Gelatin nanoparticle-mediated intranasal delivery of substance P protects against 6-hydroxydopamine-induced apoptosis: an in vitro and in vivo study
Drug Design, Development and Therapy
gelatin nanoparticle-mediated intranasal delivery of substance P protects against 6-hydroxydopamine- induced apoptosis: an in vitro and in vivo study
0 apoptosis , Parkinson's disease
1 Ying-Zheng Zhao school of Pharmaceutical sciences, Wenzhou Medical University , Wenzhou, Zhejiang 325035, People's republic of china
8 1 0 2 - l u J - 2 1 n o 8 4 . 6 9 . 7 3 1 . 9 7 y b / m o c . s s e r p e v o .dw l.y from rsoe PowerdbyTCPDF(ww.tcpdf.org) Background: The aim of this study was to investigate the protective role of intranasally administered substance P-loaded gelatin nanoparticles (SP-GNPs) against 6-hydroxydopamine (6-OHDA)-induced apoptosis in vitro and in vivo, and to provide a new strategy for treating brain pathology, such as Parkinson's disease. Methods: SP-GNPs were prepared by a water-in-water emulsion method, and their stability, encapsulating efficiency, and loading capacity were evaluated. PC-12 cells were used to examine the enhancement of growth and inhibition of apoptosis by SP-GNPs in vitro using MTT assays. In the in vivo study, hemiparkinsonian rats were created by intracerebroventricular injection of 6-OHDA. The rats then received intranasal SP-GNPs daily for 2 weeks. Functional improvement was assessed by quantifying rotational behavior, and the degree of apoptosis was assessed by immunohistochemical staining for caspase-3 in the substantia nigra region. Results: PC-12 cells with 6-OHDA-induced disease treated with SP-GNPs showed higher cell viability than their untreated counterparts, and cell viability increased as the concentration of substance P (SP) increased, indicating that SP could enhance cell growth and inhibit the cell apoptosis induced by 6-OHDA. Rats with 6-OHDA-induced hemiparkinsonism treated with SP-GNPs made fewer rotations and showed less staining for caspase-3 than their counterparts not treated with SP, indicating that SP protects rats with 6-OHDA-induced hemiparkinsonism from apoptosis and therefore demonstrates their functional improvement. Conclusion: Intranasal delivery of SP-GNPs protects against 6-OHDA-induced apoptosis A hydroxyl derivative of catecholamine, 6-hydroxydopamine (6-OHDA) is a neurotoxicant
gelatin nanoparticles; intranasal delivery; substance P; 6-hydroxydopamine
open access to scientific and medical research
O r i g i n a l r e s e a r c h
cui-Tao lu 1,2
rong-rong Jin 2
Yi-n a Jiang2
Qian lin 2
Kai-li Mao 2
Fu-rong Tian 2
1The second affiliated hospital,
Wenzhou Medical University, 2s chool
of Pharmaceutical s ciences, Wenzhou
Medical University, Wenzhou,
People’s r epublic of c hina
*These authors contributed equally
to this work
that activates apoptotic and proapoptotic factors, eg, caspase proteins, as well as
transduction of Bax factor, leading to apoptosis and degeneration of dopamine neurons.1–3 Studies
have shown that 6-OHDA can induce apoptosis in PC-12 (adrenal pheochromocytoma)
cells by activating apoptotic factors.2 Also, rats intracerebroventricularly injected with
6-OHDA show apoptosis, degeneration, and death of dopaminergic neurons in the
substantia nigra. The apoptosis occurs mainly due to a caspase family member-mediated
protease cascade, and caspase-3 has a vital role in this process. If large numbers of
dopaminergic neurons undergo apoptosis, the result is irreversible degenerative brain disease,
ie, Parkinson’s disease (PD), for which there is still no effective therapy.4
Substance P (SP), a member of the tachykinin peptide family, is involved in
the regulation of many biological processes in the central and peripheral nervous
systems (Figure 1).5 SP-containing neurons are widely
distributed throughout the central and peripheral nervous
systems, especially in the substantia nigra region.6 Most SP
receptors are located within dopaminergic and cholinergic
neurons in the basal ganglia, suggesting that SP may have a
physiologically regulating effect on these neurons.7 Therefore,
018 SP and its receptor may have a therapeutic use in PD, which is
l-u2 characterized by impaired dopaminergic transmission.
-J21 It has been reported that SP and dopamine are regulated
no via a positive feedback mechanism whereby binding of SP
.486 to its tachykinin neurokinin-1 receptor on dopamine neurons
.379 causes striatal release of dopamine, and by binding to its
.971 D1 receptor on striatal projection neurons, dopamine
poten/yb tiates the release of SP within the substantia nigra.8 Previous
com research has shown that expression of SP is significantly
.sse decreased in the basal nuclei in both hemiparkinsonian
rvpe rats and PD patients, indicating probable involvement
.dow l.y of SP in regulating the pathogenesis of PD.7 In vitro
ww no experiments have demonstrated that SP can reduce
:s s Fas-induced apoptosis in human tenocytes via regulation of
h na neurokinin-1-specific and Akt-specific pathways.9 An vivo
from rsoe study suggested that SP can reduce apoptotic cell death by
modulating the immune response in the early stages after
spinal cord injury.10
Intracerebroventricular administration of SP to rats with
6-OHDA-induced disease can increase the dopamine content
in the brain and help to restore the dopamine deficit, with
the positive effects seen being more prominent in the
nigrostriatal system than in the mesocorticolimbic dopaminergic
system.11 Further, hemiparkinsonian rats pretreated with SP
fragments12 or an SP receptor antagonist13 show increased
levels of dopamine and its metabolites in the corpus striatum,
as well as clear functional recovery. However, the current
research focuses on the pharmacological effects of SP given
by invasive intracerebroventricular injection, which can
result in a high local concentration of SP in the brain, and it
has been confirmed that a high level of SP in the brain can
induce serious neuroinflammation and further aggravate
illness,8,14,15 so intracerebroventricular injection is not a safe
or practical strategy for PD patients who need continuous
Intranasal administration has been reported to be an
efficient and noninvasive way to delivery biologics directly
into the brain.16 Gelatin nanoparticles (GNPs) are a type
of gelatin-cored nanostructured lipid carrier prepared by a
water-in-water emulsion method and have good stability
and strong penetrating ability, encapsulating efficiency, and
loading capacity, as well as bioactivity.17 It has been reported
that GNPs are a suitable carrier for targeted delivery, making
it possible to delivery therapeutics to a focal zone effectively
without compromising drug stability or concentration.18,19
Theoretically, a novel strategy combining GNP-loaded
therapeutics and the nasal olfactory pathway might maximize the
potential efficacy of SP in the treatment of PD.
In the present study, we investigated whether intranasally
administered SP-GNPs could maximize the ability of SP
to protect against 6-OHDA-induced apoptosis in vitro and
in vivo. SP-GNPs were prepared by a water-in-water
emulsion method and were found to have good stability,
encapsulating efficiency, and loading capacity. The protective
effect of SP-GNPs on PC-12 cells with 6-OHDA-induced
disease was assessed by MTT
[3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay, and the inhibition
of apoptosis and neuroprotective effect of SP-GNPs in rats
with 6-OHDA-induced hemiparkinsonism were evaluated by
behavioral assessment and immunohistochemical staining
Materials and methods
Materials and animals
All the materials and equipment used in this study were
commercially available. SP was purchased from GL Biochem
Ltd (Shanghai, People’s Republic of China). The protocols
and procedures were approved by the local animal
experimentation ethics committee. Male Sprague-Dawley rats weighing
around 300–320 g were provided by the Laboratory Animal
Services Centre at our university. Two to three animals were
housed per stainless steel cage on a 12-hour light/12-hour dark
cycle in an air-conditioned room at 22°C, and checked daily by
the animal care staff. Standard commercial rat chow (Prolab
RMH 2500, PMI Nutrition International LLC, Brentwood,
MO, USA) and water were available ad libitum.
Preparation and characterization
of sP-gnPs and blank gnPs
SP-GNPs and blank GNPs were prepared using
waterin-water emulsion and freeze-drying techniques.20,21 Briefly,
a high concentration of SP was dissolved in 1 mL of 20%
w/v Poloxamer 188-grafted heparin copolymer solution.
This solution was added to 2 mL of 2.0% w/v gelatin
solution to produce a homogeneous mixture. Under sonication
(110 W, 15°C) using a probe sonicator, d ,l -glyceraldehyde
was injected into the mixed solution until its final
concentration reached 0.1% w/v to initiate the cross-linking reaction.
The mixture was bathed at 5°C under magnetic stirring at
2,500 rpm for 5 hours to form a suspension of SP and GNPs.
The suspension was lyophilized to obtain a powder
containing SP and polymeric GNPs. Next, the lyophilized powder
was dispersed in a solution containing soy
phosphatidylcholine, trehalose, and cholesterol. By sonication (90 W,
20 seconds) at 25°C, the suspension was then lyophilized to
obtain a powder containing SP-GNPs, which were
reconstituted in double-distilled water to form a 2 mg/mL SP-GNP
suspension for administration. Blank GNPs (using gelatin
solution instead of SP gelatin solution during preparation)
was also prepared for the subsequent experiment.
The morphologies of the SP-GNPs and blank GNPs
were determined using a scanning electron microscope
(X-650, Hitachi, Tokyo, Japan). The particle size and zeta
potential were determined by dynamic light scattering using
a Nicomp™ 380 ZLS zeta potential/particle sizer (PSS
Nicomp, Santa Barbara, CA, USA).
To determine the encapsulating efficiency of the
SPGNPs and blank GNPs, approximately 1.5 mL of the SP-GNP
dispersion were placed in a microtube and centrifuged at
10,000 g for 40 minutes. The supernatant was then
collected and diluted for determination of SP content using an
enzyme-linked immunosorbent assay kit; this experiment was
performed in triplicate. Drug encapsulation efficiency (%) =
(total amount of drug − amount of drug in supernatant)/total
amount of drugs added initially ×100%.
experiment in vitro
Male rat PC-12 cells were used for the in vitro study. The
PC-12 cells were cultured at 37°C in high-glucose
Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine
serum and 1% penicillin–streptomycin in a humidified
incubator containing 5% CO2. Cells in the logarithmic
growth phase were harvested with trypsin for further
The ability of SP-GNPs to impede the growth of PC-12 cells
with 6-OHDA-induced disease was confirmed by MTT assay
(run in triplicate). PC-12 cells were cultured in a 96-well
plate for 24 hours at a density of 5,000 cells per well. With
blank PC-12 cells as the control, 100 μM of 6-OHDA was
added to the cells for 24 hours to induce cell apoptosis, after
which blank GNPs and different concentrations of SP-GNPs
were incubated for another 24 hours. Next, 10 μL of MTT
5 mg/mL were added to each well and incubated for 4 hours;
100 μL of formazan solution was then added to each well,
followed by incubation for a further 4 hours to dissolve the
crystals that developed in each well. The plate was then put
into a microplate reader to measure the optical density at
526 nm and quantify the extent of cell viability. The higher
the amount of cell viability in each well, the less the degree
experiment in vivo
rat model of hemiparkinsonism
The rats were anesthetized with pentobarbital sodium
60 mg/kg and then injected with 12 μL of 6-OHDA solution
into the right striatum (or vehicle for sham animals) using
stereotaxic apparatus (Figure 2).22,23 Gentamicin was then
given to prevent infection.
Four weeks after injection of the 6-OHDA solution,
rodent behavior was evaluated by counting the number of
apomorphine-induced rotations to determine if the rat model
of hemiparkinsonism had been successfully created. The rats
were injected with apomorphine 0.5 mg/kg subcutaneously,
and both contralateral and ipsilateral full-body rotations were
recorded in the following 30 minutes. At least seven full-body
contralateral rotations per minute were considered to indicate
a successful hemiparkinsonian (PD) model, and these rats
were used in the following experiment.
As one of the endpoint shear enzymes in apoptosis, caspase-3
plays a critical role in the apoptotic cascade.24
Immunohistochemical staining with anti-caspase-3 antibody was used to
evaluate levels of apoptosis in the substantia nigra region in
hemiparkinsonian rats treated or not treated with SP.
ImagePro Plus version 6.0 was used to quantify the number of cells,
the areas stained, and the degree of staining. The better the
protective effect against 6-OHDA-induced apoptosis, the
lower rates of caspase-3 staining and apoptosis in brain
sections from the PD rats.
Statistically significant differences across multiple groups
were determined using one-way analysis of variance with the
Newman–Keuls post hoc test. Statistically significant
differences between individual groups was determined using the
Mann–Whitney U-test. All testing was done using Statistical
Package for the Social Sciences version 19 software (SPSS
Inc, Chicago, IL, USA). A difference was considered to be
statistically significant at P0.05.
Physicochemical properties and
bioactivity of sP-gnPs and blank gnPs
Scanning electron micrographs showed that the SP-GNPs
and blank GNPs were uniform in shape and size (Figure 3).
Characterization for the SP-GNPs and blank GNPs is shown
in Table 2. Dynamic light scattering showed the average
particle size of the blank GNPs to be 136±1.32 nm.
The polydispersity index (PDI) indicates the
distribution of particle size. Low PDI values were observed for the
SP-GNPs and the blank GNPs (Table 2), indicating that
both were monodispersed stable systems. After loading with
SP, the mean diameters of the nanoparticles and liposomes
increased, but were still below 200 nm (Table 2).
The zeta potential is an important indicator of the physical
stability of nanoparticles. Nanoparticles with a high absolute
zeta potential value are electrically stable while those with a
low absolute zeta potential value tend to be less electrically
stable. As shown in Table 2, both the SP-GNPs and the blank
GNPs had a strong negative surface charge, indicating that
coating with phospholipids makes these nanoparticles more
The encapsulation efficiency and loading capacity of
the SP-GNPs were 93.3±1.4% and 5.2±0.02%, respectively
Table 3 shows the ability of different concentrations of
SP-GNPs to limit the growth of PC-12 cells with
6-OHDAinduced disease. When compared with untreated PC-12 cells
with 6-OHDA-induced disease, those treated with blank
GNPs showed slightly higher but not significantly
different cell viability, whereas their counterparts treated with
SP-GNPs did demonstrate significantly higher cell viability
(P0.05), indicating that SP-GNPs can decrease the extent
of apoptosis caused by 6-OHDA and enhance cell growth.
In the meantime, cell viability increased as the concentration
of SP increased, suggesting that within a certain range of
concentrations, the degree of inhibition of apoptosis achieved
by SP is concentration-dependent.
Behavioral evaluation of PD rats after
2 weeks of treatment
The number of apomorphine-induced rotations following
2 weeks of daily treatment with SP-GNPs in each
experimental group were consistent with the dopamine levels in the
diseased brain. As seen in Table 4, the PD rats that received
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801 intranasal SP-GNP treatment made fewer rotations than
l-2u those that received blank GNPs, demonstrating that SP could
-J21 protect dopaminergic neurons against 6-OHDA-induced
on apoptosis and aid recovery of diseased dopaminergic neurons
.684 in rats with PD. Meanwhile, the PD rats that received daily
.937 intranasal treatment with 75 μg or 100 μg of SP showed a
.971 significant decrease in the number of apomorphine-induced
/yb rotations when compared with hemiparkinsonian rats
com (P0.05), while PD rats that received 50 μg of SP per day
.sse intranasally showed a trend towards improvement (P0.05),
rvpe indicating that the extent of the protective effect of SP against
.dow l.y 6-OHDA-induced apoptosis has a positive relationship with
/ww no the concentration of SP.
h na immunohistochemical staining
from rsoe of caspase-3 in the substantia nigra
deda ropF As seen in Figure 4, immunohistochemical staining for
lno caspase-3 in the diseased substantia nigra was limited in the
dow sham group but extensive in the PD group, indicating that
ryap caspase-3 is rarely expressed in normal circumstances but is
heT expressed in large amounts in the presence of PD. Less
stainand ing was seen in the SP-GNP groups than in the blank GNP
tne group, suggesting that SP-GNPs can inhibit the expression
lopm of caspase-3 and reduce neuronal apoptosis, thus helping
vee the diseased neurons to recover. Further, PD rats receiving
,nD 75 μg or 100 μg of intranasal SP per day showed significantly
ise less caspase-3 staining than PD rats that did not receive SP
ugD (P0.05), while PD rats receiving 50 μg of intranasal SP per
D day showed slightly lower level of caspase-3 staining than
PD rats (P0.05), indicating that the higher the concentration
of SP, the better the effect in protecting against
6-OHDAinduced neuronal apoptosis.
6-OHDA is a neurotoxin that activates the apoptotic
cascade in the central nervous system, leading to apoptosis and
degeneration of dopaminergic neurons, which culminates
in cell apoptosis in vitro and PD in vivo. SP, a member of
the tachykinin peptide family, has been shown to play an
important role in protecting against neurotoxin-induced
Studies show that drugs or particles smaller than 300 nm
can bypass the blood–brain barrier, can be absorbed through
the mucous membrane in the nasal olfactory region, and can
be delivered into the brain directly through the cribriform
plate, beyond which they exert their therapeutic effects
in specific regions inside the brain.25–27 In earlier studies,
nanoparticles were used as intranasal carriers for
therapeutics to enable effective treatment of brain disorders, such as
cerebral ischemia28 and PD.29 It is reported that nanoparticles
administered intranasally can penetrate the brain through
several pathways: the olfactory pathway, in which particles
are taken up by the olfactory epithelium and the olfactory
bulb; the trigeminal pathway, in which particles are delivered
along the trigeminal nerve system; the vascular pathway, in
which particles are absorbed into the capillaries underlying
the nasal mucosa; and other pathways, such as cerebrospinal
fluid and the lymphatic system.30–32 However, because of
the mucociliary clearance mechanism in the nose, particles
cannot be lodged in the nasal cavity for a long period, which
limits the application of intranasal administered drug-loaded
In recent years, gelatin and nonionic surfactants (such
as Poloxamer 188) have been used to prepare nanoparticles
due to their biocompatibility, biodegradability, low
immunogenicity, and amenability for surface modification.34–36
Nanoparticles modified with gelatin have a negative charge
that can reduce mucociliary clearance, extend the residence
time at the site of delivery, and enhance the therapeutic effect
when administered intranasally.17–19 In a previous study,
we found that gelatin nanostructured lipid carrier-mediated
intranasal delivery of basic fibroblast growth factor could
enhance functional recovery in hemiparkinsonian rats.37
PC-12 cells, a monoamine cell line derived from a
pheochromocytoma in the adrenal medulla of a male rat,
can express tyrosine hydroxylase and synthesize dopamine
intracellularly, so are widely used in the study of PD models in
vitro.38 In our in vitro experiment, we used 6-OHDA to trigger
apoptosis and then added SP-GNPs at different concentrations
to investigate the effect of SP-GNPs on growth of PC-12 cells.
It is evident from the results of these investigations that SP
can decrease apoptosis and enhance cell growth to a
considerable degree. Further, within a certain range of concentrations,
the degree of inhibition of cell apoptosis increases as the
concentration of SP increases, with cells growing better and
in larger numbers at higher SP concentrations.
In our in vivo experiment, SP-GNPs were administered
intranasally to rats with 6-OHDA-induced
hemiparkinsonism, and these rats showed more functional improvement
and less apoptosis than their counterparts that were not
treated with intranasal SP-GNPs. Intranasal administration of
SP-GNPs inhibited 6-OHDA-induced apoptosis and
improved symptoms of hemiparkinsonism. With increasing
concentrations of SP, rats with hemiparkinsonism showed
more functional improvement, with further decreases in
levels of apoptosis, indicating that the strength of the
neuroprotective effect had a positive relationship with the SP
concentration in the brain.
As a noninvasive strategy, GNP-mediated intranasal
delivery of SP protects against 6-OHDA-induced apoptosis,
and might constitute a practical therapy for PD patients in
This research was supported by grants from the National
Natural Science Funds of China (81301982, 81360195,
81272160, 81302726, 81101570), the Major Scientific
Project of Guangdong Province (2012A080201010) and the
science and technology research of college students in Zhejiang
(Xinmiao research, 2014R413069)
The authors report no conflicts of interest in this work.
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