Optimization and Design of Magnetic Ferrite Nanoparticles with

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Optimization and Design of Magnetic Ferrite Nanoparticles with Uniform Tumor Distribution for Highly Sensitive MRI/MPI Performance and Improved Magnetic Hyperthermia Therapy Yang Du, Xiaoli Liu, Qian Liang, Xing-Jie Liang, and Jie Tian Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00630 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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Optimization and Design of Magnetic Ferrite Nanoparticles with Uniform Tumor Distribution for Highly Sensitive MRI/MPI Performance and Improved Magnetic Hyperthermia Therapy Yang Du,#,†,‡ Xiaoli Liu,#,‡,§ Qian Liang,#,†,‡ Xing-Jie Liang,*,‡,§ Jie Tian,*,†,‡,║,┴ †CAS

Key Laboratory of Molecular Imaging, The State Key Laboratory of

Management and Control for Complex Systems, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China ‡University § CAS

of Chinese Academy of Sciences, Beijing 100049, China

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS

Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, No. 11, First North Road, Zhongguancun, Beijing 100190, China ║Beijing

Advanced Innovation Center for Big Data-Based Precision Medicine, School

of Medicine, Beihang University, Beijing, 100190, China ┴Engineering

Research Center of Molecular and Neuro Imaging of Ministry of

Education, School of Life Science and Technology, Xidian University, Xi’an, Shaanxi, 710126, China

# These authors contribute equally to this work.

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ACS Paragon Plus Environment

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Correspondence to Prof. Jie Tian, email: [email protected], phone: +86 10 82618465; Fax: +86 10 62527995; Prof. Xing-Jie Liang, email: [email protected], Phone: +86 10 82545569.

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ABSTRACT Two major technical challenges of magnetic hyperthermia are quantitative assessment of agent distribution during and following administration and achieving uniform heating of the tumor at the desired temperature without damaging the surrounding tissues. In this study, we developed a multimodal MRI/MPI theranostic agent with active biological targeting for improved magnetic hyperthermia therapy (MHT). Firstly, by systematically elucidating the magnetic nanoparticle magnetic characteristics and the magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) signal enhancement effects, which are based on the magnetic anisotropy, size, and type of nanoparticles, we found that 18 nm iron oxide NPs (IOs) could be used as superior nanocrystallines for high performance of MRI/MPI contrast agents in vitro. To improve the delivery uniformity, we then targeted tumors with the 18 nm IOs using a tumor targeting peptide, CREKA. Both MRI and MPI signals showed that the targeting agent improves the intratumoral delivery uniformity of nanoparticles in a 4T1 orthotopic mouse breast cancer model. Lastly, the in vivo antitumor MHT effect was evaluated, and the data showed that the improved targeting and delivery uniformity enables more effective magnetic hyperthermia cancer ablation than otherwise identical, non-targeting IOs. This pre-clinical study of image-guided MHT using cancer-targeting IOs and a novel MPI system paves the way for new MHT strategies.

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KEYWORDS Magnetic hyperthermia therapy (MHT), magnetic resonance imaging (MRI), magnetic particle imaging (MPI), magnetic ferrite nanoparticles (MFNPs), theranostics

Magnetic ferrite nanoparticles (MFNPs) with unique size and composition-tunable physical properties have been widely studied as distinctive diagnostic, therapeutic, or theranostic agents for diverse biomedical applications.1-5 Engineered MFNPs represent a cutting-edge multi-functional tool in medicine because they can achieve magnetic resonance imaging (MRI) or magnetic particle imaging (MPI) contrast enhancement as well as therapy by local hyperthermia generated by absorbing energy from an alternating magnetic field (AMF).5-7 Integrative therapeutic and diagnostic applications, such as MRI-guided cell replacement therapy and MRI-based imaging of cancer-specific gene delivery, have emerged with the use of MFNPs.8 Recently, development of MFNPs as a highly sensitive imaging contrast agent and an efficient hyperthermia agent for the early cancer diagnosis, prevention, and treatment has gained much interest.9-11 MFNPs-mediated magnetic hyperthermia is expected to be a new breakthrough in cancer treatment.12 Based on the mechanism that ferromagnetic/ferrimagnetic MFNPs administered into the tumor site heat up under AMF, MFNP-mediated hyperthermia can lead to direct killing of local tumors quickly and without affecting the adjacent 4


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healthy tissues.13 Moreover, it can be used as a monotherapy or in combination with radiotherapy and/or chemotherapy to enhance their effectiveness.14 Currently, experimental and clinical studies show that the preferred administration route of MFNPs for MFNP-mediated hyperthermia is by intratumoral injection, allowing for high concentrations of MFNPs in the target site, while avoiding the toxicity to normal tissues commonly associated with systemic delivery.15,

16

To distribute the

administered MFNPs uniformly in solid tumors and generate heat evenly within the tumor, the common strategy is to apply multiple injections of MFNPs at different sites of the tumor. However, the repeated needle penetration may largely increase the risk of local tumor spread through metastasis.17 Therefore, it is particularly attractive to develop magnetic hyperthermia agents for magnetic-medicated hyperthermia with the following traits: (1) simultaneously possessing strong contrast enhancement for both MRI and MPI for image-guided therapy; (2) generating well-distributed and sufficient temperature to effectively kill cancer cells; and (3) requiring the fewest number of intratumoral injections to avoid possible tumor recurrence risk. Taking advantage of high-resolution MRI and highly sensitive MPI combined with MFNPs yields more accurate and feasible image-guided therapy. The emergence of MPI offers a new approach for accurate quantification of nanoparticle (NP) quantity and distribution in solid tumors as part of the hyperthermia treatment planning process. Unlike MRI, which indirectly detects iron oxides, MPI directly images MFNP biodistribution with exceptional sensitivity and specificity. Because MPI 5


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directly detects MFNPs, an MPI image can be used to predict the specific absorption rate (SAR) dose to ensure the safety and efficacy of magnetic hyperthermia treatment (MHT). MPI images resemble nuclear medicine images, and this technique enables the visualization of only tracer but not tissue. MPI was first introduced by Gleich and Weizenecker in 200518, and has been demonstrated across a wide array of promising biomedical applications, including angiography, stem cell tracking, lung ventilation, cancer imaging, gut bleed detection, and hyperthermia therapy.19-37 Previous studies have focused on designing and optimizing the size, composition, and structure of MFNPs to maximize the performance of MRI, magnetic hyperthermia, or both.9, 11, 38, 39

However, modulating MFNPs for highly sensitive MRI/MPI contrast agent and an

efficient hyperthermia agent for the early cancer diagnosis, prevention, and treatment is still challenging. In addition to the imaging guidance, it is also possible to biologically target tumors with NPs. In particular, the coating of MFNPs has a substantial effect on their behavior in vivo. Different coatings can make a NP hide from the immune system 40-42,

effectively label cells 42, 43, and even selectively target specific cell types such as

cancer cells.44-46 One particular approach that has garnered much interest is peptide targeting of magnetic particles. In peptide targeting, a NP is studded with peptide sequences known to bind to proteins that are frequently overexpressed in cancers. For example,

previous

studies

have

shown

that

the

penta-peptide

CREKA

(Cys-Arg-Glu-Lys-Ala) selectively binds to fibrin–fibronectin complexes that are 6


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overexpressed in breast cancer cells and interstitial cells.47 By attaching CREKA to the outside of imaging agents such as NPs, it can enhance the delivery to the tumor site.47 In the present study, we aimed to improve the uniformity of hyperthermia in solid tumors by combining two concepts, MRI and MPI guidance with a biological tumor-targeting peptide. We developed CREKA-modified MFNPs to harness their advantages and the potential of dual-mode MRI/MPI and magnetic hyperthermia for precision imaging and cancer therapy (Figure 1). Namely, fibrin–fibronectin complexes are reported abundantly expressed in breast cancer cells and interstitial cells.47 The interaction between fibronectin and its ligand CREKA has been proposed for the development of antibody-targeted vehicles for specific and effective delivery of imaging agents and therapeutic drugs to the tumor site.47 First, highly monodisperse polyethylene glycol-coated manganese iron oxide NPs (MIOs) and iron oxide NPs (IOs), 8 nm or 18 nm in diameter, have been prepared to systematically investigate the effects of their size and composition on the imaging efficacy of MPI and MRI. The sizes of both MIO and IO cores were 8 nm and 18 nm in diameter, indicating their superparamagnetism. After optimization, 18 nm IOs were chosen as a superior nanocrystalline for high performance of MRI/MPI contrast agents. We further studied the efficiency of MPI, MRI, and magnetic hyperthermia mediated by 18 nm IOs, before and after conjugation with CREKA, on a mouse orthotopic 4T1-Luc breast cancer model. The goal of this study was to gain insight into the 7


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intricate interplay of particle size, composition, and magnetic properties on the effect of the performance of dual-mode MPI/MRI, and as well as provide a general strategy for modifying the surface of MFNPs for inducing even temperature, which would allow for high-performance hyperthermia in biomedical applications. To the best of our knowledge, this work represents the first example of the modulation of MFNPs in dual-mode MRI/MPI and improving the efficiency of magnetic hyperthermia by generating uniform temperature inside a tumor, all of which enable more efficient MPI- and MRI-guided magnetic hyperthermia in vivo. Monodispersed IOs and MIOs of different sizes were fabricated by means of the well-established high-temperature thermal decomposition of metal acetylacetonate in the presence of oleic acid as a surfactant. The size of the IOs and MIOs was tuned by controlling the concentration of metal acetylacetonate and the growth temperature. As the resulting hydrophobic ligand-coated NPs are insoluble in aqueous media, we exchanged the ligand with DHCA. The DHCA-coated IOs and MIOs have high monodispersity. The IOs and MIOs were highly uniform in morphology and size, and no appreciable agglomeration was observed (Figure 2a–d). The average sizes of IOs and MIOs were 8 nm and 18 nm. TEM images clearly showed IOs with a mean diameter of 8.0 ± 1.4 nm (Figure 2a) and 18.0 ± 2.3 nm (Figure 2b) and MIOs with a mean diameter of 8.0 ± 1.8 nm (Figure 2c) and 18.0 ± 2.7 nm (Figure 2d). The composition of the so-synthesized MIOs was analyzed by energy-dispersive X-ray spectrum (EDS). From the EDS (Figure 2e-f), we observed that both Mn and Fe were 8


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present in the MIOs, and the Fe/Mn atomic ratio was around 2.10 for both the 8 nm and the 18 nm samples, which was further confirmed by inductively coupled plasma (ICP) elemental analysis (Fe/Mn ratio, 2.08) (Table 1). Since the colloidal stability of the magnetic NPs in biomedical applications plays an important role, the stability of the modified IOs and MIOs of different sizes was examined using dynamic light scattering (DLS). DLS measurements were carried out to evaluate the hydrodynamic size of the modified IOs and MIOs. The measured hydrodynamic sizes in aqueous solution were slightly larger than those obtained by TEM observation, which verified the existence of the polymer coating on the NPs: 8-IOs and 8-MIOs had a hydrodynamic size of 13.6 nm and 12.4 nm, respectively, which is considerably smaller than that of the 18-IOs (33.1 nm) and 18-MIOs (31.9 nm), respectively (Figure 3a-b). The surface charge properties of samples were studied by measuring the zeta potentials in DI water (Figure 3c). All samples are negatively charged. These negative surface charges were due to the dissociation of the carboxyl groups at the chain end. Testing the stability of samples of different sizes was conducted in 0.9% NaCl solution and cell culture medium containing 10% FBS, simulating in vivo blood plasma. The hydrodynamic size of all samples determined by DLS did not change significantly upon incubation in these media at room temperature for 24 days (Figure 3d-f), further verifying the excellent colloidal stability of these samples as theranostic agents under physiological conditions. These characterizations clearly suggested that monodispersed IOs and MIOs of different sizes were 9


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successfully synthesized, and the DHCA-modified samples possessed a narrow size distribution and excellent colloidal stability that ensured the feasibility of model study of determining the effects of size and composition optimization in MPI and MRI measurements. Magnetic measurements were carried out using a vibrating sample magnetometer (VSM). M-H curves of all samples taken at 300 K are shown in Figure 4a. None of the samples showed remanence or coercivity, indicating superparamagnetic behavior at room temperature.9 For both IOs and MIOs, the reduced saturation magnetization (MS) with decreasing size is observed due to the finite size effect.9 The MS of 8 nm IOs was 35.8 emu/g, which is lower than that of MIOs of the same size (43 emu/g). Similarly, the MS of 18-IOs (60.4 emu/g) increases to 75 emu/g for MIOs, which may have resulted from the reduced magnetocrystalline anisotropy of MIOs. In order to assess the MRI T2 relaxivity and MPI properties of IOs and MIOs, we performed in vitro MRI and MPI measurements. We prepared five samples of different diameters and compositions, Vivotrax, 8-IOs, 8-MIOs, 18-IOs, and 18-MIOs, at concentrations of 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, and 0.0625 mM confirmed by ICP-MS to investigate the correlation of MRI and MPI signals. Figure 4b-c shows T2-weighted MRI of NPs with respect to sample concentration. The T2-weighted MR images of all the samples tend to become darker with increasing sample concentration (Figure 4b), showing that all samples can effectively reduce the spin-spin relaxation time of water protons as T2 negative contrast agents.48 The 10


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comparison of r2 relaxivity of samples is presented in Figure 4c. The r2 of Vivotrax, 8-IOs, 8-MIOs, 18-IOs, and 18-MIOs was 171.9, 124.8, 162.1, 241.3, and 195.4 mM-1s-1, respectively. Larger IOs or MIOs had higher r2 values, which can be attributed to the higher MS for larger-sized samples. The 18 nm samples induced an apparent contrast enhancement under the same MRI parameters, regardless of composition. Moreover, the 18-IOs had an r2 relaxivity of 241.3 mM-1s-1, which is 40% greater than that of the commercial Vivotrax (171.9 mM-1s-1) (Figure 4c). The MPI images and the linear regression results are shown in Figure 4d-4e. Interestingly, we found that most samples had MPI signals except the 18-MIOs at the above concentrations (Figure 4d). At the same concentration, the observed MPI signal intensity was in the following order: 18-IOs > 8-IOs > Vivotrax > 8-MIOs. 18-IOs and 8-IOs had an MPI signal of 134.2 and 94.73, which is 2.68 and 1.89 times larger than that of commercially available Vivotrax (50.11), respectively (Figure 4e). Although MIOs showed higher MS and reduced magnetocrystalline anisotropy compared to the IOs of the same core size, which is beneficial to the performance of T2-weighted MRI and magnetic hyperthermia, MIOs have extremely low MPI signal. In general, to achieve dual-modal MRI/MPI imaging in vivo, we found that 18-IOs are the best candidate because of the superior MPI and MRI signal (Figure 4f). From our in vitro results with MPI and MRI, we decided to concentrate our efforts on the functionalizing of the 18-IOs, which showed both the highest sensitivity in MRI and MPI. We applied the CREKA peptide with a standard functionalization 11


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process to the 18-IOs. The resultant 18-IOs provided abundant carboxyl groups (– COOH) on their surface, facilitating the subsequent bioconjugation process. The hydrophilic

18-IOs

were

conjugated

with

CREKA

through

a

standard

EDC/NHS-coupling reaction. The measured zeta potential of the IOs was −10.34 mV, which is attributed to the abundant surface carboxyl groups. After conjugation with CREKA, the zeta potential changed to -4.68 mV (Figure S1a), confirming successful CREKA conjugation to their surface. In addition, quantitative analysis of the amount of CREKA loaded onto the surface of IOs was calculated to be around 7%, using thermogravimetric analysis data (Figure S1b). We further tested the 18-IOs for the targeted MPI and MRI in vivo on an orthotopic 4T1 breast tumor-bearing mouse model. In our experiment, 50 μL of 0.5 mg/mL 18-IOs or Vivotrax were injected intratumorally with equal Fe content. MPI and MRI were then performed to longitudinally monitor the signal changes. The synthesized IOs exhibited significantly higher MPI signal relative to the Vivotrax 4 h to 14 days post injection (Figure 5a and 5b). Moreover, we found the MPI signal enhancement of the non-functionalized IOs is confined to the injection site, instead of distributed evenly throughout the tumor. This is apparent in Figure 5a, wherein it can be seen that MPI shows the injection site and little distribution of the NP inside the tumor. To improve the intratumoral delivery, we used our CREKA-conjugated NPs. The CREKA peptide targets fibronectin, which is highly expressed in the breast tumor microenvironment. The fibronectin protein expression in 4T1 tumors was verified 12


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using western blot (Figure S2). The specific targeting of CREKA peptide to the 4T1 breast tumor was examined using in vivo fluorescence imaging. The fluorescence labeled CREKA-CY7 was intravenously injected into the tumor bearing mice, and the data showed that the CREKA can specifically target to the 4T1 tumors (Figure S3). There was no fluorescence signal in the blocking group 2 h pre-injected with excess CREKA peptide. Moreover, the specific binding of CREKA to the fibronectin protein was further confirmed using immunofluorescence staining. The 4T1 breast tumor bearing mice was intravenously injected with CREKA-FITC and the control peptide CERAK-FITC, respectively, and, 4 h later, the tumors were dissected out and preceded for the fibronectin immunofluorescence staining. The data showed that the CREKA was co-expressed with fibronectin in the tumor tissues but not for the control peptide (Figure S4). As seen in Figure 5a, we observed that the targeted IOs-CREKA NPs showed higher and more uniformly distributed MPI signal in the whole tumor region since 4 h post injection compared to the non-conjugated IOs. For example, at day 14 post-injection, the MPI signal intensity of the functionalized IOs-CREKA group was 1.526 ± 0.119-fold higher than that of the non-targeting IOs group and 5.700 ± 0.581-fold higher compared with the Vivotrax group (Figure 5b). To further validate and compare our MPI observation, T2 MRI was performed simultaneously to investigate the biodistribution of Vivotrax, IOs, and IOs-CREKA NPs over time. The intratumoral distribution pattern of these probes as observed by T2 MRI was almost consistent with the MPI signal distribution (Figure 6a). The T2 MRI 13


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showed the darkening of the tumor in the IOs-CREKA group was more uniform compared to that of the IOs and Vivotrax groups (Figure 6a). The T2 darkening signal evidently diffused from the dark spot to the peripheral site over time in the IOs-CREKA treated group. The signal gradually became uniform, indicating that IOs, after modification by CREKA, can be distributed evenly inside the tumor. CREKA can specifically accumulate in breast cancer and specifically bind to the abundant fibrin–fibronectin complexes present in the tumor microenvironment. As such, although the IOs distribution was confined to the injection site at the beginning, IOs modified by CREKA can be specific, effective, and uniformly delivered inside the tumor, which is a result of the driving force between the CREKA and fibrin– fibronectin complexes inside the tumor microenvironment. However, the intrinsic dark signals present in T2-weighted MRI are often confronted with ambiguities derived from MRI artifacts such as bleeding, calcification, or metal deposition.49 To confirm the in vivo MPI and MRI observations, Prussian blue staining was performed after in vivo imaging of the tumor, and the data showed that there was more Fe-positive staining inside the tumor in the IOs-CREKA group than in the IOs and Vivotrax group (Figure 6b). Moreover, the tissue transmission electron microscopy (TEM) was performed to further confirm that the IOs were distributed both inside and outside of the tumor cells (Figure S5). The currently prevailing strategy is the design of MFNPs with switchable T1-T2 dual-modal MRI abilities, allowing them to enhance the accuracy of tumor detection 14


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and effectively eliminate potential false results from mono-mode contrast agents.50-52 However, this design is complicated and uncontrollable, since the switchability relies on microenvironmental changes inside the tumor, such as the pH. The emergence of MPI allowed for more accurate diagnostics. MPI represents an emerging molecular imaging technique that is very promising in tumor detection and guidance of therapy.19-30 Compared with MRI, the superiority of MPI lies in the ability to directly detect MFNP amounts rather than indirectly via hydrogen proton signal attenuation, as in the MRI model. Hence, MPI can predict the SAR from an image of the MFNPs, making sure that the amount of administrated MFNPs for magnetic hyperthermia is appropriate, i.e., both safe and effective. Compared to the gold-standard tracer imaging, MPI has no safety concerns regarding radiation or the half-life of radionuclides for longitudinal imaging. MPI and MRI provide complementary roles in this application. The MPI is demonstrated useful for the rapid assessment of the distribution of MFNPs in tumors, with data comparable to those of the established MRI. Besides the standard MRI, using MPI as a point-of-care device for imaging could be an optimal alternative. In MRI, the high resolution of the technique allows assessing qualitative biodistribution in the tumor. However, due to the inherently non-quantitative negative contrast of MFNPs in MRI, the signal enhancement of the tumor as we have observed with MPI is difficult to visualize with MRI. MPI directly measures the MFNP concentration, and it is expected to be more sensitive than MRI. Moreover, because of the imaging 15


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speed of MPI being similar to that of ultrasound, it can be used for real-time imaging. The IOs-CREKA NPs, combined with the MPI and MRI dual modality imaging, provide a powerful tool for the guidance of MHT. In the future, we believe IOs-CREKA NPs tailored to specifically target the tumor site, coupled with MPI/MRI-guided magnetic hyperthermia, will be able to focus heat onto the area within the field-free point, ensuring that tumor destruction is maximized and damage to normal tissues is prevented. Based on the above results, we then investigated the in vivo antitumor activity of the unmodified IOs, IOs-CREKA NPs, and controls under AMF on 4T1 tumors. The orthotopic 4T1 tumor-bearing mice were intratumorally injected with samples (IOs or IOs-CREKA NPs), and AMF treatment was performed every 2 days for a total of 3 treatments. As monitored by an infrared thermal camera, the tumor temperature of mice injected with IOs-CREKA NPs under AMF exposure quickly rose to ~43 °C (Figure 7a), which can effectively kill tumor cells. BLI was carried out for dynamic and accurate monitoring of the therapeutic effects between the different treatment groups (Figure 7b), and the bioluminescence light signal intensity was further measured and compared (Figure 7c). The tumor BLI signal continued to increase throughout the 15-day observation period in the control, AMF-only, and IOs-only groups. On days 15 post-treatment, bioluminescence light intensity reached (1.293 ± 0.273) × 109,

(1.185

±

0.294) × 109,

and

(0.7890

±

0.056) × 109

photons/s/cm2/sr in the control, AMF-only, and IOs-only groups, respectively. For the 16


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tumor treated with Vivotrax+AMF, the bioluminescence light signal increased at a relatively slower rate compared to the aforementioned 3 groups, and the signal intensity was (0.513 ± 0.052) × 109 photons/s/cm2/sr on day 15. In contrast, IOs-mediated hyperthermia had a better treatment response in the early stage of the treatment, although some residual tumor cells at the edge of the tumor area gradually re-established. We believe this occurred due to that the IOs distribution was mainly confined to the injection site and the heat induced by the IOs was not uniform. Hence, the heat could not cover the whole tumor area. Interestingly, we found that tumors almost disappeared, and there was no bioluminescence light signal detected after IOs-CREKA NPs-mediated hyperthermia treatment, which may be attributed to the uniform distribution of NPs and an even increase of temperature inside the tumor. In addition, the tumor volume was also measured (Figure 7d), and the data were almost consistent with the BLI observation. Rapid tumor growth and greater tumor volume were observed in the control, AMF-only, and IOs-only groups, the tumor volumes on day

15

post-treatment,

respectively.

The

Vivotrax+AMF,

IOs+AMF,

and

IOs-CREKA+AMF treatment slowed down the tumor growth compared with the aforementioned groups. The IOs-CREKA+AMF treatment dramatically inhibited tumor growth, with no tumor recurrence found in this group during the observation period. IOs-CREKA+AMF showed the best MHT effect. We believe that the uniform heat inside the tumor is another critical factor for achieving effective MHT.

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In conclusion, in this study, we have successfully developed theranostic MFNPs, which induce a significant potentiation of magnetic heat by generating uniform temperature inside the tumor and work as a new strategy for cancer thermotherapy. To the best of our knowledge, this work is the first example of MFNPs optimized for dual-mode MRI/MPI image-guided MHT in vivo. We have systemically investigated the diameters and components of MFNPs on the performance of MPI and MRI signal properties, and we found that 18 nm IOs exhibited superior MPI and MRI signal properties compared to other developed MFNPs, including commercially available Vivotrax. To improve delivery uniformity, we targeted tumors using a targeting peptide, CREKA. We showed that the combination of the multimodality imaging (MPI and MRI) and the targeting agent can improve NP delivery uniformity in an orthotopic 4T1 mouse breast tumor model. We then demonstrated that the improved targeting and delivery uniformity enables more effective cancer ablation via magnetic hyperthermia than otherwise identical non-targeting IOs. The findings obtained in this work provide a general strategy for the design and development of novel MRI/MPI-guided hyperthermia agents for advanced Nano biotechnology applications.

ASSOCIATED CONTENT Supporting information Experimental procedures and supporting figures (PDF)

COMPETING INTEREST: 18


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The authors declare no competing financial interest.

ACKNOWLEDGEMENTS General: We would like to thank Patric W Goodwill (Magnetic Insight, Inc.) and Elaine Y.Yu (Department of Bioengineering, University of California) for their kindly support with the MPI experiment and data analysis. Funding: This paper is supported by Ministry of Science and Technology of China under Grant No. 2017YFA0205200; National Natural Science Foundation of China under Grant No. 81871514, 81227901, 81470083; Natural Science Foundation key project 31630027 and 31430031 and NSFC-DFG project 31761133013; Chinese Academy of Sciences under Grant No. GJJSTD20170004 and QYZDJ-SSW-JSC005; Beijing Municipal Science & Technology Commission No. Z161100002616022 ， Z171100000117023. The authors would like to acknowledge the instrumental and technical support of Multi-modal biomedical imaging experimental platform, Institute of Automation, Chinese Academy of Sciences.

ABBREVIATIONS CREKA, Cys-Arg-Glu-Lys-Ala pentapeptide; DHCA, 3,4-dihydroxyhydrocinnamic acid; DLS, dynamic light scattering; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; energy-dispersive X-ray spectrum (EDS); FBS, fetal bovine serum; ICP, inductively coupled plasma; MFNP, magnetic ferrite nanoparticle; MHT, magnetic hyperthermia treatment; MPI, magnetic particle imaging; MRI, magnetic resonance imaging; MIO, manganese iron oxide; NHS, N-hydroxysuccinimide; IO,

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iron oxide; NP, nanoparticle; RPMI, Roswell Park Memorial Institute; MS, saturation magnetization; SAR, specific absorption rate; THF, tetrahydrofuran

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Figure 1. Schematic illustration of the developed CREKA-modified iron oxide (IO) NPs with different sizes and compositions for the potential application on dual-mode MRI/MPI and magnetic hyperthermia therapy for the precision cancer imaging and therapy on 4T1 breast tumor mouse model.

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Figure 2. TEM images of samples of different sizes. (a) 8 nm IOs, (b) 18 nm IOs, (c) 8 nm MIOs, and (d) 18 nm MIOs dispersed in water by DHCA coating. EDS of MIOs: (e) 8 nm and (f) 18 nm.

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Figure 3. Hydrodynamic diameter of (a) IOs and (b) MIOs. (c) Surface charges (zeta-potential) of the corresponding modified samples at neutral pH value (pH = 7). All samples measured as a function of time upon incubation in (d) distilled water, (e) 0.9% NaCl, and (f) DMEM containing 10% FBS.

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Figure 4. In vitro MRI and MPI characterization of IOs and MIOs. (a) Vibrating sample magnetometer (VSM) measurement for all samples. (b) T2-weighted MRI phantom images, and (c) Plot of 1/T2 MRI signal over samples with series concentrations. The slope indicates the specific relaxivity (r2). (d) MPI images of all samples at series concentrations. (e) Plot of MPI signals of all samples. (f) The comparison of r2 values and the slope of MPI signal.

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Figure 5. MPI image of NP distribution in 4T1 orthotopic breast tumor mouse model (n = 3). (A) In vivo dynamic MPI of mice intratumorally injected with Vivotrax, IOs, and IOs-CREKA NPs, respectively. (B) MPI signal calculation and comparison between groups. *** p < 0.001, IOs-CREKA NPs or IOs compared with Vivotrax group; # p < 0.05, IOs vs. IO-CREKA NPs.

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Figure 6. (a) In vivo MRI of Vivotrax, IOs, and IOs-CREKA NPs in an orthotopic breast tumor mouse model. MRI was performed at different time points to monitor the tracer dynamics. (b) Prussian blue staining was carried out to confirm the Fe distribution at the tumor sites. Scale bar, 50 µm.

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Figure 7. Assessment of the therapeutic efficacy of the MHT with different treatments in a 4T1 orthotopic tumor-bearing mouse model (n = 5). (a) The increase in the temperature induced by the MHT with different NPs treatment. (b) The therapeutic efficacy of the magnetic hyperthermia dynamically monitored by BLI in different groups. The calculation and comparison of the tumor (c) bioluminescence intensity and (d) tumor volume changes. * p < 0.05; ** p < 0.01; *** p < 0.001.

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Table 1. Summary of parameters for samples. Diameter (from Hydrodynamic TEM observation) Diameter (DLS)

Ms (VSM)

Composition (EDS and ICP)

8-IOs

8.0 ± 1.4 nm

13.6 ± 2.4 nm

35.8

Fe3O4

18-IOs

18.0 ± 2.3 nm

33.1 ± 4.5 nm

60.4

Fe3O4

8-MIOs

8.0 ± 1.8 nm

12.4 ± 1.5 nm

43.0

Fe/Mn ratio of 2.08

18-MIOs

18.0 ± 2.7 nm

31.9 ± 4.8 nm

75.0

Fe/Mn ratio of 2.08

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For Table of Contents Only

Schematic illustration of the optimized CREKA-modified iron oxide (IO) NPs with uniform tumor distribution for the potential application on dual-mode MRI/MPI and improved magnetic hyperthermia therapy for the precision cancer imaging and therapy on 4T1 breast tumor mouse model.

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Figure 1. Schematic illustration of the developed CREKA-modified iron oxide (IO) NPs with different sizes and compositions for the potential application on dual-mode MRI/MPI and magnetic hyperthermia therapy for the precision cancer imaging and therapy on 4T1 breast tumor mouse model. 140x106mm (300 x 300 DPI)


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Figure 2. TEM images of samples of different sizes. (a) 8 nm IOs, (b) 18 nm IOs, (c) 8 nm MIOs, and (d) 18 nm MIOs dispersed in water by DHCA coating. EDS of MIOs: (e) 8 nm and (f) 18 nm. 119x167mm (300 x 300 DPI)


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Figure 3. Hydrodynamic diameter of (a) IOs and (b) MIOs. (c) Surface charges (zeta-potential) of the corresponding modified samples at neutral pH value (pH = 7). All samples measured as a function of time upon incubation in (d) distilled water, (e) 0.9% NaCl, and (f) DMEM containing 10% FBS. 140x83mm (300 x 300 DPI)


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Figure 4. In vitro MRI and MPI characterization of IOs and MIOs. (a) Vibrating sample magnetometer (VSM) measurement for all samples. (b) T2-weighted MRI phantom images, and (c) Plot of 1/T2 MRI signal over samples with series concentrations. The slope indicates the specific relaxivity (r2). (d) MPI images of all samples at series concentrations. (e) Plot of MPI signals of all samples. (f) The comparison of r2 values and the slope of MPI signal.


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Figure 5. MPI image of NP distribution in 4T1 orthotopic breast tumor mouse model (n = 3). (A) In vivo dynamic MPI of mice intratumorally injected with Vivotrax, IOs, and IOs-CREKA NPs, respectively. (B) MPI signal calculation and comparison between groups. *** p < 0.001, IOs-CREKA NPs or IOs compared with Vivotrax group; # p < 0.05, IOs vs. IO-CREKA NPs.


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Figure 6. (a) In vivo MRI of Vivotrax, IOs, and IOs-CREKA NPs in an orthotopic breast tumor mouse model. MRI was performed at different time points to monitor the tracer dynamics. (b) Prussian blue staining was carried out to confirm the Fe distribution at the tumor sites. Scale bar, 50 µm. 119x202mm (300 x 300 DPI)


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Figure 7. Assessment of the therapeutic efficacy of the MHT with different treatments in a 4T1 orthotopic tumor-bearing mouse model (n = 5). (a) The increase in the temperature induced by the MHT with different NPs treatment. (b) The therapeutic efficacy of the magnetic hyperthermia dynamically monitored by BLI in different groups. The calculation and comparison of the tumor (c) bioluminescence intensity and (d) tumor volume changes. * p < 0.05; ** p < 0.01; *** p < 0.001.


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Schematic illustration of the optimized CREKA-modified iron oxide (IO) NPs with uniform tumor distribution for the potential application on dual-mode MRI/MPI and improved magnetic hyperthermia therapy for the precision cancer imaging and therapy on 4T1 breast tumor mouse model. 47x34mm (300 x 300 DPI)


Optimization and Design of Magnetic Ferrite Nanoparticles with

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