HOD, Department of Biotechnology, Raipur Institute of Technology, Raipur (CG)
*Corresponding Author E-mail: email@example.com
Today, clinical medicine possesses an extremely long list of different pharmaceutical products and every year many new drugs are added to the list with the understanding of molecular mechanisms of diseases. Scientists and physicians are never satisfied only with a favorable drug action against the disease under treatment. The task of avoiding undesirable drug actions on normal organs and tissues and minimizing side effects of the therapy is very important. Thus, screening of biologically active compounds became necessary, permitting the choice of drug with selective action on the appropriate organs or tissues. A liposome is an artificially prepared vesicle composed of a lipid bilayer. The liposome can be used as a vehicle for administration of nutrients and pharmaceutical drugs. Liposomes can be prepared by disrupting biological membranes such as by sonication.
Liposomes are composed of natural phospholipids, and may also contain mixed lipid chains with surfactant properties. A liposome design may employ surface ligands for attaching to unhealthy tissue. The major types of liposomes are the multilamellar vesicle (MLV), the small unilamellarvesicle (SUV), and the large unilamellar vesicle (LUV).
KEYWORDS: Liposomes, drug delivery, target therapy, encapsulation.
Liposomes are sub-micron particles that are finding important applications in fields such as biotechnology (in applications like siRNA delivery, antibody delivery), cosmetology (emulsions and creams etc.) and the pharmaceutical industry (chemotherapeutic delivery, altering the PK/PD of drug). Liposomes are composed of phospholipids that have a polar end attached to a non-polar chain. When these phospholipids are introduced into an aqueous medium, they self-assemble into bilayer vesicles with the polar ends facing the aqueous medium and non-polar ends forming a bilayer. In pharmaceutical applications the active molecule (drug) is usually incorporated into the liposome either into the hydrophilic pocket or sandwiched between the bilayers depending on the hydrophilicity or lipophilicity of the drug. Chemotherapeutics such as Doxorubicin and Paciltaxel have been used to treat cancers of various kinds for over two decades.
The main disadvantage with these or any other chemotherapeutic drug is their inability to differentiate between a normal cell and a cancer cell. This leads to unwanted side effects such as loss of hair, stomach ulcers and loss in body weight. Drug delivery systems or targeting systems that would channel these and other therapeutics to the area of interest (tumors etc.) have garnered tremendous interest.
DOXIL, a reformulated version of Doxorubicin, was one of the first drugs to be approved that is delivered using a liposome. The Doxorubicin drug lies within the hydrophilic pocket of a liposome coated with PEG (polyethylene glycol) to evade detection and destruction by the immune system. The PEG coating improves the stability and lengthens the half-life in circulation. A PEG coated liposome is often referred to as a sterically enhanced or stealth liposome. For solid tumors, liposomes are of particular interest because of their easy manipulation and optimum size. It has been known for some time that the tumor areas have leaky vasculature and poor lymphatic drainage. This can be used as a targeting tool with what has been described as Enhanced Permeation and Retention effect (EPR). This refers to the permeation and retention of molecules of sizes 50 to 150 nm in to the tumor area. Hence size measurement is of prime most importance in liposomal drug delivery systems.
Liposomes were first described by British haematologist Dr Alec D BanghamBanghamand Horne1964; Horne et al., 1963; Bangham et al., 1962 in 1961 at the Babraham Institute, in Cambridge. It were discovered when Bangham and R. W. Horne were testing the institute's new electron microscope by adding negative stain to dry phospholipids. The resemblance to the plasmalemma was obvious, and the microscope pictures served as the first real evidence for the cell membrane being a bilayer lipid structure. The word liposome derives from two Greek words: lipo ("fat") and soma ("body"); it is so named because its composition is primarily of phospholipid.
Liposomes are lyotropic liquid crystals composed of relatively biocompatible and biodegradable materials and consist of an aqueous core entrapped by one or more bilayers of natural and/or synthetic lipids. Drugs with widely varying lipophilic nature can be encapsulated in liposomes either in the phospholipid bilayer, in the entrapped aqueous core or at the bilayer interface. Reformulation of drugs in liposomes has provided an opportunity to enhance the therapeutic indices of various agents mainly through the alteration of bio distribution. They are versatile drug carriers, which can be used to control retention of entrapped drugs in the presence of biological fluids, controlled vesicle residence in the systemic circulation or other compartments in the body and enhanced vesicle uptake by target cells (Pozanansky and Juliano 1984). Liposomes composed of natural lipids are biodegradable, biologically inert, weakly immunogenic (Gregoriadis and Florence 1993; van Rooijen and van Nieuwmegen 1980), produce no antigenic or pyrogenic reactions and possess limited intrinsic toxicity (Campbell 1983). Therefore, drugs encapsulated in liposomes are expected to be transported without rapid degradation and minimum side effects to the recipients. Moreover, efforts have been made to assess the specificity of drug carriers to the target organs, cells or compartments within the cells (Gregoriadis 1977). Liposomes are better suited for assessing their targetable properties because of the ease of modifying their surface when compared to other drug carriers such as nanoparticles (Grislain et al., 1983; Illum et al., 1983) and microemulsions (Hashida et al., 1977; Mizushima et al., 1982). Many approaches have been attempted to achieve targetable properties, including noncovalent association of cell specific antibodies with liposomes (Gregoriadis and Neerunjun 1975), coating of liposomes with heat aggregated immunoglobulins M (IgM) (Weissmann et al., 1975), covalent attachment of poly and monoclonal antibodies to the liposomes, glycoprotein bearing liposomes (Banuo et al., 1983) and natural (Szoka and Mayhew 1983; Spanzer and Scherphof 1983; Soriano et al., 1983) and synthetic glycolipid containing liposomes. The compounds entrapped into the liposomes are protected from the action of external media, particularly enzymes (Chaize et al., 2004) and inhibitors. Moreover, liposomes afford a unique opportunity to deliver the drugs into cells by fusion or endocytosis mechanism and practically any drug can be entrapped into liposomes irrespective of its solubility.
It should be noted that formation of liposomes and nanoliposomes is not a spontaneous process. Lipid vesicles are formed when phospholipids such as lecithin are placed in water and consequently form one bilayer or a series of bilayers, each separated by water molecules, once enough energy is supplied. Liposomes can be created by sonicating phospholipids in water are employed to produce materials for human use. Liposomes are used for drug delivery due to their unique properties. A liposome encapsulates a region on aqueous solution inside a hydrophobic membrane; dissolved hydrophilic solutes cannot readily pass through the lipids. Hydrophobic chemicals can be dissolved into the membrane, and in this way liposome can carry both hydrophobic molecules and hydrophilic molecules. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bilayers such as the cell membrane, thus delivering the liposome contents. By making liposomes in a solution of DNA or drugs (which would normally be unable to diffuse through the membrane) they can be (indiscriminately) delivered past the lipid bilayer.
Liposomes are used as models for artificial cells. Liposomes can also be designed to deliver drugs in other ways. Liposomes that contain low (or high) pH can be constructed such that dissolved aqueous drugs will be charged in solution (i.e., the pH is outside the drug's pI range). As the pH naturally neutralizes within the liposome (protons can pass through some membranes), the drug will also be neutralized, allowing it to freely pass through a membrane. These liposomes work to deliver drug by diffusion rather than by direct cell fusion. Another strategy for liposome drug delivery is to target endocytosis events. Liposomes can be made in a particular size range that makes them viable targets for natural macrophage phagocytosis. These liposomes may be digested while in the macrophage's phagosome, thus releasing its drug. Liposomes can also be decorated with opsonins and ligands to activate endocytosis in other cell types. The use of liposomes for transformation or transfection of DNA into a host cell is known as lipofection.
In addition to gene and drug delivery applications, liposomes can be used as carriers for the delivery of dyes to textiles, pesticides to plants, enzymes and nutritional supplements to foods, and cosmetics to the skin. The use of liposomes in nano cosmetology also has many benefits, including improved penetration and diffusion of active ingredients, selective transport of active ingredients, longer release time, greater stability of active, reduction of unwanted side effects, and high biocompatibility. Another interesting property of liposomes are their natural ability to target cancer. The endothelial wall of all healthy human blood vessels is encapsulated by endothelial cells that are bound together by tight junctions. These tight junctions stop any large particle in the blood from leaking out of the vessel. Liposomes of certain sizes, typically less than 400 nm, can rapidly enter tumour sites from the blood, but are kept in the bloodstream by the endothelial wall in healthy tissue vasculature. Anti-cancer drugs such as Doxorubicin (Doxil), Camptothecin and Daunorubicin (Daunoxome) are currently being marketed in liposome delivery systems. Further advances in liposome research have been able to allow liposomes to avoid detection by the body's immune system, specifically, the cells of reticuloendothelial system (RES).These liposomes are known as "stealth liposomes", and are constructed with PEG (Polyethylene Glycol) studding the outside of the membrane. The PEG coating, which is inert in the body, allows for longer circulatory life for the drug delivery mechanism. However, research currently seeks to investigate at what amount of PEG coating the PEG actually hinders binding of the liposome to the delivery site. In addition to a PEG coating, most stealth liposomes also have some sort of biological species attached as a ligand to the liposome in order to enable binding via a specific expression on the targeted drug delivery site. These targeting ligands could be monoclonal antibodies (making an immunoliposome), vitamins, or specific antigens. Targeted liposomes can target nearly any cell type in the body and deliver drugs that would naturally be systemically delivered. Naturally toxic drugs can be much less toxic if delivered only to diseased tissues. Polymersomes, morphologically related to liposomes can also be used this way.
The Pharmaceutical Industry is one of the most potent industries all over the world, facing high risks and important challenges. Both, the cost of bringing a New Molecular Entity (NME) to market is between 500-1000 million Euros and the time required to launch a NME is 10-15 years. One of the challenges to rationalise and further improve these NME will be the development of appropriate drug delivery systems, namely liposomes. During the past 30 years liposomes have received increased attention from the scientific community, as well as from the Industry, due to the possibility of being a pharmaceutical carrier for numerous problematic drugs. This success seems to drive the present interest on the field with more than 2000 papers and reviews per year. Intracellular infections, namely those localized in mononuclear phagocytic system (MPS) are very difficult to eradicate due to the low access of drugs to the sites of infection resulting in sub- therapeutic local drug concentration. Liposomes are ideal carriers to transport drugs to infected macrophages, as they have the tendency to accumulate in MPS cells by passive targeting. This capability was exploited for the treatment of leismaniasis and tuberculosis, with liposomes incorporating dinitroanilines and rifamycins respectively.
Rifamycins are candidates to be associated to liposomes for treating mycobacterial infections specially M. aviumand M. tuberculosis. We have developed different types of liposomes incorporating a rifamycin, Rifabutin (RFB) and tested inappropriate models of M. tuberculosis and M. avium infection. RFB encapsulated in conventional liposomes showed a superior therapeutic effect, than the commercial antibiotic (free RFB) This superior effect has been observed either through the reduction on the number of viable colony forming units in liver, spleen and lungs as well as through histological and immunological studies (Gaspar et al., 2005; Gaspar et al., 2000). Macromolecules, namely enzymes with therapeutic activity, were also incorporated in liposomes in view of reducing some of the common drawbacks such as low circulation time, immunogenic reactions and reduced therapeutic activity. The enzyme L- Asparaginase (L-ASNase) is currently a standard agent for the treatment of children with acute lymphobastic leukaemia. Due to hypersensitivity reactions the treatment needs discontinuation in up to 25% of patients. These disadvantages were overlapped by means of incorporating L-ASNase into liposomes.
Superoxide dismutase (SOD) incorporated in liposomes also presented advantages over the free from of the enzyme in the treatment of rat adjuvant arthritis.Liposomes increased the short half-life of SOD, targeted the enzyme to the inflamed sites and increased its anti-inflammatory activity. Long circulating liposomes incorporating SOD are superior to conventional liposomes, in terms of anti-inflammatory activity (Cruz et al., 1993; Gaspar et al., 1996). These are examples of the potential of Liposomes as delivery system for NME or even to recue problematic old drugs.
1. Bangham, A. D.; Horne, R. W. (1964). "Negative Staining of Phospholipids and Their Structural Modification by Surface-Active Agents As Observed in the Electron Microscope". Journal of molecular biology8: 660668.
5. Chaize B., J. P. Colletier, M. Winterhalter and D. Fournier, Encapsulation of enzymes in liposomes: high encapsulation efficiency and control of substrate permeability, Artif. Cells Blood Substit. Immobil. Biotechnol. 32 (2004) 6775.
8. Gaspar, M.M., Penha, A.F., Sousa, A.C., Eleutιrio, C.V., Domingues, S.A., Cruz, A., Pedrosa, J., Cruz, M.E.M. 2005. Proceed.7thLiposomes Advances, Progress in Drug and Vaccine Delivery, Londres, Inglaterra, p. 50.
14. Hashida M., Y. Takahashi, S. Muranishi and H. Sezaki, An application of water in oil and gelatin microsphere in oil emulsions to specific delivery of anticancer agents into stomach lymphatics, J. Pharmacokin. Biopharmacol. 5 (1977) 241244.
16. Illum L., P. D. E. Gones, J. Kreuker, R. W. Daldwin and D. D. Davis, Adsorption of monoclonal antibodies to polyhexylcyanoacrylate nanoparticles and subsequent immunospecific binding to tumor cells, Int. J. Pharm. 17 (1983) 6569.
19. Soriano P., J. Dijkstra, A. Legrand, H. Spanjer, D. Londos-Gagliardi, F. Roerdink, G. Scherphof and C. Nicolau, Targeted and non-targeted liposomes for in vivo transfer to rat liver cells of a plasmid containing the preproinsulin I gene, Proc. Natl. Acad. Sci. USA 80 (1983) 71287131.
23. vanRooijen N. and R. van Nieuwmegen, Liposomes in immunology: multilamellarphosphatidylcholine liposome as a simple biodegradable and harmless adjuvant without any immunogenic activity of its own, Immunol. Commun. 9 (1980) 243256
24. Weissmann G., D. Bloomgerden, R. Kaplan, C. Cohen, S. Hoffstein, T. Collins, A. Gotlieb and D. Nagle, A general method for introduction of enzymes, by means of immunoglobulin coated liposomes into lysosomes of deficient cells, Proc. Natl. Acad. Sci. USA 72 (1975) 8894.