Delivering Tamoxifen within Solid Lipid Nanoparticles

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.

Cell line. 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 equations:

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 (IC₅₀). 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.

Results

All data were subjected to one-way analysis of variance followed by post hoc multiple comparison and Duncan tests after verifying that data were distributed normally. The authors used high-pressure homogenization to prepare SLNs because the matrix lipid composed of palm oil (i.e., a triglyceride mixture of natural, hydrogenated, and unbranched fatty-acid chains) was suitable for the incorporation of lipophilic drugs, such as tamoxifen (10). Soy lecithin was the most useful surfactant in SLN dispersions. SLNs and tamoxifen-loaded SLNs were characterized in vitro for particle size, particle-size distribution, and zeta potential (see Table I). In this study, the average size of tamoxifen-loaded SLNs was significantly larger than that of the free SLNs, and the surfaces of tamoxifen-loaded SLNs carried a positive charge.

Table I: Particle sizes, particle-size distribution (PI), specific surface area (Aspec), and zeta potential of solid lipid nanoparticle formulations of tamoxifen.

The transmission electron microscopy (TEM) image of tamoxifen-loaded SLNs is shown in Figure 1, where the particles have a round and uniform shape. The DSC thermogram and melting point of tamoxifen-loaded SLNs is shown in Figure 2. The melting point of the bulk lipid matrix was 58.88 °C. Drug-free SLNs prepared using lecithin and oleyl alcohol had a melting point of 57.88 °C, and incorporating tamoxifen into the SLNs reduced the melting point to 56.56 °C. The WAXD pattern for the bulk lipid was different from that of the nanoparticle, showing relatively sharper peaks compared with SLN and tamoxifen-loaded SLNs (see Figure 3). The WAXD pattern also showed that the typical peak shape associated with free tamoxifen was absent in the tamoxifen-loaded SLN.

Figure 1: Micrographs of tamoxifen-loaded solid lipid nanoparticles by transmission electron microscope (bar = 500 nm). (ALL FIGURES ARE COURTESY OF THE AUTHORS)

The EE and DL of tamoxifen-loaded SLN were 89.98 ± 1.5% and 17.99 ± 1.9%, respectively (see Table I). The release profile of tamoxifen-loaded SLNs in human plasma is shown in Figure 4. The release rate remained low for the first 8 h, increasing by a mere 2% for that period. Immediately after 8 h, there was a sudden burst of drug release approaching 10% by 11 h after drug incorporation.

Figure 2: Thermograms of bulk lipid, solid lipid nanoparticles (SLNs), and tamoxifen-loaded SLNs (TAM-SLN). The thermograms were recorded within seven days of preparation (scan rate: 5 °C/min).

The cytotoxicity test suggested that tamoxifen-loaded SLNs had an equally efficient cytotoxic effect on MCF-7 cells compared with free tamoxifen. The IC₅₀ of tamoxifen-loaded SLNs on breast-cancer cell lines was generally lower than those for free tamoxifen (see Table II).

Figure 3: Wide-angle X-ray diffraction patterns of bulk lipid, solid lipid nanoparticle (SLN), tamoxifen (TAM), and TAM-loaded SLN.

Discussion

Particle size is an important characteristic for pharmaceutical applications because it significantly affects in vitro and in vivo studies (11). When tamoxifen was incorporated into SLNs, the increase in particle size suggested that loaded tamoxifen was either adsorbed onto the particle surface or entangled in the aliphatic chains of triglycerides. Zeta potential is also an important factor when evaluating the stability of colloidal systems (12). In the presence of 1 mg of tamoxifen, some of the negative charges were neutralized by the complex formation, thus leading to a less negative or positive zeta potential (see Table I). The positive charge also might be raised by the tamoxifen amino group and by tamoxifen localization on the surface of SLNs (13). The TEM image shows that some particles were in the 40–100-nm diameter range (see Figure 1). This particle-size distribution could allow regional drainage if it is directed into or close to the primary tumor or surrounding tissues attacked by cancer cells (14).

Figure 4: Entrapment efficiency of three batches of tamoxifen-loaded solid lipid nanoparticles. Mean values are represented (n = 3).

With regard to the melting point and crystallization behavior of SLNs, incorporating tamoxifen reduced the melting point from 57.88 to 56.56 °C. Tamoxifen thus is probably in an amorphous state and not crystalline (see Figure 2). A substance with a less ordered crystal or amorphous state requires less energy to overcome lattice forces, and perfect crystalline substances require high melting enthalpy. Hence, the lipid-phase of SLN is less-ordered crystal than the bulk lipid (15). The shape of the bulk-lipid DSC thermogram was a sharp trough, whereas those of the drug-free SLNs and tamoxifen-loaded SLNs were broader (see Figure 2). The shape of the latter could be associated with the surfactant and the dispersion of lipid, and the DSC thermogram of tamoxifen-loaded SLNs displayed only one endothermic melting point (see Figure 2), also indicating dispersion (9). The WAXD pattern for the bulk lipid was different from that of the nanoparticle, showing relatively sharper peaks than for SLN (see Figure 3). In the tamoxifen-free and tamoxifen-loaded SLNs, less-ordered crystals were predominant, and the amorphous state may contribute to high drug-loading capacity (15). This study showed that the tamoxifen was fully entrapped into the SLN during preparation.

Table II: The half maximal inhibitory concentrations (IC50) of tamoxifen (TAM) and tamoxifen-loaded solid lipid nanoparticle (SLN) formulations on MCF-7 cells.

The EE of tamoxifen-loaded SLNs was quite high (i.e., 89.98%). The palm oil used to prepare the SLN dispersion produced the highest entrapment efficiency. Triglycerides with various fatty acids offer relatively better drug solubilization (16). In 2004, Wong et al. used doxorubicin, verapamil HCl, propranolol HCl, and quinidine sulfate in SLNs stabilized with tween 80, and showed that increasing drug concentration led to a significant increase (p < 0.05) in DL and significant decrease (p < 0.05) in EE (17). These effects were a result of reduced SLN dispersions and high solubility of the drug in high lipid concentrations. Other researchers showed a positive correlation between particle size and drug loading (18, 19).

Figure 5: Drug loading of three batches of tamoxifen-loaded solid lipid nanoparticles (SLN). Mean values are represented (n = 3).

Among the factors that support fast drug release from SLNs are a large surface area, small molecular size, low matrix viscosity, and short diffusion distance of the drug (20). In the authors' study, however, the release of tamoxifen from SLN, which contains a lipid matrix, was retarded. Characterization of tamoxifen-loaded SLNs suggested that tamoxifen was either encapsulated within the matrix and membrane of palm oil or entrapped in aliphatic chains. This situation could explain the burst release of the drug from SLNs after eight hours. Considering that SLN is solid at room temperature and that the incorporated drug is released relatively slowly, SLNs have potential as a sustained-release drug carrier. When tamoxifen was incorporated into the SLN carrier system, its antitumoral activity was maintained, suggesting that SLN is a good drug carrier. Tamoxifen-loaded SLNs showed an equally efficient cytotoxic activity against MCF-7 cells, compared with free tamoxifen, and the IC₅₀ of tamoxifen-loaded SLNs was generally lower than that of free tamoxifen. This result indicates that tamoxifen's cytotoxicity may result from improved drug internalization through encapsulation into the SLN matrix and endocytosis (21). A previous study had a similar finding, with reduced MCF-7 cell viability in the presence of tamoxifen-loaded SLN (1). It seems that the improved cytotoxicity of the incorporated drug did not depend on the composition of the SLN. In fact, the IC₅₀ value of drug-loaded SLNs composed of different materials was lower than that of the free drug solution (18).

Figure 6: Human plasma-concentration profile of tamoxifen-loaded solid lipid nanoparticles (SLN). Mean values are represented (n = 3) ± standard deviation.

Conclusion

The tamoxifen-loaded SLNs showed high entrapment efficiency and drug loading. Characterization of tamoxifen-loaded SLNs indicated that the drug was encapsulated within the membrane of palm oil or entrapped in aliphatic chains. The authors concluded that SLN can serve as a good drug carrier for the sustained released of tamoxifen.

Acknowledgment

The authors would like to thank Condea and Lipoid for their kind support with materials, and the Universiti Putra Malaysia for financial support and facilities.

Roghayeh Abbasalipourkabir* is a biochemistry lecturer, and Aref Salehzadeh is an associate professor of toxicology, both at Hamadan University of Medical Science, Hamadan, Iran, tel. +98 811 251 4227, fax +98 811 827 6299, rpourkabir@hotmail.com. Rasedee Abdullah is a professor of biochemistry at University Putra Malaysia.

*To whom all correspondence should be addressed.

Submitted: Apr. 9, 2010. Accepted: Nov. 22, 2010.

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Citation: When referring to this article, please cite it as "R.Abbasalipourkabir, .Salehzadeh, R.Abdullah, "Delivering Tamoxifen Within Solid Lipid Nanoparticles,"Pharmaceutical Technology 35 (4) 74-80 (2011)."