Acta Biomaterialia 6 (2010) 11401148

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Brief communication

Controlled and extended drug release behavior of chitosan-based

nanoparticle carrier
Q. Yuan a, J. Shah a, S. Hein b, R.D.K. Misra a,*
a

Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, PO Box
44130, Lafayette, LA 70504-4130, USA
b
Interdisciplinary Nanoscience Center and Department of Molecular Biology, University of Aarhus, C.F. Moellers All 1130, 8000 Aarhus C, Denmark

a r t i c l e

i n f o

Article history:
Received 13 May 2009
Received in revised form 22 July 2009
Accepted 19 August 2009
Available online 21 August 2009
Keywords:
Biodegradable polymer
Chitosan
Nanocomposite
Drug response

a b s t r a c t
Controlled drug release is presently gaining signicant attention. In this regard, we describe here the synthesis (based on the understanding of chemical structure), structural morphology, swelling behavior and
drug release response of chitosan intercalated in an expandable layered aluminosilicate. In contrast to
pure chitosan, for which there is a continuous increase in drug release with time, the chitosanaluminosilicate nanocomposite carrier was characterized by controlled and extended release. Drug release from
the nanocomposite particle carrier occurred by degradation of the carrier to its individual components or
nanostructures with a different composition. In both the layered aluminosilicate-based mineral and
chitosanaluminosilicate nanocomposite carriers the positively charged chemotherapeutic drug strongly
bound to the negatively charged aluminosilicate and release of the drug was slow. Furthermore, the pattern of drug release from the chitosanaluminosilicate nanocomposite carrier was affected by pH and the
chitosan/aluminosilicate ratio. The study points to the potential application of this hybrid nanocomposite
carrier in biomedical applications, including tissue engineering and controlled drug delivery.
2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Silicate minerals are characterized by a layered structure and
exhibit properties such as good water absorption, swelling, adsorbability and cation exchange ability that are considered benecial
from the viewpoint of synthesis of pharmaceutical products, as
both inactive and active substances [1,2]. In this regard, clay minerals have been used as stabilizers or emulsifying agents for the
formulation of liquid drugs in this case it was observed that
the bioavailability of drugs was reduced [3,4]. This led to the suggestion that an interaction between the drug and clay inhibited or
delayed release of the drug. Clay minerals are natural cationic
exchangers and thus can bind with cationic drugs in solution via
electrostatic interaction. Depending on the cation exchange capacity of the clay, the cationicity of the drug and pH of the release
medium determine the kinetics of drug release. Apart from electrostatic force, there also exist the possibility of other interactions,
including hydrophobic, hydrogen bonding, ligand exchange and
water bridging. These properties have encouraged the use of clay
minerals for sustained release of drugs and improved drug dissolution [38].
Colloidal clay particles are preferred because they provide a
reproducible pattern of controlled release based on drugclay
* Corresponding author. Tel.: +1 337 482 6430; fax: +1 337 482 1220.
E-mail address: dmisra@louisiana.edu (R.D.K. Misra).

interaction and the swelling property of clay minerals [39]. Clay

also has the ability to form a hydrogel or sol by spontaneous dispersion in water, such that they swell on coming into contact with
water and the exchangeable cations diffuse into the water phase.
This results in deocculation of the clay and individual platelets
detached from the tactoid (a stack of platelets) by ionic repulsion
of negatively charged surfaces [5,10,11]. Given that the drug molecules are bound to the clay through cation exchange, deocculation of the clay is expected to reduce this interaction, with
consequent benets for release of the drug. Thus, the aforementioned properties of clay, including formation of complexes (interaction between drug and clay) and swelling, are benecial for drug
release.
However, in spite of the benecial effects of clay, there are some
inherent drawbacks associated with the use of clay for drug delivery. Under physiological conditions clay dispersions are unstable
and tend to occulate and precipitate in ion containing solutions,
because of the high salt concentration and the presence of polyelectrolytes such as proteins. Stability of dispersion is an important
requirement for drug carriers because it plays a determining role
with regard to adsorption and bioavailability. Furthermore, the
ability of clay particles to adsorb negatively charged or neutral
drugs is low, restricting their application as carriers of negatively
charged or neutral drugs [5]. In this regard, it is believed that the
synthesis of a composite nanocomposite drug carrier would alleviate some of the above disadvantages by exploiting the properties of

1742-7061/$ - see front matter 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2009.08.027

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Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

clay and polymer in such a way that the behavior of the clay is
modied (see below).
Chitosan is a biodegradable copolymer of N-acetylglucosamine
and D-glucosamine that is useful in biomedical applications
[12,13]. For instance, it nds application in battleeld bandages
that stop hemorrhaging in seconds. Non-toxic and non-allergenic
with anti-microbial properties, chitosan has the ability to rapidly
clot blood. Furthermore, it can be used as a matrix material to build
a three-dimensional composite scaffold for tissue engineering. In
the context of the proposed research, chitosan can exchange the
metal interlayer cations of clay [1416] via an ion exchange process [13,17], as schematically illustrated in Fig. 1. Fig. 1 depicts
our fundamental understanding of the structures of clay and chitosan. The cationic exchange mechanism involves interaction between the positive NH3+ groups of chitosan and negatively
charged sites in the clay structure, and mainly controls the adsorption process and generates a strong cross-linked structure in the
hybrid composite [12,13,1719] with a higher anion exchange
ability [14,16].
The benets that can be envisaged for a chitosanclay nanocomposite carrier include: (a) the intercalation of cationic chitosan
in the expandable aluminosilicate structure of clay is expected to
neutralize the strong binding of cationic drug by anionic clay; (b)
the solubility of chitosan at the low pH of gastric uid will decrease
and premature release of the drug in the gastric environment can

be minimized; (c) cationic chitosan provides the possibility of efciently loading negatively charged drugs compared with clay; and
(d) the presence of reactive amine groups on chitosan provides ligand attachment sites for targeted delivery. The limited solubility
of a chitosanclay nanocomposite drug carrier at gastric pH offers
signicant advantages for colon-specic delivery because some
drugs are destroyed in the stomach, at acidic pH and in the presence of digestive enzymes. Furthermore, the mucoadhesive property of chitosan can enhance the bioavailability of drugs in the
gastrointestional tract.
Based on the above discussion, a chitosanclay nanocomposite
drug carrier in the form of nanoparticles was prepared to investigate the release of a model cationic chemotherapeutic drug, doxorubicin. The expandable layered aluminosilicate structure of
nanoclay, consisting of stacks of plate-like layers of 12 nm
thickness separated by an interlayer distance of 13 nm, depends
on the degree to which the polymer penetrates between the individual clay layers during melt compounding, referred to as intercalation. The platelets with an aspect ratio in the range 20100 nm
have an extremely large surface area of 750 m2 g1. Given the
cationic exchange capacity of 120 meq per 100 g Na+ of layered
smectic clay [20], this would allow the adsorption of a similar
number of NH3+ equivalents of polycationic chitosan [14]. In order
to develop an unambiguous understanding of the drug release
behavior of the chitosanclay nanocomposite carrier, drug-loaded

Tetrahedral
~ 1 nm

Octahedral

OH
O

Tetrahed ral

Na

Na

Na+ ~ 1.20 nm

HO

NH COCH3
O
HO
O
OH

NH3+X- HO

Tetrahedra l

OH
O

NH3+X-

o
NH3+X- HO

~ 1 nm

Octahedral

Chitosan

Tetrahedral

nNa X

Tetrahedral
~ 1 nm

Octahedral
Tetrahedral
OH
O
HO

o
NH3+X- HO

NH COCH3
O
HO
O
OH

OH
O
o
+ - HO
NH 3 X

NH 3+X -

~ 1.86 nm

O
OH

Tetrahedral
Octahedral

~ 1 nm

Tetrahedral
Fig. 1. Schematic illustration of intercalation of chitosan in the interplate space between the silicate layers of clay.

O
OH

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Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

chitosan and clay were also examined under identical experimental conditions.
2. Materials and experimental procedures
2.1. Materials
The nanoclay used in this study was montmorillonite from
Nanocor, USA. Chitosan (molecular weight 310 kDa) with a 75
85% degree of deacetylation, ethanol (P99.5%), acetic acid
(P99.7%), sodium hydroxide (98.1%), sodium chloride (99.0%),
anhydrous sodium phosphate dibasic (P99.0%), potassium phosphate monobasic (99.99%) and dialysis membranes (molecular
weight cut-off 6 12,400) were obtained from SigmaAldrich, USA.
Hydrochloric acid was obtained from Fisher Scientic and doxorubicin hydrochloride (DOX) from Tecoland Corp., USA.
2.2. Preparation of the drug carrier
2.2.1. The chitosanclay nanocomposite carrier
Preparation of the chitosanclay nanocomposite particle carrier
involved two steps: (i) dispersion of ethanolic clay suspension in
0.2% (w/v) chitosan solution and (ii) centrifuging, washing and drying of the nanocomposite particles. The 0.2% (w/v) chitosan solution was prepared by diluting 1.0% (w/v) chitosan solution in
1.0 vol.% acetic acid with deionized water. Then, the pH of the
chitosan solution was adjusted to 5.5 using 1 N NaOH. The ethanolic clay suspension was prepared by dispersing clay in deionized
water for 12 h followed by 2 h sonication and addition of ethanol
to the aqueous clay suspension in a 1:1 volume ratio. Finally, the
chitosan solution and the clay suspension were mixed and stirred
for 4 h at 500 r.p.m. Two different chitosan/clay weight ratio of
5:1 and 10:1 were examined. These ratios were selected based
on a recent study with a chitosanmagnetite nanocarrier for targeted drug delivery that indicated non-agglomeration of nanoparticles [21]. The pH of the suspension was kept at 5.5 to minimize
hydrolysis of the clay while ensuring complete solubility of the
chitosan. A washing step was carried out to remove free chitosan
and was carried out by spinning the colloidal suspension at
15,000g for 10 min (Sorvall RC6, Thermo Fisher Scientic, USA)
and redispersing the nanoparticle pellet in deionized water. This
procedure was repeated ve times and the nal pellet was
freeze-dried to collect the chitosanclay nanocomposite particle
carrier.

The drug-containing chitosanclay nanocomposite particle carrier was prepared by mixing the chitosan solution with drug
loaded ethanolic clay suspension. The DOX (20 wt.% with respect
to chitosan) was dissolved in deionized water and added drop by
drop to the ethanolic clay suspension while being sonicated. Washing and drying of the drug-loaded nanocomposite carrier was carried out by repeated centrifuging and redispersion until the
supernatant solution became colorless. Finally, the DOX-loaded
chitosanclay nanocomposite particles were freeze-dried. To avoid
photodegradation of DOX the experiment was performed in the
dark. A schematic illustration of the process is depicted in Fig. 2.
2.2.2. The chitosanDOX carrier
Drug-free and drug-containing chitosan carriers were prepared
using a procedure similar to that described above for the chitosan
clay drug carrier. DOX (20 wt.% with respect to chitosan) was
added to a solution of 0.2% (w/v) chitosan in water at pH 5.5. The
amount of loaded drug was maintained constant to that of chitosanclay. The solution was magnetically stirred for 24 h at room
temperature and then dialyzed against deionized water and the
pH lowered to 5 with 1 N HCl for 48 h.
2.2.3. The clayDOX carrier
First, clay was dispersed in deionized water for 12 h and ultrasonicated for 2 h. This was followed by addition of ethanol
(1:1 v/v) and DOX (4 wt.% with respect to clay) to the clay dispersion. The dispersion was magnetically stirred for 24 h at room temperature. Subsequently, the resulting colloidal solution was
centrifuged at 15,000g for 10 min and the nanoparticles redispersed in deionized water by sonication and further centrifugation. The process was continued until the solution became
colorless and particles settled at the bottom of the glass container.
The collected particles were freeze-dried (Labconco Freezone 6L,
USA) to obtain DOX-loaded clay pellets. A similar procedure was
adopted to prepare a drug-free clay carrier.
The objective of synthesizing drug-free chitosanclay nanocomposite, chitosan and clay particle carriers together with their drugloaded counterparts was to conrm conjugation of drug via Fourier
transform infrared (FTIR) spectroscopy of individual materials.
2.3. Drug loading efciency
To determine the free DOX during preparation of the chitosan
clayDOX and clayDOX carriers the centrifuged solution was col-

Dissolution of chitosan (1%

Dispersion of clay in deionized water
w/v) in 1 vol.% acetic acid
for 12 h and ultrasonicated for 2 h
Dilution with deionized water
I. Add ethanol (1:1 v/v)
0.2 % chitosan solution
II. Add DOX water solution
Adjustment of pH to 5.5
(50 wt.% of chitosan)
with NaOH

Centrifuging
Re-dispersion with deionized water
Centrifuging
Freeze-dried
DOX-loaded chitosan-clay drug carrier
Fig. 2. Flow chart for the preparation of the DOX-loaded chitosanclay nanocomposite particle carrier.

Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

lected, while for the chitosanDOX carrier the dialyzed solution

was collected. The weight of free DOX (Wfree DOX) in the solution
was determined by UVvis spectrophotometry (V-630, Jasco,
USA) using a wavelength of 260 nm. The DOX loading efciency
was calculated as follows:

DOX loading efficiency% 100  W feed DOX  W free DOX =W feed DOX
1
Wfeed DOX is the amount of added DOX. The DOX loading efciency
was estimated to be similar at 79 2%, 75 3%, 84 2% and 84 2%
for chitosanclay (5:1), chitosanclay (10:1), chitosan and clay systems, respectively.

2.4. Swelling behavior

Dried clay, chitosan or chitosanclay (5:1) particles of known
weights were immersed in buffer solutions (pH 1.2, 5.3 and 7.4)
(see Section 2.5) at room temperature. After allowing them to swell
for different times, the weight of the swollen samples was measured after removing excess surface water by gently blotting with
lter paper. The degree of swelling was determined using the following relationship:

Swelling ratio% 100  ms  md =md

where ms and md are the weights of the swollen and dried samples,
respectively. All the swelling experiments were repeated at least
three times.

2.5. Drug release response

The drug release responses of the chitosan, clay and chitosan
clay nanocomposite drug carriers were studied at the physiological
temperature of 37 C and pH 1.2, 5.3 and 7.4. The buffer solution
with pH values of 5.3 and 7.4 was prepared using Na2HPO4 and
KH2PO4, while the buffer solution with a pH value of 1.2 was prepared with NaCl, HCl and deionized water. The pH of 1.2 was used
to mimic the gastric uid; however, the nanocomposite drug carrier need not stay at pH 1.2 for long, because the transition time
for the drug is low. The pH values of 5.3 and 7.4 were selected to
closely mimic the pH gradient from the stomach to the intestine.
In each experiment 2.0 mg of the drug carrier were sealed in a dialysis membrane tube (molecular weight cut-off 6 12,400). The dialyses tube was submerged in 10 ml of buffer solution of pH 1.2, 5.3
or 7.4 and placed in a test tube with a closure. The test tube with a
closure was placed in a water bath maintained at 37 C. An aliquot
of the release medium (2 ml) was withdrawn every hour for the
rst 12 h and thereafter every 12 h until 60 h. The amount of DOX
(Wfree DOX) in the buffer solution was quantied using UVvis spectrophotometry [Eq. (1)] using a wavelength of 261 nm. After each
measurement the withdrawn medium was put back into the system. Given that the measurement time was very short, while the
predetermined drug release time interval was signicantly larger,
the inuence of the returned medium on drug release during the
measurement time was insignicant. All the drug release experiments were repeated three times.
Control experiments using drug solution only were conducted
at 37 C and pH 1.2, 5.3 and 7.4 using the above described membrane method. This is important because at pH 1.2 the free drug
may display a very similar diffusion behavior to the pure chitosan
formulation. After drug release the chitosanclay drug carrier was
collected and dried at 50 C for 24 h to obtain an insight into the
release process by FTIR spectroscopy.

1143

2.6. Characterization of the chitosanclay nanocomposite particle drug

carrier
The morphology and dimensional changes of the chitosanclay
nanocomposite drug carrier before and after drug release were studied via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) Hitachi H-7600). The chitosanclay
particles before and after drug release were placed on a stub and
sputter coated with gold and examined at 10 keV in a JEOL JSM
6300 eld emission scanning electron microscope. The particles
were dispersed in deionized water and a drop of the liquid containing the dispersed nanoparticles were placed on the copper grid for
TEM examination.
The incorporation of clay in the chitosan polymer matrix and
conjugation of drug to the nanocomposite particle was studied
by recording FTIR spectra (FT/IR-480) of clay, chitosan, chitosan
clay (5:1), DOX and chitosanclayDOX (5:1) at 4 cm1 resolution.
3. Results and discussion
3.1. Morphology of the chitosanclay nanocomposite drug carrier
It is important to examine the nanoparticle drug carrier before
and after drug release because any dimensional change may provide a basis for understanding the mechanism of drug release.
Transmission electron micrographs of the chitosanclay nanocomposite drug carrier at identical magnications before and after drug
release at the selected pH of 7.4 are presented in Fig. 3. Fig. 3a suggests near monodispersion of as prepared chitosanclay nanocomposite particle drug carrier with an average diameter of 150 nm
(Fig. 3a), while Fig. 3b implies that the size of the chitosanclay
nanocomposite particles after drug release was signicantly reduced to 30 nm. A similar reduction in size was apparent at pH
5.3. The reason for the decrease in size after drug release is believed to be a consequence of detachment or separation of the
chitosan and clay and is discussed below.
3.2. Characterization of the chitosanclay nanocomposite and
conjugation with the drug
FTIR was used to conrm the incorporation of clay into the host
polymer matrix and loading of drug in the nanocomposite particle
carrier. The FTIR spectra of clay, chitosan, DOX, chitosanclay nanocomposite particle and DOX-loaded chitosanclay before and after
drug release are presented in Fig. 4. The assigned characteristic FTIR
absorption bands derived from Fig. 4 are summarized in Table 1.
Fig. 4a is the FTIR spectrum of clay. The characteristic absorption band at 3632 cm1 [m(OAH)] is assigned to the stretching
vibration of AlAOH. The symmetrical SiAOASi band [m(SiAOASi)]
is characterized by the stretching band at 1160 cm1. Other characteristic absorption bands of pure clay are at 914 [d AlAAlAO)],
886 [d AlAFeAO)] and 848 cm1 [d AlAMgAO)].
The FTIR spectrum of chitosan (Fig. 4b) shows a broad band at
3440 cm1 corresponding to the stretching vibration of NAH.
The peaks at 2924 and 2846 cm1 are typical of CAH stretch vibration, while peaks at 1647, 1597 and 1317 cm1 are characteristic
of amides I, II and III, respectively. The sharp peaks at 1420 and
1383 cm1 are assigned to the CH3 symmetrical deformation mode
and 1153 and 1088 cm1 are indicative of CAO stretching vibrations [m(CAOAC)]. The small peak at 900 cm1 corresponds to
wagging of the saccharide structure of chitosan.
The FTIR spectrum of the chitosanclay nanocomposite shows
the characteristic absorption bands of both clay and chitosan
(Fig. 4c), conrming preparation of the chitosanclay nanoparticle
carrier.

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Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

f. Chitosan-clay-DOX after drug release

1637

e. Chitosan-clay-DOX before drug release

Transmittance (arb. units)

3630

3440

2852
2927

810

1730
1420
15871385

887
624
917
526
1121
467
1047

1622

d. DOX

3334

2925

871 764
816

1732
1545

1115

1583

c. Chitosan-clay

1620

2846
2924

1639

1071

1414
1383

3632
3440

886

1420
1263

1540

920

626
522

1153

464

1080

b. Chitosan

1263

3440

a. Clay

2846
2924

1317
900
1383
1597 1420 1153
1088

1647

3632

1160 914 848

886

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber cm

-1

Fig. 4. FTIR spectra of: (a) clay; (b) chitosan; (c) chitosanclay nanoparticles; (d)
DOX; (e) chitosanclayDOX before drug release; and (f) chitosanclayDOX after
drug release at pH 7.4.

Fig. 3. (a) Low and (b) high magnication transmission electron micrographs of the
chitosanclay nanocomposite drug carrier.

The FTIR spectrum for pure DOX (Fig. 4d) shows multiple peaks
at 3334, 2925, 1732, 1620, 1414 and 1071 cm1. These different
peaks correspond to the different quinone and ketone carbonyls
of DOX. However, it is difcult to delineate the different bands
for the quinine and ketone because both have carbonyl groups.
The peak at 1545 cm1 is due to the stretching bands of NAH.
The peak at 816 cm1 is due to the stretching bands of CAOACH3.
The peaks at 871 and 764 cm1 are due to the primary amine NH2
wag and NAH deformation bonds, respectively.
Comparing the FTIR spectrum of DOX-loaded chitosanclay
with that of chitosanclay, there are additional absorption bands
at 1730, 1121 and 810 cm1 corresponding to the CAOACH3
stretching bands of DOX (highlighted by the box in Fig. 4e), conrming the successful loading of DOX on the chitosanclay nanocomposite particle carrier.

FTIR spectroscopy is also an appropriate technique to study the

polymerclay interaction [22]. It is suggested that when chelation
of transition metal ions by chitosan occurs there is a shift in the
NY vibration [23]. In this regard, the small peak at 1597 cm1
(Fig. 4b) corresponding to the deformation vibration m(NAH) amide
II of the amine group shifted to a lower frequency at 1540 (Fig. 4c)
and 1587 cm1 (Fig. 4e) in the chitosanclay and DOX-conjugated
chitosanclay nanocomposite particles, respectively, indicating the
possibility of an electrostatic interaction between the negatively
charged structure of clay and the amine groups of chitosan. Additionally, compared with pure clay and chitosan, there were three
peaks at 626, 522 and 464 cm1 (highlighted by the box in
Fig. 4c) in chitosanclay. These peaks were of low intensity in chitosan and suggest the possibility of a strong interaction between chitosan and clay.
If we compare the FTIR spectra before and after drug release
(Fig. 4e and f), it seems that the absorption peaks became broad
and were not sharp after drug release. Secondly, the band at
1622 cm1 corresponding to the combined contribution of chitosan
in chitosanclay (1639 cm1, Fig. 4c) and DOX (1620 cm1,
Fig. 4d) became broad and was shifted to 1637 cm1 after drug release at pH 7.4. The spectra after drug release (Fig. 4f) resembled
chitosanclay (Fig. 4c). This observation leads us to suggest that
the drug was released and pointed to the possibility of degradation
of the nanocomposite particle carrier into its individual components
or a nanostructure consistent with a reduction in the size of the
nanoparticle carrier after release of the drug, as implied by TEM
(Fig. 3b).

3.3. Drug release response

The drug release response of pure DOX, pure clay, pure chitosan
and the chitosanclay nanocomposite particle carrier in buffer
solutions with the three different pH values 1.2, 5.3 and 7.4 was

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Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

Table 1
Assignment of FTIR spectra of clay, chitosan, chitosanclay and chitosanclay
conjugated with DOX presented in Fig. 4.
IR absorption Descriptiona
band (cm1)

(a) Clay

3632
1160
914
886
848

m(OAH) for AlAOH and SiAOH

m(SiAO) out of plane

3440
2924
2846
1647
1597
1420, 1383
1317
1263
1153, 1088
900

ms(NAH)
mas(CAH)
ms(CAH)
m(AC@OA) amide I

3632
3440
2924
2846
1639
1540
1444, 1383
1263
1153, 1080
920
886
626
522
464

m(OAH) for AlAOH and SiAOH

ms(NAH)
mas(CAH)
ms(CAH)
m(AC@OA) amide I
m(NAH) amide II

d(AlFeOH)
(FeAO) out of plane vibration
d(SiAOAAl)
d(SiAOASi)

33341071
1530
871
810
764

Quinone and ketone carbonyls

m(NAH) amide I
x(NAH)
m(CAOACH3)
d(NAH)

(e) ChitosanclayDOX
3630
(5:1) before drug release 3440
2927
2852
1730
1622
1587
1420, 1385
1121
1047
917
887
810
624
526
467

m(OAH) for AlAOH and SiAOH

ms(NAH)
mas(CAH)
ms(CAH)

(c) Chitosanclay (5:1)

(d) DOX

(f) ChitosanclayDOX
(5:1) after drug release

3630
3440
2927
2852
1730
1637
1587
1420, 1385
1121
1047
917
887
810
624
526
467

Amine
d(CAH)
m(ACH3) amide III
m(CAOAH)
mas(CAOAC) and ms(CAOAC)
x(CAH)

Chitosan

80

pH = 1.2

70
60
Chitosan-Clay (10:1)

50
40

Chitosan-Clay (5:1)

30
20
10

Clay

0
0

10

20

d(CAH)

m(CAOAH)
mas(CAOAC) and ms(CAOAC)
d(AlAlOH), x(CAH)

Absorption band for DOX

m(AC@OA) amide I
m(NAH) amide II
d(CAH)
Absorption band for DOX
mas(CAOAC) and ms(CAOAC)
x(CAH)
d(AlFeOH)
m(CAOACH3) for DOX
(FeAO) out of plane vibration
d(SiAOAAl)
d(SiAOASi)

m(OAH) for AlAOH and SiAOH

ms(NAH)
mas(CAH)
ms(CAH)
Absorption band for DOX
m(AC@OA) amide I
m(NAH) amide II
d(CAH)
Absorption band for DOX
mas(CAOAC) and ms(CAOAC)
x(CAH)
d(AlFeOH)
m(CAOACH3) from DOX
(FeAO) out of plane vibration
d(SiAOAAl)
d(SiAOASi)

m = stretching vibration; ms = symmetric stretching vibration; mas = asymmetric

stretching vibration; d = bending vibration; x = wagging.

30
40
Time (h)

50

60

Fig. 5a. Cumulative DOX release (%) from the chitosanclay, pure clay and pure
chitosan drug carriers at 37 C. (a) In phosphate buffer solution pH 1.2. At pH 1.2 in
the control experiment using only drug solution the drug was completely released
within 1 h, hence the data points are not shown for pure drug.

studied at the physiological temperature of 37 C (Figs. 5ac). In

control experiments using only drug solution (pure DOX without
carrier) the drug was completely released within 1, 5 (Fig. 5b)
and 12 h (Fig. 5c) at pH 1.2, 5.3 and 7.4, respectively. Compared
with the nanocomposite particle carrier, the release rate of pure
drug was very fast, conrming the ability of controlled drug release
by the nanocomposite drug carrier, as described below.
The dependence of percentage cumulative DOX release from the
nanocomposite particle carrier at a temperature of 37 C in the
HClNaCl buffer solution (pH 1.2) and phosphate buffer solutions

100
DOX

90

pH = 5.3

80
% Cumulative DOX Release

(b) Chitosan

d(AlAlOH)
d(AlFeOH)
d(AlMgOH)

90

% Cumulative DOX Release

Sample

70
60
50

Chitosan

40
Chitosan-Clay (10:1)

30
Chitosan-Clay (5:1)

20
10

Clay

0
0

10

20

30
40
Time (h)

50

Fig. 5b. In phosphate buffer solution pH 5.3.

60

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Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

100

pH = 7.4

% Cumulative DOX Release

90

DOX

80
70
60
50

Chitosan

40
Chitosan-Clay (10:1)

30

Chitosan-Clay (5:1)

20
10
Clay

0
0

10

20

30
40
Time (h)

50

60

at pH 7.4. This means that DOX binds even more strongly to clay
and, therefore, DOX release from clay after 10 h dropped by more
than half at pH 5.3 and 7.4. Given that the clay was loaded with
DOX before the chitosanclay nanocomposite was prepared, drug
release from the nanocomposite particle carrier was primarily controlled by the clay. However, the presence of chitosan in the nanocomposite particle carrier undermined the attractive force
between DOX and the clay. This is corroborated by the observation
of faster release of the drug (Figs. 5ac) with increasing chitosan
content in the nanocomposite particle carrier. Thus, DOX release
was comparatively faster from the nanocomposite carrier than
from pure clay at all three pH values.
Moreover, the presence of chitosan in the nanocomposite particle carrier resulted in mucoadhesion and promoted bioavailability
of the drug by interacting with the gastric and intestinal mucosa.
Thus, increasing the chitosan content of the chitosanclay nanocomposite could increase the release rate. The release of drug from
the nanocomposite could be tuned by controlling the amount of
chitosan in the nanocomposite. It may be noted from Figs. 5ac
that at pH 1.2 the drug release rate at times (t) greater than
20 h was nearly constant, while at pH 5.3 and 7.4 the drug release
rate at t > 20 h continued to increase at a rate of 0.002 h1 at pH 5.3
and 0.004 h1 at pH 7.4 for chitosan10 wt.% clay. This implies that

Fig. 5c. In phosphate buffer solution pH 7.4. The data points are averages of at least
three experiments.

1000

pH = 5.3

Clay

Swelling Ratio (%)

800

600

Chitosan

400

Chitosan-Clay

200

0
0

3
Time (h)

1000

pH = 7.4

Clay

800

Swelling Ratio (%)

(pH 5.3 and 7.4) are presented in Figs. 5ac. There was an initial
burst release and then a gradual release of DOX in all the investigated drug carriers at different pH values. The initial burst release
was attributed to diffusion of the drug due to rapid swelling and
was also partially related to drug adsorbed on the surface. However, the release rates were affected by pH and the weight ratio
of chitosan to clay. The burst release of drug was unlikely to be
non-encapsulated drug because the nanocomposite carrier was
centrifuged and thoroughly washed to remove any non-encapsulated drug (see drug loading efciency data in Section 2.3). A significant nding was that cumulative release from the chitosanclay
nanocomposite particle carrier was intermediate between chitosan
and clay, i.e. greater than pure clay and signicantly lower than
pure chitosan. The percentage cumulative drug release followed
the sequence chitosan > chitosanclay (10:1) > chitosanclay
(5:1) > clay at all three investigated pH values (pH 1.2, 5.3 and
7.4). An identical sequence was found when the pH was increased
from 1.2 to 7.4. At the high pH of 7.4 chitosan is insoluble, while at
pH 5.3 it is partially soluble and at pH 1.2 completely soluble.
Drug release from pure chitosan was very rapid at pH 1.2. In
contrast, drug release was far less rapid for both the chitosanclay
nanocomposite and pure clay. Drug release from these matrices
was signicantly slower and controlled release (Fig. 5a). The study
at pH 1.2 suggested that the carrier would release drug in gastrointestinal uid following oral administration. In the presence of
digestive enzymes and the microora inside the stomach a faster
release rate would be expected, because the enzymes degrade
chitosan. In clay and chitosanclay nanocomposite particle carriers
the positively charged DOX bound strongly to the negatively
charged clay and the release of DOX is very slow. For a similar reason, when the clay content was high in the nanocomposite carrier
(chitosanclay 5:1) less drug was released.
With an increase in pH to 5.3 the solubility of chitosan was limited, and it was insoluble at pH 7.4, leading to a signicant decrease
in the burst release of drug. The release of DOX after 10 h from the
pure chitosan matrix dropped from 90% at pH 1.2 to 20% and
15% at pH 5.3 and 7.4, respectively (Figs. 5b and c). On the other
hand, the negative charge on clay increases with increasing pH,
while DOX (weak base, pKa  8.3) is still positively charged even

600

400

Chitosan

200

Chitosan-Clay

0
0

Time (h)
Fig. 6. Swelling behavior of clay, chitosan and chitosanclay nanoparticles at pH 5.3
and 7.4.

Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

1147

a higher cumulative amount of drug would be released at pH 7.4

compared with pH 5.3 and 1.2 at t > 100 h. Furthermore, the
low drug release from the nanocarrier in contrast to pure chitosan
may be considered an advantage because in the nanocomposite
carrier the solubility of chitosan in low pH gastric uid will be reduced and premature release of the drug in the gastric environment will be avoided. The continued and higher release of drug
at t > 20 h at pH 5.3 and 7.4 from the nanocomposite carrier could
be an advantage for colon-specic drug release when controlled
and extended release is preferred. Another potential application
area where drug-loaded chitosanclay can be considered is in the
preparation of tissue engineering scaffolds.
Using the chitosanclay nanocomposite synthesis approach described here one can prepare an implant capable of prolonged release of drug up to several days. Furthermore, the drug loading
capacity of chitosanclay will be higher than normal chitosan scaffolds. In this study the drug loading capacities of clay, chitosan
clay composite (5:1), chitosanclay composite (10:1) and chitosan
were high at 0.21, 0.19, 0.16 and 0.12 mg DOX per mg matrix,
respectively.
Fig. 6 describes the swelling behavior of clay, chitosan and
chitosanclay (5:1) as a function of time at pH 5.3 and 7.4. Experiments at pH 1.2 were not conducted because of the high dissolution of pure chitosan and chitosanclay and consequent nonavailability of data for pure chitosan and chitosanclay for comparison with clay, even though the clay was stable at pH 1.2. It is
intriguing that the swelling ratios were similar at pH 5.3 and 7.4
and within the experimental scatter for all three systems. However, chitosanclay experienced less swelling than pure clay and

pure chitosan under identical experimental conditions, but drug

release was greater than from clay but less than from pure chitosan. The addition of clay to chitosan builds a strong cross-linking
structure because of the negatively charged clay and positively
charged NH3+ groups of chitosan [17]. This inuences the swelling
behavior of the nanocomposite and consequently inuences diffusion of the drug through the bulk entity.
From this study on chitosanclay nanocomposite, pure clay and
pure chitosan drug carriers we propose that the electrostatic interaction between the positive charge of DOX and negatively charged
sites on clay and a similar interaction between clay and chitosan
are responsible for the lower release of drug as compared with
pure chitosan. These interactions between DOX, clay and/or clay
chitosan must be stable, such that intercalation of the polymer between the clay layers permanently separates these layers.
The drug carrier was further subjected to examination by SEM
before and after drug release. A comparison of the micrographs
suggests detachment of the drug, clay and chitosan during drug release, with a consequent increase in the size of pores (Figs. 7 and
8). The pore size increased from 1.1 0.1 lm before drug release
(Fig. 7) to 2.3 0.2 lm after drug release (Fig. 8). In addition, a
reduction in the size of the nanocomposite carrier was observed
by TEM (Fig. 1), implying detachment of the drug and carrier.
TEM (Fig. 1) and SEM observations of drug release (Figs. 7 and 8)
and swelling behavior (Fig. 6) suggest that drug release occurred
by degradation of the nanocomposite carrier to its individual components or nanostructures with different composition and was
controlled by ionic interaction between the drug molecules and
chitosan and/or clay.

Fig. 7. Scanning electron micrographs of DOX-loaded chitosanclay particles before

drug release.

Fig. 8. Scanning electron micrographs of DOX-loaded chitosanclay particles after

drug release at pH 5.3 and 37 C.

1148

Q. Yuan et al. / Acta Biomaterialia 6 (2010) 11401148

As discussed above, by optimizing the content of chitosan in the

composite one can control drug release. It is, however, important
to bear in mind [13,18] that additional chitosan results in intercalation of the biopolymer as a bilayer, with the thickness of two layers of chitosan together with that of the acetate anion. The second
layer of biopolymer is adsorbed by means of hydrogen bonding,
since the cationic exchange capacity (CEC) of the clay has already
been balanced by the NH3+ groups of the rst layer. Thus, the
NH3+ groups of the second layer interact electrostatically with acetate ions from the starting chitosan solution, available as anionic
exchange sites, which will be useful in the encapsulation of anionic
drugs. This unique characteristic will enable the nanocomposite to
encapsulate either cationic or anionic drugs for controlled drug
delivery. In summary, chitosanclay nanocomposite is a versatile
polymer nanocomposite for biomedical applications, including tissue engineering and controlled drug delivery.
4. Conclusions
Chitosanclay nanocomposites are potential polymer nanocomposites of interest in biomedical applications, including tissue
engineering and controlled drug delivery. The controlled release
of drug from a chitosanclay nanocomposite drug carrier, in contrast to pure chitosan, is controlled by electrostatic interaction between the positive charge of DOX and negatively charged sites in
the clay. The factors governing the drug release prole include
swelling behavior and drugcarrier interactions. The drug release
behavior is inuenced by pH and the chitosan/clay ratio. Drug release occurs by degradation of the nanocomposite particle carrier
to its individual components or nanostructures of different
composition.
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