Dihydroartemisinin (purity > 99%) was kindly provided by Beijing Zhongyan Tongrentang Medicine R&D Co., Ltd. (Beijing, China). Poloxamer 188 was purchased from BASF Corporation (Ludwigshafen, Germany), cholesterol from Pharmacia Biothech (Piscataway, NJ, USA), and soya phosphatidylcholine from Lipoid GmbH (Ludwighafen, Germany). Mannitol, sucrose, and glyceryl monostearate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Hepatic H22 cell line was kindly provided by Zhejiang University of Traditional Chinese Medicine. All other chemicals were of analytical grade.
Preparation of DHA N/O/W emulsion
DHA (150 mg) and lecithin (900 mg) were dissolved in 30-ml alcohol and then mixed with soybean oil (5,000 mg) and glyceryl monostearate (150 mg). The mixture was used as oily phase and warmed up to 60°C and then dropwise added to the aqueous phase containing glycerol (1,100 mg) and poloxamer 188 (900 mg) with agitation at high speed and temperature (8,000 to 10,000 rpm and 60°C). After adjusting the pH from 6 to 7, the primary emulsion was homogenized by microfluid technology at a pressure of 200 to 1,000 bar (8 to 10 cycles) to obtain the DHA N/O/W emulsion with DHA concentration about 3.0 mg/ml. In order to maintain DHA stability, the freeze-dried emulsion was prepared with a complex of sucrose and mannitol (4:1) as lyoprotectant.
Meanwhile, the DHA O/W emulsion was prepared as follows. DHA (49 mg) and lecithin (1,000 mg) were dissolved in alcohol and attached to a rotary evaporator to remove the organic solvent. Soybean oil (5,000 mg) was then added to dissolve the dried film as oil phase. Aqueous phase contained glycerol (1,100 mg); poloxamer, 188 (1,000 mg). The DHA O/W emulsion was prepared as described above.
Characterization of the DHA N/O/W emulsion
Size and zeta potential of the DHA N/O/W emulsion were determined by laser scattering technique using Malvern ZS90 (Malvern Instruments Ltd., Malvern, Worcestershire, UK) after appropriate dilution with double distilled water.
The primary N/O/W emulsion with a size of about 10 μm (before microfluid process), through light microscopy (MPS-30, Leica Microsystems, Wetzlar, Hesse, Germany), was employed to observe the encapsulation of particles in O/W emulsion. Furthermore, morphology of the mid-product of DHA N/O/W emulsion (about 1 μm) was studied with transmission electron microscopy (TEM) (JEM-1200EX, JEOL Ltd., Akishima, Tokyo, Japan). The sample for TEM was dropped onto a 400-mesh copper grid coated with carbon film. The sample was dried in air before TEM observation. The sample for freeze-fracture transmission electron microscopy (FF-TEM) was prepared as follows. A droplet of microemulsion was sandwiched between two copper electron microscopy grids, snapfrozen by immersion in liquid ethane, and then loaded into a double-replica device and immersed in liquid nitrogen (−196°C). The double replica device was mounted on the rotary sample stage of a Balzers BAF-400D freeze etch device (Balzers AG, Balzers, Liechtenstein) cooled to −110°C. Fracturing was carried out at a vacuum of 3 × 10−3 mbar. The fractured surfaces were shadowed with platinum (45°) and carbon (90°). The replicas were then washed in chloroform, methanol, and finally, distilled water. Dried replicas were examined with TEM operating at an accelerating voltage of 80 kV.
Drug loading capacity
The DHA N/O/W emulsion was dissolved in methanol and then injected to the high-performance liquid chromatography (HPLC) system. Chromatographic separation was achieved using a C18 column (150 × 4.6 mm, 5 μm, Agilent Technologies, Palo Alto, CA, USA) with column temperature at 40°C. The mobile phase consisted of 40:60 double-distilled water containing 0.1% acetic acid (Millipore Corporation, Milford, MA, USA)/acetonitrile (HPLC grade, Merck & Co., Inc., Whitehouse Station, NJ, USA). The flow rate was set to 1.0 ml/min, and UV detection wavelength was 210 nm.
The experimental protocols were approved by the Animal Ethics Review Committee of Zhejiang University. The male rabbits (2.0 ± 0.2 kg) were divided into three groups (6 per group). DHA solution, DHA O/W emulsion or DHA N/O/W emulsion was injected via ear vein at a dose of 6.2 mg/kg. Blood samples were collected from the marginal vein on another ear before administration and at predetermined time points after intravenous administration. Plasma sample (100 μl) was spiked with 10 μl of the IS solution (artemisinin, 1 μg/ml), and the mixture was vortexed for 10 s. The sample was extracted with 2-ml ethyl acetate, vortexed for 3 min, and centrifuged for 15 min at 3,500 rpm. The upper layer was transferred into another test tube and evaporated to dryness under a gentle stream of N2 gas at 37°C. The residue was reconstituted in 100 μl of methanol/water (60:40). The concentration of DHA in plasma samples was determined by LC-MS method (ESI, positive mode, Agilent 1100 LC-MSD, Agilent Technologies, Palo Alto, CA, USA). Pharmacokinetic analysis was conducted using Kinetica Software (Version 4.4, Thermo Electron Corporation, Waltham, MA, USA).
In vivo antitumor efficacy of DHA N/O/W emulsion
tumor cells (5 to 6 × 106
cells per mouse) were inoculated subcutaneously to the Institute for Cancer Research (ICR) mice (6 to 8 weeks, 22 to 26 g) at the right axilla. Tumors were measured by a vernier caliper, and its volume (V
) was calculated as follows:
where d and D are the shortest and the longest diameter of the tumor in mm, repectively.
When the tumor grew to a mean size of approximately 300 mm3, mice were treated intravenously (i.v.) with DHA solution, DHA O/W emulsion, or N/O/W emulsion.
Hemolysis test and vascular irritation test
Blood sample was collected from New Zealand white rabbit (2.5 kg) ear vein. A 10-ml aliquot of defibrinated blood and 10 ml of saline were mixed and centrifuged. The supernatant was discarded, and the wash step was repeated three times until no red color was observed in the supernatant. Erythrocytes were diluted with saline to 2% (v
). The DHA N/O/W emulsion at varying concentrations was mixed with 2% erythrocyte solution in equal volume. The morphology of the erythrocytes was observed under the microscope, and the color change of the solution was monitored with spectrophotometry (λ
= 414 nm) for hemolysis evaluation. Hemolysis effect was calculated as follows:
where Asample is the absorbance of the tested sample; Anegative, the absorbance of the negative control (i.e., mixture of equal volumes of 2% erythrocytes and saline); and Apositive, the absorbance of the positive control (i.e., mixture of equal volumes of 2% erythrocytes and distilled water).
For vascular irritation test, DHA N/O/W emulsion with a DHA dose of 6.2 mg/kg was injected to the marginal ear vein once per day for 7 days. At the eighth day, the rabbits were killed by aortic bleeding under pentobarbital anesthesia. The vascular irritation was investigated by histological examination of the injection site.
Each experiment was performed in triplicate, and the values were expressed as mean ± S.D. Statistical analysis was performed using Student's t test. Significant differences are demonstrated by symbols (*, **).