Vesicular Drug Delivery Systems

Liposomes

What is a Liposome?
Spherical vesicles with a phospholipid bilayer
Liposomes are concentric
bilayered vesicles in which an aqueous volume is entirely
(Bangham et al. 1965)

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

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 Solubility Improved pharmacokinetic effects Reduction in toxicity of the encapsulated agent Suitable 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

PROBLEMS - Physicochemical instability

- Prone to degradation by oxidation and hydrolysis

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.

Cross-Section of a Liposome

Polar Lipids (Phospholipid) Lipid Soluble ingredients (Drugs, Nutrients & vitamins) Water Soluble ingredients (Drugs, Nutrients & vitamins)

H2O Layer

Lipid Layer

Basic Components of Liposomal System

Vesicle Former Structural Lipid Charge Inducer

PHOSPHOLIPIDS

Polar Head Groups

Three carbon glycerol

Natural Source Eggs for PC, PE, Sphingomyelin Soy bean for PC, PE, PI Modified natural Phospholipids Hydrogenation to reduce degree of unsaturation Synthetic 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

Small Unilamellar Vesicle (SUV)

Large Unilamellar Vesicle (LUV)

Multilamellar Vesicle (MLV)

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

Formation of a Liposome

PREPARATION OF LIPOSOMES
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)

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

Detergent Removal Technique

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.

CHARACTERIZATION OF LIPOSOMES

Mean Size & Size distribution

Electron Microscopy Dynamic Light Scattering (PCS)

Surface Potential & Surface pH No of lamellae -

Microelectrophoresis
Small angle X ray Scattering, NMR, Electron microscopy DSC, ESR, NMR XPS, SIMS, NMR

Structural & Motional behavior

of lipids Surface Chemical Analysis -

Quality Control Assays of Liposome Formulation

REMOVAL OF UNENCAPSULATED DRUG

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

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

IN VIVO FATE OF LIPOSOME

I.V Injection

Uptake RES (Release in cell)

Disintegration in blood

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

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).

LONG-CIRCULATING LIPOSOMES
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. The hydrophilic barrier also retards disintegration of the liposomes through exchange and/or transfer of liposomal phospholipids to high

density lipoproteins. PEG = polyethylene glycol.

Cationic liposomes
Positively charged lipid heads

positively charged lipid droplets can interact with negatively charged DNA to wrap it up and deliver to cells Inside liposomes DNA is resistant to degradation

Lipofectin, lipofectamine, lipofectase.

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

APPLICATIONS
Cancer 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 tissuespecific 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

Liposome Factors
Long circulation time Stability (drug retention) Small vesicle size

Phagocytic Activity

RES Function

List of Liposome products

Thank You