Liposomes

1
Liposomes

• NANO52
• Foothill College

2
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.
• Liposome is a lipid vesicle suspending in the
  hydro-phase with a diameter around 0.00253.5um.
  The membrane of liposome is made of
  phospholipids, which have phosphoric acid sides
  to form the liposome bilayers.

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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.

• Presentation by
• Shilpi Bhatnagar
• M.Pharm (Q.A.) 1st semester

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Liposome Structure
PHOSPHOLIPID BILAYER
AQUEOUS CAVITY
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Composition of liposomesA. 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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Phospholipid Molecule

• Phosphatidylcholine is an amphipathic molecule in
  which exists
• A hydrophilic polar head group, phosphocholine.
• A glycerol bridge
• A pair of hydrophobic acyl hydrocarbon chains

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Liposome Composition

• A liposome is an artificially-prepared vesicle
  composed of a lipid bilayer. The liposome can be
  used as a vehicle for administration of nutrients
  and pharmaceutical drugs.2 Liposomes can be
  prepared by disrupting biological membranes (such
  as by sonication).
• Liposomes are composed of natural phospholipids,
  and may also contain mixed lipid chains with
  surfactant properties (e.g., egg
  phosphatidylethanolamine). A liposome design may
  employ surface ligands for attaching to unhealthy
  tissue.3
• The major types of liposomes are the
  multilamellar vesicle (MLV), the small
  unilamellar vesicle (SUV), and the large
  unilamellar vesicle (LUV).

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Advantages of Liposomes

• Biocompatible, completely biodegradable,
  non-toxic, flexible, nonimmunogenic.
• Liposomes supply both a lipophilic environment
  and aqueous milieu interne in one system. Can
  protect encapsulated drug.
• 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 encapsulate micro and
  macromolecules such as haemoglobin,
  erythropoeitin, interferon g etc.
• Can be formulated into multiple dosage forms.

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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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Types of Liposomes

• Liposomes are classified on the basis of
• Structural parameters
• Methods of preparation
• Composition and applications
• 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.

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Types of Liposomes
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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 gt 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.
• 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 gt1um.

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Types of Preparation
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Composition - Applications
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General Structure of various types of liposomes
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General Method of Liposome Preparation
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Method of Preparation
Passive loading techniques
Active loading techniques

• Mechanical dispersion methods
• Solvent dispersion methods
• Detergent removal methods

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Method of Preparation of Liposomes
Passive Loading Techniques

• Mechanical dispersion methods
• Lipid film hydration by hand shaking, non-hand
  shaking or freeze drying
• Microemulsification
• Sonication
• French pressure cell
• Membrane extrusion
• Dried reconstituted vesicles
• Freeze-thawed liposomes
• Solvent dispersion methods
• Ethanol injection
• Ether injection
• Double emulsion vesicles
• Reverse phase evaporation vesicles
• Stable plurilamellar vesicles
• Detergent removal methods
• Dialysis
• Column chromatography
• Dilution
• Reconstituted Sendai Virus enveloped vesicles

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Physical Dispersion

• There are four basic methods of physical
  dispersion
• Hand shaken multilamellar vesicles.
• Non shaking vesicles.
• Pro liposomes.
• Freeze drying.

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Hand Shaking Method
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Non Shaking Method Pro - Liposomes
Buchi Rotatory Evaporator
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Mechanical Treatments of MLVs

• 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

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Micro Emulsification Liposomes (MEL)
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Sonicated Unilamellar Vesicles
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VESICLES PREPARED BY EXTRUSION TECHNIQUES (VETs)
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Dried Reconstituted Vesicles (DRV) and Freeze
Thaw Sonication (FTS)
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Ethanol / Ether Injection Method
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Reverse Phase Evaporation Vesicles
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Mechanism of Incorporationof Drug in liposomes

1. Encapsulation
2. Partitioning
3. Reverse loading

http//en.wikipedia.org/wiki/Liposome
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1. Physical Characterization
Characterization parameters Characterization parameters Analytical method/Instrument
1. Vesicle shape and surface morphology Transmission electron microscopy, Freeze-fracture electron microscopy
2. Mean vesicle size and size distribution (submicron and micron range) Dynamic light scattering, zetasizer, Photon correlation spectroscopy, laser 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
5. Lamellarity Small angle X-ray scattering, 31P-NMR, Freeze-fracture electron microscopy
6. Phase behavior Freeze-fracture electron microscopy, Differential scanning colorimetery
7. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange chromatography,  radiolabelling
8. Drug release Diffusion cell/ dialysis
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2. Chemical Characterization
Characterization parameters 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
4. Phospholipid hydrolysis, Cholesterol auto-oxidation. HPLC and TLC
5. Osmolarity Osmometer
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3. Biological Characterization
Characterization parameters Characterization parameters Analytical method/Instrument
1. Sterility Aerobic or anaerobic cultures
2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test
3. Animal toxicity Monitoring survival rates, histology and pathology
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Therapeutic applications of liposomes
Drug Route of administration Application Targeted Diseases
Amphotericin-B Oral delivery Ergosterol membrane Mycotic infection
 Insulin Oral, Ocular, Pulmonary and Transdermal delivery Decreaase glucose level Diabetic mellitus
 Ketoprofen Ocular delivery Cyclo-oxygenase enzyme inhibitor Pain muscle condition
Pentoxyfylline Pulmonary delivery Phosphodiesterase Asthma
Tobramycin Pulmonary delivery Protein synthesis inhibitor Pseudomonas infection, aeruginosa
Salbutamol Pulmonary delivery ß2- adrenoceptor antagonist Asthma
Cytarabin Pulmonary delivery DNA-polymerase inhibition Acute-leukemias
Benzocain Transdermal Inhibition of nerve impulse from sensory nerves ulcer on mucous surface with pain
Ketoconazole Transdermal Inhibit ergosterol membrane Candida- albicans
Levonogesterol Transdermal Rhamnose receptor Skin disorder
Hydroxyzine Transdermal H1- receptor antagonist Urtecaria, allergic skin disorder
Ibuprofen Oral delivery Chemoreceptor, free nerve ending Rheumatoid arthritis
Triamcinolone Ocular delivery Transdermal Inhibition of prostaglandin Anti-inflammatory
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List of marketed products
Marketed product Drug used Target diseases Company
DoxilTM or CaelyxTM Doxorubicin Kaposis sarcoma SEQUUS, USA
DaunoXomeTM Daunorubicin Kaposis sarcoma, breast lung cancer NeXstar, USA
AmphotecTM Amphotericin-B  fungal infections, Leishmaniasis SEQUUS, USA
Fungizone Amphotericin-B  fungal infections, Leishmaniasis Bristol-squibb, Netherland
VENTUSTM Prostaglandin-E1 Systemic inflammatory diseases The liposome company, USA
ALECTM Dry protein free powder of DPPC-PG Expanding lung diseases in babies Britannia Pharm, UK
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
VincaXome Vincristine Solid Tumours NeXstar, USA
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Summary

• Liposomes over the years have been investigated
  as the major drug delivery systems due to their
  flexibility to be tailored for varied desirable
  purposes.
• The flexibility in their behaviour can be
  exploited for the drug delivery through any route
  of administration and for any drug or material
  irrespective of its physicochemical properties.
• The uses of liposomes in the delivery of drugs
  and genes to tumour sites are promising and may
  serve as a handle for focus of future research.

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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/2010/09/l
  ipo1.jpg (accessed on 15-04-2011)
• 3.      www.nanobiotec.iqm.unicamp.br/download/lip
  osomas-3.ppt accessed on 15-04-2011)
• 4.      http//www.uni-magdeburg.de/imos/mea_sen/i
  mg/pictures/Lipo.jpg (accessed on 15-04-2011)
• 5.      http//www.nanolifenutra.com/images/image_
  liposome_01.jpg (accessed on 15-04-2011)
• 6.      http//www.azonano.com/work/bFgW9FjRw248U2
  PRC2In_files/image002.gif (accessed on
  15-04-2011)
• 7.      http//upload.wikimedia.org/wikipedia/en/2
  /28/Liposome.jpg (accessed on 15-04-2011)
• Biopharmaceutics and pharmacokinetics a treatise
  by D.M.Bramhankar and sunil B. jaiswal (first
  edition reprint 2005) pg.no- 360-361.
• 9.    Liposomes preparation methods a review by
  Mohammad riaz in Pakistan journal of
  pharmaceutical sciences vol.19(1), January 1996,
  pp.65-77.