Neuroprotective effect of gold nanoparticles composites in Parkinson's disease model

Article abstract of DOI:10.1016/j.nano.2018.01.020

Parkinson's disease (PD) is second most common neurodegenerative disorder worldwide. Although drugs and surgery can relieve the symptoms of PD, these therapies are incapable of fundamentally treating the disease. For PD patients, over-expression of α-synuclein (SNCA) leads to the death of dopaminergic neurons. This process can be prevented by suppressing SNCA over-expression through RNA interference. Here, we successfully synthesized gold nanoparticles (GNP) composites (CTS@GNP-pDNA-NGF) via the combination of electrostatic adsorption and photochemical immobilization, which could load plasmid DNA (pDNA) and target specific cell types. GNP was transfected into cells via endocytosis to inhibiting the apoptosis of PC12 cells and dopaminergic neurons. Simultaneously, GNP composites are also used in PD models in vivo, and it can successfully cross the blood-brain barrier by contents of GNP in the mice brain. In general, all the works demonstrated that GNP composites have good therapeutic effects for PD models in vitro and in vivo.

Full text of DOI:10.1016/j.nano.2018.01.020

Accepted Manuscript
Neuroprotective Effect of Gold Nanoparticles Composites in
Parkinson's Disease Model
Kaikai Hu, Xiaohui Chen, Wuya Chen, Lingkun Zhang, Jian Li,
Jialin Ye, Yuxiao Zhang, Li Zhang, Chu-Hua Li, Liang Yin, Yan-
Qing Guan
PII:
S1549-9634(18)30032-7
doi:10.1016/j.nano.2018.01.020
NANO 1755
DOI:
Reference:
To appear in:
Received date:
Revised date:
Accepted date:
11 November 2017
24 January 2018
30 January 2018
Please cite this article as: Kaikai Hu, Xiaohui Chen, Wuya Chen, Lingkun Zhang, Jian
Li, Jialin Ye, Yuxiao Zhang, Li Zhang, Chu-Hua Li, Liang Yin, Yan-Qing Guan ,
Neuroprotective Effect of Gold Nanoparticles Composites in Parkinson's Disease Model.
The address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Nano(2018), doi:10.1016/j.nano.2018.01.020
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ACCEPTED MANUSCRIPT
Neuroprotective Effect of Gold Nanoparticles Composites in Parkinson's Disease Model
1
,2*
3*
3
3
1,2
3
Kaikai Hu , Xiaohui Chen , Wuya Chen , Lingkun Zhang , Jian Li , Jialin Ye , Yuxiao
3
3
3
3
1,2,3 a)
Zhang , Li Zhang , Chu-Hua Li , Liang Yin , and Yan-Qing Guan
1MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South
China Normal University, Guangzhou 510631, China
2
Joint Laboratory of Laser Oncology with Cancer Center of Sun Yet-sen University, South China Normal
University, Guangzhou 510631, China
3
School of Life Science, South China Normal University, Guangzhou 510631, China
Abbreviations: PC12 cells, Rat adrenal pheochromocytoma cells; SNCA，α-synuclein；pDNA，plasmid DNA；
BBB, blood–brain barrier; GNP，gold nanoparticles； CTS, chitosan; DTPA, 3,3'-dithiodipropionic acid; NHS,
N-hydroxysuccinimide; DTSP, dithiobis-(succinimidyl propionate); THF, Tetrahydrofuran; DMF,
N,
N-dimethylformamide; DCC, Dicyclohexylcarbodiimide; AAH, Azide aniline hydrochloride; DMSO, dimethyl
sulfoxide; β-ME, 2-mercaptoethanol; NGF, Nerve growth factor; MPTP, 1-methyl-4-phenyl-
+
1
,2,3,6-tetrahydropyridine;
MPP ,
1-methyl-4-phenylpyridinium;
DTT,
Dithiothreitol;
TEMED,
N,N,N',N'-Tetramethylethylenediamine; PMSF, Phenylmethanesulfonyl fluoride;
Source of funding: this work was supported by the National Natural Science Foundation of China (31370967,
1170919), the Guangdong Province Universities and Colleges Pearl River Scholar Fund Scheme (2014), China,
3
the Science and Technology Planning Project of Guangdong Province (No.2015A020212033), China.
Conflict of interest: The authors declare no conflict of interest.
a) Corresponding author at: School of Life Science, South China Normal University, Guangzhou 510631, P. R.
China. Tel.: (+86-20)85211241; E-mail address: guanyq@scnu.edu.cn (Y. Q. Guan)
*
These authors contributed equally to this work and should be considered co-first authors.

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[Abstract]
Parkinson’s disease (PD) is second most common neurodegenerative disorder worldwide.
Although drugs and surgery can relieve the symptoms of PD, these therapies are incapable of
fundamentally treating the disease. For PD patients, over-expression of α-synuclein (SNCA)
leads to the death of dopaminergic neurons. This process can be prevented by suppressing
SNCA over-expression through RNA interference. Here, we successfully synthesized gold
nanoparticles (GNP) composites (CTS@GNP-pDNA-NGF) via the combination of
electrostatic adsorption and photochemical immobilization, which could load plasmid DNA
(pDNA) and target specific cell types. GNP was transfected into cells via endocytosis to
inhibiting the apoptosis of PC12 cells and dopaminergic neurons. Simultaneously, GNP
composites are also used in PD models in vivo, and it can successfully cross the blood-brain
barrier by contents of GNP in the mice brain. In general, all the works demonstrated that GNP
composites have good therapeutic effects for PD models in vitro and in vivo.
Keywords: parkinson's disease; gold nanopaiticles; plasmid DNA; nerve growth factor
(NGF), neuroprotective effect

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Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative disorder
1
-4
worldwide.
Degenerative loss of dopaminergic neurons in the substantia nigra pars
compacta is the major characteristic of PD, resulting in reduced levels of neurotransmitter
5
,6
dopamine (DA), bradykinesia, rigidity and tremor. With the increasing size of the aging
population, PD has been threatening the health of thousands of people.
Currently, the therapies of drugs and surgery cannot cure PD fundamentally, which is
1
only symptomatic treatment. Consequently, the development of new treatments for PD is
very desirable. The α-synuclein (SNCA) plays a significant role in the pathophysiology of PD,
7
and its over-expression can produce toxicity for PD models in vivo and in vitro. The
formation of intracellular toxic SNCA oligomers in PD is attributed to an accumulation of
SNCA, which results in SNCA aggregation, Lewy body formation and even the loss of
dopaminergic neurons. All these above are relative to the pathogenic missense mutations of
2
SNCA gene. Thus, suppression of the over-expression of SNCA could be used to protect
dopaminergic neurons.
Development of RNAi as a therapeutic approach is expected to provide novel
opportunities for treating a wide range of disorders. Exogenous interfering RNAs can be
administered as synthetic small interfering (si)RNAs, usually delivered using vectors. The
siRNAs incorporate into the cellular RNAi machinery at the RNA inhibitory silencing
8
complex and have only transient effects. The pDNA as a tool for SNCA gene silencing, the
role of exogenous RNA interference, can suppress the over-expression of SNCA by silencing
it.

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9
Gene therapy strategies for PD have recently been extensively investigated. The vectors
that deliver specific genes into neurons of the patient can be divided into two types: viral and
non-viral vectors. The viral vectors, such as lentivirus and adeno-associated virus, however,
may induce autoimmune reactions and inflammation, and even the risk of mutations during
treatment of the disease. In addition, lentivirus and adeno-associated virus cannot cross the
5
blood-brain barrier (BBB). Hence, viral vectors are not an ideal vehicle for gene therapy
strategies for PD. Nevertheless, non-viral vectors-functional nanomaterial can traverse the
1
0
BBB. In studies of non-viral gene transfer vectors for PD, PEGylated liposomes and
PEG-PEI have demonstrated specific limitations, such as the need for repeated treatments and
5
,11
low transfection efficiency.
In contrast, GNP has a number of desirable properties that
make them excellent candidates for delivery applications.
Actually Wang et al. and Gunaratne et al. demonstrated that AuNPs are capable of
delivering microRNAs and small interfering RNAs into cells and efficiently down-regulated
1
2,13
target genes and modulated cell functions.
Moreover, Peng et al. used gold nanoparticles
as unique non-viral vectors for gene delivery mesenchymal stem cells with antibacterial
1
4
15
activity. Bishop et al. used gold nanoparticles for co-delivery of DNA and siRNA. There
are some advantages for GNP as gene delivery system. First, the gold core is essentially inert,
non-toxic, and biocompatible, which is an ideal starting point for carrier construction.
Secondly, AuNPs with a wide range of core sizes (1-150 nm) can be fabricated easily with
controlled dispersity. Both their size and dispersity are key aspects for drug delivery systems.
AuNPs can be readily fabricated with sizes commensurate with biomolecules such as proteins
and DNA, facilitating their integration into biological systems. Furthermore, the high surface

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area-to-volume ratio of NPs provides dense loading of functionalities incorporating targeting
1
6,17
and therapeutic materials.
Therefore, gold nanoparticles, comparing to PEGylated
liposomes and PEG-PEI, has obvious advantages as carrier. GNP used as gene transfer
vectors are rarely seen in gene therapy strategies for PD.
Research investigating GNP with unique physical and chemical advantages has expanded
in several areas, such as sensors, catalysis, the detection of Enzyme, medical imaging,
1
8-22
antibody detection,
fluorescence detection, quantification of neurochemicals, etc.
CTS) modified GNP, which functionalized with cationic groups bind plasmid DNA via
immunoassays, the absorption and release of DNA, protease
2
3-31
The application of chitosan
(
electrostatic interaction and can inhibit the transcription of DNA. Positively charged GNP
bind to DNA efficiently and protect it from enzymatic digestion. These non-covalent
1
6,32,33
DNA-AuNP conjugates can effectively transfect cells.
suppressing neuronal cell apoptosis.
This has not been investigated for
Here, we successfully synthesized GNP composites (CTS@GNP-pDNA-NGF) via the
combination of electrostatic adsorption and photochemical immobilization, by which the
pDNA could load and specific cell types can be targeted. CTS@GNP-pDNA-NGF was
transfected into cells via NGF receptor-mediated endocytosis to suppress the expression of
SNCA, inhibiting the apoptosis of PC12 cells and substantia nigra striatum dopaminergic
neurons. These data demonstrated that CTS@GNP-pDNA-NGF have good therapeutic effects
for PD models in vitro and in vivo.
Methods

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The details of the experiment about preparation, characterization of
CTS-GNP-pDNA-NGF, experiment in vitro and in vivo, and statistical analysis can be found
in the Supplymentary Materials.
Results
Synthesis and characterization of CTS@GNP-pDNA-NGF nanoparticles
The experiment design was illustrated in Figure 1. We obtained two different sizes of
gold nanoparticles by drop addition and mixing at 5 nm and 10 nm. The results of X-ray
photoelectron spectroscopy was shown in Figure 2, A, and we analyzed the percentages of the
contents of C, N, O and S in dithiobis-(succinimidyl propionate)(DTSP). The results
demonstrated that DTSP contained N and S, as a result of the dehydration reaction between
DTAP and N-hydroxysuccinimide (NHS). Compared with CTS, thiolated chitosan (TCTS)
contains S because of the presence of thiol. Figure 2, B-C showed typical Fourier transform
infrared (FTIR) spectrum of corresponding compounds. The FTIR spectrum of
-1
3
,3'-dithiodipropionic acid (DTPA) exhibited a strong absorption peak at 1698 cm , which
-1
-1
was caused by C=O carbonyl stretching. The peaks of C=O stretching at 1651 cm , 1705 cm ,
-
1
-1
-1
1
1
780 cm were present in the spectrum of NHS. The typical feature at 1668 cm , 1740 cm ,
-
1
-1
783 cm and 1813 cm in the spectrum of DTSP was also attributed to C=O stretching,
caused by ester formation between DTPA and NHS during the reaction. Compared with CTS,
-1
-1
the spectrum of TCTS displayed a typical peak at 2509 cm and 1572 cm , respectively,
which was attributed to S-H stretching and N-H deformation vibration. These observations
demonstrated that TCTS was successfully synthesized. In addition, FT-IR spectrum of TCTS,

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GNP and CTS@GNP revealed that GNP was capped by CTS. Compared with CTS@GNP,
-1
-1
-1
-1
CTS@GNP-NGF had four additional peaks at 1303 cm , 1419 cm , 1457 cm and 1541 cm ,
which were ascribed to the modification of NGF.
Transmission electron microscopy (TEM) images were shown the morphology and size
distribution of the prepared GNP composites (Figure 3, A, B). The CTS@GNP exhibited the
potentials of uniform spherical morphology, good dispersion, small particle size and great
surface area for 5 nm and 10 nm group. UV-visible spectrum was applied to detect the GNP,
CTS@GNP (5 nm and 10 nm group) (Figure 3, C). The GNP without capping CTS exhibited
a characteristic surface plasmon resonance (SPR) band at around 517 nm, indicating synthesis
of GNP. However, the SPR band of CTS@GNP (5 nm and 10 nm group) was observed at
approximately 525 nm and 527 nm, indicating slight red-shifts of its SPR absorption peak as a
result of the modification of CTS, which was probably attributed to an increase in the average
size of GNP. Furthermore, the grafting ratio of NGF was tested (Figure 3, D). The results
illustrated that the grafting ratio of NGF in the 10 nm group was larger than that in the 5 nm
group.
To further confirm the successful synthesis of GNP, we determined the Zeta potential of
nanoparticles, respectively (Figure 3, E). In the 5 nm group, the Zeta potential of
CTS@GNP-pDNA (-22.5 mv) was lower than that of CTS@GNP, which was attributed to the
interaction of the negatively charged pDNA and the positively charged CTS@GNP. To
evaluate the formation of CTS@GNP-pDNA, we first used different weight/weight (w/w)
ratios of GNP to pDNA in an agarose gel retardation assay (Figure 3, F -F ). pDNA was
1
3
clearly completely retarded, when the w/w ratio of GNP (5 nm group) to pDNA was greater

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than 2.0, and that of GNP (10 nm group) to pDNA was above 0.5. The results indicated that
CTS@GNP could absorb negatively charged pDNA to completely or partially prevent the
migration of pDNA via electrostatic interaction. Figure 3, F showed the electrophoretic
3
mobility of CTS@GNP-pDNA and CTS@GNP-pDNA-NGF. After NGF was grafted onto the
surface of CTS@GNP-pDNA, no desorption of pDNA was observed. Thus,
CTS@GNP-pDNA was successfully synthesized and the adsorption of pDNA was not
affected by NGF grafting.
Inhibition of PC12 cells apoptosis by CTS@GNP-pDNA-NGF
To construct the PD model in vitro, PC12 cells were treated with different concentrations
+
of MPP (1-methyl-4-phenylpyridinium) for 48 h, the cells viability was evaluated using the
MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay (Figure 4,
A). The viability was greatly reduced when PC12 cells were treated with increasing
+
+
concentrations of MPP . PC12 cells treated with 300 μM MPP for 48 h was selected for
subsequent experiments because their viability was reduced by nearly 45% compared with the
control (P<0.001). The MTT assay was used to evaluate the viability of PC12 cells treated
with different concentrations of CTS@GNP (0, 15.6, 62.5, 250, 1000, 4000 ng) and incubated
for 24 h. (Figure 4, B), the results showed that the viability of PC12 cells declined with
increasing concentrations of CTS@GNP. A 24-h treatment with 250μg/ml CTS@GNP was
used for PC12 cells in subsequent experiments because the cell viability was reduced by
nearly 12% compared with the control (P<0.001) and higher levels of pDNA could be
adsorbed.

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Bright field and fluorescence images of PC12 cells showed the green fluorescent protein
gene in the pDNA (Figure 4, C). The rate of endocytosis of PC12 cells treated with
CTS@GNP, CTS@GNP-pDNA, CTS@GNP-pDNA-NGF (5 nm and 10 nm group) was
masured by ICP-MS (Inductively coupled plasma mass spectrometry) (Figure 4, D). Among
the three nanoparticles, we detected the highest rate of endocytosis in PC12 cells treated with
CTS@GNP-pDNA-NGF. Furthermore, we also detected the stability of CTS@GNP-pDNA in
the simulated biological buffer in 5 nm and 10 nm groups (Figure 4, E). The time it takes for
3
4
particles to transport to the brain after intraperitoneal injection only needs 2-4 h. And the
results show that within 12h among the buffers in the stirred state, the pDNA content showed
a rapid downward trend at the beginning and stabilized after a period of time. That means it
can maintain good integrity before they reach the brain.
To evaluate the ability of GNP composites to inhibit PC12 cells apoptosis, PC12 cells
were pretreated with PBS (control), PBS (I), CTS@GNP (II), CTS@GNP-pDNA (III) or
CTS@GNP-pDNA-NGF (IV), and then PBS (control) continue processed using PBS when
+
the others processed using MPP for 48 h. Subsequently, the cell nuclei were stained with
DAPI (blue fluorescence), and the SNCA were detected with Cy5 (red fluorescence) by the
confocal laser scanning microscope (Figure 5, A). PC12 cells treated only with PBS can
+
maintain good cell morphology and minimal apoptosis, then it treated alone with MPP , the
prominent morphology of the nucleus was semilunar and polygonal, and the emergence of
apoptotic bodies was evident. The PC12 cells which were treated with II group, displayed
similar features. However, the shape of some nuclei for PC12 cells treated with III group
returned to normal, and fewer apoptotic bodies were observed. Because the PC12 cells were

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treated with IV group, their nuclei tended to exhibit a more normal morphology and became
elliptical and circular, this could be attributed to CTS@GNP-pDNA-NGF suppressing
MPP+-induced PC12 cells apoptosis. Moreover, in comparison to different groups, the PC12
cells treated with CTS@GNP-pDNA-NGF only showed a small amount of the red SNCA.
These results suggested that CTS@GNP-pDNA-NGF played a major role in inhibiting cell
apoptosis, potentially due to the suppression of SNCA.
To further verify whether the inhibition of SNCA expression caused by
+
CTS@GNP-pDNA-NGF would modify MPP -induced PC12 cells apoptosis, the apoptosis
rate of PC12 cells transfected with the above-described formulations was examined by flow
cytometry (Figure 5, B -B ). The results exhibited that 2.6% of PC12 cells apoptosis occurred
1
2
when treated only with PBS in control group, and 43.64% of PC12 cells treated only with
+
MPP were undergoing apoptosis, it can be shown that we are modeling the cellular level is a
success. However, the apoptotic rate of PC12 cells (5 nm group) was only 18.62% when the
cells were pre-treated with CTS@GNP-pDNA-NGF and subsequently supplemented with
+
medium containing MPP . The apoptotic rate of PC12 cells was greatly reduced compared
+
+
with the MPP control and the CTS@GNP-pDNA plus MPP group. The 10 nm group
displayed similar characteristics. In general, these results demonstrated that the
+
CTS@GNP-pDNA-NGF could effectively prevent apoptosis induced by MPP in PC12 cells.
We detected the expression of SNCA by western blot analysis to demonstrate silencing
+
of the SNCA gene by the GNP composites (Figure 5, C -C ). Compared with MPP ,
1
2
CTS@GNP and CTS@GNP-pDNA, SNCA expression was significantly reduced in PC12
cells treated with CTS@GNP-pDNA-NGF.

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Ability of GNP composites to rescue PD model mice in vivo
To evaluate the effect of CTS@GNP-pDNA-NGF in the PD model in vivo, we treated
C57bl/6 mice with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and then injected
CTS@GNP-pDNA-NGF (2 mg Au/kg) into the abdomen of the mice. The morphological
characteristics and body weight variation within 14 days was shown in Figure 6, A -A . As
1
2
shown in Figure 6, A , during moving, the tails in IV and V were significantly lower than that
1
in II. Intraperitoneal injection of MPTP induced a slow rise in body weight within 7 days
compared with the initial value. Similarly, body weight also increased following injection of
CTS@GNP-pDNA-NGF. Furthermore, percentage of body weight increase after injecting of
CTS@GNP-pDNA-NGF (10 nm) was higher than that of 5 nm (Figure 6, A ). These findings
2
suggested that 10 nm of CTS@GNP-pDNA-NGF had a better therapeutic effect in the PD
model in vivo.
The mouse nigra-striatum system was analyzed by Magnetic Resonance Imaging (MRI)
(
Figure 6, B -B ). The substantia nigra-striatum system edge (yellow arrow) density was
1 2
significantly reduced after injection. By the GNP therapy, the nigra-striatum system density
has recovered, and the recovery rate of GNP composite effect in 10 nm group was preferable
to the one in 5 nm group.
To observe the distribution of the GNP composites in mouse brain and to determine
whether the GNP can cross the BBB, we employed TEM for observation of the nigra-striatum
system in mice’s brain and employed ICP-MS to measure the Au concentration of brain in I,
II, III, IV and V (Figure 6, C). In MPTP-injected animals, the nuclear membrane of neurons in
nigra-striatum system appeared shrunken and incomplete (black arrow), and chromatin

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condensation and disruption of the cytomembrane were also confirmed, which compared with
the group I and III. But after the treatment of GNP composites, the nuclear membrane returns
to normal, chromatin condensation phenomenon has basically eliminated. Moreover, we can
also find the presence of small amounts of GNP (Figure 6, E). There was significant
accumulation of Au. These results suggested that GNP might be able to cross the BBB.
The analysis of nissl bodies for the brain in I, II, III, IV and V was operated (Figure 6,
D ). Mice treated MPTP plus SN showed small amounts of nissl bodies (black arrow), that
1
means the nerve cells of the mice have been severely damaged. However, mice treated with
CTS@GNP-pDNA-NGF exhibited normal amounts of nissl bodies compared to control
groups, and the number of 10 nm group were higher than 5 nm group. These results indicated
that the GNP composites have a significant therapeutic effect for neurological diseases and 10
nm GNP composites have better effects than 5 nm groups. According to quantitative results of
nissl bodies (Figure 6, D -D ), the number of nissl bodies and average of blackness in
2
3
pathogenic group was significantly lower than that of the control group. Thus, after GNP
treatment, the number of nissl bodies in the cytoplasm increased significantly and the average
blackness became normal.
Furthermore, at the molecular level, the results of Western blot analysis and quantitative
results were presented in Figure 7, A -A . SNCA expression in III, IV and V was significantly
1
2
lower than that in II. And IHC revealed a similar result (Figure 7, B -B ). Thus, it was
1
2
concluded that SNCA expression was suppressed after injection of CTS@GNP-pDNA-NGF,
and 10 nm group had a better effect than that 5 nm, which was also been found clearly for the
gait test of mice (Video).

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In order to study possible toxic effects, histological examination of hearts, livers, spleens,
lungs, kidneys and brains in I, II, III, IV and V was done (Figure 7, C -C ). As the results of
1
2
III, IV and V, Mice treated with CTS@GNP-pDNA-NGF exhibited no tissue damage in any
of the sections. While the mice treated with MPTP only exhibiting severe brains atrophy, the
neurons and glial cells in their brains were showing a clear separation. By GNP composites
treatment, the lesions tissue appeared to be normal and the treatment effects of 10 nm group
was better than those of 5 nm. The results show that GNP composites have a significant effect
for the treatment of PD. Finally, we employed ICP-MS to measure the Au concentration of
hearts, livers, spleens, lungs, kidneys and brains in III, IV and V (Figure 7, D(1)-(3)).were
shown that the concentration of Au in spleen was the highest. These results supported
CTS@GNP-pDNA-NGF was effectively cleared through spleen.
Discussion
In recent years, nanocarriers have been developed for the targeted transport of genes in
gene therapy. RNAi has been widely applied for tumors, virus infection, nervous system
3
5,36
disease and other diseases as an effective gene therapy technique.
Therefore, the
successful design of a targeted gene therapy system will provide a highly efficient and
low-toxicity treatment method for PD.
The GNP nanoparticles were synthesized according to a procedure described in previous
3
7,38
reports.
Because of the strong interaction between sulfur and gold, GNP capped by a
thiol-modified polymer possesses several advantages, such as ease of preparation, significant
3
9
stability and ease of functionality. When the surface of GNP is reasonably modified, it could

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4
0
have benefit potential as a perfect vector carrying drugs/genes. Thus in this work, we first
used TCTS to modify GNP. And because the compounds with free amino groups can react
with DTSP to generate a stable peptide bond, TCTS was synthesized via a substitution
3
7
reaction. Finally, because the NGF can be internalized into cells by binding to its
4
1-43
receptor.
Thus, CTS@GNP modified with NGF was supposed to be more easily taken
into cells than CTS@GNP, and the CTS has good biocompatibility to cells, and can promote
4
4
the transport of siRNA. As the previous research, different sizes influence the optical,
mechanical, and electrical properties of the AuNPs. And 5 nm groups reflects the very small
1
7
size of particles, 10nm group represents slightly larger particles, respectively, which were
then employed in the latter study.
Compared with CTS@GNP, CTS@GNP-pDNA and CTS@GNP-pDNA-NGF did not
exhibit significant changes in morphology, particle size. It was indicated that CTS@GNP
almost maintained its original features and had no obvious agglomeration after absorbing
pDNA and grafting NGF. Moreover, the rate of endocytosis of 10 nm group was higher than
that of 5 nm. This result was likely because the grafting quantity of NGF for CTS@GNP (10
nm group) was higher than that of 5 nm group.
It is also known that positively charged vectors can adsorb negatively charged pDNA via
electrostatic interaction. Thus, the positively charged vectors are capable of partially or
completely neutralizing the negatively charged pDNA, potentially leading to the retardation
4
5
of pDNA during gel electrophoresis. Our results indicated that CTS@GNP could absorb
negatively charged pDNA to completely or partially prevent the migration of pDNA via
electrostatic interaction. Moreover the expression of GFP might indecates that pDNA became

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4
6
the shRNA.
+
A toxic metabolite of MPTP, MPP , has been widely used to construct models of PD in
4
7
vitro. Because they exhibit features similar to midbrain dopaminergic neurons, and damages
mitochondrial complex I of dopaminergic neurons after transfer by the dopamine transporter,
and increased calcium permeability of the mitochondrial membrane induces free radicals.
+
MPP has been used to induce selective degeneration of dopaminergic neurons in an
+
experimental model of PD, and the metabolite of MPTP in vivo was MPP , and
MPTP-induced symptoms of PD and changes in brain structure are very similar, therefore
48
MPTP can be used to simulate PD. PC12 cells have played an important role in studying the
+
neurotoxicity of MPP and the PD model in vitro, and PD model in mice was established by
49,50
MPTP.
High concentrations of GNP are toxic to many mammalian cell lines, and the different
5
1
size of which lead to various degrees cytotoxicity. Thus, it is imperative that an appropriate
concentration of GNP is chosen to reduce cytotoxicity and absorb more pDNA. In our study,
we found a concentration balance alllowed the particle absorb more pDNA in the case of low
toxicity with PC12 cells. However, this concentration of GNP induced much greater toxicity
5
1
in AGS cells and A549 cells. It is indicated that GNP modified with CTS was less toxic than
simple GNP.
GNP has great potential to overcome current drug delivery limitations because of its
5
2
biocompatibility, low toxicity, and modifiable surface functionalities. At the same time,
4
1
NGF can be internalized into cells by binding to its receptor. Considering preliminary
evaluation of the transfection efficiency of GNP composites for PC12 cells, we used

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nanoparticles to treat PC12 cells for 48 h. The results suggested that the GNP successfully
carried pDNA into the cells, and avoided enzymatic degradation. The PC12 cells in the 10 nm
group have more SNCA expression represented by more detectable flurescence. We assumed
that this is because of the higher grafting rate of NGF in the 10 nm group, which leads to
5
3
more GNP and pDNA targeting and recovering PD model nerve cells in vitro.
Actually, Yue and Zhang have shown that for a single spherical nanoparticle, both extent
and dynamics of the receptormediated endocytosis are strongly dependent on nanoparticle
54,55
size, ligand density and membrane surface tension.
The nanoparticles we have been
obtained, 5 nm and 10 nm, with high specific surface area. That means the 5 nm group
nanoparticles can have more NGF binding to the receptor on the cell membrane, thereby
enhancing the endocytosis efficiency. But in other words, smaller particles with greater
possibility of aggregation, which affects the process of cell endocytosis. So 5nm and 10nm
group nanoparticles had similar results in cells endocytosis.
In fact, SNCA is a gene responsible for encoding α-synuclein, a protein found to be the
major component of Lewy body and Lewy neurite which are the pathognomonic hallmarks of
PD. SNCA is also a brain-rich neuromorphin maintaining the normal synaptic function and
protecting those cells from a variety of neurodegenerative diseases. In the brain of patients
with PD, SNCA is the most important component of Lewy bodies in dopaminergic
5
6,57
neurons.
SNCA may be involved in PD pathogenesis at least in 2 ways: α-synuclein
overexpression and its mutation types via different mechanisms. Associations between SNCA
mutations and other Lewy body disorders, such as dementia with Lewy bodies and multiple
5
6
system atrophy, are also mentioned. The data, in Figure 5, confirmed that after it delivered

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into cells, the GNP composites were able to inhibit the expression of SNCA. Moreover, the
level of expression of SNCA reflects the transcription and translation of SNCA genes.
Furthermore, the gene expression is the process of DNA to protein, that is, the transcription
46
and translation of the gene. We may observe the expression of gene by protein expression. It
also demonstrated that the GNP composites were internalized by PC12 cells and the pDNA
escaped from the lysosome and expressed shRNA which then associated with the
RNA-interfering silencing complex (RISC) to suppress SNCA expression via mRNA
5
6-60
degradation (Figure 8).
Consequently GNP composites, could effectively inhibit PC12
+
cells apoptosis induced by MPP . Similarly, the expression of NGFR increased in the cells
treated with GNP composites, further supporting the reduced rate of apoptosis in these cells.
5
0
The PD models in vivo were established according to our previous work. For the
sharper drop in weight in the group I, that might be the injection process showed different
effects due to individual differences in mice. More than this, the results in vivo also proved
our hypothesis about the PD model treatment in vitro. When more NGF was grafted on the
surface, more pDNA can be transported into the neurons, and thus more suppressions of the
molecular expressions would be occurred with more SNCA being silence. This is also the
reason why the nanoparticles in the 10 nm group more better treatment effects than the ones
in the 5 nm, which although containing a greater rate of drug delivery, hardly had the same
therapeutic effects.
The next questions remained were the locations of the GNP composites in mouse brain
and capability of them to cross the BBB. For the results, there was significant accumulation of
Au in brain of the mice, reaching quantities much higher than the reported physiological

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6
1
levels . Results in Figure 6 also suggested that GNP might be able to cross the BBB, which
was supported by a previous study and the microstructural changes can still reflect the
6
2
condition of the disease . Results of nissl staining, Western blotting and IHC fully
demonstrated that the abilities of GNP composites in protection of the nissl bodies and the
inhibition of SNCA expression. Finally, results of ICP-MS shown the only organ that
CTS@GNP-pDNA-NGF concentrating in was spleen, which was the same as a previous
6
1,63
study.
These results supported CTS@GNP-pDNA-NGF was effectively cleared through
spleen. We can make a judgement that there was no obvious toxic side effect in mice body
from the results which we have been obtained.
In summary, The GNP composites of two sizes were successfully synthesized by the
combination of physical and chemical methods and it as a non-viral vector could play an
important role in carrying pDNA into cells. The GNP composites were successfully delivered
into PC12 cells through NGF and NGF receptor-mediated endocytosis, and they effectively
+
inhibited MPP -induced cellular apoptosis. For the PD model in vitro, SNCA expression in
+
PC12 cells treated with GNP composites plus MPP was greatly down-regulated compared
+
with cells treated with MPP only. Similar results were observed for the PD model in vivo.
Considering the results of all of these studies, GNP composites loading pDNA and targeting
specific cell types were successfully synthesized. In addition, the GNP composites were
transfected into cells via endocytosis to suppress SNCA expression, inhibiting the apoptosis of
PC12 cells and substantia nigra striatum dopaminergic neurons. We anticipate that the
aforementioned GNP composites may serve as a good starting point for the fabrication of
multifunctional nanomaterial with huge potential for the treatment of PD.

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Figure Legends
Figure 1. Schematic illustration: (A) the preparation of CTS@GNP-pDNA-NGF; (B) the
synthesis of CTS@GNP-pDNA-NGF.
Figure 2. (A) Element analysis of XPS from DTPA, NHS, DTSP, CTS and TCTS. (B) FTIR
spectra of DTPA, NHS, DTSP, CTS and TCTS. (C) FTIR spectra of GNP, CTS@GNP and
CTS@GNP-NGF.
Figure 3. (A) TEM images of the different sizes of CTS@GNP, CTS@GNP-pDNA and
CTS@GNP-pDNA-NGF. (B) Histograms size distribution of the CTS@GNP,
CTS@GNP-pDNA and CTS@GNP-pDNA-NGF. (C) UV-vis spectra of GNP and CTS@GNP.
(D) Grafting rate of NGF. (E) Zeta potentials of CTS@GNP, CTS@GNP-pDNA and
CTS@GNP-pDNA-NGF (5 nm and 10 nm group). (F) Electrophoretic mobility retardation
assay of the complexes of (F1) pDNA with CTS@GNP for the 5 nm group, (F2) pDNA with
CTS@GNP for the 10 nm group, (F3) CTS@GNP-pDNA and CTS@GNP-pDNA-NGF.
+
Figure 4. (A) Dose-and time-dependent neurotoxicity of MPP in PC12 cells determined
using the MTT assay. (B) Dose-dependent toxicity of CTS@GNP in PC12 cells using the
MTT assay. ***p<0.001, compared with control group. (C) Bright field and fluorescence
imaging of PC12 cells treated with CTS@GNP, CTS@GNP-pDNA and
CTS@GNP-pDNA-NGF. (D) Rate of endocytosis by PC12 cells treated with CTS@GNP,
CTS@GNP-pDNA and CTS@GNPpDNA-NGF. (E) Stability of pDNA in the
CTS@GNPpDNA-NGF in 10nm and 5nm group at 72h. Error bars represent the standard
deviation of three repeated measurements. ***p<0.001, compared with non-grafted NGF
group.

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Figure 5. (A) Confocal fluorescence images of PC12 cells treated with control (PBS plus
+
+
+
PBS), I (PBS plus MPP ), II (CTS@GNP plus MPP ), III (CTS@GNP-pDNA plus MPP )
+
and IV (CTS@GNP-pDNA-NGF plus MPP ). SNCA was stained with Cy5.0-labeled
antibody (red). The nuclei of cells were stained with DAPI (blue). (B1-B2) Flow cytometric
analysis and corresponding histograms of PC12 cells treated with different groups. (C1-C2)
Expression of NGFR and SNCA after treatment with different groups. Error bars represent the
standard deviation of three repeated measurements. **p<0.005, compared with control group.
Figure 6. (A1) Morphological changes of PD mice before and after treatment. (A2) Body
weight changes for C57bl/6 mice within at 14 days. C57bl/6 mice were treated with SN,
MPTP, SN, MPTP and MPTP for 7 days, followed by I (SN), II (SN), III, IV and V
(
CTS@GNP-pDNA-NGF for the 10 nm, 5 nm and 10 nm group) for additional 7 days.
B1-B2) MRI analysis for C57bl/6 mice nigra-striatum system. Mice after intraperitoneal
(
injection of the drug treatment in different groups. (C) TEM analysis for mice substantia nigra
and the presence of GNP (black arrow). Scale bar: 1 µm. (D1-D3) Nissl staining of mice nissl
body in nerve cell axons in different groups and amplification result of axons. Scale bar: 10
µm and 20 µm.The quantitative analysis of the substantia nigra area and the average of
blackness. (E) ICP-MS analysis of brain in different groups.

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Figure 7. (A1-A2) Expression levels of SNCA in the substantia nigra pars compacta of
C57bl/6 mice and the quantitative results of ratio. (B1) IHC images of the substantia nigra
pars compacta of C57bl/6 mice. (B2) Quantitative results of the SNCA expression. (C1-C2)
HE staining of hearts, livers, spleens, lungs, kidneys and brain tissues in different groups.
Scale bar: 20 µm. (D) ICP-MS analysis of hearts, livers, spleens, lungs, kidneys and brains
different groups. Error bars represent the standard deviation of three repeated measurements.
*
**p<0.001, compared with model group.
Figure 8. Schematic diagram showing the process of GNP composites treatment: GNP
composites were internalized by PC12 cells and that the pDNA escaped from the lysosome
and expressed shRNA which then associated with the RNA-interfering silencing complex
(RISC) to suppress SNCA expression via mRNA degradation.