ReviewNovel methods for liposome preparation
Introduction
Liposomes are spherical vesicles having an aqueous core enclosed by one or more phospholipid bilayers or lamellae. Liposomes are most frequently classified on the basis of their size (small, large and giant vesicles), number of bilayers (uni-, oligo- and multi-lamellar) (Vemuri and Rhodes, 1995, Vuillemard, 1991) and phospholipid charge (neutral, anionic or cationic) (Storm and Crommelin, 1998). Recently, liposomes have also been categorized with respect to their function such as conventional, stealth, ligand-targeted, long-release, and triggered-release (Sharma and Sharma, 1997, Storm and Crommelin, 1998). Multi-functional liposomes possessing a combination of these features have also been reported (Kale and Torchilin, 2010, Perche and Torchilin, 2013, Xiang et al., 2013). Even though liposomes were first reported close to half a century ago (Bangham et al., 1965), it was three decades after their discovery that the first liposomal drug Ambisome® entered the market (Davidson et al., 1994, Hann and Prentice, 2001). Since then, the list of liposomal drugs has continued to increase (Allen and Cullis, 2013).
Over the years, different recipes for liposome preparation at the laboratory scale have been developed and optimized. Liposome preparation techniques may be divided into (a) bulk methods, where liposomes are obtained by transfer of phospholipids from an organic phase into an aqueous phase, and (b) film methods, in which lipid films are first deposited on a substrate and subsequently hydrated to give liposomes. The preparation methods have also been classified based on mean size, polydispersity and lamellarity of liposomes obtained, because control over these parameters remains a challenge with almost all preparation methods. This problem is exacerbated when moving from the laboratory to industrial scale (Mozafari, 2005, Riaz, 1996, Wagner and Vorauer-Uhl, 2011). For drug delivery applications the desirable size of liposomes ranges between 50 and 200 nm (Harashima et al., 1994, Woodle, 1995). Therefore, reduction of size and lamellarity of liposomes is typically carried out by subjecting them to homogenization, sonication, extrusion or freeze–thaw cycles (Brandl et al., 1990, Furukawa, 2010, Hope et al., 1985, Hope et al., 1986, Johnson et al., 1971).
Several exhaustive reviews have been published describing and comparing conventional methods for liposome preparation (Akbarzadeh et al., 2013, Dua et al., 2012, Mansoori, 2012, Mozafari, 2005, Riaz, 1996, Sharma and Sharma, 1997, Shashi et al., 2012, Storm and Crommelin, 1998, Wagner and Vorauer-Uhl, 2011). The present review covers these methods only briefly and further details may be found in cited literature. The review also does not cover the vast literature on synthetic modifications to phospholipids that are aimed at avoiding liposome detection and clearance by the immune system, targeting of liposomes to afflicted sites and controlled drug release. The focus of this review is on novel methods of liposome preparation that have become possible due to recent developments in technologies enabling fabrication and pattern formation on materials at the micro- and nano-scale. The review also discusses innovative strategies that have been used to overcome the limitations faced in conventional methods of liposome preparation. However, one should bear in mind that the new liposome preparation methods discussed in this review have only been demonstrated at the laboratory scale and remain to be developed for industrial use. Prior to describing the liposome preparation techniques we mention some commonly used liposome characterization methods and briefly discuss the forces and mechanisms governing liposome formation.
Section snippets
Energetics and kinetics of liposomes formation
Phospholipids have a hydrophilic head group and two long hydrophobic tails which make them poorly soluble in water unless they self-assemble into bilayers. A finite patch of the phospholipid bilayer has an energy associated with its edge where the hydrophobic tails are exposed to water and is proportional to the perimeter of the patch (Fig. 1A). This energy may be minimized by eliminating the edge if the bilayer patch closes to form a spherical vesicle. However, there is also an energy penalty
Liposome characterization methods
Since liposome function is strongly dependent on properties such as liposome size, shape, lamellarity and surface charge (Hunt et al., 1979, Juliano and Stamp, 1975, Park et al., 1992), accurate estimation of these properties is vital. Electron microscopic techniques not only enable visualization of liposomes to study their morphology and lamellarity but also facilitate accurate estimation of size of individual liposomes (Egerdie and Singer, 1982, Larrabee et al., 1978). However, electron
Conventional methods for preparing giant uni-lamellar vesicles (GUVs)
A. Gentle hydration of a phospholipid film. The method involves deposition of phospholipids, from a solution in an organic solvent such as chloroform or ethanol, onto a substrate. The film consisting of stacked phospholipid bilayers is subsequently hydrated over a couple of days in the absence of hydrodynamic flow to obtain an aqueous suspension of GUVs (Reeves and Dowben, 1969). Though a significant fraction of the vesicle population also comprises of MLVs (Kuroiwa et al., 2009, Reeves and
Conventional methods for preparing multilamellar vesicles (MLVs)
A. Hydration of a phospholipid film under hydrodynamic flow. MLVs are formed when a dry phospholipid film of stacked bilayers, deposited on a substrate, is rehydrated under strong hydrodynamic flows for a couple of hours (Bangham et al., 1967). The resulting MLV suspension contains vesicles that are heterogeneous in size and lamellarity.
B. Solvent spherule method. Vigorous mixing of an organic phase containing phospholipids and an aqueous phase for ∼1 hr under low vacuum yields an oil-in-water
Conventional methods for preparing small and large unilamellar vesicles (SUVs and LUVs)
A. Reverse phase evaporation. Similar to the solvent spherule method used for preparation of MLVs, reverse phase evaporation also involves hydration of phospholipids dissolved in an organic phase by addition of water with vigorous mixing. In contrast, to the solvent spherule method, a water-in-oil emulsion is formed in this case and evaporation of the organic phase results in an aqueous suspension containing LUVs (Deamer and Bangham, 1976, Szoka Jr and Papahadjopoulos, 1978) as well as MLVs (
Microfluidic methods for liposome formation
Microfluidics involves fluid flow in channels having cross-sectional dimensions, typically in the range of 5–500 μm. In the last decade, several novel microfluidics-based techniques have been developed to produce liposomes. The features of microfluidic systems that can be used to advantage in liposome production include ability to accurately dispense nanoliter volumes, precise control over the position of the interface, diffusion-dominated axial mixing and continuous mode of operation at low
Supercritical fluids for liposome formation
Supercritical fluids (SCFs) possess some desirable properties of both liquids and gases. For instance, a small change in pressure or temperature leads to large changes in density of the SCF and solubility of various species in the SCF. SCFs are increasingly replacing organic solvents as they enable more efficient separation and purification. The use of SCFs in liposome preparation has been reviewed by Meur and coworkers (Meure et al., 2008). The SCF used in most studies is carbon dioxide.
Castor
Modified electroformation methods for preparation of giant vesicles
The conventional electroformation protocol yields GUVs only under certain constraints. For instance, in the presence of physiological ionic solutions, GUVs are obtained only if charged phospholipids are used (Akashi et al., 1996). It was shown that, by incorporation of 10 mol% of negatively charged phospholipids, GUVs could be obtained in 100 mM potassium chloride solutions (Akashi et al., 1996). It was also shown that 2–5 times more GUVs were electroformed, when uniform thin films of
Size reduction of MLVs and GUVs
To obtain highly mono-disperse liposomes having size in the range 40–200 nm for use in drug delivery applications, MLV suspensions are typically subjected to extrusion or sonication (Hope et al., 1985, Huang, 1969). Richardson and coworkers suggested that the primary mechanism size reduction of liposomes by ultrasound waves was due to a shear-mediated elongation and subsequent rupture of cylindrical liposomes, caused by micro-streaming around the bubbles, and not a result of the collapse of
Other novel methods for preparing liposomes
A. Freeze drying of double emulsions. Freeze drying of liposome-forming lipids and water-soluble carrier materials dissolved in tert-butyl alcohol/water co-solvent systems results in cakes of an isotropic monophasic solution. On addition of water, the freeze-dried product spontaneously forms a homogenous dispersion of MLVs which may then be downsized by extrusion. However, one of the limitations of freeze drying was the relatively low encapsulation efficiency of freeze dried liposomes. Wang and
Conclusions and perspectives
Several liposomal formulations are already on the market, while quite a few are still in the pipeline. Conventional techniques for liposome preparation and size reduction remain popular as these are simple to implement and do not require sophisticated equipment. However, not all laboratory scale techniques are easy to scale-up for industrial liposome production. Many conventional methods, for preparing small and large unilamellar vesicles, involve use of either water miscible/immiscible organic
Acknowledgments
The authors thank Ms. Vibha Jayaraj for technical help. The authors are grateful to Department of Science and Technology, Government of India, for financial support.
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