Supporting Information

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1 Supporting Information Targeted Imaging of Brain Tumors with a Framework Nucleic Acid Probe Tian Tian, Jiang Li, Cao Xie, # Yanhong Sun, * Haozhi Lei, Xinyi Liu, Jiaoyun Xia, Jiye Shi, Lihua Wang, Weiyue Lu, #* Chunhai Fan * Division of Physical Biology & Bioimaging Center, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai , China Institute of Interdisciplinary Integrative Biomedical Research, Shanghai University of Traditional Chinese Medicine, Shanghai , China # Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai , China School of Chemistry and Biology Engineering, Changsha University of Science and Technology, Changsha , China UCB Pharma, Slough, SL1 14EN Berkshire, UK Corresponding Authors * fchh@sinap.ac.cn * sunyanhong@sinap.ac.cn * wylu@shmu.edu.cn S-1

2 EXPERIMENTAL SECTION Synthesis of TDNs and ANG-TDNs. All TDNs with or without arms were synthesized as previously reported by mixing equimolar quantities (1 µm) of oligonucleotides in TM buffer (10 mm Tris base, 5 mm MgCl 2, ph 8.0), heating to 95 C for 5 min, and then rapidly cooling to 4 C. 1,2 Angiopep-2 modified oligonucleotides were synthesized by the help of click action kit. Angiopep-2 with alkynyl and oligonucleotide with azide were mixed in click kit buffer at a ratio of 6:1. The mixture was incubated at room temperature overnight. After incubation, the mixture was desalted by Zeba spin desalting columns, and was freeze-dried into power. The oligonucleotide linked angiopep-2 was called Ang-ssDNA. ANG-TDNs was obtained by the mixing of Ang-ssDNA and armed TDNs at a ratio of 3:1 in TM buffer at room temperature for 2 h. And then removed unreacted Ang-ssDNA by a centrifuge tube. Gel Electrophoresis. 8% Polyacrylamide Gel Electrophoresis (PAGE) was performed in 1 TAE buffer (4 mm Tris base, 2 mm acetic acid, 0.2 mm EDTA) including 1.25 mm MgAc 2 at 4 C for about 1.5 h. Then TDNs and ANG-TDNs were stained with GelRed for 15 min for further analysis on a chemiluminescence imaging system (G: Box Chemi-XL). Stability Analysis of TDNs and ANG-TDNs. The solutions of TDNs and ANG-TDNs at a same concentration (1 μt) were separately mixed with non-heat-inactivated FBS at a ratio of 1:1 and incubated at 37 C for 0, 2, 4, 8, 12 or 24 h. After incubation, the mixtures were run on a 1% AGE stained with GelRed. MTT Assay. The cytotoxicity of TDNs and ANG-TDNs were estimated using a MTT assay. bend.3 cells and U87MG cells were separately seeded in 96-well plates and cultured overnight to reach 80% confluency. Fresh media containing DNA nanostructures (0, 25, 50 and 100 nm) were incubated with cells for 24 h (each sample was repeated six times). Then, 20 µl of 5 mg/ml thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich, USA) solution was added to each well, followed by 4 h incubation at 37 C. Next, cells were lysed with DMSO. After sufficient mixing, a microplate reader (Bio-Rad 680, USA) was used to measure the absorbance at 570 nm. Confocal Fluorescence Microscopic Imaging. bend.3 cells and U87MG cells were separately seeded on confocal culture plates at a density of cells/ml and incubated at 37 C overnight. Then cells were washed twice with phosphate buffer (PBS) and incubated with Cy3 labeled TDNs and ANG-TDNs in fresh medium for 4 h at 37 C. After incubation, cells were washed twice with PBS, fixed with 4% paraformaldehyde, and the nuclei were stained using 5 μg/ml Hoechst Then cell images were taken with a Leica confocal microscope setup (Leica TCS SP8, Germany). Flow Cytometry. bend.3 cells and U87MG cells were separately seeded on 24-well culture plates at a density of cells/ml and cultured for 24 h. Then washed twice with PBS and incubated with Cy3 labeled TDNs and ANG-TDNs for 0, 0.5, 2, and 4 h at 37 C. After incubation, cells were harvested, and washed three times with PBS. Then, the fluorescence intensity of the cells was determined by flow cytometry (FACSArray, BD Biosciences, USA). Transport ability across the BBB in vitro. The in vitro BBB model was constructed with bend.3 cells using a transwell cell culture system as described previously. 3 Briefly, bend.3 cells ( cells/well) were seeded onto gelatin solution-coated the upper chamber of transwells (3 µm pore size, 6.5 mm diameter and 0.33 cm 2 membrane surface area, Corning, S-2

3 USA), and cultured with the medium containing 10% FBS for 7 days. The culture medium was changed every other day. The integrity of the cell monolayer was evaluated by measuring the TEER values using a Millicell-ERS voltohmmeter (Millipore). The cell monolayers with TEER values higher than 300 Ω cm 2 were used as the BBB model for the experiments. Brain glioma U87MG cells were seeded at the lower chamber of transwells at a density of cells/well. To evaluate the penetrating efficiency of TDNs and ANG-TDNs across the BBB, TDNs or ANG-TDNs labeled with Cy3 were added to the upper chamber of transwells at a concentration of 50 nm. After incubation for 6 h, the inserts were moved away, and brain glioma U87MG cells were further incubated until for 24 h. The penetrating efficienty of TDNs or ANG-TDNs across the BBB model was evaluated by measuring the fluorescent indensity of brain glioma cells in the lower chamber under the BBB insert. In vivo Pharmacokinetics and Biodistribution. ICR mice (4 weeks old, 18~20 g) were used in evaluating the pharmacokinetics of TDNs and ANG-TDNs. The mice were divided into two groups (n=6 in each group), and 200 ls at a concentration of 50 nm. After incubation for 6 h, the inserts were moved away, and was drawn from orbit at 0, 2, 5, 10, 15, 20, 25, 30, 45 min post injection. The total fluorescent counts of each blood were measured. Then the plasma half-life of TDNs and ANG-TDNs were calculated by the DAS 2.0 software. In order to assess the in vivo biodistribution of TDNs and ANG-TDNs, mice were sacrificed after TDNs or ANG-TDNs injection for 2h, the main tissues (e.g. heart, liver, spleen, lung, kidney and brain) were collected to measure the total fluorescent counts by using a small animal in vivo imaging system (Berthold NightOWL LB 983, Germany). In vivo and Ex vivo Intracranial Glioblastoma Imaging and Slice distribution. BALB/c nude mice were anesthetized with chloral hydrate and U87 MG cells ( cells suspended in 5 anesthetized with chlorato the right brain (1.8 mm lateral, 0.6 mm anterior to the bregma, and 3 mm of depth) with the help of a stereotactic fixation device. After inoculation for 18 days, the models were used for imaging. The solutions of TDNs and ANG-TDNs labeled with Dlight 755 (2 µm, 200 µl) were separately injected in tumor bearing mice via tail vein. After 90 min, mice were anesthetized and used in imaging experiment. 3D fluorescent images were acquired with IVIS Lumina XRMS Series III Multi-species Optical and X-ray Imaging System (PerkinElmer, USA). Then, the mice were sacrificed, and brain were subjected to ex vivo fluorescence imaging. After fixing with 4% paraformaldehyde, brain were further dehydrated by 15% sucrose followed with 30% sucrose. Consecutive frozen sections of 30µm thicknesses were prepared and then stained by 5 µg/ml Hoechst for 5 min. The brain slices were observed by a Leica confocal microscope setup (Leica TCS SP8, Germany). All animal care and experimental procedures were conducted using institutionally approved protocols. All in vivo experiments were conducted under the authority of project and personal licenses granted by the institutionally approved protocols. Statistical Analysis. All data were represented as the mean value with a standard deviation. Statistical analysis was analyzed using a student t-test. A value of p< 0.05 was considered to be statistically significant. S-3

4 Table S1. Oligonucleotide sequence used in this work. Oligo Sequence A B C D T20-A T20-B T20-C 5 -ATTGCTGTATTGGCTCTGGTGATGCGTTAAAGGATCTCGTA TAGCAGCTCAGTCCACTCGAAC-3 5 -CATAGTCAATAACGCATCACCAGAGCCAAATGACGACATC TGTGCGATGAAACCTAGCAGACC-3 5 -GAGATCCTATGACTATGGGTCTGCTAGGTACAGTCTGTCGC TTATGCACTAGAGCTGCTATAC-3 5 -TGTCGTCAAACAGCAATGTTCGAGTGGACAAGTGCATAAG CGACAGACTGATCATCGCACAGA-3 5 -TTTTTTTTTTTTTTTTTTTTATTGCTGTATTGGCTCTGGTGAT GCGTTAAAGGATCTCGTATAGCAGCTCAGTCCACTCGAAC-3 5 -TTTTTTTTTTTTTTTTTTTTCATAGTCAATAACGCATCACCA GAGCCAAATGACGACATCTGTGCGATGAAACCTAGCAGACC-3 5 -TTTTTTTTTTTTTTTTTTTTGAGATCCTATGACTATGGGTCTG CTAGGTACAGTCTGTCGCTTATGCACTAGAGCTGCTATAC-3 Cy3-D 5 -Cy3-TGTCGTCAAACAGCAATGTTCGAGTGGACAAGTGCATAA GCGACAGACTGATCATCGCACAGA D 5 -Dlight755-TGTCGTCAAACAGCAATGTTCGAGTGGACAAGTGC ATAAGCGACAGACTGATCATCGCACAGA-3 A20 5 -AAAAAAAAAAAAAAAAAAAA-3 A20-N 3 5 -AAAAAAAAAAAAAAAAAAAA-N 3-3 ANG-ssDNA was assembled with Angiopep-2 and A20-N 3. TDNs was assembled with A, B, C and D. TDNs with Cy3 fluorophore was assembled with A, B, C and Cy3-D. TDNs with Dylight755 fluorophore was assembled with A, B, C and 755-D. ANG-TDNs was assembled with T20-A, T20-B, T20-C, D and ANG-ssDNA. ANG-TDNs with Cy3 fluorophore was assembled with T20-A, T20-B, T20-C, Cy3-D and S-4

5 ANG-ssDNA. ANG-TDNs with Dylight755 fluorophore was assembled with T20-A, T20-B, T20-C, 755-D and ANG-ssDNA. Video S1. 3D reconstruction of in vivo imaging of TDNs treated mice. Video S2. 3D reconstruction of in vivo imaging of ANG-TDNs treated mice. Figure S1. (a) DLS measurement statistics of TDNs. (b) AFM images of TDNs. (c) HPLC analysis of TDNs and ANG-TDNs. S-5

6 Figure S2. Cell uptake of TDNs and ANG-TDNs. (a) Confocal images after DNA nanoparticles incubating with bend.3 cells for 4 h, red pointed DNA nanostructures, blue pointed cell nucleus. (b) Flow cytometry analysis of cell uptake efficiency of nanostructures in bend.3 cells. (c) Confocal images after DNA nanostructures incubating with U87MG cells for 4 h, red pointed DNA nanostructures, blue pointed cell nucleus. (d) Flow cytometry analysis of cell uptake efficiency of nanostructures in U87MG cells. *P<0.05, **P<0.01. S-6

7 Figure S3. Flow cytometry analysis of cell uptake efficiency of nanostructures in bend.3 cells (a) or U87MG cells (b), after incubation for 0.5 h, 1 h, 2 h and 4 h. *P<0.05, **P<0.01. Figure S4. Quantitative analysis of the total fluorescent intensity of liver, kidney and brain. S-7

8 Figure S5. (a) 3D reconstruction of in vivo imaging of TDNs treated mice. (b) 3D reconstruction of in vivo imaging of ANG-TDNs treated mice. REFERENCES (1) Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J., Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication. Science 2005,310, (2) Jiang, D. W.; Sun, Y. H.; Li, J.; Li, Q.; Lv, M.; Zhu, B.; Tian, T.; Cheng, D. F.; Xia, J. Y.; Zhang, L.; Wang, L. H.; Huang, Q.; Shi, J. Y.; Fan, C. H., Multiple-Armed Tetrahedral DNA Nanostructures for Tumor-Targeting, Dual-Modality in Vivo Imaging. Acs Appl. Mater. Interfaces 2016,8, (3) Xue, J. W.; Zhao, Z. K.; Zhang, L.; Xue, L. J.; Shen, S. Y.;Wen, Y. J.; Wei, Z. Y.; Wang, L.; Kong, L. Y.; Sun, H. B.; Ping, Q. N.; Mo, R.; Zhang, C., Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence. Nat. Nanotechnol. 2017,12, S-8