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Journal of Microencapsulation, August 2007; 24(5): 457475

Testosterone solid lipid microparticles for transdermal drug


delivery. Formulation and physicochemical characterization

AMAL H. EL-KAMEL, IMAN M. AL-FAGIH, & IBRAHIM A. ALSARRA

Faculty of Pharmacy, Department of Pharmaceutics, King Saud University, Riyadh, Saudi Arabia

(Received 31 October 2006; revised 27 March 2007; accepted 27 March 2007)

Abstract
Purpose: The main objective of the study was to formulate and characterize testosterone (TS) solid lipid
microparticles (SLM) to be applied as a transdermal delivery system.
Methods: Testosterone SLMs were formulated using an emulsion melt homogenization method.
Various types and concentrations of fatty materials, namely glyceryl monostearate (GM), glyceryl
distearate (GD), stearic acid (SA) and glyceryl behanate (GB) were used. The formulations contained
2.5 or 5 mg TS g1. Morphology, particle size, entrapment efficiency (EE), rheological properties and
thermal behaviour of the prepared SLM were examined. In vitro release characteristics of TS from
various prepared SLM were also evaluated over 24 h using a vertical Franz diffusion cell. In addition,
the effect of storage and freeze-drying on particle size and release pattern of TS from the selected
formulation was evaluated.
Results: The results indicated that the type of lipid affected the morphology and particle size of SLM. A
relatively high drug percentage entrapment efficiency ranging from 80.795.7% was obtained.
Rheological studies showed plastic flow characteristics of the prepared formulations. DSC examination
revealed that TS existed in amorphous form in the prepared SLM. Release studies revealed the
following rank order of TS permeation through cellophane membrane after application of various
formulations: 5% GM < 5% GD < 5% SA < 5% GB < 2.5% GM < 2.5% SA < 10% GD < 10% GB.
The drug permeation through excised abdomen rat skin after application of 10% GB2.5 mg TS g1
SLM was lower than that permeated through cellophane membrane. Moreover, SLM containing 10%
GB2.5 mg TS g1 stored at 5 C showed good stability as indicated by the release study and particle
size analysis. Trehalose showed high potential as a cryoprotectant during freeze drying of the selected
SLM formulation.
Conclusions: The developed TS SLM delivery system seemed to be promising as a TS transdermal
delivery system.

Keywords: Testosterone, solid lipid microparticles, release, stability, freeze-drying

Correspondence: Amal H. El-Kamel, Associate Professor of Pharmaceutics, Department of Pharmaceutics, Faculty of Pharmacy,
King Saud University, PO Box 2452, Riyadh 11495, Saudi Arabia. E-mail: amalelkamel@yahoo.com
ISSN 02652048 print/ISSN 14645246 online 2007 Informa UK Ltd.
DOI: 10.1080/02652040701368865
458 A. H. El-Kamel et al.

Introduction
Solid lipid microparticles (SLMs) attract increasing attention as alternative delivery systems.
SLMs combine the advantages of different traditional carriers; for example, they can be
produced on a large industrial scale and they are toxicologically highly acceptable and allow
the control of drug release (Jaspart et al. 2005). SLMs appear promising as drug carrier
systems for topical application (Gavini et al. 2005). Occlusion properties as a result of film
formation on the skin which can enhance the penetration of drugs through the stratum
corneum have been reported for SLM (Jenning et al. 2000).
Testosterone (TS) is an anabolic/androgenic hormone. A variety of conditions has now
been identified in which TS production is diminished and replacement therapy may be
beneficial (Mazer 2000, Somboonporn 2006). Various dosage forms have been developed
and utilized clinically, including oral formulations of alkylated esterified TS, intra-mascular
TS esters and a subcutaneous implant of TS pellets (Cantrill et al. 1984). These delivery
systems for TS are problematic, uncomfortable to administer, produce wide fluctuations in
testosterone levels and hepatotoxic.
Currently, testosterone transdermal systems are marketed internationally. Clinical studies
of transdermal systems demonstrated their efficacy in providing adequate testosterone
replacement therapy. However, skin irritation is usually associated with the use of
transdermal patches. Consequently, it is important to develop transdermal systems with
minimal adverse effects on the skin.
Recent approaches were reported to deliver TS by transdermal routes such as gels (Alberti
et al. 2005), spray (Leichtnam et al. 2006), ethosoms (Ainbinder and Touitou 2005), a
reservoir-type transdermal delivery system (Kim et al. 2001) and patches (Gooren and
Bunck 2003). However, no study has reported the development of testosterone encapsulated
in SLM for transdermal delivery.
The aim of the study was to formulate and characterize TS SLM to be applied as a
transdermal delivery system. Two classes of lipids were used, namely glycerides and fatty
acids. Shape, particle size, viscosity, thermal behaviour, drug entrapment efficacy and
release characteristics of the prepared SLM were evaluated. The effect of storage and freeze-
drying on the selected formulation was also investigated.

Materials and methods


Materials
Testosterone (Fluka Chemie GmbH), poloxamer 188 (Pluronic F-68) (BASF, Germany),
glycerol monostearate (GM), trehalose, chloroform, acetonitrile and ethanol HPLC grade
(BDH, Poole, UK), stearic acid (SA) and propylene glycol (Winlab, UK), compritol 888
ATO (glycerol dibehenate, GB) and precirol ATO 5 (glycerol distearate, GD) (Gattefosse,
Lyon, France).

Methods
High performance liquid chromatography assay of testosterone. Testosterone was assayed by an
HPLC method proposed by Morgan et al. (1998). The HPLC system consisted of a delivery
pump (LC-10 Schimadzu, Japan) and UV detector (SPD-10 Schimadzu, Japan).
Testosterone was detected at 241 nm. The data were recorded using Shimadzu VP
chromatography software (version: 6.12 SP5, Shimadzu Corporation). The mobile
phase was 55% acetonitrile in water eluted at a flow rate 1 ml min1. The injection
Formulation and physicochemical characterization of TS SLM 459

volume was 20 ml. The retention time of the drug was 5.2  0.5 min. The column used was
reversed phase C18 m-Shimpack TM (150  4.6 mm) operated at ambient temperature. The
calibration curve was constructed to cover a concentration range of 0.530 mg ml1. The
minimum detected concentration was 0.07 mg ml1.

Preparation of testosterone solid lipid microparticles. Solid lipid microparticles dispersion was
prepared by a hot homogenization technique on a weight basis (Cortesi et al. 2002). In the
hot homogenization technique, a lipid matrix content of 2.5% or 5% w/w GM or 5% or 10%
w/w GD or 5% or 10% w/w GB or 2.5% or 5% w/w SA (based on the total weight of SLM
dispersion) was melted at 10 C above its melting point. Testosterone 2.5% or 5% w/w
calculated as a percentage of lipid matrix (could be also expressed as 2.5 or 5 mg TS g1
SLM dispersion), e.g. for 100 g of a 10% w/w GB SLM dispersion loaded with 5% w/w drug
contained 10 g solid consisting of 9.5 g GB and 0.5 g drug) was added to the melted lipid.
The dispersion was kept at the same temperature until it appeared optically clear. A chemical
enhancer (1% w/w OA or DA based on the total weight of SLM dispersion) was dissolved in
the melted lipid when required. Poloxamer 188 (2.5% w/w used as stabilizer) was dissolved
in distilled water and heated to the same temperature as the lipid mixture. Hot poloxamer
solution was added to the melted lipiddrug mixture and emulsified by an Ultra-Turrax
Ika T18 basic at 8000 rpm for 1 min. The formulation was then removed from the water
bath and mixed until cool.

Freeze-drying of selected SLM formulation. The selected SLM formulation (10% GB


containing 5 mg TS g1) was freeze-dried in the presence of a cryoprotectant (trehalose).
Trehalose was added to the selected formulation by one of the following methods: it was
added to the aqueous phase before homogenization in a ratio of 3:1 w/w sugar : lipid (Heiati
et al. 1998) or the selected formulation was diluted in (1:1 v/v) with 15% w/w trehalose
solution (Cavalli et al. 1997). SLMs were frozen in a deep freezer at 70 C overnight and
then lyophilized in a freeze dryer (Christ alpha 12, Osterodea. H., Germany) for 24 h.
Reconstitution of the lyophilized products was performed using distilled water and a vortex
for 5 min (to give a concentration equivalent to 5 mg of TS g1).

Scanning electron microscopy (SEM). Solid lipid microparticle suspensions were deposited
on metallic stubs then placed in liquid nitrogen and dried under vacuum. The freeze-dried
microparticles were coated uniformly with gold. All samples were examined for morphology
and surface properties using a scanning electron microscope (Joel, SEM, JSM-25 SII,
Tokyo, Japan).

Particle size and zeta potential measurement. Particle size, polydispersity index and zeta
potential were initially measured by a laser particle size analyser (Submicron Particle Size
Analyser 90 plus, Brookhaven Instrument Co., Holtsville, NY, USA). An aliquot of 200 ml of
SLM was diluted with 3 ml of deionized water. The diluted samples were loaded into a 4 ml
cuvette and the particle size and zeta potential measurement were conducted at ambient
temperature. Measurements were performed in triplicate.

Rheological studies. Rheological properties of the prepared SLM formulations were


measured using a Brookfield viscometer (Brookfield Model RVDV-II Middleboro,
USA) using spindle no. 61, 62 and 64. The spindle speed rate was increased in ascending
order speed from 0.550 rpm and then in descending order speed setting from 500.5 rpm.
460 A. H. El-Kamel et al.

The viscosity was read directly from the viscometer display. The sample was equilibrated at
25  0.1 C prior to each measurement. All measurements were made in triplicate.

Differential scanning calorimetry (DSC). Thermal analysis was performed using DSC
(Perkin Elmer, Shelton, USA). About 5 mg of TS, bulk lipid, lyophilized SLM after
separation of aqueous phase were heated from 25 C to 250 C at a heating rate of 5 C
per min.

Determination of testosterone entrapment efficiency (EE). Testosterone entrapped in SLM was


determined by HPLC assay after drug extraction from SLM. SLM was centrifuged at
19 000 rpm and 5 C for 2 h. Then supernatant was removed and the sediment was washed
with distilled water. The sediment was freeze-dried for 8 h. A weight of 10 mg of the freeze-
dried SLM was dissolved in 9 ml ethanol while heating at 60 C. The volume was then
completed to 10 ml with water. Concerning GB, 10 mg of freeze-dried SLM were dissolved
in 1 ml dichloromethane and the volume was completed to 10 ml with ethanol. After
filtration through a PTEF filter a suitable dilution was performed. An aliquot of 20 ml was
injected into HPLC to determine TS concentration. Entrapment efficiency was calculated
according to the following equation:
amount of TS determined in SLM
EE% 100
amount of TS added during preparation of SLM

Preparation of rat skin. Male rats (150170 g) were sacrificed by an overdose of chloroform
inhalation. Abdomen skin was excised after clipping of the hair and removal of subcutaneous
fat by means of blunt dissection. The excised skin was used freshly or kept in normal saline
at 5 C for a maximum period of 24 h.

Release of testosterone from different prepared SLM formulations. Jacketed vertical Franz
diffusion cells were placed in a V-3 station vertical cell stirrer and connected to a circulating
water bath heated to 32  0.5 C. Excised abdomen rat skin or cellophane membrane
(molecular weight cut-off 60008000, Spectra/Por) previously soaked in receptor medium
overnight was clamped by an O ring between the donor and the receptor chamber of the
diffusion cell. The area of diffusion in the case of cellophane membrane was 4.9107 cm2 and
1.7679 cm2 in the case of excised abdomen rat skin. A suitable aliquot of the formulation
(equivalent to 2.5 mg of testosterone) was added to the donor chamber of the diffusion cell
and was occluded with a parafilm. The receptor cell was filled with 20 or 12 ml normal saline
solution containing 40% propylene glycol (Morgan et al. 1998), in the cases of cellophane
membrane and excised abdomen rat skin, respectively, and stirred by magnetic bar. Aliquots
(200 ml) were withdrawn from the receptor compartment at scheduled intervals of 2, 4, 6, 8,
18, 20, 22 and 24 h and were replaced by the same volume of fresh receptor fluid. Samples
were analysed by HPLC. The concentration of TS was corrected for sampling effects. Each
experiment was carried out at least in triplicate.

Stability study. Testosterone SLM (10% GB containing 2.5 mg TS g1) was stored in the
dark in glass containers at temperatures of 5 C and 30  0.5 C. The particle size, zeta
potential and drug release were evaluated after 16 weeks.
Formulation and physicochemical characterization of TS SLM 461

Statistics. Statistical analyses of data were performed using the statistical package for social
sciences (SPSS ver. 10.0). Analysis of variance ANOVA was applied; a p-value of less than
0.05 was considered significant.

Results and discussion


Scanning electron microscopy (SEM)
Different lipid types might influence the surface morphology or shape of the particles
(Cortesi et al. 2002). Micrographs of SLM containing 5 and 10% GB and 5% and 10%
GD revealed the formation of spherical microparticles with irregular surface, as shown in
Figure 1. However, GD particles showed a large population of small particles. Glyceryl
monostearate microparticles (2.5 and 5% GM SLM) (Figure 1) showed relatively smaller
microparticles with rough and irregular surfaces. On the other hand, SA microparticles were
small and somewhat spherical although they had imperfections on their surfaces. It is worth
mentioning that during SEM sample preparation, both water of the bulk phase and water
present in the particles were completely removed. Such drying apparently may cause the
observed shrinking of the surface of the particles.

Particle size and zeta potential measurement


The effect of lipid type and concentration on the size of SLM was investigated. Regarding
the effect of lipid concentration on the particle size, statistical analysis followed by the
Duncan Multiple comparisons post-hoc test revealed that there was no significant difference
in the size of GD and GB SLM as the concentration of lipid increased from 5% to 10%.
Similarly, there was no significant difference in the size of SA SLM as the lipid concentration
increased from 2.5% to 5%. This was in agreement with Lippacher et al. (2001), who found
that, despite the increasing lipid content of SLN, their particle size did not change. Contrary,
the size of GM SLM increased as the lipid concentration increased from 2.5% to 5%. The
increase in particle size with increasing lipid concentration can be explained by the decrease
in homogenization efficiency with increasing content of dispersed phase (lipid phase)
(Lippacher et al. 2004). Similarly, Souto et al. (2004a) reported that the particle size of SLN
increased with the increase of lipid concentration from 9.5% to 19%. However, it has been
reported that increasing the lipid content over 510% in most cases results in larger particles
and broader size distribution (Siekmann and Westesen 1994a).
On the other hand, the type of lipid may affect the size of microparticles. ANOVA
followed by post-hoc Duncan test revealed the following rank order for the size of SLM at
lipid concentration of 5%: SA  GD < GB  GM. This was in agreement with Cavalli et al.
(1997), who reported that the choice of the lipid could affect SLM diameter. The data in
Table I show PI values which were below 0.49 except for 5% SA SLM that have a PI value
of 0.6. The high PI value for 5% SA SLM could be due to high viscosity of SLM dispersion
(as shown in rheological studies) that could affect the homogenization efficiency resulting
in a wide range of particle size distribution.
Zeta potential is an important and useful indicator of particle surface charge, which can be
used to predict and control the stability. The zeta potentials of SLN are presented in Table I.
The zeta potentials of all the formulations ranged from 10.6 to 25.6 mV. In general,
particles could be dispersed stably when the absolute value of zeta potential was above
30 mV due to the electric repulsion between particles (Muller et al. 2001). Accordingly, the
measured zeta potential was not sufficiently high to stabilize the dispersion solely by
462 A. H. El-Kamel et al.

Figure 1. Scanning electron micrographs of various formulations of TS SLM. All formulations


containing 2.5 mg TS g1.
Formulation and physicochemical characterization of TS SLM 463

Table I. Mean particle size, polydispersity index (PI), zeta potential and entrapment efficiency (% EE) of various
SLM formulations containing 2.5 mg TS g1.

SLM formulation Mean particle size  SD (mm) PI Zeta potential  SD (mV) % EE

2.5% GM 19.1  14.7c 0.49 25.6  2.2 88.5  7.0


5% GM 66.1  27.5ab 0.49 19.0  1.4 82.2  7.3
5% GD 22.7  17.5c 0.45 21.6  2.3 83.2  4.2
10% GD 43.6  19.4abc 0.12 15.7  3.2 83.3  3.4
5% GB 32.1  18.5ab 0.24 22.3  5.1 91.7  1.8
10% GB 69.8  30.5a 0.01 18.3  8.6 80.7  0.3
2.5% SA 46.8  17.6abc 0.15 11.8  1.8 90.3  2.1
5% SA 18.1  14.3c 0.60 22.2  2.9 95.7  0.0
10% GB* 31.2  9.8ab 0.46 10.6  2.0 82.2  3.9

Means of same symbols are statistically equivalent, a > b.


*NB: it contains 5 mg TS g1.

electrostatic repulsion. However, pre-requisites to allow the stability were reported as a


minimum of 89 mV zeta potential in combination with a steric stabilization (Zimmermann
and Muller 2001). Consequently, poloxamer 188 which was used as a steric stabilizer can
easily compensate for the missing electrostatic repulsion. This phenomenon has been also
observed by Schwarz and Mehnert (1999).

Rheological studies
The prepared formulations showed plastic flow characteristics, where the viscosity decreased
with increasing shear rate. Ascending and descending flow curves overlapped and show no
time effects like, e.g. thixotropy. The lipid particles in the dispersion tend to align with
increasing shear stress which is alleviating the flow. Unlike other formulations, the ascending
and descending curves of 5% SA and 5% GM SLM formulations did not overlap, showing
thixotropy. In other words, increasing the lipid content of SA and GM from 2.5% to 5% lead
to different flow characteristics. The same observation was documented for cetylpalmitate
and tripalmitin solid lipid nanoparticles, which showed plastic flow with thixotropic
characteristics up on increasing the lipid concentration (Lippacher et al. 2000, Souto et al.
2004b).
By comparing the viscosity of various formulations at 0.5 rpm (Figure 2), it was observed
that, for each type of lipid, as the concentration of lipid increased the viscosity increased.
In addition, the type of lipid affected the viscosity of the final product. At fixed lipid
concentration (5%) the rank order of the viscosity of SLM according to the type of lipid was
GB < GD < GM < SA.
The change in viscoelastic behaviour can be attributed to the presence of different
particleparticle interactions since there are different particle sizes and particle size
distributions. Decreasing the particle size, as in the case of SA, is accompanied by a huge
increase of surface. Therefore, the number of contact points increases and so particle
particle interactions are more pronounced, leading to a three-dimensional network structure
and hence increasing the viscosity (Lippacher et al. 2002).

Differential scanning calorimetry (DSC)


With lipid drug delivery systems, polymorphic transformation may occur during the
preparation of the dosage form (Eldem et al. 1991). Therefore, the DSC thermograms of
microparticles and bulk lipid were investigated. The thermograms of freeze-dried SLM
464 A. H. El-Kamel et al.

20000

15000
Viscosity (cps)

10000

5000

0
5% GB 10% GB 2.5% GM 5% GM 5% GD 10% GD 2.5% SA 5% SA
Formula

Figure 2. Viscosity of various formulations of TS SLM measured at speed of 0.5 rpm.


All formulations containing 2.5 mg TS g1.

containing TS did not show the melting peak of TS (Figures 36). This suggested that
TS was dispersed in SLM in an amorphous state. Venkateswarlu and Manjunath (2004)
reported similar results by stating that rapid quenching of the formed microemulsion during
the preparation process prevents the drug crystallizing. In addition, the presence of
poloxamer as a surfactant may not allow TS to crystallize.
Melting points of GM (59.41 C and 57.26 C for 2.5% and 5%, respectively) and SA
(53.09 C and 55.83 C for 2.5% and 5%, respectively) in SLM formulations were slightly
depressed when compared to the melting points of corresponding bulk lipid (60.89 C and
61.20 C for GM and SA, respectively), as shown in Figures 3 and 4. This might suggest that
these lipids might exist in stable  form in SLM. The observed melting point depression
could be due to small size effect as predicted by the Thomson equation (Siekmann and
Westesen 1994b, Hou et al. 2003). The small particle size and therefore high surface being
an energetically state leads to a decrease of crystallization point (Freitas and Muller 1999).
The second peak appeared at 47 C in the thermogram of GM and 2.5% SA SLM
may indicate the presence of a 0 polymorph.
Concerning GD, the melting endotherm of bulk lipid was at 64.1 C with a shoulder at
58 C, as shown in Figure 5, indicating the presence of  and 0 form, respectively. In GD
SLM thermograms this shoulder disappeared and gave a broad peak at 61 C as the lipid
concentration increased from 5 to 10% in SLM. This may indicate the transition of unstable
0 polymorph to stable  form.
Figure 6 shows a melting endotherm for GB as a bulk lipid and SLM at 71.5  1 C. This
was in agreement with Bunjes et al. (1995), who reported that SLN which are composed of
glycerides with heterogeneous composition, as in the case of GB, possess a less pronounced
melting point depression. In general, GB showed highest thermal stability among the
examined lipids and this was in agreement with the documented literature which reported
that the polymorphic transitions after crystallization of lipid in SLM are slower for longer
Formulation and physicochemical characterization of TS SLM 465

Figure 3. DSC thermograms of TS, GM, 2.5% GM SLM and 5% GM SLM.

Figure 4. DSC thermograms of TS, SA, 2.5% SA SLM and 5% SA SLM.


466 A. H. El-Kamel et al.

Figure 5. DSC thermograms of TS, GD, 5% GD SLM and 10% GD SLM.

Figure 6. DSC thermograms of TS, GB, 5% GB SLM and 10% GB SLM.


Formulation and physicochemical characterization of TS SLM 467

chain glyceride (GB, C22) than for shorter chain glycerides (SA, C18) (Hamdani et al. 2003).
Therefore, it was assumed that stability problems can be avoided by using compritol as a
lipid matrix.

Drug entrapment efficiency (EE)


The chemical nature of the lipid is an important factor in determining the EE of drug in the
SLM because lipid which forms highly crystalline particles with a perfect lattice lead to drug
expulsion (Westesen et al. 1997). On the other hand, the imperfection (lattice defects) of the
lipid structure could offer space to accommodate the drug. Table I listed the entrapment
efficiency of TS in various SLM formulations. The percentage EE ranged from 80.795.7%.
The lost or unentrapped drug could be due to the solubility of the drug in the water
poloxamer phase. Schwarz and Mehnert (1999) also reported a reduction in drug
entrapment in the presence of poloxamer.
Complex lipids, e.g. GB, GD and GM being mixtures of mono-, di- and triglycerides and
also containing fatty acids of different chain length form less perfect crystals with many
imperfections offering space to accommodate the drug (Mehnert et al. 1997). In addition,
the presence of mono- and diglycerides in the lipid used as matrix material promotes drug
solubilization in lipid.

In vitro release of testosterone from SLM


Testosterone had a poor water solubility and high lipophilicity that made it an excellent
candidate for SLM. Poloxamer was chosen as a surfactant since it has been reported that
it has a stabilizing effect by inhibiting lipid transition and crystallization (Freitas and Muller
1999). Furthermore, poloxamer has been reported to stabilize SLN by steric stabilization
(Schwarz and Mehnert 1999). The effect of the following three different factors on the
release characteristics of TS from SLM was examined:

Effect of type and concentration of the lipid. Figure 7(a and b) shows the cumulative amounts
of TS permeated across Spectra/por membrane after application of low and high lipid
content SLM formulations, repectively, which contained 2.5 mg TS g1 assuming that
an artificial membrane could be an alternative to a biological membrane.
The release data were fitted into Ficks equation and the Higuchi equation. Almost all the
SLM formulations followed Ficks law better than the Higuchi model as indicated by the
higher values of coefficient of determination shown in Table II.
The in vitro release data were then treated in accordance with Ficks law to calculate the
flux ( J, mg cm2 h1) and the permeability coefficient (P, cm h1), as shown in Table II.
Statistical analysis of release data using two-way ANOVA followed by the Duncan test
revealed the following order for the release of TS from various formulations: 10% GB > 10%
GD > 2.5% SA > 2.5% GM > 5% GB > 5% SA > 5% GD > 5% GM.
It seemed that the release and transport of TS were affected by not only the concentration
of lipid but also by the type of lipid used in the formulation. The dependence of transdermal
drug transport on the carrier matrix is well documented in the literature (Barry 2001). The
different effect of each carrier relates specifically to solubility of the drug within the carrier,
only solubilized drug can diffuse within the matrix and contribute significantly to release rate
(Mills et al. 2006).
In addition, within 24 h (the release experiment period) the applied fluid SLM dispersion
slowly turned into a semi-solid gel due to water evaporation. Gel formation of SLM could be
correlated with polymorphic transition of lipid matrix that could affect the release of TS
468 A. H. El-Kamel et al.

(a) 250

permeated (mg / cm2) 200


Cumulative amount

150

100 2.5% GM

5% GD

50 5% GB

2.5% SA

0
0 5 10 15 20 25 30
Time (h)

(b) 250

200
permeated (mg / cm2)
Cumulative amount

150

100 5% GM

10% GD
50 10% GB

5% SA

0
0 5 10 15 20 25 30
Time (h)

Figure 7. Permeation profiles of TS through cellophane membrane after application of various


SLM formulations containing (a) low lipid concentrations and (b) high lipid concentrations. All
formulations containing 2.5 mg TS g1. Each value is the mean value of three different
experiments  SD.

from various lipid microparticles (Jenning et al. 2000). Since different polymorphic forms
differ in their ability to include host molecules in their lattice (Bunjes et al. 1996), drug
expulsion as a consequence of this transition was likely to occur.
The mechanism of permeation of drug could be explained as follows: TS which is in
amorphous form (as shown in DSC) dissolves in lipid, diffuses to the surface and undergoes
Formulation and physicochemical characterization of TS SLM 469

Table II. In vitro permeation parameters of testosterone through cellophane membrane after application of various
SLM formulations containing 2.5 mg TS g1.

SLM formulation J (mg cm2 h1) P (cm h1)  102 R2 Ficks R2 Higuchi

2.5% GM 8.038  0.6 0.320  0.0002 0.9988 0.9854


5% GM 5.012  0.6 0.200  0.0002 0.9924 0.9944
5% GD 7.071  0.8 0.282  0.0003 0.9909 0.9733
10% GD 8.292  0.2 0.331  0.0001 0.9985 0.9888
5% GB 7.598  0.3 0.304  0.0001 0.9969 0.9916
10% GB 8.778  0.1 0.351  0.0000 0.9985 0.9832
2.5% SA 8.418  0.3 0.336  0.0001 0.9984 0.9826
5% SA 7.100  0.3 0.284  0.0001 0.9997 0.9880
10% GB 0.934  0.1 0.037  0.0001 0.9825 0.9384
10% GD* 0.431  0.1 0.017  0.00002 0.9758 0.9324
10% GB** 0.951  0.1 0.038  0.00004 0.9918 0.9384

* Permeation was performed through rat skin.


**SLM containing 5 mg TS g1 and the permeation was performed through rat skin.

partitioning between lipid and aqueous phase. Soluble drug is partitioned into aqueous
phase from which it is permeated to the receptor medium.
The obtained results showed no relation between the viscosity of the formulation and the
TS transport. For example, stearic acid SLMs showed intermediate permeation behaviour in
spite that they have the highest viscosity. This result indicated that the permeation process is
consistent with skin-controlled mechanism, since the viscosity of lipid formulation will play
an important role in controlling the permeation of the drug if the diffusion of drug through
the polymer matrix is the rate-determining step (Ho et al. 1994).

Effect of transporting membrane. Based on the flux values and on the total amount permeated
through the cellophane membrane two formulations were chosen to be tested using excised
abdominal rat skin. These formulations were 10% GB and 10% GD containing 2.5 mg
TS g1 of SLM formulation.
The permeability of TS through cellulose membrane was higher than that obtained
through rat skin (p  0.0001), as indicated by the flux and permeability coefficient values
shown in Table II. The higher permeability can be due to the less dense structure of
cellulose membrane relative to the skin. Considering the structure of cellulose membrane,
the molecular weight cut off of the membrane is 60008000, suggesting that there
are water-filled pores or channels for drug molecules to diffuse freely. Accordingly, the
penetration of drug may depend on the speed of drug partitioning from the lipid into
receptor phase (Ho et al. 1994).
On the other hand, the total cumulative amount of TS released from 10% GB
(20.2  1.9 mg cm2) was higher than 10% GD microparticles (11.57  0.44 mg cm2)
using excised abdomen rat skin (p  0.0001). The effect of matrix on drug penetration
through skin cannot be considered in isolation because the fat matrix itself may have an
enhancing effect on drug penetration or permeation (Mei et al. 2003).

Effect of drug loading. The permeation rate of drug can be altered by changing the drug
concentration in the lipid matrix. It has been reported that the increase of drug
concentration up to a certain level may enhance the permeability of drugs due to an
increase in the drug thermodynamic activity (Rao et al. 1998).
470 A. H. El-Kamel et al.

Based on the total cumulative amount of TS permeated through rat skin, 10% GB SLM
was chosen to test the effect of changing drug loading on TS permeation. SLMs (10% GB)
containing either 2.5 or 5 mg TS g1 were evaluated. In the present study, statistical analysis
revealed no significant change in percentage drug permeated or TS flux (p > 0.05) through
excised abdomen rat skin as the drug loading was increased from 2.5 to 5 mg g1 SLM. This
indicated that the conducting pathways of the skin have reached saturation (Brand et al.
1997). Contrarily, Rao et al. (1998) found that the permeation of indomethacin increased
as the initial drug concentration increased from 5 to 20%.
On the other hand, Souto et al. (2004a) reported that the release rate of clotrimazole
decreased as the drug concentration increased due to a steric hindrance effect of the drug
molecules at higher drug concentration.
The amount of TS permeated through excised abdomen rat skin was plotted against the
amount permeated through a cellophane membrane, after application of 10% GB SLM
formulation containing 5 mg g1, at the same time points over 24 h in an attempt to find a
correlation between them. Regression analysis revealed high correlation between the amount
of TS permeated through the excised abdomen rat skin and that permeated through
a cellophane membrane over 24 h, as indicated by the high value of coefficient of
determination (0.997). This relation was best fitted into the following cubic equation:
y 0:247 0:048x 0:00036x2  3:257x3
where y is TS amount permeated through the excised abdomen rat skin (mg cm2) and x is
TS amount permeated through cellophane membrane (mg cm2).
Consequently, the amount of TS permeated can be predicted from in vitro permeation
studies under the adapted experimental conditions without the need for sacrificing animals
using the above equation. Pearson correlation was also performed and revealed significant
(p  0.0001) positive correlations (direct relationship) as indicated by its value of 0.995.

Stability study
In order to evaluate the physical stability of TS loaded SLM, release behaviour, particle size
and zeta potential were evaluated after storage for a period of 16 weeks at 5 C and 30 C
in the dark.
The cumulative amount of TS permeated through excised abdomen rat skin over 24 h
after application of 10% GB SLM containing 2.5 mg TS g1 was significantly decreased
at the 16th week of storage at 5 and 30 C, respectively, as shown in Figure 8.
In an attempt to correlate the change in release characteristics with the particle size of
SLM, particle size measurement was performed after 16 weeks storage at 5 C and 30 C.
Statistically, the particle size increased significantly after storage for 16 weeks at 30 C.
Consequently, the exposed surface area was expected to decrease that could affect the drug
release negatively. In addition, during storage, rearrangement of the lipid crystal lattice
might occur in favour of thermodynamically stable configurations and this is often
connected with expulsion of the drug molecules (Mehnert and Mader 2001). This could be
resulted in enhanced drug release after storage of SLM. This effect is opposed by gelling of
SLM that could be observed at the16th week of storage at 30 C. Gelling of SLM may lead to
increase in microviscosity and retard drug diffusion and consequently its release. The final
consequences of the last mentioned two effects may be the decrease in overall amount drug
release.
Contrarily, there was no significant increase in particle size of SLM stored at 5 C. It was
proposed that the partial glycerides like GB (15% monoglycerides and 70% diglycerides
Formulation and physicochemical characterization of TS SLM 471

25
5 C

Cumulative amount permeated (mg / cm2)


30 C
20

15

10

0
Fresh 16 weeks

Figure 8. Effect of storage of 10% GB SLM containing 2.5 mg TS g1 at 5 C and 30 C for 16 weeks
on the total cumulative amount of TS permeated through abdomen rat skin.

possess improved surfactant properties (HLB 25). The surfactant film around the
microparticles prevents aggregation (Westesen et al. 1993). Borgia et al. (2005), who
documented that SLN composed of 10% GB did not show change in particle size or particle
size distribution after storage for almost 12 weeks at 8 C reported similar results.
Consequently, other factors were expected to affect the release of drug from SLM after
storage for 16 weeks at 5 C.
After 16 weeks storage at 5 C and 30 C there was no significant change in zeta potential.
These data suggested that the particle size but not the zeta potential appears to be an efficient
indicator of particle stability in this case.
Based on the stability results, it could be recommended to store the prepared formulation
at 5 C in the dark to maintain its stability as long as possible.

Effect of freeze-drying
Although lyophilization has been used widely to improve the chemical and physical stability
of SLM over an extended period of time, it may damage the surfactant film around the
microparticles due to a freezing out effect and may also cause particle aggregation during the
redispersion process (Mehnert and Mader 2001). Various cryoprotectants have been used
to prevent these problems associated with lyophilization.
Preliminary experiments were conducted to select the cryoprotactant of highest potential.
Sucrose and trehalose were examined as cryoprotectant.
The use of sucrose as cryoprotectant resulted in the formation of sticky glassy dried mass
that is very difficult to handle. On the other hand, addition of trehalose as cryoprotectant
gave dried free flowing powder. Consequently, trehalose was chosen for further studies. This
results was in a good agreement with that reported by Mehnert and Mader (2001), who
documented that trehalose was more efficient as a cryoprotectant than sucrose.
The method of addition of trehalose influenced the quality of the final formulation. The
results showed that the formulation to which trehalose was added to the aqueous phase
before homogenization, in a ratio of 3:1 w/w (sugar : lipid), the freeze-dried powder was
rapidly reconstituted after addition of water and the lipid phase was homogenously dispersed
472 A. H. El-Kamel et al.

in the aqueous phase. On the other hand, when 15% trehalose solution was used to dilute
the final formulation in a ratio of 1:1 v/v before freeze-drying, the reconstituted formulation
needs longer time to be homogenously dispersed and large aggregates were macroscopically
observed in SLM suspension. Similarly, it has been also reported that the best results were
obtained when cryoprotectant was added to the sample prior to homogenization
(Zimmermann et al. 2000).
Figure 9 shows the cumulative TS released from selected formulation (10% GB
containing 5 mg TS g1 SLM) to which trehalose was added to the aqueous phase before
homogenization or by dilution of the final formulation by 15% trehalose in a ratio of 1:1 in
comparison with the freshly prepared formulation. Statistical analysis revealed the following
order of TS release: fresh SLM  SLM diluted with 15% trehalose > SLM-containing
trehalose before homogenization.
25 a
a
Cumulative amount permeated

20
after 24 hr (mg / cm2)

b
15

10

0
Fresh Diluted with 15 % Containing trehalose
trehalose (3:1)

Method of addition of trehalose

Figure 9. Effect of freeze-drying in presence and in absence of trehalose on the cumulative TS


permeated through abdomen rat skin after application of 10% GB SLM containing 5 mg TS g1. Same
symbols are statistically equivalent, a > b.

Statistical analysis revealed the following rank order for the mean particle size of the
reconstituted freeze-dried powder in comparison with freshly prepared formulation: SLM
containing no trehalose  SLM diluted with 15% trehalose (1 : 1) > fresh SLM > SLM-
containing trehalose in aqueous phase (3 : 1 sugar: lipid) before homogenization.
It could be concluded that dilution of SLM formulation with trehalose has no stabilizing
effect on the mean particle size of the reconstituted formulation and resulted in SLM
suspension of larger particle compared to the freshly prepared SLM. Similarly, Cavalli et al.
(1997) observed increase particle sizes after lyophilization. However, addition of trehalose in
the aqueous phase before homogenization resulted in SLM suspension of smaller mean
particle size compared with the freshly prepared SLM.
The cryoprotectant effect of trehalose may arise from the formation of a protective capping
layer around SLM (Jenning et al. 2000). It can be considered as placeholders which prevent
the contact between discrete lipid particles. Furthermore, trehalose interacts with the polar
head group of the surfactant and serves as a kind of pseudo hydration shell (Mobley and
Schreier 1994).
Formulation and physicochemical characterization of TS SLM 473

The lower cumulative amount of TS released from SLM that contains trehalose in
aqueous medium in spite of smaller mean particle size could be due to possible H-bond
formation between TS and hydroxyl functions of trehalose.

Conclusion
All things considered, the choice of type and lipid concentration can affect the final
physicochemical and release characteristics of SLM formulation. The selected SLM
formulation (10% compritol2.5 mg TS g1) showed high potential for application as a
transdermal delivery system for TS by virtue of its high encapsulation efficiency, release
characteristics, thermal behaviour and stability during storage. Further studies are underway
to improve TS skin permeation.

Acknowledgements

The authors are grateful to the Research Center, King Saud University and to King
Abdullaziz City for Science and Technology for the financial support.

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