Targeted Drug Delivery System

LIPOSOMES

Learning Objectives

• Introduction• Mechanism of liposome formation• Classification of liposomes• Material used in preparation of liposomes• Methodes of liposome preparation• Characterization of liposomes• Stability • Therapeutic application• References

What are Liposomes?

• They are simply vesicles or ‘bags’ in which an aqueous volume is entirely enclosed by a membrane composed of lipid (fat) molecules, usually phospholipids.

• These vesicles can encapsulate water-soluble drugs in their aqueous spaces and lipid soluble drug within the membrane itself.

• The unique property of liposomes, namely their versatile, biodegradable, hypoallergenic nature, along with their similarity to biological membranes are the important factors in the continued efforts to develop liposomal drug delivery froms.

•Structurally, liposomes are bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids.

Advantages of liposome :

• Provides selective passive targeting to tumor tissues

• Increased efficacy and therapeutic index• Increased stability via encapsulation• Reduction in toxicity of the encapsulated agent.• Improved pharmacokinetic effects• Used as carriers for controlled and sustained

drug delivery• Can be made into variety of sizes.

Disadvantages of liposome :

• Leakage of encapsulated drug during storage.• Uptake of liposomes by the reticuloendothelial

system• Batch to batch variation• Difficult in large scale manufacturing and

sterilization• Once administered, liposomes can not be removed• Possibility of dumping, due to faulty administration

Liposomes Evolution :

• 1965 First description of closed lipid bilayer vesicles.

• 1967 introduction of the term liposomes to describe closed lipid bilayer vesicles

• 1972 liposomes first used as delivery systems of drugs

• 1974 first patients to be injected with liposomes• 1979 liposomes first used as delivery systems

of nucleic acids to cells• 1980 first monoclonal anti body targeted

liposomes termed imunoliposomes

• 1987 first synthetic cationic liposomes deliver genes to cells

• 1987 first sterically stabilized long circulating liposomes system introduced

• 1992 first lioposome based non viral vecrtor gene therapy clinical trail on cystic fibrosis patients

• 1993 frist liposome based vaccine against hepatitis A is marketed

• 1995 first long circulating immunoliposomes• 1995 the liposomes encapsulated from of the

anticancer drug doxorubicin and daunorubicin approved for human use

• 1997 first liposomes based DNA vaccine

Mechanism of liposome formation• In order to understand why liposomes are formed

when phospholipids are hydrated, it requires a basic understanding of physiochemical features of phospholipids.

• Phospholipids are amphipathic molecules (having affinity for both aqueous and polar moieties) as they have a hydrophobic tail is composed of two fatty acids containing 10-24 carbon atoms and 0-6 double bonds in each chain.

• In aqueous medium the phospholipid molecules are oriented in such a way that the polar portion of the molecule remains in contact with the polar environment and at the same shields the non-polar part.

• They align themselves closely in planer bilayer sheets to minimize the interaction between the bulky aqueous phase and long hydrocarbon fatty acyl chains.

• This alignment requires input of sufficient amount of energy (in the form of shaking, sonication, homogenization, heating, etc).

• Interactions are completely eliminated when these sheets fold over themselves to form closed, sealed and continuous bilayer vesicles.

Classification of liposomes

1. Multilamellar vesicles (MLVs) : consist of several bilayers and having size ranging from 100nm-20m

2. Small unilamellar vesicles (SUVs) : composed of single lipid bilayer with dimeter ranging from 20-100nm

3. Large unilamellar vesicles (LUVs) : consist of single bilayer with diameter ranging from 0.1-1m

4. Multivesicular vesicles (MVVs) : consist of vesicles with size ranging from 100nm-20m

Materials used in preparation of liposomes

A.Phospholipids :

• It is the major component of the biological membrane.

• Two types of phospholipids are used natural and synthetic phospholipids.

• The most common natural phospholipid is the phospatidylcholine (PC) is the amphipathic molecule and also known as lecithin.

• It is originated from animal (hen egg) and vegetable (soyabean).

B. Steroids :

• Cholesterol is generally used steroid in the formulation of liposomes.

• It improves the fluidity of the bilayer membrane and reduces the permeability of bilayer membrane in the presence of biological fluids such as blood / plasma.

• Cholesterol appers to reduce the interactions with blood proteins.

Methods of liposomespreparations

Passive loading technique

Active loadingtechnique

Mechanical dispersion methods

Solvent dispersion methods

Detergent removal methods

• Mechanical dispersion methods

Lipid is solublised in organic solvent, drug to be entrapped is solubilise in aqueous solvent, the lipid phase is hydrated at high speed stirring due to affinity of aqueous phase to polar head it is entrapped in lipid vesicles.

e.g. Lipid film hydration, Micro-emulsification ( Microfluidizer ), Sonication, Dried reconstituted vesicle

• Solvent dispersion methods

in this method, lipids are first dissolved in organic solvent, which then brought in to contact with aqueous phase containing material which is to be entrapped in liposome under rapid dilution and rapid evaporation of organic solvent.

e.g.

Ethanol injection

Ether injection

De-emulsification

• Detergent removal methods

Surface charge Free-flow electrophoresis

Electrical surface potential and surface pH

Zetapotential measurements & pH sensitive probes

Percent of free drug/ percent capture

Drug release Diffusion cell/ dialysis

Parameter Characterization method

Vesicle shape and surface morphology

Mean vesicle size and size distribution

Dynamic light scattering, zetasizer, Photon correlation spectroscopy, laser light scattering, gel permeation and gel exclusion

Minicolumn centrifugation, ion-exchange chromatography,  radiolabelling

Transmission electron microscopy, Freeze-fracture electron microscopy

Physical Characterization

Phopholipid peroxidation UV absorbance, Iodometric  and GLC

Phospholipid hydrolysis, Cholesterol auto-oxidation HPLC and TLC

Osmolarity

Parameter Characterization method

Phospholipid concentration

Cholesterol concentration Cholesterol oxidase assay and HPLC

Osmometer

Barlett assay, stewart assay, HPLC

Chemical Characterization

Animal toxicity Monitoring survival rates, histology and pathology

Parameter Characterization method

Sterility

Pyrogenicity Limulus Amebocyte Lysate (LAL) test

Aerobic or anaerobic cultures

Biological Characterization

Stability • Physical stability :

Once liposomes are formed, they behave similar to the other colloidal particles suspended in water.

Neutral particles tend to aggregate or flocculate and sediment with increase in size on storage. Adding charged lipids such as stearyl amine, diactyl phosphate and phosphatidyl serine can control the aggregation.

The addition of charged lipids causes repulsion and prevents major changes in the overall size of liposomes.

• Chemical stability :Phospholipids, especially those derived from natural sources, are subject to two major degradative reaction :

A. Lipid peroxidation : most phospolipid liposomes contain unsaturated acyl chains as part of their molecular structure and susceptible to oxidative degradation. It can be minimized by the use of animal derived lipids like egg PC, which has less saturated lipids, use of light resistant containers, use of antioxidants are useful in minimizing oxidation.

B. Lipid hydrolysis :

hydrolysis in phospolipids results in the formation of free fatty acids and lyso-lecithin. Selecting a good source of lipid, temperature, pH, and minimizing oxidation.

• Biological stability :

liposomes release entrapped molecules rapidly when incubated with blood or plasma. This instability is attributed to the transfer of bilayer lipids to albumin and high density liposomes.

Therapeutic applications of liposomes

1. Liposomes as drug / protein delivery vehicles• Controlled and sustain release in situ• Enhanced drug solubilization• Enzyme replacement therapy and lysosomal

storage disorders• Altered pharmacokinetics and biodistribution2. Liposomes in antimicrobial, antifungal and

antiviral therapy3. Liposomes in tumour therapy• Carrier of small cytotoxic molecules • Vehicle for macromolecules as cytokines and

genes

4. Liposomes in gene delivery • Genes and antisense therapy • Genetic (DNA) vaccination

5. Liposomes in immunology

6. Liposomes as radiopharmaceutical and radio diagnostic carrier

7. Liposomes in cosmetic and dermatology

8. Liposomes in enzyme immobilization and bioractor technology

Drug Route of administration

Targeted Diseases

Amphotericin-B Oral delivery Mycotic infection

 Insulin Oral, Ocular, Pulmonary and Transdermal delivery

Diabetic mellitus

 Ketoprofen Ocular delivery Pain muscle condition

Pentoxyfylline Pulmonary delivery Asthma

Tobramycin Pulmonary delivery Pseudomonas infection, aeruginosa

Drug Route of administration

Targeted Diseases

Salbutamol Pulmonary delivery Asthma

Benzocain Transdermal ulcer on mucous surface with pain

Ibuprofen Oral delivery Rheumatoid arthritis

Adrenaline Ocular delivery Glucoma, Conjectivitis

Penicillin G Pulmonary delivery Meningococal, staphylococcal

Methotrexate Transdermal Cancer

Marketed product

Drug used Target diseases

Company

DoxilTM or CaelyxTM

Doxorubicin Kaposi’s sarcoma

SEQUUS, USA

DaunoXomeTM Daunorubicin Kaposi’s sarcoma, breast & lung cancer

NeXstar, USA

AmphotecTM Amphotericin-B fungal infections, Leishmaniasis

SEQUUS, USA

VENTUSTM Prostaglandin-E1 Systemic inflammatory

diseases

The liposome company, USA

ALECTM Dry protein free powder of DPPC-PG

Expanding lung diseases in babies

Britannia Pharm, UK

Marketed product

Drug used Target diseases

Company

Topex-Br Terbutaline sulphate

Asthma Ozone, USA

Depocyt Cytarabine Cancer therapy Skye Pharm, USA

Novasome® Smallpox vaccine Smallpox Novavax, USA

Avian retrovirus vaccine

Killed avian retrovirus

Chicken pox Vineland lab, USA

Epaxal –Berna Vaccine

Inactivated hepatitis-A Virions

Hepatitis A Swiss serum & vaccine institute, Switzerland

Conclusion • Liposomes are one of the unique drug delivery

system, which can be of potential use in controlling and targeting drug delivery.

• Liposomes are administrated orally, parenterally and topically as well as used in cosmetic and hair technologies, sustained release formulations, diagnostic purpose and as good carriers in gene delivery.

• Nowadays liposomes are used as versatile carriers for targeted delivery of drug.

Reference

• Targeted and controlled drug delivery, S.P.Vyas and R.K.Khar, CBS Publication 2008,

Nanoparticles – page no 331 to 386.Resealed Erythrocytes – page no 387 to 416.Liposomes – page no 173 to 248.

• Text book of Industrial Pharmacy, Shobha Rani Hiremath, Orient Longman Private ltd.

Nanoparticles – 129 to 142.Liposomes – 97 to 110

• Medical applications of nanotechnology by Timothy E. Morey, University of Florida.

• Liposome: A versatile platform for targeted delivery of drugs by Sanjay S. Patel

• Resealed Erythrocytes : A Review by A.V.Gothoskar MIT Campus, Pune.

• www.pharmainfo.net

• www.google.com

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