Ultrasound-Excited Protoporphyrin IX-Modified Multifunctional

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Biological and Medical Applications of Materials and Interfaces

Ultrasoundexcited Protoporphyrin IX Modified Multifunctional Nanoparticles as a Strong Inhibitors of Tau Phosphorylation and #-Amyloid Aggregation Mengmeng Xu, Hui Zhou, Yanan Liu, Jing Sun, Wenjie Xie, Ping Zhao, and Jie Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08230 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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ACS Applied Materials & Interfaces

Ultrasoundexcited Protoporphyrin IX Modified Multifunctional Nanoparticles as a Strong Inhibitors of Tau Phosphorylation and β-Amyloid Aggregation Mengmeng Xua, †, Hui Zhoua, †, Yanan Liua,*, Jing Suna, Wenjie Xiea, Ping Zhaob and Jie Liua,* a Department of Chemistry, Jinan University, Guangzhou 510632, China b Department of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University,Guangzhou 510006, China * Corresponding authors, E-mail:[email protected]. E-mail:[email protected]. Tel/Fax: +86-20-85220223 † These authors contributed equally to the work. ABSTRACT Alzheimer's disease (AD) has become one of the most serious societal problems globally, with no effective treatments. Parenchymal accumulation of amyloid beta (Aβ) plaques and the formation of neurofibrillary tangles are the hallmarks of AD. Their possible interactions and synergistic effects in AD have been gradually elucidated. The failure of many clinical trials suggests that it is difficult to treat AD with a focus on a single target. Instead, multiple targets may be an important direction for AD drug research. In this study, we used protoporphyrin IX (PX) modified oxidized mesoporous carbon (OMCN) nanospheres (PX@OMCN@PEG(OP)@RVGs) as a novel AD multifunctional nanodrug having multiple targets. The nanodrug efficiently inhibitors of Tau Phosphorylation. In addition, the use of PX with focused ultrasound （US）triggered the production of ROS that significantly inhibited Aβ aggregation. Both approaches notably increased the cognitive level of APP/PS1 transgenic (Tg) mice and ultimately achieved dual-target inhibition of AD. Furthermore, the safe and effective delivery of PX across the blood-brain barrier (BBB) due to modification of the RVG peptide was demonstrated in vivo and in vitro. The favorable photothermal effect of the nanoparticles improved the BBB permeability of PX@OP@RVGs under near-infrared irradiation. The results demonstrated that the novel PX@OP@RVGs multifunctional nanomedicine has a dual target treatment capability for AD and can traverse the BBB, indicating the potential for the effective treatment of alzheimer's

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disease.

Keywords: Protoporphyrin IX; Focused ultrasoun; Amyloid-beta; Phosphorylated Tau; Photothermal effect; Multiple targets.

1. INTRODUCTION Alzheimer's disease (AD) is the most prevalent progressive neurodegenerative disorder and seriously affects human health and quality of life, with no cure.1,2 The two major neuropathological hallmarks of AD are the accumulation of extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles formed by pathological forms of tau protein.3,4 Aβ activates glycogen synthase kinase 3 beta (GSK3β)

via

the

phosphoinositol-3-kinase/protein

kinase

B/GSK3β

(PI3K/AKT/GSK3β) signaling pathways lead to tau phosphorylation in neurons.5 Inhibiting abnormal levels of GSK3β is a hopeful treatment strategy for AD therapy.6 However, the treatment of a single target has not produced good clinical efficacy due to the complex pathogenic mechanism of AD.7,8 Thus, dual-targeted AD drugs that can reduce Aβ plaque deposition and inhibit the hyperphosphorylation of Tau protein mediated by Aβ and GSK3β are an attractive prospect. Sonodynamic therapy (SDT) is a minimally invasive treatment strategy that can be applied in a precisely focused manner, with deep tissue penetration in the brain. Ultrasonic waves pass through the skull and focus on a rice grain-sized region in the thalamus, which minimizes the energy applied to the surrounding non-target tissues.9 The United States Food and Drug Administration (FDA) recently approved focused ultrasound (US) thalamotomy as a treatment for people with essential tremors that have been otherwise ineffectively treated.10 Ultrasound can also control brain function through targeted drug delivery. In this approach, drug-loaded nanoparticles enter the brain after intravenous administration and release the cargo upon US radiation.11 SDT also requires good sonosensitizers as triggers. In these sonosensitizers, protoporphyrin IX (PX) can result in the accumulation of reactive oxygen species (ROS) when used 2


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in the presence of US treatment of tumors. This approach produces a good clinical effect in the local treatment of tumors.12,13 In addition, the ROS generated by focused US stimulation of protoporphyrins can further react with the liposomal membrane to achieve personalized local anesthesia.14 PX also has photosensitizer properties. Photoexcited PX leads to the production of ROS, which strongly suppresses Aβ aggregation and synaptic toxicity.15 Catalytic oxygenation of Aβ might be an valid way to therapy AD.16 More importantly, PX has no obvious toxic and side effects on the liver, kidneys, and blood system and so has value as a therapeutic approach.17,18 Given this background, we explored the use of focused US to stimulate PX. The idea was that the resulting ROS that was produced would inhibit Aβ accumulation and neurotoxicity. Concurrently, PX can also specifically inhibit GSK3β. As a substrate inhibitor of GSK3β, PX can effectively reduce the phosphorylation of Tau protein mediated by Aβ and GSK3β. Thus, the novel strategy is a dual-target treatment of AD. PX cannot cross the blood-brain barrier (BBB) and so cannot be diametrically injected into the brain. In seeking a solution, we considered nanoparticles, which have been used for the target-specific supply of drugs through the BBB.19-23 The mesoporous carbon nanosphere (MCN) has been widely developed as a carrier because of its suitable particle size, good biocompatibility, and high drug loading capacity.24,25 Oxidized mesoporous carbon nanosphere (OMCN) have relatively higher water solubility and biocompatibility than MCNs, as well as an excellent photothermal effect, and can improve the permeability of the BBB of nanoparticles during near-infrared (NIR) irradiation.26,27 However, safe and sustained delivery of nanoparticles across the BBB is a major barrier for successful treatment of AD.28-30 The 29 amino-acid brain-targeting peptide termed RVG, which was derived from rabies virus glycoprotein, can truss up the specific n-acetylcholine receptors that are widely detected in the brain parenchyma cells and BBB. The RVG peptide connects to the core surface via an amido bond and plays a gatekeeper role. The use of RVG peptide can increase the targeting of drugs to a specific site in the brain and allows passage across the BBB.27 Tailoring of a drug with prolonged circulation could enhance the accumulation of 3



the drug in the brain via an enhanced permeability effect and result in improved efficacy. To this end, we synthesized targeting peptide, RVG-modified OMCN containing a PX payload and successfully delivered the nanoparticles across the BBB and internalized the nanoparticles in the brain after intravenous administration. A multinuclear Ru (II) complex can make a imaging agent for real-time tracking drugs transport.31-33 In a previous study we explore the real-time imaging of drug-loaded nanoparticles by substituting PX with a dinuclear Ru(II) complex as an imaging tracking agent (Ru@OP@RVG) with a stable fluorescent property. In this research, we have been used in vivo and in vitro experiments to study the influence of PX@OP@RVG on lowering of the Tau phosphorylated, and on the inhibition of Aβ aggregation in the presence of focused US. We report the therapeutic potential of this novel nano vehicle that displays robust biocompatibility and brain bioavailability. Its excellent photothermal effect provides a new way to improve BBB penetration. The use of PX@OP@RVG can permit dual targeting therapy of AD.

Scheme 1.(A) Schematic illustration of the fabrication of PX@OP@RVGs nanosystem. (B) The NPs cross the BBB by receptor-mediated transcytosis and double targeted treating process in vitro and in vivo. 4


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2. MATERIALS AND METHODS 2.1. Materials All reagents and chemicals were purchased commercially and without further purification if not otherwise mentioned, and Ultrapure Milli-Q water (18.2 MW) was used in all experiments. β-amyloid peptides (Aβ40) and RVG peptides (RVG29 sequence: YTIWMPENPRPGTPCDIFTNSRGKRASNGC) were obtained form GL Biochem Ltd. (Shanghai, China). Pluronic F127 (Mw=12,600,PEO106PPO70PEO106), Phenol and formalin aqueous solution were obtained from Aladdin. Dulbecco modified Eagle’s medium (DMEM) was obtained from Invitrogen Corp. A terminal transferase dUTP nick end labeling (TUNEL) assay kit were purchased from Roche Applied Science (Basel, Switzerland). Lithiumchloride(LiCl), Sodiumazide (NaN3), thioflavine-T(ThT),3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetraz-oliumbromide(M TT),4′,6-diamidino-2-phenylindole (DAPI)were from Sigma (St. Louis, MO).Fetal bovine serum (FBS) and horse serum were obtained from Gibco (Life Technologies AG,Zug,Switzerland). ROS Assay Kit were from Beyotime Institute of Biotechnology, China. Antibodies Tau pS199/pS202,Tau-5, Tau pS396, GSK3β and pS9GSK3β, were purchased from Shanghai BestBio Biotech. Co. Ltd. Shanghai, China. β-Actin antibody were purchased from GenScript (Piscataway, NJ, USA).

2.2. Preparation of MCN and OMCN The MCN was synthesized on the basis of previous article and with appropriate modification. Concisely, the mixture phenol (0.6 g), Aqueous NaOH solution(0.1 5



M,15 mL) and formalin aqueous solution (37 wt%,2.1 mL) were heated to 70 °C for 0.5 h. Then, Pluronic F127 (Mw=12,600,PEO106PPO70PEO106) (0.96 g) dissolved in Ultrapure Milli-Q water(15 mL) and then transferred into the above mixture. After successive stirring for 2 h, Accurate measurement Ultrapure Milli-Q water(50 mL) was added to the above solution. Continuous reaction for 18 h and then collected solution (17.7 mL) was added into an autoclave,use Ultrapure Milli-Q water (56 mL) to diluted, and heated at 130 °C for 24 h. Collected products by centrifugation and washed by Ultrapure Milli-Q water and naturally dried at room temperature. The carbonization was conduct at 700 °C in N2 atmosphere for 3 h and then collect the product put into H2O2 solution, and treated 100 rpm agitation for 12 h. Then, washed with Ultrapure Milli-Q water to remove the residual solution.

2.3. Preparation of OMCN-PEG(OP),PX@OP and PX@OP@RVG Frist,10 mg of NH2-PEG-NH2 was dissolved in Ultrapure Milli-Q water (10 mL),and then 10 mg OMCN were added to the above solution stirred for 2 h under room temperature. The carboxyl group on the surface of the OMCN specifically reacts with the -NH2 of the PEG. Centrifugation and lyophilization, the powder was collected to obtain PEG-modified OMCN-PEG (OP). 5 mg of OP was dispersed in 1 mL of PX solution (2 mg/mL), and stirred at room temperature for 12 hours. and then the RVG peptide (20µL, 5mg/mL) was transferred into the above solution, shaken for 4 hours. Thereafter, centrifuge at 10,000 rpm for 10 minutes and then by Milli-Q water washed three times. Then,we were obtained the PX@OP@RVG NPs.

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2.4. Characterization of the Nanoparticles Transmission electron microscope (TEM) use Hitachi H-7650 to imaged morphology.Raman spectra were carried out by a Labram-1B (Dilor, France). Zeta potential and size distribution by Malvern Nanosizer Nano ZS instrument to measured. Digital photographs of RVG peptide conjugation to OMCN surfaces via amido bonds were obtained by BCA assay kits. Fourier transform-infrared spectra (FT-IR) were conducted on a FT-IR Spectrometer (Nicolet 6700). Using a Philips XL-30 scanning electron microscope (SEM) to obtain micrographs. X-ray diffraction patterns (XRD) and Small-angle X-ray scattering (SAXS) were taken on Nanostar U SAXS system (Bruker, Germany) and D/MAX-RB diffractometer (Rigaku).

2.5. Reactive Oxygen Species (ROS) Level Assessment The generation of ROS in SH-SY5Y cells were taken though an oxidationsensitive fluorescent probe (DCFH-DA), which a commercially available for the detection of ROS. The SH-SY5Y cells were cultured with PBS, OP@RVG and PX@OP@RVG (20 µg/mL) for 4 h in 6-well culture plates, and treating with or without of 3 min focused ultrasound (3 W/cm2, 1 MHz) in SH-SY5Y cells, respectively. The SH-SY5Y cells were incubated for 30 min with 5 µM/L DCFH-DA according the manufacturer’s protocol. and then assessment ROS level by laser scanning confocal microscopy (LSM,Leica TCS SP5, Leica Microsystems, Wetzlar, Germany) the excitation wavelength at 488 nm and the emission wavelength at 535 nm.H2O2 as positive

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control.

2.6. Atomic Force Microscopy (AFM) Aβ was dissolved in hexafluoroisopropanol and in order to further dissolve,the Aβ sample shaken at 4 °C for 2 hour. Then, equilibrate into centrifugetubes, dried Aβ films in vacuo environment for further use.Fresh Aβ were dissolved in PBS(50 mM ) then filtered by a syringe filter(0.22 mm) to filtered the aggregate that has formed. Then the Aβ sample(30 µM) was incubated with OP@RVGs or PX@OP@RVGs in the absence or presence of focused US and in eppendorf tubes(0.2 mL) with PBS (pH 7.4) for 3 days at 37 °C.The inhibition Aβ monomers aggregation were further confirmed by AFM. For the sampling of Aβ was put onto mica substrate then dried in the N2 atmosphere. Three different regions of the surface were examined. The AFM images were acquired by the Nanoscope V software.

2.7. NIR-Mediated Photothermal Analyze In order to investigate the photothermal effect of nanoparticles under 750 nm NIR irradiation. An aqueous suspension of MCN, OMCN and PX@OP@RVGs (20 µg/ml) was placed in an EP tube and irradiated with 750 nm NIR laser (1 W/cm2) for 10 min,respectively. Thermocouple microprobe immersed in suspension to record temperature. The photothermal effects of different concentrations of PX@OP@RVGs were performed at the same power density. Using thermal imaging cameras (Fluke T125) to record different nanoparticles suspensions and PBS infrared thermal images.

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2.8. Ability for Nanoparticles to Cross the BBB in Vitro Use of transwell plate to set up BBB model in vitro. Briefly, the bEnd.3 cells were cultured in the top chambers to form a tight monolayer,based on previous experience and then the SH-SY5Y cells was grown into the bottom chambers for 24h. Measured the transendothelial electric resistance (TEER) of the BBB model were obtained by Millicell ERS (Millipore,USA). Then, ruthenium-labeled NPs were added into the top chambers and coincubated for 12 h. What’s more, by six times NIR irradiation during this period, then observed the cells were using LSM excitation at 488 nm.

2.9. 8-Anilinonaphthalene-1-Sulfonic Acid ( ANS ) Measurement ANS was dissolved in 4 mL of PBS (20 mM). The Aβ sample was diluted into the final concentration. Measured ANS fluorescence use a Cary eclipse fluorescence spectrophotometer (Malcom, Japan) after incubated for 72 h. The excitation wavelength at 350 nm and emission wavelength were set between 400 nm and 600 nm and slit width were recorded at 5 and 10 nm, respectively.

2.10. Thioflavin-T (ThT) Fluorescence Assay The fibrillation of Aβ inhibition experiment were taken by ThT with a spectrofluorometer (Malcom, Japan). Aβ alone or was coincubated with OP@RVG, PX@OP@RVG in PBS buffer and 100 rpm stirred at 37°C for 72 h with or without

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ultrasound. The aggregation Aβ fibers was performed use ThT fluorescence (480 nm) at different time gap (0, 6 12, 18, 36, 48 and 72 h) and recorde the emission. For ROS inhibition Aβ aggregation experiment, the excitation wavelength at 440 nm and emission wavelength was between 460 nm and 600 nm. The amyloid aggregation curve were recorded using ThT emission peak measured at 488 nm with time (h).

2.11. Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) and 4',6-Diamidino-2-Phenylindole (DAPI）Assay SH-SY5Y cells was grown into 6-well and cocultivated with the sample of Aβ. In brief, Frist Aβ coincubated with different nanoparticles (PX@OP@RVGs or OP@RVGs) at 37°C with or without ultrasound for 72 h, and then incubated with SH-SY5Y cells ， which were fixed by 4.0% paraformaldehyde,what’s more, permeabilized using Triton X-100(0.1%) in PBS. Afterwards, SH-SY5Y cells was cultured with the reaction mixture solution of TUNEL about 1 h, then using DAPI to stained the nucleus. Samples was washed with PBS.The images of SH-SY5Y cells were obtained by fluorescence microscope.

2.12. Mitochondrial Membrane Potential (MMP) Assay MMP were detected by means of JC-1 mitochondrial membrane potential assay kit (Beyotime) according the manufacturer’s synopsis. SH-SY5Y cells were seeded in 6-well plate, Aβ incubated with different OP@RVG,PX@OP@RVG with or without ultrasound for 72 h, and then incubated with SH-SY5Y cells. Subsequently, collect

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SH-SY5Y cells and re-suspended in cell culture media(0.5 ml), next add the staining solution JC-1(0.5 ml). SH-SY5Y cells were centrifuged for 4 min at 25°C by high speed centrifuge, after incubation at 37°C for 30 min, remove the suspension, and then washed twice by staining buffer. The fluorescence intensity were conducted

by

laser scanning confocal microscopy excited at 525 nm, emission at 590 nm (red) and 490 nm excitation wavelength, 530 nm emission wavelength(green).

2.13. Western Blot Analysis SH-SY5Y cells was grown into each well of 6-well plates and incubated with Aβ fibrils for 72 h, then incubated with LiCl, OP@RVG and PX@OP@RVG for 48 h. Extract the protein by lysed with phosphate protein extraction buffer (115 mM NaCl,20 mM Na4P2O7, pH 7.4 ) containing PMSF and a protease inhibitor cocktail(1 mM),

then

were

separated

by

Sodium

Dodecyl

Sulfate

PolyAcrylamide Gel Electrophoresis (SDS-PAGE) frist at 80 V for half an hour then at 130 V for 2 h using Mes-SDS running buffer (Invitrogen) and Bis-Tris gels(12% ). Gels were then transferred to nitrocellulose membrane, incubated in a blocking solution containing 5% milk proteins in TBST for 1h at room temperature.The membranes hybridized with primary antibodies against Tau pS396,Tau-5 (total tau), Tau pS199/pS202, GSK3β pS9, GSK3β (total GSK3β),or β-actin overnight at 4°C.Anti-horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, 1:12,000) were used and bands were then visualized by ECL according to the manufacturers' instructions (Luminata Crescendo; Millipore).

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2.14. Uptake of Nanodrugs by SH-SY5YCells SH-SY5Y cells was grown into a laser confocal 35 mm2 Petri dish at 37 °C for 48 h,afterwards were incubated with Ru@OP, Ru@OP@RVG and Ru@OP@RVG (20 µg/mL) for 10 h. Subsequently, Use PBS washed the SH-SY5Y cells for three times and fixed with paraformaldehyde(4.0%). The resulting samples was calendar using LSM excitation at 488 nm.

2.15. In Vivo Distribution and Imaging. The transport of ruthenium-labeled Ru@OP or Ru@OP@RVG cross over the BBB was inquire into by a realtime imaging system (Maestro). Injection Ru@OP or Ru@OP@RVG (1mg/kg) into mice’s by tail intravenous. Subsequently, the mice were placed in the chamber to imaging at set time points. for the other group, at 24 h postinjection, the mice were sacrificed and then collecte the organs for fluorescent imaging. Afterwards, the results were get (emission wavelength at 620 nm,excitation wavelength at 488 nm). In addition, Histological tissue preparation and immunocytochemistry brains were dissected into forebrain, hindbrain and cerebellum before processing by paraffin embedding according to previous literature report. Sections were analysed using immunohistochemistry by the phospho-specific antibody AT180.Nude and APP/PS1 Tg mice were obtained from Guangdong Medical Laboratory Animal Center.

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2.16. Morris Water Maze (MWM) Test The MWM test is a classic method for evaluating spatial learning ability and widely used.Thus, trained and then tested the mice by MWM to investigate the NPs effect on rescued memory deficits. Briefly, the MWM setting was composed of a circular pool (diameter, 150 cm;height, 50 cm ) with a 9 cm platform 1 cm below the surface of the water in the middle of a quadrant and in sometime milk was added to the circular pool water to conceal the platform. While making it easier for mice to be tracked. Put the mice in the circular pool and freely swim until found the conceal platform. Training experiment lasted 7 d.The positions around the edge of the circular pool maze have different sequences on every day. The deadline time is 60 s for the latency to found the platform. Guided the mice to the platform and stay on the platform for 30 s,if the mice do not found the platform in the stipulated time (60 s). Using a tracking system to recorded the swimming path and escape latencies. Probe trails were taken on the 6 day, removed the platform, and the mice were ordered to be put into water from not the same points away from the platform.Spatial learning ability is the percentage of time that the mice spent in the quadrants of escape platform.

2.17. Nest Construction Performing impaired function and decreased daily live activities (DLA) are common symptoms of Alzheimer's disease. Therefore using nest construction test measured PX@OP@RVGs with ultrasoundat improved APP/PS1 Tg mice cognitive

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decline is necessary. The mice were kept alone for a week with corn cob supplies,before the nesting test.For the first day, 5 pieces of paper(5×5 cm2) were used in the cage was implemented the nesting test. Respectively, in 0 day, 2 day, 4 day according to the following grading standards for scoring and statistical analysis: 1 points, did not touch the papers; 2 points, There is no obvious bit/tear, and have an identifiable nest site; 3 points, There is partial bit/tear of the paper, and have an identifiable nest address; 4 points,There is the most bit/tear of the paper, and have an identifiable nest site.

2.18. Animal Studies and Treatment Regimen Nude mice for in vivo imaging investigation. APP/PS1 Tg mice for MWM and Nest Test. In brief, Twenty-five, 30 weeks age APP/PS1 Tg mice, and stochastic divided into 5 groups then tail vein injection OP@RVG or PX@OP@RVG (5.0 mg/kg) with or without of 3 min focused ultrasound and the same volume of saline (5 mice in each group). Experimental Animal Center of Jinan University SPF level of feeding. All animal experiments were conducted out in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (Document NO. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of Jinan University.

2.19. Statistical Analysis The data were express through three independent experiments, the statistical

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analyses were performed by Student’s test. Statistical significance were showed by *p < 0.05, **p < 0.01 and ***p < 0.001 respectively.

3. RESULTS AND DISCUSSION 3.1. Preparation and Identification of Nanoparticles The nanodrugs composite process were illustrated in Scheme 1A. Frist, the MCNs were synthesized by hydrothermal method have some modification in previous reports.24,25 Synthesized MCNs were spherical with a diameter of 89±2 nm (Figure S1). To synthesize OMCNs, MCNs was treated with hydrogen peroxide (H2O2) to introduce abundant carboxylic groups on the MCN surface.SEM and high-resolution transmission electron microscopy (HRTEM) images revealed the highly monodisperse spherical morphology of the OMCN with a particle size is 90 nm (Figure 1A(a) and inset). TEM also indicated the spherical morphology of OMCN (Figure 1A(b)) with consistent particle size revealed by dynamic light scattering (DLS; inset in Figure 1A(b)). What’s more, DLS also showed that the particle had sizes distributed over a narrow range about 90 nm with and the highest occurrences number precent about 35%. To endow OMCNs with a combined drug multifunctional therapeutic function, PX was loaded and then polyethylene glycol (PEG) was conjugated to OMCN surfaces via amido bonds to constitute PX@OMCN-PEG composite (PX@OP). The RVG peptide was then also connected to the surface of OMCN-PEG(OP) via amide bond formation to generate PX@OP@RVGs. After OMCN conjugation, the PX@OP@RVGs also displayed a nanosphere morphology (Figure 1A(c)). TEM also confirmed the successful synthesis of the PX@OP@RVGs composite,which was compared to the HRTEM image, it was found that the mesopores disappeared after the OMCN load PX and linked RVG. Furthermore, the NPs were very monodisperse with a uniform size of approximately 110 nm. Conjugation of the RVG peptide was examined by a bicinchoninic acid (BCA) assay. The BCA working solution changed to purple after mixing with the NPs from colorless, while the noconjugation RVG NPs

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remain the same unchanged (Figure 1B). These observations afford further evidence of the successful synthesis of PX@OP@RVG. The zeta (ζ)-potential analysis revealed the negative charges of the MCNs and OMCNs (-10.8 mV and −37.8 mV, respectively; (Figure 1C). The potential changes demonstrated the successful production of carboxyl groups on the OMCNs. The ζ-potential results revealed a gradual increase from -37.8 mV of OMCNs to -24.9 mV of (OP), which could be attributed to the shielding effect of PEGylation. Modification of the OPs with the positively-charged RVG peptide caused a change in the potential from -24.9 to +6.4 mV. The size change was examined to validate the stability of the PX@OP@RVGs in the biological environment (Figure 1D). When the nanoparticles were dispersed in water, 10% FBS in DMEM, and serum for more than 120 h, there was only marginal change in the size distribution of PX@OP@RVGs, suggesting well stability on biological media. This provided optimism in subsequent in vitro and in vivo experiments. Small-angle Xray scattering (SAXS) pattern of OMCN is exhibit in Fig. 1E, the appearance of wellresolved peaks value is 0.68 nm-1 and 1.29 nm-1 at q, respectively, demonstrated the OMCN possessed ordered Im3m mesostructure. X-ray powder diffraction (XRD) pattern of OMCN is shown that the broad and the typical peaks value of 24.5° and 42.9° at 2q, respectively,all the result indicate the graphitization carbon, instruction OMCN had the graphitic domains (inset in Figure 1E ). The Raman spectrum (Figure. S2) of the OMCN confirmed the graphitic domains in OMCNs on account of have two broad peaks at around 1579 cm-1 (G band ) and 1344 cm-1 (D band ) similar to E2g and A1g modes of graphitic carbon atoms. These consequences further demonstrate that OMCN has successfully synthesized. Further, the appear of characteristic peaks of carboxyl groups and the RVG peptides on PX@OP@RVG by FT-IR spectroscopy (Figure 1F) further confirmed RVG peptides successful conjugation to the surface of OMCN. Nitrogen adsorption/desorption isotherm measurements further demonstrated that OMCNs had a larger pore size than MCNs, indicating the potential for a higher drug load efficiency (Figure 1G).We use UV-Vis analysis to calculate the load rate of OMCN of PX and RVG,After calculation, the load rate is 52.5% and 2.7%, respectively.(Figure S3). 16


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Figure 1. Characterization of the nanoparticles. (A) SEM and HR-TEM (inset) of OMCNs (a); TEM and DLS histogram (inset) of OMCNs (b); TEM image of PX@OP@RVG (c); (B) Use digital photographs to prove RVG peptide successful load of the nanoparticles; (C) Zeta Potential of different nanoparticles; (D) The stability of PX@OP@RVG in 10% FBS,aqueousr, and serum; (E) SAXS and XRD (inset) patterns of OMCN; (F) FT-IR spectra of PX, OMCN, OP, PX@OP@RVG; (G) N2 sorption isotherms and the corresponding BJH pore size distribution curves (inset) of MCN and OMCN.

3.2. In Vitro Cell Biocompatibility Assay. To analyze the selectivity of assimilation to SH-SY5Y cells, we observed the fluorescence of nano drug labeled with the Ru complex (Ru@OP@RVGs) by laser confocal microscopy. The presence of conjugated RVG peptide allowed better penetration of SH-SY5Y cell membranes, which enhances the selective cellular uptake by SH-SY5Y cells compared to other BBB-targeting Pen peptides (Figure 2A). 3-dimensional (3D) cell experiments further determine the distribution of 17



nanoparticles in SH-SY5Y cells (Figure 2B). Quantitative analysis of the fluorescence intensity (Figure 2C) provided support for the results in panel A. Dose-dependent selective uptake of the RVG-conjugated peptide was evident (Figure 2D). By vitro experiments were conducted to define the bioavailability and biosafety influence of the NPs. Biosafety was explored by investigating the cell toxicity of NPs with or without of focused US using the MTT-based viability assay. PX@OP@RVGs (20 µg/mL) were nontoxic against during a 24 h treatment with or without of focused US (Figure 2E-F). Except for the facilitative uptake efficiency, the cellular retention time of nanodrugs is also a crucially importance parameter for effective brain accumulation.34 The retention efficacy of 20 µg/mL Ru@OP@RVGs or Ru was assessed for 8 h in SH-SY5Y cells. As shown in Figure S4, Ru@OP@RVGs effectively hindered the diffusion of internalized Ru outside the cells during the observation period. The prolonged retention time of Ru@OP@RVGs might be attributed to the connection of the RVG peptide to the core surface via amido bonds, which would effectively block the pore, in turn promoting higher incorporation and cellular retention efficacy of drugs. Overall, these results suggested that Ru@OP@RVGs are an effective treatment due to their facilitated accumulation in SH-SY5Y cells. Considering the inflammatory response mediated by pro-inflammatory cytokines are associated with T cells.31,35,36 Therefore,we detected the level of interleukin (IL)-6 to evaluate whether PX@OP@RVGs applied with focused US could injure peripheral blood mononuclear cells (PBMCs). In contrast to lipopolysaccharide (LPS), which

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activates the inflammatory reaction, an increased IL-6 level was observed after treatment with PX@OP@RVGs in the absence or presence of focused US (Figure 2G). Consistent with this, the serum, IL-1β, and TNF-α levels of mice treated with the nanoparticles treatment of mice mice were similar to the levels in saline-treatment of mice (Figure S5). These results demonstrated that the virus-derived peptide RVG-modified NPs have low immunogenicity and further confirmed their lack of cytotoxicity. Subsequently, we studied in vitro whether erythrocytes treated PX@OP@RVGs in the presence of focused US could cause thrombogenic responses. Measurement of free hemoglobin of the blood exposed to PX@OP@RVGs with or without focused US was done to evaluate the hemolysis ratio. Blood compatibility of the nanodrug was evident (Figure 2H). The results provided evidence that reasonably designed nanodrugs could be used in subsequent in vitro and in vivo experiments to explore their clinical application. what's more, to research the latent side effects of the PX@OP@RVGs, collecte the main organs and been stained with H&E.Compared to the control groups no obvious impairment or inflammation has been discovered after injection for the tested dose range(Figure S6). It also with potential applied as an appropriate nanovehicle in medicine in further, being highly biocompatible and highly biosafety.

19



Figure 2. Cellular uptake and Biocompatibility assay. (A) Cellular uptake of nanoparticles in SH-SY5Y cells(scale bar=50 µm); (B) 3D image of cellular uptake Ru@OP@RVG; (C) Quantitative analysis of fluorescenc intensity of Ru; (D) By ICP-AES method quantitative analysis the concentrations of Ru in SH-SY5Y cells; (E) and (F) by an MTT assay the SH-SY5Y cells viability after incubated with OMCN, OP, PX@OP and PX@OP@RVG with or without focused ultrasound for 24 h; (G) IL-6 level. (H) Hemolysis assay.

3.3. Effect of PX@OP@RVGs on Inhibition Aβ Monomers Aggregation Assay. Memory loss in individuals with AD is related to the gradual change of β-amyloid protein from a soluble monomer to highly-ordered aggregates of protein fibrils (amyloid cascade hypothesis).37-39 We examined the effect of PX@OP@RVGs on 20


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inhibition Aβ monomers aggregation in the presence or absence of focused US (3 W/cm2, 1M Hz) using atomic force microscopy (AFM). Typical Aβ fibrils were seen in Aβ-treated samples after cultivate 3 days, even in the presence of focused US (Figure 3A). This indicated that focused US does not inhibition Aβ monomers aggregation. Under the same conditions, Aβ monomers incubated with OP@RVGs remained observed Aβ fibrils. When incubated with PX@OP@RVGs in the absence of focused US massive Aβ fibrils were clearly apparent. However, after incubation with PX@OP@RVGs in the presence of focused US, fewer amorphous aggregates were observed. These results demonstrated that PX@OP@RVGs could effectively cause the inhibition Aβ monomers aggregation in the presence of focused US. Protein misfolding can expose hydrophobic leftover on the surface can triggers aggregation through hydrophobic interactions. In addition, higher value of surface hydrophobicity can further triggers Aβ aggregation by means hydrophobic interactions. Thus, the surface hydrophobicity is also an influential factor in Aβ monomers aggregation.40 We next studied the surface hydrophobicity using a protein-bound 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence assay. PX@OP@RVGs applied in the presence of 3 min focused US significantly reduced ANS fluorescence compared to pure amyloid and other control groups (Figure 3B), the result confirmed that the decreased value of amyloid surface hydrophobicity, When incubated with OP@RVGs, even under the same conditions, the ANS fluorescence remained very high. This indicated that PX@OP@RVGs can reduce the formation of new hydrophobic regions and effectively prevented Aβ monomers

21



aggregation upon focused US. Thioflavin-T (ThT) has been extensively used to measure Aβ aggregation because it can be acquainted with the β-sheet structures linked to Aβ fibrils. The reduced fluorescence intensity is being related to inhibition Aβ monomers aggregation.41,42 ThT fluorescence was measured to assess the influence of PX@OP@RVGs on the aggregation of Aβ monomers upon 3 min of focused US. Under appropriate conditions，Aβ monomers in PBS (pH 7.4) were incubated at 37 °C and ThT fluorescence was measured at defined times. The higher ThT fluorescence that was observed (Figure 3C) indicated the formation of Aβ monomers aggregates. The focused US did not affect the aggregation of Aβ monomers. When PX@OP@RVGs incubated with Aβ monomers in solution in with 3 min focused US, the ThT fluorescence intensity decreased by approximately 71%. In contrast, the decrease was not observed upon incubation with OP@RVGs with or without 3 min focused US. These results demonstrated that PX@OP@RVGs prevented Aβ monomers aggregation, while OP@RVGs did not,in the presence of 3 min focused US.

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Figure 3. Effect of NPs on inhibition Aβ monomers aggregation. (A) AFM images of Aβ, alone or incubated with OP@RVG, PX@OP@RVG (20 µg/mL) with or without of 3 min focused ultrasound (3 W/cm2 ,1 M Hz); (B) ANS fluorescence changes of Aβ, alone or incubated with OP@RVG, PX@OP@RVG with or without of 3 min focused ultrasound; (C) ThT fluorescence changes of Aβ, alone or incubated with OP@RVG, PX@OP@RVG with or without of 3 min focused ultrasound (scale bars=1µm).

3.4. In Vivo and in Vitro Experiments Demonstrate that ROS Hindrance Aβ Aggregation Certain concentrations of ROS may positively influence intracellular signaling mechanisms and could regulate many cellular processes.30,43 We explored the idea that ROS-mediated damage of Aβ peptides can occur by focused US excitation of PX@OP@RVGs. A DCFH-DA fluorescence research were taken to evaluate the effect of ROS accumulation in SH-SY5Y cells. In this experiment H2O2 treated samples served as a positive control. By LSM images can obviously observed the endocellular ROS displayed baseline values when SH-SY5Y cells were incubated with OP@RVGs, even in the presence of focused US (Figure 4A). In contrast, a 23



certain increase in the induction of the manufacture of ROS was evident in the presence of PX@OP@RVGs upon focused US. Quantitative analysis of ROS levels provided a more intuitive explanation (Figure 4B). To further support our conjecture, we study the influence of different nanoparticles on Aβ monomers aggregation by measuring ThT fluorescence. The Aβ samples were diluted to a appropriate concentration and incubated with ThT with or without focused US for 20 min before fluorescence analysis. Importantly, during this period, used an Argon-purged buffer including 2 mM NaN3 as a singlet oxygen quencher in the anaerobic experiment. The ThT emission peak at 488 nm was recorded and an Aβ growth curve was plotted. The circular dichroism (CD) spectroscopy clearly demonstrated that oxygenation prevented the conformational transition of Aβ from a random coil to a β-sheet by PX@OP@RVGs under focused US (Figure S7 ).Furthermore, the hindrance effect of focused US excited PX@OP@RVGs in the aerobic condition markedly increased (Figure 4C) compared to the anaerobic condition (Figure 4D). The results indicate that ROS generation contributes to the inhibitory effect of focused US excited PX@OP@RVGs on Aβ aggregation. The finding was meaningful considering that Aβ aggregation in the brain is a crucial factor to AD pathologies features.In order to further determine the influence of nanodrugs on the accumulation effects of Aβ in APP/PS1 Tg mice, Aβ deposition immunostaining was performed. Before collecting the brain, PX@OP@RVGs or OP@RVGs were intravenously injected into 11-month-old, large APP/PS1 mice. Some mice also received focused US during this period. After 1 months, Aβ immunostaining was obvious in the hippocampus and the

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region of Aβ accumulation was evidented round the dentate gyrus (DG) of the mice (Figure 4E). When treated with OP@RVG in the absence or presence of focused US, no significant alteration in Aβ deposition was observed. The consequence illustrated that the nanodrugs could not influence the accumulation of Aβ. However, after treated APP/PS1Tg mice of PX@OP@RVGs in the presence of focused US, the decreased number of Aβ was clearly evident in DG region of APP/PS1Tg mice. These data clearly demonstrated that the PX@OP@RVG in the presence of focused US can enhanced decrease the accumulation of Aβ in vitro experiments.

25



Figure 4. The impact of ROS on the structural of Aβ fibrils. (A) Evaluated ROS production by DCFH-DA in intracellular after treatment with alone or treated with OP@RVG, PX@OP@RVG (20 µg/mL) with or without of 3 min focused ultrasound (3 W/cm2, 1 M Hz) , H2O2 was used as positive control; (B) Quantitative analysis of ROS levels; (C) and (D) Thioflavin-T fluorescence assay of Aβ aggregation formed under aerobic (C) and anaerobic (D) conditions with 3 min focused ultrasound (3 W/cm2, 1 M Hz). scale bar=50 µm; (E) PX@OP@RVGs damaged the aggregation of Aβ with focused ultrasound in the brains of APP/PS1Tg mice; scale bar = 200 µm.

3.5. Effects of Nanodrugs on Apoptosis of SH-SY5Y Cells 26


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When a cell undergoes apoptosis, a number of DNA endonucleases will be activated that cut inter-nucleosome genomic DNA and cause DNA fragmentation and cause cell apoptosis. Therefore, we study Aβ-mediated apoptosis in a via TUNEL experiment, using in vitro DAPI (blue) and TUNEL (green) enzymatic labeling co-staining experiment. After SH-SY5Y cells incubated with along Aβ showed significant

green

fluorescence

(Figure

5A),

implying

strengthened

DNA

fragmentation and widespread apoptosis in SH-SY5Y cells. The green fluorescence decreased gradually,in the presence of 3 min focused US with PX@OP@RVGs, confirming decreased Aβ-mediated cellular toxicity. However, treatment with PX@OP@RVGs in the absence of focused US did not produce significant effects. Treatment with OP@RVGs produced similar results, even in the presence of focused US, demonstrating that PX@OP@RVGs in the presence of focused US could effectively suppression DNA fragmentation and weaken Aβ-mediated apoptosis. The changes of MMP is also an significant indicator of apoptosis.44,45 We measured the MMP in SH-SY5Y cells using the JC-1 fluorescence probe. Control cells emitted strong red fluorescence, compared to the control group of cells incubated with Aβ , which displayed distinct green fluorescence (monomeric form of JC-1, for abnormal dissipation of MMP) in the absence or presence of focused US (Figure 5B). The results implied that Aβ fibrils can induce mitochondrial-mediated apoptosis in SH-SY5Y cells. After incubation with PX@OP@RVG in the presence of focused US, the green fluorescence decreased gradually, while the red fluorescence (aggregate form of JC-1, for normal intact MMP) increased. However, under similar conditions,

27



only slight changes were observed in the absence of focused US. Treatment with OP@RVGs was ineffective, even in the presence of focused US. Thus, PX@OP@RVG upon focused US could effectively inhibition Aβ monomers aggregation into toxic forms in SH-SY5Y cells.

Figure 5. In vitro cell viability assay. (A) The effect of OP@RVG, and PX@OP@RVG (20 µg/mL) in the presence or absence of focused ultrasound on cell apoptosis induced by Aβ aggregation; scale bar=25 µm; (B) SH-SY5Y cells were 28


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stained by JC-1 to study the MMP under OP@RVG, and PX@OP@RVG (20 µg/mL) in the presence or absence of focused ultrasound.scale bar = 50 µm.

3.6. The Photothermal of NPs Influences BBB Permeability in Vivo or Vitro and the Localization in Rat Brain The transit of nanodrugs across the BBB is extremely challenging. The efficient passage of nanodrugs across the BBB of nanodrugs is crucial for the treatment of brain diseases.46-49 To verify that nanodrugs have the potential of enhancing the permeability of the BBB by the photothermal effect, PX@OP@RVGs and the control group were exposed to NIR irradiation at 808 nm (1 W/cm2). The photothermal effect of nanodrugs was research by thermal imaging. After a 10-min irradiation, an excellent photothermal effect was evident for PX@OP@RVGs compared to the control group (Figure 6A). The temperature of the PX@OP@RVGs solution increased markedly under laser irradiation, and the temperature increase was dose-dependent (Figure 6B). Next, the photothermal effect of MCN, OMCN, and PX@OP@RVGs was investigated. After a 10-min NIR irradiation, the temperature of the MCN and OMCN solutions was increased by 41°C and 43°C, respectively, while the temperature of the PX@OP@RVGs solution was increased by 44.5°C (Figure 6C). These results indicated that peptide RVG improved the photothermal effect of OMCN. Thermal imaging substantiated these findings (Figure 6D).

29



Figure 6. (A) Thermal images showing the temperature of PBS, MCN, OMCN and PX@OP@RVG solutions during NIR irradiation; (B) Thermal images showing the temperature of different concentration of PX@OP@RVG solutions and ultrapure water during NIR irradiation; (C) The temperature curve of the heating process for PBS, MCN, OMCN or PX@OP@RVG; (D) The rate of temperature rise of PX@OP@RVG in various concentration under 808 nm laser irradiated for 10 min. The feasibility of the photothermal activity of NPs to improve the BBB permeability was studied by Transwell experiments. First, Ru-labeled NPs were incubated in a Transwell plate containing a cell monolayer of bEnd3 cells. SH-SY5Y cells were placed into the lower chamber as a model to evaluate BBB permeability in vitro to research the influence of brain targeting peptide RVGs and NIR on the permeability of BBB by inductively coupled plasma atomic emission spectroscopy (Figure 7A). Quantitative study of Ru concentration changes in the upper chamber enabled the exploration of the BBB permeability efficiency of Ru-labeled NPs. Compared with the control group, the brain targeting peptide RVGs greatly increased 30


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the permeability efficiency of nanodrugs (Figure 7B). Under NIR irradiation, the penetration efficiency of nanodrugs further increased, and the permeability efficiency reached 42.4% after 15 h. A slight fluorescence signal was observed by confocal microscopy after coincubation with Ru@OP (Figure 7C), indicating that Ru@OP was rarely transmitted through single-layer bEnd3 cells. However, in the same period, significantly greater fluorescence signal could be observed when attached to peptide RVG, meaning that the Ru@OP@RVG possessed the ability to the BBB. Moreover, the Ru-fluorescence signal was significantly increased, especially after the irradiation of the heads of the mice by a 808 nm laser. The fluorescence distribution and the thermal imaging of the brain provided further proof of the above results (Figure 7D). Furthermore, considering the aggregation of Aβ was obvious around the rat hippocampus and parietal cortex region is the pathological features of alzheimer disease.50 We next used confocal microscopy to observe the fluorescence distribution of Ru-labeled NPs and DAPI, to investigate the cellular localization of nanodrugs in the rat brain and to explore whether the photothermal effect could further improve the BBB permeability of NPs. Eight hours following intravenous administration, brains of mice were collected and brain sections were prepared and processed as previously described. Ru fluorescence was weak in the rat hippocampus and parietal cortex after the administration of Ru@OP (Figure 7E). In contrast, The fluorescence of Ru have a significant increase was detected in same areas in the rats administered Ru@OP@RVGs. Ru fluorescence was mainly located in the cytosol, with a perinuclear distribution pattern discerned in high magnification images. Importantly,

31



when under NIR light, the Ru fluorescence further increased in the same region. Taken together, these results reveal the important role of RVG peptide and excellent photothermal effect on the BBB permeability of NPs. In particular, the photothermal effect of the nanodrugs further increased the permeability. The findings implicate Ru@OP@RVGs could be an appropriate candidate for AD treatment. In addition, Ru@OP@RVGs enhanced the visualization of the distribution of nanodrugs in the brain, indicating the ability to precisely deliver drug combinations.

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Figure 7. The effect of NIR irradiation across the BBB in vitro and in vivo . (A) The 33



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cartoon illustrates the ability for Ru@OP@RVG with or without NIR irradiation across the BBB in vitro; (B) Transfer of Ru@OP, Ru@OP@RVG or Ru@OP@RVG under NIR irradiation in SH-SY5Y cells on the BBB model were taken by a flow cytometer; (C) The transport efficiency of Ru@OP, Ru@OP@RVG or Ru@OP@RVG (20 µg/mL) under NIR irradiation cross BBB. The results were obtain by LSM; (D) The in vivo photothermal effect of Ru@OP, Ru@OP@RVG or Ru@OP@RVG under 808 nm laser irradiation and the fluorescence signals and thermal images were investigated after the NPs injection for 8 h; (E) In vivo localization of NPs after intravenous in the normal rat brain. scale bar=25 µm.

3.7. Nanodrugs Inhibits GSK 3β or Aβ Mediated Tau Hyperphosphorylation Assay In AD, abnormal phosphorylation of tau attenuates its binding to microtubules in neurons, leading to microtubule destruction and neuronal dysfunction.51,52 To demonstrate if PX@OP@RVGs can efficiently lower the ratio of phosphorylated Tau to total Tau, Next,we examine the expression of AD closely related to tau and GSK3β specific phosphorylation sites pS199/pS396 and pS202 were taken western blot analysis. GSK3β overactivity resulting from the introduction of exogenous GSK3β (Figure 8A) obviously up-regulated tau phosphorylation at pS199/pS396 and pS202 sites about 2.5-fold (Figure 8B) compared to the basal phosphorylation levels. On the contrary, treatment with PX@OP@RVGs or LiCl significantly down-regulated tau phosphorylation levels at the specific sites mediated by GSK3β. The results indicated that PX@OP@RVGs inhibit GSK3β-mediated phosphorylation of tau protein, as does LiCl, revealing a promising application for AD treatment. LiCl is a specific inhibitor of GSK3β that is being evaluated in Phase II clinical trials. Furthermore, more and more

evidence

demonstrate

that

Aβ

activates

GSK3β

via

the

phosphoinositol-3-kinase/protein kinase B/GSK3β (PI3K/AKT/GSK3β) signaling pathways in alzheimer disease, and induces downstream tau hyperphosphorylation in 34


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neurons.53-55 Therefore,Our group investigated the impact of PX@OP@RVGs on tau hyperphosphorylation associated with AD pathology in a cell model. SH-SY5Y cells were induced by Aβ (15 µM) for 72 h(Figure 8C). During that time, the level of tau phosphorylation was up-regulated at the pS199/pS396 and pS202 sites by about 3.3-and 2.5 fold, respectively, compared to control group cells (Figure 8D). After treatment of SH-SY5Y cells with PX@OP@RVGs, tau phosphorylation at the same phosphorylation sites was significantly down-regulated. More importantly, the anti-tau phosphorylation ability of PX@OP@RVGs was comparable to that of LiCl at the same concentration.

Figure 8. PX@OP@RVG inhibition tau phosphorylation assay. (A) and (C)PX@OP@RVG attenuates GSK3β-mediated or Aβ-induced tau phosphorylation in 35



a SH-SY5Y by western blot analysis. (B)and(D) Quantitative analysis of associated protein expression. **p < 0.01, and ***p < 0.001, relative to control. Although in vitro analysis confirmed the potential of PX@OP@RVGs anti-tau phosphorylation, the demonstration of in vivo efficacy was still necessary. To characterize the GSK3β-mediated Tau hyperphosphorylation pattern in the lateral amygdala and hippocampus in a mice model, we make use of the tau phosphorylation-specific antibody AT180 to further study. One day after the intravenous injection of PX@OP@RVGs or OP@RVGs in AD mice, AT180 staining was obviously visible compared to the sham mice. As expected,treatment with PX@OP@RVG slightly decreased the level of AT180 immunoreactivity at the site of the lateral amygdala and hippocampus in AD mice 7 days. However, under the same conditions of injection of OP@RVGs, AT180-positive staining at the lateral Amygdala and Hippocampus was still clearly visible. The results supported the in vitro observations and confirmed the inhibition of tau hyperphosphorylation through GSK3β or Aβ by PX@OP@RVG.

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Figure 9. Nano-drug(Nanoparticles) effect in mouse models of AD. Representative images of AT180 immunoreactivty of Hippocampus and Amygdala, respectively collected from AD mouse brain after different treatments for 1 days and 7 days by intravenously injected. After treatments PX@OP@RVG for 7 days relatively little tau phosphorylation visible in Hippocampus and Amygdala. scale bar = 50 µm.

3.8. Rescue of Memory Deficits in AD Model Mice. AD is a type of dementia that causes memory loss and cognitive dysfunction. Therefore, we examined the nanodrugs associated with memory deficit mice via the morris water maze (MWM). APP/PS1 Tg mice display neuropsychiatric symptoms featuring unequivocal learning deficits and cognitive dysfunction due to Aβ deposition in the region of hippocampus from 11 to 17 months of age ,were used as the animal models.56 Therefore, treatment of APP/PS1 Tg mice with saline,

37



OP@RVGs, or PX@OP@RVGs in the absence or presence of focused US adjuvant treatment began at 11 months of age. Typical swimming path of APP/PS1 Tg mice after treatment with different nanoparticles 2 weeks are presented in Figure 10A(12 for each group). After injection with saline or OP@RVGs, the swimming trails of APP/PS1 Tg mice remained untargeted even with focused US assisted therapy, and exhibited the unequivocal learning deficits. In contrast, for APP/PS1 Tg mice treated with PX@OP@RVGs and focused US, the number of swimming tracks that crossed the target quadrant platform was no different from normal mice, clearly indicating improved learning ability. In the absence of focused US, a certain increase in spatial memory was evident. Figure 10 B-C show that the PX@OP@RVGs-treated APP/PS1 Tg mice who additionally received focused US displayed progressively shorter escape latency and exhibited favouritism toward the target quadrant, therefore, all the results clearly confirm memory have significant improvement compared to the other group. The above results were also convinced by nest construction experiments, and obtain similar results (Figure 10 D-E). Particularly, memory retention in APP/PS1 Tg mice express by the PX@OP@RVG with focused US treated was higher than that associated with PX@OP@RVGs alone. The results indicate that inhibition Aβ accumulation and p-tau associated fibrils block of the progression of AD and improved abilities of learning and memory.

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Figure 10. PX@OP@RVGs improved the cognitive function of APP/PS1 Tg mice by focused ultrasound. Morris water maze test (A-C). (A) Representative swimming path; (B) Escape latency; (C) The percentage time in the targeted quadrant; (D and E)The nest behavior test, Representative images were taken from 0 to 4 days. The data represent the mean ± S.E. *, p < 0.1; **, p < 0.01; ***, p < 0.001 compared to the sham mice.###, p < 0.001 with respect to the APP/PS1 mice.

3.9. Real-Time Monitoring of Nanoparticles Distribution In Vivo. We expected that the peptide RVG modified nanodrugs would solve the problem 39



of shorter circulation of the drug and also enhance the brain-targeting delivery efficiency. To verify these expectations, the fluorescent of Ru-labeled NPs was observed and distribution at the brain site to further verify the transport efficiency and biodistribution of Ru@OP@RVGs in vivo. After intravenous administration of Ru@OP@RVGs, the mice were imaged at various times. The fluorescent intensity of Ru@OP@RVG and Ru@OP increased gradually in the brain from 2 to 12 h after injection (Figure 11A). The fluorescence of Ru@OP@RVG in the brain was distinctly intensify in contrast with Ru@OP at various times, with a peak in the fluorescence signal at 10 h. The results indicated that the nanodrugs had better drug circulation and retention efficiency in vivo. Exvivo imaging revealed considerable accumulation of Ru@OP@RVGs in the brain. In contrast, for the group treated with Ru@OPs without peptide RVG, the fluorescence signals were insignificant in the brain. The brain showed the highest-level fluorescence signals among the dissected organs (Figure 11B-C). The collective results demonstrate that RVG peptide can mediate active targeting to the brain, promoting the movement across the BBB and brain infiltration, as well as circulation efficiency in vivo.

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Figure 11. Biodistribution of Ru@OP and Ru@OP@RVG after tail vein . (A) In vivo imaging of mice from 0 to 72 h; (B) Fluorescence signals discover in the kidneys, hearts, spleens, livers, lungs and brains 8 h, 10 h and 12 h after the NPs injection; (C) and (D) Quantitative analysis for the fluorescence in vivo and organ site. The difference with control was showed by *p < 0.05, **p < 0.01 and ***p < 0.001, respectively. 4. CONCLUSIONS In conclusion, we successfully developed an focused US-triggered nanosystem (PX@OP@RVGs) that inhibits Aβ aggregation and attenuates cytotoxicity through a 41



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simple acoustic stimulation. Experimental studies demonstrated that the efficacy of focused US-driven anti-Aβ aggregation was attributed to PX ultrasonic oxidatio in vitro and in vivon. Furthermore, NPs carrying the potentially therapeutic PX can reduce the ratio of phosphorylated Tau to total Tau mediated by Aβ or GSK3β. More importantly, the amide linkage of RVG peptides can virtually increase the transport of nanoparticles into the brain and increase the efficiency of intracellular retention, overcoming major limitations in the treatment of brain diseases. At the same time, the excellent photothermal performance of the NPs further increases the capacity to traverse the BBB during NIR irradiation. The findings open up new avenues for the development of nanomedicines that efficiently infiltrate the BBB. These novel, multi-functional, dual-targeted PX@OP@RVGs are potentially therapeutic for AD under focused US stimulation. Further studies are needed before clinical applications can be contemplated.

ASSOCIATED CONTENT Supporting Information Available: Structures of MCN, Raman spectrum of OMCN,quantitative

analysis

of

the

mean

cellular

retention

of

Ru

and

Ru@OP@RVGs inSH-SY5Ycells, IL-1β and TNF-a level, saline as positive control, The major organs H&E-stained slice images. Aβ fibrils structure analysis by CD spectra.The standard curve of PX (A) and RVG (B).

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ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21877051, 81803027，21371075), the Natural Science Foundation of Guangdong Province (2014A030311025，2018A030310628), and the Planned Item of Science and Technology of Guangdong Province (2016A020217011).

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Ultrasound-Excited Protoporphyrin IX-Modified Multifunctional

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