Superparamagnetic polycrystalline iron oxide nanoparticles for magnetic hyperthermia

Abstract

This invention relates to the utilization of superpararmagnetic polycrystalline iron oxide nanoparticles for magnetic hperthermia applications and methods. The present invention provides a magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites.

Description

SUPERPARAMAGNETIC POLYCRYSTALLINE IRON OXIDE NANOPARTICLES FOR MAGNETIC HYPERTHERMIA CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No.63 / 546,115 filed October 27, 2023, and U.S. Provisional Patent Application No.63 / 655,201 filed June 3, 2024, both of which are incorporated herein by reference in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Nos. FA9550-20- 1-0349 and FA9550-23-1-0581 awarded by the U.S. Air Force Office of Scientific Research, and Grant No. DE-FG02-07ER46438 awarded by the U.S. Department of Energy. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] This invention relates to the utilization of superparamagnetic polycrystalline iron oxide nanoparticles for magnetic hyperthermia applications and methods. BACKGROUND

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] Current technologies predominantly focus on the utilization of single-domain magnetic iron oxide nanoparticles for magnetic hyperthermia applications. However, these single- domain magnetic nanoparticles exhibit two primary disadvantages: (1) weak magnetization and (2) susceptibility to genotoxicity, biochemical toxicity, and the capacity to penetrate deeply into 4876-0545-6626.1 Page 1 of 72094876-000021WOPTsensitive areas due to their small size (typically < 25 nm). While increasing the size of nanoparticles to the ferrimagnetic regime can enhance heating efficiency, it often gives rise to nanoparticle aggregation due to strong magnetic dipole interactions. Therefore, there is an ongoing need for magnetic iron oxide nanoparticles for magnetic hyperthermia applications that overcome these disadvantages. The embodiments of the present invention address these needs. SUMMARY OF THE INVENTION

[0006] In various embodiments, the present invention provides a magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; wherein the plurality of iron oxide primary crystals does not have a core- shell structure; and wherein the at least one superparamagnetic nanoparticle does not have a core- shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

[0007] In some embodiments, the iron oxide is Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the particle size is less than 500 nm, and wherein the crystallite size is less than 15 nm. In some embodiments, the crystallite size is 10 nm to 15 nm. In some embodiments, the particle size is 150 nm to 400 nm, or 160 nm to 375 nm. In some embodiments, the magnetic field is an alternating current magnetic field. In some embodiments, the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject. In some embodiments, the subject is a human subject. In some embodiments, the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof. In some embodiments, the condition is cancer. In some embodiments, the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject. In some embodiments, the subject is a human subject. In some embodiments, the region is a tumor, tissue, organ, limb,4876-0545-6626.1Page 2 of 72094876-000021WOPTor body cavity, or any combination thereof. In some embodiments, the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of cancer cells in the subject. In some embodiments, the subject is a human subject. In some embodiments, the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0009] FIG. 1A – FIG. 1D depicts in accordance with various embodiments of the invention, (FIG. 1A) the qualitative behavior of the coercivity and the domain boundaries in nanoparticles with particle size and crystallite size, (FIG. 1B) Hysteresis loop of superparamagnetic (SPM) particles with no coercivity (HC= 0) and remanent magnetization (Mr= 0), (FIG. 1C) Hysteresis loop of ferromagnetic (FM) multidomain particles with non-zero coercivity (HC≠ 0) and remanent magnetization (Mr≠ 0), and (FIG.1D) Hysteresis loop, domain states, and a 3D visualization of a superparticle showing SPM characteristic.

[0010] FIG. 2A – FIG. 2D depicts in accordance with various embodiments of the invention, (FIG. 2A) SEM image of sample S3 and the inset showing a selected area electron diffraction (SAED) pattern of this sample, (FIG.2B) TEM image of sample S3, (FIG.2C) XRD patterns of all the samples with peaks corresponding to Fe3O4, and (FIG.2D) Raman spectra of all the samples with peaks corresponding to Fe3O4(major phase) and a-Fe2O3(minor phase).

[0011] FIG. 3A – FIG. 3C depicts in accordance with various embodiments of the invention, Magnetic characteristics of sample S6: (FIG. 3A) ZFC and FC M(T) curves measured in an applied field of 0.05 T, (FIG.3B) M(H) curve at 300 K, and (FIG.3C) Coercivity change with temperature.

[0012] FIG. 4A – FIG. 4D depicts in accordance with various embodiments of the invention, temperature vs. time measurements or heating curves for all six samples at 1 mg / ml, and 0.5 mg / ml concentrations in the presence of 600 Oe and 800 Oe AC fields.

[0013] FIG. 5A – FIG. 5B depicts in accordance with various embodiments of the invention, (FIG. 5A) SAR values of the samples in 2% agar at 0.5 mg / ml concentration in the4876-0545-6626.1Page 3 of 72094876-000021WOPTpresence of 400, 600, and 800 Oe AC fields, and (FIG.5B) SAR values of all the samples in 2% agar at 0.5 mg / ml, and 1 mg / ml concentrations in the presence of 800 Oe applied magnetic field.

[0014] FIG. 6A – FIG. 6D depicts in accordance with various embodiments of the invention, SEM (FIG. 6A, FIG. 6C) and TEM (FIG. 6B, FIG. 6D) images of samples 4 and 6, respectively.

[0015] FIG. 7A – FIG. 7D depicts in accordance with various embodiments of the invention, magnetic properties of samples 3 and 4.

[0016] FIG. 8A – FIG. 8B depicts in accordance with various embodiments of the invention, heating curves of the samples in water and their SAR values measured at 800 Oe in both water and agar.

[0017] FIG. 9A – FIG. 9B depicts in accordance with various embodiments of the invention, SAR values measured at 600 and 800 Oe for 0.5 and 1.0 mg / ml in agar.

[0018] FIG.10A – FIG.10B depicts various embodiments of the invention, Superparamagnetic (SPM) Region. Observed in sub-20 nm nanostructures (S ≤ SSPM; Typically, single domain; Zero / negligible coercivity; and Easily controllable via a magnetic field.

[0019] FIG.11A – FIG.11B depicts various embodiments of the invention, Ferromagnetic Single Domain: Smaller than the critical size (SC); Larger coercivity which peaks at SC; Anisotropy, especially shape can cause higher HC; Defects and multiple phases may cause higher HC; and Interparticle interactions may cause higher HC.

[0020] FIG.12A – FIG.12B depicts various embodiments of the invention, Ferromagnetic Multi-Domain: Grain boundary shifts with applied magnetic field; High HCexists with the grain / domain boundaries.

[0021] FIG.13A – FIG.13C depicts various embodiments of the invention, superparticle (SUPA): Large spherical structure; Particle size 150-400 nm; Crystallite size 10-40 nm; and Uniformly distributed sizes.

[0022] FIG.14A – FIG.14B depicts various embodiments of the invention, supa: structure and phase. Biphase existence; Significant Fe3O4phase; Presence of the α-Fe2O3phase observed via Raman Spectra; and Uniformly distributed sizes.

[0023] FIG.15A – FIG.15C depicts various embodiments of the invention, supa: magnetic property, Superparamagnetic-like behavior (Mr~0, HC~0) is observed around room temperature.4876-0545-6626.1Page 4 of 72094876-000021WOPT

[0024] FIG.16A – FIG.16B depicts various embodiments of the invention, magnetic hyperthermia: measured in Measured in DI water and agar; Agar 2% w / w mimics an environment similar to the inside of the body; SUPA indicated higher stability and performance in agar; and Performance was observed to optimize at 0.5 mg / ml.

[0025] FIG.17A – FIG.17B depicts various embodiments of the invention, magnetic hyperthermia: Measured in DI water and agar; Agar 2% w / w mimics an environment similar to the inside of the body; SUPA indicated higher stability and performance in agar; and Performance was observed to optimize at 0.5 mg / ml.

[0026] FIG.18 depicts various embodiments of the invention, Measured at 400, 600, and 800 Oe applied magnetic fields; The SAR improved with the applied field; Highest SAR registered with S1 and can be tuned by varying particle size; and Performance was observed to optimize at 0.5 mg / ml.

[0027] FIG. 19 depicts in accordance with various embodiments of the invention, using different additives in solvothermal synthesis to control the crystallinity and magnetic properties of iron oxide nanoparticles.

[0028] FIG.20 depicts in accordance with various embodiments of the invention, synthesis procedure for highly crystalline iron oxide nanospheres.

[0029] FIG. 21A – FIG. 21D depicts in accordance with various embodiments of the invention, (FIG. 21A) The qualitative behavior of the coercivity and the domain boundaries in nanoparticles with size, (FIG.21B) Hysteresis loop of superparamagnetic (SPM) particles with no coercivity (Hc= 0) and remanent magnetization (Mr= 0), (FIG. 21C) Hysteresis loop of ferro / ferrimagnetic (FM or FiM) multidomain particles with non-zero coercivity (Hc≠ 0) and remanent magnetization (Mr≠ 0), and (FIG. 21D) Hysteresis loop, domain states, and a 3D visualization of a superparticle showing SPM characteristics.

[0030] FIG. 22A – FIG. 22F depicts in accordance with various embodiments of the invention, SEM images of samples (FIG. 22A) S2 and (FIG. 22B) S3; TEM images of samples (FIG.22C) S2, (FIG.22D) S3, and (FIG.22E) S5. The inset of (FIG.22C) shows a selected area electron diffraction (SAED) pattern of sample S2; (FIG.22F) XRD patterns for all samples. The positions of the hkl reflections for bulk Fe3O4are marked for reference.

[0031] FIG. 23A – FIG. 23D depicts in accordance with various embodiments of the invention, Magnetic characteristics of samples: (FIG.23A) ZFC and FC M(T) curves measured in4876-0545-6626.1Page 5 of 72094876-000021WOPTan applied field of 0.05 T for sample S1; (FIG. 23B) Coercivity change with temperature for sample S1 and sample S5 (inset). Experimental and fitted M-H curves at 300 K, with the M(H) loops at 300 K shown in the insets, for (FIG.23C) sample S1 and (FIG.23D) sample S5. The fits were conducted utilizing the SPM model (Eq. (1)).

[0032] FIG. 24A – FIG. 24D depicts in accordance with various embodiments of the invention, Temperature vs. time measurements or heating curves for all five samples at concentrations of 1 mg / mL and 0.5 mg / mL in the presence of 600 Oe and 800 Oe AC fields at a constant frequency of 310 kHz.

[0033] FIG. 25A – FIG. 25B depicts in accordance with various embodiments of the invention, (FIG.25A) SAR values of all SUPA samples in 2% agar at 0.5 mg / mL concentration in the presence of 400, 600, and 800 Oe AC fields, and (FIG.25B) SAR values of all the samples in 2% agar at concentrations of 0.5 and 1 mg / mL in an 800 Oe AC field.

[0034] FIG. 26A – FIG. 26F depicts in accordance with various embodiments of the invention, (FIG.26A – FIG.26C) Dynamic hysteresis loops measured at various frequencies and (FIG.26D – FIG.26F) the corresponding SAR vs. field curves for sample S5 at a concentration of 1.4 mg / mL.

[0035] FIG.27 depicts various embodiments of the invention.

[0036] FIG. 28A – FIG. 28C depicts in accordance with various embodiments of the invention, SEM images of SUPA samples S1 (FIG.28A), S4 (FIG.28B), and S5 (FIG.28C).

[0037] FIG. 29 depicts in accordance with various embodiments of the invention, XPS spectra of SUPA samples S1 and S5. A detailed analysis of these spectra confirms the presence of Fe3O4as a major phase and γ-Fe2O3as a minor phase.

[0038] FIG. 30A – FIG. 30D depicts in accordance with various embodiments of the invention, The temperature-dependent magnetization M(T) curves under ZFC and FC protocols for samples S2 (FIG.30A), S3 (FIG.30B), S4 (FIG.30C), and S5 (FIG.30D). The observation of consistent magnetic behavior suggests that the magnetic properties of the superparticles are predominantly determined by the size of the nanocrystals composing SUPAs.

[0039] FIG. 31A – FIG. 31C depicts in accordance with various embodiments of the present invention, The magnetic field-dependent magnetization M(H) curves, measured at 300 K, for samples S2 (FIG.31A), S3 (FIG.31B), and S4 (FIG.31C). The absence of magnetic hysteresis in these curves confirms the superparamagnetic characteristics of the samples.4876-0545-6626.1Page 6 of 72094876-000021WOPT

[0040] FIG. 32A – FIG. 32B depicts in accordance with various embodiments of the present invention, The M(H) results obtained from the experiment and fitting of the Langevin Function (Eq. (1)) for samples (FIG.32A) S3 and (FIG.32B) S4.

[0041] FIG. 33A – FIG. 33B depicts in accordance with various embodiments of the present invention, (FIG.33A) Heating curves for the samples at a concentration of 1 mg / mL and (FIG.33B) their corresponding SAR values measured at an 800 Oe AC field and a frequency of 310 kHz in both water and agar.

[0042] FIG. 34A – FIG.34B depicts in accordance with various embodiments of the present invention, SAR values of all samples measured at 600 and 800 Oe AC fields for concentrations of (FIG.34A) 0.5 and (FIG.34B) 1.0 mg / mL in agar. DETAILED DESCRIPTION OF THE INVENTION

[0043] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0045] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The definitions4876-0545-6626.1Page 7 of 72094876-000021WOPTand terminology used herein are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

[0046] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, systems, articles of manufacture, apparatus, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open- ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”

[0047] Unless stated otherwise, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” No language in the specification should be construed as indicating any non- claimed element essential to the practice of the application.

[0048] “Optional" or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.4876-0545-6626.1Page 8 of 72094876-000021WOPT

[0049] In some embodiments, the numbers expressing quantities of reagents, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0050] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0051] In various embodiments, compounds of the present invention as disclosed herein may be synthesized using any synthetic method available to one of skill in the art. Non-limiting examples of synthetic methods used to prepare various embodiments of compounds of the present invention are disclosed in the Examples section herein.

[0052] In the context of this disclosure, the term particle size, abbreviated as “ps” or “PS”, refers to “average diameter” for spherical nanoparticles and the “average edge length” for cubic nanoparticles. The term “crystallite size” is abbreviated as “cs” or “CS”, denoting the average crystallite size, also known as the “grain size” of polycrystalline nanoparticles. In some embodiments, the average diameter may be abbreviated by the letter “D”.

[0053] Chapter 14876-0545-6626.1Page 9 of 72094876-000021WOPT

[0054] Iron oxide nanoparticles are promising for a variety of biomedical applications ranging from magnetic hyperthermia therapy to drug delivery and bio-detection, owing to their outstanding superparamagnetic characteristics, non-toxicity, and bio-degradability. While particles of small particle size (below a critical size, for example, ~30 nm) retain superparamagnetic characteristics, these particles tend to penetrate highly sensitive areas of the body such as the Blood Brain Barrier (BBB) leading to unwanted complexities and problems. For example, without being bound by theory, when particles get smaller, they also possess a higher probability of retention leading to genotoxicity and biochemical toxicity. Increasing the particle size (PS) seems to solve these problems, but takes away the desirable superparamagnetic property. Herein, we overcome this particle size-limit challenge by exploring the novel synthesis of superparamagnetic nanoparticles with average particle size (PS) in the range of 150 nm - 400 nm. These so-called “superparticles” are composed of magnetite and maghemite and consist of nanocrystallites of crystallite size 10 nm - 20 nm, allowing us to achieve both superparamagnetic properties and hyperthermia properties. Unexpectedly, the heating efficiency can also be tuned by varying the size of the particles and / or the crystallites, while preserving the superparamagnetic property. We found that the particles of average particle size (PS), for example ~160 nm, with the smallest crystallite size (CS) of 10 nm showed the highest potential for hyperthermia therapy, reaching the potentially promising 40-440C mark. The significant enhancement of the Specific Absorption Rate (SAR > 330 W / g) with a very low 0.5 mg / ml concentration used indicates that a minimal amount of particles is needed in the treatment of cancer. The work disclosed herein paves a new pathway for harnessing the excellent magnetic properties and hyperthermia properties in iron oxide superparticles for advanced biomedical applications.

[0055] The innovative science of nanomaterials is utilized in many day-to-day applications and discoveries. From the initial primary goal to miniaturize, the applications of nanostructures have enabled their use in many connected fields which have in turn facilitated their use in many environments and under a variety of conditions. Dimensions, shapes, and compositions that initially played key roles have shifted to become secondary parameters while phase-tunability and structural ordering have become more prominent topics in fine-tuning nanostructures for applications in the future. Hyperthermia therapy has been investigated as a secondary cancer treatment process with minimal adverse effects on the body while ensuring a higher efficacy in battling cancers via direct and indirect involvement. The authorization obtained from the U.S.4876-0545-6626.1Page 10 of 72094876-000021WOPTFood and Drug Administration (FDA), especially for certain iron oxide compositions and spherical nanostructures has allowed for their use in many therapeutic and treatment processes. The use of such therapeutic technologies on a large scale due to less invasive, minimal side-effects, etc., has further given hope in making them more effective and safer, thereby paving the way for acceptance and innovation. The use of nanostructures in highly sensitive areas within the body such as the remarkably sensitive blood-brain barrier (BBB), a semi-permeable membrane, and the gateway to the central nervous system, tends to carry an imminently high risk to the life of a patient. This is particularly true as many nanostructures possess the ability to cross the blood-brain barrier.

[0056] Without being bound by theory, superparamagnetism (SPM), generally tends to be a characteristic found in structures that are less than 20 nm in particle size. Superparamagnetic structures are easily detected, possess high efficacy and controllability. Superparamagnetic structures have been explored in hyperthermia treatment processes, drug delivery and targeting, and magnetic memory devices. The synergistic approach required to protect SPM while mitigating the risk in vulnerable areas has led us to scrutinize the superparamagnetic structures more closely and propose a new, innovative approach (FIG.1A – FIG.1D) to solve the aforementioned issues, through the concept of “Superparticle” (SUPA) structures of 100 nm - 400 nm particle size. These superparticle (SUPA) structures eliminate potential hazards while retaining the SPM nature within the 10 nm - 20 nm crystallite size range. These 3D comparatively large spherical structures of iron oxide are projected to conform to FDA guidelines with minimal curation. The efficiency and effectiveness of these superparticle (SUPA) structures were evaluated using hyperthermia measurements which showed significantly high specific absorption rates (SARs). In the work disclosed herein, we focused on SUPAs in 2% agar, since these conditions tend to mimic similar conditions found in the actual biological environment. The structures have certain uniformity in the process of the measurement which was carried out for 900 s to get an idea of the potential. The initial slope method, which is typically used to determine SAR values, was not employed with the SUPAs since the slope showed a concave up, increasing graph in the first ~60 s as the SUPA interactions established stability. Such fascinating features of this novel approach have shown the potential of SUPAs for the benefit of humankind, where more than 10 million fatalities are caused by cancer and cancer-related effects annually.

[0057] Novel approach to overcoming the particle size limit.4876-0545-6626.1Page 11 of 72094876-000021WOPT

[0058] Magnetic nanostructures ranging from a couple of nanometers to a few hundred nanometers can be altered to have various properties of size, shape, and composition. The SPM behavior found in ferromagnetic (FM) and ferrimagnetic (FiM) materials is a characteristic with zero coercivity (HC) and remanence magnetization (Mr) above the blocking temperature (TB). Upon size reduction, it has been shown that HCvaries with particle size / grain size in a complex manner. Without being bound by theory, Hcincreases with decreasing particle size / grain size for multidomain materials (particle size / grain size > SC), reaches a maximum at the critical size (SC) at which the system transforms from the multiple domain (MD) state to the single domain (SD) state), declines to zero when particle size / grain size decreases to SSPM, and retains zero as particle size / grain size below SSPM(FIG. 1A). Without being bound by theory, generally, in the multidomain regime, FM materials possess a significant HCsince grain boundaries inhibit the propagation of magnetization reversal or magnetic domain boundary movement which depends on the magnetic anisotropy, and nucleation and growth of reverse domains. As the structure morphs into a single domain from a multi-domain system, this makes the resulting single domain difficult to align with the applied field, yielding the maximum HCat the SC. Without being bound by theory, with further size decreases, the surface area and the volume which depend on the exchange interactions and thermal energy decimate, enabling the applied field to govern the spins via thermal energy.

[0059] This mechanism enables the structures to show SPM behavior with no coercivity and remanent magnetization (FIG.1B), as the crystallite size (CS) of the structure falls below the SPM size limit (SSPM). Hyperthermia therapy, drug delivery applications, contrast agents in MRI, etc. which heavily depend on the controllability of the nanostructure, favor structures to closely behave to the applied field as observed in SPM materials which require a relatively smaller field to reach MS. Research approaches have been carried out to ensure the biocompatibility and ease of usage of these therapeutic procedures. General safety of the composition and stability does not necessarily speak for the application safety since SPM nanostructures which are generally tiny overshadow safety aspects due to their potential to penetrate sensitive organs and systems, for example, like the blood-brain-barrier (BBB). Usage of controlled amounts, functionalizing, and ensuring the nanoparticles (NP) are localized and extracted after application are a few of the methods used to counter their potentially adverse effects on such sensitive areas. Such measures may be effective, but could potentially lead to harmful and adverse effects in normal brain4876-0545-6626.1Page 12 of 72094876-000021WOPTfunction, localized and systemic inflammation, etc. The BBB which consists of a majority endothelial cell-cell diffusion barrier, selectively blocks the penetration of most blood-borne substances from entering the brain. To ensure the controllability and effectiveness of SPM NPs along with the guarantee of impenetrability across highly critical and sensitive organs and environments, the concept of “Superparticles or SUPAs” can be a possible solution. Unlike the case of multidomain FM particles (FIG.1C), SUPAs possess a CS smaller than the SSPM, securing SPM features but with PSs above the SCwarrantying impassability through vital barriers (FIG. 1D). The experimental results presented and discussed herein below prove our hypothesis.

[0060] Structural Characterization

[0061] The structural evaluation of the SUPA samples was initially carried out using SEM to determine general morphology and uniformity. The particle sizes of the SUPA system enabled us to categorize them to varying PS (particle size) ranging from 160 nm – 375 nm, as summarized in Table 1. The uniformity of the structures in all six samples was consistent with no significant structural deviations (FIG.6A – FIG.6B).

[0062] Furthermore, TEM was devised to evaluate the structural integrity, interparticle adjacency, and superficial crystallite distribution. A closer look via the subsequent qualitative analysis yielded a crystallite size (CS) distribution which stayed below 15 nm in all the structures. The selected area electron diffraction (SAED) shown in FIG.2A inset obtained using TEM shows the crystal nature of the structure, with amorphous materials showing a diffused ring pattern with no significant ring structures, which is not the case in SUPAs. This reflects the long-range order inherent in crystalline structures. The planes can be indexed back to the composition, and the presence of dislocations in the structure has led to the streaks, and partial rings speaking for the preferred orientations in the structures. During post-qualitative analysis, the composition was examined using XRD spectroscopy, which positively concluded the presence of magnetite (Fe3O4), with peaks perfectly aligning, but further analysis was expanded using RS to determine subordinate iron oxide phases. This indeed assisted in ascertaining the hematite (a-Fe2O3) phase which is understood to be less significant in the structures. The volume fraction of such structures has been understood to be tunable via annealing and structural changes which expands the potential of the SUPAs to a new paradigm. The comparison of nanostructures shown in Table 2 confirms the absence of SPM behavior in ordinary nanostructures with the increment of the PS. The4876-0545-6626.1Page 13 of 72094876-000021WOPTfollowing in Table 2 are iron oxide-based nanostructures of different shapes and particle sizes for comparison with SUPAs.

[0063] Table 1: Magnetic and hyperthermia properties of the iron oxide superparticles. )

[0064] Table 2: Magnetic and hyperthermia properties of some candidate materials. Ref. Sample Particle Concentration SAR in MSat 300 K HC) 04876-0545-6626.1Page 14 of 72094876-000021WOPT2 Nanorods 41 1 350 86 ~0 0

[0065] Magnetic Properties The temperature dependence of magnetization, M(T) with zero-field cooled warming (ZFCW), field-cooled cooling (FCC), and field-cooled warming (FCW) protocols shown in FIG. 3A were measured sequentially over 10 K ≤ T ≤ 350 K range in the presence of a μ0H = 0.05 T applied field. The ZFCW showed saturation with warming, with a significantly high slope until ~45 K which is reported in the literature as the spin freezing temperature (TF) found in Fe3O4. Further warming showed a slope change around ~200 K which without being bound by theory can be understood to be due to the Morin transition (MT) with the presence of a-Fe2O3, which arises due to the gradual change from low temperature antiferromagnetic (AFM) to high temperature weakly ferromagnetic as the spin alignment is along the rhombohedral z-axis with zero net magnetic to perpendicular to the rhombohedral z-axis. MT which is usually found around ~260 K, especially in bulk a-Fe2O3is found to get suppressed, reaching the lows of 165 K with the high surface-to-volume ratio in SUPAs. Without being bound by theory, the expected Verwey transition was absent in the structure possibly due to the significantly small crystallite sizes along with defects more prominent in spherical structures compared to its counterparts. Without being bound by theory, the decreasing slope of the FC curves in all the samples at low temperatures below the crystallite blocking temperature (^^^) can be accounted for by the interparticle interactions, below which the sample showed significant FM features. The bifurcation of the ZFCW and FCC curves, represented by the blocking temperature (TB) stayed below 300 K in all the samples indicating room-temperature SPM features. Sample S6, the largest particle size (PS) sample, clearly showed SPM features at room temperature as shown in FIG.3B, and thus SPM features are expected to be observed in other samples which possess smaller particle size (PS) compared to sample S6. When closely analyzing the HCvs. T plot in FIG.3C, a significant decrease in HCbelow TBCis observed, which shows that the SUPAs attain an SPM-like behavior above^^^ distancing itself from general FM features.4876-0545-6626.1Page 15 of 72094876-000021WOPT

[0066] Without being bound by theory, the FC curves which tend to flatten out when reaching lower temperatures indicate that the sample has kept its magnetization constant, while upwards or downward shifting of the FC curve at low temperatures hints at the interparticle interactions which actively take part as the thermal energy decreases. Here, as the thermal energy decreases the magnetization drops since the interparticle attractions have made the SUPAs difficult to be aligned with the applied magnetic field. Similar observations were made for other samples (FIG.7).

[0067] Magnetic Hyperthermia Properties

[0068] The evaluation of the heating efficiency of the SUPAs for potential hyperthermia therapy was evaluated in both water and 2% weight agar solution at 0.5 mg / mL and 1 mg / mL which are comparatively lower concentration levels. The agar solution enabled us to restrict the physical movement of SUPAs, which also mimicked the cell environment such as cell cytoplasm and extracellular matrix. The samples showed quite consistent results with sample S1 with the smallest particle size (PS) and crystallite size (CS) showing significantly higher heating capabilities in all the concentrations measured. The heating capabilities of the samples for the hyperthermia treatment process were evaluated for a maximum of 15 minutes (900 s) which is low in comparison to the treatment which typically ranges from 30 minutes to 1 hour. The natural cooling observed after the removal of HACindicated satisfactory retainment of heat, which is a promising feature.

[0069] To further understand the potential of the samples, Specific Absorption Rate (SAR) which is an index that enables comparison and extrapolation of the absorption energy, usually using the initial rate of temperature rise, as SAR tends to decrease with time. Unexpectedly, in the case of SUPAs this standard behavior is not seen but the sample tends to register a higher SAR after some time. In our calculations we measured the SAR after 60 seconds since the SUPAs tend to become stabilized with time. Without being bound by theory this lag can be understood to be due to the changes within the SUPA with the initial application of the field. ^^^^ ^^^ ൌ^^ ൈ^4876-0545-6626.1Page 16 of 72094876-000021WOPT

[0070] Here the DT / Dt represents the change in temperature with time, and CPrepresents the heat capacity of the liquid solvent (in this case, water with 4.186 J / g∙K). φ represents a unitless quantity: mass of magnetic material per unit mass of liquid solvent. FIG.5A shows all the samples at 0.5 mg / ml concentration in 2% Agar of which the applied field (HAC) of 800 Oe showed the highest SAR values, all of which were comfortably above 200 W / g, with sample S1 above 300 W / g. The SUPAs were initially evaluated with de-ionized water (FIG. 8) which led to drops in temperature with the HAC. Without being bound by theory, this is understood to be unique to SUPAs due to their large particle size (PS) and small crystallite size (CS), even though they showed higher SAR values when calculated with the initial rate of temperature, the inconsistency caused due to the temperature drops led them not to be considered for the SAR calculations. Similar to the peculiar observation related to water, the concentration increments which usually lead to higher SAR values were not observed in SUPAs but rather a drop. Without being bound by theory, this may be due to the interparticle interactions which intensify with the increased presence of SUPAs per unit volume. This is clearly shown in FIG. 5B. Furthermore, extensive field-dependent and concentration-dependent measurements were carried out which further reinforced the prior observations (FIG.9).

[0071] It is worth noting in Table 1 and Table 2 that while the conventionally synthesized iron oxide nanoparticles with average particle sizes of 40 nm or more showed FM behavior, our superparticles with particle sizes up to 400 nm retain SPM characteristics. The SPM properties together with the tunable SARs make the iron oxide superparticles of the present invention compelling candidates for a wide range of biomedical applications.

[0072] In various embodiments of the present invention, iron oxide superparticles of particle sizes 160 nm and up with 10-15 nm crystallite sizes showed superparamagnetic features at room temperature. All the samples studied except sample S6 showed a specific absorption rate of more than 250 W / g at 800 Oe applied field, with the highest at 333 W / g in sample S1 with the smallest particle size and crystallite size of all the samples. All the samples studied displayed exceptional stability in the 2% by-weight agar solution at both 0.5 mg / ml and 1 mg / ml concentrations, which concurs with the feasibility of using the superparticles in in vivo studies, particularly as the composition and structures conform with internationally accredited safety agencies such as the U.S Food and Drug Administration (FDA). Proposed future work includes without limitation mechanistic studies, and cytotoxicity and biocompatibility studies.4876-0545-6626.1Page 17 of 72094876-000021WOPT

[0073] In various embodiments the present invention describes the utilization of superparamagnetic polycrystalline iron oxide nanoparticles for magnetic hyperthermia applications. The magnetic iron oxide nanoparticles, characterized by their larger size (ranging from 150 to 400 nm) and smaller crystallite size (grain size of <15 nm), exhibit excellent superparamagnetic properties, rendering them suitable for use in both magnetic hyperthermia and drug delivery applications. Experimental results demonstrate that these magnetic nanoparticles, when dispersed in agar medium at concentrations of 0.5 mg / mL and 1.0 mg / mL, exhibit rapid and efficient heating efficiency upon the application of an alternating current magnetic field. The Specific Absorption Rates (SAR) can reach up to 330 W / g at low concentration of 0.5 mg / mL indicate the capability in cancer treatment with minimum dose. This innovation underscores the potential of large polycrystalline iron oxide nanoparticles with superparamagnetic properties for advanced biomedical applications, particularly in the realms of magnetic hyperthermia and drug delivery.

[0074] Magnetic hyperthermia therapy utilizes magnetic nanoparticles to generate heat through the induction of an alternating current (AC) magnetic field. When exposed to this AC magnetic field, magnetic nanoparticles can elevate their temperature from the body's natural level to the effective hyperthermia range of 40 – 45°C, making them highly efficient for cancer treatment. Additionally, these magnetic nanoparticles can be coated with heat-responsive polymers for controlled drug delivery applications. Traditionally, magnetic hyperthermia has primarily focused on the use of single-domain iron oxide nanoparticles with superparamagnetic properties. However, due to their small size, these nanoparticles exhibit limited heating efficiency, arising from their low magnetization. Transitioning to larger nanoparticles with greater magnetization can enhance heat generation efficiency, but it often gives rise to the emergence of ferrimagnetic properties, leading to magnetic clustering due to strong magnetic dipole interactions. Moreover, the smaller size of nanoparticles raises concerns about their ability to penetrate highly sensitive areas, which can give rise to potential issues related to genotoxicity and biochemical toxicity. The use of large polycrystalline nanoparticles with superparamagnetic properties offers an efficient solution to address these existing problems of single-domain nanoparticles.

[0075] Current technologies predominantly focus on the utilization of single-domain magnetic iron oxide nanoparticles for magnetic hyperthermia applications. However, these single- domain magnetic nanoparticles exhibit two primary disadvantages: (1) weak magnetization and4876-0545-6626.1Page 18 of 72094876-000021WOPT(2) susceptibility to genotoxicity, biochemical toxicity, and the capacity to penetrate deeply into sensitive areas due to their small size (typically < 25 nm). While increasing the size of nanoparticles to the ferrimagnetic regime can enhance heating efficiency, it often leads to nanoparticle aggregation due to strong magnetic dipole interactions. Therefore, this invention aims to overcome the current limitations of single-domain superparamagnetic nanoparticles with weak magnetization capabilities by employing larger superparamagnetic nanoparticles. These larger superparamagnetic nanoparticles, with sizes ranging from 150 to 400 nm and small crystallite sizes (<15 nm), exhibit excellent superparamagnetic properties suitable for magnetic hyperthermia. Moreover, they alleviate concerns related to deep penetration into highly sensitive areas, genotoxicity, and biochemical toxicity associated with single-domain nanoparticles.

[0076] Iron-oxide-based magnetic nanostructures are well-known for their non-toxicity and biodegradability, along with their potential applications in different fields ranging from cancer treatments to spintronics devices. On the contrary, due to the small size of these nanoparticles, they tend to penetrate highly sensitive areas of the body such as the Blood Brain Barrier (BBB), leading to complexities. Additionally, the smaller nanoparticles, with their inherent higher reactivity, have a higher probability of retention, which can lead to genotoxicity and biochemical toxicity. However, with an increase in particle size (> ~30 nm), the excellent superparamagnetic properties that are desirable for biomedical applications including magnetic hyperthermia therapy and drug delivery are lost. These grand challenges have motivated us to explore the development of large superparamagnetic nanoparticles with average diameters in the range of 150-400 nm comprised of small nanocrystallites 10–15 nm in diameter. These so-called "superparticles" contain both magnetite and maghemite phases. Here, we have systematically studied the influence of the size of the superparticles and their crystallite size on their magnetic and hyperthermia responses. We found that the particles having an average diameter of ~160 nm with the smallest crystallite diameter of 10 nm showed the highest potential, reaching the promising 40-44 °C mark. The significant enhancement of the Specific Absorption Rates (SAR > 330 W / g) generated with a low 0.5 mg / ml concentration indicates that a minimal amount of nanoparticles is needed for cancer treatment. We also found that for similar crystallite sizes, the smaller superparticles were more efficient than the larger ones. While all samples showed nearly the same saturation magnetization, the coercivity decreased with both superparticle size and crystallite size. This study enables us to determine the ideal particle-to-crystallite size combination to harness the superparamagnetic and4876-0545-6626.1Page 19 of 72094876-000021WOPThyperthermia properties for advanced biomedical applications of magnetic iron oxide nanoparticles.

[0077] Chapter 2

[0078] Iron oxide (e.g., Fe3O4or ^J-Fe2O3) nanoparticles are promising candidates for a variety of biomedical applications ranging from magnetic hyperthermia therapy to drug delivery and bio-detection, due to their superparamagnetism, non-toxicity, and biodegradability. While particles of small size (below a critical size, ~20 nm) display superparamagnetic behavior at room temperature, these particles tend to penetrate highly sensitive areas of the body such as the Blood- Brain Barrier (BBB), leading to undesired effects. In addition, these particles possess a high probability of retention, which can lead to genotoxicity and biochemical toxicity. Increasing particle size is a means for addressing these problems but also suppresses the superparamagnetism. We have overcome this particle size limit by synthesizing unique polycrystalline iron oxide nanoparticles composed of multiple nanocrystals of 10 to 15 nm size while tuning particle size from 160 to 400 nm. These so-called superparticles preserve superparamagnetic characteristics and exhibit excellent hyperthermia responses. The specific absorption rates (SAR) exceed 250 W / g (HAC= 800 Oe, f = 310 kHz) at a low concentration of 0.5 mg / mL, indicating their capability in cancer treatment with minimum dose. Our study underscores the potential of size-tunable polycrystalline iron oxide superparticles with superparamagnetic properties for advanced biomedical applications and sensing technologies.

[0079] Introduction

[0080] Nanotechnology, the science of material manipulation at the atomic level, continues to play a significant role in everyday applications (I. Chauhan, M. Yasir, M. Verma, A. P. Singh, Adv. Pharm. Bull.2020, 10, 150; J. Conde, R. Langer, J. Rueff, Nat. Nanotechnol.2023, 18, 537; T. I. Janjua, Y. Cao, A. Ahmed-Cox, A. Raza, M. Moniruzzaman, D. T. Akhter, N. L. Fletcher, M. Kavallaris, K. J. Thurecht, A. Popat, J. Control. Release 2023, 357, 161; M. Omidiyan, P. Srinoi, P. Tajalli, T. R. Lee, ACS Appl. Nano Mater.2024, 7, 8377; R. Medhi, P. Srinoi, N. Ngo, H.-V. Tran, T. R. Lee, ACS Appl. Nano Mater. 2020, 3, 8557). Its impact spans across diverse fields, including electronics, computing, medicine, healthcare, energy, and environmental sectors. Traditionally, the dimensions, shapes, and compositions of materials have been central to nanotechnology research (N. M. Ngo, H.-V. Tran, T. R. Lee, ACS Appl. Nano Mater. 2022, 5, 14051 ; R. Medhi, M. D. Marquez, T. R. Lee, ACS Appl. Nano Mater.2020, 3, 6156). However,4876-0545-6626.1Page 20 of 72094876-000021WOPTthese parameters have become secondary to the control of phase-tunability and structural ordering, which allows for the fine-tuning of nanostructures for current and future applications (S. B. Attanayake, A. Chanda, T. Hulse, R. Das, M.-H. Phan, H. Srikanth, Nanomaterials 2023, 13, 1340; L. Chen, R. Luque, Y. Li, Chem. Soc. Rev.2017, 46, 4614; F. A. Jung, M. Schart, L. Bührend, E. Meidinger, J.-J. Kang, B.-J. Niebuur, S. Ariaee, D. S. Molodenskiy, D. Posselt, H. Amenitsch, C. Tsitsilianis, C. M. Papadakis, Adv. Funct. Mater.2021, 31, 2102905; M. D. Nguyen, H.-V. Tran, S. Xu, T. R. Lee, Appl. Sci.2021, 11, 11301; Y.-T. Chen, R. Medhi, I. Nekrashevich, D. Litvinov, S. Xu, T. R. Lee, Anal. Chem.2018, 90, 6749; A. G. Kolhatkar, Y.-T. Chen, P. Chinwangso, I. Nekrashevich, G. C. Dannangoda, A. Singh, A. C. Jamison, O. Zenasni, I. A. Rusakova, K. S. Martirosyan, D. Litvinov, S. Xu, R. C. Willson, T. R. Lee, ACS Omega 2017, 2, 8010)

[0081] Magnetic hyperthermia therapy utilizes magnetic nanoparticles (magnetite Fe3O4or maghemite J-Fe2O3) to generate heat through the induction of an alternating current (AC) magnetic field (E. A. Périgo, G. Hemery, O. Sandre, D. Ortega, E. Garaio, F. Plazaola, F. J. Teran, Appl. Phys. Rev.2015, 2, 041302; X. Liu, Y. Zhang, Y. Wang, W. Zhu, G. Li, X. Ma, Y. Zhang, S. Chen, S. Tiwari, K. Shi, S. Zhang, H. M. Fan, Y. X. Zhao, X.-J. Liang, Theranostics 2020, 10, 3793; D. Chang, M. Lim, J. A. C. M. Goos, R. Qiao, Y. Y. Ng, F. M. Mansfeld, M. Jackson, T. P. Davis, M. Kavallaris, Front. Pharmacol.2018, 9, DOI 10.3389 / fphar.2018.00831; G. C. Lavorato, R. Das, J. A. Masa, M.-H. Phan, H. Srikanth, Nanoscale Adv. 2021, 3, 867; H. Gavilán, S. Kumar Avugadda, T. Fernández-Cabada, N. Soni, M. Cassani, B. T. Mai, R. Chantrell, T. Pellegrino, Chem. Soc. Rev.2021, 50, 11614). When exposed to this AC magnetic field, magnetic nanoparticles can elevate the temperature of the body from the natural level to the effective hyperthermia range of 40 to 43°C, making them highly efficient for cancer treatment (E. A. Périgo, G. Hemery, O. Sandre, D. Ortega, E. Garaio, F. Plazaola, F. J. Teran, Appl. Phys. Rev. 2015, 2, 041302; X. Liu, Y. Zhang, Y. Wang, W. Zhu, G. Li, X. Ma, Y. Zhang, S. Chen, S. Tiwari, K. Shi, S. Zhang, H. M. Fan, Y. X. Zhao, X.-J. Liang, Theranostics 2020, 10, 3793). Hyperthermia therapy has been extensively explored as a secondary cancer treatment process with minimal adverse effects on the body while ensuring high efficacy in combating cancers via direct and indirect involvement (E. A. Périgo, G. Hemery, O. Sandre, D. Ortega, E. Garaio, F. Plazaola, F. J. Teran, Appl. Phys. Rev.2015, 2, 041302; G. Y. Yi, M. J. Kim, H. I. Kim, J. Park, S. H. Baek, Antioxidants 2022, 11, 625; M. IJff, J. Crezee, A. L. Oei, L. J. A. Stalpers, H. Westerveld, Int. J. Gynecol. Cancer 2022, 32, DOI 10.1136 / ijgc-2021-002473; A. Ademaj, D. P. Veltsista, P. Ghadjar, D.4876-0545-6626.1Page 21 of 72094876-000021WOPTMarder, E. Oberacker, O. J. Ott, P. Wust, E. Puric, R. A. Hälg, S. Rogers, S. Bodis, R. Fietkau, H. Crezee, O. Riesterer, Cancers 2022, 14, 625; T. Mantso, S. Vasileiadis, I. Anestopoulos, G. P. Voulgaridou, E. Lampri, S. Botaitis, E. N. Kontomanolis, C. Simopoulos, G. Goussetis, R. Franco, K. Chlichlia, A. Pappa, M. I. Panayiotidis, Sci. Rep.2018, 8, 10724). The U.S. Food and Drug Administration (FDA) has approved certain iron oxide compositions and spherical nanostructures for the usability in many treatment processes (F. Soetaert, P. Korangath, D. Serantes, S. Fiering, R. Ivkov, Adv. Drug Deliv. Rev. 2020, 163–164, 65; A. S. Thakor, J. V. Jokerst, P. Ghanouni, J. L. Campbell, E. Mittra, S. S. Gambhir, J. Nucl. Med.2016, 57, 1833; D. Bobo, K. J. Robinson, J. Islam, K. J. Thurecht, S. R. Corrie, Pharm. Res. 2016, 33, 2373; R. A. Revia, M. Zhang, Mater. Today 2016, 19, 157). The large-scale usage of such therapeutic technologies, due to their less invasive nature and minimal side effects, has further given hope in making them more effective and safer (H. Peeters, E. M. van Zwol, L. Brancato, M. G. M. C. da Cunha, J. Bogers, Int. J. Hyperth.2022, 39, 806). The application of nanostructures in highly sensitive areas of the body such as the remarkably sensitive blood-brain barrier (BBB) -- a semi-permeable membrane and the gateway to the central nervous system -- tends to carry an imminent high risk to the life of a patient as particles in the nano-level possess the caliber to pass through (C. H. Sutton, F. B. Carroll, Radio Sci.1979, 14, 329; M. Segarra, M. R. Aburto, A. Acker-Palmer, Trends Neurosci.2021, 44, 393; R. R. Shivers, J. A. Wijsman, in Progress in Brain Research (Eds.: H. S. Sharma, J. Westman), Elsevier, 1998, pp. 413–424). Therefore, it is essential to exploit magnetic nanoparticles with tunable sizes for such applications.

[0082] Unlike ferro / ferri-magnetism, superparamagnetism, which generally tends to establish in structures less than 20 nm (with zero remanent magnetization, Mr~ 0 and coercivity, Hc~ 0), is advantageous due to its easy manipulation, detectability, higher efficacy, and controllability, and is attractive for use in hyperthermia treatment processes, drug delivery and targeting, biodetection, and magnetic memory devices (H. Mok, M. Zhang, Expert Opin. Drug Deliv. 2013, 10, 73; I. S. Caizer, C. Caizer, Int. J. Mol. Sci. 2022, 23, 4350; G. Koplovitz, G. Leitus, S. Ghosh, B. P. Bloom, S. Yochelis, D. Rotem, F. Vischio, M. Striccoli, E. Fanizza, R. Naaman, D. H. Waldeck, D. Porath, Y. Paltiel, Small 2019, 15, 1804557; D. Vodenicarevic, N. Locatelli, A. Mizrahi, J. S. Friedman, A. F. Vincent, M. Romera, A. Fukushima, K. Yakushiji, H. Kubota, S. Yuasa, S. Tiwari, J. Grollier, D. Querlioz, Phys. Rev. Appl. 2017, 8, 054045; T. Neuberger, B. Schöpf, H. Hofmann, M. Hofmann, B. von Rechenberg, J. Magn. Magn. Mater.4876-0545-6626.1Page 22 of 72094876-000021WOPT2005, 293, 483). Traditionally, magnetic hyperthermia has primarily focused on the use of single- domain iron oxide nanoparticles with superparamagnetic (SPM) properties (E. A. Périgo, G. Hemery, O. Sandre, D. Ortega, E. Garaio, F. Plazaola, F. J. Teran, Appl. Phys. Rev. 2015, 2, 041302; X. Liu, Y. Zhang, Y. Wang, W. Zhu, G. Li, X. Ma, Y. Zhang, S. Chen, S. Tiwari, K. Shi, S. Zhang, H. M. Fan, Y. X. Zhao, X.-J. Liang, Theranostics 2020, 10, 3793; G. C. Lavorato, R. Das, J. A. Masa, M.-H. Phan, H. Srikanth, Nanoscale Adv. 2021, 3, 867; V. O. Jimenez, K. Y. Hwang, D. Nguyen, Y. Rahman, C. Albrecht, B. Senator, O. Thiabgoh, J. Devkota, V. D. A. Bui, D. S. Lam, T. Eggers, M.-H. Phan, Biosensors 2022, 12, 517). The use of SPM nanostructures has been further supported in hyperthermia treatments due to the facilitation of low agglomeration levels with increased dispersibility but without remanent magnetization, as the magnetization is easily flipped by the thermal energy, which exceeds the magnetic anisotropy energy (M. Suter, O. Ergeneman, J. Zürcher, S. Schmid, A. Camenzind, B. J. Nelson, C. Hierold, J. Micromech. Microeng.2011, 21, 025023; R. Müller, R. Hergt, S. Dutz, M. Zeisberger, W. Gawalek, J. Phys. Condens. Matter 2006, 18, S2527). However, due to their small size, these nanoparticles exhibit limited heating efficiency, arising from their low magnetization. Transitioning to larger nanoparticles with greater magnetization can enhance heat generation efficiency, but often leads to the emergence of ferromagnetic (FM) or ferrimagnetic (FiM) properties, which gives rise to magnetic clustering due to strong magnetic dipole interactions (Y.-T. Chen, R. Medhi, I. Nekrashevich, D. Litvinov, S. Xu, T. R. Lee, Anal. Chem. 2018, 90, 6749; D. Chang, M. Lim, J. A. C. M. Goos, R. Qiao, Y. Y. Ng, F. M. Mansfeld, M. Jackson, T. P. Davis, M. Kavallaris, Front. Pharmacol. 2018, 9, DOI 10.3389 / fphar.2018.00831). As noted above, the smaller size of nanoparticles also raises concerns about their ability to penetrate highly sensitive areas, which can give rise to potential issues related to genotoxicity and biochemical toxicity. In this context, the use of large and size-tunable polycrystalline nanoparticles with SPM properties offers an efficient solution to address the existing problems of single-domain magnetic nanoparticles.

[0083] The synergistic approach required to protect superparamagnetism while mitigating the risk in vulnerable areas has led us to scrutinize the structures more closely and propose a novel approach as illustrated in FIG. 21A – FIG. 21D. Very large polycrystalline iron oxide nanoparticles, hereby named Superparticles (SUPAs), in the particle size range of 150 to 400 nm, can be synthesized, ruling out potential hazards while preserving the SPM nature owing to the 10 to 15 nm sizes of nanocrystals (crystallite size / grain size) within each SUPA that are smaller than4876-0545-6626.1Page 23 of 72094876-000021WOPTthe SPM size limit (~20 nm) (T. Wang, D. LaMontagne, J. Lynch, J. Zhuang, Y. Charles Cao, Chem. Soc. Rev. 2013, 42, 2804; R. K. Thapa, J. O. Kim, J. Pharm. Investig. 2023, 53, 19; F. Rodríguez, P. Caruana, N. De la Fuente, P. Español, M. Gámez, J. Balart, E. Llurba, R. Rovira, R. Ruiz, C. Martín-Lorente, J. L. Corchero, M. V. Céspedes, Biomolecules 2022, 12, 784) In this study, our emphasis was on exploring the magnetic hyperthermia characteristics of superparamagnetic SUPAs in a medium of 2% agar since it closely replicates conditions found in biological environments (J. Alonso, H. Khurshid, V. Sankar, Z. Nemati, M. H. Phan, E. Garayo, J. A. García, H. Srikanth, J. Appl. Phys. 2015, 117, 17D113; B. B. Lahiri, S. Ranoo, J. Philip, J. Magn. Magn. Mater. 2018, 451, 254; M. Salloum, R. H. Ma, D. Weeks, L. Zhu, Int. J. Hyperth. 2008, 24, 337). This novel approach has shown the potential of our SUPAs for the benefit of global cancer treatment since nearly 20 million new cancer patients and 10 million deaths are reported annually due to cancer and cancer-related effects (International Agency for Research on Cancer (IARC), “Global Cancer Observatory,” gco.iarc.fr). The preservation of superparamagnetism in SUPAs with particle size tunability up to 400 nm also highlights them as excellent candidates for many other applications including the detection of single particle-based cells and targeted drug delivery.

[0084] Novel Approach to Overcoming the Superparamagnetic Particle Size Limit

[0085] Magnetic nanostructures, ranging from a few nanometers to a few hundred nanometers, offer the capacity to tune their magnetic properties based on size, shape, crystallinity, and composition (M. D. Nguyen, H.-V. Tran, S. Xu, T. R. Lee, Appl. Sci.2021, 11, 11301; G. C. Lavorato, R. Das, J. A. Masa, M.-H. Phan, H. Srikanth, Nanoscale Adv. 2021, 3, 867; Y. Jun, J. Seo, J. Cheon, Acc. Chem.41, 179; M.-H. Phan, J. Alonso, H. Khurshid, P. Lampen- Kelley, S. Chandra, K. Stojak Repa, Z. Nemati, R. Das, Ó. Iglesias, H. Srikanth, Nanomaterials 2016, 6, 221; S. D. Shingte, A. H. Phakatkar, E. McKiernan, K. Nigoghossian, S. Ferguson, R. Shahbazian-Yassar, D. F. Brougham, Chem. Mater. 2022, 34, 10801). The SPM behavior observed in ferromagnetic (FM) and ferrimagnetic (FiM) materials implies a magnetization versus magnetic field response with zero coercivity (Hc) and zero remanence magnetization (Mr) above the blocking temperature (TB) (Y. Jun, J. Seo, J. Cheon, Acc. Chem. Res.2008, 41, 179). Upon size reduction, Hcvaries with particle / grain size in a complex manner; (Y. Jun, J. Seo, J. Cheon, Acc. Chem. Res. 2008, 41, 179; G. Herzer, IEEE Trans. Magn. 1990, 26, 1397) it increases with decreasing particle / grain size for multidomain magnetic systems (particle / grain size, D > Dc),4876-0545-6626.1Page 24 of 72094876-000021WOPTreaches a maximum at a critical size (Dc) at which the system transforms from the multidomain (MD) state to the single domain (SD) state, declines to zero when particle / grain size decreases to DSPM(the SPM size limit), and remains zero as the particle / grain size is below DSPM(FIG.21A). In the MD regime, FM or FiM materials generally possess significant Hcvalues since grain boundaries inhibit propagation of the magnetization reversal or magnetic domain boundary movement, which depends on the magnetic anisotropy and nucleation and growth of reverse domains (E. C. Stoner, E. P. Wohlfarth, Nature 1947, 160, 650; J. D. Livingston, J. Appl. Phys. 1981, 52, 2544). As the structure morphs into an SD from an MD system, the resulting SD becomes increasingly difficult to align with the applied field, yielding the maximum Hcat the Dc. With further decreases in particle / grain size, the thermal energy becomes increasingly important in governing the spin orientation above a certain critical temperature (O. Petracic, Superlattices and Microstruct.2010, 47, 569).

[0086] This mechanism enables the structures to show SPM behavior with no coercivity and no remanent magnetization (FIG.21B), as the crystallite size (dcs) of the structure falls below DSPM. Unlike the cases of large SD FM or FiM particles or multi-domain FM or FiM particles with dcs> DSPM(FIG. 21C), we propose the creation of large polycrystalline particles composed of multi-nanocrystals with dcs< DSPM, securing the SPM feature but with average particle size well above Dcoffering impassability through vital barriers (FIG. 21D). In this scenario, the average diameter of these SPM polycrystalline particles, reaching up to 400 nm, can far exceed that of the SD / MD FM or FiM particles that can span between 70 and 100 nm in diameter for magnetite, with variations based on their shape (V. Reichel, A. Kovács, M. Kumari, É. Bereczk-Tompa, E. Schneck, P. Diehle, M. Pósfai, A. M. Hirt, M. Duchamp, R. E. Dunin-Borkowski, D. Faivre, Sci. Rep.2017, 7, 45484; Q. Li, C. W. Kartikowati, S. Horie, T. Ogi, T. Iwaki, K. Okuyama, Sci. Rep. 2017, 7, 9894).

[0087] Results and Discussion

[0088] Structural characterization

[0089] The structural evaluation of SUPA samples was initially carried out using a scanning electron microscope (SEM) to determine general morphology and uniformity. The sizes of the SUPA system enabled us to categorize them at varying particle sizes up to 400 nm, as summarized in Table 3. The uniformity of the structures in all five samples was consistent with no4876-0545-6626.1Page 25 of 72094876-000021WOPTsignificant morphological deviations, as shown in FIG.22A – FIG.22B for samples S2 and S3, respectively. SEM images of samples S1, S4, and S5 are presented in FIG.28A – FIG.28C.

[0090] Furthermore, transmission electron microscopy (TEM) was employed to evaluate the structural integrity, interparticle adjacency, and superficial crystallite distribution FIG.22C – FIG. 22E. Upon closer examination through subsequent qualitative analysis, we found that the average crystallite size (dcs) remained consistently below 15 nm across all the structures. (see Table 3). The selected area electron diffraction (SAED) pattern, shown in the inset of FIG. 22C for sample S3, exhibits clear diffraction spots confirming the crystalline nature of SUPAs, (Suvorova, Buffat, J. Microsc.1999, 196, 46). which reflects the long-range order inherent in these crystalline structures. The planes can be indexed back to magnetite composition, and the presence of dislocations in the structure has led to the streaks, and the partial rings illustrate the preferred orientations in the structures (Suvorova, Buffat, J. Microsc. 1999, 196, 46). The structure and composition were further examined using X-ray diffraction (XRD). As shown in FIG. 22F, all XRD data show well-defined peaks, revealing the good crystallinity of the SUPAs.

[0091] The position and relative intensities of these peaks match well with those of magnetite structure, confirming the presence of magnetite (Fe3O4) as a major phase in all the samples (database: Fe3O4JCPDS 01-088-0315). This hypothesis has been further confirmed by X-ray photoelectron spectroscopy (XPS) analysis (FIG.29). All samples exhibited the presence of the major phase magnetite (Fe3O4) and the minor phase maghemite (γ-Fe2O3).

[0092] Table 3: Particle size (D), crystalline size (dcs), saturation magnetization (Ms), coercivity (Hc), and specific absorption rate (SAR) of the iron oxide superparticles. SAR values were measured in agar (0.5 mg / mL) at an AC field of 800 Oe and a frequency of 310 kHz.4876-0545-6626.1Page 26 of 72094876-000021WOPTSample D (nm) dcs(nm) SAR (W / g) Msat 300 K (emu / g) Hcat 300 K (Oe)

[0094] The magnetic properties of the SUPA samples were characterized by a Vibrating Sample Magnetometer (VSM) equipped within a Physical Property Measurement System (PPMS) from Quantum Design. The temperature dependence of the magnetization, M(T) with zero-field cooled (ZFC) and field-cooled (FC) curves for all samples, was measured sequentially over 10 K ≤ T ≤ 350 K range in the presence of a μ0H = 0.05 T applied field. FIG.23A shows the M(T) curves of a representative sample, S1. The data of the remaining samples are displayed in FIG.30A– FIG. 30D. It is generally understood that FC magnetization curves tend to flatten out when reaching lower temperatures, which indicates that the sample has kept magnetization constant, while upward or downward shifting of the FC magnetization curve at low temperatures hints at interactions between particles that become more prominent as thermal energy decreases (M.-H. Phan, J. Alonso, H. Khurshid, P. Lampen-Kelley, S. Chandra, K. Stojak Repa, Z. Nemati, R. Das, Ó. Iglesias, H. Srikanth, Nanomaterials 2016, 6, 221). FIG.23A shows that as the thermal energy decreases, the magnetization drops since the attractions between SUPAs have made it difficult to be aligned with the applied magnetic field. Similar behavior was observed across other samples as well (FIG.30A – FIG.30D).

[0095] We recall that a peak or maximum observed in a ZFC M(T) curve is commonly identified as the average blocking temperature (TB) of a nanoparticle system above which HCtends to approach zero with a minimal temperature-dependent change (A. G. Kolhatkar, A. C. Jamison, D. Litvinov, R. C. Willson, T. R. Lee, Int. J. Mol. Sci.2013, 14, 15977). As observed in FIG.23A and FIG.31A – FIG.31C, a conspicuous cusp or peak is evident in the FC magnetization curves at TBp~250 K across all SUPA samples. However, it should not be simply attributed to the average blocking temperature of SUPAs. Instead, it likely represents the onset temperature of magnetic4876-0545-6626.1Page 27 of 72094876-000021WOPTordering of the nanocrystals within each SUPA. Interestingly, this observation holds true regardless of particle size variations, ranging from 160 nm (S1) to 370 nm (S5). It is anticipated that all SUPA samples should manifest a SPM-like feature at T > TBp. The absence of magnetic hysteresis in the M(H) loops observed for all SUPA samples has indeed confirmed the SPM behavior at room temperature (see insets of FIG. 23C, FIG. 23D and FIG. 31A – FIG. 31C). A detailed analysis of the Hcvs. T plots for all samples, as showcased in FIG.23B and its inset for sample S1 (D = 160 nm) and sample S5 (D = 370 nm), revealed a notable increase in Hcbelow TBc~ 150 K, marking a transition from the SPM to FM or FiM (blocked) state. It is noteworthy that the significant decrease in magnetization (M) below ~150 K in the ZFC M(T) curve (FIG. 23A) correlates with the pronounced increase in Hcas the temperature drops below this threshold (FIG.23B), suggesting the blocking of nanocrystals within each SUPA occurring below TBc~150 K. Herein, TBcrefers to the average blocking temperature of nanocrystals encapsulated within SUPAs. Our findings underscore that the magnetic properties of SUPAs are primarily determined by the size of the nanocrystals composing each superparticle. The observation of broadened ZFC M(T) curves across all SUPA samples (FIG. 23A and FIG. 30A – FIG. 30D) is notable, likely stemming from competing interactions among nanocrystals within each SUPA and among different SUPAs, as well as from the size distributions of nanocrystals and SUPAs.

[0096] To provide a deeper insight into the SPM behavior, we fitted the room temperature M(H) loops for all samples using a well-known SPM model: (M. Knobel, W. C. Nunes, L. M. Socolovsky, E. De Biasi, J. M. Vargas, J. C. Denardin, J. Nanosci. Nanotechnol.2008, 8, 2836) ^ ^ ^ ^^ ^^^^ൌ ^ ^ ^ ௌ^^^ ^^^^^ ^^^^^^^^^^^^^^where V and D are the volume and diameter of the nanocrystals, respectively; ^^is the saturation magnetization reached in the experimental M–H loops; ^^^^ ^ൌ ^^^^^^^^^^ െ ^Ȁ^ is the so-called Langevin Function; and MSis the theoretical saturation magnetization of magnetite (i.e. ~450 emu cm−3for bulk magnetite at 300 K) (B. D. Cullity, C. D. Graham, Introduction to Magnetic Materials, John Wiley & Sons, 2011). The function f(D) corresponds to the particle size distribution; in our case, we have assumed a log-normal size distribution, typical for nanoparticles, as shown below:4876-0545-6626.1Page 28 of 72094876-000021WOPT^^^^ൌ^^ఉξଶగ ή ^^^ ^െ^୪୬^ି୪୬ఈ^మଶఉమ^^^^^^^^^^^^ʹ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ where ^ and ^ are fittingis ^^. From the fittings, we can average "magnetic diameter" ^ and standard deviation ^ of the nanoparticles: ଶ ^ൌ ^^^^^^^ଶ^ʹ^ǡ^^^^^^^^^^ଶൌ ^ଶ൭^^ଶെ ^^^^^^^^^^^^^^^^^ ^͵^^^^^^^^^^

[0097] A good fit was obtained for all SUPA samples, as showcased in FIG.23C and FIG. 23D for samples S1 and S5, respectively. The fitting results for other samples are also included in FIG.32A – FIG.32B. The estimated average size and standard deviation are ^ = 9.4 nm and ^ = 0.1 nm for S1, and ^ = 9.3 nm and ^ = 0.9 nm for S5, respectively. These values are close to the ones obtained by TEM for the crystallites (see Table 3), confirming that at 300 K these nanocrystallites are the ones dominating the SPM-like behavior of the SUPAs. These findings are in agreement with our aforementioned interpretation of the ZFC-FC M(T) curves. It is noteworthy that through the careful control of crystallite size to remain below the SPM threshold (dcs< SSPM), we have successfully engineered superparamagnetic SUPAs, offering the capability to tune particle sizes up to 400 nm. In other words, the SPM characteristics of SUPAs are primarily influenced by the size of the nanocrystals within each individual SUPA, rather than the size of the SUPA itself. While tuning the particle size between 160 and 400 nm, consistent values of Mswere maintained across all SUPA samples (refer to Table 3). These consistent characteristics hold significant promise for diverse biomedical applications. We note further that the optimum crystallite size range for maintaining superparamagnetic properties while preserving high saturation magnetization is 10 to 15 nm. Crystallite sizes below 10 nm retain superparamagnetism but have reduced magnetization capability, (S. Xuan, Y.-X. J. Wang, J. C. Yu, K. Cham-Fai Leung, Chem. Mater. 2009, 21, 5079) while sizes above 15 nm might exhibit ferrimagnetic or ferromagnetic behavior (Y.-T. Chen, R. Medhi, I. Nekrashevich, D. Litvinov, S. Xu, T. R. Lee, Anal. Chem.2018, 90, 6749).

[0098] Magnetic hyperthermia properties4876-0545-6626.1Page 29 of 72094876-000021WOPT

[0099] To explore the potential of superparamagnetic SUPAs for biomedical applications, we tested their magnetic hyperthermia responses. The heating efficiency of the SUPAs was evaluated in both water and 2% weight agar solution at 0.5 mg / mL and 1 mg / mL, which are comparatively lower concentration levels compared to those reported previously (P. Q. Thong, L. T. Thu Huong, N. D. Tu, H. T. My Nhung, L. Khanh, D. H. Manh, P. H. Nam, N. X. Phuc, J. Alonso, J. Qiao, S. Sridhar, H. P. Thu, M. H. Phan, N. T. Kim Thanh, Nanomedicine 2022, 17, 1677; R. Das, J. Alonso, Z. Nemati Porshokouh, V. Kalappattil, D. Torres, M.-H. Phan, E. Garaio, J. Á. García, J. L. Sanchez Llamazares, H. Srikanth, J. Phys. Chem. C 2016, 120, 10086; Z. Nemati, J. Alonso, L. M. Martinez, H. Khurshid, E. Garaio, J. A. Garcia, M. H. Phan, H. Srikanth, J. Phys. Chem. C 2016, 120, 8370). The agar solution restricted the physical movement of SUPAs, mimicking cell environments such as cell cytoplasm and extracellular matrix (S. B. Attanayake, A. Chanda, R. Das, N. Kapuruge, H. R. Gutierrez, M.-H. Phan, H. Srikanth, J. Phys.: Condens. Matter 2022, 34, 495301; V. E. Noval, J. G. Carriazo, Mat. Res.2019, 22, e20180660). FIG.24A – FIG. 24D displays the heating curves for all samples at concentrations of 1 mg / mL, and 0.5 mg / mL, subjected to AC fields of 600 Oe and 800 Oe at a constant frequency of 310 kHz. The data show that all the samples yielded consistent results, with sample S1 showing significantly higher heating capability across all concentrations. Notably, the heating capabilities of the samples for the hyperthermia treatment process were evaluated for a maximum of 15 minutes (900 s), which is lower than the typical treatment duration of 30 minutes to 1 hour (H. Kok, P. Wust, P. Stauffer, F. Bardati, G. van Rhoon, J. Crezee, Radiat. Oncol.2015, 10, 196; J. J. W. Lagendijk, Phys. Med. Biol.2000, 45, R61). The natural cooling observed after the removal of HACindicated satisfactory retention of heat, which is a promising feature.

[0100] To quantify the heating efficiency of the samples, the Specific Absorption Rate (SAR), which is a measure of the absorption energy, usually determined from the initial rate of temperature rise, was evaluated (C.-K. Chou, Int. J. Hyperth.1990, 6, 367) In our computations utilizing Eq. (4), we derived the SAR values from the heating curves after a 60-second interval, as the SUPAs tend to stabilize over time: ^^^ ൌ௱்ൈ ^^ (4)4876-0545-6626.1Page 30 of 72094876-000021WOPTHere the ΔT / Δt represents the change in temperature with time, and CPrepresents the heat capacity of the liquid solvent (in this case, water with 4.186 J / g∙K). The symbol φ represents a unitless quantity: mass of magnetic material per unit mass of liquid solvent.

[0101] FIG.25A shows the SAR values for all the samples at 0.5 mg / mL concentration in 2% agar of which the HACof 800 Oe showed the highest SAR values, with S1 at 286 W / g obtained using initial slope / time-rise protocol assuming a minimal to no heat gain / loss (H. L. Ring, A. Sharma, R. Ivkov, J. C. Bischof, Int. J. Hyperth. 2020, 37, 100). The SUPAs were initially evaluated with de-ionized water (FIG.33A – FIG.33B) which led to temperature drops with the HAC. These drops could be related to the movement or rearrangement of the SUPAs upon application of the AC field in water. Due to inconsistencies with the temperature drops, the heating responses of SUPAs in water were not considered for the SAR calculations. Similar to the peculiar observation related to water, the concentration increments that usually lead to higher SAR values were not observed in SUPAs but rather a drop, which might be due to intensified interparticle interactions with the increased presence of SUPAs per unit volume, as shown in FIG. 25B. Moreover, we conducted comprehensive field-dependent and concentration-dependent measurements, which served to deepen our aforementioned observations (FIG.34A – FIG.34B).

[0102] To validate the SAR values mentioned above, we employed an additional independent method. This approach enabled us to directly measure the AC magnetic hysteresis loops and calculate the SAR values based on the area (A) enclosed by these loops, according to the following equation: (I. Andreu, E. Natividad, Int. J. Hyperth.2013, 29, 739): ^^^^ ^^^ ^ ൌ ^ ^ ή ^ ൌ ^ ^ ή ^ ^^^௧^^௧^^^^^^^^^^^^^^^^^^^^^^^(5)with Mtbeing the instantaneous magnetization at time t, Htthe sinusoidal magnetic field of frequency f at time t, and c the magnetite weight concentration in the dispersing medium. The integration was conducted over a period of the oscillating magnetic field, T = 2π / f. AC magnetic hysteresis measurements were performed at various frequencies (f = 132, 300, and 638 kHz) on sample S5 possessing the largest average particle size of ~370 nm. The results obtained are displayed in FIG.26A – FIG.26F. As illustrated, narrow minor loops were consistently obtained at low AC fields across all applied frequencies (FIG.26A – FIG.26C). However, with increasing4876-0545-6626.1Page 31 of 72094876-000021WOPTAC field strength, both the area of the AC loops (FIG. 26A – FIG. 26C) and the corresponding SAR values (FIG.26D – FIG.26F) gradually increased. Notably, at 300 kHz and 800 Oe, a SAR value of approximately 150 W / g was achieved, aligning closely with the value previously estimated from calorimetric measurements (FIG.25B) (Z. Nemati, J. Alonso, I. Rodrigo, R. Das, E. Garaio, J. Á. García, I. Orue, M.-H. Phan, H. Srikanth, J. Phys. Chem. C 2018, 122, 2367). It is also noteworthy from FIG.26F that SAR reached a600 W / g as the frequency rose to 632 kHz, indicating the potential of SUPAs for magnetic hyperthermia therapy.

[0103] Finally, we compared the SAR values for our SUPAs with those obtained from other nanostructures (spheres, cubes, rods), highlighting their superior SPM characteristics and lower concentration requirements (Table 4). This comparison underscores the absence of SPM behavior in typical nanostructures as particle size increases. Notably, conventionally synthesized iron oxide nanoparticles with average sizes of 40 nm or larger exhibit ferrimagnetic (FiM) behavior, contrasting with our SUPAs, which maintain SPM characteristics despite sizes up to 400 nm. It is also noteworthy that only half the concentration of SUPAs (0.5 mg / mL vs.1 mg / mL) is required for magnetic hyperthermia treatment compared to other candidates (Table 4). The combination of SPM properties, large and adjustable SARs, and reduced concentration requirements positions these iron oxide superparticles as compelling contenders for a wide range of biomedical applications.

[0104] Conclusions

[0105] Polycrystalline iron oxide superparticles of particle sizes up to 400 nm, featuring crystallite sizes in the range from 10 to 15 nm, exhibit superparamagnetic features at room temperature. We have demonstrated that if crystallite sizes are alike and below the SPM size threshold, SUPAs display SPM behavior at room temperature and enter a FM or FiM (blocked) state at a comparable temperature. Across all samples, a low concentration of 0.5 mg / mL consistently yielded a SAR exceeding 250 W / g at an 800 Oe AC field. The exceptional stability of superparamagnetic SUPAs in a 2% by-weight agar solution, at both 0.5 mg / mL and 1 mg / mL concentrations, underscores their potential for in vivo studies. However, further evaluations of the cytotoxicity and biocompatibility of these superparticles are needed, even though the composition and structures conform with internationally accredited safety agencies such as the FDA. The promising performance of these superparamagnetic superparticles warrants further exploration to deepen our understanding of the underlying physics behind their mechanisms.4876-0545-6626.1Page 32 of 72094876-000021WOPT

[0106] Table 4: Particle size (D), concentration, saturation magnetization (Ms), coercivity (Hc), and SAR values of various SPM nanosystems. Sample D (nm) Concentration SAR in Msat Hc(Oe) Ref. (mg / mL) Agar at 300 K f er.Ref.4: R. Das, J. Alonso, Z. Nemati Porshokouh, V. Kalappattil, D. Torres, M.-H. Phan, E. Garaio, J. Á. García, J. L. Sanchez Llamazares, H. Srikanth, J. Phys. Chem. C 2016, 120, 10086.

[0107] Various Non-Limiting Embodiments of the Invention

[0108] Embodiments include those listed below.

[0109] Embodiment 1. A magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises iron oxide, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, and wherein the at least one superparamagnetic nanoparticle has a crystallite size of less than or equal to 15 nm; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of the subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat the condition in the subject.

[0110] Embodiment 2. The method of embodiment 1, wherein the crystallite size is less than 15 nm.

[0111] Embodiment 3. The method embodiment 1, wherein the crystallite size is 10 nm to 15 nm.4876-0545-6626.1Page 33 of 72094876-000021WOPT

[0112] Embodiment 4. The method of any one of embodiments 1-3, wherein the particle size is less than 500 nm.

[0113] Embodiment 5. The method of any one of embodiments 1-3, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.

[0114] Embodiment 6. The method of any one of embodiments 1-5, wherein the iron oxide is Fe2O3, Fe3O4, or combination thereof.

[0115] Embodiment 7. The method of any one of embodiments 1-6, wherein the magnetic field is an alternating current magnetic field.

[0116] Embodiment 8. The method of any one of embodiments 1-7, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject.

[0117] Embodiment 9. The method of any one of embodiments 1-8, wherein the subject is a human subject.

[0118] Embodiment 10. The method of any one of embodiments 1-9, wherein the region is a tumor, tissue, organ, limb, body cavity, or any combination thereof.

[0119] Embodiment 11. The method of any one of embodiments 1-10, wherein the condition is cancer.

[0120] Embodiment 12. A magnetic hyperthermia method for treating cancer in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises iron oxide, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, and wherein the at least one superparamagnetic nanoparticle has a crystallite size of less than or equal to 15 nm; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of the subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat the cancer in the subject.

[0121] Embodiment 13. The method of embodiment 12, wherein the crystallite size is less than 15 nm.

[0122] Embodiment 14. The method of embodiment 12, wherein the crystallite size is 10 nm to 15 nm.

[0123] Embodiment 15. The method of any one of embodiments 12-14, wherein the particle size is less than 500 nm.4876-0545-6626.1Page 34 of 72094876-000021WOPT

[0124] Embodiment 16. The method of any one of embodiments 12-14, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.

[0125] Embodiment 17. The method of any one of embodiments 12-16, wherein the iron oxide is Fe2O3, Fe3O4, or combination thereof.

[0126] Embodiment 18. The method of any one of embodiments 12-17, wherein the magnetic field is an alternating current magnetic field.

[0127] Embodiment 19. The method of any one of embodiments 12-18 wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject.

[0128] Embodiment 20. The method of any one of embodiments 12-19, wherein the subject is a human subject.

[0129] Embodiment 21. The method of any one of embodiments 12-20, wherein the region is a tumor, tissue, organ, limb, body cavity, or any combination thereof.

[0130] Embodiment 22. A magnetic hyperthermia method for killing cancer cells in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises iron oxide, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, and wherein the at least one superparamagnetic nanoparticle has a crystallite size of less than or equal to 15 nm; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of the subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to kill at least a portion of the cancer cells in the subject.

[0131] Embodiment 23. The method of embodiment 22, wherein the crystallite size is less than 15 nm.

[0132] Embodiment 24. The method of embodiment 22, wherein the crystallite size is 10 nm to 15 nm.

[0133] Embodiment 25. The method of any one of embodiments 22-24, wherein the particle size is less than 500 nm.

[0134] Embodiment 26. The method of any one of embodiments 22-24, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.

[0135] Embodiment 27. The method of any one of embodiments 22-26, wherein the iron oxide is Fe2O3, Fe3O4, or combination thereof.4876-0545-6626.1Page 35 of 72094876-000021WOPT

[0136] Embodiment 28. The method of any one of embodiments 22-27, wherein the magnetic field is an alternating current magnetic field.

[0137] Embodiment 29. The method of any one of embodiments 22-28, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of the cancer cells in the subject.

[0138] Embodiment 30. The method of any one of embodiments 22-29, wherein the subject is a human subject.

[0139] Embodiment 31. The method of any one of embodiments 22-30, wherein the region is a tumor, tissue, organ, limb, body cavity, or any combination thereof.

[0140] Embodiments include those listed below.

[0141] Embodiment 1A. A superparamagnetic nanoparticle for magnetic hyperthermia therapy, comprising: iron oxide, wherein the superparamagnetic nanoparticle has a particle size of 150 nm to 400 nm, and wherein the superparamagnetic nanoparticle has a crystallite size of less than or equal to 15 nm.

[0142] Embodiment 2A. The superparamagnetic nanoparticle of embodiment 1A, wherein the crystallite size is less than 15 nm.

[0143] Embodiment 3A. The superparamagnetic nanoparticle of embodiment 1A, wherein the crystallite size is 10 nm to 15 nm.

[0144] Embodiment 4A. The superparamagnetic nanoparticle of any one of embodiments 1A-3A, wherein the particle size is less than or equal to 500 nm.

[0145] Embodiment 5A. The superparamagnetic nanoparticle of any one of embodiments 1A-3A, wherein the particle size is 160 nm to 375 nm.

[0146] Embodiment 6A. The superparamagnetic nanoparticle of any one of embodiments 1A-5A, wherein the iron oxide is Fe2O3, Fe3O4, or combination thereof.

[0147] Additional embodiments include those listed below.

[0148] In some embodiments, iron oxide superparticles of the present invention showed superparamagnetic-like behavior.

[0149] In some embodiments, the iron oxide superparticles of the present invention showed more stability in agar medium which mimics the body environment.4876-0545-6626.1Page 36 of 72094876-000021WOPT

[0150] In some embodiments, the iron oxide superparticles of the present invention provide a low concentration at 0.5 mg / mL yielded large SAR, which is desirable for magnetic hyperthermia therapy.

[0151] In some embodiments, the combination of excellent superparamagnetic and large magneto-inductive heating properties (large SAR values) is achieved for the first time in the iron oxide superparticles of the present invention (particle size of up to 400 nm).

[0152] In some embodiments, superparticles of the present invention provide low-risk in penetrating sensitive areas such as the Blood-Brain barrier (BBB).

[0153] In some embodiments, the synthesized superparticles of the present invention are also promising for many other biomedical applications such as MRI, drug delivery, and biosensing.

[0154] Additional embodiments include those listed below.

[0155] In various embodiments, the present invention provides a magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises iron oxide, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, and wherein the at least one superparamagnetic nanoparticle has a crystallite size of less than or equal to 15 nm; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of the subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat the condition in the subject.

[0156] In various embodiments, the present invention provides a magnetic hyperthermia method for treating cancer in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises iron oxide, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, and wherein the at least one superparamagnetic nanoparticle has a crystallite size of less than or equal to 15 nm; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of the subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat the cancer in the subject.

[0157] In various embodiments, the present invention provides a magnetic hyperthermia method for killing cancer cells in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises iron oxide, wherein the at least one superparamagnetic nanoparticle has a particle size4876-0545-6626.1Page 37 of 72094876-000021WOPTof less than or equal to 500 nm, and wherein the at least one superparamagnetic nanoparticle has a crystallite size of less than or equal to 15 nm; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of the subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to kill at least a portion of the cancer cells in the subject.

[0158] Additional embodiments include those listed below.

[0159] Embodiment 32. A magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of Fe3O4primary crystals, wherein the plurality of Fe3O4primary crystals comprise a plurality of crystallites, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; and wherein the magnetic nanoparticle does not have a core-shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

[0160] Embodiment 33. The method of embodiment 32, wherein the Fe3O4primary crystals further comprise Fe2O3.

[0161] Embodiment 34. The method of embodiment 32, wherein the particle size is less than 500 nm, and wherein the crystallite size is less than 15 nm.

[0162] Embodiment 35. The method embodiment 32, wherein the crystallite size is 10 nm to 15 nm.

[0163] Embodiment 36. The method of embodiment 32, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.

[0164] Embodiment 37. The method of embodiment 32, wherein the magnetic field is an alternating current magnetic field.

[0165] Embodiment 38. The method of embodiment 32, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject.

[0166] Embodiment 39. The method of embodiment 32, wherein the subject is a human subject.4876-0545-6626.1Page 38 of 72094876-000021WOPT

[0167] Embodiment 40. The method of embodiment 32, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0168] Embodiment 41. The method of embodiment 32, wherein the condition is cancer.

[0169] Embodiment 42. The method of embodiment 41, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject.

[0170] Embodiment 43. The method of embodiment 42, wherein the subject is a human subject.

[0171] Embodiment 44. The method of embodiment 42, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0172] Embodiment 45. The method of embodiment 41, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of cancer cells in the subject.

[0173] Embodiment 46. The method of embodiment 45, wherein the subject is a human subject.

[0174] Embodiment 47. The method of embodiment 45, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0175] Additional embodiments include those listed below.

[0176] Embodiment 48. A magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; and wherein the magnetic nanoparticle does not have a core-shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

[0177] Embodiment 49. The method of embodiment 48, wherein the iron oxide is Fe3O4, or Fe2O3, or combination thereof.4876-0545-6626.1Page 39 of 72094876-000021WOPT

[0178] Embodiment 50. The method of embodiment 48, wherein the particle size is less than 500 nm.

[0179] Embodiment 51. The method of embodiment 48, wherein the crystallite size is less than 15 nm.

[0180] Embodiment 52. The method embodiment 48, wherein the crystallite size is 10 nm to 15 nm.

[0181] Embodiment 53. The method of embodiment 48, wherein the particle size is 150 nm to 400 nm.

[0182] Embodiment 54. The method of embodiment 48, wherein the particle size is 160 nm to 375 nm.

[0183] Embodiment 55. The method of embodiment 48, wherein the magnetic field is an alternating current magnetic field.

[0184] Embodiment 56. The method of embodiment 48, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject.

[0185] Embodiment 57. The method of embodiment 48, wherein the subject is a human subject.

[0186] Embodiment 58. The method of embodiment 48, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0187] Embodiment 59. The method of embodiment 48, wherein the condition is cancer.

[0188] Embodiment 60. The method of embodiment 59, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject.

[0189] Embodiment 61. The method of embodiment 60, wherein the subject is a human subject.

[0190] Embodiment 62. The method of embodiment 60, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0191] Embodiment 63. The method of embodiment 59, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of cancer cells in the subject.4876-0545-6626.1Page 40 of 72094876-000021WOPT

[0192] Embodiment 64. The method of embodiment 63, wherein the subject is a human subject.

[0193] Embodiment 65. The method of embodiment 63, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0194] Additional embodiments include those listed below.

[0195] In some embodiments, the particle size is less than or equal to 500 nm. In some embodiments, the particle size is 150 nm, 151 nm, 152 nm, 153 nm, 154 nm, 155 nm, 156 nm, 157 nm, 158 nm, 159 nm, 160 nm, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, 170 nm, 171 nm, 172 nm, 173 nm, 174 nm, 175 nm, 176 nm, 177 nm, 178 nm, 179 nm, 180 nm, 181 nm, 182 nm, 183 nm, 184 nm, 185 nm, 186 nm, 187 nm, 188 nm, 189 nm, 190 nm, 191 nm, 192 nm, 193 nm, 194 nm, 195 nm, 196 nm, 197 nm, 198 nm, 199 nm, 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 nm, 227 nm, 228 nm, 229 nm, 230 nm, 231 nm, 232 nm, 233 nm, 234 nm, 235 nm, 236 nm, 237 nm, 238 nm, 239 nm, 240 nm, 241 nm, 242 nm, 243 nm, 244 nm, 245 nm, 246 nm, 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm, 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm, 290 nm, 291 nm, 292 nm, 293 nm, 294 nm, 295 nm, 296 nm, 297 nm, 298 nm, 299 nm, 300 nm, 301 nm, 302 nm, 303 nm, 304 nm, 305 nm, 306 nm, 307 nm, 308 nm, 309 nm, 310 nm, 311 nm, 312 nm, 313 nm, 314 nm, 315 nm, 316 nm, 317 nm, 318 nm, 319 nm, 320 nm, 321 nm, 322 nm, 323 nm, 324 nm, 325 nm, 326 nm, 327 nm, 328 nm, 329 nm, 330 nm, 331 nm, 332 nm, 333 nm, 334 nm, 335 nm, 336 nm, 337 nm, 338 nm, 339 nm, 340 nm, 341 nm, 342 nm, 343 nm, 344 nm, 345 nm, 346 nm, 347 nm, 348 nm, 349 nm, 350 nm, 351 nm, 352 nm, 353 nm, 354 nm, 355 nm, 356 nm, 357 nm, 358 nm, 359 nm, 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm, 371 nm, 372 nm, 373 nm, 374 nm, 375 nm, 376 nm, 377 nm, 378 nm, 379 nm, 380 nm, 381 nm, 382 nm, 383 nm, 384 nm, 385 nm, 386 nm, 387 nm, 388 nm, 389 nm, 390 nm, 391 nm, 392 nm, 393 nm, 394 nm, 395 nm, 396 nm, 397 nm, 398 nm, 399 nm, 400 nm, 401 nm, 402 nm, 403 nm, 404 nm, 405 nm, 406 nm, 407 nm, 408 nm, 409 nm, 410 nm, 411 nm, 412 nm, 413 nm, 414 nm, 415 nm, 416 nm, 417 nm, 418 nm, 419 nm, 420 nm, 4214876-0545-6626.1Page 41 of 72094876-000021WOPTnm, 422 nm, 423 nm, 424 nm, 425 nm, 426 nm, 427 nm, 428 nm, 429 nm, 430 nm, 431 nm, 432 nm, 433 nm, 434 nm, 435 nm, 436 nm, 437 nm, 438 nm, 439 nm, 440 nm, 441 nm, 442 nm, 443 nm, 444 nm, 445 nm, 446 nm, 447 nm, 448 nm, 449 nm, 450 nm, 451 nm, 452 nm, 453 nm, 454 nm, 455 nm, 456 nm, 457 nm, 458 nm, 459 nm, 460 nm, 461 nm, 462 nm, 463 nm, 464 nm, 465 nm, 466 nm, 467 nm, 468 nm, 469 nm, 470 nm, 471 nm, 472 nm, 473 nm, 474 nm, 475 nm, 476 nm, 477 nm, 478 nm, 479 nm, 480 nm, 481 nm, 482 nm, 483 nm, 484 nm, 485 nm, 486 nm, 487 nm, 488 nm, 489 nm, 490 nm, 491 nm, 492 nm, 493 nm, 494 nm, 495 nm, 496 nm, 497 nm, 498 nm, 499 nm, or 500 nm.

[0196] In some embodiments, the particle size is less than 500 nm. In some embodiments, the particle size is 150 nm, 151 nm, 152 nm, 153 nm, 154 nm, 155 nm, 156 nm, 157 nm, 158 nm, 159 nm, 160 nm, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, 170 nm, 171 nm, 172 nm, 173 nm, 174 nm, 175 nm, 176 nm, 177 nm, 178 nm, 179 nm, 180 nm, 181 nm, 182 nm, 183 nm, 184 nm, 185 nm, 186 nm, 187 nm, 188 nm, 189 nm, 190 nm, 191 nm, 192 nm, 193 nm, 194 nm, 195 nm, 196 nm, 197 nm, 198 nm, 199 nm, 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 nm, 227 nm, 228 nm, 229 nm, 230 nm, 231 nm, 232 nm, 233 nm, 234 nm, 235 nm, 236 nm, 237 nm, 238 nm, 239 nm, 240 nm, 241 nm, 242 nm, 243 nm, 244 nm, 245 nm, 246 nm, 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm, 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm, 290 nm, 291 nm, 292 nm, 293 nm, 294 nm, 295 nm, 296 nm, 297 nm, 298 nm, 299 nm, 300 nm, 301 nm, 302 nm, 303 nm, 304 nm, 305 nm, 306 nm, 307 nm, 308 nm, 309 nm, 310 nm, 311 nm, 312 nm, 313 nm, 314 nm, 315 nm, 316 nm, 317 nm, 318 nm, 319 nm, 320 nm, 321 nm, 322 nm, 323 nm, 324 nm, 325 nm, 326 nm, 327 nm, 328 nm, 329 nm, 330 nm, 331 nm, 332 nm, 333 nm, 334 nm, 335 nm, 336 nm, 337 nm, 338 nm, 339 nm, 340 nm, 341 nm, 342 nm, 343 nm, 344 nm, 345 nm, 346 nm, 347 nm, 348 nm, 349 nm, 350 nm, 351 nm, 352 nm, 353 nm, 354 nm, 355 nm, 356 nm, 357 nm, 358 nm, 359 nm, 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm, 371 nm, 372 nm, 373 nm, 374 nm, 375 nm, 376 nm, 377 nm, 378 nm, 379 nm, 380 nm, 381 nm, 382 nm, 383 nm, 384 nm, 385 nm, 386 nm, 387 nm, 388 nm, 389 nm,4876-0545-6626.1Page 42 of 72094876-000021WOPT390 nm, 391 nm, 392 nm, 393 nm, 394 nm, 395 nm, 396 nm, 397 nm, 398 nm, 399 nm, 400 nm, 401 nm, 402 nm, 403 nm, 404 nm, 405 nm, 406 nm, 407 nm, 408 nm, 409 nm, 410 nm, 411 nm, 412 nm, 413 nm, 414 nm, 415 nm, 416 nm, 417 nm, 418 nm, 419 nm, 420 nm, 421 nm, 422 nm, 423 nm, 424 nm, 425 nm, 426 nm, 427 nm, 428 nm, 429 nm, 430 nm, 431 nm, 432 nm, 433 nm, 434 nm, 435 nm, 436 nm, 437 nm, 438 nm, 439 nm, 440 nm, 441 nm, 442 nm, 443 nm, 444 nm, 445 nm, 446 nm, 447 nm, 448 nm, 449 nm, 450 nm, 451 nm, 452 nm, 453 nm, 454 nm, 455 nm, 456 nm, 457 nm, 458 nm, 459 nm, 460 nm, 461 nm, 462 nm, 463 nm, 464 nm, 465 nm, 466 nm, 467 nm, 468 nm, 469 nm, 470 nm, 471 nm, 472 nm, 473 nm, 474 nm, 475 nm, 476 nm, 477 nm, 478 nm, 479 nm, 480 nm, 481 nm, 482 nm, 483 nm, 484 nm, 485 nm, 486 nm, 487 nm, 488 nm, 489 nm, 490 nm, 491 nm, 492 nm, 493 nm, 494 nm, 495 nm, 496 nm, 497 nm, 498 nm, or 499 nm.

[0197] In some embodiments, the particle size is 150 nm to 400 nm. In some embodiments, the particle size is 150 nm, 151 nm, 152 nm, 153 nm, 154 nm, 155 nm, 156 nm, 157 nm, 158 nm, 159 nm, 160 nm, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, 170 nm, 171 nm, 172 nm, 173 nm, 174 nm, 175 nm, 176 nm, 177 nm, 178 nm, 179 nm, 180 nm, 181 nm, 182 nm, 183 nm, 184 nm, 185 nm, 186 nm, 187 nm, 188 nm, 189 nm, 190 nm, 191 nm, 192 nm, 193 nm, 194 nm, 195 nm, 196 nm, 197 nm, 198 nm, 199 nm, 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 nm, 227 nm, 228 nm, 229 nm, 230 nm, 231 nm, 232 nm, 233 nm, 234 nm, 235 nm, 236 nm, 237 nm, 238 nm, 239 nm, 240 nm, 241 nm, 242 nm, 243 nm, 244 nm, 245 nm, 246 nm, 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm, 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm, 290 nm, 291 nm, 292 nm, 293 nm, 294 nm, 295 nm, 296 nm, 297 nm, 298 nm, 299 nm, 300 nm, 301 nm, 302 nm, 303 nm, 304 nm, 305 nm, 306 nm, 307 nm, 308 nm, 309 nm, 310 nm, 311 nm, 312 nm, 313 nm, 314 nm, 315 nm, 316 nm, 317 nm, 318 nm, 319 nm, 320 nm, 321 nm, 322 nm, 323 nm, 324 nm, 325 nm, 326 nm, 327 nm, 328 nm, 329 nm, 330 nm, 331 nm, 332 nm, 333 nm, 334 nm, 335 nm, 336 nm, 337 nm, 338 nm, 339 nm, 340 nm, 341 nm, 342 nm, 343 nm, 344 nm, 345 nm, 346 nm, 347 nm, 348 nm, 349 nm, 350 nm, 351 nm, 352 nm, 353 nm, 354 nm, 3554876-0545-6626.1Page 43 of 72094876-000021WOPTnm, 356 nm, 357 nm, 358 nm, 359 nm, 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm, 371 nm, 372 nm, 373 nm, 374 nm, 375 nm, 376 nm, 377 nm, 378 nm, 379 nm, 380 nm, 381 nm, 382 nm, 383 nm, 384 nm, 385 nm, 386 nm, 387 nm, 388 nm, 389 nm, 390 nm, 391 nm, 392 nm, 393 nm, 394 nm, 395 nm, 396 nm, 397 nm, 398 nm, 399 nm, or 400 nm.

[0198] In some embodiments, the particle size is 160 nm to 375 nm. In some embodiments, the particle size is 160 nm, 161 nm, 162 nm, 163 nm, 164 nm, 165 nm, 166 nm, 167 nm, 168 nm, 169 nm, 170 nm, 171 nm, 172 nm, 173 nm, 174 nm, 175 nm, 176 nm, 177 nm, 178 nm, 179 nm, 180 nm, 181 nm, 182 nm, 183 nm, 184 nm, 185 nm, 186 nm, 187 nm, 188 nm, 189 nm, 190 nm, 191 nm, 192 nm, 193 nm, 194 nm, 195 nm, 196 nm, 197 nm, 198 nm, 199 nm, 200 nm, 201 nm, 202 nm, 203 nm, 204 nm, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, 225 nm, 226 nm, 227 nm, 228 nm, 229 nm, 230 nm, 231 nm, 232 nm, 233 nm, 234 nm, 235 nm, 236 nm, 237 nm, 238 nm, 239 nm, 240 nm, 241 nm, 242 nm, 243 nm, 244 nm, 245 nm, 246 nm, 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, 265 nm, 266 nm, 267 nm, 268 nm, 269 nm, 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, 275 nm, 276 nm, 277 nm, 278 nm, 279 nm, 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm, 290 nm, 291 nm, 292 nm, 293 nm, 294 nm, 295 nm, 296 nm, 297 nm, 298 nm, 299 nm, 300 nm, 301 nm, 302 nm, 303 nm, 304 nm, 305 nm, 306 nm, 307 nm, 308 nm, 309 nm, 310 nm, 311 nm, 312 nm, 313 nm, 314 nm, 315 nm, 316 nm, 317 nm, 318 nm, 319 nm, 320 nm, 321 nm, 322 nm, 323 nm, 324 nm, 325 nm, 326 nm, 327 nm, 328 nm, 329 nm, 330 nm, 331 nm, 332 nm, 333 nm, 334 nm, 335 nm, 336 nm, 337 nm, 338 nm, 339 nm, 340 nm, 341 nm, 342 nm, 343 nm, 344 nm, 345 nm, 346 nm, 347 nm, 348 nm, 349 nm, 350 nm, 351 nm, 352 nm, 353 nm, 354 nm, 355 nm, 356 nm, 357 nm, 358 nm, 359 nm, 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm, 371 nm, 372 nm, 373 nm, 374 nm, or 375 nm.

[0199] In some embodiments, the crystallite size is less than 15 nm. In some embodiments, the crystallite size is 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, or 15 nm. In some embodiments, the crystallite size is 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, or 14 nm.4876-0545-6626.1Page 44 of 72094876-000021WOPT

[0200] In some embodiments, the crystallite size is 10 nm to 15 nm. In some embodiments, the crystallite size is 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, or 15 nm.

[0201] In some embodiments, the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof. In some embodiments, the region is a tumor. In some embodiments, the region is a tissue. In some embodiments, the region is an organ. In some embodiments, the region is a limb. In some embodiments, the region is a body cavity.

[0202] Additional embodiments include those listed below.

[0203] In various embodiments, the superparamagnetic nanoparticles of the present invention do not comprise FeO. In various embodiments, the superparamagnetic nanoparticles of the present invention do not contain FeO. In various embodiments, the superparamagnetic nanoparticles of the present invention do not consist of FeO. In various embodiments, the superparamagnetic nanoparticles of the present invention do not consist essentially of FeO.

[0204] In some embodiments, the superparamagnetic nanoparticle is not a core-shell magnetic nanoparticle. In some embodiments, the superparamagnetic nanoparticle is not a core / shell magnetic nanoparticle. In some embodiments, the superparamagnetic nanoparticle is not a core-shell superparamagnetic nanoparticle. In some embodiments, the superparamagnetic nanoparticle is not a core / shell superparamagnetic nanoparticle. In some embodiments, the superparamagnetic nanoparticle is not a core-shell structure. In some embodiments, the superparamagnetic nanoparticle is not a core / shell structure. In some embodiments, the superparamagnetic nanoparticle does not have a core-shell structure. In some embodiments, the superparamagnetic nanoparticle does not have a core / shell structure.

[0205] In some embodiments, the iron oxide primary crystal comprises Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the iron oxide primary crystal is Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the iron oxide primary crystal comprises Fe3O4. In some embodiments, the iron oxide primary crystal is Fe3O4. In some embodiments, the iron oxide primary crystal comprises Fe2O3. In some embodiments, the iron oxide primary crystal is Fe2O3.

[0206] In some embodiments, the crystallite comprises Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the crystallite is Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the crystallite comprises Fe3O4. In some embodiments, the crystallite is Fe3O4. In4876-0545-6626.1Page 45 of 72094876-000021WOPTsome embodiments, the crystallite comprises Fe2O3. In some embodiments, the crystallite is Fe2O3.

[0207] In some embodiments, the at least one superparamagnetic nanoparticle comprises Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the at least one superparamagnetic nanoparticle is Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the at least one superparamagnetic nanoparticle comprises Fe3O4. In some embodiments, the at least one superparamagnetic nanoparticle is Fe3O4. In some embodiments, the at least one superparamagnetic nanoparticle comprises Fe2O3. In some embodiments, the at least one superparamagnetic nanoparticle is Fe2O3. In some embodiments, the at least one superparamagnetic nanoparticle further comprises Fe3O4. In some embodiments, the at least one superparamagnetic nanoparticle further comprises Fe2O3.

[0208] In some embodiments, the at least one Fe3O4nanosphere further comprises Fe2O3. In some embodiments, the Fe3O4primary crystals further comprise Fe2O3.

[0209] In various embodiments, the primary crystals of the present invention do not comprise FeO. In various embodiments, the primary crystals of the present invention do not contain FeO. In various embodiments, the primary crystals of the present invention do not consist of FeO. In various embodiments, the primary crystals of the present invention do not consist essentially of FeO.

[0210] In various embodiments, the crystallites of the present invention do not comprise FeO. In various embodiments, the crystallites of the present invention do not contain FeO. In various embodiments, the crystallites of the present invention do not consist of FeO. In various embodiments, the crystallites of the present invention do not consist essentially of FeO.

[0211] In some embodiments, the at least one Fe3O4nanosphere does not have a core-shell structure.

[0212] In some embodiments, the Fe3O4nanosphere does not comprise FeO. In some embodiments, the Fe3O4nanosphere does not contain FeO. In some embodiments, the Fe3O4nanosphere does not consist of FeO. In some embodiments, the Fe3O4nanosphere does not consist essentially of FeO. In some embodiments, the Fe3O4nanosphere is not FeO.

[0213] In some embodiments, the primary crystal does not have a core-shell structure. In some embodiments, the iron oxide primary crystal does not have a core-shell structure. In some embodiments, the crystallite does not have a core-shell structure. In some embodiments, the iron4876-0545-6626.1Page 46 of 72094876-000021WOPToxide crystallite does not have a core-shell structure. In some embodiments, the nanosphere does not have a core-shell structure.

[0214] In some embodiments, the at least one primary crystal does not have a core-shell structure. In some embodiments, the plurality of primary crystals does not have a core-shell structure. In some embodiments, the at least one crystallite does not have a core-shell structure. In some embodiments, the plurality of crystallites does not have a core-shell structure.

[0215] In some embodiments, the iron oxide is Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the iron oxide is Fe3O4. In some embodiments, the iron oxide is Fe2O3. In some embodiments, the iron oxide is a combination of Fe3O4and Fe2O3.

[0216] In some embodiments, the iron oxide is not FeO. In some embodiments, the iron oxide does not comprise FeO. In some embodiments, the iron oxide does not contain FeO. In some embodiments, the iron oxide does not consist of FeO. In some embodiments, the iron oxide does not consist essentially of FeO.

[0217] In some embodiments, the primary crystal comprises iron oxide. In some embodiments, the crystallite comprises iron oxide. In some embodiments, the plurality of primary crystals comprise iron oxide. In some embodiments, the plurality of crystallites comprise iron oxide. In some embodiments, the at least one primary crystal comprises iron oxide. In some embodiments, the at least one crystallite comprises iron oxide.

[0218] In some embodiments, the primary crystal is an iron oxide primary crystal. In some embodiments, the plurality of primary crystals is a plurality of iron oxide primary crystals. In some embodiments, the at least one primary crystal is at least one iron oxide primary crystal.

[0219] In some embodiments, the crystallite is an iron oxide crystallite. In some embodiments, the plurality of crystallites is a plurality of iron oxide crystallites. In some embodiments, the at least one crystallite is an iron oxide crystallite.

[0220] In some embodiments, the superparamagnetic nanoparticle is iron oxide. In some embodiments, the superparamagnetic nanoparticle comprises iron oxide. In some embodiments, the superparamagnetic nanoparticle consists of iron oxide. In some embodiments, the superparamagnetic nanoparticle consists essentially of iron oxide.

[0221] In some embodiments, the superparamagnetic nanoparticle is Fe3O4. In some embodiments, the superparamagnetic nanoparticle comprises Fe3O4. In some embodiments, the superparamagnetic nanoparticle consists of Fe3O4. In some embodiments, the superparamagnetic4876-0545-6626.1Page 47 of 72094876-000021WOPTnanoparticle consists essentially of Fe3O4. In some embodiments, the superparamagnetic nanoparticle further comprises Fe2O3.

[0222] In some embodiments, the plurality of primary crystals is a plurality of iron oxide primary crystals. In some embodiments, the plurality of crystallites is a plurality of iron oxide crystallites.

[0223] In some embodiments, the at least one primary crystal is at least one iron oxide primary crystal. In some embodiments, the at least one crystallite is at least one iron oxide crystallite.

[0224] In some embodiments, the primary crystal does not comprise FeO. In some embodiments, the primary crystal does not contain FeO. In some embodiments, the primary crystal does not consist of FeO. In some embodiments, the primary crystal does not consist essentially of FeO. In some embodiments, the primary crystal is not FeO.

[0225] In some embodiments, the plurality of primary crystals does not comprise FeO. In some embodiments, the plurality of primary crystals does not contain FeO. In some embodiments, the plurality of primary crystals does not consist of FeO. In some embodiments, the plurality of primary crystals does not consist essentially of FeO. In some embodiments, the plurality of primary crystals is not FeO.

[0226] In some embodiments, the crystallite does not comprise FeO. In some embodiments, the crystallite does not contain FeO. In some embodiments, the crystallite does not consist of FeO. In some embodiments, the crystallite does not consist essentially of FeO. In some embodiments, the crystallite is not FeO.

[0227] In some embodiments, the plurality of crystallites does not comprise FeO. In some embodiments, the plurality of crystallites does not contain FeO. In some embodiments, the plurality of crystallites does not consist of FeO. In some embodiments, the plurality of crystallites does not consist essentially of FeO. In some embodiments, the plurality of crystallites is not FeO.

[0228] In some embodiments, the iron oxide primary crystal comprises Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the iron oxide primary crystal is Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the iron oxide primary crystal comprises Fe3O4. In some embodiments, the iron oxide primary crystal is Fe3O4. In some embodiments, the iron oxide primary crystal comprises Fe2O3. In some embodiments, the iron oxide primary crystal is Fe2O3.4876-0545-6626.1Page 48 of 72094876-000021WOPT

[0229] In some embodiments, the crystallite comprises Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the crystallite is Fe3O4, or Fe2O3, or combination thereof. In some embodiments, the crystallite comprises Fe3O4. In some embodiments, the crystallite is Fe3O4. In some embodiments, the crystallite comprises Fe2O3. In some embodiments, the crystallite is Fe2O3.

[0230] In some embodiments, the at least one Fe3O4nanosphere further comprises Fe2O3.

[0231] In some embodiments, the primary crystal has a spherical shape. In some embodiments, the crystallite has a spherical shape.

[0232] In some embodiments, the superparamagnetic nanoparticle has a spherical shape. In some embodiments, the superparamagnetic nanoparticle is a nanosphere.

[0233] In some embodiments, the terms “trihydrate sodium acetate” and “sodium acetate trihydrate” have the same meaning and can be used interchangeably. In some embodiments, the terms “anhydrous sodium acetate” and “sodium acetate anhydrous” have the same meaning and can be used interchangeably.

[0234] In various embodiments, the present invention provides a magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises at least one iron oxide primary crystal, wherein the at least one iron oxide primary crystal comprises at least one crystallite, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the at least one crystallite has a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; and wherein the superparamagnetic nanoparticle does not have a core-shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

[0235] In various embodiments, the present invention provides a superparamagnetic nanoparticle for magnetic hyperthermia therapy, comprising: a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the4876-0545-6626.1Page 49 of 72094876-000021WOPTsuperparamagnetic nanoparticle has a spherical shape; and wherein the superparamagnetic nanoparticle does not have a core-shell structure.

[0236] In various embodiments, the present invention provides a superparamagnetic nanoparticle for magnetic hyperthermia therapy, comprising: a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the superparamagnetic nanoparticle has a spherical shape; wherein the plurality of iron oxide primary crystals does not have a core-shell structure; and wherein the superparamagnetic nanoparticle does not have a core-shell structure.

[0237] In various embodiments, the present invention provides a superparamagnetic nanoparticle for magnetic hyperthermia therapy, comprising: at least one iron oxide primary crystal, wherein the at least one iron oxide primary crystal comprises at least one crystallite, wherein the superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the at least one crystallite has a crystallite size of less than 15 nm, wherein the superparamagnetic nanoparticle has a spherical shape; wherein the at least one iron oxide primary crystal does not have a core-shell structure; and wherein the superparamagnetic nanoparticle does not have a core-shell structure.

[0238] In various embodiments, the present invention provides a superparamagnetic nanoparticle for magnetic hyperthermia therapy, comprising: at least one iron oxide primary crystal, wherein the at least one iron oxide primary crystal comprises at least one crystallite, wherein the superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the at least one crystallite has a crystallite size of less than 15 nm, wherein the superparamagnetic nanoparticle has a spherical shape; and wherein the superparamagnetic nanoparticle does not have a core-shell structure.

[0239] In some embodiments, the superparamagnetic nanoparticle does not comprise FeO. In some embodiments, the superparamagnetic nanoparticle does not contain FeO.

[0240] In some embodiments, the present invention provides for use of a superparamagnetic nanoparticle of the present invention as a medicament. In some embodiments, the present invention provides for use of a superparamagnetic nanoparticle of the present invention as a medicament for treating a condition in a subject. In some embodiments, the condition is4876-0545-6626.1Page 50 of 72094876-000021WOPTcancer. In some embodiments, the present invention provides for use of a superparamagnetic nanoparticle of the present invention as a medicament for treating a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides for use of a superparamagnetic nanoparticle of the present invention as a medicament for treating cancer.

[0241] In some embodiments, the present invention provides for use of at least one superparamagnetic nanoparticle of the present invention as a medicament. In some embodiments, the present invention provides for use of at least one superparamagnetic nanoparticle of the present invention as a medicament for treating a condition in a subject. In some embodiments, the condition is cancer. In some embodiments, the present invention provides for use of at least one superparamagnetic nanoparticle of the present invention as a medicament for treating a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides for use of at least one superparamagnetic nanoparticle of the present invention as a medicament for treating cancer.

[0242] In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia therapy. In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia. In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia for treating a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia therapy for treating a condition. In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia for treating cancer.

[0243] In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia therapy. In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia. In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia for treating a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of4876-0545-6626.1Page 51 of 72094876-000021WOPTthe present invention for use in magnetic hyperthermia therapy for treating a condition. In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia for treating cancer.

[0244] In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia treatment. In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia treatment of a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia treatment of cancer.

[0245] In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia treatment. In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia treatment of a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia treatment of cancer.

[0246] In some embodiments, the present invention provides a superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia therapy for the treatment of a condition in a subject. In some embodiments, the condition is cancer.

[0247] In some embodiments, the present invention provides at least one superparamagnetic nanoparticle of the present invention for use in magnetic hyperthermia therapy for the treatment of a condition in a subject. In some embodiments, the condition is cancer.

[0248] In some embodiments, the present invention provides for use of a superparamagnetic nanoparticle of the present invention for treating a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides for use of a superparamagnetic nanoparticle of the present invention for the treatment of cancer.

[0249] In some embodiments, the present invention provides for use of at least one superparamagnetic nanoparticle of the present invention for treating a condition. In some embodiments, the condition is cancer. In some embodiments, the present invention provides for use of at least one superparamagnetic nanoparticle of the present invention for the treatment of cancer.4876-0545-6626.1Page 52 of 72094876-000021WOPT

[0250] In some embodiments, the plurality of iron oxide primary crystals does not have a core-shell structure. In some embodiments, each of the iron oxide primary crystals in the plurality of iron oxide primary crystals does not have a core-shell structure. In some embodiments, each of the iron oxide primary crystals in the plurality of iron oxide primary crystals does not comprise FeO. In some embodiments, each of the iron oxide primary crystals in the plurality of iron oxide primary crystals does not contain FeO.

[0251] In some embodiments, each of the crystallites in the plurality of crystallites does not have a core-shell structure. In some embodiments, each of the crystallites in the plurality of crystallites does not comprise FeO. In some embodiments, each of the crystallites in the plurality of crystallites does not contain FeO.

[0252] In some embodiments, the at least one iron oxide primary crystal does not have a core-shell structure. In some embodiments, the at least one iron oxide primary crystal does not comprise FeO. In some embodiments, the at least one iron oxide primary crystal does not contain FeO.

[0253] In some embodiments, the at least one crystallite does not have a core-shell structure. In some embodiments, the at least one crystallite does not comprise FeO. In some embodiments, the at least one crystallite does not contain FeO.

[0254] Additional embodiments include those listed below.

[0255] In some embodiments, the condition is a disease or disorder. In some embodiments, the condition is cancer. In some embodiments, the disease is cancer. In some embodiments, the disorder is cancer.

[0256] In some embodiments, the cancer is carcinoma, sarcoma, melanoma, lymphoma, or leukemia.

[0257] In some embodiments, the cancer is lung cancer, prostate cancer, kidney cancer, brain cancer, brain tumor, oral cancer, liver cancer, head and neck cancer, breast cancer, skin cancer, cervical cancer, pancreatic cancer, bone cancer, blood cancer, ovarian cancer, colorectal cancer, bladder cancer, anal cancer, bile duct cancer, or endometrial cancer.

[0258] In some embodiments, the cancer is cancer cells.

[0259] In some embodiments, the cancer is a tumor or tumor cells.

[0260] In some embodiments, administering refers to the placement of at least one superparamagnetic nanoparticle into a region of a subject. In some embodiments, administering4876-0545-6626.1Page 53 of 72094876-000021WOPTrefers to the placement of at least one superparamagnetic nanoparticle adjacent to a region of a subject.

[0261] In some embodiments, therapeutically effective amount refers to an amount that will elicit the response sought. In some embodiments, therapeutically effective amount refers to an amount that results in, for example, improved treatment, healing, prevention, reduction, elimination, at least partial reduction, at least partial elimination, reversal, at least partial reversal, or amelioration of a condition, disease, or disorder, or in the rate of advancement of a condition, disease, or disorder.

[0262] As used herein, a “subject” refers to a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, “patient”, “individual” and “subject” are used interchangeably herein. In an embodiment, the subject is mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In addition, the methods described herein can be used to treat domesticated animals and / or pets. In various embodiments, the subject is human. In some embodiments, the subject is a human subject.

[0263] “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex.

[0264] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments or prophylactic or preventative measures, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition, disease, or disorder. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder described herein. Treatment is generally “effective" if one or more symptoms are reduced. Alternatively, treatment is “effective" if the progression of a condition, disease, or disorder is reduced or halted. That is, “treatment" includes not just the improvement of symptoms, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected4876-0545-6626.1Page 54 of 72094876-000021WOPTin absence of treatment. Also, “treatment” may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition, disease, or disorder even if the treatment is ultimately unsuccessful. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s) of the condition, disease, or disorder; diminishment of extent of the condition, disease, or disorder; stabilized (i.e., not worsening) state of the condition, disease, or disorder; delay or slowing of the progression of the condition, disease, or disorder; amelioration or palliation of the condition, disease, or disorder; and remission (whether partial or total), whether detectable or undetectable, of the condition, disease, or disorder. The term "treatment" of a condition, disease, or disorder also includes providing relief from the symptoms or side-effects of the condition, disease, or disorder (including palliative treatment). Those in need of treatment (e.g., a subject in need of treatment) include those already with the condition, disease, or disorder as well as those prone to have the condition, disease, or disorder or those in whom the condition, disease, or disorder is to be prevented.

[0265] Additional embodiments include those listed below.

[0266] Embodiment 66. A magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; and wherein the magnetic nanoparticle does not have a core-shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

[0267] Embodiment 67. The method of embodiment 66, wherein the iron oxide is Fe3O4, or Fe2O3, or combination thereof.

[0268] Embodiment 68. The method of embodiment 66 or embodiment 67, wherein the particle size is less than 500 nm, and wherein the crystallite size is less than 15 nm.

[0269] Embodiment 69. The method any one of embodiments 66-68, wherein the crystallite size is 10 nm to 15 nm.4876-0545-6626.1Page 55 of 72094876-000021WOPT

[0270] Embodiment 70. The method of any one of embodiments 66-69, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.

[0271] Embodiment 71. The method of any one of embodiments 66-70, wherein the magnetic field is an alternating current magnetic field.

[0272] Embodiment 72. The method of any one of embodiments 66-71, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject.

[0273] Embodiment 73. The method of any one of embodiments 66-72, wherein the subject is a human subject.

[0274] Embodiment 74. The method of any one of embodiments 66-73, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0275] Embodiment 75. The method of any one of embodiments 66-74, wherein the condition is cancer.

[0276] Embodiment 76. The method of embodiment 75, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject.

[0277] Embodiment 77. The method of embodiment 76, wherein the subject is a human subject.

[0278] Embodiment 78. The method of embodiment 76, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0279] Embodiment 79. The method of embodiment 75, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of cancer cells in the subject.

[0280] Embodiment 80. The method of embodiment 79, wherein the subject is a human subject.

[0281] Embodiment 81. The method of embodiment 79, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0282] Additional embodiments include those listed below.

[0283] Embodiment 82. A magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of iron oxide primary4876-0545-6626.1Page 56 of 72094876-000021WOPTcrystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; and wherein the at least one superparamagnetic nanoparticle does not have a core-shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

[0284] Embodiment 83. The method of embodiment 82, wherein the iron oxide is Fe3O4, or Fe2O3, or combination thereof.

[0285] Embodiment 84. The method of embodiment 82, wherein the particle size is less than 500 nm, and wherein the crystallite size is less than 15 nm.

[0286] Embodiment 85. The method embodiment 82, wherein the crystallite size is 10 nm to 15 nm.

[0287] Embodiment 86. The method of embodiment 82, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.

[0288] Embodiment 87. The method of embodiment 82, wherein the magnetic field is an alternating current magnetic field.

[0289] Embodiment 88. The method of embodiment 82, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject.

[0290] Embodiment 89. The method of embodiment 82, wherein the subject is a human subject.

[0291] Embodiment 90. The method of embodiment 82, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0292] Embodiment 91. The method of embodiment 82, wherein the condition is cancer.

[0293] Embodiment 92. The method of embodiment 91, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject.

[0294] Embodiment 93. The method of embodiment 92, wherein the subject is a human subject.4876-0545-6626.1Page 57 of 72094876-000021WOPT

[0295] Embodiment 94. The method of embodiment 92, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0296] Embodiment 95. The method of embodiment 91, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of cancer cells in the subject.

[0297] Embodiment 96. The method of embodiment 95, wherein the subject is a human subject.

[0298] Embodiment 97. The method of embodiment 95, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0299] Additional embodiments include those listed below.

[0300] Embodiment 98. A magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; wherein the plurality of iron oxide primary crystals does not have a core-shell structure; and wherein the at least one superparamagnetic nanoparticle does not have a core-shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

[0301] Embodiment 99. The method of embodiment 98, wherein the iron oxide is Fe3O4, or Fe2O3, or combination thereof.

[0302] Embodiment 100. The method of embodiment 98, wherein the particle size is less than 500 nm, and wherein the crystallite size is less than 15 nm.

[0303] Embodiment 101. The method embodiment 98, wherein the crystallite size is 10 nm to 15 nm.

[0304] Embodiment 102. The method of embodiment 98, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.4876-0545-6626.1Page 58 of 72094876-000021WOPT

[0305] Embodiment 103. The method of embodiment 98, wherein the magnetic field is an alternating current magnetic field.

[0306] Embodiment 104. The method of embodiment 98, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject.

[0307] Embodiment 105. The method of embodiment 98, wherein the subject is a human subject.

[0308] Embodiment 106. The method of embodiment 98, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0309] Embodiment 107. The method of embodiment 98, wherein the condition is cancer.

[0310] Embodiment 108. The method of embodiment 107, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject.

[0311] Embodiment 109. The method of embodiment 108, wherein the subject is a human subject.

[0312] Embodiment 110. The method of embodiment 108, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

[0313] Embodiment 111. The method of embodiment 107, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of cancer cells in the subject.

[0314] Embodiment 112. The method of embodiment 111, wherein the subject is a human subject.

[0315] Embodiment 113. The method of embodiment 111, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof. EXAMPLES

[0316] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.4876-0545-6626.1Page 59 of 72094876-000021WOPT

[0317] Chapter 1 - Experiments

[0318] Materials.

[0319] The chemicals used for synthesis of polycrystalline superparamagnetic nanoparticles were iron (III) chloride hexahydrate (97%, Alfa Aesar), ethylene glycol (99%, Sigma Aldrich), diethylene glycol (99%, ACROS Organics), sodium acetate anhydrous (99%, ACROS Organics), sodium acetate trihydrate (99%, Fisher), sodium acrylate (97%, Sigma Aldrich), and polyethylene glycol (400) (Olin Mathieson Chemical Corporation). A pressure vessel 65-mL volume was purchased from Chem Glass. For the synthesis of iron oxide nanoparticles (particle size < 20 nm), 1-octadecene (90%, Sigma Aldrich) and sodium oleate (97%, TCI) were used as reagents. In addition, common solvents such as deionized water with a resistance of 18 MΩ-cm (Academic Milli-Q Water System, Millipore Corporation), ethanol (200 proof, Decon Labs), and n-hexane (99%, Oakwood) were used consistently.

[0320] Example 1

[0321] Synthesis of iron oxide superparticles. The synthesis of polycrystalline superparamagnetic nanoparticles was achieved through solvothermal synthesis in a binary solvent system containing ethylene glycol and diethylene glycol. The binary solvent system consisted of 15 / 25 ml or 20 / 20 ml of ethylene glycol / diethylene glycol. In this method, the use of anhydrous sodium acetate (2.17 or 3.6 g) or the addition of small amounts of sodium acrylate (0.1 to 0.2 g) was found to be effective in reducing the crystallite size (CS) (the size of the primary crystal) while allowing for the fabrication of large particles with diameters ranging from 150 nm to 400 nm. Initially, a pressure vessel was charged with 1.35 g of FeCl3·6H2O and 20 ml of the binary solvent mixture. After the complete dissolution of the chemical, an additional 20 ml of the binary solvent was added, along with the appropriate quantities of additives (sodium acetate anhydrous, water, sodium acrylate). The mixture was stirred for 30 minutes to ensure complete dissolution, followed by the addition of 1.2 mL of PEG (400) surfactant. The Teflon cap was securely fastened, and the pressure vessel was heated to 188 °C for refluxing over 5 hours. A safety shield was used to cover the synthesis setup. After synthesis, the product was cooled to room temperature and washed with ethanol, combined with magnetic separation for at least 3 cycles. It is important to note that faster stirring speed and slower ramping rate can increase the average particle size of nanoparticles.

[0322] Characterizations.4876-0545-6626.1Page 60 of 72094876-000021WOPT

[0323] Scanning electron microscope (SEM) FEI Dual Beam 235 Focused Ion Beam at operating voltage 15 kV was used for imaging the nanoparticles. Samples were dissolved in ethanol and drop-casted on a clean silicon wafer. Transmission electron microscope (TEM) JEOL JEM-2010 was used with an accelerating voltage of 200kV. Samples were prepared on TEM grids 300-mesh holey carbon-coated copper grids (TED Pella). X-ray Diffractometer (Smart Lab, Rigaku) using Cu Kα irradiations operated at 45 kV and 44 mA with a 0.01° step size was used for characterization of all samples.

[0324] Raman spectra were obtained in the backscattering geometry using a confocal microscope Raman spectrometer (LabRAM HR Evolution, Horiba Scientific) dispersed by an 1800 gr / mm grating with a 532 nm wavelength laser as the excitation source. The magnetic measurements were executed using a Physical Property Measurement System (PPMS) by Quantum Design, Inc., utilizing the vibrating sample magnetometer option between 10-350 K at a maximum applied magnetic field of 2 T. Magnetic hyperthermia experiments were carried out using a 4.2 kW Ambrell Easyheat Li3542 equipment with varying AC magnetic fields (0-800 Oe) at a constant 310 kHz frequency starting at 200C for 900 seconds with 0.5 mg / mL and 1 mg / mL nanoparticles in a 2% by weight agar solution prepared with deionized water.

[0325] Additional Materials and Additional Examples

[0326] Additional Materials (Examples A-B). Iron (III) chloride hexahydrate (97%, Alfa Aesar) was used as precursor for synthesis of Iron oxide nanospheres. Ethylene glycol (99%, Sigma Aldrich) and Diethylene glycol (99%, ACROS Organics) were used as solvents. Sodium acetate (99%, anhydrous) was purchased from ACROS. Organic and sodium acetate trihydrate (99%) was purchased from Fisher. Sodium acrylate (97%) was purchased from Sigma Aldrich. Surfactant polyethylene glycol-400 were purchased from Olin Mathieson Chemical Corporation. Deionized water with resistance of 18 MΩ-cm (Academic Milli-Q Water System, Millipore Corporation). Pressure vessel used for synthesis of spherical IONSs was used with volume of 65 mL from ChemGlass. All chemicals were used for synthesis without any further purification.

[0327] Example A. Synthesis of Highly Crystalline Iron Oxide Nanospheres. A synthetic solvothermal method for fabricating highly crystalline Fe3O4nanospheres was developed. FIG.20 illustrates the procedure for synthesizing highly crystalline Fe3O4nanospheres. Pressure vessels used for the synthesis of iron oxide nanospheres were cleaned in an aqua regia solution (3:1 HCl:HNO3) and then dried overnight at 60°C. Initially, a 40 mL mixture of a binary4876-0545-6626.1Page 61 of 72094876-000021WOPTsolvent system containing ethylene glycol (EG) and diethylene glycol (DEG) was prepared. To produce nanoparticles with different particle sizes (average diameters), various ratios of the bi- solvent system EG / DEG (mL) were used: 10 / 30, 15 / 25, 20 / 20, and 30 / 10, giving nanospheres with average diameters (particle sizes) of approximately 50 nm, 120 nm, 220 nm, and 390 nm, respectively. The process involved charging 1.35 g of FeCl3·6H2O into a pressure vessel with 20 mL of the solvent mixture and stirring for 15 minutes to dissolve the chemicals. Subsequently, 3.6 g of NaAc·3H2O was added to the remaining half of the solvent mixture, and vigorous stirring continued for 30 minutes to form a homogeneous mixture. An aliquot of 1200 μL PEG(400) was then injected into the mixture, followed by heating it to 188°C. Only under the synthetic condition using 10 / 30 EG / DEG mL was 1 mL of PEG(400) surfactant employed. The Teflon cap of the pressure vessel was securely screwed on, and a safety shield was used to cover the surroundings throughout the reaction. The ramping rate was maintained at approximately 4.8°C / min, and the reaction time was set to 5 hours under vigorous stirring at least 540 rpm. The resulting product, which had a black color, was allowed to cool naturally to room temperature and was washed three times with ethanol, followed by magnetic separation. During each washing cycle, the iron oxide nanoparticles in ethanol were sonicated for 15 minutes. In addition to solvent compositions, we observed that stirring speed and ramping rate were two factors affecting the particle size of the nanoparticles, as well as the homogeneity of their spherical morphology. By increasing the stirring speed by 20%, the conditions for synthesizing IONSs with an average particle size of 120 nm and 220 nm were adjusted to yield average particle sizes of 140 nm and 255 nm, respectively. Conversely, by increasing the ramp rate, the synthesis conditions could be altered to reduce the average particle size of the nanoparticles. For instance, conditions to prepare nanoparticles with a particle size of 120 nm (ramping rate ~ 4.8°C / min) were adjusted to produce IONSs with an average particle size of 89 nm by using a ramping rate of 11°C / min. Therefore, the combination of stirring speed adjustments, ramp rates, and variations in solvent mixtures (EG & DEG) allowed for the production of IONSs with desired average particle sizes. Overall, this synthetic procedure efficiently yielded spherical nanoparticles with homogeneous, spherical-like morphology, characterized by large crystallite sizes (ranging from 24 nm to 37 nm) and a uniform particle size distribution. Among different solvent compositions, we noted that a 15 / 25 (mL) EG / DEG ratio produced IONSs with the best crystalline structure and an average crystallite size of approximately 34 nm to 37 nm.4876-0545-6626.1Page 62 of 72094876-000021WOPT

[0328] Additionally, we noticed that the amount of water contained in trihydrate sodium acetate plays an important role in controlling the average particle size of NPs and their crystallinity. Instead of using 3.6 g of NaAc.3H2O (which contains 2.17 g of NaAc and 1.43 mL of water), we achieved similar results (particle size and crystallite size) by using a combination of 2.17 g of NaAc (anhydrous chemical) and 1.43 mL of deionized water. Varying the amount of water can be used to change the particle size and crystallite size of NPs. For example, by adding an extra 0.54 mL of water, the synthetic conditions for preparing 220-nm nanospheres yielded Fe3O4NPs with an average particle size of 175 nm and the crystallite size of 34 nm. Similarly, adding 0.54 mL of DI water to the synthetic conditions for a target size of 120 nm gave NPs with a particle size of 93 nm and a crystallite size of 34 nm. Therefore, adding water can be used to fabricate IONSs with different particle sizes and crystallite sizes. Experiments involving the addition of different amounts of water or the use of anhydrous NaAc are presented in Table 5.

[0329] Table 5. Control Experiments Involving Addition of Water and Anhydrous NaAc. EG DEG Additives Added H2O (mL) Average Particle Crystallite Size4876-0545-6626.1Page 63 of 72094876-000021WOPT15 25 3.6 g 0.18 118 ± 9 38 NaAc·3H2Oite Sizes. To reduce the crystallinity of IONSs, we employed two approaches: replacing NaAc·3H2O with anhydrous NaAc or adding an extra amount of sodium acrylate (Na-acrylate) to decrease the average crystallite size. Generally, when we replaced 3.6 g of NaAc·3H2O with 3.6 g of anhydrous NaAc while keeping other parameters constant, the average particle size of NPs increased, but the crystallite size was significantly reduced to less than 20 nm, as shown in Table 5. Similarly, to synthesize highly crystalline IONSs, we used anhydrous sodium acetate in combination with variations in the EG / DEG ratio and adjustments to stirring speed and ramping rate, affording various NP particle sizes with small crystallite sizes (less than 20 nm). We prepared different sets of IONSs with similar particle sizes around 175 nm, 230 nm, and 380 nm but with varying crystallite sizes. Additionally, the addition of extra sodium acrylate, such as 100 mg or 200 mg, proved effective in finely reducing the average crystallite size of IONSs while maintaining a consistent average particle size. For instance, when we added 100 mg or 200 mg of sodium acrylate to the synthetic conditions for IONSs (particle size 160 nm, crystallite size 26 nm), we were able to produce IONSs with similar particle sizes while reducing the average crystallite sizes to 19 nm and 10 nm, respectively. However, the addition of sodium acrylate led to a broadening of the particle size distribution and deviations from spherical morphology in the synthesized nanoparticles.

[0331] Materials Characterization (Examples A-B). Scanning electron microscope (SEM) LEO-1525 and FEI Dual Beam 235 Focused Ion Beam was used for imaging the nanoparticles at operating voltage 15 kV. All samples were dissolved in ethanol and drop-casted on a clean surface of silicon wafer. Transmission electron microscope (TEM) JEOL JEM-2010 was used for imaging nanoparticles and Selected Area Electron Diffraction of TEM was used to analyze the crystalline nature of the samples. The accelerating voltage of TEM was 200 kV. TEM4876-0545-6626.1Page 64 of 72094876-000021WOPTgrids 300-mesh holey carbon-coated copper grids were used. X-ray Diffractometer (Smart Lab, Rigaku) using Cu Kα irradiations operated at 45 kV and 44 mA with a 0.01° step size was used for characterization of phase composition and determine crystallinity. Samples were drop-casted on a clean glass slide surface for measurement from 5 to 90° (2θ) in 20 mins. Full width at half maximum (FWHM) and θ of the most prominent peak of (311) planes at diffracted angle 2θ at 35.5° was used to calculate the average crystallite size. The average crystallite size was calculated by Debye-Scherrer formula, ^ ൌ^^ஒୡ୭^^with K = 0.9 for spherical particles, λ = 0.15406 nm, β = FWHM (radians), and θ is the Bragg angle (radians). X-ray photoelectron spectroscopy (XPS) - PHI 5700 X-ray photoelectron spectrometer with a monochromatic Al Kα X-ray source was used to characterize the iron oxide powder samples. C 1s at binding energy 284.8 eV is used for calibration. Dried powder samples with known mass (from 10 – 12 mg) were loaded in capsule for measure magnetic properties by vibrating sample magnetometer (VSM) (LakeShore VSM 7300 Series with Controller LakeShore 735 and Gaussmeter LakeShore 450 and Software Version 3.8.0). Magnetization versus field (M-H) measurements were swept in the range of field ± 3k Oe. A superconducting quantum interference device (SQUID) MPMS3 by Quantum Design in DC mode was used to record the magnetization versus temperature (M-T) of superparamagnetic samples. The M-T measurement was recorded following the common zero-field-cooling / field- cooling (ZFC / FC) protocol at field 500 Oe.

[0332] Chapter 2 – Experiments

[0333] Experimental Methods

[0334] Materials. The chemicals used for synthesis of polycrystalline superparamagnetic nanoparticles were iron(III) chloride hexahydrate (97%, Alfa Aesar), ethylene glycol (99%, Sigma Aldrich), diethylene glycol (99%, ACROS Organics), sodium acetate anhydrous (99%, ACROS Organics), sodium acetate trihydrate (99%, Fisher), sodium acrylate (97%, Sigma Aldrich), and polyethylene glycol (400) (Olin Mathieson Chemical Corporation). A 65-mL volume pressure vessel was purchased from Chem Glass. In addition, common solvents such as deionized water with a resistance of 18 MΩ-cm (Academic Milli-Q Water System, Millipore Corporation), ethanol (200 proof, Decon Labs), and acetone (99%, Oakwood) were used.

[0335] Synthesis of iron oxide superparticles. The polycrystalline superparamagnetic nanoparticles were synthesized by solvothermal methods in a binary solvent system containing4876-0545-6626.1Page 65 of 72094876-000021WOPTethylene glycol and diethylene glycol, with modifications in the technical set-up and the uses of chemical additives (Y.-T. Chen, R. Medhi, I. Nekrashevich, D. Litvinov, S. Xu, T. R. Lee, Anal. Chem.2018, 90, 6749; H. Deng, X. Li, Q. Peng, X. Wang, J. Chen, Y. Li, Angew. Chem. Int. Ed. 2005, 44, 2782; S. Xuan, Y.-X. J. Wang, J. C. Yu, K. Cham-Fai Leung, Chem. Mater.2009, 21, 5079; Y. Liu, T. Cui, Y. Li, Y. Zhao, Y. Ye, W. Wu, G. Tong, Mater. Chem. Phys. 2016, 173, 152). In this method, the use of anhydrous sodium acetate (2.17 or 3.6 g) in the synthesis was found to be effective in maintaining the small crystallite size (the size of the primary crystal) at the range from 10 to 15 nm while allowing for the fabrication of large particles with diameters larger than 160 nm (Nguyen, M. D.; Deng, L.; Lee, J. M.; Resendez, K. M.; Fuller, M.; Hoijang, S.; Robles Hernandez, F. C.; Chu, C.-W.; Litvinov, D.; Hadjiev, V. G.; Xu, S.; Phan, M.-H.; Lee, T. R. Magnetic Tunability via Control of Crystallinity and Size in Polycrystalline Iron Oxide Nanoparticles. Small 2024, 2402940). Initially, a pressure vessel was charged with 1.35 g of FeCl3·6H2O and 20 mL of the binary solvent mixture (see below). After the complete dissolution of the iron chloride, an additional 20 mL of the binary solvent was added to anhydrous sodium acetate. The binary solvent system used for the synthesis consisted of 15 / 25 mL or 20 / 20 mL of ethylene glycol / diethylene glycol. The mixture was stirred for 30 minutes to ensure complete dissolution, followed by the addition of 1.2 mL of PEG (400) surfactant. The Teflon cap was securely fastened, and the pressure vessel was heated to 188 °C for refluxing over 5 hours. A safety shield was used to cover the synthesis setup. After synthesis, the product was cooled to room temperature and washed with ethanol combined with magnetic separation for at least 3 cycles. It is important to note that technical parameters such as stirring speed or slower heating rate can be used to alter the size of nanoparticles. Particularly, a faster stirring speed or slower ramping rate can increase the average size of nanoparticles. By adjusting the technical parameters, varying the solvent composition, and utilizing different amounts of sodium acetate additives, the size of polycrystalline iron oxide nanoparticles can be tuned from 160 to 400 nm (Nguyen, M. D.; Deng, L.; Lee, J. M.; Resendez, K. M.; Fuller, M.; Hoijang, S.; Robles Hernandez, F. C.; Chu, C.-W.; Litvinov, D.; Hadjiev, V. G.; Xu, S.; Phan, M.-H.; Lee, T. R. Magnetic Tunability via Control of Crystallinity and Size in Polycrystalline Iron Oxide Nanoparticles. Small 2024, 2402940).

[0336] Characterizations. Scanning electron microscope (SEM) FEI Dual Beam 235 Focused Ion Beam at operating voltage 15 kV was used for imaging the nanoparticles. Samples were dissolved in ethanol and drop-cast onto a clean silicon wafer. Transmission electron4876-0545-6626.1Page 66 of 72094876-000021WOPTmicroscopy (TEM) JEOL JEM-2010 was used with an accelerating voltage of 200 kV. Samples were prepared on TEM grids 300-mesh holey carbon-coated copper grids (TED Pella). X-ray diffraction data (Smart Lab, Rigaku) were collected using Cu Kα irradiation operated at 40 kV and 44 mA with a 0.01° step size was used for all samples. The crystallite size was calculated by the Scherrer formula from the diffracted peak of the (311) plane at 2θ of 35.5°. X-ray photoelectron spectroscopy (XPS) analysis was conducted on a dried sample drop-cast onto a silicon wafer using a PHI 5700 X-ray photoelectron spectrometer with monochromatic Al Kα X-rays. Calibration was performed using the C 1s peak at 284.8 eV.

[0337] The magnetic measurements were performed using a Physical Property Measurement System (PPMS) by Quantum Design, Inc., utilizing the vibrating sample magnetometer option between 10-350 K at a maximum applied magnetic field of 2 T. Calorimetric magnetic hyperthermia experiments were carried out using a 4.2 kW Ambrell Easyheat Li3542 equipment with varying AC magnetic fields (0-800 Oe) at a constant 310 kHz frequency starting at 200C for 900 seconds with 0.5 mg / mL and 1 mg / mL nanoparticles in a 2% by weight agar solution prepared with deionized water. AC magnetometry measurements were carried out using a homemade setup to record the AC hysteresis loops (I. Rodrigo, I. Castellanos-Rubio, E. Garaio, O. K. Arriortua, M. Insausti, I. Orue, J. Á. García, F. Plazaola, Int. J. Hyperth.2020, 37, 976). The AC magnetic field amplitude was tuned between 0 and 80 mT, and 3 different frequencies were employed, 149 kHz, 300, and 638 kHz. Samples were again prepared in a 2% agar solution.

[0338] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0339] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above,4876-0545-6626.1Page 67 of 72094876-000021WOPTas well as other known equivalents for each such element, feature, or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0340] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0341] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0342] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0343] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in4876-0545-6626.1Page 68 of 72094876-000021WOPTaccordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

[0344] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0345] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention is not limited to the particular embodiments disclosed for carrying out the invention.

[0346] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.4876-0545-6626.1Page 69 of 72094876-000021WOPT

Claims

CLAIMS What is claimed is:

1. A magnetic hyperthermia method for treating a condition in a subject, the method comprising: obtaining at least one superparamagnetic nanoparticle, wherein the at least one superparamagnetic nanoparticle comprises a plurality of iron oxide primary crystals, wherein the plurality of iron oxide primary crystals comprise a plurality of crystallites, wherein the at least one superparamagnetic nanoparticle has a particle size of less than or equal to 500 nm, wherein the plurality of crystallites have a crystallite size of less than 15 nm, wherein the at least one superparamagnetic nanoparticle has a spherical shape; wherein the plurality of iron oxide primary crystals does not have a core-shell structure; and wherein the at least one superparamagnetic nanoparticle does not have a core-shell structure; administering a therapeutically effective amount of the at least one superparamagnetic nanoparticle to a region of a subject; and subjecting the at least one superparamagnetic nanoparticle to a magnetic field to treat a condition in the subject.

2. The method of claim 1, wherein the iron oxide is Fe3O4, or Fe2O3, or combination thereof.

3. The method of claim 1, wherein the particle size is less than 500 nm, and wherein the crystallite size is less than 15 nm.

4. The method claim 1, wherein the crystallite size is 10 nm to 15 nm.

5. The method of claim 1, wherein the particle size is 150 nm to 400 nm, or 160 nm to 375 nm.

6. The method of claim 1, wherein the magnetic field is an alternating current magnetic field.4876-0545-6626.1Page 70 of 72094876-000021WOPT7. The method of claim 1, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the condition in the subject.

8. The method of claim 1, wherein the subject is a human subject.

9. The method of claim 1, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

10. The method of claim 1, wherein the condition is cancer.

11. The method of claim 10, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat treats the cancer in the subject.

12. The method of claim 11, wherein the subject is a human subject.

13. The method of claim 11, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.

14. The method of claim 10, wherein the at least one superparamagnetic nanoparticle converts electromagnetic energy from the magnetic field to heat, and wherein the heat kills at least a portion of cancer cells in the subject.

15. The method of claim 14, wherein the subject is a human subject.

16. The method of claim 14, wherein the region is a tumor, tissue, organ, limb, or body cavity, or any combination thereof.4876-0545-6626.1Page 71 of 72094876-000021WOPT

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