Many o f the techniques described above may require a further processing step after manufacture to reduce liposome size or enhance the aqueous entrapment. In the context of using liposomes as carriers for parenterals, one of the main concerns is to reduce the size o f the liposomes. Therefore, most manufacturing protocols employ two stage procedures to generate liposomes suitable for intravenous administration.
The basis of all the following size reduction techniques is to generate energy to fragment the liposome bilayer. This induces the break up of the liposome and subsequently reduces the liposome size after reformation. For efficient size reduction, the processing temperature has to be above the phase transition of the phospholipid (section 2.1.3), i.e. the phospholipid molecules in the liposomes have to be in a relatively fluid state. The
Chapter one- Introduction
various methods by which this liposome size reduction can be achieved are outlined below:
1.1.4.1 French press
French presses are used to disrupt cells under high pressure. In the case of liposomes, the mechanical ram o f the press forces the liposome dispersion through a narrow orifice, which ruptures the vesicles by shearing. The resultant translucent dispersion consists predominantly of SUVs (Barenholz et al., 1979; Hamilton et al., 1980).
1.1.4.2 High pressure homogenisation
This technology was originally developed for reducing the size o f emulsion droplets in the food industry to prevent creaming in dairy products. It was later employed in pharmaceutics in an allied technology to produce fat emulsions for parenteral nutrition. This technology is the current commercial technique of choice to reduce coarse liposome dispersions to small unilamellar liposomes. The homogeniser reduces the size o f the liposomes via cavitation and shearing during laminar and turbulent flow (Brandi et al., 1990; Bachmann et al., 1993). This is achieved by passing the dispersion through a small knife edged gap under high pressure. After optimising the process, it is possible to generate homogeneous liposomes with average diameters of less than 50 nm within a very narrow size band. These translucent dispersions are eminently suited to aseptic filtration. This direct method of producing liposomes is amenable to large scale processing. Furthermore, scaling up the production is easy: once the parameters for small scale production have been established and optimised, the information can usually be directly transferred to larger scale manufacturing equipment for larger batches.
The main disadvantage is that during use, particulates may be shed from the interaction chamber. Additionally, there is little control over the size of the liposome, only small unilamellar vesicles can be generated.
1.1.4.3 Microfluidisation
This process is similar to high pressure homogenisation, the main difference being the interaction chamber, which relies upon head on collision of the liquid at right angles against a plate of the interaction chamber (Mayhew et al., 1984). The advantage is that high amounts of lipid (up to 20% w/v) can be handled. The disadvantages are the same as high pressure homogenisation.
1.1.4.4 Membrane extrusion
Originally described by Olson et al. (1979) and Szoka et al. (1980), the liposome dispersion is physically extruded through a filter with cylindrical channels of defined diameter. These channelled pores are created by laser etching through a polycarbonate filter. After repeated extrusions, the upper size limit of the dispersion approaches the diameter of the pores. This method is useful for producing small batches of liposomes with a defined size, ranging from 30 nm up to several microns in diameter. If the liposome diameters are 200 nm or less, the dispersions can be readily sterilised by filtration. Although this technique can be scaled up (Schneider et al., 1995), without arranging several filters in parallel, the batch volume is usually restricted to approximately 100 litres (O’Hara, personal communication). This is due to the filter diameter, which can not easily be increased above 47 mm without the filter integrity being compromised.
1.1.4.5 Sonication
There are two types of sonication: probe and bath. Probe ultrasonication employing a titanium probe can be used to produce SUVs between the lowest theoretical limit of about 25 nm up to 80 nm (Huang, 1969). For size reproducibility between batches, it is important to maintain the exact position of the probe. The main disadvantages are possible oxidative and hydrolytic lipid damage, which can occur if the process is not controlled. This is due to the highly energy intensive nature of the process. Secondly, the probe sheds titanium into the dispersion, resulting in fine grey deposits which have to be removed from the dispersion by centrifugation. Bath sonication is less intense than probe sonication and, therefore, the rate o f the size reduction is inferior and final liposome diameter is generally larger.
1.1.4.6 Freeze thaw sonication
This technique involves alternating cycles o f sonication and freezing of a liposome dispersion (Pick, 1981). For large scale production this technique is impractical and is normally applied to enhance the aqueous entrapment of water soluble materials by creating larger unilamellar vesicles (LUVs).
The adoption o f the mechanical disruption techniques, particularly microfluidisation and high pressure homogenisation, have greatly aided the commercialisation of liposome products. However, these techniques are expensive and may require large capital
Chapter one- Introduction
investment. The size reduction step should be appreciated in the context of the manufacturing process as a whole. It is just a part of the manufacturing process, further processing steps, such as sterilisation and stabilisation, may also be required.