Liposomes

1

OUTLINE

• WHAT ARE LIPOSOMES?• BASIC LIPOSOME STRUCTURE.• WHY USE LIPOSOMES IN DRUG DELIVERY?• ADVANTAGES OF LIPOSOMES.• STRUCTURAL COMPONENTS OF 

LIPOSOMES.• CLASSIFICATION OF LIPOSOMES.• PREPARATION OF LIPOSOMES.

2

3

Liposomes are concentric bilayered vesicles Liposomes are concentric bilayered vesicles

in which an aqueous core is entirely enclosed in which an aqueous core is entirely enclosed

by a membranous lipid bilayer mainly by a membranous lipid bilayer mainly

composed of natural or synthetic composed of natural or synthetic

phospholipids.phospholipids.

Liposomes were first produced in England in Liposomes were first produced in England in

1961 by Alec D. Bangham, who was 1961 by Alec D. Bangham, who was

studying phospholipids and blood clotting.studying phospholipids and blood clotting.

The size of a liposome ranges from some The size of a liposome ranges from some

20 nm up to several micrometers.20 nm up to several micrometers.

Liposomes (= vesicles)Liposomes (= vesicles)

The lipid moecules are ususally phospholipids- amphipathic moieties with a

hydrophilic head group and two hydrophobic tails.

On addition of excess water, such lipidic moieties spontaneously originate to give

the most thermodynamically stable conformation.

In which polor head groups face outwords into the aqueous medium, and the lipidic

chains turns inwords to avoid the water phase, giving rise to double layer or bilayer

lamellar stractures.

Hydrophobic

Hydrophilic cavity

5

Basic liposome structure

6

What is a lamella?

– A Lamella is a flat plate like structure that appearsduring the formation of liposomes. The phospholipidsbilayer first exists as a lamella before getting converted into spheres.

– Several lamella of phospholipids bilayers are stacked one on top of the other during formation of liposomes  to form a multilamellar structure.

7

Multilamellar vesicles

Unilamellar vesicles

8

Structural Components of Liposomes

• THE MAIN COMPONENTS OF LIPOSOMES ARE :-

1. Phospholipids

2. Cholesterol

9

Phospholipids • Phospholipids are the major structural compone

nts of biological membranes such as  the cell membrane.

Phosphoglycerides

Two Types Of  Phospholipids(Along With Their Hydrolysis Products)

Two Types Of  Phospholipids(Along With Their Hydrolysis Products)

Sphingolipids

10

Phosphatidylcholine

• Most common phospholipids used is 

phosphatidylcholine (PC).

• Phosphatidylcholine is an amphipathic

molecule in which exists:-

– a hydrophilic polar head group, 

phosphocholine.

– a glycerol bridge.

– a pair of hydrophobic acyl hydrocarbon 

chains.

11

Generally phospholipids are  represented as follows:-

follows:

12

PhospholipidsPhospholipids

Polar Head Groups

Three carbon glycerol

13

14

ROLE OF CHOLESTEROL IN BILAYER FORMATIONROLE OF CHOLESTEROL IN BILAYER FORMATION

• Cholesterol by itself does not form bilayer structure.Cholesterol by itself does not form bilayer structure.

• Cholesterol act as fluidity bufferCholesterol act as fluidity buffer

• After intercalation with phospholipid molecules alter the freedom After intercalation with phospholipid molecules alter the freedom of motion of carbon molecules in the acyl chainof motion of carbon molecules in the acyl chain

• Restricts the transformations of Restricts the transformations of trans trans to to gauchegauche conformations conformations

• Cholesterol incorporation increases the separation between Cholesterol incorporation increases the separation between choline head group & eliminates normal electrostatic & hydrogen choline head group & eliminates normal electrostatic & hydrogen bonding interactionsbonding interactions

Some other commonly used phospholipids

Naturally occurring phospholipids:

– PC : Phosphatidylcholine

– PE : Phosphatidylethanolamine

– PS : Phosphatidylserine

Synthetic phospholipids:

– DOPC : Dioleoylphosphatidylcholine

– DSPC : Distearoylphosphatidylcholine

16

Why Use Liposomes in Drug Delivery?

Inactive: Unmodified liposomes gather in specific tissue reticuloendothelial system

Active: alter liposome surface with ligand (antibodies, enzymes, protein A, sugars)

Directly to site

Physical: temperature or pH sensitive liposomes

Drug Targeting

17

ProtectionDecrease harmful side effects

Pharmacokinetics - efficacy and toxicityChanges the absorbance and biodistribution

Change where drug accumulates in the body

Protects drug

Deliver drug in desired formMultidrug resistance

Why Use Liposomes in Drug Delivery?

18

ReleaseAffect the time in which the drug is released

Prolong time -increase duration of action and decrease administration

Dependent on drug and liposome properties

Liposome composition, pH and osmotic gradient, and environment

Why Use Liposomes in Drug Delivery?

19

ADVANTAGES OF LIPOSOMESADVANTAGES OF LIPOSOMES

• Provides selective passive targeting to tumor 

tissues.• Increased efficacy and therapeutic index.• Increased stability of encapsulated drug.• Reduction in toxicity of the encapsulated agent.• Site avoidance effect (avoids non-target tissues).• Improved pharmacokinetic effects (reduced 

elimination increased circulation life times).• Flexibility to couple with site specific ligands to

achieve active targetting.

20

DISADVANTAGESDISADVANTAGES PHYSICAL/ CHEMICAL STABILITYPHYSICAL/ CHEMICAL STABILITY

VERY HIGH PRODUTION COSTVERY HIGH PRODUTION COST

DRUG LEAKEGE/ ENTRAPMENT/ DRUG FUSIONDRUG LEAKEGE/ ENTRAPMENT/ DRUG FUSION

STERILIZATION STERILIZATION

SHORT BIOLOGICAL ACTIVITY / t SHORT BIOLOGICAL ACTIVITY / t ½½

OXIDATION OF BILAYER …LIPIDS AND LOW SOLUBILITYOXIDATION OF BILAYER …LIPIDS AND LOW SOLUBILITY

RATE OF RELEASE and ALTERED BIODISTRIBUTIONRATE OF RELEASE and ALTERED BIODISTRIBUTION

LOW THEARAPEUTIC INDEX and DOSE EFFECTIVENESSLOW THEARAPEUTIC INDEX and DOSE EFFECTIVENESS

OVERCOMING RESISTANCEOVERCOMING RESISTANCE

EXTENCIVE CLINICAL AND LABORATORY RESEARCH TO EXTENCIVE CLINICAL AND LABORATORY RESEARCH TO

ACERTAIN LONG CERCULATING LIPOSOMESACERTAIN LONG CERCULATING LIPOSOMES

REPEATED IV ADMINISTRATION PROBLEMESREPEATED IV ADMINISTRATION PROBLEMES

CLASSIFICATION OF LIPOSOMES

• Liposomes are classified on the basis of 

– Structural parameters

– Method of preparation

– Composition and applications

22

1. Based on structural parameters

MLVMultilamellar

Large vesicles

(>0.5 um)

OLV oligolamellar

vesicles(>0.1-1.0 um)

UV UnilamellarVesicles (all  size ranges)

MVVMultivesicular

vesicles(> 1.0 um)

MUV Medium Unilamellar Vesicles

GUV Giant Unilamellar Vesicles>1um

SUV Small Unilamellar Vesicles 20-100nm

LUV Large Unilamellar Vesicles>100nm

Based on structural parameters

23

24

25

3. Based on composition & application

1- Conventional.

2- Stealth.(PEG, increase blood circulation time and decrease phagocytic attack).

3- Cationic.(lipoplex)

4- Targeted.(antibody)

26

PREPARATION OF LIPOSOMES

Methods of liposome preparation

Passive loading:Involves loading of  the 

entrapped agents before or during the 

manufacturing procedure

Active or remote loading:certain types of compounds with

ionisable groups and those with both 

manufacturing procedurelipid and water solubility can beintroduced into the liposomes 

after the formation of the intact vesicles27

Methods  of liposome preparation

Solvent dispersion

  methods

Ethanol injectionEther injectionDouble emulsion  vesiclesStable plurilamellar VesiclesReverse phase  evaporation vesicles

Detergent removal methods

 

Passive loading techniques

Detergent(Cholate, Alkyl glycoside, Triton X-100) removal from mixed micelles byDialysisColumn chromatographyDilutionReconstituted sendai virus enveloped vesicles

Active loading techniques

Lipid film hydration by  hand shaking non-hand shaking and freeze dryingMicro emulsificationSonicationFrench pressure cellMembrane extrusionDried reconstituted  vesiclesFreeze thawed liposomes

Mechanical dispersion

methods 

28

SOLVANT SOLVANT eg.CHCleg.CHCl33

LIPID e.g.. LIPID e.g.. LICITHIN LICITHIN

DISPERSION DISPERSION FORMATIONNFORMATIONN

Encapsulated soluteEncapsulated solute

Separate liposome's from Separate liposome's from supernatatant by supernatatant by centrifugation, gel centrifugation, gel filtration/ sonication or filtration/ sonication or dialysis or by addition of dialysis or by addition of buffers + drug.buffers + drug.

FILM OF LIPID FILM OF LIPID

OCCURS AT SIDES OCCURS AT SIDES

OF VESSELSOF VESSELS

BASIC METHOD OF FORMULATION OF LIPOSOMESBASIC METHOD OF FORMULATION OF LIPOSOMES

MethodMethod VesiclesVesiclesMechanical methodsMechanical methods

Vortex or hand shaking of phospholipid dispersions MLVExtrusion through polycarbonate filters at low or medium pressure

OLV, LUV

Extrusion through a French press cell “Microfluidizer” technique

Mainly SUV

High-pressure homogenization Mainly SUVUltrasonic irritation SUV of minimal sizeBubbling of gas BSV

Methods based on replacement of organic solvent(s) by aqueous mediaMethods based on replacement of organic solvent(s) by aqueous mediaRemoval of organic solvent(s) MLV, OLV, SUV

Use of water-immiscible solvents: ether and petroleum MLV, OLV, LUV

Ethanol injection method LUVEther infusion (solvent vaporization) LUV, OLV, MLVReverse-phase evaporation

Methods based on detergent removalMethods based on detergent removalGel exclusion chromatography SUV“Slow” dialysis LUV, OLV, MLVFast dilution LUV, OLVOther related techniques MLV, OLV, LUV, SUV

PASSIVE LOADING TECHNIQUES PASSIVE LOADING TECHNIQUES • Mechanical Dispersion method• Solvent Dispersion method• Detergent Solubilisation method

MECHANICAL DISPERSION MECHANICAL DISPERSION METHODSMETHODS

LIPID HYDRATION METHODLIPID HYDRATION METHOD

The mechanical energy required for swelling of lipids and dispersion

of casted lipid film is imparted by manual agitation (hand shaking

technique)

The % encapsulation efficiency as high as 30% is achieved due to

loss of water soluble component during swelling and entrapped only

10-15%. On other hand lipid soluble drug encapsulated 100%

efficiency.

1. Hand‐shaken multilamellar vesicles

2. Non‐shaking vesicles

3. Pro‐liposomes

4. Freeze drying 

:–AFTER THESE METHODS, OTHER PROCESSING METHODS ARE USED TO MODIFY THESE TYPE OF VESICLES THAT ARE PRODUCED SUCH AS:

• Micro emulsification liposomes (MEL)• Sonicated unilamellar vesicles (SUVs)• French Pressure Cell Liposomes• Membrane extrusion liposomes• Dried‐reconstituted vesicles (DRVs)• Freeze thaw Sonication (FTS)• pH induced vesiculation• Calcium induced fusion

:–These methods are known as “the mechanical treatment of MLVs” or “Processing of lipids by physical means

34

Hand shaken multilamellar vesicles

• Simplest and most widely used method of physical dispersion• Basic method involves

– Dissolution of the lipid mixture and charge components in chloroform:methanol solvent

– Evaporation of the solvent in a rotary evaporator or by hand shaking to form a film shaking to form a

– Further drying of the film by attaching the flask to the manifold of the lyophilizer.

– Casted film is then dispersed in an aqueous medium.

– Upon hydration, lipid swell and peel off the wall of the flask and vesiculate forming multilamellar vesicles (MLVs) 

35

PROCESS IN MORE DETAIL –

STEP 1:

• Lipid mixture of different phospholipids and charge components in chloroform:methanol solvent mixture (2:1 v/v) is prepared first and then introduced into a round bottom flask with a ground glass neck.

• This flask is then attached to a rotary evaporator and rotated at 60 rpm.

• The organic solvents are evaporated at about 30 degrees Celsius or above the transition temperature of the lipids used.

• The rotation is continued for 15 mins after the dry residue first appears.

• The evaporator is isolated from the vacuum source by closing the tap.

• The nitrogen is introduced into the evaporator and the pressure at the cylinder head is gradually raised till there is no difference between inside and outside the flask between inside and outside the flask.

• The flask is then removed from the evaporator and fixed on to the manifold of lyophilizer to remove residual solvents. 36

STEP 2‐ Hydration of lipid layer:

• After releasing the vacuum and removal from the lyophilizer, the flask is flushed with nitrogen.

• 5ml of saline phosphate buffer (containing solute to be entrapped) is added.

• The flask is attached to the evaporator again (flushed with N2) and rotated at room temperature and pressure at the same speed or below 60 rpm.

• The flask is left rotating for 30 minutes or until all lipid has been removed from the wall of the flask and has given homogenous milky‐white suspension free of visible particles.

• The suspension is allowed to stand for a further 2 hours at room temperature or at a temperature above the transition temperature of the lipid in order to complete the swelling process to give MLVs.

37

Hand shaken method in general

 

38

Non‐shaking vesicles• The procedure differs from hand shaken method in that it uses a stream

of nitrogen to provide agitation rather than the rotationary movements.• Solution of lipid in chloroform:methanol mixture is spread over the flat

bottom conical flask.• The solution is evaporated at room temperature by flow of nitrogen

through the flask without disturbing the solution.• After drying, water saturated nitrogen is passed through the flask until

the opacity of the dried lipid film disappears (15-20mins).• After hydration, lipid is swelled by addition of bulk fluid. The flask is

inclined to one side, 10‐20 ml of 0.2M sucrose in distilled water (degassed) is introduced down the side of the flask, and the flask is slowly returned to upright orientation.

• The fluid is allowed to run gently over the lipid layer on the bottom of the flask.

• The flask is flushed with nitrogen sealed and allowed to stand for 2 hrs at 37 degrees Celsius. Take care not to disturb the flask in any way.

• After swelling, the vesicles are harvested by swirling the contents of the flask gently , to yield a milky‐suspension. 39

PROLIPOSOMESPROLIPOSOMES To increase the surface area of dried lipid film and to facilitate

continuous hydration and lipid is dried over the finally divided particulate support i.e.- NaCl, Sorbitol, or other polysaccharides. These dried lipid coated particulates are called as proliposomes

Proliposomes form dispersion of MLVs on addition of water, where support is rapidly dissolved and lipid film hydrate to form MLVs

Methods overcome the stability problem and entrapment efficiency doesn’t matter when formation of stable liposome.

Freeze drying • Another method of dispersing the lipid in a finely divided

form, prior to addition of aqueous media, is to freeze dry the lipid dissolved in a suitable organic solvent.

• The solvent choice depends on the freeze point which needs to above the temperature of the condenser lyophilizers. Tertiary butanol is considered to be most ideal solvent.

• After obtaining the dry lipid which is an expanded foam like structure, water or saline can be added with rapid mixing above the phase transition temperature to give MLVs.

41

SONICATION METHODSONICATION METHOD

PROBE SONICATORPROBE SONICATOR:: is employed for is employed for dispersions, which require high energy in a dispersions, which require high energy in a small volume (e.g., high concentration of small volume (e.g., high concentration of lipids, or a viscous aqueous phase) lipids, or a viscous aqueous phase) DisadvantageDisadvantage- lipid degradation due to high - lipid degradation due to high energy and sonication tips release titanium energy and sonication tips release titanium particles into liposome dispersionparticles into liposome dispersionBATH SONICATOR: BATH SONICATOR: The bath is more The bath is more suitable for large volumes of diluted lipids. suitable for large volumes of diluted lipids. Method: Method: Placing a test tube containing the Placing a test tube containing the dispersion in a bath sonicator and sonicating dispersion in a bath sonicator and sonicating for 5-10min(1,00,000g) which yield a for 5-10min(1,00,000g) which yield a slightly hazy transparent solution. Using slightly hazy transparent solution. Using centrifugation to yield a clear SUV centrifugation to yield a clear SUV dispersiondispersion

FRENCH PRESSURE CELL FRENCH PRESSURE CELL LIPOSOMESLIPOSOMES

This techniques yields rather “uni or oligo lamellar liposomes” of intermediate size of 30-80 nm in diameter depending on the applied pressure. Dispersion of MLVs can be converted to SUVs by passage through a small orifice under high pressure. MLV dispersion are placed in the French pressure cell and extruded at about 20,000psi at 450C By multiple extrusion i.e.., 4.5 passed about 95% of MLVs get converted into SUVs which can be determined by size exclusion chromatography.

MICRO EMULSIFICATION LIPOSOMES(MEL)MICRO EMULSIFICATION LIPOSOMES(MEL)

The fluid collected can be recycled through the pump and interaction chamber until vesicles of the spherical dimension are obtained.

After a single pass, the size of vesicles is reduced to a size 0.1 and 0.2um in diameter.

“Micro Fluidizer” is used to prepare small MLVs from Concentrated lipid dispersion

The lipids can introduced into fluidizers, either as a dispersion of large MLVs or as a slurry of unhydrated lipids in organic medium.

Microfluidizer pumps the fluid at very high pressure(10,000psi, 600-700 bar) through a 5um orifice.

Then it is forced along defined micro channels, which direct two streams of fluid to collide together at right angles at a very high velocity, thereby affecting an efficient transfer of energy.

VESICLES PREPARED BY VESICLES PREPARED BY EXTRUSION TECHNIQUES EXTRUSION TECHNIQUES (VETs)(VETs)

It is used to process LUVs as well as MLVs.

Liposomes prepared by this tech. are called as membrane filter extrusion liposomes

The 30% capture volume can be obtained using high lipid conc. The trapped volume in this process is 1-2 litre /mole of lipids

It is due to their ease of production, readily selectable vesicle diameter, batch to batch reproducibility & freedom from solvent or surfactant contamination is possible

FREEZE THAW SONICATION FREEZE THAW SONICATION METHOD (FTS)METHOD (FTS)

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

SOLVENT DISPERSION SOLVENT DISPERSION METHODSMETHODS

Note:- Organic solvent miscible with aqueous phaseNote:- Organic solvent miscible with aqueous phase

SOLVENT DISPERSION METHODS FOR SOLVENT DISPERSION METHODS FOR PASSIVE LOADINGPASSIVE LOADING

• An ethanol solution of lipids is injected rapidly through a fine needle into an excess of saline or other aq. medium

• This method has low risk of degradation of sensitive lipids• The vesicles of 100 nm size may be obtained by varying the conc. Of lipid in

ethanol or by changing the rate of injection of ethanol solution in preheated aqu. solution.

• Limitation-solubility of lipids in ethanol & vol. of ethanol that can be introduced into medium(7.5%v/v max)

• Difficulty to remove residual ethanol from phospholipid membrane

• Involves mixing of organic phase into aqu. Phase at the temp. of vaporizing the organic solvent

• It has low encapsulation efficiency

Ethanol injection:-

Ether injection:-

REVERSE PHASE EVAPORATION REVERSE PHASE EVAPORATION METHODMETHOD

DETERGENT SOLUBILISATIOIN DETERGENT SOLUBILISATIOIN METHODSMETHODS

Note:- Liposome size and shape depend on chemical nature of detergent, Note:- Liposome size and shape depend on chemical nature of detergent, concentration and other lipid involved concentration and other lipid involved

• The phospholipids are brought into intimate contact with the aqueous phase via detergents which associate with phospholipid molecules

• The structures formed are called as micelles• The conc. of detergent at which micelles are formed is

called as CMC• The detergent methods are not very efficient in %

entrapment values• The methods employed for removal of detergent

include dialysis, column chromatography & use of biobeads

Detergent depletion methodDetergent depletion method

54

DialysisDialysis MethodMethodDetergent commonly use for this purpose exhibit resonably high CMC (10 to 20 mM) so that their removal is facilitated A commercial version of the dialysis system is available under the tradename LIPOREP

Column Chromatography Column Chromatography Phospholipid inthe form of either sonicated vesicle or as a dry film, at a molar ratio of 2:1 with deoxycholate form unilamellar vesicles of 100nm on removal of deoxycholate by column chromatography

• Weak amphipathic bases accumulate in the aqueous phase of lipid Weak amphipathic bases accumulate in the aqueous phase of lipid vesicles in response to a difference in pH between the inside and vesicles in response to a difference in pH between the inside and outside of the liposomes (pHin & pHout)outside of the liposomes (pHin & pHout)

• Two steps process generates this pH imbalance and active Two steps process generates this pH imbalance and active (remote) loading.(remote) loading.

• Vesicles are prapared in low pH solution, thus generating low pH Vesicles are prapared in low pH solution, thus generating low pH within the liposomal interiors, followed by addition of the base to within the liposomal interiors, followed by addition of the base to extraliposomal medium.extraliposomal medium.

• Basic compounds, carrying amino groups are relatively lipophipic Basic compounds, carrying amino groups are relatively lipophipic at high pH and hydrophilic at low pH.at high pH and hydrophilic at low pH.

• In two chambered aqueous system separated by membrane In two chambered aqueous system separated by membrane liposomes, accumulation occurs at the low pH side, under liposomes, accumulation occurs at the low pH side, under dynamic equilibrium conditions.dynamic equilibrium conditions.

• Thus the unprotonated form of basic drug can diffuse through the Thus the unprotonated form of basic drug can diffuse through the bilayerbilayer

• The exchange of external medium by gel chromatography with The exchange of external medium by gel chromatography with neutral solutionneutral solution

• Weak base doxorubicine, adriamycin and vincristine which co-Weak base doxorubicine, adriamycin and vincristine which co-exist in aqueous solutions in neutral and charged forms have been exist in aqueous solutions in neutral and charged forms have been sucessfully loaded into preformed liposomes via the pH gradient sucessfully loaded into preformed liposomes via the pH gradient method.method.

ACTIVE LOADING TECHNIQUES ACTIVE LOADING TECHNIQUES AFTER DRYING IN PROCESS AFTER DRYING IN PROCESS

FILM/CAKE OF LIPID IS FROM FILM/CAKE OF LIPID IS FROM

STACKS OF LIPID STACKS OF LIPID BILAYER FORM BILAYER FORM

SWELLING IN FLUIDSWELLING IN FLUID

SHEET IS SELF CLOSE SHEET IS SELF CLOSE

LOADING OF DRUG LOADING OF DRUG ON pH- GRADIENT TECHNIQUEON pH- GRADIENT TECHNIQUE

FORMATION OF BILAYER FORMATION OF BILAYER (LIPOSOMES) IF DRUG (LIPOSOMES) IF DRUG

56

57

58

Reverse phase evaporation vesicles

At  this  stage-

the  monolayers come 

close to each other

In rotar y evaporator close to  each other

partial  bilayer

59

Ethanol/Ether injection method

60

DRYING

• An important step involved in the preparation of liposomes is the drying of the lipid.

• Large volume of organic solution of lipids is most

easily dried in a rotary evaporator fitted with a dried a rotary evaporator fitted with cooling coil and a thermostatically controlled water bath.

• Rapid evaporation of solvent is carried out by gentle warming (20‐40 degrees) under reduced pressure (400 700 mm Hg)(400‐700 mm Hg)

• Rapid rotation of the solvent containing flask increases the surface area for evaporation. 

61

• In cases where sufficient vacuum is not attainable or if the concentration of lipids is particularly high, it may be difficult to remove the last traces of chloroform from the lipid film.

Therefore, it is recommended as a matter of routine that after rotary evaporation, some further means is employed to bring the residue to complete dryness.

Attachment of the flask to the manifold of lyophilizer, and overnight exposure to high vacuum is a good method.

62

I] Physical Dispersion orMechanical Dispersion Methods

• Aqueous volume enclosed using this method is usually 5‐10%, which is very small proportion of the total volume used for swelling.

• Therefore large quantity of water‐soluble compounds are wasted during swelling.

• On the other hand, lipid soluble compounds can be encapsulated to 100% efficacy, provided they are not present in quantities that are greater than the structural component of the membrane.

63

How LUVs Are Generated From TheSuspension?

• The suspension is centrifuged at 12,000g for 10 min. in a bench centrifuge at room temperature.

• The layer of multilamellar vesicles floating on the surface is removed. To the remaining fluid an equal volume of iso‐osmolar glucose is added and centrifuged again at 12,000g. Large unilamellar vesicles form a soft pellet which can be resuspended in any required medium of appropriate osmolarity.

64

Pro‐Liposomes

• Method devised to increase the surface of dry lipid while keeping the low aqueous volume.

• In this method, the lipids are dried down to a finely divided particulate support, such as powdered sodium particulate support, such as powdered chloride, or sorbitol or other polysaccharide – to give pro‐liposomes.

• The lipids are swelled upon adding water to dried lipid coated powder (pro‐liposomes), where the support rapidly dissolves to give a suspension of MLVs in aqueous solution.

• The size of the carrier influences the size and heterogeneity of the liposomes.

65

• This method overcomes the problems encountered when storing liposomes themselves in either liquid, dry or frozen form, and is ideally suited for preparations where the material to be entrapped incorporates into lipid membrane.

• In cases where 100% entrapment of aqueous component is not essential this method is also of value .

• For preparing pro‐liposome a special equipment i.e. Buchi rotary evaporator ‘R’ with water cooled condenser coil and a stainless steel covered thermocouple connected to a digital thermometer is required.

• The end of the glass solvent inlet tube is modified to ato fine point, so that the solvent is introduced into the flask as a fine spray.

66

BUCHI Rotary Evaporator R type

67

Method Of Preparation of Proliposomes• The lipid solution in chloroform (60mg/ml) is prepared and sorbitol powder is

introduced into 100ml flask.

• The flask is then fitted into the evaporator and rotated slowly so that the powder tumbles gently off the walls to ensure good mixing and the solvent is evaporated.

• The flask is lowered into a water bath at 50‐55 degrees Celsius when a good vacuum is developed.

• An aliquot of 5ml of lipid solution is introduced into the flask via the solvent inlet tube.

• The solvent is absorbed completely by the powder and the temperature of the bed is monitored.

• As evaporation proceeds the temperature will decrease.

• A second aliquot is introduced slowly when the temperature begins to rise again.

• The temperature is allowed to rise to 30 degrees Celsius the vacuum is released and the drying process is completed by connecting the flask containing the powder to lyophilizer, and leaving it evacuated overnight at room temperature.

• The powder is transferred into a 10ml glass vial containing 600mg solid each (100mg lipid and 500mg sorbitol support) flushed with nitrogen, and sealed well and stored.

68

Processing of the lipids hydratedby physical means, or the mechanical

treatment of MLVs• Micro emulsification liposomes (MEL)

• Sonicated unilamellar vesicles (SUVs)

• French Pressure Cell Liposomes

• Membrane extrusion liposomes

• Dried‐reconstituted vesicles (DRVs)

• Freeze thaw Sonication (FTS)

• pH induced vesiculation

• Calcium induced fusion

69

Micro emulsification liposomes (MEL) 

70

Sonicated unilamellar vesicles (SUVs)  

71

French Pressure Cell Liposomes  

72

Membrane Extrusion Liposomes

73

Liposomes Help ImproveLiposomes Help Improve

Therapeutic indexRapid metabolismUnfavorable pharmokineticsLow solubilityLack of stabilityIrritation

74

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*, Cytarabin

Triclosan, Clindamycin hydrochloride, Ampicillin, peperacillin, rifamicin

AZTcDNA - CFTR*

75

CFTRGene Therapy

Deliver cDNA of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) to epithelial tissue of respiratory system

Fuse to cell membrane and incorporate cDNA into cell

Clinical trials - no significant change in symptoms

Now trying adeno associated virus

Cationic liposome

76

DoxilChemotherapy drug doxorubin

Anemia, damage to veins and tissue at injection, decrease platelet and WBC count, toxic to

Treats Kaposi’s sarcoma lesions or cancer tumorsModifications of liposome “stealth”

keeps doxorubin in blood for 50 hours instead of 20 minutes

concentrates at KS lesions and tumors

*Just approved by FDA* 77

Amphotericin BAmphotericin B

Side effects: nephrotoxicity, chills, and fevers

Systemic fungal infections in immune compromised patients

Fungizone - AmB with deoxycholate

AmB - kills ergosterol-containing fungal cells, also kills cholesterol-containing human cells

78

No decrease in effectiveness of drug against fungi

Liposomal Formulation of AmB

Decrease in toxicity

Exact Mechanism of liposomes not understood

Cholesterol - only few %moles

Phospholipid:AmB ratio

DiffusionLipid transfer

AmB

Lipid

79

Problems with Liposomal Preparations of DrugsProblems with Liposomal Preparations of Drugs

$$$$ Fungizone $40.58 Amphotec $2334Fungizone $40.58 Amphotec $2334

Doxil $1200 per treatment, twice the cost of normal protocol Doxil $1200 per treatment, twice the cost of normal protocol of chemotherapy and drugs of chemotherapy and drugs

Lack long term stability (short shelf lifeLack long term stability (short shelf life)

Freeze dry and pH adjustmentFreeze dry and pH adjustment Low “Pay Load” - poor encapsulaLow “Pay Load” - poor encapsulation

Physical and chemical instability Physical and chemical instability

Polar drugs and drugs without opposite chargePolar drugs and drugs without opposite charge

ModificationsModifications

Possibility of new side effects

Doxil “hand and foot syndrome”

80

Studies with insulin show that liposomes may be an effective way to package proteins and peptides for use

Clinical Trials for several liposomal formulations More studies on the manipulation of liposomes

Future

81

THANK YOU

82