LIPOSOMES

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Introduction :

 Liposome was found by Alec Bangham of Babraham Institute in

Cambridge, England in 1965.

 In 1990, drugs with liposome and Amphotericin B were approved by

Ireland.

 In 1995 America F.D.A approved liposor doxodubicin.

 An artificial microscopic vesicle consisting of an aqueous core enclosed

in one or more phospholipid layers, used to convey vaccines, drugs,

enzymes, or other substances to target cells or organs.

 The membrane of liposome is made of phospholipids, which have

phosphoric acid sides to form the liposome bilayers.

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 #Definition:- Liposomes are small biocompatible and

Biodegradable lipid vesicles composed of Uni

lamellar or Multi lamellar phospholipid bilayers

surrounding aqueous compartment.

 Their Biophysical properties, such as size, surface

charge, lipid composition, and amount of cholesterol

controls the distribution, tissue uptake and drug

delivery.

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 Basically Liposomes are resultant of self

assembly of phospholipid in an aqueous core.

 The lipid soluble drug will be enclosed in the

phospholipid bilayer and the water soluble drug will

be enclosed in aqueous core.

 Liposomes are spherical in shape and they are

mainly used in the drug delivery in Tumor or cancer.

In Recent research liposomes are mainly “Brain

Targeting Drug Delivery Systems” in Brain cancer.

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Liposome Structure :

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Composition of liposomes:
A. Phospholipids

The most common natural phospholipid is the phospatidylcholine

(PC) is the amphipathic molecule and also known as lecithin.

Naturally occurring phospholipids used in liposomes are:

 Phosphatidylcholine

 Phosphatidylethanolamine

 Phosphatidylserine

Synthetic phospholipids used in the liposomes are:

 Dioleoyl phosphatidylcholine

 Disteroyl phosphatidylcholine

 Dioleoyl phosphatidylethanolamine

 Distearoyl phosphatidylethanolamine
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B. Cholesterol:
 Cholesterol can be incorporated into phospholipids membrane in

very high concentration up to 1:1 or 2:1 molar ratios of cholesterol

to phospatidylcholine.

 Being an amphipathic molecule, cholesterol inserts into the

membrane with its hydroxyl group of cholesterol oriented towards

the aqueous surface and aliphatic chain aligned parallel to the acyl

chains in the center of the bilayers and also it increase the separation

between choline head groups and eliminates the normal electrostatic

and hydrogen bonding interaction.

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 The phospholipids are arranged in such a way that

the hydrophilic head is exposed outside and the

lipophillic tails are aliened inside. This makes the

liposomes water soluble molecules.

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Overall Composition:

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Mechanism :- Liposome Formation

As liposomes are made up of phospholipids, they are

amphipathic in nature and have ability to binds both polar

and non polar moiety.

They have polar head and non polar tail.

 The polar end is mainly phosphoric acid and it will bound

to water soluble molecule.

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In aqueous medium the molecules in self-

assembled structure are oriented in such way

that the polar portion of the molecule remain in

contact with in polar environment and at same

time shields the non polar part.

However, in aqueous mixtures these molecules

are able to form various phases, some of them

are stable and other remains in metastable form.

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Types of Liposomes:

Lamella: A Lamella is a flat plate like structure that appears

during the formation of liposomes. The Phospholipid

bilayer first exists as a lamella before getting convered

into spheres.

Based on number of lamellaes there are two types:

1. Unilamellar Vesicles

2. Multilamellar Vesicles

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Classification :

 Based on Structural Parameters:

a. Multi-laminar vesicles (MLV): made up of series of

concentric bi-layer of lipid enclosing a small

internal volume with size range > 0.5um.

b. Oligolamelar vesicles (OLV): constitutes 2 to 10 bi

layer of lipids surrounding a large internal volume

with size range of 0.1 – 1um.

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c. Unilamellar vesicle (ULV): single layer of lipids. Based on

the size of the single layer they are further divide into the

following types as

 Small unilaminar vesicle: size of 20 to 40 nm

 Medium unilaminar vesicle: size of 40 to 80 nm

 Large unilaminar vesicle: size of 100 to 1000 nm

 Gaint unilaminar vesicle: size of more than 1000 nm

d. Multivesicular Vesicle(MV): constitutes for multiple vesicles

and size range >1um.

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General Structure of various types of
liposomes:

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General Method of Liposome
Preparation:

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Different Methods of Preparation:

The methods are broadly classified into
three classes according to basic modes of
dispersion:

1. Physical Dispersion

2. Solvent dispersion

3.Detergent solubilisation.

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1. Physical Dispersion:
There are four basic methods of physical dispersion:

a) Hand shaken multilamellar vesicles.

b) Non shaking vesicles.

c) Pro – liposomes.

d) Freeze drying.

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Hand shaken multilamellar vesicles.

 In this methods, the lipids are casted as stacks of film

from their organic solution using ‘Flash rotatory

evaporator’ or ‘hand shaking’

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The film formation will takes place and the film will be dried

in presence of reduced Nitrogen.

After the film stacks are dispersed in aqueous phase.

 Upon hydration, the lipids will swell and peel off from wall

of round bottom flask and vesiculate forming multi

lamellar vesicles(MLVs).

 Liposomes stored under the nitrogen umbrella store.

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Hand Shaking Method:

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Processing of the lipids hydrated by physical means
or the mechanical treatments of MLVs :

1) Micro Emulsification liposomes(MEL)
2) Sonicated unilamellar vesicles (SUVs)
3) French Pressure Cell Liposomes.
4) Membrane extrusion Liposomes
5) Dried reconstituted vesicles(DRVs)
6) Freeze thaw sonification(FTS)
7) pH induced vesiculation
8) Calcium Induced fusion

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Dried Reconstituted Vesicles (DRV) and
Freeze Thaw Sonication (FTS):

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2) Solvent Dispersion :-

1) Ethanol injection.

2) Ether injection.

3) Double emulsion vesicles.

4) Reverse phase evaporation vesicles.

5) Stable pluri lamellar vesicles

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1)Ether and Ethanol Injection :-

 Ethanol Injection:- This method has been

reported as alternatives used for preparation of SUVs

without sonication.

An ethanol solution of lipids is injected rapidly

through a fine needle into an excess of saline or other

aqueous medium.

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This procedure yields a high proportion of SUVs,

although lipid aggregates and larger vesicles may form

if the mixing is not thorough enough.

This method is very simple and very less risk for the

degradation of sensitive lipids and up to 100 nm size

can be obtained

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Ether Injection :- This method is also similar to the Ethanol

injection method.

It is somewhat different from Ethanol injection. It involves

the injecting the immiscible organic solution very slowly into

an aqueous phase through a narrow needle at the temperature

of vaporizing the organic solvent according to diagram.

 This method may also treat sensitive liquids very gently.

 It has little risk of causing oxidative degradation due to

ether.
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-: Detergent Removal Methods :-

1) Dialysis.

2) Column chromatography.

3) Detergent Adsorption:-

 Detergent/Phospholipids mixtures can form

large unilamellar vesicles upon removal of non

ionic detergent using appropriate adsorbents for

the detergent.
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Mechanism of incorporation of drug in
liposomes:

1. Encapsulation

2. Partitioning

3. Reverse

loading

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-: Application :-

 In Chemo therapy

 Anti cancer drugs are non selective so, there are

more chances of toxicity to normal cell. So,

drugs incorporated in liposome will helpful for

target of drug to neoplastic region because

liposome acts as depot.

 They are bio degradable and non toxic

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 Carriers for vaccines.

 Decreases systemic toxicities of drugs like doxorubicin.

 They are identical with lipid bio membrane due to

phospholipids used in preparation so, more absorption of

drug and also prolonged duration. E.g., Steroids , Insulin

 Used in ocular drug delivery systems.

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 Used for topical application like anti microbial, anti

infective, anti tumor, anti septic, NSAIDs

 It can carry non penetrating material like genetic

molecules so, helpful in genetic manipulation.

 Treatment of Leishmaniasis:-

 Drugs used are toxic to heart, liver, kidney but safe

when incorporated in liposomes.

 Flexible in surface characteristic.

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Advantages :
 Biocompatible, completely biodegradable, non-toxic,

flexible and nonimmunogenic.

 Liposomes supply both a lipophilic environment and
aqueous “milieu interne” in one system.

 Liposomes have the ability to protect their encapsulated
drug from the external environment.

 Liposomes help to reduce exposure of sensitive tissues to
toxic drugs.

 Alter the pharmacokinetic and pharmacodynamic
property of drugs (reduced elimination, increased
circulation life time).

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 Flexibility to couple with site-specific ligands to

achieve active targeting (Anticancer and Antimicrobial

drugs).

 Liposomes can encapsulate both micro and

macromolecules such as haemoglobin, erythropoeitin,

interferon etc.

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Disadvantages :
 Production cost is high

 Leakage and fusion of encapsulated drug / molecules.

 Sometimes phospholipid undergoes oxidation and hydrolysis

like reaction

 Short half-life

 Low solubility

 Fewer stables

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Characterisation of liposomes:
1. Physical Characterisation:

Characterization parameters Analytical method/Instrument

Transmission electron microscopy,
1. Vesicle shape and surface morphology

Freeze-fracture electron microscopy

Dynamic light scattering, zetasizer,
Mean vesicle size and size distribution

2. Photon correlation spectroscopy, laser
(submicron and micron range)

light scattering, gel permeation and gel exclusion

3. Surface charge Free-flow electrophoresis

4. Electrical surface potential and surface pH Zetapotential measurements & pH sensitive probes

Small angle X-ray scattering, 31P-NMR,
5. Lamellarity

Freeze-fracture electron microscopy

Freeze-fracture electron microscopy, Differential
6. Phase behavior

scanning colorimetery

Minicolumn centrifugation, ion-exchange
7. Percent of free drug/ percent capture

chromatography, radiolabelling

8. 38 Drug release www.DulDoMififxu.csoiomn cell/ dialysis

2. Chemical Characterisation:
Characterization parameters Analytical method/Instrument

1. Phospholipid concentration Barlett assay, stewart assay, HPLC

2. Cholesterol concentration Cholesterol oxidase assay and HPLC

3. Phopholipid peroxidation UV absorbance, Iodometric and GLC

Phospholipid hydrolysis,
4. HPLC and TLC

Cholesterol auto-oxidation.

5. Osmolarity Osmometer

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2. Biological Characterisation:
Characterization parameters Analytical method/Instrument

1. Sterility Aerobic or anaerobic cultures

2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test

Monitoring survival rates, histology and
3. Animal toxicity

pathology

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Therapeutic applications of
liposomes

Drug Route of administration Application Targeted Diseases

Amphotericin-B Oral delivery Ergosterol membrane Mycotic infection

Oral, Ocular, Pulmonary and Decreaase
Insulin Diabetic mellitus

Transdermal delivery glucose level
Cyclo-oxygenase enzyme

Ketoprofen Ocular delivery Pain muscle condition
inhibitor

Pentoxyfylline Pulmonary delivery Phosphodiesterase Asthma
Pseudomonas infection,

Tobramycin Pulmonary delivery Protein synthesis inhibitor
aeruginosa

Salbutamol Pulmonary delivery β2- adrenoceptor antagonist Asthma

Cytarabin Pulmonary delivery DNA-polymerase inhibition Acute-leukemias

Inhibition of nerve impulse ulcer on mucous surface
Benzocain Transdermal

from sensory nerves with pain

Ketoconazole Transdermal Inhibit ergosterol membrane Candida- albican’s

Levonogesterol Transdermal Rhamnose receptor Skin disorder
Urtecaria, allergic skin

Hydroxyzine Transdermal H1- receptor antagonist
disorder

Chemoreceptor, free nerve
Ibuprofen Oral delivery Rheumatoid arthritis

ending

4 Ocular delivery
Triamci1n olone Inhibition of prostaglandin Anti-inflammatory

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List of marketed products
Marketed product Drug used Target diseases Company

DoxilTM or CaelyxTM Doxorubicin Kaposi’s sarcoma SEQUUS, USA

DaunoXomeTM Daunorubicin Kaposi’s sarcoma, breast & NeXstar, USA
lung cancer

AmphotecTM Amphotericin-B fungal infections, SEQUUS, USA
Leishmaniasis

Fungizone® Amphotericin-B fungal infections, Bristol-squibb, Netherland
Leishmaniasis

VENTUSTM Prostaglandin-E1 Systemic inflammatory The liposome company, USA
diseases

ALECTM Dry protein free powder of Expanding lung diseases in Britannia Pharm, UK
DPPC-PG babies

Topex-Br Terbutaline sulphate Asthma Ozone, USA

Depocyt Cytarabine Cancer therapy Skye Pharm, USA

Novasome® Smallpox vaccine Smallpox Novavax, USA

Avian retrovirus vaccine Killed avian retrovirus Chicken pox Vineland lab, USA

Doxil® Doxorubicin Hcl Refractory ovarian cancer ALZA, USA

EvacetTM Doxorubicin Metastatic breast cancer The liposome company, USA
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VincaXome Vincristine www.DuloMSoixl.icdo Tmumours NeXstar, USA