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Anticancer Drug Delivery with Transferrin Targeted Polymeric Chitosan Vesicles


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The study reports the initial biological evaluation of targeted polymeric glycol chitosan vesicles as carrier systems for doxorubicin (Dox). Transferrin (Tf) was covalently bound to the Dox-loaded palmitoylated glycol chitosan (GCP) vesicles using dimethylsuberimidate (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. 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 increased with the use of glucose ligands, but higher uptake and increased 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. 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.
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Anticancer Drug Delivery with
Transferrin Targeted Polymeric
Chitosan Vesicles
Christine Dufes,
Jean-Marc Muller,
William Couet,
Jean-Christophe Olivier,
Ijeoma F. Uchegbu,
Andreas G. Scha¨tzlein
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.
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
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.
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 (
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).
Laboratoire de Biologie des Interactions Cellulaires, CNRS UMR
6558, Faculté des Sciences, Poitiers, France.
Laboratoire de Pharmacie Gale´nique et Biopharmacie, Faculte´de
Me´decine et de Pharmacie, Poitiers, France.
Department of Pharmaceutical Sciences, University of Strathclyde,
Strathclyde Institute for Biomedical Sciences, Glasgow, United
Cancer Research UK Department of Medical Oncology, Beatson
Laboratories, University of Glasgow, Garscube Estate, Glasgow,
G61 1BD, United Kingdom.
To whom correspondence should be addressed. (e-mail:
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 (
480 nm,
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
(A2780, A2780/AD) or 10% CO
(A431, PC3), respectively.
Confocal Microscopy
Cells were grown on cover slips (0.6 × 10
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
M Dox per
well). Cells were examined over time using confocal micros-
copy (
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
/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
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;
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
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
) 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
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
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
. The tumors were excised and weighed,
and tumor size was used as an index for in vivo antitumor
Vesicle Formulations
Dox loading into niosomes was 2.9 × 10
of Glu niosome and 2.8 × 10
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 6090 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
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
Doxorubicin IC
(3.55 × 10
to 7.44 × 10
) 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
ranging from 1.57 × 10
4.83 × 10
. 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
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 510 times more active than other vesicles in A431
cells. In the resistant A2780/AD cells, they were 34 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.
Transferrin receptors are found on the surface of most
proliferating cells and, in elevated numbers, on erythroblasts
and on many tumors (1.5 × 10
to 10
/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-
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 (2123), 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 () and of doxorubicin formulated as
Span niosomes (), Glu niosomes (), GCP vesicles (), or Tf
vesicles () in A2780, A2780AD, A431, and PC3 cells expressed as
values. n 15.
Fig. 6. Tumoricidal activity of intravenously injected Dox solution
(), Span niosomes (), Glu niosomes (), GCP vesicles (), and
Tf vesicles () against an A431 tumor subcutaneously implanted
in nude mice (n 5). Untreated animals served as controls ().
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 2030 min shows a pattern typically
observed with endocytotic uptake of particulate carriers (17).
But already after 6090 min, there is also evidence of nuclear
accumulation of doxorubicin (Fig. 1).
Between 60120 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
(2628). 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
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 m1.2 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 (), Span niosomes (), Glu niosomes
(), GCP vesicles (), and Tf GCP vesicles (). Untreated animals
served as controls (). 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.
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
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Transferrin Targeted Polymeric Chitosan Vesicles 107
... Due to their small size, NPs could be employed for targeted drug delivery via intravenous injection. By attaching the targeting molecules to the surface regarding drug-loaded NPs, the therapeutic efficiency of the medication could be improved (Dufes et al., 2004). Low molecular medications, genes, and peptides are routinely delivered by using Chitosan. ...
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By using the deacetylation method, chitin is converted into bioproduct chitosan. Deacetylation can be accomplished using chemical or biological mechanisms. Due to its biocompatibility, nontoxicity, biodegradability, natural origin, and resemblance to human macromolecules, it is useful in medicine. Chitosan may have antibacterial and antioxidant properties. Additionally, it could be used in biotechnology, agriculture, gene therapy, food technology, medication delivery, cancer therapy, and other fields. The objective of the current review was to list the most significant applications of Chitosan in the biomedical field.
... Due to the foremost advantages, chitosan has become of significant attention in a variety of domains, including biomedical [31], environmental [32], cosmetics [33,34], agriculture [35], gene delivery [36], ocular drug delivery [37], protein [38], anticancer drug delivery [39], and topical drug delivery [40]. Chitosan is soluble in dilute aqueous acids but insoluble in water at neutral or alkaline pH. ...
Natural biodegradable polymers generally include polysaccharides (starch, alginate, chitin/chitosan, hyaluronic acid derivatives, etc.) and proteins (collagen, gelatin, fibrin, etc.). In transdermal drug delivery systems (TDDS), these polymers play a vital role in controlling the device's drug release. It is possible that natural polymers can be used for TDDS to attain predetermined drug delivery rates due to their physicochemical properties. These polymers can be employed to market products and scale production because they are readily available and inexpensive. As a result of these polymers, new pharmaceutical delivery systems can be developed that is both regulated and targeted. The focus of this article is the application of a biodegradable polymeric platform based on natural polymers for TDDS. Due to their biocompatibility and biodegradability, natural biodegradable polymers are frequently used in biomedical applications. Additionally, these natural biodegradable polymers are being studied for their characteristics and behaviors.
... In recent decades, the applications of Cat-Chit in biomedicine have been well established, such as tissue adhesives [20] and drug delivery systems [21], because of its medical benefits as well as its low toxicity and biodegradability. However, its low solubility in aqueous solutions hindered its further advancement in this field [22][23][24][25][26][27][28]. In order to overcome this critical issue, chitosan-based derivatives have been synthesized via amide formation with 3,4-dihydroxyhydrocinnamic acid and amine [20,21,[29][30][31][32]. ...
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There have been numerous recent studies on improving the mechanical properties and durability of cement composites by mixing them with functional polymers. However, research into applying modified biopolymer such as catechol-functionalized chitosan to cement mortar or concrete is rare to the best of our knowledge. In this study, catechol-functionalized chitosan (Cat-Chit), a well-known bioinspired polymer that imitates the basic structures and functions of living organisms and biological materials in nature, was synthesized and combined with cement mortar in various proportions. The compressive strength, tensile strength, drying shrinkage, accelerated carbonation depth, and chloride-ion penetrability of these mixes were then evaluated. In the ultraviolet–visible spectra, a maximum absorption peak appeared at 280 nm, corresponding to catechol conjugation. The sample containing 7.5% Cat-Chit polymer in water (CPW) exhibited the highest compressive strength, and its 28-day compressive strength was ~20.2% higher than that of a control sample with no added polymer. The tensile strength of the samples containing 5% or more CPW was ~2.3–11.5% higher than that of the control sample. Additionally, all the Cat-Chit polymer mixtures exhibited lower carbonation depths than compared to the control sample. The total charge passing through the samples decreased as the amount of CPW increased. Thus, incorporating this polymer effectively improved the mechanical properties, carbonation resistance, and chloride-ion penetration resistance of cement mortar.
... Using Handjani-Vila method (A1-A5), incorporation of payload occurs when the surfactant component was heated above its phase transition temperature, followed by self-assembly of vesicles with energy input [27]. This method was shown to be effective for hydrophilic compounds such as doxorubicin and green tea polyphenol [28,29]. On the contrary, vesicles prepared using the film hydration method (B1-B5) were built upon the principles of interface concentration gradient [30]. ...
Aim: To synthesize niosomes co-encapsulating gemcitabine (GEM) and tocotrienols, and physicochemically characterize and evaluate the antipancreatic effects of the nanoformulation on Panc 10.05, SW 1990, AsPC-1 and BxPC-3 cells. Materials & methods: Niosomes-entrapping GEM and tocotrienols composed of Span 60, cholesterol and D-α-tocopheryl polyethylene glycol 1000 succinate were produced by Handjani-Vila and film hydration methods. Results: The film hydration produced vesicles measuring 161.9 ± 0.5 nm, approximately 50% smaller in size than Handjani-Vila method, with maximum entrapment efficiencies of 20.07 ± 0.22% for GEM and 34.52 ± 0.10% for tocotrienols. In Panc 10.05 cells, GEM’s antiproliferative effect was enhanced 2.78-fold in combination with tocotrienols. Niosomes produced a significant ninefold enhancement in cytotoxicity of the combination, supported by significantly higher cellular uptake of GEM in the cells. Conclusion: This study is a proof of concept on the synthesis of dual-drug niosomes and their efficacy on pancreatic cancer cells in vitro.
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In this research, Graphene oxide (GO) used as a platform, is synthesized for the purpose of nanoparticles for loading of cyclophosphamide (CYP) and the identification of the structure of this new nano drug (CYP/GO) is made using several analytical devices such as XRD, FTIR, SEM, and UV. The morphology, functionalization, stability, loading and controlled release behaviors of cyclophosphamide on GO at different pH, temperatures, contact time and concentrations were investigated. The loading and controlled release of CYP indicated strong pH dependence which is implied hydrogen-bonding interaction between GO and CYP. The equilibrium adsorption data were analyzed by Langmuir and Freundlich models. The results showed that the adsorption behavior could be fitted better to the Freundlich model. Nearly 80 % of CYP was released in simulated gastric fluid, pH 1.2, in 4 h and 68 % in simulated intestinal fluid, pH 7.4, in 30 h.
The application of gene therapy in the field of molecular medicine is an extremely promising approach to curing distinct varieties of illnesses and disorders of the human race. Currently, challenges of the gene therapy are to find secure and effective vectors which might be capable of delivering genes to the specific cells and getting them to express inside the cells. Because of safety concerns, artificial delivery systems are desired in comparison to viral vectors for gene delivery so numerous attention has been centered on the development of the effective vectors. However, Researchers are confronted with numerous problems consisting of low gene transfer efficiency, cytotoxicity, and lack of cell-targeting capability for the usage of these synthetic vectors. Chitosan, which is the biodegradable and non-toxic cationic polysaccharide, is generally preferred to the other cationic polymers as a non-viral vector mainly due to its properties of chemical versatility, excellence in transcellular transport, effectiveness as a DNA-condensing agent, and efficient and permanent transfection. The objective of this chapter is to indicate the importance and give an overview of the applications of chitosan and its derivatives as novel non-viral vectors for gene delivery.KeywordsChitosanPolymerCarrierCationicGeneTransfectionLigand
Human epidermal growth factor receptor-2 (HER-2) positive breast cancer is highly invasive with poor clinical outcomes and high risk of recurrence. Here, trastuzumab functionalized pullulan-doxorubicin nanoparticles (Tz-P-Dox) were developed and characterized as a new anticancer formulation for active targeting HER-2 overexpression of breast cancer cells. The obtained nanoparticles had the hydrodynamic diameter of 66.7 ± 2.0 nm and PDI of 0.218 ± 0.012. And the in vitro results showed significant difference of cell uptake and cytotoxic effect between Tz-P-Dox and non-targeted P-Dox against HER-2 positive cell lines (BT474 and MCF-7 cells). However, the differences between Tz-P-Dox and P-Dox were not observed in HER-2 negative cell lines (MDA-MB-231 cells). These results suggested that trastuzumab functionalized nanoparticles had great potential to be considered as a candidate drug for HER-2 positive breast cancer treatment.
Cancer is a complex disease with second leading cause of mortality all over the world. According to WHO, cancer-related deaths are projected to increase to ∼13.1 million by 2030. Although clinical management of cancer is achieved to some extent due to better understanding of tumor biology and advancement in current therapies including surgery, radiotherapy, and most importantly, in chemotherapy, all these therapies are associated with major side effects. Further, cancer is having a unique tumor-microenvironment with different types of sub-population of cells, including cancer stem cells and tumor-associated macrophages, which are responsible for tumor development, metastasis, and their recurrence. These cells are resistant to chemotherapy and radiotherapy due to non-targetability and poor availability. Chitosan, an abundant natural polymer, in its nanoformulation as nanoparticles, has been extensively studied and employed for the delivery of anticancer drugs due to its biocompatibility, biodegradability, and tunable functional groups that can be modified with targeting agent specific to these cells. In this chapter, we provide the pharmaceutical advancement of chitosan nanoparticles toward delivery of anticancer drugs with their challenges and prospects.
Cancer is a class of disease in which cells multiply irregularly and form a mass. Cancer is the most common cause of death worldwide. Prior to surgery to remove cancer, conventional chemotherapy is typically administered. Chemotherapy is a systemic therapy that causes drug-induced toxic effects and reduces patient survival time. Therefore, targeted drug delivery can be used to increase the therapeutic index by protecting normal cells from damage and preventing drug resistance. Nanoplatforms can significantly improve drug pharmacokinetic parameters and ligands, thus improving the drug’s concentration at the cancerous site. This chapter explores the various approaches for targeted drug delivery such as ligand-mediated targeting (antibodies, peptides, lectins, folates, and aptamers), physical targeting via enhanced permeation retention, and triggered targeting through external stimuli such as ultrasound, light, and magnets. The chapter will help researchers to discover new options for targeted drug delivery to overcome the challenges presented by convention systems.
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Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100–300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: (i) spatially, by growing tumors in subcutaneous or cranial locations and (ii) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 μm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: (i) delivery may be less efficient in cranial tumors than in subcutaneous tumors, (ii) delivery may be reduced during tumor regression induced by hormonal ablation, and (iii) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.
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I.F. (2000) Niosomes and polymeric chitosan based vesicles bearing transferrin and glucose ligands for drug targeting. Pharmaceutical Research, 17 (10). pp. 1250-1258. ISSN 0724-8741 Strathprints is designed to allow users to access the research output of the University of Strathclyde. Purpose. To prepare polymeric vesicles and niosomes bearing glucose or transferrin ligands for drug targeting. Methods. A glucose-palmitoyl glycol chitosan (PGC) conjugate was synthesised and glucose-PGC polymeric vesicles prepared by sonica-tion of glucose-PGC/ cholesterol. N-palmitoylglucosamine (NPG) was synthesised and NPG niosomes also prepared by sonication of NPG/ sorbitan monostearate/ cholesterol/ cholesteryl poly-24-oxyethylene ether. These 2 glucose vesicles were incubated with col-loidal concanavalin A gold (Con-A gold), washed and visualised by transmission electron microscopy (TEM). Transferrin was also con-jugated to the surface of PGC vesicles and the uptake of these vesicles investigated in the A431 cell line (over expressing the trans-ferrin receptor) by fluorescent activated cell sorter analysis. Results. TEM imaging confirmed the presence of glucose units on the surface of PGC polymeric vesicles and NPG niosomes. Transferrin was coupled to PGC vesicles at a level of 0.60 ± 0.18 g of transferrin per g polymer. The proportion of FITC-dextran positive A431 cells was 42% (FITC-dextran solution), 74% (plain vesicles) and 90% (transferrin vesicles). Conclusions. Glucose and transferrin bearing chitosan based vesicles and glucose niosomes have been prepared. Glucose bearing vesicles bind Con-A to their surface. Chitosan based vesicles are taken up by A431 cells and transferrin enhances this uptake.
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BACKGROUND Cancer cell growth is an energy-related process supported by an increased glucose metabolism. The objective of this study was to investigate the association of GLUT-1 with response to chemotherapy and outcome in patients with ovarian carcinoma.METHODS Histologic sections of formalin fixed, paraffin embedded specimens from 113 primary ovarian carcinomas were stained for GLUT-1 by using polyclonal GLUT-1 antibody (Dako Co., Carpinteria, CA) and the labeled streptavidin biotin procedure. Intensity of GLUT-1 staining was compared with disease free survival (DFS), chemotherapy response, and other clinicopathologic characteristics.RESULTSGLUT-1 cytoplasmic membrane staining was observed in 89 of 104 (85.6%) malignant tumors. Poorly differentiated tumors showed a trend to overexpress the GLUT-1 protein compared with the more differentiated counterparts (27.6% vs. 8.7%; P = 0.08). Patients who experienced a complete clinical response to chemotherapy were more frequently GLUT-1 positive than GLUT-1 negative (80% vs. 51.5%; P = 0.036). In multivariate analysis of advanced stage disease, residual tumor (P = 0.0001) and high GLUT-1 expression levels (P = 0.028) were the only independent variables that maintained a significant association with response to chemotherapy (P = 0.0001; chi-square = 38.13). In the subgroup of Stage III–IV (International Federation of Gynecology and Obstetrics patients showing a complete clinical response, GLUT-1 overexpression was associated with a shorter DFS. The median time to progression was 30 months in GLUT-1 strongly positive cases (> 50% of cancer cells positive) versus 60 months in GLUT-1 weakly positive cases (≤ 50% of cancer cells positive; P = 0.024).CONCLUSIONSGLUT-1 status is an independent prognostic factor of response to chemotherapy in advanced stage ovarian carcinoma. Moreover, patients overexpressing GLUT-1 show a significantly shorter DFS. These results suggest that the assessment of GLUT-1 status may provide clinically useful prognostic information in patients with ovarian carcinoma. Cancer 2001;92:1144–50. © 2001 American Cancer Society.
Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross-linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.
For the first time, to our knowledge, it has been demonstrated that polymeric vesicle size may be controlled by controls on polymer molecular weight. A direct relationship exists between the square root of the palmitoyl glycol chitosan molecular weight and sonicated polymeric vesicle z-average mean diameter (r = 0.95). Glycol chitosan samples of varying molecular weight were prepared by hydrolysis with 4 M hydrochloric acid and palmitoyl glycol chitosan samples of varying molecular weight synthesized by reacting glycol chitosan with palmitic acid N-hydroxysuccinimide ester. Polymer characterization was carried out by gel permeation chromatography/laser light scattering and 1H NMR. Vesicles produced from the various palmitoyl glycol chitosan samples by probe sonication in the presence of cholesterol were sized and imaged by transmission electron microscopy. Palmitoyl glycol chitosan samples of MW 276 000, 134 000, 89 000, 28 000, and 31 000 produced unilamellar polymeric vesicles with a z-average mean diameter of 481, 429, 384, 221, and 206 nm, respectively. In addition palmitoyl glycol chitosan vesicles could be prepared from the low molecular weight polymer (MW 28 000) alone in the absence of cholesterol.
Tumor vasculature was the target for tumor selective drug delivery using biocompatible macromolecular drugs or lipid formulations. These drugs selectively accumulated and were retained in tumor tissue, and for much longer periods than in normal tissue with little drainage via lymphatic clearance. Experimental evidence in support of the enhanced permeability and retention (EPR) effect of macromolecules and lipids is described as general phenomenon in solid tumors. Clinical results with such tactics using macromolecular anticancer agent SMANCS dissolved in Lipiodol®, a lipid contrast medium, are described where the drug in lipid formulation was administered via the hepatic artery using a catheter. This technique permits a drug concentration ratio of greater than 2500 in tumor vs blood plasma; more remarkable targeting than with other methods and very slow release of the drug was also accomplished resulting in unprecedented clinical results in patients with hepatocellular carcinoma.
The development of an effective anti-cancer liposomal formulation — doxorubicin in sterically stabilized liposomes — will be discussed. We shall argue that for many tumors the necessary condition for an effective anti-cancer activity of systemically administered liposomal doxorubicin formulation is the long circulation life of liposomes in blood and stable drug encapsulation. Theoretical basis for stabilization of liposomes in biological environments and for the stabilization of drug encapsulation will be shown. When a formulation with acceptable stability was obtained it was tested in pre-clinical models and simultaneously scaled-up and it entered into clinical studies. After successfully passing all these tests, doxorubicin in sterically stabilized liposomes (Doxil™ by Sequus Pharmaceuticals, Inc., Menlo Park, CA) was approved by Food and Drug Administration and is commercially available since late 1995.
Aggressive prostatic carcinomas most frequently metastasize to the skeletal system. We have previously shown that cultured human prostatic carcinoma cells are highly responsive to growth factors found in human bone marrow. To identify the factor(s) responsible for the increased prostatic carcinoma cell proliferation, we fractionated crude bone marrow preparations by using hydroxylapatite HPLC. The major activity peak contained two high molecular weight bands (M(r) = 80,000 and 69,000) that cross-reacted with antibodies to human transferrin and serum albumin, respectively. Bone marrow transferrin, purified to apparent homogeneity by using DEAE-Affi-Gel Blue chromatography, anti-transferrin affinity chromatography, and hydroxylapatite HPLC, markedly stimulated prostatic carcinoma cell proliferation, whereas human serum albumin showed no significant growth factor activity. Marrow preparations, depleted of transferrin by passage over an anti-transferrin affinity column, lost greater than 90% of their proliferative activity. In contrast to the response observed with the prostatic carcinoma cell lines, a variety of human malignant cell lines, derived from other primary sites and metastatic to sites other than bone marrow, showed a reduced response to purified marrow-derived transferrin. These results suggest that rapid growth of human prostatic carcinoma metastases in spinal bone may result from a combination of conditions that include (i) drainage of prostatic carcinoma cells into the paravertebral circulation, (ii) high concentrations of available transferrin in bone marrow, and (iii) increased sensitivity of prostatic carcinoma cells to the mitogenic activity of transferrin.
The tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cells, and this reaction is used as the end point in a rapid drug-screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantitative relationship is established between cell number and MTT-formazan production. We have shown that reduction of MTT to MTT-formazan by cells is dependent on the amount of MTT in the incubation medium. The concentration required to give maximal MTT-formazan production differs widely between cell lines. The absorption spectrum of MTT-formazan varies with cell number and with pH. At a low cell density or a high pH, the absorption maximum is at a wavelength of 560 to 570 nm. However, at a high cell density or a low pH, there are two absorption maxima; one at 510 nm and a second at about 570 nm. Measurements of absorbance at 570 nm underestimate MTT-formazan production and, hence, cell number at high cell densities. This error can result in a 10-fold underestimation of chemosensitivity. Addition of a buffer at pH 10.5 to the solubilized MTT-formazan product can overcome the effects of both cell density and culture medium on the absorption spectrum. Provided that sufficient MTT is used and the pH of the MTT-formazan product is controlled, dye reduction can be used to estimate cell numbers in a simple chemosensitivity assay the results of which agree well with a commonly used clonogenic assay.