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Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, The University of Santiago de Compostela,
15706 Santiago de Compostela, Spain
b
`
, Hospital Duran i Reynals, 08907 Barcelona, Spain
Institut de Recerca Oncologica
Received 17 August 2000; accepted 13 March 2001
Abstract
The aim of this paper was to evaluate the potential of chitosan nanoparticles as carriers for the anthracycline drug,
doxorubicin (DOX). The challenge was to entrap a cationic, hydrophilic molecule into nanoparticles formed by ionic
gelation of the positively charged polysaccharide chitosan. To achieve this objective, we attempted to mask the positive
charge of DOX by complexing it with the polyanion, dextran sulfate. This modification doubled DOX encapsulation
efficiency relative to controls and enabled real loadings up to 4.0 wt.% DOX. Separately, we investigated the possibility of
forming a complex between chitosan and DOX prior to the formation of the particles. Despite the low complexation
efficiency, no dissociation of the complex was observed upon formation of the nanoparticles. Fluorimetric analysis of the
drug released in vitro showed an initial release phase, the intensity of which was dependent on the association mode,
followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated that
those containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed to
chitosan before nanoparticle formation showed slightly decreased activity. Additionally, confocal studies showed that DOX
was not released in the cell culture medium but entered the cells while remaining associated to the nanoparticles. In
conclusion, these preliminary studies showed the feasibility of chitosan nanoparticles to entrap the basic drug DOX and to
deliver it into the cells in its active form. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Chitosan; Dextran sulfate; Nanoparticles; Doxorubicin; Adriamycin
1. Introduction
Doxorubicin (DOX) and its bioactive derivatives
are among the most widely used anticancer drugs in
chemotherapy treatment [1]. However, problems
*Corresponding author. Tel.: 134-981-594-627; fax: 134-981547-148.
E-mail address: ffmjalon@usc.es (M.J. Alonso).
0168-3659 / 01 / $ see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S0168-3659( 01 )00294-2
256
cell membranes, an attractive feature for the treatment of solid tumors. From the perspective of
intravenous administration, positively charged particles would interact with different blood components
as compared to negatively charged particles. These
changes could potentially create a different biodistribution and / or organ accumulation pattern following
intravenous administration. Additionally, a positively
charged system that would be expected to interact
with cells and / or membranes would be desirable for
testing alternative modes of administration of DOX,
i.e. mucosal administration.
We believed that an interesting candidate with
which to test these hypotheses was the cationic
polysaccharide, chitosan. This biopolymer has shown
favorable biocompatibility characteristics [19] as
well as the ability to increase membrane permeability, both in vitro [20] and in vivo [21], and be
degraded by lysozyme in serum [22]. Consequently,
the aim of this paper was to encapsulate appreciable
quantities of DOX in chitosan nanoparticles made by
ionotropic gelation with sodium tripolyphosphate
(TPP) and test the effects of DOX encapsulation
and / or release on cytotoxic activity relative to free
DOX. To achieve this aim, we tried two approaches:
ionic bridging with a coincorporated polyanion and
polymer / drug complexation.
2. Experimental section
2.1. Materials
Chitosan hydrochloride salt, Protasan CL 110
(Mw .100 kDa), was purchased from Pronova Biopolymers (Oslo, Norway). Doxorubicin hydrochloride was obtained as a 2 mg / ml solution in 0.9%
(w / v) sodium chloride from Tedec-Meiji Farma
(Madrid, Spain). TPP, type B gelatin (75 bloom),
polyphosphoric acid, and dextran sulfate (Mw 510
kDa) were all purchased from Sigma-Aldrich S.A.
(Madrid, Spain). Phosphorylated glucomannan was a
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258
The number of active cells was estimated by measuring the absorbance at 540 nm (Titertek Multiscan,
ICN, Costa Mesa, CA). The percentage of cytostasis
was calculated as follows:
A2B
Cytostasis 5 ]]
A
where A is the absorbance of cells incubated with
culture medium and B is the absorbance of cells
incubated with the different nanoparticle formulations. All samples were made in sextuplicate.
(Objekttrager,
Menzol Glaser, Germany) at a density
of 5310 4 cells / coverslip. One day later, the cultures
were washed and treated with serum-free, Ham F-12
medium containing either DOX-loaded chitosan
nanoparticles incorporating dextran sulfate (30 min
to 24 h incubation) or free DOX (30 min incubation).
All incubations were carried out at 378C with an
equivalent final concentration of DOX (5 mg / ml).
Alternatively, cells were incubated using a twocompartment Boyden chamber. Briefly, either DOXloaded chitosan nanoparticles containing dextran
sulfate or free DOX solutions were diluted in serumfree culture medium and incorporated into the upper
compartment of polycarbonate transwell filters (0.4
mm pore diameter, Costar). The cells seeded on
coverslips for 24 h were placed in the lower compartment, thereby receiving the DOX solution filtering from the upper compartment but excluding the
DOX associated to the nanoparticles (a control
experiment was performed demonstrating that the
particles did not cross the filter). Following incubation, cells were washed five times with PBS, and the
coverslips were mounted on slides. Fluorescence
observation was carried out with a confocal microscope (TCS 4D, Leica Instruments) using an argon /
krypton laser (75 mW) at 488 nm for excitation and
an LP filter of 590 nm for DOX detection. Contrast
images were simultaneously observed using the
inverted microscope equipment with a PL Apo 633 /
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3. Results
The molecular structures of DOX and the complexing agents, polyphosphoric acid and dextran
sulfate, are shown in Fig. 1. The protonable groups
in the DOX molecule were expected to interact with
the deprotonable groups of polyphosphoric acid and
dextran sulfate.
The interaction of DOX with different polyanions
and chitosan was first investigated spectrophotometrically. As seen in Fig. 2A and B, the DOX peak at
480 nm was reduced by |53% upon incubation with
either polyphosphoric acid (Fig. 2A) or dextran
sulfate (Fig. 2B). Spectral changes in the DOX peak
were also observed for the samples, with polyphosphoric acid and dextran sulfate inducing red shifts of
9 and 14 nm for DOX solutions, respectively.
Subsequent addition of chitosan increased the intensity of the 480 nm peak with polyphosphoric acid
and dextran sulfate to 110 and 83% of the original
DOX absorbance. Spectral peaks for both samples
returned to 480 nm. In control studies, no detectable
absorbance was noted for individual chitosan, polyphosphoric acid, or dextran sulfate solutions over the
chosen wavelength range at the concentrations tested
(data not shown). No significant differences in pH
were noted among any of the formulations shown
(DOX,
DOX1polyanion,
DOX1polyanion1
chitosan).
A comparison of the DOX encapsulation efficiencies for different chitosanTPP nanoparticle formulations is shown in Table 1. At 10% (w / w) polyanion
with respect to chitosan, no significant differences in
DOX encapsulation efficiencies were observed with
the coincorporation of gelatin, glucomannan, or
polyphosphoric acid relative to the control formulation, with all encapsulation efficiencies between 8
and 13%. Also, the addition of polyphosphoric acid
caused a destabilization of the suspension at this
polyanion concentration, forming visible agglomerates. Incorporation of dextran sulfate increased DOX
encapsulation efficiency approximately twofold with
respect to the control formulation.
The effect of initial DOX loading on encapsulation
efficiency for chitosanTPP nanoparticles incorporating dextran sulfate can be seen in Table 2, with
values ranging from 19 to 23%. Mean encapsulation
efficiencies decreased only slightly with increases in
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Table 1
Encapsulation efficiencies for different chitosan nanoparticle
formulations. All polyanions were incorporated at 10% (w / w)
with respect to chitosan. Theoretical DOX loading: 10% (n53)
Polyanion
incorporated
Encapsulation
efficiency (%)
No polyanion
Type B gelatin
Glucomannan
Polyphosphoric acid
Dextran sulfate
9.162.2
8.461.5
9.363.3
12.264.1
21.962.5*
* P,0.01.
Theoretical
loading (%)
Encapsulation
efficiency (%)
5
10
20
22.561.2
21.962.5
19.361.8
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0.260.8% between the normalized profiles. Conversely, encapsulated DOX exhibited an additional
emission band at 630 nm which was evident over the
range of 600700 nm.
Fig. 6 shows the cytostasis of the different
chitosan formulations in vitro for the C26 cell line.
No significant differences were noted between the
DOX loaded chitosanTPP nanoparticles incorporating dextran sulfate and the control DOX solution
over drug concentrations from 0.1 mg / ml to 100
mg / ml in any of the cell lines tested. Nanoparticles
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4. Discussion
The major goal of this work was to develop a
chitosan nanoparticulate system as a novel, positively charged, colloidal carrier for DOX. The greatest
challenge was to encapsulate appreciable quantities
of DOX, overcoming the charge repulsion between
the cationic polymer (pKa 56.5) [26] and the predominantly positively charged anthracycline drug
(pKa 58.2) [27]. To begin, we selected a chitosan
TPP nanoparticle formulation [23] which could
accommodate a large quantity of TPP (only |25%
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Fig. 7. Confocal images of (A) free DOX (30 min incubation), (B) DOX-loaded nanoparticles (overnight incubation), (C) DOX-loaded
nanoparticles in the upper compartment of a Boyden chamber (overnight incubation), and (D) DOX1blank nanoparticle mixture in the
upper compartment of a Boyden chamber (overnight incubation). All confocal studies were performed in A375 melanoma cells with 5
mg / ml equivalent DOX concentration. Magnifications: 3630 (A, B, D), 3400 (C).
264
be tightly bound to chitosan. Indeed, this phenomenon did take place. While there was only a minimal
yield for the complexation (0.43 wt.%), all of the
DOX that was complexed remained incorporated
within the nanoparticles. Therefore, it could be
induced that the entrapment efficiency of DOX
previously associated to chitosan was 100%. The
possibility that the high entrapment efficiency is
merely caused by low initial DOX loading was also
considered. As a control study, we prepared chitosan
nanoparticles with the same DOX initial 0.43 wt.%
loading, but adding DOX to the chitosan solution
prior to the nanoparticles formation. In this case the
encapsulation efficiency was far lower (23.061.0%)
than that observed for chitosanDOX complexes.
In vitro release studies were performed in acetate
buffer (pH 4). We chose this medium because DOX
is maximally stable at pH values between 3 and 5
and also because, at higher pHs we encountered
problems of fluorescence quenching or interference
for the quantification of released DOX. Obviously, in
these experiments, we did not expect to predict the
release behavior of these particles in vivo but to
compare the formulations developed and to gain
some insight about the mechanism of release.
Nanoparticles incorporating dextran sulfate showed a
burst release of 17% at 2 h, followed by an additional release of 4.5% over the next 2 days. This slow
release was quite distinct from the profiles obtained
from similar chitosan nanoparticles encapsulating
insulin, where 100% release was observed within 15
min [21]. Even less DOX was detected in PBS at 5
days (data not shown), probably due to DOX degradation in the near neutral medium. The initial phase
of release is logically attributed to the DOX located
at the surface of the particles while the remainder of
the unreleased DOX was assumed to be well entrapped within the chitosan nanoparticles and tightly
associated to the chitosan molecules, probably as an
ionic complex with dextran sulfate. Therefore, the
degradation of chitosan would be required for accomplishing the release process. Unfortunately, direct confirmation of this hypothesis by enzymatic
digestion of the nanoparticles was not possible since
chitosanase treatment of the nanoparticle suspension
also degraded DOX solutions and / or quenched
fluorescence in control studies. Nevertheless, the
effect of dextran sulfate on DOX release was apparent, as chitosan nanoparticles without the polyanion
265
5. Conclusion
In this paper, we describe the feasibility of using
chitosan nanoparticles as colloidal carriers for the
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