Journal of Controlled Release 48 (1997) 223–236

Physicochemical characterization of lipid nanoparticles and

evaluation of their drug loading capacity and sustained release
potential
a, a b
K. Westesen *, H. Bunjes , M.H.J. Koch
a
Friedrich-Schiller-University Jena, Institute for Pharmacy, Department for Pharmaceutical Technology, Lessingstrasse 8, D-07743
Jena, Germany
b
European Molecular Biology Laboratory, Hamburg Outstation, EMBL c /o DESY, Notkestrasse 85, D-22603 Hamburg, Germany

Received 16 April 1996; revised 23 December 1996; accepted 24 December 1996

Abstract

Drug carrier systems based on lipid nanosuspensions prepared by melt emulsification present a number of severe stability
problems such as a high gelation tendency, considerable particle growth and drug expulsion. Destabilization of the
emulsified lipidic carriers is related to recrystallization of the lipids. The choice of stabilizers for colloidal lipid suspensions
is, therefore, restricted. Systematic surface modifications are thus limited. In addition, the drug payload of crystalline
nanosuspension particles is generally low. Improved stability and loading capacities were found for amorphous lipid
nanoparticles which present the characteristic signals of supercooled melts in high resolution 1 H-NMR. The NMR data
indicate that such liquid but viscous carriers can, however, not immobilize the incorporated drug molecules to the same
extent as a solid matrix. Sustained release over days or weeks as in slowly biodegraded solid matrices thus seems difficult to
achieve with a supercooled melt. Attempts to combine the advantages of the solid crystalline lipids and the amorphous nature
of the supercooled melts by generating solid but amorphous lipid suspension particles with a satisfactory long-term stability
by a variation of the lipid matrix material have hitherto not been successful. Even a satisfactory stabilization of the
a-modification using complex lipid mixtures to improve the loading capacity or to slow down the drug expulsion process
could not be achieved. The rates of the polymorphic transitions were much higher in the colloidal lipid dispersions than in
the bulk for the hard fats under investigation. Despite the fact that the properties of the lipids are superimposed with colloidal
properties, significant differences between monoacid triglycerides and complex lipids were, however, found.  1997
Elsevier Science B.V.

Keywords: Solid lipid nanoparticles; Nanoparticles of supercooled melts; Loading capacity; Molecular mobility; NMR

1. Introduction with respect to intravenous (i.v.) administration.

Numerous drugs are poorly soluble or unstable in
aqueous media and thus cause formulation problems
*Corresponding author. Tel.: 149 3641 636781; fax: 149 3641
636766.
Decades ago submicron-sized vegetable oil-in-water
(O / W) emulsions were introduced as carrier systems
for poorly water-soluble drugs [1–3]. Although these
O / W emulsions are considered to be biodegradable,
biocompatible and easy to manufacture, remarkably
few drug-containing emulsions have reached the

0168-3659 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved

PII S0168-3659( 97 )00046-1
224 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
market as a consequence of severe formulation
problems. These problems arise from the low solu-
bility of most poorly water-soluble drugs in the
compositions and from the destabilizing effect of
many drugs on the emulsions. Traditional O / W
emulsions are hardly suited for sustained release in
any case since the low viscosity of the dispersed
liquid phase, combined with the high specific surface
area of colloidal dispersions, causes rapid drug
diffusion out of the droplets [4]. In contrast, the
mobility of drug molecules is drastically reduced in a
solid phase which should thus make it possible to
reduce drug leakage or hydrolysis. Drug immobiliza-
tion in a dispersed solid lipid phase might also
prevent destabilization of the colloid by the drug
often observed in emulsions. First attempts to de-
velop lipid-based colloidal suspensions by replacing
the dispersed lipid oil phase with solid triglycerides
already date back to the time when the first parenter-
al O / W emulsions became commercially available.
Attempts to formulate saturated long-chain tri-
glycerides with high melting temperatures into phos-
pholipid-stabilized aqueous suspensions by melt-
emulsification were unsuccessful because of in-
stability, leading, for instance, to the formation of
semisolid gels [5].
Since the 1980s colloidal lipid suspensions gained
renewed scientific and industrial interest as drug
carriers [6–12]. A satisfactory long-term stability of
products with lipid concentrations comparable to
parenteral emulsions was, however, either not
achieved [8,13] or not reported.
In the early 1990s colloidal lipid suspensions with
lipid concentrations of 10 wt% based on new stabi-
lizer compositions and exhibiting a satisfactory long-
term stability were described [5]. These dispersions
were prepared by melt-emulsification preferentially
using high-pressure homogenizers. The effects of
homogenization parameters and composition (matrix
constituents, volume fraction of the dispersed phase,
type and amount of emulsifying agents) on the mean
particle size of colloidal lipid suspensions turned out
to be similar to those observed for O / W emulsions
[14,15]. Since melt-emulsification processes are pref-
erably applied for the production of solid lipid
particles with narrow size distributions in the lower
nanometer size range, recrystallization of the molten
lipids must occur to obtain the desired suspension
state. Although the raw materials chosen as matrix
materials are usually crystalline at room temperature,
recrystallization does not always occur in the dis-
persed state [16,17]. Nanoparticles prepared from
trilaurin which exhibits a melting temperature around
468C in the bulk do not recrystallize above 08C as
shown by transmission electron microscopy (TEM),
X-ray diffraction studies and differential scanning
calorimetry (DSC) [16,17]. These lipidic nanoparti-
cles are emulsion droplets of supercooled melts
rather than amorphous solid particles. This is in clear
contradiction with claims from other laboratories
[18] which are, however, not substantiated by ex-
perimental evidence.
In recent studies it was found that trimyristin
nanoparticles can either be forced to transfer into the
solid (crystalline) state under controlled conditions or
be kept in the supercooled liquid state at room
temperature [16,17]. In the present study, trimyristin
dispersions were introduced as model systems to
study the influence of either the liquid or the
crystalline state on the loading capacity of (mono-
acid) triglyceride nanoparticles and on the molecular
mobility of drugs to derive information on the
sustained release potential.
Nanoparticles from mixed triglycerides may be an
interesting alternative to monoacid triglyceride
nanoparticles with respect to drug incorporation
capacity because mixed triglycerides usually have a
lower degree of crystalline order. The processing and
crystallization behavior as well as the structure of
nanoparticles prepared from several hard fat sup-
pository bases is, therefore, also investigated and
compared to that of trimyristin nanoparticles.
Since immobilization of drug molecules in
nanoparticles of supercooled melts is not expected to
occur in the same way as in solid nanoparticles, it
was also attempted to obtain information on the
formation of supercooled melts in colloidally dis-
persed lipids, on their stability and on strategies
preventing their occurrence. For this purpose, high
resolution proton nuclear magnetic resonance ( 1 H-
NMR) spectroscopy is introduced as a tool to
distinguish between amorphous solids and liquids in
dilute colloidal dispersions, to determine drug dis-
tribution within the compositions and to study the
mobility of drug molecules incorporated in the lipid
matrix.
 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236 225

2. Materials Table 1
Composition of drug free dispersions

Dynasan 114 (trimyristin, D 114) was provided by Matrix lipid Emulsifier blend Preparation temperature (8C)
¨ AG (Witten, Germany). The purity of the fatty
Huls D 114 PL / Tyl 70
acid fraction is approximately 95% and the hydroxyl D 114 PL / SGCa 70
value is below 5. The hard fat suppository bases D 114 PL / SGCb 70
D 114 PL 70
Witepsol H 35, H 42 and E 85, also provided by
E 85 PL / Tyl 80
¨ AG (Witten, Germany), consist of mixed
Huls E 85 PL 65
triglycerides with chain lengths mainly between 12 H 35 PL / Tyl 80
and 18 carbons. The maximum hydroxyl value of H 35 PL / SGCa 80
these commercial hard fats is 3 (H 35, H 42) or 15 H 35 PL 65
H 42 PL / Tyl 80
(E 85).
H 42 PL / SGCb 60
The following materials were from the indicated H 42 PL 65
sources and used without further purification: soy
Stabilizer composition: PL, Soy bean phospholipid Lipoid S100
bean lecithin Lipoid S100 (Lipoid KG, Ludwigs- (S100) 2.4%; PL / SGCa, S 100: 1.6%, sodium glycocholate:
hafen, Germany), sodium glycocholate (Sigma, St. 0.4%; PL / SGCb, S 100: 2.4%, sodium glycocholate: 0.6%; PL /
Louis, MO), menadione (Sigma, St. Louis, MO), Tyl, S 100: 2%, tyloxapol: 2%.
betamethasone valerate (Mainland Pharmazeutische
Fabrik GmbH, Frankfurt, Germany), ubidecarenone
was donated by Pharmacia AB (Uppsala, Sweden), temperature as the lipid melt was prepared by probe
prednisolone (Caelo, Hilden, Germany), tyloxapol sonication. This pre-mix was passed through a
USP (Sterling Organics), oxazepam (Thomae, thermostatized high-pressure homogenizer (Micron
Biberach, Germany), cortisone (Synopharm, Lab 40, APV Gaulin, Lubeck,¨ Germany) for five
¨
Barsbuttel, Germany), diazepam (Ph. Eur.), thiomer- cycles at 800 bars. The hot dispersions were filtered
sal (Synopharm, Barsbuttel,¨ Germany), retinol through 0.45 mm filters and allowed to cool down to
(Fluka, Buchs, Switzerland), glycerol 85% (Wasser- room temperature. The dispersions were split into
fuhr, Bonn, Germany), anhydrous glycerol (Merck, two fractions one of which was stored at room
Darmstadt, Germany), deuterated water (99.95%, temperature (20–258C), the other at 4–88C.
Energiteknik AB, Studsvik, Sweden), 3-(trimethyl- Particle sizes were analyzed by photon correlation
silyl)-propanesulfonic acid sodium salt (DSS) spectroscopy (PCS) with a Zetasizer 3 (Malvern
(Merck, Darmstadt, Germany), bidistilled water. Instruments) or an Autosizer 2 C (Malvern Instru-
ments) calculating the size distribution assuming
spherical particles applying exponential sampling
3. Methods without Mie correction. The mean particle size was
calculated from the number distribution as the diam-
The lipids were heated to at least 108C above their eter of the equivalent hydrodynamic sphere.
melting point. The phospholipid was dispersed in the Thermal analysis was performed in a Perkin Elmer
melt by probe sonication (Bandelin Desintegrator DSC 2-C calorimeter using accurately weighed
HD 200, Bandelin Electronics, Berlin, Germany) samples of 1–2 mg for bulk material or 10–20 mg
until the dispersion appeared clear. For the drug for dispersions. Heating curves were recorded with a
incorporation study, the drug was dissolved in the scan rate of 5 or 108C / min. Crystallization data was
triglyceride-phospholipid mixture. The composition obtained from thermally untreated samples or after
of the dispersions are given in Table 1 and Table 2. keeping the samples around 208C above their melting
The co-emulsifier was dissolved in the aqueous point for at least 10 min. The cooling rate was
phase containing 0.01% thiomersal as a preservative 58C / min. The melting and recrystallization tempera-
if required. Glycerol at 2.25% was added for isotoni- tures given correspond to the maxima or minima,
zation. A predispersion of the lipid in this aqueous respectively, in the DSC curves. Polymorphic forms
phase previously heated to approximately the same were assigned by correlation with the X-ray data.
226 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236

Table 2
Composition and stability of drug loaded dispersions
Matrix Drug Drug Solubility Emulsifier Phase separation
lipid concentration a in lipid blend of crystalline
(w%) melt drug (suspension)
b
D 114 Oxazepam 5 *
D 114 Diazepam 5 1 PL / SGCb *
D 114 Diazepam 3 1 PL / SGCb *
b
D 114 Cortisone 5 *
D 114 Betamethasone 5 1 PL / SGCb 1 Drug crystals also
valerate observed in emulsion
b
D 114 Prednisolone 5 *
D 114 Retinol 5 1 PL / SGCb *
D 114 Menadione 2 1 PL / Tyl 1 c Macroscopic drug
crystals after 2 days
of cold storage
D 114 Menadione 1 1 PL / Tyl *
E 85 Menadione 2 1 PL / Tyl 1 c Macroscopic drug
crystals after 4 days
of cold storage
H 42 Menadione 2 1 PL / Tyl 1 c Macroscopic drug
crystals after 6 days
of cold storage
D 114 Ubidecarenone 50 1 PL / SGCb *
D 114 Ubidecarenone 10 1 PL / SGCb *
D 114 Ubidecarenone 5 1 PL / SGCb *
a
Drug concentration is related to the lipid phase (matrix lipid1drug).
b
No dispersions were prepared if the incorporated drug was insoluble in the lipid melt.
c
Macroscopic and microscopic investigations were performed regularly within several days after preparation.
All dispersions were prepared at 708C applying five homogenization cycles at 800 bar.

X-ray diffraction measurements were performed
on the double focusing monochromator mirror ca-
mera X33 of the EMBL in HASYLAB on the
storage ring DORIS of the Deutsches Elektronen
Synchrotron (DESY) at Hamburg as described previ-
ously [17].
High resolution proton nuclear magnetic resonance
( 1 H-NMR) spectra of samples prepared in deuterium
oxide were obtained on a WM-400 (drug-free disper-
sion) and a DRX-400 (drug-loaded dispersions)
¨
NMR spectrometer (Bruker, Rheinstatten, Germany)
operating at 400 MHz and ambient temperature.
Drug-containing dispersions were investigated with-
out adding an internal reference using 308 pulse
widths. In case of the drug-free D 114 nanodisper-
sions, 3-(trimethylsilyl)-propanesulfonic acid sodium
salt (DSS) (d50.00 ppm) was used as an internal
reference for quantitative analysis. DSS was dis-
solved in D 2 O (about 25 mg DSS accurately weighed
in 10 ml D 2 O) and added in 1:1 portions to the
samples. 908 pulse widths were used with a pulse
repetition time of 14 s allowing complete relaxation
of the system. The relaxation time T 1 was deter-
mined by the inversion recovery method to be 3.2 s
for the trimethylsilyl signal of DSS. FIDs were
Fourier transformed without line broadening.
The drug loaded dispersions were examined in a
Zeiss Laboval 4 microscope (Zeiss, Oberkochen,
Germany) equipped with crossed polarizers and a
l-sheet. The investigations were performed after 1
day of storage if not otherwise specified.
4. Results and discussion
After melt-emulsification the dispersions were
split into two fractions one of which was stored at
room temperature (20–258C), the other one at 4–
88C. If the dispersions were stabilized with either the
phospholipid / tyloxapol blend or with the phos-
 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
pholipid / bile salt blend both fractions exhibit ap-
proximated mean particle sizes between 60 and 150
nm and are thus in the lower colloidal size range.
Stabilization with the phospholipid / tyloxapol blend
usually yields smaller particles than with the phos-
pholipid / bile salt blend. The particle sizes in the
drug-containing dispersions are comparable to those
of the corresponding drug-free dispersions.
Dispersions stabilized with the phospholipid only
deviate in their behavior. The purely phospholipid
stabilized dispersions of E 85 and H 42 kept at room
temperature form highly viscous or soft gel-like
systems within one day of storage. More rigid gels
are formed at refrigerator temperature. D 114 and H
35 dispersions remain liquid at room temperature.
Whereas the D 114 dispersion also turns rapidly into
a semisolid gel at 58C the H 35 dispersions remains
liquid even at this low temperature at least for
several days. Despite the fact that there are obvious
similarities between lipid nanosuspensions and col-
loidal oil-in-water (o / w) emulsions with respect to
the preparation method and the chemical composi-
tion, all suspension formulations under investigation
stabilized with phospholipids only exhibit a pro-
nounced tendency to form semisolid, water-rich gels
especially at low temperatures. The critical tempera-
ture depends on the matrix material. The gel forma-
tion indicates clear differences in the properties of
colloidal lipid emulsions and suspensions but can be
circumvented by the addition of a co-surfactant.
To evaluate the loading capacity and the sustained
release potential of lipid nanoparticle dispersions
based on monoacid triglycerides or complex
glyceride mixtures, a selection of poorly water
soluble model drugs were added in various con-
centrations to the basic compositions. The composi-
tion of the drug-loaded systems and the observed
instability phenomena are given in Table 2.
Ubidecarenone is the only drug that can be added to
the basic compositions even in high concentrations
without destabilizing the systems. Several model
drugs cannot even be dissolved in the lipid melts in
concentrations as low as 5 wt% related to the lipid
— demonstrating the low solubilization capacity of
the molten lipids for foreign poorly water-soluble
compounds already in the heat. Many of the few
successfully drug-loaded dispersions exhibit a poor
storage stability resulting in the formation of crystals
227
enriched in phase separated drug (Table 2). The
detection of these drug crystals in the milky disper-
sions is often difficult. The use of X-ray diffraction
is, in most cases, not possible due to problems in the
preparation of homogenous samples (sedimentation
of drug crystals) and detection limits of the method.
Detection and quantification of phase separated drug
by DSC is usually also impossible because the high
water content of the dispersions limits the maximum
temperature of the heating runs. Moreover, the first
order transition endotherms of the matrix materials
are extremely broadened for the colloidally dispersed
materials stabilized with emulsifier blends. This
holds especially true for the complex lipids (Fig. 8).
In contrast, polarizing light microscopy is an appro-
priate technique for the detection of expelled, crys-
tal-forming drugs. The crystals of the model drugs
can be identified in the dispersions via their optical
anisotropy and simple geometric shape. In some
cases, the drug crystals can also be detected by
macroscopic observation of the dispersions. The
phase separation of incorporated drug may occur
after various time intervals depending on the com-
position of the matrix material. For menadione-
loaded dispersions, the phase separation is considera-
bly retarded in systems based on complex glycerides.
At this stage comprehensive physicochemical
characterization of the colloidal dispersions seemed
essential to understand the phenomena and to check
the conclusions drawn from bulk properties of lipids.
In order to be able to generalize the results and to
correlate the physical state of the lipids in the
dispersions with the drug loading capacity and their
sustained release potential, initial studies were per-
formed on the drug free compositions.
None of the drug free dispersions stabilized with
one of the stabilizer blends stored at room tempera-
ture for more than three months displays any small
or wide angle reflection in their X-ray patterns (Fig.
1). They also do not display any thermal event
(melting endotherms or glass transitions) in DSC
heating runs (Fig. 2). Whereas X-ray diffraction data
only allow to differentiate between crystalline and
amorphous material, DSC data can be used to
differentiate between amorphous solids and liquids.
The absence of a glass transition in the DSC
thermograms hints at a liquid state of the dispersed
lipid phase although it cannot be excluded in case of
228 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
Fig. 1. Small angle X-ray scattering patterns of phospholipid /
tyloxapol stabilized nanoparticle dispersions stored at room tem-
perature for more than 4 months (10 days in case of H 35) (s52
sin Q / l, 2 Q: scattering angle). The curves have been displaced
vertically for better visualization.
aqueous colloidal dispersions of amorphous material,
that a glass transition would occur below the de-
tection limit. Additional investigations on the phys-
ical state of the submicron-sized D 114 were,
therefore, performed by 1 H-NMR spectroscopy.
Spectra taken after 1 day of storage at room tempera-
Fig. 2. DSC heating curves of phospholipid / tyloxapol stabilized
nanoparticle dispersions stored at room temperature for more than
3 months. The curves have been displaced along the ordinate for
better visualization.
ture display strong signals which can be assigned to
the glycerides (Fig. 3) indicating the high mobility of
these molecules characteristic of the liquid state.
This result confirms earlier assumptions [16,17] that
the colloidal dispersion kept at room temperature is
an emulsion of a supercooled melt. To our knowl-
edge, this novel type of colloidal drug carrier has not
been described by others so far.
In contrast, cooling the freshly melt-emulsified
dispersions to refrigerator temperature results in
dispersions that display X-ray reflections in the small
and wide angle regions pointing to the crystalline
nature of the particles (Fig. 4). These dispersions
also exhibit melting endotherms in DSC heating
runs. DSC and X-ray diffraction data indicate that
solidification of all colloidally dispersed lipids under
investigation results in crystalline material. The
corresponding 1 H-NMR spectrum of the D 114
dispersion is dominated by signals which can be
assigned to the stabilizers including glycerol (Fig. 3).
This is indicative of a highly restricted mobility of
the glyceride molecules typical of the solid state.
Obviously, cooling to refrigerator temperatures in-
duces solidification of the glycerides. Since the
formation of crystalline material upon cooling was
also observed in earlier studies for a series of melt-
emulsified monoacid triglycerides [16,17], the results
may be considered in more general terms indicating
that solidification of colloidally dispersed lipids
commonly results in crystalline material and that the
chance to obtain an amorphous but solid lipid matrix
via supercooling of colloidally dispersed lipid melts
seems low. The line broadening of the reference
signal (d50 ppm) in the NMR spectrum of the D
114 suspension (Fig. 3) may result from the surface
activity of DSS. Adsorption of DSS molecules on
solid particles might lead to a more restricted
molecular mobility than adsorption at the liquid-
liquid interface of emulsion droplets. The phenom-
enon is currently under investigation.
The crystallization temperature of H 35 nanoparti-
cles is so low that solidification does not always
occur, not even upon cold storage. Therefore, sys-
tematic DSC studies were performed to determine
the crystallization temperature of the supercooled
particles (Fig. 5). The crystallization temperature
decreases in the order E 85, H 42, D 114, H 35. This
is in accordance with the macroscopic observations
 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236 229

Fig. 3. 1 H-NMR spectra of a drug free trimyristin dispersion (stabilized with 2.4% S 100 and 0.6% sodium glycocholate) stored 1 day at
208C (left) or 58C (right).

on the gel formation tendencies of the various matrix nanosuspensions. Comparison between the melting
materials and explains the deviating stabilities on temperature of the cold stored nanoparticles and the
either room or refrigerator temperature of the differ- crystallization temperature (Fig. 5) reveals a pro-
ent compositions if stabilized with phospholipids nounced tendency towards supercooling. The differ-
only. The stabilization requirements of emulsions of
supercooled melts are obviously more similar to
those of conventional o / w emulsions than to those of

Fig. 5. DSC melting and crystallization temperatures of nanoparti-

Fig. 4. Small (SAXS) and wide (WAXS) angle X-ray scattering cle dispersions stabilized with the phospholipid / tyloxapol blend.
curves of phospholipid / tyloxapol stabilized dispersions cold The melting temperatures were obtained from samples cold stored
stored for about 1 month (3 months in case of H 35). The curves for at least several days (H 35 nanoparticles had been previously
have been displaced vertically for better visualization. crystallized at 08C).
230 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236

ence between melting and crystallization temperature

is, however, distinctly smaller for dispersed hard fats
than for trimyristin or other monoacid triglycerides
[16,17]. This phenomenon may be attributed to the
fact that longer chain components in the complex
hard fat mixtures may serve as nucleation centers.
Hard fat bulk materials display two DSC ex-
otherms upon cooling of the melts whereas hard fat
nanoparticles crystallize in a single thermal event at
lower temperatures than the bulk (Fig. 6). The
processes during crystallization of the particles must
thus be different from those in bulk material. X-ray
crystallization studies on E 85 indicate that the
polymorphic transition into the more stable b9-poly-
morph occurs faster in the bulk than in nanoparticles
(Fig. 7). This phenomenon may among others be
attributed to the higher crystallization temperature of
the bulk material.
The course of polymorphic transitions is distinctly
different in hard fat nanoparticles than in trimyristin.
In contrast to the trimyristin dispersion freshly
crystallized hard fat particles tend to display several
diffuse melting maxima in the DSC (Fig. 8). The
melting endotherm with the highest melting point is
the one that remains after storage. This behavior
points to slow polymorphic transitions within the
particles. Time-resolved X-ray crystallization studies

Fig. 7. X-ray crystallization (cooling rate 0.31258C / min) studies

Fig. 6. DSC cooling curves of Witepsol E 85 melts in the bulk and of Witepsol E 85 in the bulk (top), in colloidal dispersion (middle)
in colloidal dispersion (stabilized with the phospholipid / tyloxapol and colloidal D 114 (bottom). The colloidal dispersions were
blend). The curves have been displaced vertically for better stabilized with the phospholipid / tyloxapol blend. The curves have
visualization. been displaced vertically for better visualization.
 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
Fig. 8. DSC heating curves of freshly crystallized dispersions
stabilized with the phospholipid / tyloxapol blend. The curves have
been displaced vertically for better visualization.
confirm that the transition rates of hard fat nanoparti-
cles are lower than those of trimyristin dispersions
(Fig. 7). These differences may be due to the more
complex structure of the mixed fats impeding order-
ing of the molecules.
Compared to their bulk material, however, the
hard fat nanoparticles seem to transform more rapid-
ly into the stable b-polymorph. While the thermally
unstressed hard fat raw materials under investigation
are in the metastable b9-form the solidified dispersed
materials display wide angle X-ray reflection patterns
similar to those of the stable b-form of monoacid
triglycerides after one or three months of storage
(Fig. 4). The faster polymorphic transitions in lipid
nanoparticles have been attributed to the small size
of the crystallites [20]. Compared to those of the
trimyristin dispersion the X-ray reflections of the
hard fat dispersions are less intense, especially for H
35 which may be due to lattice imperfections or
incomplete crystallization of the dispersed hard fats.
Due to the different polymorphic forms of the hard
fat nanoparticles and their bulk material it is not
possible to quantify the crystallinity of these
nanoparticles by comparison of their melting en-
thalpy with that of the bulk (DSC recrystallization
index according to [20]) since different transition
enthalpies are expected for the different polymorphs.
Interpretation of DSC data for colloidally dispersed
complex glyceride mixtures without further infor-
231
mation on the structure of the nanoparticles, e.g.
from X-ray diffraction, is thus questionable.
For hard fat suppository bases in the bulk, an
intermediate b-form has been described as the
‘stable’ polymorph [21]. The wide angle reflections
of this intermediate b-form were also found in an
earlier study on dispersions of the hard fat Witepsol
W 35 [19]. In contrast, none of the hard fat
nanoparticle dispersions in the present study display
the strong reflection at 0.42 nm which is characteris-
tic of this intermediate b-form. Instead, the reflec-
tions arising from colloidally dispersed E 85, H 35
and H 42 resemble those of the regular b-form of
triglycerides. The comparison of the behavior of
Witepsol W 35 with that of the three hard fats under
investigation in the present study indicates that even
within the group of hard fats there is no consistent
behavior. Nevertheless, certain conclusions can be
drawn taking the chemical composition of the four
complex lipids into account. The three hard fats of
the present study contain only comparatively small
amounts of partial glycerides and differ mainly with
respect to the fatty acid pattern. In contrast, Witepsol
W 35 (hydroxyl value between 40 and 50) is a
mixture of mono-, di- and triglycerides therefore
differing also significantly in physicochemical prop-
erties from the other three.
The X-ray long-spacings of hard fat nanoparticles
are larger than those of the corresponding bulk
materials although they are due to a more stable
polymorph (Fig. 9). This may result from lattice
Fig. 9. X-ray long-spacings of the bulk materials compared to
those of the phospholipid / tyloxapol stabilized nanosuspensions
cold stored for at least 1 month.
232 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
imperfections in the region of the methyl end group
planes or to a different chain tilt in bulk material and
nanoparticles. Defects in the crystal lattice may also
contribute to the depression of the DSC melting
temperature in these hard fat nanoparticles (b-modi-
fication) compared to their bulk material (b9-modi-
fication) which is much larger in hard fats than in
nanoparticles from simple triglycerides, e.g. trimyris-
tin (Fig. 10). The small size (below 100 nm) of the
phospholipid / tyloxapol stabilized hard fat nanoparti-
cles may be an additional reason for their low
melting temperature.
The comparison of the physicochemical properties
of the bulk and the colloidally dispersed lipids
indicate that fundamental differences exist which
make it impossible to derive the properties of
dispersions from those of the bulk material in a
simple way. Colloidal dispersions of supercooled
melts are not only formed by monoacid triglycerides
such as trimyristin or trilaurin [16,17] but also by
complex lipid mixtures and represent an unexpected
novel type of colloidal lipid drug carrier systems.
The crystallization and melting behavior of solid
nanoparticles prepared from hard fat suppository
masses is, however, different from those of dispersed
trimyristin resulting among others in a different time
course of drug expulsion (Table 2). Supercooling
effects are less pronounced in hard fat dispersions
Fig. 10. DSC melting temperatures of the lipid in the bulk and in
colloidal dispersions (stabilized with the phospolipid / tyloxapol
blend) cold stored for at least several days (H 35 nanoparticles
had been previously crystallized at 08C).
than in dispersed monoacid triglycerides but colloi-
dal dispersions of supercooled hard fats still display
a considerable storage stability.
The cold stored solidified hard fat nanoparticles
investigated here transform into a more stable poly-
morph than their bulk material within a comparative-
ly short time of storage. Nevertheless, their structure
appears to be less ordered than in bulk material
which should be favorable with respect to the drug
incorporation capacity. All colloidal hard fat disper-
sions have, however, to be considered as highly
dynamic systems which complicates an early evalua-
tion of the final drug loading capacities. The chances
for efficient incorporation of foreign compounds is
known to decrease with increasing crystal lattice
order. Annealing of the crystal lattice and poly-
morphic transitions can, therefore, result in sustained
expulsion of the drug upon storage as demonstrated
for menadione (Table 2). Such retarded destabilizing
processes can almost be excluded for the rapidly
transforming monoacid triglyceride trimyristin
(Table 2) and are typically not observed for tradi-
tional emulsions. Nanoemulsions of supercooled
melts are, therefore, less critical in this respect than
nanosuspensions.
The drug loading characteristics observed for the
model drugs may be explained on the basis of the
physicochemical data for the colloidally dispersed
lipids. Several of the successfully drug-loaded dis-
persions exhibit a poor storage stability. Colloidal D
114 particles exhibit a higher loading capacity for
menadione if kept at room temperature i.e. in the
liquid state of a supercooled melt. Solidification of D
114 results in crystallization and drug expulsion
indicating that the drug is mainly located in the lipid
phase of the emulsions. The time course of phase
separation from a solid lipid carrier is, however,
dependent on the composition of the matrix material
since drug expulsion occurrs after different time
intervals when different types of matrices (D 114, E
85, H 42) are used (Table 2). This behavior is related
to the different time course of polymorphic transi-
tions of the matrix lipids. Crystallization in the a-
modification and slow polymorphic transitions result
in a delayed expulsion of drug from the hard fat
nanoparticles. Another reason may be different an-
nealing properties of the lipids.
High resolution NMR spectroscopy was applied as
 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
a non-destructive method to the native drug-loaded
systems since neither X-ray diffraction nor DSC
experiments allow a localization of the drug in
colloidal dispersions among others due to detection
limits (see above). The 1 H-NMR spectra on the
colloidal drug free D 114 dispersions illustrate that
significant changes in molecular mobility due to
phase transitions can be detected (Fig. 3). The 1 H-
NMR spectra of colloidal D 114 dispersions loaded
with menadione (1 wt% related to the dispersed
phase), diazepam (3 wt%), and ubidecarenone (5, 10
or 50 wt%) were also recorded. These nanodisper-
sions were studied by NMR in the state of a
supercooled melt and after solidification of the lipid.
The detection of drug signals in the spectra is, of
course, easier for high drug loads and for drugs
exhibiting signals clearly separated from those of the
carrier dispersion. The drugs have a comparatively
high molecular mobility in the emulsions of super-
cooled D 114 as demonstrated for diazepam (Fig.
11). The considerable line widths of the drug signals
indicate, however, that the signals result from mole-
cules which are not dissolved in the aqueous phase
but in a viscous phase, i.e. mainly in the dispersed
lipid phase. This is especially interesting in the case
of diazepam which has a solubility in aqueous media
around 0.3 wt% [22], i.e. in the concentration range
of the loading experiment. Diazepam obviously
Fig. 11. 1 H-NMR spectra of a trimyristin emulsion (left) and suspension (right) containing 3% diazepam related to the dispersed phase. The
dispersions were stabilized with 2.4% S 100 and 0.6% sodium glycocholate. The arrows indicate the position of the drug signals.
233
distributes preferentially towards the liquid lipid
phase in this two-phase system. The high molecular
mobility observed for all drugs under investigation
within the liquid lipid phase indicates that such an
emulsion of a supercooled melt cannot be used to
obtain a sustained release of drugs over days or
weeks under sink conditions.
Solidification of D 114 not only results in a loss of
the strong triglyceride signals but also in a consider-
able broadening or loss of the drug signals as
demonstrated for diazepam (Fig. 11). The mobility
of the drug molecules in the nanosuspensions is
drastically reduced indicating that these drugs are
associated with D 114 since no other structure in the
systems is observed to transfer irreversibly into a
much more rigid state on cooling to refrigerator
temperature despite final storage of the dispersion at
room temperature. Even the fairly water soluble
diazepam is not expelled from the recrystallizing
lipid matrix into the water phase. The NMR data
indicate that such a glyceride suspension may serve
as a real sustained release system and that the model
drugs may be released more slowly from the D 114
nanosuspensions than from the D 114 nanoemulsions
of supercooled melts.
In contrast to all other poorly water soluble drug
substances tested so far, ubidecarenone is the only
one that can be added to the basic composition even
234 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
in high concentrations without any destabilization of
the system. Its specific feature is reflected in the
NMR spectra of D 114 nanodispersions loaded with
5, 10 or even 50 wt% ubidecarenone (Fig. 12). The
shape of the ubidecarenone signals in the spectra of
the nanoemulsions indicates a similarly restricted
drug mobility as in the other drug loaded D 114
nanoemulsions suggesting that no significant
amounts of the drug are dissolved in the continuous
phase. The spectra of the nanosuspensions containing
the high drug loads of either 10 or 50% still contain
signals of ubidecarenone. The intensity of the signals
is, however, reduced in comparison with the corre-
sponding drug signals in the emulsions suggesting
Fig. 12. 1 H-NMR spectra of trimyristin dispersions stored at
20–258C (left column) and 4–88C (right column) containing 5%
(top), 10% (middle) and 50% (bottom) ubidecarenone related to
the dispersed phase. All dispersions were stabilized with 2.4% S
100 and 0.6% sodium glycocholate.
that a fraction of the drug is strongly bound to the
solidified triglyceride and that ubidecarenone is
distributed between two different locations in the
colloidal system. The intensity of the remaining drug
signals in the ubidecarenone loaded nanosuspensions
increases with the drug load of the system. The line
widths of the signals indicates that the drug mole-
cules still have a mobility similar to that in the
nanoemulsion. In contrast to the dispersions loaded
with 5 and 10% ubidecarenone, signals of liquid
triglyceride are detected in the spectrum of the cold
stored dispersion containing 50% ubidecarenone in
the lipid phase suggesting that not all triglyceride has
crystallized in this dispersion due to eutectic be-
havior with ubidecarenone. This assumption is sup-
ported by DSC measurements indicating that drugs
may alter the physicochemical properties of lipid
nanoparticles dramatically.
5. Conclusions
Important physicochemical properties of lipids in
the bulk and in the colloidal state differ significantly.
Physicochemical characterization demonstrated that
colloidal drug carriers based on melt-emulsified solid
glycerides are complex systems due to their crys-
tallization behavior, the rate of polymorphic transi-
tions and the potential formation of supercooled
melts. The formation of emulsified supercooled melts
which are stable on storage depends on the con-
ditions chosen for the cooling step of the hot melt-in-
water nanoemulsions. Nanoemulsions of supercooled
melts which deviate substantially from solid lipid
nanoparticles in their properties represent a new
alternative drug carrier system.
The drug loading capacity of both types of colloi-
dal lipidic carriers is limited owing to the generally
low solubilization capacity of the molten lipids for
many poorly water-soluble drugs. The drug-loading
capacity of nanoparticles representing supercooled
melts was shown to be higher than that of crys-
tallized nanoparticles. Crystallization of the dis-
persed glycerides can lead to phase separation of
incorporated drugs. Neither amorphous solids nor
any sufficiently stabilized a -modification could be
obtained by variations of the lipid matrices. Complex
glyceride mixtures such as hard fats may, however,
 K. Westesen et al. / Journal of Controlled Release 48 (1997) 223 – 236
possess a higher drug-loading capacity in the crys-
talline state due to their lower crystallinity as com-
pared to pure monoacid triglycerides. Nanodisper-
sions of complex glycerides represent highly dy-
namic systems on storage which complicates an early
evaluation of the final drug loading capacity.
All poorly water-soluble drugs investigated in
NMR studies were distributed towards the lipid
phase in emulsions of supercooled melts. Drug
molecules incorporated in particles of supercooled
melts have a high mobility making these systems
unsuitable for sustained release over days, weeks or
months. Drug mobility is drastically reduced in
solidified (crystallized) nanoparticles. If crystalliza-
tion does not lead to drug expulsion from the crystal
lattice, these carriers may have a good potential as
sustained release systems.
Trimyristin nanoparticles provide an excellent
model system to study the effects of supercooling
and crystallization on the molecular mobility of
drugs incorporated in melt-emulsified glyceride
nanoparticles as well as on stability of these systems.
At a more general experimental level it was shown
that high resolution proton NMR is a powerful tool
to distinguish between amorphous solids and liquids
in dilute colloidal systems, to determine drug dis-
tribution within the systems and to study the mobility
of drug molecules incorporated in the lipid
nanoparticles.
Acknowledgments
¨ AG, D-Witten, and Lipoid
The authors thank Huls
KG, D-Ludwigshafen, for supply of raw materials
and Pharmacia Oncology Immunology AB, S-Hel-
singborg for financial support. We would like to
thank Dr. V. Wray, C. Kakoschke and B. Jaschok-
Kentner, National Research Center for Biotechnol-
¨
ogy, Braunschweig, Germany, and Dr. W. Gunther,
Institute for Organic Chemistry and Macromolecular
Chemistry, Jena, Germany, for support in the 1 H-
NMR experiments.
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