Novel Liposomal Gefitinib (L-GEF) Formulations

Materials and Methods

Reagents. Egg phosphatidylcholine (EPC) and hydrogenated soy phosphatidylcholine (HSPC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (Chol), chloroform, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Monomethoxy polyethylene glycol 2000-distearoyl phosphatidylethanolamine (mPEG-DSPE) was purchased from Genzyme Pharmaceuticals (Cambridge, MA, USA). Gefitinib was purchased from LC Laboratories (Woburn, MA, USA). Phosphate-buffered saline (PBS) was purchased from Fisher Scientific (Pittsburgh, PA, USA). HPβCD, Sepharose CL-4B chromatography media and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture. A549 human lung cancer cells were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Preparation of L-GEF. L-GEF was prepared by two different methods, with and without pH gradient-assisted loading. A mixture of EPC or HSPC, cholesterol, and mPEG-DSPE (55/40/5 mol/mol), and gefitinib at a lipid to drug weight ratio of 20:1 were dissolved in chloroform and subsequently evaporated at 35°C to form a thin film. The resulting lipid film was rehydrated with PBS (pH 7.4), 0.3 M (NH4)₂SO₄ solution, or 0.3 M (NH₄)₂SO₄ plus 0.1 M HPβCD, and incubated at room temperature for 30 min. The generated multilamellar vesicles (MLV) were freeze-thawed three times and extruded five times through 0.2 μm pore size polycarbonate membranes on a Lipex extruder (Northern Lipids Inc., Vancouver, BC, Canada). In order to form a transmembrane gradient, solution outside of the liposomes was removed by size exclusion chromatography (SEC) or by tangential flow diafiltration against PBS, pH 7.4, using a MicroKros Hollow Fiber Module (Spectrum Laboratories, Rancho Dominguez, CA, USA) with a molecular weight cut-off (MWCO) of 50 kDa.

Particle size determination. The size distribution of the liposomes was determined by dynamic light scattering on a NICOMP Submicron Particle Sizer Model 370 (NICOMP, Santa Barbara, CA, USA). All particle size data refer to volume-weighted averages. The zeta potentials (ζ) of the liposomes were determined on a ZetaPALS instrument (Brookhaven Instruments Corp., Holtsville, NY, USA).

Determination of DLE. The free drug was removed by SEC on a Sepharose CL-4B column. The amount of gefitinib incorporated into the liposomes was determined at 252 nm using a Shimadzu UV-160U UV-VIS Recording Spectrophotometer (17). In short, the liposomes were dissolved with 80% ethanol containing 0.1 M HCl. The DLE was calculated using the following equation (18):

Gefitinib release experiments. The in vitro release of gefitinib from the liposomes was determined using cellulose membrane dialysis (MWCO 3 kDa) (19). Briefly, 0.5 ml of liposomal suspension combined with 0.5 ml of human plasma was placed in a cellulose dialysis membrane bag (Viskase Co., Chicago, IL, USA), which had been pre-hydrated by soaking in water overnight and washed with deionized water. The dialysis bag was then placed in 500 ml PBS. Drug release was monitored at 37°C and drug release from the liposomes at 1, 2, 4, 8, 12, 24, 36, 48 and 72 h was determined at 252 nm. At the given time points, 20 μl aliquots were withdrawn from the dialysis bag for analysis. At 24 h and 48 h, the external medium was removed and replaced with fresh buffer in order to maintain sink condition.

Stability of the liposomal formulation. Formulation stability was evaluated at 4°C for a period of 30 days. The DLE, particle size, and zeta potential of the liposomes were determined on days 0 and 30.

Cytotoxicity assay. In brief, A549 cells (0.5×10⁴) were plated in 96-well cell culture plates in RPMI-1640 medium containing 10% fetal bovine serum for a total volume of 100 μl per well. Free gefitinib was dissolved in dimethyl sulfoxide (DMSO). Subsequently, 100 μl medium containing 1:4 serial dilutions of L-GEF and free gefitinib were added to the cells. The cells were incubated at 37°C for 48 h before adding 20 μl MTS solution to each well and the cells with incubation at 37°C for an additional hour. The absorbance was measured at 490 nm on an automated plate reader. The half-maximal inhibitory concentration (IC₅₀) of L-GEF and free gefitinib were calculated.

Pharmacokinetic studies. Imprinting control region (ICR) mice (Charles River Lab, Wilmington, MA, USA) were treated with free gefitinib or L-GEF at 10 mg/kg body weight via tail vein injection. The free gefitinib was dissolved in DMSO and further diluted with 4.5% glucose (pH 4) to 3.5 mg/ml for i.v. administration. Blood samples were collected in heparin-containing tubes at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h. Plasma was isolated by centrifugation at 4000 ×g for 10 min and stored at −20°C. Gefitinib in plasma was extracted and analyzed by electrospray ionization liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with a method previously reported (20). WinNonlin Version 3.2 (Pharsight Co., CA, USA) was used to determine pharmacokinetic parameters, including area under the curve (AUC), total body clearance (CL), and plasma half-life.