Sulfur Nanoparticles with Novel Morphologies Coupled with Brain

Subscriber access provided by READING UNIV

Article

Sulfur Nanoparticles with Novel Morphologies Coupled with Brain-Targeting Peptides RVG as a New Type of Inhibitor Against Metal-Induced A# Aggregation Jing Sun, Wenjie Xie, Xufeng Zhu, Mengmeng Xu, and Jie Liu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00312 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

1

Sulfur Nanoparticles with Novel Morphologies Coupled with

2

Brain-Targeting Peptides RVG as a New Type of Inhibitor

3

Against Metal-Induced Aβ Aggregation

4

Jing Suna,1, Wenjie Xiea,1, Xufeng Zhua, Mengmeng Xua, and Jie Liua,*

5

a

6

*

7

E-mail address: [email protected] (J. Liu).

Department of Chemistry, Jinan University, Guangzhou 510632, China Corresponding author. Tel: +86 20 85220223; fax: +86 20 85221263.

8 9

Abstract

10

Functionalized nanomaterials, which have been applied widely to inhibit

11

amyloid-β protein (Aβ) aggregation, show an enormous potential in the field of

12

prevention and treatment of Alzheimer's disease (AD). A significant body of data has

13

demonstrated that the morphology and size of nanomaterials have remarkable effects

14

on their biological behaviors. In this paper, we proposed and designed three kinds of

15

brain-targeting sulfur nanoparticles (RVG@Met@SNPs) with novel morphologies

16

(volute-like, tadpole-like and sphere-like), and investigated the effect of different

17

RVG@Met@SNPs on the Aβ-Cu2+ complex aggregation and their corresponding

18

neurotoxicity. Among them, the sphere-like nanoparticles (RVG@Met@SS) exhibited

19

the most effective inhibitory activity due to their unique mini size effect, and they

20

reduced 61.6% of the Aβ-Cu2+ complex aggregation and increased 92.4% SH-SY5Y

21

cells viability in a dose of 10 µg/mL. In vitro and in vivo, the abilities of different

22

morphologies of RVG@Met@SNPs crossing blood-brain barrier (BBB) and targeting

23

the brain parenchymal cells were significantly different. Moreover, improvements in

24

learning disability and cognitive lose were shown in the transgenic AD mice model

25

using the Morris water maze test after multiple dosage of RVG@Met@SNPs

26

treatment. In general, the purpose of this research is to develop a biological

27

application of sulfur nanoparticles and to provide a novel, functionalized nanomaterial 1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

to treat AD.

2

Keywords: Alzheimer’s disease; amyloid-β protein; sulfur nanoparticles; blood-brain

3

barrier;

4

1. Introduction

5

Alzheimer’s disease (AD) is one of most common neurodegenerative disorders.1

6

A great deal of studies have demonstrated that the extracellular amyloid-β protein (Aβ)

7

aggregation is the main pathological hallmarks of AD.2 The Aβ aggregation can

8

induce the crosstalk of various molecular signaling pathways, cause the loss of neuron,

9

and produce synaptic dysfunction in brains.3,4,5 Therefore, therapeutic strategies

10

aiming at the initial stage of Aβ aggregation will be viable to prevent or delay the

11

onset of AD.6

12

In the brains of AD patients, the high concentrations of redox-active metal ions,

13

especially Cu2+ ion have been demonstrated that they can accelerate the fibrillation of

14

Aβ significantly.7,8,9,10 The toxic Aβ-Cu2+ aggregation can contribute to abnormal

15

formations of reactive oxygen species (ROS), as well as oxidative stresses, triggering

16

a vicious cycle as a result.11,12 Thus, metal-chelating agents such as clioquinol (CQ)

17

and 8-hydroxyquinoline-2-carboxylic acid (HQC), have been applied for targeting and

18

controlling Aβ aggregation, which are shown to be able to capture Cu2+ and inhibit the

19

Cu2+-induced Aβ aggregation process.13,14 However, most of metal-chelating agents

20

are limited in their further application in AD because of their poor brain-targeting

21

ability, inefficient permeability to blood-brain barrier (BBB), and toxic effect. 15,16,17

22

Nowadays, nanomaterials (also known as nanoparticles) have been applied

23

successfully for diagnosing and treating diseases due to their excellent

24

properties.18,19,20,21 Physicochemical characteristics of nanoparticles such as size and

25

morphology have profound influences on their biological behaviors.22 Recent

26

researches had found that the AgNPs show various results on their biological

27

applications due to their sizes, and such applications include antibacterial activity and 2


Page 2 of 53

Page 3 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

ecological toxicity.23 Furthermore, it was proven that the special morphology of

2

cerium oxide nanoparticles has the potential to influence antioxidant activity.24

3

Moreover, through the assembly of functionalized biomolecules, nanoparticles can

4

target the brain and penetrate BBB effectively.25,26 Inspired by the discussions above,

5

in order to overcome the limitation of metal chelators, we decided to design

6

nanoparticles with special morphology which are able to effectively decrease Aβ-Cu2+

7

complex and penetrate BBB, and this may be a promising therapeutic strategy for

8

curing AD.

9

Sulfur nanoparticles have excellent efficacies such as removal of Cu2+ ions and

10

radicals, antioxidant and antibacterial activity, and these functions could all be

11

enhanced by their minimal sizes and surface energy effects.27,28 With different

12

morphologies and sizes, sulfur nanoparticles show distinct chemical properties and

13

biological activities. Therefore, selecting the activity of different modifiers and

14

heterogeneous reactions could be adjusted and optimized by regulating the size and

15

morphology of sulfur nanoparticles.29,30,31,32 In our work, methionine was selected as a

16

modifier to regulate the morphology of nanoparticles. By adjusting the proportion of

17

methionine and sodium thiosulfate in the synthesis process, three kinds of sulfur

18

nanoparticles with novel morphologies were obtained (volute-like, tadpole-like and

19

sphere-like; called as Met@VS, Met@TS and Met@SS respectively).In recent years,

20

sulfide nanosheets in neurodegenerative diseases are reported that the sulfur play a

21

key role to bind with Aβ and interfere the formation of hydrogen bonds in Aβ

22

aggregation.33,34 However, to the best of our knowledge, there is no existing report on

23

the utilization of sulfur nanoparticles as therapeutic agents for AD treatments. Thus,

24

whether different morphologies of sulfur nanoparticles could remove the

25

dyshomeostasis of Cu2+ ion and inhibit the aggregation of Aβ-Cu2+ or not require

26

further investigation.

27

Designing BBB-penetrable nanoparticles or brain-targeting molecules is a key

28

process to the success of AD treatment.35 RVG, a 29 amino-acid peptide isolated from 3



1

the coat protein of rabies virus, was applied as a promising candidate to improve the

2

drug’s ability to target the brain and its penetrability to BBB.36,37 Utilizing the rich

3

carboxyl groups in methionine offered plenty of modification sites. We successfully

4

conjoined RVG with Met@SNPs surface (call as RVG@Met@VS, RVG@Met@TS

5

and RVG@Met@SS, respectively). In this work, characteristics of nanoparticles were

6

investigated. Meanwhile, we compared and explored initially the abilities of

7

RVG@Met@SNPs with different morphologies to inhibit Aβ self-fibrillation and to

8

decrease Cu2+-induced Aβ aggregation. In vitro, it is demonstrated that various

9

RVG@Met@SNPs showed morphological effects on their biological applications, in

10

which they penetrated BBB, and were absorbed by the brain parenchyma cells

11

differently. Moreover, RVG@Met@SNPs could scavenge the Aβ-Cu2+ mediated

12

formation of reactive oxygen species (ROS) and protect SH-SY5Y cells from the

13

corresponding neurotoxicity. Among them, spherical-like RVG@Met@SS was the

14

most effective one as an inhibitor, and it also significantly increased cells viability. As

15

expected, after multiple-dosing treatments, AD mouse in the Morris water maze test

16

showed a rescue of learning and memory loss.

17

2. Results and discussion

18

Design and Characterization of NPs

19

In this experiment, by adjusting the synthetic ratios of methionine and Na2S2O3,

20

we obtained sulfur nanoparticles with novel morphologies: volute-liked, tadpole-liked

21

and sphere-liked (called as Met@VS, Met@TS and Met@SS, respectively). Figure

22

S1 show the synthesis procedure of RVG@Met@SNPs. And the detailed

23

morphological structure of Met@SNPs could be recorded by transmission electron

24

microscopy (TEM) (Figure 1A). It could be observed that NPs dispersed nicely, and

25

their specific morphologies are presented as the following: around 150 nm for

26

Met@VS, 100 nm for Met@TS and 50 nm for Met@SS.

27

FT-IR was performed to confirm the success of the formation of methionine 4


Page 4 of 53

Page 5 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

modified nanoparticles. As illustrated in Figure 1B, Met@SNPs exhibits different

2

infrared peaks to sulfur but shows similar peaks to methionine. These peaks include

3

the stretching vibration of N-H at 2910 cm-1, the NH3+ deformation vibration at 2100

4

cm-1 and the S-CH2 deformation vibration at 1450 cm-1. These data indicated

5

methionine has been successfully modified on the sulfur surface. The weaker

6

absorbance peak of NH3+ and the high frequency-shifted S-CH2 vibration may imply

7

that these groups formed coordination with the sulfur surface.

8

Moreover, the elemental composition analysis of different nanoparticles was

9

characterized by EDX. This data is illustrated in Figure 1C, in which an increased C

10

element is shown in nanoparticles. That is the case because the C element in

11

methionine has an increased methionine modification on sulfur surface.

12

In order to enhance the brain-targeting ability and the permeability of Met@SNPs,

13

we coated the RVG peptide with the Met@SNPs surfaces (RVG@Met@VS,

14

RVG@Met@TS, RVG@Met@SS). Herein, we used high Resolution Transmission

15

Electron Microscopy (HRTEM) to determine the conjugation of RVG peptides on

16

Met@SNPs. As shown in Figure S2, the thickness of conjugated RVG peptides on

17

Met@SNPs could be clearly observed: RVG@Met@VS is about 2.5 nm,

18

RVG@Met@TS about 1.6 nm, and RVG@Met@SS about 2.2 nm. These data implied

19

that the RVG peptides might have conjugated successfully with the surfaces of

20

Met@VS, Met@TS and Met@SS, respectively. Moreover, the brain-targeting RVG

21

peptides which were attached on the surfaces of Met@SNPs got detected by the BCA

22

assay.38 As shown in Figure S3A and B, after NPs mixed with the BCA working

23

solution, the color of the brain-targeted nanoparticles changed from colorless to

24

purple while the no-brain-targeted NPs system remain unchanged. Meanwhile, there

25

was an absorption peak from the mixed solution at 560 nm in the UV absorption

26

spectrum, indicating that RVG peptides were detected in the brain-targeted

27

nanoparticles. The concentration of RVG peptides in the supernatant was further

28

determined by BCA assay. The total concentration of RVG peptides on the 5



1

Met@SNPs were about: RVG@Met@VS: 34.2 µg/mL, RVG@Met@TS: 33.8 µg/mL,

2

RVG@Met@SS: 35.1 µg/mL.

3

The properties of various nanoparticles were investigated by the Nano-ZS

4

instrument. The size distribution and zeta potential values of nanoparticles are shown

5

in Figure S4 and Table S1. The size of RVG@Met@SNPs was increased slightly after

6

the RVG peptide coated with the Met@SNPs surfaces, corresponding with the results

7

of TEM and HRTEM. The zeta potential values of all nanoparticles were greater than

8

-30 mV, indicating that both Met@SNPs and RVG@Met@SNPs have high stability.

9

Dynamic light scattering (DLS) measurements were carried out over 7 days, which

10

indicated that both Met@SNPs and RVG@Met@SNPs were maintained stable

11

(Figure S5).

12

Inhibitory effect on NPs of Aβ self-fibrillation

13

ThT fluorescence assay is used widely to assess Aβ aggregation.39 Thus, we

14

firstly investigated whether different concentrations: 1, 2.5, 5, and 10 µg/mL of NPs

15

have inhibitory effects on Aβ self-fibrillation by using the ThT fluorescence assays.

16

As a positive control, we used the strongest ThT fluorescence intensity obtained from

17

pure Aβ incubation. As shown in Figure 2A, after respectively incubating Aβ with the

18

RVG@Met@VS in four different concentrations, RVG@Met@VS exhibited a weak

19

effect in inhibiting Aβ aggregation, confirmed by the data that even under the

20

strongest concentration, 10 µg/mL, only 13% of the Aβ aggregation was inhibited.

21

RVG@Met@TS showed moderate Aβ aggregation inhibitions, in which the

22

aggregation had an 8% inhibition with 2.5 µg/mL, a 20% inhibition with 5 µg/mL and

23

a 28% inhibition with 10 µg/mL. These data show that RVG@Met@TS inhibited

24

Aβ self-fibrillation in a dose-dependent manner, and it had a better inhibitory ability

25

than RVG@Met@VS. It should be noted that after co-incubation of Aβ and freshly

26

added RVG@Met@SS, the ThT fluorescence intensity of Aβ was declined

27

remarkably by 25% at 2.5 µg/mL, by 43% at 5 µg/mL, and by 55% at 10 µg/mL,

28

which suggested that RVG@Met@SS could effectively inhibit the aggregation of Aβ, 6


Page 6 of 53

Page 7 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

and it has the strongest inhibitory capacity among the three of them when same

2

concentrations were used. To further investigate the effect of NPs on the inhibition of

3

Aβ, we monitored the kinetics of Aβ in the absence or presence of RVG@Met@SNPs

4

(Figure S6). Aβ monomer was incubated alone in PBS at 37 °C and exhibited a

5

remarkable ThT fluorescence, which indicated that Aβ aggregation was formed. As

6

expected,

7

RVG@Met@SNPs was added to Aβ solution. Especially, on the presence of

8

RVG@Met@SS, the most significant decline of ThT fluorescence intensity was

9

observed. As shown in the control experiment, RVG@Met@SNPs could not affect

10

we

found

different

degrees

of

decline

in

fluorescence

when

ThT fluorescence under the experimental condition (Figure S7).

11

In addition, to further investigate the degradation effect of different NPs on Aβ,

12

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

13

(MALDI-TOF MS) was performed. As illustrated in Figure 2B, after co-incubation of

14

different NPs and fresh Aβ at 37 ℃ for 72 h respectively in groups, all

15

RVG@Met@SNPs degraded the Aβ with different extents. Among them,

16

RVG@Met@SS was the most effective one in which the particular peaks of Aβ were

17

declined and some low molecular peaks were observed. The details of the molecular

18

peaks were showed in Figure S8.

19

Effect of NPs on kinetics of the Aβ-Cu2+ complex aggregation

20

We explored the abilities of three kinds of RVG@Met@SNPs to inhibit Aβ

21

self-fibrillation at the first stage of our research. However, whether NPs could repress

22

the Aβ aggregation induced by Cu2+ requires further investigation. Firstly, we

23

investigated the direct effect of nanoparticles' ability to scavenge Cu2+ on Aβ

24

aggregation. As shown in Figure 3A, when Aβ monomers were being added into 60

25

µM Cu2+ solution, and left to a 72 h incubation, the kinetic data showed that the Aβ

26

monomers were first in a negligible lag phase (＜1 h), and then in an accelerated

27

growth phase (＜18 h), and finally in a steady equilibrium phase after 18 h. The

28

formation of more Aβ fibril was proven by the evidence that the fluorescence intensity 7



1

increased by 38.8%, comparing to that of the Aβ samples alone. When Aβ monomers

2

were treated respectively with the supernatants of three kinds of RVG@Met@SNPs

3

incubated individually with 60 µM Cu2+ solution for 72 h, the lag-phase did not with

4

change but the intensity of ThT fluorescence of Aβ-Cu2+ complex was decreased,

5

depending on the efficiency of their capacities to scavenge Cu2+. This indicated that

6

three kinds of RVG@Met@SNPs could repress the Aβ-Cu2+ complex by adsorbing

7

Cu2+. Next, we examined the influence of the kinetics of RVG@Met@VS,

8

RVG@Met@TS, RVG@Met@SS with different concentrations on Aβ-Cu2+

9

aggregation. As shown in Figure 3B, C, and D, RVG@Met@VS with all kinds of

10

concentrations did not inhibit Aβ-Cu2+ aggregation significantly and exhibited similar

11

kinetic profiles as the Aβ-Cu2+ samples. On the other hand, while the RVG@Met@TS

12

did not change the process of Aβ-Cu2+complex aggregation, it could reduce the

13

Aβ-Cu2+ complex aggregation in a dose-dependent manner. Among them,

14

RVG@Met@SS had the strongest inhibiting effects under the same concentrations.

15

Interestingly, we found that RVG@Met@SS could prolong the lag time of Aβ-Cu2+

16

aggregation significantly, indicating that its mechanism to inhibit Aβ-Cu2+

17

aggregation may be different from its precursors.

18

In addition, we used dynamic light scattering (DLS) to evaluate the

19

hydrodynamic distribution size of Aβ-Cu2+ complex when treating it with or without

20

different NPs. As shown in Figure 3E, the samples of the Aβ incubation alone show

21

that the aggregation sizes of Aβ increases gradually as time went on: from small

22

oligomer (≈300 nm) after 12 h to large aggregation (≈800 nm) after 72 h. In the

23

presence of Cu2+, a wide range of peaks from 200 to 800 nm appeared after 12 h, and

24

gradually shifted to 1300 nm after 72 h with no small peaks. This indicated that Cu2+

25

could accelerate the Aβ initial aggregation and increase the formation of mature fibril.

26

In contrast, in the treatment of Aβ-Cu2+ with RVG@Met@SNPs, the small peaks of

27

40~150 nm were first observed, which agreed with the hydration diameters of

28

individual RVG@Met@SNPs. As the incubation time increased, the intensity of the

29

peak was weakened, but the size was almost invariant. This indicated that many 8


Page 8 of 53

Page 9 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

monodispersed NPs decreased and some of them the monodisperse NPs did not

2

integrate further with Aβ species. In comparison with the size distribution of Aβ-Cu2+

3

treated with different NPs, all of them could inhibit Aβ-Cu2+ aggregation in different

4

extent. In the RVG@Met@SS samples, the large peaks of mature Aβ-Cu2+

5

aggregation (＞800 nm) almost disappeared, which indicated that RVG@Met@SS

6

could hinder the Aβ-Cu2+fibrillation process effectively and produce Aβ-Cu2+

7

aggregation with different sizes. The inhibitory effect of different nanoparticles on Aβ

8

self- fibrosis was also investigated by DLS (Figure S9). Compared with the Aβ-alone

9

groups, large fibrils peaks (≈800 nm) did not appear, which indicated that the fibrosis

10

of Aβ was effectively inhibited by RVG@Met@SS.

11

Morphology of Aβ-Cu2+ aggregation investigated by AFM and TEM

12

Based on the ThT results, we further investigated the morphological evidences of

13

inhibition of RVG@Met@SNPs to Aβ-Cu2+ complex using TEM and AFM. As

14

illustrated in Figure 4A and B, Aβ peptides formed a typical network structure with

15

long, thick, and branched fibrils. When Aβ peptides were incubated with Cu2+, the

16

fibrils became thicker, and large aggregation could be observed. When incubating

17

Aβ-Cu2+ with RVG@Met@VS, the aggregation was still visible, but the entanglement

18

of Aβ fibrils was weakened. RVG@Met@TS showed an increased but moderate

19

inhibitory effect on Aβ-Cu2+aggregation, in which the large plaques were hardly

20

found. Especially after the presence of RVG@Met@SS, the Aβ-Cu2+ complexes

21

transformed into small spherical particles and amorphous oligomers, showing the

22

RVG@Met@SS had a more significant inhibitory effect on the Aβ-Cu2+ complex

23

aggregation.

24

The ThT fluorescence, DLS, TEM and AFM results indicated that

25

RVG@Met@TS and RVG@Met@SS could inhibit the Aβ aggregation induced by

26

Cu2+. Between them, RVG@Met@SS was more effective than RVG@Met@TS.

27

Cabaleiro Lago showed that nanoparticles with a large particle surface area could

28

inhibit Aβ aggregation.40 Due to their special morphologies and smaller particle sizes, 9



Page 10 of 53

1

RVG@Met@SS provided larger particle surface areas than RVG@Met@VS and

2

RVG@Met@TS under the same concentrations. The larger particle surface area of

3

RVG@Met@SS not only weakened the interaction between Aβ monomer and Cu2+

4

but also perturbed the formation of hydrogen bonding of Aβ fibril growth in the early

5

stage, which led to a more effective inhibition on Aβ-Cu2+ aggregation.

6

Biocompatibility assay

7

To assess the biosafety of different NPs, we used a methyl thiazolyl tetrazolium

8

(MTT) assay to investigate cytotoxicity at different concentrations. As illustrated in

9

Figure 5A, there was no obvious influence on SH-SY5Y cells survival after

10

incubating them with NPs with concentrations from 50 µg/mL to 200 µg/mL for 48 h,

11

and this proved that they were suitable for biological experiment. The cytotoxicity of

12

all NPs on bEnd.3 cells also showed that the cells viability was above 92% at all NPs

13

even at concentration of 200 µg/mL. (Figure 5B) The result ensured the integrity of

14

the bEnd.3 cell-layer as in vitro BBB model in the follow-up transcytosis study.

15

Moreover, to further validate the biocompatibility of NPs, we carried out the

16

apoptosis of SH-SY5Y cells after treating them with different NPs at a concentration

17

of 50 µg/mL using the Annexin V/PI assay. As shown in Figure 5C, less than 1% cell

18

apoptosis was caused by nanoparticles. The result indicated that all NPs could not

19

induce significant SH-SY5Y cell apoptosis.

20

Cellular uptake of nanoparticles

21

The NPs modified by the RVG peptides take neurons as their target cells.41 Thus,

22

we used SH-SY5Y cells as research models to investigate the RVG@Met@SNPs

23

internalization in the brain neuron. The cellular uptakes of RVG-modified NPs and

24

none RVG-modified NPs were evaluated by laser-scanning confocal microscopy after

25

incubating for 6 h. As shown in Figure 6A, the fluorescence intensity of

26

ruthenium-labeled

27

ruthenium-labeled none-RVG-modified NPs, implying that RVG enhanced cell to

RVG-modified

NPs

were

remarkably

10


higher

than

Page 11 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

uptake of nanoparticles significantly. Meanwhile, we could see the fluorescence

2

intensity of RVG@Met@SS was higher than the other two morphologies, and this

3

indicated RVG@Met@SS had the best permeability to reach neuron than

4

RVG@Met@VS and RVG@Met@TS.

5

Inductively coupled plasma atomic emission spectrometry (ICP-AES) experiment

6

helped to confirm the uptakes of different NPs inside the cells. Figure 6B reveals that

7

the cellular uptakes of RVG-modified NPs were increased by many times (evaluated

8

by the Ru concentration) comparing with none-RVG-modified NPs. When the

9

concentration of NPs was 10 µg/mL, RVG@Met@VS was 1.7 times more than

10

Met@VS, RVG@Met@TS was 3.8 times more than Met@TS, and RVG@Met@SS

11

was 4.1 times more than Met@SS. Moreover, morphology and particle size also

12

played significant roles in the uptake of NPs: RVG@Met@SS was 1.2 times more

13

than RVG@Met@TS, and was 2.2 times more than RVG@Met@VS. These results

14

indicated that RVG-modified NPs were more efficient than none-RVG-modified on

15

the cellular uptake, and the RVG@Met@SS was benefited from its sphericity-shape

16

and small size such that it had the highest penetration efficiency comparing with the

17

others. Meanwhile, accumulations of NPs inside the SH-SY5Y cells were enhanced

18

by increasing co-incubation concentration with 5, 10, 20 µg/mL progressively,

19

revealing that the process of cellular on NPs depended on concentration. The results

20

above showed that RVG-modified NPs targeted the neurons effectively and provided

21

the possibility to cross through the blood-brain barrier.

22

Clearance of Aβ-Cu2+ -induced ROS in SY-SY5Y cells

23

Previous studies showed that the ROS is a pathological feature of AD, in which it

24

influences the oxidation modification to lipids, proteins, RNA and DNA in the

25

cortex.42,43 Meanwhile, aggregation of Aβ is often accompanied with abnormal

26

production of ROS which can cause damage to neurons. Therefore, clearance of ROS

27

helps to protect neurons from the neurotoxicity of Aβ aggregation. Fluorometric

28

analysis by DCFH-DA experiment was performed to evaluate the elimination of 11



1

cellular ROS on NPs.44 As shown in the Figure 7A, the fluorescence intensities of the

2

Aβ-Cu2+ samples were significantly stronger comparing to the Aβ samples alone,

3

which implied that Aβ-Cu2+ complex could increase intracellular ROS severely. In

4

contrast, co-incubation of the SH-SY5Y cells with RVG@Met@SNPs, respectively,

5

the ROS induced by Aβ-Cu2+ was scavenged in different extent, among which

6

RVG@Met@SS caused cellular ROS decreased to normal levels. Based on the results,

7

quantification of intracellular ROS was carried out by flow cytometry. As illustrated

8

in Figure 7B, Aβ-Cu2+ caused remarkably ROS, enhancing 2.2 times level of ROS

9

than control group while the Aβ alone samples with 1.6 times. When treated

10

respectively NPs with Aβ-Cu2+ in the culture cells, the degree of ROS induced by

11

Aβ-Cu2+ complex was decreased by 19.3% of RVG@Met@VS, by 33.0% of

12

RVG@Met@TS, by 42.9% of RVG@Met@SS. The data above confirmed that the

13

RVG@Met@SNPs could prohibit the Cu2+-induced Aβ aggregation and reduce the

14

Aβ-Cu2+-induced ROS in different degree.

15

The scavenging capacity of nanoparticles on the Aβ-induced ROS was measured

16

by flow cytometry (Figure S10). Because of the similar mechanism of nanoparticles

17

on scavenging Aβ-Cu2+-induced ROS, nanoparticles also have unequal ability to

18

scavenge Aβ-induced ROS. After co-incubation with RVG@Met@SS, the

19

Aβ-induced ROS level was close to that of the control groups, indicating that

20

intracellular ROS were significantly scavenged.

21

Nanoparticles protected SH-SY5Y cells against Aβ-Cu2+ complex’s cytotoxicity

22

All RVG@Met@SNPs could inhibit Aβ-Cu2+ aggregation differently, but whether

23

RVG@Met@SNPs could reduce the neurotoxicity induced by Aβ-Cu2+complex

24

requires further investigation. SH-SY5Y cells were selected as indicators of their

25

morphological changes and cellular viability. First, we used scanning electron

26

microscope (SEM) to determine the morphological changes of SH-SY5Y cells

27

directly. As shown in Figure 8A, compared with the control group, the Aβ group cells

28

showed obvious atrophy after treating with Aβ fibers alone. On the other hand, cells 12


Page 12 of 53

Page 13 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

shrunk more and the cell membranes wrinkled more intensely in the Aβ-Cu2+ complex

2

group, which indicated that Aβ-Cu2+ complex could interact with cell membranes and

3

damage them with stronger neurotoxicity comparing with the Aβ fibers. Interestingly,

4

after incubation with RVG@Met@SS, the cells restored the neurite’s growth and

5

branching significantly, and the wrinkle of cell membranes disappeared. To further

6

confirm cytoprotective effect of nanoparticles, we calculated the average branching

7

length of SH-SY5Y cells. As numbered in Figure 8B, RVG@Met@SS could recover

8

93% of neurite cells to normal condition, whereas RVG@Met@TS could recover

9

72%, and RVG@Met@VS could only recover 63%. Moreover, to investigate the

10

cellular ulterior viability, the MTT assay was used. The result showed that

11

RVG@Met@SS increased 92% of SH-SY5Y cells viability by precluding the

12

cytotoxicity induced by Aβ-Cu2+ complex from cells. Results above indicated that the

13

protection of RVG@Met@SS was remarkably better than that of RVG@Met@VS

14

and RVG@Met@TS.

15

Using the BBB model to study the permeability of NPs

16

The permeability of NPs through BBB was assessed by transwell experiments.

17

Herein, we selected SH-SY5Y cells and bEnd.3 to establish the BBB model in vitro.45

18

After adding different NPs to bEnd.3 cells monolayer and incubate the mixture for 8 h,

19

we detected the fluorescence of SH-SY5Y cells in the basolateral side using the flow

20

cytometry, and the fluorescence intensity could be used to explain the penetrability of

21

different NPs (Figure 9A). The average fluorescence intensity of RVG-modified NPs

22

was about 1.5 times higher than the none-RVG-modified NPs, demonstrating that

23

RVG peptide could effectively enhance the ability of NPs to penetrate the

24

blood-brain-barrier. Moreover, in order to calculate the efficiency of different

25

morphologies NPs to transport across the blood-brain barrier, we added the ruthenium

26

complexes alone to the bEnd.3 cells monolayer as a positive control. In Figure 9B, the

27

permeability of RVG@Met@SS is 1.4 times than RVG@Met@TS and 2.3 times than

28

RVG@Met@VS, indicating that the BBB-crossing efficacy of RVG@Met@SS is 13



1

significantly higher than the RVG@Met@VS and RVG@Met@TS. As shown in

2

Figure 9C, the RVG@Met@SNPs with different morphologies could be observed

3

respectively in the vesicles by endocytosis of bEnd.3 cells. Experimental results

4

above indicated strongly that the RVG peptide modified NPs could transport across

5

the blood-brain-barrier effectively through the endocytic pathway.46

6

In vivo imaging of NPs

7

The real-time imaging system combined with X-ray location test was performed

8

to detect the distribution of ruthenium-labeled NPs in vivo. As shown in Figure 10A,

9

an intense fluorescence signal was detected in the brain of the mice which was

10

injected intravenously with RVG-modified NPs after 12 h. In contrast, no obvious

11

fluorescence signal was detected in the brain of the mice with an intravenous injection

12

with none RVG-modified NPs. These results indicated that the brain-targeting

13

efficiency of RVG-modified NPs were remarkably higher than that of the

14

none-modified NPs. Figure 10B and C demonstrate that the distributions of

15

RVG-modified NPs were mainly concentrated in the brain, liver and kidney. The

16

fluorescence signal in the brain of the mice treated with RVG@Met@SS was the

17

highest among itself, the mice treated with RVG@Met@VS and RVG@Met@TS,

18

which confirmed that RVG@Met@SS could travel more efficiently through the

19

blood-brain barrier and thus accumulate in the brain. Based on the results above, it

20

can be speculated that due to its smallest particle size, RVG@Met@SS (about 50 nm)

21

exhibits the strongest BBB permeability. Moreover, the relatively uniformed spherical

22

structure of RVG@Met@SS may help RVG peptides to bind with the n-acetylcholine

23

receptors of BBB more effectively, thus resulting a higher delivery efficiency.

24

Morris Water Maze for behavioral tests

25

Alzheimer’s disease is the most common type of neurodegenerative diseases

26

which correlates with memory deficiencies and cognitive dysfunctions.47 The

27

neuropsychiatric impairment caused by amyloid-β proteins (Aβ) and neurofibrillary 14


Page 14 of 53

Page 15 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

mainly occurs in the hippocampus and cortex, so memory and cognitive abilities can

2

be accessed by the Morris Water Maze Test.48 It has been reported that the APP/PS1

3

mouse would show neuropsychiatric symptoms resulted from memories loss and

4

cognitive impairment resulted from Aβ accumulation in hippocampus area from 11 to

5

17 months of their ages.49 Therefore, we performed the Morris water maze test to

6

evaluate the learning ability in AD mouse by injecting them with multiple doses of

7

different NPs. Representative swimming trails of AD mice treated with different NPs

8

were shown in Figure 11A. AD mice treated with RVG@Met@VS presented similar

9

behaviors as the control AD mice, indicating that RVG@Met@VS was unable to

10

improve the memory and cognition ability of AD mouse. When AD mice were treated

11

with RVG@Met@TS and RVG@Met@SS respectively, both groups showed an

12

improvement of AD symptoms, proven by the decreased time spent to reach the

13

platform comparing with RVG@Met@VS groups and control AD groups. Figure 11B

14

shows the result of time spent to search for the platform and the percentage of time

15

spent in the target quadrant. Normal mice had the best performance and

16

RVG@Met@SS groups were better than the RVG@Met@TS groups. In summary,

17

our results demonstrate that RVG@Met@SS can lessen memory symptoms in AD

18

mouse more effectively.

19

3. Conclusion

20

In this study, we designed three kinds of brain-targeted sulfur nanoparticles with

21

special morphologies that were first employed as nano-sized inhibitors for Cu2+

22

induced Aβ aggregation and toxicity. With their different morphologies, three kinds of

23

RVG@Met@SNPs could inhibit Aβ self-assembly and dissociate Aβ-Cu2+ complex

24

aggregation differently. Particularly, among the RVG@Met@SNPs, the smallest and

25

spherical-like RVG@Met@SS was the most effective one to inhibit the Cu2+-induced

26

Aβ aggregation and to protect the SH-SY5Y cells from neurotoxicity. Moreover, the

27

brain-targeting RVG@Met@SS could penetrate the blood-brain barrier efficiently.

28

And the Morris Water Maze Test showed there was a rescue of learning memory loss 15



Page 16 of 53

1

in AD mice after treating them with RVG@Met@SS. For the brighter future, our

2

results expand the biological applications for sulfur nanoparticles and provide a novel

3

nanomaterial for AD treatment.

4

4. Methods

5

Materials

6

Sodium thiosulfate (Na2S2O3) and L-ascorbic acid (VC), Methionine were

7

purchased from Sigma-Aldrich Chemical Co. Amyloid-β (Aβ1–42) was synthesized by

8

solid-phase Fmoc chemistry at GL Biochem Ltd. (Shanghai, China). RVG peptides

9

(YTIWMPENPRPGTPCDIFTNSRGKRASNGC) were synthesized by Nanjing Leon

10

biological technology Co. LTD (Nanjing, China). 1-ethyl-3-[3-dimethlyaminopropyl]

11

carbodiimide hydrochloride (EDC or EDAC) and N-hydroxysulfosuccinimide (NHS)

12

were purchased from Aladdin Reagent Co., Ltd. Fetal bovine serum (FBS) was

13

purchased from Gibco (Life Technologies AG, Switzerland). Dulbecco Eagle Medium

14

(DMEM)

15

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased

16

from Beyotime. 4,6-diamidino-2-phenylindole (DAPI) and thioflavine T (ThT), were

17

purchased from Sigma (St. Louis,MO). All other chemicals are analytical reagent

18

grade and used as received.

19

Synthesis of Met@SNPs and RVG@Met@SNPs

was

purchased

from

Invitrogen

Corporation.

20

Methionine-modified sulfur nanoparicles production process: sodium thiosulfate

21

was first reduced by vitamin C (VC), and the resulting elemental sulfur was

22

immediately modified by methionine. Methionine played a role in stabilizing the

23

elemental sulfur during the synthesis process. Therefore, depending on the amount of

24

methionine in the solution, elemental sulfur would spontaneously form the

25

nanoparticles of different morphologies. Methionine modified sulfur nanoparticles

26

(Met@SNPs) were synthesized with the following steps. 2 mL 0.1 M VC was added

27

into 5 mL 10 mM methionine solution, and then 5 mL of 10 mM Na2S2O3 solution 16


Page 17 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

was added slowly under ultrasound. The color of the solution changed from

2

transparent to light white, and continued to react with ultrasound for 20 min. After

3

centrifugation and washing with Milli-Q water for three times, volute-like sulfur

4

nanoparticles (Met@VS) were obtained. Then, the molar ratio of methionine to

5

sodium thiosulfate was 2: 1 and 5: 1 by adjusting the methionine concentrations to 20

6

mM and 50 mM. By repeating the synthetic steps above, tadpole-like sulfur

7

nanoparticles (Met@TS) and spherical-like sulfur nanoparticles (Met@SS) were

8

obtained.

9

RVC peptide conjugating to Met@SNPs surface carried out by the EDC-NHS

10

coupling reaction is shown as below. Firstly, 100 µL of Met@VS, Met@TS and

11

Met@SS

12

1-ethyl-3-[3-dimethlyaminopropyl] carbodiimide hydrochloride (EDC or EDAC) and

13

200 µL of 100 mM N-hydroxysulfosuccinimide (NHS). The mixture was gently

14

shaken for 1 h at 25 ℃. Then, 20 µL RVG peptides (5 mg/ml) were added to the

15

mixture and kept shaking for 4 h. After that, the solution was centrifuged at 13000

16

rpm for 15 min and washed with Milli-Q water for three times. Finally, the

17

RVG@Met@SNPs would be obtained.

were

pretreated

individually

with

200

µL

of

400

mM

18

Ruthenium(II) complexes were employed as fluorescent probes to modify on

19

nanoparticles surface in this study. For ruthenium-labelled nanoparticles, the

20

Ruthenium (II) complexes (denoted as [Ru(phen)2(p-HPIP)]2+) were obtained from

21

our previous work and modified on nanoparticles surface by the electrostatic bonding

22

effect.50 The preparation method of ruthenium-labelled nanoparticles is consistent as

23

the above. The only difference is to add 100 µg Ruthenium (II) complexes into the

24

reaction system before joining of VC.

25

Characterization of Met@SNPs and RVG@Met@SNPs

26

Different morphologies of Met@SNPs were recorded by transmission electron

27

microscopy (TEM, Hitachi, H-7650). Fourier transform infrared spectroscopy (FT-IR) 17



1

spectra of samples were recorded on a Fourier transform infrared spectroscopy

2

(Equinox) with KBr pellet technique in an effective range from 400 to 4000 cm -1. The

3

elemental composition of Met@SNPs was detected by HRTEM-EDX analysis on an

4

EX-250 system (Horiba). The morphology structures of RVG@Met@SNPs were

5

recorded by high-resolution transmission electron microscopy (HRTEM, JEM-2100).

6

RVG peptides coating on the surfaces of nanoparticles were detected by BCA assay

7

kits. The nanoparticles were centrifuged at 13000 rpm for 15 min and washed with

8

Milli-Q water for three times. BCA assay kits were used to detect the presence of

9

peptides in washed nanoparticles. Size distribution of nanoparticles and zeta potential

10

were performed by Nano-Zetasizer (Malvern Instruments Ltd.).

11

Preparation of Aβ aggregation

12

Firstly, Aβ proteins were dissolved in hexafluoroisopropanol (HFIP) to a final

13

concentration of 400 µM, and then, Aβ solutions were shaken at 4 ℃ for 2 h to

14

dissolve further. Thereafter, the solutions were equilibrated into centrifuge tubes,

15

dried in vacuoand stored at -80 ℃. Aβ were dissolved in 50 mM phosphate buffer

16

solution (PBS) before use and filtered through a 0.22 mm syringe filter to remove the

17

aggregation. The Aβ monomers (30 µM) were treated with or without Cu2+ (60 µM)

18

and various nanoparticles (10 µg/mL) in phosphate buffer solution (PBS, pH 7.4)

19

from 0 to 3 days at 37 ℃ in 0.2 mL eppendorf tubes.

20

ThT Fluorescence assay

21

The extension of aggregation of Aβ was evaluated by ThT fluorescence assay with

22

a JASCO FP6500 spectrofluorometer. Aβ monomers (30 µM) were incubated with

23

various concentrations of nanoparticles in PBS from 0 to 3 days. On the third day, 50

24

µL of the Aβ solutions were incubated with 200 µL ThT (15 µM) for 15 min in the

25

dark, and then, the fluorescence intensity of ThT was measured on a fluorescence

26

spectrophotometer. The excitation wavelength of ThT is 440 nm and the emission

27

wavelength is 490 nm. 18


Page 18 of 53

Page 19 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

In kinetics of Aβ-Cu2+ aggregation experiment, different nanoparticles (1 mg)

2

were respectively treated with 100 mL 60 µM Cu2+ in PBS for 3 days at 37 ℃. After

3

that, the solution was centrifuged at 15000 rpm for 15 min to remove the

4

nanoparticles and the suspensions were collected. Aβ monomers (30 µM) were

5

incubated with the collected suspension or 60 µM Cu2+ solution in PBS for 3 days.

6

The effect of nanoparticles on ThT fluorescence of Aβ-Cu2+ aggregation was also

7

checked. In this experiment, Aβ monomers (30 µM) in PBS were incubated with Cu2+

8

(60 µM) and various concentrations of nanoparticles from 0 to 3 days at 37 ℃. The

9

ThT ﬂuorescence intensities were detected by a plate reader at 25 °C. Above ThT

10

fluorescence experiments were repeated three times and the standard deviation was

11

averaged along.

12

MALDI-TOF MS

13

The degradation of Aβ was analyzed by Matrix-assisted laser desorption

14

ionization time-of-flight mass spectrometry (MALDI-TOF MS). The Aβ peptides (30

15

µM) were untreated or treateded with different nanoparticles (10 µg/mL) in PBS (50

16

µM, pH 7.4). After 3 days of incubation at 37 ℃, the samples were detected at 25 ℃

17

by MALDI-TOF MS (bruker Ltd).

18

Dynamic Light Scattering (DLS)

19

The hydrodynamic size distribution of Aβ-Cu2+ complex or Aβ in the absence or

20

presence of different NPs was analyzed by Dynamic Light Scattering (DLS). The Aβ

21

peptides (30 µM) were treated or untreated with Cu2+ (60 µM) and different

22

nanoparticles (10 µg/mL) in PBS (50 µM, pH 7.4). After co-incubation at 37 ℃ for 3

23

days, the samples were diluted to the appropriate concentration, transferred to the dish

24

zeta cell and detected at 25 ℃ by Nano-Zetasizer (Malvern Instruments Ltd).

25

Transmission electron microscopy (TEM)

26

The fresh Aβ peptides (30 µM) in PBS (50 µM, pH 7.4) were incubated with Cu2+

27

(60 µM) or nanoparticles (10 µg/mL) at 37 ℃ for 3 days. 10 µL of each solution was 19



1

dropped onto the carbon-coated copper grids and air-dried for 10 min. Then, the

2

samples were examined by TEM (Hitachi, H-7650).

3

Atomic force microscopy (AFM)

4

Morphologies of Aβ aggregates were further recorded by AFM. The Aβ samples

5

for AFM were prepared in the same way as of TEM image. 10 µL of each solution

6

was mounted onto the freshly cleaved mica for 15 min, then gently washed with

7

deionized water three times, and then air-dried overnight. Images were obtained under

8

an atmosphere using the silicon probe in a tapping mode packaged in a multi-mode

9

SPM Nanoscope IV system (Veeco Metrology Group, USA).

10

Cytotoxicity assay

11

SH-SY5Y cells or bEnd.3 cells were plated at a density of 5×103 cells per well in

12

96 well culture clusters to grow for 24 h at 37 ℃. To investigate the biocompatibility

13

of NPs, the cells were treated with various concentrations of Met@SNPs and

14

RVG@Met@SNPs for 48 h at 37 ℃. In the Aβ cytotoxicity experiments, Aβ peptides

15

(30 mM) in cell culture medium were treated with or without Cu2+ (60 mM) and

16

nanoparticles (10 µg/mL) for 72 h at 37 ℃. After that, SH-SY5Y cells were incubated

17

with these samples for another 48 h. Control groups were only treated with the vehicle.

18

The cell vitality was tested by MTT assay.

19

Apoptosis analysis

20

The early and late apoptosis of was detected by annexin V-FITC/PI staining. The

21

SH-SY5Y cells were incubated with Met@SNPs or RVG@Met@SNPs (50 µg/mL)

22

for 48 h at 37 ℃. Subsequently, the suspension and adherent cells were collected and

23

centrifuged at 0.2 mL PBS. 10 µM Annexin V-FITC and 5 µM PI dyes were added

24

into each cell suspension respectively, and incubated for 15 min in the dark and then

25

analyzed by flow cytometry (BD, Bioscience, USA).

26

Cellular Uptake of nanoparticles 20


Page 20 of 53

Page 21 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

SH-SY5Y cells were plated into 24-well plates for 48 h in 37 ℃ and then

2

incubated with ruthenium-labeled NPs in 12 h. After that, SH-SY5Y cells were

3

washed in PBS three times and fixed in 4.0% paraformaldehyde for 20 min in room

4

temperature, and then incubated with 1 mg/mL DAPI for 10 min. The results were

5

observed by Zeiss LSM meta 510 multiphoton laser scanning confocal microscope.

6

(excitation at 488 nm, emission at 620 nm).

7

ICP-AES detection of nanoparticles uptake

8

SH-SY5Y cells were grown to attain a confluence of 107 cells and then incubated

9

with ruthenium-labeled NPs in 12 h. After the incubation, the cells were rinsed with

10

PBS for three times to remove all the unbound nanoparticles. The collected cells were

11

digested in an infrared rapid digestion system (Gerhardt) with 1 mL of H2O2 and 3 mL

12

of nitric acid for 1.5 h at 180 ℃. The digested solution was diluted to 10 mL with

13

Milli-Q water and analyzed by ICP-AES.

14

Measurement of intracellular reactive oxygen species (ROS)

15

The intracellular reactive oxygen species (ROS) were assessed by DCFH-DA

16

probe. Briefly, Aβ peptides (30 mM) in cell culture medium were treated with or

17

without Cu2+ (60 mM) and nanoparticles (10 µg/mL) for 72 h at 37 ℃. Subsequently,

18

SH-SY5Y cells were incubated with these samples for another 48 h at 37 ℃. After

19

incubation, the cells were incubated with 5 µM DCFH-DA for 30 min, the

20

intracellular ROS level was observed by laser scanning confocal microscope. For

21

quantitative analyzing intracellular ROS in SH-SY5Y cells, SH-SY5Y cells were

22

pretreated as above. After incubation, the adherent cells were digested with trypsin

23

and harvested by centrifugation, rinsed with PBS for twice and incubated with

24

DCFH-DA (10 mM) at 37 ℃ for 15 min. And then, the cells were collected by

25

centrifugation and suspended in PBS. The intracellular ROS was detected by flow

26

cytometry.

27

Scanning electron microscopy (SEM) study 21



1

SH-SY5Y cells were plated into 24-well plates with glass cover slips (10 mm in

2

diameter) and pretreated as above. The SH-SY5Y cells were washed third times with

3

PBS and immobilized with 2% glutaraldehyde and 2% paraformaldehyde for 30 min

4

at 4 ℃. Subsequently, the cells were washed three times with PBS. The cells were

5

dehydrated in ascending series of ethanol (50%, 70%, 80%, 90%, and 100%). Finally,

6

cells were dried at the critical point of CO2 for 4 h and then sprayed gold. The effect

7

of Aβ-Cu2+ complex on morphology of SH-SY5Y cells was observed by scanning

8

electron microscopy (SEM).

9

BBB model in vitro

10

Transwell plates were used to establish in vitro model of BBB. The brain

11

endothelial cells (bEnd.3) cells were grown to form a tight monolayer (TEER

12

achieved 200 Ω) in the apical side chamber and then SH-SY5Y cells were seeded in

13

the bottom closet. Ruthenium-labelled nanoparticles were added into the apical side

14

chamber. After incubation for 12 h, the uptake of nanoparticles on SH-SY5Y cells was

15

analyzed by flow cytometry. The bEnd.3 cells were collected and routinely

16

paraffin-embeded. The distribution of nanoparticles in bEnd.3 cells were observed by

17

TEM.

18

In vivo imaging

19

Nude mouse were purchased from Guangdong Medical Laboratory Animal

20

Center. All animal experiments were approved by the Animal Care and Use

21

Committee of Jinan University. Permeability of NPs on BBB was investigated by a

22

Realtime imaging system (Maestro). Ruthenium-labeled NPs (1 mg/kg) was injected

23

into mice via the tail vein. After 12 h of injection, the anesthetized mice were placed

24

in the chamber and the fluorescent images were detected. The other mice were

25

sacrificed by cervical dislocation at the time point and the organs were obtained.

26

Subsequently, the distribution of fluorescence of major organs was acquired.

27

(excitation, 488 nm, emission, 620 nm). 22


Page 22 of 53

Page 23 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Treatment regimen in AD mice

2

20 AD mice, 30 weeks old, were randomly divided into four groups (5 mice in

3

each group) and intravenously administered with RVG@Met@VS (5.0 mg/kg),

4

RVG@Met@TS

5

volume of saline. The mice were injected twice weekly (Monday and Thursday) for a

6

total of 8 injections in 4 weeks.

7

Morris water maze test

(5.0

mg/kg),

RVG@Met@SS

(5.0

mg/kg)

and

equal

8

The learning and cognitive abilities of AD mice after 4 weeks treatment were

9

assessed by the Morris water maze test. Experimental details are described in

10

Supporting Information. After training, the mice were tested to search the platform in

11

the Morris water maze apparatus which they were permitted to swim freely for 60 s.

12

The time spent to reach the platform and the ratio of time in target quadrant was

13

recorded. The daily test data were analyzed statistically.

14

Statistical analysis

15

The data were presented by three independent experiments and the analysis of

16

variance was performed by Students T- test and p < 0.05 or less was represented as the

17

statistical significance.

18

Author contribution

19 20 21 22

The manuscript was written through the contribution of all the authors. Conflict of Interest Authors declare no conflict of interest. ACKNOWLEDGMENTS

23

This work was supported by the National Natural Science Foundation of China

24

(21171070 and 21371075), the Natural Science Foundation of Guangdong Province

25

(2014A030311025), and the Planned Item of Science and Technology of Guangdong 23



1

Province (2016A020217011).

2

References

3

1. Song, Q., Huang, M., Yao, L., Wang, X., Gu, X., Chen, J., Chen, J., Huang, J., Hu,

4

Q., and Kang, T. (2014) Lipoprotein-based nanoparticles rescue the memory loss of

5

mice with Alzheimer's disease by accelerating the clearance of amyloid-beta, Acs

6

Nano 8, 2345-2359.

7

2. Jiang, Y., and Sabatini, D. D. (2010) Alzheimer's-related endosome dysfunction in

8

Down syndrome is A -independent but requires APP and is reversed by BACE-1

9

inhibition, Proceedings of the National Academy of Sciences of the United States of

10

America 107, 1630-1635.

11

3. Kamenetz, F., Tomita, T., Hsieh, H., Seabrook, G., Borchelt, D., Iwatsubo, T.,

12

Sisodia, S., and Malinow, R. (2003) APP processing and synaptic function, Neuron 37,

13

925.

14

4. Tu, S., Okamoto, S., Lipton, S. A., and Xu, H. (2014) Oligomeric Abeta-induced

15

synaptic dysfunction in Alzheimer's disease, Molecular Neurodegeneration 9, 1-12.

16

5. Geng, J., Li, M., Ren, J., Wang, E., and Qu, X. (2011) Inside Cover:

17

Polyoxometalates as Inhibitors of the Aggregation of Amyloid β Peptides Associated

18

with Alzheimer’s Disease (Angew. Chem. Int. Ed. 18/2011), Angew Chem Int

19

Ed Engl 50, 4184-4188.

20

6. Huang, Y., and Mucke, L. (2012) Alzheimer Mechanisms and Therapeutic

21

Strategies, Cell 148, 1204-1222.

22

7. Lincoln, K. M., Richardson, T. E., Rutter, L., Gonzalez, P., Simpkins, J. W., and

23

Green, K. N. (2012)An n-heterocyclic amine chelate capable of antioxidant capacity

24

and amyloid disaggregation,Acs Chemical Neuroscience3, 919-927.

25

8. Miller, Y., Ma, B., and Nussinov, R. (2012) Metal binding sites in amyloid

26

oligomers: Complexes and mechanisms, Coordination Chemistry Reviews 256, 24


Page 24 of 53

Page 25 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

2245-2252.

2

9. Yako, N., Young, T. R., Cottam Jones, J. M., Hutton, C. A., Wedd, A. G., and Xiao,

3

Z. (2017) Copper binding and redox chemistry of the Aβ16 peptide and its variants:

4

insights into determinants of copper-dependent reactivity, Metallomics Integrated

5

Biometal Science 9, 278-291.

6

10. Yuan, S., Chen, S., Xi, Z., and Liu, Y. (2017) Copper-finger protein of Sp1: the

7

molecular basis of copper sensing, Metallomics Integrated Biometal Science 9,

8

1169-1175.

9

11.Tiiman, A., Palumaa, P., and Tougu, V. (2013) The missing link in the amyloid

10

cascade of Alzheimer's disease - metal ions, Neurochemistry International 62,

11

367-378.

12

12. Folk, D. S., and Franz, K. J. (2010) A prochelator activated by beta-secretase

13

inhibits Abeta aggregation and suppresses copper-induced reactive oxygen species

14

formation, Journal of the American Chemical Society 132, 4994-4995.

15

13. Adlard, P. A., and Bush, A. I. (2012) Metal chaperones: a holistic approach to the

16

treatment of Alzheimer's disease, Frontiers in Psychiatry 3, 15-15.

17

14. Geng, J., Li, M., Wu, L., Chen, C., and Qu, X. (2012) Mesoporous silica

18

nanoparticle-based H2O2 responsive controlled-release system used for Alzheimer's

19

disease treatment, Advanced Healthcare Materials 1, 332-336.

20

15. Grasso, G., Santoro, A. M., Lanza, V., Sbardella, D., Tundo, G. R., Ciaccio, C.,

21

Marini, S., Coletta, M., and Milardi, D. (2017) The double faced role of copper in Aβ

22

homeostasis: a survey on the interrelationship between metal dyshomeostasis, UPS

23

functioning and autophagy in neurodegeneration, Coordination Chemistry Reviews

24

347, 1–22.

25

16. Sharma, A. K., Pavlova, S. T., Kim, J., Finkelstein, D., Hawco, N. J., Rath, N. P.,

26

Kim, J., and Mirica, L. M. (2012) Bifunctional compounds for controlling 25



1

metal-mediated aggregation of the aβ42 peptide, Journal of the American Chemical

2

Society 134, 6625-6636.

3

17. Pardridge, W. M. (2005) The Blood-Brain Barrier: Bottleneck in Brain Drug

4

Development, Neurorx 2, 3-14.

5

18. Wang, Y., Miao, L., Satterlee, A., and Huang, L. (2015) Delivery of

6

Oligonucleotides with Lipid Nanoparticles, Advanced Drug Delivery Reviews 87,

7

68-80.

8

19. Sun, T., Zhang, Y. S., Pang, B., Hyun, D. C., Yang, M., and Xia, Y. (2014)

9

Engineered nanoparticles for drug delivery in cancer therapy, Angewandte Chemie 53,

10

12320-12364.

11

20. Dube, T., Chibh, S., Mishra, J., and Panda, J. J. (2017) Receptor Targeted

12

Polymeric Nanostructures Capable of Navigating across the Blood-Brain Barrier for

13

Effective Delivery of Neural Therapeutics, Acs Chemical Neuroscience 8, 2105-2117.

14

21. Shi, H., Fang, T., Tian, Y., Huang, H., and Liu, Y. (2016) A dual-fluorescent

15

nano-carrier for delivering photoactive ruthenium polypyridyl complexes, Journal of

16

Materials Chemistry B 4, 4746-4753.

17

22. Gatoo, M. A., Naseem, S., Arfat, M. Y., Dar, A. M., Qasim, K., and Zubair, S.

18

(2014) Physicochemical Properties of Nanomaterials: Implication in Associated Toxic

19

Manifestations, BioMed Research International 2014, 498420-498420.

20

23. Yokoyama, K., Cho, H., Cullen, S. P., Kowalik, M., Briglio, N. M., Hoops, H. J.,

21

Zhao, Z., and Carpenter, M. A. (2009) Microscopic investigation of reversible

22

nanoscale surface size dependent protein conjugation, International Journal of

23

Molecular Sciences 10, 2348-2366.

24

24. Tamaki, and Naganuma. (2017) Shape design of cerium oxide nanoparticles for

25

enhancement of enzyme mimetic activity in therapeutic applications, Nano Research

26

10, 199-217. 26


Page 26 of 53

Page 27 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

25. Xu, L., Zhang, H., and Wu, Y. (2014)Dendrimer advances for the central nervous

2

system delivery of therapeutics, Acs Chemical Neuroscience5, 2-13.

3

26. Gao, W., Liu, Y., Jing, G., Li, K., Zhao, Y., Sha, B., Wang, Q., and Wu, D. (2016)

4

Rapid and efficient crossing blood-brain barrier: Hydrophobic drug delivery system

5

based on propionylated amylose helix nanoclusters, Biomaterials 113, 133-144.

6

27. Nishida, M., Hada, T., Kuramochi, K., Yoshida, H., Yonezawa, Y., Kuriyama, I.,

7

Sugawara, F., Yoshida, H., and Mizushina, Y. (2008) Diallyl sulfides: Selective

8

inhibitors of family X DNA polymerases from garlic (Allium sativum L.), Food

9

Chemistry 108, 551-560.

10

28. Deshpande, A. S., Khomane, R. B., Vaidya, B. K., Joshi, R. M., Harle, A. S., and

11

Kulkarni, B. D. (2008) Sulfur Nanoparticles Synthesis and Characterization from H2S

12

Gas, Using Novel Biodegradable Iron Chelates in W/O Microemulsion, Nanoscale

13

Research Letters 3, 221-229.

14

29. Xie, X., Li, L., Zheng, P., Zheng, W., Bai, Y., Cheng, T., and Liu, J. (2012) Facile

15

synthesis, spectral properties and formation mechanism of sulfur nanorods in

16

PEG-200, Materials Research Bulletin 47, 3665-3669.

17

30. Chaudhuri, R. G., and Paria, S. (2010) Synthesis of sulfur nanoparticles in aqueous

18

surfactant solutions, Journal of Colloid and Interface Science 343, 439-446.

19

31. Guo, Y., Zhao, J., Yang, S., Yu, K., Wang, Z., and Zhang, H. (2006) Preparation

20

and

21

microemulsions technique, Powder Technology 162, 83-86.

22

32. Buranakic, E., Margus, M., Jurasin, D., Milanovic, I., and Cigleneckijusic, I.

23

(2015) Chronoamperometric study of elemental sulphur (S) nanoparticles (NPs) in

24

NaCl water solution: new methodology for S NPs sizing and detection, Geochemical

25

Transactions 16, 1-1.

26

33. Fuchtbauer, H. G., Tuxen, A. K., Moses, P. G., Topsoe, H., Besenbacher, F., and

characterization

of

monoclinic

sulfur

nanoparticles

27


by

water-in-oil


1

Lauritsen, J. V. (2013) Morphology and atomic-scale structure of single-layer WS2

2

nanoclusters, Physical Chemistry Chemical Physics 15, 15971-15980.

3

34. Li, M., Zhao, A., Dong, K., Li, W., Ren, J., and Qu, X. (2015) Chemically

4

exfoliated WS2 nanosheets efficiently inhibit amyloid β-peptide aggregation and can

5

be used for photothermal treatment of Alzheimer’s disease, Nano Research 8,

6

3216-3227.

7

35. Gao, N., Sun, H., Dong, K., Ren, J., Duan, T., Xu, C., and Qu, X. (2014)

8

Transition-metal-substituted polyoxometalate derivatives as functional anti-amyloid

9

agents for Alzheimer’s disease, Nature Communications 5, 3422.

10

36. Kumar, P., Wu, H., Mcbride, J. L., Jung, K., Kim, M. H., Davidson, B. L., Lee, S.

11

K., Shankar, P., and Manjunath, N. (2007) Transvascular delivery of small interfering

12

RNA to the central nervous system, Nature 448, 39-43.

13

37. Kwon, E. J., Skalak, M., Bu, R. L., and Bhatia, S. N. (2016) Neuron-Targeted

14

Nanoparticle for siRNA Delivery to Traumatic Brain Injuries, ACS Nano 10,

15

7926-7933.

16

38. Huang, Y., He, L., Liu, W., Fan, C., Zheng, W., Wong, Y., and Chen, T. (2013)

17

Selective cellular uptake and induction of apoptosis of cancer-targeted selenium

18

nanoparticles, Biomaterials 34, 7106-7116.

19

39. Yang, L., Chen, Q., Liu, Y., Zhang, J., Sun, D., Zhou, Y., and Liu, J. (2014) Se/Ru

20

nanoparticles as inhibitors of metal-induced Aβ aggregation in Alzheimer's disease,

21

Journal of Materials Chemistry B 2, 1977-1987.

22

40. Cabaleirolago, C., Quinlanpluck, F., Lynch, I., Dawson, K. A., and Linse, S. (2010)

23

Dual effect of amino modified polystyrene nanoparticles on amyloid β protein

24

fibrillation, ACS Chemical Neuroscience 1, 279-287.

25

41. Oyewumi, M. O., Yokel, R. A., Jay, M., Coakley, T., and Mumper, R. J. (2004)

26

Comparison of cell uptake, biodistribution and tumor retention of folate-coated and 28


Page 28 of 53

Page 29 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

PEG-coated gadolinium nanoparticles in tumor-bearing mice, Journal of Controlled

2

Release 95, 613-626.

3

42. Hou, Y., Ghosh, P., Wan, R., Ouyang, X., Cheng, H., Mattson, M. P., and Cheng, A.

4

(2014) Permeability transition pore-mediated mitochondrial superoxide flashes

5

mediate an early inhibitory effect of amyloid beta1−42 on neural progenitor cell

6

proliferation, Neurobiology of Aging 35, 975-989.

7

43. Butterfield, D. A., Reed, T., and Sultana, R. (2011) Roles of 3-nitrotyrosine- and

8

4-hydroxynonenal-modified brain proteins in the progression and pathogenesis of

9

Alzheimer's disease, Free Radical Research 45, 59-72.

10

44. Mastroeni, D., Khdour, O. M., Arce, P. M., Hecht, S. M., and Coleman, P. D.

11

(2015)Novel antioxidants protect mitochondria from the effects of oligomeric

12

amyloid beta and contribute to the maintenance of epigenome function, Acs Chemical

13

Neuroscience6, 588-598.

14

45. Yin, T., Xie, W., Sun, J., Yang, L., and Liu, J. (2016) Penetratin (Pen)

15

Peptide-Functionalised Gold Nanostars: Enhanced BBB Permeability and NIR

16

Photothermal Treatment of Alzheimer's Disease Using Ultralow Irradiance, Acs

17

Applied Materials & Interfaces 8, 19291.

18

46. Kim, H. R., Gil, S., Andrieux, K., Nicolas, V., Appel, M., Chacun, H., Desmaele,

19

D., Taran, F., Georgin, D., and Couvreur, P. (2007) Low-density lipoprotein

20

receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial

21

cells, Cellular and Molecular Life Sciences 64, 356-364.

22

47. Grant, K. B., and Kassai, M. (2007) Major Advances in the Hydrolysis of Peptides

23

and Proteins by Metal Ions and Complexes, Cheminform 38, 1035-1049.

24

48. Ashe, K. H. (2001) Learning and Memory in Transgenic Mice Modeling

25

Alzheimer's Disease, Learning & Memory 8, 301-308.

26

49. Pistell, P. J., Zhu, M., and Ingram, D. K. (2008) Acquisition of conditioned taste 29



1

aversion is impaired in the amyloid precursor protein/presenilin 1 mouse model of

2

Alzheimer's disease, Neuroscience 152, 594-600.

3

50. Yin, T., Yang, L., Liu, Y., Zhou, X., Sun, J., and Liu, J. (2015) Sialic acid

4

(SA)-modified selenium nanoparticles coated with a high blood-brain barrier

5

permeability peptide-B6 peptide for potential use in Alzheimer's disease, Acta

6

Biomaterialia 25, 172-183.

7 8

9 10

Graphical Abstract. Schematic representation of the novel morphology sulfur

11

nanoparticles with brain-targeting peptides used for AD treatment. RVG@Met@SNPs

12

could effectively penetrate the blood brain barrier to protect neuron from the ROS and

13

neurotoxicity which was induced by Aβ-Cu2+complex.

30


Page 30 of 53

Page 31 of 53

Met@VS

Met@TS

200 nm

100 nm

50 nm

100 nm

50 nm

B Methionine Met@TS

3500

1

3000

Met@SS

200 nm

C

Met@VS Met@SS

2500

2000

1500

C S

60

Sulfur

Atomic Precent (%)

A

Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


46 36

40

38 32

18

20 8 0

1000

Met@VS Met@TS

Wavenumber (nm)

Met@SS

2

Figure 1. Characterization of NPs. (A) TEM image of Met@SNPs. The white square

3

of the above images was magnified into the below image. (B) FT-IR spectra of

4

Met@SNPs and Methionine. (C) EDX analysis of Met@SNPs.

5 6 7 8 9 10

31



1 2

Figure 2. Inhibition effects of the NPs on Aβ aggregation. (A) ThT fluorescence

3

changes for Aβ treated with different concentrations of RVG@Met@VS,

4

RVG@Met@TS and RVG@Met@SS after the incubation of at 37 ℃ and pH 7.4 for

5

72 h. ThT fluorescence of mature Aβ aggregation in the absence of inhibitors was

6

defned as 100%. [Aβ] = 30 µM. *p < 0.05 and **p < 0.01, respectively, indicate

7

significant and very significant, comparing to the Aβ-alone groups. (B) MALDI-TOF

8

MS of Aβ aggregation in the absence and presence of RVG@Met@VS,

9

RVG@Met@TS and RVG@Met@SS, [NPs]=10 µg/mL.

10 11 12 13 14 15 16

32


Page 32 of 53

Page 33 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 3. kinetics of Cu2+-induced Aβ aggregation. (A) ThT fluorescence with

3

temporal change are shown in pure Aβ, Cu2+-induced Aβ aggregation and Aβ-Cu2+

4

treated with: (a) supernatant of Cu2+ in the absence and presence of RVG@Met@VS,

5

RVG@Met@TS

6

RVG@Met@VS, RVG@Met@TS and RVG@Met@SS; (E) Size distribution of pure

7

Aβ, Cu2+-induced Aβ aggregation and Aβ-Cu2+ treated with RVG@Met@VS,

8

RVG@Met@TS and RVG@Met@SS after incubation for 0, 12, 24 and 72 h. [Aβ] =

9

30 µM, [Cu2+] = 60 µM, [NPs]=10 µg/mL, pH7.4.

and

RVG@Met@SS;

(B)-(D)

different

10 33


concentrations

of


1

2 3

Figure 4. Disaggregation of Cu2+-induced Aβ aggregation by NPs. (A) TEM images

4

of morphology change of pure Aβ and Aβ-Cu2+ treated without and with

5

RVG@Met@VS, RVG@Met@TS and RVG@Met@SS after 72 h incubation; (B)

6

AFM images of morphology change of pure Aβ and Aβ-Cu2+ treated without and with

7

RVG@Met@VS, RVG@Met@TS and RVG@Met@SS after 72 h incubation; [Aβ]=

8

30 µM, [Cu2+]= 60 µM, [NPs]=10 µg/mL. Scale bar: 500 nm.

9 10 11 12 13 14 15 16 17

34


Page 34 of 53

Page 35 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 5. Biocompatibility assay. (A) The cell viability of SH-SY5Y cells after 48 h

3

treatment with different NPs below different concentration in the culture medium. (B)

4

The cell viability of bEnd.3 cells after 48 h treatment with different NPs below

5

different concentration in the culture medium. (C) The apoptosis of SH-SY5Y cells

6

was detected by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit

7

(Invitrogen). The mean ± SD is shown (n=3).

35



1 2

Figure 6. Cellular association and uptake. (A) Cellular association and uptake of

3

various NPs (labelled by ruthenium) at a concentration of 10 µg/mL in SH-SY5Y

4

under a confocal microscopy after 12 h incubation at 37 ℃, scale bar=20 nm. (B)

5

Quantitative analysis of Ru concentrations in SH-SY5Y cells treated with various NPs

6

at different concentrations for 12 h by ICP-AES method. The data were presented as

7

mean ± SD (n = 3) and *p < 0.05 and **p < 0.01, respectively, indicate significant and

8

very significant, comparing to the control groups.

9 10 36


Page 36 of 53

Page 37 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

2 3

Figure 7. Clearance of Aβ-Cu2+ induced intracellular ROS by NPs in vitro. (A)

4

Confocal fluorescence images of intracellular ROS in SH-SY5Y cells were obtained

5

by DCFH-DA. SH-SY5Y cells were pre-treated with Aβ, Aβ-Cu2+ and Aβ-Cu2+ in

6

present of various NPs. After incubation the cells were stained using 5 µM DCFH-DA;

7

[Aβ] = 30 µM, [Cu2+]= 60 µM and [NPs]=10 µg/mL, scale bar=20 µm. (B) DCFH-DA

8

fluorescence intensity was measured in SH-SY5Y cells by flow cytometry. The data

9

were presented as mean ± SD (n = 3) and *p < 0.05, **p < 0.01 and ***p < 0.001,

10

respectively, indicate significant and very significant, comparing to the Aβ-Cu2+

11

groups.

12

37



1 2

Figure 8. Effect of NPs on protecting neuronal cells from Aβ-Cu2+complex induced

3

neurotoxicity. (A) SEM imagines ofordinarySH-SY5Y cells, SH-SY5Y cells treated

4

with Aβ, Aβ-Cu2+ andAβ-Cu2+ in present of various NPs; (B) The mean neurite length

5

of SH-SY5Y cells; (C) Viability of SH-SY5Y cells. The control groups were defined

6

as 100%. *p < 0.05, **p < 0.01 and ***p < 0.001, indicate significant and very

7

significant, comparing to the Aβ-Cu2+ groups. Scan bar: 5 µm.

8 9 10 11 12 13 14 15

38


Page 38 of 53

Page 39 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 9. Transwell assay for NPs in vitro. (A) The basolateral side (BS) fluorescence

3

intensity detection of various ruthenium labelled NPs (10 µg/mL) in SH-SY5Y cells by

4

a flow cytometry analysis, after 12 h incubation, at 37 ℃.(B) Efficiency ofvarious NPs

5

on crossing through the blood-brain barrier. The fluorescence intensity of pure

6

ruthenium which was detained in cellular monolayer (CM) defined as positive control.

7

(C) TEM images of bEnd.3 cells respectively treated with RVG@Met@VS,

8

RVG@Met@TS and RVG@Met@SS for 12 h incubation. The white square of the

9

above images was magnified into the below images. Arrows indicates the location of

10

the RVG@Met@SNPs in cells.

11 39



A

RVG@Met@ RVG@Met@ RVG@Met@ VS TS SS

B

Kindney Lung Heart Spleen Liver

Brain

High

RVG@Met @VS RVG@Met @TS RVG@Met @SS Low

C RVG@Met@VS RVG@Met@TS RVG@Met@SS

80 60 40

*** **

*

*

20

ey

g

n

Lu n

Sp lee

Ki dn

1

ar t

r

0 He

Met@SS

Li ve

Met@TS

Br ain

Met@VS

Flurorescence intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 53

2

Figure 10. Biodistribution of NPs in nude mice following intravenous administration.

3

(A) In vivo fluorescent images of nude mice given various NPs following intravenous

4

administration at 12 h. (B) Fluorescence signals detected in the major organs in 12 h

5

after the NPs injection. (C) Semiquantitative results of the fluorescence intensity in

6

the major organs. *p < 0.05, **p < 0.01 and ***p < 0.001, indicate significant and

7

very significant, comparing to the RVG@Met@VS groups.

8 9 10 11 12 13 14 40


Page 41 of 53 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 11. Thebehavioral evaluation results of Morris water maze test. (A)

3

Representative swimming trails of APP/PS1 mice without or with treatment of

4

different NPs. (I) +saline; (II) +RVG@Met@VS; (III) +RVG@Met@TS; (IV)

5

+RVG@Met@SS; (V) non-treated abnormal mice. (B) the average time spent to reach

6

the platform in 60 s. (C) The proportion of time in the target quadrant in 60 s. *p

Sulfur Nanoparticles with Novel Morphologies Coupled with Brain

Recommend Documents