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ORIGINAL RESEARCH
open access to scientific and medical research
Open Access Full Text Article
http://dx.doi.org/10.2147/IJN.S120939
Intranasal delivery of rotigotine to the brain with
lactoferrin-modied PEG-PLGA nanoparticles for
Parkinson’s disease treatment
Chenchen Bi
1,
*
Aiping Wang
1,
*
Yongchao Chu
1
Sha Liu
1
Hongjie Mu
1
Wanhui Liu
1
Zimei Wu
1
Kaoxiang Sun
1
Youxin Li
1,2
1
School of Pharmacy, Collaborative
Innovation Center of Advanced
Drug Delivery System and Biotech
Drugs in Universities of Shandong,
Key Laboratory of Molecular
Pharmacology and Drug Evaluation
(Yantai University), Ministry of
Education,
2
State Key Laboratory
of Long-Acting and Targeting Drug
Delivery System, Shandong Luye
Pharmaceutical Co, Ltd., Yantai,
People’s Republic of China
*These authors contributed equally
to this work
Abstract: Sustainable and safe delivery of brain-targeted drugs is highly important for successful
therapy in Parkinson’s disease (PD). This study was designed to formulate biodegradable
poly(ethylene glycol)–poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NPs), which
were surface-modified with lactoferrin (Lf), for efficient intranasal delivery of rotigotine to the
brain for the treatment of PD. Rotigotine NPs were prepared by nanoprecipitation, and the effect
of various independent process variables on the resulting properties of NPs was investigated by
a Box–Behnken experimental design. The physicochemical and pharmaceutical properties of the
NPs and Lf-NPs were characterized, and the release kinetics suggested that both NPs and Lf-NPs
provided continuous, slow release of rotigotine for 48 h. Neither rotigotine NPs nor Lf-NPs
reduced the viability of 16HBE and SH-SY5Y cells; in contrast, free rotigotine was cytotoxic.
Qualitative and quantitative cellular uptake studies demonstrated that accumulation of Lf-NPs
was greater than that of NPs in 16HBE and SH-SY5Y cells. Following intranasal administration,
brain delivery of rotigotine was much more effective with Lf-NPs than with NPs. The brain
distribution of rotigotine was heterogeneous, with a higher concentration in the striatum, the
primary region affected in PD. This strongly suggested that Lf-NPs enable the targeted delivery
of rotigotine for the treatment of PD. Taken together, these results demonstrated that Lf-NPs
have potential as a carrier for nose-to-brain delivery of rotigotine for the treatment of PD.
Keywords: rotigotine, lactoferrin-modified PEG-PLGA nanoparticles, brain targeting, intranasal
delivery, Parkinson’s disease
Introduction
Parkinson’s disease (PD), the second most common neurodegenerative disorder in
the world, manifests in approximately 1%–1.5% of individuals older than 60 years
of age.
1
Because of the aging population and longer life expectancy, the incidence
of PD is expected to increase.
2
Currently available antiparkinsonian drugs include
dopamine agonists (eg, apomorphine), monoamine oxidase type B inhibitors and
cholinesterase inhibitors (eg, donepezil).
3
Among these drugs, the most commonly
used is levodopa, which alleviates the symptoms and improves the quality of life
of patients for several years. However, patients gradually develop tolerance to the
therapeutic benefits of levodopa, and its use is related to some harmful side effects,
including response fluctuations (wearing off) and levodopa-induced dyskinesia
(LID).
2,4
Therefore, novel strategies that focus on sustained drug release are required to
decrease the occurrence of LID and attain continuous dopaminergic stimulation (CDS).
5
Rotigotine was initially developed as an adjuvant treatment for early, secondary and
advanced PD. It is a non-ergoline, enantioselective dopamine agonist, with preference
Correspondence: Kaoxiang Sun
School of Pharmacy, Yantai University,
No 30 Qingquan road, Yantai, 264005
Shandong Province, People’s Republic of
China
Tel +86 535 394 6400
Email sunkx@ytu.edu.cn
Youxin Li
Shandong Luye Pharmaceutical Co,
Ltd, No 9 Baoyuan Road, Yantai
264003, Shandong Province, People’s
Republic of China
Tel +86 535 394 6187
Email liyouxin@luye.cn
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Bi et al
for D
3
D
2
D
1
receptors, which is considered to be of
particular importance for its efficacy in PD.
6
The blood–brain barrier (BBB) is composed of micro-
vascular endothelial cells sealed by tight junctions. It plays
a central role in regulating intra- and intercellular signaling
pathways and maintaining central nervous system homeo-
stasis, but it also restricts the delivery of therapeutic agents
to the brain.
7
To overcome this issue, a potential route is the
direct transport of drugs from the nose to the brain via the
olfactory and trigeminal nerve pathways, thereby bypass-
ing the BBB.
3
Over the past decades, research efforts have
been reported on the design of nasal drug carriers for brain
delivery, including nanoparticles (NPs), liposomes, micro-
spheres and microemulsions.
8–11
Poly(ethylene glycol)–poly(lactic-co-glycolic acid)
(PEG-PLGA) NPs constitute a promising strategy for brain-
targeted delivery of rotigotine following intranasal admin-
istration. PEG-PLGA is biocompatible and biodegradable,
and it has long-circulating behavior. NPs mask the physico-
chemical properties of drugs, in addition to providing con-
trolled drug release, lower drug toxicity, as well as improved
bioavailability, therapeutic efficacy and biodistribution.
12,13
However, NPs contain surface PEG chains, which are likely
to inhibit cell surface interactions.
14
To achieve more efficient
delivery from nose to brain, one approach is to modify NPs
with biological ligands.
Lactoferrin (Lf) is a naturally occurring, iron-binding gly-
coprotein of the transferrin family, with a molecular weight of
80 kDa.
15
Based on previous studies, the Lf receptor (LfR) is
highly expressed on the apical surface of respiratory epithe-
lial cells, as well as brain endothelial cells and neurons.
16,17
Furthermore, LfR is overexpressed in the capillaries and neu-
rons associated with age-related neurodegenerative diseases,
including PD, Alzheimer’s disease and amyotrophic lateral
sclerosis.
18
Compared with transferrin (Tf) and OX-26, an
anti-Tf receptor antibody, Lf displays higher brain uptake.
19
Therefore, Lf may improve nose-to-brain delivery of NPs
following intranasal administration.
Accordingly, the aim of this study was to prepare Lf-
modified PEG-PLGA NPs (Lf-NPs) as a carrier for brain
delivery of rotigotine, following intranasal administration
for the treatment of PD. Experiments with mice and cells
were conducted to evaluate the brain-targeting ability and
toxicity of this system.
Materials and methods
Materials and animals
Bovine Lf, 2-iminothiolane (Traut’s reagent) and 5,
5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent) were
purchased from Sigma-Aldrich (St Louis, MO, USA).
Rotigotine was kindly provided by Luye Pharma Group
(Shandong, People’s Republic of China). PEG-PLGA-
maleimide (mal-PEG-PLGA; 3,400–20,000 Da, 50:50
LA:GA, w/w) and methoxy-PEG-PLGA (mPEG-PLGA;
2,000–20,000 Da, 50:50 LA:GA, w/w) were purchased
from Polyscitech (West Lafayette, IN, USA). Sephadex G25
and Sepharose CL-4B were purchased from GE Healthcare
Bio-Sciences (Piscataway, NJ, USA). Furthermore, 1,1′-
dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide
(DiR) and Hoechst 33342 were purchased from Fanbo
Biochemicals Co (Beijing, People’s Republic of China);
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT reagent) and 3-(2′-benzothiazolyl)-7-
diethylaminocoumarin (coumarin-6) were purchased from
Aladdin Industrial Corp (Shanghai, People’s Republic of
China). A bicinchoninic acid (BCA) protein quantitation kit
was purchased from Beyotime Biotechnology (Shanghai,
People’s Republic of China). All other reagents were of
laboratory grade.
The cell lines 16HBE and SH-SY5Y and male Kunming
(KM) mice (age: 4–5 weeks; weight: 20±2 g) were kindly
provided by Luye Pharma Group (Shandong, People’s
Republic of China). All animal studies were performed
according to the Guide for the Care and Use of Laboratory
Animals. The China Animal Care and Use Committee
approved the study protocols.
Preparation of NPs and Lf-NPs
Preparation of NPs
Nanoprecipitation was used to prepare the NPs.
20
In brief,
accurately weighed mal-PEG-PLGA, mPEG-PLGA and
rotigotine were dissolved in the organic phase (acetone)
and then added dropwise into the aqueous phase under ice-
cooling, with continued stirring to completely volatilize the
organic solvent. The rotigotine NP was obtained. The same
approach was applied for the coumarin-6 NP and DiR NP.
All NPs were washed three times with ultrapure water, con-
centrated and collected by ultrafiltration.
Box–Behnken experimental design
To optimize the preparation process and to determine the
impact of various factors on the encapsulation efficiency and
particle size of NPs, a four-factor, two-level Box–Behnken
design was used, based on preliminary experimental data.
20
A total of 29 confirmatory formulation runs were gener-
ated with five center points using Design-Expert
®
software
(version 8.0.6; Stat-Ease Inc, Minneapolis, MN, USA).
Levels of the independent and dependent variables are shown
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Intranasal delivery of rotigotine with lactoferrin-modied PEG-PLGA NPs
in Table 1. The effect of the independent process variables
on the dependent responses was studied using second-order
polynomial equations.
Preparation of Lf-NPs
Lf was thiolated by reaction with a 20:1 M excess of
2-iminothiolane (Traut’s reagent) for 60 min, according
to Huwyler’s method.
21,22
The products were purified via
a Sephadex G25 column. The quantity of introduced thiol
groups was measured by ultraviolet (UV) spectrophotometry
(λ=412 nm) (Metash Instruments Co, Ltd, Shanghai, People’s
Republic of China) with Ellmann’s reagent. To generate the
Lf-NPs, purified, thiolated Lf was mixed with maleimide-
functionalized NPs and incubated at room temperature for
9 h, enabling the maleimide groups to react with the thiol
groups via a maleimide–thiol coupling reaction. The product
was eluted with 0.01 M phosphate-buffered saline (PBS)
buffer (pH 7.4) using a 1.5×20 cm Sepharose CL-4B column
to remove the unconjugated protein. The concentration of
protein and the protein conjugation efficiency were assessed
with the BCA kit.
Characterization of NPs and Lf-NPs
Morphology, particle size and zeta potential
Morphological examinations of NPs were conducted by
transmission electron microscopy (TEM) (H-600; Hitachi,
Tokyo, Japan) following negative staining with sodium
phosphotungstate solution (1%, w/v). The average particle
size and zeta potential of the NPs were determined with
a particle analyzer (Delsa Nano C; Beckman Coulter Inc,
Brea, CA, USA).
Encapsulation efciency and drug loading capacity
The encapsulation efficiency was determined by ultrafiltra-
tion. The samples were placed in an ultrafiltration device
(100 kMWCO; Sartorius, Göettingen, Germany) and centri-
fuged at 3,500 rpm for 10 min at room temperature to isolate
the free drug. The same volume of each sample was dissolved
in acetonitrile to obtain the total amount of drug. The con-
centrations of rotigotine and coumarin-6 were measured with
a high-performance liqid chromatography (HPLC) system
(LC-20A VP system; Shimadzu, Kyoto, Japan), while the con-
centration of DiR was measured by UV spectrophotometry.
The encapsulation efficiency and drug loading capacity of the
NPs were calculated using the following equation:
Entrapment efficiency
Total amount of drug Amount of fr
(%) =
−eee drug
Drug dosage
×100%
Drug loading
Total amount of drug Amount of free drug
We
(%) =
−
iight of NPs
×100%
In vitro drug release study
Cellulose membrane dialysis tubing was used to investigate
the release kinetics.
23
In brief, 2 mL of NPs and Lf-NPs
samples were encased in dialysis bags (molecular weight
cutoff: 7 kDa), which were incubated in 30 mL phosphate
buffer (pH 7.4) containing 0.5% (w/v) sodium dodecyl sul-
fate, with shaking in a water bath at 37°C. At prespecified
time intervals, 1 mL of phosphate buffer was withdrawn from
outside the dialysis bag, and an equivalent volume of release
medium was supplemented. The rotigotine content was ana-
lyzed by HPLC at 223 nm. The amount of rotigotine released
was calculated using a standard curve of rotigotine.
Cellular studies of NPs and Lf-NPs
Cell culture of SH-SY5Y and 16HBE cells
Cells of the human bronchial epithelial cell line 16HBE as
well as SH-SY5Y cells, derived from the human neuroblas-
toma cell line SK-N-S, were incubated at 37°C and 5% CO
2
in Dulbecco’s Modified Eagle’s Medium supplemented
with 10% fetal bovine serum, 100 IU/mL penicillin and
100 mg/mL streptomycin sulfate.
Cytotoxicity studies
The MTT assay was used to determine the cytotoxicity of
NPs, Lf-NPs, rotigotine NPs and rotigotine Lf-NPs. The
16HBE cells and SH-SY5Y cells were seeded into a 96-well
plate at a density of 10
4
cells per well. After culturing for
24 h, the cells were treated with different concentrations
of free rotigotine (12.5–200 μg/mL), or corresponding
concentrations of rotigotine NPs or rotigotine Lf-NPs. The
plates were incubated at 37°C in 5% CO
2
for either 24 h or
48 h, followed by 4 h exposure to 20 μL of MTT solution
Table 1 Different levels of variables in the Box–Behnken design
Variables Levels
Low Medium High
Independent variables
A = polymer concentration, mg/mL
0.20 0.85 1.50
B = aqueous/organic phase ratio
5.00 10.00 15.00
C = theoretical drug loading, %
1.00 5.50 10.00
D = ice water bath duration, h
0.00 1.00 2.00
Dependent variables Desired constraints
Y
1
= diameter of particles, nm
Minimize
Y
2
= entrapment efciency, %
Maximize
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(5 mg/mL) per well. To solubilize the resultant formazan
crystals, 200 μL of dimethyl sulfoxide was added to each
well. Then, the absorbance of each well was measured at
570 nm after gentle shaking for 10 min.
Cellular uptake of coumarin-6-labeled NPs
and Lf-NPs
Qualitative analyses of cellular internalization of coumarin-6-
labeled NPs and Lf-NPs were performed using fluorescence
microscopy (Eclipse E400; Nikon Corporation, Tokyo,
Japan), while the quantitative analyses were performed
using flow cytometry (BD FACSAria III; BD Biosciences,
San Jose, CA, USA).
For fluorescence microscopy, 16HBE cells and SH-
SY5Y cells were seeded into 24-well plates at a density
of 4,000 cells per well and incubated at 37°C in 5% CO
2
.
On Day 2, the cells were treated with NPs or Lf-NPs
containing different concentrations of coumarin-6 for 2 h.
Furthermore, the cells were incubated with NPs or Lf-NPs
(0.60 μg/mL) for different time intervals. Subsequently,
the cells were washed three times with cold PBS and fixed
with 4% paraformaldehyde for 10 min. To label the cell
nucleus for locating NPs and Lf-NPs within cells, the cells
were further incubated with Hoechst 33342 for 10 min.
Finally, the cells were washed three times and visualized
by fluorescence microscopy.
For flow cytometry, 16HBE cells and SH-SY5Y cells
were seeded into six-well plates at a density of 10
5
cells
per well and treated as described earlier. After incubation
with NPs or Lf-NPs containing different concentrations of
coumarin-6 (0.05–1 μg/mL) for 2 h, the cells were washed
three times with cold PBS. Then, cells were trypsinized and
centrifuged at 1,500 rpm for 5 min. After resuspension in
PBS, the cells were analyzed by flow cytometry.
In vivo study of NPs and Lf-NPs
Brain accumulation of DiR-labeled NPs and Lf-NPs
after intranasal administration
To examine the brain distribution of Lf-NPs and NPs fol-
lowing intranasal administration, in vivo real-time fluores-
cence imaging analysis (Care stream In Vivo FX; Bruker,
Madison, WI, USA) was used. Mice were randomized into
three groups and treated with DiR solution, DiR NPs or
DiR Lf-NPs as a single dose of 0.25 mg DiR/kg of body
weight. Before administration, mice were anesthetized by
intraperitoneal injection of chloral hydrate. For intranasal
administration, a polyethylene 10 (PE 10) tubing attached
to a microliter syringe was inserted ~10 mm deep into the
nares. Mice were anesthetized and imaged at 0.5, 1, 2, 4 and
6 h postadministration. To further verify the brain targeting
of NPs and Lf-NPs, mice were sacrificed at 4 h postadmin-
istration and dissected to obtain the whole brain, heart, liver,
spleen, lungs and kidneys. Images were captured with an in
vivo imaging system.
Brain distribution of rotigotine NPs and Lf-NPs
following intranasal administration
Forty mice were randomly divided into two groups for intra-
nasal administration of either rotigotine NPs, as a control
group, or rotigotine Lf-NPs. At prespecified time points
(0.25, 1, 2, 4 and 8 h), blood samples were obtained by
removing the eyeball, and the mice were sacrificed for dis-
section of the olfactory bulb, striatum, cerebrum with striatum
removed and cerebellum. Blood samples were centrifuged
at 5,000 rpm for 10 min to collect plasma. Other tissues
were homogenized after adding three volumes of saline. To
extract the drugs, two volumes of acetonitrile were added
to plasma and the tissue homogenates. The concentration of
drug was measured via liquid chromatography–tandem mass
spectrometry (LC-MS/MS) (AB Sciex Triple Quad™ 4500;
Sciex, Framingham, MA, USA) (the LC-MS/MS conditions
were as follows: chromatographic column, Shim-pack XR-
ODS III C18; mass spectrometer ion source, electrospray
ionization source; rotigotine ion pair, 316.0/147.1).
Statistical analysis
Data are presented as the mean ± standard deviation (SD).
Statistical comparisons were performed using a two-tailed
Student’s t-test. Differences were considered statistically
significant for a P-value 0.05 and highly significant for a
P-value 0.01.
Results
Preparation and characterization of NPs
and Lf-NPs
Box–Behnken design
According to the principles of response surface methodol-
ogy (RSM), using the Box–Behnken design, 29 tests were
designed, and the corresponding dependent variables are
shown in Table 2. Design-Expert 8.0.6 software was used
to perform regression analysis using various indexes, as
well as to delineate the interaction between the four inde-
pendent factors.
Polynomial equations between independent variables and
dependent variables were constructed using Design-Expert
8.0.6 software.
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Intranasal delivery of rotigotine with lactoferrin-modied PEG-PLGA NPs
YABCDAB
AC AD
1
55 52 20 88 15 43 13 11 6381490
11 95 615
=++−−+
−−
.. ....
..++++
++++
335065 667
14 47 10 07 10 56 541
2222
...
....
BC BD CD
ABCD
The P-value of the regression equation for the particle
size was 0.0023 (P0.01), indicating an extremely sig-
nificant level. Moreover, lack-of-fit analysis (P=0.2038)
revealed that the equation was a good fit and predictive of
the relationship between the factors and particle size. Among
the factors, A, B, C and A
2
had a significant effect on the
particle size.
YABC DAB
AC AD
2
89 05 488466 9671654070
28 88 17610
=.. .. ..
..
−−−++
−−−.. ..
.. ..
71 2181075
19 99 0191590093
22 22
BC BD CD
AB CD
−−
−−−+
The regression equation for entrapment efficiency was
significant (P=0.0303), whereas the lack of fit was not
significant (P=0.0515). Factors C, D, AD, A
2
and C
2
had a
significant effect on entrapment efficiency.
Based on real-life clinical practice and trial requirements,
particle size tends to be minimum, while the encapsulation
efficiency tends to be maximum. The regression model
was further processed to determine the level of interaction
between the four factors. The impact of the interaction
between the various factors on the desirability of encapsula-
tion efficiency and particle size was intuitive, as reflected in
Figure 1. The oval and steep slopes in the response surface
correspond to a significant interaction between two factors.
As indicated by the flag at the highest point of the response
surface, the highest response value was within the selected
range of each factor.
The optimum formulation of rotigotine NP was selected
according to the desired constraints within the range for
independent variables, as well as the minimized and maxi-
mized constraints for the diameter of particles and entrap-
ment efficiency, respectively. The optimized formulation
Table 2 Effect of independent variables on dependent variables
Run Independent variables Dependent variables
Polymer concentration,
mg/mL (A)
W/O ratio
(B)
Theoretical drug
loading, % (C)
Ice water bath
duration, h (D)
Diameter of
particles, nm (Y
1
)
Entrapment
efciency, % (Y
2
)
1 1.50 10.00 5.50 2.00 76.10 84.58
2 0.85 10.00 10.00 0.00 55.70 42.09
3 0.85 15.00 5.50 0.00 93.80 92.14
4 0.85 10.00 5.50 1.00 45.40 90.59
5 0.85 10.00 5.50 1.00 67.80 84.16
6 0.20 5.00 5.50 1.00 42.50 88.56
7 0.85 10.00 1.00 2.00 68.20 104.84
8 1.50 15.00 5.50 1.00 141.70 27.86
9 1.50 5.00 5.50 1.00 50.70 58.78
10 0.85 5.00 10.00 1.00 58.00 73.95
11 0.85 5.00 5.50 0.00 67.40 85.53
12 0.85 10.00 10.00 2.00 65.00 61.15
13 1.50 10.00 10.00 1.00 82.40 36.35
14 0.20 10.00 1.00 1.00 57.90 25.21
15 1.50 10.00 5.50 0.00 106.50 44.36
16 0.85 15.00 1.00 1.00 90.20 103.12
17 0.85 15.00 10.00 1.00 68.40 54.63
18 0.85 5.00 1.00 1.00 93.20 79.61
19 0.20 15.00 5.50 1.00 73.90 54.83
20 0.85 10.00 5.50 1.00 54.70 79.40
21 0.20 10.00 5.50 0.00 65.00 61.62
22 0.85 10.00 5.50 1.00 46.10 88.67
23 1.50 10.00 1.00 1.00 139.90 102.87
24 0.85 10.00 1.00 0.00 85.60 42.77
25 0.85 15.00 5.50 2.00 79.00 102.70
26 0.20 10.00 10.00 1.00 48.20 74.23
27 0.20 10.00 5.50 2.00 59.20 108.88
28 0.85 10.00 5.50 1.00 63.60 102.44
29 0.85 5.00 5.50 2.00 50.00 104.82
Abbreviation: W/O, water:organic phase.
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