METHODS OF PREPARATION OF LIPOSOMES

Under guidance of A. Anil kumar ,Asst. professor, Dept .of Pharmaceutics. Vikas college of pharmacy By k.VijayRanjan,118YISO306 M.pham (pharmaceutics) Vikas college of pharmacy

Contents
1. Introduction 2. Basic steps in preparation 3. Method of preparation A. Passive loading B. Active loading 4. Industrial production of liposomes i.detergent dialysis ii.microfluidizer iii.proliposomes iv.lyophilization 5. Marketed equipments

Introduction
Liposomes are small, spherical vesicles which consist of amphiphilic lipids, enclosing an aqueous core. These are phospholipids which form bilayers similar to those found in biomembranes. An artificial microscopic vesicle consisting of an aqueous core enclosed in one or more phospholipid layers, used to convey vaccines, drugs, enzymes, or other substances to target cells or organs. In most cases the major component is phosphatidyl choline. Depending on the processing conditions and the chemical composition, liposomes are formed with one or several concentric bilayers.

BASIC STEPS IN PREPARATION

CHOLESTEROL

LECITHIN

CHARGE

Organic Solven Dissolve in organic solvent Drying down lipid from organic solvent( Vaccum ) Dispersion of lipid in aqueous media (Hydration) Purification of resultant Liposomes Analysis of final product

Method of Preparation of Liposomes

A.Passive loading: A. Passive loading Involves loading of entrapped agents before or during the manufacturing procedure

B. Active or remote loading Certain types of compounds with ionizable groups and those with both lipid and water solubility can be introduced into the liposomes after the formation of the intact vesicle

A. Passive loading technique

i Mechanical dispersion methods
1.Lipid film hydration by hand shaking,non-hand shaking or freeze drying 2.Microemulsification 3.Sonication 4.French pressure cell 5.Membrane extrusion 6.Dried reconstituted vesicles 7.Freeze-thawed liposomes

ii Solvent dispersion methods

1.Ethanol injection 2.Ether injection 3.Double emulsion vesicles 4.Reverse phase evaporation vesicles 5.Stable plurilamellar vesicles

iii Detergent removal methods

1.Dialysis 2.Column chromatography 3.Adsorption by Bio-beads

a)Hand shaken method in general

b) Pro-Liposomes
In order to increase the surface area of dried lipid film and to facilitate instantaneous hydration, the lipid is dried over a finely divided particulate support like powdered sodium chloride, or sorbitol These dried lipid coated particulates are called pro-liposomes. Pro-liposomes form dispersion of MLVs on adding water ,support is rapidly dissolved and lipid film hydrates to form MLVs. This method overcomes the stability problems of liposomes encountered during the storage of dispersion, dry or frozen form. It is ideally suited for preparations where the material incorporates into lipid membrane.

Freeze-drying
Freeze-drying (lyophilization) involves the removal of water from products in the frozen state at extremely low pressures. The process is generally used to dry products that are thermolabile and would be destroyed by heat-drying. The technique has a great potential as a method to solve long term stability problems with respect to liposomal stability. It is exposed that leakage of entrapped materials may take place during the process of freeze- drying and on reconstitution.

I(B) Mechanical treatments of MLVs :

1) 2) 3) 4) 5) 6) 7)

Micro Emulsification liposomes(MEL) Sonicated unilamellar vesicles (SUVs) French Pressure Cell Liposomes. Membrane extrusion Liposomes Dried reconstituted vesicles(DRVs) Freeze thaw sonification(FTS) Calcium Induced fusion

Micro Emulsification Liposomes (MEL):

First MLV were prepared by these were passed through a Microfluidizer at 40 psi inlet air pressure. The size range was 150-160 nm after 25 recylces. In the Microfluidizer,the interaction of fluid streams takes place at high velocities (pressures) in a precisely defined microchannels which are present in an interaction chamber. In the chamber pressure reaches up to 10,000 psi this can be cause partial degradation of lipids.

Sonicated Unilamellar Vesicles: PROBE SONICATOR: The probe is employed for dispersions, which require high energy in a small volume(e.g., high concentration of lipids, or a viscous aqueous phase) BATH SONICATOR: The bath is more suitable for large volumes of diluted lipids. - Method: Placing a test tube containing the dispersion in a bath sonicator and sonicating for 5-10min(1,00,000g) which yield a slightly hazy transparent solution. -Using centrifugation to yield a clear SUV dispersion. French pressure cell The method involves the extrusion of MLV at 20,000 psi at 4C through a small orifice The resulting liposomes are larger than sonicated SUVs The drawbacks of the method are that the temperature is difficult to achieve and the working volumes are relatively small (about 50 mL maximum)

Bath sonicator

Probe sonicator

French Pressure Cell

VESICLES PREPARED BY EXTRUSION TECHNIQUES (VETs)

The technique can be used to process LUVs as well as MLVs. The size of liposomes is reduced by gently passing them through membrane filter of defined pore size achieved at much lower pressure(<100psi). In this process, the vesicles contents are exchanged with the dispersion medium during breaking and resealing of phospholipids bilayers as they pass through the polycarbonate membrane. The liposomes produced by this technique have been termed LUVETs. This techniques is most widely used method for SUV and LUV production for in vitro and in vivo studies.

 Dried Reconstituted Vesicles (DRV) This method starts with freeze drying of dispersion of empty SUVs and then rehydrating with aqueous fluid containing material to be entrapped It leads to dispersion of solid lipids in finely subdivided form . Liposomes obtained are uni or oligo lamellar Advantages are high entrapment of water soluble component ,loading of bioactives Freeze-Thaw Sonication(FTS) FTS is extension of DRV method. It is freezing of unilamellar dispersion and then thawing by standing room temp for 15 min. SUVs are rapidly frozen and followed by slow thawing.

Dried Reconstituted Vesicles (DRV) and Freeze Thaw Sonication (FTS):

Calcium-Induced Fusion Method

This method is used to prepare LUV from acidic phospholipids. The procedure is based on the observation that calcium addition to SUV induces fusion and results in the formation of multilamellar structures in spiral configuration (Cochleate cylinders). The addition of EDTA to these preparations results in the formation of LUVs The main advantage of this method is that macromolecules can be encapsulated under gentle conditions.

ii. SOLVENT DISPERSION METHODS

Ethanol / Ether Injection Method

4.Reverse Phase Evaporation Vesicles

3.Double Emulsion Vesicles

MLVs obtained In this method outer half of the liposome membrane is created at interface b/w two phases by emulsification of an organic solution in water An organic solution already contain water droplet introduced into excess aqeous medium by mechanical dispersion which forms w/o/w emulsion or double emulsion The vesicles with aqeous core is suspended in aqeous medium two aquous compartments seperated by pair of phospholipid layer whose hydrophobic surfaces face each other across a thin layer of organic solvent

5.Stable Plurilamellar Vesicles SPVs (stable plurilamellar vesicles) formed. A w/o dispersion is prepared with excess lipid as described earlier but drying is accompanied with sonication Redistribution and equilibration of aqeous solvent and solute occurs in b/w each plurilamellar vesicle Internal structure of SPVs is different from MLVREVs which lacks large aqeous core ,entrapped aqeous medium located in b/w adjacent lamellae

iii. DETERGENT REMOVAL MEHODS

1.Dialysis: A commercial device called LIPOPREP (Diachema AG, Switzerland) which is a version of dialysis system is available for the removal of detergents. 2.Column chromatography : by using Gel Chromatography involving a column of Sephadex G-25 3.Use of Biobeads:
-by adsorption or binding of Triton X-100 (a detergent) to Bio-Beads SM-2. -by binding of octyl glucoside (a detergent) to Amberlite XAD-2 beads.

B. ACTIVE LOADING TECHNIQUE Advantages over the passive loading methods: A high encapsulation efficiency and capacity. A reduced leakage of the encapsulated compounds. The technique in general applies the concept of improved loading of drugs due to various transmembrane gradients, such as electrical, ionic or specific salt gradients. This technique brings about improved loading into preformed liposomes using pH gradients and potential difference across liposomal membranes. A concentration difference in proton concentration across the membrane of liposomes can drive the loading of amphipathic molecules.

pH Gradient Method Weak amphipathic bases accumulate in the aqueus phase of lipid vesicles in response to difference in pH b/w inside and outside of liposomes (pHinpHout). pH gradient is created by preparing liposmes with low pH inside and out side the vesicles,followed by the addition of the base to the extraliposomal medium. At low pH side the molecules are predominantly protonated which lowers the concentration of drug in the unprotonated form and thus promote the diffusion of more drugmolecules at the low-pH side of the bilayer. Weak bases like Doxorubicin , adriyamycin vincristine which coexist in aqueous solutions in neutral and charged forms have been successfully loaded into preformed liposomes via pH gradient method

Active loading 0f drug into preformed liposomes by pH Gradient obtained by (NH4)2SO4 Gradient

INDUSTRIAL PRODUCTION OF LIPOSOMES

Of the several preparation methods described in the literature, only a few have potential for large scale manufacture of liposomes. The main issues faced to formulator and production supervisor are presence of organic solvent residues, physical and chemical stability, pyrogen control, sterility, size and size distribution and batch to batch reproducibility.

Sterility of liposomes : Liposomes for parenteral use should be sterile and pyrogen free. For animal experiments adequate sterility can be achieved by the passage of liposomes through up to approximately 400 nm pore size Millipore filters. For human use, precautions for sterility must be taken during the entire preparation process: that is, (1) the raw materials must be sterile and pyrogen free, (2) preparation in sterile system: working areas equipped with laminar flow and (3) use of sterile containers.

(i) Detergent Dialysis A pilot plant under the trade name of LIPOPREPR II-CIS is available from Diachema, AG, Switzerland. The production capacity at higher lipid concentration (80 mg/ml) is 30 ml liposomes/minute. But when lipid concentration is 10-20 mg/ml 100 mg/ml then up to many litres of liposomes can be produced. In USA, LIPOPREPR is marketed by Dianorm-Geraete (Maierhofer, 1985). (ii) Microfluidization A method based on microfluidization / microemulsiftcation/ homogenization was developed for the preparation of liposomes. MICROFLUIDIZERR is available from Microfludics Corporation, Massachusetts, USA. A pilot plant based on this technology can produce about 20 gallon/minute of liposomes in 50-200 nm size range. The encapsulation efficiency up to 75% could be obtained (Mayhew et al, 1985).

Aqeous dispersion of liposomes tends to aggregate fuse or degrades.two solutions are proposed. (iiia) Proliposomes In proliposomes, lipid and drug are coated onto a soluble carrier to form free-flowing granular material which on hydration forms an isotonic liposomal suspension. The proliposome approach may provide an opportunity for costeffective large scale manufacture of liposomes containing particularly lipophilic drugs. (iiib) Lyophilization. Cryoprotectants like trehalose are used in freeze drying ,retains 100% original contents. Freeze-driers range in size from small laboratory models to large industrial units are available from Pharmaceutical Equipment Suppliers.

Mini LipoPrep
The Mini Lipoprep is well suited for the preparation of small volumes of liposomes (0.5 ml to 1.0 ml). It consists of a sample chamber, which is used with a membrane, and a motor to rotate the sample chamber at constant speed. Liposomes are formed during dialysis of the detergent in the lipid/detergent micelles through the membrane. Using the Mini Lipoprep, unilamellar homogeneous liposomes can be prepared in 2 to 3 hours.

Liposomat
Used for preparation of liposomes of volumes from 3 ml to 50 ml or higher. It has two serpentine channels superimposed on each other and separated by a membrane. Each channel has a volume of 3 ml and a length of 3 meters. The mixed lipid/detergent micelles run through one of the channels while the buffer flows through the other channel. Due to controlled dialysis and the high surface area in the system, liposomes can be formed within 30 minutes. The serpentine chambers can also be immersed in a water bath for liposome production at constant temperature.

liposomat

Avanti Mini-Extruder It allows to prepare large, unilamellar vesicles by extrusion in an efficient, rapid manner. The holder/heating block allows the extrusion of unilamellar vesicles at elevated temperatures, which is critical for the production of vesicles from phospholipids with a phase transition temperature above room temperature. Constructed of stainless steel and Teflon, which allows rapid cleaning of all wetted parts, which reduces the "down-time" between production of vesicles from different lipid species.

The Avanti Mini-Extruder

OptiMixTM
LIPPOMIX has developed a novel, patent manufacturing technology for the production of locally acting liposome-encapsulated drugs. Producing consistent and high quality liquid and semi-solid liposomal products Improving manufacturing yields and reducing the cost of goods Eliminating the use of volatile solvents and thus the need for elaborate and very costly solvent abatement systems Allowing manufacturing processes to be scaled-up faster and more efficiently

OptiMixTM

REFERENCES
Target and Controlled Drug delivery Novel Carrier Systems by S.P.Vyas and R.K.Khar. Controlled and Novel Drug Delivery Systems by Sanjay K. Jain and N.K.Jain. Biopharmaceutics and pharmacokinetics a treatise by D.M.Bramhankar and sunil B. jaiswal.

Liposomes preparation methods by Mohammad riaz ,Pakistan Journal of Pharmaceutical Sciences Vol.19(1), January 1996, pp.65-77
Liposome- as drug carriers-International Journal of Pharmacy & life sciencesHimanshu Anwekar*, Sitasharan Patel and A.K Singhai http://www.wikipedia.com/liposomes http://www.avantilipids.com

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