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1 Supporting Information Size-Tuning Ionization to Optimize Gold Nanoparticles for Simultaneous Enhanced CT Imaging and Radiotherapy for Yan Dou, Yanyan Guo, Xiaodong Li, Xue Li, Sheng Wang, Lin Wang, Guoxian Lv, Xuening Zhang, Hanjie Wang, Xiaoqun Gong, Jin Chang School of Material Science and Engineering, School of Life Sciences, Tianjin University, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, , P.R. China. Department of Radiation Oncology and Department of Radiology, The Second Hospital of Tianjin Medical University, Tianjin, , P.R. China. Address correspondence to Supporting Figures Supporting Figure S1. DLS results for as-prepared AuNPs with different particle sizes. Supporting Figure S2. TEM for AuNPs with different particle sizes after PEGylation. Supporting Figure S3. Statistics histograms of particle size and particle-size distribution 1

2 of AuNPs after PEGylation. Supporting Figure S4. DLS results of particle size and particle-size distribution of AuNPs after PEGylation. Supporting Figure S5. UV-visible absorption spectra of AuNPs before and after PEGylation for all seven types of AuNPs. Supporting Figure S6. UV-visible absorption spectra of PEGylated AuNPs under PH conditions. Supporting Figure S7. UV-visible absorption spectra of PEGylated AuNPs under different temperature conditions. Supporting Figure S8. The colloidal stability of PEGylated AuNPs though evaluation of UV-visble maximum absorption wavelength with the increase of storage time for 240 days. Supporting Figure S9. Cell viability tests by MTT assay. Supporting Figure S10. X-ray attenuation intensity of different-sized PEGylated AuNPs and Idohexol. Supporting Figure S11. Relative electron density of different-sized PEGylated AuNPs. Supporting Figure S12. Cellular radiosensitization of different-sized PEGylated AuNPs. Supporting Figure S13. The sensitization enhancement ratio (SER) of different-sized PEGylated AuNPs. Supporting Figure S14. Flow cytometry plots of cellar apoptosis and necrosis induced by different-sized AuNPs without and with radiation. 2

3 Supporting Figure S15. Dose absorption distribution of simulated modeling vesicle for different-sized AuNPs. Supporting Figure S16. Monte Carlo simulations verification for modeling vesicle containing only one particle. Supporting Figure S17. Dose absorption distribution of simulated modeling vesicle containing only one particle. Supporting Figure S18. Cell morphologies observation with incubation of PEGylated AuNPs with the optimal sizes and Idohexol. Supporting Figure S19. Cell viability induced by the optimized AuNPs compared with CMNa before and after radiotherapy. Supporting Figure S20. Intratumoral CT imaging of PEGylated AuNPs with the optimal sizes compared with Idohexol. Supporting Figure S21. Real-time in vivo CT contrast phantom images and quantitative analysis of major organs and tumors. Supporting Figure S22. The disparity in CT contrast values of various organs with circulation time of PEGylated AuNPs compared with Idohexol. Supporting Figure S23. In vivo biodistribution of gold and iodine element in different organs. Supporting Figure S24. The photographs of the HeLa xenograft tumor-bearing nude mice for different in vivo radiosensitization groups. Supporting Figure S25. Histology (hematoxylin and eosin staining (H&E)) of major 3

4 organs and tumors of mice after different treatment to assess safety in vivo. Supporting Tables Supporting Table S1. Particle size (D), Uv maximum absorption wavelength (λmax) and Surface zeta-potentials charges of gold nanoparticles before and after PEGylation. Supporting Table S2. The Correspondence between CT value results (CVR), Dose monitoring results (DMR) and Relative electron density results (REDR) of gold nanoparticles. 4

5 Supporting Figures Supporting Figure S1. DLS results for as-prepared AuNPs with different particle size. a-g, DLS results corresponding with statistics results of as-prepared AuNPs with seven types: 3.9nm ± 0.6 nm (a), 9.1 nm ± 0.7 nm (b), 13.2 nm ± 0.7 nm (c), 17.5 nm ± 1.1 nm (d), 28.3 nm ± 2.2 nm (e), 34.8 nm ± 2.2 nm (f), 41.1 nm ± 1.9 nm (g). 5

Supporting Figure S2. TEM for AuNPs with different particle size after PEGylation. a-g, TEM images showing seven types of PEGylated AuNPs: 4.0 nm ± 0.8 nm (a), 9.

6 Supporting Figure S2. TEM for AuNPs with different particle size after PEGylation. a-g, TEM images showing seven types of PEGylated AuNPs: 4.0 nm ± 0.8 nm (a), 9.2 nm ± 0.8 nm (b), 14.1 nm ± 0.9 nm (c), 18.4 nm ± 0.8 nm (d), 31.2 nm ± 1.8 nm (e), 37.3 nm ± 1.8 nm (f), 46.2 nm ± 2.2 nm (g). The 100 nm scale bar applies to all images. 6

7 Supporting Figure S3. Statistics histograms of particle size and particle-size distribution of AuNPs after PEGylation. a-g, statistics results by measuring the diameter of particles for seven types through TEM images: 4.0 nm ± 0.8 nm (a), 9.2 nm ± 0.8 nm (b), 14.1 nm ± 0.9 nm (c), 18.4 nm ± 0.8 nm (d), 31.2 nm ± 1.8 nm (e), 37.3 nm ± 1.8 nm (f), 46.2 nm ± 2.2 nm (g). 7

8 Supporting Figure S4. DLS results of particle size and particle-size distribution of AuNPs after PEGylation. a-g, DLS results corresponding with statistics results of as-prepared AuNPs with seven types: 4.0 nm ± 0.8 nm (a), 9.2 nm ± 0.8 nm (b), 14.1 nm ± 0.9 nm (c), 18.4 nm ± 0.8 nm (d), 31.2 nm ± 1.8 nm (e), 37.3 nm ± 1.8 nm (f), 46.2 nm ± 2.2 nm (g). 8

9 Supporting Figure S5. UV-visible absorption spectra of AuNPs before and after PEGylation for all seven types of AuNPs. a-g, we can see little red shift after PEGylation in range of 0.8 nm nm: 4.0 nm ± 0.8 nm (a), 9.2 nm ± 0.8 nm (b), 14.1 nm ± 0.9 nm (c), 18.4 nm ± 0.8 nm (d), 31.2 nm ± 1.8 nm (e), 37.3 nm ± 1.8 nm (f), 46.2 nm ± 2.2 nm (g). 9

10 Supporting Figure S6. UV-visible absorption spectra of PEGylated AuNPs under PH conditions. a-g, The aqueous solution of PEGylated AuNPs (0.5 mg/ml) was adjusted using HCl or NaOH (0.1 M) to have a ph range of 5-8 and maintained for 20 min before measurements: 4.0 nm ± 0.8 nm (a), 9.2 nm ± 0.8 nm (b), 14.1 nm ± 0.9 nm (c), 18.4 nm ± 0.8 nm (d), 31.2 nm ± 1.8 nm (e), 37.3 nm ± 1.8 nm (f), 46.2 nm ± 2.2 nm (g). 10

11 Supporting Figure S7. UV-visible absorption spectra of PEGylated AuNPs under different temperature conditions. a-g, The PEGylated AuNPs dispersed in water were also kept at different temperatures (4, 25, 37,and 50, respectively) for 30 min before measurements: 4.0 nm ± 0.8 nm (a), 9.2 nm ± 0.8 nm (b), 14.1 nm ± 0.9 nm (c), 18.4 nm ± 0.8 nm (d), 31.2 nm ± 1.8 nm (e), 37.3 nm ± 1.8 nm (f), 46.2 nm ± 2.2 nm (g). 11

time for 240 days. PEGylated AuNPs for seven types were assessed by dispersing the particles deionized water (a).

12 Supporting Figure S8. The colloidal stability of PEGylated AuNPs though evaluation of UV-visble maximum absorption wavelength with the increase of storage time for 240 days. PEGylated AuNPs for seven types were assessed by dispersing the particles deionized water (a). This trend for other conditions have similar phenomenon: phosphate buffered saline (PBS, b), the blank cell culture medium (c) and cell culture medium (d). It can be found that the particles do not precipitate at least four month's storage at room temperature. 12

2 nm ± 0.8 nm (d), 14.1 nm ± 0.9 nm (e), 18.4 nm ± 0.8 nm (f), 31.2 nm ± 1.8 nm (g), 37.3 nm ± 1.8 nm (h), 46.2 nm ± 2.2 nm (i).

13 Supporting Figure S9. Cell viability tests by MTT assay. HeLa cells treated with PEGylated AuNPs, Idohexol and CMNa with PBS as blank control: PBS (a), CMNa (b), 4.0 nm ± 0.8 nm (c), 9.2 nm ± 0.8 nm (d), 14.1 nm ± 0.9 nm (e), 18.4 nm ± 0.8 nm (f), 31.2 nm ± 1.8 nm (g), 37.3 nm ± 1.8 nm (h), 46.2 nm ± 2.2 nm (i). The disparity in cell viability between CMNa and different-sized PEGylated AuNPs were especially compared to demonstrate cytocompatibility. Error bars shown in the figure correspond to standard errors (n = 5). 13

14 Supporting Figure S10. X-ray attenuation intensity of different-sized PEGylated AuNPs and Idohexol. We can see attenuation intensity (HU) varying as a function of the molar concentration of gold (Au) or iodine (I) from 0 to 0.06 M: 4.0 nm ± 0.8 nm (a), 9.2 nm ± 0.8 nm (b), 14.1 nm ± 0.9 nm (c), 18.4 nm ± 0.8 nm (d), 31.2 nm ± 1.8 nm (e), 37.3 nm ± 1.8 nm (f), 46.2 nm ± 2.2 nm (g). Error bars shown in the figure correspond to standard errors (n = 3). 14

15 Supporting Figure S11. Relative electron density of different-sized PEGylated AuNPs. (a) The distribution of measured CT values was calibrated on accelerator dose calibration straight indicated the accuracy of the measured data. (b) CT contrast (up) and radiosensitization (medium) showed the similar variation corresponded to the curves of relative electron density (down). Error bars shown in the figure correspond to standard errors (n = 3). 15

16 Supporting Figure S12. Cellular radiosensitization of different-sized PEGylated AuNPs. Cell survivability of HeLa cells treated for different-sized PEGylated AuNPs changing with various concentrations (5, 10, 25, 50 and 100 µm) through MTT assay under radiation with X-ray doses of 2 Gy (a), 4 Gy (b), 6 Gy (c) and 8 Gy (d). HeLa cells treated with PBS buffer under radiation were used as control for all graphs. Error bars shown in the figure correspond to standard errors (n = 5). *P <

17 Supporting Figure S13. The sensitization enhancement ratio (SER) of different-sized PEGylated AuNPs. To evaluate radiotherapy enhancement efficiency, SER was calculated by SER = Cell viability (AuNPs)/ Cell viability (RT alone) from cell viability data of Supporting Figure S 10. Different changes of SER under various concentrations responding to X-ray radiation doses: 2 Gy (a), 4 Gy (b), 6 Gy (c) and 8 Gy (d). Error bars shown in the figure correspond to standard errors (n = 5). *P < 0.05, **P <

18 Supporting Figure S14. Flow cytometry plots of cellar apoptosis and necrosis induced by different-sized AuNPs without and with radiation. Hela cells were treated with different-sized AuNPs compared with CMNa and PBS as blank contro without (a-i) (n = 3) and with (j-r) (n = 3) 6 Gy X-ray irradiation to quantitatively analyze the size-dependent capability through cellar apoptosis and necrosis. 18

19 Supporting Figure S15. Dose absorption distribution of simulated modeling vesicle for different-sized AuNPs. There were two kinds of dose absorption peaks changing with particle sizes: 2 nm (a), 5 nm (b), 10 nm (c), 14 nm (d), 18 nm (e), 22 nm (f), 28 nm (g), 34 nm (h) and 40 nm (i). The lower peaks were for secondary effects which could compete with the higher peaks for primary effects when these energies with the same order of magnitudes because of sizes changed to nm (c, d, e). 19

20 Supporting Figure S16. Monte Carlo simulations verification for modeling vesicle containing only one particle. (a) A simulated modeling vesicle containing only one particle representative of the system homogeneity based on particle sizes. (b) CT imaging simulation by X-ray attenuation mainly from Photoelectric effect. Total emitted photon fluence variation with simulated particle sizes compared to water absence of particles under incident fluence level. (c) Total emitted photon fluence and attenuation enhancement ratio of simulated particle sizes, indicating no size-dependent effects. (d) Radiosensitization simulation by dose absorption mainly from Electron pair effect. Dose absorption levels (left) and dose enhancement ratios (DER) (right) of simulated particle sizes. Inset, DER disparity is highlighted to show no size-dependent effects for sizes over 5 nm. (e) Absorbed dose per specific surface area to present persistent enhancement without changes for simulated particle sizes with water as control. Simulations were repeated at least three times using the Monte Carlo code EGSnrc. Error bars, mean ± s.d. *P <

There were only one significant energy region for all particle sizes: 2 nm (a), 5 nm (b), 10 nm (c), 14

21 Supporting Figure S17. Dose absorption distribution of simulated modeling vesicle containing only one particle. There were only one significant energy region for all particle sizes: 2 nm (a), 5 nm (b), 10 nm (c), 14 nm (d), 18 nm (e), 22 nm (f), 28 nm (g), 34 nm (h) and 40 nm (i), indicating that secondary effects for this modeling vesicle could not compete with primary effects which energies were much higher. 21

22 Supporting Figure S18. Cell morphologies observation with incubation of PEGylated AuNPs with the optimal sizes and Idohexol. We observed normal cell morphologies maintained after 24 h incubation of PEGylated AuNPs (a), as measured by microscope, against abnormal changes for Idohexol (b), suggesting cytotoxicity-induced cell death. 22

23 Supporting Figure S19. Cell viability induced by the optimal AuNPs compared with CMNa before and after radiotherapy. To assess the ability of PEGylated AuNPs with the optimal size as efficient cellular radiosensitizers, Hela cells were treated with the optimized AuNPs or CMNa at a high concentration over 50 µm followed without or with RT compared with RT alone. Cell viability declined more greatly after RT with AuNPs than with CMNa which were all lower than RT alone. Error bars shown in the figure correspond to standard errors (n = 5). *P <

Balb/c female nude mice before and after injected subcutaneously with the optimized AuNPs (13.2 nm ± 0.

24 Supporting Figure S20. Intratumoral CT imaging of PEGylated AuNPs with the optimal size compared with Idohexol. CT contrast phantom images (a) and the corresponding quantitative analysis of CT contrast value histogram (b) of Hela tumor xenograft-bearing Balb/c female nude mice before and after injected subcutaneously with the optimized AuNPs (13.2 nm ± 0.7 nm) in left shoulder (red arrows) and Idohexol in right shoulder (blue arrows) at 0.6nmol/g from axial view (a, up) and coronal view (a, down). Error bars shown in the figure correspond to standard errors (n = 3). 24

25 Supporting Figure S21. Real-time in vivo CT contrast phantom images and quantitative analysis of major organs and tumors. The images from heart (a), bladder (b), brain (c), liver (d), tumor (e) and kidney (f) were collected at 0, 5, 15, 30, 45, 60 and 90 min after intravenous injection the optimized AuNPs (up, red arrows) and Idohexol (down, blue arrows) respectively of HeLa xenograft tumor-bearing balb/c nude mice with the same dose of 0.6 nmol/g ([Au or I]) at different time points post-injection. Arrows indicate the interesting regions. Error bars shown in the figure correspond to standard errors (n = 3). *P < 0.05, **P <

Supporting Figure S22. The disparity in CT contrast values of various organs with circulation time of PEGylated AuNPs compared with Idohexol.

26 Supporting Figure S22. The disparity in CT contrast values of various organs with circulation time of PEGylated AuNPs compared with Idohexol. CT contrast values (HU) histogram of different organs between PEGylated AuNPs (a) and Idohexol (b) at same doses of 0.6 nmol/g ([Au or I]) for different circulation time (-5, 5, 15, 30, 45, 60 and 90 min). Error bars shown in the figure correspond to standard errors (n = 3). *P < 0.05, **P <

27 Supporting Figure S23. In vivo biodistribution of gold and iodine element in different organs. After 24 h post intravenous injection of the PEGylated AuNPs and Idohexol, the contents of gold (Au) and iodine (I) in the major organs of the mouse were measured, including heart, liver, spleen, lung, kidney, brain, and bladder. The mouse with injection of PBS was used as blank control. The injected condition is same dose of 0.6nmol/g ([Au or I]). Error bars shown in the figure correspond to standard errors (n = 3). *P <

28 Supporting Figure S24. The photographs of the HeLa xenograft tumor-bearing nude mice for different in vivo radiosensitization groups. The images showed visibly different tumor inhibition of the tumors by evaluation before (black arrows) and after 30-day treatment (yellow arrows) for the groups as follows: Control with PBS injection (a), Radiotherapy alone (b), Radiotherapy within 30 min after intravenous injection of CMNa (0.6 nmol/g) (c), Radiotherapy within 30 min after intravenous injection of AuNPs (0.6 nmol/g) (d), Radiotherapy over 30 min after intravenous injection of AuNPs (0.6 nmol/g) (e) and Radiotherapy over 30 min after intravenous injection of AuNPs (0.6 nmol/g) (f). Arrows indicate the tumor regions. 28

Supporting Figure S25. Histology (hematoxylin and eosin staining (H&E)) of major organs and tumors of mice after different treatment to assess biosafety in vivo.

29 Supporting Figure S25. Histology (hematoxylin and eosin staining (H&E)) of major organs and tumors of mice after different treatment to assess biosafety in vivo. H&E-stained tissue sections from mice to monitor the histological changes in heart, liver, spleen, lung, kidney, brain and bladder of mice receiving single intravenous injection of PBS (control), Idohexol, the optimized AuNPs and CMNa with the same doses of 0.6 nmol/g followed by dissections in 30 days post injection. Scale bar represents 10 µm. 29

30 Supporting Tables Supporting Table S1. Particle size (D), Uv maximum absorption wavelength (λmax) and Surface zeta-potentials charges of gold nanoparticles before and after PEGylation. * The D values were obtained from measuring average diameters by TEM statistics results. Here, before and after refer to samples before and after PEGylation, respectively. After PEGylated incubation for 24 h, the nanoparticles were centrifuged, collected and re-dispersed in deionized water. The difference between after and before corresponds to the thickness of PEG adsorbed around the particle surface. # The λ max were obtained from measuring the maximum absorption wavelength by UV-vis spectra. Here, before and after refer to samples before and after PEGylation, respectively. Surface charges were measured while the nanoparticles were dispersed in deionized water. The errors are standard errors (n 3). Sizes were measured from literature methods. 30

31 Supporting Table S2. The Correspondence between CT value results (CVR), Dose monitoring results (DMR) and Relative electron density results (REDR) of gold nanoparticles. 31

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