Liposomes

1. LIPOSOMES

2. 22
Liposomes
Definition:
Liposomes are simple microscopic
vesicles in which an aqueous volume
is entirely enclosed by a membrane
composed of lipid molecule.
Structurally, liposomes are concentric
bilayered vesicles in which an
aqueous volume is entirely enclosed
by a membranous lipid bilayers
mainly composed of natural or
synthetic phospholipids.

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Advantages
Provide selective passive targeting to tumour tissues
(liposomal doxorubicin).
Increased efficacy and therapeutic index.
Increased stability via encapsulation.
Reduction in toxicity of the encapsulated agent.
Improved pharmacokinetic effects (reduced
elimination, increased circulation life times).
Flexibility to couple with site-specific ligand to
achieve active targeting.

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STRUCTURE AND COMPOSITION OF LIPOSOMES

There are number of components of There are number of components of
liposomes however liposomes however phospholipids phospholipids and and
cholesterolcholesterol being main components. being main components.

Phospholipids are the major structural Phospholipids are the major structural
components of biological membranes, components of biological membranes,
where two types of phospholipids exist – where two types of phospholipids exist –
phosphodiglycerides and sphingolipids, phosphodiglycerides and sphingolipids,
together with their corresponding together with their corresponding
hydrolysis products. hydrolysis products.

The most common phospholipid is The most common phospholipid is
phosphatidylcholine (PC) molecule. phosphatidylcholine (PC) molecule.

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
Commonly used synthetic Phospholipids:Commonly used synthetic Phospholipids:

DOPC = Dioleoyl PhosphatidylcholineDOPC = Dioleoyl Phosphatidylcholine

DOPE = Dioleoyl phosphatidylethanolamineDOPE = Dioleoyl phosphatidylethanolamine

DSPC = Distearoyl phosphatidylcholineDSPC = Distearoyl phosphatidylcholine

DSPE = Distearoyl phosphatidylethanolamineDSPE = Distearoyl phosphatidylethanolamine

DLPC = Dilauryl phosphatidylcholineDLPC = Dilauryl phosphatidylcholine

DMPC = Dimyristoyl phosphatidylcholineDMPC = Dimyristoyl phosphatidylcholine

DLPE = Dilauryl phosphatidylethanolamineDLPE = Dilauryl phosphatidylethanolamine

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Cholesterols:
Incorporation of sterols in liposome
bilayer can bring about major changes in
the preparation of these membranes.
 Cholesterol does not by itself form
bilayer structure, but can be incorporated
into phospholipid membranes in very
high concentration unto 1:1 or even 2:1
molar ratios of PC.
Cholesterol incorporation increases the
separation between the cholin head
groups and eliminates the normal
electrostatic and hydrogen-bonding
interactions.

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Classification Based on structural parameters
Multivesicular vesicles->1 μm MV
Giant unilamellar vesicles->1 μm GUV
Large unilamellar vesicles->100nm LUV
Medium sized unilamellar vesicles MUV
Small unilamellar vesicles-20-100nm SUV
Unilamellar vesicles (all in size) UV
Oligolamellar vesicles- 0.1-1 μm OLV
Multilamellar large vesicles- >0.5 μm MLV
SPECIFICATIONS TYPE

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Classification Based on the method of liposome
preparation
TYPE SPECIFICATIONS
REV Single or oligolamellar vesicles made by
reverse-phase evaporation method
MLV-REV Multilamellar vesicles made by reverse-
phase evaporation method
SPLV Stable plurilamellar vesicles
FATMLV Frozen and thawed MLV
VET Vesicles prepared by extrusion technique
DRV Dehydration-rehydration method

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Chemical Characterization
Characterization parameters Analytical methods
Phospholipid concentration Lipid phosphorous content using
Barlett assay,HPLC
Cholesterol concentration Cholesterol oxidase assay and
HPLC
Drug concentration Appropriate method given in
monograph
Phospholipid peroxidation UV absorbance,
TBA,indometric and GLC
Phospholipid hydrolysis HPLC and TLC
Cholesterol auto-oxidation HPLC and TLC
Anti-oxidant degradation HPLC and TLC
pH pH meter
osmolarity Osmometer

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Physical Characterization
Characterization parameters Analytical methods
Vesicle shape and surface
morphology
Transmission electron
microscopy, freeze-fracture
electron microscopy.
Surface charge Free-flow electrophoresis.
Lamellarity Small angle X-ray scattering,
freeze-fracture electron
microscopy,
31
P-NMR.
Phase behavior Freeze-fracture electron
microscopy, differential
scanning calorimetry
Percent capture/percent of free
drug
Minicolumn centrifugation, gel
exclusion, ion-exchange
chromatography, radiolabelling

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Biological Characterization
Characterization
parameters
Analytical methods
sterility Aerobic or anaerobic
cultures
pyrogenicity Limulus amebocyte
lysate (LAL) test
Animal toxicity Monitoring survival
rates, histology and
pathology

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TECHNIQUES OF LIPOSOMES PREPARATION
(A) (A) physical dispersion
a) Hand-shaken multilamellar vesicles (MLVs)
b) Non-shaking vesicles
c) Pro-liposomes
d) Freeze drying
(B) Processing of lipids hydrated by physical means
a) Micro emulsification liposomes (MEL)
b) Sonicated unilamellar vesicles (SUVs)
c) French pressure cell liposomes
d) Membrane extrusion liposomes
e) Dried-reconstituted vesicles (DRVs)
f) Freeze thaw sonication (FTS)
g) pH induced vesiculation
h) Calcium induced fusion

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(C )(C ) Solvent dispersion methods
Ethanol Injection
Ether injection
Water in organic phase
Reverse phase evaporation vesicles
Stable plurilamellar vesicles (SPVs)
Double emulsion vesicles
(D) DETERGENT SOLUBILIZATION

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All the methods of preparing liposomes involve three or four basic
stages:
•Drying down lipids from organic solvent,
•Dispersion of lipids in aqueous media,
•Purification of resultant liposomes, and
•Analysis of final product.

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Physical dispersion:
There are four basic method of physical dispersion. I.e. hand shaking,
non-shaking, freeze dry and proliposomes.

Hand-shaken multilamellar vesicles (MLVs) :

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Sonicated Unilamellar Vesicles (SUVs) :

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French pressure cell liposomes : :
Type of liposomes : ULV or OLV
Pressure :20000 or 40000
Sample volume : maximum 40ml
Minimum 4ml
Out flow : 0.5-1 ml / min.

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Membrane extrusion liposomes :
Type of liposomes : MLV or
LUV
Pressure : 100 psi
Type membranes : Tortuous path,
Nucleation track (polycarbonate )

19. 1919
Solvent dispersion methods:
In this methods, lipids are first dissolved in an organic
solution, which is then brought into contact with the aqueous
phase containing material to be entrapped within the
liposomes.
Methods employing solvent dispersion fall into one of three
categories.
The organic solvent is miscible with the aqueous phase.
The organic solvent is miscible with the aqueous phase, the
latter being in a large excess.
Organic solvent is in large excess, and is again immiscible

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Ether injection &ethanol injection

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DETERGENT SOLUBILIZATION :
In this method, the phospholipids are brought into intimate contact
with the aqueous phase via the intermediary of detergents, which
associate with phospholipid molecules and serve to screen the
hydrophobic portions of the molecule from water.
The structures formed as a result of this association are known as
micelles, and can be composed of several hundred component
molecules.
Their shape and size depends on chemical nature of the detergent,
the concentration and other lipids involved.
As a general rule, membrane-solubilizing detergents have a higher
affinity for phospholipid membranes than for the pure detergent
micelles.

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ACTIVE LOADING TECHNIQUE

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Active loading method have following advantages
over passive encapsulation techniques: :
A high encapsulating efficiency and capacity.
A reduced leakage of the encapsulated compounds.
“Bed side” loading of drug thus limiting loss of retention of
drug by diffusion or chemical degradation during storage.
Flexibility for the use of constitutive lipids, as drug is
loaded after the formation of carrier units.
Avoidance of biological compound during preparation
steps in the dispersion thus reducing safety hazards.
The transmembrane pH gradient can be developed using
various method depending upon the nature of the drug be
encapsulated.
For amphipathic weak bases by active loading procedures
such as using a proton gradient or an ammonium sulphate
gradient or calcium acetate gradient.

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STABILITY OF LIPOSOMES
The stability studies could be broadly covered under
two main sections.
1.The stability in vitro, which covers the stability
aspects prior to the administration of the formulation
and with regard to the stability of the constitutive
lipids.
2.The stability in vivo, which covers the stability
aspects once the formulation, is administered via
various routes to the biological fluids. These include
stability aspects in blood (serum) if administered by
systemic route or in gastrointestinal tract, if
administered by oral or preoral routes.

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Stability in vitro
Lipid oxidation an peroxidation
Lipid hydrolysis
Long term and accelerated stability
Stability after systemic administration
Stability in vivo after oral administration

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APPLICATION OF LIPOSOMES
Liposomes as drug/protein delivery vehicles
Controlled and sustained drug release in situ.
Enhanced drug solubilization
Altered pharmacokinetics and biodistribution
Enzyme replacement therapy and lysosomal storage
disorders
Liposomes in antimicrobial, antifungal and antiviral therapy
Liposomal drugs
Liposomal biological response modifiers
Liposomes in tumour therapy
Carrier of small cytotoxic molecules
Vehicle for macomolecules as cytokines o genes

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Liposomes in gene delivery
Gene and antisense therapy
Genetic vaccination
Liposomes in immunology
Immonoadjuvant
Immunomodulator
Immunodiagnosis
Liposomes as artificial blood surrogates
Liposomes as radiophamaceutical and radiodiagnostic cariers
Liposomes in cosmetics an dermatology
Liposomes in enzyme immobilization and bioreactor
technology

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Some liposomal formulation of Amphotericin B
System Target diseaseBrand nameProduct
Liposomes
(i.v)
Systemic fungal
infection,
Visceral
leishmaniasis
AmBisomeNeXstar, USA
Liposomes
(i.v)
Systemic fungal
infection
AmphocilSEQUUS, USA
Liposomes
(i.v)
Systemic fungal
infection
ABELECT The Liposome
company,
USA

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Liposomes in gene therapy:
Type of
vector
Advantages Disadvantages
Viral
vector
·Relative high transfection
efficiency
·Immunogenicity, presence of
contaminants and safety
·Vector restricted size limitation
for recombinant gene
·Unfavourable p’ceutical issue-
large scale production, GMP,
stability and cost
Non-viral·Favourable p’ceutical issue-
large scale production, GMP,
stability and cost
·Plasmid independent structure
·Low immunogenicity
·Opportunity for
chemical/physical
manipulation
·Relative low transfection
efficiency

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Various liposomal product in dermatology and cosmetics
(launched or investigated)
Vesicular
system
Marketed by Liposomes and
ingredients
Capture
TM
Christian DiorLiposomes in gel with
ingredients
Plentitude
TM
L’Oreal Tanning agent in
liposomes
Dermosome
TM
Microfluidics Skin care, loaded
liposomes
Penta
TM
Pentapharm Humectant pentavitin R in
liposomes
Coatsome NC
TM
Nichiya liposomes
Co
Liposomes with humectant

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Imaging modality and required concentration of diagnostic
moieties:
Imaging modalityDiagnostic moiety Concentrat
ion
g-scintigraphy Diagnostic radio-nucleus
such as
111
In,
99m
Tc,
67
Ga
10
-10
M
Magnetic
resonance(MR)
Po-magnetic ions such as
Gd, Mn and iron oxide
10
-4
M
Computed
tomography(CT)
Iodine, Bromine an Barium10
-2
M
Ultrasound imaging
or
Ultrasonography(
US)
Gas (Air, Argon and
Nitrogen)

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COMMERCIAL MANUFACTUING OF LIPSOMAL DRUG
NOProblems Remedies
1Poor quality of the
raw material
mainly the
phospholipids.
High quality products with improved
purification protocol and validated
analytical techniques are available
2Pay load is too
low
Use either lipophilic drug/lipophilic
prodrug of hydrophilic drug or using
active techniques.
3Poor
characterization of
the
physicochemical
properties of the
liposomes
Quality control assay can be performed
using sophisticated instruments and batch
to batch variability can be checked.

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NOProblems Remedies
4 Shelf life is too
short
Improved by appropriate cryoprotectant
and lyoprotectant and product can be
successfully freeze dried.
5 Scale up related
problems
Scaling up can be improved by
carefully selecting method of
preparation, sterilization by autoclaving
or membrane filtration coupled with
aseptic processing and pyrogen removal
using properly validated LAL test
6 Absence of any
data on safety of
these carrier
systems on
chronic use.
By choosing candidate potent drugs
with narrow therapeutic window the
drug elated safety problems can be
alleviated.

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