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ARTICLE IN PRESS

Biomaterials 24 (2003) 42834300

Review

Physico-chemical stability of colloidal lipid particles


Be! atrice Heurtault, Patrick Saulnier, Brigitte Pech, Jacques-Emile Proust,
Jean-Pierre Benoit*
Inserm ERIT-M 0104, Ing!enierie de la Vectorisation Particulaire, Immeuble IBT, 10, rue A. Boquel, 49100 Angers Cedex, France
Received 8 November 2002; accepted 8 April 2003

Abstract
Recent advances in nanoparticle systems for improved drug delivery display a great potential for the administration of active
molecules. Generally, the lipid systems presented the advantage of their low toxicity due to their composition of physiological lipids
compared to polymeric particles. The physico-chemical stability of the lipid carriers showed variations due to their numerous
compositions and structures. This review consequently focuses on the physico-chemical stability of dispersions in the nanometer
range where the lipids are the main or the only components. It highlights on the destabilization mechanisms, the techniques used to
detect this destabilization and the inductors of the destabilization. Finally, the methods used to optimize the stability of lipid
nanoparticle systems are described in the last part.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Review; Physico-chemical stability; Lipid; Drug delivery systems; Nanoparticles

Contents
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4284

2.

Physico-chemical mechanisms implied in


2.1. Chemical stability . . . . . . . .
2.1.1. Phospholipid stability . .
2.1.2. Triglyceride stability . . .
2.2. Physical stability . . . . . . . . .
2.2.1. Lipid modications . . .
2.2.2. Dispersion modications .
2.3. Interfacial stability . . . . . . . .

3.

Stability measurements . . . . . .
3.1. Physical stability . . . . . .
3.1.1. Crystallization . . .
3.1.2. Modication of size
3.1.3. Modication of zeta
3.2. Interfacial stability . . . . .

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Abbreviations: BSA, Bovine serum albumin; CETP, Plasma cholesteryl ester transfer protein; DMPG, Dimyristoylphosphatidylglycerol; DOPC,
Dioleoylphosphatidylcholine; DOPE, Dioleoylphosphatidylethanolamine; DPPC, Dipalmitoylphosphatidylcholine; DPPG, Dipalmitoylphosphatidylglycerol; DPPE, Dipalmitoylphosphatidylethanolamine; DSC, Differential scanning calorimetry; DSPC, Distearoylphosphatidylcholine; DSPG,
Distearoylphosphatidylglycerol; FFF, Field-ow fractionation; HDL, High-density lipoprotein; HPLC, High performance liquid chromatography;
LD, Laser diffraction; LDL, Low-density lipoprotein; NMR, Nuclear magnetic resonance; NTU, Nephelometric Turbidity Units; PCS, Photon
correlation spectroscopy; PEG, Polyethyleneglycol; PLs, Phospholipids; PLA 50, poly(d,l-lactic acid) containing 50% l-repeating units; SAXS, Xray scattering; SLN, Solid lipid nanoparticle; TL, Trilaurin; USP, United States Pharmacopeia
*Corresponding author. Tel.: +33-241-735-855; fax: +33-241-7358-53.
E-mail address: jean-pierre.benoit@univ-angers.fr (J.-P. Benoit).
0142-9612/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0142-9612(03)00331-4

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B. Heurtault et al. / Biomaterials 24 (2003) 42834300

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4.

Destabilization inductors . . . . . . . . . . .
4.1. Process parameters . . . . . . . . . . .
4.1.1. Formulation method . . . . . .
4.1.2. Sterilization . . . . . . . . . .
4.2. Composition . . . . . . . . . . . . . .
4.2.1. Particle composition . . . . . .
4.2.2. Continuous phase composition:
4.2.3. Encapsulation of drugs . . . .
4.3. Storage parameters . . . . . . . . . . .
4.3.1. Inuence of light . . . . . . . .
4.3.2. Inuence of temperature . . . .
4.3.3. Inuence of packing material .

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electrolyte concentration and pH
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5.

Stability optimization . . . . . . . . . . . . . . .
5.1. Chemical stability . . . . . . . . . . . . .
5.1.1. Addition of anti-oxidants . . . . .
5.1.2. Water elimination . . . . . . . . .
5.2. Physical stability . . . . . . . . . . . . . .
5.2.1. Steric stabilization . . . . . . . . .
5.2.2. Electrostatic stabilization/repulsion
5.2.3. Lipid composition . . . . . . . . .
5.2.4. Cream or hydrogel incorporation .
5.2.5. Optimization of storage parameters

6.

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4296

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4297

1. Introduction
Recent advances in nanoparticle systems for improved drug delivery display a great potential for the
administration of active molecules [1]. These drug
carriers allow the properties of the drug being carried
to be hidden and then to control and target its
release. It allows drug protection against chemical and
biological degradation related to the administration route. Therefore, the physico-chemical characteristics of the carriers themselves govern the types of
application.
Inside the numerous nanoparticle systems, lipid
structures were largely developed for various administration routes [2]. Liposomes, micelles, nanoemulsions,
microemulsions, and solid lipid nanoparticles (SLNs)
were the main lipid colloidal structures studied due to
their low toxicity, their ability to carry hydrophilic or
lipophilic drugs, their ability to control and localize the
release of the active drug, and their small size. All of
them were potential drug carriers for oral, topical, and
parenteral administrations.
Generally, the lipid systems presented the advantage
of their low toxicity due to their composition of
physiological lipids compared to polymeric particles.
However, a chemical transformation of these lipids
might change the structure of the system, the load and/
or release capacity, the interfacial properties, and their
in vivo fate. Physical modications can also occur. The

consequences on size control and nanoparticle growth


are important considerations in preparing dispersions,
and particular attention has to be focused on their
evolution. For drug release, the homogeneity of the
administration depends on the homogeneity of the
initial product. If precipitation occurs during storage,
the quantity of drug delivered for each administration is
unknown. Furthermore, the drug itself may sometimes
destabilize the initial system.
The physico-chemical stability of the lipid carriers
showed variations due to their numerous compositions
and structures. Micelles are amphiphilic aggregates in an
aqueous solution (inverted micelles in apolar solvents)
leading to micellar solutions [3]. In terms of stability,
micelles are uniformly distributed, dynamic entities,
rapidly exchanging molecules with the continuous
surrounding region [3]. Microemulsion is dened as a
system of water and oil, stabilized by amphiphilic
molecules and which is optically isotropic and thermodynamically stable [4,5]. In this case, the thermodynamic
stability is due to the low interfacial tension between the
two immiscible liquids [6]. Co-surfactant is usually used
to decrease this interfacial tension. Neither case is
developed in this work due to their original properties
which have been widely studied in the literature [3,57].
Lipoproteins are particles in which a hydrophobic core
containing triglycerides and cholesteryl esters is surrounded by a shell composed of phospholipids (PLs),
unesteried cholesterol, and one or several specic

ARTICLE IN PRESS
B. Heurtault et al. / Biomaterials 24 (2003) 42834300

proteins, named apolipoproteins. In the current work,


their stability results will be compared to other
structures.
This review consequently focuses on the physicochemical stability of dispersions in the nanometer range
where the lipids are the main or the only components.
The term lipid is taken in the general sense to include
wax, glycerides and PL. They can constitute the external
membrane, such as the PL surrounding an aqueous core
of liposome structure. Lipids, mainly triglycerides, can
also constitute the core. In nanoemulsions, they are in
the liquid state; in the SLNs they form a solid matrix
core. PL can be used as stabilizing agents in both cases.
If PLs are present under the form of a rigid membrane
and surround a triglyceride core, lipid nanocapsules are
formed [8].
In order to optimize particle stability during the
formulation process, storage and when in the biological
medium, the mechanisms of destabilization should be
known. They are sorted out in the rst part of this
review in chemical, physical, and interfacial mechanisms. Particle interfacial behavior has been particularly
well developed due to the numerous steps related to
interfacial phenomena during the destabilization processes, the formulation (emulsication process), and the
applications of these carriers. The techniques used to
detect this destabilization are described in Section 2. In
Section 3, the inductors of the destabilization mechanisms are presented as a function of the composition, of
the storage, and of the process parameters. Finally, the
last section highlights the methods used to optimize the
stability of nanoparticle systems.
DESTABILIZATION INDUCTORS
pH
Light

Ionizing radiation
Sonication
Surfactants
Electrolytes
pH
Drug
Temperature
Light
Packing material
Lipid matrix
Surfactants
Drug
Temperature
Light
Formulation process
Temperature

EFFECTS

Hydrolysis
Oxidation

Zeta potential

4285

2. Physico-chemical mechanisms implied in the


destabilization of lipid colloidal structures
The various mechanisms described below and implied
in the destabilization processes are summarized in Fig. 1
with their direct consequences on particle stability.
2.1. Chemical stability
2.1.1. Phospholipid stability
Liposome membranes are mainly constituted of PL,
which are sensitive to hydrolysis from ester bonds [9]. As
a consequence of chemical hydrolysis of the liposomal
PL, the organization of the lipid assembly can change
from a lamellar to a micellar system [10]. When such a
transformation occurred, lysophosphatidylcholine and
fatty acids were formed [11] and membrane permeability
increased [12].
The peroxidation of unsaturated acyl chains, if
present, was the other way of PL degradation [12].
Lipid peroxidation caused an increase in the permeability of the bilayer. The degradation process produced
a number of products with highly different chemical
natures [9,12].
These degradation processes create very serious
limitations to PL use. They have been exchanged with
non-ionic surfactants in niosome formulations to avoid
these degradation problems [13].
2.1.2. Triglyceride stability
Triglyceride hydrolysis can also occur. It leads to
mono- or di-glycerides with free fatty acids. However,
CONSEQUENCES
Membrane permeability
Chemical products
Drug release

Creaming
Gelling
Size increase
Drug release

Crystallisation
Polymorphism

Drug release
Crystalline drug structure

Surfactant film rigidity


Organization

Size increase
Membrane permeability
Drug release
Particle aggregation

Phase inversion

Structure modification

Interfacial stability
Drug location
Mobility

Destruction
Drug release

PHYSICO-CHEMICAL
INSTABILITY

Surfactant
Temperature
Packing material

Drug

Fig. 1. Destabilization mechanisms and consequences on lipid particles. Bold, normal or underlined scripts were used for the composition, the
storage and the formulation process inductors, respectively.

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B. Heurtault et al. / Biomaterials 24 (2003) 42834300

due to their internal location in the studied particles


(SLN), they were less susceptible to hydrolysis than
external PL.
2.2. Physical stability
2.2.1. Lipid modifications
2.2.1.1. Crystallization-polymorphism of glycerides. Crystalline solids, unlike liquids and gases, have an orderly
arrangement of units. SLN suspensions consequently
presented complex additional stability aspects compared
to other lipid systems due to their crystallization kinetics
and the polymorphism of the dispersed lipid. Crystalline
solids show denite melting points, passing from the solid
to the liquid state [14]. This should be taken into
consideration in SLN formulation [15].
These physical properties were used in the highpressure hot homogenization and warm microemulsion
methods to formulate SLNs from melt lipid-dispersed
systems. SLNs were consequently made from lipids
which are solid at room temperature [16]. Glycerides
frequently composed the matrix of SLNs. With long
chain triglycerides (tristearin, tripalmitin), fusion of the
b form (dened in the next paragraph) occurred at
higher temperatures (68 C and 60 C, respectively) than
for low chain triglycerides (trimyristin, trilaurin, 53 C
and 43 C, respectively).
However, the size characteristics of the structure can
alter the solidication phenomenon [17,18]. As a result
of their small particle size, lipid nanocrystals melt at
about 35 C lower than the corresponding bulk
material. It is noted that the fusion temperature of bulk
lipids is 73 C, 64 C, 56 C, and 47 C for tristearin,
tripalmitin, trimyristin, and trilaurin, respectively. The
recrystallization of the melted bulk lipid occurs at a
lower temperature leading to the a form that will be
dened in the next paragraph (51 C, 42 C, 28 C, 11 C,
respectively). In the same way, recrystallization of the
molten lipids was delayed by about 20 C in the colloidal
dispersions compared to the bulk lipids (30 C, 21 C,
9 C, 8 C, respectively) [18]. Consequently, nanoparticles prepared from triglycerides which are solid at
room temperature did not necessarily crystallize on
cooling to common storage temperatures. The particles
can remain liquid for several months without crystallization [19]. Westesen and Bunjes [18] observed that
colloidally dispersed trimyristin and trilaurin particles
remain in the liquid state for at least several months of
storage at room temperature. Cooling the dispersion
below a critical crystallization temperature did not result
in particle crystallization. The particles can thus remain
in a supercooled liquid state for a long period of time. In
this case, the emulsions of supercooled melts were
formulated instead of SLNs [18]. The supercooled state
of the droplets was not thermodynamically stable.
Gradual crystallization upon long-term storage cannot

be excluded and the product properties will change. The


crystallization process might lead to stability problems
such as gelling or the expulsion of the incorporated drug
[20]. These aspects will be developed later on.
Furthermore, crystalline re-orientation can result in
changes of the charges on the particle surface and
subsequently the measured zeta potential. In addition,
the crystal can possess different charge densities. It led
to an increase in zeta potential from approximately 25
to 15 mV for glyceryl tribehenate SLN [21]. At this
point the system started to gel as can be seen by the
increase in diameter from 0.7 to 52 mm.
Polymorphism is dened as the ability to reveal
different unit cell structures in crystals, originating
from a variety of molecular conformations and molecular packing [22]. Polymorphism is one of the
important physical degradation routes which affects
the stability of solid dosage forms because, even
though they are chemically identical, polymorphs
generally have different thermodynamic properties such
as melting points, X-ray diffraction patterns, and
solubility [14].
The main polymorph forms in glycerides are the a; b0
and b forms described by Hagemann [22].
a: Hexagonal (H) subcell with a lattice spacing of
0.42 nm.
b0 : Orthorhombic perpendicular (O) subcell with wide
lattice spacings of 0.420.43 and 0.370.40 nm.
b: Triclinic parallel (T) subcell with a wide lattice
spacing of 0.46 nm.
The a form has the tendency to be transformed
quickly to a form with better chain packing such as the
b0 form. The most stable form is the b form. Therefore,
the transition of liquid (melt) from a to b via b0 was the
pathway for triglycerides to the optimum packing form
of the molecules [22]. This unstable form gradually
transformed toward the most stable form during storage
at elevated temperatures while losing the initial spherical
surface structures [23]. This led to crystalline aggregate
growth [23].
Some consequences of lipid polymorphism on drug
encapsulation were described. Polymorphism inuenced
the nanoparticle drug content. The presence of oil
allowed higher drug loads [24,25]. Furthermore, the
highly disordered state in the glyceryl behenate nanoparticles due to liquid medium chain triglycerides
delayed crystallization that led to long-term physical
stability and inuenced the release mechanism that
depended on the transformation rate. The release
mechanism of active substances from the cetylpalmitate matrix was inuenced by the crystal structure [26].
A correlation between polymorph transitions and
increased drug release was observed with SLNs.
Sustained release was often related to the metastable
b0 polymorph. Drug expulsion during polymorph

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B. Heurtault et al. / Biomaterials 24 (2003) 42834300

transition was explained by a reduction of amorphous


regions in the carrier lattice due to a b0 to bi polymorph
transition [27]. bi is an intermediate triclinic and
orthorhombic polymorph. By increasing the formation
of the more stable modications, the lattice became
more perfect and the number of imperfections decreased
[16]. A medium chain triglyceride containing oil was
incorporated in a matrix of a solid long chain glyceride
(glyceryl behenate) to obtain SLNs based on binary
mixtures [24,28].
2.2.1.2. Gelling phenomena. Gelling phenomena describe the transformation of a low-viscosity system into
a viscous gel. Particle growth generally precedes the
gelling step [21]. A recent review on SLN has largely
described the origin and the consequences of SLN
dispersion gelling [15]. Various studies [21] indicated
that the gelling phenomena presented some inductors
similar to crystallization phenomena (high temperatures, light) and the degree of gelling was correlated to
the degree of crystallization of the lipid phase. Westesen
and Siekmann [29] related both processes. They
suggested that the crystallization of tripalmitate in
SLN suspension was associated with an increase in the
specic interfacial area because platelet-like colloidal
crystals with structured surfaces were formed. Due to
the limited number of PL molecules in excess, they are
not able to immediately cover the newly created
interface. Crystal interfaces with low concentrations of
adsorbed emulsier molecules represented preferred
sites of particle aggregation over which gel formation
can proceed [29]. In most cases, gel formation is an
irreversible process, which involves the loss of the
colloidal particle structure.
2.2.2. Dispersion modifications
Nanoemulsion, liposome or SLN organization frequently changed over time implying various processes. It
is related to the difference between the chemical
potentials of each species in all the dispersion phases
(thermodynamic instability). The next presentation is
organized following the degree of gravity or irreversibility of the process.
2.2.2.1. Inversion phase. The inversion phase of an
emulsion corresponds to the inversion of the continuous
and dispersed phases. It appears as a destabilization
process. However, the phenomenon of phase inversion
can be used to produce nanoemulsions [30,31]. In this
case, the initial emulsion is heated up and cooled down
above the temperature range of the phase inversion in
order to produce ne dispersal and therefore long-term
stable oil-in-water emulsions.
2.2.2.2. Ostwalt ripening. This phenomenon depends on
both the granulometry and on the Laplace surpressure

4287

being higher for lower size droplets. A species ux


occurs from small to large droplets via the continuous
phase. The average diameter consequently increases.
For SLN, it corresponded to a particle size increase due
to the dissolution of smaller crystals and deposition of
the dissolved material on larger surfaces, resulting in the
growth of large particles at the expense of smaller ones.
2.2.2.3. Flocculation. Attraction forces between droplets cause them to cluster. Flocculation results in the
formation of a larger structure by bridging. These
processes are commonly used to remove suspended
matter from water because the occulate nally sediments. It is a reversible phenomenon.
The formation of small liposomes required the input
of considerable energy and created a thermodynamically
unfavorable packing status. Aggregation (and/or fusion)
of these liposomes was a mechanism to dissipate the
excess surface energy originating from the distorted
molecular packing [32]. The aggregation of liposomes
led to larger units. These units were still composed of
individual liposomes. In principle, this process is
reversible by applying mild shear forces, by changing
the temperature, or by binding metal ions that initially
induced aggregation [12]. In general, the origin of vesicle
aggregation is van der Waals attraction [33].
2.2.2.4. Creaming and sedimentation. The reversible
creaming process describes how emulsion droplets tend
to rise to the top of a vial or to sink to the bottom as
sedimentation [34]. Competition between Brownian
agitation and gravity can lead to non-homogeneous
dispersion. It is generally due to the density difference
between the two phases. Robins recently reviewed the
mechanisms of creaming and phase separation due to its
commercial importance in food emulsion [34].
2.2.2.5. Coalescence. Coalescence is an irreversible rupture of the emulsion leading to phase separation. On
collision between two drops, the interfaces distort, up to
a certain critical thickness before it nally ruptures. It
allows droplets to merge. In most interesting operations,
coalescence must be delayed. Rigid solid particles are
expected to be stable against coalescence. In contrast,
SLN dispersions tend to cream or gel after particle
contact.
2.2.2.6. Fusion. Liposome fusion (see [33] for a review)
corresponds to membrane reorganization with the
relocation of individual lipid molecules between adjacent lipid layers in liposome aggregates [33]. Liposome
fusion corresponds to the formation of new colloidal
structures. Fusion is an irreversible process and the
original liposome structure is denitively lost [12]. Many
physical or chemical parameters can induce membrane
fusion. Intracellular trafc inside macrophages is one of

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the biological processes based on membrane fusion. In


some cases, in order to increase the entrance of vesicles
inside cells, the fusion process between biological
membranes and vesicles is favored in order to carry
drugs inside the cytoplasma [35]. In this case, controlled
vesicle fusion can be considered as an interesting
parameter, unlike the other destabilization processes
previously described.
2.3. Interfacial stability
The rst results concerning the spreading of lipid
structures at the air/water interface (Table 1) were
obtained by Pattus et al. [36] with liposomes, showing a
slow transformation of closed bilayer structures into a
surface lm. Vesicle disintegration was observed by
recording the increase in surface pressure against time
after their spreading. At zero surface pressure (without
compression), there was a slow transformation of
the closed bilayered structure into a lipid monolayer.
The internal content of the liposomes was released into
the aqueous subphase. In contrast, when multilamellar
liposomes were spread and compressed, they retained
their internal content at the air/water interface by
forming multilayered structures [36]. A theoretical
approach was described by Schindler [37] and explained
by a simple kinetic mechanism in two steps. Thus, after
the spreading of a liposomal suspension at the air/water
interface, the kinetics of the surface lm formation can
be described in two simultaneous processes: the irreversible diffusion of liposomes into or from the liquid phase
(Fig. 2c) and the irreversible transformation of closed
vesicles into destroyed ones which form the surface lm
[38,39]. A diffusion-controlled process applies for small
initial amounts of liposomes and a transformationcontrolled process predominates for large initial
amounts of liposomes [39]. Fig. 2 corresponds to the
schematic presentation of these phenomena. In the
simplest case, the unilamellar liposomes disintegrate to
form an insoluble monomolecular layer at the interface
[37,40] (Fig. 2a). However, in many cases, after an initial
disintegration of liposomes, surface mesophases (aggregates) were formed in addition to a monomolecular
layer (Fig. 2b) [4144]. The mechanisms and kinetics of
transformation of the intact liposomes at the airwater
interface were mainly related to short-range hydrophobic molecular interactions [44].
However, vesicle destabilization at the air/water
interface was used as a tool to study membrane
biological behavior. The formation of a proteolipidic
interfacial lm with pre-established proteinlipid
interactions improved the stability of the proteoglycolipidic assemblies [45]. Consequently, glycolipidic vesicles associated with protein were previously formulated
and spread at the air/water interface [40,4547]. It was
an alternative to the classical way which consists of the

Fig. 2. (a) Intact liposomes disintegrate to form a monomolecular


surface lipid layer. (b) Intact liposomes partially disintegrate to form
an insoluble monolayer and insoluble surface aggregates (process 12).
If the surface is perturbed by expansion the metastable aggregates can
act as sources of lipid molecules (process 23). (c) Diffusion of
liposomes into or from the bulk phase predominates for small initial
amounts [44], with permission from Elsevier.

isolation of the cell membrane which required considerable care to maintain structure and biochemical
activity [48].
IntralipidTM is composed of emulsion droplets with a
mean diameter of 360 nm containing a triacylglycerol
core emulsied by a PL monolayer, and of PL
unilamellar liposomes with a diameter of 85 nm. The
interfacial destabilization of both emulsion and liposomal particles leads to a rapid spreading of triacylglycerol
molecules from the core of the emulsion and to the
formation of a true monomolecular lm. The observed
higher global rate constant of spreading of IntralipidTM
in comparison with liposomes was attributed to the
higher spreading capacity of the core of the emulsion
particles [49] (Table 1).
HDL lipoproteins consist of a core of neutral lipids
encapsulated by a monolayer of polar lipids and
apolipoproteins. Surface balance and monolayer techniques have also been used to investigate the surface
properties of human lipoproteins at air/liquid interfaces
[56,57]. A rst study compared LDL and HDL3 surface
pressure/area isotherms (HDL3 has a density between
1.125 and 1.21 g/ml). Considering the molecular area
obtained for lipids, it indicated that LDL phospholipids
form more condensed monolayers than HDL3 phospholipids. The closer packing in the LDL phospholipids
monolayer has been attributed to the greater content of
saturated phosphatidylcholines and sphingomyelin with
respect to HDL3 content. Furthermore, the inuence of
lipid molecular packing on the afnity of human
apolipoprotein A1 for HDL3 and LDL surface lipids
was evaluated by monitoring the adsorption of apo A1
to monolayers of these lipids spread at various initial
surface pressures.
The spreading of HDL3 at the air/water interface led
to a breaking of these particles and to the liberation of
their core content. A schematic model describing the

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4289

Table 1
Lipid structures studied at the interfaces
Ref.

Lipid structures

Experimental eld

Interface

[37]
[50]
[36]
[51]
[39]

Spreading theory
Spreading process
Spreading process
Spreading kinetics
Spreading kinetics
Adsorption/spreading comparison
Spreading kinetics
Spreading kinetics

Air/water
Air/liquid
Air/water
Air/water
Air/water

[44]
[52]
[42]
[53]
[46]
[45]
[47]
[54]

Liposomes DOPC
Liposomes DMPC
Liposomes
Liposomes DOPC
Liposomes DPPC
DPPC:DSPC:SL
Liposomes (proteo-glycolipidic)
Liposomes
Emulsion
Liposomes DMPC
Liposomes DMPC
Liposomes
Liposomes
Liposomes (proteo-glycolipidic)
Liposomes (proteo-glycolipidic)
Liposomes PC
Liposomes DOPC

[55]
[56]
[57]

DMPC
DPPC
Liposomes
HDL3 and LDL
HDL3

[58]
[59]

HDL3
Oil-in-water emulsion

[40]
[49]

Spreading kinetics
Spreading kinetics
Surface pressure hysteresis lms
PL/drug interactions
Biological membrane modelization
Biological membrane modelization
Biological membrane modelization
Spreading process with enzymatic action (phospholipase A2)
on liposomes

Spreading process with enzymatic action


PL surface organization
Surface organization
Spreading process
CETP/ HDL3 interactions
PLA50/BSA interactions

Air/aqueous buffer
Air/water
Air/water
Air/water
Air/water
Air/aqueous solution of drug
Air/aqueous buffer
Air/aqueous buffer
Air/water
Air/water

Air/water
Air/water
Air/water
Air/water

Note: PLA 50, poly(d,l-lactic acid) containing 50% l-repeating units; BSA, bovine serum albumin; HDL, high-density lipoprotein; CETP, plasma
cholesteryl ester transfer protein.

Fig. 3. Schematic model describing the two processes involved in the formation of the surface lm from a spread HDL3 suspension: diffusion of
intact structures (1) or adsorption of partially open HDL3 (1a) and lm formation (1b). From [57], with permission from Elsevier.

two processes involved in the formation of the surface


lm from a spread HDL3 suspension is presented in
Fig. 3. It appears similar to the observed liposome
behavior. The rst phenomenon corresponded to the
diffusion of intact structures whereas the adsorption of
partially open HDL3 followed by lm formation
corresponded to the second one. This resulted in the

formation of a very stable, complex, mixed proteinlipid


lm at the air/water interface [57].
Finally, the spreading of lipid structures like liposomes, oil-in-water emulsion, and HDL at the air/water
interface can lead to their destruction (Figs. 1 and 2).
Their components, mainly PL molecules, formed an
interfacial layer. The stability of the initial structure was

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thus determined. The properties of this interfacial layer


provide information about the organization of the initial
structure. It allows extrapolations to determine the
structure in volume.

3. Stability measurements
At the end of the destabilization phenomena, most of
the previously described processes can be directly
observed (creaming, coalescence, gelling). The nal
product becomes a non-homogeneous system and can
change in viscosity properties. However, the beginning
of the destabilization process could be detected earlier
using appropriate tools as described herewith. Chemical
instability can be observed by classical methods. HPLC
or thin layer chromatography is the main technique used
to measure the production of new chemical entities due
to the peroxidation or hydrolysis of PL. However,
physical instability can be detected directly by the
mechanism implied (crystallization) or by the consequences on the dispersion. The method to detect
interfacial instability is developed at the end of this
chapter.
3.1. Physical stability
3.1.1. Crystallization
3.1.1.1. Differential scanning calorimetry. Thermoanalysis of the SLNs provides information about crystallization behavior, the timing of polymorphic transitions,
fusion temperature, enthalpy, and the degree of crystallinity of melt-homogenized glyceride nanoparticle dispersions [60,61].
3.1.1.2. X-rays. Crystals diffract X-rays in the same
way that visible light is dispersed into a color spectrum
by a ruled grating (i.e., a piece of glass with ne parallel
lines of equal width drawn on it). This is due to the fact
that X-rays have wavelengths of about the same
magnitude as the distance between the atoms or
molecules of crystals. The X-ray diffraction pattern is
photographed on a sensitive plate arranged behind the
crystal, and by such a method the structure of a crystal
may be investigated. It has become possible to determine
the distances of the various planes of the crystal lattice.
The structure of various compounds can be determined
in this way [14]. X-ray scattering (SAXS) often
completes the study. These techniques were used to
investigate crystallization tendency and polymorphic
transitions of triglyceride nanoparticles [25,26,62].
Crystallization directly concerned the lipids constituting the structure. The two next paragraphs describe
the consequences of the destabilization process on the
complete structures with modications according to
their size or zeta potential.

3.1.2. Modification of size


The study of size was generally used as a characterization tool. When the composition, the technique, or
the process parameters (time, temperature, pressure,
equipment type, sterilization and lyophilization conditions) were modied in an attempt to optimize the
conditions, particle sizing was chosen as a quality
response parameter [6368].
Numerous factors had an inuence on the average
particle diameter of SLNs. The excipient composition
[66,69], the drug composition [62,66,6974], the formulation process, the process factors (temperature,
homogenizing equipment) [19,63,65,67,7577], the sterilization [66,78], the dispersing medium [77,78], the
freeze-drying [64,68], the presence of stealth agent [79],
the storage conditions [80,81] and the lipid composition
[16,19,24,77,82,83] are some of the common parameters
implicated in SLN size characteristics. These various
factors, as previously described, were related to crystallization processes such as coalescence that induced size
growth. In most cases, the particle sizes increased before
macroscopic changes appeared. It was consequently a
good indicator of instability.
3.1.2.1. Importance of size in pharmaceutical applications. The denition of colloidal particles is based on
the size characteristic of the structure, i.e. a structure
with a size below 1 mm [84] as liposomes, nanospheres,
nanocapsules. Well-formulated systems should display a
narrow particle size distribution in the submicron range.
Furthermore, particles greater than 1 mm and the
increase of their number in time can be an indicator of
physical instability [85]. Intravenous injection of particles with an average diameter above 5 mm might cause
death due to embolism. Thus, size control and the
avoidance of nanoparticle growth are important considerations in preparing dispersions and particular
attention is paid to size. Furthermore, particle size can
modulate the capture mechanism by macrophages and
inuence their biological stability. It appeared that the
quantitative contribution of pinocytosis declined, and
that of phagocytosis increased, with increasing particle
size [86]. These results may inuence the biodistribution
behavior of the particles.
3.1.2.2. Hydrodynamic size measurement methods. In
the size range studied here, a random movement of the
particle occurs, on which the size determination
techniques are often based. Photon Correlation Spectroscopy (also known as dynamic light scattering) is the
most powerful technique for routine measurements of
nanoparticle system size [87] and most of the studies of
lipid systems use PCS for size determination [15]. PCS
measures the uctuation of the intensity of scattered
light caused by particle movement. However, PCS is
not able to detect particles under 3 mm. The calculations

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of size with PCS are made by considering particles as


spherical systems. This characteristic should be veried
by other techniques like microscopy before measurement. To visualize particles larger than 3 mm, laser
diffraction (LD) could be used simultaneously. This
method is based on the dependence of the diffraction
angle on the particle radius. Particle radius has been
also determined for SLNs from a relationship between
the particle radius and the rotational correlation time
obtained by NMR investigations [88]. This technique is
complex compared to PCS and is probably more
appropriate to structure determination of any type of
component. Rapid progress in the development of
eld-ow fractionation (FFF) has been observed
during recent years [89]. This technique has demonstrated the ability to separate particles based on their
size and has been applied to the investigation of
submicron lipid emulsions with great success [85]. FFF
corresponds to a separation technology for which
particles are separated based upon their relative position
in a laminar ow associated to a perpendicular eld.
However, until now, only a few determinations of lipid
particles have been reported using this promising
technique [90].
3.1.2.3. Turbidity measurements. Turbidity quanties
the degree to which light travelling through a sample is
scattered by the suspended particles. Turbidity depends
essentially on the entities of colloidal size present in the
liquid phase, on the difference in the refractive index
between the particles and the medium, and on the particle
size distribution [91]. Microbiological instability quickly
increases the cloudiness of the liquid as well. Turbidity is
commonly measured in Nephelometric Turbidity Units
(NTU). It is a quantitative way of measuring turbidity;
but it does not directly give the size of the particles or the
quantity of particles.
3.1.3. Modification of zeta potential
3.1.3.1. Definition. Almost all particles in contact with
a liquid acquire an electric charge on their surface. The
electric potential at the shear plane is called the zeta
potential. The shear plane is an imaginary surface
separating the thin layer of liquid (liquid layer constituted of counter-ions) bound to the solid surface in
motion. Zeta potential is an important and useful
indicator of particle surface charge, which can be used
to predict and control the stability of colloidal suspensions or emulsions. The measurement of zeta potential is
often the key to understanding dispersion and aggregation processes in applications. Laser Doppler anemometry calculated from the mean electrophoretic mobility
of particles quanties the particle charge.
3.1.3.2. Stability consequences. The greater the zeta
potential the more likely the suspension is to be stable

4291

because the charged particles repel one another and thus


overcome the natural tendency to aggregate. The
measurement of the zeta potential allows predictions
to be made about the storage stability of a colloidal
dispersion. It is currently admitted that zeta potentials
under |30| mV, optimum >|60| mV, are required for full
electrostatic stabilization; potentials between |5| and
|15| mV are in the region of limited occulation; and
between |5| and |3| mV of maximum occulation [92].
Thus, particle aggregation is less likely to occur for
charged particles (high zeta potential) due to electric
repulsion. However, this rule cannot strictly be applied
for systems which contain steric stabilizers, because the
adsorption of steric stabilizers will decrease the zeta
potential due to the shift in the shear plane of the
particle. Moreover, one has to take care about strong
dependence of the zeta potential with ions present in the
medium.
This phenomenon has been observed by Schwarz et al.
[93] with lipid structures. They measured a zeta potential
of SLNs constituted of trilaurin of approximately
30 mV. It was not sufciently high to stabilize the
dispersion solely by electrostatic repulsion but Poloxamers 188 was used as a steric stabilizer which can
easily compensate for the missing electrostatic repulsion
[93]. The zeta potentials observed with soybean lecithin
(Lipoids S75) as a surfactant (40 to 50 mV) were
sufciently high for electrostatic stabilization probably
because of the charge of phosphatidic acid present in the
Lipoids product (phosphatidic acid) [94]. In the same
way, during SLN lyophilization, for the neutral trilaurin-DPPC SLN, trehalose was effective at a higher sugar/
lipid weight ratio (3.9) compared to a lower value (2.6)
when negatively charged trilaurin-DPPC-DMPG SLN
was lyophilized. The need to use steric stabilization with
trehalose as a cryoprotectant appeared clearly in this
study [95].
3.2. Interfacial stability
In the biological environment of the blood stream,
particles interact with various interfaces. An appropriate
model for studying the behavior of these particles at
interfaces was their spreading at the air/liquid interface.
Various articles on this subject have been published with
various aims (Table 1). The studied interfacial parameters corresponded mainly to surface pressure (g) and
surface potential. The rst one was generally determined
using a Wilhelmy plate method with a thin platinum
plate and an electromicrobalance. The surface potential
was measured using an electrometer. A thermostated
Langmuir lm balance generally generated surface
pressure/surface area isotherms (Fig. 4). It consists of
a trough, usually made up of hydrophobic materials like
Teons to contain the subphase water and movable
barrier(s) that span over the water surface (Fig. 4). A

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4292
Computer
Electrobalance
Fixed
Barrier
Force transducter

Wilhelmy
plate

Movable barrier

Substrate
Subphase

Trough

safe and convenient sterilization technique, more studies


will be necessary [98].
In various studies an autoclaving approach was
preferred because it did not change the zeta potential
of particles [93,95]. No change in the amount of
incorporated drug or in size characteristics of SLN
formulated via hot microemulsion has been observed
[73]. The sterile SLN dispersions were stable for more
than 18 months stored at 4 C. These results were
paradoxical considering the previously discussed possible effect of temperature.

Fig. 4. Schematic representation of a Langmuir trough that contains


the Wilhelmy plate for measuring surface pressure using an electrobalance and a surface potential probe.

4.2. Composition

known amount of amphiphilic material dissolved in a


water-immiscible, volatile organic solvent such as
chloroform is placed on the water surface.

4.2.1. Particle composition


4.2.1.1. Lipid matrix. Considering the evolution of the
crystallized structure from metastable to stable polymorphs and its consequences on drug release, the choice
of the initial crystal lattice is of primary importance.

4. Destabilization inductors
During the development of nanoparticulate structures, the formulation process is the rst step which
might play a role on stability. The composition has to be
critically controlled for optimal stability without forgetting that the drug might also be a possible destabilizing
inductor. Finally, external parameters such as temperature and light appear to be of primary importance for
long-term stability.
4.1. Process parameters
4.1.1. Formulation method
The formulation process is an important parameter
acting on the initial polymorphic form of the lipid
nanoparticles. In the case of spray-drying, unstable
polymorphic forms were obtained due to rapid solvent
evaporation. The same consequence was observed with
the spray-congealing process of micropellets [23,61].
In the same way sonication has been described as an
inductor of PL oxidation [12].
4.1.2. Sterilization
Gamma-irradiation is a current sterilization technique
for pharmaceutical products. However, chemical degradation of the PL took place during irradiation [96].
Peroxidation of unsaturated PL has been observed as
well as hydrolysis and degradation processes such as
dehydrogenation, chain rupture and dimerization [97]. It
might lead to more negative zeta potentials, changes in
phase transition behavior, or smaller liposomes as
described by Stensrud [96], on distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol
(DSPG) liposomes. Ionizing radiation has consequently
been excluded or at least, before it will be accepted as a

4.2.1.2. Surfactant. As previously described, the surfactant used in the formulations can be a lipid molecule like
PL; however, amphiphilic polymers or bile salts are also
used.
The paper of Ahlin et al. [99] showed the effect of PL
on zeta potential of glyceryl tripalmitate SLN dispersions at various concentrations of between 0.25% and
5%. At a pH value of around 7, the particles carried
negative charges due to the ionization of various minor
components in PL: phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. The zeta potential decreased as the emulsier concentration
increased up to 2% (w/w). These results showed that
an increase in the amount of negatively charged PL
brought about a decrease in the zeta potential, which
reached a plateau region when the surface of SLN was
completely covered with PL molecules. The increased
negative surface charges might be a consequence of the
increased surface curvature of smaller particles, resulting in higher package density of PL molecules on the
particle surface [99].
In the case of Lipo.ds S75, the major component
(phosphatidylcholine) could not explain the stabilization induced. In fact, the impurities in the surfactant
sample acted as stabilizers and could be considered
as co-surfactants. As a consequence, the less tendency
of the Lipo.ds S75 stabilized systems to gell compared
to those stabilized by pure Lipo.ds S100 or E80
might be explained by the participation of minor
components (such as glycolipids) in the steric or
electrostatic stabilization as described by Westesen and
Siekmann [29].
Schwarz and Mehnert [100] compared the effect
of soybean lecithin (Lipoids) and Poloxamers 188 on
zeta potential. The charge of the Lipoids stabilized Witepsols (glyceride mixture) and Dynasans

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(triglycerides) SLN were mainly in the range of 40 to


50 mV compared to the range of 18 to 27 mV for
Poloxamers 188 stabilized SLN [100]. In this second
case, the zeta potential value was not sufcient to
achieve electrostatic stabilization, however, the Poloxamers could simultaneously induce steric stabilization
(see stability optimization) [100].
In SLNs, the surfactant composition of the mixture
can favor one of the various possible polymorphic forms
of triglycerides. In Witepsols solid lipid particles, bile
salts delayed crystallization to a stable form (bi ), and
metastable forms such as a and b0 ; were obtained.
However, PL led to fast bi crystallization [19].

4293

4.2.1.3. Co-surfactant. The inuence of the co-surfactant composition of lipospheres on the polymorphism of
lipid matrices has been also demonstrated by Aquilano
et al. [101] on stearic acid particles. Lipospheres were
obtained from a warm microemulsion process. Stearic
acid was crystallized in three polymorphic forms named
A, B, and C depending on their melting points: 46 C,
54 C, and 74 C, respectively. It appeared that the
polymorph B is favored by the presence of small
amounts of butanol [101]. The solvent mainly acted as
a single molecule placed sideways to the crystallizing
chains, forming perpendicular bonds to the chain
development axis. During the formulation processes,
butanol acted as an impurity, promoting both the
lowering of the surface tension of polymorph B and its
nucleation frequency [101].
The presence of n-butanol in the formulation of lipid
particles led to smaller particles (of stearic acid) due to
the stabilizing effect on the microemulsion interface.
And probably as a consequence of its effect on steric
crystallization, growth over time was minimal [102].

preferred to the use of a buffer in order to minimize zeta


potential decreases due to the addition of an electrolyte.
For phosphatidylcholine liposomes, the hydrolysis of
the ester bonds depended on the pH. In general, a lower
hydrolysis rate was obtained for pH levels of around 6.5
[12,102]. In a similar way, nanoparticle growth and
charge density changed when the pH of the dispersing
medium was close to the pKa value of lipid nanospheres
composed of stearic acid [102].
Both parameters (pH and electrolytes) act on the
particle zeta potential and they are characteristic of a
physiological medium. Many consequences concerning
stability on particle applications and particularly in
biological media should arise from this effect. In this
way, Zimmermann et al. [81] optimized the electrolyte
and pH-stability of aqueous SLN dispersions in
articial gastrointestinal media. The stability depended
on the specic interactions of the lipid matrix with the
emulsier [81]. It appeared in this study that the
inuence of a low pH was, in general, stronger than
the inuence of the electrolyte concentration.
From the biological point of view, the primary route
of internalization of liposomes by cells is the endocytic
pathway where the liposomes are in contact with a very
low pH. If ionizable lipids are incorporated into bilayer
phases, the stability of the bilayer is conditional on the
pH. With acidication of the medium, a protonation of
various molecules incorporated in liposome membrane
occurs. It leads to a suppression of the charge repulsion
in the bilayer and promotes liposome destabilization.
The efciency of these pH-sensitive liposomes is directly
related to their fusogenic properties. The consequence is
the release of their content in the cell cytoplasm. Various
studies have been developed on these interesting pHsensitive liposomes [104,105].

4.2.2. Continuous phase composition: electrolyte


concentration and pH
It seems that the observed effects of electrolytes and
pH with SLN dispersions were similar to those reported
for emulsions. Therefore, explanation mechanisms could
be derived from emulsion theory. Thus, SLN dispersions
were mainly stabilized by electrostatic repulsion. The
addition of electrolytes generally reduced the zeta
potential, i.e. electrostatic repulsion. Zeta potential
determination proved to be suitable to assess the longterm stability of electrolyte-containing systems. Higher
concentrations and higher valence of the ion enhanced
zeta potential reduction and colloid destabilization
[103]. Depending on the nature of the lipid, electrolytes
could cause destabilization and subsequent gelling by
reducing the zeta potential [103]. Considering the
inuence of the concentration of electrolytes on the
zeta potential, the pH was adjusted by the addition of
HCl and NaOH to study the effect of pH on the zeta
potential of SLNs, instead of using a buffer. This was

4.2.3. Encapsulation of drugs


The incorporation of drugs like etomidate or tetracaine below 10% had little effect on the zeta potential of
SLN (mixture of mono-, di- and triglycerides, cetylpalmitate, glyceryl behenate, triglycerides) stabilized with
Poloxamers or soybean lecithin [100]. The same result
was observed by Heiati et al. [95] with azidothymidine
palmitate incorporated in trilaurin SLNs. In the same
way, the incorporation of diazepam in SLN did not
inuence the stability of SLN [66].
However, the presence of drugs in particles can induce
a major decrease of the crystallization temperature.
When high quantities of drug were encapsulated, the
particles corresponded to supercooled melts at room
temperature instead of solid particles; this is the case
with ubidecarenone incorporated at 3050% in tripalmitin particles [20]. When the dispersions were stored at
4 C with the same high percentage of drug, the structure
appeared biphasic. The particles consisted of a plateletlike basis similar to unloaded triglyceride nanoparticles

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with a cap adhering to one of the two large faces of each


platelet. The cap was formed by excess drug, which was
expelled from the particle matrix upon crystallization of
the triglyceride. If less drug was encapsulated, no cap
appeared [20].
Furthermore, the polymorphic transition can also be
accelerated by the presence of a drug in the carrier. This
phenomenon led to a higher stability compared to nonloaded particles. These results were attributed to high
interactions between lipids and drug [106]. In the same
way, the structures of incorporated drugs led to different
polymorphic forms of stearic acid depending also on the
composition of the initial microemulsion. Polymorph C
was generally favored for initial microemulsion containing nonionic (Tweens 20 and butanol) or ionic
(taurodeoxycholate, monooctylphosphate and Epidurons 200) molecules as surfactants and co-surfactants.
However, in the presence of oxygenate drug derivatives,
the crystallization polymorph B or C was obtained. This
phenomenon also seemed to act on the structure of the
active drug. The drug was in an amorphous state or in a
crystalline state depending on the crystalline structure of
the lipid matrix [69].
In drug delivery applications, liposomes are of great
interest to carry and target drugs into cells [107]. In
order to understand the interactions between drug
(antiacneic drug: RU 5884) and PL, the mixed drug/
PL monolayer obtained by spreading liposomes at the
air/aqueous surface of drug solutions was studied using
the lm balance technique. From the study of the
isotherms and the rheological behavior of mixed
monolayers at low surface pressures, it was deduced
that the drug could be localized between the PL polar
headgroups. In the opposite case, at high surface
pressures, the drug molecule was believed to be trapped
in the hydrophobic part of the phospholipidic layer and
behave as if it were insoluble. Finally, from a liposome
spreading study, the destruction of loaded liposomes
appeared to occur earlier and with greater effect than for
placebo liposomes [53].
4.3. Storage parameters
4.3.1. Influence of light
The peroxidation of PL increased with light exposure
[12].
The light exposure of the SLN caused changes in the
system leading to a reduction of the zeta potential (due
to a modication of the crystallization form; see
preceding paragraph). With sufcient reduction of
repulsive forces, particles can interact to form a gel
network [21].
Freitas and Muller
.
showed rapid gelling of the initial
SLN suspension (composed of glyceryl behenate) when
it was stored in white glass under articial light. Data
prove that light radiation has a destabilizing effect.

Furthermore, increase in the intensity of light radiation


accelerated particle growth and gelling. Moreover, high
energetic radiation (UV, short wavelengths) increased
destabilization [21,80]. The mechanism implied was
related to the modication of the zeta potential (see
preceding paragraph).
Indeed, lipid dispersions such as emulsions for
parenteral nutrition, closed to SLN, were not destabilized by light. The physical stability of lipid suspensions
for more than 3 years has been reported when stored at
room temperature and in daylight. They were consequently stored in white glass bottles [108].
4.3.2. Influence of temperature
Temperature, like light exposure, corresponds to
energy input and can lead to changes in the crystalline
structure of lipids. Due to the previously explained
mechanism, a destabilization process was generally
induced by high temperatures due to changes in zeta
potential. Freitas and co-workers observed rapid growth
of particles when stored at 50 C, associated with a
decrease in their zeta potential [21,80]. 4 C was
generally the most favorable storage temperature.
However, in some cases, long-term storage at 20 C did
not result in drug-loaded SLN aggregation or loss of
drug, compared to 4 C storage conditions [95].
Furthermore, in some cases, the authors showed faster
transformation of tripalmitate SLN from the b0 to the
more stable b polymorphic form for lower temperatures
(4 C) compared to room temperature [19].
Furthermore, a high level of lm rigidity of the
emulsier (also named microviscosity) prevented fusion
of the lm layers after particle collision. Microviscosity
was a temperature-dependent factor. When temperature
increased, microviscosity decreased, leading to destabilization of the particles [21].
Temperature can modify the packing arrangement
of PL molecules on the surface of the SLN. Heiati
et al. [83] produced SLN composed of PL coating
the surface and packed in multilayer structures containing dissolved trilaurin (TL) from the SLN core. Their
incubation at 4 C may decrease the solubility of TL
and result in the perturbation of the PL membrane [83].
This temporary non-homogeneity in the PL bilayers
may be responsible for particle aggregation and drug
release from the SLN [95]. In the same way, changes in
the uidity of the PL membrane and non-homogeneous phase structure occurred at 37 C which affected
PL continuity on the surface of SLN and might be
responsible for particle aggregation and an increase in
mean diameter [95]. Such a temperature corresponded to the physiological temperature and had to be
considered in the choice of the initial lipid composition [62].
In the same way, a linear relationship existed between
the phospholipid log of hydrolysis rate and the

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B. Heurtault et al. / Biomaterials 24 (2003) 42834300

inverse of the temperature for liposomes consisting of


phosphatidylcholine. A prediction of the stability as
a function of storage temperature can thus be established [12].
If non-ionic surfactant emulsions are exposed to high
temperatures, a phase inversion process can occur but
this is unusual during storage [109,110].
Inverted hexagonal phase-adopting lipids such as
dioleoyl phosphatidylethanolamine (DOPE) or protonated phosphatidylserine (PS) promote fusion of lipid
bilayers. The hexagonal form of DOPE exists at
temperatures above 10 C whereas it forms a non-bilayer
form at physiological temperatures. The incorporation
of DOPE consequently induces the fusion of liposomes
with biological membranes.
4.3.3. Influence of packing material
The effect of storing material is less pronounced on
the stability of SLNs than the temperature. But an
aggregation and subsequent gelling can be promoted
by the inner surface. However, as for emulsions, the
zeta potential was slightly higher and the size lower in
siliconized glass vials [21,111]. Furthermore, a possible effect of the packing material on the induction
of a local phase inversion of emulsion has also been
reported [109].

5. Stability optimization
5.1. Chemical stability
5.1.1. Addition of anti-oxidants
Oxidation can be prevented by excluding oxygen from
the vial, by the addition of an anti-oxidant such as atocopherol and butyl hydroxy toluene, or by the
selection of saturated acyl-chains in the PL. Peroxidation of PL in liposomes can be minimized by the use of
high-quality raw materials which are puried from
hydroperoxides and transition metal ions. Storage at
low temperatures reduced the rate of oxidation as well.
Furthermore, the use of partially saturated PL could be
a better choice than the PL carrying polyunsaturated
fatty acyl chains (natural egg phosphatidylcholine) [9].
Minimizing hydrolysis was possible by selecting an
environmental pH of 6.5, where PL have their maximum
stability, and low temperatures [112].
5.1.2. Water elimination
Another way to decrease the hydrolysis phenomenon
was the elimination of water in the sample by
lyophilization or spray-drying. Afterwards, the
powders should be reconstituted with the same particle
size distribution as the original dispersion. It gave
successful results on liposomes and SLN formulations [64].

4295

5.1.2.1. Lyophilization. Changes in particle size distribution during lyophilization were observed but were
minimized by optimizing the parameters of the lyophilization process, i.e. freezing velocity and redispersion
method (redispersion medium, for example). A longer
drying treatment on SLN suspensions gave no better
results [68]. In some cases, slower freezing proved to be
the best method [68], in contrast to other studies [64].
The thermal treatment should be adjusted for each
formulation [68]. Furthermore, the cryoprotector favored the redispersion of the freeze-dried SLNs [73].
Trehalose proved to be most effective cryoprotectant in
preventing particle growth during the freeze-drying
process [64,68]. Dextrose was suitable if high concentrations were used. Mannitol and lactose were not efcient
in protecting SLN dispersions [95]. Furthermore, the
presence of electrolytes in water reduced the zeta
potential with increasing concentration, i.e. with the
progression of the freezing process. The reduction in
zeta potential was considered to be one cause of
aggregation. Taking these factors into consideration, it
might be advantageous to remove the protonated
molecules which were present in the continuous phase
before lyophilization. Schwarz and Mehnert [64], thus
optimized the SLN freeze-drying by removing the
charged non-encapsulated drug in water, etomidate
and tetracaine, before lyophilization.
5.1.2.2. Spray-drying. Spray-drying was an alternative
method of lyophilization, to convert a liquid dispersion
into a dry product with the same goal: long-term stability.
Due to shear forces and temperature during the process,
destabilization of the systems can occur. Furthermore,
the redispersion properties have to be optimized. Freitas
and Muller
.
studied these parameters on three exemplary
SLN formulations composed of cetylpalmitate, Compritols 888 ATO (glyceryl behenate) or Synchrowaxs
HRSC (glyceroltribehenate and calcium behenate). Various parameters showed their ability to minimize the
temperature effect as well as the shear forces effect. By
addition of carbohydrates, the sugar layer around the
particles prevented the coalescence of molten lipid
droplets. A reduction of temperature effects can be
achieved by spraying alcoholic instead of aqueous SLN
dispersions, due to lower necessary inlet temperatures.
The inuence of shear forces could also be reduced by the
addition of carbohydrates. The sugar layer around the
particles protected the emulsier lm against shearing off
from the particle surface. Furthermore, a lower lipid
particle content reduced the probability of particle
contact and subsequent aggregation [77].
5.2. Physical stability
In the 1940s, two groups of scientists developed a
theory of colloidal stability now known as the

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B. Heurtault et al. / Biomaterials 24 (2003) 42834300

DLVO theory. For particles, an electrostatic repulsion


occurs when the diffuse layers overlap while attraction is due to van der Waals energy. In the DLVO
theory, it was assumed that these long-ranged forces
(van der Waals and electrostatic) control the stability of
colloids. However, the DLVO theory does not always
provide accurate predictions of colloid stability [113].
The rst forces occur when the interface is charged. It
corresponds to the electrostatic stabilization. The
second one corresponds to the steric stabilization when
polymeric chains take a great deal of room at the
interface. These two mechanisms lead to droplet
repulsion and are discussed below. However, the
prediction on the physical stability by the DLVO
theory did not always correlate with the experimentally
observed stability. The physical stability of positively
charged liposomes was not explained by the DLVO
theory [114].
5.2.1. Steric stabilization
Steric stabilization is currently used in the synthesis of
polymer particles. In the same way steric stabilization
has been obtained by coating the SLNs (glycerol
behenate) with Poloxamers 188. It led to sterically
stabilized particles with high stability against electrolytes [115]. The zeta potential also decreased [73].
Moreover, this structure led to a decrease of the
macrophage uptake (due to the hydrophilic properties)
and subsequently, to a prolonged circulation time in vivo
[115]. The same consequences were observed by Gasco,
with DPPE-PEG or PEGylated stearic acid incorporated in stearic acid SLNs [79]. In both cases, by analogy
to polymer particles, these drug carriers were considered
as stealth systems.
Electrolyte and pH induced change in the particle zeta
potential and size lead to a lack of stability of numerous
formulations. However, pre-requisites to allow the
stability were found: a minimum of 89 mV zeta
potential in combination with a steric stabilization
[81]. To limit destabilization, steric stabilization using
a surfactant was often used. However, in gastrointestinal media, additional steric stabilization through nonionic surfactants, e.g. Poloxamers 188, was not always
sufcient to provide stability [81].

5.2.3. Lipid composition


As previously described, dispersions with a highly
recrystallized lipid phase (high recrystallization index)
showed increased growth in particle size. However, the
retardation of the crystallization can be achieved by the
addition of inhibitors to the lipid matrix. Garti and Sato
[116] reported the effects of surfactants on the inhibition
of the crystallization and polymorphic transformation
of fats and fatty acids. Thus, SLN sensitive to gelling
were stabilized by inhibiting lipid transition using
Poloxamers [60,111].
5.2.4. Cream or hydrogel incorporation
Drug expulsion can be correlated to crystal transformation. However, two studies have shown that the SLN
incorporation into creams allowed the crystallization
phenomenon to be controlled; Thus, it can be possible to
stop this evolution during storage. The rst one was
based on retinol encapsulation in SLN [117]. The same
results were observed with cyclosporin which was stable
for 6 months in these conditions [118]. By being
incorporated into a cream, the system was maintained
in a high-energy state. Thus, the release rate for the
topical route was adjustable [27].
The inuence of hydrogels on liposome stability was
studied due to their favorable visco-elastic properties for
topical application. Contrary to SLN incorporation in
cream, destabilization can occurred as Gabrijelcic and
Sentjurc [119] observed with liposomes incorporated in
xanthan gum. The carboxymethylcellulose gel did not
inuence stability. The physical stability of liposomes
should be studied for each liposome preparation.
5.2.5. Optimization of storage parameters
In general, the introduction of energy into the system
(temperature, light) led to particle growth and subsequent gelling. At no light exposure and low temperatures, the zeta potentials remained practically
unchanged and the SLN dispersions were stable. By
optimizing storage conditions (8 C, darkness, siliconized vials), stability of the less-stable aqueous
Compritols (glyceryl behenate) SLN over 3 years was
achieved [21].

6. Conclusion
5.2.2. Electrostatic stabilization/repulsion
Ionic surfactants like sodium dodecyl sulfate were
usually used to stabilize the emulsions. The polar group
was negative in water and a counter-ion was released.
The interface was consequently charged. An ion layer
was consequently formed preventing the coalescence of
the drops. However, the modication of the charge has
multiple consequences on biological applications (membrane interactions, protein interaction) and has to be
taken into account.

Determination of stability should be the rst step in


the development of a new lipid carrier. Considering all
these results, a stability study of lipid systems will
involve multidisciplinary research due to the numerous
parameters implied. The physico-chemical stability is
directly related to the composition showing the importance of the initial choice of components and their
chemical structure. However, the required stability of a
new system is a compromise between essential long-term

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B. Heurtault et al. / Biomaterials 24 (2003) 42834300

storage stability and its destabilization necessary for


drug release in a biological environment. A controlled
destabilization process could be of interest to improve
the latter point.

[21]

[22]

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