Mitochondria as a Targeted Delivery Platform

Abstract

The present invention focuses on methods for delivering various payloads including nucleic acids (such as oligonucleotides), polypeptides (such as proteins), drugs, or combinations thereof. Thus, the present invention relates, inter alia, to mitochondria containing one or more payloads added to the outer membrane of the mitochondria, the payloads being electrostatically added indirectly or directly to the outer membrane of the mitochondria. The present invention further involves providing a further delivery platform by combining mitochondria containing one or more payloads added to the outer membrane of the mitochondria with a protective layer that surrounds / encloses and / or coats the mitochondria and the payloads. This methodology is particularly effective for enhancing the uptake and efficiency of one or more payloads for therapeutic purposes.

Description

Technical Field

[0001] The present invention focuses on methods for delivering various payloads including nucleic acids (such as oligonucleotides), polypeptides (such as proteins), drugs, or combinations thereof. Thus, the present invention relates, inter alia, to mitochondria comprising one or more payloads attached to the outer mitochondrial membrane, the payloads being electrostatically attached indirectly or directly to the outer mitochondrial membrane. The present invention further involves providing a further delivery platform by combining mitochondria comprising one or more payloads attached to the outer mitochondrial membrane with a protective layer that surrounds / encloses and / or coats the mitochondria and the payload. This methodology is particularly effective in enhancing the uptake and efficiency of one or more payloads for therapeutic purposes.

Background Art

[0002] The delivery of nucleic acid molecules such as DNA and RNA, polypeptides such as proteins, drugs, or combinations thereof to cells and tissues remains a major challenge in the field of biotechnology. Direct injection of naked DNA and RNA has been shown to have low transfection efficiency in vitro, ex vivo, and in vivo (Non-Patent Document 1). DNA and RNA molecules are large in size, have low stability in biological media, and are susceptible to degradation by nucleases. Viral vectors combined with synthetic lipids or nanoparticles have been used as delivery platforms, but most of these combination products often induce undesirable immune responses, have low transfection efficiency, and can be toxic in the long term (Non-Patent Documents 2 and 3). Furthermore, when interacting with blood, the formation of protein coronas can lead to abnormal biodistribution, mistargeting, unexpected toxicity, and low therapeutic efficacy (Non-Patent Document 4).

[0003] Isolated mitochondria have been found to be biocompatible and non-toxic materials and can be effectively taken up by cells via endocytosis, as reported in the study by Pacak et al. (Non-Patent Document 5). These organelles also have a specific distribution targeting specific organs (e.g., but not limited to, the heart, lungs, or kidneys) (Non-Patent Document 6). Mitochondria are also immune-silent (Non-Patent Document 7) and can thus be an attractive delivery platform. However, mitochondria have not been well utilized as vehicles for delivering various payloads.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, there is an urgent need to develop biocompatible vectors or delivery platforms that can overcome the above limitations.

Means for Solving the Problems

[0005] The technical problems are solved by the embodiments provided herein and presented in the claims.

[0006] Therefore, the present invention relates, inter alia, to the following items.

[0007] 1. Mitochondria comprising one or more payloads attached to the outer membrane of the mitochondria, wherein the payload is electrostatically attached to the outer membrane of the mitochondria either indirectly or directly.

[0008] 2. The mitochondria according to item 1, wherein the payload is i) a nucleic acid molecule, ii) a polypeptide, iii) a drug, or iv) one or more combinations of (i) to (iii) and is one or more of the above.

[0009] 3. The mitochondrion according to item 1 or 2, wherein the payload is charged.

[0010] 4. The mitochondrion according to any one of items 1 to 3, wherein the payload has the same net charge as the net charge of the mitochondrion.

[0011] 5. The mitochondrion according to item 4, wherein both the payload and the mitochondrion have a net negative charge, and the payload is added to the mitochondrion via a positively charged species.

[0012] 6. The mitochondrion according to item 5, wherein the positively charged species is a polycationic species.

[0013] 7. The mitochondrion according to item 6, wherein the polycationic species is a linear or branched polycationic polymer.

[0014] 8. The mitochondrion according to item 7, wherein the linear or branched polycationic polymer is polylysine, histidyl-polylysine, polyornithine, polyarginine, high mobility group protein (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivative, diethylaminoethyl (DEAE)-dextran, poly(N-alkyl-4-vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose, or a combination thereof.

[0015] 9. The mitochondrion according to item 5, wherein the positively charged species is a positively charged nanoparticle.

[0016] 10. The mitochondrion according to item 5, wherein the positively charged species is a positively charged particle.

[0017] 11. The mitochondrion according to item 9, wherein one or more nucleic acid molecules are attached to the surface of a positively charged nanoparticle or encapsulated in a positively charged nanoparticle.

[0018] 12. The mitochondrion according to item 10, wherein one or more nucleic acid molecules are attached to the surface of a positively charged particle or encapsulated in a positively charged particle.

[0019] 13. The mitochondrion according to any one of items 9 to 12, wherein the positively charged nanoparticle and / or particle is a lipid nanoparticle / particle, dendrimer nanoparticle / particle, micelle nanoparticle / particle, protein nanoparticle / particle, liposome, non-porous silica nanoparticle / particle, mesoporous silica nanoparticle / particle, silicon nanoparticle / particle, gold nanoparticle / particle, gold nanowire, silver nanoparticle / particle, platinum nanoparticle / particle, palladium nanoparticle / particle, titanium dioxide nanoparticle / particle, carbon nanotube, carbon dot nanoparticle / particle, polymer nanoparticle / particle, zeolite nanoparticle / particle, aluminum oxide nanoparticle / particle, hydroxyapatite nanoparticle / particle, quantum dot nanoparticle / particle, zinc oxide nanoparticle / particle, zirconium dioxide nanoparticle / particle, graphene or graphene oxide nanoparticle / particle.

[0020] 14. The mitochondrion according to any one of items 1 to 3, wherein the payload has a net charge different from the net charge of the mitochondrion.

[0021] 15. The mitochondrion according to item 14, wherein the payload and the mitochondrion are added via zwitterionic species.

[0022] 16. The mitochondrion according to item 14, wherein the payload is uncharged and the payload is added to a positively charged species.

[0023] 17. The mitochondrion according to item 16, wherein the positively charged species is as defined in any one of items 6 to 13.

[0024] 18. One or more nucleic acid molecules are electrostatically linked to an antibody, optionally the antibody is a modified antibody, and optionally the modified antibody has one or more positive charges, the mitochondrion according to item 2.

[0025] 19. One or more nucleic acid molecules are encapsulated in nanoparticles, the nanoparticles are electrostatically linked to an antibody, optionally the antibody is a modified antibody, and optionally the modified antibody has one or more positive charges, the mitochondrion according to item 2.

[0026] 20. The antibody specifically binds to an antigen contained in the outer membrane of the mitochondrion, and the antigen is OPA1, TOM70, TOMM20, mitofusin 1, mitofusin 2 or VDAC1, the mitochondrion according to item 18 or 19.

[0027] 21. The mitochondrion is linked to a protective layer and / or surrounded by a protective layer, the mitochondrion according to any one of items 1 to 20.

[0028] 22. The protective layer is a protective polymer, the mitochondrion according to item 21.

[0029] 23. The protective polymer is a linear or branched cationic polymer, and optionally the linear or branched cationic polymer is electrostatically linked to one or more payloads, the mitochondrion according to item 22.

[0030] 24. The protective polymer is a linear or branched cationic block copolymer, and optionally the linear or branched cationic block copolymer is electrostatically linked to one or more payloads, the mitochondrion according to item 22.

[0031] 25. The protective polymer is a cationic graft (g) copolymer, and optionally the cationic graft (g) copolymer is electrostatically linked to one or more payloads, the mitochondrion according to item 22.

[0032] 26. The mitochondrion according to item 22, wherein the protective polymer is a linear or branched pegylated (PEG) cationic polymer, and optionally, the linear or branched pegylated (PEG) cationic polymer is electrostatically linked to one or more payloads.

[0033] 27. The mitochondrion according to item 21, wherein the protective layer is a lipid formulation, optionally the lipid formulation is a cationic lipid formulation, and further optionally, the cationic lipid formulation is electrostatically linked to one or more payloads.

[0034] 28. The mitochondrion according to any one of items 21 to 27, wherein the protective layer is linked to a targeting moiety.

[0035] 29. The mitochondrion according to any one of items 21 to 28, wherein the protective layer is linked to an antibody, and optionally, the protective layer linked to the antibody is electrostatically linked to one or more payloads or the protective layer linked to the antibody is covalently linked to one or more payloads.

[0036] 30. The mitochondrion according to any one of items 21 to 28, wherein the protective layer is linked to a carbohydrate, and optionally, the protective layer linked to the carbohydrate is electrostatically linked to one or more payloads or the protective layer linked to the carbohydrate is covalently linked to one or more payloads.

[0037] 31. The mitochondrion according to item 23, wherein the linear or branched cationic polymer is polyethyleneimine, RGD-modified polyethyleneimine, polylysine, RGD-modified polylysine, polyornithine, RGD-modified polyornithine, polyarginine, RGD-modified polyarginine, polypropyleneimine, RGD-modified polypropyleneimine, polyallylamine, RGD-modified polyallylamine, chitosan, RGD-modified chitosan, poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(2-(dimethylamino)ethyl methacrylate), poly(amidoamine), RGD-modified poly(amidoamine), or a combination thereof.

[0038] 32. The mitochondria according to item 24, wherein the cationic block copolymer is poly(ethylene glycol)-block-polyethyleneimine, RGD-modified poly(ethylene glycol)-block-polyethyleneimine, poly(ethylene glycol)-block-polylysine, RGD-modified poly(ethylene glycol)-block-polylysine, poly(ethylene glycol)-block-polyornithine, RGD-modified poly(ethylene glycol)-block-polyornithine, poly(ethylene glycol)-block-polyarginine, RGD-modified poly(ethylene glycol)-block-polyarginine, poly(ethylene glycol)-block-polypropyleneimine, RGD-modified poly(ethylene glycol)-block-polypropyleneimine, poly(ethylene glycol)-block-polyallylamine, RGD-modified poly(ethylene glycol)-block-polyallylamine, poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-block-poly(amidoamine), RGD-modified poly(ethylene glycol)-block-poly(amidoamine), or a combination thereof.

[0039] 33. The mitochondrial of item 25, wherein the cationic graft (g) copolymer is poly(ethylene glycol)-g-polyethyleneimine, RGD-modified poly(ethylene glycol)-g-polyethyleneimine, poly(ethylene glycol)-g-polylysine, RGD-modified poly(ethylene glycol)-g-polylysine, poly(ethylene glycol)-g-polyornithine, RGD-modified poly(ethylene glycol)-g-polyornithine, poly(ethylene glycol)-g-polyarginine, RGD-modified poly(ethylene glycol)-g-polyarginine, poly(ethylene glycol)-g-polypropyleneimine, RGD-modified poly(ethylene glycol)-g-polypropyleneimine, poly(ethylene glycol)-g-polyallylamine, RGD-modified poly(ethylene glycol)-g-polyallylamine, poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-g-poly(amidoamine), RGD-modified poly(ethylene glycol)-g-poly(amidoamine), or a combination thereof.

[0040] 34. The mitochondrial of item 26, wherein the pegylated (PEG) cationic polymer is pegylated polyethyleneimine, RGD-modified pegylated polyethyleneimine, pegylated polylysine, RGD-modified pegylated polylysine, histidyl polylysine, pegylated polyornithine, RGD-modified pegylated polyornithine, pegylated polyarginine, RGD-modified pegylated polyarginine, pegylated polypropyleneimine, RGD-modified pegylated polypropyleneimine, pegylated polyallylamine, RGD-modified pegylated polyallylamine, pegylated chitosan, RGD-modified pegylated chitosan, pegylated poly(2-(dimethylamino)ethyl methacrylate), RGD-modified pegylated poly(2-(dimethylamino)ethyl methacrylate), pegylated poly(amidoamine), RGD-modified pegylated poly(amidoamine), or a combination thereof.

[0041] 35. The mitochondrial of item 27, wherein the lipid preparation comprises DC-cholesterol (3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DLinDMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleyloxy-3-dimethylaminopropane), DOGS (dioctadecylamidoglycyl spermine), DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium), DOTAP (1,2-dioleoyl-3-trimethylammonium propane chloride), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane chloride), UGG (unsaturated guanidinium glycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), Lipofectamine or a combination thereof.

[0042] 36. The mitochondrial of item 35, wherein the lipid preparation further comprises another lipid, preferably the lipid is cholesterol, substituted or unsubstituted cholesterol, cholesterol derivative, such as hydroxylated cholesterol derivative (e.g., hydroxy cholesterol), PEG-lipid, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DDA (dimethyldioctadecylammonium), 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, bis(monooleoylglycero)phosphate or a combination thereof.

[0043] 37. The mitochondrial of item 22, wherein the mitochondrial is linked to and / or surrounded by an amphoteric protective polymer, and optionally, the amphoteric protective polymer is electrostatically linked to one or more payloads.

[0044] 38. The mitochondria according to item 37, wherein the zwitterionic protective polymer is selected from the co-assembly of cationic (carboxyl-functionalized) and anionic (amino-functionalized) copolyesters based on poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyethyleneimine-g-poly(2-methacryloyloxyethyl phosphorylcholine) (PEI-g-PMPC), poly(ε-caprolactone)-block-poly(butylene fumarate)-block-poly(ε-caprolactone) (PCL-b-PBF-b-PCL), poly(lactic acid-co-glycolic acid) (PLGA)-PCB block copolymer (PLGA-b-PCB).

[0045] 39. A composition comprising a plurality of mitochondria according to any one of items 1 to 38.

[0046] 40. A pharmaceutical composition comprising a plurality of mitochondria according to any one of items 1 to 38 and a pharmaceutically acceptable carrier.

[0047] 41. The pharmaceutical composition according to item 40, wherein the pharmaceutical composition is formulated as a solution.

[0048] 42. The pharmaceutical composition according to item 40, wherein the pharmaceutical composition is formulated as an aerosol.

[0049] 43. The mitochondria according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 42 for use as a medicament.

[0050] 44. The mitochondria according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 42 for use in gene therapy.

[0051] 45. The mitochondrion according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 42 for use in the treatment of cardiovascular diseases, in particular for use in the treatment of ischemic heart diseases, ischemia-reperfusion injuries or atherosclerosis.

[0052] 46. The mitochondrion according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 42 for use in the treatment of aging-related diseases, in particular for use in the treatment of sarcopenia, Parkinson's disease or Hutchinson-Gilford progeria syndrome.

[0053] 47. The mitochondrion according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 42 for use in the treatment of renal diseases, in particular for use in the treatment of autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis or Fabry disease.

[0054] 48. The mitochondrion according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 42 for use in the treatment of cancer.

[0055] 49. The mitochondrion according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 42 for use in in vitro, ex vivo, or in vivo genome editing.

[0056] 50. The mitochondrion according to any one of items 1 to 38, the composition according to item 39, or the pharmaceutical composition according to any one of items 40 to 43 for use in radiotherapy.

[0057] 51. A method for delivering a payload to a target organ, comprising the step of administering the pharmaceutical composition according to any one of items 40 to 42 into the bloodstream of a subject in need thereof, wherein the pharmaceutical composition is administered into the bloodstream upstream of the target organ.

[0058] 52. A method for delivering a payload to the lungs, comprising administering to a subject in need of the pharmaceutical composition according to item 42, wherein the pharmaceutical composition is administered by inhalation, the method for delivering a payload to the lungs.

[0059] 53. A method for attaching a payload to the outer membrane of mitochondria, comprising: a) providing a preparation of mitochondria; and b) contacting the mitochondria provided in step (a) with at least one payload in the presence of a positively charged species; and c) attaching at least one payload to the mitochondria via the positively charged species. A method for attaching a payload to the outer membrane of mitochondria, comprising the steps of:

[0060] 54. a) contacting at least one payload simultaneously with a positively charged species and mitochondria; b) contacting at least one payload with a positively charged species to form a positively charged complex before contacting the positively charged complex with mitochondria, or c) contacting mitochondria with a positively charged species and subsequently with at least one payload. The method according to item 53.

[0061] 55. The method according to item 52 or 54, wherein the mitochondria are contacted with at least one payload and a positively charged species in a suitable buffer.

[0062] 56. The buffer comprises, or consists of, HEPES, EGTA, trehalose, CHES and sodium phosphate dihydrate, preferably, the buffer comprises, or consists of, a mixture of solution X which comprises, or consists of, HEPES, EGTA and trehalose and solution Y which comprises, or consists of, CHES and sodium phosphate dihydrate, more preferably, the buffer comprises, or consists of, a 4:1 mixture of solution X which comprises, or consists of, 20 mM HEPES, 1 mM EGTA and 300 mM trehalose (pH 7.2) and solution Y which comprises, or consists of, 0.1 M CHES (pH 10) and 0.2 M sodium phosphate dihydrate, the method according to item 55.

[0063] 57. The method according to any one of items 53 to 56, wherein the mitochondria are contacted with at least one payload and a positively charged species at room temperature for at least 5 minutes, such as at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes or 120 minutes.

[0064] 58. The method according to any one of items 53 to 57, wherein the mitochondria are contacted with at least one payload and a positively charged species in the dark.

[0065] 59. The method according to any one of items 53 to 58, wherein the payload is a nucleic acid molecule which is DNA or RNA.

[0066] 60. The method according to any one of items 53 to 59, wherein the positively charged species is a polycationic species, the polycationic species is a linear or branched polycationic polymer, and optionally the linear or branched polycationic polymer is electrostatically linked to at least one payload.

[0067] 61. The method according to item 60, wherein the linear or branched polycationic polymer is polylysine, histidyl - polylysine, polyornithine, polyarginine, high - mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2 - (dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivative, diethylaminoethyl (DEAE) - dextran, poly(N - alkyl - 4 - vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose or a combination thereof.

[0068] 62. The method according to any one of items 53 - 59, wherein the positively charged species is a positively charged nanoparticle.

[0069] 63. The method further comprises the step a) adding at least one payload to the surface of the positively charged nanoparticle, or b) encapsulating at least one payload within the positively charged nanoparticle The method according to item 62.

[0070] 64. The method according to item 62 or 63, wherein the positively charged nanoparticle is a lipid nanoparticle, dendrimer nanoparticle, micelle nanoparticle, protein nanoparticle, liposome, non - porous silica nanoparticle, mesoporous silica nanoparticle, silicon nanoparticle, gold nanoparticle, gold nanowire, silver nanoparticle, platinum nanoparticle, palladium nanoparticle, titanium dioxide nanoparticle, carbon nanotube, carbon dot nanoparticle, polymer nanoparticle, zeolite nanoparticle, aluminum oxide nanoparticle, hydroxyapatite nanoparticle, quantum dot nanoparticle, zinc oxide nanoparticle, zirconium dioxide nanoparticle, graphene or graphene oxide nanoparticle.

[0071] 65. A method for preparing mitochondria containing a payload, comprising a) providing a preparation of mitochondria, and b) subjecting the mitochondria to i) When both the payload and the mitochondria have a net negative charge, a positively charged species, ii) When the payload has a net charge different from the net charge of the mitochondria, the payload, optionally further a zwitterionic species, or iii) When the payload is uncharged, the payload added to a positively charged species contacting step; c) obtaining the mitochondria according to any one of items 1 to 20, A method for preparing mitochondria containing a payload, comprising:

[0072] 66. After step c), further comprising the step of contacting the mitochondria with a component to form a protective layer, and obtaining the mitochondria according to any one of items 22 to 38, The method according to item 65.

[0073] 67. Contacting 50 μg to 200 μg of mitochondria with 0.1 to 50 pmol of payload and 0.02 to 10 μg, preferably 0.02 to 5 μg of a positively charged species, The method according to any one of items 53 to 66.

[0074] 68. The mitochondria contain a positively charged species, the positively charged species is a polycationic polymer according to any one of the previous items, and the ratio of the polycationic polymer to the protective layer is about 1:2, The method according to any one of items 53 to 66.

[0075] 69. Contacting 50 μg to 200 μg of mitochondria with 0.1 to 50 pmol of payload and 0.2 to 10 μg of a protective layer, The method according to any one of the previous method items.

[0076] 70. A method for delivering a payload to the kidney, comprising administering to the renal artery of a subject in need of the pharmaceutical composition according to items 40 to 50, A method for delivering a payload to the kidney.

[0077] 71. A method for delivering a payload to the heart, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the coronary artery of a subject in need thereof.

[0078] 72. A method for delivering a payload to the liver, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the hepatic artery or portal vein of a subject in need thereof.

[0079] 73. A method for delivering a payload to the pancreas, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the hepatic artery of a subject in need thereof.

[0080] 74. A method for delivering a payload to the duodenum, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the hepatic artery of a subject in need thereof.

[0081] 75. A method for delivering a payload to the spleen, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the splenic artery of a subject in need thereof.

[0082] 76. A method for delivering a payload to the lung, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the pulmonary artery of a subject in need thereof.

[0083] 77. A method for delivering a payload to the intestine, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the superior mesenteric artery of a subject in need thereof.

[0084] 78. A method for delivering a payload to the bladder, comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the superior and inferior vesical arteries of a subject in need thereof.

[0085] A method for delivering a payload to a target organ, comprising administering to a subject in need of the pharmaceutical composition according to items 40 to 50, wherein the pharmaceutical composition is administered to the kidney or bladder or intestine or pancreas or duodenum or liver or lung or spleen by direct injection, a method for delivering a payload to a target organ.

[0086] Thus, in its broadest aspect, the present invention relates to mitochondria to which one or more payloads such as one or more nucleic acid molecules, one or more polypeptides, and / or one or more drugs are added by innovative means.

[0087] More specifically, the present invention relates to and / or utilizes mitochondria complexed with and / or conjugated to oligonucleotides, nucleic acids such as DNA or RNA (e.g., mRNA and / or siRNA), polypeptides, proteins, drugs, or combinations thereof as a platform for targeted and safe delivery to cells and tissues (FIGS. 1-2). The process of producing this mitochondria complex involves functionalizing mitochondria with a cationic species such as a cationic polymer, followed by oligonucleotides, nucleic acid molecules, polypeptides, proteins and / or drugs. The mitochondria complex of the present invention may include a protective polymer layer composed of a cationic copolymer linked to or surrounding the mitochondria to protect one or more additional payloads (e.g., nucleic acid molecules such as oligonucleotides, polypeptides such as proteins and / or drugs) from degradation and to enable efficient internalization of, for example, mitochondria-oligonucleotide complexes or mitochondria-protein complexes. The mitochondria-payload complexes of the present invention, such as mitochondria-oligonucleotide complexes, can escape from digestive organelles (i.e., lysosomes) upon internalization. The isolated mitochondria of the mitochondria-based system of the present invention transport different nucleic acids such as DNA / RNA molecules or proteins and are ideal for maintaining low cytotoxicity while enabling high biological activity of DNA / RNA (e.g., translation, transcription, protein expression, knockdown) when released into cells. This demonstrates that mRNA translation efficiency exceeds 70% in various cell types including human epithelial lung cells (A549, 79%) and human heart fibroblasts (HCF, 70%) compared to Lipofectamine (100%) commonly used as a control. Furthermore, mitochondrial delivery of siRNA containing a protective layer is exemplified to result in greater protein knockdown compared to Lipofectamine-siRNA and also compared to previous generation products as described in European Patent Applications Nos. 22178524.9 and 22211826.7.Furthermore, provided is the use of mitochondria for the co-delivery of one or more, such as two or more, different oligonucleotides (e.g., mRNA and siRNA) or an oligonucleotide and a drug (e.g., an anionic drug) or one or more, such as two or more, oligonucleotides and one or more drugs, such as two or more anionic drugs (e.g., siRNA and PX-12) for oncological use.

[0088] Thus, the delivery platform for mitochondria of the present invention provides several advantages.

[0089] 1. A natural and safe method for delivering payloads such as nucleic acid molecules such as DNA, RNA, polypeptides or drugs.

[0090] 2. It has been shown to be successful in in vivo, ex vivo, or in vitro delivery, as demonstrated by high levels of transcription, translation, and protein expression / knockdown.

[0091] 3. The payload-mitochondria complex has a stabilizing effect on the payload, particularly DNA or RNA, in contrast to naked nucleic acids, which is further improved by adding a protective layer to the mitochondria-oligonucleotide complex.

[0092] 4. The platform does not cause an immune response or cytotoxicity when internalized into cells, in contrast to commonly used delivery systems such as viral vectors.

[0093] 5. It can be administered to cells, tissues, or the whole body via different routes such as injection or aerosol. Furthermore, the mitochondria can be administered as a single dose or as at least two or more doses.

[0094] 6. Delivery of payloads such as nucleic acid molecules, polypeptides or drugs with high colloidal stability via mitochondria.

[0095] 7. A new generation of mitochondrial delivery platforms containing a protective layer is effective in delivering payloads such as nucleic acids, polypeptides, drugs, etc., achieving higher transcription of mRNA and / or higher protein knockdown by siRNA compared to previously available means.

[0096] 8. Enables combination therapies in which various payloads such as at least two different nucleic acid molecules, polypeptides, drugs, oligonucleotides, etc. can be simultaneously delivered by a single mitochondrion.

[0097] 9. Enables combination therapies in which at least two or more different payloads such as nucleic acid molecules, polypeptides, drugs, etc. can be simultaneously delivered by a single mitochondrion.

[0098] 10. Enables combination therapies in which at least one or more different payloads such as nucleic acid molecules, polypeptides, drugs, and especially their combinations, etc. can be simultaneously delivered by a single mitochondrion.

[0099] 11. Enables combination therapies in which at least one or more different nucleic acid molecules and one or more drugs can be simultaneously delivered by a single mitochondrion.

[0100] 12. Enables combination therapies in which at least two or more different polypeptides can be simultaneously delivered by a single mitochondrion.

[0101] 13. Enables combination therapies in which at least two or more different proteins can be simultaneously delivered by a single mitochondrion.

[0102] 14. Enables combination therapies in which at least one or more different polypeptides and one or more drugs can be simultaneously delivered by a single mitochondrion.

[0103] 15. Enables combination therapies in which at least one or more different proteins and one or more drugs can be simultaneously delivered by a single mitochondrion.

[0104] 16. Enables combination therapies in which at least one or more different nucleic acid molecules (e.g., oligonucleotides) having at least one or more polypeptides (e.g., proteins) can be simultaneously delivered by a single mitochondrion.

[0105] 17. Enables combination therapies in which at least one or more different nucleic acid molecules (e.g., oligonucleotides) having at least one or more polypeptides (e.g., proteins) can be simultaneously delivered by a single mitochondrion together with at least one or more drugs.

[0106] 18. Enables combination therapies in which at least one or more siRNAs and at least one or more anionic drugs can be simultaneously delivered by a single mitochondrion.

[0107] 19. Enables combination therapies in which at least two or more drugs, such as anionic drugs, can be simultaneously delivered by a single mitochondrion.

[0108] The disclosure in the context of the present invention described herein is applicable to the corresponding uses and vice versa.

[0109] In one aspect, the present invention provides a mitochondrion comprising one or more nucleic acid molecules attached to the outer membrane of the mitochondrion, wherein the one or more nucleic acid molecules are: a) electrostatically attached to the outer membrane of the mitochondrion via a positively charged species; or b) covalently bound to the outer membrane of the mitochondrion; or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion; or d) linked to a mitochondrion-targeting small molecule.

[0110] Mitochondria have a negatively charged surface, which, according to one aspect of the present invention, can be functionalized with cationic molecules, changing the surface charge of the outer mitochondrial membrane to a fully positive value (i.e., a net positive value) or a partially positive value. That is, mitochondria generally have a negative surface charge, but part or all of the surface can be masked / added by the positively charged molecules provided herein. Thus, the surface charge of mitochondria recognized by another molecule can be positive. Positively charged mitochondria, i.e., mitochondria having a net surface charge that is positive, or mitochondria having a positively charged surface region, can conjugate with negatively charged payload molecules such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0111] Mitochondria are double-membrane bound organelles found in most eukaryotes. Thus, the mitochondria of the present invention can be the mitochondria of any eukaryote. The mitochondria can be those of animals, plants, yeast or fungi. The mitochondria can be those of humans. The mitochondria of the present invention can be obtained by any means, such as cell culture. Thus, the mitochondria of the present invention can be obtained by in vitro cell culture. Preferably, the mitochondria can be obtained from in vitro 2D or 3D cell culture. The mitochondria of the present invention can also be obtained from tissues. The mitochondria obtained from tissues can be obtained from any tissue of a eukaryote. Thus, the mitochondria can be obtained from eukaryotic cells or tissues maintained in culture. The mitochondria can be obtained from animal, plant, yeast or fungal cells maintained in in vitro cell culture. Preferably, the mitochondria are obtained from human tissues or cell cultures. More preferably, the mitochondria are obtained from human in vitro cell cultures. In a preferred embodiment, the mitochondria are obtained from animal tissues or cell cultures, particularly mouse tissues or cell cultures. Preferably, the mitochondria are obtained from mouse in vitro cell cultures. The mitochondria can be obtained from mouse embryonic fibroblasts (MEF). The mitochondria can be obtained from MEF maintained in in vitro cell culture. The mitochondria can be obtained from MEF maintained in in vitro cell culture containing Dulbecco's Modified Eagle Medium (DMEM). In some embodiments, the mitochondria are obtained from, for example, human cardiac fibroblasts (HCF). The mitochondria can be obtained from HCF maintained in in vitro cell culture. The mitochondria can be obtained from HCF maintained in in vitro cell culture containing fibroblast medium-2. In a further embodiment, the mitochondria can be obtained from HepG2 cells. The mitochondria can be obtained from HepG2 maintained in in vitro cell culture. The mitochondria can be obtained from HepG2 maintained in in vitro cell culture containing Roswell Park Memorial Institute (RPMI) medium.

[0112] The mitochondria of the present invention can also be newly obtained, for example, by isolating mitochondria from eukaryotic cell cultures or tissues. Mitochondria obtained from tissues can be derived from placenta, liver, muscle or porcine tissues. Thus, mitochondria can be obtained by fresh isolation from animal, plant, yeast or fungal cell cultures or tissues. Preferably, mitochondria can be obtained by fresh isolation from human cell cultures or tissues. Mitochondria can be obtained by fresh isolation from HCF or HepG2. Mitochondria can be obtained by fresh isolation from HCF or HepG2 maintained in in vitro cell culture. Mitochondria can be obtained by fresh isolation from HCF maintained in in vitro cell culture containing fibroblast medium - 2. Mitochondria can be obtained by fresh isolation from HepG2 maintained in in vitro cell culture containing RPMI medium. Furthermore, mitochondria can be obtained by fresh isolation from mouse cell cultures or tissues. Mitochondria can be obtained by fresh isolation from mouse in vitro cell cultures. Mitochondria can be obtained by fresh isolation from MEF. Mitochondria can be obtained by fresh isolation from MEF maintained in in vitro cell culture. Mitochondria can be obtained by fresh isolation from MEF maintained in in vitro cell culture containing DMEM medium.

[0113] Mitochondria can be self-derived (i.e., autologous or autogenic). In some embodiments, the mitochondria are self-mitochondria or self-derived mitochondria having genetic modifications. In some other embodiments, the mitochondria are self-derived and are linked to imaging, diagnostic, or pharmaceutical agents (e.g., nucleic acid molecules, polypeptides, and / or drugs). In some other embodiments, the agent is embedded or incorporated into the self-mitochondria. In some other embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are allogeneic mitochondria having genetic modifications. In some other embodiments, the mitochondria are allogeneic mitochondria linked to imaging, diagnostic, or pharmaceutical agents. In some other embodiments, the agent is embedded or incorporated into the allogeneic mitochondria. In some other embodiments, the mitochondria are xenogeneic. In some embodiments, the mitochondria are xenogeneic mitochondria having genetic modifications. In some other embodiments, the mitochondria are xenogeneic mitochondria linked to imaging, diagnostic, or pharmaceutical agents. In some other embodiments, the agent is embedded or incorporated into the xenogeneic mitochondria. In certain aspects, a method is contemplated herein in which mitochondria are obtained / isolated from a subject (patient) and modified by adding one or more payloads, such as nucleic acid molecules (e.g., oligonucleotides), and / or one or more polypeptides (e.g., proteins) and / or one or more drugs to the outer membrane of the mitochondria as described by the methods provided herein, and subsequently administered to the same subject (patient), specifically, in a therapeutic setting, e.g., as an ex vivo method.

[0114] The mitochondria of the present invention can also be obtained from a frozen stock of mitochondria. Therefore, the mitochondria obtained from a frozen stock of mitochondria are thawed before being used in the means and methods of the present invention. The mitochondria can be obtained from a frozen stock containing mitochondria of any eukaryote such as an animal, a plant, yeast or a fungus. The mitochondria can be obtained from a frozen stock containing human mitochondria. The mitochondria can be obtained from a frozen stock containing mitochondria obtained by fresh isolation or cell culture or tissue culture. The mitochondria can be obtained from a frozen stock containing human mitochondria obtained by fresh isolation or cell culture or tissue culture. The mitochondria can be obtained from a frozen stock containing human mitochondria obtained from HCF by fresh isolation or cell culture or tissue culture. Preferably, the mitochondria can be obtained from a frozen stock containing human mitochondria obtained from an HCF in vitro cell culture. More preferably, the mitochondria can be obtained from a frozen stock containing human mitochondria obtained from an HCF in vitro cell culture containing fibroblast medium-2.

[0115] The mitochondria of the present invention may or may not be labeled. Labeled mitochondria enable subsequent detection, and unlabeled mitochondria reflect their native properties. The mitochondria can be labeled by any means known to those skilled in the art. Therefore, the mitochondria can be labeled with a dye. The mitochondria can be labeled with a dye including rhodamine, tetramethylrhodamine, X-rhodamine, dihydrotetramethylrhodamine, dihydro-X-rhodamine, carbocyanine or a derivative thereof. The mitochondria can also be labeled with small molecules or small particles. Therefore, the mitochondria of the present invention can be 18 labeled with F-rhodamine 6G or iron oxide nanoparticles or gold nanoparticles or gold nanostars or silver nanoparticles.

[0116] As used herein, "mitochondria" refers to viable mitochondria that (essentially) do not contain eukaryotic cell material such as, for example, exogenous eukaryotic cell material isolated / purified from cells or cell cultures. Thus, only a minimal amount of cell components (other than mitochondria) are present in the mitochondria (composition) used herein. Preferably, cell components other than mitochondria are not present in the mitochondria (composition) used herein. In this sense, "mitochondria" as used herein is "isolated mitochondria", and the terms "mitochondria" and "isolated mitochondria" can be used interchangeably. For example, intracellular fractionation by repeated differential centrifugation (DC) or density gradient centrifugation (DGC), or any currently known technique in the art such as differential filtration (McCully, WO 2015 / 192020) can be used for the isolation of mitochondria. The mitochondria of the present invention are useful for delivering nucleic acids (such as oligonucleotides), polypeptides (such as proteins) and / or drugs to cells. Thus, the mitochondria of the present invention are preferably viable or viable and have a negative membrane potential. In the context of the present invention, "viable" means having or maintaining a metabolism or another biological function or structure.

[0117] As used herein, the term "viable mitochondria" is used herein to describe viable mitochondria that are intact, active, functional and respiration competent. According to some embodiments, "viable mitochondria" refers to mitochondria that exhibit biological functions such as, for example, respiration and ATP and / or protein synthesis.

[0118] As used herein, the term "intact mitochondria" is used throughout this specification to describe mitochondria that include an integral outer membrane and inner membrane, an integral intermembrane space, integral cristae (formed by the inner membrane), and an integral matrix. Alternatively, intact mitochondria are mitochondria that preserve their structure and ultrastructure. In another aspect, intact mitochondria include active respiratory chain complexes I-V embedded in the inner membrane and maintain the ability to synthesize membrane potential and ATP.

[0119] The mitochondria of the present invention can be functionalized with a targeting molecule (e.g., a small molecule targeting molecule, a targeting aptamer, a targeting peptide, a carbohydrate, a sugar, and a targeting antibody), a drug, a reporter molecule / nanoparticle (e.g., a fluorescent molecule, a metal nanoparticle, a magnetic nanoparticle, etc.), or a constrictor, a contrast agent, a diagnostic agent, or a pharmaceutical agent.

[0120] In the context of the present invention, the terms "nanoparticle", "nanopreparation" and "nanobody" can be used interchangeably. In some embodiments, the nanoparticle is a lipid nanoparticle. Exemplary nanoparticles of the present invention include lipid nanoparticles, dendrimer nanoparticles, micelle nanoparticles, protein nanoparticles, liposomes, non-porous silica nanoparticles, mesoporous silica nanoparticles, silicon nanoparticles, gold nanoparticles, gold nanowires, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, titanium dioxide nanoparticles, carbon nanotubes, carbon dot nanoparticles, polymer nanoparticles, zeolite nanoparticles, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, quantum dot nanoparticles, zinc oxide nanoparticles, zirconium dioxide nanoparticles, graphene or graphene oxide nanoparticles. Those skilled in the art will recognize that nanoparticles can contain different charges or can be functionalized to have a specific charge. In the context of the present invention, a nanoparticle can be functionalized with a positively charged functional group (e.g., a quaternary ammonium group) or a positively charged species such as a polycationic species to result in a positively charged nanoparticle. Further, in some embodiments, the nanoparticle may be chemically modified to be positively charged, and the chemical modification may be, for example, protonation of a chemical group contained in the nanoparticle. In the context of the present invention, "functionalized" can mean "adding to any part or compound having a function" such as a biological function, for example, a targeting function, a protecting function or a regulating function. Thus, mitochondria can be functionalized by adding different agents that carry a desired function such as changing the charge of the nanoparticle.

[0121] In the context of the present invention, the term "particle" or "positively charged particle" preferably refers to lipid particles, dendrimer particles, micelle particles, protein particles, liposomes, non-porous silica particles, mesoporous silica particles, silicon particles, gold particles, gold wires, silver particles, platinum particles, palladium particles, titanium dioxide particles, carbon tubes, carbon dot particles, polymer particles, zeolite particles, aluminum oxide particles, hydroxyapatite particles, quantum dot particles, zinc oxide particles, zirconium dioxide particles, graphene or graphene oxide particles.

[0122] The mitochondria of the present invention are particularly useful because they can be stored stably for a long period of time without deterioration and / or disintegration. Accordingly, the present invention provides mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are a) electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) linked to a mitochondria-targeting small molecule, Preferably, the mitochondria are stored at a low temperature such as -80°C or -20°C in a conjugation buffer. The mitochondria of the present invention can be stored in a conjugation buffer at a low temperature, for example, -20°C, preferably -80°C, for at least 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 4 months, for example, 6 months or more, without disintegration or degradation. Mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane can be stored in a conjugation buffer to maintain high colloidal stability (e.g., no aggregation / agglomeration or disintegration). Mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane are preferably stored in a conjugation buffer at a low temperature (e.g., -20°C, preferably -80°C) in the dark, for example, for at least 2 months after complex formation.

[0123] The mitochondria of the present invention can be encapsulated inside an alginate / hydrogel capsule. Encapsulation in an alginate / hydrogel capsule can increase the storage time of the mitochondria of the present invention, i.e., avoid disintegration and increase stability.

[0124] The mitochondria of the present invention can be contacted with a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in a solution such as a buffer. The buffer of the present invention is preferably an aqueous solution of any compound capable of performing conjugation with the payload and the mitochondria. The buffer used in the contacting step is preferably a conjugation buffer. The conjugation buffer of the present invention can be an aqueous solution. The solvent used in the aqueous solution of the present invention can be an aqueous solvent such as water, deionized water, redistilled water, water free of DNAse and RNAse, deionized water free of DNAse and RNAse, redistilled water free of DNAse and RNAse, and a buffer. The conjugation buffer of the present invention can contain a mixture of Solution X and Solution Y. Solution X can contain or consist of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (i.e., HEPES), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (i.e., EGTA), and trehalose. Solution Y can contain or consist of N-cyclohexyl-2-aminoethanesulfonic acid (i.e., CHES) and disodium hydrogen phosphate dihydrate. Solution X and Solution Y may be aqueous solutions.

[0125] In the context of the present invention, the compositions of solutions X and Y can be used in any amount and at any pH that is feasible to achieve the successful conjugation of mitochondria and its payload, such as nucleic acids or polypeptides. In some embodiments, solution X comprises or consists of 5 to 150 mM HEPES, 0.1 to 10 mM EGTA, and 150 to 500 mM trehalose (pH 6 to 9) and optionally an aqueous solvent.In some embodiments, solution X comprises, consists of, or consists essentially of HEPES at 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149 or 150 mM, EGTA at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 mM, trehalose at 150, 157, 164, 171, 178, 185, 192, 199, 206, 213, 220, 227, 234, 241, 248, 255, 262, 269, 276, 283, 290, 297, 304, 311, 318, 325, 332, 339, 346, 353, 360, 367, 374, 381, 388, 395, 402, 409, 416, 423, 430, 437, 444, 451, 458, 465, 472, 479, 486, 493 or 500 mM (pH 6, 6.5, 7, 7.5, 8, 8.5 or 9), and optionally an aqueous solvent. Preferably, solution X comprises, consists of, or consists essentially of 20 mM HEPES, 1 mM EGTA, 300 mM trehalose (pH 7.2), and optionally an aqueous solvent.

[0126] In some embodiments, solution Y comprises or consists of 0.01 M to 0.2 M CHES (pH 8 to 12), 0.02 M to 0.6 M disodium phosphate dihydrate, and optionally an aqueous solvent. In some embodiments, solution Y comprises or consists of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2 M CHES (pH 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12) and optionally an aqueous solvent. Preferably, solution Y comprises or consists of 0.1 M CHES (pH 10), 0.2 M disodium phosphate dihydrate, and optionally an aqueous solvent.

[0127] In the context of the present invention, solutions X and Y can be mixed at any rate feasible to achieve successful conjugation of mitochondria and their payloads, such as nucleic acids or polypeptides. The mixture of solutions X and Y can result in the conjugation buffer of the present invention. Thus, in some embodiments, the conjugation buffer comprises a 2:1 to 10:1 mixture of solution X and solution Y. In some embodiments, the conjugation buffer comprises a 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 mixture of solution X and solution Y. Preferably, the conjugation buffer comprises a 4:1 mixture of solution X and solution Y.

[0128] In some embodiments, the conjugation buffer of the present invention has a pH of 7.5 to 11. In some embodiments, the conjugation buffer of the present invention has a pH of 7.5, 8, 8.5, 9, 9.5, 10, 10.5 or 11.

[0129] In a preferred embodiment, the conjugation buffer of the present invention comprises a solution X containing or consisting of 20 mM HEPES + 1 mM EGTA + 300 mM trehalose (pH 7.2), a solution Y containing or consisting of 0.1 M CHES (pH 10) + 0.2 M sodium phosphate dihydrate, and optionally a mixture with an aqueous solvent. In a more preferred embodiment, the conjugation buffer of the present invention comprises a 4:1 mixture of a solution X containing or consisting of 20 mM HEPES, 1 mM EGTA and 300 mM trehalose (pH 7.2), a solution Y containing or consisting of 0.1 M CHES (pH 10) and 0.2 M sodium phosphate dihydrate, and optionally an aqueous solvent.

[0130] The conjugation buffer of the present invention can be used to preserve the mitochondria, its compositions and pharmaceutical compositions of the present invention. The conjugation buffer used for preservation is referred to as a preservation buffer and contains components as defined above herein. Thus, in a preferred embodiment, the preservation buffer of the present invention comprises a 4:1 mixture of a solution X containing or consisting of 20 mM HEPES, 1 mM EGTA and 300 mM trehalose (pH 7.2), and a solution Y containing or consisting of 0.1 M CHES (pH 10) and 0.2 M sodium phosphate dihydrate.

[0131] The nucleic acid molecules of the present invention can be preserved in a buffer containing or consisting of an aqueous solvent and solution X. The nucleic acid molecules of the present invention can be preserved in a DNA / RNA buffer containing DNase / RNase-free water, PBS, and solution X containing or consisting of 20 mM HEPES + 1 mM EGTA + 300 mM trehalose (pH 7.2).

[0132] The mitochondria of the present invention can form a complex with one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof attached to the outer membrane of the mitochondria.

[0133] In some embodiments, the invention provides mitochondria comprising one or more nucleic acid molecules, wherein the nucleic acid molecule is DNA or RNA.

[0134] Generally, the nucleic acids of the present invention can be any nucleic acids, such as naturally occurring nucleic acids or synthetic nucleic acids. The nucleic acid can be an endogenous or exogenous nucleic acid. A nucleic acid is a polymer composed of nucleotides, which are monomers containing a five-carbon sugar, a phosphate group, and a nitrogenous base, such as adenine, cytosine, guanine, thymine, and uracil. It is also contemplated herein that the nucleic acid can be a modified nucleic acid. The nucleic acids of the present invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, the nucleic acids of the present invention can be oligonucleotides containing DNA or RNA of any length. Thus, the term "nucleic acid molecule" or any grammatical variation thereof used herein can be used interchangeably with the term "oligonucleotide". In some embodiments, the oligonucleotides of the present invention can contain 10 to 15,000 base pairs. The nucleic acid can be single-stranded DNA (ssDNA). In some embodiments, the single-stranded DNA can contain 10 to 15,000 nucleotides. The nucleic acid can be double-stranded (dsDNA). The nucleic acids of the present invention can be linear. The nucleic acid can be circular. Thus, the nucleic acid can be circular DNA (cDNA). The nucleic acid can be plasmid DNA (pDNA). The nucleic acids of the present invention can have different structural forms. Thus, the nucleic acid can be A-DNA, B-DNA (Watson-Crick), Z-DNA, C-DNA, D-DNA or E-DNA. The nucleic acids of the present invention can contain different segments. Thus, the nucleic acid can be DNA containing a sense segment having a translatable sequence. The nucleic acid can be DNA containing an antisense segment complementary to the sense segment. The nucleic acids of the present invention can be of natural origin or synthetic. Thus, the DNA can be derived from any natural source, such as an organism such as a eukaryote. The DNA can be derived from an animal, a plant, a bacterium or a yeast. Preferably, the DNA is human or substantially similar to human DNA.

[0135] The nucleic acid of the present invention may be RNA. Thus, the nucleic acid of the present invention can be an oligonucleotide containing RNA of any length. In some embodiments, the RNA of the present invention can contain from 10 to 10,000 nucleotides. The nucleic acid can be single-stranded RNA (ssRNA). The nucleic acid can be double-stranded (dsRNA). The RNA of the present invention can be linear. The RNA can be circular. The RNA molecule can contain protein-coding RNA such as mRNA or non-coding RNA such as siRNA. Thus, the RNA of the present invention can be messenger RNA (mRNA). The RNA of the present invention may be a non-coding RNA involved in RNA interference (RNAi) such as small interfering RNA (siRNA) and microRNA (miRNA). The RNA can also be other small RNAs selected from the group of small nuclear RNA (snRNA) including small nucleolar RNA (snoRNA), U1 spliceosomal RNA, U2 spliceosomal RNA, U4 spliceosomal RNA, U5 spliceosomal RNA, and U6 spliceosomal RNA, exRNA, scaRNA, and long ncRNAs such as Xist and HOTAIR. The RNA can also be non-coding RNA (ncRNA) such as transfer RNA (tRNA) or ribosomal RNA. The RNA of the present invention can be complementary to a DNA sequence of an animal, plant, bacterium, or yeast. Preferably, the RNA can be complementary to a human DNA sequence. Preferably, the RNA can be complementary to a DNA sequence within a human gene. The RNA of the present invention can also be complementary to an RNA sequence of an animal, plant, bacterium, or yeast. Preferably, the RNA can be complementary to an RNA sequence in a human. Preferably, the RNA can be complementary to a human mRNA sequence.

[0136] The RNA of the present invention may be of natural origin or synthetic. Thus, the RNA can be artificial such as short hairpin RNA (shRNA). The RNA can be derived from any natural source. The RNA can be endogenous or exogenous. The RNA can be derived from animals, plants, bacteria or yeast. The RNA may be human RNA or may be substantially similar to human RNA. The RNA can be human mRNA. The RNA can be siRNA complementary to human mRNA. Preferably, the RNA is glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, optionally siRNA complementary to human GAPDH mRNA. Preferably, the RNA is MDM2 mRNA, optionally siRNA complementary to human MDM2 oncogene (MDM2) mRNA. In one embodiment, the RNA can be hexokinase 1 mRNA, optionally siRNA complementary to the mRNA of human hexokinase 1 mRNA. Preferably, the RNA of the present invention is mRNA encoding a human peptide such as a human polypeptide and / or protein.

[0137] The nucleic acid molecule of the present invention can be functionalized with a targeting molecule (e.g., a small molecule targeting molecule, a targeting aptamer, a targeting peptide, a carbohydrate, a sugar, and a targeting antibody), a drug, a reporter molecule / nanoparticle (e.g., a fluorescent molecule, a metal nanoparticle, a magnetic nanoparticle, etc.), or a contrast agent.

[0138] In some embodiments, a payload such as the nucleic acid molecule, polypeptide, drug, or combination thereof of the present invention is formulated into a nanoparticle, a particle, a cationic lipid formulation (e.g., a lipid nanoparticle formulation), a block copolymer, a cationic lipid, or a cationic polymer. The payload such as the nucleic acid molecule, polypeptide, drug, or combination thereof may be attached to the surface of the nanoparticle or particle or may be encapsulated in the nanoparticle or particle.

[0139] The present invention provides a mitochondrion - payload complex useful for delivery to cells, particularly a complex of a nucleic acid molecule, polypeptide, drug, or a combination thereof. The present invention also provides for the addition of a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to mitochondria. One or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof can be electrostatically added to the outer membrane of mitochondria, particularly when the payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof has an overall positive surface charge. When the payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof has an overall negative surface charge, electrostatic binding can be facilitated via a positively charged species. In one embodiment, the positively charged species is a polycationic species. In another embodiment, the positively charged species is a positively charged nanoparticle or particle. Electrostatic interactions include attractive or repulsive interactions between charged molecules and / or surfaces of intracellular organelles such as the membrane surface of mitochondria. In the context of the present invention, mitochondria can electrostatically interact with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to form a complex comprising mitochondria and one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof. Thus, electrostatic interactions can be used to add a positively charged entity to a negatively charged entity. In this regard, isolated mitochondria are known to have a negative net surface charge. In the context of the present invention, mitochondria can be positively or negatively charged or neutral, depending on the complex of mitochondria and the various agents provided herein. In the context of the present invention, a nucleic acid can be positively or negatively charged. Any of the above constellations can result in successful addition via electrostatic interactions, provided that the mitochondria and the payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof have opposite charges or do not have the same charge. In this regard, it is understood that the charge can be pH - dependent. Those skilled in the art know how to handle pH - dependent charges. Generally, the mitochondrial surface has a negative surface charge profile. Similarly, DNA and RNA are generally negatively charged molecules.According to the present invention, the mitochondrial surface can also be functionalized with a positively charged species in order to establish electrostatic binding of a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof. Thus, one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof can be electrostatically added to the outer membrane of mitochondria via the positively charged species. One or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof may be electrostatically added to the outer membrane of mitochondria via a polycationic species, and the polycationic species is a linear or branched polycationic polymer. As used herein, the term "polycat ion" refers to a moiety having a positive charge at a plurality of sites and having an overall positive charge. One or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof may be electrostatically added to the outer membrane of mitochondria via a linear or branched polycationic polymer, and the linear or branched cationic polymer is polylysine, histidyl - ated polylysine, polyornithine, polyarginine, high - mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2 - (dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivative, diethylaminoethyl (DEAE) - dextran, poly(N - alkyl - 4 - vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose, or a combination thereof.

[0140] In the context of the present invention, the negative surface charge profile of mitochondria can also be useful for electrostatically attaching one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, to the outer membrane of mitochondria via positively charged nanoparticles. Thus, positively charged nanoparticles containing one or more nucleic acid molecules can electrostatically attach to the negatively charged surface of mitochondria. Thus, one or more nucleic acid molecules can be electrostatically attached to the outer membrane of mitochondria via positively charged nanoparticles. One or more nucleic acid molecules can be electrostatically attached to the outer membrane of mitochondria via positively charged particles. As those skilled in the art will recognize, the difference between nanoparticles and particles generally relates to a size difference, where nanoparticles typically have a size in the range of 1 nm to 100 nm, while particles typically have a size in the range of 100 nm to 2.5 μm. However, as those skilled in the art will also recognize, the distinction between nanoparticles and particles based on their size is not consistently maintained in the art, and different size distinctions can be made depending on the class of particles. In some embodiments, the particles of the present invention can be microparticles or microspheres. A payload, such as a nucleic acid molecule, polypeptide, drug, or combination thereof, may be attached to the surface of a positively charged nanoparticle or, for example, a positively charged particle, or may be encapsulated by a positively charged nanoparticle or, for example, a positively charged particle. Thus, one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, may be electrostatically attached to the outer membrane of mitochondria via positively charged nanoparticles, and one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, may be attached to the surface of a positively charged nanoparticle or encapsulated by a positively charged nanoparticle. One or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, may be electrostatically attached to the outer membrane of mitochondria via positively charged particles, and one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, may be attached to the surface of a positively charged particle or encapsulated by a positively charged particle.

[0141] Generally, the present invention is not limited to any particular nanoparticles or particles, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, or their encapsulation, for addition to mitochondria and addition of payloads. Thus, one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof can be added to the surface of lipid nanoparticles, dendrimer nanoparticles, micelle nanoparticles, protein nanoparticles, liposomes, non-porous silica nanoparticles, mesoporous silica nanoparticles, silicon nanoparticles, gold nanoparticles, gold nanowires, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, titanium dioxide nanoparticles, carbon nanotubes, carbon dot nanoparticles, polymer nanoparticles, zeolite nanoparticles, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, quantum dot nanoparticles, zinc oxide nanoparticles, zirconium dioxide nanoparticles, graphene or graphene oxide nanoparticles, or can be encapsulated therein. Further, one or more nucleic acid molecules can be added to the surface of lipid particles, dendrimer particles, micelle particles, protein particles, liposomes, non-porous silica particles, mesoporous silica particles, silicon particles, gold particles, gold wires, silver particles, platinum particles, palladium particles, titanium dioxide particles, carbon tubes (e.g., carbon microtubes), carbon dot particles, polymer particles, zeolite particles, aluminum oxide particles, hydroxyapatite particles, quantum dot particles, zinc oxide particles, zirconium dioxide particles, graphene or graphene oxide particles, or can be encapsulated therein.

[0142] Those skilled in the art will recognize that the above means of electrostatic addition or encapsulation into nanoparticles or particles can be applied to all products, methods, devices, or uses described herein.

[0143] In the context of the present invention, a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof can also covalently bind to the outer membrane of mitochondria. A covalent bond or covalent linkage or covalent interaction is formed by a chemical bond that involves the sharing of electron pairs between atoms. A payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, particularly a polypeptide, such as a protein, can be attached to mitochondria via a peptide bond, such as an amide bond (e.g., a carboxamide bond or a carbamide bond). The mitochondria of the present invention having an amino group of a mitochondrial membrane-associated protein / peptide can covalently bind to an N-hydroxysuccinimide ester (NHS)-functionalized nanoparticle / particle, an NHS-modified nucleic acid molecule, or an NHS-modified molecule that forms a covalent bond and a more stable conjugate. Alternatively, the mitochondria of the present invention having a carboxyl group as part of a mitochondrial-associated protein may be covalently bound to an amine group contained in a nanoparticle / particle or a nucleic acid molecule. Generally, mitochondria may be covalently bound via any chemical group that can form a chemical bond, preferably with a primary amine, for example, by acylation or alkylation. The present invention is not particularly limited to any such group. Exemplary chemical groups useful in the context of the present invention are isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, and fluorophenyl ester. Thus, in some embodiments, the mitochondria of the present invention may be covalently bound to isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, or fluorophenyl ester.Thus, in some embodiments in mitochondria containing a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, the payload may be covalently linked to an isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imido ester, carbodiimide, anhydride, or fluorophenyl ester. Thus, in some embodiments, the polypeptide of the present invention may be covalently linked to an isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imido ester, carbodiimide, anhydride or fluorophenyl ester. One skilled in the art will recognize that the choice of chemical groups (i.e., functional groups) included in the payload, such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, the nanoparticle or particle to which the payload is linked, such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, the particle or nanoparticle, may be determined by the available chemical groups on the surface of the mitochondria (e.g., on the polypeptide or protein contained in the outer membrane of the mitochondria), and vice versa.

[0144] Accordingly, one or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, may be covalently attached to the outer membrane of mitochondria. Preferably, one or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, may be linked to a polypeptide of the outer membrane of mitochondria via an amide bond. One or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, may be linked to a polypeptide of the outer membrane of mitochondria via an amide bond, and one or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, are modified to undergo formation of an amide bond with an amine functional group contained in a polypeptide of the outer membrane of mitochondria. One or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, may be linked to a polypeptide of the outer membrane of mitochondria via an ester bond, and one or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, are modified to undergo formation of an ester bond with a carboxylic acid functional group (e.g., contained in a polypeptide of the outer membrane of mitochondria). Payloads such as nucleic acids, polypeptides, drugs, or combinations thereof may also be added to mitochondria by covalently attaching nanoparticles containing the payload, such as nucleic acids, polypeptides, drugs, or combinations thereof, to mitochondria. The nanoparticles may be any nanoparticles known to those skilled in the art, may be charged (i.e., positively charged or negatively charged), or may not be charged (i.e., have a neutral charge). In a preferred embodiment, the nanoparticles are positively charged nanoparticles as described above herein. Accordingly, one or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, may be linked to a polypeptide of the outer membrane of mitochondria via an amide bond, and one or more payloads, such as nucleic acids, polypeptides, drugs, or combinations thereof, are encapsulated in nanoparticles, and the nanoparticles contain functional groups that enable covalent attachment of the nanoparticles to a polypeptide of the outer membrane of mitochondria.Accordingly, one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof may be linked to a polypeptide of the outer mitochondrial membrane via ester addition, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are encapsulated in a nanoparticle, and the nanoparticle contains a functional group that enables covalent bonding of the nanoparticle to a polypeptide of the outer mitochondrial membrane. Payloads such as nucleic acids, polypeptides, drugs, or combinations thereof may also be added to or encapsulated in positively charged nanoparticles such as polycationic nanoparticles. Nanoparticles such as positively charged nanoparticles containing a payload such as nucleic acids, polypeptides, drugs, or combinations thereof may be covalently bound to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane. Nanoparticles such as positively charged nanoparticles containing a payload, for example nucleic acids, polypeptides, drugs, or combinations thereof, may contain a phospholipid having a reactive group that enables covalent bonding to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane. Accordingly, one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof can be encapsulated in a nanoparticle, and the nanoparticle is covalently bound to an antibody.

[0145] In the context of the present invention, a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof can also be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria. Such an antibody containing a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof preferably binds to an antigen contained in the outer membrane of mitochondria, thereby facilitating the formation of a delivery platform. The present invention is not limited to any specific antigen, and generally, the present invention can be practiced using an antibody that specifically binds to any antigen contained in the outer membrane of mitochondria, thereby facilitating the formation of a mitochondria-nucleic acid complex (i.e., mitochondria containing one or more payloads such as a nucleic acid molecule, polypeptide, drug, or combination thereof). As used herein, the term "antibody" (used interchangeably in plural forms) is an immunoglobulin molecule that can specifically bind to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Preferred targets herein are antigens contained in the outer membrane of mitochondria, particularly human mitochondria. As used herein, the term "antibody" includes not only intact (i.e., full-length) monoclonal antibodies, but also antigen-binding fragments (e.g., Fab, Fab’, F(ab’)2, Fv, single-chain variable fragment (scFv)), variants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, single-domain antibodies (e.g., camelid or llama VHH antibodies), multispecific antibodies (e.g., bispecific antibodies), as well as any other modified configuration of an immunoglobulin molecule containing an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody includes antibodies of any class such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the antibody's constant domain of the heavy chain, the immunoglobulin can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0146] An antibody that "specifically binds" to a target or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to "specifically bind" if it reacts or associates with a particular target antigen more frequently, more rapidly, for a longer duration, and / or with a higher affinity than it does with other targets. An antibody "specifically binds" to a target antigen if it binds with a higher affinity, binding activity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an epitope is an antibody that binds to this epitope with a higher affinity, binding activity, more readily, and / or for a longer duration than it binds to other epitopes. By reading this definition, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it can include it). In general, although not always, a reference to binding means preferential binding.

[0147] Generally, one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, can be linked to any antibody that specifically binds to an antigen contained in mitochondria. Exemplary antigens include, but are not limited to, AIF, GCSH, MRPL40, TIMM23, ATP5A, HSP60, OPA1, TOM70, ATP5F1, OXA1L, TOMM20, BCS1L, mitofilin, prohibitin, TUFM, COX4, mitofusin 1, SDHB, UQCRC1, COX5b, mitofusin 2, SSBP1, VDAC1.

[0148] Preferably, one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, can be linked to any antibody that specifically binds to an antigen contained in the outer membrane of mitochondria. Thus, one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, can be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, and preferred antigens are any one of OPA1, TOM70, TOMM20, mitofusin 1, mitofusin 2, VDAC1.

[0149] A payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof can be covalently linked to an antibody that forms a payload-antibody complex capable of binding to an antigen of mitochondria. Thus, a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof can be covalently linked to an antibody that forms a payload-antibody complex capable of binding to an antigen contained in the outer membrane of mitochondria. In some embodiments, a DNA or RNA molecule can be covalently linked to an antibody that forms a payload-antibody complex capable of binding to an antigen of mitochondria. In some embodiments, a DNA or RNA molecule can be covalently linked to an antibody that forms a payload-antibody complex capable of binding to an antigen contained in the outer membrane of mitochondria.

[0150] Payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof can also be electrostatically linked to antibodies that specifically bind to antigens contained in the outer membrane of mitochondria. Thus, one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are electrostatically linked to a modified antibody that has one or more positive charges.

[0151] Payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof can be electrostatically linked to a modified antibody, such as an antibody containing a positive charge, to form a payload-antibody complex that can bind to mitochondrial antigens. Thus, payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof can be electrostatically linked to a modified antibody, such as an antibody containing a positive charge, to form a payload-antibody complex that can bind to an antigen contained in the outer membrane of mitochondria. In some embodiments, a DNA or RNA molecule can be electrostatically linked to a modified antibody, such as an antibody containing a positive charge, that forms a nucleic acid-antibody complex capable of binding to a mitochondrial antigen. In some embodiments, a DNA or RNA molecule can be electrostatically linked to a modified antibody, such as an antibody containing a positive charge, that forms a nucleic acid-antibody complex capable of binding to an antigen contained in the outer membrane of mitochondria.

[0152] Using an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, nanoparticles such as lipid nanoparticles containing a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof are added, thereby promoting the addition of a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to mitochondria. Accordingly, one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof are encapsulated in nanoparticles, and the nanoparticles are covalently bound to the antibody. The nanoparticles may be any nanoparticles known to those skilled in the art, and may be charged (i.e., positively or negatively charged) or uncharged (i.e., having an overall neutral charge). One or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, the nanoparticles are electrostatically linked to a modified antibody, and the modified antibody has one or more positive charges. When the nanoparticles are electrostatically linked to a modified antibody having one or more positive charges, the nanoparticles preferably have a negative charge. In some embodiments, one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, the nanoparticles are electrostatically linked to a modified antibody, and the modified antibody has one or more negative charges. When the nanoparticles are electrostatically linked to a modified antibody having one or more negative charges, the nanoparticles preferably have a positive charge. The positively charged nanoparticles are preferably the positively charged nanoparticles as described above herein.

[0153] Payloads such as nucleic acids, polypeptides, drugs, or combinations thereof can also be linked to an entity and then to an antibody. Such an entity can be biotin linked to an avidin-conjugated antibody. Thus, one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are covalently bound to biotin, and the biotin is linked to an avidin-conjugated antibody. Further, a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof can also be linked to an entity that is then linked to an antibody when encapsulated in a nanoparticle. Thus, one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are encapsulated in a nanoparticle, and the nanoparticle is covalently bound to biotin, and the biotin is linked to an avidin-conjugated antibody. The present invention is not limited to avidin-conjugated antibodies, and as those skilled in the art will recognize, avidin can be replaced with a structural analog such as streptavidin or neutravidin. Streptavidin typically has about 30% sequence identity with avidin but has nearly identical secondary, tertiary, and quaternary structures. Neutravidin is a deglycosylated analog of avidin.

[0154] Payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof can also be linked to an entity and then to an antibody. Such an entity can be an activated ester linked to the antibody. Thus, one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are covalently bound to an activated ester, and the activated ester is linked to the antibody via an amide bond. Further, a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof can also be linked to an entity that is then linked to an antibody when encapsulated in a nanoparticle. Thus, one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, the nanoparticle is covalently bound to an activated ester, and the activated ester is linked to the antibody via an amide bond.

[0155] Single-stranded nucleic acid molecules such as ssDNA or ssRNA can hybridize with an antibody that specifically binds to mitochondria or one or more complementary single-stranded nucleic acid molecules added to the antibody, thereby promoting the addition of nucleic acid and the formation of a delivery platform. Thus, one or more nucleic acid molecules may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, where the nucleic acid molecule is a single-stranded nucleic acid molecule (ssDNA or ssRNA), and where the single-stranded nucleic acid molecule hybridizes with one or more complementary single-stranded nucleic acid molecules on or added to the modified antibody.

[0156] In the context of the present invention, an antibody may be "modified with one or more complementary single-stranded nucleic acid molecules", which means that the antibody is modified with a single-stranded nucleic acid molecule capable of hybridizing with another single-stranded nucleic acid molecule, i.e., the nucleic acid can be added to the antibody via hybridization.

[0157] In the context of the present invention, "modified antibody" also means that the antibody is modified such that it has one or more positive charges, for example by adding a negatively charged nucleic acid.

[0158] In another aspect of the present invention, a payload such as a nucleic acid molecule, polypeptide, drug or combination thereof can be linked to a mitochondrially targeted small molecule to facilitate the addition and delivery platform formation of the payload such as a nucleic acid molecule, polypeptide, drug or combination thereof. Thus, one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof can be added to the outer membrane of mitochondria, where the one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof are linked to the mitochondrially targeted small molecule. In the context of the present invention, any mitochondrially targeted small molecule can be used to facilitate the addition. Exemplary mitochondrially targeted small molecules are selected from triphenylphosphonium (TPP), decalinium (DQA), E-4-(1H-indol-3-ylvinyl)-N-methylpyridinium iodide (F16), rhodamine 19, biguanide and guanidine. Thus, one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof can be linked to a mitochondrially targeted small molecule, where the mitochondrially targeted small molecule is selected from triphenylphosphonium (TPP), decalinium (DQA), E-4-(1H-indol-3-ylvinyl)-N-methylpyridinium iodide (F16), rhodamine 19, biguanide and / or guanidine. In a preferred embodiment, one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof can be linked to triphenylphosphonium (TPP).

[0159] As used herein, a "targeting moiety" refers to a moiety that can specifically bind to (i) a molecule on the surface of a target cell or (ii) a molecule on the surface of a target cell such as a cell within a target tissue of a subject. A molecule that specifically binds to a targeting moiety (e.g., a cell surface molecule) is also referred to herein as a "binding partner". In some embodiments of the copolymers and related compositions and methods described herein, the targeting moiety specifically binds to a molecule on the surface of a target cell.

[0160] The present invention is based, at least in part, on electrostatic interactions. The charge of a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof, or a mitochondrion, can be modified, for example, with a cationic molecule or polymer. Thus, the charge of a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof, or a mitochondrion, can be reversed, for example. In view of the above, one of ordinary skill in the art will understand that the products, methods, devices, and uses provided herein can also be carried out when the charge of a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof, and a mitochondrion is adjusted, such as by inversion. Accordingly, the present invention also provides a mitochondrion comprising one or more payloads such as a nucleic acid molecule, polypeptide, drug, or combination thereof added to the outer membrane of the mitochondrion, wherein the one or more payloads such as a nucleic acid molecule, polypeptide, drug, or combination thereof are electrostatically added to the outer membrane of the mitochondrion, (a) a polycation or a positively charged species is added to the outer surface of the mitochondrion to provide a positively charged mitochondrial surface, and (b) one or more payloads such as a nucleic acid molecule, polypeptide, drug, or combination thereof are electrostatically added to the positively charged mitochondrial surface via the positively charged species. A mitochondrion comprising one or more payloads such as a nucleic acid molecule, polypeptide, drug, or combination thereof added to the outer membrane of the mitochondrion, wherein the one or more payloads such as a nucleic acid molecule, polypeptide, drug, or combination thereof are electrostatically added to the outer membrane of the mitochondrion and the surface of the mitochondrion is positively charged, (a) One or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, are added to or encapsulated within positively charged nanoparticles, and (b) Positively charged nanoparticles containing one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, are electrostatically added to mitochondria.

[0161] The present invention is not particularly limited to nanoparticles, and any nanoparticles as described above may be used.

[0162] In some embodiments, the mitochondria according to the present invention containing one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, may be linked to and / or surrounded by a protective layer.

[0163] As used herein, the term "protective layer" refers to a layer that partially or wholly covers, coats, and / or encapsulates (i.e., surrounds) the mitochondria according to the present invention. The protective layer of the present invention is used to modify the mitochondria in order to improve the pharmacokinetic and pharmacodynamic properties of the mitochondria. In particular, the protective layer can, inter alia, increase the plasma half-life of the mitochondria and protect the payload of the mitochondria, such as one or more nucleic acid molecules, from degradation, i.e., from being broken down into its component parts when administered in vivo, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof. Furthermore, the protective layer can enhance the stability of the payload, for example, the protective layer can have a stabilizing effect on one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof. The protective layer also prevents or reduces an immune response or cytotoxicity when the mitochondria are internalized within a cell. As can be seen from the attached examples (see, for example, Examples 24 - 29), a mitochondria delivery platform comprising a protective layer is, for example, more effective in delivering a payload, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, and achieves higher transcription of mRNA by siRNA and higher protein knockdown compared to previous approaches.

[0164] The protective layer of the present invention preferably comprises a polymer or lipid component or molecule. In some embodiments, the protective layer surrounds the mitochondria according to the present invention and forms particles surrounded by the mitochondria. In further embodiments, the protective layer partially coats or covers the mitochondria according to the present invention and forms mitochondria having a protective layer surface coating. The protective layer can be linked to the mitochondria electrostatically or covalently, either directly (e.g., directly linked to the outer membrane of the mitochondria) or indirectly (e.g., linked to the outer membrane of the mitochondria via another entity). In a preferred embodiment, the protective layer surrounds mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of the mitochondria, and preferably one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof are electrostatically added to the outer membrane of the mitochondria via a polycationic species, particularly a linear or branched polycationic polymer according to the present invention. The protective layer surrounding the mitochondria can also be linked to the mitochondria directly, for example, by electrostatic interaction with the outer membrane of the mitochondria or by covalent bonding to the outer membrane of the mitochondria, and the covalent bond can be, for example, to a polypeptide in the outer membrane of the mitochondria via an amide bond. The protective layer can also be covalently linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria. In further embodiments, the protective layer is linked to the outer membrane of the mitochondria without surrounding the mitochondria. The protective layer is particularly linked to the outer membrane of the mitochondria when the mitochondria contain one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to or encapsulated within the surface of nanoparticles, particles, positively charged particles or positively charged nanoparticles. In embodiments where the mitochondria contain one or more positively charged particles, positively charged nanoparticles, particles or nanoparticles, those skilled in the art will recognize that the surface of the outer membrane of the mitochondria may not be fully accessible to molecules containing the protective layer, thus preventing complete encapsulation (i.e., surrounding) of the mitochondria.

[0165] In some embodiments, the mitochondria according to the present invention comprising one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof can be linked to and / or surrounded by a protective layer, where the protective layer is a protective polymer.

[0166] As used herein, the term "polymer" as defined by FW Billmeyer, JR. in Textbook of Polymer Science, second edition, 1971 refers to a relatively large molecule composed of smaller chemical repeating units that have undergone a polymerization reaction to provide a polymer product. Chemical substances that react with each other to form the repeating units of a polymer are known herein as "monomers", and a polymer is said herein to be made from the "polymerization units" of monomers that react to form repeating units. The chemical reaction or reactions by which monomers react to become the polymerization units of a polymer are known herein as "polymerization" or "polymerization reaction". Typically, a polymer contains 11 or more monomers. A polymer can have a structure that is linear, branched, star-shaped, loop-shaped, hyperbranched, cross-linked, or a combination thereof, and a polymer can have a single type of repeating monomer unit (a "homopolymer"), or a polymer can have two or more types of repeating monomer units (a "copolymer"). A copolymer can have various types of repeating monomer units arranged randomly, in a sequence, in blocks, in other arrangements, or any mixture or combination thereof. Generally, the weight average molecular weight (Mw) of a polymer is 1,000 or more. Polymer molecular weight can be measured by standard methods such as size exclusion chromatography or intrinsic viscosity. The broadest range of values for Mw is from 1,000 (one thousand) daltons to 2,000,000 (two million) daltons, preferably from 1,000 to 500,000 daltons. The preferred range for polycationic species is an Mw of 10,000 to 70,000 daltons. For the protective polymer, the preferred Mw is about 15,000 daltons.

[0167] In some embodiments, the protective polymer is a linear or branched cationic polymer, and optionally, the linear or branched cationic polymer is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof. In some embodiments, the protective polymer is a linear or branched cationic polymer, and optionally, the linear or branched cationic polymer is covalently bound to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0168] As used herein, the term "linear or branched cationic polymer" refers to a linear or branched cationic homopolymer. As used herein, the term "linear polymer" refers to a polymer containing repeating monomer units that add to each other to form a linear structure, and a "branched polymer" includes a linear polymer chain substituted with one or more polymer chains (either short or long polymer chains). As defined herein, a cationic homopolymer is a polymer containing one or more cationic monomers as polymerization units. In some embodiments, one or more cationic monomers containing cations that are present in cationic form when in solution at some range of pH values useful for the practice of the invention are used, although the cations may be in neutral form at some other pH values. In some embodiments, at least one cationic monomer that is in neutral form during polymerization is used, and in such embodiments, after polymerization, the conditions surrounding the polymer (e.g., pH, etc.) are changed such that the polymerization units resulting from the cationic monomer obtain a positive charge. Independently, in some embodiments, one or more cationic monomers containing cationic groups that are permanently in cationic form (i.e., cations that remain in cationic form at all pH values less than 9) are used. Examples of cations that are permanently in cationic form include, for example, quaternary ammonium salts. In some embodiments, one or more cationic polymers are used and all cationic groups are permanently in cationic form. In some embodiments, all cationic groups in all cationic polymers used are permanently in cationic form. The anion(s) corresponding to the cation(s) may be located in solution, in a complex with the cation (such as a nucleic acid-cationic polymer complex or a mitochondria-polymer complex, etc.), at other locations on the polymer, or a combination thereof. The anion corresponding to the cation of a suitable cationic monomer can be any type of anion. Suitable anions include, but are not limited to, halides (e.g., including chloride, bromide, or iodide), hydroxide, phosphate, sulfate, bisulfate, ethyl sulfate, methyl sulfate, formate, acetate, or any mixture thereof.Furthermore, the anion may be replaced in the process of forming the mitochondria and the protective layer, that is, the polymer containing the protective layer may have one type of anion before contacting the mitochondria, and then be replaced by another type of anion, such as a payload of a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, or the mitochondria of the present invention.

[0169] In some embodiments, the linear or branched cationic polymer may be electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, and thus one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof may be bound to the inner surface of the protective cationic polymer layer. In some embodiments, the linear or branched cationic polymer may be covalently bound to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, and thus one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof may be bound to the inner surface of the protective cationic polymer layer.

[0170] The linear or branched cationic polymer is not particularly limited and may be any suitable linear or branched cationic polymer. In preferred embodiments, the linear or branched cationic polymer is polyethyleneimine, RGD-modified polyethyleneimine, polylysine, RGD-modified polylysine, polyornithine, RGD-modified polyornithine, polyarginine, RGD-modified polyarginine, polypropyleneimine, RGD-modified polypropyleneimine, polyallylamine, RGD-modified polyallylamine, chitosan, RGD-modified chitosan, poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(2-(dimethylamino)ethyl methacrylate), poly(amidoamine), RGD-modified poly(amidoamine), or a combination thereof.

[0171] In some embodiments, the protective polymer is a linear or branched cationic copolymer, and optionally, the linear or branched cationic copolymer is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0172] The term "copolymer" refers to the copolymers described above. The copolymers of the present invention may be linear (e.g., block copolymers, alternating copolymers, periodic copolymers, statistical copolymers, stereoblock copolymers, or gradient copolymers) or branched (e.g., graft or star copolymers).

[0173] In some embodiments, the protective polymer is a linear or branched cationic block copolymer, and optionally, the linear or branched cationic block copolymer is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof. In some embodiments, the protective polymer is a linear or branched cationic block copolymer, and optionally, the linear or branched cationic block copolymer is covalently bonded to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0174] As used herein, the term "block copolymer" is a copolymer that contains two or more monomers, and the monomers are present in blocks. Each block of monomers contains a repeating sequence of monomers. Further, a block is a part of a polymer that contains repeating monomer units having at least one characteristic that is not present in adjacent blocks. The formula representing a block copolymer is -(A) a -(B) b -(C) c -(D) d …-(Z) z- wherein A, B, C, D to Z represent monomer units, and the subscripts "a", "b", "c", "d" to "z" represent the number of repeating units of A, B, C, D to Z respectively. The representative formula does not mean to limit the structure of the block copolymer used in the present invention. The block copolymer of the present invention may be a diblock, triblock, tetrablock or other copolymers. Furthermore, the block copolymer may be a linear or branched block copolymer.

[0175] In some embodiments, the cationic block copolymer is poly(ethylene glycol)-block-polyethyleneimine, RGD-modified poly(ethylene glycol)-block-polyethyleneimine, poly(ethylene glycol)-block-polylysine, RGD-modified poly(ethylene glycol)-block-polylysine, poly(ethylene glycol)-block-polyornithine, RGD-modified poly(ethylene glycol)-block-polyornithine, poly(ethylene glycol)-block-polyarginine, RGD-modified poly(ethylene glycol)-block-polyarginine, poly(ethylene glycol)-block-polypropyleneimine, RGD-modified poly(ethylene glycol)-block-polypropyleneimine, poly(ethylene glycol)-block-polyallylamine, RGD-modified poly(ethylene glycol)-block-polyallylamine, poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-block-poly(amidoamine), RGD-modified poly(ethylene glycol)-block-poly(amidoamine) or a combination thereof.

[0176] In some embodiments, the protective polymer is a cationic graft (g) copolymer, and optionally, the cationic graft (g) copolymer is electrostatically linked to one or more nucleic acid molecules. In some embodiments, the protective polymer is a cationic graft (g) copolymer, and optionally, the cationic graft (g) copolymer is covalently bound to one or more nucleic acid molecules.

[0177] As used herein, the term "graft copolymer" refers to a branched polymer formed when a polymer or copolymer chain is chemically attached to a polymer backbone as a side chain. Typically, the side chain has a different polymer composition than the backbone. Graft copolymers have unique properties, including, for example, mechanical film properties resulting from the thermodynamically driven microphase separation of the polymers.

[0178] In some embodiments, the cationic graft (g) copolymer is poly(ethylene glycol)-g-polyethyleneimine, RGD-modified poly(ethylene glycol)-g-polyethyleneimine, poly(ethylene glycol)-g-polylysine, RGD-modified poly(ethylene glycol)-g-polylysine, poly(ethylene glycol)-g-polyornithine, RGD-modified poly(ethylene glycol)-g-polyornithine, poly(ethylene glycol)-g-polyarginine, RGD-modified poly(ethylene glycol)-g-polyarginine, poly(ethylene glycol)-g-polypropyleneimine, RGD-modified poly(ethylene glycol)-g-polypropyleneimine, poly(ethylene glycol)-g-polyallylamine, RGD-modified poly(ethylene glycol)-g-polyallylamine, poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-g-poly(amidoamine), RGD-modified poly(ethylene glycol)-g-poly(amidoamine), or a combination thereof.

[0179] In further embodiments, the protective polymer is a linear or branched pegylated (PEG) cationic polymer, and optionally, the linear or branched pegylated (PEG) cationic polymer is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof. In further embodiments, the protective polymer is a linear or branched pegylated (PEG) cationic polymer, and optionally the linear or branched pegylated (PEG) cationic polymer is covalently bound to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0180] As used herein, the term pegylated cationic polymer refers to a cationic polymer modified with poly(ethylene glycol) (PEG) or a derivative thereof via a covalent or non-covalent binding force (such as ionic interaction or hydrogen bonding). Modification of a material with a group derived from PEG (also called polyethylene oxide) is known as PEGylation. PEGylation of a bioactive entity can, in particular, prevent degradation of the entity by proteolytic enzymes. Other advantages of PEGylation include, but are not limited to, increased water solubility, increased bioavailability, increased blood circulation, decreased aggregation, decreased immunogenicity, decreased toxicity, and decreased frequency of administration.

[0181] In some embodiments, the pegylated (PEG) cationic polymer is pegylated polyethyleneimine, RGD-modified pegylated polyethyleneimine, pegylated polylysine, RGD-modified pegylated polylysine, histidyl polylysine, pegylated polyornithine, RGD-modified pegylated polyornithine, pegylated polyarginine, RGD-modified pegylated polyarginine, pegylated polypropyleneimine, RGD-modified pegylated polypropyleneimine, pegylated polyallylamine, RGD-modified pegylated polyallylamine, pegylated chitosan, RGD-modified pegylated chitosan, pegylated poly(2-(dimethylamino)ethyl methacrylate), RGD-modified pegylated poly(2-(dimethylamino)ethyl methacrylate), pegylated poly(amidoamine), RGD-modified pegylated poly(amidoamine), or combinations thereof.

[0182] In some embodiments, the protective layer is a lipid formulation, optionally the lipid formulation is a cationic lipid formulation, and further optionally the cationic lipid formulation is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof.

[0183] The lipid formulation includes lipid particles (such as liposomes) or lipid molecules forming a lipid layer. The lipid formulation of the present invention can be linked to and / or surround the mitochondria of the present invention. The lipid formulation can partially cover or coat the mitochondria of the present invention or can surround the mitochondria of the present invention. In some embodiments, the lipid formulation surrounding the mitochondria of the present invention is a liposome.

[0184] As used herein, the term "liposome" is a structure having one or more lipid membranes surrounding an aqueous interior containing, inter alia, the mitochondria of the present invention. The present invention can include both unilamellar liposomes (which are referred to as monolayers) and multilamellar liposomes (which are referred to as multilayers). The selection of the lipid formulation and the lipids contained therein depends on various considerations including, inter alia, stability, physicochemical properties, payload loading efficiency, payload release efficiency and toxicity. The lipids contained in the lipid formulation of the present invention can be any lipids capable of binding to and / or surrounding the mitochondria of the present invention, including but not limited to fatty acids, glycerolipids, glycerophospholipids, sphingolipids and sterols. The lipids contained in the lipid formulation can be amphiphilic lipids containing both hydrophilic (polar) groups and hydrophobic (non-polar) groups. Amphiphilic lipids include but are not limited to phospholipids, aminolipids and sphingolipids. The lipids contained in the lipid formulation of the present invention can include one or more saturated or unsaturated acyl groups of various carbon chain lengths. In a preferred embodiment, one or more lipids contained in the lipid formulation include one or more saturated, monounsaturated or polyunsaturated fatty acids having a carbon chain length of C14 - C22. The lipids of the present invention can also include mixtures of saturated and unsaturated fatty acid chains.

[0185] As used herein, the term "cationic lipid" as included in a cationic lipid formulation refers to a lipid having one or more fatty acids or fatty alkyl chains and a cationic or cationic ionizable group (i.e., a functional group) such as an amino group (including alkylamino, dialkylamino, trialkylamino, and quaternary alkylamino groups). A cationic group refers to a group that is positively charged at physiological pH (e.g., at a pH of about 7.4). A cationic ionizable group refers to a group that can be protonated at a pH below physiological pH, for example, below about 6.5, which is a typical pH within an endosome, to form a cationic lipid. One advantage of protonation of the cationic ionizable group in an endosome is to promote membrane fusion and subsequent release into the cytosol. In certain embodiments, the cationic ionizable lipid has a pKa of protonatable groups in the range of about 6 to about 7. The overall pKa of a lipid formulation depends not only on the pKa of each lipid but also on the molar ratio of the lipids. Each lipid has a distinct pKa that can be varied by modifying its ionizable group. Thus, one strategy for adjusting the overall pKa of a lipid formulation is to chemically modify the lipid. Another strategy is to use a mixture of two or more lipids having different pKa values and adjust their ratios to achieve a desired apparent pKa. A cationic lipid formulation may also be electrostatically linked to one or more nucleic acid molecules. Cationic lipids include, but are not limited to, DOSPA (2,3-dioleyloxy-N-[2(speminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium), DC-cholesterol (3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DOTAP (1,2-dioleoyl-3-trimethylammonium propane chloride), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane chloride), UGG (unsaturated guanidinium glycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), and Lipofectamine. Lipofectamine (also called Lipofectamine 2000) typically contains a 3:1 mixture of DOSPA and DOPE.

[0186] The lipid formulation may also contain one or more neutral lipids, and the neutral lipid molecules are either uncharged or neutral zwitterionic at physiological pH. Neutral lipids include, but are not limited to, DLinDMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleyloxy-3-dimethylaminopropane), and DOGS (dioctadecylamidoglycyl spermine). As will be understood by those skilled in the art, the neutral lipid may be an ionizable cationic lipid under conditions where the neutral lipid is protonated.

[0187] The lipid formulation of the present invention may also contain one or more anionic lipids. Anionic lipids suitable for the lipid formulation of the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-acylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, and lysylphosphatidylglycerol.

[0188] In some embodiments, the lipid formulation comprises DC-cholesterol (3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DLinDMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleyloxy-3-dimethylaminopropane), DOGS (dioctadecylamidoglycyl spermine), DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium), DOTAP (1,2-dioleoyl-3-trimethylammonium propane chloride), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane chloride)), UGG (unsaturated guanidinium glycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), Lipofectamine, or a combination thereof.

[0189] Furthermore, the lipid formulation of the present invention may further comprise one or more other lipids, preferably the lipid is cholesterol, substituted or unsubstituted cholesterol, a cholesterol derivative, such as a hydroxylated cholesterol derivative (e.g., hydroxy cholesterol), a PEG-lipid, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DDA (dimethyldioctadecylammonium), 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, bis(monooleoylglycerol)phosphate, or a combination thereof.

[0190] The lipid formulation of the present invention may further comprise one or more additional lipids. Additional lipids can be included in the lipid formulation for various purposes such as prevention of lipid oxidation, addition of ligands to the surface of the lipid formulation, stabilization of the lipid formulation, or improvement of payload delivery. The additional lipids included in the lipid formulation can be any lipid including, but not limited to, amphiphilic, neutral, cationic, and anionic lipids. Stabilizing a lipid can, in the context of the present invention, refer to a lipid that renders the lipid formulation resistant to chemical change. Examples of stabilizing lipids include sterols such as cholesterol, substituted or unsubstituted cholesterol, cholesterol derivatives such as hydroxylated cholesterol derivatives (e.g., hydroxy cholesterol), PEG-lipids such as PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramide, and lipids selected to reduce aggregation of lipid molecules during formation, but are not limited thereto, and can result from the steric stabilization of particles that prevent charge-induced aggregation during formation. Examples of molecules that can be conjugated to lipids to reduce aggregation of particles during formation include PEG, monosialoganglioside (Gm1), polyamide oligomers (PAO) such as ATTA. It should be noted that the aggregation-preventing compound does not necessarily require lipid conjugation to function properly. Free PEG or free ATTA in solution may be sufficient to prevent aggregation. If the lipid formulation is stable after formation, PEG or ATTA can be removed by dialysis prior to administration to the subject.

[0191] In some embodiments, the lipid formulation of the present invention comprises a mixture of any one of the above lipids, and exemplary lipid formulations can include a cationic lipid, a neutral lipid (other than the cationic lipid), a sterol (e.g., cholesterol), and a PEG-modified lipid.

[0192] In some embodiments, the mitochondria of the present invention are linked to and / or surrounded by a zwitterionic protective polymer, and optionally, the zwitterionic protective polymer is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0193] The zwitterionic protective polymer can be a homopolymer or copolymer as described above herein. The zwitterionic polymer contains one or more positive charges and one or more negative charges, and the overall (i.e., net) charge of the polymer is substantially electronically neutral. In one embodiment, the zwitterionic polymer is a zwitterionic copolymer, and the ratio of the number of positively charged repeating units to the number of negatively charged repeating units is from about 1:1.1 to about 1:0.5. In one embodiment, the ratio of the number of positively charged repeating units to the number of negatively charged repeating units is from about 1:1.1 to about 1:0.7. In one embodiment, the ratio of the number of positively charged repeating units to the number of negatively charged repeating units is from about 1:1.1 to about 1:0.9.

[0194] In a preferred embodiment, the zwitterionic protective polymer is selected from the co-assemblies of cationic (carboxyl-functionalized) and anionic (amino-functionalized) copolyesters based on poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyethyleneimine-g-poly(2-methacryloyloxyethyl phosphorylcholine) (PEI-g-PMPC), poly(ε-caprolactone)-block-poly(butylene fumarate)-block-poly(ε-caprolactone) (PCL-b-PBF-b-PCL), poly(lactic acid-co-glycolic acid) (PLGA)-PCB block copolymer (PLGA-b-PCB).

[0195] In some embodiments, the protective layer is linked to the targeting moiety, and optionally, the protective layer linked to the targeting moiety is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof. In preferred embodiments, the targeting moiety is an antibody or a carbohydrate molecule. In more preferred embodiments, the targeting moiety is included on the outer surface of the protective layer. The outer surface of the protective layer is the surface that contacts the environment, and the inner surface is in proximity to or in contact with the mitochondria and the payload contained therein. In some embodiments, the protective layer is linked to the targeting moiety, and optionally, the protective layer linked to the targeting moiety is covalently bound to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof. In preferred embodiments, the targeting moiety is an antibody or a carbohydrate molecule. In more preferred embodiments, the targeting moiety is included on the outer surface of the protective layer.

[0196] In further embodiments, the protective layer is linked to an antibody, and optionally, the protective layer linked to the antibody is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0197] Furthermore, in some embodiments, the protective layer is linked to a carbohydrate, and optionally, the protective layer linked to the carbohydrate is electrostatically linked to one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof.

[0198] The carbohydrate or antibody linked to the protective layer of the present invention is preferably a targeting moiety, i.e., a moiety that targets a cell or tissue or a molecule contained therein via an affinity-type interaction. The targeting mechanism generally requires that the targeting moiety be disposed on the surface of the protective layer such that the targeting moiety is available for interaction with a target, such as a cell surface receptor. The targeting moieties enhance the association of the entities to which they are linked with target cells, tissues, specific cell types or molecules contained therein, such as cell surface molecules. The targeting moiety can be a carbohydrate such as lactose, galactose, N-acetylgalactosamine (NAG), mannose, mannose-6-phosphate (M6P) or derivatives thereof, but is not limited to these examples. As used herein, the term "antibody" includes not only intact (i.e., full-length) monoclonal antibodies, but also antigen-binding fragments (e.g., Fab, Fab’, F(ab’)2, Fv, single-chain variable fragment (scFv)), variants thereof, fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, single-domain antibodies (e.g., camelid or llama VHH antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules containing an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody does not have to be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known. Exemplary targeting antibodies include, but are not limited to, monoclonal antibodies, whole antibodies or antibody fragments.The antibody as a targeting antibody can be any antibody as defined above herein. Standard methods for linking the targeting moiety, such as a carbohydrate or an antibody, may be used. For example, the targeting moiety may be covalently bound to the protective layer via an amide bond, thioester addition, disulfide bond, or hydrazone bond. The covalent bonding of the targeting moiety may be carried out by covalently bonding the targeting moiety to the polymer or lipid contained in the protective layer before the formation of the protective layer, or alternatively, the targeting moiety may be covalently bonded to the protective layer after it is formed. The methods for attaching the targeting moiety are well understood by those skilled in the art and are described in a number of review articles (e.g., Z. Zhao et al., Cell, 2020, 181, p151-167; M. J. Mitchell et al., Nature Reviews Drug Discovery, 2021, 20, p101-124). The targeting moiety as part of the present invention is not particularly limited and may include molecules other than carbohydrates or antibodies, such as peptides, proteins, vitamins, and small molecules. The targeting moiety may be electrostatically linked to, for example, a payload or a protective polymer.

[0199] In another aspect, any of the mitochondria described herein can be incorporated into a composition. Accordingly, the present invention provides a composition comprising the mitochondria of the present invention, wherein the mitochondria comprise one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are a) electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) linked to a mitochondrial targeting small molecule.

[0200] The composition can include any of the mitochondria described herein and any additional compounds useful for promoting delivery.

[0201] The mitochondria and compositions of the present invention can be formulated into a pharmaceutical composition comprising an acceptable carrier such as a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues, organs, and / or body fluids of a subject without excessive toxicity, irritation, allergic reaction, or other problems or complications within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. As used herein, the term "pharmaceutically acceptable carrier" refers to physiologically compatible solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and the like. The composition can include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0202] Accordingly, the present invention relates to a pharmaceutical composition comprising the mitochondria of the present invention described above and a pharmaceutically acceptable carrier. The pharmaceutical composition can include the mitochondria described herein and a pharmaceutically acceptable carrier, and the pharmaceutical composition is formulated as a solution. The pharmaceutical composition can include the mitochondria described herein and a pharmaceutically acceptable carrier, and the pharmaceutical composition is formulated as an aerosol.

[0203] The products, compositions, and mitochondria described herein can be used for treatment. The mitochondria used in the present application for treatment can be used in allogeneic or autologous manners. The present invention provides mitochondria, compositions, and pharmaceutical compositions for use in the treatment of diseases that can benefit from the use of healthy mitochondria and combinations of healthy mitochondria and nucleic acid molecules. For example, it is envisioned to increase the expression of a specific target protein via the delivery of messenger RNA (mRNA), or to decrease a specific target protein via the delivery of small interfering RNA (siRNA). Accordingly, the present invention provides for the treatment of cardiovascular diseases (CVD) in humans such as ischemic heart disease, ischemia-reperfusion injury, and atherosclerosis, the treatment of age-related diseases such as sarcopenia, Parkinson's disease, and Hutchinson-Gilford progeria syndrome (HGPS), the treatment of kidney diseases such as autosomal dominant polycystic kidney, Alport syndrome, nephronophthisis, and Fabry disease, methods and treatments using in vitro / in vivo gene transfection and editing using CRISPR-Cas9, gene therapy for diseases such as cystic fibrosis, and cancer treatment.

[0204] The terms "pharmaceutical" and "pharmaceutical composition" are used interchangeably herein. Accordingly, the definitions and descriptions provided herein in relation to "pharmaceutical composition" apply to the term "pharmaceutical" with the necessary modifications. Accordingly, the present invention provides mitochondria for use as a pharmaceutical, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of the mitochondria via positively charged species, or b) are covalently bound to the outer membrane of the mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) are linked to a mitochondria-targeting small molecule.

[0205] The present invention provides a composition for use as a pharmaceutical comprising mitochondria and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of the mitochondria, wherein the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of the mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) are linked to a mitochondria-targeting small molecule.

[0206] The present invention provides a pharmaceutical composition for use as a pharmaceutical comprising mitochondria and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of the mitochondria, wherein the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of the mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) are linked to a mitochondria-targeting small molecule.

[0207] The mitochondria, compositions, and pharmaceutical compositions of the present invention can be used for gene therapy. The present invention provides a delivery platform for payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, which is particularly useful for in vivo, ex vivo, or in vitro gene therapy. Those skilled in the art will recognize that in vivo gene therapy or gene editing methods may be relevant to treatment or gene editing methods in a subject, ex vivo gene therapy or gene editing methods may be relevant to treatment or gene editing methods in, for example, an organ artificially maintained outside the subject, and in vitro gene therapy or gene editing methods may be relevant to treatment or gene editing methods in, for example, cells or tissues in culture. As used herein, "gene therapy" relates to the modification of a subject's genes for treating or curing a disease. The terms "gene editing" and "genome editing" may be used interchangeably herein. Accordingly, the present invention provides mitochondria for use in gene therapy, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, attached to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) electrostatically attached to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, d) linked to a mitochondria-targeting small molecule.

[0208] The present invention provides a composition for use in gene therapy comprising mitochondria, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, attached to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) electrostatically attached to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeting small molecule.

[0209] The present invention provides a pharmaceutical composition for use in gene therapy, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeting small molecule.

[0210] a) are electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeting small molecule.

[0211] The present invention provides a composition for use in in vitro, ex vivo, or in vivo genome editing, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) electrostatically attached to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondrially targeted small molecule.

[0212] The present invention provides a pharmaceutical composition for use in in vitro, ex vivo or in vivo genome editing, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of mitochondria, wherein the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof a) electrostatically attached to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondrially targeted small molecule.

[0213] The mitochondria, compositions and pharmaceutical compositions of the present invention can be used for the treatment of a condition or disease of a subject. The term "subject" generally relates to any individual such as an animal. In the context of the present invention, the individual is preferably a mammal, most preferably a human. The terms "individual", "subject" and / or "patient" can be used interchangeably.

[0214] A disease can be any medical condition or state of an individual lacking health. A disease can also be a state of discomfort or malaise. According to the present invention, the disease can preferably be a cardiovascular disease, an age-related disease, a kidney disease or cancer. In a preferred embodiment, the disease is ischemic heart disease, atherosclerosis, muscular dystrophy, Parkinson's disease, or Hutchinson-Gilford progeria syndrome.

[0215] Terms such as "treatment", "treating", etc. are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in that it completely or partially prevents a disease or its symptoms and / or prevents the progression of a disease or its symptoms. The term "treatment" as used herein can be understood to relate to any form of therapy.

[0216] Accordingly, the present invention provides mitochondria for use in the treatment of cardiovascular diseases, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeted small molecule.

[0217] Preferably, the present invention provides mitochondria for use in the treatment of ischemic heart disease, ischemia-reperfusion injury or atherosclerosis, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeted small molecule.

[0218] The present invention provides a composition for use in the treatment of cardiovascular diseases, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeted small molecule.

[0219] Preferably, the present invention provides a composition for use in the treatment of ischemic heart disease, ischemia-reperfusion injury, or atherosclerosis, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeted small molecule.

[0220] The present invention provides a pharmaceutical composition for use in the treatment of cardiovascular diseases, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeting small molecule.

[0221] Preferably, the present invention provides a pharmaceutical composition for use in the treatment of ischemic heart disease, ischemia-reperfusion injury or atherosclerosis, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof are a) electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeting small molecule.

[0222] Therefore, the present invention provides mitochondria for use in the treatment of aging-related diseases, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof are a) electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeting small molecule.

[0223] Preferably, the present invention provides mitochondria for use in the treatment of sarcopenia, Parkinson's disease or Hutchinson-Gilford progeria syndrome, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof are a) electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) linked to a mitochondria-targeted small molecule.

[0224] The present invention provides a composition for use in the treatment of age-related diseases, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof are a) electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) linked to a mitochondria-targeted small molecule.

[0225] Preferably, the present invention provides a composition for use in the treatment of sarcopenia, Parkinson's disease or Hutchinson-Gilford progeria syndrome, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof are a) electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeted small molecule.

[0226] The present invention provides a pharmaceutical composition for use in the treatment of age-related diseases, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeted small molecule.

[0227] Preferably, the present invention provides a pharmaceutical composition for use in the treatment of sarcopenia, Parkinson's disease, or Hutchinson-Gilford progeria syndrome, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeted small molecule.

[0228] Accordingly, the present invention provides mitochondria for use in the treatment of kidney diseases, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeting small molecule.

[0229] Preferably, the present invention provides mitochondria for use in the treatment of autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis, or Fabry disease, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) are linked to a mitochondria-targeting small molecule.

[0230] The present invention provides a composition for use in the treatment of kidney diseases, comprising a plurality of mitochondria, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) are covalently bound to the outer membrane of mitochondria, or c) are linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondrion-targeted small molecule.

[0231] Preferably, the present invention provides a composition for use in the treatment of autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis or Fabry disease, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof a) electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) linked to a mitochondrion-targeted small molecule.

[0232] The present invention provides a pharmaceutical composition for use in the treatment of kidney diseases, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof a) electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) linked to a mitochondrion-targeted small molecule.

[0233] Preferably, the present invention provides a pharmaceutical composition for use in the treatment of autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis or Fabry disease, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof a) electrostatically attached to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondrially targeted small molecule.

[0234] Accordingly, the present invention provides mitochondria for use in the treatment of cancer, comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof, etc., added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof, etc. are a) electrostatically attached to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondrially targeted small molecule.

[0235] The present invention provides a composition for use in the treatment of cancer, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof, etc., added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs or combinations thereof, etc. are a) electrostatically attached to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondrially targeted small molecule.

[0236] The present invention provides a pharmaceutical composition for use in the treatment of cancer, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, etc., added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, etc. are a) electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeting small molecule.

[0237] As described above herein, the mitochondria, compositions or pharmaceutical compositions of the present invention are for use in the treatment of various diseases including cardiovascular diseases, ischemia-reperfusion injury, kidney diseases, cancer, mitochondrial dysfunction, metabolic disorders, autoimmune disorders, infectious diseases, inflammatory diseases, muscle diseases and age-related diseases.

[0238] Cardiovascular diseases are preferably selected from ischemic heart diseases, myocardial ischemia, atherosclerosis, myocardial infarction, acute coronary syndrome heart failure, and hypertensive heart diseases.

[0239] Ischemia-reperfusion injury can be any disease associated with ischemia, and preferably, ischemia-reperfusion injury is selected from liver ischemia-reperfusion injury, ischemic injury-compartment syndrome, chronic ischemia, hypertension and any injury associated with ischemia, such as myocardial infarction, stroke, organ transplantation, etc.

[0240] Kidney diseases are preferably selected from autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis and Fabry disease.

[0241] The cancer is preferably selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., glioblastoma), breast cancer, anal, anal canal, or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B chronic lymphocytic leukemia, hairy cell leukemia, Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, reticulum cell cancer, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0242] The autoimmune disorder is preferably selected from multiple sclerosis, diabetes, irritable bowel syndrome (IBS), celiac disease, Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, autoimmune vasculitis, myasthenia gravis, pernicious anemia, Hashimoto's thyroiditis, type 1 diabetes, autoimmune Addison's disease, Graves' disease, Sjogren's syndrome, psoriasis, and celiac disease.

[0243] The inflammatory disease is preferably selected from rheumatoid arthritis, inflammatory skin diseases such as psoriasis, inflammatory bowel diseases such as colitis, and inflammatory lung diseases such as asthma and bronchitis.

[0244] Mitochondrial dysfunction is preferably selected from diseases caused by mutations in mtDNA such as Kearns-Sayre syndrome, mitochondrial encephalomyopathy, lactic acidosis-stroke-like episodes (MELAS) syndrome, Leber's hereditary optic neuropathy, Pearson syndrome, progressive external ophthalmoplegia, mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leigh syndrome, "neuropathy, ataxia, retinitis pigmentosa, and ptosis" (NARP), myoneurogastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with ragged red fibers (MERRF syndrome), encephalomyopathy, lactic acidosis-Parkinson's disease-stroke-like symptoms (MELAS syndrome), etc., MERRF syndrome, MELAS syndrome, Leber's disease, Barth syndrome, and diabetes mellitus.

[0245] The metabolic disorder is preferably selected from obesity and its related metabolic diseases (e.g., type 2 diabetes). The metabolic disorder can be treated or prevented by administering the mitochondria, composition or pharmaceutical composition of the present invention to the white adipose tissue of the subject. White adipose tissue or white fat is one of two types of adipose tissue found in mammals. It is often used by the body as an energy store and contains many white adipocytes. The other type of adipose tissue is brown adipose tissue. The function of brown adipose tissue is to transfer energy from food to heat. White adipocytes often contain a single lipid droplet. In contrast, brown adipocytes contain numerous smaller droplets and far more mitochondria. Due to the recognition that adults have a significant ability to dissipate energy in brown adipose tissue, targeting the thermogenesis of brown adipose tissue is now considered a method for treating or preventing metabolic disorders such as obesity and its related metabolic diseases (e.g., type 2 diabetes). The use of brown adipose tissue for treating obesity and diabetes is described, for example, in Cypess, Aaron M., and C. Ronald Kahn. “Brown fat as a therapy for obesity and diabetes.” Current opinion in endocrinology, diabetes, and obesity 17.2 (2010): 143, which is incorporated herein by reference in its entirety. Since one of the main differences between brown adipocytes and white adipocytes is the number of mitochondria in the cells, the present disclosure provides a method for treating and preventing metabolic disorders by administering mitochondria, a composition containing mitochondria or a pharmaceutical composition to the white adipose tissue of the subject. By administering the mitochondria of the present invention to white adipocytes, the white adipocytes can be converted into brown adipocytes, and thus, white adipose tissue can be converted into brown adipose tissue.

[0246] The infectious disease is preferably selected from viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections and sepsis.

[0247] The muscle disorder is preferably selected from Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Outlier muscular dystrophy (OMD), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD, also known as Landouzy-Dejerine), myotonic dystrophy (MMD, also known as Steinert's disease), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), and congenital muscular dystrophy (CMD). The muscle disorder may also include diseases or disorders (including myositis disorders, polymyositis, dermatomyositis, and inclusion body myositis, as well as myopathy) that are accompanied by or may be accompanied by voluntary muscle cell death or inflammation.

[0248] The age-related diseases are preferably selected from neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, Huntington's disease, dementia, etc.), sarcopenia, Hutchinson-Gilford progeria syndrome, osteopenia, osteoporosis, arthritis, atherosclerosis, cardiovascular diseases, hypertension, cataract, presbyopia, glaucoma, type 2 diabetes, metabolic syndrome, alopecia, chronic inflammation, immunosenescence, and age-related visual impairment.

[0249] The mitochondria, compositions, and pharmaceutical compositions of the present invention can be used for radiotherapy. In particular, the mitochondria of the present invention can be used to deliver a radioactive agent that can be used in radiotherapy. Such a radioactive agent for radiotherapy can be delivered to solid tumors by the delivery system of the present invention. The present invention is not particularly limited to the agents for radiotherapy. Iodine-131 is an exemplary agent for radiotherapy of thyroid cancer. Accordingly, the present invention provides mitochondria for use in radiotherapy, which include one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof added to the outer membrane of the mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) are electrostatically added to the outer membrane of the mitochondria via a positively charged species, or b) Covalently bound to the outer membrane of mitochondria, or c) Linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) Linked to a mitochondrially targeted small molecule.

[0250] The present invention provides a composition for use in radiotherapy, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) Electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) Covalently bound to the outer membrane of mitochondria, or c) Linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) Linked to a mitochondrially targeted small molecule.

[0251] The present invention provides a pharmaceutical composition for use in radiotherapy, comprising a plurality of mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of mitochondria, and the one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof a) Electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) Covalently bound to the outer membrane of mitochondria, or c) Linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) Linked to a mitochondrially targeted small molecule.

[0252] The present invention provides mitochondria for use in radiotherapy, comprising one or more radiopharmaceuticals added to the outer membrane of mitochondria, and the one or more radioactive substances a) Electrostatically added to the outer membrane of mitochondria via a positively charged species, or b) covalently bound to the outer mitochondrial membrane, or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane, or d) linked to a mitochondrially targeted small molecule.

[0253] In yet another aspect, the present invention provides a method of delivering a payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, to a target organ by administering the delivery platform of the present invention to a subject. The terms "administering," "introducing," and "delivering" are used interchangeably in the context of the present invention. For example, the delivery platform of the present invention, i.e., the mitochondrial payload complex, can be introduced into a subject by a method or route that results in at least partial localization of the introduced complex at a desired site, such as a site understood to produce a desired effect, such as treatment or therapy. The mitochondria, compositions, or pharmaceutical compositions of the present invention can be administered enterally (into the intestine), via the gastrointestinal tract, epidurally (into the dura), orally (through the mouth), transdermally, epidurally, intracerebrally (into the cerebrum), intraventricularly (into the ventricle), topically (application to the skin), intradermally, (to the skin itself), subcutaneously (under the skin), intranasally (through the nose), intravenously (into the vein), intravenous bolus, intravenous drip, intraarterially (into the artery), intramuscularly (into the muscle), intracardially (into the heart), intraosseously (into the bone marrow), intrathecally (into the spinal canal), intraperitoneally, (injection or infusion into the peritoneal cavity), intravesically, intravitreally, (through the eye), intracavitary (into the lesion cavity), intracavity (into the base of the penis), intravaginally, intrauterinely, extraamniotically, transdermally (diffusion through intact skin for systemic distribution), transmucosally (diffusion through the mucosa), transvaginally, insufflation (snort), sublingually, sublabially, enema, eyedrops (onto the conjunctiva), otic, auricular (into or through the ear), buccal (directed towards the cheek), conjunctival, cutaneous, dental (to one or more teeth), electrostomatognathia, endocervically, intraluminally, intratracheally, extracorporeally, hemodialysis, infiltration, interstitial, intraperitoneal, intraamniotic, intraarticular, intrahepatic, intratracheal, intracapsular, intracartilaginous (into cartilage), intrasacral (into the cauda equina), intracapsular (into the cerebellomedullary cistern), intracorneal (into the cornea), intradental, intracoronary (into the coronary artery), intracavernosally (into the expandable space of the corpus cavernosum of the penis), intradiscal (into the intervertebral disc), intraductal (into the glandular duct), intraduodenal (into the duodenum), intradural (into the dura or subdura), intraepidermal (into the epidermis), intraesophageal (into the esophagus), intragastric (into the stomach), intramuscular (into the gingiva), intraluminal (into the lumen), intralymphatic (into the lymph), intramedullary (into the intramedullary cavity of bone), intrathecal (into the meninges), intramyocardial (into the myocardium), intraocular (into the eye), intraovarian (into the ovary), epicardial (into the epicardium),Intrapleural (into the pleura), intraprostatic (into the prostate), intrapulmonary (into the lung or its bronchi), intranasal (into the nose or periorbital cavities), intraspinal (into the vertebral column), intrasynovial (into the synovial cavity of a joint), intratendinous (into a tendon), intratesticular (into the testis), intrathecal (into the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (into the thorax), intrarenal (into the renal tubules of an organ), intratumoral (into a tumor), intratympanic (into the tympanic membrane), intravascular (into a blood vessel or within a blood vessel), intraventricular (into a ventricle of the brain), iontophoresis (by an electric current that moves ions of a soluble salt into body tissues), irrigation (penetrating or water-washing an open wound or body cavity), laryngeal (directly onto the larynx), nasogastric tube (into the nose and stomach), occlusive dressing technique (local route administration (which is then covered by a dressing that occludes the area)), ophthalmic (to the external eye), oropharyngeal (directly into the mouth and oropharynx), parenteral, transdermal, perijoint, epidural, perineural, periodontal, rectal, respiratory (into the airways by oral or nasal inhalation for local or systemic effects), retrobulbar (behind the bridge or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, local, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (through or across the tympanic cavity), intrarenal (into the renal tubules), urethral (into the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, and the spinal cord, etc., but not limited to these, can be administered via the routes.

[0254] Modes of administration include injection, infusion, drip, and / or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intrathecal, and intrasternal injection and infusion. In some examples, the route is intravenous. The mitochondria, composition, or pharmaceutical composition of the present invention is administered as a single dose or as at least two or more consecutive doses. Preferably, the mitochondria, composition, or pharmaceutical composition of the present invention is administered by intravenous injection or inhalation. Preferably, the mitochondria, composition, or pharmaceutical composition of the present invention is administered into the bloodstream upstream of the target organ. Preferably, the mitochondria, composition, or pharmaceutical composition of the present invention is administered to an organ. Preferably, the mitochondria, composition, or pharmaceutical composition of the present invention is directly administered to a target organ such as an organ where treatment is desired. Preferably, the mitochondria, composition, or pharmaceutical composition of the present invention is directly administered to the target organ by injecting the mitochondria, composition, or pharmaceutical composition into the target organ. Preferably, the mitochondria, composition, or pharmaceutical composition of the present invention is administered by inhalation.

[0255] In certain embodiments, the target organ is the kidney. In certain embodiments, the mitochondria, composition, or pharmaceutical composition of the present invention is delivered to the kidney of a subject. To that end, the mitochondria, composition, or pharmaceutical composition of the present invention is preferably administered upstream of the kidney, i.e., into the renal artery of the subject. Alternatively, the mitochondria, composition, or pharmaceutical composition of the present invention is directly injected into the kidney.

[0256] In certain embodiments, the target organ is the heart. In certain embodiments, the mitochondria, composition, or pharmaceutical composition of the present invention is delivered to the heart of a subject. To that end, the mitochondria, composition, or pharmaceutical composition of the present invention is preferably administered upstream of the heart, i.e., into the coronary artery of the subject. Alternatively, the mitochondria, composition, or pharmaceutical composition of the present invention is directly injected into the heart.

[0257] In certain embodiments, the target organ is the liver. In certain embodiments, the mitochondria, composition or pharmaceutical composition of the present invention is delivered to the liver of the subject. For this purpose, the mitochondria, composition or pharmaceutical composition of the present invention is preferably administered upstream of the liver, i.e., into the hepatic artery or portal vein of the subject. Alternatively, the mitochondria, composition or pharmaceutical composition of the present invention is directly injected into the liver.

[0258] In certain embodiments, the target organ is the pancreas. In certain embodiments, the mitochondria, composition or pharmaceutical composition of the present invention is delivered to the pancreas of the subject. For this purpose, the mitochondria, composition or pharmaceutical composition of the present invention is preferably administered upstream of the pancreas, i.e., into the hepatic artery of the subject. Alternatively, the mitochondria, composition or pharmaceutical composition of the present invention is directly injected into the pancreas.

[0259] In certain embodiments, the target organ is the duodenum. In certain embodiments, the mitochondria, composition or pharmaceutical composition of the present invention is delivered to the duodenum of the subject. For this purpose, the mitochondria, composition or pharmaceutical composition of the present invention is preferably administered upstream of the duodenum, i.e., into the hepatic artery of the subject. Alternatively, the mitochondria, composition or pharmaceutical composition of the present invention is directly injected into the duodenum.

[0260] In certain embodiments, the target organ is the spleen. In certain embodiments, the mitochondria, composition or pharmaceutical composition of the present invention is delivered to the spleen of the subject. For this purpose, the mitochondria, composition or pharmaceutical composition of the present invention is preferably administered upstream of the spleen, i.e., into the splenic artery of the subject. Alternatively, the mitochondria, composition or pharmaceutical composition of the present invention is directly injected into the spleen.

[0261] In certain embodiments, the target organ is the lung. In certain embodiments, the mitochondria, composition or pharmaceutical composition of the present invention is delivered to the lung of the subject. For this purpose, the mitochondria, composition or pharmaceutical composition of the present invention is preferably administered upstream of the lung, i.e., into the pulmonary artery of the subject. Alternatively, the mitochondria, composition or pharmaceutical composition of the present invention is directly injected into the lung.

[0262] In certain embodiments, the target organ is the intestine. In certain embodiments, the mitochondria, composition or pharmaceutical composition of the present invention is delivered to the intestine of a subject. To that end, the mitochondria, composition or pharmaceutical composition of the present invention is preferably administered upstream of the intestine, i.e., to the superior mesenteric artery of the subject. Alternatively, the mitochondria, composition or pharmaceutical composition of the present invention is directly injected into the intestine.

[0263] In certain embodiments, the target organ is the bladder. In certain embodiments, the mitochondria, composition or pharmaceutical composition of the present invention is delivered to the bladder of a subject. To that end, the mitochondria, composition or pharmaceutical composition of the present invention is preferably administered upstream of the bladder, i.e., to the superior and inferior vesical arteries of the subject. Alternatively, the mitochondria, composition or pharmaceutical composition of the present invention is directly injected into the bladder.

[0264] For delivery of the mitochondria, composition or pharmaceutical composition, injection or administration by injection can be performed. The mitochondria, composition or pharmaceutical composition can be systemically administered. The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" refer to administering the mitochondria, composition or pharmaceutical composition other than directly to the target site, cell, tissue or organ, such that as a result it enters the subject's circulatory system and thus undergoes metabolism and other similar processes. The mitochondria, composition or pharmaceutical composition of the present invention is preferably delivered to cells via direct incubation with the cells in cell culture medium. In a further preferred embodiment, the mitochondria, composition or pharmaceutical composition of the present invention is directly delivered to the site where treatment is desired by injection. In a further preferred embodiment, the mitochondria, composition or pharmaceutical composition of the present invention is systemically delivered by intravenous injection. In a further preferred embodiment, the mitochondria, composition or pharmaceutical composition of the present invention is delivered by injection into the blood flow upstream of the target organ where treatment is desired. In a further preferred embodiment, the atomized mitochondria, atomized composition or atomized pharmaceutical composition of the present invention is delivered by inhalation.

[0265] The mitochondria, composition or pharmaceutical composition of the present invention can be administered into the bloodstream upstream of the target organ. Accordingly, the present invention provides a method for delivering a nucleic acid molecule to a target organ, the method comprising a mitochondrion comprising one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof attached to the outer membrane of the mitochondrion, wherein the one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof are a) electrostatically attached to the outer membrane of the mitochondrion via a positively charged species, or b) covalently bound to the outer membrane of the mitochondrion, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion, or d) linked to a mitochondrion-targeting small molecule, administering to the bloodstream of a patient in need of a pharmaceutical composition comprising the mitochondrion and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered into the bloodstream upstream of the target organ.

[0266] The present invention provides a method for delivering a payload such as a nucleic acid molecule, polypeptide, drug or combination thereof to a target organ, the method comprising a mitochondrion comprising one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof attached to the outer membrane of the mitochondrion, wherein the one or more payloads such as a nucleic acid molecule, polypeptide, drug or combination thereof are a) electrostatically attached to the outer membrane of the mitochondrion via a positively charged species, or b) covalently bound to the outer membrane of the mitochondrion, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion, or d) linked to a mitochondrion-targeting small molecule, administering a pharmaceutical composition comprising the mitochondrion and a pharmaceutically acceptable carrier into the bloodstream of a subject having a cardiovascular disease, age-related disease, kidney disease or cancer, wherein the pharmaceutical composition is administered into the bloodstream upstream of the target organ.

[0267] The present invention provides a method for delivering a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to a target organ. The method involves a mitochondrion comprising one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, which is attached to the outer membrane of the mitochondrion, and the one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are a) electrostatically attached to the outer membrane of the mitochondrion via a positively charged species, or b) covalently bound to the outer membrane of the mitochondrion, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion, or d) linked to a mitochondrion-targeting small molecule, administering a pharmaceutical composition comprising the mitochondrion and a pharmaceutically acceptable carrier into the bloodstream of a subject having ischemic heart disease, atherosclerosis, sarcopenia, Parkinson's disease, Hutchinson-Gilford progeria syndrome, or cancer, wherein the pharmaceutical composition is administered into the bloodstream upstream of the target organ.

[0268] The mitochondrion, composition, or pharmaceutical composition of the present invention can be administered by inhalation. Accordingly, the present invention provides a method for delivering a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the lung. The method involves a mitochondrion comprising one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, which is attached to the outer membrane of the mitochondrion, and the one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are a) electrostatically attached to the outer membrane of the mitochondrion via a positively charged species, or b) covalently bound to the outer membrane of the mitochondrion, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion, or d) linked to a mitochondrion-targeting small molecule, administering a pharmaceutical composition comprising the mitochondrion and a pharmaceutically acceptable carrier to a subject in need thereof, wherein the pharmaceutical composition is administered by inhalation.

[0269] The present invention provides a method for delivering a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the lung, the method comprising a mitochondrion comprising one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof attached to the outer membrane of the mitochondrion, wherein the one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are a) electrostatically attached to the outer membrane of the mitochondrion via a positively charged species, or b) covalently bound to the outer membrane of the mitochondrion, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion, or d) linked to a mitochondrion-targeting small molecule, administering to a subject having a cardiovascular disease, an age-related disease, a kidney disease, or cancer a pharmaceutical composition comprising the mitochondrion and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered by inhalation.

[0270] The present invention provides a method for delivering a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the lung, the method comprising a mitochondrion comprising one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof attached to the outer membrane of the mitochondrion, wherein the one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are a) electrostatically attached to the outer membrane of the mitochondrion via a positively charged species, or b) covalently bound to the outer membrane of the mitochondrion, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion, or d) linked to a mitochondrion-targeting small molecule, administering to a subject having ischemic heart disease, atherosclerosis, muscular dystrophy, Parkinson's disease, Hutchinson-Gilford progeria syndrome, or cancer a pharmaceutical composition comprising the mitochondrion and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered by inhalation.

[0271] In certain embodiments, a mitochondrion containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, or a composition or pharmaceutical composition containing a mitochondrion containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, is delivered to the kidney of a subject. In certain embodiments, delivery to the kidney is achieved by injection into the renal artery or direct injection into the kidney. In certain embodiments, delivery to the kidney is achieved by injection into the renal artery or direct injection into the kidney, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are electrostatically added to the outer membrane of the mitochondrion via a positively charged species. In certain embodiments, delivery to the kidney is achieved by injection into the renal artery or direct injection into the kidney, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are covalently bound to the outer membrane of the mitochondrion. In certain embodiments, delivery to the kidney is achieved by injection into the renal artery or direct injection into the kidney, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion. In certain embodiments, delivery to the kidney is achieved by injection into the renal artery or direct injection into the kidney, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to a mitochondrion-targeting small molecule.

[0272] In certain embodiments, a mitochondrion comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, or a composition or pharmaceutical composition comprising a mitochondrion comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, is delivered to the heart of a subject. In certain embodiments, delivery to the heart is achieved by intracoronary injection or direct injection into the heart. In certain embodiments, delivery to the heart is achieved by intracoronary injection or direct injection into the heart, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are electrostatically attached to the outer membrane of the mitochondrion via a positively charged species. In certain embodiments, delivery to the heart is achieved by intracoronary injection or direct injection into the heart, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are covalently bound to the outer membrane of the mitochondrion. In certain embodiments, delivery to the heart is achieved by intracoronary injection or direct injection into the heart, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion. In certain embodiments, delivery to the heart is achieved by intracoronary injection or direct injection into the heart, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to a mitochondrion-targeting small molecule.

[0273] In certain embodiments, a mitochondrion comprising one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof, or a composition or pharmaceutical composition comprising a mitochondrion comprising one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof, is delivered to the liver of a subject. In certain embodiments, delivery to the liver is accomplished by injection into the hepatic artery or portal vein, or by direct injection into the liver. In certain embodiments, delivery to the liver is accomplished by injection into the hepatic artery or portal vein, or by direct injection into the liver, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are electrostatically attached to the outer membrane of the mitochondrion via a positively charged species. In certain embodiments, delivery to the liver is accomplished by injection into the hepatic artery or portal vein, or by direct injection into the liver, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are covalently bound to the outer membrane of the mitochondrion. In certain embodiments, delivery to the liver is accomplished by injection into the hepatic artery or portal vein, or by direct injection into the liver, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion. In certain embodiments, delivery to the liver is accomplished by injection into the hepatic artery or portal vein, or by direct injection into the liver, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are linked to a mitochondrion-targeting small molecule.

[0274] In certain embodiments, a composition or pharmaceutical composition comprising mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, or mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, is delivered to the pancreas of a subject. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or direct injection into the pancreas. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or direct injection into the pancreas, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are electrostatically attached to the outer membrane of the mitochondria via positively charged species. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or direct injection into the pancreas, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are covalently bound to the outer membrane of the mitochondria. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or direct injection into the pancreas, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or direct injection into the pancreas, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to a mitochondria-targeting small molecule.

[0275] In certain embodiments, a composition or pharmaceutical composition comprising a mitochondrion containing one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, or a mitochondrion containing one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof is delivered to the duodenum of a subject. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or direct injection into the duodenum. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or direct injection into the duodenum, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are electrostatically attached to the outer membrane of the mitochondrion via a positively charged species. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or direct injection into the duodenum, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are covalently bound to the outer membrane of the mitochondrion. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or direct injection into the duodenum, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or direct injection into the duodenum, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are linked to a mitochondrion-targeting small molecule.

[0276] In certain embodiments, a composition or pharmaceutical composition comprising mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, or mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, is delivered to the spleen of a subject. In certain embodiments, delivery to the spleen is accomplished by injection into the splenic artery or direct injection into the spleen. In certain embodiments, delivery to the spleen is accomplished by injection into the splenic artery or direct injection into the spleen, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are electrostatically attached to the outer membrane of the mitochondria via positively charged species. In certain embodiments, delivery to the spleen is accomplished by injection into the splenic artery or direct injection into the spleen, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are covalently bound to the outer membrane of the mitochondria. In certain embodiments, delivery to the spleen is accomplished by injection into the splenic artery or direct injection into the spleen, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria. In certain embodiments, delivery to the spleen is accomplished by injection into the splenic artery or direct injection into the spleen, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to a mitochondria-targeting small molecule.

[0277] In certain embodiments, a mitochondrion comprising one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, or a composition or pharmaceutical composition comprising a mitochondrion comprising one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, is delivered to the lung of a subject. In certain embodiments, delivery to the lung is accomplished by injection into the pulmonary artery or direct injection into the lung. In certain embodiments, delivery to the lung is accomplished by injection into the pulmonary artery or direct injection into the lung, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are electrostatically appended to the outer membrane of the mitochondrion via a positively charged species. In certain embodiments, delivery to the lung is accomplished by injection into the pulmonary artery or direct injection into the lung, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are covalently bound to the outer membrane of the mitochondrion. In certain embodiments, delivery to the lung is accomplished by injection into the pulmonary artery or direct injection into the lung, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondrion. In certain embodiments, delivery to the lung is accomplished by injection into the pulmonary artery or direct injection into the lung, and one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof are linked to a mitochondrion-targeting small molecule.

[0278] In certain embodiments, a composition or pharmaceutical composition comprising mitochondria containing one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof, or mitochondria containing one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof is delivered to the intestine of a subject. In certain embodiments, delivery to the intestine is achieved by injection into the superior mesenteric artery or direct injection into the intestine. In certain embodiments, delivery to the intestine is achieved by injection into the superior mesenteric artery or direct injection into the intestine, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are electrostatically attached to the outer membrane of the mitochondria via positively charged species. In certain embodiments, delivery to the intestine is achieved by injection into the superior mesenteric artery or direct injection into the intestine, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are covalently bound to the outer membrane of the mitochondria. In certain embodiments, delivery to the intestine is achieved by injection into the superior mesenteric artery or direct injection into the intestine, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria. In certain embodiments, delivery to the intestine is achieved by injection into the superior mesenteric artery or direct injection into the intestine, and one or more payloads such as nucleic acids, polypeptides, drugs, or combinations thereof are linked to a mitochondria-targeting small molecule.

[0279] In certain embodiments, a composition or pharmaceutical composition comprising mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, or mitochondria containing one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, is delivered to the bladder of a subject. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or direct injection into the bladder. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or direct injection into the bladder, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are electrostatically attached to the outer membrane of the mitochondria via positively charged species. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or direct injection into the bladder, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are covalently bound to the outer membrane of the mitochondria. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or direct injection into the bladder, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or direct injection into the bladder, and one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are linked to a mitochondria-targeting small molecule.

[0280] In one aspect, the present invention provides a method of making the mitochondria of the present invention by adding a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof to the mitochondria. As used herein, "contacting" means physically bringing a first substance into proximity to a second substance such that both can undergo a reaction. For example, the mitochondria can be contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof in the presence of a positively charged species in a solution such as a buffer.

[0281] In the context of the present invention, any payload such as a nucleic acid, polypeptide, or drug can be added to mitochondria. The payload can be added to the mitochondria by contacting the mitochondria with the payload, such as a nucleic acid, drug, or polypeptide. In the context of the present invention, the mitochondria and the payload can be contacted in the presence of a positively charged species such as a polycationic species. The step of contacting the mitochondria with a nucleic acid, drug, or polypeptide and / or a positively charged species can be carried out under any reaction conditions that are feasible for the successful formation of a complex, i.e., the successful addition of the nucleic acid, drug, or polypeptide.

[0282] The mitochondria can be contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof in a buffer such as a conjugation buffer. The mitochondria can be contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof and a positively charged species such as a polycationic species in a buffer such as a conjugation buffer. The mitochondria can be contacted with a polypeptide in a buffer such as a conjugation buffer. The mitochondria can be contacted with a polypeptide and a positively charged species such as a polycationic species in a buffer such as a conjugation buffer.

[0283] In some embodiments, the concentration of mitochondria is 0.1 to 5 mg / mL. In some embodiments, the concentration of mitochondria in the conjugation buffer is 0.1 to 5 mg / mL. In some embodiments, the concentration of mitochondria is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 mg / mL. In some embodiments, the concentration of mitochondria in the conjugation buffer is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 3.8, 4.9 or 5 mg / mL. In a preferred embodiment, the concentration of mitochondria in the conjugation buffer is 2 mg / mL. In a preferred embodiment, the concentration of mitochondria in the conjugation buffer is 4 mg / mL.

[0284] In some embodiments, the concentration of mitochondria is 0.5 to 30 billion / mL. In some embodiments, the concentration of mitochondria in the conjugation buffer is 0.5 to 30 billion / mL. In some embodiments, the concentration of mitochondria is 0.5 billion, 0.6 billion, 0.7 billion, 0.8 billion, 0.9 billion, 1 billion, 1.1 billion, 1.2 billion, 1.3 billion, 1.4 billion, 1.5 billion, 1.6 billion, 1.7 billion, 1.8 billion, 1.9 billion, 2 billion, 2.1 billion, 2.2 billion, 2.3 billion, 2.4 billion, 2.5 billion, 2.6 billion, 2.7 billion, 2.8 billion, 2.9 billion, 3 billion, 3.1 billion, 3.2 billion, 3.3 billion, 3.4 billion, 3.5 billion, 3.6 billion, 3.7 billion, 3.8 billion, 3.9 billion, 4 billion, 4.1 billion, 4.2 billion, 4.3 billion, 4.4 billion, 4.5 billion, 4.6 billion, 4.7 billion, 4.8 billion, 4.9 billion, 5 billion, 5.2 billion, 5.5 billion, 5.8 billion, 6 billion, 6.2 billion, 6.4 billion, 6.8 billion, 7 billion, 7.2 billion, 7.5 billion, 7.8 billion, 8 billion, 8.2 billion, 8.4 billion, 8.6 billion, 8.8 billion, 9 billion, 9.2 billion, 9.4 billion, 9.6 billion, 9.8 billion, 10 billion, 10.2 billion, 10.4 billion, 10.6 billion, 10.8 billion, 11 billion, 11.2 billion, 11.4 billion, 11.6 billion, 11.8 billion, 12 billion, 12.2 billion, 12.5 billion, 12.9 billion, 13.2 billion, 13.4 billion, 13.6 billion, 13.8 billion, 14 billion, 15 billion, 16 billion, 17 billion, 18 billion, 20 billion, 22 billion, 24 billion, 26 billion, 28 billion, or 30 billion / mL. In some embodiments, the concentration of mitochondria in the conjugation buffer is 0.5 billion, 0.6 billion, 0.7 billion, 0.8 billion, 0.9 billion, 1 billion, 1.1 billion, 1.2 billion, 1.3 billion, 1.4 billion, 1.5 billion, 1.6 billion, 1.7 billion, 1.8 billion, 1.9 billion, 2 billion, 2.1 billion, 2.2 billion, 2.3 billion, 2.4 billion, 2.5 billion, 2.6 billion, 2.7 billion, 2.8 billion, 2.9 billion, 3 billion, 3.1 billion, 3.2 billion, 3.3 billion, 3.4 billion, 3.5 billion, 3.6 billion, 3.7 billion, 3.8 billion, 3.9 billion, 4 billion, 4.1 billion, 4.2 billion, 4.3 billion, 4.4 billion, 4.5 billion, 4.6 billion, 4.7 billion, 4.8 billion, 4.9 billion, 5 billion, 5.2 billion, 5.5 billion, 5.8 billion, 6 billion, 6.2 billion, 6.4 billion, 6.8 billion, 7 billion, 7.2 billion, 7.5 billion, 7.8 billion, 8 billion, 8.2 billion, 8.4 billion, 8.6 billion, 8.8 billion, 9 billion, 9.2 billion, 9.4 billion, 9.6 billion, 9.8 billion, 10 billion, 10.2 billion, 10.4 billion, 10.6 billion, 10.8 billion, 11 billion, 11.2 billion, 11.4 billion, 11.6 billion, 11.8 billion, 12 billion, 12.2 billion, 12.5 billion, 12.9 billion, 13.2 billion, 13.4 billion, 13.6 billion, 13.8 billion, 14 billion, 15 billion, 16 billion, 17 billion, 18 billion, 20 billion, 22 billion, 24 billion, 26 billion, 28 billion, or 30 billion / mL.In a preferred embodiment, the concentration of mitochondria in the conjugation buffer is 6 billion / mL. In a preferred embodiment, the concentration of mitochondria in the conjugation buffer is 12 billion / mL. In a preferred embodiment, the concentration of mitochondria in the conjugation buffer is 15 billion / mL.

[0285] In some embodiments, the mitochondria are contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, in an amount of from 0.002 pmol to 5000 pmol. In some embodiments, the mitochondria are contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, in conjugation buffer, in an amount of from 0.002 pmol to 5000 pmol. In some embodiments, the mitochondria are optionally contacted, in conjugation buffer, with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, in an amount of 0.002, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, or 5000 pmol. In some embodiments, the mitochondria are optionally contacted, in conjugation buffer, with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, in an amount of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 pmol.In a preferred embodiment, the mitochondria are contacted with a payload such as 0.1 pmol to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof. In a preferred embodiment, the mitochondria are contacted with a payload such as 0.1 pmol to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof in a conjugation buffer.

[0286] In some embodiments, mitochondria are contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof at 0.002 μg / μL to 5 μg / μL. In some embodiments, mitochondria are contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof at 0.002 μg / μL to 5 μg / μL in a conjugation buffer. In some embodiments, the mitochondria are optionally contacted with a nucleic acid molecule at 0.002, 0.004, 0.008, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 μg / μL in a conjugation buffer. In some embodiments, the mitochondria are optionally contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof at 0.002, 0.004, 0.008, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 μg / μL. In preferred embodiments, the mitochondria are contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof at 0.1 μg / μL to 2 μg / μL. In preferred embodiments, the mitochondria are contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof at 0.1 μg / μL to 2 μg / μL in a conjugation buffer.

[0287] In some embodiments, the mitochondria are contacted with a positively charged species at 0.004 mg / mL to 40 mg / mL. In some embodiments, the mitochondria are contacted with a positively charged species at 0.004 mg / mL to 40 mg / mL in a conjugation buffer. In some embodiments, the mitochondria are contacted with a positively charged species at 0.004, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mg / mL. In some embodiments, the mitochondria are contacted with a positively charged species at 0.004, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mg / mL in a conjugation buffer. In preferred embodiments, the mitochondria are optionally contacted with a positively charged species at 0.02 to 1.0 mg / mL in a conjugation buffer. In preferred embodiments, the mitochondria are optionally contacted with a positively charged species at 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg / mL in a conjugation buffer.

[0288] In some embodiments, the mitochondria are contacted with a protective polymer at a concentration of 0.004 to 40 mg / mL. In some embodiments, the mitochondria are contacted with a protective polymer at a concentration of 0.004 to 40 mg / mL in a conjugation buffer. In some embodiments, the mitochondria are contacted with a protective polymer at a concentration of 0.004, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mg / mL. In some embodiments, the mitochondria are contacted with a protective polymer at a concentration of 0.004, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mg / mL in a conjugation buffer. In preferred embodiments, the mitochondria are optionally contacted with a protective polymer at a concentration of 0.02 to 2 mg / mL in a conjugation buffer. In preferred embodiments, the mitochondria are optionally contacted with a protective polymer at a concentration of 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.8, 2.0 mg / mL.

[0289] In a more preferred embodiment, the mitochondria are contacted with a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof at 0.002 to 5000 pmol and a positively charged species at 0.004 to 40 mg / mL, wherein the concentration of the mitochondria is optionally 0.1 to 5 mg / mL in a conjugation buffer.

[0290] In a more preferred embodiment, mitochondria are contacted with a payload such as 0.1 to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof, and a positively charged species at a concentration of 0.02 to 1.0 mg / mL, wherein the concentration of mitochondria is optionally 1 mg / mL in the conjugation buffer.

[0291] In a more preferred embodiment, mitochondria are contacted with a payload such as 0.1 to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof, and a positively charged species at a concentration of 0.02 to 1.0 mg / mL, and the concentration of mitochondria is 1 mg / mL in the conjugation buffer. In a more preferred embodiment, 50 μg of mitochondria are contacted with a payload such as 0.1 to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof, and a positively charged species at a concentration of 0.02 to 1.0 mg / mL, and the concentration of mitochondria is 1 mg / mL in the conjugation buffer.

[0292] In some embodiments, 50 μg of mitochondria are contacted with a payload such as 0.1 to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof or a peptide, and a positively charged species at a concentration of 0.02 to 1.0 mg / mL. In some embodiments, 50 μg of mitochondria are contacted with a plurality of payloads such as 0.1 to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof linked to a mitochondrion-targeting small molecule. In a preferred embodiment, 50 μg of mitochondria are contacted with a plurality of payloads such as 0.1 to 50 pmol of a nucleic acid molecule, polypeptide, drug, or a combination thereof, and a positively charged species at a concentration of 0.02 to 1.0 mg / mL.

[0293] In some embodiments, 50 μg of mitochondria are contacted with 0.1 - 50 pmol of siRNA or mRNA. In some embodiments, 50 μg of mitochondria are contacted with 0.1 - 50 pmol of fluorescently labeled ssDNA or ssRNA or plasmid DNA. In some embodiments, 50 μg of mitochondria are contacted with 0.1 - 2 μL of 10 mg / mL poly-L-lysine. The concentration of mitochondria is preferably 1 mg of mitochondria per 1 mL of conjugation buffer, i.e., 1 mg / mL. Those skilled in the art recognize that the above embodiments can be combined to facilitate the successful conjugation of payloads such as nucleic acids or polypeptides to mitochondria.

[0294] In some embodiments, an amount of 50 μg - 200 μg of mitochondria is contacted with 0.1 - 50 pmol of a nucleic acid molecule and 0.02 - 10 μg, preferably 0.02 - 5 μg of a positively charged species.

[0295] In some embodiments, an amount of 50 μg - 200 μg of mitochondria is contacted with 0.1 pmol - 50 pmol of a nucleic acid molecule linked to a mitochondria-targeting small molecule.

[0296] - 50 micrograms of mitochondria corresponds to approximately 150 million mitochondria. 1 mg / mL of mitochondria (based on Qubit protein assay) corresponds to approximately 3B mitochondria / mL (based on particle counter).

[0297] - The concentration of the preparation of the protective polymer used in the preparation of the mitochondria of the present invention is 1 mg / mL. The amount of the protective polymer is 0.1 mg - 10 mg.

[0298] - The concentration of the preparation of the nanoparticles used in the preparation of the mitochondria of the present invention is 1 mg / mL. The amount of the protective polymer is 0.1 mg - 10 mg.

[0299] Accordingly, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a positively charged species; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged species. The method includes steps (a), (b), and (c).

[0300] At least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof can be contacted with the positively charged species and the mitochondria simultaneously, or at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof can be first contacted with the positively charged species to form a positively charged complex, and then the positively charged complex can be contacted with the mitochondria, or the mitochondria can be first contacted with the positively charged species and then contacted with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof. Accordingly, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a positively charged species, wherein at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof is contacted with the positively charged species and the mitochondria simultaneously, or at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof is contacted with the positively charged species to form a positively charged complex, and then the positively charged complex is contacted with the mitochondria, or The mitochondria are contacted with a positively charged species and subsequently with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof. a process, c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged species; and includes.

[0301] Within the scope of the method of the present invention, the method may include contacting the mitochondria with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a positively charged species, where a) contacting at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof with the positively charged species and the mitochondria simultaneously; b) at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof is contacted with the positively charged species to form a positively charged complex before contacting the positively charged complex with the mitochondria, or c) contacting the mitochondria with the positively charged species and subsequently with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof.

[0302] In a preferred embodiment, the method of the present invention includes contacting the mitochondria with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a positively charged species, where the mitochondria are contacted with the positively charged species and subsequently with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof.

[0303] The step of contacting the mitochondria with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof and the positively charged species can be carried out in a suitable buffer. Accordingly, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, and the method is a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, in the presence of a positively charged species; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged species; comprising; contacting the mitochondria with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof and a positively charged species in a suitable buffer.

[0304] Preferably, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, in the presence of a positively charged species; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged species; comprising; contacting the mitochondria with one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof and a polycationic species in a buffer comprising a 4:1 mixture of solution X comprising 20 mM HEPES, 1 mM EGTA, and 300 mM trehalose (pH 7.2) and solution Y comprising 0.1 M CHES (pH 10) and 0.2 M sodium phosphate dihydrate.

[0305] The contacting step of the present invention is not particularly limited in terms of reaction conditions, time, and reaction time. Generally, any reaction conditions can be used that promote the addition of a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to mitochondria via a positively charged species, thereby promoting the formation of a delivery complex. However, it is preferred to contact the mitochondria with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof and a positively charged species at room temperature for more than 5 minutes, preferably in the dark. Accordingly, the present invention provides a method for adding a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof in the presence of a positively charged species, the step of contacting the mitochondria with a plurality of nucleic acid molecules and a positively charged species at room temperature for at least 5 minutes, for example at least 10 minutes, 20 minutes, or 30 minutes; c) adding at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the mitochondria via the positively charged species; and comprising.

[0306] The present invention provides a method for adding a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof in the presence of a positively charged species, the step of contacting the mitochondria with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof and a positively charged species in the dark; c) adding at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the mitochondria via the positively charged species; and comprising.

[0307] The present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria. The method comprises: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a positively charged species, the contacting being carried out at room temperature for at least 5 minutes, for example in the dark for at least 10 minutes, 20 minutes, or 30 minutes, with a plurality of payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof and the positively charged species; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged species. The method includes the steps above.

[0308] Preferably, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria. The method comprises: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a positively charged species, the contacting being carried out at room temperature for 30 minutes in the dark with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof and the positively charged species; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged species. The method includes the steps above.

[0309] As described above herein, the present invention provides mitochondria comprising one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, added to the outer membrane of the mitochondria. The nucleic acid molecule is preferably DNA or RNA. Thus, the present invention also provides a method for adding a DNA molecule to the outer membrane of mitochondria, a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one DNA molecule in the presence of a positively charged species; c) adding at least one DNA molecule to the mitochondria via the positively charged species; and comprising.

[0310] The present invention also provides a method for adding an RNA molecule to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one RNA molecule in the presence of a positively charged species; c) adding at least one RNA molecule to the mitochondria via the positively charged species; and comprising.

[0311] As described above, the present invention provides for the addition of nucleic acid molecules to mitochondria via positively charged species, preferably polycationic species. Thus, the present invention provides a method for adding a nucleic acid molecule to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one nucleic acid molecule in the presence of a polycationic species; c) adding at least one nucleic acid molecule to the mitochondria via the polycationic species; and comprising.

[0312] In the context of the present invention, the polycationic species can be a linear or branched polycationic polymer. The linear or branched polycationic polymer can be electrostatically linked to a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, for example, a DNA or RNA molecule. The present invention is not particularly limited to polycationic polymers. Generally, any polycationic polymer that promotes the addition of a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to mitochondria, thereby promoting the formation of a delivery complex, can be used. However, the linear or branched polycationic polymer is preferably polylysine, histidyl-polylysine, polyornithine, polyarginine, high mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivatives, diethylaminoethyl (DEAE)-dextran, poly(N-alkyl-4-vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose, or a combination thereof. Accordingly, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a linear or branched polycationic polymer; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the polycationic species; and comprising.

[0313] The present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a linear or branched polycationic polymer electrostatically linked to the payload; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via a positively charged species; comprising.

[0314] The present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of a polycationic polymer, wherein the polycationic polymer is polylysine, histidyl - polylysine, polyornithine, polyarginine, high - mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2 - (dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivative, diethylaminoethyl (DEAE) - dextran, poly(N - alkyl - 4 - vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose, or a combination thereof, and optionally, the polycationic polymer is electrostatically linked to at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via a positively charged species; comprising.

[0315] As described above herein, the negative surface charge profile of mitochondria can also be useful for electrostatically adding one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, to the outer membrane of mitochondria via positively charged nanoparticles or positively charged particles. The payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, may be added to the surface of the positively charged nanoparticles / particles or encapsulated therein. The present invention is not particularly limited to any nanoparticles or particles. Generally, any positively charged nanoparticles / particles that facilitate the addition of payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof to mitochondria, thereby facilitating the formation of delivery complexes, can be used. However, the positively charged nanoparticles / particles are preferably lipid nanoparticles / particles, dendrimer nanoparticles / particles, micelle nanoparticles / particles, protein nanoparticles / particles, liposomes, non-porous silica nanoparticles / particles, mesoporous silica nanoparticles / particles, silicon nanoparticles / particles, gold nanoparticles / particles, gold nanowires / wires, silver nanoparticles / particles, platinum nanoparticles / particles, palladium nanoparticles / particles, titanium dioxide nanoparticles / particles, carbon nanotubes / tubes, carbon dot nanoparticles / particles, polymer nanoparticles / particles, zeolite nanoparticles / particles, aluminum oxide nanoparticles / particles, hydroxyapatite nanoparticles / particles, quantum dot nanoparticles / particles, zinc oxide nanoparticles / particles, zirconium dioxide nanoparticles / particles, graphene or graphene oxide nanoparticles / particles. Accordingly, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of positively charged nanoparticles; c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged species; and including.

[0316] The present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria. The method comprises: a) providing a preparation of mitochondria; and b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of positively charged nanoparticles; and c) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via the positively charged nanoparticles. The method includes:

[0317] Within the scope of the method of the present invention, the method can include contacting the mitochondria with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of positively charged nanoparticles. The method further includes: a) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the surface of the positively charged nanoparticles; or b) encapsulating at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof within the positively charged nanoparticles. The method further includes:

[0318] Accordingly, the present invention provides a method for adding a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria. The method comprises: a) providing a preparation of mitochondria; and b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof in the presence of positively charged nanoparticles, wherein before step (b), a further step: a’) adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the surface of the positively charged nanoparticles; or b’) encapsulating at least one payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, within positively charged nanoparticles; which is carried out, c) adding at least one payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, to mitochondria via positively charged nanoparticles; and includes.

[0319] The present invention provides a method for adding a payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, to the outer membrane of mitochondria, and the method includes a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, in the presence of positively charged nanoparticles, and prior to step (b), a further step: a’) adding at least one payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, to the surface of positively charged nanoparticles, or b’) encapsulating at least one payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, within positively charged nanoparticles; which is carried out, c) adding at least one payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, to mitochondria via positively charged nanoparticles; and the positively charged nanoparticles are lipid nanoparticles, dendrimer nanoparticles, micelle nanoparticles, protein nanoparticles, liposomes, non-porous silica nanoparticles, mesoporous silica nanoparticles, silicon nanoparticles, gold nanoparticles, gold nanowires, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, titanium dioxide nanoparticles, carbon nanotubes, carbon dot nanoparticles, polymer nanoparticles, zeolite nanoparticles, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, quantum dot nanoparticles, zinc oxide nanoparticles, zirconium oxide nanoparticles, graphene, or graphene oxide nanoparticles.

[0320] In another aspect, the present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria. As described above, the present invention provides a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof that can covalently bind to the outer membrane of mitochondria, either directly or indirectly, for example via an intermediate. Exemplary intermediates include activated esters such as N-hydroxysuccinimide (NHS) esters. Accordingly, the present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) providing a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof modified to include an activated ester; c) adding the payload such as the nucleic acid molecule, polypeptide, drug, or combination thereof provided in step (b) to an amine contained in a polypeptide in the outer membrane of the mitochondria; and comprising.

[0321] Preferably, the present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) providing a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof modified to include an N-hydroxysuccinimide (NHS) ester; c) adding the payload such as the nucleic acid molecule, polypeptide, drug, or combination thereof provided in step (b) to an amine contained in a polypeptide in the outer membrane of the mitochondria; and comprising.

[0322] In another aspect, the present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) providing a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, modified to contain a chemical group; c) adding the payload such as the nucleic acid molecule, polypeptide, drug, or a combination thereof provided in step (b) to an amine contained in a polypeptide of the outer membrane of mitochondria via a chemical group; comprising.

[0323] The present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) providing a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, modified to contain a chemical group selected from isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imido ester, carbodiimide, anhydride, and fluorophenyl ester; c) adding the nucleic acid molecule provided in step (b) to an amine contained in a polypeptide in the outer membrane of mitochondria via a chemical group; comprising.

[0324] As described above herein, a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof can be added to or encapsulated within nanoparticles and then covalently attached to mitochondria via a covalent bond (e.g., an amide bond).

[0325] The present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) encapsulating a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof within nanoparticles, wherein the surface of the nanoparticles contains an activated ester; c) adding the nanoparticles provided in step (b) to the amine contained in the polypeptide in the outer membrane of the mitochondrion; comprises.

[0326] Preferably, the present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof to the outer membrane of a mitochondrion, the method comprising: a) providing a preparation of mitochondria; b) encapsulating a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof in nanoparticles, wherein the surface of the nanoparticles comprises N-hydroxysuccinimide (NHS) ester; c) adding the nanoparticles provided in step (b) to the amine contained in the polypeptide in the outer membrane of the mitochondrion comprises.

[0327] The nanoparticles are not particularly limited and can be any nanoparticles known to those skilled in the art. In some embodiments, the nanoparticles are positively charged nanoparticles as described above herein. In another aspect, the present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof to the outer membrane of a mitochondrion, the method comprising: a) providing a preparation of mitochondria; b) encapsulating a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof in nanoparticles, wherein the surface of the nanoparticles comprises a chemical group; c) adding the nanoparticles provided in step (b) to the amine contained in the polypeptide in the outer membrane of the mitochondrion via the chemical group; comprises.

[0328] The present invention provides a method for covalently attaching a payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof to the outer membrane of a mitochondrion, the method comprising: a) providing a preparation of mitochondria; b) Encapsulating a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof into nanoparticles, wherein the surface of the nanoparticles contains a chemical group selected from isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imido ester, carbodiimide, anhydride, and fluorophenyl ester; c) Attaching the nanoparticles provided in step (b) to an amine contained in a polypeptide in the outer membrane of mitochondria via the chemical group; and comprising.

[0329] In a further embodiment, the present invention provides a method for attaching a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, wherein the nucleic acid molecule is linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria. Accordingly, the present invention provides a method for attaching at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) Providing a preparation of mitochondria; b) Contacting the mitochondria provided in step (a) having an antigen in its outer membrane with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof linked to an antibody; c) Attaching at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the mitochondria via the antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of mitochondria; and comprising.

[0330] In some embodiments, the present invention provides a method for attaching one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) Providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) that contain the antigen on their outer membrane with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof encapsulated in nanoparticles, wherein the nanoparticles are covalently bound to an antibody; c) adding at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria; comprising.

[0331] In some embodiments, the present invention provides a method for adding one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) that contain the antigen on their outer membrane with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, wherein the nucleic acid molecule is electrostatically linked to a modified antibody, and the modified antibody has one or more positive charges; c) adding at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria; comprising.

[0332] In some embodiments, the present invention provides a method for adding one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) that contain the antigen on their outer membrane with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof, wherein the nucleic acid molecule is covalently bound to biotin, and the biotin is linked to an avidin-conjugated antibody; c) a step of adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria comprising.

[0333] In some embodiments, the present invention provides a method for adding one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) containing an antigen in its outer membrane with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, wherein the nucleic acid molecule is covalently bonded to an activated ester, and the activated ester is bonded to the antibody via an amide bond; c) a step of adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria comprising.

[0334] In some embodiments, the present invention provides a method for adding one or more nucleic acid molecules, which are single-stranded nucleic acid molecules (ssDNA or ssRNA), to the outer membrane of mitochondria, the method comprising a) providing a preparation of mitochondria; b) contacting the mitochondria containing an antigen in its outer membrane provided in step (a) with at least one single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule is capable of hybridizing with one or more complementary single-stranded nucleic acid molecules on or attached to the antibody; c) a step of adding at least one single-stranded nucleic acid molecule to mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria comprising.

[0335] In some embodiments, the present invention provides a method for adding one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a), which contain an antigen on their outer membrane, with at least one payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof, wherein one or more nucleic acid molecules are encapsulated in nanoparticles, the nanoparticles are electrostatically linked to a modified antibody, and the modified antibody has one or more positive charges; c) adding at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria; and comprising.

[0336] In some embodiments, the present invention provides a method for adding one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, to the outer membrane of mitochondria, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a), which contain an antigen on their outer membrane, with at least one payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof, wherein one or more payloads such as nucleic acid molecules, polypeptides, drugs, or combinations thereof are encapsulated in nanoparticles, the nanoparticles are electrostatically linked to a modified antibody, and the modified antibody has one or more negative charges; c) adding at least one payload such as a nucleic acid molecule, polypeptide, drug, or combination thereof to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria; and comprising.

[0337] In some embodiments, the present invention provides a method for adding one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, to the outer membrane of mitochondria, the method comprising: a) Providing a preparation of mitochondria; b) Contacting the mitochondria provided in step (a) that contain an antigen on their outer membrane with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, wherein one or more nucleic acid molecules are encapsulated in nanoparticles, the nanoparticles are covalently bound to biotin, and the biotin is linked to an avidin-conjugated antibody; c) Adding at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria. In some embodiments, the present invention provides a method for adding one or more payloads such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof to the outer membrane of mitochondria, the method comprising: a) Providing a preparation of mitochondria; b) Contacting the mitochondria provided in step (a) that contain an antigen on their outer membrane with at least one payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, wherein one or more nucleic acid molecules are encapsulated in nanoparticles, the nanoparticles are covalently bound to an activated ester, and the activated ester is linked to an antibody via an amide bond; c) Adding at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria. Including.

[0338] The antigen contained in the outer membrane of the mitochondria can be any antigen that can bind to an antibody linked to a payload such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof. In preferred embodiments, the antigen is OPA1, TOM70, TOMM20, mitofusin 1, mitofusin 2, or VDAC1.

[0339] The present invention also provides a method for adding a payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the outer membrane of mitochondria via a mitochondrially targeted small molecule, the method comprising: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof linked to a mitochondrially targeted small molecule; c) adding at least one payload such as a nucleic acid molecule, polypeptide, drug, or a combination thereof to the mitochondria via the mitochondrially targeted small molecule. The method includes the above steps.

[0340] The mitochondrially targeted small molecule can be any mitochondrially targeted small molecule. Preferably, the mitochondrially targeted small molecule is selected from triphenylphosphonium (TPP), decalinium (DQA), E-4-(1H-indol-3-ylvinyl)-N-methylpyridinium iodide (F16), rhodamine 19, biguanide, and guanidine.

[0341] In the context of the present invention, the nucleic acid molecule is not necessarily related to the same nucleic acid molecule, i.e., a molecule of the same sequence. Although it is understood that in some embodiments, nucleic acid molecules of the same sequence are delivered, in other embodiments of the present invention, at least two or more different nucleic acid molecules may be added to the outer membrane of mitochondria.

[0342] In some embodiments, the method of the present invention further includes linking and / or surrounding the mitochondria containing one or more payloads such as a nucleic acid molecule, polypeptide, drug, or a combination thereof with a protective layer. The mitochondria containing one or more nucleic acid molecules can be any of the above-mentioned mitochondria and any of the above-mentioned protective layers. The method of linking and / or surrounding the mitochondria with a protective layer preferably includes contacting the mitochondria with a component forming the protective layer, such as the above-mentioned protective polymer or protective lipid layer. In a preferred embodiment, the present invention is a method for adding a nucleic acid molecule to the outer membrane of mitochondria. a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one nucleic acid molecule in the presence of a positively charged species; c) adding at least one nucleic acid molecule to the mitochondria via the positively charged species; d) linking and / or surrounding the mitochondria provided in steps (a) to (c) with a protective layer.

[0343] In some embodiments, the protective layer is a protective polymer. The protective polymer is as described above herein.

[0344] In some embodiments of the method of the present invention, the protective polymer is a linear or branched cationic polymer, and optionally, the linear or branched cationic polymer is electrostatically linked to one or more nucleic acid molecules. Preferably, the linear or branched cationic polymer is polyethyleneimine, RGD-modified polyethyleneimine, polylysine, RGD-modified polylysine, polyornithine, RGD-modified polyornithine, polyarginine, RGD-modified polyarginine, polypropyleneimine, RGD-modified polypropyleneimine, polyallylamine, RGD-modified polyallylamine, chitosan, RGD-modified chitosan, poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(2-(dimethylamino)ethyl methacrylate), poly(amidoamine), RGD-modified poly(amidoamine), or a combination thereof.

[0345] In some embodiments of the method of the present invention, the protective polymer is a linear or branched cationic block copolymer, and optionally, the linear or branched cationic block copolymer is electrostatically linked to one or more nucleic acid molecules. Preferably, the cationic block copolymer is poly(ethylene glycol)-block-polyethyleneimine, RGD-modified poly(ethylene glycol)-block-polyethyleneimine, poly(ethylene glycol)-block-polylysine, RGD-modified poly(ethylene glycol)-block-polylysine, poly(ethylene glycol)-block-polyornithine, RGD-modified poly(ethylene glycol)-block-polyornithine, poly(ethylene glycol)-block-polyarginine, RGD-modified poly(ethylene glycol)-block-polyarginine, poly(ethylene glycol)-block-polypropyleneimine, RGD-modified poly(ethylene glycol)-block-polypropyleneimine, poly(ethylene glycol)-block-polyallylamine, RGD-modified poly(ethylene glycol)-block-polyallylamine, poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-block-poly(amidoamine), RGD-modified poly(ethylene glycol)-block-poly(amidoamine) or a combination thereof.

[0346] In some embodiments of the method of the present invention, the protective polymer is a linear or branched cationic graft (g) copolymer, and optionally, the linear or branched cationic graft (g) copolymer is electrostatically linked to one or more nucleic acid molecules. Preferably, the cationic graft (g) copolymer is poly(ethylene glycol)-g-polyethyleneimine, RGD-modified poly(ethylene glycol)-g-polyethyleneimine, poly(ethylene glycol)-g-polylysine, RGD-modified poly(ethylene glycol)-g-polylysine, poly(ethylene glycol)-g-polyornithine, RGD-modified poly(ethylene glycol)-g-polyornithine, poly(ethylene glycol)-g-polyarginine, RGD-modified poly(ethylene glycol)-g-polyarginine, poly(ethylene glycol)-g-polypropyleneimine, RGD-modified poly(ethylene glycol)-g-polypropyleneimine, poly(ethylene glycol)-g-polyallylamine, RGD-modified poly(ethylene glycol)-g-polyallylamine, poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-g-poly(amidoamine), RGD-modified poly(ethylene glycol)-g-poly(amidoamine) or a combination thereof.

[0347] In some embodiments of the method of the present invention, the protective polymer is a linear or branched pegylated (PEG) cationic polymer, and optionally, the linear or branched pegylated (PEG) cationic polymer is electrostatically linked to one or more nucleic acid molecules. Preferably, the pegylated (PEG) cationic polymer is pegylated polyethyleneimine, RGD-modified pegylated polyethyleneimine, pegylated polylysine, RGD-modified pegylated polylysine, histidyl polylysine, pegylated polyornithine, RGD-modified pegylated polyornithine, pegylated polyarginine, RGD-modified pegylated polyarginine, pegylated polypropyleneimine, RGD-modified pegylated polypropyleneimine, pegylated polyallylamine, RGD-modified pegylated polyallylamine, pegylated chitosan, RGD-modified pegylated chitosan, pegylated poly(2-(dimethylamino)ethyl methacrylate), RGD-modified pegylated poly(2-(dimethylamino)ethyl methacrylate), pegylated poly(amidoamine), RGD-modified pegylated poly(amidoamine) or a combination thereof.

[0348] In some embodiments of the method of the present invention, the protective layer is a lipid formulation, optionally the lipid formulation is a cationic lipid formulation, and further optionally the cationic lipid formulation is electrostatically linked to one or more nucleic acid molecules. Preferably, the lipid formulation comprises DC-cholesterol (3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DLinDMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoeylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleyloxy-3-dimethylaminopropane), DOGS (dioctadecylamidoglycyl spermine), DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium), DOTAP (1,2-dioleoyl-3-trimethylammonium propane chloride), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane chloride), UGG (unsaturated guanidinium glycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), Lipofectamine or a combination thereof. In a further embodiment, the lipid formulation further comprises another lipid, preferably the lipid is cholesterol, substituted or unsubstituted cholesterol, a cholesterol derivative, such as a hydroxylated cholesterol derivative (e.g., hydroxy cholesterol), a PEG-lipid, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DDA (dimethyldioctadecylammonium), 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, bis(monooleoylglycero)phosphate or a combination thereof.

[0349] In some embodiments of the method of the present invention, the protective polymer is a zwitterionic protective polymer, and optionally, the zwitterionic protective polymer is electrostatically linked to one or more nucleic acid molecules. Preferably, the zwitterionic protective polymer is selected from the co-assembly of cationic (carboxyl-functionalized) and anionic (amino-functionalized) copolyesters based on poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyethyleneimine-g-poly(2-methacryloyloxyethyl phosphorylcholine) (PEI-g-PMPC), poly(ε-caprolactone)-block-poly(butylene fumarate)-block-poly(ε-caprolactone) (PCL-b-PBF-b-PCL), poly(lactic acid-co-glycolic acid) (PLGA)-PCB block copolymer (PLGA-b-PCB).

[0350] In some embodiments of the method of the present invention, the protective layer is linked to the targeting moiety, and optionally, the protective layer linked to the targeting moiety is electrostatically linked to one or more nucleic acid molecules. The linking, targeting, and targeting moiety are as described above. Preferably, the targeting moiety is an antibody or a carbohydrate molecule.

[0351] In some embodiments of the method of the present invention, the protective layer is linked to an antibody, and optionally the protective layer is linked to the antibody, and the antibody is electrostatically linked to one or more nucleic acid molecules.

[0352] In some embodiments of the method of the present invention, the protective layer is linked to a carbohydrate, and optionally the protective layer linked to the carbohydrate is electrostatically linked to one or more nucleic acid molecules.

[0353] In a preferred embodiment, the present invention is a method in which mitochondria contain a positively charged species, the positively charged species is a polycationic polymer, and the weight ratio of the polycationic polymer to the protective layer is between about 1:2.

[0354] The method according to claim 100, wherein 50 μg to 200 μg of mitochondria are contacted with 0.1 pmol to 50 pmol of nucleic acid molecules and 0.2 μg to 10 μg of a protective layer.

[0355] - 50 micrograms of mitochondria corresponds to approximately 150 million mitochondria. 1 mg / mL of mitochondria (based on the Qubit protein assay) corresponds to approximately 3B mitochondria / mL (based on the particle counter).

[0356] - The concentration of the preparation of the protective polymer used in the preparation of the mitochondria of the present invention is 1 mg / mL. The amount of the protective polymer is from 0.1 mg to 10 mg.

[0357] - The concentration of the preparation of the nanoparticles used in the preparation of the mitochondria of the present invention is 1 mg / mL. The amount of the protective polymer is from 0.1 mg to 10 mg.

[0358] In a further embodiment, the method of the present invention may include a centrifugation step. In the context of the present invention, the centrifugation step enables the removal of components of the mitochondrial delivery vehicle, such as an unadded payload, such as a nucleic acid molecule, a positively charged species or a protective layer, facilitating the formation of the delivery vehicle. As will be appreciated by those skilled in the art, the centrifugation step may be carried out after any step that requires the removal of excess components of the delivery vehicle, such as excess payload, excess positively charged species, excess protective layer.

[0359] Thus, the method of the present invention a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one nucleic acid molecule in the presence of a positively charged species; c) adding at least one nucleic acid molecule to the mitochondria via the positively charged species; d) centrifuging the mitochondria provided in step (c); e) optionally, linking and / or surrounding the mitochondria provided in step (d) with a protective layer; may include.

[0360] In a further embodiment, the method of the present invention comprises: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with a positively charged species; c) optionally, centrifuging the mitochondria provided in step (b); d) contacting the mitochondria provided in steps (a) to (c) with at least one nucleic acid molecule; e) adding at least one nucleic acid molecule to the mitochondria via the positively charged species; f) optionally, centrifuging the mitochondria provided in step (d); g) optionally, linking and / or surrounding the mitochondria provided in step (d) with a protective layer. It may include.

[0361] In a further embodiment, the method of the present invention comprises: a) providing a preparation of mitochondria; b) contacting at least one nucleic acid molecule with a positively charged species to form a positively charged complex; c) contacting the mitochondria of (a) with the positively charged complex of (b); d) adding at least one nucleic acid molecule to the mitochondria via the positively charged species; f) optionally, centrifuging the mitochondria provided in step (d); g) optionally, linking and / or surrounding the mitochondria provided in step (d) with a protective layer. It may include.

[0362] In some embodiments, the method of the present invention comprises: a) providing a preparation of mitochondria; b) providing a nucleic acid molecule modified to contain an activated ester; c) adding the nucleic acid molecule provided in step (b) to an amine contained in a polypeptide in the outer membrane of the mitochondria. d) centrifuging the mitochondria provided in step (c); e) optionally, linking and / or surrounding the mitochondria provided in step (d) with a protective layer; and may include.

[0363] In some embodiments, the method of the present invention comprises: a) providing a preparation of mitochondria; b) encapsulating a nucleic acid molecule in a nanoparticle, wherein the surface of the nanoparticle comprises a chemical group capable of covalently binding to a polypeptide in the outer membrane of the mitochondria; c) adding the nucleic acid molecule provided in step (b) to a polypeptide in the outer membrane of the mitochondria; d) centrifuging the mitochondria provided in step (c); e) optionally, linking and / or surrounding the mitochondria provided in step (d) with a protective layer; and may include.

[0364] In some embodiments, the method of the present invention comprises: a) providing a preparation of mitochondria; b) contacting the mitochondria containing an antigen on the outer membrane of the mitochondria provided in step (a) with at least one nucleic acid molecule linked to an antibody; c) adding at least one nucleic acid molecule to the mitochondria via the antibody, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria; d) centrifuging the mitochondria provided in step (c); e) optionally, linking and / or surrounding the mitochondria provided in step (d) with a protective layer; and may include.

[0365] In some embodiments, the method of the present invention comprises: a) providing a preparation of mitochondria; b) contacting the mitochondria provided in step (a) with at least one nucleic acid molecule linked to a mitochondrially targeted small molecule; c) adding at least one nucleic acid molecule to the mitochondria via the mitochondrially targeted small molecule; d) centrifuging the mitochondria provided in step (c); e) optionally, linking and / or surrounding the mitochondria provided in step (d) with a protective layer; may be included.

[0366] The present invention also provides mitochondria comprising one or more polypeptides added to the outer membrane of the mitochondria. Thus, the products, methods, devices and uses of the present invention can be carried out by adding polypeptides to the mitochondria instead of, or together with, nucleic acid molecules. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length that may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. In some embodiments, the polypeptides of the present invention comprise from 3 to 38,000 amino acids. As used herein, the term "protein" refers to a macromolecule comprising one or more polypeptide chains. Proteins may also include non-peptidic components such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to the protein by the cell in which the protein is produced and vary depending on the cell type. Some proteins are defined herein with respect to their amino acid backbone structure. As used herein, the term "peptide" refers to a polypeptide having from 2 to 100 amino acid monomers.

[0367] The present invention is not particularly limited to any polypeptide. Any polypeptide of interest can be used as a payload added to the outer membrane of mitochondria. Thus, the present invention provides a polypeptide added to the outer membrane of mitochondria that is useful, for example, in therapy and / or gene editing. Generally, any polypeptide of interest may be added to the outer membrane of mitochondria. In the context of the present invention, the mitochondria may be positively charged or negatively charged. In the context of the present invention, the polypeptide may be positively charged or negatively charged. A positively charged polypeptide may be added to a negatively charged mitochondrion or entity. A negatively charged polypeptide may be added to a positively charged mitochondrion or entity. Any of the above constellations can lead to successful addition via electrostatic interaction as long as the mitochondria and the polypeptide have opposite charges or do not have the same charge at the respective pH of the environment in which the polypeptide contacts the mitochondria or entity, such as physiological pH (about 7.2). In a preferred embodiment, the positively charged polypeptide contains lysine, arginine or histidine. In a further embodiment, the negatively charged polypeptide contains aspartate or glutamate.

[0368] Accordingly, the present invention provides mitochondria comprising one or more polypeptides added to the outer membrane of mitochondria, wherein the one or more polypeptides are a) electrostatically added to the outer membrane of mitochondria, or b) covalently bound to the outer membrane of mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, or d) linked to a mitochondria-targeting small molecule.

[0369] In some embodiments, the polypeptide is negatively charged. In other embodiments, the polypeptide is positively charged.

[0370] The present invention provides a mitochondrion-polypeptide complex useful for the delivery of polypeptides to cells, tissues or organs. The present invention also provides the addition of polypeptides to mitochondria, and such polypeptides can be charged. One or more polypeptides can be electrostatically added to the outer membrane of mitochondria. One or more polypeptides can be electrostatically added to the outer membrane of mitochondria via a positively charged species. One or more positively charged polypeptides can be electrostatically added to the negatively charged outer membrane of mitochondria. One or more negatively charged polypeptides can be electrostatically added to the outer membrane of mitochondria via a positively charged species.

[0371] Mitochondria can electrostatically interact with polypeptides, thereby forming a complex comprising mitochondria and one or more polypeptides. Thus, electrostatic interactions can be used to add a positively charged entity to a negatively charged entity. In the context of the present invention, mitochondria can be either positively or negatively charged. In the context of the present invention, polypeptides can be either positively or negatively charged. Any of the above constellations can result in successful addition via electrostatic interactions as long as the mitochondria and the polypeptides have opposite charges or do not have the same charge. Mitochondria have a negatively charged surface to which a positively charged polypeptide can be electrostatically added. In one aspect, the present invention provides mitochondria comprising one or more polypeptides added to the outer membrane of mitochondria. The polypeptides can be electrostatically added to the outer membrane. The polypeptides can be charged polypeptides. The polypeptides can be positively charged polypeptides.

[0372] Mitochondria have a negatively charged surface that can be functionalized with cationic molecules, changing the surface charge of the outer mitochondrial membrane to a positive value (i.e., either partially or entirely positive). Subsequently, the positively charged mitochondria can be conjugated with a negatively charged polypeptide. In one aspect, the present invention provides mitochondria comprising one or more polypeptides added to the outer membrane of the mitochondria, and the one or more polypeptides are electrostatically added to the outer membrane of the mitochondria via a positively charged species. The present invention provides mitochondria comprising one or more polypeptides added to the outer membrane of the mitochondria, and the one or more polypeptides are electrostatically added to the outer membrane of the mitochondria via a positively charged species, and the polypeptides are negatively charged.

[0373] The polypeptide of the present invention is preferably positively or negatively charged. As used herein, "charge" or "charged" relates to the overall or net charge on a peptide or protein, i.e., the sum of the charges in the peptide or protein. One skilled in the art is aware of methods for determining the net charge of a given polypeptide at a given pH (e.g., physiological pH (about 7.2)). In the context of the present invention, the net charge of the polypeptide of the present invention is preferably negative or positive when in contact with mitochondria. Thus, one or more polypeptides can be electrostatically added to the outer membrane of mitochondria via a positively charged species. One or more polypeptides may be electrostatically added to the outer membrane of mitochondria via a polycationic species, which is a linear or branched polycationic polymer. One or more polypeptides may be electrostatically added to the outer membrane of mitochondria via a linear or branched polycationic polymer, such as polylysine, histidyl-polylysine, polyornithine, polyarginine, high mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivatives, diethylaminoethyl (DEAE)-dextran, poly(N-alkyl-4-vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose, or combinations thereof.

[0374] In the context of the present invention, the negative surface charge profile of mitochondria may also be useful for electrostatically adding one or more polypeptides to the outer membrane of mitochondria via positively charged nanoparticles or particles. Thus, positively charged nanoparticles or particles comprising one or more polypeptides can be electrostatically added to the negative surface of mitochondria.

[0375] Thus, one or more polypeptides can be electrostatically attached to the outer membrane of mitochondria via positively charged nanoparticles. One or more polypeptides can be electrostatically attached to the outer membrane of mitochondria via positively charged particles. For complex formation, the polypeptide may be attached to the surface of the positively charged nanoparticle or positively charged particle, or may be encapsulated by the positively charged nanoparticle or positively charged particle. Thus, one or more polypeptides may be electrostatically attached to the outer membrane of mitochondria via positively charged nanoparticles, and one or more polypeptides may be attached to the surface of the positively charged nanoparticle or encapsulated by the positively charged nanoparticle. One or more polypeptides may be electrostatically attached to the outer membrane of mitochondria via positively charged particles, and one or more polypeptides may be attached to the surface of the positively charged particle or encapsulated by the positively charged particle.

[0376] Generally, the present invention is not limited to any particular nanoparticle or particle for addition to mitochondria and addition or encapsulation of polypeptides. Thus, one or more polypeptides can be attached to or encapsulated by the surface of lipid nanoparticles, dendrimer nanoparticles, micelle nanoparticles, protein nanoparticles, liposomes, non-porous silica nanoparticles, mesoporous silica nanoparticles, silicon nanoparticles, gold nanoparticles, gold nanowires, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, titanium dioxide nanoparticles, carbon nanotubes, carbon dot nanoparticles, polymer nanoparticles, zeolite nanoparticles, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, quantum dot nanoparticles, zinc oxide nanoparticles, zirconium oxide nanoparticles, graphene or graphene oxide nanoparticles.

[0377] In addition, one or more polypeptides may be added to the surfaces of lipid particles, dendrimer particles, micelle particles, protein particles, liposomes, non-porous silica particles, mesoporous silica particles, silicon particles, gold particles, gold wires, silver particles, platinum particles, palladium particles, titanium dioxide particles, carbon nanotubes (such as carbon microtubes), carbon dot particles, polymer particles, zeolite particles, aluminum oxide particles, hydroxyapatite particles, quantum dot particles, zinc oxide particles, zirconium oxide particles, graphene or graphene oxide particles.

[0378] Those skilled in the art will recognize that the above means of electrostatic addition can be applied to all products, methods, devices or uses described herein.

[0379] The mitochondria of the present invention are particularly useful because they can be stored stably for a long time without disintegrating. Therefore, the present invention provides mitochondria comprising one or more polypeptides added to the outer membrane of the mitochondria, wherein the one or more polypeptides are a) electrostatically added to the outer membrane of the mitochondria, optionally via a positively charged species, or b) covalently bound to the outer membrane of the mitochondria, or c) linked to an antibody that specifically binds to an antigen contained in the outer membrane of the mitochondria, or d) linked to a mitochondrial targeting small molecule, The mitochondria are stored at -80 °C in a conjugation buffer. The mitochondria of the present invention can be stored at -80 °C in a conjugation buffer without disintegrating for at least 2 months, 1 month, 3 weeks, 2 weeks, 1 week, or at least 5 days. Mitochondria containing one or more polypeptides added to the outer membrane can be stored in a conjugation buffer to maintain high colloidal stability (e.g., no aggregation / agglomeration or disintegration). Mitochondria containing one or more polypeptides added to the outer membrane are stored in a conjugation buffer at low temperature (e.g., -80 °C) in the dark for storage up to 4 months after complex formation.

[0380] The polypeptide of the present invention can be functionalized with a targeting molecule (e.g., a small molecule targeting molecule, a targeting aptamer, a targeting peptide, a carbohydrate, a sugar, and a targeting antibody), a drug, a reporter molecule / nanoparticle (e.g., a fluorescent molecule, a metal nanoparticle, a magnetic nanoparticle, etc.), or a constrictor.

[0381] The polypeptide of the present invention can be formulated into nanoparticles, cationic lipid nanomedicines, block copolymers, cationic lipids, or cationic polymers.

[0382] In the context of the present invention, the polypeptide can also covalently bind to the outer membrane of mitochondria. A covalent bond or covalent linkage or covalent interaction is formed by a chemical bond involving the sharing of an electron pair between atoms. The polypeptide can be added to mitochondria via a peptide bond such as an amide bond. The mitochondria of the present invention having an amino group of a mitochondrial membrane-related protein can covalently bind to an N-hydroxysuccinimide ester (NHS)-functionalized nanoparticle, an NHS-modified oligonucleotide, or an NHS-modified molecule that forms a covalently bound ligand and a more stable conjugate.

[0383] Thus, one or more polypeptides may be covalently attached to the outer membrane of mitochondria. One or more polypeptides may be linked to a polypeptide of the outer membrane of mitochondria via an amide bond. One or more polypeptides may be linked via an amide bond to a polypeptide in the outer membrane of mitochondria, and one or more polypeptides are modified to undergo formation of an amide bond with an amine functional group contained in a polypeptide in the outer membrane of mitochondria. By covalently attaching nanoparticles containing polypeptides to mitochondria, polypeptides can also be added to mitochondria. Thus, one or more polypeptides may be linked via an amide bond to a polypeptide of the outer membrane of mitochondria, where one or more polypeptides are encapsulated in a nanoparticle (such as a lipid nanoparticle), and the nanoparticle contains a functional group that enables covalent attachment of the nanoparticle to a second polypeptide of the outer membrane of mitochondria. One or more polypeptides may be covalently attached to an N-hydroxysuccinimide ester. One or more polypeptides may be covalently attached to an N-hydroxysuccinimide ester, and the N-hydroxysuccinimide ester facilitates addition of the polypeptide to an amine contained in a second polypeptide of the outer membrane of mitochondria. One or more polypeptides encapsulated in a nanoparticle containing an N-hydroxysuccinimide ester, where the N-hydroxysuccinimide ester facilitates binding of the polypeptide to an amine contained in a second polypeptide in the outer membrane of mitochondria via the nanoparticle containing the N-hydroxysuccinimide ester.

[0384] In the context of the present invention, a polypeptide can also be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria. Such an antibody containing a polypeptide binds to mitochondria, thereby facilitating formation of a delivery platform. The present invention is not limited to any particular antigen or antibody, and generally, the present invention can be practiced using an antibody that specifically binds to any antigen contained in the outer membrane of mitochondria, thereby facilitating formation of a mitochondria-polypeptide complex.

[0385] Generally, one or more polypeptides can be linked to any antibody that specifically binds to an antigen contained in mitochondria. Exemplary antigens are AIF, GCSH, MRPL40, TIMM23, ATP5A, HSP60, OPA1, TOM70, ATP5F1, OXA1L, TOMM20, BCS1L, mitofilin, prohibitin, TUFM, COX4, mitofusin 1, SDHB, UQCRC1, COX5b, mitofusin 2, SSBP1, VDAC1.

[0386] Preferably, one or more polypeptides can be linked to any antibody that specifically binds to an antigen contained in the outer membrane of mitochondria. Thus, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, and preferred antigens are any one of OPA1, TOM70, TOMM20, mitofusin 1, mitofusin 2 or VDAC1.

[0387] The polypeptide can be covalently linked to an antibody that forms a polypeptide-antibody complex capable of binding to an antigen of mitochondria. Thus, the polypeptide can be covalently linked to an antibody that forms a polypeptide-antibody complex capable of binding to an antigen contained in the outer membrane of mitochondria.

[0388] The polypeptide can be electrostatically linked to a modified antibody such as an antibody containing a positive or negative charge that forms a polypeptide-antibody complex capable of binding to an antigen of mitochondria. Thus, the polypeptide can be electrostatically linked to a modified antibody such as an antibody containing a positive or negative charge that forms a polypeptide-antibody complex capable of binding to an antigen contained in the outer membrane of mitochondria.

[0389] By using an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, nanoparticles such as lipid nanoparticles containing a polypeptide can be added, thereby promoting the addition of the polypeptide to mitochondria. Accordingly, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, one or more polypeptides are encapsulated in a nanoparticle (such as a lipid nanoparticle), and the nanoparticle is covalently bound to the antibody. One or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, the nanoparticle is electrostatically linked to the antibody, the antibody is a modified antibody, and the modified antibody has one or more positive charges. Further, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, the nanoparticle is electrostatically linked to the antibody, the antibody is a modified antibody, and the modified antibody has one or more negative charges.

[0390] The nanoparticles containing a nucleic acid molecule are not particularly limited and may be any of the above nanoparticles. In some embodiments, the nanoparticles are substantially neutral (i.e., net neutral) nanoparticles. In some embodiments, the nanoparticles are charged nanoparticles such as positively or negatively charged nanoparticles. In some embodiments, the nanoparticles are charged nanoparticles, for example, nanoparticles having a positive "zeta potential" or a positive "surface charge", or nanoparticles having a negative "zeta potential" or a negative "surface charge".

[0391] The polypeptide can also be electrostatically linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria. The binding is facilitated by the opposite charges of the antibody and the polypeptide in the environment where the antibody contacts the polypeptide. Accordingly, a positively charged polypeptide may be electrostatically linked to a negatively charged antibody, and vice versa. Accordingly, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, one or more polypeptides are electrostatically linked to a modified antibody, and the modified antibody has one or more positive or negative charges.

[0392] The polypeptide can also be linked to an entity and then linked to an antibody. Such an entity can be biotin linked to an avidin-conjugated antibody. Thus, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, one or more polypeptides are covalently linked to biotin, biotin is linked to an antibody, and the antibody is an avidin-conjugated antibody. Further, the polypeptide can also be linked to an entity that is linked to an antibody when added to or encapsulated in lipid nanoparticles. Thus, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, one or more polypeptides are added to or encapsulated in nanoparticles, the nanoparticles are covalently bound to biotin, biotin is linked to an antibody, and the antibody is an avidin-conjugated antibody.

[0393] The polypeptide can also be linked to an entity and then linked to an antibody. Such an entity can be an activated ester linked to an antibody. Thus, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the outer membrane of mitochondria, one or more polypeptides are covalently bound to an activated ester, and the activated ester is linked to the antibody via an amide bond. Further, the polypeptide can also be linked to an entity that is linked to an antibody when added to or encapsulated in nanoparticles (such as lipid nanoparticles). Thus, one or more polypeptides may be linked to an antibody that specifically binds to an antigen contained in the ou...

Claims

1. A mitochondria comprising one or more payloads attached to the outer membrane of a mitochondria, wherein the payloads are indirectly attached to the outer membrane of the mitochondria electrostatically.

2. The aforementioned payload, i) a nucleic acid molecule; ii) polypeptide, iii) Drugs, or iv) One or more combinations of (i) to (iii) The mitochondria according to claim 1, which is one or more of the following.

3. The mitochondria according to claim 1, wherein the payload is charged.

4. The mitochondria according to claim 1, wherein the payload has the same net charge as the net charge of the mitochondria.

5. The mitochondria according to claim 4, wherein both the payload and the mitochondria have a net negative charge, and the payload is attached to the mitochondria via positively charged species.

6. The mitochondria according to claim 5, wherein the positively charged species is a polycation species, or the positively charged species is a positively charged nanoparticle or a positively charged particle.

7. The mitochondria according to claim 6, wherein the polycation species is a linear or branched polycationic polymer.

8. The mitochondria according to claim 7, wherein the linear or branched polycationic polymer is polylysine, histidylated polylysine, polyornithine, polyarginine, high mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivative, diethylaminoethyl (DEAE)-dextran, poly(N-alkyl-4-vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose, or a combination thereof.

9. The one or more nucleic acid molecules described above The positively charged nanoparticles or positively charged particles are attached to the surface of the aforementioned positively charged nanoparticles or particles, The positively charged nanoparticles or positively charged particles encapsulated, and / or, The mitochondria according to claim 6, wherein the positively charged nanoparticles and / or particles are lipid nanoparticles, dendrimer nanoparticles, micellar nanoparticles, protein nanoparticles, liposomes, nonporous silica nanoparticles, mesoporous silica nanoparticles, silicon nanoparticles, gold nanoparticles, gold nanowires, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, titanium dioxide nanoparticles, carbon nanotubes, carbon dot nanoparticles, polymer nanoparticles, zeolite nanoparticles, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, quantum dot nanoparticles, zinc oxide nanoparticles, zirconium oxide nanoparticles, graphene, or graphene oxide nanoparticles.

10. The payload has a net charge different from the net charge of the mitochondria, or The payload has a net charge different from the net charge of the mitochondria, and the payload and mitochondria are attached via a zwitterionic species, or The aforementioned payload is uncharged, or The mitochondrion according to claim 1, wherein the payload is uncharged and is attached to a positively charged species, where the positively charged species is as defined in claim 6.

11. The one or more nucleic acid molecules (i) electrostatically bound to the antibody, (ii) Encapsulated in nanoparticles electrostatically linked to the antibody, Mitochondria according to claim 2.

12. The mitochondrion according to claim 11, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondrion, and the antigen is OPA1, TOM70, TOMM20, mitofucin1, mitofucin2, or VDAC1.

13. The mitochondria according to claim 1, wherein the mitochondria are connected to and / or surrounded by a protective layer.

14. The protective layer is a protective polymer, or The protective layer is a lipid preparation, or The protective layer is linked to the targeting portion, antibody, or carbohydrate. Mitochondria according to claim 13.

15. The aforementioned protective polymer (i) Linear or branched cationic polymers, (ii) linear or branched cationic block copolymers, or (iii) cationic graft (g) copolymers, or (iv) linear or branched PEG cationic polymers. That is, Mitochondria according to claim 14.

16. (i) The linear or branched cationic polymer is polyethyleneimine, RGD-modified polyethyleneimine, polylysine, RGD-modified polylysine, polyornithine, RGD-modified polyornithine, polyarginine, RGD-modified polyarginine, polypropyleneimine, RGD-modified polypropyleneimine, polyallylamine, RGD-modified polyallylamine, chitosan, RGD-modified chitosan, poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(2-(dimethylamino)ethyl methacrylate), poly(amideamine), RGD-modified poly(amideamine), or a combination thereof. (ii) The cationic block copolymer is poly(ethylene glycol)-block-polyethyleneimine, RGD-modified poly(ethylene glycol)-block-polyethyleneimine, poly(ethylene glycol)-block-polylysine, RGD-modified poly(ethylene glycol)-block-polylysine, poly(ethylene glycol)-block-polyornithine, RGD-modified poly(ethylene glycol)-block-polyornithine, poly(ethylene glycol)-block-polyarginine, RGD-modified poly(ethylene glycol)-block-polyarginine, poly(ethylene glycol)-block-poly Polypropyleneimine, RGD-modified poly(ethylene glycol)-block-polypropyleneimine, poly(ethylene glycol)-block-polyallylamine, RGD-modified poly(ethylene glycol)-block-polyallylamine, poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-block-poly(amideamine), RGD-modified poly(ethylene glycol)-block-poly(amideamine), or combinations thereof. (iii) The cationic graft (g) copolymer is poly(ethylene glycol)-g-polyethyleneimine, RGD-modified poly(ethylene glycol)-g-polyethyleneimine, poly(ethylene glycol)-g-polylysine, RGD-modified poly(ethylene glycol)-g-polylysine, poly(ethylene glycol)-g-polyornithine, RGD-modified poly(ethylene glycol)-g-polyornithine, poly(ethylene glycol)-g-polyarginine, RGD-modified poly(ethylene glycol)-g-polyarginine, poly(ethylene glycol)-g-polypro Pyreneimine, RGD-modified poly(ethylene glycol)-g-polypropyleneimine, poly(ethylene glycol)-g-polyallylamine, RGD-modified poly(ethylene glycol)-g-polyallylamine, poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), RGD-modified poly(ethylene glycol)-g-poly(2-(dimethylamino)ethyl methacrylate), poly(ethylene glycol)-g-poly(amideamine), RGD-modified poly(ethylene glycol)-g-poly(amideamine), or a combination thereof, or (iv) The PEG-modified cationic polymer is PEG-modified polyethyleneimine, RGD-modified PEG-modified polyethyleneimine, PEG-modified polylysine, RGD-modified PEG-modified polylysine, histidylated polylysine, PEG-modified polyornithine, RGD-modified PEG-modified polyornithine, PEG-modified polyarginine, RGD-modified PEG-modified polyarginine, PEG-modified polypropyleneimine, RGD-modified PEG-modified polypropyleneimine, PEG-modified polyallylamine, RGD-modified PEG-modified polyallylamine, PEG-modified chitosan, RGD-modified PEG-modified chitosan, PEG-modified poly(2-(dimethylamino)ethyl methacrylate), RGD-modified PEG-modified poly(2-(dimethylamino)ethyl methacrylate), PEG-modified poly(amideamine), RGD-modified PEG-modified poly(amideamine), or a combination thereof. Mitochondria according to claim 15.

17. The aforementioned lipid preparations are DC-cholesterol (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DLinDMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleylmethyl-4-dimethylaminobutyric acid), DODMA (1,2-dioleyloxy-3-dimethylaminopropane), DOGS (dioctadecylamideglycylspermine), DOSPA (2,3-dioleyloxy This includes -N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium), DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane chloride), DOTMA (1,2-di-Ooctadecenyl-3-trimethylammoniumpropane chloride), UGG (unsaturated guanidinium glycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), lipofectamine, or combinations thereof, or The mitochondria according to claim 14, wherein the lipid preparation further comprises another lipid.

18. The mitochondria according to claim 14, wherein the mitochondria are linked to and / or surrounded by a zwitterionic protective polymer.

19. The mitochondria according to claim 18, wherein the zwitterionic protective polymer is selected from a coassembly of cationic (carboxyl-functionalized) and anionic (amino-functionalized) copolyesters based on poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyethyleneimine-g-poly(2-methacryloyloxyethyl phosphorylcholine) (PEI-g-PMPC), poly(ε-caprolactone)-block-poly(butylene fumarat)-block-poly(ε-caprolactone) (PCL-b-PBF-b-PCL), and poly(lactic acid-co-glycolic acid) (PLGA)-PCB block copolymer (PLGA-b-PCB).

20. A composition comprising a plurality of mitochondria as described in claim 1.

21. A pharmaceutical composition comprising a plurality of mitochondria and a pharmaceutically acceptable carrier as described in claim 1.

22. The pharmaceutical composition according to claim 21, wherein the pharmaceutical composition is formulated as a solution or an aerosol.

23. A pharmaceutical composition according to claim 20, for use as a pharmaceutical or in gene therapy.

24. (i) cardiovascular disease, (ii) Age-related diseases, (iii) kidney disease, or (iv) cancer A pharmaceutical composition according to claim 21 for use in the treatment of [condition].

25. The pharmaceutical composition according to claim 21 for use in in vitro, ex vivo, or in vivo genome editing.

26. A pharmaceutical composition according to claim 21 for use in radiotherapy.

27. A pharmaceutical composition according to claim 21 for delivering a payload to a target organ, wherein the pharmaceutical composition is administered into the bloodstream of an object requiring the pharmaceutical composition, wherein the pharmaceutical composition is administered into the bloodstream upstream of the target organ, or the pharmaceutical composition is administered by inhalation.

28. A method for attaching a payload to the outer membrane of mitochondria, a) A step of providing a mitochondrial preparation, b) A step of bringing the mitochondria provided in step (a) into contact with at least one payload in the presence of positively charged species, c) A step of indirectly electrostatically attaching the at least one payload to the mitochondria via the positively charged seeds, A method for attaching a payload to the outer membrane of mitochondria, including [specific example].

29. a) The at least one payload is in contact with the positively charged species and the mitochondria simultaneously. b) The at least one payload comes into contact with the positively charged species before bringing the positively charged complex into contact with the mitochondria to form a positively charged complex, or c) The mitochondria are brought into contact with the positively charged species, and then into contact with the at least one payload. The method according to claim 28.

30. The mitochondria are brought into contact with the at least one payload and the positively charged species in a buffer, where, The method according to claim 28, wherein the buffering agent comprises or consists of HEPES, EGTA, trehalose, CHES, and sodium phosphate dihydrate.

31. The mitochondria are brought into contact with the at least one payload and the positively charged species at room temperature for at least 5 minutes, for example, at least 10, 20, 30, 40, 50, 60 or 120 minutes, and / or The method according to claim 28, wherein the mitochondria are brought into contact with the at least one payload and the positively charged species in the dark.

32. The method according to claim 28, wherein the payload is a nucleic acid molecule that is DNA or RNA.

33. The method according to claim 28, wherein the positively charged species is a polycationic species, and the polycationic species is a linear or branched polycationic polymer, or the positively charged species is a positively charged nanoparticle.

34. The method according to claim 33, wherein the linear or branched polycationic polymer is polylysine, histidylated polylysine, polyornithine, polyarginine, high mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), polyallylamine derivative, diethylaminoethyl (DEAE)-dextran, poly(N-alkyl-4-vinylpyridinium), poly(amideamine), cationic gelatin, cationic cellulose, or a combination thereof.

35. The above method further steps a) A step of adding the at least one payload to the surface of the positively charged nanoparticles, or b) The step of encapsulating the at least one payload within the positively charged nanoparticles. The method according to claim 33, including the method described in claim 33.

36. The method according to claim 33, wherein the positively charged nanoparticles are lipid nanoparticles, dendrimer nanoparticles, micelle nanoparticles, protein nanoparticles, liposomes, nonporous silica nanoparticles, mesoporous silica nanoparticles, silicon nanoparticles, gold nanoparticles, gold nanowires, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, titanium dioxide nanoparticles, carbon nanotubes, carbon dot nanoparticles, polymer nanoparticles, zeolite nanoparticles, aluminum oxide nanoparticles, hydroxyapatite nanoparticles, quantum dot nanoparticles, zinc oxide nanoparticles, zirconium oxide nanoparticles, graphene, or graphene oxide nanoparticles.

37. A method for preparing mitochondria containing a payload, a) A step of providing a mitochondrial preparation, b) Mitochondria, i) If both the payload and the mitochondria have a net negative charge, positively charged species, ii) If the payload has a net charge different from the net charge of the mitochondria, the payload, or iii) If the payload is uncharged, the payload attached to a positively charged species The process of bringing it into contact with, c) A step of obtaining mitochondria according to any one of claims 1 to 12, A method for preparing mitochondria containing a payload, including [a specific component].

38. The method of claim 37, further comprising, after step c), a step of bringing the mitochondria into contact with a component to form a protective layer, and a step of obtaining the mitochondria described in claim 13.

39. The method according to claim 37, wherein 50 μg to 200 μg of mitochondria are brought into contact with a payload of 0.1 to 50 pmol and 0.02 to 10 μg of the positively charged species.

40. The method according to claim 28, wherein the mitochondria include positively charged species, the positively charged species are a polycationic polymer, and the ratio of the polycationic polymer to the protective layer is about 1:

2.

41. The method according to claim 38, wherein 50 μg to 200 μg of mitochondria are brought into contact with a payload of 0.1 to 50 pmol and 0.2 to 10 μg of the protective layer.

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