PEGylated organelle complex

Abstract

This specification discloses methods, compositions, and kits suitable for use in enhancing the stability, functionality, pharmacodynamic control, and targeting of populations including mitochondria (e.g., organelle complexes). In some embodiments, coated organelle complex populations are provided. The surface of the coated organelle complex may contain one or more lipid-polymer conjugates. One or more lipid-polymer conjugates comprise a lipid and a conjugated polymer (e.g., a biosoluble polymer and / or a biodegradable polymer).

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts the interests under Section 119(e) of U.S. Provisional Patent Application No. 63 / 493,686, filed on 31 March 2023, which is incorporated herein by reference in its entirety.

[0002] field This disclosure relates, in general terms, to methods for generating coated organelle complexes, coated organelle complexes obtained by such methods, and uses of coated organelle complexes obtained by such methods. [Background technology]

[0003] Explanation of related technologies Mitochondria are intracellular organelles responsible for several metabolic transformations and regulatory functions. They produce the majority of the ATP used by eukaryotic cells. The folded inner and outer membranes, as well as the electron transport system located within the inner membrane, play crucial roles in mitochondrial function. The inner membrane forms highly folded structures called crystals, which are thought to maintain a high proton concentration by holding a supercomplex of the electron transport system within the crystal membrane and trapping excited protons in the crystal space. The electrochemical proton gradient created by the electron transport system enables anion transport, as well as ATP synthesis and cation transport.

[0004] Mitochondria are highly dynamic organelles that move across cells, undergo structural transfers, and change their length, morphology, shape, and size. Furthermore, mitochondria are continuously removed and regenerated in a process known as mitochondrial biogenesis. While most mitochondrial genes are transferred to the nuclear genome, the mitochondrial genome still encodes rRNA, tRNA, and 13 subunits of the electron transport chain (ETC). Therefore, functional transfer between the nuclear and mitochondrial genomes is essential for mitochondrial biogenesis, efficient oxidative phosphorylation, and normal health. Mitochondria are also a major source of free radicals and reactive oxygen species (ROS) that cause oxidative stress. In addition, mitochondria play a crucial role in intracellular signaling, as well as in the regulation of cell death, including apoptosis and necrosis.

[0005] There is growing evidence linking mitochondrial dysfunction to a wide range of human diseases. Mitochondrial dysfunction, such as respiratory chain complex dysfunction, is a major cause of mitochondrial diseases and aging. When mitochondrial function declines, it affects cells in many organs, primarily those involved in mitochondrial diseases or age-related disorders. Introducing exogenous mitochondria into target cells where needed is a promising approach to treat or prevent multiple diseases and disorders. However, there is a need for populations containing mitochondria (e.g., organelle complexes) with enhanced stability, functionality, pharmacodynamic control, and targeting. [Overview of the Initiative]

[0006] The disclosed herein includes a population of coated organelle complexes. In some embodiments, the coated organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the surface of the coated organelle complex comprises one or more lipid-polymer conjugates. In some embodiments, the one or more lipid-polymer conjugates comprises a polymer conjugated with a lipid. In some embodiments, the polymer is a biosoluble polymer and / or a biodegradable polymer. In some embodiments, the one or more lipid-polymer conjugates are 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol 2000 (DMG-PEG 2000), and / or distearoyl-sn-glycero-3-phosphoethanolamine-N-,methoxypolyethylene glycol 2000 (DSPE-PEG 2000). In some embodiments, the one or more lipid-polymer conjugates do not contain triphenylphosphonium (TPP), cholesterol, oleic acid, or any combination thereof.

[0007] In some embodiments, the coated organelle complex includes a coated first organelle complex, a coated second organelle complex, or a combination of the coated first organelle complex and the coated second organelle complex. In some embodiments, the coated first organelle complex and the coated second organelle complex deplete cytoplasmic macromolecules. In some embodiments, the coated first organelle complex is derived from (i) frozen cells, (ii) suspension cells, and / or (iii) cells that have come into contact with a surfactant at or above the critical micelle concentration (CMC) of the surfactant. In some embodiments, the coated second organelle complex is derived from (i) adherent cells, and / or (ii) cells that have come into contact with a surfactant at or below the critical micelle concentration (CMC) of the surfactant. In some embodiments, the cytoplasmic macromolecules include cytoplasmic proteins, and the abundance of one or more cytoplasmic proteins is depleted by at least about 90% compared to the cells from which the organelle complex population originates. In some embodiments, the cytoplasmic protein is p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

[0008] In some embodiments, the coated organelle complex may include one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM), and / or citrate synthase (CS)); one or more mitochondrial outer membrane proteins (e.g., mitochondrial outer membrane complex subunit 20 (TOMM20)); one or more lysosomal proteins (e.g., lysosomal-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosomal-associated membrane protein 1 (LAMP1)); and one or more peroxisome proteins (e.g., catalase, and / or ATP receptor). The complex comprises: composite cassette transporter 1, subfamily D, type 3 (ABCD3); one or more mitochondrial inner membrane proteins (e.g., respiratory chain proteins); mitochondrial DNA, mitochondrial RNA, or both; one or more Golgi apparatus proteins (e.g., Golgin-97, Syntaxin-6, TGOLIN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha class 2A member 1 (MAN2A1)); and / or one or more endoplasmic reticulum proteins (e.g., calreticulin, and / or calnexin). In some embodiments, the organelle complex is derived from cells treated with a mitochondrial activator (e.g., resveratrol).

[0009] In some embodiments, the lipids include amphiphilic lipids having hydrophobic and hydrophilic moieties. In some embodiments, the amphiphilic lipids are selected from the group including phospholipids, aminolipids, and sphingolipids. In some embodiments, the phospholipids include dimyristoyl phosphatidylglycerol (DMG), distearoyl phosphatidyl ethanolamine (DSPE), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), diarachidoyl phosphatidylcholine (DAPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1,2 Distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleylphosphatidylcholine (POPC), 1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroylphosphatidylglycerol (DLPG), diarachidoylphosphatidylglycerol (DAPG), dipalmito Ilphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleylphosphatidylethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylserine (DMPS),The group is selected from diarachidoylphosphatidylserine (DAPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroylphosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), and dioleoylphosphatidylinositol (DOPI). In some embodiments, the phospholipids include saturated fatty acids having a C14-C20 carbon chain and / or unsaturated fatty acids having a C14-C20 carbon chain. In some embodiments, the lipid is selected from the group comprising phosphatidylethanolamine (e.g., phosphatidylethanolamine), having a carbon chain length of 10 to 20, comprising saturated fatty acids, comprising unsaturated fatty acids, comprising saturated and unsaturated fatty acids, and / or comprising distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), and dioleoylphosphatidylethanolamine (DOPE).

[0010] In some embodiments, the polymer has molecular weights of approximately 100 to 20,000 Daltons (Da), approximately 100 to 1,000 Da, approximately 1,000 to 3,500 Da, approximately 3,500 to 7,000 Da, and / or approximately 200, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, and 8,000. It has 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000Da. In some embodiments, the polymer is poly(acrylate), poly(methacrylate), poly(acrylic acid), poly(acrylamide), poly(vinylpyridine), poly(vinylpyrrolidone), poly(vinyl alcohol), naturally derived polymers, poly(ether), poly(maleic anhydride), poly(styrene sulfonate), poly(allylamine hydrochloride), poly(sulfone), poly(ethersulfone), poly(ethylene glycol), copolymers thereof, or any combination thereof, or comprising them. In some embodiments, the surface of the coated organelle complex contains one or more lipid-polymer conjugates in a molar ratio of at least about 0.1%, about 0.5%, about 1%, or more than about 5% of the mass of the organelle complex.

[0011] In some embodiments, the presence of one or more lipid-polymer conjugates on the surface of a coated organelle complex does not reduce mitochondrial function. In some embodiments, the presence of one or more lipid-polymer conjugates on the surface of a coated organelle complex reduces mitochondrial function by about 10 percent, about 5 percent, or less than 1 percent compared to organelle complexes that do not contain one or more lipid-polymer conjugates. In some embodiments, mitochondrial function includes one or more of the following: ATP production, integrity of the mitochondrial outer membrane structure, and cytochrome c oxidase (COX) activity.

[0012] In some embodiments, the polydispersity index (PDI) of the coated organelle complex population is within about 5 percent, about 10 percent, about 15 percent, or about 20 percent of the PDI of the organelle complex population that does not contain one or more lipid-polymer conjugates. In some embodiments, the zeta potential of the coated organelle complex population is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent more positive than the zeta potential of the organelle complex population that does not contain one or more lipid-polymer conjugates. In some embodiments, the mean diameter of the coated organelle complex population is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent smaller than the mean diameter of the organelle complex population that does not contain one or more lipid-polymer conjugates. In some embodiments, the stability of a population of coated organelle complexes in solution is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent greater than that of a population of organelle complexes that do not contain one or more lipid-polymer conjugates. In some embodiments, the physical stability of a population of coated organelle complexes to internal and / or external stimuli is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent greater than that of a population of organelle complexes that do not contain one or more lipid-polymer conjugates. In some embodiments, at least about 70 percent, about 80 percent, about 90 percent, or about 100 percent of a population of coated organelle complexes remains functional after the population has undergone one or more freeze-thaw cycles.

[0013] In some embodiments, one or both ends of the polymer are functionalized with a functional group selected from the group including vinyl, carboxylate, hydroxyl, epoxide, sulfhydryl, amide, acrylate, thiol, azide, maleimide, isocyanate, aziridine, carbonate, N-hydroxysuccinimide ester, imide ester, carbodiimide, anhydride, succinimidyl carbonate, and amine, or any combination thereof. In some embodiments, one or more lipid-polymer conjugates further comprise a targeting agent (e.g., polyarginine). In some embodiments, when the coated organelle complex population is brought into contact with a population of cells, the coated organelle complex has at least about 1.1 times better integration ability into cells compared to organelle complexes that do not contain one or more lipid-polymer conjugates containing the targeting agent. In some embodiments, the targeting agent is configured to bind to a ligand on the surface of a target cell (e.g., a target cell of the desired object). In some embodiments, the binding of the targeting agent to the ligand causes the coated organelle complex to be incorporated into the target cell. In some embodiments, the ligand is differentially expressed between target cells and non-target cells, is absent on non-target cells, and / or is overexpressed on target cells. In some embodiments, the target cells are commensal to a target tissue, which is a site of cancer, inflammation, injury, dysfunction, infection, disease, or impairment, and / or is in close proximity to a site of disease or impairment. In some embodiments, the tissues include adrenal tissue, appendiceal tissue, bladder tissue, bone, intestinal tissue, brain tissue, breast tissue, bronchi, coronary tissue, ear tissue, esophageal tissue, eye tissue, gallbladder tissue, genital tissue, heart tissue, hypothalamic tissue, kidney tissue, large intestine tissue, intestinal tissue, laryngeal tissue, liver tissue, lung tissue, lymph nodes, oral tissue, nasal tissue, pancreatic tissue, parathyroid tissue, pituitary tissue, prostate tissue, rectal tissue, salivary gland tissue, skeletal muscle tissue, skin tissue, small intestine tissue, spinal cord, spleen tissue, stomach tissue, thymus tissue, tracheal tissue, thyroid tissue, ureteral tissue, urethral tissue, soft tissue and connective tissue, peritoneal tissue, vascular tissue, and / or adipose tissue. In some embodiments, the targeting agent is configured to bind to axons.In some embodiments, the targeting agent is, or comprises, a peptide, an antigen-binding domain, a cytokine, a chemokine, an aptamer, a growth factor, a hormone, a cytokine, an interleukin, a receptor, or any combination thereof. In some embodiments, the antigen-binding domain comprises an antibody, an antibody fragment, scFv, Fv, Fab, (Fab′)2, single domain antibody (SDAB), VH or VL domain, camelid VHH domain, Fab′, F(ab′)2, Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single domain antibody (sdAb), anti-complementary scFv (tandem scFv) or single chain, Fv construct, disulfide-linked Fv, dual variable domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, affibody, affilin, affitin, affimer, alphabody, anticalin, avimer, DARPin, finomer, knotted domain peptide, monobody, or any combination thereof.

[0014] In some embodiments, one or more lipid-polymer conjugates further comprise a detectable moiety configured to detect coated organelle complexes in vivo and / or in vitro. In some embodiments, the detectable moiety comprises a fluorescent molecule (e.g., fluorescein amidite (FAM), fluorescein dye, carbocyanine, merocyanine, styryl dye, oxonol dye, phycoerythrin, erythrosine, eosin, rhodamine dye, coumarin, coumarin dye, Oregon Green dye, Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dye, fluorescent dye, BODIPY dye, derivatives thereof, or any combination thereof). In some embodiments, the detectable moiety comprises a fluorescent protein (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), derivatives thereof, or any combination thereof). In some embodiments, the detectable moiety comprises a radioisotope detectable by single photon emission computed tomography (SPECT) and / or positron emission tomography (PET). In some embodiments, the radioisotope is iodine-131( 111 I), iodine-125( 125 I), bismuth-212( 212 Bi), bismuth-213( 213 Bi), astatine-211( 211 At), copper-67( 67 Cu), copper-64( 64 Cu), rhenium-186( 186 Re), rhenium-188( 188 Re), phosphorus-32( 32 P), samarium-153( 153 Sm), lutetium-177( 177 Lu), technetium-99m( 99m Tc), gallium-67(<0​​​201 It is selected from the group comprising Tl). In some embodiments, the detectable moiety comprises quantum dot (Qdot) fluorescent particles (e.g., Qdot525, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705, Qdot800, their derivatives, or any combination thereof).

[0015] In some embodiments, one or more lipid-polymer conjugates further comprise one or more secondary agents, such as therapeutic agents (e.g., small molecule drugs). In some embodiments, the one or more secondary agents are anti-cancer agents, anti-inflammatory agents, anti-infective agents, regenerative agents, relaxants, apoptosis inhibitors, apoptosis inducers, anticoagulants, antioxidant molecules, autophagy inducers, dermatological agents, growth stimulants, vasodilators, vasoconstrictors, analgesics, and anti-allergy agents, condensing modifiers (c-MODS), or combinations thereof. In some embodiments, the one or more secondary agents are chemotherapeutic agents, nucleic acids, polysaccharides, peptides, polypeptides, or any combination thereof. In some embodiments, the one or more secondary agents are protein phosphatase inhibitors, kinase inhibitors, cytokines, inhibitors of immune inhibitory molecules, immunomodulators, anti-metastatic, chemotherapeutic, hormonal or growth factor antagonists, alkylating agents, TLR agonists, cytokine antagonists, cytokine antagonists, or any combination thereof. In some embodiments, the one or more secondary agents are agonist antibodies or antagonist antibodies specific for checkpoint inhibitors or checkpoint stimulatory substance molecules such as PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, TIM-3, etc.

[0016] This specification discloses methods for generating populations of coated organelle complexes. In some embodiments, the method includes contacting organelle complexes in a first solution with one or more lipid-polymer conjugates to generate coated organelle complexes, and recovering the coated organelle complexes from the first solution to generate populations of coated organelle complexes. This specification also discloses populations of coated organelle complexes obtained by the methods provided herein. This specification also discloses populations of host cells comprising coated organelle complexes generated according to the methods provided herein.

[0017] In some embodiments, the method further includes incubating the first solution after the contact step for, for example, about 1 minute to about 120 minutes (e.g., about 15 minutes). In some embodiments, the contact step includes applying a physical stimulus to the first solution (e.g., shaking, mixing, pipetting, and / or stirring). In some embodiments, the incubation step is carried out at a first temperature. In some embodiments, the organelle complex is present in the first solution at a concentration of about 0.01 mg / mL to about 10 mg / mL. In some embodiments, the organelle complex is present in the first solution at a concentration of about 0.1 mg / mL to about 1 mg / mL (e.g., about 1 mg / mL). In some embodiments, the contact step involves contacting a first solution with a solution containing about 1 μL to about 1000 μL of one or more lipid-polymer conjugates, where the one or more lipid-polymer conjugates are present at a concentration of about 0.1 mM to about 10 mM (e.g., 100 μL of a 1 mM solution). In some embodiments, recovering the coated organelle complexes from the first solution involves one or more centrifugation steps. In some embodiments, recovering the coated organelle complexes from the first solution involves centrifugating the first solution with a first centrifugal force, collecting the pellet, and recovering the coated organelle complexes. In some embodiments, the first centrifugal force is about 100 g to about 10000 g (e.g., about 3000 g). In some embodiments, collecting the pellet involves resuspending the population of coated organelle complexes in a second solution. In some embodiments, the second solution has a volume of about 50 μL to about 50 mL. In some embodiments, the second solution has a volume of about 100 μL to about 1000 μL (e.g., about 1000 μL). In some embodiments, the centrifugation step is carried out for at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, or about 20 minutes. In some embodiments, the centrifugation step is carried out at a second temperature.In some embodiments, the first and / or second temperature is about 0°C to about 50°C. In some embodiments, the first temperature is about 20°C to about 25°C and the second temperature is about 0°C to about 4°C. In some embodiments, the recovery step depletes one or more lipid-polymer conjugates that are not incorporated into the surface of the organelle complex. In some embodiments, one or more lipid-polymer conjugates that are not incorporated into the surface of the organelle complex exist as micelles.

[0018] The disclosed herein includes methods for introducing coated organelle complexes into host cells. In some embodiments, the method includes contacting a population of coated organelle complexes disclosed herein with a population of host cells, and upon contact of the coated organelle complexes with host cells, the coated organelle complexes can be incorporated into the host cells.

[0019] In some embodiments, the host cells include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia, neurons, spleen cells, lymphocytes, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, theca cells, cementoocytes, chief cells, chondrocytes, chondrocytes, chromaffin cells, chromophobic cells, adrenocorticotropic hormone-producing cells, delta cells, Langerhans cells. Ns cells, follicular dendritic cells, enteric chromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional cells, erythroblasts, erythrocytes, fibroblasts, fibrous cells, follicular cells, germ cells, gametes, eggs, sperm, oocytes, primary oocytes, secondary oocytes, spermatids, spermatocytes, primary spermatocytes, secondary spermatocytes, germ epithelium, giant cells, glial cells, astroblasts, astrocytes, oligodendroglioblasts, oligodendroglioblasts Glial cells, glioblasts, goblet cells, gonadotropin-secreting cells, granulosa cells, hematoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, keratocytes, fibrous sheath cells, leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B lymphoblasts, T lymphoblasts, lymphocytes, B lymphocytes, T lymphocytes, helper-induced T lymphocytes, Th1 T lymphocytes, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphocytic stem cells, lymphocytes, lymphocytic stem cells, macroglia, mammotropin-producing cells, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanin-forming blasts, melanin-forming cells, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxocervular cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts The organism comprises one or more mammalian cells selected from the group including cells, neuroblasts, neuroepithelial cells, neurons, odontoblasts, osteoblasts, osteoclasts, osteocytes, acid-secreting cells, parafollicular cells, paraluteal cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronoblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, enzyme-forming cells, or any combination thereof. In some embodiments, stem cells include embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells / progenitor cells (HSPCs), or any combination thereof. In some embodiments, host cells are cells of a subject, e.g., a subject suffering from a disease or disorder. In some embodiments, the disease or disorder is a blood disorder, an immune disorder, cancer, an infection, a genetic disorder, a disorder caused by abnormal mtDNA, a metabolic disorder, a disorder caused by an abnormal cell cycle, a disorder caused by abnormal angiogenesis, a disorder caused by abnormal DNA damage repair, or any combination thereof. In some embodiments, the contact is carried out for a period of at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, or about 24 hours.

[0020] In some embodiments, the method involves conjugating a targeting agent, a detectable moiety, and / or one or more secondary agents with one or more lipid-polymer conjugates. In some embodiments, the polymer, the targeting agent, the detectable moiety, and / or one or more secondary agents are functionalized with functional groups selected from the group including vinyl, carboxylate, hydroxyl, epoxide, sulfhydryl, amide, acrylate, thiol, azide, maleimide, isocyanate, aziridine, carbonate, N-hydroxysuccinimide, ester, imide ester, carbodiimide, anhydride, succinimidyl carbonate, and amine, as well as combinations thereof. In some embodiments, conjugation involves contacting a targeting agent, a detectable moiety, and / or one or more secondary agents with one or more lipid-polymer conjugates for at least about 1 minute, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 30, about 40, about 50, or about 60 minutes. In some embodiments, conjugation is carried out at a temperature of about 0°C to about 50°C (e.g., about 20°C to about 25°C or about 0°C to about 4°C). In some embodiments, when a population of coated organelle complexes is brought into contact with a population of host cells, the coated organelle complexes have a superior ability to be incorporated into host cells compared to organelle complexes that do not contain one or more lipid-polymer conjugates. In some embodiments, the one or more lipid-polymer conjugates include a targeting agent configured to bind to a ligand on the surface of the host cells.

[0021] The disclosed herein includes compositions comprising (i) a population of coated organelle complexes disclosed herein, and / or (ii) a population of host cells comprising the coated organelle complexes disclosed herein. The disclosed herein also includes pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises (i) a population of coated organelle complexes disclosed herein, and / or (ii) a population of host cells comprising the coated organelle complexes disclosed herein. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers, and / or one or more secondary agents.

[0022] The disclosed herein includes methods for treating or preventing a disease or disorder in a subject. In some embodiments, the method includes contacting cells of a subject requiring treatment with an effective amount of (i) a population of coated organelle complexes provided herein, (ii) a population of host cells comprising the coated organelle complexes provided herein, (iii) a composition provided herein, and / or (iv) a pharmaceutical composition provided herein, thereby treating or preventing a disease or disorder in a subject.

[0023] In some embodiments, contact is performed ex vivo, in vitro, or in vivo. In some embodiments, the effective dose comprises at least about 1 ug to about 1 mg of coated organelle complex population. In some embodiments, the subject is mammalian. In some embodiments, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the coated organelle complex population is incorporated into the target cell(s) and / or target tissue(s) of the subject. In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or less than 100% of the coated organelle complex population is incorporated into the target non-target cells and / or non-target tissues.

[0024] In some embodiments, the disease or disorder is selected from the group consisting of diabetes mellitus (types I and II), metabolic disorders, ocular disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic drugs, mitochondrial dysfunction associated with space travel, cardiotoxicity associated with chemotherapy or other therapeutic drugs, mitochondrial dysfunction, and migraines. In some embodiments, the disease or disorder is selected from the group consisting of mitochondrial myopathy, diabetes mellitus and deafness (DAD) syndrome, Barth syndrome, Leber hereditary optic neuropathy (LHON), Leigh syndrome, NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis syndrome), myocardial gastroenteropathy (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episode) syndrome, myoclonus epilepsy with red rag fibers (MERRF) syndrome, Kearns-Sayre syndrome, and mitochondrial DNA depletion syndrome. In some embodiments, the disease or disorder is an ischemic disease or disorder, a genetic disorder, an age-related disease or disorder, a neurodegenerative state, a cardiovascular state, cancer, an autoimmune disease, an inflammatory disease, a fibrous disorder, or any combination thereof. In some embodiments, the ischemic disease or disorder is selected from the group consisting of cerebral ischemic-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, hepatic ischemic-reperfusion injury, ischemic compartmental injury syndrome, vascular occlusion, wound healing, spinal cord injury, sickle cell disease, severe limb ischemia, and reperfusion injury of transplanted organs. In some embodiments, the neurodegenerative state is selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonus epilepsy with red ragged fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addiction, anxiety disorders, attention deficit disorder, personality disorders, autism, and Asperger's syndrome. In some embodiments, cardiovascular conditions are selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmias, stroke, deep vein thrombosis, and pulmonary embolism.In some embodiments, the disease or disorder is acute respiratory distress syndrome (ARDS), or pre-eclampsia, or intrauterine growth restriction (IUGR), or fetal growth restriction (FGR).

[0025] In some embodiments, the disease or disorder is associated with the expression of a tumor antigen, and the disease associated with the expression of a tumor antigen is selected from the group consisting of proliferative disorders, precancerous conditions, cancers, and non-cancer-related indications associated with the expression of a tumor antigen. In some embodiments, cancer is colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell lung cancer, mesothelioma, small intestine cancer, esophageal cancer, malignant melanoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's disease The group is selected from the following: lymphoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of the above cancers, and metastatic lesions of the above cancers. In some embodiments, cancer is defined as chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, pilocytic cell leukemia It is a hematological cancer selected from one or more of the following: disease, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström hypergammaglobulinemia, or preleukemia.

[0026] In some embodiments, contact includes systemic administration, subarachnoid administration, intracranial injection, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracisional delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, inhalation, intrapulmonary delivery, and intraocular delivery, or any combination thereof. In some embodiments, systemic administration is intravenous, intramuscular, intraperitoneal, or intraarticular. [Brief explanation of the drawing]

[0027] [Figure 1] A non-limiting illustrative schematic diagram of coated mitochondria provided herein is shown. [Figure 2A] The size distribution data for isolated mitochondria alone (PEG(-); Figure 2A), DMG-PEG 1mM (Figure 2B), DMG-PEG 3mM (Figure 2C), and DMG-PEG 5mM (Figure 2D) are shown. [Figure 2B] The size distribution data for isolated mitochondria alone (PEG(-); Figure 2A), DMG-PEG 1mM (Figure 2B), DMG-PEG 3mM (Figure 2C), and DMG-PEG 5mM (Figure 2D) are shown. [Figure 2C] The size distribution data for isolated mitochondria alone (PEG(-); Figure 2A), DMG-PEG 1mM (Figure 2B), DMG-PEG 3mM (Figure 2C), and DMG-PEG 5mM (Figure 2D) are shown. [Figure 2D] The size distribution data for isolated mitochondria alone (PEG(-); Figure 2A), DMG-PEG 1mM (Figure 2B), DMG-PEG 3mM (Figure 2C), and DMG-PEG 5mM (Figure 2D) are shown. [Figure 3A] The size distribution data for isolated mitochondria alone (PEG(-), Figure 3A), DSPE-PEG 1mM (Figure 3B), DSPE-PEG 3mM (Figure 3C), and DSPE-PEG 5mM (Figure 3D) are shown. [Figure 3B] The size distribution data for isolated mitochondria alone (PEG(-), Figure 3A), DSPE-PEG 1mM (Figure 3B), DSPE-PEG 3mM (Figure 3C), and DSPE-PEG 5mM (Figure 3D) are shown. [Figure 3C] The size distribution data for isolated mitochondria alone (PEG(-), Figure 3A), DSPE-PEG 1mM (Figure 3B), DSPE-PEG 3mM (Figure 3C), and DSPE-PEG 5mM (Figure 3D) are shown. [Figure 3D] The size distribution data for isolated mitochondria alone (PEG(-), Figure 3A), DSPE-PEG 1mM (Figure 3B), DSPE-PEG 3mM (Figure 3C), and DSPE-PEG 5mM (Figure 3D) are shown. [Figure 4A] Data regarding the size distribution (Figure 4A) and zeta potential distribution (Figure 4B) are shown. [Figure 4B] Data regarding the size distribution (Figure 4A) and zeta potential distribution (Figure 4B) are shown. [Figure 5A] Data for diameter (Figure 5A), PdI (Figure 5B), and ζ potential (Figure 5C) before and after the centrifugation step are shown. *: p<0.05 by independent two-sample t-test (mean ± SDn=3). [Figure 5B] Data for diameter (Figure 5A), PdI (Figure 5B), and ζ potential (Figure 5C) before and after the centrifugation step are shown. *: p<0.05 by independent two-sample t-test (mean ± SDn=3). [Figure 5C] Data for diameter (Figure 5A), PdI (Figure 5B), and ζ potential (Figure 5C) before and after the centrifugation step are shown. *: p<0.05 by independent two-sample t-test (mean ± SDn=3). [Figure 6A] Data for diameter (Figure 6A), PdI (Figure 6B), and zeta potential (Figure 6C) before and after PEG modification are shown. *: p<0.05, **: p<0.01 by independent two-sample t-test (mean ± SDn=3). [Figure 6B]Data for diameter (Figure 6A), PdI (Figure 6B), and zeta potential (Figure 6C) before and after PEG modification are shown. *: p<0.05, **: p<0.01 by independent two-sample t-test (mean ± SDn=3). [Figure 6C] Data for diameter (Figure 6A), PdI (Figure 6B), and zeta potential (Figure 6C) before and after PEG modification are shown. *: p<0.05, **: p<0.01 by independent two-sample t-test (mean ± SDn=3). [Figure 7] Data on the PEG content of the control second organelle complex (Q+tris) and the coated second organelle complex (Q+PEG) are shown. **: p<0.01 by unpaired two-sample t-test. Mean ± SD (n=3). [Figure 8A] Data on ATP levels (Figure 8A) and outer membrane integrity (Figure 8B) for intact (untreated second organelle complex (Q)), PEG-coated second organelle complex (PEG-Q), and control second organelle complex (Tris-Q) are shown. nrANOVA, followed by SNK test. ns (mean ± SDn = 3). [Figure 8B] Data on ATP levels (Figure 8A) and outer membrane integrity (Figure 8B) for intact (untreated second organelle complex (Q)), PEG-coated second organelle complex (PEG-Q), and control second organelle complex (Tris-Q) are shown. nrANOVA, followed by SNK test. ns (mean ± SDn = 3). [Figure 9A] This is a non-restrictive, illustrative schematic diagram showing PEGylated mitochondria, where the PEG-lipid conjugate is peptide-modified (Figure 9A) and the maleimide (mal) group is bound to PEG (Figure 9B). [Figure 9B] This is a non-restrictive, illustrative schematic diagram showing PEGylated mitochondria, where the PEG-lipid conjugate is peptide-modified (Figure 9A) and the maleimide (mal) group is bound to PEG (Figure 9B). [Figure 10A]Data are shown for the unmodified second organelle complex (Q) reacted at 25°C for 1 hour in the presence and absence of the peptide (Figure 10A), and for the coated second organelle complex (PEG(mal)-Q) reacted at the indicated PEG:peptide molar ratio (Figure 10B). [Figure 10B] Data are shown for the unmodified second organelle complex (Q) reacted at 25°C for 1 hour in the presence and absence of the peptide (Figure 10A), and for the coated second organelle complex (PEG(mal)-Q) reacted at the indicated PEG:peptide molar ratio (Figure 10B). [Figure 11A] Data on peptide modification of the second organelle complex at 4°C are shown. Figure 11A shows the coated second organelle complex (PEG(mal)-Q) reacted with the indicated PEG:peptide molar ratio. Figure 11B shows the coated second organelle complex (PEG(mal)-Q) reacted with the peptide or without the peptide for the indicated period. [Figure 11B] Data on peptide modification of the second organelle complex at 4°C are shown. Figure 11A shows the coated second organelle complex (PEG(mal)-Q) reacted with the indicated PEG:peptide molar ratio. Figure 11B shows the coated second organelle complex (PEG(mal)-Q) reacted with the peptide or without the peptide for the indicated period. [Figure 12A] Data for the diameter (Figure 12A), PdI (Figure 12B), and zeta potential (Figure 12C) of PEG(mal)-Q (DMG-PEG 2000 maleimide + Q), Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q), Pep PEG-Q (peptide + DMG-PEG 2000 + Q), and Pep-Q (peptide + Q) are shown. nrANOVA followed by SNK test. *: p<0.05, **: p<0.01. Mean ± SD (n=3~9). [Figure 12B]Data for the diameter (Figure 12A), PdI (Figure 12B), and zeta potential (Figure 12C) of PEG(mal)-Q (DMG-PEG 2000 maleimide + Q), Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q), Pep PEG-Q (peptide + DMG-PEG 2000 + Q), and Pep-Q (peptide + Q) are shown. nrANOVA followed by SNK test. *: p<0.05, **: p<0.01. Mean ± SD (n=3~9). [Figure 12C] Data for the diameter (Figure 12A), PdI (Figure 12B), and zeta potential (Figure 12C) of PEG(mal)-Q (DMG-PEG 2000 maleimide + Q), Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q), Pep PEG-Q (peptide + DMG-PEG 2000 + Q), and Pep-Q (peptide + Q) are shown. nrANOVA followed by SNK test. *: p<0.05, **: p<0.01. Mean ± SD (n=3~9). [Figure 13A] FACS data are shown for PEG(mal)-Q (DMG-PEG 2000 maleimide + Q) vs. Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q) (Figure 13A), PEG(mal)-Q vs. Pep PEG-Q (peptide + DMG-PEG 2000 + Q) (Figure 13B), PEG(mal)-Q vs. Pep-Q (peptide + Q) (Figure 13C), and all four modification conditions (Figure 13D). nrANOVA followed by SNK test. *: p<0.05, **: p<0.01. Mean ± SD (n=3~9). [Figure 13B] FACS data are shown for PEG(mal)-Q (DMG-PEG 2000 maleimide + Q) vs. Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q) (Figure 13A), PEG(mal)-Q vs. Pep PEG-Q (peptide + DMG-PEG 2000 + Q) (Figure 13B), PEG(mal)-Q vs. Pep-Q (peptide + Q) (Figure 13C), and all four modification conditions (Figure 13D). nrANOVA followed by SNK test. *: p<0.05, **: p<0.01. Mean ± SD (n=3~9). [Figure 13C] FACS data are shown for PEG(mal)-Q (DMG-PEG 2000 maleimide + Q) vs. Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q) (Figure 13A), PEG(mal)-Q vs. Pep PEG-Q (peptide + DMG-PEG 2000 + Q) (Figure 13B), PEG(mal)-Q vs. Pep-Q (peptide + Q) (Figure 13C), and all four modification conditions (Figure 13D). nrANOVA followed by SNK test. *: p<0.05, **: p<0.01. Mean ± SD (n=3~9). [Figure 13D] FACS data are shown for PEG(mal)-Q (DMG-PEG 2000 maleimide + Q) vs. Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q) (Figure 13A), PEG(mal)-Q vs. Pep PEG-Q (peptide + DMG-PEG 2000 + Q) (Figure 13B), PEG(mal)-Q vs. Pep-Q (peptide + Q) (Figure 13C), and all four modification conditions (Figure 13D). nrANOVA followed by SNK test. *: p<0.05, **: p<0.01. Mean ± SD (n=3~9). [Figure 14A] A schematic diagram (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide + PEG-modified HEKQ by HEK cells are shown. nrANOVA followed by SNK test. Figure 14B shows FACS data for peptide (-) versus peptide (+) modification conditions using HEKQ / RFP. Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). Figures 14D-14E show FITC-A (FAM) data, and Figures 14F-14G show PE-A (RFP) FACS data. *: p<0.05, **: p<0.01. Mean ± SD (n=3). [Figure 14B]A schematic diagram (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide + PEG-modified HEKQ by HEK cells are shown. nrANOVA followed by SNK test. Figure 14B shows FACS data for peptide (-) versus peptide (+) modification conditions using HEKQ / RFP. Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). Figures 14D-14E show FITC-A (FAM) data, and Figures 14F-14G show PE-A (RFP) FACS data. *: p<0.05, **: p<0.01. Mean ± SD (n=3). [Figure 14C] A schematic diagram (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide + PEG-modified HEKQ by HEK cells are shown. nrANOVA followed by SNK test. Figure 14B shows FACS data for peptide (-) versus peptide (+) modification conditions using HEKQ / RFP. Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). Figures 14D-14E show FITC-A (FAM) data, and Figures 14F-14G show PE-A (RFP) FACS data. *: p<0.05, **: p<0.01. Mean ± SD (n=3). [Figure 14D]A schematic diagram (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide + PEG-modified HEKQ by HEK cells are shown. nrANOVA followed by SNK test. Figure 14B shows FACS data for peptide (-) versus peptide (+) modification conditions using HEKQ / RFP. Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). Figures 14D-14E show FITC-A (FAM) data, and Figures 14F-14G show PE-A (RFP) FACS data. *: p<0.05, **: p<0.01. Mean ± SD (n=3). [Figure 14E] A schematic diagram (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide + PEG-modified HEKQ by HEK cells are shown. nrANOVA followed by SNK test. Figure 14B shows FACS data for peptide (-) versus peptide (+) modification conditions using HEKQ / RFP. Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). Figures 14D-14E show FITC-A (FAM) data, and Figures 14F-14G show PE-A (RFP) FACS data. *: p<0.05, **: p<0.01. Mean ± SD (n=3). [Figure 14F]A schematic diagram (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide + PEG-modified HEKQ by HEK cells are shown. nrANOVA followed by SNK test. Figure 14B shows FACS data for peptide (-) versus peptide (+) modification conditions using HEKQ / RFP. Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). Figures 14D-14E show FITC-A (FAM) data, and Figures 14F-14G show PE-A (RFP) FACS data. *: p<0.05, **: p<0.01. Mean ± SD (n=3). [Figure 14G] A schematic diagram (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide + PEG-modified HEKQ by HEK cells are shown. nrANOVA followed by SNK test. Figure 14B shows FACS data for peptide (-) versus peptide (+) modification conditions using HEKQ / RFP. Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). Figures 14D-14E show FITC-A (FAM) data, and Figures 14F-14G show PE-A (RFP) FACS data. *: p<0.05, **: p<0.01. Mean ± SD (n=3). [Figure 15A] Data on the effect of incubation time on uptake for HEKQ (Figure 15A), PEG-HEKQ (Figure 15B), and PepPEG-HEKQ (Figure 15C) are shown. nrANOVA followed by SNK test. **: p<0.01. Mean ± SD (n=3). [Figure 15B] Data on the effect of incubation time on uptake for HEKQ (Figure 15A), PEG-HEKQ (Figure 15B), and PepPEG-HEKQ (Figure 15C) are shown. nrANOVA followed by SNK test. **: p<0.01. Mean ± SD (n=3). [Figure 15C]Data on the effect of incubation time on uptake for HEKQ (Figure 15A), PEG-HEKQ (Figure 15B), and PepPEG-HEKQ (Figure 15C) are shown. nrANOVA followed by SNK test. **: p<0.01. Mean ± SD (n=3). [Figure 16A] Data on mitochondrial function of peptide-modified PEG-Q are shown with respect to ATP production (Figures 16A-16B), outer membrane integrity (Figure 16C), and cytochrome C oxidase activity (Figure 16D). [Figure 16B] Data on mitochondrial function of peptide-modified PEG-Q are shown with respect to ATP production (Figures 16A-16B), outer membrane integrity (Figure 16C), and cytochrome C oxidase activity (Figure 16D). [Figure 16C] Data on mitochondrial function of peptide-modified PEG-Q are shown with respect to ATP production (Figures 16A-16B), outer membrane integrity (Figure 16C), and cytochrome C oxidase activity (Figure 16D). [Figure 16D] Data on mitochondrial function of peptide-modified PEG-Q are shown with respect to ATP production (Figures 16A-16B), outer membrane integrity (Figure 16C), and cytochrome C oxidase activity (Figure 16D). [Figure 17] Data regarding the effect of administration time on R8-PEG-HEKQ uptake are presented. [Figure 18A] Data on the respiratory function of peptide-modified PEG-Q are shown. [Figure 18B] Data on the respiratory function of peptide-modified PEG-Q are shown. [Figure 18C] Data on the respiratory function of peptide-modified PEG-Q are shown. [Figure 19A] Data on the respiratory function of peptide-modified PEG-Q are shown. [Figure 19B] Data on the respiratory function of peptide-modified PEG-Q are shown. [Figure 19C] Data on the respiratory function of peptide-modified PEG-Q are shown. [Figure 20A]Data on the respiratory function of peptide-modified PEG-Q are shown. [Figure 20B] Data on the respiratory function of peptide-modified PEG-Q are shown. [Modes for carrying out the invention]

[0028] The following detailed description refers to the accompanying drawings which form part of this specification. In the drawings, similar symbols typically identify similar components unless otherwise indicated in the context. The exemplary embodiments described in the modes of carrying out the invention, drawings, and claims are not intended to limit the invention. Other embodiments may be used and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of this disclosure generally described herein and shown in the drawings can all be arranged, substituted, combined, separated, and designed in a wide variety of different configurations which are expressly intended herein and form part of this disclosure.

[0029] All patents, published patent applications, other publications, and sequences from GenBank, as well as other databases referenced herein, are incorporated by reference in their entirety with respect to the relevant technology.

[0030] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994) and Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below.

[0031] As used herein, “isolated” shall have its usual meaning and shall also refer to a substance or entity that (1) has been isolated from at least a portion of the associated components when it was first produced (in nature or in an experimental environment), and / or (2) has been produced, prepared, and / or manufactured by human hands. In some embodiments, isolated mitochondria or isolated organelle complex populations are processed to obtain them from the cellular environment via methods provided herein.

[0032] As used herein, the term “mitochondria” has its usual meaning and refers to an organelle present in eukaryotic cells having a bilayer lipid membrane, an inner membrane and an outer membrane, and a matrix surrounded by the cristae membrane and inner membrane. Mitochondria (one or more mitochondria) have enzymes on their inner membrane, such as the respiratory chain complex, which is involved in oxidative phosphorylation. The inner membrane has a membrane potential due to the in-and-out-in In some embodiments, the organelle complex does not contain cytoplasmic macromolecules. In some embodiments, the organelle complex population includes homogenized mitochondria. As used herein, the term “population” shall give its ordinary meaning and shall also refer to a group of multiple identical or different substances. For example, “organelle complex population” is a group of at least multiple identical or different organelle complexes. The population may not always be homogeneous and may have physical, chemical, and / or physiological distributions. Physical distributions include, for example, particle size and polydispersity index. Chemical distributions include, for example, zeta potential distribution and lipid composition distribution. Physiological distributions include, for example, differences in physiological function (e.g., respiratory activity). The organelle complex population may include a first organelle complex, a second organelle complex, homogenized mitochondria, or any combination thereof.As used herein, the term “homogenized mitochondria” shall have its usual meaning and shall refer to mitochondria isolated via a method comprising one or more homogenization steps.

[0033] As used herein, the term “surfactant” shall have its usual meaning and refer to a molecule having both a hydrophilic and a hydrophobic portion in a single molecule. Surfactants play a role in mixing polar and nonpolar substances by reducing surface tension at the interface or by forming micelles. Surfactants are broadly classified into nonionic surfactants and ionic surfactants. Nonionic surfactants have a hydrophilic portion that is not ionized, while ionic surfactants have a hydrophilic portion that is cationic, anionic, or both cationic and anionic. As used herein, the term “critical micelle concentration” (CMC) shall have its usual meaning and refer to the concentration at which a surfactant forms micelles, and at which further surfactant added to the system contributes to micelle formation, particularly in bulk. Adding a surfactant to a system at a concentration exceeding the critical micelle concentration ideally increases the amount of micelles, particularly the number of micelles.

[0034] As used herein, “subject” refers to an animal that is the subject of treatment, observation, or experimentation. “Animals” include cold-blooded and warm-blooded vertebrates, as well as invertebrates, such as fish, mollusks, reptiles, and mammals in particular. As used herein, “mammal” refers to an individual belonging to the class mammal, including, but not limited to, humans, domesticated and livestock animals, zoo animals, sports and pet animals. Examples of mammals include, but not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, as well as primates such as monkeys, chimpanzees and apes, and humans in particular. In some embodiments, the mammal is a human. However, in some embodiments, the mammal is not a human. As used herein, the term “host cell” is given its usual meaning and also refers to in vivo cells, in vitro cells, and / or ex vivo cells in which the incorporation of exogenous mitochondria and / or coated organelle complexes is intended.

[0035] As used herein, the term “treatment” refers to an intervention undertaken in response to a disease, disorder, or physiological condition manifesting in a patient. The objectives of treatment include, but are not limited to, one or more of the following: alleviation or prevention of symptoms, delay or cessation of the progression or worsening of the disease, disorder, or condition, and remission of the disease, disorder, or condition. The terms “to treat” and “treatment” include, for example, therapeutic measures, preventive treatments, and uses that reduce the risk that a subject will develop a disorder or other risk factor. Treatment does not require a complete cure of the disorder and encompasses embodiments that reduce symptoms or underlying risk factors. In some embodiments, “treatment” refers to both therapeutic measures and preventive measures or preventive means. Subjects requiring treatment include subjects already affected by a disease or disorder or an undesirable physiological condition, as well as subjects for whom the disease or disorder or undesirable physiological condition is to be prevented. As used herein, the term “prevention” refers to any activity that reduces the burden on individuals who later develop those symptoms. This can be done at the primary, secondary, and / or tertiary prevention levels, where a) primary prevention avoids the onset of symptoms / disorders / conditions, b) secondary prevention targets the initial stages of treatment for conditions / disorders / symptoms, thereby increasing opportunities for interventions that prevent progression of conditions / disorders / symptoms and the appearance of symptoms, and c) tertiary prevention reduces the adverse effects of already established conditions / disorders / symptoms, for example, by restoring function and / or reducing any conditions / disorders / symptoms or associated complications. The term “prevents” does not require 100% elimination of the possibility of an event. Rather, it indicates that the likelihood of the event occurring is reduced in the presence of the compound or method. Where used herein, the term “effective dose” refers to a quantity sufficient to produce beneficial or desirable biological and / or clinical outcomes.

[0036] As used herein, the term “contact” shall have its usual meaning and also refer to placing two or more entities in such close proximity that they actually come into physical contact with each other, for example, by combining two or more entities (e.g., coated organelle complexes and host cells). Contacting may include co-incubation. Contacting may be done in vitro, in sights, or in vivo. In some embodiments, contacting two entities includes the incorporation (e.g., transplantation) of one entity into another entity that is in physical contact. Contacting coated organelle complexes with a population of host cells may include contacting a population of coated organelle complexes with a population of host cells. Contacting coated organelle complexes with a population of host cells can generate a population of host cells containing exogenous organelle complexes. By contacting coated organelle complexes with a population of host cells, the coated organelle complexes provided herein can be incorporated into the host cells. In some embodiments, the integration of a coated organelle complex (e.g., transplanted) into a host cell involves co-localization and / or fusion with endogenous mitochondria within the host cell. The host cell can be in vivo, in vitro, or ex vivo. In some embodiments, the coated organelle complex integrated into (e.g., transplanted) a host cell can be detected (e.g., distinguished from endogenous organelles in the host cell) for at least a certain period of time (e.g., 6 hours, 12 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or any number or range between any two of these values). The beneficial effects of transplanting a coated organelle complex provided herein can persist beyond the time for which the coated organelle complex is detectable within the host cell population.

[0037] The disclosed herein includes a population of coated organelle complexes. In some embodiments, the coated organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the surface of the coated organelle complex comprises one or more lipid-polymer conjugates. In some embodiments, the one or more lipid-polymer conjugates comprises a polymer conjugated with a lipid. In some embodiments, the polymer is a biosoluble polymer and / or a biodegradable polymer. In some embodiments, the one or more lipid-polymer conjugates are 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol 2000 (DMG-PEG 2000), and / or distearoyl-sn-glycero-3-phosphoethanolamine-N-,methoxypolyethylene glycol 2000 (DSPE-PEG 2000). In some embodiments, the one or more lipid-polymer conjugates do not contain triphenylphosphonium (TPP), cholesterol, oleic acid, or any combination thereof.

[0038] This specification discloses methods for generating populations of coated organelle complexes. In some embodiments, the method includes contacting organelle complexes in a first solution with one or more lipid-polymer conjugates to generate coated organelle complexes, and recovering the coated organelle complexes from the first solution to generate populations of coated organelle complexes. This specification also discloses populations of coated organelle complexes obtained by the methods provided herein. This specification also discloses populations of host cells comprising coated organelle complexes generated according to the methods provided herein.

[0039] The disclosed herein includes methods for introducing coated organelle complexes into host cells. In some embodiments, the method includes contacting a population of coated organelle complexes disclosed herein with a population of host cells, and upon contact of the coated organelle complexes with host cells, the coated organelle complexes can be incorporated into the host cells.

[0040] The disclosed herein includes compositions comprising (i) a population of coated organelle complexes disclosed herein, and / or (ii) a population of host cells comprising the coated organelle complexes disclosed herein. The disclosed herein also includes pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises (i) a population of coated organelle complexes disclosed herein, and / or (ii) a population of host cells comprising the coated organelle complexes disclosed herein. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers, and / or one or more secondary agents.

[0041] The disclosed herein includes methods for treating or preventing a disease or disorder in a subject. In some embodiments, the method includes contacting cells of a subject requiring treatment with an effective amount of (i) a population of coated organelle complexes provided herein, (ii) a population of host cells comprising the coated organelle complexes provided herein, (iii) a composition provided herein, and / or (iv) a pharmaceutical composition provided herein, thereby treating or preventing a disease or disorder in a subject.

[0042] The methods, compositions, systems, and kits provided herein may, in some embodiments, be used in combination with the methods, compositions, systems, and kits described in PCT Patent Publications WO2018 / 092839, WO2017 / 090763, WO2020 / 230601, WO2019 / 164003, WO2020 / 054824, WO2020 / 203961, WO2020 / 054829, WO2021 / 015298, and WO2021 / 132735, the entire contents of which are incorporated herein by reference. The methods, compositions, systems, and kits provided herein may, in some embodiments, be used in conjunction with the methods, compositions, systems, and kits described in U.S. Patent Application No. 63 / 359,108, “REDOX-MODULATING ORGANELLE COMPLEXES,” filed July 7, 2022, and PCT Patent Application Publication No. WO2024 / 010866, and U.S. Patent Application No. 63 / 406,022, “METHODS OF IMPROVING CELLULAR THERAPY WITH ORGANELLE COMPLEXES,” filed September 13, 2022, and PCT Patent Application Publication No. WO2024 / 030441, the entire contents of which are incorporated herein by reference.

[0043] Coated organelle complex The disclosed herein includes a population of coated organelle complexes. A coated organelle complex may include mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The surface of a coated organelle complex may include one or more lipid-polymer conjugates. One or more lipid-polymer conjugates may include a polymer conjugated with a lipid. The polymer may be a biosoluble polymer and / or a biodegradable polymer. One or more lipid-polymer conjugates may be 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol 2000 (DMG-PEG 2000), and / or distearoyl-sn-glycero-3-phosphoethanolamine-N-,methoxypolyethylene glycol 2000 (DSPE-PEG 2000). In some embodiments, one or more lipid-polymer conjugates do not include triphenylphosphonium (TPP), cholesterol, oleic acid, or any combination thereof. One or both ends of the polymer can be functionalized with a functional group selected from the group including vinyl, carboxylate, hydroxyl, epoxide, sulfhydryl, amide, acrylate, thiol, azide, maleimide, isocyanate, aziridine, carbonate, N-hydroxysuccinimide ester, imide ester, carbodiimide, anhydride, succinimidyl carbonate, and amine, or any combination thereof. Also disclosed herein are a population of coated organelle complexes obtained by the methods provided herein.

[0044] Lipids can include amphiphilic lipids having hydrophobic and hydrophilic portions. Amphiphilic lipids can be selected from the group including phospholipids, aminolipids, and sphingolipids. Phospholipids include dimyristoyl phosphatidylglycerol (DMG), distearoyl phosphatidyl ethanolamine (DSPE), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), diarachidoyl phosphatidylcholine (DAPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1,2 Distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleylphosphatidylcholine (POPC), 1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroylphosphatidylglycerol (DLPG), diarachidoylphosphatidylglycerol (DAPG), dipalmitoylphosphatidylglycerol Roll (DPPG), Distearoyl Phosphatidyl Glycerol (DSPG), Dioleoyl Phosphatidyl Glycerol (DOPG), Dimyristoyl Phosphatidic Acid (DMPA), Dipalmitoyl Phosphatidic Acid (DPPA), Distearoyl Phosphatidic Acid (DSPA), Diarachidoyl Phosphatidic Acid (DAPA), Dimyristoyl Phosphatidyl Ethanolamine (DMPE), Dipalmitoyl Phosphatidyl Ethanolamine (DPPE), Dioleyl Phosphatidyl Ethanolamine (DOPE), Diarachidoyl Phosphatidyl Ethanolamine (DAPE), Dilinoleyl Phosphatidyl Ethanolamine (DLPE), Dimyristoyl Phosphatidylserine (DMPS), Diarachidoyl Phosphatidylserine (DAPS),The group can be selected from those comprising dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoyl sphingomyelin (DSSP), dilauroyl phosphatidylinositol (DLPI), diarachidoyl phosphatidylinositol (DAPI), dimyristoyl phosphatidylinositol (DMPI), dipalmitoyl phosphatidylinositol (DPPI), distearoyl phosphatidylinositol (DSPI), and dioleoyl phosphatidylinositol (DOPI). The phospholipids may include saturated fatty acids having C14-C20 carbon chains and / or unsaturated fatty acids having C14-C20 carbon chains. Lipids may contain phosphatidylethanolamine (e.g., phosphatidylethanolamine). Lipids may have a carbon chain length of 10 to 20. Lipids may contain saturated fatty acids. Lipids may contain unsaturated fatty acids. Lipids may contain both saturated and unsaturated fatty acids. Lipids can be selected from the group including distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), and dioleoylphosphatidylethanolamine (DOPE).

[0045] Polymers have molecular weights of approximately 100 to 20,000 Daltons (Da), approximately 100 to 1,000 Da, approximately 1,000 to 3,500 Da, approximately 3,500 to 7,000 Da, and / or approximately 200, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, and 10,000. It can be 0, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000Da, or a number or range between any two of these values. The polymer may be poly(acrylate), poly(methacrylate), poly(acrylic acid), poly(acrylamide), poly(vinylpyridine), poly(vinylpyrrolidone), poly(vinyl alcohol), naturally derived polymers, poly(ether), poly(maleic anhydride), poly(styrene sulfonate), poly(allylamine hydrochloride), poly(sulfone), poly(ethersulfone), poly(ethylene glycol), copolymers thereof, or any combination thereof, or may contain them. The surface of the coated organelle complex may contain one or more lipid-polymer conjugates in a molar ratio exceeding at least about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, or a number or range between any two of these values, based on the mass of the organelle complex.

[0046] In some embodiments, the presence of one or more lipid-polymer conjugates on the surface of a coated organelle complex does not reduce mitochondrial function. In some embodiments, the presence of one or more lipid-polymer conjugates on the surface of a coated organelle complex reduces mitochondrial function by about 10 percent, about 5 percent, or about 1 percent, or a number or range between any two of these values, compared to organelle complexes that do not contain one or more lipid-polymer conjugates. Mitochondrial function can include one or more of the following: ATP production, integrity of the mitochondrial outer membrane structure, and cytochrome c oxidase (COX) activity.

[0047] The polydispersity index (PDI) of a population of coated organelle complexes may be approximately 5%, approximately 10%, approximately 15%, approximately 20%, or any two of these values, or within a range, of the PDI of a population of organelle complexes that do not contain one or more lipid-polymer conjugates. The zeta potential of a population of coated organelle complexes may be more positive than the zeta potential of a population of organelle complexes that do not contain one or more lipid-polymer conjugates, by at least approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 100%, or in a range between any two of these values. The average diameter of a population of coated organelle complexes can be smaller than the average diameter of a population of organelle complexes that do not contain one or more lipid-polymer conjugates by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or a number or range between any two of these values.

[0048] The stability of a population of coated organelle complexes in solution can be greater than that of a population of organelle complexes that do not contain one or more lipid-polymer conjugates by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or a number or range between any two of these values. The physical stability of a coated organelle complex population to internal and / or external stimuli can be greater by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or a number or range between any two of these values, compared to a population of organelle complexes that do not contain one or more lipid-polymer conjugates. In some embodiments, at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or a number or range between any two of these values, of the coated organelle complex population can function after the population has undergone one or more freeze-thaw cycles.

[0049] The coated organelle complexes provided herein may include a first organelle complex, a coated second organelle complex, or a combination of the coated first organelle complex and the coated second organelle complex. The coated organelle complexes provided herein (e.g., a coated first organelle complex, a coated second organelle complex) may include mitochondria, as well as one, two, three, or four of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The coated first organelle complex and the coated second organelle complex can deplete cytoplasmic macromolecules. The coated first organelle complex may be derived from (i) frozen cells, (ii) suspension cells, and / or (iii) cells that have come into contact with a surfactant at or above the critical micelle concentration (CMC) of the surfactant. The coated second organelle complex can originate from (i) adherent cells and / or (ii) cells that have come into contact with the surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant. The cytoplasmic macromolecule may contain cytoplasmic proteins, and the abundance of one or more cytoplasmic proteins can be depleted by at least about 90% compared to the cell from which the organelle complex population originates. The cytoplasmic proteins may be p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).The coated organelle complex contains one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM), and / or citrate synthase (CS)); one or more mitochondrial outer membrane proteins (e.g., mitochondrial outer membrane complex subunit 20 (TOMM20)); one or more lysosomal proteins (e.g., lysosomal-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosomal-associated membrane protein 1 (LAMP1)); and one or more peroxisome proteins (e.g., catalase, and / or ATP-binding cassette trans). It may include: Porter 1, subfamily D, type 3 (ABCD3); one or more mitochondrial inner membrane proteins (e.g., respiratory chain proteins); mitochondrial DNA, mitochondrial RNA, or both; one or more Golgi apparatus proteins (e.g., Golgin-97, Syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha class 2A member 1 (MAN2A1)); and / or one or more endoplasmic reticulum proteins (e.g., calreticulin, and / or calnexin).

[0050] Coated organelle complexes (e.g., coated first organelle complex, coated second organelle complex) include: (i) mitochondria and endoplasmic reticulum, (ii) mitochondria and peroxisomes, (iii) mitochondria and lysosomes, (iv) mitochondria and Golgi apparatus, (v) mitochondria, endoplasmic reticulum, and peroxisomes, (vi) mitochondria, endoplasmic reticulum, and lysosomes, (vii) mitochondria, endoplasmic reticulum, and Golgi apparatus, (viii) mitochondria, endoplasmic reticulum, and peroxisomes. The population of coated organelle complexes may include (ix) mitochondria, endoplasmic reticulum, peroxisomes, and Golgi apparatus, (x) mitochondria, endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus, (xi) mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus, (xii) mitochondria, peroxisomes, and lysosomes, (xiii) mitochondria, peroxisomes, and Golgi apparatus, (xiv) mitochondria, peroxisomes, lysosomes, and Golgi apparatus, and / or (xv) mitochondria, lysosomes, and Golgi apparatus. The ratio of mitochondria to additional organelles (e.g., endoplasmic reticulum, peroxisomes, lysosomes, and / or Golgi apparatus) in the population of coated organelle complexes may vary.

[0051] Provided herein are coated first organelle complexes produced from a first organelle complex population using the method provided herein. Also provided herein are coated second organelle complexes produced from a second organelle complex population using the method provided herein. Disclosed herein includes a method for producing the first organelle complex population. In some embodiments, the method includes incubating cells in a first solution containing a surfactant at a first temperature, removing the surfactant to form a second solution, and recovering the first organelle complexes from the second solution. The first organelle complexes may be derived from (i) frozen cells, (ii) suspension cells, and / or (iii) cells that have been in contact with a surfactant at or above the critical micelle concentration (CMC) of the surfactant. The production of the first organelle complexes may include (step A) providing adherent cells, suspension cells, and / or frozen cells, thawing them, and placing them in tubes. The generation of the first organelle complex may include (Step A) providing adherent cells, suspension cells, and / or frozen cells, centrifugating, and collecting the precipitate. The generation of the first organelle complex may also include (Step A) providing adherent cells, aspirating, adding a solution (e.g., PBS(-)), aspirating, adding TrypLE, incubating, adding a solution (e.g., PBS(-)), placing the cell suspension in a tube, centrifugating, and collecting the precipitate.The generation of the first organelle complex may include one or more of the following steps: (Step B) adding Tris buffer, centrifugating, and collecting the precipitate; (Step C) adding Tris buffer and vortexing; (Step D) adding a solution containing a surfactant and incubating; (Step E) centrifugating and collecting the precipitate; (Step F) adding Tris buffer, centrifugating, and collecting the precipitate; (Step G) adding Tris buffer and pipetting; (Step H) transferring to another tube, collecting the buffer back into the original tube, and rinsing; (Step I) centrifugating and collecting the supernatant; (Step J) centrifugating and collecting the precipitate; and (Step K) pipetting. One or more of the above steps may include an incubation period. One or more of the above steps may include a centrifugation step, followed by collection of the supernatant and / or precipitate. One or more of the above steps may be omitted, and one or more additional steps may be included. Depending on the embodiment, the time, volume, concentration, and centrifugal force may be varied. Methods for obtaining organelle complexes from cells and organelle complexes obtained by such methods are disclosed in U.S. Patent Application No. 63 / 359,110, entitled "ORGANELLE COMPLEXES," filed July 7, 2022, and PCT Patent Application Publication No. WO2024 / 010862, the entire contents of which are incorporated herein by reference. In some embodiments, a second organelle complex is provided. In some embodiments, a method for isolating a second organelle complex from cells comprises treating cells in a first solution with a surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant, removing the surfactant to form a second solution, incubating the cells in the second solution, and recovering the second organelle complex from the second solution. The second organelle complex may be derived from (i) adherent cells and / or (ii) cells that have been in contact with the surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant. In some embodiments, Q mitochondria are provided.The second organelle complex may or may not contain Q mitochondria. A method for obtaining Q mitochondria from cells and Q mitochondria obtained by such a method are disclosed in PCT Patent Application Publication WO / 2021 / 015298, the entire contents of which are incorporated herein by reference. The organelle complex population may be derived from cells treated with a mitochondrial activator (e.g., resveratrol). The organelle complex may be depleted of cytoplasmic macromolecules. Cytoplasmic macromolecules may not be present in the organelle complex population provided herein. The organelle complex population provided herein (e.g., first organelle complex, second organelle complex) may contain negligible and / or undetectable amounts of cytoplasmic macromolecules. Cytoplasmic macromolecules may include cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). The first and second organelle complexes may be derived from cells treated with a mitochondrial activator. Homogenized mitochondria, a first organelle complex, and / or a second organelle complex can be encapsulated in lipid membrane-based vesicles. A method for encapsulation in lipid membrane-based vesicles is disclosed in PCT Patent Application Publication WO2021 / 132735, the entirety of which is incorporated herein by reference.

[0052] Targeting agent In some embodiments, one or more lipid-polymer conjugates further comprise a targeting agent (e.g., polyarginine). In some embodiments, when the coated organelle complex population is brought into contact with a population of cells, the coated organelle complexes have at least about 1.1 times (e.g., 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or any number or range between these values) superior integration ability into host cells compared to organelle complexes that do not contain one or more lipid-polymer conjugates containing the targeting agent. In some embodiments, the targeting agent can be configured to bind to a ligand on the surface of a target cell (e.g., the target cell of the desired object). The binding of the targeting agent and ligand allows the coated organelle complex to be incorporated into the target cell.

[0053] In some embodiments, the ligand is differentially expressed between target cells and non-target cells, is absent on non-target cells, and / or is overexpressed on target cells. The target cells may be commensal to the target tissue. The target tissue may be a site of cancer, inflammation, injury, dysfunction, infection, disease, or impairment, and / or may be in close proximity to a site of disease or impairment. The tissues may include adrenal tissue, appendix tissue, bladder tissue, bone, intestinal tissue, brain tissue, breast tissue, bronchi, coronary tissue, ear tissue, esophageal tissue, eye tissue, gallbladder tissue, reproductive tissue, heart tissue, hypothalamic tissue, kidney tissue, large intestine tissue, intestinal tissue, laryngeal tissue, liver tissue, lung tissue, lymph nodes, oral tissue, nasal tissue, pancreatic tissue, parathyroid tissue, pituitary tissue, prostate tissue, rectal tissue, salivary gland tissue, skeletal muscle tissue, skin tissue, small intestine tissue, spinal cord, spleen tissue, stomach tissue, thymus tissue, tracheal tissue, thyroid tissue, ureteral tissue, urethral tissue, soft tissue and connective tissue, peritoneal tissue, vascular tissue, and / or adipose tissue. The targeting agent may be configured to bind to axons.

[0054] The targeting agent may be a peptide, antigen-binding domain, cytokine, chemokine, aptamer, growth factor, hormone, cytokine, interleukin, receptor, or any combination thereof, or may contain them. The antigen-binding domain may include antibodies, antibody fragments, scFv, Fv, Fab, (Fab′)2, single-domain antibodies (SDAB), VH or VL domains, camelid VHH domains, Fab′, F(ab′)2, Fv, scFv, dsFv, diabodies, triabodies, tetrabodies, multispecific antibodies formed from antibody fragments, single-domain antibodies (sdAb), single chains containing anti-complementary scFv (tandem scFv) or bispecific tandem scFv, Fv constructs, disulfide-linked Fv, bivariable-domain immunoglobulin (DVD-Ig) binding proteins or nanobodies, aptamers, afibodies, affin, afitin, affimer, alphabodies, antikalin, avimer, DARPin, finomer, Knitz domain peptides, monobodies, or any combination thereof.

[0055] Detectable portion In some embodiments, one or more lipid-polymer conjugates further include a detectable moiety configured to detect in vivo and / or in vitro coated organelle complexes. The detectable moiety may include fluorescent molecules (e.g., fluorescein amidite (FAM), fluorocein dyes, carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes, coumarin, coumarin dyes, Oregon Green dye, Texas Red, Texas Red-X, Spectrum Red®, Spectrum Green®, cyanine dyes, fluorescent dyes, BODIPY dyes, their derivatives, or any combination thereof). The detectable moiety may also include fluorescent proteins (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), blue fluorescent protein (BFP), cyanide fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), their derivatives, or any combination thereof). The detectable portion may include radioactive isotopes detectable by single-photon emission computed tomography (SPECT) and / or positron emission tomography (PET). The radioactive isotope is iodine-131( 131 I) Iodine-125 125 I) Bismuth-212 ( 212 Bi), Bismuth-213 213 Bi), Astatine-211( 211 At), Copper-67( 67 Cu), Copper-64( 64 Cu), Rhenium-186 ( 186 Re), Rhenium-188 188 Re), Lin-32( 32 P), Samarium-153( 153 Sm), Lutetium-177( 177 Lu), Technetium-99m( 99m Tc), Gallium-67( 67 Ga), Indium-111( 111 In), and thallium-201 ( 201The group can be selected from Tl). The detectable portion may include quantum dot (Qdot) fluorescent particles (e.g., Qdot525, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705, Qdot800, their derivatives, or any combination thereof).

[0056] Secondary drugs In some embodiments, one or more lipid-polymer conjugates further comprise one or more secondary agents, e.g., therapeutic agents (e.g., small molecule drugs). One or more secondary agents may be anticancer agents, anti-inflammatory agents, anti-infective agents, regenerative agents, muscle relaxants, apoptosis inhibitors, apoptosis inducers, anticoagulants, antioxidant molecules, autophagy inducers, dermatological agents, growth stimulants, vasodilators, vasoconstrictors, analgesics, and anti-allergic agents, condensation modifiers (c-MODS), or combinations thereof. One or more secondary agents may be chemotherapeutic agents, nucleic acids, polysaccharides, peptides, polypeptides, or any combination thereof. One or more secondary agents may be protein phosphatase inhibitors, kinase inhibitors, cytokines, inhibitors of immunosuppressive molecules, immunomodulators, anti-metastatic, chemotherapeutic, hormone or growth factor antagonists, alkylating agents, TLR agonists, cytokine antagonists, or any combination thereof. One or more secondary agents may be agonist or antagonist antibodies specific to checkpoint inhibitors or checkpoint stimulant molecules such as PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, and TIM-3.

[0057] Method for generating coated organelle complexes This specification, as disclosed herein, includes methods for generating a population of coated organelle complexes. In some embodiments, the method includes contacting organelle complexes in a first solution with one or more lipid-polymer conjugates to generate coated organelle complexes, and recovering the coated organelle complexes from the first solution to generate a population of coated organelle complexes. In some embodiments, the method further includes incubating the first solution after the contact step for, for example, about 1 minute to about 120 minutes (e.g., about 15 minutes). The contact step may include applying a physical stimulus to the first solution (e.g., shaking, mixing, pipetting, and / or stirring). The incubation step may be carried out at a first temperature. The organelle complexes may be present in the first solution at concentrations of about 0.01 mg / mL to about 10 mg / mL. The organelle complex is present in the first solution at a concentration of approximately 0.1 mg / mL to approximately 1 mg / mL (for example, approximately 1 mg / mL).The amounts of organelle complexes in the first solution are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320. ,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810 , 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 400 It can be 0, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or any number or range between any two of these values ​​in ug, mg, ug / mL, and / or mg / mL, or it can be a number of those, or at least that number, or at most that number.

[0058] The contact step may include contacting the first solution with a solution containing about 1 μL to about 1000 μL of one or more lipid-polymer conjugates, where the one or more lipid-polymer conjugates may be present at concentrations of about 0.1 mM to about 10 mM (e.g., 100 μL of a 1 mM solution). The one or more lipid-polymer conjugates may be present at concentrations of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a number or range of mM between any two of these values. Recovering the coated organelle complexes from the first solution may include one or more centrifugation steps. In some embodiments, recovering the coated organelle complexes from the first solution includes centrifugating the first solution with a first centrifugal force, collecting the pellet, and recovering the coated organelle complexes. The first centrifugal force can be approximately 100g to 10,000g (for example, approximately 3,000g).The first centrifugal force is 100g, 110g, 120g, 128g, 130g, 140g, 150g, 160g, 170g, 180g, 190g, 200g, 210g, 220g, 230g, 240g, 250g, 260g, 270g, 280g, 290g, 300g, 310g, 320g, 330g, 340g, 350g, 360g, 370g, 380g, 390g, 400g, 410g, 420g, 430g, 440g, 450g, 460g, 4 70g, 480g, 490g, 500g, 510g, 520g, 530g, 540g, 550g, 560g, 570g, 580g, 590g, 600g, 610g, 620g, 630g, 640g, 650g, 660g, 670g, 680g, 690g, 700g, 710g, 720g, 730g, 740g, 750g, 760g, 770g, 780g, 790g, 800g, 810g, 820g, 830g, 840g, 850g, 860g, 870g, 880g, 890g, 900g, 910g, 920g, 930g, 940g, 950g, 960g, 970g, 980g, 990g, 1000g, 1100g, 1200g, 1300g, 1400g, 1500 g, 1600g, 1700g, 1800g, 1900g, 2000g, 2100g, 2200g, 2300g, 2400g, 2500g, 2600g, 2700g, 2800g, 2900g, 3000g, 3250g, 35 00g, 3750g, 4000g, 4250g, 4500g, 4750g, 5000g, 5500g, 6000g, 6500g, 7000g, 7500g, 8000g, 8500g, 9000g, 9500g, 10000g, or any number or range between any two of these values, or a degree of those numbers, or at least those numbers, or at most those numbers.

[0059] Collecting the pellet may involve resuspending the coated organelle complex population in a second solution. The second solution may have a volume of about 50 μL to about 50 mL. The second solution may have a volume of about 100 μL to about 1000 μL (e.g., about 1000 μL). The first solution, a solution containing one or more lipid-polymer conjugates, and / or the second solution are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 2 70, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 32 It can be 50, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or any number or range between any two of these values ​​in μL and / or mL, or to the extent of those numbers, or at least those numbers, or up to those numbers.

[0060] The centrifugation step can be performed for at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or any number or range between any two of these values. The centrifugation step can be performed at a second temperature. The first and / or second temperatures can be about 0°C to about 50°C. The first temperature can be about 20°C to about 25°C, and the second temperature can be about 0°C to about 4°C. The first temperature and / or the second temperature may be 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or any number or range between any two of these values, or a degree of those numbers, or at least those numbers, or at most those numbers. In some embodiments, the recovery step depletes one or more lipid-polymer conjugates that are not incorporated into the surface of the organelle complex. In some embodiments, the one or more lipid-polymer conjugates that are not incorporated into the surface of the organelle complex exist as micelles.

[0061] Method for introducing coated organelle complexes into host cells The disclosed herein includes methods for introducing coated organelle complexes into host cells. In some embodiments, the method includes contacting a population of coated organelle complexes disclosed herein with a population of host cells, and upon contact of the coated organelle complexes with the host cells, the coated organelle complexes can be incorporated into the host cells. The disclosed herein also includes a population of host cells containing coated organelle complexes produced according to the method provided herein. Host cells include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia, neurons, spleen cells, lymphocytes, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, theca cells, cementoocytes, chief cells, chondroblasts, chondrocytes, chromaffin cells, chromophobic cells, adrenocorticotropic hormone-producing cells, delta cells, Langerhans cells, and filtration cells. vesicular dendritic cells, enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional cells, erythroblasts, red blood cells, fibroblasts, fibrocytes, follicular cells, germ cells, gametes, ova, Sperm, oocyte, primary oocyte, secondary oocyte, spermatocyte, spermatocyte, primary spermatocyte, secondary spermatocyte, reproductive epithelium, giant cell, glial cell, astroblast, astrocyte, oligodendroglioblast, oligodendrocyte Cells, glioblasts, goblet cells, gonadotropin-secreting cells, granulosa cells, hematoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, keratocytes, fibrous sheath cells, leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B lymphoblasts, T lymphoblasts, lymphocytes, B lymphocytes, T lymphocytes, helper-induced T lymphocytes, Th1 T lymphocytes, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphocytic stem cells, lymphocytes, lymphocytic stem cells, macroglia, mammotropin-producing cells, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanin-forming blasts, melanin-forming cells, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxocervular cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts, The cells may include one or more mammalian cells selected from the group including neuroblasts, neuroepithelial cells, neurons, odontoblasts, osteoblasts, osteoclasts, osteocytes, acid-secreting cells, parafollicular cells, paraluteal cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronoblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, and enzyme-forming cells, or any combination thereof. Stem cells may include embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells / progenitor cells (HSPCs), or any combination thereof.

[0062] The host cell can be the cell of a subject, for example, a subject suffering from a disease or disorder. The disease or disorder can be a blood disorder, an immune disorder, cancer, an infection, a genetic disorder, a disorder caused by abnormal mtDNA, a metabolic disorder, a disorder caused by an abnormal cell cycle, a disorder caused by abnormal angiogenesis, a disorder caused by abnormal DNA damage repair, or any combination thereof. Contact is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 2 It can be implemented for a period of minutes of 40, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, or any number or range of minutes between any two of these values.

[0063] The method may include conjugating a targeting agent, a detectable moiety, and / or one or more secondary agents with one or more lipid-polymer conjugates. The polymer, targeting agent, detectable moiety, and / or one or more secondary agents may be functionalized with functional groups selected from the group including vinyl, carboxylate, hydroxyl, epoxide, sulfhydryl, amide, acrylate, thiol, azide, maleimide, isocyanate, aziridine, carbonate, N-hydroxysuccinimide, ester, imide ester, carbodiimide, anhydride, succinimidyl carbonate, and amine, as well as combinations thereof. Conjugation may involve contacting a targeting agent, a detectable moiety, and / or one or more secondary agents with one or more lipid-polymer conjugates for at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, or a number or range between any two of these values. Conjugation may be carried out at temperatures between about 0°C and about 50°C (e.g., about 20°C to about 25°C or about 0°C to about 4°C). The temperature at which the coupling is carried out may be 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or any number or range between any two of these values, or to the extent of those numbers, or at least those numbers, or at most those numbers.

[0064] In some embodiments, when a population of coated organelle complexes is brought into contact with a population of host cells, the coated organelle complexes exhibit superior integration into the host cells compared to organelle complexes that do not contain one or more lipid-polymer conjugates. The one or more lipid-polymer conjugates may include targeting agents configured to bind to ligands on the surface of the host cells.

[0065] Treatment method In some embodiments provided herein, methods are provided for treating or preventing a disease or disorder of interest. In some embodiments, the method includes contacting cells of interest that require it with an effective amount of (i) a population of coated organelle complexes provided herein, (ii) a population of host cells comprising the coated organelle complexes provided herein, (iii) a composition provided herein, and / or (iv) a pharmaceutical composition provided herein, thereby treating or preventing a disease or disorder in interest. The contact may be carried out ex vivo, in vitro, or in vivo.The effective amount of coated organelle complex is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 8 10, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 40 It can be 00, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or any number or range between any two of these values ​​in ug, mg, ug / mL, and / or mg / mL, or it can be a number of those, or at least that number, or at most that number.

[0066] The subjects may be mammals. At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a number or range between any two of these values, may be incorporated into the target cells and / or target tissues of the subject. Approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or less than any two of these values ​​or ranges, can be incorporated into the target non-target cells and / or non-target tissues.

[0067] The disease or disorder may be selected from the group consisting of diabetes mellitus (type 1 and type 2), metabolic disorders, ocular disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic drugs, mitochondrial dysfunction associated with space travel, cardiotoxicity associated with chemotherapy or other therapeutic drugs, mitochondrial dysfunction, and migraines.

[0068] The disease or disorder may be selected from the group consisting of mitochondrial myopathy, diabetes mellitus and deafness (DAD) syndrome, Barth syndrome, Leber hereditary optic neuropathy (LHON), Leigh syndrome, NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis syndrome), myocardial gastroenteropathy (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episode) syndrome, myoclonus epilepsy with red rag fibers (MERRF) syndrome, Kearns-Sayre syndrome, and mitochondrial DNA depletion syndrome.

[0069] The disease or disorder may be ischemic-related disease or disorder, hereditary disorder, age-related disease or disorder, neurodegenerative state, cardiovascular state, cancer, autoimmune disease, inflammatory disease, fibrous disorder, or any combination thereof. Ischemia-related disease or disorder may be selected from the group consisting of cerebral ischemic-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, hepatic ischemic-reperfusion injury, ischemic compartmental injury syndrome, vascular occlusion, wound healing, spinal cord injury, sickle cell disease, severe limb ischemia, and reperfusion injury of transplanted organs. Neurodegenerative states may be selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial encephalopathy, lactic acidosis, and stroke-like episode (MELAS), myoclonus epilepsy with red ragged fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addiction, anxiety disorders, attention deficit disorder, personality disorders, autism, and Asperger's syndrome. Cardiovascular conditions may be selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmias, stroke, deep vein thrombosis, and pulmonary embolism. The disease or disorder may be acute respiratory distress syndrome (ARDS), or pre-eclampsia, or intrauterine growth restriction (IUGR), or fetal growth restriction (FGR).

[0070] A disease or disorder may be associated with the expression of tumor antigens, and diseases associated with the expression of tumor antigens may be selected from the group consisting of proliferative disorders, precancerous conditions, cancers, and non-cancer-related indications associated with the expression of tumor antigens.

[0071] Cancers include colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell lung cancer, mesothelioma, small intestine cancer, esophageal cancer, malignant melanoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, and endocrine system cancers. The following can be selected from the group consisting of thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of the above cancers, and metastatic lesions of the above cancers.

[0072] Cancer is a type of cancer that includes chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, pilocytic cell leukemia, small cell or It may be one or more hematological cancers selected from large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström hypergammaglobulinemia, or preleukemia.

[0073] Pharmacologically acceptable compositions and methods of administration In some embodiments, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises (i) a population of coated organelle complexes as disclosed herein, and / or (ii) a population of host cells comprising the coated organelle complexes as disclosed herein. The pharmaceutical composition may include one or more pharmaceutically acceptable carriers, and / or one or more secondary agents. Disclosed herein are compositions comprising (i) a population of coated organelle complexes as disclosed herein, and / or (ii) a population of host cells comprising the coated organelle complexes as disclosed herein.

[0074] This disclosure also provides the use of coated organelle complexes in the manufacture of pharmaceuticals for treating diseases and disorders provided herein. In some embodiments, the coated organelle complexes are administered to a target in conjunction with one or more additional agents and / or additional therapies designed to treat a disease or disorder.

[0075] Contact with target cells may include administration routes selected from the group including intravenous, intra-arterial, intratracheal, subcutaneous, intramuscular, inhalation, intrapulmonary, and intraocular administration. The coated organelle complex population can be administered locally or systemically.

[0076] As used herein, the terms “local administration” or “topic administration” refer to a route of administration in which a population of coated organelle complexes comes into contact with the body of an individual, and the resulting location of the coated organelle complexes within the body is local (limited to a specific tissue, organ, or other body part for which imaging is desired). Exemplary routes of topic administration include injection into a specific tissue with a needle, gastric tube feeding into the gastrointestinal tract, and spreading a solution containing a population of coated organelle complexes on a skin surface.

[0077] As used herein, the term “systemic administration” refers to a route of administration in which the coated organelle complex comes into contact with the body of an individual, and the resulting location of the coated organelle complex within the body is systemic (i.e., not limited to a specific tissue, organ, or other body part for which imaging is desired). Systemic administration includes enteral and parenteral administration. Enteral administration is a systemic route of administration in which the substance is delivered via the gastrointestinal tract and includes, but is not limited to, oral administration, administration via gastrofeeding tube, administration via duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration. Parenteral administration is a systemic route of administration in which the substance is delivered via a route other than the gastrointestinal tract and includes, but is not limited to, intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical instillation.

[0078] In another aspect, the Disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of the coated organelle complex disclosed herein. As described in detail below, the pharmaceutically acceptable compositions of the Disclosure may be specifically formulated for administration in solid or liquid forms, including dosage forms suitable for (1) oral administration, for example, in the form of an infusion (aqueous or nonaqueous solution or suspension), tablet, bolus, powder, granules, or paste; (2) parenteral administration, for example, as a sterile solution or suspension, for example, by subcutaneous, intramuscular, or intravenous injection; (3) topical application, for example, as a cream, ointment, or spray applied to the skin; (4) intravaginal or rectal administration, for example, as a pessary, cream, or foam; or (5) solid or liquid forms, including dosage forms suitable for aerosols, for example, as an aqueous aerosol, liposome preparation, or solid particles containing the coated organelle complex. The pharmaceutically acceptable compositions may contain one or more pharmaceutically acceptable carriers. As used herein, the term “therapeutic dose” may refer to the amount of coated organelle complex disclosed herein that is effective in producing some desired therapeutic effect, for example, cancer treatment, in a reasonable benefit / risk ratio.

[0079] In this specification, the term “pharmaceutically acceptable” is used to mean, within reasonable medical judgment, those drugs, materials, compositions, and / or forms of administration that are suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0080] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, vehicle, excipient, solvent, or encapsulating material, that is involved in transporting or carrying the chemical substance of interest from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) bakuga; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository wax; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil. Examples include sorghum oil and soybean oil, (10) glycols, such as propylene glycol, (1) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic salts, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic, suitable substances used in pharmaceutical formulations.

[0081] Useful formulations in the methods of this disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and / or parenteral administration. The formulations may, conveniently, be presented in unit dosage forms and may be prepared by any method well known in the field of pharmaceuticals. The amount of active ingredient (e.g., a population of coated organelle complexes) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of coated organelle complexes that produce the therapeutic effect. Generally, out of 100 percent, this amount is in the range of about 1% to about 99%, preferably about 5% to about 70%, and most preferably about 10% to about 30% of the active ingredient.

[0082] In addition to the active agent, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0083] Dosage forms for topical or transdermal administration of coated organelle complexes include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active element can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives, buffers, or propellants.

[0084] Ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0085] Ophthalmic preparations, ophthalmic ointments, powders, solutions, etc., are also intended to be within the scope of this disclosure.

[0086] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0087] These compositions may contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial activity can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin can result in prolonged absorption of the injectable drug form.

[0088] The actual dose levels of the active ingredients in the pharmaceutical compositions of this disclosure may be determined by the methods of this disclosure to obtain the amount of the active ingredients, which are non-toxic to the subject and effective in achieving the desired therapeutic response for a particular subject, composition, and mode of administration.

[0089] Kits comprising one or more compositions described herein (e.g., formulations comprising a population of coated cytoplasmic complexes) in suitable packaging are also provided herein and may further include written materials, which may include instructions for use, discussions of clinical trials, and lists of adverse events. Such kits may also include information such as references to scientific literature, package inserts, clinical trial results, and / or summaries thereof, which demonstrate or establish the activity and / or benefits of the composition and / or describe dosage, administration, adverse events, drug interactions, or other information useful to healthcare providers. Such information may be based on the results of various studies, e.g., studies using experimental animals, including in vivo models, and studies based on human clinical trials. The kits may contain one or more unit doses described herein. [Examples]

[0090] Some aspects of the embodiments described above are disclosed in more detail in the following embodiments and are not intended to limit the scope of this disclosure.

[0091] Example 1 Coated organelle complex strategy To increase the selectivity of organelle complexes for organs / cells and to promote the stability of isolated organelle complexes, this embodiment evaluates coating of the mitochondrial outer membrane. For example, in some embodiments, lipids conjugated with biosoluble polymers (PEGs) are inserted into the mitochondrial outer membrane. Figure 1 shows a non-limiting exemplary schematic diagram of coated mitochondria provided herein. Coating with lipid-polymer conjugates disclosed herein can increase physical stability against external stimuli. In some embodiments, without being constrained by any particular theory, the use of lipid-polymer conjugates provided herein can improve the stability of mitochondria in solution by increasing dispersion. Furthermore, coating the mitochondrial membrane with specific ligands such as specific tissues, cell surfaces, and cell penetration-oriented molecules can, in some embodiments, lead to mitochondrial pharmacodynamic control.

[0092] Various methods for inserting lipid-polymer conjugates (e.g., PEG-lipids) into the mitochondrial outer membrane were investigated. The approach tested was pipetting (a gentle mixing method), which could include gently mixing an excess amount of PEG-conjugate lipid with mitochondria at room temperature for 15 minutes. This approach allows for the passive insertion of the lipid portion of the PEG-conjugate lipid into the mitochondrial outer membrane (e.g., an approach similar to liposome (lipid nanoparticle) modification). In this example, the use of DMG-PEG2000(C14) and DSPE-2000(C18) was evaluated. 1,2-Dimiristoyl-sn-glycerol,methoxypolyethylene glycol 2000 (DMG-PEG 2000) has DMG as a lipid anchor, which has two anchors, a carbon chain of 14, and no double bond. Distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypolyethylene glycol 2000 (DSPE-PEG 2000) has DSPE as a lipid anchor, which has two anchors, an 18-carbon chain, and no double bonds. Measurements were performed regarding the diameter for maintaining the original mitochondrial size and the zeta potential for insertion of the PEG lipid into the mitochondria.

[0093] Evaluation of DMG-PEG A gentle mixing method using a pipetting base was employed. Mitochondria were gently mixed with an excess amount of PEG-conjugated lipid at room temperature for 15 minutes. 1–5 mM DMG-PEG (25 μL) was placed in a tube containing 0.1 mg / mL mitochondria (25 μL). After three pipettes using a wide-mouth tip, a 15-minute room temperature incubation was performed to fix the PEG-lipid. Tables 1–2 and Figures 2A–2D show data on the size distribution of isolated mitochondria alone (PEG(-); Figure 2A), DMG-PEG 1 mM (Figure 2B), DMG-PEG 3 mM (Figure 2C), and DMG-PEG 5 mM (Figure 2D). A peak at approximately 10 nm is present, which may be due to PEG-lipid micelles. There was a reduction in zeta potential due to the PEG coating with mitochondria. The detected small particles were hypothesized to be PEG-lipid micelles. [Table 1] [Table 2]

[0094] Evaluation of DSPE-PEG A gentle mixing method using a pipetting base was employed. Mitochondria were gently mixed with an excess amount of PEG-conjugate lipids at room temperature for 15 minutes. 1–5 mM DSPE-PEG (25 μL) was added to a tube containing 0.1 mg / mL mitochondria (25 μL). After three pipettes using a wide-mouth tip, a 15-minute room temperature incubation was performed to fix the PEG-lipids. Tables 3–4 and Figures 3A–3D show data on the size distribution of isolated mitochondria alone (PEG(-), Figure 3A), DSPE-PEG 1 mM (Figure 3B), DSPE-PEG 3 mM (Figure 3C), and DSPE-PEG 5 mM (Figure 3D). A peak was observed at approximately 10 nm, which may be due to PEG-lipid micelles. A slight reduction in zeta potential was observed. The detected small particles were assumed to be PEG-lipid micelles. [Table 3] [Table 4]

[0095] Evaluation of the purification process A purification process to remove excess PEG-lipids (micelles) was evaluated. 1 mM DMG-PEG (100 μL) was added to a tube containing 0.1 mg / mL mitochondria (100 μL). After pipetting three times using a wide-mouth tip, the mixture was incubated at room temperature for 15 minutes to fix the PEG-lipids. After incubation, the mixture was centrifuged at 10,000 g for 10 minutes at 4°C to remove the micelles. After centrifugation, the pellet contained PEG-coated mitochondria, and the supernatant contained PEG-lipid micelles. The supernatant was removed, and the pellet was resuspended in Tris buffer (100 μL). Figures 4A-4B show data regarding the size distribution (Figure 4A) and zeta potential distribution (Figure 4B). A diameter of 375 nm and a zeta potential of -5.8 mV were observed. The zeta potential shifted to neutral due to the fixation of PEG.

[0096] The physicochemical properties of the PEG-coated second organelle complex (second OC;Q) were compared before and after centrifugation. Table 5 and Figures 5A–5C show data for diameter (Figure 5A), PdI (Figure 5B), and zeta potential (Figure 5C) before and after the centrifugation step. [Table 5]

[0097] Next, we compared the physical properties of the PEG-coated second organelle complex (purified) with those of the second organelle complex (control). Table 6 and Figures 6A-6C show data for diameter (Figure 6A), PdI (Figure 6B), and zeta potential (Figure 6C) before and after PEG coating. [Table 6]

[0098] Evaluation of different PEG coating conditions PEG coating was attempted at different temperatures and under different post-coating centrifugation conditions. The mitochondrial concentration was changed from 0.1 mg / mL to 1 mg / mL. In addition, the PEG coating temperature was changed from room temperature to 4°C. Furthermore, the centrifugation speed was changed from 10,000 g to 3,000 g, and the resuspend volume was changed from 100 μL to 1,000 μL. 1 mM DMG-PEG (100 μL) was added to a tube containing 1 mg / mL mitochondria (100 μL). After pipetting three times using a wide-mouth tip, an incubation step was performed at 4°C for 15 minutes to fix the PEG-lipids. After incubation, centrifugation was performed at 3,000 g for 10 minutes at 4°C to remove micelles. After centrifugation, the pellet contained PEG-coated mitochondria, and the supernatant contained PEG-lipid micelles. The supernatant was removed, and the pellet was resuspended in Tris buffer. The reproducibility of the physical properties of PEG-coated mitochondria was confirmed using the above protocol.

[0099] Evaluation of PEG quantity The amount of incorporated PEG was determined using ELISA. PEG content was measured using the ELIZA kit (ENZO PEGylated Protein ELISA Kit) (competitive assay). The amount of PEG coated in mitochondria was estimated. Figure 7 shows data on the PEG content of the control second organelle complex (Q+tris) and the coated second organelle complex (Q+PEG). The following series of calculations were performed: PEG wt / Q wt → 466 nM (50 μL) / 0.1 mg / 1000 μL (50 μL) → 466 × 10⁻¹⁶ -6 nmol / μL(50μL) / 0.1mg / 20→50×466×10 -6 nmol / 5×10 -3 mg → 23.3 × 10 -3 nmol / 5×10 -3mg → 4.66 nmol / mg → 2489 × 4.66 ng / mg → 11598.74 ng / mg → approximately 10 μg / mg. It was determined that the amount of PEG fixed to mitochondria is approximately 10 μg of PEG per 1 mg of mitochondria, and therefore, 1% of the given PEG was fixed to mitochondria by mass.

[0100] Evaluation of mitochondrial function Mitochondrial function was evaluated in intact organelle complex 2 (untreated Q), PEG-coated organelle complex 2 (PEG-Q), and control organelle complex 2 (Tris-Q). The control organelle complex 2 received tris buffer instead of PEG-lipid and underwent centrifugation, supernatant removal, and resuspension. Figures 8A–8B show data on ATP levels (Figure 8A) and outer membrane integrity (Figure 8B) for intact organelle complex 2 (untreated Q), PEG-coated organelle complex 2 (PEG-Q), and control organelle complex 2 (Tris-Q). It was determined that PEG coating did not reduce mitochondrial function.

[0101] Investigation of peptide modifications of pegylated organelle complex 2 Figures 9A-9B are non-limiting, exemplary schematic diagrams showing PEGylated mitochondria, where the PEG-lipid conjugate is peptide-modified (Figure 9A) and the maleimide (mal) group is bound to PEG (Figure 9B). A peptide (e.g., 5-FAM-RRRRRRRRC-NH2) can be added to the lipid-polymer conjugate using the method provided herein. Functional elements (e.g., R8) can be added by utilizing specific chemical reactions between the maleimide functional group in PEG and the SH group in the peptide. Conditions, including temperature, time, and amount of peptide modification during the reaction, were investigated. The physical properties of the peptide-treated second organelle complex were evaluated, and the peptide-treated second organelle complex was assessed by FACS.

[0102] Peptide modifications of the second organelle complex were first investigated at 25°C for 1 hour. Tables 7-8 and Figures 10A-10B show data for the uncoated second organelle complex (Q) (Figure 10A) reacted at 25°C for 1 hour in the presence / absence of the peptide, and for the coated second organelle complex (PEG(mal)-Q) (Figure 10B) reacted with the indicated PEG:peptide molar ratio. No peptide modifications were observed without PEG(mal). However, peptide modifications were observed in PEG(mal)Q (coated second organelle complex). In addition, more peptide modifications were observed with higher doses (molar ratios) of the peptide used. [Table 7] [Table 8]

[0103] Next, peptide modification of PEGylated second organelle complexes at 4°C for 15 minutes to 1 hour was investigated. Tables 9-10 and Figures 11A-11B show data on peptide modification of second organelle complexes at 4°C. Figure 11A shows coated second organelle complexes (PEG(mal)-Q) reacted with the indicated PEG:peptide molar ratio. Figure 11B shows coated second organelle complexes (PEG(mal)-Q) reacted with or without the peptide for the indicated duration. Peptide modification occurred when the reaction was carried out at 4°C. In addition, the higher the dose (molar ratio) of the peptide used, the more peptide modification was observed at 4°C. However, it was found that adjusting the peptide modification time from 15 minutes to 1 hour did not affect the amount of modification at 4°C. [Table 9] [Table 10]

[0104] Next, the physical properties of peptide-modified PEG-Q were investigated. The peptide modification was performed at a ratio of 1:10. 4 The experiment was conducted at 4°C for 15 minutes using the specified PEG:peptide molar ratio. Table 11 and Figures 12A-12C show data for diameter (Figure 12A), PdI (Figure 12B), and zeta potential (Figure 12C) of PEG(mal)-Q (DMG-PEG 2000 maleimide + Q), Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q), Pep PEG-Q (peptide + DMG-PEG 2000 + Q), and Pep-Q (peptide + Q). The zeta potential was found to shift to neutral after the addition of the peptide. [Table 11]

[0105] FAM (fluorescence) detection was performed in a second organelle complex treated with PEG(mal)+ peptide using FAM-conjugate peptides. Table 12 and Figures 13A-13D show FACS data for PEG(mal)-Q (DMG-PEG 2000 maleimide + Q) vs. Pep PEG(mal)-Q (peptide + DMG-PEG 2000 maleimide + Q) (Figure 13A), PEG(mal)-Q vs. Pep PEG-Q (peptide + DMG-PEG 2000 + Q) (Figure 13B), PEG(mal)-Q vs. Pep-Q (peptide + Q) (Figure 13C), and all four modification conditions (Figure 13D). [Table 12]

[0106] Evaluation of uptake by HEK cells Tables 13-15 and Figures 14A-14G show schematic diagrams (Figure 14A) and data (Figures 14B-14G) regarding the uptake of peptide-+PEG coated HEKQ by HEK cells using nrANOVA, followed by SNK test. We first evaluated whether Q (HEKQ / RFP) derived from RFP-labeled mitochondria in HEK cells could be coated with PEG and peptides. Peptide modification was performed at a 1:10 ratio. 4 The procedure was performed at 4°C for 15 minutes using the specified PEG:peptide molar ratio. Figure 14B shows FACS data for peptide(-) versus peptide(+) modification conditions using HEKQ / RFP. [Table 13]

[0107] Next, HEKQ / RFP was coated with PEG and peptides, and its uptake into HEK cells was evaluated (in FBS(+) medium for 6 hours after addition). Figure 14C shows FACS data for pepPEG-HEKQRFP (PEG + peptide HEKQ / RFP), PEG-HEKQRFP (PEG HEKQ / RFP), HEKQRFP (HEKQ / RFP), and untreated (NT). [Table 14] [Table 15]

[0108] Figures 14D-14E show FITC-A(FAM) data, and Figures 14F-14G show PE-A(RFP)FACS data. It was found that peptide-+PEG coated HEKQ was significantly taken up by HEK cells. In addition, uncoated HEKQ was also taken up, but in small amounts. In contrast, PEG-coated HEKQ was not taken up.

[0109] Next, we investigated the effect of incubation time on peptide-modified PEG-Q. Figures 15A-15C show data on the effect of incubation time on the uptake of HEKQ (Figure 15A), PEG-HEKQ (Figure 15B), and PepPEG-HEKQ (Figure 15C). The XG mean of PE-A(RFP) was calculated relative to each other, with untreated (NT) set to 1. Pep PEG-HEKQ uptake increased with longer administration times up to 6 hours, but the XG mean decreased at 24 hours. HEKQ uptake increased with longer administration times, and PEG-HEKQ showed increased uptake 24 hours after administration.

[0110] Finally, the mitochondrial function of peptide-modified PEG-Q was evaluated. ATP was measured using 0.5 mg of protein, and the outer membrane was measured using 1 mg of protein. Protein levels were determined by the Pierce method and found to be as follows: Q, 77.6 ug / mL; PEG-Q, 77.6 ug / mL; peptide-PEG-Q, 103.3 ug / mL. Figures 16A-16D show data on the mitochondrial function of peptide-modified PEG-Q with respect to ATP production (Figures 16A-16B), outer membrane integrity (Figure 16C), and cytochrome C oxidase activity (Figure 16D). It was found that peptide modification did not reduce mitochondrial function. The protein values ​​used for protein correction are high due to the effect of the peptide, which is why ATP appears to decrease with peptide-modified PEG-Q.

[0111] Evaluation of R8-PEG-HEKQ Figure 17 shows data on the uptake of peptide (R8)-modified and PEG-coated HEKQ ("R8-PEG-HEKQ") by HEK cells. Uptake of R8-PEG-HEKQ into HEK cells was examined using flow cytometry at each administration time. HEKQ contained red fluorescent protein (RFP), and the geometric mean (cellular uptake value) of RFP at each administration time (1 hour, 2 hours, 3 hours, 6 hours, and 24 hours) was calculated with NT (untreated) set to 1. As shown in Figure 17, R8-PEG-HEKQ uptake increased over time, reaching maximums at 3 and 6 hours, but the geometric mean decreased at 24 hours.

[0112] The effect of R8-PEG-HEKQ administration on oxygen consumption rate (OCR) was tested using a Flax analyzer (Seahorse assay). The cell seeding volume was 1.6 × 10⁶. 4 Cells were administered in 90 μL / wells, with either unmodified HEKQ or R8-PEG-HEKQ equivalent to 10% of the culture medium, over 24 hours (theoretical mt concentration after administration was 0.01 mg / ml). Overall results are shown in Figure 18A, and basal respiratory rate, respiratory reserve rate, proton leakage rate, and ATP production rate are shown in Figures 18B-18C, respectively.

[0113] The effect of R8-PEG-HEKQ administration on oxygen consumption rate (OCR) was tested using a Flax analyzer (Seahorse assay). The cell seeding volume was 1.6 × 10⁶. 4 Cells were administered in 90 μL / well doses of 0.1 mg / ml R8-PEG-HEKQ over 24 hours. The overall results are shown in Figures 19A-C, where R8-PEG-HEKQ did not increase OCR 24 hours after treatment compared to the untreated (NT) group.

[0114] Evaluation of R8-PEG-HEKQ Peptide (R8)-modified PEG-coated HeLaQ was prepared using the method described herein. R8-PEG-HeLaQ (mt concentration 1 mg / ml) and unmodified HeLaQ (mt concentration 1 mg / ml) were prepared by ultrafiltration.

[0115] The effect of R8-PEG-HeLaQ administration on oxygen consumption rate (OCR) was tested using a Seahorse XF HS Mini Analyzer. The HEK cell seeding volume was 0.9 × 10⁻⁶. 4 Cells were administered in 90 μL / well doses, and R8-PEG-HeLaQ and unmodified HeLaQ were administered at an mt concentration of 0.1 mg / ml over 24 hours. Overall results are shown in Figure 20A, and basal respiratory rate, respiratory reserve rate, proton leakage rate, and ATP production rate are shown in Figure 20B, respectively. As shown in Figures 20A-20B, administration of R8-PEG-HeLaQ significantly increased the OCR of HEK cells compared to both unmodified HeLaQ and blank (NT).

[0116] Supplementary materials and methods Mitochondrial particle size measurement and charge measurement Particle size was measured in a 50 μL suspension of mitochondria (PEG-coated and peptide-modified mitochondria). The particle size measurement conditions were as follows: RI, 1.590; absorbance, 0.010; medium, 250 mM Tris-sucrose buffer; temperature 25.0°C; and viscosity 1.1459 cP, RI 1.342. Charge measurement was performed by resuspending 50 μL of the above suspension in 500 μL of buffer. The charge measurement conditions were as follows: RI, 1.590; absorbance, 0.010; buffer, 250 mM Tris-sucrose; temperature 25.0°C; and viscosity 1.1459 cP, RI 1.342, relative permittivity 78.5. A Zetasizer Nano ZS (Malvern) was used for the measurements.

[0117] Determination of PEG coating quantity using ELISA The ENZO PEGylated Protein ELISA Kit (competitive method) was used for analysis. The 96-well plate included in the kit was used. The following steps were performed: (1) 50 μL / well of sample, followed by 50 μL / well of biotinylated PEG; (2) gently tap the plate and shake at room temperature for 1 hour; (3) aspirate and wash four times with 300 μL / well of wash buffer; (4) 100 μL / well of PEG conjugate, followed by shaking at RT for 30 minutes; (5) aspirate and wash three times with 300 μL / well of wash buffer; (6) 100 μL / well of TMB substrate, followed by shaking at RT for 30 minutes; (7) after adding 100 μL / well of stop solution 2 (1N HCl); and (8) measure the absorbance at 450 nm using a plate reader. Calibration curves were obtained by performing serial dilutions of PEG from 1 mM to 1 nM and following the same procedure as above.

[0118] Assays for ATP production and outer membrane integrity The ATP assay was performed under the following conditions: volume of mitochondrial solution added, 0.5 μg / well (protein quantification by Pierce method); reaction substrate, 10 mM malate / 5 mM glutamate / 0.1 mM ADP / 10 mM Pi / 70 mM KCl; and reaction conditions, room temperature for 10 minutes. For outer membrane integrity measurement, the cytochrome C oxidase assay was performed under the following conditions: volume of mitochondrial solution added, 3 μg / well (protein quantification by Pierce method); and reaction (measurement) temperature, 30°C. Outer membrane integrity was calculated using the following formula (where ε=21.84, L=0.625): Units / mg = (ΔA (w / surfactant)) / (ε × L × applicable protein [mg]); and Outer film integrity (%) = (ΔA (containing surfactant) - ΔA (not containing surfactant)) / (ΔA (containing surfactant)).

[0119] In at least some of the embodiments described above, one or more elements used in the embodiments may be interchangeably used in other embodiments unless such substitution is technically feasible. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to be within the scope of the subject matter as defined by the appended claims.

[0120] With regard to the use of substantially any plural and / or singular terms herein, those skilled in the art can substitute plurals for singulars and / or singulars for plurals as appropriate to the context and / or use. Various singular / plural substitutions may be expressly shown herein for clarity. Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise. Any reference to “or” herein is intended to include “and / or” unless otherwise specified.

[0121] In general, it will be understood by those skilled in the art that the terms used herein, and in particular in the appended claims (e.g., the text of the appended claims), are generally intended to be “open” terms (for example, “including” should be interpreted as “including but not limited to,” “having” should be interpreted as “having at least,” and “includes” should be interpreted as “includes but is not limited to,” etc.). It will also be understood by those skilled in the art that if a particular number of descriptions in the introduced claims are intended, such intent will be explicitly stated in the claims, and if such statement is not present, no such intent exists. For example, to aid understanding, the following appended claims may introduce the descriptions of the claims, including the use of the introductory phrases “at least one” and “one or more.” However, the use of such phrases should not be interpreted as implying that the introduction of a claim description by the indefinite article "a" or "an" limits any particular claim containing such introduced claim description to embodiments containing only one such description, even if the same claim contains the introducing phrase "one or more" or "at least one" and the indefinite article "a" or "an" (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"), that same claim still applies as in the case of the definite article used to introduce a claim description. In addition, even if a particular number of introduced claim descriptions is explicitly stated, a person skilled in the art will recognize that such a description should be interpreted as meaning at least that stated number (for example, the bare description of "two descriptions," without other modifiers, means at least two descriptions, or two or more descriptions).Furthermore, in examples where a convention similar to "at least one of A, B, and C, etc." is used, such a structure is generally intended to mean that a person skilled in the art will understand the convention (for example, "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). It will be further understood by those skilled in the art that any de facto disjunct word and / or phrase for which two or more alternative terms exist should be understood as intended to include one of those terms, either or both of those terms, whether or not they appear in the specification, claims, or drawings.

[0122] In addition, if any feature or aspect of the present disclosure is described from the perspective of the Markush Group, a person skilled in the art will recognize that the present disclosure is also described from the perspective of any individual member or subgroup of a member of the Markush Group.

[0123] As will be understood by those skilled in the art, for any or all purposes, particularly in terms of providing written explanations, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any enumerated scope can be readily recognized as sufficient to explain and enable that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope considered herein can readily be decomposed into a lower third, a middle third, and an upper third, etc. Also, as will be understood by those skilled in the art, all language such as “maximum,” “at least,” “greater than,” and “less than” includes the enumerated numbers and refers to scopes that can subsequently be decomposed into subscopes as discussed above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, the group having 1 to 3 articles refers to the group having 1, 2, or 3 articles. Similarly, the group having 1 to 5 articles refers to the group having 1, 2, 3, 4, or 5 articles, and so on.

[0124] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are illustrative and not intended to limit the scope and spirit, but are shown in the following claims.

Claims

1. A group of coated organelle complexes, The coated organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The surface of the coated organelle complex comprises one or more lipid-polymer conjugates, A group of coated organelle complexes comprising one or more lipid-polymer conjugates containing a polymer conjugated with a lipid.

2. The coated organelle complex population according to claim 1, wherein the polymer is a biosoluble polymer and / or a biodegradable polymer.

3. The coated organelle complex population according to any one of claims 1 to 2, wherein the one or more lipid-polymer conjugates is 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol 2000 (DMG-PEG 2000), and / or distearoyl-sn-glycero-3-phosphoethanolamine-N-,methoxypolyethylene glycol 2000 (DSPE-PEG 2000).

4. The coated organelle complex population according to any one of claims 1 to 3, wherein the one or more lipid-polymer conjugates does not contain triphenylphosphonium (TPP), cholesterol, oleic acid, or any combination thereof.

5. The coated organelle complex includes a coated first organelle complex, a coated second organelle complex, or a combination of a coated first organelle complex and a coated second organelle complex. The coated first organelle complex and the coated second organelle complex deplete cytoplasmic macromolecules, The coated first organelle complex originates from (i) frozen cells, (ii) suspension cells, and / or (iii) cells that have come into contact with the surfactant at or above the critical micelle concentration (CMC) of the surfactant. The coated organelle complex population according to any one of claims 1 to 4, wherein the coated second organelle complex is derived from (i) adherent cells and / or (ii) cells that have come into contact with a surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant.

6. The coated organelle complex population according to claim 5, wherein the cytoplasmic macromolecule comprises a cytoplasmic protein, the abundance of one or more cytoplasmic proteins being depleted by at least about 90% compared to the cell from which the organelle complex population originates, and optionally the cytoplasmic protein is p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

7. The coated organelle complex One or more mitochondrial matrix proteins, optionally mitochondrial transcription factor A (TFAM), and / or citrate synthase (CS), One or more mitochondrial outer membrane proteins, optionally selected from mitochondrial outer membrane complex subunit 20 (TOMM20), One or more lysosomal proteins, optionally lysosomal-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosomal-associated membrane protein 1 (LAMP1), One or more peroxisome proteins, optionally catalase, and / or ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3), One or more mitochondrial inner membrane proteins, optionally, respiratory chain proteins, Mitochondrial DNA, mitochondrial RNA, or both One or more Golgi proteins, optionally including Golgin-97, Syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha class 2A member 1 (MAN2A1), and / or A population of coated organelle complexes according to any one of claims 1 to 6, comprising one or more endoplasmic reticulum proteins, optionally calreticulin, and / or calnexin.

8. The coated organelle complex population according to any one of claims 1 to 7, wherein the organelle complex is derived from cells treated with a mitochondrial activator, optionally with resveratrol.

9. The coated organelle complex population according to any one of claims 1 to 8, wherein the lipid comprises an amphiphilic lipid having a hydrophobic portion and a hydrophilic portion.

10. The coated organelle complex population according to claim 9, wherein the amphiphilic lipid is selected from the group comprising phospholipids, aminolipids, and sphingolipids.

11. The phospholipids mentioned above are dimyristoyl phosphatidylglycerol (DMG), distearoyl phosphatidyl ethanolamine (DSPE), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), diarachidoyl phosphatidylcholine (DAPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1, 2 Distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine Zilcholine (SPPC), 1-palmitoyl-2-oleylphosphatidylcholine (POPC), 1-oleyl-2-palmitoyl-phosphatidylcholine (OPC), dilauroylphosphatidylglycerol (DLPG), diarachidoylphosphatidylglycerol (DAPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoyl-phosph Phosphatidylglycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA), diarachidoyl phosphatidic acid (DAPA), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleyl phosphatidylethanolamine (DOPE), diarachidoyl phosphatidylethanolamine (DAPE), dilinoleyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP),and a population of coated organelle complexes according to claim 10, selected from the group comprising distearoyl sphingomyelin (DSSP), dilauroyl phosphatidylinositol (DLPI), diarachidoyl phosphatidylinositol (DAPI), dimyristoyl phosphatidylinositol (DMPI), dipalmitoyl phosphatidylinositol (DPPI), distearoyl phosphatidylinositol (DSPI), and dioleoyl phosphatidylinositol (DOPI).

12. The coated organelle complex population according to any one of claims 10 to 11, wherein the phospholipid comprises a saturated fatty acid having a C14-C20 carbon chain and / or an unsaturated fatty acid having a C14-C20 carbon chain.

13. The lipid comprises phosphatidylethanolamine, and the phosphatidylethanolamine is Having a carbon chain length of 10 to 20, Contains saturated fatty acids, Contains unsaturated fatty acids, It contains saturated fatty acids and unsaturated fatty acids, and / or A population of coated organelle complexes according to any one of claims 1 to 12, selected from the group comprising distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), and dioleoylphosphatidylethanolamine (DOPE).

14. The aforementioned polymer has a molecular weight: Approximately 100 to 20,000 Daltons (Da), Approximately 100 to approximately 1000 Da, About 1000 to about 3500Da, Approximately 3500–7000 Da, and / or Approximately 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000 , 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000 A population of coated organelle complexes according to any one of claims 1 to 13, having 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 Da.

15. The coated organelle complex population according to any one of claims 1 to 14, wherein the polymer is poly(acrylate), poly(methacrylate), poly(acrylic acid), poly(acrylamide), poly(vinylpyridine), poly(vinylpyrrolidone), poly(vinyl alcohol), naturally derived polymer, poly(ether), poly(maleic anhydride), poly(styrene sulfonate), poly(allylamine hydrochloride), poly(sulfone), poly(ethersulfone), poly(ethylene glycol), copolymers thereof, or any combination thereof.

16. The coated organelle complex population according to any one of claims 1 to 15, wherein the surface of the coated organelle complex comprises one or more lipid-polymer conjugates in a molar ratio exceeding at least about 0.1%, about 0.5%, about 1%, or about 5% of the mass of the organelle complex.

17. The presence of one or more lipid-polymer conjugates on the surface of the coated organelle complex does not reduce mitochondrial function, or The presence of one or more lipid-polymer conjugates on the surface of the coated organelle complex reduces mitochondrial function by about 10 percent, about 5 percent, or less than 1 percent compared to organelle complexes that do not contain the one or more lipid-polymer conjugates. A population of coated organelle complexes according to any one of claims 1 to 16, wherein, optionally, mitochondrial function includes one or more of ATP production, integrity of the mitochondrial outer membrane structure, and cytochrome c oxidase (COX) activity.

18. The polydispersity index (PDI) of the coated organelle complex population is within approximately 5 percent, approximately 10 percent, approximately 15 percent, or approximately 20 percent of the PDI of the organelle complex population that does not contain one or more lipid-polymer conjugates. The zeta potential of the coated organelle complex population is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent more positive than the zeta potential of the organelle complex population that does not contain one or more lipid-polymer conjugates, and / or The coated organelle complex population according to any one of claims 1 to 17, wherein the average diameter of the coated organelle complex population is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent smaller than the average diameter of the organelle complex population that does not contain one or more lipid-polymer conjugates.

19. The stability of the coated organelle complex population in solution is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent greater than that of the organelle complex population that does not contain one or more lipid-polymer conjugates. The physical stability of the coated organelle complex population against internal and / or external stimuli is at least about 5 percent, about 10 percent, about 25 percent, about 50 percent, about 75 percent, or about 100 percent greater than that of the organelle complex population that does not contain one or more lipid-polymer conjugates, and / or The coated organelle complex population according to any one of claims 1 to 18, wherein at least about 70 percent, about 80 percent, about 90 percent, or about 100 percent of the coated organelle complex population functions after the population has undergone one or more freeze-thaw cycles.

20. A coated organelle complex population according to any one of claims 1 to 19, wherein one or both ends of the polymer are functionalized with a functional group selected from the group consisting of vinyl, carboxylate, hydroxyl, epoxide, sulfhydryl, amide, acrylate, thiol, azide, maleimide, isocyanate, aziridine, carbonate, N-hydroxysuccinimide ester, imide ester, carbodiimide, anhydride, succinimidyl carbonate, and amine, or any combination thereof.

21. The coated organelle complex population according to any one of claims 1 to 20, wherein the one or more lipid-polymer conjugates further optionally comprises a targeting agent, polyarginine.

22. The coated organelle complex population according to claim 21, wherein when the coated organelle complex population is brought into contact with a population of cells, the coated organelle complex has at least about 1.1 times better integration ability into the cells compared to organelle complexes that do not contain the one or more lipid-polymer conjugates containing the targeting agent.

23. The coated organelle complex population according to claim 22, wherein the targeting agent is configured to selectively bind to target cells, or to ligands on the surface of target cells of a desired type.

24. The coated organelle complex population according to claim 23, wherein the binding of the targeting agent to the ligand causes the coated organelle complex to be incorporated into the target cell.

25. The ligand is Differential expression occurs between target cells and non-target cells. Not present on non-target cells, and / or A population of coated organelle complexes according to any one of claims 23 to 24, which are overexpressed on target cells.

26. The target cells are normally present in the target tissue, the target tissue is a site of cancer, inflammation, injury, dysfunction, infection, disease or impairment, and / or is in close proximity to a site of disease or impairment, and optionally the tissue is the adrenal gland, appendix, bladder, bone, intestinal tissue, brain, breast, bronchi, coronary tissue, ear tissue, esophageal tissue, eye tissue, gallbladder tissue, reproductive organ tissue, cardiac tissue, hypothalamic tissue, kidney tissue, colon tissue, intestinal tissue, larynx A population of coated organelle complexes according to any one of claims 23 to 25, comprising tissues, liver tissue, lung tissue, lymph nodes, oral tissue, nasal tissue, pancreatic tissue, parathyroid tissue, pituitary tissue, prostate tissue, rectal tissue, salivary gland tissue, skeletal muscle tissue, skin tissue, small intestine tissue, spinal cord, spleen tissue, stomach tissue, thymus tissue, tracheal tissue, thyroid tissue, ureteral tissue, urethral tissue, soft tissue and connective tissue, peritoneal tissue, vascular tissue, and / or adipose tissue.

27. The coated organelle complex population according to any one of claims 21 to 26, wherein the targeting agent is configured to bind to axons.

28. The coated organelle complex population according to any one of claims 21 to 27, wherein the targeting agent is a peptide, an antigen-binding domain, a cytokine, a chemokine, an aptamer, a growth factor, a hormone, a cytokine, an interleukin, a receptor, or any combination thereof.

29. The antigen-binding domain is an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single-domain antibody (SDAB), VH or VL domain, camelid VHH domain, Fab', F(ab') ２ The coated organelle complex population according to claim 28, comprising Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single-domain antibody (sdAb), single chain containing anti-complementary scFv (tandem scFv) or bispecific tandem scFv, Fv construct, disulfide-linked Fv, bivariable-domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, afibody, affin, afitin, affimer, alphabody, antikalin, avimer, DARPin, finomer, Knitz domain peptide, monobody, or any combination thereof.

30. The coated organelle complex population according to any one of claims 1 to 29, wherein the one or more lipid-polymer conjugates further comprises a detectable portion configured to detect the coated organelle complex in vivo and / or in vitro.

31. The coated organelle complex population according to claim 30, wherein the detectable portion optionally comprises fluorescent molecules selected from the group including fluorescein amidite (FAM), fluorocein dye, carbocyanin, merocyanine, styryl dye, oxonol dye, phycoerythrin, erythrosine, eosin, rhodamine dye, coumarin, coumarin dye, Oregon Green dye, Texas Red, Texas Red-X, Spectrum Red (trademark), Spectrum Green (trademark), cyanine dye, fluorescent dye, BODIPY dye, derivatives thereof, or any combination thereof.

32. The coated organelle complex population according to any one of claims 30 to 31, wherein the detectable portion comprises a fluorescent protein arbitrarily selected from the group including green fluorescent protein (GFP), enhanced GFP (EGFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP), derivatives thereof, or any combination thereof.

33. The detectable portion optionally includes radioisotopes detectable by single photon emission computed tomography (SPECT) and / or positron emission tomography (PET) selected from the group consisting of iodine-131 ( １３１ I), iodine-125 ( １２５ I), bismuth-212 ( ２１２ Bi), bismuth-213 ( ２１３ Bi), astatine-221 ( ２１１ At), copper-67 ( ６７ Cu), copper-64 ( ６４ Cu), rhenium-186 ( １８６ Re), rhenium-188 ( １８８ Re), phosphorus-32 ( ３２ P), samarium-153 ( １５３ Sm), lutetium-177 ( １７７ Lu), technetium-99m ( ９９ｍ Tc), gallium-67 ( ６７ Ga), indium-111 ( １１１ In), and thallium-201 ( ２０１ Tl), and is a coated organelle complex population according to any one of claims 30 to 32.

34. The coated organelle complex population according to any one of claims 30 to 33, wherein the detectable portion optionally includes quantum dot (Qdot) fluorescent particles selected from the group including Qdot 525, Qdot 565, Qdot 585, Qdot 605, Qdot 625, Qdot 655, Qdot 705, Qdot 800, derivatives thereof, or any combination thereof.

35. The coated organelle complex population according to any one of claims 1 to 34, wherein the one or more lipid-polymer conjugates further comprises one or more secondary agents, optionally a therapeutic agent, and optionally a small molecule drug.

36. The coated organelle complex population according to claim 35, wherein one or more secondary agents are anticancer agents, anti-inflammatory agents, anti-infective agents, regenerative agents, relaxants, apoptosis inhibitors, apoptosis inducers, anticoagulants, antioxidant molecules, autophagy inducers, dermatological agents, proliferation stimulants, vasodilators, vasoconstrictors, analgesics, and anti-allergic agents, condensation modifiers (c-MODs), or a combination thereof.

37. The coated organelle complex population according to any one of claims 35 to 36, wherein one or more secondary agents are chemotherapeutic agents, nucleic acids, polysaccharides, peptides, polypeptides, or any combination thereof.

38. The coated organelle complex population according to any one of claims 35 to 37, wherein one or more secondary agents are a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor of an immunosuppressive molecule, an immunomodulator, an anti-metastatic, chemotherapeutic, hormone or growth factor antagonist, an alkylating agent, a TLR agonist, a cytokine antagonist, a cytokine antagonist, or any combination thereof.

39. A population of coated organelle complexes according to any one of claims 35 to 38, wherein one or more secondary agents are agonist antibodies or antagonist antibodies specific to checkpoint inhibitors or checkpoint stimulant molecules such as PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, and TIM-3.

40. A method for generating a population of coated organelle complexes, The first method involves contacting an organelle complex in a solution with one or more lipid-polymer conjugates to generate a coated organelle complex, A method comprising recovering the coated organelle complex from the first solution to generate a population of the coated organelle complex.

41. The method described above is The method according to claim 40, further comprising optionally incubating the first solution after the contact step for about 1 minute to about 120 minutes, and more optionally for about 15 minutes.

42. The method according to any one of claims 40 to 41, wherein the contact step comprises applying a physical stimulus to the first solution, optionally shaking, mixing, and / or stirring, and further optionally pipetting.

43. The method according to any one of claims 40 to 42, wherein the incubation step is performed at a first temperature.

44. The method according to any one of claims 40 to 43, wherein the organelle complex is present in the first solution at a concentration of about 0.01 mg / mL to about 10 mg / mL, and optionally, about 0.1 mg / mL to about 1 mg / mL, and optionally, about 1 mg / mL.

45. The method according to any one of claims 40 to 44, wherein the contacting step comprises contacting the first solution with a solution containing about 1 μL to about 1000 μL of one or more lipid-polymer conjugates, wherein the one or more lipid-polymer conjugates are optionally present in 100 μL of a 1 mM solution at a concentration of about 0.1 mM to about 10 mM.

46. The method according to any one of claims 40 to 45, wherein recovering the coated organelle complex from the first solution comprises one or more centrifugation steps.

47. The coated organelle complex can be recovered from the first solution. The first solution is centrifuged using a first centrifugal force. The method according to any one of claims 40 to 46, comprising collecting the pellets and recovering the coated organelle complex.

48. The method according to any one of claims 40 to 47, wherein the first centrifugal force is approximately 100 g to approximately 10,000 g, and optionally, the first centrifugal force is approximately 3,000 g.

49. The method according to any one of claims 40 to 48, wherein collecting the pellet comprises resuspending the coated organelle complex population in a second solution, the second solution having a volume of about 50 uL to about 50 mL, further optionally about 100 μL to about 1000 μL, and optionally about 1000 uL.

50. The method according to any one of claims 40 to 49, wherein the centrifugal separation step is carried out for at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, or about 20 minutes.

51. The method according to any one of claims 40 to 50, wherein the centrifugal separation step is performed at a second temperature.

52. The method according to any one of claims 40 to 51, wherein the first temperature and / or the second temperature is about 0°C to about 50°C, and optionally the first temperature is about 20°C to about 25°C and the second temperature is about 0°C to about 4°C.

53. The method according to any one of claims 40 to 52, wherein the recovery step depletes the one or more lipid-polymer conjugates that are not incorporated into the surface of the organelle complex, and optionally, the one or more lipid-polymer conjugates that are not incorporated into the surface of the organelle complex are present as micelles.

54. A population of coated organelle complexes obtained by the method described in any one of claims 40 to 53.

55. A method for introducing coated organelle complexes into host cells, The process involves bringing a population of coated organelle complexes according to any one of claims 1 to 39 or 54 into contact with a population of host cells, A method by which the coated organelle complex can be incorporated into the host cell by bringing the coated organelle complex into contact with the host cell.

56. The host cells include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia, neurons, spleen cells, lymphocytes, lung cells, lung epithelial cells, skin cells, keratinocytes, endothelial cells, alveolar cells, alveolar macrophages, alveolar lung cells, vascular endothelial cells, mesenchymal cells, epithelial cells, colon epithelial cells, hematopoietic cells, bone marrow cells, Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophilic cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, theca cells, cementoocytes, chief cells, chondrocytes, chondrocytes, chromaffin cells, chromophobic cells, adrenocorticotropic hormone-producing cells, delta cells, Langerhans cells, Follicular dendritic cells, enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous epithelial cells, endothelial cells, transitional cells, erythroid cells, erythrocytes, fibroblasts, fibrocytes, follicular cells, germ cells, gametes, ova , sperm, oocyte, primary oocyte, secondary oocyte, spermatocyte, spermatocyte, primary spermatocyte, secondary spermatocyte, reproductive epithelium, giant cell, glial cell, astroblast, astrocyte, oligodendroglioblast, oligodendrocyte Cells, glioblasts, goblet cells, gonadotropin-secreting cells, granulosa cells, hemoblasts, hair cells, hepatoblasts, hepatocytes, vitreous cells, stromal cells, juxtaglomerular cells, keratinocytes, keratocytes, fibrous sheath cells, leukocytes, granulocytes, basophils, eosinophils, neutrophils, lymphoblasts, B lymphoblasts, T lymphoblasts, lymphocytes, B lymphocytes, T lymphocytes, helper-induced T lymphocytes, Th1 T lymphocytes, Th2T lymphocytes, natural killer cells, thymocytes, macrophages, Kupffer cells, alveolar macrophages, foam cells, histiocytes, luteal cells, lymphocyte stem cells, lymphocyte cells, lymphocyte stem cells, macroglia cells, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanin-forming blasts, melanin-forming cells, mesangial cells, mesothelial cells, metamyelocytes, monoblasts, monocytes, myxocervular cells, myoblasts, muscle cells, muscle cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, myelocytes, myeloid cells, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblasts, neuroblasts, neuroepithelial cells, neurons, odontoblasts, osteoblasts, osteoclasts, osteocytes The method according to claim 55, comprising one or more mammalian cells selected from the group comprising acid-secreting cells, parafollicular cells, paracorpus luteum cells, digestive cells, pericytes, peripheral blood mononuclear cells, pheochromocytes, phalangeal cells, pineal cells, pituitary cells, plasma cells, platelets, podocytes, proerythroblasts, promonocytes, promyeloblasts, promyelocytes, pronoblasts, reticulocytes, retinal pigment epithelial cells, retinoblasts, small cells, growth hormone-secreting cells, stem cells, supporting cells, teloglycytic cells, enzyme-forming cells, or any combination thereof, wherein the stem cells optionally comprise embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells / progenitor cells (HSPCs), or any combination thereof.

57. The method according to any one of claims 55 to 56, wherein the host cell is, or optionally, a target cell suffering from a disease or disorder, and further optionally, the disease or disorder is a blood disorder, an immune disorder, cancer, an infectious disease, a genetic disorder, a disorder caused by abnormal mtDNA, a metabolic disorder, a disorder caused by an abnormal cell cycle, a disorder caused by abnormal angiogenesis, a disorder caused by abnormal DNA damage repair, or any combination thereof.

58. The method according to any one of claims 55 to 57, wherein the contact is carried out for a period of at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, or about 24 hours.

59. The method comprises conjugating a targeting agent, a detectable portion, and / or one or more secondary agents with the one or more lipid-polymer conjugates, The polymer, the targeting agent, the detectable portion, and / or the one or more secondary agents are functionalized with functional groups selected from the group consisting of vinyl, carboxylate, hydroxyl, epoxide, sulfhydryl, amide, acrylate, thiol, azide, maleimide, isocyanate, aziridine, carbonate, N-hydroxysuccinimide, ester, imide ester, carbodiimide, anhydride, succinimidyl carbonate, and amine, as well as combinations thereof. The method according to any one of claims 55 to 58, wherein the conjugation optionally includes contacting the targeting agent, the detectable portion, and / or the one or more secondary agents with the one or more lipid-polymer conjugate for at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes, at a temperature of about 0°C to about 50°C, optionally, at a temperature of about 20°C to about 25°C or about 0°C to about 4°C.

60. The method according to any one of claims 55 to 59, wherein when the coated organelle complex population is brought into contact with the host cell population, the coated organelle complex has superior integration ability into host cells compared to organelle complexes that do not contain the one or more lipid-polymer conjugates, and optionally comprises a targeting agent configured to bind the one or more lipid-polymer conjugates to ligands on the surface of the host cells.

61. A population of host cells comprising coated organelle complexes produced according to the method of any one of claims 55 to 60.

62. (i) a population of coated organelle complexes according to any one of claims 1 to 39 or 54, or (ii) a population of host cells comprising the coated organelle complexes according to claim 61.

63. A pharmaceutical composition, (i) a population of coated organelle complexes according to any one of claims 1 to 39 or 54, or (ii) a population of host cells comprising the coated organelle complexes according to claim 61, comprising one or more pharmaceutically acceptable carriers, A pharmaceutical composition further comprising, optionally, one or more secondary agents.

64. A method for treating or preventing a disease or disorder, The target cells that require it, in an effective amount, (i) A population of coated organelle complexes according to any one of claims 1 to 39 or 54, (ii) A population of host cells comprising the coated organelle complex according to claim 61, (iii) The composition according to claim 62, and / or (iv) Contacting with the pharmaceutical composition described in claim 63, A method comprising treating or preventing the disease or disorder in the subject thereto.

65. The method according to claim 64, wherein the contact is performed ex vivo, in vitro, or in vivo.

66. The method according to any one of claims 64 to 65, wherein the effective amount comprises at least about 1 ug to about 1 mg of the coated organelle complex population.

67. The method according to any one of claims 64 to 66, wherein the subject is a mammal.

68. The method according to any one of claims 64 to 67, wherein at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the coated organelle complex population is incorporated into the target cell(s) and / or target tissue(s).

69. The method according to any one of claims 64 to 68, wherein about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or less than about 100% of the coated organelle complex population is incorporated into the target non-target cells and / or non-target tissues.

70. The method according to any one of claims 64 to 69, wherein the disease or disorder is selected from the group consisting of diabetes mellitus (type I and type II), metabolic disorders, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic drugs, mitochondrial dysfunction associated with space travel, cardiotoxicity associated with chemotherapy or other therapeutic drugs, mitochondrial dysfunction, and migraines.

71. The method according to any one of claims 64 to 70, wherein the disease or disorder is selected from the group consisting of mitochondrial myopathy, diabetes mellitus and deafness (DAD) syndrome, Barth syndrome, Leber hereditary optic neuropathy (LHON), Leigh syndrome, NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis syndrome), myocardial gastroenteropathy (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episode) syndrome, myoclonus epilepsy with red rag fibers (MERRF) syndrome, Kearns-Sayre syndrome, and mitochondrial DNA depletion syndrome.

72. The method according to any one of claims 64 to 71, wherein the disease or disorder is an ischemia-related disease or disorder, a genetic disorder, an age-related disease or disorder, a neurodegenerative state, a cardiovascular state, cancer, an autoimmune disease, an inflammatory disease, a fibrous disorder, or any combination thereof.

73. The method according to any one of claims 64 to 72, wherein the ischemia-related disease or disorder is selected from the group consisting of cerebral ischemic reperfusion, hypoxic-ischemic encephalopathy, acute coronary artery syndrome, myocardial infarction, hepatic ischemic reperfusion injury, ischemic compartmental injury syndrome, vascular occlusion, wound healing, spinal cord injury, sickle cell disease, severe limb ischemia, and reperfusion injury of transplanted organs.

74. The method according to any one of claims 64 to 73, wherein the neurodegenerative state is selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial encephalopathy, lactic acidosis, and stroke-like episode (MELAS), myoclonus epilepsy with red ragged fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addiction, anxiety disorders, attention deficit disorder, personality disorders, autism, and Asperger's syndrome.

75. The method according to any one of claims 64 to 74, wherein the cardiovascular condition is selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmia, stroke, deep vein thrombosis, and pulmonary embolism.

76. The method according to any one of claims 64 to 75, wherein the disease or disorder is acute respiratory distress syndrome (ARDS), or pre-eclampsia, or intrauterine growth restriction (IUGR), or fetal growth restriction (FGR).

77. The method according to any one of claims 64 to 76, wherein the disease or disorder is associated with the expression of a tumor antigen, and the disease associated with the expression of the tumor antigen is selected from the group consisting of proliferative disorders, precancerous conditions, cancer, and non-cancer-related indications associated with the expression of the tumor antigen.

78. The aforementioned cancers include colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell lung cancer, mesothelioma, small intestine cancer, esophageal cancer, malignant melanoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eyeball, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, endocrine cancer, and thyroid cancer. The method according to any one of claims 64 to 77, selected from the group consisting of parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of the aforementioned cancers, and metastatic lesions of the aforementioned cancers.

79. The aforementioned cancers include chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, pilocytic cell leukemia, and small cell or large cell follicular lymphoma. The method according to any one of claims 64 to 78, wherein the hematological cancer is selected from one or more of the following: pediatric cancer, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström hypergammaglobulinemia, or preleukemia.

80. The method according to any one of claims 64 to 79, wherein the contact includes systemic administration, subarachnoid administration, intracranial injection, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracisional delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumor injection, intraperitoneal injection, intradermal injection, inhalation, intrapulmonary administration, and intraocular administration, or any combination thereof, wherein the systemic administration is optionally intravenous, intramuscular, intraperitoneal, or intraarticular.

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