Shikha Baghel Chauhan*, Vaishnavi Gupta
Assistant Professor, Department of Pharmaceutics, Amity Institute of Pharmacy, Amity University, Noida,
Uttar Pradesh, India.
*Corresponding Author E-mail: shikha.pharma@gmail.com
ABSTRACT:
Liposomes as artificial colloidal vesicular structure having hydrophilic core in a lipid bilayer membrane became one of the most tremendously explored drug delivery system. These spherical vesicles consist of amphiphilic molecule like cholesterol, sterols and can be used to deliver both hydrophilic drugs as well as lipophilic drug. These vesicles are differentiated on the basis of size, composition, method of preparation and application. Liposomes are biodegradable, nonimmunogenic, biocompatible, provide targeted and prolonged release, change pharmacokinetic and pharmacodynamic attribute of drug, they find wide clinical application as diagnostic, antimicrobial, anticancer, vaccine adjuvant and cosmetics. Expanding application show promising sign for future development in liposome. The present review provides information about liposome in drug deliver, their composition, structure, different method of preparation, various types of liposome, clinical applications of liposome, strategies, marketed formulation.
KEYWORDS: Liposome, classification, method, application, characterization.
INTRODUCTION:
Liposomes can be defined as artificial colloidal vesicular structure having hydrophilic core in a bilayer lipid membrane. These vesicles range from 0.01 um to 50 ums in size [4]. These are prepared from cholesterol and phospholipid. Due to their unique biphasic nature, these could be used for the delivery of both hydrophilic drugs as well as lipophilic drugs. Drugs which have log P value less than -0.3 and are highly hydrophilic are located inside aqueous compartment and drugs which have log P value greater than 5 and are lipophilic are located inside the lipid compartment. Liposomes formed from highly hydrophilic drugs are less stable and more prone to leakage of drug when compared with liposomes prepared from highly lipophilic drugs. Drugs having 1.7 partition co-efficient (log P< 4) show problem in loading due to their neutral behavior. Solubility of drugs play important role in liposome formulation. Drugs which have poor solubility in both the phases show difficulty in the formulation. Drug and phospholipid ratio are also important parameter for liposome formulation. It should always be kept high [4,5,6 ].
Liposomes are also used to enhance the solubility of the drugs, hydrophobic drugs like “cyclosporine A” and “Paclitaxel” are incorporated in liposome with surfactant and organic co-solvent, they protect the sensitive drug from the external environment by entrapping the drug in aqueous core or lipid bilayer and prevent degradation by the enzymes. Due to biphasic nature they act as carrier for both hydrophilic and lipophilic drugs, they provide delayed release with site specificity. They have capability to alter pharmacodynamics as well as pharmacokinetic of the drug, molecule, these are generally used to enhance the therapeutic value (actinomycetes) and decrease toxicity (antibiotics such as actinomycetes, fluconazole or also in case of anticancer drugs by minimizing the drug delivering to the targeted size. Liposome bilayer membrane show resemblance to the cell membrane they are also used to release therapeutic substance within the cell as in case of the protein and peptide delivery, Liposomes are relatively nontoxic, non-immunogenic, biocompatible, biodegradable, and flexible vesicles [8-13].
No formulation is perfect, Liposomes as drug delivery also face some problems, one of the major issues with liposome is rapid clearance by the phagocytic cell (reticule endothelial cells of liver, spleen). Large liposome is cleared more rapidly as compared to small liposome. They also show problem in sterilizations as they are prone to temperature, radiations, chemicals and it is also not possible due to stability pointy of view. They have very low entrapment value of drug because when we increase the concentration of drug, they produce toxic affect with the lipid. Active loading of drug produces liposomes with high entrapment value, chances of leakage of drug on long storage as they are physically and chemically less stable. Cost of production is also high due to expensive raw material and equipment’s involved in the manufacturing process. Large scale manufacturing of liposomes is generally not considered as they show batch to batch variation. Liposomes once administered cannot allow change in the therapy. They cause dumping when administered in wrong manner [14-18].
CLASSIFICATION OF LIPOSOME:
Liposomes have been divided on the basis of size or number of layers as multilamellar vesicles, small unilamellar vesicles or large unilamellar vesicles. Both size and number of lipid layers are helpful in determining liposome biological half-life and also affect drug encapsulation efficiency. Liposomes are also classified on the basis of composition as conventional liposome, pH sensitive liposome etc. and on the basis of method of prepetition reverse phase evaporation, French press vesicles or ether press vesicles [19,20,21].
COMPOSITION OF LIPOSOME:
The liposome is composed of phospholipid, sphingolipid, sterols, polymeric material, preservatives, surfactants and ammonium salts. Phospholipid and sphingolipid are one of the major constituents of liposome and accounts 50% by weight of liposome and these are obtained from the natural and synthetic source. The second most important ingredient is cholesterol which is used to reduce the fluidity and micro viscosity of the lipid bilayer and provide stability to it. It is incorporated in higher amount in 1:1 or 1:2 ratio. Other ingredients such as preservatives are added to prevent oxidation of lipid, quaternary ammonium salts are added to provide charge on liposome[21-25].
METHOD OF PREPARATION:
There are generally two method of preparation of liposomes. One is passive loading method and other is active loading method. In former main ingredient is entrapped by incorporating in a water phase of hydrophilic drug or organic phase of lipid loving drug before preparation of vesicle and while preparing the liposome. This method is helpful in achieving high encapsulation efficiency for lipid soluble drug. In later ionic drugs or drugs which have solubility in for both lipid and water are loaded after the formation of vesicles by creating diffusion gradient [21,27-29].
Mechanical Dispersion method:
Thin film hydration Method:
Organic liquid like chloroform, dichloromethane, ethanol, chloroform and methanol mixture are used for dissolving lipid to obtain clear solution. Usually 10-20 mg of lipid per ml is considered but higher amount of lipid can be used if its solubility is acceptable. This solution is poured into round bottom flask and solvent is removed by rotating flask method leaving behind thin layer of lipid which is obtained on side walls of flask at 40-45. C. Nitrogen and argon is used for the removal of solvent. The final step is hydration in combination with agitation for segregation of swelled layers from the vessel surface to create spherical vesicles which are sealed. The time and temperature involved is two hours at 60-70. C. The solution is left overnight for obtaining full lipid hydration. Duration of mixing and lipid hydration is effective in determining loading rate of the drug solution. The major disadvantages of this method include low encapsulation efficiency, heterogeneous size distribution, difficult in scale up [21,30]. Examples of liposome prepared by thin film hydration are MLV of doxorubicin for cancer treatment [31], Celecoxib in transdermal drug delivery [32], 5-fluorouracil for drug targeting [33].
Sonication:
This method of preparation is generally used for unilamellar vesicles ranging from15-50nm. by disruption of multilamellar vesicles (prepared by thin film hydration technique). In this MLV is sonicated by batch sonicator and probe sonicator under inert condition of nitrogen. Probe type sonicator maintains high energy to the lipid suspension and causes overheating of lipid suspension which causes mortification of lipid and also responsible of releasing titanium particles in the suspension. In batch sonication, test tube having lipid solution is placed under bath sonicator for the duration of 5-10 minute at critical temperature of lipid. The lipid solution starts to clarify and yield slightly hazy color. The particles could be removed by rotation and clear solution obtained. Composition and concentration of lipid affect the mean size and size distribution, there may be change in size variation between batches produced at different time. Major disadvantage of this method is low encapsulation efficiency and when these vesicles are store below critical phase transition temperature, they use to form large vesicles, degradation of metal tip, and metal pollution from probe tip[13,21,29,34,35].
Micro emulsification technique:
This method is used in the preparation of MLV’s. Lipid dispersion fluid is pumped at high pressure from a 5 um. Orifice of microfluidizer after that it is flowed with micro channels which manage two streams of liquid colloid with each other at right angle at high velocity therefore showing and efficient energy transfer. Thus, lipid could be incorporated in the fluidizer, as mixture of hydrated lipid in organic medium. The liquid collected can be reused with the pump and the chamber until liposomes of spherical shape are produced. Size of liposome is reduced to 0.1 to 0.2um after a single pass.
Freeze Pressure Cell:
Extrusion of MLV’s at high pressure at low temperature through a small orifice. An important feature of French pressure cell is that protein do not significantly get affected during sonication process. This method is easy, fast, and involves mild heating of less stable materials. The resultant liposomes are greater in size than sonicated SUV’s. The drawbacks of this preparation are that temperature is not easily achieved and volumes used are relatively small [13,21,37].
Freeze Thawed method:
Multilamellar vesicles have low encapsulation efficiency since it is quite heterogeneous in size and lamellarity. Freeze thawing method is usually applied to increase the encapsulation efficiency of MLV by decreasing the lamellarity. This method involves freezing and thawing to form large vesicles. This problem is overcome by incorporating larger amount of the content of phospholipid and changing the ionic strength of medium [13,21,38].
Solvent dispersion method:
Ethanol Injection method:
Required quantity of phospholipid or cholesterol is first dissolved in ethanol, then the resulting organic phase is introduced by the means of syringe pump into aqueous medium. As soon as ethanol solution comes in vicinity with aqueous medium resulting spontaneous liposome formation. The resulting liposome dispersion was kept for stirring for the duration of 15 minutes. At last ethanol and part of water evaporated by Rotatory evaporator [29]. The drawback of this method is that population is heterogeneous; Liposomes are very diluted, complete removal of ethanol is not possible [39].
Ether injection method:
This is homologous to ethanol injection method. In place of ethanol, ether or diethyl ether/methanol mixture is slowly introduced to aqueous solution phase, which is to be encapsulated [11].
Reverse phase evaporation of the vesicles:
Lipid is solubilize in organic solvent such as mixture of diethyl ether or mixture of isopropyl ether and chloroform in ratio 1:1 and mixture of chloroform and methanol in ratio of 2:1 V/V, As organic phase is miscible with water phase thus o/w type of emulsion is formed, Phosphate buffer or Citrate buffer is incorporated to aqueous phase with the objective to improve formulation. The main advantages of this method are high encapsulation efficiency [40].
APPLICATION OF LIPOSOME:
Liposomes in cancer treatment:
Liposomes as carrier have capability to alter pharmacokinetic property of the drug. The toxicity of liposomal formulation is limiting factor in the cancer chemotherapy hence liposomal formulation enhance therapeutic effect by concentrating them in tumors and preventing their circulation to the normal tissue and shows improved retention effect [43]. The first successful product in the treatment of cancer was doxil marketed in 1995 for ovarian cancer and AIDS related Kaposi sarcoma. It was first Nano sized particle which got regulatory approval. Different strategies for delivery of anticancer drugs are being used which include the stealth liposome technology, Non-PEGylated technology, Depofoam technology. In stealth technology covalent bond is formed between the polyethylene glycol (PEG) and the drug, this process is known as PEGylation and it show reduced immunogenicity and antigenicity. This technology is utilized in Doxil as an intravenous injection for the treatment of ovarian cancer, multiple myeloma, Kaposi sarcoma. Non-PEGylated technology (NPLT) eliminates the side effects associated with the PEG and provide better circulation time and also reduces toxicity, Depo Foam is extended release technology in which drug is encapsulated in multivesicular liposome without modification in their molecular structure and their release form[44,45].
Liposome in ophthalmic drug delivery:
Liposomes are biocompatible and biodegradable carrier and can improved permeability of drugs by binding on corneal surface and improve residence time. They can be used for delivery of hydrophobic as well as hydrophilic drug; they alter pharmacokinetic profile, improve therapeutic affect and reduces toxicity. Liposome can focus on delivery of drug in both anterior segment and posterior segments. Anterior segment delivery is mainly based on corneal adhesion and permeation. This can be improved by incorporating penetration enhancing and bio adhesive polymers. Delivery of drug to posterior segment is mainly based on intravitreal half -life and targeted delivery to retina. Examples of clinically used ophthalmic preparation-Verteporfin and rostaporfin.
Liposome in gene therapy:
Gene therapy, Recombinant DNA technology, study of gene function all depends on transfer of nucleic acid into the cell. Steps involved in gene transfer are- A) packaging of DNA, B) Adhesion of packed DNA to the cell surface, C) Internalization of DNA D) Escape of DNA from endosome if endocytosis is involved E) DNA expression in cell nuclei. To explore all these steps liposomes are promising approach. Different strategies are being involved in gene transfer which includes pH sensitive liposome and cationic liposomes [48,49,51]. pH sensitive liposomes were designed on the basis of vector (virus that fuses with endosome membrane) by the means of proteins at pH 5-6 delivering their genetic material to the cytosol before reaching the lysosome. Lipid which is used to design these is phosphatidyl ethanolamine (PE) {52,53].Cationic lipid strategy was developed in late 1980, The idea was to neutralize the negative charge with positively charge lipid to capture plasmid more efficiently and to deliver DNA into the cell This is a simple procedure in this cationic lipids are mixed with DNA and then added to the cell This results in formation of aggregates composed of DNA and cationic lipid . The lipid either alone or in combination with other neutral lipids, spontaneously forms MLV which may be sonicated to form SUV. Some of widely used cationic liposome formulation are- Lipofectin, Lipofetamine, LipofecTase, Trasnsfectase[53,54].
Liposomes in cosmetics:
Liposomes can be used as vehicles of cosmeceuticals material or as an active agent. If skin is damaged due to deficiency of moisture or because off eczema then liposome interact with the skin lipids proteins and carbohydrate providing nourishment to the skin and helping the skin to regains its normal state and function properly. Topical application of liposome is advantageous in improving restoring action, biodegradability, biocompatibility, extended slow release of drugs. As structure of skin is similar to liposome in having bilayer lipid membrane, thus liposome can easily cross dermal layer and also helpful in distribution of drug to epidermal and dermal layer [55,56]. Examples of marketed formulation of topical liposome includes- “Celadrin” Topical liposomal lotion help to relieve joint discomfort and Vitamin A and C entrapped in Liposome (Lipo C) used for restoring healthy skin [57].
Liposomes as vaccine delivery:
Liposomes, archaesome and virosome (Nano vesicle derived from lipid) act as important carrier in vaccine delivery. On the basis of chemical properties, water soluble compound (protein peptide and nucleic acid, carbohydrate, haptans) are incorporated in aqueous phase whereas hydrophobic compound is incorporated into lipid bilayer and antigen can be attached to liposome surface by adsorption or by chemical bond[58,59].
Liposomes as theragnostic agent:
Early detection of disease and treatment is important that’s why diagnostic and therapeutic agent are used in combination. Conventional preparation delivers insufficient amount of drug to targeted tissue and produces toxic effect. To overcome this liposome is used as they have high targeting ability and stability. Several engineering approaches are being utilized for improving the performance. For targeted delivery surface of liposome have been functionalized with disease biomarkers and ligands and for sensing application liposome have been extended with variety of synthetic phospholipid with functional group allowing stimuli response properties. These formulations also allowed in corporation of new imaging agent such as Gd3+,64Cu,18F which can be used in fluorescent imaging, magnetic resonating imaging, ultrasound imaging and nuclear imaging [60].
Liposomes in Antimicrobial Treatment:
Antibiotics generally have limited application in the treatment because off their toxicity, side effects, weak bio distribution and poor pharmacokinetic. Liposome are used for antimicrobial activity mainly for three reasons, firstly they protect the antibiotic for enzymatic degradation for example beta lactamase activity, Secondly, they enhance the therapeutic activity by promoting cellular uptake of antibiotic by microorganism as in case of amphotericin B. Liposome also overcome the bacterial resistant of antibiotic. One most common example of liposomal formulation is “Flailsome” It contain Tobramycin and DPPC/DMPG in ratio of 8:1 it fuses plasma membrane of P. aeruginosa and releases its content in periplasmic space for antimicrobial activity [61-64].
CONCLUSION:
Liposomes are nontoxic, flexible, biocompatible, nonimmunogenic vesicles which encapsulate drug and protect them from enzymatic degradation, alter pharmacokinetic and pharmacodynamics properties of drug. These as carriers have gained importance due to its various advantages and find wide application in pharmaceuticals and cosmetics. Nowadays different strategies are in development such as Pegylated liposome, Non Pegylated, cationic liposome, archaesome, virosome and many more. Development of these strategies provides additional advances. Expanding application show promising sign for future development in liposomes.
ACKNOWLEDGEMENT:
The authors are grateful to the authorities of Department of Pharmaceutics, Amity Institute of Pharmacy, Amity University.
CONFLICT OF INTEREST:
The author declares none conflict of interest
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Received on 13.08.2019 Modified on 10.10.2019
Accepted on 26.11.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(4):2051-2056.
DOI: 10.5958/0974-360X.2020.00369.8