91796286-Liposomes

Vesicular Drug Delivery Systems

Liposomes

Liposomes are concentric

bilayered vesicles in which an

aqueous volume is entirely

enclosed by a membranous lipid

bilayer mainly composed of

natural or synthetic

phospholipids.

OR

Liposomes (lipid vesicles) are

sealed sacs in the micron or

submicron range dispersed in an

aqueous environment.

Hydrophilic

Hydrophobic

Spherical vesicles with a phospholipid bilayer (Bangham et al. 1965)

What is a Liposome?

How they are formed?

Liposomes are formed by the self-assembly of phospholipid

molecules in an aqueous environment.

ADVANTAGES

Provides selective passive targeting to tumour tissues ( liposomal doxorubicin) Enhanced SolubilityImproved pharmacokinetic effectsReduction in toxicity of the encapsulated agentSuitable for delivery of hydrophobic, Amphipathic and Hydrophilic

drugs

Close resemblance with the natural membrane structure

Biocompatible, Biodegradable, Non toxic, Non-immunogenic

Can serve as a device for controlled release

Methods to overcome these limitations

Incorporation of lipids like cholesterol

Steric stabilization by high mol. wt surfactants like poloxamer.

Freeze drying

Polymerization of the vesicle in its own form.

PROBLEMS

- Physicochemical instability

- Prone to degradation by oxidation and hydrolysis

Cross-Section of a Liposome

H2O Layer Lipid Layer

Polar Lipids (Phospholipid)

LipidSoluble ingredients(Drugs, Nutrients &

vitamins)

WaterSoluble ingredients(Drugs, Nutrients &

vitamins)

Basic Components of Liposomal System

Vesicle Former

Structural Lipid

Charge Inducer

Polar Head Groups

Three carbon glycerol

PHOSPHOLIPIDS

Natural Source – Eggs for PC, PE, Sphingomyelin Soy bean for PC, PE, PIModified natural Phospholipids – Hydrogenation to reduce degree of unsaturationSynthetic Phospholipids

The parts of a phospholipid molecule. Phosphatidylcholine, represented schematically (A), in formula (B), as a space-filling model

(C), and as a symbol (D). The kink due to the cis-double bond is exaggerated in these drawings for emphasis.

Structural Lipid – Cholesterol, Tocopherol

Improves the fluidity of the bilayer

Minimizes leaching out of water soluble drug

Improves stability in biological fluids – reduce interaction

with plasma proteins

CHARGE INDUCERS – Dicetyl Phosphate, Sod. Cholate, Stearylamine

Prevents aggregation

Increases drug loading

The structure of cholesterol. Cholesterol is represented by a formula in (A), by a schematic drawing in (B),

and as a space-filling model in (C).

Classification of Liposomes

Liposome classification based on composition and mode of drug delivery

SmallUnilamellarVesicle(SUV)

LargeUnilamellarVesicle(LUV)

MultilamellarVesicle(MLV)

Typical Size Ranges: SLV: 20-50 nm – MLV:100-1000 nm

Formation of a Liposome

Mechanical methods

Hand shaking methods (MLV)

Extrusion through Polycarbonate filters (OLV)

Microfluidizer technique (mainly SUV)

High Pressure homogenization (mainly SUV)

Replacement of Organic Solvent by Aqueous medium

Removal of organic solvent (MLV, OLV, SUV)

Use of water immiscible solvents (MLV,OLV,SUV)

Reverse Phase Evaporation (LUV, OLV, MLV)

Detergent Removal Technique

Gel extrusion chromatography (SUV)

Slow dialysis (LUV, OLV, MLV)

PREPARATION OF LIPOSOMES

Hand Shaken/Film Hydration Technique (MLV) (Bangam et al, 1965)

Dehydration / Rehydration Vesicles (DRV)

• Efficient for direct loading of drugs

• Avoids use of Sonication, Organic solvents, Detergents

Solvent Evaporation

Processes for

Liposome Preparation

The lipid is initially dissolved by an aqueous solution of the detergent to form mixed lipid-detergent micelles, and the detergent is then removed by a diffusion-based process such as dialysis, diafiltration, or gel chromatography.

Uni- or oligolamellar vesicles ranging in diameter from SUV size up to several micrometers depending on the conditions used.

Ionic detergents, such as cholate and deoxycholate or nonionic detergents such as Triton X 100 and octylglucoside, have been used.

Detergent removal methods have been especially useful for functional reconstitution of membrane proteins.

Disadvantages

Encapsulation efficiency is low compared with most other methods.

Detergent removal by ordinary dialysis techniques is a tedious process.

Even traces of detergent can have pronounced effects on liposome

permeability and can greatly increase the transmembrane movement of PL.

Detergents may also have deleterious effects on the material being

encapsulated.

Detergent Removal Technique

Mean Size & Size distribution - Electron Microscopy

Dynamic Light Scattering (PCS)

Surface Potential & Surface pH - Microelectrophoresis

No of lamellae - Small angle X ray Scattering, NMR, Electron microscopy

Structural & Motional behavior

of lipids - DSC, ESR, NMR

Surface Chemical Analysis - XPS, SIMS, NMR

CHARACTERIZATION OF LIPOSOMES

Quality Control Assays of Liposome Formulation

Centrifree

- Suitable dilution is necessary

- Higher concentration of lipid blocks membrane

Adv : Rapid, requires small sample volume

Disadv : Expensive, Lipid concentration cannot exceed 5mg/mL

Gel Chromtography

- Sepharose/Sephadex

- Liposomes larger size pass through void volume

Adv : Sample recovery

Disadv : Slow and tedious, dilution of samples

REMOVAL OF UNENCAPSULATED DRUG

Dialysis

- Controlled and minimized by avoiding large dilution steps

- Several steps of small dil. vol (5-10 fold original dispersion)

Adv : Sample recovery

Disadv : Inaccurate and impossible to determine critical point

Protamine Aggregation

Adv : Economical

Disadv : Slow with neutral/positive charged liposomes

Contamination of the sample

Ultracentrifugation

- Subjected to high forces, can modify physically

Advantages and disadvantages of the different methods of separation of the entrapped from the unentrapped drug

I.V Injection

Uptake RES (Release in cell)

Disintegration in blood

Long circulation (Slow release in blood and accumulation at target site (non-MPS)

IN VIVO FATE OF LIPOSOME

Rate and Extent of MPS uptake depend on- Size- Rigidity- Hydrophilicity- Charge of the liposomes

IN VIVO BEHAVIOR OF LIPOSOMES

a. Conventional liposomes are opsonized by plasma proteins and trapped by RES. Fluid liposomes are also attacked by lipoproteins.b. Opsonins ans lipoproteins hardly attack the rigid liposomes.c. PEG coating protects liposomes against opsonization and attack of lipoproteins by

surface water layer.d. Unknown mechanisms that protect liposomes being recognized as foreign. One

possibility is that some molecules which are recognized as self are bound on the surface of liposomes and protect them.

Predominant mechanisms of intracellular drug delivery by liposomes. 1 - coated pit endocytosis of conventional, pH-sensitive and cationic liposomes; 2 - release of drug in the acidic endosome by pH-sensitive liposomes; 3 - intravascular and/or extracellular drug release from long circulating liposomes; 4 - receptor mediated endocytosis of immunoliposomes; 5 - fusion of cationic liposomes with plasma membrane.

TAILORING OF LIPOSOMES

Long Circulating Liposomes (Stealth/Sterically Stabilized)

- PEG-PE, Monosialoganglioside, Phosphoinositol

Targeted Liposomes

- Passive targeting (Conventional Liposomes)- Active targeting (Ligand Strategies: Folic acid,

Apolipoprotein E, Transferrin)

Polymerized Liposomes- Stability, artificial blood substitutes

pH & Temperature Sensitive Liposomes- Leaky in low pH (Surrounding cancerous tissue)- PL with Tc higher than body temp. (applying heat externally)

Cationic liposomes- Gene transfection (Lipoplex)

Immunoliposomes

Modified liposomes (stealth liposomes)

Hydrophilic polyoxyethylene lipids incorporated into liposome

Increased half-life is be due to a reduced coating (opsonisation) of these liposomes by plasma proteins

Coating with hydrophilic, polyethylene glycol (PEG) chains reduces the deposition of plasma proteins (by retaining water of hydration) and makes the liposomes more biocompatible (“stealthy”).

The hydrophilic barrier also retards disintegration of the liposomes

through exchange and/or transfer of liposomal phospholipids to high

density lipoproteins. PEG = polyethylene glycol.

Hydrophilic polymer coating

attracts water to the liposomes

surface, presenting a barrier to the

adherence of protein opsonins.

A decrease in opsonisation of the

liposomes in turn leads to a

decreased rate and extent of

liposome uptake into the

mononuclear phagocyte system,

resulting in increased circulation

half-lives.

LONG-CIRCULATING LIPOSOMES

Cationic liposomes

positively charged lipid dropletscan interact with negatively charged DNA

to wrap it up and deliver to cells

Positively charged lipid heads

Lipofectin, lipofectamine, lipofectase….

Inside liposomes DNA is resistant to degradation

Immunoliposomes for active targeting

Antibodies to intracellular myosin target liposomes

to infarcted areas of heart

Antibody against tumor specific molecules will target them to tumors

DNA delivery of Genes by Liposomes

Cheaper than viruses

No immune response

Especially good for in-lung delivery (cystic fibrosis)

100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector

Lipofection

APPLICATIONSCancer

Antimicrobial agents – Leishmaniasis (Amphotericin B)

Gene therapy

Immunological Adjuvants

Liposome entrapped DNA delivery

Transdermal drug delivery

Vaccine adjuvants

Enzyme replacement

Cosmetics

Topical applications

Pulmonary delivery

Leishmaniasis

Lysosomal storage diseases

Ophthalmic delivery of drugs

Pharmacological Basis for Liposome Delivery of Anti-Cancer Agents

• Slow Release: reduced peak levels of free drug and

prolonged tumor exposure

• Change in Biodistribution: avoiding drug

deposition in certain tissues will reduce tissue-

specific toxicities

• Tumor Targeting: passive accumulation by

enhanced permeability and retention (EPR) effect

Parameters Affecting Delivery of Liposomal Drugs to Tumors

Tumor Factors

• Blood Flow Rate

• Vascular

Permeability

• Interstitial Pressure

• Phagocytic Activity

Liposome Factors

• Long circulation time

• Stability (drug

retention)

• Small vesicle size

• RES Function

List of Liposome products

Thank You