J. Pharm. Pharmacol. 1999, 51: 1123±1134 # 1999 J. Pharm. Pharmacol.
Skin Delivery of Oestradiol from Deformable and Traditional
Liposomes: Mechanistic Studies
GAMAL M. M. EL MAGHRABY, ADRIAN C. WILLIAMS AND BRIAN W. BARRY
Drug Delivery Group, Postgraduate Studies in Pharmaceutical Technology, The School of Pharmacy,
University of Bradford, Bradford, BD7 1DP, UK
The drug finds it difficult to cross the barrier of skin provided by stratum corneum. The lipid vesicles have to cross this barrier which it finds difficult to overcome. Hence, most of the effect of these vesicles 1.e Liposomes is found in outer skin layers like epidermis and dermis. A new type of vesicle called transferosome is found to penetrate the intact skin, thus, giving transdermal effect. In vitro skin studies using both conventional liposomes and transferosomes is investigated in this study. Oestradiol was used as a model drug to investigate the mechanism to optimise its delivery via skin. Five mechanisms of these vesicles were investigated which are free drug mechanism, penetration enhancement, drug uptake by skin, intact vesicle permeability and . in drug free mechanism, the amount of free drug permeating the skin is calculated. Pretreating the skin with empty vesicles helped to study the penetration enhancement. Dipping stratum corneum in different formulations of liposome helped to determine the drug uptake. It was seen that vesicles of the size 200-300 nm can penetrate through the skin. The vesicles bigger than 500 is believed not to permeate through the skin. Different edge activators or surfactants like tween, span and sodium cholate are used to prepare transferosomes. Cholesterol is mixed in conventional liposomes as it is found to stabilise the vesicles
The vesicle were prepared using hand- shaking method. In this the lipid mixture was dissolved in ethanol or 2:1 ethanol : chloroform ration which were the organic solvents. These organic solvents dissolved the cholesterol and lipid. Oestradiol, the model drug was then added to this mixture which was previously radiolabelled. The mixture was then kept in rotary evaporatot. After complete evaporation of the organic phase, the thin film was hydrated with water and ethanol in 7% v/v. the vesicles were then then allowed to get swollen.
Smaller vesicles were prepared using bath sonicator at room temperaturefor 30mins. All the vesicle sizes were homogenized using 10 times extrusion using 200 and 100nm polycarbonate membranes.
Determination of entrapment efficiency
The entrapped and unentrapped drug were separated using mini column centrifuge. Sephadex gel was formed by soking Sephadex for 5h in wter. Whattman pads were used tas filter in bottom of the barrels. Centrifugation was done at 3000 rev per min for 3 minutes. Liposomes were recovered from the first and second stage of centrifugation.
Determination of drug release
The amount of free drug released by mini- column centrifugation was calculated indirectly from the amount of drug entrapped. The amont of drug entrapped helps to calculate the unentrapped amount. Unentrapped drug was calculated by considering the amount at zero time as initial amount.
Determination of vesicle size
Sizing was done using zeta sizer (photon correlation spectroscopy). Here the samples prepared in distilled water was filtered through 2µm membranes
Preparation of human skin membranes
Mid-line Caucasian skin samples were used for this purpose. These samples were stored at -20°C in vacuum. Heat separation technique was used to prepare epidermal membrane. The skin was heated at 60°C for 45 minutes in water bath which was then peeled off the underlying dermis. Stratum corneum was also prepared.
Results and discussion
This is expressed as percent entrapment of entrapped drug. The lipophilic drug estrodiol showed high entrapment efficiency. Phospholipid concentration, cholesterol and surfactant affected this entrapment efficiency. 99% was the maximum entrapment efficiency achieved.
The release of progesterone and steroid was found to be negligible for about 60h in DPPC liposomes. Steroid release was checked for diffusion lipid composition. It was found that in the formulation’s stable period negligible release was noted. In conventional liposome, 6% drug was released in 48 hours. Stabiliser like cholesterol was found to decrease drug release.
Use of deformable vesicles using surfactant showed sigmoidal shapes. The leaks were found to be greater due to leaky membranes. It was concluded that the lipid composition determines the entrapment of oestrodiol. Cholesterol decreased drug release whereas surfactants increased the drug release.
The average size of unilamellar vesicles was found to be 127 to 146nm irrespective of the liposome formulation.. the result is after sonication or manual extrusion. The sizes of multilamellar vesicles were dependant on the formulation. Vesicles with cholesterol showed greater size while with surfactants showed smaller size.
Delivery through skin
Deformable vesicles were found to be giving optimal delivery via skin. Data analysis were plotted of amount permeated against time. Deformable vesicles with cholate delivered the maximum amount of drug. The traditional liposomes only showed superficial drug delivery. deformable vesicles increased the skin permeation more than the partitioning of the compound.
In this experiment it was concluded that oestradiol delivery through vesicle is better than saturated aqueous solution. Deformable vesicles are found to be more efficient fir drug delivery via skin. It provided deeper drug penetration through stratum corneum which traditional liposomes failed to do.
Wen-Hua HUANG, Zhi-Jun YANG,* Heng WU, Yuen-Fan WONG, Zhong-Zhen ZHAO, and Liang LIU
Summary by Sneha Subramanian
In this experiment, liposome formulation containing Salbutamol sulphate (SBS) is prepared in dry powdered form. This formulation is used in DPI for the treatment of asthma. Asthma is a pulmonary disease which can be treated with the drug SBS. It is widely used for the treatment of various pulmonary diseases like bronchial asthma, chronic bronchitis and emphysema. It is given in the form of injections, oral dose and aerosol. When taken orally or as injection, SBS is digested by the enzymes in the liver. These routes are not used these days. Inhalation is the best method as drug is directly deposited in the pulmonary track. By conventional delivery of SBS by earosols, it shows the effect in 5-15 minutes, but it is not prolonged. It needs to be repeated in every 4-6 hours. There is therefore a need to improve the dosage to show a prolonged effect and improve the treatment strategy
Delivery of SBS using liposome is more preferred due to its capacity of sustained release. Liposomes have no side effect and it effects the drug’s pharmacokinetics and pharmacodynamics. It enhances the drug uptake by delaying drug clearance. It’s surface viscosity prolongs the release of entrapped drug and decreases its clearance from the pulmonary track. Ry powdered inhaler (DPI) is used for the aerosol delivery of SBS. In this study liposomal formulation is studied to treat asthma.
In this experiment the phospholipid gel was prepared using different mass ratios of SPC and aqueous SBS (160.1mg/ml) as 1:1 to 1:3. They were mixed together and allowed to swell in water bath at 60° for 2 hours. This mixture was then stirred with a homogeniser until it formed a semisolid vesicular phospholipid gel (VPG) these VPGs were again allowed to swell in water bath at 60° for 2 hours. These VPGs are hydrated with water and cryoprotectant solutions to form liposomal dispersions. The encapsulation efficiency of different concentration of SPC is shown in the graph below.
Encapsulation Efficiency of SBS in SPC VPG Liposome Suspensions, prepared Using Different SPC Concentrations
Different kinds of cryoprotectants like sucrose, mannitol, lactose and glucose were used for the lyophilisation of VPGs. Different ratios of cryoprotectants like 1:1 to 1:6 were used and frozed at -20°. These frozen mixture was then put in freeze dryer for 48 hours to get liposomal powder. The temperatire in freeze dryer is -50° and vacuum was 133×10-3 mBar. The encapsulation od SBS using different ratios of cryoprotectant was calculated and shown in the table below.
After the preparation of different kind of liposomes using various ratios of cryoprotectants, their invitro deposition profile were studied. The formulation giving different entrapment of drug SBS according to table.1 was selected. All the cryoprotectants- lactose, sucrose, mannitol and trehalose were prepared in their optimal mass ratios of SPC. The resultant porous cake was sieved through 400 mesh sieves. These sieved liposome powder was then mixed with 1:5 ratio of lactose. The invitro deposition studies of these 4 formulation of liposome and cryoprotectant was studied using twin stage impinger. The fine particle fraction (FPF) was determneind for all 4 formulations. Lactose was selected as the most appropriate cryoprotectant among the 4 cryoprotectants used.
Further the effects of different amount of lactose was studied (63-106µm). The process of preparation was similar- 1:1 to 1:7 ratios of lactose and liposome powder was used. FPF of each formukation was determined.
In this experiment, Micron centrifugal filter device was used to determine encapsulation efficiecy. The liposome preparation was centrifuged for 15mins at 17400 x g, 6°C. UV spectrophotometer at 276nm was used to determine the concentration of SBS after being diluted with 1450µl of ethanol. Encapsulated drug in liposome was determined by destroying liposomal membrane by adding 1450µl ethanol to 50µg of liposomal suspensions. The following formule was used.
Encapsulation efficiency = ( total drug- encapsulation drug) x 100%
Twin stage impinge was used for this purpose. 7ml and 30ml of capturing was used in upper and lower stage respectively. Aspinhaler was used as a delivery device with flow rate of 60/ml for SBS for 10 capsules. The SBS calculated after the washing of these chambers were used for the determination of SBS. HPLC was used for this purpose at 276nm. A 25cm x 4.6mm C18 column was used. Mobile phase consists of acetonitrile: water (10:90) with sodium dihydrogen phosphate 30mmol/l. The pH of mobile phase is 3.5 with 1.0 M phosphoric acid. The column temperature was 25°C. The amount of SBS collected in lower stage of impinge was calculated and the results were expressed in percentage of FPF of actual dose.
In this experiment, a series of SPC concentration from 250-500 mg?g was used to investigate its effect on encapsulation. It was seen that encapsulation efficiency increased with the increased concentrations of SPC. This maybe due to following reason- when VPG was hydrated with aqueous medium, the amount of drug entrapped in the control core remained entrapped while those between the vesicular layers was released. This encapsulation efficiency is determined by the ratio of core volume and not to overall aqueous space in VPG. It was also observed that after 400mg/g the encapsulation increases much as the vesicle would have reached the maximum of the total volume of all aqueous compartment. Hence, 400mg/g of SPC concentration was considered as optimum amount and was used for further studies like the inhaler test.
It was observed that the structure of liposome was best performed by 1:4 of SPC to lactose. The encapsulation efficiency of SBS was 80.71% before lyophilisation and 44.35 after dehydration rehydration. Hence, lactose was selected as cryoprotectant in the further studies. HPLC was used to determine SBS content before and after sieving and was found to be same. This indicated that drug was not lost during lyophilisation.
The optimum concentration of cryoprotectant was determined as it affected the structural and functional integrity of liposomes. It also effected the leakage of SBS from liposomes. When 400mg/g was used and then hydrated by lactose solution of 1:4 of SPC: lactose ratio, the entrapment was found to be 80.71. this was found to be very much higher than just using deionised water. This concluded that cryoprotectants helped to preserve the structural and functional integrity of liposome during hydration. In in vitro release study, it was observed that SBS in liposomal formulation showed prolonged release. It was concluded that lipsome helped the drug to be released gradually and eventually.
It was concluded by the preparation of VPG SBS liposome with a high entrapment (more than 80%). This was also stabilised by lyophilisation using cryoprotectants and used in inhalers for ashtama. 4000mg/g of SPC content in VPG and 1:5 ratio of lactose as cryoprotectant carriers was concluded as the bet formulation. Sustained release of SBS due to liposome was also achieved.
Summary 3- Sneha Subramanian
Journal of Controlled Release 84 (2002) 69–78
http://www.elsevier.com/ locate / jconrel
A facile method of delivery of liposomes by nebulization
Tejas R. Desaia, Robert E.W. Hancockb, Warren H. Finlaya ,*
aDepartment of Mechanical Engineering, Aerosol Research Laboratory of Alberta, University of Alberta, Edmonton, Alberta,
Canada T6G 2G8
bDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Received 17 May 2002; accepted 30 August 2002
In this experiment, aerosol technology is studied to treat immune mediated pulmonary disorders. Use of liposomes and its different formulations has increased the potential of aerosol technology along with an array of potent drugs. Use of liposomal delivery has many advantages over conventional delivery like reduced toxicity, sustained release, increased potency and uniform deposition. There are many devices which are used for aerosol drug delivery like dry powder inhalers, dry powder inhalers and nebulisers. Among these nebulisers are considered to be the most appropriate for liposomal drug delivery as the liposome doesn’t require any further processing in this technique. The properties of aerosol developed by the nebuliser will depend on various factors like type of nebuliser, local conditions, aerosol output rate as well as the type of phospholipid used. Various liposomal formulations have been studied in the past few years for carrying therapeutic agents like anti-inflammatory and bronchodilators.
Liposome technology has long-term stability problem. The liposome undergoes chemical changes which lead to the leakage of entrapped drug. Thus, drug delivery through liposomes and nebulisers are hampered. To overcome this stability problem, freeze drying was developed by Darwis and Kellaway which was found to overcome this stability problem. But it is also found that lyophilisation leads to loss of encapsulated drug, thus, reducing the effect of aerosol. It is also found to increase the cost of production which is not feasible from a commercial viewpoint. In the previous study, liposomes were studied to be delivered in dry powdered form and were relied on the spontaneous formation of liposome on dispersion of micronized phospholipid powders. Bronchodilator like salbutamol sulphate and microbial agent like cyproflaxin were used in dry powdered formulation containing lactose, phospholipid and drug. In this study, aqueous dispersions of liposomes are formed for their use in nebulisation. Various liposome formulations using various phospholipids and exhibiting various physiochemical properties were also studied.
- Preparation of dry powder liposomes
The dry powdered liposomes were prepared using phospholipids, lactose and drug in measured quantities. It was then followed by jet milling. The various formulations used in this study were DMPC, DMPG, DPPC and combination of EPC+DMPC and DMPC+DMPG in a molar ratio of 1:1. Drugs like ciprofloxacin, CM3 peptide and salbutamol sulphate in the contration of 7mg/ml, 1mg/ml and 2mg/ml respectively were used. Drug, phospholipids and pharmatose 325M were micronized at a pressure of 90 p.s.i. the powder was then collected in the collection vessel and was stored at a low temperature (-20°C) and low humidity environment for further experiments.
- Preparation of liposomal dispersions
Liposomal dispersions were formed by mixing phospholipid powders and saline and then vortexing them at room temperature for 1 minute. The liposomes were then hydrated and were allowed to anneal for 15mins before nebulisation. The encapsulation was studied by centrifugating the liposomal dispersions and then by assaying the supernatant and pallete by UV spectrophotometry.
Nebulisation of liposome dispersion
The nebulisation of the fresh liposome dispersion was made by PARI LC STAR jet nebuliser. 2.5ml of liposomal dispersions were used in this nebuliser unit. Tghe aerosols produced were collected by respiguard filters. Isotonic water was used to extract this aerosols from the nebuliser. The nebuliser efficiency was calculated using the following formula:
Nebulisation efficiency (%) = aerosolised drug x 100
Total drug placed in nebuliser
Nebulisation tends to loss of drug due to leakage. Hence, drug entrapment was checked after nebulisation which is called as encapsulated delivery. Here, the collected sample was centrifuged and the drug in supernatant and pallete were determined by UV spectrometry. The drug entrapment was thus calculated as the ratio of amount of drug in pallet to the sum of the amount of drug in the pallet and the supernatant.
- Sizing of aerosol droplets
For determining aerosol particle size, the nebulised was directly connected to the cascade impactor. Each sample was nebulised for about 30s and was then allowed to equilibrate. Constant humidity and temperature was maintained in the chamber. Each liposome sample was checked for aerosol size and drug entrapment.
Results and discussion
Liposomes of different formulations were prepared using diffefent concentrations and types of phospholipids. These liposomes were then tested for drug encapsulation, drug leakage, size and aerosol droplet size. Nebulisation efficiencies of different phospholipids and ciprofloxacin is shown in the graph below
It can be seen the EPC+DMPG formulation has the maximum efficiency in nebulisation while DMPC+ DMPG having the least efficiency. Formulation having DMPG is showing the maximum drug entrapment. Thus, it can be concluded that sample having DMPG shows lower drug leakage. The following graph shows the entrapment of ciprofloxacin which is derived from different liposomal formulations after nebulisation.
Again it is observed that formulations containing DMPG shows maximum entrapment delivery. This may be due to the negatively charged bilayers of DMPG that cause electrostatic separation of bilayers, thus improving drug encapsulation. The encapsulation also depends on the phase transition temperature of the phospholipid used. Phospholipids having phase transition temperature below 25°C shows better entrapment as the spontaneous liposome formation is happening in the room temperature. The figure.2 also shows that after nebulisation there is loss of drug by leakage. This may be due to the shock waves and kinematics discontinuities associated with impaction on nebuliser baffles. Leakage may also be due the dilution effect. Liposomal formulation containing DPPC shows maximum leakage. Thus, from the following results it can be concluded that liposomal formulation containing DMPG is most suitable for delivering ciprofloxacin.
The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) for the formulation containing DMPG was also calculated.
Similar studies were carried out for other drugd like CM3 peptide and salbutamol sulphate.
In this experiment, the best phospholipid formulation for drugs lige ciprofloxacin, CM3 peptide and salbutamol sulphate was calculated. The best formulations for these drugs were DMPG, DMPG+EPC and DMPG+DMPC. They showed good drug encapsulation efficiency and minimum leakage after nebulisation. Output of entrapment was also found to be dependent on phase transition temperature of the lipid.