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Anticancer Drug Delivery with
Transferrin Targeted Polymeric
Chitosan Vesicles
Christine Dufes,
1
Jean-Marc Muller,
1
William Couet,
2
Jean-Christophe Olivier,
2
Ijeoma F. Uchegbu,
3
and
Andreas G. Scha¨tzlein
4,5
Received August 4, 2003; accepted September 29 2003
Purpose. The study reports the initial biological evaluation of tar-
geted polymeric glycol chitosan vesicles as carrier systems for doxo-
rubicin (Dox).
Methods. Transferrin (Tf) was covalently bound to the Dox-loaded
palmitoylated glycol chitosan (GCP) vesicles using dimethylsuber-
imidate (DMSI). For comparison, glucose targeted niosomes were
prepared using N-palmitoyl glucosamine. Biological properties were
studied using confocal microscopy, flow cytometry, and cytotoxicity
assays as well as a mouse xenograft model.
Results. Tf vesicles were taken up rapidly with a plateau after 1–2 h
and Dox reached the nucleus after 60–90 min. Uptake was not in-
creased with the use of glucose ligands, but higher uptake and in-
creased cytotoxicity were observed for Tf targeted as compared to
GCP Dox alone. In the drug-resistant A2780AD cells and in A431
cells, the relative increase in activity was significantly higher for the
Tf-GCP vesicles than would have been expected from the uptake
studies. All vesicle formulations had a superior in vivo safety profile
compared to the free drug.
Conclusions. The in vitro advantage of targeted Tf vesicles did not
translate into a therapeutic advantage in vivo. All vesicles reduced
tumor size on day 2 but were overall less active than the free drug.
KEY WORDS: doxorubicin; glucose niosomes; glycol chitosan; poly-
meric vesicles; transferrin.
INTRODUCTION
The clinical use of the broad-spectrum anticancer drug
doxorubicin (Dox) can potentially be improved using delivery
systems (1).
To improve the specificity of polymeric vesicles, trans-
ferrin (Tf) and glucose (Glu) have been coupled to the
vesicles (2). The receptors for these ligands are expressed in
a range of tumors, but also in some healthy tissues (3,4).
Potentially, the combination of active targeting, based on the
use of ligands, and passive targeting, based on the accumula-
tion of particulate delivery systems due to the enhanced per-
meability and retention (5), should provide a tumor-selective
targeting strategy. Even without extravasation, tumor cells
that form part of the recently described “mosaic” blood ves-
sels (6) would potentially still be accessible to ligand targeted
carriers.
Another motivation for the use of drug carriers as well as
targeting ligands is their potential to overcome some acquired
mechanisms of drug resistance such as the p-glycoprotein/
MDR1 drug efflux system (7). Additionally, high levels of
transferrin expression have been linked with drug resistance
(8), offering the possibility of targeting resistant cells with
these ligand-bearing particulates.
Furthermore, oral administration of chitosan has been
shown to reduce some of the side effects of doxorubicin, in
particular the gastrointestinal mucositis after oral administra-
tion (9), and it may therefore be possible to improve the
safety profile by encapsulation within chitosan-based poly-
meric vesicles developed in our laboratories (10).
Here we report for the first time the in vivo biological
evaluation of doxorubicin formulated in transferrin targeted
polymeric vesicles made from palmitoylated glycol chitosan
(GCP). We examine whether the previously reported cou-
pling of glucose and transferrin ligands which bind to target
receptors overexpressed in some tumors (2) confers a target-
ing advantage to these systems and whether a modified up-
take mechanism could potentially help to overcome drug
transport related resistance.
MATERIALS AND METHODS
N-palmitoylglucosamine (2) and palmitoyl glycol chito-
san (11) were synthesized and characterized as previously de-
scribed. Doxorubicin hydrochloride (Dox) was supplied by
Alexis Biochemicals (UK). Glycol chitosan had a degree of
polymerization of 800, a degree of acetylation of 33 mol%,
and a degree of palmitoylation of 13 monomer units per 100
monomers. Sorbitan monostearate (Span 60), cholesterol, di-
methylsuberimidate dihydrochloride (DMSI), triethanol-
amine, phosphate-buffered saline (PBS; pH ⳱ 7.4) tablets,
iron-saturated human transferrin, Sephadex G50, polyethyl-
ene glycol (PEG) 8000, and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were all purchased from
Sigma Aldrich Co. (Poole, UK). Dialysis tubing was obtained
from Medicell International (London, UK). Isopropanol and
dimethylsulfoxide were purchased from Merck (Nottingham,
UK). Cholesteryl poly-24-oxyethylene ether (Solulan C24)
was kindly donated by D. F. Anstead (Basildon, UK). A431
cells (CRL-2592) and PC3 cells (CRL-1435) were purchased
from the American Type Culture Collection (www.attc.org)
and the A2780 cells were originally obtained from Dr. R. F.
Ozols (Fox Chase Cancer Center, PA, USA). Selection of the
A2780 variant AD has been described elsewhere (12).
Culture media were obtained from Invitrogen (Pasely,
UK). All other tissue culture reagents were obtained from
Gibco (Pasely, UK).
Formulation and Characterization
N-palmitoyl glucosamine niosomes (Glu) entrapping
doxorubicin were prepared by shaking a mixture of NPG (16
mg), Span 60 (65 mg), cholesterol (58 mg), and Solulan C24
(54 mg) in doxorubicin solution (1.5 mg/ml, 2 ml, prepared in
PBS) at 90°C for 1 h, followed by probe sonication for 10 min
(75% of max).
1
Laboratoire de Biologie des Interactions Cellulaires, CNRS UMR
6558, Faculté des Sciences, Poitiers, France.
2
Laboratoire de Pharmacie Gale´nique et Biopharmacie, Faculte´de
Me´decine et de Pharmacie, Poitiers, France.
3
Department of Pharmaceutical Sciences, University of Strathclyde,
Strathclyde Institute for Biomedical Sciences, Glasgow, United
Kingdom.
4
Cancer Research UK Department of Medical Oncology, Beatson
Laboratories, University of Glasgow, Garscube Estate, Glasgow,
G61 1BD, United Kingdom.
5
To whom correspondence should be addressed. (e-mail:
A.Schatzlein@beatson.gla.ac.uk)
Pharmaceutical Research, Vol. 21, No. 1, January 2004 (© 2004) Research Paper
101 0724-8741/04/0100-0101/0 © 2004 Plenum Publishing Corporation
Control Span 60 niosomes (Span) were prepared in the
same manner from Span 60 (73 mg), cholesterol (65 mg), and
Solulan C24 (54 mg) in doxorubicin solution (1.5 mg/ml, 2 ml,
in PBS).
Palmitoyl glycol chitosan (GCP) vesicles were prepared
as previously described (11) by probe sonicating glycol chito-
san (10 mg) and cholesterol (4 mg) in doxorubicin solution
(1.5 mg/ml). Tf was linked to the GCP vesicles by cross-
linking with DMSI as previously described (2).
For all vesicle suspensions, free drug or ligand (Dox or
Tf, respectively) were removed by size exclusion chromatog-
raphy (Sephadex G50) followed by concentration of the sus-
pensions by dialysis over PEG 8000.
The amount of conjugated transferrin was determined
using the Lowry method as previously described (2).
Doxorubicin loading of control, transferrin-, or glucose-
bearing vesicles was measured spectrofluorometrically (
ex
⳱
480 nm,
em
⳱ 560 nm) after disruption of vesicles in isopro-
panol. Vesicle sizing was performed by photon correlation
spectroscopy on a Malvern Zetasizer (Malvern Instruments,
Malvern, UK). Vesicle preparations were used immediately
after preparation and characterization.
Biological Characterization
Cell Culture
The human cell lines A431 [epidermoid carcinoma (13)],
PC3 [prostate adenocarcinoma (14)], and A2780 (ovarian car-
cinoma) and its resistant counterpart A2780/AD (15) were
grown as monolayers in DMEM (A431 cells) or RPMI 1640
medium supplemented with 10% (v/v) fetal bovine serum and
1% (v/v)
L-glutamine. Cells were cultured at 37°C in a humid
atmosphere of 5% CO
2
(A2780, A2780/AD) or 10% CO
2
(A431, PC3), respectively.
Confocal Microscopy
Cells were grown on cover slips (∼0.6 × 10
6
cells/ 35-mm
dish), washed with PBS (pH ⳱ 7.4), and then transferred to
a temperature-controlled holder/chamber (37°C). Nuclei of
the live cells were stained with DAPI (9 l, 100 M) for 10
min before vesicle suspensions were added (10
−6
M Dox per
well). Cells were examined over time using confocal micros-
copy (
Exc
⳱ 488 nm, Leica, Heidelberg, Germany, TCS SP2)
with sensitivity/photo multiplier settings being kept constant.
The sum of pixel intensity values (0–254) for each image/time
point was used to illustrate the kinetics of Dox uptake (cf.
Fig. 1).
Flow Cytometry
Cells (∼1.2×10
6
/35-mm dish) were incubated with Dox
formulated as vesicles (Tf, GCP, Glu, Span, and free Dox) or
as free drug at a final concentration of3×10
−7
M Dox per
well over 4 h at 37°C. Single cell suspensions were prepared,
washed (PBS, pH ⳱ 7.4), pelleted (600 g) twice, and exam-
ined on a FACStar flow cytometer (Becton-Dickinson Instru-
ments, Oxford, UK). Twenty thousand cells (gated events)
were counted for each sample, and Dox fluorescence was
detected with logarithmic settings (FL1;
Em
⳱ 530 ± 30 nm).
Cells were counted as positive if their fluorescence (FL1) was
higher than that of 95% of cells from an untreated cell sus-
pension. Each experiment was performed in triplicate and
analyzed statistically using one-way analysis of variance
(ANOVA), followed by Bonferroni’s post-test.
In vitro Cytotoxicity Assay
Antiproliferative activity of transferrin- or glucose-
bearing vesicles, PGC vesicles or Span 60 vesicles, all entrap-
ping Dox, were compared with Dox solution in A2780,
A2780/AD, A431, and PC3 cell lines. Cells were seeded in
quintuplicate (600 cells/96-well), and after 3 days, the medium
exchanged with medium containing the formulations at final
concentrations of1×10
-12
Mto1×10
-4
M. After 4 h, the
medium was replaced and the cells further incubated for 3
days. Cytotoxicity was evaluated by measurement of the
growth inhibitory concentration for 50% of the cell popula-
tion (IC
50
) in a standard MTT [(3-(4,5-dimethylthiazol-2-yl)-
2,5- diphenyl-tetrazolium bromide blue-indicator dye] -based
assay (16). Dose–response curves were fitted to percentage
absorbance values to obtain IC
50
values (three independent
experiments, with n ⳱ 5 for each concentration level).
In vivo Tumoricidal Activity
Mice (CD1-nu; weight, 20 g) were housed in groups of
five (19–23°C, 12 h light–dark cycle) with free access to food
(Rat and Mouse Standard Expanded, B and K Universal,
Grimston, UK) and water from the mains. Experimental
work was carried out in accordance with UK Home Office
regulations and approved by the local ethics committee.
Tumors (typical diameter 5 mm) were palpable 6 days
after implantation of A431 tumor cells (10
6
cells per flank).
The formulations (n ⳱ 5 animals/group) were intravenously
administered at a dose equivalent to 10 mg/kg Dox. Control
mice were injected either with Dox solution or received no
treatment. The tumor size was measured using callipers and
the animals weighed daily. The animals that had received the
free Dox solution had to be euthanized after 5 days (i.e.,
before the planned end of the experiment) because of weight
loss. All other animals were killed 10 days after the start of
treatment when in some animals the tumor size was already
approaching 1 cm
3
. The tumors were excised and weighed,
and tumor size was used as an index for in vivo antitumor
activity.
RESULTS
Vesicle Formulations
Dox loading into niosomes was 2.9 × 10
−3
±0.3×10
−3
g/g
of Glu niosome and 2.8 × 10
−3
±0.3×10
−3
g/g of control
niosomes (Span), respectively, corresponding to 18 ± 2% of
the initial doxorubicin. Loaded control niosomes had a z-
average mean diameter of 166 nm (polydispersity, 0.427)
whereas Glu niosomes had a z-average mean diameter of 228
nm (polydispersity, 0.148).
For the GCP vesicles with and without transferrin, the
loading was 0.049 ± 0.005 g/g, corresponding to 16.3 ± 1.7% of
the initial doxorubicin. Transferrin was successfully conju-
gated to GCP vesicles at a level of 0.6 ± 0.18 g of transferrin
per g polymer (50 ± 15% of the initial transferrin used). The
loaded GCP vesicles had a z-average mean diameter of 696
nm (polydispersity, 1) whereas Tf vesicles had a z-average
size of 889 nm (polydispersity, 0.0964).
Dufes et al.102
Biological Characterization
Confocal Microscopy
Uptake of free doxorubicin and Tf vesicles was observed
using confocal microscopy in live A2780 cells (Fig. 1). Speck-
led cytoplasmic staining patterns were visible after less than
an hour, suggesting some endocytotic uptake (17). Nuclear
staining was evident after 60–90 min, suggesting that some
Dox-derived fluorescence was either quickly released from
endocytotic vesicles or that extracellular doxorubicin leaked
from extracellular carriers with some accumulation in a cen-
tral compartment, possibly the nucleolus. Uptake kinetic was
determined in live cells using confocal microscopy (Fig. 1).
Untreated cells show a low level of autofluorescence with a
narrow distribution of fluorescence intensity. Within only 10
min, an increase of fluorescence intensity, which is not clearly
visible yet in the micrographs, can be measured as shift in the
mean pixel intensities. Uptake kinetics overall show a sigmoid
shape, apparently reaching a maximum after 120 min. Distri-
bution of fluorescence intensities is fairly broad at this point.
Flow Cytometry
In order to quantify total doxorubicin uptake for differ-
ent formulations and cell types after 4 h incubation, we used
flow cytometry (Fig. 2). The highest amount of cell-associated
fluorescence was found in cells that had been incubated with
free doxorubicin (Fig. 3). The mean cell-associated fluores-
cence for this treatment group was in general more than
double that of the best vesicular formulations. Vesicle modi-
fication with glucose did not convey any significant improve-
ment of uptake in any of the tested cell lines. The brightest
fluorescence after vesicular treatment was associated with
A431 cells incubated with the Tf vesicles (cf. Fig. 2). Tf ves-
icle uptake was significantly higher than that of the ligand-
free controls in all cell lines. In A431 cells, uptake from Tf
vesicles was significantly higher than that of any other vesicle
formulation.
In Vitro Cytotoxicity Assay
Cytotoxicity of doxorubicin formulations was assessed in
a panel of cell lines using an MTT-based cell survival assay
(Fig. 4) to derive the IC
50
.
Doxorubicin IC
50
(3.55 × 10
−8
to 7.44 × 10
−7
) is within the
range of previously observed values (15). The vesicle formu-
lations were all significantly less toxic in the individual cell
lines than the free drug with IC
50
ranging from 1.57 × 10
−7
to
4.83 × 10
−6
. In A431 cells, the Span formulation was found to
require 22 times more doxorubicin to kill 50% of cells than
the free drug.
The vesicle formulations tended to have similar levels of
cytotoxicity in all cell lines (Fig. 5). The lack of significant
differences between targeted (Glu) and nontargeted (Span)
niosomes suggests that glucose did not confer a consistent
advantage.
While the cytotoxic activity of Tf targeted GCP poly-
meric vesicles was not significantly different from the other
vesicle formulations in the sensitive A2780 cell line, they were
about 5–10 times more active than other vesicles in A431
cells. In the resistant A2780/AD cells, they were 3–4 times
more effective than GCP vesicles. Interestingly, Tf vesicles
were significantly less active than GCP vesicles in PC3 cells.
Fig. 1. Confocal microscopy of A2780 cells after incubation with doxorubicin-loaded Tf polymeric vesicles. Left panel shows distribution of
free doxorubicin (top) and TF vesicles (bottom) for qualitative comparison of intracellular distribution. The insert in the bottom left panel
highlights the speckled appearance of intracellular doxorubicin (arrows). Doxorubicin-derived fluorescence is mainly limited to the nucleus
in the case of free doxorubicin (top); scale bar ∼50 m. Right panel: The graph visualizes the kinetics of Dox uptake into A2780 cells when
incubated with doxorubicin-loaded Tf polymeric vesicles. Circles represent range of distribution of pixel intensities of the corresponding
confocal images.
Transferrin Targeted Polymeric Chitosan Vesicles 103
In Vivo Tumoricidal Activity and Toxicity
In vivo activity was tested in an A431 xenograft model
with established tumors using a single dose of the formula-
tions (Fig. 6). Starting tumor size of treated groups was not
statistically significantly different from the control group.
While the doxorubicin dose was efficient in suppressing fur-
ther tumor growth, we did not observe a reduction of tumor
size. More importantly, treatment of the animals with free
doxorubicin at this dose level induced significant signs of tox-
icity; that is, a pronounced weight loss and all animals in this
group had to be euthanized on day 5 (Fig. 7).
The response of the vesicular formulations in the A431
xenograft model were not significantly different, and treated
groups could not be distinguished from the control group,
except during the first 24 h after injection when targeted or
control vesicles were found to be significantly more active
than Dox in solution, with a reduction in size between 1 day
and 2 days after injection being significant for all the vesicle
groups (Tf, p < 0.05; GCP, p < 0.01; Span, p < 0.001; Glu,
p < 0.001).
In all vesicle groups as well as the control group, all of the
animals appeared lively throughout the study, and no weight
loss was detected. There were no signs of decreased activity,
which would indicate general toxicity. As a result, vesicles are
considered to be safe at the dosing schedule used.
DISCUSSION
Transferrin receptors are found on the surface of most
proliferating cells and, in elevated numbers, on erythroblasts
and on many tumors (1.5 × 10
5
to 10
6
/cell) (3) where they
have been linked to drug resistance (8). The facilitative glu-
cose transporter GLUT-1 has been correlated to the transi-
tion to malignancy and response to chemotherapy (4,18). We
have recently reported targeting of doxorubicin-loaded poly-
meric vesicles from palmitoylated glycol chitosan in vitro us-
ing the ligands transferrin and glucosamine, which bind to
these receptors (2). Here we report the evaluation of these
systems in vivo as potential delivery systems to solid tumors,
comparing them with well-characterized niosome formula-
tions.
Doxorubicin was chosen as a model drug with potential
Fig. 3. Flow cytometric analysis (n ⳱ 3) of the uptake of Dox solu-
tion (white bar), Dox-loaded Span 60 vesicles without glucose (light
gray), glucose-bearing vesicles (gray, black hash), GCP vesicles with-
out transferrin (dark gray), transferrin-bearing vesicles (hashed
white) by A2780, A2780/AD, A431, and PC3 cells. Untreated cells
(represented by the first bar in each group; white, black hash) are not
visible at this scale. Cells were counted as Dox positive when their
fluorescence was higher than that of 95% of cells from an untreated
cell suspension.
Fig. 2. Flow cytometric measurement of doxorubicin uptake into A431 cells. Untreated
cells (hashed, gray) served as negative control while free doxorubicin solution was used as
positive control (gray). Doxorubicin uptake from GCP vesicles (black) and transferrin
targeted GCP vesicles (white) were compared after 4 h incubation at 37°C.
Dufes et al.104
therapeutic relevance because of the ease of detection (mi-
croscopy, FACS, HPLC). Doxorubicin has been in clinical
use against a wide range of human cancers for decades. Nev-
ertheless, a number of issues critical to the therapeutic success
and safety of the drug, such as cardiotoxicity, drug resistance,
and specificity remain unresolved.
Tumor growth beyond a few millimeters requires recruit-
ment of additional blood vessels, which exhibit a large num-
ber of cellular holes and gaps (19). The resulting irregularity
in the fluid flow has been used with good effect to target
macromolecular and particulate drug carriers to tumors (20).
For a majority of tumors, the cut-off size for extravasa-
tion has been found to be below 400 nm (21–23), suggesting
that larger vesicles may be at a disadvantage. However, a
larger variation of cut-off size [the cut-off ranging from be-
tween 200 nm and 1.2 m or even up to 2 m (19)] has
recently been described. The pore size showed significant het-
erogeneity in the tumor and a strong dependence on tumor
type and microenvironment (24).
Furthermore, it has been demonstrated that a significant
proportion of the tumor vasculature in colon carcinoma xe-
nografts was made up of tumor cells themselves (6). In these
“mosaic” type vessels, a targeted carrier would potentially
have direct access to the receptor-expressing tumor cells with-
out the need for extravasation.
Both ligands, glucose and transferrin, were chosen be-
cause they could potentially modulate uptake in a broader
range of tumors. Therefore the combination of two targeting
mechanisms (active targeting using the TF ligand and passive
Fig. 5. Cytotoxicity of free Dox (䉲) and of doxorubicin formulated as
Span niosomes (䊊), Glu niosomes (䊉), GCP vesicles (䊐), or Tf
vesicles (䊏) in A2780, A2780AD, A431, and PC3 cells expressed as
IC
50
values. n ⳱ 15.
Fig. 6. Tumoricidal activity of intravenously injected Dox solution
(䉲), Span niosomes (䊏), Glu niosomes (䊐), GCP vesicles (䊉), and
Tf vesicles (䊊) against an A431 tumor subcutaneously implanted
in nude mice (n ⳱ 5). Untreated animals served as controls (䉱).
Error ⳱ SE.
Fig. 4. Cytotoxicity of Dox delivered as free drug (Dox), loaded in Span 60 vesicles without glucose
(Span), glucose-bearing vesicles (Glu), GCP vesicles without transferrin (GCP), transferrin-bearing
vesicles (Tf), against (A) A2780, (B) A2780/AD, (C) A431, and (D) PC3 cells (n ⳱ 15). Error ⳱ SE.
Transferrin Targeted Polymeric Chitosan Vesicles 105
targeting using the EPR effect) could potentially provide a
tumor-selective targeting strategy.
The cellular distribution over time seen with the vesicles
is distinctly different from the patterns observed with the free
drug (Fig. 1), and within 20–30 min shows a pattern typically
observed with endocytotic uptake of particulate carriers (17).
But already after 60–90 min, there is also evidence of nuclear
accumulation of doxorubicin (Fig. 1).
Between 60–120 min, the rate of uptake appears to slow
down, and the total amount taken up approaches a maximum
(Fig. 1). This effect was not observed with free doxorubicin
(data not shown), suggesting that self-quenching of Dox fluo-
rescence did not play a major role in this observation. The
effect may be linked to a depletion of transferrin receptors on
the cell surface possibly linked to the intracellular retention of
the receptor after multivalent binding events (25).
Free doxorubicin enters the cell by diffusion, leading to
significantly higher drug levels than found with the vesicular
formulations (Fig. 3) that are taken up via endocytosis, a
comparatively slower but potentially highly specific process.
Despite the ability of the glucose targeted vesicles to bind
concavalin A (2), there was no modulation of cellular uptake
of the vesicles compared to the unmodified control niosomes.
The Tf targeted vesicles on the other hand increased the up-
take in all cell lines with a relative improvement factor 1.37 ±
0.1 when compared to nontargeted GCP vesicles.
We then examined whether the various levels of cellular
Dox uptake are linked to similar differences in the level of
cytotoxicity of the various formulations. While glucose tar-
geted niosomes did not enhance doxorubicin cytotoxicity
when compared to plain niosomes in any of the cell lines, the
addition of transferrin to the surface of the vesicles increased
cytotoxic activity in some of the cell lines (Fig. 5). The uptake
assay clearly showed moderately increased uptake for the
transferrin-targeted vesicles by factor of 1.37, which for the
A2780 cell line was consistent with the improved cytotoxicity
observed (factor 1.6). However, in the doxorubicin-resistant
A2780/AD cell line, the activity improvement (factor 2.6) was
found to be over and above the uptake advantage. This was
even more evident for the A431 cell line where the transferrin
conferred a 5.2-fold higher activity. The A431 cells are known
to express high levels of transferrin receptor (TfR), and, in
the A2780/AD cell line, improvement may also be linked with
a relatively higher Tf receptor activity (8). Surprisingly, in the
PC3 cell line, the improved uptake is not reflected in the level
of activity; in fact, the nontargeted formulation is 3 times
more toxic than the targeted formulation in this cell line. The
reason for this is unclear.
As expected, the activity of vesicular formulations was
lower than that of the free drug. However, there were marked
differences ranging over almost one order of magnitude in the
same cell line (factor 2.67 for Tf and 22.04 for Span). How-
ever, when the formulations were tested in vivo in A431 xe-
nograft models, the results did not bear out the initial prom-
ise. None of the vesicular formulations was able to delay sig-
nificantly tumor growth after a single dose. The free drug did
not lead to tumor shrinkage but stopped further increase in
tumor size before the animals had to be killed because of the
severe weight loss.
Although the Dox dose of 10 mg/kg was not sufficient to
induce cures in any of the mice, it halted further tumor
growth. On the other hand, the dose was linked to a signifi-
cant toxicity in all the mice in this control group (Fig. 7)
despite having been used previously in another mouse strain
(26–28). As the activity of the free Dox in vitro was only 2.5
times higher than that of the Tf vesicles in vitro, it is some-
what surprising that this formulation did not show any clear
signs of therapeutic effect, suggesting problems linked to the
delivery in vivo.
Studies using doxorubicin Span 60 niosomes indicated an
improvement in tumoricidal activity with a similar single dose
on MAC 15A tumor and CH1 Dox
R
tumor (27,28). One po-
tential problem of the formulations used could lie in the rela-
tively large size of the vesicles (around 890 nm). Transvascu-
lar transport of particulate delivery systems depends on gaps
in the tumor blood vessels, which in many mouse tumor mod-
els range from 0.2 m–1.2 m (24). While still within the
range of cut-off sizes, the vesicles used in this study were
relatively large (890 nm), and their extravasation in the A431
tumor could have been impaired. The size increase from 690
nm to 890 nm induced by Tf coupling and the purification
procedure may be unavoidable, but we have recently demon-
strated that the starting size of the GCP polymeric vesicles
can be modulated by control of the molecular weight of the
starting material (29), thus offering a strategy of adjusting the
delivery system to the vascular properties of a given tumor.
Alternatively, a pharmacological augmentation or transport
may be possible (30).
In summary vesicles retain doxorubicin and altered the
uptake into cells (Fig. 1) in a fashion consistent with endo-
somal uptake of particulate carriers. The transferrin-
conjugated vesicles showed a statistically significant uptake
advantage when compared to the nontargeted vesicles (cf.
Fig. 3). The level of association after4hisconsistently in-
creased by more than 30% in contrast to the glucose targeted
niosomes, where levels are unchanged or slightly decreased.
While no significant difference in cytotoxicity was observed
between the vesicular formulations in the highly doxorubicin
sensitive cell line A2780, the Tf targeted vesicles demon-
strated a significant improvement of activity in the A2780-
Fig. 7. Daily weights of A431 tumor-bearing nude mice intravenously
injected with Dox solution (䉲), Span niosomes (䊏), Glu niosomes
(䊐), GCP vesicles (䊉), and Tf GCP vesicles (䊊). Untreated animals
served as controls (䉱). n ⳱ 5.
Dufes et al.106
derived resistant cell line and the resistant A431 cell line
(265% and 521%, respectively).
It is encouraging that in preliminary in vivo studies, use
of a single dose of systemically administered vesicles has not
only been shown to avoid the toxicity of the free drug, but
also to inhibit the growth of A431 cancer cells, if only at the
beginning of the treatment. There may be scope to improve
on the in vivo activity of these extremely safe delivery systems
by using a higher dose, reducing the size of the vesicles (29),
and improving the targeting/coupling. Thus, it may be pos-
sible to improve specificity and efficacy of these delivery sys-
tems by combination of semiselective passive and active tar-
geting strategies.
ACKNOWLEDGMENTS
C. D. would like to thank the Comité Départemental de
la Vienne of the Ligue contre le Cancer Association (France)
for its financial support. J. C. O. is a member of the emerging
Anti-Infectious Drugs and Blood Brain Barrier team of the
University of Poitiers.
REFERENCES
1. M. Winterhalter, P. M. Frederik, J. J. Vallner, and D. D. Lasic.
Stealth(R) liposomes: from theory to product. Adv. Drug Deliv.
Rev. 24:165–177 (1997).
2. C. Dufes, A. G. Schätzlein, L. Tetley, A. I. Gray, D. G. Watson,
J. C. Olivier, W. Couet, and I. F. Uchegbu. Niosomes and poly-
meric chitosan based vesicles bearing transferrin and glucose li-
gands for drug targeting. Pharm. Res. 17:1250–1258 (2000).
3. J. Goding. CD71, NCBI Proteins on the Web. Available: http://
www.ncbi.nlm.nih.gov/PROW/guide/1445562251_g.htm (1999).
4. J. D. Chandler, E. D. Williams, J. L. Slavin, J. D. Best, and S.
Rogers. Expression and localization of GLUT1 and GLUT12 in
prostate carcinoma. Cancer 97:2035–2042 (2003).
5. H. Maeda. The tumor blood vessel as an ideal target for macro-
molecular anticancer agents. J Controlled Release 19:315–324
(1992).
6. Y. S. Chang, E. di Tomaso, D. M. McDonald, R. Jones, R. K.
Jain, and L. L. Munn. Mosaic blood vessels in tumors: frequency
of cancer cells in contact with flowing blood. Proc. Natl. Acad.
Sci. U. S. A. 97:14608–14613 (2000).
7. A. K. Larsen, A. E. Escargueil, and A. Skladanowski. Resistance
mechanisms associated with altered intracellular distribution of
anticancer agents. Pharmacol. Ther. 85:217–229 (2000).
8. K. Barabas and W. P. Faulk. Transferrin receptors associate with
drug resistance in cancer cells. Biochem. Biophys. Res. Commun.
197:702–708 (1993).
9. Y. Kimura, N. Sawai, and H. Okuda. Antitumour activity and
adverse reactions of combined treatment with chitosan and doxo-
rubicin in tumor-bearing mice. J. Pharm. Pharmacol. 53:1373–
1378 (2001).
10. I. F. Uchegbu, L. Tetley, and A. G. Schätzlein. Polymeric palmi-
toyl glycol chitosan (GP) vesicles - a new drug delivery system. J.
Pharm. Pharmacol. 49:S27 (1997).
11. I. F. Uchegbu, A. G. Schätzlein, L. Tetley, A. I. Gray, J. Sludden,
S. Siddique, and E. Mosha. Polymeric chitosan-based vesicles for
drug delivery. J. Pharm. Pharmacol. 50:453–458 (1998).
12. D. A. Anthoney, A. J. McIlwrath, W. M. Gallagher, A. R. Edlin,
and R. Brown. Microsatellite instability, apoptosis, and loss of
p53 function in drug-resistant tumor cells. Cancer Res. 56:1374–
1381 (1996).
13. D. J. Giard, S. A. Aaronson, G. J. Todaro, P. Arnstein, J. H.
Kersey, H. Dosik, and W. P. Parks. In vitro cultivation of human
tumors: establishment of cell lines derived from a series of solid
tumors. J. Natl. Cancer Inst. 51:1417–1423 (1973).
14. M. C. Rossi and B. R. Zetter. Selective stimulation of prostatic
carcinoma cell proliferation by transferrin. Proc. Natl. Acad. Sci.
U. S. A. 89:6197–6201 (1992).
15. T. Minko, P. Kopeckova, V. Pozharov, and J. Kopecek. HPMA
copolymer bound adriamycin overcomes MDR1 gene encoded
resistance in a human ovarian carcinoma cell line. J Controlled
Release 54:223–233 (1998).
16. J. A. Plumb, R. Milroy, and S. B. Kaye. Effects of the pH de-
pendence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide-formazan absorption on chemosensitivity
determined by a novel tetrazolium-based assay. Cancer Res. 49:
4435–4440 (1989).
17. M. Hao and F. R. Maxfield. Characterization of rapid membrane
internalization and recycling. J. Biol. Chem. 275:15279–15286
(2000).
18. G. Cantuaria, A. Fagotti, G. Ferrandina, A. Magalhaes, M. Nadji,
R. Angioli, M. Penalver, S. Mancuso, and G. Scambia. GLUT-1
expression in ovarian carcinoma: association with survival and
response to chemotherapy. Cancer 92:1144–1150 (2001).
19. H. Hashizume, P. Baluk, S. Morikawa, J. W. McLean, G. Thur-
ston, S. Roberge, R. K. Jain, and D. M. McDonald. Openings
between defective endothelial cells explain tumor vessel leaki-
ness. Am. J. Pathol. 156:1363–1380 (2000).
20. R. Duncan. The dawning era of polymer therapeutics. Nat. Rev.
Drug Discov. 2:347–360 (2003).
21. G. Kong, R. D. Braun, and M. W. Dewhirst. Hyperthermia en-
ables tumor-specific nanoparticle delivery: effect of particle size.
Cancer Res. 60:4440–4445 (2000).
22. O. Ishida, K. Maruyama, K. Sasaki, and M. Iwatsuru. Size-
dependent extravasation and interstitial localization of polyeth-
yleneglycol liposomes in solid tumor-bearing mice. Int. J. Pharm.
190:49–56 (1999).
23. F. Yuan, M. Dellian, D. Fukumura, M. Leunig, D. A. Berk, V. P.
Torchilin, and R. K. Jain. Vascular permeability in a human tu-
mor xenograft: molecular size dependence and cutoff size. Cancer
Res. 55:3752–3756 (1995).
24. S. K. Hobbs, W. L. Monsky, F. Yuan, W. G. Roberts, L. Griffith,
V. P. Torchilin, and R. K. Jain. Regulation of transport pathways
in tumor vessels: role of tumor type and microenvironment. Proc.
Natl. Acad. Sci. U. S. A. 95:4607–4612 (1998).
25. E. W. Marsh, P. L. Leopold, N. L. Jones, and F. R. Maxfield.
Oligomerized transferrin receptors are selectively retained by a
lumenal sorting signal in a long-lived endocytic recycling com-
partment. J. Cell Biol. 129:1509–1522 (1995).
26. P. M. Loadman, M. C. Bibby, J. A. Double, W. M. Al-Shakhaa,
and R. Duncan. Pharmacokinetics of PK1 and doxorubicin in
experimental colon tumor models with differing responses to
PK1. Clin. Cancer Res. 5:3682–3688 (1999).
27. I. F. Uchegbu, J. A. Double, J. A. Turton, and A. T. Florence.
Distribution, metabolism and tumoricidal activity of doxorubicin
administered in sorbitan monostearate (Span 60) niosomes in the
mouse. Pharm. Res. 12:1019–1024 (1995).
28. I. F. Uchegbu, J. A. Double, L. R. Kelland, J. A. Turton, and
A. T. Florence. The activity of doxorubicin niosomes against an
ovarian cancer cell line and three in vivo mouse tumor models.
J. Drug Target. 3:399–409 (1996).
29. W. Wang, L. Tetley, A. M. McConaghy, and I. F. Uchegbu. Con-
trols on polymer molecular weight may be used to control the size
of palmitoyl glycol chitosan polymeric vesicles. Langmuir 17:631–
636 (2001).
30. W. L. Monsky, D. Fukumura, T. Gohongi, M. Ancukiewcz, H. A.
Weich, V. P. Torchilin, F. Yuan, and R. K. Jain. Augmentation of
transvascular transport of macromolecules and nanoparticles in
tumors using vascular endothelial growth factor. Cancer Res. 59:
4129–4135 (1999).
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