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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Liposome Classification and Preparation Methods
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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2
9. 1. Liposome
īŧ 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.
īŧ Liposomes are spherical microscopic vesicles consisting phospholipids bilayers which
enclose aqueous compartments.
īŧ The size of a liposome ranges from some 20 nm up to several micrometers.
īŧ Liposomes were first produced in England in 1961 by Alec D. Bangham, who was studying
phospholipids and blood clotting.
īŧ Small unilamellar vesicles (SUV), 25 to 100 nm in size that consist of a single bilayer
īŧ Large unilamellar vesicle (LUV), 100 to 500 nm in size that consist of a single bilayer
īŧ Multilamellar vesicle (MLV), 200 nm to several microns, that consist of two or more
concentric bilayer
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9
11. Liposome
īŧ The lipid molecules are usually phospholipids- amphipathic moieties with a hydrophilic
head group and two hydrophobic tails.
īŧ On addition of excess water, such lipidic moieties spontaneously originate to give the
most thermodynamically stable conformation.
īŧ 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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11
14. 2. Structural components of liposome's
ī´ Ther are two main components of Liposomes system they are Phospholipid and
cholesterol.
2.1 Phospholipids
īŧ Phosphatidylcholine.
īŧ Amphipathic molecule
īŧ Hydrophobic tail- 2 fatty acid chain containing 10-24 carbon atoms and 0-6
double bond in each chain
īŧ Hydrophilic polar head- Phosphoric acid bound to water soluble molecule
īŧ Self organize in ordered supramolecular structure when confronted (meet face to
face) with solvent
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27. 12/13/2015
27
2.6 Cholesterol
īŧ Cholesterol by itself does not form bilayer structure.
īŧ Cholesterol act as fluidity buffer
īŧ After intercalation with phospholipid molecules alter the freedom of motion of carbon
molecules in the acyl chain
īŧ Restricts the transformations of trans to gauche conformations
īŧ Cholesterol incorporation increases the separation between choline head group &
eliminates normal electrostatic & hydrogen bonding interactions.
īŧ its rigid steroid ring system which interferes with motion of fatty acid tails, stabilizes the
lipid bilayer and decrease the leakage of encapsulated drug
30. 12/13/2015
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, âgelâ state
(Lb phase). Transition temperature = Tc.
Liposomes are formed from aqueous dispersions
the âmoltenâ 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
32. 12/13/2015
32 īŧ 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
īŧ Biological membrane surfactants
consist of phospholipids made up of
glycerol esterified to 2 fatty acid
chains plus a phosphate derivative
polar head. Structure is
īŧ RCH2-RâCH2-CH2-OPO3-X
īŧ where R and Râ = alkyl-CO2- and the
head groups may contain a variety of
different terminal groups X.
īŧ 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
ī´ Stress induced hydration of phospholipids
ī´ Organization in to lamellar arrays
ī´ Results in to budding of lipid bilayer leading to down sizing
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34
SUV OLV
35. 4.2 bilayer phospholipids theory
ī´ Liposomes are formed when thin lipid films are hydrated
ī´ The hydrated lipid sheets detach during agitation and self-close to form large, Multilamellar
vesicles (LMV)
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35
36. 5. Advantages of Liposomes
īŧ Provides selective passive targeting to tumor tissues
īŧ Increased efficacy & therapeutic index
īŧ Increased stability via encapsulation
īŧ Reduction in toxicity of the encapsulated agent
īŧ Site avoidance effect
īŧ Improved pharmacokinetic effects
īŧ Flexibility to couple with site specific ligands to achieve active targeting
īŧ Variety of Drugs Given In Low Dose As Encapsulated For Stability
īŧ Minimum Effective Concentration And Therapeutic Index
īŧ Low Toxicity Due To Reduced Exposure To Sensitive Tissues
īŧ Minimum ADR/No Side Effects
īŧ Possible Formulation- suspension, emulsion, gel, Cream, lotion, Aerosol, reconstituted Vesicles
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37. 6. Disadvantages
īŧ Physical/ Chemical Stability
īŧ Very High Production Cost
īŧ Drug Leakage/ Entrapment/ Drug Fusion
īŧ Sterilization
īŧ Short Biological Activity / T ÂŊ
īŧ Oxidation of Bilayer âĻLipids And Low Solubility
īŧ Overcoming Resistance
īŧ Extensive Clinical And Laboratory Research To A Certain Long Circulating Liposomes
īŧ Repeated Iv Administration Problems
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38. 7. Importance of Liposomes in drug Delivery
System
ī´ Pharm kinetics - efficacy and toxicity
A. Changes the absorbance and bio distribution
B. Deliver drug in desired form
C. Multidrug resistance
ī´ Protection
A. Decrease harmful side effects
B. Change where drug accumulates in the body
C. Protects drug
ī´ 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
īļ Phospholipids are amphipathic molecules having hydrophobic tail & a hydrophilic or
polar head
īļ The hydrophilic & hydrophobic domains within the molecular geometry of amphiphilic
lipids orient & self organize in ordered supramolecular structure when confronted with
solvents
īļ Cholesterol have modulatory effect on the bilayer membrane (acts as fluidity buffer)
īļ 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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48. 12/13/2015 48
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
49. 12/13/20
15
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
51. 11. Methods of Liposome Preparation
ī´ PASSIVE LOADING TECHNIQUES
1. Mechanical Dispersion method
2. Solvent Dispersion method
3. Detergent Solubilization method
Mechanical dispersion methods of passive loading
ī´ Technique begin with a lipid solution in organic solvent & end up with lipid dispersion in water
ī´ Various components are combined by co-dissolving the lipids in organic solvent which is then
removed by film deposition under vacuum.
ī´ After solvent removal the solid lipid mixture is hydrated using aqueous buffer.
ī´ The lipids spontaneously swell & hydrate to form liposomes
ī´ The post hydration treatments include vortexing, sonication, freeze thawing & high pressure extrusion.12/13/2015
51
53. 12/13/2015
53
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
54. 12/13/2015
54 11.2 Proliposomes
īTo 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
īProliposomes form dispersion of MLVs on addition of water, where support is rapidly
dissolved and lipid film hydrate to form MLVs
īMethods overcome the stability problem and entrapment efficiency doesnât matter when
formation of stable liposome.
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.
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.
59. 12/13/2015
59 11.5 Micro Emulsification Liposomes(MEL)
īŧ âMicro Fluidizerâ is used to prepare small MLVs from Concentrated lipid dispersion.
īŧ The lipids can introduced into fluidizers, either as a dispersion of large MLVs or as a slurry
of anhydrated lipids in organic medium.
īŧ Micro fluidizer pumps the fluid at very high pressure(10,000psi, 600-700 bar) through a 5um
orifice.
īŧ 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.
īŧ The fluid collected can be recycled through the pump and interaction chamber until vesicles
of
īŧ the spherical dimension are obtained.
īŧ After a single pass, the size of vesicles is reduced to a size 0.1 and 0.2um in diameter.
62. 12/13/2015
62 11.6 Vesicles Prepared By Extrusion Techniques (Vets)
īIt is used to process LUVs as well as MLVs.
īLiposomes prepared by this tech. are called as membrane filter extrusion liposomes.
īThe 30% capture volume can be obtained using high lipid conc. The trapped volume
in this process is 1-2 litre /mole of lipids.
ī It is due to their ease of production, readily selectable vesicle diameter, batch to batch
reproducibility & freedom from solvent or surfactant contamination is possible
64. 12/13/2015
64 11.7 Freeze Thaw Sonication Method (FTS)
īą The method is based on freezing of a unilamellar dispersion & then thawing at room temp
for 15 min.
īą Thus the process ruptures & refuses SUVs during which the solute equilibrates between
inside & outside & liposomes themselves fuse & increase in size.
īą 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
66. 12/13/2015
66 11.8 Dried-reconstituted Vesicles
īą Liposomes obtained by this method are usually âuni or oligo lamellarâ of the order of 1.0um
or less in diameter.
īą 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.
īą FST method DRV method Rehydration Film stacks dispersion Aqueous phase Thawing
Sonication (15-30 sec)
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67
11.9 Solvent dispersion Method
Liposome
Formation of monolayer and bilayer of phospholipid
Excess addition of aqueous phase
Lipid dissolve in organic solvent
69. 12/13/2015
69 11.10 Detergent solubilization methods
Formation of micelles (Liposome)
By addition optimized concentration of detergent
Phospholipid brought into intimate contact with aqueous phase
70. 12/13/2015
70 11.11 Active Loading Techniques
īą 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)
īą Two steps process generates this pH imbalance and active (remote) loading.
īą 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.
īą Basic compounds, carrying amino groups are relatively lipophilic at high pH and
hydrophilic at low pH.
īą In two chambered aqueous system separated by membrane liposomes, accumulation occurs
at the low pH side, under dynamic equilibrium conditions.
īą Thus the un protonated form of basic drug can diffuse through the bilayer
īą The exchange of external medium by gel chromatography with neutral solution
īą 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.
71. 12/13/2015 71
īąLIPID FILM HYDRATION
BY HAND
SHAKING,FREEZE
DRYING OR NON
HAND
SHAKING
īąMICRO
EMULSIFICATION
īąSONICATION
īąFRENCH PRESSURE
CELL
īąETHANOL INJECTION
īą ETHER INJECTION
īą DOUBLE EMULSION
īąREVERSE PHASE
īą VAPOURATION
VESICLES
īąSTABLE PLURI
LAMELLER
īą VESICLES
īąDETERGENT
REMOVAL
FORM MIXED
MICELLES
BY DIALYSIS
īąCHROMATIGRALPY
īąDIFFUSION
īą VESICLES LIKEâĻ.
īļ 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
72. 12/13/2015
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 âMicro fluidizerâ 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
âSlowâ dialysis LUV, OLV, MLV
Fast dilution LUV, OLV
Other related techniques MLV, OLV, LUV, SUV
73. 12/13/2015
73 11.12 Rapid solvent exchange vesicles (RSEVs)
īļ Lipid mixture is transferred between pure solvent & a pure aq.environment.
īļ Organic sol. of lipids through orifice of syringe under vacuum into a tube containing
aqueous buffer. The tube is mounted on vortexed.
īļ It manifest high entrapment volumes
77. 12/13/2015
77 12. Stability of Liposomes
īļ Chemical degradation
īļ Physical degradation
īļ Prevention of chemical degradation
īļ Prevention of physical degradation
The liposomes are stable system having protection against physical, chemical and biological
degradation.
78. 12/13/2015
78 13. Liposomes in drug delivery
âĸ Protect the encapsulated drug from metabolic degradation
âĸ Increase the half-life of drug
âĸ Reduce the systemic toxicity of drugs
âĸ Could be used as sustained release vehicles
âĸ It is possible to target them to selected tissues or cell
âĸ Biodegradable and biocompatible
82. 12/13/2015
82 13.2 Liposomes in tumor therapy
īļ Targeting strategies using liposomes
īļ Natural targeting of conventional liposomes (passive vectorization)
īļ Use of long circulatory (stealth liposomes)
īļ Use of ligand mediated targeting (active targeting)
īļ The use of anti-receptor antibodies on the tumour vascular endothelium
īļ 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
83. 12/13/2015
83 13.3 Liposomes in gene therapy
īą Recombinant DNA tech., studies of gene function & gene therapy all depend on delivery of
nucleic acids( genetic material) into cells in vitro & in vivo.
īą 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
īŽImmunogenicity, presence of
contaminants & safety
īŽVector restricted size
limitation for recombinant gene
Non viral vectors
(liposomes/lipid based
systems, polymers &
peptides)
īŽ Favorable, pharmaceutical
issue-GMP, stability, cost
īŽPlasmid independent structure
īŽLow immunogenicity
īŽOpportunity for
chemical/physical manipulation
īŽ low transfection efficiency
85. 12/13/2015
85
īą PH sensitive liposomes
The PH sensitive liposomes have been reported as plasmid expression vectors for the cytosolic
delivery of DNA.
īą 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
īą 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
86. 12/13/2015
86 13.4 Liposomal vaccines
īą New vaccines that are based on recombinant protein subunits & synthetic peptide antigens
are usually non-immunogenic, hence need of immunopotentiation is realized.
īą 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.
īą 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.
87. 12/13/2015
87 13.5 Liposomes as a carrier of Immunomodulation
īą The main purpose is to activate macrophages & render them tumouricidal. They
acquire ability to recognize & destroy neoplastic cells both in vitro & in vivo.
īą 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
88. 12/13/2015
88 13.6 Liposomes in Dermatology and Cosmetology
īą Similar to biological membrane they can navigate water soluble & lipophilic substances in
different phases.
īą They mimic the lipid composition & structure of human skin, which enables them to
penetrate the epidermal barrier.
īą Liposomes are biodegradable & nontoxic, thus avoiding local/systemic side or toxic effects.
īą Moisturizing & restoring action of constitutive lipids.
īą 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.
īą Cosmetic creams, e.g. Alpha Lipoic Acid Cream
89. 12/13/2015
89
13.7 Liposomes as Radiopharmaceutical & radio diagnostic
carriers
Liposomes loaded with contrast agents are suitable for
īļ contrast agents are substances which are able to absorb certain types of signal much stronger
than surrounding tissue
īļ Radio diagnostic application include liver & spleen imaging, tumor imaging, imaging
cardiovascular pathologies, visualization of inflammation & infected sites, brain imaging,
visualization of bone marrow
īļ The RES avoidance of contrast agents can be achieved by using targeted liposomes like
immunoliposomes
90. 12/13/2015
90 13.8 Liposomes as Red cells substitutes & artificial RBCs
īą Synthetic & semisynthetic blood substitutes includes recombinant hemoglobin,
glutaraldehyde cross linked hemoglobin, hemoglobin encapsulated liposomes.
īą Liposome encapsulated hemoglobin products are being investigated as artificial RBCs.
īą 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.
91. 12/13/2015 91
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 Îŗ-scintigraphy, Magnetic resonance (MR),
Computer tomography (CT) and
Ultrasonography (US) of tumors
92. 12/13/2015
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
ī´ 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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93
96. 12/13/2015 96
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
ī´ Vesicle shape & lamellarity & Vesicle size & size distribution
ī´ Microscopic techniques
ī´ Optical Microscopy - Determination of gross size distribution of large vesicles
preparations such as MLVs & Morphological structure of liposome.
ī´ various tech. include light microscopy, fluorescent microscopy, electron
microscopy, laser light scattering, field flow fractionation, gel permeation & gel
exclusion, Zetasizer.
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97
98. Electron Microscopic Techniques
īą Freez Fracture Electron Microscopy
īą Negative Stain Electron Microscopy
īą Transmission Electron Microscopy
īą Scanning Electron Microscopy
īą Cryo-Electron Microscopy
īą Laser Light Scattering Techniques
īą Fluorescence Electron Microscopy
īą Confocal Laser Light Scanning Microscopy
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98
99. Freez Fracture Electron Microscopy
ī´ 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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99
100. Negative Stain Electron Microscopy
ī´ 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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100
101. Transmission Electron Microscopy
ī´ 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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101
102. Scanning Electron Microscopy
ī´ 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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102
103. Cryo-Electron Microscopy
ī´ Is form of transmission electron microscopy known as Cryo transmission
electron microscopy (cryo-TEM)
ī´ where the sample is studied at cryogenic temperatures (generally liquid
nitrogen temperatures).
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103
CryoEM image of GroEL suspended in
vitreous ice at 50,000X magnification
Cryo-TEM of liposome dispersion. Scale
bar is 200 nm.
105. Fluorescence Electron Microscopy
ī´ The "fluorescence microscope" refers to any microscope that uses fluorescence to generate
an image.
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105
106. Confocal Laser Light Scanning
Microscopy
ī´ Technique for obtaining high-resolution optical images with depth selectivity & use for
Penetration and Permeation Studies.
ī´ 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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106
107. Zetasizer
ī Zeta potential is an important and useful indicator of particle surface charge, which can be
used to predict and control the stability.
ī 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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107
108. Gel permeation
Preferably used for the size distribution determination of liposomes
Ultracentrifuge
Used for size distribution of liposomes
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108
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 Âĩ l/ Âĩ mol or Âĩ 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
īļ Phospholipid conc. is determined in terms of lipid phosphorus content using Barlet
assay/Stewart assay or TLC
īļ Cholesterol conc. is determined using Ferric perchlorate method/Cholesterol
oxidase assay
īļ Lysolecithin:-which is one of the major product of hydrolysis is estimated using
densitometry
īļ Phospholipid peroxidation is determined by UV absorbance, iodometric, GLC
technique.
īļ Phospholipid hydrolysis is determined using HPLC & TLC
īļ Cholesterol auto oxidation can be determined by HPLC & TLC
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114. 14.4 Stability after systemic administration
ī´ Two most frequently encountered biological events that the administered liposomal system
undergoes are phagocytosis or antigen presentation via the macrophages of the RES system
ī´ Opsonins which are proteinaceous components of serum adsorb onto the surface of
liposomes thus making these exogenous materials more palatable & conductive to
phagocytes
ī´ High density lipoprotein removes phospholipid molecules from bilayered vesicular systems
ī´ 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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116 14.6 Stability of Liposomes
Storing the vesicles at 4°C ¹ 0.5°C. Vesicle size, zeta potential, and entrapment efficiency of the
vesicles was measured after 180 days.
īą 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.
īą 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.
īą Stability in vitro:- method of formulation, nature of amphiphilic & encapsulated drug,
manipulate membrane fluidity/rigidity & permeability characteristics.
īą Storage temp. of these dispersions must be defined & controlled
īą Liposomal phospholipids can undergo degradation such as oxidation & hydrolysis
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īą Lipid oxidation & Peroxidation
īļ Lipid peroxidation measurement is based on disappearance of unsaturated fatty acids or
appearance of conjugated dienes.
īļ 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
īą 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
īą 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
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Prakashan, New Delhi.
âĸ Vyas S.P. and Khar R.K., Targeted & Controlled drug delivery- Novel Career System, CBS
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âĸ Chien Y, Novel Drug Delivery System, Mercel Decker Publications.
âĸ Lee & Robinson, Controlled Drug Delivery, Second Edition, Mercel Decker Publications.
âĸ Swarbrick J and Boylon J.C., Encyclopedia of Pharmaceutical Technology, Vol. 1-3, Mercel
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âĸ Allen, Theresa M. "Liposomal Drug Formulations: Rationale for Development and What
We Can Expect for the Future." Drugs 56: 747-756, 1998.
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