The antiestrogen molecule tamoxifen is a strong, hydrophobic endocrine drug widely used for treating breast cancers and high-risk
patients (1). The antitumoral function of tamoxifen results from its link to the intracellular estrogen receptor on breast-cancer
cells and the blocking action of the steroid hormones (2). Depending on the dose and the tissues targeted, the function of
tamoxifen can be estrogenic or antiestrogenic. The dose-dependent side-effects of tamoxifen include liver cancer, increased
blood clotting, and ocular adverse effects, such as retinopathy and corneal opacities (3). These findings suggest that small
doses given through colloidal delivery systems would be useful for long-term treatment of breast cancers. Nanoparticulate
delivery systems in the form of nanospheres and nanoparticles were used by Chawla and Amiji, in 2003 (4). The basis of this
formulation is the attainment of adequate dose of the drug at the tumor site for a known period of time and the reduction
of adverse effects on normal organs. Recently, solid lipid nanoparticles (SLNs) were recommended by Fontana et al. for drug-delivery
systems (1). The main benefit of SLNs is their lipidmatrix composition, which is physiologically tolerable and entails little
acute or chronic toxicity. Additional advantages are their widespread application, the scalability of production without the
need for organic solvents, their high bioavailability, their ability to protect drugs from degradation agents, and their ability
to control drug release (5–6).
A useful drug-delivery system should possess high capacity for incorporating drugs between fatty-acid chains or lipid layers,
or in crystal imperfections. Whether the drug is located within the core of the particles, in the shell, or as a molecular
dispersion throughout the matrix depends on the drug-to-lipid ratio and the drug's solubility within the lipid (7). The mode
of drug incorporation influences the drug release, particle size, and physical stability, and modifying the lipid matrix,
surfactant concentration, and production parameters can affect the drug-release profile (8).
The aim of this study was to prepare a tamoxifen-loaded SLN using homogenization. The authors characterized the tamoxifen-loaded
SLN and determined the optimum drug loading and in vitro release profile.
Materials and methods
Hydrogenated palm oil (Softisan 154 or S154) was a gift from Condea. Hydrogenated soybean lecithin (Lipoid S100-3, containing
90% phosphatidylcholine, including 12–16% palmitic acid, 83–88% stearic acid, oleic acid and isomers, and linoleic acid] was
a gift from Lipoid. Thimerosal, mercury((o-carboxyphenyl)thio)ethyl sodium salt, and Sorbitol, (2S,3R,4R,5R)-hexane-1,2,3,4,5,6-hexol,
were purchased from Sigma. Oleyl alcohol (octadecenol or cis-9-octadecen-1-ol) was purchased from Fluka. Tamoxifen, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] (MTT), Fetal bovine serum and Roswell Park Memorial Institute (RPMI)-1640 medium were obtained from Sigma-Aldrich.
Bidistilled water was used.
Breast cancer cell line, MCF-7, was kindly offered by Teo Guan Young (Institute Bioscience, UPM). Cells were maintained at
37 °C in a humidified atmosphere of 5% CO2 in RPMI medium supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 IU/mL penicillin.
Preparation of tamoxifen-loaded SLNs.
Tamoxifen-loaded SLNs were prepared using the high-pressure homogenization technique (9). A mixture of S154 and Lipoid S100
at a ratio of 70:30 was ground in a ceramic crucible. The mixture was then heated to 65–70 °C while being stirred with a PTFEcoated
magnet until a clear-yellowish lipid matrix (LM) solution was obtained. A solution containing 1 mL oleyl alcohol, 0.005 g
thimerosal, 4.75 g Sorbitol, and 89.25 mL bidistilled water (all w/w) at the same temperature was added to 5 g of LM. A pre-emulsion
of SLN was obtained using the homogenizer (Ultra Turrax, Ika) at 13,000 rpm for 10 min and high-pressure homogenizer (EmulsiFlex-C50
CSA10, Avestin) at 1000 bar, 20 cycles, and 60 °C. The lipophilic drug tamoxifen (1 mg) was dissolved in oleyl alcohol and
mixed with 5 mg of SLN pre-emulsion using the Ultra Turrax homogenizer at 13,000 rpm for 10 min. This mixture was then incubated
overnight at 50–60 °C, stirred periodically with a PTFEcoated magnet at 500 rpm, and finally exposed to air to solidify.
Characterization of tamoxifen-loaded SLN.
Physical characterization of tamoxifen-loaded solid lipid nanoparticles. The mean particle sizes (i.e., diameter + standard deviation) and size distribution (polydispersity index or PI) of SLNs and
tamoxifen-loaded SLNs were determined using a high-performance particle sizer (HPP5001, Malvern Instruments). Measurements
were performed at 25 °C in triplicate. Before measurement, each sample was dispersed in filtered bidistilled water by a sonic
water bath. The zeta potential (i.e., electrophoretical movement) of the SLN and tamoxifen-loaded SLN was then measured by
an analyzer (Zeta sizer, ZEN2600, Malvern) in triplicate.
Morphology and crystallinity of tamoxifen-loaded solid lipid nanoparticles.
The morphology of tamoxifen-loaded SLNs was viewed using a transmission electron microscope (Hitachi H-7100, Hitachi). After
dispersion with water, the samples were negatively stained with 1.5% (w/v) phosphotungstic acid for viewing.
The melting points of the bulk lipid and SLN formulation were measured using differential scanning calorimetry (DSC). The
DSC analysis was performed using the Mettler DSC 822e (Mettler Toledo), and thermograms were recorded in the 20–120 °C temperature
range with a heating rate of 5 °C/min. An empty aluminum sample pan was used as a reference. Wide-angle X-ray diffractometry
(WAXD) was used for the determination of the crystal characteristics of the SLN preparation and also drugs in the case of
nanoparticle samples. The WAXD analysis was performed over range 2θ, using Philips PW 3050/60 diffractometer (Kasel, Germany)
with a copper anode. Specimens of 10 mm length were placed into standard X-ray plate and exposed to 40 kV, 30 mA with scan
speed of 0.005/s, step size 2Θ and slit 100 mm.
Entrapment efficiency and drug loading.
High performance liquid chromatography was accomplished by using Waters 2695 series liquid chromatography with ultraviolet-visible
detector. The analytical column as stationary phase used was reversed-phase C18 (μ Bondpak, 5 mm, 250 × 46 μm i.d., Waters)
with the following analytical conditions: mobile phase solution consisting of methanol /0.1 M: phosphate buffer saline, PBS
(95:5, v/v, pH 7.4) and flow rate of 1 mL/min at 25 °C. The mobile phase was monitored at wavelength 250 nm. A series of tamoxifen
concentrations ranging from 10 to 150 ppm was used to obtain a calibration curve (Y = 0.239X – 0.245) with linear regression
r = 0.9994. To determine entrapment efficiency (EE) and drug loading (DL), tamoxifen-loaded SLNs were dispersed in methanol.
The dispersion was centrifuged at 40,000 g for 60 min to remove SLNs. The amount of drug in the supernatant that was not incorporated
into the SLNs was analyzed by high-performance liquid chromatography (HPLC). The EE% and DL% were obtained using the following
Tamoxifen release from nanoparticles in human plasma.
The tamoxifen release profile from SLNs was assayed in vitro. Eight similar batches of tamoxifen-loaded SLNs containing 500 μg of tamoxifen in 1 mL of human plasma were prepared. The
experiment was conducted in a 37 °C water bath with mechanical stirring. At predetermined intervals, each sample was centrifuged
at 40,000 g for 60 min and the SLN removed. Plasma protein in the supernatant was precipitated at a 1:2 ratio with methanol
and centrifuged at 1500 g for 15 min. The amount of free drug in the supernatant that was neither incorporated into the SLNs
nor linked to albumin was estimated by HPLC. Free drug at concentrations ranging from 10 to 60 ppm in plasma was used to obtain
the calibration curve.
Determination of half maximal inhibitory concentration (IC
The viability of MCF-7 breast-cancer cells in the presence of tamoxifen and tamoxifen-loaded SLNs was assessed by MTT assay.
The breast cancer cells were maintained in RPMI-1640 culture medium, supplemented with 10% fetal bovine serum at 37 °C in
a humidified incubator containing 5% CO2 and 95% air. The cells were allowed to grow to a concentration of 105 cells/mL before being seeded into a 96-well plate.
The cells were treated with tamoxifen and tamoxifen-loaded SLNs at concentrations ranging from 3.25 to 60 μg/mL for 24, 48,
and 72 h. The control wells received PBS as the vehicle. The percentage of cell viability and IC50 versus free tamoxifen and tamoxifen-loaded SLN concentrations were determined.