Article pubs.acs.org/bc
Synthesis of PEGylated Ferrocene Nanoconjugates as the Radiosensitizer of Cancer Cells Jian Tian,†,# Jie Chen,†,§,# Cuicui Ge,† Xu Liu,‡ Jinlin He,*,‡,∥ Peihong Ni,‡,∥ and Yue Pan*,‡ †
School of Radiation Medicine and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, ‡College of Chemistry, Chemical Engineering and Materials Science, and ∥State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow University, Suzhou 215123, China § The Second Affiliated Hospital of Soochow University and General Hospital of Nuclear Industry, Suzhou 215004, China S Supporting Information *
ABSTRACT: Radiation is one of the most widely used methods for cancer diagnosis and therapy. Herein, we report a new type of radiation sensitizer (Fc-PEG) by a facile one-step reaction of conjugating the hydrophilic PEG chain with hydrophobic ferrocene molecule. The chemical composition and structure of Fc-PEG have been thoroughly characterized by FT-IR, NMR, GPC, and MALDI-TOF mass spectrometry. This Fc-PEG conjugate could self-assemble in aqueous solution into spherical aggregates, and it was found that the exposure to 4 Gy of X-ray radiation have little influence on the shape and size of these aggregates. After the chemical bonding with PEG chains, the uptake level of Fe element could be enhanced via the formation of aggregates. The live/dead, CCK-8, as well as apoptosis assays, indicated that the death of cancer cells can be obviously increased by X-ray radiation after the incubation of these Fc-based nanoconjugates, which might be served as the radiation sensitizer toward cancer cells. We suggest that this radiosensitizing effect comes from the enhancement of reactive oxygen specimen (ROS) level as denoted by both flow cytometric and fluorescence microscopic analysis. The enhanced radiation sensitivity of cancer cells is contributed by the synergic effect of Fe-induced radiation-sensitizing and the increased uptake of nanoconjugates after polymeric grafting.
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INTRODUCTION Engineering artificial chemical molecules into the agent for cancer therapy and diagnosis has been a very significant topic in recent years.1−9 As one of the most commonly used tools for cancer diagnosis and therapy, radiation has the capability of selectively promoting the death and apoptosis of tumor cells caused by DNA damage, increase of reactive oxygen specimen level, and changes in cell cycles.4,10−12 It is widely understood that the elements with higher atomic numbers (high-Z) could enhance the radiation effect by generation of Auger electrons and subsequent free radicals.13−15 Ferrocene (Fc), consisting of one Fe atom with two coordinated cyclopentadiene, exhibits recyclable redox properties by releasing/binding the reactive hydrogen.16−21 Therefore, this molecule can serve as a buffer of electrons and act as a long-term and stable supplier of Auger electrons after being ionized by radiation. Besides, although many high-atomic-number elements have been employed as radiosensitizer22−24 (e.g., gold, bismuth and rare earth) with effectiveness in concentrating a greater local radiation dose within the tumor, their biosafety concerns caused by the release of ions hinders further clinic-oriented applications. Being a high-Z metal and highly abundant in human bodies, Fe is © XXXX American Chemical Society
considered to be a promising element for many biological applications with relatively higher safety.25−27 In comparison with the small molecule of Fc-COOH (for better water solubility than the Fc molecule), the cellular uptake level and biosafety could be further improved if these molecules are chemically linked with a hydrophilic polymeric chain and then form nanoconjugates28 for the “enhanced permeability and retention (EPR)” effects.29 PEGylation is an important strategy in nanoparticle design due to its unique “steric stabilization” effect, prevention of the recognition and clearance by reticuloendothelial system (RES), and function of long circulation.30−32 These long-circulating nanoparticles are capable of accumulating in various pathological areas via the EPR effect, and have been widely used for drug delivery into tumors via passive targeting.29 However, the use of PEGylated ferrocene as radiosensitizer is rarely reported. Herein, we employed a facile one-step method to obtain the PEGylated ferrocene conjugate (Fc-PEG), which can be used as Received: March 29, 2016 Revised: April 23, 2016
A
DOI: 10.1021/acs.bioconjchem.6b00168 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry a radiosensitizing agent by the increase of cellular uptake of Fe element (as shown in Scheme 1). This conjugate is expected to Scheme 1. Radiation Sensitivity of Cancer Cells Can Be Enhanced after Incubated with Fc-PEG Nanoconjugates
possess the following characteristics: (i) one-step preparation in favorable yield, (ii) increased solubility of ferrocene molecule, and (iii) enhanced cellular uptake via formation of nanoscale aggregates. The chemical structure and self-assembly behavior of this amphiphilic conjugate have been fully characterized by various kinds of techniques. We also investigated the relationships of cellular uptake and intracellular reactive oxygen specimen (ROS) level with the apoptosis of cells. It was found that the high-Z effect of Fe elements accompanied by the alleviation of hypoxic anoxia of the cancer cells could improve their radiation sensitivity for promoting further potential clinical applications.
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RESULTS AND DISCUSSION The PEGylated ferrocene conjugate (Fc-PEG) was prepared by a facile one-step esterification and the synthesis routes are shown in Scheme S1 in the SI. A relatively short PEG chain with the molecular weight of about 750 g mol−1 was selected here to endow the resultant Fc-PEG conjugate with favorable water solubility and self-assembling property. The chemical structure of Fc-PEG was confirmed by 1H NMR, FT-IR, GPC, and MALDI-TOF mass spectrometry. As seen in the 1H NMR spectrum in Figure 1A, from which one can find all the characteristic signals ascribed to the protons in Fc-PEG. The FT-IR spectra of mPEG-OH and Fc-PEG are shown in Figure S1 in the SI, it is clearly observed that the absorption peak at 3491 cm−1 attributed to the hydroxyl group of mPEG-OH completely disappeared, whereas a new absorption peak appeared at 1720 cm−1 corresponding to the carbonyl group. Figure 1B illustrates the GPC traces of mPEG-OH and FcPEG. One can find that both polymers exhibit a unimodal distribution with relatively narrow PDI values, and the GPC curve of Fc-PEG shows a major distribution that shifts toward the higher-molecular-weight side compared with that of mPEGOH. The most striking structural evidence comes from the MALDI-TOF mass spectrometry. As shown in Figure 1C, FcPEG gives a single molecular weight distribution, and the observed molecular weight is in excellent agreement with the calculated one (e.g., for [M16•Na]+ with the formula of C44H76FeO18Na, found m/z 971.47 Da vs calcd. 971.43 Da). All the above evidence unambiguously confirmed that the Fc-PEG conjugate has been successfully prepared.
Figure 1. (A) 1H NMR spectrum of Fc-PEG; (B) GPC curves of mPEG-OH (M̅ n,GPC = 880 g mol−1, PDI = 1.08) and Fc-PEG (M̅ n,GPC = 1000 g mol−1, PDI = 1.07); (C) MALDI-TOF mass spectrum of FcPEG shows one single distribution corresponding to the desired structure; the inset is the overall spectrum.
Ferrocene (Fc) is poorly soluble in water; after introducing the PEG chain, the Fc-PEG conjugate could self-assemble in aqueous solution into aggregates with hydrophobic Fc as the core and hydrophilic PEG as the shell. It is well-known that the micellization of amphiphilic molecules and the stability of resulting aggregates can be evaluated by the critical aggregation concentration (CAC).35,36 In this work, the CAC value of FcPEG was determined by the steady-state fluorescence probe method using pyrene as the probe. Figure S2 in the SI shows the relationship of I3/I1 of pyrene as a function of the logarithm concentrations of Fc-PEG, from which the CAC value was calculated to be about 0.102 mg mL−1 (∼0.105 mM) by intersecting the two straight lines. The aggregates were also characterized by TEM and DLS analysis. As shown in Figure 2A, the aggregates show spherical shape with an average diameter of 75 nm. The DLS analysis shows an average hydrodynamic diameter (Dh) of 114 nm with the polydispersity index (size PDI) of 0.285 for suspension in Mill-Q water (Figure 2C). The values of Dh and PDI show a small increase B
DOI: 10.1021/acs.bioconjchem.6b00168 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 2. TEM images of the spherical aggregates self-assembled from Fc-PEG in Milli-Q water before (A) and after (B) exposure to 4 Gy X-ray radiation. (C) and (D) show the particle size distribution curves corresponding to the samples in (A) and (B), respectively. The suspending media are Milli-Q water (red curve) and culture medium (black curve), respectively.
when the Fc-PEG was suspended in the cell culture medium (Figure 2C). This result shows that these aggregates have a relatively good distribution and water (or cell culture medium) solubility, which will be beneficial for their further utilization in raidosensitivity for cancer cells. Furthermore, the stability of FcPEG aggregates under the exposure of radiation was investigated for evaluating their capability in serving as the radiation sensitizer. It is found from Figure 2B,D that there is no obvious change in the shape and size of aggregates after their exposure to the X-ray radiation (4 Gy). The Fc-PEG nanoconjugates were incubated with 4T1 cancer cells for 24 h, which were then exposed to X-ray radiation (4 Gy). After that, the cells were further cultured for an additional 24 h and then taken for the live/dead assay. As shown in Figure 3A, the cells incubated with Fc-PEG nanoconjugates (0.6 Fe mM) show almost no cytotoxicity. After exposure to X-ray radiation at a dose of 4 Gy (Figure 3B), the cells show obvious changes in their morphologies, including their shape, emergence of cell debris, and red fluorescence by propidium iodide in live/dead kit. We have also investigated the same live/dead staining for the cells treated with free FcCOOH and PBS solution as controls, which showed almost no change before and after exposure to radiation. These results indicate that Fc-PEG gives the 4T1 cells a higher radiation sensitivity after introducing the Fc molecules into PEGylated nanoconjugates. Besides, the CCK-8 and flow cytometry assays were also used to detect the cytotoxicity of free Fc-COOH and Fc-PEG nanoconjugates under the exposure to radiation of 0 Gy (no radiation) and 4 Gy. As shown in Figure 3C, the FcPEG shows minimal cytotoxicity in the concentration range of
Figure 3. Enhancing the radiosensitivity of 4T1 cells by Fc-PEG nanoconjugates. Fluorescence microscopic images show the changes of morphology and viability of 4T1 cells after their incubation with FcPEG nanoconjugates (0.6 Fe mM) for 24 h with exposure of (A) 0 Gy and (B) 4 Gy radiations, respectively. All scale bars are 200 μm. (C) CCK-8 assay showing that Fc-PEG nanoconjugates possess good performance in enhancing the radiation sensitization of 4T1 cells.
0.1−0.6 Fe mM under the condition of 0 Gy, while for the samples exposed to 4 Gy radiation, an obvious increased cytotoxicity was observed in the same concentration range. In C
DOI: 10.1021/acs.bioconjchem.6b00168 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry addition, the flow cytometry results also reveal that the percentage of apoptosis and death is much higher for the cells under 4 Gy radiation after incubation with Fc-PEG nanoconjugates (Figure 4).
ROS level by DCFH-DA assay and the summarized ROS content results showed that the Fc-COOH and Fc-PEG could induce the increment of ROS level after exposing to 4 Gy of Xray radiation (Figure 5A,B). It is evident that the radiationinduced ROS level is much higher when the cells were exposed to X-ray radiation (4 Gy) after incubation with the Fc-PEG nanoconjugates. The ROS level is about 5−6-fold higher than that when incubating the cells with free Fc-COOH. Besides, the ROS level is twice that for the cells incubated with Fc-PEG nanoconjugates after their exposure to 4 Gy of X-ray radiation, indicating a radiation-induced increment of ROS level. It was also found that the optimized concentration was located in the range of 0.1−0.8 Fe mM. As shown in Figure S3(A) in the SI, the ROS level increases with the concentration of Fc-PEG nanoconjugates until the concentration is above 0.6 Fe mM, where the ROS level of cells with 4 Gy of radiation is obviously increased. By using the inductively coupled plasma optical emission spectrometry (ICP-OES) method, we could also determine the cellular uptake of Fe element of 4T1 cells. As shown in Figure 5C, about 60% of Fe elements are taken up by the cells after Fc is PEGylated, while the uptake level of free Fc-COOH is almost comparable with the that of the control (cells incubated with PBS buffer). The uptake contents of Fe by cells are shown in Figure S3(B) in the SI. We also investigated the relationship between the uptake percentage (as well as the absolute content) of Fe element and the initial concentration of Fc-
Figure 4. Percentage of apoptosis and death for cells that are incubated with PBS buffer (Control), free Fc-COOH, PEG, and FcPEG nanoconjugates, respectively. The concentrations of free FcCOOH and Fc-PEG are both 0.6 Fe mM. The concentration of PEG is 600 mg mL−1.
Furthermore, the mechanism of the radiation sensitization of Fc-PEG nanoconjugate was investigated in comparison with their cellular uptake level. We first investigated the intracellular
Figure 5. ROS-mediated pathway of radiation-enhanced cell death by increasing uptake level of Fe element. (A) Fluorescence images showing the intracellular ROS levels of 4T1 cells under the exposure to 0 or 4 Gy radiation after they were treated with PBS buffer (Control), free Fc-COOH, and the Fc-PEG nanoconjugates. All scale bars are 20 μm. (B) Statistical analysis of their ROS level obtained by flow cytometry. Both assays are continuous treatment of 4T1 cells including incubation with 0.6 Fe mM solutions of free Fc-COOH or Fc-PEG for 24 h and exposure to radiation of 0 or 4 Gy. (C) Cellular uptake of Fe element after the cells are incubated with PBS buffer (Control), free Fc-COOH, and Fc-PEG for 24 h. The concentrations of Fc-COOH and Fc-PEG are 0.6 Fe mM. D
DOI: 10.1021/acs.bioconjchem.6b00168 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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NMR spectra were recorded on a 400 MHz NMR spectrometer (INOVA-400) at 25 °C with CDCl 3 as solvent and tetramethylsilane (TMS) as the internal standard. The number-average molecular weights (M̅ n,GPC) and molecular weight distributions (PDIs) of polymers were recorded on a Waters 1515 GPC instrument using a PLgel 5.0 μm bead size guard column (50 × 7.5 mm), followed by two linear PLgel columns (500 Å and Mixed-C), and a differential refractive index detector. THF was used as the eluent at a flow rate of 1.0 mL min−1 at 30 °C, and the narrowly distributed polystyrene was used for the calibration standards. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were acquired on an UltrafleXtreme MALDI TOF mass spectrometer equipped with a 1 kHz smart beam-II laser. The compound trans-2-[3-(4-tert-butylphenyl)-2- methyl-2propenylidene]-malononitrile (DCTB, >99%, Sigma-Aldrich) served as the matrix and was prepared in CHCl3 at a concentration of 20 mg mL−1. The cationizing agent sodium trifluoroacetate (>99%, Sigma-Aldrich) was prepared in ethanol at a concentration of 10 mg mL−1. The matrix and cationizing salt solutions were mixed in a ratio of 10/1 (v/v). All samples were dissolved in CH2Cl2 at a concentration of 10 mg mL−1. After sample preparation and solvent evaporation, the plate was inserted into the MALDI mass spectrometer. The attenuation of the laser was adjusted to minimize undesired polymer fragmentation and to maximize the sensitivity. Data analyses were conducted with Bruker’s flexAnalysis software. Critical Aggregation Concentration (CAC). The CAC value was determined by the fluorescence probe method using pyrene as the hydrophobic probe. Typically, a predetermined pyrene solution in acetone were respectively added into a series of ampules; acetone was then evaporated and replaced with aqueous polymer solutions at different concentrations with the range (1.2−1.5) × 10−3 mg mL−1. The final concentration of pyrene in each ampule was 6 × 10−6 mol L−1. The samples were sonicated for 10 min, stirred at room temperature for 24 h, and analyzed on a spectrofluorometer (FLS920, Edinburgh) at the excitation wavelength of 335 nm and emission wavelength of 350 to 550 nm, with both bandwidths were set at 2 nm. From the pyrene emission spectra, the intensity ratio (I3/I1) of the third band (382 nm, I3) to the first band (371 nm, I1) was analyzed as a function of polymer concentration. The CAC value was defined as the point of intersection of two lines in the plot of fluorescence versus polymer concentration. Transmission Electron Microscopy (TEM). The morphologies of the aggregates were observed on a TEM instrument (HT7700, Hitachi) at 120 kV. The solution was prepared by a direct dissolution method. Briefly, 5 mg of polymer was directly dissolved in 5 mL of Milli-Q water and stirred for 10 min before use. The sample for TEM observation was then prepared by a freeze−drying method.34−36 The carbon-coated copper grid was placed on the bottom of a glass cell, which was then immediately transferred into liquid nitrogen. Subsequently, 10 μL of the polymer solution was dripped onto the grid, and the solvent in its frozen solid state was directly removed without melting in a freeze−dryer. The morphology was then imaged in a normal TEM instrument at room temperature. Dynamic Light Scattering (DLS). The average particle size (Dh) and size polydispersity indices (size PDIs) of the aggregates were measured at 25 °C using a dynamic light scattering instrument (Zetasizer Nano ZS, Malvern) equipped with a He−Ne laser (633 nm) and 90° collecting optics. All
PEG nanoconjugates that applied to the cells. As shown in Figure S4 in the SI, the uptake efficacy is higher for the concentration of 0.6 Fe mM than other concentrations, which may correspond to the favorable radiation-induced increment of ROS level at this concentration.
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CONCLUSIONS In summary, we have prepared a novel type of radiosensitizer Fc-PEG consisting of hydrophobic ferrocene moiety and hydrophilic PEG chain. The synthesis was accomplished in one step in favorable yield. The precisely defined structure was characterized by a combination of NMR, FT-IR, GPC, and MALDI-TOF mass spectrometry. The amphiphilic character enabled the Fc-PEG conjugate to self-assemble in aqueous solution into spherical aggregates, which was proven by several techniques. The as-prepared nanoconjugates were found to be effective in enhancing the radiation sensitivity of cancer cells via the increment of cellular ROS level. We believe that this study provides a facile way to achieve the radiosensitizing agent which is promising for the application in radiotherapy nanomedicine.
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EXPERIMENTAL SECTION Materials. Poly(ethylene glycol) monomethyl ether (mPEG-OH, M̅ n = 750 g mol−1, Sigma-Aldrich) was dried by azeotropic distillation just before use in the presence of anhydrous toluene. Ferrocenecarboxylic acid (Fc-COOH, 98%, Aladdin), N,N′-diisopropylcarbodiimide (DIC, 99%, SigmaAldrich), and 4-dimethylaminopyridine (DMAP, 99%, Shanghai Medpep) were used as received. Dichloromethane (CH2Cl2, A.R., Sinopharm Chemical Reagent) was refluxed with CaH2 and distilled before use. Milli-Q water (18.2 MΩ cm−1) was generated using a water purification system (Simplicity UV, Millipore). Synthesis of Poly(ethylene glycol)-ferrocenecarboxylate (Fc-PEG). As shown in Scheme S1 in the Supporting Information (SI), the PEGylated ferrocene conjugate (Fc-PEG) was prepared by a one-step esterification of mPEG-OH and FcCOOH using the method as reported previously with some modifications.28,33 A typical synthetic example is listed as follows: mPEG-OH (2.0 g, 2.67 mmol), Fc-COOH (0.736 g, 3.20 mmol), and DMAP (0.114 g, 0.93 mmol) were dissolved in 30 mL of anhydrous CH2Cl2 under nitrogen atmosphere. A solution of DIC (0.404 g, 3.20 mmol) and 5 mL of anhydrous CH2Cl2 was then added dropwise to the flask incubated at 0 °C. After addition, the reaction was performed at 25 °C for 48 h under a nitrogen atmosphere. The mixture was filtered and the filtrate was evaporated to dryness. The solid was redissolved in 30 mL of CHCl3, and the solution was extracted with a diluted NaOH solution (pH = 10, 10 mL) twice and brine (10 mL) twice to remove unreacted Fc-COOH and water-soluble byproducts. The solution was dried over anhydrous Na2SO4 for 12 h, and the filtrate was evaporated to dryness. The obtained yellow viscous product (Fc-PEG) was dried under vacuum at 25 °C for 24 h (1.50 g, yield: 57%). M̅ n,GPC = 1000 g mol−1, PDI = 1.07. 1H NMR (δ, ppm, CDCl3): 4.82, 4.39, 4.21 (s, 9H, cyclopentyl), 3.64 (m, 65H, -OCH2CH2O−), 3.35 (s, 3H, terminal CH3O−). 13C NMR (δ, ppm, CDCl3): 171.5 (-COO−), 71.8, 71.2, 69.6, 69.2 (cyclopentyl), 70.4 (-OCH2CH2O−), 63.1 (−COOCH2CH2O−), 58.8 (terminal CH3O−). Characterizations. FT-IR spectra were determined with a Nicolet 6700 spectrometer using the KBr disk method. 1H E
DOI: 10.1021/acs.bioconjchem.6b00168 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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samples were prepared by the same method as described in TEM analysis. CCK-8 Assay. 4T1 cells were maintained in RPMI-1640 medium (WISENT) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin−streptomycin (Beyotime) at 37 °C in a humidified atmosphere of 5% CO2. First, cells are plated by gently dripping 100 μL cell suspensions (3 × 104 cells mL−1) into each one of the 96-well microplates. After incubation for 24 h, cells were treated with free Fc-COOH and Fc-PEG nanoconjugates solution in a series of Fe concentrations of 0, 0.1, 0.2, 0.4, and 0.6 mM for another 24 h, respectively. For the vaibility test of cells after exposure to radiation, the cells were then irradiated with X-ray (4 Gy) after their 24 h incubation with free Fc-COOH and Fc-PEG nanoconjugates solution in a series of Fe concentrations. The CCK-8 assay was then conducted after another 24 h incubation after the radiation. The cell viability was then determined by CCK-8 assay kit (Dojindo) according to the previous report.37 Flow Cytometric Analysis. 4T1 cells were seeded in 6-well microplates by gently dripping 2 mL cell suspension (3 × 104 cells mL−1) into each one of the 6-well microplates and subsequently incubated for 24 h. Cells were incubated with free Fc-COOH and Fc-PEG aggregates (both with Fe concentration of 0.6 mM), respectively. Meanwhile, the wells without adding any samples were regarded as control. After 24 h, the cells were then irradiated with X-ray (4 Gy). After further incubation for 24 h, the cells were collected, washed three times with PBS, dyed with Annexin V-PE/7-AAD apoptosis Detection kit (BD), and then detected by flow cytometry to make clear the cell death mode. Furthermore, cells were treated with the same processing method and collected. The Cells were fixed with 75% alcohol overnight at 4 °C. Then the cells were stained with PI staining cell cycle analysis kit (Beyotime) at 37 °C for 30 min, and flow cytometry (FACSVerse, BD) was then used to detect red fluorescence and light scattering conditions at the same time at the excitation wavelength of 488 nm wavelength. Reactive Oxygen Species (ROS) Analysis. 4T1 cells were seeded in 6-well plates with a density of 5 × 104 cells mL−1 for 24 h. Cells were incubated with free Fc-COOH and Fc-PEG aggregates (both with Fe concentration of 0.6 mM), respectively. Meanwhile, the cells untreated with any samples were regarded as control. After 24 h incubation, the cells were then irradiated with X-ray. ROS levels were detected using the oxidant-sensitive dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, 97%, Sigma-Aldrich). After exposure to X-rays, a sufficient amount of 25 μM carboxy-H2 DCFDA solution was applied to cells adhering to the substrate. Cells were incubated with dye for 30 min at 37 °C, gently washed with PBS three times, then observed using fluorescence microscope (20×, DMIL LED, Leica). The cells are then rapidly collected for flow cytometric analysis.
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Article
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions #
Jian Tian and Jie Chen contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors greatly acknowledge financial support from National Natural Science Foundation of China (51402203, 21374066), China Postdoctoral Science Foundation (2015M571809), Natural Science Foundation of Jiangsu Province for Young Scholars (BK20130286, BK20140326), the Natural Science Foundation of Jiangsu Higher Education Institutions (13KJB150034, 14KJB430021), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20133201120007), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection. The Project is also sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00168. Synthetic route and FT-IR spectra of the Fc-PEG conjugate, CAC data, ROS levels, and cellular uptake levels of iron elements (PDF) F
DOI: 10.1021/acs.bioconjchem.6b00168 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.6b00168 Bioconjugate Chem. XXXX, XXX, XXX−XXX
