liposme manufacturing.pdf

CELLULAR & MOLECULAR BIOLOGY LETTERS Volume 10, (2005) pp 711 719

http://www.cmbl.org.pl Received 15 July 2005 Accepted 6 October 2005

* e-mail: [email protected]

LIPOSOMES: AN OVERVIEW OF MANUFACTURING TECHNIQUES

M. REZA MOZAFARI *

Riddet Centre, Massey University, Private Bag 11 222, Palmerston North, New Zealand

Abstract: During the last few decades liposomes have attracted great interest as ideal models for biological membranes as well as efficient carriers for drugs, diagnostics, vaccines, nutrients and other bioactive agents. The extensive and ever increasing literature covering the field of liposomology written by researchers with diverse backgrounds is an indication of increasing interest in this field. Many techniques and methodologies have evolved for the manufacture of liposomes, on small and large scales, since their introduction to the scientific community around 40 years ago. This article intends to provide an overview of the advantages and disadvantages of liposome preparation methods in general with particular emphasis on the heating method, developed in our laboratory, as a model technique for fast production of the lipid vesicles. Key Words: Carrier Systems, Heating Method, Lipid Vesicles, Liposomology, Manufacturing Techniques INTRODUCTION

Liposome science and technology is one of the fastest growing scientific fields contributing to areas such as drug delivery, cosmetics, structure and function of biological membranes and investigations of the origin of life to name a few. This is due to several advantageous characteristics of liposomes such as ability to incorporate not only water soluble but also lipid soluble agents, specific targeting to the required site in the body and versatility in terms of fluidity, size, charge and number of lamellae. While the use of liposomes as models for biomembranes is confined to the research laboratory, their successful application in the entrapment and delivery of bioactive agents will depend not only on a demonstration of the superiority of the liposome carrier for the intended purpose, but also upon technical and economic feasibility of the formulation. For delivery applications liposomal formulations should have high entrapment efficiencies, narrow size distributions, long-term stabilities and ideal release properties (based on the intended application). These require the preparation method to have the potential to produce liposomes using a wide range of ingredient molecules, e.g.

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lipids/phospholipids that promote liposome stability. In addition to the above properties, for the delivery of sensitive molecules/compounds such as proteins and nucleic acids, liposomes should also be able to protect the incorporated agents from degradation. Despite the enormous research and development works on liposomes, only a small number of liposomal products have been approved for human use so far. This may be due to many reasons including: toxicity of some liposomal formulations, low entrapment of molecules and compounds into liposomes, instability of the liposomal carriers, and high cost of liposome production especially on large scales. There are numerous lab-scale and a few large-scale techniques for liposome preparation giving rise to vesicles of different sizes ranging from 20nm to several microns in diameter and composed of one or more bilayers (e.g. see: [1-5]). However, most of these techniques are not suitable for the encapsulation of sensitive substances because of their exposure to mechanical stresses (e.g. sonication, high-shear homogenisation, or high pressures), potentially harmful chemicals (e.g. volatile organic solvents and detergents) or low/high values of pH during the preparation. Conventional liposome preparation techniques were discussed extensively by Gregoriadis [1, 2] and New [4]. More novel techniques are being introduced for liposome preparation, each with its own advantages and possible limitations. The key point to grasp in considering the manufacture of liposomes is that lipid/phospholipid membranes form as a result of unfavourable interactions between lipids/phospholipids and water molecules. Thus the emphasis in making liposomes is not towards assembling the membranes, but towards getting the membranes to form vesicles of the right size and structure, and to entrap materials with high efficiency and in such a way that these materials do not leak out of the liposome randomly. The present article aims at providing a concise review of liposome preparation techniques. Technical and practical details of these techniques are not given here, for which readers are referred to the respective references provided. However, as a model liposome manufacture method, a fast technique recently developed by the author of this article is explained in more details in the final section of the manuscript. MECHANISM OF LIPOSOME FORMATION

The detailed mechanism of liposome formation by different techniques is out of the scope of this article and requires a thorough biophysical review. Almost for each liposome preparation method a different liposome formation mechanism can be suggested. Lasic [6] for example proposed that liposomes form when hydrated bilayered phospholipid flakes (BPF) are generated and subsequently vesiculate into larger liposomes. Gould-Fogerite and Mannino [7] proposed the involvement of structures called cochleate cylinders as intermediates in the formation of large unilamellar vesicles (LUV) by calcium-EDTA chelation, agarose plug diffusion and rotary dialysis techniques. Talsma and co-workers

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[8], for their bubble method, postulated that the continuous generation of gas/water interfacial areas might produce lipid monolayers at these interfaces that could be the starting material for further vesicle formation. The basic underlying principle for the formation of liposomes, regardless of the preparation methodology, is the hydrophilic/hydrophobic interactions between lipid-lipid and lipid-water molecules. Input of energy (e.g. in the form of sonication, homogenisation, shaking, heating, etc.) results in the arrangement of the lipid molecules, in the form of bilayered vesicles, to achieve a thermodynamic equilibrium in the aqueous phase. Lasic et al [9] have proposed that symmetric membranes prefer to be flat (spontaneous curvature CO = 0) and energy is required to curve them. Whether spherical lipid membranes form in one stage (with curvatures initially as they form) or in two stages, as suggested by Lasic and co-workers for the above-mentioned case, it seems that energy in one form or another is a requirement for the production of liposomes. In the heating method of Mozafari and colleagues [10-12], for instance, the main form of energy for the formation of liposomes is heating while stirring is used to facilitate homogeneous distribution of the ingredients. There are indications that this heating method simulates the formation of the early cell membranes under the conditions of the primordial earth [13] (Fig. 1). This is another evidence of the crucial role of liposomes in the origin of life.

Fig. 1. A possible mechanism of the formation of primitive biomembranes from the lipid precursors (simple, single-tailed lipid molecules) by some energy input. Considering hydrothermal vents as the potential location for the emergence of first life forms, this energy is in the form of heat as is the case with the heating method of liposome preparation (from Ref. [13]).


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CONVENTIONAL LIPOSOME PREPARATION METHODS

Conventional methods of liposome manufacture can be said to involve four basic stages: drying down of lipids from organic solvents, dispersion of the lipids in aqueous media, purification of the resultant liposomes, and analysis of the final product [4]. However, organic solvent residues, remaining in the lipid and/or aqueous phases of the liposomes during their preparation, could result in toxicity [14] (explained more in the next section). Involvement of the volatile organic solvents in liposome preparation is one of the disadvantages that nearly all conventional liposome manufacture methods suffer from. Recent developments in the field of liposome technology have made it possible to prepare lipid vesicles without using any volatile organic solvent or detergent, examples of which are the polyol dilution method [15], the bubble method [8] and the heating method developed in our laboratory [10-12]. It should be noted that only a few of the conventional liposome preparation procedures are capable of entrapping large quantities of water-soluble agents [16]. Bioactive agents can be entrapped in lipid vesicles by the conventional methods of reverse-phase evaporation technique [17], ether injection/vaporisation technique [18, 19], and freeze-thaw method [20], just to name a few. These techniques yield large-unilamellar vesicles (LUV) or multilamellar vesicles (MLV) based on the selected method. Without employing further steps (e.g. extrusion through polycarbonate filters [21-22]) almost all the above methods produce heterogeneous mixture of lipid vesicles. The original method of Bangham et al [23] appears to be a simpler method compared with the techniques mentioned above. This method, however, has limited use because of its low encapsulation ability and impossibility to scale to larger batch sizes. VOLATILE ORGANIC SOLVENTS: A TRADITIONAL PROBLEM IN LIPOSOME PREPARATION

The majority of liposome preparation techniques involve the application of volatile organic solvents (mainly chloroform, ether, or methanol), as a first step, to dissolve or solubilise the lipids. These solvents not only affect the chemical structure of the entrapped substance but will also remain in the final liposome formulation and contribute to toxicity and influence the stability of the vesicles [14, 16, 24]. In general, residual solvents in pharmaceuticals, known as organic volatile impurities (OVIs), have no therapeutic benefits but may be hazardous to human health as well as the environment [25]. It has been suggested that organic solvents can exert toxicity towards cells via two types of mechanism: a) at the molecular level, or b) at the phase level. Molecular toxicity represents the effects caused by organic solvents that are dissolved within the aqueous phase and include enzyme inhibition, protein denaturation and membrane modifications such as membrane expansion, structure disorders and permeability changes. Phase toxicity effects include the extraction of nutrients, disruption of the cell wall (extraction of outer cellular


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components), and the limited access to nutrients caused by cell attraction to interfaces, the formation of emulsions and the coating of cells [26, 27]. A correlation between the ability of some organic solvents to destabilise membrane proteins and their cytotoxicity has recently been shown by Ivanov [28]. In addition to the above-mentioned disadvantages, application of volatile organic solvents or detergents necessitates performance of two additional steps in the preparation of liposomes: i) removal of these solvents/detergents, and ii) assessment of the level of residual organic solvents or detergents remained in the liposomal formulations (e.g. see [16]). Hence avoiding the utilisation of these solvents/detergents will potentially bring down the time and cost of liposome preparation. Several techniques have been suggested for the removal of detergent and solvent traces from liposomes. These techniques include gel filtration, vacuum and dialysis. It has been reported that even after removal of ether residues, by gel filtration, from liposomes prepared by the reverse phase evaporation method, trace amounts of ether still remained in the formulation and this was responsible for REVs (vesicles made by reverse-phase evaporation method) being more leaky to entrapped solutes when compared with liposomes prepared in the absence of ether [29]. Additionally, Weder and Zumbuehl [30] have reported, for liposomes prepared by the detergent dialysis method, that after dialysis 3 to 4% residual detergent was still present in the final preparation. Therefore, a method which would produce lipid vesicles avoiding the use of hazardous chemicals and solvents will be very useful in entrapment and delivery of the bioactive agents. LARGE-SCALE LIPOSOME MANUFACTURE

An important point in the development of a liposomal dosage form and evaluation of a suitable preparation process is whether it is possible to prepare, isolate and characterise the particular liposomal formulation on an industrial scale with clearly defined and reproducible properties. Another crucial point in this regard is the stability of the produced lipid vesicles [31]. Nevertheless, in order to prepare liposomes on a large scale for clinical applications, it is necessary to employ techniques that will meet the requirements of the pharmaceutical industry. One of the most important steps in the manufacturing of liposomes for human and animal use is sterilisation. It is generally known that lipid vesicles can only be sterilised by filtration and that any other method involving chemical or physical treatments, especially heating, would destroy the liposomal structure and consequently release the encapsulated material. However, most viruses can not be removed by filtration, so that intensive microbiological control is necessary, and furthermore, the filtering process is so time-consuming that it is preferably avoided when dealing with large batches of the liposomal products. A promising breakthrough regarding liposome sterilisation came with the work of Kikuchi and co-workers [32]. This group reported that it is possible to apply an


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ordinary heat sterilisation (121C, 20 min) and obtain liposomes which retain their structural integrity with high encapsulation efficiency after the heat treatment. Another major point regarding mass manufacture of liposomes is the production cost. An ideal preparation method, especially on industrial scales, would be the one necessitating minimum number of steps and equipment. Many of the conventional preparation techniques of liposomes do not meet these criteria. In the development of any new liposome preparation method the possibility of mass production of lipid vesicles in a fast and reproducible manner, utilising minimum number of equipment, should be seriously taken into account. HEATING METHOD

Recently, a new method for fast production of liposomes without the use of any hazardous chemical or process has been described. This method involves the hydration of the liposome components in an aqueous medium followed by the heating of these components, in the presence of glycerol (3% v/v), up to c. 120C [10-12]. Glycerol is a water-soluble and physiologically acceptable chemical with the ability to increase the stability of lipid vesicles and does not need to be removed from the final liposomal product. Since heating is the main step in this methodology it is termed Heating Method and the resultant liposomes are referred to as HM-liposomes [10-12]. It was confirmed by TLC that no degradation of the lipids occurred at the abovementioned temperatures [11]. Employment of heat, in fact, abolishes the need to carry out any further sterilisation procedure hence reducing the time and cost of liposome production by the heating method. Application of glycerol in the preparation of the HM-liposomes has the following advantages: i) glycerol is a bioacceptable, non-toxic agent already in use in many pharmaceutical products and can serve as an isotonising agent in the liposomal preparations; ii) unlike the volatile organic solvents employed in the manufacture of conventional liposomes, there is no need for the removal of glycerol from the final preparation; iii) it serves as dispersant and prevents coagulation or sedimentation of the vesicles thereby enhancing the stability of the liposome preparations; iv) it also improves the stability of the liposome preparations against freezing, thawing etc. Therefore, HM-liposomes are also ideal for freeze-drying e.g. in the preparation of dry powder inhalation products. HM-liposomes have been employed as gene transfer vectors [11, 12] as well as drug delivery vehicles using 5-FU [33] and glutathione [Mozafari et al., unpublished data] as model drugs. Incorporation of plasmid DNA molecules, which are sensitive to high temperatures, to the HM-liposomes was carried out at room temperature by incubation of DNA with the empty, pre-formed, HM-liposomes [11, 12]. Incorporation of drugs into the HM-liposomes can be achieved by several routes including: i) adding the drug to the reaction medium along with the liposomal ingredients and glycerol; ii) adding the drug to the reaction medium when


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temperature has dropped to a point not lower than the transition temperature (Tc) of the lipids; and iii) adding the drug to the HM-liposomes after they are prepared e.g. at room temperature (incorporation of DNA to the HM-liposomes was performed by this route as explained above). It is known that formation of liposomes requires heating the liposomal components at temperatures not lower than the Tc of the lipids. This is because below Tc lipids are in the gel state and can not usually form closed continuous bilayered structures. When cholesterol (or any other sterol) is used as a liposomal component, HM-liposomes are prepared successfully at 120C. Since the majority of the phospholipid molecules employed as liposomal constituents have transition temperatures below 60C, in the absence of cholesterol (or other sterols which require high temperatures to be dissolved) HM-liposomes can be prepared for example at 60-70C. The antineoplastic drug 5-FU was incorporated to cholesterol-containing HM-liposomes efficiently at two temperatures of 60C and 120C [Mozafari et al, unpublished data]. This indicates that, even in the presence of sterols, incorporation of drugs sensitive to high temperatures to the HM-liposomes can be achieved with high efficiencies by adding the drug to the reaction medium when the temperature has decreased to 60C or 70C for example. It seems that once sterols are dissolved at high temperatures, at a lower temperature (above the Tc of the lipid ingredients) liposomes are either still not formed or are highly dynamic and able to incorporate drug molecules efficiently. It appears that, HM-liposomes possess the afore-mentioned required characteristics to be employed both as model biomembranes and carrier systems for bioactive agents successfully. Acknowledgements. The financial support of the Riddet Centre, Massey University, towards this publication is gratefully acknowledged. REFERENCES

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diffusion. in: Liposome Technology. Vol. 1, Liposome preparation and Related Techniques, (Gregoriadis, G. Ed.), CRC press, Boca Raton, Florida, 1993, 67-79.

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30. Weder, H.G. and Zumbuehl, O. The preparation of variably sized homogenous liposomes for laboratory, clinical, and industrial use by controlled detergent dialysis. in: Liposome Technology. Vol. 1, Preparation of liposomes, (Gregoriadis, G. Ed.), CRC press, Boca Raton, Florida, 1984, 79-107.

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