International Journal of Pharmaceutics 307 (2006) 194–200
Hydroxyethylstarch microcapsules: A preliminary study
for tumor immunotherapy application
J. Devy a,b , E. Balasse b , H. Kaplan b , C. Madoulet b , M.-C. Andry a,∗
a
b
Laboratoire de Pharmacotechnie, FRE CNRS 2715, IFR53, Faculté de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France
Laboratoire de Biochimie et Biologie Moléculaire, EA 3796-IPCM, IFR53 Faculté de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France
Received 11 September 2005; accepted 30 September 2005
Available online 28 November 2005
Abstract
The objective of this work was to prepare microcapsules which would allow protection and slow release of antigens used for melanoma
immunotherapy treatment. Hydroxyethylstarch (HES) microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride
(TC). They were characterized with respect to morphology (microscopy) and size (in the 4–15 m range). Bovine serum albumin (BSA) was
used as model protein for loading and release studies. Microcapsules were loaded with solutions at different protein concentrations (0.5–5%). The
maximum loading efficiency (20%) was observed with the concentration of 2.5%, which allowed a loading capacity near 100%. Confocal laser
scanning microscopy (CLSM) visualization showed that BSA was entrapped within the microcapsules and not only associated to their outer surface.
BSA-release studies showed a 20% BSA release within 30 min while 80% remained entrapped in the microcapsules for 4 days. Microcapsules
were degraded by ␣-amylase and addition of esterase to ␣-amylase enhanced slightly their degradation. In vitro studies on melanoma cells showed
that HES microcapsules were non-toxic. Preliminary in vivo studies demonstrated that microcapsules were biodegradable after intraperitoneal
injection (i.p.). The observation of peritoneal wash showed a complete degradation within 7 days, indicating a possible application as an in vivo
drug delivery system especially to enhance the presentation of antigens.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Hydroxyethylstarch; Biodegradable microcapsules; Confocal microscopy; Loading efficiency; Cytotoxicity; Enzymatic degradation
1. Introduction
The relative resistance of melanoma cells to chemotherapy has led to search for alternative treatment options, including immunotherapy (Komenaka et al., 2004). This technique
involves the treatment of cancer through manipulation of the
immune system (Pardoll, 1998; Nawrocki and Mackiewicz,
1999; Vasey, 2000; Hoffman et al., 2000; Jager et al., 2001).
Such an approach needs an appropriate presentation of tumorspecific antigens (Thumann et al., 2003). The use of biodegradable, polymeric systems for tumor immunotherapy has received
limited consideration as compared to more conventional cellbased approaches (Golumbek et al., 1993; Egilmez et al., 2000;
Kuriakose et al., 2000; Denis-Mize et al., 2000). Microparticulate antigen delivery systems are of special interest as stable
∗
Corresponding author. Tel.: +33 3 26 91 37 22; fax: +33 3 26 91 37 44.
E-mail address: mc.andry@univ-reims.fr (M.-C. Andry).
0378-5173/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2005.09.035
carriers and effective adjuvants for the delivery of vaccines (Thomasin et al., 1996; Raychaudhuri and Rock, 1998;
Strindelius et al., 2002). Attraction is based on different advantages, such as the enhancement of drug stability and the controlled drug release (Pappo et al., 1991; Eldridge et al., 1991;
Degling and Stjärnkvist, 1995; Men et al., 1996; Putney and
Burke, 1998). We focused our attention on the development
of such devices to enhance the presentation of antigens in
immunotherapy. Starch-based microparticles are suitable carriers for protein delivery systems due to their biocompatibility,
shelf-life stability, high loading capacity, biodegradability, and
controlled release of the encapsulated drug (Laakso et al., 1987;
Heritage et al., 1996; Larionova et al., 1999; Sturesson and
Wikingsson, 2000). Native starch may not be appropriate to
prepare parenteral controlled drug delivery systems, since it
is rapidly degraded in vivo and many drugs are released too
quickly from such unmodified starch systems (Henrist et al.,
1999; Pereswetoff-Morath, 1998; Michailova et al., 2001). In
contrast, hydroxyethylstarch (HES) is a less quickly degraded
J. Devy et al. / International Journal of Pharmaceutics 307 (2006) 194–200
starch derivative, widely used for therapy and prophylaxis of
all kinds of volume deficiencies. Because of its major attractions including biocompatibility and biodegradation properties,
HES was chosen for the production of microcapsules intended
to further vectorization in vivo. Moreover, we have previously
shown that HES leads to stable microparticles by interfacial
cross-linking (Levy and Andry, 1990). These HES microparticles had never been used for the encapsulation of large molecules
like proteins and they had never been tested in vivo. So different
studies were realised to characterize these particles. The morphology and the size of HES microparticles were evaluated using
light microscopy, confocal laser scanning microscopy (CLSM),
scanning electron microscopy (SEM) and laser diffraction granulometry. Loading and release characteristics were investigated
with a model protein: bovine serum albumin (BSA). Degradation was evaluated in vitro in presence of amylase and esterase.
Furthermore, cytotoxicity studies on melanoma cell line, and in
vivo biodegradation were evaluated.
2. Materials and methods
2.1. Materials
Hydroxyethylstarch (Voluven® ) was purchased from Fresenius Kabi (Sèvres, France) and terephthaloyl chloride from
Acros Organics. Esterase (19 IU/mg) from porcine liver, ␣amylase (19.5 IU/mg) from porcine pancreas and bovine serum
albumin were from Sigma (St. Quentin Fallavier, France). Chloroform, cyclohexane and ethanol, were of analytical grade and
provided by SDS (Peypin, France). Texas red labeled BSA
was purchased from Invitrogen (Cergy–Pontoise, France). Surfactants were polysorbate (Tween® 20) and sorbitan trioleate
(Span® 85) from Seppic (Paris, France). Methylene Blue and fluoresceinamine were from Fluka (St. Quentin Fallavier, France).
2.2. Preparation of HES microparticles
Microcapsules were prepared by the interfacial cross-linking
method according to our protocol (Levy and Andry, 1990).
Briefly, a 20% (w/v) HES solution was prepared in carbonate
buffer pH 9.8. This aqueous phase (6 ml) was emulsified under
mechanical agitation (5000 rpm) in cyclohexane (30 ml) containing 5% (v/v) Span® 85. After 5 min, 40 ml of a 5% (w/v)
solution of terephthaloyl chloride in chloroform/cyclohexane
(1/4, v/v) were added to the emulsion and stirring was prolonged
for 30 min. The reaction was stopped by dilution with 40 ml of
chloroform/cyclohexane (1/4, v/v). Then, microparticles were
washed with cyclohexane (4×), with ethanol 95% (v/v) containing 2% (v/v) Tween® 20 (1×), then with ethanol 95%(v/v) (2×)
and with water (4×). Finally, microcapsules were re-suspended
in water and lyophilized.
2.3. Microparticles characterization
2.3.1. Morphology and size
Light microscopy (Olympus BH-2, Olympus, Shibuya-Ku,
Tokyo, Japan) was performed for initial visualization of the
195
HES microcapsules. Further morphology studies were carried
out by means of confocal laser scanning microscopy (CLSM)
and scanning electron microscopy (SEM). For CLSM study, fluorescent microparticles were prepared by incorporating 30 mg of
fluoresceinamine in the HES solution before the emulsification
step. A Bio-Rad MRC 1024 system (Bio-Rad, Hercules, CA,
USA) mounted on Olympus IX70 Axioplan optical microscope
(Olympus, Shibuya-Ku, Tokyo, Japan) was used. All acquisitions were made using UPlan FI×63, 1.4 numerical aperture
objective. Acquisitions were performed by exciting fluoresceinamine with the 488-nm line of an air-cooled argon ion laser.
Eighty sections per microparticle were recorded with a 0.30-m
z-step to collect the whole volume with a sufficient z-sampling.
Files were then transferred to a Sun Sparc 20 workstation (Sun
Microsystems, Mountain View, CA, USA) for further processing.
For SEM, lyophilized microparticles were deposited on
double-faced adhesive and coated with palladium/gold before
observation. Samples were observed under a scanning electron
microscope (JEOL 5400 LV) (JEOL, Schiphol, The Netherlands) at 15 kV to study the shape and surface morphology.
The samples for Fourrier transform-infrared (FT-IR) study
were prepared according to the standard technique: 1 mg of
lyophilized microcapsules or HES was ground with 190 mg
of kBr. The mixture was compressed in tablets, 1 mm thick,
under a pressure of 10 kPa. FT-IR spectra were obtained from
a Perkin-Elmer Spectrum BXII spectrometer (Perkin-Elmer,
Courtaboeuf, France).
Particles were sized by a laser diffraction technique (Coulter
Particle Sizer, type LS 200, Coultronics, France). Size distribution was displayed in terms of volume versus particle
size.
2.3.2. Enzymatic degradation of HES microparticles
Lyophilized microcapsules (10 mg) were suspended in 5 ml
phosphate buffer (20 mM sodium phosphate, 6 mM NaCl, pH 7)
containing either ␣-amylase (19.5 IU/ml), esterase (19 IU/ml) or
a mixture of both enzymes. The samples were incubated under
shaking at 37 ◦ C. The microparticle morphology was observed
by light microscopy and the concentration was evaluated on
Malassez cell in aliquots withdrawn at appropriate time intervals.
2.3.3. Bovine serum albumin loading
The BSA loading of microparticles was obtained by incubating 5 mg of HES microparticles in 1 ml of a 0.5–5% (w/v)
BSA solution in phosphate buffer saline (PBS) pH 7.4. Tubes
were incubated 3 h under shaking at 37 ◦ C. Then, they were centrifuged (1400 rpm for 30 min) to remove the unloaded BSA.
The unloaded BSA in the supernatant was quantified with
the Bradford protein assay method (Bradford, 1976). Loading efficiency (LE) was determined as: LE = [(total amount of
BSA)−(unloaded BSA)]/total amount of BSA (Van der Lubben
et al., 2001).
CLSM was used to demonstrate effective BSA loading. Microparticles were incubated with a 2.5% Texas red
labeled-BSA solution (Molecular Probes, Invitrogen) as afore-
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J. Devy et al. / International Journal of Pharmaceutics 307 (2006) 194–200
mentioned. Acquisitions were performed by exciting with the
568 nm line of an air-cooled krypton/argon ion laser.
2.3.4. Bovine serum albumin release
For BSA release, 5 mg of microparticles were loaded as previously described. After centrifugation, loaded microcapsules
were re-suspended in PBS pH 7.4 to make a 1% (w/v) microparticles suspension. Samples were incubated under gentle shaking
at 37 ◦ C during 30, 60, 90, 120, 180 and 240 min, 24 and 48 h
and 4 days (one tube for each time). The tubes were given
a spin-off and unbound BSA present in the supernatant was
determined with the Bradford protein assay method (Bradford,
1976).
2.4. Cell and culture conditions
A murine melanoma cell subline, denoted B16-R, resistant
to 3.5 × 10−7 M doxorubicin, derived from the ATCC stock was
isolated at the National Tumor Institute in Milan (Mariani and
Supino, 1990) by stepwise selection in increasing concentrations of doxorubicin. B16-R cells were grown in a 5% CO2
atmosphere at 37 ◦ C, in RPMI 1640 medium supplemented with
10% heat-inactivated foetal bovine serum (Invitrogen). To generate spheroids, exponentially monolayer growing B16-R cells
were detached by trypsinization and 100 l culture medium
containing 50 × 103 cells were added to each well of a 96well microplate, previously coated with 40 l 1.33% agarose
(Sigma). Microplates were placed on a 3-D stirring machine
(Polymax 1040, Heidolph, Germany) for overnight incubation.
After 3 days at 37 ◦ C, 5% CO2 , the medium was changed and
the cytotoxicity assays could be initiated.
was administered by intraperitoneal injection (i.p.) on a group
of 21 female B6D2F1 mice. Daily, three mice were sacrificed,
the intraperitoneal cavity was washed twice with 10 ml of physiological saline and the microparticle degradation was assessed
by light microscopy.
3. Results and discussion
3.1. Morphology and size of microparticles
The morphology of the microparticles prepared from a 20%
(w/v) HES solution with 5% (w/v) terephthaloyl chloride concentration was analyzed by light microscopy after suspension in
water and staining with Methylene Blue. They appeared transparent, spherical and well individualised (Fig. 1a). The size of
these HES microparticles was found to range from 4 to 15 m
with an average of 8.3 ± 1.9 m. Microcapsule lyophilization
gave white and free-flowing powders. Moreower, the particles
were intact and easily recovered their spherical shape after rehydratation in aqueous solutions. These particles exhibited a
continuous and smooth surface, as shown by scanning electron microscopy (Fig. 1b). Three-dimensional reconstruction
of the microparticles, using CLSM, allowed us to visualize a
cross-section and perfectly illustrated that particles were hollow
spheres with a concave membrane (Fig. 2a).
2.5. Microparticle cytotoxicity
A MTT (Sigma) colorimetric assay was performed. Cell
viability was determined by measuring the optical density differences between 550 and 650 nm using the 550 microplate reader
model (Bio-rad, Marnes la Coquette, France). The cell surviving fraction was determined by dividing the mean absorbance
values of treated samples by the mean absorbance of untreated
control samples.
2.6. Animals
Female B6D2F1 mice (6-week-old) were obtained from
Charles River Laboratories (Iffa Credo, L’Arbresle, France) and
housed at the maintenance facility of the School of Pharmacy
of Reims. All experiments were carried out in compliance with
the regulations of the Animal Care and Use at the School of
Pharmacy of Reims.
2.7. In vivo biodegradation studies
Blank lyophilized microparticles (5 mg) were re-suspended
in 200 l of sterile physiological saline (a 0.9% NaCl (w/v) aqueous solution) and incubated at 37 ◦ C with permanent shaking
until adequate re-swelling. Then the microparticle suspension
Fig. 1. HES microparticles: (a) optical photomicrograph (scale bar = 10 m)
and (b) SEM (scale bar = 10 m).
J. Devy et al. / International Journal of Pharmaceutics 307 (2006) 194–200
197
progressive degradation activity: 10% of microcapsules were
degraded after 2 h and 50% after 24 h. Addition of esterase
(19 IU/ml) to ␣-amylase enhanced slightly the rate of microcapsules degradation; after incubation times of 6 and 24 h, the
degradation of microcapsules (%) were, respectively, 40% versus 50% and 50% versus 70% with amylase alone versus both
enzymes (Table 1). The same observation was performed for
microcapsules prepared with soluble starch (Larionova et al.,
1999). This phenomenon is to link with the esterase activity
which is only possible after partial degradation of HES by amylase.
3.3. Bovine serum albumin loading efficiencies of HES
microparticles
Fig. 2. Three-dimension reconstruction of the maximum projection from zseries (n = 80) of confocal fluorescent image of optical section of single microcapsule: (a) labeled with fluoresceinamin; scale bar = 10 m and (b) loaded with
BSA-Texas red (2.5%); scale bar = 5 m (all sections were recorded using magnification ×63 and zoom 3×).
The IR spectra of the microcapsules were compared with the
spectrum of original HES in Fig. 3. As it was expected with
our previous studies (Andry et al., 1998), the main modifications on microcapsules spectra were two bands at 1717 and
1277 cm−1 , which reflected the formation of esters bonds from
hydroxy groups of HES.
3.2. In vitro degradation of HES microparticles
Degradation by various enzyme solutions was studied:
esterase, ␣-amylase or both enzymes. Esterase usually allows
the disruption of ester bonds involved in the microparticles wall
formation and ␣-amylase is the enzyme which degrades starch
and derivatives like HES. The enzyme concentration used in
these assays were the same that allowed the degradation of
particles prepared by cross-linking of soluble starch with terephthaloyl chloride (Larionova et al., 1999). We observed that the
microcapsules were resistant to digestion by the esterase solution (19 IU/ml). However, ␣-amylase (19.5 IU/ml) exhibited a
Five milligram of HES microcapsules were suspended in 1 ml
of BSA solution in PBS pH 7.4 (5 mg will be the quantity of
microcapsules used for further in vivo tests). Loading efficiency
was determined with 0.5–5% (w/v) BSA solutions. Fig. 4 shows
that the LE presented a maximum value for the concentration of
2.5% (w/v). The obtained loading efficiency was 19.64 ± 0.48%
(n = 5) for independently prepared batches. Therefore, 2.5%
(w/v) BSA in the loading solution was selected as the optimal concentration. Under these conditions, the loading capacity value (LC = [loaded BSA/weight microcapsules] × 100) was
98.2 ± 2.4%. With CLSM studies, it was possible to visualize
both the inside and the surface of the Texas red labeled particles.
Results obtained with this technique showed that BSA was not
only associated to the surface, but also entered within the HES
microcapsules (Fig. 2b). The repartition of BSA was homogeneous in the microcapsule and did not present aggregates. These
particles, with a thin wall allowed the encapsulation of an important amount of protein in their cavity. While microspheres in the
same range of size allow a smaller albumin loading, near 40%
(Van der Lubben et al., 2001). This high loading capacity is a
good point for further uses since large amount of protein was
encapsulated in a minimal amount of polymer. This reduces the
mass of the material to be administered (Sinha and Thehan,
2003). Moreover, HES microcapsules loading does not require
a coupling reaction as it has to be done with polyacryl starch
microparticles (Degling and Stjärnkvist, 1995).
3.4. BSA release from HES microparticles
BSA release from HES microparticles re-suspended in PBS
pH 7.4 was determined after 30, 60, 90, 120, 180 and 240 min,
24 and 48 h and 4 days. After an initial release of 20% during the
first 30 min, no BSA was released for the following 4 days. These
results indicated that 80% of the BSA remained entrapped in the
microcapsules under these conditions. These results are to be
correlated with those obtained by Van der Lubben et al. (2001);
chitosan microparticles (in the same range of size) loaded with
ovalbumin allowed a release of about 10% of the loaded protein. So, an important part of albumin will only be released after
complete degradation of the HES microcapsules since they are
degradable. The protein is likely to be protected within the particle and this would allow a progressive release in vivo.
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J. Devy et al. / International Journal of Pharmaceutics 307 (2006) 194–200
Fig. 3. FT-IR spectra: (A) original HES and (B) hydroxyethylstarch microcapsules.
Table 1
Degradation of microcapsules in presence or absence of enzymes as a function of enzymatic incubation time; percentage of degraded microcapsules counted on a
Malassez cell
Incubation time (h)
Without enzymes (%)
Amylase (19.5 IU/ml) (%)
Esterase (19 IU/ml) (%)
Amylase + Esterase
(19.5 IU/ml + 19 IU/ml) (%)
2
4
6
24
–
–
–
–
10
35
40
50
–
–
–
–
10
35
50
70
±
±
±
±
2
3
4
5
±
±
±
±
2
3
4
5
Results are the mean of three determinations ± S.D.
3.5. In vitro studies of HES microcapsules cytotoxicity on
B16-R melanoma cells
range of 0.1–1 mg/ml, did not exhibit a significant cytotoxicity
against this cell line after 72 h (all values <10%, n = 9).
The microcapsules cytotoxicity was tested on the B16-R
melanoma cell line, resistant to doxorubicin, cultivated in monolayers or as tri-dimensional models. One milliliter of microcapsule suspension was added in each well of a 96-well microplate,
each filled with 100 l of suspension containing 50 × 103 cells.
Results (Table 2) show that microcapsules, in the concentration
3.6. In vivo biodegradation studies
Administration by intraperitoneal route was used because in
our future experiments, microcapsules loaded with antigens will
be injected by this route since it was shown that it is a particularly
effective route for stimulating immune responses (Degling and
Stjärnkvist, 1995; Cleland, 1999).
In a preliminary assay, HES microcapsules (5 mg) were resuspended in 200 l of sterile physiological saline and injected
intraperitoneally in two groups of three female B6D2F1 mice.
Table 2
Growth inhibition of B16-R melanoma cells cultured in monolayers or in
spheroids (50 × 103 cells/100 l), incubated with different concentrations of
microparticles (mg/ml) after a 72 h incubation time
Microcapsules (mg/ml)
Fig. 4. The influence of BSA initial concentration on loading efficiency of HES
microcapsules. Data are expressed as mean ± S.D. of five experiments.
Growth inhibition (%)
B16-R monolayers
B16-R spheroids
0.1
0.2
0.3
0.4
0.8
1
0.5 ± 1
0
1±1
0
3±1
3±1
5±1
5±1
7±1
6±1
8±1
7±1
Results are given as means ± S.D. (n = 9).
J. Devy et al. / International Journal of Pharmaceutics 307 (2006) 194–200
199
Fig. 5. Optical photomicrographs of intraperitoneal cavity washings after injection of HES microcapsules in female B6D2F1 mice: day 1 (A), day 3 (B), day 5 (C)
and day 7 after injection (D); magnification ×40.
Animals were followed for 3 weeks and no inflammatory signs
could be observed. The microcapsules seemed subsequently
well-tolerated.
Then, the in vivo degradation study was realized: microparticles were injected in i.p. to 21 mice, and each day one group of
three mice was sacrificed to evaluate microparticles degradation
by light microscopy (Fig. 5). The day after administration, the
microparticles stained with Methylene Blue presented a lesser
smooth surface (Fig. 5A). Three days later, the microparticles
were cracked (Fig. 5B) and at day 5, only some small fragments
could be visualized (Fig. 5C). Seven days after the injection,
nothing remained observable, which meant that the totality of
microcapsules was degraded within a week (Fig. 5D). After
i.p. administration, a depot of microcapsules was formed at the
injection site which could increase the immune response comparable with the effect seen with other depot adjuvants (Sinha and
Thehan, 2003). This interesting result showed that HES microcapsules prepared by interfacial cross-linking could be used to
allow a slow release of loaded protein (e.g. antigens); most of the
associated protein would only be released after biodegradation
of the HES microcapsules.
4. Conclusion
The aim of this work was to study HES microparticles for
future in vivo applications. This study demonstrated that these
microcapsules showed an important loading capacity for the
model protein BSA. CLSM analyses fully demonstrated that
the loaded BSA was present at the surface as well as entrapped
inside the microparticles. Furthermore, a significant part of BSA
(80%) was left to be released after complete degradation of the
microparticles. From BSA loading experiments and the in vivo
degradation profile of HES microparticles, we could deduce that
high amounts of the model protein are expected to be released
with a suitable controlled release profile. Moreover, both the
absence of cytotoxicity and the observed microparticles tolerance reinforce the suitable in vitro and in vivo characteristics for
drug delivery especially to enhance the presentation of antigens.
The HES microparticles are currently under further investigations in order to increase immune response against melanoma
resistant cancer cells after loading with soluble melanoma proteins.
Acknowledgements
We would like to thank Dr. Daniel Royer of the FRE/CNRS
2715 for his help during FT-IR experiments and V.G. Roullin of
the FRE/CNRS 2715 for stimulating discussions. The “Association de la Recherche contre le Cancer” is thanked for financial
support.
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