Characterization of muco- and bioadhesive properties of chitosan, PVP, and chitosan/PVP blends and release of amoxicillin from alginate beads coated with chitosan/PVP.

Abstract

To investigate the muco/bioadhesive properties of chitosan, polyvinylpyrrolidone (PVP), and chitosan/PVP blends and the release of amoxicillin (AMX) contained in AMX-alginate beads coated with these materials.
Chitosan, PVP, and chitosan/PVP blends at various volume ratios were coated onto calcium alginate beads containing AMX. The muco/bioadhesive properties of all materials and the AMX-alginate beads coated with these materials were characterized.
Measurements of their viscosity, texture, and adhesion to HT29 cells demonstrated that chitosan/PVP at a volume ratio of 5/5 had the best muco/bioadhesive properties when compared with chitosan, PVP, and blends of other ratios. Wash-off tests indicated that the mucoadhesive property of the coated AMX-alginate beads was significantly higher than that of the uncoated beads. Diffuse reflectance infrared Fourier transform spectroscopy showed that there were interactions between chitosan-PVP, chitosan-mucin, PVP-mucin, and chitosan/PVP blend-mucin. Scanning electron microscopy revealed that the surfaces of the coated beads were smoother than those of the uncoated beads. All coated AMX-alginate beads were able to provide a controlled release of AMX with Super Case II transport properties, at a pH of 4. This was probably a result of the rapid and extensive swelling of the alginate beads. The more rapid release of AMX at pH 1 was probably because of the rapid dissolution of the drug at this pH.
From the controlled drug release and muco/bioadhesive properties of these coated AMX-alginate beads, we suggest that the alginate-coated beads might be a promising drug delivery system to assist with the eradication of Helicobacter pylori infections.

Full text

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408
Drug Development and Industrial Pharmacy, 2011, 37(4): 408–418
© 2011 Informa Healthcare USA, Inc.
ISSN 0363-9045 print/ISSN 1520-5762 online
DOI:10.3109/03639045.2010.518149
LDDIORIGINAL ARTICLE
Characterization of muco- and bioadhesive properties
of chitosan, PVP, and chitosan/PVP blends
and release of amoxicillin from alginate beads
coated with chitosan/PVP
Muco- and bioadhesive properties of chitosan/PVP blends
Krit Suknuntha1, Vimon Tantishaiyakul1, Nimit Worakul2 and Wirach Taweepreda3
1Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai,
Songkhla, Thailand, 2Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla
University, Hat-Yai, Songkhla, Thailand and 3Department of Material Science, Faculty of Science, Prince of Songkla
University, Hat-Yai, Songkhla, Thailand
Abstract
Purpose: To investigate the muco/bioadhesive properties of chitosan, polyvinylpyrrolidone (PVP), and
chitosan/PVP blends and the release of amoxicillin (AMX) contained in AMX-alginate beads coated with
these materials. Method: Chitosan, PVP, and chitosan/PVP blends at various volume ratios were coated
onto calcium alginate beads containing AMX. The muco/bioadhesive properties of all materials and the
AMX-alginate beads coated with these materials were characterized. Results: Measurements of their viscosity,
texture, and adhesion to HT29 cells demonstrated that chitosan/PVP at a volume ratio of 5/5 had the best
muco/bioadhesive properties when compared with chitosan, PVP, and blends of other ratios. Wash-off
tests indicated that the mucoadhesive property of the coated AMX-alginate beads was significantly higher
than that of the uncoated beads. Diffuse reflectance infrared Fourier transform spectroscopy showed that
there were interactions between chitosan–PVP, chitosan–mucin, PVP–mucin, and chitosan/PVP blend–
mucin. Scanning electron microscopy revealed that the surfaces of the coated beads were smoother than
those of the uncoated beads. All coated AMX-alginate beads were able to provide a controlled release of
AMX with Super Case II transport properties, at a pH of 4. This was probably a result of the rapid and extensive
swelling of the alginate beads. The more rapid release of AMX at pH 1 was probably because of the rapid
dissolution of the drug at this pH. Conclusions: From the controlled drug release and muco/bioahesive
properties of these coated AMX-alginate beads, we suggest that the alginate-coated beads might be a
promising drug delivery system to assist with the eradication of Helicobacter pylori infections.
Key words: Amoxicillin, bioadhesion, chitosan, coated alginate bead, mucoadhesion, polyvinylpyrrolidone
Introduction
Helicobacter pylori, a gram-negative bacterium, is asso-
ciated with chronic gastritis, peptic ulcer disease, and
gastric cancer1. The short residence time of antibiotics in
the stomach is the main reason for the ineffectiveness of
antibiotics for the eradication of H. pylori2. It has been
recently demonstrated that H. pylori infections can be
successfully treated if antibiotics are retained in the
stomach for 1 hour3.
Because of its mucoadhesive properties, chitosan can
improve the bioavailability of drugs when used in
mucoadhesive dosage forms4. Polyvinylpyrrolidone (PVP),
a common drug excipient, has also been used in
mucoadhesive formulations. The mucoadhesion capa-
bility of PVP is enhanced when it is blended with other
polymers5,6. In recent years, polymer blends have
attracted considerable attention, because blending is a
simple and effective method to develop new materials
with specific properties. Blends of chitosan/PVP may
Address for correspondence: Vimon Tantishaiyakul, PhD, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla
University, Hat-Yai, Songkhla 90112, Thailand. Tel: +66 7428 8864, Fax: +66 7442 8239. E-mail: vimon.t@.psu.ac.th
(Received 24 Mar 2010; accepted 19 Aug 2010)

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© 2011 Informa Healthcare USA, Inc.
Muco- and bioadhesive properties of chitosan/PVP blends 409
have enhanced mucoadhesive properties as compared
with the individual polymers.
To date, several approaches have been investigated
to increase the gastroretentive time of drugs for clear-
ance of H. pylori. These included the use of beads7
and floating mucoadhesive material-coated beads8−10,
muco/bioadhesive microspheres11−17, and mucoadhe-
sive nanoparticles18. Previous studies have demon-
strated that mucoadhesive material-coated beads
exhibited high mucoadhesive properties19,20 and could
control the release of the drug20. In this study, a new
mucoadhesive bead was prepared using an amoxicillin
(AMX)-loaded alginate core that was subsequently
coated with chitosan/PVP blends. Beads coated with
chitosan and PVP separately were also prepared for
comparison. Alginate beads can be produced simply in
the presence of calcium ions20−22. AMX, which is typi-
cally used for the treatment of H. pylori infections, was
selected as a model drug. Alginate-AMX-coated beads
could increase the residence time of AMX in the stom-
ach, thereby increasing its effectiveness in the treatment
of H. pylori infections. In addition, such coated beads
may be useful for gastroretentive delivery of various
other drugs that either act locally or are predominantly
absorbed from this site. The muco- or bioadhesive prop-
erties of chitosan, PVP, and chitosan/PVP blends were
evaluated using appropriate techniques including vis-
cosity measurement, texture analysis, and an HT29 cell
adhesion assay. Wash-off tests were performed to inves-
tigate the mucoadhesion of uncoated and coated
AMX-alginate beads. The interactions between chito-
san–PVP, chitosan–mucin, PVP–mucin, and chitosan/
PVP blends–mucin were examined using the diffuse
reflectance infrared Fourier transform spectroscopy
(DRIFTS) technique. The surface morphologies of AMX-
alginate beads were investigated using scanning elec-
tron microscopy (SEM). The AMX-loaded beads were
also subjected to a swelling study and an in vitro dissolu-
tion study and the mechanism of transport determined.
Materials and methods
Materials
Chitosan was obtained from Fluka (GmbH, Buchs,
Switzerland). Its degree of deacetylation was 75–85% and
average molecular weight was 300,000–500,000 Da. Alginic
acid sodium salt from brown algae (viscosity 250 cps, 2%
solution at 25°C), mucin type 2 from porcine stomach,
and AMX were purchased from Sigma (St. Louis, MO,
USA). PVP K-90 (Kollidon 90), with an average molecular
weight of 1,100,000, was kindly supplied by BASF,
Bangkok, Thailand. All other reagents were of analytical
grade.
Polymer blend and mucin preparation
The chitosan solution (2%, w/v) was prepared by dissolv-
ing chitosan (2.0 g) in 0.05 M hydrochloric acid (100 mL).
PVP solution (2%, w/v) was prepared by dissolving 2.0 g
of PVP in water (100 mL). Mucin solutions at concentra-
tions of 2% (w/v) and 15% (w/v) were prepared by dis-
persing the appropriate amount of mucin in water.
Polymer blends of chitosan and PVP were prepared by
mixing the 2% (w/v) solutions of the two polymers at the
volume ratios of 1/9, 3/7, 5/5, 7/3, and 9/1. All polymer
blends were mixed gently until homogeneous using a
reciprocating shaker.
Viscosity measurements
For viscosity measurements, 5 mL of mucin solution
(15%, w/v) was mixed with 2 mL of (2%, w/v) polymer
or blend solutions (chitosan, PVP, and chitosan/PVP
blends). The final volume of the mixtures was adjusted
to 8 mL with water, and the mixtures were mixed using a
reciprocating shaker until homogeneous. The final con-
centrations of polymer and mucin solutions were 0.50%
(w/v) and 9.38% (w/v), respectively. These mixtures of
mucin–polymers and mucin–polymer blends were
equilibrated at 25.0 ± 0.1°C for 1 hour before viscosity
measurements.
All viscosity measurements were performed using a
Brookfield model DV-III Ultra programmable viscome-
ter (Brookfield Engineering Laboratories, Inc., Middle-
boro, MA, USA), equipped with a SC4-18 spindle and a
small sample adaptor at 25.0 ± 0.1°C. Samples were
allowed to equilibrate for 1 minute before testing. The
apparent viscosity at a shear rate of 15.84 per second was
selected for analyses. All viscosity measurements were
performed in triplicate, and data reported as a mean ± SD.
Texture analysis
Chitosan, PVP, and chitosan/PVP blend films were pre-
pared by casting the polymer or the polymer blend solu-
tions on polystyrene plates and dried in an oven at 60°C
for 8 hours. These film samples were used for texture
analyses.
A texture analyzer, TA-XT2 (Stable Micro System,
Haslemere, UK) equipped with a 5 kg load cell and
mucoadhesive rig, was used for all tensile strength mea-
surements. All polymer blend films were cut in circular
shapes (diameter 1 cm) and fixed on the upper probe of
the instrument using double-sided adhesive tapes. Por-
cine stomach was obtained from the animal immedi-
ately after slaughter at the local slaughterhouse (Faculty
of Natural Resource, Prince of Songkla University, Hat-
Yai, Thailand). The tissue was thoroughly washed with
deionized water to remove nondigest food, kept at 4°C,
and used within 6 hours. Stomach tissue was cut into
square shapes (3 × 3 cm), and one square was fixed on a
mucoadhesive rig on the stage of the instrument with the
mucus layer upward. The film previously attached on the
upper probe was wetted with deionized water (50 μL) and
then moved lower at a constant speed of 1 mm/s to make
contact with the tissue. The contact of the film and the
tissue was maintained for 2 minutes with a 0.08 N force.

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Drug Development and Industrial Pharmacy
410 K. Suknuntha et al.
The film was then slowly moved upward at a constant
speed of 1 mm/s until the film was detached from the
mucus layer. Force versus distance curves, during the
upward movement of the film, were obtained directly
from Texture Expert software. The areas under these
curves were calculated as the work of mucoadhesion.
These analyses were replicated 6–10 times and data
reported as a mean ± SD.
In vitro cell adhesion assay
HT29 cells (passage 121–128) were obtained from American
Type Culture Collection (ATCC, Manassas, VA, USA).
The cells were grown and subcultured in Dulbecco’s
Modified Eagle’s Medium containing 10% fetal calf serum,
1% nonessential amino acids, and 1% L-glutamine. Incu-
bation was at 37°C in an atmosphere of 5% CO2, 95% O2.
Chitosan, PVP, and chitosan/PVP blends (100 μL)
were cast on an ultra low attachment 96-well plate
(Corning Costar Cat. #3474, Lowell, MA, USA). The plate
was then dried in an oven at 40°C for 8 hours. Subse-
quently, 100 μL of a trypsinized HT29 cell suspension
was added into each well. The plate was then incubated
for 3 hours to allow attachment of the cell to the poly-
mer. The cells were then washed with phosphate buffer
saline three times to remove nonattached cells. Cells
attached onto the polymer were quantified using the
MTT test [3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetra-
zolium bromide]. In brief, the MTT solution (100 μL) was
added into each well, and the plate incubated for
3 hours. Formation of the purple formazan from MTT
(catalyzed by the mitochondrial reductase in living cell)
was dissolved with DMSO (100 μL) and quantified using
a DTX-880 multimode detection microplate reader
(Beckman Coulter, Fullerton, CA, USA) at 570 nm. The
percentage relative cell attachment was calculated by
subtracting the experimental value for polymer-coated
cells from that of the corresponding value for non-polymer-
coated cells. This was then compared with the data for
non-washed cells; cell attachment with the non-washed
cells was considered to be 100%.
Uncoated and coated calcium alginate beads
preparation
Alginate beads were prepared as previously described20
with some modifications. The alginate solution was
prepared by stirring the mixture of alginate (2.0 g) and
water (100 mL) for 4 hours. AMX (5.0 g) was then dis-
persed in the alginate solution and the mixture continu-
ally stirred for 20 minutes. Subsequently, the mixture
was added dropwise using a syringe with 23-gauge needle
into a gently agitated solution of calcium chloride
(2%, w/v). After continuous stirring for 10 more minutes,
the beads were separated by filtering through a sieve
(mesh size ∼250 μm), washed with water, and then dried
in an oven at 40°C for 8 hours. The dried alginate beads
were then coated with 1.5% (w/v) of chitosan, PVP, or
chitosan/PVP blend solutions by immersing the dried
beads into these solutions for 10 minutes. The coated
beads were then dried in an oven at 40°C for 8 hours.
Wash-off test for evaluation of the mucoadhesive
property of alginate beads
The in vitro evaluation of the mucoadhesive properties
of uncoated and coated AMX-alginate beads was carried
out using the wash-off test from a porcine stomach, as
previously described23,24. The freshly slaughtered por-
cine stomach was washed with normal saline, cut into
rectangular shapes (∼2.5 × 4 cm), and attached onto a
microscopic slide. Thirty uncoated or coated beads were
brought into direct contact with the mucus layer of the
stomach tissues using a pressure of 25 g on the glass
slide for 2 minutes. The mucoadhesiveness of the beads
was determined by connecting the slide with the arm of
QC-21 disintegration test system (Hanson Research,
Chatsworth, GA, USA). The wash-off of the beads was
induced by the reciprocating motion of the disintegra-
tion apparatus in 8000 mL phosphate buffer (pH 4.0) at
37 ± 0.5°C. The remaining beads were counted every
30 minutes for 3 hours, and this data were used to calcu-
late the percentage of bead attachment. These tests were
performed in triplicate, and data reported as a mean ± SD.
DRIFTS measurement
To study the interactions between mucin and polymer as
well as the mucin and polymer blends, the solutions of
chitosan, PVP, or chitosan/PVP blends (2%, w/v) were
mixed with mucin solution (2%, w/v) in a 1/1 volume
ratio. These samples were then dried in an oven at 60°C
for 8 hours before DRIFTS measurement.
DRIFTS spectra were measured using a PerkinElmer
Spectrum One FTIR spectrometer (Perkin-Elmer,
Waltham, MA, USA) and a PerkinElmer DRIFTS acces-
sory. The samples were finely ground in an agate mortar
and placed in the microsample cup of the DRIFTS acces-
sory using the supplied sample cup holder. The spectra
were recorded from 4400 to 450 cm−1 by averaging
64 scans at a 4 cm−1 resolution. The reflectance spectra
were converted to Kubelka–Munk units using a Perki-
nElmer Spectrum for Windows version 5.02 software
package.
Scanning electron microscopy
The morphologies of uncoated and coated AMX-algi-
nate beads were observed using a JSM-6400 scanning
electron microscope (Jeol, Tokyo, Japan) at an acceler-
ated voltage of 15 kV. All samples were coated with gold
using a direct current sputter technique.
Drug loading
Drug loading was evaluated by transferring about 15 mg
of the AMX-alginate dried beads into a volumetric flask
(100 mL). Phosphate buffer (80 mL, 0.1 M, pH 4) was
added and the solution then stirred using a magnetic
stirrer for 5 hours. The magnetic bar was removed and

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© 2011 Informa Healthcare USA, Inc.
Muco- and bioadhesive properties of chitosan/PVP blends 411
thoroughly rinsed with phosphate buffer into the volu-
metric flask, and the phosphate solution was then added
to the scale. The solution was filtered and the filtrate ana-
lyzed using an 8452A HP Diode Array spectrophotometer
(Hewlett Packard, Palo Alto, CA, USA) at 230 nm. All the
experiments were carried out in triplicate. The percentage
of drug loading was calculated using Equation (1):
where Wa is the analyzed AMX content and Wb is the
theoretical content of AMX in the beads.
Swelling study of dried beads
Swelling studies of uncoated and coated AMX-alginate
dried beads were performed in 0.1 M phosphate buffer
(pH 4) at 37.0 ± 0.5°C. Accurately weighed beads (15.2–
16.40 g) were immersed in the buffer solution with slight
agitation with a shaker. The beads were removed period-
ically from the buffer solution, blotted to remove excess
liquid, and weighed on an electronic balance. The swell-
ing ratio or percentage of weight change was determined
using Equation (2) as previously described22:
where Wt is the weight of the swollen beads at time t and
Wd is the weight of the dried beads.
Dissolution study
The release of AMX from uncoated and coated beads
was determined using a VK 7000 dissolution tester
(Vankel Industries, Edison, NJ, USA) with a USP27 appa-
ratus 2 at pH 4 and also at a pH of 1.2. Phosphate buffer
(pH 4), consisting of 0.1 M KH2PO4, using either KOH or
phosphoric acid to adjust the pH to 4.0, was prepared as
the dissolution medium. The dissolution medium at pH
1.2 was prepared by dissolving 2 g NaCl in 7 mL HCl and
adding water to 1000 mL. This medium (200 mL) was
maintained at 37.0 ± 0.5°C during the test. Approxi-
mately 20 mg of beads were used in each experiment.
Samples (5 mL) were taken at 6, 12, 18, 24, 30, 40, 60, 90,
120, and 180 minutes and replaced with 5 mL of fresh
medium. The amount of AMX in collected samples at pH
4 was determined using an 8452A HP Diode Array spec-
trophotometer (Hewlett Packard) at 230 nm. High-
performance liquid chromatography (HPLC) was also
performed to check the possibility of degraded products
of AMX, which might interfere with the analysis of AMX
using the simple UV assay. However, no degradation
peaks were detected for samples at the pH of 4. Thus, at
this pH the more simple UV spectrometric method was
used for the analysis of all dissolution samples.
Degraded products were observed when using the
dissolution medium pH 1.2 and the HPLC method was
used for analysis of all samples at this pH. The HPLC
analyses were performed according to the conditions
specified by USP 30, using a Jasco PU-2080 plus an intel-
ligent HPLC pump, Jasco UV-1575 intelligent UV/Vis
detector setting at 230 nm, and a Waters 746 Data Mod-
ule. The mobile phase involved a mixture of phosphate
buffer pH 5 and acetonitrile (95:5, v/v), pumped at a flow
rate of 1.5 mL/min through the column, a Phenomenex
C18 (250 × 4.6 mm).
The amount of AMX in each type of coated beads was
determined after extracting the drug from the individual
type of beads using sonication. The AMX concentration
in each dissolution sample was determined as the per-
centage of the drug load for each type of sample and
expressed as a cumulative percentage of the released
AMX. These dissolution tests were performed in triplicate.
Statistical analysis
One-way ANOVA with LSD test using SPSS 10.0 was
applied to investigate the difference of the mean values
for data obtained from the measurements.
Results and discussion
The mucoadhesion of chitosan, PVP, and chitosan/PVP
blends was evaluated using various techniques. Mucoad-
hesion is a complex phenomenon, and its mechanisms
are not clearly understood25. The proposed mechanisms
for mucoadhesion probably involve one or more of the
following: electronic, adsorption, diffusion, and wetting
theories26. The electronic theory explains that electron
transfer between the mucus and the mucoadhesive leads
to the formation of a double electric layer at the interface
of the mucus and the mucoadhesive, resulting in an
attraction between these two components. The adsorp-
tion theory proposes that intermolecular bonding, such
as hydrogen and van der Waals bonds, causes the attrac-
tion forces between the mucus and the mucoadhesive,
these forces being higher than those involved in the elec-
tronic theory. The diffusion theory envisages the inter-
penetration and physical entanglement of the mucus
and the mucoadhesive. The wetting theory deals with
interfacial energy and correlates the surface tension of
the mucus and the mucoadhesive with the capability of
the mucoadhesive to swell and spread on the mucus
layer. In fact, not a singular mechanism, but a combina-
tion of these theories is usually applied to describe
mucoadhesion.
Viscosity synergism of polymers and mucin
Determination of mucoadhesion by viscosity measure-
ments was performed as previously described by Hassan
and Gallo27. The interactions between polymers (chitosan,
PVP, and chitosan/PVP blends) and mucin resulted in
viscosity changes; these changes can be converted into
% drug loading
a
b
= W
W
×100,
(1)
% weight change
t d
t
= W W
W
×
−
100,
(2)

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Drug Development and Industrial Pharmacy
412 K. Suknuntha et al.
mechanical energy or work. The viscosity component
caused by mucoadhesion (had) can be calculated accord-
ing to Equation (3):
where ht is the viscosity of the system, hm and hp are the
viscosity of pure mucin and polymer, respectively. All
viscosity values were measured at the same temperature
and rate of shear. Subsequently, the force of mucoadhe-
sion (F) was determined using Equation (4):
where s is shear rate (per second) and F is the force of
mucoadhesion.
The viscosities and mucoadhesion forces of chitosan,
PVP, and chitosan/PVP blends are listed in Table 1. The
had values and forces of mucoadhesion for chitosan/PVP
at volume ratio of 5/5 and 3/7 are significantly higher
than for the other blends, and for the single polymers
(P < 0.05). This indicated that the chitosan/PVP blends
at these ratios (5/5 and 3/7) are able to interact more
strongly with mucin.
In addition, the flow behavior of chitosan, PVP, and
chitosan/PVP blends in the presence or the absence of
mucin was calculated using the Ostwald–de Waele rheo-
logical model or power law model (Equation 5):
where t is shear stress, g shear rate, Kc consistency index,
and n the power law index or flow behavior index. The
values of Kc and n of all samples were determined using
Rheocalc for the Windows version 3.1 software package.
The n value describes the deviation from Newtonian
behavior. When the value of n is lower than 1, the type of
flow is shear thinning (pseudoplastic behavior). The
type of flow is shear thickening (dilatant behavior) when
n is greater than 1. As n approaches 1, the flow becomes
less shear dependent, and the n value equal to 1 indi-
cates Newtonian flow. The values of n and Kc of all sam-
ples are listed in Table 2. The n values of all samples are
less than 1 indicating that these samples exhibit a non-
Newtonian shear thinning behavior. In addition, greater
Kc values were observed for chitosan, PVP, and chito-
san/PVP blends with mucin, demonstrating good viscos-
ity synergism and interactions between these polymers
and mucin.
Texture analysis
Viscosity measurement is a relatively simple method for
evaluation of mucoadhesion. However, it does not give
any direct information on events occurring at the inter-
face. Texture analysis is based on measuring the force or
work required to detach the polymers from the mucus
layer on the tissue. Measurement of such force is there-
fore more useful because texture analysis more closely
simulated the in vivo situation28. Texture analysis was
therefore performed to confirm the mucoadhesion
properties of the polymers. For this analysis, the
mucoadhesion force of polymers on the porcine stom-
ach tissue was measured, and the work of adhesion was
subsequently evaluated. The work of adhesions of chito-
san, PVP, and chitosan/PVP blends is shown in Figure 1.
The high mucoadhesion of polymer and polymer blends
may arise from the entanglement of polymer chains and
the mucin molecules, or the formation of non-covalent
bonds between the polymers and mucin. The work of
adhesion of chitosan/PVP blend at the 5/5 volume ratio
is significantly higher than chitosan, PVP, and the other
blends (P < 0.05). PVP shows the lowest mucoadhesion
(P < 0.05), which is consistent with the results obtained
from viscosity evaluation.
Wash-off test of uncoated and coated AMX-alginate
beads
The data for the percentage attachment of uncoated and
coated AMX-alginate beads to the mucosa at pH 4.0 are
presented in Figure 2. Even though not statistically sig-
nificant, it was found that the beads coated with chito-
san/PVP at a 5/5 volume ratio exhibited the slowest
wash-off after 3 hours. The percentage of coated beads
still attached to the mucosa is higher than for the
uncoated beads (P < 0.05). The results of the wash-off
test indicated that beads coated with chitosan, PVP, and
hhhh
ad t m p
= − −
,
(3)
F = h s
ad ,
(4)
Table 1. Viscosity of a 9.38% (w/v) mucin solution (hm) plus 0.50% (w/v) polymer solution (hp), the system (ht), and viscosity due to mucoadhesion
(had) as well as the force of mucoadhesion (F) of chitosan (C), PVP (P), and C/P blends at various volume ratios at 25°C using a shear rate of 15.84
per second (mean ± SD, n = 3).
Mucin + polymer
C
C9P1
C7P3
C5P5
C3P7
C1P9
P
Viscosity F
hm + hp
120.78 ± 10.42
131.39 ± 0.14
129.14 ± 0.29
127.39 ± 0.29
126.30 ± 0.14
124.22 ± 0.25
122.72 ± 0.02
ht
had
(dyne/cm)
2.44 ± 0.11
2.21 ± 0.08
2.32 ± 0.04
4.35 ± 0.11
3.98 ± 0.30
2.54 ± 0.48
1.23 ± 0.14
136.19 ± 9.72
145.40 ± 0.05
143.78 ± 0.03
154.82 ± 0.07
151.42 ± 0.19
140.24 ± 0.30
130.58 ± 0.09
15.41 ± 0.41
13.98 ± 0.05
14.66 ± 0.03
27.46 ± 0.07
25.12 ± 0.19
16.06 ± 0.30
7.82 ± 0.09
tg= Kc
n ,
(5)

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© 2011 Informa Healthcare USA, Inc.
Muco- and bioadhesive properties of chitosan/PVP blends 413
their blends have relatively strong mucoadhesive prop-
erties. Therefore, beads coated with these single poly-
mers and their blends may be able to provide the
necessary gastroretention to facilitate local drug avail-
ability in the stomach for a sufficient length of time.
Cell adhesion study
Because HT29 cells are not covered with a mucus layer,
the adherence of polymers to this monolayer may be
considered as bioadhesion29. Attachment of HT29 cells
to chitosan, PVP, and chitosan/PVP blends is shown in
Figure 3. The single polymers or the blends exhibit
higher bioadhesion than the controls (without polymer).
The chitosan/PVP blend at 5/5 volume ratio showed the
highest cell attachment (P < 0.05). Thus, this blend dem-
onstrates both highest mucoadhesion (adherence to
mucus) and highest bioadhesion (adherence to epithe-
lium), compared with the single polymers or other
blends. This blend is therefore likely to be in intimate
contact with biological tissue for an extended period of
time and should be able to increase its gastrointestinal
residence time even in the presence of mucus turnover.
Molecular interaction study
DRIFTS was used to investigate the interactions between
chitosan and PVP in the blend, and between chitosan,
PVP, or chitosan/PVP blends and mucin. In view of the
large numbers of samples and spectra, only FTIR analyses
of chitosan, PVP, and chitosan/PVP blend at 5/5 volume
Table 2. Power law index (n) and consistency index (Kc) derived from Equation (5) of chitosan (C), PVP (P), and C/P blends at various volume
ratios with and without mucin (mean ± SD, n = 3).
Sample
Mucin
C
C9P1
C7P3
C5P5
C3P7
C1P9
P
Flow behavior index, n
Consistency index (Kc)
Polymer
–
0.68 ± 0.01
0.59 ± 0.01
0.60 ± 0.01
0.38 ± 0.01
0.51 ± 0.02
0.33 ± 0.02
0.63 ± 0.02
Polymer + Mucin
0.70 ± 0.04
0.58 ± 0.05
0.58 ± 0.08
0.64 ± 0.01
0.61 ± 0.01
0.57 ± 0.05
0.62 ± 0.07
0.65 ± 0.05
Polymer
–
35.32 ± 1.29
44.61 ± 0.98
36.44 ± 1.62
66.86 ± 3.18
36.57 ± 3.17
57.82 ± 4.04
13.95 ± 0.69
Polymer + Mucin
251.26 ± 32.94
419.19 ± 81.90
423.28 ± 120.80
335.65 ± 7.29
376.68 ± 8.30
438.38 ± 86.77
366.78 ± 91.74
316.44 ± 63.34
Figure 1. The work of adhesion for chitosan (C), PVP (P), and C/P
blends at various volume ratios on porcine stomach tissue (mean ±
SD, n = 6–10).
Work of adhesion (N/mm2 )
1.0
0.8
0.6
0.4
0.2
0
C C9P1 C7P3 C5P5 C3P7 C1P9 P
Figure 2. In vitro wash-off test for uncoated beads and coated beads with chitosan (C), PVP (P), and C/P blends at various volume ratios (mean ±
SD, n = 3).
100
80
60
0
0
50 100
Time (minutes)
Uncoated bead
C
C9P1
C7P3
C5P5
C3P7
C1P9
P
150 200
20
40
Bead remaining attached (%)

Page 7

Drug Development and Industrial Pharmacy
414 K. Suknuntha et al.
ratio (the blend that has the strongest interaction with
mucin) with and without mucin are reported here.
DRIFTS spectra of these samples are shown in Figure 4a.
DRIFTS spectra for the other blends with and without
mucin showed the same trends as for the blend at 5/5
volume ratio (data not shown). As shown in Figure 4a,
the O–H and N–H stretching vibrations of mucin and
chitosan show broad bands at 3500–3100 cm−1; this
makes spectral interpretation at this region difficult.
However, prominent peaks observed in the region of
1600–1700 cm−1 can be used for assessing possible inter-
actions. Mucin is comprised of a peptide backbone, con-
sisting of alternating oligosaccharides that terminate
with sialic acid and sulfate groups30. The DRIFTS spec-
trum of mucin showed a broad C=O stretching band of
an amide band at 1692.5 cm−1. The spectra of chitosan
and PVP show an amino band and a C=O peak at 1650.8
and 1697.8 cm−1, respectively (Figure 4b). The blend of
chitosan/PVP at 5/5 volume ratio displayed a peak at
1685.7 cm−1 (Figure 4b). This peak is due to the interac-
tion between C=O of PVP and H–O or N–H of chitosan,
as previously reported31. Because of different methods of
sample preparation, the positions of these peaks are
slightly different. The band at 1685.7 cm−1 was shifted to
1696.1 cm−1 (Figure 4c) when this blend was mixed with
mucin. This indicates that there are interactions
between the chitosan/PVP blend and mucin. The mix-
ture of PVP and mucin displayed a new peak at 1696.4
cm−1 (Figure 4d); this peak is relatively broad compared
with that of PVP. The mixture of chitosan and mucin also
displays a new peak at 1689.3 cm−1 (Figure 4e). These
peaks are not the combined spectra of PVP and mucin,
or chitosan and mucin. This indicates that there are
interactions between the PVP–mucin and the chitosan–
mucin. Mucoadhesive interactions may be due to hydro-
gen bonding between mucin and chitosan, PVP, or the
blends through their various hydrogen donor and acceptor
groups. The shift of the C=O band of PVP indicates that
this group is involved in an intermolecular hydrogen
bond with mucin. In case of chitosan, the electrostatic
interaction between the amine function of chitosan and
sialic acid or sulfonated residues of mucin may be possi-
ble. Nevertheless, due to the fact that most functional
groups of these molecules cannot be observed on
DRIFTS spectra, it is not possible to indicate the other
specific key sites for these intermolecular interactions.
Undoubtedly, DRIFTS can indicate that there are inter-
actions between polymers, or polymer blends and
mucin and these interactions may produce mucoadhe-
sion as demonstrated in the above-mentioned analyses.
Uncoated and coated bead morphology
The diameter of uncoated AMX-alginate beads mea-
sured with a Beckman Coulter LS230 (Vankel Industries,
Edison, NJ, USA) equipped with a Small-Volume Module
Plus and Beckman Coulter Particle Characterization
software version 3.29 (USA) was 1.23 ± 0.25 mm. SEM
micrographs of uncoated and coated AMX-alginate
beads are shown in Figure 5. The uncoated beads had a
rough surface (Figure 5a and b). The surfaces of beads
coated with chitosan/PVP blend, chitosan, and PVP
were smoother than the uncoated beads (Figure 5c–i).
Figure 3. Percentage relative HT29 cell attachment of chitosan (C),
PVP (P), and C/P blends at various volume ratios (mean ± SD, n = 3).
100
80
60
40
20
Relative cell attachement (%)
0
C C9P1 C7P3 C5P5 C3P7 C1P9 P
Figure 4. DRIFTS spectra of (a) chitosan (C), PVP (P), mucin (M),
C5P5, C5M5 P5M5, C5P5M5; and the enlarged spectra of (b) C5P5; (c)
C5P5M5; (d) P5M5, and (e) C5M5.
C
P
C5P5
M
P5M5
C5P5M5
(a)
(b)
(c)
(d)
(e)
1800 1750 1700
1696.4
1689.3
P
M
P5M5
C
M
C5M5
1650
cm–1
cm–1
cm–1
cm–1
1600 1550 1500 1800 1750 1700 1650 1600 1550 1500
1750 1700 1650 1600 1550 1800 1500 1800 1750 1700 1650 1600 1550 1500
3000
1685.7
1696.1
C
P
C5P5
C5P5
M
C5P5M5
2500
cm–1
2000 15003500 1000
C5M5

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© 2011 Informa Healthcare USA, Inc.
Muco- and bioadhesive properties of chitosan/PVP blends 415
There were no visible porous characteristics for uncoated
and coated beads (Figure 5a–i).
Swelling studies
The swelling behavior of the uncoated and coated AMX-
alginate beads in phosphate buffer (pH 4) (Figure 6)
shows that all beads demonstrate significant swelling.
The swelling of coated beads was higher than that of
uncoated beads (P < 0.05). The swelling of beads coated
with chitosan, PVP, or chitosan/PVP blends was similar.
The hydration of the hydrophilic groups of chitosan and
PVP may induce higher swelling of the coated beads
compared with uncoated beads, as has been previously
observed for chitosan-coated dried beads22.
Drug release study
The drug loading for the AMX bead preparation, calcu-
lated for the uncoated beads, was 76.49%. Commonly,
proton pump inhibitors or H2-receptor antagonists such
as lansoprazole, omeprazole, or ranitidine are used in
combination with antibiotics for eradication of H.
pylori32,33. These agents can raise the gastric pH to 3–534−36.
Hence, dissolution studies were investigated at pH 4.
AMX release from uncoated and coated beads was com-
pared with that from an AMX powder as shown in Figure 7a.
AMX release profiles from beads showed sustained
release characteristics when compared with the release
from the AMX powder. The AMX release profiles for
uncoated and coated beads were not statistically different
(P > 0.05). The release of AMX from these beads was
complete within 2.5 hours. The release kinetics of AMX
from the beads were evaluated using the Korsmeyer–
Peppas equation or power law37:
where Mt and M¥ are the cumulative amounts of drug
released at time t and infinite time, respectively, k is the
Figure 5. SEM micrographs of AMX beads: uncoated bead (a, b); chitosan/PVP-coated beads at the volume ratios of 1/9 (c); 3/7 (d); 5/5 (e); 7/3 (f);
9/1 (g); chitosan-coated bead (h); and PVP-coated bead (i).
(a)
SUT 20 KV
×60 39mm
00 µm
SUT
SUT
20 KV
20 KV
× 750 39 mm
10 µm
× 750 39 mm
10 µm
SUT
20 KV
× 750 39 mm
10 µm
SUT
20 KV
× 750 39 mm
10 µm
SUT
20 KV
× 750 39 mm
10 µm
SUT
20 KV
× 750 39 mm
10 µm
SUT
20 KV
× 750 39 mm
10 µm
SUT
20 KV
× 750 39 mm
10 µm
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Figure 6. Swelling profiles of uncoated dried beads and dried beads
coated with chitosan (C), PVP (P), and C/P blends at various volume
ratios at pH 4 (mean ± SD, n = 3).
1400
1200
Uncoated bead
C
C9P1
C7P3
C5P5
C3P7
C1P9
P
1000
800
600
400
200
0
0 50 100
Time (minutes)
Weight change (%)
150 200 250
M
M
t
t
n
∞
= k
(5)

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Drug Development and Industrial Pharmacy
416 K. Suknuntha et al.
release constant, and n is the release exponent charac-
terizing the release mechanism. This equation is used
for examining the first 60% of a release curve. The n and
k values were determined from the slope and intercept,
respectively, of the fitting straight line of log (Mt/M¥)
versus log (t) plots. Based on the n values, the drug trans-
port may be classified as Fickian diffusion (Case I), Case
II transport (zero order), non-Fickian (anomalous)
transport, and Super Case II transport38. As listed in
Table 3, the n values were higher than 1 indicating that
the release process is Super Case II transport. This type
of transport has been observed for alginate beads39. This
transport mechanism may be caused by rapid swelling
of the beads. This was in agreement with the swelling
studies that showed high swelling occurring in the
beads. As a result of the high swelling of these uncoated
or coated dried alginate beads, the coated materials have
not much influence on the release of AMX from the
beads. Thus for these dried beads, the release profiles
of uncoated or coated beads were similar. These phe-
nomena were previously observed for coated dried
beads20,22. According to the wash-off test and the cell
adhesion study, these coated beads have a reasonably
strong muco/bioadhesion. A high percentage of coated
beads, 84.4 ± 7.17, 78.25 ± 10.36, and 70.63 ± 10.50, still
remained adhering to the mucosa at 2, 2.5, and 3 hours,
respectively (Figure 2). For uncoated beads only 46.67%,
38.89%, 35.56%, 26.67%, and 16.67% remained adhering
to the mucosa at 1, 1.5, 2, 2.5, and 3 hours, respectively.
Thus, although AMX release from uncoated and coated
beads was somewhat similar, the coated beads are able
to be retained longer in the stomach than the uncoated
beads. Consequently, the longer presence of the drug
released from these coated beads may be able to eradi-
cate H. pylori infections.
To investigate the effect of pH on the release of AMX
from the beads, the dissolution test was also performed
at pH 1.2. The dissolution of AMX was actually rapid at
pH 1.2, at the first sampling time point (6 minutes), 99 ±
2.41% of AMX powder was detected. The release of AMX
from the uncoated and coated beads is faster at pH 1.2
compared with that at pH 4 (Figure 7a and b). In 30 min-
utes, about 90% and 82% of AMX was released from
uncoated and coated beads, respectively, at pH 1.2; but
only 32% and 26% was released from uncoated and
coated beads, respectively, at pH 4. Interestingly, at pH
1.2, AMX release from all coated beads was significantly
less than that observed for the uncoated beads in the
first 18 minutes (P < 0.05); this is probably due to the
lower swelling of the beads at this pH. According to the
measurement of the bead sizes at the end of the dissolu-
tion tests, the size of the swollen beads was about 1.58 ±
0.25 and 3.24 ± 0.21 mm at pH 1.2 and 4, respectively. As
a result of the lower swelling of the beads at pH 1.2, the
coated layer may not be destroyed during the early stage
of the test, and these coatings could control and retard
the release of AMX from the beads. The rapid release of
AMX with the change in medium pH from 4 to 1.2 is
probably due to the high solubility of AMX at in the
lower pH of the medium.
Conclusion
This study has demonstrated that chitosan, PVP, and
chitosan/PVP blends exhibit muco/bioadhesive proper-
ties. However, chitosan/PVP at a 5/5 volume ratio pro-
duced the best muco/bioadhesion compared with the
Figure 7. Dissolution profiles at pH 4 (a) and pH 1.2 (b) of AMX from
powder, uncoated alginate beads, and beads coated with chitosan
(C), PVP (P), and C/P blends at various volume ratios each loaded
with AMX (mean ± SD, n = 3).
120
(a)
(b)
100
80
60
40
20
0
100
80
60
40
20
0
0
20
Amoxicillin released (%)
Amoxicillin released (%)
40 60 80 100
AMX powder
Uncoated bead
C1P9
C3P7
C5P5
C7P3
C9P1
C
P
AMX powder
Uncoated bead
C1P9
C3P7
C5P5
C7P3
C9P1
C
P
Time (minutes)
120 140 160 180
0 20 40 60 80 100
Time (minutes)
120 140 160 180
Table 3. Kinetic analysis of the release data of amoxicillin derived
from Equation (6) for coated amoxicillin-alginate beads with chito-
san (C), PVP (P), and C/P blends at various volume ratios.
Coating materials
C
C9P1
C7P3
C5P5
C3P7
C1P9
P
n k (minute−1)
0.0016
0.0008
0.0026
0.0047
0.0026
0.0012
0.0040
r2
1.4762
1.6393
1.3478
1.1640
1.3500
1.5562
1.2774
0.9996
0.9905
0.9927
0.9835
0.9990
0.9939
0.9990

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© 2011 Informa Healthcare USA, Inc.
Muco- and bioadhesive properties of chitosan/PVP blends 417
other blends. These polymers and their blends when
coated onto alginate beads resulted in significantly high
mucoadhesion compared with the uncoated beads.
Therefore, the alginate beads coated with these materi-
als might have potential for being utilized to increase the
gastroretentive time of various drugs through muco- and
bioadhesion. Coated AMX-alginate beads may be useful
for more effective eradication of H. pylori infections,
especially because it has previously been demonstrated
that a 1-hour contact with antibiotics can effectively
remove these microorganisms.
Declaration of interest
This work was supported by Thailand Research Fund
through the Royal Golden Jubilee Ph.D. Program (Grant
no. PHD/0170/2547) and National Research Council of
Thailand (Grant no. PHA50162). The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of this paper.
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