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. A major breakthrough in the field of long circulating liposomes has been the development of PEG-grafted liposomes. These are vesicular concentric structures, range in size from a nanometer several micrometers, containing a phospholipids belayed and are biocompatible, biodegradable and non-immunogenic. Liposomes are composite structures made of phospholipids and may contain small amounts of other molecules. Though liposomes can vary in size from low micrometer range to tens of micrometers, unilamellar liposomes as pictured here, are typically in the lower size range with various targeting ligands attached to their surface allowing for their surface-attachment and accumulation in pathological areas for treatment of disease. Liposomes are artificially prepared vesicles made of lipid belayed. Liposomes can be prepared by disrupting biological membranes, for example by sonication showing in fig.1.0 and 2.0

Fig:-1.0 Liposomes formation

Fig:-2.0 Liposomes for Drug Delivery

Liposomes are microscopic vesicles composed of a belayed of phospholipids or any similar amphipathic lipids. Liposome can carry both hydrophobic and hydrophilic molecules. They can be filled with drugs and used to deliver drugs. Another interesting property of liposomes is their natural ability to target cancer by their rapid entry into tumor sites. Anti-cancer drugs such as Doxorubicin (Doxil), Camptothecin etc. are currently being marketed in liposome delivery systems. Liposomes that contain low or high pH can be constructed such that dissolved aqueous drugs will be charged in solution. Another strategy for liposome drug delivery is to target endocytosis events and can also be decorated with opsonins and ligands. The use of liposomes for transformation of DNA into a host cell is known as lipofection. In addition to these applications, liposome’s can deliver the 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 ingredients, greater stability of active, reduction of unwanted side effects, and high biocompatibility. Despite of their potential value, the major obstacles are the physical stability and manufacture of the liposomal products and these problems still remain to be overcome. More liposomes based drug formulations can be expected in the near future both for delivery of conventional drugs and for new biotechnology therapeutics such as recombinant proteins, antisense oligonucleotides and cloned genes. Liposomes have very short circulation times. This shortcoming has been overcome by the inclusion of the polyethylene glycol derivatized phosphatidylethanolamine (PE) in the liposomes membrane. Due to new developments in liposomes technology, several liposomes based drug formulations are currently in clinical trials and recently some of them have been approved for clinical use. This review presents the brief theoretical description of long circulating liposomes and the major problem associated with long circulating liposomes that is accelerated blood clearance (ABC) on repeated injection. Depending on the composition liposomes can have a positive, negative, or neutral surface charge. Lecithin can provide liposome’s with a neutral surface; stearylamine and phosphatidic acid components provide positive1 and negative surface charge, respectively. Depending on the lipid composition, methods of preparations and the nature of the encapsulated agents, many types of liposomal products can be formulated. The ideal drug candidates for liposomal encapsulation are those that have potent pharmacological activity and are either highly lipid or water soluble. If a drug is water soluble, it will be encapsulated within the aqueous compartment and its concentration in the liposomal product will depend on the volume of the entrapped water and the solubility of that drug in the encapsulated water. The lipophilic drug is usually bound to the lipid bi‐layer or ‘dissolved’ in the lipid phase. A lipophilic drug is more likely to remain encapsulated during storage due to its partition coefficient. Since the lipophilic drug is associated with the lipid bi‐layers it will not leach out as readily to the ‘external’ water phase. Generally the encapsulation efficiency is higher for lipophilic drugs than hydrophilic drugs.^[^1-3]

Biomedical applications of liposome’s:^4-7

Both hydrophilic and hydrophobic drugs can be encapsulated in liposome’s. Liposomes are also relatively non-toxic and biodegradable. They therefore have a wide range of biomedical applications.

Protection against enzymatic degradation of drugs:

Liposomes are used to protect the entrapped drug against enzymatic degradation whilst in circulation. The basis is that the lipids used in their formulation are not susceptible to enzymatic degradation the entrapped drug is thus protected while the lipid vesicles are in circulation in the extracellular fluid. Upon penetration into the cell, the entrapped drug is released either by diffusion through the microsphere shell, dissolution of the shell or degradation of the shell by lysosomal enzymes. Thus, lactamase sensitive antibiotics, e.g., the penicillins and cephalosporins have been encapsulated due to this reason to protect against the lactamase enzyme. Liposomes also offer protection for its encapsulated drugs in the environment of the gastrointestinal tract and facilitate the gastrointestinal transport of a variety of compounds. Liposomes are therefore candidates to be explored for oral delivery of insulin and proteins for use as vaccines, which are otherwise orally degradable. Liposomes offer a number of advantages as carriers of vaccine agents in that they are biodegradable and non-toxic. Drugs encapsulated in lipososmes can elicit both humoral immunity when given orally and cell-mediated immunity.50 Liposomes are now being employed as oral vaccines in numerous immunization procedures.6 Twenty five years after the discovery of the immunological adjuvant properties of liposome’s, they are now considered the major candidate as the base for oral vaccine against hepatitis A, which is being licensed for use in humans.

Drug targeting:

The need for “drug targeting” arises from a problem situation whereby a drug administered enters the blood stream and is distributed to varying extents throughout the body when the actual desire is to deliver or direct the drug selectively to its site of action. This site could be an organ structure, a cell subset, or even an intracellular region. In such a case pumping the drug throughout the whole body is not only wasteful but, more fundamentally, it is also lik ely to lead to undesirable side effects. On the other hand, restricting the distribution of the drug to the specific target site should allow for an increase in efficacy at low dose with attendant decrease in toxicity. Hence, the benefits of drug targeting include reduced drug waste, and it is possible to deliver a drug to a tissue or cell region not normally accessible to the free or untargeted drug. The approach for drug targeting via liposome’s involves the use of ligands (e.g. antibodies, sugar residues, apoproteins or hormones), which are tagged on the lipid vesicles. The ligand recognises specific receptor sites and, thus, causes the lipid vesicles to concentrate at such target sites. By this approach the otherwise preferential distribution of liposome’s into the reticuloendeothelial system RES (liver, spleen and bone marrow) is averted or minimised. The preferential distribution of liposome’s into the RES can be modified by the incorporation in the liposome membrane of protein or carbohydrates possessing specific affinity toward a target tissue or organ. A ligand selection is based on its recognition by, and specificity for, the target site. In cancer treatment, for example, one of the approaches is to target the drug to tumour cells via receptor specific ligands, which may be specific antibodies for antigens produced by the tumour cells. The first step, therefore, is to determine the antigens that are produced by the tumour cells. Also, molecules bearing oligosaccharide chains have been used as ligands for direction, and specifi attachment,to ganglion sites in cells.

Topical drug delivery:

The application of liposome’s on the skin surface has been proven to be effective in drug delivery into the skin. Liposomes increase the permeability of skin for various entrapped drugs and at the same time diminish the side effect of these drugs because lower doses are now required. Liposomes have also found an important application in cosmetics for skin care preparations. In this regard, the liposome’s are applied to the skin in the form of solution or in hydrogels. Hydrophilic polymers are suitable thickening agents for the gels. However, the liposome’s may in certain instances be trapped in the polymeric network of the hydrogels and, hence, impair bioavailability into the skin. Nevertheless, Gabrijelcic et al found enhanced transport of liposome-entrapped substances into the skin from hydrogels prepared from xanthan gum. The enhanced drug transport into the skin is attributed to the lipid nature of the vesicles, which serve as carriers for the drug.

Targeting cancer:

Another interesting property of liposome’s 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 particles in the blood from leaking out of the vessel. Tumour vessels do not contain the same level of seal between cells and are diagnostically leaky. This ability is known as the Enhanced Permeability and Retention effect. Liposomes of certain sizes, typically less than 200 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.

Treatment of human immunodeficiency virus (HIV) infections:

Several antiretroviral nucleotide analogues have been developed for the treatment of patients suffering from the acquired immunodeficiency syndromes (AIDS). These include antisense oligonucleotide, which is a new antiviral agent that ha show potential therapeutic application against HIV These antiviral agents are able to combat replication of the HIV by inhibiting reverse transcriptase and, thereby, viral DNA synthesis.68 However, these agents display a dose-related toxicity. The effective dose can be reduced by encapsulation of such drugs in liposome’s, thus reducing the incidence of toxicity. The greater efficacy of the liposomal formulation relates to the preferential uptake of the liposome’s into the virus compared with the host tissue.

Pharmacokinetic considerations:^4,6,9

Most small molecular chemotherapeutic agents have a large volume of distribution on intravenous (IV) administration of liposomes. The result of this wide distribution is often a narrow therapeutic index due to a high level of toxicity on healthy tissues Through encapsulation of drugs in liposome’s, the volume of distribution is significantly reduced and the concentration of drug at the desired site of action increased. For instance, liposomal drug delivery led to an increase in the amount of drug that can be effectively delivered to tumour sites in anticancer therapy. Liposomes are predominantly removed from circulation by phagocyte cells of the reticuloendothelial system (RES), thus accumulating to a large extent in organs like liver and spleen. This biodistribution pattern can be used for passive targeting of diagnostics to these organs. The RES should, therefore, be saturated with empty vesicles when other sites are the drug targets. Information on biodistribution is, therefore, important for drug targeting by liposome’s. Liposomes given intravenously usually interact with at least two distinct groups of plasma proteins. These are the plasma high density lipoproteins and the so-called opsonins, which bind to the surface of vesicles and mediate their endocytosis by the mononuclear phagocyte system (macrophages). The rate of liposome clearance from blood circulation will, therefore, depend on the ability of opsonins to bind to the liposome surface. The rate can be manipulated through appropriate selection of liposome characteristics. For instance, “fluid” vesicles are removed more rapidly from blood circulation than “rigid” ones. Clearance from the blood stream is also influenced by vesicle size and surface charges. The longest half-life is obtained when liposome’s are relativel small (diameter <0.05nm) and carry no net surface charge. The pharmacokinetic behaviour of liposome’s depends on the route of injection such as intraperitoneal, subcutaneous or intramuscular route. Lasic and Papahadjopoulos have shown that coating the liposome surface with polyethyleneglycol and other hydrophobic part of phospholipids substantially prolongs the half-life of liposome’s in the blood.

Liposomes in vivo:

Over the last 30 years much information has been gained concerning the behavior of liposome’s in vivo. It was found that clearance of liposomes from the circulation and their biodistribution depend on the physicochemical properties of the liposomes such as liposome size, surface charge and belayed packing, as well as on other factors such as dose and route of administration. The inter-relationships between these factors are best illustrated when considering the physiological and anatomical barriers liposomes encounter en route to a disease site, for example a solid tumour. The iv.route of administration was chosen as an example since it is the most common and universal route, which allows at the same time to target the primary tumour as well as sites of metastatic tumor growth. In order to transport drugs to or into tumour cells liposomes must avoid interactions with circulating cells and proteins in the blood, and uptake by phagocytic cells, which are responsible for their removal from the circulation.Then they must leave the vasculature (extravasate) at the site of tumour growth. Liposomes have then to cross the space between the vasculature and the tumour (interstitial space) and enter the tumour mass There, dependent on the drug being delivered, the liposome’s have to be taken up into the tumour cells and facilitate the delivery of the drug to its intracellular site of action For conventional drugs there is no absolute need for the liposome’s to associate with the tumour cells and to be taken up into the cells. Drug released within the tumour, or even in tissue nearby, can diffuse and kill target cells in adjacent areas (by-stander effect). Genetic drugs such as pDNA on the other hand have to be delivered into the target cells. These large highly charged molecules are not readily taken up by cells and lack stability in the extracellular and intracellular environments.

Liposome clearance:

Immediately after iv. injection, liposomes become coated by proteins circulating in the blood. Some of these proteins compromise the integrity of the lipid belayed causing rapid leakage of liposome contents. Others promote recognition and subsequent elimination of liposomes from the blood. For example, liposomes composed of unsaturated lipids such as EPC rapidly lose their membrane integrity through lipid transfer to lipoproteins and disintegrate This process involves insertion of ApoA1, an apolipoprotein found predominantly in the high-density lipoprotein fraction, into the lipid belayed Other proteins called opsonins, mark liposome’s for removal through phagocytic cells .Examples of opsonins include components of the complement system (C3b, iC3b), IgG, β2 glycoprotein 1 and fibronectin. The removal of foreign matter including liposome’s is carried out by the mononuclear phagocyte system (MPS), in particular the resident macrophages of the liver (Kupffer cells), spleen, lung and bone marrow. The bulk of the injected liposome’s accumulate in the liver and spleen.

Types of liposomes:^5,6,8

Liposomal vesicles were prepared in the early years of their history from various lipid classes identical to those present in most biological membranes. Basic studies on liposomal vesicles resulted in numerous methods of their preparation and characterization.

Definition:-“Liposomes are broadly defined as lipid bilayers surrounding an aqueous space.”

Multilamellar vesicles (MLV):- Multilamellar vesicles (MLV) consist of several (up to 14) lipid layers (in an onion-like arrangement) separated from one another by a layer of aqueous solution. These vesicles are over several hundred nanometers in diameter.

Small unilamellar vesicles (SUV):- Small unilamellar vesicles (SUV) are surrounded by a single lipid layer and are 25–50 nm (according to some authors up to 100 nm) in diameter.

Large unilamellar vesicles (LUV):- Large unilamellar vesicles (LUV) are, in fact, a very heterogeneous group of vesicles that, like the SUVs, are surrounded by a single lipid layer. The diameter of these liposomes is very broad, from 100 nm up to cell size (giant vesicles).

Besides the technique used for their formation the lipid composition of liposomes is also, in most cases, very important. For some bioactive compounds the presence of net charged lipids not only prevents spontaneous aggregation of liposomes but also determines the effectiveness of the entrapment of the solute into the liposomal vesicles. Natural lipids, particularly those, with aliphatic chains attached to the backbone by means of ester or amide bonds (phospholipids, sphingolipids and glycolipids) are often subject to the action of various hydrolytic (lipolytic) enzymes when injected into the animal or human body. These enzymes cleave off acyl chains and the resulting lysolipids have destabilising properties for the lipid layer and cause the release of the entrapped bioactive component(s). As a result new types of vesicles, that should merely bear the name of liposome’s as their components are lipids only by similarity of their properties to natural (phospho) lipids, have been elaborated. These vesicles, still named liposome’s, are made of various amphiphile molecules (the list of components is long). The crucial feature of these molecules is that upon hydration they are able to form aggregation structures resembling an array and have properties of natural phospholipid bilayers.

Materials used in liposomes prepration:^4,10

Polyethyleneglycol (PEG), Egg, Soyabean, Phosphatidylcholine, L-alphadipalmitoyl phosphatidylchline (DPPC), Gelatin (300 bloom), Cholesterol succinyl, Distilled water, Omega-hydroxy polyoxyethylene.

Method of preparation:

Formation of liposome’s and nanoliposomes is not a spontaneous process. Lipid vesicles are formed when phospholipids such as lecithin are placed in water and consequently form one belayed or a series of bilayers, each separated by water molecules, once enough energy is supplied .Liposomes can be created by sonicating phospholipids in water. Low shear rates create multilamellar liposome’s, which have many layers like an onion. Continued high-shear sonication tends to form smaller unilamellar liposomes. In this technique, the liposome contents are the same as the contents of the aqueous phase. Sonication is generally considered a "gross" method of preparation as it can damage the structure of the drug to be encapsulated. Newer methods such as extrusion and Mozafari method are employed to produce materials for human use.

Industrial production of liposomes

The several preparation methods described in the literature, only a few have potential for large scale manufacture of liposomes. The main issues faced to formulator and production supervisor are presence of organic solvent residues, physical and chemical stability, progeny control, sterility, size and size distribution and batch to batch reproducibility. Liposomes for parenteral use should be sterile and progeny free. For animal experiments, adequate sterility can be achieved by the passage of liposomes through up to approximately 400 nm pore size Millipore filters. For human use, precautions for sterility must be taken during the entire preparation process.

(i) Detergent Dialysis

(ii) Microlluidization

(iiia) Proliposomes

(iiia) Lyaphilization

Limitation:

In spite of these advances, the current clinically approved liposomal formulations still have some problems associated with their circulation time. Recently many researchers studied the side effects of liposomal doxorubicin which are PEG coated liposome’s as a DOX. To overcome these side effects, many researchers still attempt to further enhance the circulation time of carrier which is considered as the main reason for side effects.

CONCLUSION:

The ability of liposome’s consisting of components other than phospholipids and cholesterol or their semi synthetic derivatives to enhance the encapsulation of bioactive substances provides new promising perspectives for establishing new, efficient and stable carriers for drug delivery. Liposomes have been prepared from a variety of synthetic and naturally occurring phospholipids and generally contain cholesterol as membrane stabiliser. Several methods of preparing liposome’s were identified, which could influence the particle structure, degree of drug entrapment and leakage of the liposome’s. It was also identified that there are improved pharmacokinetic properties with liposomal drugs compared to the free drugs. Furthermore, liposome’s are tools for drug targeting in certain biomedical situations (e.g., cancer) and for reducing the incidence of dose-related drug toxicity. Instability of the preparations (particularly leakage) is a problem, which is yet to be overcome before full commercialization of the process can be realized.The ability of liposomes consisting of components other than phospholipids and cholesterol or their semisynthetic derivatives to enhance the encapsulation of bioactive substances provides new promising perspectives for establishing new, efficient and stable carriers for drug delivery. We hope that in the near future PLARosomes, the components of which are not directly toxic (unpublished data), will be used for the efficient entrapment and delivery of drugs to human or animal organisms. Resorcinolic lipids and modern studies on their biological activities are relatively new but show a tremendous potential not only as components of PLARosomes PLARosomes may come into commercial use relatively soon because of the benefits anticipated from the knowledge generated through the use of conventional liposomes.

ACKNOWLEDGEMENT:

Authors are very much thankful to the MJRP College of Heath Care and Allied Science, MJRP University, Jaipur (R.J) India, BM College of Pharmacy Education and Research for providing necessary facilities.

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Received on 19.08.2011

Accepted on 11.09.2011

Research Journal of Pharmaceutical Dosage Forms and Technology. 3(5): Sept.-Oct. 2011, 193-198