A method for changing the electrical impedance to an AC electric field

Abstract

A method for changing the electrical impedance of a target site in a subject to an alternating current electric field is disclosed, comprising: introducing nanoparticles into the target site in the subject; and applying an alternating current electric field to the target site in the subject, whereby the electrical impedance of the target site in the subject to an alternating current is changed. A method for improving transport of nanoparticles across a cell membrane is disclosed, comprising: applying an alternating current electric field to a cell for a period of time, whereby application of the alternating current electric field increases the permeability of the cell membrane; and introducing nanoparticles into the cell, whereby the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane. A method for imaging cancer cells is disclosed, comprising: applying a first alternating current electric field at a first frequency to cancer cells for a first period of time, whereby application of the first alternating current electric field to the cancer cells at the first frequency for the first period of time increases the permeability of the cell membrane of the cancer cells; introducing nanoparticles into the cancer cells, whereby the increased permeability of the cell membrane allows the nanoparticles to cross the cancer cell membrane; and imaging the cancer cells.

Description

[Technical Field]

[0001] Related Patent Cross-References This application claims priority to U.S. Provisional Patent Application No. 62 / 946,798, filed December 11, 2019, which is incorporated by reference herein in its entirety. [Background technology]

[0002] Tumor treating electric fields, or TTFields, are typically low-intensity (e.g., 1–3 V / cm) alternating electric fields in the mid-frequency range (100–300 kHz). TTFs can be delivered to tumor anatomical regions through noninvasive transducer arrays. TTFs have been established as an anti-mitotic cancer treatment modality because they disrupt proper microtubule assembly during metaphase, ultimately disrupting cells in telophase, cytokinesis, or subsequent quiescence. Due to their low intensity, TTFields have been shown not to affect the viability of non-dividing normal cells, nerves, or muscles. TTFields therapy is an approved monotherapy for recurrent glioblastoma and an approved combination therapy with chemotherapy for newly diagnosed glioblastoma and unresectable malignant pleural mesothelioma patients. These electric fields are non-invasively induced by a transducer array (i.e., an array of electrodes) placed directly on the patient's scalp. TTFields may also be beneficial for treating tumors in other parts of the body. Summary of the Invention [Means for solving the problem]

[0003] A method is disclosed for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered.

[0004] A method for altering electrical impedance to an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to the alternating current at the target site in the subject is altered.

[0005] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject and a site adjacent to the target site, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased.

[0006] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased.

[0007] A method for changing the electrical impedance to an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing nanoparticles into the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to the alternating current at the target site in the subject is changed.

[0008] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased.

[0009] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a non-target site adjacent to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased.

[0010] A method for improving transport of nanoparticles across the cell membrane of a cell is disclosed, comprising the steps of: applying an alternating electric field to the cell for a period of time, wherein the application of the alternating electric field increases the permeability of the cell membrane; and introducing nanoparticles into the cell, wherein the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane.

[0011] A method of reducing the viability of cancer cells is disclosed, comprising applying a first alternating electric field at a first frequency to the cancer cells for a first period of time, wherein applying the first alternating electric field at the first frequency to the cancer cells for the first period of time increases the permeability of a cell membrane of the cancer cells; introducing nanoparticles into the cancer cells, wherein the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane; and applying a second alternating electric field at a second frequency to the cancer cells for a second period of time, wherein the second frequency is different from the first frequency, and wherein the second alternating electric field at the second frequency reduces the viability of the cancer cells.

[0012] Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be understood from the description or may be obtained by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limitations of the invention as claimed.

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions. [Brief explanation of the drawings]

[0014] [Figure 1A] FIG. 1 shows an exemplary effect of enhancing tumor conductivity with TTFields intensity. : Axial section of an MRI of a patient with GBM with the patient's entire tumor volume area marked. [Figure 1B] FIG. 1 shows an exemplary effect of enhancing tumor conductivity with TTFields intensity: Simulation results for a computerized head model from the assignment of an enhanced tumor tissue conductivity value of 0.24 S / m. [Figure 1C] FIG. 1 shows an exemplary effect of enhancing tumor conductivity with TTFields intensity: Simulation results for a computerized head model from the assignment of an enhanced tumor tissue conductivity value of 0.3 S / m. [Figure 1D] FIG. 10 illustrates an exemplary effect of enhancing tumor conductivity with TTFields intensity: relative difference between simulation results of 0.3 S / m vs. 0.24 S / m. [Figure 2] FIG. 1 shows a histogram of mean LMiPD across the entire tumor volume of 45 head models of GBM patients treated with TTFields. [Figure 3A] Figure 1 shows the physicochemical characterization of BTNPs. FE-SEM images of BTNPs. [Figure 3B] Figure 1 shows the physicochemical characterization of BTNPs. TEM images of BTNPs. [Figure 3C] Figure 1 shows the physicochemical characterization of BTNPs: Size and zeta potential values ​​of FBS-coated BTNPs. [Figure 4A] Figure 1 shows the cytocompatibility of BTNPs in breast cancer cells. Cell proliferation in MCF-7 cells with BTNP treatment. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. NS: not significant. [Figure 4B] Figure 1 shows the cytocompatibility of BTNPs in breast cancer cells. Cell proliferation in BT-549 cells with BTNP treatment. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. NS: not significant. [Figure 4C]Figure 1 shows the cytocompatibility of BTNPs in breast cancer cells. Representative images from clonogenic assays in MCF-7 cells with BTNP treatment. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. NS: not significant. [Figure 4D] Figure 1 shows the cytocompatibility of BTNPs in breast cancer cells. Representative images from clonogenic assays in BT-549 cells with BTNP treatment. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. NS: not significant. [Figure 4E] Figure 1 shows the cytocompatibility of BTNPs in breast cancer cells. Colony counts in MCF-7 cells with BTNP treatment. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. NS: not significant. [Figure 4F] Figure 1 shows the cytocompatibility of BTNPs in breast cancer cells. Colony counts in BT-549 cells with BTNP treatment. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. NS: not significant. [Figure 5A] Figure 1 shows that BTNPs enhanced the antitumor activity of TTFields in MCF-7 cells with low TTFields sensitivity. Cell proliferation after TTFields in MCF-7, MDA-MB-231, and BT-549 cells. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. [Figure 5B] Figure 1 shows that BTNPs enhanced the antitumor activity of TTFields in MCF-7 cells with low TTFields sensitivity. Relative cell counts in MCF-7 cells treated with TTFields or TTFields and BTNPs. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. [Figure 5C]Figure 1 shows that BTNPs enhanced the antitumor activity of TTFields in low TTFields-sensitive MCF-7 cells. Colony quantification. Data represent the mean ± standard deviation of three independent experiments; **P<0.01 and *P<0.05. [Figure 6A] Figure 1 shows the cytoplasmic accumulation of BTNPs in MCF-7 and BT-549 cells in response to TTFields. Flow cytometry histograms showing the cytosolic localization of BTNPs in MCF-7 cells treated with TTFields or TTFields and BTNP. Data are representative of three independent experiments. [Figure 6B] Figure 1 shows the cytoplasmic accumulation of BTNPs in MCF-7 and BT-549 cells in response to TTFields. Flow cytometry histograms showing the cytosolic localization of BTNPs in BT-549 cells treated with TTFields or TTFields and BTNP. Data are representative of three independent experiments. [Figure 6C] Figure 1 shows the cytoplasmic accumulation of BTNPs in MCF-7 and BT-549 cells in response to TTFields. Representative images showing the cytosolic localization of BTNPs in MCF-7 cells treated with TTFields or TTFields and BTNP. Data are representative of three independent experiments. [Figure 6D] Figure 1 shows the cytoplasmic accumulation of BTNPs in MCF-7 and BT-549 cells in response to TTFields. Representative images showing the cytosolic localization of BTNPs in BT-549 cells treated with TTFields or TTFields and BTNP. Data are representative of three independent experiments. [Figure 6E] Figure 1 shows cytoplasmic accumulation of BTNPs in MCF-7 and BT-549 cells in response to TTFields. TEM images confirming cytosolic localization of BTNPs in TTFields-treated MCF-7 cells. Data are representative of three independent experiments. [Figure 7A]Figure 1 shows gene copy number changes in MCF-7 cells upon combinatorial TTFields and BTNP treatment. Heatmap with global significance score and global significance statistics for cells treated with TTFields or TTFields and BTNPs. [Figure 7B] Figure 1 shows gene copy number changes in MCF-7 cells with TTFields and BTNP combinatorial treatment. Directed global significance scores for cells treated with TTFields or TTFields and BTNPs. [Figure 8A] Figure 1 shows modulation of cell cycle apoptosis pathways by BTNPs in combination with TTFields. Gene signatures associated with cell cycle pathways are grouped in a heatmap of MCF-7 cells treated with TTFields or TTFields and BTNP. Data are representative of three independent experiments. [Figure 8B] Figure 1 shows modulation of the cell cycle apoptosis pathway by BTNPs in combination with TTFields. Western blot of MCF-7 cells treated with TTFields or TTFields and BTNP. Data are representative of three independent experiments. [Figure 9] Schematic of the proposed mechanism of cancer cell sensitization induced by BTNPs in the presence of TTFields. DETAILED DESCRIPTION OF THE INVENTION

[0015] The disclosed methods and compositions will be more readily understood by reference to the following detailed description of specific embodiments and examples contained herein, as well as the drawings and their preceding and following description.

[0016] It is understood that the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or particular reagents, as these may vary, unless otherwise specified. It is also understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0017] Disclosed are materials, compositions, and components that can be used for, used in conjunction with, used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that, although specific reference to the various individual and collective combinations and permutations of each of these compounds may not be expressly disclosed, each is specifically contemplated and described herein. Thus, when a class of molecules A, B, and C is disclosed along with a class of molecules D, E, and F, and an example combination of molecules A-D is disclosed, each is individually and collectively contemplated, even if each is not individually listed. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and example combinations A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, subgroups A-E, B-F, and C-E should be considered specifically contemplated and disclosed from the disclosure of A, B, and C; D, E, and F; and combination examples A-D. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0018] A.Definition It is understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0019] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise, so that, for example, reference to "a nanoparticle" includes a single or a plurality of such nanoparticles, reference to "the nanoparticle" is a reference to one or more nanoparticles and equivalents thereof known to those skilled in the art, and so forth.

[0020] As used herein, a "target site" is a specific site or location within or present in a subject or patient. For example, a "target site" may refer to, but is not limited to, a cell, a group of cells, an organ, a tissue, a tumor, or a cancer cell. In some aspects, organs include, but are not limited to, lung, brain, pancreas, abdominal organs (e.g., stomach, intestine), ovary, breast, uterus, prostate, bladder, liver, colon, or kidney. In some aspects, cells or group of cells include, but are not limited to, lung cells, brain cells, pancreatic cells, abdominal cells, ovarian cells, liver cells, colon cells, or kidney cells. In some aspects, a "target site" may be a tumor target site.

[0021] A "tumor target site" is a site or location within or present in a subject or patient that contains or is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells. For example, a tumor target site may refer to a site or location within or present in a subject or patient that is prone to metastasis. Additionally, a target site or tumor target site may refer to a site or location of a resection of a primary tumor within or present in a subject or patient. Additionally, a target site or tumor target site may refer to a site or location adjacent to a resection of a primary tumor within or present in a subject or patient.

[0022] As used herein, one or more "alternating electric fields" refer to very low intensity, directional, intermediate frequency alternating electric fields delivered to a subject, a sample obtained from a subject, or a specific location within a subject or patient (e.g., a target site or tumor target site). In some embodiments, the alternating electric field can be unidirectional or multidirectional.

[0023] Examples of alternating electric fields include, but are not limited to, tumor-treating electric fields. In some embodiments, TTFields can be delivered through two pairs of transducer arrays that generate perpendicular fields within the tumor being treated. For example, for the Optune™ system (a TTFields delivery system), one pair of electrodes is placed on the left and right (LR) sides of the tumor, and the other pair of electrodes is placed on the anterior and posterior (AP) sides of the tumor. Cycling the field in these two directions (i.e., LR and AP) ensures targeting of the widest range of cell orientations.

[0024] As described herein, TTFields have been established as an anti-mitotic cancer treatment modality by disrupting correct microtubule assembly during metaphase, ultimately destroying cells in telophase, cytokinesis, or subsequent quiescence. TTFields target solid tumors and are described in U.S. Patent No. 7,565,205, the teachings of TTFields being incorporated herein by reference in their entirety.

[0025] In vivo and in vitro studies have shown that the efficacy of TTFields treatment increases as the strength of the electric field increases. Therefore, optimizing array placement on the patient's scalp to increase intensity in diseased areas of the brain is standard practice for the Optune system. Optimizing array placement can be performed by "rules of thumb" (e.g., placing the array on the scalp as close to the tumor as possible), measurements representing the patient's head shape, tumor size, and / or tumor location. The measurements used as input can be derived from imaging data. Image data is intended to include any type of imaging data, such as single-photon emission computed tomography (SPECT) image data, X-ray computed tomography (X-ray CT) data, magnetic resonance imaging (MRI) data, positron emission tomography (PET) data, or data that can be captured by optical instruments (e.g., photographic cameras, charge-coupled device (CCD) cameras, infrared cameras, etc.). In certain implementations, the image data can include 3D data obtained from or generated by a 3D scanner (e.g., point cloud data). Optimization may depend on understanding how the electric field is distributed within the head as a function of array location, and in some aspects takes into account variations in the distribution of electrical properties within the heads of different patients. The term "subject" refers to the target of administration, e.g., an animal. Accordingly, the subject of the disclosed methods may be a vertebrate, such as a mammal. For example, the subject may be a human. The term does not denote a particular age or sex. Subject may be used interchangeably with "individual" or "patient." For example, the target of administration may refer to the recipient of an alternating electric field.

[0026] "Optionally" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0027] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such ranges are expressed, the range from the one particular value and / or to the other particular value is also considered to be specifically contemplated and disclosed, unless the context clearly dictates otherwise. Similarly, when values ​​are expressed as approximately, by use of the antecedent "about," it is understood that the particular value forms another specifically contemplated embodiment that should be considered disclosed, unless the context clearly dictates otherwise. It is further understood that each of the endpoints of a range is significant both in relation to the other endpoint and independently of the other endpoint, unless the context clearly dictates otherwise. Finally, it is to be understood that all individual values ​​and subranges of values ​​within an expressly disclosed range are also considered to be specifically contemplated and disclosed, unless the context clearly dictates otherwise. The foregoing applies regardless of whether some or all of these embodiments are specifically disclosed in a particular instance.

[0028] 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 the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods and compositions, particularly useful methods, devices, and materials are described. Publications cited herein and the materials for which they are cited are specifically incorporated herein by reference. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No reference is admitted to constitute prior art. The discussion of references states what their authors assert, and applicants reserve the right to verify the accuracy and pertinence of the cited documents. Although a number of publications have been referenced herein, it is expressly understood that such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0029] Throughout this description and the claims, variations of words such as "comprise" and "comprising" and "comprises" mean "including but not limited to" and are not intended to exclude, for example, other additives, ingredients, integers, or steps. In particular, in a method described as including one or more steps or operations, each step is specifically contemplated to include what is recited (unless the step includes a limiting term such as "consisting of") and each step is not intended to exclude, for example, other additives, ingredients, integers, or steps not recited in the step.

[0030] B. Nanoparticles Disclosed herein are methods that involve nanoparticles. Any of the nanoparticles described herein can be used for one or more of the disclosed methods.

[0031] In some embodiments, the nanoparticles can comprise a conductive or semiconductive material. For example, the nanoparticles can comprise carbon gold, ferrous iron, selenium, silver, copper, platinum, iron oxide, graphene, iron dextran, superparamagnetic iron oxide, boron-doped detonation nanodiamond, or a combination thereof. In some embodiments, the nanoparticles comprise an alloy selected from Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Fe, Au / Cu, or Au / Fe / Cu.

[0032] In some embodiments, the nanoparticles can be conductive nanoparticles. The conductive nanoparticles can increase the conductivity and decrease the impedance at the target site or tumor target site. Thus, in some embodiments of the disclosed methods, the impedance at the target site or tumor target site is decreased and / or the conductivity at the target site or tumor target site is increased.

[0033] In some embodiments, the nanoparticles are non-conductive nanoparticles. In some embodiments, the non-conductive nanoparticles are ferroelectric nanoparticles. Ferroelectric nanoparticles have emerged as promising tools for enhancing electrical stimulation of cells and tissues. Several nanotransducers have been shown to mediate photodynamic and thermomagnetic effect transduction and locally deliver anti-cancer drug stimuli to tumor burdens in the field of nano-oncology. The cell and tissue penetration of these nanotransducers can be controlled by remote electrical stimulation. Among ferroelectric nanoparticles, barium titanate nanoparticles (BTNPs) have high biocompatibility as well as high dielectric constant and favorable piezoelectric properties. Such non-conductive nanoparticles can be used in the methods disclosed herein to be taken up by cells via TTFields stimulation and to promote the anti-tumor effects of TTFields by enhancing cell cycle-related apoptosis in cancer cells. In some embodiments, the non-conductive nanoparticles are not ferroelectric nanoparticles. Non-conductive nanoparticles can decrease conductivity and increase impedance at the target site or tumor target site. In some aspects of the disclosed methods, the impedance at the target site or tumor target site is increased and / or the conductivity at the target site or tumor target site is decreased.

[0034] In some aspects, a population of nanoparticles can be used in the methods disclosed herein. In some aspects, a population of nanoparticles can include conductive and non-conductive nanoparticles.

[0035] It is well known that nanoparticle (NP) internalization into cells depends on particle size and its zeta potential. NPs smaller than 200 nm can be phagocytosed by cancer cells through clathrin-dependent or macropinocytosis pathways. In some embodiments, the size of nanoparticles can be between 0.5 nm and 100 nm. In some embodiments, the size of nanoparticles can be between 0.5 nm and 2.5 nm. In some embodiments, the size of nanoparticles can be between 100 nm and 200 nm. In some embodiments, the size of nanoparticles can be greater than 100 nm. In some embodiments, the disclosed methods enable the use of nanoparticles (e.g., metallic / magnetic) in the size range of 100 nm to 200 nm (preferably up to 150 nm to avoid accumulation in the liver and spleen) to target cancer cells in vivo.

[0036] In some embodiments, the nanoparticles have a three-dimensional shape, for example, the nanoparticles can be nanocubes, nanotubes, nanobipyramids, nanoplates, nanoclusters, nanochains, nanostars, nanoshuttles, nanohollows, dendrimers, nanorods, nanoshells, nanocage, nanospheres, nanofibers, or nanowires, or combinations thereof.

[0037] In some aspects, the nanoparticles may be mesoporous or non-porous.

[0038] In some embodiments, nanoparticles may be coated with polysaccharides, polyamino acids, or synthetic polymers. Suitable coatings for nanoparticles can be selected to reduce the toxicity of the nanoparticles and provide them with the ability to selectively interact with various types of cells and biological molecules. Suitable coatings for nanoparticles can be selected to improve nanoparticle biocompatibility and solubility in water and biological fluids by reducing their aggregation potential or increasing their stability. Suitable coatings for nanoparticles can be selected to alter the pattern of nanoparticles and / or their distribution and accumulation in the body, affecting nanoparticle pharmacokinetics.

[0039] In some embodiments, nanoparticles can be incorporated into the scaffold before introducing the nanoparticles into the subject. In some embodiments, nanoparticles can be loaded into or into the scaffold before or after introducing the scaffold into the subject. For example, a scaffold can be surgically provided to a subject, and then one or more nanoparticles described herein can be administered to the subject under conditions that allow the nanoparticles to be incorporated into the scaffold. Alternatively, nanoparticles can be incorporated into the scaffold outside the subject, and then the nanoparticle-loaded scaffold can be surgically provided to the subject.

[0040] Exemplary scaffolds include, but are not limited to, scaffolds comprising natural polymers such as hyaluronic acid, fibrin, chitosan, and collagen, etc. Exemplary scaffolds include, but are not limited to, scaffolds comprising synthetic polymers such as polyethylene glycol (PEG), polypropylene fumarate (PPF), polyanhydrides, polycaprolactone (PCL), polyphosphazene, polyetheretherketone (PEEK), polylactic acid (PLA), and poly(glycolic acid) (PGA).

[0041] In some embodiments, the nanoparticles are conjugated to one or more ligands. In some embodiments, the one or more ligands are conjugated to the nanoparticles via a linker. In some embodiments, the linker comprises a thiol group, a C2 to C12 alkyl group, a C2 to C12 glycol group, or a peptide. In some embodiments, the linker comprises a thiol group represented by the general formula HO-(CH)n,-SS-(CH2)m-OH, where n and m are independently between 1 and 5. In some embodiments, the one or more ligands are small molecules, nucleic acids, carbohydrates, lipids, peptides, antibodies, antibody fragments, or therapeutic agents. For example, the one or more ligands may be, but are not limited to, an anti-cancer drug, a cytotoxic drug, a pain management drug, Pseudomonas exotoxin A, a non-radioactive isotope (e.g., boron-10 for boron neutron capture therapy), or a photosensitizer (e.g., photofrin, foscan, 5-aminolevulinic acid, mono-L-aspartyl chlorin e6, phthalocyanine, metatetra(hydroxyphenyl)porphyrin, texaphyrin, ethyl etiopurinse).

[0042] In some embodiments, the nanoparticles can be labeled nanoparticles. In some embodiments, the labeled nanoparticles can be magnetic nanoparticles, nanoparticles decorated with Gd3+, nanoparticles decorated with radioactive isotopes (e.g., technetium-99m, iodine-123, iodine-131, fluorine-18, carbon-11, nitrogen-13, oxygen-15, gallium-68, zirconium-89, and rubidium-82), nanoparticles decorated with fluorescent labels (e.g., quantum dots), nanoparticles decorated with photosensitizers (e.g., photofrin, phoscan, 5-aminolevulinic acid, mono-L-aspartyl chlorin e6, phthalocyanine, metatetra(hydroxyphenyl)porphyrin, texaphyrin, ethyl etiopurinse), or nanoparticles decorated with dyes. In some embodiments, the nanoparticles can be coated with a labeled antibody, thereby indirectly labeling the nanoparticles. In some embodiments, if there are size constraints, the nanoparticles can be small enough to accommodate the larger moiety when decorated with or conjugated to the larger moiety.

[0043] Other examples of nanoparticles include, but are not limited to, silica nanoparticles, hydrophilic polymers (e.g., polyacrylamide (PAA), polyurethane, poly(hydroxyethylmethacrylamide) (pHEMA), certain poly(ethylene glycol)s, and hydrophobic polymers (e.g., polystyrene nanoparticles).

[0044] In some embodiments, the nanoparticles may be introduced to a target site. In some embodiments, the nanoparticles may be introduced to a tumor target site. In some embodiments, the nanoparticles may be introduced to tumor or cancer cells. In some embodiments, the nanoparticles may be introduced to a location in a subject suspected of containing one or more tumor cells. In some embodiments, the nanoparticles may be introduced to a site or location within or present in a subject or patient prone to metastasis. In some embodiments, the nanoparticles may be introduced to a site or location of resection of a primary tumor within or present in a subject or patient. In some embodiments, the nanoparticles may be introduced to tumor or cancer cells via injection after primary tumor resection. In some embodiments, the nanoparticles may be introduced to a site adjacent to the target site. In some embodiments, the nanoparticles may be introduced to a site adjacent to a location in a subject suspected of containing one or more tumor cells.

[0045] In some embodiments, nanoparticles may be introduced into a tumor target site adjacent to a tumor target site. In some embodiments, nanoparticles may be introduced into a tumor target site adjacent to a tumor or cancer cells. In some embodiments, nanoparticles may be introduced into a tumor target site adjacent to a location in a subject suspected of containing one or more tumor cells. In some embodiments, nanoparticles may be introduced into a tumor target site adjacent to a site or location within or present in a subject or patient prone to metastasis. In some embodiments, nanoparticles may be introduced into a tumor target site adjacent to a site or location of resection of a primary tumor within or present in a subject or patient. In some embodiments, nanoparticles may be introduced into a tumor target site adjacent to a tumor or cancer cells via injection after primary tumor resection. In some embodiments, nanoparticles may be introduced into a tumor target site adjacent to a tumor or cancer cells via intratumoral injection (e.g., under computed tomography guidance during surgery or biopsy).

[0046] In some aspects, nanoparticles can be introduced intratumorally, intracranially, intracerebroventricularly, intrathecally, epidurally, intradurally, intravascularly, intravenously (targeted or non-targeted), intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, via intratumoral injection (e.g., under computed tomography guidance during surgery or biopsy), or via inhalation. In some aspects, nanoparticles can be targeted to tumors or tumor target sites using tumor-targeting moieties. Tumor-targeting moieties can be, but are not limited to, folic acid, transferrin, aptamers, antibodies, nucleic acids, and peptides. Thus, in some aspects, nanoparticles can be introduced into a subject in a targeted or non-targeted manner.

[0047] In some embodiments, nanoparticles can be introduced at a concentration based on tumor volume, method of delivery, constraints of the device applying the AC electric field, patient weight, patient age, tumor size, tumor or cancer type, tumor or cancer cell location, patient age, or any other physical or genotypic characteristics of the patient, cancer cells, or tumor. In some embodiments, the size of the nanoparticles can be used to determine the concentration of nanoparticles to be introduced. In some embodiments, nanoparticles can be introduced at a concentration that is ... cell location, patient age, or any other physical or genotypic characteristics of the patient, cancer cells, or tumor. 3The antibody may be introduced at about 0.001 to 0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5 to 5, 5 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 ng per antibody. In some embodiments, the nanoparticles may be introduced at about 0.001 to 0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5 to 5, 5 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 μg.

[0048] In some embodiments, the nanoparticles may be introduced into the subject one, two, three or more times.

[0049] 1. Pharmaceutical Composition Disclosed herein is a pharmaceutical composition comprising one or more nanoparticles as described herein.In some embodiments, the nanoparticles as described herein can be provided in pharmaceutical compositions.For example, the nanoparticles as described herein can be formulated with a pharmaceutically acceptable carrier.

[0050] In some embodiments, the pharmaceutical composition may include a chemotherapeutic agent. In some embodiments, the pharmaceutical composition may include a chemotherapeutic agent and one or more nanoparticles described herein. For example, disclosed herein are pharmaceutical compositions comprising one or more nanoparticles described herein and an anticancer drug, a cytotoxic drug, a pain management drug, Pseudomonas exotoxin A, a non-radioactive isotope (e.g., boron-10 for boron neutron capture therapy), and / or a photosensitizer (e.g., Photofrin, Foscan, 5-aminolevulinic acid, mono-L-aspartylchlorin e6, phthalocyanine, metatetra(hydroxyphenyl)porphyrin, texaphyrin, ethyl etiopurinse).

[0051] Disclosed herein are compositions comprising one or more nanoparticles described herein, further comprising a carrier, such as a pharmaceutically acceptable carrier. For example, disclosed are pharmaceutical compositions comprising the nanoparticles disclosed herein and a pharmaceutically acceptable carrier.

[0052] For example, the nanoparticles described herein may include a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material or carrier well known to those skilled in the art, selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. Examples of carriers include dimyristoylphosphatidylcholine (DMPC), phosphate-buffered saline, or multivesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide may be used as a carrier in the present invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution can be about 5 to about 8 or about 7 to about 7.5. Other carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, for example, films, stents (implanted in blood vessels during revascularization procedures), liposomes, or microparticles.It will be apparent to those skilled in the art that certain carriers may be more preferable, for example, depending on the route of administration and the concentration of nanoparticles administered.Most typically, these are standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0053] Pharmaceutical compositions may also contain carriers, thickeners, diluents, buffers, preservatives, etc., so long as the intended activity of the polypeptides, peptides, nucleic acids, and vectors of the invention is not impaired. Pharmaceutical compositions may also contain one or more active ingredients (in addition to the compositions of the invention), such as antibacterial agents, anti-inflammatory agents, anesthetics, etc. Pharmaceutical compositions may be administered in a number of ways depending on whether local or systemic treatment is desired and on the area to be treated.

[0054] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases.

[0055] Formulations for optimal administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0056] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some compositions may also be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl, and aryl amines and substituted ethanolamines.

[0057] In the methods described herein, delivery (or administration or introduction) of the nanoparticles or pharmaceutical compositions disclosed herein to a subject can be via a variety of mechanisms.

[0058] C. Changes in impedance at the target site A method is disclosed for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered.

[0059] Disclosed is a method for altering electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered, thereby changing the current density and / or power loss density of the alternating current at the target site in the subject.

[0060] Disclosed is a method for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered and the conductivity at the site adjacent to the target site is reduced.

[0061] Disclosed is a method for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered and the impedance at the site adjacent to the target site is increased.

[0062] Disclosed is a method for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered and conductivity at the target site is increased.

[0063] Disclosed is a method for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered, resulting in a decrease in impedance at the target site.

[0064] Disclosed is a method for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered, and the non-conductive nanoparticles are not ferroelectric nanoparticles.

[0065] Disclosed are methods for altering electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered, and the method further comprises introducing nanoparticles to the target site in the subject and applying an alternating current electric field to the target site in the subject. In some embodiments, the impedance at the target site is decreased and / or the conductivity at the target site is increased.

[0066] Disclosed are methods for altering electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered and the nanoparticles are non-conductive nanoparticles. In some embodiments, the impedance at the target site increases and / or the conductivity at the target site decreases.

[0067] A method is disclosed for altering the electrical impedance to an alternating current electric field at a site adjacent to a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance to the alternating current at the site in the subject is altered, and the alternating current electric field is a tumor treatment electric field.

[0068] A method for altering the electrical impedance of a target site in a subject to an alternating current electric field at a site adjacent to the target site is disclosed, the method comprising the steps of: introducing non-conductive nanoparticles into the site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject, wherein the electrical impedance of the site in the subject to the alternating current is altered, and the target site is a tumor target site. In some embodiments, the alteration in the electrical impedance of the target site in the subject to the alternating current results in an increase in the mitotic effect of the alternating current electric field at the target site.

[0069] A method for altering electrical impedance to an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to the alternating current at the target site in the subject is altered.

[0070] Disclosed is a method for altering electrical impedance to an alternating current electric field at a target site in a subject, the method comprising the steps of: introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to the alternating current at the target site in the subject is altered, the current density and / or power loss density to the alternating current at the target site in the subject is altered, the impedance at the target site is reduced, and / or the conductivity at the target site is increased.

[0071] Disclosed are methods for altering electrical impedance to an alternating current electric field at a target site in a subject, comprising: introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to the alternating current at the target site in the subject is altered, and the current density and / or power loss density to the alternating current at the target site in the subject is altered, and the method further comprises introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the site adjacent to the target site in the subject. In some embodiments, the current density and / or power loss density to the alternating current at the target site in the subject is altered. In some embodiments, the electrical conductivity at the site adjacent to the target site is decreased. In some embodiments, the impedance at the site adjacent to the target site is increased. In some embodiments, the electrical conductivity at the target site is increased. In some embodiments, the impedance at the target site is decreased.

[0072] Disclosed are methods for altering electrical impedance at a target site in a subject to an alternating current electric field, the method comprising: introducing conductive nanoparticles into the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance at the target site in the subject to the alternating current is altered, and the current density and / or power loss density at the target site in the subject to the alternating current is altered, and the non-conductive nanoparticles are not ferroelectric nanoparticles. In some embodiments, the impedance at the target site increases and / or the conductivity at the target site decreases.

[0073] Disclosed are methods for altering electrical impedance to an alternating current electric field at a target site in a subject, the method comprising: introducing conductive nanoparticles into the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to the alternating current at the target site in the subject is altered, and the current density and / or power loss density to the alternating current at the target site in the subject is altered, and the alternating current electric field is a tumor-treating electric field. In some embodiments, the nanoparticles are nanoparticles that increase tissue permittivity. In some embodiments, the target site is a tumor target site. In some embodiments, the alteration in electrical impedance to the alternating current at the tumor target site in the subject results in an increase in the mitogenic effect of the alternating current electric field at the tumor target site.

[0074] A method for changing the electrical impedance to an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing nanoparticles into the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to the alternating current at the target site in the subject is changed.

[0075] A method for altering electrical impedance to an alternating current electric field at a tumor target site in a subject is disclosed, comprising the steps of: introducing nanoparticles into a tumor target site in a subject; and applying an alternating current electric field to the tumor target site in the subject, wherein the electrical impedance to the alternating current at the tumor target site in the subject is altered.

[0076] Disclosed are methods for altering electrical impedance to an alternating current electric field at a tumor target site in a subject, the methods comprising: introducing nanoparticles to a tumor target site in a subject; and applying an alternating current electric field to the tumor target site in the subject, wherein the electrical impedance to an alternating current at the tumor target site in the subject is altered, the tumor target site being adjacent to one or more cancer cells, previously containing one or more tumor cells, or suspected of containing one or more tumor cells. Disclosed are methods for altering electrical impedance to an alternating current electric field at a target site in a subject, the methods comprising: introducing nanoparticles to a target site in a subject; and applying an alternating current electric field to the target site in the subject, wherein the electrical impedance to an alternating current at the target site in the subject is altered, resulting in a change in current density and / or power loss density for the alternating current at the target site in the subject. Disclosed are methods for altering electrical impedance to an alternating current electric field at a tumor target site in a subject, the methods comprising: introducing nanoparticles to a tumor target site in a subject; and applying an alternating current electric field to the tumor target site in the subject, wherein the electrical impedance to an alternating current at the tumor target site in the subject is altered, resulting in a change in current density and / or power loss density for the alternating current at the tumor target site in the subject.

[0077] Disclosed is a method of altering electrical impedance to an alternating current electric field at a tumor target site in a subject, the method comprising the steps of: introducing nanoparticles into a tumor target site in a subject; and applying an alternating current electric field to the tumor target site in the subject, wherein the electrical impedance to the alternating current at the tumor target site in the subject is altered, and the current density and / or power loss density to the alternating current at the tumor target site in the subject is altered, and the tumor target site is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells.

[0078] In some aspects, the current density at the target site or tumor target site is increased. In some aspects, the current density is decreased at the target site or tumor target site. In some aspects, the power loss density at the target site or tumor target site is increased. In some aspects, the power loss density at the target site or tumor target site is decreased.

[0079] In some embodiments, the nanoparticles are conductive nanoparticles. In some embodiments, the conductive nanoparticles can increase the conductivity and decrease the impedance at the target site or tumor target site. Thus, in some embodiments of the disclosed methods, the impedance at the target site or tumor target site is decreased and / or the conductivity at the target site or tumor target site is increased.

[0080] In some embodiments, the nanoparticles are non-conductive nanoparticles. In some embodiments, the non-conductive nanoparticles are not ferroelectric nanoparticles. In some embodiments, the non-conductive nanoparticles can decrease the conductivity and increase the impedance at the target site or tumor target site. Thus, in some embodiments of the disclosed methods, the impedance at the target site or tumor target site increases and / or the conductivity at the target site or tumor target site decreases.

[0081] In some aspects, a population of nanoparticles can be used in the methods disclosed herein. In some aspects, a population of nanoparticles can include conductive and non-conductive nanoparticles.

[0082] In some embodiments, the alternating electric field used in the methods disclosed herein is a tumor-treating electric field. In some embodiments, the alternating electric field (e.g., tumor-treating electric field) may vary depending on the type of cancer or tumor being treated. In some embodiments, the cancer cells are glioblastoma cells, uterine sarcoma cells, breast cancer cells, pancreatic cancer cells, non-small cell lung cancer cells, liver cells, gastric cancer cells, brain cancer cells, kidney cancer cells, neuroblastoma cells, colon cancer cells, bladder cancer cells, prostate cancer cells, or thymic cancer cells. In some embodiments, the frequency of the alternating electric field may be 200 kHz. The frequency of the alternating electric field may be, but is not limited to, about 200 kHz, between 50 and 500 kHz, between 100 and 500 kHz, between 25 kHz and 1 MHz, between 50 and 190 kHz, between 25 and 190 kHz, or between 210 and 400 kHz.

[0083] In some embodiments, the field strength of the alternating electric field can be 1 to 4 V / cm RMS. In some embodiments, a range of field strengths can be used (e.g., between 0.1 and 10 V / cm).

[0084] In some embodiments, the AC electric field can be applied for various intervals ranging from 0.5 hours to 72 hours. In some embodiments, various durations can be used (e.g., between 0.5 hours and 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied daily for a duration of 2 hours.

[0085] In some aspects, the nanoparticles are nanoparticles that increase the tissue or cell dielectric constant.

[0086] In some aspects, the change in electrical impedance to alternating current at the subject target site or tumor target site results in an increased antimitotic effect of the alternating electric field at the target site. For example, an increased antimitotic effect can refer to disruption of correct microtubule assembly during metaphase, which can ultimately destroy cells (e.g., cancer cells) within or present at the target site during telophase, cytokinesis, or subsequent quiescence.

[0087] Disclosed are methods of altering electrical impedance at a target site or tumor target site with one frequency to allow nanoparticles to enter cells at the target site or tumor target site, and then applying a second frequency to the target site or tumor target site, where the electrical impedance at the target site or tumor target site changes to the second frequency.Disclosed are methods of altering electrical impedance at a target site or tumor target site with one frequency (a first frequency) to allow nanoparticles to enter cells at the target site or tumor target site, and then applying a second frequency to the target site or tumor target site, where the electrical impedance at the target site or tumor target site changes to the second frequency, and further comprising applying the first and second frequencies multiple times. For example, disclosed are methods for altering electrical impedance to an alternating current electric field at a target site or tumor target site of a subject, the methods including: applying a first alternating current electric field at a first frequency to the target site or tumor target site for a first period of time, wherein application of the first alternating current electric field at the first frequency to the target site or tumor target site for the first period of time increases the permeability of a cell membrane of cells present at the target site or tumor target site; introducing nanoparticles to the target site or tumor target site, wherein the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane; and applying a second alternating current electric field at a second frequency to the target site or tumor target site for a second period of time, wherein the second frequency is different from the first frequency, thereby altering the impedance of the second alternating current electric field at the second frequency at the target site or tumor target site of the subject. In some embodiments, the current density and / or power loss density of the alternating current at the target site or tumor target site of the subject is altered. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are glioblastoma cells, uterine sarcoma cells, breast cancer cells, pancreatic cancer cells, non-small cell lung cancer, hepatocellular carcinoma cells, gastric cancer cells, or brain cancer cells. In some embodiments, the cancer cells include glioblastoma cells, and the first frequency is between 250 kHz and 350 kHz, and the second frequency is between 150 kHz and 250 kHz. In some embodiments, the cancer cells include uterine sarcoma cells, and the first frequency is between 125 kHz and 175 kHz, and the second frequency is between 75 kHz and 125 kHz.In some embodiments, the cancer cells include breast adenocarcinoma cells, the first frequency is between 75 kHz and 175 kHz, and the second frequency is between 100 kHz and 300 kHz. In some embodiments, the step of introducing the nanoparticles begins at a given time, and the step of applying the first alternating electric field ends at least 12 hours after the given time. In some embodiments, the step of applying the first alternating electric field begins at least 1 hour before the given time. In some embodiments, the second period includes multiple non-consecutive intermissions when the second alternating electric field is applied to the cancer cells at the second frequency, the multiple non-consecutive intermissions totaling at least one week.

[0088] Disclosed are methods of altering electrical impedance in response to an alternating electric field at a target site or tumor target site of a subject, the methods comprising: applying a first alternating electric field at a first frequency for a first period of time to the target site or tumor target site, wherein application of the first alternating electric field to the target site or tumor target site at the first frequency for the first period of time increases the permeability of a cell membrane of cells present at the target site or tumor target site; introducing nanoparticles to the target site or tumor target site, wherein the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane; and applying a second alternating electric field at a second frequency to the target site or tumor target site for a second period of time, wherein the second frequency is different from the first frequency, and wherein the second frequency of the second alternating electric field alters the impedance at the target site or tumor target site of the subject, wherein the target site or tumor target site is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells. In some embodiments, the current density and / or power loss density of the alternating current at the target site of the subject or tumor target site is varied. In some embodiments, the second alternating electric field is a tumor-treating electric field. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are glioblastoma cells, uterine sarcoma cells, breast adenocarcinoma cells, pancreatic cancer cells, non-small cell lung cancer cells, hepatocellular carcinoma cells, gastric cancer cells, or brain cancer cells. In some embodiments, the cancer cells comprise glioblastoma cells, and the first frequency is between 250 kHz and 350 kHz, and the second frequency is between 150 kHz and 250 kHz. In some embodiments, the cancer cells comprise uterine sarcoma cells, and the first frequency is between 125 kHz and 175 kHz, and the second frequency is between 75 kHz and 125 kHz. In some embodiments, the cancer cells comprise breast adenocarcinoma cells, and the first frequency is between 75 kHz and 175 kHz, and the second frequency is between 100 kHz and 300 kHz. In some embodiments, the step of introducing the nanoparticles begins at a given time, and the step of applying the first alternating electric field ends at least 12 hours after the given time, hi some embodiments, the step of applying the first alternating electric field begins at least 1 hour before the given time.In some embodiments, the second period of time includes multiple non-consecutive intermittent periods when the second alternating electric field is applied to the cancer cells at the second frequency, the multiple non-consecutive intermittent periods totaling at least one week.

[0089] Also contemplated herein are methods that use heat or hyperthermia to kill or eliminate cells at a target site or tumor target site. For example, the methods disclosed herein can use one or more nanoparticles disclosed herein, where the nanoparticles are introduced into cells at a target site or tumor target site and then exposed to an alternating electric field or magnetic field (AMF). Exposing the cells at the target site or tumor target site to the alternating electric field or magnetic field (AMF) can heat the nanoparticles (e.g., reach a temperature of over 100 degrees Fahrenheit), resulting in the death of cells at the target site or tumor target site.

[0090] Disclosed is a method for killing or removing cells at a target site or tumor target site by using a frequency that allows nanoparticles to enter cells at the target site or tumor target site, and then applying an alternating electric or magnetic field to the target site or tumor target site, wherein the nanoparticles convert the alternating electric or magnetic field into thermal energy, thereby killing or removing cells at the target site or tumor target site. In some aspects, the methods disclosed herein can further include applying the first and second frequencies multiple times.

[0091] For example, disclosed are methods for ablating or killing cells at a target site or tumor target site in a subject, the methods including: applying a first alternating electric field at a first frequency for a first period of time to the target site or tumor target site, where application of the first alternating electric field at the first frequency to the target site or tumor target site for the first period of time increases the permeability of the cell membrane of cells present at the target site or tumor target site; introducing nanoparticles to the target site or tumor target site, where the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane; and applying a second alternating electric field at a second frequency or an alternating magnetic field to the target site or tumor target site for a second period of time, whereby one or more cells present at the target site or tumor target site are killed or ablated. In some embodiments, the second alternating electric field is a tumor-treating electric field. In some embodiments, the target site or tumor target site is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells. In some embodiments, the current density and / or power loss density of the alternating current at the target site of the subject or tumor target site is varied. hi some embodiments, the second alternating electric field is a tumor-treating electric field.

[0092] In any of the methods disclosed herein, the method may further include administering to the subject an anti-cancer drug, a cytotoxic drug, a pain management drug, Pseudomonas exotoxin A, a non-radioactive isotope (e.g., boron-10 for boron neutron capture therapy), a photosensitizer (e.g., Photofrin, Foscan, 5-aminolevulinic acid, mono-L-aspartyl chlorin e6, phthalocyanine, metatetra(hydroxyphenyl)porphyrin, texaphyrin, ethyl etiopurinse), or applying or exposing the subject to an electronic device to affect cellular function. For example, in any of the methods disclosed herein, the subject may be exposed to, or a system may be applied to, the subject, wherein the system includes one or more controllable low-energy HF (high frequency) carrier signal generating circuits, one or more data processing devices for receiving control information, one or more amplitude-modulated control signal generators, and one or more amplitude-modulated frequency control signal generators. In some aspects, the amplitude modulation frequency control signal generator is adapted to precisely control the frequency of the amplitude modulation relative to one or more set or predetermined reference amplitude modulation frequencies to an accuracy of at least 1000 ppm, and most preferably within about 1 ppm. Additional embodiments and specific frequencies for particular cancers are described in U.S. Patent No. 8,977,365, which is incorporated herein by reference in its entirety for its teachings of systems and methods useful for affecting cellular function or dysfunction in a subject.

[0093] D. Increasing the antitumor activity of TTF by altering the distribution of electric fields using nanoparticles A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject and a site adjacent to the target site, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased.

[0094] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject and a site adjacent to the target site, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased and the magnitude of the current density of the alternating current electric field is increased at the target site.

[0095] Disclosed is a method for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject and a site adjacent to the target site, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased and impedance at the target site is reduced.

[0096] Disclosed is a method for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site and a site adjacent to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, the conductivity at the target site is increased, and / or the impedance at the site adjacent to the target site is increased.

[0097] A method of increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site and a site adjacent to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, and the alternating current electric field is a tumor treating electric field.

[0098] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject and a site adjacent to the target site, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, and the target site is a tumor target site.

[0099] Disclosed is a method for increasing the effectiveness of an alternating current electric field at a target site in a subject, the method comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; introducing conductive nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site and a site adjacent to the target site in the subject, wherein the increased effectiveness of the alternating current electric field at the target site results in an increased anti-mitotic effect of the alternating current electric field at the target site.

[0100] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased.

[0101] A method of increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased and the nanoparticles are non-conductive nanoparticles.

[0102] Disclosed is a method of increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, and impedance at non-target sites adjacent to the target site is increased and / or conductivity at non-target sites adjacent to the target site is decreased.

[0103] Disclosed is a method of increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, the impedance at the target site is reduced, and / or the conductivity at the target site is increased.

[0104] Disclosed is a method for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased and the magnitude of the current density of the alternating current electric field is decreased at non-target sites adjacent to the target site.

[0105] Disclosed are methods for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, and the method further comprises introducing conductive nanoparticles to the target site in the subject. In some aspects, the impedance at the target site is reduced.

[0106] A method of increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, and the alternating current electric field is a tumor treating electric field.

[0107] Disclosed are methods for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to or at the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, and the target site is a tumor target site. In some embodiments, increasing the effectiveness of the alternating current electric field at the target site results in an increase in the mitotic effect of the alternating current electric field at the target site. In some embodiments, the nanoparticles are introduced into a tumor. In some embodiments, the nanoparticles are introduced into a tumor via injection after primary tumor resection. In some embodiments, the nanoparticles are introduced into a tumor via intratumoral injection (e.g., under computed tomography guidance during surgery or biopsy).

[0108] Disclosed is a method of increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to or at the target site in the subject, wherein the nanoparticles are introduced intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intradurally, intravascularly, intravenously (targeted or non-targeted), intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, via intratumoral injection (e.g., under computed tomography guidance during surgery or biopsy), or via inhalation.

[0109] Disclosed is a method for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles are introduced to the subject in a targeted or non-targeted manner.

[0110] 1. A method for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles increase the effectiveness of an alternating current electric field at a site adjacent to the target site or to a site adjacent to the target site in the subject, wherein the nanoparticles increase the effectiveness of an alternating current electric field at a site adjacent to the target site or to a site adjacent to the target site in the subject, wherein the nanoparticles increase the effectiveness of an alternating current electric field at a site adjacent to the target site or to a site adjacent to the target site in the subject, wherein the alternating current electric field increases ... 3Disclosed are methods in which the antibody is introduced at about 0.001 to 0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5 to 5, 5 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 ng per antibody.

[0111] Disclosed is a method of increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles are introduced at about 0.001 to 0.01, 0.01 to 0.1, 0.1 to 0.5, 0.5 to 5, 5 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 μg.

[0112] Disclosed is a method of increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles are introduced once, twice, three times, or more.

[0113] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying the alternating current electric field to a site adjacent to or at the target site in the subject, wherein the conductive nanoparticles comprise or consist of carbon gold, ferrous iron, selenium, silver, copper, platinum, iron oxide, graphene, iron dextran, superparamagnetic iron oxide, boron-doped detonation nanodiamond, or a combination thereof. In some embodiments, the conductive nanoparticles comprise an alloy selected from Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Fe, Au / Cu, or Au / Fe / Cu.

[0114] A method of increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles are between 0.5 nm and 100 nm in size.

[0115] A method of increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles are between 0.5 nm and 2.5 nm in size.

[0116] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles are greater than 100 nm in size.

[0117] A method of increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to a site adjacent to the target site or to the target site in the subject, wherein the nanoparticles are between 100 nm and 200 nm in size.

[0118] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying the alternating current electric field to a site adjacent to or at the target site in the subject, wherein the nanoparticles have a three-dimensional shape. In some embodiments, the nanoparticles are nanocubes, nanotubes, nanobipyramids, nanoplates, nanoclusters, nanochains, nanostars, nanoshuttle, nanohollows, dendrimers, nanorods, nanoshells, nanocages, nanospheres, nanofibers, or nanowires, or combinations thereof. In some embodiments, the nanoparticles are mesoporous or nonporous. In some embodiments, the nanoparticles are coated with polysaccharides, polyamino acids, or synthetic polymers.

[0119] In some embodiments, the nanoparticles are incorporated into a scaffold prior to introducing the nanoparticles into a subject.

[0120] In some aspects, nanoparticles are loaded onto or within the scaffold.

[0121] In some aspects, the nanoparticles are provided in a pharmaceutical composition.

[0122] In some aspects, the pharmaceutical composition further comprises a chemotherapeutic agent.

[0123] In some embodiments, the nanoparticles are conjugated to one or more ligands. In some embodiments, the one or more ligands are conjugated to the nanoparticles via a linker. In some embodiments, the linker comprises a thiol group, a C2 to C12 alkyl group, a C2 to C12 glycol group, or a peptide. In some embodiments, the linker comprises a thiol group represented by the general formula HO-(CH)n,-SS-(CH2)m-OH, where n and m are independently between 1 and 5.

[0124] In some embodiments, the one or more ligands are small molecules, nucleic acids, carbohydrates, lipids, peptides, antibodies, antibody fragments, or therapeutic agents, hi some embodiments, the one or more ligands are anti-cancer or cytotoxic drugs.

[0125] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased.

[0126] A method for increasing the effectiveness of an alternating electric field at a tumor target site in a subject is disclosed, comprising the steps of: introducing nanoparticles to the tumor target site in the subject; and applying an alternating electric field to the tumor target site in the subject, wherein the effectiveness of the alternating electric field at the tumor target site in the subject is increased.

[0127] Disclosed is a method for increasing the effectiveness of an alternating electric field at a tumor target site in a subject, the method comprising the steps of: introducing nanoparticles to a tumor target site in the subject; and applying an alternating electric field to the tumor target site in the subject, wherein the effectiveness of the alternating electric field at the tumor target site in the subject is increased, and the tumor target site is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells.

[0128] A method for increasing the effectiveness of an alternating current electric field at a target site in a subject is disclosed, comprising the steps of: introducing nanoparticles into the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased and the magnitude of the current density of the alternating current electric field is increased at the target site.

[0129] Disclosed is a method for increasing the effectiveness of an alternating electric field at a tumor target site in a subject, comprising the steps of: introducing nanoparticles into a tumor target site in a subject; and applying an alternating electric field to the tumor target site in the subject, wherein the effectiveness of the alternating electric field at the tumor target site in the subject is increased and the magnitude of the current density of the alternating electric field is increased at the tumor target site.

[0130] Disclosed is a method for increasing the effectiveness of an alternating current electric field at a tumor target site in a subject, comprising the steps of: introducing nanoparticles to a tumor target site in the subject; and applying an alternating current electric field to the tumor target site in the subject, wherein the effectiveness of the alternating current electric field at the tumor target site in the subject is increased and the magnitude of the current density of the alternating current electric field is increased at the tumor target site, and wherein the tumor target site is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells.

[0131] Disclosed are methods for increasing the effectiveness of an alternating current electric field at a target site in a subject, comprising: introducing nanoparticles to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased, and further comprising introducing non-conductive nanoparticles to a non-target site in the subject adjacent to the target site. In some embodiments, the target site is a tumor target site. In some embodiments, the tumor target site is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells.

[0132] Disclosed is a method for increasing the effectiveness of an alternating current electric field at a target site in a subject, the method comprising: introducing non-conductive nanoparticles to a site adjacent to the target site in the subject; and applying an alternating current electric field to the target site in the subject, wherein the effectiveness of the alternating current electric field at the target site in the subject is increased. In some embodiments, the target site is a tumor target site. In some embodiments, the tumor target site is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells.

[0133] In some embodiments, the nanoparticles are conductive nanoparticles. The conductive nanoparticles can increase the electrical conductivity and decrease the impedance at the target site or tumor target site. Thus, in some embodiments of the disclosed methods, the impedance at the target site or tumor target site is decreased and / or the electrical conductivity at the target site or tumor target site is increased.

[0134] In some embodiments, the nanoparticles are non-conductive nanoparticles. In some embodiments, the non-conductive nanoparticles are not ferroelectric nanoparticles. The non-conductive nanoparticles can decrease the conductivity and increase the impedance at the target site or tumor target site. In some embodiments of the disclosed methods, the impedance at the target site or tumor target site increases and / or the conductivity at the target site or tumor target site decreases. In some embodiments, the impedance at the site adjacent to the target site or tumor target site increases and / or the conductivity at the site adjacent to the target site or tumor target site decreases.

[0135] In some aspects, any of the nanoparticles disclosed herein can be used in the methods disclosed herein to increase the effectiveness of an alternating electric field at a target site in a subject or at a tumor target site.

[0136] In some aspects, the alternating electric field is a tumor-treating electric field disclosed herein.

[0137] In some embodiments, the AC electric field can be applied for various intervals ranging from 0.5 hours to 72 hours. In some embodiments, various durations can be used (e.g., between 0.5 hours and 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied daily for a duration of 2 hours.

[0138] In some aspects, increasing the effectiveness of the AC electric field at the target site or tumor target site results in an increased mitotic effect of the AC electric field at the target site or tumor target site. For example, increased mitotic effect can refer to disruption of correct microtubule assembly during metaphase, which can ultimately destroy cells (e.g., cancer cells) within or present at the target site or tumor target site during telophase, cytokinesis, or subsequent quiescence.

[0139] In some aspects, the magnitude of the current density of the alternating electric field is reduced at the target site or at a site adjacent to the tumor target site.

[0140] E. Increased nanoparticle uptake Disclosed are methods for improving transport of nanoparticles across a cell membrane, comprising applying an alternating current electric field to a cell for a period of time, wherein the application of the alternating current electric field increases the permeability of the cell membrane; and introducing nanoparticles into the cell, wherein the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane. In some aspects, the cell is a cancer or tumor cell. In some aspects, the cell is not a cancer or tumor cell.

[0141] In some embodiments, the methods of PCT / US19 / 40479, filed July 3, 2019, can be used in the methods disclosed herein. For example, PCT / US19 / 40479, filed July 3, 2019, the teachings of which are incorporated herein by reference, describes methods and processes for delivering a substance across a cell membrane of a cell. For example, PCT / US19 / 40479, filed July 3, 2019, describes methods and processes including applying an alternating current electric field to a cell for a period of time, where application of the alternating current electric field increases the permeability of the cell membrane; and introducing a substance into the vicinity of the cell, where the increased permeability of the cell membrane allows the substance to cross the cell membrane.

[0142] In some embodiments, the nanoparticles delivered across the cell membrane are conductive nanoparticles. In some embodiments, the nanoparticles are non-conductive nanoparticles. In some embodiments, the non-conductive nanoparticles are not ferroelectric nanoparticles. Thus, in some embodiments, the nanoparticles may be non-ferroelectric nanoparticles.

[0143] In some aspects, any of the nanoparticles disclosed herein can be used in the disclosed methods for improving transport of nanoparticles across the plasma membrane of a cell.

[0144] In some embodiments, a method for improving transport of nanoparticles across a cell membrane includes, in part, applying an alternating current electric field to cells for a period of time. In some embodiments, the alternating current electric field can be used to introduce nanoparticles only to cancer cells. In some embodiments, the alternating current electric field can be applied at a frequency of about 200 kHz. In some embodiments, the alternating current electric field can be applied at a frequency between 50 and 190 kHz. In some embodiments, the alternating current electric field can be applied at a frequency between 210 and 400 kHz. In some embodiments, the alternating current electric field has a field strength of at least 1 V / cm RMS. In some embodiments, the alternating current electric field has a frequency between 50 and 190 kHz. In some embodiments, the alternating current electric field has a frequency between 210 and 400 kHz. In some embodiments, the alternating current electric field has a field strength of at least 1 V / cm RMS. In some embodiments, the alternating current electric field has a field strength between 1 and 4 V / cm RMS.

[0145] In some embodiments, the step of introducing nanoparticles begins at a given time, and the step of applying an AC electric field ends at least 12 hours after the given time. In some embodiments, the step of applying an AC electric field begins at least 1 hour before the given time. In some embodiments, the step of applying an AC electric field begins at least 1 to approximately 24 hours before the given time.

[0146] F. Increasing Nanoparticle Uptake and Treatment Disclosed are methods of increasing the cell permeability of cells (e.g., tumor or cancer cells) using one frequency that allows nanoparticles to enter the cell, and then applying a second frequency for treatment via a tumor-treating electric field based on the presence of the nanoparticles in the cell. In some aspects, the methods disclosed herein may further include applying the first and second frequencies multiple times. In some aspects, the first frequency may be selected to maximize openings in the cell membrane to allow nanoparticles to pass through. In some aspects, the second frequency may be selected to enhance the effect of TTFields on the cell.

[0147] Disclosed is a method of reducing cell viability, comprising applying a first alternating electric field at a first frequency to a cell for a first period of time, wherein applying the first alternating electric field at the first frequency to the cell for the first period of time increases the permeability of a cell membrane of the cancer cell; introducing nanoparticles into the cell, wherein the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane; and applying a second alternating electric field at a second frequency to the cell for a second period of time, wherein the second frequency is different from the first frequency, and wherein the second alternating electric field at the second frequency reduces the viability of the cell.

[0148] A method of reducing the viability of cancer cells is disclosed, comprising applying a first alternating electric field at a first frequency to the cancer cells for a first period of time, wherein applying the first alternating electric field at the first frequency to the cancer cells for the first period of time increases the permeability of a cell membrane of the cancer cells; introducing nanoparticles into the cancer cells, wherein the increased permeability of the cell membrane allows the nanoparticles to cross the cell membrane; and applying a second alternating electric field at a second frequency to the cancer cells for a second period of time, wherein the second frequency is different from the first frequency, and wherein the second alternating electric field at the second frequency reduces the viability of the cancer cells.

[0149] A method for reducing cell viability is disclosed, comprising: applying a first alternating current electric field at a first frequency to a target site or tumor target site containing cells for a first period of time, wherein applying the first alternating current electric field to the target site or tumor target site for the first period of time at the first frequency increases the permeability of the cell membrane; introducing first nanoparticles into the cells, wherein the increased permeability of the cell membrane allows the first nanoparticles to cross the cell membrane; and applying a second alternating current electric field at a second frequency to the target site or tumor target site for a second period of time, wherein the second frequency is different from the first frequency and the second alternating current electric field at the second frequency reduces the viability of the cells, and further comprising introducing the second nanoparticles into a site adjacent to the target site or tumor target site in a subject. In some embodiments, the site adjacent to the target site or tumor target site in a subject may be any site adjacent to cells that does not contain cells. For example, a target site or a site adjacent to a tumor target site in a subject can be any site adjacent to the cancer or tumor cells that does not contain cancer or tumor cells.

[0150] A method for reducing the viability of cancer cells is disclosed, comprising: applying a first alternating current electric field at a first frequency to cancer cells for a first period of time, wherein application of the first alternating current electric field to the cancer cells at the first frequency for the first period of time increases the permeability of the cell membrane of the cancer cells; introducing first nanoparticles into the cancer cells, wherein the increased permeability of the cell membrane allows the first nanoparticles to cross the cell membrane; and applying a second alternating current electric field at a second frequency to the cancer cells for a second period of time, wherein the second frequency is different from the first frequency and the second alternating current electric field at the second frequency reduces the viability of the cancer cells, and further comprising introducing the second nanoparticles into a non-target site adjacent to the cancer cells in a subject. In some embodiments, the non-target site may be any site adjacent to the cancer cells that does not contain cancer cells.

[0151] In some embodiments, the nanoparticle or first nanoparticles are conductive nanoparticles. The conductive nanoparticles can increase the electrical conductivity and decrease the impedance at the cell, target site, or tumor target site. Thus, in some embodiments of the disclosed methods, the impedance at the cell, target site, or tumor target site is decreased and / or the electrical conductivity at the cell, target site, or tumor target site is increased.

[0152] In some embodiments, the second nanoparticles are non-conductive nanoparticles. In some embodiments, the non-conductive nanoparticles are not ferroelectric nanoparticles. The non-conductive nanoparticles can decrease the conductivity and increase the impedance at a cell, target site, or tumor target site. Thus, in some embodiments of the disclosed methods, the impedance at non-target sites adjacent to the cell, target site, or tumor target site is increased and / or the conductivity at sites adjacent to the cell, target site, or tumor target site is decreased.

[0153] In some aspects, the current density and / or power loss density of the alternating current at the cell, target site, or tumor target site may be changed. In some aspects, the current density at the cell, target site, or tumor target site is increased. In some aspects, the current density at the cell, target site, or tumor target site is decreased. In some aspects, the power loss density at the cell, target site, or tumor target site is increased. In some aspects, the power loss density at the cell, target site, or tumor target site is decreased.

[0154] In some embodiments, the second period of time includes multiple non-consecutive intermittent periods when the second alternating electric field is applied to the cancer cells at the second frequency, the multiple non-consecutive intermittent periods totaling at least one week.

[0155] In some embodiments, the cells are placed in the body of a living subject, and the first alternating current electric field is applied to the cells (e.g., tumor or cancer cells) by applying the first alternating current electric field to the subject's body, and the second alternating current electric field is applied to the cells by applying the second alternating current electric field to the subject's body, where the introducing step comprises administering nanoparticles to the subject. In some embodiments, the application of the first and second alternating current fields occurs at locations in the subject's body based on the type and location of the cancer in the subject. For example, for glioblastoma, the first and second alternating current fields can be applied to the head.

[0156] In some embodiments, the first alternating electric field has a field strength of at least 1 V / cm RMS, hi some embodiments, the first alternating electric field has a field strength of between 1 and 4 V / cm RMS.

[0157] In some embodiments, the disclosed methods further include introducing second nanoparticles to a site adjacent to the cancer cells in the subject. In some embodiments, the site can be any site adjacent to the cancer cells that does not contain cancer cells. In some embodiments, the second nanoparticles are non-conductive nanoparticles. In some embodiments, the non-conductive nanoparticles are not ferroelectric nanoparticles. The non-conductive nanoparticles can decrease conductivity and increase impedance within or at the site adjacent to the cancer cells, thereby increasing impedance at the site adjacent to the cancer cells and / or decreasing conductivity at the site adjacent to the cancer cells in some embodiments of the disclosed methods.

[0158] G.Kit The materials described above and other materials can be packaged together in any suitable combination as a useful kit for carrying out or assisting in carrying out the method of the present disclosure. The components of a given kit are useful when designed and adapted for combined use in the method of the present disclosure. For example, kits for imaging and / or treatment are disclosed. In some embodiments, the kit can include one or more nanoparticles of the present disclosure. The kit can also include an apparatus for applying an alternating electric field.

[0159] Disclosed herein are kits comprising one or more nanoparticles described herein and a device capable of applying an alternating electric field. For example, disclosed herein are kits comprising one or more nanoparticles described herein and a TTFields device (e.g., Optune®, Novocure Ltd.).

[0160] (Example) Use of nanoparticles to increase tumor conductivity and enhance alternating electric field strength in tumors Preclinical studies have demonstrated a correlation between the effectiveness of tumor treating fields (TTFields) in killing cancer cells and field strength [1]. A recent study [2] showed that TTFields strength in the tumor region corresponds to outcomes in newly diagnosed glioblastoma patients. In this study, TTFields strength was calculated using computer simulations of patient-specific head models for 317 patients treated with TTFields (at 200 kHz). The dielectric properties assigned to the models in these simulations were based on values ​​from the literature [3-4], and the metric for TTFields strength was defined as the minimum power density (LMiPD) of the two values ​​derived from each pair of transducer arrays used to deliver TTFields to the patient's head.

[0161] To demonstrate the impact of enhanced tumor conductivity on TTFields intensity in tumors, 45 patient models were utilized from a computational study [2], and the electrical conductivity throughout the tumor volume was increased by 25% (0.3 S / m instead of 0.24 S / m) and the simulations were re-run. The results of these studies demonstrate that for all 45 patients, increased tumor conductivity enhanced mean LMiPD throughout the tumor volume by a percentage similar to or even higher than the relative increase in conductivity, as shown in Figure 1A (26%-46%, median = 32%, std = 4%). The increase in intensity was significant across a range of tumor volumes (206-850 mm). 3) were observed. These results indicate that increased tumor conductivity can lead to enhanced TTFields efficacy.

[0162] A study investigating the effect of gold nanoparticles (GNPs) on tissue conductivity showed that incorporating GNPs enhanced tissue conductivity.[6] The study reported that at a frequency of 10 kHz, the average conductivity of divided adipose tissue increased from 0.0191 S / m to 0.0198 S / m, and in divided muscle tissue from 0.55 S / m to 0.57 S / m.

[0163] A study investigating the effect of nano-titanium dioxide (nano-TiO2) on electrical impedance tomography (EIT) signals showed that injecting nano-TiO2 into tumors inoculated into the axilla of mice enhanced EIT signals.[7] The study reported that tumor impedance at 40 kHz decreased from 12.5 kOhm to 11.2 kOhm after injection of nano-TiO2 particles, a 12% increase in conductivity.

[0164] Studies have also been carried out to investigate the conductivity of nanoparticles in solution. A study investigating the electrical conductivity of iron oxide nanoparticles dispersed in an ethylene glycol-based liquid showed that the electrical conductivity of the nanoliquid increased from 0.39 μS / cm to 2.419 mS / cm at 4 vol% iron oxide loading at 25°C [8].

[0165] Polyethylene glycol (PEG) is often used as an encapsulating agent due to its non-toxic properties, which can increase the dispersibility of nanoparticles. PEG is advantageous for preventing aggregation and increasing the penetration of nanoparticles into the cellular environment. Studies investigating the dielectric properties of PEG-encapsulated Mn0.5Zn0.5Fe2O4 nanoparticles (MNPs) have reported that the MNPs remain conductive in the frequency range from 5 kHz to 120 kHz after encapsulation [9].

[0166] (References) [1] Kirson, E. D., Dbaly, V., Tovarys, F., Vymazal, J., Soustiel, J. F., Itzhaki, A., Mordechovich, D., Steinberg-Shapira, S., Gurvich, Z., Schneiderman, R., Wasserman, Y., Salzberg, M., Ryffel, B., Goldsher, D., Dekel, E., Palti, Y. (2007). Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors. Proceedings of the National Academy of Sciences of the United States of America, 104(24), 10152-7. [2] M Ballo, Z Bomzon, N Urman, G Lavy-Shahaf, S A Toms; P01.113 Increasing TTFields dose to the tumor bed improves overall survival in newly diagnosed glioblastoma patients,°Neuro-Oncology, Volume 20, Issue suppl_3, 19 September 2018, Pages iii257,°https: / / doi.org / 10.1093 / neuonc / noy139.155 [3] N Urman, S Levy, A Frenkel, A Naveh, H S Hershkovich, E Kirson, C Wenger, G Lavy-Shahaf, D Manzur, O Yesharim, Z Bomzon; P04.57 Creating patient-specific computational head models for the study of tissue-electric field interactions using deformable templates,°Neuro-Oncology, Volume 20, Issue suppl_3, 19 September 2018, Pages iii292,°https: / / doi.org / 10.1093 / neuonc / noy139.291 [4] Wenger C, Salvador R, Basser PJ, Miranda PC. The electric field distribution in the brain during TTFields therapy and its dependence on tissue dielectric properties and anatomy: a computational study.°Phys Med Biol. 2015;60(18):7339-57. [5] Hershkovich HS, Bomzon Z, Wenger C, Urman N, Chaudhry A, Garcia-Carracedo D, Kirson ED, Weinberg U, Wassermann Y, Palti Y., “First steps to creating a platform for high throughput simulation of TTFields”, Conf Proc IEEE Eng Med Biol Soc. 2016 Aug; 2016 :2357-2360. doi: 10.1109 / EMBC.2016.7591203. [6] Ostovari M, Riahi Alam, Zabihzadeh, Gharibvand, Hoseini-Ghahfarokhi; “The Effect of Gold Nanoparticles on Electrical Impedance of Tissue on Low Frequency Ranges”, J Biomed Phys Eng. 2018 Sep 1;8(3):241-250. eCollection 2018 Sep. [7] Liu R1, Jin C, Song F, Liu J. “Nanoparticle-enhanced electrical impedance detection and its potential significance in image tomography”, Int J Nanomedicine. 2013; 8:33-8. doi: 10.2147 / IJN.S37275. Epub 2013 Jan 3. [8] Jamilpanah, P., Pahlavanzadeh, H. & Kheradmand, A. “Thermal conductivity, viscosity, and electrical conductivity of iron oxide with a cloud fractal structure”, Heat Mass Transfer (2017) 53: 1343. https: / / doi.org / 10.1007 / s00231-016-1891-5 [9] L. Armitasari°et al. “Effect of Polyethylene Glycol (PEG-4000) on Dielectric Properties of Mn0.5Zn0.5Fe2O4 Nanoparticles”, 2018°IOP Conf. Ser.: Mater. Sci. Eng.°367°012035.

[0167] Barium titanate nanoparticles sensitize therapy-resistant breast cancer cells to the antitumor effects of tumor-treating electric fields. Introduction: Numerous preclinical and clinical studies have demonstrated that TTFields can be applied to other tumor types, including breast, lung, pancreatic, and ovarian cancers. Early clinical trials showed that TTFields treatment alone was not significantly better than conventional chemotherapy for patients with GBM. However, recent preclinical studies suggest that TTFields combined with conventional treatments, including chemotherapy, immunotherapy, and radiation therapy, is more effective than TTFields monotherapy in GBM. Despite the promise shown by TTFields as a viable cancer treatment, little is known about TTFields-responsive sensitizers.

[0168] Ferroelectric nanomaterials (non-conductive nanoparticles) have emerged as promising tools for enhancing electrical stimulation of cells and tissues. Among ferroelectric materials, barium titanate nanoparticles (BTNPs) possess a high dielectric constant and favorable piezoelectric properties, along with high biocompatibility. Recent reports suggest that BTNPs can be used in a wide range of applications in nanomedicine, including nonlinear imaging, drug delivery, tissue engineering, and biostimulation. For example, BTNPs have been shown to promote enhanced internalization of doxorubicin in human neuroblastoma cells, and BTNPs containing polyethyleneimine have been shown to improve cellular uptake for cell imaging and DNA delivery. Disclosed herein is a study investigating whether BTNPs can enhance the antitumor effects of TTFields in response to TTFields. Data herein demonstrate that BTNPs alone are cytocompatible with breast cancer cells, but in response to TTFields, they can sensitize TTFields-resistant breast cancer cells to the antitumor effects of TTFields. Furthermore, the data herein demonstrate that BTNPs can be internalized upon TTFields stimulation and that they enhance the anti-tumor effects of TTFields by promoting cell cycle-related apoptosis in breast cancer cells. Thus, this study demonstrates BTNPs as a TTFields-responsive sensitizer in breast cancer cells. The studies described herein can also be performed with other cancer cells to provide additional evidence that BTNPs can be internalized upon TTFields stimulation and that they enhance the anti-tumor effects of TTFields by promoting cell cycle-related apoptosis in other cancer cell types.

[0169] result: Characteristics and cytocompatibility of BTNPs in breast cancer cells Since the dielectric constant of BTNPs can be maximized depending on their size, we prepared two different sizes of FBS (fetal bovine serum)-coated BTNPs (100 nm and 200 nm). SEM images of the 100 nm and 200 nm BTNPs showed the typical circular and uniform size of nanoparticles (Figures 3a and 3b). The hydrodynamic radii of the 100 nm and 200 nm BTNPs were 110 ± 35 nm and 224 ± 63 nm, respectively, and the zeta potentials of the 100 nm and 200 nm BTNPs were -14.1 ± 10.4 mV and -14.5 ± 12.8 mV (Figure 3c), indicating that the BTNPs were relatively stable in aqueous dispersions. Next, the cytocompatibility of the 100 nm and 200 nm BTNPs was investigated by cell viability and clonogenicity assays in two breast cancer cell lines, MCF-7 and BT-549. Ethanol was used as a positive control in these assays. Cell viability assays showed that treatment with 100 and 200 nm BTNPs up to 20 μg / ml did not affect cell viability in MCF-7 and BT-549 cells (Figures 4A and 4B). Additionally, clonogenic assays showed that treatment with 100 and 200 nm BTNPs up to 100 μg / ml did not affect colony formation in MCF-7 and BT-549 cells (Figures 4C-F). Together, these results demonstrate that BTNPs are cytocompatible with breast cancer cells without cytotoxic effects.

[0170] BTNPs sensitize TTFields-resistant breast cancer cells in response to TTFields. TTFields efficacy was examined in three breast cancer cell lines: MCF-7, MDA-MB-231, and BT-549. Among these, MCF-7 cells were the most resistant to TTFields (Figure 5A), consistent with previous reports. Therefore, the combinatorial effects of BTNPs and TTFields were investigated in MCF-7 cells. Cell viability and clonogenicity assays demonstrated that treatment with 100 nm and 200 nm BTNPs enhanced the antitumor activity of TTFields in TTFields-resistant MCF-7 cells (Figures 5B-D). Notably, 200 nm BTNPs were more potent than 100 nm BTNPs (Figures 5B-D), indicating that size may be an important factor in the antitumor activity of BTNPs in the presence of TTFields. These results therefore demonstrated that BTNPs sensitized TTFields-resistant breast cancer cells in response to TTFields.

[0171] TTFields induce cytosolic accumulation of BTNPs in breast cancer cells. To investigate the mechanism of BTNPs-mediated sensitization in the presence of TTFields, we investigated whether BTNPs accumulate in breast cancer cells in response to TTFields. Fluorescence-activated cell sorting (FACS) analysis was first performed to determine cell size and granularity in TTFields- and BTNP / TTFields-treated cells. These parameters were similar between control and TTFields-treated MCF-7 and BT-549 cells (Figures 6A and 6B, left panels). Cell size and granularity were increased in BTNP / TTFields-treated MCF-7 and BT-549 cells (Figures 6A and 6B, center and right panels). Additionally, methylene blue staining demonstrated cytosolic accumulation of BTNPs in response to TTFields in MCF-7 and BT-549 cells (Figures 6C and 6D). Additionally, transmission electron microscopy (TEM) analysis showed that BTNPs accumulated in the cytoplasm of TTFields-treated MCF cells (Figure 6E). Therefore, these results indicated that BTNPs accumulated in the cytoplasm of breast cancer cells in response to TTFields.

[0172] TTFields combined with BTNPs modulates the cell cycle apoptosis pathway. To further explore the regulatory effects of the TTFields / BTNPs combination approach, NanoString nCounter™ Pan-Cancer pathway analysis, containing probes targeting 700 transcripts associated with 13 cancer pathways, was performed on MCF-7 cells exposed to TTFields and untreated or treated with 200 nm BTNPs for 48 hours. MCF-7 cells treated with BTNPs without TTFields exposure were also included as controls. While gene expression patterns were similar between control and BTNPs-treated MCF-7 cells, TTFields treatment induced dramatic changes in nine different cancer pathways (Figure 7A). Among them, cell cycle apoptosis, Wnt, transcriptional migration, transforming growth factor beta (TGF-β), driver genes, Notch, Janus kinase-signaling and activator of transcription (JAK-STAT), and Ras signaling were significantly regulated in TTFields-treated and BTNP / TTFields-treated MCF-7 cells (Figure 7B), demonstrating that BTNPs / TTFields may have the ability to regulate several cancer signaling pathways. Cell cycle pathways were further analyzed. Data showed that several cell cycle-regulated transcripts, including cyclin-dependent kinase 4 (CDK4), RB1, tumor protein TP53, cyclin-dependent kinase 6 (CDK6), MDM2, and CDKN1A / 2A, were regulated in BTNP / TTFields-treated MCF-7 cells (Figure 8A). In addition, Western blot analysis of cell cycle regulatory genes also showed that TTFields in combination with BTNPs inhibited cell cycle progression, as determined by a significant decrease in CDK6 and the transcription factor E2F1 (Figure 8B). These results indicate that TTFields in combination with BTNPs exerts anticancer activity in breast cancer cells by modulating cancer-related pathways and specifically inhibiting cell cycle progression.

[0173] Consideration BTNPs enhanced the antitumor activity of TTFields-resistant breast cancer cells in response to TTFields without cytotoxic effects in breast cancer cells. Furthermore, TTFields were found to induce the accumulation of BTNPs, which promoted cell cycle-related apoptotic pathways. These data indicate that biocompatible nanomaterials such as BTNPs can be used as TTFields-responsive sensitizers in cancer cells.

[0174] These results demonstrate that BTNPs had no cytotoxic effects on breast cancer cells, even at high concentrations (100 μg / ml), indicating their biocompatibility. Consistent with these results, other reports have shown that treatment with BTNPs has minimal adverse effects, as evidenced by several assays, including metabolic activity, viability / cytotoxicity, early apoptosis, and reactive oxygen species (ROS) generation, in multiple cell types, including human neuroblastoma SH-SY5Y cells, HeLa cells, and rat mesenchymal stem cells. The results also showed that treatment with BTNPs alone did not significantly alter 13 key cancer pathway and cell cycle regulatory proteins (Figure 7). BTNPs and their coating complexes can be used as biocompatible sensitizers for TTFields.

[0175] The data indicate that TTFields efficacy depends on the cell doubling time in various cancer cell lines. However, despite similar cell doubling times between MCF-7 and MDA-MB-231 cells, MCF-7 cells were more resistant to TTFields than MDA-MB-231 and BT-549 cells. The data also indicate that BTNPs sensitized TTFields-resistant MCF-7 breast cancer cells in response to TTFields, suggesting that TTFields-responsive sensitizers such as BTNPs can enhance TTFields efficacy in TTFields-resistant cancer cells. In this context, several studies have shown that chemotherapy or radiation therapy enhances the efficacy of TTFields in various cancer cells. However, these combined treatments did not respond to TTFields, indicating that conventional chemotherapy or radiation therapy are not specific sensitizers for TTFields, a physical treatment modality. Therefore, the data indicate that BTNPs can be used as a TTFields-responsive sensitizer for TTFields-resistant tumors.

[0176] The data show that BTNPs accumulated in the cytoplasm of breast cancer cells in response to TTFields. It is well known that cellular nanoparticle (NP) internalization depends on particle size and its zeta potential. NPs smaller than 200 nm can be phagocytosed by cancer cells through the clathrin-dependent pathway or macropinocytosis pathway. However, specific inhibitors of these pathways, such as chlorpromazine, amiloride, and cytochalasin D, did not modulate the accumulation of BTNPs in the cytoplasm in response to TTFields, indicating that BTNP accumulation in the cytoplasm is not mediated by the clathrin-dependent pathway or macropinocytosis pathway. The data herein demonstrate that increased membrane permeability by TTFields can induce BTNP accumulation in the cytoplasm of cancer cells.

[0177] The data herein demonstrate that TTFields in combination with BTNPs significantly modulated the cell cycle apoptosis pathway beyond other relevant pathways. Because cells with mitotic defects undergo mitotic cell death or G1-arrest senescence, the data indicate that TTFields in combination with BTNPs can induce mitotic cell death or G1-arrest senescence by modulating the cell cycle apoptosis pathway, as evidenced by the reduction of G1 cell cycle regulators, including CDK4 / 6, p-RB, and E2F1, in BTNPs / TTFields-treated cells. In addition to the cell cycle apoptosis pathway, significant modulation of several cancer pathways, including Wnt, transcriptional migration, transforming growth factor beta (TGF-β), driver genes, Notch, Janus kinase-signaling and activator of transcription (JAK-STAT), and Ras signaling, was observed in TTFields-treated and BTNPs / TTFields-treated MCF-7 cells. The data also demonstrate that BTNPs, characterized by high biocompatibility and ferroelectric properties, act as TTFields-responsive sensitizers for breast cancer cells by modulating the cell cycle apoptosis pathway (Figure 9). Therefore, electro-responsive nanomaterials such as BTNPs can be used as TTFields-responsive sensitizers to enhance the therapeutic efficacy of TTFields in cancer cells.

[0178] method Cell Culture: MCF-7 and BT-549 breast cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Both cell lines were confirmed by their karyotype, imaging, and detailed gene expression, as confirmed by information provided by the ATCC. Both cell lines were stored and passaged for less than two months according to ATCC protocols and tested for mycoplasma infection by polymerase chain reaction (PCR) once a week. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Corning, NY, USA), and BT-549 cells were cultured in RPMI (Corning, NY, USA). All media were supplemented with 10% fetal bovine serum (FBS, Corning, NY, USA) and 1% penicillin / streptomycin (Sigma-Aldrich, MO, USA). All cell lines were maintained at 37°C in a humidified 5% CO2 incubator.

[0179] TTFields applied: MCF-7 (1.5×10 4 pcs) and BT-549 (1 x 10 4 Cells were seeded onto 18 mm glass coverslips (Marienfeld-Superior, Mediline, Lauda-Königshofen, Germany) or 22 mm plastic coverslips (Thermo Fisher Scientific, MA, USA) for 24 hours, and then transferred to ceramic in vitro dishes (NovoCure, Haifa, Israel) using autoclaved tweezers. For TTFields treatment, we applied an invitro system (NovoCure, Haifa, Israel) for 72 hours as previously described. Briefly, cells on coverslips were exposed to a 150 mA current generated at 1 V / cm and 150 kHz by an invitro TTFields generator (NovoCure, Haifa, Israel), and the plate temperature was maintained at 37°C in a refrigerated incubator (ESCO Technologies, USA) at 19°C.

[0180] Preparation and physicochemical characterization of BTNPs: Barium titanate nanoparticles (100 nm and 200 nm) were purchased from US Research Nanomaterials Inc. (TX, USA) and used without further purification. BTNPs were dispersed in ethanol and sonicated to reduce aggregation. Additionally, 5% FBS was added to coat the BTNPs' surfaces with a protein corona before addition to cells. The nanostructure and morphology of the prepared BTNPs were examined by field-emission scanning electron microscopy (FE-SEM) using a Sirion-400 (FEI, OR, USA) and transmission electron microscopy (TEM) using a JEM-2100F (JEOL, Japan). The zeta potential of the FBS-coated BTNPs was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK).

[0181] Cell viability assay: Cell viability assay was performed using WST-8 reagent (Cyto X; LPS solution, Daejeon). MCF-7 cells (0.5 × 10 4 Cells (number of cells) were seeded into 96-well plates and treated with medium containing increasing concentrations of BTNPs or ethanol as a vehicle control. After 72 h, WST-8 reagent (10 μl) was added to each well, and the plates were incubated for 2 h at 37°C. Absorbance was then measured at 450 nm using a VersaMax microplate reader (Molecular Devices, CA, USA).

[0182] Clonogenic assay: Clonogenic assays were performed as previously described. MCF-7 or BT-549 cells (500 cells) were seeded onto 22 mm plastic cover glasses in 6-well plates for 24 hours. Using autoclaved tweezers, the cover glasses were transferred to ceramic in vitro dishes and incubated in an in vitro TTFields generator for 72 hours. After TTFields treatment, the cover glasses were transferred to 6-well plates and incubated at 37°C. After 7 days, colonies were fixed and stained with 1% crystal violet (Sigma-Aldrich) and 40% methanol, and the number of colonies was counted.

[0183] Cell counting: To assess the number of viable cells in the same volume, absolute cell counts were obtained using a BD Accuri™ C6 flow cytometer (BD Biosciences, CA, USA) as previously described. Briefly, detached MCF-7 and BT-549 cells in fresh medium (500 μl) were stained with propidium iodide (50 μg / ml; PI; Sigma-Aldrich, MO, USA), and the number of cells in the PI-negative population was counted in a 100 μl volume.

[0184] Cell cycle analysis: Cell cycle analysis was performed. Briefly, cells treated under the conditions described above were trypsinized, washed twice in PBS, and fixed in ice-cold 70% ethanol. Fixed cells were incubated with PI (50 μg / ml) and RNase (100 μg / ml) at 37°C for 30 minutes and then analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences, CA, USA).

[0185] Apoptosis analysis: Detached cells were collected and apoptosis was detected using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, CA, USA) according to the manufacturer's protocol. Samples were analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences, CA, USA).

[0186] Methylene blue staining: Cells were fixed with 4% paraformaldehyde and then stained with 0.1% methylene blue (Sigma-Aldrich, MO, USA) dissolved in Dulbecco's phosphate-buffered saline (DPBS) for 5 min. After several washes with DPBS, slides were mounted with glycerol, and images were taken using an LSM 710 confocal microscope (Carl Zeiss, Germany).

[0187] TEM imaging: TTFields-treated MCF-7 cells with or without BTNP treatment were detached and fixed overnight at 4°C in 2.5% glutaraldehyde (Sigma-Aldrich, MO, USA) and 0.1 M phosphate buffer (pH 7.3). After fixation, cells were treated with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M phosphate buffer (pH 7.3) for 1 hour at 4°C in the dark. They were then dehydrated with ethanol and propylene oxide treatment cycles and embedded in Epon 812 (Sigma-Aldrich, MO, USA). Polymerization was performed using pure resin at 70°C for 2 days. Ultrathin samples were obtained using an UltraCut-UCT ultramicrotome (Leica, Austria) and collected on 150-mesh copper grids. After staining with 2% uranyl acetate for 10 min and lead citrate for 5 min, the samples were examined in a Tecnai G2 Spirit Twin TEM instrument at 120 kV.

[0188] RNA isolation and NanoString analysis: Total RNA was isolated from treated cells using QIAzol reagent (Qiagen, Hilden, Germany). 100 ng of RNA was used to hybridize to the probes using nCounter XT CodeSet Gene Expression Assays (NanoString Technologies, WA, USA) according to the protocol provided by the manufacturer.

[0189] Western blot analysis: Western blots were performed as previously described. Briefly, proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected using specific antibodies. The following antibodies were used: rabbit polyclonal anti-CDK4, rabbit monoclonal CDK6, rabbit monoclonal phospho-RB, rabbit polyclonal RB (Santa Cruz Biotechnology, CA, USA); mouse monoclonal p21, mouse monoclonal E2F1, mouse monoclonal MDM2, mouse polyclonal anti-β-actin (Santa Cruz Biotechnology, CA, USA), and mouse monoclonal p53 (Merck, NJ, USA). Blots were developed using peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence detection system (Amersham Life Science, Buckinghamshire, UK).

[0190] Statistical analysis: A two-tailed Student's t-test was performed to analyze statistical differences between groups. A P value of less than 0.05 was considered statistically significant. Statistical analysis was performed using Microsoft Excel and XLSTAT software.

[0191] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method of operating an alternating current electric field delivery system that alters the electrical impedance to alternating current at a target site of a subject by conductive nanoparticles introduced into the target site of the subject, said method comprising: the alternating current electric field delivery system includes generating an alternating current electric field through two pairs of transducer arrays arranged to generate a perpendicular field at a target region of a subject; The method wherein the alternating electric field has a frequency between 50 kHz and 500 kHz and lasts for 0.5 hours to 14 days.

2. 10. The method of claim 1, wherein a current density and / or a power loss density for the alternating current at the target site of the subject is varied.

3. The method of claim 1 or 2, wherein the impedance at the target site is reduced.

4. 4. The method of claim 1, wherein the electrical conductivity at the target site is increased.

5. A method described in any one of claims 1 to 4, wherein the alternating current electric field delivery system further comprises a step of generating an alternating current electric field through the two pairs of transducer arrays arranged to generate a perpendicular field at a site adjacent to the target site where non-conductive nanoparticles have been introduced.

6. The method of claim 5 , wherein the electrical conductivity at a site adjacent to the target site is decreased.

7. 7. The method of claim 5 or 6, wherein the impedance is increased at a site adjacent to the target site.

8. A method described in any one of claims 5 to 7, wherein the non-conductive nanoparticles are not ferroelectric nanoparticles.

9. 9. The method of claim 1, wherein the alternating electric field is a tumor treatment electric field.

10. 10. The method of claim 5, wherein the non-conductive nanoparticles are nanoparticles that increase the tissue permittivity.

11. 11. The method of any one of claims 1 to 10, wherein the target site is a tumor target site.

12. 12. The method of any one of claims 1 to 11, wherein the change in electrical impedance to the alternating current at the tumor target site of the subject results in an increase in the mitogenic effect of the alternating electric field at the tumor target site.

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