Solid Lipid Nanoparticles - Concepts, Procedures, and Physicochemical Aspects
Solid Lipid Nanoparticles - Concepts, Procedures, and Physicochemical Aspects
Solid Lipid Nanoparticles - Concepts, Procedures, and Physicochemical Aspects
1 Solid Lipid
Nanoparticles —
Concepts, Procedures,
and Physicochemical
Aspects
Karsten Mäder and Wolfgang Mehnert
CONTENTS
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© 2005 by CRC Press LLC 1
1.1 SUMMARY
Solid lipid nanoparticles (SLN) have attracted increasing scientific and commercial
attention during the last few years. This chapter highlights the main features of SLN,
including the concept of SLN, different methods of production, and their applications.
Special attention is paid to the relation among drug incorporation, the heterogeneity
of the lipid particle, and the presence of other colloidal species. Strategies of SLN
stabilization to avoid particle growth or gelation are discussed. The biological fate
of the particles and the suitability of SLN for drug targeting are reviewed.
CDC are defined only by their size (most scientists agree on sizes below 1 µm;
others set 0.5 µm as the upper limit). CDC are very heterogeneous in all other aspects
(e.g., thermodynamic stability, chemical composition, and the physical state, includ-
ing solid, liquid, or liquid-crystalline dispersions) [1]. The most prominent examples
are nanoparticles, nanoemulsions, nanocapsules, liposomes, nanosuspensions, (mixed)
micelles, microemulsions, and cubosomes. Some CDC have reached the commercial
market. Probably the best known example is the microemulsion preconcentrate of
cyclosporine (Sandimmun-Neoral), which minimized the high variability of phar-
macokinetics of the Sandimmun formulation. In addition, intravenous injectable
CDC have been on the commercial market for many years. Examples include
nanoemulsions of etomidate (Etomidat-Lipuro) and diazepam (Diazepam-Lipuro)
[2–4], mixed micelles (Valium-MM, Konakion), and liposomes (AmBisome) [5].
However, overall only a very limited number of CDC has reached the marketplace.
It is expected that this number will increase as a result of requirements for drug
safety and of the increasing number of poorly soluble compounds in the pipeline.
More and more molecules have to be formulated with a sophisticated drug delivery
system to achieve predictable pharmacokinetics. Patents of other molecules (e.g.,
cyclosporine) have expired, and the first generics of cyclosporine microemulsion
preconcentrates have entered the market. Companies develop strategies to protect
or to get market shares based on formulation technology using CDC. Therefore, the
modification of existing CDC (to circumvent existing patents) or the development
of new CDC (preferably with new advantages and patent protection) is considered
crucial by many companies.
The main efforts to improve current CDC are related to:
High-shear homogenization and ultrasound were initially used for the production of
solid lipid nanodispersions [7,8]. Both methods are widespread and easy to handle.
However, in many cases, bimodal size distributions are obtained with one population
in the micrometer range. In addition, metal contamination has to be considered if
ultrasound is used.
Ahlin et al. used a rotor-stator homogenizer to produce SLN by melt-emulsifi-
cation [21]. They investigated the influence of different process parameters —
including emulsification time, stirring rate, and cooling conditions — on the particle
size and the zeta potential. In most cases, average particle sizes in the range of 100 to
200 nm were obtained using stirring rates of 20,000 to 25,000 rpm for 8 to 10 min
and controlled cooling with a stirring rate of 5,000 rpm.
HPH has emerged as a reliable and powerful technique for the preparation of SLN.
HPH has been used for years for the production of nanoemulsions for parenteral
nutrition. In contrast to other techniques, scaling up represents no or minor problems
in most cases. High-pressure homogenizers push a liquid with high pressure (10 to
200 MPa) through a narrow gap (in the range of few microns). The fluid accelerates
on a very short distance to very high velocity (over 1000 km/h). Very high-shear
forces disrupt the particles down to the submicron range. Typical lipid contents range
between 5 to 10% of the fluid and represent no problem to the homogenizer. Even
lipid concentrations up to 40% have been homogenized to lipid nanodispersions [22].
Two general approaches of the homogenization step, the hot and the cold homog-
enization techniques, can be used for the production of SLN [13,23,24]. In both
cases, a preparatory step involves incorporating the drug into the bulk lipid by
dissolving or dispersing the drug in the lipid melt.
1.4.2.2.1 Hot Homogenization
Hot homogenization is carried out at temperatures above the melting point of the
lipid and can therefore be regarded as the homogenization of an emulsion. A preemul-
sion of the drug-loaded lipid melt and the aqueous emulsifier phase (same temper-
ature) is obtained by a high-shear mixing device (Ultra-Turrax). The quality of the
preemulsion affects the quality of the final product to a large extent, and obtaining
droplets in the size range of a few micrometers is desirable. HPH of the preemulsion
is carried out at temperatures above the melting point of the lipid. In general, higher
temperatures result in lower particle sizes because of the decreased viscosity of the
inner phase [25]. However, high temperatures may also increase the degradation rate
of the drug and the carrier. Furthermore, many surfactants have decreased solubilities
and HLB values at a higher temperature, which might have a negative impact on
homogenization efficacy. The homogenization step can be repeated several times. It
should always be kept in mind that HPH increases the temperature of the sample
(approximately 10˚C for 500 bar [26]). In most cases, 3 to 5 homogenization cycles
at 500 to 1500 bar are sufficient. Increasing the homogenization pressure or the
number of cycles often results in an increase of the particle size because of particle
coalescence, which occurs as a result of the high kinetic energy of the particles [27].
The primary product of the hot homogenization is a nanoemulsion resulting
from the liquid state of the lipid. Solid particles are expected to be formed by the
cooling of the sample to room temperature or below. Because of the small particle
size and the presence of the emulsifiers, lipid crystallization may be highly retarded,
and the sample may remain as a supercooled melt (nanoemulsion) for several months
[28]. Westesen and Bunjes found that purported “SLN” data published by another
group were, in fact, measurements from supercooled melts [29].
1.4.2.2.2 Cold Homogenization
Cold homogenization is carried out with the solid lipid and can therefore by regarded
as a high-pressure milling of a lipid suspension. Effective temperature control and
regulation is needed to ensure the unmolten state of the lipid because of the increase
in temperature during homogenization [26]. Cold homogenization has been developed
to overcome the following three problems of the hot homogenization technique:
The first preparatory step is the same as in the hot homogenization procedure
and includes the solubilization or dispersing of the drug in the melt of the bulk lipid.
However, different steps follow. The drug-containing melt is cooled very rapidly
(e.g., by means of dry ice or liquid nitrogen). The high cooling rate favors a homog-
enous distribution of the drug within the lipid matrix. The solid, drug-containing
lipid is milled by means of ball or mortar milling in the range of 50 to 100 µm.
Low temperatures increase the fragility of the lipid and, therefore, favor particle
disruption. The solid lipid microparticles are dispersed in a chilled emulsifier solution.
The presuspension is subjected to HPH at or below room temperature. In general,
compared with hot homogenization, larger particle sizes and a broader size distri-
bution are observed in cold-homogenized samples [30]. The method of cold homog-
enization minimizes the thermal exposure of the sample, but it does not avoid it
because of the melting of the lipid/drug mixture in the initial step. Most investigators
use the hot homogenization process because of its higher efficacy and the avoidance
of the cold milling process. It must be also mentioned that the rapid cooling of the
lipid melt in the first step favors metastable lipid modifications (with higher drug
loading capacity), which might transform with time into more stable polymorphs
(with the expulsion of the incorporated drug).
acetone, ethanol, isopropanol, methanol), whereas larger particle sizes were obtained
with more lipophilic solvents. The process also can be easily used for the production
of lipid nanodispersions [17]. A requirement is the solubility of the lipid in the polar
organic solvent, which limits the application range of this procedure. A further
disadvantage is the low concentration of the lipid nanoparticles (typically 1% or
less). Higher amounts of the organic solvent increase the solubility of the lipid in
the aqueous phase and lead to an increase in particle size resulting from Ostwald
ripening. The main advantage of the method is the avoidance of thermal stress.
to Small [20], into nonpolar and different classes of polar lipids. This classification
is very helpful for understanding the interplay among drug, lipid emulsifier, and
water. The general lipid composition (mixed chain lengths or triglycerides made
from one fatty acid) will have different crystallinities and capacities for accommo-
dating foreign molecules. In addition, pH levels may change the behavior of lipids
considerably. Fatty acids are, in the protonated form (e.g., myristic acid), insoluble,
nonswelling lipids, and they behave similarly to triglycerides. Unprotonated fatty
acids (e.g., sodium myristate) can form micelles and are soluble amphiphiles with
lyotropic mesomorphism. Around the pKa (which varies in strong dependency of the
environment), fatty acid–fatty acid salt complexes form lyotropic liquid crystalline
lamellar phases and represent insoluble swelling lipids. The interaction between the
fatty acid and the drug will be very different for each species.
Furthermore, even triglycerides with the same fatty acid composition will behave
differently, depending on the localization of the fatty acid on the glycerol. For
example, cacao butter has a rather sharp melting point because of the defined
localization of the oleic (2 position) and palmitic and stearic acids (1 and 3 positions).
Random localization of the fatty acids leads to a broadening of the melting point to
a melting range, which means that a certain amount of liquid lipids will be present
over a large temperature range.
Jenning and Gohla found that high crystallinity of lipid matrices was linked with
good physical stability but a low degree drug incorporation, whereas lipid matrices
with low crystallinity were able to accommodate higher amounts of drug and showed
poor physical stability [39]. However, further parameters for nanoparticle formation
will be different for different lipids. Examples include the melting point, the velocity
of lipid crystallization, and the shape of the lipid crystals (and therefore the surface
area). Higher-melting lipids led to an increase in particle size [11,30]. These results
are in agreement with the general theory of HPH [26] and can be explained by the
higher viscosity of the dispersed phase.
It is also noteworthy that most of the lipids used represent a mixture of several
chemical compounds. The composition might, therefore, vary among different suppli-
ers and might even vary for different batches from the same supplier. Small differences
in the lipid composition (e.g., impurities) might have a significant effect on the quality
of SLN dispersion (e.g., by changing the zeta potential or retarding crystallization
processes). For example, lipid nanodispersions made with cetyl palmitate from dif-
ferent suppliers had different particle sizes and storage stabilities (A. Lippacher,
personal communication).
The influence of lipid composition on particle size was also confirmed for SLN
produced via high-shear homogenization [21]. The average particle size of Witepsol
W35 SLN was found to be significantly smaller (117.0 ± 1.8 nm) than the average
particle size of Dynasan 118 SLN (175.1 ± 3.5 nm). Witepsol W35 contains shorter
fatty acid chains and considerable amounts of mono- and diglycerides, which possess
surface-active properties.
Increasing the lipid content over 10% leads to larger particles (including micro-
particles) and broader particle size distributions [27,30]. Both a decrease of the
homogenization efficiency and an increase in particle agglomeration cause this
phenomenon.
1.4.3 STERILIZATION
Parenteral administration requires sterile products. Aseptic production, filtration,
γ-irradiation, and heating are normally used to achieve sterility. Filtration sterilization
of dispersed systems requires high pressure and is not applicable to particles larger
than 0.2 µm.
The impact of different sterilization techniques (steam sterilization at 121˚C
[15 min] and 110˚C [15 min], γ-sterilization) on SLN characteristics has been
the increase in particle size after γ-irradiation was lower, but was comparable to
steam sterilization at 110˚C.
Unfortunately, most investigators did not search for steam sterilization– or irra-
diation-induced chemical degradation. It should be kept in mind that degradation
does not always cause increased particle sizes. In contrast, the formation of species
like lysophosphatides or free fatty acids might even preserve small particle sizes,
but might also cause toxicological problems (e.g., hemolytic activity). Detailed
studies that involve the aspects of chemical stability are clearly necessary to permit
valid statements of the possibilities of SLN sterilization.
Mean particle sizes and size distributions of Compritol and Dynasan 112 SLN
were only slightly increased after freeze-thaw experiments [50].
The results of unloaded SLN do not predict the quality of drug-loaded lyo-
philizates. Even low concentrations of 1% tetracaine or etomidate caused a signifi-
cant increase in particle size, which excludes an intravenous administration [50].
Siekmann and Westesen investigated the lyophilization of tripalmitin SLN (sur-
factants: 4.5% tyloxapol and 3% soybean lecithin [Lipoid S100]) [52]. Glucose,
sucrose, maltose, and trehalose were used as cryoprotective agents in concentrations
of 5, 10, and 20%. Handshaking of redispersed samples was an insufficient method,
whereas bath sonification produced better results. Average particle sizes of all lyo-
philized samples with cryoprotective agents were 1.5 to 2.4 times higher than the
original dispersions. Cryoprotector-free samples showed very high particle aggre-
gation. Samples with a lipid content below 10% showed less aggregation than more
highly concentrated samples. The efficiency of the cryoprotectors decreases in the
following order: trehalose > sucrose > glucose and maltose. Surprisingly, it was
found that the time at which the cryoprotector is added influences the quality of the
final formulation. Best results were obtained when the cryoprotector was added to
the sample before homogenization. Under these circumstances, average particle size
remained almost unchanged, though storage over 1 year caused significant increases
in particle sizes. Average particle sizes were 4 to 6.5 times larger than in the original
dispersion. In contrast to the lyophilizates, the aqueous dispersions of tylox-
apol/phospholipid-stabilized tripalmitin SLN exhibited remarkable storage stability.
The average particle size increased only very slightly, from 56 to 65 nm, over 1 year.
The instability of the SLN lyophilizates can be explained by the sintering of the
particles.
Cavalli et al. also observed increased particle sizes (2.1 to 4.9 times) after
lyophilization [46]. A trehalose concentration of 2% was insufficient to prevent
lyophilization-induced particle aggregation. Increasing the concentration of trehalose
to 15% resulted in average particle sizes around 100 nm and in polydispersity indices
of 0.25 after reconstitution.
Heiati compared the influence of four cryoprotectors (trehalose, glucose, lactose,
and mannitol) on the particle size of azidothymidine palmitate–loaded SLN lyo-
philizates [47]. Trehalose was found to be the most effective cryoprotector for
preventing aggregation during lyophilization and subsequent reconstitution of SLN.
A sugar/lipid weight ratio of 2.6 to 3.9 was recommended.
The freezing process has an effect on the product quality. Rapid freezing in
liquid nitrogen was suggested by Schwarz and Mehnert [50]. In contrast, other
researchers observed the best results after a slow freezing process. Zimmermann
et al. found that optimization of the lyophilization parameters results in formulations
that are intravenously injectable, with regard to particle size [53]. Again, best results
were obtained with samples of low lipid content and with the cryoprotector trehalose.
In contrast to the results of Schwarz, slow freezing in a deep freeze (–70˚C) was
superior to rapid cooling in liquid nitrogen. Furthermore, introduction of an addi-
tional thermal treatment to the frozen SLN dispersion (2 h at –22˚C followed by a
2-h temperature decrease to –40˚C) was found to improve the quality of the product.
Recent studies of Gasco’s group indicate that drying with a nitrogen stream at
low temperatures (3 to 10˚C) might be superior to lyophilization [54]. Compared to
lyophilization, the advantages of this process are the avoidance of freezing and the
energy efficiency resulting from the higher vapor pressure of water.
Spray drying might be an alternative procedure to lyophilization to transform
an aqueous SLN dispersion into a dry product. This method has been used scarcely
for SLN formulation, although spray drying is cheaper than lyophilization. By spray
drying, Freitas and Müller obtained a redispersable powder that complies with the
general requirements regarding particle size and selection of ingredients for intra-
venous injections [55]. Spray drying might potentially cause particle aggregation as
a result of high temperatures, shear forces, and partial melting of the particles. Freitas
and Müller recommend the use of lipids with melting points greater than 70˚C for
spray drying. Furthermore, the addition of carbohydrates and low lipid contents
favor the preservation of the colloidal particle size in spray drying. The melting of
the lipid can be minimized by using ethanol/water mixtures as a dispersion medium
instead of pure water because of the lower inlet temperatures. The best result was
obtained with SLN concentrations of 1% in solutions of 30% trehalose in water or
20% trehalose in ethanol/water mixtures (10/90 v/v).
Lipids and fats, as soft condensed material in general, are very complex systems, not
only in their static structures but also with respect to their kinetics of supramolecular
formation. Hysteresis phenomena or supercooling can gravely complicate the task of
defining the underlying structures and boundaries in a phase diagram (p. 334).
This is especially true for lipids in the colloidal size range. Many analytical tools
do not permit direct measurement in the undiluted SLN dispersion. Therefore, sample
preparation might cause artifacts (e.g., removal of emulsifier from particle surface
by dilution, induction of crystallization processes, changes of lipid modifications).
Pushing SLN dispersions though a syringe needle might result in the immediate
transformation of the low, viscous SLN dispersion into a viscous gel. In this case,
the artifact caused by sample preparation is clearly visible, although in many other
cases it will not be.
The most important parameters include:
• Particle size
• Degree of crystallinity and lipid modification
• Coexistence of additional colloidal structures (micelles, liposomes, super-
cooled melts, drug nanoparticles) and timescale of distribution processes
• Zeta potential
Photon correlation spectroscopy (PCS) and laser diffraction (LD) are the most
frequently applied techniques for measurements of particle size. PCS (also known
as dynamic light scattering) measures the fluctuation of the intensity of the scattered
light caused by particle movement and covers a size range from a few nanometers to
about 3 µm. New developments (back scattering, cross polarization) permit measure-
ment in undiluted or less diluted samples. Microparticles are not detected by PCS,
but they can be visualized by means of LD measurements. This method is based on
the dependence of the diffraction angle on the particle radius. Smaller particles cause
more intense scattering at high angles than do larger ones. New developments of
LD expanded the lower limit of measurable particle sizes from 40 to 100 nm.
However, despite this progress, it is highly recommended that both PCS and LD
be used simultaneously. It should be kept in mind that both methods are not “mea-
suring” particle sizes. Rather, they detect light-scattering effects that are used to
calculate particle size. For example, uncertainties may result from nonspherical
particle shapes and from the assumption of certain parameters that are used to
calculate the particle size. Platelet structures commonly occur during lipid crystal-
lization [57] and have also been observed for SLN [11,40,58]. The presence of
several populations and other colloidal structures adds further difficulties.
The use of additional techniques is recommended. Light microscopy is not
sensitive to the nanometer size range but gives a fast indication of the presence of
microparticles. Electron microscopy provides, in contrast to PCS and LD, direct
information on the particle shape. However, the investigator should pay special
attention to possible artifacts that may be caused by the sample preparation. For
example, solvent removal may cause modification changes that will influence the
particle shape [57].
Atomic force microscopy (AFM) has also been applied to image the morpho-
logical structure of SLN [59]. The size of the visualized particles was of the same
magnitude as the results of PCS measurements. The AFM investigations revealed
the disklike structure of the particles. Dingler and others investigated cetyl palmitate
SLN (stabilized by polyglycerol methylglucose distearate, Tegocare 450) by electron
microscopy and AFM and observed spherical forms of the particles [60,61]. Different
SLN shapes were reported by Westesen and others for SLN made of well-defined
lipids of high purity (e.g., pure triglycerides) [11,62]. A disadvantage of AFM is the
required fixation of the particles (by removal of water), which changes the status of
the emulsifier and might also cause polymorphic transitions of the lipid.
The particle sizing by field flow fractionation (FFF) is based on the different
effect of a perpendicular applied field on particles in a laminar flow [63–66]. The
separation principle corresponds to the nature of the perpendicular field and may,
for example, be based on different mass (sedimentation FFF), size (cross-flow FFF),
or charge (electric-field FFF). Cross-flow FFF has been applied recently to investi-
gate nanoemulsions, SLN, and nanostructured lipid carriers (NLC, particles com-
posed of liquid and solid lipids) [58]. Although all samples had comparable particle
sizes in PCS, their retention in the FFF was very different. Compared to the spherical
droplets of the nanoemulsion, SLN and NLC were pushed more efficiently to the
bottom of the channel because of their anisotropic shape. Their very different shapes
have been confirmed by electron microscopy.
supercooled melt > alpha modification > beta′ modification > beta modification
Because of the small size of the particles and the presence of emulsifiers, lipid
crystallization and modification changes might be highly retarded. For example, it
has been observed that polymorphic transitions might occur very slowly and that
Dynasan 112 SLN, if crystallization is not artificially induced, may remain a super-
cooled melt over several months [29,62]. Q10 nanodispersions remain also stable as
supercooled melts over several months [1].
Differential scanning calorimetry and x-ray scattering are widely used to inves-
tigate the status of the lipid. Differential scanning calorimetry is based on the fact
that different lipid modifications possess different melting points and melting enthal-
pies. By means of x-ray scattering, it is possible to assess the length of the long and
short spacings of the lipid lattice. Measuring the SLN dispersions themselves is
highly recommended because solvent removal will lead to modification changes.
Sensitivity problems and long measurement times of conventional x-ray sources
might be overcome by synchrotron irradiation [62]. In addition, this method allows
for conducting time-resolved experiments and allows the detection of intermediate
states of colloidal systems that will be undetectable by conventional x-ray methods
[56]. Unfortunately, this source has limited accessibility for most investigators.
NMR is a very useful tool for investigating colloidal systems. NMR active nuclei
of interest are 1H, 13C, 19F, and 31P. Because of the different chemical shifts, it is
possible to attribute the NMR signals to particular molecules or their segments.
Simple 1H-NMR spectroscopy permits an easy and very rapid detection of super-
cooled melts caused by the low line widths of the lipid protons [67]. This method
is based on the different proton relaxation times in the liquid and semisolid/solid
states. Protons in the liquid state give sharp signals with high signal amplitudes,
whereas semisolid/solid protons give weak and broad NMR signals under these
circumstances. Simple 1H-NMR spectroscopy also allows the characterization of
lipid particles composed of solid and liquid lipids (NLC) [68,69]. The great potential
of NMR, with its variety of different approaches (solid-state NMR, determination
of self-diffusion coefficients, etc.), has scarcely been used in the SLN field, although
it will provide unique insights into the structure and dynamics of SLN dispersions.
Electron spin resonance (ESR) is, as is NMR, a noninvasive method that does
not require dilution of the sample. Paramagnetic spin probes are used as model drugs
to investigate SLN dispersions. A large variety of spin probes is commercially
available. The corresponding ESR spectra give information about the microviscosity
and micropolarity. ESR permits the direct, repeatable, and noninvasive characteriza-
tion of the distribution of the spin probe between the aqueous and the lipid phases.
Experimental results demonstrate that storage-induced crystallization of SLN leads
to an expulsion of the probe out of the lipid into the aqueous phase [43]. Furthermore,
high drug loading caused by the liquid lipid and controlled release caused by the
solid lipid). Unfortunately, the proposed structures were not backed up by experi-
mental data. Recent studies demonstrate that NLC possess no advantages over
nanoemulsions. It has been shown that the liquid lipid forms a half drop on the solid
platelet [58,69].
The presence of several colloidal species is an important point that has been
overlooked by many scientists. Stabilizing agents are not localized exclusively on
the lipid surface but also in the aqueous phase in different forms, which might serve
as an alternative location to host the drug molecules. Sometimes the amount of
stabilizers exceeds the amount of the lipid phase. For example, stearic acid (as lipid
phase), Epikuron 200 (lecithin), and taurocholate have been formulated in the ratio
of 3:4:6 [54].
Published NMR spectra of diazepam indicate a high mobility of the drug, which
indicates a localization of the drug in other colloidal species of high mobility [74]
(an association with the solid lipid would cause extensive line broadening [69]).
Therefore, micelle-forming surfactant molecules (e.g., SDS) will be present in
three different forms, namely, on the lipid surface, as micelles, and as monomeric
surfactant molecules in solution. Lecithin will form liposomes, which have also been
detected in nanoemulsions for parenteral nutrition [77]. Mixed micelles have to be
considered in glycocholate/lecithin-stabilized and -related systems. Micelles, mixed
micelles, and liposomes are known to solubilize drugs, and are therefore attractive
alternative drug-incorporation sites (especially with respect to the low incorporation
capacity of lipid crystals).
A more detailed investigation of the SLN and NLC — including the appropriate
characterization of drug incorporation, the presence of other colloids, and their in vivo
fate — is necessary to understand and judge the real potential of these colloids.
ACKNOWLEDGMENTS
We thank K. Jores (Novartis, Basle) and H. Bunjes for helpful discussions and
scientific cooperation.
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