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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
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ACS Chemical Neuroscience
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Sulfur Nanoparticles with Novel Morphologies Coupled with
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Brain-Targeting Peptides RVG as a New Type of Inhibitor
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Against Metal-Induced Aβ Aggregation
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Jing Suna,1, Wenjie Xiea,1, Xufeng Zhua, Mengmeng Xua, and Jie Liua,*
5
a
6
*
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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.
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Abstract
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Functionalized nanomaterials, which have been applied widely to inhibit
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amyloid-β protein (Aβ) aggregation, show an enormous potential in the field of
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prevention and treatment of Alzheimer's disease (AD). A significant body of data has
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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
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brain-targeting sulfur nanoparticles (RVG@Met@SNPs) with novel morphologies
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(volute-like, tadpole-like and sphere-like), and investigated the effect of different
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RVG@Met@SNPs on the Aβ-Cu2+ complex aggregation and their corresponding
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neurotoxicity. Among them, the sphere-like nanoparticles (RVG@Met@SS) exhibited
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the most effective inhibitory activity due to their unique mini size effect, and they
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reduced 61.6% of the Aβ-Cu2+ complex aggregation and increased 92.4% SH-SY5Y
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cells viability in a dose of 10 µg/mL. In vitro and in vivo, the abilities of different
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morphologies of RVG@Met@SNPs crossing blood-brain barrier (BBB) and targeting
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the brain parenchymal cells were significantly different. Moreover, improvements in
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learning disability and cognitive lose were shown in the transgenic AD mice model
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using the Morris water maze test after multiple dosage of RVG@Met@SNPs
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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
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to treat AD.
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Keywords: Alzheimer’s disease; amyloid-β protein; sulfur nanoparticles; blood-brain
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barrier;
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1. Introduction
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Alzheimer’s disease (AD) is one of most common neurodegenerative disorders.1
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A great deal of studies have demonstrated that the extracellular amyloid-β protein (Aβ)
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aggregation is the main pathological hallmarks of AD.2 The Aβ aggregation can
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induce the crosstalk of various molecular signaling pathways, cause the loss of neuron,
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and produce synaptic dysfunction in brains.3,4,5 Therefore, therapeutic strategies
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aiming at the initial stage of Aβ aggregation will be viable to prevent or delay the
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onset of AD.6
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In the brains of AD patients, the high concentrations of redox-active metal ions,
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especially Cu2+ ion have been demonstrated that they can accelerate the fibrillation of
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Aβ significantly.7,8,9,10 The toxic Aβ-Cu2+ aggregation can contribute to abnormal
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formations of reactive oxygen species (ROS), as well as oxidative stresses, triggering
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a vicious cycle as a result.11,12 Thus, metal-chelating agents such as clioquinol (CQ)
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and 8-hydroxyquinoline-2-carboxylic acid (HQC), have been applied for targeting and
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controlling Aβ aggregation, which are shown to be able to capture Cu2+ and inhibit the
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Cu2+-induced Aβ aggregation process.13,14 However, most of metal-chelating agents
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are limited in their further application in AD because of their poor brain-targeting
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ability, inefficient permeability to blood-brain barrier (BBB), and toxic effect. 15,16,17
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Nowadays, nanomaterials (also known as nanoparticles) have been applied
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successfully for diagnosing and treating diseases due to their excellent
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properties.18,19,20,21 Physicochemical characteristics of nanoparticles such as size and
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morphology have profound influences on their biological behaviors.22 Recent
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researches had found that the AgNPs show various results on their biological
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applications due to their sizes, and such applications include antibacterial activity and 2
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ecological toxicity.23 Furthermore, it was proven that the special morphology of
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cerium oxide nanoparticles has the potential to influence antioxidant activity.24
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Moreover, through the assembly of functionalized biomolecules, nanoparticles can
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target the brain and penetrate BBB effectively.25,26 Inspired by the discussions above,
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in order to overcome the limitation of metal chelators, we decided to design
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nanoparticles with special morphology which are able to effectively decrease Aβ-Cu2+
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complex and penetrate BBB, and this may be a promising therapeutic strategy for
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curing AD.
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Sulfur nanoparticles have excellent efficacies such as removal of Cu2+ ions and
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radicals, antioxidant and antibacterial activity, and these functions could all be
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enhanced by their minimal sizes and surface energy effects.27,28 With different
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morphologies and sizes, sulfur nanoparticles show distinct chemical properties and
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biological activities. Therefore, selecting the activity of different modifiers and
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heterogeneous reactions could be adjusted and optimized by regulating the size and
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morphology of sulfur nanoparticles.29,30,31,32 In our work, methionine was selected as a
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modifier to regulate the morphology of nanoparticles. By adjusting the proportion of
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methionine and sodium thiosulfate in the synthesis process, three kinds of sulfur
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nanoparticles with novel morphologies were obtained (volute-like, tadpole-like and
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sphere-like; called as Met@VS, Met@TS and Met@SS respectively).In recent years,
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sulfide nanosheets in neurodegenerative diseases are reported that the sulfur play a
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key role to bind with Aβ and interfere the formation of hydrogen bonds in Aβ
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aggregation.33,34 However, to the best of our knowledge, there is no existing report on
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the utilization of sulfur nanoparticles as therapeutic agents for AD treatments. Thus,
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whether different morphologies of sulfur nanoparticles could remove the
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dyshomeostasis of Cu2+ ion and inhibit the aggregation of Aβ-Cu2+ or not require
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further investigation.
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Designing BBB-penetrable nanoparticles or brain-targeting molecules is a key
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process to the success of AD treatment.35 RVG, a 29 amino-acid peptide isolated from 3
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the coat protein of rabies virus, was applied as a promising candidate to improve the
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drug’s ability to target the brain and its penetrability to BBB.36,37 Utilizing the rich
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carboxyl groups in methionine offered plenty of modification sites. We successfully
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conjoined RVG with Met@SNPs surface (call as RVG@Met@VS, RVG@Met@TS
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and RVG@Met@SS, respectively). In this work, characteristics of nanoparticles were
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investigated. Meanwhile, we compared and explored initially the abilities of
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RVG@Met@SNPs with different morphologies to inhibit Aβ self-fibrillation and to
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decrease Cu2+-induced Aβ aggregation. In vitro, it is demonstrated that various
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RVG@Met@SNPs showed morphological effects on their biological applications, in
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which they penetrated BBB, and were absorbed by the brain parenchyma cells
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differently. Moreover, RVG@Met@SNPs could scavenge the Aβ-Cu2+ mediated
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formation of reactive oxygen species (ROS) and protect SH-SY5Y cells from the
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corresponding neurotoxicity. Among them, spherical-like RVG@Met@SS was the
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most effective one as an inhibitor, and it also significantly increased cells viability. As
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expected, after multiple-dosing treatments, AD mouse in the Morris water maze test
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showed a rescue of learning and memory loss.
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2. Results and discussion
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Design and Characterization of NPs
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In this experiment, by adjusting the synthetic ratios of methionine and Na2S2O3,
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we obtained sulfur nanoparticles with novel morphologies: volute-liked, tadpole-liked
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and sphere-liked (called as Met@VS, Met@TS and Met@SS, respectively). Figure
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S1 show the synthesis procedure of RVG@Met@SNPs. And the detailed
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morphological structure of Met@SNPs could be recorded by transmission electron
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microscopy (TEM) (Figure 1A). It could be observed that NPs dispersed nicely, and
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their specific morphologies are presented as the following: around 150 nm for
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Met@VS, 100 nm for Met@TS and 50 nm for Met@SS.
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FT-IR was performed to confirm the success of the formation of methionine 4
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modified nanoparticles. As illustrated in Figure 1B, Met@SNPs exhibits different
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infrared peaks to sulfur but shows similar peaks to methionine. These peaks include
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the stretching vibration of N-H at 2910 cm-1, the NH3+ deformation vibration at 2100
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cm-1 and the S-CH2 deformation vibration at 1450 cm-1. These data indicated
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methionine has been successfully modified on the sulfur surface. The weaker
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absorbance peak of NH3+ and the high frequency-shifted S-CH2 vibration may imply
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that these groups formed coordination with the sulfur surface.
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Moreover, the elemental composition analysis of different nanoparticles was
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characterized by EDX. This data is illustrated in Figure 1C, in which an increased C
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element is shown in nanoparticles. That is the case because the C element in
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methionine has an increased methionine modification on sulfur surface.
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In order to enhance the brain-targeting ability and the permeability of Met@SNPs,
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we coated the RVG peptide with the Met@SNPs surfaces (RVG@Met@VS,
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RVG@Met@TS, RVG@Met@SS). Herein, we used high Resolution Transmission
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Electron Microscopy (HRTEM) to determine the conjugation of RVG peptides on
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Met@SNPs. As shown in Figure S2, the thickness of conjugated RVG peptides on
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Met@SNPs could be clearly observed: RVG@Met@VS is about 2.5 nm,
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RVG@Met@TS about 1.6 nm, and RVG@Met@SS about 2.2 nm. These data implied
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that the RVG peptides might have conjugated successfully with the surfaces of
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Met@VS, Met@TS and Met@SS, respectively. Moreover, the brain-targeting RVG
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peptides which were attached on the surfaces of Met@SNPs got detected by the BCA
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assay.38 As shown in Figure S3A and B, after NPs mixed with the BCA working
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solution, the color of the brain-targeted nanoparticles changed from colorless to
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purple while the no-brain-targeted NPs system remain unchanged. Meanwhile, there
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was an absorption peak from the mixed solution at 560 nm in the UV absorption
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spectrum, indicating that RVG peptides were detected in the brain-targeted
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nanoparticles. The concentration of RVG peptides in the supernatant was further
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determined by BCA assay. The total concentration of RVG peptides on the 5
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Met@SNPs were about: RVG@Met@VS: 34.2 µg/mL, RVG@Met@TS: 33.8 µg/mL,
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RVG@Met@SS: 35.1 µg/mL.
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The properties of various nanoparticles were investigated by the Nano-ZS
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instrument. The size distribution and zeta potential values of nanoparticles are shown
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in Figure S4 and Table S1. The size of RVG@Met@SNPs was increased slightly after
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the RVG peptide coated with the Met@SNPs surfaces, corresponding with the results
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of TEM and HRTEM. The zeta potential values of all nanoparticles were greater than
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-30 mV, indicating that both Met@SNPs and RVG@Met@SNPs have high stability.
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Dynamic light scattering (DLS) measurements were carried out over 7 days, which
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indicated that both Met@SNPs and RVG@Met@SNPs were maintained stable
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(Figure S5).
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Inhibitory effect on NPs of Aβ self-fibrillation
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ThT fluorescence assay is used widely to assess Aβ aggregation.39 Thus, we
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firstly investigated whether different concentrations: 1, 2.5, 5, and 10 µg/mL of NPs
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have inhibitory effects on Aβ self-fibrillation by using the ThT fluorescence assays.
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As a positive control, we used the strongest ThT fluorescence intensity obtained from
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pure Aβ incubation. As shown in Figure 2A, after respectively incubating Aβ with the
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RVG@Met@VS in four different concentrations, RVG@Met@VS exhibited a weak
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effect in inhibiting Aβ aggregation, confirmed by the data that even under the
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strongest concentration, 10 µg/mL, only 13% of the Aβ aggregation was inhibited.
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RVG@Met@TS showed moderate Aβ aggregation inhibitions, in which the
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aggregation had an 8% inhibition with 2.5 µg/mL, a 20% inhibition with 5 µg/mL and
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a 28% inhibition with 10 µg/mL. These data show that RVG@Met@TS inhibited
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Aβ self-fibrillation in a dose-dependent manner, and it had a better inhibitory ability
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than RVG@Met@VS. It should be noted that after co-incubation of Aβ and freshly
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added RVG@Met@SS, the ThT fluorescence intensity of Aβ was declined
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remarkably by 25% at 2.5 µg/mL, by 43% at 5 µg/mL, and by 55% at 10 µg/mL,
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which suggested that RVG@Met@SS could effectively inhibit the aggregation of Aβ, 6
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and it has the strongest inhibitory capacity among the three of them when same
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concentrations were used. To further investigate the effect of NPs on the inhibition of
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Aβ, we monitored the kinetics of Aβ in the absence or presence of RVG@Met@SNPs
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(Figure S6). Aβ monomer was incubated alone in PBS at 37 °C and exhibited a
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remarkable ThT fluorescence, which indicated that Aβ aggregation was formed. As
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expected,
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RVG@Met@SNPs was added to Aβ solution. Especially, on the presence of
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RVG@Met@SS, the most significant decline of ThT fluorescence intensity was
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observed. As shown in the control experiment, RVG@Met@SNPs could not affect
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we
found
different
degrees
of
decline
in
fluorescence
when
ThT fluorescence under the experimental condition (Figure S7).
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In addition, to further investigate the degradation effect of different NPs on Aβ,
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Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
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(MALDI-TOF MS) was performed. As illustrated in Figure 2B, after co-incubation of
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different NPs and fresh Aβ at 37 ℃ for 72 h respectively in groups, all
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RVG@Met@SNPs degraded the Aβ with different extents. Among them,
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RVG@Met@SS was the most effective one in which the particular peaks of Aβ were
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declined and some low molecular peaks were observed. The details of the molecular
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peaks were showed in Figure S8.
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Effect of NPs on kinetics of the Aβ-Cu2+ complex aggregation
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We explored the abilities of three kinds of RVG@Met@SNPs to inhibit Aβ
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self-fibrillation at the first stage of our research. However, whether NPs could repress
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the Aβ aggregation induced by Cu2+ requires further investigation. Firstly, we
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investigated the direct effect of nanoparticles' ability to scavenge Cu2+ on Aβ
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aggregation. As shown in Figure 3A, when Aβ monomers were being added into 60
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µM Cu2+ solution, and left to a 72 h incubation, the kinetic data showed that the Aβ
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monomers were first in a negligible lag phase (＜1 h), and then in an accelerated
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growth phase (＜18 h), and finally in a steady equilibrium phase after 18 h. The
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formation of more Aβ fibril was proven by the evidence that the fluorescence intensity 7
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increased by 38.8%, comparing to that of the Aβ samples alone. When Aβ monomers
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were treated respectively with the supernatants of three kinds of RVG@Met@SNPs
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incubated individually with 60 µM Cu2+ solution for 72 h, the lag-phase did not with
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change but the intensity of ThT fluorescence of Aβ-Cu2+ complex was decreased,
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depending on the efficiency of their capacities to scavenge Cu2+. This indicated that
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three kinds of RVG@Met@SNPs could repress the Aβ-Cu2+ complex by adsorbing
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Cu2+. Next, we examined the influence of the kinetics of RVG@Met@VS,
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RVG@Met@TS, RVG@Met@SS with different concentrations on Aβ-Cu2+
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aggregation. As shown in Figure 3B, C, and D, RVG@Met@VS with all kinds of
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concentrations did not inhibit Aβ-Cu2+ aggregation significantly and exhibited similar
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kinetic profiles as the Aβ-Cu2+ samples. On the other hand, while the RVG@Met@TS
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did not change the process of Aβ-Cu2+complex aggregation, it could reduce the
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Aβ-Cu2+ complex aggregation in a dose-dependent manner. Among them,
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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.
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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
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oligomer (≈300 nm) after 12 h to large aggregation (≈800 nm) after 72 h. In the
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presence of Cu2+, a wide range of peaks from 200 to 800 nm appeared after 12 h, and
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gradually shifted to 1300 nm after 72 h with no small peaks. This indicated that Cu2+
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could accelerate the Aβ initial aggregation and increase the formation of mature fibril.
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In contrast, in the treatment of Aβ-Cu2+ with RVG@Met@SNPs, the small peaks of
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40~150 nm were first observed, which agreed with the hydration diameters of
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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
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monodispersed NPs decreased and some of them the monodisperse NPs did not
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integrate further with Aβ species. In comparison with the size distribution of Aβ-Cu2+
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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+
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aggregation (＞800 nm) almost disappeared, which indicated that RVG@Met@SS
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could hinder the Aβ-Cu2+fibrillation process effectively and produce Aβ-Cu2+
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aggregation with different sizes. The inhibitory effect of different nanoparticles on Aβ
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self- fibrosis was also investigated by DLS (Figure S9). Compared with the Aβ-alone
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groups, large fibrils peaks (≈800 nm) did not appear, which indicated that the fibrosis
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of Aβ was effectively inhibited by RVG@Met@SS.
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Morphology of Aβ-Cu2+ aggregation investigated by AFM and TEM
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Based on the ThT results, we further investigated the morphological evidences of
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inhibition of RVG@Met@SNPs to Aβ-Cu2+ complex using TEM and AFM. As
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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
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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
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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
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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.
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Biocompatibility assay
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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
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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.
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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.
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Cellular uptake of nanoparticles
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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
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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.
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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
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Met@VS, RVG@Met@TS was 3.8 times more than Met@TS, and RVG@Met@SS
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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1
Province (2016A020217011).
2
References
3
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Graphical Abstract. Schematic representation of the novel morphology sulfur
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nanoparticles with brain-targeting peptides used for AD treatment. RVG@Met@SNPs
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could effectively penetrate the blood brain barrier to protect neuron from the ROS and
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neurotoxicity which was induced by Aβ-Cu2+complex.
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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 (%)
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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
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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,
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RVG@Met@TS and RVG@Met@SS, [NPs]=10 µg/mL.
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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
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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
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concentrations
of
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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
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RVG@Met@VS, RVG@Met@TS and RVG@Met@SS after 72 h incubation; [Aβ]=
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30 µM, [Cu2+]= 60 µM, [NPs]=10 µg/mL. Scale bar: 500 nm.
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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).
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Figure 6. Cellular association and uptake. (A) Cellular association and uptake of
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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)
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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.
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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
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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.
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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.
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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
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Figure 10. Biodistribution of NPs in nude mice following intravenous administration.
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(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.
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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 <
7
0.05, **p < 0.01,***p < 0.001 respectively indicate significant and very significant,
8
comparing to saline groups.
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Figure 1. Characterization of NPs. (A) TEM image of Met@SNPs. The white square of the above images was magnified into the below image. (B) FT-IR spectra of Met@SNPs and Methionine. (C) EDX analysis of Met@SNPs. 238x230mm (300 x 300 DPI)
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Figure 2. Inhibition effects of the NPs on Aβ aggregation. (A) ThT fluorescence changes for Aβ treated with different concentrations of RVG@Met@VS, RVG@Met@TS and RVG@Met@SS after the incubation of at 37 ℃ and pH 7.4 for 72 h. ThT fluorescence of mature Aβ aggregation in the absence of inhibitors was defned as 100%. [Aβ] = 30 µM. *p < 0.05 and **p < 0.01, respectively, indicate significant and very significant, comparing to the Aβ-alone groups. (B) MALDI-TOF MS of Aβ aggregation in the absence and presence of RVG@Met@VS, RVG@Met@TS and RVG@Met@SS, [NPs]=10 µg/mL. 330x182mm (300 x 300 DPI)
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Figure 3. kinetics of Cu2+-induced Aβ aggregation. (A) ThT fluorescence with temporal change are shown in pure Aβ, Cu2+-induced Aβ aggregation and Aβ-Cu2+ treated with: (a) supernatant of Cu2+ in the absence and presence of RVG@Met@VS, RVG@Met@TS and RVG@Met@SS; (B)-(D) different concentrations of RVG@Met@VS, RVG@Met@TS and RVG@Met@SS; (E) Size distribution of pure Aβ, Cu2+-induced Aβ aggregation and Aβ-Cu2+ treated with RVG@Met@VS, RVG@Met@TS and RVG@Met@SS after incubation for 0, 12, 24 and 72 h. [Aβ] = 30 µM, [Cu2+] = 60 µM, [NPs]=10 µg/mL, pH7.4. 316x341mm (300 x 300 DPI)
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Figure 4. Disaggregation of Cu2+-induced Aβ aggregation by NPs. (A) TEM images of morphology change of pure Aβ and Aβ-Cu2+ treated without and with RVG@Met@VS, RVG@Met@TS and RVG@Met@SS after 72 h incubation; (B) AFM images of morphology change of pure Aβ and Aβ-Cu2+ treated without and with RVG@Met@VS, RVG@Met@TS and RVG@Met@SS after 72 h incubation; [Aβ]= 30 µM, [Cu2+]= 60 µM, [NPs]=10 µg/mL. Scale bar: 500 nm. 394x195mm (300 x 300 DPI)
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Figure 5. Biocompatibility assay. (A) The cell viability of SH-SY5Y cells after 48 h treatment with different NPs below different concentration in the culture medium. (B) The cell viability of bEnd.3 cells after 48 h treatment with different NPs below different concentration in the culture medium. (C) The apoptosis of SHSY5Y cells was detected by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Invitrogen). The mean ± SD is shown (n=3). 359x393mm (300 x 300 DPI)
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Figure 6. Cellular association and uptake. (A) Cellular association and uptake of various NPs (labelled by ruthenium) at a concentration of 10 µg/mL in SH-SY5Y under a confocal microscopy after 12 h incubation at 37 ℃, scale bar=20 nm. (B) Quantitative analysis of Ru concentrations in SH-SY5Y cells treated with various NPs at different concentrations for 12 h by ICP-AES method. The data were presented as mean ± SD (n = 3) and *p < 0.05 and **p < 0.01, respectively, indicate significant and very significant, comparing to the control groups. 445x457mm (300 x 300 DPI)
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Figure 7. Clearance of Aβ-Cu2+ induced intracellular ROS by NPs in vitro. (A) Confocal fluorescence images of intracellular ROS in SH-SY5Y cells were obtained by DCFH-DA. SH-SY5Y cells were pre-treated with Aβ, Aβ-Cu2+ and Aβ-Cu2+ in present of various NPs. After incubation the cells were stained using 5 µM DCFHDA; [Aβ] = 30 µM, [Cu2+]= 60 µM and [NPs]=10 µg/mL, scale bar=20 µm. (B) DCFH-DA fluorescence intensity was measured in SH-SY5Y cells by flow cytometry. The data were presented as mean ± SD (n = 3) and *p < 0.05, **p < 0.01 and ***p < 0.001, respectively, indicate significant and very significant, comparing to the Aβ-Cu2+ groups. Scan bar: 20 µm. 418x196mm (300 x 300 DPI)
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Figure 8. Effect of NPs on protecting neuronal cells from Aβ-Cu2+complex induced neurotoxicity. (A) SEM imagines ofordinarySH-SY5Y cells, SH-SY5Y cells treated with Aβ, Aβ-Cu2+ andAβ-Cu2+ in present of various NPs; (B) The mean neurite length of SH-SY5Y cells; (C) Viability of SH-SY5Y cells. The control groups were defined as 100%. *p < 0.05, **p < 0.01 and ***p < 0.001, indicate significant and very significant, comparing to the Aβ-Cu2+ groups. Scan bar: 5 µm. 358x222mm (300 x 300 DPI)
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Figure 9. Transwell assay for NPs in vitro. (A) The basolateral side (BS) fluorescence intensity detection of various ruthenium labelled NPs (10 µg/mL) in SH-SY5Y cells by a flow cytometry analysis, after 12 h incubation, at 37 ℃.(B) Efficiency ofvarious NPs on crossing through the blood-brain barrier. The fluorescence intensity of pure ruthenium which was detained in cellular monolayer (CM) defined as positive control. (C) TEM images of bEnd.3 cells respectively treated with RVG@Met@VS, RVG@Met@TS and RVG@Met@SS for 12 h incubation. The white square of the above images was magnified into the below images. Arrows indicates the location of the RVG@Met@SNPs in cells. 416x427mm (300 x 300 DPI)
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Figure 10. Biodistribution of NPs in nude mice following intravenous administration. (A) In vivo fluorescent images of nude mice given various NPs following intravenous administration at 12 h. (B) Fluorescence signals detected in the major organs in 12 h after the NPs injection. (C) Semiquantitative results of the fluorescence intensity in the major organs. *p < 0.05, **p < 0.01 and ***p < 0.001, indicate significant and very significant, comparing to the RVG@Met@VS groups. 302x224mm (300 x 300 DPI)
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Figure 11. The behavioral evaluation results of Morris water maze test. (A) Representative swimming trails of APP/PS1 mice without or with treatment of different NPs. (I) +saline; (II) +RVG@Met@VS; (III) +RVG@Met@TS; (IV) +RVG@Met@SS; (V) non-treated abnormal mice. (B) the average time spent to reach the platform in 60 s. (C) The proportion of time in the target quadrant in 60 s. *p < 0.05, **p < 0.01,***p < 0.001 respectively indicate significant and very significant, comparing to saline groups. 300x197mm (300 x 300 DPI)
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Graphical Abstract. Schematic representation of the novel morphology sulfur nanoparticles with braintargeting peptides used for AD treatment. RVG@Met@SNPs could effectively penetrate the blood brain barrier to protect neuron from the ROS and neurotoxicity which was induced by Aβ-Cu2+complex. 238x154mm (300 x 300 DPI)
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