Journal of Microencapsulation

Micro and Nano Carriers

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SLN and NLC for topical delivery of ketoconazole

E. B. Souto & R. H. Müller

To cite this article: E. B. Souto & R. H. Müller (2005) SLN and NLC for topical delivery of
ketoconazole, Journal of Microencapsulation, 22:5, 501-510, DOI: 10.1080/02652040500162436

To link to this article: https://doi.org/10.1080/02652040500162436

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Journal of Microencapsulation, August 2005; 22(5): 501–510

SLN and NLC for topical delivery of ketoconazole

E. B. SOUTO1 & R. H. MÜLLER1,2

1
Department of Pharmaceutical Technology, Biotechnology & Quality Management, The Free
University of Berlin, Berlin, Germany, and 2PharmaSol GmbH, Berlin, Germany

(Received 6 September 2004; accepted 10 January 2005)

Abstract
The clinical use of ketoconazole has been related to some adverse effects in healthy adults, specially
local reactions, such as severe irritation, pruritus and stinging. The purpose of the present work is
the assessment of ketoconazole stability in aqueous SLN and NLC dispersions, as well as the phy-
sicochemical stability of these lipid nanoparticles, which might be useful for targeting this drug into
topical route, minimizing the adverse side effects and providing a controlled release. Lipid particles
were prepared using CompritolÕ 888 ATO as solid lipid. The natural antioxidant -tocopherol was
selected as liquid lipid compound for the preparation of NLC. Ketoconazole loading capacity was
identical for both SLN and NLC systems (5% of particle mass). SLN were physically stable as
suspensions during 3 months of storage, but the SLN matrix was not able to protect the chemically
labile ketoconazole against degradation under light exposure. In contrast, the NLC were able to
stabilize the drug, but the aqueous NLC dispersion showed size increase during storage. Potential topi-
cal formulations are light-protected packaged SLN or NLC physically stabilized in a gel formulation.

Keywords: Solid lipid nanoparticles, nanostructured lipid carriers, -tocopherol, ketoconazole

Introduction
During the last decade, considerable attention has been paid to the development of new
controlled delivery systems, in order to supply a long-term drug release and, therefore,
increase patient’s therapeutic compliance and acceptance. Solid lipid nanoparticles
(SLNTM) and nanostructured lipid carriers (NLCTM) are interesting systems for the
present purpose due to their solid matrix which might avoid the burst release obtained in
conventional topical formulations (Müller et al. 2002a, b). These lipid nanoparticles differ
from each other because in NLC a controlled nanostructuring of the lipid matrix is
performed due to the mixture of solid and liquid lipids, in order to increase drug-loading
and prevent its expulsion. In addition, the nanostructured lipid matrix gives more flexibility
in modulation of drug release (Müller et al. 2002a, b).

Correspondence: R. H. Müller, Department of Pharmaceutical Technology, Biotechnology & Quality Management,

The Free University of Berlin, Kelchstr. 31, D-12169 Berlin, Germany. Tel: 49 30 83850678. Fax: 49 30 83850616.
E-mail: mpharma@zedat.fu-berlin.de
ISSN 0265-2048 print/ISSN 1464-5246 online # 2005 Taylor & Francis
DOI: 10.1080/02652040500162436
502 E. B. Souto and R. H. Müller

The literature describes several methods for production of SLN and NLC. The
microemulsion technique was first described and patented by Gasco (1993). Sjöström
and Bergenståhl (1992) described a production method for preparing SLN dispersions
by solvent evaporation in o/w emulsions. Methods used for production of polymeric
nanoparticles made from pre-formed polymers have also been tested for preparation
of SLN: (i) the solvent displacement technique (Dubes et al. 2003a, b; Schubert
and Müller-Goymann 2003), described and patented by Fessi et al. (1992), and (ii) the
emulsification–diffusion technique (Hu et al. 2002; Shahgaldian et al. 2003; Trotta et al.
2003), described and patented by Quintanar-Gerrero et al. (1999).
However, high pressure homogenization (HPH) technique is still the main procedure
adopted for production of lipid nanoparticles, which can be performed either at hot or
cold temperature (Müller et al. 1995, 2000). A current limitation of this technique is the
low loading capacity obtained for hydrophilic drugs (Almeida et al. 1997). Additionally,
in the case of highly temperature sensitive molecules, a second drawback can potentially
arise during the nanoparticles manufacturing process because of the temperatures required
to melt the lipid phase. The aim of the present study was the development of SLN and NLC
for topical delivery of ketoconazole using the hot HPH technique.
Ketoconazole (cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-
1,3-dioxalan-4-yl]methoxy]phenyl] piperazine) is an imidazole anti-fungal agent, which is
clinically administered both in orally and topical formulations. Due to its high permeability
but low aqueous solubility, this drug is classified as a Class II active substance, since its
dissolution properties in gastro-intestinal tract is insufficient under normal conditions
(Van der Mooter et al. 2001). Concerning topical formulations, this drug is the active
ingredient of NizoralTM cream, as well as of an anti-dandruff shampoo (Nguyet et al.
2003). It is a weak base with high lipo-solubility, used for the treatment of human mycotic
infections (Karasulu et al. 2004).
This paper reports the development of new systems based on physiologic lipids for
ketoconazole targeting in topical route. For the production of ketoconazole-loaded lipid
nanoparticles, CompritolÕ 888 ATO (glyceryl behenate) has been selected as the solid
lipid. The lipid-soluble compound -tocopherol has been chosen as liquid lipid for
preparation of NLC, due to its increasing use for protection of substances which are
sensitive to oxidation, its miscibility with glyceryl behenate at high temperatures and also
due to the high solubility of ketoconazole in the obtained mixture. This natural anti-oxidant
is able to protect from auto-oxidation lipids present in the lipid phase of foods and in
the membrane of living cells (Machlin 1980, Elmadfa and Bosse 1985).

Materials
CompritolÕ 888 ATO (Gattefossé, Weil am Rhein, Germany) was the selected lipid for the
core formation. -Tocopherol was purchased from Sigma Aldrich (Deisenhofen, Germany).
Poloxamer 188 (BASF AG, Ludwigshafen, Germany) and sodium deoxycholate
(Fluka, Buchs, Switzerland) were used as surfactant and co-surfactant, respectively.
Microfine ketoconazole was a generous gift of Chemo Iberica S.A. (Madrid, Spain).

Methods
Preparation of lipid nanoparticles
The method chosen for preparation of SLN and NLC was the hot high pressure
homogenization technique described by Müller and Lucks (1996). Briefly, the lipid phase
 SLN and NLC for topical delivery of ketoconazole 503

was melted at 90
C and dispersed in a hot surfactant aqueous solution heated at the
same temperature, using an Ultra-Turrax T25 ( Janke & Kunkel GmbH and Co KG,
Staufen, Germany) at 8000 rpm for 1 min. The obtained pre-emulsion was homogenized
at 90
C using an APV Micron Lab 40 (APV Deutschland GmbH, Lübeck, Germany),
applying a pressure of 500 bar and three homogenization cycles. For the preparation
of ketoconazole-loaded lipid nanoparticles, 5% (m/m) of the lipid matrix was replaced with
drug. Ketoconazole was dissolved in the melted lipid phase prior to dispersion in the
surfactant solution.

Particle size and zeta potential analysis

The particle size of aqueous SLN and NLC dispersions was performed by photon
correlation spectroscopy (PCS) with a Zetasizer IV (Malvern Instruments, Malvern, UK)
and by laser diffractometry (LD), using a CoulterÕ LS 230 (Bechmann–Coulter, Krefeld,
Germany). PCS yields the mean particle size and the polydispersity index (PI) as a measure
of the width of the distribution. The LD data were evaluated using the diameter d 90%,
which means that 90% (volume distribution) of the measured particle is below the obtained
value. LD analysis was performed using 1.456 35 as the real refractive index and 0.01 as
the imaginary refractive index applying the Mie theory. The zeta potential was determined
by Laser Doppler Anemometry (Zetasizer IV, Malvern Instruments, UK) using the
Helmholtz–Smoluchowsky equation. Samples were diluted with bidistilled water adjusted
to a conductivity of 50 mS cm1 with a solution of 0.9% NaCl (if not otherwise stated).
The pH was in the range 5.5–6.0 and the field strength was 20 V cm1.

Thermal analysis
Differential scanning calorimetry. Differential scanning calorimetry (DSC) measurements
were performed on a Mettler DSC 821e (Mettler Toledo, Giessen, Germany), in order
to determine the degree of crystallinity of the lipid nanoparticles. Samples containing 15 mg
SLN or NLC aqueous dispersions, i.e. 1–3 mg solid lipid or 1–2 mg of bulk material,
were accurately weighed in 40 ml aluminium pans and cold sealed. The reference pan
remained empty and was sealed in the same manner. Heating curves were recorded with a
scan rate of 5 K min1 from 25–85
C and cooled from 85–25
C under liquid nitrogen. The
crystallization index was calculated using the following equation (Freitas and Müller 1999):

HSLN or NLC aqueous dispersion
CIð%Þ ¼  100

Hbulk material  Concentrationlipid phase
Polymorphic forms were assigned by correlation with X-ray data.

Thermal gravimetry analysis. Thermal gravimetry analysis (TGA) was carried out using
a Mettler TG-DTA analyser (Mettler Toledo, Giessen, Germany). Samples of 15 mg
were heated in a aluminium oxide crucible from 20–200
C at 10 K min1 and the loss of
weight was recorded.

X-ray diffraction
X-ray measurements were performed by wide-angle X-ray scattering (WAXS, 2  ¼ 4–40
)
on a Philips PW 1830 X-ray generator (Philips, Amedo, The Netherlands) with a
504 E. B. Souto and R. H. Müller

copper anode (Cu-K radiation,  ¼ 0.154 18 nm). Measurements were performed with an
anode voltage of 40 kV, a current of 25 mA and a scan rate of 0.5
per minute. Data of the
scattered radiation were detected with a blend local-sensible detector. Aqueous dispersions
were transformed into a paste using locust bean gum as a thickening agent. Briefly, a small
amount of gum was primarily mixed to 1 ml of the aqueous dispersion obtaining a paste
which was placed into a thin X-ray glass fibre and then transferred to the camera for analysis.
This procedure induces no changes in the recorded diffraction patterns as ensured
by comparison with X-ray curves of liquid preparations.

Results and discussion

Characterization of the developed formulations
Table I shows the composition of the developed SLN and NLC formulations. Preliminary
experiments of the present work showed that lipid nanoparticles containing ketoconazole
were sensitive to light exposure and high temperatures of storage. Only z-average (z-ave)
values, polydispersity indices (PI) and zeta potential () values of samples stored at room
temperature (i.e. 20
C) and at 4
C are, therefore, reported here. Measurements of particle
sizes right after production revealed little difference between the developed formulations;
however, their long-term stabilities were quite different. After 90 days of storage,
ketoconazole-loaded SLN formulation yielded approximately a mean particle size between
210–260 nm (Table II). It was possible to detect a pronounced increase in z-ave and
PI of NLC formulations. Ketoconazole-free SLN stored at 20
C revealed a mean particle
size higher than 3 mm. The  values of cold stored CompritolÕ 888 ATO nanoparticles were
lower than of those stored at RT. The ketoconazole-loaded NLC formulation stored
at RT under light exposure showed a positive  value in contrast to the sample protected
from light.
PCS is limited to detection of particles undergoing Brownian motion and particles
larger than 3 mm are, therefore, not detectable (Müller and Schuhmann 1996).
However, the presence of microparticles and/or liquid lipid droplets could be determined
by LD measurements. Figure 1 shows the volume distribution of ketoconazole-loaded
SLN and NLC formulations obtained by LD analysis after 90 days of storage, using
PIDS and Mie calculation.
Drug-loaded SLN formulations stored at both temperatures were fitted to a perfect
log-normal distribution by cumulative analysis. In contrast to SLN stored at 4
C, the
storage at 20
C slightly increased the particle size; however, protection against light
exposure could avoid particle growth (data not shown). Concerning NLC formulation
stored at 4
C, as shown in Figure 1, the size distribution shifted to bigger particles showing
a broadened distribution pattern. In comparison to SLN, the observed destabilization

Table I. Composition of the developed SLN and NLC formulations % (m/m).

Sodium
System Compritol Tocopherol Ketoconazole Poloxamer deoxycholate Water ad

Ket-free SLN 15.00 – – 2.50 0.125 100

Ket-free NLC 10.50 4.50 – 2.50 0.125 100
Ket-loaded SLN 14.25 – 0.75 2.50 0.125 100
Ket-loaded NLC 10.125 4.125 0.75 2.50 0.125 100
 SLN and NLC for topical delivery of ketoconazole 505

8 Ket-loaded SLN (4°C)

7
6 Ket-loaded SLN (20°C/dark)
Volume (%)

Ket-loaded NLC (4°C)

5
4
3
2
1
0
0.04 0.6 1 2 60 400 1000
Particle diameter (µm)

Figure 1. LD curves of ketoconazole-loaded SLN at 4
C and at 20
C/dark and of ketoconazole-

loaded NLC formulation at 4
C, recorded after 90 days of storage.

Table II. Mean particle size (z-ave), polydispersity index (PI) and zeta potential () values of
developed SLN and NLC formulations recorded after 90 days of storage at 4
C and at room
temperature (20
C) (RT, storage at room temperature under daylight exposure).

Storage
System conditions z-avea (nm) PIa b (mv)

Ket-free SLN 4
C 211.6  4.3 0.159  0.08 21.6  0.5

RT >3 mm 1.0  0.0 10.7  1.6
Ket-free NLC 4
C >3 mm 1.0  0.0 17.4  0.8
RT 244.9  11.6 0.282  0.05 14.4  0.8
Ket-loaded SLN 4
C 212.3  7.4 0.157  0.06 24.0  1.7
RT 214.1  7.2 0.241  0.05 18.3  0.5
RT/dark 261.9  9.3 0.278  0.05 17.6  0.1
Ket-loaded NLC 4
C >3 mm 1.0  0.0 22.3  0.7
RT >3 mm 1.0  0.0 2.8  1.5
RT/dark >3 mm 1.0  0.0 5.9  0.3
a
Standard deviation of n ¼ 10 measurements; b Standard deviation of n ¼ 3 measurements.

of NLC seems to result from the presence of a liquid component inside the lipid matrix of
this carrier, i.e. -tocopherol, which might cause aggregation of the particles. Furthermore,
in contrast to ketoconazole-loaded NLC under light exposure, drug-loaded SLN showed a
purple colour. This result shows the effect of the anti-oxidant -tocopherol used as matrix
material of glyceryl behenate-based NLC. As shown in Table II, the  of
ketoconazole-loaded SLN stored at 20
C in light and dark was close to zero. This lack
of electrostatic repulsion can explain the aggregation of these samples. However, the  of
ketoconazole-loaded SLN stored at 4
C is 20 mV, i.e. similar to the values of the various
SLN which proved to be physically stable. This clearly indicates the effect of the lipid matrix
material, i.e. the admixture of -tocopherol. Particle aggregation and even gel formation
have been described by lecithin-mediated effects (Westesen and Siekmann 1997), a similar
effect is assumed in this case. It should be pointed out that the final formulation
would be a cream loaded with SLN/NLC or a viscosity enhanced particle dispersion
(gel). In these systems, even physically unstable, SLN and NLC show a high stability
506 E. B. Souto and R. H. Müller

(Souto et al. 2004). From this, the size result with NLC does not make them necessarily
unstable for a topical ketoconazole preparation.

Thermal analysis
Table III shows the melting and crystallization parameters of CompritolÕ 888 ATO based
SLN and NLC obtained by DSC analysis after 90 days of storage at 4
C and 20
C.
The difference between the registered onset and melting point values can be taken as
a measure for the width of melting peaks of each formulation. As expected, the crystallinity
index (CI) values obtained for NLC formulations were lower than SLN formulations with
the same lipid content. The presence of drug decreased the crystallinity of the lipid
nanoparticles. The effect of temperature of storage was more pronounced for drug-free
samples.
DSC allows determination of thermotropic phase transitions in a quantitative manner
and is especially useful for the investigation of the complex behaviour of polymorphic triacyl-
glycerols (Westesen and Siekmann 1997). The thermal curves of lipid nanoparticles
recorded during DSC analysis display pronounced melting peaks (Figure 2). With regard
to ketoconazole-loaded SLN a peak shoulder at higher temperatures is obtained. In general,
crystal ordering and density of NLC decreases in comparison to the recorded DSC
run for SLN with the same lipid content. This is in agreement with the theory that NLC
are characterized by a less ordered, less crystalline structure as their special feature
(Müller et al. 1998).
As shown in Figure 2, ketoconazole-loaded SLN yields a narrower peak than
ketoconazole-loaded NLC, which shows a very broad transition stretching. As the fatty
acid profiles of both are comparable, this difference must be a result of the higher content
of minor components present in the NLC formulation. The difference in the DSC thermo-
grams observed here was attributed to the presence of -tocopherol in NLC formulation,
which may well account for the measurable lowering of the transition enthalpy. This oil
distorts the crystal formation, as intended in the NLC concept. No peaks arising from
ketoconazole could be observed, revealing that the drug is soluble in the lipid matrix of
both formulations.
Besides characterization of particle size distribution and crystallinity of lipid nanoparticles,
a complementary observation of the chemical stability of ketoconazole at high temperatures

Table III. DSC results of ketoconazole-free and ketoconazole-loaded SLN and NLC formulations, recorded after
90 days of storage at 4
C and 20
C.

Storage Melting
System conditions point (
C) Onset (
C) Integral (mJ ) Enthalpy ( J/g) CI (%)

Ket-free SLN 4
C 70.65 68.45 455.27 17.75 85.6

RT 71.17 71.11 339.91 12.97 62.5
Ket-free NLC 4
C 67.05 55.44 307.86 12.35 59.5
RT 65.39 38.29 342.33 16.86 81.3
Ket-loaded SLN 4
C 70.52 67.96 614.31 15.69 75.6
RT 70.64 68.06 424.97 16.21 78.1
RT/dark 71.04 68.63 510.68 16.23 78.2
Ket-loaded NLC 4
C 69.11 62.77 189.06 12.59 60.7
RT 66.99 56.36 356.86 14.02 67.6
RT/dark 66.13 55.37 308.67 10.49 50.6
 SLN and NLC for topical delivery of ketoconazole 507

0.6 Ket-loaded
Ket-loaded SLN
Enthalpy (W/g) 0.5 NLC

0.4

0.3

0.2

0.1

0
25
28
30
33
35
38
41
43
46
48
51
53
56
59
61
64
66
69
72
74
77
79
82
84
°C
Figure 2. DSC curves of ketoconazole-loaded SLN and NLC formulations obtained after 90 days
of storage at 4
C.

100
99
98
97
96
%m

95
94
93
92
91
90
100
115
130
145
160
175
190
195
180
165
150
135
120
105
25
40
55
70
85

90
75
60
45
30
°C
Figure 3. Thermal gravimetry analysis of microfine ketoconazole powder.

has been obtained by thermal gravimetry analysis. As Figure 3 reveals, at temperatures above
the melting point of ketoconazole (m.p. 146
C) the pure drug is melted, no mass depending
decomposition occurs in the temperature range of 25–200
C, at a heating rate of 10 K min1.
This result supports that ketoconazole should be chemically stable under the production
conditions applied for preparation of these lipid carriers by HPH technique. Chemical
degradation of ketoconazole is easy to detect by the change in colour to purple. In general,
the human eye is highly sensitive to even slight colour changes. Therefore, for this first basic
study, to identify the most suitable formulation principle (SLN vs. NLC), this criterion
was sufficient. Directly after production, all aqueous SLN and NLC dispersions remained
unchanged white. In the case of homogenized dispersions being unstable, colour changes
are detectable (e.g. omeprazole nanosuspensions (Möschwitzer et al. 2004)). From this,
the result of white lipid suspensions is in agreement with the TGA data. During storage,
light exposed ketoconazole-loaded SLN turned purple, indicating clearly the less suitability
of this carrier system for ketoconazole (of course, the alternative would be light-excluding
packaging!).
508 E. B. Souto and R. H. Müller

Ket-loaded NLC (20°C)

450
400 d = 0.46 nm
350
I/counts
300
250
200
150
Ket-loaded SLN (20°C)
100
50
0
1
2
4
6
7
9
11
13
14
16
18
20
21
23
25
26
28
30
32
33
35
37
38
40
2 theta

Figure 4. X-ray patterns of ketoconazole-loaded SLN and NLC formulations obtained after 90 days
of storage at 20
C.

X-ray diffraction
X-ray diffraction investigations have been most valuable in the elucidation of the manner of
arrangement of lipid molecules, their multiple-melting phenomena, phase behaviour and the
characterization and identification of the structure of lipid and drug molecules (Bunjes et al.
2000). The bulk material, as well as drug-loaded and drug-free SLN prepared by HPH,
revealed diffraction patterns very similar, showing the typical signals of the triacylglycerols
(data not shown) (Thoma et al. 1983; Garti 1988), whereas NLC formulations showed
a small third peak at 0.46 nm, indicating the partial formation of an intermediate i form
(Precht 1988) (Figure 4).
Lower transition temperatures observed by DSC for NLC formulations were confirmed
by X-ray diffraction analysis. In fact, the broader peaks obtained for this formulation
result from less crystal order and increased number of crystal defects.
With regard to the ketoconazole stability assessed by X-ray diffraction analysis, the WAXS
patterns of this drug have been recorded before and after its tempering for 1 h at 90
C. This
is the temperature selected for production of CompritolÕ 888 ATO based SLN and NLC
formulations. No decomposition took place when tempering the drug at 90
C, even when
exposing the drug to this temperature for 1 h, which is more than 10-times longer than
heat exposure during production (data not shown). Both TGA and X-ray diffraction analysis
reveal the high stability of this drug to be incorporated into SLN and NLC.

Conclusions
In conclusion, CompritolÕ 888 ATO based SLN formulations can be characterized by
relatively narrow particle size distribution (PI) and physical long-term stability, also when
ketoconazole is incorporated into these nanoparticles. Glyceride SLN and NLC are both
suitable for the incorporation of ketoconazole. However, in light exposed SLN the
ketoconazole shows chemical instability, a problem which can be solved by adequate
packaging. In contrast to this, ketoconazole has a higher chemical stability in NLC, but the
CompritolÕ 888 ATO based formulations showed a significant increase in particle size
during storage time. Obviously this is due to the presence of -tocopherol, which decreases
the crystal ordering of the lipid nanoparticles but increases the chemical stability of the drug
under light exposure. This physical instability of NLC should not occur anymore when they
 SLN and NLC for topical delivery of ketoconazole 509

are formulated, e.g. in gels, as discussed above. To summarize, in principle both types
of lipid particles appear suitable for a topical formulation, whereas the SLN require special
packaging and the NLC physical stabilization by a gel network. The final device will,
therefore, depend on the in vivo treatment efficiency.

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