LIPOSOME: A NOVEL DRUG DELIVERY SYSTEM

         Saikat Santra, 
       Student,B.pharm 

         Bengal School of Technology(A college of pharmacy)

B

• v  INTRODUCTION: 
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•          Paul erchlich in 1906 initiated the era of development for targeted drug delivery when he envisaged  a drug delivery mechanism that that would target drug directly to diseased cell what he called as magic bullets.[1]
•     Liposome were discovered in the early 1960s by bingham and co workers and subsequently become the most extensively explored drug delivery system structurally liposome are phospholipids based collidial vesicular structure in which hydrophilic core is entirely enclosed by membranous lipid by layers.
•    Liposome are manufactured using various procedures in which water soluble materials are entrapped by using aqueous solution of these materials as hydrating fluid or by the addition of drug solution at some stage during the manufacturing. The lipid soluble materials are solubilized in the organic solution of the constitutive lipid and then evaporated to a dry drug containing lipid film followed by its hydration.
•    Liposome due to their biphasic environment can act as carriers for both lipophilic and hydrophilic drugs highly hydrophilic drugs are located exclusively in the aqueous domains where as highly lipolphilic drugs are entrapped within the lipid bilayers of the liposomes. It is interesting to note that the loss of drug on long term storage is maximal with the former, and minimal or none with the latter. Drugs with intermdiary partion coefficients, Log p<4 impos eproblem for loading bas they equilibrate between the lipid and aqueous domains and are prone to appreciable degree of leakageon storage.
•    The delivery aspect of liposomes could also be exploited for drug that must penetrate the plusma membrane in order to be therapeutically beneficial. The use of liposomal carrier system assists to surpass the membrane barriers and promote the non specific entry of drugs into the cellular interiors.
•    Liposomes are relatively non toxic,non immunogenic,Biocompatible and biodegradable;an and can deliver the drug systemically with an increased therapeutic index and miniimized toxicity.Liposomes have also been successfully used for several other particles in drug delivery such as Solubilization of water insoluble drug ,  protection of sensitive drug molecule,alteration of pharmacokinetics and biodistribution and enhancing in tracellular uptake. To increase the taegeting
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•    Potential of liposome and decreases RES uptake, stealth liposome (pegaylated) are the recent innovation in the field of drug delivery DOXII, is the marketed product doxorubicin based on liposome. It is being used for the targeted delivery of anticancer agent. Doxorubicm, in the treatment of AIDS related kaposts sarcoma [3]
•   ADVANTAGES:
•         I.          Liposome are bio compatible, completely biodegradable, nontoxic and non-immunogenic.
•       II.          Liposome are suitable for delivery of hydrophobic amphiphatic and hydrophilic drugs.
•      III.          Liposome are protecting the encapsulated drug from the external environment.
•     IV.          Liposome are reduced the toxicity and increased the theraputicaleffect of the drugs.
•    DISADVANTAGES:
•         I.          The production cost is high.
•       II.          Leakage and fusion of encapsulated drug/molecules can occur.
•      III.          It has short half life-In reticuloendothelial system, particularly the Chuffer cells in the liver remove leptosomes from the circulation.
•     IV.          Stability problem.
•    CLASSIFICATION OF LIPOSOMES:
•    Liposome 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 liposomes vesicles resulted in numerous methods of their reparation and characterization. Liposome is broadly defined as lipid bilayer surrounding an aqueous space. Multilamellar vesicles (MLV) consist of several (up to 14) lipid layers (in an onion-like arrangement) separated nanometers in diameter. Small unilamellar vesicles (SUV) are surrounded by a single lipid layer and are 25-50nm.Based on structural parameters leptosomes are-
•   1. Small unilamellar vesicles (SUV): Size range from 20-40nm.
•    2. Medium unilamellar vesicles (MUV): Size range from 40-80nm. Large unilamellar vesicles (LUV): Size range from 100-1000nm.
•    3. Oligolamellar vesicles (OLV) these are made up of 2-10 bilayers of lipids surrounding a large internal volume.
•   4. Multilamellar vesicles (MLU). They have several bilayers. They can compartmentalize the aqueous volume in infinite members of ways. They differ according to way by which they are prepared. The arrangements can be onion like arrangements of concentric spherical bilayer of LUV/MLV enclosing a large number of SUVs.
•     METHOD OF PREPARATION
•        All the methods of preparing the leptosomes involve four basis stages:
•   1        Drying down lipids from organic solvent.
•   2        Dispersing the lipid in aqueous media.
•    3        Purifying the resultant liposome.
•    4        Analyzing the final product.[5]
•    GENERAL METHODS OF PREPARATION:
•    The lipid is dissolved in organic solvent. The solvent is evaporated leaving a small film of lipids on the wall of container. An aqueous solution of drug is added. In first procedure the mixture is agitated to produce multi lamellar vesicle and then sonicated to get SUVs. In the second procedures the mixture is sonicated and the solvent is evaporated to get LUVs after extrusion SUVs are formed. Drug can be incorporated into the aqueous or buffer if it is water soluble or included in organic solvent if it is hydrophobic.
•    SPECIFIC METHODS:  
•   These are classified into 3 types based on the mode of dispersion:
•   1.      Physical dispersion method.
•    2.      Solvent dispersion method.
•   3.      Detergent solubilization method.
•   1.  PHYSICAL DISPERSION METHOD:
•    In this method the aqueous volume enclosed within lipid membrane is about 5-10% which is very small proportion of total volume used for preparation. So large amount of water soluble drug is wasted during preparation. But lipid soluble drug can be encapsulated to high percentage. In these methods MLVs are formed and further treatment is required for preparation of unilamellar vesicle.[6]The following  are types of physical dispersion methods:
•   A.      Sanitation.
•    B.      French pressure cell: extrusion.
•    C.      Freeze-thawed leptosomes.
•   D.     Lipid film hydration by hand shaking, non-hand. Shaking or freeze drying.
•   E.      Micro-emulsification.
•   F.      Membrane extrusion.
•   G.     Dried reconstituted vesicles.[7]·      
•     Sonication:
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•   Sonication is perhaps the most extensively used method for the preparation of SUV. Here, MLVs are sancated either with a bath type sonicator or a probe sonicator under a passive atmosphere. The main disadvantage of this method are very low internal volume/encapsulation efficiency, possible degradation of phospholipids and compounds to be encapsulated, elimination of large molecules, metel pollution from probe tip, and presence of MLV along with SUV[4]. There are two sonication techniques:
• ·        Probe sonication : The tip of a sonicator is directly engrossed into the liposome dispersion. The energy input into lipid dispersion is very high in this method. The coupling of energy at the tip result in local hotness, therefore, the vessel must be engrossed into a water/ice bath. Thoroughout the sonication upto 1h, more than 5 % of the lipids can be de-esterified. Also, with the probe sonicator, titanium will slough off and pollute the solution.
• ·        Bath sonication : The liposome dispersion in a cylinder is placed into a bath sonicator.
•   Controlling the temperature of the lipid dispersion is usually easier in this method, in contrast to
•   Sonication by dispersal directly using the tip. The material being sonicated can be protected in a
•   Sterile vessel, dissimilar the probe units, or under the inert atmosphere[6
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• ·       French pressure cell: extrusion:
•   French pressure cell involves the extraction of MLV through a small orifice [4]. An important feature of the French press vesicle method is that the proteins do not seem to be significantly pretentious during the procedure as there sonication. An interesting comment is that French press vesicle appears to recall entrapped solutes significantly longer than SUV’s do, produced by sonication or detergent removal [6]
•   2. Solvent dispersion method:
•   The method involves gentle handing of unstable materials. The method has several advantages over sonication method [9].The resulting liposomes are rather larger than sonicated SUVs. The drawbacks of the method are that the high temperature is difficult to attain, and the working volumes are comparatively small (about 50 mL as the maximum) [4].
• ·        Ether injection (solvent vaporization ):
•   A solution of lipids dissolved in diethyl ether or ether-methanol mixture is gradually injected to an aqueous solution of the material to be encapsulated at 55ºC to 65ºC or under reduced pressure. The consequent removal of ether under vacuum leads to the creation of liposomes. The main disadvantages of the technique are that the population is heterogeneous (70 to 200 nm) and the exposure of compounds to be encapsulated to organic solvents at high temperature [10][11].
• ·        Ethanol injection:
•   A lipid solution of ethanol is rapidly injected to a huge excess of buffer. The MLVs are at once formed. The disadvantages of the method are that the population is heterogeneous (30 to 110 nm), liposomes are very dilute, the removal all ethanol is difficult because it forms into zoetrope with water, and the probability of the various biologically active macromolecules to inactivate in the presence of even low amounts of ethanol is high[12]
•   3. Detergent removal method (removal of non-encapsulated material):
• ·        Dialysis:
•   The detergents at their critical micelle concentration (CMC) have been used to solubilize lipids. As the detergent detached, the micelles become increasingly better-off in phospholipid and lastly combine to form LUVs. The detergents were removed by dialysis [13-15]. A commercial device called LipoPrep (Diachema AG, Switzerland), which is a version of dialysis system, is obtainable for the elimination of detergents. The dialysis can be performed in dialysis bags engrossed in large detergent free buffers (equilibrium dialysis) [16]
• ·        Detergent(chocolate, alkyl glycoside, Triton X-100) removal of mixed micelles(absorption):
•    Detergent absorption is attained by shaking mixed micelle solution with beaded organic polystyrene adsorbers such as XAD-2 beads (SERVA Electrophoresis GmbH, Heidelberg, Germany) and Bio-beads
•   SM2 (Bio-RadLaboratories, Inc, Hercules, USA). The great benefit of using detergent absorbers is that they can eliminate detergents with a very low CMC, which are not entirely depleted.
• ·        Gel-permeation chromatography:
•    In this method, the detergent depleted by size special chromatography. Sephadex G-50, Sephadex G-100 (Sigma-Aldrich, MO, USA), Sepharose 2B-6B, and Sephacryl S200-S1000 (General Electric Company, Tehran, Iran) can be used for gel filtration. The liposomes do not penetrate into the pores of the             
•    Bedes packed in column. They parculate through the inter-bead spaces. At slow flow rates , The separation of liposomes from detergent monomers is very good. This swollen polysaccharide beads adsorb substantial amounts of amphilic lipids, therefore, pre-treatment is necessary. The pre-treatment is done by pre-saturton of the gel filtration column by lipids using empty liposome suspensions, [16]
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•    CHARSATERSATION OF LIPOSOMES:
•   1.      Sized: shape of liposome  vesicles is assumed to be spherical, and there mean diameter can be determined by using lasel light scattering method (17). Also, diameter of these vesicles can be ultracentrifugation, photon correlation mycroscepi,  optical microscopy freezed fracture electron microscopy (18,19) freeze thawing (keeping vesicles suspension at -20 ̊C for 24 hrs and then hiting to ambient temperature ) of Liposomes incereasers the vesicle diameter, which might be attributed to fusion if vesicles during the cycle
•   2.       Bilayer formation : assembly of non-ionic surfactants to form a by lawer vesicle is characterized by and X-cross formation under light polarization microscopy (20).
•   3.      Number of Lamellae: This is determined by using nuclear magnetic resonance (NMR) spectroscopy, small angel exray cattering and electron microscopy (190.
•   4.      Membrane Rigidity: Membrain rigidity can be measured by means of mobility of fluorescence probe as function of temperature (20).
•   5.      Entrapment Efficiency: After preparing nisomal dispersion, unentrapped drug is sepreted  by dialiasis, centrifugation, orgel filtration as described above and the drug remained entrapped in Liposome  is determined by complete vesicle disruption nusing 50% n-propanol or 01% Triton X-100 and analyzing the resultant solution by appropriate assay method of drug (21).
•   Entrapment efficiency= (amount entrapped/total amount) x 100
•   In Vivo Release Study: albino rats are used for this study. These raps are sub divided with groups Niosomal suspension used for in vivo study is injected intravenously (through tail vein) using appropriate disposal syringe.
•   In Vitro Release Study: A method of in vitro released rate study has been reported with the help of dialysis tubing (22). A dialysis sac is washed and soaked in distilled water. The vesicle suspension is pipette into a bag med up of the tubing and sealed. The bag containing the vesicles is then placed in 200 ml buffer solution in a 250ml bakear with constant shaking at 25̊ C or 37° C. At various time intervals, the buffer is analyzed for the drug containt by an apprate assay method. In another method isoniazed –encapsulated Liposome are separated by gel filtration on Sepheadex G-50 powder kept
•    In double distilled water for 48 h for swelling (23). At first, 1 ml of prepared niosome suspension is placed on the top of the column and elution is carried out using normal saline. Liposome encapsulated isoniazid elutes out first as
•    a slightly dense, why opalescent suspension followed by free drug.  Separated Liposome are filled in a Diolysis tubeto which a sigma dialysis sac is attached to one end. The dialysis tube is suspended in
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• v APPLICATIONS OF LIPOSOMES IN MEDICINE AND PHARMACOLOGY:
•   Applications of liposomes in medicine and pharmacology can be devided into diagnostic and therapeutic applications of liposomes containing of various markers of drugs, and their use as a tool, a model,  or reagent in the basic studies of cell interactions, recognition processes, and mode of action of certain substances[28]
•   Unfortunately, many drugs have a very narrow therapeutic window, meaning that the therapeutic concentration is not much lower than the toxic one. In several cases, the toxicity can be reduced or the efficacy can be enhanced by the use of a suitable drug carrier which alters the temporal and spatial delivery of the drug, i.e., its biodistribution and pharmacokinetics. It is clear from many pre-clinical and clinical studies that drugs, for instance antitumor drugs, parceled in liposome demonstration reduced toxicities, which retentive enhanced efficacy.
•   1.       Liposomesin bio technology :
•    Advances in liposome design are leading to new applications for the delivery of new biotechnology proteins. A vast literature define the viability of formulating wide range of conservative drugs of liposomes, frequently resultant in improved therapeutic activity and/or reduced toxicity compared with the free drug. As a whole, changed pharmacokinetics for liposomal drugs can lead to improved drug bioavailability to particular target cells that live in the circulation, or more prominently, to extravascular disease sites, for example, tumors. Recent improvements include liposomal formulations of all-transretinoic acid[29,30] and daunorubicin[31,32,33,34], which has received Food and Drug Administration consent as a first-line treatment of AIDS-related advanced Kaposi's sarcoma. Distinguished examples are vincristine, doxorubicin, and amphotericinB[35] products, for example antisense oligonucleotides, cloned genes and recombinant technology.
•   2. Liposomes in parasitic diseases and infections :
•      From the time when conventional liposomes are digested by phagocytic cells in the body after intravenous management, they are  ideal vehicles for the targeting drug molecules into these marcophages. The best known instances of this ‘Torjan horse-like’ mechanism are several parastics diseases which normally exit in the cell of MPS. They comprise leishmaniasis and several fungal infections. Leishmaniasis is a parasitic infection of  macrophages which affects over 100 million people in topical regions and is often deadly. The effectual dose of drugs, mostly different antimonials, is not much lower than the toxic one. Liposomes accumulate in the very same cell pop[ulation which is infected, and so an ideal drug delivery vehicle was proposed[36]. Ccrtainly, the therapeutic index was increased in rodents as much as several hundred times upon administration of the drug in various liposomes. Unexpectedly, and unfortunately , there was not much interest to scale up the formulations and clinically approve them after several very encouraging studies dating back to 1978. Only now, there are several continuing studies with various anti-parasitic liposome formulations in humans. These formulations use mostly ionosphere amphotericin B and are transplanted from very successful and prolific area of liposome formulatrions in antifungal therapy.
•   The best results reported so far in human therapy are probably liposomes as carriers foramphotericim B in antifungal therapies.This is the drug of choice in dispersed fungal infection which often in parallel work together with chemotherapy, immune system, or AIDS, and is frequently fatal. Unfortunately , the drug.
•   Itself is very toxic and its dosage due to its ionosphere and neurotoxicity. These toxicities are normally related with the size of the drug molecule or its complex. Obviously, liposome encapsulation inhibits the accumulation of drug in these organs are and radically reduces toxicities. Furthermore, often, the fungus exits in the cells of the mononuclear phagocyte system, therefore, encapsulation result in reduced toxicity and passive targeting. These benefits, however, can be associated with any colloidal drug career. Certainly, similar improvements in therapy were observed with stable mixed micellar formulation and micro-emulations [38]. Additionally, it seems that many of the early liposomal preparations were in actual fact liquid crystalline colloidal practices rather than self-closed MLV. Since the lives of the first terminally ill patients (who did not rely to all the conventional therapies) wear saved [36].  Many patients were very effectively treated with diverse of amphotericim B formulations.
• Comparable methods can be achieved antiviral and antibacterial therapies [39]. Most of the antibiotics, however, are orally available, liposome encapsulations can be considered only in the case of very potent and toxic ones which are administered parenterally. The preparation of antibiotic-loaded liposome at sensibly high drug-to-lipid rations may not be easy because of the infractions of these molecules with bilayers and high densities of there aqueous solutions which often force liposomes to float as a creamy layer on the top of the tube. Several, other ways for instance, topical or pulmonary (by inhalations) administration are being considered also. Liposome encapsulated antivirals (for example ribavirin, azdothymidme, or acyclovir) have also shown to reduced toxicity, currently, more detail experiments are being performed in relation to their efficacy
• 1.      Liposomes in anticancer therapy:
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• Numerous different liposome formulations of memerous anticancer against were shown to be less toxic than the free drug[41,42,43]. Anthracyclines are drugs which stop the growth of dividing cells. These cells are not only in tumors but are also in hair, gastrointestinal mucosa, and blood cells, therefore, these cells of drug is very toxic. The most used and studied as Adriamycin (commercial name for doxorubicin HCI, Ben Venue Laboratories, Bedford, Ohio). In addition to the above-mentioned acute toxicities, its dosage is limited by its increasing cardio toxicity. Numerous diverse formulations were tiried. In most case, the toxicity was reduced to about 50%. These include both acute and choronic toxicities because liposome
•  Encapsulation reduces the delivery of the drugs molecules towards those tissues. For the same reason, the efficiency was in many cases compromised due to the reduced bioavailability of the drug, especially if the tumour was not phagocytes or located in the organs of mononuclear phagocytes system.  In some cases, such as systemic lymphoma, the effect of liposome encapsulation showed enhanced efficacy due to the continued release effect, i.e. longer presence of therapeutic concentrations in the circulation[44, 45,46], while in several other cases, the sequestration of the drug into tissues of mononuclear phagocytes system actually reduced its efficacy.
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• Applications in man showed, in general, reduced toxicity in better tolerability of administration with not too encouraging efficacy. Several different formulations are in different phases of clinical studies and show mixed results.
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• v  FUTURE SCOPE :
• 1.      Liposomes represent a promising drug delivery module.
• 2.      There is lot of scope to encapsulate toxic anti-cancer drugs, anti infective drugs, anti AIDS drugs, anti-inflammatory drugs, anti viral drugs etc. in Liposome and to use them as promising drug carries to achieve better bioavailability and targeting properties and for reducing the toxicity and effects of the drugs.
• 3.      The ionic drugs carries are relatively toxic and unsuitable whereas noisome carriers are safer.
• 4.      In addition, handling and storage of Liposome requires no special conditions.
• 5.       Vesicular drugs carriers like Liposome can be transported by macrophages which are known to infiltrate tumour cells.
• 6.      It may be possible to take advantage of these activated macrophage system in delivering anti tumour agents within vesicles more quantitavely to tumour sites.
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• v  CONCLUTION:  Liposomes have been used in a broad range of pharmaceutical applications. Liposomes are showing particular promise as intracellular delivery system for anti-sense molecules, ribosome, proteins/peptides, and DNA. Liposomes with enhanced drug delivery to disease locations, by ability of long circulation residence times, are now achieving clinical acceptance. Also, Liposomes promote targeting of particular diseased cells within the disease site. Finally liposomal drugs exhibit reduced toxicities and retain enhanced efficacy compared with free complements. However, based on the pharmaceutical applications and available products, we can say that liposome's have definitely established their position in modern delivery systems. The use of liposomes in the delivery of drugs and genes are promising and is sure to undergo further developments in future. The flexibility of their behaviour can be exploited for the drug delivery through any route administration and for any drug material irrespective of their solubility properties. There is even greater promise in future for marketing of more sophisticated are highly stabilized liposomal formulations. The use of liposomes in the delivery of the drugs and genes are promising and is sure to undergo further development in future. The liposomal drug delivery system will revolutionize the vesicular systems with wide applications especially in the treatment of cancer.
•  v REPRESENCE:
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