Large-scale production of exosome mimics and their uses

Abstract

Provided herein are methods for generating exosome mimics that are uniform in size and similar in structure and / or biological function to natural exosomes. Also provided herein is the use of EM as a delivery vehicle for delivering an agent (eg, a therapeutic agent, or a diagnostic agent) for the treatment or diagnosis of disease.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 887,876, filed August 16, 2019, entitled "LARGE SCALE PRODUCTION OF EXOSOME MIMETICS AND USES THEREOF," the entire contents of which are incorporated herein by reference.

[0002] Government support This invention was made with government support under Grant No. CA185530 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Cell-derived extracellular vesicles (EVs), such as exosomes, have been used as natural drug delivery tools for the diagnosis and treatment of various diseases. EVs are advantageous as delivery vehicles due to various benefits, such as their lack of immunogenicity and ability to efficiently target various organs. However, robust and reproducible methods for large-scale production of exosomes are lacking. Traditional methods for isolating exosomes (e.g., ultracentrifugation) are inefficient, time-consuming, and often require expensive equipment. Summary of the Invention

[0004] overview In some aspects, described herein is a novel magnetic extrusion method for producing endosome-derived nanoscale vesicles (herein referred to as "exosome mimics (EM)") from various cultured cell lines in a large-scale and reproducible manner. EMs produced using the methods described herein exhibit similar biological functions as natural exosomes. In some embodiments, therapeutic agents are encapsulated in the engineered EMs with high encapsulation efficiencies (e.g., greater than 95%, greater than 90%, greater than 85%, greater than 80%, greater than 75%, or greater than 70%). The methods described herein can be used for the industrial production of GMP-grade exosome-based drug delivery systems. In some embodiments, EMs produced using the methods described herein can be used for the delivery of agents (e.g., therapeutic or diagnostic agents) for the treatment or diagnosis of a wide variety of diseases.

[0005] Accordingly, some aspects of the present disclosure provide methods for producing exosome mimics, the methods including: (i) incubating cells with magnetic nanoparticles such that the magnetic nanoparticles enter endosomes in the cells; (ii) lysing the cells to produce a cell lysate containing endosomes; (iii) isolating endosomes encapsulating the magnetic nanoparticles from the cell lysate in step (ii); and (iv) extruding the isolated endosomes obtained in step (iii) through a nanoporous membrane to produce exosome mimics. In some embodiments, the cells are selected from stem cells, bone marrow-derived cells, immune cells, erythrocytes, epithelial cells, stem cells, and endothelial cells. In some embodiments, the magnetic nanoparticles are iron oxide nanoparticles. In some embodiments, the nanoparticles enter endosomes within the cell via endocytosis. In some embodiments, the cells are lysed via homogenization. In some embodiments, step (iii) is carried out using a magnetic separator. In some embodiments, the nanoporous membrane has a pore diameter of 100 nm. In some embodiments, the method further comprises: (v) removing unencapsulated magnetic nanoparticles. In some embodiments, step (v) is carried out via size exclusion chromatography. In some embodiments, the method further comprises: (vi) removing the magnetic nanoparticles from the exosome mimic. In some embodiments, the magnetic nanoparticles are conjugated to a targeting moiety, therapeutic agent, or diagnostic agent.

[0006] Another aspect of the present disclosure provides an exosome mimic produced by the methods described herein. In some embodiments, the exosome mimic comprises a magnetic nanoparticle. In some embodiments, the exosome mimic further comprises an agent. In some embodiments, the agent is a therapeutic agent or a diagnostic agent. In some embodiments, the agent is conjugated to a magnetic nanoparticle. In some embodiments, the magnetic nanoparticle is an iron oxide nanoparticle. Further provided are compositions comprising the exosome mimics described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0007] Another aspect of the present disclosure relates to a method of treating a disease, comprising administering to a subject in need thereof an effective amount of an exosome mimetic or a composition described herein. In some embodiments, the disease is cancer, cardiovascular disease, brain disease, immunodeficiency, autoimmune and infectious disease, respiratory disease, or endocrine system disease.

[0008] Another aspect of the present disclosure relates to a method of diagnosing a disease, the method comprising administering to a subject in need thereof an effective amount of an exosome mimetic or a composition described herein, wherein the exosome mimetic comprises a diagnostic agent. In some embodiments, the disease is cancer, cardiovascular disease, brain disease, immune deficiency, autoimmune and infectious disease, respiratory disease, or endocrine system disease.

[0009] Further provided herein is an in vivo imaging method, comprising administering to a subject in need thereof an effective amount of an exosome mimic described herein, and visualizing the exosome mimic in the subject via magnetic resonance imaging (MRI), fluorescence imaging, PET imaging, bioluminescence imaging, and ultrasound imaging. In some embodiments, the exosome mimic is visualized via MRI.

[0010] In some embodiments, the exosome mimetic comprises a diagnostic agent. In some embodiments, the diagnostic agent is a targeting moiety. In some embodiments, the targeting moiety targets a cancer biomarker. In some embodiments, the cancer is breast cancer. In some embodiments, the biomarker is ICAM1 or HER2. [Brief explanation of the drawings]

[0011] Brief description of the drawings The accompanying drawings are not intended to be drawn to scale.

[0012] In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by the same numeral. For purposes of clarity, not every component may be labeled in every figure. In the drawings: Figures 1A-1I show the characterization of IONP-EM derived from MDA-MB-231 cells by magnetic extrusion. [Figure 1A-1B]Figure 1A shows a representative TEM image of IONPs internalized by MDA-MB-231 cells, and Figure 1B shows IONPs encapsulated in endosomes. [Figure 1C-1D] Figure 1C shows endosomes encapsulating IONPs after purification and magnetic separation. Figure 1D shows endosomes encapsulating purified IONPs extruded into engineered IONP-EM. Arrows indicate encapsulated IONPs. [Figure 1E] Figures 1E and 1F show hydrodynamic size and immunoblot of Alix protein expression of IONP-EMs and native exosomes, respectively. [Figure 1F-1G] Figures 1E and 1F show hydrodynamic size and immunoblot of Alix protein expression in IONP-EM and native exosomes, respectively. Figure 1G shows the production of IONP-EM and total EM. [Figure 1H-1I] Figure 1H shows the protein concentration and hydrodynamic size of IONP-EM in five independent replicates. Figure 1I shows the increased cell proliferation of MDA-MB-231 cells with IONP-EM. [Figure 2] Figure 2 shows the doxorubicin encapsulation efficiency of EM. Doxorubicin was loaded into EM from mouse fibroblast 3T3 cells using the direct encapsulation (left) and ammonium sulfate gradient loading (right) methods. [Figure 3] FIG. 3 shows the anticancer activity of Dox-EM in treating human breast cancer MDA-MB-231 cells. [Figure 4] FIG. 4 shows the anticancer activity of Dox-EM in treating human breast cancer MDA-MB-436 cells. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description of Specific Embodiments Large-scale production of extracellular vesicles (e.g., exosomes) is extremely expensive, time-consuming, and labor-intensive. Furthermore, the variability among different preparations of exosomes is high, and the resulting formulations are often not suitable for clinical applications. Furthermore, exosomes produced via extrusion of cell plasma membranes in known methods are similar in size to natural exosomes, but have different compositions and biological functions from natural exosomes.

[0014] The magnetic extrusion method described herein produces endosome-derived nanoscale vesicles (herein referred to as "exosome mimics (EMs)") that are uniform in structure and function and retain the biological functions of natural exosomes. Endosome-derived EMs have a unique composition enriched in endosomal proteins, making them more similar to natural exosomes in composition and biological function. As shown herein, the method is suitable for consistent and large-scale production of EMs that retain the biological properties of exosomes, and these EMs are suitable as delivery tools for agents (e.g., therapeutic or diagnostic agents). Furthermore, agents can be loaded into EMs during production with high encapsulation efficiency.

[0015] Accordingly, some aspects of the present disclosure provide methods for producing exosome mimics, the methods including: (i) incubating cells with magnetic nanoparticles such that the magnetic nanoparticles enter endosomes in the cells; (ii) lysing the cells to produce a cell lysate containing endosomes; (iii) isolating endosomes encapsulating the magnetic nanoparticles from the cell lysate in step (ii); and (iv) extruding the isolated endosomes obtained in step (iii) through a nanoporous membrane to produce exosome mimics.

[0016] "Exosomes" are small cell-derived vesicles of endocytic origin. Exosomes are considered to be vehicles for the removal of unwanted cellular proteins and important drivers of intercellular communication. Exosomes are found in all biological fluids, including blood, milk, urine, sweat, tears, and culture supernatants.

[0017] During exosome biogenesis, early endosomes loaded with ubiquitinated proteins upon recognition by ESCRT (Endosomal Sorting Complex Required for Transport) allow the formation of intraluminal vesicles (ILVs), which in turn become multivesicular bodies (MVBs), some of which are degraded in lysosomes. Fusion of MVBs with the plasma membrane triggers the release of exosomes into the extracellular space.

[0018] Exosomes contain a complex composition of molecules, including proteins, lipids, microRNAs, and mRNAs, which are listed in the EXoCarta database (exocarta.org). The most common exosomal proteins are membrane transporters and fusion proteins (annexins, GTPases, and flotillins), heat shock proteins, tetraspanins (CD9, CD63, and CD81), MVB synthesis proteins (alix and TSG101), lipid-associated proteins, and phospholipases. Proteins such as CD9, CD63, CD81, TSG101, alix, and HSP70 are common to most exosomes. Exosomes are rich in lipids, such as cholesterol, sphingolipids, ceramides, glycolipid GM3, and glycerophospholipids containing long saturated fatty acyl chains.

[0019] Exosomes play a key role in intercellular communication by binding to recipient cells, where they either remain tightly associated with the plasma membrane or are internalized via endocytic pathways and release their contents.

[0020] The biological properties of the target cells can then be altered at the genetic (exosomal RNA), epigenetic (exosomal miRNA), or protein levels. Beneficial (e.g., enhancing immune status) or harmful (e.g., spreading pathology) outcomes can result from these interactions.

[0021] As used herein, "exosome mimics" refer to nanoscale membranous vesicles originating from the endosomal system of cells. The exosome mimics of the present disclosure are similar to natural exosomes in their structure and biological function. For example, the exosome mimics of the present disclosure are vesicles comprising a lipid bilayer. In some embodiments, the exosome mimics possess one or more known biomarkers of natural exosomes (for example, but not limited to, Alix, TSG101, CD9, CD63 and CD81, and HSP70). Cells from which exosome mimics can be generated include, but are not limited to, bone marrow-derived cells, immune cells, erythrocytes, epithelial cells, stem cells, and endothelial cells.

[0022] "Magnetic nanoparticles" refer to nanoparticles that can be manipulated using a magnetic field. Such particles generally consist of two components: a magnetic material, often iron, nickel, or cobalt, and a functionalized chemical component. Magnetic nanoparticles can be iron-based, cobalt-based, nickel-based, or manganese-based (e.g., as described in Kudr et al. (Nanomaterials (Basel). 2017 Sep; 7(9)); incorporated herein by reference). Non-limiting examples of magnetic nanoparticles that may be used in accordance with the present disclosure include: Ferrite nanoparticles (also called iron oxide nanoparticles), ferrite nanoparticles with a shell, metal nanoparticles, and metal nanoparticles with a shell.

[0023] Ferrite nanoparticles, or iron oxide nanoparticles (iron oxide in the crystalline structure of maghemite or magnetite), are the most explored magnetic nanoparticles to date. Once ferrite particles are smaller than 128 nm, they become superparamagnetic, preventing self-agglomeration, as they exhibit magnetic behavior only when an external magnetic field is applied. The magnetic moment of ferrite nanoparticles can be greatly increased by the controlled clustering of many individual superparamagnetic nanoparticles into superparamagnetic nanoparticle clusters, i.e., magnetic nanobeads. Upon switching off the external magnetic field, the remanence recedes to zero. Like nonmagnetic oxide nanoparticles, the surface of ferrite nanoparticles is often modified with surfactants, silica, silicon, or phosphate derivatives to increase their stability in solution.

[0024] The surfaces of maghemite or magnetite magnetic nanoparticles are relatively inert and do not tolerate the strong covalent bonds that typically accompany functionalized molecules. However, the reactivity of magnetic nanoparticles can be improved by coating a silica layer on their surface. The silica shell can be easily modified with various surface functional groups via covalent bonding between organosilane molecules and the silica shell. In addition, some fluorescent dye molecules can be covalently bonded to functionalized silica shells (e.g., ferrite nanoparticles with shells).

[0025] Metal nanoparticles can be made smaller than their oxide counterparts, which may be beneficial for some technological applications. Metal nanoparticles are pyrophoric and reactive to oxidizing agents to varying degrees. The metal cores of magnetic nanoparticles may be passivated by mild oxidation, surfactants, polymers, and precious metals. In an oxygen environment, Co nanoparticles form an antiferromagnetic CoO layer on the surface of the Co nanoparticles (e.g., metal nanoparticles with a shell). Nanoparticles with magnetic cores composed of elemental iron or cobalt and non-reactive shells made of graphene have been synthesized.

[0026] In some embodiments, the magnetic nanoparticles used in the methods described herein are iron oxide nanoparticles (IONPs). "Iron oxide nanoparticles (IONPs)" typically have a diameter between about 1 and 100 nanometers. The two main forms of IONPs are magnetite (Fe3O4) and its oxidized form, maghemite (γ-Fe2O3). Magnetite has an inverse spinel structure, with oxygen forming a face-centered cubic crystal system. In magnetite, all tetrahedral moieties are Fe 3+ and the octahedral part is occupied by Fe 3+ and Fe 2+ Maghemite is a mineral in which all or most of the iron is in the trivalent state (Fe 3+ Maghemite differs from magnetite in that it has a cubic unit cell with cations randomly distributed over 8 tetrahedral and 16 octahedral sites (see, e.g., Laurent et al., Chemical Reviews. 108(6):2064-110, incorporated herein by reference). IONPs (e.g., magnetite and maghemite) are biocompatible and potentially non-toxic to humans. Iron oxides are readily degradable and therefore useful for in vivo applications.

[0027] To produce exosome mimics, cells are incubated with magnetic nanoparticles (e.g., IONPs) for a period of time. The incubation may be under conditions suitable for the maintenance and / or growth of the cells used. Those skilled in the art can determine the incubation conditions, such as temperature, duration, and / or medium. In some embodiments, cells are incubated with magnetic nanoparticles (e.g., IONPs) at 25°C to 37°C (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C). In some embodiments, cells are incubated with magnetic nanoparticles (e.g., IONPs) at 37°C. In some embodiments, the cells are incubated with the magnetic nanoparticles (e.g., IONPs) for 1 to 24 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours). In some embodiments, the cells are incubated with the magnetic nanoparticles (e.g., IONPs) for less than 1 hour. In some embodiments, the cells are incubated with the magnetic nanoparticles (e.g., IONPs) for more than 24 hours.

[0028] In some embodiments, the magnetic nanoparticles enter cells via endocytosis. "Endocytosis" is a cellular process by which substances are brought into cells. The internalized material is surrounded by a region of the cell membrane, which then develops inside the cell to form a vesicle containing the internalized material. Endocytosis is a form of active transport.

[0029] Magnetic nanoparticles (e.g., IONPs) that enter cells via endocytosis are contained in endosomes. "Endosome" refers to a membrane-bound compartment within eukaryotic cells. It is a compartment of the endocytic membrane trafficking pathway originating from the trans-Golgi membrane. Endosomes can be classified into early endosomes, recycling endosomes, and late endosomes. Early endosomes are the first compartment of the endocytic pathway. Early endosomes are often located at the periphery of the cell and receive most types of vesicles coming from the cell surface. Early endosomes have a characteristic tubulovesicular structure and are primarily sorting organelles where many plasma membrane-invaginated ligands dissociate from their receptors at the compartment's acidic pH, and many of the receptors recycle (via tubules) to the cell surface. Early endosomes are also the site of sorting for cellular trafficking pathways to later compartments (such as late endosomes or lysosomes). Recycling endosomes are often considered a subcompartment of early endosomes that recycle cargo internalized at the plasma membrane. Late endosomes typically receive plasma membrane-invaginated material from early endosomes in the endocytic pathway, from the trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway en route to lysosomes. Late endosomes often contain proteins characteristic of nucleosomes, mitochondria, and mRNA, including lysosomal membrane glycoproteins and acid hydrolases. Late endosomes are acidic (approximately pH 5.5) and are part of the mannose-6-phosphate receptor transport pathway. Late endosomes appear to mediate a final set of sorting events prior to delivery of material to lysosomes.

[0030] After the incubation is completed, the cells are lysed. "Lysing" a cell means disrupting the cell's plasma membrane so that the contents of the cell are released. Any suitable method for lysing cells may be used, for example, mechanical disruption, liquid homogenization, high frequency sound, freeze / thaw cycles, sonication, or manual trituration. In some embodiments, the cells are lysed via homogenization.

[0031] After homogenization, endosomes containing magnetic nanoparticles (e.g., IONPs) are separated from the cell lysate. In some embodiments, endosomes containing magnetic nanoparticles (e.g., IONPs) are separated using a magnetic separator. The magnetic separator can exert a magnetic force to extract magnetically sensitive materials (e.g., endosomes containing magnetic nanoparticles) from the cell lysate. After endosomes containing magnetic nanoparticles (e.g., IONPs) have been extracted, the remaining cell lysate may be discarded. In some embodiments, the separated endosomes containing magnetic nanoparticles (e.g., IONPs) are subjected to several steps of washing to remove any impurities (e.g., proteins, nucleic acids, or other materials typically present in cell lysates).

[0032] The separated endosomes are then extruded through a nanoporous membrane to produce EM. A "nanoporous membrane" is a membrane containing an ordered, organic or inorganic framework that supports an ordered, porous structure. In some embodiments, the nanoporous membrane is a track-etched polycarbonate (PCTE) nanoporous membrane. In some embodiments, the pores of the nanoporous membrane are 20 to 400 nm (e.g., 20, 50, 100, 150, 200, 250, 300, 350, or 400 nm). In some embodiments, the pores of the nanoporous membrane are 100 nm. Nanoporous membranes (e.g., track-etched polycarbonate (PCTE) nanoporous membranes) are commercially available, for example, from SterliTech Corporation (WA, USA). In some embodiments, the extrusion step is carried out using a Lipex™ extruder, which is commercially available, for example, from Transferra Nanosciences Inc. (Canada).

[0033] In some embodiments, the methods described herein further include removing unencapsulated magnetic nanoparticles (e.g., IONPs) from the resulting EM after the extrusion step. In some embodiments, unencapsulated magnetic nanoparticles (e.g., IONPs) are removed from the EM via size exclusion chromatography. Size exclusion chromatography (SEC), also known as gel filtration, separates molecules based on differences in size as they pass through an SEC resin packed in a column. During SEC, molecules do not bind to the chromatography resin. SEC resins consist of a porous matrix of spherical particles that lack reactive and adsorbent properties. After a sample is applied, molecules larger than the pores cannot diffuse into the beads, so they elute first. Molecules ranging in size from very large to very small can permeate the pores differently based on their size. If a molecule is smaller than the smallest pore in the resin, it can enter the entire pore volume. Molecules that enter the entire pore volume are eluted last. Unencapsulated magnetic nanoparticles (e.g., IONPs) have a size smaller than the EM, and they can be separated by SEC.

[0034] In some embodiments, the methods described herein further comprise isolating the EM encapsulating the magnetic nanoparticles (e.g., IONPs) from the empty EM. In some embodiments, this step is performed using a magnetic separator.

[0035] In some embodiments, the resulting isolated EM encapsulating the magnetic nanoparticles (e.g., IONPs) may be further processed to remove the magnetic nanoparticles (e.g., IONPs) from the EM.

[0036] In some embodiments, EMs generated using the methods described herein have a diameter of 20-400 nm. For example, EMs generated using the methods described herein have a diameter of 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 20-50, 50-400, 50-350, 50-300, 50-250, 50-200, 50-150, 50-100, 100-400, 100-350, 100-3 EMs may be 20, 50, 100, 150, 100, 250, 100, 200, 100, 150, 150, 150, 400, 150, 350, 150, 300, 150, 250, 150, 200, 200, 250, 250, 400, 250, 350, 250, 300, 300, 350, or 350-400 nm in diameter. In some embodiments, EMs generated using the methods described herein are 20, 50, 100, 150, 200, 250, 300, 350, or 400 nm in diameter. In some embodiments, EMs generated using the methods described herein are 100 nm in diameter.

[0037] In some embodiments, EMs generated using the methods described herein retain the composition (e.g., biomarkers) and / or biological function of naturally occurring exosomes. For example, in some embodiments, EMs generated using the methods described herein contain known exosome markers (e.g., Alix). In some embodiments, EMs generated using the methods described herein contain known exosome markers (e.g., Alix) at levels comparable to those of naturally occurring exosomes (e.g., by less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% difference).

[0038] In some embodiments, the methods described herein can be used to generate EMs with encapsulated agents (e.g., therapeutic or diagnostic agents). For example, magnetic nanoparticles (e.g., IONPs) can be conjugated to agents (e.g., therapeutic or diagnostic agents). A "therapeutic agent" refers to an agent that has a therapeutic effect on a disease or disorder. Therapeutic agents may be, but are not limited to, proteins, peptides, nucleic acids, polysaccharides and carbohydrates, lipids, glycoproteins, small molecules, gene editing agents (e.g., CRISPR / Cas9 systems, ZNFs, or TALENs), or synthetic organic and inorganic drugs. In some embodiments, the therapeutic agent is an anti-inflammatory agent, a vaccine antigen, a vaccine adjuvant, an antibody, an ScFv, a nanobody, an enzyme, an anti-cancer or chemotherapeutic agent, a clotting factor, a hormone, a steroid, a cytokine, an antibiotic, or a drug for the treatment of cardiovascular disease, lung disease, kidney disease, infectious disease, autoimmune disease, immunodeficiency, allergy, blood disease, metabolic disorder, skin disease, eye disease, brain disease, respiratory disease, endocrine system disease, or cancer.

[0039] In some embodiments, the therapeutic agent is a vaccine antigen. A "vaccine antigen" is a molecule or moiety that, when administered to a subject, activates or increases the production of antibodies that specifically bind to the antigen. In some embodiments, the antigen is a protein or polysaccharide. Pathogen antigens are well known to those skilled in the art and include, but are not limited to, parts of bacteria, viruses, and other microorganisms (e.g., outer shells, capsules, cell walls, flagella, pili, and toxins).

[0040] Vaccines typically contain an antigen and are intentionally administered to a subject to induce an immune response in the recipient subject. The antigen may be from a pathogenic virus, bacteria, or fungus.

[0041] Examples of pathogenic viruses include, but are not limited to, Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also referred to as HTLV-III, LAV, or HTLV-III / LAV, or HIV-III; and other isolates such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hantaan viruses, Bunyaviridae, Phleboviruses, and Nairoviruses; Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B viruses); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex viruses (HSV) 1 and 2, varicella-zoster virus, cytoplasmic viruses, and avian viruses); megalovirus (CMV), herpesviruses); Poxviridae (variola virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and non-typeable viruses (e.g., agents responsible for the pathogenesis of spongiform encephalopathy, agents of delta hepatitis (thought to be an incomplete satellite of hepatitis B virus), agents of non-A, non-B hepatitis (class 1 = internally transmitted; class 2 = orally transmitted (i.e., hepatitis C)); Norwalk virus and related viruses, and astroviruses).

[0042] Examples of pathogenic bacteria include, but are not limited to, Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, and Actinomyces israelli.

[0043] Examples of pathogenic fungi include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Other infectious microorganisms (i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.

[0044] In some embodiments, the therapeutic agent is an agent that induces immune tolerance. Immune tolerance is a state of immune insensitivity specific to a particular antigen or set of antigens induced by previous exposure to that antigen or set of antigens. In some embodiments, the immune tolerance is oral tolerance. Oral tolerance is a state of local and systemic immune insensitivity induced by oral administration of a harmless antigen, such as a food protein. In some embodiments, the therapeutic agent is an agent for inducing immune tolerance for the treatment of allergies or autoimmune diseases (e.g., multiple sclerosis).

[0045] Other non-limiting examples of agents that may be conjugated to magnetic nanoparticles (e.g., IONPs) and encapsulated in EMs generated using the methods described herein are provided.

[0046] Non-limiting exemplary chemical pharmaceutical compositions include actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

[0047] Examples of anti-neoplastic compounds include, but are not limited to, nitrosoureas, such as carmustine, lomustine, semustine, and streptozotocin; methylhydrazines, such as procarbazine and dacarbazine; steroid hormones, such as glucocorticoids, estrogens, progestins, androgens, tetrahydrodesoxycalycosterone, cytokines, and growth factors; and asparaginase.

[0048] Examples of immunoactive compounds include, but are not limited to: immunosuppressants, such as pyrimethamine, trimethopterin, penicillamine, cyclosporine, azathioprine; immunostimulants, such as levamisole, diethyldithiocarbamate, enkephalins, endorphins.

[0049] Examples of antibacterial compounds include, but are not limited to: antibiotics, such as beta-lactams, penicillins, cephalosporins, carbapenems and monobactams, beta-lactamase inhibitors, aminoglycosides, macrolides, tetracyclines, spectinomycin; antimalarials, amoebicides, antiprotozoals, antifungals, such as amphotericin beta or clotrimazole; antivirals, such as acyclovir, idoxuridine, ribavirin, trifluridine, vidarabine, ganciclovir. Examples of parasiticides include, but are not limited to: anthelmintics, radiopharmaceuticals, gastrointestinal drugs.

[0050] Examples of hematological compounds include, but are not limited to: immunoglobulins; blood clotting proteins; e.g., antihemophilic factor, Christmas factor complex; anticoagulants, e.g., dicoumarol, heparin sodium; fibrinolysis inhibitors, tranexamic acid.

[0051] Examples of cardiovascular drugs include, but are not limited to, peripheral antiadrenergics, centrally acting antihypertensives, e.g., methyldopa, methyldopa HCl; antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl; drugs affecting the renin-angiotensin system; peripheral vasodilators, phentolamine; antianginal drugs; cardiac glycosides; cardiac vasodilators; e.g., amrinone, milrinone, enoximone, phenoximone, imazodan, sulmazole; antiarrhythmic drugs; calcium entry blockers; drugs affecting blood lipids; ranitidine, bosentan, rezulin.

[0052] Examples of respiratory drugs include, but are not limited to, the following: sympathomimetic drugs: albuterol, bitolterol mesylate, dobutamine HCl, dopamine HCl, ephedrine SO, epinephrine, fenfluramine HCl, isoproterenol HCl, methoxamine HCl, norepinephrine bitartrate, phenylephrine HCl, ritodrine HCl; cholinergic drugs, e.g., acetylcholine Cl; anticholinesterases, e.g., edrophonium Cl; cholinesterase reactivators; adrenergic blocking drugs, e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl, metoprolol, nadolol, phentolamine mesylate, propanolol HCl; antimuscarinic drugs, e.g., anisotropine methylbromide, atropine SO, clinidium Br, glycopyrrolate, ipratropium Br, scopolamine HBr.

[0053] Examples of neuromuscular blocking agents include, but are not limited to: depolarizing agents, such as atracurium besylate, hexafluorenium Br, methocrine iodide, succinylcholine Cl, tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, such as baclofen.

[0054] Examples of neurotransmitters and neurotransmitter agents include, but are not limited to, acetylcholine, adenosine, adenosine triphosphate, amino acid neurotransmitters, such as excitatory amino acids, GABA, and glycine; biogenic amine neurotransmitters, such as dopamine, epinephrine, histamine, norepinephrine, octopamine, serotonin, and tyramine; neuropeptides, nitric oxide, and potassium channel toxins. Examples of anti-Parkinson drugs include, but are not limited to: Amartidine HCl, benztropine methanesulfonate, e.g., carbidopa. Examples of diuretics include, but are not limited to: dichlorphenamide, methazolamide, bendroflumethiazide, polythiazide. Uterine, antimigraine medications include, but are not limited to: carboprost tromethamine mesylate, methysergide maleate.

[0055] Examples of hormones include, but are not limited to, pituitary hormones, such as chorionic gonadotropin, cosyntropin, menotropin, somatotropin, iorticotropin, protirelin, thyrotropin, vasopressin, and lypressin; adrenal hormones, such as beclomethasone dipropionate, betamethasone, dexamethasone, and triamcinolone; pancreatic hormones, such as glucagon and insulin; parathyroid hormones, such as dihydrochysterol; and thyroid hormones. Hormones, such as calcitonin etidronate disodium, levothyroxine sodium, liothyronine sodium, liotrix, thyroglobulin, teriparatide acetate; antithyroid drugs; estrogens; female hormones and antagonists, hormonal contraceptives, testicular hormones; gastrointestinal hormones: cholecystokinin, enteroglycan, galanin, gastric acid inhibitory hormone, epidermal growth factor-urogastron, gastric acid inhibitory hormone, gastrin-releasing peptide, gastrin, pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactive intestinal peptide, sincalide.

[0056] Examples of enzymes include, but are not limited to, lysosomal storage enzymes, hyaluronidase, streptokinase, tissue plasminogen activator, urokinase, PGE-adenosine deaminase, oxidoreductases, transferases, polymerases, hydrolases, lyases, synthases, isomerases and ligases, digestive enzymes (e.g., proteases, lipases, carbohydrases, and nucleases). In some embodiments, the enzyme is selected from the group consisting of lactase, beta-galactosidase, pancreatic enzymes, oil-degrading enzymes, mucinase, cellulase, isomaltase, alginase, digestive lipases (e.g., lingual lipase, steapsin, phospholipase), amylase, cellulase, lysozyme, protease (e.g., pepsin, trypsin, chymotrypsin, carboxypeptidase, elastase), esterase (e.g., sterol esterase), disaccharides (e.g., saccharase, lactase, beta-galactosidase, maltase, isomaltase), DNase, and RNase.

[0057] Examples of intravenous anesthetics include, but are not limited to: droperidol, etomidate, fetanyl citrate / droperidol, hexobarbital, ketamine HCl, methohexital Na, thiamylal Na, thiopental Na.

[0058] Examples of antiepileptic drugs include, but are not limited to, carbamazepine, clonazepam, divalproex sodium, ethosuximide, mephenytoin, paramethadione, phenytoin, and primidone.

[0059] Examples of peptides and proteins that may be used as therapeutic agents include, but are not limited to, ankyrin, arrestin, bacterial membrane proteins, clathrin, connexin, dystrophin, endothelin receptors, spectrin, selectins, cytokines; chemokines; growth factors, insulin, erythropoietin (EPO), tumor necrosis factor (TNF), neuropeptides, neuropeptide Y, neurotensin, transforming growth factor alpha, transforming growth factor beta, interferons. (IFN) and hormones, growth inhibitors, such as genistein, steroids, etc.; glycoproteins, such as ABC transporters, platelet glycoproteins, GPIb-IX complex, GPIIb-IIIa complex, vitronectin, thrombomodulin, CD4, CD55, CD58, CD59, CD44, lymphocyte function-associated antigen, intercellular adhesion molecule, vascular cell adhesion molecule, Thy-1, antiporters, CA-15-3 antigen, fibronectin, laminin, myelin-associated glycoprotein, GAP, GAP-43, exendin-4, and GLP-1.

[0060] Examples of cytokines and cytokine receptors include, but are not limited to, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor , IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-18 receptor, lymphokine inhibitory factor, macrophage colony-stimulating factor, platelet-derived growth factor, stem cell factor, tumor growth factor beta, tumor necrosis factor, lymphotoxin, Fas, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-alpha, interferon-beta, interferon-gamma.

[0061] Examples of growth factors and protein hormones include, but are not limited to, erythropoietin, angiogenin, hepatocyte growth factor, fibroblast growth factor, keratinocyte growth factor, nerve growth factor, tumor growth factor-alpha, thrombopoietin, thyroid-stimulating factor, thyroid-releasing hormone, neurotrophins, epidermal growth factor, VEGF, ciliary neurotrophic factor, LDL, somatomedin, insulin growth factor, insulin-like growth factor I and II.

[0062] Examples of chemokines include, but are not limited to, ENA-78, ELC, GRO-alpha, GRO-beta, GRO-gamma, HRG, LIF, IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP-1alpha, MIP-1beta, MIG, MDC, NT-3, NT-4, SCF, LIF, leptin, RANTES, lymphotactin, eotaxin-1, eotaxin-2, TARC, TECK, WAP-1, WAP-2, GCP-1, GCP-2; alpha-chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7; beta-chemokine receptors: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7.

[0063] In some embodiments, antibodies that may be used as therapeutic agents in accordance with the present disclosure include, but are not limited to: (a) anticluster of differentiation antigens CD-1 through CD-166 and ligands or counter-receptors for these molecules; (b) anti-cytokine antibodies, e.g., anti-IL-1 through anti-IL-18 and receptors for these molecules; (c) anti-immune receptor antibodies, antibodies against T cell receptors, major histocompatibility complexes I and II, B cell receptors, selectins, killer inhibitory receptors, killer activating receptors, OX-40, MadCA M-1, Gly-CAM1, integrins, cadherins, sialoadhesins, Fas, CTLA-4, Fc gamma receptors, Fc alpha receptors, Fc epsilon receptors, Fc mu receptors, and their ligands; (d) anti-metalloproteinase antibodies, such as collagenases, MMP-1 to MMP-8, TIMP-1, TIMP-2; anti-cytolytic / pro-inflammatory molecules, such as perforin, complement components, prostanoids, nitrone oxide, thromboxane; and (e) anti-adhesion molecules, such as carcinoembryonic antigen, lamin, fibronectin.

[0064] Other non-limiting exemplary antibodies and fragments thereof include: bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), alemtuzumab (CAMPATH®, indicated for B-cell chronic lymphocytic leukemia), gemtuzumab (MYLOTARG®, hP67.6, anti-CD33, indicated for leukemias such as acute myeloid leukemia), rituximab (RITUXAN®), tositumomab (BEXXAR®, anti-CD20, indicated for B-cell malignancies), MDX-210 (anti-HER-2 / neu oncogene protein product and type I immunoglobulin G (IgG) (Fc gamma RI)). bispecific antibody that simultaneously binds to Fc receptors), oregovomab (OVAREX®, indicated for ovarian cancer), edrecolomab (PANOREX®), daclizumab (ZENAPAX®), palivizumab (SYNAGIS®, indicated for respiratory symptoms such as RSV infection), ibritumomab tiuxetan (ZEVALIN®, indicated for non-Hodgkin's lymphoma), cetuximab (ERBITUX®), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD1), IOR EGF / R3, cerogovab (ONCOSCINT® OV103), epratuzumab (LYMPHOCIDE®), pemtumomab (THERAGYN®), and gliomab-H (indicated for brain cancer (melanoma)). Other antibodies and antibody fragments are contemplated and may be used in accordance with the disclosure.

[0065] In some embodiments, the therapeutic agent is a nanobody. "Nanobodies" are therapeutic proteins based on single-domain antibody fragments that contain the unique structural and functional properties of naturally occurring heavy-chain-only antibodies.

[0066] In some embodiments, the therapeutic agent is a ligand for a cellular receptor (for example, but not limited to, a growth factor receptor, a G protein-linked receptor, or a toll-like receptor).

[0067] In some embodiments, the regulatory protein that can be used as a therapeutic agent described herein may be a transcription factor or an immunomodulatory protein. Non-limiting exemplary transcription factors include: NFκB family members such as Rel-A, c-Rel, Rel-B, p50 and p52; AP-1 family members such as Fos, FosB, Fra-1, Fra-2, Jun, JunB and JunD; ATF; CREB; STAT-1, -2, -3, -4, -5 and -6; NFAT-1, -2 and -4; MAF; thyroid factor; IRF; Oct-1 and -2; NF-Y; Egr-1; and USF-43, EGR1, Sp1 and E2F1.

[0068] Examples of antiviral agents include, but are not limited to, reverse transcriptase inhibitors and nucleoside analogues, such as ddI, ddC, 3TC, ddA, AZT; protease inhibitors, such as Invirase, ABT-538; inhibitors of RNA processing, such as ribavirin. Other non-limiting examples of known therapeutics that may be delivered by coupling to the magnetic nanoparticles (e.g., IONPs) described herein include:

[0069] (a) Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil, Duricef / Ultracef, Azactam, Videx, Zerit, Maxime, VePesid, Paraplatin, Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS, Estrace, Glucophage (Bristol-Myers Squibb);

[0070] (b)Ceclor、Lorabid、Dynabac、Prozac、Darvon、Permax、Zyprexa、Humalog、Axid、Gemzar、Evista(Eli Lily));

[0071] (C)Vasotec / Vaseretic、Mevacor、Zocor、Prinivil / Prinizide、Plendil、Cozaar / Hyzaar、Pepcid、Prilosec、Primaxin、Noroxin、Recombivax HB、Varivax、Timoptic / XE、Trusopt、Proscar、Fosamax、Sinemet、Crixivan、Propecia, Vioxx, Singulair, Maxalt, Ivermectin(Merck & Co.);

[0072] (d)Diflucan、Unasyn、Sulperazon、Zithromax、Trovan、Procardia XL、Cardura、Norvasc、Dofetilide、Feldene、Zoloft、Zeldox、Glucotrol XL、Zyrtec、Eletriptan、Viagra、Droloxifene、Aricept、Lipitor(Pfizer);

[0073] (e)Vantin、Rescriptor、Vistide、Genotropin、Micronase / Glyn. / Glyb.、Fragmin、Total Medrol、Xanax / alprazolam、Sermion、Halcion / triazolam、Freedox、Dostinex、Edronax、Mirapex、Pharmorubicin、Adriamycin、Camptosar、Remisar、Depo-Provera、Caverject、Detrusitol、Estring、Healon、Xalatan、Rogaine(Pharmacia & Upjohn);

[0074] (f) Lopid, Accrupil, Dilantin, Cognex, Neurontin, Loestrin, Dilzem, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef, FemHRT, Suramin, Clinafloxacin (Warner Lambert).

[0075] Non-limiting examples of therapeutic agents for ocular diseases include: anti-infectives (e.g., acyclovir, chloramphenicol, ciprofloxacin, gentamicin, neomycin, polymyxin B); anti-inflammatory drugs (e.g., betamethasone, dexamethasone, emedastine, nedocromil sodium, prednisolone, sodium cromoglycate); artificial tears (e.g., carmellose, hydroxyethylcellulose, hypromellose, polyvinyl alcohol); and mydriatics (e.g., atropine, cyclopentolate, phenylephrine).

[0076] Further non-limiting examples of therapeutic agents may be found in Goodman and Gilman's The Pharmacological Basis of Therapeutics. 9th ed. McGraw-Hill 1996, incorporated herein by reference.

[0077] "Diagnostic agent" refers to an agent used for diagnostic purposes, e.g., by detecting another molecule in a cell or tissue. In some embodiments, a diagnostic agent is an agent that targets (e.g., binds to) a known biomarker (e.g., a nucleic acid biomarker, a protein biomarker, or a metabolite biomarker) to be associated with a disease in a subject and generates a detectable signal, which can be used to determine the presence / absence of the biomarker and thus diagnose the disease. For example, a diagnostic agent may be, but is not limited to, an antibody or an antisense nucleic acid.

[0078] In some embodiments, the diagnostic agent contains a detectable molecule. A detectable molecule refers to a moiety, such as a protein or polypeptide, or other entity, that has an incorporated structure or functional group that allows for the detection of at least one element, isotope, or molecule, to which the diagnostic agent is attached. In some embodiments, the detectable molecule falls into one (or more) of five classes: a) agents containing an isotopic moiety, which may be radioactive or a stable isotope, including, but not limited to, H, H, C, C, N, F, P, P, S, Ga, Br, Tc (Tc-99m), In, I, I, I, Gd, Yb, and Re; b) agents containing an immunological moiety. , they may be antibodies or antigens, and they may be conjugated to an enzyme (e.g., horseradish peroxidase, etc.); c) agents containing a colored, luminescent, phosphorescent, or fluorescent moiety (e.g., fluorescently labeled fluorescein isothiocyanate (FITC), etc.); d) agents having one or more photoaffinity moieties; and e) agents that are ligands for one or more known binding partners (e.g., biotin-streptavidin, His-NiTNAFK506-FKBP). In some embodiments, the detectable molecule comprises a radioisotope. In some embodiments, the detection agent comprises a fluorescent moiety. In some embodiments, the detectable molecule comprises a dye, e.g., a fluorescent dye, e.g., fluorescein isothiocyanate, Texas Red, rhodamine, Cy3, Cy5, Cy5.5, Alexa 647, and derivatives. In some embodiments, the detectable molecule comprises biotin. In some embodiments, the detectable molecule is a fluorescent polypeptide (e.g., GFP or a derivative thereof, such as enhanced GFP (EGFP) or luciferase (e.g., firefly, Renilla, or Gaussia luciferase)). In some embodiments, the detectable molecule may react with a suitable substrate (e.g., luciferin) to generate a detectable signal.Non-limiting examples of fluorescent proteins include proteins containing chromophores that emit light of various colors, such as GFPs and their derivatives, red, yellow, and cyan fluorescent proteins, etc. Exemplary fluorescent proteins include, by way of example, Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, and mKeima. For a discussion of GFP and myriad other fluorescent or luminescent proteins, see, e.g., Chalfie, M. and Kain, S.R. (eds.) Green fluorescent protein: properties, applications, and protocols (Methods of biochemical analysis, v. 47, Wiley-Interscience, and Hoboken, NJ, 2006, and / or Chudakov, D.M., et al., Physiol Rev. 90(3):1103-63, 2010, which are incorporated herein by reference. In some embodiments, the detectable molecule comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.

[0079] In some embodiments, therapeutic and or diagnostic agents that can be conjugated to magnetic nanoparticles (e.g., IONPs) and encapsulated in EMs generated using the methods described herein are for treating or diagnosing brain diseases (e.g., but not limited to, brain cancer, neurological disorders, psychological disorders, cerebrovascular disorders (e.g., cerebrovascular incidents, vascular malformations and abnormalities, Moyamoya disease, venous angiomas), brain trauma, and brain infections).

[0080] In some embodiments, the therapeutic agent is for treating brain cancer (e.g., primary brain cancer and / or metastatic brain cancer). "Primary brain cancer" refers to cancer that begins in the brain. "Metastatic brain cancer" refers to cancer that begins elsewhere (e.g., breast cancer, prostate cancer, lung cancer, colorectal cancer, skin cancer). In some embodiments, the therapeutic agent for treating brain cancer is a chemotherapeutic agent. "Chemotherapeutic agent" refers to a chemical agent or drug that is selectively harmful to malignant cells and tissues. Non-limiting exemplary chemical pharmaceutical compositions that may be used in accordance with the present disclosure include neratinib or lapatinib, actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epinephrine, erythropoietin, erythrocytopenic acid, erythropoietin ... These include rubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

[0081] In some embodiments, the therapeutic agent for treating brain cancer is an immunotherapeutic agent. "Immunotherapeutic agent" refers to an agent that modulates (for example, suppresses or activates) immune response to treat disease.Immunotherapeutic agents are known to those skilled in the art, and examples include those listed at www.ncbi.nlm.nih.gov / medgen / 2637.

[0082] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. "Immune checkpoint" refers to a protein in the immune system that enhances or reduces immune response signals (costimulatory molecules). Many cancers protect themselves from the immune system by utilizing inhibitory immune checkpoint proteins to inhibit T cell signals. Exemplary inhibitory checkpoint proteins include, but are not limited to, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death 1 receptor (Pd-1), T-cell immunoglobulin and mucin domain 3 (TIM3), lymphocyte-activation gene-3 (LAG3), V-set domain-containing inhibitor of T-cell activation 1 (VTVN1 or B7-H4), cluster of differentiation 276 (CD276 or B7-H3), B- and T-lymphocyte attenuator (BTLA), galectin-9 (GAL9), checkpoint kinase 1 (Chk1), adenosine A2A receptor (A2aR), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte-activation gene-3 (LAG3), and V-domain Ig suppressor of T-cell activation (VISTA).

[0083] Some of these immune checkpoint proteins require their cognate binding partners or ligands for their immunoinhibitory activity. For example, A2AR is a receptor for adenosine A2A, and the binding of A2A to A2AR activates a negative immune feedback loop. As another example, Pd-1 downregulates the immune system by binding to its two ligands, Pd-L1 and Pd-L2, and preventing T cell activation. Pd-1 promotes the programmed cell death of antigen-specific T cells in lymph nodes and simultaneously reduces the programmed cell death of suppressor T cells, thus achieving its immunoinhibitory function. As yet another example, CTLA4 is present on the surface of T cells, and when bound to its binding partners CD80 or CD86 on the surface of antigen-presenting cells (APCs), it transmits an inhibitory signal to T cells, thereby reducing the immune response.

[0084] An "immune checkpoint inhibitor" is a molecule that prevents or attenuates the activity of an immune checkpoint protein; for example, an immune checkpoint inhibitor may inhibit binding of an immune checkpoint protein to its cognate binding partner, e.g., Pd-1, CTLA-4, or A2aR. In some embodiments, the immune checkpoint inhibitor is a small molecule. In some embodiments, the immune checkpoint inhibitor is a nucleic acid aptamer (e.g., an siRNA targeting any one of the immune checkpoint proteins). In some embodiments, the immune checkpoint inhibitor is a recombinant protein. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the antibody comprises an anti-CTLA-4, anti-Pd-1, anti-Pd-L1, anti-TIM3, anti-LAG3, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-GAL9, anti-Chk, anti-A2aR, anti-IDO, anti-KIR, anti-LAG3, anti-VISTA antibody, or a combination of any two or more of the foregoing antibodies. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody. In some embodiments, the immune checkpoint inhibitor comprises anti-PD1, anti-Pd-L1, anti-CTLA-4, or a combination of any two or more of the foregoing antibodies. For example, the anti-Pd-1 antibody is pembrolizumab (Keytruda®) or nivolumab (Opdivo®), and the anti-CTLA-4 antibody is ipilimumab (Yervoy®). Thus, in some embodiments, the immune checkpoint inhibitor comprises pembrolizumab, nivolumab, ipilimumab, or any combination of two or more of the foregoing antibodies. The examples described herein are not meant to be limiting, and any immune checkpoint inhibitor known in the art, and any combination thereof, may be used in accordance with the present disclosure.

[0085] In some embodiments, the therapeutic agent for treating brain cancer is an oligonucleotide (e.g., an siRNA, shRNA, or miRNA targeting an oncogene). An "oncogene" is a gene that can, under certain circumstances, transform a cell into a tumor cell. An oncogene may be a gene encoding a growth factor or mitogen (e.g., c-Sis), a receptor tyrosine kinase (e.g., EGFR, PDGFR, VEGFR, or HER2 / neu), a cytoplasmic tyrosine kinase (e.g., Src family kinase, Syk-ZAP-70 family kinase, or BTK family kinase), a cytoplasmic serine / threonine kinase or its regulatory subunit (e.g., Raf kinase or cyclin-dependent kinase), a regulatory GTPase (e.g., Ras), or a transcription factor (e.g., Myc). In some embodiments, the oligonucleotide targets lipocalin (Lcn2) (e.g., Lcn2 siRNA). Those skilled in the art are familiar with genes that may be targeted for the treatment of cancer.

[0086] In some embodiments, the therapeutic agent is a gene editing agent. A "gene editing agent" refers to an agent that can insert, delete, or replace a nucleotide(s) in the genome of a biological system. In some embodiments, the genome editing agent is an engineered nuclease that can generate a site-specific double-strand break (DSB) at a desired location in the genome. The induced double-strand break is repaired through non-equivalent end joining (NHEJ) or homologous recombination (HR), resulting in a targeted mutation ("editing"). Therefore, engineered nucleases suitable for genome editing can be programmed to target any desired sequence in the genome and are also referred to herein as "programmable nucleases." Suitable programmable nucleases for genome editing that may be used in accordance with the present disclosure include, but are not limited to, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and CRISPR / Cas systems. Those skilled in the art are familiar with programmable nuclease and the method that they are used for genome editing.For example, the method that uses ZFN and TALEN for genome editing is described in Maeder et al., Mol.Cell 31 (2): 294-301, 2008;Carroll et al., Genetics Society of America, 188 (4): 773-782, 2011;Miller et al., Nature Biotechnology 25 (7): 778-785, 2007;Christian et al., Genetics 186 (2): 757-61, 2008;Li et al., Nucleic Acids Res 39 (1): 359-372, 2010;And Moscou et al., Science 326 (5959): 1501, 2009, and are incorporated herein by reference.

[0087] In some embodiments, the genome editing agent is a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system (e.g., Cas9 and guide RNA). "CRISPR / Cas system" refers to the adaptive immune system of prokaryotes that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).

[0088] In type II CRISPR systems, correct processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein.

[0089] The tracrRNA serves as a guide for RNase 3-assisted processing of the pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand not complementary to the crRNA is first endonucleolytically cleaved, and then exonucleolytically cleaved 3'-5'. In nature, DNA binding and cleavage typically require a protein and both RNAs. However, single guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek et al., Science 337:816-821 (2012), incorporated herein by reference.

[0090] The anti-cancer agent for treating brain cancer used in accordance with the present disclosure can be any anti-cancer agent known to one of skill in the art, such as those listed at www.cancer.gov / about-cancer / treatment / drugs.

[0091] In some embodiments, the therapeutic agent is for treating a neurological disorder. "Neurological disorder" refers to any disorder of the nervous system (e.g., the central nervous system or the peripheral nervous system). Structural, biochemical, or electrical abnormalities in the brain, spinal cord, or other nerves can result in a variety of symptoms. Examples of symptoms include paralysis, muscle weakness, poor coordination, sensory loss, seizures, confusion, pain, and altered levels of consciousness. And there are many recognized neurological disorders, including, but not limited to, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, amyotrophic lateral sclerosis (ALS), prion diseases, and motor neuron diseases), neurobehavioral diseases, and developmental disorders.

[0092] Those skilled in the art are familiar with therapeutic agents for treating neurological disorders.For example, therapeutic agents for treating neurological disorders that may be used according to the present disclosure include, but are not limited to, dopaminergic agents (e.g., dopamine receptor agonists), cholinesterase inhibitors, antipsychotics, anti-inflammatory agents, and brain stimulants.Any known agent for treating neurological disorders can be used according to the present disclosure.

[0093] In some embodiments, the therapeutic agent is for treating a psychogenic disorder. "Psychogenic disorder" is also referred to as a mental disorder or a psychiatric disorder. A psychogenic disorder is a behavioral or mental pattern that causes significant distress or impairment of personal functioning. Such features may be persistent, relapsing and remitting, or occur as a single episode. Many disorders have been described, with signs and symptoms that vary widely among specific disorders. Non-limiting examples of psychogenic disorders include post-traumatic stress disorder (PTSD), depressive disorder, major depressive disorder, postpartum depression, bipolar disorder, acute stress disorder, generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, schizophrenia, and trichotillomania.

[0094] Those skilled in the art are familiar with therapeutic agents (e.g., psychiatric medications) for treating psychogenic disorders. Non-limiting examples of psychiatric medications include antidepressants, antipsychotics, mood stabilizers, brain stimulants, and anti-anxiety agents. In some embodiments, the therapeutic agent is for treating brain trauma (also called "traumatic brain injury"). "Brain trauma" refers to a form of acquired brain damage that occurs when a sudden trauma causes damage to the brain. Symptoms of brain trauma can be mild, moderate, or severe, depending on the extent of the damage to the brain. Subjects with mild brain trauma may remain conscious or experience loss of consciousness for a few seconds or minutes. Other symptoms of mild brain trauma include headache, confusion, dizziness, vertigo, blurred or tired vision, tinnitus, an unpleasant taste in the mouth, fatigue or lethargy, changes in sleep patterns, changes in behavior or mood, and trouble with memory, concentration, attention, or thinking. Subjects with moderate or severe brain trauma may exhibit these same symptoms, but may also have worsening or persistent headaches, repeated vomiting or nausea, convulsions or seizures, difficulty waking from sleep, dilated pupils of one or both eyes, slurred speech, weakness or numbness in the limbs, loss of coordination, increasing confusion, restlessness, or agitation.

[0095] Those skilled in the art are familiar with therapeutic agents for treating brain trauma. Non-limiting examples of therapeutic agents for treating brain trauma include anti-inflammatory agents, corticosteroids, and blood clotting agents.

[0096] Non-limiting examples of dopaminergic agents include apomorphine, bromocriptine, cabergoline, dihydrexidine (LS-186,899), dopamine, phenordopam, piribedil, lisuride, pergolide, pramipexole, ropinirole, and rotigotine.

[0097] Cholinesterase inhibitors (also called "acetylcholinesterase inhibitors") are agents that block the breakdown of acetylcholine in the body. Cholinesterase inhibitors have been used to treat neurological disorders (e.g., Alzheimer's disease and dementia). Non-limiting examples of cholinesterase inhibitors include organophosphates (e.g., echothiophate, diisopropylfluorophosphate, cadusafos, chlorpyrifos, cyclosarin, dichlorvos, dimethoate, metrifonate, sarin, soman, tabun, diazinon, malathion, parathion, carbamates), carbamates (e.g., aldicarb, bendiocarb, bufencarb, carbaryl, carbendazim, carbetamide, carbofuran, carbosulfan, chlorbufam, chlorpropham, ethiofencarb, formetanate, methiocarb, methomyl, oxazolidinone, thiazolin ... Mil, phenmedipham, pirimicarb, pirimicarb, propamocarb, propham, propoxur), onquidar, coumarin, physostigmine, neostigmine, pyridostigmine, ambenonium, demecarium, rivastigmine, phenanthrene derivatives, galantamine, caffeine, rosmarinic acid, alpha-pinene, piperidine, donepezil, tetrahydroaminoacridine (THA), edrophonium, huperzine a, ladostigil, ungelemine, lactucopicrin, acotiamide, hybrid / bitopic ligands, dyflos, echothiophate, and parathion. Cholinesterase inhibitors in clinical use include, but are not limited to: Cognex, Namzaric (Pro), Razadyne ER, Aricept ODT (Pro), Reminyl, Exelon (Pro), Aricept (Pro), and Razadyne (Pro).

[0098] Any known antipsychotic drug may be used in accordance with the present disclosure. Non-limiting examples of antipsychotics include aripiprazole (Abilify), asenapine (Saphris), cariprazine (Vraylar), clozapine (Clozaril), lurasidone (Latuda), olanzapine (Zyprexa), quetiapine (Seroquel), risperidone (Risperdal), and ziprasidone (Geodon), fluoxetine, citalopram, sertraline, paroxetine, escitalopram, clonazepam, alprazolam, lorazepam, methylphenidate, amphetamine, dextroamphetamine, lisdexamfetamine dimesylate; typical antipsychotics include: chlorpromazine, haloperidol, perphenazine, fluphenazine, aripiprazole, paliperidone, lurasidone, carbamazepine, lamotrigine, and oxcarbazepine.

[0099] Anti-inflammatory agents are substances that reduce inflammation (redness, swelling, and pain) in the body. Any known anti-inflammatory agent may be used in accordance with the present disclosure, for example, the anti-inflammatory agents described in Maroon et al., Surg Neurol Int. 2010; 1: 80; and Dinarello et al., Cell 140, 935-950, March 19, 2010, which are incorporated herein by reference.

[0100] Any known brain stimulant may be used according to the present disclosure.Cerebral stimulants may be divided into three categories: short-acting, medium-acting, and long-acting.Non-limiting examples of short-acting brain stimulants include: amphetamine / dextroamphetamine (Adderall), dextroamphetamine (Dexedrine, ProCentra, Zenzedi), dexmethylphenidate (Focalin) and methylphenidate (Ritalin).Non-limiting examples of medium-acting brain stimulants include: amphetamine sulfate (Evekeo) and methylphenidate (Ritalin SR, Metadate ER, Methylin ER). Non-limiting examples of long-acting brain stimulants include: amphetamine (Adzenys XR-ODT, Dyanavel XR), dexmethylphenidate (Focalin XR), dextroamphetamine (Adderall XR), lisdexamfetamine (Vyvanse), methylphenidate (Concerta, Daytrana, Jornay PM, Metadate CD, Quillivant XR, Quillichew ER, RitalinLa), and mixed salts of simple amphetamine products (Mydayis).

[0101] Any known antidepressant may be used in accordance with the present disclosure. Non-limiting examples of antidepressants include citalopram (Celexa), escitalopram (Lexapro), fluoxetine (Prozac, Sarafem, Selfemra, Prozac Weekly), fluvoxamine (Luvox), paroxetine (Paxil, Paxil). CR, Pexeva), sertraline (Zoloft), vortioxetine (Trintellix, formerly known as Brintellix), vilazodone (Viibryd), duloxetine (Cymbalta), venlafaxine (Effexor), desvenlafaxine (Pristiq, Khedezla), levomilnacipran (Fetzima), amitriptyline (Elavil and Endep are discontinued brands in the US), amoxapine, clomipramine (Anafranil), desipramine (Norpramin), doxepin (Sinequ and Adapin are discontinued brands in the US), imipramine (Tofranil), nortriptyline (Pamelor; Aventyl is a discontinued brand in the US), protriptyline (Vivactil), trimipramine (Surmontil), mirtazapine (Remeron), bupropion (Wellbutrin), trazodone (Desyrel), trazodone extended-release tablets (Oleptro), vortioxetine (Trintellix, formerly known as Brintellix), and vilazodone (Viibryd).

[0102] Mood stabilizers are psychiatric medications used to treat mood disorders characterized by intense and sustained dysthymia (e.g., as typically seen in patients with bipolar disorder type I or type II, borderline personality disorder (BPD), and schizoaffective disorder). Any known mood stabilizer may be used in accordance with the present disclosure. Non-limiting examples of mood stabilizers include: lithium (lithium carbonate or lithium citrate), divalproex (valproic acid or valproic acid), carbamazepine, oxcarbazepine (Trileptal), and lamotrigine.

[0103] Any known anti-anxiety medication may be used in accordance with the present disclosure. Non-limiting examples of anti-anxiety agents include: benzodiazepines, citalopram (Celexa), escitalopram (Lexapro), fluoxetine (Prozac), fluvoxamine (Luvox), paroxetine (Paxil, Pexeva), sertraline (Zoloft), duloxetine (Cymbalta), venlafaxine (Effexor XR), amitriptyline (Elavil), imipramine (Tofranil), nortriptyline (Pamelor), isocarboxazid (Marplan), phenelzine (Nardil), selegiline (Emsam), and tranylcypromine (Parnate). Exemplary benzodiazepines include, but are not limited to, alprazolam (Xanax), clonazepam (Klonopin), chlordiazepoxide (Librium), diazepam (Valium), and lorazepam (Ativan).

[0104] Any known corticosteroid may be used in accordance with the present disclosure. Non-limiting examples of corticosteroids include: bethamethasone (Celestone), prednisone (Prednisone Intensol), prednisolone (Orapred, Prelone), triamcinolone (Aristospan Intra-Articular, Aristospan Intralesional, Kenalog), methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), dexamethasone (Dexamethasone Intensol, DexPak 10 Day, DexPak 13Day, DexPak 6Day), hydrocortisone (Cortef), cortisone, ethamethasone (ethamethasoneb, Celestone), methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), and fludrocortisone (Florinef).

[0105] Any known blood clotting agent may be used in accordance with the present disclosure. Non-limiting examples of blood clotting agents include hemostatic agents, zeolite, desmopressin, clotting factor concentrates, prothrombin complex concentrates, cryoprecipitate and fresh frozen plasma, recombinant activated human factor VII, tranexamic acid, and aminocaproic acid.

[0106] In some embodiments, the therapeutic agent is for treating a brain infection. A "brain infection" can be caused by a virus, bacteria, fungus, protozoan, or parasite. Another group of brain disorders (called spongiform encephalopathies) is caused by abnormal proteins called prions. Brain infections often involve other parts of the central nervous system, including the spinal cord. In some instances, the infection can cause inflammation of the brain (encephalitis). Viruses are the most common cause of encephalitis. Infections can also cause inflammation of the layers of tissue (meninges) that cover the brain and spinal cord, called meningitis. Often, bacterial meningitis spreads to the brain itself and causes encephalitis. Similarly, viral infections that cause encephalitis often also cause meningitis. Technically, when the brain and meninges are infected, the disorder is called meningoencephalitis.

[0107] However, infections that primarily affect the meninges are usually called meningitis, and infections that primarily affect the brain are usually called encephalitis. In encephalitis and meningitis, the infection is usually not limited to one area. It may occur throughout the brain, or within the meninges along the entire length of the spinal cord and throughout the entire brain.

[0108] In some embodiments, the therapeutic agent for treating the brain infection is selected from known anti-infective agents (e.g., antibiotics for treating bacterial infections, antiviral agents for treating viral infections, or antifungal agents for treating fungal infections, or antiparasitic agents for treating parasitic infections). In some embodiments, the brain infection is a prion disease, and the therapeutic agent for treating the prion disease is an anti-prion antibody.

[0109] Any known antibacterial compound may be used in accordance with the present disclosure. Non-limiting examples of antibacterial compounds include, but are not limited to, antibiotics (e.g., beta-lactams, penicillins, cephalosporins, carbapenems and monobactams, beta-lactamase inhibitors, aminoglycosides, macrolides, tetracyclines, spectinomycin), antimalarials, antiamoebics, antiprotozoal agents, antifungal agents (e.g., amphotericin beta or clotrimazole), and antivirals (e.g., acyclovir, idoxuridine, ribavirin, trifluridine, vidarubin, ganciclovir). Examples of parasiticides include, but are not limited to, anthelmintics, radiopharmaceuticals, and gastrointestinal drugs.

[0110] In some embodiments, the magnetic nanoparticles (e.g., IONPs) are conjugated to a targeting moiety. A "targeting moiety" refers to a molecule that can target a specific cell (e.g., a cancer cell) or tissue (e.g., a muscle) with a magnetic nanoparticle (e.g., an IONP) and / or an EM encapsulating the magnetic nanoparticle (e.g., an IONP). In some embodiments, the targeting moiety is a molecule that specifically binds to a target in a specific cell (e.g., a cancer cell) or tissue (e.g., a muscle). For example, the targeting moiety may be an antibody that targets a cancer-specific antigen or a ligand for a cell surface receptor. In some embodiments, the targeting moiety targets cancer cells. In some embodiments, the targeting moiety is an ICAM-1 antibody and / or a HER2 antibody. Methods for conjugating magnetic nanoparticles (e.g., IONPs) to agents (e.g., therapeutic or diagnostic agents) or targeting moieties are known in the art. Conjugation can be covalent or non-covalent. For example, conjugation methods are provided in PGuo et al., Proc Natl Acad Sci US A. 2014 Oct 14;111(41):14710-5; Huang et al., International Journal of Nanomedicine 07 Jul 2016, 11:3087-3099; Cho et al., Small (Weinheim an der Bergstrasse, Germany) 06 Jan 2013, 9(11):1964-1973; Chorny et al., FASEB Journal, 02 Apr 2007, 21(10):2510-2519; and Hryhorowicz et al., Mol Biotechnol. 2019 Mar;61(3):173-180, which are incorporated herein by reference.

[0111] It should be understood that in encapsulating any one of an agent or targeting moiety into an EM generated using the methods described herein, the agent or targeting moiety can be conjugated to magnetic nanoparticles (e.g., IONPs) prior to EM generation, or can be loaded into the EM after its generation using unconjugated magnetic nanoparticles (e.g., IONPs).

[0112] In some aspects, the present disclosure further provides any one of the EMs produced using a composition comprising any one of the methods and EMs described herein. In some embodiments, the EMs comprise magnetic nanoparticles (e.g., IONPs). In some embodiments, the EMs comprise magnetic nanoparticles (e.g., IONPs) conjugated (e.g., covalently or non-covalently) to an agent (e.g., a therapeutic or diagnostic agent) or a targeting moiety. In some embodiments, the EMs are empty EMs (e.g., when the magnetic nanoparticles are removed from the EMs after their production). In some embodiments, the empty EMs are later loaded with an agent (e.g., a therapeutic or diagnostic agent).

[0113] In some embodiments, EMs produced using the methods described herein can be used as a delivery vehicle to deliver an agent (e.g., a therapeutic or diagnostic agent) to a cell (e.g., an in vitro cultured cell or an in vivo cell in a subject). In some embodiments, EMs can be used as a delivery vehicle to deliver an agent (e.g., a therapeutic or diagnostic agent) to a subject, e.g., for the treatment or diagnosis of a disease.

[0114] In some embodiments, the composition is formulated as a pharmaceutical composition for administration to a subject. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. A "pharmaceutically acceptable carrier" may be a composition or vehicle, such as a pharmaceutically acceptable material, liquid or solid filler, diluent, additive, solvent, or encapsulating material, involved in carrying or transporting the subject agent from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient's tissues (e.g., physiologically compatible, sterile, physiological pH, etc.). The term "carrier" refers to an organic or inorganic ingredient, natural or synthetic, with which an active ingredient is combined to facilitate application. The components of the pharmaceutical compositions are capable of being commingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) additives such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols such as ethanol; and (23) other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation.

[0115] The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. When used in connection with the pharmaceutical compositions of the present disclosure, the term "unit dose" refers to physically discrete units suitable as single dosages for subjects, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect together with the required diluent, i.e., carrier, or vehicle.

[0116] The formulation of the pharmaceutical composition may depend on the route of administration. Injectable preparations suitable for parenteral administration or intratumoral, peritumoral, intralesional, or perilesional administration include, for example, sterile injectable aqueous or oily suspensions, which may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3 propanediol or 1,3 butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, bland fixed oils, including synthetic monoglycerides or diglycerides, may be used. In addition, fatty acids such as oleic acid are used in injectable preparations. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid components which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, or an emulsion.

[0117] In some embodiments, pharmaceutical compositions used for therapeutic administration must be sterile. Sterility is readily achieved by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Pharmaceutical compositions will usually be stored in lyophilized form, or, if highly stable to heat and oxidative degradation, as an aqueous solution. The pH of the preparation will typically be about 6 to 8, although higher or lower pH values ​​may be appropriate in certain cases.

[0118] Thus, further provided herein are methods for diagnosing a disease (e.g., cardiovascular disease, lung disease, kidney disease, infectious disease, autoimmune disease, immunodeficiency, allergy, blood disorder, metabolic disorder, skin disease, eye disease, brain disease, respiratory disease, endocrine system disease, or cancer), the methods comprising administering to a subject in need thereof any one of the EMs produced using the methods described herein, wherein the EM comprises any one of the diagnostic agents described herein. In some embodiments, the method further comprises detecting a signal. In some embodiments, the disease is a brain disease (e.g., brain cancer, neurological disorder, psychogenic disorder, cerebrovascular vascular disorder, brain trauma, or brain infection).

[0119] Also provided herein are methods for treating a disease (e.g., cardiovascular disease, pulmonary disease, renal disease, infectious disease, autoimmune disease, immunodeficiency, allergy, blood disorder, metabolic disorder, skin disease, eye disease, brain disease, respiratory disease, endocrine system disease, or cancer), comprising administering to a subject in need thereof any one of the EMs produced using the methods described herein, wherein the EM comprises any one of the therapeutic agents described herein. In some embodiments, the disease is a brain disease (e.g., brain cancer, neurological disorder, psychogenic disorder, cerebrovascular vascular disorder, brain trauma, or brain infection).

[0120] In some embodiments, the brain disease is brain cancer (primary brain cancer or metastatic brain cancer). In some embodiments, the brain disease is a neurological disorder (e.g., a neurodegenerative, neurobehavioral, or developmental disorder such as Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, amyotrophic lateral sclerosis (ALS), prion disease, and motor neuron disease). In some embodiments, the brain disease is a psychogenic disorder (e.g., post-traumatic stress disorder (PTSD), depressive disorder, major depressive disorder, postpartum depression, bipolar disorder, acute stress disorder, generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, schizophrenia, or trichotillomania). In some embodiments, the brain disease is brain trauma. In some embodiments, the brain disease is a brain infection.

[0121] In treating or diagnosing brain diseases, EM may be administered to a subject via injection or infusion. In some embodiments, EM is administered intravenously, subcutaneously, intraperitoneally, or intracerebrally. In some embodiments, the disease is a cardiovascular disease.

[0122] In some embodiments, the disease is cancer. The term "cancer" refers to a class of diseases characterized by the development of abnormal cells that have the ability to proliferate exponentially and invade and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers that may be treated using the methods described herein include, but are not limited to, hematological malignancies. Additional exemplary cancers include lung cancer (e.g., bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); kidney cancer (e.g., nephroblastoma, commonly known as Wilms' tumor, renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelioma, hemangioendothelioma); appendix cancer; benign monoclonal gammopathy; biliary tract cancer (e.g., cholangiocarcinoma); bladder cancer; and breast cancer. (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, breast cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchial cancer; carcinoid tumor; cervical cancer (e.g., adenocarcinoma of the cervix); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon carcinoma, rectal carcinoma, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endothelial sarcoma (e.g., Cancers of the endometrium (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familial hypereosinophilia; gallbladder cancer; gastric cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma), oral cancer (e.g., oral tonsillar carcinoma) squamous cell carcinoma); throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer); heavy chain diseases (e.g., alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic tumor; immune cell amyloidosis; liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS);Mesothelioma; Myeloproliferative Disorders (MPDs) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), essential myeloid metaplasia (AMM), commonly known as myelofibrosis (MF) (chronic idiopathic myelofibrosis), chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibromas (e.g., neurofibromatosis (NF) type 1 or type 2, multiple schwannomatosis); neuroendocrine cancers (e.g., gastroenteropancreatic neuroendocrine tumors (GEP-NETs), carcinoid tumors); osteosarcomas (e.g., bone cancer); ovarian cancers (e.g., cystadenocarcinoma, embryonal carcinoma of the ovary, adenocarcinoma of the ovary); papillary carcinoma; pancreatic cancers (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumor); penile cancers (e.g., Paget's disease of the penis and scrotum); pinealoma; undifferentiated These include, but are not limited to, metastatic neuroectodermal tumors (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasia; prostate cancer (e.g., prostatic adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small intestine cancer (e.g., appendix cancer); soft tissue sarcomas (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (phenylthiourea), medullary thyroid carcinoma); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

[0123] In some embodiments, the disease is an autoimmune disease. Non-limiting examples of autoimmune diseases include: multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease (IBD), lupus, and ankylosing spondylitis. Some of these disorders are discussed below. In one aspect, the present invention provides a method for the treatment of cancer. Other disorders that can be treated with FcRn-binding antibodies still include: scleroderma, Sjögren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue syndrome-immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, dermatomyositis, adolescent Dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, hyperthyroidism, Guillain-Barré syndrome, Hashimoto's disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus (type 1), juvenile arthritis, Meniere's disease, mixed connective tissue disease, myasthenia gravis, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, stiff-man syndrome, Takayasu's arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo.

[0124] As used herein, a "therapeutically effective amount" refers to the amount of each therapeutic agent of the present disclosure (e.g., a therapeutic agent for treating any of the brain disorders described herein) required to confer a therapeutic effect on a subject, alone or in combination with one or more other therapeutic agents. As will be recognized by those skilled in the art, effective amounts will vary depending on the particular condition being treated, the severity of the condition, individual subject parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of concomitant treatment (if any), the particular route of administration, and factors within the knowledge and experience of the medical practitioner. These factors are well known to those skilled in the art and can be addressed using only routine experimentation. It is generally preferred that maximum amounts of individual components or combinations thereof be used, i.e., the highest safe doses according to sound medical judgment.

[0125] However, it will be understood by one of skill in the art that a subject may insist on a lower dose or tolerated dose for medical reasons, psychological reasons, or for virtually any other reason.

[0126] Empirical considerations such as half-life generally contribute to determining dosage. For example, therapeutic agents compatible with the human immune system, such as polypeptides containing regions from humanized antibodies or fully human antibodies, may be used to extend the half-life of the polypeptide and protect it from attack by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment, generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the disease. Alternatively, sustained continuous release formulations of polypeptides may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0127] In some embodiments, administration is daily, every 2 days, every 3 days, every 4 days, every 5 days, or every 6 days. In some embodiments, administration frequency is once every 1 week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every 1 month, every 2 months, or every 3 months, or longer. The progress of this treatment is easily monitored by conventional techniques and assays. Administration regimen (including the anticancer drug used) can be changed over time.

[0128] In some embodiments for normal weight adult subjects, doses ranging from about 0.01 to 1000 mg / kg may be administered. In some embodiments, the dose is between 1 and 200 mg. The specific dosing regimen, i.e., dose, timing, and repetition, will depend on the particular subject and their medical history, as well as the characteristics of the anti-cancer drug (including half-life of the anti-cancer drug and other considerations well known in the art).

[0129] For purposes of this disclosure, appropriate dosing of the therapeutic agents described herein will depend on the particular agent (or composition thereof) used, the administration formulation and route, the type and severity of the disease, whether the anti-cancer agent is administered for prophylactic or therapeutic purposes, previous treatments, the subject's clinical history, and response to the antagonist, and the discretion of the attending physician. Typically, a clinician will administer the anti-cancer agent until the dosage achieves the desired result. Administration of one or more anti-cancer agents can be continuous or intermittent, depending, for example, on the recipient's physiological condition, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the anti-cancer agent may be essentially continuous over a preselected period of time, or in a series of spaced doses, e.g., before, during, or after disease progression.

[0130] As used herein, the term "treatment" refers to the application or administration of an anti-cancer agent to a subject in need thereof. A "subject in need thereof" refers to an individual having a disease, a symptom of a disease, or a tendency toward a disease, with the goal of curing, relieving, alleviating, ameliorating, altering, fixing, improving, ameliorating, or affecting the disease, symptom of a disease, or tendency toward a disease.

[0131] A "subject" to which administration is intended refers to a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a toddler, or an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)), or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a rodent (e.g., a mouse or a rat), a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird such as a chicken, a duck, a goose, or a turkey)). The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic or a transgenic animal.

[0132] In some embodiments, the subject is a companion animal (pet). "Companion animal," as used herein, refers to pets and other livestock. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cows, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a research animal. Non-limiting examples of research animals include: rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.

[0133] Alleviating disease includes delaying the progression or progression of disease or reducing the severity of disease. Alleviating disease does not necessarily require a curative result. As used in this context, "delaying" disease progression means extending, preventing, slowing, preventing, stabilizing, and / or postponing the progression of disease. This delay can vary in time depending on the disease being treated and / or the medical history of the individual. A method of "delaying" or alleviating disease progression or delaying the onset of disease is a method that reduces the probability of developing one or more symptoms of disease in a given time frame and / or reduces the severity of symptoms in a given time frame when compared with not using the method. Such comparisons are typically based on clinical trials using a large number of subjects that are sufficient to provide statistically significant results.

[0134] "Progression" or "progression" of a disease refers to the initial appearance and / or subsequent progression of the disease. Disease progression can be detectable and can be assessed using standard clinical techniques, as is well known in the art. However, progression also refers to progression that may be undetectable. For the purposes of this disclosure, progression or progression refers to the biological course of symptoms. "Progression" includes appearance, recurrence, and development. As used herein, "development" or "development" of a disease includes initial appearance and / or recurrence.

[0135] Conventional methods known to those skilled in the art of medicine can be used to administer anticancer drugs to a subject, depending on the type or site of the disease being treated. EM can also be administered via other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In some embodiments, EM is administered via intravenous infusion or infusion. In addition, it can be administered to a subject via an injectable depot route of administration, such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0136] Another aspect of the present disclosure provides an in vivo imaging method, comprising administering to a subject in need thereof an effective amount of EM produced using the methods described herein, and visualizing the exosome mimic in the subject via magnetic resonance imaging (MRI), fluorescence imaging, PET imaging, bioluminescence imaging, and ultrasound imaging.For use in the imaging methods described herein, the EM comprises magnetic nanoparticles (e.g., IONPs).In some embodiments, the imaging method is MRI.

[0137] In some embodiments, the magnetic nanoparticles (e.g., IONPs) are conjugated to a targeting moiety (e.g., any targeting moiety described herein or known in the art). In some embodiments, the targeting moiety targets cancer (e.g., an ICAM-1 antibody and / or a HER2 antibody for targeting breast cancer). The imaging methods described herein are non-invasive and allow visualization of the distribution of EM throughout a subject's body and assessment of EM uptake by specific tissues (e.g., cancer tissues).

[0138] example Development of a novel magnetic extrusion method for producing EM from cultured cells We developed a novel magnetic extrusion method to generate EMs from various cultured cell lines (e.g., MDA-MB-231, MDA-MB-436, and 3T3) in a large-scale and reproducible manner. Here, human triple-negative breast cancer (TNBC) MDA-MB-231 cells demonstrate EM generation using the magnetic extrusion method. Cultured MDA-MB-231 cells were first incubated overnight with 30 nm magnetic iron oxide nanoparticles (IONPs), allowing endocytosis and transfer of IONPs to endosomes, as confirmed by transmission electron microscopy (TEM) (Figures 1A and 1B).

[0139] Next, the IONP-loaded cells were subjected to the established hypotonic treatment. 1 The cells were subjected to a homogenization step followed by lysis of the whole cells and release of the organelles into suspension. A magnetic separator was used to isolate the IONP-encapsulated endosomes from other organelles, and these endosomes were purified. They were clearly visible under TEM (Figure 1C). The purified IONP-encapsulated endosomes were extruded using a Lipex extruder. 2~8 Using a fluoropolymer, IONPs were extruded through a track-etched polycarbonate (PCTE) nanoporous membrane (100 nm diameter). After extrusion, the IONP-encapsulated endosomes were formulated into nanoscale vesicles with a diameter of 100 nm and passed through a size-exclusion column to remove unencapsulated IONPs. These endosome-derived nanoscale vesicles were termed "exosome mimics (EMs)" because they share several key characteristics with natural exosomes, such as size, morphology, and structure, and share the same biological origin as exosomes.

[0140] Next, a magnetic separator was used to isolate IONP-encapsulated exosomes (IONP-EM) from the empty exosomes. TEM analysis of IONP-EM revealed a lipid bilayer structure similar to that of native exosomes (Figure 1D). Based on dynamic light scattering (DLS) measurements, IONP-EM exhibited a homogeneous hydrodynamic diameter of 100 nm with a significantly narrower size distribution than native exosomes (Figures 1E, 1G), providing more consistent and reproducible biodistribution and circulation characteristics. IONP-EM production by magnetic extrusion was confirmed using DLS measurements at 3 × 10 10 particles / 10 6 Significantly, this was significantly higher than that of natural exosomes prepared by conventional ultracentrifugation (approximately 5–8 × 10 8 particles / 10 6 cells) is over 30 times higher 9~11 Native exosomes and IONP-EM expressed ALIX, an established exosome marker, as determined by immunoblotting. 9~11 The exosomes expressed equivalent levels of IONP-EM (Figure 1F). Notably, this magnetic extrusion method is highly reproducible, as evidenced by the fact that the hydrodynamic size and protein concentration of IONP-EM remained unchanged in five independent experiments (Figure 1H). It was further demonstrated that breast cancer cell-derived IONP-EM promoted host cell proliferation (Figure 1I). Such biological functions have also been reported for natural exosomes. 12 .

[0141] Innovative MRI-based molecular imaging of breast tumors IONPs are not only used as magnetic beads for EM preparations, but also function as highly efficient MRI contrast agents. Notably, IONPs have already been approved by the US Food and Drug Administration (US FDA) as MRI contrast agents for clinical applications. 13 It has previously been shown that 30 nm IONPs can be easily modified with various targeting ligands to facilitate molecular-specific MR imaging of breast tumors in vivo. 14ICAM1-targeted IONPs bound and penetrated TNBC tumors more strongly than HER2-targeted IONPs did in vivo, suggesting that ICAM1 is highly overexpressed in these TNBC tumors. The ICAM1 and HER2 expression determined in these in vivo MRI results correlated closely with their in vitro cell membrane expression characterized by flow cytometry. Therefore, IONP-loaded EM derived from various cell types, including immune cells, can be used to monitor the tumor microenvironment through imaging.

[0142] Activity of engineered exosome mimics (EMs) The potential of engineered exosome mimics (EMs) for drug delivery applications was evaluated. The chemotherapeutic drug doxorubicin was successfully loaded into EMs engineered from mouse fibroblast 3T3 cells using two cargo loading methods: direct encapsulation and ammonium sulfate gradient loading. As shown in Figure 2, the encapsulation efficiency of doxorubicin in EMs was determined to be 23.9% for direct encapsulation and 67.4% for ammonium sulfate gradient loading. The results indicate that the ammonium sulfate gradient loading method is more efficient than the direct encapsulation method.

[0143] The 67.4% encapsulation efficiency of EMs by ammonium sulfate gradient loading was not achieved with native exosomes. Next, the anticancer activity of doxorubicin-encapsulated EMs (Dox-EMs) was evaluated in two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436.

[0144] As shown in Figures 3 and 4, Dox-EM effectively eliminated MDA-MB-231 and MDA-MB-436 cells in in vitro cytotoxicity assays. The half-maximal inhibitory concentration (IC50) of Dox-EM was 1.677 μg / ml for MDA-MB-231 cells and 0.378 μg / ml for MDA-MB-436 cells. These IC50 values ​​of Dox-EM were not achieved by natural exosomes. These studies demonstrate that the engineered EMs described herein can be used as nanoscale drug delivery systems for therapeutic applications. References [Table 1]

[0145] Equivalence and Scope Those skilled in the art will be able to ascertain, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but is as defined in the appended claims.

[0146] Articles such as "a," "an," and "the" may mean one or more than one, unless specifically stated or otherwise clear from the context. A claim or description containing "or" between two or more members of a group is considered satisfied when one, more than one, or all of the group members are present, unless indicated to the contrary or clear from the context. The disclosure of a group containing "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all of the group members are present. For brevity, these embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may or may not be specifically claimed.

[0147] It should be understood that the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms from one or more claims, or from the relevant portion of the description, are introduced into another claim. For example, a claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends from the same base claim. Furthermore, where a claim recites a composition, it should be understood that methods of making or using the composition according to any of the methods of making or using the composition disclosed herein or according to methods known in the art are included, unless otherwise indicated or unless it is clear to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0148] Where elements are presented as a list, for example in Markush group format, it should be understood that each possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term "comprising" is intended to be open-ended and allow for the inclusion of additional elements or steps. In general, where an aspect, product, or method is referred to as including a particular element, feature, step, or aspect, product, or method consisting of or consisting essentially thereof, it should be understood that such element, feature, or step is also provided. For brevity, these aspects have not been individually spelled out herein, but it will be understood that each of these aspects may or may not be provided and claimed herein.

[0149] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and / or the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume any specific value within the stated range in some embodiments, down to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0150] For brevity, each range value has not been spelled out individually herein, but it will be understood that each of these values ​​is provided herein and may or may not be specifically claimed. Unless otherwise indicated or otherwise apparent from the context and / or the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can contemplate any subrange of a given range, where the endpoints of the subrange are expressed to the same accuracy as one-tenth of the unit of the lower limit of the range.

[0151] Where websites are provided, URL addresses are provided as non-browser executable code, with each web address period within parentheses. The actual web address does not contain the parentheses. In addition, it is to be understood that any particular aspect of the present disclosure may be explicitly excluded from any one or more claims.

[0152] Where a range is given, any value within that range may be explicitly excluded from any one or more claims. Any aspect, element, feature, application, or aspect of the compositions and / or methods of the present disclosure may be excluded from any one or more claims. For purposes of brevity, not all of the embodiments in which one or more elements, features, applications, or aspects are excluded are explicitly set forth herein.

Claims

1. A method of producing an exosome mimic, comprising: (i) incubating cells with magnetic nanoparticles so that the magnetic nanoparticles enter endosomes in the cells; (ii) lysing the cells to produce a cell lysate containing endosomes; (iii) isolating endosomes encapsulating magnetic nanoparticles from the cell lysate in step (ii); and (iv) extruding the isolated endosomes obtained in step (iii) through a nanoporous membrane to generate exosome mimics.

2. 10. The method of claim 1, wherein the cells are selected from stem cells, bone marrow-derived cells, immune cells, erythrocytes, epithelial cells, and endothelial cells.

3. The method of claim 1 or 2, wherein the magnetic nanoparticles are iron oxide nanoparticles.

4. The method of any one of claims 1 to 3, wherein the nanoparticles enter endosomes within the cell via endocytosis.

5. The method of any one of claims 1 to 4, wherein the cells are lysed via homogenization.

6. The method of any one of claims 1 to 5, wherein step (iii) is carried out using a magnetic separator.

7. The method according to any one of claims 1 to 6, wherein the nanoporous membrane has a pore diameter of 100 nm.

8. The method of any one of claims 1 to 7, further comprising: (v) Removing unencapsulated magnetic nanoparticles.

9. 9. The method of claim 8, wherein step (v) is carried out via size exclusion chromatography.

10. The method of any one of claims 1 to 9, further comprising: (vi) Removal of magnetic nanoparticles from exosome mimics.

11. The method of any one of claims 1 to 9, wherein the magnetic nanoparticles are conjugated to a targeting moiety, a therapeutic agent, or a diagnostic agent.

12. An exosome mimetic produced by the method of any one of claims 1 to 11.

13. 13. The exosome mimetic of claim 12, comprising magnetic nanoparticles.

14. 14. The exosome mimetic of claim 12 or 13, further comprising an agent.

15. 15. The exosome mimetic of claim 14, wherein the agent is a therapeutic or diagnostic agent.

16. 16. The exosome mimetic of claim 14 or 15, wherein the agent is conjugated to a magnetic nanoparticle.

17. The exosome mimetic of any one of claims 13 to 16, wherein the magnetic nanoparticles are iron oxide nanoparticles.

18. A composition comprising the exosome mimetic of any one of claims 12 to 17.

19. 20. The composition of claim 18, further comprising a pharmaceutically acceptable carrier.

20. 20. The composition of claim 18 or 19 for a method of treating a disease, wherein the treatment comprises administering an effective amount of the exosome mimetic of any one of claims 12 to 17 or the composition of claim 18 or 19 to a subject in need thereof.

21. 20. The composition of claim 18 or 19 for a method for diagnosing a disease, wherein the diagnosis comprises administering an effective amount of the exosome mimetic of any one of claims 12 to 17, or the composition of claim 18 or 19, to a subject in need thereof.

22. 22. The composition of claim 20 or 21, wherein the disease is: Cancer, cardiovascular disease, brain disease, immune deficiency, autoimmune and infectious diseases, respiratory disease, or endocrine system diseases.

23. 13. An in vivo imaging method comprising visualizing an exosome mimic in a subject in need thereof administered an effective amount of the exosome mimic of claim 12 via magnetic resonance imaging (MRI), fluorescence imaging, PET imaging, bioluminescence imaging, and ultrasound imaging.

24. 24. The method of claim 23, wherein the exosome mimic is visualized via MRI.

25. 25. The method of claim 23 or claim 24, wherein the exosome mimetic further comprises a diagnostic agent.

26. 26. The method of claim 25, wherein the diagnostic agent is a targeting moiety.

27. 27. The method of claim 26, wherein the targeting moiety targets a cancer biomarker.

28. 28. The method of claim 27, wherein the cancer is breast cancer.

29. 29. The method of claim 27 or 28, wherein the biomarker is ICAM1 or HER2.

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