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LIPOSOMES A NOVEL DRUG DELIVERY SYSTEM

LIPOSOMES A NOVEL DRUG DELIVERY SYSTEMM. PraveenHH, Rahul Soman, Vimal Mathew

National College of Pharmacy, Manassery, Calicut

INTRODUCTION

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 aqueousphase 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.

DEFINITION

Liposomes are defined as structure consisting of one or more concentric spheres of lipid

bilayers separated by water or aqueous buffer compartments.

Or simply,

Liposomes are simple microscopic vesicles in which an aqueous volume is entirelyenclosed by a membrane composed of lipid bilayers.

Figure: Liposomes

ADVANTAGES


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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

DISADVANTAGES

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

CLASSIFICATION

I. Based on composition and mode of drug delivery

1. Conventional liposomesComposed of neutral or negatively charged phospholipids and cholesterol. Subject to

coated pit endocytosis, contents ultimately delivered to Lysosomes if they do not fuse


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with the endosomes, useful for E.E.S targeting; rapid and saturable uptake by R.E.S;

short circulation half life, dose dependent pharmacokinetics.

1. pH sensitive liposomesComposed 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.

1. Cationic LiposomesComposed 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

1. Long circulating or stealth liposomesComposed 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

1. Immuno liposomesConventional 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.

1. Magnetic Liposomes


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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.

1. Temperature (or) heat sensitive liposomesComposed 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

1. 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.

2. 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.


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3. 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.

STRUCTURAL COMPONENTS

PhospholipidsGlycerol 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

o Phosphatidyl choline (Lecithin) PCo Phosphatidyl ethanolamine (cephalin) PEo Phosphatidyl serine (PS)o Phosphatidyl inositol (PI)o Phosphatidyl Glycerol (PG)

For stable liposomes, saturated fatly acids are used. Unsaturated fatty

acids are not used generally.

SphingolipidsBackbone is sphingosine or a related base. These are important constituents of

plant and animal cells. This contain 3 characteristic building blocks


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o A mol of F.Ao A mol of sphingosineo 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.

SterolsCholesterol & its derivatives are often included in liposomes for

o decreasing the fluidity or microviscocity of the bilayero reducing the permeability of the membrane to water soluble

molecules

o Stabilizing the membrane in the presence of biological fluids suchas 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 phospholipidsE.g.: for saturated phospholipids are

o Dipalmitoyl phosphatidyl choline (DPPC)o Distearoyl phosphatidyl choline (DSPC)o Dipalmitoyl phosphatidyl ethanolamine (DPPE)


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o Dipalmitoyl phosphatidyl serine (DPPS)o Dipalmitoyl phosphatidic acid (DPPA)o Dipalmitoyl phosphatidyl glycerol (DPPG)

E.g.: for unsaturated phospholipids

1. Dioleoyl phosphatidyl choline (DOPC)2. Dioleoyl phosphatidyl glycerol (DOPG)

Polymeric materialsSynthetic 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 lipidsStability 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.


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Cationic lipidsE.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 SubstancesVariety of other lipids of surfactants are used to form liposomes

o Many single chain surfactants can form liposomes on mixingwith cholesterol

o Non ionic lipidsA variety of Polyglycerol and Polyethoxylated mono and dialkylamphiphiles used mainly in cosmetic preparations

o Single and double chain lipids having fluoro carbon chains canform very stable liposomes

o Sterylamine and Dicetyl phosphateIncorporated into liposomes so as to impart either a negative

or positive surface charge to these structures

o A number of compounds having a single long chainhydrocarbon 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

2) Ultrasonication3) French Pressure cell

4) Solvent injection method

a) Ether injection method

b) Ethanol injection

5) Detergent removal

Detergent can be removed by


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a) Dialysis

b) Coloumn chromatography

c) Bio-beads

6) Reverse phase evaporation technique

7) High pressure extrusion

8) Miscellaneous methods

a) Slow swelling in Non electrolyte solution

b) Removal of Chaotropic ion

c) Freeze-Thawing

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.Cfound to be 4.6 1010 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 hydrophobiccore in order to achieve the lowest energy level, hence the highest stability for the

aggregate structure.


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PHARMACOKINETICS OF LIPOSOMESLiposomal 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.

o endocytosis by Phagocytotic cells of the R.E.S such asmacrophages and Neutrophils

o adsorption to the cell surface either by non specific weakhydrophobic or electrostatic forces or by specific interaction

with cell surface components

o Fusion with the plasma cell membrane by insertion of lipidbilayer of liposome into plasma membrane with simultaneous

release of liposomal contents into the cytoplasm.

o Transfer of liposomal lipids to cellular or sub cellular membraneor 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.

Plasma Interaction

If cholesterol is not present, liposomes interact rapidly with plasma proteins such

as albumin, transferrin and macroglobulin. These proteins extract bulkphospholipids 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 liposomesclearance. 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.


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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 chargeLarge 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


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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 DISTRIBUTIONPresence of any barrier should be considered while formulating a drug in

liposome. Generally or mainly three barriers are much effective internally. They

are

1. The endothelial barrier of the vasculature2. The phagocytic cells of the R.E.S3. Cellular barrier by complex compartmentalized organization of cells

1. Endothelial barrierThe 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 theunderlying 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 ispenetrated by transverse openings of about 600-800 A0 with an

underlying continuous basement membrane.


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Most common is a continuous endothelium where the cells are closelybuilt 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.

1. R.E.S barrierThe 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 aprotein 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


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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

2. Cellular barrierThe 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 channelswhich 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 ENCAPSULATEDDRUGS

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


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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.

For e.g.

SUV injected into the skin can persist interact at the site for 600 hrs. Andrelease 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 lungfor 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 42o

C which causes drug release exactly in thetumor without any toxic effect.

The liposomal drug tends to have the following Pharmacodynamic effects

1. Retardation of drug clearance from the circulation2. High drug accumulation in tissues rich on RES especially in liver and

spleen

3. Retention of drug in tissues for large period4.

Protection of drug against metabolic degradation.

METABOLIC FATE OF BILAYER FORMING LIPIDSThe 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


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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

1. Phospholipase A1 removes F.A from C1 of Glycerin

2. Phospholipase A2 removes F.A from C2 of Glycerin

3. Phospholipase B mix of P A1 & PA2 remove both F.A. chains4. Phopholipase C Catalyse hydrolysis of bond of PC and Glycerol5. 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 LIPOSOMESTwo 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 stepin 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


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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 aparticular 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.

APPLICATIONS

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


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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 tumorse.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.


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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.

Disadvantages

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

Drug Route of administration

1. Methotrexate Transdermal

2. Doxorubicin Oral, i.v.

3. Daunorubicin i.v.

4. Cytarabin Pulmonary

Table 3: Some drugs which are in clinical trial

Drug Status Indication

1. Annamycin Phase II Breast cancer

2. Tretinion Phase I Blood cancer

3. HLA-B7 Plasmid Phase II Gene therapy of metastatic cancer

4 Lymphokinine Phase II B 16 melanoma


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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


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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.

Name Composition

Lipofectin DOTMA: DOPE (1: 1)

Lipofectamine DOSMA: DOPE (3: 1)

Lipofectace DODAB: DOPE (1: 2.5)

DTAD DOTAD

Cellfectin TMTPSP: DOPE (11.5)

DC chol Dc chol: DOPE 3:2TFX-50 TDA: DPE (1: 1)

3. Liposomes as carriers for vaccines

a)

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

1.

A non immunogenic substance may be converted to immunogenic2. Hydrophobic antigens may be reconstituted.3. Small amount of antigen may be suitable as immunogen4. Multiple antigen may be incorporated into single liposomes5. Adjuvant may be incorporated with antigens into liposomes6. Longer duration for functional antibody activity may be achieved7. Toxic and allergic reactions of antigens may be reduced or eliminated by

inclusion in liposomes

8. Soluble synthetic antigen may be presented as membrane associated antigen inan 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.


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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

1. Rate of uptake of liposome by RES, must be minimised by using small, neutral,ULV having higher Transition temperature and cholesterol.

2. By coating the surface of liposome, this would render liposomes less recognizableby RES.

3. Coupling appropriate molecules (legends) on the liposomes surface which canbind 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

AntigetnLiposome composition

and natureMajor observations

Plasmodium falciparum

merojoiteenriched antigen

DPPC:CH Neutral

MLVs

All immunized monkey survived the challenge only with

the

Mycobacterium leprae

antigen

PC:CH:Ganglioside,

ULVs

Liposomised antigen clicited both early and late delayed

type hypersensitivity, unlike the soluble antigen alone

which elicits only early reaction.

Tetanus toxoidVarious 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 antigenVarious phospholipids

CH:DCPAdjuvant effect

Poliovirus peptideVarious

phospholipids:CHAdjuvant effect

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

a) Arthritis


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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

B) Diabetes

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

applications

Drug Results

TriamcinoloneIn epidermis and dermis 4 times higher conc. than control ointment. Decreased urinary excretion

of drug.

Progesterone Reduces the rate of hair growth in idiopathic hixsutims

Methotrexate Reduce percutaneous absorption of drug was obtained. Retention of methotrexate in skin was 2-3fold higher than free form.

Hydrocortisone Higher conc. of drug in the individual layers of human skin than control ointment.

Diclofenac gelIncrease 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.

Drug Results

Cytosine Free Ara-C was rapidly absorbed into the (Ara-C) systemic circulation whilst liposome


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arabinoside encapsulated drug remained within the lung for a considerable time, hence reduces the adverse

effects.

PentamidineNo significant difference in organ distribution on comparing free Vs liposome enccapulated

drug. Aerosolized product produced substantially higher deposition in the alveolar.

Sodiumcromoglycate

Free drug produced peak plasma level more than seven fold higher than the liposomal drug.Showed extended duration of drug plasma level.

Metepreterenol 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.

E.g. Desferrioxamine

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.


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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

Drug Results

Idoxuridine Improved efficacy of liposomes encapsulated drug.

Triamcinolone acetonide Observed significant higher conc. of drug in ocular tissues.

Benzyl penicillin indoxol Ocular bioavailability enhanced by delivery in liposomes.

Inulin Absorption greatly enhanced.


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Penicillin G Flux was enhanced.

CONCLUSION

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.

REFERENCES

1. Remington, The science and practice of pharmacy PgNo: 9192. The Pharma Review, June 2005, Liposome a magic bullet concept. PgNo: 53-583. Lasic et al, Liposome a controlled drug delivery system. PgNo: 44-854. Rudy L.Juliano, Micro particulate drug carriers, Liposomes, Microspheres and

cells PgNo: 555-5735. http//en.wikipedia.org/wiki/liposome6. Alving C.R Macrophages, as targets for delivery of liposome encapsulated

antimicrobial agents. Adv Drug Delivery Rev, 2(1998)

7. Sharma. A. and Sharma Liposomes in drug delivery progress and Limitations IntJ.Pharm

8. 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

9. N.K Jain Controlled and novel drug delivery10.Indian Journal of Pharmaceutical science; Vesicular systems-An overview PgNo:

141-152

11.http//en .ijpsonline/Liposomes

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