was proven to enhance the therapeutic effectiveness of many drugs in the
treatment of different diseases, among which is cancer ; since they allow
either passive or active targeting to the tumor tissues. Recently, antioxidant
nutraceuticals were reported as promising molecules for treatment of cancer,
owing to their efficacy and considerable safety. Among the promising
nutraceuticals in this regard is oleuropein, which is a bioactive phenolic
compound present in the leaves of olive tree . It was reported to be
effective against several types of cancer , among which is colon cancer. Its
exact mechanism of anticancer activity in colorectal cancers was verified in
HT-29 cell lines, in which it was reported to activate p53 pathway which causes
cellular apoptosis .
of the effectiveness of oleuropein, it was reported to be exhibit poor oral
absorption owing to its high polarity . Therefore, it can be hypothesized
that the incorporation of oleuropein in a surfactant-based nanoparticulate
system might be a feasible means for enhancing its delivery and increasing its
anticancer activity in colon cancer. Nanoparticles were reported to potentiate
the anticancer activity of drugs [6-8], owing to their enhanced internalization
within cancer cells because of their unique small size . Among the promising
and recently reported nanoparticles are nanocapsules, which are mainly composed
of an oily core and a polymeric or surfactant based shell . Lipidic
nanocapsules possess a lipoprotein-like composition, and were reported to
enhance the anticancer activity of cytotoxic drugs, with reduced side effects
. Therefore in the current work, oleuropein was encapsulated in
surfactant-based nanocapsules and characterized for its physicochemical
properties, and was further tested for its cytotoxic effect against HCT-166
colon cancer cells. To authors’ knowledge, only one published paper attempted
the encapsulation of oleuropein in nanoparticles, and no papers reported the
anticancer activity of oleuropein against HCT-116 cancer cell line.
MATERIALS AND METHODS
Soybean lecithin (Epikuron
200) was kindly obtained from Cargill Co., Germany. Labrafac Lipophile was
kindly obtained from Gattefosse’ Co., France. Solutol HS15, disodium hydrogen
phosphate, potassium dihydrogen phosphate, dimethylsulfoxide, MTT dye and
dialysis membrane (molecular weight cut off 12000-14000) were purchased from
Sigma Aldrich Co., USA. Oleuropein was purchased from Skin Actives company,
USA. Fetal bovine serum, DMEM,
RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25%
trypsin-EDTA were purchased from Lonza, Belgium. HCT-116 cancer cells were
obtained from the American type culture collection (ATCC, USA).
Preparation of oleuropein
oleuropein nanocapsules were prepared by the phase inversion method .
Briefly, 20 mg of oleuropein and 100 mg soybean lecithin were dispersed in 2.5
gram labrafac lipophile oil by magnetic stirring for 10 minutes, followed by
addition of 2.5 gram solutol HS15 and 3 gram distilled water. Magnetic stirring
was continued for 5 minutes followed by heating of the dispersion during mixing
till the temperature reaches 85°C for the formation of a water in oil emulsion.
Cooling of the emulsion was then performed to a temperature of 55°C, in which
the emulsion was inverted to oil in water type. The cycle was repeated two more
times, followed by addition of distilled water at 4°C till a total weight of 10
Determination of the particle size, polydispersity index and zeta
potential of oleuropein nanocapsules
particle size, zeta potential and polydispersity index (PDI) of the prepared
oleuropein nanocapsules were measured using the Zetasizer device (model ZS3600,
using transmission electron microscopy
The prepared oleuropein nanocapsules was
examined for its morphology using transmission electron microscopy without
staining, after drying on a carbon grid (VERSA 3D, USA).
In vitro release
of oleuropein from the prepared nanocapsules
The release of oleuropein from the nanocapsules
was performed using a dialysis-based method [13-15], and compared to the
release of oleuropein from solution control. One ml of either the drug-loaded
nanocapsules or the drug solution was placed in a plastic cylinder of 8 cm
height, attached to the shaft of USP dissolution device (Pharma Test, Germany)
rotating at 50 rpm and temperature of 37±0.5°C. The release medium was 200 ml
phosphate buffer of pH 7.4 containing 2% tween 20, from which three ml samples
were drawn at definite time intervals (0.25, 0.5, 1, 2, 3, 4, 6, 24 hours).
Finally, the amount of oleuropein released was measured spectrophotometrically
at wavelength of 288 nm (SPUV UV/VIS double beam spectrophotometer, SCO TECH,
Assessment of the storage
stability of oleuropein nanocapsules
The physicochemical properties of the
oleuropein nanocapsules (particle size, PDI and zeta potential) were measured
after 3 months storage at refrigeration temperature, to assess the stability of
the prepared formulation [6,16].
Evaluation of the cytotoxicity
of oleuropein and its nanocapsules against HCT-116 cancer cell line
HCT-116 cells were grown on RPMI-1640
medium containing 10% inactivated fetal calf serum and 50 µg/ml gentamycin at
37ºC in a humidified atmosphere with 5% CO2, and were sub-cultured two to three
times weekly. Cells were then placed in culture medium at a concentration of
50,000 cells per well (Corning® 96-well tissue culture plates) then incubated
for twenty four hours. Either oleuropein or oleuropein nanocapsules were added
to the cells at different concentrations (0.5-500 µg/ml). The viability
percentage values were calculated after 24 hours incubation period, in which
the media was replaced with another fresh media containing MTT dye followed by
incubation for 4 hours and further addition of dimethyl sulfoxide. The optical density was measured at 590 nm
with a microplate reader (SunRise, TECAN Inc, USA) to calculate cell viability according to the
following equation [8, 17]:
Viability % =
Measurements were done in triplicate
and reported as mean±S.D. Student T-test was performed using Graphpad® Instat
software, at significance of P=0.05. The IC50 values were calculated
using Graphpad Prism software (San Diego, CA. USA)
RESULTS AND DISCUSSION
Measurement of particle size,
PDI and zeta potential of oleuropein nanocapsules
Oleuropein nanocapsules displayed a particle
size of 151.45±9.97 nm, a PDI value of 0.286±0.08 and a zeta potential value of
-0.059±0.004 mV. The small particle size of the nanocapsules is expected to
provide better tumor uptake by the enhanced permeation and retention EPR effect
. The low polydispersity index value of the nanocapsules (less than 0.4)
indicates the presence of a homogenous population of nanocapsules, and the
effective solubilization of oleuropein within the nanocapsules. The almost
neutral charge of the prepared nanocapsules is caused by the non-ionic nature
of the utilized surfactant Solutol HTS15.
Morphological examination of
oleuropein nanocapsules using transmission electron microscopy
As shown in Figure 1, the oleuropein
nanocapsules displayed homogenous non-aggregated spherical droplets, displaying
an oily core and a surfactant shell. The particle size obtained concurred with
the particle size obtained with the Zetasizer.
Figure 1. Transmission electron microscopy pictures of the prepared
In vitro release
of oleuropein from the nanocapsules
Oleuropein showed a sustained release pattern
over 24 hours from the nanocapsules, reaching complete release (100%) as
displayed in Figure 2. Meanwhile, the release from oleuropein solution was
complete after only 2 hours. The sustained release of oleuropein from
nanocapsules is ascribed to the consequent partitioning of oleuropein from the
oily phase of nanocapsules into the aqueous medium, and the considerable
viscosity of the formulation, creating hindrance for oleuropein release.
Figure 2. Cumulative percent released of oleuropein for 24
hours from the nanocapsules compared to oleuropein solution.
Figure 3. The viability percentage of HCT-116 colon cancer
cells as a function of oleuropein concentration.
Stability of oleuropein
After three months storage of the nanocapsules
at refrigeration temperature, oleuropein nanocapsules displayed a particle size
of 153.85±33.87, a PDI value of 0.28±0.04 and a zeta potential value of
-0.031±0.002. It was found that these values were statistically insignificant
compared to the values obtained for the freshly prepared nanocapsules
(P>0.05), suggesting the stability of the nanocapsules.
Evaluation of cytotoxicity of
oleuropein nanocapsules in HCT-116 cancer cell line
Till current date, no studies were published on
the cytotoxicity of oleuropein in HCT-116 cells. The cytotoxicity of oleuropein
compared to oleuropein nanocapsules was assessed and displayed in Figure 3. As
observed in the previous figure, oleuropein exhibited an IC50 value
of 185±8.7 µg/ml when administered as free drug, while displaying an IC50
value of 6.52±0.7 µg/ml in the nanocapsules form. Oleuropein was about 28 times
more potent as an anticancer with significant decrease (P<0.05) in the cellular viability when encapsulated
in nanocapsules. This could be attributed to the small size of the nanocapsules
and their surfactant-based shell, which allows better cellular uptake, and
hence enhanced anticancer activity. The reported IC50 value for
oleuropein in HT-29 cancer cells ranged from 108-216 µg/ml , which concurred
with the value obtained in this work. The aforementioned results delineate
nanocapsules as promising carrier for oleuropein.
Oleuropein nanocapsules were proven to
be provide high loading of the drug, a sustained release nature in addition to
exhibiting physicochemical stability. They were also successful in enhancing
its anticancer activity, suggesting that they can be pharmacologically tested
in animal models as a futuristic step.
Declaration of interest
The authors report no conflict of
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