LIPOSOMES-MICROPARTICULATE LIPOSOMES-MICROPARTICULATE DRUG CARRIERSDRUG CARRIERS

PART 1PART 1

DIGPATI ROY M Pharm.DIGPATI ROY M Pharm.Assistant ProfessorAssistant Professor

KVSR SIDDHARTHA COLLEGE OF KVSR SIDDHARTHA COLLEGE OF PHARMACEUTICALPHARMACEUTICAL

SCIENCES,VIJAYAWADA 520010 SCIENCES,VIJAYAWADA 520010

CONTENTSCONTENTS

• INTRODUCTIONINTRODUCTION

• PASSIVE LOADING TECHNIQUESPASSIVE LOADING TECHNIQUES

LIPOSOMESLIPOSOMES

• Concentric bilayered vesicles-an Concentric bilayered vesicles-an aqueous volume enclosed by a lipid aqueous volume enclosed by a lipid bilayer membranebilayer membrane

• The membrane composed of natural The membrane composed of natural or synthetic phospholipidsor synthetic phospholipids

• Versatile drug delivery and targeting Versatile drug delivery and targeting systemsystem

• Discovered by Dr Alec D Bangham in Discovered by Dr Alec D Bangham in 1961(Published in 1964)1961(Published in 1964)

BENEFITS OF LIPOSOMESBENEFITS OF LIPOSOMES

• Increased stability via encapsulationIncreased stability via encapsulation

• Reduction in toxicity of encapsulated agentReduction in toxicity of encapsulated agent

• Selective passive targeting to tumour tissuesSelective passive targeting to tumour tissues

• Active targeting by coupling with site specific Active targeting by coupling with site specific ligandsligands

• Site avoidance effectSite avoidance effect

• Improved pharmacokinetic effects-increased Improved pharmacokinetic effects-increased circulation, decreased eliminationcirculation, decreased elimination

• Increased efficacy and therapeutic indexIncreased efficacy and therapeutic index

MECHANISM OF LIPOSOME FORMATIONMECHANISM OF LIPOSOME FORMATION

• Phospholipids are amphipathic in nature.Phospholipids are amphipathic in nature.

• The polar part consists of phosphoric acid, The polar part consists of phosphoric acid, bound to a water soluble molecule.bound to a water soluble molecule.

• The non-polar part contains two fatty acid The non-polar part contains two fatty acid chains having 10-24 carbon atoms and 0-6 chains having 10-24 carbon atoms and 0-6 double bonds in each chain.double bonds in each chain.

• The polar lipids transform into stable colloidal The polar lipids transform into stable colloidal nanoconstructs referred to as liposomes,upon nanoconstructs referred to as liposomes,upon dilution with an excess of water .dilution with an excess of water .

TYPICAL PHOSPHOLIPID STRUCTURE

• Phosphatidylcholine is a natural phospholipid.Phosphatidylcholine is a natural phospholipid.• A glycerol bridge links to pair of hydrophobic A glycerol bridge links to pair of hydrophobic

acyl hydrocarbon chains with a hydrophilic acyl hydrocarbon chains with a hydrophilic polar head group,phosphocholine.polar head group,phosphocholine.

• In aqueous medium, the molecules align In aqueous medium, the molecules align themselves closely in planner bilayer sheets.themselves closely in planner bilayer sheets.

• These sheets fold over themselves to form These sheets fold over themselves to form closed, sealed and concentric vesicles.closed, sealed and concentric vesicles.

• This distinctive behavior derives in the lowest This distinctive behavior derives in the lowest free energy state.free energy state.

LIPOSOMES FROM PHOSPHATIDYLCHOLINE

PARAMETERS AFFECTING BILAYER PARAMETERS AFFECTING BILAYER FORMATIONFORMATION

• The large free energy difference between The large free energy difference between the aqueous and hydrophobic environment the aqueous and hydrophobic environment promotes the bilayer structures in order to promotes the bilayer structures in order to achieve the lowest free energy level.achieve the lowest free energy level.

• The driving force for bilayer configuration of The driving force for bilayer configuration of liposomes is the hydrophobic interaction liposomes is the hydrophobic interaction coupled with the amphiphilic nature of the coupled with the amphiphilic nature of the principal phospholipid molecules.principal phospholipid molecules.

• Supramolecular self-assemblages mediated Supramolecular self-assemblages mediated through specific molecular geometry. through specific molecular geometry.

PHASE TRANSITION OF LIPID BILAYERS INTO LIPOSOMESPHASE TRANSITION OF LIPID BILAYERS INTO LIPOSOMES

Homogeneous,parallel lipid bilayers,closely packed,more Homogeneous,parallel lipid bilayers,closely packed,more ordered, ordered,

lyotropic state(Gel phase)lyotropic state(Gel phase)

Phase transition temperature WaterPhase transition temperature Water

Heterogeneous,multilamellar /unilamellar liposomes,loosely Heterogeneous,multilamellar /unilamellar liposomes,loosely packed,packed,

less ordered,liquid crystal state(Fluid phase)less ordered,liquid crystal state(Fluid phase)

RIGIDIZATION OF FLUID PHASE VESICLES WITH RIGIDIZATION OF FLUID PHASE VESICLES WITH CHOLESTEROLCHOLESTEROL

CHOLESTEROLCHOLESTEROL

• Inserts into the bilayer membrane formed by Inserts into the bilayer membrane formed by the phospholipid in very high concentrations.the phospholipid in very high concentrations.

• Orients its hydroxyl group towards the Orients its hydroxyl group towards the aqueous surface and the aliphatic chain aqueous surface and the aliphatic chain aligned parallel to the acyl chains in the aligned parallel to the acyl chains in the center of the bilayer membrane.center of the bilayer membrane.

• Tends to condense and rigidize the membrane Tends to condense and rigidize the membrane by suppressing the tilt of the acyl chains by suppressing the tilt of the acyl chains

ROLE OF CHOLESTEROL IN BILAYER ROLE OF CHOLESTEROL IN BILAYER FORMATIONFORMATION

• Acts as a fluidity buffer.Acts as a fluidity buffer.

• After intercalation with phospholipid After intercalation with phospholipid molecules alters the freedom of molecules alters the freedom of motion of carbon molecules in the motion of carbon molecules in the acyl chain.acyl chain.

• Restricts the transformations of Restricts the transformations of trans- to gauche- conformations.trans- to gauche- conformations.

CHOLESTEROL ORIENTATION IN PHOSPHOLIPID BILAYERS

MOLECULAR GEOMETRY & LIPOSOME FORMATIONMOLECULAR GEOMETRY & LIPOSOME FORMATION

(Israelachvili Hypothesis)(Israelachvili Hypothesis)

• Micelle-forming amphiphilesMicelle-forming amphiphiles

(e.g. surfactants)(e.g. surfactants)

-CMC about 10-CMC about 10-3-3 mol L mol L-1-1

-High solubility in water-High solubility in water

• Membrane-forming amphiphilesMembrane-forming amphiphiles

(e.g.phospholipids)(e.g.phospholipids)

-CMC about 10-CMC about 10-8-8 mol L mol L-1-1

-Less solubility in water -Less solubility in water

CRITICAL PACKING CRITICAL PACKING PARAMETER(CPP)PARAMETER(CPP)

CPP=v /Ic Ap=Ahp/ApCPP=v /Ic Ap=Ahp/ApWhere Where v=hydrophobic group volume,v=hydrophobic group volume,Ic =the critical hydrophobic group length ,Ic =the critical hydrophobic group length ,Ap =the cross-sectional area of the hydrophilic head Ap =the cross-sectional area of the hydrophilic head

group and group and Ahp =the cross-sectional area of hydrophobic groupAhp =the cross-sectional area of hydrophobic groupA CPP below 0.5 (indicating a large contribution from A CPP below 0.5 (indicating a large contribution from the hydrophilic group area) is reported to give spherical the hydrophilic group area) is reported to give spherical

micellesmicelles and above 1(indicating a large contribution from theand above 1(indicating a large contribution from the hydrophobic group volume) should produce inverted hydrophobic group volume) should produce inverted

micelles. micelles.

FORMATION OF LIPOSOMESFORMATION OF LIPOSOMES

CLASSIFICATION OF LIPOSOMES CLASSIFICATION OF LIPOSOMES

METHODS OF LIPOSOME PREPARATION METHODS OF LIPOSOME PREPARATION -Water soluble(hydrophilic) materials entrapped inside the liposomes by -Water soluble(hydrophilic) materials entrapped inside the liposomes by

using aqueous solution of these materials as hydrating fluid orusing aqueous solution of these materials as hydrating fluid or by the addition of drug/drug solution at some stage during the by the addition of drug/drug solution at some stage during the

manufacturing of the liposomes.manufacturing of the liposomes.

-Lipid soluble (lipophilic) materials solubilized in the organic solution of -Lipid soluble (lipophilic) materials solubilized in the organic solution of the constitutive lipid(s) and then evaporated to a dry drug containingthe constitutive lipid(s) and then evaporated to a dry drug containing lipid film followed by its hydration.lipid film followed by its hydration.

-These methods involve the loading of the entrapped agents before or-These methods involve the loading of the entrapped agents before or during the manufacturing procedure (PASSIVE LOADING).during the manufacturing procedure (PASSIVE LOADING).

-Certain types of compounds with ionizable groups and those which-Certain types of compounds with ionizable groups and those which display both lipid and water solubility can be introduced into the display both lipid and water solubility can be introduced into the liposomes after the formation of intact vesicles(ACTIVE LOADING). liposomes after the formation of intact vesicles(ACTIVE LOADING).

METHODS OF LIPOSOMES PREPARATION

PASSIVE LOADING TECHNIQUESPASSIVE LOADING TECHNIQUES

MECHANICAL DISPERSIONSOLVENT DISPERSIONDETERGENT DEPLETION

MECHANICAL DISPERSION METHODSMECHANICAL DISPERSION METHODS

- Co-dissolve the lipids in an organic solvent.-Remove the organic solvent by film deposition under vacuum.

-Hydrate the solid lipid mixture using an aqueous buffer.

-The lipids spontaneously swell and form liposomes.

THIN FILM HYDRATION USING HAND THIN FILM HYDRATION USING HAND SHAKING (MLVs) SHAKING (MLVs)

-Lipids casted as stacks of film .-Lipids casted as stacks of film .-The casted film dispersed in an aqueous -The casted film dispersed in an aqueous medium medium by hand shakingby hand shaking-Upon hydration, the lipids swell and peel off -Upon hydration, the lipids swell and peel off -MLVs are formed-MLVs are formed

  -Expose the film to a stream of water saturated nitrogen for 15 min-Swell in an aqueous medium without hand shaking-ULVs are formed

NON-SHAKING METHODS(ULVs)

HAND SHAKING TECHNIQUEHAND SHAKING TECHNIQUE

PRO-LIPOSOMESPRO-LIPOSOMES

-Lipids are dried over a finely -Lipids are dried over a finely divided particulate support (e.g. divided particulate support (e.g. Sodium chloride or Sorbitol in Sodium chloride or Sorbitol in powder form)powder form)

-These lipid coated particulates are -These lipid coated particulates are called pro- liposomes.called pro- liposomes.

-In water,the support is rapidly -In water,the support is rapidly dissolved.dissolved.

-The lipid film is hydrated to form -The lipid film is hydrated to form MLVs.MLVs.

MECHANICAL TREATMENTS OF MECHANICAL TREATMENTS OF MLVsMLVs

MICRO-EMULSIFICATIONMICRO-EMULSIFICATION--Force out an MLV dispersion through a 5 µm orifice Force out an MLV dispersion through a 5 µm orifice in a in a

microfluidizer at very high pressure.microfluidizer at very high pressure.

-Then separate the fluid into two streams.-Then separate the fluid into two streams.

-Allow the streams to collide together at right -Allow the streams to collide together at right angles in an interaction chamber.angles in an interaction chamber.

-Collect SUVs.-Collect SUVs.

MICRO-EMULSIFICATIONMICRO-EMULSIFICATION

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SONICATIONSONICATION

--Place test tube containing an MLV dispersion in a Place test tube containing an MLV dispersion in a bath sonicator or place the tip of the sonicatorin bath sonicator or place the tip of the sonicatorin the test tube in a probe sonicator.the test tube in a probe sonicator.

-Sonicate for 5-10 min above the transition -Sonicate for 5-10 min above the transition temperature of the constituent lipid.temperature of the constituent lipid.

-Centrifuge to remove residual large particles.-Centrifuge to remove residual large particles.

-Collect a clear SUV dispersion.-Collect a clear SUV dispersion.

BATH/PROBE SONICATIONBATH/PROBE SONICATION

FRENCH PRESSURE CELLFRENCH PRESSURE CELL-Extrude MLVs in a French press under high -Extrude MLVs in a French press under high pressure.pressure.

-Get ULVs or OLVs.-Get ULVs or OLVs.

MEMBRANE EXTRUSIONMEMBRANE EXTRUSION-Pass an MLV dispersion through a polycarbonate membrane filter .

-Get SUV s / LUV s from MLVs.

Dried Reconstituted Vesicles Dried Reconstituted Vesicles (DRV)(DRV)

-Freeze and lyophilize empty SUVs.-Freeze and lyophilize empty SUVs.

-Re-hydrate them with the aqueous fluid containing -Re-hydrate them with the aqueous fluid containing

the material to be entrapped.the material to be entrapped.

-Get ULVs or OLVs.-Get ULVs or OLVs.

  

-Freeze a SUV dispersion and thaw it at room -Freeze a SUV dispersion and thaw it at room temperaturetemperature

for 15 min.for 15 min.

-Subject it to a brief sonication cycle-Subject it to a brief sonication cycle

-Obtain ULVs. -Obtain ULVs.

Freeze Thawed Sonication (FTS) Method

DRV vs FTS METHODDRV vs FTS METHOD

  SOLVENT DISPERSION METHODS FOR PASSIVE SOLVENT DISPERSION METHODS FOR PASSIVE

LOADINGLOADING

-Dissolve lipids in an organic solution-Dissolve lipids in an organic solution

-Bring the solution into contact with an -Bring the solution into contact with an aqueous phase containing materials to be aqueous phase containing materials to be entrapped within the liposomes.entrapped within the liposomes.

Ethanol InjectioEthanol Injectionn

--Inject an ethanolic lipid solution Inject an ethanolic lipid solution rapidly ,through a fine needle into an rapidly ,through a fine needle into an excess of saline.excess of saline.

-Collect a SUV dispersion.-Collect a SUV dispersion.

Ether InjectionEther Injection-Inject an ethereal lipid solution very -Inject an ethereal lipid solution very slowly ,through a narrow needle into an slowly ,through a narrow needle into an aqueous phase, at the temperature of aqueous phase, at the temperature of vaporizing the organic solvent.vaporizing the organic solvent.

-Collect a LUV dispersion.-Collect a LUV dispersion.

SOLVENT DISPERSION METHODSSOLVENT DISPERSION METHODS

DOUBLE EMULSIONDOUBLE EMULSION-Inject organic solution containing water -Inject organic solution containing water droplets rapidly into hot aqueous solution of droplets rapidly into hot aqueous solution of Tris -buffer by a 22-gauge hypodermic Tris -buffer by a 22-gauge hypodermic needle.needle.

-Evaporate the organic solvent using strong -Evaporate the organic solvent using strong jet of nitrogen , thus forming double jet of nitrogen , thus forming double emulsion (W/O/W system).emulsion (W/O/W system).

-Obtain ULVs.-Obtain ULVs.

DOUBLE EMULSIONDOUBLE EMULSION

REVERSE PHASE EVAPORATION VESICLESREVERSE PHASE EVAPORATION VESICLES

-Sonicate a mixture of two phases in a bath -Sonicate a mixture of two phases in a bath sonicator.sonicator.

-Dry down the emulsion to a semisolid gel in a -Dry down the emulsion to a semisolid gel in a rotaryrotary

evaporator under reduced pressure.evaporator under reduced pressure.

-Collapse a certain proportion of the water -Collapse a certain proportion of the water dropletsdroplets

by vigorous mechanical shaking using a vortex by vigorous mechanical shaking using a vortex mixture.mixture.

-Get MLVs or LUVs.-Get MLVs or LUVs.

REVERSE PHASE EVAPORATIONREVERSE PHASE EVAPORATION

STABLE PLURIMELLAR VESICLESSTABLE PLURIMELLAR VESICLES

-Prepare water-in-organic phase dispersion with an -Prepare water-in-organic phase dispersion with an

excess of lipid.excess of lipid.

-Dry it under continued batch sonication with an -Dry it under continued batch sonication with an

intermittent stream of nitrogen.intermittent stream of nitrogen.

-Obtain plurilamellar vesicles-entrapped aqueous -Obtain plurilamellar vesicles-entrapped aqueous

medium being located in the compartment in medium being located in the compartment in

between adjacent lamellae.between adjacent lamellae.

DETERGENT DEPLETION METHODS DETERGENT DEPLETION METHODS OF OF

PASSIVE LOADINGPASSIVE LOADING-Bring the phospholipids intimate contact with the -Bring the phospholipids intimate contact with the aqueous aqueous phase via detergents.phase via detergents.

-Get micelles containing other participating -Get micelles containing other participating components components known as mixed micelles.known as mixed micelles.

-Remove the detergent by dialysis or by column -Remove the detergent by dialysis or by column chromatography or by the use of biobeads.chromatography or by the use of biobeads.

-Collect ULVs.-Collect ULVs.

ACTIVE LOADINGACTIVE LOADING