LIPOSOMES

Presensted by : Kiran Kumar

M.Pharm(pceutics)

AKRG College of

Definition :
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.

Introduction :
Liposomewas 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. Liposomeis a lipid vesicle suspending in the hydro-phase with a diameter around 0.0025~3.5um. The membrane of liposome is made of phospholipids, which have phosphoric acid sides to form the liposome bilayers.

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 Dioleoyl phosphatidylcholine Disteroyl phosphatidylcholine Dioleoyl phosphatidylethanolamine Distearoyl phosphatidylethanolamine

Synthetic phospholipids used in the liposomes are:

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. 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.

Overall Composition:

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). Flexibility to couple with site-specific ligands to achieve active targeting (Anticancer and Antimicrobial drugs). Liposomes can be formulated into multiple dosage forms. Liposomes can encapsulate both micro and macromolecules such as haemoglobin, erythropoeitin, interferon g etc.

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

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

Classification :
Based on Structural Parameters:
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.
a.

c. Unilamellar vesicle (ULV): single layer of lipids. Based on the size of the single layer they are further divide into the following types with in ULV 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.

General Structure of various types of liposomes:

Types :
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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.

1. Physical Dispersion:
There are four basic methods of physical dispersion: Hand shaken multilamellar vesicles. Non shaking vesicles. Pro liposomes. Freeze drying.

a) b) c) d)

Hand Shaking Method:

Non Shaking Method & Pro - Liposomes:

Buchi Rotatory Evaporator

Buchi Rotatory Evaporator

Freeze Thaw Method:

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Processing of the lipids hydrated by physical means or the mechanical treatments of MLVs :
1)

Micro Emulsification liposomes(MEL) Sonicated unilamellar vesicles (SUVs) French Pressure Cell Liposomes. Membrane extrusion Liposomes Dried reconstituted vesicles(DRVs) Freeze thaw sonification(FTS) pH induced vesiculation Calcium Induced fusion

2)

3)

4)

5)

6)

7)

8)

Micro Emulsification Liposomes (MEL):

Sonicated Unilamellar Vesicles:

Membrane Extrusion Liposomes:

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

Ethanol / Ether Injection Method

Reverse Phase Evaporation Vesicles:

Reverse Phase Evaporation Vesicles:

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Mechanism of incorporation of drug in liposomes:

1.

Encapsulati on Partitioning Reverse loading

2.

3.

Characterisation of liposomes: Analytical method/Instrument Characterization 1. Physical parameters Characterisation:

1. Vesicle shape and surface morphology Transmission electron microscopy, Freeze-fracture electron microscopy Dynamic light scattering, zetasizer, Photon correlation spectroscopy, laser light scattering, gel permeation and gel exclusion Free-flow electrophoresis Zetapotential measurements & pH sensitive probes Small angle X-ray scattering, 31P-NMR, Freeze-fracture electron microscopy Freeze-fracture electron microscopy, Differential scanning colorimetery Minicolumn centrifugation, ion-exchange chromatography, radiolabelling Diffusion cell/ dialysis 2. 3. 4. 5. 6. 7. 8. Mean vesicle size and size distribution (submicron and micron range) Surface charge Electrical surface potential and surface pH Lamellarity Phase behavior Percent of free drug/ percent capture Drug release

2. Chemical Characterisation:
Characterization parameters
1.

Analytical method/Instrument

Phospholipid concentration

Barlett assay, stewart assay, HPLC

2. 3. 4. 5.

Cholesterol concentration Phopholipid peroxidation Phospholipid hydrolysis, Cholesterol auto-oxidation. Osmolarity

Cholesterol oxidase assay and HPLC UV absorbance, Iodometric and GLC HPLC and TLC Osmometer

2. Biological Characterisation:
Characterization parameters
1.

Analytical method/Instrument

Sterility

Aerobic or anaerobic cultures

2.

Pyrogenicity Animal toxicity

Limulus Amebocyte Lysate (LAL) test Monitoring survival rates, histology and pathology

3.

Therapeutic applications of liposomes

Drug
Amphotericin-B Insulin Ketoprofen Pentoxyfylline Tobramycin Salbutamol Cytarabin Benzocain Ketoconazole Levonogesterol Hydroxyzine Ibuprofen

Route of administration

Application

Targeted Diseases
Mycotic infection Diabetic mellitus Pain muscle condition Asthma Pseudomonas infection, aeruginosa Asthma Acute-leukemias ulcer on mucous surface with pain Candida- albicans

Oral delivery Ergosterol membrane Oral, Ocular, Pulmonary Decreaase and Transdermal glucose level delivery Cyclo-oxygenase Ocular delivery enzyme inhibitor Pulmonary delivery Phosphodiesterase Protein synthesis Pulmonary delivery inhibitor 2- adrenoceptor Pulmonary delivery antagonist DNA-polymerase Pulmonary delivery inhibition Inhibition of nerve Transdermal impulse from sensory nerves Inhibit ergosterol Transdermal membrane Transdermal Rhamnose receptor Transdermal Oral delivery

Skin disorder Urtecaria, allergic skin H1- receptor antagonist disorder Chemoreceptor, free Rheumatoid arthritis nerve ending

List of marketed products

Marketed product DoxilTM or CaelyxTM DaunoXomeTM AmphotecTM Fungizone VENTUSTM ALECTM Topex-Br Depocyt Novasome Avian retrovirus vaccine Doxil EvacetTM VincaXome Drug used Doxorubicin Daunorubicin Amphotericin-B Amphotericin-B Prostaglandin-E1 Dry protein free powder of DPPC-PG Terbutaline sulphate Cytarabine Smallpox vaccine Killed avian retrovirus Doxorubicin Hcl Doxorubicin Vincristine Target diseases Kaposis sarcoma Kaposis sarcoma, breast & lung cancer fungal infections, Leishmaniasis fungal infections, Leishmaniasis Systemic inflammatory diseases Expanding lung diseases in babies Asthma Cancer therapy Smallpox Chicken pox Refractory ovarian cancer Metastatic breast cancer Solid Tumours Company SEQUUS, USA NeXstar, USA SEQUUS, USA Bristol-squibb, Netherland The liposome company, USA Britannia Pharm, UK Ozone, USA Skye Pharm, USA Novavax, USA Vineland lab, USA ALZA, USA The liposome company, USA NeXstar, USA

References :
Target and Controlled Drug delivery Novel Carrier Systems by S.P.Vyas and R.K.Khar.

Controlled and Novel Drug Delivery Systems by Sanjay K. Jain and N.K.Jain. 2. http://noopurmandrek.files.wordpress.com/ (accessed on 15-04-2011)

3. www.nanobiotec.iqm.unicamp.br/download

Thank You