A glucocorticoid steroidal drug Beclomethasone dipropionate (BDP) is used as prophylaxis for asthma and was used for pulmonary delivery to minimise the pulmonary infection. BDP is highly hydrophilic drug and that’s why it entrapped into the lipophilic part of the DPPC containing a long diglyceride fatty acid chain with a phase transition temperature of 41 0C. However, it is observed that the entrapment efficiency of the steroid esters is very low in phospholipids. In addition, it was also reported that the crystalline structure of BDP has improper steric fit in the bilayers and it may leads to the low entrapment and the drug leaks out by extrusion in the liposome. Waldrep use a high ratio of Dilaurylphosphotidylcholine (DLPC) to BDP (21:1) and got the good entrapment of the drug it may be due to the shorter fatty acid chain with having low melting point. For the maximum calculation it is important to completely separate the unentraped drug from the entrapped one, and this process could be harder if the drug is hydrophobic in nature like in case of BDP. The size of the free drug can be eliminated once there is a marked difference between the drug crystal and liposomes, an ultra centrifugation can be operated for the separation of both entrapped and unentraped phases and can combine with filters for complete separation. However, the problem got to noticed was that both liposomes and BDP crystals have the same size and density, in such situation a density gradient medium was introduced for their separation.
In the stage one study, liposomal drop was placed onto the glass slide and a cover slip was placed properly over it and checked it by the microscopy (either from the DIC or cross-polarised microscopy) and the BDP crystals were detected in the aqueous medium. However, the more accurate measurement or analysis was done by using HPLC method. In this procedure methanol and water (75:25) was used as a mobile phase with a flow rate of 2 ml/min and 20 µl injection volume. Sample absorbance was detected by using UV at 238 nm. A calibration curve of BDP from 0 – 20 µg/ ml was prepared and measured by HPLC using methanol as a solvent. For the accurate measurement for the fact that the size and density of liposomes and BDP crystals are same, two different dispersion mediums were used. This concept of different gradient were initially introduced by Fraley et al., that on the basis of difference in densities of dispersion medium the entrapped and unentraped phases can be separated, the density of water at 20 0C is 0.9982 g/ml and deuterium oxide is 1.053 g/ml. But the method of separation in HPLC or deionised water was used by many authors like Meisner, Kevin Taylor and Ma et al.
Proliposome sample was hydrated in water and D2O separately and then left for annealing, a sample was taken from each medium and centrifugation was done using bench centrifuge for specific time at specific rotation per minute. It was found that in water liposomes go to the bottom of the eppendrof while unentraped drug remains in the supernatant but in D2O liposomes were found suspended in the supernatant and BDP crystals at the bottom of eppendrof. Both parts were separated from each other and dissolved in methanol. Samples were analysed and found that water had got more entrapment than D2O and the reason behind that was that in water both liposomes and BDP crystals go to the bottom of eppendrof due to their densities and only some crystals remains in the supernatant, while in D2O the separation was more precise and showed less entrapment in the liposomes.