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Synergistic Theranostics of Magnetic Resonance Imaging and Photothermal Therapy of Breast Cancer Based on the Janus Nanostructures Fe3O4-Aushell-PEG

Authors Kang X , Sun T, Zhang L , Zhou C, Xu Z, Du M, Xiao S, Liu Y, Gong M , Zhang D

Received 1 June 2021

Accepted for publication 3 September 2021

Published 16 September 2021 Volume 2021:16 Pages 6383—6394

DOI https://doi.org/10.2147/IJN.S322894

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Mian Wang



Xun Kang, Tao Sun, Liang Zhang, Chunyu Zhou, Zhongsheng Xu, Mengmeng Du, Shilin Xiao, Yun Liu, Mingfu Gong, Dong Zhang

Department of Radiology, Xinqiao Hospital, Army Medical University, Chongqing, People’s Republic of China

Correspondence: Mingfu Gong; Dong Zhang
Department of Radiology, Xinqiao Hospital, Army Medical University, No. 183, Xinqiao Street, Shapingba District, Chongqing, People’s Republic of China
Tel +86 2368763843
Fax +86 2368755306
Email [email protected]; [email protected]

Background: Satisfactory prognosis of breast cancer (BC) is limited by difficulty in early diagnosis and insufficient treatment. The combination of molecular imaging and photothermal therapy (PTT) may provide a solution.
Methods: Fe3O4-Aushell nanoparticles (NPs) were prepared by thermal decomposition, seeded growth and galvanic replacement and were comprehensively characterized. After conjugated to PEG, NPs were used as MRI and PTT agents in vitro and in vivo.
Results: Fe3O4-Aushell NPs which had uniform Janus-like morphology were successfully synthesized. The Fe3O4 had a size of 18 ± 2.2 nm, and the Aushell had an outer diameter of 25 ± 3.3 nm and an inner diameter of 20 ± 2.9 nm. The NPs showed superparamagnetism, a saturation magnetization of 36 emu/g, and an optical absorption plateau from 700 to 808 nm. The Fe3O4-Aushell NPs were determined to possess good biocompatibility. After PEG coating, the zeta potential of NPs was changed from − 23.75 ± 1.37 mV to − 13.93 ± 0.55 mV, and the FTIR showed the characteristic C–O stretching vibration at 1113 cm− 1. The NPs’ MR imaging implied that the T2 can be shortened by Fe3O4-Aushell NPs in a concentration-dependent manner, and the Fe3O4-Aushell NPs coated with PEG at the molar ratio of 160 (PEG: NPs) showed the highest transverse relaxivity (r2) of 216 mM− 1s− 1. After irradiation at 0.65 W/cm2 for 5 min, all cells incubated with the Fe3O4-Aushell-PEG160 NPs (Fe: 30 ppm, Au: 70 ppm) died. After administrated intratumorally, Fe3O4-Aushell-PEG160 notably decreased the signal intensity of tumor in T2WI images. Under the same irradiation, the temperature of tumors injected with Fe3O4-Aushell-PEG160 quickly rose to 54.6°C, and the tumors shrank rapidly and were ablated in 6 days.
Conclusion: Fe3O4-Aushell-PEG NPs show good r2 and PTT performance and are promising synergistic theranostic agents of MRI and PTT for BC.

Keywords: magnetic resonance imaging, photothermal therapy, nanoparticle, early breast cancer theranostics, gold nanoshells

Introduction

Breast cancer (BC) has the highest morbidity and mortality among women worldwide.1–3 The difficulty of early diagnosis and insufficient treatment are believed to be the two most important factors leading to this situation.4 In clinical practice, BC is usually diagnosed by an imaging examination.4,5 Imaging screening is critical for the diagnosis and staging of BC and includes mammography, ultrasound, and magnetic resonance imaging (MRI).6 However, BC can only be detected when the BC mass can be measured in millimeters. At that time, the BC cells may have already metastasized, resulting in the patient missing their optimal treatment window.7 Detecting serum tumor markers from the blood test can also be used for BC diagnosing and recurrence monitoring, including cancer antigen (CA) 27.29, CA 15–3, CA 125, and carcinoembryonic antigen (CEA).8,9 However, patients with ulcerative colitis, pancreatitis, cirrhosis, or even healthy individuals can show positive results, which makes this BC diagnosis test unreliable.9

Benefiting from molecular probes, molecular imaging can visualize biological events at the cellular and molecular levels in vivo, thus having the potential to diagnose cancer at its early stage.10,11 Some preliminary studies have shown the preclinical diagnosis of cancer using molecular imaging.10 Among all the molecular imaging modalities, MRI is believed to be a promising approach because of its high spatial resolution, good soft tissue contrast, and non-ionizing radiation.12 Moreover, with the progress of nanotechnology, superparamagnetic nanoparticles (NPs)—the key moiety of MR molecular probe with higher relaxivity—are emerging.13 As a result, the sensitivity and specificity of MR molecular imaging (MRMI) keeps improving, making it a more powerful and promising alternative for the early diagnosis of BC.14

The inefficiency of traditional therapy is another important factor contributing to the high fatality of BC. Upon diagnosis, the patient will undergo surgery, radiotherapy, or chemotherapy according to the BC stage.4,15 If the patient is estrogen receptor- (ER) or human epidermal growth factor receptor-2 (HER-2) positive, endocrine therapy (ET) or anti-HER-2 therapy will benefit the patient’s recovery.16 However, surgery can only benefit if the BC has no metastasis. In addition, surgery can result in extensive trauma that can harm the immune balance and delay tumor healing.17 Because of their rapid proliferation, BC cells can be inhibited and killed by radiotherapy and chemotherapy. At the same time, other fast-growing cells that are essential for normal physiological homeostasis will be non-selectively killed and result in the side effects including emesis, alopecia, and emaciation.18 As a result, the dose of chemotherapy and radiotherapy is strictly limited in clinical practice, which may abate the killing effect and induce BC cell resistance to chemotherapy and radiotherapy.18 ET and anti-HER-2 therapy have recently been widely applied in the clinic and showed promising results.19,20 Nevertheless, about 15–20% of patients express none or few of these receptors, which greatly compromises the therapeutic effect. In addition, some ER- or HER-2-positive cancer cells can become resistant to the treatment during the course of therapy.20 Photothermal therapy (PTT) is an emerging and effective tumor treating strategy, having attracted great attention in recent years due to its noninvasive nature, controllability, and minimal side effects.21 Cells usually perform their physiological functions well at 37°C. As the temperature increases to 42–45°C, proteins aggregate and denaturize, resulting in the cell activity being significantly reduced. Further increasing the temperature to 48–60°C will cause cells to suffer irreversible damage and lead to apoptosis.22 Tumor cells are also more sensitive to high temperature than normal cells because of their rapid proliferation. Hyperthermia within cancer lesions can induce the release of CAs and proinflammatory cytokines to promote anticancer immunity, which kills cancer cells synergistically. It is known that photothermal agents (PTAs) are essential for PTT and the efficacy of PTT is closely related to the photothermal conversion efficiency (PCE). The higher the PCE of PTAs, the less the time and energy required for inducing cell death.21 Non-specific damage to surrounding tissues induced by the PTAs with higher PCE can be effectively minimized. Thus, PTAs with high PCE are desired for PTT.

Therefore, combining MRMI and PTT is expected to diagnose and treat BC early enough to improve its prognosis. The combination of MRMI and PTT can be theoretically achieved by injecting MRI and PTT agents simultaneously. Still, it is difficult to keep the two agents at the same pharmacokinetic and biodistribution profiles using the above strategy, leaving the combination of MRMI and PTT a challenge. Alternatively, using a Janus nanostructure composed of two functional moieties that can both shorten the relaxation time and convert light energy to thermal energy as the theranostic agents can, in practice, achieve the combination of MRMI and PTT. We previously synthesized a multifunctional Janus NP Fe3O4-Aushell composed of two functional parts: Fe3O4 nanospheres that shorten the transverse relaxation time (T2) and an Aushell that serves as a PTA.23 Fe3O4 NPs are the most widely studied and applied superparamagnetic NPs for MRMI. They show satisfactory relaxivity and good biocompatibility, with some iron oxide-based NPs (Resovis®, Feridex I.V.®) approved for clinical use by the FDA.24,25 The Fe3O4 NPs are also easily modified by various ligands and biomacromolecules on the NP surface for multi-functionalization. Aushell NPs are widely used PTA for PTT and have shown good biocompatibility and high PCE. Benefitting from its hollow shell structure, the Aushell has a high PCE and an absorption peak in the near-infrared (NIR) region, with minimal biological tissue absorption. This allows the laser energy to be more effectively absorbed and minimizes collateral damage to adjacent normal tissue.26 Accordingly, Fe3O4-Aushell Janus NPs may be considered an ideal candidate for the combination of MRMI and PTT.

Herein, we synthesized Fe3O4 NPs by thermolysis and Janus Fe3O4-Ag nanoparticles by reducing Ag+ on the surface of Fe3O4 nanoparticles followed by the seeded growth of Ag NPs. Based on the galvanic chemistry, Ag NPs were replaced with Au nanoshells, and Fe3O4-Aushell Janus NPs were obtained. Fe3O4-Aushell NPs were then characterized by transmission electron microscopy (TEM), high-resolution TEM (HRTEM), magnetic property measurement system (MPMS), microplate reader, electrophoretic light scattering (ELS), Fourier-transform infrared spectroscopy (FTIR), and inductively coupled plasma optical emission spectroscopy (ICP-OES). The cytotoxicity of Fe3O4-Aushell NPs was measured using CCK-8 and hemolysis analysis. The relaxivity of Fe3O4-Aushell coated by varied amounts of polyethylene glycol (PEG) was evaluated, and the in vitro PTT efficiency was determined. Finally, Fe3O4-Aushell-PEG NPs were administered intratumorally for MR imaging and PTT in vivo to determine its feasibility for BC diagnosis and treatment.

Methods

Materials

Carbonyl iron (Fe(CO)5) was obtained from the Xindingpengfei Technology Development Co., Ltd (Beijing, China). Chloroauric acid (HAuCl4) and oleic acid (OA) were delivered by Sigma-Aldrich (USA). The 1-octadecene (ODE) was purchased from Acros (Shanghai, China). Tetramethyl ammonium hydroxide (TMAH), silver nitrate, and hydroxylamine hydrochloride were purchased from Adamas-beta (Shanghai, China). Trisodium citrate (TSC) was purchased from Sinopharm Chemical Reagent Co. Ltd (Beijing, China). H2O2 was purchased from Chuandong Chemical Co. Ltd (Chongqing, China). Thoil-PEG2K was bought from the Xi’an Ruixi Biological Technology Co., Ltd (Xi’an, China). The Cell Counting Kit-8 (CCK8), Calcein Acetoxymethyl (AM)/Propidium Iodide (PI) Cell Viability Assay Kit, and Penicillin–Streptomycin Solution were purchased from Beyotime Biotechnology (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from HyClone (Shanghai, China). Phosphate buffer saline (PBS) powder was purchased from Boster Biotechnology (Wuhan, China). All the chemicals were used as delivered and all glassware engaged in synthesis were rinsed by aqua regia and deionized water before use.

Nanoparticle Synthesis

Fe3O4-Agseed: The Fe3O4-Aushell nanostructure was synthesized according to a previous method with some modifications that were initiated with the synthesis of Fe3O4-Agseed.23 First, hydrophobic Fe3O4 nanoparticles were synthesized by thermolysis of Fe(CO)5. Briefly, a mixture of 2.4 mL OA and 20 mL ODE was heated to 100°C with magnetic stirring and Ar gas bubbling. 0.5 h later, 0.4 mL of (3.04 mmol) Fe(CO)5 was injected, and the system was heated to 295°C. After reacting for 1 h, the heating source was removed to cool down the dispersion to room temperature. The prepared NPs were washed with isopropanol three times and dispersed in 20 mL of hexane. To make the nanoparticles hydrophilic, the OA on the Fe3O4 surface was exchanged with TMAH. The synthesized Fe3O4 NPs were precipitated and dried to obtain a black-colored powder, followed by dispersed in 20 mL TMAH (10%) and sonicated until clear. The hydrophilic Fe3O4 NPs were then centrifuged and finally dispersed in 20 mL DI water. The Fe3O4-Agseed was further synthesized by reducing Ag+ on the surface of Fe3O4 NPs with the help of ferrous ion and citrate. A mixture of 40 mL DI water, 1 mL hydrophilic Fe3O4, and 1 mL 1% (w/v) TSC was heated to 60°C with magnetic stirring. Then, 500 μL of 0.17% (w/v) AgNO3 was slowly added and reacted for 2 h. The dispersion was then irradiated under 254 nm ultraviolet light for 0.5 h at room temperature to ripen the Ag NPs. Afterwards, 4 mL of 1% TSC was added, and the system was reheated to 60°C, followed by the addition of 3.75 mL 0.17% AgNO3 within 1 h. The solution was heated for another 0.5 h, and Fe3O4-Agseed was obtained.

Fe3O4-Aushell: To make the absorption peak locate in the NIR region, the cavity to thickness ratio of Aushell must be suitably fixed. Hence, we increased the Ag NPs’ size by seeded-growth. A mixture of 5 mL Fe3O4-Agseed, 6.4 mL 1% (w/v) TSC, and 80 mL DI water was heated to 60°C under magnetic stirring. Then, 6.4 mL of 0.17% (w/v) AgNO3 was added within 3 h followed by a reaction of 1 h to obtain Fe3O4-Ag nanoparticles (Fe3O4: 18 nm, Ag: 22 nm). Finally, Fe3O4-Aushell NPs were synthesized by coating an Au shell on the Ag core and then cavitating the Ag core based on the galvanic replacement chemistry.27 Briefly, 5 mL of synthesized Fe3O4-Ag NPs and 6.25 mM of hydroxylamine hydrochloride were mixed under magnetic stirring. Next, 5 mL of 0.465 mM HAuCl4 was added within 12.5 min. When the color was stabilized, the dispersion was mixed with H2O2 (30%) in a volume ratio of 50:1 for 2 h to obtain Fe3O4-Aushell. To improve the biocompatibility of the obtained Fe3O4-Aushell, PEGs were conjugated on their surface by thiol-Au coordination. Equal volumes of Thiol-PEG2K and Fe3O4-Aushell were mixed at different molar ratios (PEG to Fe3O4-Aushell: 0, 10, 40, 160, 640), followed by incubation at room temperature overnight. Then, unlinked PEG was removed by centrifugation. The obtained nanoparticles were termed as Fe3O4-Aushell-PEG0, Fe3O4-Aushell-PEG10, Fe3O4-Aushell-PEG40, Fe3O4-Aushell-PEG160, and Fe3O4-Aushell-PEG640, accordingly.

Nanoparticle Characterization

The morphology and size distribution of NPs were analyzed using a TEM (HT7700, Hitachi, Japan). The element distribution was identified by EDS mapping (FEI Tecnai G2 F30, USA). Hysteresis loop was recorded by a MPMS3 (Quantum Design, USA). The Fe and Au content in nanoparticle were measured by an ICP-OES (Agilent ICPOES730, USA). The UV-Vis absorption spectrum of NPs was measured by a multi-mode microplate reader (Varioskan Flash, Thermo Scientific, USA). NPs’ zeta potential was determined using ELS (Z3000, NICOMP, USA). We also verify the linking of PEG on the surface of Fe3O4-Aushell using FTIR (ALPHA II, BRUKER, USA).

Cells and Animals

Mouse breast cancer cells 4T1 were used for in vitro biocompatibility and photothermal efficiency analysis in the present study and purchased from Procell Co. Ltd (Wuhan, China). Cells were routinely cultured with complete medium, which was composed of 89% DMEM medium, 10% FBS, and 1% streptomycin and penicillin in a 5% CO2 incubator at 37°C.

With permission from the Animal Welfare and Ethics Committee of the Army Medical University (No. 2019366), 106 4T1 cells were injected subcutaneously into the right thigh of nude mice to establish a xenograft tumor model for in vivo MRI and PTT in this study. All the BALB/c nude mice (female, 5 weeks old) were purchased from HFK BIOSCIENCE Co. Ltd. (Beijing, China) and hosted in a specific pathogen free (SPF) environment at 20°C with a 12:12 dark/light cycle in the experimental animal center of Xinqiao Hospital, Army Medical University. All animal experiments were performed in accordance with the guidelines of Care and Use of Laboratory Animals of Ministry of Science and Technology of the People's Republic of China.

Cytotoxicity and Hemolysis

The cytotoxicity of Fe3O4-Aushell NPs was determined using a CCK-8 analysis. 4T1 cells were seeded into 96-well plates at 5000 cells per well and incubated with complete medium overnight to allow cell adhesion. Then, the complete medium in each well was replaced with media containing Fe3O4-Aushell of different concentrations (Fe: 0, 10, 20, 40, 80 ppm). After 24 h of incubation, the media containing Fe3O4-Aushell NPs were removed and the wells were washed three times with PBS. Afterwards, 100 μL of fresh complete medium and 10 μL of CCK8 working solution were added to each well, followed by 2 h of incubation. Finally, the absorbance of each well was measured with a multi-mode microplate reader (Varioskan Flash, Thermo Scientific, USA) at 450 nm.

For the hemolysis analysis, fresh red blood cells (RBCs) were harvested from a healthy BALB/c mouse and then washed 3 times and re-dispersed in 2 mL PBS for further use. About 0.2 mL RBCs-PBS dispersions were then added to 1 mL PBS (negative control), 1 mL DI water (positive control), 1 mL Fe3O4-Aushell dispersions in PBS with various Au concentrations (25, 50, 100 ppm). Then, all the samples were incubated in a 5% CO2 incubator at 37°C for 2 h. After centrifuged, the supernatants were collected and measured at 545 nm with the multi-mode microplate reader to calculate the hemolysis rate.

MRI and PTT in vitro

The Fe3O4-Aushell-PEG0, Fe3O4-Aushell-PEG10, Fe3O4-Aushell-PEG40, Fe3O4-Aushell-PEG160, and Fe3O4-Aushell-PEG640 dispersions with different concentrations (Fe: 0, 0.02, 0.04, 0.09, 0.18, 0.36 ppm) were MR-imaged in a Philips Ingenia 3 T MRI system with a head coil. The imaging parameters were as followed: turbo spin-echo (TSE) T2 mapping: repetition time (TR) = 2000 ms, echo time (TE) = 13, 26, 39, 52, 65, and 78 ms; slice thickness/spacing = 2.5 mm/0.25 mm, field of view (FOV) = 164×164 mm2. Linear regression of transverse relaxation rates (1/T2) and Fe concentrations was conducted to obtain the slope as transverse relaxivity (r2) values. The region of interest (ROI) was 40 mm2 for both the signal intensity and relaxation time measurements.

To measure the heating performance, 0.5 mL of Fe3O4-Aushell-PEG160 (Fe: 30 ppm, Au: 70 ppm) were irradiated under 808 nm laser at different power densities (0.65, 1.2, 2.0 W/cm2) for 5 min. Then, 0.5 mL of DI water, Fe3O4 (Fe: 30 ppm), Fe3O4-Aushell-PEG160 (Fe: 10 ppm, Au: 23 ppm), and Fe3O4-Aushell-PEG160 (Fe: 30 ppm, Au: 70 ppm) were irradiated under a 1.2 W/cm2 laser for 5 min. A thermal camera (E8, FLIR, USA) was used to record the temperature every 30 s. We further evaluated the Fe3O4-Aushell-PEG160 killing effect under PTT using CCK-8 assay and fluorescence staining. Cells (104) were seeded into a 96-well plate overnight for adhesion. Afterwards, cells were classified into four groups and were treated differently as follows: group 1 and 3 cells were cultured with complete medium, while group 2 and 4 cells were incubated with complete medium containing Fe3O4-Aushell-PEG160 (Fe: 30 ppm, Au: 70 ppm). Group 3 and group 4 cells were irradiated with a laser for 5 min at a power density of 0.65 W/cm2, and groups 1 and 2 did not undergo any irradiation. At 24 h after treatment, the media containing Fe3O4-Aushell-PEG160 NPs were removed, and the wells were washed three times with PBS. Afterwards, 100 μL of fresh complete medium and 10 μL of CCK8 working solution were added to each well, followed by 2 h of incubation. Finally, all the wells were measured at 450 nm under a microplate reader. Meanwhile, 100 μL of Calcein-AM/PI working solution was added to each well to stain all the cells with fluorescence for 30 min, and then all the cells were photographed under an inverted fluorescence microscope (IX83, Olympus, Japan).

MRI and PTT in vivo

At 7–8 days post-subcutaneous injection, the xenograft tumors were about 80 mm3 and used for MRI and PTT in vivo. The mice were anesthetized with isoflurane and maintained in a stereotaxic frame (R510IP, RWD Life Science, Shenzhen, China). Before and after Fe3O4-Aushell-PEG160 (100 μL, Fe concentration: 40 ppm) administration intratumorally, the T2 weighted images (T2WI) were acquired using a special mouse coil (MS40, Suzhou Medcoil Healthcare Co., Ltd) in a 3 T clinical GE MRI scanner according to the following parameters: TE= 70.5 ms, TR= 3040 ms, FOV = 60×60 mm2, matrix = 288×192, slice thickness/spacing = 2.0 mm/0.2 mm, and number of excitation (NEX) = 6.

To determine the potential of Fe3O4-Aushell-PEG160 for BC PTT in vivo, 12 tumor-bearing mice were equally divided into four groups and treated as follows: mice in group 1 received no additional treatment except for the routine feeding; mice in group 2 were injected with of 100 μL PBS intratumorally and then irradiated with 0.65 W/cm2 808 nm laser for 5 min; group 3 mice received intratumoral injections of 100 μL Fe3O4-Aushell-PEG160 (Au: 70 ppm); and group 4 mice were injected with 100 μL of Fe3O4-Aushell-PEG160 (Au: 70 ppm) intratumorally and then irradiated with a 0.65 W/cm2 808 nm laser for 5 min. For groups 2 and 4, the temperature of the mice’s tumor was recorded every 0.5 min with a thermal imaging camera (E8, FLIR, USA) during laser irradiation. Tumor volume and body weight of all the mice were recorded every two days for 14 days. The tumor volume was calculated following the equation: length × width2 × 0.5, in which the length and width are the largest longitudinal and transverse diameter. The relative tumor volume was defined as the ratio of current tumor volume compared with the same mouse’s original volume and used for the comparison between different groups. At 14 days after treatment, all mice were humanely killed, and their main organs including the heart, lung, liver, left kidney, spleen were fixed with 4% formaldehyde for H&E staining.

Statistical Analysis

All values are expressed as the mean ± standard deviation (SD). Significant differences between different groups were assessed by a Student’s unpaired t-test. The statistical significance is shown as: NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Results and Discussion

Nanoparticle Synthesis and Characterization

The morphology and crystallinity of magnetic nanoparticles are closely related to their physical and chemical properties. Compared with coprecipitation and microemulsion, the NPs synthesized by thermal decomposition of carbonyl iron are usually of high uniformity and crystallinity, making the obtained NPs preferred for biomedical applications.28,29 The TEM image of the acquired Fe3O4 is shown in Figure 1A, indicating an average diameter of 18 ± 2.1 nm and narrow size distribution. Benefitting from the reducibility of TSC and ferrous ions, silver nanoparticles could anchor on the surface of Fe3O4 to form a stable heterodimer structure (Figure 1B, Ag: 9.3 ± 1.8 nm). After seeded growth of Ag NPs, the Fe3O4-Ag NPs with a typical Janus nano structure were successfully synthesized. By averaging 300 NPs using Nano Measurer, we were told that the Janus NPs had a uniform morphology with the size of 18 ± 2.2 nm (Fe3O4) and 22 ± 3.2 nm (Ag) (Figure 1D). According to galvanic replacement theory, Au3+ can be reduced and form an Au layer on Ag nanoparticle’s surface by introducing the Au3+ ion to the Ag nanoparticle with the assistance of hydroxylamine hydrochloride.30 Then, in the presence of H2O2, the Ag core will be further oxidized to Ag+ and drift away, leaving a hollow Au shell. As Figure 1E shows, the original Ag nanoparticle cores in the Janus structure were replaced by a hollow shell. The inner diameter of the hollow shell is about 20 ± 2.9 nm, which is about the same size as silver NPs, and the outer diameter was about 25 ± 3.3 nm, suggesting that the Fe3O4-Aushell is formed. It has been repetitively determined that Ag nanoparticles have a strong absorption cross section at about 400 nm, while Au nanosphere absorbs mostly at 520 nm.31,32 In addition, the hollowing of the Au nanosphere can significantly change its absorption profile. With the increase of the cavity-to-shell ratio, the absorption peak of the Au nanostructures would be red-shifted and located within the NIR region when the ratio is 5:1.26 This study showed consistent results (Figure 1C). Fe3O4 nanoparticles, which do not have a surface plasmon resonance (SPR) effect, exhibited a downward curve. After seeded growth, NPs showed a pointed peak at 412 nm, which was consistent with the Ag NPs’ absorption, verifying the existence of Ag NPs. When the NPs were further galvanically replaced and etched, the 412 nm peak faded away and a gentle slope centered at 720 nm emerged, which represented the absorption pattern of Au shell and implicated the successful synthesis of Fe3O4-Aushell. To further identify the Janus NPs with nano Au shell visually, EDS mapping was performed. As Figure 1F shows, the nanoparticles were a typical Janus nanoparticle composed of two moieties. The solid spheres overlapped with the iron element and the hollow shells overlapped the Au element, confirming the results from TEM and absorption spectrum. We also determined the element concentration using ICP-OES, which showed that the synthesized Janus NPs contained both Fe and Au and their molar ratio was 1.5:1.

Figure 1 (A, B, D and E) TEM of Fe3O4, Fe3O4-Agseed, Fe3O4-Ag, Fe3O4-Aushell NPs, scale bar: 200 nm. (C) Absorbance of Fe3O4, Fe3O4-Ag, Fe3O4-Aushell NPs. (F) EDS mapping of Fe3O4-Aushell NP, scale bar: 20 nm.

Cytotoxicity and Hemolysis

Good biocompatibility is a prerequisite of all inorganic nanoparticles for bio applications. Theoretically, iron is safe for organisms because of its essentiality for physiology homeostasis.33 Au is an inert element, which will rarely react with other chemicals.34 Hence, the synthesized Fe3O4-Aushell NPs were considered to be of good biocompatibility. The results from the cytotoxicity assay confirmed our expectation as shown in Figure 2A. When the cells were co-incubated with Fe3O4-Aushell NPs no higher than 20 ppm (5, 10, and 20 ppm), their viability was not different from the control cells. With the concentrations increasing, the viability decreased gradually. However, the cells still showed a high viability of 87% at 80 ppm, indicating good biosafety and paving the way for further bio application. The result of hemolysis analysis also confirmed the good biocompatibility of synthesized NPs (Figure 2B). As nanoparticles’ concentration increased, hemolysis rate of RBCs rose from 1.6 ± 0.15% (25 ppm) to 3.0 ± 0.26% (50 ppm). However, even incubated with 100 ppm NPs, hemolysis rate of RBC was far below 5% (3.9 ± 0.45%) which was regarded as a safety requirement of NPs, demonstrating the biosafety and competency of Fe3O4-Aushell NPs for further applications.35

Figure 2 (A) Relative cell viability of 4T1 cells incubated with different concentrations of Fe3O4-Aushell for 24 h. (B) Hemolysis rate of RBC incubated with Fe3O4-Aushell dispersions in PBS of various concentrations.

MRI and PTT in vitro

r2 is believed to be the key to measuring the enhanced performance of the MRI contrast agents in T2WI.36 According to the outer-sphere theory, the r2 of superparamagnetic NPs is positively correlated with their saturation magnetization.37 Therefore, we first recorded the hysteresis loop of the Fe3O4-Aushell to evaluate their MRI enhancing performance. As shown in Figure 3A, the saturation magnetization of Fe3O4-Aushell was 36 emu/g (mass measured from Fe3O4), which was consistent with the results from previous studies.38 When the external magnetic field was absent, there was no residual magnetism, indicating their superparamagnetism, which is extremely essential to the in vivo bioapplications of magnetic NPs.

Figure 3 (A) Hysteresis loop of Fe3O4-Aushell nanoparticles. (B) MRI T2WI images of Fe3O4-Aushell NPs with different PEG coating. (C) Linear regression of relaxation rate over different Fe concentrations of Fe3O4-Aushell NPs with different PEG coating. (D) Calculated r2 of Fe3O4-Aushell NPs with different PEG coating. (E, F) Zeta potential and FTIR spectrum of NPs measured before and after PEG coating.

PEG is commonly used for improving inorganic nanoparticles’ biocompatibility, retention time, and preventing NP aggregates.39–41 The NPs with more PEG usually show better biocompatibility and longer retention times.39,42,43 However, too much PEG on the nanoparticles will hinder the adjacent water accessibility, which may dampen its contrast efficiency. To obtain Fe3O4-Aushell with enough r2 and excellent biosafety simultaneously, we coated Fe3O4-Aushell NPs with different amounts of PEG and studied them with MRI. To prove the successful modification of Fe3O4-Aushell NPs with PEG, zeta potential analyses and FT-IR spectrum were applied. As Figure 3E showed, zeta potential of NPs changed from −23.75 ± 1.37 mV to −13.93 ± 0.55 mV after incubated with PEG. The zeta potential change could be contributed from the partly replacement of negatively charged citrate ligand on the surface of Fe3O4-Aushell Janus NPs by PEG which was of electroneutrality, indirectly proving the successful modification of Fe3O4-Aushell nanoparticles by PEG.44 FTIR showed an intense peak at 1113 cm−1 after PEG incubation, which matches the characteristic C-O stretching vibration and confirms the successful linking of PEG on the NPs decisively (Figure 3F). The T2WI images of NPs were illustrated as Figure 3B. Due to NPs’ shortening T2 ability, the images became darker as the concentration increased. However, Fe3O4-Aushell-PEG10, Fe3O4-Aushell-PEG40, Fe3O4-Aushell-PEG160 manifested a darker appearance compared with Fe3O4-Aushell-PEG0, Fe3O4-Aushell-PEG640 at the same concentration. To study this observation quantitatively, the T2 from each sample was used to calculate r2 through linear regression. As Figure 3C and D shows, when the PEG amount increased, r2 of MNPs gradually increased from 170 mM−1s−1 to 216 mM−1s−1. When the incubation ratio reached 640 (Fe3O4-Aushell-PEG640), r2 decreased to 190 mM−1s−1. This phenomenon may result from that PEG can improve the accessibility of surrounding water molecules to Fe3O4 NPs and result in a notable elevation of r2. However, too many PEGs attaching to Fe3O4-Aushell surface will not only impede water accessibility but also increase the NPs’ size, leading to a decrease in the r2 of Fe3O4-Aushell NPs. These results suggested that Fe3O4-Aushell-PEG160 was optimal for MR T2 imaging and was consequently used in the following experiment.

The killing effect of PTT is mainly derived from the elevated temperature induced by laser irradiation.45 Therefore, the photothermal efficiency of a certain PTA under various experimental conditions is essential for optimizing the PTT strategy. As shown in Figure 4A, after 5 min of exposure, different powered lasers led to different temperature rises of Fe3O4-Aushell-PEG160 at a fixed concentration (Fe: 30 ppm, Au: 70 ppm). The dispersion reached 75°C with a ΔT of nearly 50°C at 2.0 W/cm2, while the dispersion showed much lower temperatures of 64.6°C and 52.5°C after 1.2 W/cm2 and 0.65 W/cm2 laser irradiation. When the laser power was fixed at 1.2 W/cm2, the Fe3O4-Aushell-PEG160 dispersions showed different temperatures as shown in Figure 4B. NPs with higher concentration (Fe: 30 ppm, Au: 70 ppm) heated the water more efficiently than the lower ones (Fe: 10 ppm, Au: 23 ppm) (64.6°C VS 60.1°C). By contrast, the DI water containing Fe3O4 NPs and no NPs were not heated and showed no temperature rise. Because cells can be efficiently killed at 50°C,46 the concentration of Fe3O4-Aushell-PEG160 was fixed at 100 ppm (Fe + Au), and the laser power was fixed at 0.65 W/cm2 for the following study to minimize non-specific damage to PTT. Subsequently, in vitro PTT was performed to verify this assumption. After 5 min of irradiation and overnight incubation, all the cells were analyzed by CCK-8 assay for viability evaluation and stained with Calcein-AM/PI for fluorescence imaging. As Figure 4C shows, Fe3O4-Aushell-PEG160 NPs had ignorable influence in cell viability (93.5 ± 6.9%). The viability of cells from laser+PBS group was slightly lowered (84 ± 8.0%) due to the minor temperature rising after laser irradiation. In contrast, only few cells (5.1 ± 0.8%) were alive after treatment in the NPs+laser group, which indicated a drastic PTT effect of Fe3O4-Aushell-PEG160. Fluorescence microscope imaging showed a consistent result. Dead cells were hardly seen in the untreated group (Figure 4D), while some sporadic red dots were observed in the Fe3O4-Aushell-PEG160 group (Figure 4E) and laser group (Figure 4F). In contrast, the dead cells were present in every view of the Fe3O4-Aushell-PEG160+laser group (Figure 4G), demonstrating the good PTT effect of Fe3O4-Aushell-PEG160 in vitro.

Figure 4 (A) Photothermal curves of Fe3O4-Aushell NPs irradiated under 808 nm laser of 0.65 W/cm2, 1.2 W/cm2, 2.0 W/cm2. (B) Photothermal curves of H2O, Fe3O4, Fe3O4-Aushell (Au: 70 ppm) and Fe3O4-Aushell (Au: 23 ppm) under irradiation 1.2 W/cm2 808 nm laser. (C) Relative cell viability of 4T1 cells from different groups. Fluorescence microscope images of Calcein-AM/PI stained cells from groups of (D) untreated, (E) Fe3O4-Aushell-PEG160, (F) laser and (G) Fe3O4-Aushell-PEG160+laser. Green means live cells and red indicate dead cells, scale bar: 100 μm.

MRI and PTT in vivo

To further determine the potential of Fe3O4-Aushell-PEG as MRI CAs, Fe3O4-Aushell-PEG160 NPs were administered intratumorally and then imaged in a 3 T MRI scanner. As shown in Figure 5A and B, xenograft tumors exhibited a signal intensity of 1487 ± 249 in T2WI before NPs injection. After intratumoral injection (Figure 5C and D), the signal intensity rapidly decreased to 586 ± 233. This significant signal intensity drop of 60.6% in T2WI suggested that Fe3O4-Aushell-PEG160 NPs are qualified to serve as MRI T2 contrast agent.

Figure 5 T2WI images of tumor before (A) and after (C) Fe3O4-Aushell injection, signal intensity rapidly decreased from 1487 ± 249 to 586 ± 233. Pseudo color was added by ImageJ (B and D).

Preferred PTT requires that the cancer cells be effectively killed and adjacent normal tissue damage was avoided. Accordingly, the tumor should be irradiated under the laser with a lower power in a shorter time period with the help of PTAs with superb PCE.21,22 Herein, all the tumors were irradiated under 0.65 W/cm2 laser for 5 min. The tumor temperature of the PBS+laser group rose to only 43.7°C, which is far below the killing threshold (50°C). In contrast, the tumor temperature of the Fe3O4-Aushell-PEG+laser group rose rapidly, reaching 50°C at 2.5 min and finally reaching 54.6°C at 5 min (Figure 6B and D). After treatment, tumors of Fe3O4-Aushell-PEG+laser group shrink rapidly, and the relative volume dropped from 1.0 at the beginning to 0.55 in the 2nd day to 0.24 in the 3rd day (Figure 6C). Six days after treatment, there was no visible tumor except a black scar. The tumors in the other groups (untreated, laser+PBS, Fe3O4-Aushell-PEG) grew fast. The relative volume of these tumors (untreated, laser+PBS, Fe3O4-Aushell-PEG) reached 3.63, 3.82, 3.85 in the 8th day and rose to 5.79, 5.05, 5.41 in the 14th day. In the H&E photos, cells of the laser, NPs, and untreated groups all showed regular morphology. There were no obvious necrotic or inflammatory cells that could be seen in any view of the section (Figure 6EG). The biosafety of Fe3O4-Aushell-PEG160 was also evaluated. As shown in Figure 6A and H, the mice neither lost weight nor showed abnormal behavior. As expected, organs from mice treated with Fe3O4-Aushell-PEG showed no observable inflammation or other changes (Figure 6I). Based on these promising results, it is reasonable to conclude that Fe3O4-Aushell-PEG NPs are an effective PTA for BC PTT in vivo.

Figure 6 (A) Mice weight, (C) mice tumor volume and (H) tumor photograph at different time points after treatment. Mice tumor’s thermal images (D) and temperature change (B) under 0.65 W/cm2 laser irradiation for 5 min. (EG) H&E staining of tumor from laser, NPs and untreated groups (scale bar: 50 μm). (I) H&E staining of vital organs harvested from different group mice at 14th day after treatment (scale bar: 50 μm).

Conclusion

In conclusion, to address the diagnosis and therapeutic straits in BC, we successfully synthesized Fe3O4-Aushell NPs which had uniform Janus-like morphology and explored its theranostic potential. After synthesis and characterization, Fe3O4-Aushell showed a favorable biocompatibility from the cell viability and hemolysis test, which is prerequisite for bioapplications. PEG linking did not harm the nanoparticle’s MRI performance. In the contrast, enhanced r2 could be achieved by a suitable incubation ratio. In vitro measurements verified the r2 and PTT efficacy of Fe3O4-Aushell-PEG, which fulfills the demand for simultaneously effective MR imaging and PTT treatment. Based on this promising result, Fe3O4-Aushell-PEG NPs were injected in 4T1 xenograft models and then were MR imaged and irradiated under 808 nm laser. Obvious T2 enhancement and tumor killing effect were observed, while no noticeable side effect was seen until the end of experiment. Given the exciting outcomes, Fe3O4-Aushell-PEG manifested its competences in MR imaging and PTT treatment, and thus provides a promising alternative for future BC theranostics.

Acknowledgments

The research was supported by the Research Project of Army Medical University (2018XYY12), Miaopu Foundation of Army Medical University (No. 2019R059), Natural Science Foundation of Chongqing (No. cstc2018jcyjAX0321, cstc2017jcyjBX0038 and cstc2020jcyj-msxmX1017) and Basic Research Project of Army Medical University (2021-2018-070).

Disclosure

The authors report no conflicts of interest in this work.

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