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http://informahealthcare.com/drt
ISSN: 1061-186X (print), 1029-2330 (electronic)
J Drug Target, Early Online: 1–8
!2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.878941
ORIGINAL ARTICLE
Magnetic Catechin–Dextran conjugate as targeted therapeutic for
pancreatic tumour cells
Orazio Vittorio
1,2
, Valerio Voliani
3,4
, Paolo Faraci
3
, Biswajit Karmakar
3,5
, Francesca Iemma
6
, Silke Hampel
7
,
Maria Kavallaris
1,2
, and Giuseppe Cirillo
6,7
1
Children’s Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, New South Wales, Australia,
2
Australian Centre
for Nano Medicine, University of New South Wales, New South Wales, Australia,
3
NEST Scuola Normale Superiore and Istituto Nanoscienze-CNR,
Piazza San Silvestro, Pisa, Italy,
4
Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy,
5
Saha Institute of Nuclear
Physics, Kolkata, India,
6
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende (CS), Italy, and
7
Leibniz Institute for Solid State and Materials Research Dresden, Dresden, Germany
Abstract
Background: Catechin–dextran conjugates have recently attracted a lot of attention due to their
anticancer activity against a range of cancer cells. Magnetic nanoparticles have the ability to
concentrate therapeutically important drugs due to their magnetic-spatial control and provide
opportunities for targeted drug delivery.
Purpose: Enhancement of the anticancer efficiency of catechin–dextran conjugate by
functionalisation with magnetic iron oxide nanoparticles.
Methods: Modification of the coating shell of commercial magnetic nanoparticles (Endorem)
composed of dextran with the catechin–dextran conjugate.
Results: Catechin–dextran conjugated with Endorem (Endo–Cat) increased the intracellular
concentration of the drug and it induced apoptosis in 98% of pancreatic tumour cells placed
under magnetic field.
Discussion: The conjugation of catechin–dextran with Endorem enhances the anticancer activity
of this drug and provides a new strategy for targeted drug delivery on tumour cells driven by
magnetic field.
Conclusion: The ability to spatially control the delivery of the catechin–dextran by magnetic
field makes it a promising agent for further application in cancer therapy.
Keywords
Anticancer activity, catechin, dextran,
magnetic nanoparticles, polymer
therapeutics
History
Received 30 August 2013
Revised 18 December 2013
Accepted 22 December 2013
Published online 16 January 2014
Introduction
Many existing drugs stem from plants and are used in
traditional medicine, and among them polyphenols are
currently used as dietary supplements because of their
known preventive properties against ageing, neurodegenera-
tion and cardiovascular risk [1,2]. Interestingly, several
different reports highlight the involvement of polyphenols in
cancer prevention and treatment [3–5]. Although the specific
mechanism responsible for the anticancer activity remains
unclear, several different tumour models have been explored.
It was found that the biological activity of this class of
compounds (e.g. antioxidative, anti-inflammatory, etc.)
contributes to their cancer chemopreventive potential [6].
More specifically, polyphenols have the ability to abrogate
various biochemical processes induced or mediated by the
tumour promoters, to induce apoptosis in premalignant or
cancerous cells, and to suppress the growth and proliferation
of various types of tumour cells via induction of apoptosis or
arrest of a specific phase of the cell cycle [7–9]. Among the
different polyphenols, green tea flavonoids and catechins in
particular, have shown potential preventive activity for oral
leukoplakia, prostate cancer and colorectal cancer [10–12].
However, polyphenols are generally not yet used in clinical
practise due to their pharmacokinetic concerns [13]. To
overcome this limitation, naturally occurring polyphenols
have been coupled to protein and/or polysaccharide materials
with the aim to improve both the pharmacokinetic and
pharmacodynamic profiles [14]. Recently, this specific class
of conjugates has been shown to be effective anticancer
therapeutics in HeLa, prostate, renal and pancreatic cancer
cells [15,16]. In particular, a flavonoid–polysaccharide con-
jugate based on the flavonoid catechin and the polysaccharide
dextran (Cat–Dex) was found to be highly effective in the
treatment against the pancreatic ductal adenocarcinoma cells
Address for correspondence: Orazio Vittorio, Children’s Cancer Institute
Australia for Medical Research, Lowy Cancer Research Centre,
University of New South Wales, PO Box 81 Randwick, New South
Wales 2031, Australia. Tel: +6102 9385 2572. E-mail:
OVittorio@ccia.unsw.edu.au
Giuseppe Cirillo, Department of Pharmacy, Health and Nutritional
Sciences, University of Calabria, I-87036 Arcavacata di Rende (CS),
Italy. Tel/Fax +39 0984 493011. E-mail: giuseppe.cirillo@unical.it
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[17]. In vitro Cat–Dex induced significant cytotoxicity
(apoptosis) against MIA PaCa-2 and PL45 cancer cells
lines, with low side effects against non-malignant cells.
Moreover, one of the most important factors for the failure of
cancer chemotherapy in the clinic is the lack of tumour
specificity of the therapeutics [18]. Thus, one of the main
challenges in cancer therapy is the development of new
strategies to eliminate, or at least significantly reduce, the
side-effects of chemotherapeutics on healthy tissues and cells;
thus several strategies have been developed in order to
enhance the therapeutic efficacy of drugs against tumours that
in turn, have low systemic side effects in non-cancerous
tissues [19,20]. The recent development of innovative chem-
ical strategies in the synthesis of polymers and nano-materials
has been exploited to modify chemotherapy agents for
their targeted delivery in cancer cells [21–24]. Using these
approaches different features can be achieved, including
increased drug concentration to the target cells, the reduction
of early clearance from blood, the minimisation of
non-specific cell interactions and thus of the side effects,
the enhancement of internalisation efficiency within
target cells and thus the reduction of the total dose required
[25–27].
Among the different strategies employed for passive
targeting, the preparation of magnetic responsive materials
is one of the most widely explored. The concept of using
magnetism in medicine and biology was firstly introduced by
Freeman et al., in 1960 [28], and since then, much effort has
been applied in this area, leading to the design of various
magnetic particles and vectors [29] for applications such as
targeted drug delivery, magnetic thermotherapy (hyperther-
mia), magnetic resonance imaging (MRI), and separation
of proteins and cells [30,31]. In particular, superparamagnetic
magnetite (Fe
3
O
4
) has attracted attention as a responsive
element for the preparation of drug delivery vehicles, thanks
to its retention of magnetic properties after modification
with polymers or chemical reactions, and the high stability
at physiological conditions [32–34]. To increase the bio-
compatibility of these nanostructures with cells and tissues,
they are generally coated with hydrophilic polymers such as
starch or dextran because of their low toxicity and high
affinity for iron oxide (chelation and hydrogen bonding) [35],
while the therapeutic agent of interest is either covalently
conjugated or ionically bound to the outer layer of the
polymer [36].
In this study, we demonstrate the enhancement in the
antitumour efficiency of Cat–Dex on pancreatic tumour cells
as a result of its conjugation with magnetic nanoparticles. For
this purpose, we selected commercial dextran-coated iron
oxide nanoparticle (Endorem) as a magnetic responsive
element, because they are an FDA-approved nanostructure
used as magnetic resonance imaging (MRI) contrast
agents [37]. Here, a simple one-step process to prepare
magnetic Cat–Dex obtained by substitution of the dextran
layer of Endorem with the Cat–Dex conjugate is proposed.
The resulting nanocomposite was characterised and its
anticancer efficiency in the treatment of pancreatic cancer
was tested, confirming the high versatility of the magnetic
nanocarrier and its practical applicability in targeted antic-
ancer therapy.
Materials and methods
Synthesis and characterisation of catechin conjugate
We previously described the synthesis of the Cat–Dex
conjugate by a free radical grafting process as follows: in a
50 mL glass tube, 0.5 g of Dex from Leuconostoc spp (Dex,
Sigma, St. Louis, MO, Mw 4 kDa) was dissolved in 50 mL
distilled water. Then, 1 mL of the redox initiator system H
2
O
2
5.0 M (Sigma, St. Louis, MO) and 0.25 g of ascorbic acid
(Sigma, St. Louis, MO) was added and the solution was
maintained at 25 C under atmospheric air for 2 h [17]. Then,
100 mg of catechin (Cat) was introduced into the reaction
flask and the solution was maintained at 25 C under stirring
in atmospheric air for 24 h. The resultant conjugated polymer
was purified by dialysis process in distilled water (dialysis
tubes of 6–27/3200, Medicell International LTD (London,
UK), MWCO: 3.5–5000 D) for 48 h. The resulting solutions
were frozen and dried with a Micro Modulyo freeze-dryer
(Edwards Lifesciences, Irvine, CA) to afford vaporous solids.
At the end of the purification step, the conjugate was checked
to be free of unreacted flavonoid and any other compounds
through analysis of the washing media by a HPLC system
consisting of a Jasco PU-2089 Plus liquid chromatography
equipped with a Rheodyne 7725i injector (fitted with a 20 mL
loop), a Jasco UV-2075 HPLC detector and Jasco-Borwin
integrator (Jasco Europe s.r.l., Milan, Italy). A Tracer Excel
120 ODS-A column particle size 5 mm, 15 0.4 cm
(Barcelona, Spain) was employed as stationary phase, while
the eluent consists of a mixture of methanol/water/orthophos-
phoric acid (20/79.9/0.1) (HPLC grade, Carlo Erba, Milan,
Italy) at a flow rate of 1.0 mL min
1
, and the detector was set
at 260 nm [38]. A control sample (Dex), which acts as a
control, was prepared with the same protocol but without
adding CT. The conjugate was characterised by UV-Vis and
1
H-NMR. A Perkin Elmer Lambda 900 spectrophotometer
(Perkin Elmer, Waltham, MA) was used for obtaining the
absorption spectra, while the corrected emission spectra, all
confirmed by excitation ones, were recorded with a Perkin
Elmer LS 50B spectrofluorimeter, equipped with Hamamatsu
R928 photomultiplier tube. The
1
H-NMR spectra were run on
Bruker VM-300 ACP using DMSO-d
6
as a solvent.
Evaluation of catechin content
The amount of catechin equivalents conjugated to the dextran
was evaluated by means of the Folin–Ciocalteu reagent
procedure, according to the literature with some modifica-
tions [15]. Briefly, Cat–Dex conjugate (5.0 mg) was dissolved
in 6.0 mL of distilled water and 1.0 mL of the Folin–Ciocalteu
reagent (Sigma, St. Louis, MO) was added. The mixture was
mixed thoroughly, and after 3 min of incubation, 3.0 mL of
Na
2
CO
3
solution (2.0%, w/w) was added, and the mixture
allowed to stand in the dark for 2h with intermittent shaking.
The absorbance was measured at 760 nm against a control
prepared using the blank polymer under the same reaction
conditions. The Cat content in the conjugate was expressed as
mg per g of dry material by using the equation of the
calibration curve for the free flavonoid, which was recorded
by employing five different Cat standard solutions. 0.5 mL
amount of each solution was added to the Folin–Ciocalteu
2O. Vittorio et al. J Drug Target, Early Online: 1–8
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system to raise the final concentration of 8.0, 16.0, 24.0, 32.0
and 40.0 10
6
mol L
1
. After 2 h, the absorbance of the
solutions was measured to record the calibration curve and the
correlation coefficient (R2); slope and intercept of the
regression equation obtained were calculated by the method
of least squares.
Preparation of magnetic nanocomposite
For a fast and complete encapsulation of End (11.2 mg of
Fe/mL, Guerbet, Milan, Italy), the substitution process was
performed in a great excess of Cat–Dex. In order to obtain a
molar ratio between Endorem nanoparticles and catechin–
dextran molecules higher than 300, 100 mL of the colloid was
added to 1 mL 0.14 mol L
1
Cat–Dex solution in Phosphate
Buffered Saline (PBS 0.01 mol L
1
, Sigma, St. Louis, MO).
The resulting solution was kept under gentle agitation for 24 h
at 37 C. The encapsulated nanostructures were purified from
non-conjugated molecules by three washing steps in PBS
solution with Amicon centrifugal filters (Sigma, St. Louis,
MO) at 13 400 rpm for 15 min. The colloid was resuspended
in 1 mL PBS solution, sonicated for 10 min, and then dialysed
for 1 day (SpectraPore, 10 K membranes) against PBS.
We obtained a final solution with 10
15
nanoparticles/mL,
containing 1.10 mg Fe/mL and 3 mg/mL of Catechin,
characterized by DLS and TEM measurements, were stored
at +4 C and used within 1 week.
Dynamic light scattering (DLS) measurements
Measurements were performed on a Zetasizer nano ZS DLS
(Malvern Instrument, Malvern, UK) following manufacturer’s
instructions and in PBS solutions in standard capillary cells
DTS 1060 or 700 mL standard plastic cuvette for, respectively,
z-potential and size measurements. A concentration of
1013 Np mL
1
was used for all measurements, and each
reported value is the average of five consecutive analysis.
Cell culture
The human pancreatic cancer cell line (MIA PaCa-2) was
obtained from American Type Culture Collection (ATCC,
Rockville, MD). These cell lines were grown in a complete
culture medium consisting of DMEM (Lonza, Milan, Italy)
supplemented with 2 mM L-glutamine, 100 IU mL
1
penicil-
lin, 100 mgmL
1
streptomycin and 10% heat inactivated fetal
bovine serum (FBS, Lonza, Milan, Italy). Cells were main-
tained at 37 C in a saturated humidity atmosphere of 95% air
and 5% CO
2
.
Measurement of cell viability by calcein-AM
MiaPaCa cells were stained with 2 M calcein-AM solution
(Molecular Probe, Eugene, OR) at room temperature for
10 min, and, after that, the cells were treated with Cat–Dex
solution at 150 mgmL
1
for 30 min under magnetic field.
Calcein AM is a cell-permeant dye that can be used to
determine cell viability in most eukaryotic cells [39]. In live
cells the non-fluorescent calcein AM is converted to green-
fluorescent calcein (ex/em 495/515 nm), after acetoxymethyl
ester hydrolysis by intracellular esterases. A complete leak of
calcein AM fluorescence signal caused by the cellular death is
observed in the area where the magnetic field was applied.
This would be caused by a higher Cat–Dex concentration
directly in those areas.
Cell viability after Endorem incubation
WST-1 (tetrazolium salt 2-(4-iodophenyl)-3-(4-nitophenyl)-
5-(2,4-disulfophenyl)-2H-tetrazoilium) cell proliferation
assay was performed on MIA PaCa-2 cultured with
Endorem nanoparticles at different concentration for 24 h.
25 10
3
cells were seeded into a 96-well plate and then
incubated with the culture media. After cell adhesion (six
hours), we incubated the cells with different concentrations of
Endorem for 24 h (Figure 4B). After that, 10 mLofWST-1
solution (as described in quick cell proliferation assay kit,
BioVision, Milpitas, CA) was added to the culture media and
the plate was incubated for 1 h in standard conditions. The
absorbance was measured on a Versamax microplate reader
(Molecular Devices, Sunnyvale, CA) at a wavelength of
450 nm with background subtracted at 650 nm. The results are
given relative to the untreated control cells.
Evaluation of apoptotic cell death
After the evaluation of drug activity in the presence of the
magnetic field by Calcein AM, we assessed cell death by
Hoechst 33258 (Invitrogen, Milan, Italy) 0.5 mgmL
1
staining
of cell nuclei (excitation 346 nm; emission 460 nm), which
enables determination of the presence of DNA condensates
indicative of apoptosis [40]. After washing with PBS, cells
were fixed with 40 g L
1
para-formaldehyde in PBS,
incubated for 30 min at 4 C and then washed three times
with PBS before further incubation for 20 min at 37 C with
0.5 mgmL
1
Hoechst 33258 in PBS. The cells were
then washed again with PBS and imaged. The pyknotic
nuclei were examined with an epifluorescence microscope
(Nikon TI, Amsterdam, the Netherlands). The number of
apoptotic nuclei was counted on five randomly selected areas
from each experimental group. Results are expressed as
number of apoptotic cells as a percentage of the total number
of cells.
Statistical analysis
Each experiment was carried out in triplicate. For the
antioxidant experiments, data are expressed as means
(±SD). For the calculation of the fluorescence assays, five
random microscopic fields were counted under and far-off the
magnetic field. Statistical significance was assessed by the
one-way analysis of variance (ANOVA) followed by post-hoc
comparison test (Tukey). Significance was set at 5%.
Results and discussion
Synthesis and characterisation of End–Cat
The system objective of this study (End–Cat) is prepared
by modification of commercially available magnetic nano-
particles by Cat–Dex. Specifically, the coating of End
(composite by dextran) was substituted by Cat–Dex
(19.9 mg of CT per g Folin–Ciocalteu test) with the aim to
couple the main features of iron magnetic nanoparticles,
DOI: 10.3109/1061186X.2013.878941 Magnetic Cat–Dex conjugate 3
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dextran and catechin: (i) the ability to be quickly internalised
in cells; (ii) the magnetic responsivity [41]; and (iii) the
therapeutic activity against pancreatic tumoural cells [17].
According to our previous work [17], Cat–Dex is synthe-
sised by a protocol developed and successfully employed for
the covalent functionalisation of different natural biomacro-
molecules (e.g. protein and polysaccharides) with antioxidant
compounds (e.g. polyphenols and flavonoids). The reaction
mechanism involves a complex free radical-driven process in
which free radical species are formed in situ and react with
the side chain of the polysaccharide, such as the hydroxyl
(OH) and the a-methylene (CH
2
) positions, by action of the
redox initiator pair (H
2
O
2
and AA). Thus, a covalent coupling
occurs with the ortho- and para-positions relative to the
hydroxyl groups of the flavonoid.
The reaction conditions were optimised elsewhere [17],
and the most significant characterisations of the polymeric
conjugate were obtained by means of HPLC,
1
H-NMR and
UV-Vis measurements. In particular, HPLC was used to
confirm that no free Cat is present in the sample after the
purification process, while UV-Vis spectroscopy (both
absorption and emission, Figure 1A) proved the formation
of a covalent bond between the polymeric backbone and the
flavonoids by virtue of the observed bathochromic shift of the
Cat-relative peaks moving from the free to the conjugated
form.
The presence of Cat within the polymeric conjugate was
also proved by the presence of the typical signal of Cat
aromatic region in the
1
H-NMR spectrum (Figure 1B).
Furthermore, other significant information comes from the
Figure 1. (A) Cat–Dex characterisation. UV-Vis absorption spectra showing the bathochromic shift of the Cat relative peaks moving from the free to
the conjugated form. (B)
1
H-NMR spectra of Cat–Dex proving the presence of the Cat aromatic region into the conjugate.
4O. Vittorio et al. J Drug Target, Early Online: 1–8
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comparison of the
1
H-NMR spectrum of the flavonoid in the
native form with that of the Cat recovered after the dialysis
process. Specifically, no difference can be detected between
the two spectra, and this was used as a confirmation that the
conjugation process prevents Cat from oxidation. This is of
great importance because it supports the hypothesis that the
whole of biological properties of the conjugate are related to
the Cat equivalent conjugated to Dex, and thus a quantitative
measurement of this value can give information about the
conjugate efficiency.
The subsequent step is the loading of the Cat–Dex onto the
End nanoparticles, with the formation of the End–Cat
nanocomposite. This was reached by a straight-forward
procedure involving the substitution of the End coating by a
simple mixing/purification step at 37 C. This methodology
was chosen by considering the chemical properties of Cat (in
particular, instability of flavonoid species over thermal
treatment at 70–90 C) and the common strategies employed
for the preparation of dextran-coated magnetic iron nanopar-
ticles [42,43]. Furthermore, this process addresses another
key requirement for the preparation of magnetic nanocompo-
site materials, such as the homogeneous water dispersibility
of the iron core [44].
The encapsulation of End was performed with a molar
ratio between magnetic nanoparticles and Cat–Dex molecules
higher than 300, in order to obtain a fast and complete
substitution of the surfactant (dextran). The success of the
substitution was confirmed by DLS measurements. Both the
hydrodynamic diameter and the zeta-potential values, indeed,
increased after the substitution process (Figure 2). End shows
a hydrodynamic diameter of 91 nm and a zeta-potential of
5.7 mV, while for End–Cat these values were 105 nm and
13.9 mV, respectively. This was in agreement with data
reported in the literature [41], and the increased End–Cat
values could be associated to the higher acidity of the Cat
phenolic groups in respect to that of the Dex aliphatic
alcoholic hydroxyl groups.
The substitution process (and thus the loading step) was
optimised and the maximum substitution degree was raised:
by extending the reaction time, indeed, no further increases in
hydrodynamic diameter and the zeta-potential were recorded.
After the encapsulation process, the preservation of the
magnetic features of End–Cat were tested by applying a
permanent magnet to the colloidal solution (Figure 2),
confirming the retention of the magnetic behaviour.
The method of directed drug delivery was based on the
magnetic separation of nano-particle in a fluid medium in
which a magnetic field gradient attracts the nanoparticles
towards a permanent magnet. The translation force F
m
acting
on a magnetic nanoparticle with magnetic moment munder
Figure 2. DLS measurements, (A) size and (B) zeta-potential, of commercial End and End–Cat. The increase in both hydrodynamic diameter
and surface potential of End–Cat compared to End is associated to the presence of catechin on the coating, and thus to the success of the
surface substitution. (C) Left panel: a PBS solution of Endorem nanoparticles coated by Cat–Dex. Right panel: the magnetic particles are attracted
from the applied magnetic field (circle) in less than 20, demonstrating that the substitution process does not change the magnetic features of the
nanoparticles.
DOI: 10.3109/1061186X.2013.878941 Magnetic Cat–Dex conjugate 5
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an inhomogeneous magnetic field Bcan be written according
to the following Equation (1):
Fm¼mrBð1Þ
For effective magnetic separation, the force F
m
must
compete with the hydrodynamic drag force (F
d
) according to
Equation (2) [45]
Fd¼3Dvdð2Þ
where, is the viscosity of water, Dis the diameter of the
nano-particle and v
d
is the drag velocity. Balancing the above
two competing forces, the magnetic drag velocity of the
nanoparticle can be estimated as reported in Equation (3):
vd¼mrB
3Dð3Þ
To estimate the magnetic drug velocity, we consider the
physical dimensions of the nanoparticles and physical param-
eters of our setup as follows: End–Cat has a magnetite (Fe
3
O
4
)
core of diameter d5 nm and with Cat–Dex coating the
diameter is nearly d105 nm [45]. Because of small core
diameter d, the particles behave as a superparamagnetic and
saturation of magnetisation occurs at relatively low applied
magnetic field nearly 125 mT [46]. In our experiment, a
Neodymium permanent magnet with an edge field of 150 mT in
the plane of the fluid (Figure 3) is used. The field strength
decays rapidly with a rate of 30 T/m and at a distance 2 cm away
from the magnet, it becomes 200 mT. Considering, saturation
magnetisation of magnetite core [47] 26.64 emu/g and density
2.25 g/cm
3
, the particle velocity v
d
¼130 mm/s is estimated at
the edge of the magnet. Away from the magnet field strength B
as well as field gradient rBdecreases and hence the
magnetisation of nano-particle reduces from its saturation
value. As a result, the magnetic drag velocity v
d
decreases away
from the magnet and magnetic drag become negligible at far
distances 2.0 cm from the magnet. However, microscope stage
movement during experiment helps in staring the liquid, as a
result, this staring brings the nano-drug particles in the range of
the magnet and magnetic separation becomes efficient. In our
experiment, nearly uniform deposition of End–Cat occurs
above the magnet (Figure 3).
Anticancer activity
Recently, great effort was devoted on the controlled delivery
and release of drugs from nanoparticles [48]. The specific
delivery is usually accomplished by the conjugation of the
nanostructure to antibodies or by exploiting peculiar features
of the nanoparticles themselves, such as magnetic
behaviour. On the other hand, the release can be triggered
by: (i) desorption of the drugs from the nanostructures, or
(ii) interaction of the nanostructure with external stimuli such
as light. In the system, we propose here the spatial control of
the anticancer activity is exploited by the magnetic behaviour
of End, the cellular uptake is reached by passive endocytosis
[48], and the release of catechin is triggered by desorption of
Cat–Dex from the End surface.
In our experimental protocol, 250 10
3
cells were seeded
in a 35 mm petri dish 24 h before performing the experiments.
Subsequently a Calcein-AM solution was added to the cells
10 min before to add 150 mg/mL of End–Cat in the cell culture
media. Then the petri dish was placed in agitation for 10 s and
after that a permanent magnet was placed under the petri dish
and it was incubated at 37 C and 5% of CO
2
for 30 min.
Afterwards the entire system was placed under a fluorescence
microscope as showed in Figure 3 in order to study the
efficiency of End–Cat as a magnetic-responsive carrier. Our
results showed that the End–Cat was attracted from the
magnetic field increasing the local concentration of the drug
Figure 3. Experimental setup composite by a Petri dish with a layer of cultured pancreatic tumoural cells after the application of a magnet for 24 h.
(A) Cells far from the magnetic field. (B) Bubbling cells (early stage of apoptosis) only nearly the magnet. (C) Picnotic cells (late stage of apoptosis)
only nearly the magnet.
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which was able to target and kill 98% of tumour cells in 24 h
when exposed to the magnet (Figure 4). This strong effect is
due the increased intracellular concentration of the Cat–Dex
because we previously observed that the concentration used in
the conjugation with Endorem (150 mg/mL) was able to kill
90% of pancreatic tumour cells only after 72 h of incubation.
Moreover, when the same experiments where performed
by using free Endorem no toxicological effects were observed
after 24 h of incubation. Figure 4(B) shows the effect of
different concentrations of free Endorem in MIA PaCa-2
cells. The highest tested concentration is 10 times higher than
the nanoparticle concentration we used for the conjugation
with the drug. Endorem nanoparticles did not show any
toxicity on the cells after 24 h of incubation.
These results indicate that the conjugation of Endorem
with Cat–Dex represents an effective magnetic responsive
carrier.
Conclusion
A novel polymer therapeutic was synthesised by the modi-
fication of the Endorem by means of a Catechin–Dextran
conjugate showing high anticancer activity. By this approach,
an enhancement of the anticancer efficiency of the flavonoid–
polysaccharide conjugate was achieved and 98% of pancreatic
cancer cells were killed within 24 h in the presence of the
magnet as a consequence of the local concentration of the
drug under the magnetic field. The straight-forward procedure
employed involves the simple substitution of the Endorem
external layer and the combined innovative approach allows
the obtainment of an efficient and targeted therapeutic which
can be used in cancer therapy with high efficiency and
selectivity.
Declaration of interest
The authors report no declarations of interest.
The Vice-Chancellor’s Postdoctoral Research Fellowships
of University of New South Wales (O.V.) and Regional
Operative Program (ROP) Calabria ESF 2007/2013 – IV Axis
Human Capital – Operative Objective M2 – Action D.5
(G.C.) are gratefully acknowledged.
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