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.
Chitosan is natural biodegradable polymer and highly mucoadhesion properties which are used to enhance the residence time of drug particles in the nasal cavity, until now the detail of degradation of chitosan microspheres have not been studied in detail. For this purpose four formulation of ropinirole loaded chitosan microspheres were prepared using Mini B-290 Buchi spray dryer in various drug to polymer ratio.
Summary by Sneha Subramanian – 22/06/2012
Current Drug Delivery, 2008, 5, 207-214 207
1567-2018/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.
Sanjay K. Jain*, Yashwant Gupta, Anekant Jain and Sadia Amin
In this experiment, elastic liposomes are used for the delivery of Meloxicam-β-Cyclodextrin vie skin.
Meloxicam has anti-inflammatory activity which is used for the treatment of ostereostasis and rheumatoid arthritis. This drug is poorly soluble in water; hence, its absorption from GI track is prolonged. This leads to many side effects like ulceration and bleeding. Cyclodextrin has hydrophobic central cavity and hydrophilic outer surface. They form inclusion complex with the drug and improves stability, solubility and dissolution rate of the drug. Recently various vesicles are used for the effective delivery of drug via Transdermal route, they include liposomes and niosomeswhich have their own mechanism to deliver drug. These vesicles are obstructed by the layer of the skin called stratum corneum which is very thick. Hence, a specialised vesicle which is elastic is developed to overcome this problem. They vary from liposomes and niosomes by their characterisctic fluid membrane and can easily pass through the tough stratum corneum. They have the ability to sqeeze and bend, hence, can pass though small intracellular space. Transdermal water gradient aids it in this movement of elastic liposomes. This work aims at he delivery of Meloxicam-β-Cyclodextrin complex along with the elastic vesicle to exploit the characteristics of both the carriers.
Meloxicam-β-Cyclodextrin complex was taken in the molar ratio 1:2. This complex was watted in water and was kneaded to paste. This method is called kneading method. This paste was stirred continuously till it peeled off the walls of the mortar. This preparation was then dried in rotary evaporator at 45° which was then sieved. The particles below 50-100µm was used for this experiment.
Solubility studies – This was carried out according to the method given by Higuchi and Connors. In this method excess amount of Meloxicam was transferred into 25ml of aqueous solution containing β –Cyclodextrin from 0-10M/L concentration. It is also shaken for 48hours at room temperature. After filtration, their spectrophotometric studies were carried out at 361nm to estimate the quantity of Meloxicam. Their mixtures were shaken till three consecutive samples estimated same amount of drug.
Differential scanning calorimetry – All samples were taken in 0.5-1mg size range and scanned in temperature 100-270°C/10°/min under nitrogen atmosphere.
X- ray powder Diffractometry (XRD) – was used for the interval of 10-60°/20. The conditions were taken as follows: voltage, 50 kV; current , 200mA; angular speed, 3°/min and angular step of 0.02°
FTIR studies – In this technique, the samples are vacuum drived for 12 hours before studies. The samples- Meloxicam, β-Cyclodextrin and Meloxicam-β-Cyclodextrin mixed with potassium bromine (100mg) and compressed with palate.
Elastic liposomes are prepared by conventional rotary evaporation sonication method. Here HSPC and span80 (85:15w/w) was taken in a clean dry round bottom flask. To this organic solvent- chloroform:methanol (2:1 v/v) was used to dissolve the lipid phase. This organic solvent was removed rotary evaporation above phase transition temperature. This thin film was hydrated with PBS (pH 6.5) containing Meloxicam/ Meloxicam-β-Cyclodextrin complex by rotation at 60rpm for 1hour at 55°C. This was kept for annealing for 2 hours to form multilamellar vesicles (MLVs) and was then was sonicated using probe sonicator for 20mins at 40W. This sonicated vesicles were extruded between 100-200nm polycarbonate membranes. The final lipid drug concentration was 5% wv and 1%wv and similarly Rhodaminered loaded elastic liposomes were prepared.
Morphology was visualised using TEM at voltage of 100KV. For this a drop of sample was kept of the carbon-coated copper and to leave a thin film and before drying was negatively stained with 1% PTA. After the sample was stained and dried and was viewed under TEM. The size and size distribution was determined using DLS method-dynamic light scattering . All measures were conducted in triplicates
Unentrapped drugs were separated using minicolumn centrifuge method. The separated liposomes were separated using 0.1% triton X-100 and was analysed for drug content at 361nm.
These elastic vesicles were extruded through the polycarbonate filter of 50nm diameter with 200ml capacity barrel at 2.5 bar pressure for 10mins. Vesicular sizes were compared before and after extrusion and were repeated.
The in-vitro studies were carried out using dialysis membrane and Franz diffusion cell.
The maximum drug solubility was at 4mM/L concentration of Meloxicam-β-Cyclodextrin. The stability constant (Kc) of Meloxicam and Meloxicam-β-Cyclodextrin complex was calculated
The thermal behaviour of Meloxicam and Meloxicam-β-Cyclodextrin was studies using DSC to confirm the formation of solid complex. The thermogram of Meloxicam-β-Cyclodextrin shows an endothermic peak at 173.8°C while the DSC pattern showed a very diminished melting endotherm of Meloxicam which was shifted to 249°C suggesting a complex formation in 1:2 molar ratio and indicating that the drug has been engulfed in the Cyclodextrin cavity.
The X-ray pattern of Meloxicam and β-Cyclodextrin are shown in the above figure.The XRD of pure of Meloxicam and β-Cyclodextrin are intense and sharp. Thus, inclusion complex indicates their crystalline nature. The inclusion complex shows a completly different pattern which cannot be distinguished from the peak of of Meloxicam. This confirms the existence of new compound at 1:2 molar ratio.
TEM revealed that they are unilamellar and spherical in shape. Precipitation of dry crystals was observed in drug loaded liposomes. This was due to the lower stability of the complex of Meloxicam with native cyclodextrin, part of the drug, less strongly complexed with Cyclodextrin was recrystalized in the aqueous solution during the elastic liposome preparation.
It is observed that the size of drug loaded liposome and empty liposome are almost similar. Larger vesicles are obtained in the presence of Meloxicam-β-Cyclodextrin complex. Polydispersion index is less than 1 indicating narrow size distribution of both formulations. After the entrapment studies , high entrapment of Meloxicam was observed due to its small size as compared to Meloxicam-β-Cyclodextrin complex. This may be due to the lower stability of the complex of Meloxicam with mature cyclodextrin.
The combined effect of using both elastic liposomes and cyclodextrin for Transdermal delivery was studies in this experiment Anti-inflamatory drug Meloxicam was used as a sample drug. Use of cyclodextrin increased the drug’s dissolution property. This increase in water solubility increased its entrapment in the internal aqueous phase of elastic vesicle. This in turn improved the permeability of drug through the skin.
This post by Iftikhar Khan
Corticosteroid drugs are oftenly used as a prophylaxis for asthmatic patients and the method of administration is m ore important for its proper activity. Beclomethasone dipropionate (BDP) produce s some systemic side effects like skin changes, adrenocortical suppression and cataract formation by using dry formulation. In addition, pulmonary delivery of BDP causes Candida infection. For minimising the side effect the drug formulation should be applied to the specific side of action. However, it was noticed that only 10% of the dry powder formulation reaches to the deep lung using Rotahaler, Spinhaler and Diskhaler because most of the drug deposit to the upper respiratory tract from where they swallowed and absorbed systemically through gastrointestinal tract (GIT). So, by preparing the better formulation and drug delivery method reduces most of the side effects. And in this experiment a drug Beclomethasone dipropionate (BDP) was mixed with carrier lactose in two different forms i.e. coarse and fine lactose. The formulation of all three content as a ternary mixture was checked for their homogeneity and with the delivery of BDP. And it was found that the combination of coarse lactose with fine lactose and BDP minimise the aggregation and maximise the dispersion. However, the controlled amounts of both lactose (CL and FL) play a key role in the flowability of powder, dispersion and stability of the powder formulation.
Coarse lactose (CL) was prepared using air jet sieve and the lactose particles were collected from 63- 90 um in size after 15 minutes of shaking. Air-jet mill was used for the micronisation of fine lactose (FL) and particle less than 63 um was passed once through the jet mill. Both the sample were collected and stored at 50 C for 24 hours and then placed in dedicator over silica gel to protect it from moisture and to use for further investigation. Particle size characterization was done by using Scanning electron microscopy (SEM), where sample was placed on the stub and then coated with gold. Six formulation were prepared using CL, FL and BDP altogether with different mixing sequence and using two different ratios i.e. 64.1 : 3.4 : 1 and 65.8 : 1.7 : 1 w/w. A Turbula mixer was used for powder mixing at 90-95 rpm. In each formulation the first two components were mixed for a predetermined time (15 or 60 minutes) and than a third ingredient was added and mixed for the same time (15 or 60 minutes) for both ratios. BDP and CL in an only ratio of 67.1 : 1 was used as control.
High performance liquid chromatography (HPLC) was used for the analysis of BDP. Methanol and water (7:3) ratio was used as a mobile phase with 0.8 ml/min of flow rate and UV detector was set at 239nm. A calibration curve was prepared from standard solution between 0.1-20.0 ug/ml in range. Each formulation containing a sample of 33 + mg was taken and assayed for by HPLC for the content of BDP.
Particle size of aerosolised BDP was measured using twin stage impinger (TI), a Rotahaler was used containing 33 + mg of powder as a delivery device. A mobile phase (7 ml) was placed in each of the upper and lower parts of the twin impinger. Capsule was placed in the Rotahaler and a rubber moulded mouthpiece was used to attach Rotahaler with throat piece of the impinger. A vacuum pump was set at 60 l/min of flow rate and the dose was released for 5 seconds under these conditions and re-run for the further 7 seconds. The same procedure was repeated for 5 capsules in total for each formulation. The capsule shells, mouth piece and the Rotahaler were washed with the mobile phase to analyse it for the retained amount of drug. The samples from both upper and lower parts of TI were collected and analysed using HPLC method.
In conclusion the addition of fine lactose to the mixture of BDP with coarse lactose as a binary mixture did not affect the mixing uniformity. The addition of BDP into the binary mixture of CL and FL (became ternary mixture) however showed a reduced in the homogeneity. And the presence of 2.5 % w/w fine lactose in the formulation exhibited an increase dispersion and deaggregation of BDP during aerosolization with an improved fine particle fraction (FPF) and fine particle dispersion (FPD). In addition, it was observed that 60 minutes mixing is better regardless of the mixing pattern of the ingredients than 15 minutes mixing.
Summary by Sneha Subramanian on the paper
Proniosomes used as a drug delivery
Proniosome is basically a dry formulation in which a carrier is coated with a suitable non-ionic surfactant (2000. Hu and Rhodes) and cholesterol (increase the rigidity of niosomal membrane) by dissolving them into an organic solvent. They can be prepared by slurry method using rotary evaporator. In this method a carrier (sucrose, lactose, trehalose and mannitol) is taken in a round bottom flask and a non-ionic surfactant (span 20, 60, 80) with cholesterol and drug (hydrophobic) is dissolved in organic solvent (ethanol or chloroform). Then this solvent is poured into the round bottom flask to make slurry, if the solvent is not enough to make slurry then extra organic solvent can be used to get the desire material. A vacuum and water bath is used in the rotary evaporator to evaporate the organic solvent and allow the solvent phase to make a layer of lipid phase over the carrier. The proniosome can be flushed with nitrogen to remove all the organic solvent. This method can be used to increase the stability of the formulation. Non-ionic surfactant are used instead of phospholipids to get the higher physical and chemical stability (2000. Hu and Rhodes). These substances are biodegradable, biocompatible and have low toxicity (2003. Youan) (2008. Abdelbary) for example sucrose stearate consists of both polar and non-polar group and the combination is due to the ester formation between these two molecules. Sucrose belongs to carbohydrates class and Stearate from fatty acid (palmitic, lauric, stearic acid). So, it acts the same as phospholipid and can entrapped both hydrophilic and lipophilic drugs (2008.Abdelbary).
It is converted into niosomal dispersion by hydration with aqueous solution before administration (2010. Sankar et al), (1998. Vora et al). Niosomes derived from Proniosomes are better (size distribution) than the niosomes prepared from conventional method (2010.Sudhamani.) (2010. Sankar et al). The Proniosomes-based niosomes are more stable both during storage and sterilization. Proniosomes decreases the problems associated with niosomes like fusion, aggregation and leakage of drug (2008.Abdelbary) (2000.Hu and Rhodes). Proniosomes (dry formulation) enhance the transportation, measurement, distribution and storage (2007. Solanki et al) (2010. Sudhamani), (2008. Abdelbary). And this is the property which makes it potentially more suitable for active pharmaceutical Ingredients (2007.Solanki).
One of the advantages is that proniosomes can be converted immediately into niosomes aerosol by hydration in hot water (2001a. Blazek), (2010. Sudhamani), (2007. Solanki.) and form multilamellar niosomes (2000. Hu and Rhodes.). Proniosomes act as a reservoir (carrier) and have been used for the targeted delivery to control the drug release and to achieve a prolong action. As proniosomes-derived niosomes have the ability to encapsulate both hydrophilic (within bilayer) and lipophilic (in central compartmental core) drug (2000.Hu and Rhodes.) so, verity of drug can be delivered to the targeted area (2010.Sudhamani).
The preparation of proniosomes and their evaluation was done by a number of authors (1998. Vora), (2000. Hu and Rhodes), (2001a and 2001 b. Blazek-welsh), (2001. Fang), (2005.Alsara), (2007. Gupta), (2007. Solanki), (2008. Azeem) and (2008. Abdelbary) for their different drug delivery system. Most of them used Proniosomes for the transdermal studies. Proniosomes are potentially very important because of their stability and large scale production. They were highly prepared for the transdermal application due to the non-toxicity and high penetration of non-ionic surfactant through skin (2010.Sankar. last conclusion). However, in future, they will be using for a verity of drug and target areas.
This is a post by Urwashi Sharad Naik on the paper:
Nanosized ethosmes bearing ketoprofen for improved transdermal delivery
Manish K. Chourasia, Lifeng Kang, Sui Yung Chan
Transdermal delivery of the drug provides excellent route of administration of the drug to enhance the oral absorption of the drug. Our human skin contains the major barrier called stratum corneum layer which is a greatest challenge for transdermal delivery of drugs. Various approaches have been adopted to pass this major barrier such as micro needles, chemicals, surfactants, iontophoresis, chemicals and lipid based systems. The lipid based systems are of the best mechanism to carry out transdermal drug delivery as the lipids are biocompatible to our skin lipids. Conventional liposomes are used for transdermal drug delivery. However, these conventional liposomes are unable to cross the major stratum corneum barrier due to which the drug is unable to delivery deep inside the skin. These conventional liposomes have been modified by introduction of two novel carriers called as transfersomes and ethosomes. Transfersomes are ultradeformable lipid vesicles which consist of one layer of phospholipid and other layer of surfactants which allows them to squeeze through the stratum corneum layer and enhances the transdermal delivery of the drug. One the other hand, ethosomes are the vesicles contain higher concentration of the ethanol which acts as penetration enhancer allowing them to be more flexible than liposomes. The preparations of ethosomes are similar to liposomes but in liposomal preparation cholesterol and phospholipids are present but in ethosomes higher concentration of ethanol is present in place of cholesterol. As reported by Elsayed et al., ethosomes seems to be more superior than tansferosomes with respect to increase the permeability of entrapped ketotifen.
Ketotifen is non-steroidal anti-inflammatory drug. This drug have been tried to pass through other modes of transdermal delivery such as gels/patches which are available in the market. However, the drug delivery has been poorly observed previously. In this experiment, ketotifen have been entrapped in the ethosomal bilayer and the invitro and invivo transdermal delivery of this drug have been studied using varies techniques of characterization such as laser diffraction, zeta charger, HPLC (for drug entrapment), confocal laser scanning microscopy and micro column centrifugation. The in vivo studies were performed using the skin samples of female adult mice.
For in vitro studies, the experimental procedure was setup by placing the cellophone membrane dialysis tubing with both the ends sealed and suspended into a beaker containing PBS solution (pH 7.4)of 100 ml at 37 ± 1 ᵒC. The buffer of the solution was stirred at 45 mins intervals and sample was collected at 24,6,8,10,12,18 and 24 hr time intervals by replacing with equal quantities of fresh buffer and drug contain was analysed using HPLC. Different ethosomal formutions were evaluated for drug release and compared with hydroaloholic drug solutions.
For in vitro studies, the permeation was checked through diffusion cell system consisting of 16 channel peristaltic cassette pump, a circulating water bath, a fraction collector and flow through diffusion cells. Adult Chinese female skin was used in the experiment, the skin was thawed and hydrated with saline solution containing 1% v/v antibiotic antimitotic solution. For experiment the skin was immersed into water bath (60ᵒC) for 2 mins, peeled off and then stored at -80ᵒC.The receptor solution was pumped into the peristaltic cassette continuously through receptor compartment and drained into sample collection tubes. Sample collection was performed at various time intervals.
Penetration of ethosomes were confirmed using confocal laser scanning microscopy. The formulations were loaded with dye called fluorescent probe Rhodamine 123 insread of ketoprofen. Skin sample was mounted between the donar and the receiver compartment and either 1 ml of either hydroalcoholic probe solution was placed in the donar compartment and covered with paraflim to prevent contamination and evaporation at 37oC. Skin was removed and washed after 4 hours and scanned at different increments through Z- axis of confocal laser scanning microscopy.
Drug quantitative studies was performed using HPLC using C18 column (Agilent, 5µm 4.0 x 250 mm) and using a mobile phase of phosphate buffer (pH 3.5) and acetonitrile in ratio of 50:50 at wavemenght 254 nm. The experiments were statistically performed using ANOVA. at p < 0.05 with mean ± standard deviation. The size of the vesicles were seems to be decrease with increase in concentration of alcohol contained. Higher concentration of ethanol confers net negative charges to the vesicular systems. As the keptoprofen is hydrophobic drug, it was expected to be encapsulated within the non-polar region of the bilayer as the bilayer amount increases the drug holding capacity also increases.
|SPC (1%)||Ethanol (20%)||42.9 ±3.7 %|
|(3%)||Ethanol (20%)||63.1 ±5.8 % SPC|
Entrapment efficiency of vesicles
The above table show that the entrapment efficiency of the vesicles increases with increase in phospholipid concentration even though keeping the concentration of the alcohol constant.
In vitro drug release through cellophane membrane shows that the hydroacoholic drug release was 3-4 hours slower than the drug release from the ethosomal preparation indicating that the drug diffusion in ethosomes is rate limiting step. The increase drug release may be due to increase in alcohol contain within the bilayer membrane.