Drug Development and Industrial Pharmacy, 2013; 39(4): 540–547

© 2013 Informa Healthcare USA, Inc.

ISSN 0363-9045 print/ISSN 1520-5762 online
DOI: 10.3109/03639045.2012.666978

Research article

Chitosan based mucoadhesive nanoparticles of ketoconazole

for bioavailability enhancement: formulation, optimization,
in vitro and ex vivo evaluation
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Jigar Modi, Garima Joshi, and Krutika Sawant

TIFAC Centre of Relevance and Excellence in NDDS, Centre of PG Studies and Research, Pharmacy Department,
The M. S. University of Baroda, Fatehgunj, Vadodara, Gujarat, India

Abstract
Purpose: The conventional dosage form of Ketoconazole (KZ) shows poor absorption due to rapid gastric emptying.
Chitosan based mucoadhesive nanoparticles (NPs) of KZ were developed to efficiently release drug at its absorption
window i.e. stomach and the site of action i.e. esophagus.
Method: The NPs were prepared by ionic gelation method. Concentration of polymer, cross-linking agent and ratio of
drug/polymer as well as polymer/cross linking agent were optimized.
For personal use only.

Results: NPs had 69.16 ± 5.91% mucin binding efficiency, particle size of 382.6 ± 2.384 nm, ζ potential of +48.1 mv
and entrapment efficiency of 59.84 ± 1.088%. DSC thermogram indicated absence of any drug polymer interaction.
The drug release was by controlled, non-fickian diffusion mechanism. Ex vivo diffusion studies were performed by
emptying the stomach contents after 2 h to simulate in vivo gastric emptying. The results showed that drug diffusion
from the solution across stomach mucosa stopped after emptying whereas that from the NPs continued upto 5 h.
Hence we could conclude that the NPs must have adhered to the stomach mucosa and thereby would have been
retained at this absorption site even after gastric emptying.
Conclusion: The orally delivered KZ loaded mucoadhesive NPs can be used as an efficient carrier for delivering drug at
its absorption window i.e. the stomach and the site of action i.e. esophagus even after gastric emptying.
Keywords:  Chitosan, ionic gelation, ketoconazole, Ex vivo, nanoparticles

Introduction
Candidiasis has emerged as a significant medical prob-
lem associated with indiscriminate long-term use of
antibiotics, immunosuppressive and cytotoxic therapies,
immune-defects and acquired immunodeficiency syn-
drome (AIDS)1. The GI tract is the major site of dissemi-
nated candidiasis2. The esophagus is the most common
site of colonization of fungi. Candida esophagitis is most
common type of infective esophagitis found to occur in
patients with predisposing factors including malignancy,
corticosteroid use, diabetes mellitus, human immuno-
deficiency virus/acquired immunodeficiency syndrome
(HIV/AIDS), acid suppression, esophageal dysmotility,
gastric surgery and antibiotic use3.
Systemically active therapy is needed for effective
treatment of Candida esophagitis, for which Azole based
antifungal agents are drugs of choice. Ketoconazole
(KZ) is a broad spectrum antifungal agent useful in the
treatment of candidal esophagitis. However, it requires
an acidic pH for absorption and its absorption is depen-
dent on gastric acidity and gastric emptying time, lead-
ing to variable bioavailability. Though it is an orally
active drug, its clinical use is sometimes limited due to
its erratic absorption. Often, the major drawback of the

Address for Correspondence:  Krutika Sawant, TIFAC Centre of Relevance and Excellence in NDDS, Centre of PG Studies and Research,
Pharmacy Department, G. H. Patel Pharmacy Building, Donor’s Plaza, The M. S. University of Baroda, Fatehgunj, Vadodara-390002, Gujarat,
India. Tel: +91 265 2434187. Fax: +91 265 2418927. E-mail: dr_krutikasawant@rediffmail.com
(Received 08 September 2011; revised 20 December 2011; accepted 13 February 2012)

540

Abbreviations
NPs: Nanoparticles
CS: Chitosan
KZ: Ketoconazole
conventional dosage form is that due to rapid gastric
emptying, the absorption of the drug is poor (Cmax after
a single oral dose of 200 mg is reached within 2 h and is
3.5 mcg/mL)4.
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Polymeric nanoparticles (NPs) have been extensively
researched as drug delivery carriers5. NPs are suitable
carriers to deliver drugs which are unstable in the biolog-
ical fluids and cannot readily traffic across the mucosal
barrier6. Polymeric NPs from biodegradable polymers
are effective drug carriers because they are expected to
be absorbed intact after oral administration7. Oral NPs
are drug delivery systems of choice due to enhanced
bioavailability, mucoadhesion and controlled release of
drugs in GIT8,9. Mucoadhesion has become the area of
interest of researchers for its potential to optimize local-
ized drug delivery, by retaining a dosage form at the
absorption window in the GIT. However, the significant
problem with large mucoadhesive solid dosage forms
For personal use only.
like tablets is the poor adhesion to the mucosal surfaces
due to their large size and the vigorous movement of
the GIT10. Hence, NPs are considered to be much better
alternative to overcome such problem.
NPs may be used for oral administration of gut-
labile drugs or those with low aqueous solubility11. The
NPs are expected to adhere to the gastric mucosa and
remain in the gastrointestinal tract, while protecting the
entrapped drug from enzymatic degradation, until the
release of the loaded drug or their absorption as intact
particles12. Chitosan (CS) has the potential of serving
as an absorption enhancer across intestinal epithelia
due to its mucoadhesive and permeability enhancing
property13. It has biocompatibility, biodegradability,
high charge density and non toxicity14. CS NPs loaded
with insulin showed increased oral bioavailability of
the peptide and prolonged reduction in blood glucose
levels by protecting the labile drug from enzymatic deg-
radation in GI tract15. CS is soluble at acidic pH but has
limited solubility at neutral as well as basic pH 16. Hence,
it can be used as absorption enhancer for drugs which
are more soluble in the acidic environment of stomach.
Ionic gelation technique has attracted considerable
attention as this process is organic solvent free, con-
venient and controllable17. Sodium tripolyphosphate
(TPP) is the most extensively used ion cross-linking
agent due to its non-toxic and multivalent properties18.
A recent study by Palmeira-de Oliveira et  al reported
that TPP had an inhibitory species dependent and
concentration dependent cytotoxicity against several
Candida spp. Strains19.
© 2013 Informa Healthcare USA, Inc.
Orally delivered chitosan nanoparticles of ketoconazole  541
TPP: Tripolyphosphate
PDE: Percentage drug entrapped
TEM: Transmission electron microscopy
DSC: Differential scanning calorimetry
Thus it was hypothesized that TPP cross-linked
Chitosan nanoparticles would improve absorption and
bioavailability of KZ which requires acidic pH for absorp-
tion and whose conventional dosage form often shows
poor absorption of drug due to rapid gastric emptying.
Also, use of TPP in chitosan NPs may synergize the anti-
fungal activity of Ketoconazole.
Therefore, the present study was aimed to develop and
optimize orally delivered mucoadhesive chitosan nano-
particles of KZ which will efficiently release drug at its
absorption window i.e. stomach and near its site of action
(esophagus). The optimized formulation was character-
ized for particle size, % drug entrapment, ζ potential,
Differential scanning calorimetry (DSC) and TEM. The
formulations were evaluated for mucin binding capac-
ity, drug diffusion study through dialysis membrane and
through rat stomach and obtained data were fitted to
Korsmeyer Peppas model to understand the mechanism
of drug release.
Materials and methods
Materials
KZ was obtained as gift sample from Gufic Bioscience Pvt.
Ltd, Mumbai, India. Low molecular weight CS (molecu-
lar weight less than 60 kDa) (deacetylation value >85%),
viscosity 30–50 cps was acquired from Ample Effect Sdn
Bhd, Malaysia, and was used without any modification
and purification. Sodium Tripolyphosphate was pur-
chased from Loba Chemie, Mumbai, India. Pig Mucin
was acquired from Sigma-Aldrich, St Louis, USA. Glacial
acetic acid, Mannitol and all other chemicals used were
of analytical grade.
Preparation of NPs
The ionic gelation method was used for preparation of
KZ loaded CS NPs20. In brief, CS (30 mg) was dissolved
in 1% acetic acid solution and hydrated overnight with
stirring. KZ (6 mg) was dissolved in the CS solution with
stirring. Aqueous solution of TPP (7.5 mL, 1 mg/mL) was
then added drop wise to the CS solution (10 mL, 3 mg/
mL) under constant stirring on magnetic stirrer (Remi
Equipment Pvt. Ltd., Mumbai, India), which led to the
formation of dispersion of NPs. The nanodispersion
was then centrifuged at 25,000 rpm for 30 min (3K 30,
Sigma Laboratory Centrifuge, Osterode, Germany) and
the pellet of the NPs was redispersed in water. The NPs
were lyophilized for 24 h (Lyophillizer, Heto Dry Winner,
Allerod, Denmark) using mannitol as cryoprotectant
 542  J. Modi et al.
[NPs (1 parts): cryoprotectant (3 parts)] to get free flow-
ing powder of NPs.
The various process parameters were optimized sys-
tematically. Only one parameter was changed at a time
in each set of experiments. The parameters studied
included concentration of polymer, concentration of
cross-linking agent and ratios of drug/polymer as well as
polymer/cross-linking agent. The effect of these variables
on particle size and entrapment efficiency was evaluated
to obtain optimized nanoparticulate formulation with
minimum possible size and maximum possible entrap-
ment. All batches were prepared in triplicate.
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Characterization of NPs
Particle size
Size of the CS NPs was measured by dynamic light
scattering using Malvern Zetasizer Nano ZS (Malvern
Instruments, Malvern, Worcestershire, UK). A suitable
amount of NPs was dispersed in distilled water creat-
ing a total concentration of 1% w/v. Each sample was
measured in triplicate and particle size was expressed as
mean diameter ± standard deviation.
ζ potential
ζ potential distribution was measured using a Zetasizer
(Nano ZS, Malvern Instruments, Malvern, Worcestershire,
UK). Each sample was suitably diluted with filtered dis-
For personal use only.
tilled water creating a concentration of 1% w/v and placed
in a disposable ζ cell. The electrophoretic mobility (µm/
sec) was converted to ζ potential by in-built software using
Helmholtz-Smoluchowski equation. Three measurements
of each sample were used to derive average ζ potential.
Transmission electron microscopy
The morphology of drug loaded NPs was ascertained by
Transmission electron microscopy (TEM). The studies
were performed using Transmission electron microscope
(Phillips Technai-20, Hillsboro, OR) at 50000 X. NPs were
dispersed in distilled water and one drop of the reconsti-
tuted NPs was incubated on carbon coated copper grid.
The copper grid was fixed into sample holder and placed
in the vacuum chamber of the transmission electron
microscope and observed under low vacuum.
Differential scanning calorimetry
The physical state of KZ loaded in NPs was investigated
using DSC. The analysis was carried out using a DSC
(DSC 60, Shimadzu, Kyoto, Japan) at a heating rate of
20°C per min in the range of 30°C to 300°C under inert
nitrogen atmosphere at a flow rate of 40 mL/min. DSC
thermograms were recorded for CS, KZ and KZ loaded
CS NPs.
Entrapment efficiency
The entrapment efficiency was determined after sepa-
rating the NPs from the aqueous medium (containing
non-entrapped KZ) by centrifugation at 25000 rpm for
30 min (3K 30, Sigma Laboratory Centrifuge, Osterode,
 Drug Development and Industrial Pharmacy
Germany). The supernatant was diluted with appropri-
ate amount of 0.1 N HCl and analyzed for the amount
of unentrapped drug by UV-Visible spectrophotometer
(UV-1601, Shimadzu, Kyoto, Japan) at 270 nm. Similarly,
the pellet was dissolved in 0.1 N HCl and analyzed for the
entrapped drug content by UV-Visible spectrophotom-
eter at 270 nm21.
The percentage drug entrapped (PDE) was calculated
according to following formula:
PDE ( % ) = ( TD-FD/TD ) ×100
 Where TD is total amount of drug added and FD is
amount of free drug in supernatant.
Measurement of mucoadhesive force
The mucoadhesive properties of drug loaded CS NPs
were evaluated by measuring the amount (% weight) of
Pig Mucin (Sigma-Aldrich, St Louis, MO, USA) bound to
the NPs as per the method reported by Yin et al. (2006)22.
In brief, 2 mL of mucin solution (100 µg/mL) was mixed
with 2 mL NPs suspension, incubated at 37 °C for 30 min
and centrifuged at 25000 rpm for 30 min (3K 30, Sigma
Laboratory centrifuge, Osterode, Germany). The super-
natant was separated and the free mucin content was
determined by UV spectrophotometry at 251 nm. The %
mucin binding efficiency of the NPs was calculated using
the following formula:
% mucin binding efficiency = (Co − Cs ) /Co  ×100
 Where,
Co = Initial concentration of mucin
CS = Concentration of free mucin in the supernatant
In vitro diffusion study
In vitro diffusion study was carried out in order to deter-
mine the kinetics and mechanism of drug release from
the CS NPs. The study was carried out in phosphate buf-
fer saline (PBS) (pH 7.4) containing 1% sodium lauryl
sulphate (SLS) for a period of 5 h, as it is reported that the
mucus layer is continuously renewed and replaced by a
fresh layer; the turnover time of the gastric mucus is of
4–5 h from production to digestion23.
The suspension of NPs equivalent to 2 mg drug was
placed in a previously activated dialysis bag (cut off
molecular weight: 12000 Daltons, Himedia, India) and
was sealed at both the ends. The bag was then placed in
a beaker containing 20 mL of diffusion medium which
was maintained at 37 ± 2°C and stirred continuously at
100 rpm24. At regular intervals, samples were withdrawn
from the receptor phase and replaced with the same
quantity of fresh medium to maintain sink conditions25.
All samples were analyzed spectroscopically at 270 nm to
determine the amount of drug released. All the experi-
ments were repeated thrice and the average values were
recorded. The same process was carried out for the plain
drug by taking 2 mL of KZ solution (1 mg/mL in 0.1 N
HCl) as control.

The kinetic analysis of the release data was done using
Korsmeyer and Peppas or power law equation (Peppas
et al. 1987)26
M t / M ∞ = K ⋅t n
Log ( M t / M ∞ ) = Log K + n log t
 Where, time t
Mt/M∞ is the fractional drug release, K is a kinetic con-
stant incorporating structural and geometrical character-
istic of the drug/polymer system, n is diffusion exponent
that characterizes the mechanism of drug release, t is the
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Orally delivered chitosan nanoparticles of ketoconazole  543
was filled into the stomach. The esophageal connection
was then tied and the stomach was mounted in the
organ tube containing 20 mL PBS (pH 7.4) with 1% SLS.
Temperature was maintained at 37 ± 0.5°C using heat-
ing coil. Oxygen was supplied to the tissue through aer-
ator. Samples were withdrawn from the organ tube at
predetermined intervals and replaced with same quan-
tity of fresh media. After 2 h, the stomach was removed
from the organ bath, opened and the contents were
emptied (to simulate in vivo gastric emptying, which is
2 h). The stomach was then filled with 0.1 N HCl, tied
and again mounted in the organ bath and the diffu-
sion was continued till 5 h. All samples were analyzed

time. spectroscopically at 270 nm to determine the amount

Ex vivo diffusion study through rat stomach
This study was conducted to obtain idea about drug dif-
fusion across stomach mucosa under conditions simu-
lating in vivo gastric emptying. The ex vivo study was
carried out for a period of 5 h. However, as the in vivo
gastric emptying time is 2 h, the stomach contents were
emptied after 2 h and diffusion studies were continued
upto 5 h. The studies were carried out according to the
guidelines of Committee for the Purpose of Control
and Supervision of Experiments on Animals, Ministry
of Social Justice and Empowerment, Government
of India, New Delhi, India and were approved by the
For personal use only.
of drug diffused. The same procedure was repeated for
plain drug solution27.
Results and discussion
Preparation and optimization of NPs
The basic principle behind the formation of CS NPs
in Ionic Gelation method28 is based on electrostatic
interaction between CS and TPP. The NPs form due to
intermolecular linkages between amino groups of CS
and phosphate groups of TPP29. CS and TPP concentra-
tions play an important role in the formation of NPs.
Furthermore, CS reacts with TPP in fixed ratios only and

Institutional Animal Ethics Committee of Pharmacy
Department. Male albino rats weighing 200–250 g were
fasted overnight, sacrificed by spinal dislocation and
the stomach was isolated immediately. The stomach
contents were emptied and it was cleaned with normal
saline. Then the lower ends of the stomach (duodenal
connection) were tied with thread and from the esoph-
ageal connection, 2 mL nanoparticulate formulation
it should be within the range of 4:1–6:1 to obtain NPs30.
Hence, these formulation parameters were optimized
on the basis of particle size and PDE and the results are
shown in Tables 1–3. Various concentrations of CS (2,
2.5, 3 and 4 mg/mL) were used. At a fixed weight ratio28
of CS/TPP, ionic gelation between CS and TPP is affected
by concentration of CS, thus affecting the particle size
of the NPs31. With increasing CS concentration, particle

Table 1.  Optimization of CS concentration.

CS/TPP weight ratio CS/Drug ratio CS conc. (mg/mL) TPP conc. (mg/mL) Particle size* (nm) PDE* (%)
4:1 4:1 2.0 1.0 177.2 ± 3.164 20.22 ± 1.950
4:1 4:1 2.5 1.0 284.7 ± 1.352 28.64 ± 1.339
4:1 4:1 3.0 1.0 364.0 ± 4.218 42.31 ± 1.136
*Data represents mean ± S.D. (n = 3).

Table 2.  Optimization of TPP concentration.

CS/TPP weight ratio CS/Drug ratio CS conc. (mg/mL) TPP conc. (mg/mL) Particle size* (nm) PDE* (%)
4:1 4:1 3.0 0.5 316.4 ± 2.155 30.52 ± 1.451
4:1 4:1 3.0 0.75 348.2 ± 2.463 34.291 ± 0.311
4:1 4:1 3.0 1.0 364.0 ± 4.218 42.311 ± 0.136
*Data represents mean ± S.D. (n = 3).

Table 3.  Optimization of CS/TPP weight ratio and CS/Drug ratio.

CS/TPP weight ratio CS/Drug ratio CS conc. (mg/mL) TPP conc. (mg/mL) Particle Size* (nm) PDE* (%)
4:1 4:1 3.0 1.0 364.0 ± 4.218 42.31 ± 1.136
4:1 5:1 3.0 1.0 382.6 ± 2.384 59.84 ± 1.088
5:1 4:1 3.0 1.0 312.52 ± 0.116 34.13 ± 1.225
5:1 5:1 3.0 1.0 358.4 ± 3.912 44.59 ± 1.455
*Data represents mean ± S.D. (n = 3).

© 2013 Informa Healthcare USA, Inc.

 544  J. Modi et al.
size was found to increase which could be attributed to
increase in viscosity of CS solution. Very high concentra-
tion of CS (4 mg/mL) resulted in formation of micropar-
ticles, because of very high viscosity of the CS solution.
The entrapment efficiency also increased with increase
in CS concentration. This was due to increase in amount
of polymer available for both drug entrapment and for
gelation.
When various concentrations of TPP from 0.5 to
1.25 mg/mL were investigated, it was observed that as
concentration of TPP increased, particle size increased.
However, at 1.25  mg/mL, there was formation of
microparticles. Simultaneously, entrapment increased
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with increase in TPP concentration. This could be due
to increase in number of TPP ions available to cross-link
amino groups on CS chains20,29, thereby improving gela-
tion and drug incorporation.
The size and the size distribution of the NPs depend
upon the weight ratio between CS and TPP32. For this
reason, the effect of CS/TPP weight ratio on the size char-
acteristics of the NPs was studied in order to find the opti-
mum ratio that yields NPs of moderate size and narrow
size distribution. However, it was observed that though
there was a reduction in size due to more compact parti-
cle structure, entrapment efficiency also decreased upon
increasing the CS/TPP weight ratio from 4:1 to 5:1, prob-
ably due to inefficient crosslinking having taken place at
For personal use only.
higher CS contents. There are less TPP ions available to
cross-link the higher content of Chitosan. Particle size
and entrapment efficiency was also dependent on CS/
drug ratio. As CS/drug ratio increased, both, particle size
and entrapment increased due to greater amount of CS
available for incorporating the drug. CS/drug ratio of 5:1
provided maximum drug entrapment (59.84 ± 1.088%)
with moderate particle size (382.6 ± 2.384 nm). Hence,
the batch containing CS/TPP weight ratio of 4:1 and CS/
Drug ratio of 5:1 was considered as optimized batch (at
CS conc. 3 mg/mL and TPP conc. 1 mg/mL).
Characterization of NPs
Particle size, morphology and ζ potential
Particle size of the optimized batch was found to
be 382.6 ± 2.384 nm. Drug loaded NPs must have an
appropriate particle size along with ζ potential. The
size characteristics have been found to affect the bio-
logical performance of CS NPs33. TEM image showed
that NPs were spherical in shape without aggregation
(Figure 1). The ζ potential value is important as it can
influence particle stability as well as mucoadhesivity.
The more positive and negative values of ζ potential
tend to stabilize particle suspension to a greater extent
as the electrostatic repulsion between particles of like
charges prevents the aggregation of particles34. The
ζ potential of the drug loaded NPs was found to be
+48.1 mv indicating physical stability of the system. The
high positive ζ was attributed to the positively charged
CS (due to protonation of its amino groups in acetic
acid). ζ potential not only governs the stability of the
 Drug Development and Industrial Pharmacy
Figure 1. Transmission electronic micrographs of optimized
ketoconazole loaded chitosan nanoparticles.
particulate formulation in vitro, but also affects the
interactions with biomolecules in vivo. The high posi-
tive ζ value is advantageous as it indicates high muco-
adhesive interaction with mucin which contains sialic
acid and sulphonic acid residues that are negatively
charged35. Thus, the ζ potential of the NPs was suitable
for providing physical stability as well as for mucoad-
hesion which would improve in vivo drug permeation
across the mucosal membrane.
Differential scanning calorimetry
Differential scanning calorimetry (DSC) is an impor-
tant technique for investigation of thermal properties
of a formulation and provides information about the
physicochemical state of drug in the system. There is no
detectable endotherm if the drug is present in a molec-
ular dispersion or solid solution state in the polymeric
NPs36. The DSC curves of CS, KZ and drug loaded CS NPs
were obtained. It was seen that the DSC thermograms
of drug loaded CS NPs showed a broad endothermic
peak at 102.81°C which was due to the glass transition
temperature of CS. However, the melting peak of drug
was absent in the thermogram of NPs indicating that KZ
was incorporated in amorphous form in the CS matrix
in the NPs.
Measurement of mucoadhesive force
The mucoadhesive properties of CS NPs were evalu-
ated by measuring the pig mucin binding efficiency on
the NPs which was found to be 69.16 ± 5.91%. From this
value, we expect that the NPs will adhere to the mucosal
membrane in vivo and thereby improve absorption and
bioavailability of KZ. The high value of binding efficiency
can be attributed to highly positive ζ potential, the
bioadhesive nature of CS and the nano size of the NPs.
Studies on isolated porcine gastric mucosa preparations
have shown that hydrated CS has a positive charge and
can adhere to negatively charged mucus gel layers. This
ability could result in CS containing formulations being

retained in the stomach. Adhesion would be expected to
be particularly marked under the acidic conditions in the
stomach, where cationic CS would be highly charged37.
Therefore, it implies that the formulation will be retained
at the absorption window in the stomach and KZ will be
better absorbed from there.
In vitro drug release study
The efficiency of conventional dosage form of KZ is
limited due to short gastric residence time. As CS has
mucoadhesive potential, the drug can be better retained
at absorption window for prolonged period. Comparison
of release profile of plain drug solution and NPs formu-
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lation showed that sustained release of KZ was obtained
from the NPs (Figure 2) due to the cross-linking between
amino groups of CS and the phosphate ions of TPP29.
Sustained drug release from mucoadhesive NPs is impor-
tant as it would allow for a prolonged residence time at
the absorption window. Thus, 99.63 ± 1.23% of drug was
released from the drug solution while 80.36 ± 2.15% of
drug was released from NPs by the end of 5 h. Although
the drug release from plain drug solution was more than
90%, it should be noted that the plain drug formula-
tion would leave the absorption window (i.e. stomach)
at around 2 h because of gastric emptying and hence
the effective absorption would be much less than the
observed data. On the other hand, the nanoparticulate
For personal use only.
formulation will remain adhered to the gastric mucosa
and will release the drug in controlled manner for a pro-
longed period at the absorption window. Hence, we can
hypothesize that the entire drug from NPs will be avail-
able for absorption at the absorption window and there-
fore its bioavailability will be enhanced. Moreover, the
presence of the drug near its site of action (esophagus)
for a prolonged time is expected to improve its therapeu-
tic efficacy
Theoretically, the drug release from NPs may take
place by several mechanisms including surface ero-
sion, disintegration, diffusion and desorption38. Hence
to understand the exact mechanism of release, the data
obtained from in vitro drug release studies were fitted to
Korsmeyer–Peppas model. The regression coefficient of
Figure 2. Release profile of KZ from plain drug solution and
nanoparticulate formulation through dialysis bag in PBS (7.4).
© 2013 Informa Healthcare USA, Inc.
Orally delivered chitosan nanoparticles of ketoconazole  545
the plot of log Mt/M∞ versus log t for NPs was found to be
0.9791, indicating a good model fit26. The nanoparticulate
formulation exhibited a non-fickian diffusion mecha-
nism as the value of release exponent ‘n’ was found to
be more than 0.5 (n = 0.9042). The release of drug from
NPs was initially by diffusion followed by erosion. As CS
is a hydrophilic, swellable polymer, it can be assumed
that the drug release was controlled by combination of
polymer swelling, diffusion through the hydrated matrix
and erosion.
Ex-vivo diffusion study
The ex vivo studies are very important to understand
the real drug release characteristics through natural
organs and tissues38. Hence, we designed ex vivo diffu-
sion studies aimed at simulating actual in vivo condi-
tions wherein any ingested substance is subjected to
average gastric emptying after 2 h.Thus, the stomach
contents were emptied after 2 h of starting the ex vivo
diffusion studies. The data showed that 53.22 ± 2.19% of
drug was released from plain drug solution at the end
of 2 h (Figure 3). However, when the stomach contents
were emptied after 2 h, the drug release from plain drug
solution stopped, indicating its complete removal from
the stomach due to emptying. On the other hand, drug
release continued from the NPs formulation even after
emptying the contents after 2 h, with 78.37 ± 2.24% drug
release at the end of 5 h. It can thus be concluded from
this study that the NP formulation will remain adhered
to the absorption site i.e. stomach for a prolonged
period even after gastric emptying of the stomach
contents, and thereby improve the absorption and bio-
availability of KZ. The data obtained from ex vivo drug
release studies was fitted to Korsmeyer–Peppas model.
The regression coefficient of the plot of log Mt/M∞ ver-
sus log t for NPs was found to be 0.9901, indicating a
good model fit. As expected due to the swelling and
erodible nature of CS, the nanoparticulate formulation
exhibited a non-fickian diffusion mechanism as the
value of release exponent n was found to be more than
0.5 (n = 0.8401).
Figure 3. Release profile of KZ from plain drug solution and
nanoparticulate formulation through Rat stomach, in PBS (pH
7.4) for a period of 5 h.
 546  J. Modi et al.
Conclusion
CS NPs of KZ were prepared by ionic gelation method
using sodium tripolyphosphate as cross-linking agent.
The concentration of CS, concentration of TPP and CS
/TPP weight ratio were optimized to obtain minimum
possible size and maximum drug entrapment. The NPs
showed good entrapment, moderate particle size and
high positive ζ potential. DSC studies confirmed the
absence of any interaction between drug and polymer
and indicated the incorporation of drug in polymer
matrix in an amorphous form. Mucoadhesion studies
revealed good mucin binding efficiency and hence
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good mucoadhesion capacity of NPs. In vitro and ex
vivo drug release studies through rat stomach for 5 h
showed that the drug release was sustained and fol-
lowed non-fickian transport of drug from NPs. Results
of ex vivo diffusion studies designed to simulate actual
in vivo conditions wherein any ingested substance is
subjected to average gastric emptying showed that the
drug release from plain drug solution stopped after 2 h,
indicating its complete removal from the stomach due
to emptying. On the other hand, drug release continued
from the NPs formulation even after emptying the con-
tents. It can thus be concluded from this study that the
NP formulation will remain adhered to the absorption
site i.e. stomach and the site of action i.e. esophagus for
For personal use only.
a prolonged period even after gastric emptying of the
stomach contents, and thereby improve the absorption,
bioavailability and therapeutic efficacy of KZ. These
findings suggest the suitability of orally delivered KZ
loaded mucoadhesive NPs as efficient carrier system
for delivering drug at absorption window even after
gastric emptying.
Declaration of interest
The authors report no conflict of interests.
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