Liposome as a Drug Carrier
Ms. Tabasum Siraj Mulla*, Ms. Monika Sanjay Thorat, Ms. Yogita Rayate, Dr. Manoj Nitalikar
Rajarambapu College of Pharmacy, Kasegaon
One of various talented new drug delivery systems, liposomes characterize an advanced technology to deliver active molecules to the site of action, and at present, several formulations are in clinical use. The liposome consist of phospholipids, which have phosphoric acid sides to form the liposome players. Liposomes can be prepared in different lipid compositions or by different method. The present review will shortly explain the characteristics, advantages, disadvantages, method of preparation applications of liposomes in food, cosmetics, gen genetic engineering, immunology, cancer therapy, infection, and also the diagnosis.
Liposomes were discovered in the early 60’s, by Bangham who was studying phospholipids and blood clotting. Liposomes, defined as microscopic spherical-shaped vesicles, consist of an internl aqueous compartment entrapped by one or multiple concentric lipidic bilayer . Liposomes membrane is composed of natural and/or synthetic lipids which are relatively biocompatible, biodegradable and non-immunogenic material due to their unique bilayer-structure properties, liposomes are used as carriers for both lipophilic and water-soluble molecules.
Hydrophilic substances are encapsulated in the interior aqueous compartments. Lipophilic drugs are mainlyentrapped within lipid bilayers.  Liposomes are extensively used as carriers for numerous molecules in cosmetic and pharmaceutical industries.
Fig 1: Basic liposome structure 
Fig 2: Structure of liposome
· Provides selective passive targeting to tumor tissues.
· Increased stability of encapsulated drug also therapeutic index via encapsulation. 
· Reduction in toxicity of the encapsulated agent.
· Flexibility to add site specific ligands to get active targeting.
· It should be biodegradable also biocompatible, and flexible, non ionic. 
· Suitable for delivery of hydrophobic, hydrophilic and amphipatic drugs and agents also acts as reservoir of drugs.
· It provides controlled hydration, sustained release
· It provides the distribution of targeted drugs or the distribution of a particular drug in the site.
· Stabilization of entrapped drug from hostile environment.
· Alter pharmacokinetics and pharmacodynamics of drugs. 
· It can be administered through various routes and site avoidance is occurred.
· Repeated Iv Administration Problems.
· Difficult in large scale manufacturing and sterilization 
· Once administered, liposomes can not be removed
· Focus on certain cells or cell types by changing lipid types in liposomes.
· Release their contents at a specific pH value.
· Very High Production Cost.
· Drug Leakage/ Entrapment/ Drug Fusion.
· Distribute fat-soluble (oil-like) compounds, such as some vitamins, antioxidants, antibiotics, flavors, etc. which can not be mixed with many products, including water-based products.
· Protect the compounds from the stomach and intestine with acid and enzymatic degradation using some atoms to coat the liposome. 
· Protect compounds like vitamins and antioxidants from oxidation.
· Inceases the intestinal absorption of compounds by coating with certain molecules.
CLASSIFICATION OF LIPOSOMES:
A. Based on structural parameters 
· UV-unilamellar vesicles (all size range)
· SUV-small unilamellar vesicles(-20-100nm)
· MUV-medium sized unilamellar vesicles
· LUV-large unilamellar vesicles>100nm
· MLV- Multilamellar large vesicles>0.5um
· OLV- oligolamellar vesicles,0.1-1um
· GUV-giant unilamellar vesicles>1um
· MV-multivesicular vesicles>1um
B. Based on method of liposome preparation [3,6]
a) REV-OLV made by reverse phase evaporation method
· MLV-REV-MLV made by reverse phase evaporation method
· SPLV-stable plurilamellar vesicle
· VET-vesicles prepared by extrusion technique
· DRV-dehydration-rehydration method 
C. Based on composition and applications 
· Conventional liposomes-neutral or negatively charged phospholipids and chol.
· PH sensitive liposomes- phospholipid such as PE or DOPE
· Immuno liposomes-CL with attached monoclonal antibody
· Cationic liposomes-cationic lipids with DOPE
· Fusogenic liposomes-Reconstituted Sendai virus envelops
COMPOSITION OF LIPOSOMES:
The Main Components of Liposomes are
1. Phospholipids 
Phospholipids are the important structural components of biological membranes, there are two types of phospholipids exist–phosphodiglycerides and sphingolipids, together with their corresponding
hydrolysis products. The mostly used phospholipid is phosphatidylcholine (PC) molecule
Naturally occurring phospholipids: 
Incorporation of sterols in liposome bilayer can bring about major changes in the preparation of these membranes. Cholesterol itself not form bilayer structure, acts as fluidity buffer. It can be incorporated into phospholipid membranes in very high concentration unto 1:1 or even 2:1 molar ratios of PC. Cholesterol incorporation increases the separation between the cholin head groups and eliminates the normal electrostatic and hydrogen-bondinginteractions. Enhances the stability of the membrane Enhances the rigidity of the phospholipid bilayer. Reduces the permeability of watersoluble substance.
MECHANISM: LIPOSOME FORMATION:
In aqueous media phospholipids as they are not soluble align themselves closely in planar bilayer sheets or lipid cakes which is thermodynamically stable.  In which polar head groups face outwards into the aqueous medium, and the lipidic chains turns inwards to avoid the water phase, giving rise to double layer or bilayer. This structure is also called as lamella. For the liposomes to be formed, upon further hydration, the lipid cakes(lamella) swells eventually they curve to form a closed vesicles in the form of spheres. This spheres are called as liposomes. 
Fig 3 Schematic drawing of liposomes structure and lipophilic or hydrophilic drug entrapment models 
Mechanism of Action of Liposomes:
A liposome consists of a part of aqueous solution within a hydrophobic membrane. Hydrophobic chemicals can be easily immersed into the lipid membranes; in this way liposomes are able to carry both hydrophilic and hydrophobic molecules
Steps involved in liposome action of drug delivery: 
The cell membrane of the liposomes receives contact on the cell membrane due to the adsorption of the cell membrane.
Adsorption of liposomes on the cell surface membrane carried by engulfment and internalization into the liposomes
lipid bilayers of liposomes are fused with the lipoidal cell membrane by lateral diffusion and intermingling of lipids results in direct delivery of liposomal contents in the cytoplasm.
4. Lipid exchange:
Because of the similarity of liposomal lipid membrane with cell membrane phospholipids, lipid transfer proteins in the cell membrane easily recognize liposomes and cause lipid exchange.
For example, in case of cancer cells; they consume large amounts of fats to fill the requirement of rapid growth, they recognize the liposomes (loaded with anti-cancer drug) as a potential source of nutrition. When they are targeted by liposome, they get absorbed. Once the anti-cancer drugs are released from the liposome into the site, cancer cells are killed by the drug.
METHOD OF PREPARATION BASED ON THE METHOD OF DISPERSION:
A. Passive loading techniques:
It includes three different groups of methods working on different principles i.e mechanical dispersion, solvent dispersion and detergent solubilization.
1.Mechanical dispersion methods:
[I] Lipids film hydration by hand shaking, non hand shaking and freeze drying:
In these methods,the lipids are casted as stacks of film from their organic solution using flash rotary evaporator under reduced pressure(or by hand shaking)and then casted film is dispersed in an aqueous medium.Upon hydration the lipids swell and peel off the round bottom flask and vesiculate forming MLVs and by non shaking methods forming LUVs.The mechanical energy required for the swelling of lipids and dispersion of casted lipid film is imparted by manual agitation(hand shaking methods)or by exposing the film to a stream of water saturated nitrogen for 15 min. followed by swelling in aq.medium without shaking(non shaken vesicles 
Fig 4 Method of preparation of Liposomes by Lipid Film Hydration by hand shaking method
II] Sonication unicellular Liposomes:
Sonication is most widely used method for the preparation of SUV. There are two sonication techniques: 
Is employed for dispersions, which require high energy in asmall volume e.g., high concentration of lipids, or a viscous aqueous phase 
Disadvantage- Due to high energy lipid degradation occurs and sonication tips release titanium particles into liposome dispersion
Bath sonication: 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 which yield a slightly hazy transparent solution. Using centrifugation to yield a clear SUV dispersion 
Fig 5 Method of preparation of Liposomes by sonication
III ] Micro-emulsification Liposomes:
From Concentrated lipid Dispersion small MLVs are prepared by “Micro Fluidizer” The lipids can introduced into fluidizers, either as a dispersion of large MLVs or as a slurry of unhydrated lipids in organic medium. Microfluidizer pumps the fluid through a 5um orifice at very high pressure (10,000psi, 600-700 bar). Then it is forced with defined micro channels, which direct two streams of fluid to collide  together at right angles at a very high velocity, thereby affecting an efficient transfer of energy. The collected fluid can be recycled through the pump and interaction chamber until vesicles of the spherical dimension are obtained. After a single pass, the size of vesicles is reduced to a size 0.1 and 0.2um in diameter.
Fig 6 Method of preparation of liposome by micro emulsification
IV] French Pressure Cell Liposomes:
Through a small orifice the extrusion of MLV occurs by using French pressure cell. The method has several advantages over sonication method. The forming liposomes are rather larger than sonicated SUVs. The drawbacks of the method are that the high temperature is difficult to attain, and the working volumes are comparatively small (about 50 mL as the maximum) This techniques forms rather “uni or oligo lamellar liposomes” of intermediate size of 30-80 nm in diameter depending on the applied pressure.
Fig 7 French Pressure Cell
V] Membrane Extrusion Liposomes:
It is used to process LUVs also MLVs. Liposomes formed by this tech. are called as membrane filter extrusion liposomes. obtained by using high lipid conc. the 30% volume can be captured. 1-2 litre /mole of lipids can be trapped by this process. It is because to their ease of production, randomly selectable vesicle diameter, batch to batch reproduction and freedom from solvent or surfactant contamination is possible 
Fig 8 Membrane Extrusion Method
VI] Dried reconstituted vesicles and Freeze-thawed Liposomes:
DRV-Liposomes obtained by this method are usually “uni or oligo lamellar” of the order of 1.0um
or less in diameter. This method starts with freeze drying of a dispersion of empty SUV and then rehydrating it with the aq. fluid containing the material to be entrapped.
FTS-The method is based upon freezing of a unilamellar dispersion and then thawing at room temp for 15 min. So the process ruptures and refuses SUVs during which the solute equilibrates between inside and outside and liposomes themselves fuse and increase in size. Entrapment volume can be upto 30% of the total vol. of dispersion. Because of Sucrose(cryoprotectant), divalent metal ions (which can neutralize the surface charge) and high ionic strength salt solutions can not be entrapped efficiently
Fig 9 Method of preparation of Liposomes by DRVs and Freeze Thaw Sonication
VII] PH induced vesiculation:
It is used to convert MLVs to LUVs using change in the pH of the dispersion thus avoiding the use of sonication or high pressure application. The change in pH brings about an increase in the surface charge density of the lipid bilayer, which induces spontaneous vesiculation.
2. Solvent dispersion methods:
I] Ethanol injection and Ether injection:
Ethanol injection-. Through a fine needle an ethanol solution of lipids is injected rapidly into an excess of saline or other aq. medium. This method has less risk of degradation of sensitive lipids The vesicles of 100 nm size may be little modification in this method i.e by altering conc. of lipid in ethanol or by changing the rate of injection of ethanol solution in preheated aqu. solution.
Limitation-Solubility of lipids in ethanol and vol. of ethanol that can be introduced into medium (7.5%v/v max) so dispersion gets diluted. Tough to remove residual ethanol from phospholipid membrane
Ether injection- It is based on injecting the immiscible org. solution very slowly into an aqu. phase through a narrow needle at the temp. of vapourizing the org. solvent. It has less encapsulation efficiency.It has little risk of causing oxidative degradation,provided ether is free from peroxide.
Fig 10 Ethanol and Ether injection Method
II] Double emulsion vesicles:
It is prepared by rapidly injecting the dispersion of micro-droplets into hot aqu. solution of Tris-buffer with the help of 22 gauge hypodermic needle under vigorous stirring. The org.solvent is evaporated using strong jet of nitrogen thus forming double emulsion.
III] Reverse phase evaporation vesicles:
The droplets are formed by brief sonication of a two phase system containing phospholipids in organic solvent (diethylether or isopropylether or mixture of isopropyl ether and chloroform) and aqueous buffer.  The formation of a viscous gel by removing organic solvents under reduced pressure. With the help of continued rotary evaporation under reduced pressure liposomes are formed. The encapsulation efficiency up to 50%.
Fig 11 Reverse phase evaporation Methopd
IV] Stable plurilamellar vesicles:
It involves preparation of W/O dispersion with an excess of lipid followed by drying under continued bath sonication with the intermittent stream of nitrogen. the percent entrapment normally ranges around 30%.
3.Detergent removal methods:
Detergent commonly use for this purpose exhibit reasonably high CMC (10 to 20 mM) so that their removal is facilitated A commercial version of the dialysis system is available under
the tradename LIPOREP 
II] Column Chromatography:
Phospholipid in the form of sonicated vesicle or as a dry film, at a molar ratio of 2:1 with deoxycholate form unilamellar vesicles of 100nm on removal of deoxycholate by column chromatography.
B. Active loading techniques:
Fig 12 Active loading technique
1. Physical Characterization :
Vesicle shape and surface morphology
Transmission electron microscopy,
Freeze-fracture electron microscopy
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
Electrical surface potential and surface pH
Zeta potential measurements and pH sensitive probes
Small angle X-ray scattering, 31P-NMR,
Freeze-fracture electron microscopy
Freeze-fracture electron microscopy, Differential
Percent of free drug/ percent capture
Minicolumn centrifugation, ion-exchange
2. Chemical characterization 
Barlett assay, Stewart assay, HPLC
Cholesterol oxidase assay and HPLC
UV absorbance, Iodometric and GLC
3.Biological characterization 
Aerobic or anaerobic cultures
Limulus Amebocyte Lysate (LAL) test
Monitoring survival rates, histology and pathology
· Liposomes as drug/protein delivery vehicles.
· Liposomes in antimicrobial, antifungal and antiviral therapy. 
· Liposomes in tumour therapy.
· Liposomes in gene delivery. 
· Liposomes in immunology.
· Liposomes as artificial blood surrogatesn.
· Liposomes as radiophamaceutical and radiodiagnostic cariers.
· Liposomes in cosmetics an dermatology.
· Liposomes in enzyme immobilization and bioreactor technology.
It was concluded from the review that liposomes are one of the unique drug delivery system, which can be of potential use in controlling and improving targeted drug delivery. Liposomes are administered orally, parenterally and topically as well as used in cosmetic and hair technologies, sustained release formulations, diagnostic purpose and as good carriers in gene delivery i.e. antimicribial agents, drugs against cancer, antigungal drugs, peptide harmones, enzymes, vaccines and genetic materials. Nowdays liposomes are used as resourseful carriers for targeted delivery of drug.
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