Liposomes

NANO52
Foothill College
 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 0.0025~3.5um. The
membrane of liposome is made of phospholipids,
which have phosphoric acid sides to form the
liposome bilayers.
 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
Liposome Structure

PHOSPHOLIPID
BILAYER

AQUEOUS
CAVITY
 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
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
 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).
 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.
 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
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.
Types of Liposomes
 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.
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.
Types of Preparation
REV SUVs/OLV
made by
reverse-phase
evaporation MLV-REV
method MLVs made by
DRV Dehydrated reverse phase
– rehydration evaporation
method method

Based on
Method of
Preparation
SPLV
VET
Stable
Vesicles prepared
plurilamellar
by extrusion
vesicles
technique

FATMLV
Frozen and
Thawed MLVs
Composition - Applications
General Structure of various
types of liposomes
General Method of Liposome Preparation
 Method of Preparation

Passive loading techniques Active loading techniques

 Mechanical dispersion methods

 Solvent dispersion methods
 Detergent removal methods
 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
 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.
Hand Shaking Method
Non Shaking Method &
Pro - Liposomes

Buchi Rotatory Evaporator

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

1. Encapsulation

2. Partitioning

3. Reverse
loading

http://en.wikipedia.org/wiki/Liposome
 1. Physical Characterization:
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. Drug release Diffusion cell/ dialysis

 2. Chemical Characterization

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
 3. Biological Characterization
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
 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
Ocular delivery
Triamcinolone Inhibition of prostaglandin Anti-inflammatory
Transdermal
 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,
diseases USA
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

The liposome company,
EvacetTM Doxorubicin Metastatic breast cancer
USA
VincaXome Vincristine Solid Tumours NeXstar, USA
 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.
 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/lipo1.jpg (accessed on 15-04-2011)
 3.      www.nanobiotec.iqm.unicamp.br/download/liposomas-3.ppt accessed on 15-04-2011)
 4.      http://www.uni-magdeburg.de/imos/mea_sen/img/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/bFgW9FjRw248U2PRC2In_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.