Penetratin Peptide-Functionalized Gold Nanostars: Enhanced BBB

Jul 14, 2016 - The structural changes of amyloid-beta (Aβ) from nontoxic monomers into neurotoxic aggregates are implicated with pathogenesis of Alzh...
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Penetratin Peptide-Functionalized Gold Nanostars: Enhanced BBB Permeability and NIR Photothermal Treatment of Alzheimer’s Disease Using Ultralow Irradiance Tiantian Yin,⊥ Wenjie Xie,⊥ Jing Sun, Licong Yang, and Jie Liu* Department of Chemistry, Jinan University, Guangzhou 510632, China S Supporting Information *

ABSTRACT: The structural changes of amyloid-beta (Aβ) from nontoxic monomers into neurotoxic aggregates are implicated with pathogenesis of Alzheimer’s disease (AD). Over the past decades, weak disaggregation ability and low permeability to the blood−brain barrier (BBB) may be the main obstacles for major Aβ aggregation blockers. Here, we synthesized penetratin (Pen) peptide loaded poly(ethylene glycol) (PEG)-stabilized gold nanostars (AuNS) modified with ruthenium complex (Ru@Pen@ PEG-AuNS), and Ru(II) complex as luminescent probes for tracking drug delivery. We revealed that Ru@Pen@PEG-AuNS could obviously inhibit the formation of Aβ fibrils as well as dissociate preformed fibrous Aβ under the irradiation of near-infrared (NIR) due to the NIR absorption characteristic of AuNS. More importantly, this novel design could be applied in medicine as an appropriate nanovehicle, being highly biocompatible and hemocompatible. In addition, Ru@Pen@PEG-AuNS had excellent neuroprotective effect on the Aβ-induced cellular toxicity by applying NIR irradiation. Meanwhile, Pen peptide could effectively improve the delivery of nanoparticles to the brain in vitro and in vivo, which overcame the major limitation of Aβ aggregation blockers. These consequences illustrated that the enhanced BBB permeability and efficient photothermolysis of Ru@Pen@PEGAuNS are promising agents in AD therapy. KEYWORDS: Alzheimer’s disease, amyloid-beta, gold nanostars, blood−brain barrier, penetratin peptide

1. INTRODUCTION

these photothermal agents, different types of gold nanoparticles (AuNP) as a kind of Aβ aggregation inhibitors exhibit facile synthesis, biocompatibility, and strong visibility to near-infrared (NIR) absorbance owing to the surface plasmon resonance (SPR) effect, making them powerful agents in biomedical applications.12 Gold nanostars (AuNS), which feature a high NIR absorption-scattering ratio (700−1300 nm) and hornlike structures favorably for producing heat, have potential for photothermal AD treatment.13 Meanwhile, AuNS possess significantly higher loading capacity due to their large specific surface area.14 With this background, we synthesized AuNS, which were then functionalized with a hydrophilic polymer, poly(ethylene glycol) (PEG), to acquire physiological stability,

Alzheimer’s disease (AD), the most common form of dementia, is now affecting over 36 million people worldwide.1 This neurodegenerative disease, clinically characterized by cognitive and memory impairment, can be identified by remarkable pathological hallmarks such as the accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles.2,3 Recent studies have demonstrated that the aggregation of amyloid-beta (Aβ) in brain is believed to be a crucial factor to AD pathologies.4−6 Therefore, considerable researches have been conducted to develop a new therapeutic strategy to inhibit the aggregation process of Aβ and dissociate the preformed Aβ fibrils in Alzheimer’s disease.7−9 Photothermal therapy (PTT), which utilizes light-induced heating to destabilize the preformed Aβ fibrils, has given rise to extensive attention recently as a minimally invasive strategy without damaging surrounding healthy tissues.10,11 Among © 2016 American Chemical Society

Received: April 28, 2016 Accepted: July 14, 2016 Published: July 14, 2016 19291

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

Research Article

ACS Applied Materials & Interfaces

2.3. Photothermal Effect Investigation. Different concentrations of AuNS solutions in quartz cuvette were irradiated by an 808 nm NIR laser at a power density of 0.75 W cm−2 for 10 min. The temperature changes were recorded by a thermocouple (Tes Instrument, China), and PBS was a control. 2.4. Measurement of Soluble Aβ. Aβ was coincubated with PEG-AuNS (20 μg/mL−1), Ru@Pen@PEG-AuNS (10 μg/mL−1), and Ru@Pen@PEG-AuNS (20 μg/mL−1), respectively, in PBS buffer with 100 rpm of agitation at 37 °C for 72 h. The concentration of nonaggregated Aβ was determined with bicinchoninic acid assay (BCA) protein assay after centrifugation at 20 000 rpm for 20 min. 2.5. Turbidity Assay. PEG-AuNS (20 μg/mL−1), Ru@Pen@PEGAuNS (10 μg/mL−1), and Ru@Pen@PEG-AuNS (20 μg/mL−1) were added to Aβ sample solutions, respectively. After 72 h, samples were then mixed with a buffer (900 μL, 20 mM Tris−HCl, 150 mM NaCl, pH = 7.4). The turbidity of the solutions was measured by the UV−vis absorbance at 405 nm. Aβ sample alone was used as controls. 2.6. ThT Fluorescence Assessment. For inhibition experiment, Aβ was coincubated with NPs for 72 h in PBS buffer with 100 rpm of agitation. Samples (20 μL) were withdrawn at different time points, and 180 μL of 10 μM ThT buffer solution was added to the well. ThT fluorescence was recorded at 480 nm with a spectrofluorometer (Malcom, Japan). For disaggregation experiment, preformed Aβ fibrils were treated with PEG-AuNS (20 μg/mL−1), Ru@Pen@PEG-AuNS (20 μg/mL−1) without NIR irradiation and with 3 min of NIR irradiation after preincubated with Ru@Pen@PEG-AuNS(20 μg/ mL−1) for 30 min to maximize the targeting effect. Then, Aβ solutions were taken out at predetermined time points for fluorescence assay. 2.7. Transmission Electron Microscopy. Aβ solutions were incubated for 72 h to achieve abundant Aβ fibrils. For TEM measurements, the preformed Aβ fibrils coincubated with PEG-AuNS (20 μg/mL−1), Ru@Pen@PEG-AuNS(20 μg/mL−1) without NIR irradiation and with 3 min of NIR irradiation after preincubated with Ru@Pen@PEG-AuNS(20 μg/mL−1) for 30 min, then further incubated for 24 h. The sample solutions (10 μL) were spotted on a carbon-coated copper grid, dried for 10 min, and then negatively stained with 2% uranyl acetate. Samples were analyzed by a Hitachi transmission electron microscope. 2.8. Atomic Force Microscopy. The samples were dropped on freshly cleaved mica substrates and dried with N2. The atomic force microscopy (AFM) images were taken by a Nanoscope Multimode scanning probe. Results were examined by three independent regions to confirm uniformity. 2.9. Intracellular Reactive Oxygen Species Level Measurement. The reactive oxygen species (ROS) generation in SH-SY5Y cells was assessed by DCFH-DA probe. The cells were treated with Ru@Pen@PEG-AuNS (20 μg/mL−1) without NIR irradiation and with 3 min of NIR irradiation after cells were preincubated with Ru@ Pen@PEG-AuNS (20 μg/mL−1) for 3 h, and then they were further incubated for 24 h. H2O2-treated sample was used as positive control. Subsequently, the cells were treated with 5 μM DCFH-DA for 30 min, and then intracellular ROS level was monitored qualitatively by fluorescent microscopy (Nikon Eclipse). 2.10. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to monitor cell toxicity of NPs upon NIR irradiation. SH-SY5Y cells were incubated in 96-well plates for 24 h. Ru@Pen@PEG-AuNS were added with or without NIR irradiation, and the cells were further incubated for 24 and 48 h, then treated with MTT (10 mg/mL−1) for 4 h. After the culture media was removed, 150 μL of dimethyl sulfoxide was added to each well. Samples were measured at 570 nm with a microplate reader. Similarly, SH-SY5Y cells were incubated in 96-well plates for 24 h and then preincubated with Aβ fibrils for 6 h. To study if NPs could attenuate Aβ-induced cytotoxicity upon NIR irradiation, the cells were treated with different NPs in the absence or presence of NIR irradiation. Then samples were conducted as above. 2.11. Immunofluorescence. Control and treated cells were seeded on plates and immobilized with paraformaldehyde after treatments. The cells were then were rinsed in PBS and permeabilized

excellent water solubility, and biocompatibility. Much previous work indicated that the PEGylated nanoparticles exhibited excellent in vivo biodistribution and pharmacokinetics properties.15 Over the past decades, a majority of Aβ aggregation blockers showed a moderate suppression effect and weak degradation capacity. Insufficient intracellular particle delivery and low permeability to the blood−brain barrier (BBB) may be a key factor.16 One of the most efficient ways is to overcome the limitation through binding with cell penetrating peptide (CPPs). 17,18 CPPs could enter the cells by different mechanisms that have opened a novel way for drug delivery.19,20 Penetratin (Pen) peptide is one of the most promising CPPs whose sequence contains 16 amino acid residues (RQIKIWFQNRRMKWKK), which has been used to promote cellular internalization of nanoparticles and cross the BBB.21,22 Pen peptide has a powerful ability to conquer the poor BBB permeability and transmit its cargoes without causing significant cytotoxicity.23,24 Given these issues, we synthesized PEG-stabilized AuNS conjugated with Pen peptide (Pen@ PEG-AuNS), which show high permeability across the BBB, and so could overcome the major limitation of Aβ aggregation blockers. Considering Pen@PEG-AuNS alone lack fluorescent property, this would limit the application of Pen@PEG-AuNS in real-time imaging. In our previous work, we have synthesized a dinuclear ruthenium (Ru(II)) complex that exhibits stable fluorescence properties.25 Some studies have shown that multinuclear Ru(II) complex could be applied to act as imaging agents for real-time tracking during drug delivery.26−28 Moreover, Ru(II) complex could inhibit aggregation of Aβ and its neurotoxicity.29,30 Thus, a dinuclear Ru(II) complex modified Pen@PEG-AuNS (Ru@ Pen@PEG-AuNS) were synthesized as a multifunctional nanomaterials during drug delivery. In this study, we performed in vitro experiments to investigate the inhibition effect of Ru@Pen@PEG-AuNS on Aβ aggregation and the effect of destabilization of preformed Aβ fibrils in the presence of Ru@Pen@PEG-AuNS by applying NIR irradiation. Meanwhile, we found that this novel design could be applied in medicine as an appropriate nanovehicle, being highly biocompatible and hemocompatible. Afterward, we evaluated the neuroprotective effect of Ru@Pen@PEGAuNS on the Aβ-induced cellular toxicity upon NIR irradiation and the transport efficiency of Ru@Pen@PEG-AuNS across the BBB in vitro and in vivo. The findings demonstrated that Ru@ Pen@PEG-AuNS could serve as a kind of novel multifunctional nanomaterials for AD therapeutics.

2. EXPERIMENTAL SECTION 2.1. Characterization of the Nanoparticles. Morphology was imaged by Hitachi transmission electron microscope (TEM). Size distribution and zeta potential were measured by Malvern Nanosizer Nano ZS instrument. Further characterizations were performed by UV/visible spectroscopy and Nicolet 6700 Fourier transform-infrared spectra (FT-IR). 2.2. Ru(II) Complex Release from Nanoparticles. To confirm Ru(II) complex could steadily bind with NPs for 24 h, we measured Ru(II) complex release from the NPs by UV−vis spectrum. Ru@PEGAuNS and Ru@Pen@PEG-AuNS were, respectively, dispersed in pH 4.0 and pH 7.4 phosphate-buffered saline (PBS) with or without laser irradiation. Samples were taken at predetermined time intervals and centrifuged for 5 min. Then supernatants were measured at 456 nm. 19292

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

Research Article

ACS Applied Materials & Interfaces

Figure 1. Characterization of nanoparticles. TEM images of (A) PEG-AuNS and (B, C) Ru@Pen@PEG-AuNS; (D) UV spectra of AuNS, PEGAuNS, Pen@PEG-AuNS, and Ru@Pen@PEG-AuNS; (E) FT-IR spectra of AuNS, PEG-AuNS, Pen@PEG-AuNS, and Ru@Pen@PEG-AuNS.

Figure 2. Fibrils inhibition and disaggregation assay. (A) The soluble percentage of Aβ, alone or treated with PEG-AuNS (10 μg/mL) and Ru@ Pen@PEG-AuNS (10 or 20 μg/mL). (B) Turbidity of Aβ (20 μM), alone or treated with PEG-AuNS (10 μg/mL) and Ru@Pen@PEG-AuNS (10 or 20 μg/mL). (C) Relative ThT fluorescence intensity of Aβ solution, alone or treated with PEG-AuNS (10 μg/mL) and Ru@Pen@PEG-AuNS (10 or 20 μg/mL). (D) Relative ThT fluorescence intensity of Aβ fibrils, alone or treated with PEG-AuNS and Ru@Pen@PEG-AuNS (20 μg mL−1) in the absence or presence of NIR irradiation. The difference with control cells was showed by *p < 0.05 and **p < 0.01, respectively. °C for 6 h with or without stimulus. Ten microliters of 20% Triton X100 as positive control. After centrifugation, the supernatant of samples was measured by a microplate reader at 490 nm. The percentages of hemolysis were expressed relative to the mean absorbance value, which indicated 100% hemolysis. 2.13. AO/EB Staining. The Live/Dead cells viability assay was performed with SH-SY5Y cells stained using AO&EB fluorescent dyes.

with 0.1% Triton X-100 for 15 min. The samples were treated with 200 μL of anti-β-tubulin III (1:250) for 60 min in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Results were observed by confocal microscope (Leica, Germany). 2.12. Hemolysis Assay. For the hemolysis assay, 200 μL of erythrocytes from a human donor was pipetted into a 96-well plate, and NPs were added to the well to coincubate with erythrocytes at 37 19293

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

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ACS Applied Materials & Interfaces

Figure 3. Structural changes of Aβ fibrils. TEM (A) and AFM (B) images of Aβ fibrils, alone or treated with PEG-AuNS and Ru@Pen@PEG-AuNS (20 μg mL−1) in the absence or presence of 3 min NIR irradiation (0.75 W cm−2, 808 nm). After treatment, the culture medium of cells was removed and washed twice with PBS. Afterward, the cells were stained with 200 μL of AO/ EB (5 μg/mL) for 5 min and rinsed in PBS three times. AO could stain total cell nuclei green, but EB only stains the impaired cell nuclei red. Finally, the photographs were obtained by fluorescent microscope. 2.14. TUNEL and DAPI Costaining Assay. SH-SY5Y cells were grown on plates and precultivated with Aβ fibrils. Afterward, cells were cultured with different NPs with or without NIR irradiation, which were fixed with 4.0% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Subsequently, cells were placed in the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and nucleotides for 1 h, and then nuclei were stained with DAPI. Samples were rinsed in PBS three times. Images were captured using a fluorescence microscope. 2.15. Morphology Changes. SH-SY5Y cells were seeded on coverslips and precultivated with Aβ fibrils. The cells were rinsed in PBS three times and immobilized with paraformaldehyde after treatments. Coverslips were examined by an inverted microscope. 2.16. Intracellular Aβ Fibrils Investigation. To evaluate whether NPs under NIR irradiation could degrade Aβ fibrils in SH-SY5Y cells, first the cells were coincubated with Aβ fibrils for 6 h.31 After treatments, cells were immobilized with paraformaldehyde and costained by ThT and DAPI solution. The samples then were rinsed with PBS extensively, and results were observed by fluorescent microscope. 2.17. Transwell Experiment. The brain endothelial cells (bEnd3) cells were cultured to form a tight monolayer (TEER achieved 200 Ω) in the top closet of transwell plates, and then SH-SY5Y cells were seeded in the bottom closet overnight. Subsequently, addition of ruthenium-labeled NPs to the top closet and further incubated for 12 h, SH-SY5Y cells were observed using a confocal microscopy (excitation at 488 nm). 2.18. Inductively Coupled Plasma-Atomic Emission Spectrometry Analysis. SH-SY5Y cells were coincubated with various concentrations of ruthenium-labeled NPs for 12 h. After incubation, cells were rinsed in PBS three times and digested with concentrated

HNO3 and H2O2 at appropriate does. The samples were analyzed by inductively coupled plasma-atomic emission spectrometry (ICP-AES). 2.19. Cellular Uptake of Nanoparticles. SH-SY5Y cells were plated into 24-well plates for 24 h and then were treated with ruthenium-labeled NPs at different time points and temperatures. Afterward, SH-SY5Y cells were rinsed in PBS three times and immobilized with paraformaldehyde. The results were observed under fluorescent microscope (excitation at 488 nm). 2.20. Uptake Inhibition Experiment. The NPs cellular uptake pathways were studied by using several uptake inhibitors: chlorpromazine (10 μg/mL; inhibits clathrin-mediated endocytosis), methyl-β-cyclodextrin (5 μg/mL; inhibits lipid raft), colchicine (4 μg/ mL; inhibits macropinocytosis). The uptake of SH-SY5Y cells was quantitatively analyzed by flow cytometry after treating with different inhibitors for 30 min (BD, FACS Aria). 2.21. In Vivo Distribution. Nude mice were provided by Guangdong Medical Laboratory Animal Center. The transport of ruthenium-labeled NPs across the BBB was investigated using a realtime imaging system (Maestro). Ru@PEG-AuNS and Ru@Pen@ PEG-AuNS (1 mg/kg) were injected into mice’s caudal vein, and the organs were obtained at predetermined time points. Subsequently, results were obtained (excitation, 488 nm, emission, 620 nm). 2.22. Statistical Analysis. Data were presented by three times independent experiments, and all statistical analyses were conducted using Student’s t test. The differences of significant and very significant were showed by *p < 0.05 and **p < 0.01, respectively.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Nanoparticles. Figure S1 shows the synthesis strategy for Ru@Pen@ PEG-AuNS. Briefly, the AuNS were synthesized using seedmediated growth method, and PEG was conjugated to AuNS via Au−S bonds to obtain hydrophilic polymer-stabilized AuNS (PEG-AuNS). Then, Pen peptide was loaded on the surface of PEG-AuNS by amide formation. And ruthenium-labeled NPs 19294

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

Research Article

ACS Applied Materials & Interfaces

Figure 4. Biocompatibility assay. (A) Intracellular ROS production was evaluated with DCFH-DA after treating with Ru@Pen@PEG-AuNS (20 μg mL−1) with or without NIR irradiation in SH-SY5Y cells. H2O2 as positive control; scale bar, 30 μm. (B) Viability of SH-SY5Y cells incubated for 24 and 48 h with different concentrations of Ru@Pen@PEG-AuNS with or without NIR irradiation. (C) Fluorescence images of SH-SY5Y cells after incubation with Ru@Pen@PEG-AuNS (20 μg mL−1) upon NIR irradiation. Costaining was performed by β-tubulin (red) and DAPI (blue); scale bar, 10 μm. (D) IL-6 level. LPS as positive control. (E) Hemolysis assay. Triton-X as positive control. The difference with control cells was shown by *p < 0.05 and **p < 0.01, respectively.

that adding Ru(II) complex to Pen@PEG-AuNS led to the red shift from 678 to 686 nm and generated new absorption peaks at 456 nm, which suggested that Ru(II) complex successfully synthesized the surface of Pen@PEG-AuNS. Next, FT-IR spectroscopy of NPs was shown in Figure 1D; the naked AuNS had no characteristic peaks. The IR spectrum of PEG-AuNS showed a weak and broad band at 3200−3400 cm−1 belonging to −OH groups of PEG-AuNS and a peak at 2810 cm−1 corresponding to CH stretching of SH-PEGCOOH, which further supported the presence of PEG. Furthermore, the appearance of absorption peaks at 1649 cm−1 was assigned to CO stretching in the CO-NH of Pen@ PEG-AuNS. The result suggested that Pen peptide was successfully conjugated to the surface of PEG-AuNS. Similarly, the additional weak peak at 3083 cm−1 corresponding to CH stretching of pyridine ring in the spectrum of Ru@Pen@PEGAuNS confirmed the existence of Ru(II) complex on the surface of Pen@PEG-AuNS. To validate the stability of ruthenium-labeled NPs as luminescent probes for real-time tracking during drug delivery, we conducted the in vitro release study at 37 °C in pH 4.0 and pH 7.4 PBS (Figure S3), which implied the pH in endolysosomal and physiological pH, respectively. During 24 h of incubation, the accumulated Ru(II) complex released from Ru@PEG-AuNS and Ru@Pen@PEG-AuNS gradually increased with time under endolysosomal and physiological conditions but less than 8%. Even after irradiation with the NIR laser, no more than 25% of Ru(II) complex was released from

were fabricated through the electrostatic interactions. The branched morphology of PEG-AuNS and Ru@Pen@PEGAuNS was characterized by TEM. As shown in Figure 1, large quantities of PEG-AuNS and Ru@Pen@PEG-AuNS were obtained. The images of high-power TEM further illustrated the structure of PEG-AuNS as shown in the lower right corner of Figure 1A, displaying its polycrystalline nature. The size distribution and stability of PEG-AuNS and Ru@Pen@PEGAuNS were then studied by Nano-ZS analyzer. Figure S2A,B shows that the average size of PEG-AuNS was 78 nm, which increased to 105 nm when loaded with Pen peptide and Ru(II) complex. The stability of NPs was investigated by dynamic light scattering (DLS), which displayed Ru@Pen@PEG-AuNS that were able to stably exist within 15 d and that the size of Ru@ Pen@PEG-AuNS remained more stable than PEG-AuNS. Then, after 20 d, both PEG-AuNS and Ru@Pen@PEG-AuNS exhibited obvious aggregation (Figure S2C). As shown in Figure S2D, the zeta potentials of AuNS, PEG-AuNS, Pen@ PEG-AuNS, and Ru@Pen@PEG-AuNS were measured. Further characterization of NPs was performed by using UV spectroscopy (Figure 1C). The spectroscopy showed AuNS and PEG-AuNS had similar maximum absorption peaks (667 and 669 nm, respectively), which just appeared redshift of 2 nm. After it was conjugated with the Pen peptide, the maximum absorption peaks of PEG-AuNS underwent a red shift from 669 to 678 nm, indicating the Pen peptide conjugation process could lead to modest aggregation owing to PEG serving as a stabilizer when absorbed on the surface of AuNS. We noticed 19295

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

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ACS Applied Materials & Interfaces

Figure 5. Cell viability study. (A) The cytotoxicity of Aβ fibrils, alone or treated with PEG-AuNS and Ru@Pen@PEG-AuNS (20 μg mL−1) in the absence or presence of NIR irradiation. (B) The Live/Dead cells viability assay was performed with SH-SY5Y cells stained using AO&EB fluorescent dyes; scale bar, 50 μm. (C) The effect of PEG-AuNS and Ru@Pen@PEG-AuNS (20 μg mL−1) with or without NIR irradiation on DNA fragmentation induced by disrupted Aβ fibrils; scale bar, 50 μm. The difference with control cells was shown by *p < 0.05 and **p < 0.01, respectively.

soluble monomers to insoluble fibrils cause neurodegeneration.33,34 We first studied the inhibition effect of NPs on Aβ aggregation by the BCA protein assay. The soluble content of Aβ solution alone incubated for 72 h reached 26% of its original amount (Figure 2A), indicating that it formed Aβ aggregates. After addition of PEG-AuNS and Ru@Pen@PEG-AuNS (10 μg mL−1), the soluble Aβ content increased to 47% and 73%, respectively, which suggested that PEG-AuNS and Ru@Pen@ PEG-AuNS could slow the aggregation process of Aβ. Meanwhile, we noticed that Ru@Pen@PEG-AuNS showed a significantly higher inhibition effect than PEG-AuNS. Additionally, the inhibition effect of Ru@Pen@PEG-AuNS was concentration dependent, with the amount of soluble Aβ increased to 86% at the concentration (20 μg mL−1) of Ru@ Pen@PEG-AuNS. The results demonstrated that Aβ aggregation could be greatly suppressed by Ru@Pen@PEG-AuNS at a low concentration.

the NPs. We speculated that both electrostatic interactions and delocalized π-electrons may be important for strong dye adsorption.32 This means that the Ru(II) complex could remain associated with the NPs for real-time tracking during drug delivery. The AuNS solutions were exposed to an 808 nm NIR laser at a power density of 0.75 W cm−2 to study the potential use of AuNS in photothermal therapy and PBS as the control. As shown in Figure S4, the PBS sample did not show a significant response to irradiation at 808 nm NIR laser. In contrast, when the AuNS solution (5 μg·mL−1) was exposed to an 808 nm laser, which caused apparent temperature rises with the extension of irradiation time, reaching 46 °C in 7 min. In addition, the solutions of AuNS showed a dose-dependent (from 5 to 50 μg·mL−1) photothermal effect. 3.2. Fibrils Inhibition and Disaggregation Assay. It is widely recognized that the changes of Aβ gradually from 19296

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

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ACS Applied Materials & Interfaces

Figure 6. Cellular morphology changes and intracellular Aβ aggregates detection. (A) Representative photographs of cell morphology by living cell picture, scale bar, 30 μm; (B) Aβ aggregation detection in SH-SY5Y cells by ThT (green) and DAPI (blue) costaining, scale bar, 20 μm.

Figure 7. Transwell experiment and cellular uptake. (A) The transport efficiency of Ru@ PEG-AuNS and Ru@Pen@PEG-AuNS (20 μg mL−1) through BBB. The results were observed by confocal microscopy; scale bar, 25 μm. (B) Quantitative analysis of Ru concentrations in SH-SY5Y cells exposed to Ru@ PEG-AuNS and Ru@Pen@PEG-AuNS at different concentrations for 12 h by ICP-AES method. (C) Cellular uptake detection of Ru@PEG-AuNS and Ru@Pen@PEG-AuNS in SH-SY5Y cells under a fluorescent microscopy following 1 and 3 h of incubation at 4 or 37 °C, respectively; scale bar, 200 μm. The difference with control cells was shown by *p < 0.05 and **p < 0.01, respectively.

due to its large surface area.35,36 A full understanding of the mechanism is still underway. Given the above results, it is plausible that Ru@Pen@PEG-AuNS are relatively more effective in inhibiting Aβ aggregation than PEG-AuNS. It is possible that one reason Pen peptide possesses more hydrogenbonding sites, resulting in better interaction with Aβ. Further researches are needed to confirm this assumption.

Previous researches have intensively explored the effect of AuNP on Aβ aggregation. The possible mechanisms for the interactions between AuNP and Aβ were proposed: (1) the hydrophobic nature of Aβ could bind to the AuNP surface through hydrogen-bonding or other interactions, and thus block the formation of Aβ fibrils. (2) AuNP could intervene potential aggregation sites on the neuronal isomer molecule 19297

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

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fibrils.39−41 ThT dye could specifically bind to aggregated βsheet fibrils, resulting in ThT fluorescence strengthening.42 As seen in Figure 2C, Aβ solution was incubated alone in PBS at 37 °C and displayed a significant ThT fluorescence, which suggested that Aβ aggregates were formed. As expected, we observed a tremendous decrease in fluorescence when Ru@ Pen@PEG-AuNS were added to Aβ solution, in particular, on doubling the concentration of Ru@Pen@PEG-AuNS, the decreases of ThT fluorescence intensity to 23% were consistent with our observations using the BCA protein and turbidity assay. In contrast, the phenomenon was not observed in the presence of PEG-AuNS. These results demonstrated that Ru@ Pen@PEG-AuNS could more effectively hinder Aβ aggregation than PEG-AuNS. Having established the inhibitory effect of NPs, we next investigated the ability of the NPs to degrade mature Aβ fibrils upon NIR irradiation. As seen in Figure 2D, the relative ThT fluorescence intensity for Aβ alone was 99.4% after a 96 h period, and the sample incubation at room temperature was followed for 72 h, but the intensity of fluorescence did not increase, which demonstrated that the fibrillogenic process had reached a maximum. However, decreases in fluorescence intensity to 68% or 47% were observed when PEG-AuNS or Ru@Pen@PEG-AuNS were added to Aβ fibrils, respectively, suggesting that PEG-AuNS and Ru@Pen@PEG-AuNS could degrade Aβ fibrils to some extent and that the disaggregation efficiency of Ru@Pen@PEG-AuNS was higher than that of PEG-AuNS on Aβ aggregation. Inspired by the NIR absorption characteristic of AuNS, we performed a photothermal experiment to investigate the influence on Aβ fibrils. After Ru@Pen@ PEG-AuNS−Aβ aggregates were irradiated by a 808 nm laser for 3 min at the power density of 0.75 W cm−2, and further incubation, the ThT fluorescence signal markedly decreased compared to that of PEG-AuNS or Ru@Pen@PEG-AuNS alone, demonstrating that Ru@Pen@PEG-AuNS could generate local heat to dissociate Aβ fibrils upon NIR laser irradiation, leading to Aβ fibrils losing their amyloidogenic potential. The results clearly showed that Ru@Pen@PEGAuNS could more effectively disaggregate Aβ aggregation with NIR laser irradiation. In contrast, for Aβ fibrils with NIR irradiation alone, the fibril degradation process did not occur.

Figure 8. Investigation of internalization pathway of nanoparticles. Relative cellular uptake of Ru@PEG-AuNS and Ru@Pen@PEG-AuNS in SH-SY5Y cells in the presence of different endocytosis inhibitor. The difference with control cells was shown by *p < 0.05 and **p < 0.01, respectively.

The achieved inhibitory effect of NPs on Aβ aggregation was further verified by measuring the turbidity at the 405 nm wavelength. The optical density change of Aβ solution was revealed by turbidity, which was an index of Aβ aggregates.37 As shown in Figure 2B, NPs and pure Aβ alone caused a very small turbidity increase. After Aβ alone was incubated for 72 h, it exhibited a rapid increase in turbidity (0.072 units), in agreement with the described in previous literature.38 However, the turbidity of Aβ solution decreased to 0.053 units in the solution of Aβ containing PEG-AuNS (10 μg mL−1), which indicated that PEG-AuNS could inhibit Aβ aggregation to some extent. Under similar conditions, aggregation of Aβ in the presence of Ru@Pen@PEG-AuNS (10 or 20 μg mL−1) showed a sharp reduction (0.035 or 0.028 units, respectively), suggesting that Ru@Pen@PEG-AuNS could significantly exert an inhibitory function on Aβ fibrils formation in a dosedependent manner, to which they were more effective than PEG-AuNS. To investigate the effect of NPs on the inhibition of Aβ fibrillogenesis and the degradation of preformed Aβ fibrils upon NIR irradiation, thioflavin T (ThT) dye has been used for quantitative determination of the amount of aggregated β-sheet

Figure 9. Biodistribution of Ru@PEG-AuNS and Ru@Pen@PEG-AuNS following intravenous administration. (A) Fluorescence signals detected in the kidneys, lungs, hearts, spleens, and livers 12 and 24 h after the NPs injection. (B) Fluorescent signals detected in the brains 12 and 24 h after the NPs injection. 19298

DOI: 10.1021/acsami.6b05089 ACS Appl. Mater. Interfaces 2016, 8, 19291−19302

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with the control, indicating they did not induce cytotoxicity as confirmed by MTT. On the basis of these results, further experiments were performed, such as IL-6 measurement (Figure 4D) and hemolysis (Figure 4E) assay. T cells are highly relevant with inflammatory response mediated by various pro-inflammatory cytokines.45 To this end, PBMC cells were used to evaluate whether Ru@Pen@PEG-AuNS upon NIR irradiation could produce injurious effects by measuring the level of IL-6 production. Coincubation with LPS, an activator of inflammatory reaction, increased the IL-6 level, whereas no enhancement in the production of IL-6 was observed after treating with Ru@ Pen@PEG-AuNS in the absence or presence of NIR irradiation. This suggested that the novel design would not disturb the immune system, thus confirming its noncytotoxicity. Meanwhile, we investigated whether erythrocytes upon treatment of NPs with NIR irradiation could induce thrombogenic responses by in vitro test. The hemolysis ratio was evaluated by measuring free hemoglobin in the supernatant of the blood mixtures exposed to Ru@Pen@PEG-AuNS with or without NIR irradiation. The data revealed that the hemolysis percentage stimulated with Ru@Pen@PEG-AuNS in the absence or presence of NIR irradiation was similar in comparison to negative (PBS) and untreated controls, demonstrating high blood compatibility. In conclusion, these tests suggested that the novel design could be applied in medicine as an appropriate nanovehicle, being highly biocompatible and highly hemocompatible. 3.5. In Vitro Cell Viability Assay. The success in inhibiting Aβ aggregate formation and degrading amyloid aggregates upon laser irradiation prompted us to examine whether Ru@Pen@ PEG-AuNS could attenuate Aβ-induced cytotoxicity upon NIR irradiation. An MTT test was operated to detect cellular survival. As shown in Figure 5A, a decrease of 52% in cellular reduction arose when applying treatment with Aβ fibrils alone. Incubation of the cells with Aβ fibrils in the absence of Ru@ Pen@PEG-AuNS under NIR laser irradiation for 3 min did not increase cell viability, suggesting that NIR laser irradiation alone could not reduce the cytotoxicity of Aβ fibrils. In contrast, Aβ fibrils treated with Ru@Pen@PEG-AuNS under NIR laser irradiation showed low cytotoxicity, resulting in a marked enhancement of cell viability (86%), which demonstrated that Ru@Pen@PEG-AuNS were effective in dissolving the existing Aβ fibrils upon NIR laser irradiation. Comparatively, in the presence of PEG-AuNS or Ru@Pen@PEG-AuNS, there was less effectiveness at decreasing Aβ-mediated cellular toxicity. A live/dead cells viability assay was performed with SH-SY5Y cells stained using AO/EB fluorescent dyes to further confirm the neuroprotective effect of NPs by applying NIR irradiation (Figure 5B). With AO/EB staining, normal cells appeared green, and apoptotic cells appeared orange. After treatment of Ru@Pen@PEG-AuNS upon NIR irradiation led to a remarkable reduction in orange fluorescence, compared with treated with PEG-AuNS and Ru@Pen@PEG-AuNS alone, demonstrating that Ru@Pen@PEG-AuNS with NIR irradiation could prominently prevent SH-SY5Y cells from apoptosis induced by Aβ fibrils, consistent with our observations using MTT. DNA fragmentation is an important biochemical hallmark of cell apoptosis, which can be detected in the early stages of apoptotic cells before more apparent changes in the cellular morphology by using an in vitro TUNEL (green) enzymatic labeling and DAPI (blue) costaining test (Figure 5C). Thus, we

This meant that NIR laser irradiation alone could not affect the Aβ morphology. 3.3. Investigation of Morphology Changes in Aβ Fibrils. On the basis of ThT results, we used TEM to better ascertain morphological changes of Aβ aggregates (Figure 3A). Aβ formed long and networked fibrils after incubation for 72 h: a typical structure for amyloid fibrils. When incubated with PEG-AuNS and Ru@Pen@PEG-AuNS, similar Aβ fibrils were still present, although some of them were broken. However, after irradiation with the NIR laser in the presence of Ru@ Pen@PEG-AuNS and further incubated for 24 h, a majority of defined and lengthy fibrils were changed into relatively smaller structures, demonstrating the excellent disaggregation effect of Ru@Pen@PEG-AuNS on mature Aβ fibrils with laser irradiation. Conversely, exposure to the NIR laser alone without Ru@Pen@PEG-AuNS, the structure of Aβ aggregates was not converted, which meant NIR laser irradiation alone barely altered the assembly of Aβ. These results indicated that Ru@Pen@PEG-AuNS could be used to effectively dissociate Aβ fibrils upon NIR irradiation. Next, we further investigated the degradation efficiency of NPs for Aβ aggregates under NIR irradiation using AFM. As shown in Figure 3B, representative amyloid fibrils were seen in Aβ-treated sample alone. Similarly, incubation with NIR laser irradiation alone or PEG-AuNS retained the existing Aβ fibrils. When incubated with Ru@Pen@PEG-AuNS, a large amount of amorphous aggregates connected by fibrillar structures remained visible. Whereas, after irradiation with the NIR laser in the presence of Ru@Pen@PEG-AuNS and further incubated for 36 h, fewer amorphous aggregates were observed, while the fibrillar bridges all but disappeared as a result of photothermal ablation. These results further supported the above results and illustrated that Ru@Pen@PEG-AuNS could effectively degrade mature Aβ fibrils upon NIR irradiation. 3.4. Biocompatibility Assay. It has been reported that NIR irradiation of NPs can induce the production of ROS.43 Thus, we next evaluated the biocompatibility of NPs, which is crucial to guarantee secure application of NPs in medicine. As shown in Figure 4A, fluorimetric analysis by DCFH-DA assay demonstrated that an obvious increased fluorescence signal was detected in H2O2 treated sample used as a positive control. Whereas, the endocellular ROS was consistent with baseline when Ru@Pen@PEG-AuNS were added to cells. Comparatively speaking, there was a slight increase at inducing the production of ROS in the presence of Ru@Pen@PEG-AuNS upon NIR laser irradiation. The results revealed that Ru@ Pen@PEG-AuNS upon NIR laser irradiation would generate low concentrations of ROS. Increasing evidence showed that low concentrations of ROS may have the positive consequence of regulating many cellular processes and intracellular signaling mechanisms.44 To be assured of its biosafety, we further performed an MTT assay to investigate cell toxicity of NPs upon NIR irradiation at different dosages. As shown in Figure 4B, Ru@Pen@PEG-AuNS had no influence on cellular survival for 24 and 48 h below 30 μg·mL−1, even irradiated by NIR laser. Furthermore, confocal fluorescence images of SH-SY5Y cells were stained with β-tubulin tracker (red), a marker of early neuronal differentiation, and DAPI (blue) was shown (Figure 4C): β-tubulin mainly localizes at the cytoplasmatic and neurite levels, while DAPI localizes at the nuclei. After cells were subjected to Ru@Pen@PEG-AuNS upon NIR irradiation treatment the neurites underwent no changes if compared 19299

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coincubated with 5, 10, and 20 μg/mL of Ru@Pen@PEGAuNS, relative to those treated with Ru@PEG-AuNS. It can be concluded that the presence of Pen peptide was capable of enhancing drug uptake, consistent with transwell experiment results. We continued to investigate cellular uptake by visualizing the fluorescence of ruthenium-labeled NPs using fluorescence microscopy. As shown in Figure 7C, the fluorescence intensity displayed both time- and temperature-dependent behaviors. Enhanced fluorescence intensity was seen as time increased. Obviously, the fluorescence intensity of the cells treated with Ru@Pen@PEG-AuNS was stronger than that of the cells treated with Ru@PEG-AuNS. It was worth noting that the uptake of Ru@PEG-AuNS by SH-SY5Y cells was inhibited at 4 °C, whereas cellular uptake was not inhibited when treated with Ru@Pen@PEG-AuNS at 4 °C, which was indicative of energyindependent uptake. These results suggested that Ru@Pen@ PEG-AuNS internalization in SH-SY5Y cells may be mediated by two tapes of cellular uptake mechanism, including energydependent and energy-independent pathways. 3.8. Cellular Uptake Inhibition Experiment. To further elucidate the NPs internalization pathway, we studied the cellular uptake of Ru@PEG-AuNS and Ru@Pen@PEG-AuNS in the presence of several different inhibitors (Figure 8). We found that Ru@PEG-AuNS and Ru@Pen@PEG-AuNS internalization were obviously blocked by methyl-β-cyclodextrin (lipid raft inhibition) but not by chlorpromazine (clathrin inhibition), indicating that the lipid raft-mediated endocytosis pathway played an important role in the cellular interaction with both of the NPs. Furthermore, cellular uptake of Ru@ Pen@PEG-AuNS was also inhibited by colchicine (macropinocytosis inhibition), suggesting that the macropinocytosismediated pathway was also involved by Pen peptide loading. 3.9. In Vivo Distribution of Nanoparticles. The real-time imaging system was used to further confirm transport efficiency of NPs in vivo. After intravenous injection of the rutheniumlabeled NPs, the fluorescent images were detected at predetermined time points. As shown in Figure 9, we found that the fluorescence signal of Ru@Pen@PEG-AuNS remaining in brain was markedly strengthened in comparison to those of Ru@PEG-AuNS at 12 and 24 h, respectively. In contrast, the fluorescence signal in the peripheral organs of those animals treated with Ru@Pen@PEG-AuNS was even lower than that of those treated with Ru@PEG-AuNS. These results indicated that Pen peptide could effectively improve the brain delivery of NPs.

investigated Aβ-mediated apoptosis by assessing DNA fragmentation levels. As expected, incubation of SH-SY5Y cells with Aβ fibrils aroused obvious strengthening of DNA fragmentation. Whereas, after irradiation with the NIR laser in the presence of Ru@Pen@PEG-AuNS, we observed a remarkable decrease of DNA fragmentation as exhibited by decreased green fluorescence, suggesting a decrease in the Aβmediated cellular toxicity. Although treatment with PEG-AuNS and Ru@Pen@PEG-AuNS also had certain effects, they were less effective than Ru@Pen@PEG-AuNS upon NIR laser irradiation; in particular, PEG-AuNS hardly had influence on weakening DNA fragmentation. The results demonstrated that Ru@Pen@PEG-AuNS upon NIR laser irradiation could effectively convert Aβ fibrils into nontoxic forms in SH-SY5Y cells. 3.6. Cellular Morphology Changes and Intracellular Aβ Aggregates Detection. The morphology change of SHSY5Y cells is an indicator of cell toxicity.46 As shown in Figure 6A, SH-SY5Y cells treated with Aβ fibrils alone experienced shrinkage, leading to less outgrowth and branching of neurites relative to control cells. Application of Ru@Pen@PEG-AuNS upon NIR laser irradiation rather than PEG-AuNS or Ru@ Pen@PEG-AuNS alone could dramatically recover neurite outgrowth and branching, which suggested that Ru@Pen@ PEG-AuNS under NIR laser irradiation could significantly attenuate cellular toxicity as well as protect the neurites against Aβ-mediated neuronal damage. To directly determine whether NPs upon NIR laser irradiation could effectively degrade endocellular Aβ fibrils, we used SH-SY5Y cells coincubated with Aβ aggregates for 6 h and then treated with NPs with or without NIR laser irradiation. After 48 h, the cells were costained with ThT (green) and DAPI (blue) to reveal Aβ fibrillation levels by means of fluorescence microscope. As shown in Figure 6B, the cells treated with Aβ aggregates alone showed obvious ThT fluorescence, despite a reduced trend of ThT fluorescence that occurred after treatment with either PEG-AuNS or Ru@Pen@ PEG-AuNS, which still could be seen clearly. Whereas, treatment with Ru@Pen@PEG-AuNS under NIR laser irradiation caused distinct disruption on mature Aβ aggregates, with ThT fluorescence disappearance. The results showed that Ru@Pen@PEG-AuNS upon NIR laser irradiation could effectively disaggregate intracellular Aβ fibrils. 3.7. Transwell Experiment and Cellular Uptake Study. The penetrating capacity of NPs through BBB was evaluated by transwell experiments. We used bEnd3 and SH-SY5Y cells coculture to mimic the BBB in vivo.47 Observed by a confocal microscope (Figure 7A), small amounts of fluorescence signal were acquired after coincubation with Ru@PEG-AuNS. Instead, the fluorescence signal from Ru@Pen@PEG-AuNS could been markedly seen in the same period, proving that Ru@Pen@PEG-AuNS possessed the ability to cross the BBB. The results demonstrated that the increased permeation of NPs across an in vitro BBB model could be attributed to Pen peptide loading, which suggested that Ru@Pen@PEG-AuNS could be a suitable candidate for AD treatment. Next, we explored the effect of various concentrations, time, and temperature on cellular uptake of NPs. Inductively coupled plasma atomic emission spectrometry (ICP-AES) was applied to quantitatively reveal the cellular uptake of different concentrations of NPs in SH-SY5Y cells. Figure 7B revealed that the uptake process of NPs was dose-dependent, and the Ru concentration increased many times when SH-SY5Y cells were

4. CONCLUSIONS In conclusion, the results of our in vitro study suggested that Ru@Pen@PEG-AuNS could obviously inhibit the formation of Aβ fibrils as well as dissociate preformed fibrous Aβ under the irradiation of NIR due to the NIR absorption characteristic of AuNS. More importantly, this novel design could be applied in medicine as an appropriate nanovehicle, being highly biocompatible and highly hemocompatible. In addition, Ru@ Pen@PEG-AuNS under NIR irradiation had excellent neuroprotective effect against cellular toxicity triggered by Aβ fibrils. Meanwhile, Pen peptide could effectively improve the delivery of NPs to the brain in vitro and in vivo, which overcame the major limitation of Aβ aggregation blockers. On the basis of all these findings, we believed that Ru@Pen@PEG-AuNS may serve as an innovative multifunctional nanovehicle for the treatment of AD. Further experiments are required to validate 19300

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ACS Applied Materials & Interfaces the findings, thereby better demonstrating that Ru@Pen@ PEG-AuNS could be proposed for AD therapy.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b05089. Experimental section including materials, reagents, and synthesis of the nanoparticles. Schematic representation of the synthesis of the Ru@Pen@PEG-AuNS. Size distribution of PEG-AuNS and Ru@Pen@PEG-AuNS in PBS (pH = 7.4) buffer. The size stability of PEGAuNS and Ru@Pen@PEG-AuNS in PBS (pH = 7.4) buffer. Zeta potential of AuNS, PEG-AuNS, Pen@PEGAuNS, and Ru@Pen@PEG-AuNS in PBS (pH = 7.4) buffer. In vitro release of Ru(II) complex from Ru@PEGAuNS and Ru@Pen@PEG-AuNS in PBS buffer at pH 7.4 and 4.0 with or without 3 min of irradiation (0.75 W cm−2, 808 nm), respectively. The temperature change curves of the AuNS with various concentrations exposed to the 808 nm laser at a power density of 0.75 W cm−2. PBS as control. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 20 85220223. Fax: +86 20 85220223. E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21171070, 21371075), the Natural Science Foundation of Guangdong Province (2014A030311025), and the Planned Item of Science and Technology of Guangdong Province (2016A020217011).

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REFERENCES

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