Chemicals and reagents
A standard CA preparation (> 98% purity, 110783–200503) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). CA used for the preparation was purchased from Nanjing Zelang Medical Technological Development Co. Ltd in China (> 98% purity). Glyceryl monostearate was obtained from Hebei Gaobeidian Chunguang Chemical Reagent (China). Poloxamer 188 (F-68) was obtained from Sigma Chemicals (St Louis, MO, USA). Lecithin (Qrs95, Lot: A014989601) was purchased from Shanghai Taiwei Limited, Shanghai, China.
HPLC-grade methanol was purchased from Fisher Scientific Co. Ltd. (Dubuque, IA, USA). Absolute alcohol was of analytical reagent grade (Beijing Shiji, Beijing, China). All other reagents were of analytical grade (Beijing Chemicals, Beijing, China).
The exact drug content of the SLNs was determined using a pump L7100 and a Shimadzu SPD-10AVP UV-detector (Shimadzu, Japan). An SLC-10AVP (Shimadzu) equipped with a C-18 reversed-phase chromatographic column purchased from Dikma (China) (250 × 4.6 mm; 5 μm particle size) was used. The column was maintained at 30°C throughout the elution process, using a mobile phase comprising methanol and water 4:6 (v/v) at a flow rate of 1 mL/min. The detection wavelength was 228 nm. The presence of other materials did not interfere with the analysis of CA.
GC-MS was used to determine the concentration of CA in plasma. The GC/EI/MS system consisted of a Trace GC with a Thermo DSQ MSD (Thermo Finnigan, CA, USA). Samples were injected in splitless mode on a DB-5MS analytical column (30 m × 0.25 mm i.d. with 0.25 μm film thickness, J. & W. Scientific, USA). The carrier gas was helium with 99.999% purity at a flow rate of 1 mL/min. The injector temperature was set at 250°C. The column temperature started at 60 °C, was held for 1 min and then initially increased at 6°C/min to 220°C and then to 280°C at 20°C/min. The temperatures of both the source and MS Quad were at 250°C. The MS was operated in single ion monitoring (SIM) mode with electron impact ionization. CA and internal standard (IS; clofibrate) ion fragments monitored were m/z 128.
Male Sprague–Dawley rats (SCXK (jing) 2007–0001; 200 – 250 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) for the animal experiments. Animals were kept in a normally controlled breeding room with standard laboratory food and water for one week prior to experiments. The rats were maintained according to internationally accepted principles of laboratory animal use and the study was approved by the Beijing Animal Care Committee. Twelve rats were randomly divided into two groups for the pharmacokinetic study. Another twelve rats were randomly divided into four groups for the irritation study.
Preparation of the SLNs
Various samples of 2 mg cantharidin, 4 mg lecithin, 10 mg glyceryl monostearate and 4 mg cholesterol were dissolved in 10 mL alcohol, and the solution evaporated under reduced pressure to form a thin layer of uniform film at the bottom of the bottle. The residue of the aqueous phase, containing F-68 (0.5%) and Tween-80 (2%), were added to allow the film to expand and disperse for 30 min, and the mixture further dispersed ultrasonically for 60 min to obtain the CA-SLN suspension. On the basis of optimization for single factors, a central composite design was used for further optimization. The amounts of CA, F-68, lecithin, and glyceryl monostearate were used as the tested variables or independent variables, and encapsulation yield (EY), drug content (DC) as the evaluation indexes according to the following equations:
where WT and WF are the weights of total drug in SLNs and free drug in the ultra filtrate after centrifugation, respectively; and WO is the weight of nanoparticles.
Transmission electron microscopy
SLN dispersions (10 μL) were placed on a copper network layer on a carbon film. The sample was dried under room conditions before imaging the SLNs with a transmission electron microscope (JEM-1400 Transmission Electron Microscope, JEOL Ltd., Japan), operating at an acceleration voltage of 200 kV.
Determination of mean diameter and surface charge
The mean particle size (z-average) of the SLNs was measured by photon correlation spectroscopy (Malvern Mastersizer 2000, U.K.) by a helium-neon laser with a wavelength of 633 nm. Photon correlations of spectroscopic measurements were carried out at a scattering angle of 90°. A 1:50 dilution of the formulations was made with double distilled water before measurement. The zeta potential of CA-SLNs was determined with a zeta potential instrument (32BIT, Brookhaven, MA, USA).
Drug encapsulation efficiency and drug loading content determination
Free CA (not loaded within the CA-SLNs) was separated by an ultrafiltration centrifugation technique (HERAEUS Labofuge 400R, Thermo Scientific, U.S.A.). 2 mL of SLN solution loaded CA was added to the centrifugation technique and centrifuged at 12500 × g for 15 min at 4°C and the supernatant was then transferred to a clean tube and filtered with a 0.45 μm membrane. TrionX-100 (0.5 mL, 10%) was added into 2 mL of CA-SLNs colloidal solution and vortexed for 5 min to obtain the total CA. Free and total CA concentrations in the CA-SLNs were measured by HPLC.
The CA-SLNs colloidal solution was withdrawn by ultracentrifugation and freeze-dried to determine the drug loading content by accurately weighing the residue. The EY and DC were then calculated using equations (1) and (2), respectively.
Mucous membrane irritation experiment
Four groups of three rats had free access to water but were fasted for 24 h before drug administration and for 4 h after drug administration. All samples including physiological saline (negative control group), salicylic acid, free CA and CA-SLN, were dispersed separately in 2 mL of physiological saline and then administered orally. The rats were killed via CO2 inhalation. The intact stomach was removed and cut into pathological sections.
In vitro drug release
CA release from SLNs was performed in phosphate buffered saline (PBS; pH 6.8) by a Franz diffusion cell. A cellulose membrane was mounted between the donor and receptor compartments. The donor medium consisted of 2 mL CA-SLNs solution. The receptor medium consisted of 8 mL of 1.5% PBS in pH 6.8 buffer to maintain sink conditions during the experiments. The available diffusion area between cells was 2.84 cm2. The stirring rate and temperature were kept at 100 rpm and 37 ± 5°C, respectively. At appropriate intervals, 200 μL aliquots of the receptor medium were withdrawn and immediately replaced with an equal volume of fresh buffer. The amount of drug released was determined by HPLC. All operations were carried out in triplicate.
In vivo administration studies
Two groups of six Sprague–Dawley rats were allowed free to access to water but were fasted for 24 h before drug administration and for 6 h after drug administration. CA and CA-SLNs were dispersed in 2 mL of physiological saline and then administered orally. The doses were 0.1 mg·kg-1 as CA. Blood samples were withdrawn from the jugular vein of rats at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 h after dosing. Aliquots of 0.2 mL of plasma samples from rats in a disposable Eppendorf tube, were combined with 200 μL HCl (6 M), 28 ng of IS (2.8 μg·mL-1) and 2 mL ethyl acetate. After vortexing for 90 s, the tube was centrifuged at 12500 × g for 15 min. The supernatant (0.8 mL) was transferred to a clean tube and evaporated to dryness under a stream of nitrogen gas at 30°C. The residue was dissolved in 50 μL ethyl acetate and then injected into the GC-MS system for analysis.
The AUC (area under the time-concentration curve) was calculated using the linear trapezoidal rule (Microsoft Office Excel 2007, U.S.A.) from zero to the last plasma concentration. The maximum plasma concentration, Cmax, and the time at which it occurred, Tmax, were obtained directly from the actual observed data.