Fe3O4 Nanoparticles and Paraffin Wax as Phase-Change Materials Embedded in Polymer Matrixes for Temperature-Controlled Magnetic Hyperthermia

Moshe Cohen-Erner,*   Raz Khandadash,*†  Raphael Hof,  Ofer Shalev,  Adam Antebi,  Arnoldo Cyjon,  Dian Kanakov, Abraham Nyska, Glenwood Goss, || John Hilton, || and, Dan Peer §

† New-Phase Nanotechnology Ltd., Petah Tikvah, 4934829, Israel

‡ Oncology Department, Shamir Medical Center, Zerifin 70300, Israel

∇ Internal Noninfectious Diseases Department, Trakia University, 6000 Stara Zagora, Bulgaria

Consultant in Toxicologic Pathology, Sackler School of Medicine, Tel Aviv University 69978, Israel

|| Division of Medical Oncology, University of Ottawa, ON K1N 6N5, Canada

§ Laboratory of Precision Nanomedicine, Shmunis School for Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel


magnetic hyperthermia, nanoclusters, phase change material, superparamagnetic iron oxide nanoparticles, specific absorption rate, theranostic, magnetic resonance imaging


Over the last two decades, magnetic hyperthermia (MH) has been recognized as a promising concept for efficient cancer treatment. Recently, it has been receiving further growing attention due to its procedure simplicity, noninvasive nature and effective solid tumor heating with minimal damage to healthy surrounding normal tissues. In this paper, we report about the development of theranostic superparamagnetic iron-oxide nanoclusters, demonstrating efficient heating in hyperthermia and good response under magnetic resonance (MR) scan. Multiple cores of 25 ± 2 nm superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs) and paraffin wax based on 24-hydrocarbon chains (tetracosane), used as phase change material (PCM), were co-encapsulated in an interior core of self-assembled PEO-PPO-PEO polymer and subsequently covalently coated by 20 kDa branched polyethylene glycol (PEG), resulting in 135 ± 10 nm hydrodynamic diameter nanoclusters. The synthesized nanoclusters were found to have good stability in phosphate-buffered saline. The physicochemical and magnetic properties of the nanoclusters exhibit an efficient magnetic-to-thermal energy conversion with self-regulation of the hyperthermia temperature. Under irradiation to an alternating magnetic field (AMF) of 33 kA/m at frequency of 300 kHz, the nanoclusters demonstrate specific absorption rate (SAR) of 475 ± 17 W/g. The nanoclusters also exhibit high transverse relaxivity of 68 (mM s)-1, at 1.5T MRI. In preclinical studies, nanoclusters were intravenously injected to mice bearing 4T1 triple negative breast carcinoma lung metastases.  Mice were irradiated by AMF, to demonstrate antitumor efficacy, with 66% reduction in the number of metastases, which pave the route for application of effective hyperthermia treatment for metastatic cancer model.


Lung cancer is one of the most aggressive types of cancer worldwide.(1) The aggressive invasiveness characteristics of lung cancer contribute to short-term survival of patients and presents a therapeutic challenge. Significant advances have been made in cancer therapy, although selective elimination of cancer cells remains challenging. Therefore, it is vital that effective methods for selective treatment with improved safety and efficacy are to be developed, to enhance conventional methods that will prolong survival, control symptoms and improve patient’s quality of life.

One of the promising therapeutic technologies for cancer treatment is hyperthermia. Heat exposure of malignant cancer cells to temperatures between 42 to 46oC for at least 30 minutes, leads to apoptosis in vivo and in vitro, rendering it as a preferable strategy for tumor eradication compared to necrosis mechanism caused at temperatures above 46oC.‎(1) Moreover, at this temperature range, tumor cells are more sensitive to heat than normal cells. This phenomenon is related to the vasculature architecture complexity of solid tumors, which includes hypoxic and low-pH regions.‎(2) 

Magnetic hyperthermia (MH) and photothermal therapy (PTT) techniques are widely explored innovative cancer hyperthermia therapies which elevate the local malignant tissue temperature by heat generation, using magnetic and optical characteristics of distinct or composite nanoentities. These nanoentities are located within, or stay close to the tumor lesion, and induce cell death upon exposure to non-invasive external force such as magnetic field or NIR-IR radiation.‎(3) Unlike the PTT used to treat superficial solid tumors which are up to the depth of 3-4 mm from the skin surface,‎(4) MH can deal with deep-seated tumors and metastases due to excellent tissue penetration ability of alternating magnetic field (AMF).(5) Clinical research developments demonstrated the feasibility of MH, using 12 nm superparamagnetic iron-oxide (Fe3O4) nanoparticles (SPIONs) activated by AMF of 2.5-18 kA/m at a frequency of 100 kHz, as a safe and effective stand-alone therapy for prostate carcinoma(6) and glioblastoma multiforme (GBM).‎(7) These results were further corroborated by phase 2 clinical trials, examining MH technique as GBM adjuvant therapy.‎(8) Despite the main advantage of this technique to easily treat localized and superficial, or easily accessible tumors without damaging healthy tissue, this approach is limited to specific types of cancer and not applicable to a cancer in advanced stage. Furthermore, direct tumor injection results in a non-homogeneous SPIONs distribution within the tumor, which makes the complete regression of the tumor difficult and entails a high risk of metastases development, unsuitable for local conventional MH treatment. These drawbacks can be overcome by systemic delivery of magnetic nanoparticles (MNPs) through intravenous (IV) administration. It allows the technique to be employed to tumors in various sizes, locations and distribution.‎(9)

Previous reports suggest that the relatively moderate heating efficiency of SPIONs, indicated by specific absorption rate (SAR) parameter, combined with their low tumor accumulation, present some limitations on the clinical realization in systemically delivered MH.9In addition, in order to minimize potential side effects, the dosage of SPIONs administered during the MH, should be kept to a minimum‎(10). Ideally, one would desire to increase the SAR as much as possible in order to achieve an efficient hyperthermia treatment with low amount of SPIONs. In the last decades, different strategies have been proposed in order to synthesize SPIONs that possess enhanced SAR values while retaining their inherent benefits (e.g., biocompatibility). These schemes include tuning the particle size, magnetic anisotropy, coating and magnetization,‎(11),‎(12) which are the parameters identified as the determinants of both, the static and dynamic behavior of single-domain nanoparticles.‎‎(13) At present, most existing MNPs require a high frequency or high AMF intensity to deliver an adequate thermal dose to the tumor while there are clinical upper limits for the magnetic field intensity and frequency values the human body can withstand.‎(14) A new type of colloidal clusters composed of multicore MNP have been developed to overcome these aforementioned drawbacks and produce higher heating rates for the same concentration of MNP or, alternatively, obtain similar temperature rises with smaller MNPs concentrations. This phenomenon was ascribed to “spin glass” dynamics of the magnetic moments within a cluster, strongly correlated by the exchange interaction influencing the higher magnetic susceptibility at low magnetic field strength.‎(14) This has significance for MH applications, where the SAR in the currently studied materials has not been sufficiently high at reasonable concentrations to target small tumors.‎(15) Thus, the ability to achieve temperature rise at lower concentrations allows for novel means of MNPs delivery to the tumor site by IV administration. Moreover, the translational research of MH with systematically administrated NPs is limited because of lack of control mechanism over temperature rise of the NPs, in both healthy and cancer tissues, when exposed to AMF.‎(16) 

Recently, we proposed a new method to control and regulate the temperature of NPs in tissues by using tetracosane (paraffin wax) as phase change material (PCM), incorporated in the NPs structure. (17)  The tetracosane was selected as a PCM to provide high latent energy and high specific heat capacity, in both liquid and solid phases, with phase change temperature required by hyperthermia (47–53 oC), and biocompatibility with biological tissue. (17)  

In this paper, we present a MH scheme which is based on temperature-controlled SPION nanoclusters at size of 135 ± 10 nm, activated by AMF (8-33 kA/m, 300 kHz). The nanoclusters are co-encapsulating multiple 25 nm SPION cores and 275 J/g latent-heat tetracosane which serves as PCM. (17) Their outer shell is based on biocompatible material polyethylene glycol (PEG). We were able to demonstrate an IV administration and localization of the nanoclusters to be targeted passively on cancer cells. The nanoclusters, called Sarah nanoparticles (SaNP), have been characterized in terms of physiochemical properties, magnetic-to-thermal conversion efficiency and energy absorption capability. Here, we present the nanoclusters use and performance in terms of MH efficiency and therapeutic efficacy in vivo. 

    1.  Materials 

The 25nm oleic-acid capped SPIONs (Fe3O4) were purchased from Imagion Biosystems (San-Diego, CA USA). poly(ethylene-oxide)-poly(propylene-oxide)-poly(ethylene-oxide) (PEO-PPO-PEO) triblock copolymer (Pluronic F127, Mw = 12,600 g/mol) was purchased from Sigma Aldrich (Rehovot, Israel). p-Nitrophenyl chloroformate (p-NPC) (97%) and tetracosane (> 99%) were purchased from Acros Organics (USA and Czech Republic). Amine- functionalized 6-arm- branched PEG (Mw = 20 kD) was purchased from Sunbio Inc. (Gunpo-si, Gyeonggi-do, South Korea). 50% glucose w/v was purchased from B. Braun (Germany).  Hydrochloric acid (36.5-38 %) and Nitric acid (69-70 %) for trace metal analysis were purchased from Avantor Performance Materials Inc. (PA, USA). All other chemicals were of analytical grade.

  1.  Synthesis of Water-Dispersible SaNP Nanoclusters 

To synthesize the SaNP nanocluster, the chemically inert Pluronic F127 was first activated with p-NPC at its two terminal hydroxyl groups following the preparation procedure referred in literature.‎(18) The activation efficiency was determined using 1H-NMR. The SaNP nanoclusters comprising an inner core encapsulating SPIONs and tetracosane and a hydrophilic Pluronic/ amine-functionalized 6-arm-branched PEG polymer shell layer were prepared using a modified emulsification/solvent evaporation method.‎(19) Briefly, the mixture of of p-NPC-activated Pluronic F127 (5.2 g), tetracosane (36 mg) and oleic acid capped SPIONs (15 mg) dissolved in 6 ml dichloromethane was added in dropwise manner to the 36 ml aqueous solution (pH = 8.4) containing 93 mg Amine- functionalized 6-arm- branched PEG. The oil-in-water mixture is then sonicated using a 20 kHz Vibra cell ultrasonication (VCX-750) for 4 min followed by neutralizing the pH of solution with 37 % HCl (120 µl) to quench the reaction. The organic solvent (dichloromethane) in the emulsion was then removed by rotary evaporation with a water bath set at 40 °C until the solution became clear. The resultant dispersion containing the SaNP nanoclusters is washed several times by centrifugation (4 hr at 18,000 rcf) precipitation-redispersion cycles using water for irrigation (WFI) and filtered through a sterile 0.22 µm filter (Sartorius, Israel) After filtration, the SaNP nanoclusters undergo additional centrifugation (2 hrs. at 14000 rcf) in order to concentrate the final product followed by aseptic filling. In the preclinical studies, the SaNP nanoclusters dispersion was diluted with 50% glucose w/v to produce isotonic solution (5% glucose concentration).  

  1.  SaNP Nanocluster Characterization

The hydrodynamic diameter, zeta-potential and polydispersity index (PDI) of the obtained SaNP nanoclusters were determined by Zetasizer Nano Series ZS (Nano-ZS, Malvern Instrument Ltd., UK). The nanoclusters morphology was characterized by Tecnai G2 Cryo-transmission electron microscopy (TEM) system. The total mass and iron content in the synthesized nanoclusters was determined using an AAS analysis. The SaNP nanoclusters concentration (mg/mL) in the purified aqueous dispersion was determined by weighing the remaining solid after removing the water from a given dispersion volume by lyophilization process using a Labconco FreeZone -105°C 4.5 Liter Benchtop Freeze Dry System. The ratio of organic to inorganic material in the SaNP nanoclusters formulation in a given volume was calculated by subtracting the SPION mass determined by AAS from the dry weight of SaNP nanoclusters obtained after dryfreezing process. The chemical composition of SaNP nanocluster was characterized with a Nicolet 8700 spectrometer fitted with a DTGS detector (Thermo Scientific inc., USA) and equipped with attenuated total reflectance (ATR) accessory with a diamond crystal. The magnetization curves of nanoclusters were determined with  a superconducting quantum interference device (SQUID) (Quantum Design MPMS) at room temperature. The magnetic fluid heating experiments were performed with a 6.6 kW high frequency induction heating system model No. WUH-06A (Shenquia yongda high frequency Equipment Co., Inc, China), which could generate an AMF at a frequency of 290 kHz ± 10%.  For the SAR measurement, the average slope of the temperature versus time plot during the first 2 min of heating was drawn and the heating rate was calculated by forward linear fitting of each sample, using the first 30 sec data, and subtracted by that of solvent alone to compensate for the heat exchange with the environment. The SAR was calculated using Eq. (1).  Detailed characterization procedures are provided in Supplementary Notes S1-S7, within Supporting Information data file.

  1. SaNP Nanoclusters Stability Study in Physiological Media Over Time

Stability study of SaNP nanoclusters in physiological media was carried out using 6 ml of SaNP nanoclusters dispersed in WFI, concentrated to 2 mg/ml (2 hours centrifugation at 14000 RCF), conducted in class-10000 clean-room facility. The solution is then re-suspended in 6 ml of Dulbecco’s PBS under aseptic conditionsThe SaNP nanoclusters were transferred into 10 ml glass vials (OMPI (Stevanato)) sealed with rubber stopper (West) and aluminium cap (West) to avoid contamination of samples during incubation period. Finally, the SaNP nanoclusters were stored for 14 days at controlled temperature of 15-25oC and humidity of 50%-60%. Analyses for hydrodynamic size, Zeta potential and heating rate measurements were carried out at two time points, 7 and 14 days after start, and compared to those measured at start point (control).

  1. Evaluation of SaNP Nanoclusters Detection by MRI   

In vivo biodistribution of IV-administered SaNP nanoclusters was determined using MRI modality. It is used to characterize the SPIONs accumulation in vital organs and to adjust the optimal dosage (mg/kg) in the body. The MR tests were conducted on 1.5-T whole-body MR scanner (OPTIMATM MR-450W, GE Healthcare’s premium 1.5-T, 70 cm wide bore MRI system). A thirty-two-channel anterior array coil (AAC) was used to scan Torso part of 32-cm length. T2-weighted images were acquired using multi-slice, multi-echo (MSME) pulse sequence. Scan parameters are 33 axial slices placed in a field of view (FOV) of 320mm, image spatial resolution 256×256 pixels (350mmx350mm), slice thickness 7.5mm, slice spacing 2.5mm. T2 weighting was obtained using repetition time (TR) of 3000ms and 8 echo times (TE = 9.7, 19.4, 29.1, 38.8, 48.5, 58.2, 67.9, and 77.6 ms). The total scan time is 13min. The T2-weigthed images were analyzed using a region of interest (ROI) tool (ImageJ software). The relaxation rate values (1/T2) were plotted versus the SPION concentrations in the dilutions. The relaxivity was determined by a linear fit.

  1. Cell Culture

The 4T1 Triple negative breast cancer (TNBC) mouse mammary carcinoma cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) FBS, 1.0 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in a humidified atmosphere containing 5% CO2 at 370C. To generate a metastatic lung cancer model, mouse 4T1 TNBC cells were grown to 70% confluency and metastatic tumors were established by harvesting early passage 4T1 TNBC cells with 0.25% Trypsin-EDTA, centrifuged at 500×g for 5 min, and resuspended in ice cold HBSS at 2.5×104 cells/200μl solution. The cell suspension was IV injected via the lateral tail vein of BALB/c mice.  

  1. Animals 

BALB/c mice, 7-8 weeks old, were purchased from Envigo (Nes-Ziona, Israel). All animal experiments were reviewed and approved by an Institutional Animal Care and Use Committee (IACUC), followed officially approved procedures for the care and use of laboratory animals, and all protocols met the requirements of the local ethical committee of Technion – Israel Institute of Technology, Israel (ethical approval No. IL-0800617). 45 kg-weight swine, one male and two females, were purchased from Trakia University (Stara Zagora Bulgaria). The animal handling was compliant with guidelines of the National Institute of Health (NIH). All animals were euthanized at the end of the experiments.

  1. Evaluation of Therapeutic Efficiency of Systemic In Vivo Magnetic Heating Therapy

Eighteen BALB/c female mice, in a bearing lung metastasized 4T1 TNBC model, were weighted and randomly divided into to 3 groups (n=6). Each group received a different treatment. 1st group: 5% Glucose solution, 2nd group: SaNP + AMF (f = 300 kHz; H=10.4 kA/m), 3rd group: (f = 300 kHz; H=13 kA/m). To evaluate the MH efficiency of SaNP nanoclusters with AMF application, groups 2 and 3 were IV bolus injected with the aqueous dispersion of SaNP nanoclusters diluted with 5% glucose at a dose of 20 mg/kg in terms of mass of fraction Fe3O4, using insulin syringe and a 27G needle. Group 1 was IV injected with 5% glucose and used as control. Eight hours post injection, groups 2 and 3 were exposed, for 30 min time period, to continuous 300kHz AMF at strength of 10.4 kA/m and 13 kA/m, respectively. The treatment is composed of 3 repeated MH cycles, at day 14th, 16th and 18th post cell inoculation. At day 21st, the animals were sacrificed following by lungs excision and visual counting of the number of metastases nodules and by histopathology efficacy evaluation. The number of tumors/nodules in the lungs was reported, expressed either as single, or multiple nodules. A two-dimensional morphometric measurement and average area quantitation (mm2) were done on the largest nodule (i.e., tumor) in the lung sections. The morphometric evaluation was performed using the Augmentiqs system (https://www.augmentiqs.com/).‎(20)

For the large animal pre-clinical study, swine, weighting ~10 kg, were IV administrated with a dose of 2.6 mg/kg. Then, 4-hours post-injection, were subjected to AMF application using clinical system with an AMF strength of 12 kA/m at frequency 300 kHz. The AMF application was conducted in the target area which included the heart, lungs, stomach, liver, spleen and kidneys. The irradiated animals were monitored for 5 days after AMF exposure. The follow up included monitoring of clinical signs, behavioral changes, blood and coagulation analyses. 

  1.  Histopathology Analysis

Histological slides were prepared by Patho-Logica Lab., Nes-Ziona, Israel. Tissues harvested for microscopic examination were fixed in 4% formaldehyde. Tissues were trimmed in a standard position and sectioned into five different longitudinal cross sections with an interval of 100 microns each. Per lung, five cross section levels were prepared: #1 (most dorsal level), #2 (dorsal level), #3 (middle level), #4 (ventral level), #5 (most ventral level), mounted on glass slides and stained with Hematoxylin & Eosin (H&E). 

  1. Statistical Analysis

All data are expressed as the mean ± S.D. The statistical significance of differences between groups was analyzed by Student’s t test and a p value of < 0.05 was considered to be statistically significant.  Comparison of results among groups was carried out by one-way analysis of variance (ANOVA).

    1.  Design and Synthesis of Pegylated SPION-Based Nanoclusters for Temperature-Controlled MH   

Enhanced SAR for MH applications is a challenge which has been addressed by many studies over the years.‎(21),‎(22),‎(23) Recently, magnetic nanoclusters consisting of SPIONs cores, co-doped with zinc/manganese or cobalt/manganese‎(24) were demonstrated to be used as heat mediator for systemically delivered MH owing their high heating efficiency. However, this approach does not stand in line with regulatory safety requirements related to a clinical use of NPs containing heavy metals in their structure. There is currently insufficient data concerning the exposure and degradability of heavy metals NPs in vivo. SPIONs have been explored traditionally for application in MH due to their biocompatibility and biodegradability.‎(25) Moreover, SPIONs with sizes within the range of 20 – 40 nm were demonstrated to have high SAR values when irradiated by an AMF at a frequency range of 325-341 kHz.‎(26),‎(27),‎(28) In an attempt to develop biocompatible IV systemic delivered nanoclusters comprising both high heating efficiency and controllable temperature functionality, we synthesized a new class of NPs which contain tetracosane and multiple cores of narrow-sized spherical-shaped 25 nm SPIONs coated with oleic acid in their reservoir structure, and functionalized with PEG at their outer surface. The tetracosane is a paraffin wax-based material with 24 carbon atoms in its backbone. Owing its high latent heat of fusion, obtained by undergoing a phase change, >275 J/g, we used it as a PCM component of SaNP nanoclusters to enable control of the NP temperature within the range of 47 – 53oC. (29)

A PEO-PPO-PEO block copolymer which undergoes self-assembly in aqueous media, is used to encapsulate the tetracosane and the SPIONs, to form highly stable aqueous nanocapsules. It also serves as a delivery vehicle of the tetracosane and SPIONs through the surrounding medium, by using its well-known ability to facilitate the solubilization of poorly water-soluble materials in drug delivery systems.‎(30)Above the critical micelle temperature and concentration, the PEO-PPO-PEO copolymer spontaneously form polymeric micelles with a distinct core–shell structure in which a hydrophobic inner core (PPO middle block) is surrounded by a hydrophilic shell (PEO flank blocks) exposed to the water phase. The PPO core can incorporate water insoluble molecules and protects the interior agent from exterior components.‎(31) Therefore, the tetracosane and SPIONs (the water insoluble components) were loaded into the hydrophobic interior of a PEO-PPO-PEO polymeric micelles while the outer hydrophilic shell of micelles (PEO flank blocks) preactivated with amine reactive moieties were crosslinked with amine-functionalized 6-arm branch PEG (Figure 1). The SaNP nanoclusters were prepared according to the previously reported emulsification/solvent evaporation approach‎(19) confirmed as an efficient encapsulation strategy of poorly water-soluble drugs/materials into the water soluble PEO-PPO-PEO based polymeric nanoplatform. Briefly, this approach is based on the dispersion of mixture containing hydrophobic components (tetracosane and SPIONs) and p-NPC-activated PEO-PPO-PEO copolymer in a dichloromethane (DCM), and subsequently, emulsification in a basic buffered aqueous solution containing an amine-functionalized 6-arm-branched PEG (Mw=20kDa) by ultrasonication to form an oil in water emulsion (Figure 1(A)). 

Figure 1. Illustration of the chemistry and procedures to synthesize the SaNP nanoclusters. (A) Ultrasonic (US) emulsification of organic oil phase mixture of p-NPC-activated PEO-PPO-PEO copolymer, tetracosane and SPIONs in an aqueous phase (B) Shell cross-linking between the PEO-PPO-PEO and PEG polymers. (C) Solvent evaporation to form SaNP nanocluster encapsulation of SPIONs and tetracosane and a hydrophilic amine-functionalized 6-arm-branched PEG polymer shell layer.

The terminal groups of the PEO-PPO-PEO copolymers preactivated with p-NPC were conjugated covalently with the primary amine groups of amine-functionalized 6-arm-branched PEG polymer at the interface of oil/water emulsion droplets to generate stable carbamate bonding, resulting in shell cross-linking between the PEO-PPO-PEO and PEG polymers (Figure 1(B)). Once the residual DCM was evaporated, the SaNP nanocluster, with an inner core composed with PPO segments of PEO-PPO-PEO encapsulating SPIONs and tetracosane and a hydrophilic amine-functionalized 6-arm-branched PEG polymer shell layer, is formed (Figure 1(C)). Following the filtration process of non-encapsulated hydrophobic large aggregates of tetracosane and free oleic acid capped SPIONs, a stable SaNP nanoclusters dispersion in water is obtained.

  1. MH Temperature-Controlled by PCM 

To verify the temperature control property, the magneto-thermal conversion behavior of SaNP nanoclusters was evaluated by applying AMF irradiation with a field amplitude of 33.4 kA/m at 300 kHz for 30 min, continuously. The sample was thermally equilibrated to a stable 36.5±0.5oC temperature range for 3 min, to simulate human core temperature, prior to the application of AMF irradiation. Care was taken to set stable and close to adiabatic conditions of the test sample with the environment.‎(32) 

Figure 2 illustrates the temperature profiles of water-dispersible individual 25nm SPIONs particles and SaNP nanoclusters, for two different SPION concentrations, 1.81 and 2.56 g/L. We found that the heating rate was higher for higher SPIONs dispersion concentration.‎(33)

For the reference line, the heating profile of individual 25 nm pegylated SPIONs was measured. In Figure 2, the thermal equilibrium temperature of SPIONs solution over 30 min of continuous AMF irradiation was found to be 56oC (orange line) and 63oC (green line) for concentrations of 1.81 and 2.56 g/L, respectively. Thus, with a higher concentration, a higher equilibrium plateau temperature is reached. 

To demonstrate the PCM functionality, SaNP nanoclusters samples were heated under the same setup. The profiles shown in Figure 2 (blue and red lines) depicts temperature plateau within the range of 48–52 oC, independent of their SPIONs mass contents. These profile plateaus are in agreement with the melting phase transition temperature range of the tetracosane component.‎(28) The water temperature for both SaNP nanoclusters samples reached 47oC, the phase-change melting onset of tetracosane, within 2-3 min from start, and moderately increased to around 48-52oC. The absence of a phase-transition temperature peak above 52°C in the SaNP nanoclusters profiles, confirms that the solid-to-liquid phase-transition was not completed and the tetracosane indeed remains in its solid-liquid state, acting as a PCM temperature control. 

Figure 2. Temperature profiles of SaNP nanoclusters vs. pegylated SPIONs, demonstrating PCM functionality for controlling the thermodynamic equilibrium temperature under continuous AMF irradiation (33.4 kA/m at frequency of 300kHz). 

The PCM also shows good reversibility of energy storage and release. A magnetic-to-thermal energy conversion cycling tests of 10 successive heating and cooling cycles were performed using a SaNP nanoclusters sample, irradiated under AMF of 33.4 kA/m and 300kHz. Figure S1 depicts the profiles for the 1st, 5th and 10th cycles. The profiles of the 5th and 10th heating cycles coincide with the 1st baseline cycle. The repeatability of this profile demonstrates the high solid-to-liquid phase transition reversibility of the tetracosane-based nanoclusters.

These results demonstrate the magnetic-to-thermal conversion of SaNP nanoclusters with thermal heating control capability, owing to PCM physical properties, which is key function to allow efficient temperature control, compared to previously reported approaches, and to prevent healthy tissue overheating MH application. ‎(34),‎(35),‎(36),‎(37)

  1.  FTIR Spectrum Analysis

Attenuated-Total-Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy analysis was performed to verify the presence of PEO-PPO-PEO block copolymer in the SaNP nanocluster composition. The spectra are presented in Figure 3. In the tetracosane spectrum (green line plot in Figure 3), the peaks at 2,958, 2,922 and 2,853 cm-1 wavenumbers are attributed to C–H stretching peaks, the peaks at 1,470 and 1376 cm-1 are –CH2 asymmetric and –CH3 symmetric deformation peaks, and the peak at 720 cm-1 is –(CH2)n– swinging in-plane peaks. As noticed in SaNP nanoclusters spectrum (red line plot in Figure 3), the appearance of the above-mentioned spectral peak components indicates a well-integration of the n-tetracosane into the SaNP nanocluster. When comparing the FTIR spectrum of PEO-PPO-PEO to SaNP nanocluster (blue and red line plots, respectively in Figure 3), similar principal absorption peaks mainly at 2,882 cm−1 (C–H stretch aliphatic), 1,343 cm−1 (in-plane O–H bend) and 1,100 cm−1 (C–O stretch) are observed on both materials spectra.  Moreover, the presence of the asymmetric stretching band of carbonyl (-CO-), located at 1,704 cm-1, is noticeable only for the SaNP nanocluster, indicating the formation of carbamate bonding. In summary, all of these observations indicate the proper integration of PEO-PPO-PEO block copolymer and tetracosane in the SaNP nanoclusters composition. 

Figure 3. ATR-FTIR spectra of tetracosane, Pluronic F127 and SaNP nanocluster.

  1. SaNP Nanoclusters Chemical Composition

The mass weight ratio of SPION to the whole freeze-dried SaNP nanocluster, evaluated by AAS analysis, is about 58 wt%. The tetracosane to the nanocluster mass weight ratio, as found by GCMS analysis, is about 6 wt%. Accordingly, the weight percentage of remaining organic content in SaNP nanoclusters composition is 36 wt% which is attributed to the presence of a mixture of PEG polymer and PEO-PPO-PEO copolymers. SPIONs to SaNP nanoclusters encapsulation yield is as high as 77% (by mass). The presence of tetracosane and oleic acid SPIONs within the SaNP nanocluster and its excellent water dispersibility, demonstrate the core-shell structure of SaNP nanocluster with a cross-linked PEO-PPO-PEO/PEG as a shell structure. To the best of our knowledge, this is the first time reported, that an efficient encapsulation of long-chain hydrocarbon (C24) was processed to generate stable NPs. 

  1.  Hydrodynamic Particle Size

The hydrodynamic size of the synthesized SaNP nanoclusters and the surface charge zeta-potential (ZP) were characterized using DLS. Figure 4 presents the DLS measurement which indicates the synthesized SaNP nanoclusters exhibit a mean hydrodynamic diameter of 135±10.5 nm with a narrow size distribution (PDI ≤ 0.15). Notably, the SaNPs’ surface electric charge is measured to be negative value (ZP = -9.7±1.2 mV) at neutral pH as depicted by Figure S4

Figure 4. Hydrodynamic diameter of SaNP nanoclusters analyzed by DLS.

Generally, one should expect to observe a positive surface electric charge due to the presence of unreacted primary amines, naturally protonated at neutral pH, originating from the PEG chains of amine-functionalized 6-arm-branched PEG polymer, which did not participate in the surface cross-linking reaction. We attribute the negative electric charge to a shift of the diffusive layer slipping plane away from nanocluster surface; this shift is caused by PEG polymer chains as described previously,‎(38) and thus the terminal amino groups presented on PEG chains exist beyond the measurable shear plane used for zeta-potential analysis.‎(39),‎(40) These results provide indirect evidence for the chemical integration of amine-functionalized 6-arm-branched PEG polymer at the outer layer of SaNP nanoclusters, generating a pegylated corona layer. This indicates that its role in the synthesis is not used only as a cross-linker agent, but also as a sterically stabilizing additive.

  1.  SaNP Nanoclusters Morphology Characterization

The SaNP nanoclusters morphology was evaluated by Cryogenic TEM (cryo-TEM) analysis which preserves the size and morphology of the polymer assemblies in the hydrated state and allows SaNP nanoclusters imaging in their actual form. In cryo-TEM conditions, it was revealed that the samples consisted of well-dispersed nanosized clusters without significant aggregation, indicating that they were effectively stabilized by a cross-linked hydrophilic polymer shell in aqueous solution, as depicted in Figure S5. It was likely that the dispersion stability was partially attributed to the charge repulsion phenomena exerted by cationic amino groups present on cross-linked PEG or grafted PEG chains on the nanocapsules. Cryo-TEM analyses demonstrate a unique nanoreservoir structure of SaNP nanoclusters capable of enclosing diverse inorganic nanomaterials in the interior with a surrounding polymer shell layer. Since the shell cross-linking reaction occurs primarily at the interface of the oil/water emulsion droplets containing the SPIONs, it is conceivable that the encapsulation process does not affect the crystalline structure and magnetization properties as well as the size of the SPIONs. Therefore, it is safe to say that the current encapsulation process is potentially applicable for stabilization and functionalization of a variety of inorganic nanomaterials which are poorly soluble and processable in aqueous solutions. 

  1. Magnetic Properties 

The magnetization property in the SaNP nanoclusters with respect to the oleic acid coated SPIONs, was evaluated by recording the field-dependent magnetization M(H) curve, using a superconducting quantum interference device (SQUID) at 298 K, as depicted in Figure S6. The SaNP nanoclusters curve (blue line) presents negligible coercivity and remanence, suggesting superparamagnetic characteristic at room temperature is comparable to that of 25 nm SPIONs (green line), despite the large size of 130 nm for SaNP nanocluster.  The magnetic saturation, Ms (in emu/g), of the oleic acid coated SPIONs dropped from 75.2 emu/g (which was closer to that of bulk magnetite (92 emu/g)) to 56.4 emu/g (for SaNP nanoclusters) after mini-emulsion process and encapsulation in organic matrix, with alignment to Ms value, commonly reported in the literature.‎(13),‎(24),‎(28),‎(41),‎(42) The decrease in magnetic saturation Ms (magnetic moment per weight unit) is partly attributed to the decreased effective weight fraction of the magnetic core. These results are in good agreement with the estimated organic components composition, showing 40% diminution at Ms, attributed to ~40 wt% of organic mass fraction in SaNP nanoclusters sample, as previously described.‎(43)  

  1.  SaNP Nanocluster Heating Potency for MH Clinical Application 

The heating capability of the SaNP nanoclusters was evaluated by measuring their SAR value. Samples of 2 mg/mL were irradiated by an AMF in the range of 8 to 33 kA/m at a fixed frequency of 300 kHz. The SAR value, in W/g, is calculated using the following equation:  


where w is water density (g/mL), CIO is the SPIONs concentration in the dispersion, Cp is the heat capacity of water (J/(kg oC)) and ∆T/∆t is the heating rate (oC/s) in the first 30 seconds of AMF exposure of SaNP nanoclusters. Figure S2 and Figure S3 depict the heating profiles for pegylated SPIONs and for SaNP nanoclusters, for various magnetic field strengths. The pegylated SPIONs and the nanoclusters exhibit heating rates of 14-16 oC/min and 10-12 oC/min, respectively, which point out of approximately an 80% energy conversion consistency. It is generally assumed that significant heating occurs only in a very close vicinity of the iron oxide-base nanoclusters surface.(44),(45)Several works have accumulated indirect proofs that, even if no macroscopic temperature changes under AMF are recorded, the local temperature at the nanoparticle surface might be significantly different from that of the surroundings. It was shown for instance that, even if the concentration of the MNPs at the tumor cells did not produce macroscopic heating, MNPs could induce the apoptosis of tumor cells under AMF.‎(45) Consequently, we estimate that even at concentration below 2 mg/ml of SaNP nanoclusters present in the malignant tissue, a local temperature increase of 10-12 oC/min can be occurred close to vicinity of SaNP nanoclusters surface, and so, can potentially leads to an in-vivo cellular heating up to temperature 46-48 oC within the 1 min post AMF radiation.(43). (44).(45),(46) 

Figure 5. Heating profile for the nanoclusters was evaluated: SAR (W/gSPION) for SaNP nanoclusters and pegylated SPIONs, as function of applied AMF.

The SAR obtained by the SaNP nanoclusters is improved and optimized by specific design of core material type, size, morphology, coating thickness and magnetic properties. Additionally, the nanoclusters synthesis was designed to have optimal size, zeta-potential and flexible morphology‎(16) to utilize the EPR effect efficiently, enabling accumulation of SaNP nanoclusters at the target site, with the purpose of increasing the SAR per unit volume. The SAR value was measured as a function of the AMF amplitude and shown in Figure 5.  Nanoclusters samples of 200 µL volume, with SPION concentration of CSPION=1.8 g/L, exhibit heating rate of ∆T/∆t = 12.3oC/min, when AMF of 33.4 kA/m at frequency of 300 kHz is applied. Using Eq. (1), with water heat capacity of Cp=4190  J/(kg °C) and w=1 g/mL, a SAR value of 475 ± 17 W/gSPION is obtained. The number of SPIONS cores in the sample is estimated using the relation, N=mSPION/[(π×D3/6)×SPION]. Assume 1 mL volume of 2 mg/mL pegylated SPIONs concentration, mSPION=0.002g, D=25 nm diameter and density of SPION=5.2 g/cm3, we obtained number of SPIONs cores  NSPION=4.7×1013. The same calculation for SaNP nanoclusters, we take mSaNP=0.0034g (0.002g SPIONs and 0.0014g organic contents) with D=135 nm, and same density of 5.2 g/cm3, we obtained NSaNP=5.1×1011 nanoclusters. The SAR per SaNP nanocluster, SAR/N,  is higher by ~90 folds, when compared to the SAR per SPION core. In other words, a more efficient treatment may be attained with nanoclusters in relation to SPION cores, per dose.

  1.  SaNP Nanocluster Stability

One of the key characteristic parameters design of nanoclusters is its stability over time.  We tracked the SaNP nanoclusters characteristics, dispersed in phosphate-buffered saline (PBS), at three time points, analysing their hydrodynamic diameter, zeta-potential and heating rate variability as a function of storage time. No statistically significant differences were observed throughout the sampling period. Notably, the hydrodynamic size remains stable over time (Figure S7). For zeta-potential results, SaNPs nanoclusters were revealed as highly stable with no significant aggregation (Figure S8). These results provide evidence that the PEG encapsulates the SaNP nanoclusters in a way that the charged ammonium groups are exposed to the aqueous medium as mentioned earlier, which allows a robust colloidal stability to be obtained. The thermal property of SaNP nanoclusters under AMF application of 33.4 kA/m at 300 kHz was analysed. Results in Figure 6 indicate that heating profile and potency of SaNP nanoclusters are preserved during the storage period time. Both, the good colloidal and thermal stability denote their clinical translation potential.

Figure 6. Heating-rate profile of SaNP nanoclusters, dispersed in PBS, analyzed induction heating system, before and after storage of 7 and 14 days at room temperature. Polydispersity index (PDI) parameter was found to be 0.117, 0.101, and 0.099 for storage of 0, 7, and 14 days, respectively.

  1. MH Metastatic 4T1 Breast Cancer Treatment with SaNP Nanoclusters in Mice

SaNP nanoclusters effectiveness for MH application was demonstrated in mice bearing lung-metastasized through in vivo studies, as assessed by histopathology analysis and gross pathology nodules count. First, we evaluated the tissues distribution of SaNP nanoclusters in mice bearing 4T1 TNBC lung-metastasized (Figure S9). It was shown that SaNPs accumulate mainly in the liver and spleen which are known to be the main RES organs. The results show that 8 hours nanoclusters post injection is an optimal time window for hyperthermia while the SaNP nanoclusters accumulation in normal tissue is low. In order to evaluate the MH efficacy, SaNP nanoclusters were IV administered at a dose of 20 mg/kg to mice bearing 4T1 TNBC lung-metastasized and irradiated by AMF, 8 hours post injection.

The study group was randomly divided into three groups (n=6): Group 1 was assigned as control, injected with 5% glucose; group 2 and 3 were injected with SaNP nanoclusters and exposed to AMF strength of 10.4 kA/m and 13 kA/m, at 300 kHz, for 30 min, respectively. The treatment includes of 3 successive cycles, with two days between the cycles and termination at the 8th day, sacrificing the mice for visual counting of the number of metastases lung nodules. As can be seen in Figure 7, the average number of 4T1 TNBC metastatic pulmonary nodules for treatment group 3 (N3=13±8) was significantly smaller than the number found in group 1 (N1=38±15), which corresponds to 66% reduction in the number of metastases (p value <0.0001, Student’s t-Test). For the moderate AMF exposure level, a similar trend of 45% reduction in the number of metastases nodules was observed (group 2). Establishment of the primary mode of action of SaNP treatment was performed to demonstrate the effect of both elements, SaNP and AMF, incorporation, as shown in Figure S10

Figure 7. Metastatic nodules in lungs are depicted by black-colored arrows, in the 3 photos above: (A) Control untreated group. (B) Treated group with SaNP and AMF amplitude application of 10.4 mT (C) Treated group with SaNP and AMF amplitude of 13mT. (D) Number of metastatic lung nodules determined by gross pathology count for each of the cases in photos (A), (B) and (C).  

***Statistically significant difference compared to the control (p value <0.0001). Statistical analysis was performed by Student’s t-Test.

To better validate the treatments impact on the pulmonary tissue, a histopathological assessment of the mice lungs was performed, using H&E staining. Histopathological assessment showed that mice treated with the SaNP nanoclusters and AMF developed reduced number and size metastatic nodules when compared to the control group, as demonstrated in Figure 8

Figure 8. Representative histopathology images of lung tissues from 3 study groups. (A) Control untreated group. (B) Treated group with SaNP and AMF amplitude of 10.4 mT (C) Treated group with SaNP and AMF amplitude of 13mT. H&E staining, Magnification, X4.  Lung tissues were removed 3 days post last AMF cycle treatment. Tumor metastases in lung tissues are marked with yellow demarcation.

A whole slide scanning and digital 2-D morphometric analysis‎(20) of control and treatment mice lungs was employed to estimate the number of metastases nodules and quantitate the percentage of metastatic surface areas in the lungs. As shown in Figure 9, the mean percentage of lung metastatic lesions area which was found in both treatment groups, 2 and 3, was measurably lower than in the control group (17.8% ± 18.03 and 16% ± 19.66 vs 38.2% ± 14.34, respectively), indicating a reduction of 53% and 58% of the metastatic area on the lung of treated mice groups 2 and 3, respectively. Finally, these findings were in concordance with the results obtained by the macro visual counting, suggesting that the SaNP nanocluster as heat mediator interacts with AMF magnetic field to induce hyperthermia to treat lung-metastasized 4T1 TNBC in a murine model.

Figure 9. Percentage of metastases lesion surface area as determined by whole slide scanning and digital 2-D morphometric analysis method.

Both groups were found with statistically significant difference compared to the control (p value <0.05). Statistical analysis was performed by Student’s t-Test.

  1. SaNP Nanoclusters Identification and Quantification by MRI Modality

Magnetic resonance imaging (MRI) is a non-invasive technique widely used to diagnose diseases or injuries based on its high soft tissue contrast, with no penetration limit and spatial resolution.‎(47) MRI uses a powerful magnetic field and radio frequency pulses to produce detailed images of organs, tissues and other internal body structures. Swine model was used to study the biodistribution of a single dose of SaNP nanoclusters, using MRI technique, qualitatively and quantitatively. 

T2-weighted MR imaging enhancement by the SaNP nanoclusters was evaluated using 1.5-T MR scanner. T2-weighted MR images for different SPION concentrations of SaNP nanoclusters dispersion were acquired on the MRI instrument. A set of tubes (50mL-volume, 30mm-diameter) filled with SPIONs dispersion, each at a pre-determined concentration, ranging from 1:75 down to 1:4000, were scanned by the MRI system using a T2-weighted protocol. Basically, SPIONs shorten the transverse relaxation time by increasing the spin phase coherence loss. Consequently, in T2 weighted image, low SPION concentration region appears bright and higher SPION concentration region appears darker, as shown in Figure 10(A).

Figure 10. (A)T2-weighted MR image of set of tubes containing solutions of water and SaNP nanoclusters at different SPION concentration at 1.5T field strength. (B) T2-weighted relaxation rates of SaNP nanoclusters dispersion with various SPIONs concentration. This calibration line is used to quantify SPION concentration within in vivo tissue.

The plot in Figure 10(B) depicts the T2-weighted mapping results. A good linear fit between the relaxation rate  R2=1/T2 and the SPION concentration was established, demonstrating contrast variations for the different SPION concentration. The signal intensity decays (short relaxation time) with the increase of the SPION concentration, following the equation:


where 1/T2 is the measured relaxation rate in the presence of SPIONs, (1/T2)w is the relaxation rate of a pure water, CSPION is the concentration of the SPION, and r2 is the transverse relaxivity. Under the current system, the relaxation rate of SaNP nanoclusters is found to be 68 (mM s)-1.

In ex vivo swine biodistribution studies, using particle electron paramagnetic resonance (pEPR) technique‎(48) we found that SaNP nanoclusters mainly accumulated in lungs, liver, and spleen tissues, the main RES organs. In order to evaluate the ability of the MRI to detect the SaNP nanoclusters in vivo and its applicability to MH, a swine model was used, where biodistribution mapping of SaNP within the body was evaluated. Figure S11 depicts the in vivo MRI scan of a swine torso transverse cross-section. Figure S11(A), Figure S11(B) and Figure S11(C), demonstrate T2-weighted mapping images which depict the pre-injection, 4h post-injection and 8h post-injection of the SaNP, respectively. The lungs are darkened 4h after SaNP nanoclusters injection (Figure S11(B)) as a result of the SPIONs presence in the lungs tissue. The lungs get brighter 8h post-injection (Figure S11(C)), which indicate SaNP nanocluster’s evacuation from the lungs.

The SaNP nanoclusters bio-distribution accumulation is organ-dependent and is indicated by MR signal contrast. Quantitative analysis [unpublished experiments], based on signals received in treated tissues, in relation to the signals from untreated tissues, shows accumulation of more than 60%, out of the total injected volume, in the lungs tissue, during the first 4 h after IV administration. This clearly points on the potential applicability of the present SaNP nanoclusters for hyperthermia clinical procedure for cancer treatment, with special emphasis on metastases in the lungs [unpublished experiments].               

  1. Hyperthermia procedure – Animal Model 

In previous sections, we demonstrated the hyperthermia efficiency of magnetic SaNP nanoclusters on a mice model. Another preclinical study in a swine model was conducted to explore the SaNP PCM-based nanoclusters thermodynamic response, the heating control management and the potential of thermotherapeutic capability when administering SaNP nanoclusters. The SaNP nanoclusters were IV administered and irradiated by an AMF strength, operating within the range of 8-13 kA/m and frequency of 300 kHz. Eight pigs, with an average weight of 10 kg, received a single dose of 2.6 mg/kg SaNP nanoclusters and were irradiated 4 h post-injection. The dose was calculated according to the FDA guideline.‎(49) Eight fiberoptics thermometry probes were located around the chest circumference and one probe was set rectally for core body temperature recording. The core temperature was kept in control, during the whole procedure, within the range of 35-37oC. Upon applying the AMF, the temperature in metastases was estimated to reach 45-47°C and kept stable. This averaged, steady-state of tissue and body core temperature is obtained through a unique AMF irradiation profile which is composed of 3 intervals, 10 min duration each and 3 min break. The irradiation cycle profile that was used, enables heat diffusion and conduction from the body core towards the external surface skin, allowing efficient heat removal by a cooling system. The thermodynamic cycling profile, shown in Figure S12, demonstrates a controlled thermal state of the body. 

To demonstrate the scalability, stability and repeatability of the presented hyperthermia irradiation profile, larger pig of 45 kg weight was radiated. In this preclinical study, the thermal equilibrium over 3×10 min cycles for a total of half hour radiation profile was demonstrated under exposure parameters of 8 kA/m at 300 kHz. Control over the thermal heat generation is depicted in Figure S13.

To evaluate the total power heat that was applied by the magnetic field on the animal’s torso, the SAR induced was calculated using the relation for the absorbed power density in tissue due to eddy currents, given by 

SAR=2E2=222r2f2H2     [w/kg](3)

   where E is the induced electrical field, and are the electrical conductivity and the magnetic permeability of the tissue, respectively, and r is the radial distance of the tissue from the center. f is the frequency and  H is the magnetic strength of the applied AMF. The SAR distribution within the animal body versus the radial distance, was calculated and depicted in Figure S14

Hergt et al.‎(13) showed that in order to minimize side effects of AMF on normal tissue, the irradiated area of the human body shall be limited to a field-frequency product meeting the biological upper limit criterion of H×f<5×106  kA∙m-1s-1. Clinical studies with H×f=1.8×106  kA∙m-1s-1 (18 kA/m at 100 kHz) showed safe AMF application to GBM  lesions, in 6, 10 min sessions (60 min total treatment duration).‎(50) While the hyperthermia community is still discussing the safe and tolerable level of the maximum safe levels, with no clear agreement, intensive efforts are applied by many groups to reduce eddy currents heating by various strategies.‎(51) The frequency to field strength product used in the present in vivo preclinical studies was 2.4×106  kA∙m-1s-1 (8 kA/m at 300 kHz) which is 48% lower than the biological limit (5×106  kA∙m-1s-1) and showed complete control of the thermodynamic state of the pig during treatment. In the experimental setting, the core temperature profile (bold red line in Figure S12) was found to have a total increase of 1.5 °C, during the entire hyperthermia treatment without any side effects. Clinical signs or indications of complications were not observed throughout the study. Moreover, no significant hematology, chemistry or coagulation changes were observed. 

In summary, this demonstrates the ability of the high heating-rate SPIONs-based SaNP nanoclusters, encapsulating PCM, to effectively enable a MH treatment, when metastases temperature is increased to 45-47°C under application of AMF in a controlled thermally safe level. The treatment procedure described here can be further enhanced by increasing the heating efficiency in the tumor. The SaNPs, IV injected, target cancer cells through a passive mechanism, using the EPR effect. By utilizing glucose-based active targeting mechanism, we can potentially double the accumulated SPIONs in metastases ‎(52),‎(53), owing the high glucose consumption by cancer cells, known as Warburg effect.‎(54)     


In summary, we have designed and synthesized safe and effective PCM-based theranostic SPIONs nanoclusters which offer high heating conversion efficiency in MH, with thermodynamic control and high T2-weighted relaxivity signal to enable detection and quantification of SPION in organs when exposed and scanned by a MR system. Under irradiation to AMF of 33 kA/m at frequency of 300 kHz, the nanoclusters demonstrated SAR of 475 ± 17 W/g and high transverse relaxivity of 68 (mM s)-1, at 1.5T. The SaNP nanoclusters presented here, showed stability and integrity, in terms of physicochemical characteristics and heating profile, when tested 7 and 14 days after production release. The nanoclusters also showed high heat resistivity and magnetic reversibility when subjected to multiple cycles of AMF irradiation intervals. The designed nanoclusters were used in preclinical animal studies (BALB/c mice with 4T1 TNBC tumors and large size swine treated in a preclinical system) and found to be safe and with no toxicity effects or any adverse event findings and with highly efficient accumulation of SPIONS in cancer tumors target organs (e.g., lungs), validated by pEPR ex vivo and MRI in vivo methods. In these preclinical studies, we demonstrated a scheme of 3 successive AMF irradiation intervals, with thermodynamic balance and control of the core and the skin temperatures, the temperature in metastases area were estimated to reach 45-47oC, and resulting in a notable reduction in number of metastatic nodules and cancer lesion area in mice. These nanoclusters that showed their capability to reach the operational temperature threshold to destroy cancer cells of tumors, using a single intravenous administration, have encouraged us to further advance the concept and to design a human clinical study, demonstrating the potential of MH for an efficient therapeutic treatment of cancer.  


The Supporting Information is given in appended PDF file:

Note S1 – S7: Detailed Procedures for SaNP nanocluster characterization

Figures S1-S14.


Corresponding Author

Moshe Cohen-Erner – New-Phase Ltd. Petah Tikvah, 4934829, Israel

* E-mail: moshece@newphase.co.il   Phone: +972-35411938; 

ORCID: orcid.org/0000-0001-7245-4261


Raz Khandadash – New-Phase Ltd., Petah Tikvah, 4934829, Israel. 

Raphael Hof – New-Phase Ltd., Petah Tikvah, 4934829, Israel.

 Ofer Shalev – New-Phase Ltd., Petah Tikvah, 4934829, Israel.

Adam Antebi- New-Phase Ltd., Petah Tikvah, 4934829, Israel. ORCID: https://orcid.org/0000-0003-1586-2933

Arnoldo Cyjon – Oncology Department, Shamir Medical Center, Zerifin 70300, Israel.

Dian Kanakov – Internal Noninfectious Diseases Department, Trakia University, 6000 Stara Zagora, Bulgaria. ORCID: https://orcid.org/0000-0002-0506-4439.

Abraham Nyska – Consultant in Toxicologic Pathology, Sackler School of Medicine, Tel Aviv University 69978, Israel.

Glenwood Goss – Division of Medical Oncology, University of Ottawa, ON K1N 6N5, Canada.

John Hilton – Division of Medical Oncology, University of Ottawa, ON K1N 6N5, Canada.

Dan Peer – Laboratory of Precision Nanomedicine, Shmunis School for Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. ORCID: https://orcid.org/0000-0001-8238-0673.

Author Contributions

MCE and RK contributed equally to this work.

Conflict of Interest

The authors MCE, RK, RH, OS, and AA are employees at New-Phase Ltd. The other authors declare that they do not have any affiliations that would lead to competing financial interest or any conflict of interest.

This work was supported by New-Phase Ltd.


The authors are thankful to Ricarina Rabinovitz, Ekaterina Sigalov, Pazit Rukenstein, Rana Kassem, Manzooma Kablan, Neta Buskila, Eddie Sharaga, Moshe Eltanani, Ivgeni Ram, Sasha Shlyapentokh and Andrey Kaznasab for their great support and contributions to this work. 

  1. Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2020. Ca-Cancer J. Clin. 2017, 67, 7-30.
  2. Gordon, R. T.; Hines, J. R.; Gordon, D. Intracellular Hyperthermia. A Biophysical Approach to Cancer Treatment via Intracellular Temperature and Biophysical Alterations. Med. Hypotheses 1979, 5, 83-102.
  3. Siemann, D. W. The Unique Characteristics of Tumor Vasculature and Preclinical Evidence for its Selective Disruption by Tumor-Vascular Disrupting Agents. Cancer Treat. Rev. 2011, 37, 63-74.
  4. Sharma, S. K.; Shrivastava, N.; Rossi, F.; Tung, L. D.; Thanh, N. T. K. Nanoparticles-Based Magnetic and Photo Induced Hyperthermia for Cancer Treatment. Nano Today 2019, 29, 100795.
  5. Derfus, A. M.;  von Maltzahn,  G.; Harris, T. J.; Duza, T.;  Vecchio, K. S.;  Ruoslahti, E.; Bhatia, S. N. Remotely Triggered Release from Magnetic Nanoparticles. Adv. Mater. 2007, 19, 3932-36.
  6. Johannsen, M.; Gneveckow, U.; Eckelt, L.; Feussner, A.; Waldöfner, N.; Scholz, R.; Deger, S.; Wust, P.; Loening, S. A.; Jordan, A.  Clinical Hyperthermia of Prostate Cancer Using Magnetic Nanoparticles: Presentation of a New Interstitial Technique. Int. J. Hyperthermia 2005, 21, 637-47.
  7. Maier-Hauff, K.; Rothe, R.; Scholz, R.; Gneveckow, U.; Wust, P.; Thiesen, B.; Feussner, A.; von Deimling, A.; Waldoefner, N.; Felix, R.; Jordan, A. Intracranial Thermotherapy Using Magnetic Nanoparticles Combined with External Beam Radiotherapy: Results of a Feasibility Study on Patients with Glioblastoma Multiforme. J. Neuro-Oncol. 2007, 81, 53– 60. 
  8. Maier-Hauff, K.; Ulrich, F.; Nestler, D.; Niehoff, H.; Wust, P.; Thiesen, B.; Orawa, H.; Budach, V.; Jordan, A. Efficacy and Safety of Intratumoral Thermotherapy Using Magnetic Iron-Oxide Nanoparticles Combined with External Beam Radiotherapy on Patients with Recurrent Glioblastoma Multiforme. J. Neuro-Oncol. 2011, 103, 317–324.
  9. Huang, H. S.;  Hainfeld, J. F.  Intravenous Magnetic Nanoparticle Cancer Hyperthermia. Int. J. Nanomed. 2013, 8 , 2521-32.
  10. Dutz, S.; Hergt, R.  Magnetic Nanoparticle Heating and Heat Transfer on a Microscale: Basic Principles, Realities and Physical Limitations of Hyperthermia for Tumour Therapy. Int. J. Hyperthermia 2013, 29, 790-800.
  11. Martinez-Boubeta, C.; Simeonidis, K.; Makridis, A.; Angelakeris, M.; Iglesias, O.; Guardia, P.; Cabot, A.; Yedra, L.; Estradé, S.; Peiró, F.; Saghi, Z.; Midgley, P. A.; Conde-Leborán, I.; Serantes, D.; Baldomir, D. Learning from Nature to Improve the Heat Generation of Iron-Oxide Nanoparticles for Magnetic Hyperthermia Applications. Sci. Rep. 2013, 3, 1-8.
  12. Suriyanto; Ng, E. Y. K.; Kumar, S. D. Physical Mechanism and Modeling of Heat Generation and Transfer in Magnetic Fluid Hyperthermia through Néelian and Brownian Relaxation: a Review. Biomed. Eng. Online. 2017, 16, 36.
  13. Cervadoro, A.; Cho, M.; Key, J.; Cooper, C.; Stigliano, C.; Aryal, S.; Brazdeikis, A.; Leary J. F.; Decuzzi, P. Synthesis of Multifunctional Magnetic Nanoflakes for Magnetic Resonance Imaging, Hyperthermia, and Targeting. ACS Appl. Mater. Interfaces 2014, 6, 12939–46.
  14. Hergt, R.; Dutz, S. Magnetic Particle Hyperthermia—Biophysical Limitations of a Visionary Tumour Therapy. J. Magn. Magn. Mater. 2007, 311, 187-92.
  15. Wetterskog, E.; Castro, A.; Zeng, L.; Petronis, S.; Heinke, D.; Olsson, E.; Nilsson, L.; Gehrke, N.; Svedlindh, P. Size and Property Bimodality in Magnetic Nanoparticle Dispersions: Single Domain Particles vs. Strongly Coupled Nanoclusters. Nanoscale 2017, 9, 4227-35.
  16. Pandey, S.; Quetz, A.; Aryal, A.; Dubenko, I.; Mazumdar, D.; Stadler, S.; Ali, N. Thermosensitive Ni-based Magnetic Particles for Self-Controlled Hyperthermia Applications. J. Magn. Magn. Mater. 2017, 427 ,200-205. 
  17. Hof, R.; Khandadash, R.; Fremder, E. Phase-Change Nanoparticle, US Patent 10,172,939 B2, 2019.
  18. Cho, K. C.; Choi, S. H.; Park, T. G. Low Molecular Weight PEI Conjugated Pluronic Copolymer: Useful Additive for Enhancing Gene Transfection Efficiency. Macromol. Res. 2006, 14, 348-353.
  19. Zhang, W.; Gilstrap, K.; Wu, L.; Bahadur K. C., R.; Moss, M. A.; Wang, Q.; Lu, X.; He, X. Synthesis and Characterization of Thermally Responsive Pluronic F127-Chitosan Nanocapsules for Controlled Release and Intracellular Delivery of Small Molecules. ACS Nano 2010, 4, 6747-59.
  20. Zarella, M. D.; Bowman, D.; Aeffner, F.; Farahani, N.; Xthona, A.; Absar, S. F.; Parwani A.; Bui, M.; Hartman, D. J. A Practical Guide to Whole Slide Imaging: A White Paper from the Digital Pathology Association. Arch. Pathol. Lab. Med. 2019, 143, 222-34. 
  21. Lee, J. h.; Jang, J. t.; Choi, J. s.; Moon, S. H.; Noh, S. h.; Kim, J. w.; Kim, J-G.; Kim, I. s.; Park, K. I.; Cheon, J. Exchange-Coupled Magnetic Nanoparticles for Efficient Heat Induction. Nat. Nanotechnol. 2011, 6, 418-22.
  22. Das, R.; Alonso, J.; Nemati Porshokouh, Z.; Kalappattil, V.;  Torres, D.; Phan, M. H.; Garaio, E.; Garcia, J. A.; Sánchez Llamazares, J. L.; Srikanth, H. Tunable High Aspect Ratio Iron Oxide Nanorods for Enhanced Hyperthermia. J. Phys. Chem. C 2016, 120, 10086-93.
  23. Liu, X.; Zheng, J.; Sun, W.; Zhao, X.; Li, Y.; Gong N.; Wang, Y.; Ma, X.; Zhang, T.; Zhao, L-Y.; Hou, Y.; Wu, Z.; Du, Y.; Fan, H.; Tian, J.; and Liang, X-J. Ferrimagnetic Vortex Nanoring-Mediated Mild Magnetic Hyperthermia Imparts Potent Immunological Effect for Treating Cancer Metastasis. ACS Nano 2019, 13, 8811-8825.
  24. Albarqi, H. A.; Wong, L. H.; Schumann, C.; Sabei, F. Y.; Korzun, T.; Li, X.; Hansen M. N.; Dhagat, P.; Moses, A. S.; Taratula, O.; and Taratula, O. Biocompatible Nanoclusters with High Heating Efficiency for Systemically Delivered Magnetic Hyperthermia. ACS Nano 2019, 13, 6383-95.
  25. Sun, C.;  Du, Kim .; Fang, C.;  Bhattarai, N.;  Veiseh, O.;  Kivit, F.; Stephen, Z.; Lee, D.;  Ellenbogen, R. G.;  Ratner, B.;  Zhang, M.  PEG-Mediated Synthesis of Highly Dispersive Multifunctional Superparamagnetic Nanoparticles: Their Physicochemical Properties and Function In Vivo. ACS Nano 2010, 4, 2402-10.
  26. Tong, S.; Quinto, C. A.; Zhang, L.; Mohindra, P.; Bao, G. The Size-Dependent Heating of Magnetic Iron Oxide Nanoparticles. ACS Nano 2017, 11, 6808-16.
  27. Vreeland, E. C.; Watt, J.; Schober, G. B.; Hance, B. G.; Austin, M. J.; Price, A. D.; Fellows, B. D.; Monson, T. C.; Hudak, N. S.; Maldonado-Camargo, L.; Bohorquez, A. C.; Rinaldi, Carlos.; Huber, D. L. Enhanced Nanoparticle Size Control by Extending LaMer’s Mechanism. Chem. Mater. 2015, 27, 6059-66.
  28. Lanier, O. l.; Korotych, O. I.; Monsalve, A. G.; Wable, D.; Savliwala, S.; Grooms, N. W. F.; Nacea, C.; Tuitt, O. R.; Dobson, J. Evaluation of Magnetic Nanoparticles for Magnetic Fluid Hyperthermia. Int. J. Hyperthermia 2019, 36, 686-700.
  29. Sarı, A.; Alkan, C.; Kahraman, D. K.; Cınar Kızıl. Micro/Nano Encapsulated n-Tetracosane and n-Octadecane Eutectic Mixture with Polystyrene Shell for Low-Temperature Latent Heat Thermal Energy Storage Applications. Sol. Energy 2015, 115-195-203. 
  30. Russo, E.; Villa, C.  Poloxamer Hydrogels for Biomedical Applications. Pharmaceutics 2019, 11, 671.
  31. Bohorquez, M.; Koch, C.; Trygstad, T.; Pandit, N. A Study of the Temperature-Dependent Micellization of Pluronic F127. J. Colloid Interface Sci. 1999, 216, 34-40.
  32. Jean-Paul, F.; Claire, W.; Jacques, S.; Christine, M.; Jean-Claude, B.; Florence, G. Size-Sorted Anionic Iron Oxide Nanomagnets as Colloidal Mediators for Magnetic Hyperthermia J. Am. Chem. Soc. 2007, 129, 2628-2635
  33. de la Presa, P.; Luengo, Y.; Multigner, M.; Costo, R.; Morales, M. P.;  Rivero, G.; Hernando, A. Study of Heating Efficiency as a Function of Concentration, Size, and Applied Field in γ Fe2O3 Nanoparticles. J. Phys. Chem. C 2012, 116, 25602−10.
  34. Ito, A.; Tanaka, K.; Kondo, K.; Shinkai, M.; Honda, H.; Matsumoto, K.; Saida, T.; Kobayashi, T. Tumor Regression by Combined Immunotherapy and Hyperthermia Using Magnetic Nanoparticles in an Experimental Subcutaneous Murine Melanoma. Cancer Sci. 2005, 94, 308-13.
  35. Ortgies, D. H.; Teran, F. J.; Rocha, U.; de la Cueva, L.; Salas, G.; Cabrera, D.; Vanetsev, A. S.; Rähn, M.; Sammelselg, V.; Orlovskii, Y. V.; Jaque, D.  Optomagnetic Nanoplatforms for In Situ Controlled Hyperthermia. Adv. Funct. Mater. 2018, 28, 1704434. 
  36. Ebrahimi, M. On the Temperature Control in Self-Controlling Hyperthermia Therapy. J. Magn. Magn. Mater. 2016, 416, 134-140.
  37. Zhang, W.; Zuo, X.; Niu, Y.; Wu, C.; Wang, S.; Guanb, S.; Silva, S. R. P. Novel Nanoparticles with Cr3+ Substituted Ferrite for Self-Regulating Temperature Hyperthermia. Nanoscale 2017, 9, 13929-37. 
  38. Rabanel, J. -M.; Hildgen, P.; Banquy, X. Assessment of PEG on Polymeric Particles Surface, a Key Step in Drug Carrier Translation. J. Controlled Release 2014, 185, 71-87.
  39. Burns, N. L.; Van Alstine, J. M.; Harris, J. M. Poly(ethylene glycol) Grafted to Quartz: Analysis in Terms of a Site-Dissociation Model of Electroosmotic Fluid Flow. Langmuir 1995, 11, 2768–2776.
  40. Khandadash, R.; Machtey, V.; Shainer, I.; Gottlieb, H. E.; Gothilf, Y.; Ebenstein, Y.; Weiss, A.; Byk, G. Novel biocompatible hydrogel nanoparticles: generation and size-tuning of nanoparticles by the formation of micelle templates obtained from thermo-responsive monomers mixtures J. Nanopart. Res. 2014, 16, 2796. 
  41. Fan, X.; Xiao, J.; Wang, W.; Zhang, Y.; Zhang, S.; Tang B. Novel Magnetic-to-Thermal Conversion and Thermal Energy Management Composite Phase Change Material. Polymers 2018, 10, 585.
  42. Liu, X.; Zheng, J.; Sun, W.; Zhao, X.; Li, Y.; Gong, N.; Wang, Y.; Ma, X.; Zhang, T.; Zho, L. Y.; Hou, Y.; Wu, Z.; Du, Y.; Fan, H.; Tian, J.; Liang, X.-J. Ferrimagnetic Vortex Nanoring-Mediated Mild Magnetic Hyperthermia Imparts Potent Immunological Effect for Treating Cancer Metastasis. ACS Nano 2019, 13, 8811-25.
  43. Sasikala, A. R. K.; Thomas, R. G.; Unnithan, A. R.; Saravanakumar, B.; Jeong, Y. Y.; Park, C. H.; Kim, C. S. Multifunctional Nanocarpets for Cancer Theranostics: Remotely Controlled Graphene Nanoheaters for Thermo-Chemosensitisation and Magnetic Resonance Imaging. Sci. Rep. 2016, 6, 20543. 
  44. Keblinski, P.; Cahill, D. G.; Bodapati, A.; Sullivan, C. R.; Taton, T. A. Limits of Localized Heating by Electromagnetically Excited Nanoparticles. J. Appl. Phys. 2006, 100, 054305. 
  45. Riedinger, A.; Guardia, P.; Curcio, A.; Garcia, M. A.; Cingolani, R.; Manna, L.; Pellegrino, T. Subnanometer Local Temperature Probing and Remotely Controlled Drug Release Based on Azo-Functionalized Iron Oxide Nanoparticles. Nano Lett 2013 13, 2399-406. 
  46. Creixell, M.; Bohorquez, A. C.; Torres-Lugo, M.; Rinaldi, C. ́ EGFR-Targeted Magnetic Nanoparticle Heaters Kill Cancer Cells without a Perceptible Temperature Rise. ACS Nano 2011, 5, 7124−7129.
  47. Clinical Magnetic Resonance Imaging. Edelman, R. R.; Hesselink, J. R.; Zlatkin, M. B., Eds.; Saunders Publishing Co: Philadelphia, 1996; pp 2196.
  48. Gobbo, O. L.; Friedrich, W.; Vaes, P.; Teughels, S.; Markos, F.; Edge, D.; Shortt, C.; Crosbie-Staunton, C.; Radomski, M. W.; Volkov, Y.; Prina-Mello, A. Biodistribution and Pharmacokinetic Studies of SPION Using Particle Electron Paramagnetic Resonance, MRI and ICP-MS. Nanomedicine 2015, 10, 1751–1760.
  49. FDA’s Center for Drug Evaluation and Research (CDEER), Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers,  July 2005, Corpus ID: 17535338.
  50. Mahmoudi, K.; Bouras, A.; Bozec, D.; Ivkov, R.; Hadjipanayis, C. Magnetic Hyperthermia Therapy for the Treatment of Glioblastoma: A Review of the Therapy’s History, Efficacy and Application in Humans. Int. J. Hyperthermia 2018, 34, 1316−1328.
  51. Stigliano, R. V.; Shubitidze, F.; Petryk, J. D.; Shoshiashvili, L.; Petryk, A. A.; Hoopes, P. J. Mitigation of Eddy Current Heating During Magnetic Nanoparticle Hyperthermia Therapy. Int. J. Hyperthermia 2016, 32, 735−748.
  52. Sykes, E. A.; Chen, J.; Zheng, G.; Chan, W. C. Investigating the Impact of Nanoparticle Size on Active and Passive Tumor Targeting Efficiency. ACS Nano 2014, 8, 5696−5706.
  53. Dai, Q.; Wilhelm, S.; Ding, D.; Syed, A. M.; Sindhwani, S.; Zhang, Y.; Chen, Y. Y.; MacMillan, P.; Chan, W. C. W. Quantifying the Ligand-Coated Nanoparticle Delivery to Cancer Cells in Solid Tumors. ACS Nano 2018, 12, 8423−8435.
  54. Ganapathy-Kanniappan, S.; Geschwind, J. Tumor Glycolysis as a Target for Cancer Therapy: Progress and Prospects. Mol Cancer 2013, 12, 4598-12. 

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