LIPOSOMES – A NOVEL DRUG DELIVERY SYSTEM
M. PraveenHH, Rahul Soman, Vimal Mathew
National College of Pharmacy, Manassery, Calicut
The goal of any drug delivery system is the spatial placement and temporal delivery of the medicaments. Research works are going on to prepare an ideal drug delivery system which satisfies these needs. Researches carried out by Alec Bingham lead to the development of a new drug delivery system called as Liposomes.
This was an accidental discovery, when he dispersed Phosphatidyl choline molecules in water; he found that it was forming a closed bilayer structure containing an aqueous phase entrapped by lipid bilayers.
Liposomes are now used to deliver certain vaccines, enzymes and drugs to the body. When used in the delivery of certain cancer drugs, liposomes help to shield healthy cells from the drugs toxicity and prevent their concentration in vulnerable tissues (e.g., kidney, liver), lessening or eliminating the common side effects of nausea, fatigue and hair lose.
Liposomes are especially effective in treating diseases that effect phagocytes. Also used to carry genes into cells and can be administered by various routes.
Liposomes are defined as structure consisting of one or more concentric spheres of lipid bilayers separated by water or aqueous buffer compartments.
Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid bilayers.
- Provide controlled drug delivery
- Biodegradable, biocompatible, flexible
- Non ionic
- Can carry both water and lipid soluble drugs
- Drugs can be stabilized from oxidation
- Improve protein stabilization
- Controlled hydration
- Provide sustained release
- Targeted drug delivery or site specific drug delivery
- Stabilization of entrapped drug from hostile environment
- Alter pharmacokinetics and pharmacodynamics of drugs
- Can be administered through various routes
- Can incorporate micro and macro molecules
- Act as reservoir of drugs
- Therapeutic index of drugs is increased
- Site avoidance therapy
- Can modulate the distribution of drug
- Direct interaction of the drug with cell
- Less stability
- Low solubility
- Short half life
- Phospho lipid undergoes oxidation, hydrolysis
- Leakage and fusion
- High production cost
- Quick uptake by cells of R.E.S
- Allergic reactions may occur to liposomal constituents
- Problem to targeting to various tissue due to their large size
I. Based on composition and mode of drug delivery
- Conventional liposomes
Composed of neutral or negatively charged phospholipids and cholesterol. Subject to coated pit endocytosis, contents ultimately delivered to Lysosomes if they do not fuse with the endosomes, useful for E.E.S targeting; rapid and saturable uptake by R.E.S; short circulation half life, dose dependent pharmacokinetics.
- pH sensitive liposomes
Composed of phospholipids such as phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine.
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 similar to conventional liposomes.
- Cationic Liposomes
Composed of cationic lipids
Fuse with cell or endosome membranes; suitable for delivery of negatively charged macromolecules (DNA, RNA); ease of formation, structurally unstable; toxic at high dose, mainly restricted to local administration
- Long circulating or stealth liposomes
Composed of neutral high transition temperature lipid, cholesterol and 5-10% of PEG-DSPE.
Hydrophilic surface coating, low opsonisation and thus low rate of uptake by RES long circulating half life (40 hrs); Dose independent Pharmacokinetics
- Immuno liposomes
Conventional or stealth liposomes with attached Antibody or Recognition Sequence.
Subject to receptor mediated endocytosis, cell specific binding (targeting); can release contents extra cellularly near the target tissue and drugs diffuse through plasma membrane to produce their effects.
- Magnetic Liposomes
Composed of P.C, 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. Can be made use by an external vibrating magnetic field on their deliberate, on site, rapture and immediate release of their components.
- Temperature (or) heat sensitive liposomes
Composed of Dipalmitoyl P.C.
These are vesicles showed maximum release at 41o, the phase transition temperature of Dipalmitoyl P.C. Liposomes release the entrapped content at the target cell surface upon a brief heating to the phase transition temperature of the liposome membrane.
II Based on Size and Number of Lamellae
- Multi lamellar vesicles (M.L.V)
Size ® 0.1 - 0.3 micro meter
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 & MLV
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.
- Large Unilamellar Vesicles (L.U.V)
Size ® 0.1 - 10 micro meter
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.
- Small Unilamellar Vesicles (S.U.V)
Size £ 0.1 micro meters
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.
Glycerol containing phospholipids are most common used component of liposome formulation and represent greater than 50% of weight of lipid in biological membranes. These are derived from Phosphatidic acid. The back bone of the molecule is glycerol moiety. At C3 OH group is esterified to phosphoric acid. OH at C1 & C2 are esterified with long chain. Fatty acid giving rise to the lipidic nature. One of the remaining OH group of phosphoric acid may be further esterified to a wide range of organic alcohols including glycerol, choline, ethanolamine, serine and inositol. Thus the parent compound of the series is the phosphoric ester of glycerol.
Examples of phospholipids are
- Phosphatidyl choline (Lecithin) – PC
- Phosphatidyl ethanolamine (cephalin) – PE
- Phosphatidyl serine (PS)
- Phosphatidyl inositol (PI)
- Phosphatidyl Glycerol (PG)
For stable liposomes, saturated fatly acids are used. Unsaturated fatty acids are not used generally.
Backbone is sphingosine or a related base. These are important constituents of plant and animal cells. This contain 3 characteristic building blocks
- A mol of F.A
- A mol of sphingosine
- A head group that can vary from simple alcohols such as choline to very complex carbohydrates.
Most common Sphingolipids – Sphingomyelin. Glycosphingo lipids.
Gangliosides – found on grey matter, used as a minor component for liposome production
This molecule contain complex saccharides with one or more Sialicacid residues in their polar head group & thus have one or more negative charge at neutral pH. These are included in liposomes to provide a layer of surface charged group.
Cholesterol & its derivatives are often included in liposomes for
- decreasing the fluidity or microviscocity of the bilayer
- reducing the permeability of the membrane to water soluble molecules
- Stabilizing the membrane in the presence of biological fluids such as plasma.( This effect used in formulation of i.v. liposomes)
Liposomes without cholesterol are known to interact rapidly with plasma protein such as albumin, transferrin, and macroglobulin. These proteins tend to extract bulk phospholipids from liposomes, there by depleting the outer monolayer of the vesicles leading to physical instability. Cholesterol appears to substantially reduce this type of interaction. Cholesterol has been called the mortar 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. The OH group at 3rd position provides small Polar head group and the hydrocarbon chain at C17 becomes non polar end by these molecules, the cholesterol intercalates in the bilayers.
- Synthetic phospholipids
E.g.: for saturated phospholipids are
- Dipalmitoyl phosphatidyl choline (DPPC)
- Distearoyl phosphatidyl choline (DSPC)
- Dipalmitoyl phosphatidyl ethanolamine (DPPE)
- Dipalmitoyl phosphatidyl serine (DPPS)
- Dipalmitoyl phosphatidic acid (DPPA)
- Dipalmitoyl phosphatidyl glycerol (DPPG)
E.g.: for unsaturated phospholipids
- Dioleoyl phosphatidyl choline (DOPC)
- Dioleoyl phosphatidyl glycerol (DOPG)
- Polymeric materials
Synthetic phospholipids with diactylenic group in the hydrocarbon chain polymerizes when exposed to U.V, leading to formation of polymerized liposomes having significantly higher permeability barriers to entrapped aqueous drugs.
E.g.: for other Polymerisable lipids are – lipids containing conjugated diene, Methacrylate etc
Also several Polymerisable surfactants are also synthesized.
- Polymer bearing lipids
Stability of repulsive interactions with macromolecules is governed mostly by repulsive electrostatic forces. This repulsion can be induced by coating liposome surfaces with charged polymers.
Non ionic and water compatible polymers like polyethylene oxide, polyvinyl alcohol, and Polyoxazolidines confers higher solubility. But adsorption of such copolymers containing hydrophilic segments with hydrophobic part leads to liposome leakage, so best results can be achieved by covalently attaching polymers to phospholipids
E.g.: Diacyl Phosphatidyl ethanolamine with PEG polymer linked via a carbon at or succinate bond.
The degree of polymerization varies from 15-120 units. Longer polymers give rise to aqueous solubility of polymer lipids and their first removal from membranes in non equilibrium conditions. While shorter polymers do not offer enough repulsive pressure because Vanderwaal's attraction is a long range force.
- Cationic lipids
E.g.: DODAB/C – Dioctadecyl dimethyl ammonium bromide or chloride
DOTAP – Dioleoyl propyl trimethyl ammonium chloride – this is an analogue of DOTAP and various others including various analogues of DOTMA and cationic derivatives of cholesterol
- Other Substances
Variety of other lipids of surfactants are used to form liposomes
- Many single chain surfactants can form liposomes on mixing with cholesterol
- Non ionic lipids
A variety of Polyglycerol and Polyethoxylated mono and dialkyl amphiphiles used mainly in cosmetic preparations
- Single and double chain lipids having fluoro carbon chains can form very stable liposomes
- Sterylamine and Dicetyl phosphate
Incorporated into liposomes so as to impart either a negative or positive surface charge to these structures
- A number of compounds having a single long chain hydrocarbon and an ionic head group found to be capable of forming vesicles. These include quaternary ammonium salts of dialkyl phosphates.
METHODS OF PREPARATION OF LIPOSOMES
1) Hydration of lipids in presence of solvent
3) French Pressure cell
4) Solvent injection method
a) Ether injection method
b) Ethanol injection
5) Detergent removal
Detergent can be removed by
b) Coloumn chromatography
6) Reverse phase evaporation technique
7) High pressure extrusion
8) Miscellaneous methods
a) Slow swelling in Non electrolyte solution
b) Removal of Chaotropic ion
MECHANISM OF FORMATION OF LIPOSOMES
Lipids capable of forming liposomes exhibit a dual chemical nature. Their head groups are hydrophilic and their fatty acyl chains are hydrophobic.
It has been estimated that each Zwitter ionic head group of Phosphatidyl choline has on the order of 15 molecules of water weakly bound to it, which explain it's over whelming preference for the water phase. The hydrocarbon fatty acid chains on the other hand vastly prefer each others company to that of H2O. This can be understood by taking the CMC of P.C into account. The CMC of Dipalmitoyl P.C found to be 4.6 ´ 10–10 M in water, which is a small number indicating the over whelming preference of this molecule for a hydrophobic environment such as that found in the core of micelle or bilayer.
The free energy of transfer from water to micelle is 15.3K cal/mol for Dipalmitoyl PC and 13.0K cal/mol for Dimyristoyl P.C. These results clearly point out the thermodynamic basis for bilayer assembly that has been termed the hydrophobic effect. The large free energy change between a water and a hydrophobic environment explains the over whelming preference of typical lipids to assemble in bilayer structures, including water as much is possible from the hydrophobic core in order to achieve the lowest energy level, hence the highest stability for the aggregate structure.
PHARMACOKINETICS OF LIPOSOMES
Liposomal drugs can be applied through various routes, but mainly i.v and topical administration is preferred. After reaching in the systemic circulation or in the local area, a liposome can interact with the cell by any of the following methods.
- endocytosis by Phagocytotic cells of the R.E.S such as macrophages and Neutrophils
- adsorption to the cell surface either by non specific weak hydrophobic or electrostatic forces or by specific interaction with cell surface components
- Fusion with the plasma cell membrane by insertion of lipid bilayer of liposome into plasma membrane with simultaneous release of liposomal contents into the cytoplasm.
- Transfer of liposomal lipids to cellular or sub cellular membrane or vice versa without any association of the liposome contents.
It is often difficult to determine what mechanism is operative and more than one may operate at the same time.
If cholesterol is not present, liposomes interact rapidly with plasma proteins such as albumin, transferrin and macroglobulin. These proteins extract bulk phospholipids from liposomes, there by depleting outer monolayer of vesicles leading to physical instability. Liposomes with different surface charges bind different arrays of plasma proteins.
Clearance and Distribution of Liposomes
Liposomes injected into circulation are gradually sequestered in various tissues, probably in the intact form.
The size and surface charge of liposomes are 2 major determinants of liposomes clearance. Thus small U.L.V persist in the circulation for longer periods than large multilammellar vesicles of the same composition. If administered homogenous liposomes the clearance can be described by exponential functions and if heterogeneous, some of exponential is needed, indicating that clearance of liposomes is a single type size depended process.
The charge also affects clearance. SUV with positive and negative charge are returned in the circulation for long periods, whereas small negative vesicles are rapidly cleared. Liposomes with alternative surface changes bind different arrays of plasma proteins.
After the clearance from circulation liposomes are sequestered in various tissues of organs. In case of MLVs, the primary sites of uptake are the liver & spleen which are rich in phagocytic R.E. cells, and blood flow is through open sinusoids rather than through capillaries, so the liposomes can leave the circulation and be taken up by Hepatic and splenic R.E. cells
Large MLVs are preferentially retained in the lung which may be due to physical entrapment of liposomes into the capillary beds of this organ
SUVs have a broader tissue distribution than MLVs
From studies, it was observed that, liposomes are taken up primarily by kupffer cells and perhaps by other liver sinusoidal cells possibly followed by slow redistribution of some material to hepatocytes
The destination can be modified by synthetic aminoglycolipids
For e.g. the positive charged methyl 2 amino 6 palmitoyil - glucosides can increase the residence in circulation
Hall life (T1/2 )
Behavior of encapsulated drug is largely determined by the behaviour of liposomes and thus T1/2 may be affected. i.e., liposome combination remained intact for longer period, thus the T1/2 increases.
For e.g. Daunorubicin ® T1/2 of conventional drug is 2 min. while that of liposomal is 2 hr
Encapsulated drug themselves tends to accumulate in the liver, spleen and other areas rich in R.E elements. Encapsulation puts the drugs in shattered or cryptic form and thus the rate of metabolism of encapsulated drug is less than that of free drug. This may be beneficial where metabolic degradation occurs.
Factors affecting clearance and distribution
1. Particle size and charge
Large liposomes are cleared more rapidly than small ones and negatively charged vesicles are cleared more for rapidly than neutral or positive ones.
2. Chemical composition
If cholesterol is not present, liposomes can bind to plasma proteins. If membrane stabilizers against serum lipoproteins are added then decreases clearance.
3. Dose or load of liposome administered
The particle clearance rate of RES is inversely proportional to the load of liposomes i.e., rate of clearance for larger dose is smaller than for smaller dose.
4. Structure of capillary endothelium
5. Phagocytotic capabilities of RES
6. Fluidity of liposomal membrane
BARRIERS FOR DISTRIBUTION
Presence of any barrier should be considered while formulating a drug in liposome. Generally or mainly three barriers are much effective internally. They are
- The endothelial barrier of the vasculature
- The phagocytic cells of the R.E.S
- Cellular barrier by complex compartmentalized organization of cells
- Endothelial barrier
The cells which separate the vascular and extra vascular compartments act as a barrier. The barrier is mainly based on size and which regulate flow of solute between these compartments. Most of the exchange takes place on capillary endothelium which is having a surface area of 60m2 in an adult.
The nature of capillary endothelial differs in different tissues
- In liver and spleen, sinusoidal vessels, both the endothelial cell layer at the underlying basement membrane are "Fenestrated" which allow passage of molecules up to 1000A0 into the tissue spaces of these organs
- In renal glomerulus and in some glandular tissues a thin cellular layer is penetrated by transverse openings of about 600-800 A0 with an underlying continuous basement membrane.
- Most common is a continuous endothelium where the cells are closely built one upon another, joined by height occluding functions and are subtended on a continuous basement membrane at 200-500 A0 thickness.
Macro molecules can cross by transcytosis. But liposomes are excluded from this. So they are either remaining on the luminal side of the capillary endothelium or may exit from the circulation in specialized sites such as the sinusoidal vessels of liver and spleen.
- R.E.S barrier
The R.E.S is composed of mononuclear phagocytic cells which care essential part of the defense functions of the body. A primitive but crucial function of the macrophages of the R.E. system is to monitor the blood stream and to remove and engulf circulating pathogens, tissue debries of damaged macromolecules.
Like wise they will also very effectively capture liposomes and clear them from the circulation. Examples of macrophages which are effectively involved are kupffer cells of the liver and the macrophages which border the splenic sinusoidal vessels ideally positioned to intercept circulating particles.
The non specific phagocytic capabilities of macrophages are highly developed and these cells readily take up a variety of microparicles including liposomes. In addition, macrophages possess specific receptor mediated endocytic mechanisms of high efficiency. The most macrophages express surface receptors for the F.C domain of IgG, for complement components, for mannosyl or fucosyl terminated glycoproteins and for fibronectin. Particle uptake via these specific systems can often exceed basal uptake by a factor 100 or more. These specific receptor mediated endocytotic system may sometimes come into play in the clearance of circulating liposomes. For instance, repeated use of a drug coupled to a protein micro carrier may elicit an immune response. The antibodies produced would then bind to the micro particle and promote rapid uptake via the F.C receptor of macrophages. Highly charged anionic particle can trigger the alternate path way of complement activation, there by causing complement components to bind to the particle and hastening its uptake via macrophage receptors. Alternatively micro particulates may simply adsorb certain serum proteins such as fibronectin, which can interact with macrophage receptors and thus promote particle clearance.
In summary an understanding of the cells of the R.E.S seems a prerequisite to the intelligent design of liposomal drug carriers. This is because the fate of such carriers will largely be determined by the phagocytic activities of the RES cells
- Cellular barrier
The cells are highly organized entities so it is difficult to target a drug to receptors with in a cell. The task of drug delivery system then is not so much to get the drug to a given tissue or cell type but rather to get the drug to the appropriate receptor.
So the cell itself acts as a barrier to the drug delivery system. The simple fact that a drug carrier entity binds to a particular cell does not ensure that the drug will have an affect on that cell.
E.g. 1. When monoclonal antibodies are bound to liposomes containing a cytotoxic drug, specifically to lymphoid tumor cells – when the antibodies were directed against certain cell surface antigens, the liposomes were internalized and the drug was able to exert its effect on the cell. On the other hand, with antibodies directed against other determinants, the liposomes were bound to cells but not internalized and thus the drug was without effect.
E.g. 2. Amphotericin B is ordinarily very toxic to mammalian cells because it interacts with cholesterol in the plasma membrane to form pores or channels which then leads to osmotic lysis of the cell. We have found that when AMB is incorporated in liposomes, it is much less toxic to mammalian cells, even when cells take up quantities of liposomal drug. Thus macrophage can internalize substantial amount of liposomal AMB, presumably into an endosome compartment without any appreciable cytotoxicity. The drug is with in the cell, but not at the critical place for an effect to occur. Since liposomal drug is lipophilic it can cross the ordinary barriers to the polar drug. Thus it reaches brain, CNS,etc
PHARMOCODYNAMICS OF LIPOSOME ENCAPSULATED DRUGS
To continue the action of drugs to a particular site in the body, the general approach is to deposit drug bearing liposome directly into the site where therapy is desired. Since liposomes are large and do not easily cross epithelial or connective barriers, they are likely to remain at the site of local administration. The liposomes would then slowly released into the target site or perhaps create a local drug level higher than the systemic level. Alternatively the drug loaded liposomes might interact directly with cells in the target site, without producing release. The goal of this approach is to maximise the amount of effective drug at the target site, while minimizing the drug levels at other sites and thus decreasing systemic toxicity.
- SUV injected into the skin can persist interact at the site for 600 hrs. And release of entrapped markers from the liposomes occurs only after cellular uptake and intracellular space remain intact.
- In rats Ara-C when liposomal drug injected directly to lungs, persisted in lung for long time while free drug given on same manner enters the systemic circulation.
The liposomal Ara-C inhibit DNA synthesis with little effect on other tissues (normally sensitive) such as get and bone marrow, where as free drug depresses DNA synthesis throughout the body.
For treating superficial tumors the liposome encapsulated drug [Methotrexate] was prepared with transition temperature just above the normal body temperature. These drugs are injected and tumors heated to 42oC which causes drug release exactly in the tumor without any toxic effect.
The liposomal drug tends to have the following Pharmacodynamic effects
- Retardation of drug clearance from the circulation
- High drug accumulation in tissues rich on RES especially in liver and spleen
- Retention of drug in tissues for large period
- Protection of drug against metabolic degradation.
METABOLIC FATE OF BILAYER FORMING LIPIDS
The liposomal membrane, from the body (i.e., lipids and cholesterol) is broken down by enzyme systems into natural intermediates like glycerol phosphate, fatty acids, ethanolamine cholose and acyl COA and these either metabolites to provide energy, or enter lipid pool; which are drawn to build new lipids and replace those that naturally turnover in biological membrane.
Phospho lipids are hydrolysed by phospholipases
- Phospholipase A1
® removes F.A from C1 of Glycerin
- Phospholipase A2
® removes F.A from C2 of Glycerin
- Phospholipase B ® mix of P A1 & PA2 remove both F.A. chains
- Phopholipase C ® Catalyse hydrolysis of bond of PC and Glycerol
- Phosphlipase D ® Clears off the polar alcohol head group to leave phosphatidic acid
Fatty acid generated enters the F.A pool and may be used as precursors to regenerate new phospholipids or triglyceroids and converted to Acyl CoA and oxidised to CO2 and water via Beta oxidation to yield energy.
Glycerol phosphate remains as such and serve as the back bone for the formation of new phospholipids or triglycerides. Cholesterol get deposited on liver. A chief portion of cholesterol gets excreted in bile. In Lumina of gut cholesterol broken into Coprastanol by intestinal bacteria. 80-% of cholesterol taken up by liver and transformed into bile acids
TARGETING OF LIPOSOMES
Two types of targeting.
1. Passive targeting
As a mean of passive targeting, such usually administered liposomes have been shown to be rapidly cleared from the blood stream and taken up by the RES in liver spleen. Thus capacity of the macrophages can be exploited when liposomes are to be targeted to the macrophages. This has been demonstrated by successful delivery of liposomal antimicrobial agents to macrophages.
Liposomes have now been used for targeting of antigens to macrophages as a first step in the index of immunity. For e.g. In rats the i.v administration of liposomal antigen elicited spleen phagocyte mediated antibody response where as the non liposome associated antigen failed to elicit antibody response.
2. Active targeting
A pre requisite for targeting is the targeting agents be positioned on the liposomal surface such that the interaction with the target i.e., the receptor is tabulated such as a plug and socket device. The liposome physically prepared such that the lipophilic part of the connector is anchored into the membrane during the formation of the membrane. The hydrophilic part on the surface of the liposome, to which the targeting agent should be held in a stericaly correct position to bond to the receptor on the cell surface.
The active targeting can be brought about the using
i. Immuno liposomes
These are conventional or stealth liposomes with attached Antibodies or other recognition sequence [e.g. Carbohydrate determinants like glycoprotein]
The antibody bound, direct the liposome to specific antigenic receptors located on a particular cell. Glycoprotein or Glycolipid cell surface component that play a role in cell-cell recognition and adhesion
ii. Magnetic liposomes
Contain magnetic iron oxide. These liposomes can be directed by an external vibrating magnetic field in their delivery sites.
iii. Temperature or heat sensitive liposomes
Made in such a way that their transition temperature is just above body temperature. After reaching the site, externally heated the site to release the drug.
Table 1: Major modes of liposomal action and related application
The following are some properties which make liposomes applicable in various fields
1. Cell -liposome interaction
a) Stable adsorption - Association of intact vesicles with cell surface, mediated by non specific electrostatic, hydrophobic or other forces or by specific components present in the vesicle or on the cell surface.
b) Endocytosis - Uptake of intact vesicles
c) Fusion - Merging of the vesicle bilayer with plasma membrane with concomitant release of vesicle contents to the cytoplasm.
d) Lipid exchange - Transfer of individual lipid molecules between vesicles and cell surface, without the cell association of aqueous vesicle content
2. Localized drug effect
Liposomes help in depositing the drug within selected sites or selected cell. Due to their larger size and low degree of penetration in epithelial and connective tissue barriers which tend to remain at the site
3. Enhanced drug uptake
By vesicle cell fusion or via endocytosis
4. Molecules with wide range of solubility and molecular weight can be accommodated
5. Flexibility in structural characteristics
1. Cancer chemotherapy
Liposomes by virtue of their ability to be modified on the surface can be used as excellent delivery vehicles for anti tumor drugs.
Liposomes are used to:
- Target drugs to the tumors
e.g. a) The liposomal Ara -C inhibit DNA synthesis in the lungs
b) For targeted drug delivery for blood born Neoplasms
c) By active targeting using monoclonal antibodies, by magnetosomes or by temperature sensitive liposomes
d) By passive targeting to liver, spleen, R.E.S cancers.
- Reduction of Toxicity
This is usually due to targeted or site specific delivery.
e.g. Hydrophobic drugs including alkylating agents, antimitoticagents, anthracyclines
Liposomal encapsulation of doxorubicin can reduce does limiting to toxicity to myocardium without loss of antitumor potency, Also it reduces toxicity to skin. This may be due to low uptake of the drug by the myocardium. Also the following actions were observed.
- Reduction of immuno suppressive actions
- Enhanced tumoricidal effects in certain organs such as liver
- Enhancement of membrane directed actions
Generally by incorporating into liposomes the following objectives are obtained
- Increase circulation life time. i.e., drug tends to deposit in the tissue.
- Protects from the metabolic degradation of drug.
- Altered tissue distribution of drugs with enhanced uptake in organs rich in mono nuclear phagocytic cells (liver, spleen, and bone marrow) and decreased uptake in kidney, myocardium or brain.
Most of the anticancer potency of encapsulated drug has been concentrated in particular specific phase of the cell cycle. They are called as cell specific drugs.
The capillary endothelium of R.E.S tend to prevent selective delivery of liposomal drugs to solid neoplasms
Table 2: Anticancer drugs used in liposomes
Route of administration
Table 3: Some drugs which are in clinical trial
3. HLA-B7 Plasmid
Gene therapy of metastatic cancer
B 16 melanoma
2. Gene therapy
Liposomes can be used to deliver DNA into the cell. This is because of the ability of liposomes to enhance intracellular accumulation i.e., facilitate transfer of large & charged molecules across rather impermeable cell membranes.
Cationic liposomes (C.L s) are used as gene vectors (carriers) in nonviral gene therapy. These lipid gene complexes were bound to have the potential for transferring large pieces of DNA of up to 1 million base pairs into cells.
C.Ls can also be used for the delivery of RNA, antisense oligonucleotides, ribozymes, proteins and other negatively charged molecules.
Liposomes have larger carrying capacity and lack of immunogenicity and safer so preferred over other usual vectors.
Lipids used for this purpose are
- Dioctadecyl dimethyl ammonium bromide (DODAB)
- 1.2 di acyl 3 trimethyl ammonium propane (DOTAD)
- 2.3 bis (oleoyl) propyl trimethyl ammonium chloride (DOTMA)
In this fatty acids are attached to propyl back bone via ether derivatives of these are prepared by attaching polyelectrolyte (poly lysine) and polycations (spermine, spermidine) onto the diacylated back bone of the sterol group.
E.g. 2, 3 di oleoyl org N [spermine carboxamino ethyl] NN dimethyl 1 - propanaminium trifluorate (DOSPA) and poly lysine lipid.
An alternative approach is to impose a positive charge on cholesterol, and a series of such molecules were synthesized. Natural zwitterionic lipid can be rendered cationic by reacting of thus eliminating the negative charge on the phosphate group& the zwitter ionic & positively charged amino acids can be (di) acylated to form positively charged acylesters or diacylated basic amino acids. Novel approaches are to exploit longer polyelectrolyte, lipopolyelectrolytes and other polymer s such as dendrimers and block copolymers.
The properties of C.Ls like (1) length and saturation of fatty acids, (2) nature of chemical bonds between various parts of the molecule, (3) the space length between the charge and the hydrophobic part of the molecule, (4) presence and nature of back bone, (5) nature of the charge and its Pk value, (6) charge density or number of charges per molecule, (7) hydroxylations, ethylhydroxylations, methylations etc of polar head can influence transfection efficiency.
These lipids are mixed with DOPE normally at equimolar ratio. Only rarely are other neutral lipids such as lecithin or cholesterol or no lipid used. Pure cationic lipid liposomes most notably DOTAD, transfect better than their mixtures with DOPE. Cholesterol was found to be more effective than DOPE.
Lipofectin DOTMA: DOPE (1: 1)
Lipofectamine DOSMA: DOPE (3: 1)
Lipofectace DODAB: DOPE (1: 2.5)
Cellfectin TMTPSP: DOPE (11.5)
DC chol Dc chol: DOPE 3:2
TFX-50 TDA: DPE (1: 1)
3. Liposomes as carriers for vaccines
Liposomes as immunological adjuvant
Can be used as an adjuvant for protein antigens (diphtheria toxoid)
Advantages in use of liposomes as carriers for vaccines include
- A non immunogenic substance may be converted to immunogenic
- Hydrophobic antigens may be reconstituted.
- Small amount of antigen may be suitable as immunogen
- Multiple antigen may be incorporated into single liposomes
- Adjuvant may be incorporated with antigens into liposomes
- Longer duration for functional antibody activity may be achieved
- Toxic and allergic reactions of antigens may be reduced or eliminated by inclusion in liposomes
- Soluble synthetic antigen may be presented as membrane associated antigen in an insoluble liposomal matrix.
Natural negatively charged liposomised diphtheria toxoids were equally responsible to produce the same immune response where as positive charged produced reduced responses, indicating that responses are unpredictable with charge.
In comparing the size, U.L.Vs are more effective than MLVs to entrap BSA.
Liposomes whose transition temperature higher than ambient temperature (e.g. DPPC & DSPC) is more effective.
Routes of immunization ® i.v. i.p; i.m; s.c.
b) Liposomes as carrier of antigens
For effective use, the following points should be considered
- Rate of uptake of liposome by RES, must be minimised by using small, neutral, ULV having higher Transition temperature and cholesterol.
- By coating the surface of liposome, this would render liposomes less recognizable by RES.
- Coupling appropriate molecules (legends) on the liposomes surface which can bind to their receptors on the surface of target cells.
Proteins (Antigens) may be distributed either on the outer surface of lipid bilayer or within the bilayer.
Also liposome can incorporate lipopolysacchrides (LPs) muramyl di peptide (MDP), lymphokines etc. to trigger immune response.
Table 4: Liposomes for gene delivery
Liposome composition and nature
Plasmodium falciparum merojoiteenriched antigen
DPPC:CH Neutral MLVs
All immunized monkey survived the challenge only with the
Mycobacterium leprae antigen
Liposomised antigen clicited both early and late delayed type hypersensitivity, unlike the soluble antigen alone which elicits only early reaction.
Various phospholipids, CH
Adjuvant effect dependent on liposomal characteristics and source, amount and formulation of IL-2; demonstration of receptor mediated targeted adjuvanticity.
Hepatitis B surface antigen
Various phospholipids CH:DCP
4 Liposomes as carrier of drug in oral treatment
Oral route is used not only for convenience, but it is important that drugs enter the periphery via portal circulation
Treated with steroids using MLVs prepared by DPPC and P.A.
- since steroids are destroyed by their peripheral effect and on local administration into joints due to diffusion, only transient action on inflamed area , so liposomes are used as carriers.
e.g. for Drugs are Ibuprofen, cortisol palmitate
Alternation in blood glucose level in diabetic animals was obtained by oral administration of liposome encapsulated insulin (PC: CH liposomes)
Liposomes can protect insulin in gastric and intestinal areas [i.e., proteolytic digestive enzymes - Pepsin and Pancreatin].But this is not effective in presence of bile acids
Also liposome can increase the intestine uptake of macromolecules
5. Liposomes for topical applications
table 5: Liposomal
drugs for topical
In epidermis and dermis 4 times higher conc. than control ointment. Decreased urinary excretion of drug.
Reduces the rate of hair growth in idiopathic hixsutims
Reduce percutaneous absorption of drug was obtained. Retention of methotrexate in skin was 2-3 fold higher than free form.
Higher conc. of drug in the individual layers of human skin than control ointment.
Increase conc. of the drug in the subcutaneous tissue as well as increase permeation through the skin.
6. Liposomes for pulmonary delivery
Size of liposome is a critical particulate parameter determining the deposition site with in the lung. Variation in lipid composition provided the opportunity of controlling the release rate of entrapped solute usually applied by a Nebuliser.
Table 6: Liposomal drugs for pulmonary delivery.
Free Ara-C was rapidly absorbed into the (Ara-C) systemic circulation whilst liposome encapsulated drug remained within the lung for a considerable time, hence reduces the adverse effects.
No significant difference in organ distribution on comparing free Vs liposome enccapulated drug. Aerosolized product produced substantially higher deposition in the alveolar.
Free drug produced peak plasma level more than seven fold higher than the liposomal drug. Showed extended duration of drug plasma level.
Shorter duration of effect from free sulfate drug compared to the same dose of liposomal drug.
7. against Leishmaniasis
A parasitic disease if severe affects liver and spleen. Drugs contain high level of arsenic, so are highly toxic. So encapsulation into liposomes reduces toxicity and provides site specific delivery.
8. Lysosomal storage disease
These are Heterogeneous group of disorders due to genetically determined defects of lysosomal hydrolytic enzymes. This include Beta glucosidase deficiency and Pomp's disease (Alpha glucosidase deficiency)
In former catabolite accumulation via RES and in later primary infected tissues are liver and muscle. So lysosomal enzymes are incorporated in liposomes and administered. Usually immunoglobulin coated liposomes are used for better results.
9) Cell biological application
For manipulating the status of membrane lipid, by liposomes through lipid exchange particularly cholesterol.
Here also uses capacity of liposome to carry DNA & RNA to cells
Also used to insert regulatory molecules such as (AMP, CGMP and enzymatic co factors into the cell)
10) Metal storage disease
Many chelating agents in its original form (EDTA, DTAA) can not cross cell membranes.
In these several diseases, metal accumulates in the lysosomes of the cells; the lysosomotropic action of liposomes renders this carrier a hopeful approach to therapy.
Liposomal DTPA was capable of removing significant amounts of Plutonium from liver of Mice loaded with metal.
When EDTA incorporated in liposome it diffuses and lost during circulation. So 14C labeled EDTA phosphatidyl ethanolamine complex was incorporated in liposomes composed of egg PC, cholesterol and P.A. The EDTA- Phospholpid complex has capability of forming liposome by its self, from blood than that exhibited by labeled EDTA entrapped in similar liposomes
11) Ophthalmic delivery of drugs
In order to maintain optimal drug concentration at the site of action liposomes are used as carriers or vehicles
E.g. Treatment of Keratitis by Idoxuridine Also increases (2 times) the Trans corneal flux of penicillin G, indoxol and carbachol.
Major advantage of liposomes is their ability to intimately contact with the corneal and conjunctival surfaces thereby increasing drug absorption
Also by varying the Phospholipid composition or by incorporating legands for receptors, can control degree of liposome accumulation.
Also liposome protect drug from its metabolism
The effectiveness of liposomes in ocular, drug delivery depends on
- Drug encapsulation efficiency
- Size and charge of liposomes
- Distribution of a drug within liposomes
- Stability of liposomes in the conjunctival sac and ocular tissues
- Retention of liposomes in the conjunctival sac
- Affinity of liposomes exhibited towards corneal surface
Table 7: Liposomal drugs for ophthalmic delivery
Improved efficacy of liposomes encapsulated drug.
Observed significant higher conc. of drug in ocular tissues.
Benzyl penicillin indoxol
Ocular bioavailability enhanced by delivery in liposomes.
Absorption greatly enhanced.
Flux was enhanced.
Considering the advantages of this drug delivery system – Liposomes and also its modifications or upgraded versions like Enzymosomes, Hemosomes, Virosomes, Erythrosomes, Virosomes, etc, Liposomes have emerged as a dynamic mode for Targeted Drug Delivery.
- Remington, The science and practice of pharmacy PgNo: 919
- The Pharma Review, June 2005, Liposome a magic bullet concept. PgNo: 53-58
- Lasic et al, Liposome a controlled drug delivery system. PgNo: 44-85
- Rudy L.Juliano, Micro particulate drug carriers, Liposomes, Microspheres and cells PgNo: 555-573
- Alving C.R Macrophages, as targets for delivery of liposome encapsulated antimicrobial agents. Adv Drug Delivery Rev, 2(1998)
- Sharma. A. and Sharma Liposomes in drug delivery progress and Limitations Int J.Pharm
- Su.D.et.al The role of Macrophages in the immune adjuent action of liposomes, Immune response against intravenously injected liposome associated albumin antigen. Immunology
- N.K Jain Controlled and novel drug delivery
- Indian Journal of Pharmaceutical science; Vesicular systems-An overview PgNo: 141-152
- http//en .ijpsonline/Liposomes