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LIPOSOMES :A NOVEL DRUG DELIVERY SYSTEM

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INTRODUCTION

Liposomes were discovered in the early 1960’s by British Haemalogist Dr. A.D.Bangham.

Subsequently studied as cell membrane models.

The particle size of liposomes ranges from 20nm to10μm in diameter.

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LIPOSOMESLiposomes are simple microscopic vesicle in which an

aqueous volume is entirely enclosed by membrane composed of a lipid molecules.

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WHY USE LIPOSOMES IN DRUG DELIVERY SYSTEM

Pharmacokinetics:- Efficacy & toxicity.1. Changes the Absorbance & Bio-distribution.2. Deliver drug in desired form.3. Multidrug resistance.

Protection:-1. Decreases harmful side effects.2. Change where drug accumulates in the body.3. Protects drug.

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WHY USE LIPOSOMES IN DRUG DELIVERY SYSTEMRelease:-1. Affect the time in which the drug is released. 2. Prolong time- Increases duration of action &

Decreases administration interval period of time.

Dependent on drug & liposome properties:-1. Liposomes composition, pH, osmotic gradient &

environment.

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MODES OF LIPOSOME/CELL INTERACTION

Adsorption Endocytosis

Fusion Lipid transfer

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LIPOSOMES HELPS TO IMPROVE……

Therapeutic Index.Rapid metabolismUnfavorable Pharmacokinetics.Low solubility.Lack of stability. Irritation.

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ADVANTAGESTargeting drug delivery or site specific drug delivery. Increased efficacy and therapeutic index. Increased stability via encapsulation.Reduction in toxicity of the encapsulated agents.Site avoidance effect. Improved pharmacokinetic effects .Administered by various routes.Can act as reservoir for drug.

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DISADVANTAGESProduction cost is high Leakage and fusion of encapsulated drug /

molecules. Sometimes phospholipid undergoes reaction like

oxidation and hydrolysis Short half-life Low solubility Lower stables.Allergic reactions may occur due to liposomal

constituents

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COMPOSITION OF LIPOSOMES

The main components of liposomes are:

1) Phospholipids

2) Cholesterol

3) Sphingolipids

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PHOSPHOLIPIDS Phospholipids are the major structural

components of biological membranes such as the cell membrane.

Natural Synthetic

Phospholipids

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GENERALLY PHOSPHOLIPIDS ARE REPRESENTED AS FOLLOWS,

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MECHANISM OF PHOSPHOLIPIDS:

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CHOLESTEROLCholesterol by it self does not form a bilayer.

However, cholesterol acts as a fluidity buffer. i.e. below the phase transition temperature, it makes the membrane less ordered & slightly more permeable, while above the phase transition temperature, it makes the membrane more ordered & stable.

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SPHINGOLIPIDSBackbone sphingolipid is sphingosine or related

base.

This contains 3 characteristic building blocks…1. A mol of fatty acid2. A mol of sphingosine3. A head group that can vary from simple alcohols

such as choline to very complex carbohydrates.

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PHYSICAL CHARACTERIZATION OF LIPOSOME

Sr. no.

Characterization parameters

Analytical method/Instrument

1. Vesicle shape and surface morphology

Transmission electron microscopy,

Freeze-fracture electron microscopy

2. Vesicle size and size distribution

Dynamic light scattering, zeta sizer, laser light scattering

3. Surface charge Free-flow electrophoresis

4. Electrical surface potential and surface pH

Zeta-potential measurements & pH sensitive probes

5. Drug release Diffusion cell/ dialysis

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CHEMICAL CHARACTERIZATIONSr. No

Characterization parameters

Analytical method/Instrument

1… Phospholipid concentration

HPLC

2. Cholesterol concentration Cholesterol oxidase assay and HPLC

3. Phopholipid peroxidation UV absorbance, Iodometric and GLC

4. Phospholipid hydrolysis, Cholesterol auto-oxidation.

HPLC and TLC

5. Osmolarity Osmometer

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BIOLOGICAL CHARACTERIZATION

Sr. No

Characterization parameters

Analytical method/Instrument

1. Sterility Aerobic or anaerobic cultures

2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test

3. Animal toxicity Monitoring survival rates, histology and pathology

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CLASSIFICATION OF LIPOSOMES

Liposomes are classified on the basis of….1. Structural parameters. 2. Method of preparation.3. Composition and application.4. Conventional liposome.

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Based on structural parameter

MLVMultilamellar vesicle(>0.5u

m)

OLVOligolamellar vesicle(0.1-

1um)

UVUnilamellar vesicle(all

size ranges)

MUVMediam

unilamellar vesicle

SUVSmall unilamellar vesicle

GUVGiant unilamllar vesicle

LUVLarge unilamellar vesicle

MVV/MVMultivesicula

r vesicle(>1um)

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Based on Method of

Preparation

REV SUVs/OLVs

Reveres-Phase

Evaporation Method

MLV-REVMLV’s made by Reverse

Phase Evaporation

Method

SPLV Stable

Plurilamellar Vesicle

FAT-MLVFrozen and

Thawed MLVs

VETVesicle

Prepared by Extrusion Technique

DRVDehydrated - Rehydration

Method

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Based on composition & application

pH Sensitive Liposome

Immuno-Liposome

Long Circulatory Liposome

(LCL)

Convention-al Liposome

Fusogenic Liposome

Cationic Liposome

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Based on Conventi

onal Liposom

e

Stabilize Natural

lacithin (PC) Mixture

Synthetic Identical

chain Phospholipi

d

Glycolipid containing Liposome

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PREPARATION OF LIPOSOMESMethod of Liposome Preparation

Passive LoadingInvolves loading of

entrapped agent before or during the mfg procedure

Active or remote Loading:Certain types of compounds with ionized groups & those

with both lipid & water solubility, can be introduced into the liposomes after the

formation of the intact vesicle

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Method of preparation of Liposomes

Passive Loading Techniques Active Loading Techniques

Mechanical Dispersion Methods

Solvent Dispersion Methods

Detergent Removal Methods

•Lipid Film Hydration•Micro Emulsification• Sonication Methods•French Pressure cell•Membrane Extrusion•Dried reconstituted vesicles•Freeze thawed Liposome

•Ethanol Injection•Ether Injection•Double Emulsion Vesicles•Reverse Phase Evaporation Vesicles•Stable Plurilamellar Vesicles

•Detergent•Dialysis•Column Chromatography•Dilution•Reconstituted Sendal Virus Enveloped Vesicles

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GENERAL METHOD OF PREPARATION OF LIPOSOMEAll the methods of preparing liposomes involve 3-4

basic steps:

Drying down lipids from organic solvent

Dispersion of lipid in aqueous medium

Purification of resultant Liposomes

Analysis of final product

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Cholesterol Lacithin Charge

Solution in Organic Solvent

Thin Film

Liposome Suspension

Drying

Dispersion (Hydration)

Fig: Common Stages of all the Method of Preparation of Liposomes

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HANDLING OF LIPOSOMES

Unsaturated lipids:- Susceptible to oxidation.

Volatile solvents:- Tend to evaporate from the container, such as chloroform.

Store:- In an inert atmosphere of nitrogen & in dark place, in glass vessels.

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1) METHOD OF PHYSICAL DISPERSION/ MECHANICAL DISPERSION: Basic methods of physical dispersion… 1. Hand-Shaken Multilamellar Vesicles2. Non-Shaking Vesicles3. Pro-liposomes4. Freeze Drying

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HAND SHAKEN METHOD IN GENERAL

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NON-HAND SHAKING METHOD: Method described by:- Reeves & Dowben in 1969 for

LUVs.

Lipid

Chloroform:Methanol

Agitation

Stream of Nitrogen

Spread over bottom of

flaskEvaporate at room temp.

Flow Through Nitrogen

Passed Through

Saturated Nitrogen

Hydration

(Drying)

LUVs liposomes

Swelling Milky suspension

2hrs at 37⁰C

+

Add bulk fluid

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PRO-LIPOSOMES:Method increases surface of the dry lipid (at low

aqueous volume). s

Lipid +

Chloroform

Powdered sorbitol,

NaCl,

Swelling Lipid

Pro-Liposome

Transfer to flask

Evaporation

50-55⁰C

Add 5ml lipid

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FREEZE DRYING:

The solvent choice depend on the freezing point drug which is to be entrapped.

Finely divided lipid

Freeze dried lipid

Organic solvent

Aqueoussolvent Dissolve in

With rapid mixing above phase

transition temp.

Add water or saline

MLVs liposomes

Milky suspension

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PROCESSING OF HYDRATED LIPID BY PHYSICAL METHOD OR MECHANICAL METHOD:

Sonicated unilamellar vesicles (SUVs).French Pressure cell method.Membrane Extrusion method.Dried-reconstitution vesicles (DRVs).Freeze and thawed technique ((FAT).pH Induced Vesiculation.Calcium induced Fusion.

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SONICATED UNILAMELLAR VESICLES (SUVS):

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FRENCH PRESSURE CELL LIPOSOMES:

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MEMBRANE EXTRUSION LIPOSOMES:

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DRIED RECONSTITUTED VESICLES (DRVS) & FREEZE THAW SONICATION (FTS):

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PH INDUCED VESICULATION:

In this method ULVs is prepared from MLVs without sonication or high pressure application.

It is an electrostatic phenomenon.Exposure of phospholipid to high pH is less than 2

mins.

Dry film Minimum quantity of

water

Material added to be entrapped

HydrationLiposomes15⁰C

NaCL

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2) SOLVENT DISPERSION METHOD:

Methods depends on one of three categories….1. Organic solvent miscible with aqueous phase.

E.g - Ethanol.2. Organic solvent immiscible with aqueous phase, the

latter being in large excess.3. Organic solvent is in large excess & again with

immiscible aqueous phase

Lipid

Organic solvent

+Aqueous phase

containing materialLiposome

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ETHANOL/ETHER INJECTION METHOD:

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DOUBLE EMULSION VESICLES:The outer portion of the liposome membrane is

created at a second interface between two phases by emulsification of an organic solution in water.

Also described as W/O/W system or double emulsion.

These vesicles are suspended in aqueous medium.

Organic solvent

Water droplets

Multi-compartment

vesicle+

Mechanical

Dispersion

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REVERSE PHASE EVAPORATION METHOD:

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STABLE PLURILAMELLA VESICLES (SPVS) (SONICATION METHOD):

In this method, drying in accompanied by continued bath sonication with the stream of nitrogen.

The internal structure of SPVs is different from MUV.

The percent entrapment is normally 60%.

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Their shape and size depend upon chemical nature of detergent, the concentration & other lipids involved.

The concentration of detergent in water at which micelles just start to form is known as critical micelle concentration (CMC).

3) DETERGENT REMOVAL METHOD:

Phospholipid

Aqueous Phase

Micelles+Detergents

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REMOTE OR ACTIVE LOADINGLipid bilayer membrane is impermeable to ions or

large hydrophilic molecules.

Advantages:1. Provides high encapsulation efficiency & capacity.2. Reduce leakage of encapsulated compounds.

Weak bases such as…3. Doxorubacin4. Vincristine5. Modified peptides & insulin

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PHARMACEUTICAL APPLICATIONS 1. Cell -liposome interaction2. Localized drug effect3. Enhanced drug uptake4. Flexibility in structural characteristics 5. Enzyme Replacement.6. Diagnostic imaging of tumors.7. Study of membranes.8. Targeted gene delivery to specific cells.9. Ligand mediated targeted drug delivery by long

circulating liposomes.

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MEDICAL APPLICATION OF LIPOSOMES:

AIDS Therapy Malaria Therapy Infectious Diseases Dermatology Heavy metal poisoning

(chelation therapy).

Cancer Therapy Lung therapyCosmetology Radiodiagnostic Carriers

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MARKETED PREPARATION: Product Drug Uses

Doxil Doxorubicin Brest & ovarian cancer

Marqiobo Vincristine Cancer

Arikace Amikacin Bacterial Infection

Amphotec Amphotericin B Fungal Infection

Lipoplastin Cisplastin Lung, head ,neck Ovarian Cancer

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REFERENCES: D.M.Bramhankar & Sunil B. Jaiswal Biopharmaceutics &

Pharmacokinetics(First edition 2005) pg.no- 360-361

International Journal of Pharmaceutical Studies and Research.

S.P. Vyas and R.K. Khar “Target and Oontrolled Drug Delivery - Novel Carrier Systems”.

Sanjay K. Jain & N. K. Jain “Controlled and Novel Drug Delivery Systems”

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