Biologics labeling system and methods thereof

Abstract

To provide techniques for labelling low-phagocytic cells and low-sensitive cells in a clinically applicable way.SOLUTION: The present invention discloses: nanoparticles that are intended for cellular tracking, drug encapsulation, and biologic tracking, and comprises heparin, protamine, an iron-based imaging agent, and a contrast agent conjugated to the heparin and / or the protamine; compositions; and methods for manufacturing and using the nanoparticles and compositions.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] Priority This application claims priority to U.S. Provisional Patent Application No. 62 / 787,042, filed December 31, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] As the field of cell-based therapeutics continues to advance, there is a need for safe and effective prognostic and diagnostic tools that can be used in the clinic for non-invasive monitoring of cell-based therapeutic products and cell-derived biologics (e.g., vesicles). Specifically, this need necessitates overcoming the challenges associated with labeling and / or manipulating cell-based therapeutic products and cell-derived biologics that arise due to the physiological properties and susceptibility to foreign substances of these cell-based therapeutic products and cell-derived biologics.

[0003] An important consideration in the development of cell-based therapeutics and cell-derived biologics for regenerative medicine and immuno-oncology is the need to understand what happens to the cells after they are implanted in patients in order to guide treatment strategies. One approach to achieving this goal is to develop cell tracking tools that are safe and equipped with imaging modalities that provide observation and visibility of the cells once implanted in the patient.

[0004] Popular cell-based therapeutics, such as mesenchymal stem cells for regenerative medicine applications and chimeric antigen receptor (CAR)-modified T lymphocyte (CAR-T) cell therapy for treating hematological malignancies and more recently solid tumors, have progressed from the laboratory to the market. However, there remains a critical unmet need to develop labeling techniques for these poorly phagocytes and less sensitive cells in a clinically applicable manner that would further enable integration of these cell therapy developers into the standard workflow of manufacturing and point-of-care processes. Furthermore, it is important that the technology be highly performant, using standard imaging modalities and conventional imaging protocols available in most clinics, in order to be smoothly adopted and integrated into the workflow of clinical imaging practices. [Summary of the Invention]

[0005] In some embodiments, the present disclosure relates to nanoparticles comprising heparin, protamine, an iron-based imaging agent, and a contrast agent, wherein the contrast agent is bound to the heparin and / or the protamine.

[0006] In another aspect, the present disclosure relates to a composition comprising nanoparticles of heparin, protamine, and an iron-based contrast agent, wherein the composition is free or substantially free of unbound heparin, protamine, or iron-based contrast agent.

[0007] In some embodiments, the nanoparticles have an iron content of about 0.1 femtogram (fg) to about 1 fg. In certain embodiments, the nanoparticles are uniform in size and have an average diameter of 120 nm to 195 nm.

[0008] In another embodiment, the iron-based contrast agent is ferumoxytol.

[0009] In some embodiments, the contrast agent is a fluorescent probe, a PET agent, a CT agent, or an MRI agent.

[0010] In some embodiments, the composition is free or substantially free of unbound heparin-protamine complexes, protamine-iron-based contrast agent complexes, or heparin-iron-based contrast agent complexes.

[0011] In some embodiments, the composition further comprises an excipient, hi another embodiment, the composition is lyophilized or frozen.

[0012] In one aspect, the disclosure relates to a method of labeling a cell or a cell-derived product comprising contacting the cell or cell-derived product with a composition comprising nanoparticles comprised of heparin, protamine, and an iron-based contrast agent, wherein the composition is free or substantially free of unbound heparin, protamine, or iron-based contrast agent.

[0013] In some embodiments, the method further comprises applying a magnetic field while contacting the cells or cell-derived products with the composition comprising the nanoparticles.

[0014] In some embodiments, the composition is free or substantially free of unbound heparin-protamine complexes, protamine-iron-based contrast agent complexes, or heparin-iron-based contrast agent complexes.

[0015] In certain embodiments, the method further comprises labeling the cells in serum-containing cell culture while contacting the cells or cell-derived products with the composition, hi another embodiment, the method further comprises labeling the cells in serum-starved cell culture while contacting the cells or cell-derived products with the composition.

[0016] In some embodiments, the cells or cell-derived products are labeled for less than 4 hours. In some embodiments, the cells or cell-derived products are labeled for more than 4 hours.

[0017] In one embodiment, the cell is a mammalian cell. In some embodiments, the mammalian cell is a mouse cell, a rat cell, a dog cell, a pig cell, a bovine cell, a horse cell, a primate cell, or a human cell. In another embodiment, the cell is a cancer cell, an immune cell, or a blood cell. In some embodiments, the cell is a stem cell, an embryonic cell, a fetal cell, or a somatic cell. In yet another embodiment, the stem cell is a mesenchymal stem cell, a pluripotent stem cell, an induced pluripotent stem cell, an adipose stem cell, a neural stem cell, or an adult stem cell.

[0018] In some embodiments, the cell-derived product is a vesicle. In some embodiments, the method further comprises contacting the cell-derived product with a cell to label the cell with the cell-derived product.

[0019] In another embodiment, the method further comprises tracking the cells or cell-derived products for a period of time after contacting the cells. In one embodiment, the cell tracking is ex vivo, in vitro, or in vivo tracking. In some embodiments, tracking is performed by imaging the cells.

[0020] In some embodiments, the method further comprises tracking a cell-derived product, the cell-derived product being either inside or outside the cell.

[0021] In one embodiment, imaging is performed by fluorescence microscopy, MRI, FL, in vivo optical imaging, PET, SPECT, and / or ultrasound. In some embodiments, imaging is performed at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or more after the cells are contacted with the nanoparticles or composition.

[0022] In some embodiments, the method is a method for in vitro or in vivo labeling of cells.

[0023] In another embodiment, the method further comprises administering the composition to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In yet another embodiment, the composition is administered intravenously, intraarterially, by direct tissue injection, and / or intratumorally.

[0024] In some embodiments, the method further comprises encapsulating the nucleic acid in the nanoparticles prior to contacting the composition comprising the nanoparticles with the cells or cell-derived products.

[0025] Another aspect of the present disclosure relates to a method of preparing magnetically separated nanoparticles, the method including: (a) combining heparin, protamine, and an iron-based contrast agent, where at least a portion of the heparin, protamine, and iron-based contrast agent form nanoparticles comprising heparin, protamine, and iron-based contrast agent; and (b) exposing the nanoparticles to a magnetic field to separate the nanoparticles based on their magnetic moment.

[0026] In some embodiments, exposing the solution to a magnetic field further separates unbound protamine, heparin, or iron-based labeling agent that did not form nanoparticles through complexation.

[0027] In some embodiments, the nanoparticles are further separated based on size.

[0028] In another embodiment, exposing the solution to a magnetic field comprises applying a magnet to the solution, hi some embodiments, the magnet has a magnetic field strength of about 300 mT to about 600 mT.

[0029] In some embodiments, the magnetic field is uniform and applied perpendicular to the nanoparticles, hi other embodiments, the magnetic field is non-uniform and directionally varying.

[0030] A method of labeling a cell from a cell-derived product, the method comprising: (a) contacting a first cell with a composition comprising nanoparticles of heparin, protamine, and an iron-based contrast agent, where the contacting step labels the cell-derived product within the cell; (b) removing the labeled cell-derived product from the cell; and (c) contacting a second cell with the cell-derived product, where the second cell takes up the labeled cell-derived product. In some embodiments, the method further comprises directly tracking the labeled cell-derived product from the first cell. In some embodiments, the method further comprises tracking the second cell.

[0031] These and other features, aspects, and advantages of the present teachings will become better understood with reference to the following description, examples, and appended claims.

[0032] This application contains at least one drawing executed in color. Copies of this application including color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0033] [Figure 1A] FIG. 1A is a representative graph showing the size distribution of standard HPF nanoparticles, not prepared by the magnetic separation process of the present disclosure. [Figure 1B] 1B and 1C are representative graphs showing the size distribution of VMI NCE 1 nanoparticles synthesized according to the magnetic separation process of the present disclosure. [Figure 1C] 1B and 1C are representative graphs showing the size distribution of VMI NCE 1 nanoparticles synthesized according to the magnetic separation process of the present disclosure. [Figure 1D]FIG. 1D is a representative image showing that standard HPF nanoparticles have unbound ferumoxytol, whereas VMI NCE 1 nanoparticles synthesized according to the magnetic separation process of the present disclosure have no residual unbound ferumoxytol. [Figure 2A] 2A and 2B are representative images of Prussian blue staining of cells showing iron loading in cells labeled with nanoparticles of the present disclosure. [Figure 2B] 2A and 2B are representative images of Prussian blue staining of cells showing iron loading in cells labeled with nanoparticles of the present disclosure. [Figure 3A] 3A and 3B are representative graphs showing increased cell viability upon labeling of cells with VMI NCE 1 nanoparticles of the present disclosure compared to standard HPF nanoparticles. [Figure 3B] 3A and 3B are representative graphs showing increased cell viability upon labeling of cells with VMI NCE 1 nanoparticles of the present disclosure compared to standard HPF nanoparticles. [Figure 3C] FIG. 3C shows by TEM analysis that VMI NCE 1 nanoparticles synthesized according to the magnetic separation process of the present disclosure retain more electron-dense magnetically active MRI-responsive iron agents compared to standard HPF nanoparticles. [Figure 4A] FIG. 4 shows representative images of Prussian blue staining of cells labeled with nanoparticles of the present disclosure at various concentrations. [Figure 4B] FIG. 4 shows representative images of Prussian blue staining of cells labeled with nanoparticles of the present disclosure at various concentrations. [Figure 5A] FIG. 5A is a representative image of Prussian blue staining of cells highlighting the difference in cell labeling efficiency of VMI NCE 1 nanoparticles prepared by magnetic separation according to the method of the present disclosure. [Figure 5B] FIG. 5B is a representative graph showing the viability of cells labeled with particles of the present disclosure. [Figure 5C]5C and 5D are representative graphs showing particle size distribution of standard HPF and VMI NCE 1 produced by magnetic separation according to the methods of the present disclosure. [Figure 5D] 5C and 5D are representative graphs showing particle size distribution of standard HPF and VMI NCE 1 produced by magnetic separation according to the methods of the present disclosure. [Figure 6] FIG. 6 is a representative image of Prussian blue staining of cells, showing that the concentration of VMI NCE 1 nanoparticles of the present disclosure can be increased 10-fold without reducing cell viability, which is required to label non- or poorly phagocytic cells. [Figure 7] FIG. 7 is a representative image of Prussian blue staining of cells, demonstrating that cells can be labeled with the VMI NCE 1 nanoparticles of the present disclosure under serum-containing conditions without the need for serum starvation. [Figure 8A] 8A and 8B are representative images showing the ability to label cells under serum-starved and serum-containing conditions in less than 3 hours using the magnetic labeling system of the present disclosure. [Figure 8B] 8A and 8B are representative images showing the ability to label cells under serum-starved and serum-containing conditions in less than 3 hours using the magnetic labeling system of the present disclosure. [Figure 9A] 9A-9C are representative images showing Prussian blue staining and fluorescence activity of dual-probe VMI NCE 1 nanoparticles of the present disclosure. [Figure 9B] 9A-9C are representative images showing Prussian blue staining and fluorescence activity of dual-probe VMI NCE 1 nanoparticles of the present disclosure. [Figure 9C] 9A-9C are representative images showing Prussian blue staining and fluorescence activity of dual-probe VMI NCE 1 nanoparticles of the present disclosure. [Figure 10A] FIG. 10A is a representative graph showing increased cell viability upon labeling of cells with dual-probe VMI NCE 1 nanoparticles of the present disclosure compared to standard dual-probe HPF particles. [Figure 10B]10B and 10C are representative graphs showing the average diameter of dual-probe VMI NCE 1 nanoparticles synthesized according to the method of the present disclosure. In FIG. 10C, the dual-probe VMI NCE 1 residual curve shows the unbound residual components that are present in excess and are toxic to cells, while the dual-probe VMI NCE 1 curve shows the removal of unbound toxic substances. [Figure 10C] 10B and 10C are representative graphs showing the average diameter of dual-probe VMI NCE 1 nanoparticles synthesized according to the method of the present disclosure. In FIG. 10C, the dual-probe VMI NCE 1 residual curve shows the unbound residual components that are present in excess and are toxic to cells, while the dual-probe VMI NCE 1 curve shows the removal of unbound toxic substances. [Figure 11] FIG. 11 is a representative image showing the use of a magnetic labeling system to facilitate uptake of the nanoparticles of the present disclosure to efficiently and effectively label cells with desired iron loading. [Figure 12A] 12A and 12B are representative images of MRI signal of cells labeled with nanoparticles of the present disclosure in a 7T small animal scanner using conventional T2-weighted MRI and a 3T clinical scanner using conventional MRI T2*-weighted MRI. [Figure 12B] 12A and 12B are representative images of MRI signal of cells labeled with nanoparticles of the present disclosure in a 7T small animal scanner using conventional T2-weighted MRI and a 3T clinical scanner using conventional MRI T2*-weighted MRI. [Figure 13] FIG. 13 is a representative color map of various MR parameters (e.g., (a) QSM map (b) T1 map (c) T2 map (d) T2* map) (top images) and their correlation fitting between the density of nanoparticles of the present disclosure and the MR parameters in a 3T clinical scanner (bottom images). [Figure 14] FIG. 14 shows a representative TEM image of vesicles labeled with nanoparticles of the present disclosure. [Figure 15A]15A and 15B are representative images of vesicles labeled with nanoparticles of the present disclosure in a 7T small animal scanner using conventional T2 and T2* weighted MRI. [Figure 15B] 15A and 15B are representative images of vesicles labeled with nanoparticles of the present disclosure in a 7T small animal scanner using conventional T2 and T2* weighted MRI. [Figure 16] FIG. 16 is a representative image of cells labeled with nucleic acid embedded nanoparticles of the present disclosure. [Figure 17] 17-19 are schematic diagrams of the protocols disclosed and described herein. [Figure 18] 17-19 are schematic diagrams of the protocols disclosed and described herein. [Figure 19] 17-19 are schematic diagrams of the protocols disclosed and described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Provided herein are nanoparticles, compositions, and methods for making and using the nanoparticles and compositions for cell tracking, drug encapsulation, and biological tracking.

[0035] There is a need to develop techniques for cell labeling of many cell types, including poorly phagocytes and non-phagocytes, via non-invasive magnetic resonance imaging (MRI) detection. The present disclosure describes a magnetic separation process to synthesize and generate a population of heparin (H), protamine (P), ferraheme (F) particles (HPF particles) that have enriched iron content and increased magnetic properties to improve MRI signal (VMI NCE 1 nanoparticles) for use with conventional MRI sequences available in most clinical settings. Importantly, the results of this study provide magnetically enriched HPF particles with high cell labeling efficiency and a favorable safety profile. The results further demonstrate that HPF particles generated and manufactured using the methods disclosed herein can be used for cell labeling and tracking. The present disclosure also provides a magnetic labeling system that allows for abbreviated (e.g., less than 3 hours) labeling of cells in both serum and serum-starved conditions.

[0036] After reading this description, it will be apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, not all various embodiments of the present invention are described herein. It will be understood that the embodiments presented herein are presented only as examples, not as limitations. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention, as described below.

[0037] The detailed description has been divided into various sections for the convenience of the reader only, and the disclosure in any section may be combined with the disclosure in another section. Titles or subtitles are used herein for the convenience of the reader without intending to affect the scope of the disclosure.

[0038] It is specifically intended that the various features of the invention described herein can be used in any combination, unless the context dictates otherwise.

[0039] definition All numerical designations are approximations that vary (+) or (-) by increments of 1.0 or 0.1, as appropriate, or by variances of + / - 15%, or 10%, or 5%, or 2%. It is to be understood, although not always expressly stated, that all numerical designations are preceded by the word "about." It is to be understood that such range formats are used for convenience and brevity, and that they should be flexibly understood to include not only the numerical values ​​expressly specified as limits of the range, but also all individual numerical values ​​or subranges contained within the range, as if each numerical value and subrange were expressly specified. For example, a percentage in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also individual percentages such as about 2, about 3, and about 4, as well as subranges such as about 10 to about 50, about 20 to about 100, etc. It is also to be understood, although not always expressly stated, that the reagents described herein are merely exemplary, and that equivalents of such are known in the art.

[0040] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0041] As used herein, the phrase "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit-risk ratio. Nanoparticles

[0042] Aspects of the present disclosure relate to nanoparticles comprising heparin, protamine, and an iron-based contrast agent. In some embodiments, the nanoparticles further comprise an additional contrast agent and / or a nucleic acid.

[0043] In some embodiments, the nanoparticles of the present disclosure include heparin. Heparin may be synthetic or biologically sourced and is incorporated into medical and biological applications involving nanoparticles to improve overall biocompatibility. In some embodiments, the heparin is in the form of a salt. For example, in some embodiments, the heparin is in the form of a sodium, calcium, potassium, or lithium salt. In some embodiments, the heparin is heparan sulfate. In some embodiments, the heparin is sodium heparin. In some embodiments, sodium heparin is more suitable compared to heparan sulfate due to the number of sulfate groups present in heparan sulfate, which gives rise to the more anionic character of heparin. In some embodiments, the heparin has a low molecular weight.

[0044] In some embodiments, heparin can be used to form complexes with protamine and iron-based contrast agents (synthetically from any manufacturer or biologically sourced) without the need to know the exact molecular weight and chain length of heparin. The exact molecular weight and chain length of heparin is highly variable due to its biologically sourced nature and cannot be obtained without significant expense and time. In some embodiments, the ratio is agnostic as it depends on the ability to extract the desired VMI NCE 1 particles after standard HPF particles are formed at different initial ratios. In some embodiments, the total yield of the desired VMI NCE 1 particles can vary, and thus there are recommended ranges as disclosed herein.

[0045] In some embodiments, heparin is provided in an amount of about 0.01 U / ml to about 10 U / ml. In some embodiments, heparin is provided in an amount of about 0.25 U / ml, about 0.5 U / ml, about 0.75 U / ml, about 1 U / ml, about 1.25 U / ml, about 1.5 U / ml, about 1.75 U / ml, about 2 U / ml, about 2.25 U / ml, about 2.5 U / ml, about 2.75 U / ml, about 3 U / ml, about 3.25 U / ml, about 3.5 U / ml, about 3.75 U / ml, about 4 U / ml, about 4.25 U / ml, about 4.5 U / ml, about 4.75 U / ml, The amount is about 5 U / ml, about 5.25 U / ml, 5.5 U / ml, about 5.75 U / ml, about 6 U / ml, about 6.25 U / ml, about 6.5 U / ml, about 6.75 U / ml, about 7 U / ml, about 7.25 U / ml, about 7.5 U / ml, about 7.75 U / ml, about 8 U / ml, 8.25 U / ml, about 8.5 U / ml, about 8.75 U / ml, about 9 U / ml, about 9.25 U / ml, about 9.5 U / ml, about 9.75 U / ml, or about 10 U / ml.

[0046] In some embodiments, the nanoparticles of the present disclosure further comprise protamine. Protamine is synthetically or biologically sourced and used in therapy to neutralize the highly negatively charged heparin by complexing with heparin to form a stable salt. In some embodiments, the protamine is obtained from salmon or other fish species sperm. In some embodiments, the protamine is in the form of a sulfate, phosphate, or hydrochloride salt. In some embodiments, the protamine is a sulfate salt from salmon. In some embodiments, the protamine is protamine sulfate. In some embodiments, the protamine is low molecular weight.

[0047] In some embodiments, protamine can be used to form complexes with heparin and iron-based drugs (synthetically or biologically sourced from any manufacturer) without the need to know the exact molecular weight and chain length of protamine (which is highly variable due to its biologically sourced nature and impossible to obtain without significant expense and time). In some embodiments, the ratio is agnostic due to the ability to extract the desired nanoparticles after the standard particles are formed at different starting ratios. There is no point in the total yield of the desired nanoparticles being different, so there are recommended ranges disclosed herein.

[0048] In some embodiments, protamine is provided in an amount of about 1 μg / ml to about 200 μg / ml. In some embodiments, protamine is provided in an amount of about 10 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, about 50 μg / ml, about 60 μg / ml, about 70 μg / ml, about 80 μg / ml, about 90 μg / ml, about 100 μg / ml, about 120 μg / ml, about 130 μg / ml, about 140 μg / ml, about 150 μg / ml, about 160 μg / ml, about 170 μg / ml, about 180 μg / ml, about 190 μg / ml, or about 200 μg / ml.

[0049] In some embodiments, the nanoparticles of the present disclosure further comprise a contrast agent. The contrast agent functions to allow the nanoparticles themselves to be visualized or, once incorporated into biological material such as cells, to be visualized via imaging modalities. In some embodiments, the contrast agent is a superparamagnetic metal complex. In some embodiments, the contrast agent is an iron-based drug. In some embodiments, the contrast agent is any superparamagnetic iron-containing compound. In some embodiments, the contrast agent is a superparamagnetic iron oxide. In other embodiments, the contrast agent is an ultra-small superparamagnetic iron oxide. In some embodiments, the same iron-based contrast agent is used for magnetic separation and production of VMI NCE 1 nanoparticles. Non-limiting examples of suitable contrast agents include ferumoxytol (feraheme), ferumoxide, ferucarbotran, ferumoxtran-10, and iron oxide.

[0050] In some embodiments, the iron-based agent is provided in an amount of about 1 μg / ml to about 800 μg / ml. In some embodiments, the labeling agent is provided in an amount of about 10 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, about 50 μg / ml, about 60 μg / ml, about 70 μg / ml, about 80 μg / ml, about 90 μg / ml, about 100 μg / ml, about 120 μg / ml, about 130 μg / ml, about 140 μg / ml, about 150 μg / ml, about 160 μg / ml, about 170 μg / ml, about 180 μg / ml, about 190 μg / ml, about 200 μg / ml, about 250 μg / ml, about 300 μg / ml, about 350 μg / ml, about 360 μg / ml, about 370 μg / ml, about 380 μg / ml, about 390 μg / ml, about 400 μg / ml, about 410 μg / ml, about 420 μg / ml, about 430 μg / ml, about 440 μg / ml, about 450 μg / ml, about 460 μg / ml, about 470 μg / ml, about 480 μg / ml, about 490 μg / ml, about 500 μg / ml, about 510 μg / ml, about 520 μg / ml, about 530 μg / ml, about 540 μg / ml, about 550 μg / ml, about 560 μg / ml, about 570 μg / ml, about 580 μg / ml, about 590 μg / ml, about 600 μg / ml, about 6 10μg / ml, about 220μg / ml, about 230μg / ml, about 240μg / ml, about 250μg / ml, about 260μg / ml, about 270μg / ml, about 280μg / ml, about 290μg / ml, about 300μg / ml, about 31 0μg / ml, approximately 320μg / ml, approximately 330μg / ml, approximately 340μg / ml, approximately 350μg / ml, approximately 360μg / ml, approximately 370μg / ml, approximately 380μg / ml, approximately 390μg / ml, approximately 400μg / ml, approximately 410μ g / ml, approximately 420μg / ml, approximately 430μg / ml, approximately 440μg / ml, approximately 450μg / ml, approximately 460μg / ml, approximately 470μg / ml, approximately 480μg / ml, approximately 490μg / ml, approximately 500μg / ml, approximately 510μg / ml, approximately 520μg / ml, approximately 530μg / ml, approximately 540μg / ml, approximately 550μg / ml, approximately 560μg / ml, approximately 570μg / ml, approximately 580μg / ml, approximately 590μg / ml, approximately 600μg / ml, approximately 610μg / m 1, about 620 μg / ml, about 630 μg / ml, about 640 μg / ml, about 650 μg / ml, about 660 μg / ml, about 670 μg / ml, about 680 μg / ml, about 690 μg / ml, about 700 μg / ml, about 710 μg / ml, about 720 μg / ml, about 730 μg / ml, about 740 μg / ml, about 750 μg / ml, about 760 μg / ml, about 770 μg / ml, about 780 μg / ml, about 790 μg / ml, or about 800 μg / ml.

[0051] Those skilled in the art will appreciate that the various amounts of each of heparin, protamine, and iron-based contrast agent will depend in part on the desired concentration and / or amount of nanoparticles to be produced. In some embodiments, the nanoparticles contain heparin, protamine, and ferumoxytol in different ratios. In some embodiments, heparin is present in an amount of about 2U / ml, protamine is present in an amount of about 60μg / ml, and ferumoxytol is present in an amount of about 50μg / ml. In some embodiments, heparin is present in an amount of about 2U / ml, protamine is present in an amount of about 40μg / ml, and ferumoxytol is present in an amount of about 100μg / ml.

[0052] In some embodiments, heparin is present in an amount of about 0.01 to about 10 U / ml, protamine is present in an amount of about 1 to about 200 μg / ml, and ferumoxytol is present in an amount of about 1 μg to about 800 μg / ml.

[0053] In some embodiments, the VMI NCE 1 nanoparticle further comprises a contrast agent. In some embodiments, the contrast agent is encapsulated in the nanoparticle or is bound to one component of the nanoparticle, such as protamine and / or heparin, or is bound to the nanoparticle. Non-limiting examples of suitable contrast agents include fluorescent probes, positron emission tomography (PET) agents, computed tomography (CT) agents (e.g., gold), ultrasound agents, or magnetic resonance imaging (MRI) agents.

[0054] In some embodiments, the fluorescent probe is indocyanine green (ICG), fluorescein, photofrin, or 5-aminolevulinic acid (ALA).

[0055] In some embodiments, the PET agent comprises a radionucleotide (e.g., a radioactive tracer) selected from the group consisting of carbon-11, nitrogen-13, oxygen-15, fluorine-18, gallium-68, zirconium-89, fluorine-18, and rubidium-82. In some embodiments, the PET agent is fluorodeoxyglucose (FDG). In some embodiments, the PET agent is zirconium-89.

[0056] In some embodiments, the CT agent is iodine or a barium-based compound or gold.

[0057] In some embodiments, the ultrasound agent is a fluorinated hydrocarbon, such as a perfluorocarbon or octafluoropropane.

[0058] In some embodiments, the MRI agent is a gadolinium agent, an iron oxide agent, an iron platinum agent, or a magnesium agent.

[0059] In some embodiments, the nanoparticles comprise dual labeling agents for multimodal imaging. Each imaging modality has its own unique characteristics, so a single imaging modality does not provide overall structural, functional, and molecular information. However, a combination of two imaging modalities can combine the unique characteristics of each imaging modality, thereby improving the diagnostic and therapeutic monitoring capabilities of the nanoparticles. In some embodiments, the nanoparticles further comprise an additional imaging agent. In some embodiments, the additional imaging agent is a fluorescent dye selected from the group consisting of fluorescein-5-isothiocyanate (FITC), Cy5, IRDye560, Cy7, ZW800, IRDye800, ICG, and CH1055. In some embodiments, the additional imaging agent allows monitoring of the nanoparticles via a fluorescent microscope.

[0060] In some embodiments, the nanoparticle further comprises a gene editing tool, e.g., a nucleic acid-based molecule. In some embodiments, the drug is encapsulated or bound to the nanoparticle. In some embodiments, the gene editing tool is encapsulated or bound to the nanoparticle, allowing the gene editing tool to be taken up into the cell. In some embodiments, the gene editing tool is RNA, e.g., siRNA, miRNA, and mRNA. In some embodiments, the gene editing tool is episomal in nature. In some embodiments, the gene editing tool is DNA, e.g., plasmid, double-stranded DNA, single-stranded DNA. In some embodiments, the gene editing tool is a plasmid vector, e.g., the CRISPR-Cas9 system. In some embodiments, the gene editing tool is genetic material for the expression of a particular protein or receptor, e.g., a chimeric antigen receptor (CAR) for cells such as T lymphocytes and natural killer cells.

[0061] In some embodiments, the nanoparticles are uniform in size or substantially uniform in size. In some embodiments, the nanoparticles have an average diameter of about 125 nm to about 195 nm, and the average diameter is 95 nm or more. For example, in some embodiments, the nanoparticles have an average diameter of about 125 nm to about 130 nm, about 125 nm to about 180 nm, about 130 nm to about 160 nm, about 140 nm to about 160 nm, about 140 nm to about 190 nm, about 140 nm to about 150 nm, about 125 nm to about 145 nm, about 130 nm to about 140 nm, about 130 nm to about 150 nm, about 140 nm to about 160 nm, about 140 nm to about 170 nm, about 160 nm to about 175 nm, or about 130 nm to about 180 nm. In some embodiments, the nanoparticles have an average particle size of about 130 nm or less, about 135 nm or less, about 140 nm or less, about 145 nm or less, about 150 nm or less, about 155 nm or less, about 160 nm or less, about 165 nm or less, about 170 nm or less, about 175 nm or less, about 180 nm or less, about 190 nm or less, or about 195 nm or less.

[0062] In some embodiments, the nanoparticles have an average diameter of about 125 nm to about 195 nm, and in synthetic samples, the number of nanoparticles having an average diameter of 95 nm or greater is at least about 0.5×10 9 particles / mL, approximately 1×10 9 particles / mL, approximately 2x10 9 particles / mL, approximately 3x10 9 particles / mL, approx. 4x10 9 particles / mL, approximately 5x10 9 particles / mL, approximately 6x10 9 particles / mL, approximately 7x10 9 particles / mL, approximately 8x10 9 particles / mL, or approximately 9x10 9 Contains particles / mL.

[0063] In some embodiments, the nanoparticles are magnetic nanoparticles, including many iron-based nanoparticles with a single or a few magnetic domains. A magnetic domain is a region of a nanoparticle where the magnetic moments of the iron atoms are aligned with each other. In some embodiments, the nanoparticles have more than 1000 magnetic domains. In some embodiments, the nanoparticles have more than about 1500 magnetic domains, more than about 2000 magnetic domains, more than about 2500 magnetic domains, more than about 3000 magnetic domains, more than about 3500 magnetic domains, more than about 4000 magnetic domains, more than about 4500 magnetic domains, or more than about 5000 magnetic domains.

[0064] In some embodiments, the iron content of the nanoparticles is greater than about 0.1 fg (femtogram). In some embodiments, the iron content is greater than about 0.15 fg, about 0.20 fg, about 0.25 fg, about 0.30 fg, about 0.35 fg, about 0.40 fg, about 0.45 fg, about 0.50 fg, about 0.55 fg, about 0.60 fg, about 0.65 fg, about 0.70 fg, about 0.75 fg, about 0.80 fg, about 0.85 fg, about 0.90 fg, about 0.95, or about 1 fg. In some embodiments, the iron content of the nanoparticles is between about 0.1 fg and about 1 fg. For example, in some embodiments, the iron content of the nanoparticles is between about 0.1 fg and about 0.50 fg, about 0.1 fg and about 0.60 fg, about 0.50 fg and about 1 fg, about 0.3 fg and about 0.80 fg, about 0.4 fg and about 0.70 fg, about 0.80 fg and about 1 fg, about 0.1 fg and about 0.4 fg, about 0.1 fg and about 0.65 fg, or about 0.15 fg and about 0.35 fg.

[0065] In some embodiments, the nanoparticles have r, r, and r2 that are about 3-10 times greater than the iron contrast agent (i.e., free iron contrast agent not incorporated into a nanoparticle). * For example, in some embodiments, nanoparticles encapsulated with ferumoxytol have magnetic properties r1, r2, and r2 that are about 3 to 10 times greater than the magnetic properties of the ferumoxytol nanoparticles themselves. * (r1=7.5 and r2=92 mM in water at 37°C and 3 T) -1 seconds -1 and 7T, r1 = 2 and r2 = 95mM. -1 sec -1 ).

[0066] Nanoparticle Composition Aspects of the present disclosure relate to compositions that include nanoparticles.

[0067] In some embodiments, nanoparticles comprising heparin, protamine, and an imaging agent may be formulated as a composition. In some embodiments, the imaging agent is ferumoxytol.

[0068] In some embodiments, the composition further comprises an excipient, diluent or carrier. In some embodiments, the composition further comprises a pharma- ceutically acceptable excipient, diluent or carrier.

[0069] In some embodiments, the composition may include pharma- ceutically acceptable excipients, pharma-ceutically acceptable salts, diluents, carriers, vehicles, and other such inactive agents. Vehicles and excipients commonly used in pharmaceutical preparations include, for example, talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, and the like. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2-propylene glycol, polyglycols, dimethyl sulfoxide, fatty alcohols, triglycerides, partial esters of glycerin, and the like. Parenteral compositions can be prepared using conventional techniques that may include sterile isotonic saline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol, polyglycols mixed with water, Ringer's solution, and the like. In one aspect, colorants are added to facilitate placement and proper positioning of the composition at the desired site.

[0070] In some embodiments, the composition further comprises a stabilizer and / or preservative. Non-limiting examples of preservatives include methyl-, ethyl-, propyl-paraben, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, propionic acid, benzalkonium chloride, benzyl alcohol, thimerosal, phenylmercuric salts, chlorhexidine, phenol, 3-cresol, quaternary ammonium compounds (QACs), chlorbutanol, 2-ethoxyethanol, and imidurea.

[0071] In some embodiments, the composition can include a physiological salt, such as a sodium salt, to control tonicity. In some embodiments, the physiological salt is sodium chloride (NaCl), potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride, and calcium chloride. In some embodiments, the physiological salt is present in an amount between about 1 mg / ml and about 20 mg / ml. For example, in some embodiments, the physiological salt is present in an amount of about 1 mg / ml to about 5 mg / ml, about 1 mg / ml to about 10 mg / ml, about 5 mg / ml to about 20 mg / ml, about 2 mg / ml to about 5 mg / ml, about 6 mg / ml to about 10 mg / ml, about 7 mg / ml to about 20 mg / ml, about 3 mg / ml to about 18 mg / ml, about 4 mg / ml to about 10 mg / ml, about 6 mg / ml to about 15 mg / ml, or about 5 mg / ml to about 15 mg / ml.

[0072] In some embodiments, the composition further comprises one or more buffers. Non-limiting examples of buffers include phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, or citrate buffer. In some embodiments, the composition comprises a buffer, and the buffer has a concentration ranging from about 5 mM to about 20 mM. For example, in some embodiments, the buffer has a concentration of about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 5 mM to about 15 mM, about 10 mM to about 15 mM, or about 8 mM to about 20 mM. In some embodiments, the pH of the composition will be between about 5 and about 8. For example, in some embodiments, the pH of the composition will be between about 5 and about 7.5, about 6 to about 8, about 6.5 to about 7.5, or between 7.0 and 7.8.

[0073] In some embodiments, the composition may further comprise a cryoprotectant. The cryoprotectant protects biological tissue from freezing damage due to ice formation. Non-limiting examples of cryoprotectants include glycols (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combination thereof.

[0074] In some embodiments, the composition is free or substantially free of unbound heparin. In some embodiments, the composition comprises less than about 10% unbound heparin, about 9% unbound heparin, about 8% unbound heparin, about 7% unbound heparin, about 6% unbound heparin, about 5% unbound heparin, about 4% unbound heparin, about 3% unbound heparin, about 2% unbound heparin, about 1% unbound heparin, about 0.5% unbound heparin, about 0.1% unbound heparin, about 0.05% unbound heparin, about 0.01% unbound heparin, or less total heparin in the composition. In some embodiments, a lower percentage of unbound heparin reduces the toxicity of the composition.

[0075] In some embodiments, the composition is free or substantially free of unbound protamine.In some embodiments, the composition comprises less than about 10% unbound protamine, about 9% unbound protamine, about 8% unbound protamine, about 7% unbound protamine, about 6% unbound protamine, about 5% unbound protamine, about 4% unbound protamine, about 3% unbound protamine, about 2% unbound protamine, about 1% unbound protamine, about 0.5% unbound protamine, about 0.1% unbound protamine, about 0.05% unbound protamine, about 0.01% unbound protamine, or less total protamine in the composition.In some embodiments, a lower percentage of unbound protamine reduces the toxicity of the composition.

[0076] In some embodiments, the composition does not contain or is substantially free of unbound ferumoxytol.In some embodiments, the composition contains less than about 10% unbound ferumoxytol, about 9% unbound ferumoxytol, about 8% unbound ferumoxytol, about 7% unbound ferumoxytol, about 6% unbound ferumoxytol, about 5% unbound ferumoxytol, about 4% unbound ferumoxytol, about 3% unbound ferumoxytol, about 2% unbound ferumoxytol, about 1% unbound ferumoxytol, about 0.5% unbound ferumoxytol, about 0.1% unbound ferumoxytol, about 0.05% unbound ferumoxytol, about 0.01% unbound ferumoxytol, or less total ferumoxytol in the composition.In some embodiments, a lower percentage of unbound ferumoxytol reduces the toxicity of the composition.

[0077] In some embodiments, the compositions are free or substantially free of unbound protamine and heparin. In some embodiments, the composition comprises about 10% or less unbound protamine and heparin, about 9% unbound protamine and heparin, about 8% unbound protamine and heparin, about 7% unbound protamine and heparin, about 6% unbound protamine and heparin, about 5% unbound protamine and heparin, about 4% unbound protamine and heparin, about 3% unbound protamine and heparin, about 2% unbound protamine and heparin, about 1% unbound protamine and heparin, about 0.5% unbound protamine and heparin, about 0.1% unbound protamine and heparin, about 0.05% unbound protamine and heparin, about 0.01% unbound protamine and heparin, or less total protamine and heparin in the composition. In some embodiments, a low percentage of unbound heparin-protamine complex reduces toxicity of the composition.

[0078] In some embodiments, water is removed from the composition, hi some embodiments, the composition is lyophilized, dried, freeze-dried, and / or dehydrated to remove water.

[0079] In one embodiment, less than about 5% of the nanoparticles in the composition have a particle size of more than 200 nm, more than 300 nm, more than 400 nm, more than 500 nm, more than 600 nm, more than 700 nm, or more than 800 nm. Less than about 0.01% of the nanoparticles in the composition have a particle size of more than 200 nm, more than 300 nm, more than 400 nm, more than 500 nm, more than 600 nm, more than 700 nm, or more than 800 nm. In another embodiment, less than about 0.5% of the nanoparticles in the composition have a particle size of more than 200 nm, more than 300 nm, more than 400 nm, more than 500 nm, more than 600 nm, more than 700 nm, or more than 800 nm. In some embodiments, less than about 0.05% of the nanoparticles in the composition have a particle size of more than 200 nm, more than 300 nm, more than 400 nm, more than 500 nm, more than 600 nm, more than 700 nm, or more than 800 nm.

[0080] In certain embodiments, the nanoparticles in the composition have an average diameter of about 120 nm to about 190 nm. In some embodiments, the nanoparticles in the composition have an average diameter of about 125 nm to about 195 nm, and the average diameter is 95 nm or more. For example, in some embodiments, the nanoparticles have an average diameter of about 125 nm to about 130 nm, about 125 nm to about 180 nm, about 130 nm to about 160 nm, about 140 nm to about 160 nm, about 140 nm to about 190 nm, about 140 nm to about 150 nm, about 125 nm to about 145 nm, about 130 nm to about 140 nm, about 130 nm to about 150 nm, about 140 nm to about 160 nm, about 140 nm to about 170 nm, about 160 nm to about 175 nm, or about 130 nm to about 180 nm. In some embodiments, the nanoparticles have an average particle size of about 130 nm or less, about 135 nm or less, about 140 nm or less, about 145 nm or less, about 150 nm or less, about 155 nm or less, about 160 nm or less, about 165 nm or less, about 170 nm or less, about 175 nm or less, about 180 nm or less, about 190 nm or less, or about 195 nm or less.

[0081] kit Aspects of the present disclosure relate to kits comprising nanoparticles or compositions comprising nanoparticles.

[0082] In some embodiments, the kit comprises the nanoparticles or a composition comprising the nanoparticles, and instructions describing a protocol for preparing the nanoparticles, hi some embodiments, the kit further comprises instructions for administering the nanoparticles or a composition comprising the nanoparticles to a cell or a subject in need thereof.

[0083] In some embodiments, the kit comprises nanoparticles and compositions comprising the nanoparticles, the nanoparticles being comprised of heparin, protamine, and a contrast agent. In some embodiments, the contrast agent is ferumoxytol.

[0084] In some embodiments, the kit comprises a dual-probe nanoparticle or a composition comprising the dual-probe particle, and instructions describing a protocol for preparing the dual-probe particle, in some embodiments, the kit further comprises instructions for administering the dual-probe nanoparticle or a composition comprising the dual-probe nanoparticle to a cell or a subject in need thereof.

[0085] In some embodiments, the kit comprises a magnetic labeling system for labeling cells with nanoparticles or a composition comprising nanoparticles in less than 3 hours, and instructions describing protocols for preparing the nanoparticles and using them with the magnetic labeling system, In some embodiments, the kit further comprises instructions for administering the nanoparticles or a composition comprising nanoparticles to cells or a subject in need thereof.

[0086] Manufacturing method Aspects of the present disclosure relate to methods of preparing magnetically separated nanoparticles.

[0087] In some embodiments, the method includes preparing magnetically separated nanoparticles comprising heparin, protamine, and an iron-based contrast agent. In some embodiments, the method includes (a) combining heparin, protamine, and an iron-based contrast agent, where at least a portion of the heparin, protamine, and iron-based contrast agent complexes to form nanoparticles comprising heparin, protamine, and the iron-based contrast agent, and (b) exposing the nanoparticles to a magnetic field to separate the nanoparticles based on their magnetic moment. In some embodiments, the separation is based on the iron content of the nanoparticles, resulting in separation of the nanoparticles based on size and magnetic moment.

[0088] In some embodiments, the method further comprises separating the nanoparticles from unbound material. In some embodiments, the heparin, protamine, and contrast agent are combined in a medium, and at least a portion of the heparin, protamine, and iron-based contrast agent are complexed to form nanoparticles comprising heparin, protamine, and iron-based contrast agent. In some embodiments, the medium is a buffer, serum, nutrient-enriched medium, or basal medium. The unbound material includes any compounds, substances, debris, or by-products that did not complex to form nanoparticles, including heparin, protamine, and iron-based contrast agent. In some embodiments, the unbound substances are heparin, protamine, ferumoxytol, and / or heparin-protamine complexes. In some embodiments, the method further comprises separating the nanoparticles from unbound heparin, unbound protamine, unbound ferumoxytol, and / or unbound heparin-protamine complexes. Application of a magnetic field selectively separates nanoparticles comprising heparin, protamine, and an iron-based contrast agent from unbound heparin, unbound protamine, unbound ferumoxytol, and / or unbound heparin-protamine complexes based on the iron content of the nanoparticles and, therefore, the magnetic properties of the nanoparticles. In some embodiments, application of a magnetic field excludes unbound heparin, unbound protamine, unbound ferumoxytol, and / or unbound heparin-protamine complexes, allowing for selective separation of the nanoparticles.

[0089] In some embodiments, the method includes (a) combining heparin, protamine, and an iron-based contrast agent, where at least a portion of the heparin, protamine, and iron-based contrast agent complexes form nanoparticles comprising heparin-protamine and the iron-based contrast agent; and (b) exposing the nanoparticles to a magnetic field to separate unbound protamine and / or heparin that did not complex to form nanoparticles.

[0090] In some embodiments, the solution is exposed to the magnetic field 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In some embodiments, the solution is exposed to the magnetic field until no or substantially no unbound heparin, unbound protamine, unbound ferumoxytol and / or unbound heparin-protamine complex is present in the solution.

[0091] In some embodiments, the method further comprises separating the nanoparticles based on size. In some embodiments, the iron content and strength of the magnetic dipole moment associated with the nanoparticles correlate with the size of the nanoparticles, providing a method for selectively separating the nanoparticles based on size.

[0092] In some embodiments, an alternative method for extracting nanoparticles from unbound material includes separating the nanoparticles by ultrafiltration or dialysis. In some embodiments, the heparin, protamine, and contrast agent are combined in a medium, and at least a portion of the heparin, protamine, and contrast agent are complexed to form nanoparticles comprising heparin, protamine, and contrast agent. In some embodiments, the medium is a buffer, serum, nutrient-enriched medium, or basal medium. In some embodiments, after the nanoparticles are formed, the nanoparticles in the medium are collected from the filtrate to form a preparation comprising nanoparticles. In some embodiments, following ultrafiltration or dialysis, the medium (i.e., supernatant) is decanted from the preparation comprising nanoparticles. In some embodiments, the decanted medium comprises unbound heparin, unbound protamine, and / or unbound heparin-protamine complexes.

[0093] In some embodiments, exposing the nanoparticles to a magnetic field comprises applying a magnet to the nanoparticles.

[0094] In some embodiments, the magnetic field strength is a magnetic flux density of about 300 mT to about 600 mT. In some embodiments, the magnetic flux density is about 300 mT to about 400 mT, about 300 mT to about 500 mT, about 400 mT to about 500 mT, about 400 mT to about 600 mT, or about 500 mT to about 600 mT.

[0095] In some embodiments, the applied external magnetic field is uniform and perpendicular to the solution and collection surface. In certain embodiments, the applied external magnetic field is non-uniform. In some embodiments, separation can be achieved with the south and / or north poles of a permanent magnet. In yet another embodiment, separation can also be achieved at the interface of two magnets in contact with each other. In another embodiment, separation can be achieved by applying an external magnetic field with a certain strength that is generated by a current-carrying wire of a desired shape.

[0096] In some embodiments, the method selectively isolates nanoparticles with an iron content of about 0.1 fg, with more than 1,000 atoms encapsulated per nanoparticle and with magnetic properties (r1, r2 and r2*) that are 3-10 times greater than the iron-based contrast agent (e.g., ferumoxytol) itself. In some embodiments, the method selectively isolates nanoparticles with an iron content of greater than about 0.15 fg, about 0.20 fg, about 0.25 fg, about 0.30 fg, about 0.35 fg, about 0.40 fg, about 0.45 fg, about 0.50 fg, about 0.55 fg, about 0.60 fg, about 0.65 fg, about 0.70 fg, about 0.75 fg, about 0.80 fg, about 0.85 fg, about 0.90 fg, about 0.95, or about 1 fg. In some embodiments, the iron content of the nanoparticles is between about 0.1 fg and about 1 fg. For example, in some embodiments, the iron content of the nanoparticles is between about 0.1 fg and about 0.50 fg, about 0.1 fg and about 0.60 fg, about 0.50 fg and about 1 fg, about 0.3 fg and about 0.80 fg, about 0.4 fg and about 0.70 fg, about 0.80 fg and about 1 fg, about 0.1 fg and about 0.4 fg, about 0.1 fg and about 0.65 fg, or about 0.15 fg and about 0.35 fg.

[0097] In some embodiments, the method selectively separates particles having an average diameter of about 120 nm to about 195 nm. For example, in some embodiments, the nanoparticles have an average diameter of about 125 nm to about 130 nm, about 125 nm to about 180 nm, about 130 nm to about 160 nm, about 140 nm to about 160 nm, about 140 nm to about 190 nm, about 140 nm to about 150 nm, about 125 nm to about 145 nm, about 130 nm to about 140 nm, about 130 nm to about 150 nm, about 140 nm to about 160 nm, about 140 nm to about 170 nm, about 160 nm to about 175 nm, or about 130 nm to about 180 nm. In some embodiments, the nanoparticles have an average particle size of about 130 nm or less, about 135 nm or less, about 140 nm or less, about 145 nm or less, about 150 nm or less, about 155 nm or less, about 160 nm or less, about 165 nm or less, about 170 nm or less, about 175 nm or less, about 180 nm or less, about 190 nm or less, or about 195 nm or less.

[0098] In some embodiments, nanoparticles isolated by magnetic separation have about 30% to about 200% greater iron loading than nanoparticles not isolated by magnetic separation. In some embodiments, nanoparticles have about 30% to about 100%, about 50% to about 200%, about 40% to about 80%, about 60% to about 200%, about 50% to about 150%, about 40% to about 120%, about 100% to about 200%, about 150% to about 200%, about 100% to about 180%, about 90% to about 190%, or about 30% to about 60% greater iron loading than nanoparticles not isolated by magnetic separation.

[0099] In some embodiments, the nanoparticles are concentrated by between about 2-fold and about 10-fold without significantly increasing the amount of unbound material. In some embodiments, the nanoparticles are concentrated by between about 0.5×10 5 Particles / mL, approximately 1.0×10 5 particles / mL, approximately 0.5×10 6 Particles / mL, approximately 1.0×10 6 particles / mL, approximately 0.5×10 7 Particles / mL, approximately 1.0×10 7particles / mL, approximately 0.5×10 8 Particles / mL, approximately 1.0×10 8 particles / mL, approximately 0.5×10 9 Particles / mL, approximately 1.0×10 9 particles / mL, approximately 0.5×10 10 Particles / mL, approximately 1.0×10 10 particles / mL, approximately 0.5×10 11 Particles / mL, approximately 1.0×10 11 particles / mL, approximately 0.5×10 12 Particles / mL, approximately 1.0×10 12 In some embodiments, the particles are concentrated to about 1.0×10 particles / mL or more. 8 ~Approx. 1.0×10 11 The nanoparticles are concentrated to a specific amount of particles / mL. In some embodiments, the nanoparticles are concentrated to a specific amount without significantly increasing the amount of unbound material. In some embodiments, the unbound material comprises unbound heparin, unbound protamine, unbound ferumoxytol, and / or unbound heparin-protamine complex.

[0100] How to use Aspects of the present disclosure relate to methods of labeling cells, comprising contacting the cells with nanoparticles or compositions comprising nanoparticles, hi some embodiments, the methods further comprise labeling a cell-derived product, such as a vesicle.

[0101] In some embodiments, the method further comprises labeling the cells under serum starvation and / or serum deprivation conditions while contacting the cells with the nanoparticles or a composition comprising the nanoparticles. In some embodiments, the method comprises labeling the cells under serum starvation and / or serum deprivation conditions during the contacting step. In some embodiments, the cells remain serum starved and / or serum deprived for a period of time and then allowed to recover under normal cell culture conditions for an additional period of time.

[0102] In some embodiments, the method includes labeling a cell from a cell-derived product, the method includes: (a) contacting a first cell with a composition comprising nanoparticles of heparin, protamine, and an iron-based imaging agent, where the contacting step labels the cell-derived product within the cell; (b) removing the labeled cell-derived product from the cell; and (c) contacting a second cell with the cell-derived product, where the second cell takes up the labeled cell-derived product. In some embodiments, the method further includes tracking the second cell.

[0103] In some embodiments, the method further comprises labeling the cells in serum-containing medium during contact with the composition. The ability to label cells for several hours without the need for serum starvation as part of the labeling protocol allows users to adhere to existing cell culture standard operating procedures (SOPs) without substantial alterations to the cells.

[0104] In some embodiments, the cell is a mammalian cell, such as a mouse cell, a rat cell, a dog cell, a pig cell, a cow cell, a horse cell, a primate cell, or a human cell.

[0105] In some embodiments, the cell is a cancer cell, an immune cell, or a blood cell.In other embodiments, the cell is a stem cell, an embryonic cell, a fetal cell, or a somatic cell.Non-limiting examples of suitable stem cells include mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, adipose stem cells, neural stem cells, T lymphocytes, natural killer cells, or adult stem cells.

[0106] In some embodiments, the method further comprises tracking the cells for a period of time after contacting the cells with the nanoparticles or a composition comprising the nanoparticles. Some non-limiting methods for imaging cells include by fluorescence microscopy, MRI, in vivo optical imaging, PET and / or single photon emission computed tomography (SPECT), computed tomography (CT) and / or ultrasound.

[0107] In some embodiments, the method further comprises tracking the cells over a period of time using standard MRI imaging sequences. The specific magnetic properties of the particles (e.g., cluster effect, high iron loading) allow the use of standard MRI imaging sequences, which allows for highly efficient labeling of the particles.

[0108] In some embodiments, cells are labeled with nanoparticles or compositions comprising nanoparticles in less than about 3 hours using a magnetic labeling system. The magnetic labeling system allows for shortening the labeling time by promoting rapid uptake of nanoparticles or compositions comprising nanoparticles by applying a magnetic field to the magnetically labeled nanoparticles. In some embodiments, the rapid uptake occurs by changing the magnetic flux density by applying a permanent magnet or a current-driven temporary magnet to the nanoparticles. The force applied by the magnet promotes efficient uptake of the particles to label the cells. For example, in some embodiments, cells are labeled with nanoparticles or compositions comprising nanoparticles in less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, or less than about 0.5 hours.

[0109] In some embodiments, imaging is performed at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or more after the cells have been contacted with the nanoparticles or composition.

[0110] In some embodiments, the method includes administering the nanoparticles or a composition comprising the nanoparticles to a subject. In some embodiments, the subject is a mammalian subject, such as a human, a non-human primate, or an experimental non-human mammalian subject, such as a mouse or rat. In some embodiments, the mammalian subject is a human.

[0111] In some embodiments, the method comprises administering the nanoparticles or compositions comprising the nanoparticles by any suitable route depending on the use and / or disease or condition being treated. In some embodiments, the method comprises administering the nanoparticles or compositions comprising the nanoparticles by intravenous, intraarterial, intramuscular, subcutaneous, intracranial, intranasal or intraperitoneal administration. In some embodiments, the nanoparticles or compositions comprising the nanoparticles are administered intravenously, intraarterially, by direct tissue injection, and / or intratumorally.

[0112] In some embodiments, the method further comprises programming, genetically modifying, and / or tracking the cells for a period of time following administration to the subject. In some embodiments, tracking is performed by imaging at least a portion of the subject. In some embodiments, imaging is performed by fluorescence detection, MRI, FL, in vivo optical imaging, PET / SPECT, CT, and / or ultrasound.

[0113] In some embodiments, imaging is performed at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or longer after the subject is administered the nanoparticles or composition.

[0114] In some embodiments, the method of labeling cells allows for imaging by MRI due to an increase in iron loading in the nanoparticles of about 30% to about 300% compared to non-magnetically separated nanoparticles. In some embodiments, the nanoparticles have an iron loading of between about 30% to about 100%, about 50% to about 300%, about 40% to about 80%, about 60% to about 300%, about 50% to about 150%, about 40% to about 120%, about 100% to about 300%, about 150% to about 300%, about 100% to about 180%, about 90% to about 190%, or between about 30% to about 60% compared to non-magnetically separated particles.

[0115] In some embodiments, the methods include ex vivo, in vitro, and / or in vivo cell tracking.

[0116] In some embodiments, the method further comprises encapsulating the nucleic acid in nanoparticles prior to introduction into the cells. Encapsulation of the nucleic acid in nanoparticles provides safer and more effective introduction of the nucleic acid into cells. Introduction of nucleic acids such as RNA, plasmid DNA, DNA, peptides / proteins is difficult with low efficiency and has undesirable adverse effects on cells using currently available methods such as, for example, electroporation or delivery via lipofectamine or transfection agents. In some embodiments, the method further comprises modifying the cells with nanoparticles having encapsulated nucleic acids. The cells can be modified with nanoparticles having encapsulated nucleic acids by reprogramming the cells, differentiating the cells, or increasing the transfection of the cells.

[0117] In some embodiments, the nanoparticles can be used to label and track cell-derived biologics, such as vesicles, which can then be further used in diagnostic and / or therapeutic applications.

[0118] In some embodiments, the nanoparticles can be used for three-dimensional (3D) printing. In some embodiments, the 3D printing is bioprinting. In some embodiments, the method includes creating a 3D scaffold containing nanoparticle-labeled cells for applications ranging from biosensing, tissue regeneration, environmental sensing, drug discovery and clinical implementation. EXAMPLES

[0119] The above and the following examples are intended to merely illustrate various embodiments of the present invention. The specific modifications discussed above should not be interpreted as limitations on the scope of the present invention. It is clear to those skilled in the art that various equivalents, changes and modifications can be realized without departing from the scope of the present invention, and it will be understood that such equivalent embodiments are included in this specification.

[0120] There is a need to develop techniques for cell labeling and tracking via non-invasive MRI detection. Towards this end, a magnetic separation process has been developed to synthesize and generate a population of herapin (H), protamine (P), and ferraheme (F) particles (HPF particles) with enriched iron content and enhanced magnetic properties (VMI NCE 1 nanoparticles) for improved MRI signal for use with conventional MRI sequences available in most clinics. Importantly, the results of this study provide magnetically enriched HPF particles with high cell labeling efficiency and a favorable safety profile. The results further demonstrate that HPF particles generated and manufactured using the methods disclosed herein can be used for cell labeling, cell tracking, biologic labeling, biologic tracking, and cell reprogramming.

[0121] Example 1: SOP for standard HPF formation The objective of the following study was to develop a standard operating procedure (SOP) for standard HPF (Std HPF) formation.

[0122] The following reagents were used to prepare standard HPF particles: heparin (H, sodium injection, USP, 1000 units / ml), protamine sulfate (P, injection, USP, 50 mg / ml), and ferraheme (F, injection, USP, 510 mg / 17 ml).

[0123] To form standard HPF particles, a 2 U / ml solution of H was prepared by first adding a 30-500 mL aliquot of basal medium (e.g., Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F-12)) to a sterile tube and / or container. For every 10 mL of basal medium, 20 µL of H was added to the sterile tube and / or container containing the basal medium (e.g., add 60 µL of H to 30 mL of basal medium). The resulting basal medium and H solution were then mixed thoroughly.

[0124] As the second step in preparing standard HPF, 60 μL of P solution was slowly added to each 10 mL of basal medium containing 2 U / mL H, swirling the tube effectively during the addition (e.g., adding 180 μL of P to 30 mL of solution), at which point the solution appeared cloudy.

[0125] Within approximately 15-30 seconds of adding P, 16.67 μL of F solution was added to 10 mL of each of the HP solutions above and mixed in the tube and / or container (e.g., 50.01 μL of F was added to 30 mL of HP solution).

[0126] The above procedure results in the formation of Std HPF particles.

[0127] Example 2: SOP for dual-probe standard HPF formation Because a single imaging modality does not provide complete structural, functional, and molecular information, combining two imaging modalities in a single nanoparticle can improve the diagnostic and therapeutic monitoring capabilities of the nanoparticle. Therefore, the aim of the following study was to develop an SOP for generating dual-labeled agents for multimodal imaging. Toward this end, FITC-conjugated heparin was mixed with protamine and ferraheme to form dual-probe standard HPF particles.

[0128] To form dual-probe standard HPF particles, 1 mg of H-FITC reconstitution was added to 1 mL of sterile water to obtain a solution with an H-FITC concentration of 70-100 U / ml. Then, 200 µl of the H-FITC solution was transferred to 10 mL of basal medium (DMEM / F-12). Following this procedure, H-FITC solutions with concentrations of 1.4-2 U / ml can be obtained.

[0129] An aliquot with the appropriate amount of F was then added to a separate sterile tube and / or container and set aside. To prepare the dual-probe standard HPF particles, 16.67 μL of F was required for every 10 mL of base medium.

[0130] An aliquot with the appropriate amount of P was then added to a separate tube and / or container. 60 μL of P is needed for every 10 ml of final solution.

[0131] With separate solutions of each component (e.g., H, F, and P solutions) prepared, the final step was the preparation of a dual-probe HPF particle solution with a concentration ratio of 2 U / ml H-FITC, 60 μg / ml P, and 50 μg / ml F. A 60 μg / ml aliquot of P solution was slowly added to 10 ml of H-FITC solution. The solution was then mixed (e.g., swirled), at which point the solution appeared cloudy. Within approximately 15-30 seconds of adding the aliquot of P solution, 16.67 μL of F solution was added and mixed in the tube and / or container.

[0132] The above procedure results in the formation of dual-probe Std HPF particles.

[0133] Example 3: SOP for HPF particles using magnetic separation process Once the SOPs for Std HPF and Dual-Probe Std HPF particle formation have been established, the next step will be to develop SOPs for generating HPF particles via a magnetic separation process to generate / manufacture a population of HPF particles with enriched iron content, improved magnetic properties, and no residual unbound material (H or P or F or HP), thereby improving the overall labeling efficiency of the resulting HPF particles, their safety profile, and MRI activity.

[0134] Std HPF particles formed in Example 1 were transferred to a 15 mL conical tube. After transfer, a permanent magnet with a magnetic strength of 300 mT-600 mT was applied to the side of the tube for approximately 25 minutes at room temperature. Separation can be performed simultaneously with both the north and south poles of the permanent magnet. The tube was inverted every 10 minutes while the magnet was applied for 25 minutes. After the magnet was applied for 25 minutes, a solid pellet was formed, and then the supernatant (i.e., the sediment-free liquid remaining above the solid material), referred to as VMI NCE 1 residue, was transferred to a new tube.

[0135] After transferring the supernatant, the pellet was washed with an equal volume of basal medium (DMEM / F-12). A permanent magnet with a magnetic strength of 300mT-600mT was then applied again to the side of the tube holding the reconstituted pellet (nanoparticles) for approximately 25 minutes at room temperature to extract HPF particles with enriched iron content and increased magnetic properties. Separation can be performed simultaneously with both the north and south poles of the permanent magnet. During the application of the Visicell magnetic separation system for 25 minutes, the tube was inverted every 10 minutes. The supernatant was then removed. Each step was then repeated 3-6 times. Similar populations of HPF particles with enriched iron content and increased magnetic properties can be extracted using a uniformly or non-uniformly applied external magnetic field (Figure 20).

[0136] To generate different formulations of the new HPF particles with enriched iron content and improved magnetic properties (e.g., different concentrations of particles (different number of particles / ml), an equal volume of basal medium was added to the pellet to form a solution with 1x magnetic concentration, VMI NCE 1 (1x) particles, or 1 / 5 the volume of basal medium was added to the pellet to form a more concentrated solution with 5x magnetic concentration, VMI NCE 1 (5x) particles. The same procedure was used for magnetic separation of dual contrast agents to form dual-probe VMI NCE 1 (1x) and dual-probe VMI NCE 1 (5x) particles.

[0137] The above procedure results in the formation of VMI NCE 1 (e.g., VMI NCE 1(1x) and VMI NCE 1(5x)) particles and dual-probe VMI NCE 1 (e.g., dual-probe VMI NCE 1(1x) and dual-probe VMINCE1(5x)) particles that are enriched in iron content compared to Std HPF and dual-probe Std HPF particles.

[0138] Example 4: Ultrafiltration purification In addition to developing a magnetic separation process as described above, an ultrafiltration purification process was also developed to isolate HPF particles with increased iron content, a desired size, and reduced unbound material.

[0139] The Std HPF particles formed in Example 1 were transferred to a Corning SpinX filter with a MWCO of 100 Kda for centrifugation. The Std HPF particles were then centrifuged in a swing-back centrifuge at 2,000 RPM for 15-20 minutes or until only 0.25 mL of volume remained. After filtration through the Corning SpinX filter, the filtrate, referred to as VMI NCE 2 retentate, was transferred to a 15 mL conical tube and the retentate (i.e., material that does not pass through the filter), referred to as VMI NCE 2, was washed with an equal volume of basal medium. The retentate washed with basal medium was then recentrifuged at 2,000 RPM for 15-20 minutes until only 0.25 mL remained. The filtrate was then removed. Each step was repeated 4-6 times.

[0140] To produce different formulations of new HPF particles with enriched iron content, desired size, and reduced unbound material (e.g., different concentrations of particles (different numbers of particles / ml), an equal volume of basal medium was added to VMI NCE2 to form a solution with 1x magnetic concentration, VMI NCE 2 (1x) particles, or 1 / 5 of the volume of basal medium was added to the remainder to form a solution with 5x magnetic concentration, VMI NCE 2 (5x) particles.

[0141] The above procedure results in the formation of VMI NCE 2 particles.

[0142] Example 5: Labeling of extracellular vesicles (EVs). Having developed a method to isolate HPF particles enriched in iron and with improved magnetic properties, the next step was to develop a method for labeling extracellular vesicles with HPF particles.

[0143] Mesenchymal stem cells (MSCs), neural stem cells (NSCs), and amniotic fluid stem cells (AFSCs) were incubated with any of the HPF particles described in Examples 1-3 for 4 hours in the absence of fetal bovine serum (FBS), and then allowed to recover overnight in medium containing 10% FBS. The medium of the labeled cells was then aspirated. After aspiration, the cells were washed twice with PBS. EV-depleted medium was then added to the cells. The labeled cells were then incubated for 48-72 hours.

[0144] After incubation, the medium in which the cells were suspended (i.e., conditioned medium) was collected. The conditioned medium was then centrifuged at 2,000 x g for 20 min to remove cell debris. After centrifugation, the supernatant was transferred to an ultracentrifuge tube and centrifuged at 120,000 x g for approximately 2 h. After 2 h of centrifugation, the supernatant was removed and the collected EVs were resuspended in PBS. Each centrifugation step was performed at 4 °C.

[0145] Extracellular vesicles were labeled with HPF particles from Examples 1-3 following the steps outlined above.

[0146] Example 6: Labeling efficiency, safety profile, and physicochemical properties of HPF particles Having isolated and then labeled cells with HPF particles, the next step was to examine the efficiency of labeling, the safety profile, and how the physicochemical properties of Std HPF, VMI NCE 1, and dual-probe VMI NCE1 particles affect the ability to label cells.

[0147] Labeling efficiency Nanoparticle tracking analysis (NTA) was used to compare the particle size distribution of Std HPF and VMI NCE 1 (1x). Std HPF prepared as described in Example 1 had a mean diameter of 121 nm. (Figure 1A, Table 1). In comparison, VMI NCE 1 (1x) prepared as described in Example 3 was larger with a mean diameter of 140 nm, while VMI NCE 1 retentate from the remaining supernatant from magnetic separation had a mean diameter of 86 nm (Figure 1B, Table 1). VMI NCE2 isolated via the ultrafiltration purification process described in Example 4 had a mean diameter of 117 compared to VMI NCE 2 retentate with a mean diameter of 83 nm. (Figure 1C, Table 1). VMI NCE 1 (1x), which contains more iron, was enriched during the magnetic separation process, whereas unbound H, unbound P, unbound HP, and the smaller free ferumoxytol were not retained and were likely enriched in the VMI NCE 1 residual fraction, as shown in Figure 1D , which indicates the smaller average particle size of VMI NCE1 residual.

[0148] [Table 1]

[0149] Figures 2A-2B demonstrate that HPF particles generated and manufactured using magnetic separation methods have better labeling efficiency and intracellular iron loading as determined by Prussian blue staining (i.e., iron signal) in low phagocytes such as mesenchymal stem cells (MSCs). In particular, Figures 2A-2B show the labeling efficiency of unlabeled MSCs (i.e., Mock cells), Std HPF labeled MSCs, VMI NCE 1 (1x) labeled MSCs, VMI NCE 1 residual labeled MSCs, VMI NCE 2 labeled MSCs, and finally VMI NCE2 residual labeled MSCs. As shown in Figures 2A-2B, MSCs labeled with VMI NCE 1 (1x) were more efficiently labeled with iron compared to MSCs labeled with Std HPF.

[0150] Therefore, this data confirms that HPF particles prepared by magnetic separation method have high labeling efficiency, as evidenced by intracellular iron loading.

[0151] Safety Profile Next, the safety profile of HPF particles prepared by the method described in Examples 1-4 was evaluated by labeling MSCs with those particles and by their mitochondrial dehydrogenase activity. MSCs labeled with VMI NCE 1 (1x) showed minimal toxicity compared to Mock-labeled cells, as determined by mitochondrial dehydrogenase activity. In contrast, MSCs labeled with Std HPF showed a moderate decrease in cell viability, while VMI NCE1 residue had the most toxic effect on MSCs. (Figure 3A). The results show that the novel VMI NCE 1 (1x) particles have an excellent safety profile, increasing cell viability by approximately 20% compared to that of Std HPF. The results also show that unbound materials such as unbound H, unbound P, and unbound HP in the VMI NCE 1 residue are responsible for the adverse effects in cells (40% decrease in viability compared to VMI NCE 1 (1x) and 20% decrease in viability compared to Std HPF). Moreover, it is noteworthy that VMI NCE 1 (5x) exhibits a superior safety profile compared to Std HPF or VMI NCE 1 residues. Similar results are shown in Figure 3B, where VMI NCE 1 (1x) and VMI NCE 2 exhibited a more favorable safety profile compared to Std HPF. Figure 3C further demonstrates that VMI NCE 1 nanoparticles are not only safer but also more potent for cell labeling compared to Std HPF. Notably, Figure 3C shows that VMI NCE 1 retains more electron-dense magnetically active MRI-responsive iron agent compared to Std HPF by TEM analysis.

[0152] Furthermore, the total iron concentration of the resulting VMI NCE 1 particles can be adjusted by varying the volume of basal medium added to the pellet after magnetic separation, as described in Example 3. Notably, adding an equal volume of basal medium to the pellet after magnetic separation produces VMI NCE 1 (1x) particles. However, adding 1 / 5 the volume of basal medium to the pellet after magnetic separation results in a VMI NCE 1 (5x) pellet with 5x the magnetic concentration. MSCs labeled with VMI NCE 1 (5x) had higher iron content, more intense Prussian blue staining, and only a slight increase in HPF-related toxicity compared to VMI NCE 1 (1x). (Figures 4A-4B, respectively). The stability of the HPF particles was also examined, and there was no significant change in the labeling efficiency or safety profile after storage of Std HPF or VMI NCE 1 particles at 4 °C for 1 week and at 0 °C for up to 6 months.

[0153] Thus, and as described above, HPF particles prepared by magnetic separation have a favorable safety profile, exhibiting minimal toxicity to labeled cells.

[0154] Physicochemical composition of HPF particles HPF particles prepared by magnetic separation were evaluated to determine whether the physicochemical composition of the particles was suitable for the cellular environment.

[0155] VMI NCE 1 particles were first tested for labeling efficiency in a cellular environment and compared with Std HPF particles. Std HPF particles with various HPF ratios of 2:60:50 HPF and 2:40:100 HPF and VMI NCE 1 particles prepared via magnetic separation process were prepared as described in Examples 1 and 3. Figure 5A displays images highlighting the difference in cell labeling efficiency of Std HPF particles with variable HPF ratios compared to VMI NCE 1 particles. As shown in Figure 5A, the Prussian blue staining of unlabeled cells, Std HPF labeled cells with HPF ratio of 2:60:50, and Std HPF labeled cells with HPF ratio of 2:40:100 is less than that of VMI NCE1 labeled cells, suggesting that VMI NCE 1 particles label cells more efficiently compared to Std HPF particles. Figure 5B shows the cell viability of Std HPF labeled cells with a 2:60:50 HPF ratio, Std HPF labeled cells with a 2:40:100 HPF ratio, VMI NCE 1 labeled cells, and cells labeled with 2:40:100 HPF particles generated using method 1 (magnetic separation). Figures 5C and 5D show the size distribution of Std HPF particles and VMI NCE 1 particles. As listed in Table 3, HPF particles enriched with iron during magnetic separation have a larger particle size.

[0156] [Table 2]

[0157] Next, HPF particles were evaluated to determine whether the total number of particles exposed to cells, and therefore the total iron content and / or particle concentration, could be adjusted (i.e., increased) without affecting cell health. Figure 6 shows differential labeling of the disclosed HPF particles in neural stem cells (NSCs) (top image) and low phagocytic cells such as MSCs (bottom image). Importantly, Figure 6 demonstrates that the concentration of HPF particles introduced to cells can be increased while maintaining comparable cell health. Notably, these results show that the HPF particle concentration can be increased ten-fold, increasing labeling efficiency and iron loading without increasing unwanted residual toxic components in the formulation. Additionally, there is evidence of increased intracellular iron loading as indicated by more intense Prussian blue staining without compromising cell health for each cell type, NSCs and MSCs, respectively (see Figure 6, images of labeled NSCs at VMI NCE 1 original intensity vs. NSCs at VMI NCE 1 intermediate intensity and MSCs at VMI NCE 1 original intensity vs. MSCs at VMI NCE 1 verified intensity). The strengths verified are 4x to 6x for MSCs, although 5x is usually a good strength for MSCs.

[0158] Next, HPF particles were tested to determine whether they could effectively label cells under serum-containing conditions without the need for serum starvation. This is important because serum starvation can affect cell health or disrupt already established SOPs and workflows. Figure 7 shows that not only can the VMI NCE-1 particle concentration be adjusted, but the particles enable efficient and effective labeling of cells under serum-containing conditions without the need for serum starvation in both cell types, namely MSCs (top image) and MSCs (bottom image) compared to Std HPF-labeled NSCs (top, far left image) and MSCs (bottom, far right image).

[0159] Having determined that VMI NCE 1 particles effectively labeled cells overnight under serum-containing conditions, we assessed their ability to increase labeling rates (reduced total labeling time) under both serum-starved and serum-containing conditions using a combination of VMI NCE 1 particles and the VMI magnetic labeling system (Figure 11). The degree of labeling efficiency of VMI NCE 1 particles was determined under standard serum-starved conditions (Figure 8A-8B, top images) and serum-containing conditions (Figure 8A-8B, bottom images) for NSCs (8A, top and bottom) and MSCs (8B, top and bottom) under each condition within a 1-3 hour time frame. As shown in Figures 8A and 8B, labeling at each time point (e.g., 1 hour, 2 hours, and 3 hours) was successful as indicated by the high intensity of PB staining under both serum-starved and serum-containing conditions for both types of cells compared to without the use of the labeling system (left-most panels (top and bottom) of 8A and 8B).

[0160] This study demonstrated that the physicochemical composition of VMI NCE-1 particles prepared by magnetic separation is suitable for highly efficient cell labeling under serum-containing conditions. Furthermore, when used in combination with the VMI magnetic labeling system, VMI NCE-1 particles shorten the labeling time and are suitable for point-of-care use.

[0161] Example 7: Dual Imaging VMI NCE1 Nanoparticles Having established the efficiency of using nanoparticles prepared by magnetic separation for cell labeling, the next step in the study was to further explore the ability to label cells with the dual-probe nanoparticles described in Examples 2 and 3.

[0162] As described in Examples 2 and 3, dual imaging agents for multimodal imaging were prepared by mixing FITC-conjugated heparin with protamine and ferraheme to form dual-probe Std HPF particles. After magnetic separation, dual-probe VMI NCE 1 was generated and subsequently used to label MSCs. Dual-probe VMI NCE 1-labeled cells did not show a statistically significant decrease in cell viability compared to unlabeled cells.

[0163] With a fluorescent probe (i.e., FITC), the labeling efficiency of dual-probe VMI NCE 1 was assessed by fluorescence activity as well as by determining iron loading using Prussian blue staining (Figures 9A-9C). Figures 9A and 9B show the Prussian blue staining and fluorescence activity of dual-probe VMI NCE 1 particles, respectively, and Figure 9C shows the overlaid images of Figures 9A and 9B.

[0164] FIG. 10A shows the cell viability of unlabeled cells, dual-probe Std HPF labeled cells, and dual-probe VMI NCE 1 labeled cells. As shown in FIG. 10A, the viability of cells labeled with dual-probe VMI NCE 1 particles is superior (about 25% increase in viability) compared to cells labeled with dual-probe Std HPF particles. FIG. 10B and 10C show the size distribution of standard dual-probe Std HPF particles and dual-probe VMI NCE 1 particles. As listed in Table 4, HPF particles enriched with iron during magnetic separation have a larger particle size.

[0165] [Table 3]

[0166] Importantly, the data show that dual-probe VMI NCE-1 particles efficiently label cells, as evidenced by Prussian blue staining and fluorescent activity.

[0167] Example 8: MRI activity of VMI NCE1 nanoparticles The objective of the next study was to determine the performance of VMI NCE 1 particles for magnetic resonance imaging (MRI).

[0168] A representative diagram of a magnetic labeling system that facilitates rapid uptake of VMI NCE 1 particles generated and manufactured using the methods disclosed herein, thereby allowing for shorter labeling times (e.g., less than 3 hours), is shown in Figure 11. The VMI magnetic labeling system utilizes changes in magnetic flux density generated by permanent magnets or current-driven temporary magnets (electromagnets) to exert forces on the VMI NCE 1 magnetic particles through interaction, causing deflection of the particles from their original position in suspension, helping to facilitate the uptake of these nanoparticles and helping to efficiently and effectively label cells with the desired iron loading in a shortened time frame of less than 3 hours. Changes in magnetic flux density (in direction and / or magnitude greater than 400 mT) can be exposed to the desired region of cells to selectively label cells in the desired region. Cells should be positioned between 0.00 mm and 5 mm from the surface of the applied magnet. Additionally, the particle collection surface should be within 0.00 mm to 4 mm from the surface of the applied magnet for the magnetic separation process. Figure 11 shows that changes in magnetic flux density with a permanent magnet or a current-driven temporary magnet can be used to drive VMI NCE-1 particle uptake to efficiently and effectively label cells at both the desired ratio of cells and iron loading per cell (see step (i) in Figure 11). Step (ii) in Figure 11 shows particle accumulation in the desired area of ​​labeling induced by the VMI magnetic labeling system and images of labeled cells. Prussian blue staining in the images shows the intensity of labeling when exposed to a concentration of VMI NCE-1 particles appropriate for the cell type of interest while using the VMI magnetic labeling system.

[0169] Next, VMI NCE-1 particles were evaluated for their ability to provide enhanced signals in MRI compared to Std HPF particles. MRI activity was measured by an animal MRI scanner (7T Bruker PharmaScan) and a clinical MRI scanner (3T GE-MR750) using conventional and quantitative MRI sequences. As shown in Figure 12A, the in vitro T 2 and T 2 *MRI signals were determined for (a) unlabeled cells, (b) standard Std HPF labeled cells, (c) VMI NCE 1 labeled cells at original intensity (1x), (d) VMI NCE 1 labeled cells at intensity validated for low-calorie MSCs, (e) dual-probe Std HPF labeled cells, and (f) dual-probe VMI NCE 1 labeled cells. Figure 12B shows representative T of the clinical scanner (phantom at 3T GE-MR750). 2 * Figure 12B shows the weighted images. In Figure 12B, layer 0 was unlabeled stem cells, and layers 1-5 were magnetically labeled cells with concentrations of 1500 cells / ul, 2500 cells / ul, 5000 cells / ul, 10,000 cells / ul, and 20,000 cells / ul. Figure 13 shows the color maps of different MR parameters (e.g., (a) QSM map (b) T 1 Map (c) T 2 Map(d)T 2 * Map) (top image). Correlation fitting between density and MR parameters (bottom image). Finally, Table 1 lists the quantitative MR results of unlabeled and VMI NCE 1 labeled stem cells at various densities.

[0170] [Table 4]

[0171] Having confirmed that the VMI NCE 1 particles had a favorable magnetic profile, the particles were then evaluated to determine whether they could provide in vivo tracking of cell-derived particles. Figure 14 shows TEM images of HPF particle labeling of cell-derived particles, which are vesicles derived from NSCs, MSCs, and amniotic fluid stem cells (AFSCs). As shown in Figure 14, TEM analysis of the unlabeled vesicles shows that the unlabeled vesicles have a hollow, disc-like shape. In contrast, the labeled vesicles appear round and have high electron density. Both unlabeled and labeled vesicles were introduced into a glioblastoma cell line (U251) to determine whether the cells would take up the labeled vesicles. Importantly, and as shown in Figure 14, the cells efficiently took up the labeled vesicles, as evidenced by high iron loading in the cells by Prussian blue staining. (Figure 14, bottom right image).

[0172] VMI NCE 1 particle labeling of cell-derived particles was further investigated by MRI activity in diseased kidneys of mice. * FIG. 15B shows VMI NCE 1-labeled vesicles in a T2-weighted image of the diseased kidney using a 7T Burker Pharmascan, and FIG. 15B shows VMI NCE 1-labeled vesicles in a T2-weighted image of the diseased kidney using a clinical MRI scanner (3T GE-MR750) using a T2-weighted conventional MRI sequence.

[0173] In Figure 15A, one can observe the difference in contrast-enhanced signal intensity, and the increase in signal intensity with increasing density of VMI NCE 1-labeled vesicles. In Figure 15A, (a) and (e) show VMI NCE 1 particles (positive control), (b) and (f) show 2.5x10 vesicles from NSCs and AFSCs, respectively. 10 (c) and (g) show 2.5x10 unlabeled vesicles (negative control) from NSCs and AFSCs, respectively. 10 Shown are labeled vesicles, and finally, (d) and (h) show 1x10 11 Labeled vesicles are shown.

[0174] In Figure 15B, (i) shows phantom images of VMI NCE 1 labeled vesicles (left, tubular), reference (middle, circular) and unlabeled vesicles (right, tubular), and the right images show mouse left kidney (j) before injection of VMI NCE 1 labeled vesicles, (k) 20 min after injection of VMI NCE 1 labeled vesicles, and (l) 3 h after injection of VMI NCE 1 labeled vesicles. White arrows in images labeled (k) and (l) indicate low intensity signals indicating the presence of VMI NCE 1 labeled vesicles in the diseased kidney.

[0175] Finally, VMI NCE 1 particles were evaluated to determine whether nucleic acid cargo could be encapsulated within the particles to safely and more effectively introduce such nucleic acids into cells for therapeutic or reprogramming applications. To evaluate the ability of VMI NCE 1 particles to encapsulate nucleic acids, bright field (BF) and fluorescence (i.e., Hoescht (nuclear) and DY-547-nucleic acid) assays were performed on unlabeled NSCs (Mock), NSCs labeled with control particles carrying nucleic acids that have known toxic effects on cells due to residual H or P components, and NSCs labeled with VMI NCE 1 particles carrying nucleic acids. As shown in Figure 16, the physicochemical composition of VMI NCE 1 particles allows for efficient encapsulation of nucleic acids, providing a more effective and safer method for introducing nucleic acids into stem cells.

[0176] All publications, patents, patent applications, and other references cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of a reference herein should not be construed as an admission that it is prior art to the present invention.

[0177] Specifically intended to be within the scope of the present invention and incorporated herein by reference in their entirety are the following publications: Thu MS, Bryant LH, Coppola T, Jordan EK, Budde MD, Lewis BK, Chaudhry A, Ren J, Varma NR, Arbab AS, Frank JA. Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging. Nat Med. 2012;18(3):463-7. Epub 2012 / 03 / 01. doi:10.1038 / nm.2666. PubMed PMID:22366951; PMCID:PMC3296876.

Claims

1. A nanoparticle comprising heparin, protamine, an iron-based imaging agent, and a contrast agent, wherein the contrast agent is bound to the heparin and / or the protamine.

2. 10. The nanoparticle of claim 1, wherein the nanoparticle has an iron content of about 0.1 femtogram (fg) to about 1 fg.

3. The nanoparticles of claim 1 or 2, wherein the iron-based contrast agent is ferumoxytol.

4. The nanoparticle of any one of claims 1 to 6, wherein the contrast agent is a fluorescent probe, a PET agent, a CT agent, or an MRI agent.

5. The nanoparticles according to any one of claims 1 to 7, wherein the nanoparticles are uniform in size and have an average diameter between 120 nm and 195 nm.

6. A composition comprising nanoparticles comprised of heparin, protamine, and an iron-based contrast agent, the composition being free or substantially free of unbound heparin, protamine, or iron-based contrast agent.

7. 7. The composition of claim 6, wherein the composition is free or substantially free of unbound heparin-protamine complexes, protamine-iron-based contrast agent complexes, or heparin-iron-based contrast agent complexes.

8. The composition of claim 6 or 7, further comprising an excipient.

9. The composition of any one of claims 6 to 8, wherein the composition is lyophilized or frozen.

10. 1. A method for labeling a cell or cell-derived product, comprising contacting the cell or cell-derived product with a composition comprising nanoparticles of heparin, protamine, and an iron-based contrast agent, wherein the composition is free of, or substantially free of, unbound heparin, protamine, or iron-based contrast agent.

11. 11. The method of claim 10, further comprising applying a magnetic field while contacting the cells or cell-derived products with the composition comprising nanoparticles.

12. 11. The method of claim 10, wherein the composition is free or substantially free of unbound heparin-protamine complexes, protamine-iron-based contrast agent complexes, or heparin-iron-based contrast agent complexes.

13. 12. The method of claim 10 or 11, further comprising labeling the cells in serum-containing cell culture while contacting the cells or cell-derived products with the composition.

14. 12. The method of claim 10 or 11, further comprising labeling the cells in serum-starved cell culture while contacting the cells or cell-derived products with the composition.

15. The method of any one of claims 10 to 13, wherein the cells or cell-derived products are labeled for more than 4 hours.

16. The method of any one of claims 12 to 14, wherein the cells or cell-derived products are labeled in less than 4 hours.

17. The method of any one of claims 10 to 15, wherein the cell is a mammalian cell.

18. 17. The method of claim 16, wherein the mammalian cell is a mouse cell, a rat cell, a dog cell, a pig cell, a bovine cell, a horse cell, a primate cell, or a human cell.

19. The method of any one of claims 10 to 17, wherein the cell is a cancer cell, an immune cell, or a blood cell.

20. The method of any one of claims 10 to 17, wherein the cell is a stem cell, an embryonic cell, a fetal cell, or a somatic cell.

21. 20. The method of claim 19, wherein the stem cells are mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, adipose stem cells, neural stem cells, or adult stem cells.

22. The method of claim 10, wherein the cell-derived product is a vesicle.

23. 23. The method of claim 22, further comprising contacting said cell-derived product with a cell to label said cell with said cell-derived product.

24. 24. The method of any one of claims 10 to 23, further comprising tracking the cell or cell-derived product for a period of time after contacting the cell or cell-derived particle.

25. 25. The method of claim 24, wherein the tracking is ex vivo, in vitro, or in vivo tracking.

26. 25. The method of claim 24, wherein the tracking is performed by imaging the cells.

27. 26. The method of claim 25, wherein the imaging is performed by fluorescence microscopy, MRI, FL, in vivo optical imaging, PET, SPECT, and / or ultrasound.

28. 28. The method of claim 26 or 27, wherein the imaging is performed at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or more after the cells have been contacted with the nanoparticles or composition.

29. The method according to any one of claims 10 to 28, wherein the method is for in vitro or in vivo labelling of the cells.

30. The method of any one of claims 10 to 28, wherein the composition is administered to a subject.

31. 31. The method of claim 30, wherein the subject is a mammal.

32. 32. The method of claim 31 , wherein the mammal is a human.

33. The method of any one of claims 30 to 32, wherein the composition is administered intravenously, intraarterially, by direct tissue injection, and / or intratumorally.

34. 34. The method of any one of claims 10 to 33, further comprising encapsulating nucleic acid within the nanoparticles prior to contacting the composition comprising nanoparticles with the cells or cell-derived products.

35. 1. A method for preparing magnetically separated nanoparticles, comprising: (a) combining heparin, protamine, and an iron-based contrast agent, wherein at least a portion of the heparin, protamine, and iron-based contrast agent forms nanoparticles comprising heparin, protamine, and iron-based contrast agent; (b) exposing the nanoparticles to a magnetic field to separate the nanoparticles based on their magnetic moment.

36. 36. The method of claim 35, wherein exposing the solution to the magnetic field further separates unbound protamine, heparin, or iron-based labeling agents that have not complexed to form the nanoparticles.

37. 37. The method of claim 35 or 36, wherein the nanoparticles are further separated based on size.

38. The method of any one of claims 35 to 37, wherein exposing the solution to a magnetic field comprises applying a magnet to the solution.

39. 39. The method of claim 38, wherein the magnet has a magnetic field strength of about 300 mT to about 600 mT.

40. 39. The method of claim 38, wherein the magnetic field is uniform and applied perpendicular to the nanoparticles.

41. 40. The method of claim 38, wherein the magnetic field is non-uniform and directionally varying.

42. 1. A method for labeling cells from cell-derived products, comprising: (a) contacting a first cell with a composition comprising nanoparticles of heparin, protamine, and an iron-based imaging agent, said contacting step labeling the cell-derived product in said cell; (b) removing the labeled cell-derived product from the cell; and (c) contacting a second cell with the cell-derived product, wherein the second cell takes up the labeled cell-derived product.

43. 43. The method of claim 42, further comprising tracking the labeled cell-derived product from the first cell.

44. 43. The method of claim 42, further comprising tracking the second cell.

Images