Nanoscale Metal–Organic Framework with an X-ray Triggerable Prodrug for Synergistic Radiotherapy and Chemotherapy

As heavy-metal-based nanoscale metal–organic frameworks (nMOFs) are excellent radiosensitizers for radiotherapy via enhanced energy deposition and reactive oxygen species (ROS) generation, we hypothesize that nMOFs with covalently conjugated and X-ray triggerable prodrugs can harness the ROS for on-demand release of chemotherapeutics for chemoradiotherapy. Herein, we report the design of a novel nMOF, Hf-TP-SN, with an X-ray-triggerable 7-ethyl-10-hydroxycamptothecin (SN38) prodrug for synergistic radiotherapy and chemotherapy. Upon X-ray irradiation, electron-dense Hf12 secondary building units serve as radiosensitizers to enhance hydroxyl radical generation for the triggered release of SN38 via hydroxylation of the 3,5-dimethoxylbenzyl carbonate followed by 1,4-elimination, leading to 5-fold higher release of SN38 from Hf-TP-SN than its molecular counterpart. As a result, Hf-TP-SN plus radiation induces significant cytotoxicity to cancer cells and efficiently inhibits tumor growth in colon and breast cancer mouse models.


S1. Materials and Methods
All starting chemicals for the synthesis of nanoscale metal-organic frameworks (nMOFs) were purchased from Sigma-Aldrich or Fisher (USA) and used directly without purification.Phosphate buffered saline (PBS) for cell culture and RPMI-1640 medium were purchased from Corning, USA.Trypsin-EDTA solution and HyClone penicillin-streptomycin 100X solution were purchased from Cytiva USA.2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), dead cell apoptosis kit with annexin V Alexa Fluor 488 & propidium iodide (PI), hydroxyphenyl fluorescein (HPF), and aminophenyl fluorescein (APF) were purchased from Invitrogen.Chlorpromazine was purchased from Thermo Scientific.Nystatin, and rottlerin were purchased from MedChemExpress.CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega, USA.Phospho-histone H2A.X (Ser139) (γ-H2AX) rabbit monoclonal antibody and anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) were purchased from Cell Signaling Technology.Murine colorectal carcinoma CT26 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).MC38 and 4T1 cells were obtained from Dr. Weichselbaum and Prof. Kron at the University of Chicago, respectively.
Transmission electron microscopy (TEM) was carried out on an FEI Spirit 120kV LaB6 Electron Microscope.Powder X-ray diffraction (PXRD) data were collected on a Bruker D8 Venture diffractometer using a Cu Kα radiation source (λ = 1.54178Å) and processed with PowderX software, or collected on a Small Angle X-ray Scattering instrument, GANESHA.UV-Vis spectra were collected using a Shimadzu UV-2600 UV-Vis spectrophotometer.Fluorescence emission and excitation spectra were obtained using a Shimadzu RF-5301PC spectrofluorophotometer. Dynamic light scattering (DLS) and ζ-potential measurements were performed on a Malvern Zetasizer Nano ZS instrument.Inductively coupled plasma-mass spectrometry (ICP-MS) data were collected using an Agilent 7700x ICP-MS and analyzed using an ICP-MS Mass Hunter version 4.6 C.01.06.Samples were diluted in a 2% HNO3 matrix and analyzed with 159 Tb as internal standards against a 10-point standard curve between 1 ppb and 500 ppb (R>0.999 for 178 Hf and 232 Th).Data collection was performed in Spectrum Mode with triplicates per sample and 100 sweeps per replicate. 1H NMR spectra were recorded on a Bruker Ultrashield Plus spectrometer at 400 MHz and referenced to the proton resonance resulting from incomplete deuteration of CDCl3 (δ = 7.26) or DMSO-d6 (δ = 2.50).SN38 concentration was quantified using HPLC (Shimadzu Prominence UFLC/HPLC system with LC-20AT pump, Shimadzu Corporation, Japan) or LC-MS (Agilent 6540, Agilent Technologies, USA).
For test tube and in vitro X-ray irradiation experiments, an RT250 orthovoltage X-ray machine (Philips, USA) with fixed setting at 250 kVp, 15 mA and a built-in 1 mm Cu filter was used.For animal irradiation and computerized tomography (CT) scans, an X-RAD 225 image-guided biological irradiator (Precision X-ray Inc., USA) was used with voltage at 225 kVp, current at 13 mA, a 0.3 mm Cu filter, and a 15 mm collimator.The X-ray dose rate of X-RAD 225 was 0.04167 Gy/second.The Xray dosimetry of both instruments was calibrated with an ionization chamber regularly by the Department of Radiation Oncology at the University of Chicago.Flow cytometry data were collected on an LSR-Fortessa + Scheme S1.Synthetic route for H2TP-OH (9).

S3.1 Synthesis of Hf-TP-OH
HfCl4 and H2TP-OH were separately dissolved in dimethylformamide (DMF) at a concentration of 2 mg/mL.500 μL HfCl4 solution and 500 μL H2TP-OH were then combined in a 1-dram vial with the addition of 1 μL trifluoracetic acid and 5 μL water as modulators.The mixture was heated in an oven at 80 o C for 1 day, after which the white precipitate was collected by centrifugation and sequentially washed with DMF, 1% triethylamine (TEA) in ethanol (EtOH) (v/v), and EtOH to afford Hf-TP-OH in 88% yield based on H2TP-OH.

S3.2 Synthesis of Hf-TP-SN via post-synthetic modification
10 mL Hf-TP-OH was washed with dry acetonitrile (ACN) twice and dispersed in ACN with a ligand concentration of 3.0 mM.4-nitrophenyl chloroformate (15.1 mg, 75 μmol), and TEA (12.5 μL, 90 μmol) were then added, and the solution was stirred for 2 days to afford Hf-TP-NO2 nMOF.The mixture was then centrifuged, washed with ACN for 3 times and redispersed in ACN before the addition of SN38 (11.8 mg, 30 μmol) and TEA (6.3 μL, 45 μmol); the solution was then stirred for 2 more days.The assynthesized Hf-TP-SN was washed with 10% dimethyl sulfoxide (DMSO) in ACN 6 times and dispersed in EA for storage.

S4.1 Digestion of Hf-MOFs for 1 H NMR Characterization
Hf-TP-OH was dried under vacuum overnight.500 µL DMSO-d6 and 50 µL D3PO4 were added to the resulting solid.The mixture was sonicated for 1 hour, followed by the addition of 50 µL D2O for 1 H NMR analysis.

S4.3 Digestion of Hf-TP-SN for quantification of SN38 by LC-MS
100 µL 1M NaHCO3 solution was added to 100 µL Hf-TP-SN dispersion in water (total SN concentration: 100 µM).The mixture was sealed and sonicated for 20 minutes, after which 100 µL PBS (200 mM, pH 4.0) was added to adjust the pH to 7. 100 µL saturated NaCl solution and 150 µL ethyl acetate (EA) were then added before vortexing the mixture for 1 minute.The EA layer after centrifugation was analyzed with LC-MS.For HPLC, absorption of SN38 at 380 nm was used to calculate the concentration of free SN38 after comparison with the standard curve of SN38 in EA.For LC-MS, extracted ion chromatogram (EIC) at m/z value of 393.1450 was used to calculate the concentration of free SN38 after comparison with the standard curve of SN38 in EA.

S4.4 Stability of Hf-TP-SN in PBS
Hf-TP-SN was dispersed in 1 mL PBS (1 mM) with a Hf concentration of 5.2 mM.200 uL suspension was taken after incubation for 1, 2, 4, 8, and 24 hours and centrifuged for PXRD measurement.

S4.5 Stability of Hf-TP-SN after irradiation and long-term storage
Hf-TP-SN was dispersed in 1 mL water with a Hf concentration of 2 mM and then irradiated with 10 Gy X-ray.The morphology, crystallinity, and number-averaged size of the irradiated Hf-TP-SN were examined using TEM, PXRD and DLS, respectively.The Hf-TP-SN nMOF retained the crystallinity and morphology after irradiation as indicated by the same nanoplate morphology in TEM, consistent PXRD pattern, and similar number-averaged sizes.The number-averaged sizes of Hf-TP-SN before and after 10 Gy irradiation were 164 ± 6 nm and 156 ± 5 nm, respectively.
Hf-TP-SN was stored in EA with a Hf concentration of 2 mM for 1 year.The morphology, crystallinity, number-averaged size, and ζ-potential of Hf-TP-SN after long-term storage were examined using TEM, PXRD and DLS.Hf-TP-SN retained the nanoplate morphology under TEM and good crystallinity evidenced by PXRD.The number-averaged size of Hf-TP-SN was 133 ± 8nm, while its ζ -potential was -15.7 ± 0.4 mV, consistent with those of as-synthesized Hf-TP-SN.

S4.6 ROS generation in test tubes
Total ROS generation under irradiation was detected by the 2',7'-dichlorodihydrofluorescein (DCFH) assay following reported procedures. 2 1 mL DCFH-DA (1mM) in DMSO was hydrolyzed by 4 mL NaOH (0.01 M) solution in the dark for 30 minutes and stopped by adding 20 mL PBS (25 mM, pH 7.4).The freshly-prepared DCFH was then added to the PBS suspension (pH 7, 10 mM) of Hf-TP-OH or Hf-TP-SN at the same Hf concentration.In the final mixture, the concentration of DCFH was 10 μM while the concentration of Hf was 40 μM.The PBS solution with the same DCFH concentration served as a blank control.100 μL of each suspension was added to 96-well plates (n = 6) and then irradiated with Xray at 0, 1, 2, 3, 5, or 10 Gy, respectively.The fluorescence signal (em.520/20 nm) was collected with a Synergy HTX microplate reader (ex.485/20 nm).
Hydroxyl radical (•OH) generation under irradiation was detected by the APF assay.APF assay was added to the PBS suspension of Hf-TP-OH or Hf-TP-SN at the same Hf concentration.In the final mixture, the concentration of APF was 5 μM while that of Hf was 40 μM.The PBS solution with the same APF concentration served as a blank control.100 μL of each suspension was added to 96-well plates (n = 6) and then irradiated with X-ray at 0, 1, 2, 3, 5, or 10 Gy, respectively.The fluorescence signal (em.520/20 nm) was collected with a Synergy HTX microplate reader (ex.485/20 nm).

S4.7 Hydroxyl radical triggered SN38 release in test tubes
Hf-TP-SN or MeO-SN was dispersed in H2O at the same concentration of total SN38 (100 μM).FeCl3, (+)-Sodium L-ascorbate, Na2(EDTA)•2H2O (ethylenediaminetetraacetic acid, disodium salt dihydrate) was firstly dissolved in water to reach a concentration of 10 mM separately (10.5 mM for Na2(EDTA)•2H2O).Then, 100 μL of each solution and 50 μL H2O2 (200 mM in H2O) was added to Hf-TP-SN or MeO-SN solution to generate hydroxyl radical in situ and to trigger the release of SN38.For control group, only H2O2 was added.After 8h incubation at RT, additional 100 μL 1M NaHCO3 was added to Hf-TP-SN to digest the nMOF, after which the mixture was sealed and sonicated for 20 minutes before the addition of 100 μL PBS (200 mM, pH 4) to adjust the pH to 7. 100 µL saturated NaCl solution and 150 µL ethyl acetate (EA) were added before vortexing the Hf-TP-SN or MeO-SN mixture for 1 minute.The EA layer after centrifugation was analyzed by LC-MS.

S4.8 X-ray triggered release in test tubes
Hf-TP-SN or MeO-SN were dispersed in H2O at the same concentration of total SN38 (100 μM). 100 μL Hf-TP-SN or MeO-SN suspension was irradiated with 10 Gy X-ray. 100 μL 1M NaHCO3 was added to Hf-TP-SN to digest the nMOF, after which the mixture was sealed, sonicated for 20 minutes, and sat 15h before the addition of 100 μL PBS (200 mM, pH 4.0) to adjust the pH to 7. 100 µL saturated NaCl solution and 150 µL ethyl acetate (EA) were added before vortexing the Hf-TP-SN or MeO-SN mixture for 1 minute.The EA layer after centrifugation was analyzed by HPLC.

S4.9 Time-dependent release of SN38 from Hf-TP-SN with or without X-ray irradiation
Hf-TP-SN were dispersed in PBS (0.1 ×, pH = 7.4) at a total SN38 concentration of 100 μM.Half of Hf-TP-SN suspension was irradiated with 10 Gy X-ray.At different timepoints, 100 µL saturated NaCl solution and 150 µL ethyl acetate were added to a 100 µL Hf-TP-SN dispersion stored at 4 o C (with or without irradiation) before vortexing the mixture for 1 minute.The EA layer after centrifugation was analyzed with LC-MS.Hf-TP-SN without irradiation slowly released the entrapped SN38 (~2.9% after 24 h; the entrapped SN38 for this sample was determined to be 2.6%), while Hf-TP-SN after 10 Gy irradiation released more SN38 (by 0.70% ~ 1.56%) due to X-ray triggered release of SN38.

S5.2. Clonogenic assay
CT26 cells were seeded in 6-well plates at a density of 1.5 × 10 5 cells/well and cultured overnight.The cells were incubated with PBS, Hf-TP-OH, or Hf-TP-SN at an equivalent metal concentration of 50 μM for 4 hours, and then irradiated with 0, 2, 4, 6 and 8 Gy X-ray (n = 3).The cells were washed with PBS twice and then trypsinized to afford single cell suspensions.The cells were counted and diluted, then 200 cells were seeded in each well of 6-well plates and cultured in 2 mL medium for another 7 days.Then, the plates were rinsed once with PBS, fixed by 4% paraformaldehyde for 20 minutes, and washed with PBS twice at RT.After that, the 6-well plates were scanned with IncuCyte S3 in the whole well mode with a 4× objective.Clonogenic assay of 4T1 and MC38 cells was also conducted according to the same method as mentioned above.The colonies were identified with IncuCyte 2021A software in a cellular resolution and the confluence was used as a parameter to calculate the plating efficiency (PE) and surviving fraction (SF):

S5.3. Hydroxyl radical generation
To demonstrate •OH generation, CT26 cells were seeded in cell culture dishes at a density of 1.5×10 5 and cultured overnight.Hf-TP-OH was added at an equivalent Hf concentration of 50 µM and further incubated in a 37℃ incubator for 4 hours.The cells were washed with PBS solution 3 times and 1 mL cell culture medium containing with 10 µM HPF was added and incubated at 37℃ for another 30 minutes.Then the cells were irradiated with X-ray (3 Gy) and cultured in the cell incubator for another 24 h.After that, the cells were washed with PBS for three times and further incubated with Hoechst 33342 (10 μg mL -1 ) in PBS for 10 minutes in the cell incubator.Finally, the cells were washed with PBS 3 times and observed on a Leica Stellaris 8 confocal microscope.

S5.4. Dark toxicity
The cytotoxicities of SN38 and Me2TP-SN on CT26 cells were detected by MTS assay.CT26 cells were seeded in 96-well plates at a density of 2500 cells/well.Different concentrations of SN38 and Me2TP-SN were added and 48 hours later, 10% (v/v) of MTS reagent was added to each well.90 minutes later the absorbance of each well at 490 nm was read by a Synergy HTX plate reader to calculate cell viability.

S5.5. Cellular uptake
The cellular uptake of Hf-TP-SN (Hf: 50 µM) was evaluated on CT26 cells.The cells were seeded in 6well plates at a density of 2 × 10 5 /well and cultured overnight.Hf-TP-SN was added at a Hf concentration of 50 µM into the medium (n = 3).The cells were incubated in a 37 ℃ incubator for 1, 2, and 6 hours.At each time point, the medium was aspirated, the cells were washed with PBS three times, collected by centrifugation, and counted with a hemocytometer.The cell pellets were digested with nitric acid for 24 h and the concentration of Hf was detected by ICP-MS.
To probe the endocytosis mechanism, cells were seeded in 6-well plates at a density of 2 × 10 5 /well and cultured overnight.100 μM chlorpromazine, 270 μM nystatin, or 5 μM rottlerin was added to CT26 cells and incubated for 1 h to inhibit clathrin, caveolae, or macropinocytosis-mediated endocytotic pathway, respectively.Then, Hf-TP-SN was added at a Hf concentration of 25 µM into the medium (n = 3).The cells were incubated in a 37 ℃ incubator for 24 hours.Cell pellets were digested with nitric acid for 24 h and the concentration of Hf was detected by ICP-MS.The inhibition by chlorpromazine and rottlerin indicates that the uptake of Hf-TP-SN into CT26 cells is through clathrin-dependent pathway and macropinocytosis.

S5.6. ROS generation
To demonstrate the generation of ROS, CT26 cells were seeded in cell culture dishes at a density of 1.5×10 5 and cultured overnight.Hf-TP-SN or Hf-TP-OH was added at a Hf concentration of 50 µM and further incubated in a 37℃ incubator for 4 hours.The cells were washed with PBS solution 3 times and 1 mL cell culture medium containing with 30 µM DCFH-DA was added and incubated at 37℃ for another 30 minutes.Then the cells were irradiated with X-ray (3 Gy) and cultured in the cell incubator for another 24 h.After that, the cells were washed with PBS three times and further incubated with Hoechst 33342 (10 μg mL -1 ) in PBS for 10 minutes.Finally, cells were washed with PBS 3 times and observed on a Leica Stellaris 8 confocal microscope.The DCF + cells were also quantified by flow cytometry (FITC channel).Table S1.Percentage of DCF + CT26 cells after different treatments.X-ray dose was 3 Gy (Figure S15b).

S5.7. DNA damage
For CLSM imaging, CT26 cells were seeded in cell culture dishes at a density of 1.5× 10 5 .The cells were treated in the same way as in the clonogenic assay.24 hours after radiation, the cells were washed with PBS and fixed with 4% paraformaldehyde at RT for 20 minutes.The cells were again rinsed with PBS, blocked and permeabilized with 5% FBS + 0.3% Triton-X in PBS at RT for 1 hour.After blocking, cells were incubated with the γ-H2AX primary antibody (1:500) in 1% BSA + 0.3% Triton-X in PBS at RT for 1 hour.The cells were then washed with PBS and incubated with the Alexa Fluor 488 conjugated secondary antibody (1:3000) in 1% BSA + 0.3% Triton-X in PBS at RT for 1 hour.Afterwards, the cells were washed with PBS and further incubated with Hoechst 33342 (10 μg mL -1 ) in PBS for 10 minutes at 37 o C to visualize cell nuclei, respectively.Finally, cells were washed with PBS 3 times and observed on a Leica Stellaris 8 confocal microscope.

S5.8. Apoptotic cell death
To quantify apoptosis, CT26 cells were seeded in 6-well plates at a density of 1.5 × 10 5 /well and cultured overnight.Hf-TP-OH or Hf-TP-SN was added at an equivalent metal concentration of 50 µM for 4 hours and irradiated with X-ray (3 Gy).24 hours later, the cells were washed with PBS, trypsinized to afford single cell suspensions, cells were stained with the dead cell apoptosis kit with annexin V Alexa Fluor 488 & PI following the manufacturer's protocol and resuspended in the binding buffer for flow cytometric analysis (Annexin-V in FITC channel, PI in PE-dazzle 594 channel).

S5.9. In vitro drug release
The release of SN38 from Hf-TP-SN on CT26 cells was detected by MTS assay.CT26 cells were seeded in 96-well plates at a density of 2500 cells/well.Different concentrations of Hf-TP-SN were dispersed in cell culture medium and irradiated by X-ray with different doses (6 and 60 Gy).After that, 100 mL of above Hf-TP-SN containing culture medium was added to CT26 cells.48 hours later, 10% (V/V) of MTS reagent was added to each well.90 minutes later the absorbance of each well at 490 nm was read by a Synergy HTX plate reader to calculate cell viability.To evaluate the in vivo therapeutic efficacy, CT26 tumor models were established on BALB/c mice by inoculating 2 × 10 6 cells/mouse subcutaneously onto the right flanks at day 0, respectively.When CT26 tumors reached ~85 mm 3 , the mice were randomized for treatments.PBS, irinotecan, Hf-TP-OH, or Hf-TP-SN was intratumorally injected with an equivalent metal dose of 0.5 μmol in 20 μL PBS and SN38 dose of 0.047 μmol in 20 μL PBS.The dose of irinotecan was 0.047 μmol in 20 μL PBS.6-8 hours later, the mice were anaesthetized with 2.5% (v/v) isoflurane/O2 and mounted onto the X-Rad 225 irradiator.The CT26 tumors were irradiated with 2 Gy X-ray/fraction for 3 consecutive days.
4T1 tumor models were established on BALB/c mice by inoculating 2 × 10 6 cells/mouse subcutaneously onto the right flanks at day 0, respectively.When 4T1 tumors reached ~100-120 mm 3 , the mice were randomized for treatments.PBS, Hf-TP-OH, or Hf-TP-SN was intratumorally injected with an equivalent metal dose of 0.5 μmol in 20 μL PBS and SN38 dose of 0.047 μmol in 20 μL PBS.The dose of irinotecan was 0.047 μmol in 20 μL PBS.6-8 hours later, the mice were anaesthetized with 2.5% (v/v) isoflurane/O2 and mounted onto the X-Rad 225 irradiator.The 4T1 tumors were irradiated with 2 Gy X-ray/fraction for 3 consecutive days.
The length and width of tumor tissues were measured with an electronic caliper and body weights were monitored with an electronic scale.At the endpoint of the experiments, the mice were euthanized, and the tumors and major organs were sectioned for hematoxylin-eosin (H&E) staining to evaluate general toxicity.The tumor growth inhibition index (TGI) was defined as the equation below:         Owing to the enhanced RT effect and successful release of SN38, Hf-TP-SN(+) treated tumor showed the highest ratio of TUNEL + cancer cells due to the synergistic chemotherapy and radiotherapy.
4-15 HTS (BD Biosciences, USA) at the Cytometry and Antibody Technology Facility at the University of Chicago and analyzed by FlowJo software (Tree Star, USA).Confocal laser scanning microscopy (CLSM) images were collected on a Leica Stellaris 8 laser scanning microscope at the Integrated Light Microscopy Facility at the University of Chicago, and analysis was done with Image J software (NIH, USA).Live cell images were recorded and analyzed by IncuCyte S3 (Essen BioScience) at Cellular Screening Center at the University of Chicago.The histological slides were scanned on a CRi Pannoramic SCAN 40x whole slide scanner by Integrated Light Microscopy Core at the University of Chicago and analyzed with the QuPath-0.2.3 software.The absorbance and fluorescence intensities from well plates were read by a BioTek Synergy HTX microplate reader.P value was calculated by student's two-tailed t test, *, p<0.05, **, p<0.01, ***, p<0.001, and ****, p<0.0001 were used in all figures to show the statistical significance.

S4. 2
Digestion of Hf-MOFs for UV-Vis spectroscopic measurements 50 µL Hf-TP-OH or Hf-TP-SN solution, 900 µL DMSO and 50 µL H3PO4 were mixed and sonicated for 1 hour and let stand overnight, after which the mixture was diluted to a proper concentration for UV-Vis measurement.The absorbance of H2TP-OH at 304 nm was used to calculate the concentration of H2TP-OH in Hf-TP-OH or Hf-TP-SN after comparison with the standard curve of H2TP-OH in DMSO, while the absorbance of SN38 at 390 nm was used to calculate the concentration of SN38 in Hf-TP-SN after comparison with the standard curve of SN38 in DMSO.

Figure S2 .
Figure S2.UV-Vis absorption spectra of (a) H2TP-OH and (c) SN38 and the fitted standard curves of (b) H2TP-OH and (d) SN38 in DMSO.

Figure S3 .
Figure S3.The fitted standard curves of SN38 in EA by (a) HPLC or (b)LC-MS.(c) Representative EIC at m/z value of 393.1450 for quantification of entrapped SN38 in Hf-TP-SN.

Figure S6 .
Figure S6.Concentration of SN38 released from MeO-SN or Hf-TP-SN (a) after 10 Gy of X-ray irradiation or (b) after reacting with hydroxyl radical generated by Fenton reaction.Total concentration of SN38 in MeO-SN and Hf-TP-SN was 100 µM.(+) represents 10 Gy X-ray irradiation while (-) represents no irradiation in a. Entrapped SN38 was subtracted.

Figure S12 .
Figure S12.CLSM images of CT26 cells stained by hydroxyphenyl fluorescein (HPF, green) and Hoechst 33342 (blue, cell nucleus) for detecting the generation of •OH.

Figure S16 .
Figure S16.(a) Gating strategy for the analysis of apoptotic cell death in PBS(-) group without staining.(b) Percentage of early apoptotic, late apoptotic, and necrotic CT26 cells after different treatments.X-ray dose was 3 Gy.

Figure S17 .
Figure S17.Cell viability of CT26 cells treated by Hf-TP-SN pre-irradiated with different doses of Xray.

4 V
L, W, ms and mc represent tumor volume, tumor length, tumor width, average tumor weight of treated mice at endpoint, control mice at endpoint, respectively.

Figure S18 .
Figure S18.Schematic illustration of the dosing schedule on subcutaneous CT26 and 4T1 tumors in BALB/c mice.

Figure S23 .
Figure S23.Statistical analysis of positive rates of γ-H2AX staining in CT26 tumor slides.

Figure S24 .
Figure S24.Statistical analysis of positive rates of Ki67 staining in CT26 tumor slides.

Figure S25 .
Figure S25.Statistical analysis of positive rates of TUNEL staining in CT26 tumor slides.