Structural Biochemistry/Liposomes

Liposomes are artificially constructed vesicles consisting of a phospholipid bilayer. First discovered in 1961 by Alec Bangham, a British scientist studying blood clotting, liposomes are now being studied for their potential in both laboratory techniques as well as medical applications. Of particular interest are their ability to cross cell membranes and to transport certain types of drugs to pre-designated locations within the human body.

Structure of a liposome formed by a phospholipid bilayer.

Structure

Liposomes are spherical structures, usually between 15nm and 1000nm in diameter. Various targeting ligands can be attached to their surface to direct them to the appropriate sites within cells; these include, but are not limited to, membrane proteins. It is important to differentiate liposomes from micelles; even though both of these macromolecular complexes are spherical and consist of lipids, a micelle is normally formed from ionized fatty acids, whereas a liposome consists of phospholipids. Furthermore, micelles consist of only a single layer of lipids, with their non-polar carbon tails clustered together at the center (therefore not allowing any water soluble compounds on the interior), whereas liposomes are constructed from a bilayer that does allow charged molecules on the inside. This is due to the presence of the hydrophilic glycerol-phosphate-alcohol heads of phospholipids, which define both the outer and inner surfaces of liposomes.

Composition of Liposomes

The Major Structural Components of Liposomes are:

1. Phospholipids - Phospholipids are the main component of the liposome's membrane. The phospholipids used in liposomes are further categorized into natural and synthetic phospholipids. The most common phospholipid used is known as lecithin (also known as phosphatidylocholine) and is amphipathic.
2. Cholestorol - Cholesterol molecules in the membrane increases separation between choline head groups which reduces the normal hydrogen bonding and electrostatic interaction.

Classification of Liposomes

Diagram of Liposome Types - Classification

Liposomes can be classified into several types according to their following features:

1. Size
2. Number of Lamellae

Liposome Types	Size	Number of Lamellae
Small Unilamellar Vesicles (SUV)	20 nm - 100 nm	Single
Large Unilamellar Vesicles (LUV)	100 nm - 400 nm	Single
Giant Unilamellar Vesicles (GUV)	1 µm and Larger	Single
Large Multilamellar Vesicles (MLV)	200 nm - ~3 µm	Multiple
Multivesicular Vesicles (MVV)	200 nm - ~3 µm	Multiple

1. Note: Lamellae refers to the lipid bilayers.
2. Note: Refer to Diagram to Right for Better Idea.

Synthesis

Liposomes are frequently synthesized by mixing and dissolving the phospholipids in organic solvent, such as chloroform or a chloroform-methanol mixture. A clear lipid film is subsequently formed by removal of the solvent, and hydration of this film eventually leads to formation of large, multilamellar vesicles (LMVs). An LMV consists of more than one bilayer, creating a complex the structure of which has several layers, analogous to the structure of an onion. Each bilayer is separated from the next by water. Smaller liposomes are produced by disrupting LMVs using sonication (agitation by sound-waves). This process yields small, unilamellar liposomes (SUVs) between 15nm and 50nm in diameter. These are not very stable and tend to form larger vesicles. Storing them above their phase transition temperature can help prevent formation of those larger vesicles. In order to synthesize larger liposomes, the method of extrusion is commonly used. Following several freeze-thaw cycles, the lipid suspension (the LMV suspension) is forced through polycarbonate filters containing pores, which leads to formation of liposomes with diameters similar to the size of the pores. This technique, if employed with pores of approximately 100nm in diameter, allows for the formation of large, unilamellar vesicles (LUVs) approximately 120nm – 140nm in size. These sizes are significantly more reproducible than those achieved through sonication.

Scientific Use

The study of phospholipid bilayers can help clarify several of their characteristics, such as their permeability under varying pH and temperature, their fluidity, and their electro conductivity. This is usually done with sheets of bilayers, which can be more easily generated and are more stable, not vesicles such as liposomes. However, liposomes have been found useful in studies of phase transitions and lattice spacing and were thus used for such purposes in the late 1960s and early 1970s.

Pharmaceutical and Medical Applications

The greatest potential of liposomes lies in the medical field, where their ability to deliver drugs and other compounds to specific areas of an organism are under active investigation. The basis for this ability is that the hydrophilic compounds contained by a liposome cannot pass through the hydrophobic core of the lipid bilayer, and are thus trapped on the inside. The attachment of glycosylated membrane proteins to the outside or the vesicle can help direct the liposome to the desired cells. These binding ligands may also be responsible for the fusion of the vesicle with the diseased cell. This process of drug delivery often lessens the toxicity of the drugs, which are sheltered from interaction with other non-target cells. Additionally, this mode of delivery can be more efficient, as long circulating liposomes may accumulate in a region of higher than average blood circulation, such as an inflammation site, a tumor, or other diseased areas.

Factors other than the membrane protein mediated fusion of vesicles to cellular membranes can contribute to the release of the compound contained in the liposome. An example of this is the pH-triggered permeability change of a heterogeneous liposome. Liposomes under certain pH conditions can become “leaky” and thereby release the compounds contained within. Doxorubicin, a cancer drug, is delivered to tumor cells by this mechanism, which does not affect overall liposome stability.

Advantages of Liposomes in Drug Delivery

Since the 1960's Liposomes and their use in the medicinal field has been greatly explored by pharmaceutical companies. Liposomes have many advantages as a method of drug delivery. These advantages are as follows:

1. Liposomes are biocompatible, completely biodegradable, non-toxic, flexible, and nonimmunogenic.
2. Liposomes have both a lipophilic and aqueous environment making it useful for delivering hydrophobic, amphipathic, and hydrophilic medicines.
3. Liposomes with their layers encapsulates the drug and serves as a protection of the drug from the environment as well as acting as a sustained release mechanism. This encapsulation also serves to protect sensitive areas from the drug as well.
4. Liposomes are extremely versatile in the form which they may be administered. These forms include suspension, aerosol, gel, cream, lotion, and powder which can then by administered through most common routes of medicinal administration.
5. Liposomes are also flexible in their size, and as such they can enclose a wide size range of molecules.
6. Liposomes can aide with active targeting as it has flexibility in coupling with site-specific ligands.

Disadvantages of Liposomes in Drug Delivery

Despite all the wonderful advantages, Liposomes do have some disadvantages when compared with other methods of drug delivery.

1. Liposomes encapsulated drugs require a high production cost.
2. Liposomes may have leakage and fusion of encapsulated drugs.
3. The liposome phospholipid may undergo oxidation and hydrolysis.
4. Liposomes have a shorter half-life.
5. Liposomes have lower solubility.

Where Drugs Interact with Liposome

List of Drugs Using Liposomes

Marketed Drug	Targeted Disease
DoxilTM	Kaposi’s Sarcoma
DaunoXomeTM	Kaposi’s Sarcoma, Breast & Lung Cancer
AmphotecTM	Leishmaniasis, Fungal Infections
Fungizone®	Leishmaniasis, Fungal Infections
VENTUSTM	Inflammatory Diseases
ALECTM	Expanding lung diseases in babies
Topex-Br	Asthma
Depocyt	Cancer therapy
Novasome®	Smallpox
Avian Retrovirus Vaccine	Chicken pox
Epaxal –Berna Vaccine	Hepatitis A
Doxil®	Refractory Ovarian Cancer
NyotranTM	Fungal Infections

References

• 1. http://www.nanopharmaceuticals.org/Liposomes.html
• 2. http://www.fasebj.org/content/24/5/1308.full
• 3. http://www.ncbi.nlm.nih.gov/pubmed/17092599
• 4. http://www.avantilipids.com/index.php?option=com_content&view=article&id=1384&Itemid=372
• 5. http://www.bioportfolio.com/resources/pmarticle/264214/Heterogeneous-Liposome-Membranes-With-Ph-triggered-Permeability-Enhance-The-In-Vitro-Antitumor.html
• 6. D Papahadjapoulos and N Miller."Phospholipid Model Membranes I. Structural characteristics of hydrated liquid crystals." Biochimica et Biophysica Acta. 135. (1967) 624-638.
• 7. H Trauble and D H Haynes."The volume change in lipid bilayer lamellae at the crystalline-liquid crystalline phase transition." Chem. Phys. Lipids. 7. (1971) 324-335.
• 8. Berg, Jeremy (2012). Biochemistry. New York: W. H. Freeman and Company. ISBN 978-1-4292-2936-4.