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Ligand Mediated and Enzyme-Directed Precise Targeting and Retention for Enhanced Treatment of Glioblastoma Shaobo Ruan, Wei Xiao, Chuan Hu, Huajin Zhang, Jingdong Rao, Sihan Wang, Xiao Wang, Qin He, and Huile Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017

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ACS Applied Materials & Interfaces

Ligand Mediated and Enzyme-Directed Precise Targeting and Retention for Enhanced Treatment of Glioblastoma Shaobo Ruan, Wei Xiao, Chuan Hu, Huajin Zhang, Jingdong Rao, Sihan Wang, Xiao Wang, 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

ACS Paragon Plus Environment


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ABSTRACT: Glioblastoma (GBM), one of the most lethal cancers, remains a hard task to handle. The major hurdle of nanostructured therapeutic agents comes from the limited retention at GBM site and poor selectivity. In this study, we reported dual-functional gold nanoparticles (AuNPs) to figure out the biological barrier and improve their accumulation in GBM. The nanoparticles, AuNPs-A&C-R, were composed of two functional particles, one was Ala-Ala-Asn-Cys-Asp (AK) and

R8-RGD

co-modified

AuNPs

(AuNPs-AK-R)

and

the

other

was

2-cyanoo-6-amino-benzothiazole and R8-RGD co-modified AuNPs (AuNPs-CABT-R). The AuNPs-A&C-R could aggregate in the presence of legumain, resulting in a size increase from 41.4 ± 0.6 nm to 172.9 ± 10.2 nm after 8 h incubation. After entering circulatory system, the AuNPs-A&C-R actively targeted integrin αvβ3 receptor on BBB and mediated particles transcytosis across BBB, then targeted the receptor on GBM cells. Once the AuNPs-A&C-R entered into GBM, the AuNPs-A&C-R further formed aggregates with increased size extracellularly or intracellularly due to the overexpressed legumain, which in turn blocked their back-flow to bloodstream or limited their exocytosis by cells. In vivo optical imaging demonstrated that AuNPs-A&C-R efficiently delivered to GBM site and retained with high selectivity. We further confirmed AuNPs-A&C-R acquired a higher accumulation at GBM site compared with AuNPs-A&C and AuNPs-R due to the synergistic effect. More importantly, the doxorubicin loaded AuNPs-A&C-R showed an improved chemotherapeutic effect to C6 GBM-bearing mice, which significantly prolonged the median survival time by 1.22-fold and 1.27-fold compared with doxorubicin loaded AuNPs-A&C and doxorubicin loaded AuNPs-R respectively. These results suggested the dual-functional nanoplatform is promising for the GBM treatment.


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Keywords: enhanced accumulation; gold nanoparticles; click cycloaddition; legumain; R8-RGD 1. INTRODUCTION GBM, one of the most malignant brain tumors, is characterized by highly proliferative, aggressive and distinctive pathological heterogeneities.1,2 The life expectancy of GBM baring patients is less than 2 years after maximal treatment since diagnosis.3 Despite the progress in surgical resection, chemotherapy and radiotherapy in the past few decades, the treatment outcome for GBM remains unsatisfied. The extensive development of nanomaterials and nanotechnology provides a promising approach for the delivery of therapeutic and diagnostic agents to GBM.4 However, the major hurdle of nanostructured therapeutic agents comes from the insufficient accumulation at GBM site and poor selectivity. The blood-brain barrier (BBB) is a dynamic barrier that modulates the passage of molecules from systemic circulation to brain parenchyma and also protects brain against harmful substances.5,6 Pharmacological treatment of GBM is limited by the lack of delivery platforms that can transport across BBB efficiently.7 It has been well revealed that there are several transport pathways to mediate water-soluble molecules or therapeutic agents across BBB. Among these, receptor-mediated transcytosis (RMT) has received much attention.8,9 Kinds of well-characterized receptors are utilized for brain targeting, e.g. lipoprotein receptors, integrin receptors, and transferrin receptors.10 Despite various ligands are designed with high affinity to corresponding receptors, the transcytosis efficiency of the ligands across BBB is still modest because of the saturation phenomenon of ligand-receptor interaction. Here, we introduced a tandem peptide, R8-RGD, which was conjugated octaarginine with RGD. Octaarginine is a kind of cell penetrating peptide that possesses superior membrane permeability.11 While RGD could specifically target



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integrin αvβ3 receptor that expressed on both BBB and GBM cells.12 Our previous study had confirmed its excellent targeting efficiency and BBB penetrating capacity.13 Active targeting delivery to GBM is influenced by the complexity of biological and pathological systems. Particles with proper surface modification can avoid clearance by reticuloendothelial system (RES) in bloodstream. However, the high interstitial fluid pressure (IFP) of GBM impedes the particles extravasation from neovessels after they transport across BBB, resulting in insufficient internalization by GBM cells. Small-size particles have been widely reported with efficient diffusion into interstitial matrix, however, the application shadows by back-flowing to bloodstream, which depleted the amount retained within tumors.14-16 In contrast, large-size particles cannot penetrate into deep tumor but are able to dock in the extracellular matrix.17,18 Therefore, it is great important to improve GBM targeting delivery and enhance penetration and retention at GBM for nanostructured therapeutic agents simultaneously. Our previous study has demonstrated a functional nanoplatform, AuNPs-A&C, could deliver drugs to GBM site via the enhance permeability and retention (EPR) effect and aggregate in the presence of legumain.19 In this study, we further conceived a dual-functional nanoplatform, AuNPs-A&C-R, which combined the better GBM retention ability of AuNPs-A&C and the better GBM targeting ability of R8-RGD. The nanoplatform composed by two functional compartments, one was AK peptide and R8-RGD co-modified AuNPs (AuNPs-AK-R), the other was 2-cyano-6-amino-benzothiazole and R8-RGD co-modified AuNPs (AuNPs-CABT-R). The AuNPs-A&C-R could specifically target the integrin αvβ3 receptor on BBB and mediated it transcytosis across BBB, then targeted the receptor on GBM cells. Once they were transited to GBM site, they could further undergo a click cycloaddition between two particles after activation


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by legumain, resulting in the formation of aggregates in the extracellular matrix or in the endosome/lysosome. These aggregates with large size in turn blocked their back-flow to bloodstream and restricted their exocytosis by cells.16,18 In vitro investigation confirmed the effectiveness of legumain-triggered aggregation of AuNPs-A&C-R. In vivo, AuNPs-A&C-R could efficiently deliver to GBM site and retain at there with precise localization. ICP-OES analysis further demonstrated AuNPs-A&C-R could accumulate higher than AuNPs-A&C and AuNPs-R due to the synergistic effect of GBM targeting delivery and enhanced retention. Importantly, this dual-functional nanoplatform showed an improved chemotherapeutic outcome of C6 GBM-bearing mice.



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2. MATERIALS AND METHODS 2.1. Materials. Chloroauric acid was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Cy5.5-NHS ester was bought from Lumiprobe Corporation (Hallandale Beach, USA). Doxorubicin hydrochloride was bought from Beijing Huafeng United Technology Co., Ltd (Beijing, China). R8-RGD peptide was custom-made by Sangon Biotech Co., Ltd (shanghai, China) and AK peptide was custom-made by PHTD Peptide Co., Ltd (Zhengzhou, China). 2-Cyano-6-aminobenzothiazole

(CABT)

was

obtained

from

Shanghai

Chemical

Pharm-Intermediate Tech. Co., Ltd (Shanghai, China). Rabbit anti-legumain antibody (H-300) was bought from Santa Cruz Biotechnology, lnc. (Santa Cruz, USA) and rabbit anti-integrin αvβ3 antibody was bought from Abcam Ltd (Hong Kong, China). All chemical reagents were analytical grade or better. Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were bought from Gibco (Grand Island, USA). Penicillin-streptomycin and trypsin were bought from Solarbio life science (Beijing, China). Plastic cell culture dishes and plates were obtained from Wuxi NEST Biotechnology Co., Ltd (Wuxi, China). Male Kunming mice and male nude mice were obtained from Chengdu experimental animal center (Chengdu, China) and maintained under standard housing conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Sichuan University. 2.2. Preparation of AuNPs-AK-R and AuNPs-CABT-R. 20 nm AuNPs were synthesized and characterized as described previously in our literature.20 SH-PEG-AK, SH-PEG-CABT and SH-PEG-R8-RGD were synthesized according to our previous procedures.13,19 To get AuNPs-AK-R, 1 mL AuNPs solution was co-incubated with 5 µL SH-PEG-AK (1 mg mL-1) and 3


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µL SH-PEG-R8-RGD (1 mg mL-1) at 25 °C and 150 rpm min-1 for 8 h. Similarly, 1 mL AuNPs solution was incubated with 5 µL SH-PEG-CABT (1 mg mL-1) and 3 µL SH-PEG-R8-RGD (1 mg mL-1) under the same condition to obtain AuNPs-CABT-R. Preparation of SH-PEG-AK modified AuNPs (AuNPs-AK), SH-PEG-CABT modified AuNPs (AuNPs-CABT), SH-PEG-R8-RGD modified AuNPs (AuNPs-R) and SH-PEG modified AuNPs (AuNPs-P) was described in Supporting Information. 2.3. Characterization of legumain-responsive AuNPs-A&C-R. 1 mL AuNPs-AK-R solution and 1 mL AuNPs-CABT-R solution were centrifuged respectively at 12000 rpm min-1 for 10 min, then AuNPs-AK-R and AuNPs-CABT-R were re-suspended together in 1 mL HEPES buffer (pH 5.0). The hydrodynamic size of AuNPs-A&C-R, AuNPs-A&C, AuNPs-R and AuNPs-P incubated with 2.5 µL legumain (1 mg mL-1) were monitored using dynamic light scattering (DLS) analysis. The morphology of AuNPs-A&C-R and AuNPs-R was observed using H-600 transmission electron microscopy (TEM) at 200 kV (Hitachi, Japan). 2.4. Confocal laser microscopy. Murine C6 cells were continuously cultured in complete high glucose DMEM cell culture containing 10% FBS, 100 U mL-1 of penicillin and 100 U mL-1 of streptomycin, and were maintained at 37 °C in 5% CO2 humidified atmosphere and all experiments were carried out in the logarithmic phase of cell growth. Before in vitro cellular stduy, we firstly conjugated a pH sensitive DOX probe onto the surface of AuNPs as described previously (see methods in Supporting Information).20 C6 cells were seeded onto 400 mm2 coverslip pre-placed in 6-well plate at a density of 5×104 per well and allowed to grow until 60% confluent. Then medium was replaced with fresh medium containing DOX-AuNPs-A&C-R, DOX-AuNPs-A&C, DOX-AuNPs-R or DOX-AuNPs-P (at equal DOX



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dose of 5 µg mL-1), and further incubated for 1 h, 4 h and 24 h. At 30 min before end, LysoTracker Red (DND-99, 100 nM) was introduced. After incubation, cells were washed with PBS (pH 7.4) twice, fixed with fresh 4% paraformaldehyde for 20 min at room temperature and then counterstained with 4´,6-diamidino-2-phenylindole (DAPI, 0.5 µg mL-1, 5 min). The coverslips were transferred onto glass microscope slides with a drop of antifade mounting media (Beyotime Biotechnology, China). Fluorescence was captured using an Olympus Fluoview FV 1000 confocal laser microscopy (CLSM) (Japan). 2.5. Flow cytometer. C6 cells were seeded into 12-well plate at a density of 5×104 per well and cultured for 24 h. Then the medium was replaced with fresh medium containing DOX-AuNPs-A&C-R, DOX-AuNPs-A&C, DOX-AuNPs-R or DOX-AuNPs-P (at equal DOX dose of 5 µg mL-1). The cells were further incubated for designed periods of time, and then washed with PBS (pH 7.4) twice, harvested, and suspended in 350 µL of PBS for analysis using Cytomics FC 500 Flow Cytometer CXP (Beckman Coulter, USA). 2.6. In vitro BBB model transcytosis study. Millicell Hanging Cell Culture Inserts were applied for establishing in vitro BBB model. Briefly, bEnd.3 cells were seeded into the 12-well cell culture inserts pre-placed in 12-well plate at a density of 2 × 105 per well and cultured for 2 days. The transendothelial electric resistance (TEER) of the bEnd.3 monolayers was determined by millicell ERS (Millipore, USA) and bEnd.3 monolayers were allowed for further study in the case TEER over 200 Ω. Meanwhile, C6 cells were seeded into another 12-well plate at a density of 5×104 per well and were cultured for 24 h. The cell culture inserts with bEnd.3 monolayers were transferred to 12-well plate containing C6 cells and co-cultured for 12 h. Then different DOX-tethered nanoparticles were introduced into cell culture inserts (donor chamber). After


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incubation for 2 h, the C6 cells in plate were washed with PBS twice, harvested, and suspended in 350 µL of PBS respectively for analysis using Cytomics FC 500 Flow Cytometer CXP (Beckman Coulter, USA). 2.7. Uptake mechanism study. C6 cells were seeded into 12-well plate at a density of 5×104 per well and cultured for 24 h. Then C6 cells were pre-incubated with various endocytosis inhibitors, including free RGD (200 µg mL-1), poly-lysine (400 µg mL-1), sodium azide (3.25 mg mL-1), amiloride (31.18 µg mL-1), Genistein (56 µg mL-1) and Nystain (23 µg mL-1). After 1 h pre-incubation with the inhibitors, the C6 cells were further incubated with DOX-AuNPs-A&C-R and DOX-AuNPs-R for another 2 h. Then cells were treated as described above for analysis using Cytomics FC 500 Flow Cytometer CXP (Beckman Coulter, USA). Meanwhile, C6 cells pre-incubated under the condition of 4 ºC were also evaluated. 2.8. In vivo living fluorescent imaging. Before in vivo optical study, we firstly conjugated a pH sensitive Cy5.5 probe onto the surface of AuNPs as described previously (see methods in Supporting Information).13 The C6 GBM-bearing nude mice were established as described in our previous literature.13 Briefly, male nude mice were first anesthetized by intraperitoneal injection with 5% chloral hydrate. C6 cells (2.5 × 105 cells suspended in 5 µL PBS) were implanted into right striatum (1.8 mm lateral, 0.6 mm longitudinal, 3 mm of depth) of the mice using a brain stereotactic fixation device with mouse adapter. Two weeks after implantation, the intracranial C6 GBM bearing mice were randomly divided into four groups (3 mice for each group): Cy5.5-AuNPs-A&C-R, Cy5.5-AuNPs-A&C, Cy5.5-AuNPs-R and Cy5.5-AuNPs-P. Each mouse was intravenously injected with corresponding formulations at equal Cy5.5 dose of 1 mg kg-1 through the tail vein. Then the fluorescent distribution and radiograph of whole body was acquired



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by an In-vivo Xtreme Imaging System (Xtreme BI, Bruker, USA). Associated default conditions for fluorescence imaging: Exposure time = 2 s, the camera was set to a f-stop = 2, a binning factor of 2 × 2. Filter setting: excitation = 650 nm and emission = 700 nm. Associated default conditions for X-Ray imaging: Exposure time = 1.2 s, the camera was set to a f-stop = 2, a binning factor of 1 × 1. 2.9. In vivo photoacoustic (PA) imaging. The C6 GBM-bearing Kunming mice were established as described above. After two weeks implantation, mice were intravenously injected with AuNPs-A&C-R, AuNPs-A&C, AuNPs-R and AuNPs-PEG respectively (0.6 mg AuNPs in 0.2 mL PBS). For in vivo PA imaging, the C6 GBM-bearing Kunming mice were induced anesthesia with 3% isoflurane and maintained anesthesia with 2% isoflurane. Subsequently, ultrasound gel was applied on the surface of the mice skin, especially on the brain skin. The PA imaging of mice was acquired under the same condition with a step size of 0.3 mm distance along the long axis of brain at 690, 715, 730, 760, 800, 815 and 850 nm. All these PA signals of multi-wavelength were collected and reconstructed at 690 nm. The in vivo PA imaging was performed using a fast multispectral optoacoustic tomography (MSOT) imaging system (in Vision 128, iThera medical, Germany). PA signal was acquired using a 128-element concave transducer assay spanning a circular arc of 270 form 680 nm 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 mice was translated via the transducer array along its axis across the volume region of interest.


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2.10. ICP-OES analysis. In vitro cellular study: C6 cells were seeded into 12-well plate with a density of 5 × 104 per well and cultured for 24 h. Then the medium was replaced with fresh serum-free medium containing DOX-AuNPs-A&C-R, DOX-AuNPs-A&C, DOX-AuNPs-R and DOX-AuNPs-P (AuNPs incubation dose: 50.31, 51.68, 51.19, 50.82 µg mL-1). After incubation for 4 h and 24 h, the cells were washed with PBS (pH 7.4) twice, harvested and then digested in 1 mL 68% concentrated nitric acid and 0.5 mL 30% hydrogen peroxide under heating condition of 110 °C for 12 h. All of the digestion solution was further diluted 20-fold by 2% nitric acid and the gold content was detected by 2100DV inductively coupled plasma optical emission spectrometer (ICP-OES) (PE, USA). In vivo accumulation at GBM: The C6 GBM-bearing Kunming mice were intravenously injected with AuNPs-A&C-R, AuNPs-A&C, AuNPs-R and AuNPs-P respectively at an equal dose of 25 mg kg-1. At 24 h, the brains were peeled off from the mice and washed in iced PBS (pH 7.4), and then digested in 1 mL 68% nitric acid and 0.5 mL 30% hydrogen peroxide under the heating condition of 110 °C for 12 h. All of the digestion solution was further diluted 20-fold by 2% nitric acid and the gold content was detected by 2100DV ICP-OES (PE, USA). 2.11. In vivo anti-GBM evaluation. C6 GBM-bearing Kunming mice were established as described above. 10 days after implantation, the mice were randomly divided into 6 groups (13 mice for each group): DOX-AuNPs-A&C-R group, DOX-AuNPs-A&C group, DOX-AuNPs-R group, DOX-AuNPs-P group, free DOX group and normal saline group. At day 10, 12, 14, 16 and 18, mice were intravenously injected with corresponding formulations with a DOX dose of 3 mg kg-1. For all groups, the overall survival time of mice was monitored. One day after the last dose,



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three mice from each group were sacrificed, brain and major organs were then sampled for hematoxylin-eosin (H&E) staining. 2.12. Statistical analysis. Comparison between two groups was evaluated by the paired Student’s t test. The statistical analysis for survival was determined using Kaplan-Meier survival plot (SPSS 16.0) and the comparison of survival time was analyzed using the log-rank (Mantel-Cox) test. p < 0.05, 0.01, 0.001 were considered a statistical significance difference and represented with *, **, *** respectively.


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3. RESULTS Legumain triggered aggregation of AuNPs-A&C-R. The hydrodynamic size of AuNPs-A&C-R and control particles had slight increase than the bare AuNPs, suggesting these formulations were successfully prepared through the chelation of “S-Au” (Table S1).21 Our previous study has confirmed the AuNPs-A&C could aggregate in the presence of legumain, which was induced by the click cycloaddition between 1,2-thiolanmio group and cyano group.19 In this study, our aim was to validate the functional nanoplatform could also aggregate after R8-RGD modification. We thus incubated AuNPs-A&C-R, AuNPs-A&C, AuNPs-R and AuNPs-P with legumain in pH 5.0 HEPES buffer, the hydrodynamic size of AuNPs-R and AuNPs-P slightly increased with the extend of time and presented in similar trend, which was mainly due to the protein absorption (Figure 1A). In comparison, the hydrodynamic size of AuNPs-A&C-R increased from 41.4 ± 0.6 nm to 172.9 ± 10.2 nm after 8 h incubation, which was much larger than that of AuNPs-R and AuNPs-P, suggesting the successful aggregation between AuNPs-AK-R and AuNPs-CABT-R (Figure 1B). However, the size increase was slightly lower than that of AuNPs-A&C, indicating R8-RGD modification minorly hindered the click cycloaddition. To further investigate the aggregation of AuNPs-A&C-R, we next evaluated the intensity distribution of size. The intensity distribution of AuNPs-A&C-R shifted from 39.4 nm (peak 1, 100%) to 64.2 nm (peak 1, 58.1%) and 745.7 nm (peak 2, 41.9%) after 8 h incubation with legumain (Figure 1C). In contrast, the distribution of AuNPs-R shifted from 30.60 nm (peak 1, 100%) to 31.26 nm (peak 1, 78.2%) and 452.5 nm (peak 2, 21.8%) after incubation (Figure S1). Additionally, the TEM images clearly showed the aggregate of AuNPs-A&C-R, which was consisted of abundant particles compared with their initial morphology.



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Figure 1. Characterization of legumain-triggered aggregation of AuNPs-A&C-R. A: Hydrodynamic size of AuNPs-A&C-R, AuNPs-A&C, AuNPs-R and AuNPs-P after incubation with legumain for different time intervals, data represent mean SD (n = 3). B: Scheme of the molecule mechanism of legumain-activated click cycloaddition and aggregation process of AuNPs-A&C-R. C: Intensity distribution of AuNPs-A&C-R after incubation with legumain at 0 h and 8 h using DLS analysis, inner graph indicated the corresponding TEM images of AuNPs-A&C-R, bars represent 100 nm.

In vitro cellular uptake. C6 cells were chosen due to its high expression of integrin αvβ3 receptor and legumain.19,22 Confocal images of C6 cells treated with DOX-AuNPs-A&C-R and DOX-AuNPs-R showed slight higher fluorescent signal than that of DOX-AuNPs-A&C and DOX-AuNPs-P at 1 h (Figure 2A), suggesting the R8-RGD enhanced the cellular internalization at short incubation time. As time went on, the fluorescent intensity of each group increased because of continueous internalization, while the fluorescence in C6 cells treated with


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DOX-AuNPs-A&C-R displayed stronger intensity than control nanoparticles at 24 h, indicating DOX-AuNPs-A&C-R accumulated more in C6 cells. In addition, the mean fluorescent intensity measured by imageJ directly confirmed that the cellular internalization of DOX-AuNPs-A&C-R was highest after 24 h incubation (Figure S2). Next, we determined the cellular internalization using flow cytometry and ICP-OES analysis respectively. For flow cytometry analysis, the cellular uptake of DOX-AuNPs-A&C-R and DOX-AuNPs-R by C6 cells at 0.5 h, 2 h, 4 h and 8 h was higher than that of DOX-AuNPs-A&C and DOX-AuNPs-P (Figure 2B), which was mainly due to that the R8-RGD could specifically recognize the integrin αvβ3 receptors expressed on C6 cells and mediate endocytosis.22 With incubation time extended to 24 h, C6 cells treated with DOX-AuNPs-A&C-R showed highest intensity among all groups. The results validated the effectiveness of synergistic effect: namely receptor-mediated endocytosis followed by the legumain-induced intracellular aggregation and restricted exocytosis. ICP-OES analysis showed the percentage incubated dose of cellular uptake of DOX-AuNPs-A&C-R, DOX-AuNPs-A&C, DOX-AuNPs-R and DOX-AuNPs-P was 3.60 ± 0.55%, 3.81 ± 0.49%, 4.22 ± 0.23% and 2.67 ± 0.52% respectively after 2 h incubation (Figure 2C). These percentages increased to 15.34 ± 1.23%, 13.12 ± 0.92%, 12.32 ± 1.06% and 8.82 ± 0.89% at 24 h, which further supported that the cellular internalization of DOX-AuNPs-A&C-R by C6 cells was higher than control particles. Although the uptake study demonstrated higher cellular internalization of R8-RGD modified nanoparticles, it is still important to investigate the transcytosis mechanism across BBB. bEnd.3 cells exhibied endothelial properties and were thus used for establishing in vitro BBB model and determining the transcytosis efficiency.23-25 Due to the αvβ3 integrin receptor was overexpressed on bEnd.3 cells,22 the cellular uptake of different particles by bEnd.3 cells displayed similar trend to



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that of C6 cells (Figure S3). Next, we further incubated the DOX-AuNPs-A&C-R in a transwell model and collected the C6 cells for flow cytometry analysis. The C6 cells showed a much stronger cellular uptake of DOX-AuNPs-A&C-R

and

DOX-AuNPs-R than

that of

DOX-AuNPs-A&C and DOX-AuNPs-P (Figure 2D). The results directly indicated that DOX-AuNPs-A&C-R and DOX-AuNPs-R possessed stronger ability to cross BBB model, which was mainly due to RGD domain of R8-RGD mediated nanoparticles endocytosis and R8 domain promoted nanoparticles penetration through cellular membrane of bEnd.3 monolayers.

Figure 2. In vitro cellular uptake study. A: Confocal images of C6 cells treated with DOX-AuNPs-A&C-R and control particles for 1 h, 4 h and 24 h, bars represent 20 µm. B: Flow cytometer analysis of C6 cells treated with DOX-AuNPs-A&C-R and control particles for 0.5 h, 2


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h, 4 h, 8 h and 24 h, *p < 0.05 and **p < 0.01. C: ICP-OES analysis of C6 cells treated with DOX-AuNPs-A&C-R and control particles for 2 h and 24 h, *p < 0.05 and **p < 0.01. D: Cellular uptake of C6 cell treated with DOX-AuNPs-A&C-R and control particles for 2 h in the presence of BBB model using flow cytometry analysis, inner graph depicts the schematic diagram of in vitro BBB model, * represents statistical significance vs DOX-AuNPs-A&C and DOX-AuNPs-P (p < 0.05), & represents statistical significance vs DOX-AuNPs-A&C and DOX-AuNPs-P (p < 0.05). Internalization mechanism. Various endocytosis inhibitors were pre-incubated with C6 cells and the inhibition rate was calculated to evaluate the uptake mechanism. The cellular uptake of DOX-AuNPs-A&C-R was decreased to 78.29% after pre-incubation with free RGD, indicating that the RGD domain of R8-RGD mediated the cellular internalization via specifically recognition to integrin receptors expressed on C6 cells (Figure S4). Poly-lysine showed an uptake of DOX-AuNPs-A&C-R down to 90.3%, suggesting the R8 domain of R8-RGD promoted cellular internalization through positive charge mediated absorption. 4 ºC and sodium azide inhibited uptake down to 64.5% and 88.5%, indicating energy-dependent receptor-mediated endocytosis were involved in primary internalized pathway. In addition, the amiloride, genistein and nystatin inhibited the cellular uptake down to 80.9%, 82.1% and 90.2% respectively, indicating the cellular internalization of DOX-AuNPs-A&C-R involved several pathways. However, the R8 domain and RGD domain of R8-RGD contribute to the dominant effect to enhance cellular uptake of DOX-AuNPs-A&C-Rand DOX-AuNPs-R. To understand why the DOX-AuNPs-A&C-R showed higher the cellular accumulation than DOX-AuNPs-A&C and DOX-AuNPs-R, we further engaged two types of fluorescence dyes



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(DOX and Cy5.5) to conjugate onto AuNPs-AK-R and AuNPs-CABT-R respectively, namely Cy5.5-AuNPs-AK-R and DOX-AuNPs-CABT-R. After incubated with C6 cells for 24 h, obvious fluorescence of DOX (green) and fluorescence of Cy5.5 (megenta) could be observed in the same cells (Figure 3A), and both kinds of fluorescence were well colocalized with the fluorescence of lyso-tracker (red), suggesting both particles were internalized into cells through the receptor-mediated endocytosis pathway and localized in the same compartments.26 Additionally, due to the legumain is a kind of well conserved lysosomal cysteine protease and found abundant in membranous vesicles, this internalized behavior may benefit for the intracellular aggregation after entering endosome/lysosome.27 Although they could be internalized into same compartment, it remained unclear whether both particles were internalized with similar rate. We further exchange the conjugation of DOX and Cy5.5 onto AuNPs-AK-R and AuNPs-CABT-R, namely DOX-AuNPs-AK-R and Cy5.5-AuNPs-CABT-R. After incubation for 24 h, similar phenomenon was found that they could be internalized through receptor-mediated endocytosis and localized in same cellular compartment (Figure 3B). More importantly, the mean fluorescent intensity of DOX and Cy5.5 measured in cells treated with Cy5.5-AuNPs-AK-R and DOX-AuNPs-CABT-R was close to corresponding fluorescence dye in cells treated with DOX-AuNPs-AK-R and Cy5.5-AuNPs-CABT-R, suggesting the internalization rate of AuNPs-AK-R and AuNPs-CABT-R by C6 cells was similar (Figure A-i and B-i). All these results supported that R8-RGD mediated internalization and legumain-trigged aggregation could efficiently enhance the accumulation of nanoparticles in cells.


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Figure 3. Internalized mechanism of AuNPs-A&C-R. A: Confocal images of C6 cells treated with Cy5.5-AuNPs-AK-R and DOX-AuNPs-CABT-R for 24 h, bar represents 20 µm. A-i: Mean fluorescent intensity (MFI) of DOX and Cy5.5 measured from the cells in A using ImageJ software.

B:

Confocal

images

of

C6

cells

treated

with

DOX-AuNPs-AK-R

and

Cy5.5-AuNPs-CABT-R for 24 h, bar represents 20 µm. B-i: Mean fluorescent intensity (MFI) of DOX and Cy5.5 measured from the cells in B using ImageJ software.

In vivo GBM distribution. We next evaluated the distribution of Cy5.5-AuNPs-A&C-R in C6 GBM-bearing mice by fluorescence imaging and CT imaging. After intravenous injection for 24 h,



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obvious fluorescence could be observed at the brain region of mice treated with Cy5.5-AuNPs-A&C-R, Cy5.5-AuNPs-A&C and Cy5.5-AuNPs-R respectively, which was much stronger than that of mice treated with Cy5.5-AuNPs-P. In comparison, the Cy5.5-AuNPs-A&C-R exhibited highest intensity at the right region of brain, where the GBM located (Figure 4A). Ex vivo fluorescence imaging further demonstrated Cy5.5-AuNPs-A&C-R could target GBM with higher intensity (Figure 4B). The fluorescence of Cy5.5-AuNPs-A&C-R was located mainly at the GBM site where its mean fluorescent intensity was 1.35-fold, 1.72-fold and 2.58-fold higher than that of Cy5.5-AuNPs-A&C, Cy5.5-AuNPs-R and Cy5.5-AuNPs-P groups respectively (Figure 4D). Then ratio of fluorescent intensity at GBM site to normal brain parenchyma (G/B ratio) was introduced to evaluate the target specificity.28 The G/B ratio of Cy5.5-AuNPs-A&C-R, Cy5.5-AuNPs-A&C, Cy5.5-AuNPs-R and Cy5.5-AuNPs-P was 1.81, 1.36, 1.21 and 1.04 respectively, suggesting Cy5.5-AuNPs-A&C-R possessed a better selectivity to GBM cells. Besides, we also evaluated the ex vivo fluorescence imaging of major organs, the fluorescence was mainly distributed in kidney (Figure 4C and E), suggesting these particles may be eliminated by the kidney.


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Figure 4. In vivo distribution of Cy5.5-AuNPs-A&C-R in C6 GBM-bearing mice. A: Living imaging

and

CT imaging

of GBM-bearing

mice

after

intravenous injection

with

Cy5.5-AuNPs-A&C-R and control particles for 24 h, fluorescence bars represent the intensity range from 0.5 × 103 to 2.5 × 103, X-ray bars represent the radiant efficiency from 0.02 X.D. to 1.20 X.D.. B: Ex vivo imaging of brain peeled off from GBM-bearing mice treated with different formulations for 24 h, 1 represents Cy5.5-AuNPs-A&C-R, 2 represents Cy5.5-AuNPs-A&C, 3 represents Cy5.5-AuNPs-R, and 4 represents Cy5.5-AuNPs-P, fluorescence bars represent the intensity range from 0.5 × 103 to 2.5 × 103. C: Ex vivo imaging of major organs peeled off from



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GBM-bearing mice treated with different formulations for 24 h, fluorescence bars represent the intensity range from 0.5 × 103 to 5 × 103. D: Left column indicates ROI analysis of fluorescent signal in GBM site and normal brain from B, right column indicates G/B ratio, * represents statistical significance vs Cy5.5-AuNPs-A&C, Cy5.5-AuNPs-R and Cy5.5-AuNPs-P (p < 0.05). E: ROI analysis of fluorescent signal in major organs from C, * represents statistical significance vs kidney (p < 0.05).

Immunofluorescence. To further explore the mechanism underlying the enhanced accumulation of Cy5.5-AuNPs-A&C-R, in vivo immunofluorescence was performed. After staining the GBM section with anti-integrin αvβ3 antibody, the fluorescence of integrin αvβ3 was obvious observed in GBM site and normal brain site (Figure 5), which was due to that integrin αvβ3 receptor was overexpressed on both GBM cells and luminal side of the brain endothelial cells.29 The fluorescence of particles was barely observed at the GBM site treated with Cy5.5-AuNPs-P for 24 h. However, the fluorescence of Cy5.5-AuNPs-A&C and Cy5.5-AuNPs-R at GBM site showed much stronger signal, suggesting both Cy5.5-AuNPs-A&C and Cy5.5-AuNPs-R could efficiently deliver to GBM site and retained at there. Furthermore, the fluorescence of Cy5.5-AuNPs-A&C-R in GBM site showed strongest signal at 24 h, indicating the improved accumulation at GBM site. Moreover, the fluorescence of Cy5.5-AuNPs-A&C-R and Cy5.5-AuNPs-R at GBM site was well colocalized with the fluorescence of integrin αvβ3 receptor, suggesting the delivery of Cy5.5-AuNPs-A&C-R and Cy5.5-AuNPs-R to GBM site was mediated by R8-RGD’s specific recognition to integrin αvβ3 receptor. Additionally, we also stained the GBM section with anti-legumain antibody after treatment with these particles for 24 h. As expected, the


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fluorescence showed the similar distribution as described above and the fluorescence signal of legumain was obvious at GBM site but inconspicuous at normal brain tissue, suggesting the expression of legumain in GBM was up-regulated (Figure 6 and Figure S5). Importantly, the fluorescence of Cy5.5-AuNPs-A&C-R and Cy5.5-AuNPs-A&C at GBM site exhibited well colocalization with the fluorescence of legumain, indicating Cy5.5-AuNPs-A&C-R and Cy5.5-AuNPs-A&C could aggregate after entering GBM site in the presence of legumain. All these results clearly elucidated the enhanced accumulation of Cy5.5-AuNPs-A&C-R was due to the synergistic effect of R8-RGD mediated targeted delivery and AK&CABT induced aggregation.



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Figure 5. In vivo distribution of Cy5.5-AuNPs-A&C-R and control particles at GBM site after staining with anti-integrin αvβ3 antibody and DAPI, dash line represents the boundary between GBM site and normal brain site, and bars represent 100 µm.

Figure 6. In vivo distribution of Cy5.5-AuNPs-A&C-R and control particles at GBM site after staining with anti-legumain antibody and DAPI, dash line represents the boundary between GBM site and normal brain site, and bars represents 100 µm.


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In vivo PA imaging of AuNPs-A&C-R. Our previous study has verified that AuNPs-based formulations could be served as a contrast agent, which provided the feasibility for in vivo PA imaging.19 The PA imaging of cross-sectional anatomical brain through GBM region was captured over time. After intravenous injection for 4 h, the PA signal of AuNPs-P showed very low distribution at GBM site but mainly at the sagittal sinus and submandibular gland (Figure 7A). In comparison, the PA signal of AuNPs-A&C-R, AuNPs-A&C and AuNPs-R exhibited more distribution at GBM site, suggesting R8-RGD modification and AK&CABT modification could efficiently mediate GBM targeting delivery of nanoparticles. After 24 h, the PA signal of AuNPs-A&C-R at GBM site was much stronger than that of AuNPs-A&C and AuNPs-R, indicating the AuNPs-A&C-R could retain more at GBM site. Next, we further evaluated the distribution of these particles by three-dimensional (3D) mimetic analysis of the PA signal of AuNPs collected at 24 h. The 3D mimetic PA signal of AuNPs-A&C-R obviously located at the GBM region (Figure 7B). Besides, the amplifying imaging at cross section, vertical section and sagittal section all supported that the PA signal of AuNPs-A&C-R was located at the GBM site with much stronger intensity, suggesting AuNPs-A&C-R efficiently accumulate at GBM site with high selectivity. The mean pixel intensity of region-of-interest (ROI) further validated the comparison (Figure 7C). We then measured the actual AuNPs content in GBM site using the ICP-OES analysis. After intravenous injection for 24 h, the percentage injected dose (%ID) per gram GBM of AuNPs-P was 4.28 ± 1.48%, which was much lower than that of AuNPs-A&C (10.30 ± 1.27%) and AuNPs-R (6.66 ± 2.37%). In comparison, the %ID per gram GBM of AuNP-A&C-R was as high as 17.73 ± 1.39%, which was 1.72-fold, 2.66-fold, and 4.14-fold higher than that of AuNPs-A&C, AuNPs-R, and AuNPs-P respectively.



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Figure 7. In vivo PA imaging of AuNPs-A&C-R in GBM-bearing mice. A: PA imaging of cross-sectional GBM-bearing brain after intravenous injection with AuNPs-A&C-R and control particles for 4 h and 24 h, gray bars represent ultrasound signal of brain, blue bars represent PA signal of Hb, red bars represent PA signal of HbO2, jet bars represent PA signal of AuNPs. B: 3D analysis of PA imaging of GBM-bearing brain after injection with AuNPs-A&C-R and control particles for 24 h, dotted region represents the GBM site. C: ROI analysis of PA signal in GBM site from B. D: ICP-OES analysis of gold content in GBM site after injection with AuNPs-A&C-R and control particles for 24 h, *p < 0.05, error bars represent SD (n = 3).


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Anti-GBM effect. Comparing with saline group and free DOX group, all AuNPs-based treatment showed prolonged survival time, especially the DOX-AuNPs-A&C-R treatment (Figure 8). In detail, the median survival time of DOX-AuNPs-A&C-R group was 56 days (Table 1), which was greatly longer than that of DOX-AuNPs-A&C group (46 days, 1.22-fold), DOX-AuNPs-R group (44 days, 1.27-fold), DOX-AuNPs-P group (36 days, 1.55-fold), free DOX group (23 days, 2.43-fold), and saline group (24 days, 2.33-fold).

Figure 8. Kaplan-Meier plots of the survival in C6 GBM-bearing mice treated with different formulations (n = 10), the administration dose of DOX was 3 mg kg-1.



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Table 1. Mean and median survival time of C6 GBM-bearing mice treated with different formulations (n = 10). Group

Mean survival

Median survival

Standard error

significance

(day)

(day)

(day)

DOX-AuNPs-A&C-R

65.3

56

15.3

-

DOX-AuNPs-A&C

50.9

46

12.6

0.172

DOX-AuNPs-R

50.1

44

13.4

0.157

DOX-AuNPs-P

37.2

36

12.4

0.027

Free DOX

28.8

23

7.9

0.006

N.S.

26.6

24

2.4

0.003

To give an overall assessment for the treatment activity of different formulations, the H&E staining of GBM-bearing brain were further evaluated. In saline group and free DOX group, compact nuclei of GBM cells were clearly observed, suggesting free DOX could not efficiently deliver to GBM site and cause GBM cell apoptosis, resulting in short survival time (Figure 9). However, in brains treated with DOX-AuNPs-A&C-R, DOX-AuNPs-A&C and DOX-AuNPs-R, the density of GBM cells reduced greatly. In particular, the DOX-AuNPs-A&C-R demonstrated relatively smaller density of nuclei compared to DOX-AuNPs-A&C and DOX-AuNPs-R, indicating the DOX-AuNPs-A&C-R could induce more apoptosis of GBM cells, leading to an improved anti-GBM effect. Besides, we also performed the H&E staining of major organs to evaluate the systemic toxicity of DOX. In free DOX group, obvious myocardial injury related with acute neutrophil accumulation was found in the myocardium, which denoted the acute cardiotoxicity of free DOX (Figure S6). However, no obvious damages were found in the heart of


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groups treated with AuNPs based formulations, as well as the liver, spleen, lung and kidney, suggesting these AuNPs based formulations could reduce the accumulation of DOX in heart.

Figure 9. H&E staining of GBM-bearing brain collected at the day after last administration, the dotted region represents GBM cells and bars represent 200 µm.

4. DISCUSSION Nanocarriers within the size range of 10-100 nm can passively target and access the brain tumor due to the EPR effect.30 Importantly, the EPR effect in the brain tumor opens many opportunities for nanocarriers. Gao et al showed that docetaxel-loaded nanoemulsion and albumin NPs could passively target brain tumors via EPR effect, resulting in prolonged median survival time relative to docetaxel solution.31,32 However, the brain tumor delivery efficiency of these drug-loaded nanocarriers faced the problem of insufficient accumulation. The high intratumoral IFP causes the nanocarriers hard to extravaste from leaky vasculature and easy to back-flow to bloodstream. Therefore, in our previous study, we have developed a functional nanoplatform (AuNPs-A&C) to deliver DOX to GBM site, which was also based on EPR effect.19 The AuNPs-DOX-A&C could click cycloaddited in the activation of legumain, resulting in the in situ



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formation of AuNPs aggregates. The AuNPs aggregates in turn blocked their back-flow to bloodstream and restricted their exocytosis by cells, which enhanced their retention at tumor site. The nanoplatform exhibited excellent biological performance of site-specific localization and enhanced retention, resulting in improved treatment outcome. Other strategies, i.e. biological orthogonal reactions, charge absorption and thermal-sensitivity, were also reported with the ability to induce aggregation. The most common and typical click reaction is Cu(I)-catalyzed azide/alkyne cycloaddition.33 Xianyu et al. have proposed a azide/alkyne functionalized AuNPs, which can occur aggregation in the presence of alkaline phosphatase.34 Besides, similar enzymatic aggregation behavior has been showed by Gu’s lab. They developed a transformable core-shell based nanoplatform (CS-NG), which can form extracellular depots with the assistance of hyaluronidase (HAase).35 The CS-NG was proved to possess a 1.3-fold accumulation in tumor site compared to non-HAase sensitive CS-NG. However, all these enzymatic chemical reactions are general not sensitive enough to provide rapid response to the tumor-associated enzyme, which have the potential to be improved. Unlike enzymatic aggregation, AuNPs can also form mutual aggregation via pH-induced change of interparticle interaction, which exhibited fast, ultrasensitive and reversible response.17,36 Additionally, Jones et al. have engineered a thermo-responsive polymer-coated AuNPs (PNIPAm-AuNPs), which aggregated upon heating in pure water.37 However, most of these particles could only distribute into tumor through EPR effect, which attenuate the tumor targeting efficiency. Receptor-mediated drug delivery is a common mechanism for brain tumor targeting delivery of therapeutic and diagnostic agents. In particular, cyclic RGD is promising ligand for targeting the integrin αvβ3/αvβ5 receptor, which are overexpressed on the BBB,22 neovessels38,39 and GBM


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cells.40,41 RGD could mediate nanostructured therapeutics targeting brain and transporting across BBB via the receptor-mediated transcytosis pathway. Octaarginine is a common cell penetrating peptide that promotes the penetration of its cargo through BBB.11 Our previous study showed RGD-decorated fluorescent carbonaceous nanospheres has a 1.3-fold accumulation compared to PEGylated nanospheres in GBM site.42 Moreover, we have also demonstrated that the distribution of R8-RGD decorated gelatin-gold fabricated nanoparticles at GBM site was 3.1-fold higher than that of PEGylated gelatin-gold fabricated nanoparticles.13 In this study, in vivo imaging demonstrated the fluorescent signal of AuNPs-R in GBM site was 1.5-fold higher than that of AuNPs-P after 24 h injection, PA imaging demonstrated the signal of AuNPs-R in GBM site was 1.9-fold higher and ICP-OES analysis showed a %ID per gram GBM of 6.7% after 24 h injection. These results suggested a relatively better accumulation of AuNPs-R in GBM site, which was mainly due to the specific targeting of RGD and cell penetrating ability of octaarginine. Until today, RGD has been widely used for targeting integrin receptor associated with different malignant tumors by decorating onto different nanocarriers or by different methods. Yataka et al. has proposed a cyclic RGD-linked polymeric micelle, which could targeted deliver the platinum to GBM site with a %ID/g GBM of 7% after 24 h injection.43 Beyond the application of RGD connected with cell penetrating peptide, the grafting amount of RGD is also an important factor. Guo et al. developed three RGD modified nanocarriers with different intervals that received an enhanced therapeutic efficacy to B16F10 tumor compared to monomeric RGD and dimeric RGD modification.44 As a consequence, we combined the better tumor penetration and retention ability of AuNPs-A&C with the better GBM targeting ability of R8-RGD in this study. One of important



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things is to confirm whether the introduction of R8-RGD has influence on the stereospecific click cycloaddition. In vitro size investigation indicated the AuNPs-A&C-R could aggregate in the presence of legumain, despite the size increase of AuNPs-A&C-R was lower than that of AuNPs-A&C. This result gave the possibility of synergistic capacity of targeting delivery and legumain-induced aggregation. In vivo living imaging has demonstrated the fluorescent signal of AuNPs-A&C in GBM site was 1.9-fold higher than that of AuNPs-P after 24 h injection. PA imaging demonstrated the signal of AuNPs-A&C in GBM site was 1.9-fold higher than that of the AuNPs-P and ICP-OES analysis showed a %ID per gram GBM of 10.3% after 24 h injection. In combination with R8-RGD and AK&CABT, optical imaging showed a much higher accumulation of AuNPs-A&C-R in GBM site and the %ID per gram GBM was as high as 17.7%. Most importantly, the DOX-loaded AuNPs-A&C-R greatly enhanced anti-GBM effect and the median survival time was 120.7%, 127.3%, 155.6%, 243.5% and 233.3% longer than that of DOX-AuNPs-A&C, DOX-AuNPs-R, DOX-AuNPs-P, free DOX and normal saline group respectively. In addition, the developed dual-functional nanoplatform may also provide the chance to carry other chemotherapeutic drugs, e.g. temozolomide,45 the standard of care for GBM.

5. CONCLUSION In summary, we developed a dual-functional nanoplatform which possessed high receptor affinity and tumor microenvironment responsibility. The AuNPs-A&C-R, consisted of AuNPs-AK-R and AuNPs-CABT-R, could aggregate in the presence of legumain, resulting in size increase. In vitro cellular study and in vivo optical imaging showed AuNPs-A&C-R could target GBM site and retain at there with high precise location. ICP-OES analysis demonstrated


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AuNPs-A&C-R could obtain an enhanced accumulation in GBM site compared with AuNPs-A&C and AuNPs-R. Importantly, this functional presented an improved treatment outcome to C6 GBM-bearing mice. The validity of this dual-functional nanoplatform may open up a new avenue for conceiving more efficient nanotherapeutics to achieve preferential accumulation in tumor site and provide the opportunities to carry other chemotherapeutic drugs. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthesis of SH-PEG-AK, SH-PEG-CABT and SH-PEG-RRGD; Synthesis of pH sensitive DOX probe and Cy5.5 probe; Preparation procedure of AuNPs-AK, AuNPs-CABT, AuNPs-R and AuNPs-P;

Preparation

procedure

of

DOX-AuNPs-AK-R,

DOX-AuNPs-CABT-R,

DOX-AuNPs-AK, DOX-AuNPs-CABT, DOX-AuNPs-R and DOX-AuNPs-P; Preparation of Cy5.5-AuNPs-AK-R,

Cy5.5-AuNPs-CABT-R,

Cy5.5-AuNPs-AK,

Cy5.5-AuNPs-CABT,

Cy5.5-AuNPs-R and Cy5.5-AuNPs-P; Physiochemical characterization of different formulations; Intensity distribution and TEM images of AuNPs-R after incubation with legumain. Mean fluorescent intensity (MFI) of DOX measured from the cells in figure 2A; Cellular uptake of DOX-AuNPs-A&C-R, DOX-AuNPs-A&C, DOX-AuNPs-R and DOX-AuNPs-P by bEnd.3 cells; Uptake mechanism study of DOX-AuNPs-A&C-R and DOX-AuNPs-R by C6 cells; Fluorescent distribution of Cy5.5-AuNPs-A&C-R and control particles at the cerebral cortex after staining with anti-legumain antibody; H&E staining of major organs collected at the day after last administration. AUTHOR INFORMATION



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Corresponding Author *E-mail: [email protected]; Fax/Tel: 86 28 85502532 *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31571016, 81402866).


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Graphical Abstract 93x41mm (300 x 300 DPI)


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Ligand-Mediated and Enzyme-Directed Precise ... - ACS Publications

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