Liposomes are concentric bilayered vesicles in which an aqueous core is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. Liposomes are spherical microscopic vesicles consisting phospholipids bilayers which enclose aqueous compartments. The size of a liposome ranges from some 20 nm up to several micrometers. Liposomes were first produced in England in 1961 by Alec D. Bangham, who was studying phospholipids and blood clotting. Small unilamellar vesicles (SUV), 25 to 100 nm in size that consist of a single bilayer Large unilamellar vesicle (LUV), 100 to 500 nm in size that consist of a single bilayer Multilamellar vesicle (MLV), 200 nm to several microns, that consist of two or more concentric bilayer

1. LIPOSOME
1
Department of Pharmacy (Pharmaceutics) | Sagar savale
Mr. Sagar Kishor Savale
[Department of Pharmacy (Pharmaceutics)]
2015-016
avengersagar16@gmail.com
12/13/2015

2. Contents
1. Liposomes
2. Structural components of liposome's
3. Formation of liposomes
4. Theory of Liposomes
5. Advantages of Liposomes
6. Disadvantages
7. Importance of Liposomes in drug Delivery System
8. Mechanism Of Liposome Formation And Subsequent Processing To Generate Types Of
Liposomes
9. Classification of liposomes
10. Conventional liposome preparation methods
11. Methods of Liposome Preparation
12. Stability of Liposomes
13. Liposomes in drug delivery
14. Characterization of liposomes
15. Application Of Liposomes
16. References
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3. 3
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9. 1. Liposome
 Liposomes are concentric bilayered vesicles in which an aqueous core is entirely enclosed
by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids.
 Liposomes are spherical microscopic vesicles consisting phospholipids bilayers which
enclose aqueous compartments.
 The size of a liposome ranges from some 20 nm up to several micrometers.
 Liposomes were first produced in England in 1961 by Alec D. Bangham, who was studying
phospholipids and blood clotting.
 Small unilamellar vesicles (SUV), 25 to 100 nm in size that consist of a single bilayer
 Large unilamellar vesicle (LUV), 100 to 500 nm in size that consist of a single bilayer
 Multilamellar vesicle (MLV), 200 nm to several microns, that consist of two or more
concentric bilayer
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11. Liposome
 The lipid molecules are usually phospholipids- amphipathic moieties with a hydrophilic
head group and two hydrophobic tails.
 On addition of excess water, such lipidic moieties spontaneously originate to give the
most thermodynamically stable conformation.
 In which polar head groups face outwards into the aqueous medium, and the lipidic
chains turns inwards to avoid the water phase, giving rise to double layer or bilayer
lamellar structures.
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14. 2. Structural components of liposome's
 Ther are two main components of Liposomes system they are Phospholipid and
cholesterol.
2.1 Phospholipids
 Phosphatidylcholine.
 Amphipathic molecule
 Hydrophobic tail- 2 fatty acid chain containing 10-24 carbon atoms and 0-6
double bond in each chain
 Hydrophilic polar head- Phosphoric acid bound to water soluble molecule
 Self organize in ordered supramolecular structure when confronted (meet face to
face) with solvent
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15
Polar Head Groups Three carbon glycerol

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18

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2.2 Flow chart of Lecithin extraction process

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2.3 Phase Transition Temperature Tm

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2.4 Membrane permeability

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26 2.5 Some other conventional used phospholipids
•Naturally occurring phospholipids
- PC: phosphatidylcholine
- PE: Phosphatidylethanolamine
- PS: Phosphatidylserine
• Synthetic phospholipids
- DOPC: Dioleoylphosphatidylcholine
- DSPC: Ditsearoylphosphatidylcholine
- DOPE: Dioleoylphosphatidylethanolamine
- DSPE: Ditsearoylphosphatidylethanolamine

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2.6 Cholesterol
 Cholesterol by itself does not form bilayer structure.
 Cholesterol act as fluidity buffer
 After intercalation with phospholipid molecules alter the freedom of motion of carbon
molecules in the acyl chain
 Restricts the transformations of trans to gauche conformations
 Cholesterol incorporation increases the separation between choline head group &
eliminates normal electrostatic & hydrogen bonding interactions.
 its rigid steroid ring system which interferes with motion of fatty acid tails, stabilizes the
lipid bilayer and decrease the leakage of encapsulated drug

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2.7 Non Structural Components

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30 3. Formation of liposomes
Surfactants self assemble in water to make micelles
and a variety of lipotropic liquid crystalline phases.
Liposomes are generally formed from 2 phase
mixtures of a lamellar phase with water. Depending
on temperature, the lamellar phase can either be in
the molten state (La phase) or solid, “gel” state
(Lb phase). Transition temperature = Tc.
Liposomes are formed from aqueous dispersions
the “molten” La phase.
Surfactant molecular shape/interactions mainly
determines aggregate geometry.
Critical packing factor = v/aolc (unit less), where:
v = molecular volume of surfactant chain
ao = area per surfactant head
lc = length of surfactant chain

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32. 12/13/2015
32  For stable liposomes, we need
surfactant molecules with long
chains (for strong aggregation) plus
tendency to form flat sheets, i.e.
packing parameter = 0.5-1
 Biological membrane surfactants
consist of phospholipids made up of
glycerol esterified to 2 fatty acid
chains plus a phosphate derivative
polar head. Structure is
 RCH2-R’CH2-CH2-OPO3-X
 where R and R’ = alkyl-CO2- and the
head groups may contain a variety of
different terminal groups X.
 For example, in phosphatidylcholine,
X = -CH2CH2-N(CH3)3
+

33. 4. Theory of Liposomes
1. The budding theory
2. The bilayer phospholipids theory
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34. 4.1 budding theory
 Stress induced hydration of phospholipids
 Organization in to lamellar arrays
 Results in to budding of lipid bilayer leading to down sizing
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SUV OLV

35. 4.2 bilayer phospholipids theory
 Liposomes are formed when thin lipid films are hydrated
 The hydrated lipid sheets detach during agitation and self-close to form large, Multilamellar
vesicles (LMV)
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36. 5. Advantages of Liposomes
 Provides selective passive targeting to tumor tissues
 Increased efficacy & therapeutic index
 Increased stability via encapsulation
 Reduction in toxicity of the encapsulated agent
 Site avoidance effect
 Improved pharmacokinetic effects
 Flexibility to couple with site specific ligands to achieve active targeting
 Variety of Drugs Given In Low Dose As Encapsulated For Stability
 Minimum Effective Concentration And Therapeutic Index
 Low Toxicity Due To Reduced Exposure To Sensitive Tissues
 Minimum ADR/No Side Effects
 Possible Formulation- suspension, emulsion, gel, Cream, lotion, Aerosol, reconstituted Vesicles
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37. 6. Disadvantages
 Physical/ Chemical Stability
 Very High Production Cost
 Drug Leakage/ Entrapment/ Drug Fusion
 Sterilization
 Short Biological Activity / T ½
 Oxidation of Bilayer …Lipids And Low Solubility
 Overcoming Resistance
 Extensive Clinical And Laboratory Research To A Certain Long Circulating Liposomes
 Repeated Iv Administration Problems
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38. 7. Importance of Liposomes in drug Delivery
System
 Pharm kinetics - efficacy and toxicity
A. Changes the absorbance and bio distribution
B. Deliver drug in desired form
C. Multidrug resistance
 Protection
A. Decrease harmful side effects
B. Change where drug accumulates in the body
C. Protects drug
 Release
A. -Affect the time in which the drug is released
B. -Prolong time -increase duration of action and decrease administration
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39. 8. Mechanism Of Liposome Formation And Subsequent
Processing To Generate Types Of Liposomes
 Phospholipids are amphipathic molecules having hydrophobic tail & a hydrophilic or
polar head
 The hydrophilic & hydrophobic domains within the molecular geometry of amphiphilic
lipids orient & self organize in ordered supramolecular structure when confronted with
solvents
 Cholesterol have modulatory effect on the bilayer membrane (acts as fluidity buffer)
 Below phase transition it tends to make the membrane less ordered while above the
transition it tends to make the membrane more ordered.
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41
8.1 Mechanism Of Liposome

42. 9. Classification of liposomes
 Based on structural parameters
1. MLV- Multilamellar large vesicles>0.5um
2. OLV- oligolamellar vesicles,0.1-1um
3. UV-unilamellar vesicles( all size range)
4. SUV-small unilamellar vesicles(-20-100nm)
5. MUV-medium sized unilamellar vesicles
6. LUV-large unilamellar vesicles>100nm
7. GUV-giant unilamellar vesicles>1um
8. MV-multivesicular vesicles>1um
 Based on method of liposome preparation
1. REV-OLV made by reverse phase evaporation method
2. MLV-REV-MLV made by reverse phase evaporation method
3. SPLV-stable plurilamellar vesicle
4. VET-vesicles prepared by extrusion technique
5. DRV-dehydration-rehydration method
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 Based on composition & applications
1. Conventional liposomes-neutral or negatively charged phospholipids& chol.
2. PH sensitive liposomes- phospholipid such as PE or DOPE
3. Immuno liposomes-CL with attached monoclonal antibody
4. Cationic liposomes-cationic lipids with DOPE
5. Fusogenic liposomes-Reconstituted Sendai virus envelops

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9.1 Types of vesicles on bases of lamella

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Based on structural
parameters
MLV
Multilamell
ar Large
vesicles
(>0.5 um)
OLV
oligolame
llar
vesicles
(>0.1-1.0
um)
UV
Unilame
llar
Vesicles
MVV
Multivesi
cularvesi
cles
(> 1.0 UM)
MUV
GUV
>1um
SUV
20-
100nm
LUV
>100nm

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Based on
method of
preparatio
n
REV, SUV made
by reverse
phase
evaporation
method
SPLV
Stable
plurilamenar
vesicles
FATMLV
Frozen &
thawed MLV
VET
Vesicles
prepared by
extrusion
tech.

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Based
on
composi
tion &
applicati
on
conven
tial
fusoge
nic
pH
sensiti
ve
cation
ic
Long
circulat
ory
immu
no

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Passive
loading
techniqu
e
Active/re
mote
loading
techniqu
e
Loading of the entrapped
agents before/ during the
manufacture procedure.
Certain types of
compounds with ionizable
groups & those with both
lipid & water solubility
can be Introduced into
liposomes after the
formation of intact
vesicles.
MethodS of Liposome Preparation
21

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49
10. Conventional liposome preparation methods
Phospholipids
Cholesterol
Antioxidant
Lipid component
compounding
Lipid solvent
Pyrogen Ultra filter
yes
No
Filter
Solvent
removal
Drug ,Salt
Antioxidant
Buffer
WFI
Filter
Hydration
Solvent
recovery
Extrusion
Down sizing
Free drug removal
Prefilter
Sterile filter
Vial filling
Free drug
recovery
Aseptic processing
Lyophollization
Seal / package

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51. 11. Methods of Liposome Preparation
 PASSIVE LOADING TECHNIQUES
1. Mechanical Dispersion method
2. Solvent Dispersion method
3. Detergent Solubilization method
Mechanical dispersion methods of passive loading
 Technique begin with a lipid solution in organic solvent & end up with lipid dispersion in water
 Various components are combined by co-dissolving the lipids in organic solvent which is then
removed by film deposition under vacuum.
 After solvent removal the solid lipid mixture is hydrated using aqueous buffer.
 The lipids spontaneously swell & hydrate to form liposomes
 The post hydration treatments include vortexing, sonication, freeze thawing & high pressure extrusion.12/13/2015
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Post Hydration vortexing, sonication, freeze thawing & high
pressure extrusion
Liposome
Lipid spontaneously swell & Hydrate
Solid lipid mixture is hydrated by using aqueous buffer
Film deposition
Remove organic solvent under vacuum
Lipid dissolve in organic solvent/co-solvent
11.1 MECHANICAL DISPERSION METHODS

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54 11.2 Proliposomes
To increase the surface area of dried lipid film and to facilitate continuous hydration and lipid
is dried over the finally divided particulate support i.e.- NaCl, Sorbitol, or other
polysaccharides. These dried lipid coated particulates are called as Proliposomes
Proliposomes form dispersion of MLVs on addition of water, where support is rapidly
dissolved and lipid film hydrate to form MLVs
Methods overcome the stability problem and entrapment efficiency doesn’t matter when
formation of stable liposome.

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56. 12/13/2015
56 11.3 Sonication Method
Probe Sonicator: is employed for dispersions, which require high energy in a small volume
(e.g., high concentration of lipids, or a viscous aqueous phase)
Disadvantage- lipid degradation due to high energy and sonication tips release titanium
particles into liposome dispersion
Bath Sonicator: The bath is more suitable for large volumes of diluted lipids.
Method: Placing a test tube containing the dispersion in a bath sonicator and sonicating for 5-
10min(1,00,000g) which yield a slightly hazy transparent solution. Using centrifugation to yield
a clear SUV dispersion.

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58. 12/13/2015
58 11.4 French pressure cell liposomes
The ultrasonic radiation degrades the lipids, other sensitive compounds, macromolecules for
this extrusion of preformed larger liposomes in a French press under very high pressure is done
This tech. yields unit or oligo lamellar liposomes of size (30-80nm in dia.)
Includes high cost of press that consists of electric hydraulic press & pressure cell
Liposomes prepared by this method are less likely to suffer from structural defects &
instabilities as observed in sonicated vesicles.

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59 11.5 Micro Emulsification Liposomes(MEL)
 “Micro Fluidizer” is used to prepare small MLVs from Concentrated lipid dispersion.
 The lipids can introduced into fluidizers, either as a dispersion of large MLVs or as a slurry
of anhydrated lipids in organic medium.
 Micro fluidizer pumps the fluid at very high pressure(10,000psi, 600-700 bar) through a 5um
orifice.
 Then it is forced along defined micro channels, which direct two streams of fluid to collide
together at right angles at a very high velocity, thereby affecting an efficient transfer of
energy.
 The fluid collected can be recycled through the pump and interaction chamber until vesicles
of
 the spherical dimension are obtained.
 After a single pass, the size of vesicles is reduced to a size 0.1 and 0.2um in diameter.

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62 11.6 Vesicles Prepared By Extrusion Techniques (Vets)
It is used to process LUVs as well as MLVs.
Liposomes prepared by this tech. are called as membrane filter extrusion liposomes.
The 30% capture volume can be obtained using high lipid conc. The trapped volume
in this process is 1-2 litre /mole of lipids.
 It is due to their ease of production, readily selectable vesicle diameter, batch to batch
reproducibility & freedom from solvent or surfactant contamination is possible

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64 11.7 Freeze Thaw Sonication Method (FTS)
 The method is based on freezing of a unilamellar dispersion & then thawing at room temp
for 15 min.
 Thus the process ruptures & refuses SUVs during which the solute equilibrates between
inside & outside & liposomes themselves fuse & increase in size.
 Entrapment volume can be upto 30% of the total vol. of dispersion. Sucrose, divalent metal
ions & high ionic strength salt solutions can not be entrapped efficiently

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66 11.8 Dried-reconstituted Vesicles
 Liposomes obtained by this method are usually “uni or oligo lamellar” of the order of 1.0um
or less in diameter.
 SUVs in aqueous phase SUVs with solutes to be entrapped Freeze dried membrane Solutes
in uni lamellar vesicles Solutes in uni or oligo lamellar vesicles.
 FST method DRV method Rehydration Film stacks dispersion Aqueous phase Thawing
Sonication (15-30 sec)

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11.9 Solvent dispersion Method
Liposome
Formation of monolayer and bilayer of phospholipid
Excess addition of aqueous phase
Lipid dissolve in organic solvent

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69 11.10 Detergent solubilization methods
Formation of micelles (Liposome)
By addition optimized concentration of detergent
Phospholipid brought into intimate contact with aqueous phase

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70 11.11 Active Loading Techniques
 Weak amphipathic bases accumulate in the aqueous phase of lipid vesicles in response to a
difference in pH between the inside and outside of the liposomes (pHin & pHout)
 Two steps process generates this pH imbalance and active (remote) loading.
 Vesicles are prepared in low pH solution, thus generating low pH within the liposomal
interiors, followed by addition of the base to extra liposomal medium.
 Basic compounds, carrying amino groups are relatively lipophilic at high pH and
hydrophilic at low pH.
 In two chambered aqueous system separated by membrane liposomes, accumulation occurs
at the low pH side, under dynamic equilibrium conditions.
 Thus the un protonated form of basic drug can diffuse through the bilayer
 The exchange of external medium by gel chromatography with neutral solution
 Weak base doxorubicin, Adriamycin and vincristine which co-exist in aqueous solutions in
neutral and charged forms have been successfully loaded into preformed liposomes via the
pH gradient method.

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LIPID FILM HYDRATION
BY HAND
SHAKING,FREEZE
DRYING OR NON
HAND
SHAKING
MICRO
EMULSIFICATION
SONICATION
FRENCH PRESSURE
CELL
ETHANOL INJECTION
 ETHER INJECTION
 DOUBLE EMULSION
REVERSE PHASE
 VAPOURATION
VESICLES
STABLE PLURI
LAMELLER
 VESICLES
DETERGENT
REMOVAL
FORM MIXED
MICELLES
BY DIALYSIS
CHROMATIGRALPY
DIFFUSION
 VESICLES LIKE….
 RECONSTITUTED
&
Methods of liposome preparation
Passive loading tec
hniques
Active loading tech
niques
Mechanical disp
ersion
methods
Solvent dispersi
on
methods
Detergent
removal
technique
22

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72
Method Vesicles
Mechanical methods
Vortex or hand shaking of phospholipid dispersions MLV
Extrusion through polycarbonate filters at low or medium pressure OLV, LUV
Extrusion through a French press cell “Micro fluidizer” technique Mainly SUV
High-pressure homogenization Mainly SUV
Ultrasonic irritation SUV of minimal size
Bubbling of gas BSV
Methods based on replacement of organic solvent(s) by aqueous media
Removal of organic solvent(s) MLV, OLV, SUV
Use of water-immiscible solvents: ether and petroleum MLV, OLV, LUV
Ethanol injection method LUV
Ether infusion (solvent vaporization) LUV, OLV, MLV
Reverse-phase evaporation
Methods based on detergent removal
Gel exclusion chromatography SUV
“Slow” dialysis LUV, OLV, MLV
Fast dilution LUV, OLV
Other related techniques MLV, OLV, LUV, SUV

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73 11.12 Rapid solvent exchange vesicles (RSEVs)
 Lipid mixture is transferred between pure solvent & a pure aq.environment.
 Organic sol. of lipids through orifice of syringe under vacuum into a tube containing
aqueous buffer. The tube is mounted on vortexed.
 It manifest high entrapment volumes

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74 11.13 Reverse phase evaporation method

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75 11.14 Purification of Liposomes
Gel Filtration Column Chromatography

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76

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77 12. Stability of Liposomes
 Chemical degradation
 Physical degradation
 Prevention of chemical degradation
 Prevention of physical degradation
The liposomes are stable system having protection against physical, chemical and biological
degradation.

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78 13. Liposomes in drug delivery
• Protect the encapsulated drug from metabolic degradation
• Increase the half-life of drug
• Reduce the systemic toxicity of drugs
• Could be used as sustained release vehicles
• It is possible to target them to selected tissues or cell
• Biodegradable and biocompatible

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80 13.1 Stealth or PEG-Liposomes

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82 13.2 Liposomes in tumor therapy
 Targeting strategies using liposomes
 Natural targeting of conventional liposomes (passive vectorization)
 Use of long circulatory (stealth liposomes)
 Use of ligand mediated targeting (active targeting)
 The use of anti-receptor antibodies on the tumour vascular endothelium
 Use of stealth liposomes & ligands mediated targeting in combination
Drug Target
disease
Status Product
Doxorubicin Kaposi's sarcoma Approved SEQUUS
Daunosome Breast cancer Approved NeXstar,USA
Nystatin Systemic fungal
infections
Phase II Aronex, USA
Amikacin Serious bacterial
infections
Phase II NeXstar,USA
Vincristin Solid tumours Preclinical dev. NeXstar,USA

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83 13.3 Liposomes in gene therapy
 Recombinant DNA tech., studies of gene function & gene therapy all depend on delivery of
nucleic acids( genetic material) into cells in vitro & in vivo.
 Gene can be viral (adenovirus, retrovirus) & non viral( liposomes & lipid based systems,
polymers & peptides)
Type of vectors Advantages Disadvantages
Viral vectors
(Adenovirus, retrovirus
& adeno-associated
virus)
Relatively high transfection
efficiency
Immunogenicity, presence of
contaminants & safety
Vector restricted size
limitation for recombinant gene
Non viral vectors
(liposomes/lipid based
systems, polymers &
peptides)
 Favorable, pharmaceutical
issue-GMP, stability, cost
Plasmid independent structure
Low immunogenicity
Opportunity for
chemical/physical manipulation
 low transfection efficiency

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Liposomes in gene therapy

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 PH sensitive liposomes
The PH sensitive liposomes have been reported as plasmid expression vectors for the cytosolic
delivery of DNA.
 PH sensitive immunoliposomes
PH sensitive liposomes have been developed to release their contents in response to an acid
machinery within endosomal system following receptor mediated endocytosis of the
immunological targeting ligand
 Fusogenic liposomes & Virosomes
They fuse & merge with cell membranes & directly introduce molecules (entrapped or
anchored) into cytoplasm & avoiding route followed by conventional liposomes. Fusion can be
mediated by PEG, glycerol & Polyvinyl alcohol or by reconstituted fusogenic viral membrane
based liposomes are termed as Virosomes

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86 13.4 Liposomal vaccines
 New vaccines that are based on recombinant protein subunits & synthetic peptide antigens
are usually non-immunogenic, hence need of immunopotentiation is realized.
 The first liposome based vaccine (against hepatitis A) that has been licensed for use in
human is an IRIV vaccine which are spherical, unilamellar vesicles with a diameter of
150nm.
 IRIVs are prepared by detergent removal of influenza surface glycoproteins & a mixture of
natural & synthetic phospholipids containing 70% egg yolk phosphatidylcholine,20 %
synthetic PE & 10 % envelop phospholipids originating from H1N1 influenza virus.

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87 13.5 Liposomes as a carrier of Immunomodulation
 The main purpose is to activate macrophages & render them tumouricidal. They
acquire ability to recognize & destroy neoplastic cells both in vitro & in vivo.
 Liposomes in Immunodiagnosis
1. LILA assays (liposome immune lysis assay) has been implicated in the detection of serum
components such as carcinoembryonic antigen,C-reactive protein & other serum protein
which serve as diagnostic tools for cancer
2 . LILA sandwich method has been used to detect many important antigens in serum, which
are useful indicators of various abnormalities

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88 13.6 Liposomes in Dermatology and Cosmetology
 Similar to biological membrane they can navigate water soluble & lipophilic substances in
different phases.
 They mimic the lipid composition & structure of human skin, which enables them to
penetrate the epidermal barrier.
 Liposomes are biodegradable & nontoxic, thus avoiding local/systemic side or toxic effects.
 Moisturizing & restoring action of constitutive lipids.
 Liposomes may act as localized drug depots in skin resulting in sustained release of drug,
thus improving therapeutic index of drug at target site while reducing toxicity profile to
minimum.
 Cosmetic creams, e.g. Alpha Lipoic Acid Cream

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89
13.7 Liposomes as Radiopharmaceutical & radio diagnostic
carriers
Liposomes loaded with contrast agents are suitable for
 contrast agents are substances which are able to absorb certain types of signal much stronger
than surrounding tissue
 Radio diagnostic application include liver & spleen imaging, tumor imaging, imaging
cardiovascular pathologies, visualization of inflammation & infected sites, brain imaging,
visualization of bone marrow
 The RES avoidance of contrast agents can be achieved by using targeted liposomes like
immunoliposomes

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90 13.8 Liposomes as Red cells substitutes & artificial RBCs
 Synthetic & semisynthetic blood substitutes includes recombinant hemoglobin,
glutaraldehyde cross linked hemoglobin, hemoglobin encapsulated liposomes.
 Liposome encapsulated hemoglobin products are being investigated as artificial RBCs.
 Researchers reported completely synthetic amphiphilic heme derivative (lipid heme) &
incorporated them into the hydrophobic center of the bilayer membrane of the phospholipid
vesicles, which has excellent oxygen carrying & transporting abilities.

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LIPOSOMES THERAPY DRUGS /USE
AIDS Azidotymidine
Cancer Cisplatin,Taxol,Doxorubicin
Malaria Primaquine, Chloroquine, Artemisinin
Gramicidin A
lung Isoniazid, Rifampicin, Budesonide
Infectious Diseases e.g. skin Amphotericin, Antimony, Pentamidine
DRUGS Antibiotics, Antifungal Disinfectant,
Immunosuppressive agents
Dermatology and Cosmetology Local anesthetic e.g. Lidocaine and
Benzocaine, Gentamycin, Cefazolin
immunological (Vaccine)
Adjuvant
Hepatitis A rabies virus, Measles virus,
influenza virus Herpes virus, HIV-1 and
Vesicular stomatitis
DIEBETIS INSULIN / Hypoglycemic
Radiodiagnostic Carriers γ-scintigraphy, Magnetic resonance (MR),
Computer tomography (CT) and
Ultrasonography (US) of tumors

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92
14. Characterization of liposomes
Liposomes
Size Number of
lamellae
Charge Stability
Preparation Raw
materials
Protection
Sizing
method
Hydration
methods
Degree of
saturation
Head
group
Presence
of sterols
Protecting
agents
Characterized by
Determined by
Classified by

93. Characterization of liposomes
 There are three main types of Characterization technique of liposomes
1. Physical Characterization
1. Vesicles size/shape/morphology
2. Surface -charge/electrical potential
3. Phase bahaviour/ lamellarity
4. Drug release
5. % capture /free drug
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2. Chemical Characterization
1. Phospholipids /lipid concentration
2. Drug concentration
3. PH / Osmolality
4. Antioxidant degradation
5. Phospholipids / cholesterols
6. peroxidation/oxidation/hydrolysis

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3. Biological Characterization
1. Sterility
2. Pyrogenisity
3. Animal toxicity
4. Plasma Stability

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Characterization parameters Analytical methods/instrumentation
Chemical characterization
Phospholipid conc.
Cholesterol conc.
Drug conc.
Phospholipid peroxidation
Osmolality
Barlet assays/Stewart assays, HPLC
HPLC
Monograph
UV absorbance, iodometric & GLC
Ohmmeter
Physical characterization
Vesicle shape & surface morphology
Size & size distribution
Submicron range
Micron range
TEM, Freeze fracture electron microscopy
TEM
TEM,FFEM, photon correlation spectroscopy,
laser light scattering, gel permeation
Biological characterization
Sterility
Pyrogenisity
Animal toxicity
Aerobic or anaerobic cultures
LAL test
Monitoring survival rates, histology &
pathology

97. 14.1 Physical Characterization
 Vesicle shape & lamellarity & Vesicle size & size distribution
 Microscopic techniques
 Optical Microscopy - Determination of gross size distribution of large vesicles
preparations such as MLVs & Morphological structure of liposome.
 various tech. include light microscopy, fluorescent microscopy, electron
microscopy, laser light scattering, field flow fractionation, gel permeation & gel
exclusion, Zetasizer.
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98. Electron Microscopic Techniques
 Freez Fracture Electron Microscopy
 Negative Stain Electron Microscopy
 Transmission Electron Microscopy
 Scanning Electron Microscopy
 Cryo-Electron Microscopy
 Laser Light Scattering Techniques
 Fluorescence Electron Microscopy
 Confocal Laser Light Scanning Microscopy
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99. Freez Fracture Electron Microscopy
 The freeze-fracture/freeze etch technique starts with rapid freezing of a cell. Then
the frozen cells are cleaved along a fracture plane. This fracture plane is in
between the leaflets of the lipid bilayer , The two fractured sections are then
coated with heavy metal (etched) and a replica is made of their surfaces. This
replica is then viewed in an electron microscope.
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100. Negative Stain Electron Microscopy
 Negative stain electron microscopy visualizes electron transparent liposomes as bright
areas against a dark background. Negative stains used in the TEM analysis is ammonium
molybdate.
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101. Transmission Electron Microscopy
 Transmission electron microscopy (TEM) is a microscopy technique whereby a beam
of electrons is transmitted through an ultra thin specimen, interacting with the specimen as
it passes through. An image is formed from the interaction of the electrons transmitted
through the specimen; the image is magnified and focused onto an imaging device, such as
a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as
a CCD camera.
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102. Scanning Electron Microscopy
 A scanning electron microscope (SEM) is a type of electron microscope that images a
sample by scanning it with a high-energy beam of electrons in a raster scan pattern. The
electrons interact with the atoms that make up the sample producing signals that contain
information about the sample's surface topography, composition, and other properties such
as electrical conductivity.
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103. Cryo-Electron Microscopy
 Is form of transmission electron microscopy known as Cryo transmission
electron microscopy (cryo-TEM)
 where the sample is studied at cryogenic temperatures (generally liquid
nitrogen temperatures).
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CryoEM image of GroEL suspended in
vitreous ice at 50,000X magnification
Cryo-TEM of liposome dispersion. Scale
bar is 200 nm.

104. Laser Light Scattering Technique
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105. Fluorescence Electron Microscopy
 The "fluorescence microscope" refers to any microscope that uses fluorescence to generate
an image.
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106. Confocal Laser Light Scanning
Microscopy
 Technique for obtaining high-resolution optical images with depth selectivity & use for
Penetration and Permeation Studies.
 Confocal microscopy is an optical imaging technique used to increase optical
resolution and contrast of a micrograph by using point illumination and a spatial pinhole to
eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the
reconstruction of three-dimensional structures from the obtained images.
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107. Zetasizer
 Zeta potential is an important and useful indicator of particle surface charge, which can be
used to predict and control the stability.
 In general, particles could be dispersed stably when the absolute value of zeta potential was
above 30mV due to the electric repulsion between particles
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108. Gel permeation
Preferably used for the size distribution determination of liposomes
Ultracentrifuge
Used for size distribution of liposomes
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109. Encapsulation efficiency - Determines % of the aq. Phase & hence % of water
soluble drug which is entrapped & expressed as % entrapment/mg lipid.
Trapped volume - The internal or trapped volume is the aqueous entrapped volume per
unit quantity of lipid & expressed as µ l/ µ mol or µ l/mg of total lipid. Radioactive markers
are used to determine the internal volume.
Vesicle fusion measurements - It has been studied in case of cationic liposomes, PH
sensitive liposomes. fusion has been monitored using a fluorescence resonance energy
transfer (RET) between two lipid analogues originally placed in separate vesicle population
that measures intermixing of membrane lipids
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Phase response & transitional behavior
Lipid bilayers can exists in a low temperature solid ordered phase & above certain temp in a
fluid disordered phase. Phase behavior of liposomal membrane determines prop. such as
permeability, fusion, aggregation & protein binding Thermodynamic methods:-In differential
scanning micro calorimeter, the heat required by liposomes to maintain a steady upward rise in
temp is plotted as a function of temperature

111. Elasticity Measurement of Liposomes
Extrusion Method
Liposomal formulations were extruded through filter membrane (pore diameter 50 nm), using a
stainless steel filter holder having 25-mm diameter, by applying a pressure of 2.5 bar. The
quantity of vesicle suspension, extruded in 5 minutes was measured.
Skin Permeation Study
Franz Diffusion Cell
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112. 14.2 Chemical Characterization
 Phospholipid conc. is determined in terms of lipid phosphorus content using Barlet
assay/Stewart assay or TLC
 Cholesterol conc. is determined using Ferric perchlorate method/Cholesterol
oxidase assay
 Lysolecithin:-which is one of the major product of hydrolysis is estimated using
densitometry
 Phospholipid peroxidation is determined by UV absorbance, iodometric, GLC
technique.
 Phospholipid hydrolysis is determined using HPLC & TLC
 Cholesterol auto oxidation can be determined by HPLC & TLC
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113. 14.3 Biological Characterization
 Sterility
Aerobic or anaerobic cultures
 Pyrogenisity
LAL test
 Animal toxicity
Monitoring survival rates, histology & pathology
 Plasma Stability
Cytotoxicity Assay, HPLC Assay
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114. 14.4 Stability after systemic administration
 Two most frequently encountered biological events that the administered liposomal system
undergoes are phagocytosis or antigen presentation via the macrophages of the RES system
 Opsonins which are proteinaceous components of serum adsorb onto the surface of
liposomes thus making these exogenous materials more palatable & conductive to
phagocytes
 High density lipoprotein removes phospholipid molecules from bilayered vesicular systems
 The molecular origin of these interactions are mostly long range electrostatic, Vander waals
& short range hydrophobic interactions of particulate surface with macromolecules in the
serum
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115. 14.5 Liposome-cell interaction
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Storing the vesicles at 4°C ± 0.5°C. Vesicle size, zeta potential, and entrapment efficiency of the
vesicles was measured after 180 days.
 The stability in vitro which covers the stability aspects prior to the administration of the
formulation & with regard to the stability of the constitutive lipids.
 The stability in vivo which covers the stability aspects once the formulation is administered
via various routes to the biological fluids. It includes stability aspects in blood if
administered by systemic route or in gastrointestinal tract if administered by oral or per oral
routes.
 Stability in vitro:- method of formulation, nature of amphiphilic & encapsulated drug,
manipulate membrane fluidity/rigidity & permeability characteristics.
 Storage temp. of these dispersions must be defined & controlled
 Liposomal phospholipids can undergo degradation such as oxidation & hydrolysis

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 Lipid oxidation & Peroxidation
 Lipid peroxidation measurement is based on disappearance of unsaturated fatty acids or
appearance of conjugated dienes.
 It can be prevented by minimizing use of unsaturated lipids, use of oxygen, argon or
nitrogen environment, use of antioxidant such as Alpha tocopherols or BHT or use of light
resistant containers for storage of liposomal preparations
 Lipid hydrolysis
It leads to Lysolecithin formation The inclusion of charged molecule in the bilayer shifts the
electrophoretic mobility & makes it positive with addition of stearylamine or negative with
dicetyl phosphate thus prevents liposomal fusion/swelling or aggregation
 Long term & Accelerated stability
High temp. testing(>250C) is universally used for heterogeneous products. Various laboratories
store their products at temp ranging from 40C to 500 C.

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1.Liposomes as drug/protein delivery vehicles
Controlled & sustained drug release in situ
Enhanced drug solubilization
Altered pharmacokinetics & bio distribution
Enzyme replacement therapy & liposomal storage disorders
2.Liposomes in antimicrobial, antifungal & antiviral therapy
Liposomal drugs
Liposomal biological response modifiers
3.Liposomes in tumor therapy
Carrier of small cytotoxic molecules
Vehicle for macromolecules as genes
4.Liposomes in gene delivery
Gene & antisense therapy
Genetic vaccination
5.Liposomes in immunology
Immunoadjuvant
15. Application Of Liposomes

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Immunomodulation
Immunodiagnosis
6. Liposomes as artificial blood surrogates
7.Liposomes as radiopharmaceutical & radio diagnostic carriers
8.Liposomes in cosmetics & dermatology
9.Liposomes in enzyme immobilization & bioreactor technology

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16. References
• Jain N.K., Controlled & Novel Drug Delivery, CBS Publications, New Delhi
• Jain N.K., Advances in Controlled & Novel Drug Delivery, CBS Publications, New Delhi.
• Vyas S.P. and Khar R.K., Controlled drug delivery- Concepts & Advances, Vallabh
Prakashan, New Delhi.
• Vyas S.P. and Khar R.K., Targeted & Controlled drug delivery- Novel Career System, CBS
Publications, New Delhi.
• Chien Y, Novel Drug Delivery System, Mercel Decker Publications.
• Lee & Robinson, Controlled Drug Delivery, Second Edition, Mercel Decker Publications.
• Swarbrick J and Boylon J.C., Encyclopedia of Pharmaceutical Technology, Vol. 1-3, Mercel
Decker Inc.
• Allen, Theresa M. "Liposomal Drug Formulations: Rationale for Development and What
We Can Expect for the Future." Drugs 56: 747-756, 1998.
• Allen, Theresa M. "Long-circulating (serially stabilized) liposome for targeted drug
delivery." Tips 15: 214-219, 1994.
• Vesicular drug delivery system by R.S.R. Murthy.

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• Allen, Theresa M. "Liposomal Drug Formulations: Rationale for Development and What
We Can Expect for the Future." Drugs 56: 747-756, 1998.
• Allen, Theresa M. "Long-circulating (sterically stabilized) liposomes for targeted drug
delivery."TiPs 15: 214-219, 1994.
• Allen, Theresa M. "Opportunities in Drug Delivery." Drugs 54 Suppl. 4: 8-14, 1997
Janknegt, Robert. "Liposomal and Lipid Formulations of Amphotericin B." Clinical
Pharmacokinetics.23(4): 279-291, 1992.
• Kim, Anna et al. "Pharmacodynamics of insulin in polyethylene glycol-coated
liposomes."International Journal of Pharmaceutics. 180: 75-81, 1999.
• Quilitz, Rod. "Oncology Pharmacotherapy: The Use of Lipid Formulations of
Amphotericin B in Cancer
• Patients." Cancer Control.5:439-449, 1998.
• Ranade, Vasant V. "Drug Delivery Systems: Site-Specific Drug Delivery Using Liposomes
as Carriers."
• Pharmacology. 29: 685-694, 1989.
• Storm, Gert and Daan J.A. Crommelin. "Liposomes:quo vadi?" PSTT 1: 19-31, 1998.
• Taylor, KMG and JM Newton. "Liposomes as a vecicle for drug delivery." British Journal
of Hospital
• Medicine. 51: 55-59, 1994

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• Navone, NM, et al. p53 mutations in prostate cancer bone metastases suggest that selected
p53 mutants in th eprimary site define foci with metastatic potential. J Urol 161(1):304-8,
1/99.
• Novahealth@earthlink.net www.prostatematters.com 1999
• Pienta, K., Goodson, J., & Esper, P. Epidemiology of Prostate Cancer: Molecular and
Environmental Clues. http://www.cancer.med.umich.edu/prostcan/articles/clues.html
• Smith, J, et al. Major Susceptibility Locus for Prostate Cancer on Chromosome 1 Suggested
by a Genome-Wide Search. Science 274: 1371-4, 11/22/96
• Veterinary Genetics Laboratory, School of Veterinary Medicine University of California,
Davis. Microsatellites. http://www.vgl.ucdavis.edu/service/canine/micros.htm 12/30.97
• Wolf, G. University Hospital Charite Institute of Pathology. http://amba.charite.de/cgh
1/15/99
• Xu, J., et al. Evidence for a prostate cancer susceptibility locus on the X chromosome.
Nature Genet 20: 175-179, 1998.

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