Letter pubs.acs.org/NanoLett
Casp3/7-Instructed Intracellular Aggregation of Fe3O4 Nanoparticles Enhances T2 MR Imaging of Tumor Apoptosis Yue Yuan,† Zhanling Ding,† Junchao Qian,‡ Jia Zhang,† Jinyong Xu,‡ Xuejiao Dong,† Tao Han,† Shuchao Ge,§ Yufeng Luo,† Yuwei Wang,† Kai Zhong,*,‡ and Gaolin Liang*,† †
CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ‡ High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei, Anhui 230031, China § School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China S Supporting Information *
ABSTRACT: Large magnetic nanoparticles or aggregates are advantageous in their magnetic resonance properties over ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs), but the former are cleared faster from the blood pool. Therefore, the “smart” strategy of intracellular aggregation of USPIO NPs is required for enhanced T2-weighted MR imaging. Herein, employing an enzyme-instructed condensation reaction, we rationally designed a small molecule Ac-Asp-Glu-Val-Asp-Cys(StBu)-LysCBT (1) to covalently modify USPIO NPs to prepare monodispersive Fe3O4@1 NPs. In vitro results showed that Fe3O4@1 NPs could be subjected to caspase 3 (Casp3)-instructed aggregation. T2 phantom MR imaging showed that the transverse molar relaxivity (r2) of Fe3O4@1 NPs with Casp3 or apoptotic HepG2 cells was significantly larger than those of control groups. In vivo tumor MR imaging results indicated that Fe3O4@1 NPs could be specifically applied for enhanced T2 MR imaging of tumor apoptosis. We propose that the enzyme-instructed intracellular aggregation of Fe3O4 NPs could be a novel strategy for the design of “smart” probes for efficient T2 MR imaging of in vivo biomarkers. KEYWORDS: Caspase 3/7, aggregation, ultrasmall superparamagnetic iron oxide, transverse relaxation, magnetic resonance imaging
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demonstrated an increase in R2 with the increase of the magnetization of SPIO NP, and Mn- or Zn-doped ferrites with high magnetizations showed significant increase in R2 values compared with undoped ferrites.18,19 However, doping of SPIO NPs with metals might risk in vivo toxicity due to the leakage of the metal ions.20 Large iron oxide nanoparticles (IONPs) and magnetic nanoparticle aggregates have improved MRI properties, and SPIO NP aggregates have shown dramatically higher relaxation rates compared to monodispersive SPIO NPs.21,22 However, the NP aggregates are more quickly cleared from the blood pool by the mononuclear phagocyte system than single NP.23 Thus, intracellular aggregation of SPIO NPs seems to be a “smart” and optimal strategy for enhanced T2 MRI in tissues.24 Previous methods for NP aggregation mostly rely on electrostatic or noncovalent interactions.25,26 To the best of our knowledge, there is only one covalent SPIO NP aggregation method reported which uses azide and alkyne [3 + 2] cycloaddition click reaction.24 However, that method
agnetic resonance imaging (MRI) is one of the best noninvasive methods in medical imaging for assessing function of tissues or diagnosis of diseases. It has several advantages, such as excellent temporal and spatial resolution, long effective time window of imaging, and fine signal contrast.1,2 However, due to the limited range of relaxation time, contrast agents (CAs) are usually required to enhance the MRI contrast.3−6 Among the numerous experimental MR CAs, superparamagnetic iron oxide (SPIO) nanoparticles (NPs) are emerging as one category of CAs widely used to improve MRI contrast, mostly in blood but also in cellular tissues.7−9 Among the SPIOs, ultrasmall superparamagnetic iron oxide (USPIO) NPs are advantageous over other SPIO NPs such as microparticles of iron oxide NPs due to their higher biocompatibility and safety for in vivo applications.10−12 To date, USPIO-enhanced MRI has been employed to study tissue inflammation and dynamic tracking in both experimental and clinical studies.13,14 A number of theoretical and experimental studies have shown that the magnetization and the size are two key factors of a SPIO NP as CA to influence the transverse relaxation rate (R2) of surrounding water protons, and larger R2 values could be obtained at higher magnetic field.15−17 Studies have © XXXX American Chemical Society
Received: January 26, 2016 Revised: March 28, 2016
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time (T2) of surrounding water protons, resulting in enhanced T2 MR signal. Casp3-Instructed Aggregation of Fe3O4 NPs in Vitro. After the syntheses and characterizations of compound 1 and 1Scr (Schemes S1−S2 and Figures S1−S4 in Supporting Information), using high performance liquid chromatography (HPLC) and mass spectral analyses, we first validated that 1 could be subjected to GSH-reduction and Casp3-cleavage to yield the cyclized dimer (1-Dimer), while 1-Scr was only subjected to GSH-reduction but impervious to Casp3 (Figures S5−S8). We then prepared Fe3O4@1 NPs and Fe3O4@1-Scr NPs using a facile 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/ NHS) method. First, 10 μL of carboxyl modified Fe3O4 NPs (5 mg/mL Fe, average diameter of 10 nm), 1.75 μmol of EDC, and an equivalent amount of NHS were added into 500 μL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (50 mM, pH 6) and stirred for 2 h. Following centrifugation (15 000 rpm) and washing with MES buffer for three times at 4 °C to remove excess EDC and NHS, Fe3O4 NP−NHS ester was obtained and then redispersed into 500 μL of phosphatebuffered saline (PBS, pH 7.4) buffer. Afterward 87.5 nmol 1 or 1-Scr (optimized amount, Figure S9) was added into the above Fe3O4 NP−NHS dispersion and stirred overnight at room temperature (RT). Fe3O4@1 NPs and Fe3O4@1-Scr NPs were then obtained respectively by centrifugation and washing with 1 mL of Casp3 buffer for three times at 4 °C. Compared with the −4.2 mV zeta potential of Fe3O4 nanoparticles, as prepared Fe3O4@1 NPs and Fe3O4@1-Scr NPs had respective zeta potentials of −18.5 mV and −18.0 mV (Table S2), suggesting the organic modifications are successful. To validate the Casp3instructed condensation and the aggregation of Fe3O4 NPs, the above prepared Fe3O4@1 NPs or Fe3O4@1-Scr NPs were dispersed in 200 μL Casp3 buffer to a final concentration of 8.5 μg/mL (0.15 mM) of Fe (measured by atomic absorption spectroscopy, AAS). The solution was equally divided into two parts, one for GSH-controlled reduction and the other for both GSH-reduction and Casp3 cleavage. After addition of 1 mM GSH and incubation for 2 h at 37 °C, the disulfide bonds on Fe3O4@1 NPs or Fe3O4@1-Scr NPs were reduced by GSH. Transmission electron microscopy (TEM) images of the NPs showed that they remain monodispersive in Casp3 buffer (Figure 2a), similar to those untreated NPs (Figure S10). When Fe3O4@1 NPs dispersion was treated with 1 mM GSH and 5 units of Casp3 for 8 h at 37 °C, the reduction product of 1 on Fe3O4@1 NPs was gradually cleaved by Casp3 to yield the active intermediate which instantly condenses with another, cross-linking the Fe3O4@1 NPs to form nanoparticle aggregates (Figure 2b). In comparison, 1-Scr on Fe3O4@1-Scr NPs was not cleaved by Casp3, and the NPs remained monodispersive (Figure 2d). Zeta potential measurements showed that Casp3treated Fe3O4@1-Scr NPs had zeta potential of −18.8 mV, closing to that of the nanoparticles before enzyme treatment (i.e., −18.0 mV). But zeta potential of Casp3-treated Fe3O4@1 NPs increased to −8.5 mV from that −18.5 mV before enzyme treatment (Supporting Information, Table S2), still lower than that of naked Fe3O4 NPs (i.e., −4.2 mV). This suggests that above aggregation was induced by condensation reactions but not electrostatic interactions among the nanoparticles after Casp3 cleaving the DEVD substrates from Fe3O4@1 NPs. To additionally validate that above aggregation was induced by cross-linking among the nanoparticles, we designed and synthesized another control compound Ac-Asp-Glu-Val-Asp-
requires two complementary IONPs, each of which is modified with one peptide substrate for enzyme cleavage and one functional group (azide or alkyne) for click reaction, thus making the whole system complicated. Caspase 3/7 (Casp3/7) are important biomarkers for tumor apoptosis. A number of MRI probes (or CAs) including an activatable thulium-based paramagnetic chemical exchange saturation transfer (PARACEST) probe, 27 19 F MRI probes,28−30 gadolinium (Gd)-based CA,31 and SPIO-annexin conjugates32,33 have been reported for imaging of Casp3/7 activity in apoptotic cells or living mice. But to the best of our knowledge, there has been no report of intracellular covalent aggregation of single type of SPIO NPs for T2 MR imaging of Casp3/7 activity. Herein, we report a “smart” strategy of Casp3/7-instructed intracellular covalent aggregation of USPIO NPs for enhanced T2 MR imaging of apoptotic cells and tumors. As shown in Figure 1a, based on a biocompatible
Figure 1. Chemical structures and schematic illustration of Fe3O4 NPs aggregation. (a) Chemical structures of 1 and 1-Scr. (b) Schematic illustration of intracellular Casp3/7-instructed aggregation of Fe3O4@ 1 NPs.
condensation reaction recently developed by Rao and Liang,28,34−37 we first designed a small molecule Ac-Asp-GluVal-Asp-Cys(StBu)-Lys-CBT (1), which could undergo Casp3/ 7-controlled condensation. A scrambled control compound AcLys-Asp-Glu-Asp-Val-Cys(StBu)-CBT (1-Scr) was used for parallel study. Then, the “smart” MR contrast agent Ac-AspGlu-Val-Asp-Cys(StBu)-Lys(Fe3O4 NP)-CBT (Fe3O4@1 NPs) containing following components was synthesized: Ac-Asp-GluVal-Asp (DEVD) substrates for Casp3/7 cleavage,38 2cyanobenzothiazole (CBT) motifs, and latent cysteine (Cys) motifs for CBT-Cys condensation, an Fe3O4 NP conjugating to the side chains of the lysine (Lys) motifs (Figure 1b). After Fe3O4@1 NP entering into Casp3/7-activated cells (e.g., apoptotic HepG2 cells), its disulfide bonds on the cysteine motifs are reduced by intracellular glutathione (GSH), and the DEVD peptide substrates are cleaved by Casp3/7 to expose the reactive 1,2-aminothiol groups. Then the condensation reaction between the free 1,2-aminothiol groups and the cyano groups of the CBT motifs occurs to yield cross-linked Fe3O4 NPs aggregations, which greatly shorten the transverse relaxation B
DOI: 10.1021/acs.nanolett.6b00331 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 2. TEM images of Fe3O4@1 NPs treated with 1 mM GSH for 2 h at 37 °C (a) or 1 mM GSH and 5 unit Casp3 for 8 h at 37 °C (b). TEM images of Fe3O4@1-Scr NPs treated with 1 mM GSH for 2 h at 37 °C (c) or 1 mM GSH and 5 unit Casp3 for 8 h at 37 °C (d). (e) T2-weighted MR phantom images (echo time: 75 ms), and T2 relaxation times of solutions in Figure 2a-d on a 9.4 T MR scanner (TR 5,500 ms, TE 15−180 ms). (f) Transverse relaxation rates (1/T2) of Fe3O4@1 NPs or Fe3O4@1-Scr NPs at different concentrations in the presence/absence of Casp3. Each error bar represents the standard deviation of three parallel experiments.
time of GSH-treated Fe3O4@1 NPs, the T2 value of Casp3treated Fe3O4@1 NPs decreased to 36.4 ms, suggesting that Casp3-instructed aggregation of Fe3O4 NPs greatly enhances the transverse relaxations of water protons. However, as expected, the T2 value of Casp3-treated Fe3O4@1-Scr NPs (55.2 ms) was not different from that of GSH-treated Fe3O4@ 1-Scr NPs (56.0 ms) (Figure 2e). Similarly, T2 value of Casp3treated Fe3O4@1-Ctrl NPs (53.1 ms) was close to that of GSHtreated Fe3O4@1-Ctrl NPs (56.2 ms) (Figure S17). At an echo time of 75 ms, T2-weighted MR phantom image of Casp3treated Fe3O4@1 NPs showed much lower intensity compared to the control groups (Figure 2e). After that, we diluted the incubations to obtain serial dilutions for each group. Analyses of the T2-weighted MR phantom images of the serial dilutions gave their corresponding T2 values. Then the transverse molar relaxivity (r2) of each incubation mixture was calculated according to the equation r2 = ΔR2/Δ[Fe], where the transverse relaxation rate R2 equals to 1/T2. As shown in Figure 2f, at 9.4 T and RT, the r2 values were determined to be 112 s−1 mM−1 for Fe3O4@1 NPs with GSH, 185 s−1 mM−1 for Fe3O4@1 NPs with GSH and Casp3, 116 s−1 mM−1 for Fe3O4@1-Scr NPs with GSH, and 119 s−1 mM−1 for Fe3O4@1Scr NPs with GSH and Casp3, respectively. This indicates that Casp3-instructed aggregation of Fe3O4@1 NPs induces approximately 65.2% enhancement of the transverse relaxivity (r2) of the nanoparticles. Before validating the efficacy of Fe3O4@1 NPs for enhancing T2 MR imaging of tumor apoptosis, we first tested them on apoptotic cancer cells. Following a method previously reported, we exposed 4 × 106 healthy HepG2 cells to 254 nm UV light at 10 000 μJ cm−2 and waited 30 min for the activation of Casp3/7 in the cells.39 Then the cells were suspended in 2 mL of serum-free culture medium, added with Fe3O4@1 NPs or Fe3O4@1-Scr NPs at 20 μg Fe/mL, and incubated at 37 °C for 1.5 h. Then the cells
Lys-CBT (1-Ctrl) (Figure S11), which can be cleaved by Casp3 but cannot take condensation reaction. Therefore, 1-Ctrlmodified Fe3O4 NPs (i.e., Fe3O4@1-Ctrl NPs) can be subjected to Casp3-cleavage but not cross-linking. TEM images showed that, compared with those Casp3-treated Fe3O4@1 NPs, Casp3-treated Fe3O4@1-Ctrl NPs did not aggregate but exhibited similar dispersiveness to that of other three control groups (Figure S12). To chemically validate the aggregation of Fe3O4@1 NPs was induced by Casp3-instructed condensation reaction, we conducted high resolution matrix-assisted laser desorption/ionization mass spectroscopic (HR-MALDI/MS) analyses on the aggregates. Clearly we found a molecular ionic peak of the cyclic dimer of 1 (i.e., 1-Dimer) and a peak of its fragment on the spectrum (Figure S13a), which in principle testified our hypothesis in Figure 1b. Besides, we also collected the supernatant of Casp3-treated Fe3O4@1 NPs mixture for mass spectral analysis. Indeed we found Ac-DEVD, the Casp3cleavable peptide substrate in 1, existing in the supernatant (Figure S14), which reaffirmed above Casp3-cleavage of AcDEVD from Fe3O4@1 NPs. Dynamic light scattering (DLS) measurement showed that the average hydrous dynamic diameter of the aggregates of Fe3O4@1 NPs was 128.4 ± 15.3 nm, significantly larger than those in control groups (Figure S15). In Vitro Aggregation of Fe3O4@1 NPs Enhances T2 MR Imaging in Phantoms of Casp3 or Apoptotic HepG2 Cells. T2-weighted MR phantom images of Fe3O4@1 NPs with GSH, Fe3O4@1 NPs with GSH and Casp3, Fe3O4@1-Scr NPs with GSH, and Fe3O4@1-Scr NPs with GSH and Casp3 at different echo times were acquired on a 9.4 T MR scanner (Figure S16). Analyses of signal intensities (or gray values) of the phantoms versus echo times in Figure S16 gave the corresponding T2 relaxation times, as shown in Figure 2e. Figure 2e indicated that, compared to a 56.0 ms T2 relaxation C
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Figure 3. (a) Low magnification TEM image of apoptotic HepG2 cells after incubation with Fe3O4@1 NPs at 37 °C for 1.5 h. (b) High magnification TEM image of the white rectangle area in a. (c) T2-weighted MR phantom images (echo time: 70 ms), and T2 relaxation times of Fe3O4@1 NPs incubated with healthy cells or apoptotic cells, Fe3O4@1-Scr NPs incubated with healthy cells or apoptotic cells at 37 °C for 1.5 h on a 9.4 T MR scanner (TR 5,500 ms, TE 10−120 ms). (d) Transverse relaxation rates (1/T2) of Fe3O4@1 NPs or Fe3O4@1-Scr NPs at different concentrations in the healthy/apoptotic HepG2 cells. Each error bar represents the standard deviation of three parallel experiments.
Figure 4. In vivo aggregation of Fe3O4@1 NPs enhances T2 MR imaging of tumor apoptosis. (a) In vivo T2-weighted coronal MR images of Fe3O4@ 1 NPs-injected saline-treated mice, Fe3O4@1 NPs-injected DOX-treated mice, Fe3O4@1-Scr NPs-injected saline-treated mice, and Fe3O4@1-Scr NPs-injected DOX-treated mice at 0 h (top) or 3 h post injection (bottom). (b) In vivo dynamic T2-weighted coronal MR images of Fe3O4@1 NPsinjected DOX-treated mice. Injection dose: 9.8 mg Fe/kg. Tumors in the mice were marked with false color to show the significant difference in MR signal intensity.
phantoms with almost identical concentration around 41 μg/ mL, suggesting that Fe3O4@1 NPs or Fe3O4@1-Scr NPs were indiscriminately uptaken by healthy or apoptotic HepG2 cells at an efficiency of 15.3%. After that, we conducted TEM observations on the cell samples. Clearly we found a large area of Fe3O4 NPs aggregates in apoptotic HepG2 cells incubated
were washed for four times with PBS to remove the remaining probe and resuspended in 150 μL of serum-free culture medium for T2 phantom MRI. Healthy HepG2 cells without irradiation were also treated with Fe3O4@1 NPs or Fe3O4@1Scr NPs as above-described for T2 phantom MRI. AAS was used to quantitate the Fe concentrations in these four groups of D
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of that at 0 h) injected with same nanoparticles (Figure 4a and Figure S25 in Supporting Information), suggesting Fe3O4@1Scr NPs are impervious to apoptotic tumors. Time course T/M values in mice of above four groups were summarized in Figure S26 (Supporting Information). Dynamic MRI indicated that the apoptotic tumors in Fe3O4@1 NPs-injected mice had T/M value of 2.23 ± 0.07 at 0 h, reached their lowest of 1.55 ± 0.09 at 3 h, and recovered to 1.74 ± 0.12 at 5 h post injection (Figure S27). In order to confirm the aggregation of Fe3O4@1 NPs in apoptotic tumors (i.e., tumors in DOX-injected mice), after imaging at 5 h post injection, these four types of tumors (i.e., normal and apoptotic tumors in Fe3O4@1 NPs- or Fe3O4@1-Scr NPs-injected mice) were sliced for Prussian blue staining. The results indicated that only the apoptotic tumors in Fe3O4@1 NPs-injected mice showed large area of aggregated blue staining of Fe, while small area of scattered blue staining was seen in other three groups (Figure S28). Inductively coupled plasma atomic emission spectroscopy (ICP-AES) analyses of the tumor tissues indicated that the apoptotic tumors of Fe3O4@1 NPs-injected mice had a Fe concentration of 92.1 μg/g tumor tissue while that of the normal tumors of Fe3O4@1 NPs-injected mice was 45.3 μg/g tumor tissue (Figure S29). The Fe concentration difference in apoptotic and normal tumors showed good correlation with the difference of quantitative Casp3/7 activity in these two types of tumors in Figure S23 (Figure S29). In conclusion, employing an enzyme-instructed condensation reaction, we rationally designed a small molecule 1 and its scrambled control 1-Scr and used them to covalently modify the surface of USPIO NPs to obtain monodispersive Fe3O4@1 NPs and Fe3O4@1-Scr NPs. TEM results indicated that Fe3O4@1 NPs could subject to Casp3-instructed covalent aggregation, while Fe3O4@1-Scr NPs could not. T2 phantom MR imaging indicated that, after 8 h incubation at 37 °C, T2 relaxation time of Fe3O4@1 NPs buffer with Casp3 was significantly shorter than that of Fe3O4@1 NPs buffer with GSH while those of Fe3O4@1-Scr NPs buffers with Casp3 or GSH were close to each other. Casp3-instructed aggregation of Fe3O4@1 NPs induces approximately 65.2% enhancement of the transverse relaxivity (r2) of the nanoparticles, compared with the GSH-treated group. Cell studies indicated that, while Fe3O4@1 NPs or Fe3O4@1-Scr NPs were indiscriminately uptaken by healthy or apoptotic HepG2 cells, only apoptotic cells incubated Fe3O4@1 NPs showed much shorter T2 relaxation time than those of the other three groups. In vivo tumor MR imaging results suggested that Fe3O4@1 NPs have specificity for T2 enhanced MR imaging in tumor apoptosis. We anticipate that our Fe3O4@1 NPs might be applied to MRI chemotherapeutic efficiency of tumors in routine preclinical studies in near future. And we propose that the enzymeinstructed intracellular covalent aggregation of Fe3O4 NPs could be a novel strategy for the design of “smart” probes for efficient T2 MR imaging of in vivo biomarkers.
with Fe3O4@1 NPs (Figure 3a and b, Figure S18 in Supporting Information), but not in other three groups (Figures S19− S20). T2-weighted MR phantom images of Fe3O4@1 NPs with healthy or apoptotic cells, Fe3O4@1-Scr NPs with healthy or apoptotic cells were acquired on a 9.4 T MR scanner (Figure S21). Analyses of signal intensity versus echo time in Figure S21 gave the corresponding T2 relaxation times, also shown in Figure 3c. Figure 3c indicated that, compared to 61.5 ms T2 relaxation time of healthy HepG2 cells treated with Fe3O4@1 NPs, the T2 value of apoptotic HepG2 cells treated with Fe3O4@1 NPs decreased to 42.5 ms, suggesting that Casp3/7 in apoptotic cells induced the aggregation of the nanoparticles. However, the T2 value of Fe3O4@1-Scr NPs-treated apoptotic HepG2 cells (57.2 ms) was similar to that of Fe3O4@1-Scr NPs-treated healthy HepG2 cells (57.6 ms), suggesting Fe3O4@1-Scr NPs are impervious to Casp3/7. Similar to in vitro tests, we also diluted the cell samples and conducted their T2-weighted MR phantom imaging. Then the r2 values of the cell samples at 9.4 T and RT were calculated to be 22.7 s−1 mM−1 for Fe3O4@1 NPs with healthy cells, 35.1 s−1 mM−1 for Fe3O4@1 NPs with apoptotic cells, 23.6 s−1 mM−1 for Fe3O4@ 1-Scr NPs with healthy cells, and 23.2 s−1 mM−1 for Fe3O4@1Scr NPs with apoptotic cells, respectively, as shown in Figure 3d. All of the above r2 values were summarized in Table S3. Cytotoxicity studies of as-prepared Fe3O4@1 NPs or Fe3O4@1Scr NPs on HepG2 cells indicated that, up to 50 μg Fe/mL and 48 h, neither Fe3O4@1 NPs (84.7 ± 9.4% cell viability) nor Fe3O4@1-Scr NPs (82.6 ± 4.1% cell viability) induced obvious cytotoxicity to the cells (Figure S22). In Vivo Aggregation of Fe3O4@1 NPs Enhances T2 MR Imaging of Tumor Apoptosis. Nude mouse was subcutaneously xenografted with HepG2 tumor in the left thigh. Until a single aspect of tumor grew to 0.7−0.9 cm (approximately 20 days), 8 mg/kg doxorubicin (DOX) or saline was initiated through tail vein administration, once every 4 days for a total of three times.40 Two days after the final treatment, immunohistochemical TUNEL staining (Vazyme Biotech Co., Ltd.) of tumor slices of DOX- or saline-injected mice were performed at this time point. From Figure S23 in Supporting Information, we found that the number of apoptotic cells in tumors of DOXinjected mice was significantly larger than that in tumors of saline-injected mice, confirming that Casp3/7 was highly activated in DOX-injected mice. Each mouse was intravenously injected with 9.8 mg Fe/kg Fe3O4@1 NPs or Fe3O4@1-Scr NPs (concentration: 2.6 mg Fe/mL in PBS) and T2 MR imaging of each mouse was acquired on a 9.4 T MR scanner. Three mice were imaged for each group of total four groups (i.e., normal mice or apoptotic mice injected with Fe3O4@1 NPs or Fe3O4@1-Scr NPs). Three hours post injection, we found that apoptotic tumors in Fe3O4@1 NPs-injected mice showed strong enhancement of T2 MR signal than that of normal tumors in mice injected with same nanoparticles (Figure 4a). Original coronal T2 MR images of mice were shown in Figure S24, and clearly we could observe tumor heterogeneity at 9.4 T. Quantitative analysis indicated that the average contrast change of apoptotic tumors in Fe3O4@1 NPsinjected mice (the tumor-to-muscle contrast (T/M) at 3 h was 69.8% of that at 0 h) was much larger than that of normal tumors in mice injected with the same nanoparticles (the T/M at 3 h was 86.3% of that at 0 h) (Figure S25). T2 MR imaging of apoptotic tumors in Fe3O4@1-Scr NPs-injected mice showed comparable MR contrast enhancement (T/M at 3 h was 88.2% of that at 0 h) to that of normal tumors (T/M at 3 h was 90.0%
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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.nanolett.6b00331. General methods; syntheses and characterizations of 1 and 1-Scr; HPLC conditions; Schemes S1, S2; Figures S1−S29; Tables S1−S3 (PDF) E
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AUTHOR INFORMATION
Corresponding Authors
*Tel.: +86-186-5513-7193. E-mail: kzhong@hmfl.ac.cn. *Tel.: +86-551-6360-7935. E-mail:
[email protected]. Author Contributions
Y.Y. and Z.D. contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the Major Program of Development Foundation of Hefei Center for Physical Science and Technology, Hefei Science Center CAS (2015HSC-UP012), the Fundamental Research Funds for the Central Universities (WK2060190054), and National Natural Science Foundation of China (Grants U1532144, 21375121, U1232212, and U1332142).
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DOI: 10.1021/acs.nanolett.6b00331 Nano Lett. XXXX, XXX, XXX−XXX
