Increased Gold Nanoparticle Retention in Brain Tumors by in Situ

Nov 11, 2016 - AuNPs-AK could be hydrolyzed to expose the 1,2-thiolamino groups on AuNPs-AK in the presence of legumain, which occurs by a click cyclo...
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Increased Gold Nanoparticle Retention in Brain Tumors by in Situ Enzyme-Induced Aggregation Shaobo Ruan, Chuan Hu, Xian Tang, Xingli Cun, Wei Xiao, Kairong Shi, Qin He, and Huile Gao* Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China S Supporting Information *

ABSTRACT: The treatment of brain tumors remains a challenge due to the limited accumulation of drugs and nanoparticles. Here, we triggered the aggregation of gold nanoparticles (AuNPs) using legumain to enhance the retention of chemotherapeutics in brain tumors. This nanoplatform, AuNPs-A&C, is comprised of Ala-Ala-AsnCys-Lys modified AuNPs (AuNPs-AK) and 2-cyano-6-aminobenzothiazole modified AuNPs (AuNPs-CABT). AuNPs-AK could be hydrolyzed to expose the 1,2-thiolamino groups on AuNPs-AK in the presence of legumain, which occurs by a click cycloaddition with the contiguous cyano group on AuNPs-CABT, resulting in formation of AuNPs aggregates. This strategy led to an enhanced retention of the AuNPs in glioma cells both in vitro and in vivo due to the blocking of nanoparticle exocytosis and minimizing nanoparticle backflow to the bloodstream. After conjugation of doxorubicin (DOX) via a pH-sensitive linker to AuNPs-A&C, the efficiency for treating glioma was improved. The median survival time for the DOX-linked AuNPs-A&C increased to 288% in comparison to the saline group. We further show the use of the AuNPs-A&C for optical imaging applications. In conclusion, we provide a strategy to increase nanoparticle tumor accumulation with the potential to improve therapeutic outcome. KEYWORDS: brain tumors, legumain, click cycloaddition, gold nanoparticles, tumor microenvironment effectiveness of an active targeting process.12 Other methods to increase brain tumor accumulation and retention include: (1) transient disruption of the BBB can be achieved with intracarotid administration of hyperosmolar agents-coated nanoparticle;16 (2) implantation of biodegradable polymer walls in the resection cavity after excision;17,18 (3) convectionenhanced delivery of drug-load nanocarrier;19 and (4) intranasal delivery of nanostructured therapeutic.20 However, these approaches are restricted by poor selectivity, potential brain damage, and the tumor’s infiltrative nature.21 Therefore, there is a need to develop methods to increase nanocarrier accumulation in brain tumors with high selectivity. To address this, a variety of bioresponsive functional nanocarriers that can target the heterogeneities of tumor microenvironment, such as altered redox,22 acidic pH,23,24 hypoxic,25,26 or up-regulated enzyme,27−29 have been proposed.

B

rain tumors are a diverse group of primary and metastatic neoplasms in the central nervous system (CNS).1,2 Approximately 24,000 cases are diagnosed annually with half of the patients presenting a fatal brain tumor type.2,3 Malignant brain tumors, including diffusely infiltrating glioma (mostly affect children with a high cellular differentiation and slow growth), anaplastic astrocytomas, glioblastoma (GBM), and brain metastasis, are the most common and aggressive.4−7 The prognosis for most brain tumor patients remains unsatisfactory, despite the use of surgical resection, radiotherapy, and/or chemotherapy for treatment. Nanocarriers have been demonstrated in the diagnosis and treatment of the central nervous system (CNS) disease.8−10 A major challenge is to retain these nanocarriers in a brain tumor. There are currently a number of mechanisms of mediating nanocarriers accumulation in brain tumors.11,12 Receptormediated delivery (RMT) involves the active targeting of nanocarriers to receptors expressed on blood−brain barrier (BBB),13 tumor cells,14 as well as neovessels.15 Nevertheless, differential distribution and expression of receptors in different pathological state of brain tumor may influence the © 2016 American Chemical Society

Received: July 28, 2016 Accepted: November 7, 2016 Published: November 11, 2016 10086

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Figure 1. (A) Diagram depicting the legumain-triggered aggregation and composition of AuNPs-DOX-A&C. The AuNPs-DOX-AK undergoes a cleavage by legumain to expose 1,2-thiolamino group of cysteine, where click cycloaddition further occurs with the contiguous cyano group of AuNPs-DOX-CABT to form aggregates. (B) Schematic illustrations of in vivo behavior of AuNPs-DOX-A&C after intravenous injection for increased accumulation.

showed an improved therapeutic outcome due to the increased accumulation in glioma.

Nanocarriers modified with bioresponsive functional moieties are expected to selectively recognize unique molecular characteristics of the tumor microenvironment, resulting in tumor-specific localization of nanocarriers in vivo. Here, we proposed an aggregation-based strategy to increase the accumulation of chemotherapeutic in the brain tumor. Figure 1 describes the overall strategy and mechanism. In this context, we validated that the AK modification be spliced by the legumain to expose the 1,2-thiolamino groups on cysteine, which occurred by a click cycloaddition with the contiguous cyano groups on CABT modification, resulting in aggregation of gold nanoparticles (AuNPs). In vitro, the size of AuNPsA&C increased from 35.6 to 309.6 nm after incubation with legumain for 12 h. In vivo fluorescence imaging and photoacoustic (PA) imaging suggested AuNPs-A&C selectively accumulated in the orthotopic C6 glioma site with precise localization. Inductively coupled plasma atomic mass spectrometry (ICP-MS) analysis further demonstrated AuNPs-A&C reached a much higher accumulation in the glioma site. Most significantly, the doxorubicin (DOX)-linked AuNPs-A&C

RESULTS AND DISCUSSION Characterization of Legumain Triggered Size Increase of AuNPs-A&C. We prepared citrated-stabilized AuNPs with a size of around 20 nm and then synthesized SH-PEG-AK and SH-PEG-CABT by conjugating the AK and CABT to SH-PEGCM (Figures S1 and S2). AuNPs-A&C consisting of AuNPsAK and AuNPs-CABT was prepared via metal coordination of “S−Au” with an initial size of 35.6 ± 2.1 nm using dynamic light scattering (DLS) analysis (Table S1). Considering the pHdependent proteolytic capacity of legumain,30 we thus incubated AuNPs-A&C in HEPES buffer at a pH of 7.4, 6.5, 5.5, 5.0, and 4.0 to evaluate the efficiency of legumain-triggered size increase. Indeed, our results showed legumain could trigger aggregation of AuNPs-A&C in the aqueous phase, especially with a low-pH condition (Figures 2A and S3). It was worth noting that in pH 5.0, the AuNPs-A&C showed the highest size increase to 309.6 ± 16.3 nm after incubation with legumain for 10087

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Figure 2. In vitro study of legumain-triggered size increase. (A) Hydrodynamic diameter of AuNPs-A&C incubated with legumain (1 mg· mL−1) in different pH values after DLS measurements using DLS analysis; error bars indicate SD (n = 3). (B) Hydrodynamic diameter of AuNPs-PEG, AuNPs-AK and AuNPs-CABT incubated with legumain in pH 5.0 HEPES buffer; error bars indicate SD (n = 3). (C) TEM images of AuNPs-A&C incubated with legumain in different pH conditions for 12 h and control nanoparticles in pH 5.0 for 12 h; bar represents 100 nm. (D) Hydrodynamic diameter of AuNPs-A&C pre-incubated in pH 5.0 HEPES buffer containing 20% mouse plasma for 24 h, then incubated with legumain (1 mg·mL−1) and without legumain for another 24 h; error bars indicate SD (n = 3), **p < 0.01. (E) Hydrodynamic diameter of AuNPs-A&C pre-incubated in pH 5.0 HEPES buffer containing 50% mouse plasma for 24 h, then incubated with legumain (1 mg·mL−1) and without legumain for another 24 h; error bars indicate SD (n = 3), **p < 0.01.

114.5 ± 5.5, and 127.6 ± 4.9 nm respectively, which was mainly owing to the protein absorption and subdued electrostatic repulsion (Figures S4 and S5). These results indicated the click cycloaddition could not be triggered in the absence of either the proper surface coating or legumain. Furthermore, transmission electron microscopy (TEM) further confirmed the

12 h. Further control experiments were conducted to validate the role of nanoparticle design in mediating legumain aggregation. We incubated AuNPs-PEG, AuNPs-AK, and AuNPs-CABT in pH 5.0 HEPES buffer with legumain (Figure 2B). After 12 h incubation, the sizes of AuNPs-PEG, AuNPsAK, and AuNPs-CABT had a slight increase to 134.2 ± 4.5, 10088

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Figure 3. Evaluation of legumain responsive AuNPs-DOX-A&C at cellular level. (A) Confocal images of C6 cells incubated with AuNPs-DOXA&C and other control formulations for 4 and 24 h at an equal DOX concentration of 5 μg·mL−1, the concentration of Ato was 20 μM; bars indicate 50 μm. (B) Cellular uptake of C6 cells incubated with AuNPs-DOX-A&C and control formulations for 1, 4, 8, and 24 h using flow cytometer; error bars indicate SD (n = 3), **p < 0.01 vs control DOX-tethered nanoparticles. (C) Percentage incubated dose (%ID) of cellular uptake of C6 cells incubated with AuNPs-DOX-A&C and control formulations for 4 and 24 h using ICP-MS analysis; error bars indicate SD (n = 3), *p < 0.05. (D) %ID of cellular exocytosis of C6 cells incubated with AuNPs-DOX-A&C and control formulations for 4 and 24 h using ICP- MS analysis; error bars indicate SD (n = 3). (E) TEM images of C6 cells incubated with AuNPs-DOX-A&C and control formulations for 24 h; bar indicates 100 μm. (F) Legumain expression level by Western blotting analysis: left to right are C6, U87, bEnd.3, HepG2, LO2, B16F10, 4T1, and MDA-MB-231 cells; L indicates legumain and G indicates GAPDH. (G) Cellular uptake of AuNPs-DOX-A&C and control formulations for 24 h by different cells, 1 indicates AuNPs-DOX-A&C, 2 indicates AuNPs-DOX-PEG, 3 indicates AuNPs-DOX-AK, and 4 indicates AuNPs-DOX-CABT; error bars indicate SD (n = 3), *p < 0.05 vs control DOX-tethered nanoparticles. (H) Cytotoxicity evaluation of AuNPs-DOX-A&C and control formulations using MTT assay, 5 indicates free DOX; error bars indicate SD (n = 3), *p < 0.05 vs control DOX-tethered nanoparticles. (I) Flow cytometer analysis of C6 cells after staining with Annexin V-FITC/PI. Cells were incubated with AuNPs-DOX-A&C and control formulations for 24 h. 10089

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was mainly because AuNPs-DOX-A&C formed intracellular aggregates which may block exocytosis of particles. The fluorescence of C6 cells co-incubated with AuNPs-DOX-A&C and Ato showed a much lower intensity than that of AuNPsDOX-A&C, suggesting legumain was crucial for the intracellular aggregation of AuNPs. These results were further identified by flow cytometer analysis (Figure 3B). Additionally, subcellular colocalization investigation showed the nanoparticles were trapped in endosomes (Figure S13), which might benefit pH-dependent release of the payload because endosomes are in the pH range of 5.0−5.5.23,30,36 Although cellular uptake had demonstrated that AuNPs-A&C could improve the DOX accumulation in cells, it was still difficult to distinguish between the populations of AuNPs-AK and AuNPsCABT in same cells. It also became unclear whether both types of particles entered the same cell at a similar rate and localized in same intracellular compartments. To figure this out, we conjugated two types of fluorescence dyes onto AuNPs-AK and AuNPs-CABT, namely AuNPs-DOX-AK and AuNPs-Cy5.5CABT. Confocal image showed fluorescence overlap between DOX (green) and Cy5.5 (red) in the same C6 cells after coincubation with AuNPs-DOX-AK and AuNPs-Cy5.5-CABT for 4 h, indicating both particles could enter the cells and localized in same intracellular compartments (Figure S14). Meanwhile, we further exchange the conjugation of Cy5.5 and DOX onto AuNPs-AK and AuNPs-CABT to obtain AuNPs-Cy5.5-AK and AuNPs-DOX-CABT. After 4 h incubation, there was a fluorescence overlap in the same cells. Most importantly, the fluorescent intensity of DOX and Cy5.5 in C6 cells treated with AuNPs-DOX-AK and AuNPs-Cy5.5-CABT was similar to the corresponding type of fluorescence in C6 cells treated with AuNPs-Cy5.5-AK and AuNPs-DOX-CABT. Additionally, the fluorescent intensity of DOX and Cy5.5 in C6 cells after 24 h incubation was much stronger than that of PEGylated fluorescence dye-loaded AuNPs (Figure S15). To directly determine the amount of AuNPs internalized by C6 cells, we further measured the AuNPs in cells using inductively coupled plasma atomic mass spectrometry (ICPMS). As shown in Figure 3C, the percentage incubated dose (% ID) of cellular uptake of AuNPs-DOX-A&C, AuNPs-DOXPEG, AuNPs-DOX-AK, and AuNPs-DOX-CABT were 1.20 ± 0.15%, 0.81 ± 0.03%, 1.22 ± 0.30%, and 0.69 ± 0.05%, respectively, after incubation for 4 h, demonstrating a similar cellular uptake for each particle. Over time, the %ID of cellular uptake of AuNPs-DOX-A&C reached as high as 10.88 ± 1.6% at 24 h, which was much higher than that of AuNPs-DOX-PEG (4.11 ± 1.19%), AuNPs-DOX-AK (2.52 ± 0.61%), and AuNPsDOX-CABT (4.82 ± 1.19%). We also assessed the exocytosis of different formulations by cells to explain the high internalization of AuNPs-DOX-A&C. The %ID of cellular exocytosis of AuNPs-DOX-A&C, AuNPs-DOX-PEG, AuNPsDOX-AK, and AuNPs-DOX-CABT were 2.17 ± 0.15%, 2.73 ± 0.08%, 2.72 ± 0.42%, and 1.73 ± 0.85%, respectively, after incubation for 4 h. After 24 h incubation, the %ID of cellular exocytosis of corresponding nanoparticles were 2.52 ± 0.98%, 4.19 ± 0.94%, 2.93 ± 1.22%, and 2.97 ± 0.67%, respectively, with a confidence value of 0.0686, 0.3429, and 0.2233 for % ID of cellular exocytosis of AuNPs-DOX-A&C vs AuNPs-DOXPEG, AuNPs-DOX-AK, and AuNPs-DOX-CABT, respectively. It could be implied that intracellular aggregation of AuNPsDOX-A&C has more restriction on cellular exocytosis compared to other particles.37,38 All these results suggested that this strategy could enhance the cellular accumulation by

aggregation of AuNPs-A&C in different pH conditions relative to control particles after incubation with legumain for 12 h (Figure 2C). AuNPs-A&C incubated with legumain showed an increased intensity in size from 37.05 nm (peak 1, 100%) at 0 h to 37.5 nm (peak 1, 22.9%) and 438.5 nm (peak 2, 77.1%) at 12 h in pH 5.0 (Figure S6). In comparison, AuNPs-PEG showed an increase to 417.5 nm (peak 2, 47.5%), AuNPs-AK showed an increase to 313.7 nm (peak 2, 45.6%), and AuNPsCABT showed an increase to 227.3 nm (peak 2, 51.9%). Although the control particles showed an approximate 50% increase of intensity to 300−400 nm size range, the increase of number was no more than 0.5%, which was much lower than that of AuNPs-A&C (12%). The size increase of AuNPs-A&C by legumain could be inhibited by the atorvastatin (Ato)31 and simvastatin (Siv)32 over time, indicating that the legumain was essential for the click cycloaddition (Figures S7 and S8). Since cysteine is present in blood,33 it may react with the CABT on the surface of the AuNPs. This suggests the need to evaluate whether AuNPs-A&C could still induce aggregation in glioma after circulating in blood. Therefore, we further preincubated AuNPs-A&C in pH 5.0 HEPES buffer containing 20% mouse plasma, and the hydrodynamic size was measured. We showed that AuNPs-A&C incubated with plasma and legumain demonstrated a significant size increase relative to that incubated without legumain (Figure 2D). However, AuNPs-PEG, AuNPs-AK, and AuNPs-CABT incubated in the same condition and procedure with or without legumain displayed close size increases to that of AuNPs-A&C incubated in 20% plasma-containing HEPES for 48 h without legumain (Figure S9). Moreover, AuNPs-A&C pre-incubated with 50% mouse plasma also showed a significant size increase despite protein absorption also causing a size increase (Figures 2E and S10). In Vitro Evaluation of Cellular Uptake by Brain Glioma Cells. We further conjugated DOX, a chemotherapeutic, onto the surface of AuNPs via a pH-sensitive bond described in our previous research,34 namely AuNPs-DOX-A&C. We evaluated whether legumain-triggered aggregation of AuNPs affected the pH-dependent release of DOX. AuNPs-DOX-A&C and AuNPs-DOX-PEG were both incubated with legumain in pH 5.0 HEPES buffer, and the cumulative release of DOX from AuNPs-DOX-A&C and AuNPs-DOX-PEG was measured. There was a similar release trend, which was 92.5 ± 1.3% and 90.7 ± 2.1%, respectively, suggesting the legumaintriggered aggregation of AuNPs has a negligible influence on the pH-dependent release of DOX (Figure S11). Before cellular investigation, we further determined the stability of AuNPsDOX-A&C and control particles in serum-containing and serum-free DMEM culture (Figure S12). All particles in DMEM containing 10% fetal bovine serum displayed a slight size increase compared to that in serum-free DMEM even after 48 h of incubation, which was due to the protein absorption. Next we incubated our nanoparticles with C6 cells, a murine glioma cell line, which was chosen for this study due to its high malignancy and invasion.35 Confocal laser microscopy images of C6 cells showed that cellular uptake of AuNPs-DOX-A&C (without or with Ato), AuNPs-DOX-PEG, AuNPs-DOX-AK, AuNPs-DOX-CABT had no obvious differences after incubation for 4 h. Our results indicated that these particles modified without any active-targeted moieties presented a similar cellular internalization level (Figure 3A). With the time extended to 24 h, the fluorescence intensity of C6 cells incubated with AuNPsDOX-A&C was much stronger than that of other groups, which 10090

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Figure 4. In vivo distribution of AuNPs-Cy5.5-A&C in orthotopic C6 glioma-bearing mice. (A) Living imaging of BALB/c mice after intravenous injection with AuNPs-Cy5.5-A&C and control formulations for different time intervals; bar indicates radiant efficiency from 0.5 × 107 to 2.5 × 107. (B) Ex vivo imaging of brains after injection with different formulations for 24 h, 1 indicates AuNPs-Cy5.5-A&C, 2 indicates AuNPs-Cy5.5-PEG, 3 indicates AuNPs-Cy5.5-AK, 4 indicates AuNPs-Cy5.5-CABT, and 5 indicates free Cy5.5; bar indicates radiant efficiency from 0.5 × 107 to 2.5 × 107. (C) Left column indicates semi-quantitative data of signals in glioma and normal brain after injection with different formulations for 24 h, and right column indicates G/B ratio of different formulations; error bars indicate SD (n = 3), *p < 0.05. (D) Ex vivo imaging of main organs after injection with different formulations for 24 h; bar indicates radiant efficiency from 0.5 × 107 to 7.5 × 107. (E) Corresponding semi-quantitative data of signal in organs; error bars indicate SD (n = 3). (F) Fluorescence distribution of different formulations in glioma section after staining with anti-legumain antibody; bars indicate 100 μm. (G) Fluorescence distribution of different AuNPs-Cy5.5-A&C in the glioma section after staining with anti-CD34 antibody; bars indicate 100 μm.

blocking cellular exocytosis. Moreover, TEM observation at cellular level was carried out to evaluate the internalization behavior of these particles. Obvious aggregates of AuNPs could be found in TEM image of C6 cells incubated with AuNPsDOX-A&C for 24 h. In comparison, only smaller AuNPs aggregates composed of several particles could be found in groups of AuNPs-DOX-PEG, AuNPs-DOX-AK, and AuNPsDOX-CABT (Figure 3E). This result strongly validated the effectiveness of legumain-triggered aggregation of AuNPsDOX-A&C within cellular cytoplasm and explained intuitively why it possessed lower cellular exocytosis after 24 h incubation. To validate the legumain-related cellular uptake of AuNPsDOX-A&C, we further applied this nanoplatform to other cells.

Western blotting analysis showed a different level of legumain expression in several cells including cancer cells and normal cells (Figure 3F). Subsequently, different cells were incubated with AuNPs-DOX-A&C and control particles for 24 h, and fluorescence intensity of different cells incubated with AuNPsDOX-A&C were generally higher than that of AuNPs-DOXPEG, AuNPs-DOX-AK, and AuNPs-DOX-CABT (Figure 3G). It was noticeable that the difference of cellular uptake between AuNPs-DOX-A&C and control particles was relevant to its expression level of legumian, meaning that the higher legumain expression in cells, the higher the cellular accumulation of AuNPs-DOX-A&C compared to control particles. This result 10091

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Figure 5. Accumulation evaluation of AuNPs-A&C in orthotopic C6 glioma-bearing mice. (A) Elucidation of tissue mimicking phantom setup (left), and photoacoustic transverse image acquired at the indicated red dash line (right). (B) PA images of AuNPs-A&C incubated with legumain in the absence and presence of Ato acquired from phantom at 690 nm, and AuNPs-PEG incubated with legumian; gray bars indicate ultrasound image of phantom, red bars indicate PA signal of phantom, and jet bars indicate the PA signal of AuNPs. (C) Time course mean pixel intensity of PA signal measured from B; error bars indicate SD (n = 3). (D) Corresponding transverse anatomic image of the male mouse atlas provided by View MSOT 3.6 software. (E) Overlayed MSOT images of orthotopic C6 glioma-bearing brains after intravenous injection with AuNPs-A&C and control particles for different time intervals; gray bars indicate ultrasound image of brain, blue bars indicate PA signal of blood deoxygenation, red bars indicate PA signal of blood oxygenation, and jet bars indicate the PA signal of AuNPs. (F) Spectrum resolution of overlaid PA images at 48 h from (E); HbO2 represents oxygenated hemoglobin and Hb represents deoxyhemoglobin. (G) Time course mean pixel intensity of PA signal measured from (E); error bars indicate SD (n = 3), ***p < 0.001. (H) %ID/g glioma of AuNPs-A&C and control particles using ICP-MS analysis; error bars indicate SD (n = 3), *p < 0.05. (I) TEM images of C6 cells from glioma after injection with AuNPs-A&C and control particles for 24 h; bars indicate 100 nm.

addition, the Annexin-V/PI staining assay was performed to qualitatively and quantitatively evaluate the in vitro apoptosisinducing capacity of AuNPs-DOX-A&C. The early, late apoptosis and necrosis of C6 cells were 24.8%, 44.2%, and 16.5%, respectively, after incubation with AuNPs-DOX-A&C for 24 h, which were much higher than that of AuNPs-DOXPEG, AuNPs-DOX-AK, and AuNPs-DOX-CABT (Figures 3I and S17). In Vivo Distribution of AuNPs-Cy5.5-A&C at Glioma Site. We next evaluated the distribution of AuNPs-Cy5.5-A&C after intravenous injection to orthotopic C6 glioma-bearing mice using the IVIS imaging system. 0.15 h after injection, whole body images showed no difference between AuNPs-

suggested that this legumain-responsive AuNP-DOX-A&C possessed general applicability in legumain-positive cells. In vitro cytotoxicity of C6 cells incubated with AuNPs-DOXA&C and other formulations were evaluated by MTT assay. After 12 h incubation, the AuNPs-DOX-A&C showed no obvious difference of cytotoxicity between the different nanoparticle designs. With time extended to 24, 36, and 48 h, C6 cells treated with AuNPs-DOX-A&C began to exhibit higher cytotoxicity relative to other DOX-tethered particles (Figure 3H). In comparison, the cell viability of C6 cells treated with these particles without loading DOX reached approximately 90% after 48 h incubation, suggesting that these particles were of good biocompatibility (Figure S16). In 10092

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ACS Nano Cy5.5-A&C and control groups (Figure 4A). The fluorescence signal of AuNPs-Cy5.5-A&C began emerging in the glioma site of the right brain with an increasing intensity over time, which was much stronger than that of the control groups, suggesting that AuNPs-Cy5.5-A&C could target glioma sites selectively. The distribution of AuNPs-Cy5.5-A&C was further confirmed by three-dimensional (3D) reconstruction using the signal collected at 24 h (Figure S18). Ex vivo imaging of brains clearly showed the fluorescent intensity of AuNPs-Cy5.5-A&C in the glioma site was much stronger than that of control groups with precise localization (Figure 4B). The semi-quantitative data were further measured to determine the accumulation, which was consistent with the qualitative result (Figure 4C). Herein, we introduced the ratio of fluorescence at the glioma site to fluorescence at the normal brain site (G/B ratio) to evaluate the capacity of glioma selective accumulation of AuNPs-Cy5.5A&C.39 The G/B ratio of AuNPs-Cy5.5-A&C was 1.53, which was higher than that of AuNPs-Cy5.5-PEG (1.07), AuNPsCy5.5-AK (1.12), AuNPs-Cy5.5-CABT (1.11), and free Cy5.5 (1.04) groups, respectively. This result indicated the AuNPsCy5.5-A&C could selectively accumulate in glioma site due to the overexpressed legumain triggered aggregation of AuNPs, which in turn impeded their back-flow out of the target site.40,41 Additionally, ex vivo imaging showed a high signal in the kidney among the major organs (Figure 4D), which is confirmed by the semi-quantitative data (Figure 4E), suggesting that they may be eliminated in the kidney. To validate whether the selective accumulation of AuNPsCy5.5-A&C in glioma site was due to the overexpression of legumain, we further investigated the legumain expression by staining the frozen section of glioma with anti-legumain antibody. High intensity of green fluorescence was observed in the glioma site, while the fluorescence in normal brain was much weaker, indicating the overexpression of legumain was accompanied by the proliferation and progression of glioma cells (Figure 4F). Importantly, the fluorescence intensity of Cy5.5 showed the highest distribution of AuNPs-Cy5.5-A&C in the glioma region, which further validated the responsiveness of AuNPs-Cy5.5-A&C to legumain. Meanwhile, the fluorescence distribution of AuNPs-Cy5.5-A&C in sections of main organs showed similar fluorescent intensity to that of control groups, which was consistent with the ex vivo imaging (Figure S19). Prerequisite for this legumain-induced accumulation of AuNPsCy5.5-A&C in glioma site is leaky vasculatures which enable the particles to be transported via the enhanced permeability and retention (EPR) effect.42,43 After staining with anti-CD34 antibody, clear neovessels were found in glioma region (Figures 4G and S20). Our result supported the idea that high accumulation of AuNPs-Cy5.5-A&C was due to legumaintriggered aggregation of AuNPs, which trapped the nanoparticles within the glioma site and cells. In Vitro and in Vivo Photoacoustic Imaging of AuNPsA&C. Owing to the high density and extinction coefficients, AuNPs can be employed as contrast agents for CT, dark-field imaging, and PA imaging.44,45 We examined whether AuNPsA&C can be used as a contrast agent for fast multispectral optoacoustic tomography (MSOT) imaging. First, we evaluated the light absorbing and photoacoustic properties of the AuNPsA&C in a tissue-mimicking phantom. Figure 5A elucidated the phantom and a typical image obtained after identification of the spectral components contained in the phantom. The PA signal of AuNPs-A&C was similar to AuNPs-PEG as well as AuNPAK and AuNPs-CABT (Figures 5B and S21). After

introduction of legumain, the PA signal of AuNPs-A&C showed the highest intensity with a 1.82-times increase at 24 h, which was much stronger than the 0.47-times increase of AuNPs-PEG. However, the increase of PA signal of AuNPsA&C reduced to 0.98-times after co-incubation with legumain and Ato (Figure 5C). These results indicated that these AuNPsbased formulations possessed the capacity of PA imaging and identified the PA signal of these particles was close at equivalent concentrations of AuNPs. After incubation with legumain, the PA signal of AuNPs-A&C improved greatly relative to control particles. The formation of AuNPs aggregates led to an increasing absorption in near-infrared region between 650 and 900 nm (Figure S22). These performances provided the possibility for in vivo PA imaging using AuNPs-A&C as the contrast agent. Subsequently, we applied AuNPs-A&C for PA imaging at the cellular level. As expected, the PA signal of C6 treated with AuNPs-A&C for 24 h exhibited a higher intensity than that of C6 cells treated with control particles (Figure S23). To investigate the in vivo MSOT imaging capacity and monitoring the accumulation behavior in glioma site, orthotopic glioma-bearing mice were intravenously injected with AuNPs-A&C and control particles. The cross-sectional anatomical PA images through the brain at the glioma area were captured over time. The PA images overlaid with ultrasound signal of tissue, blood oxygenation signal, and blood deoxygenation signal were employed for anatomic guidance. At 2 h after injection, the PA signal of AuNPs-A&C was mainly distributed around the sagittal sinus and submandibular gland but rarely in the glioma site, which was close to that of control groups (Figure 5D, E). At 24 h after injection, the PA signal of AuNPs-A&C emerged precisely in the position referring to the glioma site, in which the signal got stronger at 48 h. However, there was still no obvious signal in glioma site of control groups even after 48 h injection (Figures 5F and S24). The comparison of PA signal in glioma site was further supported by the mean pixel intensity (Figure 5G). These results validated that the legumain-triggered aggregation of AuNPs-A&C was retained in glioma site after systemic administration and was in accordance with the results of in vivo imaging. Delivery of nanostructed agents without targeting ligand to the glioma site in brain is mediated by the EPR effect.46,47 However, this process has its inherent limitations. After systemic administration, nanoparticles are suspectible to opsonization and removal by the reticuloendothelial system.48 Meanwhile, when reaching the brain, the high intratumoral interstitial pressure created by the leaky tumor vasculature limits small-size nanoparticle penetration.40,49 Therefore, to measure the AuNP accumulation in the glioma site, we used ICP-MS analysis. The glioma was excised and prepared for the measurement. The glioma-bearing mice treated with AuNPsA&C for 24 h had the highest percentage injected dose per gram glioma (%ID) of 7.52%. While the %ID of glioma-bearing mice treated with AuNPs-PEG, AuNPs-AK, and AuNPs-CABT were only 3.22%, 2.14%, and 3.89%, respectively (Figure 5H). This suggested that AuNPs-A&C could obtain about 2 times higher accumulation in the glioma site over the PEGylated AuNPs at 24 h after systemic injection, which might provide the possibility for improved therapeutic efficiency of DOX.50 Besides, this retention behavior of each particles in glioma site was observed by TEM. At 24 h after injection, the AuNPs-A&C trapped in glioma cells presented obvious aggregates, which was much larger than that of AuNPs-PEG, AuNPs-AK, and AuNPsCABT in glioma cells (Figure 5I). This phenomenon might 10093

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Figure 6. In vivo anti-glioma effect and pharmacokinetic perform of AuNPs-DOX-A&C. (A) Survival curve of brain glioma-bearing mice treated with different formulations (n = 10). (B) Plasma concentration−time curve of DOX in the mice after intravenous injection with different DOX-loaded formulations. The administration dose of DOX was 3 mg·kg−1; error bars indicate SD (n = 5). (C) H&E staining assays of glioma-bearing brain collected on the day after the last administration; the dashed area indicates glioma site and bars indicate 100 μm.

days, respectively, demonstrating a better therapeutic effect than free DOX group. The median survival time of AuNPsDOX-A&C group was 66 days, which was greatly prolonged and 288% longer than that of the saline group. The pharmacokinetic behaviors of DOX were investigated after intravenous injection of the five formulations (AuNPsDOX-A&C, AuNPs-DOX-PEG, AuNPs-DOX-AK, AuNPsDOX-CABT, and free DOX). The corresponding pharmacokinetic parameters were calculated and recorded (Table 2). The area under the curve (AUC) of free DOX was lower than that of DOX-loaded nanoparticles, suggesting that nanoparticles, as delivery carriers of chemotherapeutics, played an important role in reducing the blood clearance and prolonging the circulating time. AuNPs-DOX-A&C exhibited similar pharmacokinetic profiles with AuNPs-DOX-PEG, AuNPs-DOX-AK, and AuNPs-DOX-CABT (Figure 6B). Meanwhile, the plasma elimination half-life (t1/2) and mean residence time (MRT) of AuNPs-DOX-A&C showed a significant increase compared with free DOX, which manifested that AuNPs-DOX-A&C improved blood circulation time of DOX and thus improved the distribution to glioma site. To understand the potential mechanism that enabled prolonged survival, the glioma-bearing brain slices were prepared and examined by hematoxylin-eosin (H&E) staining assay. In brains treated with saline and free DOX, obvious compact nuclei of glioma cells were observed, which was consistent with their short median survival time. In brains treated with AuNPs-DOX-PEG, AuNPs-DOX-AK, and AuNPsDOX-CABT, the density of glioma cells decreased because PEGylated DOX-loaded nanoparticles were passively delivered

clearly explain the reason why AuNPs-A&C could retain in glioma site, which was due to the large size aggregates in glioma cells, and interstitial matrix blocked their exocytosis and backflow to bloodstream. In Vivo Chemotherapy of AuNPs-DOX-A&C for Glioma. Encouraged by the in vitro and in vivo investigation, we further assessed the anti-glioma efficiency of AuNPs-DOXA&C after systemic administration using a survival study (Figure 6A and Table 1). The median survival time of gliomabearing mice treated with free DOX was prolonged from 17 days to 25 days, which was similar to the saline group. The median survival times of those treated with AuNPs-DOX-PEG, AuNPs-DOX-AK, and AuNPs-DOX-CABT were 28, 31, and 32 Table 1. Median Survival Time of Glioma-Bearing Mice Treated with Different Therapeutic Groups (n = 10) group saline free DOX AuNPs-DOXCABT AuNPs-DOXAK AuNPs-DOXPEG AuNPs-DOXA&C

median (day)

standard error (day)

17.0 25.0 32.0

1.6 3.2 7.1

31.0

18.1

a

82%

28.0

7.4

a

64%

66.0

17.4

significance − − a

a,b,c,d,e

increased survival time − 47% 88%

288%

a

p < 0.05 vs saline. bp < 0.05 vs free DOX. cp < 0.05 vs AuNPs-DOXCABT. dp < 0.05 vs AuNPs-DOX-AK. ep < 0.05 vs AuNPs-DOX-PEG. 10094

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ACS Nano Table 2. Pharmacokinetic Parameters of DOX after Intravenous Injection with Different Formulationsa group

AuNPs-DOX-A&C

AuNPs-DOX-PEG

AuNPs-DOX-AK

AuNPs-DOX -CABT

free DOX

dose (mg/kg) Cmax (ng/mL)a Tmax (h) AUC (h·μg/mL) t1/2 (h) MRT (h) CL (L/h/kg)

3 0.80 ± 0.11 2 84.434* 85.088* 158.28* 0.036

3 0.72 ± 0.09 2 53.175 79.106* 100.39 0.056

3 0.73 ± 0.15 2 72.43* 86.258* 153.96 0.041

3 0.79 ± 0.23 2 48.906 70.047* 89.19 0.061

3 1.15 ± 0.22 0.25 33.356 17.498 69.80 0.090

a Data represent mean data ± SD, n = 5. Cmax represents maximum plasma concentration, Tmax represents time to maximum plasma concentration, AUC represents area under the curve, t1/2 represents plasma elimination half-life, MRT represents mean residence time, and CL represents clearance rate; *p < 0.05 vs free DOX.

Subsequently, 2 mL 1% sodium citrate solution was added rapidly into the mixture. The solution was kept boiling until the color of solution turned to wine red and the concentration of AuNPs was approximately 53 μg·mL−1. One mL AuNPs was incubated with 5 μL SH-PEG-AK (1 mg·mL−1) or SH-PEG-CABT (1 mg·mL−1) for at least 8 h under the condition of 150 rpm·min−1 and 37 °C to harvest AuNPs-AK or AuNPs-CABT. Preparation of AuNPs-DOX-AK, AuNPs-DOX-CABT, and Cy5.5-Loaded Formulations. One mL AuNPs was first incubated with 5 μL pH-sensitive DOX probe dissolved in methanol (1 mg· mL−1). After 8 h incubation at 150 rpm·min−1 and 37 °C, AuNPsDOX were obtained. Then, AuNPs-DOX was further incubated with SH-PEG-AK or SH-PEG-CABT at a dose of 5 μg·mL−1 to harvest AuNPs-DOX-AK or AuNPs-DOX-CABT (Table S2). Using the same procedure, Cy5.5 was applied to these nanoplatforms to obtain AuNPs-Cy5.5-AK or AuNPs-Cy5.5-CABT with an incubation dose of 2 μg·mL−1 (Table S3). Additionally, the AuNPs-DOX-PEG or AuNPsCy5.5-PEG served as the control, whose incubation dose of mPEG-SH was 10 μg·mL−1. Characterization of Legumain-Triggered Aggregation of AuNPs-A&C. One mL AuNPs-AK and 1 mL AuNPs-CABT were centrifuged at 12,000 rpm·min−1 for 10 min. After removing the supernatant, the AuNPs-AK and AuNPs-CABT were resuspended together in 1 mL HEPES buffer at a pH of 7.4, 6.5, 5.5, 5.0, and 4.0. The concentration of AuNPs was 44.75 μg·mL−1. The hydrodynamic diameter of AuNPs-A&C (1 mL) incubated with 2.5 μL legumain (1 mg·mL−1) in different pH conditions was determined over time using DLS analysis. Meanwhile, AuNPs-PEG, AuNPs-AK, and AuNPsCABT at similar concentrations of AuNPs incubated with legumain in pH 5.0 HEPES were determined as the control. Furthermore, TEM images of AuNPs-A&C incubated with legumain for 24 h at the conditions described above were observed using a Hitachi H-600 (Japan) at 200 kV. Cellular Uptake and Exocytosis of AuNPs by ICP-MS. C6 cells were seeded into a 12-well plate with 5 × 104 cells per well and incubated at 37 °C in 5% CO2 humidified atmosphere for 24 h. After washed twice with PBS, the C6 cells were treated with AuNPs-A&C, AuNPs-PEG, AuNPs-AK, and AuNPs-CABT for 4 and 24 h (AuNPs concentration: 45.26, 44.13, 45.33, and 45.2 μg·mL−1). After incubation, the media was removed and washed twice with PBS, and then 500 μL fresh media was added into each well for an extra 2 h incubation. Finally, cell culture and cells of each group were collected at the end of incubation. Cells were digested in 5 mL 70% concentrated nitric acid using a microwave digestion instrument (MARS, U.S.A.). A 400 μL cell culture was digested in 5 mL 70% concentrated nitric acid. All of the digestion solution was further diluted 20-fold by 2% nitric acid, and the gold content was analyzed by ICP-MS (ICAP Q, Thermal, U.S.A.). In Vitro and in Vivo TEM Observation. C6 cells were cultured in culture dish (ΦA = 100 mm) until a density of 3 × 107 cells. Then C6 cells were treated with AuNPs-DOX-A&C, AuNPs-DOX-PEG, AuNPs-DOX-AK, and AuNPs-DOX-CABT for 24 h (AuNPs concentration: 45.53, 46.01, 45.65, and 45.87 μg·mL−1). Cells were washed twice with PBS, harvested, and then centrifuged at 1500 rpm· min−1 for 15 min. After removing the supernatant, 1 mL 0.5%

to glioma site and released DOX in response to acidic tumor microenvironment. However, the density of glioma cells in brains treated with AuNPs-DOX-A&C was much lower than that of control formulations, demonstrating the enhanced antiglioma effect. Additionally, the systemic toxicity of DOX and biocompatibility of AuNPs were further evaluated by the H&E staining of major organs (Figure S25). Obvious typical myocardial injury associated with acute neutrophil accumulation was found in the heart of mice treated with free DOX, suggesting acute cardiotoxicity of free DOX. In comparison, there were no neutrophils in AuNPs-based groups, probably due to the reducing accumulation in the heart (see Figure 4E). H&E staining of liver, spleen, lung, and kidney treated with these AuNP-based formulations also demonstrated no obvious damage compared with the saline group, indicating the excellent biocompatibility of AuNPs in vivo.

CONCLUSION In summary, we developed a tumor microenvironment sensitive platform, AuNPs-A&C, which consisted of AuNPs-AK and AuNPs-CABT. The AuNPs-A&C could undergo a legumaintriggered click cycloaddition, resulting in an obvious size increase both in vitro and in vivo. This AuNPs-based functional nanoplatform was stable with a stealth surface in systemic circulation and was able to aggregate in a rapid motion upon entering into the glioma site. In vitro cellular study suggested that AuNPs-DOX-A&C showed an enhanced accumulation in the cell, leading to elevated cell-induced apoptosis. In vivo fluorescence imaging and PA imaging demonstrated that AuNPs-A&C could selectively accumulate in the glioma site, enabling the capacity to precisely diagnosis the glioma. Additionally, ICP-MS analysis quantitatively demonstrated that this legumain-triggered aggregation could acquire a higher level accumulation in the glioma site compared with the PEGylated nanoparticles. More importantly, this strategy showed a positive preclinical significance in improving the therapeutic outcome of glioma with reduced systemic toxicity of DOX. The median survival time of orthotopic C6 gliomabearing mice treated with AuNPs-DOX-A&C was much longer than that of control groups. Our strategy may provide a prospective to engineer a nanoplatform for improving accumulation of therapeutic agents in tumor site, leading to an enhanced treatment outcome. MATERIALS AND METHODS Preparation of AuNPs-AK and AuNPs-CABT. AuNPs were prepared using the sodium citrate as a reductive agent. Briefly, 2.5 mL HAuCl4 solution (2.2 mg·mL−1) was diluted to 50 mL using deionized water and heated to boiling under vigorous magnetic stirring. 10095

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ACS Nano glutaraldehyde was introduced slowly to infiltrate cells, followed by 10 min storage at 4 °C. Next, the cells were centrifuged at 12,000 rpm· min−1 for 15 min, and the supernatant was abandoned. Finally, 1 mL 3% glutaraldehyde was introduced slowly to infiltrate cells. The samples were then polymerized at 60 °C for 24 h, cut into ultrathin sections using an ultramicrotome (leica), and placed on 150-mesh copper grids. The ultrasection of cell was stained with uranyl acetate (2% in ethanol) for 10 min and then with lead citrate for 5 min. TEM images of C6 cells were observed using a Hitachi H-600 (Japan) at 200 kV. After intravenous administration of AuNPs-A&C, AuNPs-PEG, AuNPs-AK, and AuNPs-CABT at an equivalent administration dose of 25 mg·kg−1 for 24 h, the glioma was excised from brain and cut into a 1 mm3 cube. The cube was further fixed with 3% glutaraldehyde solution and embedded in epoxy resin. The sample was cut into ultrathin sections using an ultramicrotome and placed on 150-mesh copper grids. The ultrasection of glioma was stained with uranyl acetate (2% in ethanol) for 10 min and with lead citrate for 5 min and then observed under TEM. In Vivo Fluorescent Imaging. The orthotopic C6 glioma-bearing mice were established by injecting C6 cells into the right brain of mice with a density of 2.5 × 105 cells in 5 μL PBS.51 After 10 days implantation, the C6 glioma-bearing mice were intravenously administrated with AuNPs-Cy5.5-A&C and control Cy5.5-loaded AuNPs formulations, and the administration dose of Cy5.5 was 1.2 mg·kg−1. Fluorescence distribution of whole body was acquired at different time intervals after administration using an in vivo imaging system (IVIS Spectrum, Caliper, U.S.A.). In vivo imaging conditions: IVIS Imaging System with filter sets (emission = 615−665 nm, excitation = 695−770 nm) was used to perform optical imaging studies. The scanning parameters include: excitation wavelength = 650 nm, emission wavelength = 720 nm, field of view = 13.5 cm and fluency rate = 2 mW/cm2. The camera was set to a maximum gain, a binning factor of 4, and a luminescent exposure time of 4 s. Immunofluorescence. The isolated brains and major tissues were first fixed within 4% paraformaldehyde overnight, following dehydration by 15% and 30% sucrose solution for another 24 h in sequence. The frozen sections of brains were stained with primary anti-legumain antibody (dilution ratio 1:100) at 4 °C overnight and anti-CD34 antibody (dilution ratio 1:100). The sections were further stained with corresponding FITC-conjugated secondary antibodies (dilution ratio 1:200) at room temperature for 2 h. Tris-buffered saline was utilized for dilution and washing through all experimental procedures. Images were captured using a confocal microscope (Olympus FV1000, Japan). Glioma-Specific Accumulation of AuNPs by ICP-MS in Vivo. After intravenous administration of AuNPs-A&C and control AuNPsbased formulations for 24 h (the administration dose of AuNPs was 25 mg·kg−1), the glioma was excised from brains and washed in iced PBS. Subsequently, the gold content in glioma of each group was further examined using ICP-MS following the same procedures described above. In Vitro and in vivo PA Imaging. In vitro phantom and in vivo PA imaging investigations were carried out using the MSOT imaging system (MSOT in Vision 128, iTheramediacal, Germany). PA signal was acquired using a 128-element concave transducer array spanning a circular arc of 270° from 680 to 980 nm with average pulse duration of about 10 ns and repetition rate of 10 Hz. The transducer array has a central frequency of 5 MHz, which is used to provide a transverse spatial resolution of 150 μm. Image acquisition of sample was translated via the transducer array along its axis across the volume region of interest. Phantom preparation and imaging: Cylindrical phantom with diameter of 2 cm was prepared using agar and intralipid. Briefly, 1.3 g agar and 5 mL of 20% intralipid were added into 94 mL of deionized water and then heated to boiling. When the mixture cooled to 50°, it was filled into a 20 cc syringe. A straw (white plastic, inner diameter of 2−3 mm) was cut to a 3−4 cm length, and each side was sealed with glue. Two straws in the center of the phantom were parallelly submerged but not to be completely submerged. The straws were

stabilized until the phantom solidified so that a different straw containing the contrast agent could replace this air-filled straw. The phantom was imaged, and the straws replaced in between scans. The concentrations of AuNPs-A&C, AuNPs-PEG, AuNPs-AK, and AuNPsCABT for PA imaging using phantom were 44.85, 45.59, 45.32, and 45.53 μg·mL−1. For in vivo PA imaging of mice intravenous injected with AuNPsA&C, AuNPs-PEG, AuNPs-AK, and AuNPs-CABT (0.6 mg AuNPs in 0.2 mL PBS), the orthotopic C6 tumor-bearing nude mice were first anaesthetized with 3% isoflurane, and ultrasound gel was applied on the surface of the mouse skin. Subsequently, multiwavelength PA imaging was acquired under the same condition with a step size of about 0.3 mm step distance along the long axis of brain at 690, 715, 730, 760, 800, 815, and 850 nm, and the maximum contrast PA signal could be obtained when the excitation wavelength is 680 nm during the PA imaging experiments. In Vivo Survival Monitoring. C6-glioma-bearing mice were established as described above. Ten days after implantation, the mice were randomly divided into 6 groups (13 mice per group). At days 10, 12, 14, 16, and 18, each group of mice was treated with AuNPs-DOXA&C, AuNPs-DOX-AK, AuNPs-DOX-CABT, AuNPs-DOX-PEG, and free DOX, and the administration dose of DOX was 3 mg/kg. The saline-treated group was settled as the control. For all groups, 10 mice/ group were monitored, and the overall survival and body weight was determined. Three mice/group were sacrificed on day 19, and brains were sampled for H&E staining. Additionally, major organs were also sampled for H&E staining. Statistics Analysis. A comparison between experimental and control groups was analyzed by the unpaired Student’s t test. The statistical analysis for survival was determined using Kaplan−Meier survival plot (SPSS 16.0), and the survival curves were compared using the log-rank (Mantel−Cox) test. p < 0.05, 0.01, and 0.001 were considered a statistically significant difference and remarked with *, **, ***, respectively.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b05070. Materials, more detail methods and supporting figures (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Huile Gao: 0000-0002-5355-7238 Author Contributions

H.G., Q.H., and S.R. designed the experiments. S.R., C.H., X.T, X.C., W.X., and K.S. were involved in experiments. S.R. and H.G. analyzed the data and wrote the paper. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS National Natural Science Foundation of China (31571016, 81402866) and the National Basic Research Program of China (973 Program, 2013CB932504). REFERENCES (1) Bondy, M. L.; Scheurer, M. E.; Malmer, B.; Barnholtz-Sloan, J. S.; Davis, F. G.; Il’yasova, D.; Kruchko, C.; McCarthy, B. J.; Rajaraman, P.; Schwartzbaum, J. A.; Sadetzki, S.; Schlehofer, B.; Tihan, T.; Wiemels, J. L.; Wrensch, M.; Buffler, P. A. Brain Tumor Epidemiology: Consensus 10096

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DOI: 10.1021/acsnano.6b05070 ACS Nano 2016, 10, 10086−10098

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DOI: 10.1021/acsnano.6b05070 ACS Nano 2016, 10, 10086−10098