Preparation and evaluation of warfarin-β-cyclodextrin loaded chitosan nanoparticles for transdermal delivery

Article abstract of DOI:10.1016/j.carbpol.2012.06.056

The main objective of the present work was to prepare warfarin-β- cyclodextrin (WAF-β-CD) loaded chitosan (CS) nanoparticles for transdermal delivery. CS is a hydrophilic carrier therefore, to overcome the hydrophobic nature of WAF and allow its incorporation into CS nanoparticles, WAF was first complexed with β-cyclodextrin (β-CD). CS nanoparticles were prepared by ionotropic pre-gelation using tripolyphosphate (TPP). Morphology, size and structure characterization of nanoparticles were carried out using SEM, TEM and FTIR, respectively. Nanoparticles prepared with 3:1 CS:TPP weight ratio and 2 mg/ml final CS concentration were found optimum. They possessed spherical particles (35 ± 12 nm diameter) with narrow size distribution (PDI = 0.364) and 94% entrapment efficiency. The in vitro release as well as the ex vivo permeation profiles of WAF-β-CD from the selected nanoparticle formulation were studied at different time intervals up to 8 h. In vitro release of WAF-β-CD from CS nanoparticles followed a Higuchi release profile whereas its ex vivo permeation (at pH 7.4) followed a zero order permeation profile. Results suggested that the developed WAF-β-CD loaded CS carrier could offer a controlled and constant delivery of WAF transdermally.

Full text of DOI:10.1016/j.carbpol.2012.06.056

Carbohydrate Polymers 90 (2012) 1244–1253
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Preparation and evaluation of warfarin-␤-cyclodextrin loaded chitosan
nanoparticles for transdermal delivery
Safaa K.H. Khalila,∗, Gina S. El-Fekyb, Sally T. El-Bannaa, Wafaa A. Khalilc
a Advanced Materials and Nanotechnology Group, Center of Excellence for Advanced Sciences (CEAS), Department of Spectroscopy, Division of Physics, National Research Center,
Cairo, Egypt
b Department of Pharmaceutical Technology, Division of Pharmaceutics, National Research Center, Cairo, Egypt
c Depatment of Biophysics, Faculty of Science, University of Cairo, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The main objective of the present work was to prepare warfarin-␤-cyclodextrin (WAF-␤-CD) loaded
chitosan (CS) nanoparticles for transdermal delivery. CS is a hydrophilic carrier therefore, to overcome
the hydrophobic nature of WAF and allow its incorporation into CS nanoparticles, WAF was first com-
plexed with ␤-cyclodextrin (␤-CD). CS nanoparticles were prepared by ionotropic pre-gelation using
tripolyphosphate (TPP). Morphology, size and structure characterization of nanoparticles were carried
out using SEM, TEM and FTIR, respectively. Nanoparticles prepared with 3:1 CS:TPP weight ratio and
2 mg/ml final CS concentration were found optimum. They possessed spherical particles (35 12 nm
diameter) with narrow size distribution (PDI = 0.364) and 94% entrapment efficiency. The in vitro release
as well as the ex vivo permeation profiles of WAF-␤-CD from the selected nanoparticle formulation were
studied at different time intervals up to 8 h. In vitro release of WAF-␤-CD from CS nanoparticles followed
a Higuchi release profile whereas its ex vivo permeation (at pH 7.4) followed a zero order permeation
profile. Results suggested that the developed WAF-␤-CD loaded CS carrier could offer a controlled and
constant delivery of WAF transdermally.
Received 23 February 2012
Received in revised form 19 May 2012
Accepted 21 June 2012
Available online 29 June 2012
Keywords:
Chitosan
Cyclodextrin
Nanoparticles
Transdermal
Warfarin
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
successfully by introducing CDs (Grenha et al., 2008; Oyarzun-
Ampuero, Brea, Loza, Torres,
& Alonso, 2009). The CS/CD
Considerable research is being directed towards developing
polymeric nanoparticles for drug delivery (Panyam & Labhasetwar,
2003; Shenoy & Amiji, 2005). Among water-soluble polymers
used, CS is the most extensively studied (Morishita & Peppas,
2006; Wilson et al., 2009) due to the ideal properties it pos-
sesses such as biocompatibility, biodegradability, low cost, positive
charge and absorption enhancing effect (De Campos, Sanchez,
& Alonso, 2001; Illum, Jabbal-Gill, Hinchcliffe, Fisher, & Davis,
2001; Muzzrelli, 2012). In addition to the great interest CS
has received recently as transdermal drug carrier due to its
bioadhesion, permeability-enhancing properties and interesting
physico-chemical characteristics (Langoth et al., 2006; Mei, Mao,
Sun, Wang, & Kissel, 2008; Ozcan et al., 2009). However, its
hydrophilic nature has limited its use to delivering hydrophilic
drugs. Currently, new nanocarriers of CS/CD were synthesized;
the novel nanoparticles could encapsulate hydrophobic drugs
nanoparticles with the advantages of good solubilization and per-
meability would improve the bioavailability of hydrophobic drugs.
CDs have a hydrophobic central cavity and hydrophilic outer sur-
face and can encapsulate substrates to form host guest complexes
(Loftsson & Brewster, 1996; Szejtli, 1988; Uekama, Hirayama, & Irie,
1998). This complexation process normally results in a modula-
tion of the properties of the guest molecule (i.e. the drug), such
as increasing solubility, improving chemical and physical stability
and enhancing absorption. Besides these capabilities, some studies
have discussed their ability to optimize systemic dermal delivery
through enhancement of drug release and/or permeation and sus-
taining drug release from vehicle (Matsuda & Arima, 1999). Due to
its price, availability and cavity dimension, the ␤-CD is the most
widely used.
The most popular approach for the formation of CD/hydrophilic
polymer nanoparticles consists of the incorporation of a previously
formed drug–CD inclusion complex into polymeric nanoparticles.
In these systems, the presence of CDs generally results in an
increased loading capacity for lipophilic drugs (Ceschel et al., 2003).
WAF is the most widely prescribed anticoagulant drug in North
America (Holbrook et al., 2005). However, problems associated
with oral WAF makes clear the continuing need for a new promising
∗
Corresponding author. Tel.: +20 1005406479; fax: +20 233370931.
E-mail addresses: safaakhk@yahoo.com (S.K.H. Khalil), gelfeky@hotmail.com
(G.S. El-Feky), sallyelbanna77@yahoo.com (S.T. El-Banna),
profwafaa khalil@yahoo.com (W.A. Khalil).
0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2012.06.056

S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
1245
technology for its delivery in order to overcome the inconvenience
of WAF therapy related to the variable effects resulting either from
the high inter- and intra-individual variability in dose response
due to its narrow therapeutic index or from its extensive inter-
actions with food and medications and the related requirement for
coagulation monitoring. Also, being a hydrophobic drug, WAF is
practically insoluble in water and presents a very slow dissolution
rate in acidic media.
a mortar until a homogeneous paste was obtained. The resulting
paste was dried in an oven at 45 ◦C for 48 h and the solid was finally
ground and sieved through a 250 ␮m sieve. Co-evaporated complex
was obtained by dissolving equimolar amounts of ␤-CD and WAF
in 250 ml of 50% aqueous ethanol. The solution was stirred up to
complete dissolution of the powders; the solvent was then removed
at reduced pressure using a rotary evaporator at 45 ◦C. The obtained
solid was ground, sieved through a 250 ␮m sieve and placed in an
oven at 45 ◦C for 48 h.
Transdermal delivery avoids many problems associated with the
oral route including drastic pH changes, the presence of enzymes,
variable transit times and fluctuating drug plasma concentra-
tions. In addition, it offers longer duration of action resulting
in a reduction in dosing frequency, improved bioavailability,
uniform plasma levels, reduced side effects as well as flexibil-
ity of terminating the drug administration by simply removing
the patch from the skin (Bayarski, 2005). For transdermal ther-
apy, the key point of dosage design is to enhance the drug
solubility in the vehicle and improve its permeability via skin
without causing irritation. Thus, our approach is to combine the
benefits of CS as a penetration enhancing, biocompatible and
rate controlling polymer of the nanoparticle generation together
with those of CD as a solubility enhancer having the capabil-
ity of bettering the solubility and stability of hydrophobic drugs
like warfarin. Thus allowing their efficient encapsulation into
hydrophilic carriers like chitosan nanoparticles to provide
enhanced transdermal delivery of WAF capable of offering effective,
non-toxic and controlled anti-coagulation.
2.2.3. Characterization
2.2.3.1. Scanning electron microscopy (SEM). Complexes were gold
coated using a Hitachi coating unit IB-2 coater under a high vac-
uum, 0.1 Torr, high voltage, 1.2 kV and 50 mA. Coated samples were
examined using SEM (JEOL JXA-840A SEM, Japan) to characterize
the changes of the drug surface before and after complexation for
indicating complexation.
2.2.3.2. Fourier-transform infrared (FTIR) spectroscopy. FTIR spectra
of the complexes were obtained on a 6100 JASCO FTIR spectrom-
eter, Japan. A 1% of each dried complex was mixed with KBr and
compressed to form tablets. The IR spectra were scanned over the
wave number range of 4000–400 cm−1 using a resolution of 4 cm−1
and 64 co-added scans.
2.2.4. Analysis of drug content in the binary mixtures
Drug content was assayed by dissolving weighed amounts of
each binary mixture in 10 ml of ethyl alcohol. The drug content
was determined spectrophotometrically at 307 nm.
2. Materials and methods
2.1. Materials
2.3. Preparation and characterization of blank CS nanoparticles
CS was purchased from Mallinckrodt, USA [M.W = 400,000,
degree of deacetylation 95%]. Tripolyphosphate was supplied
by Mallinckrodt, The Netherland. Glacial acetic acid 99.8% was
obtained from El Nasr Pharmaceutical Chemicals Co., Egypt. WAF
was purchased from Sigma–Aldrich, USA. Pharmaceutical grade ␤-
CD was supplied by Roquette, France. Other chemicals and solvents
were of analytical grade.
2.3.1. Preparation
CS nanoparticles preparation was based on the ionic gelation
of CS with TPP anions (Calvo, Remunan-Lopez, Vila-Jato, & Alonso,
1997). CS nanoparticles were prepared in 2 groups, the first used
different ratios of CS:TPP (1:1, 2:1, 3:1, 4:1, 5:1 and 6:1) with final CS
concentration of 2 mg/ml. The second used different concentrations
of CS (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/ml) within the 3:1 CS:TPP
ratio. In both groups, CS was first dissolved in acetic acid aqueous
solution. The concentration of acetic acid in the aqueous media was
1.75 times that of CS. TPP aqueous solution was then added drop
wise under magnetic stirring onto the corresponding amount of the
CS solution at room temperature; 25 1 ◦C.
2.2. Preparation of WAF-ˇ-CD complex
2.2.1. Phase solubility studies
Solubility study was carried out according to the method of
Higuchi and Connors (1965). An excess drug (WAF) was added to
distilled water containing different concentrations of ␤-CD. The
formed suspensions were shaken at 25 0.5 ◦C till equilibrium and
then filtered through a millipore filter (0.45 ␮m). An aliquot portion
of the filtrate was analyzed by UV spectrophotometer (UV-240 1PC,
Shimadzu, Japan) at ꢀmax = 307 nm against blank. Experiment was
performed in triplicate. The apparent stability constant (K1:1) was
calculated from the linear fit of the curve according to the following
equation:
2.3.2. Characterization
2.3.2.1. FTIR spectroscopy. See Section 2.2.3.2.
2.3.2.2. Transmission electron microscopy (TEM). TEM was used to
determine the size and morphology of prepared nanopaticles. One
drop of the nanoparticle colloidal suspension was spread onto a
carbon film 200 mesh copper grid and allowed to stand for 10 min.
The samples were stained with one drop of 3% phosphotungestic
acid before viewing under microscope (JEOL JEM-1230, Japan).
slope
D0(1 − slope)
K1:1
=
where slope is the value found in the linear regression and D0 is
the intrinsic drug solubility derived from the intercept of the phase
solubility relationship (Marcus, Brewster, & Loftsson, 2007).
2.4. Loading WAF-ˇ-CD complex into CS nanoparticles
To load WAF into CS nanoparticles, the pH of the CS solution
was adjusted to 5.4 using 0.1 N NaOH and a weighed amount of
WAF-␤-CD complex was then dissolved under magnetic stirring at
room temperature. After the complete dissolution of WAF complex
in the media, tripolyphosphate solution was added dropwise to the
WAF-␤-CD/CS solution. The mixture was stirred then sonicated.
2.2.2. WAF-ˇ-CD complexation
Binary mixtures were prepared in a 1:1 molar ratio of WAF:␤-CD
based on the results obtained from the phase solubility study.
Physical mixture was prepared by simple mixing of the drug and
the ␤-CD in a mortar for 10 min. Kneaded complex was obtained
by adding small amount of water to WAF-␤-CD mixture placed in

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S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
2.4.1. Yield and entrapment efficiency CS nanoparticles
The yield of nanoparticles fabrication process was calculated by
weighing dried samples of the isolated nanoparticles and referring
them to the initial amounts of CS, tripolyphosphate and CD (Adlin,
Nesalin, Gowthamarajan, & Somashekhara, 2009). The yield was
calculated as follows:
weight of nanoparticles
total amount of the components
yeild =
× 100
The nanoparticles were separated from the aqueous dispersion
medium by centrifugation at 9000 rpm for 1 h. Entrapment effi-
ciency (EE) was calculated based on the ratio of the amount of drug
present in the nanoparticles to the initial amount of drug used in
the loading process (Anitha et al., 2011).
Fig. 1. Effect of ␤-cyclodextrins on the solubility of warfarin.
2.6. Ex vivo WAF permeation of the prepared nanoparticles
The permeation of WAF through excised rat skin was carried out
using Franz diffusion cells. The receptor compartment was filled
with phosphate buffer, pH 7.4, stirred continuously using a mag-
netic stirrer. The temperature of the receptor compartment was
kept at 37 ◦C. The skin was mounted between the donor and recep-
tor compartments of the Franz diffusion cell. The prepared formula
was placed in the donor compartment. At predetermined time
intervals, samples were taken from the receptor compartment and
the cell refilled with an equivalent amount of fresh buffer solution.
total amount of drug in the nanoparticles
initial amount of drug taken for loading studies
EE(%) =
× 100
2.4.2. Preparation of nanoparticle powder
Dry powder of WAF-␤-CD loaded CS nanoparticle was obtained
by spray-drying the aqueous suspension of the nanoparticles
using a laboratory-scale spray-dryer (Büchi® Mini Spray Dryer,
B-290, Switzerland), under the following conditions; a feed rate
of 2.5 ml/min, inlet and outlet temperatures of 120 2 ◦C and
85 2 ◦C, respectively, an air flow rate and aspirator kept constant
at 400 Nl/h and 80%, respectively. Nanoparticles were collected
and stored in a dessicator at room temperature until use (Grenha,
Remunan-Lopez, Carvalho, & Seijo, 2008).
3. Results and discussion
3.1. WAF-ˇ-CD complex formation
3.1.1. Phase solubility study
The apparent solubility of WAF increased linearly as a function of
␤-CD concentration over the studied concentration range (Fig. 1)
which is a characteristic of AL-type system (Higuchi & Connors,
1965). The slope value was lower than one indicating an inclusion
complex in the molar ratio of 1:1 between the guest (WAF) and host
(␤-CD) molecules (Arima et al., 2001; Zingone & Rubessa, 2005).
The solubilizing power of ␤-CD towards WAF was found to be
1.39 × 10−3 mM and the apparent stability constant, K1:1, obtained
from the slope of linear phase solubility diagram was found to be
138.59 M−1. Large K1:1 value suggests that the CD cavity is large
which in turn favors the inclusion of WAF (Lin & Yang, 1986).
2.4.3. Characterization of WAF-ˇ-CD loaded CS nanoparticles
2.4.3.1. FTIR spectroscopy. See Section 2.2.3.2.
2.4.3.2. TEM. See Section 2.4.2.
2.4.3.3. X-ray diffraction (XRD). The XRD experiments were car-
ried out using X-ray diffractometer (Empyrean, Pixcel3D, Holland).
Diffraction patterns of kneaded WAF-␤-CD complex, blank CS NP
and WAF-␤-CD loaded CS NP were obtained at room temperature
˚
with powder diffractometer using a Cu K␣ radiation (ꢀ = 1.54060 A),
a current of 30 mA and voltage of 45 kV to study their chemical
structures. Fine powdered samples, were analyzed over the 5–80
2ꢁ range with a scan step size of 0.0260 and time of acquisition of
18.8700 s.
3.1.2. Characterization of WAF-ˇ-CD complex
3.1.2.1. SEM. SEM demonstrated ␤-CD as parallelogram like struc-
tures having smooth surface with some irregularities (Fig. 2a) and
WAF as apparent large rods with different sizes (Fig. 2b). WAF-␤-
CD physical mixture appeared as irregular rods of WAF adhered
on the crystal surfaces of ␤-CD (Fig. 2c). The kneaded complex
appeared as agglomerates clumping to each other (Fig. 2d), whereas
the co-evaporation complex showed a new homogeneous inter-
locked crystal mass (Fig. 2e). These drastic changes reported with
the kneaded and co-evaporated complexes indicate complete com-
plex formation where the presence of a new single solid phase could
be simply a consequence of a crystalline pattern change in these
systems due to WAF-␤-CD complex formation (Fernandes, Viera, &
Veiga, 2002).
2.4.3.4. Zeta potential. The zeta potential of nanoparticles was
measured using a Laser Zetameter (Malvern Instruments model
“Zetasizer 2000”, UK). Freshly prepared particles’ suspension
(0.01 g) was placed in 50 ml double distilled water at ionic strength
of 2 × 10−2 M NaCl. The pH was adjusted to the required value. The
sample was shaken for 30 min. After shaking, equilibrium pH was
recorded and zeta potential of the particles measured.
2.5. In vitro release of WAF from the prepared nanoparticles
Release of WAF from its ␤-CD complex alone as well as from
the CS loaded WAF-␤-CD complex nanoparticles was compared at
32 0.5 ◦C using USP Dissolution Tester, Apparatus II, filled with
900 ml phosphate buffer of pH 5.5 at a rotation rate of 50 rpm. In
addition drug release from CS loaded WAF-␤-CD complex nanopar-
ticles was compared at pH 5.5 and 7.4. Aliquots, each of 5 ml, were
withdrawn at different time intervals up to 8 h and replenished
by equal volumes of fresh dissolution medium. WAF content was
measured spectrophotometrically at 307 nm. The percentage drug
released and release profile were evaluated.
3.1.2.2. FTIR spectroscopy. The absorbance peak of WAF at
1074 cm−1 (C O) was observed in the physical mixture but in
the kneaded and co-evaporated mixtures it was slightly shifted to
1079 cm−1. This was considered an indication of complex forma-
tion. The solid form of WAF has an IR absorption band at 998 cm−1
associated with the hemiketal ring (Stella, Mooney, & Pipkin, 1984).
This peak was found in the physical mixture at 995 cm−1 and dis-
appeared when inclusion complexes were formed by kneading and
co-evaporation techniques (Fig. 3). This implies that when WAF

S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
1247
Fig. 2. SEM micrographs of (a) ␤-CD, (b) WAF, (c) WAF-␤-CD physical mixture, (d) WAF-␤-CD kneaded mixture and (e) WAF-␤-CD co-evaporated mixture.
was included into the cavity of CD, it was changed from cyclic
diastereometric hemiketal tautomer to open-chained tautomer in
the solid complex state. These results are in good agreement with
those previously reported (Lin & Yang, 1986).
3.2. Preparation and characterization of blank CS nanoparticles
Formation of chitosan nanoparticles depends on the ionic inter-
action between the positively charged CS and the polyanion TPP and
the ability of CS to form a gel through inter- and intra-molecular
linkages (Calvo et al., 1997).
3.1.2.3. WAF content in the drug–CD binary mixtures. Physical and
kneaded mixtures showed a good agreement between theoretical
and actual drug content (23 mg vs. 25 mg and 23 mg vs. 17.12 mg,
respectively), whereas, in co-evaporated mixtures the actual drug
content was much lower than that of the theoretical (23 mg
vs. 3.96 mg). Based on this finding together with those obtained
from SEM and FTIR, the kneading method was used for preparing
drug–CD binary mixture.
3.2.1. FTIR spectroscopy
FT-IR spectroscopy was adopted to characterize the molecular
structure of CS before and after nanoparticle formation. Careful
investigation of the spectra of the CS nanoparticle in comparison to
that of the pure CS revealed the following; the amino group (C
O
amide I) absorption at 1646 cm−1 was shifted to 1644 cm−1 at the
Fig. 3. FT-IR spectra of WAF, ␤-CD, WAF-␤-CD physical, kneaded and co-evaporated complexes.

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S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
Fig. 4. The relationship between the absorbance ratio A 1550/A 895 with the different CS:TPP ratios (a) and different CS concentration (b).
Table 1
CS:TPP ratios 1:1 and 2:1, and was shifted to 1640 cm−1 at the ratio
3:1. The CS:TPP ratios 4:1, 5:1 and 6:1 produced shifting of the
amino group (amide I) absorption to 1635 cm−1. The absorption
peak at 1596 cm−1 specific to the amino groups (N H amide II) of
CS with high deacetylation degree, disappeared completely in the
spectra of the CS:TPP ratios 1:1, 2:1 and 3:1 and a new absorption
peak appeared at 1550 cm−1 as a result of nanoparticle formation
with a gradually increased intensity from ratios 1:1 up to 3:1. For
the ratios 4:1, 5:1 and 6:1, the spectra showed both; the peaks
of the pure CS (1646 and 1596 cm−1) and the new characteristic
peak of the formed nanoparticles (1550 cm−1). In order to quan-
tify the CS nanoparticles formation along the studied CS:TPP ratios,
the absorbance ratio (A 1550/A 895) was calculated using man-
ual 2-point baseline method. The absorption peak at 1550 cm−1
was taken as a measure of the newly formed CS nanoparticle. The
absorption peak at 895 cm−1 assigned to C H stretching vibra-
tion of saccharine structure was taken as a reference band. The
absorbance ratio was plotted against the different CS:TPP ratios
(Fig. 4a). It is obvious that CS nanoparticle formation increased
by increasing the CS:TPP ratio reaching a maximum value with
CS:TPP weight ratio 3:1, this was followed by a gradual decline
through the 4:1 and 5:1 ratios hitting its minimal value with the
6:1 ratio. This might be attributed to the insufficiency in the num-
ber of TPP molecules required to bond with the CS molecules
with increasing CS concentration more than triple TPP concentra-
tion. Also, increasing the CS concentration could lower the free
space available for the molecules to collide therefore decreasing
the amount of ionizing bonds and subsequently the amount of
nanoparticles formed. Thus, the 3:1 CS:TPP weight ratio was found
ideal for nanoparticle formation; this comes in agreement with pre-
vious studies (Calvo et al., 1997; Zhang, Oh, Allen, & Kumacheva,
2004).
FT-IR spectra was then performed for pure CS and CS nanopar-
ticles prepared with different CS concentrations (from 0.5 up to
3 mg/ml final CS concentration) within the CS:TPP weight ratio
of 3:1. The amide I band 1646 cm−1 was shifted to 1640 cm−1
(0.5 mg/ml), 1644 cm−1 (1–2.5 mg/ml) and 1642 cm−1 (3 mg/ml).
Furthermore, the absorption band at 1596 cm−1 in all CS concen-
trations (0.5, 1, 1.5, 2, 2.5 and 3 mg/ml) disappeared and new bands
were formed at (1553, 1549, 1550, 1550, 1549 and 1547 cm−1),
respectively. FTIR results indicated that the amino group in all
studied ratios and concentrations interacted with TPP creating
ionic bonds. These interactions reduced CS solubility and were
responsible for CS separation from solution in the form of nanopar-
ticles. Furthermore, the new absorption band at 1550 cm−1 which
appeared after the conjugation reaction with TPP, indicated the
formation of new amide bonds through ionic interaction with
TPP (Papadimitriou, Bikiaris, Avgoustakis, Karavas, & Georgarakis,
2008). Also, results showed that the CS:TPP ratio not the CS con-
centration is what plays the important role in determining the
amount of nanoparticles formed where there was no obvious
Diameter of chitosan nanoparticles prepared by different ratios of CS:TPP and dif-
ferent concentrations of chitosan within the CS:TPP weight ratio of 3:1.
CS concentrations (mg/ml)
Mean diameter (D in nm)
S.D.
21.0
30.0
39.0
12.7
142.0
65.7
PDI
0.5
1.0
1.5
2.0
2.5
3.0
72
130
77
35
405
596
0.291
0.230
0.506
0.364
0.350
0.110
CS:TPP ratios
Mean diameter (D in nm)
S.D.
PDI
1:1
2:1
3:1
4:1
5:1
6:1
13
30
35
32
19
13
2.7
8.4
12.7
11.0
3.6
0.207
0.280
0.364
0.343
0.189
0.153
2.0
Notes: D is the mean value of at least 40 particles, S.D. is the standard deviation and
PDI is the polydispersity index.
relationship between the concentration of CS and the amount
CS nanoparticles formed (Fig. 4b), since the absorbance ratio (A
1550/A 895) appeared more or less constant with increasing CS
concentration.
3.2.2. TEM
The ratio between CS and TPP is critical and controls the size and
distribution of the nanoparticles and might in turn affect the biolog-
ical performance of CS nanoparticles (Pan et al., 2002). Therefore,
the effect of different CS:TPP weight ratios (1:1, 2:1, 3:1, 4:1, 5:1
and 6:1) on the size characteristics of prepared nanoparticles was
studied. Results are represented in Fig. 5.1a–f. It could be observed
that nanoparticles formed with ratios 1:1, 2:1, 4:1, 5:1 and 6:1 are
roughly spherical and agglomerated while those formed with the
ratio 3:1 (Fig. 5.1c) are separate and distinctly spherical. Table 1
shows that the particle size increased from 13 nm to 35 nm with
increasing CS:TPP weight ratio from 1:1 up to 3:1, respectively,
this was followed by a gradual decrease with further increasing
CS:TPP weight ratio up to 6:1. It could be assumed that the decrease
in the particle size at higher CS:TPP ratios is correlated with the
reduction in the ionic bond formation (Calvo et al., 1997) indicat-
ing that the ideal particle size results when the availability of the
functional groups on the two interacting polymers is in stoichio-
metric proportion. This contradicts the findings of Papadimitriou
et al. (2008).
The effect of changing CS final concentrations (0.5, 1, 1.5, 2,
2.5 and 3 mg/ml); within the 3:1 CS:TPP weight ratio; on the par-
ticle size of the produced nanoparticles, was then investigated.
The TEM micrographs of the formed nanoparticles showed distinct
homogenous spherical morphology (Fig. 5.2a–f). Table 1 shows that
the particle size was independent on the CS concentration up to

S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
1249
Fig. 5. TEM micrographs of CS nanoparticles prepared by different CS:TPP weight ratios (5.1); (a) 1:1, (b) 2:1, (c) 3:1, (d) 4:1, (e) 5:1 and (f) 6:1 and those prepared by different
concentration of CS within the 3:1 CS:TPP weight ratio (5.2); (a) o.5 mg/ml, (b) 1 mg/ml, (c) 1.5 mg/ml, (d) 2 mg/ml, (e) 2.5 mg/ml and (f) 3 mg/ml.
2 mg/ml final CS concentration however, a remarkable increase in
the particle size was reported with the 2.5 and 3.0 mg/ml final
CS concentration (405 and 596 nm, respectively). This could be
attributed to the increased possibility of each TPP molecule bond-
ing to five CS molecules; through its five Na+; with increasing the
concentration of CS per unit volume. This might be the cause behind
the formation of large sized particles. TEM results could be corre-
lated to those of the quantitative FT-IR analysis (Fig. 4a and b).
mainly attributed to the primarily inclusion of the drug into the
␤-CD cavity which resulted in larger amounts of WAF available for
association with CS nanoparticles.
Results of FT-IR, TEM and EE% demonstrated that the 3:1 CS:TPP
ratio with 2 mg/ml final CS concentration offered the optimum
conditions in the production of homogenous distinct smooth spher-
ical CS nanoparticles (35 nm) with narrow particle size distribution
range (PDI = 0.364). This finding coincides with that reported by
Zhang in 2004 where he stated that, the CS:TPP (3:1) ratio lead
to the most efficient cross-linkage of amino groups producing the
most compact particle structure (Zhang et al., 2004). Therefore,
drug-loaded nanoparticle formulation prepared at these conditions
was adopted for intensive characterization using TEM, FT-IR, zeta-
potential, in vitro release and ex vivo permeation.
3.3. WAF-ˇ-CD loaded CS nanoparticles
3.3.1. Loading WAF-ˇ-CD complex into CS nanoparticles
WAF-loaded CS nanoparticles were prepared in a two step pro-
cedure. The WAF-␤-CD inclusion complex was prepared first (as
previously described) then the complex was entrapped into the CS
nanoparticles possessing the highest entrapment efficiency via the
cross linking method.
3.3.3. Characterization of selected WAF-ˇ-CD complex loaded CS
nanoparticle
3.3.3.1. TEM. Using TEM, WAF complex loaded CS nanoparticles
appeared as distinct, spherical and regular (Fig. 6a) with a mean
particle size diameter of 63 23 nm and a relatively narrow parti-
cle size distribution (PDI = 0.368). It is noted that, the incorporation
of WAF into CS nanoparticles led to an increase in their size com-
pared to the blank nanoparticles (Chen, Mohanraj, & Parkin, 2003;
Kim et al., 2006; Papadimitriou et al., 2008).
3.3.2. Yield and entrapment efficiency of CS nanoparticles
The yield of all studied WAF loaded CS nanoparticles ranged
from 62 to 88%. High nanoparticle recovery is favorable for reduc-
ing manufacturing costs (Douglas, Davis, & Lllum, 1987; Poovi,
Lekshmi, Narayanan, & Reddy, 2011).
The entrapment efficiency (EE%) of nanoparticles, generally,
depends on many factors, such as CS molecular weight, TPP con-
tent and CS concentration (Deng, Zhou, & Luo, 2006). The EE% of CS
nanoparticles was found to increase from 73% to 94% with increas-
ing the CS:TPP weight ratio from 1:1 up to 3:1, respectively. Further
increase in the CS:TPP weight ratio did not result in any signif-
icant changes in drug entrapment. Hence, a series of increasing
CS concentrations (0.5–3 mg/ml final CS concentration) within the
weight ratio 3:1 was prepared and their entrapment efficiencies
were determined.
A gradual increase in the entrapment efficiency was observed
along the studied range of concentrations up to 2 mg/ml final CS
concentration (from 30% up to 94% with 0.5 up to 2 mg/ml final CS
concentration, respectively). Further increase in CS concentration
led to a significant decrease in the entrapment of WAF reaching
24% with 3 mg/ml final CS concentration. It has been previously
reported that the gradual and low increase in the viscosity of CS
at lower concentrations (1–2 mg/ml) promotes the entrapment of
WAF and gelation between CS and TPP (Wu, Yang, Wang, Hu, & Fu,
2005), until a certain limit above which the viscosity of the gela-
tion medium highly increases with subsequent hindrance of drug
entrapment (Kumar, Dharmendra, Jhansee, Shrikant, & Pandey,
2011; Vandenberg, Drolet, Scott, & No¨ue, 2001; Wu et al., 2005).
The general trend of efficient entrapment reaching 94% of the
hydrophobic drug into the hydrophilic CS nanoparticles could be
3.3.3.2. FT-IR. FT-IR spectra of WAF, blank CS nanoparticles and
WAF-␤-CD complex loaded CS nanoparticles revealed spectral
changes resulting from WAF encapsulation (Fig. 6b). The C
O
amid I peak at 1644 cm−1 of the blank CS nanoparticle was
shifted to 1646 cm−1 and its intensity decreased markedly chang-
ing into a shoulder. The new absorption peak at 1550 cm−1 (due to
nanoparticles formation) was shifted to 1571 cm−1 after drug load-
ing. The major characteristic peaks of warfain; 1682 cm−1 (C O)
and 1614 cm−1 (C C aromatic ring) disappeared while 1074 cm−1
(C O) remained. FT-IR changes indicated the strong interaction
developed between the C O group of WAF and the NH2 group of
CS/TPP matrix with drug loading (Anitha et al., 2011; Chen et al.,
2003; Papadimitriou et al., 2008).
3.3.3.3. XRD. Blank CS NP diffraction pattern showed some major
peaks at approximately 11◦, 19◦, 22.5◦, 29◦, 44.5◦ and 72.5◦ with
strongest intensity at 2ꢁ = 44.5◦. The kneaded WAF-␤-CD binary
mixture presented several peaks implying their crystalline nature.
The diffraction pattern of WAF-␤-CD loaded CS NP exhibited the
characteristic peaks of the blank CS NP together with only one
intense peak due to the drug–CD complex (at 9◦), indicating
that WAF may present as molecular dispersion in the polymeric
nanoparticle (Jingou et al., 2011).

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S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
Fig. 6. Characteristics of warfarin-␤-CD loaded chitosan nanoparticles, (a) TEM micrographs, (b) FT-IR spectra and (c) X-ray diffraction.
3.3.3.4. Zeta potential. The incorporation of WAF into CS nanopar-
ticles had significant effect on the zeta potential values, where
an increase in the mean particle size of the blank and drug
loaded nanoparticles from 35 nm to 63 nm, respectively was
accompanied by a decrease in the zeta potential value from 32.4 mV
to 19.8 mV, respectively. Similar results were reported when hep-
arin was incorporated into CS/␥-PGA nanoparticles (Tang et al.,
2010) and when quercetin was incorporated into CS particles (Liu
& Guo, 2006). Positive zeta potential values could be attributed to
the residual amino groups of CS molecules entangled onto the sur-
face of the nanoparticles (Oyarzun-Ampuero et al., 2009; Jingou
et al., 2011). This positive charge is desirable in order to prevent
particles aggregation and to promote electrostatic interaction with
the overall negative charge of the cell membrane (Schillers et al.,
2004).
burst that lasted for 2 h at which approximately 40–50% of the drug
content was released from the nanoparticles this was followed by
a slower and more gradual drug release pattern reaching approx-
imately 65% at 8 h (Fig. 7a). In 2006, Maestrelli et al., reported a
typical release profile characterized by an initial rapid release phase
that reached a plateau in a relatively short time (1.5 h) due to an ini-
tially released drug–CD complex by simple diffusion through the CS
network followed by drug release due to degradation of the poly-
meric matrix (Maestrelli, Garcia-Fuentes, Mura, & Alonso, 2006).
This comes also in agreement with recent studies in drug loaded-CS
nanoparticles (Boonsongrit, Mitrevej, & Mueller, 2006). The ini-
tial fast release might be the result of the dispersion of drug–CD
molecules close to the nanoparticles surface, thus allowing easy dif-
fusion in the initial incubation time through the porous surface of
the nanoparticles. After the burst release period, the rate of release
decreased as the dominate release mechanism might have changed
to drug–CD complex diffusion through the CS matrix followed by
drug exposure due to polymer erosion (Papadimitriou et al., 2008;
Zhang, Wu, Tao, Zang, & Su, 2010).
3.3.3.5. In vitro release of WAF from the prepared nanoparticles. An
important requirement of successful transdermal therapy is that
a drug carried by a vehicle be able to reach the skin surface at an
adequate rate and in sufficient amounts. This makes studying the
release profile of WAF from nanoparticles crucial in order to predict
the behavior of the drug during its transdermal delivery.
The release data were fitted to different kinetic equations and
showed that WAF release from the CS nanoparticles followed a
Higuchi release model. WAF release showed a very rapid initial
The presence of CD within the drug loaded nanoparticle for-
mulation could have contributed to the sustained release effect
exhibited by the CS nanoparticles. In a recent study by Jingou et al.,
the release rate of calcium folinate (CaF) from the CS/CD nanoparti-
cles was found to be higher and more sustained than methotrexate
(MTX) from CS only nanoparticles (Jingou et al., 2011).

S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
1251
Fig. 7. In vitro drug release profile of warfarin-␤-CD complex and warfarin-␤-CD loaded chitosan nanoparticles at pH 5.5 (a) and pH 7.4 (b). Ex vivo drug permeation of
warfarin-␤-CD loaded chitosan nanoparticles at pH 7.4 (c).
Comparing the in vitro drug release profile from WAF-␤-CD
complex with that of WAF-␤-CD loaded chitosan nanoparticles at
pH 5.5 a different monophasic release pattern was observed, where,
gradual slower zero order release profile was reported (R2; 0.9456),
total percentage drug released at 8 h was found to be 51.56% i.e.
about 14% less than the total percentage released from CS loaded
warfarin-␤-CD complex (Fig. 7a). This could be attributed to what
was previously reported by Zerrouk et al., in 2006 that the pres-
ence of CS strongly reduces the drug–Cd complex stability, thus
increasing the amount of free drug released and in turn available
for permeation (Zerrouk et al., 2006).
Furthermore, comparing the drug release profile from WAF-
␤-CD loaded CS nanoparticles at pH 5.5 and 7.4 (Fig. 7b), it was
found that the drug release at pH 7.4 followed the same bipha-
sic release pattern (initial burst followed by subsequent gradual
release) as at pH 5.5, however, the release rate during both phases
was lower and during an 8 h experiment, the amount drug released
reached a total percentage of 46.87% compared to 65.78 at pH 5.5,
this might be attributed to the reduced solubility of chitosan at pH
above 6.8 (Wen, Xianxi, Lihai, & Feng, 2009). This finding supported
by the hypothesis that chitosan carriers release drugs on the skin
surface and binds covalently with the negatively charged stratum
corneum increasing its fluidity and enhancing drug permeability
(Taveira, Nomizo, & Lopez, 2009) goes with our selection of pH 5.5
as the most suitable media for assessing the release profile of the
drug.
3.3.3.6. Ex vivo WAF permeation of the prepared nanoparticles. Ex
vivo permeation experiments were carried out through excised rat
skin. It was shown that permeation studies using rat skin is capa-
ble of providing information useful for manipulating the design of
transdermal drug systems so that the desired permeation of the
drug across human skin would be achieved (Al-Saidan, 2004).
As shown in (Fig. 7c), a steady increase in the amount of perme-
ated drug in the receptor compartment with time was observed. It
was found that WAF permeation through excised rat skin followed
a typical zero order kinetics (R2 = 0.991) with approximately 60% of
the drug released after 8 h. This could be a result of CS nanoparti-
cle acting as drug reservoir, where drug is released from the inner
phase to the outer phase and then further into the skin (Peltola,
Saarinen-Savolainen, Kiesvaara, & Suhonen, 2003). This reservoir
effect makes depletion of WAF in the external phase due to its
permeation into the skin constantly replenished by the release of
WAF from the internal phase, thus leading to zero order permeation
profile. The facilitated permeability of WAF from rat skin could be
attributed to the favorable synergistic effect from the simultaneous
presence of the two types of carriers; ␤-CD and CS where ␤-CD is
known to act as a permeation enhancer in transdermal drug deliv-
ery (Krauland & Alonso, 2007) while, CS and its derivatives could
significantly change the secondary structure of keratin in stratum
corneum, increase the water content in stratum corneum, decrease
HaCaT cell membrane potential and enhance cell membrane fluid-
ity to various degrees.

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S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
Also, such results could be due to the marked decrease of the
Illum, L., Jabbal-Gill, I., Hinchcliffe, M., Fisher, A. N., & Davis, S. S. (2001). Chitosan as
a novel nasal delivery system for vaccines. Advanced Drug Delivery Reviews, 51,
81–96.
Jingou, Ji, Shilei, H., Weiqi, L., Danjun, W., Tengfei, W., & Yi, Xu. (2011). Preparation,
characterization of hydrophilic and hydrophobic drug in combine loaded chi-
tosan/cyclodextrin nanoparticles and in vitro release study. Colloids and Surfaces
B: Biointerfaces, 83, 103–107.
WAF–CD complex stability constant resulting from the presence
of CS leading to an increase of the free drug amount available for
permeation, and thus, rendering the enhancing effect of CD on WAF
permeation evident (Zerrouk et al., 2006).
Release and permeation studies of WAF from CS nanoparticles
indicated that the percentage drug released from the prepared
nanoparticles correlates well with the percentage permeated
(approximately 60% in 8 h) through the skin. This suggests that
nanoparticles structures allow high drug mobility in the vehicle
leading to faster drug diffusion to the skin surface, and thus, a higher
transdermal delivery (Kreilgaard, 2002).
Kim, D. G., Jeong, Y. I., Choi, C., Roh, S. H., Kang, S. K., Jang, M. K., et al. (2006). Retinol
encapsulated low molecular water-soluble chitosan nanoparticles. International
Journal of Pharmaceutics, 319, 130–138.
Krauland, A. H., & Alonso, M. J. (2007). Chitosan/cyclodextrin nanoparticles as macro-
molecular drug delivery system. International Journal of Pharmaceutics, 340,
134–142.
Kreilgaard, M. (2002). A ssessment of cutaneous drug delivery using microdialysis.
Advanced Drug Delivery Reviews, 1, 99–121.
Kumar, D. A., Dharmendra, S., Jhansee, M., Shrikant, N., & Pandey, S. P. (2011). Devel-
opment and characterization of chitosan nanoparticles loaded with Amoxycilin.
International Research Journal of Pharmacy, 2, 145–151.
4. Conclusion
Langoth, N., Kahlbacher, H., Schöffmann, G., Schmerold, I., Schuh, M., Franz, S.,
et al. (2006). Thiolated chitosans: Design and in vivo evaluation of a mucoad-
hesive buccal peptide drug delivery system. Pharmaceutical Research, 23,
573–579.
WAF-␤-CD loaded CS nanoparticles were successfully prepared
by ionic gelation method. CS nanoparticles were found to be spher-
ical, smooth and with narrow size distribution. They showed high
drug entrapment efficiency and well accepted yield. The release
profile of WAF-␤-CD complex from nanoparticles showed an ini-
tial burst effect followed by a slow and continuous release phase.
The nanoparticle formulation enhanced the permeation of WAF
through excised rat skin in a constant and continuous profile. There-
fore, it could be concluded that combining the virtues of ␤-CD
and CS was a good tool for enhancing its controlled release and
successful permeation, thus, offering a promising system for the
transdermal delivery of WAF.
Lin, S. Y.,
& Yang, J. C. (1986). Inclusion complexation of WARFARIN with
cyclodextrins to improve some pharmaceutical characteristics. Pharmaceutisch
Weekblad-Scientific Edition, 8, 223–228.
Liu, W., & Guo, Y. (2006). Interaction between flavonoid, quercetin and surfactant
aggregates with different charges. Journal of Colloid and Interface Science, 302,
625–632.
Loftsson, T., & Brewster, M. E. (1996). Pharmaceutical applications of cyclodextrins.
I. Drug solubilization and stabilization. Journal of Pharmaceutical Sciences, 85,
1017–1025.
Maestrelli, F., Garcia-Fuentes, M., Mura, P., & Alonso, M. J. (2006). A new drug
nanocarrier consisting of chitosan and hydoxypropylcyclodextrin. European
Journal of Pharmaceutics and Biopharmaceutics, 63, 79–86.
Marcus, E., Brewster, M. E., & Loftsson, T. (2007). Cyclodextrins as pharmaceutical
solubilizers. Advanced Drug Delivery Reviews, 59, 645–666.
Matsuda, H., & Arima, H. (1999). Cyclodextrins in transdermal and rectal delivery.
Advanced Drug Delivery Reviews, 36, 81–99.
References
Mei, D., Mao, S., Sun, W., Wang, Y.,
& Kissel, T. (2008). Effect of chitosan
structure properties and molecular weight on the intranasal absorption of
tetramethylpyrazine phosphate in rats. European Journal of Pharmaceutics and
Biopharmaceutics, 70, 874–881.
Adlin, J., Nesalin, J., Gowthamarajan, K., & Somashekhara, C. N. (2009). Formulation
and evaluation of nanoparticles containing flutamide. International Journal of
ChemTech Research, 1, 1331–1334.
Al-Saidan, S. M. (2004). Transdermal self-permeation enhancement of ibuprofen.
Journal of Controlled Release, 100, 199–209.
Morishita, M., & Peppas, N. A. (2006). Is the oral route possible for peptide and protein
drug delivery. Drug Discovery Today, 11, 905–910.
Muzzrelli, R. A. A. (2012). Chemical and technological advances in chitins and chi-
tosans useful for the formulation of biopharmaceuticals. In B. Sarmento, &
DasNeves (Eds.), Chitosan-based systems for biopharmaceuticals (pp. 3–22). New
York: Wiley.
Oyarzun-Ampuero, F. A., Brea, J., Loza, M. I., Torres, D., & Alonso, M. J. (2009).
Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment
of asthma. International Journal of Pharmaceutics, 381, 122–129.
Ozcan, I., Abaci, O., Uztan, A. H., Aksu, B., Boyaciog˘lu, H., Güneri, T., et al. (2009).
Enhanced topical delivery of terbinafine hydrochloride with chitosan hydrogels.
AAPS Pharmaceutical Science and Technology, 10, 1024–1031.
Anitha, A., Maya, S., Deepa, N., Chennazhi, K. P., Nair, S. V., & Tamura, H. (2011).
Efficient water-soluble biodegradable polymeric nanocarrier for the delivery of
curcumin to cancer cells. Carbohydrate Polymers, 83, 452–461.
Arima, H., Yunomae, K., Miyake, K., Irie, T., Hirayama, F., & Uekama, K. (2001). Com-
parative studies of the enhancing effects of cyclodextrins on the solubility and
oral bioavailability of tacrolimus in rats. Journal of Pharmaceutical Sciences, 90,
690–701.
Bayarski, Y. Transdermal drug delivery, transdermal patches. (2005). Available
from:
http://ezinearticles.com/?Transdermal-Drug-Delivery,-Transdermal-
Patches&id=155961.
Pan, Y., Li, Y. J., Zhao, H. Y., Zheng, J. M., Xu, H., Wei, G., et al.
Boonsongrit, Y., Mitrevej, A., & Mueller, B. W. (2006). Chitosan drug binding by
ionic interaction. European Journal of Pharmaceutics and Biopharmaceutics, 62,
267–274.
Calvo, P., Remunan-Lopez, C., Vila-Jato, J. L., & Alonso, M. J. (1997). Chitosan and chi-
tosan/ethylene oxide–propylene oxide block copolymer nanoparticles as novel
carriers for proteins and vaccines. Pharmacal Research, 14, 1431–1436.
Ceschel, G., Bergamante, V., Maffei, P., Borgia Lambardi, S., Calabrese, V., Bis-
erni, S., et al. (2003). Solubility and transdermal permeation properties of a
dehydroepiandrosterone cyclodextrin complex from hydrophilic and lipophilic
vehicles. Drug Delivery, 12, 275–280.
(2002).
tem:
Bioadhesive
Chitosan nanoparticles
polysaccharide
improve
in
protein
the intestinal
delivery
sys-
absorption
of insulin in vivo. International Journal of Pharmaceutics, 249,
139–147.
Panyam, J.,
& Labhasetwar, V. (2003). Biodegradable nanoparticles for drug
and gene delivery to cells and tissue. Advanced Drug Delivery Reviews, 55,
329–347.
Papadimitriou, S., Bikiaris, D., Avgoustakis, K., Karavas, E., & Georgarakis, M. (2008).
Chitosan nanoparticles loaded with dorzolamide and pramipexole. Carbohydrate
Polymers, 73, 44–54.
Peltola, S., Saarinen-Savolainen, P., Kiesvaara, J., & Suhonen, T. M. (2003). Microemul-
sions for topical delivery of estradiol. International Journal of Pharmaceutics, 254,
99–107.
Poovi, G., Lekshmi, U. M. D., Narayanan, N., & Reddy, N. (2011). Prepration and char-
acterization of repaglinide loaded chitosan polymeric nanoparticles. Research
Journal of Nanoscience and Nanotechnology, 1, 12–24.
Schillers, J., Naji, L., Trampel, R., Ngwa, W., Knauss, R., & Arnold, K. (2004). Pulsed-
field gradient-nuclear magnetic resonance (PFG NMR) to measure the diffusion
of ions and polymers in cartilage. In F. De Ceuninck, P. Pastoureau, & M. Saba-
tini (Eds.), Osteoarthritis: Methods and protocols (pp. 287–302). Totowa: Humana
Press.
Shenoy, D. B., & Amiji, M. M. (2005). Poly (ethyleneoxide)-modified poly (epsilon
caprolactone) nanoparticles for targeted delivery of tamoxifrnin breast cancer.
International Journal of Pharmaceutics, 293, 261–270.
Stella, V. J., Mooney, K. G., & Pipkin, J. D. (1984). Dissolution and ionization of WAR-
FARIN. Journal of Pharmaceutical Sciences, 73, 946–948.
Szejtli, J. (1988). Introduction and general overview of cyclodextrin chemistry. Chem-
ical Reviews, 98, 1743–1753.
Chen, Y., Mohanraj, V. J., & Parkin, J. E. (2003). Chitosan-dextran sulfate nanoparti-
cles for delivery of an antiangiogenesis peptide. International Journal of Peptide
Research and Therapeutics, 10, 621–629.
De Campos, A. M., Sanchez, A., & Alonso, M. J. (2001). Chitosan nanoparticles: A
new vehicle for the improvement of the delivery of drugs to the ocular surface
application to cyclosporine. International Journal of Pharmaceutics, 224, 159–168.
Deng, Q. Y., Zhou, C. R., & Luo, B. H. (2006). Preparation and characterization of chi-
tosan nanoparticles containing lysozyme. Pharmaceutical Biology, 44, 336–342.
Douglas, S. J., Davis, S. S., & Lllum, L. (1987). Nanoparticles in drug delivery. CRC
Critical Reviews in Therapeutic Drug Carrier Systems, 3, 233–261.
Fernandes, C., Viera, M. T., & Veiga, F. (2002). Physicochemical chraracterization and
in vitro dissolution behavior of nicardipinecyclodextrins inclusion compounds.
European Journal of Pharmaceutical Sciences, 15, 79–88.
Grenha, A., Remunan-Lopez, C., Carvalho, E. L., & Seijo, B. (2008). Microspheres
containing lipid/chitosan nanoparticles complexes for pulmonary delivery of
therapeutic proteins. European Journal of Pharmaceutics and Biopharmaceutics,
69, 83–93.
Higuchi, T., & Connors, K. A. (1965). Phase-solubility techniques. Advances in Analyt-
ical Chemistry and Instrumentation, 4, 212–217.
Tang, D. W., Yu, S. H., Ho, Y. C., Mi, F. L., Kuo, P. L., & Sung, H. W. (2010). Heparinized
chitosan/poly(g-glutamic acid) nanoparticles for multi-functional delivery of
fibroblast growth factor and heparin. Biomaterials, 31, 9320–9332.
Holbrook, A. M., Pereira, J. A., Labiris, R., McDonald, H., Douketis, J. D., Crowther, M.,
et al. (2005). Systematic overview of WARFARIN and its drug and food interac-
tions. Archives of Internal Medicine, 165, 1095–1106.

S.K.H. Khalil et al. / Carbohydrate Polymers 90 (2012) 1244–1253
1253
Taveira, S. F., Nomizo, A., & Lopez, R. F. V. (2009). Effect of the iontophoresis of a chi-
tosan gelon doxorubicin skin penetration and cytotoxicity. Journal of Controlled
Release, 134, 35–40.
Wu, Y., Yang, W., Wang, C., Hu, J., & Fu, S. (2005). Chitosan nanoparticles as a novel
delivery system for ammonium glycyrrhizinate. International Journal of Pharma-
ceutics, 295, 235–245.
Uekama, K., Hirayama, F., & Irie, T. (1998). Cyclodextrin drug carrier systems. Chem-
Zerrouk, N., Courti, G., Ancillotti, S., Maestrelli, F., Cerri, M., & Mura, P. (2006). Influ-
ence of cyclodextrins and chitosan, separately or in combination, on glyburide
solubility and permeability. European Journal of Pharmaceutics and Biopharma-
ceutics, 62, 241–246.
Zhang, H., Oh, M., Allen, C., & Kumacheva, E. (2004). Monodisperese chitosan
nanoparticles for mucosal drug delivery. Biomacromolecules, 5, 2461–2468.
Zhang, H. L., Wu, S. H., Tao, Y., Zang, L. Q., & Su, Z. Q. (2010). Preparation and char-
acterization of water-soluble chitosan nanoparticles as protein delivery system.
Journal of Nanomaterials, 2010, 5.
ical Reviews, 98, 2045–2076.
Vandenberg, G. W., Drolet, C., Scott, S. L., & No¨ue, J. D. (2001). Factors affect-
ing protein release from alginate-chitosan coacervate microcapsules during
production and gastric/intestinal simulation. Journal of Controlled Release, 77,
297–307.
Wen, H., Xianxi, G., Lihai, X., & Feng, M. (2009). Study on the mechanisms of chitosan
and its derivatives used as transdermal penetration enhancers. International
Journal of Pharmaceutics, 382, 234–243.
Wilson, B., Sammanta, M. K., Santhi, K., Kumar, K. P., Ramasamy, M., & Suresh, B.
(2009). Chitosan nanoparticles as a new delivery system for anti-Alzheimer’s
drug tacrine. Nanomedicine, 6, 144–152.
Zingone, G., & Rubessa, F. (2005). Preformulation study of the inclusion com-
plex WARFARIN-␤-cyclodextrin. International Journal of Pharmaceutics, 291,
3–10.