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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.
0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S0168-3659( 01 )00294-2
256 K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267
solid, accessible tumors, these particles are too large cell membranes, an attractive feature for the treat-
to be endocytosed by most cells or circulate freely in ment of solid tumors. From the perspective of
the bloodstream. Consequently, the association of intravenous administration, positively charged par-
DOX to submicron carriers, such as liposomes [7], ticles would interact with different blood components
nanoparticles [8], or micelles [9], has drawn greater as compared to negatively charged particles. These
interest. changes could potentially create a different biodistri-
The majority of attempts to associate DOX to bution and / or organ accumulation pattern following
nanoparticulate carriers have used anionic or neutral intravenous administration. Additionally, a positively
polymers. Akiyoshi et al. [10] achieved DOX en- charged system that would be expected to interact
capsulation in cholesterol-bearing pullulan hydrogel with cells and / or membranes would be desirable for
nanoparticles, though loading levels were very low testing alternative modes of administration of DOX,
(,0.1 wt.%) and cytotoxic effects of the nanoparti- i.e. mucosal administration.
cles were lower than that of free DOX. Combining We believed that an interesting candidate with
prodrug and encapsulation strategies, Yoo et al. [11] which to test these hypotheses was the cationic
covalently linked DOX to the terminus of poly( D,L- polysaccharide, chitosan. This biopolymer has shown
lactic-co-glycolic acid) (PLGA), then formed favorable biocompatibility characteristics [19] as
nanoparticles with the conjugate by an emulsion- well as the ability to increase membrane permeabili-
solvent diffusion method. The group was able to ty, both in vitro [20] and in vivo [21], and be
obtain considerable loadings (3.45 wt.%), achieve a degraded by lysozyme in serum [22]. Consequently,
controlled release of DOX over nearly 1 month, and the aim of this paper was to encapsulate appreciable
maintain antiproliferative activity relative to free quantities of DOX in chitosan nanoparticles made by
DOX, though these results were possible only by ionotropic gelation with sodium tripolyphosphate
forming the covalent linkage between the polymer (TPP) and test the effects of DOX encapsulation
and the drug. and / or release on cytotoxic activity relative to free
The vast majority of work involving nanoparticu- DOX. To achieve this aim, we tried two approaches:
late DOX association, however, has been with ionic bridging with a coincorporated polyanion and
polyacrylates, exploiting charge interactions of the polymer / drug complexation.
polymer with the drug to achieve high association
efficiencies. Polymethacrylate nanoparticles with ad-
sorbed DOX were administered intravenously to
hepatoma patients and demonstrated prolonged plas- 2. Experimental section
ma levels, as well as reduced total clearance of DOX
relative to a control DOX solution [12]. DOX
associated to polyalkylcyanoacrylate nanoparticles 2.1. Materials
[13] have demonstrated reduced cardiotoxicity fol-
lowing intravenous administration in mice [14] as Chitosan hydrochloride salt, Protasan CL 110
well as increased cytoxicity against multidrug resis- (Mw .100 kDa), was purchased from Pronova Bio-
tant cell lines in vitro [15]. Later work showed that polymers (Oslo, Norway). Doxorubicin hydrochlo-
coating of these particles with polysorbate 80 sig- ride was obtained as a 2 mg / ml solution in 0.9%
nificantly increased DOX accumulation in brain (w / v) sodium chloride from Tedec-Meiji Farma
tissue [16]. However, these DOX loaded particles (Madrid, Spain). TPP, type B gelatin (75 bloom),
have demonstrated acute renal toxicity [17] as well polyphosphoric acid, and dextran sulfate (Mw 510
as decreased permeability of the drug across artificial kDa) were all purchased from Sigma-Aldrich S.A.
membranes with respect to free DOX [18]. (Madrid, Spain). Phosphorylated glucomannan was a
An alternative approach would be to entrap DOX ´
gift from Industrial Farmaceutica Cantabria (Madrid,
into a positively charged carrier. Cell adhesion and Spain). Unless otherwise mentioned, water was
potentially cell uptake of such particles should be ´
ultrapure grade (Milli-Q plus, Millipore Iberica,
favored due to their attraction to negatively charged Spain). All other chemicals were reagent grade.
K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267 257
sampling). Calibration curves were made with the The number of active cells was estimated by measur-
incubation medium, and all measurements were ing the absorbance at 540 nm (Titertek Multiscan,
performed in triplicate. ICN, Costa Mesa, CA). The percentage of cytostasis
was calculated as follows:
2.8. Fluorimetric analysis
A2B
Cytostasis 5 ]]
DOX emission spectra were recorded in water A
from 500 to 750 nm at a fixed excitation of 480 nm where A is the absorbance of cells incubated with
with excitation and emission slit widths of 5 nm. culture medium and B is the absorbance of cells
Spectra were read at a scan speed of 200 nm / min incubated with the different nanoparticle formula-
and normalized with respect to peak emission. tions. All samples were made in sextuplicate.
2.9. In vitro cytostasis assays
2.10. Confocal microscopy analysis
Human melanoma A375 cells (ATCC, Rockville,
MD) and C26 murine colorectal carcinoma cells DOX accumulation in treated cells was localized
were grown in Ham F-12 medium (GIBCO, Grand by confocal microscopy. Briefly, cells (Human
Island, NY) supplemented with 10% fetal bovine melanoma A375 cells (ATCC)) from exponential
serum, sodium pyruvate, non-essential amino acids, cultures were grown on 1.13-cm 2 glass coverslips
L-glutamine, and twofold vitamin solution (GIBCO). ¨
(Objekttrager, Menzol Glaser, Germany) at a density
The cultures were maintained in plastic flasks and of 5310 4 cells / coverslip. One day later, the cultures
incubated in 5% CO 2 / 95% air at 378C in a were washed and treated with serum-free, Ham F-12
humidified incubator. The cell lines were examined medium containing either DOX-loaded chitosan
and found to be free of mycoplasma, as assayed by nanoparticles incorporating dextran sulfate (30 min
the Gen-Probe Mycoplasma T.C. (Gen-Probe Inc., to 24 h incubation) or free DOX (30 min incubation).
San Diego, CA). All incubations were carried out at 378C with an
The antiproliferative effect of DOX was analyzed equivalent final concentration of DOX (5 mg / ml).
by the MTT method [24]. Cells from exponential Alternatively, cells were incubated using a two-
cultures were seeded onto 96-well tissue culture compartment Boyden chamber. Briefly, either DOX-
plates (TPP, Switzerland) at a density of 5310 3 loaded chitosan nanoparticles containing dextran
cells / well for a 0.36-cm 2 well (optimal seeding sulfate or free DOX solutions were diluted in serum-
density that avoids full confluency for the length of free culture medium and incorporated into the upper
the 4-day experiment). One day later, the cultures compartment of polycarbonate transwell filters (0.4
were washed and incubated for 2 h with the different mm pore diameter, Costar). The cells seeded on
samples at various dilutions in serum-free, Ham F-12 coverslips for 24 h were placed in the lower com-
medium. The different preparations were adjusted to partment, thereby receiving the DOX solution fil-
maintain the same drug concentration, and experi- tering from the upper compartment but excluding the
ments were performed in parallel wells with increas- DOX associated to the nanoparticles (a control
ing DOX concentrations of 0.1 mg / ml to 100 mg / ml. experiment was performed demonstrating that the
Following incubation, the cell monolayers were particles did not cross the filter). Following incuba-
washed five times with PBS and left 3 days in tion, cells were washed five times with PBS, and the
complete media. After this time, 50 ml / well of PBS coverslips were mounted on slides. Fluorescence
containing 1 mg / ml MTT (tetrazolium salt, Sigma- observation was carried out with a confocal micro-
Aldrich S.A., Madrid) was added, and the plates scope (TCS 4D, Leica Instruments) using an argon /
were incubated an additional 4 h. The intracellular krypton laser (75 mW) at 488 nm for excitation and
formazan crystals resulting from the reduction of the an LP filter of 590 nm for DOX detection. Contrast
tetrazolium salts present only in metabolically active images were simultaneously observed using the
cells were solubilized with DMSO (Sigma-Aldrich). inverted microscope equipment with a PL Apo 633 /
K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267 259
3. Results
Table 2
Effect of DOX loading on encapsulation efficiency for chitosan
nanoparticles incorporating dextran sulfate (n53)
Theoretical Encapsulation
loading (%) efficiency (%)
5 22.561.2
10 21.962.5
20 19.361.8
0.260.8% between the normalized profiles. Con- made from DOX complexed to chitosan showed
versely, encapsulated DOX exhibited an additional slightly decreased cytostatic activity relative to the
emission band at 630 nm which was evident over the DOX solution over this same concentration range.
range of 600–700 nm. However, for most concentrations assayed, the dif-
Fig. 6 shows the cytostasis of the different ferences were not statistically significant. The blank
chitosan formulations in vitro for the C26 cell line. nanoparticle suspension showed no significant cyto-
No significant differences were noted between the stasis. Very similar results were obtained using
DOX loaded chitosan–TPP nanoparticles incorporat- human melanoma A375 cells (results not shown).
ing dextran sulfate and the control DOX solution DOX localization of different nanoparticle / control
over drug concentrations from 0.1 mg / ml to 100 formulations in human melanoma A375 cells is
mg / ml in any of the cell lines tested. Nanoparticles shown in Fig. 7. Within 30 min incubation, free
DOX was localized within the cell nucleus (Fig. 7A),
Fig. 4. In vitro release profile for: (– + –) DOX loaded chitosan– Fig. 5. Fluorescence emission spectra of: (———) DOX solution
TPP nanoparticles incorporating dextran sulfate (DOX loading: in water, (— — —) DOX encapsulated in chitosan–TPP
4%) and (– j –) DOX complexed nanoparticles (DOX loading: nanoparticles, (- - -) DOX released from chitosan–TPP nanoparti-
0.4%) (n53). cles.
262 K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267
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).
chitosan solution led to the formation of aggregates, chitosan nanoparticles for the encapsulation of DOX.
rather than nanoparticles, upon addition of TPP. As could be anticipated, effects of the polyanion on
Sulfonic acid groups have been shown to bind the UV-VIS spectrum of DOX were readily visible.
DOX in considerable quantities when incorporated in More importantly, however, the DOX–dextran sul-
ion-exchange resins [28]. Additionally, dextran sul- fate complex appeared to only be partially disso-
fate has been used successfully to augment the ciated by the addition of chitosan (Fig. 2B), opening
encapsulation of DOX in albumin microspheres [29]. the possibility of using the complex to draw more
With these reports in mind, we decided to test the DOX into the nanoparticles. Indeed, this entrapment
feasibility of incorporating dextran sulfate into did occur, as the formulation incorporating dextran
264 K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267
sulfate was the only one to achieve encapsulation be tightly bound to chitosan. Indeed, this phenom-
efficiencies significantly above the control formula- enon did take place. While there was only a minimal
tion. DOX encapsulation was also visibly apparent yield for the complexation (0.43 wt.%), all of the
with these nanoparticles, which formed a dense red DOX that was complexed remained incorporated
pellet upon centrifugation. within the nanoparticles. Therefore, it could be
The difference between DOX–dextran sulfate and induced that the entrapment efficiency of DOX
DOX–polyphosphoric acid interactions is intriguing, previously associated to chitosan was 100%. The
in that one would expect coulombic forces to be possibility that the high entrapment efficiency is
stronger with the acid, owing to its higher charge merely caused by low initial DOX loading was also
density on a per weight basis. However, another considered. As a control study, we prepared chitosan
important consideration is the interaction of these nanoparticles with the same DOX initial 0.43 wt.%
polyanions with chitosan. Using the same argument loading, but adding DOX to the chitosan solution
of charge density, polyphosphoric acid should ex- prior to the nanoparticles formation. In this case the
hibit far greater avidity to chitosan relative to dextran encapsulation efficiency was far lower (23.061.0%)
sulfate. Hence, more DOX would be displaced from than that observed for chitosan–DOX complexes.
polyphosphoric acid than from dextran sulfate upon In vitro release studies were performed in acetate
addition of chitosan. buffer (pH 4). We chose this medium because DOX
Interestingly, DOX encapsulation efficiency in the is maximally stable at pH values between 3 and 5
formulation containing dextran sulfate appeared and also because, at higher pHs we encountered
minimally dependent upon theoretical DOX loading problems of fluorescence quenching or interference
over the range of 5–20% (w / w). At 10% DOX for the quantification of released DOX. Obviously, in
loading, there remains a 3.7-fold molar charge excess these experiments, we did not expect to predict the
of negatively-charged sulfonic acid groups relative to release behavior of these particles in vivo but to
DOX amino groups, so it is quite feasible that, due to compare the formulations developed and to gain
this excess, the saturation level of DOX association some insight about the mechanism of release.
is not reached. Under these conditions, therefore, it is Nanoparticles incorporating dextran sulfate showed a
likely that the formation of DOX–dextran sulfate burst release of 17% at 2 h, followed by an addition-
complexes is favored by higher quantities of DOX al release of 4.5% over the next 2 days. This slow
incubated, with real loading increasing almost linear- release was quite distinct from the profiles obtained
ly with theoretical loading. from similar chitosan nanoparticles encapsulating
An entirely different approach was taken with insulin, where 100% release was observed within 15
nanoparticles containing DOX complexed to min [21]. Even less DOX was detected in PBS at 5
chitosan. As an amphoteric drug (protonable amino days (data not shown), probably due to DOX degra-
group and deprotonable phenolic group), there con- dation in the near neutral medium. The initial phase
tinually exists an equilibrium between the positively of release is logically attributed to the DOX located
charged, negatively charged, neutral, and zwit- at the surface of the particles while the remainder of
terionic species of DOX (Fig. 1). Additionally, there the unreleased DOX was assumed to be well en-
are other factors (hydrophobic / hydrophilic interac- trapped within the chitosan nanoparticles and tightly
tions, resonance effects, etc.) which could allow associated to the chitosan molecules, probably as an
small quantities of DOX to complex with chitosan, ionic complex with dextran sulfate. Therefore, the
despite the overwhelming charge repulsion between degradation of chitosan would be required for ac-
the two molecules, as has been noted previously complishing the release process. Unfortunately, di-
between DOX and positively charged transition rect confirmation of this hypothesis by enzymatic
metal ions [30,31]. We tested the extent of this digestion of the nanoparticles was not possible since
association by incubating DOX and chitosan in chitosanase treatment of the nanoparticle suspension
solution, dialyzing to remove non-associated DOX, also degraded DOX solutions and / or quenched
and lyophilizing to promote polymer–drug interac- fluorescence in control studies. Nevertheless, the
tions. We did not expect considerable association to effect of dextran sulfate on DOX release was appar-
occur, but that the DOX which did associate would ent, as chitosan nanoparticles without the polyanion
K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267 265
showed over twice the burst effect after 2 h under the than to the release of free drug in the cell culture
same conditions (36.760.3%). medium. To confirm this hypothesis we investigated
Nanoparticles containing DOX complexed with the mechanism of in vitro cytotoxicity for DOX-
chitosan displayed an even smaller release over the loaded chitosan nanoparticles, using human
same period, an observation that is easily explained melanoma A375 cells, via confocal microscopy.
by the aforementioned interactions binding the drug Comparable fluorescence localization, visualized as
to the polymers. Indeed, since the interaction DOX– red, was seen for DOX-loaded nanoparticles relative
chitosan seems to be quite stable, any drug released to free DOX (Fig. 7A and B), however, a sig-
would be a result of degradation of chitosan or by nificantly longer incubation time was required for the
release of DOX complexed on the particle surface. nanoparticles to display the intracellular fluorescence
Notwithstanding, spectral analysis of the DOX signals. This suggests that these particles might enter
released from chitosan nanoparticles incorporating the cells, and that this internalization process occurs
dextran sulfate showed that the released DOX was over a much longer time than the diffusion of the
fluorimetrically identical to that of the native DOX free drug. Indeed, from these observations it could be
solution, as seen in Fig. 5. This preservation of the inferred that, after short incubation times, the
fluorescence signature supports the claim that DOX nanoparticles are not sufficiently associated with the
structure is retained following encapsulation in cells and are thus easily removed in the subsequent
chitosan nanoparticles, though it is not a definitive washes.
indication in itself. Conversely, the spectrum of An alternative explanation of these results, how-
associated DOX showed an entirely new, longer ever, could be that the longer incubation time is
wavelength fluorescence band. This same band has needed simply to allow the particles to release an
been previously reported for DOX in environments amount of DOX in the culture medium comparable
with high dielectric constant [32], and suggests that to that of the control DOX solution. To exclude this
DOX is mostly associated with the nanoparticles (via possibility, we used a two-compartment in vitro set-
encapsulation, adsorption, or both), rather than up where the DOX-loaded nanoparticles were placed
nanoprecipitated outside of the particles. in the donor compartment and the cells in a receptor
The retention of DOX bioactivity was best demon- compartment, separated via a polycarbonate mem-
strated by the in vitro cytostasis assays, shown in brane. After 24 h incubation, no DOX could be
Fig. 6. Despite that only about one fifth of the detected in the cell culture compartment (Fig. 7C).
encapsulated DOX was released from chitosan–TPP This led us to conclude that no significant amounts
nanoparticles in vitro, this formulation was equally of DOX were released from the nanoparticles into
able to slow tumor cell proliferation relative to DOX the cell culture medium. This was also confirmed by
solutions for the C26 and human melanoma A375 the fact that a control mixture of free DOX and blank
cell lines, indicating that DOX must maintain its nanoparticles under the same experimental condi-
bioactivity within these nanoparticles. The same was tions showed significant DOX accumulation (Fig.
the case with the nanoparticles using DOX–chitosan 7D), indicating that the drug can freely pass through
complexes, though the formulation did show lesser the membrane. The results of the in vitro release and
cytostasis at certain drug concentrations relative to confocal studies combined strongly support our
the control DOX solution. This could be due to an hypothesis that DOX-loaded chitosan nanoparticles
excessively tight interaction between the drug and are internalized by cells and degraded intracellularly
chitosan, which might impede its transit to the to release the drug. However, more thorough con-
nucleus. However, one cannot discard the possibility focal studies are needed to ultimately prove this
that partial damage to the molecular structure of mechanism.
DOX occurred during its complexation with
chitosan.
Given the limited DOX release exhibited by the 5. Conclusion
two formulations over this period, we hypothesized
that the cytotoxic action exhibited by these In this paper, we describe the feasibility of using
nanoparticles would be due to endocytosis, rather chitosan nanoparticles as colloidal carriers for the
266 K. A. Janes et al. / Journal of Controlled Release 73 (2001) 255 – 267
delivery of the small, cationic anthracycline drug, Drug Delivery Systems, Plenum, New York, 1984, pp. 77–
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We would like to thank Monica ´
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