Suppressing Nanoparticle-Mononuclear Phagocyte. System Interactions of Two-Dimensional Gold. Nanorings for Improved Tumor Accumulation and

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1 Supporting information Suppressing Nanoparticle-Mononuclear Phagocyte System Interactions of Two-Dimensional Gold Nanorings for Improved Tumor Accumulation and Photothermal Ablation of Tumors Yijing Liu, 1 Zhantong Wang, 1 Yi Liu, 2,3 Guizhi Zhu, 1 Orit Jacobson, 1 Xiao Fu, 1 Ruiliang Bai, 4 Xiaoying Lin, 2 Nan Lu, 1 Xiangyu Yang, 1 Wenpei Fan, 1 Jibin Song, 1 Zhe Wang, 1 Guocan Yu, 1 Fuwu Zhang, 1 Heather Kalish, 5 Gang Niu, 1 Zhihong Nie, 2* Xiaoyuan Chen 1*

2 Supplementary Information Supplementary Figures Figure S1. TEM images of different intermediate steps during the synthesis of Au nanorings and corresponding UV-Vis absorptions. (a) Ag nanoplates were used as initial templates. (b) nanoplates were prepared after coating Au on the edge of Ag nanoplates. (c) Au nanorings were formed after Ag templates being etched away. (d) The UV-Vis absorption spectrum of Ag nanoplates, nanoplates, and Au nanorings. Scale bars: 50 nm.

3 Figure S2. DLS spectrum of Au nanorings with different sizes. Figure S3. TEM images of Au nanorings with different thicknesses. The thicknesses from (a) to (e) are 2, 4, 9, 13, and 20 nm. Scale bares: 50 nm.

4 Figure S4. Photothermal characterizations of Au nanorings. (a) Representative TEM images of Au nanorings with 4, 9, and 20 nm thicknesses. Scale bars: 20 nm. (b) UV-Vis extinction spectrum of Au nanorings with different thicknesses. (c) Representative thermal images of solutions of Au nanorings with different thicknesses under 808 nm laser irradiation (0.5 W/cm 2 ). (d) Heating curves and (e) photothemal conversion efficiency of Au nanorings with different thicknesses in solutions under 808 nm laser irradiation. The photothermal conversion efficiency was calculated via a method reported previously. 1

5 Figure S5. Photothermal stability of 50 nm Au nanorings with different thicknesses. Figure S6. TEM images of Au nanospheres and Au nanoplates. The average diameters of Au nanospheres and Au nanoplates are 55.0 nm and 54.6 nm. Scale bars: 100 nm

6 Figure S7. Relative viability of cells incubated with different concentrations of Au nanorings, Au nanoplates, and Au nanopsheres. Figure S8. TEM images of (a,d) Au nanorings, (b,e) Au nanoplates, and (c,f) Au nanopsheres before and after incubation with cell culture medium for 48h. The upper row

7 and lower row are the TEM images of Au nanostructures before and after incubation with cell culture medium, respectively. Figure S9. The absorption spectra of (a) Au naonplates, (b) Au nanorings, and (c) Au nanospheres at the beginning of and 48 h after their incubation with cell culture medium.

8 Figure S10. Polyacrylamide Gel Electrophoresis (PAGE) analysis of the composition of protein adsorbed on three different Au nanostructures. Complex band patterns of proteins were shown, indicating the adsorptions of broad range of serum proteins on the surfaces of Au NPs.

9 Figure S11. Biodistributions of 50 nm (a) Au nanorings, (b) Au nanospheres, and (c) Au nanoplates in tumors and primary organs at 48 h postinjection. Mean values ± s.d.; n = 4 tumor uptake:p<0.05. Figure S12. Cross-section PET images of mice and tumor tissues for the analysis the distributions of Au nanostructures within tumors at 24 h postinjection. In group (a), 50 nm Au nanospheres were injected. In group (b), 50 nm Au nanorings were injected. In group (c), 50 nm Au nanoplates were injected. The uniform PET signals in different cross-section images of group (b) and (c) indicate more uniform distribution and stronger diffusivity of Au nanostructures.

10 Figure S13. Study of size effect of Au nanorings on the in vivo biodistributions and tumor uptake. (a,b) Representative whole-body coronal PET images of mice after intravenous injection of 64 Cu labeled Au nanorings with diameter of (a) 25 nm and (b) 130 nm at 1 h, 3 h, 8 h, 11 h, 24 h, and 48 h (from left to right) post injection.

11 Figure S14. Study of size effect of Au nanorings on the in vivo biodistributions and tumor uptake. (a-d) Time activity curves of the mean uptake of 64 Cu labeled Au nanostructures with different sizes in (a) livers, (b) spleens, (c) hearts, and (d) tumors, derived from the ROI analysis of the PET images. Mean values ± s.d.; n = 3

12 Figure S15. Biodistributions of 130 nm (a) and 25 nm (b) Au nanorings in tumors and primary organs 48 h postinjection. Mean values ± s.d.; n = 4 tumor uptake:p < 0.05.

13 Table S1 DLS characterizations of Au naonrings, Au nanoplates, and Au nanospheres at the beginning of and 48 h after their incubation with cell culture medium. Table S2. DLS and zeta potential characterizations of Au nanorings, Au nanoplates, and Au nanospheres in water without or with the adsorption of serum proteins.

14 Supplementary Methods Characterizations. The structures of different AuNPs were analyzed by a TEM (FEI, Hillsboro, OR) equipped with a Gatan Ultrascan 1000 CCD camera (Gatan, Pleasaton, CA). SEM images were obtained via a Hitachi SU-70 Schottky field emission gun scanning electron microscope (FEG-SEM). UV-Vis absorption were measured by a Genesys 10S UV-vis spectrophotometer. HAADF image and EDS line scan measurement were obtained from a JEM 2100 FEG TEM. The hydrodynamic diameters of AuNPs were measured by a Zetasizer Nano series. The concentrations of Au or Ag in different NPs were characterized by ICP-OES. PA/US imaging was performed on a Visual Sonics Vevo LAZR system (VisualSonics Inc. New York, NY). PET images were obtained by a micro PET (Siemens Inveon) scanner. Photothermal Imaging was monitored by a SC300 infrared camera. In vitro evaluation of the toxicity of Au nanorings. U87MG cells were seeded in 96-well plates at the density of cells/well. 24 h later, Au nanorings with different concentrations were added into each well and incubated with cells for 24 h. Then Minimum Essential Medium (MEM) was removed and new MEM was used to wash the wells. After that a 100 µl MEM with 5 mg/ml MTT was added into each well and the plates were incubated at 37 C for another 4 h. Then, the MEM was removed again and each well was filled with 100 µl DMSO. The absorbance at 570 nm from each well was measured by a plate reader, and the cell viability with different concentrations of Au nanorings was calculated by comparing with wells free of Au nanorings. Adsorption of serum proteins on NP. Human serum (150 µl, Sigma H45222) were added to 50 µl aqueous solution of PEGylated gold nanoparticles (Au NPs) (1.2 nm). The mixture was

15 shaking for 2 h at 37ºC. The free proteins or the loosely bound proteins can be removed via centrifugation and wash for 3 times. Extraction of surface proteins for Polyacrylamide Gel Electrophoresis (PAGE) analysis The NPs were concentrated to 20 µl through centrifugation. To extract the proteins from NP surfaces, 2 µl 1M DTT and 4 µl NuPAGE LDS Sample Buffer were added and the solution mixture were incubated at 70ºC for 1h. Then the NPs were precipitated via centrifugation and the supernatant was collected and analyzed by PAGE to determine the components of extracted proteins. In vitro evaluation of the macrophage cell uptake of different Au nanostructures. RAW cells were seeded in 6-well plates at the density of cells/well in Dulbecco's Modified Eagle's Medium (DMEM) plus 10% serum for 24 h. After removing the old DMEM, each well was filled with 1.5 ml of DMEM containing nm of AuNPs with different shapes. After 8 h incubation, the DMEM from each well was removed and each well was washed with PBS for three times. Then the cells were detached and concentrated via centrifugation. The AuNP containing cells were digested by aqua regia and the concentrations of Au were characterized by ICP-OES. In vitro evaluation of the NP-cell binding for different Au nanostructures. RAW cells were seeded in 12-well plates at the density of cells/well in DMEM plus 10% serum for 24 h. AuNPs with different shapes were incubated with cell culture media for 1 h at 4 0 C before use. The cell seeded plates were also incubated at 4 0 C for 30 min before use. After removing the old DMEM in 12-well plates, each well was filled with 1.0 ml of cold DMEM containing nm AuNPs with different shapes. The 12-well plates were then incubated at 4 0 C for 1h. Then, the DMEM from each well was removed and each well was washed with PBS for two times. Then the cells were detached and concentrated via centrifugation. The AuNP containing cells were digested by aqua regia and the concentrations of Au were characterized by ICP-OES.

16 Animal Models. All animal experiments were performed under the National Institutes of Health Animal Care and Use Committee (NIHACUC) approved protocol. Nude mice (Harlan) were subcutaneously injected of U87MG cells into the right flanks. PET imaging was performed when tumor sizes reached about 200 mm 3. PA imaging and photothermal therapy were performed when the tumor size reached about 100 mm 3 In vivo PET imaging and ex vivo biodistribution study. The preparation of 64 Cu labeled AuNPs was previously reported. 2 Briefly, 3 µl of 64 CuCl 2 (ph 1-2) was added to a solution containing mg Sodium Ascorbate in 40 µl 1M Borate buffer ph 8. The mixture was shortly vortexed and incubate for 3-5 min at room temperature. Then 200 µl of Au-NPs was added. The reaction was agitated for 0.5 h at 37ᴼC. Radiochemical purity was determined using itlc plates (Fisher Scientific), developed in 0.1 M Citric acid ph 5 (Rf of 64 Cu-Au-NPs ~ 0.1, Rf of uncomplexed 64 Cu ~ 0.9). The U87MG tumor-bearing mice were intravenously injected with 100 µci 64 Cu labelled AuNPs with different sizes or shapes. Three mice were used in PET imaging analysis and four mice were used in ex vivo study of biodistributions. PET imaging analysis was performed on a micropet scanner at different postinjection time points. Three-dimensional region of interests (ROIs) were drawn over the tumors and primary organs in the PET images to obtain the mean pixel values within the ROI volume. The percent injected dose per gram (%ID/g) of average radioactivity from tumors and other primary organs were calculated from the mean pixel values within the ROI volume. After 48 h, the mice were sacrificed and primary organs from mice were collected and weighted. The signal of 64 Cu of primary organs was measured in a well gamma-counter (Wallach Wizard, PerkinElmer, Waltham, MA, USA) and was calculated as the % ID/g of tissue. Calculations of the diffusivity of different Au nanostructures. The diffusivity of the different Au nanostructures can be calculated a via a simplified mathematical model following the Stokes-Einstein equation by

17 where k B, T, and ζ represent boltzmann s constant, temperature, and Stoke s drag coefficient, respectively. For Au nanospheres, the diffusivity can be calculated by 6 6 Where R(sphere) represents the radius of the Au nanosphere. For the calculation of diffusivity of Au nanoplates and Au naonrings, we simplified the mathematical model by not considering their thickness. The left scheme can represent Au nanoplates, if the R i = 0 nm. The boltzmann s constant of Au nanoplates can be calculated via the following equations.,, 32 3, 16 nanoplate. where R(plate) represents the radius of the Au The diffusivity of Au nanoplate can be calculated via the following equation., The above scheme can represent Au nanoring in our study if R i = 16 nm. The boltzmann s constant of Au nanorings can be calculated via the following equations,, 1 4/3 /,, 1 4/3 / The ratio of diffusivity between Au nanoplate and Au nanosphere can be calculated as following equation., 1.51

18 The ratio of diffusivity between Au ring and Au nanosphere can be calculated as following equation., 1.59 Evaluation of photothermal treatment. The tumor-bearing nude mice were divided into four groups with 3 mice in each group, including the group injected of Au nanorings with laser treatment, the group injected of Au nanorings only, the group injected of PBS with laser treatment, and the group injected of PBS only. For each group, mice were intravenously injected with 100 µl of Au nanorings or PBS and the laser irradiation were performed 48 h after the injection. 808 nm laser with density of 0.75 W/cm 2 was irradiated the tumor for 5 min. Tumor volumes and mice body weight were measured every two days. Calculation of photothermal conversion efficiency. The photothermal conversion efficiency (ƞ) of the Au nanorings was calculated via a method reported previously. 1 ƞ 1 10 Where s stands for the surface area between solution and container, h represents the heat transfer coefficient, is the difference between the highest temperature of the solution and temperature of the environment, I stands for the laser intensity, A represents the absorption intensity at 808 nm, and Q 0 represents the dissipation of heat from the solution other than from the NPs after laser irradiation. Q 0 can be calculated by measuring the temperature change curve of the pure water with the same volume and container of the samples. To calculate sh, we define a new parameter α where T is the temperature of the solution at different time points during the cooling process and T surrounding represents the environment temperature. A linear relationship between time and ln can be obtained, described by the following equation t = -κ * In (α),,

19 in which κ represents the thermal time constant. sh can be calculated by the following equation: sh =, in which m and C represent the mass and heat capacity of the solution, respectively. 1. Huang, P.; Lin, J.; Li, W.; Rong, P.; Wang, Z.; Wang, S.; Wang, X.; Sun, X.; Aronova, M.; Niu, G.;et al., Biodegradable Gold Nanovesicles with an Ultrastrong Plasmonic Coupling Effect for Photoacoustic Imaging and Photothermal Therapy. Angew. Chem. Int. Ed. 2013, 52, Sun, X.; Huang, X.; Yan, X.; Wang, Y.; Guo, J.; Jacobson, O.; Liu, D.; Szajek, L. P.; Zhu, W.; Niu, G.; et al., Chelator-Free 64Cu-Integrated Gold Nanomaterials for Positron Emission Tomography Imaging Guided Photothermal Cancer Therapy. ACS Nano 2014, 8,