Table 1 Classification of commonly known lipid vesicles according to their structures and/or preparation.

Identification	Definition
Archeosomes	Archeosomes are vesicles consisting of arch bacteria lipids which are chemically distinct from eukaryotic and prokaryotic species. They are less sensitive to oxidative stress, high temperature, and alkaline pH.^7,8
Cochleates	Cochleates are derived from liposomes which are suspended in an aqueous two-phase polymer solution, allowing the logic partitioning of polar molecule-based structures by phase separation. The liposome containing two-phase polymer solution treated with positively charged molecules such as Ca⁺²or Zn²⁺forms a cochleate precipitate of a particle dimension less than 1 um^.9
Dendrosomes	Dendrosomes represent a family of novel, nontoxic, neutral, biodegradable, covalent or self-assembled, hyper branched, dendritic, spheroidal nanoparticles which are easy to prepare, inexpensive, highly stable as well as easy to handle and apply, compared with other existing synthetic vehicles for gene delivery.¹⁰
Dried reconstituted vesicles (DRV)	By this preparation technique, small, “empty” unilamellar vesicles, containing different lipids or mixtures of them, are prepared. After mixing those SUV’s with the solubilized drug, dehydration is performed. By addition of water, rehydration leads to the formation of large quantities of rather inhomogeneous, multilamellar vesicles which need further processing.¹¹
Ethosomes	Ethosomal systems are much more efficient at delivering to the skin, in terms of quantity and depth, than either conventional liposomes or hydro alcoholic solutions. Ethosomal drug permeation through the skin was demonstrated in diffusion cell experiments. Ethosomal systems composed of soy phosphatidylcholine and about 30% of ethanol was shown to contain multilamellar vesicles by electron microscopy.¹²
Immunoliposmes	Liposomes modified with antibodies, Fab’s, or peptide structures on the bilayer surface were established for in vitro and in vivo application.^13,14
Immunosomes	Immunosomes are prepared by the anchorage of glycoprotein molecules to performed liposomes. Under the electron microscope, immunosomes look like homogenous spherical vesicles (50-60nm) evenly covered with spikes. Immunosomes have structural and immunogen characteristics closer to those of purified and inactivated viruses than any other form of glycoprotein lipids association.¹⁵
Immune stimulating complex(ISCOM)	ISCOM’s are spherical, micellar assemblies of about 40nm. They are made of the saponin mixture Quil A, cholesterol, and phospholids. They contain amphiphilic antigens like membrane proteins. ISCOM’s already have a built-in adjuvant, Quillaja saponin, which is a structural part of the vehicle.¹⁶
Lipoplexes	Cationic lipid-DNA complexes are efficient carriers for cell transfection but have certain drawbacks due to their toxicity. These toxic effects may result from either cationic lipids or nucleic acids.^17,18
LUVET’s	LUVETs are large unilamellar vesicles prepared by extrusion technique, mainly performed with high-pressure systems.²⁰
Niosomes	Niosomes are small unilamellar vesicles made from nonionic surfactants also called Novasomes. Their chemical stability is comparable to that of archeosomes.²¹
pH-sensitive liposomes	Four basic classes of pH-sensitive liposomes have been described previously. The first class combines polymorphic lipids, such as unsaturated phosphatidylethanolamines, with mild acidic amphiphiles that act as stabilizers at neutral pH. This class of pH-sensitive liposomes has been the most intensively studied. The second class includes liposomes composed of lipid derivatives resulting in increased permeability to encapsulated solutes. A third class of pH-sensitive liposomes utilizes pH-sensitive peptide or reconstituted fusion proteins to destabilize membranes following change of the polymer conformation at low pH.²²
Polymerized liposomes	Polymerized phosphotidyl choline vesicles (35-140 nm) have been synthesized from lipids bearing one or two methacrylate groups per monomer. Compared to nonpolymeric analogues, these vesicles exhibited improved stability and controllable time-release properties.^23,24
Proliposomes	Proliposomes are defined as dry, free-flowing particles that immediately form a liposomal dispersion on contact with water.²⁵
Proteosomes	Vesicles of bacterial origin were solubilized, followed by ammonium sulphate precipitation and dialysis against detergent buffer. Proteins and peptides are noncovalently complexed to the membrane, making them highly immunogenic.²⁶
Reverse-phase evaporation vesicles (REV) Stealth liposomes	Vesicles are prepared by evaporation of oil in water emulsions resulting in large unilamellar liposomes. In the early 1990s, this liposomes engineering process culminated with the observation that coating of liposomes with the polyethylene glycol (PEG), a synthetic hydrophilic polymer, would improve their stability and lengthens their half-lives in circulation, rendering the use of glycolipids obsolete. PEG coating inhibits protein adsorption and opsonization of liposomes, thereby avoiding or retarding liposome recognition by the reticuloendothelial system (RES). These PEG-coated liposomes are also referred to as sterically stabilized or stealth liposomes. The PEG stabilizing effects results from local surface concentration of highly hydrated groups that sterically inhibit both hydrophobic and electrostatic interactions of a variety of blood components at the liposome surface.^{27,28,29,30,31,32,33}
Temperature- sensitive liposomes	Temperature-sensitive liposomes are considered to be a promising tool to achieve site-specific delivery of drugs. Such liposomes have been prepared using lipids which undergoes a gel-to-liquid crystalline phase transition a few degrees above physiological temperature. However, temperature sensitization of liposomes has been attempted whose content release behavior, surface properties, and affinity to cell surface can be controlled in a temperature-dependent manner.^34,35
Transferosomes	Transferosomes consist of phosphatidylcholine and cholate and are ultra deformable vesicles with enhanced skin-penetration properties.³⁶
Virosomes	Virosomes are small unilamellar vesicles consisting influenza hem agglutinin, by which they became fusogenic with endocytic membranes. Co incorporation of other membrane antigens induces enhanced immune responses.³⁷

In general liposomes are defined as spherical vesicles with particle sizes ranging from 30nm to several micrometers. They consist of one or more lipid bilayers surrounding aqueous phases. However, self-aggregation of polar lipids is not restricted to conventional bilayer structures which depend on temperature, molecular shape, and environmental and preparation conditions but may self-assemble into various kinds of colloidal particles.⁵ Due to this fact, the liposome family includes various kinds of colloidal particles and structures which hamper systemic classification. However, they can be classified by structure, composition, and preparation, as shown in table 1.

Technology and application are driven by two major facts. First, the transfer from academic bench to highly regulated, high technology industry was difficult for liposome technology because of the lack of appropriate methods to produce large quantities in a controlled and reproducible manner. Although several methods are suitable for large-scale production, their development, implementation, and quality control needed a certain time. Second, early clinical trials were not as successful as expected because the stability of conventional liposomes was low, caused by insufficient preparation, physical properties, and unfavorable choice of lipids. Furthermore, they were to a great extent cleared by liver and spleen very rapidly so that neither a prolonged biological half-life nor specific targeting was achieved. More stable conventional liposomes and second generation formulations, such as the stealth technology, gave new impulses to the industry as well as to clinicians with the development of industrial processes in 1990s.⁶

Classification of Liposomes:³⁸

When liposomes are described based on the number of bilayers, they are described as unilamellar vesicles (ULV) or multilamellar vesicles (MLV); reverse phase evaporation vesicles (REV) and French press vesicles (FPV) and ether injection vesicles (EIV) are the descriptions based on the method of preparation; or when liposomes are described based on their size, they are large unilamellar vesicles (LUV) and small unilamellar vesicles (SUV). The description of liposomes by the lamellarity and size are more common than by the method of their preparation. (See table no.2)

Table 2: Nomenclature and approximate sizes of various liposomes

Liposome	Classification	Approximate size (um)
By size	SUV LUV	0.025-0.05 0.1
By lamellarity	MLV ULV	0.05-10 0.025-0.1
By method	REV FPV EIV	0.5 0.05 0.02

Multilamellar vesicles (MLV):

Liposomes prepared and described by Banghum are a heterogeneous mixture of MLV and ULV. They are the widely studied vesicles. MLV form spontaneously when an excess volume of aqueous buffer is added to dry lipids. In many cases MLV have not been characterized with respect to size, polydispersibility, number of lamellae, encapsulated volume and stability on the shelf. Because of their ease of the preparation, many investigators have simply prepared the MLV without taking time to characterize the. Often times this cause minor change in the preparation could lead to differences in the liposomes and in turn their in vitro and in vivo behavior. When the molecular mixture of lipids are prepared in an organic solvent and subsequently removed by rotary evaporation in a round bottom flask, under vacuum, lipids are deposited as a thin film on the flask. The time allowed for the hydration of the lipid layer with the aqueous buffer/drug solution determines the amount of drug entrapment into vesicles. This procedure also influences the size of the MLV.

Small unilamellar vesicles (SUV):

Dispersions of phospholipids in water were mixed by sonication method at 60°C to prepare liposome. Subsequently, other investigators used high pressure techniques to prepare larger volumes of liposomes. Usually, the MLV preparation is subjected to sonication under nitrogen or argon to reduce size and prepare SUV. Sonicator can be of a bath type or probe type instrument. A problem associated with sonicator probe is its metal particle shedding. The bath sonicator has its advantages. For instance, the temperature of the product can be controlled during process by controlling the temperature of the water in the bath, and the product can be processed aseptically in a sealed container. Conversion of MLV to SUV can be achieved by passing through the narrow orifice under high pressure. It was reported that multiple passes through the device are needed to obtain uniform vesicle size, at high pressure of 20,000 psi and 4^°C. This method yields a vesicle size range of 0.3um to 0.5um. SUV can also be prepared by solvent injection methods such as ether injection and ethanol injection. In ether injection method, lipids dissolved in diethyl ether are slowly injected into warm water, typically with the help of a syringe type infusion pump. Subsequently the ether is removed from the preparation by applying the vacuum. The resultant product is single layers of liposome vesicles. An alternative method of preparing SUV is by the ethanol injection method. This method requires neither sonication nor the high vacuum environment. In this method, the lipids dissolved in ethanol are rapidly injected into an aqueous media resulting in SUV spontaneously. Removal of residual ethanol from the preparation can present problems, since the alcohol forms the azeotrope with water and its difficult to remove under vacuum or by distillation procedures.

Large unilamellar vesicles (LUV):

Large unilamellar vesicles (LUV) are capable of holding larger volumes of solution in their cavity and thus they have higher encapsulation efficiency compared to MLV. Other advantages of LUV are economy of lipids that can result from larger quantity of drug encapsulation in lesser quantity of lipid (mg of drug per mg of lipid) and reproducible drug release rates. ‘Large’ in the context of liposomes usually means vesicles structure larger than 100nm. LUV’s are liposome vesicles that are bounded by a single bilayer of lipids and are greater than 100 nm in size. The size of LUV is a debatable topic. Unilamellar liposomes of 50-100 nm were also referred as LUV by some investigators. LUV can be produce by reverse phase evaporation technique and detergent dialysis technique. Unilamellar liposomes vesicles of less than 100 nm can be produced from MLV by sequential extrusion through small size polycarbonate membranes under high pressure. A number of methods of preparing LUV appeared in the scientific literature. Some of them are described in this review.

Preparation Techniques:³⁸

Lipid molecules have to be introduced into an aqueous environment for the preparation of liposomes independent of liposome size and structure. A general overview representing the correlation of the way of lipid hydration, respectively, the way of primary liposome formation with the resulting liposome structure, was originally developed by Lasic. Several ways of treating the lipids are known to support the hydration of these molecules, as lipid molecules themselves are poorly soluble in aqueous compartments. These procedures can be categorized as shown in Table no.3.

Table 3: Methods of liposome preparation and the resulting product.³⁹

Method

Vesicles

Mechanical methods

Vortex or hand shaking of phospholipids dispersion

Extrusion through polycarbonate filters at low or medium pressure

Extrusion through a French press cell “Micro fluidizer” technique

High-pressure homogenization

Ultrasonic irrigation

Bubbling gas

MLV

OLV,LUV

Mainly SUV

SUV of minimal size

BSV

Methods based on replacement of organic solvent (s) by aqueous media

Removal of organic solvent (s)

Use of water-immiscible solvents: ether and petroleum

Ethanol injection method

Ether infusion (solvent vaporization)

Reverse-phase evaporation

MLV,OLV,SUV

MLV,OLV,LUV

LUV

LUV,OLV,MLV

Methods based on detergent removal

Gel extrusion chromatography

“Slow” dialysis

Fast dilution

Other related techniques

SUV

LUV,OLV,MLV

LUV,OLV

MLV,OLV,LUV,SUV

Mechanical Methods

Preparation by Film Methods⁴⁰

Properties of lipid formulations can vary depending upon the composition (cationic, anionic and neutral lipid species). However, the same preparation method can be used for all lipid vesicles regardless of composition. The general steps of the procedure are preparation of the lipids hydration, hydration with agitation, and sizing to a homogenous distribution of vesicles. Several downsizing techniques have been established in order to make the heterogeneous vesicles more uniform. The first published method was sonication. A very high energy input based on cavitations is applied to the liposomal dispersion either directly with a tip or indirectly in a bath sonicator.⁴¹ the most defined method for downsizing is the extrusion technique whereby liposomes are forced through filters with well defined pores.

Homogenization Techniques:⁴²

Similar to the ultrasound methods, homogenization techniques have been used in biology and microbiology for breaking up the cells. Therefore, many scientists have been used them for reducing the size and number of lamellae of multilamellar liposomes⁴³. The French press originally was established for breaking up cells under milder and more appropriate conditions compared to the ultrasound techniques, because lipids as well as proteins or other sensitive compounds might be degraded during the sonication procedure. This system is normally used in the volume of 1 to 40 ml and therefore not suitable for large-scale production. This continuous and scalable variation of the French press technique^{44, 45} enforces downsizing of liposomes by collision of larger vesicles at high pressure in the interaction chamber of the micro fluidizer. The main disadvantage of this method is the long-lasting preparation starting with preformed liposomes, eventually an additional freeze-thaw step, and finally the extrusion. In these entire procedures, high product losses may be generated, especially if clogging of the extrusion membranes occurs, which may cause technical limitations with large scale production of high-priced goods.⁴⁶

Methods Based on Replacement of Organic Solvents by Aqueous Media

The Ethanol Injection Method:⁴⁷

This technique was first reported in the early 1970s by Batzri and Korn as one of the first alternatives for the preparation of SUVs without sonication. By the immediate dilution of the ethanol in the aqueous media, the lipid molecules precipitates and form bilayer planer fragments which themselves form into liposomal systems, thereby encapsulating aqueous phase. This method has many advantages as the technique is in principle easy to scale up, and ethanol is a very harmless solvent, accepted by the authorities also for the injectables at the maximum of 0.1%. Some other solvents might also be used, but one has to keep in mind the regulations for residual solvents classified into different categories by the European or US Pharmacopoeia. Another important advantage of this method is the suitability of the entrapment of many different drug substances such as large hydrophilic proteins by passive encapsulation, small amphiphilic drugs by a one-step remote loading technique, on membrane association of antigens for vaccines.

Proliposome-Liposome Method:⁴⁸

The Proliposome-liposome method is based on the conversion of the initial proliposome preparation into liposome dispersion by dilution with an aqueous phase. This method is suitable for the encapsulation of a wide range of drugs with varying solubility in water and alcohol and has extremely high encapsulation efficiencies when compared with other methods based on passive entrapment. Turanek and coworkers have developed a sterile liposome production procedure based on this method.

Reverse-Phase Evaporation (REV):⁴⁹

Similarly to the above presented injection methods, lipid is hydrated via solubilization in an organic phase followed by introduction into an aqueous phase. The organic phase should be immiscible with the aqueous phase, thus an oil/water emulsion is created, which is diluted with the further aqueous phase for liposome formation. The advantage of this very popular preparation technique is very high encapsulation rate up to 50%. One variation of the micro emulsion technique, the double emulsion technique, further improves the encapsulation rates and results in multilamellar liposomes. A possible drawback of this efficient method is the remaining solvent or the proof of their absence especially for using them for pharmaceutical purposes. The another important issue is large-scale production which might be feasible if appropriate shear mixing devices for the creation of the micro emulsion and pumps for the dilution step are available.

Method Based on Detergent Removal:⁵⁰

In this group of liposome preparation procedures, detergents, such as bile salts or alkyl glycosides, are used for the solubilization of lipids in micellar systems. In contact to lipids, detergents are highly soluble in both aqueous and organic media. There is equilibrium between the detergent molecules in the aqueous phase and the lipid environment of the micelle. The size and shape of the resulting vesicles are depending on the chemical nature of the detergent, their concentration, and the lipids used. To date, the most frequently applied method for the membrane proteins reconstitution involves the cosolubilization of membranes proteins and phospholipids. Common procedures of detergent removal from the mixed micelle are dilution, gel chromatography, and dialysis through hollow fibers or through membrane filters. Additionally, detergents can also be removed by adsorption to hydrophobic resins or cyclodextrins.

Evaluation of Liposomes:⁵¹

Entrapment Efficiency:

Drug associated with liposome was separated from unentrapped drug using centrifugation method. Liposomes were centrifuged at 20000 rpm for 1 h at controlled temperature of 4 C. Supernatant containing unentrapped drug was withdrawn and measured UV spectrophotometrically against phosphate buffer saline (pH 7.4). The amount of drug entrapped in liposome was determined as follow EE (%) = [(Cd–Cf)/Cd] 100 Where Cd is concentration detected of total drug and Cf is concentration of free drug. The entrapment efficiency was obtained by repeating the experiment in triplicate and the values were expressed as mean standard deviation.

Size Distribution:

Prepared liposomal batches were monitored for their morphological attributes using optical microscope. Mean vesicle size and size distribution profile of liposome was determined by using Malvern particle size analyzer model SM 2000, which follows Mie's theory of light scattering. Diluted liposome suspension was added to the sample dispersion unit containing stirrer and stirred at 2000 rpm in order to reduce the inter particle aggregation, and laser obscuration range was maintained between 10-20%. The average particle size was measured after performing the experiment in triplicate.

Zeta Potential (z) Determination:

Charge on empty and drug loaded vesicles surface was determined using Zetasizer 300HSA (Malvern Instruments, Malvern, UK). Analysis time was kept for 60 s and average zeta potential and charge on the liposome was determined.

Oscillation Stress Sweep:

Dynamic oscillation stress sweep was performed to determine the linear viscoelastic region (LVR). LVR is the region where the elastic modulus (G') was independent of applied stress because destruction in the structure of gels occurs at high shear stress. Analysis of viscoelastic material was designed not to destroy the structure so that measurement can provide the information about intermolecular and inter particle forces in the material. This test gives idea about the critical stress beyond which the sample may show significant structural changes, and therefore the consequent choice of the stress value to be used in other in other oscillation tests. The samples were exposed to increasing stress (0.5 to 150 Pa) at a constant frequency of 0.1 Hz. The three main parameters determined in this test were the storage modulus G', loss modulus G” and loss tangent tan! The end point of the linear viscoelastic region was determined as astress, when the G' value was dropped 10% from the linear level that indicated a significant change in the structure gel samples.

Marketed Formulation of Topical Liposome:⁵¹

a) Celadrin® b) Optisome^TM– Encapsulated Tetracaine.c) Lipo C^TM Liposome -encapsulated Active Vitamin C with Vitamin E and Zinc.d) Lipo-Gest™ Natural Balancing cream) Liposome progesterone based cream.

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Received on 26.10.2015 Accepted on 18.11.2015

Asian J. Pharm. Tech. 2015; Vol. 5: Issue 4, Oct. - Dec., Pg 231-237

DOI: 10.5958/2231-5713.2015.00033.1