Inhibition of trained immunity with therapeutic nanobiological compositions

Abstract

To provide compositions and methods for treating a patient affected by trained immunity.SOLUTION: A nanobiological composition for reducing the long-term hyperresponsiveness of myeloid cells and their stem cells and progenitors as a result of trained immunity comprises a nanoscale assembly, and an inhibitor drug incorporated in the nanoscale assembly. The nanoscale assembly is a multi-component carrier composition comprising a phospholipid and apoA-I or a peptide mimetic of apoA-I. The drug is an inhibitor of an inflammasome, an inhibitor of a metabolic pathway or an inhibitor of an epigenetic pathway within a hematopoietic stem cell (HSC), a CMP or a myeloid cell. In an aqueous environment, the nanobiological composition is a self-assembled nanodisc or nanosphere with size between about 8 nm and about 400 nm in diameter.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-references to related cases This application claims priority to U.S. Patent Application No. 62 / 588,790, filed November 20, 2018, and U.S. Patent Application No. 62 / 734,664, filed September 21, 2018, which are incorporated by reference herein in their entireties.

[0002] Declaration of Federally Funded R&D This invention was made with government support under grant number R01 HL118440 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present invention relates to therapeutic nanobiological compositions and methods for treating patients who have undergone organ transplantation or who suffer from atherosclerosis, arthritis, inflammatory bowel disease including Crohn's disease, autoimmune disease, and / or autoinflammatory conditions, or following a cardiovascular event including stroke and myocardial infarction, by inhibiting trained immunity, a long-term hyperresponsiveness secondary to increased cytokine output resulting from metabolic and epigenetic rewiring upon restimulation following the initial invasion of myeloid cells and their precursors and stem cells in the bone marrow, spleen, and blood. [Background technology]

[0004] Current treatments for patients suffering from autoimmunity and immune system dysfunction are inadequate. Patients who have undergone organ transplants or suffer from autoimmune diseases and / or autoinflammatory conditions, including atherosclerosis, arthritis, inflammatory bowel disease, including Crohn's disease, diabetes, or have suffered from cardiovascular events, including stroke and myocardial infarction, need a treatment paradigm that is sustainable and does not pose side effects compared to the primary treatment itself. Summary of the Invention

[0005] Thus, to address these and other deficiencies of the prior art, in preferred embodiments of the present invention, methods are provided for treating a patient in need thereof with a therapeutic agent for inhibiting trained immunity.

[0006] Trained immunity is defined by a secondary, long-term hyperresponsiveness, manifested by increased cytokine secretion, induced by metabolic and epigenetic rearrangements upon restimulation of myeloid cells and their precursors and stem cells in the bone marrow, spleen, and blood following an initial insult. Trained immunity (also called innate immune memory) is also defined by a secondary, long-term hyperresponsiveness (e.g., significant cytokine production) upon restimulation of myeloid innate immune cells, induced by an initial insult that stimulates these cells or their precursors and stem cells in the bone marrow and spleen, and mediated by epigenetic, metabolic, and transcriptional rearrangements.

[0007] Treatment of patients affected by trained immunity In a preferred, non-limiting embodiment of the present invention, there is provided a method of treating a patient susceptible to trained immunity to reduce the innate immune response in said patient, comprising: administering to the patient a nanobiological composition in an amount effective to reduce a hyperresponsive innate immune response; the nanobiology composition comprises (i) a nanoscale construct; and (ii) an inhibitor drug incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising (a) a phospholipid and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs (common myeloid progenitors), or myeloid cells; the nanoscale construct delivers the drug to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen, thereby reducing a hyperresponsive innate immune response caused by trained immunity in the patient; Including steps, A method is provided.

[0008] In a preferred, non-limiting embodiment of the present invention, there is provided a method of treating a patient susceptible to trained immunity to reduce the innate immune response in said patient, comprising: The nanoscale structure is Phospholipids and apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or a combination thereof; A multi-component carrier composition comprising: A method is provided.

[0009] In another non-limiting preferred embodiment of the present invention, there is provided a method of treating a patient susceptible to trained immunity to reduce a hyperresponsive innate immune response in said patient, comprising: The nanoscale structure is Phospholipids and apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof; Cholesterol and A multi-component carrier composition comprising: A method is provided.

[0010] Promoting allograft tolerance In a preferred, non-limiting embodiment of the present invention, there is provided a method for promoting allograft tolerance in a transplant recipient patient, comprising: administering to said patient a nanobiological composition in an amount effective to induce durable allograft tolerance; the nanobiology composition comprises (i) a nanoscale construct; and (ii) an inhibitor drug incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising (a) a phospholipid or a mixture of phospholipids, and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; the nanoscale construct delivers the drug to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen, thereby inducing durable allograft acceptance in the transplant recipient patient; A method is provided that includes the steps.

[0011] In a preferred, non-limiting embodiment of the present invention, there is provided a method for promoting allograft tolerance in a transplant recipient patient, comprising: The nanoscale structure is a phospholipid or a mixture of phospholipids; apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a matrix lipid selected from one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; A multi-component carrier composition comprising: A method is provided.

[0012] In a preferred, non-limiting embodiment of the present invention, there is provided a method for enhancing allograft tolerance in a transplant recipient patient, the method comprising the steps of: a phospholipid or a mixture of phospholipids; apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a matrix lipid selected from one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; Cholesterol and A multi-component carrier composition comprising: A method is provided.

[0013] Lasting effect In non-limiting preferred embodiments of the present invention, in any one of the methods herein, the hyperresponsive innate immune response is reduced for at least 7 to 30 days.

[0014] In non-limiting preferred embodiments of the present invention, in any one of the methods herein, the hyperresponsive innate immune response is reduced for at least 30 to 100 days.

[0015] In non-limiting preferred embodiments of the present invention, in any one of the methods herein, the long-term hyperresponsiveness of myeloid cells, their stem cells and progenitors as a result of trained immunity (hyperresponsive innate immune response) is reduced for at least 100 days up to several years.

[0016] In a non-limiting preferred embodiment of the present invention, in any one of the methods herein, the nanobiology composition is administered once and reduces long-term hyperresponsiveness of myeloid cells, their stem cells and progenitors as a result of trained immunity for at least 30 days.

[0017] In a non-limiting preferred embodiment of the present invention, in any one of the methods herein, the nanobiology composition is administered at least once per day in a multiple dose regimen, and long-term hyperresponsiveness of myeloid cells, their stem cells and progenitors as a result of trained immunity is reduced for at least 30 days.

[0018] In a non-limiting preferred embodiment of the present invention, in any one of the methods herein, trained immunity is defined by a secondary, long-term hyperresponsiveness characterized by increased cytokine secretion caused by metabolic and epigenetic rearrangements of myeloid cells and their precursors and stem cells in the bone marrow, spleen, and blood upon restimulation following an initial invasion.

[0019] In a non-limiting preferred embodiment of the present invention, in any one of the methods herein, trained immunity is defined by a prolonged increase in responsiveness from significant cytokine production following restimulation with a secondary stimulation of myeloid innate immune cells induced by an initial insult that stimulates myeloid innate immune cells or their precursors and stem cells in the bone marrow, and carried out via epigenetic, metabolic, and transcriptional rearrangements.

[0020] Diseases, disorders, and conditions In non-limiting preferred embodiments of the present invention, the patient affected by trained immunity in any one of the methods herein is an organ transplant recipient or suffers from atherosclerosis, arthritis, inflammatory bowel disease including Crohn's disease, autoimmune disease including diabetes, autoinflammatory conditions, or has suffered from a cardiovascular event including stroke and myocardial infarction.

[0021] In non-limiting preferred embodiments of the present invention, in any one of the methods herein, the patient is a transplant recipient or suffers from atherosclerosis, arthritis, or inflammatory bowel disease, or has suffered from a cardiovascular event.

[0022] In a non-limiting preferred embodiment of the present invention, in any one of the methods herein, the patient has undergone a transplant, and the transplanted tissue is lung tissue, heart tissue, kidney tissue, liver tissue, retinal tissue, corneal tissue, skin tissue, pancreatic tissue, intestinal tissue, reproductive tissue, ovarian tissue, bone tissue, tendon tissue, bone marrow, or vascular tissue.

[0023] In non-limiting preferred embodiments of the present invention, in any one of the methods herein, the method is performed prior to transplantation to restore cytokine production to naive, non-hyperresponsive levels and to induce long-lasting, naive, non-hyperresponsive levels of cytokine production, preferably reducing the ratio of inflammatory to immunosuppressive myeloid cells to said patient for post-transplant acceptance.

[0024] In a non-limiting preferred embodiment of the present invention, in any one of the methods herein, the nanobiology composition is administered in a treatment regimen comprising one or more administrations to said patient to result in accumulation of the drug in myeloid cells, myeloid progenitor cells, and hematopoietic stem cells in the bone marrow, blood, and / or spleen.

[0025] inhibitors In non-limiting preferred embodiments of the present invention, in any one of the methods herein, the inhibitor is an inflammasome inhibitor or an inhibitor of a metabolic or epigenetic pathway, including, but not limited to, NOD2 receptor inhibitors, mTOR inhibitors, ribosomal protein S6 kinase beta-1 (S6K1) inhibitors, HMG-CoA reductase inhibitors (statins), histone H3K27 demethylase inhibitors, BET bromodomain blockade inhibitors, inhibitors of histone methyltransferases and acetyltransferases, inhibitors of DNA methyltransferases and acetyltransferases, serine / threonine kinase Akt inhibitors, inhibitors of hypoxia inducible factor 1-alpha, also known as HIF-1-alpha, and mixtures of one or more thereof.

[0026] In a non-limiting preferred embodiment of the present invention, any one of the methods herein is provided that comprises co-administration of an immunotherapeutic drug as a combination therapy with the nanobiological composition.

[0027] Nanobiology Composition In a preferred, non-limiting embodiment of the present invention, there is provided a nanobiological composition for inhibiting trained immunity, comprising: (ii) an inhibitor drug incorporated into said nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising (a) a phospholipid or a mixture of phospholipids, and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; Nanobiology compositions are provided.

[0028] In a preferred, non-limiting embodiment of the present invention, there is provided a nanobiological composition for inhibiting trained immunity, comprising: The nanoscale structure is a phospholipid or a mixture of phospholipids; apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; A multi-component carrier composition comprising: Nanobiology compositions are provided.

[0029] In a preferred, non-limiting embodiment of the present invention, there is provided a nanobiological composition for inhibiting trained immunity, comprising: The nanoscale structure is a phospholipid or a mixture of phospholipids; apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; Cholesterol and A multi-component carrier composition comprising: Nanobiology compositions are provided.

[0030] In a preferred, non-limiting embodiment of the present invention, there is provided a nanobiological composition for inhibiting trained immunity, comprising: inhibitors of metabolic or epigenetic pathways, including NOD2 receptor inhibitors, mTOR inhibitors, ribosomal protein S6 kinase beta-1 (S6K1) inhibitors, HMG-CoA reductase inhibitors (statins), histone H3K27 demethylase inhibitors, BET bromodomain blockade inhibitors, inhibitors of histone methyltransferases and acetyltransferases, inhibitors of DNA methyltransferases and acetyltransferases, inflammasome inhibitors, serine / threonine kinase Akt inhibitors, inhibitors of hypoxia inducible factor 1-alpha, also known as HIF-1-alpha, and mixtures of one or more thereof; Nanobiology compositions are provided.

[0031] Manufacturing Process In a preferred, non-limiting embodiment of the present invention, there is provided a process for producing a nanobiological composition for inhibiting trained immunity, comprising: incorporating an inhibitor drug into the nanoscale construct, the nanoscale construct is a multi-component carrier composition comprising (a) a phospholipid or a mixture of phospholipids, and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; A process is provided that includes steps.

[0032] In a preferred, non-limiting embodiment of the present invention, there is provided a process for producing a nanobiological composition for inhibiting trained immunity, comprising: The nanoscale structure is a phospholipid or a mixture of phospholipids; apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; A multi-component carrier composition comprising: A process is provided.

[0033] In a preferred, non-limiting embodiment of the present invention, there is provided a process for producing a nanobiological composition for inhibiting trained immunity, comprising: The nanoscale structure is a phospholipid or a mixture of phospholipids; apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; Cholesterol and A multi-component carrier composition comprising: A process is provided.

[0034] In a non-limiting preferred embodiment of the present invention, a process for manufacturing is provided wherein the constructs are combined using microfluidics, high pressure homogenization scale-up microfluidizer technology, sonication, organic-to-aqueous infusion, or lipid film hydration.

[0035] Radiolabeled nanobiology compositions and methods of use In a non-limiting preferred embodiment of the present invention, there is provided a nanobiological composition for imaging accumulation in bone marrow, blood, and spleen, comprising: (ii) an inhibitor drug incorporated into the nanoscale construct; and (iii) a positron emission tomography (PET) imaging radioisotope incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising (a) a phospholipid or a mixture of phospholipids, and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; The PET imaging radioisotope is 89 Zr, 124 I, 64 Cu, 18 F, and 86 Y is selected from The PET imaging radioisotope is complexed with the nanobiological composition using a chelating agent suitable for forming a stable nanobiological composition-radioisotope chelate. Nanobiology compositions are provided.

[0036] In a further non-limiting preferred embodiment of the present invention, there is provided a nanobiological composition for imaging accumulation in bone marrow, blood, and spleen, comprising: (ii) an inhibitor drug incorporated into the nanoscale construct; and (iii) a positron emission tomography (PET) imaging radioisotope incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising: (a) a phospholipid or a mixture of phospholipids; (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; and (c) a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; The PET imaging radioisotope is 89 Zr, 124 I, 64 Cu, 18 F, and 86 Y is selected from The PET imaging radioisotope is complexed with the nanobiological composition using a chelating agent suitable for forming a stable nanobiological composition-radioisotope chelate. Nanobiology compositions are provided.

[0037] In a further non-limiting preferred embodiment of the present invention, there is provided a nanobiological composition for imaging accumulation in bone marrow, blood, and spleen, comprising: (ii) an inhibitor drug incorporated into the nanoscale construct; and (iii) a positron emission tomography (PET) imaging radioisotope incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising: (a) a phospholipid or a mixture of phospholipids; (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; (c) a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; and (d) cholesterol; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; The PET imaging radioisotope is 89 Zr, 124 I, 64 Cu, 18 F, and 86 Y is selected from The PET imaging radioisotope is complexed with the nanobiological composition using a chelating agent suitable for forming a stable nanobiological composition-radioisotope chelate. Nanobiology compositions are provided.

[0038] In a preferred, non-limiting embodiment of the present invention, there is provided a positron emission tomography (PET) method for imaging the accumulation of a nanobiological composition in the bone marrow, blood, and / or spleen of a patient affected by trained immunity, comprising: administering to the patient a nanobiology composition for imaging accumulation in bone marrow, blood, and spleen, the nanobiology composition comprising a nanoscale construct, (ii) an inhibitor drug incorporated into the nanoscale construct, and (iii) a positron emission tomography (PET) imaging radioisotope incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising (a) a phospholipid or a mixture of phospholipids, and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; The PET imaging radioisotope is 89 Zr, 124 I, 64 Cu, 18 F, and 86 Y is selected from the PET imaging radioisotope is complexed with the nanobiological composition using a chelating agent suitable for forming a stable nanobiological composition-radioisotope chelate; Steps and (2) performing PET imaging of the patient to visualize the biodistribution of the stable nanobiology composition-radioisotope chelate in the bone marrow, blood, and / or spleen of the patient's body; A method is provided, comprising:

[0039] In a further non-limiting preferred embodiment of the present invention, there is provided a positron emission tomography (PET) method for imaging accumulation of a nanobiological composition in bone marrow, blood, and / or spleen of a patient affected by trained immunity, comprising: administering to the patient a nanobiology composition for imaging accumulation in bone marrow, blood, and spleen, the nanobiology composition comprising a nanoscale construct, (ii) an inhibitor drug incorporated into the nanoscale construct, and (iii) a positron emission tomography (PET) imaging radioisotope incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising: (a) a phospholipid or a mixture of phospholipids; (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; and (c) a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; The PET imaging radioisotope is 89 Zr, 124 I, 64 Cu, 18 F, and 86 Y is selected from the PET imaging radioisotope is complexed with the nanobiological composition using a chelating agent suitable for forming a stable nanobiological composition-radioisotope chelate; Steps and (2) performing PET imaging of the patient to visualize the biodistribution of the stable nanobiology composition-radioisotope chelate in the bone marrow, blood, and / or spleen of the patient's body; A method is provided, comprising:

[0040] In a preferred, non-limiting embodiment of the present invention, there is provided a positron emission tomography (PET) method for imaging the accumulation of a nanobiological composition in the bone marrow, blood, and / or spleen of a patient affected by trained immunity, comprising: administering to the patient a nanobiology composition for imaging accumulation in bone marrow, blood, and spleen, the nanobiology composition comprising a nanoscale construct, (ii) an inhibitor drug incorporated into the nanoscale construct, and (iii) a positron emission tomography (PET) imaging radioisotope incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising: (a) a phospholipid or a mixture of phospholipids; (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; (c) a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters; and (d) cholesterol; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; the drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells; The PET imaging radioisotope is 89 Zr, 124 I, 64 Cu, 18 F, and 86 Y is selected from the PET imaging radioisotope is complexed with the nanobiological composition using a chelating agent suitable for forming a stable nanobiological composition-radioisotope chelate; Steps and (2) performing PET imaging of the patient to visualize the biodistribution of the stable nanobiology composition-radioisotope chelate in the bone marrow, blood, and / or spleen of the patient's body; A method is provided, comprising: [Brief explanation of the drawings]

[0041] transplant [Figure 1] Figure 1 is an immunostaining panel of four images of vimentin and HMGB1 expression in donor and nontransplanted hearts (n = 3 / mice per group from three independent experiments, t test; **P < 0.01), demonstrating that vimentin and HMGB1 are upregulated after organ transplantation and promote the training of graft-infiltrating macrophages. [Figure 2] Figure 2 is a graph of the fold expression of mRNA in real-time PCR of vimentin and HMGB1 expression in donor and nontransplanted hearts (n=3 / mice per group of three independent experiments, t-test; **P<0.01), showing that vimentin and HMGB1 are upregulated after organ transplantation and promote the training of graft-infiltrating macrophages. [Figure 3] Figure 3 shows a panel of four images of Western blot analysis next to two panels of bar graphs of vimentin and HMGB1 expression in donor and nontransplanted hearts (n = 3 / mice per group of three independent experiments, t test; **P < 0.01), demonstrating that vimentin and HMGB1 are upregulated after organ transplantation and promote the training of graft-infiltrating macrophages. [Figure 4] FIG. 4 is a four-panel depiction of flow cytometry analysis showing the expression of Dectin-1 and TLR4 in graft-infiltrating macrophages (n=3 mice per group from two independent experiments). [Figure 5]Figure 5 is a three-panel depiction of flow cytometry analysis showing Ly-6C expression in graft-infiltrating macrophages from WT, Dectin1KO, and TLR4KO untreated recipient mice (n = 3 mice per group from two independent experiments). [Figure 6] Figure 6 is a four-panel bar graph depiction showing the production of inflammatory cytokines and chromatin immunoprecipitation in monocytes from mice trained with vimentin and HMGB, as well as β-glucan and LPS (n = 3 independent experiments, one-way ANOVA, **P < 0.01; dashed line represents the control untrained condition). [Figure 7] Figure 7 is a three-panel bar graph depiction showing cytokine and lactate production by graft-infiltrating macrophages (n = 4 mice per group of two independent experiments, one-way ANOVA, **P < 0.01). [Figure 8] Figure 8 is a four-panel bar graph depiction showing immunoprecipitation of chromatin from graft-infiltrating macrophages (n = 4 mice per group from two independent experiments, one-way ANOVA, *P<0.05; **P<0.01). [Figure 9] Figure 9 is a diagram of the components and constructs of one non-limiting example of an inhibitor-HDL complex, apolipoprotein A1 (apoA1, also referred to as apolipoprotein AI or apoA-I) + a mixture of double- and single-chain phosphocholine compounds (DMPC / MHPC) + mammalian target of rapamycin inhibitor (mTORi), to form the inhibitor-HDL complex as mTORi-HDL, in a 50 nm-scale transmission electron microscope (TEM) image of the mTORi-HDL nanobiology composition. Figure 9 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy inhibits trained immunity to naive cell levels in vitro, inhibits avidity for myeloid cells in the blood and stem and progenitor cells in the bone marrow and spleen, and is systemically distributed in vivo. [Figure 10]Figure 10 is a three-panel graph showing cytokine and lactate production in in vitro-trained human macrophages. (n=3 independent experiments, t-test, *P<0.05; dashed line represents control, non-beta-glucan-trained condition.) Figure 10 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy inhibits trained immunity to naive cell levels in vitro, inhibits avidity for myeloid cells in blood and stem and progenitor cells in bone marrow and spleen, and is systemically distributed in vivo. [Figure 11] Figure 11 is a four-panel graph showing chromatin immunoprecipitation of in vitro-trained human macrophages (n=3 independent experiments, t-test, *P<0.05; dashed line represents control non-β-glucan-trained condition). Figure 11 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy prevents trained immunity to naive cell levels in vitro, prevents avidity for myeloid cells in blood and stem and progenitor cells in bone marrow and spleen, and is systemically distributed in vivo. [Figure 12] Figure 12 is a diagram of the components and construct labeling of one non-limiting example of a labeled inhibitor-HDL complex. mTORi-HDL is labeled with either the radioisotope Zr or the fluorescent dyes DiO or DiR. Figure 12 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy inhibits trained immunity to naive cell levels in vitro, inhibits avidity for myeloid cells in the blood and stem and progenitor cells in the bone marrow and spleen, and is systemically distributed in vivo. [Figure 13]Figure 13 is a graphic illustration of the micro-PET / CT and cell specificity of mTORi-HDL nanobiology compositions. Figure 13 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy inhibits trained immunity to naive cell levels in vitro, inhibits avidity for myeloid cells in blood and stem and progenitor cells in bone marrow and spleen, and is systemically distributed in vivo. [Figure 14] Figure 14 shows representative micro-PET / CT 3D fusion images, PET maximum intensity projection (MIP), and a graph of these results (mean ± SEM, n = 3). Figure 14 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy inhibits trained immunity to naive cell levels in vitro, inhibits avidity for myeloid cells in blood and stem and progenitor cells in bone marrow and spleen, and is systemically distributed in vivo. [Figure 15] Figure 15 is a four-panel graphic representation of the uptake of fluorescently labeled DiO mTORi-HDL by myeloid and lymphoid cells (n=5 mice / group, one-way ANOVA, **P<0.01). Figure 15 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy prevents trained immunity to naive cell levels in vitro, prevents avidity for myeloid cells in the blood and stem and progenitor cells in the bone marrow and spleen, and is systemically distributed in vivo. [Figure 16] Figure 16 is a graph of one panel (mean ± SEM, n = 5) of fluorescently labeled DiO mTORi-HDL uptake by bone marrow progenitors. Figure 16 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy prevents trained immunity to naive cell levels in vitro, prevents avidity for myeloid cells in the blood and stem and progenitor cells in the bone marrow and spleen, and is systemically distributed in vivo. [Figure 17]Figure 17 is a diagram of a BALB / c donor heart (H2d) transplanted into a fully allogeneic C57BL / 6 recipient (H2b). Figure 17 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in the allograft and prevents trained immunity. [Figure 18] Figure 18 is a series of panel images that are 3D fusion images of micro-PET / CT 24 hours after intravenous administration of 89Zr-mTORi-HDL (n = 3 mice per group from two independent experiments). Figure 18 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in the allograft and prevents trained immunity. [Figure 19] Figure 19 shows a pair of images and graphs of ex vivo autoradiography of native (N) and transplanted (Tx) hearts 24 hours after intravenous administration of 89Zr-mTORi-HDL (n = 3 mice per group of two independent experiments, t-test, *P < 0.05). Figure 19 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in allografts and prevents trained immunity. [Figure 20] Figure 20 is a bar graph of fluorescently labeled DiO mTORi-HDL uptake by myeloid and lymphoid cells in allografts (n=4 mice per group of 3 independent experiments; one-way ANOVA, *P<0.05; **P<0.01). Figure 20 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in allografts and prevents trained immunity. [Figure 21]Figure 21 is a pair of pie charts of the ratio of Ly-6Chi / Ly-6CloMφ in allografts from recipients treated with either placebo or mTORi-HDL at day 6 post-transplant (n=4 mice per group of 3 independent experiments; one-way ANOVA, *P0.05; **P<0.01). Figure 21 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in allografts and prevents trained immunity. [Figure 22] Figure 22 is one of a pair of graphs from GSEA gene array analysis of mTOR and glycolysis pathways in Mφ in grafts from recipients treated with placebo or mTORi-HDL (n=3 mice / group). Figure 22 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in allografts and prevents trained immunity. [Figure 23] Figure 23 is the second of a pair of graphs from GSEA gene array analysis of mTOR and glycolysis pathways in intragraft Mφs from recipients treated with placebo or mTORi-HDL (n=3 mice / group). Figure 23 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in allografts and prevents trained immunity. [Figure 24] Figure 24 is a three-panel bar graph of cytokine and lactate production of graft-infiltrating macrophages from recipients treated with placebo or mTORi-HDL (n=4 mice per group from three independent experiments, t-test, *P<0.05, **P<0.01). Figure 24 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in allografts and prevents trained immunity. [Figure 25]Figure 25 is a four-panel bar graph depiction of chromatin immunoprecipitation of graft-infiltrating macrophages from recipients treated with placebo or mTORi-HDL (n=4 mice per group from 3 independent experiments, t-test, *P<0.05; **P<0.01). Figure 25 shows that, in one embodiment, mTORi-HDL nanoimmunotherapy targets myeloid cells in allografts and prevents trained immunity. [Figure 26] Figure 26 is a nine-panel graphic representation of the functional characteristics of graft-infiltrating Mφ from placebo-treated and mTORi-HDL-treated recipients using CD8 T cell suppression assays and CD4 Treg proliferation assays. (n=4 mice per group from 3 independent experiments, t-test, **P<0.01). Figure 26 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and CD40 activation of T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 27] Figure 27 is a pair of pie charts of the percentage of graft-infiltrating CD4+CD25+ Treg cells from placebo-treated and mTORi-HDL-treated recipients (n=4 mice per group from 3 independent experiments, t-test, **P≦0.01). Figure 27 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and CD40 activation of T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 28]Figure 28 is a five-panel graphic representation of the loss of CD169+ graft-infiltrating Mregs in placebo-treated and mTORi-HDL-treated recipients (n=5 mice per group from 3 independent experiments, t-test, **P<0.01). Figure 28 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and CD40 activation of T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 29] Figure 29 is a line graph of graft survival after loss of CD169+ graft-infiltrating Mregs (n=5 mice / group, Kaplan-Meier **P<0.01). Figure 29 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and activation of CD40 on T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 30] Figure 30 is a line graph of graft survival after loss of CD11c+ cells in CCR2-deficient recipient mice (n=5 mice / group, Kaplan-Meier, **P<0.01). Figure 30 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and CD40 activation of T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 31] Figure 31 is a line graph of graft survival in recipients treated with mTORi-HDL administered with in vivo agonistic CD40 mAb, with or without TRAF6i-HDL nanoimmunotherapy (n=5 mice / group, Kaplan-Meier, **P<0.01). Figure 31 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and CD40 activation of T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 32]Figure 32 is a line graph of graft survival in recipients treated with placebo, vehicle HDL, mTORi-HDL, TRAF6i-HDL, and mTORi-HDL / TRAF6i-HDL (n=7-8 mice / group, Kaplan-Meier, **P<0.01). Figure 32 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and CD40 activation of T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 33] Figure 33 shows two panels of immunohistochemistry images of cardiac allografts from mTORi-HDL / TRAF6i-HDL-treated recipients 100 days post-transplant (n=5 mice / group; magnification ×200). Figure 33 shows that, in one embodiment, the combination of nanoimmunotherapy of trained immunity with mTORi-HDL and CD40 activation of T cells (but not trained immunity) as a synergistic treatment promotes organ transplant acceptance. [Figure 34] Figure 34 is a series of bar graphs of four panels of chromatin immunoprecipitation assays (ChIP) of graft-infiltrating bone marrow monocytes from untreated, rejecting recipients on day 6 post-transplant. ChIP was performed to assess histone H3K4 trimethylation. The abundance of four trained immunity-related genes was examined by qPCR (n=3, Wilcoxon signed-rank test, **P<0.01. Results from one experiment). Figure 34 shows the development and in vivo distribution of mTORi-HDL in one embodiment. [Figure 35] FIG. 35 is a diagram of the chemical structure of the mTOR inhibitor (mTORi) rapamycin. [Figure 36] FIG. 36 is an image of a transmission electron micrograph showing the discoid morphology of the mTORi-HDL nanobiology composition. [Figure 37]Figure 37 is a schematic bar graph depicting images of mTORi-HDL biodistribution in wild-type C57 / B16 mice. Representative near-infrared fluorescence (NIRF) images of organs injected with either PBS control (first row of organs) or DiR-labeled mTORi-HDL show accumulation in the liver, spleen, lung, kidney, heart, and muscle. Figure 37 shows, in one embodiment, the development and in vivo distribution of mTORi-HDL. [Figure 38] Figure 38 is a bar graph in which the ratio of mTORi-HDL-DiR accumulation to control in each organ is represented by a bar, calculated by dividing the total signal in each organ in the control and mTORi-HDL-DiR groups (n=4 mice / group. Results from 3 experiments). Figure 38 shows, in one embodiment, the development and in vivo distribution of mTORi-HDL. [Figure 39] Figure 39 is a bar graph of PET-quantified uptake values ​​by mean ID (%) / g in transplanted hearts, kidneys, livers, and spleens (n=3 mice. Results from 3 experiments). Figure 39 shows the development and in vivo distribution of mTORi-HDL in one embodiment. [Figure 40] Figure 40 is a 21-panel diagram of a flow cytometry gating strategy for distinguishing myeloid cells in blood, spleen, and transplanted hearts. The gray histograms show the distribution of immune cells in mice injected with DiO-labeled mTORi-HDL compared to controls (black histograms). Figure 40 shows, in one embodiment, the cellular targeting of mTORi-HDL in vivo. [Figure 41] Figure 41 is a two-panel bar graph of mean fluorescence intensity (MFI) of neutrophils, monocytes / macrophages, Ly-6Clo and Ly-6Chi monocytes / macrophages, dendritic cells, and T cells in blood and spleen (n=4 mice / group, One-way ANOVA, *P<0.05; **P<0.01. Results from 3 experiments). Figure 41 shows, in one embodiment, cellular targeting of mTORi-HDL in vivo. [Figure 42] Figure 42 is a three-panel diagram with a nine-panel view of flow cytometry gating strategies for differentiating T cells in blood, spleen, and transplanted hearts. The gray histogram (right) shows T cell distribution in mice injected with DiO-labeled mTORi-HDL compared to the distribution in control animals (black histogram). Figure 42 shows cellular targeting of mTORi-HDL in vivo in one embodiment. [Figure 43] Figure 43 shows three panels of mean fluorescence intensity (MFI) of monocytes / macrophages, CD3+T, CD4+T, and CD8+T cells in blood and transplanted hearts (n=4 mice / group, One-way ANOVA, **P<0.01. Results from three experiments). Figure 43 shows cellular targeting of mTORi-HDL in vivo in one embodiment. [Figure 44] Figure 44 shows 12 panels of flow cytometry analysis of cell suspensions collected from the allograft, blood, and spleen of allograft recipients treated with placebo, oral rapamycin (5 mg / kg), and mTORi-HDL (5 mg / kg) on ​​day 6 after transplantation. The total numbers of leukocytes, neutrophils, macrophages (Mφ), and dendritic cells (DC) are shown (n=4 mice / group, one-way ANOVA, *P<0.05; **P<0.01. Results from three experiments). Figure 44 shows that, in one embodiment, mTORi-HDL rebalances the myeloid and Treg compartments in vivo. [Figure 45] Figure 45 is a nine-panel diagram of the ratios of Ly-6Chi to Ly-6Clo monocytes in blood, spleen, and cardiac allografts from allograft recipients treated with placebo, oral rapamycin (5 mg / kg), and mTORi-HDL (5 mg / kg) (n=4 per group, one-way ANOVA, *P<0.05; **P<0.01. Results from three experiments). Figure 45 shows that, in one embodiment, mTORi-HDL rebalances the myeloid and Treg compartments in vivo. [Figure 46]Figure 46 is a three-panel pie chart depiction of the percentage of graft-infiltrating CD4+CD25+ vs. CD4+CD25- T cells from allograft recipients treated with placebo, oral rapamycin (5 mg / kg), and mTORi-HDL (5 mg / kg) (n=4 mice / group, one-way ANOVA, **P<0.01. Results from three experiments). Figure 46 shows that, in one embodiment, mTORi-HDL rebalances the myeloid and Treg compartments in vivo. [Figure 47] FIG. 47 is a diagram of the chemical structure of a TRAF6 inhibitor, which is the untrained immune part of a synergistic combination therapy with trained immune nanoimmunotherapy. [Figure 48] Figure 48 is a transmission electron microscopy image showing the discoid morphology of TRAF6i-HDL. The nanoparticles have an average hydrodynamic radius of 19.2±3.1 nm and an average drug incorporation efficiency of 84.6±8.6%, as determined by DLS and HPLC, respectively. [Figure 49] Figure 49 is a line graph of graft survival curves for oral rapamycin, intravenous rapamycin, and oral rapamycin + TRAF6i-HDL (n = 8 mice per group). This background shows the graft survival curves for placebo, HDL vehicle, TRAF6i-HDL, mTORi-HDL, and mTORi-HDL / TRAF6i-HDL combination therapy from Figure 23. Figure 49 shows the therapeutic effect of a nanobiology composition of mTORi-HDL and TRAF6i-HDL combined in one embodiment. [Figure 50]Figure 50 shows six panels of representative hematoxylin / eosin (H&E), periodic acid-Schiff (PAS), and Masson's trichrome immunohistochemistry images of kidneys and livers from a transplant recipient treated with mTORi / TRAF6i-HDL harvested 100 days after transplantation. The kidneys show no significant changes in the three compartments of the renal parenchyma. There is no evidence of glomerulosclerosis, and the glomeruli appear normal. The tubules do not show significant atrophy or any evidence of epithelial cell damage, including vacuolization, brush border loss, or mitosis. The liver has normal acinar and lobular architecture. There is no evidence of inflammation or fibrosis in the portal tracts or hepatic parenchyma. Hepatocytes are normal, with no evidence of cholestasis, inclusions, or apoptosis (n = 4 mice; magnification × 200). FIG. 50 shows the therapeutic effect of a combined mTORi-HDL and TRAF6i-HDL nanobiology composition in one embodiment. [Figure 51] Figure 51 is a pair of bar graphs depicting toxicity associated with mTORi-HDL treatment. Recipient mice received either an mTORi-HDL treatment regimen (5 mg / kg on days 0, 2, and 5 post-transplant) or oral rapamycin (5 mg / kg daily for 15 days) to achieve the same therapeutic outcome (100% allograft survival for 30 days). mTORi-HDL had no significant effect on blood urea nitrogen (BUN) or serum creatinine, but renal toxicity parameters showed statistical differences between oral rapamycin and mTORi-HDL. No differences were observed between syngenic and mTORi-HDL recipients (n = 4 mice / group, one-way ANOVA, *P < 0.05; **P < 0.01; results from three experiments). Figure 51 shows the therapeutic effect of a combined mTORi-HDL and TRAF6i-HDL nanobiological composition in one embodiment. Atherosclerosis [Figure 52]Figure 52 is a schematic diagram of the different components of mTORi-HDL, constructed by combining human apolipoprotein AI (apoA-I), the phospholipids DMPC and MHPC, and the mTOR inhibitor rapamycin. Figure 52 shows that, in one embodiment, mTORi-HDL targets atherosclerotic plaques and accumulates in macrophages and inflammatory Ly6Chi monocytes. Apoe- / - mice developed atherosclerotic plaques on a 12-week high-cholesterol diet. [Figure 53] Figure 53 shows three panels of IVIS images of whole aortas from Apoe- / - mice injected with PBS (control) or DiR-labeled mTORi-HDL. Aortas were harvested 24 hours after injection. [Figure 54] Figure 54 is a nine-panel diagram of the gating strategy for flow cytometry of CD45+ cells in whole aortas. Identification of Lin+ cells, macrophages, and Ly6Chi monocytes (top), representative histograms of each cell type (middle), and quantification of DiO signal (bottom). Aortas were harvested 24 hours after injection of DiO-labeled mTORi-HDL. Figure 54 demonstrates that, in one embodiment, mTORi-HDL targets atherosclerotic plaques and accumulates in macrophages and inflammatory Ly-6Chi monocytes. In all figures, data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001). P values ​​were calculated using a Mann-Whitney U test (two-tailed). [Figure 55] FIG. 55 is a six panel histological image and two panel pie chart comparing control groups with mTORi-HDL. [Figure 56] On the right are four-panel plots of plaque area, collagen content, Mac3-positive area, and Mac3 to collagen ratios comparing control and mTORi-HDL. Figures 55-56 show mTORi-HDL atherosclerotic plaque inflammation in one embodiment. Apoe- / - mice were subjected to a high-cholesterol diet for 12 weeks, followed by one week of treatment while continuing the high-cholesterol diet. [Figure 57] Figure 57 is a pair of parallel fluorescence molecular tomography images with X-ray computed tomography showing reduced protease activity in the aortic root of mice treated with mTORi-HDL versus control mice. The mTORi-HDL mice showed a significant reduction. [Figure 58] Figure 58 is a graph of protease activity. [Figure 59] FIG. 59 is a schematic diagram of the different components of the S6K1i-HDL nanobiology composition constructed by incorporating human apolipoprotein AI (apoA-I), phospholipids POPC and PHPC, and the S6K1 inhibitor PF-4708671. [Figure 60] Figure 60 is a diagram of IVIS imaging of organs from Apoe- / - mice injected with DiR-labeled S6K1i-HDL. Organs were harvested 24 hours after injection. [Figure 61] FIG. 61 is a five panel diagram of quantification of DiO signals of different leukocyte subsets in aortic plaques after intravenous injection of DiO-labeled S6K1i-HDL (n=2-4 per group). [Figure 62] Figure 62 is a pair of graphs of quantification of macrophages and Ly6C(hi) monocytic cells in whole aortas, comparing control, rHDL only, mTORi-HDL, and S6K1i-HDL treatments. Apoe- / - mice were subjected to a high-cholesterol diet for 12 weeks, followed by 1 week of treatment while continuing the high-cholesterol diet. [Figure 63] Figure 63 shows an in vitro analysis of human adherent monocytes in which trained immunity induced by oxLDL led to amplified TNFα cytokine production when the cells were restimulated with LPS 5 days later. This response was attenuated by mTORi-HDL and S6K1i-HDL (n=6). Figure 63 is a pair of graphs of TNFα values ​​in pg / mL for RPMI and oxLDL-invasion, comparing RPMI alone vs. mTORi-HDL and RPMI alone vs. S6K1i-HDL. [Figure 64]Figure 64 is a graphical representation of various prodrug formulations by size over time. [Figure 65] Figure 65 is a graphical representation of the magnitude of the prodrug over time. [Figure 66] Figure 66 is a graphical representation of the mean dispersity over time for various prodrugs. [Figure 67] Figure 67 is a graphic representation of percent drug recovery for various prodrugs. [Figure 68] Figure 68 is a graphical representation of the percent hydrolysis of various prodrugs. [Figure 69] Figure 69 is a graphic representation of percent apoA-I recovery for various prodrugs. [Figure 70] FIG. 70 is a graphical representation of the zeta potential of various prodrugs. [Figure 71] Figure 71 is a graphical representation of the fraction of drug (malonate) incorporated into aliphatic vs. cholesterol matrices. [Figure 72] FIG. 72 is a graphical representation of the fraction of drug incorporated into the aliphatic (JQ1) versus cholesterol matrix (JQ1). [Figure 73] Figure 73 is a graphic representation of the fraction of drug (GSK-J4) alone vs. drug (GSK-J4) incorporated into aliphatic vs. drug (GSK-J4) incorporated into cholesterol. [Figure 74] Figure 74 is a graphical representation of the fraction of drug (rapamycin) alone versus drug (rapamycin) incorporated into aliphatics. [Figure 75] Figure 75 is a graphical representation of the fraction of drug (PF-4708671 S6K1i) incorporated over time. [Figure 76] Figure 76 is a diagram of the radioisotope labeling process. [Figure 77]FIG. 77 is an illustration of PET imaging using a radioisotope delivered by a nanobiological composition, showing accumulation of the nanobiological composition in the bone marrow and spleen in mouse, rabbit, monkey, and pig models. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention is directed to nanobiological compositions for inhibiting trained immunity, methods for making the nanobiological compositions, methods for incorporating drugs into the nanobiological compositions, and prodrug formulations incorporating drugs with functionalized linker moieties such as phospholipids, aliphatic chains, and sterols.

[0043] Inflammation is induced by innate immune cells as a defense mechanism against tissue injury. The ancient mechanism of immune memory, termed trained immunity and also called innate immune memory, is induced by an initial insult that stimulates myeloid innate immune cells or their precursors and stem cells in the bone marrow, blood, and / or spleen, and is defined by a long-term increase in responsiveness (e.g., significant cytokine production) after restimulation with a secondary stimulus of myeloid innate immune cells, mediated by epigenetic, metabolic, and transcriptional rearrangements.

[0044] Trained immunity is defined by a secondary, long-term hyperresponsiveness to restimulation following an initial invasion of myeloid cells, myeloid progenitors, and hematopoietic stem cells in the bone marrow, blood, and / or spleen, characterized by increased cytokine secretion caused by metabolic and epigenetic rearrangements.

[0045] In one preferred embodiment, the present invention is directed to myeloid cell-specific nanoimmunotherapy based on the delivery of nanobiological compositions (mTORi-HDL) carrying or having incorporated the mTOR inhibitor rapamycin, which interferes with the epigenetic and metabolic modifications underlying trained immunity. The present invention relates to therapeutic nanobiological compositions and methods for treating patients who have undergone organ transplantation, or who suffer from autoimmune diseases, including atherosclerosis, arthritis, inflammatory bowel diseases including Crohn's disease, diabetes, and / or autoinflammatory conditions, or who have suffered from cardiovascular events, including stroke and myocardial infarction, by inhibiting trained immunity with increased long-term responsiveness, which is the result of metabolic and epigenetic rearrangements of myeloid cells and their stem cells and progenitors in the bone marrow, spleen, and blood, induced by an initial insult and characterized by increased cytokine secretion after restimulation with one or more secondary stimuli.

[0046] definition Nanobiology Composition The term "nanobiologic composition" refers to a composition for inhibiting trained immunity, (ii) an inhibitor drug incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising (a) a phospholipid or a mixture of phospholipids, (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I, and optionally (c) a hydrophobic matrix composed of one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters, and also optionally (d) cholesterol; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, the inhibitor drug is a prodrug of a hydrophobic drug or a hydrophilic drug derivatized with an attached aliphatic chain or cholesterol or phospholipid; The drug is an inhibitor of an inflammasome, metabolic pathway, or epigenetic pathway in hematopoietic stem cells (HSCs), CMPs, or myeloid cells. Represents a composition.

[0047] For proof of concept, an inhibitor of mTOR incorporated into HDL (mTORi-HDL) or an inhibitor of S6K1 incorporated into HDL (S6K1i-HDL) served as the nanobiology compositions for data generation herein.

[0048] Nanoscale Constructs The term "nanoscale construct" (NA) refers to a multi-component carrier composition for carrying a useful payload, such as a drug.

[0049] In one preferred embodiment, the nanoscale construct comprises a multi-component carrier composition for carrying a useful payload, having subcomponents: (a) a phospholipid and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I.

[0050] In another preferred embodiment, a "nanoscale construct" (NA) refers to a multi-component carrier composition for carrying an effective payload, e.g., a drug, that inhibits trained immunity, having subcomponents: (a) a phospholipid, (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I, and (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters.

[0051] In another preferred embodiment, a "nanoscale construct" (NA) refers to a multi-component carrier composition for carrying an effective payload, e.g., a drug, that inhibits trained immunity, having subcomponents: (a) a phospholipid, (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I, (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters, and (d) cholesterol.

[0052] phospholipids The term "phospholipid" refers to an amphipathic compound consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are linked together by a glycerol molecule. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine.

[0053] Choline represents an important bioactive nutrient with the chemical formula R-(CH2)2-N-(CH2)4. When the phospho-moiety is R-, it is called phosphocholine.

[0054] Examples of suitable phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, or other ceramides, and phospholipid-containing oils, such as lecithin oil. Combinations of phospholipids or mixtures of phospholipids with other substances may be used.

[0055] Non-limiting examples of phospholipids that can be used in the present compositions include phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid / ester (PA), and lysophosphatidylcholine.

[0056] Specific examples include DDPC CAS-3436-44-0 1,2-didecanoyl-sn-glycero-3-phosphocholine, DEPA-NA CAS-80724-31-8 1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt), DEPC CAS-56649-39-9 1,2-dierucoyl-sn-glycero-3-phosphocholine, DEPE CAS-988-07-2 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, DEPG-NA 1,2-dierucoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DLOPC CAS-998-06-1 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPA-NA 1,2-Dilauroyl-sn-glycero-3-phosphate (sodium salt), DLPC CAS-18194-25-7 1,2-Dilauroyl-sn-glycero-3-phosphocholine, DLPE 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine, DLPG-NA 1,2-Dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol...)(sodium salt), DLPG-NH4 1,2-Dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol...)(ammonium salt), DLPS-NA 1,2-Dilauroyl-sn-glycero-3-phosphoserine (sodium salt), DMPA-NA CAS-80724-3 1,2-Dimyristoyl-sn-glycero-3-phosphate (sodium salt), DMPC CAS-18194-24-6 1,2-Dimyristoyl-sn-glycero-3-phosphocholine, DMPE CAS-988-07-2 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPG-NA CAS-67232-80-8 1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DMPG-NH4 1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (ammonium salt), DMPG-NH4 / NA 1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol...)) (sodium / ammonium salt), DMPS-NA 1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt), DOPA-NA 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt), DOPC CAS-4235-95-4 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPE CAS-4004-5-1 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPG-NA CAS-62700-69-0 1,2-dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DOPS-NA CAS-70614-14-1 1,2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt), DPPA-NA CAS-71065-87-7 1,2-Dipalmitoyl-sn-glycero-3-phosphate (sodium salt), DPPC CAS-63-89-8 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, DPPE CAS-923-61-5 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPG-NA CAS-67232-81-9 1,2-Dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DPPG-NH4 CAS-73548-70-6 1,2-Dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (ammonium salt), DPPS-NA 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt), DSPA-NA CAS-108321-18-2 1,2-Distearoyl-sn-glycero-3-phosphate (sodium salt), DSPC CAS-816-94-4 1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPE CAS-1069-79-0 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, DSPG-NA CAS-67232-82-0 1,2-Distearoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DSPG-NH4 CAS-108347-80-4 1,2-Distearoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (ammonium salt), DSPS-NA 1,2-distearoyl-sn-glycero-3-phosphoserine (sodium salt), EPC Egg-PC, HEPC Hydrogenated Egg PC, HSPC Hydrogenated Soy PC, LYSOPC MYRISTIC CAS-18194-24-6 1-Myristoyl-sn-glycero-3-phosphocholine, LYSOPC PALMITIC CAS-17364-16-8 1-Palmitoyl-sn-glycero-3-phosphocholine, LYSOPC STEARIC CAS-19420-57-6 1-Stearoyl-sn-glycero-3-phosphocholine, milk-derived sphingomyelin, MPPC 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, MSPC 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, PMPC 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, POPC CAS-26853-31-6 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPG-NA CAS-81490-05-3 1-Palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)...] (sodium salt), PSPC 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, SMPC 1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, SOPC Examples include 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and SPPC 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine.

[0057] In some preferred embodiments, specific non-limiting examples of phospholipids include dimyristoylphosphatidylcholine (DMPC), soybean lecithin, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dilaurylolyphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), dilaurylolylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (D SPG), dioleoylphosphatidylglycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl sphingomyelin (DPSP), distearoyl sphingomyelin (DSSP), and mixtures thereof.

[0058] In certain embodiments, when the composition comprises (consists essentially of, or consists of) two or more phospholipids, the weight ratio of the two phospholipids can be in the range of about 1:10 to about 10:1, about 2:1 to about 4:1, about 1:1 to about 5:1, about 2:1 to about 5:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 7:1 to about 9:1, or about 8:1 to about 9:1. For example, the weight ratio of the two phospholipids can be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0059] In one embodiment, (a) the phospholipids of the nanoscale constructs of the present invention comprise (consist essentially of or consist of) a mixture of double-chain diacyl-phospholipids and single-chain acyl-phospholipids / lysolipids.

[0060] In one embodiment, the phospholipid in (a) is a mixture of phospholipids and lysolipids, which are (DMPC), and (MHPC).

[0061] The weight ratio of DMPC to MHPC can be in the range of about 1:10 to about 10:1, about 2:1 to about 4:1, about 1:1 to about 5:1, about 2:1 to about 5:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 7:1 to about 9:1, or about 8:1 to about 9:1. The weight ratio of DMPC to MHPC can be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0062] In one embodiment, the phospholipid in (a) is a mixture of phospholipids and lysolipids, which are (POPC) and (PHPC).

[0063] The weight ratio of POPC to PHPC can be in the range of about 1:10 to about 10:1, about 2:1 to about 4:1, about 1:1 to about 5:1, about 2:1 to about 5:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 7:1 to about 9:1, or about 8:1 to about 9:1. The weight ratio of DMPC to MHPC can be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0064] It should be noted that phospholipids ranging in chain length from C4 to C30, saturated or unsaturated, cis or trans, unsubstituted or substituted with 1 to 6 side chains, and with or without added lysolipids are all contemplated for use in the nanoscale constructs or nanoparticle / nanobiology compositions described herein.

[0065] Additionally, other synthetic variants and variants with other phospholipid head groups are contemplated.

[0066] Lysolipids The term "lysolipid" as used herein includes, in non-limiting embodiments, (acyl-, single chain), such as 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC), 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine (PHPC), and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (SHPC).

[0067] Apolipoprotein AI (apoA-I) (apoA1) The term "apolipoprotein AI" or "apoA-I," also "apoliprotein A1" or "apoA1," refers to the protein encoded by the human APOA1 gene and, as used herein, also includes peptide mimetics of apoA-I. Apolipoprotein A1 (apoA-I) is a subcomponent in (b) nanoscale constructs.

[0068] Hydrophobic matrix The term "hydrophobic matrix" refers to the core, bulking agent, or structural modifier of a nanobiology composition. Structural modulation includes (1) using a hydrophobic matrix to increase or engineer the particle size of nanoscale constructs made solely from (a) phospholipids and (b) apoA-I, (2) increasing or decreasing (engineering) the size and / or shape of nanoscale construct particles, (3) increasing or decreasing (engineering) the hydrophobic core of nanoscale construct particles, (4) increasing or decreasing (engineering) the properties and / or miscibility of nanobiology compositions incorporating hydrophobic drugs, and (5) increasing or decreasing the biodistribution properties of nanoscale construct particles.

[0069] The particle size, hardness, viscosity, and / or biodistribution of nanoscale constructs can be adjusted by the amount and type of hydrophobic molecules added. As a non-limiting example, nanoscale constructs made solely from (a) phospholipids and (b) apoA-I can have a diameter of 10 nm to 50 nm. (c) Adding hydrophobic matrix molecules such as triglycerides can expand the nanoscale constructs from a minimum of 10 nm to at least 30 nm. Adding more triglycerides can increase the diameter of the nanoscale constructs to at least 50 nm, at least 75 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 300 nm, and up to 400 nm, within the scope of the present invention.

[0070] The production method can prepare nanoscale structure particles of uniform size, or it can prepare a mixture of nanoscale structure particles of non-uniform size, either by not filtering or by preparing nanoscale structure particles of a range of different sizes and then recombining them in a post-production step. Increasing the size of the nanoscale structure particles allows for more drug to be incorporated. However, large sizes, such as greater than 120 nm, can limit, hinder, or delay the diffusion of the nanoscale structure particles into the tissues of the treated patient. Small nanoscale structure particles do not carry as much drug per particle, but can access the bone marrow, blood, or spleen, or other localized tissues affected by trained immunity, such as transplanted tissues and surrounding tissues, atherosclerotic plaques, etc. (biodistribution). Using a non-uniform nanoparticle size mixture in a single dose or regimen can result in an immediate reduction in innate immune hyperresponsiveness, as well as a sustainable long-term reduction in innate immune hyperresponsiveness that can last for days, weeks, months, and years, where the nanobiological composition restores, alters, or re-regulates metabolic, epigenetic, and inflammasome pathways in hematopoietic stem cells (HSCs), CMPs, and myeloid cells, such as monocytes, macrophages, and other short-lived circulating cells.

[0071] The addition of other (c) hydrophobic matrix molecules, such as cholesterol, fatty acid esters, hydrophobic polymers, sterol esters, and different types of triglycerides, or specific mixtures thereof, can further engineer nanoscale construct particles to emphasize characteristics particularly desirable for specific purposes. Size, rigidity, and viscosity can affect packing and biodistribution.

[0072] As a non-limiting example, the maximum loading capacity can be determined by dividing the internal volume of the nanoscale construct particle by the volume of the drug-loaded spheroid.

[0073] Particles: Assume 100 nm spherical particles with 2.2 nm to 3.0 nm phospholipid walls, resulting in an inner diameter of 94 nm and a volume (L) @ 4 / 3π(r).

[0074] Drug: Sirolimus (rapamycin) is assumed as a 12 x 12 x 35 Angstrom or 1.2 x 1.2 x 3.5 nm cylinder (where multiple, e.g., 7 or 9, drug molecule cylinders, or multiple drugs plus a hydrophobic matrix carrier such as triglyceride, can assume a 3.5 nm diameter spheroid with a 1.75 nm radius and Vol(small)@4 / 3π(r)).

[0075] Maximum packing capacity (calc): Approximately 19,372 3.5 nm spheroids in a 100 nm particle.

[0076] Biologically relevant lipids include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, glycolipids (saccharolipids), and polyketides. A complete list of over 42,000 lipids is available at https: / / www.lipidmaps.org.

[0077] triglycerides "Triglyceride" and similar terms refer to an ester derived from glycerol and three fatty acids. The notation used herein to describe triglycerides is the same as that used below to describe fatty acids. Triglycerides can contain glycerol with any combination of the following fatty acids: C18:1, C14:1, and C16:1, both polyunsaturated and saturated. The fatty acids can be bonded to the glycerol molecule in any order; for example, any fatty acid can react with any of the hydroxyl groups on the glycerol molecule to form an ester bond. A triglyceride of C18:1 fatty acids simply means that the fatty acid component of the triglyceride is derived from or based on a C18:1 fatty acid. That is, a C18:1 triglyceride is an ester of glycerol and three fatty acids of 18 carbon atoms each, each with one double bond. Similarly, a C14:1 triglyceride is an ester of glycerol and three fatty acids of 14 carbon atoms each, each having one double bond. Similarly, a C16:1 triglyceride is an ester of glycerol and three fatty acids of 16 carbon atoms each, each having one double bond. A triglyceride of C18:1 fatty acids in combination with C14:1 and / or C16:1 fatty acids means that (a) the C18:1 triglyceride is mixed with either a C14:1 triglyceride or a C16:1 triglyceride, or both; or (b) at least one of the fatty acid components of the triglyceride is derived from or based on a C18:1 fatty acid, and the other two are derived from or based on a C14:1 fatty acid and / or a C16:1 fatty acid.

[0078] fatty acid "Fatty acid" and similar terms refer to carboxylic acids with a long aliphatic tail, either saturated or unsaturated. Fatty acids can be esterified into phospholipids and triglycerides. As used herein, fatty acid chain lengths include C4 to C30, saturated or unsaturated, cis or trans, and unsubstituted or substituted with one to six side chains. Unsaturated fatty acids have one or more double bonds between carbon atoms. Saturated fatty acids contain no double bonds. The notation used herein to describe fatty acids includes a capital letter "C" for carbon atom, followed by a number describing the number of carbon atoms in the fatty acid, followed by a colon, and another number for the number of double bonds in the fatty acid. For example, C16:1 refers to a 16-carbon fatty acid containing one double bond, such as palmitoleic acid. The number after the colon in this notation does not indicate the position of the double bond in the fatty acid, nor does it indicate whether the hydrogen atoms attached to the carbon atoms of the double bond are cis to each other. Other examples of this designation include C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C18:3 (α-linolenic acid), and C20:4 (arachidonic acid).

[0079] Sterols and sterol esters The term "sterol," such as, but not limited to, cholesterol, may also be utilized in the methods and compounds described herein. Sterols are animal or plant steroids containing only a hydroxyl group at the C-3 position and no other functional groups. Sterols generally contain 27-30 carbon atoms and a double bond at the 5 / 6 position, and optionally at the 7 / 8, 8 / 9, or other positions. Other sterols, other than these unsaturated species, are saturated compounds obtained by hydrogenation. An example of a suitable animal sterol is cholesterol. Typical examples of suitable phytosterols that are preferred from an application standpoint include ergosterol, campesterol, stigmasterol, brassicasterol, preferably sitosterol or sitostanol, more preferably β-sitosterol or β-sitostanol. In addition to the phytosterols listed above, their esters are preferably used. The acid component of this ester can be reduced to a carboxylic acid corresponding to formula (I): RCO-OH(I), where RCO is an aliphatic, straight- or branched-chain acyl group containing 2 to 30 carbon atoms and 0 and / or 1, 2, or 3 double bonds. Typical examples include acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, 2-ethylhexanoic acid, capric acid, lauric acid, isotridecanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroselic acid, linoleic acid, conjugated linoleic acid (CLA), linolenic acid, eleostearic acid, arachic acid, gadoleic acid, behenic acid, and erucic acid.

[0080] Hydrophobic polymer The hydrophobic polymer(s) used to make the matrix can be selected from the group of polymers approved for human use (i.e., biocompatible and FDA approved), including, for example, but not limited to, the following polymers, their derivatives, copolymers, block copolymers, branched polymers, and polymer blends: polyalkenedicarboxylates, polyanhydrides, poly(aspartic acid), polyamides, polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), poly(ε-caprolactone) (PCL), polycarbonates (PC), including poly-alkylene carbonates, fats, and the like. Polyesters, including aromatic polyesters and polyester-amides, polyethylene succinate (PES), polyglycolide (PGA), polyimines and polyalkyleneimines (PI, PAI), polylactides (PLA, PLLA, PDLLA), polylactic-co-glycolic acid (PLGA), poly(l-lysine), polymethacrylates, polypeptides, polyorthoesters, poly-p-dioxanone (PPDO), (hydrophobically) modified polysaccharides, polysiloxanes and poly-alkyl-siloxanes, polyureas, polyurethanes, and polyvinyl alcohol.

[0081] biohydrolyzable As used herein, unless otherwise specified, the terms “biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzable carbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,” and “biohydrolyzable phosphate” mean, respectively, an amide, ester, carbamate, carbonate, ureide, or phosphate of a compound that 1) does not interfere with the biological activity of the compound but may provide the compound with favorable properties in vivo, such as uptake, duration of action, or onset of action; or 2) is biologically inactive but is converted in vivo to a biologically active compound. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, lower acyloxyalkyl esters (e.g., acetoxylmethyl, acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, and pivaloyloxyethyl esters), lactonyl esters (e.g., phthalidyl and thiophthalidyl esters), lower alkoxyacyloxyalkyl esters (e.g., methoxycarbonyloxymethyl, ethoxycarbonyloxyethyl, and isopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters, and acylaminoalkyl esters (e.g., acetamidomethyl ester). Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, α-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic amines, and aromatic heterocyclic amines, and polyether amines.

[0082] Methods for generating nanoscale constructs The methods are described below and there are related variants to these methods.

[0083] Method 1 - Film The phospholipid, (pro)drug, and optional triglyceride or polymer are dissolved (usually in chloroform, ethanol, or acetonitrile). The solution is then evaporated under vacuum to form a film of these components. A buffer solution is then added to hydrate the film, creating a vesicle suspension.

[0084] The phospholipid, (pro)drug, and optional triglyceride or polymer are dissolved (usually in chloroform, ethanol, or acetonitrile), and this solution is then poured or added dropwise to a gently heated buffer solution with stirring until the organic solvent has completely evaporated, creating a vesicle suspension.

[0085] Apolipoprotein AI (apoA-I) (note that apoA-I may also already be in B) is added dropwise to avoid denaturation, and the resulting mixture is sonicated for 30 minutes using a tip sonicator while being thoroughly cooled using an external ice-water bath. The resulting solution containing the nanobiological composition and other by-products is transferred to a Sartorius Vivaspin tube with a molecular weight cutoff corresponding to the estimated size of the nanobiological composition (typically, Vivaspin tubes with a cutoff of 10,000-100,000 kDa are used). These tubes are centrifuged until approximately 90% of the solvent volume passes through the filter. A volume of buffer roughly equivalent to the volume of the remaining solution is then added, and the tube is spun again until approximately half of the volume passes through the filter. This process is repeated twice until the remaining solution passes through a 0.22 μm polyethersulfone syringe filter, yielding the final nanobiological composition solution.

[0086] Method 2 - Microfluidics In another approach, phospholipids, (pro)drugs, and optional triglycerides, cholesterol, steryl esters, or polymers are dissolved (typically in ethanol or acetonitrile) and loaded into a syringe. A solution of apolipoprotein AI (apoA-I) in phosphate-buffered saline is then loaded into a second syringe. A microfluidics pump is used to mix the contents of both syringes using a microvortex platform. The resulting solution containing the nanobiological composition and other by-products is transferred into Sartorius Vivaspin tubes with a molecular weight cutoff corresponding to the estimated particle size (typically, Vivaspin tubes with a cutoff of 10,000-100,000 kDa are used). These tubes are centrifuged until approximately 90% of the solvent volume passes through the filter. A volume of phosphate-buffered saline roughly equivalent to the volume of the remaining solution is then added, and the tubes are spun again until approximately half of the volume passes through the filter. This is repeated twice until the remaining solution is passed through a 0.22 μm polyethersulfone syringe filter to obtain the final nanobiological composition solution.

[0087] Method 3 - Microfluidizer In another preferred method according to the present invention, microfluidizer technology is used to prepare the nanoscale constructs and final nanobiological compositions.

[0088] A microfluidizer is a device for preparing small particle size materials that operates on the submerged jet principle. To obtain nanoparticles, a microfluidizer forces a premixed stream through a so-called interaction chamber, which consists of a system of channels in a ceramic block that separates the stream into two. Precisely controlled shear, turbulence, and cavitation forces are introduced into the interaction chamber during microfluidization. The two streams are combined at high speed to produce shear. The resulting product can be recycled back into the microfluidizer to obtain even smaller particles.

[0089] Advantages of microfluidization over conventional milling processes include a substantial reduction in contamination of the final product and ease of scale-up production.

[0090] Microfluidizer Example 1-1L Nanoscale constructs and formation of rapamycin nanobiological compositions This example illustrates the preparation of a pharmaceutical composition comprising rapamycin and nanoscale constructs, where the concentration of rapamycin is 4-8 mg / mL in the nanoscale construct / emulsion and the formulation is made at a 1 L scale.

[0091] Rapamycin (7,200 mg) was dissolved in 36 mL of chloroform / t-butanol. This solution was then added to 900 mL of a nanoscale construct solution (3% w / v) containing a POPC / PHPC phospholipid mixture, apoA-I, tricaprylin, and cholesterol. The mixture was homogenized (Vitris homogenizer model Tempest IQ) at 10,000-15,000 rpm for 5 minutes to form a crude emulsion, which was then transferred to a high-pressure homogenizer. Emulsification was performed at 20,000 psi with emulsion recirculation. The resulting system was transferred to a rotary evaporator, and the solvent was rapidly removed under reduced pressure (25 mmHg) at 40°C. The resulting dispersion was translucent. This dispersion was then filtered through multiple filters in a stepwise fashion. The particle size of the filtered formulation ranged from 8 to 400 nm.

[0092] Microfluidizer example 2-5L Formation of nanoscale constructs and nanobiological compositions of rapamycin This example illustrates the preparation of a pharmaceutical composition containing rapamycin and nanoscale constructs, the formulation being made at a 5 L scale.

[0093] Rapamycin is dissolved in chloroform / t-butanol. This solution is then added to a nanoscale construct solution (1-5% w / v) containing a POPC / PHPC phospholipid mixture, an apoA-I peptidomimetic, a C16-C20 triglyceride mixture, a cholesterol and one or more steryl esters, and a hydrophobic polymer. The mixture is homogenized (Vitris homogenizer model Tempest IQ) at 10,000-15,000 rpm for 5 minutes to form a crude emulsion, which is then transferred to a high-pressure homogenizer. Emulsification is performed at 20,000 psi with emulsion recirculation. The resulting system is transferred to a rotary evaporator, and the solvent is rapidly removed under reduced pressure (25 mmHg) at 40°C. The resulting dispersion is translucent. This dispersion is then filtered through multiple filters in a stepwise fashion. The particle size of the filtered formulation is 35-100 nm.

[0094] Microfluidizer Example 3 - Freeze Drying A nanobiology composition is formed as in any of the above examples. The dispersion is then freeze-dried for an additional 60 hours (FTS Systems, Dura-Dry μP, Stone Ridge, NY). The resulting freeze-dried cake can be easily reconstituted to the original dispersion by adding sterile water or 0.9% (w / v) sterile saline. The particle size after reconstitution is the same as before freeze-drying.

[0095] Prodrug As used herein, unless otherwise specified, the term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compound. Examples of prodrugs include, but are not limited to, derivatives of the nanobiology compositions of the present invention that contain biohydrolyzable moieties, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable ethers, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs. Other examples of prodrugs include moieties that are non-biohydrolyzable but nonetheless provide stability and functionality. Other examples of prodrugs include derivatives of the nanobiology compositions of the present invention that contain -NO, -NO2, -ONO, or -ONO2 moieties. Prodrugs can generally be prepared using well-known methods, such as those described in 1 Burger's Medicinal Chemistry and Drug Discovery, 172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995), and Design of Prodrugs (H. Bundgaard ed., Elselvier, NY 1985).

[0096] Increasing drug compatibility with nanobiological compositions can be achieved using the strategies described below: Drugs are covalently attached to hydrophobic moieties such as cholesterol. Optionally, a prodrug approach can be achieved via a labile bond, resulting in, for example, an enzymatically cleavable prodrug.

[0097] The derivatized drug is then incorporated into lipid-based nanobiological compositions used for in vivo drug delivery. The primary goal of drug derivatization is to form a drug-conjugate with increased hydrophobicity compared to the parent drug. As a result, the retention of the drug-conjugate in the nanobiological composition is enhanced compared to that of the parent drug, thereby reducing leakage and improving delivery to target tissues. In the case of prodrug strategies, different types of hydrophobic moieties can result in different cleavage rates in vivo, affecting the rate at which the effective drug is delivered and thus the overall therapeutic efficacy of the nanobiological composition-drug construct.

[0098] In particular, lipids, sterols, polymers, and aliphatic side chains can be used as hydrophobic moieties. Optimal derivatization of mTORi HDL nanobiology compositions with carbon chains to increase hydrophobicity has been synthesized by these methods. Furthermore, in a further embodiment, the inclusion of triglycerides in HDL creates a larger, more miscible hydrophobic core for loading effective agents such as mTOR inhibitors.

[0099] Combination with a second active agent The nanobiological compositions may be combined with other pharmacologically active compounds ("second active agents") in the methods and compositions of the invention. Certain combinations are believed to act synergistically in the treatment of certain types of transplants, atherosclerosis, arthritis, inflammatory bowel disease, and certain diseases and conditions associated with or characterized by unwanted autoimmune activity.

[0100] Nanobiological compositions may also act to mitigate adverse effects associated with certain second effective agents, and some second effective agents may be used to mitigate adverse effects associated with nanobiological compositions.

[0101] Small molecule secondary agents Small molecule drugs that may be used in combination therapy with the nanobiology compositions of the present invention include prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, acetylsalicylic acid, phenylbutazone, indomethacin, diflunisal, sulfasalazine, acetaminophen, mefenamic acid, meclofenamic acid, flufenamic acid, ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, piroxicam, tenoxicam, salicylate, nimesulide, celecoxib, rofecoxib, valdecoxib, lumiracoxib, parecoxib, etoricoxib, methotrexate, leflunomide, sulfasalazine, azathioprine, cyclophosphamide, the antimalarials hydroxychloroquine and chloroquine, d-penicillamine, and cyclosporine.

[0102] dose Doses generally range from 5 μg to 100 mg per kg of recipient (mammal) body weight per day, usually in the range of 5 μg to 10 mg / kg body weight per day. This amount may be provided in a single daily dose, or more commonly, in multiple (e.g., 2, 3, 4, 5, or 6) subdoses per day so that the total daily dose is the same. The effective amount of the salt or solvate may be determined based on the ratio of the effective amount of the compound to a nanobiology composition containing the inhibitor, where the inhibitor or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, or prodrug thereof is formulated as a nanobiology composition using nanoscale constructs (IMPEPi-NA).

[0103] In another preferred embodiment, the inhibitor may include an mTOR inhibitor (mTORi-NA), an S6K1 inhibitor (S6K1i-NA), diethylmalonate (DMM), 3BP, 2-DG (DMM-NA) (-Gly-NA, which generally inhibits glycolysis), or camptothecin (Hif-1a), or tacrolimus plus a nanoscale construct.

[0104] Combination therapy The compounds of the present invention for inhibiting trained immunity, as well as their salts and solvates, and physiologically functional derivatives thereof, may be used alone or in combination with other therapeutic agents for treating diseases and conditions. Combination therapy of nanobiology compositions with secondary therapeutic agents may include co-administration with known immunosuppressant compounds. Exemplary immunosuppressants include, but are not limited to, statins; mTOR inhibitors, such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase (HDAC) inhibitors; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitors; proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; PPAR (peroxisome proliferator-activated receptor) antagonists; PPAR agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors, and oxidized ATP.

[0105] Immunosuppressants also include IDO, vitamin D3, cyclosporin A, aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine, 6-mercaptopurine, aspirin, niflumic acid, estriol, triptolide, interleukins (e.g., IL-1, IL-10), cyclosporin A, siRNA targeting cytokines or cytokine receptors, and the like. Examples of statins include atorvastatin (LIPITOR®, TORVAST®), cerivastatin, fluvastatin (LESCOL®, LESCOL®XL), lovastatin (MEVACOR®, ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®, PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin (CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

[0106] transplant "Transplantable graft" refers to biological materials, such as cells, tissues, and organs (whole or partial), that can be administered to a subject. Transplantable grafts can be autografts, allografts, or xenografts of biological materials, such as organs, tissues, skin, bone, nerves, tendons, neurons, blood vessels, fat, cornea, pluripotent cells, and differentiated cells (obtained or derived in vivo or in vitro). In some embodiments, transplantable grafts are formed from, for example, cartilage, bone, extracellular matrix, or collagen matrix. Transplantable grafts can also be single cells, cell suspensions, and cells in tissues and organs that can be transplanted. Transplantable cells typically have a therapeutic function, e.g., a function that is missing or diminished in the recipient subject. Non-limiting examples of some transplantable cells include islet cells, beta cells, hepatocytes, hematopoietic stem cells, neural stem cells, neurons, glial cells, or myelin-forming cells. Transplantable cells can be unmodified cells, e.g., cells obtained from a donor subject and usable for transplantation without any genetic or epigenetic modification. In other embodiments, transplantable cells can be modified cells, e.g., cells obtained from a subject with a genetic defect in which the genetic defect has been corrected, or cells derived from reprogrammed cells, e.g., differentiated cells derived from cells obtained from the subject.

[0107] "Transplantation" refers to the process of transferring (transferring) a transplantable graft (e.g., from a donor subject, from an in vitro source (e.g., from differentiated autologous or xenogeneic native or induced pluripotent cells) into a recipient subject, and / or from one bodily location to another bodily location in the same subject.

[0108] In one embodiment, the transplant tissue is lung tissue, heart tissue, kidney tissue, liver tissue, retinal tissue, corneal tissue, skin tissue, pancreatic tissue, intestinal tissue, reproductive tissue, ovarian tissue, bone tissue, tendon tissue, or vascular tissue.

[0109] In one embodiment, the transplant tissue is transplanted as an intact organ.

[0110] As used herein, a "recipient subject" is a subject that receives or has received transplanted cells, tissues, or organs from another subject.

[0111] As used herein, a "donor subject" is a subject from which cells, tissues, or organs to be transplanted are removed prior to transplantation of the cells, tissues, or organs into a recipient subject.

[0112] In one embodiment, the donor subject is a primate. In a further embodiment, the donor subject is human. In one embodiment, the recipient subject is a primate. In one embodiment, the recipient subject is human. In one embodiment, both the donor and recipient subjects are human. Thus, the present subject matter includes xenotransplantation embodiments. As used herein, "immune rejection" refers to a hyperacute, acute, and / or chronic response of the recipient subject's immune system that recognizes the transplanted cells, tissues, or organs from the donor as non-self, and the resulting immune response events.

[0113] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another if their genes are not identical at one or more loci.

[0114] The term "autologous" refers to any material derived from the same individual that is later reintroduced into the same individual.

[0115] As used herein, an "immunosuppressant" is a pharmaceutically acceptable drug used to suppress the immune response of a recipient subject. A non-limiting example includes rapamycin.

[0116] Pharmaceutical Delivery As used herein, a "prophylactically effective" amount is an amount of a substance effective to prevent or delay the onset of a given condition in a subject to which the substance is administered. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose is used in a subject prior to or at an early stage of disease, and therefore the prophylactically effective amount is less than the therapeutically effective amount.

[0117] As used herein, a "therapeutically effective" amount is an amount of a substance effective to treat, ameliorate, or attenuate the symptoms or causes of a given condition in a subject suffering from that condition to which the substance is administered.

[0118] In one embodiment, the therapeutically or prophylactically effective amount is from about 1 mg of agent / kg subject to about 1 g of agent / kg subject per administration. In another embodiment, the therapeutically or prophylactically effective amount is from about 10 mg of agent / kg subject to 500 mg of agent / kg subject. In a further embodiment, the therapeutically or prophylactically effective amount is from about 50 mg of agent / kg subject to 200 mg of agent / kg subject. In a further embodiment, the therapeutically or prophylactically effective amount is about 100 mg of agent / kg subject. In a further embodiment, the therapeutically or prophylactically effective amount is selected from 50 mg of agent / kg subject, 100 mg of agent / kg subject, 150 mg of agent / kg subject, 200 mg of agent / kg subject, 250 mg of agent / kg subject, 300 mg of agent / kg subject, 400 mg of agent / kg subject, and 500 mg of agent / kg subject.

[0119] Treatment and prevention methods The methods of the present invention encompass methods of treating, preventing, and / or managing various types of transplants, atherosclerosis, arthritis, inflammatory bowel disease, and diseases and conditions associated with or characterized by unwanted autoimmune activity. As used herein, unless otherwise specified, the term "treating" refers to the administration of a compound of the present invention or other additional effective agent after the onset of symptoms of a particular disease or disorder.

[0120] The words "treating" or "treatment" of a condition, disorder, or pathology are used to refer to: Preventing or delaying the onset of clinical symptoms of a condition, disorder, or condition in a person who may be susceptible to or affected by the condition, disorder, or condition but who has not yet experienced or exhibited clinical symptoms of the condition, disorder, or condition; or Inhibition of a condition, disorder, or pathological state, i.e., arresting, reducing, or delaying the onset of a disease or its recurrence (in the case of maintenance treatment) or at least one clinical symptom, sign, or test thereof; or Relief of disease, i.e., causing regression of a condition, disorder, or pathology, or at least one of its clinical or subclinical symptoms or signs Includes.

[0121] As used herein, unless otherwise specified, the term "preventing" refers to administration prior to the onset of symptoms, particularly to patients at risk of transplantation, atherosclerosis, arthritis, inflammatory bowel disease, and other diseases and disorders associated with or characterized by unwanted autoimmune activity. The term "prevention" includes the inhibition of symptoms of a particular disease or disorder. Patients with a family history of transplantation, atherosclerosis, arthritis, inflammatory bowel disease, and diseases and disorders associated with or characterized by unwanted autoimmune activity are preferred candidates for preventative regimens.

[0122] As used herein, unless otherwise specified, the term "managing" includes preventing the recurrence of a particular disease or disorder in a patient who has had the particular disease or disorder and / or increasing the amount of time that a patient who has had the disease or disorder remains in remission.

[0123] In another embodiment, the present invention encompasses methods of treating, preventing, and / or managing transplantation, atherosclerosis, arthritis, inflammatory bowel disease, comprising administering a nanoscale particle of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, in combination with (e.g., before, during, or after) conventional therapy, including but not limited to surgery, immunotherapy, biological therapy, radiation therapy, or other non-drug based therapy currently used to treat, prevent, or manage transplantation.

[0124] Radiolabeling for PET imaging of drug accumulation within the body In a preferred, non-limiting embodiment of the present invention, there is provided a radiopharmaceutical composition and method for radiopharmaceutical imaging of nanobiological composition accumulation in bone marrow, blood, and / or spleen of a patient affected by trained immunity, comprising: administering to said patient a nanobiological composition in an amount effective to promote a hyperresponsive innate immune response; the nanobiology composition comprises: (i) a nanoscale construct; (ii) an inhibitor drug incorporated into the nanoscale construct; and (iii) a positron emission tomography (PET) imaging agent incorporated into the nanoscale construct; the nanoscale construct is a multi-component carrier composition comprising: (a) a phospholipid; (b) apoA-I or a peptidomimetic of apoA-I; and optionally (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof; and optionally (d) cholesterol; the inhibitors of metabolic or epigenetic pathways comprise NOD2 receptor inhibitors, mTOR inhibitors, ribosomal protein S6 kinase beta-1 (S6K1) inhibitors, HMG-CoA reductase inhibitors (statins), histone H3K27 demethylase inhibitors, BET bromodomain blockade inhibitors, inhibitors of histone methyltransferases and acetyltransferases, inhibitors of DNA methyltransferases and acetyltransferases, inflammasome inhibitors, serine / threonine kinase Akt inhibitors, inhibitors of hypoxia inducible factor 1-alpha, also known as HIF-1-alpha, and mixtures of one or more thereof; the PET contrast agent 89 Zr, 124 I, 64 Cu, 18 F, and 86 Y, wherein the PET imaging agent is complexed with the nanobiology composition using a suitable chelating agent to form a stable drug-agent chelate; The nanobiology composition is a self-assembled nanodisk or nanosphere having a diameter of about 8 nm to about 400 nm in an aqueous environment, The nanoscale constructs deliver stable drug-agent chelates to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen. Steps and (ii) performing PET imaging of the patient to visualize the biodistribution of the stable drug-agent chelate in the bone marrow, blood, and / or spleen of the patient's body; Including, A method is provided.

[0125] Additionally, gamma counting or autoradiography can be used to verify imaging results. 89 Ex vivo methods can be used to quantify tissue uptake of Zr-labeled nanoparticles.

[0126] It also provides a novel approach to autoradiography-based histology, allowing assessment of the regional distribution of nanomaterials within tissues of interest by comparing the accumulation patterns of radioactivity obtained by autoradiography with histological and / or immunohistochemical staining in the same or adjacent sections.

[0127] Currently, the most used methods for assessing the in vivo events of nanotherapeutics rely on fluorescent dyes. However, these techniques are not quantitative due to autofluorescence, quenching, FRET, and the high sensitivity of fluorophores to the environment (e.g., pH or solvent polarity). The integration of magnetic resonance imaging contrast agents as nanoparticle labels has been attempted, but requires significant payload and dosing, compromising the integrity of the nanoparticle formulation. Nuclear contrast agents do not suffer from these drawbacks, especially 89 Zr is suitable due to the positron emission required for PET imaging and its relatively long physical half-life (78.4 hours), allowing for long-term studies of slowly cleared substances and eliminating the need for a nearby cyclotron.

[0128] The method of the present inventors is as follows: 89 This provides an excellent method for functionalizing nanobiology compositions using Zr. DSPE-DFO represents a stable method for immobilizing the DFO chelator within lipid monolayers or bilayers. Furthermore, because DFO is present outside the nanoparticle platform, the nanoparticles can be labeled after formulation. This eliminates the need to perform these formulations under radiation-shielded conditions, reducing the amount of activity that needs to be used. Finally, incorporating DSPE-DFO, 89 The mild conditions for Zr incorporation are compatible with a wide variety of nanoparticle types and formulation methods.

[0129] In yet another preferred embodiment of the present invention, when further stability is desired in the formulation, 34 -DFO 6A lipophilic DFO derivative, termed , can be incorporated following the same protocol.

[0130] In a further non-limiting preferred embodiment of the present invention, the present invention provides a method for first formulating particles, then functionalizing protein components using commercially available p-NCS-Bz-DFO, and finally, using our general methodology. 89 These include radiolabeled protein-coated nanoparticles prepared by incorporating Zr.

[0131] Example Transplant Immunization Results - Examples 1-13 Example 1 - Transplant Immunity - Donor allografts express vimentin and HMGB1 and promote local recruitment of macrophages. To elucidate the macrophage activation pathways that promote allograft immunity, we assessed the functional state of macrophages, which are accompanied by increased inflammatory cytokine production caused by non-permanent epigenetic reprogramming associated with trained immunity. The roles of dectin-1, which may be present under sterile inflammation, and the TLR4 agonists vimentin and HMGB1 (high mobility group box 1) were demonstrated.

[0132] BALB / c (H2d) hearts were transplanted into fully allogeneic C57BL / 6 (H2b) recipients as described, and the data in Figures 1-3 show that these proteins were upregulated in the donor allograft after organ transplantation, indicating that vimentin and HMGB1 can promote the training of macrophages to locally infiltrate the graft.

[0133] To confirm this, flow cytometry analysis of Dectin-1 and TLR4 expression in graft-infiltrating macrophages is shown in Figure 4. Absence of Dectin-1 and TLR4 expression using deficient recipient mice prevented the accumulation of inflammatory Ly6Chi macrophages infiltrating the graft (Figure 5). Conversely, deficiency of Dectin-1 or TLR4 promoted the accumulation of Ly6Clo macrophages in allografts, which promoted allograft acceptance.

[0134] We demonstrated that donor allografts upregulated vimentin and HMGB1, indicating that vimentin and HMGB1 promote macrophage training. Using an established in vitro trained immune model in which purified monocytes were exposed to β-glucan and then restimulated with LPS, a similar increase was observed in the production of the pro-inflammatory cytokines TNFα and IL-6 after stimulation with vimentin and HMGB1 (Figure 6), demonstrating the training-inducing properties of these proteins. To verify that vimentin and HMGB1 induced local training of graft-infiltrating macrophages, these cells were flow-sorted from cardiac allografts and evaluated for their ability to produce pro-inflammatory cytokines and glycolytic products. We demonstrated that deficiency of Dectin-1 or TLR4 significantly reduced the expression of pro-inflammatory cytokines TNFα and IL-6 and lactate production by graft-infiltrating macrophages after ex vivo LPS stimulation (Figure 7). Consistent with protein expression, the absence of Dectin-1 or TLR4 prevented epigenetic changes of H3K4me3 at the promoters of the proinflammatory cytokines TNFα and IL-6 and the glycolytic enzymes hexokinase (HK) and phosphofructokinase (PFKP) in graft-infiltrating macrophages (Figure 8). Collectively, the data indicate that bone marrow monocytic precursors (Figure 34) migrate to the allograft early after transplantation and become trained after local exposure to vimentin / HMGB1.

[0135] Example 2 - Transplantation Immunity - mTORi-HDL Nanoimmunotherapy Prevents Trained Immunity In Vitro. In another preferred embodiment of the present invention, a nanoimmunotherapy based on a nanobiological composition of high-density lipoprotein (HDL) was developed to target myeloid cells. Because the mammalian target of rapamycin (mTOR) regulates cytokine production (signal 3) via trained immunity, the mTOR inhibitor rapamycin (Figure 35) was encapsulated in a corona of natural phospholipids and apolipoprotein AI (apoA-I) isolated from human plasma to provide an mTORi-HDL nanobiological composition.

[0136] The resulting nanobiology composition had a drug encapsulation efficiency of 62±1:1% and a mean hydrodynamic diameter of 12.7±4.4 nm, as determined by high-performance liquid chromatography and dynamic light scattering, respectively. Transmission electron microscopy revealed that mTORi-HDL had a discoidal structure (Figures 9 and 36; STAR method).

[0137] Example 3 - Transplantation Immunity - Immunization Model Using an established in vitro model of trained immunity in which purified human monocytes were exposed to β-glucan, we observed increased cytokine and lactate production after restimulation with LPS. Conversely, β-glucan-trained human monocytes treated with mTORi-HDL during the training period exhibited significantly less cytokine and lactate production after LPS restimulation (Figure 10). This result indicated that trained immunity is dependent on mTOR. Because significant cytokine and glycolytic responses may be the result of epigenetic reprogramming of macrophages, histone H3K4 trimethylation, which represents open chromatin, was assessed (Figure 11; STAR assay). mTORi-HDL treatment prevented epigenetic changes at the promoter levels of four inflammatory genes associated with trained immunity in human monocytes.

[0138] Example 4 - Transplant Immunity - Biodistribution The biodistribution and immune cell specificity of fluorescently stained (DiO or DiR) or zirconium-89 radiolabeled mTORi-HDL has been demonstrated using computed tomography (PET-CT) imaging, ex vivo near-infrared fluorescence (NIRF) imaging, and a combination of flow cytometry and in vivo positron emission tomography in wild-type C57BL / 6 mice (Figure 13). 89 Zr-mTORi-HDL; Figure 12; STAR method). These figures show that it preferentially binds to myeloid cells, but not to T or B cells (Figure 15), in the kidney, liver, and spleen. 89 Detection of Zr-mTORi-HDL accumulation is shown (FIG. 14 and FIGS. 37 to 38).

[0139] Importantly, robust mTORi-HDL accumulation in the bone marrow was observed (Figures 14-15), promoting the induction of long-term therapeutic effects, and it bound to several myeloid cells and their precursors (Figure 16).

[0140] Example 5 - Transplantation Immunity - mTORi-HDL Nanoimmunotherapy Prevents Trained Immunity In Vivo. mTORi-HDL treatment was applied to an experimental heart transplant mouse model (Figure 17) to determine allograft targeting and immune cell specificity, as described above. Six days after receiving heterotopic heart transplants, mice were 89 Mice were treated intravenously with Zr-mTORi-HDL. After 24 hours of circulation and distribution of this nanoimmunotherapy, the mice were subjected to PET-CT. The image shows significant changes in the cardiac allograft. 89 The results show the presence of Zr-mTORi-HDL (Figures 18 and 39; STAR method). After sacrificing the mice, the native hearts and allografts were cultured ex vivo. 89 The radioactivity in the cardiac allografts (Tx) was 2.3-fold higher (25.2±2.4×103 counts / unit area) compared to the native hearts (N) (11.1±1.9×103 counts / unit area) (FIG. 19).

[0141] Example 6 - Transplant Immunity - Immune Cell Specificity Because the nanoimmunotherapy demonstrated favorable organ distribution patterns and cardiac allograft uptake, we evaluated the immune cell specificity of mTORi-HDL, labeled with the fluorescent dye DiO. Twenty-four hours after intravenous administration, cardiac allografts, as well as blood and spleen, were collected and the distribution of mTORi-HDL in DCs, macrophages, neutrophils, and T cells was measured by flow cytometry. The cellular preference of mTORi-HDL toward myeloid cells is demonstrated in the figures, where significant uptake by macrophages over either DCs or neutrophils is observed in the allograft, blood, and spleen (Figures 20 and 40-41). T cells exhibited poor uptake of mTORi-HDL (Figures 42 and 43), highlighting the preferential targeting of mTORi-HDL to myeloid cells.

[0142] Example 7 - Transplantation Immuno-Treatment Regimen A treatment regimen was evaluated that included three intravenous injections of mTORi-HDL at 5 mg / kg per dose of rapamycin on the day of transplantation and on post-surgery days 2 and 5. The myeloid cell compartments in the allograft, blood, and spleen of mice receiving either mTORi-HDL treatment or placebo were profiled. Consistent with the targeting data, the overall numbers of macrophages, neutrophils, and DCs were significantly lower in the allograft, blood, and spleen of mTORi-HDL-treated recipients compared with placebo-treated mice or mice treated with oral rapamycin (5 mg / kg on post-surgery days 0, 2, and 5) (Figure 44).

[0143] Example 8 - Transplantation Immunity - Macrophage Subsets The effects of mTORi-HDL nanoimmunotherapy on the distribution of two distinct macrophage subsets (Ly-6Chi and Ly-6Clo), which have distinct immunoregulatory properties, are also provided in the figures. Six days after transplantation, untreated recipient mice had increased numbers of inflammatory Ly-6Chi macrophages in the allograft, blood, and spleen (Figures 21 and 45). In contrast, recipients treated with mTORi-HDL had increased numbers of Ly-6Clo macrophages. This data indicates that Ly-6Chi macrophages constitute the majority of macrophages during graft rejection, but our mTORi-HDL nanoimmunotherapy promoted the accumulation of Ly-6Clo macrophages. This change was not observed in animals treated with oral rapamycin (Figure 45).

[0144] Example 9 - Transplantation Immunity - Molecular Pathways Gene Set Enrichment Analysis (GSEA) of mRNA isolated from flow-sorted macrophages derived from allografts of animals treated with either placebo or mTORi-HDL was used to demonstrate the molecular pathways targeted by mTORi-HDL nanoimmunotherapy. Gene array results indicated that the mTOR and glycolysis pathways, which are associated with trained immunity, were negatively regulated by mTORi-HDL (Figures 22-23). ​​Cardiac allograft-derived macrophages were flow-sorted and evaluated to demonstrate their ability to produce proinflammatory cytokines (signal 3) and glycolytic products. mTORi-HDL treatment significantly reduced TNFα and IL-6 protein expression and lactate production by graft-infiltrating macrophages after ex vivo LPS stimulation (Figure 24). Consistent with in vitro observations (FIGS. 10 and 11), mTORi-HDL treatment also prevented epigenetic changes of H3K4me3 in graft-infiltrating macrophages (FIG. 25; STAR method).

[0145] Example 10 - Transplant Immunity - Organ Transplant Tolerance Figures 26-33 demonstrate that mTORi-HDL nanoimmunotherapy promotes organ transplant tolerance. Figures 26-33 show the immune function of graft-infiltrating macrophages. The suppressive function of Ly-6Clo macrophages was measured by their ability to inhibit the in vitro proliferation of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD8+ T cells. Ly-6Clo macrophages obtained from allografts of mTORi-HDL-treated recipient mice were observed to inhibit T cell proliferation in vitro (Figure 26). Ly-6Clo macrophages from the same mTORi-HDL-treated allografts expanded regulatory T cells (Tregs) expressing the immunosuppressive Foxp3. Consistent with these data, significantly more CD4+CD25+ T cells were observed in the allografts of mTORi-HDL-treated recipients (Figure 27). These results suggest that mTORi-HDL treatment supports transplant acceptance by promoting the development of Ly-6Clo regulatory macrophages (Mregs).

[0146] Example 11 - Transplant Immunity - Transplant Recipients As shown in the figures, the functional role of Ly-6Clo Mregs in transplant recipients was demonstrated by depleting Ly-6Clo Mregs in vivo. Briefly, BALB / c (H2d) donor cardiac allografts were transplanted into fully allogeneic C57BL / 6 CD169 diphtheria toxin (DT) receptor (DTR) (H2b) recipient mice treated with mTORi-HDL. On the day of transplantation, regulatory Ly-6Clo Mregs were depleted by DT administration (Figure 28), leading to early graft rejection (12.3 ± 1.8 days) despite mTORi-HDL treatment (Figure 29).

[0147] Adoptive transfer of wild-type monocytes restored allograft survival, indicating that nanoimmunotherapy exerts its effect via Mregs (Figure 29). This was further confirmed by using CD11c-DTR mice as transplant recipients, in which administration of DT in these mice depletes CD11c+DCs. This indicated that prolonged graft survival is independent of CD11c+DCs. In contrast, graft survival in CCR2-deficient recipient mice, which have fewer Ly-6Chi circulating monocytes, was not sustained for a long time (Figure 30). Overall, these experiments demonstrate that macrophages are required for organ transplant acceptance promoted by mTORi-HDL nanoimmunotherapy.

[0148] Example 12 - Transplantation Immunity - Blockade of Costimulation Activated macrophages produce large amounts of IL-6 and TNFα, which promote T cell graft-responsive alloimmunity. The absence of IL-6 and TNFα in the recipient synergizes with CD40-CD40L costimulatory blockade to induce durable allograft acceptance. This was demonstrated by simultaneous costimulatory blockade (signal 2) to enhance the efficacy of mTORi-HDL. To illustrate, a second nanoimmunotherapy treatment consisting of CD40-TRAF6-inhibitory HDL (TRAF6i-HDL) was used (Figures 47 and 48). The specificity of CD40 signaling inhibition was demonstrated using an agonistic CD40 mAb (clone FGK4.5) to induce rejection in mTORi-HDL-treated recipients. Treatment with the TRAF6i-HDL nanobiology composition was shown to prevent the adverse effects of stimulatory CD40 mAb and restored mTORi-HDL-mediated allograft survival (Figure 31).

[0149] Example 13 - Transplant Immunity - Fully Allogeneic Donor Hearts The prolonged survival properties of nanoimmunotherapy for fully allogeneic donor heart grafts are shown in the figures. Using the three dose regimens described above of 5 mg / kg per dose on days 0, 2, and 5 after surgery, mTORi-HDL treatment significantly increased cardiac allograft survival compared with placebo, HDL vehicle, and oral / intravenous rapamycin treatment (Figures 32 and 49). Subsequently, treatment regimens were tested by combining nanobiological compositions of mTORi-HDL (signal 3) and TRAF6i-HDL (signal 2). This mTORi-HDL / TRAF6i-HDL treatment synergistically promoted organ transplant acceptance, resulting in >70% allograft survival at 100 days after transplantation. This combination treatment was far superior to mTORi-HDL and TRAF6i-HDL monotherapy (Figure 32) and was not associated with histopathological evidence of toxicity or chronic allograft vasculopathy (Figures 33 and 50).

[0150] Collectively, this data demonstrated that HDL-based nanoimmunotherapy prevented the production of macrophage-derived proinflammatory cytokines associated with trained immunity. Furthermore, HDL-based nanoimmunotherapy exhibited less toxicity than oral rapamycin and provided long-term therapeutic benefits without off-target side effects (Figure 51).

[0151] Example 14 - Transplantation Immunization - Materials and Methods mouse Female C57BL / 6J (B6 WT, H-2b) and BALB / c (H-2d) mice were purchased from Jackson Laboratory. Eight-week-old C57BL / 6J (Foxp3tm1Flv / J), CCR2-deficient, and CD11c-DTR mice were purchased from Jackson Laboratory. C57BL / 6J CD169DTR mice were obtained from Masato Tanaka (Kawaguchi, Japan) (Miyake et al., 2007). Animals were enrolled at 8–10 weeks of age (body weight, 20–25 g). All experiments were performed on matched 8–12-week-old female mice according to protocols approved by the Mount Sinai Animal Care and Utilization Committee.

[0152] Human samples Buffy coats were obtained from unspecified healthy donors who were recruited after providing informed consent (Sanquin blood bank, Nijmegen, The Netherlands). The gender and age of the healthy donors were not collected and therefore unavailable.

[0153] Learn more about how Vascularized heart transplant BALB / c hearts were transplanted into C57BL / 6 mice as fully vascularized heterotopic grafts, as previously described (Corry et al., 1973). The hearts were transplanted into the recipient peritoneal cavity by establishing end-to-side anastomoses between the donor and recipient aorta and between the donor pulmonary trunk and the recipient inferior vena cava. Cardiac allograft survival was then assessed via daily palpation. Rejection, defined as the complete cessation of cardiac contractions, was confirmed by direct visualization via laparotomy. Graft survival was compared between groups using Kaplan-Meier survival analysis.

[0154] Isolation of apolipoprotein AI (apoA-I) Human apoA-I was isolated from human HDL concentrate (Bioresource Technology) using a previously described method (Zamanian-Daryoush et al., 2013). Briefly, potassium bromide solution (density: 1.20 g / mL) was layered on top of the concentrate, and purified HDL was obtained by ultracentrifugation. The purified fraction was added to a chloroform / methanol solution for delipidation. The resulting milky solution was filtered, and the apoA-I precipitate was dried overnight. The protein was reconstituted with 6 M guanidine hydrochloride, and the resulting solution was dialyzed against PBS. Finally, the apoA-I PBS solution was filtered through a 0.22 μm filter, and the identity and purity of the protein were confirmed by gel electrophoresis and size-exclusion chromatography.

[0155] Synthesis of nanobiological compositions mTORi-HDL nanoparticles were synthesized using a modified lipid film hydration method. Briefly, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) (both purchased from Avanti Polar Lipids), and rapamycin (Selleckchem) were dissolved in a chloroform / methanol (10:1 v / v) mixture at a weight ratio of 3:1:0.5. After evaporation of the solvent, human apoA-I in PBS was added at a 5:1 weight ratio of phospholipid:apoA-I to hydrate the lipid film and incubated in an ice bath for 20 minutes. The resulting mixture was homogenized in an ice bath using a probe sonicator for 15 minutes to obtain mTORi-HDL nanoparticles. mTORi-HDL was washed and concentrated by centrifugal filtration using a 10 kDa molecular weight cutoff (MWCO) filter tube. Aggregates were removed using centrifugation and filtration (0.22 μm). For therapeutic studies, animals were orally administered or tail vein injected (mTORi-HDL or intravenous Ra) with rapamycin at a dose of 5 mg / kg on the day of transplantation and on days 2 and 5 after transplantation.

[0156] The size and surface charge of the HDL nanobiology composition were determined by dynamic light scattering (DLS) and Z-potential measurements. The final composition after purification was determined by standard protein and phospholipid quantification methods (bicinchoninic acid assay and malachite green phosphate assay), and the drug concentration was established by HPLC by comparison with a calibration curve of a reference compound. A batch-to-batch variation of ±15% was considered acceptable.

[0157] Radiolabeling of mTORi-HDL nanoparticles mTORi-HDL was radiolabeled with 89Zr according to a previously described method (Perez-Medina et al., 2015). Briefly, ready-to-label mTORi-HDL was obtained by adding 1 mol% of the phospholipid chelator DSPE-DFO at the expense of DMPC in the initial formulation. Radiolabeling with 89Zr was achieved by reacting DFO-loaded nanoparticles with 89Zr-oxalate in PBS (pH = 7.1) at 37 °C for 1 h. 89Zr-mTORi-HDL was isolated by centrifugal filtration using 10 kDa MWCO tubing. The radiochemical yield was 75 ± 2% (n = 2).

[0158] Micro-PET / CT imaging and biodistribution studies Mice (n = 6; 3 bearing cardiac grafts [weight: 18.8 ± 1.0 g]) were injected with a single dose of 89Zr-mTORi-HDL (0.17 ± 0.01 mCi, approximately 0.25 mg apoA-I) in 0.2 mL of PBS solution via the lateral tail vein 6 days after graft implantation. 24 h later, animals were anesthetized with an isoflurane (Baxter Healthcare, Deerfield, USA) / oxygen gas mixture (2% induction, 1% maintenance) and then scanned using an Inveon PET / CT system (Siemens Healthcare Global, Erlangen, Germany). A 15-minute, whole-body PET static scan was performed, recording a minimum of 30 million simultaneous events. The energy and timing window for the match were 350–700 keV and 6 ns, respectively. Image data were normalized to correct for PET response heterogeneity, dead-time count-down, positron branching ratios, and physical decay over injection time; no corrections for attenuation, scattering, or partial volume averaging were applied. Measured ratios in reconstructed images were converted to activity concentrations (percentage of injected dose per gram of tissue [ID (%)]) using a system calibration factor derived from imaging a mouse-sized water-equivalent phantom containing 89Zr. Images were analyzed using ASIPro VM™ software (Concorde Microsystems, Knoxville, USA) and Inveon Research Workplace (Siemens Healthcare Global, Erlangen, Germany) software. Standard low-magnification CT scans of the whole body were performed with an X-ray tube set at 80 kV voltage and 500 μA current. CT scans were acquired using 120 rotation steps for a total of 220 degrees, resulting in an estimated scan time of 120 s with an exposure of 145 ms per frame.Immediately after the PET / CT scan, the animals were sacrificed, and the tissues of interest—kidney, heart, liver, spleen, blood, bone, skin, and muscle—were collected, weighed, and counted in a Wizard2 2480 automated gamma counter (Perkin Elmer, Waltham, USA) to determine radioactivity content. This value was corrected for decay and converted to a percentage of the injected dose per gram (ID(%) / g). To determine the distribution of radioactivity within the transplanted heart, the native and transplanted specimens were placed in film cassettes and exposed to phosphorimaging plates (BASMS-2325, Fujifilm, Valhalla, USA) at −20°C for 4 hours. The plates were read using a Typhoon 7000 IP plate reader (GE Healthcare, Pittsburgh, USA) at a pixel resolution of 25 μm. Images were analyzed using ImageJ software.

[0159] Immunofluorescence microscopy The transplanted hearts were harvested, minced, and directly frozen in Tissue-Tek OCT (Sakura) and stored at -80°C in preparation for immunoassays. Eight-micrometer sections were mounted on polylysine-coated slides, cut using a Leica 1900CM freezing microtome, fixed in acetone (-20°C for 20 minutes), and then incubated with a blocking buffer containing 1% BSA and 5% goat or rabbit serum. The slides were then incubated overnight at 4°C with 1 / 100 dilution of rat anti-mouse dectin-1 (clone 2A11) or rabbit anti-mouse vimentin (clone EPR3776) from Abcam. After overnight incubation, the slides were washed with PBS and then incubated with conjugated goat monoclonal anti-rabbit Cy-3 (1 / 800) or goat monoclonal anti-rabbit Cy-2 (1 / 500) purchased from Jackson Immunoresearch. All slides were mounted with Vectashield (Vector Laboratories) containing Dapi to preserve fluorescence. Images were acquired using a Leica DMRA2 fluorescence microscope (Wetzlar) and a digital Hamamatsu charge-coupled device camera. Separate green, red, and blue images were collected and analyzed with ImageJ software (NIH).

[0160] Isolation of graft-infiltrating leukocytes Mouse hearts were rinsed in situ with HBSS containing 1% heparin. The explanted hearts were cut into small pieces and digested with 400 U / ml collagenase A (Sigma-Aldrich), 10 mM HEPES (Cellgro), and 0.01% DNase I (MP Biomedicals) in HBSS (Cellgro) for 40 minutes at 37°C. The digested suspension was passed through a nylon mesh and centrifuged. The cell pellet was resuspended in complete HBSS, stained, and analyzed by flow cytometry (BD LSR-II; BD Biosciences).

[0161] Flow cytometry and cell sorting For myeloid cell staining, fluorochrome-conjugated mAbs specific for mouse CD45 (clone 30-F11), CD11b (clone M1 / 70), CD11c (clone N418), F4 / 80 (clone CI:A3.1), Ly-6C (clone HK1.4), and the corresponding isotype controls were purchased from eBioscience. Ly-6G (clone 1A8) mAb was purchased from Biolegend. For T cell staining, antibodies against CD3 (clone 2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), and CD25 (clone PC61.5) were purchased from eBioscience. Absolute cell counts were performed using countbright beads (Invitrogen). For staining of progenitor, myeloid, and lymphoid cells in bone marrow, spleen, kidney, and liver, fluorochrome-conjugated mAbs specific for mouse B220 / CD45R (clone RA3-6B2), CD34 (clone RAM34), CD16 / 32 (clone 93), CD90 (clone 53-2.1), CD19 (clone 1D3), CD115 (clone AFS98), and CD135 (clone A2F10) were purchased from eBioscience; CD49b (clone DX5), MHCII (clone M5 / 114.15.2), and Sca-1 (clone D7) were purchased from Biolegend; CD64 (clone X54-5 / 7.1), CD117 (clone 2B8), and CD172α (clone P84) were purchased from BD Biosciences. Flow cytometry analysis was performed on an LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc.). Results were expressed as the percentage of cell staining above background or cell count (cells per milliliter). To purify graft-infiltrating myeloid cells, single-cell suspensions from donor hearts were sorted using an InFlux cell sorter (BD) at the Icahn School of Medicine at Mount Sinai's Flow Cytometry Shared Resource Facility, achieving a purity of >96%.

[0162] Experiments with trained immunity of human monocytes Human monocytes were isolated and trained as described previously. PBMCs were isolated by diluting blood in pyrogen-free PBS and performing differential density centrifugation on Ficoll-Paque (GE Healthcare, UK). Monocytes were then isolated by high osmolality density gradient centrifugation on Percoll (Sigma). Monocytes (1 × 107) were plated in 10 cm Petri dishes (Greiner) in a 10 ml medium volume and incubated for 24 hours (in 10% pooled human serum) with culture medium alone as a negative control or with 5 μg / ml β-glucan with or without mTORi-HDL (1 μg / ml). On day 6, cells were detached from the plates, and 1 × 10 macrophages were replated into 96-well flat-bottom plates. After restimulation with either 200 μl of RPMI or E. coli LPS (serotype 055:B5, Sigma-Aldrich, 10 ng / ml) for 24 hours, the supernatants were collected and stored at -20°C. Cytokine production was determined in the supernatants using commercially available ELISA kits for TNFα and IL-6 (R&D systems) according to the manufacturer's instructions. The remaining cells were fixed with 1% methanol-free formaldehyde and sonicated. Immunoprecipitation was performed using an antibody against H3K4me3 (Diagenode, Seraing, Belgium). DNA was isolated using a MinElute PCR purification kit (Qiagen) and further processed for qPCR analysis using the Sybr Green method. Samples were analyzed by the comparative Ct method according to the manufacturer's instructions.

[0163] Experiments with trained immunity of mouse monocytes Bone marrow monocytes were isolated using a monocyte isolation kit (Miltenyi). Monocyte precursors (1 × 10 cells / well in a 48-well plate) were differentiated in vitro for 6 days with 10 ng / ml recombinant mouse GM-CSF (Peprotech). On day 6, either 10 μg / ml β-glucan (Sigma) or 100 μg / ml vimentin (R&D systems) was added to the culture for 24 hours. After a 3-day rest period, macrophages were restimulated with either 10 ng / ml LPS (Sigma) or 20 μg / ml HMGB1 (R&D systems) for 24 hours. Cytokine production was determined in the supernatant using commercially available ELISA kits for TNFα and IL-6 (R&D systems), and the remaining cells were used for chromatin immunoprecipitation (ChIP) assays.

[0164] Mouse chromatin immunoprecipitation (ChIP) In vitro bone marrow-derived trained macrophages or graft-infiltrating macrophages were used in this assay. The following antibodies were used: anti-H3K4me3 (39159; Active Motif) and anti-IgG (ab171870; Abcam). For ChIP followed by qPCR, crosslinking was performed for 10 minutes. For sonication, we used a Bioruptor (Diagenode) with cooling, which we optimized to generate DNA fragments of approximately 200–1,000 base pairs (bp). Lysates were precleared for 2 hours using an appropriate isotype-matched control antibody (rabbit IgG; Abcam). Specific antibodies were coupled to magnetic beads (Dynabeads® M-280 Sheep Anti-Rabbit IgG; ThermoFisher Scientific) overnight at 4°C. The antibody-bound beads and chromatin were then immunoprecipitated overnight at 4°C with rotation. After washing, reverse crosslinking was performed overnight at 65°C. After digestion with RNase and proteinase K (Roche), DNA was isolated using the MinElute kit (Qiagen) and used for downstream applications. qPCR was performed using iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions. Primers were designed using the Primer3 online tool and cross-referenced to the mouse mm10 genome visualized on the Integrated Genomics Viewer (IGV; Broad).

[0165] Suppression assay Spleens from C57BL / 6 (H-2b) mice were gently dissociated into single-cell suspensions, and red blood cells were removed using hypotonic ACK lysis buffer. Splenocytes were labeled with 5 μM CFSE (using Invitrogen Molecular Probes) and then stained with anti-CD8 mAb for 30 minutes on ice. Responder CFSE+ CD8+ T cells were sorted to greater than 98% purity using a FACS Aria II (BD Biosciences). CFSE+ CD8+ T cells were used with anti-CD3 / CD28 microbeads as stimulators. Stimulated CFSE+ CD8+ T cells were cultured with graft-infiltrating Ly-6Clo macrophages, mTORi-HDL, or placebo for 72 hours at 37°C in a 5% CO2 incubator. T cell proliferation was measured by flow cytometry analysis of CFSE dilution in CD8+ T cells.

[0166] Treg proliferation assay Spleens from C57BL / 6-Foxp3tm1Flv / J (H-2b) mice were gently dissociated into single-cell suspensions, and red blood cells were removed using hypotonic ACK lysis buffer. Splenocytes were stained with anti-CD4 mAb for 30 minutes on ice. Responder CD4+ cells were sorted to >98% purity using a FACS Aria II (BD Biosciences). CD4+ T cells were stimulated with anti-CD3 / CD28 microbeads. Stimulated CD4+ T cells were cultured with graft-infiltrating Ly-6Clo macrophages, mTORi-HDL, or placebo for 72 hours at 37°C in a 5% CO2 incubator. Treg proliferation was measured by flow cytometry analysis of Foxp3-RFP in CD4+ T cells.

[0167] Enzyme-linked immunosorbent assay (ELISA) Bone marrow-derived macrophages were trained as described above. Graft-infiltrating macrophages were isolated as described above. TNF-α and IL-6 cytokines produced by in vitro-trained and graft-infiltrating macrophages were assessed by ELISA (R&D Systems) according to the manufacturer's protocol.

[0168] Microarray analysis Graft-infiltrating recipient Ly-6Clo macrophages were sorted from mTORi-HDL-treated and placebo-rejecting recipients on day 6 after transplantation. To achieve >98% purity, cells were sorted twice using a FACS Aria II sorter (BD Biosciences). Microarray analysis of sorted cells was performed using a total of six Affymetrix Mouse Exon GeneChip 2.0 arrays (Thermo Fisher Scientific), with each sample run in triplicate. Raw CEL file data were normalized using Affymetrix Expression Console software. Gene expression was filtered based on an IQR (0.25) filter using the gene filter package. Log2-normalized and filtered data (adjusted to P < 0.05) were used for further analysis. Gene signatures were compared between Ly6Clo macrophages in grafts from mTORi-HDL-treated recipients and placebo-treated recipients. GSEA was performed using GSEA version 17 from Gene pattern version 3.9.6. The parameters used for the analysis were as follows: Gene sets c2.cp.biocarta.v5.1.symbols.gmt; c2.cp.kegg.v5.1.symbols.gmt; c2.cp.reactome.v5.1.symbols.gmt; c6.all.v5.1.symbols.gmt (Oncogenic Signatures); c7.all.v5.1.symbols.gmt (Immunologic Signatures), and h.all.v5.1.symbols.gmt (Hallmarks) were used to run GSEA. An fdr q value of 0.25 was used as a cutoff to select significant pathways from each gene set result. Only genes that contribute to core enrichment were considered.

[0169] Macrophage loss in vivo To deplete CD169-expressing Ly-6Clo macrophages, heterozygous CD169-DTR recipients were intraperitoneally injected with 10 ng / g body weight of DT (Sigma-Aldrich) at 24, 48, and 72 hours after transplantation.

[0170] Quantification and statistical analysis statistical analysis Results are expressed as mean ± SEM. Statistical comparisons between two groups were assessed using the Mann-Whitney test or the Wilcoxon signed-rank test for paired measurements. Comparisons between three or more groups were analyzed using the Kruskal-Wallis test followed by Dunn's multiple comparison test. Kaplan-Meier curves were plotted for allograft survival analysis, and differences between groups were assessed using the log-rank test. A P value of 0.05 or less was considered statistically significant. GraphPad Prism 7 was used for statistical analysis.

[0171] Data and Software Availability The microarray data discussed in this publication have been deposited at NCBI, GEO Series accession number GSE119370: https: / / urldefense.proofpoint.com / v2 / url?u=https-3A_www.ncbi.nlm.nih.gov_geo_query_acc.cgi-3Facc-3DGSE119370&d=DwIEAg&c =shNJtf5dKgNcPZ6Yh64b-A&r=UQzd7yXCG-7V6o6EdZSeY_KvCshJgQzt0LAtZPqCh9Q&m=cuA3YUXFJvxExRDD8AweBNKmcjdYXoyMojyj9IZeQf8&s=f1i6P2_K57m-i40hkuoOxGuMsZH_IKcvtAi3C-9QfmQ&e=

[0172] Atherosclerosis Results - Examples 15-17 Example 15 - mTORi-HDL and Monocyte and Macrophage Targeting Referring to Figures 52-61, in addition to the role of monocytes and macrophages, other cell types, including T cells, endothelial cells, and smooth muscle cells, play important roles in the pathogenesis of atherosclerosis. Because mTOR signaling is broadly cell-associated, systemic mTOR inhibition would affect all cell types involved in atherogenesis. The present inventors specifically investigated the effects of inhibiting the mTOR pathway in monocytes and macrophages. To achieve this, the present inventors developed an HDL-based nanobiology composition that facilitates drug delivery to monocytes and macrophages with remarkable targeting efficiency.

[0173] mTORi-HDL was constructed from human apolipoprotein AI (apoA-I) and the phospholipids 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), and incorporated with the mTOR inhibitor rapamycin (Figure 52). mTORi-HDL measured 23 nm ± 9 nm (PDI = 0.3) as determined by dynamic light scattering. mTORi-HDL variants incorporating fluorescent dyes (DiO or DiR) were synthesized for detection by fluorescence techniques. Ex vivo near-infrared fluorescence (NIRF) imaging performed 24 hours after intravenous administration showed that DiR-labeled mTORi-HDL accumulated primarily in the liver, spleen, and kidney of Apoe- / - mice. Significant DiR uptake was observed in the aortic sinus region, the site of preferential plaque development in this mouse model (FIG. 53).

[0174] Cell specificity was assessed by flow cytometry. For this purpose, DiO-labeled mTORi-HDL was formulated and intravenously injected. We observed that DiO-labeled mTORi-HDL was taken up by 91% of macrophages and 93% of Ly6Chi monocytes present in the aorta. Furthermore, we found that 50% of dendritic cells and 73% of neutrophils contained the mTORi-HDL nanobiology composition (Figure 54). Minimal to negligible mTORi-HDL uptake was observed in non-myeloid (Lin+) cells. These results mirror our findings in blood, spleen, and bone marrow, demonstrating that myeloid cells, particularly Ly6Chi monocytes and macrophages, exhibit significant mTORi-HDL uptake.

[0175] Example 16 - mTORi-HDL reduces plaque inflammation. To evaluate the effect of mTORi-HDL on plaque inflammation, we used 20-week-old Apoe- / - mice that had been fed a high-cholesterol diet for 12 weeks to develop atherosclerotic lesions. While still on the high-cholesterol diet, all mice were treated with four intravenous injections of PBS (control, n = 7) or mTORi-HDL (containing 5 mg / kg rapamycin, n = 10) for 1 week. Twenty-four hours after the final injection, mice were euthanized. Quantitative histological analysis of plaques in the aortic sinus region showed no differences in plaque size or collagen content compared with controls (Figure 55). We observed a 33% (P = 0.02) decrease in plaque macrophage content. The ratio of Mac3 to collagen in plaques was reduced by 35% (P = 0.004), indicating a more stable plaque phenotype in the mTORi-HDL group (Figure 55).

[0176] Next, we performed fluorescent molecular tomography (FMT-CT) to visualize protease activity in the aortic root region. We used the same mouse model and treatment regimen described above. Twenty-four hours before imaging, control mice (n = 8) and Apoe- / - mice treated with mTORi-HDL (n = 10) received a single injection of an activatable pan-cathepsin protease sensor. This protease sensor is internalized by activated macrophages, cleaved in endolysosomes, and fluorescence is recovered as a function of enzymatic activity. mTORi-HDL reduced protease activity by 30% (P = 0.03, Figure 58). Together, these data provided clear evidence that inhibition of the mTOR signaling pathway in monocytes and macrophages results in a rapid reduction in atherosclerotic inflammatory activity. This motivated us to elucidate the mechanism by which this occurs.

[0177] Example 17 - S6K1i-HDL and targeting of plaque monocytes and macrophages To understand the mechanism by which the mTOR signaling pathway controls monocyte and macrophage dynamics in atherosclerosis, we focused on the mTOR-S6K1 (S6K1: ribosomal protein S6 kinase β-1) signaling system. Although S6K1 signaling is known to regulate fundamental cellular processes, including transcription, translation, cell proliferation, and cellular metabolism, little is known about its role in controlling the innate immune response in atherosclerosis. To this end, we constructed an HDL nanobiology composition (S6K1i-HDL) containing a specific S6K1 inhibitor, PF-4708671 (Figure 59). This nanobiology composition was constructed from human apolipoprotein AI (apoA-I) and the phospholipids 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), incorporating PF-4708671 (Figure 59). S6K1i-HDL measured 34 nm ± 10 nm (PDI = 0.3) as determined by dynamic light scattering.

[0178] Ex vivo near-infrared fluorescence (NIRF) imaging performed 24 hours after injection into Apoe- / - mice showed that DiR-labeled S6K1i-HDL accumulated primarily in the liver, spleen, and kidney (Figure 60). Furthermore, significant DiR uptake was observed in the aortic sinus region (Figure 60), which is very similar to what we found with mTORi-HDL. Cell specificity was analyzed by flow cytometry of whole aortas using DiO-labeled S6K1i-HDL (Figure 61). The percentages of DiO-positive cells were 87% for macrophages, 84% for Ly6Chi monocytes, 64% for dendritic cells, and 71% for neutrophils (Figure 61). Uptake by non-myeloid (Lin+) cells was negligible. These results demonstrate that the nature of the nanobiological composition is independent of the therapeutic payload, allowing us to specifically test mTOR and S6K1 inhibition in atherosclerosis. One week of S6K1i-HDL treatment showed a similar trend in reducing plaque inflammation compared to mTORi-HDL (Figure 62).

[0179] Next, in vitro experiments were performed on adherent human monocytes in which trained immunity was induced with oxLDL as previously described (Bekkering et al., 2018). We investigated whether treatment with the mTORi-HDL and S6K1i-HDL nanobiological compositions inhibited oxLDL-induced trained immunity. Indeed, we found a decrease in cytokine production upon restimulation with lipopolysaccharide (LPS) via TLR-4 and TLR-2 (Figure 63).

[0180] Example 18 - Atherosclerosis Overview and Discussion Monocytes and macrophages constitute important components of the host defense mechanism. Upon recognition of a foreign pathogen, these phagocytes become activated and initiate an inflammatory response to resolve the infection. Sterile substances can also be perceived as danger signals and stimulate an inflammatory response. This can be appropriate in some cases, but equally maladaptive, for example in atherosclerosis.

[0181] Oxidized low-density lipoprotein cholesterol (oxLDL) and cholesterol crystals are major stimuli for the pathogenic innate immune response in atherosclerosis. OxLDL induces transcriptional reprogramming of granulocyte-monocyte precursor cells and stimulates the production and release of bone marrow-derived proinflammatory monocytes. This increases the recruitment of inflammatory monocytes into the plaque, where they differentiate into macrophages. Importantly, plaque inflammation is sustained in part by localized proliferation of macrophages.

[0182] OxLDL and cholesterol crystals have also been implicated in the activation of macrophage inflammation. OxLDL cholesterol can stimulate macrophages through activation of a signaling complex formed by the heterodimer of Toll-like receptor 4 (TLR4) and TLR6 together with scavenger receptor class B member 1 (SRB1), which activates nuclear factor kappa B (NF-κB). Cholesterol crystals induce activation of the NLRP3 inflammasome by damaging phagolysosomes in macrophages.

[0183] Another mechanism by which cholesterol stimulates ongoing innate immune cell activation in atherosclerosis is "trained immunity." Trained immunity, also known as innate immune memory, induces nonspecific immune memory constructed through epigenetic modifications. This process can be induced by oxLDL and results in a macrophage phenotype characterized by a long-lasting pro-inflammatory response. oxLDL-induced trained immunity is mediated through activation of the NLRP3 inflammasome. Thus, trained immunity contributes to the persistence of inflammatory activity in atherosclerosis.

[0184] The epigenetic reprogramming of myeloid cells that occurs during trained immunity is associated with significant alterations in cellular machinery. A metabolic shift toward aerobic glycolysis induces trained immunity. Not only glucose metabolism but also other metabolic pathways, particularly glutaminolysis and cholesterol synthesis, are involved. Interestingly, the induction of trained immunity through either of these metabolic pathways depends on activation of the target of rapamycin (mTOR), making it a compelling target for preventing trained immunity. The mTOR signaling pathway plays a critical role in innate immune cell function by acting as an intrinsic sensor of cellular nutritional status and metabolically coordinating macrophage inflammatory activity.

[0185] The effects of inhibiting the mTOR signaling pathway in atherosclerotic monocytes and macrophages were investigated in apolipoprotein E-deficient (Apoe- / -) mice, focusing on the mTOR-S6K1 system. To achieve specific inhibition in myeloid cells, we intravenously administered two different high-density lipoprotein (HDL) nanobiology compositions incorporating mTOR or S6K1, respectively. We observed a rapid reduction in plaque inflammation through a combination of reduced macrophage proliferation and inflammatory activity.

[0186] The mTOR signaling network is important for balancing anabolism and catabolism in response to nutritional status in all eukaryotic cells. It plays a major role in controlling cell activity, growth, and division. In this study, we provide evidence for a mechanistic framework in which mTOR and S6K1 signaling influence mononuclear phagocyte proliferation and inflammatory activity in atherosclerosis, an energy-demanding process.

[0187] As claimed and discussed, the inventors demonstrate that cell-specific inhibition of mTOR and S6K1 achieved through the use of HDL nanobiology compositions rapidly suppresses plaque inflammation. The inventors observed this as a result of reduced local macrophage proliferation and suppression of the inflammatory state. Transcriptome analysis of monocytes and macrophages isolated from plaques revealed key cellular processes affected by mTOR and S6K1 inhibition. These included processes related to cell growth and proliferation, metabolism, and phagocytic function.

[0188] Tissue macrophages can self-sustain through local proliferation. This self-renewal ability contributes significantly to the proliferation of macrophages in advanced plaques. Our data demonstrate that pharmacological inhibition of macrophage proliferation by blocking mTOR and S6K1 signaling resulted in a rapid reduction in plaque inflammation.

[0189] Transcriptome analysis revealed alterations in the expression of genes related to pathways regulating transcription and translation, as well as cell growth and division. Our findings are similar to those made in other activated macrophages. In a mouse model of helminth-induced infection in which macrophage activation is preferentially induced by interleukin 4 (IL-4), massive localized proliferation of macrophages was observed. It was subsequently shown that the IL-4 receptor targets the phosphatidylinositide 3-kinase (PI3K)-Akt signaling pathway, which contributes to IL-4-induced proliferation. Because the PI3K-Akt pathway directly regulates mTOR activation, mTOR was likely involved in mediating these effects.

[0190] In addition to their effects on proliferation, we observed that mTORi-HDL and S6K1i-HDL distracted myeloid cells from initiating innate immune memory responses. While the dependence of trained immunity on mTOR activation has been firmly established, our data reveal that this also applies to S6K1 signaling. Interestingly, however, S6K1 is not simply a downstream target of mTOR, as this ribosomal protein can inhibit the phosphorylation of insulin receptor substrate 1 (IRS1). This allows S6K1 to suppress insulin-like growth factor 1 receptor (IGFR) and phosphatidylinositide 3-kinase (PI3K)-Akt signaling, both of which are upstream in the regulation of mTOR.

[0191] The epigenetic reprogramming that occurs in trained immunity is closely associated with significant changes in cellular metabolism: in vitro, trained monocytes switch to aerobic glycolysis, presumably to prepare them for metabolic requirements after reactivation.

[0192] Metabolic changes influence epigenetic processes, and it is clear that metabolites such as acetyl-coenzyme A, succinate, and α-ketoglutarate can directly affect histone acetylation and methylation. In this context, it is interesting to note that we observed a significant downregulation of oxidative phosphorylation. This may place macrophages in a state of low ATP production, as mTOR-S6K1 inhibition is known to also suppress glycolysis. This low energy state would negatively impact macrophage performance in orchestrating inflammatory responses. The extent to which this metabolic reprogramming influences trained immunity has not been investigated here and is beyond the scope of the current study.

[0193] Atherosclerosis is a lipid-driven inflammatory disease that elicits a complex immune response, and macrophages are thought to be a major contributor to this. The data we present in this study provide novel insights into the pathogenesis of this disease by demonstrating that mTOR signaling underlies the chronic, maladaptive inflammatory response of macrophages. Both the activation of inflammation, a form of trained immunity, and macrophage proliferation have been shown to be under the auspices of the mTOR signaling network. These novel mechanistic insights offer novel therapeutic opportunities to alleviate the dysfunction of the innate immune response in atherosclerosis.

[0194] Example 19 - Atherosclerosis Materials and Methods mouse Female Apoe- / - mice (B6.129P2-Apoetm1Unc) were used in this study. Animal care and procedures were in accordance with institutional protocols approved by the Icahn School of Medicine at Mount Sinai. Eight-week-old Apoe- / - mice were purchased from The Jackson Laboratory. All mice were fed a high-cholesterol diet (0.2% cholesterol by weight; 15.2 kcal% protein, 42.7 kcal% carbohydrate, 42.0 kcal% fat; Harlan TD. 88137) for 12 weeks. Littermates were randomly assigned to treatment groups.

[0195] In vitro experiments were performed with either the RAW264.7 cell line or bone marrow-derived macrophages (BMDM). RAW264.7 cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) (Gibco Life Technologies) in T-shaped 75 cm flasks (Falcon). BMDM were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 15% L929 cell-conditioned medium in cell culture dishes. All cells were incubated at 37°C in a 5% CO2 atmosphere.

[0196] Human subjects For in vitro studies on human monocytes, buffy coats were obtained from healthy donors after written informed consent (Sanquin blood bank, Nijmegen, The Netherlands). For histological analysis, human atherosclerotic plaque samples were obtained from four patients. All four patients underwent carotid endarterectomy. Although the sex of subjects included in both studies was known, gender associations could not be analyzed due to small group sizes. Allocation of subjects to groups was not applicable.

[0197] Synthesis of nanobiological compositions rHDL nanobiology formulations were synthesized as described herein. For mTORi-HDL, the mTORC1 complex inhibitor rapamycin (3 mg, 3.3 μmol) was combined with 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) (6 mg, 12.8 μmol) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (18 mg, 26.6 μmol) (Avanti Polar Fipids). For S6Ki-HDL, the S6K1 inhibitor PF-4708671 (1.5 mg, 4.6 μmol) was combined with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (18 mg, 23.7 μmol) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC) (6 mg, 12.1 μmol). The compound and lipids were dissolved in methanol and chloroform, mixed, and then dried under vacuum to form a thin lipid film. A PBS solution of human apolipoprotein A1 (apoA-I) (4.8 mg in 5 ml) was added to the lipid film. The mixture was incubated in an ice-cooled sonication bath for 15–30 min. The solution was then sonicated for 20 min at 0°C using a tip sonicator to form the rHDL-based nanobiology composition. The resulting solution was concentrated by centrifugal filtration using a 100 MWCO Vivaspin tube at 3000 rpm to obtain a volume of approximately 1 ml. PBS (5 ml) was added, and the solution was concentrated to approximately 1 ml. PBS (5 ml) was added again, and the solution was concentrated to approximately 1 ml. The remaining solution was filtered through a 0.22 μm PES syringe filter to obtain the final nanobiology composition solution. For targeting and biodistribution experiments, analogs of mTORi-HDL and S6K1i-HDL were prepared by incorporating the fluorescent dyes DiR or DiO (Invitrogen).

[0198] Treatment with Nanobiological Compositions Twenty-week-old Apoe- / - mice were administered either PBS, empty rHDL nanobiology composition, mTORi-HDL (5 mg / kg mTORi), or S6Ki-HDL (5 mg / kg S6K1i) via unilateral tail vein injection.

[0199] Mice were treated with four injections over a seven-day period while on a high-cholesterol diet. For targeting and biodistribution studies, mice received a single intravenous injection. All animals were euthanized 24 hours after the last injection.

[0200] Fluorescence molecular tomography / X-ray computed tomography After treatment with the nanobiological composition, mice were injected with 5 nmol of a pan-cathepsin protease sensor (ProSense 680, PerkinElmer, Cat no. NEV10003). Twenty-four hours later, animals were placed in a custom-made cartridge and sedated for imaging using continuous isoflurane administration as previously described (ref). Animals were first scanned using a high-resolution CT scanner (Inveon PET-CT, Siemens) with continuous infusion of CT contrast agent (Isovue-370, Bracco Diagnostics) at a rate of 55 μL / min via the tail vein catheter. Animals were then scanned in the same cartridge using an FMT scanner (PerkinElmer). The CT X-ray source was operated at 80 kVp and 500 mA with an exposure time of 370–400 ms. High-resolution contrast-enhanced CT images were used to localize the aortic root and were used to guide the location of volumes of interest for quantitative FMT protease activity maps. Image fusion relied on registration markers. Image fusion and analysis were performed using OsiriX v.6.5.2 (The Osirix Foundation, Geneva).

[0201] Near-infrared fluorescence imaging Mice received a single intravenous injection of DiR (0.5 mg / kg)-labeled mTORi-HDL (5 mg / kg) or S6K1i-HDL (5 mg / kg). Liver, spleen, lung, kidney, heart, and muscle tissues were collected for NIRF imaging. Fluorescence images were acquired using an IVIS 200 system (Xenogen) with a 2-second exposure time and a 745 nm excitation filter and an 820 nm emission filter. ROIs were generated for each tissue using software provided by the supplier, and quantitative analysis was then performed using the average radiant efficiency within these ROIs.

[0202] Preparation of single-cell suspensions Blood was collected by cardiac puncture, and the mice were then perfused with 20 mL of chilled PBS. The spleen and femur were collected. The aorta was gently cleaned of fat from the aortic root to the iliac bifurcation and collected. The aorta was digested for 60 min at 37 °C using an enzymatic digestion solution containing Liberase TH (4 U / ml) (Roche), deoxyribonuclease (DNase) I (40 U / ml) (Sigma-Aldrich), and hyaluronidase (60 U / ml) (Sigma-Aldrich) in PBS. Cells were filtered through a 70 μm cell strainer and washed with serum-containing medium. Blood was incubated with lysis buffer for 4 min and washed with serum-containing medium. The spleen was mashed, filtered through a 70 μm cell strainer, incubated with lysis buffer for 4 min, and washed with serum-containing medium. Bone marrow was flushed from femurs with PBS, filtered through a 70 μm cell strainer, incubated with lysis buffer for 30 seconds, and washed with serum-containing medium.

[0203] Flow cytometry Single-cell suspensions were stained with the following monoclonal antibodies: anti-CD11b (clone M1 / 70), anti-F4 / 80 (clone BM8); anti-CD11c (clone N418), anti-CD45 (clone 30-F11), anti-Ly6C (clone AL-21), and a lineage cocktail (Lin) containing anti-CD90.2 (clone 53-2.1), anti-Ter119 (clone TER119), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-CD45R (clone RA3-6B2), and anti-Ly6G (clone 1A8). The contribution of newly generated cells to the different populations was determined by in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU). The anti-BrdU antibody was used according to the manufacturer's protocol (BD APC-BrdU Kit). Macrophages were identified as CD45+, CD11bhi, Lin- / low, CD11clo, and F4 / 80hi. Ly6Chi monocytes were identified as CD45+, CD11bhi, Lin- / low, CD11clo, and Ly6Chi. Data were acquired using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo v10.0.7 (Tree Star).

[0204] Histology and immunohistochemistry Tissues for histological analysis were harvested, fixed in formalin, and embedded in paraffin. Mouse aortic roots were sectioned into 4-μm sections, resulting in a total of 90–100 cross sections per aortic root. Eight cross sections were stained with hematoxylin and eosin (H&E) and used to measure atherosclerotic plaque size. Sirius red staining was used to analyze collagen content. For immunohistochemical staining, mouse aortic root and human carotid endarterectomy (CEA) sections were deparaffinized, blocked with 4% FCS in PBS for 30 minutes, and incubated in antigen retrieval solution (DAKO) at 95°C for 10 minutes. Mouse aortic root sections were immunostained with rat anti-mouse Mac3 monoclonal antibody (1:30, BD Biosciences). Prosaposin in both mouse aortic root and CEA samples was stained using a rabbit anti-human prosaposin primary antibody (1:500, Abcam) in combination with a biotinylated goat anti-rabbit secondary antibody (1:300, DAKO). Macrophages in CEA samples were stained using a donkey anti-mouse CD68 primary antibody (1:300, Abcam) in combination with a biotinylated donkey anti-mouse secondary antibody (1:300; Jackson ImmunoResearch). Antibody staining was visualized with either Impact AMEC red (Vectorlabs) or diaminobenzylidene (DAB). Sections were analyzed using a Leica DM6000 microscope (Leica Microsystems) or a VENTANA iScan HT slide scanner (Ventana).

[0205] Laser Capture Microdissection Laser capture microdissection was performed on 24 aortic root sections (6 μm). Frozen sections were dehydrated in graded ethanol solutions (70% twice, 95% twice, and 100% once), washed in diethylpyrocarbonate (DEPC)-treated water, stained with Mayer's H&E, and cleared in xylene. One out of every eight sections was used for CD68 staining (Abd Serotec, 1:250 dilution) to guide laser capture microdissection. CD68-rich regions within the plaque were identified and retrieved using the ArcturusXT LCM System.

[0206] RNA sequencing CD68+ cells collected by laser capture microdissection were used for RNA isolation (PicoPure RNA Isolation Kit, Arcturus), followed by RNA amplification and cDNA preparation according to the manufacturer's protocol (Ovation Pico WTA System, NuGEN). The quality and concentration of the collected samples were measured using an Agilent 2100 Bioanalyzer. For RNA sequencing, paired-end libraries were prepared and validated. The purity, fragment size, yield, and concentration were determined. During cluster generation, library molecules were hybridized on an Illumina flow cell. Hybridized molecules were then amplified using bridge amplification, resulting in a heterogeneous population of clusters. This dataset was obtained using an Ilumina HiSeq 2500 sequencer.

[0207] Cell proliferation ELISA To quantify cell proliferation, a colorimetric immunoassay based on the incorporation of BrdU during DNA synthesis was used (Roche, Switzerland). RAW264.7 cells were seeded at 2.5 × 10 cells per well in 96-well clear flat-bottom culture plates (Falcon) and allowed to adhere overnight. The adherent cells were incubated with either mTORi or S6K1i for 24 hours. After incubation, BrdU labeling solution was added to each well (1:1000) and incubated at 37°C for 2 hours. Cells were fixed and incubated with anti-BrdU POD for 1.5 hours according to the manufacturer's instructions. After adding the substrate solution, the absorbance of the samples was measured at 450 nm using a GloMax-Multi+ plate reader (Promega).

[0208] Metabolic extracellular flux analysis BMDMs were plated at 2.5 × 10 cells / well on XF-96 cell culture plates (Seahorse Bioscience) and allowed to adhere. BMDMs were incubated with either mTORi or S6K1i for 16 hours. Oxygen consumption rate (OCR) was measured using an XL-96 Flux Analyzer (Seahorse Bioscience). Responses to the addition of oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone were used to calculate all respiratory characteristics. Upon completion, DNA content was measured using CyQuant to compensate for differences in cell number.

[0209] Preparation of oxidized LDL LDL was isolated from serum from healthy volunteers using KBr-density gradient ultracentrifugation. Plasma density was adjusted to d = 1.100 g / mL with KBr. Samples were centrifuged at 32,000 rpm for 22 hours in an SW41 Ti rotor. Oxidized LDL was prepared by incubating LDL with 20 μmol CuSO4 / L in a shaking water bath at 37°C for 15 minutes, as previously described (Tits et al., 2011).

[0210] Isolation of human PBMCs and monocytes PBMC isolation was performed by diluting blood with pyrogen-free PBS and performing differential density centrifugation on Ficoll-Paque. Cells were washed three times with PBS. Percoll isolation of monocytes was performed as previously described (Repnik et al., 2003). Briefly, 150–200 × 10 PBMCs were placed on a hyperosmolar Percoll solution (48.5% Percoll, 41.5% sterile water, 0.16 M filter-sterilized NaCl) and centrifuged at 580 g for 15 min. The interphase layer was isolated, and cells were washed once with chilled PBS. Cells were resuspended in RPMI culture medium supplemented with 50 μg / ml gentamicin, 2 mM glutamax, and 1 mM pyruvate and counted using a Beckman Coulter counter. A further purification step was added by allowing Percoll-isolated monocytes to adhere to polystyrene flat-bottom plates (Corning, NY, USA) for 1 hour at 37°C, followed by a washing step with warmed PBS to obtain maximum purity (this increases purity to only 3% T cell contamination, as described in Bekkering et al., 2016).

[0211] Monocyte training and inhibition experiments Human monocytes were trained as previously described (Bekkering et al., 2016). Briefly, 100,000 cells were added to a flat-bottom 96-well plate. After washing with warmed PBS, monocytes were incubated for 24 hours (in 10% pooled human serum) with either culture medium alone as a negative control, 2 μg / mL β-glucan, 10 μg / mL oxLDL, or 10–5000 ng / mL prosaposin. Cells were washed once with 200 μL of warmed PBS and incubated for 5 days in culture medium containing 10% pooled human serum, with one medium change. Cells were restimulated with 200 μL of RPMI, LPS (10 ng / mL), or Pam3Cys (10 μg / mL). After 24 hours, supernatants were collected and stored at -20°C until cytokine measurements. In some experiments, cells were preincubated with nanobiology compositions (rHDL as a control, 10 μM mTORi-HDL, or 0.1 μM S6K1i-HDL) for 1 hour (prior to oxLDL training). Training stimuli were added to the cells and inhibitors after 1 hour, and the inhibitors were left in place for the remainder of the training period. After 24 hours, both stimuli and inhibitors were washed out, and the cells were allowed to rest for 5 days as described above.

[0212] Cytokine and lactate measurements Cytokine production was determined in the supernatants using commercially available ELISA kits for human TNFα and IL-6 according to the manufacturer's instructions.

[0213] RNA isolation and qPCR For qRT-PCR, monocytes were trained as described above, but the amount of cells required for RNA extraction was adjusted. 500,000 cells / well were seeded in duplicate in 24-well plates. On days 0 (after 1 hour of adhesion and washing), 1 (after training and washing), 2, 3, and 6, the supernatant was removed and the cells were stored in TRIzol reagent. Total RNA was purified according to the manufacturer's instructions. RNA concentration was measured using NanoDrop software, and the isolated RNA was reverse transcribed using the iScript cDNA Synthesis Kit according to the manufacturer's instructions. qPCR was performed using the Sybr Green method. The genes measured were 18S and prosaposin. Samples were analyzed by a quantification method with efficiency correction, and 18S was used as a housekeeping gene. The relative mRNA expression value of the unstimulated sample on day 0 was used as a reference.

[0214] Quantification and statistical analysis RNA sequencing analysis Paired-end sequencing reads were assigned to the human genome hg19 using the TopHat aligner (bowtie2) (Langmead and Salzberg, 2012). Next, HTSeq (Anders et al., 2015) was used to quantify gene expression at the gene level based on GENCODE gene model release 22 (Mudge and Harrow, 2015). Raw read counts for gene expression were normalized as counts per million using the trimmed mean of M-values ​​normalization method to adjust for differences in sequencing library size between samples. DE genes between drug treatments and controls were identified using the Bioconductor package limma (Ritchie et al., 2015). To correct for multiple testing issues, limma was used to calculate random sample statistics and P values ​​after label permutation. This procedure was repeated 1,000 times to obtain a t-statistic of 0 and a distribution of P values ​​to estimate the false positive rate (FDR) value for all genes. DE genes in cells isolated from aortic plaques were identified using a cutoff with a corrected P value of less than 0.2. DE genes in RAW264.7 cells were identified using a cutoff with a corrected P value of less than 0.05. A weighted gene co-expression analysis was constructed to identify groups (modules) of genes involved in various activated pathways according to a previously described algorithm (Zhang and Horvath, 2005). Briefly, Pearson correlations were computed between each pair of genes to retrieve a similarity (correlation) matrix (sij). The power function

number

[0215] statistical analysis Results of in vivo experiments were expressed as mean ± SD. The significance of differences was calculated using the nonparametric Mann-Whitney U test and Kruskal-Wallis test.

[0216] In vitro human monocyte experiments were performed at least six times, and normality was confirmed using visual analysis of histograms and box plots, as well as normality assays using Graphpad Prism. Nonparametric parameters were analyzed using two-tailed Wilcoxon signed-rank tests. Data are presented as mean ± SEM.

[0217] A p-value of less than 0.05 was considered statistically significant. All data were analyzed using Graphpad Prism 5.0 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0218] Example 20 - Prodrugs - General Materials and Methods All chemicals were purchased from Sigma-Aldrich, Medchem Express, or Selleckchem. PES syringe filters were obtained from Celltreat. A World Precision Instrument NE-1002X model microfluidic pump was used in combination with a Microfluidic-chipshop Zeonor herringbone mixer (#14-1038-0187-05). Particles were purified using a 100 kDa MWCO 20 mL Vivaspin centrifugal filter. Dialysis bags were from Thermo Scientific. ApoA-I protein was purified in-house using a literature method. Spectroscopic quantification of ApoA-I was performed on a BioTek Cytation 3 imaging plate reader using the Bradfort assay. DLS and zeta potential measurements were performed on a Brookhaven Instrument Corporation ZetaPals analyzer. The mean of the number distribution was used to determine particle size. 1 H and 13 C NMR samples were analyzed using a Bruker 600 ultrashield magnet coupled to a Bruker advance 600 console, and data were processed using Topspin version 3.5 pl 7.

[0219] Quantitative analysis of all drugs except dimethyl malonate and its derivatives was performed using C 18HPLC analysis was performed using a Shimadzu UFLC instrument equipped with either a 1000-milliliter (1000-milliliter) or 1000-milliliter (1000-milliliter) column. Acetonitrile and water were used as the mobile phase, and compounds were detected with an SPD-M20a diode array detector. Dimethyl malonate was analyzed using an Agilent tech 5977B MSD 7890B GC-MS equipped with an HP5MS 30 m, 0.25 mm, 0.25 μm column. Aliphatic and cholesterol-derivatized malonates were analyzed using a Waters acquity UPC2 SFC-MS using an isopropanol / water mixture as the mobile phase and a 1-aminoanthracene column. Radiolabeling of nanoparticles was performed using a method previously reported by the inventors.

[0220] Example 21 - Synthesis of Prodrugs - Malonate Derivatives [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ethyl malonate Cholesterol (194 mg, 0.50 mmol) was dissolved in DCM (30 mL), pyridine (60 μL, 0.75 mmol) was added, and the mixture was cooled to 0 °C. Ethyl 3-chloro-3-oxopropanoate (80 μL, 0.75 mmol) was added in small portions, and the mixture was stirred at 0 °C for 2 h, warmed to room temperature, and stirred for an additional 16 h. Water (60 mL) was added, the phases were separated, and the aqueous phase was washed twice with DCM (50 mL). The combined organic fractions were dried under vacuum using MgSO4. The crude product was purified using column chromatography (hexane:ethyl acetate 1:1) to recover the product as a yellowish solid. Yield: 243 mg, 49 mmol. η = 97%. 1H NMR(600MHz,CDCl3)δ=5.41(br,1H),4.69(m,1H),4.22(q,J=7.1Hz,2H),3.37(s,2H),2.37(m,2H),2.1-1.1( m,26H),1.30(t,J=7.2Hz,3H),1.03(s,3H),0.92(d,J=6.5Hz,3H),0.87(dd,J=6.5,2.6Hz,6H),0.69(s,3H). 13 C NMR(150MHz,CDCl3)δ=166.88,166.20,139.52,123.07,75.40,61.61,56.85,56.30,50.17,42.48,42.16,39.89,39.70,38.05,37 .09,36.74,36.36,35.97,32.07,32.02,28.41,28.19,27.76,24.46,24.01,23.01,22.75,21.21,19.48,18.90,14.28,12.04.Mass calc.for C 32 H 52 O4=500.39D,mass found:501.67[M+H + ], 369.63 [fragment with malonate-cholesterol bond separated].

[0221] Example 22 - Synthesis of Prodrug - Ethyl Octadecyl Malonate [ka] 1-Octadecanol (250 mg, 1.08 mmol) was dissolved in dry chloroform (30 mL) at 40 °C, and trimethylamine (165 μL, 119 mmol) was added, followed by ethyl 3-chloro-3-oxopropanoate (140 μL, 1.30 mmol). The mixture was stirred for 2 h, cooled to room temperature, and washed with water (3 × 30 mL). The organic phase was dried under vacuum using MgSO4, and the crude product was purified by column chromatography (3% methanol in chloroform) to recover the product as a yellowish wax. Yield = 314 mg, 0.82 mmol. η = 76%. 1H NMR(600MHz,CDCl3)δ=4.14(q,J=7.2Hz,1H),4.07(t,J=6.7Hz,1H),3.30(s,2H),1 .61-1.44(m,4H),1.36-1.01(m,30H),1.21(t,J=7.2Hz,6H),0.81(t,J=6.8Hz,1H). 13 C NMR(150MHz,CDCl3)δ=166.77,65.84,61.65,41.85,32.10,29.87,29.74,29.68,29.54,29.38,28.63,25.96,22.86,14.28.Mass calc.for C 23 H 44 O4=384.32D, mass found.386[M+H + ],408[M+Na + ].

[0222] Example 23 - Synthesis of Prodrug - GSK-J1-Cholesterol [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-((2-(pyridin-2-yl)-6-(1,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate GSK-J1 (25 mg, 64.2 μmol) was dissolved in dry chloroform (3 mL), EDC.HCl (16.0 mg, 83.3 μmol) and 4-(dimethylamino)pyridine (2.3 mg, 18.8 μmol) were added, and the mixture was stirred for 30 minutes. Cholesterol (27 mg, 69.8 μmol) was added, and the mixture was stirred at room temperature overnight. The mixture was washed with water (3 × 5 mL) and dried under vacuum using MgSO4. The crude product was purified using preparative TLC (6% methanol in chloroform) to give the product as a white solid. Yield = 17.2 mg, 22.7 μmol. η = 35%. 1 H NMR(600MHz,CDCl3)δ=8.75(b,1H),8.45(d,J=7.3,1H),7.83(b,1H),7.36(b,1H) ),7.15(s,4H),5.57(s,1H),5.36(b,1H),4.64(m,1H),3.95(b,4H),3.63(q,J=6. 2Hz,2H),3.03(m,4H),2.65(t,J=6.4,2H),2.33(d,J=7.5Hz,2H),2.1-1.0(m,26 H),1.01(s,3H),0.92(d,J=6.5Hz,3H),0.86(dd,J=6.6,2.7Hz,6H),0.67(s,3H). 13 C NMR(150MHz,CDCl3)δ=171.45,163.60,162.45,161.40,155.17,149.88,140.95,139.68, 137.02,130.19,126.67,124.83,123.74,122.96,79.68,74.77,56.86,56.31,50.18,47. 68,42.49,39.90,39.70,38.29,37.80,37.14,37.07,36.76,36.37,35.97,34.63,32.08, 29.90,28.41,28.20,27.96,24.47,24.01,23.02,22.76,21.21,19.48,18.90,12.04.Mass calc.for C 49 H 67 N5O2=757.53D,mass found.758.77[M+H + ],1516.27[2M+H + ].

[0223] Example 24 - Synthesis of Prodrug - GSK-J1-Octadecyl [ka] Octadecyl 3-((2-(pyridin-2-yl)-6-(1,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate GSK-J1 (20 mg, 51.4 μmol) was dissolved in dry chloroform (3 mL), EDC.HCl (12.8 mg, 66.6 μmol) and 4-(dimethylamino)pyridine (1.8 mg, 14.8 μmol) were added, and the mixture was stirred for 30 minutes. 1-Octadecanol (15.4 mg, 66.6 μmol) was added, and the mixture was stirred at room temperature overnight. The mixture was washed with water (3 × 5 mL) and dried under vacuum using MgSO4. The crude product was purified using preparative TLC (6% methanol in chloroform) to give the product as a white solid. Yield = 19.3 mg, 30.9 μmol. η = 60%. 1 H NMR(600MHz,CDCl3)δ=8.75(s,1H),8.45(d,J=7.7Hz,1H),7.81(t,J=7.1Hz, 1H),7.35(b,1H),7.15(s,4H),5.55(s,1H),5.42(b,1H),4.10(t,J=6.8Hz,2 H),3.95(s,4H),3.63(q,J=6.4Hz,2H),3.05-3.00(m,4H),2.66(t,J=6.6Hz, 2H),1.62(dt,J=14.7,6.8Hz,4H),1.37-1.13(m,28H),0.88(t,J=7.0Hz,3H). 13C NMR(150MHz,CDCl3)δ=172.13,163.74,162.54,156.41,149.39,141.03,136.80,130.17,126.64,124.48,123.60,120.0 7,79.65,65.29,47.64,37.74,37.09,34.36,32.11,29.89,29.79,29.71,29.55,29.46,28.77,26.11,22.88,14.32.Mass calc.for C 40 H 59 N5O2=641.47D,mass found.642.73[M+H+].

[0224] Example 25 - Synthesis of Prodrug - (+)JQ-1 [ka] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (+)-JQ1 (90 mg, 0.20 mmol) was dissolved in 5 mL of 5% TFA in chloroform and stirred at 40° C. for 16 h, after which the solvent was evaporated. Chloroform (5 mL) was added and evaporated under vacuum, which was repeated twice to give the product, which was used without further characterization. Yield = 78 mg, 0.20 mmol. η = >99%.

[0225] Example 26 - Synthesis of prodrug - (+)JQ-1-octadecyl [ka] Octadecyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (78 mg, 0.20 mmol) was dissolved in dry chloroform (5 mL), EDC.HCl (45 mg, 0.23 mmol) and 4-(dimethylamino)pyridine (37 mg, 0.30 mmol) were added, and the mixture was stirred for 30 min. 1-Octadecanol (63 mg, 0.23 mmol) was added, and the mixture was stirred at room temperature for 16 h. The mixture was washed with water (3 × 5 mL) and dried under vacuum using MgSO. The crude product was purified using preparative TLC (6% methanol in chloroform) to give the product as a white wax. Yield = 40 mg, 61 μmol. η = 31%. 1 H NMR(600MHz,CDCl3)δ=7.40(d,J=8.2Hz,2H),7.32(d,J=8.6Hz,2H),4.60(m,1H),4.16(t,J=6.7Hz,2H),3.65-3.59(m,2H) ,2.67(s,3H),2.41(s,3H),1.74(s,3H),1.73-1.62(m,2H),1.39-1.32(m,2H),1.32-1.17(m,28H),0.87(t,J=6.9Hz,3H). 13 C NMR(150MHz,CDCl3)δ=171.87,163.91,155.57,150.05,136.92,136.79,132.45,131.04,130.87,130.54,130.01,128.8 5,65.15,53.99,37.08,32.11,29.89,29.81,29.75,29.55,29.49,28.85,26.13,22.88,14.60,14.32,13.29,12.06.Mass calc.for C 37 H 53 ClN4O2S=652.36D,mass found=653.6[M+H + ].

[0226] Example 27 - Synthesis of Prodrug - (+)JQ-1-Cholesterol [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (75 mg, 0.19 mmol) was dissolved in dry chloroform (5 mL), EDC.HCl (50 mg, 0.26 mmol) and 4-(dimethylamino)pyridine (40 mg, 0.33 mmol) were added, and the mixture was stirred for 30 min. Cholesterol (92 mg, 0.23 mmol) was added, and the mixture was stirred at room temperature for 16 h. The mixture was washed with water (3 × 5 mL) and dried under vacuum using MgSO4. The crude product was purified using preparative TLC (6% methanol in chloroform) to give the product as a white powder. Yield = 30 mg, 39 μmol. η = 21%. 1 H NMR(600MHz, CDCl3)δ=7.40(d,J=8.3Hz,2H),7.32(d,J=8.6Hz 2H),5.36(d,J=4.1Hz,1H),4.69(m,1H),4.60(t,1H),3.59(t,J=6.5Hz,2H),2.67(s,3H),2.41(s,3H),2.36( d,J=6.9Hz,2H),2.1-0.9(m,19H),1.68(s,3H),1.03(s,3H),0.91(d,J=6.5Hz,3H),0.87(m,3H),0.68(s,3H). 13C NMR(150MHz,CDCl3)δ=171.21,163.87,155.58,150.03,139.81,136.91,136.80,132. 47,131.02,130.87,130.54,130.00,128.87,122.84,74.70,56.89,56.32,54.08,50. 23,42.50,39.93,39.70,38.28,37.29,37.22,36.81,36.37,35.97,32.10,32.03,29. 89,28.03,24.47,24.01,23.01,22.75,21.23,19.52,18.91,14.58,13.30,12.05.Mass calc.for C 46 H 61 ClN4O2S=768.42D,mass found=769.82[M+H + ].

[0227] Example 28 - Prodrug Synthesis - Rapamycin Prodrug - C17H35 [ka] Rapamycin-C 18 synthesis Rapamycin (100 mg, 110 μmol) and vinyl stereate (170 mg, 548 μmol) were dissolved in dry toluene (40 mL) and Novozyme 435 (50 mg) was added. The mixture was stirred on a rotavapor under slight vacuum at 45°C for 3 days. Additional toluene was added if necessary. The Novozyme beads were filtered, the solvent evaporated, and the crude product purified using column chromatography (0-6% MeOH in chloroform) to give the pure product. Yield = 108 mg, 89.4 μmol. η = 84%. Conversion was monitored via monitoring the signal corresponding to the proton adjacent to the esterified alcohol group, present at 2.73 ppm and 4.67 ppm in unfunctionalized and functionalized rapamycin, respectively. 1H NMR (600 MHz, CDCl3) was used for monitoring. Mass calculation for C 69 H 113 NO 14 1179.82D,mass found 1131.0[M-OCH3-H2O],1149.0[M-OCH3],1203.0[M+Na + ]D (A similar fragmentation pattern was observed with non-functionalized rapamycin.) Purity was further confirmed by HPLC and TLC.

[0228] Example 29 - Synthesis of approximately 35 nm nanobiological compositions From a 10 mg / ml stock solution in chloroform, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 250 μL), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC, 65 μL), cholesterol (15 μL), tricaprylin (1000 μL), and (pro)drug (65 μL) were combined in a 20 ml vial and dried under vacuum. The resulting film was redissolved in an acetonitrile:methanol mixture (95%:5%, 3 mL total volume). Separately, a solution of ApoA-I protein (0.1 mg / ml) in PBS was prepared. Using a microfluidics setup, both solutions were simultaneously injected into a herringbone mixer at a flow rate of 0.75 ml / min for the lipid solution and 6 ml / min for the ApoA-I solution. The resulting solution was concentrated by centrifugal filtration using a 100 MWCO Vivaspin tube at 4000 rpm to obtain a volume of 5 mL. PBS (5 mL) was added, and the solution was concentrated to 5 mL. PBS (5 mL) was added again, and the solution was concentrated to approximately 3 mL. The remaining solution was filtered through a 0.22 μm PES syringe filter to obtain the final nanobiology composition solution. To obtain the nanobiology composition for FACS measurement, 3,3'-dioctadecyloxacarbocyanine perchlorate (DIO-C) was added. 18 , 0.25 mg) was added to the acetonitrile solution. 89To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to an acetonitrile solution (made in-house). To scale up the synthesis of the nanobiological composition, the above procedure was simply repeated until sufficient quantities were produced.

[0229] For the PF-4708671 drug (S6K1i), less than 1% drug recovery was observed using the above procedure, likely due to its high solubility in water and acetonitrile. To further incorporate this drug into our nanobiology composition library, we integrated it using a sonication method. Here, a film of the same lipid and drug was formed by drying an acetonitrile solution. To this film, 10 mL of PBS containing 2.4 mg of ApoA-I was added, and the solution was sonicated in an ultrasonic bath for 5 minutes. The resulting suspension was then sonicated for 30 minutes at 0°C using a tip sonicator. The resulting clear solution was purified using the same Vivaspin and syringe filter techniques as for the nanobiology compositions generated by microfluidics.

[0230] Example 30 - Synthesis of approximately 15 nm nanobiological compositions For the synthesis of 15 nm nanoparticles, a microfluidic approach similar to that used for 35 nm particles was used. Here, an acetonitrile mixture (again from a 10 mg / mL stock solution) contained POPC (250 μL), PHPC (15 μL), and cholesterol (13 μL). This acetonitrile solution was infused at a rate of 0.75 mL / min. ApoA-I solution (0.1 mg / mL in PBS) was infused at 3 mL / min. To obtain the nanobiology composition for FACS analysis, DiO-C was used. 18 (0.25 mg) was added to the acetonitrile solution. 89 To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to the acetonitrile solution.

[0231] Example 31 - Synthesis of approximately 65 nm nanobiological compositions For the synthesis of 65 nm nanoparticles, a microfluidic approach similar to that used for 35 nm particles was used. Here, an acetonitrile mixture (again from a 10 mg / mL stock solution) contained POPC (250 μL), cholesterol (12 μL), and tricaprylin (1400 μL). This acetonitrile solution was infused at a rate of 0.75 mL / min. ApoA-I solution (0.1 mg / mL in PBS) was infused at 4 mL / min. To obtain the nanobiology composition for FACS analysis, DiO-C was used. 18 (0.25 mg) was added to the acetonitrile solution. 89 To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to the acetonitrile solution.

[0232] Example 32 - Synthesis of approximately 120 nm nanobiological compositions For the synthesis of 120 nm nanoparticles, a microfluidic approach similar to that used for 35 nm particles was used. Here, an acetonitrile mixture (again from a 10 mg / mL stock solution) contained POPC (100 μL), cholesterol (10 μL), and tricaprylin (4000 μL). The acetonitrile solution was infused at a rate of 0.75 mL / min. ApoA-I solution (0.1 mg / mL in PBS) was infused at 1.5 mL / min. To obtain the nanobiology composition for FACS analysis, DiO-C was used. 18 (0.25 mg) was added to the acetonitrile solution. 89 To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to the acetonitrile solution.

[0233] Example 33 - Determination of particle size and dispersity by DLS Aliquots (10 μL) of the final particle solution were dissolved in PBS (1 mL), filtered through a 0.22 μm PES syringe filter, and analyzed by DLS to determine the mean number average size distribution. Samples were then analyzed directly after particle synthesis and on days 2, 4, 6, 8, and 10.

[0234] Figure 64 shows the size and stability of the four different nanoparticles developed. To address the issues with radiolabeling the two larger particles, we are also investigating radiolabeling particles with DFO-functionalized APAO1 instead of the traditionally used DSPE-DFO. Based on the results obtained with DIO-loaded particles and their good reproducibility, we have now turned to 35 nm particles to generate a library of nanobiology compositions.

[0235] Figure 65 shows the average size of each nanobiology composition over the 10-day measurement period, where two different batches were analyzed for each type of particle. The average size over time for all nanobiology compositions is also plotted, showing that these sizes remain constant over time.

[0236] Figure 66 shows the average dispersibility of each nanobiology composition over the 10-day measurement period, where two different batches were analyzed for each type of particle. The average dispersibility over time for all nanobiology compositions is also plotted, showing that these dispersibility values ​​remain constant over time.

[0237] Example 34 - Drug Recovery and Hydrolysis by HPLC The recovery and hydrolysis of the (pro)drug was determined using the following procedure: an aliquot (200 μL) of the particle solution was dried under vacuum, acetonitrile (600 μL) was added, and the suspension was sonicated for 20 min. The suspension was centrifuged to precipitate any solids, and the remaining solution was analyzed using HPLC, except for the malonate derivative, which was analyzed using SFC-MS, and dimethyl malonate, which was analyzed using GC-MS.

[0238] Figure 67 shows the recovery of (pro)drugs in nanobiological compositions. Two batches of each type of nanobiological composition were analyzed in duplicate. This was measured again for in vitro samples.

[0239] Figure 68 shows the hydrolysis of (pro)drugs of nanobiology compositions over time in PBS at 4°C. Rapamycin and C 18 Hydrolysis was observed only in the nanobiological compositions loaded with rapamycin, and in these cases, only hydrolysis of the ester in the macrocycle was observed. Two batches of each type of nanobiological composition were analyzed. Hydrolysis of the nanobiological compositions loaded with dimethyl malonate and PF-4708671 was not determined because these drugs had 0% recovery or contained no biohydrolyzable moieties, respectively.

[0240] Example 35 - Determination of ApoA-I recovery ApoA-I recovery was determined spectrophotometrically using the Bradfort assay. Nanobiological composition solutions (10 μL) and calibration solutions (bare ApoA-I in PBS) were placed in a 96-well plate, Bradfort reagent (150 μL) was added, and the mixture was incubated at room temperature for 5 minutes before measuring absorbance at 544 nm. The average ApoA-I recovery of two different batches of each type of nanobiological composition is plotted. All calibration and analyte samples were prepared in duplicate.

[0241] Figure 69 shows the average ApoA-I recovery for two different batches of each type of nanobiology composition. All calibration and analyte samples were generated in duplicate. We replicate this with samples generated in in vitro experiments. The large error bars are more likely a result of poor reproducibility of the methods used than representing actual differences in ApoA-I recovery.

[0242] Example 36 - Zeta potential determination Samples for zeta potential analysis were prepared by dissolving an aliquot (50 μL) of the final particle solution in MilliQ water (1 mL) and filtering it through a 0.22 μm PES syringe filter. All samples were analyzed in triplicate.

[0243] Figure 70 shows the zeta potential of each type of nanobiology composition in MilliQ water. Samples were analyzed in triplicate. We will repeat this with samples generated in in vitro experiments.

[0244] Example 37 - Determination of drug release under in vivo-like conditions To compare the stability of nanobiological compositions under in vivo-like conditions, nanoparticles were dialyzed in fetal bovine serum at 37°C. The particle solution (0.5 mL) was placed in a 10 kDa dialysis bag and suspended in fetal bovine serum (45 mL) at 37°C. At predetermined time points (0, 15, 30, 60, 120, and 360 minutes after synthesis), aliquots (50 μL) were withdrawn from the dialysis bag. The aliquots were dried under vacuum, acetonitrile (100 μL) was added, and the solution was sonicated for 20 minutes. The remaining suspension was then centrifuged and analyzed by HPLC. This dialysis experiment was performed in duplicate using the same batch of nanobiological composition. The obtained kinetic data were fitted using a bi-exponential decay after removing outliers (shown in red, 5 out of 144 data points) and then normalized using the y-axis intercept of the fit. In some cases, significant amounts of hydrolysis products were observed. We assumed that such hydrolyzed (pro)drugs had not yet diffused out of the dialysis bag but had already leaked out of the nanobiological composition, and for this reason, they were not included in our calculations of the amount of drug retained in the nanobiological composition over time.

[0245] Figure 71 shows that release of unfunctionalized dimethylmalonate, a malonate derivative, from the nanobiology composition provided 0% drug recovery and was therefore not dialyzed. The nanobiology composition in PBS (0.5 mL) was dialyzed against fetal bovine serum (45 mL) at 37°C using a 10 kDa dialysis bag. Experiments were performed in duplicate. The resulting time-dependent drug concentrations were fitted using a biexponential decay and then normalized.

[0246] Figure 72 shows the release of (+)JQ-1 and its derivatives from the nanobiology composition. The nanobiology composition in PBS (0.5 mL) was dialyzed against fetal bovine serum (45 mL) at 37°C using a 10 kDa dialysis bag. Experiments were performed in duplicate. The resulting time-dependent drug concentrations were fitted using a biexponential decay after removing outliers (red) and then normalized.

[0247] Figure 73 shows the release of GSK-J4 and its derivatives from the nanobiology composition. The nanobiology composition in PBS (0.5 mL) was dialyzed against fetal bovine serum (45 mL) at 37°C using a 10 kDa dialysis bag. Experiments were performed in duplicate. The resulting time-dependent drug concentrations were fitted using a biexponential decay after removing outliers (red) and then normalized.

[0248] Figure 74 shows the release of rapamycin and its derivatives from the nanobiology composition. The nanobiology composition in PBS (0.5 mL) was dialyzed against fetal bovine serum (45 mL) at 37°C using a 10 kDa dialysis bag. Experiments were performed in duplicate. The resulting time-dependent drug concentrations could not be adequately fitted using a biexponential decay; instead, the data were fitted according to the data point at 0 min.

[0249] Figure 75 shows the release of PF-4708671 from the nanobiology composition. The nanobiology composition in PBS (0.5 mL) was dialyzed against fetal bovine serum (45 mL) at 37°C using a 10 kDa dialysis bag. Experiments were performed in duplicate. The resulting time-dependent drug concentrations were fitted using a biexponential decay and then normalized.

[0250] Example 38 - Radiolabeling for PET imaging of inhibitor accumulation of trained immunity Referring now to Figure 76, this shows a diagram of the radioisotope labeling process.

[0251] In a non-limiting example, radiopharmaceutical labeling of trained immune inhibitor drugs / molecules can be achieved by using various types of chelators, primarily via three hydroxamic acid groups. 89 This can be achieved via deferoxamine B (DFO), which can form a stable chelate with Zr.

[0252] Generally, a phospholipid is conjugated with a chelating agent compound, the nanobiological composition is prepared with a promoter drug or molecule, and finally, a radioisotope is complexed with the nanobiological composition (which already has a chelating agent attached).

[0253] protocol This protocol is 89 This protocol teaches the modular radiolabeling of the nanobiological compositions described herein with Zr. The protocol describes the synthesis of DSPE-DFO, obtained via the reaction of the phospholipid DSPE and the isothiocyanate derivative of the chelator DFO (p-NCS-Bz-DFO), its formulation into nanobiological compositions, and nanoemulsification, as well as the subsequent dissolution of these nanoformulations. 89 Includes radiolabeling with Zr.

[0254] radioactive isotope 89 Zr was chosen because of its physical decay half-life of 3.3 days, which eliminates the need for a nearby cyclotron and allows for agents to be tested, such as antibodies, to be slowly cleared from the body. Although both are contemplated as workable herein, 89 The relatively low positron energy of Zr is 124 It allows for higher imaging resolution compared to other isotopes such as I.

[0255] Our nanotherapeutic materials 89 Labeling with Zr allows the non-invasive study of events in vivo by positron emission tomography (PET) in patients.

[0256] This protocol includes the following steps: conjugation of the chelator deferoxamine B (DFO) to the phospholipid DSPE to form a lipophilic chelator (DSPE-DFO) that readily integrates in different lipid nanoparticle platforms (approximately 0.5 wt%); L SEP Preparation of nanoscale construct formulations incorporating DSPE-DFO (using sonication, nanoemulsification using hot dripping, or using microfluidics); and In PBS at pH 7 and 30-40°C, 89 This is done by mixing the nanoparticles with Zr-oxide for 30-60 minutes. 89 Labeling of DSPE-DFO-containing lipid nanoparticles with Zr.

[0257] Furthermore, the purification and characterization methods are such that radiochemically pure 89 This method can be used to obtain Zr-labeled lipid nanoparticles. Purification can be typically performed using either centrifugal filtration or a PD-10 desalting column, followed by evaluation using size-exclusion radio-HPLC. Radiochemical yields are typically greater than 80%, and radiochemical purities of greater than 95% are typically obtained.

[0258] The common imaging strategy is PET / CT or PET / MRI. 89 It is used to test the in vivo behavior of Zr-labeled nanobiological compositions.

[0259] FIG. 77 shows PET imaging using a radioisotope delivered by the nanobiology composition, demonstrating accumulation of the nanobiology composition in the bone marrow and spleen in mouse, rabbit, monkey, and pig models.

[0260] Details of the embodiments herein and their various properties and advantages are more fully described with reference to the non-limiting embodiments shown in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as not to unnecessarily obscure the embodiments herein. The examples used herein are intended solely to facilitate understanding of how the embodiments herein can be practiced and to further enable those skilled in the art to practice the embodiments herein. Therefore, these examples should not be construed as limiting the scope of the embodiments herein.

[0261] Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0262] The terminology used herein is merely for the purpose of describing particular embodiments and is not intended to limit the complete scope of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, it is understood that the terms "comprises" and / or "comprising," when used herein, specify the presence of stated features, integers, steps, implementations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, implementations, elements, components, and / or groups thereof.

[0263] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure should be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used in this document, the term "comprising" means "including, but not limited to."

[0264] As will be apparent to those skilled in the art, many modifications and variations can be made without departing from the spirit and scope of the present invention. In addition to the methods and devices recited herein, functionally equivalent methods and devices within the scope of the present disclosure will be apparent to those skilled in the art from the above description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is limited only by the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that the present disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, which may, of course, vary. Similarly, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Where substantially any plural and / or singular form is used herein, one of ordinary skill in the art may translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural variations may be specified for clarity.

[0265] It will be understood by those skilled in the art that, generally, the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally interpreted as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Furthermore, it will be within the purview of those skilled in the art that any disjunctive term and / or phrase that in fact presents two or more separate terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either one of those terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0266] Furthermore, where a particular or subject of the disclosure is described in terms of a Markush group, one skilled in the art will understand that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0267] As will be understood by one of ordinary skill in the art, for all purposes, including in terms of providing a specification, all ranges disclosed herein also encompass all possible subranges and combinations of subranges. All recited ranges are fully descriptive and can be readily recognized as being divisible into at least equal subdivisions. As will be understood by one of ordinary skill in the art, a range includes each individual member.

[0268] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications, and various substitutions, modifications, variations, or improvements thereon, not presently anticipated or anticipated, may be made by those skilled in the art, and each of these is intended to be encompassed by the disclosed embodiments.

[0269] While embodiments of the invention have been described herein, it should be noted that modifications and variations can be made by those skilled in the art in light of the above teachings. It is therefore to be understood that changes can be made in the particular embodiments of the invention disclosed within the scope and spirit of the invention as defined by the appended claims. Having thus described the invention with the detail and specificity required by the patent laws, what is claimed, desired and protected by Letters Patent is set forth in the appended claims.

Claims

1. 1. A nanobiological composition for inhibiting trained immunity, comprising: (i) comprising a nanoscale construct; (ii) having an inhibitor drug incorporated into said nanoscale construct; The nanoscale structure is (a) phospholipids and lysophospholipids; (b) human apolipoprotein AI (apoA-I); and (c) a hydrophobic matrix core comprising triglycerides; a multi-component carrier composition comprising: the inhibitor drug is rapamycin derivatized with an aliphatic chain; Nanobiological compositions.

2. 2. The nanobiology composition of claim 1, wherein the triglyceride is tricaprylin.

3. The nanobiology composition of claim 1 or 2, wherein the composition is a nanosphere having a diameter of about 35 nm to about 65 nm.

4. The nanobiology composition of claim 3, wherein the nanospheres have a diameter of about 35 nm.

5. The nanobiology composition according to any one of claims 1 to 4, wherein the average dispersity of the nanobiology composition is 0.1 to 0.

2.

6. The nanobiology composition of any one of claims 1 to 5, wherein the phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the lysophospholipid is 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC) or 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC).

7. The nanobiology composition of claim 6, wherein the phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the lysophospholipid is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC).

8. 8. The nanobiology composition of claim 7, wherein the POPC and the PHPC are present in a weight ratio of about 2:1 to about 4:

1.

9. The nanoscale structure is (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC), (b) human apo A-I; (c) tricaprylin, Including, The nanoscale structures are about 35 nm in diameter. The nanobiological composition according to any one of claims 1 to 8.

10. The nanobiology composition of any one of claims 1 to 9, wherein the inhibitor drug is an ester of rapamycin.

11. The inhibitor drug is C of rapamycin ４～３０ The nanobiological composition according to any one of claims 1 to 10, which is a saturated fatty acid ester.

12. The inhibitor drug is C of rapamycin １８ The nanobiological composition according to any one of claims 1 to 11, which is a saturated fatty acid ester.

13. The inhibitor drug is 【Chemistry 1】 The nanobiology composition according to any one of claims 1 to 12, wherein

14. The nanobiology composition of any one of claims 1 to 13, adapted for intravenous administration.

15. The nanobiology composition of any one of claims 1 to 14, wherein the nanoscale construct delivers the inhibitor drug to myeloid progenitor cells, the cells being located in bone marrow.

16. The nanobiological composition of any one of claims 1 to 15 for use in a method for promoting allograft tolerance in a patient in need thereof.

17. 17. The nanobiology composition of claim 16, wherein the patient has undergone a transplant and the transplanted tissue is lung tissue, heart tissue, kidney tissue, liver tissue, retinal tissue, corneal tissue, skin tissue, pancreatic tissue, intestinal tissue, reproductive tissue, ovarian tissue, bone tissue, tendon tissue, bone marrow, or vascular tissue.

18. 20. The nanobiology composition of claim 17, wherein the method comprises co-administering an immunosuppressant as a combination therapy with the nanobiology composition.

19. 16. The nanobiology composition of any one of claims 1 to 15 for treating atherosclerosis, arthritis, inflammatory bowel disease, autoimmune disease, autoinflammatory conditions, stroke or myocardial infarction in a patient in need thereof.

20. Use of the nanobiological composition of any one of claims 1 to 15 in the manufacture of a medicament for use in a method for promoting allograft acceptance in a patient.

21. 21. The use of claim 20, wherein the patient has undergone a transplant and the transplanted tissue is lung tissue, heart tissue, kidney tissue, liver tissue, retinal tissue, corneal tissue, skin tissue, pancreatic tissue, intestinal tissue, reproductive tissue, ovarian tissue, bone tissue, tendon tissue, bone marrow, or vascular tissue.

22. The use of claim 20, wherein the method comprises co-administering an immunosuppressant as a combination therapy with the nanobiological composition.

23. Use of the nanobiology composition of any one of claims 1 to 15 in the manufacture of a medicament for treating atherosclerosis, arthritis, inflammatory bowel disease, autoimmune diseases, autoinflammatory conditions, stroke or myocardial infarction.

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