Using Gelatin Nanoparticle Mediated Intranasal Delivery of Neuropeptide Substance P to Enhance Neuro-Recovery in Hemiparkinsonian Rats
Using Gelatin Nanoparticle Mediated Intranasal Delivery of Neuropeptide Substance P to Enhance Neuro-Recovery in Hemiparkinsonian Rats
Ying-Zheng Zhao 0 1
Rong-Rong Jin 0 1
Wei Yang 0 1
Qi Xiang 0 1
Wen-Ze Yu 0 1
Qian Lin 0 1
Fu-Rong Tian 0 1
Kai-Li Mao 0 1
Chuan-Zhu Lv 0 1
Yi-Xiáng J. Wáng 0 1
Cui-Tao Lu 0 1
0 Editor: Hélder A. Santos, University of Helsinki , FINLAND
1 1 College of Pharmaceutical Sciences, Wenzhou Medical University , Wenzhou, Zhejiang 325035, China , 2 The Second Affiliated Hospital of Wenzhou Medical University , Wenzhou, Zhejiang 325000, China , 3 Hainan Medical College , Haikou, Hainan 570102, China , 4 Taizhou Traditional Chinese Medical Hospital , Taizhou, Zhejiang 318000, China , 5 Biopharmaceutical R&D Center of Jinan University , Guangzhou, Guangdong 510000, China , 6 Department of Imaging and Interventional Radiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital , Shatin, New Territories , Hong Kong SA
Data Availability Statement: All relevant data are
within the paper and its Supporting Information file.
PC-12 cells under SP-GNP treatment showed better cell viability and lower degree of apo
ptosis than those under SP solution treatment. Hemiparkinsonian rats under intranasal
GNP administration demonstrated better behavioral improvement, higher level of TH in SN along with much lower extent of p-c-Jun and Cas-3 than those under intranasal SP solution administration and intravenous SP-GNP administration.
Project of Guangzhou, (201508020001). The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared
that no competing interests exist.
Abbreviations: PD, Parkinson Disease; GNP, gelatin
nanoparticles; SP, Substance P; SP-GNP, Substance
P-loaded gelatin nanoparticles; MTT, 3-(4,
bromide; FCM, Flow cytometry; 6-OHDA,
6hydroxydopamine; TH, Tyrosine hydroxylase;
p-cJun, Phosphorylated c-Jun protein; Cas-3,
Caspase3; SN, Substantia nigra; JNK, c-Jun N-terminalkinase;
CNS, Central nervous system; DA, Dopamine;
MAPK, Mitogen-activated protein kinase; BBB,
Bloodbrain barrier; GN, Gelatin polymeric nanoparticles;
SP-GN, Substance P-loaded gelatin polymeric
nanoparticles; L-DOPA, L-3,
4dihydroxyphenylalanine; PDI, Polydispersibility index.
With the advantages of GNP and nose-to-brain pathway, SP can be effectively delivered
into the damaged SN region and exhibit its neuro-recovery function through the inhibition on
JNK pathway and dopaminergic neuron apoptosis.
Parkinson’s disease (PD) is a chronic disorder of the central nervous system (CNS). PD is
caused primarily by progressive loss of dopaminergic cells in the substantia nigra (SN) region,
which leads to bradykinesia, muscular rigidity, resting tremor, and postural instability[
PD affects 1%-2% of the population above the age of 60 . Despite the progress for therapy of
Parkinson’s disease, it remains a difficult disease for clinical management.
Neuropeptide Substance P (SP) is a member of the tachykinin peptide family that is
involved in the regulation of many biological processes. SP is a major mediator of
neuroimmunomodulatory activities and neurogenic inflammation within the central and peripheral
nervous system [
]. SP-containing neurons are widely distributed throughout the central and
peripheral nervous systems, especially in the SN region [
]. From in vitro experiments, SP can
protect dopamine (DA) neurons from neurotoxicity, decrease neuron apoptosis and enhance
cell growth [
]. From in vivo studies, the expression of SP as well as DA is significantly reduced
in the substantia nigra (SNc) of hemiparkinsonian rats and PD patients, which results in
increased DA deaths and limited expression of tyrosine hydroxylase (TH) [
]. Researchs have
also shown that SP plays an important regulatory role on dopaminergic pathways, particularly
the nigrostriatal pathway [
]. After SP or SP receptor antagonist treatment for the rat model of
Parkinson's disease, the content of striatal dopamine and its metabolites increase, and PD
symptoms improve [
]. In rats with substantia nigra partially damaged by 6-0HDA,
systemic administration of SP before and after the damage promotes functional recovery of
Parkinson's disease [
Abnormally activated c-Jun N-terminalkinase (JNK) pathway is one of the important
mechanisms leading to DA neuronal apoptosis in SN region [
]. JNK is one of the important
members of mitogen-activated protein kinase, MAPK family. It has an important regulatory role for
a variety of cells including nerve cells for their growth, differentiation, survival, and apoptosis
]. Studies have shown that abnormal activation of JNK signaling pathway can activate
downstream signaling pathways, leading to the death of DA neurons. Thus JNK signaling
pathway plays an important role in the dopaminergic neuron apoptosis in Parkinson's disease [
By inhibiting the abnormal activation of JNK pathway, SP can execute certain therapeutic
effects for Parkinson’s disease [
Dopaminergic neuron apoptosis is also one of major causes of Parkinson’s disease [
apoptotic process mainly results from protease cascade process mediated by Caspase family
member. Caspase-3 has a vital role in the reaction process. From previous studies, 6-OHDA
can induce apoptosis in PC12 cells by activating caspase and pro-apoptotic factor as well as
transduction of Bax factor [
]. 6-OHDA injection into the rat brain can be used to induce the
apoptosis of DA neurons in substantia nigra [
]. Researchs have also found that SP can
effectively regulate the expression of caspase family proteins and cell apoptosis, thereby inhibit the
symptoms of many diseases including cancer [
], spinal cord injury [
] and Parkinson’s
]. Meanwhile, JNK signaling pathway downstream mitochondrial malfunction can lead
to over-expression of Caspase-3 protein, ultimately leading to apoptosis or degenerative death
of cells [
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SP can play an important role in the recovery of diseased dopaminergic neuron through the
deactivation of JNK pathway and decreasing neuron apoptosis. However, as a macromolecule
neurokinin, it is not likely that SP administrated orally or intravenously could cross the
bloodbrain barrier (BBB) and its role in the treatment of brain diseases is therefore significantly
Intranasal delivery of biologics has been proved as a noninvasive and efficient strategy [
From previous studies, drugs or particles with a size below 300 nm could bypass BBB and play
therapeutic roles at certain regions inside the brain [
]. With intranasal pathway, these
drugs or particles can be absorbed through mucous membrane inside nasal olfactory region,
and then delivered into the brain directly through the cribriform plate. However, what cannot
be overlooked is the short resident time of biologics in the nasal cavity for absorption, which
decreases the concentration of drug in brain and compromises the therapeutic effect for
diseases like Parkinson’s disease [
]. With the development of nanomedicine, nanoparticles
have emerged as a promising approach for delivering therapeutic agent across the BBB [
]. Lipid nanoparticles with solid matrix possess some advantages over other colloidal systems
such as liposomes, emulsion, micro/nanoparticles, which include possibility of controlled
release, drug targeting, increased stability, high payload, incorporation of both hydrophilic and
hydrophobic drugs, and low biotoxicity [
]. The gelatin nanoparticle used in this study is
a type of gelatin-cored nanostructured lipid carrier with high stability, strong penetration,
encapsulating efficiency, loading capacity as well as bioactivity [
]. It is reported that gelatin
nanoparticles are excellent carriers for targeted delivery, making it possible to delivery
therapeutics to special tissue effectively without compromising drug stability and concentration [
The current study investigated whether SP-GNP can be delivered into CNS to play a
neuroprotective role on hemiparkinsonian rats by inhibiting activation of JNK pathway and decrease
dopaminergic neuron apoptosis. In this study, physicochemical properties of the prepared
gelatin nanoparticles, including micromorphology, particle size, Zeta potential, encapsulating
efficiency and loading capacity, were characterized. The growth promoting effect of SP solution
and SP-GNP on 6-OHDA diseased PC-12 cells were assessed by MTT assays and flow
cytometry analysis. The neuroprotective effect and the inhibition of JNK pathway activation and
dopaminergic neuron apoptosis by intranasally administrated SP-GNP on hemiparkinsonian rats
were evaluated by behavioral assessment, immumohistochemical staining and Western Blot
Materials and Methods
Materials and animals
Consent and approval for this investigation were obtained from the Laboratory Animal Ethics
Committee of Wenzhou Medical University & Laboratory Animal Centre of Wenzhou Medical
University (Wenzhou, Zhejiang, China). Rats were provided by the Laboratory Animal Centre
of Wenzhou Medical University. Male SD rats weighed around 300~320g were used in the
study. Two to three animals were housed per stainless steel cage on a 12-h light/12-h dark cycle
in an air-conditioned room at 22°C, and check daily by the animal care staff. A standard
commercial rat chow and water were available ad libitum.
Preparation and characterization of SP-GNP and blank GNP
All reagents used in this study were commercially available. SP was purchased from GL
Biochem (Shanghai) Ltd. Its molecular structure is shown in Fig 1 and its molecule weight was
1347. The SP-GNP and blank GNP were prepared using water-in-water emulsion and
freeze3 / 18
Fig 1. Molecular structure of SP.
drying technique [
]. Briefly, high concentration SP was dissolved in 1 ml of 20% w/v
Poloxamer 188–grafted heparin copolymer solution. The solution was added into 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 mixture until its final
concentration reached 0.1% w/v to initiate the cross-linking reaction. The mixture solution was bathed at
5°C under magnetic stirring at 2500 rpm for 5 h to form the SP gelatin nanoparticles
suspension. The suspension was lyophilized to gain SP gelatin polymeric nanoparticles (SP-GNs)
powder. Then lyophilized SP-GNs powder was dispersed in solution containing HSPC,
trehalose and cholesterol. By sonication (90 w, 20 s) at 25°C, the mixture suspension was then
lyophilized to gain lyophilized powder containing SP-GNP, which was reconstituted in
doubledistilled water to form SP-GNP suspension for administration.
Blank GNP (using gelatin solution instead of SP gelatin solution in preparation) and SP
solution (SP dissolved in 0.9% NaCl solution) were also prepared for the following
experiments. The final SP concentration in SP-containing solution was 2 mg/mL.
The morphologies of loaded or unloaded gelatin polymeric nanoparticles and gelatin
nanoparticles were determined using a scanning electron microscopy (SEM) (X-650, Hitachi Co.
Ltd., Tokyo, Japan). Their size and zeta potential values were determined by dynamic light
scattering using a Zeta Potential/Particle Sizer Nicomp™ 380 ZLS (PSS. Nicomp, Santa Barbara,
To determine the encapsulating efficiency of SP-GNs and SP-GNP, approximately 1.5 mL
of the SP-GNs or SP-GNP dispersion was placed in microtubes and was centrifuged at 10,000 g
for 40 min. The supernatant was then collected and diluted for content determination of SP
using an ELISA kit. The drug encapsulation efficiency was calculated as indicated below. The
analyses were performed in triplicate.
Encapsulation efficiency (%) = (total amount of drug − amount of drug in supernatant) /
total amount of drugs added initially × 100%
Experiments in vitro
Cell culture. Male rat adrenal pheochromocytoma PC-12 cell was used for the in vitro
studies. PC-12 cell was cultured in DMEM high-glucose medium with 10% FBS and 1%
penicillin as well as streptomycin in humidified incubator (Thermo) containing 5% CO2 at 37°C.
The cells in their logarithmic growth phase were harvested with trypsin for further
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MTT assay. The growth enhancement of SP on 6-OHDA diseased PC-12 cells was
confirmed by MTT assay. First of all, the suitable concentration of 6-OHDA to induce the disease
model was determined. All cells were cultured in 96 well plates for 24 hours with a density of
5000 cells per well. With blank PC-12 cells as the control, four levels of 6-OHDA
concentrations (25, 50, 75, 100 μmol/L) were incubated with PC-12 cells for 24 hours before 10μL of
5mg/mL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] was added
into each well and incubated for 4 hours. Then 100 μL of Formanzan solution was added into
each well and incubated for another 4 hours to dissolve the crystals occurred in each well.
Next, the plate was put in a microplate reader to measure the optical density at 526 nm and
quantify the extent of cell viability. The lower extent of cell viability, the more damage
6-OHDA was done to the cells. The concentration causing the lowest extent of cell viability
was chosen to set up the diseased cell model in the following assays.
Next was to choose the suitable SP concentration that could enhance cell growth. All cells
were cultured in 96 well plates for 24 hours with a density of 5000 cells per well. With blank
PC-12 cells as the control, six levels of SP concentrations (0. 1, 1, 10, 100, 1000, 10000 nmol/L)
were incubated with PC-12 cells for 24 hours before 10μL of 5mg/mL of MTT
[3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] was added into each well and
incubated for 4 hours. Then 100 μL of Formanzan solution was added into each well and
incubated for another 4 hours, dissolving the crystals occurred in each well. Next, the plate was put
in a microplate reader to measure the optical density at 526 nm and quantify the extent of cell
viability. The higher extent of cell viability, the better effect SP was on cells. The concentrations
raised the extent of cell viability were proved to enhance cell growth and were used in the
The final step was to investigate if SP solution and SP-GNP could enhance growth on
diseased cells. All cells were cultured in 96 well plates for 24 hours with a density of 5000 cells per
well. With blank PC-12 cells as the control, 6-OHDA in the most suitable concentration was
used on cells for 24 hours to induce the diseased cell model. Then, different concentrations of
SP solution and SP-GNP were incubated with PC-12 cells for 24 hours before 10μL of 5mg/mL
of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] was added into each
well and incubated for 4 hours. Then 100 μL of Formanzan solution was added into each well
and incubated for another 4 hours, dissolving the crystals occurred in each well. Next, the plate
was put in a microplate reader to measure the optical density at 526 nm and quantify the extent
of cell viability. The higher extent of cell viability, the better effect SP solution or SP-GNP was
Flow cytometry. All cells were cultured in 12 well plates for 24 hours with a density of
50000 cells per well. With blank PC-12 cells as the control, 6-OHDA was added to cells for 24
hours to induce the diseased cell model. Different concentrations of SP solution and SP-GNP
were then incubated with PC-12 cells for 24 hours before the cells were resuspended with
195μL binding buffer. Next, 5 μL of Annexin V-FITC was added and incubated for 10 minutes
at room temperature in the dark. Then, the cells were resuspended with 190μL binding buffer
again into tubes and 10 μL of Propidium Iodide (PI) was added. Finally, the tubes were placed
in ice and the extent of cell apoptosis was detected in the Annexin V-FITCL and PI channels of
the flow cytometer. Apoptotic cells were stained red with Annexin V-FITC, and necrotic cells
were stained green with PI, while normal cells were not stained. Lower extent of cell apoptosis
suggests better enhancement of SP solution or SP-GNP on cells.
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Fig 2. Creation of hemiparkinsonian rat model. The anesthetized SD rats were placed on stereotaxic apparatus and then injected with 12 μL 6-OHDA
solution (or vehicle for sham animals) in the right-side striatum. AP: Distance after the fontanelle; R: Movement to the right side; DV: Depth value.
Experiments in vivo
Hemiparkinsonian rat model. As seen in Fig 2, SD rats were anesthetized with
pentobarbital sodium (60 mg/kg) and then injected with 12 μL 6-OHDA solution in the right-side
striatum (or vehicle for sham animals) by use of the stereotaxic apparatus [
]. Gentamicin was
given after those procedures to prevent possible infection.
Four weeks after 6-OHDA solution injection, the behavior evaluation of
apomorphineinduced rotations was conducted to determine if the hemiparkinsonian rat model was
successfully set up. The rats were injected with apomorphine (0. 5 mg/kg) subcutaneously, and then
both contra- and ipsi-lateral full-body rotations in the following 30 minutes were recorded.
The rats had over 7 full-body contralateral rotations per minute were considered as successful
hemiparkinsonian model rats (PD rats), which were used in the following experiments.
Treatment of hemiparkinsonian rats. After the behavior evaluation, sham SD rats and
PD rats were divided into six groups randomly (n = 6/group) and started daily treatment for
two weeks. As seen in Table 1, group 1 was sham rats with intranasal PBS treatment. Group
2–4 were PD rats which received intranasal PBS, blank GNP and SP solution respectively.
Group 5 and group 6 were PD rats received SP–GNP intranasally and intravenously.
Note: in. represents intranasal administration; iv. represents intravenous administration.
After the two-week daily treatment, the experimental rats were injected subcutaneously
with apomorphine (0.5 mg/kg) to evaluate their neuro-recovery extent. During the 30 minutes
after apomorphine injection, rats’ all contra- and ipsi-lateral full-body rotations were
measured. The rat groups with low rotations suggested some neuro-recovery.
The rats were then all sacrificed and their brains were collected for coronal sectioning across
the striatum region as well as the SNc region. Half of them were embedded in paraffin and
sectioned for histopathological study and immunohistochemical staining, while the rest were
dissected and homogenized in cold lysis buffer for Western blot assessment.
Histopathological study. The paraffin sections were handled with conventional
deparafinization and hydration steps before incubating in hematoxylin for 7 min. Next, the sections
were washed with cold running water for 9 min and soaked in 95% ethanol for 5 sec, and
incubated in eosin for 30 sec. Then a drop of permount and a glass slip were added to the sections.
Finally, the sections were placed under optical microscope to study the microstructure.
Immunohistochemical staining. The immunohistochemical staining with anti-TH
antibody, anti-p-c-Jun antibody and anti-Caspase-3 antibody was conducted to evaluate
therapeutic effect of different SP preparations on hemiparkinsonian rats.
Tyrosine hydroxylase (TH) is the rate-limiting enzyme to catalyze the hydroxylation of
Ltyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor to dopamine,
norepinephrine and epinephrine. TH is considered a marker for dopaminergic neurons [
]. Higher ratio
of TH staining suggests less loss of dopaminergic neurons. Phosphorylated c-Jun protein
(p-cJun) is a vital protein in JNK pathway. As the primary endpoint shear enzyme in the process of
cell apoptosis, Caspase-3 plays an irreplaceable part during cell apoptosis. The ratio of p-c-Jun
and Caspase-3 staining suggests their levels in SN regions.
Western blot assessment. Western blot assessment was carried out to evaluate the TH,
pc-Jun and Caspase-3 levels in SN area of experimental rats. Proteins were extracted in an ice
bath and their levels were assessed using standard biochemical procedures [
]. The band
densities were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA).
Statistically significant difference for multiple groups was determined using a one way
ANOVA with a Newman-Keuls post-hoc test. Statistical significance between individual
groups was determined using a Mann-Whitney U test. All testing was done with Statistical
Program for Social Sciences (Spass) 19. Difference was considered statistically significant when the
p-value was less than 0. 05.
Physicochemical properties and bioactivity of gelatin nanoparticle
From SEM micrographs, the blank GNP and SP-GNP were uniform in shape and size (Fig 3).
Characterizations of GNs and GNPs loaded with or without SP were shown in Table 2. The
dynamic light scattering results demonstrated that the average particle size of blank GNs and
GNPs were 87±1.01 nm and 136±1.32 nm respectively. The polydispersibility index (PDI)
represents the distribution of particle size. Low PDI values were observed for both of blank GNP
and SP-GNP (Table 2), indicating blank GNP and SP-GNP approached a monodisperse stable
system. After SP loading, the mean diameters of nanoparticle increased, not exceeding 200 nm
(Table 2). The zeta potential is an important index evaluating the physical stability of
nanoparticles. Nanoparticles with high absolute value of zeta potential are electrically stable while those
with low absolute value of zeta potential tend to be less stable. As shown in Table 2, both of the
blank GNP and SP-GNP possessed stronger negative charge on the surface than blank GNs
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Fig 3. Scanning electron micrographs of blank GNP (A) and SP-GNP (B). SEM micrographs showed both
blank GNP and SP-GNP were uniform in shape, and the dynamic light scattering demonstrated that both
blank GNP and SP-GNP were uniform in size.
and SP-GNs, indicating the stabilization of phospholipid-coated nanoparticles. Furthermore,
SP-GNP showed better encapsulation efficiency and loading capacity than SP-GNs (Table 2).
The growth inhibition of 6-OHDA in different concentrations on PC-12 cells was shown in
Fig 4A. The cell viability decreased with the increased concentration of 6-OHDA, indicating
the role of 6-OHDA in cell apoptosis. Cells treated with 6-OHDA concentration of 100μmol/L
showed significantly lower cell viability (p<0. 05) than the sham cells.
Fig 4B showed the growth enhancement of SP solution in different concentrations on
PC12 cells. With the concentration of 6-OHDA increasing, the cell viability showed increasing
trend when SP concentration was lower than 100nM, but showed a declined trend when SP
concentration was up to 1μM or 10μM. From the result, SP can enhance cell growth in low
dose while inhibit cell growth in high dose.
Fig 4C revealed the growth enhancement of SP solution and SP-GNP on 6-OHDA treated
PC-12 cells. These PC-12 cells treated with SP solution or SP-GNP treatment demonstrated
higher cell viability (p<0. 05) than those without SP treatment. This illustrated that SP of
certain concentration could reduce cell apoptosis caused by 6-OHDA and enhance cell growth.
Furthermore, the cell viability with SP-GNP treatment was much higher than those with SP
solution treatment (p<0.01). Compared with SP solution, SP-GNP can play a better role to
mediate SP entering the cells and execute its role in the inhibition of apoptosis, showing a
better therapeutic effect on 6-OHDA treated cells.
Zeta Potential (mV)
Encapsulation Efficiency (%)
Load Capacity (%)
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Fig 4. Results of MTT assay on PC-12 cells. (A) Growth inhibition of 6-OHDA in different concentrations on PC-12 cells. * represents p <0.05 vs sham
cells. (B) Growth enhancement of SP solution in different concentrations on PC-12 cells. * represents p <0.05 vs sham cells. ** represents p <0.01 vs sham
cells. (C) Growth enhancement of SP solution and SP-GNP in different concentrations on 6-OHDA diseased PC-12 cells.** represents p<0.01 vs 6-OHDA
diseased cells without SP treatment.
Fig 5 revealed the ratio of apoptosis cells and growth enhancement of SP administrated groups
on 6-OHDA treated PC-12 cells. With SP treatment, the ratio of apoptosis in 6-OHDA treated
cells decreased, compared with those without SP treatment. Furthermore, the 6-OHDA treated
cells with SP-GNP achieved lower ratio of apoptosis than those with SP solution treatment.
Compared with SP solution, SP-GNP showed better enhancement in SP penetration into the
cells, and exhibited stronger inhibition on cell apoptosis.
Behavioral evaluation of PD rats after two-week treatment
The apomorphine-induced rotations following two-week daily treatment of SP in each
experimental group were consistent with the DA levels in the diseased brain. As seen in Table 3, the
order of the reduction in apomorphine-induced rotations was: intranasal SP-GNP >
intravenous SP-GNP > intranasal SP solution > intranasal Blank GNP > intranasal PBS
9 / 18
Fig 5. Flow cytometry results on PC-12 cells. (A) Blank PC-12 cells. (B) 6-OHDA diseased PC-12 cells without treatment. (C) 6-OHDA diseased PC-12
cells with 1nM SP solution treatment. (D) 6-OHDA diseased PC-12 cells with 10nM SP solution treatment. (E) 6-OHDA diseased PC-12 cells with 100nM SP
solution treatment. (F) 6-OHDA diseased PC-12 cells with 1nM SP-GNP treatment. (G) 6-OHDA diseased PC-12 cells with 10nM SP-GNP treatment. (H)
6-OHDA diseased PC-12 cells with 100nM SP-GNP treatment.
Note: in. represents intranasal administration; iv. represents intravenous administration.
* represents p<0.05 vs hemiparkinsonian group;
** represents p<0.01 vs hemiparkinsonian group.
7. 8±0. 6
7. 4±0. 8
6. 8±1. 5
6. 2±1. 3*
4. 7±1. 9**
10 / 18
group. The hemiparkinsonian rats received intranasal blank GNP treatment showed little
difference from those with intranasal PBS treatment. However, the rotations of the
hemiparkinsonian rats with SP administration were lower than those without SP administration,
demonstrating that SP could enhance the recovery of diseased dopaminergic neurons in
hemiparkinsonian rats. In the meantime, hemiparkinsonian rats treated with intranasal SP-GNP
(p = 0.001) and intravenous SP-GNP (p = 0.02) showed a significant decrease on
apomorphine-induced rotation than hemiparkinsonian rats with intranasal PBS solution treatment,
while those with intranasal SP solution and blank GNP treatment showed a trend of
improvement without statistical significance (p = 0.15 and p = 0.35 respectively). From the result,
SP-GNP could play a better role on the recovery of diseased dopaminergic neurons in
hemiparkinsonian rats than intravenous SP-GNP and intranasal SP solution.
The HE staining of the right substantia nigra in each group of rats was shown in Fig 6.
Compared with normal group (Fig 6A), hemiparkinsonian model group and the group receiving
blank GNP group (Fig 6B and 6C) showed apparently reduced cell numbers, with large
number of glial cell proliferation, inflammatory cell infiltration and connective tissue
proliferation observed. After SP treatment, there was an obvious increase of neuron number and a
reduction in glial cell proliferation, inflammatory cell infiltration and connective tissue
hyperplasia. Compared with the rats with SP solution treatment and intravenous SP-GNP
Fig 6. Histopathological HE staining. (a) Sham SD rats. (b) Hemiparkinsonian rats with intranasal PBS treatment. (c) Hemiparkinsonian rats with intranasal
blank GNP treatment. (d) Hemiparkinsonian rats with intranasal SP solution treatment. (e) Hemiparkinsonian rats with intravenous SP-GNP treatment. (f)
Hemiparkinsonian rats with intranasal SP-GNP treatment.
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treatment (Fig 6D and 6E), rats with intranasal SP-GNP treatment (Fig 6F) possessed a larger
number of neurons with regular arrangement. The glial cell proliferation, inflammatory cell
infiltration and connective tissue proliferation were obviously limited in intranasal SP-GNP
group, following with intravenous SP-GNP and intranasal SP solution groups. Result from
the histopathological observation further supported the data of behavioral evaluation
mentioned in Table 3.
Immunohistochemical staining of TH, p-c-Jun and Caspase-3 levels in diseased SN region
TH immunohistochemical staining was shown in Fig 7A. Image-Pro Plus 6. 0 was used to
quantify the cells, the staining area and the staining degrees. TH immunohistochemistry in
diseased SN region revealed severe loss of dopaminergic cells in hemiparkinsonian group and in
the blank GNP treated group, compared with sham group. More TH-positive staining was seen
in SP administration groups (p<0. 05) than in hemiparkinsonian group, illustrating the
enhancement of SP on the recovery of diseased dopaminergic neurons. Among SP
administrated groups, intranasal SP-GNP group (p<0. 01) showed more TH-positive staining than
intranasal SP solution group and intravenous SP-GNP group. From the result, intranasal
SP-GNP can effectively mediate SP into the brain through the nose and exhibit its
neuro-recovery function in the damaged areas of the brain.
Fig 7. Immunohistochemical staining. (a) Sham SD rats with intranasal PBS treatment. (b)PD rats with
intranasal PBS treatment. (c)PD rats with intranasal blank GNP treatment. (d) PD rats with intranasal SP
solution treatment. (e) PD rats with intravenous SP-GNP treatment. (f) PD rats with intranasal SP-GNP
treatment. * represents p<0.05 vs hemiparkinsonian group (Group b), ** represents p<0.
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From p-c-Jun immunohistochemistry in diseased SN region (Fig 7B), sham-PBS/IN group
showed little staining, while hemiparkinsonian group and the blank GNP group showed
extremely high levels of staining. It indicated that p-c-Jun was rarely expressed in normal
circumstances but expressed in large amount when PD occurred. Less staining was seen in SP
administration groups than hemiparkinsonian group (p<0.05), indicating that SP might block
the JNK pathway activation, inhibit the expression of p-c-Jun, and thus slow down the PD
disease process. Among SP administration groups, intranasal SP-GNP group showed less staining
than intranasal SP solution group and intravenous SP-GNP group (p<0.01), demonstrating
the effective inhibition of intranasal SP-GNP on the JNK pathway and the p-c-Jun expression
in the brain.
As shown in Fig 7C, Caspase-3 immunohistochemistry in diseased SN region revealed little
staining in sham-PBS/IN group and extremely high levels of staining in hemiparkinsonian
group and the blank GNP group. From the result, Caspase-3 was rarely expressed in normal
circumstances but expressed in large amount when PD occurred. Less staining was observed in
SP administration groups than in hemiparkinsonian group (p<0.05). Therefore, SP could
reduce neuron apoptosis, inhibit the expression Caspase-3, and promote the recovery of the
diseased neurons. Among SP administration groups, intranasal SP-GNP group showed less
staining than intranasal SP solution group and intravenous SP-GNP group, further supporting
the efficiency SP-GNP for SP delivery into the diseased SN region.
Western Blot assessment of TH, p-c-Jun and Caspase-3 levels in diseased SN region
Western Blot was conducted to analyze the TH, p-c-Jun and Caspase-3 levels in diseased SN
region quantitatively. As shown in Fig 8, TH levels in hemiparkinsonian group and the blank
GNP group were much less than those in the sham group. Compared with hemiparkinsonian
group, SP treatment groups showed increased TH levels (p<0.05), proving the SP therapeutic
Fig 8. Western blot assessment. PD rats with intranasal PBS treatment. (c)PD rats with intranasal blank GNP treatment. (d) PD rats with intranasal SP
solution treatment. (e) PD rats with intravenous SP-GNP treatment. (f) PD rats with intranasal SP-GNP treatment. * represents p<0.05 vs hemiparkinsonian
group (Group b), **01vs hemiparkinsonian group (Group b).
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action for diseased dopaminergic neurons. Among SP administration groups, intranasal
SP-GNP group showed higher TH level than the intranasal SP solution group and intravenous
SP-GNP group, suggesting the effective neuro-recovery function of intranasal SP-GNP in the
As for the p-c-Jun and Caspase-3 levels in diseased SN region, the sham group showed both
very low concentration of p-c-Jun and Caspase-3 while hemiparkinsonian group and the blank
GNP group revealed quite high level of them. From the result, p-c-Jun and Caspase-3 were
rarely expressed in normal circumstances but expressed in large amount when PD developed.
Compared with hemiparkinsonian group, SP treated groups showed decreased levels in
p-cJun and Caspase-3 (p<0.05). It suggested that SP could play a role in reducing neuron
apoptosis and inhibiting the expression of p-c-Jun and Caspase-3. Among SP treated groups, the
p-cJun and Caspase-3 levels in intranasal SP-GNP group were much lower than intranasal SP
solution group and intravenous SP-GNP group, demonstrating the enhancement of the
intranasal SP-GNP for SP penetration into the brain. In the meantime, the relative destiny of
p-cJun level was lower than that of Caspase-3 level in hemiparkinsonian group and other groups
with treatment. All the results from Western Blot and immunohistochemical staining were
consistent with the results of histopathological observation and behavioral evaluation of PD
Current therapies for Parkinson’s disease have limitations due to the presence of BBB which
prevents therapeutic chemicals or biologics entering the brain. As a member of the tachykinin
peptide family, SP can play an important role in treating diseased dopaminergic neuron
through deactivation of JNK pathway and inhibition of neuron apoptosis. But SP is a
macromolecule which can not penetrate the BBB. In this study, a novel gelatin nanoparticle
encapsulating SP was prepared to investigate the role of SP in enhancing neuro-recovery on
hemiparkinsonian rats via the noninvasive intranasal route.
Gelatin nanoparticle mediated intranasal delivery of therapeutics to CNS
Therapeutic biologic particles with a size below 300 nm can bypass BBB and gain access to the
brain directly through the cribriform plate, and then execute their therapeutic effects [
They are believed to gain access to the CNS after intranasal administration via the following
pathways: the systemic circulation in which the drug has to cross the BBB; the olfactory
pathway in which the drug is taken up by the olfactory epithelium and enter the olfactory bulb; and
the trigeminal pathway in which the drug is transported via the trigeminal nerve system [
]. However, their residence time at the delivery site is quite limited due to the mucociliary
clearance mechanisms, which restricts their concentration level in CNS and their biological
activity as well.
Researchers have demonstrated that lipid nanoparticles mediated intranasal delivery of
therapeutics can gain access directly into the brain and achieve effective treatment on brain
diseases such as cerebral ischemic [
] and Parkinson’s disease [
]. It has been reported that
nonionic surfactants, like Poloxamer 188, might increase transcellular transport of olanzapine
by reduction of the barrier function of the mucous layer due to their ability to reduce the
mucous viscosity and elasticity, or to modulate the tight junction [
].] In recent years, gelatin
is adopted for nanoparticle preparation due to its biocompatibility, biodegradability, low
immunogenicity and surface modification. Studies have shown that modified gelatin mixed
with Poloxamer 188 can generate nanoparticles that may be used for intranasal drug delivery
]. Intranasal administrated gelatin nanoparticle, which is prepared with Poloxamer 188 and
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gelatin, can bypass BBB and find their way right into CNS through the olfactory pathway and
the trigeminal pathway, and then the loaded drugs can play therapeutic role in CNS. In the
meantime, gelatin nanoparticle possesses stronger negative charge, which can reduce
mucociliary clearance, extend the resident time at the delivery site and enhance the therapeutic effect
when intranasally administrated [
The role of SP in treating diseased dopaminergic neuron through deactivation of JNK pathway and inhibition of neuron apoptosis
From previous studies, SP administrated intracerebroventricularly in a suitable concentration
can inhibit both of the activation of JNK pathway and the expression of caspase-3 [
showing significant functional improvement of Parkinson’s disease [
]. However, its
therapeutic effect in vivo is limited due to the poor permeability into BBB when administered
PC—12 cell, a monoamine cell derived from pheochromocytoma of the male rat adrenal
medulla which expresses Tyrosine hydroxylase (TH) and synthesise dopamine inside of cell, is
widely used to study Parkinson’s disease model in vitro [
]. In this study, PC-12 cells were
used to examine the growth enhancement of SP in vitro. From the result, SP within a certain
range of concentrations can enhance cell growth and decrease apoptosis, supporting the
therapeutic application for Parkinson’s disease.
For in vivo experiment, 6-OHDA was injected into the right-side striatum of rats to create
hemiparkinsonian rat model. Those rats were then divided into 6 groups and administrated SP
solution or SP-loaded gelatin nanoparticle intranasally or intravenously. Finally, the rats were
sacrificed and the levels of TH, p-c-Jun and caspase-3 were evaluated through
immunohistochemical study and Western Blot analysis. From this study, SP can play an important role in
deactivation of JNK pathway and inhibition of expression of capase-3, thus recovering the
diseased dopaminergic neurons and decreasing the neuron apoptosis in hemiparkinsonian rats.
Though the levels of p-c-Jun and caspase-3 decrease when hemiparkinsonian rats are
administrated with SP, it is still unknown whether the reduction of caspase-3 level is mainly
caused by deactivation of JNK pathway or by the inhibition of expression of capase-3.
Researchers showed that JNK signaling pathway downstream mitochondrial malfunction can
lead to over-expression of Caspase-3 protein, ultimately leading to apoptosis or degenerative
death of cells [
]. SP can regulate p38 MAPK pathway, JNK pathway, and activate ERK
pathway, thereby regulating the expression of caspase family of proteins and cell apoptosis, and
improving the symptoms of various brain diseases [
]. It remains unclear whether
deactivation of JNK pathway or inhibition of expression of capase-3 is the dominating factor in the
role for decreasing the neuron apoptosis.
Therapeutic effect of SP-GNP on 6-OHDA induced PC-12 cell and hemiparkinsonian rats
In this study, novel SP-GNP was prepared and administrated to 6-OHDA induced PC-12 cells
and hemiparkinsonian rats via the noninvasive intranasal route. Then the therapeutic effects of
SP-GNP and pure SP solution on 6-OHDA induced hemiparkinsonian rats were assessed by
quantifying rotational behavior and the levels of tyrosine hydroxylase (TH), phosphorylated
cJun protein (p-c-Jun) and Caspase-3 (Cas-3) expressed in substantia nigra (SN) region.
From the in vitro experiment, PC-12 cells under SP-GNP treatment showed better cell
viability (Fig 4C) and lower degree of apoptosis (Fig 5) than those under SP solution treatment.
Hemiparkinsonian rats under intranasal SP-GNP administration demonstrated better
behavioral improvement (Table 3), neuron regeneration (Fig 6), higher TH levels in SN (Fig 7) along
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with much lower extent of p-c-Jun and Cas-3 (Fig 8) than those under intranasal SP solution
administration. Compared with SP solution, SP-GNP showed more effective penetration for
PC-12 cells and BBB, along with the stronger inhibition of cell apoptosis both in vitro and in
vivo. Histopathological study also illustrated the good biocompatibility of SP-GNP with
6-OHDA treated neuron.
To sum up, SP can enhance 6-OHDA induced dopaminergic neuron recovery in
hemiparkinsonian rats through deactivation of JNK pathway and reduction of dopaminergic neuron
apoptosis (S1 Fig). SP-GNP administrated intranasally may be an effective therapy choice for
S1 Fig. Shows the mechanism diagram of using gelatin nanoparticle mediated intranasal
delivery of neuropeptide substance P to enhance neuro-recovery in hemiparkinsonian rats.
Conceived and designed the experiments: YZZ CZL CTL. Performed the experiments: RRJ WY
QL WZY FRT KLM. Analyzed the data: RRJ. Contributed reagents/materials/analysis tools:
QX. Wrote the paper: YXW YZZ.
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