Phytosome

LIPOSOME AND PHYTOSOMENOVEL TARGETED DRUG

DELIVERY SYSTEM

Ruchi Shakya

DEBAJYOTI BHATTACHARYA

1st year M. Pharrm

Industrial pharmacy

Under the guidance of Prof. SATEESHA S.B

2

LIPOSOMES Liposomes were first produced in

England in 1961 by Alec D. Bangham.

Definition: Liposomes are simple microscopic

vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule.

Structurally, liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayers mainly composed of natural or synthetic phospholipids.

Hydrophilic

Hydrohobic

phospholipid

Hydrophobic tail

2 fatty acid chain

containing 10-20 carbon

atoms

0-6 double bond in each

chain

Hydrophillic head or polar

head

Phosphoric acid bound to water soluble

molecule

PHOSPHOLIPID- Major component of biological cell membrane

COMMONLY USED OTHER PHOSPHOLIPIDS

Natural phospholipid PC- phosphatidyl choline PE- phosphatidyl ethanolamine PS –phosphatidyl serineSynthetic phosholipid DOPC = Dioleoyl Phosphatidylcholine DOPE = Dioleoyl phosphatidyl ethanolamine DSPC = Distearoyl phosphatidyl choline DSPE = Distearoyl phosphatidyl

ethanolamine DLPC = Dilauryl phosphatidyl choline

PHOSPHATIDYLCHOLINE PC

Phosphatidylcholine is amphipathic molecule containing

A hydrophillic polar head group phosphocholine

A glycerol bridge A pair of hydrophobic acyl hydrocarbon chain

The most common phospholipid use is phosphatidylcholine PC

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

RATIONALE 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.

Advantages Provide selective passive targeting to tumor

tissues (liposomal doxorubicin). Increased efficacy and therapeutic index. Increased stability via encapsulation. Reduction in toxicity of the encapsulated agent. Improved pharmacokinetic effects (reduced

elimination, increased circulation life times).

Flexibility to couple with site-specific ligand to achieve active targeting

CHOLESTEROL

Incorporation of sterols in liposome bilayer can bring about major changes in preparation of these membranes.

Cholesterol by itself does not form a bilayer structure

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 interactions.

MICROSCOPIC VIEW OF LIPOSOME

WHAT IS LAMELLA? Lamella are flat plate like structure that

appear during the formation of liposome Phospholipid bilayer appear as lamella

before getting converted to sphere

Several lamella stack one upon other during formation of liposome to form a multilamellar structure

Multi lamellar vesicleSeveral phospholipid bilayer

Uni lamellar vesicleSingle phospholipid bilayer

CLASSIFICATION OF LIPOSOMESBased on structural parameter

MVMulti

vesicular vesicle>1µm

GUVGiant Unilamellarvesicles >1 µm

LUV Large Unilamellar vesicles >100 nm

MUV Medium sized Unilamellar

vesicles

SUVSmall Unilamellar vesicles 20-100 nm

UVUnilamellar

vesicles (all size range)

OLOOligolamellar

vesicle 0.1-1 µm

MLVMultilamellar

large vesicles >0.5

nm

REV-reverse-phase evaporation method

Single or oligolamellar vesicles made

MLV-REVMultilamellar vesicles made by reverse-phase evaporation method

SPLVStable plurilamellar vesicles

FATMLV Frozen and thawed MLV

VET-Vesicles prepared by extrusion technique

DRV-dried reconstituted vesicleDehydration-rehydration method

On the basis of preparation

BASED UPON COMPOSITION AND APPLICATION

CLConventional

liposme

Neutral or negatively charged phospholipid

Fusogenic liposome

pH sensitive liposome

Cationic liposome

Immuno liposome

RSVE-Reconstituted sendai virus envelop

Phospholipid such as PE or DOPE with either CHEMS

high temp made with cholesterol and 5-10% of PEG-DSPE

CL or LCL with attached monoclonal antibody or recognition sequence

LCLLong circularly (stealth)liposom

e

Cationic ion with DOPE

METHODS OF LIPOSOME PREPARATIONS

Passive loading techniqueInvolves loading of entrapped agent before or during manufacturing process

Active loading techniqueCertain type of compound with ionizable group and those with both lipid and water solubility can be introduce in liposome after formation of vesicle

Mechanical dispersion method

Solvent dispersion method

Detergent removal method

•Lipid film hydration•Micro emulsification•Sonication•French pressure cell•Membrane extrution•Dried reconstituted vesicle•Freeze thawed liposomes

•Ethanol injection•Ether injection•Double emulsion vesicles•Reverse phase evaporation vesicle•Stable plurilamellar vesicle

•

•Detergent - like cholate alkyl glycoside triton x 100 removal from mixed micelle by•column chromatography•Dialysis •Dilution •Reconstituted sendai virus envelop

MECHANICAL DISPERSION METHOD-1.THIN FILM HYDRATION

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.

2. MICRO EMULSIFICATION LIPOSOME Microfluidizer 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 orifice.

Then this dispersion is forced along micro channels, which make two streams of fluid to collide with each other at right angles at a high velocity.

Due to this transfer of energy takes place resulting in formation of multilamellar vesicles.

Collision at right angle

Vesicles of required dimension

Reservoir of MLVs

air in air out

Filter5

Separation into two streams

Interaction chamber

3. SONICATED UNILAMELLAR VESICLES

In 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 diluted lipids.

In probe a high energy is used which may result lipid degradation and also titanium particles may be released into dispersion.

For these reasons bath sonicators are used for preparing MLVs. In this method dispersion is placed in a test tube which is placed In a sonicator

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 get SUVs.

PRO-LIPOSOMES 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.

SOLVENT DISPERSION METHOD-1.ETHANOL INJECTION In this method an ethanol solution of

lipids is injected rapidly into an excess of saline or other aqueous medium, through a fine needle.

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 in medium.

This procedure yields high proportion of SUVs (25 nm).

This is a simple method with low risk of degradation of sensitive lipids

A major limitation is the solubility of lipids in ethanol.

2. ETHER INJECTION This 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.

3. DOUBLE EMULSION VESICLES 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 phospholipid monolayer.

Organic solvent is evaporated using strong jet of nitrogen into double emulsion.

ULV is formed

DETERGENT SOLUBILIZATION 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 Concentration

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 in CMC

Mixed micelle-two or more detergent

1. DIALYSIS In contrast to phospholipids detergents are highly

soluble in both aqueous and organic media. Equilibrium is indicated by critical micelle

concentration 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 solution removal by dialysis easy

Commonly used detergents are sodium cholate and sodium deoxycholate.

Commercial version of dialysis system is LIPOREP.

2. COLUMN CHROMATOGRAPHY Phospholipids 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 chromatography.

This can be achieved by passing the dispersion over a Sephadex G-25 column presaturated with lipids and pre equilibrated eith hydrating buffer.

CHARACTERIZATION OF LIPOSOMES Shape, size and its distribution Surface charge Percentage drug entrapment Entrapped volume Lamellarity Phase behavior of liposome Percentage drug release

1. Size and its distribution-

Laser light scattering

Gel permeation

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 examination

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

then examined

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 min ,

plate is dried and phospholipids are visualized by molybdenum blue

reagent

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

standard ,method.

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 filter

paper

Spun in centrifuge tube at 2000 rpm for 3 min to remove excess saline

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

4. Entrapped volume-• 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 untrapped material

• Invalid in two-phase method• Measure by NMR- adding spectroscopically inert

substance and measure water signal.

5. .Lamellarity

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 liposomes

• 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 of liposomes.

• Tc gives good clue regarding liposome stability,permeability,and drug entrapped

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

.

HANDLING OF LIPOSOME Lipid use in preparation of liposome are

unsaturated and highly prone to oxidation

Volatile solvent such as chloroform which are very susceptible to evaporate from container.

So liposome should be store in inert atmosphere of nitrogen and in dark, glass vessel with securely fastened cap

APPLICATIONS

1.Cancer chemotherapy: Liposomes are successfully used to entrap

anticancer drugs. This increases circulation life time, protects from metabolic degradation.

2.Liposomes as carrier of drug in oral treatment:

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 liposomes.

5.Ophthalmic delivery: 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 conjuctival surfaces

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.

CURRENT LIPOSOMAL DRUG PREPARATIONS

Type of Agents ExamplesAnticancer Drugs

Anti bacterial

AntiviralDNA materialEnzymes

RadionuclideFungicidesVaccines

*Currently in Clinical Trials or Approved for Clinical Use

Malaria merozoite, Malaria sporozoiteHepatitis B antigen, Rabies virus glycoprotein

Amphotericin B*In-111*, Tc-99m

Hexosaminidase A Glucocerebrosidase, Peroxidase

Duanorubicin, Doxorubicin*, Epirubicin Methotrexate, Cisplatin*, CytarabinTriclosan, Clindamycin hydrochloride, Ampicillin, peperacillin, rifamicinAZTcDNA - CFTR*

PHYTOSOMES 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 lipid-rich biomembranes and reach circulation.

As they are better absorbed and produces better results

Applied to standardized plant extracts, water-soluble 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 complexes

STRUCTURE 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 complex.

Molecules are anchored through chemical bonds to the polar choline head of the PC, it can be demonstrated by specific spectroscopic techniques.

PHYTOSOMES VS LIPOSOMES

PHYTOSOMES LIPOSOMES

In phytosomes active chemical constituents molecules areanchored through chemical bonds to the polar head of the phospholipids.

In liposomes, the active principle is dissolved in the medium of the cavity or in the layers of themembrane. 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

BENEFITS OF PHYTOSOMES 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 membrane.

METHOD OF PREPARATION

Phytosomes 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 of phytosomes

CHARACTERIZATION OF PHYTOSOMES

The behavior of phytosomes in both physical and biological system is governed by the factors such as

Physical size Membrane permeability Percent entrapped solute Chemical composition as well as quality

and purity of the starting material

PHYSICAL PROPERTY 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 resemble liposome.

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 molecule.

CHEMICAL PROPERTIES In the 1H-NMR spectrum, the signals of the

complexes substances undergo a strong broadening .

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 complex formation.

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.

EVALUATION OF PHYTOSOMES 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 phytosomes

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 study.

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

HERBAL DRUG

PHYTOSOME

PHYTOCONSTITUENTS

INDICATION

Ginkgobiloba

GinkgoselectPhytosome

-Dimeric flavonoids-terpenoids(gikgolides andBilobalide

(a) Vasoactive agent(b)Anti-inflammatory agents

Silybummarianum

SilybinPhytosome

-Flavolignan Silybin-Flavanolignan(Silymarin

(a)Antioxidant andHepatoproptective(b)Anti-inflammatoryAnti-aging

Crataegusoxyacantha

HawthornePhytosomes

Flavonoids Antioxidant, cardioprotective,Food product

CamelliaSinensis

GreenselectPhytosome

Catechins and theirgallate derivatives.

Antioxidant,cardio protective,food product

PanaxGinseng

Ginselect TM

Saponins Anti-aging

VacciniumMyrtillus

Mirtoselect Phytosome

Antcinocide Antioxidant

Vitis vinifera Leucoselect Phytosome

Monomeric flavan-3-ols (catechins andepicatechins and their gallate derivatives)

Cardiovascular protectant,antiinflammatory antioxidant.

REFERENCE

Target and controlled drug delivery-novel carrier system by S.P Vyas, R.K Khar

Controlled and Novel Drug Deliver system, chapter 15,liposomes as a drug carrier by Sanjay k Jain and N.K. Jain