Chemicals and reagent
Standard curcumin and emodin (as the internal standard) were purchased from Fluka (Sigma-Aldrich, Buchs, Switzerland). Poloxamer 188 was purchased from Chemie (Sigma-Aldrich Chemie, Munich, Germany) and glyceryl monostearate was purchased from Tokyo Chemical Industry Co, Ltd (Tokyo, Japan). Soy lecithin was purchased from Shanghai Taiwei Medical Group Co, Ltd (Shanghai, China). Medium chain triglycerides (Lexol®) were sourced from Zhejiang WuMei Chemical Products Co, Ltd (Zhejiang, China). Methanol and acetonitrile (chromatographic grade) were obtained from Fisher Scientific Inc (Waltham, MA). Water for high-performance liquid chromatography (HPLC) was double-distilled. All other chemicals and reagents were of analytical grade and were used without further purification.
Healthy Sprague-Dawley rats weighing 190–220 g were purchased from the Animal Institution, Zhongnan Hospital, Wuhan University. The rats were housed under standard conditions, and provided with fresh water and a commercial diet ad libitum. The animal tests were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. All animal experiments were approved by the local animal ethics committee.
Preparation of CUR-NLCs
Nanostructured lipid carriers, empty or loaded with curcumin, were prepared using an ethanol dripping method.26
Briefly, an aqueous surfactant solution consisting of 1% (w/w) Poloxamer 188 was prepared and heated to 75°C prior to addition of the lipid phase. To prepare the lipid phase, the selected solid and liquid lipid mixture was heated to 5°C–10°C above its melting point. An ethanolic solution of soy lecithin (0.64 mmol) was added under stirring to the melted lipid (1.18 mmol). For obtaining drug-loaded nanoparticles, curcumin (0.32 mmol) was added under mechanical stirring. To obtain nanostructured lipid carriers, the ethanol solution containing lipid, lecithin, and curcumin was dripped into an aqueous surfactant solution with high-speed homogenization (A200-18G-S; Shanghai Angyi Instruments, Shanghai, China) at 10,000 rpm for 15 minutes. The ratio of the ethanolic lipid phase to the aqueous phase was 1:3 (v/v) in the final suspension. The suspension was then dispersed in cold twice-distilled water (100 mL at 2°C–3°C) containing 5% (w/w) sucrose under continuous magnetic stirring at 1000 rpm for 30 minutes. Successively, the nanostructured lipid carriers were submitted to exhaustive dialysis using a Visking 18/32 membrane (molecular cutoff 12,000–14,000 Da), freeze-dried by a lyophilizer (Alpha 1–4; Martin Christ, Osterode am Harz, Germany) and stored at −20°C in a freezer. The dry powder was kept at room temperature for successive characterization. Ethanol was completely removed from the nanoparticles during purification.
Characterization of nanostructured lipid carriers
The mean particle size, polydispersity index, and zeta potential of the nanostructured lipid carriers were determined by dynamic light scattering using a nanoparticle analysis instrument (Delsa™; Beckman Coulter, Fullerton, CA). Morphological examination of the nanostructured lipid carriers was performed using a transmission electron microscope (Hitachi Ltd, Tokyo, Japan).
Differential scanning calorimetry experiments can be used to determine thermodynamic variations related to morphological changes as a result of melting points and melting enthalpies varying with lipid modification. In the present study, crystalline state evaluation of blank nanostructured lipid carriers, curcumin, CUR-NLCs, and a physical mixture of blank nanostructured lipid carriers and curcumin (with same ratio as that of the CUR-NLCs) was conducted using a differential scanning calorimeter (Diamond; PerkinElmer Instruments, Boston, MA). A heating rate of 10°C per minute was used, and the temperature range was 50°C–220°C. According to our previous work, no melting peak is detected in the range of 30°C–50°C.
LC% and EE% were determined by HPLC.27
Briefly, the CUR-NLC suspensions were first dissolved and diluted with anhydrous methanol. The suspensions were then centrifuged at 11,000 rpm for 15 minutes, after which the supernatant was determined by the HPLC method and the total amount of curcumin in the lipid nanoparticles was calculated. To determine the amount of curcumin contained in the nanostructured lipid carriers, equal volumes of the CUR-NLC formulation were accurately dispensed into a 0.9% NaCl solution at a ratio of 5:1 (v/v) to salt out the nonincorporated drug, and the suspensions were centrifuged at 18,000 rpm for 30 minutes. The upper portions of the suspensions were then diluted with anhydrous methanol, sonicated, and centrifuged at 11,000 rpm for 15 minutes. Finally, the supernatant was analyzed under the same HPLC conditions. For sample analysis, chromatography was performed using a Waters Alliance HPLC 2695 series (Waters Technologies, Milford, MA) with separation on a Thermo Hypersil ODS C18 Column (250 mm × 4.6 mm, 5 μm; Hypersil, Thermo Scientific, Waltham, MA). The mobile phase consisted of acetonitrile and water (58:42, v/v) at a flow rate of 1 mL per minute. The run time for the analysis was 10 minutes and the detection wavelength was set at 423 nm. The sample injection volume was 20 μL and the column temperature was maintained at 30°C. The EE% and LC% of the CUR-NLCs were calculated as follows:
In vitro release study of DXMA-NLCs
The dialysis membrane method was used to investigate the in vitro release of curcumin from the CUR-NLC formulation. First, 2 mL of CUR-NLC solution (equivalent to 1.5 mg of curcumin) was transferred into dialysis bags with a molecular cutoff of 3.5 kDa. The bags were suspended in 250 mL of pH 6.8 phosphate buffer (containing ethanol 15% v/v) maintained at 37°C ± 0.5°C in a shaking water bath at 100 rpm. At designated time intervals, 1 mL samples of the dialysis medium were taken for measurement of curcumin by HPLC, and the same volume of fresh medium was then added. The release experiments were performed in triplicate.
Pharmacokinetics and tissue distribution studies of CUR-NLCs
Sprague-Dawley rats received a single intragastric dose of the CUR-NLC formulation or curcumin suspension containing an equivalent curcumin dose of 80 mg/kg. The rats were sacrificed at predetermined time points (15, 30, and 45 minutes, and at 1, 2, 4, 6, 8, 12, and 24 hours) after dosing (five mice were sacrificed at each time point). Blood samples were collected from the orbital plexus into heparin-treated (10 μL, 500 IU/mL) tubes and immediately centrifuged at 3000 rpm for 15 minutes at 4°C. Tissue samples from the heart, lung, liver, spleen, kidney, and brain were collected, washed, weighed, and homogenized. All of the samples were stored at −80°C until later analysis.
Curcumin concentrations in plasma and tissues were determined by HPLC assay. Plasma or tissue homogenates (300 μL) were mixed with 10 μL of emodin (20 μg/mL) as the internal standard. The mixture was vortexed with 3 mL of acetic ether and centrifuged at 4000 rpm for 10 minutes to precipitate the proteins. The supernatant was collected into clean test tubes and evaporated under a stream of nitrogen at 45°C. The residues were dissolved in 100 μL of mobile phase, and 20 μL aliquots were injected into the HPLC system as described above.
Pharmacokinetics and statistical analysis
Pharmacokinetic analysis was carried out using 3P97 pharmacokinetic software (Chinese Pharmacological Association, Beijing, China). The statistical significance of differences in pharmacokinetic parameters between the treatment groups was determined with the Student’s t-test using the Statistical Package for Social Sciences version 12.0 (IBM Corporation, Armonk, NY). Statistical significance was defined to be P < 0.05 or P < 0.01.