About Author:
Patel Chirag J*, Asija Rajesh, Asija Sangeeta, Mangukia Dhruv
Maharishi Arvind Institute of Pharmacy,
Department of pharmaceutics, Jaipur,
Rajasthan, India.
*chirag.bangalore@gmail.com
ABSTRACT
Amongst the various carriers, few drug carriers reached the stages of clinical trials where liposome shows strong potential for effective drug delivery to the site of action. Liposomes are vesicles having concentric phospholipid bilayers. Molecules from low molecular weight to high molecular weight have been incorporated in liposomes. The water soluble compounds/drugs are present in aqueous compartments while lipid soluble compounds/drugs and amphiphilic compounds/drugs insert themselves in phospholipid bilayers. Drug encapsulated in liposomes include doxorubin, cisplatin, vincristin, melphalan, sarcolycin, daunorubicin, etoposide, etc. The liposomes containing drugs can be administrated by many routes (intravenous, oral inhalation, local application, ocular) and these can be used for the treatment of various diseases. Their predominance in drug delivery and targeting has enabled them to be used as therapeutics tool in fields like tumour targeting, gene and antisense therapy etc. This review discusses the advantages, disadvantages, mechanism, classification, method of preparation, characterization and application of liposomes.
Reference Id: PHARMATUTOR-ART-1352
INTRODUCTION
Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule. The name liposome is derived from two Greek words: 'Lipos' meaning fat and 'Soma' meaning body. A liposome can be formed at a variety of sizes as unilamellar or multi-lamellar construction, and its name relates to its structural building blocks, phospholipids, and not to its size1, 2.
The liposomes have emerged as most practically useful carriers for in-vivo drug delivery as majority of reports has concentrated on the use of phospholipid vesicles or liposomes as potential drug carrier systems. Liposomes or lipid based vesicles are microscopic (unilamellar or multilamellar) vesicles that are formed as a result of self-assembly of phospholipids in an aqueous media resulting in closed bilayered structures3. The assembly into closed bilayered structures is a spontaneous process and usually needs some input of energy in the form of physical agitation, sonication, heat etc. Since lipid bilayered membrane encloses an aqueous core, both water and lipid soluble drugs can be successfully entrapped into the liposomes. The lipid soluble or lipophilic drugs get entrapped within the bilayered membrane whereas water soluble or hydrophilic drugs get entrapped in the central aqueous core of the vesicles4.
The use of liposomes for transformation or transfection of DNA into a host cell is known as lipofection. Liposomes can be created by sonicating phospholipids in water. Low shear rates create multilamellar liposomes, which have many layers like an onion. Continued high shear sonication tends to form smaller unilamellar liposomes3, 4.
ADVANTAGES2, 5, 6
1. Liposomes are biocompatible, completely biodegradable, non-toxic, flexible and nonimmunogenic for systemic and non-systemic administrations.
2. Can carry both water and lipid soluble drugs
3. Provide controlled and sustained release
4. Stabilization of entrapped drug from hostile environment
5. Controlled hydration
6. Help to reduce exposure of sensitive tissues to toxic drugs.
7. Drugs can be stabilized from oxidation
8. Targeted drug delivery or site specific drug delivery
9. Alter pharmacokinetics and pharmacodynamics of drugs
10. Can incorporate micro and macro molecules
11. Can be administered through various routes
12. Act as reservoir of drugs
13. Can modulate the distribution of drug
14. Therapeutic index of drugs is increased
15. Can modulate the distribution of drug
DISADVANTAGES5, 6
1. Less stability
2. Low solubility
3. Problem to targeting to various tissue due to their large size
4. Short half life
5. Sometimes phospholipid undergoes oxidation and hydrolysis like reaction.
6. Leakage and fusion of encapsulated drug / molecules
7. High production cost
8. Quick uptake by cells of R.E.S
9. Allergic reactions may occur to liposomal constituents
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MATERIALS USED FOR LIPOSOMES2, 5, 7, 8
1. Membrane forming components
Phospholipids that are the major components of the biological membranes are the building blocks of the liposomes. The phospholipids have tubular shape owning to the presence of two acyl chains attached to a polar head and on hydration, results into a bilayered membrane. Two types of phospholipids are there i.e. phosphodiglycerides and sphingolipids along with their corresponding hydrolysis products.
Classification of phospholipids
a. Neutral phospholipids e.g. Sphingomyelin, Phosphatidylethanolamine and Phosphatidylcholine.
b. Negatively charged phospholipids e.g. Dipalmitoyl phosphatidylcholine, Dipalmitoyl phosphatidyl acid (DDPA), Distearoyl phosphatidyl choline (DSPC), Dioleoyl phosphatidyl choline (DOPC) etc.
c. Positively charged phospholipids e.g. 1, 2-dihexadecyl-N, N-dimethyl–N-trimethyl amine methyl ethanol amine etc.
2. Membrane Additives (Sterols)
Cholesterol is the most commonly used sterol, which is included in the liposomal membranes. It has been called as the ‘motar’ of bilayers because by virtue of its molecular shape and solubility properties, it fills in empty spaces among the phospholipid molecules, anchoring them more strongly into the structure. Cholesterol is an amphipathic molecule and inserts itself into the membrane with its hydroxyl groups oriented towards the aqueous phase and aliphatic chain aligned parallel to acyl chains of the phospholipid molecules. In other words, cholesterol increases the transition temperature of the system by making the membrane more ordered. Cholesterol reduces this type of interaction to a great extent and provides both physical and biological stability.
3. Charge inducers and Steric stabilizers
Stearylamine, dicetylphosphate, solulan C-24 and diacylglycerol are commonly used to impart either a negative or a positive surface charge. Since it is a well-known fact that negatively charged and positively charged liposomes are more rapidly uptaken by the reticulo-endothelial system as compared to neutral liposomes, charge inducers are used to overcome this problem. Also they proved to be useful in reducing aggregation as neutral liposomes show higher tendency to undergo aggregation.
4. Other substances
In case, the drug is very prone to oxidation, antioxidants e.g. tocopherol, butylated hydroxy toluene and stabilizers are used. The use of preservatives is very common to increase the shelf-life of liposomal formulations.
MECHANISM OF TRANSPORTATION THROUGH LIPOSOMES2, 5
Liposome can interact with cells by four different mechanisms:
1. Endocytosis by phagocytic cells of the reticulo endothelial system such as macrophages and neutrophils.
2. Adsorption to the cell surface either by nonspecific weak hydrophobic or electrostatic forces or by specific interactions with cell-surface components.
3. Fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal content into the cytoplasm
4. Transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine what mechanism is operative and more than one may operate at the same time.
CLASSIFICATION5, 6, 7
1. Based on composition and mode of drug delivery
A. Conventional liposomes
These types of liposomes arecomposed of neutral or negatively charged phospholipids and cholesterol. It is useful for E.E.S targeting; rapid and saturable uptake by R.E.S; short circulation half life, dose dependent pharmacokinetics.
B. pH sensitive liposomes
These types of liposomes arecomposed of phospholipids such as phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine. These are subjected to coated pit endocytosis at low pH, fuse with cell or endosomes membrane and release their contents in cytoplasm; suitable for intra cellular delivery of weak base and macromolecules. Biodistribution and pharmacokinetics are similar to conventional liposomes.
C. Cationic Liposomes
These types of liposomes arecomposed of cationic lipids. These are mainly suitable for delivery of negatively charged macromolecules (DNA, RNA); ease of formation, structurally unstable; toxic at high dose, mainly restricted to local administration
D. Temperature or heat sensitive liposomes
These types of liposomes arecomposed of dipalmitoyl phosphotidyl choline. These are vesicles showed maximum release at 41?C, the phase transition temperature of dipalmitoyl phosphotidyl choline. Liposomes release the entrapped content at the target cell surface upon a brief heating to the phase transition temperature of the liposome membrane.
E. Immuno liposomes
These are conventional or stealth liposomes with attached antibody or recognition sequence. These are subjected to receptor mediated endocytosis. It has cell specific binding (targeting) and can release contents extra cellularly near the target tissue and drugs diffuse through plasma membrane to produce their effects.
F. Long circulating or stealth liposomes
These types of liposomes arecomposed of neutral high transition temperature lipid, cholesterol and 5-10% of PEG-DSPE. These are subjected to hydrophilic surface coating, low opsonisation and thus low rate of uptake by R.E.S. So, it has long circulating half life (40 hrs) and dose independent Pharmacokinetics.
G. Magnetic Liposomes
These types of liposomes arecomposed of phosphotidyl choline, cholesterol and small amount of a linear chain aldehyde and colloidal particles of magnetic iron oxide. These are liposomes that indigenously contain binding sites for attaching other molecules like antibodies on their exterior surface. These can be made use by an external vibrating magnetic field on their deliberate, on site, rapture and immediate release of their components.
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2. Based on Size and Number of Lamellae
A. Multi Lamellar Vesicles (M.L.V.)
Multi lamellar vesicles have more than one bilayer; moderate aqueous volume to lipid ratio 4: 1 mole lipid. Greater encapsulation of lipophilic drug, mechanically stable upon long term storage, rapidly cleared by R.E.S, useful for targeting the cells of R.E.S, simplest to prepare by thin film hydration of lipids in presence of an organic solvent.
a. Oligo lamellar vesicles or Paucilamellar vesicles: Intermediate between L.U.V. & M.L.V.
b. Multi vesicular liposomes: Separate compartments are present in a single M.L.V.
c. Stable Pluri lamellar vesicles: Have unique physical and biological properties due to osmotic compression.
B. Large Unilamellar Vesicles (L.U.V.)
Large unilamellar vesicles have single bilayer, high aqueous volume to lipid ratio (7: 1 mole lipid), useful for hydrophilic drugs, high capture of macro molecules; rapidly cleared by R.E.S. Prepared by detergent dialysis, ether injection, reverse phase evaporation or active loading methods.
C. Small Unilamellar Vesicles (S.U.V.)
Single bilayer, homogeneous in size, thermodynamically unstable, susceptible to aggregation and fusion at low or no charge, limited capture of macro molecules, low aqueous volume to lipid ratio (0.2 : 1.5 : 1 mole lipid) prepared by reducing the size of M.L.V. or L.U.V. using probe sonicator or gas extruder or by active loading or solvent injection technique.
METHOD OF PREPARATION2, 5, 9, 10
The preparation of all types of vesicular systems requires the input of energy. Generally all the methods of liposome preparation involve three basic stages:
1. Dissolved cholesterol and lecithin in organic solvent.
2. Dispersion of lipids in aqueous media.
3. Separation and purification of resultant liposomes.
Various methods used for the preparation of liposome:
1. Passive loading techniques
Passive loading techniques include three different methods:
A. Mechanical dispersion method
a. Lipid film hydration by hand shaking, non-hand shaking or freeze drying
b. Micro-emulsification
c. Sonication
d. French pressure cell
e. Membrane extrusion
f. Dried reconstituted vesicles
g. Freeze-thawed liposomes
B. Solvent dispersion method
a. Ether injection
b. Ethanol injection
c. Double emulsion vesicles
d. Reverse phase evaporation vesicles
e. Stable plurilamellar vesicles
C. Detergent removal method
a. Detergent (cholate, alkylglycoside, Triton X-100) removal form mixed micelles
b. Dialysis
c. Column chromatography
d. Dilution
2. Active loading
CHARACTERIZATION OF LIPOSOMES11, 12
Liposome prepared by one of the preceding method must be characterized. The most important parameters of liposome characterization include visual appearance, turbidity, size distribution, lamellarity, concentration, composition, presence of degradation products, and stability.
1. Visual appearance
Liposome suspension can range from translucent to milky, depending on the composition and particle size. If the turbidity has a bluish shade this means that particles in the sample are homogeneous; a flat, gray color indicates that presence of a nonliposomal dispersion and is most likely a dispersed, inversed hexagonal phase or dispersed microcrystallites. An optical microscope (phase contrast) can detect liposome > 0.3 μm and contamination with larger particles.
2. Determination of liposomal size distribution
Size distribution is normally measured by dynamic light scattering. This method is reliable for liposomes with relatively homogeneous size distribution. A simple but powerful method is gel exclusion chromatography, in which a truly hydrodynamic radius can be detected. Sephacryl-S100 can separate liposome in size range of 30-300nm. Sepharose -4B and -2B columns can separate SUV from micelles.
3. Surface charge
Liposomes are usually prepared using charge imparting constituting lipids and hence it is imparting to study the charge on the vesicle surface. In general two methods are used to assess the charge, namely free flow electrophoresis and zeta potential measurement. From the mobility of the liposomal dispersion in a suitable buffer, the surface charge on the vesicles.
4. Determination of lamillarity
The lamellarity of liposomes is measured by electron microscopy or by spectroscopic techniques. Most frequently the nuclear magnetic resonance spectrum of liposome is recorded with and without the addition of a paramagnetic agent that shifts or bleaches the signal of the observed nuclei on the outer surface of liposome. Encapsulation efficiency is measured by encapsulating a hydrophilic marker.
5. Entrapped volume
The entrapped volume of a population of liposome (in μL/ mg phospholipid) can often be deduced from measurements of the total quantity of solute entrapped inside liposome assuring that the concentration of solute in the aqueous medium inside liposomes is the same after separation from unentrapped material. For example, in two phase method of preparation, water can be lost from the internal compartment during the drying down step to remove organic solvent.
6. Liposome stability
Liposome stability is a complex issue, and consists of physical, chemical, and biological stability. In the pharmaceutical industry and in drug delivery, shelf life stability is also important. Physical stability indicates mostly the constancy of the size and the ratio of lipid to active agent. The cationic liposomes can be stable at 4°C for a long period of time, if properly sterilized.
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APPLICATIONS2, 5, 13, 14
1. Liposome as drug/protein delivery vehicles
a. Controlled and sustained drug release
b. Altered pharmacokinetics and biodistribution
c. Enhanced drug solubilization
d. Enzyme replacement therapy and biodistribution
e. Altered pharmacokinetics and biodistribution
2. Liposome in antimicrobial, antifungal and antiviral therapy
a. Liposomal drugs
b. Liposomal biological response modifiers
3. Liposome in tumour therapy
a. Carrier of small cytotoxic molecules
b. Vehicle for macromolecules as cytokines or genes
4. Liposome in gene delivery
a. Gene and antisense therapy
b. Genetic (DNA) vaccination
5. Liposome in immunology
a. Immunoadjuvant
b. Immunodiagnosis
c. Immunomodulator
6. Liposome as artificial blood surrogates
7. Liposome in enzyme immobilization and bioreactor technology
8. Liposome in cosmetics and dermatology
9. Liposome as radiopharmaceutical and radio diagnostic carriers
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