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Novel Drug Delivery Systems II Introduction to Liposomes
Paper Coordinator Principal Investigator
Dr. Vijaya Khader
Former Dean, Acharya N G Ranga Agricultural University
Content Writer
Prof. Farhan J Ahmad Jamia Hamdard, New Delhi Paper No. : 08 Novel Drug Delivery Systems II
Module No : 11 Introduction to Liposomes
Development Team
Dr. Sushama Talegaonkar Jamia Hamdard, New Delhi
Content Reviewer Dr Harish Dure ja, Associate
Professor, MD University, Rohtak Dr. Sushama Talegaonkar
Jamia Hamdard, New Delhi
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Novel Drug Delivery Systems II Introduction to Liposomes
Description of Module Subject Name Pharmaceutical Sciences
Paper Name Novel Drug Delivery Systems II Module
Name/Title
Introduction to Liposomes
Module Id Pre-requisites
Objectives Introduction, Advantages and Types of Liposomes
Components of Liposomes
Modifications of Liposomes
Limitations of Liposomes
Keywords Liposomes, Components, Mechanismof formation , Lamellar, Phospholipids Content Reviewer
Dr. Vijaya Khader Dr. MC Varadaraj
Prof A K Tiwarey Punjabi University, Patiala
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Novel Drug Delivery Systems II Introduction to Liposomes
INTRODUCTION
Liposomes are vesicular, spherical colloidal particles which are composed of lipid bilayer encapsulating an aqueous core, as shown in figure 1. They were discovered by Bangham and co- workers about 40 years ago. Since then, liposomes have progressed commendably. Liposomes are successful drug delivery systems in which hydrophilic drugs can be enc apsulated in the aqueous core and the hydrophobic drugs can be dispersed in the bilayer of the lipid. Thus, liposomes are one of those very few drug delivery systems in which drugs of both the nature (hydrophilic and hydrophobic) can be loaded in the same particle.
Figure 1: Liposome
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Novel Drug Delivery Systems II Introduction to Liposomes
ADVANTAGES OF LIPOSOMES
Liposomes possess a number of advantages, which are as follows:
Biodegradable and biocompatible: Liposomes are completely biodegradable and biocompatible as they are made up of biodegradable and bioco mpatible lipids.
Non immunogenic and non toxic: Liposomes usually do not trigger any immunogenic or toxic reaction in the body, that is, they are non immunogenic and non toxic.
Natural and easily available lipids: Most of the lipids that are used for the formulation of the liposomes are natural and are easily available.
Drugs of different nature can be loaded: Hydrophobic, hydrophilic, and as well as amphiphilic drugs can be loaded.
Co administration of drugs: Combination of drugs having different nature (hydrophilic or hydrophobic) can be administered through same drug delivery system, i.e. liposomes.
Protection of drug: Encapsulated drug remains protected from the external environment.
Safety of sensitive and non target tissues: Sensitive and non target tissues are not exposed to the toxic drugs when drug loaded liposomes are administered in the body.
Improved stability of drug: The drugs which are unstable or are prone to inactivation in certain conditions, such as acidic pH of stomach, remains protected a nd stable inside of the liposomes until they reach the target site.
Targeting: Targeting of the potent and toxic drugs to the target tissues or cells is possible. This can be achieved by attaching targeting ligands onto the surface onto the liposomes (active targeting), or through Enhanced Permeation Retention (EPR) effect (passive targeting).
Reduction in dose of the drug: Since the drug is targeted to the specific site, wastage of drug at non target sites is significantly reduced. This results in the reduction in dose of the drug which is to be administered.
MECHANISM OF LIPOSOME FORMATION
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Novel Drug Delivery Systems II Introduction to Liposomes
The mechanism of liposome formation is based on the nature of the lipid that is the main component of the liposomes. Lipid molecule is amphiphilic in nature, that is, it has a hydrophilic polar head, and a hydrophobic tail of hydrocarbon chains.
When these lipid molecules are exposed to an aqueous environment, and some energy is dissipated into the system in form of agitation, extrusion, sonication or homogenisation, the lipid molecules arrange themselves as closed bilayered structure as shown in figure 2. This way, lipid molecules avoid the encounter of hydrophobic moiety with the aqueous molecule. The hydrophobic chains of the two layers remain facing each other, while the hydrophilic polar moieties stay in contact with the water molecules.
Thus, the basic structure of a liposome consists of a bilayered wall made up of amphiphilic lipid molecules. This lipid bilayer surrounds an aqueous core. The hydrophilic drugs are dispersed in the aqueous core, whereas the lipophilic drugs are embedded in the hydrophobic tails of the lip id molecules, i.e. in between both the layers of lipid.
Figure 2: Mechanis m of liposome formation
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Novel Drug Delivery Systems II Introduction to Liposomes
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TYPES OF LIPOSOMES
Liposomes are classified on the basis of structural parameters. They are classified as Unilamellar vesicles, Oligolamellar vesicles, and Multilamellar vesicles.
1. Unilamellar Vesicles (ULV): These liposomes have only one bilayer of lipid membrane which encapsulates the aqueous core. Unilamellar liposomes have a very wide range of size, from 20 nm to 1000 nm. Based on their size, unilamellar liposomes can be further divide as :
a. Small Unilamellar Vesicles (SUV): These are very small in size. Their size ranges between 20 nm to 40 nm.
b. Medium Unilamellar Vesicles (MUV): These are larger and have size in the range of 40 nm to 80 nm.
c. Large Unilamellar Vesicles (LUV): These are very large uni lamellar liposomes having size in the range of 100 nm to 1000 nm.
2. Oligo Lamellar Liposomes (OLV): These liposomes are made up of 2-10 bilayers of lipid which surround a large volume of aqueous core. The size of the oligo lamellar liposomes lies in the range of 100 nm to1000 nm.
3. Multi Lamellar Vesicles (MLV): These liposomes are made up of several bilayers of lipid.
The aqueous core can be divided into a number of compartments by these bilayers. Multi lamellar vesicles may also be encapsulating several unilamellar liposomes. The size of these liposomes is greater than 500 nm.
The classification of liposomes is summarized in table 1.
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Table 1: Classification of Liposomes
S. No. Type Number of
Bilayer
Sub Types Size range
1. Unilamellar Vesicles (ULV)
1 Small Unilamellar
Vesicles (SUV)
20-40 nm
Medium Unilamellar Vesicles (MUV)
40-80 nm
Large Unilamellar Vesicles (LUV)
100-1000 nm
2. Oligo Lamellar
Vesicles (OLV)
2 – 10 100-1000 nm
3. Multi Lamellar
Vesicles (MLV)
More than 10 >500 nm
COMPONENTS OF LIPOSOMES
Essential components of liposomes are mainly the lipids and cholesterol.
Lipid
Lipids form the main body of the liposomes, that is, the bilayer which surrounds the aqueous core.
The typical characteristic of the lipids is their amphiphilic nature. Amphiphilic nature means the co- existence of both hydrophilic and hydrophobic moieties in the same molecule. A lipid molecule consists of a polar head which is hydrophilic in nature. This polar head is covalently attached to one or two hydrocarbon chains which form the hydrophobic tail part. The structure of a lipid molecule is depicted in figure 3.
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Novel Drug Delivery Systems II Introduction to Liposomes
Figure 3: Structure of lipid
Following are common types of lipids that are used to formulate liposomes:
1. Phospholipids: Phospholipids are one of the most common lipids used for liposomes formation.
Glycerol containing phospholipids constitute more than half of the total lipids present in the biological membranes. Phospholipids are derived from phosphatidic acid.
Glycerol moiety forms the back bone of the phospholipid molecule. The hydroxyl group present at C3 is esterified to phosphoric acid. The hydroxyl groups of C1 and C2 are esterified with long chain fatty acids. These chains lead to the lipidic nature of the molecule. The remaining hydroxyl groups of the phosphoric acid may further get esterified to various different alcohols such as choline, serine, ethanolamine etc. leading to the formation of a wide range of different phospholipids.
Following are the different types of phospholipids:
Phosphatidyl choline (Lecithin)
Phosphatidyl ethanolamine (Cephalin)
Phosphatidyl glycerol
Phosphatidyl serine
Phosphatidyl inositol
2. Sphingolipids: Sphingolipids are made up of sphingosine which forms the back bone of the molecule. The three components of a sphingolipid molecule are:
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Novel Drug Delivery Systems II Introduction to Liposomes
A molecule of fatty acid
A molecule of sphingosine
A head group which can be anything from a simple alcohol (such as c holine) to complex carbohydrates
Examples of sphingolipids include sphingomyelin and glycosphingo lipids.
3. Synthetic lipids: Synthetic lipids, as the name suggests, are the lipids which are synthesized in laboratory.
Examples of saturated synthetic phospholipids include: Dipalmitoyl phosphatidyl choline (DPPC), Dipalmitoyl phosphatidyl ethanolamine (DPPE), Dipalmitoyl phosphatidyl serine (DPPS), Dipalmitoyl phosphatidyl glycerol (DPPG), Dipalmitoyl phoshatidic acid (DPPA), Distearoyl phosphatidyl choline (DSPC).
Examples of unsaturated synthetic phospholipids include: Dioleoyl phosphatidyl choline (DOPC), Dioleoyl phosphatidyl glycerol (DOPG)
Cholesterol
Cholesterol (or derivatives of cholesterol) also forms an essential component of liposomes. The main function of the cholesterol is to impart stability to the liposomes. It does so by interacting with the lipid bilayer of liposomes. The structure of the cholesterol is shown in figure 4.
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Novel Drug Delivery Systems II Introduction to Liposomes
Figure 4: Structure of Cholesterol
Cholesterol has a hydroxyl group (OH) at 3rd position which forms a small polar head. The non polar tail is formed by the hydrocarbon chain which is present at 17th position. Thus, cholesterol too has an amphiphilic structure like lipids, which makes its interaction easy and strong.
Cholesterol performs the following functions in the formation of the liposomes:
To stabilize the lipid membrane when liposomes are in fluids, such as biological fluids like plasma
To decrease the fluidity and microviscosity of lipid bilayer
To reduce the permeability of the lipid bilayer towards water soluble agents
Cholesterol performs these functions by acting as a mortar for lipid bilayer and filling in the empty spaces that are present among the molecules of the lipids. Thus, cholesterol fixes the molecules of lipids more strongly in the complete structure of the liposomes.
If cholesterol is not included in the structure of the lipid bilayer, the liposomes would interact aggressively with plasma proteins such as albumin and transferrin. These plasma proteins have a tendency to extract the bulk lipids from the liposomes and consequently depleting the outer layer of liposomes which will eventually result in to the instability of the liposo mes. Because of its solubility properties and molecular shape, cholesterol significantly reduces these interactions between liposomal lipids and plasma proteins, thus imparting stability to the liposomes.
MODIFICATION OF LIPOSOMES
Conventional liposomes can be modified also to serve other purposes, such as targeting, and to overcome the limitations of conventional liposomes, such as their early removal from blood circulation. Following are the common types of modified liposomes:
1. Stealth Liposomes:
The term “stealth” means cautious and surreptitious action or movement with a purpose to accomplish something which cannot be accomplished through usual route. Thus, liposomes are made stealth in order to increase their residence time in the blood circulation. Stealth liposomes
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Novel Drug Delivery Systems II Introduction to Liposomes
are thus also known as long circulating liposomes. Conventional liposomes usually bind with opsonin proteins which are present in blood and are identified by cells of Reticulo Endothelial System (RES) as antigens (foreign bodies). Consequently, liposomes are uptaken by RES and are removed from the blood circulation. Conventional liposomes are made long circulating by coating their surface with poly ethylene glycol (PEG) or PEG derivatives, as shown in figure 5.
This imparts hydrophilicity to the surface of liposomes, hence they escape their uptake by the cells of RES. Thus, clearance of liposomes from blood decreases and circulation time increases.
Figure 5: Stealth Liposome
2. Magnetic liposomes:
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Novel Drug Delivery Systems II Introduction to Liposomes
Magnetic liposomes are those which respond to external magnetic fields. These are a type of external stimuli responsive liposomes. Magnetic liposomes are formulated by encapsulating a suitable magnetic responsive material, such as iron oxide (magnetite and maghemite) is encapsulated in the core of the liposomes. In the presence of an external magnetic field, magnetic responsive material responds to it, and moves inside the body as the magnetic field moves outside the body. This enables the targeting of the liposomes to the cells or tissue where the action is desired. An area of application of magnetic liposomes is the treatment of cancer.
Magnetic liposomes are introduced into the body and an external magnetic field is created around the tumor. The magnetic liposomes, attracted by the external magnetic field reach the tumor and act on the tumor mass specifically.
3. Temperature responsive liposomes:
Temperature responsive liposomes are another type of modified liposomes which respond to a change in the temperature of the surrounding tissues (usually responds to a comparatively higher temperature). These liposomes are a type of internal stimuli responsive liposomes.
Temperature sensitive liposomes can be formulated by using either lysolipids or synthetic polymers which are temperature sensitive, such as poly(N- isopropylacrylamide). Temperature sensitive lipids or polymers sense the change in the temperature of surroundings and change their conformation. This results into disruption of the structure of the liposomal assembly and the drug is released. Temperature responsive liposomes have been mainly explored for the treatment of cancers, as cancer tissues bear a temperature slightly higher than the normal body temperature.
4. pH responsive liposomes:
pH responsive liposomes are another type of internal stimuli responsive liposomes. These liposomes respond to a change in pH of different tissues of body, and at a specific pH, release the loaded drug. To impart pH responsive properties, these liposomes are formulated with some pH responsive lipid. pH responsive lipids undergo conformational changes at the specific pH.
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Novel Drug Delivery Systems II Introduction to Liposomes
This leads to destabilization of the liposomal structure and thus the drug gets released into the surrounding tissue. Phosphatidylethanolamine is an example of pH sensitive lipid.
Phosphatidylethanolamine exhibits a change in its conformation at acidic pH.
5. Immuno responsive liposomes:
Immuno responsive liposomes are those which are formulated in such a way that they respond to the antigens present in the body or the diseased tissue. Immuno responsive liposomes are prepared by attaching antibodies to the surface of conventional or stealth liposomes, as shown in figure 6. These antibodies recognize and attach to the specific receptors (antigens) present on the surface of the cells (such as cancer cells). This whole phenomenon enables active tissue targeting through binding to the specific receptors of the cells.
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Novel Drug Delivery Systems II Introduction to Liposomes
Figure 6: Immuno Responsive Liposome
6. Cationic liposomes:
Conventional liposomes are either neutral or negatively char ged. Cationic liposomes can be prepared by using cationic lipids for formulating liposomes. Cationic liposomes are formulated to enable the attachment of negatively charged targeting ligands such as hyaluronic acid.
Dioctadecyl dimethyl ammonium bromide is an example of cationic lipid.
7. Targeting Liposomes:
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Novel Drug Delivery Systems II Introduction to Liposomes
Liposomes can be modified to target a specific tissue or organ by attaching targeting ligands onto their surface (figure 7). Targeting ligands recognize and bind to the specific receptors which are present on the surface of target cells. These receptors are specifically present or are over expressed on the surface of the target tissue or the diseased tissue. Thus the targeting liposomes having ligands on their surface recognize these target receptors and selectively bind to them, thereby sparing the other tissues of the body. This helps in reducing the unwanted effects of the toxic drugs and reducing the dose to be administered. For example, hyaluronic acid present on the liposomal surface binds to CD44 receptors which are over expressed in cancer cells, and folic acid can be attached to liposomes to make them preferably bind to folate receptors over expressed on diseased tissues.
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Novel Drug Delivery Systems II Introduction to Liposomes
Figure 7: Targeting Liposomes
LIMITATIONS OF LIPOSOMES
Despite of the several advantages, commercial success and utility, liposomes do suffer from few limitations also. Limitations of liposomes are:
1. Liposomes tend to be unstable
Stability is a major concern with liposomal formulations. They exhibit both physical and chemical instability.
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Novel Drug Delivery Systems II Introduction to Liposomes
Physical instability may manifest itself in the following forms:
Liposomes may fuse with each other to form larger sized vesicles or may aggregate to form lumps. This will affect their delivery to the target tissues and in vivo performance of liposomes, and thus will ultimately affect the therapeutic index of the drug.
On storage, drugs may leach out of the liposomes and move into surrounding media. This will result in the failure of purpose of the drug delivery system.
Liposomes usually lose their stability when stored for long time.
Liposomes may have short shelf lives.
Che mical instability may manifest itself in the following ways:
Ester bonds present in the lipids may get hydrolysed.
Oxidation of unsaturated acyl chains of lipids may take place.
The problem of physical instability can be minimized by taking the following measures:
Leakage or fusion of liposomes can be a result of lattice defects of the membrane. This can be minimized by incubating the vesicles at a temperature above the membrane phase transition temperature to balance differences in packing density between opposite sides of the bilayers by trans membrane flip flop. This process is called annealing.
Aggregation of neutral liposomes is a consequence of vander waals interaction and is more prominent in large vesicles. This problem can be overcome by imparting small negative charge to the lipids.
Very small unilamellar vesicles tend to fuse because of relieving stress arising from the high curvature of the membrane. Including sufficient cholesterol in the membrane helps to overcome this problem.
High amount of cholesterol also helps in minimizing the leakage of drugs from the liposomes.
Lyophilization of liposomes helps in minimizing both physical and c hemical instability by converting liposomes into solid anhydrous form.
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Novel Drug Delivery Systems II Introduction to Liposomes
The problem of chemical instability can be minimized by taking the following measures:
The procedures which involve use of high temperature should be avoided.
Preparation of liposomes should be carried out in the absence of oxygen.
The aqueous solutions should be deoxygenated by using nitrogen.
Liposomes should be stored in an inert atmosphere.
An anti oxidant agent should be added as a component of the lipid membrane.
2. Poor Encaps ulation efficiency
Encapsulation efficiency of liposomes is generally low (approximately 30%). To administer therapeutic doses, high amount of liposomes may have to be administered. However this will also expose the body tissues to very high concentration of lipids. This may lead to non linear (saturable) pharmacokinetics of liposomal formulation. Active loading techniques give much better encapsulation efficiencies than passive loading.
3. Uptake by RES:
Though composition of lipids is similar to the composition of cellular membrane, the reticuloendothelial system (RES) of our body recognizes liposomes as foreign bodies. This RES uptake of liposomes is due to the binding of opsonin protein which is present in blood, and is recognized by the cells of RES. This causes the rapid elimination of liposomes from the blood circulation, which ultimately results in unsatisfactory therapeutic response. Uptake of liposomes by the cells of RES can be minimized by rendering the liposomal surface hydrophilic through coating it with some hydrophilic polymer such as polyethylene glycol (PEG) or its derivatives.
1. Sterilization:
Since phospholipids are thermo labile compounds and are sensitive to heat, chemicals and radiations; selecting a suitable method for sterilization of liposomes comes as a major challenge. Generally, liposomal formulations are sterilized by filtration through sterile
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membranes having pore size of 0.22 μm. But filtration method is not suitable for vesicles having size larger than 0.2 μm, and also, is not able to remove viruses. However, liposomes made up of thermo stable phospholipids can be sterilized through autoclaving under certain conditions.
LIPOSOME PREPARATIONS IN MARKET
There are several liposome formulations which are already in market, and many more are in clinical trials. Marketed liposomal preparations are enlisted below in the table 2.
Table 2: Marketed Liposome Preparations S.
No.
Liposome Formulation
Drug Route of Administration Manufacturer
1. Ambisome Amphotericin B Intravenous infusion Gilead Sciences, NeXstar Pharmaceuticals
2. DaunoXome Daunorubicin Intravenous infusion Gilead Sciences, NeXstar Pharmaceuticals
3. Depocyt Cytarabine Intrathecal (intraventricular or lumbar puncture)
SkyePharma
4. Doxil/Caelyx Doxorubicin Intravenous infusion Sequus Pharmaceuticals
5. Myocet Doxorubicin Intravenous infusion Elan Pharma
SUMMARY
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Novel Drug Delivery Systems II Introduction to Liposomes
Liposome is one of the most viable and commercialized colloidal drug delivery systems. Liposomal formulations such as doxil and daunoxome are commercially available for the treatment of diseases like cancer. Liposomes offer several advantages such as biocompatibility and ease of loading both hydrophilic and hydrophobic drugs, even in the same formulation. But liposomes suffer from few limitations also, such as unstable nature and poor encapsulation efficiency. Measures need to be taken to improve the encapsulation efficiency and stability of liposomes, thus making them more valuable tool for drug delivery and other biomedical applications.
