Title: Manganese–Fluorouracil Metallodrug Nanotheranostic for MRI-Correlated Drug Release and Enhanced Chemoradiotherapy
Abstract: Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2021Manganese–Fluorouracil Metallodrug Nanotheranostic for MRI-Correlated Drug Release and Enhanced Chemoradiotherapy Chan Yang‡, Guosheng Song‡, Haifeng Yuan, Yue Yang, Yuqi Wang, Deju Ye, Hongmin Meng, Shuangyan Huan and Xiao-Bing Zhang Chan Yang‡ State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 , Guosheng Song‡ State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 , Haifeng Yuan State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 , Yue Yang State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 , Yuqi Wang State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 , Deju Ye State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 , Hongmin Meng College of Chemistry, Zhengzhou University, Zhengzhou 450001. , Shuangyan Huan State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 and Xiao-Bing Zhang *Corresponding author: E-mail Address: [email protected] State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 https://doi.org/10.31635/ccschem.020.202000188 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail For cancer therapy, drug delivery systems are often limited by insufficient drug loading capacity, which usually results in systemic toxicity and heavy metabolic burden to excrete the carriers. Herein, we reported a "one-pot" method for constructing metal (Mn2+)–fluorouracil (FU)-coordinated nanotheranostics (Mn-FU) by self-assembly of FU (as bridging ligands) and Mn2+ (as metal nodes) through Mn–N/O coordination interactions. Importantly, owing to the effective coordination between Mn and FU, Mn-FU exhibits high drug loading efficacy (47.7 wt %), encapsulation efficacy (82.6%), and relatively large yield (1 g/pot). In acidic tumor microenvironments, efficient release of FU and Mn2+ is realized because of nitrogen protonation. The released FU and Mn2+ from Mn-FU are used for chemotherapy and turn on magnetic resonance imaging (MRI), respectively, achieving MRI-correlated drug release. After PEG modification, Mn-FU displays high tumor homing ability via enhanced permeability and retention effects and quick renal clearance owing to the disassembly in acidic biological conditions. As a result, Mn-FU substantially enhances the synergistic effects of chemoradiotherapy. Meanwhile, the systemic toxic side effects of free FU-based chemoradiotherapy were greatly reduced through this nanotheranostic. Our strategy offers a facile way to construct metallodrug nanotheranostics for efficient cancer theranostics. Download figure Download PowerPoint Introduction Chemotherapy remains one of the most commonly used techniques for cancer treatment in clinics, even though it shows limited efficacy due to low tumor accumulation, multidrug resistance, and inevitable lethal side effects.1,2 Fluorouracil (FU) is a clinically used small molecule drug for antitumor chemotherapy.3 As a uracil analog, FU exhibits cytotoxicity through interfering with DNA synthesis of live cells.4 However, such misincorporated processes exhibit poor selectivity between cancer cells and normal proliferative cells, which results in inevitable toxic side effects, including myelosuppression, mucositis, dermatitis, diarrhea, and cardiac toxicity.5,6 Besides, antitumor efficacy of FU is often limited by insufficient accumulation into tumor sites as well as inadequate cellular uptake,7–10 and thereby excessive dosage of FU is usually required, which might cause toxic side effects and severe drug resistance.11 Moreover, the combination of FU and radiotherapy (RT) has been widely used for antitumor treatment in clinics.12 Although a low dosage of FU has been achieved for effective treatment, such chemoradiotherapy has caused much more severe toxic side effects, due to the lack of selectivity of both X-ray and FU.13 Thus, novel strategies that can promote therapeutic efficacy and reduce the toxic side effects for chemotherapy or chemoradiotherapy of FU are highly desired for clinical cancer treatment. Recently, a variety of nanotechnology-based drug delivery systems have been employed to deliver FU, which have greatly improved antitumor efficacy and attracted great attention in clinical or preclinical research.14–17 For example, gold and Se nanoparticles have been employed as carriers to load FU through a photoresponsive linkage or coordination interaction of Se–O/N.18,19 However, such covalent linkages lead to unsatisfactory drug loading efficacy (14.82% for gold nanoparticles). Furthermore, graphene or covalent organic frameworks with more accessible surfaces can effectively load FU through either H-bond or π–π stacking interaction with drug loading efficiencies of 12% and 35%, respectively.20,21 Besides, various mesoporous nanocarriers (e.g., hollow mesoporous silica and zeolitic imidazole framework) have been attempted to encapsulate FU through physical absorption owing to abundant porous structures10,22,23 with a relatively high loading capacity (21.1% of zeolitic imidazole framework) and controlled release profiles. Unfortunately, most of the aforementioned drug delivery systems have been difficult to be metabolized, which may induce heavy burdens for body clearance and ultimately cause unavoidable side effects. Excitingly, FU as a nucleoside analog has been recently integrated into DNA strands and further self-assembled into well-defined DNA nanostructures with definite drug embedding rates (18.36% for DNA polyhedra) as well as tunable size and morphology.24,25 Although such programmable DNA nanostructures serve as promising candidates for combinatorial chemo and gene therapy,26 they still suffer from extremely high cost for future extensive applications in clinical and unstable structures during blood circulation because of the existing various hydrolases in biological environments. Various imaging agents such as near-infrared fluorochromes and magnetic resonance contrast agents have been integrated with nanocarriers that do not only deliver FU but also localize the carriers for guiding FU delivery, such as layered double hydroxide covered upconversion nanoparticles or covalent organic framework-based fluorescent nanovehicles and Gd3+-doped mesoporous hydroxyapatite nanoparticles by either fluorescence imaging or magnetic resonance imaging (MRI).21,27,28 However, changes in fluorescence or MRI signal are independent of the release of FU, which is incapable of monitoring the drug release processes. Besides, most fluorescence imaging techniques often suffer from low imaging resolution, autofluorescence, photobleaching, and low tissue penetration depth.29 Furthermore, complicated designs and tedious synthetic steps are usually needed for constructing such highly complex nanoplatforms, which may further hinder their clinical translational application. Therefore, it is desired to develop more accessible strategies for efficiently enhancing synergetic chemoradiotherapy and monitoring drug release, which is beneficial to evaluate therapeutic results. Different from various nanocarrier-based theranostics, nanoplatforms constructed from drug-based building blocks demonstrate incomparable advantages including high drug loading, biodegradability, and stimuli-responsiveness. Herein, we developed a "one-pot" method for constructing Mn-FU metallodrug nanotheranostics (Mn-FU), through a facile coordination self-assembly strategy (Scheme 1). Compared with other drug delivery systems, higher loading efficiency of FU (47.7 wt %) and relatively large yields (1 g/pot) were achieved by embedding FU as bridging ligands into such metal–drug-coordinated nanotheranostics. Notably, Mn-FU exhibited ultrasensitive release of FU and Mn2+ within the acidic tumor microenvironment (TME), resulting in high selectivity toward cancer cells, so as to reduce unwanted toxicity toward normal tissues. Moreover, the accompanying release of Mn2+ produced gradually elevate T1 and T2 MRI contrast, and the responsive MRI signal was correlated with the release of FU, which could achieve MRI-correlated drug release. Hence, compared with the existing complicated nanoplatforms, the simple Mn-FU achieved complete functional utilization of each component. After proper surface modification, Mn-FU was able to accumulate into the tumor site, which significantly improved the utilization rate of FU and, thus, reduced the required dosage by 67% for tumor growth inhibition. Thereby, Mn-FU could effectively amplify the combined chemoradiotherapeutic outcomes, with synergistic antitumor efficacy. Besides, the quick renal clearance of Mn-FU could avoid the chronic toxicity induced by the long-term accumulation of nanotheranostics within the body. Scheme 1 | Schematic illustration of the construction of metallodrug nanotheranostics and its further application for antitumor therapy. Download figure Download PowerPoint Experimental Section Materials and instruments Manganese chloride tetrahydrate (MnCl2·4H2O), FU, triethylamine [TEA, (C2H5)3N], NaN3, sucrose, dopamine, and N,N-dimethylformamide (DMF), were purchased from Tianjin Heowns Biochemical Technology Co., Ltd.,Tianjin, China. Methyl-β-cyclodextrin (M-β-CD) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. Amine-mPEG (Mw = 5000) was purchased from Shanghai Tuoyang Biological Science & Technology Co., Ltd., Shanghai, China. Transmission electron microscopy (TEM) measurements were carried out with a JEM-2100Plus (JEOL) transmission electron microscope. More detailed information of materials and instruments can be found in the Supporting Information. Preparation of Mn-FU About 0.2 g of MnCl2·4H2O (1 mmoL) and 0.13 g of FU (1 mmoL) were dissolved in 25 mL of DMF and sonicated until a clear solution was obtained. Then, the mixture solution was added with 0.25 mL of TEA and quickly transferred to a flask and sonicated for 1 h. After sonication, the sample was heated to 100 °C in an oven and kept for 1 h. After cooling down to room temperature, the obtained suspension was sonicated for another 20 min. The mixture was centrifuged and washed with DMF and H2O three times. The drug loading efficacy and drug encapsulation efficiency were calculated according to the following equations: Drug loading efficiency ( % ) = Mass of FU Total mass of nanotheranostics * 100 % Encapsulation efficiency ( % ) = Mass of loaded FU Mass of initial added FU * 100 % PEGylation of Mn-FU About 8 mL of Mn-FU suspension in Tris buffer (10 mM, pH 8.5) was added with 0.2 mL of freshly prepared dopamine solution (2 mg/mL). After being stirred under air at room temperature for 1 h, the product was separated by centrifugation and dispersed into 8 mL of water. Next, 100 mg of amine-terminated PEG (mPEG-NH2, 5 kDa) was added into the aforementioned solution at pH 12.0, followed by 30 min of sonication. After stirring overnight, the final product (Mn-FU-PEG) was separated and purified by centrifugation, and stored at 4 °C. In vitro FU and Mn2+ release Mn-FU was introduced into 1×HEPES solutions at pH 5.5 or 7.4. At different time intervals, 0.2 mL of solution was taken out and centrifuged. The supernatants were used for measuring FU by using UV–Vis absorbance spectrometer and Mn2+ concentrations by using inductively coupled plasma–mass spectrometry (ICP-MS). Cellular uptake of Mn-FU Fluorescein-labeled Mn-FU (F-Mn-FU) was prepared by mixing polydopamine-covered Mn-FU (1 mg/mL) with fluorescein isothiocyanate (FITC) (2 mg) in anhydrous DMF, stirred in dark at room temperature for 24 h, and followed by washing with DMF/H2O to remove excessive FITC. HeLa cells were incubated with F-Mn-FU (10 μg/mL) for 3 h. Cell nuclei were stained by 4',6-diamidino-2-phenylindole for 10 min. Fluorescence images were obtained by confocal laser scanning microscopy (TI-E+A1 SI, Nikon) with excitation at 488 nm. To evaluate the endocytosis pathway, experiments were carried out according to literature30,31 with details provided in the Supporting Information. In vitro cytotoxicity of Mn-FU The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, cell apoptosis and necrosis, and DNA damage evaluation assay were carried out according to the literature32 with minor modifications, and the details are presented in the Supporting Information. In vivo antitumor therapy All animal protocols were approved by the Animal Care and Use Committee of Hunan University. Mice bearing a 4T1 xenograft tumor were used for antitumor chemoradiotherapy with similar experimental procedures to literature,32 and the details are presented in the Supporting Information. Results and Discussion Synthesis and characterization of Mn-FU Mn-FU was prepared through coordination-driven self-assembly strategy by simply mixing FU and manganese chloride under sonication in one-pot (Figure 1a). The TEM image (Figure 1b) shows Mn-FU with fusiform morphology and homogeneous size distribution (about 35 nm in length and 15 nm in width), which was further confirmed by dynamic light scattering (DLS) ( Supporting Information Figure S1). High-resolution TEM showed Mn-FU with a highly crystalline structure (Figure 1c), which is consistent with powder X-ray diffraction (PXRD) (Figure 1e). High-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping showed a good mixture of Mn, O, F within the nanoparticles (Figure 1d). UV–Vis spectra ( Supporting Information Figure S2) and 19F nuclear magnetic resonance (19F-NMR) spectra ( Supporting Information Figure S3) showed Mn-FU with typical spectral peaks of FU. In Fourier transform infrared (FTIR) spectroscopy, the absorption band at 3142 cm−1 was attributed to the N–H stretching vibration in FU, which disappeared in Mn-FU (Figure 1f), demonstrating the coordination between Mn and N.33 In addition, the absorption band at 1662 cm−1 was attributed to the C=O stretching vibration in FU, which changed to 1670 cm−1 and 1540 cm−1 in Mn-FU, demonstrating the coordination between Mn and O. As demonstrated by X-ray photoelectron spectroscopy (XPS), the slightly shifted peaks of Mn2p (from 641.48 to 641.28 eV), N1s (from 400.58 to 398.88 eV), and O1s (from 531.58 to 531.28 eV) (Figures 1g, 1h, and 1i and Supporting Information Figure S4) confirmed that the construction of Mn-FU nanotheranostics was driven by the coordination between divalent manganese ion with nitrogen and oxygen in FU molecules, with the simulated coordination structure in Figure 1a.34 The FU-to-Mn ratio was determined to be 1.18, based on the ICP-MS measurement and thermogravimetric analysis (TGA) results (Figure 1j). Due to the facile preparation, as high as 47.7% of drug loading efficacy (based on the TGA result) and 82.6% encapsulation efficacy were achieved, and more than 1 g product could be obtained in one-pot synthesis ( Supporting Information Figure S5). To further improve the biostability of Mn-FU, PEGylation was employed to afford Mn-FU-PEG nanoparticles. From TEM imaging, we found that Mn-FU-PEG showed the same fusiform morphology with Mn-FU, indicating a well-maintained structure. Both TEM imaging (Figure 1k) and DLS profiles (Figure 1l) showed Mn-FU-PEG with homogeneous size distribution (about 49 nm in length and 29 nm in width). The UV–Vis spectrum (Figure 1m) shows Mn-FU-PEG with typical spectral peaks of surface PEG layer, which demonstrated successful surface modification. Figure 1 | Synthesis and characterization of Mn-FU. (a) Illustration of the construction of Mn-FU and its coordination structure. Blue: N, gray: C, green: O, yellow: F, light gray: H. (b) TEM image of Mn-FU. (c) High-resolution TEM image of Mn-FU. (d) Scanning transmission electron microscope (STEM) images of Mn-FU and the corresponding STEM-energy dispersive spectrometer (STEM-EDS) elemental mapping of Mn, O, and F. (e) PXRD profile of Mn-FU. (f) FTIR spectra of FU and Mn-FU nanodrugs. (g) XPS spectra of Mn 2p in MnCl2 and Mn-FU nanodrugs. (h) XPS spectra of N 1s in FU and Mn-FU nanodrugs. (i) XPS spectra of O 1s in FU and Mn-FU nanodrugs. (j) Thermogravimetric curves of FU and Mn-FU nanodrugs. (k) TEM image of Mn-FU-PEG. (l) DLS profile of Mn-FU-PEG. (m) UV–Vis spectra of Mn-FU and Mn-FU-PEG. Download figure Download PowerPoint MRI-correlated drug release Inspired by the unstable coordination between Mn and N under acidic conditions due to the protonation of nitrogen,35 Mn-FU is expected to decompose and release FU and Mn2+ synchronously under acidic stimulation (Figure 2a and Supporting Information Figure S6). First, we tested the release dynamics of FU and Mn from Mn-FU under pH 5.5 and 7.4, using standard absorption curves and ICP-MS, respectively. The dynamic curves showed 80% of FU and 84% of Mn2+ released at pH 5.5, while <10% of FU and Mn2+ was released at pH 7.4 within 4 h (Figures 2b and 2c and Supporting Information Figure S7). Next, we investigated the release of FU and Mn2+ at different pH conditions after 12-h incubation. The release profiles of FU and Mn2+ exhibited obvious acidity-triggered release behaviors (Figures 2d and 2e). Specifically, the release percentage of FU and Mn2+ increased with pH reductions, while stable at neutral environment, which indicated Mn-FU working as acidity-sensitive nanotheranostics. Furthermore, the simultaneous release of FU and Mn2+ inspired us to investigate their MRI performance. Surprisingly, both T1 and T2 relaxivity of Mn-FU was greatly enhanced as the pH value decreased to pH 6.0 and 5.5 after 12-h incubation (Figures 2f and 2g). For T1 or T2 mode, the relaxivity showed 31- or 29-fold enhancement, respectively, which indicated ultrasensitive MRI signal responses along with Mn2+ release. Moreover, the gradually brighter T1 MRI images and gradually darker T2 MR images were obtained as the pH decreased (Figure 2h), which verified that Mn-FU could serve as a TME-responsive T1- and T2-weighted MRI contrast agent. Because of the synchronous release of FU and Mn2+ from Mn-FU presented positive correlation (Figure 2i), we further investigated the performance of MRI-correlated drug release. Excitingly, both T1 and T2 relaxation rates exhibited good correlation with FU release (Figures 2j and 2k), which could be further employed to monitor drug release. Figure 2 | MRI-correlated drug release. (a) Illustration of pH-responsive drug and Mn2+ release and MRI visualizing drug release. (b) FU release percentage at different time points at pH 5.5 and 7.4. (c) Mn2+ release percentage at different time points at pH 5.5 and 7.4. (d) FU release percentage under different pH at 12 h. (e) Mn2+ release percentage under different pH at 12 h. (f) T1 relaxivity of Mn-FU under different pH at 12 h. (g) T2 relaxivity of Mn-FU under different pH at 12 h. (h) T1- and T2-weighted MRI images of Mn-FU at different pH. (i) The correlation between FU and Mn2+ release percentages of Mn-FU at different pH. (j) The correlation between FU release percentage of Mn-FU and T1 relaxivity. (k) The correlation between FU release percentage of Mn-FU and T2 relaxivity. Error bars denote the standard deviation (n = 3). Download figure Download PowerPoint Anticancer activity of Mn-FU via chemo-and radiotherapy in vitro Cellular fluorescent imaging experiments indicated that Mn-FU was mainly located in cytoplasm ( Supporting Information Figure S8), and the uptake of Mn-FU by tumor cells was energy-dependent and mainly via clathrin-mediated endocytosis pathways ( Supporting Information Figure S9).30,31 We investigated the therapeutic effects of Mn-FU in vitro via the relative cellular viability. Mn-FU caused more inhibition toward both 4T1 murine breast cancer cells and HeLa cells after 24- or 48-h incubation, compared with free FU or free Mn + FU (Figures 3a and 3b and Supporting Information Figure S10). When the concentration of Mn-FU was 70 μg/mL, the viability of HEK293 cells was above 80%, while the viability of 4T1 cells was about 30%, which demonstrated higher toxicity of Mn-FU toward cancer cells than normal cells ( Supporting Information Figure S11). We further explored the cell apoptosis or necrosis induced by Mn-FU, via flow cytometry using an Alexa Fluor 488 Annexin V/dead cell apoptosis kit. Both 4T1 cells and HeLa cells treated with Mn-FU exhibited more cellular apoptosis or necrosis than those treated with free FU (Figure 3c). Such higher inhibition of cancer growth and more severe cellular apoptosis/necrosis confirmed the excellent anticancer activity of Mn-FU, which was superior to free FU. Due to the high activity as a chemodrug, Mn-FU was further combined with RT. To further investigate synergistic chemoradiotherapeutic effects (Figure 4a), the immunofluorescent labeling of γ-H2AX was carried out to evaluate DNA damages.32 From confocal images (Figure 4b) and corresponding quantification (Figure 4c), Mn-FU caused higher levels of DNA damage than free FU only, which is consistent with the aforementioned higher cellular apoptosis or necrosis induced by Mn-FU. Importantly, Mn-FU + X-ray produced much higher levels of DNA damage, compared with that induced by RT alone, Mn-FU alone, and even free FU + X-ray, which indicated Mn-FU was able to greatly enhance the response of cancer cells toward RT. Figure 3 | Cytotoxicity and apoptosis assay. (a) Cell viability of 4T1 cells incubated with different concentration of Mn-FU, FU, or free Mn + FU for 24 h. (b) Cell viability of HeLa cells incubated with different concentration of Mn-FU, FU, or free Mn + FU for 24 h. (c) Annexin V/PI analysis of 4T1 and HeLa cells incubated with PBS, FU, or Mn-FU for 24 h. The quadrants from lower left to upper left (counterclockwise) represent healthy, early apoptotic, late apoptotic, and necrotic cells, respectively. The percentage of cells in each quadrant are shown on the graphs. Error bars denote the standard deviation (n = 3). Download figure Download PowerPoint Figure 4 | Synergetic chemoradiotherapy in vitro. (a) Scheme for synergetic chemoradiotherapy. (b) γ-H2AX-stained 4T1 cancer cells treated with PBS, FU (50 µg/mL), Mn-FU (50 µg/mL of FU), X-ray (6 Gy), FU (50 µg/mL) + X-ray (6 Gy) or Mn-FU (50 µg/mL of FU) + X-ray (6 Gy). Scale bar is 50 μm. (c) Quantitative analysis of γ-H2AX foci density (γ-H2AX foci/100 µm2) for n > 100 cells in each treatment group. (1) PBS, (2) FU (50 µg/mL), (3) Mn-FU (50 µg/mL of FU), (4) X-ray (6 Gy), (5) FU (50 µg/mL) + X-ray (6 Gy), and (6) Mn-FU (50 µg/mL of FU) + X-ray (6 Gy). Download figure Download PowerPoint In vivo MRI and biodistribution Due to the excellent therapeutic effects in vitro, we investigated the tumor accumulation ability of nanotheranostics after successful PEGylation (Mn-FU-PEG) ( Supporting Information Figure S12). Balb/c mice bearing 4T1 xenografted tumors were intravenously (i.v.) injected with Mn-FU-PEG and scanned by an MRI scanner (Burker) via T1- and T2-weighted MRI modes (Figure 5a). From T1-MRI imaging (Figure 5b), the tumor was gradually brighter during 60-min observation, and the signals of the tumor area were increased as time extended from 0- to 60-min postinjection (Figure 5c). Such positive contrast in T1-weighted MRI and negative contrast in T2-weighted MRI (Figures 5d and 5e) confirmed the effective tumor homing ability of Mn-FU-PEG via enhanced permeability and retention (EPR) effect and the quick release of Mn2+ within the tumor area. Furthermore, to investigate whole-body biodistribution, the main organs and tumors were collected from mice at 12-h postinjection and digested for ICP-MS measurement of Mn2+. We found that the tumor uptake of Mn-FU-PEG could reach as high as 13.8% ID g−1 (Figure 5f). Surprisingly, less than 20% ID g−1 of Mn2+ within the liver and spleen was observed and 46.7% ID g−1 of Mn2+ was accumulated into the kidney at 12-h postinjection, indicating fast renal clearance of Mn-FU-PEG. Figure 5 | In vivo MRI and biodistribution. (a) Time period for MRI. (b) Dynamic contrast-enhanced T1 weight of a 4T1-xenografted tumor treated with Mn-FU-PEG (50 mg/kg of FU). (c) Dynamic enhancement curve of xenografted tumor treated with Mn-FU-PEG (50 mg/kg of FU). (d) Dynamic contrast-enhanced T2 weight of a 4T1-xenografted tumor treated with Mn-FU-PEG (50 mg/kg of FU). (e) Dynamic enhancement curve of xenografted tumor treated with Mn-FU-PEG (50 mg/kg of FU). (f) Biodistribution profiles of Mn-FU-PEG in mice. Error bars denote the standard deviation (n = 3). Download figure Download PowerPoint Chemoradiotherapy and toxicity of Mn-FU in vivo Because of the high tumor accumulation performance, we further investigated the synergistic chemoradiotherapy of Mn-FU-PEG in vivo. The mice bearing 4T1 tumors received the following treatment: (1) phosphate buffered saline (PBS), (2) X-ray (6 Gy), (3) FU (50 mg/kg), (4) FU (50 mg/kg) + X-ray (6 Gy), (5) Mn-FU-PEG (50 mg/kg of FU), (6) Mn-FU-PEG (50 mg/kg of FU) + X-ray (6 Gy), and (7) FU (150 mg/kg) + X-ray (6 Gy). The tumor was irradiated with X-ray (6 Gy) for 12-h post i.v. injection. After treatment, the tumor sizes were recorded every other day, and the tumor weights were measured at the last day of treatment. From the tumor growth curves and tumor weight profiles (Figures 6a and 6b), we found that the tumors treated with Mn-FU-PEG showed slower growth rates compared with the PBS group, indicating effective anticancer activity in vivo even after i.v. injection. However, no obvious differences in the tumor growth curves were observed for the tumor treated with X-ray and FU (50 mg/kg) + X-ray, suggesting there was little radiosensitizing effects induced by free FU (50 mg/kg). Importantly, the tumor treated with Mn-FU-PEG (50 mg/kg of FU) + X-ray exhibited much more inhibition of tumor growth, compared with that treated with X-ray alone, which demonstrated the excellent radio sensitization effects of Mn-FU-PEG. Although higher dosages of FU (150 mg/kg) + X-ray (6 Gy) were employed, the anticancer activity of free drug + RT was still lower than that of Mn-FU-PEG (50 mg/kg of FU) + X-ray (6 Gy), which demonstrated the synergetic combination of the Mn-FU-PEG nanodrug with RT, compared with the free drug. The therapeutic effect of Mn-FU-PEG was further investigated by pathological examination. Those tumors were collected at the second day postinjection for hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) staining. Notably, H&E and TUNEL staining images (Figure 6c) indicated that the tumor treated with Mn-FU-PEG + X-ray showed the most severe damages and the highest level of cell apoptosis within all groups, which further confirmed the synergistic effects of chemoradiotherapy induced by Mn-FU-PEG. Figure 6 | In vivo antitumor chemoradiotherapy. (a) Tumor growth curves of different mice groups. (1) Control, (2) X-ray (6 Gy), (3) FU (50 mg/kg), (4) FU (50 mg/kg) + X-ray (6 Gy), (5) Mn-FU-PEG (50 mg/kg of FU), (6) Mn-FU-PEG (50 mg/kg of FU) + X-ray (6 Gy), and (7) FU (150 mg/kg of FU) + X-ray (6 Gy). Error bars denote the standard deviation (n = 4). (b) Weights of tumors collected from different groups of mice at day 13 after the initiation of treatments. (1) Control, (2) X-ray (6 Gy), (3) FU (50 mg/kg), (4) FU (50 mg/kg) + X-ray (6 Gy), (5) Mn-FU-PEG (50 mg/kg of FU), and (6) Mn-FU-PEG (50 mg/kg of