A Review on Liposomes

Definition: An artificial microscopic vesicle consisting of an aqueous core enclosed in one or more phospholipids layers, used to convey vaccines, drugs, enzymes, or other substances to target cells or organs.

Introduction

¡  Liposome was found by Alec Bingham of Babraham Institute in Cambridge, England in 1965.

¡  In 1990, drugs with liposome and Amphotericin B were approved by Ireland.

¡  In 1995 America F.D.A approved liposor doxodubicin.

¡  Liposome is a lipid vesicle suspending in the hydro-phase with a diameter around 0.0025~3.5um. The membrane of liposome is made of phospholipids, which have phosphoric acid sides to form the liposome bilayers.

Composition of liposomes:
A. Phospholipids

The most common natural phospholipid is the phospatidylcholine (PC) is the amphipathic molecule and also known as lecithin.

            Naturally occurring phospholipids used in liposomes are:

▪       Phosphatidylcholine

▪       Phosphatidylethanolamine

▪       Phosphatidylserine

            Synthetic phospholipids used in the liposomes are:

▪       Dioleoyl phosphatidylcholine

▪       Disteroyl phosphatidylcholine

▪       Dioleoyl phosphatidylethanolamine

▪       Distearoyl phosphatidylethanolamine

B. Cholesterol:  

¡  Cholesterol can be incorporated into phospholipids membrane in very high concentration up to 1:1 or 2:1 molar ratios of cholesterol to phospatidylcholine.

¡  Being an amphipathic molecule, cholesterol inserts into the membrane with its hydroxyl group of cholesterol oriented towards the aqueous surface and aliphatic chain aligned parallel to the acyl chains in the center of the bilayers and also it increase the separation between choline head groups and eliminates the normal electrostatic and hydrogen bonding interaction.

¡ 

The phospholipids are arranged in such a way that the hydrophilic head is exposed outside and the lipophilic tails are aliened inside. This makes the liposomes water soluble molecules.

Advantages :

¡  Biocompatible, completely biodegradable, non-toxic, flexible and non-immunogenic.

¡  Liposomes supply both a lipophilic environment and aqueous “milieu interne” in one system.

¡  Liposomes have the ability to protect their encapsulated drug from the external environment.

¡  Liposomes help to reduce exposure of sensitive tissues to toxic drugs.

¡  Alter the pharmacokinetic and pharmacodynamic property of drugs (reduced elimination, increased circulation life time).

¡  Flexibility to couple with site-specific ligands to achieve active targeting (Anticancer and Antimicrobial drugs).

¡  Liposomes can be formulated into multiple dosage forms.

¡  Liposomes can encapsulate both micro and macromolecules such as hemoglobin, erythropoeitin, interferon g etc.

Disadvantages :

¡  Production cost is high

¡  Leakage and fusion of encapsulated drug / molecules.

¡  Sometimes phospholipids undergoes oxidation and hydrolysis like reaction

¡  Short half-life

¡  Low solubility

Classification :

¡  Based on Structural Parameters:

1. Multi-laminar vesicles (MLV): made up of series of concentric bi-layer of lipid enclosing a small internal volume with size range > 0.5um.
2. Oligolamelar vesicles (OLV): constitutes 2 to 10 bi layer of lipids surrounding a large     internal volume with size range of 0.1 – 1um.
3. Unilamellar vesicle (ULV): single layer of lipids. Based on the size of the single layer they are further divide into the following types with in ULV as
4. Small unilaminar vesicle: size of 20 to 40 nm
5. Medium unilaminar vesicle: size of 40 to 80 nm
6. Large unilaminar vesicle: size of 100 to 1000 nm
7. Gaint unilaminar vesicle: size of more than 1000 nm
8. Multivesicular Vesicle(MV): constitutes for multiple vesicles and size range >1um.

     General Structure of various types of liposomes:

General Method of Liposome Preparation:

Different Methods of Preparation

The methods are broadly classified into three classes   according to basic modes of dispersion:

1. Physical Dispersion
2. Solvent dispersion
3. Detergent solubilisation.

Physical Dispersion:

There are four basic methods of physical dispersion:

a)      Hand shaken multilamellar vesicles.

b)      Non shaking vesicles.

c)      Pro – liposomes.

d)      Freeze drying.

Hand Shaking Method:

Non Shaking Method &  Pro - Liposomes:

Buchi Rotatory Evaporator

Freeze Thaw Method:

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

Ethanol / Ether Injection Method:

Reverse Phase Evaporation Vesicles:

Mechanism of incorporation of drug in liposomes:

1. Encapsulation
2. Partitioning
3. Reverse loading

Characterisation of liposomes:
1.Physical Characterization

S.no	Characterization parameters	Analytical method/Instrument
1	Vesicle shape and surface morphology	Transmission electron microscopy, Freeze-fracture electron microscopy
2	Mean vesicle size and size distribution    (submicron and micron range)	Dynamic light scattering, zetasizer, Photon correlation spectroscopy, laser  light scattering, gel permeation and gel exclusion
3	Surface charge	Free-flow electrophoresis
4	Electrical surface potential and surface pH	Zetapotential measurements & pH sensitive probes
5	Lamellarity	Small angle X-ray scattering, 31P-NMR, Freeze-fracture electron microscopy
6	Phase behavior	Freeze-fracture electron microscopy, Differential  scanning colorimetery
7	Percent of free drug/ percent capture	Minicolumn centrifugation, ion-exchange  chromatography,  radiolabelling
8	Drug release	Diffusion cell/ dialysis

Chemical Characterization

S.No	Characterization parameters	Analytical method/Instrument
1	Phospholipid concentration	Barlett assay, stewart assay, HPLC
2	Cholesterol concentration	Cholesterol oxidase assay and HPLC
3	Phopholipid peroxidation	UV absorbance, Iodometric  and GLC
4	Phospholipid hydrolysis, Cholesterol auto-oxidation.	HPLC and TLC
5	Osmolarity	Osmometer

Biological Characterization

S.No	Characterization parameters	Analytical method/Instrument
1	Sterility	Aerobic or anaerobic cultures
2	Pyrogenicity	Limulus Amebocyte Lysate (LAL) test
3	Animal toxicity	Monitoring survival rates, histology and pathology

Therapeutic applications of liposomes

List of marketed products