Intelligent design of polymer nanogels for full-process sensitized radiotherapy and dual-mode computed tomography/magnetic resonance imaging of tumors

Rationale: Development of intelligent radiosensitization nanoplatforms for imaging-guided tumor radiotherapy (RT) remains challenging. We report here the construction of an intelligent nanoplatform based on poly(N-vinylcaprolactam) (PVCL) nanogels (NGs) co-loaded with gold (Au) and manganese dioxide (MnO2) nanoparticles (NPs) for dual-mode computed tomography (CT)/magnetic resonance (MR) imaging-guided “full-process” sensitized RT of tumors. Methods: PVCL NGs were synthesized via precipitation polymerization and in situ loaded with Au and MnO2 NPs. The created PVCL-Au-MnO2 NGs were well characterized and systematically examined in their cytotoxicity, cellular uptake, intracellular oxygen and ·OH production, and cell cycle arrest in vitro, evaluated to disclose their RT sensitization effects of cancer cells and a tumor model, and assessed to validate their dual-mode CT/MR imaging potential, pharmacokinetics, biodistribution, and biosafety in vivo. Results: The formed PVCL-Au-MnO2 NGs with a size of 121.5 nm and good stability can efficiently generate reactive oxygen species through a Fenton-like reaction to result in cell cycle distribution toward highly radiosensitive G2/M phase prior to X-ray irradiation, sensitize the RT of cancer cells under X-ray through the loaded Au NPs to induce the significant DNA damage, and further prevent DNA-repairing process after RT through the continuous production of O2 catalyzed by MnO2 in the hybrid NGs to relieve the tumor hypoxia. Likewise, the in vivo tumor RT can also be guided through dual mode CT/MR imaging due to the Au NPs and Mn(II) transformed from MnO2 NPs. Conclusion: Our study suggests an intelligent PVCL-based theranostic NG platform that can achieve “full-process” sensitized tumor RT under the guidance of dual-mode CT/MR imaging.


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Au-MnO2 NGs were measured using a Malvern zetasizer (Nano ZS model ZEN3600 with a standard 633 nm laser, Worcestershire, UK). Au and MnO2 contents in the PVCL-Au-MnO2 NGs were determined by a Leeman Prodigy inductively coupled plasma-optical emission spectroscopy (ICP-OES, Hudson, NH). The samples were digested by aqua regia and diluted with water before measurements.

·OH generation by Mn 2+ -mediated Fenton-like reaction.
First, we used MnCl2 as a model to evaluate the Mn 2+ -mediated Fenton-like reaction according to the literature [1]. In brief, methylene blue (MB) was used as a substrate (10 μg mL -1 ) to test the ·OH generation ability. In the presence of H2O2 (8 mM) dissolved in 25 mM NaHCO3/5% CO2 buffer solution (5 mL), MnCl2 was added to achieve a final concentration of 0.5 mM. The reaction was performed at 37 o C for 30 min and the changes of MB absorbance indicating the ·OH-induced MB degradation in the mixture solution at 665 nm were monitored using a Pekin-Elmer Lambda 25 UV-vis spectrophotometer (Boston, MA).
To check the concentration-dependent ·OH generation capacity, H2O2 or Mn 2+ with different concentrations were dissolved in 25 mM NaHCO3/5% CO2 buffer solution containing 10 μg mL -1 MB.

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All reactions were monitored by the absorbance change at 665 nm through UV-vis spectrometry.
X-ray attenuation property. The X-ray attenuation property of PVCL-Au-MnO2 NGs was studied using a micro-CT system (SIEMENS Inveon MM, Erlangen, Germany) at 70 kV and a slice thickness of 0.1 mm. PVCL-Au-MnO2 NGs with different Au concentrations were tested, and the Xray attenuation intensity was determined in Hounsfield units (HU). TME-responsive magnetic resonance (MR) relaxometry. For the T1 MR relaxometry study, a 0.5 T NMR analyzing and imaging system (NMI20, Niumag, Shanghai, China) was used to measure Hemolysis, cytotoxicity, and cellular uptake assays in vitro. Hemolysis assay was performed to evaluate the hemocompatibility of the PVCL-Au-MnO2 NGs according to the literature [2]. In brief, 1.5 mL of blood collected from the inner canthus vein plexus of mice was diluted with 3.5 mL of phosphate buffered saline (PBS), and then the pure red blood cells (RBCs) were obtained via repeated centrifugation/redispersion processes (2000 rpm, 10 min, 3 times). The RBCs were then diluted with 5 mL of PBS. Thereafter, 100 μL of the obtained RBC suspension was mixed with 900 μL water (positive control), PBS (negative control) and PVCL-Au-MnO2 NG dispersed in PBS at various concentrations (12.5-400 μg mL -1 ). After 2 h incubation at 37 o C, each sample was centrifuged at 13000 rpm for 15 min. UV-vis spectrometry was used to record the absorbance of the supernatant at 540 nm.
For cytotoxicity assays, L929 or Pan02 cells were seeded into 96-well plates at a density of 1 × 10 4 cells per well in 5% CO2 at 37 o C overnight to allow the attachment of cells. The next day, the medium of each well was replaced with 100 µL complete medium containing PVCL NGs, PVCL-Au NGs, PVCL-MnO2 NGs or PVCL-Au-MnO2 NGs at different NG concentrations (0, 6.25, 12.5, 25, 50, 100, 200, 300 and 400 µg mL -1 , respectively). For L929 cells, RPMI 1640 medium was used, while Pan02 cells were cultured using DMEM. The cells were incubated for additional 24 h. After that, 100 μL medium containing 10% CCK-8 agent was added to each well, and cells were incubated under S-6 regular culture conditions for 4 h. Then, the optical density of each well was examined using a Multiskan MK3 ELISA reader (Thermo Scientific, Waltham, MA).
To examine the cellular uptake of the PVCL-Au-MnO2 NGs, cells treated with NGs were observed by TEM. Pan02 cells were seeded into 6-well plates at a density of 2 × 10 5 cells per well overnight.
Then, the cells were treated with fresh complete DMEM containing PVCL-Au-MnO2 NGs (200 μg mL -1 ) for 12 h at 37 o C. Then, the cells were washed with PBS for three times, fixed with 1 mL of glutaraldehyde (2.5%) at room temperature for 30 min, washed with PBS for three times, collected with cell scraper and fixed again with 1 mL of glutaraldehyde (2.5%) at 4 o C for 12 h. Then, each cell sample was processed under standard protocols before TEM observation using a Hitachi  Clonogenic assay. Pan02 cells were cultured and treated with NGs under the same conditions described above for cell cycle analysis except the cell seeding density (5 × 10 5 cells per well). Then, the cells in each group were washed twice with PBS and received the X-ray irradiation at 0, 2, 4, 6 and 8 Gy, respectively. Next, depending on the different radiation dose (0, 2, 4, 6 or 8 Gy), the cells in each group were digested, counted, and seeded at a density of 200, 400, 800, 2000, or 4000 cells per well in 6-well plates, followed by a further incubation of 10 days. For the X-ray irradiation treatment, the total exposure irradiation time of cells during the procedure was 1.1 min. The irradiation equipment was the medical linear accelerator (Varian Clinac IX), which used 6 MV X-ray, and the size of the radiation field was 30 × 30 cm. Before irradiation, we used two equivalent solid water plates with a thickness of 5 mm to simulate the cell fluid, and the calibrated MOSFET detector was placed between these two solid water plates. Then, the accelerator with an output of 500 monitor unit (MU) was used, S-8 and the absorbed dose of the detector was recorded. On this basis, the accelerator MU value corresponding to the irradiation dose to the cells was calculated, and the absorption coefficient was also read by the detector. Colonies were stained with 1.0% crystal violet and counted to evaluate the effect of the respective treatment. The surviving fraction of each group was measured in triplicate through the formula of surviving fraction = (surviving colonies)/(cells seeded × plating efficiency).
The classical multitarget single-hit model was adopted to perform the nonlinear fitting of the cell survival fraction of each group using Graphpad prism ® 8.0 software (GraphPad Software Inc., San Diego, CA). Meanwhile, the sensitization enhancement ratio (SER) of each group was also calculated via the multitarget single-hit model. Apoptosis assay. Pan02 cells were seeded in 6-well plates at a density of 5 × 10 5 cell per well (2 mL DMEM) overnight. Then, the cells were incubated with complete DMEM containing PVCL-Au, PVCL-MnO2, or PVCL-Au-MnO2 NGs (NG concentration = 200 μg mL -1 ) for 12 h. Cells treated with PBS (200 L) were used as control. After that, the cells were exposed to X-ray irradiation (4 Gy, 3.6 Gy min -1 ), followed by further incubation for 24 h. Subsequently, the cells in each well were trypsinized, stained with Annexin V-Fluorescein Isothiocyanate/PI Kit according to the manufacturer's instruction, and analyzed by a Becton Dickinson Accuri C6 Flow Cytometer (Bedford, MA). Each sample was measured for three times.
Detection of ROS after X-ray radiation. Pan02 cells were seeded in 12-well plates at a density of 1 × 10 5 cell per well (1 mL DMEM) for 24 h. Then, the cells were incubated with complete DMEM containing PVCL-Au, PVCL-MnO2, or PVCL-Au-MnO2 NGs (200 μg mL -1 ) for 12 h. After that, the cells were exposed to X-ray irradiation (4 Gy, 3.6 Gy min -1 ), followed by further incubation for 1 h.
Cells treated with PBS only (100 L) were set as control. Subsequently, the cells were washed twice with PBS, and stained with DCFH-DA for 30 min at 37 o C and 5% CO2. After the washing and trypsinization process, the fluorescence signals of cells were detected using flow cytometry. For each sample, the mean fluorescence intensity of 1 × 10 4 cells was recorded to determine the intracellular content of ROS and each measurement was repeated for three times.

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Western blotting analysis. Pan02 cells were seeded in 6-well plates at a density of 5 × 10 5 cells per well (2 mL DMEM) for 24 h. The cells were then incubated with complete DMEM containing PVCL-Au-MnO2 NGs (200 μg mL -1 ) for 12 h, followed by washing twice with PBS before the treatment of X-ray irradiation (4 Gy, 3.6 Gy min -1 ). After that, the cells were lysed and the effects of NG treatment with or without X-ray on the expression levels of the related proteins were determined by western blotting according to our previous work [3]. Cells treated with PBS (with or without X-ray radiation) were also analyzed for comparison. were i.v. injected to each tumor-bearing C57BL/6 mouse. Then, MR scanning was performed using the Bruker Biospec 7T micro-MR imaging system as mentioned above. The detailed parameters were set as follows: TE = 11.7 ms, TR = 1658.0 ms, and slice thickness = 0.5 mm. MR images before and at 24 h post-injection were acquired, and MR signal to noise ratio (SNR) was quantified using the signal of air as the noise. Histological examinations. For histological analysis, one tumor tissue at 7 days post-treatment in each group was collected, and the major organs (heart, liver, spleen, lung and kidney) at 20 days S-12 post-treatment in each group were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned through standard protocols [5]. The pathological and morphological changes of the tumor tissues and major organs were studied by hematoxylin and eosin (H&E) staining. Additionally, the tumor cell proliferation and apoptosis were analyzed by immunohistochemical staining of proliferating cell nuclear antigen Ki67 monoclonal antibody and immunofluorescence staining with TdT-mediated dUTP Nick-End Labeling (TUNEL), respectively.

Statistical analysis.
All experimental data were given as the mean ± standard deviation (n  3).
Scientific graphing, comprehensive curve fitting, and data organization were performed by GraphPad Prism ® 8.0 software. A p value of 0.05 was selected as the significance level, and all data were marked as (ns) for p > 0.05, (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.         S-21