All-in-one multimodal nanocerenostic platform for image-guided therapy

Abstract

Disclosed is a theranostic nanocomposite comprising albumin, bismuth sulfide (Bi2S3), and superparamagnetic iron oxide (SPIO) within a single nanoplateform and its use for tracking cells, imaging cells, and / or treating a disease, condition, or disorder.

Description

Background Art

[0001] (Research or Development Supported by the Federal Government) This invention was made with government support under Grant No. EB028904 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0002] (Background Art) A wide variety of nanoparticles have been developed for magnetic resonance imaging (MRI), magnetic particle imaging (MPI), magnetically driven ultrasonic imaging (MMUS), (magnetic) photoacoustic imaging ((m)(PAI)), and computed tomography (CT). Currently, each imaging diagnostic method requires an optimized magnetic contrast agent or radiopaque (X-ray) contrast agent. Therefore, a multimodal theranostic agent is required.

Summary of the Invention

[0003] In some aspects, the subject matter of the present disclosure provides a nanocomposite comprising superparamagnetic iron oxide (SPIO) particles coated with albumin, bismuth sulfide (Bi2S3), and a natural or semi-synthetic polymer or other polymer comprising a dendrimer and / or a polypeptide.

[0004] In one aspect, the albumin comprises serum albumin. In certain aspects, the serum albumin is selected from bovine serum albumin (BSA), human serum albumin (HSA), and recombinant HSA.

[0005] In one aspect, the natural or semi-synthetic polymer is selected from dextran, carboxydextran, polyglucose sorbitol carboxymethyl ether, and a dendrimer, also referred to as a "dendron".

[0006] In one aspect, the SPIO is selected from ferumoxide, ferucarbotran, and ferumoxitol.

[0007] In one aspect, the nanoplex further comprises a targeting agent. In certain aspects, the targeting agent is selected from folic acid (FA), antibodies and fragments thereof, growth factors, vitamins, lipids, carbohydrates, cancer target ligands, proteins, nucleic acid aptamers, peptides, glycoproteins, and glycolipids.

[0008] In certain aspects, the cancer target ligand comprises a sugar. In a more particular aspect, the cancer target ligand comprises glucose. In certain aspects, the protein comprises transferrin.

[0009] In one aspect, the nanoplex further comprises cells. In a further aspect, the cells are stem cells, progenitor cells, precursor cells, or immune cells. In certain aspects, the stem cells comprise human mesenchymal stem cells (hMSCs).

[0010] In one aspect, the nanocomposite has a particle size in the range of about 50 nm to about 250 nm. In certain aspects, the particle size is about 90 nm.

[0011] In one aspect, the nanoplex has a ratio of albumin and Bi2S3 to SPIO of about 10:1 to about 1:1. In certain aspects, the nanoplex has a ratio of albumin and Bi2S3 to SPIO of 10:1, 5:1, or 1:1. In a more particular aspect, the nanoplex has a ratio of albumin and Bi2S3 to SPIO of about 5:1.

[0012] In other aspects, the subject matter of the present disclosure provides a method for tracking cells, the method comprising administering a nanocomposite of the present disclosure to a subject or cells, and monitoring the location of the cells. In one aspect, the cells are stem cells, progenitor cells, precursor cells, or immune cells. In one aspect, the locations include tumors, brain diseases, and myocardial infarctions. In one aspect, the tracking is performed in vivo. In one aspect, the tracking is performed in vitro.

[0013] In certain embodiments, the method includes monitoring the location of cells by MPI, CT, MPI / CT, MRI, MPI / MRI, MMUS, (m)PAI, and combinations thereof.

[0014] In further embodiments, the method further includes measuring the efficiency of cell delivery, the amount of cell migration, or the in vivo distribution of cells in tissue.

[0015] In other embodiments, the present invention provides a method for diagnosing a disease, condition, or disorder, the method including administering a nanoplex of the present disclosure comprising a targeting agent to a subject having or suspected of having the disease, condition, or disorder and acquiring an image. In certain embodiments, the image comprises an MPI, MRI, MMUMS, (m)PAI, or CT image. In particular embodiments, this method enables accurate co-registration of the MPI signal and the anatomical CT image. In certain embodiments, this method enables accurate cell quantification with both MPI and CT.

[0016] In other embodiments, the present disclosure provides a method for treating a disease, condition, or disorder, the method including administering a nanoplex of the present disclosure comprising a targeting agent to a subject in need thereof.

[0017] In certain embodiments, the method further includes irradiating the nanoplex. In certain embodiments, the method further includes irradiating the nanoplex with a light laser to induce photothermal heating of the tissue, subjecting the nanoplex to an alternating magnetic field to induce magnetic fluid hyperthermia (MFH) of the tissue, or treating the nanoplex with high-intensity focused ultrasound (HIFU) to induce sono-thermal heating of the tissue. In particular embodiments, the method further includes irradiating the nanoplex with a gamma-ray irradiation device. In more particular embodiments, the nanoplex acts as a radiosensitizer. In even more particular embodiments, the method includes targeted image-guided cancer therapy.

[0018] In some embodiments, the method further comprises simultaneously or sequentially tracking cells, imaging cells, and / or treating a subject with the presently disclosed nanoplexes.

[0019] Certain aspects of the subject matter of this disclosure are described above, and as the detailed description proceeds, other aspects of the subject matter of this disclosure, which are treated in whole or in part by the subject matter of this disclosure, will become apparent, as they are described in connection with the accompanying examples and figures, to the extent possible to be described below in this specification.

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication will be provided by the Office upon request and payment of the necessary fee.

[0021] Thus, to describe the subject matter of this disclosure in general terms, reference is made to the accompanying drawings, which are not necessarily drawn to scale. **Brief Description of the Drawings**

[0022]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24A

Figure 24B

Figure 25

Figure 26

Figure 27

Figure 28

[0023] The subject matter of the present disclosure is further described below with reference to the accompanying drawings, which show some but not all of the embodiments of the invention. Throughout, the same numbers refer to the same elements. The subject matter of the present disclosure may be implemented in many different forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, those of ordinary skill in the art in the technical field related to the subject matter of the present disclosure will, upon benefiting from the foregoing description and the teachings shown in the related drawings, conceive of many modifications and other embodiments of the subject matter of the present disclosure. Accordingly, it should be understood that the subject matter of the present disclosure should not be limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0024] (Abbreviations) AB: Albumin-Bi2S3 ABS: Albumin-Bi2S3-SPIO BSA: Bovine Serum Albumin CT: Computed Tomography DLS: Dynamic Light Scattering EDC: (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA: Ethylenediaminetetraacetic Acid FA: Folic Acid FTIR: Fourier Transform Infrared Spectroscopy HIFU: High-Intensity Focused Ultrasound HSA: Human Serum Albumin hMSC: Human Mesenchymal Stem Cells HR-TEM: High-Resolution Transmission Electron Microscopy HU: Hounsfield Unit LDH: Lactate Dehydrogenase mPAI: Magnetic Photoacoustic Imaging MFH: Magnetic Fluid Hyperthermia MSOT: Multispectral Photoacoustic Tomography MPI: Magnetic Particle Imaging MMUS: Magnetically Actuated Ultrasound Imaging NP: Nanoparticle PAI: Photoacoustic Imaging PCa: Prostate Cancer PLL: Poly-L-lysine PSMA: Prostate-Specific Membrane Antigen SC: Subcutaneous SPIO: Superparamagnetic Iron Oxide

[0025] The subject matter of the present disclosure provides a new hybrid nanoprobe comprising albumin, Bi2S3, and SPIO particles (this combination is referred to herein as "ABS") as a new all-in-one MRI, MPI, MMUMS, (m)PAI, and CT multimodal contrast agent or therapeutic agent.

[0026] The SPIO component enables visualization by MRI, MPI, MMUS, and mPAI. The bismuth sulfide component not only provides contrast for CT and PAI images but also functions as a sensitizer for radiotherapy, photothermal therapy, HIFU, and MFH, becoming a unique triple-modal diagnostic / therapeutic (i.e., theranostic) agent.

[0027] The ABS of the present disclosure can target specific tumors and can be used for labeling cells that accumulate in tumors. Since the particles are visualized by imaging scans, a laser (for photothermal therapy), an alternating magnetic field for MFH, or a gamma-ray (for radiotherapy) beam can be accurately irradiated only to the nanoparticles within the tumor, minimizing off-target damage to surrounding normal tissues.

[0028] In some embodiments, the present disclosure provides a nanocomposite comprising SPIO particles coated with albumin, Bi2S3, and a natural or semi-synthetic polymer, or other polymers including dendrimers and / or polypeptides.

[0029] In some embodiments, the albumin comprises serum albumin. In certain embodiments, the serum albumin is selected from BSA, HSA, and rHSA.

[0030] In one embodiment, the natural or semi-synthetic polymer is selected from dextran, carboxydextran, polyglucose sorbitol carboxymethyl ether, and dendrimers also known as "dendrons".

[0031] In one embodiment, the SPIO is selected from ferumoxide, ferucarbotran, and ferumoxitol.

[0032] In certain embodiments, the nanoplex further comprises a targeting agent. In specific embodiments, the targeting agent is selected from folic acid (FA), antibodies and fragments thereof, growth factors, vitamins, lipids, carbohydrates, cancer target ligands, proteins, nucleic acid aptamers, peptides, glycoproteins, and glycolipids. In certain embodiments, the cancer target ligand comprises a sugar. In more specific embodiments, the cancer target ligand comprises glucose. In certain embodiments, the protein comprises transferrin.

[0033] In certain embodiments, the nanoplex further comprises a cell. In yet certain embodiments, the cell is a stem cell, progenitor cell, precursor cell, or immune cell. In specific embodiments, the stem cell comprises hMSC.

[0034] In specific embodiments, the particle size of the nanocomposite ranges from about 50 to about 250. In certain embodiments, the particle size is about 90 nm.

[0035] In certain embodiments, the ratio of albumin and Bi2S3 to SPIO in the nanoplex is between about 10:1 and about 1:1. In specific embodiments, the ratio of albumin and Bi2S3 to SPIO in the nanoplex is 10:1, 5:1, or 1:1. In yet more specific embodiments, the ratio of albumin and Bi2S3 to SPIO in the nanoplex is about 5:1.

[0036] In other embodiments, the present disclosure provides a method of tracking a cell, the method comprising administering a nanocomposite according to the present invention to a subject or cell, and monitoring the location of the cell. In certain embodiments, the cell is a stem cell, progenitor cell, precursor cell, or immune cell. In certain embodiments, the location comprises a tumor, a brain disease, and a myocardial infarction. In certain embodiments, the tracking is performed in vivo. In certain embodiments, the tracking is performed in vitro.

[0037] In certain embodiments, the method includes monitoring the location of cells by MPI, CT, MPI / CT, MRI, MPI / MRI, MMUS, (m)PAI, and combinations thereof.

[0038] In further embodiments, the method further includes measuring the efficiency of cell delivery, the amount of cell migration, or the in vivo distribution of cells in tissue.

[0039] In certain embodiments, this method enables accurate co-registration of the MPI signal and anatomical CT imaging. In certain embodiments, this method enables accurate cell quantification using both MPI and CT.

[0040] In other embodiments, the present disclosure provides a method for diagnosing a disease, condition, or disorder, the method including administering a nanoplex of the present disclosure comprising a targeting agent to a subject having or suspected of having the disease, condition, or disorder and acquiring an image. In certain embodiments, the image comprises an MPI, MRI, MMUS, (m)PAI, or CT image.

[0041] In further embodiments, the method further includes measuring the efficiency of cell delivery, the amount of cell migration, or the in vivo distribution of cells in tissue.

[0042] In other embodiments, the present invention provides a method for diagnosing a disease, condition, or disorder, the method including administering a nanoplex of the present disclosure comprising a targeting agent to a subject having or suspected of having the disease, condition, or disorder and acquiring an image. In certain embodiments, the image comprises an MPI, MRI, MMUS, (m)PAI, or CT image.

[0043] In other embodiments, the present disclosure provides a method for treating a disease, condition, or disorder, the method including administering a nanoplex of the present disclosure comprising a targeting agent to a subject in need thereof.

[0044] In certain embodiments, the method further comprises irradiating the nanoplex. In certain embodiments, the method further comprises irradiating the nanoplex with an optical laser to induce photothermal heating of the tissue, exposing the nanoplex to an alternating magnetic field for MFH, or exposing the nanoplex to HIFU for ultrasonic heating of the tissue. In certain specific embodiments, the method further comprises irradiating the nanoplex with a gamma-ray irradiator. In more specific embodiments, the nanoplex acts as a radiosensitizer. In even more specific embodiments, the method includes targeted image-guided cancer therapy.

[0045] In some embodiments, the method further comprises simultaneously or sequentially tracking the cells, imaging the cells, and / or treating the subject with the nanoplexes of the present disclosure.

[0046] As used herein, the term "treatment" can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of a disease, disorder, or condition to which the term applies, or one or more of the pathological conditions or symptoms of such disease, disorder, or condition. Prevention refers to the absence of the occurrence of a disease, disorder, condition, or their symptoms or signs, or the worsening of their severity. Thus, the compounds of the present disclosure can be prophylactically administered to prevent or reduce the occurrence or recurrence of a disease, disorder, or condition.

[0047] The "subjects" treated by many embodiments of the methods of the present disclosure are preferably human subjects, but it should be understood that the methods described herein are effective for all vertebrate species intended to be included within the term "subjects". Thus, a "subject" can include a human subject for medical purposes such as the treatment of existing symptoms or diseases, or prophylactic treatment to prevent the onset of symptoms or diseases, or an animal subject for medical, veterinary, or developmental purposes. Suitable animal subjects include, but are not limited to, mammals such as humans, primates such as monkeys and apes, bovines such as cows and bulls, ovines such as sheep, caprines such as goats, suines such as pigs and domestic pigs, equines such as horses, donkeys, and zebras, felines including wild and domestic cats, canines including dogs, lagomorphs including rabbits and wild rabbits, rodents including mice and rats, etc. The animal can be a transgenic animal. In some embodiments, the subject is human, including, but not limited to, fetuses, neonates, infants, juveniles, and adult subjects. Further, a "subject" can also include a patient suffering from or suspected of suffering from a symptom or disease. Thus, the terms "subject" and "patient" are used interchangeably herein. Also, the term "subject" also refers to an organism, tissue, cell, or collection of cells from the subject.

[0048] Generally, an "effective amount" of an active agent refers to the amount necessary to induce a desired biological response. As would be understood by one of ordinary skill in the art, the effective amount of a drug can vary depending on factors such as the desired biological endpoint, the drug being delivered, the composition of the pharmaceutical composition, the drug target, etc.

[0049] The term "combination" is used in the broadest sense and means that the subject is administered one agent only, more specifically ABS nanoparticles, in combination with an applied therapy such as photothermal therapy, MFH, or HIFU. More specifically, the term "in combination" refers to the simultaneous administration of an agent and an applied therapy for the treatment of a single disease state. In the present specification, the active agent or the applied therapy can be administered simultaneously in combination, or can be administered alternately or continuously on the same day or on different days.

[0050] Furthermore, ABS nanoparticles in combination with additional therapies of the present disclosure can enhance the stability of drugs alone or in combination with drugs, facilitate the administration of pharmaceutical compositions containing them in certain embodiments, increase dissolution or dispersion, increase inhibitory activity, and can be further administered together with adjuvants (including other active ingredients) that provide adjuvant therapies and the like. Advantageously, in such combination therapies, the dosage of conventional therapeutic agents can be reduced, so that the toxicity and side effects that can occur when those agents are used as monotherapies can be avoided.

[0051] The timing of administration of the combination of the disclosed ABS nanoparticles and the additional applied therapy can be varied as long as the beneficial effects of the combination of the agent and the additional therapy are achieved. Thus, the phrase "in combination" refers to administering the ABS nanoparticles described herein and the additional therapy either simultaneously, sequentially, or in combination thereof. Thus, a subject administered the combination of the ABS nanoparticles of the present disclosure and the additional therapy can receive the ABS nanoparticles and the additional therapy at the same time (i.e., simultaneously) or at different times (i.e., in either order on the same day or different days, continuously) as long as the effects of the combination of the agent and the additional therapy are achieved in the subject.

[0052] When administered continuously, the agent and the additional therapy can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes of each other, or at intervals greater than that. In other embodiments, the agent and the additional therapy can be administered continuously and at intervals of 1, 5, 10, 15, 20 days or more from each other.

[0053] When administered in combination, the effective dosage of the agent for inducing a particular biological response can be less than the effective dosage of the agent when administered alone, thereby reducing the dosage of the agent as compared to the dosage required when the agent is administered as a single agent. The effect when the agent and the additional therapy are combined need not necessarily be additive or synergistic. The agent and / or the additional therapy can be administered in multiple doses.

[0054] In some embodiments, a synergistic effect can be obtained when the agent and the additional therapy are administered in combination. As used herein, the terms “synergism,” “synergistic,” “synergistically,” and their derivatives, such as “synergistic effect” or “synergistic combination” or “synergistic composition,” refer to a situation where the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of each agent when administered individually.

[0055] Synergism can be expressed in terms of the term “synergism index (SI).” This can generally be determined from the ratio determined by the following formula in the manner described by F.C. Kull et al., Applied Microbiology 9, 538 (1961). Q a / Q A +Q b / Q B = Synergism index (SI) Q A is the concentration of component A that acts alone and generates an endpoint in relation to component A, Q a is the concentration of component A in the mixture that generated the endpoint, Q Bis the concentration of component B that acts alone and generates an endpoint in relation to component B, Q b is the concentration of component B in the mixture that generated the endpoint.

[0056] Generally, when the sum of Qa / QA and Qb / QB is greater than 1, antagonism is shown; when the sum is equal to 1, additive action is shown; and when the sum is less than 1, synergistic action is demonstrated. The lower the SI, the greater the synergistic action shown by that particular mixture. Thus, a "synergistic combination" shows an activity higher than that expected based on the observed activities of the individual components when used alone. Further, an "amount of a component that is synergistically effective" refers to, for example, the amount of the component necessary to elicit a synergistic effect in another therapeutic agent present in a composition.

[0057] In accordance with longstanding patent law convention, the terms "a", "an", and "the", when used in this application including in the claims, refer to "one or more". Thus, for example, a reference to "a subject" includes multiple subjects unless the context clearly indicates otherwise (e.g., multiple subjects).

[0058] Throughout this specification and the claims, the terms "comprise", "comprises", and "comprising" are used in a non-exclusive sense unless the context requires otherwise. Similarly, the term "include" and its grammatical variations are intended to be non-limiting, and the listing of items in a list does not exclude other similar items that can be substituted or added in place of the listed items.

[0059] For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, sizes, dimensions, ratios, shapes, compositions, parameters, percentages, amounts, properties, and other numerical values used in this specification and the claims are to be understood as being modified in all instances by the term "about" even when the term "about" is not explicitly stated with respect to a value, quantity, or range. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximations and not exact values, and need not be exact, but rather may reflect tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art, and may be approximations and / or may vary, as appropriate, larger or smaller, depending on the desired characteristics obtainable by the subject matter of this disclosure. For example, the term "about" when referring to a value may mean that, in some embodiments, it may vary by ±100%, in some embodiments by ±50%, in some embodiments by ±20%, in some embodiments by ±10%, in some embodiments by ±5%, in some embodiments by ±1%, in some embodiments by ±0.5%, and in some embodiments by ±0.1% from the specified amount when appropriate for carrying out the disclosed method or using the disclosed composition.

[0060] Furthermore, when the term "about" is used in connection with one or more numerical values or numerical ranges, it is understood to refer to all such numerical values, including all values within the range, and to modify the range by extending the boundaries above and below the recited numerical values. The recitation of a numerical range by endpoints includes all numerical values within that range, including, for example, integers, fractions thereof (e.g., the recitation of 1 to 5 includes 1, 2, 3, 4, 5, as well as their fractions such as 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range.

Examples

[0061] The following examples are included to provide guidance to those skilled in the art for practicing representative embodiments of the subject matter of the present disclosure. Considering the present disclosure and the general level of skill in the art, those skilled in the art will understand that the following examples are intended merely as illustrations and that numerous changes, modifications, and alterations can be employed without departing from the scope of the subject matter of the present disclosure. The following description of the synthesis and the specific examples are for illustrative purposes only and should not be construed in any way as limiting the manufacture of the disclosed compounds by other methods.

Example

[0062] (BSA-Bi2S3-SPIO (ABS) nanoparticles)

[0063] (1.1. Synthesis and characterization of BSA-Bi2S3-SPIO (ABS)) (1.1.1. Synthesis of BSA-Bi2S3 (AB)) Bovine serum albumin (BSA)-Bi2S3 (AB) nanoparticles (NP) were synthesized by a biomineralization method using BSA, Bi(NO3)3, HNO3, and NaOH.

[0064] Specifically, BSA was added to double-distilled (dd) H2O to form a first solution. Bi(NO3)3·H2O was added to HNO3, followed by the addition of ddH2O to form a second solution. NaOH was dissolved in ddH2O to form a third solution.

[0065] The second solution containing the Bi salt was added to the first solution containing BSA using ultrasound. Next, the third solution containing NaOH was added with stirring, and centrifuged to obtain AB nanoparticles. The AB nanoparticles can be washed with ddH2O, for example, the washing can be repeated 3 times.

[0066] First, the incubation time for creating AB NPs was optimized. The longer the incubation time, the larger the particle size and the increased yield (see Figure 1). To optimize the size and yield, 8 hours was selected as the incubation time, and an AB NP formulation with a hydrodynamic size of approximately 90 nm was obtained.

[0067] Next, the ratio of NaOH to HNO3 was adjusted to optimize the AB formulation. It was confirmed that the more NaOH was added, the larger the particle size and the improvement in the CT effect (represented by HU value) (see Figure 2). Based on the HU values obtained from CT imaging studies, a NaOH amount of 7.5 ml was selected to create the optimal AB NPs.

[0068] Further physicochemical property evaluations of the nanocomposites were carried out by the NIH Nanotechnology Characterization Laboratory (NCL). The zeta potential, hydrodynamic size, and particle concentration of the AB nanocomposites were measured to be -20.9 mV, 87 nm, and 6.4E12 particles per mL, respectively. For the ABS nanocomposites, the respective values were -24.2 mV, 120 nm, and 2.9E12 particles per mL (1 mg Fe / mL or 17.9 μmol). Therefore, each ABS nanocomposite contains 3,695,000 iron atoms.

[0069] Referring to Figures 3A and 3B, asymmetric flow field-flow fractionation (AF4)-multi-angle light scattering (MALS) / dynamic light scattering (DLS) analysis was performed, and the formation of the protein corona on the surfaces of both AB (Figure 3A) and ABS (Figure 3B) after incubation with human plasma was studied.

[0070] Figure 4 shows the CT effect (right panel) and UV-Vis spectrum (left panel) of ABa at 1.5 m / mL of AB. Figure 5 shows the FTIR spectrum (left panel) and TEM image (right panel) of AB.

[0071] (1.1.2. Superparamagnetic iron oxide (SPIO) particles) Various SPIO formulations were tested, such as commercially available RESOVIST® (ferucarbotran (iron oxide particles coated with carboxydextran), available from Bayer Healthcare), nanoflowers, SuperSPIO20 provided based on an MTA with the University of Franche-Comté and SuperBranche. Since BSA has both NH2 and COOH functional groups in its structure, any SPIO formulation (having either a negative or positive surface charge) should be usable for manufacturing the ABS NPs of the present disclosure. Other SPIO nanoparticles include, but are not limited to, Ferumoxtran-10 (COMBIDEX®, AMAG Pharma, SINEREM®, Guerbet), NC100150 (CLARISCAN®, Nycomed), (VSOP C184, Ferropharm), Magtrace™ (Endomag), Sentimag® (Endomag), Synomag® (MicroMod), Perimag® (Micromod), Nanomag® (Micromod), Ferucarbotran (Resovist®, Meito-Sangyo), FeraTrack® (Miltenyi Biotec), Ferumoxytol (FERAHEME®).

[0072] (1.1.3. Synthesis of BSA-Bi2S3-SPIO (ABS)) BSA-Bi2S3-SPIO (ABS) NPs were synthesized by mixing AB and SPIO in the presence of ethylene diamine tetraacetic acid (EDTA) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), washing with ddH2O, centrifuging, and obtaining the final product. Any mass ratio of AB:SPIO can be used, but the final mass ratio varies depending on the final application. Formulations were made at various AB:SPIO ratios and the NPs were tested by MPI. Higher MPI signal intensities were observed with increasing amounts of SPIO. Based on the cell uptake and cytotoxicity data of the ABS NPs, a ratio of 5:1 was selected.

[0073] (1.1.4. Synthesis of FA-BSA-Bi2S3-SPIO (FA-ABS)) 30 mg of ABS was mixed with 0.6 mg of folic acid (FA) and stirred overnight at room temperature to produce FA-ABS NPs.

[0074] (1.1.5. Characteristics of ABS) Dynamic light scattering (DLS) (Figure 6), high-resolution transmission electron microscopy with elemental mapping (HR-TEM) (Figure 7), UV-Vis spectrophotometry (Figure 8), and Fourier transform infrared spectroscopy (FTIR) (Figure 9) were used to evaluate the characteristics of the synthesized AB, ABS, and FA-ABS NPs.

[0075] (1.2. In vitro tests of ABS) (1.2.1. Cytotoxicity of AB and ABS nanoparticles) hMSCs were used to test the cytotoxicity of AB NPs and ABS NPs using an LDH kit. hMSCs were labeled with or without PLL as a transfection agent. AB NPs did not have significant cytotoxicity even when cells were incubated with them at 200 μg / mL for 24 hours (see Figure 16). In the case of ABS, hMSCs were incubated with the NPs for 24 or 48 hours. Cytotoxicity was minimal when the concentration of ABS was less than 125 μg Bi / mL (or equivalent to 25 μg Fe / mL) (see Figure 17).

[0076] (1.2.2. Cellular Uptake of AB and ABS Nanoparticles) The cellular uptake of AB NPs and ABS NPs was evaluated by incubating with hMSCs for 24 hours. When testing AB NPs at various concentrations, the higher the concentration, the more uptake was observed. The NPs were taken up into the cytoplasm and found to be distributed around the nucleus, which is typical of nanoparticle endocytosis. Based on the cell uptake and cytotoxicity data, 125 μg Bi or 25 μg Fe per mL was selected as the optimal concentration for further studies.

[0077] (1.3. In vitro Imaging) The MPI imaging properties of ABS with different AB:SPIO ratios (10:1, 5:1, 1:1) were tested. The more SPIO, the stronger the MPI signal intensity (Figure 13).

[0078] The CT effects of iopamidol (clinical formulation, used as the reference gold standard), AB, and ABS were evaluated at different concentrations. AB and ABS had similar CT contrasts and both showed higher HU values than iopamidol at the same concentration (see Figure 11).

[0079] AB / ABS-labeled hMSCs were collected and dispersed in Eppendorf tubes. Approximately 520 HU values were obtained for 10,000 ABS-labeled hMSCs per μL (see Figure 12).

[0080] FA-ABS NPs were incubated with two prostate cancer cells (LNCaP and DU145) with different PSMA expression levels. PSMA is recognized as the receptor FA, which can lead to the intracellular shuttle of FA-conjugated NPs. The labeled PCa cells were evaluated by CT / MPI. A significant difference was detected in the CT / MPI signals obtained for LNCaP (high PSMA expression) and DU145 (low PSMA expression), indicating that tumor targeting is possible using the appropriate receptor / target. See Figure 18.

[0081] (1.4. In vivo cell tracking of ABS-labeled cells by MPI / CT) (1.4.1. In the case of tumors) Either plain ABS or ABS-labeled hMSCs were directly injected into SC DU145 tumors, and MPI / CT was performed in vivo at 30 minutes and 48 hours after injection. ABS-labeled hMSCs were also injected intravenously, and MPI / CT was performed in vivo at 2 hours and 24 hours after injection. The tumors were excised and ex vivo MPI / CT of all tumors was performed for further investigation.

[0082] (1.4.2. In the brain) hMSCs were labeled with ABS and directly injected into the brain. The labeled cells could be detected in the brain using CT, MRI, and MPI.

[0083] (1.5. Photothermal effect of ABS (when ABS is used as a seranostic nanoparticle)) (1.5.1. In solution) ABS (0.2 mg / mL) was irradiated with an 808 nm laser (1.5 W / cm 2 ) for 5 minutes. The temperature of the sample increased from 21.2 °C to 63.4 °C (temperature change 42.2 °C).

[0084] (1.5.2. In vitro cells) hMSCs were labeled with ABS and PCa cells (DU145 and LNCaP) were labeled with FA-ABS. The labeled cells were irradiated with a laser and the cell viability was measured using an LDH kit. For ABS, a significant in vitro PTT effect was observed.

[0085] (1.5.3. In vivo cells) FA-ABS was directly injected into SC DU145 tumors, and the tumors were irradiated with a laser 30 minutes after injection (irradiation time: 10 minutes, 2 W / cm 2)。In another SC DU145 tumor-bearing mouse, FA-ABS was intravenously injected, and the tumor was irradiated with the same laser parameters 24 hours after injection. Control mice having the same tumor model were also irradiated with a laser (without FA-ABS injection). During laser irradiation of all tumors, in vivo temperature measurements were performed using an IR camera. With FA-ABS, a significant in vivo PTT effect was observed.

[0086] (1.6 Uses) The following uses are envisioned. (a) The currently disclosed ABS can function as an excellent MPI / MRI / MMUS / (m)PAI / CT agent for in vivo cell tracking of hMSCs. Accordingly, the formulations of the present disclosure can be used for monitoring stem cell therapy or immune cell therapy in a living individual from the perspective of cell delivery, migration, and in vivo distribution of tissues. (b) PSMA-overexpressing PCa cancer cells selectively bind to FA-ABS. Accordingly, the formulations of the present disclosure can be used for cancer diagnosis. In fact, any other tumor-specific ligand can be conjugated to ABS instead of FA. (c) The ABS of the present disclosure can be used as a photothermal sensitizer when irradiated with a laser. (d) The ABS of the present disclosure can be used as an MFH agent when subjected to an alternating magnetic field. (e) The ABS of the present disclosure can be used as a radiation sensitizer when using a gamma-ray irradiation device for radiotherapy. (f) The ABS of the present disclosure can be used as an ultrasonic sensitizer when using HIFU.

[0087] Accordingly, the ABS of the present disclosure is both a diagnostic agent and a therapeutic agent, that is, a nanocerenostic agent detectable by MPI, MRI, MMUS, (m)PAI, and CT, which holds the potential for target image-guided cancer therapy.

[0088] (1.7 Summary) In summary, a new seranostic nanocomposite was synthesized that incorporates both Bi2S3 and SPIO into a single nanoplateform using albumin, such as BSA, as the matrix. Various methods for optimizing the ABS formulation for various applications, including MPI / CT and PTT, were tested.

Example

[0089] (In vivo MPI / MRI / CT imaging and tracking of mesenchymal stem cells as a delivery medium for cancer therapy using magnetic / radiopaque nanoparticles)

[0090] (2.1 Background and Scope) The passive and active targeting of therapeutic nanoparticles (NPs) to cancer cells has not yet been fully successful, and efforts are underway to establish effective targeting strategies that ensure uniform distribution of NPs throughout the tumor. The use of stem cells with intrinsic trophic properties for homing to tumors has been proposed as a new approach for NP delivery to cancer cells. (Su et al., 2021; Cheng et al., 2019; Wang et al., 2019). It is highly desirable to monitor the homing and intratumoral distribution of transplanted cells, as well as the in vivo distribution of off-target sites in other parts of the body. In vivo hybrid imaging has the potential to meet this need. (Srivastava et al., 2014; Bulte, 2019). For this purpose, the present disclosure provides a bimodal cell tracking method using a new superparamagnetic radiopaque nanocomposite that can be detected by MPI, MRI, and CT.

[0091] (2.2 Methods and Materials) An ABS nanocomposite composed of bovine serum albumin (BSA), radiopaque Bi2S3 nanoparticles, and superparamagnetic iron oxide (SPIO) was produced by a stepwise solvothermal decomposition method. The ABS nanocomposite was characterized by various techniques. hMSCs were labeled with poly-L-lysine as a secondary transfection agent, and the ABS nanocomposite was labeled for 24 hours. Naked ABS or ABS-labeled hMSCs were injected intratumorally (i.t.) or intravenously (i.v.) into the tumors of mice bearing DU145 (human prostate cancer). At 30 minutes and 48 hours after injection, the mice were imaged by MPI and CT. Two days after i.t. injection or four days after i.v. injection, the mice were euthanized, and the tumors were excised for ex vivo imaging.

[0092] (2.3 Results) Referring to Figure 7, the abs nanocomposite prepared above showed a spherical morphology with uniform distribution of bismuth, iron, and sulfur throughout the sphere (average size: 90 nm). In vivo MPI / CT images of mice that received naked ABS nanocomposite or were injected i.t. with ABS-hMSCs are shown in Figures 23A - 23D. It was shown that i.t. injection of ABS-hMSCs caused the labeled cells to migrate throughout the tumor and maintain a strong signal intensity over a 48-hour period, while the naked ABS nanocomposite remained focused near the injection site and showed a decrease in signal intensity.

[0093] Referring to Figure 23E, in the case of i.v. injection, homing of the cells to the lungs was observed at 2 hours after injection, and to the liver at 24 hours after injection. No signal was observed in the tumors in the case of i.v. injection. Ex vivo imaging showed that the highest amount of MPI signal intensity was obtained with i.t. injection of ABS-hMSCs (Figure 23F).

[0094] (2.4 Conclusions) The subject of the present disclosure demonstrates the feasibility of in vivo bimodal imaging of native ABS and ABS-labeled hMSCs using CT and MPI, and the imaging differences between native ABS and ABS-labeled hMSCs. A protocol for MPI / MRI / CT-guided hyperthermal therapy using stem cell delivery of ABS nanocomposites is currently under development.

Example

[0095] (All-in-one superparamagnetic radiopaque nanocomposite for in vivo MRI, MPI, and CT stem cell tracking)

[0096] (3.1 Background) Clinical trials using stem cells as a regenerative therapy are increasing, but in many cases, the treatment effects have been disappointing. The use of in vivo imaging techniques to track the movement of stem cells in the body has significant potential to improve treatment outcomes (Srivastava et al., 2014). However, one of the current limitations of in vivo cell tracking techniques is that a single imaging modality cannot answer all questions regarding the fate of transplanted cells, such as cell viability, quantity, and overall biodistribution. The subject of the present disclosure provides the development of a multimodal cell tracking method using a novel superparamagnetic radiopaque nanocomposite for in vivo MRI, MPI, and CT tracking of MSCs, one of the most widely used therapeutic cells in humans.

[0097] (3.2 Method) (3.2.1 Synthesis and characterization of nanocomposites) An ABS nanocomposite composed of BSA, radiopaque Bi2S3 nanoparticles, and SPIO was synthesized by a solvothermal decomposition method. The ABS nanocomposite was characterized by dynamic light scattering (DLS), Fourier transform infrared (FTIR), UV-VIS spectroscopy, and high-resolution transmission electron microscopy (HR-TEM). Elemental analysis was performed to determine the ratio of iron to bismuth in the ABS nanocomposite.

[0098] (3.2.2 Cells) Human bone marrow-derived MSCs (P2) were obtained from Rooster Bio in the United States. The MSCs were incubated with ABS at a concentration of 25 μg of Fe (about 125 μg of Bi) per 1 ml. Cell labeling was performed for 24 hours in a T-75 tissue culture flask with and without using poly-L-lysine (1125 ng / mL) as a transfection agent. The labeled cells were collected and prepared for injection into normal male Rag2 mice. The cell viability after incubation with the ABS nanocomposite was measured using the LDH assay. Prussian blue staining and a ferrozine-based spectrophotometric assay were used to evaluate the intracellular iron uptake.

[0099] (3.2.3 Cell transplantation) ABS-labeled MSCs were transplanted into the striatum of Rag2 mice under 1.5% isoflurane anesthesia. The mice were placed in a stereotaxic fixation device, and 100,000 labeled cells in 2 μL of phosphate-buffered saline were injected using a Hamilton syringe (31G, AP = 0 mm, ML = 2 mm, DV = 3 mm, 0.5 μL / min). One hour after transplantation, the mice were euthanized, the heads were removed and fixed with 4% paraformaldehyde.

[0100] (3.2.4 Imaging) The customized holders were 3D printed for use on all MRI, MPI, and CT devices. One day after fixation, the head was imaged ex vivo by MRI using a 17.6T vertical bore Bruker Biospec scanner and then by MPI using a Magnetic Insight Momentum scanner. Ex vivo CT was also performed. MR images were acquired using a FLASH sequence with TR = 8.4 ms, TE = 2.5 ms, NEX = 16, FA = 5 degrees, resolution = 0.1 mm, slice thickness = 18 mm, matrix size = 150×300×180, and FOV = 3×2×1.8 cm. The head was scanned by MPI using the same FOV as for MRI, with 55 projections, 3D high-resolution mode, and one scan per projection. Two standards containing 25,000 and 50,000 labeled MSCs were placed within the MRI / MPI / CT FOV and used for cell quantification and data co-registration using 3D slicer software.

[0101] (3.3 Results) The ABS nanocomposites showed a spherical morphology. Bismuth, iron, and sulfur were evenly distributed throughout the ABS spheres, and the average hydrodynamic diameter was 90 nm. The Fe:Bi ratio of the ABS nanocomposites was determined to be 1:5. Covalent bonds between BSA, SPIO, BSA-Bi2S3 nanoparticles, and the ABS nanocomposites were confirmed by the FTIR spectra of the BSA, SPIO, BSA-Bi2S3 nanoparticles, and the entire ABS nanocomposites. Prussian blue staining showed that the nanocomposites accumulated around the nuclei of the labeled MSCs, and the iron content was 17 pg Fe per cell. No significant cytotoxicity was observed for the ABS nanocomposites. Using ex vivo imaging data, the location of the transplanted cells could be easily identified by MRI and CT, and the cell number could be quantified using MPI.

[0102] (3.4 Discussion) The subject matter of this disclosure demonstrates the potential for multimodal imaging of transplanted cells through trimodality imaging using a single composite nanocomposite. In addition to visualizing cells in the anatomical context provided by CT and MRI, another advantage of using ABS nanocomposite as a cell labeling agent is the ability to quantify cell content with MPI (Bulte 2019; Bulte et al. 2015). Because ABS is a non-radioactive tracer, systematic injection of ABS-labeled MSCs allows for easily interpretable systemic distribution studies. Further studies are being conducted in our laboratory to evaluate the effects of ABS on stem cell differentiation into adipocytes, chondrocytes, and osteocytes.

[0103] (3.5 Summary) The presently disclosed subject matter provides novel nanocomposites for labeling and tracking stem cells using multimodal imaging. [Example]

[0104] (CT and MPI of ABS-labeled hMSCs) Figures 24A and 24B are CT and MPI images of ABS-labeled hMSCs. As shown in Figures 24A and 24B, both CT and MPI show perfect correlation, with a doubling of cell volume resulting in a doubling of signal. More specifically, Figure 24A shows that the disclosed method allows for accurate co-registration of "hot spot" MPI signals with anatomical CT imaging. Similarly, Figure 24B shows that the disclosed method allows for accurate cell quantification with both MPI and CT. [Example]

[0105] (Continuous in vivo imaging of mice receiving ABS-MSCs in the brain) In this example, ABS-labeled MSCs (100K cells, 50K cells, 25K cells, and 12.5K cells) were injected into the striatum of Rag2− / − mice. The mice were imaged at 30 minutes, 7 days, and 30 days post-injection using micro-CT (IVIS Spectrum / CT), MRI (Bruker Biospec 9.4T horizontal bore), and MPI (Magnetic Insight Momentum scanner). The mice were then euthanized, and the fixed heads were scanned using an iThera MSOT inVision 512 echo scanner. To verify the imaging data, the brain tissue was further examined using Prussian blue and anti-HuNA staining. In vivo magnetic particle imaging (MPI) and dynamic signal analysis of ABS-labeled MSCsx are shown in FIG. 25.

[0106] FIG. 26 shows sequential in vivo 3D MPI and MRI of mice that received ABS-MSCs in the brain. FIG. 27 is a quantitative in vivo 3D CT imaging on day 30 post-injection. FIG. 28 shows imaging and histological data of mice that received 100K ABS-MSCs in the brain. As shown in FIG. 28, the location of the transplanted cells could be easily visualized by MRI, MPI, CT, and MSOT. By combining Prussian blue staining and anti-HuNA staining, it was confirmed that cells containing iron were present in the brain tissue.

Example

[0107] (Magnetic hyperthermia)

[0108] Shows the magnetic heating characteristics of ABS particles within a calibration range of 6 - 15 mT (4.7 kA / m - 12.0 kA / m), obtained using the "HYPER" prototype device (Magnetic Insight, Inc.) to perform magnetic thermotherapy under MPI guidance. A "control" sample (1 mL aliquot of DI water) was placed inside the HYPER at each test magnetic field amplitude. Each sample was pulsed 20 times. As described by Carlton and Ivkov (2023), MATLAB scripts were used to analyze each pulse and compile the average specific loss power (SLP). The data shown in Figure 22 indicates that an external alternating magnetic field (AMF) above 9 mT can effectively heat the ABS composite.

[0109] (References) All publications, patent applications, patents, and other references described herein are indicative of the level of skill in the art relevant to the subject matter of the present disclosure. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference were specifically and individually indicated to be incorporated by reference. Although numerous patent applications, patents, and other references are cited herein, it should be understood that such citations do not admit that any of these documents form part of the general knowledge in the art.

Prior Art Documents

Non-Patent Documents

[0110]

Non-Patent Document 1

Non-Patent Document 2

[0111] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be made within the scope of the appended claims.

Claims

1. Albumin, bismuth sulfide (Bi ２ S ３ ), and a nanocomposite comprising superparamagnetic iron oxide (SPIO) particles coated with a natural or semi-synthetic carbohydrate or other polymer containing a dendrimer and / or polypeptide.

2. The nanocomposite according to claim 1, wherein the albumin comprises serum albumin.

3. The nanocomposite according to claim 2, wherein the serum albumin is selected from bovine serum albumin (BSA), human serum albumin (HSA), and recombinant human serum albumin (rHSA).

4. The nanocomposite according to any one of claims 1 to 3, wherein the natural or semi-synthetic carbohydrate is selected from dextran, carboxydextran, polyglucose sorbitol carboxymethyl ether, and dendrimer or dendron.

5. The nanocomposite according to any one of claims 1 to 4, wherein the SPIO is selected from ferumoxide, ferucarbotran, and ferumoxitol.

6. The nanocomposite according to claim 1, further comprising a targeting agent.

7. The nanocomposite according to claim 6, wherein the targeting agent is selected from folic acid (FA), antibodies and fragments thereof, growth factors, vitamins, lipids, carbohydrates, cancer target ligands, proteins, nucleic acid aptamers, peptides, glycoproteins, and glycolipids.

8. The nanocomposite according to claim 1, further comprising cells.

9. The nanocomposite according to claim 8, wherein the cells are stem cells, progenitor cells, precursor cells, or immune cells.

10. The nanocomposite according to claim 9, wherein the stem cells comprise human mesenchymal stem cells.

11. The nanocomposite according to any one of claims 1 to 10, having a particle size in the range of about 50 nm to about 250 nm.

12. The nanocomposite according to claim 11, wherein the particle size is about 90 nm.

13. The nanoplex has a ratio of albumin and Bi of from about 10:1 to about 1:1 ２ S ３ to SPIO as claimed in any one of claims 1 to 12.

14. The nanoplex has a ratio of albumin and Bi ２ S ３ to SPIO of 10:1, 5:1, or 1:1, the nanocomposite according to claim 13.

15. The nanoplex has a ratio of albumin and Bi of about 5:1 ２ S ３ and SPIO, the nanocomposite according to claim 14 having such a ratio.

16. A method for tracking cells, comprising administering the nanocomposite according to any one of claims 8 to 10 to a subject or cells, and monitoring the position of the cells.

17. The method according to claim 16, wherein the cells are stem cells, progenitor cells, precursor cells, or immune cells.

18. The method according to claim 16, wherein the positions include tumors, brain diseases, and myocardial infarctions.

19. The method according to claim 16, wherein the tracking is performed in vivo.

20. The method according to claim 16, wherein the tracking is performed in vitro.

21. The method according to claim 16, comprising monitoring the position of cells by magnetic particle imaging (MPI), magnetically driven ultrasonic imaging (MMUS), (magnetic) photoacoustic imaging ((m)PAI), computed tomography (CT), MPI / CT, magnetic resonance imaging (MRI), MPI / MRI / CT, and combinations thereof.

22. The method according to any one of claims 16 to 21, further comprising measuring the efficiency of cell delivery, the amount of cell migration, or the in vivo distribution of cells in a tissue.

23. The method according to claim 16, wherein the method enables accurate co-registration of the MPI signal and anatomical CT imaging.

24. The method according to claim 16, wherein the method enables accurate cell quantification using both MPI and CT.

25. A method for diagnosing a disease, symptom, or disorder, comprising administering the nanoplex according to claim 6 or 7 comprising a targeting agent to a subject having or suspected of having the disease, symptom, or disorder, and obtaining an image.

26. The method according to claim 25, wherein the image comprises an MPI, MRI, MMUS, (m)PAI, MSOT, or CT image.

27. A method for treating a disease, symptom, or disorder, comprising administering the nanoplex according to claim 6 or 7 comprising a targeting agent to a subject in need thereof.

28. The method according to claim 27, further comprising irradiating the nanoplex.

29. The method according to claim 28, further comprising irradiating the nanoplex with a light laser to induce photothermal heating of the nanoplex.

30. The method according to claim 28, further comprising subjecting the nanoplex to an alternating magnetic field to induce magnetic heating of the nanoplex.

31. The method according to claim 28, further comprising irradiating the nanoplex with a gamma-ray irradiation device.

32. The method according to claim 28, further comprising subjecting the nanoplex to HIFU to induce ultrasonic heating of the nanoplex.

33. The method according to claim 28, wherein the nanoplex acts as a radiation sensitizer.

34. The method according to claim 27, comprising target image-guided cancer therapy.

35. The method according to any one of claims 16 to 34, further comprising simultaneously or sequentially tracking cells, imaging cells, and / or treating a subject using the nanoplex according to claims 1 to 15.

Images