CELLULAR & MOLECULAR BIOLOGY LETTERS

Volume 10, (2005) pp 711 – 719

http://www.cmbl.org.pl

Received 15 July 2005

Accepted 6 October 2005

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.

* e-mail: R.Mozafari@massey.ac.nz
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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
CELLULAR & MOLECULAR BIOLOGY LETTERS 713

[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]).
714 CELL. MOL. BIOL. LETT. Vol. 10. No. 4. 2005

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
CELLULAR & MOLECULAR BIOLOGY LETTERS 715

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
716 CELL. MOL. BIOL. LETT. Vol. 10. No. 4. 2005

ordinary heat sterilisation (121°C, 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.
120°C [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
CELLULAR & MOLECULAR BIOLOGY LETTERS 717

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 120°C. Since
the majority of the phospholipid molecules employed as liposomal constituents
have transition temperatures below 60°C, in the absence of cholesterol (or other
sterols which require high temperatures to be dissolved) HM-liposomes can be
prepared for example at 60-70°C. The antineoplastic drug 5-FU was
incorporated to cholesterol-containing HM-liposomes efficiently at two
temperatures of 60°C and 120°C [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 60°C or 70°C 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.

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