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Phenylalanine-mediated Porous CuxO Nanoparticle Clusters Ameliorate Parkinson’s Disease by Reducing Oxidative Stress Changlong Hao, Aihua Qu, Liguang Xu, Maozhong Sun, Hongyu Zhang, Chuanlai Xu, and Hua Kuang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11856 • Publication Date (Web): 12 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Journal of the American Chemical Society

1

Phenylalanine-mediated Porous CuxO Nanoparticle Clusters

2

Ameliorate Parkinson’s Disease by Reducing Oxidative Stress

3

Changlong Hao, Aihua Qu, Liguang Xu, Maozhong Sun, Hongyu Zhang, Chuanlai Xu*, Hua

4

Kuang*

5 6 7

International Joint Research Laboratory for Biointerface and Biodetection, State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PRC. *Corresponding

Authors: [email protected]; [email protected]

8 9

Abstract: Reactive oxygen species (ROS)-mediated mitochondrial dysfunction is one

10

of the major pathological mechanisms of Parkinson’s disease. Using inorganic

11

nanomaterials to scavenge ROS has drawn significant interest and can prevent

12

ROS-mediated neurological disorders. We prepared uniform CuxO nanoparticle

13

clusters (NCs) with an average size of 65 ± 7 nm, using phenylalanine (Phe) as the

14

structure-directing agent. These CuxO NCs functionally mimicked the activities of

15

peroxidase, superoxide dismutase, catalase, and glutathione peroxidase. Because they

16

eliminated ROS, the CuxO NCs inhibited neurotoxicity in a cellular model of

17

Parkinson’s disease and rescued the memory loss of mice with Parkinson’s disease.

18

The biocompatibility and multiple enzyme-mimicking activities of CuxO NCs offer

19

new opportunities for the application of NCs in biomedicine, biosensing, and

20

biocatalysis.

21 22

Keywords: CuxO, Nanoparticle clusters, ROS scavenger, Biomimetic enzyme,

23

Parkinson’s disease

24 1

ACS Paragon Plus Environment

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Introduction

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In living organisms, reactive oxygen species (ROS), such as singlet oxygen, the

3

hydroxyl radical (·OH), the superoxide anion (O2·−), and hydrogen peroxide (H2O2),

4

are natural products of the normal intracellular metabolism.1 Other factors, such as

5

high-energy irradiation and exposure to toxic compounds, can also produce excess

6

ROS. Moderate ROS levels are important for cell signaling and many intracellular

7

functions, including the defense against pathogens. However, excessive ROS can

8

subject normal cells to oxidative stress.

9

There is evidence that oxidative stress causes neuronal death and neural dysfunction,

10

suggesting an important pathogenic role for oxidative stress in Parkinson’s disease

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(PD). But the efficiently eliminating excess ROS has the notably therapeutic effects

12

on PD,2–3 because scavenging excess ROS protects tissues from oxidative stress.

13

Under normal physiological conditions, the cellular redox balance is maintained by

14

the antioxidant system, which consists of small antioxidant molecules (such as

15

glutathione) and enzymes (catalase [CAT], superoxide dismutase [SOD], glutathione

16

peroxidase [GPx], etc.).2–3 However, these natural enzymes are sensitive to

17

environmental conditions and can become inactive under pathological conditions.

18

To solve this problem, increasing attention has been paid to nanomaterials,4–6

19

especially those with enzyme-like activities that can scavenge ROS.7–8 A large

20

number of artificial enzymes, including metal nanomaterials,9 metal oxides,10 Prussian

21

blue nanoparticles,11 and quantum dots,12 have recently been reported. These artificial

22

enzymes have been widely applied in a variety of biomedical processes, including 2


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gene editing,13 cancer therapy,14–18 delayed aging,19 and imaging.20

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Copper, an essential micronutrient in humans, is an integral part of many important

3

enzymes, such as tyrosinase and Cu-Zn SOD.21 Cu-based nanoparticles, especially

4

cuprous and cupric oxides (Cu2O and CuO),22–23 have aroused particular interest in

5

many fields, including antibacterial material development,24 biosensing,25 and

6

photocatalysis,26 because they are inexpensive and easy to synthesize.27 Cu(OH)2

7

nanomaterials have also been applied in the development of biosensors.28 However,

8

the use of these nanomaterials in living systems is often difficult because they are

9

large and highly cytotoxic.29 We report here a novel strategy for the synthesis of CuxO

10

nanoparticle clusters (NCs) with good biocompatibility and the analysis of their

11

multiple enzyme-like properties in living cells. CuxO NCs can be used as an

12

antioxidant because they function as CAT, GPx, and SOD analogues. The remarkable

13

cytoprotective effects of NCs against oxidative-stress-mediated neurotoxicity have

14

been demonstrated in a human-cell-line-based model of PD. Significantly, the PD

15

model mice has been applied to elucidate the in vivo therapeutic effects of CuxO NCs.

16 17

Results and discussion

18

Synthesis and characterization of CuxO NCs

19

To generate the NC structure,

20

structure-directing agent, polyvinylpyrrolidone (PVP) as the stabilizer, and Cu(II)

21

ions as the inorganic precursor (Scheme 1). The chemical reactions involved in

22

preparing the CuxO NCs were as follows. Phe was first coordinated to Cu(II) ions to

L-phenylalanine

(L-Phe) was chosen as the

3



1

form a Cu(II)–Phe complex,30 and Cu(OH)2–Phe formed when OH− was added to the

2

aqueous copper salt solution. This species decomposed into CuO at 120 °C. After the

3

introduction of D-(+)-glucose,31 a portion of the CuO was reduced to Cu2O, forming a

4

complex of CuO and Cu2O NCs (designated ‘CuxO NCs’). It should be noted that the

5

anions of the copper salts (CuSO4, CuCl2, Cu(NO3)2, Cu(CH3COO)2) and the cations

6

of the bases (NaOH, KOH) had no obvious affect the size or morphology of the final

7

CuxO products.

8

Analysis of the as-obtained products with transmission electron microscopy (TEM)

9

(Figures 1A) showed the formation of CuxO NCs with a mean diameter of 65 ± 7 nm,

10

which was consistent with dynamic light scattering (DLS) measurements (Figure S1).

11

A representative high-resolution TEM image and the corresponding selected area

12

electron diffraction pattern of the CuxO NCs are shown in Figure 1B. Well-resolved

13

lattice spacings of 0.242 nm and 0.251 nm corresponded to the (111) plane of Cu2O

14

and the (002) plane of CuO, respectively. Figure 1C shows a scanning TEM (STEM)

15

image of the CuxO NCs, indicating the porous structure of NCs. The corresponding

16

elemental mapping results revealed the uniform distributions of Cu and O in the NCs.

17

An electron tomographic reconstruction of the CuxO NCs showed that the

18

nanoparticle units of CuxO were tightly interconnected, forming a porous NC

19

structure (Figure 1D).

20

The structure of the CuxO NCs was also characterized with X-ray diffraction. All the

21

diffraction peaks could be indexed to CuO/Cu2O because they were consistent with

22

those simulated for CuO (Joint Committee on Powder Diffraction Standards [JCPDS] 4


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no. 45-0937) and Cu2O (JCPDS no. 05-0667) (Figure 2A). X-ray photoelectron

2

spectroscopy was used to study the surface properties and oxidation states of the Cu in

3

the NCs (Figures 2B and S2). The Cu 2p spectrum showed a doublet at binding

4

energies (BEs) of 932.6 and 952.5 eV, corresponding to the Cu 2p3/2 and Cu 2p1/2

5

lines, respectively. The Cu 2p3/2 peak could be fitted to two peaks with BEs of 932.6

6

eV and 952.1 eV, corresponding to Cu2O and CuO, respectively.

7

Formation mechanism of CuxO NCs

8

TEM images of CuxO NCs prepared with

9

displayed a similar structure to those formed with L-Phe. When no Phe ligands were

10

used in the synthesis, a sheet-like nanostructure was obtained (Figure S4). A series of

11

control experiments was performed to clarify the specificity of Phe in the synthesis of

12

the CuxO NCs (Figures S5–6). TEM images showed that the morphologies and sizes

13

of particles synthesized with five other amino acids (L-tyrosine [L-Tyr], L-aspartic

14

acid [L-Asp], L-glutamic acid [L-Glu], L-lysine [L-Lys], and L-arginine [L-Arg]). The

15

presence of L-Tyr or L-Asp in the hydrothermal synthesis led to the formation of

16

ellipsoid-shaped particles (CuxO–Tyr, CuxO–Asp) with sizes above 100 nm, and the

17

use of L-Glu (CuxO–Glu) produced rod-like nanoparticles. While the ligands L-Lys

18

and L-Arg could not form nanoparticles (CuxO–Lys, CuxO–Arg) with a uniform

19

diameter and morphology. All these CuxO structures differed from the CuxO

20

synthesized with Phe (CuxO–Phe), indicating that the Phe ligand played a crucial role

21

in determining the final NC structures.

22

The rationale for selecting this structure-directing agent can be explained as follows.

D-Phe

were shown in Figure S3, and

5



1

Phenylalanine is a convenient surface ligand for CuO and binds to it via both its

2

carboxyl group and amino group.32 In CuO, the coordination bond between Cu2+ and

3

the amino group is strong, and the oxygen atoms at the -COO- end also form covalent

4

bonds with Cu2+, replacing some of the oxygen atoms on the CuO surface. The

5

aromatic group of phenylalanine provides a strong hydrophobic attraction between the

6

nanoparticles, allowing them to form superparticle assemblies. These attractive

7

hydrophobic interactions counterbalance the repulsive electrostatic interactions33 and

8

produce nanoparticle clusters. Moreover, isothermal titration calorimetry (ITC) was

9

used to study the interactions between Cu2+ and amino acids, as shown in Figures S7–

10

12 and Table S1. The results demonstrated that the affinity between copper ions and

11

the related amino acids was different, which could be attributed to the formation of

12

CuxO with different sizes and morphologies.

13

The evolution of the formation mechanism of CuxO NCs was also monitored. As

14

shown in Scheme 1, the CuxO NCs were prepared from CuO NCs when the latter

15

were partially reduced by D-(+)-glucose. We studied the growth process of CuO NCs

16

in the presence of L-Phe (Figure S13). The small primary nanoparticles formed 1 min

17

after the metal precursor was heated (Figure S13A). As the reaction time increased,

18

these small nanoparticles became large and clustered together (Figure S13B–E),

19

suggesting that these CuO NCs grow from the primary small nanoparticles through

20

their oriental attachment. The NCs reached their maximum diameter after 20 min, and

21

no major change in their morphology occurred thereafter, even after 120 min (Figure

22

S13F). Notably, the small CuO nanoparticles orderly attached onto the primary cores 6


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within 20 min and then evolved into the CuO nanoparticle-clusters.

2

Multi-enzyme like activities of CuxO NCs

3

After the comprehensive characterization of the CuxO NCs, their multiple

4

enzyme-mimicking activities were investigated. The SOD-mimicking activity of the

5

CuxO NCs was first investigated. O2·− was generated from the reaction of xanthine

6

with xanthine oxidase. The ability of the CuxO NCs to scavenge O2·− was

7

characterized

8

-tetrazolium (WST-1), which reacts with O2·− to produce formazan, which displays

9

specific absorption at 450 nm. In the presence of CuxO NCs, the percentage of

10

formazan produced was significantly reduced, demonstrating the SOD-mimicking

11

activity of the CuxO NCs under physiologically relevant conditions (Figures 3A and

12

S14). This SOD-mimicking activity was also confirmed with electron paramagnetic

13

resonance spectroscopy (Figure S15).

14

CAT catalyzes the decomposition of H2O2 to H2O and O2. The CAT-like activity of

15

the CuxO NCs was evaluated with both fluorescence and absorbance spectroscopy.10

16

H2O2 reacts with terephthalic acid to produce 2-hydroxyterephthalic acid, which

17

displays a fluorescence peak at 425 nm. The intensity of this fluorescence peak was

18

significantly reduced by the CuxO-NC-induced decomposition of H2O2 (Figure 3B).

19

As shown in Figure S16, approximately 85% of the total H2O2 was decomposed by 30

20

μg/mL CuxO NCs, which is even more efficient than the decomposition achieved with

21

20 U/mL CAT. Importantly, the thermal stability of the CuxO NCs was significantly

22

higher than that of natural CAT (Figure S17). The CAT-like activity of the CuxO NCs

with

2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H

7



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1

was also analyzed by monitoring the absorbance of H2O2 at 240 nm. A significant

2

reduction in the absorbance of H2O2 was observed with increasing time, indicating

3

H2O2 decomposition (Figure S18). Overall, our results demonstrate that CuxO NCs act

4

as an efficient CAT analogue. A steady-state kinetic assay was performed to confirm

5

the enzymatic catalysis mechanism, by varying the concentration of H2O2 (0.05–5

6

mM) in the presence of CuxO NCs (10 μg/mL). This reaction showed typical

7

Michaelis–Menten kinetics (Figure S19).

8

In addition to their CAT- and SOD-like activities, the CuxO NCs displayed GPx-like

9

behavior, eliminating H2O2 and catalyzing the oxidation of reduced glutathione

10

(GSH) to oxidized glutathione (GSSG). GSSG could be reduced back to GSH by

11

glutathione reductase (GR) and reduced nicotinamide adenine dinucleotide phosphate

12

(NADPH). The GPx-like activity of the CuxO NCs was estimated by monitoring the

13

reduction in the absorption of NADPH at 340 nm. As shown in Figure 3C, an obvious

14

reduction in the NADPH absorbance was observed with increasing time. A

15

steady-state kinetic analysis, with H2O2 and GSH as the substrates, was used to

16

evaluate the GPx-like activity of the CuxO NCs (Figure S20). The peroxidase

17

(POD)-like

18

3,3,5,5-tetramethylbenzidine (TMB) as the chromogenic substrate (Figures 3D and

19

S21-24). We also investigated the ·OH-scavenging activity of CuxO NCs. As shown

20

in Figure S25, the signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide/·OH

21

decreased with increasing concentrations of CuxO NCs, indicating that not only do

22

CuxO NCs have antioxidant-enzyme-like activities, but they also effectively

activity

of

the

CuxO

NCs

8


was

determined

using

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1

scavenge ·OH. Based on these results, we hypothesized that CuxO NCs inhibit

2

oxidative stress and protect cells from ROS-induced cytotoxicity.

3

The sizes and shapes of nanomaterials are the vital functions of their enzyme-mimetic

4

activities. Therefore, CuxO of different sizes and morphologies were prepared using

5

different amino acids as the shape-directing agents, and their enzyme-mimetic

6

activities were compared. As shown in Figure 4A–D, compared with ellipsoids

7

(CuxO–Tyr, CuxO–Asp) and rods (CuxO–Glu), CuxO–Phe displayed the highest

8

activities in all four enzyme-mimetic experiments.

9

To understand the differences in the activities of CuxO, various shapes, sizes, surface

10

areas, pore sizes, and volumes were investigated. Nitrogen adsorption experiments

11

were used to characterize the surface areas and porosity of CuxO. As shown in Figure

12

S26, the N2 adsorption–desorption isotherm was a typical type II isotherm, and the

13

Brunauer–Emmett–Teller surface areas and the total pore volumes of the CuxO NCs

14

are summarized in Figure 4E. A porous structure was beneﬁcial for encapsulating

15

substrates and simultaneously facilitating the catalytic process. Importantly, the total

16

volume of the pores and the pore diameter in CuxO–Phe were higher than those of the

17

other materials, except CuxO–Tyr. The catalytic activity of CuxO–Tyr was much

18

lower than that of CuxO–Phe, although the size and surface area of CuxO–Tyr (186

19

nm) was larger than that of CuxO–Phe (65 nm). The higher catalytic activity of CuxO–

20

Phe may be ascribed to its higher surface area, unique structure, and the Phe ligand. A

21

larger size and greater surface area favor stronger multiple enzymatic activities, but

22

the different ligand seems to be responsible for the lower activity of CuxO–Tyr 9



1

relative to that of CuxO–Phe. Based on these experimental results, we speculated that

2

besides size, shape, and surface area, the ligand in the material was another key factor

3

dictating its catalytic activity.

4

The catalytic activity of CuxO–Phe NCs was compared with those of reported metal

5

oxides,34-35 such as Mn3O4, V2O5, Fe3O4, MnO2, Co3O4, and CeO2. As shown in

6

Figure S27, many other metal oxide nanoparticles showed no triple enzyme-like

7

activities. Although Mn3O4 displayed higher SOD-like activity than CuxO, both the

8

CAT- and GPx-like activities of Mn3O4 were lower than those of the CuxO–Phe NCs.

9

Therefore, our NCs have significant advantages over other metal-oxide-based

10

ROS-scavenging nanomaterials.

11

To test the stability and recyclability of our catalyst, the CuxO NCs were collected and

12

washed several times with water, and a second catalytic test was conducted. After

13

three cycles of washing, the catalytic activity of the NCs did not obviously decrease,

14

demonstrating the excellent catalytic stability of the CuxO (Figure S28). The good

15

stability of the CuxO catalyst may be related to its high structural stability.

16

CuxO NCs protect cells against oxidative stress in vitro

17

To assess the potential bio-applications of CuxO NCs, cell viability tests were

18

performed to study their biocompatibility. TEM images revealed that the CuxO NCs

19

were taken up by SHSY-5Y cells (Figure S29), and CCK-8 experiments showed that

20

the CuxO NCs had no obvious cytotoxicity (Figures S30 and S31). The copper content

21

of the cells was determined with inductively coupled plasma optical emission

22

spectroscopy (Figure S32), which indicated that the cellular uptake of the NCs was 10


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1

dose-dependent.

2

The

3

apoptosis in SHSY-5Y cells,36 which are then typically used as an experimental

4

model of the PD phenotype. As shown in Figure S33A, the cell viability gradually

5

decreased as the MPP+ concentration increased. In subsequent experiments, 1 mM

6

MPP+ was used to induce neurotoxicity in SHSY-5Y cells, and to test the ability of

7

CuxO NCs to reduce MPP+-induced neurotoxicity. The cell viability improved with

8

increasing concentrations of CuxO (Figure S33B), confirming the expected

9

cytoprotection afforded by CuxO NCs against MPP+-induced cell death.

neurotoxin

1-methyl-4-phenylpyridinium (MPP+)

causes

ROS-mediated

10

The flow-cytometric data shown in Figure 5A indicate that CuxO treatment

11

significantly reduced the number of apoptotic cells induced by MPP+, and cell

12

viability increased from 61.5% to 80.4%. To verify the cytoprotective effect of CuxO,

13

a western blotting analysis of caspase 3, a common effector of the apoptotic pathway,

14

was conducted (Figure 5B). Compared with the untreated sample, MPP+ exposure

15

caused an increase in caspase 3, confirming that MPP+ induces cell apoptosis.

16

However, the amount of caspase 3 in the MPP+-treated SHSY-5Y cells decreased

17

after treatment with CuxO NCs. We also investigated the morphological changes in

18

the SHSY-5Y cells. As illustrated in Figure S36, normal SHSY-5Y cells have a

19

polygonal shape, with distinct and intact edges. After MPP+ exposure, the cells

20

became shrunken, with a globular shape, confirming the toxicity of MPP+. Notably,

21

SHSY-5Y cells cotreated with CuxO and MPP+ displayed a normal morphology,

22

demonstrating the protection afforded the SHSY-5Y cells against MPP+-induced 11



1

neurotoxicity. Laser confocal fluorescence imaging was used to confirm the

2

cytoprotection conferred by CuxO. As shown in Figure 5C, treatment with MPP+

3

produced fluorescent signals indicating the apoptosis of the SHSY-5Y cells. When

4

cultured with CuxO, the fluorescence intensity greatly decreased, confirming the

5

cytoprotective effect of CuxO. Like MPP+, H2O2 also causes cell apoptosis through

6

oxidative stress. Therefore, we investigated the protection afforded SH-SY5Y cells by

7

CuxO against H2O2-induced cytotoxicity (Figures S35–37).

8

Two other cell models were established to verify the universality of CuxO NCs as

9

ROS scavengers. Using NIH-3T3 cells, we studied the inhibitory effect of the CuxO

10

NCs on UVA-induced apoptosis. As shown in Figures S38 and S39, 20 μg/mL CuxO

11

NCs effectively increased the cell viability and reduced the apoptosis of UVA-treated

12

NIH-3T3 cells. Another oxidative stress cell model was established by culturing pig

13

iliac endothelial (PIE) cells with oxidized low-density lipoprotein (OxLDL). After

14

treatment with the CuxO NCs, the viability of the OxLDL-treated PIE cells was

15

effectively improved. Significantly, ROS-mediated cell apoptosis also decreased after

16

treatment with the CuxO NCs (Figures S40 and S41). All the results presented here

17

demonstrate that CuxO NCs can be used as ROS scavengers in multiple cell models.

18

CuxO NCs rescue memory deficits in PD model mice

19

To investigate the potential safety of CuxO NCs in vivo, hematoxylin and eosin

20

(H&E)-stained images of different organs (heart, liver, kidney, spleen, and lung) and

21

substantia nigra were examined (Figure S42 and S43). Moreover, the serum

22

biochemistry tests were conducted (Figures S44). The results indicated that there were 12


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1

no obvious adverse effects in the treated mice during their long-term exposure to

2

CuxO NCs.

3

PD model mice was induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

4

(MPTP).37 To evaluate the therapeutic efficacy of the CuxO NCs as a PD therapy,

5

CuxO NCs were injected into the striata of the PD model mice. Compared with the

6

WT (wild-type) group, the number of ionized calcium-binding adapter molecule 1

7

(IBA-1)-positive cells was clearly increased in the striata of the PD group. The

8

upregulated expression of IBA-1 reflects increased neuroinflammation, which

9

activates microglia cells. In contrast, the IBA-1 levels in the CuxO-NC-treated group

10

were significantly reduced, indicating that the ROS-scavenging effects of the CuxO

11

NCs ameliorated the neuroinflammation in the PD model mice (Figure 6A and C). We

12

also examined the changes in the striatal tyrosine hydroxylase (TH) levels after

13

treatment with CuxO NC. TH is the rate-limiting enzyme in dopamine synthesis, and

14

dopamine depletion in the nigrostriatal system is a hallmark of the pathogenesis of

15

PD. As shown in Figure 6B, the MPTP-treated mice showed clearly diminished TH

16

levels. On the contrary, the CuxO-NC-treated groups showed dramatically higher TH

17

levels than the PD group. 4-Hydroxynonenal (HNE), a major aldehyde by-product of

18

lipid peroxidation, is an indicator of oxidative damage (Figure 6E). The

19

CuxO-NC-treated groups displayed much lower levels of 4-HNE than the PD group,

20

indicating that the degeneration of dopaminergic neurons was inhibited by the

21

mitigation of oxidative stress. The overall distribution of TH in the mouse brains was

22

visualized with immunohistochemistry. As shown in Figure 7A, higher levels of TH 13



1

were observed in the CuxO-NC-treated group than in the PD group, which is

2

consistent with immunohistofluorescence results in Figure 6. These TH distributions

3

were also visible on confocal-microscopic images (Figure 7B).

4

The spatial learning and memory of the PD mice after treatment were examined with

5

the Morris water maze test. As shown in Figure 8, compared with the PD mice, the

6

CuxO-NC-treated mice displayed strongly enhanced spatial learning, memory, escape

7

latency, and swimming speeds during the training process, indicating that CuxO

8

induced cognitive recovery in these mice. The PD group also spent less time in the

9

target quadrant than the other groups and showed random motion paths, reflecting the

10

memory disorder induced by MPTP. However, the CuxO-NC-treated mice showed

11

spatially oriented swimming behavior, spent longer time in the target quadrant, and

12

their motion paths were mainly focused there (Figure 8D), indicating that CuxO NC

13

significantly rescued the memory loss in the PD model mice. All the above results

14

demonstrate that CuxO NCs hold great potential in the treatment of PD.

15

Conclusion

16

In this study, we first fabricated uniform, porous CuxO NCs using Phe as the

17

structure-directing agent. Significantly, the nanomaterial not only mimicked the

18

activity of multiple enzymes, but was also highly stable, with no observable

19

cytotoxicity. ROS scavenging by the CuxO NCs was demonstrated in a PD-simulating

20

cell line and generalized to other cell types. In vivo experiments showed the excellent

21

biocompatibility and therapeutic effects of CuxO against oxidative-stress-induced

22

neurological disorders in MPTP-induced PD mice. These findings may offer 14


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opportunities for the application of inorganic nanostructures in biomedicine and other

2

biological engineering fields, such as treatment of neurodegeneration diseases, cancer

3

diagnostics and therapy.

4 5

ACKNOWLEDGMENTS

6

This work is financially supported by the National Natural Science Foundation of

7

China (21631005, 21673104, 21522102, 21503095). The authors are grateful to Prof.

8

Luis Liz-Marzán for useful discussions.

9

ASSOCIATED CONTENT

10

Supporting

11

supplementary figures. This material is available free of charge via the Internet at

12

http://pubs.acs.org.

13

References

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1 2

Scheme 1. Schematic illustration of the preparation of CuxO NCs.

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1 2

Figure 1. (A) TEM image, (B) HRTEM image and SAED pattern, (C) STEM and

3

energy dispersive X-ray spectroscopy (EDS) mapping, and (D) 3D electron

4

tomography reconstruction images (up: 3D surface rendering, down: cross-section

5

view) of CuxO NCs prepared using L-Phe.

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1 2

Figure 2. (A) XRD results and (B) High-resolution Cu 2p XPS data for CuxO NCs.

3 4 5 6 7

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1 2

Figure 3. CuxO NCs shows multi-enzyme like activities. (A) SOD-like activity, (B)

3

CAT-like activity, (C) GPx-like activity, (D) POD-like activity.

4 5 6 7 8

21



1 2

Figure 4. (A–D) Comparison of SOD, CAT, GPx, and POD-like activity of CuxO-Tyr,

3

CuxO-Asp, CuxO-Glu with CuxO-Phe. (E) Structural parameters of different

4

morphology of CuxO nanomaterials. Tyr means tyrosine, Asp means aspartic acid,

5

Glu means glutamic acid, and they are all in L-type.

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Figure 5. (A) Cell viability measured through flow cytometry. (B) Western blot

3

analysis of caspase-3 in SHSY-5Y cells. (C) Confocal microscopy images of

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SHSY-5Y cells in the presence of: (1) 0, (2) 1 mM MPP+, (3) 1 mM MPP+ and 5

5

μg/mL CuxO, (4) 1 mM MPP+ and 10 μg/mL CuxO, (5) 1 mM MPP+ and 20 μg/mL

6

CuxO. Scale bars are 50 μm. 23



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Figure 6. Confocal images of wild type mice striata, MPTP-induced PD mice striata,

6

and CuxO NCs-treated PD mice striata: (A) expression of IBA-1, (B) expression of

7

TH. Scale bars are 50 μm. Average expression levels of each treatment group (n=6):

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(C) IBA-1. (D) TH. (E) 4-HNE. One-way ANOVA was used for statistical analysis:

9

*P < 0.001.

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Figure 7. Expression of TH in the brains of control, MPTP-induced PD mice, and

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CuxO NCs-treated mice: (A) Immunohistochemistry (IHC) images. (B)

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immunohistofluorescence (IHF) images. Scale bar is 1 mm.

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Figure 8. CuxO NCs rescued memory deficits in PD mice. (A) Escape latency, (B)

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swimming speed, (C) relative time spent on the target quadrant where the escape

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platform used to located. Statistical analysis was performed using a one-way ANOVA

5

test, with ** indicating p < 0.001 and * indicating p < 0.05 compared with the OA

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group (n = 8 per group). (D) Representative path tracings of different groups.

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