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A Tau-Targeted Multifunctional Nanocomposite for Combinational Therapy of Alzheimer’s Disease Qing Chen, Yang Du, Kai Zhang, Zeyu Liang, Jinquan Li, Hao Yu, Rong Ren, Jin Feng, Zhiming Jin, Fangyuan Li, Jihong Sun, Min Zhou, Qinggang He, Xiaolian Sun, Hong Zhang, Mei Tian, and Daishun Ling ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07625 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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A Tau-Targeted Multifunctional Nanocomposite for Combinational Therapy of Alzheimer’s Disease Qing Chen,‡,# Yang Du,†,# Kai Zhang,‡,# Zeyu Liang,†,# Jinquan Li,† Hao Yu,† Rong Ren,§ Jin Feng,‡ Zhiming Jin,ο Fangyuan Li,†,∥ Jihong Sun,⊥ Min Zhou,‡ Qinggang He,§ Xiaolian Sun,ѱ Hong Zhang,‡,◊ Mei Tian,‡,◊,* Daishun Ling†,∥,* ‡

Department of Nuclear Medicine and PET/CT Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China, †

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, P. R. China,

∥

Key Laboratory of Biomedical Engineering of the Ministry of Education, College of Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou, Zhejiang 310058, P.R. China,

◊

Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, P. R. China, §

College of Chemical & Biological Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China, ⊥

Department of Radiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310016, P. R. China, ѱ

Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health, Bethesda, Maryland 20892, United States, ο

Jiangsu Huayi Technology Limited Company, Changshu, Jiangsu 215522, P. R. China.

#

These authors contributed equally to this work.

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ABSTRACT: Alzheimer’s disease (AD) remains to be an incurable disease and lacks efficient diagnostic methods. Most AD treatments have focused on amyloid-β (Aβ) targeted therapy, however, it is time to consider the alternative theranostics due to accumulated findings of weak correlation between Aβ deposition and cognition, as well as the failures of Phase III clinical trial on Aβ targeted therapy. Recent researches have shown that tau pathway is closely associated with clinical development of AD symptoms, which might be a potential therapeutic target. We herein construct a methylene blue (MB, a tau aggregation inhibitor) loaded nanocomposite (CeNC/IONC/MSN-T807),

which

not

only

possesses

high

binding

affinity

to

hyperphosphorylated tau, but also inhibits multiple key pathways of tau-associated AD pathogenesis. We demonstrate that these nanocomposites can relieve the AD symptoms by mitigating mitochondrial oxidative stress, suppressing tau hyperphosphorylation and protecting neuronal death both in vitro and in vivo. The memory deﬁcits of AD rats are significantly rescued upon treatment with MB loaded CeNC/IONC/MSN-T807. Our results indicate that hyperphosphorylated tau-targeted multifunctional nanocomposites would be a promising therapeutic candidate for Alzheimer’s disease.

KEYWORDS: Alzheimer’s disease, multifunctional nanocomposite, tauopathy, oxidative stress, synergistic effect.


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Alzheimer’s disease (AD) is featured by progressing deterioration of cognitive capacity. The number of individuals with AD increased from 21.7 million in 1990 to 46.0 million in 2015 worldwide.1 Extracellular senile plaques composed of amyloid β (Aβ) and intraneuronal neurofibrillary tangles (NFTs) constituted of hyperphosphorylated tau are considered as the characteristic pathological features of AD.2 Over the past decades, it has been hypothesized that the accumulation of Aβ peptides into toxic oligomers and amyloid plaques has played a predominant role in the etiology and the pathogenesis of AD, by which initiating the pathogenic cascade inducing tau aggregation, synaptic dysfunction, neuronal death, and consequently, loss of cognitive capacity.3 However, thus far almost all the “promising” drug candidates targeting Aβ associated pathology have been reported failure in clinical trials.4-6 Therefore, it is time to consider alternative targeting approach for AD treatment. It has been reported that tau pathology can take place independently of Aβ, and directly leads to tau aggregation via hyperphsophrorylation,7-8 which is closely correlated with cognitive deficits.7 Furthermore, tau is also involved in the axonal transport of organelles including mitochondria, the loss of tau function leads to mitochondrial dysfunction and induces oxidative stress.9-10 Thus, abnormal tau may be of utmost significance in the pathogenesis and progression of AD, and the drug candidates targeting tau pathology could be of great value for AD treatment. As a matter of fact, tau hyperphosphorylation and hyperphosphorylated tau aggregation are two most crucial processes in AD development.11-12 Tau hyperphosphorylation is mainly due to the imbalance in the regulation or activity levels of phosphatases and tau kinases.13 The increased levels of hyperphosphorylated tau is likely to enhance the chances of pathogenic conformational variety that in turn result in the fibrillization and agglomeration of tau.14 Drug candidates have been developed for these two processes respectively. However, the clinical trials


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demonstrate that treatments focusing on one target could only achieve limited success for AD treatment.4-6, 15 In recent years, nanotechnology has been widely investigated for combinational therapy of complex diseases including AD.16-19 However, these nanotechnology based AD treatments are mainly focus on Aβ pathology, including the clearance of Aβ,20-21 inhibition of Aβ aggregation and so on.22-26 To the best of our knowledge, nanotechnology based smart strategy for tautargeted treatment of AD remains to be explored. Considering that tau hyperphosphorylation and hyperphosphorylated tau aggregation are two correlative processes for AD development, combinational nanotherapeutics that can inhibit both processes represent a promising strategy for AD treatment and are highly desired. Herein, we report a hyperphosphorylated tau-targeted multifunctional nanocomposite, which is fabricated by controlled assembly of ultrasmall ceria nanocrystals (CeNCs) and iron oxide nanocrystals (IONCs) onto the surface of mesoporous silica nanoparticles (MSNs) (Scheme 1a). CeNCs of less than 5 nm exhibit high ROS-scavenging activity,27 and are ideal antioxidants for the treatment of mitochondrial oxidative-stress-induced damage in AD.28 It’s well known that oxidative stress leads to the hyperphosphorylation of tau by activation of glycogen synthase kinase 3 (GSK3).29 Moreover, persistent oxidative stress and mitochondrial dysfunction likely result in a further activation of neuronal apoptosis cascades, cognition disorders as well as behavioral functions.30 Considering the relationship between oxidative stress and tau hyperphosphorylation, we proposed to use CeNCs as tau hyperphosphorylation inhibitors. IONCs were used as high-resolution magnetic resonance imaging (MRI) agents for AD.31-36 Furthermore, methylene blue (MB),37-40 a small molecular compound inhibiting tau aggregation was loaded into the pores of MSNs. Amino-T807, an amino substituent of T807,41-42 was grafted


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onto the surface of MSNs via macrocyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),43-44 for active hyperphosphorylated tau targeting. The T807 is a tau tracer that has demonstrated significant binding to tau aggregates with high selectivity.45 The NOTA can be further labeled with

68

Ga for positron emission tomography (PET) imaging.

The overall

therapeutic strategy is as follows (Scheme 1b): Firstly, with the help of the surface anchored T807, the nanocomposites specifically targeted to hyperphosphorylated tau in neuron; secondly, surface immobilized IONCs and

68

Ga enabled MRI/PET bimodal imaging to confirm the

localization of CeNC/IONC/MSN-T807-MB in vivo; finally, tau hyperphosphoralation and hyperposphorylated tau aggregation were simultaneously inhibited by surface immobilized CeNCs and loaded MB, accompanied by the inhibition of neuronal apoptosis, resulting in synergistically enhanced therapeutic outcome.

RESULTS AND DISCUSSION Synthesis and Characterization of CeNC/IONC/MSN-T807-MB Nanocomposite. The fabrication procedure of CeNC/IONC/MSN-T807-MB is summarized in Scheme 1a. Briefly, CeNCs and IONCs were synthesized in organic phase using reported methods.27, 31 To anchor hydrophobic nanocrystals onto hydrophilic MSNs, the nanocrystals were first modified with 2bromo-2-methylpropionic acid (BMPA),46 and the surface of MSNs were modified with amino groups by reacting with 3-aminopropyltriethoxysilane (APS). Through the nucleophilic substitution reaction between the amino groups of MSNs and the bromine groups of BMPA, the modified nanocrystals were immobilized on the surfaces of MSNs.47 NOTA were conjugated onto the residual amino groups of MSNs, and then reacted with amino-T807 (see Figure S1, 2, 3 for detailed synthesis and characterization).


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The as-synthesized nanocomposites were monodispersed with an overall diameter around 51 ± 5 nm as shown in Figure 1a i. The IONCs (Figure 1a ii) and CeNCs (Figure 1a iii) immobilized on the surface of MSNs were both highly crystalized with a size around 3.2 ± 0.5 nm and 3.1 ± 0.4 nm, respectively (Figure 1a ii, iii, Figure S4). The transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy dispersive X-ray spectra (EDS) mapping (Figure 1a iv-x, Figure S5) have confirmed the successful immobilization of CeNCs and IONCs. X-ray photoelectron spectroscopy (XPS) analysis identified the nanocomposites have a mixed valence state of CeNCs (corresponding binding energy (BE) peaks at 885.0 and 903.5 eV for Ce3+, and peaks at 882.1, 888.1, 898.0, 900.9, 906.4, and 916.4 eV for Ce4+) (Figure S6), which could serve as ROS-scavenger due to the shift between Ce3+ and Ce4+.28 After conjugation of NOTA and amino-T807, the nanocomposites have a hydrodynamic size around ~131.6 nm (Figure 1b) and a zeta potential about 9.32 mV (Figure S7), suitable for in vivo AD application.48-49 The successful immobilization of T807 was confirmed by Ultraviolet–visible spectroscopy (UV) spectrum (Figure S8). The CeNC/IONC/MSN-T807 have an excellent colloidal stability, with no obvious aggregation observed over a week in both water and cell culture medium (Figure S9, 10). The superoxide dismutase (SOD) mimetic assay and autocatalytic assay have confirmed the nanocomposites have maintained the antioxidant aptitude of CeNCs (Figure 1d, Figure S11).27, 50 Besides, the nanocomposites exhibited a r1 relaxivity of 4.08 mM-1s-1 due to surface IONCs (Figure 1e). The IONCs showed a r1 relaxivity of 4.44 mM-1s-1, and a r2 relaxivity of 36.43 mM1 -1

s (Figure S12), while the CeNC/IONC/MSN nanocomposites exhibited a similar r1 relaxivity

of 4.10 mM-1s-1 (Figure S12), but an increased r2 relaxivity of 91.82 mM-1S-1. The increased r2 value of CeNC/IONC/MSN was due to assembled state of IONCs, which is consist with


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previously reported study.47 The relaxometric results showed no significant difference between CeNC/IONC/MSN

and

CeNC/IONC/MSN-T807

(Figure

1e),

indicating

that

T807

immobilization did not influence the relaxometric properties of the nanocomposites. MB was loaded into CeNC/IONC/MSN-T807 nanocomposites with a high loading ratio of ~ 36% and showed a sustained release curve (Figure 1c). The nanocomposite was highly stable, that even after 7 days of incubation in the cell culture medium, only 2.6 % of cerium and 9.2 % of iron were released (Figure S13a). Since the release rate of surface anchored nanocrystals from the nanocomposites was very slow, no significant change of the relaxivity was occurred within 7 days in biological medium (Figure S13b). The TEM results further confirmed that the IONCs and CeNCs were stably anchored on the surface of MSNs after 7 days of incubation in cell culture medium (Figure S14). Specific Hyperphosphorylated Tau Targeting Ability of CeNC/IONC/MSN-T807. The nanocomposites were labeled with fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RITC) for cellular study. Okadaic acid (OA), an important inhibitor of protein phosphatase PP-1 and PP-2A inducing aberrant hyperphosphorylation of tau and neuronal death, was used to treat SH-SY5Y cells for tauopathy cell model.51-53 The immunofluorescence staining with p-Tau antibody (S396) has proved the upregulation of tau phosphorylation in OA-treated cells (Figure 2a). We next treated normal or OA-treated SH-SY5Y cells with dye labeled nanocomposites. We found the excellent biocompatibility of CeNC/IONC/MSN-T807 (Figure S15), and the cell uptake of CeNC/IONC/MSN-T807 in the OA treated SH-SY5Y cells was significantly higher than that in normal SH-SY5Y cells. For the group treated with CeNC/IONC/MSN without T807, the fluorescence signals were negligible in both normal and OA-treated cells (Figure S16-18).


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The fluorescence signal of dye labeled CeNC/IONC/MSN-T807 was overlapped with the immunofluorescence signal of p-Tau antibody staining (Figure 2a). These data demonstrated the hyperphosphorylated tau targeting ability of CeNC/IONC/MSN-T807 in OA-treated cells. In vivo tauopathy rat models were established by microinjection of OA into the right hippocampus of rats. PET imaging with 18F-T807, a specific tau aggregation PET imaging tracer, was performed to testify the success of animal modeling and to rule out the baseline level of tau aggregation among all experimental groups. Compared with the sham group, the OA-treated group presented significantly higher tau binding potential (BPnd) (Figure S19). T1 weighted MR images were obtained in vivo before and after the injection of nanocomposites into the hippocampus of AD model rats. Compared with CeNC/IONC/MSN without T807 ligands, significantly higher MR signal was observed for CeNC/IONC/MSN-T807 treated group (Figure 2b), indicating its enhanced retention. Moreover, the NOTA segment on the T807 ligands could chelate

68

Ga with a high radiolabeling efficiency of > 99% and the stability of

68

Ga labeled

nanocomposites was over 97 % (Figure S20, 21), consequently the MRI result can be further confirmed by PET imaging using 68Ga labeled-CeNC/IONC/MSN-T807 (Figure S22). Confocal fluorescence images of brain slices further confirmed that CeNC/IONC/MSN-T807 could penetrate multi-layer cells to the diseased area in the hippocampus (Figure S23). Furthermore, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis of cerium and iron ions concentrations in unilateral hippocampus tissue further demonstrated that CeNC/IONC/MSNT807 have a higher retention due to the specific binding of T807 ligands to hyperphosphorylated tau in the hippocampus of tauopathy rats (Figure 2c). CeNC/IONC/MSN-T807-MB Alleviates OA-Induced Oxidative Stress. To demonstrate the ROS scavenging effect of CeNC/IONC/MSN-T807-MB, MitoSOX kits and flow cytometry


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study was used to evaluate the influence of mitochondrial ROS accumulation in tauopathy cells after treatment with CeNC/IONC/MSN-T807-MB. We found that OA induced up-regulation of mitochondrial ROS level, consistent with the previous studies.54-55 It is known that oxidative stress can induce the disassociation of tau from the microtubules,56 indicating mitochondrial ROS play a very important role to the development of AD. Interestingly, after treatment of CeNC/IONC/MSN-T807-MB, the intensity mitochondrial ROS in OA-treated SH-SY5Y cells was

significantly

reduced,

demonstrating

the

significant

inhibitory

aptitude

of

CeNC/IONC/MSN-T807-MB to OA-induced mitochondrial ROS (Figure 3a). The confocal images further confirmed the outstanding ROS-scavenging ability of CeNC/IONC/MSN-T807MB (Figure S24). CeNC/IONC/MSN-T807-MB Restrains Tau Hyperphosphorylation and Aggregation. We performed western blot to quantify the p-tau level at multiple sites (serine 396, 199, 404 and threonine 205) in OA-treated SH-SY5Y cells to verify whether CeNC/IONC/MSN-T807-MB colud down-regulate hyperphosphorylated tau level.57 It is known that MB can prevent tau aggregation and down-regulate p-tau level,58-59 interestingly, our results revealed that CeNC/IONC/MSN-T807 without MB can also induce the down-regulation of p-tau level likely due to its ROS scavenging effect (Figure 3b). Considering that phosphorylation of tau protein at the site of S396 is one of the earliest events in AD,60 the reducing of tau phosphorylation shall beneficial to AD treatment.61 Notably, CeNC/IONC/MSN-T807-MB showed significantly enhanced inhibition to S396, S199, S404 and T205 as compared with MB alone, indicating the synergistic effect coming from the integrated MB and ROS scavenging nanocomposites. Inhibiting tau aggregation is another crucial aspect of AD treatment. Once treated with CeNC/IONC/MSN-T807, CeNC/IONC/MSN-T807-MB or MB, the cells were tested for


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Thioflavin S (ThS) fluorescence (ThS, a fluorescent probe for detecting tau aggregation).62 As shown in Figure S25, the ThS signals were strong in the OA-treated cells, and became weak in the presence of MB due to the inhibition of tau–tau binding.63 The ThS signal almost completely diminished with the treatment of CeNC/IONC/MSN-T807-MB, demonstrating the high efficiency. To further investigate the inhibitor effect of CeNC/IONC/MSN-T807-MB on tau protein aggregation, heparin was employed to induce tau protein aggregation in vitro.62 The ThS assay was used to measure assembly inhibition degree of each group. In the ThS assay, CeNC/IONC/MSN-T807-MB could inhibit tau aggregation down to 13.3%, much lower than that of MB (25.0%) and CeNC/IONC/MSN-T807 (73.5%) (Figure 3c), which was also confirmed by electron microscopy (Figure 3d). CeNC/IONC/MSN-T807-MB Protects Neurons from Apoptosis. Tau hyperphosphorylation and ROS accumulation are known as major causes of neuronal apoptosis.64 Inhibition of tau hyperphosphorylation and clearance of ROS can benefit the cell viability in order to rescue tau pathology.65 Herein, CeNC/IONC/MSN-T807-MB was used to inhibit the cell death of OAtreated SH-SY5Y cells, with CeNC/IONC/MSN-T807 and MB only as control. As shown in Figure 4, the cell viability was merely 23.8% for the group without any treatment. The MB exhibited mild cell death inhibition with the cell viability increased to 35.2% (Figure 4a). The cell survival increased to about 54% in the group treated with CeNC/IONC/MSN-T807 (Figure 4a). In comparison with MB and CeNC/IONC/MSN-T807, the CeNC/IONC/MSN-T870-MB demonstrated synergistic protective effect on OA-treated cells with cell survival over 71.8%. In addition, flow cytometry for the quantitative study of SH-SY5Y cells apoptosis using Annexin V-FITC staining have confirmed the capability of CeNC/IONC/MSN-T807-MB to prevent OA-


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induced apoptosis (Figure 4b). The apoptosis of the SHSY5Y cells was increased to 31.65% when the cells were stimulated with OA. The CeNC/IONC/MSN-T807-MB treatment decreased the cell apoptosis rate to 10.34%, much more effective than MB treatment (23.54% apoptosis) and CeNC/IONC/MSN-T807 treatment (16.66% apoptosis). These results demonstrated that CeNC/IONC/MSN-T807-MB can effectively protect neurons from apoptosis. CeNC/IONC/MSN-T807-MB Inhibits Akt/GSK-3β Signaling and Regulates Apoptosisrelevant Proteins. We performed western blot to investigate the underlying mechanism of CeNC/IONC/MSN-T807-MB to protect cells against AD. Glycogen synthase kinase 3β (GSK3β), a proline-directed tau kinase that phosphorylate tau at serine and threonine residues and the phosphorylated protein kinase B (Akt), a upstream kinase of GSK3β are two important kinases in the tau pathology of AD.66 The inactivation of Akt and activation of GSK-3β were found in OA induced cell model.67 Our results confirmed that OA induced down-regulation of p-Akt level and p9-GSK3β level (Figure 5a). In the group treated with MB alone, both the p-Akt level and p9GSK3β level increased, indicating MB could activate the Akt/GSK-3β pathway, which consists with previous studies.68 Interestingly, after treatment with CeNC/IONC/MSN-T807, the p-Akt level and p9-GSK3β level showed the same trend with MB treatment, probably due to the ROS scavenging ability of CeNC/IONC/MSN-T807 which activate the Akt/GSK-3β pathway.69 Importantly, the p-Akt level and p9-GSK3β level significantly increased in the group treated with CeNC/IONC/MSN-T807-MB, indicating the synergistic effect for Akt/GSK-3β pathway activation. We also quantified the pro-apoptotic protein BCL2-Associated X (Bax) and downstream protein Caspase-3. The relative number of apoptotic proteins suggest whether the cells stay viable or move into apoptosis.70 Dysregulation of anti and pro-apoptotic proteins damage the


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permeability of mitochondrial membrane, and accelerates outflow of cytochrome c and other death-inducing factors to launch the apoptotic caspase cascade.71 Our results showed that bax and cleaved-Caspase-3 level was increased in OA-incubated cells, consistent with previous studies,72 indicating that OA likely to induce cell apoptosis by pro-apoptotic pathway. MB treatments

only

slightly

reduced

the

Bax

and

cleaved-

Caspase-3

level,

while

CeNC/IONC/MSN-T807-MB treatment significantly decreased both Bax and cleaved-Caspase-3 level, indicating that CeNC/IONC/MSN-T807-MB can suppress pro-apoptotic pathway by scavenging ROS and maintaining the integrity of mitochondrial function. The mRNA level of Akt, GSK-3β, Bax and Caspase-3 were further confirmed by RT-PCR. (Figure 5b). Taken all together, CeNC/IONC/MSN-T807-MB rescued neuronal cells from apoptosis by alleviating the ROS level and regulating pro-apoptotic proteins. Moreover, tau hyperphosphorylation was also inhibited by activating Akt/GSK3β pathway (Figure 5c). CeNC/IONC/MSN-T807-MB Ameliorates Learning and Memory Impairments in AD Model Rats. To evaluate the therapeutic efficiency of our nanocomposite in vivo, CeNC/IONC/MSN-T807-MB or saline were injected in the ipsilateral hippocampus 5 days after establishment of AD model rats. The in vivo therapeutic efficiency was examined by Morris water maze test at 7 days post treatment.73 As shown in Figure 6a, compared with the sham rats, AD rats took much longer time to search for the platform in the course of hidden platform tests, indicating that OA induced severe cognitive deficit to these rats. Interestingly, after treated with CeNC/IONC/MSN-T807-MB, rats showed reduced escape latency compared with the nontreated ones, especially at Day 4 and Day 5 post-treatment. On the contrary, it showed negligible difference between MB group and OA group. The insignificant therapeutic effect may due to the hydrophilic nature of MB, which diffuse fast after injection and thus limits its retention in the


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disease area in vivo.74 While the CeNC/IONC/MSN-T807-MB can specifically bind to the hyperphosphorylated tau and release MB sustainably. In addition, to rule out the motor effect in each group, the swimming speed of each group was calculated, and no significant difference was found among all groups (Figure S26). Moreover, in the probe trial, after the platform was removed, the AD model rats spent shorter time than the rats in the other groups in the target quadrant and showed a random motion paths (Figure 6b, c) due to the memory disorder induced by OA. While CeNC/IONC/MSN-T807-MB treated rats exhibited spatially oriented swimming behavior, they spent longer time in the target quadrant and their motion paths mainly focused on there (Figure 6b, c), indicating that CeNC/IONC/MSN-T807-MB could significantly rescue memory disorder in the AD model rats. CeNC/IONC/MSN-T807-MB Attenuates Microgliosis in AD Model Rats. To investigate the mechanisms by which CeNC/IONC/MSN-T807-MB mitigated cognitive impairment in AD model rats, we further analyzed the effect of CeNC/IONC/MSN-T807-MB on inflammation related astrocytic and microglial activations. Immunohistochemical staining with glial fibrillary acidic protein (GFAP, biomarker of astrocyte) and ionized calcium binding adaptor molecule-1 (Iba-1, biomarker of microglia) demonstrated significant intensive staining in the AD model rats compared to sham (Figure 6d), indicating the activation of gliosis. Microglia are the principal immune cells of the brain and play a significant role in the development of neurodegenerative disease, including AD.75-77 Recent researches in a tauopathy mouse model disclosed that microgliosis may be the earliest sign of neurodegenerative tauopathies, probably due to the impaired transport that is caused by tau hyperphosphorylation.78 Elimination of tau induced microglial activation might be therapeutically beneficial to AD.79 Furthermore, both GFAP and Iba-1 immunoreactivity were significantly decreased in the hippocampus of the AD model rats


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treated with CeNC/IONC/MSN-T807-MB. These results strongly suggest that the ROS scavenging ability and tau hyperphosphorylation inhibition effect of CeNC/IONC/MSN-MB contributed to the reduction of microglial and astrocyte activation, and consequently rescue the memory deficits of the AD model rats. The Degradation of CeNC/IONC/MSN-T807 in Physiological Environment. The degradation of CeNC/IONC/MSN-T807-MB was investigated in SH-SY5Y cells and in the brain of AD model rats. The CeNC/IONC/MSN-T807-MB were uniform and well-dispersed in water before incubation with SH-SY5Y cells or injection to living body, even after 1 day incubation with cells, most nanocomposites remained the original morphology inside the cells (Figure S27). However, the nanocomposites became shapeless after 7 days incubation, indicating the gradual degradation of the nanocomposites inside the cells. It is reported that MSNs could be gradually degraded in the cytoplasm and lysosome of the cell, and finally excluded from the cell.80 The degradation of CeNC/IONC/MSN-T807-MB was further investigated in vivo. The significant aggregation of nanocomposites was observed at 3 days after injection into the hippocampus, while the CeNCs and IONCs were partially remained within the MSNs matrix. The degradation continued and nearly no nanocrystals was observed at 7 days post-injection (Figure S28a). The degradation of IONCs and CeNCs in the hippocampus was further confirmed by ICP-MS, the cerium and iron ion concentrations were increased rapidly upon the injection of nanocomposites, and showed a gradual decrease within 3 weeks (Figure S28b, c). The ion concentrations of iron decreased more rapidly than that of cerium as confirmed by ICP-MS, indicating faster degradation of IONCs than CeNCs, such fast degradation tendency of IONCs is also reported by others.81-82 Astrocytes are considered as the key regulators of the iron metabolism in the brain, which transform into ferritin.83 The relative slow degradation of CeNCs


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is beneficial to maintain the long-term ROS-scavenging.28 All these results demonstrated that CeNC/IONC/MSN-T807 can eventually be degraded in physiological environment.

CONCLUSION In summary, we have designed and synthesized a multifunctional nanocomposite CeNC/IONC/MSN-T807 by assembling ceria and iron oxide nanocrystals on the uniform MSN, their surface was immobilized with T807 for tau protein binding, MB was loaded in the pores of MSN as tau aggregation inhibitor. The surface ligand T807 enabled the specifically hyperphosphorylated tau targeting of CeNC/IONC/MSN-T807, the tau targeted retention can be monitored in vivo under MR/PET imaging thanks to the immobilized IONC and labelled

68

Ga.

The surface decorated CeNC can mitigate mitochondrial oxidative stress and suppress tau hyperphosphorylation while the loaded MB can inhibit hyperphosphorylated tau aggregation. As a result, compared with CeNC/IONC/MSN-T807 or MB alone, a synergistic therapeutic effect is achieved by combining MB and CeNC in our multifunctional nanocomposite. To the best of our knowledge, although nanotechnology-based drug delivery approach has been investigated for tauopathy in vitro,84 our study could provide an insight into the design of in vivo tau targeted multifunctional nanoplatforms for AD theranostics. All these findings indicate that CeNC/IONC/MSN-T807-MB is an effective tau-targeted theranostic agent for AD treatment. Nevertheless, more intensive further studies, including blood brain barrier (BBB) penetrable surface modifications, will be conducted to promote the clinical translation of our nanocomposite.


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MATERIALS AND METHODS Materials: All reagents and solvents were obtained commercially and used without further purification. Tetraethyl orthosilicate (TEOS), triethanolamine (TEA), bromo-2-methylpropionic acid (BMPA), oleylamine, oleic acid, ferric trichloride hexahydrate (FeCl3·6H2O), diphenyl ether, oleyl alcohol, (3-Aminopropyl)-trimethoxysilane (APTS), fluorescein isothiocyanate (FITC),

rhodamine

B

isothiocyanate

(RITC),

dicyclohexylcarbodiimide

(DCC),

N-

hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), N, N-Dimethylformamide (DMF), Dioxane, triethyl phosphite, Acetate, zinc powder, trifluoroacetic acid were purchased from Aladdin Co. (China). Cerium(III) acetate, citric acid, xylene, methylene blue, Okadaic acid (OA) were purchased from Sigma-Aldrich Co. (USA). Sodium oleate was purchased from TCI Co. (Japan). Cetyltrimethylammoniumchloride (CTAC), ethyl alcohol, hexane, chloroform, dichloromethane, hydrochloric acid (HCl), ammonium hydroxide, sodium carbonate were purchased from Sinopharm Co. (China). 3-Bromo-4-nitropyridine, 4-Bromophenylboronic acid, terakis(triphenylphosphine) palladium (0), 5-Bromo-2-nitropyridine, hexamethylditin were purchased from J&K Chemical Co. (China). 1,4,7-triazacyclononane-1,4,7-triacetic acid was purchased from Huayi isotopes Co. (China). Synthesis of Ultra-Small Iron Oxide Nanocrystals: Iron-oleate complex was firstly synthesized by using the previously reported protocl.85 For the synthesis of ultra-small 3 nmsized iron oxide nanocrystals, 1.8 g of dry iron-oleate complex, 640 µl of oleic acid and 1.9 mL of oleyl alcohol were added to 10 g of diphenyl ether and degassed at 90 °C for 2 h. Subsequently the mixture was heated to 250 °C at a fixed heating rate of 10 °C/min followed by aging for 30 min under Ar atmosphere. The mixture was rapidly cooled to room temperature (RT) after the reaction, 100 mL of acetone was added to precipitate the as-produced


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nanocrystals. The nanocrystals were finally collected by centrifugation and dispersed in chloroform. Synthesis of Ultra-Small Ceria Nanocrystals: 0.43 g cerium(III) acetate and 3.25 g oleylamine were dissolved in 15 mL xylene followed by stirring vigorously for 24 h at RT and then heated to 90 °C at a fixed heating rate of 2 °C/min under Ar atmosphere. 1 mL of deionized water (DW) was rapidly injected into the heated mixture. The solution was then aged at 90 °C for 3 h subsequently cooled to RT. Ceria nanocrystals were precipitated by adding 100 mL of acetone, collected by centrifugation and dispersed in chloroform. Ligand Exchange of Ultra-Small Iron Oxide Nanocrystals with BMPA: Monodispersed iron oxide nanocrystals coated with oleic acid were obtained via previously reported procedures.1 BMPA (1 g) and citric acid (0.1 g) were dissolved in DMF and chloroform (50/50 v/v, 30 mL) intermixture. The as-synthesized iron oxide nanocrystals (30 mg) were added to the mixture and magnetic stirred 12 h at RT, followed by centrifugation to obtain BMPA-capped iron oxide nanocrystals and dispersed in DMF. Ligand Exchange of Ultra-Small Ceria Nanocrystals with BMPA: Citric acid (0.05 g) and BMPA (0.5 g) were dissolved in DMF and chloroform (50/50 v/v, 15 mL) intermixture. The assynthesized ceria nanocrystals (15 mg) were added to in the mixture and magnetic stirred 3 h at RT. Finally, the BMAP-capped nanocrystals were achieved by centrifugation and dispersed in DMF. Synthesis of Mesoporous Silica Nanoparticles (MSNs): For the synthesis of 50 nm-sized MSNs, 0.5 g of CTAC and 0.06 g of TEA were dissolved in 21.5 mL DW at 95°C under stirring. 1 h later, 1.5 mL of TEOS was injected dropwise and the mixture was stirred for additional 1 h. As-synthesized MSNs were separated by centrifugation and washed for 3 times with ethanol.


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Subsequently, the separated nanoparticles were incubated for 3 h with 120 µl HCl (36 %) at 60 °C for several times to remove the template agents CTAC, followed by collected and dispersed in ethanol. FITC-labeled MSNs (green) or RITC-labeled MSNs (red) was obtained by the addition of FITC-APTES or RITC-APTES, respectively, and followed by a co-condensation route using TEOS. The FITC-APTES (or RITC-APTES) was prepared by the addition reaction between FITC (4 mg) (or RITC (5 mg)) and 44 µL APTES in 1 mL methanol under light-sealed and dry conditions overnight, excessive APTES was used in order to complete the addition reaction. Surface Modification of Mesoporous Silica Nanoparticle: MSN (100 mg) was dispersed in ethanol (100 mL), then 200 µL of APTS was added and stirred for 4 h at 60 °C. After centrifugation, the collected amine-modified MSN was dispersed in DMF. Synthesis of CeNC/IONC/MSN Nanocomposite: The amine-modified MSN solution (5 mL) was mixed with 5 mL of the BMAP-capped iron oxide nanocrystals and ceria nanocrystals suspensions overnight. The desired nanocomposites obtained by centrifugation and washed with DMF to remove excrescent iron oxide and ceria nanocrystals, and then dispersed in DMSO. Synthesis of CeNC/IONC/MSN-NOTA Nanocomposite: DCC (48.6 mg), NHS (27.3 mg) and NOTA (10 mg) were dissolved in 5mL of DMSO and stirred for 3 h, then 5mL of CeNC/IONC/MSN was added. The mixture was magnetic stirred for 24 h at RT. Finally, the resulted nanocomposites were collected by centrifugation and dispersed in DMSO. Synthesis of Amino-T807: Amino-T807 is synthesized via previously reported procedures.42 The synthetic route is illustrated in Figure S1. Synthesis of CeNC/IONC/MSN-T807 Nanocomposite: DCC (50.2 mg), NHS (36.4 mg) and CeNC/IONC/MSN-NOTA (25 mg) were dissolved in 10 mL of DMSO and stirred for 3 h, then


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8.5 mg of T807 was added. The mixture was magnetic stirred for 24 h at RT. The resulted nanocomposites were collected by centrifugation and dispersed in water. Methylene Blue Loading and Release: 10 mg of nanocomposites were suspended in MB stock solution (20 mg/mL) and sonicated overnight for drug loading into the pores of the nanocomposites. MB loaded nanocomposites were centrifuged and suspended with PBS. This washing process was repeated for several times. The supernatant was kept for UV-vis absorbance measurement (UV-2600, Shimadzu, Japan) at the wavelength of 664 nm. The MB loading rate was calculated by subtracting MB in the supernatant. The concentration of MB was calculated according to MB standard curve: abs = 0.2015 c + 0.0215 (R2=0.9977). In vitro release experiments were carried out in PBS solution. The CeNC/IONC/MSN-T807-MB (about 80 µg MB) were suspended in 1 mL PBS in the dialysis membrane bag (MWCO = 3500) and the bag was immersed in 10 mL PBS and shook at the speed of 80 rpm at 37 ℃. The amount of MB released was monitored by fluorescence UV-vis measurements at different time intervals over a period of 48 h. Characterization: TEM, STEM-EDX were taken with a FEI Tecnai F20 (FEI, USA) at a voltage of 200 kV for the characterization of morphology and elements distribution. SEM, Energy-dispersive X-ray spectroscopy were performed on a Hitachi SU-70 (Hitachi, Japan). Xray powder diffraction (XRD) patterns were obtained on a Rigaku D/Max-2550 PC instrument (Rigaku, Japan). Dynamic light scattering (DLS) and zeta potentials were conducted on Zetasizer Nano ZS90 equipment (Malvern instruments, UK). The X-ray photoelectron spectroscopy (XPS) was acquired via a Thermo Scientific ESCALAB 250 Xi XPS system. SOD

Mimetic

Activity

Assay:

The

superoxide

anion

scavenging

activity

of

CeNC/IONC/MSN-T807 was measured with SOD Assay Kit-WST (Dojindo, Japan). Briefly, 20


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µL CeNC/IONC/MSN-T807 dispersion of different Ce element concentrations (0, 0.1625, 0.325, 0.65, 1.3 mM) were mixed with 200 µL of WST-1 working solution. The reaction was started by the addition of 20 mL of xanthine oxidase solution. After incubation of plate at 37°C for 20 min, the absorbance at 450 nm was measured using a microplate reader (Elx-800, Bio-Tek Instruments, USA). Since the absorbance is proportional to the amount of superoxide anion, the SOD mimetic activity could be measured by quantifying the decrease of the color development at 450nm. Autocatalytic Activity Assay: The autocatalytic activity of CeNC/IONC/MSN-T807 was assessed by treating CeNC/IONC/MSN-T807 dispersion with H2O2 solutions with various concentrations (0, 0.1, 0.5, and 1 M). Upon the addition of a drop of H2O2 solution, the color of CeNC/IONC/MSN-T807 dispersion changed from colorless to yellow, demonstrating that more Ce4+ species were generated by the addition of H2O2. During the next one month, these CeNC/IONC/MSN-T807 dispersion were kept in dark. The color of dispersion turns to colorless again as the H2O2 decomposed which indicates the regeneration of Ce3+. When a subsequent H2O2 solution was added, the color changed to yellow again. This reversible autocatalytic activity of CeNC/IONC/MSN-T807 is the key to their potential biomedical application as antioxidants in vivo. Cell Culture: In this study, SH-SY5Y cells were cultured in DMEE/F-12 medium (Gibico, USA) containing 10 % fetal bovine serum (Hyclone, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen, US) at 37 ℃ in 5% CO2. Cell Counting Kit-8 (CCK-8) Assay: Cell viability was measured by the CCK-8 Cell Counting kit (Dojindo, Japan). Okadaic acid (OA, Sigma, St Louis, MO) was dissolved in PBS and

diluted

into

sequential

concentrations.

MB,

CeNC/IONC/MSN-T807

and


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CeNC/IONC/MSN-T807-MB were dissolved and diluted in culture medium. Cells were seeded onto 96-well plates (1×104/well) for 24 h and treated with different concentrations (5-160 nmol/L) of OA for 12 h to select the appropriate concentration. Besides, SH-SY5Y cells were treated with sequential concentrations of CeNC/IONC/MSN-T807-MB for 12 h to investigate the biocompatibility. SH-SY5Y cells were pretreated with OA (40 nM/L) for 12 h, then, each group was added with different concentrations of nanocomposites and incubated for another 12 h in medium. The cells were incubated for a further 4 h at 37 °C. The final optical density (OD) was measured by the Microplate Reader (Bio-Rad, USA) at 450 nm. Flow Cytometry Analysis of Cell Apoptosis: Cells were cultured in 6-well plates (1× 106/well) before replaced with 2 mL of OA (40 nM/L), after incubation for 12 h, three experimental

groups

(MB

(2.25

µg/mL),

CeNC/IONC/MSN-T807

(4

µg/mL),

CeNC/IONC/MSN-T807-MB (6.25 µg/mL)) of cells were incubated overnight. Cells were collected and resuspended, then incubated with Annexin-V-FITC Apoptosis Detection Kit (BD Pharmingen, USA) in terms of the manufacturer’s protocol. The intensity of fluorescence was measured utilizing an Accuri C6 flow cytometer (DxFLEX, Beckman Coulter, USA). Real-Time Quantitative RT-PCR Analysis：： Cells were seeded onto 6-well plates (1× 106/well) for 24 h. Then the medium was replaced with 2 mL of OA (40nM/L), after incubation for 12 h, the medium was replaced with 2 mL of medium containing MB (2.25 µg/mL), CeNC/IONC/MSN-T807 (4 µg/mL), CeNC/IONC/MSN-T807-MB (6.25 µg/mL) overnight. Total RNA of cells was isolated by using TRIzol Reagent (GIBCO BRL; Invitrogen, Carlsbad, CA) according to the Trizol protocol. Then the extracted RNA pellets were quantified, a firststrand cDNA synthesis was performed using a Superscript First-Strand Synthesis kit (Invitrogen). All samples weredetermined by real-time PCR (Bio-Rad, USA) using the SYBR Green PCR


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Page 22 of 58

Master Mix (Applied Biosystems, USA). The DNA product was amplified by primers and measured by real-time PCR. All primer sequences were presented in Table S1. Western Blotting: SH-SY5Y cells were pretreated with OA (40 nM/L) 12 h. Then treated with MB (2.25 µg/mL), CeNC/IONC/MSNT807 (4 µg/mL), CeNC/IONC/MSN-T807-T807-MB (6.25 µg/mL) respectively. The treatments lasted for 12 h, then cells were harvested and lysed with RIPA buffer (1×PBS, 1 % NP40, 0.1 % SDS, 5 mmol/L EDTA, 0.5 % Sodium Deoxycholate, 1 % Protease inhibitor and 1 % phosphatase inhibitor). Proteins concentrations were determined using a Bio-Rad kit. The proteins were separated on 10 %-12 % SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA).86 The membranes were incubated in 5 % bovine serum albumin (BSA) for 2 h at RT, and then probed with primary antibodies at 4 ℃ overnight: phosphor (P)-tau (Ser 396/199/404, Thr 205), P-Akt, P-Gsk3β, β-Actin, Bax and cleaved-Caspase3 (Abcam, MA, USA). The blots were rinsed and incubated with appropriate secondary antibodies for 1 h at RT. Followed by chemiluminescent kit (Pierce, Rockford, IL) detection. Bands were visualized using ECL detection reagents and analyzed by Quantity One and normalized by β-Actin. Mitochondrial ROS-Scavenging Activity Analysis: SH-SY5Y cells (1×105/mL) were seeded in 2 mL of medium in a confocal dish and incubated for 24 h. Cells were pretreated with OA (40 nM/L) in the presence of MB (2.25 µg/mL), CeNC/IONC/MSN-T807 (4 µg/mL), CeNC/IONC/MSN-T807-MB (6.25 µg/mL) respectively. After incubation for 12 h, the medium was replaced with 1mL of 5 µM MitoSOXTM reagent working solution (Invitrogen-Life Technologies, Carlsbad, CA) at 37 °C for 10 min. Cells were washed with PBS before fixed with 4 % paraformaldehyde for 10 min and incubated with DAPI (0.02 %) for 5 min. Relative


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mitochondrial ROS-scavenging activity was evaluated by using confocal microscopy (OLYMPUS IX83-FV3000-OSR, Japan) Furthermore, flow cytometer was used to evaluate intracellular mitochondrial ROS accumulation. SH-SY5Y cells (1 × 105/well) were seeded in 24-well plates and cultured for 24 h. The cells were then treated with OA (40 nM/L) in the presence of MB (2.25 µg/mL), CeNC/IONC/MSN-T807 (4 µg/mL), CeNC/IONC/MSN-T807-MB (6.25 µg/mL) respectively. After incubation for 12 h, the medium was replaced with 1 mL of 5 µM MitoSOXTM reagent working solution (Invitrogen-Life Technologies, Carlsbad, CA) at 37 °C for 10 min. Then, the cells were washed with PBS before harvested by trypsinization, and fixed with 4 % formaldehyde for 10 min. The cells were fixed and collected by centrifugation and resuspended in 0.5 mL of PBS for analysis on a flow cytometer (BD Fortessa, USA). Cellular Uptake: To assess the cellular uptake of CeNC/IONC/MSN-T807 in OA treated cells. SH-SY5Y cells (1×105/mL) were seeded in 2 mL of medium in a confocal dish and incubated with or without OA overnight. RITC-labeled CeNC/IONC/MSN-T807 (10 µg/mL) or RITC-labeled CeNC/IONC/MSN (10 µg/mL) was added to the confocal dishes respectively. After incubation for 1 h, 2 h, 4 h, 6 h, 12 h and 24 h, cells were washed with PBS for three times. The samples were fixed with 4 % paraformaldehyde for 10 min and incubated with DAPI (0.02 %) for 5 min. Cellular nanocomposite uptake was monitored by confocal microscopy (OLYMPUS IX83-FV3000-OSR, Japan) at different time points. Furthermore, flow cytometer was used to quantitatively evaluate the cellular uptake SHSY5Y cells (1 × 105/well) were seeded in 24-well plates and cultured for 24 h. Then, RITClabeled CeNC/IONC/MSN-T807 (10 µg/mL) or RITC-labeled CeNC/IONC/MSN (10 µg/mL) was added, after incubation for 1 h, 2 h, 4 h, 6 h, 12 h and 24 h, cells were washed with PBS


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Page 24 of 58

before harvested by trypsinization, and fixed with 4 % formaldehyde for 10 min. Cells were fixed and collected by centrifugation and resuspended in 0.5 mL of PBS for analysis on a flow cytometer (BD Fortessa, USA). Immunofluorescence Staining: For the sake of explore the co-localization of p-tau with RITC-labeled nanocomposites in SH-SY5Y cells. The SH-SY5Y cells were prepared and grouped as the protocol above. Then, RITC-labeled CeNC/IONC/MSN (5µg/mL) and RITClabeled CeNC/IONC/MSN-T807 (5 µg/mL) were added to each confocal dish with OA incubated for 4h. The normal cells were also added RITC-labeled CeNC/IONC/MSN-T807 (5 µg/mL) and RITC-labeled CeNC/IONC/MSN (5 µg/mL) and incubation for 4 h. After incubation, the samples were fixed with 4 % paraformaldehyde for 15 min and incubated for 1 h in a blocking solution (5 % BSA, 0.5 %Triton-X in PBS), then incubated with p-tau(S396) antibody (ThermoFisher Scientific) at 4 ℃ overnight. After washing with PBS three times, samples were incubated for 1 h with a fluorescent-conjugated secondary antibody (Alexa Fluor 488 conjugated goat secondary antibody to rabbit IgG, Abcam). The samples were washed with PBS three times. Last, samples were incubated with DAPI (0.02 %) for 5 min and washed with PBS three times. The fluorescence signals were monitored by using a confocal laser scanning microscope (OLYMPUS IX83-FV3000-OSR). Inhibition of Tau Aggregation inside Cells: SH-SY5Y cells (4×105/mL) were seeded with 300 µL of culture medium in confocal dishes and incubated with 2 mL culture medium for 24 h. The SH-SY5Y cells were incubated with OA (40 nM) for 24 h in culture medium with solution of CeNC/IONC/MSN-T807 (4 µg/mL) or CeNC/IONC/MSN-T807-MB (6.25 µg/mL) or MB (2.25 µg/mL). After inbubation, the samples were fixed with 4 % paraformaldehyde for 15 min and incubated for 1 h in a blocking solution (5 % BSA, 0.5 % Triton-X in PBS), then the samples


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were washed with PBS and incubated with DAPI (0.02 %) for 5 min. Lastly, these samples were incubated with Thioflavin S (ThS 20 µM; Sigma, St Louis, MO) in PBS for 45 min in the dark at RT. Aggregation of tau in cells was measured by the fluorescence value of ThS. Fluorescence was observed by LSM-780 microscope (Carl Zeiss, Oberkochen, Germany) with excitation at 458 nm. Expression and Purification of Tau Protein: The human htau40 containing 441 residues, which was purified from the transformed recombinant E. coli strain BL21 containing expression vector with C terminal histidine-tagged tau. Cells grown in LB medium was induced (1 mM IPTG), harvested, resuspended, extracted and purified via Nickel (Ni2+)-column. After equilibration with Tris-NaCl buffer, wash with Tris-NaCl buffer with 0, 30 mM and 50 mM imidazole and elution with Tris-NaCl buffer with 200 mM and 400 mM imidazole, fractions were dialyzed, concentrated and analyzed by 10 % SDS-PAGE. Tau Filament Assembly and Inhibitor Testing by Thioflavin S Assay: Tau protein (htau40, 50 µM) and heparin (50 µM, MCE, NJ, USA) were incubated in PBS at 37 ℃, pH 7.4. To measure the inhibition of PHF assembly, CeNC/IONC/MSN-T807, CeNC/IONC/MSN-T807MB or MB (the dosage of MB was equivalent at 200 µM) was coincubated with tau protein. After 72 h, a total volume of 50 µL assay solution in a black 384-microtiter-plate well (Thermo, Dreieich, Germany) containing 10 µM of ThS and 10 µM Protein from each sample were mixed in PBS, pH 7.4 and incubated for 45 min in dark at RT. Fluorescence was measured using a spectrofluorometer (SynergyMx M5, Molecular Devices, USA) with excitation at 440 nm and emission at 520 nm, subtracting the light scattering and background fluorescence of the samples without ThS.


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Electron Microscopy: Protein solutions of each group were diluted to 0.1-10 µM and placed on copper grids (600 mesh) for 1 min, and then negatively stained with 2% uranyl acetate for 45 s. The specimens were examined in an electron microscope (H-7650, Hitachi, Japan) at 100 kV. Animal Modeling: Male Sprague-Dawley (SD) rats (n=32, 300–320 g, 3 months old) were kept in the thermo-regulated, humidity-controlled condition under a 12 h day/night light cycle and provided with food and water ad libitum. The experimental protocol was performed with the approval of the Institutional Animal Care and Use Committee of Zhejiang University School of Medicine (Protocol No. ZJU20160446) and followed the National Guidelines for Animal Protection. Rats were intraperitoneally anesthetized with 1.5 % pentobarbital sodium (50 mg/kg) and placed in a stereotaxic apparatus. Okadiac acid ammonium salt (OA, 300 ng in 1.5 µL saline) or saline (0.9 % NaCl for medical use, 1.5 µL) was microinjected stereotaxically into the right dorsal hippocampus (-3.8 mm AP, -2.5 mm ML, and -3.2 mm DV, according to the Rat Brain Paxinos Atlas). The microinjection was performed with a Hamilton microsyringe with 27G stainless steel over 5 min. Subsequently, the needle was held in place for an additional 5 min to allow for diffusion and then removed slowly over 5 min. Finally, rats were intraperitoneally administered with 80 U penicillin for consecutive three days to prevent infection. Rats injected with OA and saline were regarded as AD model group and sham group, respectively. Stereotaxic Injections: Rats were divided into five groups (n=8 per group): Sham + saline, OA + saline, OA + MB, OA + CeNC/IONC/MSN-MB, OA + CeNC/IONC/MSN-T807-MB. Unilateral hippocampus microinjection (-0.8 mm AP, -2.5 mm ML, and -3.2 DV, according to the Rat Brain Paxinos Atlas) of 10 µL CeNC/IONC/MSN-T807-MB or CeNC/IONC/MSN-MB or MB (equivalent dosage of MB (3.6 mg/mL)) or saline was performed using a stereotaxic


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apparatus 5 days after animal modeling. The detailed method of microinjection was described in “animal modeling” section. In the duration of the experimental protocol, each rat received treatment once. 18

F-T807 Miro-PET/CT Imaging and Image Analysis: An INVEON integrated modality

scanner (Siemens, Germany) was employed for microPET/CT imaging. Rats were anesthetized with 2.5% isoflurane and placed on the scanner bed. A 10-min CT scan was first performed for attention correction, followed by a 30-min dynamic PET scan with the middle skull in the center of the field of view. Subsequently, approximately 18.5 MBq (500 µCi) of

18

F-T807 was

administered to the rats via tail vein injection immediately followed by PET acquisition. Fifteen dynamic frames were reconstructed (six 10-s, four 1-min, and five 5-min frames, for a total of 30 min). Four iterations and four subsets were used for reconstruction (2-dimensional attenuationweighted ordered-subsets expectation maximization, OSEM2D). Transverse data were reformatted to a 128 × 128 matrix with 0.78-mm pixels for each dynamic frame. The summed dynamic PET frames were coregistered to a MRI rat brain template by a manual rigid-body transformation using the PMOD fusion tool (V3.3; PMOD Technologies Ltd, Zurich, Switzerland). Volumes of interest (VOIs) were drawn on the MRI rat atlas in the hippocampus as target VOI and the cerebellum as reference VOI. The non-invasive simplified reference tissue model 2 (SRTM2) was used to determine directly non-displaceable BPnd using the cerebellum as the reference region. Then the BPnd was calculated. In vitro and In vivo MRI: MRI investigations were conducted using a 3.0 T MRI system (Signa HDxt, GE Medical Systems, Milwaukee, WI, USA). Spin-lattice and spin-spin relaxation times (T1 and T2) were measured using T1 mapping IR-FSE sequence: TR= 1000 ms, TE= 13.8 ms TI=100 ms，200 ms，300 ms，400 ms，500 ms. T2 mapping FSE sequence: TR= 2000 ms,


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TE= 20 ms 40 ms 80 ms 100 ms 120 ms. The longitudinal (r1) and transverse (r2) relaxivities were calculated from ri = (1/Ti –1/Ti0)/c, where c is the Fe concentration of CeNC/IONC/MSNT807 in mM, Ti is the relaxation time at concentration c, Ti0 is the relaxation time of water, and i = 1 and 2 for T1 and T2. The in vitro MR imaging was performed using conventional spin echo acquisition (slice thickness: 2.00 mm). For T1 measurement, TR = 540 ms and TE = 15 ms were used, whereas for T2 measurement, TR = 3500 ms and TE = 80 ms were used. For the in vivo MR imaging, CeNC/IONC/MSN-T807 or CeNC/IONC/MSN (10 mg/mL) were microinjected stereotaxically into the right dorsal hippocampus (-3.8 mm AP, -2.5 mm ML, and -3.2 mm DV, according to the Rat Brain Paxinos Atlas) with 2 µL (20 mg/mL). Rats were then placed in a 3 T MR scanner, and FSE sequence was used with the followed parameters: FOV = 14 mm x 14 mm, SL = 2 mm, Flip angle (FA) = 90o, for T1 MR imaging, TR = 550 ms, TE = 12 ms. 68

Ga Radiolabeling and PET Imaging: Aliquots of CeNC/IONC/MSN-T807 (5 mg/mL) or

CeNC/IONC/MSN (5 mg/mL) in 0.1 M sodium acetate solution (pH 5.2) were reacted with an aqueous solution of 68GaCl3 (~1 mCi) for 30 min. Radiolabeled nanocomposites were purified by GPC with a PD-10 column. Instant thin-layer chromatography (ITLC) was used to assess the refined nanocomposties. After adding 4 mM (EDTA) into PBS (PH=7.4), the ITLC strips were quantified using a TLC imaging scanner (Bioscan IAR-2000, USA). Free

68

GaCl3 moved to the

solvent front (Rf = 0.9–1.0), and the nanocomposites remained at the original spot (Rf = 0.0). The radiochemical purity of all nanocomposites after GPC purification was > 99 %. The stability of the radiolabeled nanocomposites was tested in PBS and fetal bovine serum (FBS) respectively for 5 h at 37 ℃. AD model rats were anesthetized with isoflurane (2 %) and unilateral hippocampal stereotactic injected 0.62 MBq (16.67 µCi) of

68

Ga-CeNC/IONC/MSN-T807 or


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Ga-CeNC/IONC/MSN. The images were obtained by a high-resolution microPET R4 scanner

(Siemens Medical Solutions) for 0.5 h, 1.5 h, 2.5 h and 3.5 h static images acquisition. Quantification of Cerium and Iron Ion Concentration: Determination of cerium and iron content in the ipsilateral side of the brain was performed by inductively coupled plasma mass spectrometry analysis (XSENIES, Thermo, USA). Brain tissue was dissolved in HNO3 (65 %) and H2O2. The resulting solution was diluted in HNO3 (2 %) for analysis. Morris Water Maze Behavioral Test: Spatial memory function of rats was tested by Morris water maze. It was carried out for 6 days after treatment for 7 days. The maze made of circular blue opaque plastic with 50 cm depth and 120 cm width was filled with water maintained at 2426 °C using an automatic heater to avoid hypothermia. Before the task, the water was dyed with black ink. At the tank midpoint, two imaginary perpendicular lines crossed to divide the tank into four quadrants, i.e., Northwest, Southwest, Northeast, and Southeast. A small escape platform (9 × 9 cm) was placed at a fixed position in the center of Southeast quadrant with 25 cm away from the perimeter, and was hidden about 1.5 cm beneath the water surface. The maze contained a mass of fixed visual cues on the walls. Performance of each rat was recorded with a videotracking system and data were calculated with equipped software for water maze (SMART 3.0, Panlab, Spain). On the first five days, rats (n = 8 per group) received three acquisition trials daily with a 15-min inter-trial interval for consecutive 5 days. Briefly, three start points, excluded the one quadrant without platform, were randomized selected in three daily trials. Each rat facing the wall of the tank was set free into the water. If a rat failed to reach the platform within 90 s, it was guided to the platform where it remained for a further 15 s. Within the inter-trial interval, rats were towel dried in a plastic holding cage filled with additional towels. Upon completion of the daily three trials, rats were removed from the maze, towel dried and then returned to the home


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cage. The swimming speed, swimming path and the latencies to find the platform were recorded. Finally, on the sixth day, spatial memory was tested by each rat through a probe trial. Briefly, the fixed platform was removed from the maze, and each rat was released into water at the right opposite position of the escape platform and permitted to swim freely for 60 s. Spatial acuity was expressed as the percentage of time in the Southeast quadrant where the escape platform was located. Immunohistochemical Staining: Immunohistochemical staining was derived from routine formalin-fixed paraffin-embedded sections: rats were transcardially perfused with physiological saline (200 mL) next to 4 % paraformaldehyde (200 mL) under deep anesthesia with 1.5 % pentobarbital sodium. The whole brains were removed and fixed in 4 % formalin and paraffinembedded brain tissues were cut into 4 µm sections, staining for the primary antibody in 5 % BSA overnight at 4 °C. Then detected by appropriate secondary antibodies. Anti-GFAP antibody to stain mature astrocyte, and anti-Iba1 antibody to stain activated microglia. Stained brain sections were finally digitized with the use of a camera and stained section images were evaluated by using Image-ProPlus 5.0 software. In Vivo Tracking of Nanocomposites in Brain Tissues: AD model rats were anesthetized with isoflurane (2 %), and then RITC-labeled CeNC/IONC/MSN-T807 (1 mg/mL) or RITClabeled CeNC/IONC/MSN (1 mg/mL) was administered via ipsilateral side of hippocampal stereotactic injection. Rats were sacrificed 6 h post-injection, and the brains were fixed overnight with 4 % paraformaldehyde at 4 ℃, before transferred to 30 % sucrose for another 3 days. Frozen brains were cut in 20-µm-thick sections. Then the samples were stained with DAPI (0.02 %) for 5 min at RT. After washed with PBS, the sections were mounted on slides, air-dried. Images were obtained by using a confocal laser scanning microscope (OLYMPUS IX83-FV3000-OSR).


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The Degradation of CeNC/IONC/MSN-T807 in vitro and in vivo: 1×106 per well SHSY5Y cells were seeded on 6-well plates. After treated with CeNC/IONC/MSN-T807 (1 mg/mL) for 24 h, the adherent cells were preserved to passage, and the number of cells was doubled within 24 h. Adherent cells were collected at 1 day and 7 days after incubation, then fixed with 2.5 % glutaraldehyde for 1 h at RT and post-fixed with 1 % osmium tetroxide for 2 h. Subsequently, the pelleted cells were dehydrated in ethanol (30 %-100 %) and then embedded in epoxy resin. Micrometer-thick sections were cut and collected on copper grids. The samples were stained with 2 % uranyl acetate prior to TEM (H-7650, Hitachi, Japan) evaluation at 100 kV. As for in vivo assessment, AD model rats were anesthetized with isoflurane (2 %), and the CeNC/IONC/MSN-T807 (10 mg/mL, 5 µL) was administered via unilateral hippocampal stereotactic injection. The degradation was evaluated by both TEM and ICP-MS. After injection, ipsilateral hippocampal tissues of 0 day (sham group), 1 day, 3 days, 5 days, 7 days, 14 days and 21 days were cut into the size of 1 mm3 and fixed overnight with cold 2.5 % glutaraldehyde and post-fixed with 1 % osmium tetroxide for 2 h. Small pieces were dehydrated in ethanol (30 %100 %) and then embedded in epoxy resin. Micrometer-thick sections were cut and collected on copper grids. The samples were stained with 2 % uranyl acetate prior to TEM (H-7650, Hitachi, Japan) evaluation at 100 kV. The cerium and iron ion concentrations in ipsilateral hippocampal tissues at indicated times were analyzed by ICP-MS. Statistical Analysis: Experimental data were presented as mean ± SD unless otherwise indicate. For multiple-group comparison, one-way ANOVA followed by Turkey’ post hoc analysis was applied using SPSS 19.0 software. A value of probability (p) < 0.05 was considered statistically significant.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Additional results of nanoparticle characterization, in vitro/ in vivo studies, and Figures (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] Author Contributions #

Qing Chen, Yang Du, Kai Zhang and Zeyu Liang contributed equally to this work.

ACKNOWLEDGMENT D.L. acknowledges financial support by the National Key Research and Development Program of China (2016YFA0203600), the National Natural Science Foundation of China (51503180, 51703195, 51611540345), “Thousand Talents Program” for Distinguished Young Scholars (588020*G81501/048), and Fundamental Research Funds for the Central Universities (520002*172210161). M.T. acknowledges financial support by National Natural Science Foundation of China (81571711, 81725009, 81761148029), Ministry of Science and Technology of China (2015DFG32740).


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67. Jiang, W.; Luo, T.; Li, S.; Zhou, Y.; Shen, X. Y.; He, F.; Xu, J.; Wang, H. Q. Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons. PloS One 2016, 11, e0152371. 68. Xu, H.; Li, J.; Wang, Z.; Feng, M.; Shen, Y.; Cao, S.; Li, T.; Peng, Y.; Fan, L.; Chen, J. Methylene Blue Attenuates Neuroinflammation after Subarachnoid Hemorrhage in Rats through The Akt/GSK-3β/MEF2D Signaling Pathway. Brain Behav. Immun. 2017, 65, 125-139. 69. Dal-Cim, T.; Molz, S.; Egea, J.; Parada, E.; Romero, A.; Budni, J.; Saavedra, M. D.; Del, B. L.; Tasca, C. I.; López, M. G. Guanosine Protects Human Neuroblastoma SH-SY5Y Cells against Mitochondrial Oxidative Stress by Inducing Heme Oxigenase-1 via PI3K/Akt/GSK-3β Pathway. Neurochem. Int. 2012, 61, 397-404. 70. Hanson, C. J.; Bootman, M. D.; Distelhorst, C. W.; Maraldi, T.; Roderick, H. L. The Cellular Concentration of Bcl-2 Determines Its Pro- or Anti-Apoptotic Effect. Cell Calcium 2008, 44, 243-258. 71. Martin, S. J. Apoptosis: Controlled Demolition at The Cellular Level. Nat. Rev. Mol. Cell. Bio. 2008, 9, 231-241. 72. Chen, L. Q.; Wei, J. S.; Lei, Z. N.; Zhang, L. M.; Liu, Y.; Sun, F. Y. Induction of Bcl-2 and Bax was Related to Hyperphosphorylation of Tau and Neuronal Death Induced by Okadaic Acid in Rat Brain. Anat. Rec. 2005, 287A, 1236-1245. 73. Vorhees, C. V.; Williams, M. T. Morris Water Maze: Procedures for Assessing Spatial and Related Forms of Learning and Memory. Nat. Protoc. 2006, 1, 848-858.


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74. O'Leary, J. C.; Li, Q.; Marinec, P.; Blair, L. J.; Congdon, E. E.; Johnson, A. G.; Jinwal, U. K.; Koren, J.; Jones, J. R.; Kraft, C. Phenothiazine-Mediated Rescue of Cognition in Tau Transgenic Mice Requires Neuroprotection and Reduced Soluble Tau Burden. Mol. Neurodegener. 2010, 5, 45-55. 75. Mhatre, S. D.; Tsai, C. A.; Rubin, A. J.; James, M. L.; Andreasson, K. I. Microglial Malfunction: The Third Rail in The Development of Alzheimer's Disease. Trends Neurosci. 2015, 38, 621-636. 76. Maphis, N.; Xu, G.; Kokiko-Cochran, O. N.; Jiang, S.; Cardona, A.; Ransohoff, R. M.; Lamb, B. T.; Bhaskar, K. Reactive Microglia Drive Tau Pathology and Contribute to the Spreading of Pathological Tau in the Brain. Brain 2015, 138, 1738-1755. 77. El Khoury, J.; Toft, M.; Hickman, S. E.; Means, T. K.; Terada, K.; Geula, C.; Luster, A. D. Ccr2 Deficiency Impairs Microglial Accumulation and Accelerates Progression of AlzheimerLike Disease. Nat. Med. 2007, 13, 432-438. 78. Yoshiyama, Y.; Higuchi, M.; Zhang, B.; Huang, S. M.; Iwata, N.; Saido, T. C.; Maeda, J.; Suhara, T.; Trojanowski, J. Q.; Lee, V. M. Synapse Loss and Microglial Activation Precede Tangles in A P301S Tauopathy Mouse Model. Neuron 2007, 53, 337-351. 79. Asai, H.; Ikezu, S.; Tsunoda, S.; Medalla, M.; Luebke, J.; Haydar, T.; Wolozin, B.; Butovsky, O.; Kügler, S.; Ikezu, T. Depletion of Microglia and Inhibition of Exosome Synthesis Halt Tau Propagation. Nat. Neurosci. 2015, 18, 1584-1593.


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80. Zhai, W.; He, C.; Lei, W.; Yue, Z.; Chen, H.; Jiang, C.; Zhang, H. Degradation of Hollow Mesoporous Silica Nanoparticles in Human Umbilical Vein Endothelial Cells. J. Biomed. Mater. Res., Part B 2012, 100B, 1397-1403. 81. Mazuel, F.; Espinosa, A.; Luciani, N.; Reffay, M.; Borgne, R. L.; Motte, L.; Desboeufs, K.; Michel, A.; Pellegrino, T.; Lalatonne, Y. Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels. ACS Nano 2016, 10, 7627-7638. 82. Jie, W.; Ding, T.; Jiao, S. Neurotoxic Potential of Iron Oxide Nanoparticles in The Rat Brain Striatum and Hippocampus. Neurotoxicology 2013, 34, 243-253. 83. Hohnholt, M. C.; Dringen, R. Uptake and Metabolism of Iron and Iron Oxide Nanoparticles in Brain Astrocytes. Biochem. Soc. T. 2013, 41, 1588-1592. 84. Jinwal, U. K.; Groshev, A.; Zhang, J.; Grover, A.; Sutariya, V. B. Preparation and Characterization of Methylene Blue Nanoparticles for Alzheimer's Disease and Other Tauopathies. Curr. Drug Deliv. 2014, 11, 541-550. 85. Park, J.; An, K.; Hwang, Y.; Park, J. G.; Noh, H. J.; Kim, J. Y.; Park, J. H.; Hwang, N. M.; Hyeon, T. Ultra-Large-Scale Syntheses of Monodisperse Nanocrystals. Nat. Mater. 2004, 3, 891-895. 86. Benedikt, M.; Puppis, G.; Riveros, C. Endoplasmic Reticulum Stress Induced by Zinc Oxide Nanoparticles is An Earlier Biomarker for Nanotoxicological Evaluation. ACS Nano 2014, 8, 2562-2574.


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Scheme 1. Schematic illustration of designed synthetic procedure of CeNC/IONC/MSN-T807MB and its tau-targeted synergistic treatment. (a) BMPA-capped extremely small iron oxide nanocrystals and ceria nanocrystals are firstly anchored on the surface of amino groups modified MSNs. Then, the NOTA-T807 targeting ligands were conjugated to amino groups modified


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MSNs via amide bond. Finally, methylene blue was adsorbed into the pores of MSNs. (b) CeNC/IONC/MSN-T807-MB with bimodal imaging capability can specifically target to hyperphosphorylated tau, and perform a combinational therapy of ROS scavenging and methylene blue release; consequently, induce a synergistic therapeutic effect: scavenging of ROS to prevent tau hyperphosphorylation and the inhibition of hyperphosphorylated tau aggregation due to released methylene blue. Moreover, the neurons are protected from ROS mediated apoptosis.


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Figure 1. Characterization of CeNC/IONC/MSN-T807-MB. (a) TEM images of MSNs (i), ultrasmall iron oxide nanocrystals (ii), ultrasmall ceria nanocrystals (iii), CeNC/IONC/MSNT807

(iv),

high

resolution

transmission

electron

microscopy (HRTEM) image of

CeNC/IONC/MSN-T807 (v, vi), SEM images of CeNC/IONC/MSN-T807 (vii), Energy dispersive X-ray spectra (EDS) mapping of each element of CeNC/IONC/MSN-T807 (viii). Green for Fe (ix), red for Ce (x). (b) Hydrodynamic diameters of CeNC/IONC/MSN-T807 in


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aqueous solution. (c) Release profiles of methylene blue from CeNC/IONC/MSN-T807-MB in PBS solution and cell culture medium (DMEM + 10 % FBS). Error bars represent standard deviation (n = 3 per group). (d) Neutralization of superoxide anions by CeNC/IONC/MSN-T807 in a dose-dependent manner. Error bars represent standard deviation. (e) T1 weighted MR images of CeNC/IONC/MSN-T807 (left). Plot of 1/T1 over Fe ions concentration of CeNC/IONC/MSNT807, the slope indicates the specific relaxivity (r1) (right). (f) T2 weighted MR images of CeNC/IONC/MSN-T807 (left). Plot of 1/T2 over Fe ions concentration of CeNC/IONC/MSNT807, the slope indicates the specific relaxivity (r2) (right).


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Figure 2. Specific tau targeting capability of CeNC/IONC/MSN-T807. (a) Confocal fluorescence images of CeNC/IONC/MSN-T807 and CeNC/IONC/MSN in SH-SY5Y cells or OA treated SH-SY5Y cells after 4 h incubation. Cells stained with DAPI are shown in blue fluorescence. The immunofluorescence staining of SH-SY5Y cells or OA treated SH-SY5Y cells with pS396-Tau antibody are shown in green fluorescence. RITC labelled CeNC/IONC/MSNT807 and CeNC/IONC/MSN are shown in red fluorescence. (scale bar = 20 µm). (b) In vivo T1 weighted MR images of the brain of OA treated rats after administration of CeNC/IONC/MSNT807, CeNC/IONC/MSN, saline for 6 h (left) and corresponding color mapped images (right). (c) The concentration of cerium ions (upper) and iron ions (lower) in the ipsilateral hippocampus tissues were measured by ICS-MS. Error bars represent standard deviation. Statistical analysis was performed using a one-way analysis of variance (ANOVA) test with *** indicating p < 0.001, ** indicating p < 0.01, * indicating p < 0.05 (n = 3 per group).


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Figure 3. Therapeutic effect of CeNC/IONC/MSN-T807-MB in vitro. (a) Flow cytometry analysis of mitochondrial ROS accumulation in SH-SY5Y cells obtained by MitoSOX. SHSY5Y cells were stained using 5 µM MitoSOX without any treatment (Control) and after exposed to 40 nM OA (OA), to 40 nM OA and 2.25 µg MB (OA+MB), to 40 nM OA and 4 µg CeNC/IONC/MSN-T807 (OA + CeNC/IONC/MSN-T807), to 40 nM OA and 6.25 µg CeNC/IONC/MSN-T807-MB (OA + CeNC/IONC/MSN-T807-MB). The dosage of MB in MB treated group was equivalent with the dosage of MB in CeNC/IONC/MSN-T807-MB treated group, the dosage of CeNC/IONC/MSN-T807 in CeNC/IONC/MSN-T807 treated group was equivalent with the dosage of CeNC/IONC/MSNT807 in CeNC/IONC/MSN-T807-MB treated group. (b) Western blot and quantification results for hyperphosphorylated tau at serine 396, 199, 404 epitopes and threonine 205 epitopes in SH-SY5Y cells. Statistical analysis was performed using a one-way ANOVA test with, ** indicating p < 0.01, * indicating p < 0.05 compared to OA group (n = 3 per group). Error bars represent standard deviation. (c) Quantitation of tau filament formation using ThS fluorescence. Results are expressed as % fluorescence of controls (taken as 100%). Statistical analysis was performed using a one-way ANOVA test with ****indicating p < 0.0001, * indicating p < 0.05 compared to OA group (n = 3 per group). Error bars represent standard deviation. (d) Electron microscopic assessment of tau filaments formed in the absence (Control) or the presence of CeNC/IONC/MSN-T807, CeNC/IONC/MSN-T807-MB or MB (scale bar = 200 nm).


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Figure 4. Cytoprotection effect of CeNC/IONC/MSN-T807-MB in vitro. (a) Inhibition of OA induced cell death of SH-SY5Y cells after exposing to 40 nM OA 12 h, then treated with MB, CeNC/IONC/MSN-T807 and CeNC/IONC/MSN-T807-MB for another 12 h. The comparative cell death inhibition ability of MB, CeNC/IONC/MSN-T807 and CeNC/IONC/MSN-T807-MB was measured by CCK-8 assay at indicated concentrations. Error bars represent standard deviation. Statistical analysis was performed using a one-way ANOVA test with **** indicating p < 0.0001, ** indicating p < 0.01, * indicating p < 0.05 (n = 6 per group). (b) Apoptosis results of SH-SY5Y cells after treatment with MB, CeNC/IONC/MSN-T807, or CeNC/IONC/MSNT807-MB as measured by flow cytometry, cells were stained with Annexin V-FITC/PI.


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Figure 5. In vitro therapeutic mechanism of CeNC/IONC/MSN-T807-MB in OA induced cell model. (a) Western blot and quantification results of p-Akt, p9-GSK3β, Bax, c-Caspase 3 in SHSY5Y cells after treatment with MB, CeNC/IONC/MSN-T807, CeNC/IONC/MSN-T807-MB. Statistical analysis was performed using a one-way ANOVA test with ** indicating p < 0.01, * indicating p < 0.05 compared with OA group (n = 3 per group). (b) Relative ratio of genes expression of Akt, GSK3β, Bax, Caspase-3 in SH-SY5Y cells after treatment with MB, CeNC/IONC/MSN-T807 and CeNC/IONC/MSN-T807-MB. Statistical analysis was performed using a one-way ANOVA test with ** indicating p < 0.01 compared with OA group (n = 3 per group). (c) A proposed model illustrating the mechanism underlying the efficiency of CeNC/IONC/MSN-T807-MB in the amelioration of cognitive impairment in AD. CeNC/IONC/MSN-T807-MB rescued neuronal cells from apoptosis by alleviating the ROS and regulating pro-apoptotic proteins. Moreover, CeNC/IONC/MSN-T807-MB also inhibited tau hyperphosphorylation by activating Akt/GSK3β pathway.


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Figure 6. In vivo evaluation of CeNC/IONC/MSN-T807-MB therapy. (a) Hidden platform learning histograms. Day 1 indicates performance on the first trial, and subsequent histograms represent average of all daily trials. Statistical analysis was performed using a one-way ANOVA test with ** indicating p < 0.01, * indicating p < 0.05 compared with OA group (n = 7 to 8 per group). Error bars represent Standard Error of Mean. (b) Relative time spent on the target quadrant in a probe trial. Statistical analysis was performed using a one-way ANOVA test with ** indicating p < 0.001, * indicating p < 0.05 compared with OA group (n = 7 to 8 per group). Error bars represent Standard Error of Mean. (c) Representative path tracings of different groups. (d) Immunohistochemical staining with iba-1, GFAP in different groups (upper), quantification of GFAP and Iba-1 positive cells in the corresponding images (lower) (scale bar = 100 µm). Statistical analysis was performed using a one-way (ANOVA) test with ** indicating p < 0.01, * indicating p < 0.05 compared with OA group (n = 6 per group). Error bars represent Standard Error of Mean.


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