Iftikhar Khan

Two formulations of liposomes were prepared from proliposomes, one containing the lipophilic drug Beclomethasone dipropionate (BDP) and the other was empty liposomes (without drug). These proliposomes were placed in the Air-jet nebuliser and water was used for hydration, the sheered force produced during atomisation in the air-jet cause the formation of liposomes. These liposomes formation were confirmed by transmission electron microscopy (TEM) and the size analysis by mastersizer. BDP was used as a 5 mole %, and it was noticed during differential scanning calorimetery (DSC), multilamellar liposomes are capable of encapsulating this concentration of a drug and no phase separation was observed.

Differential scanning calorimetery (DSC) or high sensitivity differential scanning calorimetery (HSDSC) are well established methods and they were extensively used to study the interaction of drugs with the bilayers. In this method phospholipid molecules are converted from sub gel phase to gel phase (pretransition) which is more sensitive than the conversion of gel phase to the liquid crystalline phase (main transition) where additives are included. So in this method a change of the material (drug) is measured when they are entrapped into the liposomes. Abdelbary Elhissi mentioned in 2006 that liposomes produced by both thin film method and particulate based proliposome method the entrapment of BDP from 2.5 – 5 mol% while alcohol-based liposomes are able to entrapped only 1 mol% of the BDP within the bilayers of multilamellar liposomes. Batavia and Darwis also described 2.52 mol % and 2.24 mol% entrapment of the BDP in the bilayers of liposomes using high performance liquid chromatography (HPLC). And this advocates that DSC can be used as an alternative of HPLC for the quantification of lipophilic drug in the bilayers. In addition, DSC seems more effective and less time consuming method than HPLC as no separation is required for the entrapped and unentraped part for the liposomes and gives more information about the character of material entrapped in the bilayers. The maximum entrapment of liposomes prepared from Dimyristoylphosphatidylcholine (DMPC) was 13.2 mol % for hydrocortisone-21-palmitate described by Fildes et al and 11.25 mol% for cortisone hexadecanoate by Arrowsmith et al. Phase separation of drug in liposome is the identification of instability.

Thin film liposomes are unstable and are difficult to manufacture on a large scale however particulate-based proliposomes are more stable and can be prepared on large scale using different method because a carrier in particulate based proliposomes can be coated with ethanolic or chloroformic solution of phospholipid. The organic solvent can be removed by evaporation to get the dry proliposome powder. Liposomes are effective to be used for pulmonary diseases as they are biocompatible and biodegradable and their size range is very small as well and can be used to act on a particular site of action. Liposomes with larger sizes can be used to target the upper respiratory tract while smaller liposomes can be used for deep lung. Nebuliser can be used to deliver these micro materials into the required site of action. A number of different types of nebulisers can be used like ultrasonic and vibrating mesh nebulisers.

 In this study Air-jet nebuliser is used because proliposomes are hydrated with water within the air-jet nebuliser and the sheered forced of this device cause the formation of liposomes. These liposomes aerosolised by nebuliser passed through twin-stage impinger was compared with the liposomes remain in the nebuliser by DSC.

Particulate-based proliposome was prepared by using sucrose as a carrier and a specific size of sucrose (300-500um) was prepared by using ball mill. Sucrose was placed in a round bottom flask in a rotary evaporator using negative pressure produced by vacuum pump and the temperature was set at 35 C with maximum rotation speed of the round bottom flask in the water bath. A chloroformic solution of DMPC was injected in parts over the carrier to form a uniform film and let it dry, this procedure was repeated until all the solution was used and extra 30 minutes were given to evaporate all the chloroform. These proliposomes were stored at – 20C.

For aeroionisation, proliposomes containing 5 mol% of BDP were placed in the Air-jet nebuliser and deionised water (40 C) was used for hydration and the nebuliser was directed towards the twin-stage impinger (flow rate was adjusted as 60 L/min). Nebulisation was preceded until dryness. Size was checked and the sample was analysed by DSC and TEM. Tem showed the formation of multilamellar liposomes. HSDSC is more conventional than normal DSC as it may not detect the pretransition changes for the liposome. After nebulisation, liquid containing liposome was taken from the twin-stage impinger and from the nebuliser and bench centrifugation was used to separate the liposomal pellets from the water. Pellets were rehydrated according to the phospholipid concentration (62.5 mg/ml) with deionised water. A sample was placed in one vessel and water as a reference in the other pan. The temperature was set from 6-45 C with a rate of 1 C/min. Thermal behaviour was monitored for both pretransition and main transition. Proliposomes containing no BDP has low pre-transition for both delivered and undelivered liposomes and represents that they have similar thermal properties. The proliposome containing BDP gave the same phase transition for delivered and undelivered liposome. BDP containing liposomes cause the broadening of main transition for both delivered and undelivered liposome; it also produced a lower Tpre, ΔHpre, Tm and higher ΔHm for both undelivered and delivered liposome. The almost negligible pretransition of liposome containing 5 mol% showed that the maximum entrapment is almost reached and the broadening of main transition showed the absence of phase separation.

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