Stability Aspects of Liposomes

PREPARED BY : HARNISHA PATEL

SEMESTER :3

MPHARM(PHARMACEUTICS)

Stability testing - the primary tool used to assess expiration dating and storage conditions for pharmaceutical products.

Protocols used for stability testing in the industry are derived from the recommendations of the International Conference on Harmonization (ICH).

These guidelines were developed as a cooperative effort between regulatory agencies and industry officials from Europe, Japan, and United States.

Liposomes have been extensively investigated for drug delivery, drug targeting, controlled release and enhancing solubility.

Major Limitation:

INSTABILITY of LIPOSOMES.

CLASSIFICATION OF STABILITY OF LIPOSOMES: Liposome stability can be subdivided into physical, chemical and biological stabilities, which are all inter-

related.

The shelf-life stability of liposomes is determined by : (1). Physical Stability: By optimizing the size distribution, pH and ionic strength, the addition of antioxidants and chelating agents.

Stable liquid liposomes.

Physical processes such as aggregation/flocculation and fusion/coalescence that affect the shelf life of liposomes.

Loss of liposome associated drug and changes in size.

A). Aggregation/flocculation Formation of larger units of liposomal material;

these units are still composed of individual liposomes.

Reversible. e.g. by applying mild shears forces, by changing the temperature.

The presence of aggregation can accelerate the process of coalescence of liposomes, which indicates that new colloidal structures are formed.

There is no reduction of surface in aggregation

the small particles retain their identity.

Aggregation moves as a single unit.

B). Coalescence/fusion: Irreversible process; the original liposomes

cannot be retrieved.

A colloidal dispersion is often thermodynamically unstable.

The central feature = the total surface area is

reduced in the coarsening process of unstable liposome dispersion.

After small particles coalescence, only the new larger particle remains.

MECHANISM OF FUSION & AGGREGATION: Loosely packed head groups and tightly packed alkyl chains in the outer layer with the opposite

arrangement in the inner layer of the bilayer.

Thermodynamically unstable state

which favors aggregation and/or fusion of the vesicles

Instability

(2). Chemical stability As phospholipids form the backbone of the bilayer their chemical stability is important.

Two types of chemical degradation reactions can affect the performance of phospholipid bilayers: A).Hydrolysis of the ester bonds linking the fatty acids to the glycerol backbone and B).Peroxidation of unsaturated acyl chains (if present).

The oxidation and hydrolysis of lipids may lead to

the appearance of short-chain lipids and then soluble

derivatives will form in the membrane, resulting

in the decrease of the quality of liposome products.

C). Biological stability: Depends on :

1).The presence of agents such as proteins that interact with liposomes upon application to the subject

2).Administration route.

Strategies used to enhance biological stability of liposomes will improve liposome-mediated drug delivery by increasing circulation time in the blood stream.

Incorporating steric stability, e.g. the incorporation of PEG into liposomes has shown to increase the liposomal biological stability towards plasma components.

A COMPARISON BETWEEN VESICULAR DRUG DELIVERY SYSTEM & STABILITY

ISSUES

LIPOSOMES: For drug delivery , Unilamellar( diameter 50

– 150 nm) are used. Larger liposomes are rapidly removed from the blood circulation.

Liposomes may increase the solubility of insoluble drugs(1000X).

In the small intestine, liposomes are digested in the presence of bile and enzymes. The drug is liberated and further solubilized in bile and digested lipids.

Efficient carriers for drugs, diagnostics, vaccines, nutrients and other bioactive agents.

STABILITY ISSUES: The physical instability causes fusion &

aggregation.

Chemical instability indicates hydrolysis and oxidation of lipids.

Liposomes in plasma are prone to aggregation and exhibit leakage

The destabilization of liposomes is due to the lipid exchange between the liposomes and HDLs

NIOSOMES: Niosomes are formed of cholesterol and

nonionic surfactants with or without incorporation of cholesterol or other lipids.

Stabilized due to cholesterol and small amount of anionic surfactant such as dicetyl phosphate.

Higher chemical stability of the surfactants than that of phospholipids, which are present in liposomes.

Due to the presence of ester bond, phospholipids are easily hydrolysed.

STABILITY ISSUES:

Only chemical stability.

Stability problems such as physical stability of fusion, aggregation, sedimentation and leakage on storage.

ETHOSOMES: Noninvasive carriers that enable

drugs to reach the deep skin layers and/or the systemic circulation.

They are composed mainly of phospholipids, high concentration of ethanol and water (disturbance of skin lipid bilayer & ability to penetrate the stratum corneum)

High patient compliance due to semisolid form (gel or cream) rather than Iontophoresis and Phonophoresis

STABILITY ISSUES:

Ethosomes are used for transdermal drug delivery which can provide better skin permeation and stability than liposomes.

It improves entrapment of drug.

Overall good stability.

TRANSFERSOMES: Capable of non-invasive

transdermal delivery of low & high molecular weight drugs.

Ultradeformable (ultraflexible) lipid aggregates, which are able to penetrate the mammalian skin intact.

Incorporation of "edge activators“ such as sodium cholate, sodium deoxycholate, span 80 and Tween 80. flexibility and allows to change their membrane composition locally and reversibly & they penetrate into narrow pore.

STABILITY ISSUES: Chemically unstable because they

are prone to oxidatation.

Purity of natural phospholipids is

criterion for stability.

HERBOSOMES / PHYTOSOMES: Water soluble phytoconstituents are

poorly absorbed due to their large molecular size by passive diffusion.

Due to their poor lipid solubility

limiting their ability to pass across the lipid‐rich biological membranes, resulting in poor bioavailability.

Herbosomes enhance the absorption of active, its dose requirement is reduced.

STABILITY ISSUES: Chemical bonds are formed between

phosphatidylcholine molecule and phytoconstituent.

So the herbosomes show better stability profile with appreciable drug entrapment.

SPHINGOSOMES: Liposomal phospholipid can

undergo chemical degradation such as oxidation and hydrolysis.

The hydrolysis may be avoided by use of lipid which contains ether or amide linkage instead of ester linkage (sphingolipid).

STABILITY ISSUES: Sphingolipid are used for the

preparation of stable liposomes known as sphingosomes.

Higher cost of sphingolipids hinders the preparation and use of these vesicular systems.

They show better stability as compared to liposomes BUT they have low entrapment efficacy.

CUBOSOMES: Discrete, sub micron nanostructured

particles of bicontinuous cubic liquid crystalline phase.

Produced by high-energy dispersion of bulk cubic phase, followed by colloidal stabilization using polymeric surfactants.

STABILITY ISSUES: Cubosomes posses the simple

production procedure and have better chemico-physical stability.

They are the good option with many advantages over liposomes,

But manufacturing of cubosomes on a large scale has difficulty because of their viscosity.

METHODS FOR ENHANCEMENT OF STABILITY: Maximum encapsulation efficiency and absence of drug leakage is achieved only when the physical ,

chemical , biological stability of liposomes is enhanced. Depends on the number of factors :

1). Control of particle size and lamellarity

2). Lipid composition

3). Method of drug loading

4). Prodrug Approach

5). Lyophilization

6). Prevention from oxidation and hydrolysis

7). Pro-vesicular Drug Delivery

8). Electro steric stabilization

9). Layersome

10).Ufosomes

1). CONTROL OF PARTICLE SIZE & LAMELLARITY: Change in particle size affect targeting , RES uptake & efficacy.

Aggregation and fusion are observed with particle size < 20 nm due to excessive high stress curvature.

The lamellarity of liposomes influences encapsulation efficiency, the efflux rate of drug.

Particle size and lamellarity depends on the method of manufacturing.

Ex. liposomes prepared by the combinations of some lipids on storage at 4 and 25°C over 6-month period large unilamellar vesicles (REV) proved to be superior than multilamellar liposomes (MLV) and dehydration/rehydration liposomes (DRV) systems as far as physical stability was concerned.

2).LIPID COMPOSITION: Permeability and stability of liposomes are influenced by the rigidity/stiffness of the lipid bilayer.

Selection of lipid depends on the phase transition temperature of lipids, which depends on fatty acid side chains, degree of unsaturation, chain length and polar head groups.

Lipids with long acyl chain length are most commonly used because high phase transition temperature.

This can be achieved by combinations of lipids or incorporation of another substances such as cholesterol , provides more rigidity to those phospholipids and therefore prevent liposome aggregation.

Charge on the liposomes determines the in vivo stability. A). Liposomes with neutral charge & positive charge containing phosphatidylcholine are the most

stable B). Stability of negatively charged liposomes is depended on their composition.

3).METHOD OF DRUG LAODING:

Drug loading methods involves passive and active loading.

A).Passive loading includes mechanical dispersion, solvent dispersion and detergent solubilization.

B).Remote or active loading method load drug molecules into preformed vesicles by using pH gradient and potential difference across liposomal membranes.

Active loading give the greater encapsulation efficiency , ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of liposomes

4).PRODRUG: Physical properties of drug play a role in the stability.

Problems like poor entrapment efficiency & physical - chemical instability are associated with the liposomal entrapment of drug molecules.

Lipophilic character of prodrug improves the interaction with lipid bilayers, favoring the absorption through the lipid barriers of skin.

Liposomes work as a lipophilic carrier which is able to deliver drug near to the cell surface.

Lipid composition is also equally responsible for stability of liposomes along with partition behavior of drug.

5).LYOPHILIZATION: The problems related to lipid oxidation and hydrolysis during shelf life of the liposomal product can be

reduced by the storage of liposomal dispersion in the dry state.

Freeze-drying (Lyophilization) is used as an effective approach to render liposomes stable without compromising their physical state or encapsulation capacity.

But without appropriate stabilizers will again lead to fusion of vesicles. Cryoprotectants, including saccharides (e.g. sucrose,trehalose, and lactose) are used.

MECHANISM: Aggregation of liposomes could be prevented by the formation of stable boundaries between the

vesicles.

Cryoprotectants form these stable boundaries due to their ability to replace the bound water around the bilayer via interaction with the polar region of the lipid head group (water replacement hypothesis).

6).Prevention from oxidation and hydrolysis: Numbers of factors are responsible for chemical instability of liposomes like pH, ionic strength and

exposure to oxygen.

To prevent it:

a).Minimize use of unsaturated lipids

b).Use of argon or nitrogen environment to minimize the exposure to oxygen

c).Use of antioxidants like α-tochopherols, beta hydroxy toluene (BHT)

d).Use of light resistant container for the storage of liposomal formulations.

e).Cholesterol also protect liposomal lipids by reducing lipid bilayer hydration.

7).Pro-vesicular Drug Delivery:

Pro vesicular drug delivery developed to overcome the stability problems associated with aqueous vesicular dispersions.

They are solid carriers of drug containing liposomes.

LIPOSOMES: Unilamellar or multilamellar spheroid structures composed of

lipid molecules, often phospholipids.

They show controlled release and increased solubility.

But have tendency to aggregate or fuse,

Susceptible to hydrolysis or oxidation.

PROLIPOSOMES: an alternative forms to conventional

liposomal formulation Composed of water soluble porous

powder as a carrier phospholipids. Drugs is dissolved in organic

solvent. Lipid and drug are coated on to a soluble carrier to form free-flowing granular material.

Show controlled release, better stability, ease of handling and increased solubility.

NIOSOMES: Non-ionic surfactant based multilamellar

or unilamellar vesicles, Aqueous solution of solute is entirely

enclosed by a membrane of surfactant macro-molecules as bilayers.

They are cheap and chemically stable but possess problems related to physical stability such as fusion, aggregation, sedimentation and leakage on storage.

PRONIOSOMES: approach minimizes the problems

associated with niosomes as it is a dry and free flowing product which is more stable during sterilization and storage.

Ease of transfer, distribution, measuring and storage make it a versatile delivery system.

Proniosomes are water-soluble carrier particles that are coated with surfactant.

8).ELECTROSTERIC STABILIZATION: Steric stabilization can be achieved by covering the surface with an adsorbed layer of long, bulky

molecules to prevent the aggregation of the liposomes.

The electrostatic repulsion can be obtained by increasing the charge of a surface by lowering the ionic strength or addition of charged molecules on the bilayer.

Liposomes coated with ligands, such as PEG, polyvinyl alcohol, poloxamer , stearylamine , block copolymers have shown increase in the liposome stability.

Polymer coating done by interfacial polycondensation.

The release of drug from polymer-coated vesicles is retarded due to the effective double barrier produced by the polymeric coat

9).LAYERSOME: The layer-by-layer coating concept is one of the strategies used for the preparation or the

stabilization of nanosystems.

The layersomes are conventional liposomes coated with one or multiple layers of biocompatible polyelectrolytes in order to stabilise their structure.

The formulation strategy is based on an alternative coating procedure of positive and negative polymers on initially charged small liposomes.

Drawback of liposome : their instability during storage or in biological media which is related to surface properties.

This surface modification stabilize the structure of the liposomes and lead to stable drug delivery systems.

10).UFOSOMES: Unsaturated fatty acid liposomes.

It is colloidal suspensions of closed lipid bilayers that are composed of fatty acids and their ionized species (soap).

Fatty acid vesicles = the nonionized neutral form : the ionized form (the negatively charged soap).

The ratio of nonionized neutral form and the ionized form is critical for the vesicle stability.

Ufosome membranes are much more stable in comparison to liposomes.

Future prospective for betterment of vesicular delivery:

Type of Vesicle Description

Aquasomes Three layered compositions with ceramics carbon , nanocrystalline particulate core coated with, glassy cellobiose. Specific targeting and molecular shielding.

Cryptosmes Surface coat composed of polyoxoyethylene derivative.Capable of ligand mediated drug targeting.

Discomes Niosomes solublized with non ionic surfactant solutions

Emulsomes Lipid vesicles consisted of microscopic lipid assembly with apolar core.

Enzymosomes Liposomal constructs ,to provide a mini bioenvironmental in which enzymes are covalently immobilized or coupled to the surface of liposomes.Targeted delivery to tumor cell.

Genosomes Artificial macromolecular complexes for gene transfer

Virosomes Liposomes spiked with virus glycoprotein

Vesosomes Multiple compartments of the vesosomes give better protection to the interior contents in serum

Proteosomes High molecular weight multi-submit enzyme complexes with catalytic activity