Amphiphilic Polymer-Modified Uniform CuFeSe2 Nanoparticles for CT/MR Dual-Modal Imaging

Recently, magnetic photothermal nanomaterials have attracted much attention in the diagnosis and treatment of cancer. In this study, we developed the ultrasmall magnetic CuFeSe2 nanoparticles for CT/MR dual-modal imaging. By controlling the reaction time and condition, CuFeSe2 nanoparticles were synthesized by a simple directly aqueous method. After modification with copolymer methoxy polyethylene glycol-polycaprolactone (MPEG-PCL), the obtained MPEG-PCL@CuFeSe2 nanoparticles showed excellent water solubility, colloidal stability, and biocompatibility. In addition, they also exhibited superparamagnetism and X-ray's characteristics. For these properties, they will become ideal nanomaterials for CT/MR dual-modal imaging.


Introduction
In recent years, nanotechnology has been widely used in the field of biomedicine, such as the development of tumor therapeutic drugs and molecular imaging probes [1,2]. Among them, semiconductor nanomaterials not only have strong fluorescence characteristics but also have fine and uniform particle size, while carbon dots and organic polymer fluorescent nanomaterials have low biological toxicity and relatively stable chemical properties [3,4]. Many fluorescent nanomaterials, for example, gold-based nanomaterials [5,6], carbon-based nanomaterials [7], conjugated polymeric nanomaterials [8], and graphene [9][10][11], are extensively used in photoacoustic imaging and photothermal therapy of tumors, which achieve the purpose of the integration of diagnosis and treatment of tumors. CuFeSe 2 is classified as I-III-VI 2 ternary chalcogenide semiconductor materials, which has interesting optical, electronic, and magnetic properties. Until now, many studies about CuFeSe 2 mainly focus on the synthesis methods as well as magnetic and optoelectronic properties [12][13][14][15][16]. ere are few reports about its application in diagnosis and cancer treatment [17,18]. erefore, we will try to explore the potential value of its application in the field of imaging diagnosis.
Currently, the CuFeSe 2 nanostructures can be prepared by the solvothermal reaction [19] and the high-temperature solid phase reaction [16]. e products which are often synthesized tend to have a nonuniform size and are prone to agglomeration. erefore, in an effort to overcome their disadvantages of CuFeSe 2 nanostructures, we attempted to prepare with biodegradable copolymer loaded CuFeSe 2 nanocrystals to increase the solubility in aqueous media.
MPEG-PCL is an amphiphilic copolymer, and many studies demonstrate that MPEG-PCL copolymer can significantly improve water solubility of hydrophobic drugs and keep better stability [20][21][22]. erefore, it can be used to load CuFeSe 2 nanoparticles for molecular imaging in vivo. First, the properties of PCL, such as crystallinity, tensile strength, and hydrophobicity, can be easily modulated, and thus, the loading capacity for hydrophobic CuFeSe 2 nanoparticles can be tuned. Second, PEG is nonimmunogenic and highly hydrophilic [23]. Surface coating with PEG can prolong nanoparticle circulation time in vivo, leading to better-enhanced imaging results.
Every imaging modality has its strengths and weaknesses [24]. For instance, X-ray computed tomography (CT) owns its advantages, such as fast acquisition time, large tissue penetration depth, and high spatial resolution, but it has a poor soft-tissue contrast. Magnetic resonance (MR) imaging possesses favorable spatial and soft-tissue resolution, and it can implement multisequence, multiparameter imaging, but its limitation is low sensitivity. Nuclear imaging techniques, including single photon emission computed tomography (SPECT) and positron emission tomography (PET), exhibit high sensitivity and are quantitative, but along with a poor spatial resolution. However, multimodal imaging can improve the accuracy of cancer diagnosis by combining two or more imaging modalities into one system [25,26]. It overcomes the intrinsic limitations of single modality.
In this study, the ultrasmall magnetic CuFeSe 2 nanostructures were prepared by a simple direct aqueous method. After modification with methoxy polyethylene glycol-polycaprolactone (MPEG-PCL), the biosafety of obtained MPEG-PCL@CuFeSe 2 nanoparticles was evaluated. Lastly, the X-ray attenuation property and T 2 MR relaxometry of MPEG-PCL@CuFeSe 2 NPs in vitro/in vivo were measured to explore the potential application of these NPs as dual-modal CT/MR imaging contrast agents.

Synthesis of CuFeSe 2 Nanoparticles.
For the synthesis of CuFeSe 2 nanoparticles, 78.96 mg of Se powder was dispersed in 100 mL of Milli-Q water, and then 75.60 mg of NaBH 4 was added to reduce it at ambient conditions with protection of nitrogen flow for one hour. After Se powder was completely reduced, a colorless solution is obtained. A 10 mL mixture of CuCl 2 2H 2 O (85.24 mg), FeSO 4 7H 2 O (139.01 mg), and L-cysteine (121.20 mg) was separately prepared, and then the above mixture was added into the selenium precursor solution immediately to form a black solution. e resultant solution was collected after centrifugation with a speed of 3500 rpm for twenty minutes to remove impurities. e purified CuFeSe 2 solution was stored at 4°C for further characterization and application.

Functionalization of CuFeSe 2 Nanoparticles.
Methoxy poly (ethylene glycol)-poly (ε-caprolactone) (MPEG-PCL) used in this study was synthesized by ring-opening polymerization of ε-CL on MPEG using Sn(Oct) 2 as catalyst, according to a previous report [27]. e MPEG-PCL colloidal solution was prepared by liquid rotary evaporation method. For functionalization of CuFeSe 2 nanoparticles, the above purified CuFeSe 2 solution was slowly added into the MPEG-PCL colloidal solution under ultrasonication for 4 hours, and then the MPEG-PCL-modified CuFeSe 2 nanoparticles were obtained after centrifugation to remove excess and large impurities. e purified MPEG-PCL@CuFeSe 2 solution was stored at 4°C for future experiments.

Characterization.
e hydrodynamic diameters and zeta potentials of prepared CuFeSe 2 solution and MPEG-PCL@ CuFeSe 2 solution were measured by dynamic light scattering (DLS, NanoBrook 90Plus Zeta, Brookhaven, USA) at 25°C. e size and morphology of prepared CuFeSe 2 nanoparticles and MPEG-PCL@CuFeSe 2 nanoparticles were characterized with a transmission electron microscope (TEM, Tecnai G2 F20, USA). e crystallography structures of CuFeSe 2 nanoparticles and MPEG-PCL@CuFeSe 2 nanoparticles were characterized by using an X-ray diffractometer equipped with Cu Kα radiation (λ � 0.15406 nm). A scanning rate of 0.1°/s was applied to record the pattern in the 2θ range of 10-90°. e T2-weighted images of MPEG-PCL@CuFeSe 2 at different concentrations were scanned under a 3T clinical MRI scanner at room temperature. After the T2-weighted MR images were acquired, the signal intensity was measured by a manually drawn region-of-interest for each sample.

Cell
Culture and Cytotoxicity Assessment. 4T1 murine breast cancer cells, A549 human lung adenocarcinoma, and human normal liver cells were cultured in standard cell media supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37°C in an atmosphere of 5% CO 2 . All cell culturerelated reagents were purchased from HyClone (USA). e cytotoxicity of MPEG-PCL-CuFeSe 2 NPs was evaluated by the MTT assay. e cells were first seeded into 96-well plates (1 × 10 4 cells per well) and cultured for 24 h and then added into different concentrations of MPEG-PCL-CuFeSe 2 and continued to culture for 24 h. After this, 10 μL MTT (5 mg/ mL) was added. Four hours later, the supernatant medium was removed, and 150 μL DMSO was added into each well to dissolve the resulting formazan crystals. e absorbance was measured at 490 nm using a spectrophotometric microplate reader (iMark, MA, USA). e cytotoxicity was calculated as the percentage of cell viability.

Animal Model.
We acquired female BALB/c mice (6-8 weeks of age, 25-30 g of weight) from Chongqing Tengxin Biotechnology Co. Ltd. (Chongqing, China). To generate the 4T1 tumor murine model, 1 × 10 6 cells in the 100 μL serum-free RMPI-1640 medium were subcutaneously injected into in the right side thigh root of each mouse. All mice were selected for imaging experiments when their tumors grew to 80 mm 3 .

In Vitro CT/MR Dual-Modality
Imaging. Various concentrations of MPEG-PCL@CuFeSe 2 solution were dispensed in 5.0 mL Eppendorf tubes for CT and MR contrast imaging. e MR imaging for in vitro study was performed on a 3.0 T clinical magnetic resonance (MR) scanner (PHILIPS, Holland). e representative imaging parameters of the T2-weighted images were as follows: repetition time (TR) � 5348 ms, echo time (TE) � 70 ms, slice thickness � 1.5 mm, slice spacing � 0.15 mm, matrix � 256 × 256 pixels, field of view (FOV) � 30 cm × 60 cm × 25 cm, NSA � 4, and flip angle � 10°. e region-of-interest was selected by drawing manually to measure the signal intensity of MPEG-PCL@CuFeSe 2 solution from the T2-weighted MR images.
e CT data were acquired using a clinical CT imaging scanner (GE, USA), and X-ray attenuation values for all samples were finally calculated in Hounsfield units (HU) by averaging over the region-of-interest (ROI). CT imaging parameters were as follows: tube current � 600 mA, tube voltage � 100 kV, and slice thickness � 0.625 mm.

In Vivo CT/MR Dual-Modality Imaging.
e 4T1 tumorbearing mice were acquired before and after intratumorally (i.t.) injected with MPEG-PCL@CuFeSe 2 (250 μL, 2 mg/ mL) and imaged with a 3.0 T clinical magnetic resonance (MR) scanner (PHILIPS, Holland) equipped with a small animal coil. e representative imaging parameters of the T2-weighted images were as follows: repetition time (TR) � 5348 ms, echo time (TE) � 70 ms, slice thickness � 1.5 mm, slice spacing � 0.15 mm, matrix � 256 × 256 pixels, field of view (FOV) � 30 cm × 60 cm × 25 cm, NSA � 4, and flip angle � 10°. e region-of-interest in the tumor area of each mouse was selected by drawing manually to measure the signal intensity of tumors from the T2-weighted MR images.
e CT images were acquired before and after intratumorally (i.t.) injected with MPEG-PCL@CuFeSe 2 (250 μL, 2 mg/mL) on a clinical CT imaging scanner (GE, USA), and the CT imaging parameters were as follows: tube current � 600 mA, tube voltage � 100 kV, and slice thickness � 0.625 mm. e region-of-interest in the tumor area of each mouse was selected by drawing manually to measure the CT value of tumors from the CT images.

In Vivo Toxicity Study.
e major organs/tissues were taken from mice after intravenous injection of MPEG-PCL@ CuFeSe 2 (a dose of 20 mg/kg) at 1 day, 3 days, 7 days, and 15 days postinjection, while other mice without injection were used as the control group (four mice per group). en, the obtained major organs/tissues were fixed in 4% formalin, paraffin-embedded, sectioned, and stained with hematoxylin & eosin (H&E) and then imaged by using a digital microscope to evaluate the histological changes.

Synthesis and Characterizations of MPEG-PCL@CuFeSe 2
Nanoparticles. In our experiments, MPEG-PCL@CuFeSe 2 nanoparticles with uniform sizes and morphologies were synthesized by a simple direct aqueous method. e resultant nanoparticles were characterized by transmission electron microscopy (TEM) to determine their size and morphology. Small spherical particles with a size of 4.2 ± 0.7 nm are clearly observed (Figures 1(a), 1(c)). e crystal structure of CuFeSe 2 nanoparticles was observed by their high-resolution TEM (HR-TEM) image, which clearly shows the lattice fringes with an interplanar spacing of 0.325 nm (Figure 1(b)). e FTIR spectra showed a typical variation peak of C�O at 1727.60 cm −1 and typical variation peaks of C-H at 2891.05 cm −1 and 2949.12 cm −1 in the MPEG-PCL@CuFeSe 2 nanoparticles, verifying the successful modification of MPEG-PCL (Figure 2(a)). e X-ray powder diffraction (XRD) results show that both CuFeSe 2 and MPEG-PCL-CuFeSe 2 nanoparticles have crystal face peaks at (112) and (220), indicating that the nanoparticles are cubic crystal structures (Figure 2(b)). e content of MPEG-PCL polymer on the surfaces of CuFeSe 2 nanoparticles was determined by thermogravimetric analysis (TGA) to be approximately 40.0 wt. % (Figure 2(c)), and the TGA results demonstrate the successful coating of CuFeSe 2 nanoparticles with MPEG-PCL. For the magnetic properties of CuFeSe 2 and MPEG-PCL@CuFeSe 2 nanoparticles, the superparamagnetic properties of CuFeSe 2 and MPEG-PCL@CuFeSe 2 were illustrated by the absence of a hysteresis loop in the field-dependent magnetization measurement (Figure 2(d)). According to the results from DLS, the hydrodynamic diameters of MPEG-PCL NPs and MPEG-PCL@CuFeSe2 NPs were 25.31 ± 2.07 nm and 120.91 ± 3.44 nm, respectively. MPEG-PCL NPs had a negative surface charge of −21.03 ± 1.53 mV, and MPEG-PCL@CuF-eSe2 NPs had a negative surface charge of −10.85 ± 2.59 mV ( Table 1).

Toxicity Studies of MPEG-PCL@CuFeSe 2 Nanoparticles.
Good biosecurity is an important criterion for measuring whether nanomaterials can be applied to living organisms. us, in vitro MTT assay and in vivo pathological assay were investigated to test the cytotoxicity and biosecurity of MPEG-PCL@CuFeSe 2 NPs, respectively. e results of MTT assay showed that the viability of the above three kinds of cells still kept above 90% in the concentration range of 0-150 μg/mL for MPEG-PCL@CuFeSe 2 nanoparticles after cultured 24 h, demonstrating the cytotoxicity is not obvious (Figure 3(a)). In order to test toxicity study of MPEG-PCL@CuFeSe 2 NPs in vivo, histological assessment of tissues was performed to determine whether MPEG-PCL@-CuFeSe 2 NPs caused damage to important organs. e above representative organs including heart, liver, spleen, lung, and kidney had no apparent histopathological abnormalities or lesions, compared with those of the control group throughout the entire study. erefore, the results of in vivo toxicity indicated the good biocompatibility of MPEG-PCL@CuFeSe 2 NPs (Figure 3(b)). transplanted mice. In the first place, CT images were acquired before and after intratumoral injection, and the tumor sites showed an enhancement with a higher CT value after administration of contrast agent when compared with those before injection ( Figure 5(a)). Similar to CT imaging, MR images were acquired before and after intratumoral injection, and the tumor sites showed an enhancement with a lower T 2 WI MR signal intensity after administration of contrast agent when compared with those before injection ( Figure 5(d)). e above results indicate the development of MPEG-PCL@CuFeSe 2 has the potential to target bimodal CT/MR imaging.

Conclusions
In summary, the MPEG-PCL copolymer-modified CuFeSe 2 nanoparticles with favorable biological safety were successfully prepared by an environmentally friendly aqueous route under ambient conditions, and the MPEG-PCL@CuFeSe 2 nanoparticles perform positive CT/MR contrast effect in vitro/in vivo. ese excellent properties enable them to be a promising nanotheranostic agent for in vivo multimodal imaging.
Data Availability e laboratory experimental data used to support the findings of this study are available from the first author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.