Preparation and characterization of micelles of oligomeric chitosan linked to all-trans retinoic acid

Article abstract of DOI:10.1016/j.carbpol.2011.08.093

New amphiphilic chitosan derivatives of all trans retinoic acid-chitosan (RA-chitosan) with different molar feeding ratios of all trans retinoic acid (ATRA) were synthesized. The degree of ATRA substitution ranged from 8.72% to 18.78%. The RA-chitosan formed micelles with an average size of 142.14-208.4 nm, and zeta potential of +27.25 to 34.48 mV. The critical association concentration (CAC) was found to range from 1.3 × 10^-2 to 2.13 × 10^-2 mg ml^-1. Upon evaluation, the RA-chitosan shows no significant cytotoxicity on Hela and HepG2 cells. Analysis of micelles loaded with ATRA revealed that the size of micelles decreased by increasing loaded drug content while zeta potential did not change. ATRA was released slowly over 3-day period, and drug content had no effect on the release rate. These phenomena make RA-chitosan micelles as a candidate for drug carrier.

Full text of DOI:10.1016/j.carbpol.2011.08.093

Carbohydrate Polymers 87 (2012) 1176–1184
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Preparation and characterization of micelles of oligomeric chitosan linked to
all-trans retinoic acid
a,b,∗
, Mohammad-Ali Golozar , Jaleh Varshosazb,c,∗∗, Hamid Mirmohammad Sadeghid,
a
Ali Fattahi
a
Mohammadhossein Fathi
Biomaterials Group, Department of Materials Engineering, Isfahan University of Technology, Isfahan 84146-83111, Iran
Isfahan Pharmaceutical Sciences Research Centre, Isfahan University of Medical Sciences, Isfahan 81745-359, Iran
Department of Pharmaceutics, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran
a
b
c
d
Biotechnology Department, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2011
Received in revised form 27 August 2011
Accepted 29 August 2011
Available online 10 September 2011
New amphiphilic chitosan derivatives of all trans retinoic acid–chitosan (RA–chitosan) with different
molar feeding ratios of all trans retinoic acid (ATRA) were synthesized. The degree of ATRA substitution
ranged from 8.72% to 18.78%. The RA–chitosan formed micelles with an average size of 142.14–208.4 nm,
and zeta potential of +27.25 to 34.48 mV. The critical association concentration (CAC) was found to range
from 1.3 × 10 to 2.13 × 10 mg ml . Upon evaluation, the RA–chitosan shows no significant cytotox-
icity on Hela and HepG2 cells. Analysis of micelles loaded with ATRA revealed that the size of micelles
decreased by increasing loaded drug content while zeta potential did not change. ATRA was released
slowly over 3-day period, and drug content had no effect on the release rate. These phenomena make
RA–chitosan micelles as a candidate for drug carrier.
−2
−2
−1
Keywords:
Oligochitosan
Derivatives
All trans retinoic acid (ATRA)
Polymer micelles
Cytotoxicity
© 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
Polymeric micelles are colloidal carriers that promise enhancing
resistant (MDR) cells. It has been assumed that these micelles
reduce the production of ATP, which is indispensable for p-
glycoprotein activity. For these reasons, polymeric micelles are
optimal candidates for cancer therapy (Liu, Li, Jiang, Zhang, & Ping,
2010).
the efficacy of hydrophobic drugs. The micelles have a core–shell
structure with an internal core of hydrophobic segments sur-
rounded by hydrophilic segment in an aqueous medium (Du, Wang,
Yuan, Wei, & Hu, 2009; Nishiyama, Bae, Miyata, Fukushima, &
Kataoka, 2005). Polymeric micelles are generally more stable than
low-molecular-weight surfactant micelles, have a lower critical
association concentration (CAC), and exhibit slower dissociation
in aqueous environment (Du et al., 2009). Polymeric micelles can
improve solubility of hydrophobic drugs including most anticancer
agents. They also have a long circulation time, due to the steric hin-
drance caused by the presence of the hydrophilic shell. As a result,
they can target the encapsulated drug to specific tissues, through
either a passive or active mechanisms (Gaucher et al., 2005). They
can also overcome drug resistance that develops in multidrug
Chitosan,
a deacetylated form of chitin, consists of d-
glucosamine and N-acetyl ␤-d-glucosamine linked by ␤(1→4)
glycosidic bonds. Chitosan and its derivatives have attracted
much attention because of their abundance in nature, biocom-
patibility, low toxicity, and synthesizability, as well as their
hemostatic, bacteriostatic, fungistatic, and anticholesteremic prop-
erties. Although chitosan is insoluble in neutral aqueous medium,
and cannot form micelles in water (Zhang, Ping, Zhang, & Shen,
2003), it has been reported that modified chitosans, e.g., chi-
tosan taurocholate (Muzzarelli et al., 2006), N-alkyl-O-sulfate
chitosan (Zhang et al., 2003), N-octyl-N-(2-carboxyl cyclohexam-
ethenyl) chitosan (Liu et al., 2010), linoleic–chitosan (Liu, Chen, &
Park, 2005), cholesterol–chitosan (Yuan, Li, & Yuan, 2006), oleoy-
carboxymethyl chitosan (Li, Zhang, Meng, Chen, & Ren, 2010),
N-acetyl, and stearic-chitosan (Ye, Hu, & Yuan, 2004) can pro-
duce micelles. The low solubility and high viscosity of chitosan
hamper its capacity for systemic nanoparticle drug delivery. To
overcome these two disadvantages, many researchers have focused
on low-molecular-weight chitosan oligosaccharide (Du et al., 2009;
Hu, Ren, Yuan, Du, & Zeng, 2006; Hu, Zhao, et al., 2006; Hu, Liu,
∗ Corresponding author at: Biomaterials Group, Department of Materials Engi-
neering, Isfahan University of Technology, Isfahan 84146-83111, Iran.
Tel.: +98 311 391 2750; fax: +98 311 391 2752.
∗
han University of Medical Sciences, Isfahan 81745-359, Iran. Tel.: +98 311 792 2579;
fax: +98 311 668 0011.
∗ Corresponding author at: Isfahan Pharmaceutical Sciences Research Centre, Isfa-
E-mail address: a.fatahi.a@gmail.com (A. Fattahi).
0
144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.08.093

A. Fattahi et al. / Carbohydrate Polymers 87 (2012) 1176–1184
1177
Du, & Yuan, 2009). The fact that chitosan oligosaccharide inhibits
tumour-induced angiogenesis makes it a promising anticancer
drug carrier (Harish Prashanth & Tharanathan, 2005).
Specifically, chitosan (0.3 mmol) was dissolved in 5 ml water, after
which 40 ml DMSO was added to chitosan solution. ATRA with the
molar feeding ratios of 10%, 25%, and 50% (which mean the molar
ratio of ATRA to d-glucosamine units of chitosan), DCC (1.5× ATRA
in mol) and NHS (1.5× ATRA in mol) were dissolved in 5 ml of
dry DMSO. The solutions were stirred thoroughly for 6 h prior to
condensation reaction.
The two reactant solutions were mixed, and stirred at room
temperature for 24 h, while protected from light. After the result-
ing solution was filtered to remove insoluble by-products (e.g.,
dicyclohexylurea), it was precipitated in acetone and then washed
three times with acetone. To further increase purification, precip-
itated product was dissolved in deionized (DI) water and dialyzed
using a dialysis membrane (Sigma, cote off 2 kDa) against ethanol
for 2 days and then water for 1 day. The synthesized RA–chitosan
with 10%, 25% and 50% molar feeding ratio of ATRA were encoded
as RA–chitosan-10, RA–chitosan-25, and RA–chitosan-50, respec-
tively, for further evaluation.
Recently, polymeric micelles have attracted considerable atten-
tion to the potential advantages of hydrophobic drugs (especially
anticancer drugs) that are covalently linked to a polymer. The
conjugation of hydrophobic drugs with water-soluble polymers
improves their aqueous solubility and decreases their renal clear-
ance. Some studies have shown that covalently linked hydrophobic
drugs not only form the hydrophobic core of micelle but also func-
tion in the targeted cells (Chen, McRae, Parelkar, & Emrick, 2009;
Fan et al., 2010; Hu et al., 2009; Lee, Lee, & Park, 2008; Veronese
et al., 2005).
We hypothesize that using an anticancer drug as hydrophobic
part of the micelle can allow the co-deliver of two or even more
anticancer drugs to tumours and cancer cells. Co-delivery of anti-
cancer drugs not only can decrease side effects but also overcomes
drug resistance (Agarwal & Kaye, 2003).
All-trans retinoic acid (ATRA), the most biological active form
of retinol (vitamin A), plays an essential role in the induction of
cell differentiation and the arrest of cell proliferation. It has been
used in stem cell engineering and cancer therapy. Currently, ATRA is
effectively used in differentiation therapy for acute myelogeneous
lukemia (Lübbert, Müller Tidow, Hofmann, & Koeffler, 2008; Schug,
Berry, Shaw, Travis, & Noy, 2007; Tang & Gudas, 2011).
To prepare the micelles, RA–chitosan was suspended in water
−
1
or PBS buffer (pH 7.4) at 1 mg ml , followed by sonication using
probe type sonicator (Sonopuls HD 3200, Bandeline, Germany) at
60 W for 2 min (2 s pulse on and 2 s pulse off) (Lee, Kwon, Kim,
Jo, & Jeong, 1998). Sonication was repeated three times to obtain
optically clear solution. To avoid the heat build-up, the sonication
was done in an ice bath.
Despite all its advantages, the use of ATRA has been lim-
ited because of its disadvantages, which include poor water
solubility, physicochemical instability, drug resistance, hyper-
triglyceridermia, mucocutaneous dryness and headache (Kim, Choi,
Jeong, Jang, & Nah, 2006). Furthermore, the concentration of ATRA
in circulating blood has been shown to gradually decrease after
its oral administration or intravenous injection. This decrease
is attributed to ATRA being metabolised in the liver by an RA-
inducible P-450 cytochrome called p450AI (Coradini, 2007; Seo et
al., 2004).
2.3. Physicochemical characterization of RA–chitosan and
micelles
1
A
H NMR analysis was performed using Bruker Biospin
−
1
(AC-400, Germany). 5 mg ml of oligochitosan and RA–chitosan
dissolved in D O, and ATRA dissolved in deutriated DMSO, were
2
measured using an NMR spectrometer.
FTIR spectra were recorded on Fourier-transform infrared spec-
trometer (WQS-510/520, Raileigh, China) using KBr discs.
The degree of substitution (DS) for the RA–chitosan was assayed
using the 2,4,6-trinitrobenzene sulfonic acid (TNBS) test. Specif-
To overcome these disadvantages, ATRA has been encapsulated
in micro/nanoparticles such as chitosan micro/nanoparticles, lipo-
somes and polymeric micelles (Díaz, Vargas, & Gätjens-Boniche,
−
1
ically, 200 ␮l of 4 mg ml modified or unmodified chitosan was
incubated with 200 ␮l of 4% NaHCO3 and 200 ␮l of 0.1% TNBS
at room temperature for 8 h. UV absorbance was determined at
343 nm, using 0.033% TNBS in 1.3% NaHCO3 as a blank (Loretz &
Bernkop-Schnürch, 2006).
2
2
006; Kim et al., 2006; Li, Qi, Maitani, & Nagai, 2009; Seo et al.,
004).
In this present study, ATRA was grafted to water-soluble
chitosan oligosaccharide for the purpose of synthesizing
ATRA–chitosan conjugation with different graft ratios of ATRA,
creating a novel micellar form of chitosan that is compatible with
drug delivery.
2.3.1. Critical association concentration (CAC) of RA–chitosan
micelles
The CAC of the RA–chitosan micelles was determined by flu-
orescence measurement using pyrene as the probe (Wolszczak &
2
. Materials and methods
−
6
Miller, 2002). The pyrene solutions (6.0 × 10 M) were prepared in
◦
acetone and stored at −20 C until used. 300 ␮l of pyrene solution
2.1. Materials
was added to 3 ml of aqueous solutions containing different con-
−
3
−1
centrations of RA–chitosan micelles (2.0 × 10 to 0.5 mg ml ) and
The chitosan oligosaccharide (90% deacetylated; verified by
◦
incubated at 37 C overnight. The resulting solutions were gently
1
HNM0052, Mw = 8.6 kDa; verified by CTO-20 AC-Shimadzu high
performance liquid chromatography using refractive index detec-
tor and Ultrahydrogel 250 column, and PEG as standard) was
supplied by Yuhuan Marine Biochemistry Co., Ltd., Zhejiang, China.
Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS),
ATRA, pyrene and anhydride dimethyl sulfoxide (DMSO), as well as
other compounds were purchased from Sigma Chemical Co. (USA).
shaken to evaporate the acetone.
The fluorescence spectra of the solution were recorded on a
spectrofluorometer (LS-3B, Perkin-Elmer, USA) at room temper-
ature. The excitation wavelength was 336 nm and the emissions
were monitored at wavelengths ranging from 360 to 450 nm.
Depending on the pyrene emission spectra, the intensity ratio of
the first peak (I , 377 nm) to the third peak (I , 390 nm) was used
1
3
to calculate the CAC.
2.2. Synthesis of RA–chitosan and preparation of RA–chitosan
micelles
2.3.2. Particle size and zeta potential measurements
The particle size and zeta potential of the RA–chitosan micelles
were measured by a zetasizer (Zetasizer-ZEN3600 Malvern Instru-
ment Ltd., Worchestershire, UK). All particle-size measurements
were performed in PBS buffer (pH 7.4) using a He–Ne laser beam
The chemical conjugate of RA–chitosan was synthesized by
the coupling reaction of the carboxyl group of ATRA with the
amine group of chitosan in the presence of DCC and NHS (Fig. 1).

1
178
A. Fattahi et al. / Carbohydrate Polymers 87 (2012) 1176–1184
H
CH3
H
CH3
O
H3C CH3
OH
H
H3C CH3
CH3
H
CH3
O
H
CH3
H
H
O
N
DCC
+
H
CH3
H
H
O
O
O
NH
O
HO
HO
O
HO
NH2
HO
NH2
OH
HO
H2N
OH
OH
NH2
O
HO
O
OH
OH
O
O
HO
O
O
O
O
O
NH2
O
O
NH2
O
O
NH2
OH
HN
OH
OH
OH
OH
OH
O
H
H
+
H3C
H
O
H
H
CH3
H
CH3
O
H3C CH3
H
H3C
CH3
NH
O
N
H
H
H
O
O
O
CH3
H3C
H3C
Fig. 1. Synthesis of ATRA-grafted chitosan oligosaccharide (RA–chitosan).
◦
at 658 nm with a scattering angle of 130 . To study the colloidal
stability of the RA–chitosan micelles, their size was monitored for
2.4. Preparation and characterization of ATRA loaded
RA–chitosan micelles
2
weeks. The device calculated the zeta potential of the nanomi-
celles from their electrophoretic mobility using Smoluchowski’s
equation (Dong et al., 2009). All the experiments were performed
in triplicate.
ATRA served as a hydrophobic model drug to evaluate the
RA–chitosan micelles as carrier of anticancer drugs. The drug-
loaded RA–chitosan micelles were prepared by dialysis method.
−
1
Specifically, 800 and 200 ␮l of a 1 mg ml ATRA solution in DMSO
−
1
were added to 4 ml of a 1 mg ml of RA–chitosan-50 aqueous solu-
tion to achieve weight feeding ratios of 20% and 5%, respectively.
The mixtures were then sonicated for 2 min by a probe sonicator (2 s
pulse on and 2 s pulse off) in an ice bath. The mixture was then dia-
lyzed against water for 24 h. Next, the unloaded drug was separated
by centrifuging at 4000 rpm for 10 min (Du et al., 2009).
2.3.3. Cell culture
HepG2 and Hela cells were obtained from the Pasteur Insti-
tute in Iran. They were maintained in RPMI-1640 supplemented
by 10% (v/v) FBS (fetal bovine serum) and penicillin/streptomycin
−
1
−1 ◦
(
50 IU ml
,
500 ␮g ml
) at 37 C in a humidified atmo-
sphere of 5% CO . The cells were subcultured regularly using
2
To estimate the content of the loaded drug, 100 ␮l of the drug-
encapsulated micelle solution was added to 900 ␮l DMSO and
vortexed for 1 min to break down the micelles (Kim et al., 2006).
The ATRA content was measured by a UV–Vis spectrophotometer
(UV mini-1240 CE, Shimadzu, Japan) using the standard curve of
ATRA concentration versus absorbance at 360 nm, in a mixture of
10% DMSO to 90% water. Unloaded micelles were used as controls.
Drug content (DC) and loading efficacy (LE) were calculated by the
following equations (Li et al., 2009):
trypsin/EDTA.
2.3.4. In vitro cytotoxicity
A cytotoxicity test was used to evaluate the in vitro viability
of the RA–chitosan against HepG2 and Hela cells. Specifically, the
HepG2 and Hela cells were plated in 96-well plates and grown
for 24 h. The cells were exposed to several chitosan concentra-
◦
tions, free ATRA and RA–chitosan at 37 C for another 24 or 96 h.
At the end of the incubation period, 20 ml of MTT solution with
ꢀ
ꢁ
W
−1
a concentration of 5 mg ml was added and incubated for a fur-
DC =
LE =
× 100
(1)
(2)
◦
(Wbm + W)
ther 3 h at 37 C. Each well was then washed with 50 ml of PBS
after the medium containing the unreacted MTT was removed.
Then, 150 ml of DMSO was added to each well to dissolve the
formazan crystals. Finally, the absorbance of the dissolved for-
mazan was measured at 540 nm using an ELISA reader (Bio-Rad,
Model 680, USA) (Yokoyama, Okano, Sakurai, Suwa, & Kataoka,
W
× 100
Wo
where W is the weight of the loaded drug, Wbm is the weight of the
control micelles, and Wo is the weight of the initial feeding drug.
The FTIR, sizes and zeta potentials of the drug-loaded micelles
were assessed as described in Sections 2.3 and 2.3.2 above.
1
996; Yoo & Park, 2001). All the experiments were performed in
triplicate.

A. Fattahi et al. / Carbohydrate Polymers 87 (2012) 1176–1184
1179
Table 1
A) Characteristics of RA–chitosan micelles and (B) characteristics of ATRA loaded RA–chitosan-50 micelles.
(
(
A)
Code name
DS%
Size (nm)
Zeta (mV)
CAC (mg ml 1
−
)
−
−
2
CHRA–50
CHRA–25
CHRA–10
18.78 ± 0.86
12.77 ± 0.44
8.72 ± 0.59
142.14 ± 5.06
166.6 ± 2.45
208.4 ± 24.64
27.25 ± 6.31
31 ± 1.31
1.3 × 10
1.8 × 10
2
*
−2
34.48 ± 3.92
2.13 × 10
(
B)
Feeding ratio (WATRA/WRA–chitosan-50)
Loading efficacy
Loading content
Size
Zeta potential
*
5
2
%
0%
60.27 ± 8.57%
56.58 ± 9.7%
3.02 ± 0.43%
193.1 ± 4.2
154.1 ± 0.9
33.73 ± 2.25
32.57 ± 1.4
*
9.33 ± 2.4%
*
The main difference is significant at the 0.05 level.
Fig. 2. FTIR of chitosan and its derivatives: (a) chitosan, (b) ATRA, (c) RA–chitosan-50, (d) ATRA loaded RA–chitosan-50 micelles with 5% molar feeding amount and (e) ATRA
loaded RA–chitosan-50 micelles with 20% molar feeding amount.

1
180
A. Fattahi et al. / Carbohydrate Polymers 87 (2012) 1176–1184
Fig. 3. 1H NMR spectra: (a) ATRA, (b) RA–chitosan, and (c) chitosan.
NMR spectrum, but it disappeared in the 1H NMR spectrum
2.4.1. In vitro drug release study
The in vitro release of ATRA was achieved using PBS (pH = 7.4) as
the release medium. 2 ml of loaded micelles were placed in a dial-
of RA–chitosan. This result indicates the presence of a cova-
lence bond between ATRA and chitosan. Furthermore, the
RA–chitosan spectrum revealed the emergence of new peaks at
ı = 0.936 (–C(CH3)2– of cyclohexenyl), 5.849 (–C(CH3) CHCO–),
ysis membrane bag (cut-off 2000 Da). The whole bag was sunk in
◦
2
0 ml of release medium at 37 C while being stirred. To assure that
the sinking was adequate, the whole release medium was with-
drawn at predetermined time intervals and replaced by a fresh
medium.
6.25–6.3384
(–CH CH–C(CH3) CH–CH CH–C(CH3) CHCO–)
and 7.056 (–CH CH–C(CH3) CH–CH CH–C(CH3) CHCO–), all
attributable to ATRA.
The ATRA concentration in PBS was determined by UV spec-
trophotometry, using standard curve of ATRA in PBS at wavelength
of 360 nm.
3.2. Physicochemical properties of RA–chitosan micelles
In the aqueous solvents, the hydrophobic ATRA moieties of
the RA–chitosan were assembled themselves in hydrophobic
cores, while these cores were surrounded by hydrophilic chitosan
backbones, to achieve the lowest Gibbs free energy level. The
spontaneous self-aggregation of the RA–chitosan in the aqueous
medium was determined by the dye solubilization method using
pyrene as the probe molecule. The emission spectra of pyrene
depend strongly on the polarity of the microenvironment; in polar
3
. Results and discussion
3.1. Synthesis of RA–chitosan
RA–chitosan was successfully synthesized by the coupling reac-
tion between the amino groups of chitosan oligosaccharide and the
carboxyl group of ATRA. The degree of substitution (DS%) of the
RA–chitosan is shown in Table 1A. Conjugation of RA–chitosan was
confirmed using FTIR and H NMR spectra. A comparison of the chi-
tosan and ATRA FTIR spectra revealed in the RA–chitosan spectrum
solvents, the intensity of the first energy band (377 nm, I ) of the
1
1
pyrene emission spectrum is higher than that of the third (390 nm,
I3) whereas in a hydrophobic environment, I3 is higher than I1.
Therefore, when micelles are formed in an aqueous medium, the
pyrene tends to locate itself inside the hydrophobic core, increasing
I intensity. As a result, the ratio of I to I can be used to determine
−
1
an increase in the intensity of the amide group peak at 1635 cm
and a decrease in the intensity of the amine group at 1522 cm−1.
The peak of the protonated carboxyl groups of ATRA completely
disappeared in the RA–chitosan spectrum, whereas the intensity of
the aliphatic alkanes at 2931 peak increased (Fig. 2).
3
1
3
the CAC.
Micelle solutions are diluted in the body fluids; as a conse-
quence, they are expected to be disassociated. Thus, the lower the
CAC value, the more stable the micelles in the body fluids (Bian,
Jia, Yu, & Liu, 2009). Fig. 4a shows the variation in the fluorescence
intensity ratio (I /I ) against the logarithm of RA–chitosan micelles
1
The H NMR spectrum was also used to confirm the binding
between ATRA and chitosan oligosaccharide, as is illustrated
in Fig. 3. It is clear that the proton peak of the COOH of
ATRA (chemical shift 12.064 ppm) was observed in its
1
H
1
3

A. Fattahi et al. / Carbohydrate Polymers 87 (2012) 1176–1184
1181
Fig. 5. Particle size of RA–chitosan-50 micelles (1 mg ml−1) during the time as func-
tion of colloidal stability.
RA–chitosan concentration. It was found that the size and zeta
potential of the RA–chitosan micelles declined as the graft ratio of
the RA–chitosan increased (p < 0.05). The RA–chitosan-50 micelles
had the smallest average size and zeta potential: 142.14 ± 5.06 nm
and +27.25 ± 6.31 mV, respectively. The reduction of micelle size
resulted from the enhanced hydrophobic interaction, which was
caused by an increase in the graft ratio. In fact, with this increase
and the resulting polymer hydrophobicity, the micelles formed in
a more tightly packed core. Monitoring the size of the RA–chitosan
micelles during the time indicates that their colloidal stability
depended on the CAC. The average size of the RA–chitosan-50
micelles increased only from 142.13 to 217.93 nm in the first 24 h,
and there was no increase at all during the next 2 weeks (Fig. 5). On
the other hand, the RA–chitosan-10 and RA–chitosan-25 micelles
showed a sharp increase in size from approximately 160–200 nm
to 1 ␮m, during the same 24 h period at room temperature (data
not shown). The difference is probably attributable to the strong
ionic environment of PBS which causes the RA–chitosan-10 and
RA–chitosan-25 micelles to disassociate and re-aggregate, similar
to what López-León, Carvalho, Seijo, Ortega-Vinuesa, and Bastos-
González (2005) have found in a comparable situation. On the other
hand, the tightly packed RA–chitosan-50 micelles showed definite
stability in the buffer solution.
Fig. 4. (a) Intensity ratio plots of I1/I3 of pyrene versus log C for RA–chitosan
micelles: RA–chitosan-10 (ꢀ), RA–chitosan-25 (ꢁ), and RA–chitosan-50 (ꢂ). The
plots of ATRA molar feeding ratio versus (b) CAC and (c) DS.
concentration. At concentrations lower than the CAC, the ratios
were approximately the same, whereas in concentrations higher
than the CAC, a linear decrease in the ratios was observed with
increasing RA–chitosan micelles concentration. The CAC values
were defined as the crossover point of the two straight lines of
each RA–chitosan micelle (Na, Park, Kim, & Bae, 2000). The CAC
value of RA–chitosan micelles decreased as the graft ratio of the
polymer increased (Table 1A). Thus, there was a direct correlation
between the ATRA feeding ratio and CAC, at least for the values
used in this study. The absolute values of correlation coefficient
3.3. In vitro cytotoxicity
The viability data for the Hela and HepG2 cells at 24 h and 96 h
are shown in Fig. 6a and b. We have investigated the viability of
these cells in the presence of the RA–chitosan and free ATRA with a
concentration level similar to that of the micelles. ATRA is known to
inhibit mitosis and the proliferation of Hela and HepG2 (Díaz et al.,
2006; Mao, Liu, Chen, Li, & Zhou, 2006). Thus, to evaluate these
effects, viability was investigated at both 24 and 96 h (Díaz et al.,
2006; Kucukzeybek et al., 2008). Neither the RA–chitosan nor free
ATRA were shown to cause any significant cytotoxicity on the cells,
(
R) were larger than 0.9 suggesting a linear relationship (Fig. 4b).
The hydrophobic interaction between the ATRA moieties of the
RA–chitosan micelles increased as the graft ratio of the RA–chitosan
increased, a trend which favours micelles formation at lower
RA–chitosan concentrations. Despite, Fig. 4c shows relationship of
DS% and feeding ratio; the slope of the line is less than one which
means the graft yield decreases as the feeding ratio increases.
In this study, the RA–chitosan-50 had the lowest CAC, allow-
−
1
−1
ing micelles to be formed with a 13.5 ␮g ml
concentration,
even in concentrations as high as 100 ␮g ml . Interestingly, at 96 h,
around 170 times lower than the concentrations needed to form
micelles with low-molecular-weight surfactants micelles, e.g.,
chitosan oligosaccharide was found to create more toxicity than
RA–chitosan on Hela, although the difference was not statistically
significant. This result could be attributable to the lower positive
charge of RA–chitosan (Fig. 6A.1 and A.2). Studies of cationic carri-
ers in gene delivery show that the positive charge of these carriers
causes cytotoxicity (Lv, Zhang, Wang, Cui, & Yan, 2006). In the
−
1
sodium dodecyl sulfate (SDS) which has a CAC of 2.3 mg ml (Bian
et al., 2009). This result clearly indicates that the RA–chitosan-50
polymer can form micelles even in highly diluted solutions.
The RA–chitosan micelles were prepared by dissolving a
RA–chitosan in distilled water using a probe-type sonicator.
−
1
case of free ATRA, a concentration as small as 26.9 ␮g ml , even
not to a meaningful extent, could decrease viability. Although the
−
1
Table 1A summarizes the properties of micelles at 1 mg ml
of

1
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A. Fattahi et al. / Carbohydrate Polymers 87 (2012) 1176–1184
Fig. 6. MTT assay for cytotoxicity of RA–chitosan on Hela cell (A-1) and HepG2 cell (B-1), ATRA on Hela cell (A-2) and HepG2 cell (B-2) and chitosan oligosaccharide on Hela
cell (C-1) and Hepg2 cell (C-2).
concentration of ATRA we used was smaller than its half maximal
inhibitory concentration (IC50), it is still capable of increasing the
cytotoxicity of other anticancer drugs such as Taxol and arsenic
ionic interactions between RA–chitosan micelles and ATRA. Thus,
most of the drug was loaded in the micelle’s core rather than the
shell. According to loaded micelles FTIR, peaks at 1252, 1570, and
1685 cm are attributable to C–O stretch vibration, C O stretch,
and the COOH of free ATRA, respectively. The intensity of the
peaks was enhanced by increasing the feeding amount (Fig. 2d
and e). This result lends further supports to the above hypoth-
esis, emphasizing that most of the drug is encapsulated in the
core and that there are no ionic interactions among the micelles
shells.
−
1
trioxide (AsO ) (Kucukzeybek et al., 2008; Lin, Li, Xiao, Lin, &
3
Yang, 2005). Thus, in cancer therapy, RA–chitosan micelles may be
considered a promising targeting delivery system for drugs.
3.4. Preparation and characterization of drug-loaded
RA–chitosan micelles
The drug-encapsulated micelles were prepared by dialysis
method and then lyophilized to turn them into powder. The
chemico-physical characteristics of these drug-loaded micelles is
summarized in Table 1B. Increasing the drug feeding amount had
no significant effect on loading efficiency, although drug content
was significantly enhanced. This increase in drug content caused
an increase in molecular hydrophobicity, which resulted in more
tightly packed micelles, shrinkage of the particles, and decrease in
their water content. Collectively, these effects resulted in a decrease
in the size of the loaded micelles, along with an increase in the
feeding amount. These findings are consistent with those of other
studies (Na et al., 2000). Increasing the drug content did not signifi-
cantly change the zeta potential, which indicates that there were no
3.4.1. In vitro drug release from RA–chitosan-50 micelles
Fig. 7 shows that it took 3 days for more than 70% of the drug
to be released, and there was no significant change in the release
profile when drug content was increased. This slow and prolonged
release is attributable to the hydrophobic nature of ATRA, and per-
haps as well to crystallization inside the micelle’s core (Kim et al.,
2006; Na et al., 2000). Considering that the micelles retained the
drug inside the core due to the slow drug leakage, it can be con-
cluded that the drug is released only when the micelles are taken up
and the chitosan molecules are enzymatically degraded within the
tumoural cells. This process increases the concentration of the drug
in tumours by enhanced permeability and retention (ERP) effect.

A. Fattahi et al. / Carbohydrate Polymers 87 (2012) 1176–1184
1183
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Chen, X. J., McRae, S., Parelkar, S.
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The chemical conjugates of RA–chitosan as a novel derivative
of chitosan oligosaccharide were synthesized using a coupling
reaction of water-soluble chitosan oligosaccharide and ATRA with
different graft ratios. The self-aggregation process created nano-
sized polymeric micelles in an aqueous medium, and the resulting
micelles were stable for a long time in PBS. The size of the micelles
was controlled by DS. Colloidal stability of the RA–chitosan micelles
was increased by decreasing the CAC. The cytotoxicity of the
RA–chitosan against HepG2 and Hela cells suggests that the modi-
fied chitosan oligosaccharide was non-toxic. Loading efficiency and
drug content were 60.27% and 3.02%, respectively, for the 5% feed-
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ratio. The appearance of characteristic peaks of free ATRA in the
FTIR of the loaded micelles, along with the zeta potential, indicates
that the drug molecules were predominantly encapsulated in the
micelle’s core rather than the outer shell, because there was no
significant ionic interaction with the outer shell. The size of the
loaded micelles was decreased by increasing the feeding amount
of the loaded drug while the zeta potential did not change signif-
icantly. This result suggests that increasing DS resulted in greater
hydrophobicity and hence tightly packed hydrophobic core. This
process shrunk the micelles, i.e., reduced their size. The release of
ATRA lasted up to 3 days and there was no significant difference
in the release rate when the drug content was increased. Because
of their low CAC value, colloidal stability, lack of cytotoxicity, good
loading efficiency, and sustained release of the drug, RA–chitosan-
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Acknowledgment
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Isfahan Pharmaceutical Sciences Research Centre of the Isfahan
University of Medical Sciences, and Isfahan University of Technol-
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