International Journal of Drug Development and Research

Key words

Liposomes, Preparation, Applications

INTRODUCTION

Liposomes are the small bladder having spherical shapes which are produced from glycolipids, cholesterols, non toxic surfactants and membranous proteins [1, 2]. These are generally a drug carrier loaded with different variety of molecules like minute drug molecule, proteins and nucleotides. The liposomes were discovered in the year 1960 by British hematologist Dr. Alec D. Bangham [3, 4]. Liposomes have been manufactured and classified on the basis of size, composition, charge and speciality [5, 6, 7]. The mechanism of action of liposomes have been attributed to their binding and dispersing with cellular membrane releasing the drug into the cell; engulfment by the cell and transfer of phospholipids into the cell membrane by which the drug is released.; and the entrapment by the lipid bilayer acting upon by lysosomes and releasing the active ingredient of formulation [8, 9]. Moreover, the preparation of liposomes can be done by mechanical methods involving replacement of organic solvent; fusion of preformed vesicles; and by transformation of size [9, 10]. Further, numbers of limitations have been found to be associated with liposomes like stability, sterilization, encapsulation, efficiency, active targeting and lysosomal degradation [6]. A wide number of applications have been found to be associated with liposomes which include general application, i.e., in treatment of respiratory disorders, eye disorders, vaccine adjuvents, brain targeting, and infective agents; alongwith clinical applications, i.e., cancer therapy antimicrobial therapy and gene therapy [11, 12]. The present review article explains about the mechanisms and preparations of liposomal formulations. In addition, various applications possessed by the liposomes have been critically discussed in the present review.

BRIEF HISTORY

Liposomes were firstly discovered by British haematologist Dr Alec D Bangham FRS in the year 1960 at the Babraham institute Cambridge, during the testing of institute's new electron microscope by adding negative stain to dry phospholipids [3]. Conversely, the discovery of Dr A.D. Bangham was an accidental discovery, when he scattered the phosphatidyl choline molecule in water, during which he found that the molecule was forming a closed bilayer structure having an aqueous phase which were entrapped by a lipid bilayer [3, 4]. However, 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. In addition, the energy efficiency of liposomes as a carrier for replacement therapy in genetic disturbances of lysosomal enzyme was first noted in 1970 [6]. The liposomes have been generally used to supply certain vaccines, drugs and enzymes to the body, and can be used for the delivery of cancer drugs which helps to protect the healthy cells from the drug toxicity [3, 4, 6].

LIPOSOMES: STRUCTURE AND FUNCTION

The extended area of research makes it possible to evaluate and engineer a wide range of liposomes varying in size, phospholipid composition and surface characteristics in order to suit specific applications for which they are intended [13, 14]. It has been documented that the glycerol moiety is the back bone of molecule, and hence, the phospholipids containing glycerol were mostly used components of liposomal formulation which represent more than 50% of lipid weight in biological membrane [15, 16, 17]. Moreover, cholesterol derivatives have also been included in liposomes in order to decrease the viscosity and to provide the stabilization to the membrane. Furthermore, unsaturated phospholipids like Dioleoyl phosphatidyl choline (DOPC) and Dioleoyl phosphatidyl glycerol (DOPG); and saturated phospholipids like Dipalmitoyl phosphatidic acid (DPPA) and Dipalmitoyl phosphatidyl glycerol (DPPG) have also been applied. However, polymeric materials like Methacrylate and lipid containing conjugated diene alongwith sphingolipids have also been reported to be used in liposomes. Additionally, polymer bearing lipids like Diacyl phosphatidyl ethanolamine (DPE) with polyethylene glycol (PEG) polymer; and various cationic lipids like Dioctadecyl dimethyl ethanolamine ammonium bromide (DODAB) have also been utilized [7, 18].

A number of properties have been possessed by the liposomes which include good permeability to water, sensitivity to osmosis, and arrangement of bimolecular layer having aqueous spaces. In addition, the liposomes having negative charge on membrane are permeable to anions, whereas, positively charged membranes are impermeable to cations. On the basis of above properties, the liposomes have been noted to offer various advantages like, binding and forming complexes with both positive and negative charged molecules; providing protection to DNA by the process of degradation; ability to target specific cells and tissues; providing sustained release characterstics; and enhancement of pharmacodynamics and pharmacokinetics of drug [19, 20].

CLASSIFICATION OF LIPOSOMES

Liposomes can be classified on the basis of structure, method of preparation, composition, conventional use, and special purpose liposomes. On the basis of structure, the liposomes can be categorized as unilamellar, small unilamellar, medium unilamellar, large unilamellar, giant unilamellar, oligolamellar, multilamellar, and multivesicular vesicles [5, 6]. Further, the liposomes can be classified on the basis of method of preparation, as, single or oligolamellar vesicle made by reverse phase evaporation method (REV), multilamellar vesicle made by reverse phase evaporation method (MLV-REV), stable plurilamellar vesicle (SPLV), frozen and thawed multi lamellar vesicle (FATMLV), vesicle prepared by extrusion technique (VET), and dehydration-rehydration method (DRV) [7]. Moreover, liposomes can be categorized as conventional, fusogenic, pH-sensitive, cationic, long circulatory, and immuno liposomes on the basis of composition and applications. In addition, the liposomes can be further classifies on the basis of conventional purposes like stabilize natural lecithin (PC) mixtures, synthetic identical chain phospholipids, and glycolipids containing liposomes [5, 6, 7]. Furthermore, based upon speciality liposome, they can be differentiated as bipolar fatty acid, antibody directed liposome, methyl/methylene x-linked liposome, lipoprotein coated liposome, carbohydrate coated liposome, and multiple encapsulated liposome [2, 21].

MECHANISM OF LIPOSOMES

Initially, the liposomes were expressed as a model of cellular membrane applied to the delivery of drug cells. The mechanism of liposome action can be studied under two mechanisms, and hence classified as cationic and pH-sensitive mechanism of liposome action [8, 22]. The cationic liposomes, consisting of a positively charged lipid and a co-lipid, have been regarded as the positively charged liposomes which interact with the negatively charged DNA molecules in order to form a stable complex. The commonly used cationic lipid is lipofectin, whereas normally employed co-lipids include DOPE and DOPC. It has been shown that DNA alongwith cationic lipid lipofectin, interacts spontaneously in order to form stable complexes [8, 9]. It has been implicated that the negative charge of the DNA molecule interacts with the positively charged groups of the cationic lipid in order to form an efficient complex. Further, the most common co-lipids, also called helper lipids, have been reported to be required for the stabilization of liposome complex [23]. The pH-sensitive, or negatively-charged liposomes, have been noted to entrap DNA rather than forming stable complex with it. Some of the DNA gets entrapped within the aqueous interior of the liposomes. In some cases, the liposomes have been noted to get destabilized by the low pH and thus the term pH- sensitive has been exploited. However, the cationic liposomes possess applications in gene delivery both in vivo and in vitro, whereas, the pH-sensitive liposomes have been shown to possess the potential at in vivo DNA delivery [8, 23, 24].

PREPARATIONS OF LIPOSOMES

The accurate selection of liposome preparation method depends the physicochemical characteristics of the material to be entrapped, choice of liposomal ingredients, nature of the medium in which the lipid vesicles are to be dispersed, effective concentration of the entrapped substance, optimum size and shelf life of the vesicle, and batch-to-batch reproducibility [9, 10]. In addition, the rigidity of bilayers forms the crucial point to be kept in mind while preparing liposome. Numbers of phospholipid groups have been employed for liposome preparations which include phoshpholipids from natural source, phospholipids modified from natural source, semi synthetic phospholipids, fully synthetic phospholipids, and phospholipids with natural head groups. The addition of cholesterol to the bilayer mixture results in improvement of formulations by inhibiting the conversion of transconformation to the gauche conformation [25]. However, various methods have been used for the preparations of liposomes which include mechanical methods by film method and ultrasonic method; methods involving replacement of organic solvent; methods involving fusion of prepared vesicles or transformation of size by freeze thaw extrusion (FTE) method; and the dehydration-rehydration (DR) method [9].

The mechanical method of liposomal preparation includes film which is the simplest method for liposome preparation in which the organic solvent is used to hydrate thin lipid film. After the lipid film has been completely hydrated, the process of deposition is adapted to remove the organic solvent, which is continued until the organic solvent gets completely removed. Afterwards, an aqueous buffer is used to hydrate the solid lipid mixture, during which the lipid gets swelled and hydrated resulting in the formation of liposome [6]. Another mechanical method of liposomal preparation is by ultrasonic method, in which the process of ultrasonication is employed for liposomal formation. Generally, two types of sonicators have been used for ultrasonication of an aqueous dispersion of phospholipids which include, probe sonicator (used for small volume) and bath sonicator (used for large volume). In addition, SUVs with a diameter of 15-25 micrometer can be prepared by this method [10].

Further, the liposomes can be prepared by a method involving replacement of organic solvent, during which the cosolvation of lipid is done using an organic solution, which is later dispersed into aqueous phase containing the material to be entrapped within the liposome. The method is further is method is differentiated as reverse phase evaporation, which employes the use of rotary evaporator to remove the solvent from the lipid mixture taken in a round bottom flask. The nitrogen is introduced into the system followed by redissolvation of lipids in the organic phase, resulting in the formation of semisolid gel by evaporating the solvent under reduced pressure [26]. The material which is not encapsulated is then removed resulting in the formation of liposome called as REVs. Moreover, unilamellar and oligolamellar vesicles can be prepared by this method [6, 9]. Another method involving replacement of organic solvent is Ether vaporization method which can be further differentiated as ethanol injection method and ether injection method on the basis of solvent used. In ethanol injection method, the injection of lipid is done rapidly with the help of a fine needle into an excess of saline or other aqueous medium, whereas, ether injection method involves slow injection of lipid with the help of a fine needle into an excess of saline or other aqueous medium.

The third method of liposomal preparation involves the fusion of preformed vesicles or transformation of size, which can be further categorized as FTE method and DR method. In FTE method, the liposomes formed by film method and the solute to be entrapped are taken together and whirled continuously until the entire film is suspended [27]. The MLV thus obtained are frozen in luke warm water and then whirled again. Further, the extrusion of sample is done three times after performing two cycles of freeze thaw and whirling, accompanied by six freeze thaw cycles and eight extrusions of sample, resulting in the rupture and diffusion of SUVs. In this condition, solute attains the equilibrium between inside and outside resulting in the fusion of liposomes. As a result, they grow in size and form large unilamellar vesicles by extrusion technique (LUVET). Additionally, this method is used for encapsulation of proteins [6, 9, 10]. Furthermore, in DR method, SUVs are incorporated in an empty buffer. An aqueous fluid is taken which contains the material to be entrapped and used for rehydration of empty buffer containing SUVs. After rehydration, drying is attained resulting in solid lipids dispersing in a finely subdivided form. Rehydration of vesicles is then performed which leads to the formation of large size liposomes. Moreover, oligolamellar vesicles are prepared by this technique.

APPLICATIONS OF LIPOSOMES

Liposomes have been reported to possess a great number of advantages due to which they have been used for a variety of purposes. Several applications of liposomes have been purposed which can be categorized as general applications and clinical applications. However, the general application of liposomes may be attributed to their use in respiratory disorders, eye disorders, vaccine adjuvants, brain targeting and anti infective agents whereas clinically, the liposomes have been found to be useful for the treatment of cancer, in antimicrobial therapy and in gene therapy [11, 12].

The liposomes have been found to possess beneficial effects in the treatment of several respiratory disorders, reason being their better sustained release, improved stability and reduced toxicity than ordinary aerosols. Although, lipid composition, size, charge, drug-lipid ratio alongwith method of delivery are certain parameters that must be necessitated in order to make liposomal drug delivery effective enough. Liquid or dry form can be taken for inhalation of liposome and release of drug has been reported to occur during nebulization [28, 29]. Moreover, the liposomes have been evidenced to play a vital role in treatment of disorders of both anterior and posterior segment of eye. Dry eyes, keratitis, corneal transplant rejection, uveitis, ondopthelmitis and proliferative vitro retinopathy are the examples of eye disorders against which liposomes have been found to possess beneficial effects [30]. The drug verteprofin that is found to be effective against eye disorders has been recently approved as liposomal formulations. In addition, the liposomes have been reported to increase both cell mediated and humoral immunity which is evidenced by the fact that intramuscular injection of liposomal immunoadjuvant released encapsulated antigen which get passively accumulated in the regional lymph node; accounting for its beneficial action. The liposomes can also be used for brain targeting due to their biocompatible and biodegradable behavior, which is evidenced by the fact that inability of amitriptylline to cross the blood brain barrier (BBB) when given systemically was found to be reversed when administered as liposomal formulation; proving their application in brain targeting [31, 32]. Furthermore, diseases like leishmaniasis, candidiasis, aspergelosis, histoplasmosis, erythrococosis, gerardisis, malaria and tuberculosis were found to be cured by giving drug as liposomal formulation, accounting for their pleiotropic potential [22].

In addition, the liposomes have been suggested to exhibit various clinical applications, which is evidenced by the fact that increased circulation life time, enhanced deposition in the infected tissues, protection from the drug metabolic degradation, altered tissue distribution of drugs have been reported to be possessed by liposomal formulation over other ordinary cytotoxic drugs [11, 12]. It has been found that liposome get accumulated in the tumors in a higher concentration as compared to normal cells. It has been evidenced that the antineoplastic activity of doxorubicin is increased when given as liposomal formulation, accounting for its anticancer potential [10]. Moreover, the recurrent high grade glioma in children after surgery and progressive teratoid/rhabdoid tumor are reported to be treated effectively by liposomal formulation daunorubicin and cauboplatin+etoposide with reduced hematological toxicity. Plasma concentration of vincristrine was found to be increased when given as liposomal formulation with increased antitumor property and reduced drug toxicity, which further evidenced its anticancer potential [33, 34]. Moreover, the liposomal formulations have been noted to possess antimicrobial properties, which are evidenced from the fact that the growth of bacteria was inhibited by liposomal neomycin and penicillin [35]. Also, the plasma concentration of gentamicin has been found to be increased when given by intramuscular route due to its sustained release from injection site. The antimicrobial activity of rifabutin against Mycobaterium avium when administered as liposomal formulation has been found to be increased when compared to free rifabutin. Moreover, the antimicrobial potential of liposomes has been confirmed by the fact that both renal and haematological toxicity of conventional Amphotericin B has been reduced significantly when encapsulated in liposomes [36]. In addition, the liposomes have been widely accepted to possess potent applications in gene therapy. The defects in a gene or absence of a gene are the factors that are responsible for lack of enzymes resulting in the development and progression of various systemic diseases, and thus, the selective localization of administered therapeutic gene is the major point of concern in gene delivery systems. It has been well reported that cationic liposomes act as an extraordinary human gene delivery system [37, 38]. Further, allovectin-7tm , a gene transfer liposomal product, has been found to be effective against metastatic melanoma, renal cell and colorectal carcinoma, evidencing its potential in gene therapy [39, 40].

CONCLUSION

The liposomal drug delivery systems have expanded at a very fast rate in recent years. It has been accepted that liposomal preparations are marketed as well as clinical trials are also being performed on certain other liposomal formulations. It has been shown that liposomal formulations lead to increased therapeutic activity with decreased toxicity, and thus, possess various potential applications clinically. However, various studies have extracted the potential of liposomal formulations, but future studies are demanded in order to completely exploit the mechanisms of liposomes in order to emphasize other potential applications exhibited by them.

Conflict of Interest

NIL

Source of Support

NONE

Figures at a glance

Figure 1

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