Oral Apomorphine Delivery from Solid Lipid Nanoparticles

with Different Monostearate Emulsifiers: Pharmacokinetic

and Behavioral Evaluations
MING-JUN TSAI,1,2 YAW-BIN HUANG,3 PAO-CHU WU,3 YAW-SYAN FU,4 YAO-REN KAO,3 JIA-YOU FANG,5,6 YI-HUNG TSAI3
1
Food and Drug Administration, Department of Health, Executive Yuan, Taipei, Taiwan
2
Institute of Basic Medical Sciences, National Cheng Kung University, Taiwan
3
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
4
Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University,
Kaohsiung, Taiwan
5
Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan
6
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

Received 16 January 2010; revised 30 March 2010; accepted 8 June 2010

Published online 25 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22285

ABSTRACT: Apomorphine, a dopamine receptor agonist for treating Parkinson’s disease, has
very poor oral bioavailability (<2%) due to the first-pass effect. The aim of this work was to in-
vestigate whether the oral bioavailability and brain regional distribution of apomorphine could
be improved by utilizing solid lipid nanoparticles (SLNs). Glyceryl monostearate (GMS) and
polyethylene glycol monostearate (PMS) were individually incorporated into SLNs as emulsi-
fiers. It was found that variations in the emulsifiers had profound effects on the physicochemical
characteristics. Mean diameters of the GMS and PMS systems were 155 and 63 nm, respec-
tively. More than 90% of the apomorphine was entrapped in the SLNs. The interfacial film was
the likely location for most of apomorphine molecules. The PMS system, when incubated in
simulated intestinal medium, was found to be more stable in terms of particle size and encapsu-
lation efficiency than the GMS system. Using the GMS and PMS systems to orally administer
apomorphine (26 mg/kg) equally enhanced the bioavailability in rats. SLNs showed 12- to 13-
fold higher bioavailability than the reference solution. The drug distribution in the striatum,
the predominant site of therapeutic action, also increased when using the SLNs. The anti-
Parkinsonian activity of apomorphine was evaluated in rats with 6-hydroxydopamine-induced
lesions, a model of Parkinson’s disease. The contralateral rotation behavior was examined after
oral apomorphine delivery. The total number of rotations increased from 20 to 94 and from 20
to 115 when the drug was administered from SLNs containing GMS and PMS, respectively.
The experimental results suggest that SLNs may offer a promising strategy for apomorphine
delivery via oral ingestion. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association
J Pharm Sci 100:547–557, 2011
Keywords: apomorphine; oral absorption; bioavailability; nanoparticles; pharmacokinetics;
drug transport

INTRODUCTION striatum.1 This disease affects about 1.6% of the gen-

eral population older than 65 years, and the num-
Parkinson’s disease is characterized by progressive
ber of patients is predicted to rise as life expectancy
degeneration of dopaminergic neurons of the ni-
increases.2 Apomorphine is a mixed dopamine
grostriatal system and dopamine depletion in the
D1 /D2 receptor agonist that is potentially useful in the
Correspondence to: Jia-You Fang (Telephone: +8863-2118800
management of Parkinson’s disease. It is also used to
ext. 5521; Fax: +8863-2118236; E-mail: fajy@mail.cgu.edu.tw); Yi- treat erectile dysfunction.3 In 2004, apomorphine was
Hung Tsai (Telephone: +8867-3121101 ext. 2261; Fax: +8867- approved by the US Food and Drug Administration
3210683; E-mail: yhtsai@kmu.edu.tw)
as a rescue medication for Parkinson’s disease, par-
Journal of Pharmaceutical Sciences, Vol. 100, 547–557 (2011)
© 2010 Wiley-Liss, Inc. and the American Pharmacists Association
ticularly for treating “off” periods by a subcutaneous

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011 547

548 TSAI ET AL.
injection. The half-life of apomorphine following sub-
cutaneous administration is about 40 min, and the
duration of the benefit ranges from 40 to 90 min.4
Moreover, this administration route might not be con-
venient for all patients. Oral administration is a deliv-
ery system that is suitable for widespread clinical ap-
plication. However, apomorphine administered orally
was not successful because of the rapid degradation
in the gastrointestinal (GI) tract and first-pass effect,
resulting in bioavailability of 1.7%.5 High oral doses
of apomorphine may cause GI complications and have
been associated with nephrotoxicity.6
The oral route continues to be a challenge, even
though it is the most attractive way to adminis-
ter drugs. Incorporation of drugs into nanoparticles
opens the perspective of enhanced and less vari-
able bioavailability and prolonged plasma levels.7
Solid lipid nanoparticles (SLNs) represent an alter-
native drug carrier system to emulsions and poly-
meric nanoparticles.8 These particles are made of
crystalline solid lipids. They can overcome the mem-
brane stability and drug-leaching problems associ-
ated with emulsions and the toxicity problems of poly-
meric nanoparticles.9 Distinct advantages of SLNs
include their ability to protect chemically labile in-
gredients, the possibility of large-scale production,
their controlled-release characteristics, and improved
bioavailability.10,11
The aim of the present work was to develop SLNs
for apomorphine via the oral route, which may facil-
itate the oral bioavailability and brain targeting. If
the obtained blood profile after oral ingestion is not
optimal, it can be modulated by changing the compo-
sition of the nanoparticles, mainly the emulsifiers.12
Glyceryl monostearate (GMS) and polyethylene gly-
col monostearate (PMS) were used as emulsifiers in
SLNs to evaluate the effects of different emulsifiers
on oral apomorphine administration. The feasibility
of using apomorphine-loaded SLNs as an oral sys-
tem was demonstrated through extensive character-
ization of the size, charge, appearance of the SLNs,
and drug stability. The in vivo pharmacokinetics of
apomorphine in SLNs was evaluated to elucidate the
applicability. The intracerebral injection of the neu-
rotoxin 6-hydroxydopamine (6-OHDA) has served as
an experimental basis to develop anti-Parkinsonian
drugs and treatment strategies.13 This lesion model
has been used to investigate the behavioral functions
of the substantia nigra. 6-OHDA lesions in rats were
examined after the oral delivery of apomorphine from
SLNs with various emulsifiers.
MATERIALS AND METHODS
Materials
Apomorphine hydrochloride was kindly provided by
Standard Chemical and Pharmaceutical (Tainan,
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011
Taiwan). N-Propylnorapomorphine, Pluronic F68
(PF68), L-ascorbic acid, pepsin, and pancreatin were
purchased from Sigma-Aldrich Chemical (St. Louis,
Missouri). Tripalmitin was supplied by Tokyo Kasei
(Tokyo, Japan). Hydrogenated soybean phosphatidyl-
choline (HSPC, 90%) was from NOF (Tokyo, Japan).
GMS and PMS (C18 H35 O2 (C2 H4 O)55 H) were pur-
chased from Wako Chemical (Osaka, Japan).
Preparation of SLNs
The lipid and aqueous phases were prepared sepa-
rately. The lipid phase consisted of 200 mg of tri-
palmitin, 50 mg of HSPC, and 15 mg of apomorphine,
whereas the aqueous phase with water consisted of
30 mg of PF68, and 12 mg of L-ascorbic acid. The emul-
sifier GMS or PMS (20 mg) was added in the lipid or
aqueous phase. The total volume of the aqueous phase
was 3 mL. The reason that L-ascorbic acid was added
was to prevent apomorphine autoxidation.14 The two
phases were heated separately to 80◦ C for 20 min. The
aqueous phase was added to the lipid phase and mixed
using a probe-type sonicator (UP50H, Hielscher Ul-
trasonics, Teltow, Germany) for 20 min at 50 W.
The temperature was maintained at 80◦ C during
sonication.
Particle Size and Zeta Potential
The mean particle size (z-average) and zeta poten-
tial of the SLNs were measured by photon correla-
tion spectroscopy (Zetasizer 3000HS; Malvern Instru-
ments Ltd., Worcestershire, UK) using a helium–neon
laser with a wavelength of 633 nm at 25◦ C. The size
values are given as a volume distribution. A 1:150 di-
lution of the nanoparticulate systems was made using
double-distilled water for the measurements.
Transmission Electron Microscopy
The size and morphology of SLNs were observed us-
ing a Jeol JEM-2000EX electron microscope (Tokyo,
Japan). The system of SLNs was diluted sixfold with
double-distilled water. One drop of the diluted SLNs
was deposited on the carbon film–covered copper grid
to form a thin-film specimen, which was then stained
with 0.5% phosphotungstic acid. The sample was ex-
amined and photographed with the microscope.
Apomorphine Entrapment Efficiency
The encapsulation efficiency of apomorphine en-
trapped by SLNs was determined by an ultracen-
trifugation method. The systems were centrifuged
at 120,000 rpm (649,000 × g) at 4◦ C for 2 h in a
Hitachi CS150GXL ultracentrifuge (Tokyo, Japan) to
separate the incorporated drug from the free form.
The supernatant was analyzed by high-performance
DOI 10.1002/jps
 ORAL APOMORPHINE DELIVERY FROM SLNS WITH MONOSTEARATE EMULSIFIERS
liquid chromatography (HPLC) to determine the en-
trapment efficiency (%) of the total drug load. The
HPLC system for apomorphine was the Hitachi 7000
series. A 25 cm long, 5 mm inner diameter stainless
steel RP-18 column (Inertsil-N5 ODS; GL Sciences,
Torrance, California) was used. The mobile phase was
an acetonitrile/pH 3 sodium phosphate–ethylenedi-
aminetetraacetic acid solution (20:80) at a flow rate
of 1 mL/min. The UV wavelength was set to 280 nm.
N-Propylnorapomorphine was utilized as the internal
standard.
Storage Stability
The prepared SLNs in a solution form were stored
at room temperature in a desiccator for 3 weeks. The
relative humidity was kept at 50 ± 5%. An aliquot
of sample was taken at predetermined time intervals
to investigate the particle size and encapsulation per-
centage. The mean size was determined by photon
correlation spectroscopy as described in the previous
section. The percentage of apomorphine loading was
determined by ultracentrifugation.
Stability in Simulated Gastric and Intestinal Media
Stability studies were carried out in a pepsin solution
and pancreatin solution.15 The proteolytic enzyme so-
lution at 2.7 mL was added to 0.3 mL of apomorphine-
loaded SLNs with a stirring bar. The stirring rate and
temperature were kept at 600 rpm and 37◦ C, respec-
tively. Samples were withdrawn at 1 h. The particle
size of SLNs after incubation was determined. The re-
maining concentration of apomorphine in the sample
was determined. Apomorphine in samples (0.1 mL)
was extracted using 0.9 mL of methanol. After cen-
trifugation, the organic layer was carefully removed
and subjected to the HPLC analysis.
Animals
The in vivo experiments were performed with male
Wistar albino rats (200–250 g). The animal experi-
ment protocol was reviewed and approved by the In-
stitutional Animal Care and Use Committee of Kaoh-
siung Medical University. Animals were housed and
handled according to institutional guidelines. All ani-
mals were starved overnight prior to the experiments.
In Vivo Pharmacokinetics
Apomorphine was given orally (26 mg/kg) or the
femoral vein was exposed for an apomorphine in-
jection (0.5 mg/kg, Apo-Go

R
Pen; Britannia Pharma-
ceutical, Newbury, UK). The rats were anesthetized
with 25% (w/v) urethane at a dose of 2.5 mL/kg. Oral
formulations were delivered by an oral intubation
cannula. A parenteral solution was injected through
DOI 10.1002/jps
549
the catheter. An aliquot of a 0.3-mL blood sample
was withdrawn from the jugular vein into a heparin-
rinsed vial according to a programmed schedule at 5,
10, 15, 30, 60, 120, 240, 360, and 480 min after dosing.
Each blood sample was centrifuged at 12,000 rpm for
10 min. The resulting plasma sample (100 :L) was
vortex-mixed with 50 :L of a 0.05% L-ascorbic acid
solution with 0.5 :g/mL of N-propylnorapomorphine.
This mixture was extracted using 4 mL of ethyl ether
with shaking for 20 min. The resulting solution was
centrifuged at 3000 rpm for 10 min, and 3 mL of the
supernatant was evaporated. The residue in the tube
was added to 150 :L of the mobile phase for the HPLC
analysis.
Data Analysis
Pharmacokinetic calculations were performed on
each individual set of data, using the pharmacoki-
netic software WinNonlin Standard Edition Version
1.1 (Pharsight, Mountain View, California) using a
noncompartmental method. The maximum concen-
tration, Cmax , and the time to reach Cmax (tmax ) were
determined by observing individual animal concen-
trations versus time curves. The area under the
plasma concentration curve from the time of adminis-
tration to infinity (AUC0→∞ ) was calculated using the
trapezoidal rule, with extrapolation to infinity. The
absolute bioavailability (F) of the oral formulations
was estimated as the AUC0→∞ ratio of the oral dosage
form to the parenteral solution after dose calibration.
The clearance of the oral formulation was calculated
from the elimination rate constant (ke ) multiplied by
the volume of distribution.
In Vivo Apomorphine Content in Brain Tissues
Animals that received oral administration of 26 mg/
kg of apomorphine were decapitated at 10 min af-
ter gavage. The skull was cut open, and the olfactory
bulb, cerebellum, hippocampus, brainstem, thalamus,
striatum, and cerebral cortex were carefully isolated
and weighed. Subsequently, the regional brain tissue
was mixed with 2 mL of normal saline for homoge-
nization. The homogenate at 200 :L was withdrawn
and mixed with 50 :L of a 0.05% L-ascorbic acid solu-
tion with 0.5 :g/mL of N-propylnorapomorphine. The
sample preparation was the same as that described
for the plasma extraction method.
In Vivo Rotational Behavior in 6-OHDA-Lesioned Rats
Animals were anesthetized with sodium pentobarbi-
tal (25 mg/kg) via an intraperitoneal injection and
placed in a stereotaxic frame (Kopf Instruments,
Tujunga, California), with the incisor bar 3.3 mm be-
low the interaural line. Two microliters (12 :g) of
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011
550 TSAI ET AL.
Figure 1. Transmission electron microscopic micrographs of (a) apomorphine-loaded solid
lipid nanoparticles (SLNs) with glyceryl monostearate and (b) apomorphine-loaded SLNs with
polyethylene glycol monostearate. Original magnification: 10,000×.
6-OHDA dissolved in double-distilled water was in-
jected into the left middle forebrain bundle 4.7 mm
posterior to the bregma, 7.8 mm ventral to the skull,
and 1.4 mm left of the midline, using a 0.5 mm
diameter Hamilton syringe at a rate of 0.25 :L/min.16
Our intention was to generate partial, rather than
complete, lesions similar to the extent of nigrostriatal
dopamine depletion in patients with Parkinson’s dis-
ease. One week after surgery, the animals were tested
for contralateral rotation behavior. Apomorphine-
induced rotational behavior was measured following
an oral ingestion of apomorphine (26 mg/kg) in a con-
trol solution (0.05% L-ascorbic acid solution) or SLNs.
Animals that had completed a 360◦ circle toward the
intact (contralateral) side were continuously counted
and separately recorded.
Statistical Analysis
Statistical analysis of differences between the vari-
ous treatments was performed using unpaired Stu-
dent’s t test. The post hoc test used for checking indi-
vidual differences between the formulations was the
Newman–Keuls test. A 0.05 level of probability (p <
0.05) was taken as the level of significance. Data en-
try and analysis were completed using Winks SDA
6.0 software (Texasoft, Duncanville, TX, USA).
Table 1. The Characterization of Apomorphine-Loaded Solid Lipid Nanoparticles with Different Emulsifiers by Mean
Diameter, Polydispersity, Zeta Potential, and Encapsulation∗
Emulsifier Mean Diameter (nm)
Glyceryl monostearate 154.97 ± 2.83
Polyethylene glycol monostearate 63.20 ± 0.98
∗
Each value represents the mean ± SD (n = 3).
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011
RESULTS
Physicochemical Characterization of the SLNs
The mean diameter, polydispersity index, zeta poten-
tial, and entrapment efficiency of the resulting SLNs
with apomorphine are listed in Table 1. The results
showed that the emulsifiers were critical parameters
governing the particle size. The size of SLNs incorpo-
rating GMS was 155 nm, with a polydispersity index
of 0.33. It can be seen that the size of SLNs was sig-
nificantly reduced (p < 0.05) to 63 nm by replacing
GMS with PMS, with no influence on polydispersity.
To obtain more information about the particle size
and morphology, a transmission electron microscopic
(TEM) analysis was also performed. Figures 1a and
1b show images of SLNs with GMS and PMS, respec-
tively. TEM showed that the particles had round, uni-
form shapes. A dense, well-distributed pattern was
observed. The mean diameters of GMS- and PMS-
containing SLNs were in the ranges of approximately
130 to 200 and 50 to 80 nm, respectively. These ranges
correlated well with those found using photon corre-
lation spectroscopy.
The absolute zeta potentials of apomorphine-
loaded SLNs were 23 and 7 mV for the GMS and PMS
systems, respectively, as shown in Table 1. A positive
surface charge was observed for these two carriers.
Polydispersity Zeta Potential (mV) Encapsulation (%)
0.33 ± 0.02 23.27 ± 0.70 90.38 ± 0.04
0.31 ± 0.02 7.23 ± 0.25 91.03 ± 0.14
DOI 10.1002/jps
 ORAL APOMORPHINE DELIVERY FROM SLNS WITH MONOSTEARATE EMULSIFIERS 551

Figure 2. Changes in (a) particle size (z-average) and (b) apomorphine entrapment efficiency
(%) of solid lipid nanoparticles with glyceryl monostearate (GMS) and (c) polyethylene glycol
monostearate (PMS) following storage at room temperature for 21 days. Each value is presented
as the mean and SD (n = 4).

The zeta potential of blank SLNs without the drug
was also examined. The surface charge was −15.63 ±
0.21 and −4.57 ± 0.21 mV for the GMS and PMS sys-
tems, respectively. The encapsulation of apomorphine
in GMS- and PMS-containing SLNs was similar (90%
vs. 91%).
Storage Stability
The physical stability of SLNs was determined by
measuring particle sizes at 0, 7, 14, and 21 days, as
depicted in Figure 2a. After 21 days of storage at room
temperature, the mean diameter of GMS-containing
SLNs had gradually increased from 155 to 190 nm
(p < 0.05). The particle size of PMS-containing SLNs
remained relatively stable during this period (p >
0.05). The change in the drug entrapment efficiency
within SLNs with GMS and PMS during 21 days
was also examined as shown in Figures 2b and 2c,
respectively. Curves of the entrapment efficiency af-
ter storage were approximately the same for the two
systems. The entrapment efficiencies for both GMS
and PMS were reduced by about 12% after 21 days of
storage.
Stability in Simulated Gastric and Intestinal Media
Table 2 summarizes the particle sizes of the GMS sys-
tem and apomorphine content remaining in carriers
after incubation in simulated gastric and intestinal
fluids for 1 h. Both the mean size and polydispersity
increased (p < 0.05) in the gastric medium. A contrary
result was observed in the intestinal medium, which
showed a decreasing trend after incubation. Apomor-
phine is known to be biologically unstable. The apo-
morphine content in the aqueous solution (control)
dropped from 100% to 96.88% ± 1.25% and 85.60% ±
1.09% after a 1-h incubation period in gastric and in-
testinal media, respectively. No protective effect of the
apomorphine amount in gastric medium was found af-
ter drug incorporation in the GMS system. The resid-
ual apomorphine percentage of GMS-containing car-
riers was 98%, which was similar to that of the free
form in the aqueous solution. Table 3 gives the stabil-
ity profiles of the PMS system in gastric and intestinal
fluids. When the PMS system was kept for 1 h in gas-
tric medium, a slight increase in particle size from
63 to 71 nm (p < 0.05) was observed. The polydis-
persity also increased after this incubation. The size

Table 2. The Mean Size, Polydispersity, and Apomorphine Content of Solid Lipid Nanoparticles with
Glyceryl Monostearate After Incubation in Simulated Gastric and Intestinal Media at 1 h∗

Condition Mean Diameter (nm) Polydispersity Drug Content (%)

0h 154.97 ± 2.83 0.33 ± 0.02 100
Gastric medium 174.63 ± 3.19 0.55 ± 0.05 94.74 ± 2.32
Intestinal medium 113.97 ± 1.16 0.26 ± 0.02 98.52 ± 0.69
∗
Each value represents the mean ± SD (n = 3).

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552 TSAI ET AL.

Table 3. The Mean Size, Polydispersity, and Apomorphine Content of Solid Lipid Nanoparticles with
Polyethylene Glycol Monostearate After Incubation in Simulated Gastric and Intestinal Media at 1 h∗

Condition Mean Diameter (nm) Polydispersity Drug Content (%)

0h 63.20 ± 0.98 0.31 ± 0.02 100
Gastric medium 71.10 ± 1.00 0.45 ± 0.01 98.16 ± 1.18
Intestinal medium 62.73 ± 1.45 0.46 ± 0.04 96.11 ± 4.48
∗
Each value represents the mean ± SD (n = 3).

receiving apomorphine gavage exhibited a concen-

Figure 3. Mean plasma concentration of apomorphine
versus time curves after intravenous injection of apomor-
phine solution (0.5 mg/kg) and oral administration of apo-
morphine to rats at a dose of 26 mg/kg from a control so-
lution and solid lipid nanoparticles (SLNs) with glyceryl
monostearate (GMS) or polyethylene glycol monostearate
(PMS). Each value represents the mean and SD (n = 5).
tration–time profile characterized by an early peak
concentration followed by a continuous decay in the
plasma concentration. At all times, the mean plasma
concentrations were higher in rats treated with SLNs
than in those treated with the free control. Pharma-
cokinetic parameters obtained by a noncompartmen-
tal analysis after the oral administration of apomor-
phine are listed in Table 4. Following oral ingestion of
the control group, the GMS system, and the PMS sys-
tem, the average peak plasma concentrations (Cmax )
were 17.32 ± 1.62, 83.79 ± 5.37, and 116.58 ± 24.02
ng/mL at 29.27, 16.81, and 12.04 min, respectively.
The AUC0→∞ values of apomorphine for the GMS and
PMS systems were 238.61 ± 52.98 and 264.51 ± 70.28
(ng h)/mL, respectively, whereas for the aqueous solu-
tion, it was only 20.04 ± 1.64 (ng h)/mL. The absolute
bioavailabilities (F) of the aqueous solution, the GMS
system, and the PMS system were 2.09%, 24.85%,
and 27.55%, respectively. It can be seen that the res-
idence time of apomorphine was significantly longer
(p < 0.05) for SLNs than for the control.

of the PMS system remained constant when passing
through the intestinal medium, although the polydis-
persity significantly increased (p < 0.05). Similar to
the GMS system, the PMS system exhibited a protec-
tive effect on the residual apomorphine percentage
against enzymatic attack in the intestinal medium
but not the gastric medium.
In Vivo Pharmacokinetics
The plasma concentration versus time curves of apo-
morphine after a single oral dose (26 mg/kg) and in-
travenous injection (0.5 mg/kg) from the aqueous so-
lution and SLNs are illustrated in Figure 3. Animals
In Vivo Apomorphine Content in Brain Tissues
The apomorphine concentrations in different brain
regions were determined to assess the distribution of
the drug when orally administered as a free control
or SLNs. As shown in Figure 4, apomorphine was de-
tected in various brain regions upon oral treatment
with the aqueous solution and SLNs for 10 min. The
drug level in brain by intravenous apomorphine injec-
tion was also listed in this figure. Apomorphine con-
centrations with the SLNs in the cerebellum, brain-
stem, and striatum were significantly higher (p <
0.05) than those with the aqueous solution. There
was no significant difference (p > 0.05) between the

Table 4. Pharmacokinetic Parameters of Apomorphine (26 mg/kg) After Oral Administration of the
Control Solution, GMS System, and PMS System to Rats∗

Parameter Control GMS System PMS System

Cmax (ng/mL) 17.32 ± 1.62 83.79 ± 5.37 116.58 ± 24.02
Tmax (min) 29.27 ± 2.50 16.81 ± 3.76 12.04 ± 1.25
AUC0→∞ [(ng h)/mL] 20.04 ± 1.64 238.61 ± 52.98 264.51 ± 70.28
F (%) 2.09 ± 0.17 24.85 ± 5.52 27.55 ± 7.32
Clearance (mL/min) 243.50 ± 17.10 43.60 ± 11.61 42.51 ± 14.50
∗
Each value represents the mean ± SD (n = 5).
Cmax , maximum plasma concentration; Tmax , time to reach Cmax ; AUC0→∞ , area under the plasma concentration–time
curve; F, absolute bioavailability.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011 DOI 10.1002/jps
 ORAL APOMORPHINE DELIVERY FROM SLNS WITH MONOSTEARATE EMULSIFIERS 553

Figure 4. Apomorphine concentrations in different brain regions at 10 min after intravenous

injection of apomorphine solution (0.5 mg/kg) and oral administration of apomorphine to rats
at a dose of 26 mg/kg from a control solution and solid lipid nanoparticles (SLNs) with glyceryl
monostearate (GMS) or polyethylene glycol monostearate (PMS). Each value represents the
mean and SD (n = 4). ∗ , p < 0.05 as compared with the data of oral control solution.

apomorphine amounts from the GMS and PMS carri-
ers. SLNs did not show a benefit for enhanced brain
uptake in other regions, including the olfactory bulb,
hippocampus, thalamus, and cerebral cortex.
In Vivo Rotational Behavior in 6-OHDA-Lesioned Rats
We determined the contralateral rotation behaviors in
rats following the oral delivery of SLNs and the cor-
responding control. 6-OHDA was used to produce an
animal model of Parkinson’s disease. Apomorphine
can induce contralateral turning by lesioned rats. The
animals that were not challenged with apomorphine
did not exhibit any bias in turning behavior. Oral apo-
morphine delivery from the aqueous solution resulted
in a total rotation number of 20 as shown in Table 5.
Animals displayed a total of 94 and 115 rotations fol-
lowing administration of SLNs with GMS and PMS,
respectively. The total number with the PMS system
was significantly greater (p < 0.05) than that with the
GMS system. The rotation rate (rotations per minute)
of rats treated with SLNs was higher than that of rats
treated with the free control (p < 0.05). There was no
significant difference (p > 0.05) between the rotation
rates of rats treated with the GMS and PMS systems.
DISCUSSION
Poor oral bioavailability in patients and a short half-
life for apomorphine therapy remain clinical issues.
Apomorphine is normally clinically administered by
Table 5. Effect of Apomorphine Treatment on Rotational
Behavior in 6-Hydroxydopamine-Lesioned Rats by Control
Solution and SLNs with Different Emulsifiers∗
Total Number
Formulation of Rotations Rotations (min−1 )
Solution 20.00 ± 8.60 0.62 ± 0.14
SLNs with glyceryl 94.25 ± 9.95 0.95 ± 0.13
monostearate
SLNs with polyethylene 114.50 ± 9.33 1.08 ± 0.09
glycol monostearate
∗
Each value represents the mean ± SD (n = 4).
SLN, solid lipid nanoparticle.
a subcutaneous injection. Unfortunately, as a result
of the short duration of therapeutic effect (<1 h),17
a high injection frequency (more than 10–15 times
a day) is often required. The results of this work
have shown that loading apomorphine into SLNs of-
fers benefits in terms of increased oral absorption and
sustained drug efficacy. The selection of optimal emul-
sifiers is important to facilitate these advantages.
PMS produced a relatively smaller particle size
than GMS. The hydrophile–lipophile balances (HLBs)
of GMS and PMS are 5.5 and 18.0, respectively. This
suggests that the more hydrophilic emulsifier inter-
face contributed to smaller sized particles. PMS used
in this work has stearate groups and a polyethylene
glycol (PEG) spacer. The ability of amphiphilic PEG
to act as a hydrophilic shield in drug carriers can
reduce the surface tension of particles, further reduc-
ing the particle size.18 The emulsifier system with

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011
554 TSAI ET AL.
determined HLBs may well stabilize SLNs with spec-
ified components. One parameter for the emulsifier
film separating the water and lipid domains of the
triglyceride-based system is the spontaneous mean
curvature, H0 ,19 which expresses the natural ten-
dency of the monolayer to bend away from a flat geom-
etry. H0 is positive for emulsifiers with a large polar
head group and a small nonpolar group and decreases
with an increasing size of the nonpolar group. GMS
may be too highly lipophilic to form stable systems. In
the absence of an optimized emulsifier, this may lead
to instability of dispersions because the interface can-
not be fully covered by emulsifier molecules.20
SLNs without apomorphine loading showed a neg-
ative zeta potential. PF68 is a nonionic species. The
negative charge shown by SLNs is believed to have
resulted in HSPC. HSPC used in this study con-
tained 90% phosphatidylcholine, which that is un-
charged. The other components (10%), such as phos-
phatidylethanolamine, phosphatidic acid, and phos-
phatidylinositol, are negatively charged. Some free
fatty acids derived from the hydrolysis of monoglyc-
erides in GMS may have occurred.21 This contributed
to the greater negative charge on GMS-containing
SLNs (−16 mV) than on PMS-containing formula-
tions (−5 mV). The addition of PEG led to an initial
shielding of the negative charge. This reduction in the
case of PEG-coated nanoparticles was due to an ex-
tension in the plane of shear produced by the presence
of this polymer on the surface.22
Apomorphine hydrochloride has a pKa value (disso-
ciation constant) of 8.92. Apomorphine exhibited pos-
itive charges in the nanoparticulate systems. The in-
clusion of cationic apomorphine in the interface may
shield the negative charge of the membrane. This
indicates that the drug seemed to have a high affin-
ity for the nanoparticle interface. Most SLNs incor-
porate lipophilic drugs because of the lipidic charac-
teristics of the inner phase. SLNs were proposed as
carriers for encapsulating hydrophilic drugs such as
diminazene and methotrexate.23–25 The loading level
of approximately 90% by these SLNs is considered
very high because apomorphine is a molecule with
high hydrophilicity. Therefore, we assumed that apo-
morphine was localized within the interfacial layer
close to the aqueous phase. The crystalline tendency
of the shell near the inner core decreases with the
addition of phospholipids onto the interface.26 More-
over, the presence of phospholipids at the interface
containing PF68 causes a rearrangement of the mono-
layer membrane to a disordered form.27 The resulting
matrix of lipid particles thus produces imperfections
in the crystal lattice and leaves space to accommo-
date drugs near the shell, leading to improved drug
entrapment.
In terms of the storage stability, the GMS system
showed physical changes in particle size. But the PMS
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011
system showed better stability over 21 days. Transi-
tion of dispersed lipid from a metastable form to a
stable form may occur during storage.28,29 Particle
collisions and partial destruction of the emulsifier in-
terface can increase the possibility of aggregation of
the nanoparticles.8 GMS may produce a rapid tran-
sition from a metastable condition to a stable con-
dition for SLNs. A previous study30 suggested that
monoglycerides exhibit lipid degradation and insta-
bility in SLN systems. Replacement of GMS with PMS
improved the stability of the dispersion. The incor-
poration of PEG into the lipid matrix increases the
surface hydrophilicity, thus inhibiting nanoparticle
aggregation.18 Although the size of SLNs with GMS
increased after storage, a great fusion did not occur
in this case. Measurement of the zeta potential allows
predictions about the storage stability of colloidal dis-
persions. A high zeta potential of more than ±30 mV
can provide an electric repulsion that helps to prevent
particle aggregation.31 Our system did not fulfill this
criterion. This rule cannot be strictly applied in the
presence of steric stabilizers such as PF68 and PEG
used in this work.
The chemical nature of lipids is also important be-
cause lipids that form crystalline particles with a per-
fect lattice lead to drug expulsion during storage.31
The SLNs developed in this study exhibited a good
ability to reduce apomorphine expulsion. The location
of apomorphine in the interfacial film but not the lipid
matrix may be the predominant factor in the reduced
possibility of expulsion. Although a slight increase in
size and a decrease in the entrapment efficiency were
observed, the values were still sufficient to maintain
the stability of the SLNs.
The microclimate of the stomach favors particle fu-
sion as a result of its acidity and high ionic strength.10
Both SLNs developed in this study showed aggrega-
tion at the same level following incubation in simu-
lated gastric medium. The SLNs showed increases in
the polydispersity. This may confirm the instability of
these two systems. However, the 12% increase in size
compared with the nontreatment group can be toler-
ated. The incubation of SLNs in simulated intestinal
fluid did not alter the particle size of the PMS sys-
tem. PEG can form hydrophilic and steric barriers
against the attachment of enzymes,32 thus diminish-
ing the aggregation process. The particle size of the
GMS system was even reduced after incubation in the
intestinal medium. Lipid digestion and degradation
can occur by enzymatic attack from pancreatic lipase
and trypsin.33 This can lead to a decrease in the par-
ticle size of SLNs with GMS. The question concerning
the influence of the intestinal fluid on SLN degrada-
tion remains open. Unfortunately, only a few studies
have been performed in this regard.10 It is clear that
further research is needed to fully elucidate the mech-
anisms.
DOI 10.1002/jps
 ORAL APOMORPHINE DELIVERY FROM SLNS WITH MONOSTEARATE EMULSIFIERS
Apomorphine is known to be chemically and biolog-
ically unstable. This phenomenon was insignificant
in the acidic environment because the aqueous solu-
tion retained 97% of the apomorphine after incuba-
tion in gastric fluid. Hence, the SLNs did not exhibit
a protective effect on apomorphine in the gastric en-
vironment. Another possibility is the release of drug
molecules from inner core to outer phase by incubat-
ing in the gastric medium. Apomorphine produces dif-
ferent derivatives in acidic and alkaline solutions.14
According to the experimental results of this work,
SLNs were useful in protecting the drug against at-
tack by intestinal medium. Both the GMS and PMS
systems showed similar effects. This may be benefi-
cial for prolonging apomorphine’s half-life in the GI
tract after oral administration.
The oral bioavailability of apomorphine from the
control solution was 2.09%. The results were similar
to those obtained in a human study,5 which showed
bioavailability of 1.7%. After oral administration of
SLNs to rats, the bioavailability was considerably in-
creased by 12- to 13-fold compared with the control.
Both systems showed similar pharmacokinetic pro-
files after oral ingestion. The bioavailability of sub-
lingual apomorphine is estimated to be 16% to 18%.34
The oral delivery route by SLNs can surpass this
level. These data are very encouraging and represent
a new model of an oral delivery system for apomor-
phine devoid of unfavorable pharmacokinetics. The
ability of SLNs to enhance the transport of the drugs
through the GI tract is attributed to different mecha-
nisms including (a) lymphatic transport, (b) drug pro-
tection efficiency, and (c) sustained drug release.22,35
The inclusion of drugs into lipid nanoparticles can
enhance targeting to the lymph system.36 Nanopar-
ticles are composed of a lipid core that can stimu-
late chylomicron formation, which ultimately pulls
the carrier along with the drug following the clas-
sical transcellular mechanism of lipid absorption.25
Apomorphine should be protected from the harsh in-
testinal environments. The advantage of SLNs devel-
oped in this study is the protection of the drug by
the carriers from enzymatic degradation, thereby en-
hancing the occurrence of lymph targeting and delay-
ing metabolism. When sterically stabilizing polymers
such as PF68 are used, there is a steric hindrance
of the anchoring of the complex leading to slower
degradation.12
The benefit of the cationic charge of nanoparticles
was demonstrated and was correlated with the inter-
action between positively charged particles and the
negative charge of the GI tract.37 This may be another
reason for the oral enhancement with apomorphine-
loaded SLNs. SLNs can immobilize the drug and thus
serve as sustained-release systems. This allows the
release of apomorphine at a constant controlled rate.
Hence, the enhanced efficacy of SLNs may have been
DOI 10.1002/jps
555
the result of continuous and more sustained delivery
of apomorphine to the circulation.
The results of drug brain distribution indicated a
detectable apomorphine concentration in different re-
gions of the brain. Apomorphine from the SLNs tar-
geted the cerebellum, brainstem, and striatum with
a relatively high concentration compared with the
aqueous solution. This is important for patients with
Parkinson’s disease because the dorsal striatum is the
critical site of therapeutic action.38 Many sites of the
brain can be considered candidates for supraspinal
control of penile erection, including structures in the
cortex, the thalamus, and the limbic system, as well
as several nuclei in the medulla, the pons, and the
hypothalamus.39 Because the midbrain, pons, and
medulla are components of the brainstem, SLNs may
also be beneficial in treating erectile dysfunction.
It is possible that intact nanoparticles of SLNs can
pass through the GI tract.35,40 Bargoni et al.41 demon-
strated that the uptake and transport of SLNs parti-
cles in lymph and blood after duodenal administration
were evidenced by TEM and by ( counting of labeled
particles. Intact particles in circulation may target
brain tissues for increased therapeutic potency. Many
possible mechanisms of nanoparticle-mediated drug
transport to the brain, such as increased retention
of nanoparticles in the brain blood capillaries, higher
concentration gradients, and endocytosis of nanopar-
ticles, were reported.42 The most likely mechanism is
endocytosis by the endothelial cells lining the brain
blood capillaries. This is triggered by binding of li-
pidic SLNs to negatively charged sites on the surface
of endothelial cells so that they fuse with the cells for
transport across the biomembrane.7 SLNs less than
200 nm in size have an increased blood circulation
by avoiding the reticuloendothelial system and thus
an increased time for which the drug remains in con-
tact with the blood–brain barrier (BBB) and for the
drug to be taken up by the brain.43 Both the GMS and
PMS systems were of a size that fits this criterion. The
mechanisms underlying the brain uptake are not yet
fully understood. Further investigation is needed and
is underway to explore the mechanisms of transport.
6-OHDA lesions significantly reduced the number
of dopaminergic cells and the levels of dopamine and
its metabolites.2 Unilateral lesioning of the striatum
with 6-OHDA is an animal model for Parkinson’s dis-
ease, as it reflects both the nigrostriatal dopaminergic
loss and increased oxidative stress.38 Parkinsonian
rats exhibit reproducible behavioral asymmetry with
the administration of apomorphine, resulting from
postsynaptic dopamine receptor supersensitivity.43
An apomorphine solution and apomorphine-loaded
SLNs induced contralateral rotation after oral deliv-
ery within a certain duration. Apomorphine-induced
turning was seen in only those animals that had
nearly complete dopaminergic damage at the lesioned
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011
556 TSAI ET AL.
site.44 This indicates the successful induction of brain
lesions by 6-OHDA in our study. Higher concentra-
tions of apomorphine from SLNs in circulation and
the brain may contribute to the higher rotation num-
ber and rate than for the aqueous solution. A narrow
interindividual variability of the behavioral profiles
was also observed for the group treated with SLNs.
It can be seen that the total number of rotations for
the PMS system was greater than that for the GMS
system. Although the pharmacokinetic profiles for
both systems were similar, the PMS system showed
a higher level than the GMS system on the basis
of a comparison of the mean values. This insignifi-
cant discrepancy may have led to a meaningful ef-
fect for behavioral rotation. Nanoparticles are PEGy-
lated to form a hydrated stealth shell that protects
particles from destruction by proteins.45,46 PEG can
prolong the blood circulation time of a drug. Com-
pared with classical nanoparticles, PEGylated coun-
terparts show increased half-lives, decreased plasma
clearance rates, and a shift in distribution in favor
of diseased tissues. A previous study47 also indicated
the targeting of PEGylated nanoparticles to the brain
without damage to the BBB. Another possibility is the
smaller size of the PMS system, which had a higher
total surface area for lymphatic transport.
CONCLUSIONS
The purpose of this study was to assess the feasi-
bility of oral apomorphine delivery using SLNs as
carriers. The physicochemical properties showed that
each nanoparticulate carrier (GMS and PMS systems)
had distinguishable characteristics, which may affect
the in vivo performances of the SLNs. Both systems
increased the oral bioavailability of apomorphine to
similar levels. The clinical relevance of this is that the
dose and frequency of the drug administration can be
reduced. The in vivo drug distribution results indi-
cated that SLNs successfully targeted apomorphine
to the brain striatum and greatly improved the abil-
ity of apomorphine to treat Parkinson’s disease. In a
rat model of Parkinson’s disease, we found that the
efficacy of the PMS system was superior to that of the
GMS system in terms of a behavioral index. Carriers
with the PEG moiety also exhibited superior stabil-
ity as nanoparticles. It should be noted that SLNs
with PMS are a promising delivery system for apo-
morphine after oral administration. The oral inges-
tion of apomorphine from SLNs may provide a better
alternative to subcutaneous administration.
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