Nanoparticles containing cell membranes and uses thereof

Abstract

To provide nanoparticles containing a cellular membrane and uses thereof.SOLUTION: A nanoparticle comprises an interior compartment (or inner core) and an outer surface (or shell) comprising a cellular membrane derived from a cell, the interior compartment (or inner core) not providing a solid support to the cellular membrane in the outer surface (or shell). The present disclosure also relates to processes of making the nanoparticles. The present disclosure further relates to compositions comprising the nanoparticles and methods of using the nanoparticles.SELECTED DRAWING: None

Description

[Technical Field]

[0001] I. Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 771,561, filed November 26, 2018, the disclosure of which is incorporated by reference in its entirety for all purposes. II. FIELD OF THE INVENTION The present disclosure relates to nanoparticles containing cell membranes and their uses. The nanoparticles comprise an inner compartment (or inner core) and an outer surface (or shell) comprising a cell membrane derived from a cell, and the inner compartment (or inner core) does not provide a solid support for the cell membrane in the outer surface (or shell). The present disclosure also relates to methods for making nanoparticles. The present disclosure further relates to compositions comprising nanoparticles and methods for using nanoparticles. [Background technology]

[0002] III. Background of the Invention WO2013 / 052167(A2) relates to membrane-encapsulated nanoparticles and methods of use. Zhang et al., Angew. Chem. Int. Ed. 2017, 56:14075-14079, relates to remote loading of small molecule therapeutic agents into cholesterol-rich cell membrane-derived vesicles. Ying et al., Adv. Funct. Mater. 2018, 28, 1801032, relates to remotely loaded platelet vesicles for disease-targeted delivery of therapeutic agents. Brian et al., Nature Biotechnology, 33:81-88 (2015), relates to engineered liposomes that sequester bacterial exotoxins and protect against severe invasive infections in mice. There is a need for novel nanoparticles that contain cell membranes. The present invention addresses this need and related needs in the art. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2013 / 052167(A2) [Non-patent literature]

[0004] [Non-Patent Document 1] Zhang et al.,Angew.Chem.Int.Ed. 2017, 56:14075-14079 [Non-patent document 2] Ying et al., Adv. Funct. Mater. 2018, 28, 1801032 [Non-patent document 3] Brian et al., Nature Biotechnology, 33:81-88 (2015) Summary of the Invention [Means for solving the problem]

[0005] IV. Summary of the Invention In one aspect, the present disclosure provides nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising a cellular membrane derived from a cell, wherein the inner compartment (or inner core) does not provide a solid support for the cellular membrane in the outer surface (or shell), and wherein a) the inner compartment (or inner core) is isotonic with cellular fluids or physiological fluids, e.g., the inner compartment (or inner core) comprises a liquid that is isotonic with cellular fluids or physiological fluids, and / or b) the cellular membrane of the outer surface (or shell) comprises enhanced or enriched levels of a steroid, with the proviso that if the cellular membrane is derived from a red blood cell, the inner compartment (or inner core) is isotonic with cellular fluids or physiological fluids.

[0006] In another aspect, the present disclosure provides nanoparticles comprising an internal compartment (or internal core) and an external surface (or shell), wherein the internal compartment (or internal core) does not provide a solid support for the external surface (or shell) and / or is isotonic with cellular or physiological fluids, e.g., the internal compartment (or internal core) comprises a fluid that is isotonic with cellular or physiological fluids, and the external surface (or shell) comprises cells, cholesterol, and sphingomyelin derived from cell membranes.

[0007] In yet another aspect, the disclosure provides a method of making nanoparticles comprising: a) contacting a steroid with a cell membrane derived from a cell to form a combination; and b) applying exogenous energy to the combination in a liquid to form nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising the cell membrane comprising enhanced or enriched levels of the steroid. In yet another aspect, the disclosure provides a method of making nanoparticles comprising: a) contacting a steroid with a cell membrane derived from a cell to form a combination; and b) applying exogenous energy to the combination to form nanoparticles comprising an inner compartment (or inner core) and an outer surface comprising the cell membrane comprising enhanced or enriched levels of the steroid; and c) applying exogenous energy to the nanoparticles in a liquid isotonic to cellular fluid or physiological fluid to form nanoparticles comprising the inner compartment (or inner core) comprising the liquid isotonic to cellular fluid or physiological fluid, and the outer surface comprising the cell membrane comprising the enhanced or enriched levels of the steroid. In yet another aspect, the present disclosure provides a method of making nanoparticles, the method comprising applying exogenous energy to a cell membrane derived from a red blood cell in a fluid isotonic to a cellular fluid or physiological fluid to form a nanoparticle comprising an inner compartment (or inner core) comprising said fluid isotonic to a cellular fluid or physiological fluid and an outer surface comprising said cell membrane.

[0008] In yet another aspect, the present disclosure provides a method for making nanoparticles, the method comprising the steps of: a) contacting a cell membrane derived from a cell with a steroid and a sphingolipid dissolved in a water-miscible solvent to form a combination; and b) applying exogenous energy to the combination, e.g., the combination in a liquid, to form nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising the cell membrane, the steroid, and the sphingolipid.

[0009] Nanoparticles made by the above method are also provided.

[0010] In yet another aspect, the present disclosure provides a pharmaceutical delivery system or device comprising an effective amount of the nanoparticles described above.

[0011] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of the nanoparticles described above and a pharmaceutically acceptable carrier or excipient.

[0012] In yet another aspect, the present disclosure provides a method for treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising a cell membrane derived from a cell, wherein the inner compartment (or inner core) does not provide a solid support for the cell membrane in the outer surface (or shell). Optionally, and in some embodiments, a) the inner compartment (or inner core) of the nanoparticle is isotonic with cellular fluid or physiological fluid, e.g., the inner compartment (or inner core) comprises a liquid isotonic with cellular fluid or physiological fluid, and / or b) the cell membrane of the outer surface (or shell) of the nanoparticle comprises an enhanced or enriched level of a steroid. Additionally, optionally, and in some embodiments, when the nanoparticle comprises a cell membrane derived from a red blood cell, the inner compartment (or inner core) is isotonic with cellular fluid or physiological fluid. In some embodiments, the nanoparticles are administered using a pharmaceutical delivery system or a pharmaceutical composition comprising the nanoparticles.

[0013] In yet another aspect, the present disclosure provides the use of an effective amount of the above-described nanoparticles for the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof.

[0014] In yet another aspect, the present disclosure provides an immunogenic composition comprising an effective amount of the above-described nanoparticles, and optionally further comprising an immunogenic adjuvant or immune-enhancing substance. Vaccines comprising the above-described neoplasm-specific immunogenic compositions are also provided. Methods for treating or preventing neoplasms in a subject, using the immunogenic compositions or vaccines, are further provided. Use of an effective amount of a neoplasm-specific immunogenic composition for the manufacture of a vaccine for treating or protecting a subject from a neoplasm is further provided.

[0015] In yet another aspect, the present disclosure provides an immunogenic composition configured to treat or prevent a disease or condition associated with a cell membrane-targeting or binding moiety of a nanoparticle, the moiety comprising the outer surface of the nanoparticle. Vaccines comprising the immunogenic compositions described above are also provided. Further provided are methods for eliciting an immune response in a subject to a moiety associated with a disease or condition, using the immunogenic composition or vaccine. Further provided is the use of an effective amount of the immunogenic composition to manufacture a vaccine that protects a subject against a disease or condition associated with the moiety.

[0016] In some embodiments, the nanoparticles, pharmaceutical delivery systems, pharmaceutical compositions and methods of the present invention can be used to treat exemplary pharmaceuticals listed in the Orange Book: Approved Drug Products with Therapeutic Equivalence Evaluations published by the U.S. Food and Drug Administration (March 2012 to present), The Merck Index (a US publication, the printed 14th Edition, Whitehouse Station, NJ, USA) and its online version (The Merck Index Online SM Exemplary pharmaceuticals listed in the U.S. Food and Drug Administration's publication Biologics Products & Establishments (last day for loading on website Tuesday, May 1, 2012) can be delivered and used to treat or prevent the corresponding diseases and disorders.

[0017] In some aspects, the present disclosure is related to U.S. Application No. 13 / 827,906, filed March 14, 2013, published as US2013 / 337066(A1), International Application No. PCT / US2012 / 039411, filed May 24, 2012, published as WO2013 / 052167(A2), and U.S. Provisional Application No. 61 / 492,626, filed June 2, 2011. The entire contents of the above-mentioned applications are incorporated by reference. In certain embodiments, for example, the following items are provided: (Item 1) A nanoparticle comprising an inner compartment (or inner core) and an outer surface (or shell) comprising a cell membrane derived from a cell, wherein said inner compartment (or inner core) does not provide a solid support for said cell membrane in said outer surface (or shell); a) the inner compartment (or inner core) is isotonic with respect to cellular or physiological fluids, e.g., the inner compartment (or inner core) comprises a fluid that is isotonic with respect to cellular or physiological fluids; and / or b) the cell membrane of the outer surface (or shell) contains enhanced or enriched levels of steroids; provided that when the cell membrane is derived from a red blood cell, the inner compartment (or inner core) is isotonic with cellular or physiological fluids; Nanoparticles. (Item 2) Item 1, wherein the inner compartment (inner core) contains a liquid that is isotonic with cellular fluid. (Item 3) 3. The nanoparticle according to item 2, wherein the inner compartment (inner core) contains a liquid that is isotonic with the cellular fluid contained in the cell. (Item 4) 4. The nanoparticles according to item 3, wherein the cells are prokaryotic or eukaryotic cells. (Item 5) 4. The nanoparticle according to item 3, wherein the cell is a cell of a unicellular organism. (Item 6) Item 6. The nanoparticles according to item 5, wherein the single-cell organism is a bacterium or a fungus. (Item 7) 4. The nanoparticle according to item 3, wherein the cell is a cell of a multicellular organism. (Item 8) 8. The nanoparticles according to item 7, wherein the multicellular organism is a plant, an animal, a vertebrate, a non-human mammal or a human. (Item 9) 8. The nanoparticles according to item 7, wherein the cells are animal cells, such as non-human mammalian cells or human cells. (Item 10) Item 11. The nanoparticles according to item 9, wherein the cells are cells of connective tissue, such as blood, bone, tendon, ligament, fat or loose connective tissue, fibrous connective tissue, skeletal connective tissue, or fluid connective tissue. 10. The nanoparticles according to item 9, wherein the cells are cells of muscle tissue, such as visceral or smooth muscle, musculoskeletal or cardiac muscle. (Item 12) 10. The nanoparticles according to item 9, wherein the cells are cells of nervous tissue, such as cells in the central nervous system (CNS) or peripheral nervous system (PNS). (Item 13) 10. The nanoparticles according to item 9, wherein the cells are cells of epithelial tissue, such as simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium (also known as ciliated columnar epithelium), columnar epithelium, glandular epithelium or ciliated columnar epithelium. (Item 14) 10. The nanoparticle according to item 9, wherein the cell is a cell of the nervous system, cardiovascular system, circulatory system, vascular system, digestive system, endocrine system, immune system, integumentary system, lymphatic system, musculoskeletal system, reproductive system, respiratory system, respiratory tract, ventilatory system, urinary system or renal system or urinary tract. (Item 15) 10. The nanoparticles according to item 9, wherein the cells are blood cells, tumor cells, cancer cells, immune cells, stem cells, endothelial cells or epithelial cells. (Item 16) Item 1, wherein the inner compartment (inner core) comprises a liquid that is isotonic with physiological fluid. (Item 17) 17. The nanoparticles according to item 16, wherein the physiological fluid is a physiological fluid in a multicellular organism. (Item 18) 18. The nanoparticles according to item 17, wherein the multicellular organism is a plant, an animal, a vertebrate, a non-human mammal or a human. (Item 19) 18. The nanoparticles according to item 17, wherein the physiological fluid is a physiological fluid in an animal, a vertebrate, a non-human mammal or a human, such as circulating blood in an animal, a vertebrate, a non-human mammal or a human. (Item 20) 20. The nanoparticles according to item 19, wherein the physiological fluid is a physiological fluid of the nervous system, cardiovascular system, circulatory system, vascular system, digestive system, endocrine system, immune system, integumentary system, lymphatic system, musculoskeletal system, reproductive system, respiratory system, respiratory tract, ventilatory system, urinary system or renal system or urinary tract. (Item 21) 21. The nanoparticle according to any of items 1 to 20, wherein the inner compartment (inner core) comprises a fluid that is isotonic with cellular fluid or physiological fluid ex vivo. (Item 22) 21. The nanoparticle according to any of items 1 to 20, wherein the inner compartment (inner core) comprises a fluid that is isotonic with cellular or physiological fluids in vivo. (Item 23) 23. The nanoparticle of any of items 1 to 22, wherein the cell membrane comprises a plasma membrane. (Item 24) 23. The nanoparticle according to any of items 1 to 22, wherein the cell membrane comprises an intracellular membrane. (Item 25) 25. The nanoparticle according to any of items 1 to 24, wherein the cell membrane is derived from a prokaryotic cell. (Item 26) 26. The nanoparticle according to any of items 1 to 25, wherein the cell membrane is derived from the cell of a unicellular organism, such as a bacterium or a fungus. (Item 27) 27. The nanoparticle according to any of items 1 to 24 and 26, wherein the cell membrane is derived from a eukaryotic cell. (Item 28) 28. The nanoparticle according to any of items 1 to 24 and 27, wherein the cell membrane is derived from a cell of a multicellular organism, such as a plant, an animal, a vertebrate, a non-human mammal or a human. (Item 29) 29. The nanoparticle according to item 28, wherein the cell membrane is derived from an animal, a vertebrate, a non-human mammal or a human cell. (Item 30) 30. The nanoparticles according to item 29, wherein the cells are cells of connective tissue, such as blood, bone, tendon, ligament, fat or loose connective tissue, fibrous connective tissue, skeletal connective tissue or fluid connective tissue. (Item 31) 30. The nanoparticles according to item 29, wherein the cells are cells of muscle tissue, such as visceral or smooth muscle, musculoskeletal or cardiac muscle. (Item 32) 30. The nanoparticles according to item 29, wherein the cells are cells of nervous tissue, such as cells in the central nervous system (CNS) or peripheral nervous system (PNS). (Item 33) 30. The nanoparticles according to item 29, wherein the cells are cells of epithelial tissue, such as simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium (also known as ciliated columnar epithelium), columnar epithelium, glandular epithelium or ciliated columnar epithelium. (Item 34) 30. The nanoparticle according to item 29, wherein the cell is a cell of the nervous system, cardiovascular system, circulatory system, vascular system, digestive system, endocrine system, immune system, integumentary system, lymphatic system, musculoskeletal system, reproductive system, respiratory system, respiratory tract, ventilatory system, urinary system or renal system or urinary tract. (Item 35) 30. The nanoparticles according to item 29, wherein the cells are blood cells, tumor cells, cancer cells, immune cells, stem cells, endothelial cells or epithelial cells. (Item 36) 30. The nanoparticles according to item 29, wherein the cell membrane comprises a plasma membrane derived from a blood cell, such as a red blood cell, a white blood cell, and / or a platelet. (Item 37) 37. The nanoparticles according to any of the preceding items, wherein the steroid is a fungal steroid, an animal steroid, a plant steroid or a prokaryotic steroid. (Item 38) 38. The nanoparticles according to item 37, wherein the fungal steroid is ergosterol, ergosta-5,7,22,24(28)-tetraen-3β-ol, zymosterol, lanosterol or 5,6-dihydroergosterol. (Item 39) 38. The nanoparticles according to item 37, wherein the animal steroid is a vertebrate steroid or an insect steroid. (Item 40) 40. The nanoparticles according to item 39, wherein the insect steroid is an ecdysteroid, such as 20-hydroxyecdysone (ecdysterone or 20E). (Item 41) 40. The nanoparticles according to item 39, wherein the vertebrate steroid is a steroid hormone or cholesterol. (Item 42) 42. The nanoparticles according to item 41, wherein the vertebrate steroid is cholesterol. (Item 43) 42. The nanoparticles according to item 41, wherein the steroid hormone is a sex steroid, such as an androgen, an estrogen, or a progestogen, a corticosteroid, such as a glucocorticoid or a mineralocorticoid, or an anabolic steroid, such as testosterone or an ester thereof. (Item 44) 38. The nanoparticles according to item 37, wherein the plant steroid is an alkaloid, a cardiac glycoside, a phytosterol or a brassinosteroid. (Item 45) 38. The nanoparticles according to item 37, wherein the prokaryotic steroid is a tetracyclic steroid or a triterpene, such as a hopane. (Item 46) 37. Nanoparticles according to any of items 1 to 36, wherein the steroid is a cholestane, such as cholesterol, a cholane, such as cholic acid, a pregnane, such as progesterone, an androstane, such as testosterone, or an estrane, such as estradiol. (Item 47) 37. The nanoparticles according to any of items 1 to 36, wherein the steroid is selected from the group consisting of gonane, testosterone, cholic acid, dexamethasone, lanosterol, progesterone, medrogestone, β-sitosterol, cholesterol and 5α-cholestane. (Item 48) 48. The nanoparticle according to any of items 1 to 47, wherein the outer surface (or shell) comprises a cell membrane derived from a fungal cell, the cell membrane comprising enhanced or enriched levels of a fungal steroid. (Item 49) 48. The nanoparticle according to any of items 1 to 47, wherein the outer surface (or shell) comprises a cell membrane derived from a plant cell, the cell membrane comprising enhanced or enriched levels of a plant steroid. (Item 50) 48. The nanoparticle according to any of items 1 to 47, wherein the outer surface (or shell) comprises a cell membrane derived from a prokaryotic cell, the cell membrane comprising enhanced or enriched levels of a prokaryotic steroid. (Item 51) 48. The nanoparticle according to any of items 1 to 47, wherein the outer surface (or shell) comprises a cell membrane derived from an animal cell, the cell membrane comprising enhanced or enriched levels of an animal steroid. (Item 52) 52. The nanoparticle of item 51, wherein the outer surface (or shell) comprises a cell membrane derived from a vertebrate cell, the cell membrane comprising enhanced or enriched levels of a vertebrate steroid. (Item 53) 53. The nanoparticle according to item 52, wherein the outer surface (or shell) comprises a cell membrane derived from a mammalian cell, e.g., a human cell, and the cell membrane comprises enhanced or enriched levels of mammalian steroids, e.g., human steroids. (Item 54) 54. The nanoparticle according to item 53, wherein the outer surface (or shell) comprises a cell membrane derived from a blood cell, the cell membrane comprising enhanced or enriched levels of a mammalian steroid, such as cholesterol. (Item 55) 55. The nanoparticle according to item 53 or 54, wherein the outer surface (or shell) comprises a cell membrane derived from a red blood cell, a white blood cell or a platelet, the cell membrane comprising enhanced or enriched levels of a mammalian steroid, such as cholesterol. (Item 56) 56. The nanoparticle according to item 55, wherein the outer surface (or shell) comprises plasma membranes derived from red blood cells, white blood cells and / or platelets, the plasma membranes comprising enhanced or enriched levels of mammalian steroids, such as cholesterol. (Item 57) 57. The nanoparticle according to item 56, wherein the outer surface (or shell) comprises a plasma membrane derived from a human red blood cell, a human white blood cell and / or a human platelet, the cell membrane comprising enhanced or enriched levels of a human steroid, such as cholesterol. (Item 58) 58. The nanoparticle according to any of items 1 to 57, wherein the enhanced or enriched level of steroid in the cell membrane of the outer surface (or shell) is due to exogenously added steroid. (Item 59) 58. The nanoparticle according to any of items 1 to 57, wherein the enhanced or enriched levels of steroid in the cell membrane of the outer surface (or shell) are due to the use of cell membranes derived from cells that have been modified to contain enhanced or enriched levels of the steroid in their membranes. (Item 60) 60. The nanoparticle according to any of the preceding items, wherein the cell membrane of the outer surface (or shell) comprises a level of steroid that is at least 0.1% higher than the basal level of the steroid in the cell membrane. (Item 61) 61. The nanoparticle according to any one of items 1 to 60, wherein the cell membrane of the outer surface (or shell) comprises from about 0.1% (w / w) to about 50% (w / w) of an exogenously added steroid relative to the total membrane protein weight of the cell membrane, and optionally the cell membrane of the outer surface (or shell) comprises from about 0.1% (w / w) to about 50% (w / w) of an exogenously added steroid. (Item 62) Item 62. The nanoparticle according to Item 61, wherein the cell membrane of the outer surface (or shell) comprises a cell membrane derived from a blood cell and comprises about 0.1% (w / w) to about 50% (w / w) of an exogenously added steroid, such as cholesterol, relative to the total membrane protein weight of the cell membrane; optionally, the cell membrane of the outer surface (or shell) comprises a cell membrane derived from a blood cell and comprises about 0.1% (w / w) to about 50% (w / w) of an exogenously added steroid, such as cholesterol. (Item 63) 63. The nanoparticle according to any one of items 1 to 62, wherein the cell membrane of the outer surface (or shell) comprises about 20% (w / w) to about 100% (w / w) of the steroid relative to the total membrane protein weight of the cell membrane, and optionally the cell membrane of the outer surface (or shell) comprises about 20% (w / w) to about 100% (w / w) of the steroid. (Item 64) Item 64. The nanoparticle according to Item 63, wherein the cell membrane of the outer surface (or shell) comprises a cell membrane derived from a blood cell and comprises about 20% (w / w) to about 100% (w / w) of the steroid, for example, cholesterol, relative to the total membrane protein weight of the cell membrane, and optionally the cell membrane of the outer surface (or shell) comprises a cell membrane derived from a blood cell and comprises about 20% (w / w) to about 100% (w / w) of the steroid, for example, cholesterol. (Item 65) 65. The nanoparticle according to any of items 1 to 64, wherein the inner compartment (inner core) has a pH in the range of about 4 to about 10, such as about 6 to about 9. (Item 66) 66. The nanoparticle according to any of the preceding items, further comprising a releasable cargo. (Item 67) 67. The nanoparticle of item 66, wherein the releasable cargo is located within or on the inner compartment (inner core), between the inner compartment (inner core) and the outer surface (or shell), or within or on the outer surface (or shell). (Item 68) 68. The nanoparticle according to item 66 or 67, wherein release of the releasable cargo is triggered by contact between the nanoparticle and a target cell, tissue, organ or subject, or by a change in physical parameters surrounding the nanoparticle. (Item 69) 69. The nanoparticle of any of items 66 to 68, wherein the releasable cargo is a therapeutic agent, a prophylactic agent, a diagnostic or marker agent, a prognostic agent or a combination thereof. (Item 70) 70. The nanoparticle according to any of items 66 to 69, wherein the releasable cargo is a metal particle, a polymer particle, a dendrimer particle or an inorganic particle. (Item 71) 66. The nanoparticle according to any of items 1 to 65, which does not contain a releasable cargo. (Item 72) 72. The nanoparticles according to any one of items 1 to 71, wherein the nanoparticles have a diameter of about 10 nm to about 10 μm. (Item 73) 73. The nanoparticle according to any of items 1 to 72, wherein the nanoparticle is substantially devoid of constituents of the cell from which the cell membrane is derived. (Item 74) 74. The nanoparticle of claim 73, wherein the cell membrane comprises a plasma membrane derived from a red blood cell, and the nanoparticle is substantially devoid of hemoglobin. (Item 75) 75. The nanoparticle according to any of items 1 to 74, wherein the nanoparticle substantially maintains the native structural integrity or activity of the cell membrane or the constituents of the cell membrane. (Item 76) 76. The nanoparticle according to any of the preceding items, wherein the inner core comprises a salt, a sugar or a sugar alcohol. (Item 77) 77. The nanoparticles according to item 76, wherein the sugar is a monosaccharide or a disaccharide. (Item 78) 78. The nanoparticles according to item 77, wherein the monosaccharide is fructose, galactose or glucose. (Item 79) 78. The nanoparticles according to item 77, wherein the disaccharide is lactose, maltose or sucrose. (Item 80) 77. The nanoparticles according to item 76, wherein the sugar alcohol is selected from the group consisting of ethylene glycol, glycerol, erythritol, threitol, arabitol (or arabinitol), xylitol, ribitol (or adonitol), mannitol, sorbitol, galactitol (dulcitol), fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetriitol and polyglycitol. (Item 81) 81. The nanoparticles according to item 80, wherein the sugar alcohol is sorbitol. (Item 82) 82. The nanoparticles according to any of items 76 to 81, wherein the salt, sugar or sugar alcohol has an osmolality of about 250 mOsm / kg to about 350 mOsm / kg, such as about 275 mOsm / kg to about 295 or 300 mOsm / kg, or a concentration in the range of 250 mmol / kg to about 1,000 mmol / kg. (Item 83) 83. The nanoparticle according to any of the preceding items, wherein the nanoparticle is biocompatible or biodegradable. (Item 84) The nanoparticles are: a) an inner compartment (or inner core) that is isotonic with respect to cellular or physiological fluids, e.g., an inner compartment (or inner core) that contains a fluid that is isotonic with respect to cellular or physiological fluids, or b) an outer surface (or shell) comprising a cell membrane derived from a cell, said cell membrane of said outer surface (or shell) comprising enhanced or enriched levels of steroids. 84. The nanoparticle according to any of items 1 to 83, comprising: (Item 85) 85. The nanoparticle according to item 84, wherein the cell membrane in the outer surface (or shell) does not contain enhanced or enriched levels of steroids, such as cell membranes derived from red blood cells, white blood cells, platelets, tumor cells, cancer cells, immune cells, stem cells, endothelial cells or epithelial cells that do not contain enhanced or enriched levels of steroids. (Item 86) The nanoparticles are: a) an inner compartment (or inner core) that is isotonic with respect to cellular or physiological fluids, e.g., an inner compartment (or inner core) that contains a fluid that is isotonic with respect to cellular or physiological fluids; and b) an outer surface (or shell) comprising a cell membrane derived from a cell, said cell membrane of said outer surface (or shell) comprising enhanced or enriched levels of steroids. 84. The nanoparticle according to any of items 1 to 83, comprising: (Item 87) 87. The nanoparticle according to item 86, wherein the cell membrane is derived from a red blood cell, a white blood cell, a cell, a platelet, a tumor cell, a cancer cell, an immune cell, a stem cell, an endothelial cell or an epithelial cell. (Item 88) Item 10. The nanoparticle of item 1, wherein the inner compartment (or inner core) comprises a sugar or sugar alcohol, such as sorbitol, and the outer surface (or shell) comprises a plasma membrane derived from a red blood cell. (Item 89) 89. The nanoparticle according to any of items 1 to 88, wherein the nanoparticle has an in vivo half-life in the blood circulation of at least about 1 minute. (Item 90) 90. The nanoparticle of any of items 1 to 89, wherein the nanoparticle is substantially devoid of immunogenicity in the species or subject to which the cell membrane is derived. (Item 91) 91. The nanoparticle according to any of the preceding items, wherein the outer surface (or shell) comprises a naturally occurring cell membrane and further comprises a synthetic membrane. (Item 92) 92. The nanoparticle according to any of the preceding claims, wherein the outer surface (or shell) further comprises a sphingolipid. (Item 93) 93. The nanoparticles according to item 92, wherein the sphingolipid is a simple sphingolipid. (Item 94) 93. The nanoparticles according to item 92, wherein the sphingolipid is a complex sphingolipid, such as a sphingomyelin, a glycosphingolipid, or an inositol-containing ceramide. (Item 95) 1. A method of making nanoparticles, comprising: a) contacting a cell membrane derived from a cell with a steroid to form a combination; and b) applying exogenous energy to said combination in a liquid to form nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising said cell membrane containing enhanced or enriched levels of said steroid. A method comprising: (Item 96) 96. The method of claim 95, wherein step b) comprises applying exogenous energy to the combination in a liquid isotonic to cellular or physiological fluids to form nanoparticles comprising an inner compartment (or inner core) comprising the liquid isotonic to cellular or physiological fluids, and an outer surface comprising the cell membrane containing enhanced or enriched levels of the steroid. (Item 97) 1. A method of making nanoparticles, comprising: a) contacting a cell membrane derived from a cell with a steroid to form a combination; and b) applying exogenous energy to the combination to form nanoparticles comprising an inner compartment (or inner core) and an outer surface comprising the cell membrane containing enhanced or enriched levels of the steroid; and c) applying exogenous energy to said nanoparticles in a fluid isotonic to cellular or physiological fluids to form nanoparticles comprising said inner compartment (or inner core) comprising said fluid isotonic to cellular or physiological fluids, and said outer surface comprising said cell membrane containing enhanced or enriched levels of said steroid. A method comprising: (Item 98) A method for making nanoparticles, comprising the step of applying exogenous energy to a cell membrane derived from a red blood cell in a fluid isotonic to a cellular fluid or physiological fluid to form a nanoparticle comprising an inner compartment (or inner core) comprising said fluid isotonic to a cellular fluid or physiological fluid and an outer surface comprising said cell membrane. (Item 99) Steps below: a) contacting cell membranes derived from red blood cells with a steroid to form a combination; and b) applying exogenous energy to said combination in a fluid isotonic to cellular or physiological fluids to form nanoparticles comprising an inner compartment (or inner core) comprising said fluid isotonic to cellular or physiological fluids, and an outer surface comprising said cell membrane containing enhanced or enriched levels of said steroid. Item 99. The method of item 98, comprising: (Item 100) Steps below: a) contacting cell membranes derived from red blood cells with a steroid to form a combination; b) applying exogenous energy to said combination to form nanoparticles comprising an inner core and an outer surface comprising said cell membrane containing enhanced or enriched levels of said steroid; and c) applying exogenous energy to said nanoparticles in a fluid isotonic to cellular or physiological fluids to form nanoparticles comprising an inner compartment (or inner core) comprising said fluid isotonic to cellular or physiological fluids, and an outer surface comprising said cell membrane containing enhanced or enriched levels of said steroid. Item 99. The method of item 98, comprising: (Item 101) 101. The method of any of items 95 to 100, wherein the cell membrane derived from the cell is in the form of a cell membrane ghost. (Item 102) 102. The method of any of items 95 to 101, wherein the exogenous energy is mechanical energy, acoustic energy or thermal energy. (Item 103) 103. The method according to any of items 95 to 102, wherein the cell membrane is derived from a mammalian or human blood cell, such as a red blood cell, a white blood cell or a platelet. (Item 104) 104. The method of claim 103, wherein the steroid is a cholestane, such as cholesterol. (Item 105) 1. A method of making nanoparticles, comprising: a) contacting cell membranes derived from cells with a steroid and a sphingolipid dissolved in a water-miscible solvent to form a combination; and b) applying exogenous energy to said combination in a liquid to form nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising said cell membrane, said steroid, and said sphingolipid. A method comprising: (Item 106) 106. The method of claim 105, wherein the cell membrane is derived from an animal cell, such as a non-human mammalian cell or a human cell. (Item 107) 107. The method of claim 105 or 106, wherein the cell membrane is derived from a blood cell, a tumor cell, a cancer cell, an immune cell, a stem cell, an endothelial cell or an epithelial cell. (Item 108) 108. The method of any of items 105 to 107, wherein the cell membrane comprises a plasma membrane derived from a blood cell, such as a red blood cell, a white blood cell and / or a platelet. (Item 109) 109. The method of any of items 105 to 108, wherein the cell membrane is contained in an aqueous liquid. (Item 110) 110. The method of claim 109, wherein the aqueous liquid is water, a buffer solution having a pH in the range of about 5 to about 9, such as PBS, or an isotonic liquid or buffer solution. (Item 111) 111. The method according to any of items 105 to 110, wherein the steroid is a cholestane, such as cholesterol, a cholane, such as cholic acid, a pregnane, such as progesterone, an androstane, such as testosterone, or an estrane, such as estradiol. (Item 112) 111. The method according to any of items 105 to 110, wherein the steroid is selected from the group consisting of gonane, testosterone, cholic acid, dexamethasone, lanosterol, progesterone, medrogestone, β-sitosterol, cholesterol and 5α-cholestane. (Item 113) 113. The method of any of items 105 to 112, wherein the sphingolipid is a simple sphingolipid. (Item 114) 113. The method according to any of items 105 to 112, wherein the sphingolipid is a complex sphingolipid, such as a sphingomyelin, a glycosphingolipid or an inositol-containing ceramide. (Item 115) 115. The method according to any of items 105 to 114, wherein the steroid, such as cholesterol, and the sphingolipid, such as sphingomyelin, are dissolved in the same water-miscible solvent. (Item 116) 115. The method according to any of items 105 to 114, wherein the steroid, such as cholesterol, and the sphingolipid, such as sphingomyelin, are dissolved in different water-miscible solvents. (Item 117) 117. The method according to any of items 105 to 116, wherein dissolution of the steroid and / or the sphingolipid in the water-miscible solvent is facilitated by heating and / or mixing, e.g. stirring. (Item 118) Item 119. The method according to Item 117, wherein the heating is carried out at a temperature ranging from about 35°C to about 65°C. 119. The method of any of items 105 to 118, wherein the water-miscible solvent is an organic or inorganic compound. (Item 120) Item 119. The method according to item 119, wherein the organic compound is an alcohol, for example a C1 to C5 alcohol. (Item 121) 121. The method of claim 120, wherein the alcohol is methanol, ethanol, 1-propanol, 1,3-propanediol, 1,5-pentanediol, or isopropyl alcohol (isopropanol or IPA). (Item 122) 122. The method according to any of items 105 to 121, wherein in the combination, the water-miscible solvent, e.g., IPA, has a concentration or level ranging from about 5% (v / v) to about 40% (v / v). (Item 123) 123. The method according to any of items 105 to 122, wherein the combination has a mass concentration in the range of about 0.25 mg / ml to about 20 mg / ml, such as about 2.5 mg / ml. (Item 124) 124. The method according to any of items 105 to 123, wherein in said combination said steroid, such as cholesterol, has a concentration or level in the range of about 20% (w / w) to about 50% (w / w), such as about 40% (w / w). (Item 125) 125. The method of any of items 105 to 124, wherein in the combination, the sphingolipid, such as sphingomyelin, has a concentration or level in the range of about 20% (w / w) to about 90% (w / w), such as about 40% (w / w). (Item 126) 126. The method according to any of items 105 to 125, wherein in the combination, the cell membrane has a concentration or level in the range of about 5% (w / w) to about 50% (w / w), for example about 20% (w / w). (Item 127) 127. The method of any of items 105 to 126, wherein the step of applying exogenous energy to the combination comprises subjecting the combination to ultrasonic treatment in a liquid. (Item 128) Item 128. The method according to item 127, wherein the combination is subjected to ultrasonic treatment at a temperature ranging from about 15°C to about 50°C. (Item 129) Item 129. The method according to item 127 or 128, wherein the combination is subjected to ultrasonic treatment at a frequency ranging from about 20 kilohertz (kHz) to about 60 kHz. (Item 130) 129. The method according to any of items 127 to 129, wherein the combination is subjected to ultrasonic treatment for a time period ranging from about 5 minutes to about 50 minutes. (Item 131) 131. The method according to any of items 105 to 130, wherein the step of applying exogenous energy to the combination, for example subjecting the combination to ultrasonic treatment, is carried out to homogenize the cell membranes, steroids and sphingolipids in the combination. (Item 132) 131. The method of any of items 105 to 130, producing nanoparticles whose outer surface (or shell) comprises fused cell membranes, steroids and sphingolipids. (Item 133) 133. The method of any of items 105 to 132, producing nanoparticles having a particle distribution index (PDI) of about 0.2 or higher. (Item 134) 134. The method according to any of items 105 to 133, further comprising, after step b), subjecting the nanoparticles to high shear treatment (or high shear force treatment) in a high shear fluid processor. (Item 135) Item 135. The method of item 134, wherein the high shear fluid processor is a microfluidizer (or a microfluidizer processor) or a homogenizer that generates high shear forces. (Item 136) Item 136. The method of item 135, wherein the high shear fluid processor is a microfluidizer (or a microfluidizer processor). (Item 137) Item 137. The method of item 136, wherein the microfluidizer is configured to generate a substantially constant pressure of about 10,000 psi to about 30,000 psi. (Item 138) Item 138. The method according to Item 136 or 137, wherein the microfluidizer has a microfluidic reaction technology (MRT) configuration including, from upstream to downstream, an inlet for inputting the nanoparticles, an intensifier pump for generating static pressure, an impinging jet chamber for generating high shear pressure on the nanoparticles, and an outlet for removing the nanoparticles. (Item 139) 139. The method of any of items 136 to 138, wherein the microfluidizer comprises a Z-type interaction chamber. (Item 140) 139. The method of any of items 136 to 138, wherein the microfluidizer comprises a Y-type interaction chamber. (Item 141) 141. The method according to any of items 136 to 140, wherein the microfluidization is carried out using a single pressure or a combination of different pressures. (Item 142) 142. The method according to any of items 136 to 141, wherein the microfluidization is carried out using a single pass or multiple passes. (Item 143) 143. The method of any of items 136 to 142, further comprising the step of cooling the nanoparticles. (Item 144) 144. The method of any of items 136 to 143, wherein the nanoparticles in the channel after exiting the chamber of the microfluidizer are cooled by a product chiller containing a coolant. (Item 145) Item 145. The method of claim 144, wherein the product chiller, or the coolant in the product chiller, is set to a temperature in the range of about 4°C to about 55°C. (Item 146) 146. The method according to any of items 105 to 145, producing nanoparticles having a particle distribution index (PDI) in the range of about 0.05 to about 0.2. (Item 147) 147. The method of any of items 105 to 146, producing nanoparticles having a particle size with a Z-average value in the range of about 30 nm to about 300 nm. (Item 148) below: 1) removing or reducing the level of particles having a particle size of about 200 nm or larger; 2) removing or reducing the level of the water-miscible solvent; 3) concentrating the nanoparticles; 4) sterilizing the nanoparticles; and / or 5) Packing the nanoparticles into individual containers The method according to any one of items 105 to 147, further comprising one or more of the following: (Item 149) below: 1) removing or reducing the level of particles having a particle size of about 200 nm or larger; 2) removing or reducing the level of the water-miscible solvent; 3) concentrating the nanoparticles; 4) sterilizing the nanoparticles; and / or 5) Packing the nanoparticles into individual containers 149. The method of any of items 105 to 149, further comprising two, three, four or five of the following: (Item 150) Item 149. The method according to item 148 or 149, wherein step 1) comprises filtering the liquid containing the nanoparticles. (Item 151) Item 151. The method of item 150, wherein the filtration is performed using a filter comprising polyethersulfone (PES), polyvinylidene difluoride (PVDF), glass fiber, cellulose acetate, or a combination thereof. (Item 152) Item 152. The method according to item 150 or 151, wherein the filter has a pore size ranging from about 0.2 μm to about 1.2 μm. (Item 153) 153. The method of any of items 150 to 152, wherein the filtration is carried out using a single filter. (Item 154) 154. The method according to any of items 150 to 153, wherein the filtration is carried out using a plurality of filters having different pore sizes. (Item 155) 155. The method of any of items 150 to 154, wherein the filtration is carried out to remove or reduce the level of particles having a particle size of about 200 nm or larger and to sterilize the nanoparticles. (Item 156) 156. The method according to any of items 150 to 155, wherein step 2) and / or 3) comprises subjecting the liquid containing the nanoparticles to tangential flow filtration (TFF). (Item 157) Item 157. The method of item 156, wherein the TFF is performed using a TFF system comprising a feed reservoir, a filter device, and a collection device, wherein the feed reservoir is in fluid communication with the filter device through an inlet of the filter device, the filter device is in fluid communication with the collection device through a permeate outlet of the filter device, and the filter device is in fluid communication with the feed reservoir through a retentate outlet of the filter device. (Item 158) 158. The method of claim 157, wherein the filter device is in the form of a cartridge, cassette, or column containing a hollow fiber filter. (Item 159) Item 159. The method of item 158, wherein the filter device comprises a filtration membrane having a pore size in the range of about 100 Kd to about 300 Kd, for example 300 Kd. (Item 160) 159. The method of any of items 156 to 159, wherein the TFF is carried out by a diafiltration method. (Item 161) 161. The method of claim 160, wherein the diafiltration process is a continuous, intermittent, or sequential diafiltration process. (Item 162) 162. The method of claim 160 or 161, wherein the TFF is carried out by a single cycle or a multiple cycle diafiltration process, such as by a multiple cycle continuous diafiltration process. (Item 163) 163. The method of any of items 156 to 162, further comprising the step of collecting the nanoparticles. (Item 164) Item 164. The method of item 163, wherein the nanoparticles are collected from a retentate outlet of the filter device. (Item 165) 165. The method of any of items 156 to 164, wherein the TFF is used to reduce the amount or level of the water-miscible solvent from a composition, e.g. a liquid, containing the nanoparticles. (Item 166) 165. The method of any of items 156 to 164, wherein the TFF is used to remove the water-miscible solvent from a composition, e.g. a liquid, containing the nanoparticles. (Item 167) 167. The method of claim 165 or 166, wherein the TFF is used to remove about 50% to about 99.9999% of the water-miscible solvent from the composition. (Item 168) 167. The method of claim 165 or 166, wherein the water-miscible solvent is IPA and the TFF is used to reduce the concentration or level of the IPA in the composition to less than 5,000 ppm. (Item 169) Item 169. The method of item 168, wherein the water-miscible solvent is IPA and the TFF is used to reduce the concentration or level of the IPA in the composition to less than 1,000 ppm. (Item 170) 169. The method of any of items 156 to 169, wherein the TFF is used to concentrate and / or enrich the nanoparticles from about 1-fold to about 400-fold. (Item 171) 171. The method according to any of items 156 to 170, wherein steps 2) and 3) are combined into a single step comprising subjecting the liquid containing the nanoparticles to tangential flow filtration (TFF). (Item 172) 172. The method according to any of items 156 to 171, wherein after the TFF treatment, the composition, e.g., liquid, containing the nanoparticles is subjected to a step of sterilizing the composition containing the nanoparticles. (Item 173) Item 173. The method of item 172, wherein sterilizing the composition comprises filtering the composition. (Item 174) Item 174. The method of item 173, wherein the filtration is performed using a filter having a pore size of about 0.2 μm, and / or the filtration is performed using a filter comprising PES, PVDF, glass fiber, cellulose acetate, or a combination thereof. (Item 175) 175. The method of any of items 105 to 174, which does not include a step of removing or evaporating the water-miscible solvent to form a film comprising the steroid and the sphingolipid. (Item 176) 176. The method of any of items 105 to 175, wherein the cell membrane comprises a red blood cell derived plasma membrane, the steroid is cholesterol, the sphingolipid is sphingomyelin, and the water-miscible solvent is IPA. (Item 177) 177. Nanoparticles prepared by the method according to any one of items 95 to 176. (Item 178) 177. Nanoparticles prepared by the method according to any one of items 105 to 176. (Item 179) Item 179. The nanoparticle according to item 178, wherein the inner compartment (or inner core) of the nanoparticle does not provide a solid support for the outer surface (or shell) and / or is isotonic with cellular or physiological fluids, e.g., the inner compartment (or inner core) comprises a liquid that is isotonic with cellular or physiological fluids. (Item 180) 179. The nanoparticle according to claim 178 or 179, wherein the outer surface (or shell) comprises cells, cholesterol and sphingomyelin derived from cell membranes. (Item 181) 181. The nanoparticle according to item 180, wherein the outer surface (or shell) comprises cells, cholesterol and sphingomyelin derived from fused cell membranes. (Item 182) 182. The nanoparticle according to item 180 or 181, wherein the outer surface (or shell) comprises erythrocytes, cholesterol and sphingomyelin derived from the plasma membrane. (Item 183) 183. The nanoparticle according to item 182, wherein the outer surface (or shell) comprises about 20% (w / w) to about 50% (w / w) cholesterol, about 20% (w / w) to about 80% (w / w) sphingomyelin, and about 10% (w / w) to about 50% (w / w) plasma membrane derived from red blood cells. (Item 184) 184. The nanoparticle according to item 183, wherein the outer surface (or shell) comprises about 40% (w / w) cholesterol, about 40% (w / w) sphingomyelin, and about 20% (w / w) plasma membrane derived from red blood cells. (Item 185) A nanoparticle comprising an inner compartment (or inner core) and an outer surface (or shell), wherein the inner compartment (or inner core) does not provide a solid support for the outer surface (or shell) and / or is isotonic with cellular fluids or physiological fluids, e.g., the inner compartment (or inner core) comprises a fluid that is isotonic with cellular fluids or physiological fluids, and the outer surface (or shell) comprises cells, cholesterol, and sphingomyelin derived from cell membranes. (Item 186) 186. The nanoparticle of item 185, wherein the outer surface (or shell) comprises cells, cholesterol and sphingomyelin derived from fused cell membranes. (Item 187) 187. The nanoparticle according to item 185 or 186, wherein the outer surface (or shell) comprises erythrocytes, cholesterol and sphingomyelin derived from the plasma membrane. (Item 188) 188. The nanoparticle according to item 187, wherein the outer surface (or shell) comprises about 20% (w / w) to about 50% (w / w) cholesterol, about 20% (w / w) to about 80% (w / w) sphingomyelin, and about 10% (w / w) to about 50% (w / w) plasma membrane derived from red blood cells. (Item 189) 189. The nanoparticle according to item 188, wherein the outer surface (or shell) comprises about 40% (w / w) cholesterol, about 40% (w / w) sphingomyelin, and about 20% (w / w) plasma membrane derived from red blood cells. (Item 190) 189. The nanoparticles according to any of items 1 to 94 and 177 to 189, having better stability than comparable nanoparticles not containing said enhanced or enriched levels of steroid. (Item 191) 191. The nanoparticle of any of items 1 to 94 and 177 to 190, wherein the cell membrane of the nanoparticle is configured to bind to a cell of a certain type to which it is derived. (Item 192) 192. The nanoparticle according to any of items 1 to 94 and 177 to 191, configured to bind to or neutralize the cell membrane targeting or binding moiety of the nanoparticle. (Item 193) 193. The nanoparticle according to item 192, wherein the moiety is an agent, such as a chemical agent, a molecule or a biological agent. (Item 194) 194. The nanoparticle of item 193, wherein the moiety is a toxin, cytokine, autoantibody, or chemokine. (Item 195) 195. The nanoparticles according to item 194, wherein the toxin is a bacterial toxin, a fungal toxin, an animal toxin or a chemical toxin. (Item 196) 196. The nanoparticles according to item 195, wherein the chemical toxin is an organophosphate. (Item 197) 196. The nanoparticles according to item 195, wherein the animal toxin is a toxin in an animal venom. (Item 198) 194. The nanoparticles according to item 193, wherein the organism is a virus, bacterium, fungus or parasite. (Item 199) 199. The nanoparticle of any of items 1 to 94 and 177 to 198, having a better ability to bind to or neutralize the cell membrane targeting or binding moiety of the nanoparticle than a comparable nanoparticle that does not contain the enhanced or enriched level of steroid. (Item 200) A pharmaceutical delivery system or device comprising an effective amount of nanoparticles according to any of items 1 to 94 and 177 to 199. (Item 201) 201. The pharmaceutical delivery system or device according to item 200, further comprising another active ingredient or a medically or pharmaceutically acceptable carrier or excipient. (Item 202) A pharmaceutical composition comprising an effective amount of nanoparticles according to any of items 1 to 94 and 177 to 199, and a pharmaceutically acceptable carrier or excipient. (Item 203) 203. The pharmaceutical composition according to item 202, further comprising another active ingredient. (Item 204) 204. The pharmaceutical composition according to item 202 or 203, adapted to treat or prevent a disease or condition associated with the cell membrane-targeting or binding moiety of the nanoparticle. (Item 205) 206. The pharmaceutical composition according to any one of claims 202 to 204, wherein the outer surface of the nanoparticle comprises a plasma membrane derived from a blood cell, such as a red blood cell, a white blood cell, and / or a platelet. 1. A method of treating or preventing a disease or condition in a subject in need thereof, comprising: a) a nanoparticle comprising an inner compartment (or inner core) and an outer surface (or shell) comprising a cell membrane derived from a cell, wherein the inner compartment (or inner core) does not provide a solid support for the cell membrane in the outer surface (or shell); or b) the nanoparticles according to any of items 1 to 94 and 177 to 199, the pharmaceutical delivery system or device according to any of items 200 to 201, or the pharmaceutical composition according to any of items 202 to 205 to said subject an effective amount of (Item 207) 207. The method of claim 206, wherein the subject is a human or non-human mammal. (Item 208) 208. The method of claim 206 or 207, wherein the cell membrane in the nanoparticles is derived from a cell of the same species as the subject or derived from a cell of the subject. (Item 209) 209. The method of claim 208, wherein the cell membrane in the nanoparticles is derived from blood cells, such as red blood cells, white blood cells, and / or platelets. (Item 210) 209. The method of claim 208, wherein the cell membranes in the nanoparticles are derived from red blood cells of the same species as the subject, and the red blood cells have the same blood type as the subject. (Item 211) 211. The method of any of items 206 to 210, used to treat or prevent a disease or condition associated with the cell membrane-targeting or binding moiety of the nanoparticle. (Item 212) Item 213. The method of item 211, wherein the moiety is an agent, such as a chemical agent, molecule, or organism. 213. The method of claim 212, wherein the organism is a bacterium, a fungus, or a parasite. (Item 214) 213. The method of claim 212, wherein the moiety is a toxin, cytokine, autoantibody, or chemokine. (Item 215) 215. The method of claim 214, wherein the toxin is a bacterial toxin, a fungal toxin, an animal toxin, or a chemical toxin. (Item 216) 216. The method of claim 215, wherein the chemical toxin is an organophosphate. (Item 217) 216. The method of claim 215, wherein the animal toxin is a toxin in an animal venom. (Item 218) 216. The method of claim 215, wherein the toxin is a toxin that inserts into a cell membrane. (Item 219) 219. The method of claim 218, wherein the toxin inserts into the cell membrane or plasma membrane of a target cell of the subject as part of the toxin's natural pathological mechanism. (Item 220) 219. The method of claim 218, wherein the cell or plasma membrane at the outer surface of the nanoparticles substantially retains the toxin. (Item 221) 221. The method of any of items 206 to 220, wherein the outer surface of the nanoparticles comprises a plasma membrane derived from a blood cell, such as a red blood cell, a white blood cell and / or a platelet. (Item 222) 222. The method according to any of items 206 to 221, further comprising administering to the subject in need thereof another active ingredient or a pharmaceutically acceptable carrier or excipient, or wherein the nanoparticles are administered by a pharmaceutical delivery system or device. (Item 223) 202. Use of an effective amount of nanoparticles according to any of items 1 to 94 and 177 to 199 for the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof. (Item 224) 201. An immunogenic composition comprising an effective amount of nanoparticles according to any of items 1 to 94 and 177 to 199. (Item 225) 225. The immunogenic composition of claim 224, configured as a neoplasm-specific immunogenic composition, wherein the outer surface of the nanoparticles comprises a cell membrane derived from a neoplastic cell. (Item 226) 227. The neoplasm-specific immunogenic composition of claim 225, wherein the cell membrane is derived from a benign neoplastic cell, a potentially malignant neoplastic cell, a cancer cell, a cancer cell line, or a cancer cell of a subject. 227. The neoplasm-specific immunogenic composition according to item 225 or 226, wherein the cell membrane in the outer surface of the nanoparticles substantially retains its structural integrity for eliciting an immune response against the neoplastic cells. (Item 228) 228. The neoplasm-specific immunogenic composition according to any of items 225 to 227, further comprising an immunogenic adjuvant. (Item 229) 229. The neoplasm-specific immunogenic composition according to any of items 225 to 228, further comprising an immune enhancing substance. (Item 230) 229. The neoplasm-specific immunogenic composition according to any of items 225 to 229, wherein the nanoparticles further comprise another active ingredient or releasable cargo. (Item 231) 231. The neoplasm-specific immunogenic composition according to any of items 225 to 230, wherein the nanoparticles have a diameter of about 10 nm to about 10 μm. (Item 232) 232. The neoplasm-specific immunogenic composition according to any of items 225 to 231, wherein the nanoparticles are substantially devoid of constituents of the neoplastic cells from which the cell membrane is derived. (Item 233) 232. The neoplasm-specific immunogenic composition according to any of items 225 to 231, wherein the cell membrane of the outer surface (or shell) does not contain enhanced or enriched levels of steroids. (Item 234) 234. The neoplasm-specific immunogenic composition according to any of items 225 to 233, wherein the outer surface of the nanoparticles comprises a naturally occurring cell membrane and further comprises a synthetic membrane. (Item 235) 235. A vaccine comprising an immunogenic composition specific for a neoplasm according to any of items 225 to 234. (Item 236) 235. A method for treating or preventing a neoplasm in a subject in need thereof, comprising administering to said subject an effective amount of an immunogenic composition specific for a neoplasm according to any of items 225 to 234 or a vaccine according to item 235. (Item 237) 237. The method of claim 236, wherein the subject is a human or non-human mammal. (Item 238) 238. The method of claim 236 or 237, wherein the cell membrane is derived from a neoplastic cell of the same species as the subject or a neoplastic cell of the subject. (Item 239) 239. The method of any of items 236 to 238, further comprising administering to the subject another active ingredient or a pharmaceutically acceptable carrier or excipient. (Item 240) 235. Use of an effective amount of a neoplasm-specific immunogenic composition according to any of items 225 to 234 for the manufacture of a vaccine for treating or protecting a subject from a neoplasm. (Item 241) 225. The immunogenic composition of claim 224, configured to treat or prevent a disease or condition associated with a cell membrane-targeting or binding moiety of the nanoparticle, wherein the outer surface of the nanoparticle comprises the moiety. (Item 242) 242. The immunogenic composition according to item 241, wherein the moiety is an agent, such as a chemical agent, a molecule, an organism or an antigen thereof. (Item 243) Item 244. The immunogenic composition of Item 242, wherein the organism is a bacterium, a fungus, or a parasite. 242. The immunogenic composition of claim 241, wherein the moiety is a toxin, cytokine, autoantibody, or chemokine. (Item 245) 245. The immunogenic composition of item 244, wherein the toxin is a bacterial toxin, a fungal toxin, an animal toxin, or a chemical toxin. (Item 246) 246. The immunogenic composition of item 245, wherein the chemical toxin is an organophosphate. (Item 247) 246. The immunogenic composition of item 245, wherein the animal toxin is a toxin in an animal venom. (Item 248) 245. The immunogenic composition of item 244, wherein the toxin is a toxin that inserts into a cell membrane. (Item 249) 249. The immunogenic composition of claim 248, wherein the toxin inserts into the cell membrane or plasma membrane of a target cell of the subject as part of the toxin's natural pathological mechanism. (Item 250) 240. The immunogenic composition of item 248 or 249, wherein the cell or plasma membrane in the outer surface of the nanoparticles substantially retains the toxin. (Item 251) 251. The immunogenic composition according to any of items 241 to 250, wherein the cell membrane is a plasma membrane derived from a cell. (Item 252) 252. The immunogenic composition according to any of items 241 to 251, wherein the outer surface of the nanoparticles comprises a naturally occurring cell membrane and further comprises a synthetic membrane. (Item 253) 253. The immunogenic composition according to any of items 241 to 252, wherein the nanoparticles are biocompatible, biodegradable or comprise synthetic materials. (Item 254) 254. The immunogenic composition according to any of items 241 to 253, wherein the inner compartment (inner core) does not support the outer surface. (Item 255) 255. The immunogenic composition according to any of items 241 to 254, wherein the outer surface comprises a plasma membrane derived from an erythrocyte. (Item 256) 256. The immunogenic composition according to any of items 241 to 255, further comprising another active ingredient. (Item 257) 257. The immunogenic composition according to any of items 241 to 256, further comprising an immunogenic adjuvant or immunopotentiator. (Item 258) 258. A vaccine comprising the immunogenic composition according to any of items 241 to 257. (Item 259) 258. A method for inducing an immune response in a subject to an area associated with a disease or condition, the method comprising administering to the subject an effective amount of the immunogenic composition of any of items 241 to 257. (Item 260) 259. A method of protecting a subject from a disease or condition associated therewith, comprising administering to said subject an effective amount of the vaccine of claim 258. (Item 261) 261. The method of claim 260, wherein the subject is a human or non-human mammal. (Item 262) 262. The method of claim 260 or 261, wherein the cell membrane or plasma membrane is derived from a cell of the same species as the subject or from a cell of the subject. (Item 263) 263. The method of claim 262, wherein the plasma membrane is derived from red blood cells of the same species as the subject, and the red blood cells have the same blood type as the subject. (Item 264) 264. The method of any of items 259 to 263, further comprising administering to the subject another active ingredient or a pharmaceutically acceptable carrier or excipient. (Item 265) 265. The method of any of items 259 to 264, wherein the immune response is a T cell-mediated immune response or a B cell-mediated immune response. (Item 266) 258. Use of an effective amount of an immunogenic composition according to any of items 241 to 257 for the manufacture of a vaccine for protecting a subject against a disease or condition associated with said moiety. (Item 267) 223. The method according to any of items 206 to 222, wherein the disease or condition is an infection, such as a skin infection, sepsis, pneumonia or an autoimmune reaction, such as an autoimmune reaction due to the production of autoimmune antibodies, such as autoimmune antibodies against blood cells or red blood cells. (Item 268) 268. The method according to any of items 206 to 222 and 267, wherein the nanoparticles or the pharmaceutical composition are administered by the enteral / gastrointestinal, oral, parenteral, intravenous, rectal, nasal, topical, ocular, inhalation or intratracheal route. (Item 269) 269. The method according to item 267 or 268, wherein the disease or condition is an infection, such as a skin infection, sepsis or pneumonia, and the nanoparticles or the pharmaceutical composition are administered by the intratracheal route. (Item 270) 269. The method of claim 269, wherein the nanoparticles or the pharmaceutical composition are administered by intratracheal instillation or intratracheal inhalation. (Item 271) 239. The method of any of items 236 to 239, wherein the neoplasm-specific immunogenic composition or vaccine is administered by the enteral / gastrointestinal, oral, parenteral, intravenous, rectal, nasal, topical, ocular, inhalation or intratracheal route. (Item 272) 266. The method of any of items 259 to 265, wherein the immunogenic composition is administered by enteral / gastrointestinal, oral, parenteral, intravenous, rectal, nasal, topical, ocular, inhalation or intratracheal route. (Item 273) 272. The method of any of items 206-222, 236-239, 259-265 and 267-272, further comprising administering to the subject a second therapeutic agent. (Item 274) Item 275. The method of Item 273, wherein the second therapeutic agent is an antibiotic, an antitumor or anticancer agent, or an immune response modulator, e.g., an immune response activator or suppressor. 272. The method of any of items 206 to 222, 236 to 239, 259 to 265 and 267 to 272, not including the step of administering to the subject a second therapeutic agent.

[0018] V. Brief description of the drawings Those skilled in the art will understand that the drawings, described below, are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows the preparation and characterization of cholesterol-enriched RBC membrane vesicles (Cho-RBC-V). (A) Loading yield of exogenous cholesterol at different initial cholesterol loadings. (B) Calcein leakage from Cho-RBC-V (initial loading 10%) compared to membrane vesicles without added exogenous cholesterol. (C) Dose-dependent neutralization of MRSA culture supernatants against RBC hemolysis. For all studies, data represent mean ± standard deviation (n = 3).

[0020] [Figure 2]Figure 2 shows the preparation and characterization of cholesterol-enriched platelet membrane vesicles (Cho-PL-V). (A) Loading yield of exogenous cholesterol at different initial cholesterol loadings. (B) Calcein leakage from Cho-PL-V (initial loading 10%) compared to membrane vesicles without added exogenous cholesterol. (C) Dose-dependent neutralization of anti-platelet antibodies with Cho-PL-V. In all studies, data represent the mean ± standard deviation (n = 3).

[0021] [Figure 3] Figure 3 shows the preparation and characterization of cholesterol-enriched macrophage membrane vesicles (Cho-MΦ-V). (A) Loading yield of exogenous cholesterol at different initial cholesterol loadings. (B) Calcein leakage from Cho-MΦ-V (initial loading 10%) compared to membrane vesicles without added exogenous cholesterol. (C) Dose-dependent neutralization of LPS (endotoxin) reflected by reduced IL-6 production in culture by macrophage cells. In all studies, data represent mean ± standard deviation (n = 3).

[0022] [Figure 4] Figure 4 shows the preparation and characterization of cholesterol-enriched neutrophil membrane vesicles (Cho-Neu-V). (A) Loading yield of exogenous cholesterol at different initial cholesterol loadings. (B) Calcein leakage from Cho-Neu-V (initial loading 10%) compared to membrane vesicles without added exogenous cholesterol. (C) Dose-dependent neutralization of TNF-α. For all studies, data represent the mean ± standard deviation (n = 3).

[0023] [Figure 5]FIG. 5 illustrates an exemplary method for making Composition A, which comprises nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell), wherein the inner compartment (or inner core) does not provide a solid support for the outer surface (or shell), and the outer surface (or shell) comprises red blood cells, cholesterol, and sphingomyelin derived from the plasma membrane.

[0024] [Figure 6] FIG. 6 is a graph illustrating the particle size distribution of exemplary Composition A.

[0025] [Figure 7] Figure 7 is a graph illustrating exemplary in vivo efficacy of Composition A. A. Survival curves within the first 48 hours after bacterial challenge and formulation treatment. B. Bacterial burden in lung tissue obtained from mice surviving 48 hours after bacterial challenge and formulation treatment. Data were analyzed using a log-rank (Mantel-Cox) test (A) and a Student's t-test (B). CFU counts in lungs obtained from individual mice surviving 48 hours after challenge / treatment were log-transformed and plotted in Figure 7B. Lung bacterial counts in mice treated with Composition A were significantly lower than those in vehicle-treated mice.

[0026] [Figure 8]Figure 8 is a graph illustrating an exemplary reproducible effect of Composition A on survival. A. Survival curves from a single study in which two different lots of Composition A, 5-257-06 and 5-257-16, were administered intratracheally in mice immediately after intratracheal instillation with MRSA. *p<0.05; **p<0.01. B. Averaged survival curves from three separate studies in which three lots of Composition A, 5-257-06, 5-257-16, and 5-260-02, were administered intratracheally in mice immediately after intratracheal instillation with MRSA. Data are presented as mean ± standard error. Survival rates in vehicle- vs. lot-treated groups at each time point (19, 26, 48, or 96 hours) were analyzed using an unpaired Student's t-test. *p<0.05; **p<0.01.

[0027] [Figure 9] 9 is a graph illustrating an exemplary dose-curve effect of Composition A on survival rate. Lot 5-266-01 was used in this study. Survival dates were pooled from four independent assays using a total of 40 mice administered vehicle, 19 mg / kg, or 2 mg / kg of 5-266-01, and a total of 30 mice administered 6 mg / kg of 5-266-01.

[0028] [Figure 10] 10 is a graph illustrating an exemplary effect of Composition A on lung bacterial burden. Composition A lot 5-257-16 was administered at 22.5 mg / kg or vehicle. Mice lungs were harvested 24 hours after challenge / treatment.

[0029] [Figure 11] 11 illustrates an exemplary in vitro characterization of DiR-Composition A. The table in the top panel shows dynamic light scattering data for DiR-Composition A. The graph at the bottom shows the stability of the fluorescence intensity of DiR-Composition A after dialysis for up to 180 hours.

[0030] [Figure 12] 12 is a graph showing exemplary lung uptake and distribution of DiR-Composition A after intratracheal administration. Time indicates the number of minutes or hours after intratracheal administration of DiR-Composition A. Each sample represents the lungs of a single mouse.

[0031] [Figure 13] FIG. 13 is a graph illustrating an exemplary retention time course of DiR-Composition A in the lungs of mice following intratracheal administration.

[0032] [Figure 14] FIG. 14 is a graph illustrating an exemplary retention time course of DiR-Composition A in the lungs of mice following intratracheal administration.

[0033] [Figure 15] 15 illustrates exemplary uptake profiles of DiR-Composition A at two different concentrations. There is little or no difference between the uptake profiles of DiR-Composition A in the lungs of mice administered 20 mg / mL (22 mg / kg) or 40 mg / mL (44 mg / kg) of DiR-Composition A intratracheally. DETAILED DESCRIPTION OF THE INVENTION

[0034] VI. DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention employs, unless otherwise indicated, conventional techniques of nanotechnology, nanoengineering, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, and pharmacology, which are within the skill of those in the art. Such techniques are explained fully in the literature, such as in Molecular Cloning: A Laboratory Manual, 2 nded. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22 th ed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.All patents, applications, published applications and other publications mentioned in this specification are incorporated by reference in their entirety.If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, applications, published applications and other publications incorporated herein by reference, the definitions set forth in this section shall prevail over the definitions incorporated herein by reference. A.Definition

[0036] To facilitate understanding of the present invention, several terms and abbreviations used herein are defined below.

[0037] When introducing elements of the invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0038] The term "and / or," when used in the listing of two or more items, means that any one of the listed items can be used by itself or in combination with any one or more of the listed items. For example, the phrase "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or A and B in combination. The phrase "A, B, and / or C" is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0039] Cell membrane: As used herein, the term "cell membrane" refers to a biological membrane surrounding or separating a structure that acts as a selective barrier within or around a cell or an emerging virus particle. Cell membranes are selectively permeable to ions and organic molecules, controlling the movement of materials in and out of the cell. Cell membranes contain a single or double phospholipid layer, optionally associated with proteins and carbohydrates. As used herein, cell membrane refers to a membrane obtained from a naturally occurring biological membrane of a cell or organelle, or derived therefrom. As used herein, the term "naturally occurring" refers to something that exists in nature. As used herein, the term "derived therefrom" refers to any subsequent modification of the native membrane, such as isolating the cell membrane, generating a portion or fragment of the membrane, or removing and / or adding certain components, such as lipids, proteins, or carbohydrates, derived from or within the membrane taken from the cell or organelle. The membrane may be derived from a naturally occurring membrane by any suitable method. For example, membranes can be prepared or isolated from cells or viruses, and the prepared or isolated membranes can be combined with other substances or materials to form derived membranes. In another example, cells or viruses can be recombinantly engineered to produce "non-native" substances that are incorporated into their membranes in vivo, and cellular or viral membranes can be prepared or isolated from cells or viruses to form derived membranes.

[0040] In various embodiments, the cell membrane covering either the unilamellar or multilamellar nanoparticles can be further modified to be saturated or unsaturated with other lipid components, such as cholesterol, free fatty acids, and phospholipids, and can also contain endogenous or added proteins and carbohydrates, such as cell surface antigens. In such cases, excess amounts of other lipid components can be added to the membrane wall, which reduces their concentration in the membrane wall until an equilibrium is reached, which can depend on the nanoparticle's environment. The membrane can also contain other agents that may or may not enhance the activity of the nanoparticle. In other examples, functional groups, such as antibodies and aptamers, can be added to the outer surface of the membrane to enhance targeting sites, such as cell surface epitopes found on cancer cells. The nanoparticle membrane can also contain particles, which can be biodegradable cationic nanoparticles, including, but not limited to, gold, silver, and synthetic nanoparticles.

[0041] Synthetic or artificial membrane: As used herein, the term "synthetic membrane" or "artificial membrane" refers to a man-made membrane made from organic and inorganic materials, such as polymers and liquids. A wide range of synthetic membranes are known in the art.

[0042] Nanoparticles: In some embodiments, the term "nanoparticle," as used herein, refers to nanostructures, particles, vesicles, or fragments thereof having at least one dimension (e.g., height, length, width, or diameter) between about 1 nm and about 10 μm. For systemic use, an average diameter of about 30 nm to about 500 nm, or about 30 nm to about 300 nm, or about 50 nm to about 250 nm may be preferred. The term "nanostructure" includes, but is not necessarily limited to, particles and engineered features. Particles and engineered features can have, for example, regular or irregular shapes. Such particles are also referred to as nanoparticles. Nanoparticles can be composed of organic or other materials and, alternatively, can comprise porous particles. A layer of nanoparticles can comprise a monolayer of nanoparticles or a layer having aggregates of nanoparticles. In some embodiments, nanoparticles are discussed herein that comprise or consist of an inner compartment (or inner core) surrounded by an outer surface (or shell) comprising a membrane. The present disclosure contemplates any currently known or later developed nanoparticles that can be coated with the films described herein.

[0043] Pharmaceutically active: The term "pharmaceutically active," as used herein, refers to the beneficial biological activity of a substance on living matter, particularly the cells and tissues of the human body. A "pharmaceutical active agent" or "drug" is a pharmaceutically active substance, and a "pharmaceutically active ingredient" (API) is the pharmaceutically active substance in a drug.

[0044] Pharmaceutically acceptable: The term "pharmaceutically acceptable," as used herein, means approved by a state or national government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia, in addition to other formulations that are safe for use in animals, and more particularly, in humans and / or non-human mammals.

[0045] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to an acid or base addition salt of a compound, such as a multiple drug conjugate, in the present disclosure. A pharmaceutically acceptable salt is any salt that retains the activity of the parent nanoparticle or compound and does not cause any adverse or undesirable effects in the subject and situation in which it is administered. Pharmaceutically acceptable salts can be derived from amino acids, including, but not limited to, cysteine. Methods for producing compounds as salts are known to those skilled in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J. Pharm. Sci. 66: 1, 1977). In some embodiments, "pharmaceutically acceptable salts" is intended to mean free acid or free base salts of the nanoparticles or compounds described herein that are non-toxic, biologically tolerated, or otherwise biologically suitable for administration to a subject. See generally Berge, et al., J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without undue toxicity, irritation, or allergic reaction. The nanoparticles or compounds described herein may have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one of each type, and thus react with several inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts.

[0046] Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, hydrogen sulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dicarboxylate, hexyne-1,6-dicarboxylate, These include benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, besylate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, [gamma]-hydroxybutyrate, glycolate, tartrate and mandelate salts.

[0047] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which nanoparticles or compounds, such as multi-drug conjugates, are administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity, such as sodium chloride or dextrose, can also be carriers. Methods for combining carriers to produce compositions are known to those skilled in the art. In some embodiments, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. See, for example, Remington, The Science and Practice of Pharmacy. 20''' ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the compositions is contemplated.

[0048] Phospholipid: The term "phospholipid" as used herein refers to any of a number of lipids that contain a single organic molecule, such as diglyceride, a phosphate group, and choline. Examples of phospholipids include, but are not limited to, phosphatide acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (lecithin) (PC), phosphatidylserine (PS), and phosphoinositides, including, but not limited to, phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol triphosphate (P1P3). Additional examples of PC include DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DRPC and DEPC, as defined in the art.

[0049] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount that, when administered to a particular subject, has the desired therapeutic effect, given the nature and severity of the disease or condition of interest, e.g., an amount that cures, prevents, inhibits, or at least partially arrests or partially prevents the target disease or condition. More specific embodiments are included in the pharmaceutical preparations and methods in the Administration section below. In some embodiments, the term "therapeutically effective amount" or "effective amount" refers to an amount of a therapeutic agent that, when administered to a cell, tissue, or subject alone or in combination with an additional therapeutic agent, is effective to prevent or ameliorate a disease or condition, such as a hemolytic disease or condition, or the progression of such a disease or condition. A therapeutically effective dose further refers to such an amount of a therapeutic agent sufficient to result in symptomatic improvement, e.g., treatment, cure, prevention, or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or amelioration of such a condition. A therapeutically effective dose refers to an active ingredient administered alone when applied to an individual. A therapeutically effective dose, when applied to a combination, refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0050] "Treating" or "treatment" or "palliative" refers to therapeutic treatment whose purpose is to slow (reduce), if not cure, the targeted pathological condition or disorder or prevent the recurrence of such a condition. A subject is successfully "treated" if, after taking a therapeutic amount of a therapeutic agent, the subject shows an observable and / or measurable reduction or disappearance of one or more signs and symptoms of a particular disease. The reduction of signs or symptoms of a disease may also be experienced by the patient. A patient is also considered to be treated if the patient experiences stable disease. In some embodiments, treatment with a therapeutic agent is effective to ensure that the patient is disease-free for 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 or more years after treatment. These parameters for assessing the success of treatment and improvement in disease can be easily measured by routine procedures familiar to a physician of the appropriate skill in the art.

[0051] As used herein, "prophylactic" treatment is intended to indicate postponing the onset of a disease, symptom of a disease or medical condition, suppressing symptoms that may appear, or reducing the risk of developing or recurring a disease or condition. "Curative" treatment includes reducing the severity of an existing disease, symptom, or condition, or preventing its worsening.

[0052] The term "combination" refers to either a fixed combination in a single dosage unit form or a kit of parts for combined administration, in which the nanoparticles or compound and the combination partner (e.g., another drug described below, also referred to as a "therapeutic agent" or "co-agent") can be administered simultaneously or separately within a time interval, particularly allowing the combination partners to exhibit a cooperative effect, e.g., a synergistic effect. As used herein, the terms "co-administration" or "combined administration" and the like are intended to encompass the administration of selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include therapeutic regimens in which the agents are not necessarily administered by the same administration route or at the same time. The term "pharmaceutical combination," as used herein, refers to a product obtained by mixing or combining more than one active ingredient, and includes both fixed and loose combinations of active ingredients. The term "fixed combination" means that both the active ingredients, e.g., nanoparticles or compound, and the combination partner are administered to a patient simultaneously in the form of a single entity or single dosage. The term "unfixed combination" means that both the active ingredient, for example, nanoparticles or compounds, and the combination partner are administered to a patient as separate entities simultaneously, simultaneously, or sequentially, without any specific time limit, whereby such administration achieves therapeutically effective levels of the two moieties or compounds in the patient's body. The latter unfixed combination also applies to cocktail therapy, for example, the administration of three or more active ingredients.

[0053] It will be understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0054] Throughout this disclosure, various aspects of the present invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Thus, the description of a range should be considered to include all specifically disclosed subranges as well as individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0055] As used herein, a subject in need refers to an animal, non-human mammal, or a human. As used herein, "animal" includes pets, livestock, industrial animals, sport animals, and laboratory animals such as cats, dogs, horses, cows, oxen, pigs, donkeys, sheep, lambs, goats, mice, rabbits, chickens, ducks, geese, monkeys, and primates, including chimpanzees.

[0056] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings. B. Nanoparticles containing cell membranes

[0057] In one aspect, the present disclosure provides nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising a cellular membrane derived from a cell, wherein the inner compartment (or inner core) does not provide a solid support for the cellular membrane in the outer surface (or shell), and wherein a) the inner compartment (or inner core) is isotonic with cellular fluids or physiological fluids, e.g., the inner compartment (or inner core) comprises a liquid that is isotonic with cellular fluids or physiological fluids, and / or b) the cellular membrane of the outer surface (or shell) comprises enhanced or enriched levels of a steroid, with the proviso that if the cellular membrane is derived from a red blood cell, the inner compartment (or inner core) is isotonic with cellular fluids or physiological fluids.

[0058] The inner compartment (or inner core) of the nanoparticles of the present invention can be isotonic with cellular or physiological fluids in any suitable manner. For example, the inner compartment (inner core) can contain a liquid that is isotonic with cellular or physiological fluids. In another example, the inner compartment (inner core) can contain a dry substance that, when reconstituted with a liquid, forms a liquid that is isotonic with cellular or physiological fluids.

[0059] The inner compartment (or inner core) of the nanoparticle of the present invention can be isotonic with cellular fluids or physiological fluids in any suitable environment. For example, the inner compartment (inner core) can be isotonic with cellular fluids or physiological fluids present outside a cell or subject. In another example, the inner compartment (inner core) can be isotonic with cellular fluids or physiological fluids present within a cell or subject.

[0060] In some embodiments, the inner compartment (inner core) of the nanoparticles of the present invention can comprise a liquid that is isotonic with the cellular fluid contained in the cells. In some embodiments, the inner compartment (inner core) of the nanoparticles of the present invention can comprise a dry material that, when reconstituted with a liquid, forms a liquid that is isotonic with the cellular fluid present in the cells.

[0061] The inner compartment (inner core) of the nanoparticles of the present invention can be isotonic with the cellular fluid contained in any suitable cell. The cell can be a prokaryotic or eukaryotic cell. In some embodiments, the cell can be a unicellular organism, such as a bacterial or fungal cell. The cell can also be a multicellular organism, such as a plant, animal, vertebrate, non-human mammal, or human cell.

[0062] In some embodiments, the cell is an animal cell, such as a non-human mammalian cell or a human cell. The cell can be any suitable animal cell. For example, the cell can be a cell of connective tissue, such as blood, bone, tendon, ligament, fat or loose connective tissue, fibrous connective tissue, skeletal connective tissue, or fluid connective tissue. In another example, the cell can be a cell of muscle tissue, such as visceral muscle or smooth muscle, musculoskeletal, or cardiac muscle. In yet another example, the cell can be a cell of nervous tissue, such as a cell in the central nervous system (CNS) or peripheral nervous system (PNS). In yet another example, the cell can be a cell of epithelial tissue, such as a cell of simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium (also known as ciliated columnar epithelium), columnar epithelium, glandular epithelium, or ciliated columnar epithelium. In yet another example, the cell can be a cell of the nervous system, cardiovascular system, circulatory system, vascular system, digestive system, endocrine system, immune system, integumentary system, lymphatic system, musculoskeletal system, reproductive system, respiratory system, ventilatory system, urinary system, or renal system or urinary tract. In yet another example, the cell can be a blood cell, tumor cell, cancer cell, immune cell, stem cell, endothelial cell, or epithelial cell.

[0063] The inner compartment (inner core) of the nanoparticle of the present invention can be isotonic with physiological fluid in any suitable environment. For example, the inner compartment (inner core) can be isotonic with physiological fluid present outside a cell or subject. In another example, the inner compartment (inner core) can be isotonic with physiological fluid present inside a cell or subject.

[0064] The inner compartment (inner core) of the nanoparticle of the present invention can be isotonic with physiological fluid in any suitable subject.For example, the inner compartment (inner core) can be isotonic with physiological fluid in a multicellular organism, such as a plant, an animal, a vertebrate, a non-human mammal, or a human.In some embodiments, the inner compartment (inner core) can be isotonic with physiological fluid in an animal, a vertebrate, a non-human mammal, or a human, such as circulating blood in an animal, a vertebrate, a non-human mammal, or a human.

[0065] The inner compartment (inner core) of the nanoparticles of the present invention can be isotonic with any suitable physiological fluid, for example, the physiological fluid can be that of the nervous, cardiovascular, circulatory, vascular, digestive, endocrine, immune, integumentary, lymphatic, musculoskeletal, reproductive, respiratory, respiratory, ventilatory, urinary, or renal system or urinary tract.

[0066] In some embodiments, the inner compartment (inner core) of the nanoparticles of the invention comprises a fluid that is isotonic with cellular or physiological fluids ex vivo, hi other embodiments, the inner compartment (inner core) of the nanoparticles of the invention comprises a fluid that is isotonic with cellular or physiological fluids in vivo.

[0067] The inner compartment (internal core) of the nanoparticles of the present invention can comprise any suitable material. In some embodiments, the inner compartment (internal core) of the nanoparticles of the present invention does not support an outer surface (or shell). Any suitable material can be used. For example, the inner compartment (internal core) can contain only a liquid, e.g., a liquid isotonic with cellular or physiological fluids, and such a liquid does not support the outer surface (or shell). In another example, the inner compartment (internal core) does not contain any solid material. In another example, the inner compartment (internal core) can contain a solid material, e.g., a dry substance that, when reconstituted with a liquid, forms a liquid isotonic with cellular or physiological fluids. However, such a material is not large and / or strong enough to support the outer surface (or shell).

[0068] The nanoparticles of the present invention can comprise any suitable cell membrane derived from a cell or a cellular source, such as a red blood cell. For example, the nanoparticles can comprise a plasma membrane or intracellular membrane derived from a cell, such as a red blood cell. In some embodiments, the cell membrane comprises a plasma membrane derived from a red blood cell, such as a plasma membrane derived from a human red blood cell. In some embodiments, the nanoparticles can comprise any suitable naturally occurring cell membrane derived from a cell, such as a red blood cell. In some embodiments, the cell membrane comprises a naturally occurring plasma membrane derived from a red blood cell, such as a naturally occurring plasma membrane derived from a human red blood cell.

[0069] In some embodiments, the cell membrane can be derived from a unicellular organism (e.g., a bacterium or fungus) or a multicellular organism (e.g., a plant, an animal, a non-human mammal, a vertebrate, or a human). In other embodiments, the cell membrane can be derived from a blood cell, such as a red blood cell, a white blood cell, or a platelet. In still other embodiments, the cell membrane can be derived from an immune cell (e.g., a macrophage, a monocyte, a B cell, or a T cell), a tumor or cancer cell, and other cells, such as an epithelial cell, an endothelial cell, or a neuronal cell. In still other embodiments, the cell membrane can be derived from a non-terminally differentiated cell, such as a stem cell, including a hematopoietic stem cell, a bone marrow stem cell, a mesenchymal stem cell, a cardiac stem cell, or a neuronal stem cell. In still other embodiments, the cell membrane can be derived from a cellular component or organelle, including, but not limited to, an exosome, a secretory vesicle, a synaptic vesicle, an endoplasmic reticulum (ER), a Golgi apparatus, a mitochondria, a vacuole, or a nucleus.

[0070] In some embodiments, the cell membrane is derived from a cell of an animal, vertebrate, non-human mammal, or human. The cell membrane can be derived from any suitable type of cell. For example, the cell membrane is derived from a cell of connective tissue, such as blood, bone, tendon, ligament, fat or loose connective tissue, fibrous connective tissue, skeletal connective tissue, or fluid connective tissue. In another example, the cell is a cell of muscle tissue, such as visceral muscle or smooth muscle, musculoskeletal, or cardiac muscle. In yet another example, the cell is a cell of nervous tissue, such as a cell in the central nervous system (CNS) or peripheral nervous system (PNS). In yet another example, the cell is a cell of epithelial tissue, such as a cell of simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium (also known as ciliated columnar epithelium), columnar epithelium, glandular epithelium, or ciliated columnar epithelium. In yet another example, the cell is a cell of the nervous system, cardiovascular system, circulatory system, vascular system, digestive system, endocrine system, immune system, integumentary system, lymphatic system, musculoskeletal system, reproductive system, respiratory system, respiratory tract, ventilatory system, urinary system, or renal system or urinary tract. In yet another example, the cell is a blood cell, tumor cell, cancer cell, immune cell, stem cell, endothelial cell, or epithelial cell. In yet another example, the cell membrane comprises a plasma membrane derived from a blood cell, e.g., a red blood cell, a white blood cell, and / or a platelet.

[0071] The outer surface (or shell) of the nanoparticles of the present invention can contain any suitable steroid at an enhanced or enriched level. For example, the steroid can be a fungal steroid, an animal steroid, a plant steroid, or a prokaryotic steroid. Exemplary fungal steroids include ergosterol, ergosta-5,7,22,24(28)-tetraen-3β-ol, zymosterol, lanosterol, or 5,6-dihydroergosterol. The animal steroid can be a vertebrate steroid or an insect steroid. Exemplary insect steroids can be ecdysteroids, such as 20-hydroxyecdysone (ecdysterone or 20E).

[0072] Exemplary vertebrate steroids can be steroid hormones or cholesterol. In some embodiments, the vertebrate steroid can be cholesterol. In other embodiments, the steroid hormone can be a sex steroid, such as an androgen, estrogen, or progestogen, a corticosteroid, such as a glucocorticoid or mineralocorticoid, or an anabolic steroid, such as testosterone or an ester thereof.

[0073] Exemplary plant steroids can be alkaloids, cardiac glycosides, phytosterols, or brassinosteroids. Exemplary prokaryotic steroids can be tetracyclic steroids or triterpenes, such as hopanes.

[0074] In some embodiments, the steroid is a cholestane, such as cholesterol, a cholane, such as cholic acid, a pregnane, such as progesterone, an androstane, such as testosterone, or an estrane, such as estradiol. In other embodiments, the steroid is selected from the group consisting of gonane, testosterone, cholic acid, dexamethasone, lanosterol, progesterone, medrogestone, β-sitosterol, cholesterol, and 5α-cholestane.

[0075] In some embodiments, the outer surface (or shell) comprises a cell membrane derived from a fungal cell, wherein the cell membrane comprises enhanced or enriched levels of a fungal steroid. In other embodiments, the outer surface (or shell) comprises a cell membrane derived from a plant cell, wherein the cell membrane comprises enhanced or enriched levels of a plant steroid. In still other embodiments, the outer surface (or shell) comprises a cell membrane derived from a prokaryotic cell, wherein the cell membrane comprises enhanced or enriched levels of a prokaryotic steroid. In still other embodiments, the outer surface (or shell) comprises a cell membrane derived from an animal cell, wherein the cell membrane comprises enhanced or enriched levels of an animal steroid. In still other embodiments, the outer surface (or shell) comprises a cell membrane derived from a vertebrate cell, wherein the cell membrane comprises enhanced or enriched levels of a vertebrate steroid. In still other embodiments, the outer surface (or shell) comprises a cell membrane derived from a mammalian cell, e.g., a human cell, wherein the cell membrane comprises enhanced or enriched levels of a mammalian steroid, e.g., a human steroid.

[0076] In some embodiments, the outer surface (or shell) comprises a plasma membrane derived from a blood cell, wherein the plasma membrane comprises enhanced or enriched levels of a mammalian steroid, such as cholesterol. The outer surface (or shell) can comprise a plasma membrane derived from any suitable blood cell, such as a red blood cell, a white blood cell, or a platelet, wherein the plasma membrane comprises enhanced or enriched levels of a mammalian steroid, such as cholesterol. In some embodiments, the outer surface (or shell) comprises a plasma membrane derived from a blood cell, such as a red blood cell, a white blood cell, and / or a platelet, wherein the plasma membrane comprises enhanced or enriched levels of a mammalian steroid, such as cholesterol. In some embodiments, the outer surface (or shell) comprises a plasma membrane derived from a human blood cell, such as a human red blood cell, a human white blood cell, and / or a human platelet, wherein the plasma membrane comprises enhanced or enriched levels of a human steroid, such as cholesterol.

[0077] The outer surface (or shell) of the nanoparticles of the present invention can contain steroids, and the level can be enhanced or enriched by any suitable method. For example, the enhanced or enriched level of steroids in the cell membrane of the outer surface (or shell) can be due to exogenously added steroids. In another example, the enhanced or enriched level of steroids in the cell membrane of the outer surface (or shell) can be due to the use of cell membranes derived from cells that have been modified to contain enhanced or enriched levels of steroids in their membranes.

[0078] The cell membrane of the outer surface (or shell) of the nanoparticles of the present invention can contain any suitable level of steroid, for example, the cell membrane of the outer surface (or shell) can contain a level of steroid that is at least 0.1%, e.g., at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 4-fold, 5-fold, or more, higher than the basal level of steroid in the cell membrane.

[0079] In some embodiments, the outer surface (or shell) cell membrane can comprise from about 0.1% (w / w) to about 50% (w / w), e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% exogenously added steroid. Optionally, the outer surface (or shell) cell membrane can contain from about 0.1% (w / w) to about 50% (w / w), e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the exogenously added steroid relative to the total membrane protein weight of the cell membrane. In some embodiments, the cell membrane of the outer surface (or shell) can comprise cell membranes derived from blood cells, and can comprise from about 0.1% (w / w) to about 50% (w / w), e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 1 Including exogenously added steroids, e.g., cholesterol, which are: 1%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%Optionally, the outer surface (or shell) cell membrane can include cell membrane derived from blood cells, and can comprise from about 0.1% (w / w) to about 50% (w / w), e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 9%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% include exogenously added steroids, e.g., cholesterol.

[0080] In some embodiments, the outer surface (or shell) cell membrane can comprise about 20% (w / w) to about 100% (w / w) steroid, e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999% or 100% steroid. Optionally, the outer surface (or shell) cell membrane can comprise about 20% (w / w) to about 100% (w / w) of a steroid relative to the total membrane protein weight of the cell membrane, e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 100%. In some embodiments, "100%" refers to an equal amount or level of steroid, e.g., cholesterol, to the total membrane protein weight. In some embodiments, the cell membrane of the outer surface (or shell) can comprise cell membrane derived from blood cells and comprises about 20% (w / w) to about 100% (w / w), e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999% or 100% steroid, e.g., cholesterol. Optionally, the outer surface (or shell) cell membrane can comprise a cell membrane derived from a blood cell, and comprises from about 20% (w / w) to about 100% (w / w), e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999% or 100% steroid, e.g., cholesterol, relative to the total membrane protein weight of the cell membrane.

[0081] The inner compartment (inner core) of the nanoparticles of the invention can have any suitable pH, for example, the inner compartment (inner core) can have a pH in the range of about 4 to about 10, such as about 6 to about 9, or about 6, 6.5, 7, 7.5, 8, 8.5, or 9.

[0082] The nanoparticles of the present invention can further comprise a releasable cargo. The nanoparticles can comprise the releasable cargo at any suitable location. For example, the releasable cargo can be located within or on the internal compartment (internal core), between the internal compartment (internal core) and the outer surface (or shell), or within or on the outer surface (or shell). Release of the releasable cargo can be triggered by any suitable mechanism. For example, release of the releasable cargo can be triggered by contact between the nanoparticle and a subject or by a change in physical parameters surrounding the nanoparticle. The nanoparticles can comprise any suitable type of releasable cargo. For example, the releasable cargo can be a therapeutic agent, a preventive agent, a diagnostic or marker agent, a prognostic agent, or a combination thereof. The therapeutic agent can be a cytotoxic agent capable of killing cells. Any suitable cytotoxic agent can be used. For example, the cytotoxic agent can be an anthracycline, such as doxorubicin or daunorubicin, a taxane, such as docetaxel or paclitaxel, or an immunosuppressant, such as methotrexate or cyclosporin A. In another example, the releasable cargo can be a metallic, polymeric, dendrimeric, or inorganic particle, or the releasable cargo can be in the form of a metallic, polymeric, dendrimeric, or inorganic particle.

[0083] In some embodiments, the nanoparticles of the present invention do not contain a releasable cargo.

[0084] The nanoparticles can have any suitable size. For example, the nanoparticles can have a diameter of about 10 nm to about 10 μm. In certain embodiments, the diameter of the nanoparticles is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, and 10 μm, or any subrange within about 10 nm to about 10 μm, such as any range between any two of the above sizes.

[0085] The nanoparticles can have any suitable shape, including, but not limited to, spherical, square, rectangular, triangular, circular disc, cube-like shape, cube, rectangular prism (cuboid), cone, cylinder, prism, pyramid, right cylinder, and other regular or irregular shapes.

[0086] In some embodiments, the nanoparticles are substantially devoid of cellular constituents from which the cell membrane is derived. For example, the nanoparticles may be about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% devoid of cellular constituents, such as red blood cells from which the cell membrane is derived. In some embodiments, the nanoparticles comprise plasma membranes derived from red blood cells, and the nanoparticles are substantially devoid of hemoglobin. For example, the nanoparticles can lack about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the hemoglobin.

[0087] In some embodiments, the nanoparticles substantially maintain the native structural integrity or activity of the cell membrane or the constituents of the cell membrane. For example, the nanoparticles may retain about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native structural integrity. In some embodiments, the nanoparticles substantially maintain the native structural integrity of the cell membrane or the constituents of the cell membrane, including the primary, secondary, tertiary, and / or quaternary structure of the cell membrane or the constituents of the cell membrane. In some embodiments, the nanoparticles substantially maintain the activity or constituents of the cell membrane, including the binding activity, receptor activity, and / or enzymatic activity of the cell membrane or the constituents of the cell membrane.

[0088] The inner compartment (or inner core) of the nanoparticles of the invention can comprise any suitable substance, for example, the inner compartment (or inner core) of the nanoparticles of the invention can comprise any suitable substance that renders the inner compartment (or inner core) isotonic with cellular or physiological fluids.

[0089] In some embodiments, the inner compartment (or inner core) can include a salt, a sugar, or a sugar alcohol. The inner compartment (or inner core) can include any suitable salt, sugar, or sugar alcohol. For example, the sugar can be a monosaccharide or a disaccharide. Exemplary monosaccharides can be fructose, galactose, or glucose. Exemplary disaccharides can be lactose, maltose, or sucrose. Exemplary salts can be sodium, potassium, or magnesium salts, such as NaCl, KCl, or MgCl2.

[0090] In another example, the sugar alcohol can be ethylene glycol, glycerol, erythritol, threitol, arabitol (or arabinitol), xylitol, ribitol (or adonitol), mannitol, sorbitol, galactitol (dulcitol), fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetriitol, or polyglycitol. In some embodiments, the sugar alcohol is sorbitol.

[0091] The sugar or sugar alcohol can have any suitable osmolality or concentration. For example, the sugar or sugar alcohol can have an osmolality of about 250 mmol / kg to about 350 mmol / kg, e.g., about 275 mmol / kg to about 295 or 300 mmol / kg, or about 250 mmol / kg, 260 mmol / kg, 270 mmol / kg, 280 mmol / kg, 290 mmol / kg, 295 mmol / kg, or 300 mmol / kg. In another example, the sugar or sugar alcohol can have a concentration in the range of about 250 mmol / kg to about 1,000 mmol / kg, e.g., about 250 mmol / kg, 300 mmol / kg, 400 mmol / kg, 500 mmol / kg, 600 mmol / kg, 700 mmol / kg, 800 mmol / kg, 900 mmol / kg, or 1,000 mmol / kg.

[0092] In some embodiments, the inner compartment (or inner core) can include a salt. The inner compartment (or inner core) can include any suitable salt. For example, the salt can be a sodium, potassium, or magnesium salt, such as NaCl, KCl, or MgCl. The salt can have any suitable osmolality or concentration. For example, the salt can have an osmolality of about 250 mmol / kg to about 350 mmol / kg, e.g., about 275 mmol / kg to about 295 or 300 mmol / kg, or about 250 mmol / kg, 260 mmol / kg, 270 mmol / kg, 280 mmol / kg, 290 mmol / kg, 295 mmol / kg, or 300 mmol / kg. In another example, the salt can have a concentration in the range of about 250 mmol / kg to about 1,000 mmol / kg, e.g., about 250 mmol / kg, 300 mmol / kg, 400 mmol / kg, 500 mmol / kg, 600 mmol / kg, 700 mmol / kg, 800 mmol / kg, 900 mmol / kg, or 1,000 mmol / kg.

[0093] In some embodiments, the nanoparticles are biocompatible or biodegradable. For example, the inner core of the nanoparticle can comprise a biocompatible or biodegradable material, and the outer surface of the nanoparticle comprises a plasma membrane derived from a cell, such as a red blood cell. In another example, the inner compartment (or inner core) comprises only biocompatible or biodegradable materials, or none of the materials are biocompatible or biodegradable.

[0094] In some embodiments, the nanoparticles of the present invention comprise: a) an inner compartment (or inner core) that is isotonic with cellular fluids or physiological fluids, e.g., an inner compartment (or inner core) that contains a fluid that is isotonic with cellular fluids or physiological fluids; or b) an outer surface (or shell) that comprises a cell membrane derived from a cell, wherein the cell membrane of the outer surface (or shell) comprises enhanced or enriched levels of a steroid. Exemplary cell membranes can be derived from red blood cells, white blood cells, platelets, tumor cells, cancer cells, immune cells, stem cells, endothelial cells, or epithelial cells. In some embodiments, the cell membrane at the outer surface (or shell) does not comprise enhanced or enriched levels of a steroid, e.g., cell membranes derived from red blood cells, white blood cells, platelets, tumor cells, cancer cells, immune cells, stem cells, endothelial cells, or epithelial cells that do not comprise enhanced or enriched levels of a steroid.

[0095] In some embodiments, the nanoparticles of the present invention comprise: a) an inner compartment (or core) that is isotonic with cellular or physiological fluids, e.g., an inner compartment (or core) comprising a fluid that is isotonic with cellular or physiological fluids, and b) an outer surface (or shell) comprising a cell membrane derived from a cell, the cell membrane of the outer surface (or shell) comprising enhanced or enriched levels of a steroid. Exemplary cell membranes can be derived from red blood cells, white blood cells, cells, platelets, tumor cells, cancer cells, immune cells, stem cells, endothelial cells, or epithelial cells.

[0096] In some embodiments, the inner compartment (or inner core) comprises sorbitol and the outer surface (or shell) comprises plasma membrane derived from red blood cells.

[0097] The nanoparticles of the present invention can have any suitable half-life in vivo. For example, the nanoparticles of the present invention can have a half-life in the blood circulation in vivo of at least about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, or longer.

[0098] In some embodiments, the nanoparticles are substantially devoid of immunogenicity to the subject, mammal, non-human mammal, or human, to whom the nanoparticles are to be administered. For example, the cell membrane can be derived from a cell, such as a red blood cell, from the same species as the subject. In another example, the subject is a human, and the cell membrane is derived from a human cell, such as a human red blood cell. In some embodiments, the cell membrane can be derived from a cell, such as a red blood cell, of the subject to be treated. For example, the cell membrane can be derived from a red blood cell of the human to be treated.

[0099] The outer surface of the nanoparticles of the present invention can comprise a hybrid membrane comprising a cell-derived plasma membrane and a synthetic membrane. In some embodiments, the outer surface of the nanoparticle can comprise a hybrid membrane comprising at least about 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), or 99% (w / w) of the plasma membrane. In other embodiments, the outer surface of the nanoparticles can comprise a hybrid membrane, including at least about 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w) of a synthetic membrane. For example, the outer surface of the nanoparticle can comprise a hybrid membrane comprising about 5-10% (w / w) cell membrane and about 95-99% (w / w) synthetic membrane, about 11-25% (w / w) cell membrane and about 75-89% (w / w) synthetic membrane, about 50% (w / w) cell membrane and about 50% (w / w) synthetic membrane, about 51-75% (w / w) cell membrane and about 49-25% (w / w) synthetic membrane, or about 90-99% (w / w) cell membrane and about 1-10% (w / w) synthetic membrane.

[0100] In some embodiments, the outer surface (or shell) of the nanoparticles of the present invention can further comprise a sphingolipid.The outer surface (or shell) of the nanoparticles of the present invention can comprise any suitable sphingolipid.For example, the sphingolipid can be a simple sphingolipid.In another example, the sphingolipid can be a complex sphingolipid, such as sphingomyelin, glycosphingolipid, or inositol-containing ceramide.

[0101] In some embodiments, the outer surface (or shell) of the nanoparticles of the present invention can comprise cells, steroids, such as cholesterol, and sphingolipids, such as sphingomyelin, derived from fused cell membranes. In some embodiments, the outer surface (or shell) of the nanoparticles of the present invention can comprise cells, steroids, such as cholesterol, and sphingolipids, such as sphingomyelin, derived from fused plasma membranes. In some embodiments, the outer surface (or shell) of the nanoparticles of the present invention can comprise red blood cells, steroids, such as cholesterol, and sphingolipids, such as sphingomyelin, derived from fused plasma membranes.

[0102] In another aspect or embodiment, the present disclosure provides nanoparticles comprising an internal compartment (or internal core) and an external surface (or shell), wherein the internal compartment (or internal core) does not provide a solid support for the external surface (or shell) and / or is isotonic with cellular or physiological fluids, e.g., the internal compartment (or internal core) comprises a fluid that is isotonic with cellular or physiological fluids, and the external surface (or shell) comprises cells, cholesterol, and sphingomyelin derived from cell membranes.

[0103] In some embodiments, the outer surface (or shell) comprises fused plasma membrane-derived cells, cholesterol, and sphingomyelin. For example, the outer surface (or shell) can comprise fused plasma membrane-derived red blood cells, cholesterol, and sphingomyelin.

[0104] The outer surface (or shell) can contain any suitable level of cells, cholesterol, and sphingomyelin derived from cell membranes. For example, the outer surface (or shell) can contain about 20% (w / w) to about 50% (w / w) cholesterol, about 20% (w / w) to about 80% (w / w) sphingomyelin, and about 10% (w / w) to about 50% (w / w) of cell membrane derived from cells, e.g., plasma membrane derived from red blood cells. A cholesterol level of about 20% (w / w) to about 50% (w / w) refers to a cholesterol level that is independent of (or does not include) the cholesterol level in the cell membrane. Similarly, a sphingomyelin level of about 20% (w / w) to about 80% (w / w) refers to a sphingomyelin level that is independent of (or does not include) the sphingomyelin level in the cell membrane.

[0105] In some embodiments, the outer surface (or shell) comprises about 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), or any subrange thereof, cholesterol. In some embodiments, the outer surface (or shell) comprises about 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), or any subrange thereof, sphingomyelin. In some embodiments, the outer surface (or shell) comprises about 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), or any subrange thereof, cell membrane from a cell, e.g., plasma membrane from a red blood cell. In some embodiments, the outer surface (or shell) comprises about 40% (w / w) cholesterol, about 40% (w / w) sphingomyelin, and about 20% (w / w) plasma membrane from a cell, e.g., a red blood cell.

[0106] In some embodiments, the nanoparticles of the present invention have better stability than comparable nanoparticles that do not contain enhanced or enriched levels of steroid.

[0107] In some embodiments, the nanoparticles of the present invention are configured to bind to a cell type to which the cell membrane of the nanoparticle is directed.

[0108] In some embodiments, the nanoparticles of the present invention are configured to bind to or neutralize a moiety that targets or binds to the cell membrane of the nanoparticle. The nanoparticles of the present invention can be configured to bind to or neutralize any suitable moiety. Exemplary moieties can be agents, such as chemical agents, molecules, or organisms.

[0109] In some embodiments, the nanoparticles of the present invention are configured to bind to or neutralize toxins, cytokines, autoantibodies, or chemokines that target or bind to the cell membrane of the nanoparticle. Exemplary toxins can be bacterial toxins, fungal toxins, animal toxins, or chemical toxins. Exemplary chemical toxins can be organophosphates. Exemplary animal toxins can be toxins in animal venoms.

[0110] In some embodiments, "toxin" refers to a poisonous substance or product of a plant, animal, or microorganism (including but not limited to, a bacterium, virus, fungus, rickettsiae, or protozoan), or an infectious agent, or a recombinant or synthetic molecule, regardless of its origin or method of production. In certain embodiments, "toxin" includes bacterial, fungal, or animal toxins produced within living cells or organisms. See, e.g., paragraphs

[0131] -

[0134] of US2013 / 337066(A1).

[0111] In certain embodiments, bacterial toxins include exotoxins and endotoxins. As used herein, "exotoxins" are produced and actively secreted by bacteria, while "endotoxins" are parts of the bacteria themselves (e.g., the bacterial outer membrane) that are not released until the bacteria are killed by the immune system. The present invention contemplates any currently known or hereafter discovered exotoxins and endotoxins. The type of bacterial toxin that inserts into the cell membrane is not particularly limited. In certain embodiments, the bacterial toxin is a toxin derived from S. aureus that inserts into the cell membrane, such as alpha-hemolysin. In certain embodiments, bacterial toxins include exotoxins secreted by clostridium, streptococcus, listeria, and bacillus, such as pneumococcal toxins derived from streptococcus pneumoniae, streptolysins, e.g., streptolysin O (SLO), and streptolysin S (SLS) derived from Streptococcus pyogenes.

[0112] The present disclosure further contemplates any now known or hereafter discovered fungal toxin, including, but not limited to, aflatoxin, citrinin, ergotamine, fumonisin, ergovaline, ochratoxin, phomopsin, suraflamin, sporidesmin, trichothecenes (e.g., satratoxin, deoxynivalenol), zearalenone. The type of fungal toxin that is inserted into the cell membrane is not particularly limited.

[0113] Animal toxins contemplated in the present disclosure include any toxic substance produced by animals. Examples of animal toxins include, but are not limited to, cardiovascular toxins, gastrointestinal toxins, respiratory toxins, neurotoxins, and kidney / organ failure toxins. The present disclosure contemplates any animal toxin now known or hereafter discovered, and the type of animal toxin that is inserted into the cell membrane is not particularly limited. In certain embodiments, the animal toxin that inserts into the cell membrane is derived from arthropods such as insects, arachnids, and crustaceans, or reptiles such as crocodilia, rhynchocephalia, squamata, and testudines.

[0114] Exemplary chemical toxins include acetylcholinesterase (AChE) inhibitors, such as organophosphate poisoning. See, e.g., WO2016 / 176041(A1) and US2018 / 0140558(A1). Exemplary organophosphates or organophosphate poisons include acephate (Olsen), Aspon, Azinphos-methyl (Guthion), Carbofuran (Furadan, F formulation), Carbophenothion (Trithion), Chlorfenvinphos (Bahrain), Chlorpyrifos (Dursban, Rosban), Coumaphos (Coral), Crotoxyphos (Siodrin, Siovap), Clofomate (Luelene), Demeton (Systox), Diazinon (Spectracid), Dichlorvos (DDVP, Vapona), Dicrotophos (Vidrin), Dimethoate (Saigon, Defend), Dioxathion (Delnaf), Disulfoton (Di-Syston), EPN, Ethion, Ethoprop (Mocap), Famfur, Fenamiphos (Nemakur), Fenitrothion (Sumithion), These include fensulfothion (Dasanit), fenthion (Baytex, Tigbon), fonofos (Diphonate), isofenphos (Ophthanol, Amaze), malathion (Cythion), methamidophos (Monitor), methidathion (Supracid), methyl parathion, mevinphos (Fosdrin), monocrotophos, naled (Dibrom), nerve gases (sarin, soman, soman, VX), oxydemeton-methyl (Metasystox-R), parathion (Nilan, Fosquil), phorate (Timet), phosalone (Zolonc), phosmet (Imidan, Prolate), phosphamidon (Dimeclone), temephos (Abate), TEPP, terbufos (Counter), tetrachlorvinphos (Lavon, Lavap), and trichlorfon (Dilox, Negbon). The nanoparticles of the present invention can be used to reduce or neutralize the effects of the above-mentioned organophosphates or organophosphate poisons in a subject.

[0115] In some embodiments, the nanoparticles of the present invention are configured to bind to or neutralize bacteria, fungi, or parasites that target or bind to the cell membrane of the nanoparticle.

[0116] In some embodiments, the nanoparticles of the present invention have a better ability to bind to or neutralize the cell membrane targeting or binding moiety of the nanoparticle than comparable nanoparticles that do not contain enhanced or enriched levels of steroid.

[0117] In another aspect, the present disclosure provides a pharmaceutical delivery system or device comprising an effective amount of the nanoparticles described above. The pharmaceutical delivery system or device may further comprise another active ingredient, or a medically or pharmaceutically acceptable carrier or excipient.

[0118] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of the nanoparticles described above and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may further comprise another active ingredient.

[0119] The pharmaceutical compositions can be configured to treat or prevent diseases or conditions associated with cell membrane-targeting or binding moieties of nanoparticles. In some embodiments, the outer surface of the nanoparticles of the present invention can comprise plasma membranes derived from blood cells, e.g., red blood cells, white blood cells, and / or platelets. C. Methods for Making Nanoparticles

[0120] In yet another aspect, the disclosure provides methods of making nanoparticles, the method comprising: a) contacting a steroid with a cell membrane derived from a cell to form a combination; and b) applying exogenous energy to the combination in a liquid to form nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising the cell membrane containing enhanced or enriched levels of the steroid. In some embodiments, step b) comprises applying exogenous energy to the combination in a liquid isotonic to cellular fluid or physiological fluid to form nanoparticles comprising an inner compartment (or inner core) comprising a liquid isotonic to cellular fluid or physiological fluid, and an outer surface comprising a cell membrane containing enhanced or enriched levels of the steroid.

[0121] In yet another aspect, the disclosure provides a method of making nanoparticles comprising the steps of: a) contacting a steroid with a cell membrane derived from a cell to form a combination; and b) applying exogenous energy to the combination to form nanoparticles comprising an inner compartment (or inner core) and an outer surface comprising the cell membrane containing enhanced or enriched levels of the steroid; and c) applying exogenous energy to the nanoparticles in a fluid isotonic to cellular fluid or physiological fluid to form nanoparticles comprising an inner compartment (or inner core) comprising the fluid isotonic to cellular fluid or physiological fluid, and an outer surface comprising the cell membrane containing the enhanced or enriched levels of the steroid.

[0122] In yet another aspect, the disclosure provides a method of making nanoparticles, the method comprising the steps of imparting exogenous energy to cell membranes derived from red blood cells in a fluid isotonic to a cellular fluid or physiological fluid, to form nanoparticles comprising an inner compartment (or inner core) comprising the fluid isotonic to a cellular fluid or physiological fluid and an outer surface comprising the cell membrane. In some embodiments, the method comprises the steps of a) contacting a cell membrane derived from red blood cells with a steroid to form a combination, and b) imparting exogenous energy to the combination in a fluid isotonic to a cellular fluid or physiological fluid, to form nanoparticles comprising an inner compartment (or inner core) comprising the fluid isotonic to a cellular fluid or physiological fluid and an outer surface comprising the cell membrane with enhanced or enriched levels of the steroid. In another embodiment, the method comprises the steps of: a) contacting a steroid with a cell membrane derived from a red blood cell to form a combination; b) applying exogenous energy to the combination to form nanoparticles comprising an inner core and an outer surface comprising the cell membrane containing enhanced or enriched levels of the steroid; and c) applying exogenous energy to the nanoparticles in a fluid isotonic to cellular fluid or physiological fluid to form nanoparticles comprising an inner compartment (or inner core) comprising the fluid isotonic to cellular fluid or physiological fluid and an outer surface comprising the cell membrane containing enhanced or enriched levels of the steroid.

[0123] In yet another aspect, the present disclosure provides a method for making nanoparticles, the method comprising the steps of: a) contacting a cell membrane derived from a cell with a steroid and a sphingolipid dissolved in a water-miscible solvent to form a combination, e.g., a liquid combination; and b) applying exogenous energy to the combination, or the liquid combination, to form nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising the cell membrane, the steroid, and the sphingolipid.

[0124] The cell membrane used in the methods or nanoparticles of the present invention can be any suitable cell membrane or can be derived from any suitable cell. See, e.g., paragraphs 68-70 above. For example, the cell membrane can be derived from an animal cell, e.g., a non-human mammalian cell or a human cell. In some embodiments, the cell membrane is derived from a blood cell, tumor cell, cancer cell, immune cell, stem cell, endothelial cell, or epithelial cell. In some embodiments, the cell membrane comprises a plasma membrane derived from a cell or blood cell, e.g., a red blood cell, a white blood cell, and / or a platelet.

[0125] In the methods of the present invention, the cell membranes can be contained in any suitable liquid or aqueous liquid. For example, the aqueous liquid can be water, a buffer solution having a pH ranging from about 5 to about 9, such as PBS, or an isotonic liquid or buffer solution, such as 1X PBS. Any suitable buffer solution can be used. In some embodiments, the buffer solution can have a pH of about 5, 6, 7, 8, 9, or any subrange thereof.

[0126] The steroid used in the method or nanoparticle of the present invention can be any suitable steroid. For example, see paragraphs 71-80 above. For example, the steroid can be cholestane, e.g., cholesterol, cholane, e.g., cholic acid, pregnane, e.g., progesterone, androstane, e.g., testosterone, or estrane, e.g., estradiol. In another example, the steroid can be gonane, testosterone, cholic acid, dexamethasone, lanosterol, progesterone, medrogestone, β-sitosterol, cholesterol, and 5α-cholestane.

[0127] The sphingolipid used in the method of the present invention or the nanoparticle of the present invention can be any suitable sphingolipid.For example, see paragraph 100 above.For example, sphingolipid can be simple sphingolipid.In another example, sphingolipid can be complex sphingolipid, for example, sphingomyelin, glycosphingolipid or inositol-containing ceramide.

[0128] In some embodiments, the steroid, e.g., cholesterol, and the sphingolipid, e.g., sphingomyelin, can be dissolved in the same water-miscible solvent, hi some embodiments, the steroid, e.g., cholesterol, and the sphingolipid, e.g., sphingomyelin, can be dissolved in different water-miscible solvents.

[0129] Dissolution of the steroid and / or sphingolipid in the water-miscible solvent can be promoted by any suitable procedure or means. For example, dissolution of the steroid and / or sphingolipid in the water-miscible solvent can be promoted by heating and / or mixing, e.g., stirring. In some embodiments, heating is performed at a temperature in the range of about 35°C to about 65°C, e.g., about 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or any subrange thereof.

[0130] Any suitable water-miscible solvent can be used in the method of the present invention. For example, the water-miscible solvent can be an organic compound. Any suitable organic compound can be used. For example, the organic compound can be an alcohol, such as a C1-C5 alcohol. In some embodiments, the alcohol is methanol, ethanol, 1-propanol, 1,3-propanediol, 1,5-pentanediol, or isopropyl alcohol (isopropanol or IPA). In some embodiments, the water-miscible solvent can be one or more organic compounds selected from acetaldehyde, acetic acid, acetone, acetonitrile, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, butyric acid, diethanolamine, diethylenetriamine, dimethylformamide, dimethoxyethane, dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol, methanol, methyldiethanolamine, methyl isocyanide, N-methyl-2-pyrrolidone (NMP), 1-propanol, 1,3-propanediol, 1,5-pentanediol, isopropyl alcohol, propionic acid, propylene glycol, pyridine, tetrahydrofuran (THF), or triethylene glycol.

[0131] In another example, the water-miscible solvent can be an inorganic compound. In some embodiments, the water-miscible solvent can be one or more inorganic compounds selected from 1,2-dimethylhydrazine, asymmetric dimethylhydrazine, hydrazine, hydrofluoric acid, hydrogen peroxide, nitric acid, or sulfuric acid.

[0132] In the combination, the water-miscible solvent, e.g., IPA, can have any suitable concentration or level, for example, about 5% (v / v) to about 40% (v / v), e.g., about 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v), 25% (v / v), 30% (v / v), 35% (v / v), 40% (v / v), or any subrange thereof.

[0133] The combination can have any suitable mass concentration, for example, from about 0.25 mg / ml to about 20 mg / ml, such as about 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, or any subrange thereof.

[0134] In the combination, the steroid, for example, cholesterol, can have any suitable concentration or level. For example, in the combination, the steroid, for example, cholesterol, can have a concentration or level ranging from about 20% (w / w) to about 50% (w / w), for example, about 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w), or any subrange thereof. A level of about 20% (w / w) to about 50% (w / w) steroid, for example, cholesterol, refers to a level of steroid, for example, cholesterol, independent of (or not including) the level of steroid, for example, cholesterol, in the cell membrane.

[0135] In the combination, the sphingolipid, e.g., sphingomyelin, can have any suitable concentration or level, for example, in the combination, the sphingolipid, e.g., sphingomyelin, can have a concentration or level ranging from about 20% (w / w) to about 90% (w / w), such as about 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w), 55% (w / w), 60% (w / w), 65% (w / w), 70% (w / w), 75% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), or any subrange thereof. A sphingolipid, e.g., sphingomyelin level of about 20% (w / w) to about 90% (w / w) refers to a sphingolipid, e.g., sphingomyelin level that is independent of (or does not include) the level of sphingolipid, e.g., sphingomyelin in the cell membrane.

[0136] In the combination, cell membranes, e.g., plasma membranes derived from blood cells such as red blood cells, white blood cells, and / or platelets, can have any suitable concentration or level. For example, in the combination, cell membranes, e.g., plasma membranes derived from blood cells such as red blood cells, white blood cells, and / or platelets, can have a concentration or level ranging from about 5% (w / w) to about 50% (w / w), e.g., about 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w), or any subrange thereof.

[0137] In the methods of the present invention, exogenous energy can be applied to the combination using any suitable device, procedure, or means. For example, in the methods of the present invention, applying exogenous energy to the combination can include subjecting the combination to ultrasonic treatment in a liquid.

[0138] In the method of the present invention, the combination may be sonicated at any suitable temperature, for example, at a temperature ranging from about 15°C to about 50°C, such as about 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or any subrange thereof.

[0139] In the methods of the present invention, the combination can be sonicated at any suitable frequency, for example, the combination can be sonicated at a frequency ranging from about 20 kilohertz (kHz) to about 60 kHz, such as about 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, or any subrange thereof.

[0140] In the method of the present invention, the combination may be subjected to ultrasonic treatment for any suitable period of time, for example, for a period of time ranging from about 5 minutes to about 50 minutes, such as about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or any subrange thereof.

[0141] In the methods of the present invention, exogenous energy can be applied to the combination for any suitable purpose, for example, applying exogenous energy to the combination, such as subjecting the combination to ultrasonic treatment, can be performed to homogenize cell membranes, steroids, and sphingolipids in the combination.

[0142] The method can be used to produce any suitable nanoparticle, for example, the method can be used to produce nanoparticles where the outer surface (or shell) comprises fused cell membranes, steroids, and sphingolipids in the nanoparticle.

[0143] The nanoparticles made or prepared by the present methods can have any suitable size distribution, for example, the nanoparticles made or prepared by the present methods can have a particle distribution index (PDI) of about 0.2 or higher, such as about 0.3 or higher.

[0144] The method can further include, after step b), subjecting the nanoparticles to high shear treatment (or high shear force treatment) in a high shear fluid processor. Any suitable high shear fluid processor can be used. For example, the high shear fluid processor can be a microfluidizer (or microfluidizer processor) or a homogenizer that generates high shear force.

[0145] In some embodiments, the high shear fluid processor used in the method is a microfluidizer (or microfluidizer processor). Any suitable microfluidizer (or microfluidizer processor) can be used. In some embodiments, the microfluidizer is configured to generate a substantially constant pressure of about 10,000 psi to about 30,000 psi, e.g., about 10,000 psi, 15,000 psi, 20,000 psi, about 25,000 psi, 30,000 psi, or any subrange thereof.

[0146] In some embodiments, the microfluidizer has a microfluidic reaction technology (MRT) configuration, including, from upstream to downstream, an inlet for inputting nanoparticles, an intensifier pump for generating static pressure, an impinging jet chamber for generating high shear pressure on the nanoparticles, and an outlet for removing the nanoparticles. In some embodiments, the microfluidizer includes a Z-type interaction chamber. In some embodiments, the microfluidizer includes a Y-type interaction chamber.

[0147] Microfluidization can be performed using any suitable number of pressures. For example, microfluidization can be performed using a single pressure. In another example, microfluidization can be performed using a combination of different pressures. Microfluidization can also be performed using any suitable number of passes. For example, microfluidization can be performed using a single pass. In another example, microfluidization can be performed using multiple passes.

[0148] The method of the present invention can further include a step of cooling the nanoparticles. In some embodiments, the nanoparticles in the channel after exiting the chamber of the microfluidizer are cooled by a product chiller containing a coolant. The product chiller or the coolant in the product chiller can be set to any suitable temperature. For example, the product chiller or the coolant in the product chiller can be set to a temperature in the range of about 4°C to about 55°C, such as about 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or any subrange thereof.

[0149] The present method can be used to produce nanoparticles of any suitable size and / or size distribution. Generally, nanoparticles produced by high shear processing (or high shear force processing) have smaller sizes and a narrower range of size distributions than nanoparticles produced without high shear processing (or high shear force processing). In some embodiments, nanoparticles produced by high shear processing (or high shear force processing) have a particle distribution index (PDI) in the range of about 0.05 to about 0.2, e.g., a PDI of about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, or any subrange thereof. In some embodiments, the nanoparticles produced by high shear processing (or high shear force processing) have a particle size with a Z-average value in the range of about 30 nm to about 300 nm, e.g., a Z-average value of about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 200 nm, or any subrange thereof.

[0150] The methods of the invention can further include one or more of the following steps: 1) removing or reducing the level of particles having a particle size of about 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, or larger, 2) removing or reducing the level of water-miscible solvent, 3) concentrating the nanoparticles, 4) sterilizing the nanoparticles, and / or 5) filling the nanoparticles into individual containers. In some embodiments, the methods of the invention can further include two, three, four, or five of the following steps: 1) removing or reducing the level of particles having a particle size of about 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, or larger, 2) removing or reducing the level of water-miscible solvent, 3) concentrating the nanoparticles, 4) sterilizing the nanoparticles, and / or 5) filling the nanoparticles into individual containers.

[0151] The step of removing or reducing the level of particles having a particle size of about 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, or larger can be performed using any suitable device, procedure, or means. For example, step 1) can include filtering the liquid containing the nanoparticles. Any suitable type of filtration or filter can be used. In some embodiments, the filtration can be performed using a filter comprising polyethersulfone (PES), polyvinylidene difluoride (PVDF), glass fiber, cellulose acetate, or a combination thereof. The filter can have any suitable pore size. For example, the filter can have a pore size in the range of about 0.2 μm to about 1.2 μm, e.g., about 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.2 μm, or any subrange thereof. In some embodiments, filtration is carried out using a single filter.In some embodiments, filtration is carried out using multiple filters with different pore sizes.In some embodiments, filtration is carried out to remove or reduce the level of particles with particle sizes of about 200nm, 210nm, 220nm, 230nm, 240nm, 250nm or larger, and to sterilize nanoparticles.

[0152] The step of removing or reducing the level of water-miscible solvent and / or the step of concentrating the nanoparticles can be carried out using any suitable device, procedure, or means. For example, steps 2) and / or 3) can include subjecting the liquid containing the nanoparticles to tangential flow filtration (TFF).

[0153] TFF can be carried out using any suitable TFF device or system.For example, TFF can be carried out using a TFF system that includes a feed reservoir, a filter device and a collection device, wherein the feed reservoir is in fluid communication with the filter device through the inlet of the filter device, the filter device is in fluid communication with the collection device through the permeate outlet of the filter device, and the filter device is in fluid communication with the feed reservoir through the retentate outlet of the filter device.

[0154] The filter device can be in any suitable form. For example, the filter device can be in the form of a cartridge, cassette, or column containing a hollow fiber filter. The filter device can include a filtration membrane having any suitable pore size. For example, the filter device can include a filtration membrane having a pore size in the range of about 100 Kd to about 300 Kd, e.g., about 100 Kd, 150 Kd, 200 Kd, 250 Kd, 300 Kd, or any subrange thereof.

[0155] TFF can be carried out by any suitable type of method.For example, TFF can be carried out by diafiltration method.In some embodiments, diafiltration method is continuous, intermittent or sequential diafiltration method.TFF can be carried out by any suitable number of cycles.In some embodiments, TFF is carried out by single cycle.In some embodiments, TFF is carried out by multiple cycle diafiltration method, for example, multiple cycle continuous diafiltration method.

[0156] The method or TFF of the present invention can further comprise the step of collecting nanoparticles.Nanoparticles can be collected by any suitable device, procedure or means.For example, nanoparticles can be collected through the retentate outlet of filter device.

[0157] TFF can be performed for any suitable purpose. In some embodiments, TFF is used to reduce the amount or level of water-miscible solvent from a composition, e.g., a liquid, containing nanoparticles. In some embodiments, TFF is used to remove water-miscible solvent from a composition, e.g., a liquid, containing nanoparticles. In some embodiments, TFF is used to remove about 50% to about 99.9999% of the water-miscible solvent from the composition, e.g., about 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% of the water-miscible solvent from the composition. In some embodiments, the water-miscible solvent used in the methods of the invention is IPA, and TFF is used to reduce the IPA concentration or level in the composition to less than 5,000 ppm, e.g., to reduce the IPA concentration or level in the composition to less than 5,000 ppm, 4,000 ppm, 3,000 ppm, 2,000 ppm, 1,000 ppm, or 500 ppm.

[0158] TFF can also be used to concentrate and / or enrich nanoparticles. In some embodiments, TFF is used to concentrate and / or enrich nanoparticles by about 1-fold to about 400-fold, e.g., to concentrate and / or enrich nanoparticles 1-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, or subranges thereof.

[0159] The steps of removing or reducing the level of the water-miscible solvent and concentrating the nanoparticles can be performed as a combined step or as separate steps, for example, steps 2) and 3) can be combined into a single step that includes subjecting the liquid containing the nanoparticles to tangential flow filtration (TFF).

[0160] The method can further comprise a step of sterilizing the nanoparticles or the composition containing the nanoparticles, for example, a liquid. For example, in the method of the present invention, the composition containing the nanoparticles, for example, a liquid, is subjected to a step of sterilizing the composition containing the nanoparticles before and / or after the TFF process. In some embodiments, the composition containing the nanoparticles, for example, a liquid, is subjected to a step of sterilizing the composition containing the nanoparticles before the TFF process. In some embodiments, the composition containing the nanoparticles, for example, a liquid, is subjected to a step of sterilizing the composition containing the nanoparticles after the TFF process. In some embodiments, the composition containing the nanoparticles, for example, a liquid, is subjected to a step of sterilizing the composition containing the nanoparticles before and after the TFF process.

[0161] Sterilizing a composition containing nanoparticles can be performed using any suitable device, procedure, or means. For example, sterilizing a composition can include filtering the composition. Any suitable type of filter or filter can be used. In some embodiments, filtering can be performed using a filter comprising polyethersulfone (PES), polyvinylidene difluoride (PVDF), glass fiber, cellulose acetate, or a combination thereof. The filter can have any suitable pore size. For example, the filter can have a pore size of about 0.2 μm. In some embodiments, filtering is performed using a single filter. In some embodiments, filtering is performed using multiple filters with different pore sizes.

[0162] In some embodiments, the methods of the present invention do not include the step of removing or evaporating the water-miscible solvent to form a film comprising the steroid and sphingolipid.

[0163] In some embodiments, the method of the present invention is carried out in which the cell membrane comprises red blood cells derived from plasma membranes, the steroid is cholesterol, the sphingolipid is sphingomyelin, and the water-miscible solvent is IPA. For example, the method of the present invention can be carried out to form nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising red blood cells, cholesterol, and sphingomyelin derived from the plasma membrane. In some embodiments, the inner compartment (or inner core) of the nanoparticle does not provide a solid support for the outer surface (or shell). In some embodiments, the inner compartment (or inner core) of the nanoparticle is isotonic with cellular fluid or physiological fluid, for example, the inner compartment (or inner core) comprises a liquid isotonic with cellular fluid or physiological fluid. In some embodiments, the outer surface (or shell) of the nanoparticle comprises about 40% (w / w) cholesterol, about 40% (w / w) sphingomyelin, and about 20% (w / w) plasma membrane derived from red blood cells. A cholesterol level of about 40% (w / w) refers to a cholesterol level that is independent of (or does not include) cholesterol levels in cell membranes. Similarly, a sphingomyelin level of about 40% (w / w) refers to a sphingomyelin level that is independent of (or does not include) sphingomyelin levels in cell membranes.

[0164] The cell membrane used in the method of the present invention can be in any suitable form, for example, the cell membrane derived from the cell can be in the form of a cell membrane ghost.

[0165] Any suitable exogenous energy can be used in the methods of the present invention. For example, the exogenous energy used in the methods of the present invention can be mechanical energy, acoustic energy, or thermal energy.

[0166] Any suitable cell membrane can be used in the methods of the invention. For example, the cell membrane used in the methods of the invention can be derived from mammalian or human blood cells, such as red blood cells, white blood cells, or platelets.

[0167] Any suitable steroid can be used in the methods of the present invention. For example, the steroid used in the methods of the present invention can be cholestane, e.g., cholesterol.

[0168] Nanoparticles prepared by the methods of the present invention are also provided.

[0169] In some embodiments, the inner compartment (or inner core) of the nanoparticle does not provide a solid support to the outer surface (or shell). In some embodiments, the inner compartment (or inner core) of the nanoparticle is isotonic with cellular or physiological fluids, e.g., the inner compartment (or inner core) comprises a liquid that is isotonic with cellular or physiological fluids. In some embodiments, the inner compartment (or inner core) of the nanoparticle does not provide a solid support to the outer surface (or shell) and is isotonic with cellular or physiological fluids, e.g., the inner compartment (or inner core) comprises a liquid that is isotonic with cellular or physiological fluids.

[0170] In some embodiments, the outer surface (or shell) of the nanoparticle comprises cells, cholesterol, and sphingomyelin derived from the cell membrane. In some embodiments, the outer surface (or shell) of the nanoparticle comprises red blood cells, cholesterol, and sphingomyelin derived from the plasma membrane.

[0171] In some embodiments, the outer surface (or shell) of the nanoparticle comprises cells, cholesterol, and sphingomyelin derived from fused cell membranes. In some embodiments, the outer surface (or shell) of the nanoparticle comprises red blood cells, cholesterol, and sphingomyelin derived from fused plasma membranes.

[0172] In some embodiments, the outer surface (or shell) of the nanoparticle comprises about 20% (w / w) to about 50% (w / w) cholesterol, about 20% (w / w) to about 90% (w / w) sphingomyelin, and about 5% (w / w) to about 50% (w / w) plasma membrane derived from red blood cells. The cholesterol level of about 20% (w / w) to about 50% (w / w) refers to a cholesterol level that is independent of (or does not include) cholesterol levels in cell membranes. Similarly, the sphingomyelin level of about 20% (w / w) to about 90% (w / w) refers to a sphingomyelin level that is independent of (or does not include) sphingomyelin levels in cell membranes.

[0173] In some embodiments, the outer surface (or shell) of the nanoparticle comprises about 20% (w / w) to about 50% (w / w) cholesterol, e.g., about 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w) cholesterol, or any subrange thereof.

[0174] In some embodiments, the outer surface (or shell) of the nanoparticle comprises about 20% (w / w) to about 90% (w / w) sphingomyelin, e.g., about 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w), 55% (w / w), 60% (w / w), 65% (w / w), 70% (w / w), 75% (w / w), 80% (w / w), 85% (w / w), 90% (w / w) sphingomyelin, or any subrange thereof.

[0175] In some embodiments, the outer surface (or shell) of the nanoparticle comprises plasma membrane from about 5% (w / w) to about 50% (w / w) red blood cells, about 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w) red blood cells, or any subrange thereof.

[0176] In some embodiments, the outer surface (or shell) of the nanoparticle comprises about 40% (w / w) cholesterol, about 40% (w / w) sphingomyelin, and about 20% (w / w) plasma membrane derived from red blood cells.

[0177] Also provided is a pharmaceutical delivery system or device comprising an effective amount of the nanoparticles of the present invention. The pharmaceutical delivery system or device may further comprise another active ingredient, or a medically or pharmaceutically acceptable carrier or excipient.

[0178] Also provided is a pharmaceutical composition comprising an effective amount of the nanoparticles of the present invention and a pharmaceutically acceptable carrier or excipient. The pharmaceutical agent may further comprise another active ingredient. The pharmaceutical composition may also be configured to treat or prevent a disease or condition associated with a cell membrane-targeting or binding moiety of the nanoparticle. The outer surface of the nanoparticles in the pharmaceutical composition may comprise any suitable cell membrane, such as the plasma membrane derived from blood cells, e.g., red blood cells, white blood cells, and / or platelets. D. Methods of Treating or Preventing a Disease or Condition

[0179] In yet another aspect, the present disclosure provides a method for treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising a cellular membrane derived from a cell, wherein the inner compartment (or inner core) does not provide a solid support for the cellular membrane in the outer surface (or shell). Optionally, and in some embodiments, a) the inner compartment (or inner core) of the nanoparticle is isotonic with cellular fluid or physiological fluid, e.g., the inner compartment (or inner core) comprises a liquid isotonic with cellular fluid or physiological fluid, and / or b) the cellular membrane of the outer surface (or shell) of the nanoparticle comprises an enhanced or enriched level of a steroid. Additionally, optionally, and in some embodiments, when the nanoparticle comprises a cellular membrane derived from a red blood cell, the inner compartment (or inner core) is isotonic with cellular fluid or physiological fluid. In some embodiments, the nanoparticles are administered using a pharmaceutical delivery system or a pharmaceutical composition comprising the nanoparticles.

[0180] The method can be used to treat or prevent a disease or condition in any suitable subject. For example, the subject can be a human or non-human mammal.

[0181] The nanoparticles used in this method can contain any suitable cell membrane. For example, the cell membrane in the nanoparticles can be derived from cells of the same species as the subject, or derived from cells of the subject. The cell membrane in the nanoparticles can be derived from any suitable cell. For example, the cell membrane in the nanoparticles can be derived from blood cells, such as red blood cells, white blood cells, and / or platelets. In another example, the cell membrane in the nanoparticles can be derived from red blood cells of the same species as the subject, and the red blood cells have the same blood type as the subject.

[0182] The method can be used for any suitable purpose. For example, the method can be used to treat or prevent a disease or condition associated with a cell membrane-targeting or binding moiety of the nanoparticle. Exemplary moieties can be drugs, such as chemical agents, molecules, or organisms.

[0183] In some embodiments, the method can be used to treat or prevent a disease or condition associated with an organism that targets or binds to the cell membrane of the nanoparticle. Exemplary organisms can be viruses, bacteria, fungi, or parasites.

[0184] In some embodiments, "virus" refers to an obligate intracellular parasite of an organism, composed of DNA or RNA and a protein membrane, but noncellular in nature. Viruses range in diameter from about 20 to about 300 nm. Class I viruses (Baltimore classification) have double-stranded DNA as their genome. Class II viruses have single-stranded DNA as their genome. Class III viruses have double-stranded RNA as their genome. Class IV viruses have a positive-sense single-stranded RNA genome, which itself serves as mRNA. Class V viruses have a negative-sense single-stranded RNA genome, which serves as a template for mRNA synthesis. Class VI viruses have a positive-sense single-stranded RNA genome, but can also form a DNA intermediate during replication and mRNA synthesis. Viruses are largely recognized by the diseases they cause in plants, animals, and prokaryotes. Prokaryotic viruses are known as bacteriophages.

[0185] In some embodiments, the method can be used to treat or prevent diseases or conditions associated with pathogenic viruses. Exemplary pathogenic viruses include smallpox, influenza, mumps, measles, chickenpox, Ebola, human immunodeficiency virus (HIV), rubella, hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), and hepatitis D (HDV).

[0186] In some embodiments, "bacteria" refers to small prokaryotes (linear dimensions of about 1 micron) with non-compartmentalized circular DNA and approximately 70S ribosomes. Bacterial protein synthesis differs from that of eukaryotes. Many antibacterial antibiotics interfere with bacterial protein synthesis but do not affect the infected host. The major subclasses of bacteria include eubacteria and archaea. In some embodiments, "eubacteria" refers to the major subclasses of bacteria other than archaea. Most gram-positive bacteria, cyanobacteria, mycoplasmas, enterobacteria, pseudomonads, and chloroplasts are eubacteria. The cytoplasmic membrane of eubacteria is made up of ester-linked Contains lipids. Peptidoglycan is present in the cell wall (if present). Introns are not found in eubacteria. In some embodiments, "archaebacteria" refers to a major subclassification of bacteria other than eubacteria. There are three main orders of archaea: extreme halophiles, methanogens, and sulfur-dependent extreme thermophiles. Archaea differ from eubacteria in other characteristics, including ribosomal structure, possession of introns (in some cases), and membrane composition.

[0187] In some embodiments, the methods can be used to treat or prevent diseases or conditions associated with pathogenic bacteria. Exemplary pathogenic bacteria include bacteria that cause or are associated with tuberculosis (TB), such as Mycobacterium tuberculosis (M. tb), pneumonia, such as Streptococcus or Pseudomonas, food poisoning, such as E. coli, Shigella, Campylobacter, Salmonella, tetanus, typhoid, diphtheria, syphilis, and leprosy.

[0188] In some embodiments, "fungi" refers to eukaryotic organisms that grow in irregular masses without roots, stems, or leaves, and that lack chlorophyll or other pigments capable of photosynthesis. Each organism (thallus) is unicellular to filamentous, with branched somatic structures (hyphae) surrounded by a cell wall containing a true nucleus and containing glucan or chitin, or both.

[0189] In some embodiments, the methods can be used to treat or prevent diseases or conditions associated with pathogenic fungi. Exemplary pathogenic fungi include Candida albicans, the most common cause of candidiasis, and Cryptococcus neoformans, which can cause severe forms of meningitis.

[0190] In some embodiments, the methods can be used to treat or prevent a disease or condition associated with a parasite. Exemplary human parasites include those that cause or are associated with malaria, leishmaniasis, cryptosporidiosis, amebiasis, Chagas disease, African trypanosomiasis, schistosomiasis, ascariasis, echinococcosis, and cysticercosis.

[0191] In some embodiments, the method can be used to treat or prevent diseases or conditions associated with toxins, cytokines, autoantibodies, or chemokines that target or bind to the cell membrane of the nanoparticles. Exemplary toxins can be bacterial toxins, fungal toxins, animal toxins, or chemical toxins. Exemplary chemical toxins can be organophosphates. Exemplary animal toxins can be toxins found in animal venoms.

[0192] In some embodiments, the method can be used to treat or prevent diseases or conditions associated with toxins that insert into cell membranes. In certain embodiments, the toxin inserts into the cell membrane or plasma membrane of a target cell of a subject as part of the toxin's natural pathological mechanism. In certain embodiments, the cell membrane or plasma membrane of the outer surface of the nanoparticles used in the method substantially retains the toxin. In certain embodiments, the outer surface of the nanoparticles used in the method comprises a plasma membrane derived from a blood cell, such as a red blood cell, a white blood cell, and / or a platelet.

[0193] In some embodiments, the methods can be used to treat or prevent skin infections, sepsis, pneumonia, autoimmune reactions, e.g., autoimmune reactions due to the production of autoimmune antibodies, such as autoimmune antibodies against blood cells or red blood cells.

[0194] In some embodiments, the method can further comprise administering to a subject in need thereof another active ingredient, or a pharmaceutically acceptable carrier or excipient, hi some embodiments, the nanoparticles of the present invention can be administered by a pharmaceutical delivery system or device.

[0195] In some embodiments, the methods can be used to treat or prevent an infection, e.g., a skin infection, sepsis, pneumonia, or an autoimmune response, e.g., an autoimmune response due to the production of autoimmune antibodies, such as autoimmune antibodies against blood cells or red blood cells.

[0196] The nanoparticles or compositions comprising nanoparticles, e.g., pharmaceutical compositions comprising nanoparticles, can be administered by any suitable route or procedure, for example, the nanoparticles or compositions comprising nanoparticles, e.g., pharmaceutical compositions, can be administered by the enteral / gastrointestinal, oral, parenteral, intravenous, rectal, nasal, topical, ocular, inhalation, or intratracheal route.

[0197] In some embodiments, nanoparticles or compositions comprising nanoparticles, e.g., pharmaceutical compositions, can be administered via the intratracheal route. For example, the disease or condition to be treated or prevented can be an infection, e.g., a skin infection, sepsis, or pneumonia, and the nanoparticles or compositions comprising nanoparticles, e.g., pharmaceutical compositions, can be administered via the intratracheal route. Any suitable intratracheal administration can be used. For example, the nanoparticles or compositions comprising nanoparticles, e.g., pharmaceutical compositions, can be administered via intratracheal instillation or intratracheal inhalation.

[0198] In some embodiments, the neoplasm-specific immunogenic composition or vaccine can be administered by the enteral / gastrointestinal, oral, parenteral, intravenous, rectal, nasal, topical, ocular, inhalation, or intratracheal routes.

[0199] In some embodiments, the immunogenic compositions for generating an immune response in a subject to an area associated with a disease or condition can be administered by the enteral / gastrointestinal tract, oral, parenteral, intravenous, rectal, nasal, topical, ocular, inhalation, or intratracheal routes.

[0200] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. Any suitable second therapeutic agent can be used. For example, the second therapeutic agent can be an antibiotic, an antitumor or anticancer agent, or an immune response modulator, such as an immune response activator or suppressor.

[0201] In some embodiments, the method does not include administering to the subject a second therapeutic agent.

[0202] In yet another aspect, the present disclosure provides the use of an effective amount of a nanoparticle of the present invention for the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof. E. Immunogenic Compositions and Uses Thereof

[0203] In yet another aspect, the present disclosure provides an immunogenic composition comprising an effective amount of the nanoparticles of the present invention, and optionally further comprising an immunogenic adjuvant or immune enhancing substance.

[0204] The immunogenic compositions of the present invention can be configured for any suitable use for the application, hi some embodiments, the immunogenic compositions of the present invention can be configured as neoplasm-specific immunogenic compositions, in which case the outer surface of the nanoparticles comprises cell membranes derived from neoplastic cells.

[0205] The cell membrane of the nanoparticles in the neoplasm-specific immunogenic composition can be derived from any suitable neoplastic cell, for example, the cell membrane of the nanoparticles in the neoplasm-specific immunogenic composition can be derived from a benign neoplastic cell, a potentially malignant neoplastic cell, a cancer cell, a cancer cell line, or a cancer cell of the subject.

[0206] In some embodiments, the cell membranes at the outer surface of the nanoparticles in the neoplasm-specific immunogenic composition substantially retain structural integrity to elicit an immune response against the neoplastic cells.

[0207] In other embodiments, the inner compartment (inner core) of the nanoparticles in the neoplasm-specific immunogenic composition does not support the outer surface (or shell) of the nanoparticles.

[0208] In some embodiments, the nanoparticles in the neoplasm-specific immunogenic composition can further comprise another active ingredient or releasable cargo.

[0209] The nanoparticles in the neoplasm-specific immunogenic composition can have any suitable size. For example, the nanoparticles can have a diameter of about 10 nm to about 10 μm. In certain embodiments, the diameter of the nanoparticles is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, and 10 μm, or any subrange within the range of about 10 nm to about 10 μm, such as any range between any two of the above sizes.

[0210] In some embodiments, the nanoparticles in the neoplasm-specific immunogenic composition are substantially devoid of constituents of the neoplastic cells from which their membranes are derived. For example, the nanoparticles can be about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% devoid of constituents of the neoplastic cells from which their membranes are derived.

[0211] In some embodiments, the nanoparticles in the neoplasm-specific immunogenic composition substantially maintain the native structural integrity or activity of the cell membrane or the constituents of the cell membrane. For example, the nanoparticles may retain about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native structural integrity. In some embodiments, the nanoparticles substantially maintain the native structural integrity of the cell membrane or the constituents of the cell membrane, including the primary, secondary, tertiary, and / or quaternary structure of the cell membrane or the constituents of the cell membrane. In some embodiments, the nanoparticles substantially maintain the activity or constituents of the cell membrane, including the binding activity, receptor activity, and / or enzymatic activity of the cell membrane or the constituents of the cell membrane.

[0212] In some embodiments, the neoplasm-specific immunogenic compositions of the invention can further comprise an immunogenic adjuvant or immune enhancer.

[0213] In some embodiments, the outer surface of the nanoparticles in the neoplasm-specific immunogenic compositions of the invention comprises naturally occurring cell membranes and further comprises synthetic membranes.

[0214] Vaccines comprising the immunogenic compositions of the invention specific for neoplasms are also provided.

[0215] Also provided is a method of treating or preventing a neoplasm in a subject in need thereof, comprising administering to said subject an effective amount of an immunogenic composition of the invention specific for the neoplasm.

[0216] The method can be used to treat or prevent a neoplasm in any suitable subject. For example, the subject can be a human or non-human mammal.

[0217] The nanoparticles used in the present methods can comprise any suitable cell membrane, for example, the cell membrane in the nanoparticles can be derived from cells of the same species as the subject, or derived from neoplastic cells of the subject.

[0218] In some embodiments, the method can further comprise administering to a subject in need thereof another active ingredient or a pharmaceutically acceptable carrier or excipient, hi some embodiments, the nanoparticles of the present invention can be administered by a pharmaceutical delivery system or device.

[0219] In yet another aspect, the disclosure provides the use of an effective amount of an immunogenic composition of the invention specific for a neoplasm for the manufacture of a vaccine to treat or protect a subject from a neoplasm.

[0220] In some embodiments, the immunogenic compositions of the invention can be configured to treat or prevent a disease or condition associated with a cell membrane-targeting or binding moiety of the nanoparticle, where the outer surface of the nanoparticle comprises the moiety. Exemplary moieties can be drugs, e.g., chemical agents, molecules, or organisms.

[0221] In some embodiments, the immunogenic compositions of the present invention can be used to treat or prevent diseases or conditions associated with organisms that target or bind to the cell membrane of the nanoparticles. Exemplary organisms can be bacteria, fungi, or parasites.

[0222] In some embodiments, the immunogenic compositions of the present invention can be used to treat or prevent diseases or conditions associated with toxins, cytokines, autoantibodies, or chemokines. Exemplary toxins can be bacterial toxins, fungal toxins, animal toxins, or chemical toxins. Exemplary chemical toxins can be organophosphates. Exemplary animal toxins can be toxins found in animal venoms.

[0223] In some embodiments, the immunogenic compositions of the present invention can be used to treat or prevent diseases or conditions associated with toxins that insert into cell membranes. In certain embodiments, the toxins insert into the cell membrane or plasma membrane of target cells of interest as part of the toxin's natural pathological mechanism. In certain embodiments, the cell membrane or plasma membrane on the outer surface of the nanoparticles substantially retains the toxin.

[0224] The cell membrane on the outer surface of the nanoparticles in the immunogenic compositions of the present invention can be derived from any suitable cell. In some embodiments, the cell membrane is a plasma membrane derived from a cell. In some embodiments, the outer surface comprises a plasma membrane derived from a red blood cell.

[0225] In some embodiments, the outer surface of the nanoparticles in the immunogenic compositions of the invention can comprise naturally occurring cell membranes and can further comprise synthetic membranes.

[0226] In some embodiments, the nanoparticles in the immunogenic compositions of the invention can be biocompatible, biodegradable, or can comprise synthetic materials.

[0227] In other embodiments, the inner compartment (inner core) of the nanoparticles in the immunogenic compositions of the invention does not support the outer surface (or shell) of the nanoparticles.

[0228] In some embodiments, the immunogenic compositions of the invention can further comprise another active ingredient or immunogenic adjuvant or immune enhancing substance.

[0229] Vaccines comprising the immunogenic compositions of the invention are also provided.

[0230] Also provided is a method of inducing an immune response in a subject to an area associated with a disease or condition, comprising administering to said subject an effective amount of an immunogenic composition of the invention.

[0231] Also provided is a method of protecting a subject from a moiety associated with a disease or condition in said subject, comprising administering to said subject an effective amount of a vaccine of the invention.

[0232] The method can be used to treat or prevent a disease or condition in any suitable subject. For example, the subject can be a human or non-human mammal.

[0233] In some embodiments, the cell membrane or plasma membrane of the nanoparticles used in the methods can be derived from cells of the same species as the subject or from cells of the subject, hi some embodiments, the plasma membrane is derived from red blood cells of the same species as the subject, and the red blood cells have the same blood type as the subject.

[0234] In some embodiments, the method can further comprise administering to said subject another active ingredient, or a pharmaceutically acceptable carrier or excipient.

[0235] The method can be used to elicit any suitable type of immune response from a subject, hi some embodiments, the method can be used to elicit a T cell-mediated immune response or a B cell-mediated immune response from a subject.

[0236] In yet another aspect, the disclosure provides the use of an effective amount of an immunogenic composition of the invention to prepare a vaccine that protects a subject against a disease or condition associated with the moiety. F. Pharmaceutical Compositions and Routes of Administration

[0237] Pharmaceutical compositions containing nanoparticles alone or in combination with other active ingredients described herein can further include one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are substances that are non-toxic to a subject and otherwise biologically suitable for administration. Such excipients facilitate administration of nanoparticles alone or in combination with other active ingredients described herein and are compatible with those active ingredients. Examples of pharmaceutically acceptable excipients include stabilizers, lubricants, surfactants, diluents, antioxidants, binders, colorants, fillers, emulsifiers, or taste modifiers. In preferred embodiments, pharmaceutical compositions according to various embodiments are sterile compositions. Pharmaceutical compositions can be prepared using compounding techniques known or available to those skilled in the art.

[0238] Sterile compositions, including compositions that comply with national or local regulations governing such compositions, are within the scope of this disclosure.

[0239] The pharmaceutical compositions and nanoparticles, alone or in combination with other active ingredients described herein, can be formulated as solutions, emulsions, suspensions, or dispersions in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, sachets, dragees, granules, powders, powders for reconstitution, or capsules with solid carriers, according to conventional methods known in the art for preparing various dosage forms. The nanoparticles, alone or in combination with other active ingredients described herein, and preferably in the form of a pharmaceutical composition, can be administered by a suitable delivery route, such as oral, parenteral, rectal, nasal, topical, or ocular routes, or by inhalation. In some embodiments, the compositions are formulated for intravenous or oral administration.

[0240] For oral administration, the nanoparticles, alone or in combination with another active ingredient, may be provided in solid form, such as a tablet or capsule, or as a solution, emulsion, or suspension. To prepare oral compositions, the nanoparticles, alone or in combination with other active ingredients, may be formulated to provide a daily dose of, for example, about 0.01 to about 50 mg / kg, or about 0.05 to about 20 mg / kg, or about 0.1 to about 10 mg / kg. Oral tablets may contain the active ingredient mixed with compatible pharmaceutically acceptable excipients, such as diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugars, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Exemplary disintegrants include starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid. Binders can include starch and gelatin. Lubricants, if present, can be magnesium stearate, stearic acid, or talc. If desired, tablets can be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or can be coated with an enteric coating.

[0241] Capsules for oral administration include hard gelatin capsules and soft gelatin capsules.To prepare hard gelatin capsules, active ingredient can be mixed with solid, semi-solid or liquid diluent.Soft gelatin capsules can be prepared by mixing active ingredient with water, oil such as peanut oil or olive oil, liquid paraffin, the mixture of monoglycerides and diglycerides of short-chain fatty acids, polyethylene glycol 400 or propylene glycol.

[0242] The liquid preparation for oral administration can be in the form of suspension, solution, emulsion or syrup, or can be lyophilized, or can be provided as a dry product for reconstitution with water or other suitable vehicle before use.Such liquid compositions can optionally contain the following: suspending agent (for example, sorbitol, methylcellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, etc.) and other pharmaceutically acceptable excipients; non-aqueous vehicle, for example, oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol or water; preservative (for example, methyl p-hydroxybenzoate or propyl p-hydroxybenzoate or sorbic acid); wetting agent such as lecithin; and, if desired, flavoring agent or coloring agent.

[0243] The composition may be formulated as a suppository for rectal administration. For parenteral administration, including intravenous, intramuscular, intraperitoneal, intranasal, or subcutaneous routes, the nanoparticles, alone or in combination with other active ingredients, may be provided in a sterile aqueous solution or suspension buffered to an appropriate pH and isotonicity, or in a parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms may be provided in unit-dose forms, such as ampoules or disposable injection devices, in multi-dose forms, such as vials from which the appropriate dose can be withdrawn, or in solid forms or pre-concentrates that can be used to prepare injectable formulations. Exemplary infusion doses range from about 1 to 1,000 μg / kg / min of the drug mixed with a pharmaceutical carrier over a period ranging from several minutes to several days.

[0244] For nasal, inhaled, or oral administration, the nanoparticles, alone or in combination with other active ingredients, may be administered, for example, using a spray formulation, also containing a suitable carrier.

[0245] For topical application, the nanoparticles alone or in combination with other active ingredients are preferably formulated as a cream or ointment, or similar vehicle suitable for topical administration. For topical administration, the nanoparticles alone or in combination with other active ingredients may be mixed with a pharmaceutical carrier at a drug concentration of about 0.1% to about 10% of the vehicle. Another mode of administration of the nanoparticles alone or in combination with other active ingredients may utilize a patch formulation for transdermal delivery.

[0246] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising nanoparticles alone or in combination with other active ingredients and methylcellulose. In certain embodiments, the methylcellulose is present in a suspension at about 0.1, 0.2, 0.3, 0.4, or 0.5 to about 1%. In certain embodiments, the methylcellulose is present in a suspension at about 0.1 to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1%. In certain embodiments, the methylcellulose is present in a suspension at about 0.1 to about 1%. In certain embodiments, the methylcellulose is present in a suspension at about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.8, or 1%. In certain embodiments, the methylcellulose is present in a suspension at about 0.5%.

[0247] As used herein, "prophylactic" treatment is intended to indicate postponing the onset of a disease, symptom of a disease or medical condition, suppressing symptoms that may appear, or reducing the risk of developing or recurring a disease or condition. "Curative" treatment includes reducing the severity or preventing the worsening of an existing disease, symptom, or condition.

[0248] Those skilled in the art can modify the formulation within the scope of the teachings of this specification to provide various formulations for specific administration routes.In particular, nanoparticles alone or in combination with other active ingredients may be modified to make them more soluble in water or other vehicles.It is also well within the scope of those skilled in the art to modify the administration route and the administration regimen of specific nanoparticles alone or in combination with other active ingredients to manage the pharmacokinetics of the compound in order to maximize the beneficial effect in patients. [Example]

[0249] Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as in any way limiting the scope of the present teachings. Example 1 Preparation and characterization of cholesterol-enriched nanoparticles Experiment details

[0250] Cell membrane induction: To induce RBC membranes free of cytoplasmic content, packed RBCs from ICR mice (6-8 weeks, obtained from BioIVT) were washed in ice-cold 1X PBS at 4°C by repeated centrifugation at 800 × g for 5 minutes. To induce hypotonicity, the cells were then suspended in 0.25X PBS in an ice bath for 20 minutes and centrifuged at 800 × g for 5 minutes. Hemoglobin was removed, and the pink pellet was collected. This process was repeated three times. Membrane protein content was quantified using the Pierce BCA assay (Life Technology). To induce membranes from mammalian cells (e.g., platelets, J774 mouse macrophages, and neutrophil-like cells derived from HL-60 cells), the cells were sedimented and the pellet was washed three times with 1X PBS. Next, 15 mL of 1X PBS was added. The cell pellet was dispersed in an isolation buffer solution consisting of PBS, 0.5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; Sigma), and 50 μL of a cocktail of phosphatase and protease inhibitors (100X; Sigma). This suspension was loaded into a Dounce homogenizer, and the cells were disrupted by passing 15 times. After disruption, the suspension was sedimented at 800 × g for 5 minutes to remove large debris. The supernatant was collected and centrifuged again at 10,000 × g for 25 minutes. The pellet was then discarded, and the supernatant was centrifuged at 150,000 × g for 35 minutes. After centrifugation, the supernatant was discarded, and the plasma membranes were collected as an off-white pellet. The membrane pellet was then washed once with 1 mM ethylenediaminetetraacetic acid (EDTA; USB Corporation) in HO and resuspended by gentle sonication for subsequent experiments. Membrane protein content was quantified using the Pierce BCA assay (Life Technology).

[0251] Preparation of cholesterol-stabilized membrane vesicles: To generate cholesterol-enriched membrane vesicles, cholesterol (Avanti Polar Lipids) dissolved in chloroform at 50 mg / mL was added to membrane ghosts at various initial loadings and then gently mixed at 37°C for 10 minutes. To form vesicles, the suspension was either sonicated for 6 minutes in a Fisher Scientific FS30D bath sonicator, or extruded sequentially through polycarbonate membranes (Whatman) with pore sizes of 1 μm, 400 nm, and 200 nm using an Avanti Polar Lipids mini-extruder. To remove unincorporated cholesterol, the membrane vesicle suspension was added to hexane (approximately 1:1 volume ratio), and the mixture was left at room temperature for 30 minutes.

[0252] Cholesterol quantification: Total cholesterol content was determined using Amplex® Red Cholesterol was quantified using a Cholesterol Assay Kit (ThermoFisher Scientific) according to the manufacturer's instructions.

[0253] Preparation of MRSA300 supernatant (MS): MRSA USA300 stock solution (American Type Culture Collection, ATCC) was inoculated onto agar plates of tryptic soy broth (TSB) and incubated at 37°C for 30 hours. A single colony was then transferred from the agar plate to 4 mL of TSB broth and then incubated at 37°C for 12 hours with gentle shaking. The bacterial culture was then transferred to 200 mL of fresh TSB and incubated until the OD600 value reached approximately 7 (OD600 = 1 × 10). 8 The supernatant was collected by centrifugation at 4,500 × g for 30 minutes, and then the supernatant was passed through a 0.2 μm membrane filter. The supernatant was then lyophilized and reconstituted to 1 / 10 of its volume (equivalent to a 10× concentration). Aliquots of the MS were stored at −80°C for later experiments.

[0254] Neutralization of hemolytic activity of MRSA supernatant (MS) by cholesterol-rich RBC membrane vesicles (Cho-RBC-V): To evaluate the neutralization of MS hemolytic activity, 4.5 μL of MS was first mixed with various concentrations of Cho-RBC-V to a final volume of 60 μL in 10% w / v sucrose and 10 mM DTT. This mixture was incubated at 37°C for 30 minutes and then added to 100 μL of a 5% v / v human RBC suspension, which was then incubated at 37°C for 30 minutes. After incubation, the supernatant was collected, and the released hemoglobin was quantified by measuring the absorbance at 540 nm on a plate reader. All experiments were performed in triplicate.

[0255] Neutralization of anti-platelet antibodies by cholesterol-rich platelet membrane vesicles (Cho-PL-V): Recombinant human TNF-α (Thermo Fisher Scientific, final concentration 8.82 ± 0.80 ng ml -1) was mixed with Cho-Neu-V (final concentrations of 64, 128, 256, 512, and 1024 μg / mL). The mixture was incubated at 37°C for 2 hours and then centrifuged at 16,100g for 10 minutes to remove vesicles. Cytokine concentrations in the supernatant were quantified using a human TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Biolegend). All experiments were performed in triplicate.

[0256] Neutralization of endotoxin by cholesterol-rich macrophage membrane vesicles (Cho-MΦ-V): To determine the neutralization of lipopolysaccharide (LPS) by Cho-MΦ-V, samples of Cho-MΦ-V (64, 128, 256, 512, and 1024 μg / mL) were mixed with LPS (LPS-EK, InvivoGen, 50 μg / mL) and cultured in J774 macrophages (2 × 10 in 96-well tissue culture plates). 4 Immediately add the IL-6 solution to the culture medium (cells / well). The cells were cultured at 37°C for 5 hours. After the culture period, the concentration of IL-6 in the culture medium was quantified using a mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Biolegend). All experiments were performed in triplicate.

[0257] Neutralization of inflammatory cytokines (TNF-α) by cholesterol-rich neutrophil membrane vesicles (Cho-Neu-V): Recombinant human TNF-α (Thermo Fisher Scientific, final concentration 8.82 ± 0.80 ng ml -1 ) was mixed with Cho-Neu-V (final concentrations of 64, 128, 256, 512, and 1024 μg / mL). The mixture was incubated at 37°C for 2 hours and then filtered at 16,000 g for 5 minutes using a 1300 kDa MW cutoff centrifugal filter (Nanosep, Pall Laboratory) to separate unbound and vesicle-bound cytokines. Cytokine concentrations in the filtrate were quantified using a human TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Biolegend).

[0258] Figures 1-4 show several types of cholesterol-rich vesicles, including cholesterol-rich RBC membrane vesicles (Cho-RBC-V), cholesterol-rich platelet membrane vesicles (Cho-PL-V), cholesterol-rich macrophage membrane vesicles (Cho-MΦ-V), and cholesterol-rich neutrophil membrane vesicles (Cho-Neu-V). 1 illustrates the preparation and characterization of α-enriched plasma membrane vesicles. Example 2 Preparation of Composition A Experimental design and methods Materials and Devices

[0259] Sphingomyelin was purchased from NOF. Cholesterol was purchased from Spectrum. Isopropyl alcohol was purchased from Sigma Aldrich. Sterile PBS (1X) was purchased from Hyclone. M-110EH-30 microfluidizer was purchased from Microfluidics International Corporation. KMPi TFF pump system was purchased from Repligen. Cellulose acetate (CA) capsule filters (0.2 μm) were purchased from Sartorius. Glass capsule fibers (0.2 μm) were purchased from PALL.

[0260] Using the principles disclosed in WO2017 / 087897(A1), hRBC membranes were prepared using tangential flow filtration (TFF) to reduce the levels of hemoglobin and other impurities. Typically, TFF is used one or more times to dilute, concentrate, and diafilter lysed hRBCs to remove hemoglobin and other impurities. At the end of this process, hRBC membranes were collected in 1X PBS buffer. Test Method:

[0261] The nanoparticle (or nanosponge) size and distribution index (PDI) were measured using a Zetasizer Nano Series model: ZEN 3600. The sample was diluted with 1x PBS to a measurement concentration of 0.25 mg / ml. procedure

[0262] Calculate the amount of each component based on the starting concentration. For example, for a starting concentration of 2.5 mg / ml and 2,000 ml, the calculations for each component were as shown in Table 1 below. [Table 1] Preparation of SM / Chol IPA solution

[0263] SM and cholesterol were weighed and added to a glass bottle. IPA was added to the SM / Cholesterol bottle. SM / Chol IPA was heated until completely dissolved. Preparation of the starting working mixture for sonication

[0264] hRBC membranes in 1x PBS were mixed with SM and cholesterol IPA solvent (weight ratio of SM:cholesterol:hRBC membranes equal to 4:4:2). This mixture was mixed thoroughly using a vortex or stir bar. The mixture of hRBC membranes, SM, and cholesterol was homogenized using a sonication bath. Microfluidization for the preparation of nanoparticles (or nanosponges)

[0265] The sonicated RBC membrane / SM / cholesterol mixture was passed through a microfluidizer at 20 kpsi, and the nanoparticles (or nanosponges) were cooled and collected by a product chiller. Pre-filtration of nanosponges

[0266] To sterilize, the resulting nanosponge suspension was pre-filtered through a 0.45 μm glass fiber combined with a 0.2 μm cellulose acetate sterilizing filter to remove larger particles (>200 μm). The nanosponges were then concentrated using TFF (300 kd hollow fiber column) and IPA was removed (<5,000 ppm) by diafiltration.

[0267] The particle size distribution of an exemplary preparation of Composition A (Lot 5-270-01-S-MF-Final Average) is shown in FIG. Example 3 Effect of Composition A on survival in a murine model of pneumonia infected with methicillin-resistant Staphylococcus aureus USA300 summary

[0268] Staphylococcus aureus is a major cause of hospital-acquired and community-acquired pneumonia, with a case-fatality rate of up to 60%. Treatment of this infection is hampered by the fact that half of Staphylococcus aureus isolates from patients with pneumonia are methicillin-resistant (MRSA). Vancomycin and linezolid are currently the first-line antibiotic treatments for MRSA pneumonia. However, the case-fatality rate in pneumonia patients treated with both remains high, and the emergence of resistance and side effects limit the usefulness of these antibiotics. The consistent evolution of antibiotic resistance has led to the search for alternative methods for preventing and treating MRSA pneumonia. One such approach is to target virulence factors, such as pore-forming toxins produced by MRSA, to prevent pulmonary inflammation and epithelial cell destruction.

[0269] In this study, we developed a mouse model of pulmonary infection caused by MRSA USA300 and used this model to evaluate the effect of intratracheal administration of Composition A, a lead formulation of hRBC nanosponge, on improving the survival rate of infected mice. The results of this study show that intratracheal administration of Composition A consistently reduced the mortality rate of pneumonia-infected mice infected with MRSA USA300. The improvement in survival rate was dose-dependent, but not lot-dependent. At 2, 6, and 19 mg / kg, Composition A significantly improved mouse survival rates to 38%, 66%, and 68%, respectively, compared to vehicle-treated mice (26% survival rate). Furthermore, intratracheal administration of Composition A reduced the bacterial burden in the lungs of mice infected with MRSA USA300, potentially underlying the mechanism of action of Composition A. material and method: Animals and Husbandry

[0270] Eight-week-old female or male CD1 mice (weighing 24–28 grams) were purchased from Charles River Laboratories. Mice were group-housed (5 mice / cage) in solid-bottom micro-isolator cages on stainless steel racks and fed irradiated Teklad Global 2918 rodent chow with ad libitum access to water. Bedding was provided in the form of irradiated Teklad 1 / 8-inch corncob bedding 7902.

[0271] The environment was controlled at a temperature range of 74°±5°F and a humidity range of 30-70%. Fluorescent lighting was provided for 12 hours per day.

[0272] Mice were allowed to acclimate for a minimum of 24 hours before the start of the study. Animals were observed for general health and acceptability for use in this study. Only animals that appeared healthy were included in the study. Animals were handled in accordance with IACUC procedures and in accordance with Animal Use Protocol (AUP) No. S00227m. Bacteria, cultures and intratracheal instillation

[0273] Methicillin-resistant Staphylococcus aureus (MRSA) USA300 (TPPS1056) bacteria were grown overnight on Todd Hewitt trypticase soy agar plates supplemented with 5% sheep blood cells at 37°C in a 5% CO2 atmosphere. The cultures were aseptically wiped and transferred to liquid culture in Todd Hewitt broth for overnight growth. On the day of infection, bacteria were aliquoted at a 1:10 dilution and grown to mid-logarithmic phase at an OD600nm of 0.4. Cultures were washed several times with 1X phosphate-buffered saline (PBS) to remove toxins released by the bacteria during incubation and diluted to 1 x 10 per mouse in a volume of 30 μL. 7 ~8×10 9 Target challenge inocula were obtained in the range of CFU. The inoculum count was estimated before inoculation by optical density and confirmed after inoculation by dilution, plating, 24-hour incubation, and backcounting. The data is then recorded as "inoculum."

[0274] On the day of the experiment, mice were anesthetized with ketamine and xylazine (90 mg / kg and 10 mg / kg intraperitoneally, respectively). The anesthetized mice were held at the tip of an otoscope, and the glottis and vocal cords were visualized using the otoscope's magnifying glass and light source. Next, 30 μL of bacterial culture was delivered into the trachea using a long plastic pipette. Intratracheal Administration of the Composition

[0275] Immediately after bacterial instillation (<1 min), while the mice were still anesthetized, 30 μL of Composition A or PBS as vehicle was administered intratracheally via the tip of a long plastic pipette. The mice were allowed to recover from anesthesia and returned to their home cages. The dose per animal was calculated based on the lot concentration of Composition A used and the average body weight of the animals in the tested group. Mortality as an endpoint

[0276] Mice were closely observed throughout the study for signs of mortality and imminent death. Survival was followed for 4 days (unless otherwise noted) after bacterial challenge / nanosponge treatment. At the end of the study, surviving mice were humanely euthanized by CO2 overdose exposure. Lung bacterial count as an endpoint

[0277] After bacterial challenge / nanosponge treatment, mice were allowed to survive for 24 or 48 hours to determine lung bacterial counts. At one time point, mice were humanely euthanized by CO2 overdose exposure, and the left or right lung was harvested. Lung tissue was homogenized and plated in 96-well plates. Plates were incubated overnight at either 30°C or 37°C in an incubator. The easiest dilution to count was counted, and the dilution and count were recorded. From this data, CFU / gram of tissue were calculated. statistical analysis

[0278] Quantitative data were expressed as mean ± standard error. Survival data were analyzed using the Mantle-Cox log-rank test or Gehan-Breslow-Wilcoxon test, as appropriate (or unless otherwise stated). CFU data were analyzed using analysis of variance (ANOVA) followed by Dunnett's test or Student's t-test, as appropriate. All statistical analyses were performed using GraphPad Prism version 7.01. A P value of <0.05 was considered statistically significant. result Identification of Composition A

[0279] An initial screening study (007) was conducted to identify a lead formulation in a mouse model of MRSA pneumonia. 7Pneumonia was induced by intratracheal instillation of 30 μL of MRSA USA300 culture broth containing 10 colony-forming units (CFU) per mouse. Immediately (within 1 minute) after bacterial challenge, mice (n=10 mice per group) were intratracheally administered Composition A or 5% sorbitol / 95% phosphate buffered saline (PBS) as a vehicle. After bacterial challenge / formulation treatment, mice were allowed to survive for 48 hours. The number of mice that died within 48 hours in each treatment group was counted and survival rates were analyzed. At the 48-hour time point, the remaining surviving mice were sacrificed, and their lungs were harvested and processed for CFU enumeration. Survival data are summarized in Table 2 below and shown in Figure 7A. During the first 48 hours, five vehicle-treated mice died, while none of the Composition A-treated mice died. The survival rates were 50% and 100%, respectively. The difference in survival rates between the vehicle- and Composition A-treated groups was statistically significant. [Table 2] Reproducible effect of Composition A on improving survival rate

[0280] In the above study, we demonstrated that Composition A lot 5-257-06 significantly improved survival and reduced lung bacterial burden in a mouse model of MRSA pneumonia. To determine the reproducibility of the effect, we tested three different lots of Composition A with similar concentrations in this model. Figure 8A shows an example of these tests. In this study (011), after bacterial challenge, mice were intratracheally administered PBS (vehicle), 19 mg / kg of lot 5-257-06, or 22 mg / kg of lot 5-257-16 and observed for 96 hours after treatment. As shown in Figure 8A, at 96 hours after challenge / treatment, the survival rate in the vehicle-treated group was 55%. Treatment with lots 5-257-06 and 5-257-16 significantly improved survival (by 80% and 95%, respectively). Figure 8B plots the average survival data from studies with three different lots of Composition A (5-257-06 at 19 mg / kg, 5-257-16 at 22 mg / kg, and 5-260-02 at 25 mg / kg). Significant improvements in survival were observed after treatment at all observation time points from 19 hours to 96 hours after challenge / treatment. Dose-Curve Study of Composition A

[0281] In this study, Composition A Lot 5-266-01 was administered intratracheally in four independent experiments (022, 023, 024, and 026) at three concentrations: 19 mg / kg, 6 mg / kg, and 2 mg / kg. Overall, a total of 40 mice were administered vehicle, 19 mg / kg, and 2 mg / kg, and a total of 30 mice were administered 6 mg / kg. Pooled survival data were plotted as shown in Figure 9. At the 96-hour time point after bacterial challenge / nanosponge treatment, the vehicle-treated group had a survival rate of 26%. Survival rates improved to 68%, 66%, and 38% at the 19 mg / kg, 6 mg / kg, and 2 mg / kg doses, respectively. Significant improvements in survival were observed at all three doses. Inhibition of Lung Bacterial Burden

[0282] In the first study (007), we demonstrated that Composition A lot 5-257-06 significantly reduced bacterial burden in the lungs 48 hours after MRSA infection (Figure 7). In this study (012), we tested whether the inhibitory effect on lung bacterial burden could be reproduced using different lots of Composition A 5-257-16. Immediately after bacterial instillation, 5-257-16 was administered intratracheally at 22.5 mg / kg, and lungs of mice were harvested 24 hours after challenge / treatment. Lung homogenates were plated for CFU counts as described in the methods section. As shown in Figure 10, 5-257-16 significantly reduced bacterial burden in the lungs of mice with MRSA pneumonia. Example 4 Assessment of pulmonary exposure to fluorescent tracer-labeled composition A in naive mice summary

[0283] DiR-Composition A, a fluorescent form of Composition A, was used to study the uptake, distribution, and retention of DiR-Composition A in the lungs of mice after intratracheal administration. At the indicated time points after administration of DiR-Composition A, mice were sacrificed and lungs were harvested for ex vivo measurement of DiR-Composition A fluorescence intensity using a fluorescence imaging device or a fluorescence microplate reader. DiR-Composition A was absorbed and rapidly distributed in the lungs after intratracheal administration. Within 5 minutes, DiR-Composition A had spread to central and peripheral lung tissues with fairly uniform distribution. DiR-Composition A remained in the lungs for an extended period of time. The estimated retention half-life of DiR-Composition A in the lungs was 7-10 days. material and method: Formulation and characterization

[0284] To prepare fluorescently labeled Composition A, 0.1 weight percent DiR dye dissolved in 10 μl of DMSO was added to human RBC membranes before the addition of sphingomyelin and cholesterol. The solution was heated to 45°C and stirred for 1 hour. The bath was then sonicated for 10 minutes to incorporate all of the DiR into the lipid bilayer of the RBC membrane. These dye molecules should insert directly into the lipid bilayer in a manner similar to sterols. After labeling the RBC membrane, cholesterol and sphingomyelin were added in the same manner to prepare lots of Composition A for various efficacy studies. DiR-Composition A used in Figures 11-14 was made using the same protocol as Composition A (Lot 5-257-16). For Figure 15, the protocol for batch 5-265-01 was used.

[0285] DiR-Composition A was characterized using dynamic light scattering (Malvern Analytical) to assess size, polydispersity, and zeta potential, confirming similarity to non-DiR-labeled formulations. A dye stability test was performed to evaluate the fluorescence stability of DiR-Composition A. For release studies, 20 kDa dialysis cups were placed in a large volume (1 L) of PBS, and 200 μl of 15 mg / mL DiR-Composition A was added to each dialysis cup (n=3). The samples were capped and gently stirred at 300 rpm for one week. 10 μl samples were withdrawn at the indicated time points and diluted 10x in water. The fluorescence intensity of the samples was measured and compared to the starting time point and an internal control to assess sample stability. Animals and husbandry

[0286] Eight-week-old female CD1 mice weighing between 25 and 30 g were ordered from Charles River Laboratories, acclimated to housing conditions, and handled in accordance with Animal Use Protocol No. S00227m and IACUC procedures. Mice were group-housed (5 mice / cage) in disposable plastic cages on stainless steel racks and fed Teklad Global 2918 rodent chow with ad libitum access to water. Bedding was provided in the form of irradiated Teklad 1 / 8-inch corncob bedding 7902. The environment was controlled at a temperature range of 74°±5°F and a humidity range of 30-70%. Fluorescent lighting provided 12 hours of light per day.

[0287] Mice were allowed to acclimate for a minimum of 24 hours before the start of the study. In this study, animals were observed for general health and acceptability for use. Only animals that appeared healthy were included in the study. Nanosponge administration

[0288] Mice were anesthetized with ketamine and xylazine (90 mg / kg and 10 mg / kg intraperitoneally, respectively). The anesthetized mice were then held at the tip of an otoscope, and the glottis and vocal cords were visualized using the otoscope's magnifying glass and light source. A volume of 30 μl of DiR-Composition A was pipetted into the trachea from the tip of a long plastic gel-loading pipette. Mice were allowed to recover from anesthesia and returned to their home cages. N=3 per test group were used in this study.

[0289] Mice were closely monitored for any signs of mortality and health throughout the study. Mice were humanely euthanized at predetermined time points for this study by CO2 overexposure followed by cervical dislocation. Lungs were excised and stored in 2 mL plastic screw-top tubes wrapped in aluminum foil. In vivo imaging

[0290] Lungs were excised from mice and arranged according to group on a matte black background. Using the IVIS fluorescence imaging device and software, lung photographs were taken and the corresponding fluorescence counts were overlaid with simultaneously captured bright-field lung images. Lung homogenization and fluorescence quantification

[0291] In some studies, lung tissue was homogenized in water containing quartz and then homogenized for 60 seconds using a bead beater homogenizer, with beads (between 10 and 20 beads) added to each tube. Samples were plated in 96-well plates, and fluorescence was measured using a plate reader with excitation at 760 nm and emission at 805 nm. Background signal from lungs not treated with DiR-Composition A was subtracted from the final results. result Characterization of DiR-composition A

[0292] Figure 11 shows the size and stability analysis of DiR-Composition A. Table 3 below shows the dynamic light scattering data for DiR-Composition A. The average size of DiR-Composition A was 98.6 nm, the polydispersity index was 0.2, and the D(50) was 67.2. These size and zeta potential measurements indicate a formulation very similar to unlabeled Composition A. Size data are the average of three independent measurements. Stability data show that the fluorescence intensity maintained the same efficacy over a week and after dialysis, suggesting that the dye did not leak from the lipid layer and remained embedded in the particles throughout the study. [Table 3] Pulmonary uptake and distribution of DiR-Composition A

[0293] Figure 12 shows the uptake and distribution of DiR-Composition A over a 12-hour period following intratracheal administration. At the indicated time points after intratracheal administration, mice were sacrificed and lungs were harvested. Lung samples were imaged with an IVIS in vivo imager using an infrared fluorescence filter. At 5 minutes after administration, DiR-Composition A was distributed throughout the lung tissue at each time point, with fairly uniform distribution and notable nanoparticle accumulation in most areas of the lung. These results indicate that the nanosponges have good perfusion throughout the lung, even within 5 minutes. This indicates that there is little delay in the formulation reaching the pulmonary periphery. Pulmonary retention of DiR-Composition A after intratracheal administration

[0294] For the retention study shown in Figure 13, mice (n=3) were administered DiR-Composition A intratracheally. Three mice from each group were sacrificed at predetermined time points: 2 hours, 7 days, 14 days, and 21 days. Lungs were excised and homogenized in 900 μl of water, and fluorescence was then read using a plate reader (excitation 760 / emission 805). Quantitation of lung fluorescence showed decay of DiR-Composition A in the lungs over a 3-week period, with an estimated half-life of 7-10 days. Error bars between mice are plotted as standard error of the mean (SEM). At the 2-hour time point, the signal was normalized to 100%, and the background fluorescence of the lung tissue itself was set to 0. Effect of High Concentration DiR-Composition A on Pulmonary Delivery

[0295] In the studies shown in Figures 11-14, the nanosponges were maintained at 15 mg / mL, equivalent to batch 5-257-16. To test the effect of nanosponge concentration on lung distribution, we generated a high-concentration batch at 46 mg / mL, reflecting the same method and concentration as batch 5-265-01. This batch was then diluted to 23 mg / mL to generate a control similar to the previous study. Mice were administered 30 μl of either 46 mg / mL or 23 mg / mL nanosponges. Five minutes after administration, the mice were sacrificed, and the lungs were imaged using an IVIS system.

[0296] Figure 15 shows the distribution of nanoparticles in each group. To properly assess nanosponge spread within the lung, the 20 mg / mL group was also imaged with a longer fluorescent exposure to compensate for having half the number of fluorophores in the same volume. Comparison between these groups shows that the higher concentration of particles distributes just as evenly and rapidly as the lower concentration of nanosponges, with no clear differences observed between the groups.

[0297] Some specific references: [ka]

Claims

1. A composition comprising nanoparticles for treating or preventing a disease or condition caused by a bacterial toxin in a subject in need thereof, said nanoparticles comprising an inner compartment (or inner core) and an outer surface (or shell) comprising a cell-derived cell membrane; a) the inner compartment (or inner core) contains a fluid that is isotonic with cellular or physiological fluids; and b) said outer surface (or shell) comprises exogenously added steroids at a level of 20% (w / w) to 50% (w / w) and exogenously added sphingolipids at a level of 20% (w / w) to 70% (w / w); provided that, when the cell membrane is derived from a red blood cell, the outer surface comprises 20% (w / w) to 50% (w / w) of exogenously added cholesterol, 20% (w / w) to 70% (w / w) of exogenously added sphingolipids, and 10% (w / w) to 50% (w / w) of cell membrane derived from the red blood cell.

2. The inner compartment (inner core) contains a fluid that is isotonic with physiological fluids ex vivo; or The inner compartment (inner core) contains a fluid that is isotonic with physiological fluids in vivo; The composition of claim 1.

3. The composition described in claim 1 or 2, wherein the cell membrane comprises a plasma membrane.

4. The cell membrane Derived from eukaryotic cells, derived from a multicellular organism, such as a plant, animal, vertebrate, non-human mammal, or human cell; or derived from animal, vertebrate, non-human mammalian, or human cells; The composition according to any one of claims 1 to 3.

5. The cell membrane is derived from an animal, vertebrate, non-human mammal, or human cell, and optionally: The cells are cells of connective tissue, such as blood, bone, tendon, ligament, fat or loose connective tissue, fibrous connective tissue, skeletal connective tissue or fluid connective tissue. the cell is a muscle tissue cell, e.g., a visceral or smooth muscle, musculoskeletal or cardiac muscle cell, the cell is a cell of nervous tissue, e.g., a cell in the central nervous system (CNS) or peripheral nervous system (PNS); the cells are cells of epithelial tissue, such as simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium (also known as ciliated columnar epithelium), columnar epithelium, glandular epithelium or ciliated columnar epithelium, the cell is a cell of the nervous system, cardiovascular system, circulatory system, vascular system, digestive system, endocrine system, immune system, integumentary system, lymphatic system, musculoskeletal system, reproductive system, respiratory system, respiratory tract, ventilatory system, urinary system or renal system or urinary tract, or the cell is a blood cell, a tumor cell, a cancer cell, an immune cell, a stem cell, an endothelial cell, or an epithelial cell; The composition according to any one of claims 1 to 3.

6. A composition described in any one of claims 1 to 5, wherein the steroid is an animal steroid, such as a vertebrate steroid, a steroid hormone or cholesterol, a plant steroid, or a prokaryotic steroid.

7. A composition described in any of claims 1 to 6, wherein the outer surface (or shell) comprises a cell membrane derived from a mammalian cell, e.g., a human cell, and the cell membrane comprises an exogenously added mammalian steroid, e.g., an exogenously added human steroid, at a level of 20% (w / w) to 50% (w / w).

8. The inner compartment (inner core) has a pH in the range of about 4 to about 10, for example, about 6 to about 9. the nanoparticles further comprise a releasable cargo; the nanoparticles have a diameter of about 10 nm to about 10 μm; the nanoparticles substantially maintain the native structural integrity or activity of the cell membrane or the constituents of the cell membrane; the inner core comprises a salt, a sugar, or a sugar alcohol; the nanoparticles are biocompatible or biodegradable; the nanoparticles are substantially devoid of immunogenicity to the species or subject to which the cell membrane is derived; or the outer surface (or shell) comprises a naturally occurring cell membrane, and further comprises a synthetic membrane; The composition according to any one of claims 1 to 7.

9. A composition described in any one of claims 1 to 8, wherein the outer surface (or shell) comprises 30% (w / w) to 50% (w / w) of the exogenously added sphingolipid, such as sphingomyelin.

10. A composition described in any of claims 1 to 9, wherein the nanoparticles comprise an inner compartment (or inner core) and an outer surface (or shell), the inner compartment (or inner core) comprising a liquid isotonic with cellular fluid or physiological fluid, the outer surface (or shell) comprising cell membranes derived from cells, exogenously added cholesterol, and exogenously added sphingomyelin, the outer surface (or shell) comprising 20% ​​(w / w) to 50% (w / w) of the exogenously added cholesterol and 20% (w / w) to 70% (w / w) of the exogenously added sphingomyelin.

11. The composition of claim 10, wherein the outer surface (or shell) comprises about 20% (w / w) to about 50% (w / w) cholesterol, about 20% (w / w) to about 70% (w / w) sphingomyelin, and about 10% (w / w) to about 50% (w / w) plasma membrane derived from red blood cells.

12. Use of an effective amount of nanoparticles for the manufacture of a medicament for treating or preventing a disease or condition caused by a bacterial toxin in a subject in need thereof, wherein the nanoparticles comprise an inner compartment (or inner core) and an outer surface (or shell) comprising a cell-derived cell membrane; a) the inner compartment (or inner core) contains a fluid that is isotonic with cellular or physiological fluids; and b) said outer surface (or shell) comprises exogenously added steroids at a level of 20% (w / w) to 50% (w / w) and exogenously added sphingolipids at a level of 20% (w / w) to 70% (w / w); With the proviso that, when the cell membrane is derived from a red blood cell, the outer surface comprises 20% (w / w) to 50% (w / w) of exogenously added cholesterol, 20% (w / w) to 70% (w / w) of exogenously added sphingolipids, and 10% (w / w) to 50% (w / w) of cell membrane derived from the red blood cell.

13. A composition according to any one of claims 1 to 11, or a use according to claim 12, wherein the bacterial toxin is a pore-forming toxin.

14. The composition or use of claim 13, wherein the pore-forming toxin is alpha-hemolysin from S. aureus, such as alpha-hemolysin from methicillin-resistant Staphylococcus aureus (MRSA).

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