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Title: liposomes


1
LIPOSOMES
- as micro-particulate drug carriers
Guided by, Mrs. Samyuktha , M.Pharmacy, Departmen
t of pharmaceutics .
Presented by, K .Vinod kumar M.Pharmacy
(pharmaceutics).
ST.ANNS COLLEGE OF PHARMACY, CHIRALA
2
What are liposomes ?
  • Liposome is Greek word - lipo means fat
  • -
    soma means body
  • These are the micro-particulate drug carriers
    consisting of one or more concentric spheres of
    lipid bilayer separated by aqueous buffer
    compartments.
  • When phospholipids are dispersed in water they
    spontaneously form closed structures with
    internal aqueous compartments bounded by
    phospholipid bilayer membranes.
  • Their diameter ranges from 80nm to 100um

3
General structure of liposome
  • .

Lipid bilayer
4
  • When phospholipids dispersed in aqueous phase
    , hydration of polar head groups of lipid
    results in heterogenous mixture structure,
    referred to as vesicles ,contain
    multiple lipid bilayer which are referred to as
    Multilamellar vesicles
  • Sonication of the liposomes results in there
    size reduction to vesicles containing only a
    single bilayer with diameter 25nm to 50nm are
    referred to as Unilamellar vesicles.
  • Large unilamellar vesicles exhibit size range
    of 100 500nm.

5
Structure of the phospholipid molecule
  • .

6
Mechanisms by which liposomes act -
  • There are several mechanisms by which liposomes
    act which are as follows
  • Liposomes attaches to plasma membrane and
    appears to fuse with them,releasing their content
    into cell.
  • Liposomes are taken up by cell in some cases and
    their phospholipids are incorporated into plasma
    membrane by which drug trapped inside is
    released.
  • In case of phagocyte cell, the liposomes are
    taken up, the phospholipid walls are acted upon
    by organelles called lysosomes and the drug is
    released.

7
Drug content released into the cell
Phospholipids of liposome are incorporated into
cell membrane
Liposome
Drug releasing into cell
Cell membrane
Outside of the cell.
Cell membrane
Inside of cell.
Inside of cell.
8
Materials used in liposome preparation -
  • PHOSPHOLIPIDS
  • Glycerol containing phospholipids are commonly
    used component of liposome formulation.
  • The most abundant glycerol phosphotides in plants
    and animals are phosphotidyl
    choline(lecithin) and phosphotidyl ethanol
    amine(cephaline).
  • These two are major structural components of
    most biological membranes.
  • Fatty acids are also important constituent of
    glycerol phosphotides(triglycerides of fat cells).

9
2. SPHINGOLIPIDS
  • They contain sphingosine (or) a related base
    as their structural backbone.
  • They contain 3 characteristic building
    blocks-
  • - A molecule of fatty acid
  • - A molecule of sphingosine
  • - Head groups that can vary from simple
    alcohol as choline to very complex carbohydrates.
  • The most abundant sphingolipid in higher
    animals is sphingomyelin.

10
3. GLYCOSPHINGOLIPIDS
  • These are found mainly in grey matter of brain
    tissue of higher animals and are used in minor
    component.
  • These are included in liposomes formulation to
    provide a layer of surface charged groups.
  • STEROLS
  • Cholesterol its derivatives are quite often
    included as components of liposomal membrane.

11
  • Cholesterol can be added to bilayer mixture
    for the following purposes -

- Acts as a fluidity buffer.
- Acts as intercalator with phospholipid
molecules.
- Decreases the permeability of membrane to
water soluble molecules.
  • Liposomes without cholesterol are known to
    interact rapidly with plasma proteins like
    albumin , transferin and macroglobulins which
    lead to physical instability of liposomes.

12
5. SYNTHETIC PHOSPHOLIPIDS
  • Saturated phospholipids include dipalmitoyl
    phosphatidyl choline(DPPC) , dipalmitoyl
    phosphatidyl ethanol amine(DPPE) , dipalmitoyl
    phosphatidyl serine(DPPS) .
  • Unsaturated phospholipids have also been used
    which include dioleo-phosphatidyl choline(DOPC) ,
    dioleo-phosphatidyl glycerol(DOPG)

13
6. POLYMERIC MATERIALS
  • A large variety of polymerizable lipids which can
    form vesicles , has been synthesized.
  • These include lipids containing conjugated diene
    , methacrylate , and thiol groups as
    polymerizable moieties.

14
Special consideration for selection of lipids -
  • For non-irritant topical formulations -
  • -- non- ionic liposomes.
  • For localized infection -
  • -- ionic liposomes.
  • For other systemic applications -
  • -- liposomes with covalently
    attached polymer.
  • For nucleic acid complexation -
  • -- cationic liposomes that contain
    large fraction of neutral lipids.

15
Preparation of liposomes
  • The three different strategies for the
    preparation of liposomes include -
  • 1. Mechanical method
  • 2. Method based on replacement of organic
    solvent
  • 3. Methods based on size transformation or
    fusion of preformed vesicle

16
1. MECHANICAL METHOD
  • FILM METHOD-

Thin lipid film organic solvent
Solvent removed by film deposition under vacuum.
After all the solvent is removed.
Heterogenous Multilamellar vesicle(1u m diameter)
Lipids swell spontaneously.
Solid-lipid mixture hydrated using aqueous buffer.
17
Limitation of film method-
  • Encapsulation efficiency is low.
  • b) ULTRASONIC METHOD-
  • It is used for separation of small unilamellar
    vesicles with diameter 15 25u m.
  • Ultrasonication of an aqueous dispersion of
    phospholipids is done by 2 types of sonicators -
  • probe sonicator - for small volume
    requiring high energy.
  • bath sonicator - for large volume.

18
2. METHODS BASED ON REPLACEMENT OF ORGANIC
SOLVENTS -
  • REVERSE PHASE EVAPORATION-
  • This method is used for preparation of large
    unilamellar and oligolamellar vesicles.
  • It has the ability to encapsulate large macro
    -molecules of with high efficiency.
  • Diethyl ether and isopropyl ether are the
    solvents generally used.

19
Procedure for reverse phase evaporation-
Lipid mixture is placed in round bottom flask.
Solvent removed under reduced pressure by rotary
evaporator.
Purged with nitrogen lipids are re-dissolved
in organic phase.
  • .

Non-encapsulated material is removed.
Solvent is removed from emulsion by evaporation
under reduced pressure.
Liposomes formed are called reverse phase
evaporation vesicles.
20
b) ETHER VAPORIZATION METHOD
  • There are 2 methods according to the solvents
    used-
  • Ethanol injection method-
  • lipid is injected rapidly through fine needle
    into an excess of aqueous medium.
  • Ether injection method-
  • Lipid is injected slowly through fine needle
    into an excess of aqueous medium.

21
3. METHODS BASED ON SIZE TRANSFORMATION OR
FUSION OF PREFORMED VESICLE -
  • THE DEHYDRATION - REHYDRATION METHOD-

EMPTY BUFFER CONTAINING SUVS.
REHYDRATE WITH AQ. FLUID CONTAINING MATERIAL TO
BE ENTRAPPED.
DISPERSION OF SOLID LIPIDS IN FINELY SUBDIVIDED
FORM.
VESICLES FORMED ARE THEN REHYDRATED.
LIPOSOMES OBTAINED ARE OLIGOLAMELLAR VESICLES.
SUBJECTED TO DRYING
22
b) FREEZE THAW EXTRUSION METHOD -
This method is mainly used for encapsulation
of proteins.
  • .

Formed MLVs are frozen in luke warm water
vortexed again.
Liposomes formed by film method.
Vortexed with the solute to be entrapped until
entire film is suspended
Subjected to two cycles of freeze thaw
vortexing the sample is extruded 3 times.
Ruptures and defuses SUVs liposomes fuse and
increase in size forming LUVET
Subjected to 6 freeze thaw cycles addition 8
extrusions
23
METHODS FOR CONTROLLING THE PARTICLE SIZE SIZE
DISTRIBUTION OF LIPOSOMES
FRACTIONATION
CAPILLARY PORE MEMBRANE EXTRUSION
HOMOGENIZATION
24
1. FRACTIONATION-
  • CENTRIFUGATION
  • Liposomes sediment in a centrifugal field at
    a rate that is dependent on their size and
    density.
  • Disadvantage
  • liposomes smaller than about 0.5um tend to
    require high forces and long spinning times
  • SIZE EXCLUSION CHROMATOGRAPHY
  • It is particularly useful for separation of
    small unilamellar vesicles from large structures.

25
2. HOMOGENIZATION
  • By using high pressure homogenizers like micro
    fluidizer particle size can be reduced.
  • 3. CAPILLARY PORE MEMBRANE EXTRUSION
  • It is wide spread , simple technique for
    production of defined narrow sized liposomes.
  • The extrusion of heterogenous population of
    fairly large liposomes through polycarbonate
    membranes under reduced pressure gives uniform
    particle sized liposomes

26
Characterization of liposomes
  • a) Factors affecting drug entrapment
  • - Partition coefficient of drug.
  • - Solubility of drug.
  • - Lipid quantity used.
  • - Internal volume of liposome.
  • - Drug concentration(it should not reach the
    saturation limit).

27
b) Internal volume and encapsulation
efficiency-
  • These two parameters are used to describe
    entrapment of water soluble drugs in aqueous
    compartment of liposome.
  • These parameters depend on liposomal content ,
    lipid concentration , method of preparation and
    drug used.
  • Incorporation of charged lipids into lipids
    increases the volume of aqueous compartment by
    separating bilayer due to charge repulsion.

28
CHARACTERIZATION OF LIPOSOMES WITH THEIR QUALITY
CONTROL ASSAYSa) BIOLOGICAL CHARACTERIZATION
CHARACTERIZATION PARAMETERS INSTRUMENT FOR ANALYSIS
STERILITY AEROBIC AND ANEROBIC CULTURE
PYROGENICITY RABBIT FEVER RESPONSE
ANIMAL TOXICITY MONITORING SURVIVAL RATS
29
b) CHEMICAL CHARACTERIZATION
CHARACTERIZATION PARAMETERS INSTRUMENT FOR ANALYSIS
PHOSPHOLIPID CONCENTRATION HPLC/BARRLET ASSAY
CHOLESTEROL CONCENTRATION HPLC/CHOLESTEROL OXIDE ASSAY
DRUG CONCENTRATION ASSAY METHOD
ANTI-OXIDANT DEGRADATION HPLC/TLC
PH PH METER
OSMOLARITY OSMOMETER
PHOSPHOLIPIDS HYDROLYSIS HPLC/TLC
CHOLESTEROL OXIDATION HPLC/TLC
PHOSPHOLIPIDS OXIDATION UV ABSORBENCE
30
c) PHYSICAL CHARACTERIZATION
CHARACTERIZATION PARAMETER INSTRUMENT FOR ANALYSIS
VESICLE SHAPE SURFACE MORPHOLOGY TEM SEM
VESICLE SIZE AND SIZE DISTRIBUTION DYNAMIC LIGHT SCATTERING TEM
SURFACE CHARGE FREE FLOW ELECTROPHORESIS
ELECTRICAL SURFACE POTENTIAL SURFACE PH ZETA POTENTIAL MEASUREMENT AND PH SENSITIVE PROBES
LAMELLARITY P32 NMR
PERCENT CAPTURE MINI COLUMN CENTRIFUGATION GEL EXCLUSION
DRUG RELEASE DIFFUSE CELL / DIALYSIS
31
APPLICATIONS OF LIPOSOMES
  • LIPOSOMES IN RESPIRATORY DRUG DELIVERY SYSTEM
  • ISONIAZID RIFAMPICIN
  • - improved the effect of drugs for
    tuberculosis.
  • CYCLOSPORINS
  • - preferentially absorbed by lungs
    show sustained release
  • LIPOSOMES AS VACCINE ADJUVANTS
  • RABIES GLYCOPROTEINS
  • - interleukin 2 enhancement
  • CHOLERA TOXIN
  • - enhanced antibody level

32
LIPOSOMES FOR BRAIN TARGETING
  • Addition of sulphatide group to liposome
    composition increases their ability to cross
    blood-brain barrier.
  • Liposomes coated with mannose was found to reach
    brain tissue easily.
  • LIPOSOMES AS ANTI-INFECTIVE AGENTS
  • Active targeting approach
  • - pentamidin and anamycin for leishmaniasis
  • Passive targeting approach
  • - gentamycin for staphylococcal pneumoniasis

33
LIPOSOMES IN TUMOUR THERAPY
  • DOXARUBICIN
  • - For the treatment of refractory tumour
    and breast cancer.
  • VINCRISTINE
  • - for the treatment of solid tumour.

34
Conclusion
  • During the last years, liposomes as
    pharmaceutical drug carriers have received a lot
    of attention.
  • The new developments in liposomes is a continuous
    process which helps for better targeting with
    less toxicity.
  • Some commercially available liposomes in market
    include MIKASOME, DAUNAXOME, VERTEPORFIN.

35
REFERENCES
  • Wikipedia.org
  • Island net.com
  • Bentham science publishers ltd.
  • Journals
  • Allen, Theresa M. "Liposomal Drug Formulations
    Rationale for Development and What We Can
  • Expect for the Future." Drugs 56 747-756,
    1998.
  • Janknegt, Robert. "Liposomal and Lipid
    Formulations of Amphotericin B." Clinical
    Pharmacokinetics.
  • Kim, Anna et al. "Pharmacodynamics of insulin in
    polyethylene glycol-coated liposomes."
  • International Journal of Pharmaceutics. 180
    75-81, 1999.
  • 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.
    "Liposomesquo vadi?" PSTT 1 19-31, 1998.

36
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