Vesicular Drug Delivery Systems

Liposomes

What is a Liposome?
Spherical vesicles with a phospholipid bilayer

(Bangham et al. 1965)

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.

Hydrophilic

OR
Liposomes (lipid vesicles) are
sealed sacs in the micron or
submicron range dispersed in an
aqueous environment.

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

PolarHeadGroups

PHOSPHOLIPIDS

Threecarbonglycerol

Natural Source Eggs for PC, PE, Sphingomyelin

Soy bean for PC, PE, PI
Modified natural Phospholipids Hydrogenation to reduce degree of
unsaturation

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 -

Microelectrophoresis

No of lamellae

Small angle X ray Scattering,

NMR, Electron microscopy

of lipids

DSC, ESR, NMR

Surface Chemical Analysis

XPS, SIMS, NMR

Structural & Motional behavior

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

Rate and Extent of MPS uptake depend on

- Size
- Rigidity
- Hydrophilicity
- Charge of the liposomes

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

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

Liposome Factors

Blood Flow Rate

Long circulation time

Vascular

Stability (drug
retention)

Permeability
Interstitial Pressure

Small vesicle size

Phagocytic Activity

RES Function

List of Liposome products

Thank You