Aceclofenac was purchased from Ipca laboratories Ltd., Mumbai (India) and soyabean lecithin was purchased from Acros organics, New Delhi (India). Mannitol was purchased from Loba Chem. Pvt. Ltd., Mumbai (India). Reagents and organic solvents such as disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, sodium hydroxide, methanol and chloroform were purchased from CDH Pvt. Ltd., New Delhi (India) and were all reagent grade or better.
Preparation of aceclofenac- loaded proliposome:
The proliposome was prepared according to the method reported by Xiao et al
the film-deposition on the carrier method with minor modification. There are various process variables which could affect the preparation and properties of the proliposomes. The optimization of aceclofenac proliposomes was done by preparing the 4 different formulations which vary in amount of lecithin concentration (drug to lecithin ratios of 0.1:0.5, 0.1:1.0, 0.1:1.5, and 0.1:2.0). The effect of amount of lecithin was studied on the entrapment efficiency of drug, percent release of drug, drug permeation study and percent yield. During the preparation of a particular system, the other variables were kept constant. The best result was shown by the formulation comprised of drug to lecithin ratio of 0.1:2.0 which was selected as the optimized formulation. The optimized formulation was further evaluated for optical, scanning and transmission electron microscopy, drug-excipients interaction and for stability. The optimized formulation was prepared by taken 5 g of mannitol powder (sieved with 100 mesh) was placed in a 100 ml round bottomed flask which was held at 60-70° temperature and the flask was rotated at 80-90 rpm. The rotating flask was kept into a water bath at 70-80 rpm and mannitol was dried under vacuum for 30 min. After drying, the temperature of the water bath was adjusted to 20-30°. Aceclofenac (100 mg) and lecithin (2 g) were dissolved in mixed organic solvents (chloroform:methanol, 8:2, v/v) and a 0.5 ml aliquot of the organic solution was introduced into the round-bottomed flask at 37°, after complete drying second aliquots (0.5 ml) of the solution was used up. After the last loading, the flask containing proliposomes was connected to the lyophilizer and subsequently aceclofenac loaded mannitol powders (proliposomes) were placed in a desiccator overnight and then sieved with 100 mesh. The collected powder was transferred into a glass bottle and stored at the freeze temperature until characterization.
Optical, scanning (SEM) and transmission electron microscopy (TEM):
Surface morphology of proliposomes and plain mannitol particles were examined by a scanning electron microscope (SEM, Leo 430, England). After gold coating of proliposome and plain mannitol particles, their surface morphology was viewed and photographed. Formation of liposomes from proliposomes upon hydration was examined by optical microscopy and transmission electron microscopy (TEM). For optical microscopy, a drop of distilled water was added to the proliposome granules on a glass slide without a cover slip, and the process of liposome formation was observed through an optical microscope at 1000 X. Samples for TEM were prepared by adding a drop of distilled water to aceclofenac proliposome granules and shaking the mixture manually for 2 min. A drop of the resultant liposome suspension was placed onto a carbon –coated copper grid, forming a thin liquid film. The films on the grid were negatively stained by adding immediately a drop of 2% (w/w) ammonium molybdate in 2% (w/v) ammonium acetate buffer (pH 6.8), removing the excess staining solution with a filter paper, and followed by a thorough air-drying. The stained films were then viewed on a transmission electron microscope (TEM, FEI-Philips Tecnai 12) and photographed at 10, 000 X.
Aceclofenac content in proliposome and reconstituted liposome:
Aceclofenac content in proliposomes was assayed by an UV spectrophotometric method. Proliposomes (100 mg) were dissolved in the mixture of PBS (pH 7.4) and methanol (1:9 v/v ratio) by shaking the mixture manually for 2 min. one ml of the resultant solution was taken and diluted with methanol upto 10 ml and then absorbance was recorded at 275 nm using spectrophotometer. The entrapment efficiency (EE %) of aceclofenac in the reconstituted liposomes was determined after hydration of proliposomes with distilled water. 10 ml of distilled water was added to the 100 mg of proliposome granules and the mixture was shaken manually for 2 min. For the separation of unentrapped aceclofenac the liposomal suspension was subjected to centrifugation on a cooling ultracentrifuge at 5000 rpm for 30 min. The clear supernatant was siphoned off to separate the unentrapped drug. 1 ml of supernatant was taken and diluted with methanol upto 10 ml and absorbance was recorded at 275 nm using UV spectrophotometer. The sediment (1 ml) was resuspended in Triton X-100 (0.2% v/v) and diluted with methanol upto 10 ml and then absorbance was recorded at 275 nm. The % entrapment was determined by following formula: percentage entrapment = amount of aceclofenac in sediment/total amount of aceclofenac added × 100. Amount of aceclofenac in supernatant and sediment gave a total amount of drug present in system.
Yield of proliposomes:
After complete drying the aceclofenac loaded mannitol powders (proliposomes) were collected and weighed accurately. The yield of proliposomes was determined by formula: percentage yield= total weight of proliposomes/total weight of drug+total weight of added materials×100.
In vitro drug release studies:
The release of drug was determined by using the treated cellophane membrane mounted on the one end of open tube, containing 100 mg of proliposome granules. The dialysis tube was suspended in 100 ml beaker, containing 40 ml PBS (pH 7.4). The solution was stirred at 200 rpm with the help of magnetic stirrer at room temperature. Perfect sink conditions were maintained during the drug release testing. The samples were withdrawn at suitable time interval (at 1, 2, 3, 4, 5, 6, and 24 h). The dissolution medium was replaced with same amount of fresh PBS (pH 7.4) solution to maintain the volume 40 ml through out the experiment. The drug content in the withdrawn samples (1 ml) were estimated at 273.5 nm after making the volume upto 5 ml with PBS (pH 7.4) and cumulative % of drug released was calculated and plotted against time (t).
Drug permeation studies using rat skin:
Drug permeation study was performed after obtaining the approval of the institutional animals ethical committee of Jawaharlal Nehru Cancer Hospital, Bhopal, Madhya Pradesh, India and in accordance with disciplinary principles and guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA).
Abdominal skin of male wistar rats was used in the study. Rats (250-280 g) were anesthesized slightly by ether and hairs removed from the abdominal skin. The rats were sacrified and the abdominal skin of the rat was separated. The skin was stored at −20° until the permeation study, the skin was hydrated in normal saline at 4° and the adipose tissue layer of the skin was removed by rubbing with a cotton swab.
Permeation of aceclofenac from proliposome under occlusive condition:
Franz diffusion cell (with effective diffusion area 3.14 cm2 and 15.5 ml cell volume) was used for the drug permeation studies. Proliposomes (100 mg) are applied onto the surface of skin evenly. The skin was clamped between the donor and the receptor chamber of diffusion cell. The receptor chamber was filled with freshly prepared PBS (pH 7.4) solution to solubilize the drug. The receptor chamber was stirred by a magnetic stirrer rotating at 300 rpm and kept at 37±2°.
The samples (1.5 ml aliquots) were collected at suitable time interval. Samples were analyzed for aceclofenac content by UV/Vis spectrophotometer at 273.5 nm after making proper dilutions. Cumulative corrections were made to obtain the total amount of aceclofenac permeated at each time interval. The cumulative amount of aceclofenac permeated across the skin was determined as a function of time.
Drug-excipients interaction studies by FTIR:
The drug-excipients interaction studies were done by FTIR. Drug, lecithin, mannitol and aceclofenac proliposomes were dried and kept in desiccator until analysis. The IR spectra of drug, lecithin, mannitol and aceclofenac proliposome were recorded using Fourier transform infrared spectrophotometer (Jasco FT/IR-470 plus, Japan).
Stability of aceclofenac proliposomes (drug to lecithin ratio 0.1:2.0):
For the determination of the stability of optimized aceclofenac loaded proliposomes (drug to lecithin ratio 0.1:2.0), formulation was stored in air tight sealed, ambered colored glass containers at various temperatures 8°, room temperature (RT) and 40° for a period of 3 mo. The sample was kept in oven at 40° and in refrigerator at 8°. The aceclofenac proliposomes were sampled at regular time intervals of 30, 60 and 90 d and tested for surface morphology and color changes, residual drug content in proliposomes and entrapment efficiency of reconstituted liposomes.