclodextrin
concentration [23], higher concentration of enzyme used in this
reaction resulted in degradation of c-CD, thiolated and
S-protected c-CD just in less time.
3.5. Viscosity measurement
There is a correlation between the increase in viscosity of the
mucus and mucoadhesive properties during interaction with a
mucoadhesive oligomer [37]. Thiolated and S-protected CD were
examined for their mucoadhesive potential. Thiomers, with free
thiol moieties, form covalent bonds through oxidative coupling of
thiol moieties and/or by thiol-disulfide exchange reaction of the
thiol groups present in oligomer and cysteine-rich subdomains of
glycoproteins in the mucus layer [38].
Thiolated and S-protected c-CD were found non-mucolytic in
nature. Rheological investigations of c-CD, thiolated and
S-protected c-CD /mucus mixtures were carried out on modified
c-CD and unmodified CD as control. In detail, the viscosity of
unmodified CD/mucus mixture does not change significantly from
the viscosity of mucus/buffer mixture, used as a control.
Thiomers interact with mucin glycoproteins in many different
ways with increase in viscosity of thiomer/mucin blends, as they
interact with cysteine-rich subdomains found in mucin leading
to the formation of new disulfide bonds [39]. CDs described in this
study, however, exhibit only one thiol group and not many. Conse-
quently, they could act like mucolytic agent such as N-acetyl-
cysteine by breaking disulfide bonds via thiol/disulfide exchange
Fig. 4. Degradation of c-CD (d), c-CD-SH (j) and c-CD-SS-MNA (N) by a-amylase
in sodium phosphate buffer (pH 6.9) containing 6.7 mM NaCl at 20 °C. The results
are given as means ± standard deviation (n = 3).
 M.H. Asim et al. / Journal of Colloid and Interface Science 531 (2018) 261–268
Fig. 5. Dynamic viscosity (Pas) measurement of c-CD, c-CD-SH & c-CD-SS-MNA
with mucus at a frequency of 1 Hz on a plate-plate viscometer with test samples
(0.5%, w/v) in 200 mM phosphate buffer at pH 7 mixed with porcine intestinal
mucus in a ratio of 1:4 at 37 °C. Results are expressed as means ± SD (n = 3). * differs
from unmodified CD, p-value < 0.05.
reactions within the mucus. Unmodified CDs were not expected to
be mucolytic.
Thiolated c-CD demonstrates a 1.5- fold higher mucus viscosity,
whereas S-protected c-CD prompted 1.6- fold higher mucus vis-
cosity as compared to mucus-buffer mixture (Fig. 5). This minor
difference in viscosity between thiolated and S-protected thiomers
might be explained by free hydroxyl groups interacting with
mucosal surface. Moreover, mucoadhesion can be influenced by
several physical and chemical parameters for example electrostatic
forces. S-protected CDs interact with mucin glycoproteins in many
different ways. S-protected thiomers contain hydroxyl groups in
their structure which can easily undergo a nucleophilic attack on
the carboxyl group of aspartic acid and glutamic acid substructures
of mucus [39]. Moreover, hydroxyl groups are able to form hydro-
gen bonds with other functional groups, therefore, interaction
between the oligomer and gastric mucus is possible [18]. Results
of thiolation showed that a  1.8 hydroxyl groups per c-CD mole-
cule were thiolated whereas other OHA groups were found free.
3.6. Evaluation of mucoadhesive properties on freshly excised porcine
intestinal mucosa
The mucoadhesive properties of oligomers are subject to inter-
actions between functional groups of the mucus gel layer and the
mucoadhesive oligomer [40,41]. Most important interactions
responsible for mucoadhesive properties are ionic interactions
and chain entanglements, but CDs lack such interactions.
Fig. 6. Time course of percentage of remaining unmodified (d), thiolated (j) and S-
protected (N) c-CD on porcine intestinal mucosa continuously rinsed with 100 mM
phosphate buffer pH 6.8 at 37 °C and 100% relative humidity. Indicated values are
means ± SD of three experiments. (**P < 0.01, ***P < 0.001).
267
Thiolation is therefore perhaps the only way to enhance mucoad-
hesive properties of CDs. Non-covalent bonds also play a signifi-
cant role in mucus-oligomer interaction, so anionic thiolated
oligomers showed less pronounced adhesion time on mucosal
surface due to lack of ionic interaction [42]. In contrast to cationic
thiolated oligomers, anionic oligomers must overcome the electro-
static repulsion to attach to mucus layer [43].
Mucoadhesion study of thiolated and S-protected c-CD on por-
cine intestinal mucosa confirmed that this type of thiomer shows
strongly improved mucoadhesive properties. In detail, c-CD-SH
and c-CD-SS-MNA exhibited 44.4- and 50.9- fold higher mucoad-
hesion in comparison to unmodified c-CD, respectively.
As shown in Fig. 6, more than 44% of thiolated and more than
51% S-protected c-CD remained on the intestinal mucosa after
3 h. The unmodified c-CD washed off the mucosa in a considerable
short time period. These data demonstrate that c-CD-SH and c-CD-
SS-MNA are able to adhere and remain for longer period of time on
the intestinal mucosa.
4. Conclusion
In this study novel thiolated S-protected c-CDs with intact ring
structure were developed. These novel conjugates were biodegrad-
able, non-mucolytic and showed 60-fold improvement in mucoad-
hesion as compared to unmodified oligomer. Conjugates, were
furthermore non-toxic as compared to cationic ring-opened thio-
lated oligomers (a and b-CD) [10,44]. The ligands had no toxic
effect on cells even in high concentration (1%), while the first gen-
eration thiolated CDs showed a time and concentration dependent
toxicity [12]. These favorable properties will likely to be useful for
these novel S-protected c-CDs for localized drug delivery on muco-
sal surfaces.
Acknowledgements
This work was supported by the Higher Education Commission
(HEC), Pakistan and the Austrian Agency for International Cooper-
ation in Education and Research (OeAD), Austria.
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