ClassificatioreliesNo. of bilayer formedand diameter of resultant vesicle
Virus liposome fusion virus name sendai is incubate tiliposme at 37 c is non leaky fusion
LIPOSOME are formed when phospholipid are hydrated hydrrphillic materials are entrapped using aqueous solution of these material as hydrating fluid ,the lipophilic material are solubilized in organic solution of constitutive lipid sol and evaporate to dryness and is followed by hydration.
Under this category a lipid solution inorganic solvent and end up with lipid dispersion in water. -Hand shaking MLV non hand shaking -large ULV
Large amount of wate r soluble compound are wasted during swelling 0nly 10-15% get entrapped where as lipid soluble compound are 100%entrapped.
Lipid introduce in fludizer as dispersion of large MLVs or as slurry of unhydrated lipid in an organic medium
In this method lipid are first dissolve in organic solution which I then brought into contact with aq phase containing material to be entrapped in the liposomes
Third principle of passive loading
Laser light – simple rapid but having disadvantage of measuring average property of bulk analysis of liposomeGel permeability—expensive buffer and gel use for size range
. In liposomes, the active principles are water soluble and are hosted in the inner cavity, with little, if any, interaction taking place between the hydrophilic principle and the surrounding lipid core. In, Phytosome®s host their polyphenolic guest, generally little soluble both in water and in lipids, at their surface (Figure 1), where the polar functionalities of the lipophilic guest interact via hydrogen bonds and polar interactions with the charged phosphate head of phospholipids, forming a unique arrangement that can be evidenced by spectroscopy
1st year M. Pharrm
Under the guidance of
Prof. SATEESHA S.B
Liposomes were first produced in
England in 1961 by Alec D.
Liposomes are simple microscopic
vesicles in which an aqueous volume is
entirely enclosed by a membrane
composed of lipid molecule.
Structurally, liposomes are concentric
bilayered vesicles in which an aqueous
volume is entirely enclosed by a
membranous lipid bilayers mainly
composed of natural or synthetic
PHOSPHOLIPID- Major component of biological cell membrane
2 fatty acid
0-6 double bond
in each chain
head or polar
bound to water
The most common phospholipid use is phosphatidylcholine PC
A pair of
Molecules of PC are not soluble in water
In aqueous medium they align themselves
closely in planar bilayer sheet to
minimize the unfavorable action
between the bulk aqueous phase and
longer hydrocarbon fatty acid chain i.e.
they orient themselves such that fatty
acid chain face each other and the polar
head face the aqueous phase
This reduces the instability and close seal
vesicle is formed
The rationale of encapsulating a drug within
liposomes is to prevent its rapid metabolism and
its rapid removal from blood circulation after its
administration so that the drugs from depot
liposomes are ideally suited for drug delivery.
Provide selective passive targeting to tumor tissues
Increased efficacy and therapeutic index.
Increased stability via encapsulation.
Reduction in toxicity of the encapsulated agent.
Improved pharmacokinetic effects (reduced elimination,
increased circulation life times).
Flexibility to couple with site-specific ligand to achieve
Incorporation of sterols in liposome
bilayer can bring about major changes
in preparation of these membranes.
Cholesterol by itself does not form a
Concentration upto 1:1 or even 2:1
molar ratios of PC.
Cholesterol incorporation increases the
separation between the choline head
groups and eliminates the normal
electrostatic and hydrogen-bonding
are flat plate like structure that
appear during the formation of liposome
Phospholipid bilayer appear as lamella before
getting converted to sphere
lamella stack one upon other during
formation of liposome to form a
Uni lamellar vesicle
Single phospholipid bilayer
Multi lamellar vesicle
Several phospholipid bilayer
Based on structural parameter
vesicles (all size
Small Unilamellar vesicles 20-100 nm
Medium sized Unilamellar vesicles
Large Unilamellar vesicles >100 nm
Giant Unilamellarvesicles >1 µm
REV-reverse-phase evaporation method
Single or oligolamellar vesicles made
Multilamellar vesicles made by reverse-phase
Stable plurilamellar vesicles
On the basis of
Frozen and thawed MLV
VETVesicles prepared by extrusion technique
DRV-dried reconstituted vesicle
BASED UPON COMPOSITION AND APPLICATION
Neutral or negatively charged phospholipid
RSVE-Reconstituted sendai virus envelop
Phospholipid such as PE or DOPE with either
Cationic ion with DOPE
high temp made with cholesterol and 5-10%
CL or LCL with attached monoclonal
antibody or recognition sequence
METHODS OF LIPOSOME PREPARATIONS
Passive loading technique
Involves loading of entrapped agent
before or during manufacturing
Active loading technique
Certain type of compound with ionizable
group and those with both lipid and
water solubility can be introduce in
liposome after formation of vesicle
•Lipid film hydration
•French pressure cell
•Freeze thawed liposomes
Detergent removal method
•Detergent - like cholate
alkyl glycoside triton x 100
removal from mixed micelle
•Reconstituted sendai virus
Handshaking and non shaking
In this method a 250 ml round bottom flask is
taken containing organic solvent with lipids.
Then this beaker is attached to a rotary
evaporator and rotated at 60 rpm resulting in
formation of stacks of lipids.
Then the beaker containing stacks is dried using
nitrogen for 15 min and then the casted film is
dispersed in aqueous medium.
This results in hydration of lipids which swell and
peel of from the wall of flask resulting in
formation of multilamellar vesicles.
is used to prepare small MLVs.
In this a lipid dispersion is placed in a
microfluidizer pump which pumps the fluid
at 600-700 bar pressure through a 5 µm
Then this dispersion is forced along micro
channels, which make two streams of fluid to
collide with each other at right angles at a
Due to this transfer of energy takes place
resulting in formation of multilamellar
this method MLVs are exposed to UV
radiations to get small vesicles.
There are two methods of sonication
1. bath sonicator
2. probe sonicator
Probe is used for high concentrated lipids
while bath is used for large volumes of
In probe a high energy is used which may
result lipid degradation and also titanium
particles may be released into dispersion.
these reasons bath sonicators are used for
preparing MLVs. In this method dispersion is
placed in a test tube which is placed In a
Sonication is done for 5-10 min until a
transparent solution appears.
After sonication dispersion is placed in a
plastic centrifugation tube and centrifuged for
30 min at 20º c to get large MLVs and 3-4 hrs to
Method used to increase the surface area of dry
lipid film and to facilitate instantaneous
hydration, keeping low aqueous volume
Lipid is dried over a finely divided solid support
such as powdered sodium chloride or sorbitol or
other polysaccharides to form pro-liposomes
These dried lipid coated particulates swell upon
adding water to the support rapidly dissolves to
give a suspension of MLVs.
This method overcome the problem encountered
during lipid storage.
For preparing proliposomes Buchi rotary
evaporator is employed.
this method an ethanol solution of lipids is
injected rapidly into an excess of saline or
other aqueous medium, through a fine
The force of the injection is sufficient to
achieve complete mixing so that ethanol is
diluted instantaneously in water and
phospholipid molecules are dispersed evenly
This procedure yields high proportion of SUVs
is a simple method with low risk of
degradation of sensitive lipids
A major limitation is the solubility of lipids in
is a similar method as ethanol injection
but contrasts in some respects.
This involves injecting the immiscible organic
solution very slowly into an aqueous phase
through a narrow needle at the temperature
of vaporizing the organic solvent.
This method is used to treat sensitive lipids
very gently. Disadvantage is the long time
taken to produce a batch of liposomes.
In this method an organic solution containing
water droplets is introduced into excess aqueous
medium followed by mechanical dispersion.
By this a multi-compartment vesicle is formed
described as w/o/w system or double emulsion.
These vesicles with aqueous core are suspended in
a aqueous medium
The two compartments being separated by pair of
Organic solvent is evaporated using strong jet of
nitrogen into double emulsion.
ULV is formed
In this method phospholipids are brought into intimate
contact with the aqueous phase using detergents which
associate with phospholipid molecule and screen the
hydrophobic portions of the molecule from water.
The structure formed as a result is known as micelles.
The shape and size of the micelle depend upon chemical
nature of detergent concentration and other lipid
The concentration of detergent in water at which micelles
just start to form is known as critical micellar
Before CMC formation detergent exist in free solution.
At higher CMC concentration large amount micelle is
formed and concentration detergent in free form same as
Mixed micelle-two or more detergent
In contrast to phospholipids detergents are
highly soluble in both aqueous and organic
Equilibrium is indicated by critical micelle
lowering the concentration of detergent in the
bulk aqueous phase, the molecules of detergent
can be removed from mixed micelle by dialysis.
High CMC indicate equilibrium shifted to bulk
removal by dialysis easy
Commonly used detergents are sodium cholate
and sodium deoxycholate.
Commercial version of dialysis system is LIPOREP.
in the form of either sonicated
vesicles or as a dry film, at a molar ratio 2:1
with deoxy cholate form ULV of 100 nm on
removal of deoxy cholate by Column
This can be achieved by passing the
dispersion over a Sephadex G-25 column
presaturated with lipids and pre equilibrated
eith hydrating buffer.
size and its distribution
Percentage drug entrapment
Phase behavior of liposome
Percentage drug release
1. Size and its distribution-
Laser light scattering
Microscopic method- electron microscopy
Most precise method since it allow to view individual liposome and
obtain information about profile of liposome population over whole
range of size
size Freeze etch-is particularly used to measure small vesicle diameters
freeze fracture – a method of preparing cells for electron microscopical
a tissue specimen is frozen at −150° C,
inserted into a vacuum chamber, and fractured by a microtome, a
platinum carbon replica of the exposed surfaces is made, freed of
the underlying specimen
2. Surface charge--Electrophoresis
Lipid samples are applied to cellulose acetate plate in a
sodium borate buffer pH 8.8
electrophoresis is carried at 4ºC on a flat bed apparatus for 30
plate is dried and phospholipids are visualized by molybdenum
3. Percent entrapment
Two methods are used for this
1. protamine aggregation- (+,-)
In protamine aggregation liposome suspension 20 mg/ml in saline is
placed in conical glass centrifuge tube,
0.1 ml protamine solution is added and allowed to stand for 3 min
30 ml saline is added and then tube is spun for 20 min.
supernatant is removed and assayed for unentrapped compound by
The suspended pellets are resuspended in 0.6 ml of 10% triton X -100
and material completely dissolve.
Volume is made up and assay is done
2. mini column-
Hydrated gel filled in barrel of syringe plunge with whatman GF/B
Spun in centrifuge tube at 2000 rpm for 3 min to remove excess
Gel column is dried
Elute solution remove from collection tube
Liposome suspension is added drop wise to gel bed again spun at
2000 rpm for 3 min to remove the void volume of liposome
• The entrapped volume of liposome can be obtain by
measurement of total quantity of solute entrap in the liposome
• assuring that concentration of solute in aqueous medium
inside liposome is same as in solution that is use in
• assuming that no solute has leak out after separation from
• Invalid in two-phase method
• Measure by NMR- adding spectroscopically inert substance and
measure water signal.
Average number of bilayers present are found by freeze electron
microscopy and by 31P-NMR.
• In NMR technique broadening agent manganese ions are added it
before and after recording, it interact with outer leaflet of
bilayer. 50% reduction in NMR signal means it is unilamellar
liposome and 25 % reduction indicates presence of 2 bilayers in the
• Freeze fracture electron microscopy is nowadays very popular for
study of structural detail of aqueous lipid dispersion.
6. Phase behavior of liposomes
• An important feature of lipid membrane is the existence of a
temperature dependant, reversible phase transition, where the
hydrocarbon chain of the phospholipids undergoes a transformation
from a ordered state to more disordered fluid state.
• These changes have been documented by freeze fracturing electron
microscopy but most conveniently demonstrated by DSC.
• The physical state of the bi-layer profoundly affects permeability,
phase transition temperature (Tc), leakage rates and overall stability
• Tc gives good clue regarding liposome stability,permeability,and drug
7. Drug release:
• The mechanism of drug release from liposome can be accessed by the
use of a well calibrated in-vitro diffusion cell.
• In vitro assay of liposomal formulation is assisted to predict
pharmacokinetics and bioavailability before costly in vivo studies.
• Dilution induced drug release in buffer and plasma is employed as
predictor for pharmacokinetics of liposome
• Intra cellular drug release can also be induced by liposome
degradation in the presence of mouse-liver lysosome lysate to
determine the bioavailability of drug
use in preparation of liposome are
unsaturated and highly prone to oxidation
Volatile solvent such as chloroform which
are very susceptible to evaporate from
So liposome should be store in inert
atmosphere of nitrogen and in dark, glass
vessel with securely fastened cap
Liposomes are successfully used to entrap
anticancer drugs. This increases circulation
life time, protects from metabolic
2.Liposomes as carrier of drug in oral
Steroids used for arthritis can be incorporated
into large MLVs.
Alteration in blood glucose levels in diabetic
animals was obtained by oral administration of
liposome encapsulated insulin.
3. Liposomes for topical applications:
Drugs like triamcilone, methotrexate,
benzocaine, corticosteroids etc can be
successfully incorporated as topical liposomes
4. Liposomes for pulmonary delivery:
Inhalational devices like nebulizers are use to
produce an aerosol of droplets containing
Drugs like idoxuridine, indoxol and carbochol
are greater efficacy in the form of liposomes.
Potential advantage of ophthalmic liposome is
their intimate contact with corneal and
6. Leishmaniasis :
In this parasitic disease antimonial drugs are
used which are lethal at high concentrations as
they damage heart, liver and kidney.
Such drugs can be encapsulated in liposomes.
7. Cell biological applications:
Liposomes are used to carry functional DNA
and RNA molecules into cells. Liposomes are
used to insert enzymatic cofactors and cyclic
AMP into cells.
Type of Agents
Duanorubicin, Doxorubicin*, Epirubicin
Methotrexate, Cisplatin*, Cytarabin
Triclosan, Clindamycin hydrochloride,
Ampicillin, peperacillin, rifamicin
cDNA - CFTR*
Malaria merozoite, Malaria sporozoite
Hepatitis B antigen, Rabies virus glycoprotein
*Currently in Clinical Trials or Approved for Clinical Use
Phytosome is novel drug delivery system is a
patented technology (U.S. Patent #4,764,508) that
combines hydrophilic bioactive phytoconstituents
of herbs/ herbal extracts and bound by
phospholipids.(soybean phospholipids ,lecithin)
More bioavailable than a simple/convential herbal
extract due to its enhanced capacity to cross the lipidrich biomembranes and reach circulation.
As they are better absorbed and produces better
Applied to standardized plant extracts, watersoluble phytoconstituents and many popular herbal
extracts including , grape seed, hawthorn, olive fruits
and leaves, milk thistle, green tea, ginseng etc into
phospholipids to produce lipid compatible molecular
Phytosome structures contain the active ingredients of
the herb surrounded by the phospholipids.
The presence of a surfactant i.e. the phospholipids in
the molecule these are shielded from water-triggered
degradation while, at the same time, allows obtaining
a higher adhesion of the product itself to the surface it
comes into contact with and a better interaction of
various molecules with cell structure
Example-PC is a bifunctional compound. Specifically
the choline head (hydrophilic) binds to these
compounds while the phosphatidyl portion (lipophilic)
comprising the body and tail which then envelopes the
choline bound material and forms phyto-phospholipid
Molecules are anchored through chemical bonds to the
polar choline head of the PC, it can be demonstrated
by specific spectroscopic techniques.
In phytosomes active
anchored through chemical
bonds to the polar head of the
In liposomes, the active
principle is dissolved in the
medium of the cavity or in
the layers of the
membrane. No chemical
bonds are formed.
In phytosomes, PC and the
individual plant compound
form a 1:1or 2:1 complex
depending on the substance.
In liposoes, hundred and
thousands of phosphatidyl
choline molecules surround
the water soluble molecule
Marked enhancement of bioavailability
valuable components of the herbal extracts are
protected from destruction by digestive
secretions and gut bacteria
Assured delivery to the tissues.
No compromise of nutrient safety.
Dose requirement is reduced due to absorption
of chief constituent.
Phytosomes shows better stability profile because
chemical bonds are formed between phospholipid
molecules and phytoconstituent
Phospholipid used in the phytosome process
besides acting as a carrier also nourishes the
skin, because it is essential part of cell
are prepared by reacting natural
or synthetic phospholipids with active
components like bioflavonoid, flavolignan
and polyphenolic constituents.
Solvent Evaporation method is the most
common technique used for the preparation
The behavior of phytosomes in both physical
and biological system is governed by the
factors such as
Percent entrapped solute
Chemical composition as well as quality and
purity of the starting material
They are lipophilic substances with a definite
melting point, freely soluble in non polar and
aprotic solvents in which the hydrophilic
moiety is not.
They are moderately soluble in fats and
insoluble in water.
When treated with water, they assume a
micelle shape, forming structures which
In these complexes, the polar head of the
phospholipidis involved while the fatty acid
moieties retain a high degree of mobility
conferring marked lipophilia at the new
In the 1H-NMR spectrum, the signals of the
complexes substances undergo a strong
In the13 C-NMR spectrum, the signals of the
complex substances as well as those of the
choline and glycerin portion of the phospholipid
can no more be recorded .
The phosphorous nucleus itself undergoes a band
broadening which indicates that it is involve in
The kind of signals proves the interaction
between polar head and active sites of the
complex whereas the lipid chains are not
involved since they are free to rotate and give
complex its lipophilic character.
Various spectroscopic and in-vitro and in-vivo evaluations
are applied on phytosomes on the basis of therapeutic
activity of biologically active phytoconstituents present in
These complexes can be characterized by TEM(Transmission
Electron Microscopy), 1H-NMR,13-CNMR,31 P-NMR FT-IR.
A chemical spectral characteristic is determined in
phospholipids complexes using IR and UV spectroscopic
Liquid chromatography/atmospheric pressure chemical
ionization mass spectrometry proved to be a very powerful
tool for pharmacokinetic studies of phytochemicals
In-vivo studies are performed on Beagle dogs, rodents,
wistar rats to compare pharmacokinetics parameters
between pure extracts and its phospholipid