Controlled and Extended Drug Release Behavior of Chitosan-based

8/17/2019 Controlled and Extended Drug Release Behavior of Chitosan-based

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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, USAb Interdisciplinary Nanoscience Center and Department of Molecular Biology, University of Aarhus, C.F. Moellers Allé 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 significant attention. In this regard, we describe here the syn-

thesis (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 chitosan–alumino-

silicate 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

chitosan–aluminosilicate nanocomposite carriers the positively charged chemotherapeutic drug strongly

bound to the negatively charged aluminosilicate and release of the drug was slow. Furthermore, the pat-

tern of drug release from the chitosan–aluminosilicate 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, adsorb-

ability and cation exchange ability that are considered beneficial

from the viewpoint of synthesis of pharmaceutical products, as

both inactive and active substances [1,2]. In this regard, clay min-

erals 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 sug-

gestion 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 capac-

ity of the clay, the cationicity of the drug and pH of the release

medium determine the kinetics of drug release. Apart from electro-

static 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 dissolu-

tion [3–8].

Colloidal clay particles are preferred because they provide a

reproducible pattern of controlled release based on drug–clay

interaction and the swelling property of clay minerals [3–9]. Clay

also has the ability to form a hydrogel or sol by spontaneous dis-

persion in water, such that they swell on coming into contact with

water and the exchangeable cations diffuse into the water phase.

This results in deflocculation 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 mol-

ecules are bound to the clay through cation exchange, defloccula-

tion of the clay is expected to reduce this interaction, with

consequent benefits for release of the drug. Thus, the aforemen-

tioned properties of clay, including formation of complexes (inter-

action between drug and clay) and swelling, are beneficial for drug

release.

However, in spite of the beneficial effects of clay, there are some

inherent drawbacks associated with the use of clay for drug deliv-

ery. Under physiological conditions clay dispersions are unstable

and tend to flocculate and precipitate in ion containing solutions,

because of the high salt concentration and the presence of poly-

electrolytes 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 allevi-

ate 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

* Corresponding author. Tel.: +1 337 482 6430; fax: +1 337 482 1220.

E-mail address: [email protected] (R.D.K. Misra).

Acta Biomaterialia 6 (2010) 1140–1148

Contents lists available at ScienceDirect

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http://dx.doi.org/10.1016/j.actbio.2009.08.027mailto:[email protected]://www.sciencedirect.com/science/journal/17427061http://www.elsevier.com/locate/actabiomathttp://www.elsevier.com/locate/actabiomathttp://www.sciencedirect.com/science/journal/17427061mailto:[email protected]://dx.doi.org/10.1016/j.actbio.2009.08.027


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clay and polymer in such a way that the behavior of the clay is

modified (see below).

Chitosan is a biodegradable copolymer of N -acetylglucosamine

and D-glucosamine that is useful in biomedical applications

[12,13]. For instance, it finds application in battlefield 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 [14–16] via an ion exchange pro-

cess [13,17], as schematically illustrated in Fig. 1. Fig. 1 depicts

our fundamental understanding of the structures of clay and chito-

san. The cationic exchange mechanism involves interaction be-

tween the positive NH3+ groups of chitosan and negatively

charged sites in the clay structure, and mainly controls the adsorp-

tion process and generates a strong cross-linked structure in the

hybrid composite [12,13,17–19] with a higher anion exchange

ability [14,16].

The benefits that can be envisaged for a chitosan–clay nano-

composite 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 lowpH of gastric fluid will decrease

and premature release of the drug in the gastric environment can

be minimized; (c) cationic chitosan provides the possibility of effi-

ciently loading negatively charged drugs compared with clay; and

(d) the presence of reactive amine groups on chitosan provides li-

gand attachment sites for targeted delivery. The limited solubility

of a chitosan–clay nanocomposite drug carrier at gastric pH offers

significant advantages for colon-specific delivery because some

drugs are destroyed in the stomach, at acidic pH and in the pres-

ence of digestive enzymes. Furthermore, the mucoadhesive prop-

erty of chitosan can enhance the bioavailability of drugs in the

gastrointestional tract.

Based on the above discussion, a chitosan–clay nanocomposite

drug carrier in the form of nanoparticles was prepared to investi-

gate the release of a model cationic chemotherapeutic drug, doxo-

rubicin. The expandable layered aluminosilicate structure of

nanoclay, consisting of stacks of plate-like layers of 1–2 nm

thickness separated by an interlayer distance of 1–3 nm, depends

on the degree to which the polymer penetrates between the indi-

vidual clay layers during melt compounding, referred to as interca-

lation. The platelets with an aspect ratio in the range 20–100 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 chitosan–clay nanocomposite carrier, drug-loaded

Tetrahedral

Octahedral

Tetrahedral

~ 1 nm

~ 1.86 nm

~ 1 nmOctahedral

Tetrahedral

Tetrahedral

O

NH3+X-

OH

HO

O

NH

HOo

O

NH3+X

-

OH

HO

OH

OCOCH3

O

NH3+X-

HOo

OH

nNa+X

-

+

Tetrahedral

Octahedral

Tetrahedral

~ 1 nm

~ 1.20 nm

~ 1 nmOctahedral

Tetrahedral

Tetrahedra l

Na+

Na+Na

+O

NH3+X-

OH

HO

O

NH

HOo

O

NH3+

X-

OH

HO

OH

OCOCH3

O

NH3+X-

HOo

OH

Chitosan

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

Q. Yuan et al. / Acta Biomaterialia 6 (2010) 1140–1148 1141


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chitosan and clay were also examined under identical experimen-

tal conditions.

2. Materials and experimental procedures

2.1. Materials

The nanoclay used in this study was montmorillonite fromNanocor, 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 phos-

phate monobasic (99.99%) and dialysis membranes (molecular

weight cut-off 6 12,400) were obtained from Sigma–Aldrich, USA.

Hydrochloric acid was obtained from Fisher Scientific and doxoru-

bicin hydrochloride (DOX) from Tecoland Corp., USA.

2.2. Preparation of the drug carrier

2.2.1. The chitosan–clay nanocomposite carrier

Preparation of the chitosan–clay nanocomposite particle carrier

involved two steps: (i) dispersion of ethanolic clay suspension in0.2% (w/v) chitosan solution and (ii) centrifuging, washing and dry-

ing of the nanocomposite particles. The 0.2% (w/v) chitosan solu-

tion 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 etha-

nolic 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 chitosan–magnetite nanocarrier for tar-

geted drug delivery that indicated non-agglomeration of nanopar-

ticles [21]. The pH of the suspension was kept at 5.5 to minimizehydrolysis 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

15,000 g for 10 min (Sorvall RC6, Thermo Fisher Scientific, USA)

and redispersing the nanoparticle pellet in deionized water. This

procedure was repeated five times and the final pellet was

freeze-dried to collect the chitosan–clay nanocomposite particle

carrier.

The drug-containing chitosan–clay nanocomposite particle car-

rier 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. Wash-

ing and drying of the drug-loaded nanocomposite carrier was car-

ried out by repeated centrifuging and redispersion until the

supernatant solution became colorless. Finally, the DOX-loaded

chitosan–clay 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 chitosan–DOX 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 chito-

san–clay. 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 clay–DOX carrier

First, clay was dispersed in deionized water for 12 h and ultr-

asonicated 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 disper-

sion. The dispersion was magnetically stirred for 24 h at room tem-

perature. Subsequently, the resulting colloidal solution was

centrifuged at 15,000 g for 10 min and the nanoparticles redi-

spersed in deionized water by sonication and further centrifuga-

tion. 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 chitosan–clay nanocom-posite, chitosan and clay particle carriers together with their drug-

loaded counterparts was to confirm conjugation of drug via Fourier

transform infrared (FTIR) spectroscopy of individual materials.

2.3. Drug loading efficiency

To determine the free DOX during preparation of the chitosan–

clay–DOX and clay–DOX carriers the centrifuged solution was col-

Adjustment of pH to 5.5

with NaOH

Dilution with deionized water

Dissolution of chitosan (1%

w/v) in 1 vol.% acetic acid

0.2 % chitosan solution

I. Add ethanol (1:1 v/v)

Dispersion of clay in deionized water

for 12 h and ultrasonicated for 2 h

Centrifuging

II. Add DOX water solution

(50 wt.% of chitosan)

Re-dispersion with deionized water

Freeze-dried

DOX-loaded chitosan-clay drug carrier

Centrifuging

Fig. 2. Flow chart for the preparation of the DOX-loaded chitosan–clay nanocomposite particle carrier.

1142 Q. Yuan et al. / Acta Biomaterialia 6 (2010) 1140–1148


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lected, while for the chitosan–DOX carrier the dialyzed solution

was collected. The weight of free DOX (W free DOX) in the solution

was determined by UV–vis spectrophotometry (V-630, Jasco,

USA) using a wavelength of 260 nm. The DOX loading efficiency

was calculated as follows:

DOX loading efficiencyð%Þ¼100ðW feedDOXW freeDOXÞ=W feedDOX

ð1Þ

W feed DOX is the amount of added DOX. The DOX loading efficiency

was estimated to be similar at 79 ± 2%, 75 ± 3%, 84 ± 2% and 84 ± 2%

for chitosan–clay (5:1), chitosan–clay (10:1), chitosan and clay sys-

tems, respectively.

2.4. Swelling behavior

Dried clay, chitosan or chitosan–clay (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 mea-

sured after removing excess surface water by gently blotting with

filter paper. The degree of swelling was determined using the fol-

lowing relationship:

Swelling ratioð%Þ ¼ 100 ðms mdÞ=md ð2Þ

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 solutionwith 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 pre-

pared with NaCl, HCl and deionized water. The pH of 1.2 was used

to mimic the gastric fluid; however, the nanocomposite drug car-

rier 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 dial-

ysis membrane tube (molecular weight cut-off 6 12,400). The dial-

yses 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

first 12 h and thereafter every 12 h until 60 h. The amount of DOX(W free DOX) in the buffer solution was quantified using UV–vis spec-

trophotometry [Eq. (1)] using a wavelength of 261 nm. After each

measurement the withdrawn medium was put back into the sys-

tem. Given that the measurement time was very short, while the

predetermined drug release time interval was significantly larger,

the influence of the returned medium on drug release during the

measurement time was insignificant. All the drug release experi-

ments 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 mem-

brane 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 chitosan–clay drug carrier was

collected and dried at

50 C for 24 h to obtain an insight into therelease process by FTIR spectroscopy.

2.6. Characterization of the chitosan–clay nanocomposite particle drug

carrier

The morphology and dimensional changes of the chitosan–clay

nanocomposite drugcarrier before andafter drugrelease were stud-

ied via scanning electron microscopy (SEM) and transmission elec-

tron microscopy (TEM) Hitachi H-7600). The chitosan–clay

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 field emission scanning electron microscope. The particles

were dispersed in deionized water and a drop of the liquid contain-

ing 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 chitosan–clay–DOX (5:1) at 4 cm1 resolution.

3. Results and discussion

3.1. Morphology of the chitosan–clay nanocomposite drug carrier

It is important to examine the nanoparticle drug carrier before

and after drug release because any dimensional change may pro-

vide a basis for understanding the mechanism of drug release.

Transmission electron micrographs of the chitosan–clay nanocom-

posite drug carrier at identical magnifications before and after drug

release at the selected pH of 7.4 are presented in Fig. 3. Fig. 3a sug-

gests near monodispersion of as prepared chitosan–clay nanocom-

posite particle drug carrier with an average diameter of 150 nm

(Fig. 3a), while Fig. 3b implies that the size of the chitosan–clay

nanocomposite particles after drug release was significantly re-

duced 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 be-

lieved to be a consequence of detachment or separation of the

chitosan and clay and is discussed below.

3.2. Characterization of the chitosan–clay nanocomposite and

conjugation with the drug

FTIR was used to confirm 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, chitosan–clay nano-

composite particle and DOX-loaded chitosan–clay 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 absorp-

tion 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 char-acteristic 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 vibra-

tion, 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 vibra-

tions [m(CAOAC)]. The small peak at 900 cm1 corresponds to

wagging of the saccharide structure of chitosan.

The FTIR spectrum of the chitosan–clay nanocomposite shows

the characteristic absorption bands of both clay and chitosan

(Fig. 4c), confirming preparation of the chitosan–clay nanoparticlecarrier.



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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 difficult 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 NH2wag and NAH deformation bonds, respectively.

Comparing the FTIR spectrum of DOX-loaded chitosan–clay

with that of chitosan–clay, there are additional absorption bands

at 1730, 1121 and 810 cm1 corresponding to the CAOACH3stretching bands of DOX (highlighted by the box in Fig. 4e), con-

firming the successful loading of DOX on the chitosan–clay nano-composite particle carrier.

FTIR spectroscopy is also an appropriate technique to study the

polymer–clay 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 chitosan–clay and DOX-conjugated

chitosan–clay nanocomposite particles, respectively, indicating the

possibility of an electrostatic interaction between the negatively

charged structure of clay and the amine groups of chitosan. Addi-

tionally, compared with pure clay and chitosan, there were three

peaks at 626, 522 and 464 cm1 (highlighted by the box in

Fig. 4c) in chitosan–clay. Thesepeaks were of low intensity in chito-

sanand suggest the possibility of a strong interaction betweenchito-

san 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 chitosan–clay (1639 cm

1, Fig. 4c) and DOX (1620 cm

1,Fig. 4d) became broad and was shifted to 1637 cm1 after drug re-

lease at pH 7.4. The spectra after drug release (Fig. 4f) resembled

chitosan–clay (Fig. 4c). This observation leads us to suggest that

the drug was released and pointed to the possibility of degradation

of the nanocomposite particlecarrierinto 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 chitosan–clay nanocomposite particle carrier in buffersolutions with the three different pH values 1.2, 5.3 and 7.4 was

Fig. 3. (a) Low and (b) high magnification transmission electron micrographs of the

chitosan–clay nanocomposite drug carrier.

4000 3500 3000 2500 2000 1500 1000 500

1545

816

1420

467526

1115

28522927

34403630

764

626

464

522

886

12631317

1383

886

f. Chitosan-clay-DOX after drug release

d. DOX

1730 810

2846

1620

1153

1071

900

1088

1263

1420

1583

1597

1647

2924

3440

3632

3440

e. Chitosan-clay-DOX before drug release

c. Chitosan-clay

b. Chitosan

T r a n s m i t t a n c e ( a r b . u n i t s )

a. Clay

8489141160

1420

3632

Wavenumber cm-1

1732871

1047

920

1080

1383

1414

15401639

2846

29253334

2924

624

1153

887

917

1121

138515871622

1637

Fig. 4. FTIR spectra of: (a) clay; (b) chitosan; (c) chitosan–clay nanoparticles; (d)

DOX; (e) chitosan–clay–DOX before drug release; and (f) chitosan–clay–DOX after

drug release at pH 7.4.



6/9

studied at the physiological temperature of 37 C (Figs. 5a–c). 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, confirming 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

HCl–NaCl buffer solution (pH 1.2) and phosphate buffer solutions

Table 1

Assignment of FTIR spectra of clay, chitosan, chitosan–clay and chitosan–clay

conjugated with DOX presented in Fig. 4.

Sample IR absorption

band (cm1)

Descriptiona

(a) Clay 3632 m(OAH) for AlAOH and SiAOH

1160 m(SiAO) out of plane

914 d(AlAlOH)

886 d(AlFeOH)848 d(AlMgOH)

(b) Chitosan 3440 ms(NAH)

2924 mas(CAH)

2846 ms(CAH)

1647 m(AC@OA) amide I

1597 Amine

1420, 1383 d(CAH)

1317 m(ACH3) amide III

1263 m(CAOAH)

1153, 1088 mas(CAOAC) and ms(CAOAC)

900 x(CAH)

(c) Chitosan–clay (5:1) 3632 m(OAH) for AlAOH and SiAOH

3440 ms(NAH)

2924 mas(CAH)

2846 ms(CAH)


1540 m(NAH) amide II

1444, 1383 d(CAH)

1263 m(CAOAH)

1153, 1080 mas(CAOAC) and ms(CAOAC)

920 d(AlAlOH), x(CAH)

886 d(AlFeOH)

626 (FeAO) out of plane vibration

522 d(SiAOAAl)

464 d(SiAOASi)

(d) DOX 3334–1071 Quinone and ketone carbonyls

1530 m(NAH) amide I

871 x(NAH)

810 m(CAOACH3)

764 d(NAH)

(e) Chitosan–clay–DOX

(5:1) before drug release

3630 m(OAH) for AlAOH and SiAOH

3440 ms(NAH)

2927 mas(CAH)2852 ms(CAH)

1730 Absorption band for DOX



1420, 1385 d(CAH)


1047 mas(CAOAC) and ms(CAOAC)

917 x(CAH)

887 d(AlFeOH)

810 m(CAOACH3) for DOX


526 d(SiAOAAl)

467 d(SiAOASi)

(f) Chitosan–clay–DOX

(5:1) after drug release

3630 m(OAH) for AlAOH and SiAOH

3440 ms(NAH)

2927 mas(CAH)

2852 ms(CAH)1730 Absorption band for DOX



1420, 1385 d(CAH)


1047 mas(CAOAC) and ms(CAOAC)

917 x(CAH)

887 d(AlFeOH)

810 m(CAOACH3) from DOX


526 d(SiAOAAl)

467 d(SiAOASi)

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

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

0 10 20 30 40 50 60

0

10

20

30

40

50

60

70

80

90

Chitosan-Clay (10:1)

pH = 1.2

Clay

Chitosan-Clay (5:1)

% C

u m u l a t i v e D O X R e l e a s e

Time (h)

Chitosan

Fig. 5a. Cumulative DOX release (%) from the chitosan–clay, 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.

0 10 20 30 40 50 600

10

20

30

40

50

60

70

80

90

100

DOX

Chitosan-Clay (5:1)

Clay


pH = 5.3

% C

u m u l a t i v e D O X R e l e a s e

Time (h)

Chitosan

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



7/9

(pH 5.3 and 7.4) are presented in Figs. 5a–c. There was an initial

burst release and then a gradual release of DOX in all the investi-

gated 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. How-

ever, 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-encapsu-

lated drug (see drug loading efficiency data in Section 2.3). A signif-icant finding was that cumulative release from the chitosan–clay

nanocomposite particle carrier was intermediate between chitosan

and clay, i.e. greater than pure clay and significantly lower than

pure chitosan. The percentage cumulative drug release followed

the sequence chitosan > chitosan–clay (10:1) > chitosan–clay

(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 chitosan–clay

nanocomposite and pure clay. Drug release from these matrices

was significantly slower and controlled release (Fig. 5a). The study

at pH 1.2 suggested that the carrier would release drug in gastro-intestinal fluid following oral administration. In the presence of

digestive enzymes and the microflora inside the stomach a faster

release rate would be expected, because the enzymes degrade

chitosan. In clay and chitosan–clay 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 rea-

son, when the clay content was high in the nanocomposite carrier

(chitosan–clay 5:1) less drug was released.

With an increase in pH to 5.3 the solubility of chitosan was lim-

ited, and it was insoluble at pH 7.4, leading to a significant 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, pK a 8.3) is still positively charged even

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 chitosan–clay nanocomposite was prepared, drug

release from the nanocomposite particle carrier was primarily con-

trolled by the clay. However, the presence of chitosan in the nano-

composite particle carrier undermined the attractive force

between DOX and the clay. This is corroborated by the observation

of faster release of the drug (Figs. 5a–c) 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 parti-

cle 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 chitosan–clay nano-

composite 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. 5a–c

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 chitosan–10 wt.% clay. This implies that

0 10 20 30 40 50 600

10

20

30

40

50

60

70

80

90

100

DOX


Clay

Chitosan-Clay (5:1) % C

u m u l a t i v e D O X R

e l e a s e

Time (h)

pH = 7.4

Chitosan

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

three experiments.

0 1 2 3 4 5 60

200

400

600

800

1000

S w e l l i n g R a t i o ( % )

Time (h)

pH = 7.4

Chitosan-Clay

Chitosan

Clay

0 1 2 3 4 5 60

200

400

600

800

1000

Chitosan-Clay

Chitosan

S w e

l l i n g R a t i o ( % )

Time (h)

pH = 5.3 Clay

Fig. 6. Swelling behavior of clay, chitosan andchitosan–clay nanoparticles at pH 5.3and 7.4.



8/9

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 fluid will be re-

duced and premature release of the drug in the gastric environ-

ment 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-specific drug release when controlled

and extended release is preferred. Another potential application

area where drug-loaded chitosan–clay can be considered is in the

preparation of tissue engineering scaffolds.

Using the chitosan–clay nanocomposite synthesis approach de-

scribed here one can prepare an implant capable of prolonged re-

lease of drug up to several days. Furthermore, the drug loading

capacity of chitosan–clay will be higher than normal chitosan scaf-

folds. In this study the drug loading capacities of clay, chitosan–

clay composite (5:1), chitosan–clay 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

chitosan–clay (5:1) as a function of time at pH 5.3 and 7.4. Exper-

iments at pH 1.2 were not conducted because of the high dissolu-

tion of pure chitosan and chitosan–clay and consequent non-

availability of data for pure chitosan and chitosan–clay for compar-

ison 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. How-

ever, chitosan–clay 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 chito-

san. 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 influences the swelling

behavior of the nanocomposite and consequently influences diffu-

sion of the drug through the bulk entity.

From this study on chitosan–clay nanocomposite, pure clay and

pure chitosan drug carriers we propose that the electrostatic inter-

action 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 be-

tween 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 re-

lease, with a consequent increase in the size of pores ( Figs. 7 and

8). The pore size increased from 1.1 ± 0.1lm 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 com-

ponents 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 chitosan–clay particles beforedrug release.

Fig. 8. Scanning electron micrographs of DOX-loaded chitosan–clay particles afterdrug release at pH 5.3 and 37 C.



9/9

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 interca-

lation of the biopolymer as a bilayer, with the thickness of two lay-

ers 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 first layer. Thus, the

NH3+ groups of the second layer interact electrostatically with ace-

tate 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, chitosan–clay nanocomposite is a versatile

polymer nanocomposite for biomedical applications, including tis-

sue engineering and controlled drug delivery.

4. Conclusions

Chitosan–clay nanocomposites are potential polymer nanocom-

posites of interest in biomedical applications, including tissue

engineering and controlled drug delivery. The controlled release

of drug from a chitosan–clay nanocomposite drug carrier, in con-

trast to pure chitosan, is controlled by electrostatic interaction be-

tween the positive charge of DOX and negatively charged sites in

the clay. The factors governing the drug release profile include

swelling behavior and drug–carrier interactions. The drug release

behavior is influenced by pH and the chitosan/clay ratio. Drug re-

lease occurs by degradation of the nanocomposite particle carrier

to its individual components or nanostructures of different

composition.

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Controlled and Extended Drug Release Behavior of Chitosan-based